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BIOCHEMICAL, NUTRITIONAL AND END USE ASPECTS
OF STEVIA AS POTENTIAL NATURAL SWEETENER
By
MUHAMMAD FARHAN JAHANGIR CHUGHTAI
2007-ag-1073
M.Sc. (Hons.) Food Technology
A thesis submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
IN
FOOD TECHNOLOGY
NATIONAL INSTITUTE OF FOOD SCIENCE & TECHNOLOGY
FACULTY OF FOOD, NUTRITION & HOME SCIENCES
UNIVERSITY OF AGRICULTURE, FAISALABAD
PAKISTAN
2017
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THIS HUMBLE EFFORT IS
DEDICATED
TO
HOLY PROPHET HAZRAT MUHAMMAD
(S.A.W.W.)
MY
PARENTS
RESPECTED SUPERVISOR
LOVING BROTHER
AND
DEAREST FRIENDS
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ACKNOWLEDGEMENTS
I offer my humblest sense of gratitude to “ALMIGHTY ALLAH” the most beneficent, the merciful and the gracious,
created and blessed me to accomplish the present research project. I submit my modest gratitude from core of my
heart to HOLY PROPHET HAZRAT MUHAMMAD (Peace Be Upon Him), the source of knowledge and wisdom
for all mankind.
I am extremely thankful to my kind, affectionate, incredible and demonstrative supervisor, Dr. Imran Pasha,
Assistant Professor, National Institute of Food Science and Technology, University of Agriculture, Faisalabad for his
progressive guidance and constant encouragement during planning, research and write-up of this dissertation.
I expand my sincere admiration to Prof. Dr. Tahir Zahoor, Director General, National Institute of Food Science and
Technology, University of Agriculture, Faisalabad for his earnest support, guidance and affection throughout my
career as a student in the Institute.
I am highly indebted to Prof. Dr. Faqir Muhammad Anjum, (T.I), Vice Chancellor, University of Gambia, Gambia
for his kind and encouraging support throughout my university education carrier. His devotion, efforts and guidance
will always work as light house for me in professional career.
I am also indebted to my supervisory committee members, Prof. Dr. Masood Sadiq Butt, Dean, Faculty of Food
Nutrition and Home Sciences, University of Agriculture, Faisalabad and Prof. Dr. Muhammad Asghar, Dean
Faculty of Sciences, University of Agriculture, Faisalabad for their caring attitude and kind assistance during the
course of study.
I am very grateful to Mr. Shaukat Ali and Mr. Tahir-ur-Rehman for his guidance and assistance in the research
work. I want to express my great appreciation and sincerest gratitude to my seniors Dr. Ahmed Din, Dr. Bahzad
Afzal, Dr. Muhammad Adnan Nasir, Dr. Shabbir Ahmed and Dr. Waqas Bin Niaz for their dexterous, dynamic,
untiring help, friendly behavior and moral support during my whole study. I am indeed thankful to all my loving and
dearest friends; especially; Mr. Adnan Khaliq, Mr. Atif Liaqat, Mr. Tariq Mehmood, Mr. Husnain Raza, Misa
Hira Iftikhar, Miss. Samreen Ahsan, Miss Nazia Ali, Miss Iqra Yasmeen and Miss Saima Naz for their earnest
support throughout the course of my studies.
I am also very thankful to my Nutrition and Food Safety Lab fellows specially Mr. Muhammad Sajid Manzoor,
Miss Farah Ahmed, Miss Ayesha Riaz, Mr. Abdullah Salik, Hafiz Ahmed Toor, Mr. Ali Zaib, and Mr. Farman
Ali for their support, care and encouraging attitude with me during my studies.
Last but not the least, no acknowledgements could ever adequately express my obligation to my affectionate parents
Mr. & Mrs. Arshad Jahangir Chughtai, Mr. Shamshad Ahmed Chughtai (Late Taya Jan) & Mrs. Parveen
Mehboob (Late Phopho) whose endless efforts and best wishes sustained me at all stages of my life and encouraged
me for achieving high ideas of life and whose hands always remain raised in prayer for my success and my loving
brothers, Mr. Imran Chughtai, Mr. Muhammad Rehan Jahangir Chughtai, Mr. Ahmed Shoaib and Mr. Salman
Ali and my sweet Nephews and Nieces for their inspiring encouragement and moral support.
Muhammad Farhan Jahangir Chughtai
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TABLE OF CONTENTS 1 Chapter 1 ................................................................................................................................. 1
INTRODUCTION .......................................................................................................................... 1
2 Chapter 2 ................................................................................................................................. 5
REVIEW OF LITERATURE ......................................................................................................... 5
2.1 Global regularity status of Stevia ..................................................................................... 6
2.1 Stevia cultivation .............................................................................................................. 8
2.2 Structural expression of Steviosides ................................................................................ 9
2.3 Chemical composition of Stevia .................................................................................... 10
2.3.1 Proximate composition of Stevia ............................................................................ 10
2.3.2 Mineral composition of Stevia ................................................................................ 11
2.3.3 Fatty acid profile of Stevia ...................................................................................... 12
2.4 Functional properties of Stevia ...................................................................................... 13
2.5 Metabolism of Steviosides ............................................................................................. 14
2.6 Phytochemical and antioxidant potential of Stevia ........................................................ 15
2.6.1 Total Phenolic (TPC) and Flavonoid contents (TFC) of Stevia ............................. 16
2.6.2 Antioxidant potential of Stevia ............................................................................... 17
2.7 Extraction methods of SGs/ Steviosides ........................................................................ 18
2.8 Therapeutic remunerations of Stevia.............................................................................. 19
2.8.1 Glucoregulation....................................................................................................... 20
2.8.2 Blood pressure regulation ....................................................................................... 20
2.8.3 Anticancer benefits of Stevia .................................................................................. 21
2.8.4 Renal functions regulations by Stevia..................................................................... 22
2.8.5 Obesity control by Stevia ........................................................................................ 22
2.8.6 Inflammatory bowel disease (IBD) management ................................................... 23
2.8.7 Dental maladies management ................................................................................. 23
2.9 Product development with Stevia ................................................................................... 24
2.9.1 Temperature stability of Steviosides ....................................................................... 24
2.9.2 pH stability of Steviosides ...................................................................................... 24
2.9.3 Steviosides stability in product development ......................................................... 25
3 CHAPTER 3 ......................................................................................................................... 27
MATERIALS AND METHODS .................................................................................................. 27
3.1 Procurement of raw material .......................................................................................... 27
3.2 Preparation of raw material ............................................................................................ 27
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3.3 Proximate analysis.......................................................................................................... 27
3.3.1 Moisture Content .................................................................................................... 27
3.3.2 Ash content ............................................................................................................. 27
3.3.3 Crude fat.................................................................................................................. 28
3.3.4 Crude protein .......................................................................................................... 28
3.3.5 Crude fiber .............................................................................................................. 29
3.3.6 Nitrogen free extracts (NFE) .................................................................................. 29
3.4 Functional properties ...................................................................................................... 29
3.4.1 Bulk density ............................................................................................................ 29
3.4.2 pH ............................................................................................................................ 29
3.4.3 Swelling power ....................................................................................................... 30
3.4.4 Oil holding capacity ................................................................................................ 30
3.4.5 Water absorption capacity....................................................................................... 30
3.5 Mineral analysis ............................................................................................................. 30
3.6 Fatty acid profile ............................................................................................................ 31
3.7 Steviosides Extraction .................................................................................................... 31
3.7.1 Steviosides extraction by different solvents ........................................................... 31
3.7.2 Supercritical fluid extraction of Steviosides ........................................................... 32
3.8 Phytochemical analysis .................................................................................................. 32
3.8.1 Total phenolic content............................................................................................. 32
3.8.2 Total flavonoids content ......................................................................................... 32
3.9 In vitro Antioxidant assays ............................................................................................. 33
3.9.1 DPPH (1-1-diphenyl 2-picryl hydrazyl) free radical scavenging activity of Stevia 33
3.9.2 Ferric reducing-antioxidant power (FRAP) assay .................................................. 33
3.9.3 ABTS radical cation decolorization assay .............................................................. 33
3.10 Quantification of Steviosides through HPLC ............................................................. 34
3.11 Functional groups identification of Stevia with FT-IR .............................................. 34
3.12 Value addition of Stevia ............................................................................................. 35
3.12.1 Sensory analysis of Stevia product ......................................................................... 35
3.12.2 Physicochemical characterization of Stevia cookies .............................................. 36
3.12.3 Energy value evaluation of Stevia .......................................................................... 36
3.12.4 Color analysis.......................................................................................................... 36
3.12.5 Texture profile ........................................................................................................ 36
3.12.6 Total phenolic & Flavonoid content of Stevia cookies ........................................... 36
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3.12.7 Evaluation of in vitro radical scavenging activity of Stevia cookies ...................... 37
3.13 Selection of best treatments ........................................................................................ 37
3.13.1 Efficacy trial............................................................................................................ 37
3.13.2 Serum glucose and insulin levels ............................................................................ 39
3.13.3 Serum lipid profile analysis .................................................................................... 39
3.13.4 Liver function tests ................................................................................................. 39
3.13.5 Renal function tests ................................................................................................. 40
3.13.6 Hematological analysis ........................................................................................... 40
3.14 Statistical analysis....................................................................................................... 40
4 CHAPTER 4 ......................................................................................................................... 41
RESULTS AND DISCUSSION ................................................................................................... 41
4.1 Proximate composition of raw material ......................................................................... 41
4.2 Functional properties of Stevia ...................................................................................... 43
4.3 Mineral analysis of Stevia .............................................................................................. 46
4.4 Fatty acid profile ............................................................................................................ 49
4.5 Phytochemical analysis & antioxidant assay of Stevia .................................................. 52
4.5.1 Extraction efficiencies ............................................................................................ 52
4.5.2 Phytochemical analysis ........................................................................................... 53
4.5.3 Antioxidant activity of Stevia ................................................................................. 57
4.6 Fourier Transform Infra-red Spectrophotometric analysis (FTIR) of Stevia ................. 63
4.6.1 Stevia powder.......................................................................................................... 63
4.6.2 Stevia water extract ................................................................................................. 65
4.6.3 Stevia methanol extract ........................................................................................... 67
4.6.4 Stevia ethanol extract .............................................................................................. 69
4.7 HPLC quantification of Steviosides ............................................................................... 71
4.8 Value addition of Stevia in cookies................................................................................ 81
4.8.1 Chemical composition of Stevia cookies ................................................................ 81
4.8.2 Antioxidant activity of Stevia cookies .................................................................... 87
4.8.3 Sensory analysis of Stevia cookies ......................................................................... 92
4.8.4 Color analysis of Stevia cookies ............................................................................. 97
4.8.5 Texture analysis of Stevia cookies ........................................................................ 100
4.8.6 Calorific analysis of Stevia cookies ...................................................................... 100
4.8.7 Spread factor of Stevia cookies............................................................................. 101
4.9 Efficacy study ............................................................................................................... 104
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4.9.1 Feed intakes .......................................................................................................... 104
4.9.2 Water intake .......................................................................................................... 108
4.9.3 Body weights ........................................................................................................ 110
4.9.4 Serum profile analysis........................................................................................... 113
4.9.5 Liver functions tests .............................................................................................. 127
4.9.6 Renal function tests ............................................................................................... 131
4.9.7 Hematological analysis ......................................................................................... 134
CHAPTER 5 ............................................................................................................................... 138
SUMMARY ............................................................................................................................ 138
CONCLUSIONS......................................................................................................................... 145
RECOMMENDATIONS ............................................................................................................ 146
LITERATURE CITED ............................................................................................................... 147
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LIST OF TABLES
Table 1. Treatment plan for cookies preparation with Stevia extract ........................................... 35 Table 2. Treatment plan for efficacy trials .................................................................................... 38
Table 3. Proximate analysis (g/100g) of wheat flour and Stevia .................................................. 45 Table 4. Functional properties of Stevia ....................................................................................... 45 Table 5. Mineral profile of Stevia ................................................................................................. 48 Table 6. Heavy metals in Stevia ................................................................................................... 48 Table 7. Fatty acid profiling of Stevia .......................................................................................... 51
Table 8. Mean squares for total phenolic contents of Stevia from different extracts ................... 54 Table 9. Mean values for total phenolic contents of Stevia from different extracts ..................... 54 Table 10. Mean squares for total flavonoids contents of Stevia from different extracts .............. 56 Table 11. Mean values for total flavonoids contents of Stevia from different extracts ................ 56
Table 12. Mean squares for DPPH activity of Stevia from different extracts .............................. 58 Table 13. Mean values for DPPH activity of Stevia from different extracts ................................ 58
Table 14. Mean squares for FRAP activity of Stevia from different extracts .............................. 60 Table 15. Mean values for FRAP activity of Stevia from different extracts ................................ 60
Table 16. Mean squares for ABTS assay activity of Stevia from different extracts .................... 62 Table 17. Mean values for ABTS assay activity of Stevia from different extracts ...................... 62 Table 18. FTIR spectrum values of Stevia leaves powder............................................................ 64
Table 19. FTIR spectrum values of aqueous Stevia extract .......................................................... 66 Table 20. FTIR spectrum values of methanolic Stevia extract ..................................................... 68
Table 21. FTIR spectrum values of ethanolic Stevia extract ........................................................ 70 Table 22. Mean squares for HPLC quantification of Steviosides in Stevia extracts .................... 74 Table 23. Mean values for HPLC quantification of Steviosides in Stevia extracts (mg/kg) ........ 74
Table 24. Mean squares values for chemical composition of Stevia Cookies .............................. 86
Table 25 Mean values (percentage) for chemical composition of Stevia Cookies ....................... 86 Table 26. Mean squares values for antioxidant potential of Stevia Cookies ................................ 91 Table 27. Mean values for antioxidant potential of Stevia Cookies ............................................. 91
Table 28. Mean squares values for Sensory attributes of Stevia Cookies .................................... 96 Table 29. Mean values for Sensory attributes of Stevia Cookies ................................................. 96
Table 30. Mean square values for Color analysis of Stevia Cookies ............................................ 99 Table 31. Mean values for Color analysis of Stevia Cookies ....................................................... 99
Table 32. Mean squares values for Hardness, Spread ratio & Calorific value of Stevia Cookies
..................................................................................................................................................... 103 Table 33. Mean values for Hardness, Spread ratio & Calorific values of Stevia Cookies ......... 103 Table 34. Effect of diets and time intervals on feed, water intake & body weight of rats in
different studies ........................................................................................................................... 106 Table 35. Effect of Stevia diets on glucose (mg/dL) .................................................................. 116 Table 36. Effect of Stevia diets on Insulin (µU/mL) .................................................................. 117
Table 37. Effect of Stevia diets on Cholesterol (mg/dL) ............................................................ 120 Table 38. Effect of Stevia diets on HDL (mg/dL) ...................................................................... 124 Table 39. Effect of Stevia diets on LDL (mg/dL) ....................................................................... 125 Table 40. Effect of Stevia diets on triglycerides (mg/dL) .......................................................... 126 Table 41. Effect of Stevia diets on serum AST (IU/L) ............................................................... 129 Table 42. Effect of Stevia diets on serum ALT (IU/L) ............................................................... 130
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Table 43. Effect of Stevia diets on serum ALP (IU/L) ............................................................... 130
Table 44. Effect of Stevia diets on Urea (mg/dL) ....................................................................... 133 Table 45. Effect of Stevia diets on creatinine (mg/dL) ............................................................... 133 Table 46. Effect of Stevia diets on red blood cell indices (cells/pL) .......................................... 136
Table 47. Effect of Stevia diets on white blood cell Indices (cells/nL) ..................................... 137 Table 48. Effect of Stevia diets on Platelets count ..................................................................... 137
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LIST OF FIGURES
Figure 1: FTIR spectrum of Stevia leaves powder ....................................................................... 64
Figure 2: FTIR spectrum of aqueous Stevia extract ..................................................................... 66 Figure 3: FTIR spectrum of methanolic Stevia extract ................................................................. 68 Figure 4: FTIR spectrum of ethanolic Stevia extract .................................................................... 70 Figure 5: Steviosides/SGs concentrations from different extracts of Stevia ................................ 75 Figure 6: Calibration curve for Stevioside standard ..................................................................... 76
Figure 7: HPLC chromatogram of Stevioside standard ................................................................ 76 Figure 8: Calibration curve for Rebaudioside A standard ............................................................ 77 Figure 9: HPLC chromatogram of Rebaudioside A standard ....................................................... 77 Figure 10: Calibration curve for Steviol standard ......................................................................... 78 Figure 11: HPLC chromatogram of Rebaudioside A standard ..................................................... 78
Figure 12: HPLC chromatogram of aqueous Stevia extract ......................................................... 79 Figure 13: HPLC chromatogram of ethanolic Stevia extract ........................................................ 79
Figure 14: HPLC chromatogram of methanolic Stevia extract .................................................... 80 Figure 15: HPLC chromatogram of Stevia supercritical extract (SFE) ........................................ 80
Figure 16: Feed intakes (g) of normal rats (study I) ................................................................... 107 Figure 17: Feed intakes (g) of Hyperglycemic rats (study II) .................................................... 107 Figure 18: Feed intakes (g) of hypercholesterolemic rats (study III) ......................................... 107
Figure 19: Water intakes (mL) of normal rats (study I) .............................................................. 109 Figure 20: Water intakes (mL) of hyperglycemic rats (study II) ................................................ 109
Figure 21: Water intakes (mL) of hypercholesterolemic rats (study III) .................................... 109 Figure 22: Water intake (mL) of normal rats (Study I) .............................................................. 112 Figure 23: Water intake (mL) of hyperglycemic rats (Study II) ................................................. 112
Figure 24: Water intake (mL) of hypercholesterolemic rats (Study III) ..................................... 112
Figure 25: Percent (%) reduction in glucose as compared to control ......................................... 116 Figure 26: Percent (%) increase in Insulin as compared to control ............................................ 117 Figure 27: Percent decrease in Cholesterol as compared to control ........................................... 120
Figure 28: Percent increase in HDL as compared to control ...................................................... 124 Figure 29: Percent decrease in LDL as compared to control ...................................................... 125 Figure 30: Percent decrease in triglycerides as compared to control .......................................... 126
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ABSTRACT
Lifestyle related health matters are among grave challenges to society which are prevalent due to
sedentary contemporary habits and poor dietary patterns. Nutritional and health augmenting facets
of Stevia rebaudiana as intense natural sweetener have been studied in different parts of world.
Current research was designed to enlighten biochemical, nutritional and end use perspectives of
well adapted and cultivated indigenous Stevia. Results of this study have established that the
chemical attributes include carbohydrates, crude protein, fat, fiber and mineral content. Functional
attributes have been well observed with slightly acidic to neutral pH 6.14, exhibits good swelling
power, WHC, OHC, Bulk density. K, P, Mg, Na, Fe are found in maximum amount coinciding
their ADI. Saturated, mono and polyunsaturated fatty acids like Palmitic, Palmitoleic, Stearic,
Linoleic, Linolenic and Oleic have been identified in appreciable quantities like 28.31%, 2.17%,
2.39%, 13.65%, 25.48 and 4.95% respectively. Steviosides structure and functional groups
determination was done by FTIR and concluded alcohols, alkanes, ketones, amines, esters,
carboxylic acids, alkenes, hydroxyl groups as the major functional groups in raw powder and water
extracts of Stevia. Appreciable amount of phytochemicals extracted from different solvents
exhibited total phenolic and content ranging 24.24±0.48 to 38.22±0.05mg GAE/g and total
flavonoid content as 19.88±0.11 to 32.10±0.54 mg CE/g respectively. The antioxidant activity of
Stevia is expressed by DPPH (42.41±1.05 to 57.99±1.49% inhibition) and FRAP assay
(236.57±1.37 to 345.36±3.27µMol Fe2+/g) respectively. Extracts from water, ethanol, methanol
and supercritical were characterized for Steviosides/SGs and found to be in appreciable quantities
as Stevioside (665.34±27.27 to 1107.95±50.96 mg/kg), Rebaudioside A (383.38±17.25 to
792.15±38.02 mg/kg) and Steviol (357.26±14.64 to 485.25±22.32 mg/kg). Stevia cookies were
prepared by replacing sucrose with Stevia powder (10, 20, 30%), water extract (1, 2, 3%) and
supercritical extract (1, 2, 3%). Overall acceptability was good in 10% powder treatments, 3%
water and supercritical extracts. Bio efficacy rats modeling trials were done in order to check their
impact against hyperglycemia and hypercholesterolemia disorders. Blood glucose level was
reduced from 4.54 to 7.00% due to diets enriched with stevia powder and extracts as compared to
control. Moreover, increase in insulin level was observed as 4.27 to 6.13 as compared to control.
In hypercholesterolemic rats, up surged cholesterol level was reportedly reduced as a function of
Stevia powder and extracts from 1.28 to 5.47%. Substantial increment in HDL, reduction in LDL
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and triglycerides was recorded in Stevia leaves powder and extracts treatments as 2.79-6.66%,
2.68-4.16% and 3.12-5.36 correspondingly. Therefore it can be deduced from the outcomes that
in addition to sweetness, Stevia possess a lot to provide for health betterment. Conclusively, Stevia
based products are recommended for health sustenance and controlling the metabolic disorder.
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1 Chapter 1
INTRODUCTION
People are getting workaholic with each day passing due to work abuse that have disturbed their
lifestyle a lot making them sluggish and lethargic, ultimately impacting their health status. Out of
many health disorders, excess of sugar and fats in diet are the most prevalent issues leading to
obesity, diabetes and other diet related ailments. Therefore, less dietary options are available for
masses to meet their needs (Ceunen and Geuns, 2013). A chronic metabolic disorder; diabetes
appear when insulin production is either not enough to meet the body requirements or the body
cells are not responding in correct way. Polyuria, escalation in hunger and thirst are due to upsurge
in blood sugar. The prime reasons behind are weight increase, obesity & diet disorders. The onset
of type 2 diabetes can be avoided by adopting healthy life style, regular physical excursion, having
balanced diet and weight management. According to Global Nutrition report (2016), 8.5% (422
Million) adults of world population were suffering from diabetes. Almost 1.5 million deaths were
recorded in 2012 due to diabetes. Additional 2.2 million deaths were recorded due to rise in blood
sugar level than the normal that leads to certain chronic ailments, obesity, cardiovascular diseases,
etc. Out of these 2.2 million deaths, almost 43% occur before 70 years of age. Till 2030, diabetes
will be the 7th foremost death causing aliment as declared by WHO (WHO, 2016). Obesity is
increasing like a snowball effect globally and in Pakistan as well that leads to various health
concerns particularly diabetes that have raised to 14% of population (NNS, 2011).
People have natural inclination towards sweetened food items which is due to sensory cravings
and to fulfill the energy requirements that helps in carrying out metabolic and physical activities
of body. Foods that are naturally sweet such as fruits, honey, sugar beet, etc. contain important
health supporting nutrients. In order to make the food items, health care products, cosmetics and
medicines appealing and palatable, these are coated with different sweeteners which facilitates
their ingestion without affecting the original structure (Barriocanal et al., 2008). Foods in their
natural forms are blessings of Allah Almighty furnishing the best nutrition out of it. Glucose rich
foods including whole fruits or grains being in their natural forms, have found to be more nutrient
dense with low glycemic index as compared to refined foods with concentrated sugars in them
tends to elevate the body glucose level leading to enhancement in production of insulin as well.
Sweeteners are of various types; calorie rich natural sweeteners considered as nutritive while
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sweeteners that do not increase blood glucose level are known as non-nutritive or artificial. Honey,
sugar beet, sugarcane juice, sucrose, maltose, high fructose corn syrup, fructose, juice
concentrates, etc. are nutritive sweeteners and have been approved by FDA with respective GRAS
status. Moreover, intense natural sweeteners with zero caloric affect include Stevia, sorbitol,
Thaumatin, xylitol, Curculin, Inulins, Glycyrrhizin, Brazzein, Miraculin, lacitol, Pentadin,
Monellin, etc. However, number of permitted sucrose replacers which have been artificially
prepared are saccharin, acesulfame-K, cyclamate, aspartame, alitame, neotame, etc. but few being
extensively used in food and pharmaceutical industries (Zygler et al., 2009).
There exists quite a number of health concerns associated with the extravagant and long term usage
of artificial sweeteners as they cause serious ailments i-e headaches, fatigue, persisting depression,
intolerable migraines, anxiety, muscular pain, arthritis, ears buzzing, irritable bowel syndrome
(IBS), hallucination, nausea, vomiting, abrupt mood swings, respiratory and dermatological
disorders, etc. Metallic after taste is the most prominent disadvantage in the utilization of these
sweeteners (Brusick, 2008). With continuous changes in food preferences of people, food
industries are interested in using intense natural sweeteners with zero calorie affect dismaying the
utilization of artificial sweeteners whose regular usage leads to grave health issue. There are
different segments of society that have different preferences towards sweeteners and energy
coinage from them, therefore in order to provide wide range of sweeteners selection, it is necessary
to respect the consumer preference and maintaining their health status (Periche et al., 2015).
Stevia rebaudiana, a perennial herb, native to South America, generally famous with the name of
Stevia. In 1888 Dr. Moises Santiago Bertoni who discovered and explored it in Paraguay and
initially called it as Bert. In 1905, Dr. Rebaudi coined its scientific name as Stevia rebaudiana for
the first time (Yadav and Guleria, 2012; Urban et al., 2015). Genus Stevia has 154 members in
which species with sweetening potential are Stevia phlebophylla, S. anisostemma, S. crenata, S.
dianthoidea, S. bertholdii, S. enigmatica, S. lemmonii, S. eupatoria, S. micrantha, S. rebaudiana,
S. plummerae, S. serrata, S. salicifolia and S. viscida, however, only Stevia rebaudiana possess
highest level of sweetness (Carakostas et al., 2008).
Healthy Stevia plants can easily be grown in gardens and indoors for domestic and household
usage (Madan et al., 2010). Stevia, being a natural sweetener comprises of different sweet
bioactive moieties known as Steviol glycosides (SGs) or Steviosides. Depending on the cultivars
and sowing methods, Steviosides in dry leaves of Stevia ranges from 4-20% and more than 40
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different SGs have been reported that are concentrated in leaves (Chaturvedula and Zamora, 2014).
Most prevalent SGs that have been identified include Steviol, Stevioside, Dulcoside A,
Rebaudioside A-F and Steviol bioside (Ceunen and Geuns, 2013). However, out of these
glycosides, most important being Rebaudioside A, Dulcoside A, Stevioside and Steviol which are
industrially important. (Gasmalla et al., 2014). These glycosides have intense sweetness level with
no net gain in calories. All these SGs share the same common backbone which is an ent-kaurene
diterpene aglycone termed as Steviol with which different number of glucose or sugar molecules
are attached giving unique sweetness profile and intensity, making it 350 times sweeter than
sucrose (Purkayastha et al., 2016).
Originating from southeastern areas of Paraguay, Stevia is now mostly cultivated and abundantly
grown around the globe. Many countries like USA, Canada, China, Turkey, UK, Australia,
Thailand, South Africa, Taiwan, South Korea, India, Philippines, Norway and many other
countries that have started showing interest in its adaptation, cultivation, processing and export
(Huang et al., 2010). Therefore, in Pakistan, Stevia cultivation has been carried out across country
in different cities i-e Faisalabad, Rawalpindi, Multan, Chakwal, Gujranwala, Bahawalpur and
Ayyub Agriculture Research Institute, Faisalabad and National Agriculture Research Center,
Islamabad which are extensively working on it. Results obtained after completion of trials have
depicted that Stevia has adapted and well acclimatized to Pakistani environment (Ahmed et al.,
2007).
Different countries, after seeking regulatory permission from FDA, FDA and WHO, are
consuming Stevia in their food items (Carakostas et al., 2012). FAO and WHO have devised
JECFA (Joint Expert Committee on Food Additives) that on the basis of different researches have
established safety level of Stevia. In 2008, JECFA have entrenched the ADI as 4mg/kg body
weight/day and conferred the “Generally Recognized as Safe” status (JECFA, 2008; Urban et al.,
2015).
Compositional profile of Stevia have depicted that it is good source of protein, minerals, vitamins,
fatty acids, dietary fibers (Mondaca et al., 2012). Fatty acid profile of Stevia showed that it contain
essential fatty acids like palmitic, stearic, linolenic, linoleic and oleic acid (Tadhani and Subhash,
2006). For Steviosides extraction techniques like conventional solvent (CSE) and supercritical
fluid extraction (SFE) (Erkucuk et al., 2009; Herrero et al., 2010). Recently, plants are highlighted
due to their therapeutic potentials as antioxidants, free radical scavenging capability providing the
4
health benefits to users as well (Shukla et al., 2012; Hendawey and El-Fadl, 2014). Stevia has been
abundantly utilized as additive owing to its potential as antioxidant, promising phenolic &
flavonoids profile. Stevia extracts have been used as natural antioxidant source in fruit drinks
playing its role against inflammation and regulating immunomodulatory attributes (Carbonell-
Capella et al., 2013). Stevia is effective in lowering blood pressure and postprandial glucose level
in blood and increase insulin release in blood stream (Jeppesen et al., 2000; Nunes et al., 2007).
Nowadays, Stevia (Stevia leaf powder) is popularized as best alternate to sucrose and other
synthetic sweetener in food industries including confectionaries, beverage industries, baking, milk
processing factories, soft drinks and carbonated beverage, desserts and sauces processing (Abdel-
Salam et al., 2009). Taking aforementioned facts into consideration, instant study has been planned
to comprehensively explore the biochemical as well as nutritional profile of Pakistani grown stevia
with following objectives:
Objectives
Exploration of nutritional aspects of indigenous Stevia plant
Investigation of biochemical and end use perspectives of Stevia
Evaluation of health beneficial verdicts of Stevia through in vivo studies
5
2 Chapter 2
REVIEW OF LITERATURE
A long time ago, our ancestors were more concern about their health and diet as compared to us.
It was something normal when they ate many types of herbs as supplements to maintain their health
status and used to prepare different medicines and tonics on the basis of their experiences.
Therefore people were more healthy and protected from certain ailments (Afandi et al., 2013).
Now a days obesity and overweight are the root causes of wide number of health complications;
diabetes being the major one, followed by pulmonary and renal problems, hypertension, pregnancy
complications, surgical risks, hyperlipidemia, cardiovascular diseases, etc. Regular consumption
of food stuffs rich in calories especially sucrose sweetened snacks and beverages leads to above
mentioned metabolic disorders. In modern era, people are always busy in their daily activities and
pay much less attention towards the quality of their health. An increased demand of various
supplements and additives has been observed to fulfill the nutritional deficiencies of masses which
depicts their irregular dietary patterns (Carocho et al., 2015). People also try to justify their dietary
needs by preferring to eat junk foods, calorie dense sweetened food items that not only provide
energy to them but disturb their metabolic balance as well (Abou-Arab et al., 2010). Quite a
number of intense sweeteners either natural or artificial, mild or intense, nutritive or non-nutritive
are used by people and food industry on daily basis (Geuns, 2003). Some of the artificial sweetener
such as aspartame, saccharin, acesulfame-K, neotame and sucralose with their extensive utilization
leads to fatal maladies like cancer, phenylketonuria, etc. (Zygler et al., 2009; Liu et al., 2010).
Sweeteners provide pleasing sensations with or without calories and as food additives; their main
function is to enhance the product taste and quality. Nutritive sweeteners being natural used to
provide calories and mainly include fructose, glucose, sucrose, honey and sugar beet. They have
been given the status of Generally Recognized as Safe status (GRAS) by US Food and Drug
Administration (FDA) to these natural sources. But there exists certain health related problems
that are associated with the over consumption of nutritive sweeteners.
Intense sweeteners are either natural or synthetic compounds or their derivatives and metabolites.
Artificial sweeteners are prepared in laboratory, having taste similar to sucrose, fructose and
glucose and provide no or minimal calorie intake (Raben et al., 2002). Mostly high intensity
sweeteners are used in minute amount as they are 50-100 times sweeter than sucrose (American
6
Dietetic Association, 2004). Saccharine is very harmful for normal physiological functioning of
body causing digestive and metabolic issues when consumed on daily basis (Jaroslav et al., 2006).
Stevia (Stevia rebaudiana) is indigenous to Brazil and Paraguay and known before recorded
history. With the passage of time, it became famous globally. Based upon sweetness of its leaves,
it is known with different names viz honey leaf, Candy leaf, sweet herb and sweet leaf of Paraguay.
SGs/Steviosides; were extracted from Stevia by two French chemists in 1931 (Carakostas et al.,
2008). In 1964 and 1968, Stevia was commercially cultivated in Paraguay & Japan respectively
and used extensively by chewing gums, breads and pickle manufacturers (Bertoni, 1999). Since
1970s, with the development of extraction followed by refining & decolorization the commercial
profiteering of Stevia became more common in Japan (Huang et al., 2010). Early Stevia
formulations had depicted quite evident licorice aftertaste that hindered its industrial utilization
particularly in beverages. Sucrose provide 4kcal/g energy and used as standard for sweetness level
measurement of others sweeteners Aspartame is 200 times more sweeter than sucrose, neotame
has 700 to 1300 times more sweetness, saccharine has 300-500 times sweetness, sucralose is 600
times sweeter while aceulfame-K has sweetness level of 200 times than sucrose with no calories
(Savita et al., 2004; Barroso et al., 2016). However, the sweetness level of Stevia ranges from 250-
300 times higher as compared to sucrose with high melting point and low water solubility (Goyal
et al., 2010).
2.1 Global regularity status of Stevia
Previously, there have been great debate on giving approval to Stevia as mainstream sweetener.
There are different schools of thoughts having varied views and reservation regarding regulatory
status of Stevia. Sweeteners industry is based on various stakeholders that have their own pertinent
benefits of prime interest. It took a long time and episodes of debates by different international
governing organization that on the basis of research outcomes given the GRAS status and ADI be
given to Stevia. A brief history of such struggle is reviewed as under.
The utilization of SGs is now encouraged as food additive in order to minimize the dependence of
masses on calorie rich sugar like from sugarcane, beet sugar, honey, etc that would ultimately
lessen the incidence of diabetes and its allied diseases (Brahmachari et al., 2011; Shannon et al.,
2016). European Union (EU) regulated scientific committee on Food additives have reviewed the
safety status of Stevia and related products and declared that further research is needed to support
7
the safety status of Stevia (SCF, 1985, 1999a, b). Several petitions remained unattended at the
European Food Safety Agency (EFSA) regarding Stevia extracts. At that time Stevia powder and
extract remained restricted to few countries including Brazil, China, Korea, Japan, Paraguay,
Thailand and Israel. During 1990s, on several occasions, FDA had raised different observations
and asked food manufacturers to provide the regulatory status of their products that have Stevia as
ingredient which is not sanctioned under Dietary Supplement Health and Education Act. During
that time, FDA has also raised some points regarding the utilization of Stevia products due to lack
of safety certification that are thought to affect the various metabolic functions specially glycemic
level and fertility system. In 1994, DSHEA was approved by USA that has allowed to utilize
extracts from Stevia leaves in different supplements. While in 1995, FDA revised the restrictions
and approved Stevia as dietary supplement (FDA, 1995). In 2011, EU has declared Europe and
North America not to utilize Stevia as food additive as it is banned by FDA (US-FDA, 2009).
During the 58th, 63rd and 68th meetings of Joint Expert Committee of FAO/WHO on food
additives (JECFA) reconsidered the SGs safety limits and declared the ADI as 0-2 mg/kg body
weight/day by considering 200 as safety factor (Periche et al., 2015). In addition to detailed
specifications information, JECFA had asked to conduct various human studies to find out the
blood pressure and blood glucose lowering effect of Stevia thereby maintaining the insulin level
in body for glucose homeostasis. JECFA, after reviewing the submitted information stated to
lessen the safety factor up to 100 as permanent ADI. However in 68th meeting of JECFA, data
provided was not sufficient to meet the committee requirements regarding the potential impacts
on blood glucose and blood pressure homeostasis. In 2010, European Food Safety Authority
(EFSA) evaluated and sum up Stevia’s safe attributes and finally set 4mg/kg bw/day as the
Acceptable Daily Intake (ADI) (Logue et al., 2015).
In the 69th meeting of JECFA, 4 mg/kg bw ADI was established for SGs and NOAEL (no observed
adverse effect level) status was conferred to Stevia for its usage at commercial and domestic level.
(JECFA, 2009). JECFA have taken into consideration to give the status of sweeteners to Stevia
integral components which were Stevioside, Rubusoside, Dulcoside A, Steviolbioside and Reb.
A-F. While Stevioside and Rebaudioside A are the prime sweetening agents due to their high level
of sweetness and concentration (JECFA, 2010). United Nations have devised the committee from
its member nations for Codex Alimentarius commission (CAC) standards in order to establish their
own country level standards for Stevia. In 2011, the CAC came up with a draft determining the
8
maximum SGs level in food stuffs (Weston, 2011). According to the EU regulation No 1131/2011
from European Union SGs are now permitted to be used as sweetening agent across Europe. EU
established a criteria in which commercially available sweetener should have at least 95% of
Steviol glycoside content in it with 75% of Stevioside and total Rebaudioside A (EU,
2011;Purkayastha et al., 2016).
2.1 Stevia cultivation
Consumers increasingly demand products from natural sources. This is the driving force for
stipulation of stevia by food industry. Globally more than hundred thousand hectare area is under
Stevia cultivation, in Japan almost 2-3 billion dollar/year is the total market value, moreover China
is extensively doing Stevia cultivation. The average height of this short day plant is up to 1m at
which elliptical leaves are arranged in alternate arrangement pattern with 2-3cm length. The plant
is grown best at semi humid sub-tropical environmental adaptation of 200-400m above sea level
with average rainfall of 1500-1800 mm and temperature variation of 16-40oC. In Asia, the very
first commercial crop of Stevia was cultivated by Japan in 1968 having extensive root system with
pale purple throat of white flower and brittle stem system at which leaves are arranged in small
corymbs (Ranjan et al., 2011).
Normally, Stevia is cultivated in March-April and June-July months in different parts of world,
harvesting is done after 75-90 days (Ulbricht et al., 2010). Crude extract and steviosides content
of Stevia increases when pH of soil decreased that ultimately enhance the sweetness level of Stevia
(Das et al., 2011). The best growth of Stevia is attained at 20- 24ºC with soil pH 4-6 (Kobus &
Gramza, 2015). Single plant of Stevia can be used for more than 8 years and provide healthy green
leaves for usage. Dry weight of Stevia may be from 15-35 g per plant. Tissue culture technique
has also been extensively employed to cultivate Stevia in different soils and regions (Sharma et
al., 2015). In recent times, in vitro culture and micro propagation is the best method to tackle the
problems of output and get the ample amount of stevia crop in best possible short time. Uddin et
al. (2006) have performed in vitro propagation by using leaves, nodal and inter-nodal segments of
Stevia. Multiple shoots were obtained from nodal explants and proved it to be the best method for
large scale Stevia production (Barroso et al., 2016).
9
2.2 Structural expression of Steviosides
In 1931, Bridel and Lavieile have started working on the structural, stereochemistry, biochemical
and analytical characterization of SGs of Stevia which are refined with advancement and
sophistication of instruments. Stevia has gone through different chemical and enzymatic reactions
in order to get more than 100 different natural components which are SGs or Steviosides.
Structurally, Steviol glycoside 13oxy kaur-16-en-19-oic-acid ß-D glucopyranosyl ester) is a
glycoside with residues attached to Steviol aglycone, possessing cyclopentanon-
hydrophenanthrene skeleton. All different screened glycosides primarily differ in amount mono,
di and trisaccharide carbohydrate residues (R1 and R2), at positions C13 and C19 but share the
same backbone of Steviol (Lemus-Mondaca et al., 2012).
The major Steviol glycoside which is Stevioside has been altered by different chemical and
enzymatic operations into another major Steviol glycoside named as Rebaudioside A. Methanolic
extraction of Stevia leaves leads to the isolation Rebaudiosides B. However, Rebaudioside C, D &
E were obtained by the further fractionation of Stevia leaves extracts. Alkaline hydrolysis of both
Rebaudioside A & D separately can provide Rebaudioside B which inferred that these
Rebaudiosides are esters of each other. In the same ways, scientists have revealed that
Rebaudioside C share the same structure as that of Dulcoside A and B (Wu et al., 2012).
Steviosides as well as different Rebaudiosides were also prepared synthetically from Steviol by its
transformation using different chemicals as well enzymes (Gasmalla et al., 2014). With the
increase in number of bounded sugar units with Steviol aglycone, sweetness level enhanced
(Kovylyaeva et al., 2007). However, SGs differ in the sweetening or edulcorant properties from
each other. Significant metallic or bitter after taste is observed from pure SGs (de Oliveira et al.,
2007). Naturally, the sweetness level of different Stevia constituent is generally greater than
sucrose, which is considered as the scale with 100 as it sweetness value. The sweetness of different
steviosides is as: Rebaudioside A, B, C, D & E are is 250-450, 300-350, 50-120, 250-450 & 150-
300 times sweeter, while Dulcoside A & Steviolbioside are 50-120 & 100-125 times sweeter than
sucrose. Therefore, the sweetness level of Stevia ranges from 250-300 times higher as compared
to sucrose with high melting point and low water solubility (Goyal et al., 2010). The major
Steviosides concentration are reported as Stevioside (4-13% w/w), Dulcoside A (0.7%),
Rebaudioside A (4%) and Rebaudioside C (2%) (Gupta et al., 2016). Rebaudioside A is the
sweetest, most stable and less bitter than other steviosides. It has also been reported that synthetic
10
sweeteners like aspartame, cyclamate, neotame etc are less stable as compared to Stevioside
(Rajasekaran et al., 2008).
2.3 Chemical composition of Stevia
2.3.1 Proximate composition of Stevia
Stevia rebaudiana, is famous for its sweetening properties but there are many other aspects for its
popularity and getting importance for being the rich source of nutritional and functional
components like protein, fiber, minerals, vitamins, phenolic acids, free radical scavenging &
antioxidant capability, nutraceutical properties, etc. A number of scientists have worked on
different aspects and properties of Stevia and found some remarkable results. Fresh Stevia leaves
contain almost 80% moisture and provide 270 kcal/100g energy (Savita et al., 2004). In dried
leaves, the moisture content is usually influenced by extent and method of drying. In order to avoid
deterioration, it is recommended to dry these leaves by sun or oven drying methods. Post-harvest
drying of Stevia leaves for almost 8 hours is necessary that will concentrate the sweet glycoside
components in leaves (Samsudin and Aziz, 2013). Gasmalla et al. (2014) determined that Stevia
has considerable amount of protein and can absorb sufficient water in product development.
Protein content in Stevia leaves was recorded as 6.2-20.42% (Gisleine et al., 2006).
Fat content in extraction from Stevia was found to be only as 4.34% that is not high enough
comparing to other oil sources however fatty acid composition of Stevia present it as a good source
for optimum growth. Gasmlla et al. (2014) determined that Stevia leaves have 6.13±0.63% fat
content.. Fibers are chemically polysaccharides, oligosaccharides, lignins and their associated
plant components. Fiber are resistant starches which remained undigested during metabolism of
carbohydrates, therefore escape absorption in small intestine of humans. Regular utilization of
dietary fiber in food provides health benefits like promotes normal functioning of digestion,
reduces constipation, maintains body weight, removes extra cholesterol content and save body
from cardiovascular disorders by regulating normal blood pressure. It also helps in prevention of
cancer by providing a surface for attachment to colonic bacteria and also ease the transition of food
via intestines (Takasaki et al., 2009; Sanchez-Muniz, 2012). Stevia is source of ample amount of
dietary fibers and it has been reported that 18g/100g crude fiber is present in Stevia leaves powder
while Abdalbasit et al. (2014) reported crude fiber in Stevia as 13.56-18.5%.
11
Chemical constituents of Stevia was determined by Savita et al. (2004) and they found that
moisture content, calorific values, protein content, fat content, ash content and crude fiber were
found as 4.45-10.73%, 362.3-384.2 kcal/100g, 12.44-13.68%, 4.18-6.13%, 4.65-12.06% and
4.35-5.26% respectively. According to the research findings of Segura-Campos et al. (2014) on
Stevia, they found it to be a good source of crude protein (12.11% - 15.05%), carbohydrates
(64.06% - 67.98%) and crude fiber (5.92% - 9.52%). However, 28.61-29.12g/100g of total dietary
fiber content was found in which major share was of insoluble dietary fiber that ranges in 87.79%-
70.02%. Acid detergent lignin (2.28-8.98%), neutral detergent fiber (18.11-19.29%) and acid
detergent fiber (14.16-17.77%) have been found in impressive amount. Hemicellulose and
cellulose were 1.51%-3.96% and 8.79%-11.78% respectively (Gisleine et al., 2006).
2.3.2 Mineral composition of Stevia
Minerals are diet components inevitable for the up keeping of life and good health. Metabolic
processes need them for proper functioning, some are required in major quantity while other in
minor or trace amount. Major elements that are required include magnesium, potassium, chlorine,
sodium, phosphorous, Sulphur, calcium are classified as macronutrients. However micronutrients
include iron, cobalt, zinc, copper, selenium, iodine, molybdenum, chromium, manganese, etc.
Stevia leaves have good mineral profile with nutritionally essential elements in reasonable amount
i.e., calcium, potassium, magnesium, iron, copper, manganese, zinc and sodium in fresh as well as
dried leaves. Potassium, important mineral present in high amount followed by calcium, sodium
and magnesium being beneficial for human health as reported by many authors (Lemus-Mondaca
et al., 2012). Potassium works as an enzyme activator which is vital in making different peptide
bonds. Animal and plant based foods are rich in zinc amount, so as the Stevia has sufficient amount
of zinc which is the structural as well functional part of different enzymes like transphosphorylase,
peptidases, etc. Play an important role as anti-bacterial, anti-fungal, antiviral and anticancer
element. It is also an integral part of both DNA and RNA polymerase (Brisibe et al., 2009). Iron
is the integral part of hemoglobin and works to transport oxygen across the body for continuation
of body process. Therefore, diets missing in iron will lead to several body disorders major one
being anemia. It is a component of myoglobin protein found in muscle. Bone mineralization,
enzymatic action, proper functioning of nervous system is regulated by magnesium concentration
in body. Calcium is an integral part of teeth and bones performing vital role in normal muscle
contraction. Stevia emerged as a pronounced source of potent minerals thereby playing a
12
protecting role against diet disorders. It balances and conserve different metabolic processes
(Adotey et al., 2009).
Metabolic processes need them for proper functioning; some are required in major quantity while
other in minor or trace amount. Major elements that are required include magnesium, potassium,
chlorine, sodium, phosphorous, sulphur, calcium are classified as macronutrients. However
micronutrients include iron, cobalt, zinc, copper, selenium, iodine, molybdenum, manganese, etc
(Kesler & Simon, 2015).Trace mineral elements are needed for normal physiological functioning
of metabolic processes. Despite of this, metal elements which are present in earth’s crust in excess
amount have no role in normal functioning of metabolic working but hinders or inhibits them as
well. Heavy metals with key health concern include lead, cadmium, mercury and arsenic. These
metals may accumulate in the tissues of biological systems causing toxic issues in human system
natural functioning. Subsequently, the toxic effects that occurred are collectively termed as
bioaccumulation (Li et al., 2014). In the process of bioaccumulation, the heavy metals may
accumulate in human body via food channel i-e may come from the surrounding environment or
from food of animal origin like fish, beef, mutton, animal oil, etc. the results of this toxicity falls
from subtle to serious disease symptoms (Roohani et al., 2013). These metals may absorb in human
body for long exposure time and affect the body by delaying and stopping metabolic processes
leading to irreversible serious health distortive ailments (Jarup, 2003). By removing these toxic
heavy metals and their components from the human body can prevent crucial effects but the
removal is much difficult as it looks, so the only best possible way to avoid is awareness and
adopting safe and healthy measures.
2.3.3 Fatty acid profile of Stevia
Lipids are richest energy reserves providing 37kJ/g of energy on complete digestion thereby
providing sustainable energy for continuation of normal body function. In this regard, ingestion of
fat soluble vitamins A, D, E and K are of prime importance in health sustenance. Chemical
composition of triglycerides depicts that it constitute one unit of glycerol bounded with three same
or different fatty acids that eventually changes the chemical nature of fatty acids. Similarly
saturated or unsaturated fatty acids occur in different proportions in food furnishing body with
nutritional and medicinal benefits. Polyunsaturated fatty acids (PUFAs) have two major classes
that are omega-3 and omega-6 fatty acids. Alpha-linolenic acid, linoleic acid eicosapentaenoic acid
and docosahexaenoic acid are the essential fatty acids as human body cannot prepare them and we
13
must need these from diet (Jones et al., 2012). These essential fatty acids are interconvertible in a
limited amount with less than 15% conversion by liver action. Thereby, only way to maintain the
level of these essential fatty acids is to have them from dietary supplements and varied food sources
(Jones et al., 2014).
Fatty acid profile of food components is commonly determined by using Gas chromatography. It
is used to change over a composite mixture into volatile compounds. Essential standard of Gas
chromatography is a specimen which goes through heated column in which sample is vaporized at
elevated temperature, programmed as per protocol followed. Savita et al. (2004) have found the
concentrations of Palmitoleic acid, Stearic acid, Linolenic acid, Palmitic acid and Oleic acid as
1.27g/100g, 21.59g/100g, 1.18g/100g and 12.40g and 4.36g per 100g fat respectively. Tadhani
and Subhash (2006) identified various fatty acids primarily stearic, linoleic, linolenic, oleic,
palmitic and palmitoleic acids as 1.18g, 27.51g, 1.27g, 4.69g, 12.40g and 21.59g per 100g fat
respectively. Siddique et al. (2012) analyzed the hexane extract of Stevia rebaudiana from hexane
and determined free and bound fatty acids. They found that relative percentage of palmitic acid in
extract was highest as compared to others fatty acids and it was the 86.50%.
2.4 Functional properties of Stevia
Functional properties are the characteristic of a food which specifies quality, structure and
nutritional value of a product. These are determined by organoleptic and physico-chemical
attributes of a food e.g. pH, fat absorption capacity, water absorption capacity, bulk density,
swelling index, etc. pH is a measure of hydrogen ion concentration; measuring acidity or alkalinity.
pH of stevia powder dissolved in deionized water was found to be 6.1 which is slightly acidic and
close to neutral showing that hydrogen ions concentration is less than hydronium ion (H3O+)
concentration.
Powders, granules and other finely divided solid materials contain a property called bulk density
which is specially used regarding food stuff, food ingredients or any other matter of corpuscular
or particle nature materials. Stevia leaves powder appears to have less bulk density. In the design
of food products which are high in protein or fiber, it is important for their various properties to
control how much water is held by the food material. This water holding capacity (WHC) relates
to many sensory, health and nutritional properties. Thermal processing of protein-rich food is
accompanied by a loss of water (containing various nutritional components), so better control of
water means reducing loss of the nutritional value. Water holding capacity is directly associated
14
with the rehydration and Stevia appears to have good water holding capacity. High protein level
can be a possible reason for enhanced water holding capacity.
Swelling power is the property of thick or viscous food items like gravies, soups, doughs, etc. due
to vital role of proteins. On the other hand, proteins have a very good ability to stabilize emulsion
which is crucial in product preparation like cakes, froze desserts, coffee, batter, milk whiteners,
etc. But the composition and processing conditions to which the product is subjected, affect the
emulsion property. Fat absorption capacity can also be termed as physical binding/absorption of
oil. Stevia leaves powder is considered to have good fat absorption capacity thus making it an
efficient commodity in food processing. Fat enhances the effect of flavor retainers and improves
mouth feel of foods. Estimated fat absorption capacity of Stevia leaves powder is 4.5mL/g.
Functional properties of Stevia leave powder have been reported as 0.443g/mL bulk density,
4.7mL/g water holding capacity, 4.5 mL/g fat absorption capacity, 5.0 mL/g emulsification value,
5.01g/L swelling index, solubility was 0.365g/L and pH was 5.95 (Segura et al., 2014). Mishra et
al. (2010) calculated the Stevia powder bulk density as 0.443g/ml. Water Holding Capacity (WHC)
was found to be 4.7ml g/l, while fat absorption capacity calculated to be 4.5ml g/l and
Emulsification value of Stevia powder recorded as 5.0ml g/1. Swelling index of Stevia leaves
powder was reported as 5.01g g/1, solubility was 0.365g g/l and pH was 5.95. Savita et al. (2004)
evaluated that protein help to develop and maintain the emulsions. Development and maintenance
of emulsions in various food products is very necessary to achieve and maintain the desired
attributes of the food products such as batter, coffee, cakes, frozen desserts, milks, and whiteners.
2.5 Metabolism of Steviosides
The sweetening moieties of Stevia with backbone of Steviol including Stevioside and rebaudioside
A being the major contributors in sweetness. These SGs play an important role in body metabolism
as they only provide sweetness without any increment in body calories. Microbiota of gut
hydrolyze these SGs into diterpenoid aglycone known as Steviol and is not further metabolized by
the body therefore absorbed in blood stream from intestine for removal after filtration at kidneys.
According to Koyama et al. (2003) who worked on the human digestive tract in relation to Steviol
metabolism deduced that Steviol remained unaltered either at high or low concentrations. The
study also explained that role of liver in glucuronation of SGs in which these are absorbed and
clear from blood stream. Liver transferred the glucornated molecules of Steviol to kidneys for
15
filtration into urine. However, very small amount of these glucuronidate are not filtered and
remained in colon are excreted via feces. Rebaudioside A has lower hydrolysis rate as compared
to Stevioside. A recent mass spectrometry study have declared that Steviolepoxide is not a
microbial metabolite of SGs. Hydrolysis of glucose components is done by certain bacteria species
that utilize their β-glucosidase activity which specifically hydrolyze polymerized glycoside chains.
Similar results have been recorded by different studies conducted on human and animal mixed
fecal flora incubation which indicates that rats are pertinent models for bio efficacy studies on SGs
(Genus et al., 2007). In 2008, Renwick & Tarka, have found similarity in their study on the
microbial metabolism of rebaudioside A and Stevioside which form single hydrolysable product
known as Steviol that ultimately absorbed from the intestinal tract.
Steer et al. (2000) did an in vitro study on degradation of Steviosides and rebaudioside A by using
rat intestinal microbiota into diterpenoid aglycone, The degradation of these Steviosides into
Steviol requires different time such that complete steviosides to steviol conversion requires only 2
days when incubated in whole cell suspension, 6 days are need for Rebaudioside A transformation
under similar conditions. The metabolic fate of rats and humans is predicted to be similar and it
was confirmed from micro flora studying of rat caecum and lower human bowel qualitatively as
well as quantitatively (Renwick & Tarka, 2008). Steviol has a special metabolism mechanism in
which it is converted from steviol glycoside to Steviol only (Koyama et al., 2013). In an earlier
study, similar justification was given by Wingard et al. (1980) who gave a hypothesis about SGs
that gastric juice and digestive enzymes cannot digest or rearrange it. It was observed that all
gastric enzymes were unable to digest Stevioside but the microflora of intestine can convert it to
Steviol after hydrolyzation (Hutapea et al., 1997; Chatsudthipong and Muanprasat, 2009)
2.6 Phytochemical and antioxidant potential of Stevia
Oxidative stress is generated when production of a reactive oxygen species became too fast as it
creates imbalance and biological system loss its ability to detoxify or repair that damage produced
by reactive intermediates (Maritim et al., 2003). In cell environment, life is stable due to reducing
atmosphere which is maintained enzymatically by energy input that are metabolically attained. So,
alteration or disturbance in this reducing state can greatly affect redox potential or create toxic
effects thereby destructing the integrity of and DNA. In human prospective, oxidative stress resides
in many forms like Parkinson’s disease, Alzheimer’s, myocardial infarction, diabetes mellitus,
atherosclerosis and chronic fatigue (Elchuri et al., 2005).
16
Numerous biochemical constituents are generated by plants including phenols as well as their
oxygen substituted derivatives. These compounds have special role in plants serving as protection
against microbial attacks and infections (Johnson et al., 2010). Stevia has limitless ability to
produce phytochemicals, volatile oil components, flavonoids, sterebins A to H, triterpenes, gums,
pigments, etc. (Siddique et al., 2014). These phytochemicals have enormous potential in
minimizing risks free radicals that ultimately cause mutation, cancer and inflammation of body
organs. Moreover, phytochemicals present in Stevia have been proved to be significantly effective
for anesthetic, vasodilator cardiotonic, anti-inflammatory and austroinullin effect (Zia et al., 2011).
2.6.1 Total Phenolic (TPC) and Flavonoid contents (TFC) of Stevia
Total phenolic content (TPC) is a factor linked with countering the effects of oxidation occurred
by enzymes action (Velderrain et al., 2014). Enzymes like Peroxidase and polyphenoloxidase used
to work and reduces the antioxidant activity of food components by affecting the total phenol
contents (Aneta et al., 2007). Muanda et al. (2011) have calculated the concentration of TPC in
Stevia as 20.85mg GAE/g and have concluded that with the increase in pH of solution, oxidation
of polyphenols increases thereby high concentration of phenolate molecules are formed. Total
phenolic and total flavonoid contents in Stevia leaves and callus extracts were determined by Kim
et al. (2011) and concluded that 1mg Stevia leaves and callus extract contain 130.67mg and
43.99mg catechin. For flavonoids contents, they reported that 1mg extract of Stevia contain
15.64mg quercetin and callus extract have 1.57mg quercetin. Total phenolic and flavonoid
contents from methanolic extracts of roots, leaves, stem and flowers of Stevia were determined by
Singh et al. (2012) who concluded that methanolic leaves extract have low level of flavonoid
content as compared to root extract presented as 11.04±3.16mg/g and 16.75±0.35mg/g flavonoids.
Effect of high pressure processing treatment on the total phenolic content of Stevia based products
like fruit beverages and their antioxidant activity, amount of L. monocytogenes, peroxidase (POD)
and polyphenoloxidase (PPO) was determined by Barba et al. (2013). They first determined TPC
and antioxidant activity of samples which are not subjected to High Pressure Processing (HPP)
treatment with products subjected to HPP. Total phenolic compounds in untreated sample with no
Stevia was 185.5mg GAE/L. However Stevia products treated with HPP had had 2261.1mg
GAE/L to 4050.8mg GAE/L of TPC. They concluded that only HPP did not enhance antioxidant
capacity, it increases with the addition of Stevia and HPP break the cell wall thereby increasing
the availability of TPC.
17
2.6.2 Antioxidant potential of Stevia
Metabolic processes occurring in our body produce free radicals. Due to environmental, pathogenic,
physical, and chemical conditions the production of free radicals may increase significantly. The
free radicals are produced when internal and external factors impact on body like drugs, smoke,
pollutants, stress, etc thereby imparting deleterious effect on our body such that they can,alter the
structure of protein, lipids and DNA. These malformations may have drastic consequences on
human body such as various human disorders as well as fasten the aging process (Afify et al.,
2012; Ruiz-Ruiz et al., 2015).
Most commonly lipids are effected by free radicals which resultantly produce peroxides along with
various odorous compounds imparting foul smell. Enzymatic action are disturbed when proteins
are attacked by these free radicals. Nucleic acids when exposed to free radicals may results in
carcinogenesis and mutagenesis. Antioxidant capacity due to phenolic compounds from different
foods were assessed by the estimation of antioxidant assays like DPPH, ABTS, FRAP, etc. before
and after colonic fermentation and digestion (Tavarini and Angelini, 2013). Free radicals take part
during the oxidative stress which is a direct cause of pathogenesis of many diseases. Reduction or
elevation of anti-oxidative substances takes place when the body equilibrium shifts towards free
radicals, hence oxidative stress occurred (Kamath et al., 2004; Dlamini et al., 2007).
DPPH is widely used in order to evaluate the free radical removing antioxidant ability of food
material by stabilizing free radical with hydrogen ion donation (Kaushik et al., 2010). DPPH free
radical is a lipophilic radical and auto oxidation of lipid starts with its reaction. Owing to the least
reactive nature of this radical; they combine with each other and resultantly a stable molecule is
formed. It is shown that polyphenolic compounds when taken more than 1g/day from the diet, have
controlling effect against carcinogenesis and mutagenesis (Shukla et al., 2012). Esmat et al. (2010)
found that 10µ gm/ml Stevia leaves extract showed 3.38% and 100µgm/100ml leaf extract showed
10.15% of free radical scavenging activity. Results of various experiments showed that Stevia
leaves extract have higher DPPH free radical scavenging activity as compared to Stevia callus
extract with exception of 10µg/ml. Shukla et al. (2012) studied DPPH free radical scavenging
activity and found that 1gm of Stevia leaves extract gave 56.74mg Gallic acid which represents
phenols and 1gm of ethanolic extract gave 61.5mg gallic acid. Periche et al. (2014) determined
effect of different heat treatments (50ºC, 70ºC, 90ºC) with varying time durations (15, 20, 40
minutes) to Stevia leaves extract and antioxidant activity.
18
Various amounts of Stevia extract (20, 40, 50, 100, 200µg/ml) had antioxidant activities like 40,
46.84, 51.35, 64.26 and 72.37%, respectively. Singh et al. (2012) determined antioxidant potentials
of methanolic extracts from Stevia root, leaves, stem and flower using DPPH and ABTS assays.
ABTS radical scavenging activity assay as well as DPPH assay were used to analyze total
antioxidant activity. Root extract showed highest (64.23±8.35 mM) TEAC for ABTS free radical
scavenging activity; and leaves, stem, flower showed 56.26±16.87 mM, 49.28±12.87 mM and
46.49±13.13 mM respectively. In order to determine anti-oxidative potential of extracts, SOD,
Catalase and peroxidase enzymatic assays were carried out and the root extract was found to have
highest activities 4.84±0.22, 8.6±0.45 and 2.24±0.05 respectively (Singh et al., 2012).
2.7 Extraction methods of SGs/ Steviosides
There are many steps to obtain phytochemicals from plant such as milling, grinding,
homogenization and extraction. Among these steps, extraction is the main step for recovering and
isolating phytochemicals from plant materials. Solvent extraction is mostly employed to extract
desirable chemical constituents from food matrix (Oroian & Escriche, 2015). Extraction efficiency
is regulated by phytochemical nature, method of extraction, particle size, polarity solvent,
temperature, pH, time and sample nature (Stalikas, 2007; Do et al., 2014).Various techniques that
have been used to quantify SGs include chromatography adsorption, ion exchange, selective
precipitation, membrane processes and supercritical fluid extrction (Shi et al., 2000). Rank and
Midmore (2006) have refined by solvent (methanol & water) extraction methods followed by
precipitation with calcium hydroxide and subsequent impurities removal with CO2, while carbon
dioxide is used to remove impurities and the same protocol was followed as adopted for sugar
purification process in sugar industry. Steviosides were less soluble in hot water as compared to
Rebaudioside A (Puri et al., 2011). it has been extensively reported that usage of solvents like
propylene glycol, glycerin, methanol/chloroform and ethanol is advantageous. Liu et al. (1997)
have performed the extraction of stevioside with hot methanol from dried leaves of Stevia. They
have also used subcritical fluid extraction (Sub FE) for SGs, like Rebaudioside A, C and Dulcoside
A. An efficient Sub FE technique was developed that used methanol as modifier co-solvent that
resulted in remarkable 88% extraction efficiency.
Steviosides were also determined by extraction with supercritical fluid extraction (SFE) and
subsequent quantification with HPLC (Pol et al., 2007). SFE using ethanol as co-solvent provides
rapid extraction compared to previously used techniques. According to Erkucuk et al. (2009)
19
optimal mode of extraction elicited as 211 bar, 80oC which yielded 36.66and 17.99 mg/g of
Stevioside and Rebaudioside A. Supercritical fluid extractions (SFE) of Stevia were subjected to
liquid chromatographic analysis for estimation of Stevioside (Pol et al., 2007). There are several
known techniques for glycosides quantification in plant material specially chromatography and
spectroscopy techniques (Yoda et al., 2003). Among the numerous sophisticated high tech.
analytical instruments like HPLC, GC-MS and NMR however HPLC has been extensively
employed in different cereals and plants (Bernal et al., 2011). The quantification of Stevioside,
Steviol and Rebaudioside A was carried out carefully through various strategies as showed in the
scientific literature, including chemical detection and enzymatic hydrolysis (Gardana et al., 2010).
Near Infrared (NIR) spectroscopy model and HPLC techniques were directly employed to measure
steviosides content in leaves of Stevia rebaudiana (Yu et al., 2011).
SGs have also been estimated by a qualitative LC-TOF method along with an authorized HPTLC
procedure and densitometry detection (Jaitak et al., 2008) while quantitatively being determined
by NIRS procedure (Hearn & Subedi, 2009). Now a days, desorption electrospray ionization mass
spectrometry has been preferred for semi quantitative evaluation of SGs (Mondaca et al., 2012).
Fatty acid amides have been found discovered for very first time in Stevia. Therefore, they declared
after investigation that wide range of components have been there in Stevia that have nutraceutical
benefits ultimately benefiting health of masses (Jackson et al., 2009).
2.8 Therapeutic remunerations of Stevia
Stevia has been recommended to diabetic patients owing to its non-nutritive properties and
approved by the Food and Drug Administration (FDA) as a dietary supplement.
In ancient times, use of Stevia has been reported in treatment of various maladies. Stevia leaves
have been recommended to cure different ailments like obesity, renal diseases, CVDs, cancer,
inflammatory bowel disease,dental caries, etc (Gupta et al., 2013). Toxicological studies have
shown that Stevia play a defensive role against carcinomas, mutagenesis, teratogenesis, certain
allergic responses, cause no genetic defects in body and beyond sweetness impart anti-
hypertensive, diuretic, anti-viral, anti-diarrheal, anti-cariogenic, anti-microbial,
immunomodulatory and chemo-preventative activities (Abou-Arab et al., 2010; Yildiz-Ozturk et
al., 2015).
20
2.8.1 Glucoregulation
Diabetes mellitus is a disease described as hyperglycemia and varying degrees of an insufficient
insulin effect. There are approximately 177 million people with diabetes worldwide according to
the World Health Organization (WHO, 2004). Traditionally Stevia leaf extract has been used in
the treatment of diabetes (Megeji et al., 2005). It was observed that in both animals and humans
Stevia have the ability to increase the insulin effect on cell membranes, increase insulin production,
and stabilize glucagon secretion as well as blood sugar levels, and improved glucose tolerance to
ingested carbohydrates and lower post-prandial blood sugar levels. It can be stated that Stevia
provide a comprehensive set of mechanisms that alter the type II diabetes and its ultimate
complications. Thus SGs or stevioside of Stevia leaf can be used as a replacement of sugars to
support healthy glucose regulation (Gupta et al., 2013).
2.8.2 Blood pressure regulation
Upsurge in blood pressure from certain level or a standard measurement is known as hypertension
or high blood pressure. If a person have 140 mmHg systolic and 90 mmHg diastolic pressure,
he/she is declared as hypertensive. The high blood pressure in those veins that are already in
medium size or narrow sized arteries increases the pressure of blood and causes many problems
like they become thick and hard to pump blood towards whole body from heart, it can cause a risk
of stroke development leading to heart attack. Stevia has the ability to normalize the blood pressure
as well as regulation of heartbeat for cardiopulmonary signals. Extraction of stevia leaves by hot
water capable in regulation of blood pressure in human. A lot of studies indicated that stevia and
its compounds have the hypotensive and diuretic capacity. Stevia works just like the blood pressure
lowering medicines at membrane level, these medicines are known for their hypotensive properties
by dilating the walls of arteries to decrease the pressure of blood. Findings of many studies
expressed that Stevia has the ability of lowering the blood pressure by dilating the arteries
(Gardana et al., 2010).
Phytosterols are plant based compounds and which are present in wax that are secreted by leaves
and its properties act against defects of cardiovascular system (Markovie et al., 2008). Steviosides
and their derivatives have vasorelaxation properties (Wong et al., 2004). To evaluate these
properties of Stevia, a human trial was conducted in which 106 hypertensive patients were enrolled
and were given 750mg of Stevioside or placebo capsules daily (Chan et al., 2000). The individuals
who were taking stevioside, indicated significant reduction in blood pressure with no significant
21
side effects observed. The effect of extract of stevia was analyzed in 20 female
hypercholesterolemic patients by taking 20ml of extract in 200ml of water indicating the reduction
in cholesterol, LDL and triglycerides with increment in HDL. It shows the hypolipidemic attributes
of stevia and maintaining the cardiovascular health status of people (Sharma and Mogre, 2007;
Gupta et al., 2013).
2.8.3 Anticancer benefits of Stevia
Cancer is defined as the disorder of body cell DNA in which chemistry of DNA varies that
aggravate with the passage of time (Goyal et al., 2010). Stevia has been extensively used as
sweetener and in additive form as well. Over years nothing has been reported to be associated with
Stevia for toxicity, carcinogenicity, auto immune disorders, mutation, etc caused or with its
metabolites in mammals including certain animals and specially human, therefore can be regarded
as safe for human consumption (Periche et al., 2015). Different bioactive components have been
reported to be working against tumors, carcinomas, etc. Stevia leaves are rich in bioactive moieties
like Labdane sclareol, which has anti-cancer anti-inflammatory and cytotoxic removing attributes
(Kaushik et al., 2010).
Stevia polyphenols have been reported to possess properties inhibiting tumor initiation,
propagation and ultimately protecting the body against certain maladies. SGs particularly
Steviosides have the capability to block or minimize the activity of tumor propagation (Mizushina
et al., 2005). Rebaudioside A has been extensively investigated for its safety perspective including
carcinogenesis, mutation by employing Ames test in which bacterial reverse mutation was checked
by using S. typhimurium and E. coli as standards. The results depicted that Reb A was found to be
non-mutagenic in both bacterial strains. Another study was done on human lymphocytes in order
to establish the toxicity status of Rebaudioside-A. Male Wister rats were administrated 2000 mg/kg
body wt. in a single dose with subsequent 16h observation in order to check any toxicity signs of
Stevia. No substantial toxicity affect have been seen in rats that leads to carcinogenesis (Williams
and Burdock, 2009). Stevioside have been examined and they retards the tumor promoting agent
which promote tumor formation in mice skin. Anti-tumor and anti-carcinogenicity affect has been
recorded wen Stevioside has been administrated against urinary bladder tumor diseased cells.
Therefore neoplastic or pre-neoplastic lesions have not been seen in any of the tissue (Takahashi
et al., 2012).
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2.8.4 Renal functions regulations by Stevia
There are almost 70 million individuals present with different types of diseases in world. Due to
chronic level of renal ailments 400,000 deaths were occurred in 1990 and almost 735,000 deaths
were reported in 2010 (Lozano, 2012). Kidneys play an important role in maintaining the
environment of body by homeostasis. Many types of ailments effect the regular functioning of
renal system by disturbing the nephron structure. Shivanna et al. (2013) analyzed the effects of
stevioside on kidney functions in hypertensive and normal rats. Stevioside was found to be a
vasodilator and hypotensive properties as well as diuresis and natriuresis in hypertensive and
normal rats. In hypertensive and normal rats, glomerular filtration rate and the rate of kidney
plasma flow was found to be increased by the administration of stevioside at constant rate. Effect
of Stevia and its components on renal functioning was evaluated on transepithelial in proximal
kidney tubes of rabbits. The results were found that stevioside inhibits the transepithelial at the
dose level of 0.70 mM (Jutabha et al., 2000). Yuajit et al. (2013) conducted a study trial to evaluate
the inhibitory effects of the steviol and its derivatives on the growth of cyst. The findings revealed
that steviol is a good component in the treatment and cure of polycystic renal ailments. Steviol and
its bioactive moieties are the natural plant-based drugs for polycystic kidney disease treatment.
2.8.5 Obesity control by Stevia
Most prevalent nutritional malady worldwide is obesity in which excess fat aggregated in different
parts of body. If the excess body weight is more than 20% as compared to ideal body weight of
body, it is also defined as Obesity in clinical terms. Physical inactivity, unhealthy food selection
and habits, over consumption of food have contributed in prevalence and alarming increase in
obesity. Obesity is directly linked with calorie intakes, higher the intakes of calorie dense food
items, greater is the incidence of obesity. In this scenario, an affective weight management strategy
is to be adopted that helps in minimizing obesity chances. Low or zero calorie sweeteners are the
best choices for people who have high inclination towards desserts (Stephen et al., 2010). Stevia
being zero caloric, do not metabolize and spike in blood glucose level, have been measured in such
a way that 1g of crude extract of Stevia is 100-150 times sweeter than sucrose (Cardello et al.,
1999). Therefore Stevia can be considered to be best substitute of common table sugar, help in
weight management by restricting or minimizing calorie intake and it has also been reported that
high doses of Stevia resulted in significant weight reduction in animals (Curry and Roberts, 2008).
If daily intake of 95g (24 tsp) is regulated in diets by completely replacing sugar with Stevia
23
powder results in net deficit of 380 cal/day or weight loss of 1 pound in 9-10 days. Another
important aspect of Stevia as sweetener is that it minimizes the cravings for fatty foods and sweets,
which is also an important strategy to manage weight (Jain et al., 2007).
2.8.6 Inflammatory bowel disease (IBD) management
Inflammation of small intestine and colon is known as inflammatory bowel disease. It is basically
group of conditions and exists in two prime forms namely ulcerative colitis and Crohn’s disease.
Patients from both sexes between the ages of 15-30 years are most vulnerable. In all types of IBDs,
exact cause remained unknown, however natural phenomena involving autoimmune and genetic
predisposition play a crucial role in development and persistence of IBDs. Polyphenolic
components used to play potent role in regulation of metabolic syndrome and provide anti-
inflammatory benefits to body. Stevia being rich in polyphenols including total phenolic contents,
total flavonoids content and certain other phenolic acids, thereby defending body against harmful
diseases. Contraction of intestinal smooth muscles leads to hyper motility of intestinal microvilli
causing diarrhea. Inhibition of intestinal contraction has been observed in different animals fed on
Stevia powder along with their fodder (Shiozaki et al., 2006).
2.8.7 Dental maladies management
Dental caries (tooth decay), the most prevalent disease worldwide and individuals including babies
and elders, are prone to this during whole life span. Microorganism of oral cavity used to produce
organic acids metabolites leading to demineralization of enamel and ultimately causing proteolytic
deterioration of tooth structure. Dietary carbohydrates are fermented by various microbes
particularly Streptococcus mutans, Lactobacillus casein and Streptococcus sanguis. Utilization of
calorie dense nutritive sweeteners on daily basis furnish energy in carbohydrates form, aggravating
cavities, plaque and gingivitis formation due to microbial growth and their activity. Therefore in
order to cope up with these severe dental issues, calories dense sucrose and artificial sweeteners
needs to be replaced with other natural sources that provide zero caloric affect and are not
destructive to consumer health (Matsukubo and Takazoe, 2010). In this regard, Stevia can be
considered as best alternate to nutritive sweeteners having the properties of zero caloric and are
famous as non-nutritive sweeteners. Stevia hold the bacteriocidal and bacteriostatic attributes
thereby minimizing the chances of plaque and gingivitis. Stevia extracts along with its metabolites
are non-nutritive and tends to reduce glucan induced accumulation of cariogenic organism.
24
Therefore, Stevia have been proved to be ideal in provision of oral health perks (Gupta et al.,
2013).
2.9 Product development with Stevia
Food processing industries including confectionaries, baking industries, beverage industries, and
a number of other are replacing sucrose and other intense sweeteners with stevia powder and
extracts to cut short the price ultimately providing natural products with greater consumer
acceptability. Increasing awareness and potential perks of Stevia have urged people to use Stevia
in their daily routine food items like ready-to-eat cereals, yoghurt, beverages, sea foods, etc.
2.9.1 Temperature stability of Steviosides
Stevia can be used in various food products at high temperature and it remains stable against a
broad range of pH, it resists against fermentation and it is also acid stable. No caramelization or
browning was recorded with the addition of stevioside and rebaudioside A in food products
processed at elevated temperatures (Abou-Arab et al., 2010). Steviosides are heat stable at
temperature 95ºC as they do not decompose at this elevated temperature and utilized affectively as
sweetener in baked food stuffs. Carbonell-Capella (2013) have claimed that Stevia powder and
extracts can be employed at high processing temperatures i-e. 200ºC with proper sweetening ability
and allied potent benefits as well. Incubation of Stevioside for one hour at 120ºC was found to be
affective without any structural and functional disintegration. They have also concluded that
decomposition starts as the processing or incubation temperature raised from 200ºC. Stevia do not
exhibit specific taste and color of browning and caramelization, therefore recommended to be used
in combination with sucrose for better product quality in baked items and beverages. Product
stabilization and textural deformities have been seen when 100% Stevia incorporation have been
carried out (Abou-Arab et al., 2010).
2.9.2 pH stability of Steviosides
Steviosides have been reported to be stable in wide pH range and temperature (Virendra &
Kalpagam, 2008). They remained stable without showing any degradation under pH ranges of 1-
10 when dissolved for more than 2h at 60oC, however very minimal loss of upto 5% has been
reported when heated upto elevated temperature of 80oC with pH ranging from 2 to 10. On the
other hand, when exposed to highly acidic environment of pH 1 at temperature of 80oC for 2h
resulted in complete decomposition of Steviosides (Abou-Arab et al., 2010). In the same way
steviosides remained stable between pH ranges of 3-9 while rapid decomposition started if pH
25
raised from 9 at temperature up to 100oC for 1h (Buckenhuskers and Omran, 1997). Rebaudiside
A remained stable and gave sweetness in cola and lemon lime when it is stored for 26 weeks.
Rebaudiside A gave acceptable sweetness for 26 weeks when it is used in formulation of chewing
gum. It is observed that Rebaudiside A can tolerate pasteurization temperature (190ºF for 5 min)
and resist the fermentation process when it is used in formulation of plain yogurt and it gave
considerable sweetness when product was stored for 6 weeks (Prakash et al., 2008).
2.9.3 Steviosides stability in product development
Confectionary and bakery industries are using Stevia, solely as well as in combination with sucrose
to minimize the usage of calorie rich sweeteners. In different products, Stevia powder and extracts
have been used with good sweetness level. Stevioside and sucrose used to impart synergistic affect
to each other when added in peach juice in such a way that 160mg/L of Stevioside and 34g/L of
sucrose incorporation do not impart any bitter or metallic after taste to sensory attributes of final
product. When we compare Stevia with the saccharine in context of its metabolism it was found
that Stevia was not completely metabolized by human body providing very low calories but
saccharine is not completely metabolized and contributes to various diseases (Yadav and Guleria,
2012). Zahn et al. (2013) used Rebaudiside A, as natural sweetener to replace sugar along with
different bulking agents to provide bulkiness to muffins. Texture, color, chemical analysis and
sensory attributes showed that muffin with blend of inulin or polydextrose and 30% Rebaudiside
A exhibited good results and very close to the reference in which 100% sugar was used. When
inulin, polydextrose and 30% Rebaudiside A was added then energy of muffin was reduced by
5kJ/100kJ (Zehn et al., 2013).
Cookies have been recommended as a better utilization of flour than bread because of they are in
ready to eat form, excellent eating quality, wide consumption and extensive shelf life as compared
to bread (Okpala and Chinyelu, 2011). Abdel-Salam et al. (2009) formulated and assessed a
formulated functional yoghurt cake. They made cakes by replacing sucrose with Stevia water
extract as sweetening agent and employing functional ingredients in making of functional yoghurt
cake. They used Stevia water extract instead of sugar, olive oil instead of butter, skim milk in place
of full cream milk, egg white for whole eggs and whole wheat flour instead of 72% extraction
wheat flour. Lemon rind and orange peels incorporated to the yoghurt with Stevia water extract.
They sensory evaluated the both formulated yoghurt cake and regular yoghurt cake in which
texture, flavor, color, odor, appearance and overall acceptability was visualized. They found that
26
sensory attributes of both formulated yoghurt cake and regular yoghurt cake obtained acceptable
scores. The yoghurt cake which was made for diabetic patients showed very low food energy and
very low calorific value. Functional yoghurt cake having high amount of carbohydrates and fibers
may be very beneficial to enhance the health of blood vessels in persons which are suffering from
metabolic disorders possibly will reduce the chances of cardiovascular disease. When Stevia leaves
powder was added in cakes its texture became firmer as compared to regular yoghurt cake with its
hardness raised by 3176g as correlate with hardness of regular yoghurt cake which was the 3161g
and toughness was enhanced. Serna et al. (2014) added different proportions of stevia by replacing
the sugar and added coffee silver skin as bulking agents in cookies and separately added maltitol
by replacing sugar 100% sugar replacement with Stevia increases the moisture thereby affecting
the texture of cookies that ultimately affect the overall acceptability. This reason may cause
reduction in shelf life of the cookies. When silver skin is added in the cookies then reduction in
moisture level is observed. Thickness of Stevia cookies and maltitol cookies was similar but when
silver skin is added in combination with Stevia, thin cookies were attained.
27
3 CHAPTER 3
MATERIALS AND METHODS
3.1 Procurement of raw material
The present research experimental work was carried out in NIFSAT (National Institute of Food
Science and Technology), University of Agriculture (UAF) Faisalabad, Pakistan. Stevia
rebaudiana leaves and wheat variety Lasani 2011 were procured from Ayub Agricultural Research
Institute (AARI), Faisalabad. Chemicals and standards were purchased from RCI Labscan,
Ridelheigh, Sigma Aldrich, Fluka, etc.
3.2 Preparation of raw material
Stevia leaves were cleaned by washing and screening to remove the extraneous material. Leaves
were dried in hot air oven at 30± 5o C for 6 hours followed by conversion of dried raw leaves into
powder using high speed mixer. Afterwards, powder was kept and stored in air tight bags before
analysis. Wheat flour was obtained after sieving and milling in Quadrumate Senior mill (Kadam
et al., 2011).
3.3 Proximate analysis
Chemical composition attributes for Stevia and wheat flour were assessed by following their well-
established protocols as suggested by AOAC (2006) and AACC (2000) respectively.
3.3.1 Moisture Content
The moisture content in wheat flour and Stevia was determined by drying in Air Forced Draft
Oven (Memmert Germany). According to the method No. 44-15.02 mentioned in AOAC (2006),
10 gram of sample was taken in pre weighed china dish and placed in a hot air oven and dried at
temperature of 105±5oC till constant weight was achieved. The samples were cooled in desiccator
after being removed from oven. The moisture content was calculated according to the formula:
Moisture (%) = Wt. of raw Stevia– Wt. of dried Stevia X 100
Wt. of raw Stevia sample
3.3.2 Ash content
Ash content in Stevia and wheat samples was estimated by following the procedure given in
AOAC (2006) method No. 940.26 in which 5 gram of sample was taken in a pre-weighed porcelain
crucible and directly charred on flame till fumes stop coming out and afterwards ignited in muffle
28
furnace (MF-1/02, PCSIR, Pakistan) maintained at temperature of 550-600ºC for 5-6 hours or until
grayish white residues were obtained. The crucible was removed from the muffle furnace, cooled
in a desiccator and weighed. Ash content was calculated according to the following formula:
Ash (%) = Weight of ash residue (g) X 100
Weight of sample (g)
3.3.3 Crude fat
Stevia and wheat flour samples were analyzed for crude fat according to AOAC (2006) method
No. 920.29 in which 5 gram of moisture free sample was weighed into Whattmann No.1 filter
paper. 50 mL petroleum ether was added to cup. Both filter paper and cup were attached to Soxhlet
extraction unit (Model: H-2 1045 Extraction Unit, Hoganas, Sweden) and subjected to extraction
with solvent for 30 min. The solvent was evaporated from the cup to the condensing column.
Extracted fat in the cup was placed in an oven at 110 ºC for 1 h and fat was calculated using the
following formula:
Crude fat (%) = Extracted fat (g) X 100
Weight of sample (g)
3.3.4 Crude protein
Kjeltech apparatus (Model: D-40599, Behr Labor Technik, Gmbh-Germany) was used to
determine nitrogen percentage in Stevia powder and wheat flour samples according to AOAC
(2006) method No. 920.152. 2g sample was placed in a Kjeldahl digestion tube and digested with
25 mL concentrated H2SO4 by using digestion mixture (K2SO4:FeSO4:CuSO4 i.e. 100:5:10) until
the color was transparent or light greenish. The digested material was diluted up to 250mL in
volumetric flask. A 10mL of digested sample was taken in distillation apparatus and 10mL of 40%
NaOH was added. The liberated ammonia was collected in conical flask containing 4% boric acid
solution and methyl red as an indicator. This resultant ammonium borate was titrated with 0.1N
Sulphuric acid and volume of acid used was noted for nitrogen determination in sample. Crude
protein content was estimated by multiplying nitrogen percent (N %). The protein percentage was
calculated according to the formula given below:
N (%) = Vol. of 0.1N H2SO4 x 0.0014x Vol. of dilution (250mL) X 100
Vol. of distillate taken x Weight of sample
29
3.3.5 Crude fiber
The crude fiber was estimated by following the method No. 962.09 outlined in AOAC (2006). 2
gram defatted samples of Stevia and wheat flour were placed in a crucible and attached to the
extraction unit Labconco Fibertech apparatus (Labconco Corporation Kansas, USA). 200 mL of
1.25% Sulphuric acid solution was added. The samples were digested for 30 min and then acid
was drained out followed by samples washing with distilled water. After this, 1.25% Sodium
hydroxide solution (200 mL) was added and sample was digested for 30 min. Thereafter, the alkali
was drained out and the sample was washed again with boiling water. Finally, the sample was
placed in crucible and oven dried at 105ºC overnight. The sample was allowed to cool in a
desiccator and weighed (W1). The sample was then burnt at 550ºC in a muffle furnace (MF-1-02,
PCSIR, Pakistan) for 2 h, cooled in a desiccator and reweighed (W2). Extracted fiber was expressed
as percentage of the original defatted sample and calculated by the following formula:
Crude fiber (%) = Digested sample (W1) – Ash sample (W2) X 100
Wt. of sample
3.3.6 Nitrogen free extracts (NFE)
The nitrogen free extract (NFE) of stevia and wheat flour samples were calculated according to
the following expression:
NFE = 100 – (% moisture + % ash + % crude fat + %crude fiber + % crude protein)
3.4 Functional properties
3.4.1 Bulk density Bulk density of Stevia was calculated by adopting method of Segura-Campos et al. (2014).
According to protocol, 50g Stevia was put in 100mL measuring cylinder. The cylinder was tapped
on a laboratory bench to constant volume. The volume of sample was recorded as;
Bulk density (g/cm3) = Volume of sample after tapping
Wt. of sample
3.4.2 pH
The pH of Stevia was determined by using pH meter (Inolab). KCl solution of 7 pH was used to
standardize the equipment. Stevia powder was mixed with distilled water and then pH was
measured in triplicates.
30
3.4.3 Swelling power
Swelling power of Stevia was determined by using the method of AOAC (2000). According to
method, 1g of the Stevia was added in conical flask followed by hydration with 15mL of distilled
water and subsequently 5min shaking on mechanical shaker at low speed. Afterwards, 40min of
heating was given at 80ºC with constant stirring in a water bath followed by transfer of material in
a pre-weighed, clean and dried centrifuge tube. Centrifugation was done at 2200rpm for 20min
with 7.5mL addition of distilled water. The supernatant was transferred in pre-weighed can and
dried at 100ºC to a constant weight.
Swelling power = Weight of sediment paste (g)
Weight of dry sample (g)
3.4.4 Oil holding capacity
Oil holding capacity of Stevia was measured by using method of Segura-Campos et al. (2014). 2g
of Stevia was blended with 25 mL of distilled water for 30 s at 1600 rpm. Refined corn oil was
added when dispersion was completed and then blended until there was two layers separation of
fat and water. Oil holding capacity was expressed as mL of oil retained or hold by 1g of Stevia.
3.4.5 Water absorption capacity
According to the method of Segura-Campos et al. (2014), 5mL of distilled water was added to 1g
of Stevia in a weighed 25mL centrifuge tube. The tube was vortexed for 2min followed by 20 min
centrifugation at 4000rpm. The clear supernatant was removed carefully and then reweighed.
Water absorption capacity was calculated as the weight of water bound by 100g dried Stevia.
3.5 Mineral analysis
Mineral profile of Stevia for minerals like sodium, potassium, magnesium, phosphorous, iron,
zinc, manganese, copper, lead, mercury, cadmium, nickel, cobalt and chromium were determined
by using Flame Photometer (Sherwood Scientific Ltd., Cambridge, Model 410) and Atomic
Absorption Spectrophotometer (AA240, Varian). Samples were prepared by wet digestion method
according to AOAC (2006) method No. 965.17-968.08. A 2g sample was ignited in order to
remove the organic portion followed by wet digestion with HNO3 and HClO4 (10:3) on hot plate
till the color of the fume becomes light green or clear. After that sample was diluted with 25ml of
deionized water and filtered through Wahttman filter paper No.1 and stored for further analysis at
flame photometer and AAS.
31
3.6 Fatty acid profile
For Fatty acid profile of Stevia the fat was extracted through Soxhlet apparatus using hexane as
solvent and followed by rotary evaporation. The fatty acid methyl esters (FAME) were prepared
and stored prior to analysis at 4oC. Oil sample 100µL ±5µL was added in a test tube using an auto
pipette and then sealed with a tight cap. Oil was mixed with 5mL hexane with briefly shaking on
to dissolve lipid followed by addition of 250µL sodium methoxide reagent with vigorous vortex
shaking for 1 min pausing 10 seconds for vortex to collapse. After this, 5mL saturated NaCl
solution (Rci Labscanto) was added (5ml) and vortexed for 15 sec. The mixture was allowed to
stand for 10 min and three layers were appeared. The top layer was transferred to a clean new vial
containing small amount of Sodium Sulphate from Sigma Aldrich and stored prior to analysis.
Vials containing FAME were subjected to subsequent GC analysis. Fatty acid profile was
determined by following the protocol of Korobko et al. (2007) employing Agilent 6890N GC
Network system equipped with Flame Ionization Detector (FID). GC conditions were set as
follows: DB wax Capillary column; 60.0m×0.25mm×0.25m; oven temperature programmed: after
sample injection, column was initially held at 60ºC for 3 min, then temperature was raised to 185ºC
with 10ºC/m heating ramp for 1min and then further increased to 200ºC with 5ºC/min heating ramp
for 10min. Finally temperature was raised to 220ºC with5ºC/m in heating ramp for 20min; 250ºC
injector temperature, 275ºC detector temperature; nitrogen as carrier gas; inlet pressure, 40.65 psi;
linear gas velocity, 39cm/s; column flow rate, 2.7mL/m in; split ratio, 40:1; injected volume, 1µL.
3.7 Steviosides Extraction
Steviosides were extracted by following two methods viz. solvent extraction and the supercritical
fluid extraction.
3.7.1 Steviosides extraction by different solvents
Polyphenols from Stevia were extracted by using conventional solvent extractions technique. 5g
of dry Stevia was added separately in 100ml of water, methanol and ethanol with subsequent
boiling for 30 min. After 2-3 min. the mixture was re-boiled for short time and kept at room
temperature for one to two hours. After that, solid material or residues were removed from boiled
solvents. The extract was heated till it reduced to its half volume and then centrifuged at 10˚C,
2500rpm for a duration of 10 minutes that subsequently ease the separation of aqueous phase. Then
the phases were shifted to 25 mL volumetric flasks and filled to the mark. The solutions were
filtered through a membrane filter (0.22 μm) to remove any solid residue before further analysis
32
(Abou-Arab et al., 2010; Aranda-Gonzalez et al., 2014). The yield of respective samples was
recorded and stored at 4°C.
3.7.2 Supercritical fluid extraction of Steviosides
Supercritical Fluid Extraction (SFE) was employed for Steviosides extraction by using model No.
SFT-150 system (Super Critical Technologies, Institute, USA). The volume of extractor was
100ml and approximately 30g Stevia powder was filled in it. The temperature, pressure and ethanol
were independent variables where ethanol used as co-solvent. The extraction was carried out at
different temperature and pressure combinations i.e. 40, 60 and 80oC temperatures while the
pressures used were 15, 25 and 35 MPa. Above mentioned temperature and pressure levels were
maintained automatically throughout the extraction process of one hour. The extracted Steviosides
was recovered from separator after complete removal of CO2 gas from the extractor (Erkucuk et
al., 2009).
3.8 Phytochemical analysis
Polyphenols including total phenols and flavonoids content were analyzed quantitatively by
following their respective methods and expressed in Gallic acid, Tannic acid, or Catechin
equivalents/g of extract or dried leaves.
3.8.1 Total phenolic content
Total phenolic contents were determined in triplicate by using Folin-Ciocalteu Reagent. 5g sample
was was mixed with 1ml of 50% methanol (CH3OH) solution. Then 4ml of 50% CH3OH solution
was added again and mixture was sonicated to attain the concentration of 1mg/ml. 0.5ml of this
solution was transferred to test tube in which 3.5ml of distilled water and 0.25ml of Folin-Ciocalteu
Reagent were added. The whole mixture was incubated at room temperature for 1-8min. After this,
0.75ml of 20% Sodium carbonate (Na2CO3) was added and the test tube was incubated for 2hrs.
The absorbance was measured at 765nm. The standard solutions were run to get a standard curve
was obtained after calculations. The total phenolic compounds were calculated on w/w basis (Kim
et al., 2011).
3.8.2 Total flavonoids content
Aluminum Chloride Colorimetric Reagent was utilized for determination of total flavonoids
compounds. 0.5ml sonicated sample was taken in test tube in which 1.5ml of methanol, 0.1ml of
10% aluminum chloride, 0.1ml of 1M potassium acetate and 2.8ml of distilled water were added.
Finally, the whole solution was incubated at room temperature for 30min. The absorbance of
33
sample was measured at 415nm against blank sample. The standard curve was obtained using
quercetin solution at whose concentration ranging from 1-10µg/ml. the flavonoids content were
calculated by comparing the concentration of sample against standard curve (Shukla et al., 2012).
3.9 In vitro Antioxidant assays
Antioxidant activity must not be based on a one test model. Three in vitro test procedures were
performed to examine the antioxidant activity of different samples.
3.9.1 DPPH (1-1-diphenyl 2-picryl hydrazyl) free radical scavenging activity of Stevia
The DPPH free radical scavenging activity of Stevia extract was measured by following the
procedure described by Shukla et al. (2009). The extract (4ml) was mixed with 1 ml DPPH
and incubated at room temperature for 30 mint. Then the absorbance was measured using
spectrophotometer at 520 nm. At the end the calculation was done by suing following formula;
Reduction of absorbance =AB − AA
AB× 100
AB = absorbance of blank sample (t = 0 min)
AA = absorbance of tested extract solution (t = 30 min)
3.9.2 Ferric reducing-antioxidant power (FRAP) assay
The FRAP was done by following the procedure of Benzie and strain (1996). The stock solution
including 10mM TPTZ (2, 4, 6-tripyridyl-s-triazine) solution, 300 mM acetate buffer (3.1 g
C2H3NaO2.3H2O and 16 mL C2H4O2), pH 3.6, in 20 mM FeCl3.6H2O, 40 mM HCl solution. The
fresh working solution was prepared by mixing 2.5 mL TPTZ solution with 25 mL acetate buffer
and 2.5 mL FeCl3. 6H2O solution. After mixing, the solution was heated to 37oC before using. The
extracts of Stevia were allowed to react with FRAP solution for 30 min in dark. The absorbance
of the colored product were taken at 593 nm. The linear standard curve was formed between 25
and 800 mM. The results were expressed in mM TE (Trolox Equivalent)/g fresh mass. If the FRAP
values measured over the linear range then additional dilution was required.
3.9.3 ABTS radical cation decolorization assay
ABTS analysis was carried out by adopting the protocol of of Arnao et al. (2001) with some
modifications. Potassium persulfate (2.6 mM) and ABTSd+ (7.4 mM) solutions were used as stock
solutions. Working solution was obtained by mixing the two stock solutions in equal ratios and
keeping them in dark for 9h in order to complete the reaction. Working solution was diluted by
34
mixing 1 mL ABTSd+ solution in 60 mL methanol to record the absorbance of 1.170 units at 734
nm by using IRMECO UV-VIS U2020 spectrophotometer. 150 mL of each Stevia extract was
reacted with 2850 mL of ABTSd+ solution and kept in dark for 2h to complete the reaction.
Afterwards absorbance was recorded at 734 nm. Results were presented in mM Trolox equivalents
(TE)/g fresh mass.
3.10 Quantification of Steviosides through HPLC
Bioactive moieties of Stevia were quantified through Perkin Elmer 200 series HPLC equipped
with UV detector and C-18 column following the method of Afandi et al. (2013) with few
modifications. After derivatization, 20 µL was injected in the HPLC system on a reversed-phase
C18 column (250 × 4.6 mm ID, 5 µm particle size; Grace, Lokeren, Belgium). The HPLC system
consisted of two LC-20AT pumps and auto sampler from Perkin Elmer, USA. The derivatized
compounds were eluted with mobile phase (70:30 v/v mixture of acetonitrile: water) used at a
constant flow rate of 1 ml/min. Detection was done using a wavelength of UV detector at a
wavelength of 210 nm at ambient temperature.
3.11 Functional groups identification of Stevia with FT-IR
The FTIR spectra of samples were recorded in order to characterize various functional groups and
gather sufficient information about Stevia standard, dried powder and extracts obtained from
different solvents. A total scans of 16 scans per sample with a 4-cm-1 interval of spectral resolution
were obtained over operating in the mid–infrared region of 400 to 4000 cm-1 using Tensor 27 FTIR
by Bruker Optics GmbH, Germany with OPUS Data Collection Program. A sealed and desiccated
interferometer was fitted in the instrument having a Deuterated Triglycine Sulfate (DTGS)
detector. An ATR (attenuated total reflectance) sample cell equipped with a ZnSe single crystal
through which IR was directed to a detachable ATR and KBr beam splitter. All spectral
measurements were made at 32 cm–1 resolutions, with 256 interferograms. Background spectrum
was collected and afterwards spectra for each sample was collected. The ATR crystal was cleaned
by using methanol after each experiment and carefully checked for any impurity left in order to
ensure the authenticity of results (Kumar & Ajay, 2015).
35
3.12 Value addition of Stevia
“Stevia cookies” were prepared by replacing sucrose in the recipe with Stevia powder or extracts
according to the method No. 10-50D given in AACC (2000) method No.10-50D. Flour, sugar,
stevia powder/extract, eggs, oil and baking powder were the ingredients in cookies preparation.
Cookies were cooled at room temperature and followed by packing in polythene bags prior to
further analyses. Treatment plan is expressed in Table 1.
Table 1. Treatment plan for cookies preparation with Stevia extract
Treatments Description
To Control
T1 10% Stevia Powder
T2 20% Stevia Powder
T3 30% Stevia Powder
T4 Cookies with 1% extract
CSE
T5 Cookies with 2% extract
CSE
T6 Cookies with 3% extract
CSE
T7 Cookies with 1% extract
SFE
T8 Cookies with 2% extract
SFE
T9 Cookies with 3% extract
SFE
CSE= Conventional solvent extraction
SFE= Supercritical fluid extraction
3.12.1 Sensory analysis of Stevia product
The sensory evaluation of Stevia cookies was done for characteristics like color, flavor, taste,
crispiness, texture and overall acceptability by a panel of six judges (Lawless and Heymann, 1999).
The sensory evaluation of cookies was performed by following nine point hedonic scale system
ranging from excellent to extremely poor (09 = excellent; 01 = extremely poor) according to the
guidelines of Meilgaard et al. (2007). The detail of which is given in Appendix-I. Separate booths
equipped with fluorescent white light and white background, water and napkin were provided to
36
each panelists. Samples were randomly distributed in order to avoid any biasness. Panelists were
then asked to give scores for selected parameters of cookies.
3.12.2 Physicochemical characterization of Stevia cookies
Stevia added cookies were analyzed for moisture, crude fat, crude fiber, ash, crude protein and
nitrogen free extract were determined by following the protocols given in AACC (2000). Texture
analyzer was used for determination of textural properties especially hardness while color analysis
including L*, a* and b* were determined by using colorimeter.
3.12.3 Energy value evaluation of Stevia
Calorific values of Stevia cookies were subjected to determine calorific value by employing
Oxygen Bomb Calorimeter (IKA-WERKE, C2000 Basic, GMBH and CO. Germany) by following
the method of Krishna and Ranjhan (1981). Cookies sample was placed in metallic vial for
decomposition. A cotton thread was fastened at the middle of wire for ignition and done before
loading the sample in unscrewed vial which was tightly screwed afterwards. The vial was then
placed in measuring cell which remained till the vial fixed in its place followed by pressing the
start button that closes the cell cover. Electric spark was produced that completely burnt the sample
and the resultant heat produced was illustrated graphically on machine digital panel depicting
temperature raised or fallen with the time. Thus the output was in form calories/g of a sample.
3.12.4 Color analysis
The product color, L* (lightness or darkness), a* (+a redness or –a greenness) and b* (+b
yellowness or –b blueness) were measured using laboratory scale colorimeter (CIELAB Color
Tech-PCM, USA) by following the guidelines of Rodriguez-Garcia et al. (2012) & Krishna and
Ranjhana (1981).
3.12.5 Texture profile
Texture analysis of Stevia cookies was carried out by adopting the guidelines of Piga et al. (2005)
using Texture Analyzer (TA-XT2, Plus, Stable Microsystems, Surrey, UK) connected to computer.
Texture Expert program version 4.0.9 was used for data analysis. Average value of three repeated
measurements of force required for product breakage was recorded (Lara et al., 2011).
3.12.6 Total phenolic & Flavonoid content of Stevia cookies
Total phenolic and flavonoids contents of Stevia cookies were determined by using the method of
Tadhani et al. (2007) and Kim et al. (2011) respectively.
37
3.12.7 Evaluation of in vitro radical scavenging activity of Stevia cookies
DPPH (Free radical scavenging ability) of Stevia cookies was determined by following the method
of Mandal and Madan (2013) while FRAP assay was done by following the method of Tadhani et
al. (2007).
3.13 Selection of best treatments
On the basis of high antioxidant activity and overall acceptability three treatments were selected -
one each from powder, solvent extraction and supercritical fluid extraction for efficacy study.
3.13.1 Efficacy trial
In vivo trials were carried out by using rodent experimental model on best selected treatments.
Sprague dawley rats were used in the model, procured from National Institute of Health (NIH)
Islamabad. The study on rats was carried out in Animal Room of National Institute of Food Science
and Technology, University of Agriculture, Faisalabad. For one week period basal diet was to
acclimatize the rats. The environmental conditions like relative humidity (55±5%) and temperature
(23±2ºC) were maintained along with 12 hour light dark period throughout the trial. Normal,
hyperglycemic and hypercholesterolemic are the three groups on which the study was carried out
during efficacy trial. Baseline value was obtained in the start by sacrificing some rats randomly
selected from each module. To evaluate the restorative potential, control and selected Stevia diets
(Table 2) were given to all three groups during eight-week trial. Feed and water intakes along with
body weight were measured for the whole experimental model. At the end of the study, overnight
fastened rats were sacrificed for testing serum, lipid profile, glucose and insulin level of blood and
serum. Serum samples were collected by centrifuging blood samples at 4000 rpm for 6 min. Serum
samples were evaluated for numerous biochemical assays via Microlab 300, Merck, Germany.
38
Table 2. Treatment plan for efficacy trials
To= Control (Normal sucrose cookies)
T1= Stevia leaf powder
T2= Stevia CSE extract
T3= Stevia SFE extract
Study I: Normal rats
In this study, rats were divided into four homogeneous groups fed on normal diet along with
provision of respective diets.
Study II: Hyperglycemic rats
In study II, hyperglycemia was induced by feeding rats on 40% sucrose diet thereby recording its
effect on serum glucose and insulin.
Category III: Hypercholesterolemic rats
In study III, 1.5% cholesterol was given along with normal diet in order to induce
hypercholesterolemia in rats Customized Stevia deits were given to diseased rats and their effect
Studies
Study I
Normal rats
Groups Diet
1 To
2 T1
3 T2
4 T3
Study II
Hyperglycemic rats
1 To
2 T1
3 T2
4 T3
Study III
Hypercholesterolemic
rats
1 To
2 T1
3 T2
4 T3
39
was recorded accordingly against HDL (High density lipoproteins), LDL (Low density
lipoproteins), cholesterol and triglycerides.
Feed & water intakes
Weight gain or loss due to given diets was recorded on weekly basis during the whole study
duration in order to determine any suppressing or incremental effect of Stevia cookies feed on
weight variation. Feed intake was calculated on daily basis by subtracting the left over diet from
the total diet given during the whole study (Wolf and Weisbrode, 2003). Water was provided in
graduated bottles and daily intake was measured by recording the volume of leftover amount of
water in bottles.
3.13.2 Serum glucose and insulin levels
Glucose concentration of all groups under investigations was determined by following the GOD-
PAP protocol elaborated by Ribes et al. (1986). However, insulin level was evaluated byadopting
the method of Shivanna et al. (2013).
3.13.3 Serum lipid profile analysis
Lipid profile parameters of blood serum i-e cholesterol, HDL (high density lipoproteins), LDL
(low density lipoproteins) and triglycerides were assessed according to their protocols. Cholesterol
concentration was determined by following CHOD-PAP method (Rifai et al., 1999). HDL & LDL
levels were estimated by Cholesterol Precipitant method (Alshatwi et al., 2010) while triglycerides
concentration in sera samples were assessed by liquid triglycerides (GPO-PAP) protocol defined
by Allain et al. (1974).
3.13.4 Liver function tests
Liver functioning major variable especially enzymes like ALT (alanine aminotransferase), ALP
(alkaline phosphatase) and AST (aspartate aminotransferase) were determined according to their
respective kit methods and protocols. ALT and AST concentrations were assessed by the
dinitrophenylhydrazene (DNPH) protocol employing Sigma Kits 58-50 & 59-50, respectively.
However, ALP was determined by Alkaline Phosphates–DGKC method (Das and Kathiriya,
2012).
40
3.13.5 Renal function tests
Blood samples of Stevia fed rats were subjected to GLDH and Jaffe commercial kits methods in
order to estimate urea and creatinine level that will determine the renal functionality of different
groups (Shivanna et al., 2013).
3.13.6 Hematological analysis
Hematological parameters including red and white blood cells (RBC & WBC) and Platelets count
were assessed by adopting the protocols of Fischbach (1996) and Nikiforov and Eapen (2008).
3.14 Statistical analysis
The results obtained from this study were subjected to statistical analysis i-e completely
randomized design (CRD) and two factor factorial under CRD using Statistix 8.1 software
according to guidelines of Steel et al. (1997).
41
4 CHAPTER 4
RESULTS AND DISCUSSION
The major part of research work under discussion was carried out at National Institute of Food
Science and Technology (NIFSAT), University of Agriculture Faisalabad, Pakistan. However,
some experiments were done in Agricultural and Biological Engineering Department, Purdue
University, West Lafayette, IN, USA. The prime objectives of the study were to investigate,
characterize, and endorse the biochemical and nutritional profile of Stevia rebaudiana grown in
Pakistan. The Stevia was evaluated for its product suitability by replacing sucrose with stevia
(powder and extracts) for sweetness in cookies. Nutraceutical, safety and health potential of the
Stevia were examined against hyperglycemia and hypercholesterolemia health disorders as per
hypothesized for effective usage. Therefore, Stevia powder and extracts were examined in rat
experimental modeling; subsequent results were subjected to statistical design accordingly.
Results of this detailed study, after comprehensive analytical experiments, were segregated in main
sections to allow systematic representation of the data effectively under subheadings of nutritional
profile, antioxidant properties, steviosides quantification, product development and efficacy
studies. The discussion of various parameter has been presented under following headings and
sub-headings:
4.1 Proximate composition of raw material
The mean values for proximate composition of Stevia leaf powder and wheat flour have been
presented in Table 3. Results have depicted that moisture, ash, crude protein, crude fat, crude fiber
and carbohydrate or NFE content were found as 3.95±0.21, 8.75±0.35, 10.64±0.33, 5.47±0.36,
7.60±0.45 and 63.59±0.62 (g/100g). However, chemical composition of wheat flour have been
found as moisture content (11.08±0.5 g/100g), ash content (2.20±0.04 g/100g), crude protein
(8.62±0.13 g/100g), crude fat (1.23±0.06 g/100g), crude fiber (1.43±0.18 g/100g) and NFE
content (75.89±0.51 g/100g).
Moisture content (MC) of foods is the amount of water present or bound in any food material like
cereals, plant materials, fruits, vegetables, meat, etc. MC varies from material to material
depending on the nature, chemical composition and environmental factors. The results for MC of
Stevia leaf powder were in accordance with the research outcomes of previous work reported
42
moisture content as 4.56 (Goyal et al., 2010), 7.45-7.80 (Segura-Campos et al., 2014) and
5.37±1.12 g/100g (Abou-Arab et al., 2010).
Ash is the portion of food or any organic material which remained after complete burning at
elevated temperature. The ash content of Stevia leaf powder (8.75±0.35 g/100g) were similar to
the research investigations of Segura-Campos et al. (2014) who reported ash content in the range
of 7.73-9.25, Abdalbasit et al. (2014) reported ash content which as 4.65-12.06, however, Mishra
et al. (2010) found high ash content which was 11g/100g.
Proteins are nitrogenous organic compounds having one or more long chains of amino acids,
structural parts of body tissues such as hair, nails, enzymes, muscles, antibodies, etc. Stevia had
been found to be very impressive in protein content (10.64±0.33 g/100g) representing as a good
source. Previously various scientists have conducted research on chemical composition of Stevia
and found it to be out of the box if we consider any other plant material as a protein source. Savita
et al., (2004) conducted research on determining the biochemical attributes of Stevia grown in
India and depicted 9.8 g/100g of crude protein content while Goyal et al. (2010) have found
protein content as 11.2 g/100g, Segura-Campos et al. (2014) worked on two distinct Mexican
Stevia varieties and found high range of protein content from 12.11 to 15.05 g/100g. Variation in
results obtained from Pakistani grown Stevia may be due to environmental factors like soil &
water quality, genetic makeup, growing conditions, temperature variations and genetic makeup.
Stevia is not indigenous to Pakistan as it is native to South American countries like Paraguay,
Brazil, Canada, etc. Stevia acclimatized to Pakistani environment but the environmental
parameters are quite different as compared to South American countries.
Fats are available in sparse amount in various plant parts like leaves, stems, and fruits. Stevia leaf
powder contained 5.47±0.36 g/100g crude fat. The results of current study were in line with the
findings of Tadhani and Subash (2006a) who reported as 4.34 g/100g fat, 5.2-5.6 g/100g. Recently,
Moguel-Ordonez et al. (2015) have noted that drying conditions for Stevia leaves effect fat
content. They declared that fat content varied from 3.05 to 3.81 g/100g when dried by radiation
and shade techniques respectively. Owing to their capability of making complex matrix of
conjugated structures with starch as glycolipids and lipoprotein with proteins, ultimately helps in
regulation of structural and physiological functioning of living organisms in the form of essential
and non-essential fatty acid (Sramkova et al., 2009).
43
Crude fibers are indigestible cellulose, lignins, pentosans and other related constituents available
in foods that ultimately provide roughage and bulk diet. The crude fiber content of stevia leaf
powder found in the current research (7.60±0.45g/100g) were in accordance with the results of
Lemus-Mondaca et al. (2016) who reported crude fiber ranging from 9.52-10.65 g/100g. Atteh et
al. (2011) reported crude fiber in low range of 4.34-5.26 g/100g as compared to other research
outcomes. Fibers are direly needed for the proper digestion and smooth peristaltic movement
across intestines by resisting enzymes action. However some of their parts are digested by lower
gut microbiota. It is thought to cope with prevalent health issues including diabetes and high levels
of blood cholesterol.
Nitrogen free extract (NFE), mainly consists of carbohydrates such as starches and sugars and
major portion of hemicellulose in food and feed. These are main sources of energy and are found
as structural components of cellular elements. Stevia leaf powder had high contents of NFE
(63.59±0.62g/100g). Several scientists reported Stevia NFE in varied ranges depending upon
varietal difference, environmental parameters and cultivation practices. Abou-Arab et al. (2010)
have reported it as 61.9 g/100g, Gasmalla et al. (2014) found it in range varying from 72.42 to
79.77 g/100g. However, Moguel-Ordonez et al. (2015) reported NFE level ranging from 63.73-
66.43 g/100g.
Wheat flour have already been extensively studied by different scientists who reported varied
amounts of chemical composition parameters. Pasha et al. (2009) by using various spring wheat
varieties of Pakistan found 12.92±0.22 to 13.42±0.88 g/100g moisture content, 10.23±0.54 to
11.60±0.53 g/100g crude protein and ash content 0.41±0.04 to 0.55±0.03 g/100g as content,
respectively. Moisture content in different wheat varieties flour ranged from 11.87 to 12.66
g/100g, crude protein 12.17 to 13.07 g/100g, crude fat 1.03 to 1.28 g/100g, crude fiber 0.95 to
1.15 g/100g, whereas ash content in the range of 1.32 to 1.44 g/100g, respectively when
experimented by Ahmad (2016). In another study chemical analysis of wheat flour depicted that
10.84 g/100g protein, 1.68 g/100g fat, 1.26-2.14 g/100g fiber and 1.84 g/100g ash content (Saeed,
2012).
4.2 Functional properties of Stevia
The mean values for different functional properties of Pakistani grown Stevia and wheat flour have
been expressed in Table 4. pH depicting acidity and alkalinity for Stevia powder and wheat flour
44
are 6.14±0.38 and 6.03±0.59. Swelling power was found as 5.01±0.63 and 2.05±0.24 g/g, water
holding capacity for Stevia and wheat flour are 3.93±0.25 and 4.99±0.30 ml/g, oil holding capacity
was recorded as 5.96±0.17 and 8.17±0.67 ml/g while bulk density was calculated as 0.55±0.06 and
0.66±0.03 g/ml for Stevia powder and wheat flour respectively. Different functional properties of
Stevia have been determined by using their respective protocols including water absorption
capacity, bulk density, pH, swelling power and emulsification value.
The findings of current research were in similar trend as found by different researchers. pH for
Stevia powder was reported as 5.95 by Savita et al. (2004). However, Gasmalla et al. (2014) have
found Stevia pH in the range varying from 5.95-6.24. Bulk density of Stevia leaves powder
reported by Mishra et al. (2010) as 0.443g/mL. Water holding capacity was found to be in range
of 2.87-4.07mL/g as reported by Segura-Campos et al. (2014) and 4.7mL/g have been expressed
by Savita et al. (2004). The swelling ability of Stevia was in accordance with the results of Savita
et al. (2004) & Mishra et al. (2010) which declared it as 5.01g/g. Similarly for fat absorption
capacity current results were found to be in line with the finding of Mishra et al. (2010) which was
5.0ml/gm. However, Segura-Campos et al. (2014) found it to be in range of 6.49-6.79mL/g.
45
Table 3. Proximate analysis (g/100g) of wheat flour and Stevia
Parameter Stevia Wheat flour
Moisture 3.95±0.21 11.08±0.5
Ash 8.75±.35 2.20±0.04
Crude Protein 10.64±0.33 8.62±0.13
Crude Fat 5.47±0.36 1.23±0.06
Crude Fiber 7.60±0.45 1.43±4.18
NFE/ Carbohydrate 63.59±0.62 75.89±0.51
Values expressed are means ± standard deviation of three values
Table 4. Functional properties of Stevia
Functional property Stevia Wheat Flour
pH 6.14±0.38 6.03±0.59
Swelling power (g/g) 5.01±0.63 2.05±0.24
Water Holding Capacity (mL/g) 3.93±0.25 4.99±0.30
Oil-Holding Capacity (mL/g) 5.96±0.17 8.17±0.67
Bulk Density (g/mL) 0.55±0.06 0.66±0.03
Values expressed are means ± standard deviation of three values
46
4.3 Mineral analysis of Stevia
The mean values recorded for different mineral elements in Stevia have been expressed in Table
5. Macro minerals that have been quantified were sodium (Na), potassium (K), phosphorous (P),
magnesium (Mg), iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), nickel (Ni) and cobalt (Co).
Their values in mg/kg were recorded as 29.4, 2195.3, 372.1, 286.2, 24.29, 1.42, 10.24, 0.85, 1.26
and 0.035mg/kg, respectively. On the other hand, heavy metal such as lead, mercury, cadmium,
chromium and arsenic have also been investigated in order to provide a comprehensive mineral
profile of Stevia. The results depicted that lead, mercury and cadmium have not been detected in
Stevia samples, while chromium (0.15 µg/g) and arsenic (0.11µg/g) have been found in minute
quantities.
From nutritional point of view, the Recommended Daily Intake (RDI) of minerals like Na, K, P,
Mg, Fe, Zn, Mn, Cu, Ni and Co were 2400mg, 3500mg, 1000mg, 350mg, 15mg, 15mg, 5mg, 2mg,
<1mg and 5µg respectively per day have been reported by Biego et al. (1998). Sodium
(29.4mg/100g), iron (24.9mg/100g) and manganese (10.24mg/100g) being important in
maintaining the blood pressure, brain and nervous functions, play role in fat and carbohydrate
metabolism, blood cells production, sugar regulation and oxygen transport in the body as part of
hemoglobin and myoglobin. The similar results for sodium, iron and magnesium have been
reported by Savita et al. (2004) & Abou-Arab et al. (2010) with minor difference due to plant
growing environment specially water used for watering the plants and soil composition as they
affect the mineral composition of plants. Potassium was found in appreciable quantity have the
highest value 2195.3 mg/100g which can be due the soil fertility as in Pakistan NPK fertilizers
have been used in excess for the proper growth and soil fertility management that ultimately affect
the amount of potassium in Stevia. The results found are similar to the findings of Krasina &
Tarasenko (2016) and Mishra et al. (2010) who have reported potassium content of stevia 1585-
1915mg/100g and 1800mg/100g, respectively.
The results of Phosphorous (372.1mg/100g) and magnesium (286.2mg/100g) were coincidences
with the outcomes of Goyal and Samsher (2010) who reported them to be in range of 318mg/100g
& 349mg/100g respectively. Zinc (1.423mg/100g), nickel (1.26mg/100g), copper (0.85mg/100g)
and cobalt (0.35mg/100g) have an important impact i-e tissue repair, immune-modulation, sexual
health, DNA protection in growth regulation is maintained by Zinc. Nickel provide optimal skin
47
growth, bone strengthening, structuring and enhances zinc absorption, its deficiency leads to
dermatitis and retarded growth rate (Atteh et al., 2011; Lemus-Mondaca et al., 2016).
Heavy metals such as lead (Pb), cadmium (Cd), mercury (Hg), chromium (Cr) and arsenic (As)
have not been found in appreciable amount in Stevia leaves (Table 6). A very few studies on heavy
metals in Stevia have been done so far. Lead (Pb), cadmium (Cd), mercury (Hg), chromium (Cr)
and arsenic (As) have been reported in negligible amounts which leave it safe for consumption as
a healthy sweetener. The findings are in harmony with the work of Gasmalla et al. (2014) who
worked on profiling of Stevia dried by using oven, sunlight and microwave grown in China and
reported that Pb, Hg, As and Cd ranges from 0.14-4.77µg/g, 0.01 µg/g, 0.09-0.30 µg/g and 0.33-
0.49 µg/g. However, in another research work done in Malaysia by Hajar et al. (2014) on the heavy
metal assessment of Stevia using Inductively Coupled Plasma Mass-Spectrophotometer (ICP-MS)
revealed that Pb, As, Cd, Cr were present in ranges of 0.513-1.735, 0.053-0.438, 0.226-0.604 and
3.87-6.181ug/g respectively.
48
Table 5. Mineral profile of Stevia
Minerals Value (mg/Kg)
Na 29.4±0.12
K 2195.3±0.88
P 372.1±16.74
Mg 286.2±0.06
Fe 24.29±0.04
Zn 1.423±0.04
Mn 10.24±0.06
Cu 0.85±0.02
Ni 1.26±0.02
Co 0.035±0.02
Table 6. Heavy metals in Stevia
Minerals Value (µg/g)
Pb ND
Hg ND
Cd ND
Cr 0.15±0.02
As 0.16±0.04
ND=Not detected
49
4.4 Fatty acid profile
Fatty acids profile of Stevia powder oil has been presented in Table 7. Fatty acids including major
classes like saturated, monounsaturated and polyunsaturated were determined by comparing with
21 component fatty acids standards kit. Saturated fatty acids like Caprylic acid (C:8:0), Capric
acids (C:10:0), Lauric acid (C:12:0), Tri-decanoic acid (C:13:0), Myristic acid (C:14:0),
Pentadecanoic acid (C:15:0), Heptadecanoic acid (C:17:0), Arachidic acid (C:20:0) and Behenic
acid (C:22:0) have not been detected. However, Palmitic acid (C: 16:0) and Stearic acid (C: 18:0)
were the only two saturated fatty acids that have been found in appreciable quantities i.e.
28.31mg/g and 2.39mg/g respectively. On the other hand, monounsaturated fatty acids identified
in stevia leaves include Palmitoleic acid (C: 16:1) 2.17mg/g and Oleic acid (C: 18:1) 4.95mg/g.
Myristoleic acid (C: 14:1), Eicosaenoic acid (C: 20:1), Erucic acid (C: 22:1) are the
monounsaturated fatty acids that could not be detected in stevia extracted oil.
Linoleic acid (C: 18:2) and linolenic acid (C: 18:3), the polyunsaturated fatty acids also known as
omega-3 fatty acid alpha linolenic acid (ALA), have been found in stevia oil in quantities of
13.6mg/g and 25.48mg/g respectively. Though stevia is not a good source of lipids or oils, its fatty
acid profile makes it a plant of interest for some health point of view. Linoleic and linolenic acid
are the essential fatty acids for humans as well as other animals and they are unable to synthesize
these fatty acids within their bodies and therefore must be provided from food sources in order to
play their crucial role in life of heart cells (Sellami et al., 2011; Rezeng et al., 2014).
The results obtained in this study are found to be similar to the few previous investigations
performed on Stevia grown in diverse environmental conditions. According to the research
outcomes of Tadhani & Subash (2006b), palmitic acid, linoleic and linoleic acids have been found
to be in high amounts i.e. 27.51g/100g, 12.40g/100g and 21.59g/100g respectively. However,
Palmitoleic, Stearic and Oleic acids are in minute quantities i.e. 1.27g/100g, 1.18g/100g and
4.36g/100g respectively. Siddiqui et al. (2012) worked in Bangladesh on Stevia oil extracted by
hydro-distillation method using gas chromatography mass spectroscopy (GC-MS) reported that
the main fatty acid was Palmitic acid (86.50%) while stearic and linoleic acids were in low
proportions of 2.20% and 3.26% respectively. Recently, Lemus-Mondaca et al. (2016) have
worked on the Stevia dried from different techniques and investigated the fatty acid profile of
stevia. Palmitic, Stearic and Oleic acids found in low amount ranging from 0.46-1.47%, 0.23-
50
0.47% & 0.45-1.39% respectively. However they have discovered Linoleic and Linolenic acids in
appreciable amount ranging from 1.37-2.22% and 1.36-5.96% respectively.
51
Table 7. Fatty acid profiling of Stevia
Sr. No. Fatty acid Carbon No. Cons. of Fatty acids
(mg/g)
1 Caprylic acid C:8 ND
2 Capric acid C:10 ND
3 Lauric acid C:12 ND
4 Tri-decanoic acid C:13 ND
5 Myristic acid C:14 ND
6 Myristoleic acid C:14:1 ND
7 Pentadecanoic acid C:15:0 ND
8 Palmitic acid C:16:0 28.31
9 Palmitoleic acid C:16:1 2.17
10 Heptadecanoic acid C:17:0 ND
11 Stearic acid C:18:0 2.39
12 Oleic acid C:18:1 4.95
13 Linoleic acid C:18:2 13.65
14 Arachidic acid C:20:0 ND
15 Eicosaenoic acid C:20:1 ND
16 Linolenic acid C:18:3 25.48
17 Behenic acid C:22:0 ND
18 Erucic acid C:22:1 ND
N.D. = Not detected
52
4.5 Phytochemical analysis & antioxidant assay of Stevia
Phytochemicals play a protective role in body against chronic ailments. These are non-nutritive
plant components and also work as anti-nutritional factors in regulation of body functions. With
regards to health facets, phytochemicals from Stevia are utilized in diets as well in addition to its
usage as sweetener, therefore eliminating or inactivating the harmful effects of environment by
reducing the anti-nutritional effects (Anton et al., 2008). In current study, different solvent extracts
of Stevia leaves powder have been investigated for their phytochemical contents as well as
antioxidant potential.
4.5.1 Extraction efficiencies
In solvent extraction, all three solvents have given good extraction efficiency which ultimately
impact the phytochemical analysis results including total phenolic content and total flavonoids
content. Ethanol, methanol and water have good polarity and hence are used favorably to extract
polar compounds such as phenolics and flavonoids contents which are believed to be effective
antioxidants. Ethanol being organic and nontoxic have been extensively used for extraction.
Methanol gave the high content in solvent extraction method followed by ethanol and water.
Toxicity of methanol limits its use in extraction for food applications. Water along with ethanol
can have applications in food as they are non-toxic and extracts can be concentrated by using rotary
evaporator for solvent removal. This produces an extract dense in the biochemical constituents
(Badarinath et al., 2010).
The extracts that were obtained had different colors. Water extract had a light brownish color,
ethanol extract was dark green, methanol extract was light green, and supercritical extract had a
color in between dark and light green. Except water extract, all other extracts were quite clear with
some sedimentations and suspended particles. This may be due to non-phenol contents such as
terpenes, proteins, carbohydrate and some amount of fats as well in water extracts than in other
extracts. There is possibility that some of the phenolic compounds in extracts have formed
complexes that have the solubility in ethanol and methanol. Non-polar solvents such as ether and
low polarity solvents such as chloroform, ester, acetone etc. have been used in specific cases and
their availability also limits their use in the experiment and hence their frequency of use was found
very low (Alam et al., 2013). Therefore, based on results, utilization and application the best
extract was from supercritical method, however among solvents ethanol water mixture was the
best solvent for extraction. Extraction efficiencies increased as we moved from water to
53
supercritical. Supercritical fluid extraction gave the best results in terms of phenolics yield as
compared to other three conventional solvents. However, there are some reservations while using
supercritical fluid extraction instrument mainly the cost of the instrument which is quite high as
compared to other conventional methods. Therefore the method is less economical as compared to
the conventional solvent extraction technique.
4.5.2 Phytochemical analysis
4.5.2.1 Total phenolic content (TPC)
The results of mean squares and mean values for TPC have been expressed in Table 8 and Table
9, respectively. The values depict highly significant differences among different extract. The TPC
of all extracts was calculated by using the Folin-Ciolcateu (FC) method. The calibration curve,
y=0.007x+0.0063 with R2 = 0.9986 was obtained by running different concentrations of Gallic
acid standard solution, where x is the absorbance and y is the concentration of Gallic acid solution
expressed as mg GAE/g.
Supercritical fluid extracts contained highest TPC contents (38.22±0.05mg GAE/g) whereas
minimum TPC have been found in water extract (24.24±0.48mg GAE/g). The methanol and
ethanol extracts had higher TPC contents than the water extract but lower than the supercritical
fluid extract. The increase in total phenolic contents of other solvents as compared to water was
due to their better extraction capability which in turn related to phenolic acids formation generally
from phenolic acids precursors by non-enzymatic inter-conversion between the molecules
(Rodriguez et al., 2012).
Effect of different drying condition on nutritional and antioxidant aspects of Stevia grown in Chile
was studied by Lemus-Mondaca et al. (2016). They found that TPC extracted from aqueous
methanol were 28.76±2.94 and 55.05±2.27 mg GAE/100g d.m. in shade drying and at 50oC oven
drying, respectively. In another study conducted in South Korea, Kim et al. (2011) found that TPC
in Stevia leaves and callus by water extraction were 130.67 mg and 43.99 mg catechin equivalent
/100g, respectively. In a similar study carried out by Periche et al. (2015) have declared that TPCs
ranges from 26.5 to 76.5 mg GAE/100 g from aqueous ethanol extracts.
54
Table 8. Mean squares for total phenolic contents of Stevia from different extracts
Source DF SS MS F-value
Method 3 322.757 107.586 132**
Error 8 6.523 0.815
Total 11 329.280
**= Highly significant
* = Significant
Table 9. Mean values for total phenolic contents of Stevia from different extracts
Extraction Method TPC (mg GAE/g)
SFE 38.22±0.05a
SME 32.45±0.61b
SEE 28.21±0.28c
SWE 24.24±0.48d
Values expressed are means ± standard deviation
SWE=Stevia water extract
SEE=Stevia ethanol extract
SME=Stevia methanol extract
SFE=Supercritical extract
55
4.5.2.2 Total flavonoids content (TFC)
The mean square results of all extracts for TFC are presented in Table 10 while mean values are
expressed in Table 11. The maximum TFCs have been found in SCFE (32.10±0.54mg CE/g) and
minimum in SWE (19.88±0.11mg CE/g). However ethanol and methanol have also given the
appreciable results such as SEE was found out to be 23.30±0.63 and SME was 27.14±0.16 mg
CE/g. The difference in the TFCs from Stevia leaves powder depended upon the extraction solvent,
their solvation properties, time, temperature and technique given for the extraction.
Previously Kim et al. (2011) have concluded that TFCs in Stevia callus and leaves powder was
1.57±0.05 and 15.64±0.25 mg QE/g, respectively. While Tadhani et al. (2007) have shown
21.73mg/g TFCs from methanol extracts. However, Periche et al. (2015) have reported that TFCs
in Stevia ethanol extracts varied from 9.9 to 45.1 mg QE/g. In order to check the impact of Stevia
extracts from different solvents and methods on polyphenolic contents which are TPC and TFC, a
correlation analysis was done. The correlation was found to be 0.967. This indicates that phenols
and flavonoids are the dominating polyphenolic group in Stevia. There exists a strong linear
relationship between phenols and flavonoids depicting that if one increases the other also increases
in a linear manner (Wolwer-Rieck, 2012; Sic Zlabur et al., 2015).
56
Table 10. Mean squares for total flavonoids contents of Stevia from different extracts
Source DF SS MS F-value
Method 3 243.872 81.2908 86.5**
Error 8 7.522 0.9402
Total 11 251.394
**= Highly significant
* = Significant
Table 11. Mean values for total flavonoids contents of Stevia from different extracts
Extraction Method TFC (mg CE/g)
SFE 32.10±0.54a
SME 27.14±0.16b
SEE 23.30±0.63c
SWE 19.88±0.11d
Values expressed are means ± standard deviation
SWE=Stevia water extract
SEE=Stevia ethanol extract
SME=Stevia methanol extract
SFE=Supercritical extract
57
4.5.3 Antioxidant activity of Stevia
4.5.3.1 Free radical scavenging activity (DPPH Assay)
Mean sum of squares for free radicals scavenging ability have been presented in Table 12 while
mean values in Table 13 from all four extracts. Significant results have been observed in such a
way that Supercritical extract gave the maximum antioxidant activity of Stevia followed by
methanol, ethanol and water extracts with % inhibition as 57.99±1.49, 52.87±0.28, 47.15±0.26
and 42.41±1.05 respectively. The outcomes have shown that appreciable percentage inhibition
have been observed from all extracts that ultimately endorses Stevia as good free radical scavenger
making our defense system immune to free radical maladies that leads to carcinogenesis.
Antioxidant activities of Stevia dried at different temperatures have concluded that drying have
positive effect on the antioxidant activity of Stevia leaves powder (Lemus-Mondaca et al., 2016).
Tadhani and Subash (2006a) have concluded that % inhibition in DPPH assay in Stevia leaves
extracts was recorded to be lower as compared to callus extract. The ability to scavenge free radical
of DPPH is directly related to the phenolic and flavonoid contents. Thus the highest antiradical
activity in DPPH• assay was possessed by Stevia methanol extract (IC50 = 0.38±0.23µg/mL)
(Gawel-Bęben et al., 2015). Significant antioxidant activity have been observed by Muanda et al.
(2011) who showed that Stevia extracts from water, methanol and essential oil caused 30-40%
reduction in free radicals. High correlation coefficient has been recorded between total phenolic
contents and free radical scavenging DPPH activity (R =0.96, p < 0.005). Kumar & Pandey (2013)
have also concluded in their studies that free radical scavenging activity of Stevia leaves extracts
was in direct relation to concentration of polyphenolic components.
58
Table 12. Mean squares for DPPH activity of Stevia from different extracts
Source DF SS MS F-value
Methods 3 413.701 137.900 159**
Error 8 6.951 0.869
Total 11 420.651
**= Highly significant
* = Significant
Table 13. Mean values for DPPH activity of Stevia from different extracts
Extraction Method DPPH (% reduction)
SFE 57.99±1.49a
SME 52.87±0.28b
SEE 47.15±0.26c
SWE 42.41±1.05d
Values expressed are means ± standard deviation
SWE=Stevia water extract
SEE=Stevia ethanol extract
SME=Stevia methanol extract
SFE=Supercritical extract
59
4.5.3.2 Ferric reducing antioxidant power (FRAP assay) of Stevia
The statistical analysis have shown significant results which are expressed in Table 14 while mean
values of FRAP assay are presented in Table 15. In current study Supercritical fluid extracts have
shown maximum reducing power 345.36±3.27 µMol Fe2+/g due to their maximum extraction
ability followed by methanol, ethanol and water extracts as 324.15±1.38, 294.45±0.90 and
236.57±1.37 µMol Fe2+/g respectively. The results indicated that the Stevia extracts are capable
of donating electrons to reactive free radicals thereby stabilizing and making them unreactive
species. Positive relationship was recorded between phytochemicals and antioxidant potential of
Stevia (Luximon-Ramma et al., 2005).
In a recent study, Barroso et al. (2016) have found that aqueous methanol extracts from oven-dried
samples constitute higher ferric reducing power (i.e., lower EC50 values of 22.87 µg/mL and 28.79
µg/mL, respectively) than those of frozen fresh samples (50.66 µg/mL and 39.73 µg/mL). Moguel-
Ordonez et al. (2015) worked on the different drying conditions of Stevia followed by extraction
using ethanol and methanol. They determined antioxidant potential by FRAP assay and found that
high ferric reduction have been achieved by convection and shade drying methods, while sun and
radiation drying methods had shown lower reducing power of Ferric.
60
Table 14. Mean squares for FRAP activity of Stevia from different extracts
Source DF SS MS F-value
Methods 3 20083.2 6694.39 216**
Error 8 247.4 30.92
Total 11 20330.6
**= Highly significant
* = Significant
Table 15. Mean values for FRAP activity of Stevia from different extracts
Extraction Method FRAP
(µmol Fe2+/g)
SFE 345.36±3.27a
SME 324.15±1.38b
SEE 294.45±0.90c
SWE 236.57±1.37d
Values expressed are means ± standard deviation
SWE=Stevia water extract
SEE=Stevia ethanol extract
SME=Stevia methanol extract
SFE=Supercritical extract
61
4.5.3.3 ABTS assay for free radical scavenging
The ability of Stevia to scavenge free radicals by using different extracts was also analyzed using
ABTS•+ scavenging assay. The statistical analysis results expressing mean sum of squares and
mean values for all extracts (µM TE/L) have been shown in Table 16 and Table 17. In current
study, Stevia extracts possess significant antioxidant properties by scavenging ABTS generated
free radicals with highest value for supercritical fluid extraction (55.04±0.09µM TE/L) while
minimum was observed for water extract (25.79±0.97µM TE/L). ABTS is an excellent assay for
hydrogen donating and chain-breaking antioxidants analysis (Siow and Hui, 2013). The ability of
Stevia to scavenge free radicals by using different extracts was also analyzed using ABTS•+
scavenging assay. The medicinal benefit aids in lessening the dermatological ailments by avoiding
oxidative damage and its progression. The benefits are mainly due to the presence of different
phenolics, carotenoids, flavonoid components especially ferrulic, syringic, carvacrol,
isopinocarveol, thymol, cardinal, etc (Muanda et al., 2011; Siow and Hui, 2013).
High ABTS•+ activity was noted for ethanol and glycolic aqueous Stevia extracts 42.45%–89.27%
and 43.34%–97.23% respectively (Gawel-Beben et al., 2015). The antioxidant potential of Stevia
extracts from water, methanol and essential oils was determined by Muanda et al. (2011) and
showed that extracts have significant antioxidant potential by reducing and scavenging free
radicals in DPPH and ABTS assay. Reduction in free radicals falls in the range of 30-40% in DPPH
assay while 25-30% in ABTS assay. High correlation coefficients have been found between total
phenolic contents and free radical scavenging DPPH and ABTS activity (R =0.96, p < 0.005; R
=0.88, p < 0.01, respectively). Different pigments present in plants have been related to the
antioxidant activity. The pigments were affected by heat treatment leading to decomposition and
thermal oxidation that ultimately impact on antioxidant potential (Siow & Hui, 2013).
62
Table 16. Mean squares for ABTS assay activity of Stevia from different extracts
Source DF SS MS F-value
Method 3 1543.29 514.431 244
Error 8 16.88 2.110
Total 11 1560.17
**= Highly significant
* = Significant
Table 17. Mean values for ABTS assay activity of Stevia from different extracts
Extraction Method ABTS
(µM TE/L)
SFE 55.04±0.09a
SME 51.40±1.62b
SEE 41.12±2.19c
SWE 25.79±0.97d
Values expressed are means ± standard deviation
SWE=Stevia water extract
SEE=Stevia ethanol extract
SME=Stevia methanol extract
SFE=Supercritical extract
63
4.6 Fourier Transform Infra-red Spectrophotometric analysis (FTIR) of
Stevia
Fourier transform infrared (FTIR) spectroscopy is complementary technique that provides
information on molecular structure. Both quantitative and qualitative information can be attained
by using spectroscopy. Through unique pattern of absorption, different organic functional groups
can be distinguished and the relative concentration of these components can be calculated by
determining the absorption intensity of sampled entity (Wetzel and LeVine, 1999). High signal to
noise at spatial resolution is obtained from the output IR spectra of this special technique. Low
energy ranging from 0.05-0.5 eV of Mid-IR photons are not capable of causing ionization or
breaking any bond. The most challenging future scientific fields involve the proper knowledge and
apprehension of dynamics, structure and functionality of molecule. The Fourier transform infrared
spectroscopy (FTIR) was used in order to characterize and identification of different functional
groups present in Pakistani grown Stevia. FTIR analysis of chemical constituents and
Steviol/diterpene glycosides in stevia leaves powder and different extracts were carried out.
4.6.1 Stevia powder
The functional groups corresponding to their peaks in infrared spectra (Fig. 1) of Stevia leaves
powder has been shown in Table 18. The IR spectra for raw Stevia powder gave different bands
indicating particular functional groups at distinct IR wavelength. At 3301.05cm-1, a broad band
showed the presence of alcohols –OH groups stretching as well as secondary amides groups. This
indicated the protein availability as amide depicts defining molecular character of proteins.
Hydrogen bonding holds the secondary amides configuration. However, sp2 and sp3 hybridization
of carbon was indicated by peaks at 2848.45cm-1 and 2920.71cm-1 respectively. These indicated
the presence of compounds with alkane functional groups and configurations. Amide linkages
were basically in peptide bonds which appear with in the main chain of a protein bonds (Inamake
et al., 2010). The 1604.74cm-1 band in IR spectra of Stevia leaves powder indicated the ketone
C=O stretching group components which was attributed to flavor along with different aldehyde
groups. Alkenes and primary amines have been observed according to the C=C stretching at
1509.66cm-1 which are important components of all steviosides ranging from Stevioside to Steviol,
etc. At 1372.74cm-1 bending of –OH groups have been seen which is an important constituent of
different chemical groups including glucose attached to the Steviol which was considered as the
basic building block to all steviosides. Bands at 1022cm-1 and 809.84cm-1 in IR spectrum were
64
attributed to RCOOR` stretching of ester groups, alkanes (C-C) and carboxylic groups (ROOH)
stretching respectively.
Table 18. FTIR spectrum values of Stevia leaves powder
Figure 1: FTIR spectrum of Stevia leaves powder
Sr. No. Wave Number Functional group Vibration type
1 3301.05 Alcohols, secondary amides O-H stretching, N-H
2 2920.71 Alkane =CH2 stretching
3 2848.45 Alkane C-H stretching
4 1604.74 Ketones C=O stretching
5 1509.66 Alkene, Primary amines C=C stretching, N-H
6 1372.74 OH Bending -OH stretching
7 1022.31 Esters (RCOOR`)
8 809.84 Alkanes, Carboxylic acids C-C, O-H stretching
65
4.6.2 Stevia water extract
Stevia water extract was analyzed and different bands were obtained (Fig. 2) which coincides with
Stevia leaves powder spectrum and presented in Table 19. However, number of stretching bands
from different functional groups were less as compared to Stevia powder. These bands helped in
identification of SGs present in Stevia water extract. At 3330.0cm-1, distinct and broad band was
observed which indicate the –OH alcoholic groups stretching, however –COOH carboxylic acids
also showed their presence at this IR wavelength. Concentration of steviosides, nature of solvent
extract which was water and temperature for extraction process played an important role in
maximum absorption of IR which gave a broad and prominent band. Compounds having
carboxylic groups attached have been identified from band position. Primary amines have been
observed which indicated high protein content in Stevia powder and water extract as well. At
2358.49cm-1, stretching of thiol as well as carbon dioxide (S-H & O=C=O) group have been
observed which ultimately indicated the presence of sulphur compounds. Absorption shifts were
directly dependent on concentration of components that can clearly be observed in intermolecular
bonds of different molecules. The presence of a band at 1634cm-1 were assigned to alkenes,
inorganic phosphates, C=C groups stretching. The band at 1495.37cm-1 indicated the presence of
-CH2 stretching of alkane group, C=C stretching and –NO2 stretching representing aromatic groups
which were attached to Steviol base which was benzene ring having different functional groups
attached to it indicating the presence of diterpene glycoside in water extract of Stevia as presented
by Kumar and Kumar (2015) who had published the functional groups identification of Stevia
leaves. However, our study elaborates results on functional groups mapping of Stevia leaves
powder and stevia water extracts have been discussed.
66
Table 19. FTIR spectrum values of aqueous Stevia extract
Figure 2: FTIR spectrum of aqueous Stevia extract
Sr. No. Wave Number Functional group Vibration type
1 3330.00 Alcohols, Carboxylic acids, Amines -OH, -COOH, N-H stretching
2 2358.49 Thiol, CO2 S-H, O=C=O stretching
3 1634.28 Alkene, Inorganic phosphates C-H, P, C=C stretching
4 1495.37 Alkanes, Aromatic groups C-H, -NO2 stretching
67
4.6.3 Stevia methanol extract
The functional groups of Stevia methanol extracts have been presented in Fig. 3 and Table 20. The
IR spectra gave distinct bands indicating the particular functional groups. O-H stretching have
been observed at 3297.25cm-1, however, there was a shift in band which leads to the change in
functional group at 2924.51cm-1 & 2844.64cm-1 with =CH2 stretching representing alkane groups.
In methanol extracts of Stevia, sp2 and sp3 hybridization of carbon was indicated which showed
the availability of compounds with alkane functional groups and configurations. Ketones have
been found at 1688.42cm-1 with C=O stretching imparting characteristic fragrance to Stevia
methanol extract. At 1597.13cm-1 alkenes bending and its relative compounds have been observed
that were important constituents of SGs ranging from Stevioside to Steviol, etc. The primary
amide structure was due to their nitrogen and hydrogen bonding capabilities which were observed
at 1521.07cm-1. In a biochemical reference, amide linkages are the peptide bonds which exists in
iso-peptide bonds and in main chains of protein (Inamake et al., 2010). At 1357.52cm-1 bending
of –OH groups have been seen which was an important constituent of different chemical groups
including glucose attached to the Steviol and considered as the basic building block to all
steviosides. Bands at 1020.52cm-1 and 847.87cm-1 in IR spectrum were attributed to RCOOR`
stretching of ester groups and carboxylic groups (ROOH) stretching respectively.
68
Table 20. FTIR spectrum values of methanolic Stevia extract
Sr. No. Wave Number Functional group Vibration type
1 3297.25 Alcohols O-H stretching
2 2924.51 Alkane =CH2 stretching
3 2844.64 Alkane =CH2 stretching
4 1688.42 Ketones C=O stretching
5 1597.13 Alkene C=C stretching,
6 1521.07 Primary amines N-H
7 1357.52 OH Bending -OH stretching
8 1020.52 Esters (RCOOR`)
9 847.87 Carboxylic acids O-H stretching
Figure 3: FTIR spectrum of methanolic Stevia extract
69
4.6.4 Stevia ethanol extract
The FTIR spectrum of Stevia ethanol extract was analyzed (Fig. 4) and different bands were
obtained presented in Table 21. However, number of stretching bands/functional groups were less
as compared to Stevia powder. These bands help in identification of SGs present in indigenous
Stevia. At 3327.67cm-1, distinct and broad band representing alcoholic group –OH stretching,
while at 2924.51cm-1 alkane stretching have been observed. Concentration of steviosides, nature
of solvent and temperature for extraction process plays an important role in maximum absorption
of IR that gave a broad and prominent band. Compounds having ketonic functional groups have
been determined from their band position at 1737.86cm-1. Alkenes (C=C) and primary amine
compounds have been observed at 1597.13cm-1 indicating high protein components. At
1239.62cm-1 and 1019.02cm-1 stretching of alcoholic (-OH) and esters (RCOOR`) have been
observed which represents aromatic groups attached to Steviol base having different functional
groups attached to it indicating the presence of diterpene glycoside in ethanol extract of Stevia.
Absorption shifts were directly dependent on concentration of components that can clearly
observed in intermolecular bonds of different molecules (Kumar and Kumar, 2015)
70
Table 21. FTIR spectrum values of ethanolic Stevia extract
Figure 4: FTIR spectrum of ethanolic Stevia extract
Sr. No. Wave Number Functional group Vibration type
1 3327.67 Alcohols O-H stretching
2 2924.51 Alkane -CH2 stretching
3 1737.86 Ketones C=O stretching
4 1597.13 Alkene, Primary amines C=C stretching, N-H
5 1239.62 OH Bending -OH stretching
6 1019.02 Esters (RCOOR`)
71
4.7 HPLC quantification of Steviosides
Various analytical techniques have been adopted including thin layer chromatography (TLC), high
performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS)
in order to determine the concentration of SG in Stevia leaves. In this study, SGs or steviosides
have been quantified from different extracts including water, methanol, ethanol and Supercritical
fluid extracts of Stevia leaves. Three most prevalent SGs were individually determined by UV
detection at 210 nm and C-18 column using 70/30 acetonitrile/water mobile phase. The
chromatographic analysis run time was 15 minute. Previously it has been reported that in gradient
elution mode, analysis took more than 15 min to achieve the separation followed by equilibration
time (5 min) back to initial conditions. However, isocratic elution mode acquire 15min or less in
order to elute and separate the components of interest, without affecting resolution. Stevioside,
Rebaudioside A and Steviol standards with concentration of 10, 100, 500 and 1000pppm were
used for calibration. The calibration curves for these standards are presented in Fig. 6, 8 and 10
respectively. However, the respective chromatograms are expressed in Fig. 7, 9 and 11 for
Stevioside, Rebaudioside A and Steviol respectively. Stevioside standard with different
concentration were run and their elusions were recorded according to their retention times and
peak areas. A linear calibration curves was obtained for all three standards with their high
correlation values for Stevioside, Rebaudioside A and Steviol as R2=0.993, R2=0.998 and
R2=0.997 respectively. HPLC chromatograms of water, ethanol, methanol and supercritical were
obtained and presented in Fig. 12, 13, 14 and 15 respectively.
The results were calculated by identifying the chromatographic peaks with reference to retention
times of known standard and quantified from peak areas of known amounts of standards. The mean
squares results from different extracts have been expressed in Table 22 exhibiting substantial effect
of solvents on extraction of different steviosides. Mean values for all steviosides have been
expressed in Table 23, while the results are graphically presented in Fig. 5. Stevioside, the prime
Steviol glycoside comprised of three glucose molecules attached to an aglycone, the Steviol
moiety; ent-13-hydroxykaur-16-en-18-oic acid, very stable and 250–300 times sweeter than
sucrose. Considering the effect of solvents, maximum concentration of Stevioside were quantified
in supercritical fluid extract followed by ethanol extract, methanol and water extracts with their
respective values as 1107.9±50.9, 929.8±39.9, 822.1±36.1 and 665.3±27.3 (mg/kg or ppm).
72
The second most prevalent Steviol glycoside is Rebaudioside A having sweetening potency higher
than Stevioside (Barriocanal et al., 2008). Rebaudioside A was found maximum in supercritical
extract while minimum amount was found in water extract. Supercritical fluid extracts had
792.15±38.02 mg/kg of rebaudioside A, methanol had 456.24±21.44mg/kg, ethanol extract had
410.89±16.84mg/kg while minimum concentration 393.8±17.25 mg/kg had been found in the
water extract. Steviol is the common aglycone backbone of the all steviol glycosides (Cacciola et
al., 2011). Steviol, is almost completely metabolized from all Steviol glycosides by intestinal
microflora in the lower intestinal tract of human and rodents (Ref). Steviol concentration (Table--
--) had been found to be maximum in supercritical fluid extract (485.25±22.32mg/kg), followed
by ethanol extract (435.55±18.72mg/kg), methanol extract (379.36±16.69mg/kg) and minimum
concentration was quantified in water extract (357.26±.14.64 mg/kg).
The present results are in agreement with the investigation of Huang et al. (2010). They have
successfully worked in China on the isolation of Steviol glycosides using water, ethanol, hexane
and determined Stevioside, Rebaudioside A and Rebaudioside C by employing high speed counter
current chromatography (HSCCC). Subsequently, they subjected the fraction of HSCCC to high
performance liquid chromatography using UV detector and yielded pure Stevioside
(54mg/200mg), Rebaudioside A (36mg/200mg). In another research carried out in Singapore by
Liu et al. (1997) extracted Steviol glycosides from Stevia employing subcritical fluid extraction
technique (SubFE) using CO2 and polar co-solvent owing to their several advantages like simple,
rapid, economical and variation in extraction temperature and pressure. They employed capillary
electrophoresis (CE) and HPLC to analyze and quantify the extracts with only small amount of
samples and found more than 88% extraction efficiency of Steviol glycosides. They recommended
the wide application of SubFE coupled with CE for extraction and quantification different
bioactive moieties where HPLC is not available. A very simple and sensitive reversed-phase high-
performance liquid chromatographic method (RPHPLC) was devised by Minne et al. (2004) for
the determination of Steviol. The samples were separated on an ODS column with fluorescence
detection. From 100mg of dry Stevia plant, they determined 594ng of Steviol with accuracy and
precision. This assay was also experimented for detection and quantification of steviosides in
blood serum, plasma, urine and feces in clinical trials as well.
73
Pol et al. (2007) used supercritical fluid extractor (SCFE) for extraction of Steviol glycosides and
optimized critical temperature and pressure. They established that ethanol as co solvent worked
better and provided best extracts as compared to hot water extraction alone. The SCFE process
provide higher diffusivity and lower viscosity as compared to conventional liquid solvents. They
concluded that Stevioside separated from SCF extract ranges from 43-50% while rebaudioside B
was found in negligible amount ranging from 0.01-0.02%. Recently, Sharma et al. (2015) have
worked on in vitro production of steviosides and their molecular characterization. They found that
at the 14th day of cultivation cycle, maximal content of steviosides were found i.e. 115 mg/g of
plant dry mass. They have also reported that significant decrease in the Stevioside synthesis was
observed in suspension culture as compared to Stevioside content in Stevia callus (415 µg g/DW).
Highest yield 4.5% of Stevioside was 15.23g/L indicating the supportive role of biomass and
Stevioside content. Lorenzo et al. (2014) proposed a rapid methodology for the analysis of major
Steviol glycosides in Stevia leaves by optimizing the extraction and clarification conditions. The
quantification was achieved after ultrafiltration (UF) followed by quantification through HPLC-
DAD. They have found that the Steviol glycosides concentrations in the extracts were as follows:
3409.83 mg/L Stevioside and 1853.73 mg/L Rebaudioside A, representing 9.09% and 4.94%,
respectively, of the total mass of the starting plant material.
74
Table 22. Mean squares for HPLC quantification of Steviosides in Stevia extracts
Source DF Stevioside Rebaudioside A Steviol
Sample 3 103873** 108352** 9959.82**
Error 8 1562 622 335.54
Total 11
NS= Non-Significant
**=Highly Significant
*=Significant
Table 23. Mean values for HPLC quantification of Steviosides in Stevia extracts (mg/kg)
Values expressed are means ± standard deviation
SWE=Stevia water extract
SEE=Stevia ethanol extract
SME=Stevia methanol extract
SFE=Supercritical extract
Sample Stevioside Rebaudioside A Steviol
SWE 665.34±27.27 d 383.38±17.25 c 357.26±14.64c
SEE 929.84±39.98 b 410.89±16.84 bc 435.55±18.72b
SME 822.07±36.17 c 456.24±21.44 b 379.36±16.69c
SFE 1107.95±50.96 a 792.15±38.02 a 485.25±22.32a
75
Figure 5: Steviosides/SGs concentrations in different extracts of Stevia
0
200
400
600
800
1000
1200
1400
SWE SEE SME SCFE
Co
nc.
(p
pm
)
Steviosides Conc. in different Stevia extracts
Stevioside (ppm)
Rebaudioside A (ppm)
Steviol (ppm)
76
Figure 6: Calibration curve for Stevioside standard
Figure 7: HPLC chromatogram of Stevioside standard
y = 5530.3x - 82988R² = 0.9935
-1000000
0
1000000
2000000
3000000
4000000
5000000
6000000
0 100 200 300 400 500 600 700 800 900 1000 1100
PEA
K A
REA
CONC. (PPM)
Stevioside Standard linear curve
77
Figure 8: Calibration curve for Rebaudioside A standard
Figure 9: HPLC chromatogram of Rebaudioside A standard
y = 964.76x + 1E+06R² = 0.9998
0
500000
1000000
1500000
2000000
2500000
0 100 200 300 400 500 600 700 800 900 1000 1100
PEA
K A
REA
CONC. (PPM)
Rebaudioside A Standard linear curve
78
Figure 10: Calibration curve for Steviol standard
Figure 11: HPLC chromatogram of Rebaudioside A standard
y = 14425x - 334798
R² = 0.9977
-5000000
0
5000000
10000000
15000000
-100 100 300 500 700 900 1100
Pe
ak A
rea
Concentration (ppm)
Steviol standard linear curve
79
Figure 12: HPLC chromatogram of aqueous Stevia extract
Figure 13: HPLC chromatogram of ethanolic Stevia extract
80
Figure 14: HPLC chromatogram of methanolic Stevia extract
Figure 15: HPLC chromatogram of Stevia supercritical extract (SFE)
81
4.8 Value addition of Stevia in cookies
In developing countries like Pakistan, demand of baked products such as breads, biscuits, cookies,
etc has been enhanced in recent years to meet the needs of urban population. Owing to the inherent
nutritional and therapeutic benefits of Stevia, food industries can use Stevia as sucrose and
artificial sweetener replacer particularly in baking and beverage industries. A good deal of research
have been carried out recently by partially replacing sucrose with Stevia in different food products
(Refs). However, a little research work has been done on the utilization of Stevia powder or extract
as an alternative sweetener in cookies. Therefore, in current study, cookies have been prepared by
replacing sugar with stevia powder or extract and their consumer acceptability have been studied.
The cookies were also evaluated for biochemical and nutritional attributes.
4.8.1 Chemical composition of Stevia cookies
4.8.1.1 Moisture content
The analysis of variance (Table 24) for moisture content exhibited significant difference (P-----)
with the addition of Stevia leaves powder and extracts in cookies. The moisture content ranged
from 3.03±0.02 to 3.52±0.07% (Table 25) in different treatments with highest moisture content
being observed in T0 (3.52±0.07%) while lowest in T9 (3.03±0.02%). The results for moisture
content showed decreasing trend with the addition of Stevia leaves powder and extract. Mean
squares indicated that moisture content was substantially affected as the amount of Stevia powder
increased replacing sucrose. T1 (3.40±0.06) have maximum moisture content while T3 (3.26±0.06)
with minimum moisture content in powder treatments. The mean moisture content for Stevia
extracts treatments varied for T4 to T9 in which T4, T5 & T6 are water extract while T7, T8 & T9 are
supercritical extracts. The results depicted decreasing trend for moisture with extract increment T4
(3.19±0.04%), T5 (3.15±0.03%), T6 (3.14±0.04%), T7 (3.09±0.03%), T8 (3.06±0.03%) and T9
(3.03±0.02%) respectively.
The moisture content of any product is an important parameter from processing and technological
view point that determines the product quality and shelf life. Higher moisture content in cereal
products like cookies results in quality deterioration, promote microbial growth and ultimately
lowers the shelf life. In this study, the moisture content of Stevia cookies has decreased with the
increasing level of powder and extracts in respective treatments. In an earlier study, moisture
content of cookies prepared by substituting sucrose with different concentrations of intense
sweeteners like sorbitol, mannitol and fructose, was found to be in the range of 2.78 to 3.52%
82
(Pasha et al., 2002). The results were in line with a previous study in which development of high
protein and low calorie cookies were prepared by using different levels of Stevia leaves powder
(SLP) and defatted Soy flour (DSF). With the increase in concentration of SLP moisture content
decreased ranging from 3.7-2.4% while increasing trend was recorded with the increase in DSF
ranging from 3.5-4.5% (Kulthe et al., 2014). The results of present study showed a similar trend
when Danish cookies were prepared by replacing sucrose with erythritol at different concentration
levels varying from 1.90 to 1.86% for 25 to 100% erythritol addition respectively (Lin et al., 2010).
4.8.1.2 Ash content
The statistical analysis for ash content showed significant difference among different treatments
of Stevia leaves powder and extracts. The analysis of variance and mean values have been
presented in Table 24 and 25 respectively. Results depicted that highest amount of ash content was
observed in T3 (2.45±0.06) with 30% powder addition while lowest ash content was found in T4
(1.22±0.15%) with 1% water extract. The results have explained that with the addition of Stevia
powder, ash content enhanced significantly while non-significant increment has been observed
with the addition of Stevia water and supercritical extracts.
Appreciable amount of ash content has been found in Stevia powder cookies which was directly
associated with high mineral content in Stevia powder. The results of this study were varied and
high in ash content as compared to the findings of Lin et al. (2010) who used erythritol as sucrose
replacer and found it in the range of 0.64-0.69%. However, Kulthe et al. (2014) who used Stevia
leaves powder in their cookies and found ash content ranging from 0.9 to 1.9% which were in
harmony with this research outcomes. Cookies prepared by replacing sucrose with fructose,
mannitol and sorbitol at different level have ash content in range from 0.37 to 0.54% (Pasha et al.,
2002).
4.8.1.3 Crude Protein
Mean squares in Table 24 indicated that addition of Stevia powder and extracts have significantly
affected crude protein content. Means values for the effect of Stevia addition (Table 25) presented
that highest crude protein (15.06±0.51%) was recorded in T3 followed by T2 (13.66±0.82%), T1
(12.38±0.47%) while the lowest was observed in T0 (10.09±1.39%). Treatments in which stevia
leave powder was added have shown significant increasing trend while non-significant increase
was observed in extracts treatments. Stevia water extract treatments have maximum protein
83
content in T6 (11.55±2.08%) while minimum amount was observed in T5 (10.41±1.15%).
However, supercritical extracts treatments have shown minimum protein content in T7
(10.26±1.33%) while maximum amount was observed in T9 (11.69±0.92%).
Danish cookies prepared by replacing sucrose with intense sweetener erythritol at 25 to 100% level
showed non-significant difference among different treatments ranging from 8.69 to 8.96% (Lin et
al., 2010). The results of this study were similar to the findings of Kulthe et al. (2014) in which
sucrose was replaced with Stevia leaves powder at 15, 20, 25 and 30% level and showed significant
increase in protein content varying as 9.9, 12.8, 13.1, 13.4 and 13.5% respectively. Low calories
yoghurt cake was prepared by replacing sucrose with Stevia extracts for diabetic patients and found
12% of protein content in dietetic cakes as compared to which was 10% (Abdel-Salam et al., 2009).
4.8.1.4 Crude fat
The statistical analysis have shown that mean square values for crude fat have significant effect
among different treatments (Table 24). The crude fat content (Table 25) in Stevia powder cookies
found maximum in T3 (14.04±1.47%), followed by T2 (13.75±1.61%) and T3 (11.53±1.23%). In
Stevia water extract cookies treatments including T4 , T5 & T6 fat content increased in minute
amount expressed as 10.32±0.65, 10.58±1.15 and 11.16±2.36 respectively, compared to T0
(9.91±0.62). The highest fat content in Stevia cookies having supercritical extract replacing
sucrose was observed in T9 (12.23±2.63%) having 3% addition of extract while lowest in T7
(10.27±2.07%) with 1% level of extract. The results depicted that the replacement of sucrose with
Stevia leaves powder and extracts resulted in gradual increase in fat content. The research
outcomes of this study established that fat content slightly increased in extracts treatments while a
distinguishing increase have been observed in treatments with leaves powder due to high fat
content of raw Stevia. In an earlier study, 10% fat content have been observed in Stevia
supplemented yoghurt cakes having 100ml of extract in 500g recipe (Abdel-Salam et al., 2009). Low
fat and whole milk set type yoghurt was prepared by substituting sucrose with Stevia powder that
ultimately impact on chemical, sensory and rheological parameters. Fat content varied from 0.1-
3.5% in twelve different treatments when added from 0.04 to 0.02g/100g (Guggisberg et al., 2011).
Kulthe et al. (2014) have declared that with the increment of Stevia leaves powder in cookies from
15-30%, the fat content varied from 15.8% to 18.7%.
84
4.8.1.5 Crude fiber
Mean squares in Table 24 for crude fiber content of cookies having Stevia leaves powder and
extracts as sucrose replacer established that it was significantly different among treatments. The
mean results for crude fiber content (Table 25) depicted that incorporation of Stevia leaves powder
in cookies leads to increase in fiber content with the increase in concentration level i.e. @ T1=10%
(2.63±0.84%), @ T2=20% (3.54±0.97%) and @ T3=30% (5.65±0.72%), while lowest found in
control; T0 (1.27±0.01%), respectively. The mean fiber content for Stevia water extract calculated
to be in range of 1.95±0.10% (T4) to 2.08±0.25% (T6). The cookies having varied levels of
supercritical extracts, presented an increase in fiber content i.e. T9 (2.33±0.36%), T8 (2.02±0.39%)
and T7 (1.93±0.02%). These results have depicted that Stevia can be a good source of fiber in
cookies. A hike in crude fiber content of Stevia cookies was observed with gradual increment of
Stevia. Due to busy life style and irregular meal management, health beneficial verdicts of fiber
enriched diets are in demand and getting popular now a days (Eastwood and Kritchevsky, 2005).
The fiber content of cookies was found in range of 0.44-0.70 and 0.70-0.90 in a study involving
supplementation of defatted soy flour and stevia leaves powder in cookies respectively (Kulthe et
al., 2014). In a study performed in Spain, biscuits formulation was improved by using coffee silver
skin and stevia powder. The results established that fiber content was not significantly increased
by the addition of coffee silver skin and Stevia (Garcia-Serna et al., 2014). The results of current
study were similar to the findings of Abdel-Salam et al. (2009) who observed that when low caloric
sweetened yoghurt cakes were prepared by 50% addition of hot water extract of Stevia, 4% fiber
content was recorded.
4.8.1.6 Nitrogen free extract (NFE)
Mean squares (Table 24) for NFE in cookies prepared with stevia leaves powder and extracts stated
that the difference in NFE was significant. Mean values have been given in Table 25 revealed that
NFE content in cookies with different levels of Stevia leaves powder ranged from 65.19±1.26 to
70.61±0.91%. The highest value observed in T1 (70.61±0.91%) followed by T2 (65.19±1.26%) and
T1 (70.61±0.91%) respectively. Mean values for Stevia water extract NFE have illustrated
increasing trend as T4 (74.46±1.52%), T5 (74.54±1.73%) and T6 (72.92±3.84%) respectively.
However, in cookies with supercritical extracts of Stevia, decreasing trend was observed with the
increment of extract concentration expressed as T7 (75.11±3.07%), T8 (72.90±0.04%) and T9
85
(71.83±2.36%) respectively. The results from chemical composition of cookies prepared by
replacing sucrose with Stevia leaves powder have showed increment in crude fat, crude protein,
ash and crude fiber content, while nitrogen free content decreased (Abdel-Salam et al., 2009). The
moisture, protein, ash, fiber and fat content decreases by raising the level of stevia powder while
carbohydrates content increased, ranging from 61.9 to 65.7% (Kulthe et al., 2014). Biscuits
prepared with 100 and 50% of Stevia addition affect the NFE as 66.28±17.65% and 56.22±3.42%
respectively. NFE content decreased from 53.81 to 37.21% when sucrose replacement increased
from 25 to 100% (Lin et al., 2010). Therefore, the pattern of research findings from this study are
in accordance to research work of different scientist who have concluded that upsurge in nutritional
profile of Stevia cookies was observed with the increment in Stevia leaves powder.
86
Table 24. Mean squares values for chemical composition of Stevia Cookies
NS= Non-Significant
**=Highly Significant
*=Significant
Table 25 Mean values (percentage) for chemical composition of Stevia Cookies
Treatments Moisture
Content
Ash
Content
Crude
Protein Crude Fat
Crude
Fiber NFE
T0 3.52±0.07a 1.26±0.25b 10.90±1.39ab 9.91±0.62c 1.69±0.47b 74.41±1.14ab
T1 3.40±0.06ab 2.08±0.22a 12.38±0.47ab 11.53±1.23abc 2.63±0.84b 70.61±0.91abc
T2 3.34±0.05bc 2.15±0.23a 13.66±0.82ab 13.75±1.61ab 3.54±0.97ab 67.09±1.60bc
T3 3.26±0.06bcd 2.45±0.06a 15.06±0.51a 14.04±1.47a 5.65±0.72b 65.19±1.26c
T4 3.19±0.04cde 1.22±0.15b 10.81±0.83b 10.32±0.65c 1.95±0.10b 74.46±1.52ab
T5 3.15±0.03de 1.32±0.21b 10.41±1.15b 10.58±1.15c 2.00±0.64b 74.54±1.73ab
T6 3.14±0.04de 1.24±0.16b 11.55±2.08ab 11.16±2.36bc 2.08±0.25b 72.92±3.84ab
T7 3.09±0.03de 1.28±0.08b 10.26±1.33b 10.27±2.07c 1.93±0.02b 75.11±3.07a
T8 3.06±0.03e 1.24±0.03b 11.50±0.64ab 11.30±0.65bc 2.02±0.39b 72.90±0.04ab
T9 3.03±0.02e 1.22±0.08b 11.69±0.92ab 12.23±2.63abc 2.33±0.36b 71.83±2.36abc
To= Control (Sucrose cookies)
T1=10% Stevia Powder
T2=20% Stevia Powder
T3=30% Stevia Powder
T4= Cookies with 1% extractCSE
T5= Cookies with 2% extractCSE
T6= Cookies with 3% extractCSE
Source DF Moisture
Content
Crude
Protein
Crude
Fiber
Crude
Fat Ash NFE
Treatment 9 0.07710** 6.89596* 4.30478** 6.17571* 0.69422** 34.0261*
Error 20 0.00209 1.24619 0.31630 2.56438 0.02779 4.1369
Total 29
T7= Cookies with 1% extractSFE
T8= Cookies with 2% extractSFE
T9= Cookies with 3% extractSFE
CSE= Conventional solvent extraction
SFE= Supercritical fluid extraction
87
4.8.2 Antioxidant activity of Stevia cookies
Phytochemicals are non-nutritive plant constituents which are affiliated with protective action
against various ailments especially chronic diseases. In addition to nutritional benefits, an
important objective of food industries in product development is to impart potent health verdicts
to masses with these phytochemical supplementation. However, it has been observed that a limited
research has been done on sucrose replacement with zero caloric Stevia and evaluating the
replacement effect on stability of these phytochemicals. The phenol content of Stevia cookies was
found to be reduced as compared to raw stevia leaves powder. It might be due to the elevated
baking temperature and other processing parameters that affected the antioxidant capacity of stevia
in cookies especially its free radical scavenging and reduction of trivalent ions activity.
4.8.2.1 Total phenolic content (TPC) of Stevia cookies
The mean squares in (Table 26) represent a notable effect on TPC among different treatments
including Stevia powder and extracts. Mean values (Table 27) for cookies TPC have expressed
significant variations among treatments in such a way that T0, T1, T2, T3, T4, T5, T6, T7, T8 and T9
as 10.14±0.28, 9.36±0.57, 10.04±0.12, 11.88±0.58, 9.92±0.27, 10.28±0.06, 10.41±0.08,
10.16±0.12, 9.92±0.10 and 10.11±0.09 mg GAE/100g respectively. Significant effect of Stevia
value addition has been observed on TPC content of cookies.
The findings of current investigation for TPC in Stevia cookies have depicted that maximum TPC
was found in treatments with Stevia powder followed by control and extract cookies with non-
significant variation. The study conducted by Kim et al. (2011) have declared 43.99mg/g
polyphenols in methanol extract of Stevia leaves powder. While mean TPC calculated as ranging
from 28.76 to 55.05 mg/g GAE in cakes supplemented Stevia water extract (Kulthe et al., 2014).
The maximum content of 76.5 mg/100g GAE TPC in Stevia ethanol extract was determined by
Periche et al. (2015) and therefore establishing high antioxidant activity with high level of
polyphenol content. By increasing Stevia powder and extract addition level improvement in total
phenolic contents was observed as compared to control. The increment was basically due to the
availability of appreciable quantity of total phenolic content in raw stevia powder.
4.8.2.2 Total flavonoid content (TFC) of Stevia cookies
Analysis of variance for total flavonoids in Stevia cookies as expressed in Table 26 elucidated
notable difference in values as a function of Stevia leaves powder among different treatments.
88
Mean values obtained for total flavonoids (Table 27) for different treatments presented as control;
T0 (15.87±0.20 mg CE/g), Stevia powder cookies including T1 (17.23±0.15 mg CE/g), T2
(20.82±0.15 mg CE/g) and T3 (23.26±0.05 mg CE/g) showed maximum results with 30% stevia
powder replaced with sucrose. For stevia water extract cookies maximum amount of TFC was
observed in T6 (18.57±0.13 mg CE/g) followed by T5 (18.27±0.05 mg CE/g) and T4 (17.69±0.19
mg CE/g) respectively. On the other hand supercritical extract cookies have expressed in such a
way that maximum TFC were found as 18.76±0.02 mg CE/g (T9) and minimum as 18.44±0.02 mg
CE/g (T7).
Flavonoids are the naturally occurring polyphenols having anti-inflammatory, anti-carcinogenic,
antioxidant and different protective properties against oxidative stress in plants. Polyphenols and
flavonoids are used interchangeably considering in such a way that all flavonoids are polyphenols
however all polyphenols are not necessarily flavonoids. The defending effect of Stevia against
certain chronic maladies has been credited to antioxidant capability of flavonoids which have
flavones, flavonones, isoflavones, flavonols, anthocyanidins, saponins and alkaloids etc. (Kumar
et al., 2013). The TFC in Stevia extracts from ethanol have been reported to in range from 9.9 to
45.1 mg QE/g (Periche et al., 2015). The methanol extract of Stevia leaves powder found by Kim
et al. (2011) as 1.57±0.05 to 15.64±0.25 mg QE/g. Antioxidant properties of Stevia leaves powder
have been extensively studied and concluded that appreciable activity reported, was a function of
polyphenols and flavonoids (Wolwer-Rieck, 2012). The content of total flavonoids in Stevia
cookies is higher as compared to control having wheat flour with high calorie sucrose as major
sweetening agent declaring that Stevia is a good source of flavonoid compounds than wheat flour.
However in some treatment, TFC amount is close to control due to the effect of processing
variables like temperature and mechanical conditions that might affect flavonoids and ultimately
antioxidant profile of Stevia (Lemus-Mondaca et al, 2016).
4.8.2.3 DPPH assay for Free radical scavenging activity of Stevia cookies
Mean squares values for percent inhibition of free radical in DPPH assay in different Stevia
cookies is presented in Table 26. Significant variation was seen in cookies having Stevia replaced
with sucrose providing beneficial health verdicts in addition to sweetness. Mean values given in
Table 27 depicted that this ability has enhanced by increase in concentration level for powder and
extracts as well. In case of powder treatments maximum amount of percentage reduction was
recorded in T3 (13.15±0.09%) followed by T2 (13.01±0.05%), T1 (12.74±0.33%) while control
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treatment was calculated as T0 (9.59±0.74%) respectively. Means for different treatments of
supercritical extracts exhibited non-significant increase with maximum reduction observed in T9
(12.98±0.02%) followed by T8 (12.81±0.04%) and T7 (12.72±0.06%) with 3%, 2% and 1% level
of extract replacing sucrose respectively.
The DPPH values of different stevia extracts ranges including water and methanol were found in
the range of 54 to 68 μg/ml (Mandal & Madan, 2013). The percent inhibition of DPPH free radical
from different extracts of Stevia leaves and callus have been reported as 33.17% to 56.82%
respectively. Various factors including genetic makeup, environmental variables, solvents for
extraction and processing parameters affect antioxidant ability in food commodities (Alam et al.,
2013). In current study, ethanol extracts were prepared and have shown significant DPPH
scavenging activity in all treatments which is comparable to the outcomes earlier reported by
Shukla et al. (2012). They declared that antioxidant activity of Stevia have linear relation with
polyphenols as determined from analyzing methanol and ethanol extracts of Stevia using DPPH
assay and found the percent reduction ranging from 40.00–72.37%. High polyphenol content in
Stevia leaves powder and in its extracts is the main reason in up surging the free radical capturing
activity of Stevia cookies as compared to control cookies in this investigation.
4.8.2.4 FRAP assay of Stevia cookies
Ferric reducing ability of plasma (FRAP) is a novel method for antioxidant power determination.
Statistical analysis data regarding FRAP assay is presented in Table 26 showed highly significant
effect of sucrose replacement with Stevia powder in different treatments. Means for the given
parameter (Table 27) illustrated that T3 showed up with maximum reducing power (17.00±1.11
µmol Fe2+/g), T2 (15.06±1.36 µmol Fe2+/g), T1 (13.82±0.48 µmol Fe2+/g) and control as T0
(10.55±2.05 µmol Fe2+/g) respectively. Stevia supercritical and water extracts have also shown
some increment in reducing power in respective treatments namely T7, T8, T9 and values expressed
as (13.52±0.38 µmol Fe2+/g), (14.30±0.85 µmol Fe2+/g) and (14.55±0.32 µmol Fe2+/g). While for
treatment T4, T5 and T6 having 1%, 2% and 3% Stevia water extract with reducing power presented
as 11.88±2.25, 11.73±0.96 and 12.85±0.79 µmol Fe2+/g respectively. The results of current
investigation depicted reducing powder of all treatments enhanced with the increase in
concentration of Stevia powder and extracts.
The outcomes have illustrated that for Stevia cookies likewise raw Stevia powder possess the
ability to donate electrons establishing its reduction power thereby make stable compounds by
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neutralizing free radicals. Total antioxidant content (DPPH, FRAP) of stevia fresh and leaves was
studied by Periche et al. (2015) and have found as 52.92±0.84 mg Trolox equivalent/g. A study
was carried out by Tadhani & Subash (2006b) to check total phenols and antioxidant ability using
Gallic acid, Ascorbic acid, BHA and Trolox standards in methanol extracts of Stevia leaves and
callus using methanol extracts ranging from 1.5 to 4.05 µg/ml. Results of current investigation are
comparable with earlier findings in which effect of Stevia powder and extract was checked for
antidiabetic, antioxidant and renal protective perspectives concluding that free radical scavenging
activity and ferric reducing capability was found as 10.61±1.91 to 43.2±2.84 EC50μg (Shivana et
al., 2013). Thus, the high reducing power of Stevia cookies than control cookies having sucrose
as sweetener support the outcomes of current results that by increasing Stevia powder and extract
concentration reducing power increases.
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Table 26. Mean squares values for antioxidant potential of Stevia Cookies
NS= Non-Significant
**=Highly Significant
*=Significant
Table 27. Mean values for antioxidant potential of Stevia Cookies
Treatments TPC
(mg GAE/g)
TFC
(mg CE/g)
DPPH
(% reduction)
FRAP
(µmol Fe2+/g)
T0 10.14±0.28b 15.87±0.20f 9.59±0.74b 10.55±2.05b
T1 9.36±0.57b 17.23±0.15e 12.74±0.33a 13.82±0.48ab
T2 10.04±0.12b 20.82±0.15b 13.01±0.05a 15.06±1.36ab
T3 11.88±0.58a 23.26±0.05a 13.15±0.09a 17.00±1.11a
T4 9.92±0.27b 17.69±0.19e 12.27±0.32a 11.88±2.25b
T5 10.28±0.06b 18.27±0.05d 12.83±0.12a 11.73±0.96b
T6 10.41±0.08b 18.57±0.13cd 12.92±0.06a 12.85±0.79ab
T7 10.16±0.12b 18.44±0.02cd 12.72±0.06a 13.52±0.38ab
T8 9.92±0.10b 18.63±0.12cd 12.89±0.04a 14.30±0.85ab
T9 10.11±0.09b 18.76±0.02c 12.98±0.02a 14.55±0.32ab
To= Control (Sucrose cookies)
T1=10% Stevia Powder
T2=20% Stevia Powder
T3=30% Stevia Powder
T4= Cookies with 1% extractCSE
T5= Cookies with 2% extractCSE
T6= Cookies with 3% extractCSE
T7= Cookies with 1% extractSFE
T8= Cookies with 2% extractSFE
T9= Cookies with 3% extractSFE
CSE= Conventional solvent extraction
SFE= Supercritical fluid extraction
Source DF TPC TFC DPPH FRAP
Treatment 9 1.24988* 12.2621** 3.32607* 10.4812**
Error 20 0.08835 0.0156 0.07967 1.5121
Total 29
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4.8.3 Sensory analysis of Stevia cookies
Designer cookies are considered to be an elegant vehicle for nutrient dissemination like minerals,
vitamins, proteins, fiber, etc. for nutritionally poor masses (Chen, 2009).The sensory attributes of
product plays significant role in understanding the consumer preference and willingness towards
product. The sensory evaluation of Stevia cookies was carried out on 9-point hedonic scale by
sensory panelists who provide valuable feedback from consumer view point. Panelist’s response
was noted for different sensory character including flavor, texture, taste and overall acceptability.
Addition of Stevia powder and extracts by replacing sucrose in various treatments resulted in
change in sensory attributes of cookies. Sensory evaluation guide us in determining the most
suitable treatment for efficacy purpose. The analysis of variance results for sensory attributes of
Stevia cookie have been expressed in Table 28 indicating that Stevia powder and extracts have
imparted significant effect on color (p<0.05), taste (p<0.01), texture (p<0.01), crispiness (p<0.01),
flavor (p<0.01) and overall acceptability (p<0.01).
4.8.3.1 Color
Mean values for cookies color prepared by replacing sucrose with Stevia powder and extracts in
different concentration according to treatment plan depicted significant effect expressed in Table
28 an 29. Results established that maximum score for color was observed in T7 (7.10±0.40) with
1% supercritical extract having pale yellow to lightly greenish color while minimum score was
recorded in T3 (4.58±0.62) with 3% incorporation of raw stevia powder that imparted that to dark
green color which is less liked by panelists. However, T4, T5 and T6 which are Stevia water extract
cookies with 1%, 2% and 3% level addition have shown non-significant variation. Addition of
Stevia powder had imparted negative effect on color which is hampered with the increment of
Stevia leaves powder.
Color is foremost character in determining the quality product. It is an important variable for the
acceptability of bakery product. Consumer accept or reject product based on its sensory response
for color of product. During baking process, development of color occurs at the later stage of
baking indicating the process completion. Variation in cookies color depends on different
physicochemical parameters of batter including water content, pH, dietary fibers, different
pigments, minerals, etc. that are regulated by types of ingredients and processing variables i-e
temperature, mechanical force, mixing time and speed, relative humidity, air incorporation,
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heating method of baking process (Zanoni et al., 1995). In this study, color of cookies gets darker
with the increase in Stevia powder from T1 with less dark green to T3 with intense dark green color.
However in case of Stevia water and supercritical extracts treatments, cookies color slightly
greenish as compared to control having yellowish brown color. Similar findings were observed
when Stevia supplemented dietetic cakes were prepared with less fat and calories with 50%
replacement of sucrose with Stevia. Sensory evaluation showed that cakes have better acceptability
with good remarks for color parameter as compared to normal high calorie sucrose cake which
have very good remarks (Abdel-Salam et al., 2009).
4.8.3.2 Crispiness
Significant effect on crispiness of cookies was observed due to stevia powder that have complex
components that affects softness and hardness of cookies thereby impacting on quality of cookies.
The effect of various concentration on crispiness was analyzed and mean square results are
presented in Table 28 while mean values are expressed in Table 29. Current findings have
established that crispiness varies from 4.66±0.74 to 7.92±0.88 in which highest value was attained
by control (T0) cookies and lowest score was gained by cookies having 30% (T3) stevia powder
incorporation for sweetness. In stevia water extract treatments, maximum score was gained by T5
(6.37±0.75) and lowest was recorded in T4 (6.0±0.92) with 1% extract addition. In case of
supercritical extracts highest score was gained by T9=6.69±1.03 and minimum came in the fate of
T8=6.45±0.87.
4.8.3.3 Taste
The analysis of variance for taste expressed that treatments are affected by the concentration level
of Stevia powder and extracts which found to be highly significant (Table 28). It is evident from
the mean score results presented in Table 29 for taste of different treatments ranging from
5.16±0.81 to 7.17±0.77. Significantly the highest mean score for taste (7.71±0.52) was observed
for T0 followed by T1 (7.17±0.52), T7 (6.58±1.16) and the lowest value (5.16±0.81) was found in
T3. Cookies with Stevia powder addition were dark in color and intense sweet metallic taste,
therefore got the minimum score in maximum 30% powder treatment. However, water and
supercritical treatments have shown non-significant variation in taste. The taste intensity
evaluation involves the perception of substances which constitute the sample. Taste is considered
to be a major sensory attribute which determines product acceptability by consumer. In this study
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taste was appeared to be good but gives bitter or metallic after taste which is not desirable. From
the results it is inferred that sucrose replacement with minimum level of Stevia powder and extracts
provides acceptable results. The finding of the current study are in harmony with outcomes of
Kulthe et al. (2014) who prepared cookies with stevia leaves powder supplementation and
concluded that taste score from sensory was in range of 6.0-7.0 with control having 8.4 score.
However, supplementation level upto 50% in Stevia yoghurt cake have resulted in ++ (good) score
of the product (Abdel-Salam et al., 2009).
4.8.3.4 Flavor
Flavor is a peculiar but indiscernible quality trait, assessed by taste and smell combination. Flavor
of cookies comparing with control ranged from 5.00±1.16 to 8.00±0.25 with highest value by the
control cookies and least by T3 cookies having 30% sucrose replacement with Stevia powder
(Table 28 and Table 29). Results depicted significant variation among treatments with maximum
score obtained by cookies prepared from water (T6=6.70±0.73) and supercritical (T9=6.69±1.3)
extract cookies. On the other hand, Stevia powder cookies have acquired minimum scores from
sensory panelists; T1 (6.16±0.86) with maximum score while T3 (5.00±1.16) due to intense
sweetness and grassy flavor due to powder. Results concluded that extract cookies have better
flavor profile and acceptability as compared to powder with high concentration. With the increase
in Stevia replacement level in recipe of cookies, flavor freshness diminished significantly
(Villemejane et al., 2013). Similar trend was observed for flavor profile by using Stevia as
sweetener with the findings of Shah et al. (2010) who reported a decreasing trend ranging from
7.7 to 4.8 with the increase in concentration level.
4.8.3.5 Texture
Analysis of variance (Table 28) for cookies texture score depicted highly significant affect with
Stevia addition (p<0.05) and product quality is determined accordingly. It is found that score for
texture ranged from 5.15±0.66 to 7.55±0.38 (Table 29). The texture of cookies lessened
momentously with increasing the concentration level of Stevia in all treatment types. Among
powder treatments high score was attained by T1=6.38±0.73 while minimum score came in the
fate of T3=5.15±0.66 as compared to control (7.55±0.38). However in Stevia extract cookies high
score was grabbed by T9 (6.54±0.49) and T6 (6.05±0.37) got the low score.
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Baking time and temperature contribute in final product texture. Shah et al. (2010) declared that
texture (smoothness, firmness, and crunchiness) of bakery products having Stevia along with
sucrose for sweetness is significantly affected and a decreasing sensory trend for texture was
observed ranging from 7.8 (T0) to 5.0 (T4). Stevia supplementation from 15-30% significantly
affect the texture of cookies with sensory score decreasing from 7.0 to 6.3 respectively (Kulthe et
al., 2014). Therefore, it is inferred that Stevia powder can be supplemented up to 10% while 3%
level of Stevia extract can be used to get the better texture response from sensory point of view.
4.8.3.6 Overall acceptability
Mean values for overall acceptability of cookies depicted that concentration level of Stevia powder
and extracts have impacted significantly (Table 28).Mean scores ranges from 5.00±0.71 to
7.50±0.38 with highest score for control cookies and least score grabbed by 30% Stevia powder
cookies (Table 29) while T9 (6.43±0.76) having supercritical extract and water extract treatments
have maximum score for T6 (6.38±0.59). Significant variation was seen among all types of
treatments ranging from Stevia powder to supercritical as well as water extract. Everyone has their
own sensory perception towards food products and scores are totally dependent on their sensory
responses. Bakery items subjected to sensory analysis, panelists show likeness towards product on
the basis of good flavor, appealing taste, tempting smell, good crispiness and less hardness which
sums a good overall acceptability.
The sensory scores of current study concluded that there exists a decreasing trend for overall
acceptability of Stevia replaced cookies when compared with control one. The results of the
consumer acceptance studies obtained by Abdel-Salam et al. (2009) for low calories stevia yoghurt
cookies prepared for diabetic patients reported as ++ (good) from sensory panelists. A decreasing
trend was recorded with the increase in Stevia concentration ranging from T0 (7.9) and T4 (5.3)
(Shah et al., 2010). Intense sweetness was recorded by sensory panelists when subjected to Stevia
cookies prepared from stevia leaves supplementation with concentration level varying from 15 to
30%. The overall acceptability was recorded to be ranging from 7.2 for control and 5.8 for 30%
Stevia powder addition which depicts the decreasing trend towards the overall acceptability of
cookies (Kulthe et al., 2014). From these findings, it is established that 10% Stevia leaves powder,
1% water and 3% supercritical fluid extract can be replaced with sucrose in cookies preparation to
get the better sensory score and overall good quality product.
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Table 28. Mean squares values for Sensory attributes of Stevia Cookies
NS= Non-Significant
**=Highly Significant
*=Significant
Table 29. Mean values for Sensory attributes of Stevia Cookies
Treatments Color Taste Crispiness Flavor Texture Overall
acceptability
T0 8.42±0.32a 7.71±0.52a 7.92±0.88a 8.00±0.25a 7.55±0.38a 7.50±0.38a
T1 5.93±0.74cd 7.17±0.77ab 5.42±0.68bc 6.16±0.86bc 6.38±0.73ab 6.08±0.68bc
T2 5.43±0.50d 6.08±0.40bc 5.66±0.96bc 5.56±0.85bc 5.75±0.78b 5.50±0.37bc
T3 4.58±0.62e 5.16±0.81c 4.66±0.74c 5.00±1.16c 5.15±0.66b 5.00±0.71c
T4 6.38±0.38bc 6.58±0.58abc 6.0±0.92bc 6.36±0.93bc 6.22±0.46ab 6.45±0.34ab
T5 6.49±0.45bc 6.16±0.74abc 6.37±0.75abc 6.30±0.66bc 6.26±0.76ab 6.32±0.53ab
T6 6.73±0.28b 6.08±0.68bc 6.35±0.85abc 6.70±0.73ab 6.05±0.37b 6.38±0.59ab
T7 7.10±0.40b 6.58±1.16abc 6.51±0.29abc 6.52±0.58b 6.18±0.35b 6.25±0.45abc
T8 6.65±0.44b 6.08±0.77bc 6.53±0.37abc 6.45±0.87b 6.42±0.51ab 6.35±0.65ab
T9 6.86±0.72b 5.83±0.32bc 6.61±0.65ab 6.69±1.03ab 6.54±0.49ab 6.43±0.76ab
To= Control (Sucrose cookies) T1=10% Stevia Powder
T2=20% Stevia Powder
T3=30% Stevia Powder
T4= Cookies with 1% extractCSE
T5= Cookies with 2% extractCSE
T6= Cookies with 3% extractCSE
Source DF Color Taste Crispiness Flavor Texture Overall
acceptability
Treatment 9 6.24754* 3.02692** 4.43765* 3.64743** 2.23573** 2.58510**
Error 50 0.22610 0.41636 0.56038 0.33095 0.31341 0.28325
Total 59
T7= Cookies with 1% extractSFE
T8= Cookies with 2% extractSFE
T9= Cookies with 3% extractSFE
CSE= Conventional solvent extraction
SFE= Supercritical fluid extraction
97
4.8.4 Color analysis of Stevia cookies
Color tonality includes L*, a* and b* values. Lightness and darkness is represented by L*
explained in such a way that maximum value of for lightness is 100, while minimum value is 0
which represent darkness. b* is an indication of blueness and yellowness where negative value
represent blueness and positive as yellowness. Red to green color tone is expressed by a* in a way
that positive value shows red tone and green tone is represented by negative a* value. Mean
squares as well as mean values regarding color attributes of cookies having Stevia leaves powder
and extracts showed significant variation among treatments.
Mean square and mean values for L* regarding lightness and darkness of Stevia cookies are
presented in Table (30 & 31). Progressive increment in Stevia leaves powder gave lower L* values;
maximum value was recorded in control having bright color T0 (76.32±2.06) while minimum value
of 25.51±2.15 was observed in T3 (30% Stevia powder). Treatments having leaves powder with
concentration level as 10, 20 & 30% have decreasing trend in their respective L* values
43.53±2.20, 37.13±2.51 and 25.51±2.15 moving from less dark to highly dark tone cookies.
Maximum L* values calculated for Stevia water and supercritical extracts were T6 (62.01±2.32)
and T7 (62.16±4.06) and minimum were seen in T4 (55.32±2.84) and T8 (55.55±1.79) respectively.
Biscuits prepared by using different concentrations of sweeteners like sucrose, maltitol and Stevia
significantly affected the tonal quality of product. Lightness and darkness; L* was found in
decreasing trend with the increase in concentration levels of sweeteners exhibiting darkness in
biscuit’s tone ranging from 72.13±1.99 to 61.70±1.24 (Garcia–Serna et al., 2014). Low calorie
dietetic yoghurt cakes were prepared by adding Stevia extract along with sucrose and good
consumer acceptability was observed. Significant variation for L*, a* and b* color tonal quality
was concluded by Abdel-Salam et al. (2009).
Increasing the replacement levels of sucrose with Stevia leaves powder and extracts resulted in
marked decrease in a* (Table 30 & 31) ranging from 2.36±0.08 to 1.19±0.07 for Stevia water
extract (1-3%) expressing reddish green shade for cookies. Treatments with Stevia powder T1- T3
(10-30%) with varying results 2.23±0.11 (T1), 1.64±0.07 (T2) and 0.75±0.13 (T3) having slightly
to intense green tint with the increase in concentration level. Cookies having supercritical extract
(Table 4.8.8) resulted in slight increase in a* from 2.84±0.19 in T7 (1% extract), 2.65±0.12 in T8
(2% extract) and 2.29±0.14in T9 (3% extract). Physico-chemical analysis of chocolate cookies
having Stevia as sweetening agent along with sucrose shows a decreasing trend in a* (redness &
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darkness) values (14.26±0.36 to 9.56±0.05) as observed under colorimeter giving dark, slightly
reddish green color tone (Shah et al., 2010). However, Stevia along with maltitol and sucrose
imparted increasing green tint with the increase in concentration level of stevia. The results of this
study are in line with the findings of Garcia-Serna et al. (2014) who reported the maximum a*
value (4.00±1.12) in “A” treatment with 100 sucrose and minimum value (2.31±0.55) was
observed in “F” treatment with 100% Stevia incorporation.
Blue and yellow tonal quality for cookies was recorded in terms of a*. It is evident from results
(Table 30 & 31) that b* values decreased as a function of Stevia powder and extracts addition i.e.
36.74±3.09 in T7 (1% supercritical extract) to 10.02±1.46 in T3 (30% Stevia powder) where control
was recorded as T0 (38.45±1.87). In contrary, progressive increase in Stevia powder concentrations
gave momentous decrease; minimum value of 10.02±1.46 was recorded in T3, while maximum
(26.70±2.66) was recorded in T1. On other hand, maximum b* values for water and supercritical
extracts were recorded in T4 (34.88±3.18) and T7 (36.74±3.09). Whereas minimum values for
extracts were deduced as T6 (28.20±2.03) and T9 (31.18±3.02). Similar trend was observed and
published by Garcia-Serna et al. (2014) who checked the consumer acceptability and color
parameters of biscuits having different combinations of sweeteners with Stevia. They reported that
maximum b* (24.10±1.53) was recorded in “B” treatment having Maltitol and minimum
(16.89±0.8) was observed in “J” treatment composed of 100% stevia and coffee silver skin
combination. In another study, means depicting the effect of treatments on b* exhibited that
maximum value (15.85±0.28) was recorded in control while minimum value (8.79±0.06) was
observed in T4 (0.5% Stevia powder) (Shah et al., 2010).
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Table 30. Mean square values for Color analysis of Stevia Cookies
Source DF L* a* b*
Treatment 9 291.007** 0.83764** 144.561**
Error 20 7.538 0.01557 6.936
Total 29
NS= Non-Significant
**=Highly Significant
*=Significant
Table 31. Mean values for Color analysis of Stevia Cookies
Treatments L* a* b*
T0 76.32±2.06a 2.22±0.12a 38.45±1.87ab
T1 43.53±2.20b 2.23±0.11a 26.70±2.66bc
T2 37.13±2.51b 1.64±0.07bc 18.90±3.38c
T3 25.51±2.15b 0.75±0.13a 10.02±1.46c
T4 55.32±2.84a 2.36±0.08d 34.88±3.18ab
T5 56.46±2.88a 2.51±0.17a 30.74±2.57ab
T6 62.01±2.32a 1.19±0.07a 28.20±2.03ab
T7 62.16±4.06a 2.84±0.19cd 36.74±3.09ab
T8 55.55±1.79a 2.65±0.12b 33.75±2.34ab
T9 58.58±3.72a 2.29±0.14a 31.18±3.02a
To= Control (Sucrose cookies)
T1=10% Stevia Powder
T2=20% Stevia Powder
T3=30% Stevia Powder
T4= Cookies with 1% extractCSE
T5= Cookies with 2% extractCSE
T6= Cookies with 3% extractCSE
T7= Cookies with 1% extractSFE
T8= Cookies with 2% extractSFE
T9= Cookies with 3% extractSFE
CSE= Conventional solvent extraction
SFE= Supercritical fluid extraction
100
4.8.5 Texture analysis of Stevia cookies
Food texture measures the characteristics related to mastication in mouth. Texture of food items
can be evaluated by sensory analysis and it can be measured by using a texture analyzer. Hardness
is the peak force during the first compression cycle (first bite). It is defined as the maximum
penetration force and the force curve area is used to calculate the required penetration work. Food
products especially bakery items having definite characteristic shape and texture as desired by
consumers. Consumer acceptability is considered to be reduced significantly if any variation from
the optimal texture quality of product is observed. Mean squares for Stevia cookies hardness (Table
32) indicated highly significant variation within treatments. Mean values for texture analysis are
depicted in Table 33 which showed that for control and powder cookies i-e T0, T1, T2 and T3 were
recorded as 15.60±0.09, 18.50±0.16, 22.53±0.07 and 26.50±0.16. For Stevia water extract cookies;
T4, T5 and T6 were found as 17.90±0.18, 18.50±0.08 and 20.57±0.19 respectively. Maximum force
in value of texture of Stevia supercritical extract cookies were seen as T7 (16.57±0.22), T8
(18.60±0.13) and T9 (20.80±0.05).
Significant decrease in hardness of cookies was observed from 30.4 to 25.3 N with the progressive
increase in stevia leaves powder. Decrease in hardness reported, was due to the action of sugar and
Stevia combination which led to reduction in shortness and softness (Kulthe et al., 2014). In
another study, biscuits prepared in such a way that sugar replaced with maltitol, stevia and coffee
silverskin (Garcia-Serna et al., 2014). Results concluded that maltitol addition had not significantly
impacted on hardness. Treatment having 15% (102.26±37.39) and 60% (112.81±18.46) of stevia
addition along with sucrose have less hardness and good tenderness as compared to treatments
with high concentration of stevia, H (139.25±11.94) with 100% Stevia powder addition. Sucrose
causes the formation of a weak gluten network and disperses proteins and starch, which makes the
cookies fragile and broke them with minimum force (Rodriguez-Garcia et al., 2012). Sucrose
causes the formation of a weak gluten network and disperses proteins and starch, which makes the
biscuit fragile.
4.8.6 Calorific analysis of Stevia cookies
Calorific values of customized Stevia cookies were determined by Bomb calorimetry which
measures the combustion heat of food and give values for gross energy. Mean square values for
gross calorific value of control and optimized stevia cookies showed highly significant variation
presented in Table 32. Mean values (Table 33) depicted that calorific values in To, T1, T2, T3, T4,
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T5, T6, T7, T8 and T9 were 4106.4±21.50, 3888.8±25.41, 3790.6±63.1, 3660.6±121.05,
4035.2±45.16, 4009.2±67.91, 3989.5±66.25, 4031.9±80.39, 3915.0±72.63 and 3894.7±117.65
kcal/100g, respectively. Decreasing trend in calorific values of stevia cookies in this study was
similar to the trend observed by the findings of Kulthe et al. (2014) who checked the effect of
Stevia leaves powder addition on low calorie protein rich cookies and reported as ranging from
505.7 to 454.2 kcal/100g. Chocolate cookies made by using stevia along with sucrose as
sweetening agent and inulin with polydextrose as bulking agent when subjected to determination
of energy value, decrease in energy content was seen due to addition of stevia with maximum
value for T0 (2418kJ/100g) while minimum calorific content was recorded for T4 (1788kJ/100g)
as reported by Shah et al. (2010). In another study, Abdel-Salam et al. (2009) have compared
regular yoghurt cakes with Stevia rich yoghurt cake for physico-chemical and quality aspects.
They declared that diabetic yoghurt cakes had low calories and food energy (162 and 677 kJ/100g)
as compared to regular yoghurt cakes (252 and 1053, respectively).
4.8.7 Spread factor of Stevia cookies
Mean square values for thickness, diameter and spread ratio indicated that there is significant
difference (p<0.05) between control sample and customized treatments from T1 to T9 (Table 32).
As concentration level of Stevia powder and extracts increases, mean values for thickness,
diameter and subsequently spread ratio of cookies decreased (Table 33). Amount of protein
influences the viscosity of batter/dough due to the fact that protein expansion for gluten
development was resumed in cookies development. There exist indirect relation between amount
of protein and cookies diameter (Leon et al., 1996). On baking of cookies, protein gluten formation
from flour create a web like network. As cookies dough is heated, the gluten network goes through
different phases specially glass transition phase attain mobility which interact and create web
network. The formation of continuous web increases viscosity and stops the flow of cookie dough
(Miller et al., 1997). Lowest thickness was observed in T5 (6.33 ±0.10cm) while maximum
thickness was calculated in T8 (8.11±0.02cm). In case of diameter, T7 (26.5±0.30cm) attained
maximum diameter, however minimum diameter was calculated in T3 (22.7±0.20cm). Spread ratio
results obtained from ratio of diameter and thickness indicated that spread ratio decreases with the
increase in replacement level of Stevia with sucrose. Spread ratio decreased from 3.93±0.09 (T7)
to 2.86±0.09 (T3) which is presumed to be due to increase in dietary fiber and protein content as
the concentration level of Stevia is increased. Protein and fibers possess more water binding
102
capacity and if higher the amount of water present in dough, more sugar is expected to be dissolved
upon mixing. Therefore it lowers the dough viscosity and cookies spread factor when subjected to
baking. However, stevia components like protein and dietary fiber which have the tendency to
absorb water will ultimately impact the water availability and dissolution of sugar (Miller et al.,
1997).
103
Table 32. Mean squares values for Hardness, Spread ratio & Calorific value of Stevia
Cookies
Source DF Hardness Calorific value Thickness Diameter Spread ratio
Treatment 9 29.8265* 52500.9** 1.08832* 4.94889** 0.43694**
Error 20 0.1620 5636.2 0.01646 0.36733 0.00761
Total 29
NS= Non-Significant
**=Highly Significant
*=Significant
Table 33. Mean values for Hardness, Spread ratio & Calorific values of Stevia Cookies
Treatments Hardness (g) Calorific value
(kcal/100g)
Thickness
(cm)
Diameter
(cm) Spread ratio
T0 15.60±0.09f 4106.4±21.50a 7.13±0.04c 23.06±0.65b 3.24±0.08c
T1 18.50±0.16de 3888.8±25.41abc 7.70±0.10b 25.56±1.67a 3.32±0.18bc
T2 22.53±0.07b 3790.6±63.10bc 7.76±0.15ab 23.56±0.20b 3.04±0.03cd
T3 26.50±0.16a 3660.6±121.05c 7.91±0.03ab 22.70±0.2b 2.86±0.01d
T4 17.90±0.18e 4035.2±45.16ab 7.05±0.03cd 25.46±0.25a 3.61±0.02ab
T5 18.50±0.08de 4009.2±67.91ab 6.33±0.20e 23.60±0.10b 3.72±0.11a
T6 20.57±0.19cd 3989.5±66.25ab 7.60±0.20b 23.23±0.15b 3.06±0.10cd
T7 16.57±0.22de 4031.9±80.39ab 6.73±0.21d 26.50±0.30a 3.93±0.09a
T8 18.60±0.13cd 3915.0±72.63abc 8.11±0.02a 23.53±0.35b 2.90±0.04d
T9 20.80±0.05c 3894.7±117.65abc 8.09±0.04a 23.43±0.21b 2.89±0.01d
To= Control (Sucrose cookies)
T1=10% Stevia Powder
T2=20% Stevia Powder
T3=30% Stevia Powder
T4= Cookies with 1% extractCSE
T5= Cookies with 2% extractCSE
T6= Cookies with 3% extractCSE
T7= Cookies with 1% extractSFE
T8= Cookies with 2% extractSFE
T9= Cookies with 3% extractSFE
CSE= Conventional solvent extraction SFE= Supercritical fluid extraction
104
4.9 Efficacy study
Efficacy study was done to analyze the health beneficial verdicts of Stevia powder and extracts
against metabolic disorders employing experimental rodents. Sprague Dawley rats (Rodents) were
given supervised control and customized diets in convenient and controlled environment. In this
research, efficacy trial comprised of: Study I (normal rats), Study II (hyperglycemic rats) and
Study III (hypercholesterolemic rats) accompanied with intake of sucrose replaced diets (T0:
normal sucrose cookies, T1: Stevia powder cookies, T2: Stevia water extract cookies, T3: Stevia
supercritical extract cookies). Some rats were slaughtered to get the baseline values at the initiation
of trial, however at the end of study (56th) all rats were sacrificed to collect the blood samples for
further analysis. Fluctuations in body weights was recorded on weekly basis while feed and water
intakes were calculated on daily basis during whole study.
4.9.1 Feed intakes
Mean squares presented in Table 34 depicted that the treatments (Diet) and time intervals have
significantly impacted all studies regarding feed intake. Mean values of Study I (normal rats) was
expressed in Fig. 16 illustrated that maximum feed intake (37.67±1.04 g/rat/day) in T0 (Normal
sucrose cookies) followed by T3 (SFE cookies), T2 (Water extract cookies) and T1 (Stevia powder
cookies) as 36.25±1.34, 36.05±1.03 and 34.91±0.57 g/rat/day. The feed intake of rats was found
to be low in the start of study (28.19±1.07 g/rat/day) and it gradually increased till higher at the
completion of the study (37.67±1.04 g/rat/day) correspondingly. The feed intake increased
gradually with the passage of time and at 1st week it was recorded as 31.03±1.86, 29.83±1.22,
29.90±1.90 and 28.19±1.07 g/rat/day in T0, T1, T2 and T3 groups that afterwards marked up to
37.67±1.04, 34.91±0.57, 36.05±1.03 and 36.24±1.34 g/rat/day, respectively at the 8th week.
Similarly, in Study II maximum feed intake was calculated as; T0 showed 34.19±1.02 g/rat/day
feed intake, however for T1, T2 and T3 exhibited 33.51±1.16, 34.63±1.71 and 36.25±2.14 g/rat/day
for corresponding investigations. Time played as incremental factor on feed intake in such a way
that at start of study it was 28.49±0.50, 29.55±1.21, 30.0±1.27 and 30.05±2.06 g/rat/day (Fig. 17)
in T0, T1, T2, and T3 respectively. Increasing trend was observed up to 4th week as T0 (34.19±1.02),
T1 (33.51±1.16), T2 (34.63±1.71) and T3 (36.25±2.14) and afterwards trailed in feed intake was
recorded till the termination of studies (8th week) 30.83±1.24, 29.66±1.31, 33.25±1.02 and
32.25±2.39 g/rat/day was observed.
105
In case of Study III (hypercholesterolemic rats), T1 exhibited maximum (35.42±1.99 g/rat/day)
feed consumption succeeded by T0 (35.22±1.86 g/rat/day), T2 (35.07±0.78 g/rat/day) and T3
(34.76±1.03 g/rat/day). During the time span, it was enhanced from 28.13±0.80 to 33.15±2.56
g/rat/day at commencement to termination, respectively. Similarly, in groups rely on T0, T1, T2 and
T3 (Fig. 18) elevation in feed consumption was observed from 29.45±1.66 to 33.15±2.56,
30.07±1.69 to 31.96±1.98, 28.13±0.80 to 32.50±1.79 and 29.24±1.43 to 32.42±2.41 g/rat/day at
1st and 8th week, respectively.
Previously similar trend was recorded by Shivanna et al. (2013) who observed variation in food
intake in Streptozotocin treated different diabetic rats groups administrated with Stevia powder in
order to check the antioxidant, anti-diabetic and renal protective perspectives. They concluded that
feed intake in control group was 9.4±3.7g/day, Stevia group; 10.2±3.0g/day, 13±1.9g/day in
Streptozotocin rats, followed by decreasing trend in Stevia powder, Stevia polyphenol and Stevia
fiber diet groups as 11±3.3g/day, 9.8±2.5g/day and 9.1±2.6g/day respectively. According to the
findings of Awney et al. (2011) during the first 6 weeks of study no substantial differences were
recorded in feed intake for all groups but significant decreases in feed intakes were observed in
groups having high stevia dosage as compared to control group during the next 6 weeks. They
reported that highest decrease in feed intake was found to be 15% in groups with high stevia
dosage.
In another study, administration of Stevia powder at varied concentration levels significantly
decreased the feed intake in diabetic and cholestrolemic female rats as compared to control rats.
Inverse relation was observed between feed intake and Stevia dosage level. Rats fed with 25mg/kg
b. wt. consumed highest amount of feed which was 13.85g/day, while 12.86g/day was observed in
rats fed with 250mg/kg b and rats with highest Stevia dosage level 1000mg/kg b. wt had minimum
amount of feed intake as 7.86g/day (Elanga et al., 2016). Similarly Nikiforov & Eapen (2008)
anlyzed the toxicity affects of Rebaudoside A in sprage dawley rats for a duration of 3 months.
They found that feed intake rate gradually reduced at the end of study in various groups
administerated with different levles of Rebaudioside A as Control (34.7±2.92 to 10.7±2.73g/day),
500mg/kg/day (34.5±3.03 to 10.5±4.42g/day), 1000mg/kg/day (35.1±2.73 to 9.4±2.79g/day) and
2000mg/kg/day (35.2±2.77 to 7.3±3.0g/day).
106
Table 34. Effect of diets and time intervals on feed, water intake & body weight of rats in
different studies
** = Highly significant
* = Significant
NS = Non significant
Studies SOV Df Feed intake Water intake Body Weight
Study 1
Intervals (A) 8 97.8719** 56.297** 11.057NS
Diet (B) 3 36.6361** 100.076** 336.605NS
A x B 24 1.4611NS 3.622 2.251NS
Error 144 1.8931 0.663 294.108
Total 179
Study II
Intervals (A) 8 42.2432** 55.4995** 99.28NS
Diet (B) 3 78.0729** 37.6634** 1782.23*
A x B 24 5.2430NS 5.4198 8.10NS
Error 144 3.2131 0.2026 315.50
Total 179
Study III
Intervals (A) 8 57.6785** 85.3722** 165.31NS
Diet (B) 3 4.4658* 24.6608** 2861.11*
A x B 24 2.3142NS 0.2150NS 4.38NS
Error 144 2.9308 0.8140 155.31
Total 179
107
Figure 16: Feed intakes (g) of normal rats (study I)
Figure 17: Feed intakes (g) of Hyperglycemic rats (study II)
Figure 18: Feed intakes (g) of hypercholesterolemic rats (study III)
15.00
20.00
25.00
30.00
35.00
40.00
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
Feed
inta
ke (
g)
Feed Intakes of Normal Rats
T0 T1 T2 T3
15.00
20.00
25.00
30.00
35.00
40.00
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
Feed
inta
ke (
g)
Feed Intakes of Hypercholesterolemic Rats
T0 T1 T2 T3
15.00
20.00
25.00
30.00
35.00
40.00
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
Feed
inta
ke (
g)
Feed Intakes of Hyperglycemic Rats
T0 T1 T2 T3
108
4.9.2 Water intake
Mean squares for water intake (Table 34) presented momentous impact of treatments (diets) as
well as time intervals (weeks) during whole study. Means for water intake in Study I are
graphically expressed in Fig. 19 which shows increasing trend with time. In study I, water intake
at the beginning was 18.32±0.36, 20.41±0.20, 19.01±0.68 and 18.70±0.42 mL/rat/day,
respectively for T0, T1, T2 and T3 which subsequently increases up to 26.65±0.02, 25.16±0.47,
25.92±0.92 and 25.9±0.67 mL/rat/day respectively. However in study II, graphically expressed in
Fig. 20, mean results for water intake obtained from all treatments in hyperglycemic study were
T0, T1, T2 and T3 were 20.30±0.19, 23.48±0.10, 20.20±0.51 and 19.75±0.88 mL/rat/day,
respectively that lifted up to 23.29±0.07, 26.30±0.35, 27.33±0.37 and 26.78±0.77 mL/rat/day,
respectively during the entire trial. The mean water intake was 24.60±2.17, 25.44±2.13,
23.87±2.02 and 24.61±2.20 mL/rat/day, with T0, T1, T2 and T3 diets respectively. In the same way,
amount of water consumed by all diet groups was 20.77±0.85 mL/rat/day at start of study trial
which increased up to 27.13±1.03 mL/rat/day at termination of experiment. Rats which were given
diets with high cholesterol (study III, Fig 21) depicted that highest water intake was seen in T1
(27.96±0.57 mL/rat/day) at 8th week trailed by T3 (27.63±1.21 mL/rat/day), T2 (27.47±1.21
mL/rat/day) and T2 (25.97±0.76 mL/rat/day).
An increasing trend for water intake was recorded in all three studies. High water intake can be
considered to be due to diabetes symptom which involves polyuria, polydipsia; increased thirst.
High sweetness, powder content along with high fat in cookies results in high water intake that
ultimately. The findings of current study have depicted that a similar increasing trend was observed
in a study conducted to assess the anti-diabetic effect of Stevia on Sprague Dawley rats (Shivanna
et al., 2013). They presented that control (27.9±16.1 mL/rat/day) and stevia fed (20.8±13.3
mL/rat/day) rats had less water intake as compared to diabetic rats (59.5±28.4 mL/rat/day). A
similar trend was reported by Sclafani et al. (2010) who checked the preference of rats towards
naturally zero caloric Stevia and artificial sweetener saccharin and subsequently checked the water
intake. They found that water requirement enhanced from 5 to 22 ml/day for Stevia diets and 5.2
to 22.3 ml/day for saccharin fed rat.
109
Figure 19: Water intakes (mL) of normal rats (study I)
Figure 20: Water intakes (mL) of hyperglycemic rats (study II)
Figure 21: Water intakes (mL) of hypercholesterolemic rats (study III)
15
18
21
24
27
30
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
Wat
er in
take
(m
L)
Water Intakes of Normal Rats
T0 T1 T2 T3
15
18
21
24
27
30
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9Wat
er in
take
(m
L)
Water Intakes of Hyperglycemic Rats
T0 T1 T2 T3
15.00
18.00
21.00
24.00
27.00
30.00
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
Wat
er in
take
(m
L)
Water Intakes of Hypercholesterolemic Rats
T0 T1 T2 T3
110
4.9.3 Body weights
Mean squares (Table 34) revealed that the body weight of rats in all three studies varied
significantly due to the different treatments fed during the experiment duration (8 weeks). Body
weight illustrated (Fig.22) that at the initiation (study I), weights in different rat groups T0, T1, T2
and T3 were 156.60±7.14, 154.40±18.79, 153.90±23.18 and 156.73±10.55 g/rat respectively which
afterwards increased to 160.17±5.22 g/rat (T0), 156.80±21.59 g/rat (T1), 155.84±22.72 g/rat (T2)
and 158.47±9.30 g/rat (T3) at the completion of study. Mean values indicated weight of rats
increased up to 5th week which subsequently decreased at the end of study. Similarly, in study II
(Fig. 23), maximum weight was recorded as ranging from T3 (179.60±14.15) to T1 (171.23±5.84
g/rat), during 1st week which after wards decreased at 8th week ranging from 177.35±13.59 (T3) to
169.78±6.95 g/rat (T1). While in case of Study III, gain in weight was more pronounced in all
groups from 1st to 4th week in such a way that maximum weight gain was recorded as T0, T1, T2,
and T3 were 185.33±12.67, 178.34±11.37, 175.84±11.06 and 181.23±13.23 g/rat that afterwards
lessened to 180.20±10.98, 169.68±9.56, 170.66±10.68 & 176.32±10.84 g/rat, respectively by the
end of 8th week (Fig 24). Final mean body weight results obtained at the completion of study have
established significant difference among all groups and respective treatments. In study I, the
highest weight was calculated in T0 group (160.17±5.22 g/rat) followed by T3 (158.47±9.30 g/rat),
T1 (156.80±21.59 g/rat) and T2 (155.84±22.72 g/rat). Likewise in study II, the T3 (177.35±13.59
g/rat) group found to have maximum body weight trailed by T2 (174.36±7.99 g/rat), T0
(170.96±30.50 g/rat) and T1 (169.78±6.95 g/rat). On the other hand in study III, the T0
(180.20±10.98 g/rat) group found to have maximum body weight trailed by T3 (176.32±10.84
g/rat), T2 (170.66±10.68 g/rat) and T1 (169.68±9.56 g/rat).
Findings of current research are in accordance to some previous researches, however some
variation have also observed. A study that involved the administeration of different steviosides
(SGs) like Steviol, Rebaudiosisde A and stevioside in order to check their beneficial effect on lipid
accumulation at mice liver. They also checked the weight gain or loss of mice when given
steviosides diet and concluded that no significant weight gain or loss was observed when compard
to control (63.7±5.2g), steviol (61.3±2.8g), Rebaudioside A (63.8±5.7g) and Stevioside
(62.9±3.6g). However, in another study, Stevia sweetener given at different concentrations to
Sprague dawley rats. Positive and negative control have net body weight gain of 25.12% and
111
27.88% respectively while stevia diets with concentration 25, 250, 500, 1000mg/kg b. wt. have -
40.29, -41.38, 44.98 and -48.29% decrease in body weight respectively.
Greg Arnold (2010) have found that there was no significant difference in weight gain or loss seen
between diabetic and hypercholestrolemic mice which are fed with stevia; 21% lower in cholestrol,
18% low blood sugar level, and 35% lower insulin level was recorded. It has been reported that
increase in body weight gain may be due to nutritional components available that stimulate appetite
and feed intake enhanced that ultimately increase body weight (Broca et al., 2000), therefore better
nutrient utilization achieved. Stevia powder having sufficient amount of fiber and protein when
added in diets of hyperglycemic and hypercholesterolemic rats resulted in non-significant change
in diet intake in different groups, however a very small increment in weight gain was observed in
diabetic rats that can be attributed to its antidiabetic role (Holvoet et al., 2015). The findings of
current research established that significantly small change in mean body weight was observed as
compared to control due to very small amount of Stevia in diets. The recorded small increment in
weight is not detrimental due to very small difference from initial weight of rats at the initiation of
study. Due to the low calories Stevia diet administration, reduction in body weight in some groups
can also be due to unavailability of equivalent number of calories that rats used to consume
normally.
112
Figure 22: Water intake (mL) of normal rats (Study I)
Figure 23: Water intake (mL) of hyperglycemic rats (Study II)
Figure 24: Water intake (mL) of hypercholesterolemic rats (Study III)
150.00
155.00
160.00
165.00
170.00
175.00
180.00
185.00
190.00
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
Wat
er in
take
(m
L)
Body Weight of Hypercholesterolemic Rats
T0 T1 T2 T3
145.00147.00149.00151.00153.00155.00157.00159.00161.00163.00165.00
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
Wat
er in
take
(m
L)
Body Weight of Normal Rats
T0 T1 T2 T3
160.00
165.00
170.00
175.00
180.00
185.00
190.00
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
Wat
er
inta
ke (
mL)
Body Weight of Hyperglycemic Rats
T0 T1 T2 T3
113
4.9.4 Serum profile analysis
4.9.4.1 Glucose
The statistical analysis obtained have been presented in Table 35 which depicted that study II and
III have showed that glucose level was significantly affected with the Stevia diets in different rat
groups. However non-significant difference was observed in glucose level for Study I. Mean
values for glucose in study I had been recorded in all groups i-e T0, T1, T2 and T3 were 83.58±2.02,
78.79±7.92, 81.89±3.40 and 82.78±4.27 mg/dL respectively. In study II which have exhibited
significant variation in glucose level among all groups of this study where T0 (154.40±3.45mg/dL)
have maximum glucose level, that decreased prominently in T1 (142.29±1.44mg/dL), T2
(150.10±2.60mg/dL) and T3 (148.46±2.15mg/dL) respectively. Study III in which rats are fed with
cholesterol rich diets showed maximum glucose level 145.20±4.15mg/dL (T1) which then
significantly lessened as 140.20±2.34mg/dL (T2), followed by 138.40±2.96mg/dL (T3) and
131.80±2.38mg/dL (T2) respectively.
The results are illustrated graphically in Fig 25 which established that T1 attained the maximum
percent reduction in Study I as 2.77% whereas T2 & T3 caused showed 1.59% and 1.36 reduction
glucose level in normal rats fed on different diets. Highest percent reduction in Study II
(hyperglycemic study) was observed in T1 (-7.00%) followed by T2 (-4.90%) and T3 (-4.54). Study
III (hypercholesterolemic study) also behaved in similar reduction pattern with minimum reduction
was seen in T3 (supercritical extract cookies) as -2.05% and the highest decline was calculated T1
(-3.75%). The graphically expression and statistical results showed that cookies with stevia powder
executed in better way against glucose related anomalies as compared to diets with extracts.
Findings of Awney et al. (2011) supported current research outcomes and establishing that glucose
level was decreased in groups fed with Stevia leaves powder and extracts. They reported that
groups having high stevia dosage in their diets have significantly lowered level of glucose as
87.88±6.76 mg/dl as compared to control (150.12±4.30). However, non-significant difference was
seen among Stevia low dosage and Stevia powder with inulin fed groups with 156.04±6.78 and
156.32±6.48 mg/dl respectively. It was established that administration of Stevia powder and
extracts to diabetic as well as non-diabetic rats significantly reduces the glucose level. Stevia
absorbed in cells is readily converted into Steviol which is then excreted out of the body without
adding any calories. Another reason is the low absorption of steviosides and enhanced
114
gastrointestinal motility seen with Stevia addition. Antidiabetic activity is also supported by
retarding the carbohydrate assimilation thus improving peripheral insulin action. According to
Sharma et al. (2012), normal range of blood glucose level was recorded in normal and diabetic
rats groups fed with Stevia extracts whereas increment in glucose level was observed in diabetic
control group fed on normal diets having no Stevia. Glucose tolerance test have also established
that pancreatic ß-cells normal functioning was improved resulting in better metabolism and
glucose delivery to cells. The findings of Geuns et al. (2007) have established that SGs/Steviosides
are capable to control blood glucose levels by improving not only insulin production but its
utilization as well in insulin deficient rats.
Diabetes is aggravated due to mutual effect of oxidative stress and hyperglycemia. As Stevia
possess good antioxidant and anti-hyperglycemic activity due to high availabilities of polyphenols
including phenolics and flavonoid contents. Stevia polyphenols along with some other
biomolecules presence may trigger beta cells to release insulin and regulate enzymes production
thereby maintain normal blood glucose level (Singh and Garg, 2014). Another important regulator
in influencing glucose hemostasis is PPARγ which is a member of the ligand-activated nuclear
receptor superfamily. It is responsible for regulation of various body functions specially glucose
metabolism, inflammation and adipogenesis. Therefore, if glucose utilization is improved and
enhanced glycolytic action could be an alternative mechanism in lessening glucose level (Kim and
Ahn, 2004; Chenc et al., 2006).
4.9.4.2 Insulin
The statistical analysis results presented in Table 36 expressed that Study II and Study III were
significantly affected by diets while non-significant difference was seen in Study I rats. Means
values for insulin production in study I were 7.11±0.34, 7.19±0.16, 7.17±0.35 and 7.09±0.26
µU/mL in T0, T1, T2 and T3 groups, respectively. Maximum value for insulin production in study
II was observed in T1 (13.42±1.81µU/mL) that significantly trailed to 12.72±1.96, 10.86±1.04 and
8.76±1.94 µU/mL in T3, T2 and T0 groups, respectively.
However in study III, T0 provided lowest insulin level (10.53±3.60µU/mL) that afterwards
enhanced in T1, T2 and T3 groups as 14.21±1.99, 11.77±2.65µU/mL and 12.56±2.76µU/mL
respectively. The results are graphically depicted in Fig. 26 and it is quite obvious that non-
significant increase was observed in Study I with percent increase as expressed T1 (1.22%), T2
115
(1.07%) and T3 (0.93%). While 6.13, 4.91 and 4.27% significant increase due to diets was recorded
in treatment groups T1, T2 and T3 for study II. Similar increment level for insulin production was
observed in Study III with highest values recorded 2.88 (T1) followed by 2.02 (T2) and 1.64 (T3).
The effect of SGs aglycons especially Stevioside on insulin production and release in
mouse islets Langerhans was explained by Jeppesen et al. (2003) by using cell line INS-1. They
observed that insulin secretion was enhanced by stevioside and steviol administration in diet.
Prevailing concentration of glucose in blood stream induce the insulin secretion and regulated by
Stevioside and SGs level in diet. They concluded that SGs significantly potentiated insulin
production and secretion by directly affecting on INS-13cells and may have a potent role as
antihyperglycemic agents in the treatment of type 2 diabetes mellitus. It has been observed in
efficacy studies including rats, upsurge in postprandial glycemic index is due to calorie dense
natural sugar administration which causes certain metabolic complications including
hyperglycemia, hypertension, hyperinsulinemia and insulin resistance (Barros et al., 2007). Anton
et al. (2010) have presented the outcomes of their research report by declaring that Stevia
supplemented diets substantially lessened postprandial glucose and increases the insulin levels as
compared to sucrose and aspartame preloads. They carried out the very first study in which effect
of natural sweetener like fructose, glucose, stevia and artificial sweetener aspartame on satiety,
food intake and postprandial glucose and insulin levels in humans was directly checked.
Induction of type I diabetes in rats is mostly carried out by a compound known as Streptozotocin.
It causes very rapid depletion of β-cells thereby inducing diabetes, which ultimately reduces
insulin release. Cells that get resistant to insulin is the most important factor in the inception and
progression of type II diabetes. Reactive oxygen species are generated due to oxidative stress
caused by hyperglycemia and insufficient release of insulin (Kangralkar et al., 2010; Zhang et al.,
2009). The findings of Shivanna et al. (2013) have elaborated that rats groups fed with stevia
powder and stevia polyphenols significantly increased serum insulin level which established that
stevia could augment the β-cell number of pancreatic islets in diabetic rats which enhance the
secretion of insulin from islets of Langerhans. Stevia leaves powder and polyphenol extracts
significantly improve the insulin production level in diabetic rats and play an important in clearing
the plasma glucose level. Therefore, they conclude that stevia has the capacity to revamp glucose
tolerance and enhance insulin sensitivity.
116
Table 35. Effect of Stevia diets on glucose (mg/dL)
* = Significant
**= Highly significant
NS= Non-Significant
Figure 25: Percent (%) reduction in glucose as compared to control
Diets
Studies T0 T1 T2 T3 F value
Study I 83.58±2.02 78.79±7.92 81.89±3.40 82.78±4.27 0.87NS
Study II 154.40±3.45 142.29±1.44 150.10±2.60 148.46±2.15 19.8**
Study III 145.20±4.15 131.80±2.38 140.20±2.34 138.40±2.96 16.4*
-2.77
-7.00
-3.75
-1.59
-4.90
-2.46
-1.36
-4.54
-2.05
-8.00
-7.00
-6.00
-5.00
-4.00
-3.00
-2.00
-1.00
0.00
Normal Hyperglycemic Hypercholestrolemic
Pe
rce
nt
(%)
de
cre
ase
Glucose reduction
T1 T2 T3
117
Table 36. Effect of Stevia diets on Insulin (µU/mL)
* = Significant
**= Highly significant
NS= Non-Significant
Figure 26: Percent (%) increase in Insulin as compared to control
Diets
Studies T0 T1 T2 T3 F value
Study I 7.11±0.34 7.19±0.16 7.17±0.35 7.09±0.26 0.55NS
Study II 8.76±1.94 13.42±1.81 10.86±1.04 12.72±1.96 7.25**
Study III 10.53±3.60 14.21±1.99 11.77±2.65 12.56±2.76 4.01*
1.22
6.13
2.88
1.07
4.91
2.02
0.93
4.27
1.64
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Normal Hyperglycemic Hypercholestrolemic
Pe
rce
nt
(%)
incr
eas
e
Axis Title
Percent increase in insulin level by Stevia diets
T1 T2 T3
118
4.9.4.3 Effect of Stevia on Cholesterol (mg/dL)
The statistical analysis (Table 37) established that diets non-significantly impacted on cholesterol,
regulation in Study I groups. However significant variation in cholesterol production was recorded
in Study II and Study III. Maximum cholesterol in Study I was found as 78.24±5.06 mg/dL (T0)
which is trailed to 77.76±5.00mg/dL (T2), 76.91±4.03mg/dL (T3) and 75.01±7.30 mg/dL (T1) in
respective groups. Mean cholesterol values found for Study II in such a way that T0
(108.16±6.83mg/dL) was maximum that momentously lessened to 99.53±8.06, 101.77±8.89 and
104.80±7.35mg/dL in T1, T3 and T2, respectively. Similarly in study III, high cholesterol value
110.93±4.32mg/dL was observed in T0 followed by T2 103.59±8.45mg/dL, T3 101.60±6.78mg/dL
and T1 97.61±9.08 mg/dL.
Results graphically exhibited in Fig 27 have sown that T1 (diet having Stevia powder) induced
highest cholesterol reduction followed by T2 and T3 (diets containing Stevia water extract and
supercritical extracts). In study I, comparison with control have deduced that T1, T2, and T3
treatments presented 2.38, 1.62 and 1.18% drop in cholesterol respectively. In study II, maximum
downgrade trend was seen as T1 (3.81%) trailed by T2 (2.81%) and T3 (2.12%). Similarly, in study
III maximum reduction (5.47%) was perceived in T1 followed by T2 (3.09%) and T3 (1.28%) as
compared to control.
The current findings are in agreement with the results of Elnaga et al. (2016) who reported that
different dose levels of Stevia such as 25, 250, 500 and 1000mg/kg b. wt/day when administrated
to different rat groups for a duration of 12 weeks, have significantly reduces the total cholesterol
level was significantly reduced with the increase in Stevia concentration. Minimum level of
cholesterol 1.69mg/dl was observed in group being fed with maximum Stevia concentration,
followed by 175.38, 180.25, 185.88, 206.63 and 203mmg/dl in respective groups of 500, 250, 25
mg/kg b. wt/day, positive and negative controls respectively. Therefore it was deduced that Stevia
powder could be used as cholesterol lowering agent. The findings of Awney et al. (2010) focusing
on serum total cholesterol found that with the increase in Stevia concentration, significant increase
in total cholesterol, low density lipoprotein have been recorded. Group with low dose of Stevia
have been found to exhibit low cholesterol level 60.27±4.77mg/dL as compared to control
63.39±6.19mg/dL, while groups with high stevia dose and stevia low dose with inulin have shown
112.50±4.38mg/dL and 79.46±1.55mg/dL of cholesterol respectively.
119
In a recent study conducted by Holovet et al. (2015) proved the hypolipidemic aspect of different
SGs like Stevioside, Rebaudioside A and Steviol when administrated to hyperlipidemic rats for a
duration of 12 weeks. They found that Stevioside one of the most important Steviol glycoside have
significantly reduced the cholesterol level as 10.7±2.6mmol/L followed by control group as
12.1±2.1mmol/L, while Rebaudioside A and Steviol have shown a slight increment in cholesterol
level as 13.8±3.1mmol/L and 12.3±2.7mmol/L.
Similar findings were reported by Geeraert et al. (2010) who revealed that administration of
Stevioside dissolved in saline solution at dosage level of 10mg/kg/day for 12 weeks to rats fed on
lipid-rich diet substantially reduced total cholesterol from 13.51±3.40mmol/L in control to
10.71±2.61mmol/L in Stevioside fed group. Cholesterol lowering effect of Stevia could be due to
high content of crude fiber which helps in removal of lipid content from body and avoid plaque
formation. This is the key reason that dietary fibers are recommended in diet in order to avoid
cardiovascular diseases. In another study, total lipids and cholesterol level was significantly
reduced when experiment rats were administrated with varied level of stevia in diets in such a way
that doses at 25, 250, 500 and 1000mg/kg/b. wt and reduction calculated was 11.96%, 19.98%,
25.03% and 37.07% respectively. The reduction in lipid profile might be due to lowering of blood
glucose level with different doses of Stevia powder as it possess anti hyperglycemic, blood
pressure lowering and cholesterol lowering affects (Elanga et al., 2016).
120
Table 37. Effect of Stevia diets on Cholesterol (mg/dL)
* = Significant
**= Highly significant
NS= Non Significant
Figure 27: Percent decrease in Cholesterol as compared to control
-2.38
-3.81
-5.47
-1.62
-2.81-3.09
-1.18
-2.12
-1.28
-6.00
-5.00
-4.00
-3.00
-2.00
-1.00
0.00
Normal Hyperglycemic Hypercholestrolemic
Pe
rce
nt
(%)
de
cre
ase
Cholesterol
T1 T2 T3
Diets
Studies T0 T1 T2 T3 F value
Study I 78.24±5.06 75.01±7.30 77.76±5.00 76.91±4.03 0.34NS
Study II 108.16±6.83 99.53±8.06 104.80±7.35 101.77±8.89 2.54*
Study III 110.93±4.32 97.61±9.08 103.59±8.45 101.60±6.78 5.87**
121
4.9.4.4 High density lipoprotein (HDL)
It is obvious from the statistical analysis presented in Table 38 that diets imparted significant
differences on HDL level in all three studies. Mean HDL values recorded in study I, exhibited the
values 39.85±4.17, 44.65±5.25, 41.46±7.47 and 42.89±5.95 mg/dL for T0, T1, T2 and T3 respective
groups. However in Study II, lowest HDL value 45.05±2.68mg/dL was found in T0 that
significantly elevated in T1 (54.74±3.27mg/dL), T3 (50.73±6.29mg/dL) and T2 (48.54±2.40
mg/dL). Mean HDL level for T0 group in study III was 43.91±4.23mg/dL that momentously
increased to 50.57±6.92 mg/dL in T1, 47.78±1.68 mg/dL in T3 and 46.97±3.28 mg/dL in T2
respectively. It is obvious (Fig. 28) that in study I non-significant increase in HDL was 1.73, 1.26
& 1.13% with diets T1, T2 and T3 respectively, while in studies II & III, diets T1, T2 and T3 exhibited
substantial increase in HDL as compared to control as 2.72, 2.04, 1.74% and 7.70, 4.38 & 3.63%,
correspondingly.
4.9.4.5 Low density lipoprotein (LDL)
The statistical analysis showed non substantial effect of diets on LDL in studies I and II (Table 39)
while Study II have exhibited significant diet effect. Mean values in Study I for LDL indicated
maximum value 31.45±4.30 mg/dL in T0 that reduced to 30.82±3.45, 29.48±4.23 and 27.54±4.21
mg/dL in T1, T2 and T3 groups, respectively. Whereas in study II, LDL value 49.00±3.54 mg/dL
in T0 group was lessened significantly to 42.77±5.89mg/dL (T1), 47.16±4.50 mg/dL (T2) and
46.06±4.80 mg/dL (T3). In study III, mean values for T0, T1, T2 and T3 differed momentously i.e.
56.55±4.36, 51.39±6.84, 54.35±5.68 and 53.78±4.33 mg/dL, correspondingly.
Percent reduction in LDL values in all studies are graphically expressed in Fig. 29 for different
rats groups. In study I, non-significant decrease was seen in T0 (2.46), T1 (1.71) and T3 (1.40)
groups as compared to control. Likewise, non-substantial decrease was recorded during Study II
i.e. 4.61% in T1, 2.70% in T2 and 1.91% in T3. However in study III, diet containing Stevia powder
(T1) reduced the LDL level by 8.36%. While diet having Stevia water extract (T2) and Stevia
supercritical extract resulted in 5.29% and 4.62% decrease in LDL.
4.9.4.6 Triglycerides
The statistical analysis indicated non-significant diet effect on triglycerides in study I while
significant affect was recorded in Study II and III (Table 40). Mean values for study I depicted
80.20±7.91mg/dL as maximum triglycerides value in T0 that trailed to 75.20±4.86, 78.17±3.81 and
122
77.42±7.87mg/dL in T1, T2 and T3 groups respectively. However, in study II, maximum value for
triglycerides 89.0±6.96mg/dL was observed in T0 that afterwards significantly reduced to
81.53±5.41mg/dL (T1), 86.0±6.28mg/dL (T2) and 84.4±8.50 mg/dL (T3). Similarly for study III,
mean triglycerides values for T0, T1, T2 and T3 differed momentously i.e. 97.80±5.40, 92.18±5.85,
95.86±6.30 and 94.72±7.31mg/dL, respectively.
It is evident from graphical representation in Fig. 30 that T1 (0.83%) in study I showed maximum
reduction followed by T2 (0.63%) and T3 (0.56%). In study II, T1 delivered the highest triglycerides
reduction of 2.85% while in T2 & T3 1.83% and 1.23%. Significant reduction was observed in
study III in which diet containing Stevia leaves powder depicted a high reduction of 5.36% in T1
followed by T2 (2.09%) and T3 (2.68%) having water and supercritical extracts respectively.
The findings of Elnga et al. (2016) have established that results of current study regarding total
cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL) & triglycerides are in
similar decreasing trend in such a way that cholesterol, LDL and triglyceride levels were decreased
while HDL level was improved as compared to non-treated control group due to administration of
diseased rats with Stevia leaves powder and extracts. Similar results were described by Awney et
al. (2011) who reported that LDL, cholesterol and triglycerides were reduced in groups fed with
Stevia low dose and Stevia with inulin, while an increment was observed when high dosage of
Stevia was administrated to hypercholesterolemic rats for a duration of 12 weeks when compared
non treated control rats. On the other hand, HDL level was significantly improved with the increase
in Stevia leaves powder and extracts in diets ranging from 42.72±2.06 mg/dL in control and
80.89±4.45mg/dL in Stevia rich diets. The significant effect of Stevia on lipid profile can be due
to several aspects. Previous researches have established that dietary fibers; soluble, insoluble and
inulin can alter hepatic triacylglycerol synthesis and LDL secretion is regulated. The variation in
serum cholesterol and triglyceride levels as compared to control were also recorded in an earlier
research in which rats were fed with high dosage of Rebaudioside A that ultimately altered bile
acid homeostasis (Nikiforov and Eapen, 2008). In another study, it was found that in addition to
sweetness, Steviosides helps in plaque removal and forbids the deposition of cholesterol and low
density lipoproteins in veins and arteries, thereby decreasing the chances of strokes, heart attack
and macrophage formation (Geeraert et al., 2010).
Elevated level of high density lipoprotein (HDL) are vital for normal functioning of heart and
ensuring the blood supply in body. Liver plays an important role in catabolizing body cholesterol
123
supplied by HDL from serum to liver. Therefore, high level of HDL is beneficial for lowering the
cholesterol and finally excreted from the body. Atherosclerosis, which is affected by the ratio of
HDL and LDL in body can be minimized by adding different doses of Stevia in diets. Therefore,
if the balance of HDL and LDL disturbed in such a way that HDL lowered and LDL increase will
aggravate cardiovascular diseases. Therefore the reduction in HDL, LDL, triglycerides, cholesterol
and total lipids can be attributed to crude fiber, saponins and steviosides biochemical action (Hony
et al., 2006). Crude fiber and saponins content of Stevia substantially reduce the amount of
cholesterol, triglycerides particularly non Esterified Fatty Acids (NEFA) that are the most
important components of lipid profile. A significant increment in HDL and reduction in total lipids,
LDL and triglycerides concentrations was recorded when stevia was administrated for a long time.
It is very well established that HDL and LDL worked as antagonists to each other; increase in HDL
and decrease in LDL leads to body protection against cardiovascular diseases and atherosclerosis
(Murray et al., 2003).
124
Table 38. Effect of Stevia diets on HDL (mg/dL)
* = Significant
**= Highly significant
NS= Non Significant
Figure 28: Percent increase in HDL as compared to control
Diets
Studies T0 T1 T2 T3 F value
Study I 39.85±4.17 44.65±5.25 41.46±7.47 42.89±5.95 4.92NS
Study II 45.05±2.68 54.74±3.27 48.54±2.40 50.73±6.29 10.6*
Study III 43.91±4.23 50.57±6.92 46.97±3.28 47.78±1.68 6.95*
1.73
2.72
7.70
1.26
2.04
4.38
1.131.74
3.63
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Normal Hyperglycemic Hypercholestrolemic
Pe
rce
nt
(%)
Incr
eas
e
HDL
T1 T2 T3
125
Table 39. Effect of Stevia diets on LDL (mg/dL)
* = Significant
**= Highly significant
NS= Non Significant
Figure 29: Percent decrease in LDL as compared to control
-2.46
-4.61
-8.36
-1.71
-2.70
-5.29
-1.40-1.91
-4.62
-9.00
-8.00
-7.00
-6.00
-5.00
-4.00
-3.00
-2.00
-1.00
0.00
Normal Hyperglycemic Hypercholestrolemic
Pe
rce
nt
(%)
De
cre
ase
LDL
T1 T2 T3
Diets
Studies T0 T1 T2 T3 F value
Study I 31.45±4.30 27.54±4.21 30.82±3.45 29.48±4.23 1.89NS
Study II 49.00±3.54 42.77±5.89 47.16±4.50 46.06±4.80 2.51*
Study III 56.55±4.36 51.39±6.84 54.35±5.68 53.78±4.33 3.77*
126
Table 40. Effect of Stevia diets on triglycerides (mg/dL)
* = Significant
**= Highly significant
NS= Non Significant
Figure 30: Percent decrease in triglycerides as compared to control
Diets
Studies T0 T1 T2 T3 F value
Study I 80.20±7.91 75.20±4.86 78.17±3.81 77.42±7.87 2.96NS
Study II 89.0±6.96 81.53±5.41 86.0±6.28 84.4±8.50 3.31*
Study III 97.80±5.40 92.18±5.85 95.86±6.30 94.72±7.31 3.99*
-0.83
-8.12
-2.45
-0.63
-3.36
-1.83
-0.56
-3.12
-1.23
-9.00
-8.00
-7.00
-6.00
-5.00
-4.00
-3.00
-2.00
-1.00
0.00
Normal Hypercholestrolemic Hyperglycemic
Triglycerides
T1 T2 T3
127
4.9.5 Liver functions tests
Liver function tests comprised of aspartate transaminase (AST), alanine transaminase (ALT) and
alkaline phosphatase (ALP) were carried out for safety reasons.
4.9.5.1 Aspartate aminotransferase (AST)
The statistical analysis results (Table 41) have depicted that aspartate aminotransferase (AST)
level was non-significantly affected Study I and II, while Study I was significantly affected by
diets in all groups. Mean AST values for T0, T1, T2 and T3 groups in study I were recorded as
107.42±4.71, 105.48±3.89, 106.12±2.19 and 106.56±2.48 IU/L respectively. However, in study
II, means for AST depicted that maximum value in T0 (130.84±3.31 IU/L) as compared to T1
(126.61±5.07 IU/L), T2 (127.69±4.85 IU/L) and T3 (129.23±3.81 IU/L). On the other hand, AST
values for study III were significantly varied in such a way that T0 attained maximum level
(122.99±5.63 IU/L) which lessened in T1 (116.43±3.23 IU/L), T2 (117.91±5.52 IU/L) and T3
(120.10±5.49 IU/L).
4.9.5.2 Alanine transaminase (ALT)
Statistical analysis results presented in Table 42 depicted that non-significant variation was
observed study I, however in study II and III significant difference was found. Mean ALT values
for Study I calculated as T0 (35.64±5.91 IU/L), T1 (31.61±4.05 IU/L), T2 (33.03±3.57 IU/L) and T3
(33.27±2.76 IU/L) respectively. Study II mean ALT results have depicted significant variation and
expressed in a way that maximum value was observed T0 (54.05±4.19 IU/L) that substantially
decreased in following order T1 (49.56±1.78 IU/L), T3 (53.15±4.79 IU/L) and T2 (51.28±5.10 IU/L)
groups. Similarly in study III, maximum ALT value 52.07±3.31 IU/L was found in T0 group which
afterwards significantly lowered to 47.54±3.08, 49.60±1.80 IU/L and 50.47±5.11 IU/L in T1, T2
and T3 respectively.
4.9.5.3 Alkaline phosphatase (ALP)
Statistical analysis results for alkaline phosphatase (ALP) interpreted significant variation in all
studies (Table 43). Maximum ALP mean value in study I were recorded in T0 (141.54±3.49 IU/L)
which then significantly reduced in T1 (135.17±9.87 IU/L), T2 (138.77±5.03 IU/L) and T3
(140.95±9.53 IU/L). Similarly in study II, higher ALP value recorded was 185.12±2.64 IU/L (T0),
trailed by 179.09±6.54 IU/L (T2), 177.34±7.83 IU/L (T3) and 171.11±7.5 IU/L (T1) in respective
groups. ALP recorded in Study III all groups have depicted significant variation in such a way that
128
minimum value (193.90±6.43 IU/L) was recorded in T1 while maximum value (202.19±2.54 IU/L)
was seen in T1, while T2 and T3 have exhibited as 197.05±7.39 and 199.72±7.46 IU/L,
respectively.
Hepatic disorders are evaluated by determining the serum enzymes including ALP, AST and ALT.
Liver functioning is hindered and disturbed if the activity of these enzymes is enhanced leading to
liver damage. Liver inflammation due to diseased hepatic cells leads to extremely high levels of
transaminase in blood stream that ultimately impacts on normal functioning of body. The results
of Elnaga et al., (2016) established that high level of ALT, ALP and AST were observed in
diabetics and hypercholesterolemic diseased rats that affects the normal metabolic functioning.
They found that with the increment of Stevia leaves powder in diets of diseases rats, decrease in
hepatic enzymes was observed which were disturbed due to disorders. AST decreases from 143.33
IU/L (control) to 135 IU/L (1000mg/kg b. wt. Stevia dose), while ALT & ALP non-significantly
decreased form 116.71 IU/L to 112.80 IU/L and 73.39 IU/L to 71.66 IU/L respectively. Sharma et
al. (2012) have concluded in their study that Stevia leaves powder and extracts play potent role in
controlling oxidative stress in type-2 diabetes and maintains the liver cells integrity. They also
declared that Stevia extracts reduced the lipid infiltration and necrosis around hepatocytes which
establishes the hepatoprotective perspectives of Stevia as well.
Shivanna et al. (2013) have checked the effect of diabetes induced by Streptozotocin (STZ) and
feeding with high fructose diets on liver functioning and its enzymes regulation. They found that
serum AST and ALT level were substantially enhanced by 42% and 89% as compared to control
groups. Feeding of rats with Stevia powder and extracted polyphenols to diseased rats reduced the
AST and ALT activity to 13 & 6% and 45& 38% respectively. It can be deduced from the results
that decrease in serum enzymes levels were indicated that Stevia played the role of repairing the
plasma membrane damages due to diabetes. Therefore, Stevia laves powder and its polyphenolic
extracts significantly decreased the levels of ALT and AST, thereby imparting a hepatoprotective
effect in diseased rats. Polyphenols including total flavonoids and total phenolic contents in Stevia
have cytoprotective potential. Positive affect against lipid profile have been observed in leaves
extract of Stevia. ALP activity was substantially decreased in diabetic rats from 73.39 to 71.07
IU/L and phytochemical played a significant role in regulation of the hepatic enzymes. The
addition of Stevia leaves powder and extracts in the diets of diabetic rats, it resulted in reduction
of ALT, AST and ALP activity thereby leading towards normal functioning of liver (Wolwer-
129
Rieck, 2012). ALT, ALP and AST enzymes are biochemical indicators showing hepatic
dysfunction and maladies which are typically involved in breakdown of amino acids into a-keto
acids that are routed for metaboilsm completion via Kreb’s cycle and electron transport chain
(Shakoori et al., 1994). In anther study conducted by Awney et al. (2010), non-significant
differences in AST, ALP and ALT was observed in all groups compared with control. However,
significant decreases in ALP and acid phosphataase ACP in the grops fed with high stevia powder
dose were detected.
Table 41. Effect of Stevia diets on serum AST (IU/L)
**= Highly significant
NS= Non Significant
Diets
Studies T0 T1 T2 T3 F value
Study I 107.42±4.71 105.48±3.89 106.12±2.19 106.56±2.48 1.26NS
Study II 130.84±3.31 126.61±5.07 127.69±4.85 129.23±3.81 0.86NS
Study III 122.99±5.63 116.43±3.23 117.91±5.52 120.10±5.49 1.34*
130
Table 42. Effect of Stevia diets on serum ALT (IU/L)
* = Significant
**= Highly significant
NS= Non Significant
Table 43. Effect of Stevia diets on serum ALP (IU/L)
* = Significant
**= Highly significant
NS= Non-significant
Diets
Studies T0 T1 T2 T3 F value
Study I 35.64±5.91 31.61±4.05 33.03±3.57 33.27±2.76 1.66NS
Study II 54.05±4.19 49.56±1.78 51.28±5.10 53.15±4.79 0.32*
Study III 52.07±3.31 47.54±3.08 49.60±1.80 50.47±5.11 3.33*
Diets
Studies T0 T1 T2 T3 F value
Study I 141.54±3.49 135.17±9.87 138.77±5.03 140.95±9.53 2.48*
Study II 185.12±2.64 171.11±7.5 179.09±6.54 177.34±7.83 2.26*
Study III 202.19±2.54 193.90±6.43 197.05±7.39 199.72±7.46 1.60*
131
4.9.6 Renal function tests
In order to check the effect of stevia powder and its extracts on kidney working and structural
integrity. Renal functioning parameters including urea and creatinine were assessed and in light of
results, conclusion were drawn.
4.9.6.1 Urea
Statistical analysis results expressed in Table 44 for urea determined non-significant effect in all
three studies I, II, III. Mean values for urea in study I have maximum value for T0
(23.64±2.32mg/dL) while minimum value calculated in group T1 (20.77±1.05mg/dL) followed by
T2 and T3 groups as 22.34±0.91mg/dL and 22.80±1.91 mg/dL, respectively. In similar manner, fro
study II, serum urea value ranges from 27.94±2.65mg/dL (T1) to 30.12±1.80mg/dL (T0) group,
while T2 (28.52±3.12mg/dL) and T3 as 28.16±3.52mg/dL having non-significant variation among
groups. However Study III have maximum serum urea value for T0 trailed to T3, T2 and T1 with
their values presented as 31.10±3.73mg/dL, 29.09±2.43mg/dL, 28.79±2.28mg/dL and
27.81±3.42mg/dL, correspondingly.
4.9.6.2 Creatinine
Non-significant variation (Table 45) have been found in creatinine level among all studies. In study
I, T0 depicted the highest creatinine value as 0.67±0.06mg/dL, however, T1, T2 and T3 groups
showed decreasing trend as 0.63±0.04, 0.65±0.01 and 0.63±0.02mg/dL respectively. Similar trend
for creatinine was observed for study II with T0 as 0.96±0.08mg/dL that slightly varies in T1, T2
and T3 as 0.91±0.05, 0.93±0.02 and 0.94±0.09mg/dL respectively. In the same way momentous
variation was observed in study III which was non-significant with maximum creatinine level
1.02±0.05 found in T0 followed by 0.95±0.08, 0.97±0.07 and 0.98±0.06mg/dL in T1, T2 and T3.
Normal functioning of kidney is determined by the level of creatinine in blood stream. It
is basically a byproduct from creatinine break down. In case of any malady or disease, level of
creatinine in blood stream marked up, however it is easily filtered by kidneys during healthy state
of body. Glomerular filtration of kidneys is affected in case of abnormal functioning of body or in
diseased state. In diabetes, glycation of protein increases that will surges out the release of purine,
muscle wasting and this is the main source of uric acid (Anwar and Meki, 2003). The findings of
this study are in accordance with the outcomes of Petterino & Argentino (2006) who analyzed the
effect of Stevia powder on Sprague Dawley rats for twelve weeks duration. They recorded
132
creatinine and serum urea level in normal and diseased rats. Stevia dosage non-significantly lower
the values of urea and creatinine level. At the end of 4th week, maximum and minimum values for
creatinine and urea were found as 70.7 and 26.5µmol/L. However at the end of 12th week,
minimum and maximum creatinine level was increased as compared to intial 4 weeks as 35.4 and
79.6µmol/L. On the other hand, urea level ranges from 6.6 to 31.4 mmol/L during 4th week and by
the end of 12th week it ranges from 10.8 to 34.4mmol/L.
Similarly, serum urea level and creatinine level varies according to sex of animal as well.
Rebaudioside A administrated at different concentration ranging from 0 to 2000mg/kg b. wt/day
to male and female rats separately affect the urea level as 14.1±2.14 to 14.9±1.39mg/dl and
16.6±4.09 to 18.7±1.94mg/dl respectively. However, creatinine varied non-significantly in male
and female rats correspondingly as 0.3±0.06mg/dl to 0.4±0.05mg/dl and 0.4±0.06 to 0.4±
0.1mg/dl. Enhanced level of urea, uric acid and creatinine in diseased rat can be attributed to
metabolic maladies that leads to lipid peroxidation, increased triglycerides and cholesterol
(Madianov et al., 1999).
It has been reported on the basis of a study done in Thailand that Stevia water extract is extensively
utilized by diabetics for glucoregulation and help in improvement of renal functioning thereby
regulating urea, uric acid and creatinine production (Lailerd et al., 2004). The research outcomes
of Anton et al. (2010) have established that substantial hepatoprotective effects were obtained
against liver damage by adding Stevia powder and water extracts in rats diets. In another research
done by Shivanna et al. (2013), in which rats were converted from normal to diabetics and
hypercholesterolemic by feeding them on high fructose and cholesterol diets. In another groups,
Streptozotocin was injected for rapid induction of diabetes. Different groups were administrated
with different levels of Stevia powder, polyphenol extracts and stevia with inulin combination.
They found that there was non-significant increment creatinine level while urea level lessened
substantially in Stevia fed rats.
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Table 44. Effect of Stevia diets on Urea (mg/dL)
* = Significant
**= Highly significant
NS= Non-significant
Table 45. Effect of Stevia diets on creatinine (mg/dL)
* = Significant
**= Highly significant
NS= Non-significant
Diets
Studies T0 T1 T2 T3 F value
Study I 23.64±2.32 20.77±1.05 22.34±0.91 22.80±1.91 2.64NS
Study II 30.12±1.80 27.94±2.65 28.52±3.12 28.16±3.52 0.60NS
Study III 31.10±3.73 27.81±3.42 28.79±2.28 29.09±2.43 1.03NS
Diets
Studies T0 T1 T2 T3 F value
Study I 0.67±0.06 0.63±0.04 0.65±0.01 0.63±0.02 1.17NS
Study II 0.96±0.08 0.91±0.05 0.93±0.02 0.94±0.09 0.26NS
Study III 1.02±0.05 0.95±0.08 0.97±0.07 0.98±0.06 0.97NS
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4.9.7 Hematological analysis
4.9.7.1 Red blood cell count (RBCs)
The statistical analysis results (Table 46) showed that RBC count was non-significantly
influenced due to diets in all the three studies. In study I, maximum RBC count was
observed T1 as 6.72±0.44cells/pL succeeded by T2, T3 and T0 as 6.58±0.43, 6.44±0.57 &
5.87±0.67cells/pL respectively. In the same way, non-significant increase RBC count was
recorded in study II and study III. Maximum values in study II was found in T1 as 7.29±0.85
followed by T2, T3 and T0 having values as 7.16±0.33, 7.13±0.38 and 6.77±0.52cells/pL,
respectively. However, lower RBC content calculated in T0 (7.17±0.64cells/pL) which increase in
rest of the treatments as T1 (7.75±0.44cells/pL), T2 (7.45±0.63cells/pL) and T3 (7.37±0.32cells/pL)
respectively.
4.9.7.2 White blood cells count (WBCs)
The statistical analysis outcomes presented in Table 47 for white blood cells (WBC) count have
interpreted non momentous difference in all the three studies. In study I, higher WBC count was
found in T0 as 6.35±0.93cells/nL followed by T3 (6.32±0.52cells/nL), T2 (6.29±0.64) &
maximum was observed in T1 (6.16±0.84cells/nL) respectively. While in study II, maximum WBC
amount was seen in T0 as 13.16±1.01cells/nL and minimum was marked in T1 12.95±1.04cells/nL.
Moreover in Study III, maximum WBC count calculated in T0 (15.03±1.13) while minimum was
found in T1 (14.13±1.60) and for T2 (14.47±1.18) T3 as 14.55±1.75 respectively and 16.40±0.51
cells/nL.
4.9.7.3 Platelets count (PLC)
Non-significant variation in platelets count of rats in all three studies was found presented
in Table 48. In study I, higher plate count was calculated in T1 (7.37±0.15) trailed by T3
(7.32±0.16), T2 (7.30±0.20) & T0 (7.26±0.45) accordingly. While maximum platelets count in
Study II was calculated in T1 as 6.84±0.29 and minimum was observed in T0 (6.77±0.31). Likewise
in study III, maximum platelets count was recorded in T1 while minimum was found in T0 as
6.44±0.38 and 6.38±0.24 respectively.
The findings of this research are similar in trend to the outcomes of Elnaga et al. (2016), who
worked on experimental male and female rats by feeding them with 25, 250, 500 and 1000mg/kg
b. wt for 12 weeks and analyzed their hematological parameters. They observed that white blood
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cells (WBCs) were non-significantly decreased by Stevia in diets while platelets and red blood
cells (RBCs) were substantially improved thereby imparting health beneficial verdicts. WBCs,
RBCs and platelets volume in control rats was recorded as 8.94 103/UL, 5.96 106/mL and 529.5
103/UL respectively. However, Stevia diets with 1000mg/kg b. wt have fund to be highly affected
for all these three parameter i-e WBCs, RBCs and platelets are varied as 8.79 103/UL, 5.82 106/mL
and 521.25 103/UL respectively. Stevia powder was explored for its biosafety perspectives and
impact on hematological variable by low, high and low dosage in combination with inulin. Results
depicted that a non-significant effect of Stevia powder on white blood cells and red blood cells
was found in hyperglycemic and Stevia treated hyperglycemic groups. Results of hematological
parameter for low and low dose with inulin were found to be similar, however, Stevia high dosage
showed some variations as compared to control.
Oxidative stress effect proper functioning and structures of red blood cells. Polyphenols used to
play an important role in protecting body from harmful implications in the form of free radical
formation that ultimately impact in form of oxidative stress that leads to certain carcinomas. Stevia
being the source of very rich polyphenol profile thereby protects the body from certain damages.
In the study of Awney et al. (2010) they found that various similarities exists in all parameters of
hematology when rats were fed on low, high and low stevia dosage in combination with inulin.
They found non-significant variation in WBCs, RBCs and platelets count while significant
variation was observed in mean corpuscular volume, mean corpuscular hemoglobin, mean
corpuscular hemoglobin concentration and packed cell volume. Nikiforov and Eapen (2008) have
deduced the similar outcomes from their research that adult rats fed on varied dosage levels of
rebaudioside A depicted non-momentous variation in hematology between control and high dose-
treated rats. However, some substantial differences were observed by comparing the diabetic
groups with control. At various intervals upsurge in mean corpuscular hemoglobin, mean
corpuscular volume and mean corpuscular hemoglobin concentration and percent decrease in
basophil counts were recorded in male groups. However, female rats fed with high dose of 500
mg/kg/day resulted in low red cell count and hemoglobin values by the end of week 2.
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Table 46. Effect of Stevia diets on red blood cell indices (cells/pL)
* = Significant
**= Highly significant
NS= Non Significant
Diets
Studies T0 T1 T2 T3 F value
Study I 5.87±0.67 6.72±0.44 6.58±0.43 6.44±0.57 2.40NS
Study II 6.77±0.52 7.29±0.85 7.16±0.33 7.13±0.38 0.82 NS
Study III 7.17±0.64 7.75±0.44 7.45±0.63 7.37±0.32 1.02NS
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Table 47. Effect of Stevia diets on white blood cell Indices (cells/nL)
* = Significant
**= Highly significant
NS= Non Significant
Table 48. Effect of Stevia diets on Platelets count
* = Significant
**= Highly significant
NS= Non Significant
Diets
Studies T0 T1 T2 T3 F value
Study I 6.35±0.93 6.16±0.84 6.29±0.64 6.32±0.52 0.06NS
Study II 13.16±1.01 12.95±1.04 13.01±0.73 13.07±0.85 0.04NS
Study III 15.03±1.13 14.13±1.60 14.47±1.18 14.55±1.75 0.40NS
Diets
Studies T0 T1 T2 T3 F value
Study I 7.26±0.45 7.37±0.15 7.30±0.20 7.32±0.16 0.16NS
Study II 6.77±0.31 6.84±0.29 6.79±0.49 6.81±0.12 0.05NS
Study III 6.38±0.24 6.44±0.38 6.41±0.48 6.40±0.51 0.09NS
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CHAPTER 5
SUMMARY
The research work was carried out to evaluate the biochemical, nutritional and safety profile along
with acceptability of Stevia in baked products. The stevia leaves were washed, cleaned and dried
which afterwards converted into powder by grinding in high speed grinder. The resultant powder
was subjected to chemical analysis including proximate composition, functional properties and
mineral profiling. Stevia powder was further subjected to fatty acid profiling, FTIR mapping for
functional groups identification and stevioside characterization. Stevia was also analyzed for its
phytochemical profile and subsequent antioxidant properties determination. Cookies were
prepared by replacing sucrose with stevia powder and extracts which were then analyzed for
physicochemical properties such as antioxidant assay, color, hardness and texture analysis and
sensory evaluation. On the basis of antioxidant assay, chemical composition, sensory
characteristics and overall acceptability of prepared cookies, the best treatments, one each from
powder, water and supercritical extracts were selected for efficacy study.
Proximate analysis revealed that Stevia leaf powder contained 3.95, 8.75, 7.60, 5.47, 10.64 and
63.95g/100g of moisture content, ash content, crude fiber, crude fat, crude protein and NFE
respectively. Chemical composition of wheat flour is expressed as moisture content (11.08%), ash
content (2.20%), crude fiber (1.43%), crude fat (1.23%), crude protein (8.62%) and NFE (75.89%).
Functional properties of Stevia powder were determined for product compatibility which included
parameter like pH (6.14), swelling power (5.01 g/g), water holding capacity (3.93 ml/g),, oil
holding capacity (5.96 ml/g) and bulk density (0.55g/ml).
Mineral profile of Stevia powder has depicted that minerals like sodium, potassium, phosphorous,
magnesium, iron, zinc, manganese, copper, nickel and cobalt were present at levels 29.4, 2195.3,
372.1, 286.2, 24.29, 1.423, 10.24, 0.85, 1.26 and 0.035 mg/kg, respectively. However, heavy metal
like lead, mercury, cadmium, chromium and arsenic were present in minute quantity whereas
chromium and arsenic were found as 0.15µg/g and 0.11µg/g, respectively in Stevia mineral profile.
Fatty acids profile in Stevia oil have shown that Palmitic acid (28.31mg/kg), palmitoleic acid
(2.17mg/kg), stearic acid (2.39m/kg), oleic acid (4.95mg/kg), linoleic acid (13.65mg/kg) and
linoleic acid (25.48mg/kg) were present.
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Significant variations were observed in TPC, TFC, DPPH, FRAP and ABTS activities. TPC
was found as 38.22 mg GAE/g (SFE), 32.45 mg GAE/g (SME), 28.21 mg GAE/g (SEE) and 24.24
mg GAE/g (SWE). Total flavonoid content was found maximum in SFE (32.10 mg CE/g) followed
by SME (27.14 mg CE/g), SEE (23.30 mg CE/g) and minimum was seen in SWE (19.88 mg CE/g).
Antioxidant analysis including DPPH assay was observed in such a way that maximum percent
reduction was recorded in supercritical extract as 57.99 %, water extract as 52.87%, ethanol extrct
47.15% and water extract 42.41%. Ferric reducing ability of plasma depicting antioxidant activity
of Stevia was found in a way that water extract depicted minimum ability as 236.57 µMol Fe2+/g,
ethanol extract showed as 294.45 µMol Fe2+/g, methanol extract 324.15 µMol Fe2+/g and
maximum was showed by 345.36 µMol Fe2+/g respectively. Antioxidant perspective by using
ABTS assay was recorded as SFE (55.04 µM TE/L) with maximum activity followed by SME
(51.40 µM TE/L), SEE (41.12 µM TE/L) and SWE (25.79 µM TE/L).
FTIR analysis for functional groups identification have depicted that alcohols, secondary amides,
alkanes, alkenes, ketones, primary amines, OH bending, esters, alkanes, carboxylic acids thiols,
alkene, inorganic phosphates, aromatic groups have been identified in raw powder and different
extracts of Stevia. Steviosides quantification done by HPLC have depicted significant results for
Steviol, Rebaudioside A and Stevioside. Steviol was found in range of 485.25- 357.26mg/kg,
Rebaudioside A was calculated as 383.38- 792.15mg/kg while Stevioside content was found as
1107.95-665.34 mg/kg.
Stevia cookies prepared by replacing different levels of stevia powder, water and supercritical
extracts with sucrose have depicted that the chemical composition have improved significantly.
Significant differences in chemical composition of different cookies treatments have been recorded
like crude fiber (1.69 to 3.63%), Crude protein (10.90 to 15.06%) and crude fat (9.90 to14.04%)
increased by incrementing the amount of Stevia powder and extracts, however moisture content
(3.03 to 3.52%), ash (1.22 to 2.45%) and NFE (65.19 to 75.11%) have been found.
Treatments had substantial impact on antioxidant ability of the cookies. TPC have expressed
significant variations among treatments in such a way that T0, T1, T2, T3, T4, T5, T6, T7, T8 and T9
as 10.14±0.28, 9.36±0.57, 10.04±0.12, 11.88±0.58, 9.92±0.27, 10.28±0.06, 10.41±0.08,
10.16±0.12, 9.92±0.10 and 10.11±0.09 mg GAE/100g respectively. Increment of Stevia powder
and extracts imparted significant impact on TPC level. For total flavonoids contents, different
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treatments have shown as control; T0 (15.87±0.20 mg CE/g), Stevia powder cookies including T1
(17.23±0.15 mg CE/g), T2 (20.82±0.15 mg CE/g) and T3 (23.26±0.05 mg CE/g) showed maximum
results with 30% stevia powder replaced with sucrose. For stevia water extract cookies maximum
amount of TFC was observed in T6 (18.57±0.13 mg CE/g) followed by T5 (18.27±0.05 mg CE/g)
and T4 (17.69±0.19 mg CE/g) respectively. On the other hand supercritical extract cookies have
expressed in such a way that maximum TFC were found as 18.76±0.02 mg CE/g (T9) and minimum
as 18.44±0.02 mg CE/g (T7).
DPPH assay depicting antioxidant ability has enhanced by increase in concentration level for
powder and extracts as well. In case of powder treatments maximum amount of percentage
reduction was recorded in T3 (13.15±0.09%) followed by T2 (13.01±0.05%), T1 (12.74±0.33%)
while control treatment was calculated as T0 (9.59±0.74%) respectively. Means for different
treatments of supercritical extracts exhibited non-significant increase with maximum reduction
observed in T9 (12.98±0.02%) followed by T8 (12.81±0.04%) and T7 (12.72±0.06%) with 3%, 2%
and 1% level of extract replacing sucrose respectively. Ferric reducing antioxidant power of
different stevia cookies have been illustrated as T3 showed up with maximum reducing power
(17.00±1.11 µmol Fe2+/g), T2 (15.06±1.36 µmol Fe2+/g) and T1 (13.82±0.48 µmol Fe2+/g) and
control as T0 (10.55±2.05 µmol Fe2+/g) respectively. Stevia supercritical extracts and water extract
have also shown some increment in reducing power in respective treatments namely T7, T8, T9 and
values expressed as (13.52±0.38 µmol Fe2+/g), (14.30±0.85 µmol Fe2+/g) and (14.55±0.32 µmol
Fe2+/g). While for treatment T4, T5 and T6 having 1%, 2% and 3% Stevia water extract with
reducing power presented as 11.88±2.25, 11.73±0.96 and 12.85±0.79 µmol Fe2+/g respectively.
Sensory results obtained for maximum color score was observed in T7 (7.10±0.40) with 1%
supercritical extract having pale yellow to lightly greenish color while minimum score was
recorded in T3 (4.58±0.62) with 3% incorporation of raw stevia powder which leads to dark green
color which is less liked by panelists. Crispiness results have established that it varies from
4.66±0.74 to 7.92±0.88 in which highest value was attained by control (T0) cookies and lowest
score was gained by cookies having 30% (T3) stevia powder incorporation for sweetness.
Significantly the highest mean score for taste (7.71±0.52) was observed for T0 and minimum was
recorded in T3 (5.16±0.81). Flavor of cookies comparing with control ranged from 5.00±1.16 to
8.00±0.25 with highest value by the control cookies and least by T3 cookies with 30% sucrose
replacement with Stevia powder. The texture of cookies lessened momentously with increasing
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the concentration level of Stevia in all treatment types with maximum score attained by
T1=6.38±0.73 while minimum score came in the fate of T3=5.15±0.66 as compared to control
(7.55±0.38). Mean values for overall acceptability of cookies depicted that concentration level of
stevia powder and extracts have impacted significantly and mean scores ranges from 5.00±0.71 to
7.50±0.38 with highest score obtained by control cookies and least score grabbed by 30% Stevia
powder.
L*, a* and b* color values for cookies have been significantly affected due to progressive
increment in Stevia leaves powder that gave lower L* values; maximum value was recorded in
control having bright color T0 (76.32±2.06) while minimum value of 25.51±2.15 was observed in
T3 (30% Stevia powder). Increasing the replacement levels of sucrose with Stevia leaves powder
and extracts resulted in marked decrease in a* ranging from 2.36±0.08 to 1.19±0.07 for Stevia
water extract (1-3%) expressing reddish green shade for cookies. b* values decreased as a function
of Stevia powder and extracts addition i.e. 36.74±3.09 in T7 (1% supercritical extract) to
10.02±1.46 in T3 (30% Stevia powder) where control was recorded as T0 (38.45±1.87).
Texture analysis done by texture analyzer to determine the hardness have showed that hardness
varies from 15.60±0.09 to 26.50±0.16 (g) Gross calorific value of control and optimized stevia
cookies showed highly significant variation. Mean values depicted that calorific value ranges from
4106.4±21.50 to 3660.6±121.05 kcal/100g, respectively. Spread factor results for stevia cookies
showed that lowest thickness was observed in T5 (6.33 ±0.10cm) while maximum thickness was
calculated in T8 (8.11±0.02cm). In case of diameter, T7 (26.5±0.30cm) attained maximum
diameter, however minimum diameter was calculated in T3 (22.7±0.20cm). Spread ratio results
obtained from ratio of diameter and thickness indicated that spread ratio decreases with the
increase in replacement level of Stevia with sucrose. Spread ratio decreased from 3.93±0.09 (T7)
to 2.86±0.09 (T3) which is presumed to be due to increase in dietary fiber and protein content as
the concentration level of Stevia is increased.
Efficacy trials were done to evaluate the safety perspective of Stevia, in normal (study I),
hyperglycemic (study II) and hypercholesterolemic (study III) rats modeling. The increment in
feed intake in different rat groups recorded as; normal rats (34.91 to 37.67 g/rat/day),
hyperglycemic rats (33.51 to 36.25 g/rat/day) and hypercholesterolemic rats (34.76 to 35.42
g/rat/day) fed on different diets. Water intake was affected due to feeding treatments in all three
studies such as Study I (8.32 to 26.65 ml/rat/day), Study II (19.75 to 27.33 ml/rat/day) and Study
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III (25.97 to 27.96 ml/rat/day). Body weight of rats were affected due to the addition of stevia
powder and extracts which are expressed as 153.90 to 160.17g/rat in Study I, while Study II depicts
that weight gain ranges from 179.60g/rat at the start of study and decreases to 177.35g/rat by the
end of 8th week. However in study III, the results showed that weight decreased from 185.85 to
180.20g/rat.
It is worth mentioning that stevia leaves powder has improved increased insulin secretion,
decreased blood glucose level, LDL, cholesterol and triglycerides while HDL contents have been
enhanced non-substantially. T1 attained the maximum percent reduction in Study I as 2.77%
whereas T2 & T3 caused showed 1.59% and 1.36% reduction glucose level in normal rats fed on
different diets. Highest percent reduction in Study II (hyperglycemic study) was observed in T1
(7.0%) followed by T2 (4.90%) and T3 (4.54%). Study III (hypercholesterolemic study) also
behaved in similar reduction pattern with minimum reduction was seen in T3 (supercritical extract
cookies) as 2.05% and the highest decline was calculated T1 (3.75%). The effect of diets on insulin
secretion was observed momentous in Study I with percent increase as expressed T1 (1.22%), T2
(1.07%) and T3 (0.93%). While 6.13, 4.91 and 4.27% significant increase due to diets was recorded
in treatment groups T1, T2 and T3 for study II. Similar increment level for insulin production was
recorded in Study III with highest values recorded 2.88 (T1) followed by 2.02 (T2) and 1.64 (T3).
Results for cholesterol level have shown that T1 resulted in highest cholesterol reduction followed
by T2 and T3. In study I, comparison with control have deduced that T1, T2, and T3 treatments
presented 2.38, 1.62 and 1.18% drop in cholesterol respectively. In study II, maximum downgrade
trend was seen as T1 (4.85%) trailed by T2 (3.12%) and T3 (2.12%). Similarly, in study III
maximum reduction (5.47%) was perceived in T1 followed by T2 (2.47%) and T3 (1.28%) as
compared to control. HDL level was non-significantly increased in study I as 1.73, 1.26 & 1.13%
with diets T1, T2 and T3 respectively, while in studies II & III, diets T1, T2 and T3 exhibited
substantial increase in HDL as compared to control as 2.72, 2.04, 1.74% and 6.66, 3.60 & 2.79%,
correspondingly. However, percent reduction in LDL values in all studies for different rats groups
have depicted that in study I, non-significant decrease was seen in T0 (2.46), T1 (1.71) and T3 (1.40)
groups as compared to control. Likewise, non-substantial decrease was recorded during Study II
i.e. 3.61% in T1, 2.70% in T2 and 1.91% in T3. However in study III, diet containing Stevia powder
(T1) reduced the LDL level by 4.16%. While diet having Stevia water extract (T2) and Stevia
supercritical extract resulted in 2.09% and 2.68% decrease in LDL. Triglycerides have been seen
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to be decreased as T1 (0.83%) in study I showed maximum reduction followed by T2 (0.63%) and
T3 (0.56%). In study II, T1 delivered the highest triglycerides reduction of 2.85% while in T2 & T3
1.83% and 1.23%. Significant reduction was observed in study III in which diet containing Stevia
leaves powder depicted a high reduction of 5.36% in T1 followed by T2 (2.09%) and T3 (2.68%)
having water and supercritical extracts respectively. In study I, II and III, diets expressed
significant effect on liver function tests i.e. ALT, ALP and AST. The AST value of rats in study I
ranges from 105.48 to 107.42 IU/L. In study II varies from 126.61 to 130.84IU/L while in Study
II maximum AST level was 122.99 that lessened to 116.43IU/L.
Kidney functioning of rat was assessed by urea and creatinine results which depicts that maximum
value for urea in Study I was recorded in T0 (23.64±2.32mg/dL) while minimum value calculated
in group T1 (20.77±1.05mg/dL) followed by T2 and T3 groups as 22.34±0.91mg/dL and
22.80±1.91 mg/dL, respectively. In similar manner, fro study II, serum urea value ranges from
27.94±2.65mg/dL (T1) to 30.12±1.80mg/dL (T0) group, while T2 (28.52±3.12mg/dL) and T3 as
28.16±3.52mg/dL having non-significant variation among groups. However Study III have
maximum serum urea value for T0 trailed to T3, T2 and T1 with their values presented as
31.10±3.73mg/dL, 29.09±2.43mg/dL, 28.79±2.28mg/dL and 27.81±3.42mg/dL, correspondingly.
Non-significant variation have been found in creatinine level among all studies. In study I,
creatinine level ranges from 0.67±0.06mg/dL to 0.63±0.02mg/dL. Similar trend for creatinine was
observed for study II as 0.96±0.08mg/dL to 0.91±0.05mg/dL. In the same way momentous
variation was observed in study III ranging from 1.02±0.05 to 0.95±0.08.
Hematological results for parameter like red blood cells, white blood cells and platelets have been
non-significantly influenced due to diets in all the three studies. In study I, maximum RBC
count was observed 6.72±0.44cells/pL while minimum as 5.87±0.67cells/pL. RBC count
ranges from 7.29±0.85 to 6.77±0.52cells/pL for Study II. However, lower RBC content for study
III was recorded as 7.17±0.64cells/pL) while maximum was 7.37±0.32cells/pL. In case of WBCs,
study I have showed that higher WBC count was found 6.35±0.93cells/nL while minimum
as 6.16±0.84cells/nL. Whereas in study II, WBCs ranges from 13.16±1.01cells/nL to
12.95±1.04cells/nL. Moreover in Study III, maximum WBC count calculated as 15.03±1.13 while
minimum was found as 14.13±1.60 cells/nL. Plate count being the potent immunity parameter has
shown that higher plate count in study I, was recorded as 7.37±0.15 trailed down as
7.26±0.45. While maximum platelets count in Study II was calculated as 6.84±0.29 and minimum
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was observed as 6.77±0.31. Likewise in study III, platelets count ranged from 6.44±0.38 to
6.38±0.24.
The outcomes of current research have suggested that different parameters like chemical
composition, functional properties, antioxidant potential and product analysis are variable among
different treatments due to the addition of stevia leaves powder and different extracts. Decreasing
trend was recorded in feed intake and body weight, glucose, cholesterol, LDL and triglycerides,
while increment in HDL, RBCs and insulin secretion was recorded due to replacement of sucrose
with stevia leaves powder and extracts from solvents like water, methanol, ethanol and
supercritical fluid extract.
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CONCLUSIONS
Stevia powder and extracts are naturally non-nutritive health promoting intense sweetener
Stevia has appeared to be a good source of nutritional constituents specially protein
(10.64%), crude fiber (7.6%) ash content (8.75%), thereby exhibiting good functional
properties for value addition.
Stevia; source of promising polyphenols having TPC (24.24-38.22mg GAE/g) and TFC
(19.88-32.10mg CE/g), portray remarkable antioxidant ability by free radical scavenging
(42.41-57.99% inhibition) and ferric reducing ability (236.57-345.36 µmol Fe2+/g)
Mineral have been found to in appreciable amounts like sodium, potassium, phosphorous,
magnesium, iron, manganese, copper, nickel and cobalt as 29.4, 2195.3, 372.1, 286.2,
24.29, 10.24, 0.85, mg/kg.
Palmitic acid (28.31g/100g), linoleic acid (25.48 g/100g), linoleic acid (13.65 g/100g)
and oleic acid (4.95 g/100g) have been found in good concentration.
Alcohols, secondary amides, alkanes, alkenes, ketones, primary amines, OH bending,
esters, alkanes, carboxylic acids were the major functional that have been identified during
FTIR analysis of Stevia.
SGs like Stevioside (665.34-1107.95mg/kg), Rebaudioside A (383.38-792.15mg/kg) and
Steviol (357.26-485.25mg/kg) have been quantified from different extracts by HPLC and
found to be rich in these sweetening components that play therapeutic role as well
Stevia powder and extracts used in cookies and have received good consumer acceptance
when added up to 10% and 3% level of powder and extracts replacement with sucrose
In addition to be used as intense sweetener, Stevia help in preparation of functional and
medicinal foods that are ought to augment health status of masses
Stevia is proved to be beneficial in regulation and modulation of blood glucose (7%
reduction) and cholesterol (5.47% reduction) level and impart non-significant effect on
liver, renal and hematological parameters
146
RECOMMENDATIONS
Stevia should be used as potential replacer for synthetic and nutritive sweeteners in food
products to prevent the incidence of various physiological disorders
Stevia should be used in various food formulations to provide value added food products
Nutritionists should promote the use of Stevia powder and extracts to address lifestyle
related disorders
More avenues should be explored regarding protein characterization leading to amino acid
profiling and soluble and insoluble dietary fiber analysis
Health prevailing perspectives of Stevia regarding glucoregulatin and anticancer aspects
should be explored extensively in order to provide comprehensive information Stevia has
healing activities against wounds and ulcers along with antimicrobial and anti-cancer
properties which need to be explored and results should be disseminated.
Different varieties of Stevia should be cultivated and afterwards comprehensively explored
to portray better safety profile of Indigenous Stevia
Government should promote importers for making foreign processed Stevia available and
give encouraging incentives to farmers for its propagation
Public awareness seminars, TV and radio commercials, print and electronic media should
promote and propagate information about importance of Stevia
147
LITERATURE CITED
AACC. 2000. Approved Methods of the American Association of Cereal Chemist. 9th Ed.
Association of Official Analytical Chemist. Washington DC.
Abdel-Salam, A.M., A.S. Ammar and W.K. Galal. 2009. Evaluation and properties of formulated
low calories functional yoghurt cake. J. Food Agric. Environ. 7: 218-221.
Abou-Arab, A. E., A.A. Abou-Arab and M.F. Abu-Salem. 2010. Physico-chemical assessment of
natural sweeteners steviosides produced from Stevia rebaudiana Bertoni plant. Afr. J. Food
Sci. 4: 269-281.
Abou-Arab, E.A. and F.M. Abu-Salem. 2010. Evaluation of bioactive compounds of Stevia
rebaudiana leaves and callus. Afr. J. Food Sci. 4: 627-634.
Afandi, A., S. Sarijan and R.K. Shaha. 2013. Optimization of Rebaudioside A extraction from
Stevia rebaudiana (bertoni) and quantification by High Perfomance Liquid
Chromatography Analysis. J. Tropi. Res.Sustain. Sci. 1: 62-70.
Ahmad, S. 2016. Evaluation and optimization of mixed wheat flour in relation to end product
quality. Ph.D. Thesis, University of Agriculture, Faisalabad.
Ahmed, I. A., M.A. Mikail, M. Bin Ibrahim, N. Bin Hazali, M. Rasad and R.A. Ghani. 2015.
Antioxidant activity and phenolic profile of various morphological parts of underutilised
Baccaurea angulata fruit. Food Chem. 172: 778-787.
Ahmed, M.B., M. Salahin, R. Karim, M.A. Razvy, M.M. Hannan, R. Sultana, M. Hossain and R.
Islam. 2007. An efficient method for in vitro clonal propagation of a newly introduced
sweetener plant Steviare baudiana in Bangladesh. Am. Eurasian. J. Sci. Res. 2: 121-125.
Alam, M. N., N.J. Bristi & M. Rafiquzzaman. 2013. Review on in vivo and in vitro methods
evaluation of antioxidant activity. Saudi Pharm. J. 21: 143-152.
Allain, C.C., L.S. Poon, C.S. Chan, W. Richmond and P.C. Fu. 1974. Enzymatic determination of
total serum cholesterol. Clin. Chem. 20:470-475.
Alshatwi, A.A., M.A. Al Obaaid, S.A. Al Sedairy, A.H. Al-Assaf, J.J. Zhang and K.Y. Lei. 2010.
Tomato powder is more protective than lycopene supplement against lipid peroxidation in
rats. Nut. Res. 30:66-73.
148
Álvarez-Robles, M. J., A. Lopez-Orenes, M.A. Ferrer and A.A. Calderon. 2016. Methanol elicits
the accumulation of bioactive SGs and phenolics in Stevia rebaudiana shoot cultures. Ind.
Crops. Prod. 87: 273-279.
American Dietetic Association. 2004. Position of the American Dietetic Association: use of
nutritive and nonnutritive sweeteners. J. Am. Diet. Assoc. 104: 255.
Anton, S.D., C.K. Martin, H. Han, S. Coulon, W.T. Cefalu, P. Geiselman and D.A. Williamson.
2010. Effects of stevia, aspartame, and sucrose on food intake, satiety, and postprandial
glucose and insulin levels. Appetite. 55: 37-43.
Anwar, M.M. and A.R.M. Meki. 2003. Oxidative stress in streptozotocin-induced diabetic rats:
effects of garlic oil and melatonin. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 135:
539-547.
AOAC (Association of Official analytical chemists). 2006. Official methods of analysis of AOAC.
18th Ed. AOAC press, Arlington, VA, USA.
Arabshahi, D.S. and A. Urooj. 2007. Antioxidant properties of various solvent extracts of mulberry
(Morus indica L.) leaves. Food Chem. 102: 1233-1240.
Aranda-Gonzalez, I., Y. Moguel-Ordonez and D. Betancur-Ancona. 2014. Rapid HPLC method
for determination of rebaudioside D in leaves of stevia rebaudiana bertoni grown in the
southeast of méxico. Am. J. Analyt. Chem. 5: 813.
Asimi, O. A., N.P. Sahu and A.K. Pal. 2013. Antioxidant capacity of crude water and ethylacetate
extracts of some Indian species and their antimicrobial activity against Vibrio vulnificus
and Micrococcus luteus. J. Med. Plant Res. 7: 1907-1915.
Atteh, J., O. Onagbesan, K. Tona, J. Buyse, E. Decuypere and J. Geuns. 2011. Empleo potencial
de stevia rebaudiana en alimentación animal. Archiv. de Zootec. 60: 133-136.
Awney, H.A., M.I. Massoud and S. El‐Maghrabi. 2011. Long‐term feeding effects of stevioside
sweetener on some toxicological parameters of growing male rats. J. App. Toxi. 31: 431-
438.
149
Badarinath, A.V., K.M. Rao, C.M.S. Chetty, V. Ramkanth, T.V.S. Rajan and K. Gnanaprakash.
2010. A review on in-vitro antioxidant methods: comparisons, correlations and
considerations. Int. J. PharmTech Res. 2: 1276-1285.
Baixauli, R., T. Sanz, A. Salvador and S.M. Fiszman. 2008. Muffins with resistant starch: Baking
performance in relation to the rheological properties of the batter. J. Cereal Sci. 47: 502-
509.
Barriocanal, L., M. Palacios, G. Benitez, S. Benitez, J. Jimenez and N. Jimenez. 2008. Apparent
lack of pharmacological effect of SGs used as sweeteners in humans, a pilot study of
repeated exposures in some normatensive and hypotensive individuals and in type 1 and
type 2 diabetics. Regul. Toxicol. Pharmacol. 51: 37-41.
Barroso, M., L. Barros, M.A. Rodrigues, M.J. Sousa, C. Santos-Buelga and I.C. Ferreira. 2016.
Stevia rebaudiana Bertoni cultivated in Portugal: A prospective study of its antioxidant
potential in different conservation conditions. Ind. Crops Prod. 90: 49-55.
Basuny, A.M., A.M. Gaafar and S.M. Arafat. 2009. Tomato lycopene is a natural antioxidant and
can alleviate hypercholesterolemia. Afr. J. Biotech. 8: 6627-6633.
Benavente-Garcia, O., J. Castillo, J. Lorente, A. Ortuno and J.A. Del Rio. 2000. Antioxidant
activity of phenolics extracted from Olea europaea L. leaves. Food Chem. 68: 457-462.
Bernal, J., J.A. Mendiola, E. Ibanez and A. Cifuentes. 2011. Advanced analysis of nutraceuticals.
J. Pharma. Biomed. Anal. 55: 758-774.
Biego, G. H., M. Joyeux, P. Hartemann and G. Debry. 1998. Daily intake of essential minerals and
metallic micropollutants from foods in France. Sci. Total Environ. 217: 27-36.
Bridel, M. and R. Lavieille. Le principle sucre du Kaa-he-e (Stevia rebaudiana Bertoni) III. 2011.
Le steviol de I, hydrolyse diastasique et I‘isostevial de I‘hydrolyse acid. Bull. Soc. Chem.
Biol. 13: 409-412.
Brisibe, E., U. Umoren, F. Brisibe, P. Magalhaes, J. Ferreira, D. Luthria, X. Wu and R. Prior. 2009.
Nutritional characterisation and antioxidant capacity of different tissues of Artemisia annua
L. Food Chem. 115: 1240-1246
150
Broca, C., M. Manteghetti, R. Gross, Y. Baissac, M. Jacob, P. Petit and G. Ribes. 2000. 4-
Hydroxyisoleucine: effects of synthetic and natural analogues on insulin secretion. Europ.
J. Pharma. 390: 339-345.
Brusick, J. 2008. A critical review of the genetic toxicity of steviol and SGs. Food Chem. Toxicol.
46: 83-91.
Buckenhuskers, H. J. and Omran, H. T. 1997. Stevia rebaudiana Bertoni and stevioside. Sugar and
S. Subst. Food Proc. Nutr. Ismailia Oct. 7: 157-178.
Bushuk, W. 1997. Wheat breeding for end-product use. Wheat: Prospects for global improvement.
Springer.
Cacciola, F., P. Delmontea, K. Jaworska, P. Dugo, L. Mondello and J. Rader. 2011. Employing
ultra high pressure liquid chromatography as the second dimension in a comprehensive
two-dimensional system for analysis of Stevia rebaudiana extracts. J. Chromatogr. A. 1218:
2012-2018.
Carakostas, M. C., L.L. Curry, A.C. Boileau and D.J. Brusick. 2008. Overview: the history,
technical function and safety of rebaudioside A, a naturally occurring steviol glycoside, for
use in food and beverages. Food. Chem. Toxicol. 46: 1-10.
Carbonell-Capella, J. M., F.J Barba, M.J. Esteve and A. Frigola. 2013. High pressure processing
of fruit juice mixture sweetened with Stevia rebaudiana Bertoni: Optimal retention of
physical and nutritional quality. Innov. Food Sci. & Emerg. Technol.18: 48-56.
Carocho, M., P. Morales and I.C.F.R. Ferreira. 2015. Natural food additives: Quo vadis? Trends
Food Sci. Technol. 45: 284-295.
Ceunen, S. and J.M.C. Geuns. 2013. Glucose, sucrose, and steviol glycoside accumulation in
Stevia rebaudiana grown under different photoperiods. Biologia. Planta. 57: 390-394.
Chaturvedula, V.S.P. and J. Zamora. 2014. Isolation and structural characterization of a new minor
diterpene glycoside from Stevia rebaudiana. Nat. Prod. Commun. 9: 1677-1679.
Chen J., P.B. Jeppesen, I. Nordentoft and K. Hermansen. 2006. Steviodose counteracts the
glyburide-induces desensitization of the pancreatic beta-cell function in mice: Stud. in
vitro. Metabol. 55: 1674-1680.
151
Chen, M.F. 2009. Attitude toward organic foods among Taiwanese as related to health
consciousness, environmental attitudes, and the mediating effects of a healthy lifestyle. Br.
Food J. 111: 165-178.
Cho, B.O., H.W. Ryu, Y. So, J.K. Cho, H.S. Woo, C.H. Jin, K.I. Seo, J.C. Park and I.Y. Jeong.
2013. Anti-inflammatory effect of austroinulin and 6-O-acetyl-austroinulin from Stevia
rebaudiana in lipopolysaccharide stimulated RAW 264.7 macrophages. Food Chem.
Toxicol. 62: 638-644.
Code of Federal Regulations: 21 CFR 101.9—Nutrition labeling of food (c)(1)(i)(A)-(E)
Combined Compendium of Food Additive Specifications. (FAO JECFA Monographs 10). 73rd
Report of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), June 8-17,
2010, Geneva, Switzerland, Food and Agriculture Organization of the United Nations
(FAO) (2010).
Das, A., S. Gantait and N. Mandal. 2011. Micropropagation of an elite medicinal plant: Stevia
rebaudiana Bert. Int. J. Agri. Res. 6: 40-48.
Das, K. and A.K. Kathiriya. 2012. Hepatoprotective activity of Stevia rebaudiana Bert. leaves
against thioacetamide induced toxicity. Turk. J. Pharma. Sci. 9: 343-352.
De-Oliveira, B. H., J.F. Packer, M. Chimelli and D.A. de Jesus. 2007. Enzymatic modification of
stevioside by cell-free extract of Gibberella fujikuroi. J. Biotech. 131: 92-96.
Do, Q. D., A.E. Angkawijaya, P.L. Tran-Nguyen, L.H. Huynh, F.E. Soetaredjo, S. Ismadji and
Y.H. Ju. 2014. Effect of extraction solvent on total phenol content, total flavonoid content,
and antioxidant activity of Limnophila aromatica. J. food. drug. Ana. 22: 296-302.
Yildiz-Ozturk, A.E., Nalbantsoy, O. Tag and O. Yesil-Celiktas. 2015. A comparative study on
extraction processes of Stevia rebaudiana leaves with emphasis on antioxidant, cytotoxic
and nitric oxide inhibition activities. Industrial Crops and Products. 77:961-971.
Eastwood, M. and D. Kritchevsky. 2005. Dietary fiber: how did we get where we are? Annu. Rev.
Nutr. 25:1-8.
152
Elchuri, S., T.D. Oberley, W. Qi, R.S. Eisenstein, L.J. Roberts, H. Van Remmen and T.T. Huang.
2005. CuZnSOD deficiency leads to persistent and widespread oxidative damage and
hepatocarcinogenesis later in life. Oncogene. 24: 367-380.
Elnaga, N.A., M.I. Massoud, M.I. Yousef and H.H. Mohamed. 2016. Effect of stevia sweetener
consumption as non-caloric sweetening on body weight gain and biochemical’s parameters
in overweight female rats. Annals Agri. Sci. 61: 155-163.
Erkucuk, A., I.H. Akgun and O. Yesil-Celikta. 2009. Supercritical CO2 extraction of glycosides
from Stevia rebaudiana leaves: Identification and optimization. J. Supercrit. Fluids. 51: 29-
35.
European Food Safety Authority (EFSA). 2010. Scientific opinion of the Panel on Food
Additives and Nutrient Sources (ANS) on the safety of SGs for the
proposed uses as a food additive. EFSA J. 8: 1537.
European Food Safety Authority. 2011. Revised exposure assessment for SGs for the proposed
uses as a food additive. Eur. Food Saf. Auth. J. 9: 1-19.
Evaluation of Certain Food Additives and Contaminants. 68th Report of the Joint FAO/WHO
Expert Committee on Food Additives, Geneva, Switzerland, WHO Technical Report
Series 947 (2007), pp. 50–54 Available at:
http://apps.who.int/iris/bitstream/10665/43870/1/9789241209472_eng.pdf
FAO, 2010. JECFA Additives. SGs. Available from: http://www.fao.org/ag/agn/jecfa-
additives/specs/monograph10/additive-442-m10.pdf (accessed: 22.07.15.).
Fischbach, F. 1996. Blood studies. A manual of laboratory and diagnostic tests. Philadelphia:
Lippincott. 23-146.
Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition
(CFSAN) (2009b). Agency Response Letter GRAS Notice No. GRN 000282.
[Rebaudioside a Purified from the Leaves of Stevia rebaudiana (Bertoni) Bertoni], U.S.
Garcia-Serna, E., N. Martinez-Saez, M. Mesias, F.J. Morales and M. Castillo. 2014. Use of coffee
silverskin and stevia to improve the formulation of biscuits. Pol. J. Food. Nutri. Sci. 64:
243-251.
153
Gardana, C., M. Scaglianti and P. Simonetti. 2010. Evaluation of steviol and its glycosides in
Stevia rebaudiana leaves and commercial sweetener by ultra-high-performance liquid
chromatography-mass spectrometry. J. Chromatogr. A. 1217: 1463-1470.
Gasmalla, M. A. A., R. Yang, I. Amadou and X. Hua. 2014. Nutritional composition of Stevia
rebaudiana Bertoni leaf: effect of drying method. Trop. J. Pharm. Res. 13: 61-65.
Gawel-Beben, K., T. Bujak, Z. Niziol-Lukaszewska, B. Antosiewicz, Jakubczyk, A., M. Karas and
K. Rybczynska. 2015. Stevia rebaudiana Bert. leaf extracts as a multifunctional source of
natural antioxidants. Molecules. 20: 5468-5486.
Geeraert, B., F. Crombe, M. Hulsmans, N. Benhabiles, J.M. Geuns and P. Holvoet. 2010.
Stevioside inhibits atherosclerosis by improving insulin signaling and antioxidant defense
in obese insulin-resistant mice. Int. J. Obes. 34: 569-577.
Geuns, J.M, J. Buyse, A. Vankeirsbilck and E.H. Temme. 2007. Metabolism of stevioside by
healthy subjects. Exp. Biol. Med. 232: 164-173.
Goyal, S. K. and R.K. Goyal. 2010. Stevia (Stevia rebaudiana) a bio-sweetener: a review. Int. J.
Food Sci. Nutr. 61: 1-10.
Greg Arnold, D.C. 2010. Stevia Helps Heart Health in Mice by Affecting Insulin Levels. Int. J.
Obes. 208: 92-93.
Guggisberg, D., P. Piccinali and K. Schreier. 2011. Effects of sugar substitution with Stevia,
Actilight™ and Stevia combinations or Palatinose™ on rheological and sensory
characteristics of low-fat and whole milk set yoghurt. Int. Dairy J. 21: 636-644.
Gupta, E., S. Purwar, S. Sundaram and G.K. Rai. 2013. Nutritional and therapeutic values of Stevia
rebaudiana: A review. J. Med. Plants Res. 7: 3343-3353.
Gupta, P., S. Sharma and S. Saxena. 2016. Effect of abiotic stress on growth parameters and steviol
glycoside content in Stevia rebaudiana (Bertoni) raised in vitro. J. Appl. Res. Med. Aromat.
Plants. 3: 160-167.
Hajar, E. W. I., A.Z.B. Sulaiman and A.M. Sakinah. 2014. Assessment of heavy metals tolerance
in leaves, stems and flowers of Stevia rebaudiana plant. Procedia Environ. Sci. 20: 386-
393.
154
Hearn, L. K. and P.P. Subedi. 2009. Determining levels of SGs in the leaves of Stevia rebaudiana
by near infrared reflectance spectroscopy. J. Food Comp. Anal. 22: 165-168.
Hendawey, M.H. and R.A. El-Fadl. 2014. Biochemical studies on the production of active
constituents in stevia rebaudiana L. Callus. Global J. Biotech. Biochem. 9: 76-93.
Herrero, M., J.A. Mendiola, A. Cifuentes and E. Ibanez. 2010. Supercritical fluid extraction:
Recent advances and applications. J. Chromato. A. 1217: 2495-2511.
Holvoet, P., A. Rull, A. Garcia-Heredia, S. Lopez-Sanroma, B. Geeraert, J. Joven and J. Camps.
2015. Stevia-derived compounds attenuate the toxic effects of ectopic lipid accumulation
in the liver of obese mice: A transcriptomic and metabolomic study. Food Chem. Toxicol.
77: 22-33.
Hony, J., L. Chen and P.B. Jeppesen. 2006. Stevioside counteracts the Alpha y-cell hypersecretion
caused by long-term palmitate exposure. Am. J. Physiol. Endocrinol. Metab. 290: 416-422.
Huang, X. Y., J.F. Fu and D.L. Di. 2010. Preparative isolation and purification of SGs from Stevia
rebaudiana Bertoni using high-speed counter-current chromatography. Sep. Purif. Technol.
71: 220-224.
Inamake, M. R., P.D. Shelar, M.S. Kulkarni, S.M. Katekar and T. Rashmi. 2010. Isolation and
analytical characterization of stevioside from leaves of Stevia rebaudiana
Bert;(Asteraceae). Int. J. Res. Ayurveda. Pharm. 1: 572-581.
Jackson, A. U., A. Tata, C. Wu, R.H. Perry, G. Haas, L. West and R.G. Cooks. 2009. Direct
analysis of Stevia leaves for diterpene glycosides by desorption electrospray ionization
mass spectrometry. Analyst. 134: 867-874.
Jarup, L. 2003. Hazards of heavy metal contamination. Br. Med. Bull. 68: 167-182.
JECFA, 2005. SGs. In: 63rd Meeting of the Joint FAO/WHO Expert Committee on Food
Additives, Geneva, Switzerland. World Health Organization (WHO), Geneva,
Switzerland. WHO Technical Report Series, 928, 34e39, and 138.
http://whqlibdoc.who.int/trs/WHO_TRS_928.pdf
155
Jeppesen, P.B., S. Gregersen, S.E. Rolfsen, M. Jepsen, M. Colombo, A. Agger, J. Xiao, M.
Kruhoffer, T. Orntoft and K. Hermansen. 2003. Antihyperglycemic and blood pressure-
reducing effects of stevioside in the diabetic Goto-Kakizaki rat. Metabol. 52: 372-378.
Jeppesen, P.B., S. Gregersen, C.R. Poulsen and K. Hermansen. 2000. Stevioside acts directly on
pancreatic β cells to secrete insulin: Actions independent of cyclic adenosine
monophosphate and adenosine triphosphate—sensitivie K+-channel activity. Metabol. 49:
208-214.
Johnson, M., E.G. Wesely, H.M.I. Zahir and N. Selvan. 2010. In vivo and in vitro phytochemical
and antibacterial efficacy of Baliospermum montanum (Wïlld.) Muell. Arg. Asian Pac. J.
Trop. Med. 3: 894-897.
Joint FAO/WHO Expert Committee on Food Additives (JECFA). 2005. SGs.
In: 63rd Meeting of the Joint FAO/WHO Expert Committee on Food Additives.
Jones, P.J.H. and A.A. 2012. Papamandjaris. Lipids: cellular metabolism. In: Erdman J.W., i.A.
Macdonald, S.H. Zeisel, eds. Present Knowledge in Nutrition. 10th ed. Washington, DC:
Wiley-Blackwell; 132-48.
Jones P.J.H. and T. Rideout. 2014. Lipids, sterols, and their metabolites. In: Ross A.C., B.
Caballero, R.J. Cousins, K.L. Tucker, T.R. Ziegler, eds. Modern Nutrition in Health and
Disease. 11th ed. Baltimore, MD: Lippincott Williams & Wilkins.
Kadam, D., R.K. Goyal and M.K. Gupta. 2011. Mathematical modeling of convective thin layer
drying of basil leaves. J. Med. Plants Res. 5: 4721-4730.
Kangralkar, V., A. Shivraj, D. Patil and R.M. Bandivadekar. 2010. Oxidative stress and diabetes:
A review. Int. J. Pharmaceut. Applic. 1: 38-45.
Kesler, S.E. and A.C. Simon. 2015. Mineral resources, economics and the environment.
Cambridge University Press.
Kim, H.I. and Y.H. Ahn. 2004. Role of peroxisome proliferator-activated receptor-γ in the glucose-
sensing apparatus of liver and β-cells. Diabet. 53: S60-S65.
156
Kim, I. S., M. Yang, O.H. Lee and S.N. Kang. 2011. The antioxidant activity and the bioactive
compound content of Stevia rebaudiana water extracts. LWT-Food Sci. Technol. 44: 1328-
1332.
Kobus-Moryson, M., and A. Gramza-Michałowska. 2015. Directions on the use of Stevia Leaves
(Stevia Rebauidana) as an additive in food products. Acta Sci. Pol. Technol. Aliment. 14:
5-13.
Korobko, N.V., A. Ya, V.V. Turko, E.N. Shokun, L.M. Chernyak, O.N. Pokrovskii, B.F.
Smetankina, Kerimzhanova, and U. A. Baltaev. 2007. "GC-MS investigations. II. Lipid
composition of Stevia rebaudiana." Chemi. Nat. Compd. 44: 359-360.
Krasina, I. B. and N.A. Tarasenko. 2016. Features of a Chemical Composition of Dry Leaves of
Stevia vebaudiana. Orient. J. Chem. 32: 1171-1180.
Krishna, G. and S.K. Ranjhan. 1981. Gross Energy of the Herbage, Urine, Milk and Silage. In:
Laboratory Manual of Nutrition Research. Vikas Publishing House, New Dehli, India.
Kulthe, A.A., V.D. Pawar, P.M. Kotecha, U.D. Chavan and V.V. Bansode. 2014. Development of
high protein and low calorie cookies. J. Food Sci. Technol. 51: 153-157.
Kumar, G.P., T. Anand, D. Singsit, F. Khanum and K. Anilakumar. 2013. Evaluation of
antioxidant and anti-fatigue properties of Trigonella foenum-graecum L. in rats subjected
to weight loaded forced swim test. Pharmacogn. J. 5: 66-71.
Kumar, R. and A. Kumar 2015. "Extraction of diterpene glycoside from stevia (Stevia Rebaudiana
Bertoni)." Annals of Horticulture. 8: 185-189.
Kumar, S. and A.K. Pandey. 2013. Chemistry and biological activities of flavonoids: an overview.
The Scien. World J.
Williams, L.D. and G.A. Burdock. 2009. Genotoxicity studies on a high-purity rebaudioside: A
preparation. Food Chem. Toxicol. 47: 1831-1836.
Lailerd, N., V. Saengsirisuwan, J.A. Sloniger, C. Toskulkao and E.J. Henriksen. 2004. Effects of
stevioside on glucose transport activity in insulin-sensitive and insulin-resistant rat skeletal
muscle. Metabolism. 53: 101-107.
157
Lara, E., P. Cortes, V. Briones and M. Perez. 2011. Structural and physical modifications of corn
biscuits during baking process. LWT-Food Sci. Tech. 44: 622-630.
Lawless, H.T. and H. Heymann. 2010. Sensory evaluation of food: principles and practices (Vol.
5999). Springer Science and Business Media. New York, USA. Chapman and Hall press.
pp. 430-475.
Lemus-Mondaca, R., A. Vega-Galvez, L. Zura-Bravo and K. Ah-Hen. 2012. Stevia rebaudiana
Bertoni, source of a high-potency natural sweetener: A comprehensive review on the
biochemical, nutritional and functional aspects. Food Chem. 132: 1121-1132.
Lemus-Mondaca, R., K. Ah-Hen, A. Vega-Galvez, C. Honores and N. O. Moraga. 2016. Stevia
rebaudiana leaves: effect of drying process temperature on bioactive components,
antioxidant capacity and natural sweeteners. Plant food Hum. Nutr. 71: 49-56.
Li, Z., Ma, Z., T.J. Van Der Kuijp, Z. Yuan and L. Huang. 2014. A review of soil heavy metal
pollution from mines in China: pollution and health risk assessment. Sci. Total Environ.
468: 843-853.
Lin, S. D., C. C. Lee, J. Mau, L. Lin and S.Y. Chiou. 2010. Effect of erythritol on quality
characteristics of reduced‐calorie danish cookies. J. Food Qual. 33: 14-26.
Liu, J., C. P. Ong and S. F. Y. Li. 1997. Subcritical fluid extraction of Stevia sweeteners from
Stevia rebaudiana. J. Chromatogr. Sci. 35: 446-450.
Liu, J., J. W. Li and J. Tang. 2010. Ultrasonically assisted extraction of total carbohydrates from
Stevia rebaudiana Bertoni and identification of extracts. Food Biop. Proces. 88: 215-221.
Logue, C., S. J. Peters, A.M. Gallagher and H. Verhagen. 2015. Perspectives on Low Calorie
Intense Sweeteners with a Focus on Aspartame and Stevia. Eur. J. Food Res. Rev. 5: 104.
Lorenzo, C., J. Serrano-Díaz, Plaza, M., Quintanilla and G.L. Alonso. 2014. Fast methodology of
analysing major SGs from Stevia rebaudiana leaves. Food Chem. 157: 518-523.
Lu, Y., S.J. Suh, C.H. Kwak, K.M. Kwon, C.S. Seo, Y. Li, Y. Jin, X. Li, S.L. Hwang, O. Chang,
Y.C. Park, Y.G. Park, S.S. Son, J.K. Kim and C.H. Chang. 2012. Saucerneol F, a new
lignan, inhibits iNOS expression via MAPKs, NF-jB and AP-1 inactivation in LPS-induced
RAW 264.7 cells. Int. Immumopharmacol. 12: 175-181.
158
Madan, S., S. Ahmad, G.N. Singh, K. Kohli, Y. Kumar, R. Singh and M. Garg. 2010. Stevia
rebaudiana (Bert.) Bertoni—a review. Indian J. Nat. Prod. Resour. 183: 267–286.
Madianov, I., M. Balabolkin, D. Markov and T. Markova. 1999. Main causes of hyperuricemia in
diabetes mellitus. Terapevticheskii Arkhiv. 72: 55-58.
Mandal, B. and S. Madan. 2013. Preliminary phytochemical screening and evaluation of free
radical scavenging activity of Stevia rebaudiana Bertoni from different geographical
sources. J. Pharmacog. Phytochem. 2: 14-19.
Maritim, A. C., Sanders and A.Watkins. 2003. Diabetes, oxidative stress, and antioxidants: a
review. J. Biochem. Mol. Toxicol. 17: 24-38.
Matsukubo, T. and I. Takazoe. 2010. Sucrose substitutes and their role in caries prevention, Int.
Dent. J. 56: 119-130.
Megeji, N.W., J.K. Kumar, V. Singh, V.K. Kaul and P.S. 2005. Introducing Stevia rebaudiana, a
natural zero-calorie sweetener. Curr. Sci. 88: 801-804.
Meilgaard, M., G.V. Civille and B.T. Carr. 2007. Overall difference tests: does a sensory
difference exist between samples. Sen. Eval. Tech. 4: 63-104.
Miller, R.A. and R.C. Hoseney. 1997. Factors in hard wheat flour responsible for reduced cookie
spread. Cereal Chem. 74: 330-336.
Minne, V. J., F. Compernolle, S. Toppet and J.M. Geuns. 2004. Steviol quantification at the
picomole level by high-performance liquid chromatography. J. Agri. Food Chem. 2445-
2449.
Mizushina, Y., T. Akihisa, M. Ukiya, Y. Hamasaki , N.C. Murakami, I. Kuriyama, T. Takeuchi,
F. Sugawara and H. Yoshida. 2005. Structural analysis of isosteviol and related compounds
as DNA polymerase and DNA topoisomerase inhibitors. Life Sci. 77: 2127-2140.
Moguel-Ordonez, Y. B., D.L. Cabrera-Amaro, M.R. Segura-Campos and J.C. Ruiz-Ruiz. 2015.
Studies on drying characteristic, nutritional composition, and antioxidant properties of
Stevia rebaudiana (Bertoni) leaves. Int. Agrophys. 29: 323-331.
Molina-Calle, M., F. Priego-Capote and M.L. de Castro. 2017. Characterization of Stevia leaves
by LC–QTOF MS/MS analysis of polar and non-polar extracts. Food Chem. 219: 329-338.
159
Montgomery, D.C. 2008. Design and Analysis of Experiments. 7th Ed. John Wiley and Sons. Inc.
Hoboken, NJ, USA. pp. 162-264.
Muanda, F. N., R. Soulimani, B. Diop and A. Dicko. 2011. Study on chemical composition and
biological activities of essential oil and extracts from Stevia rebaudiana Bertoni leaves.
LWT-Food Sci. Tech. 44: 1865-1872.
Murray, R., D. Granner, P. Mayes and W. Victor. 2003. Rodwell. Harper’s Illustrated
Biochemistry, McGraw-Hill.
Nikiforov, A.I. and A.K. Eapen. 2008. A 90-day oral (dietary) toxicity study of rebaudioside A in
Sprague-Dawley rats. Int. J. Toxicol. 27: 65-80.
NNS (National Nutrition Survey of Pakistan). 2011. Planning and development Division, Planning
Commission, Pakistan.
Nunes, A.P., S.C. Ferreira-Machado, R.M. Nunes, F.J. Dantas, J.C. De Mattos and A. Caldeira.
2007. Analysis of genotoxic potentiality of stevioside by comet assay. Food Chem.
Toxicol. 45: 662-666.
Okpala, L. C. and V.A. Chinyelu. 2011. Physicochemical, Nutritional and Organoleptic evaluation
of cookies from pigeon pea (Cajanus cajan) and cocoyam (Xanthosoma Sp) flour blends.
Afr. J. Food Agric. Nutr. Dev. 11: 6.
Oroian, M. and I. Escriche. 2015. Antioxidants: characterization, natural sources, extraction and
analysis. Food Res. Int. 74: 10-36.
Pasha, I., F.M. Anjum and M.S. Butt. 2009. Genotypic variation of spring wheats for solvent
retention capacities in relation to end-use quality. LWT-Food Sci. Technol. 42:418-
423.
Pasha, I., S. Butt, F.M. Anjum and N. Shehzadi. 2002. Effect of dietetic sweeteners on the quality
of cookies. Int. J. Agric. Biol. 2: 245-248.
Periche, A., G. Koutsidis and I. Escriche. 2014. Composition of antioxidants and amino acids in
Stevia leaf infusions. Plant Food Hum. Nutr. 69: 1-7.
Periche, A., M.L. Castello, A. Heredia and I. Escriche. 2015. Influence of drying method on SGs
and antioxidants in Stevia rebaudiana leaves. Food Chem. 172: 1-6.
160
Petterino, C. and A. Argentino-Storino. 2006. Clinical chemistry and haematology historical data
in control Sprague-Dawley rats from pre-clinical toxicity studies. Experi. Toxicol. Path.
57: 213-219.
Piga, A., P. Catzeddu, S. Farris, T. Roggio, A. Sanguinetti and E. Scano. 2005. Texture evolution
of “Amaretti” biscuits during storage. Eur. Food Res. Technol. 221: 387-391.
Pol, J., E.V. Ostra, P. Karasek, M. Roth, K. Benesova, P. Kotlarikova and J. Caslavsky. 2007.
Comparison of two different solvents employed for pressurised fluid extraction of
stevioside from Stevia rebaudiana: methanol versus water. Anal. Bioanal. Chem. 388:
1847-1857.
Prakash, I., G.E. DuBois, J.F. Clos, K.L. Wilkens and L.E. Fosdick. 2008. Development of
rebiana, a natural, non-caloric sweetener. Food Chem. Toxicol. 46: 75-82.
Puri, M., D. Sharma and A.K. Tiwari. 2011. Downstream processing of stevioside and its potential
applications. Biotech. Advanc. 29: 781-791.
Purkayastha, S., A. Markosyan, I. Prakash, S. Bhusari, G. Pugh, B. Lynch and A. Roberts. 2016.
SGs in purified stevia leaf extract sharing the same metabolic fate. Regul. Toxicol.
Pharmaco. 77: 125-133.
Rajasekaran, T., A. Ramakrishna, K. Udaya Sanka, P. Giridhar and G.A. Ravishankar. 2008.
Analysis of predominant steviosides in Stevia rebaudiana Bertoni by liquid
chromatography/electrospray ionization-mass spectrometry. Food Biotech. 22: 179-188.
Ranjan, R., J. Jaiswal and J. Jena. 2011. Stevia as a natural sweetener. Int. J. Res. Pharm.Chem. 1:
1199-1202.
Renwick, A.G. and S.M. Tarka. 2008. Microbial hydrolysis of SGs. Food Chem. Toxicol. 46: 70-
S74.
Rezeng, C., C. Limao, L. Bin and S. Your. 2014. Amino acid, fatty acid, and mineral compositions
of fruit, stem, leaf and root of Rubusamabilis from the Qinghai-Tibetan Plateau. J. Food
Comp. Anal. 33: 26-31.
161
Ribes, G., Y. Sauvaire, C. Da Costa, J. Baccou and M. Loubatieres-Mariani. 1986. Antidiabetic
effects of subtractions from fenugreek seeds in diabetic dogs. Exp. Biol. Med. 182:159-
166.
Rifai, N., P.S. Bachorik and J.J. Albers. 1999. Lipids, lipoproteins and apolipoproteins. Tietz
textbook of clinical chemistry. 3rd Ed. Philadelphia: WB Saunders Company. pp. 809-861.
Rodriguez‐Garcia, J., A. Puig, A. Salvador and I. Hernando. 2012. Optimization of a sponge cake
formulation with inulin as fat replacer: structure, physicochemical, and sensory properties.
J. Food Sci. 77: 189-197.
Roohani, N., R. Hurrell, R. Kelishadi and R. Schulin. 2013. Zinc and its importance for human
health: An integrative review. J. Res. Med. Sci. 18: 144-157.
Ruiz-Ruiz, J.C., Y.B. Moguel-Ordonez, A.J. Matus-Basto and M.R. 2015. Antidiabetic and
antioxidant activity of Stevia rebaudiana extracts (Var. Morita) and their incorporation into
a potential functional bread. J. Food Sci.Tech. 52: 7894-7903.
Weston, S. 2011. PureCircle Welcomes Codex Adoption of Stevia. (Foodbev Media – Beverage)
Available at: http://www.foodbev.com/news/purecircle-welcomes-codex-adoption-of-st/
Saeed, F. 2012. Biochemical characterization of non-starch polysaccharides in relation to
end-use quality of spring wheats. Ph.D. Thesis, University of Agriculture, Faisalabad.
Segura-Campos, M., E. Barbosa-Martín, A. Matus-Basto, D. Cabrera-Amaro, M, Murguía-
Olmedo, M, Y. Moguel-Ordoñez and D. Betancur-Ancona. 2014. Comparison of chemical
and functional properties of Stevia rebaudiana (Bertoni) varieties cultivated in Mexican
Southeast. Am. J. Plant Sci. 5: 286.
Sehar, I., A. Kaul, S. Bani, H.C. Pal and A. Saxena. 2008. Immune up regulatory response of a
non-caloric natural sweetener, stevioside. Chem. Bio Int.173: 115-121.
Shakoori, A.R., A.J. Van Wijnen, R. Bortell, T.A. Owen, J.L. Stein, J.B. Lian and G.S. Stein. 1994.
Variations in vitamin D receptor transcription factor complexes associated with the
osteocalcin gene vitamin D responsive element in osteoblasts and osteosarcoma cells. J.
Cell Biochem. 55: 218-229.
162
Sharma, N., D.P. Gauchan, A. Dhakal, A. Luitel, S. Shakya and R. Shakya. 2015. Establishment
of Regenerative Callus, Cell Suspension System and Molecular Characterization of Stevia
Rebaudiana Bertoni for the Production of Stevioside in In Vitro. IJRASET. 3:133-144.
Sharma, R., R. Yadav and E. Manivannan. 2012. Study of effect of Stevia rebaudiana bertoni on
oxidative stress in type-2 diabetic rat models. Biomedi. Aging Patho. 2: 126-131.
Shiozaki, K., A. Fujii, T. Nakano, T. Yamaguchi and M. Sato. 2006. Inhibitory effects of hot water
extract of the Stevia stem on the contractile response of the smooth muscle of the guinea
pig ileum. Biosci. Biotech. Biochem. 70: 489-494.
Shivanna, N., M. Naika, F. Khanum and V.K. Kaul. 2013. Antioxidant, anti-diabetic and renal
protective properties of Stevia rebaudiana. J. Diabet. Complic. 27: 103-113.
Shukla, S., A. Mehta, J. John, S. Singh, P. Mehta and S.P. Vyas. 2009. Antioxidant activity and
total phenolic content of ethanolic extract of Caesalpinia bonducella seeds. Food Chem.
Toxicol. 47: 1848-1851.
Shukla, S., A. Mehta, P. Mehta and V.K. Bajpai. 2012. Antioxidant ability and total phenolic
content of aqueous leaf extract of Stevia rebaudiana Bert. Experim. Toxicol. Patho. 64:
807-811.
Sic Zlabur, J., S. Voca, N. Dobricevic, M. Brncic, F. Dujmic and S. Rimac Brncic. 2015.
Optimization of ultrasound assisted extraction of functional ingredients from Stevia
rebaudiana Bertoni leaves. Int. Agrophys. 29: 231-237.
Siddique, A., S.M. Rahman and M.A. Hossain. 2012. Chemical composition of essential oil by
different extraction methods and fatty acid analysis of the leaves of Stevia Rebaudiana
Bertoni. Arabi. J. Chem. S1185-S1189.
Siddique, A.B., S.M.M. Rahman, M.A. Hossain and M.A. Rashid. 2014. Phytochemical screening
and comparative antimicrobial potential of different extracts of Stevia rebaudiana Bertoni
leaves. Asian Paci. J. Tropic. Dis. 4: 275-280.
Singh, S. and V. Garg. 2014. Antidiabetic, antidyslipidymic and anti-oxidative potential of
methanolic root extract of Stevia rebaudiana (Bertoni) on alloxan induced diabetic. J.
Pharm. Sci. 3: 1859-1872.
163
Singh, S., V. Garg, D. Yadav, M.N. Beg and N. Sharma. 2012. In vitro antioxidative and
antibacterial activities of various parts of Stevia rebaudiana (Bertoni). Int. J. Pharma.
Pharmaceut. Sci. 4: 468-473.
Siow, L. and Y. Hui. 2013. Comparison on the antioxidant properties of fresh and convection oven-
dried guava (Psidium guajava L.). Int. Food Res. J. 20: 639-694.
Sramkova, Z., F. Kraic, J. Jurovata, E. Gregova and E. Sturdik. 2009. Chemometric analysis of
nutritional and bread-making quality attributes of wheat cultivars. Acta Chimica Slovaca.
2: 139-146.
Stalikas, C.D. 2007. Extraction, separation, and detection methods for phenolic acids and
flavonoids. J. Separ. Sci. 30: 3268-3295.
Steel, R., J. Torrie and D. Dickey. 1997. Principles and procedures of statistics: a biometrical
approach., 3rd edn (McGraw-Hill: New York).
Tadhani, M. and R. Subhash. 2006a. Preliminary studies on Stevia rebaudiana leaves: proximal
composition, mineral analysis and phytochemical screening. J. Med. Sci. 6: 321-326.
Tadhani, M., V. Patel and R. Subhash. 2007. In vitro antioxidant activities of Stevia rebaudiana
leaves and callus. J. Food Comp. Anal. 20: 323-329.
Tadhani, M.B. and R. Subhash. 2006b. In vitro antimicrobial activity of Stevia rebaudiana Bertoni
leaves. Tropic. J. Pharma. Res. 5: 557-560.
Takahashi, K., Y. Sun, I. Yanagiuchi, T. Hosokawa, T. Saito, M. Komori, T. Okino and M.
Kurasaki. 2012. Stevioside enhances apoptosis induced by serum deprivation in PC12
cells. Toxicol. Mechan. Meth. 22: 243-249.
Takasaki, M., T. Konoshima, M. Kozuka, H. Tokuda, J. Takayasu, H. Nishino, M. Miyakoshi, K.
Mizutani and K.H. Lee. 2009. Cancer preventive agents. Part 8: Chemopreventive effects
of stevioside and related compounds. Bioorgan. Medic. Chem.. 17: 600-605.
Tavarini, S. and L.G. Angelini. 2013. Stevia rebaudiana Bertoni as a source of bioactive
compounds: the effect of harvest time, experimental site and crop age on steviol glycoside
content and antioxidant properties. J. Sci. Food Agric. 93: 2121-2129.
164
Ulbricht, C., R. Isaac, T. Milkin, A. E. Poole, E. Rusie, M.G.J.Serrano, W. Weissner, C. R.
Windsor, and J. Woods. 2010. An evidence-based systematic review of stevia by the
Natural Standard Research Collaboration. Cardiovascular & Hematological Agents in
Medicinal Chemistry (Formerly Current Medicinal Chemistry-Cardiovascular &
Hematological Agents). 8:113-127.
Urban, J.D., M.C. Carakostas and S.L. Taylor. 2015. Steviol glycoside safety: Are highly purified
steviol glycoside sweeteners food allergens? Food Chem. Toxicol. 75: 71-78.
US-FDA (United States Food and Drug Administration). 2009. Center for Food Safety & Applied
Nutrition (CFSAN). Agency Response Letter GRAS Notice No. GRN 000278.
[Rebaudioside a Purified from the Leaves of Stevia rebaudiana (Bertoni) Bertoni]
Velderrain-Rodriguez, G.R., H. Palafox-Carlos, A. Wall-Medrano, J. F. Ayala-Zavala, C.O. Chen
and M. Robles-Sánchez. 2014. Phenolic compounds: Their journey after intake. Food
Func. 5: 189-197.
Virendra, V. and P. Kalpagam. 2008. Assessment of Stevia (Stevia rebaudiana)-natural sweetener:
A review. J. Food Sci. Tech. 45: 467-473.
WHO. 2008. World Health Organization (WHO), Geneva, Switzerland. WHO Technical Report
Series 928, pp. 34–39.
Wolf, B. and S. Weisbrode. 2003. Safety evaluation of an extract from Salacia oblonga. Food
Chem. Toxicol. 41: 867-874.
Wolwer-Rieck, U. 2012. The leaves of Stevia rebaudiana (Bertoni), their constituents and the
analyses thereof: A review. J. Agric. Food Chem. 60: 886-895.
Wu, C.S., K. Venkataraman, B.J. Nettles, F. Jaksch, D.L. Rodenburg, K.M. Alves, M.A. Ibrahim
and J.D. McChesney. 2012. New glycosides from Stevia rebaudiana. Planta Med. 78: P95.
Yadav, S.K. and P. Guleria. 2012. SGs from Stevia: Biosynthesis pathway review and their
application in foods and medicine. Critical Rev. Food Sci. Nutr. 52: 988-998.
Yoda, S.K., M.O. Marques, A.J. Petenate and M.A.A. Meireles. 2003. Supercritical fluid
extraction from Stevia rebaudiana Bertoni using CO2 and CO2 + water: Extraction kinetics
and identification of extracted components. J. Food Eng. 57: 125-134.
165
Zahn, S., A. Forker, L. Krugel and H . Rohm. 2013. Combined use of rebaudioside A and fibres
for partial sucrose replacement in muffins. LWT-Food Sci. Tech. 50: 695-701.
Zhang, M., X. Lv, J. Li, Z. Xu and L. Chen. 2009. The characterization of highfat diet and multiple
low-dose streptozotocin induced type 2 diabetes rat model. Int. J. Experi. Diab. Res. 7: 45-
53.
Zia, M.H., M. Ahmad, J.N. Mehjabeen, S. Ahmad, M. Qayum and I.K. Marwat. 2011.
Antimicrobial screening of selected flora of Pakistan. Arc. Biol. Sci. 63: 691-695.
Zygler, A., A. Wasik and J. Namiesnik. 2009. Analytical methodologies for determination of
artificial sweeteners in foodstuffs. Trends Ana. Chem. 28: 1082-1102.