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PERPUSTAKAAN UMP
I llllIffi Iffi Ill Iffi IU Villil llVIllllII 0000073686
ANTIOXIDANT ACTIVITY OF DIFFERENT FRACTIONS FROM TINOSPORA C'RISPA STEMS EXTRACTS
VIDHYAA AlP PAROOINDRAN
Report submitted in partial fulfillment of the requirements for the award of the degree of Bachelor of Applied Science (Honor) Industrial Chemistry
Faculty of Industrial Sciences & Technology UNIVERSITI MALAYSIA PAl-lANG
2012
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ABSTRACT
"Patawali (Malay) or Tinospora crispa is a plant that has many traditional uses for skin treatment. Tinospora crispa is a woody and glabrous plant in the family MenispermaCeae. In cosmetics, Tinospora crispa can be used externally for its strong antioxidant activity and' free radical scavenging properties. Therefore, the objective of the study was to fractionated all the five fractions from the Tinospora crispa stems extract, to test antioxidant activity of the extracted fractions using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay and finally to evaluate total content of phenolics in the fractions extracted from Tinospora crispa stems. The methodology of the study involves defatting of the plant material, separation and extraction of the moderately polar extract (organic extract) along with the aqueous extract. The fractions are then tested using the DPPH assay for free radical scavenging property to prove its antioxidant activity. The antioxidant activity of the plant fractions are compared with positive control ascorbic acid and butylated hydroxytoulene (BUT). On the other hand, total content of phenolics in fractions extract from the stem was determined using Folin-Ciocalteu method. Moreover, thin layer chromatography method was used to identify phenolics in fraction F3 and fraction F4 while alkaloids were also identified in fraction F4. The research carried out proved that the fractions extracted had significant antioxidant activity especially in fraction F3 and F4, 88.40 ± 0.37 % and 64.91 ± 0.58 % of inhibition repectively which was realatively comparable with the positive control ascorbic acid and butylated hydroxytoluene (BUT). Besides, the total phenolic content in F3 was reported the highest followed by F4, 73.54 ± 2.22 .tg GAE per mg of sample and 62.10 ± 1.42 tg GAB per mg of sample respectively. As an overall conclusion, the research clearly supports that the Tinospora crispa stems extract have significant antioxidant activity due to the presence of phenolics in the fractions that can be further studied. The research too emphasizes the extract from the plant stems to be a good source of herbal medication that can be produced in industries to compliment the society need for curing diseases such as cancer and rapid aging.
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I
"Patawali atau Tinospora crispa merupakan tumbuhan yang mempunyai banyak kegunaafl tradisional untuk rawatan kulit. Tinospora crispa berkayu dan glabrous dalam keluarga Menispermaceae. Dalam kosmetik, Tinospora crispa boleh digunakan secara luaran untuk aktiviti antioksidatifhya yang kuat kerana sifatnya yang boleh memerangkap radikal bebas. Oleh itu, objektif kajian adalah untuk mengasingkan dan mengumpul kesemua lima pecahan ekstrak daripada tumbuhan ini. Kemudian untuk menguji aktiviti ant ioksidatif pecahan yang diekstrak menggunakan "2,2-diphenyl- 1 -picrylhydrazyl" (DPPH) dan akhirnya untuk menilai kandungan jumlah fenolik yang diekstrak daripada batang Tinospora crispa di dalam setiap ekstrak. Metodologi kajian mi melibatkan pengasingan wax daripada batang tumbuhan dan pengeluaran ekstrak sederhana organik bersama-sama dengan ekstrak akueus. Pecahan kemudiannya diuji dengan menggunakan DPPH membuktikan aktiviti antioksidatiffiya serta membandingkannya dengan asid askorbik kawalan positif dan "butylated hydroxytoulene" (BHT). Sebaliknya, kandungan jumlah phenolics di dalam pecahan ekstrak daripada batang ditentukan dengan menggunakan kaedah "Folin-Ciocalteu". Selain itu, kaedah kromatografi digunakan untuk mengenal pasti phenolics dalam pecahan F3 dan F4 manakala alkaloid yang juga dikenal pasti dalam F4 pecahan. Penyelidikan yang dijalankan membuktikan bahawa pecahan yang diekstrak mempunyai aktiviti antioksidan yang ketara terutama dalam pecahan F3 dan F4, 88.40 ± 0.37 % dan 64.91 ± 0.58 % perencatan masing-masing yang setanding dengan kawalan positif asid askorbik dan "butylated hydroxytoluene" (BHT). Selain itu, jumlah kandungan fenolik dalam F3 dilaporkan tertinggi diikuti oleh F4 sebanyak 73.54 ± 2.22 j.tg GAE/ mg sampel dan 62.10 ± 1.42 .ig GAE/ mg sampel masing-masing. Kesimpulannya, penyelidikan mi dengan jelas menyokong bahawa ekstrak batang Tinospora crispa mempunyai aktiviti antioksidan yang ketara disebabkan oleh kandungan fenolik di dalamnya. Penyelidikan mi juga menekankan ekstrak daripada batang tumbuhan mi boleh menjadi sumber yang baik untul membuat ubat-ubatan herba di dalam indutri yang akan selaras dengan keperluan masyarakat bagi mengubati penyakit-penyakit seperti kanser dan penuaan yang cepat.
TABLE OF CONTENTS
Page
SUPERVISOR'S DECLARATION
STUDENT'S DECLARATION
ACKNOWLEDGEMENT iv
ABSTRACT
ABSTRAK
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES xi
LIST OF SYMBOLS
LIST OF ABBREVATION xiv
LIST OF APPENDICES xv
CHAPTER 1 INTRODUCTION
1.1 Background of the study I
1.2 Problem statement 3
1.3 Objective 3
1.4 Scope of research 4
1.5 Significance of the study 4
CHAPTER 2 LITERATURE REVIEW
2.1 Introduction 5
2.2 Tinospora crispa plant material 5
2.3 Antioxidants 7 2.4 Types of antioxidants 8
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viii
2.4.1 Phenolic 8 2.4.2 Ascorbic acid 15 2.4.3 Butylated hydroxytoulene 16
2.5 2, 2-Dipheñyl-1-picrylhydrazyl (DPPH) 17
CIIMTER 3 MATERIALS AND METHODS
3.1 Introduction 19
3.2 Materials 19
3.3 Apparatus 20
3.4 Instruments 20
3.5 Methodology
3.5.1 Extraction of Tinospora crispa stem extracts 20 3.5.2 2,2-Diphenyl-1-picrylhydrazyl DPPH Assay 23 3.5.3 Folin-Ciocalteu reagent method 25 3.5.4 Identification of alkaloid compounds using
Thin Layer Chromatography (TLC) 27 3.5.5 Identification of phenolic compounds using
Thin Layer Chromatography (TLC) 28 3.5.6 Statistical analysis 29
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Introduction 31
4.2 Yield of dried fractions 31
4.3 Antioxidant activity of Tinospora Crispa stems fractions,
positive controls BHT and ascorbic acid 32
4.4 Total content of phenolics in tinospora crispa stems fractions 46
4.5 Pre-Identification of alkaloid compounds 48
4.6 Pre-Identification of phenolic compounds 49
ix
CHAPTER 5 CONCLUSION AND RECOMENDATIONS
5.1 Introduction
52
5.2 Conclusion
52
5.3 Recommendations
53
REFERENCES
54
APPENDICES
61
LIST OF TABLES
Table No. Page
4.1 Table of yield of fractions extracted 31
4.2 Table of physical properties of the fractions extracted 32
4.3 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 0 min 33
4.4 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 15 min 33
4.5 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 30 min 34
4.6 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 45 min 34
4.7 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 1 h 35
4.8 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 1 h 15 min 35
4.9 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 1 h 30 min 36
4.10 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 1 h 45 min 36
4.11 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 2 h 37
4.12 Concentration and absorbance of gallic acid equivalent 46
4.13 Concentration of fraction Fl, F2, F3, F4 and F5 from gallic acid equivalent standard curve 47
4.14 Total content of phenolic compounds in fraction Fl, F2, F3, F4 and F5
x
LIST OF FIGURES
Figure No. Page
2.1 Tinospora crispa plant material 6
2.2 Schematic biochemical pathway of the synthesis of flavanone, resveratrol, and furanocoumarin. 9
2.3 Basic plant phenolic structures with examples 11
2.4 Ascorbic acid 15
2.5 Butylated hydroxytoluene. 16
2.6 2,2- Diphenyl-1- picryihydrazyl (DPPH) 18
3.1 Flow chart of extraction of fraction 1,2,3,4 and 5 from Tinospora Crispa stems. 22
3.2 Flow chart of DPPH Assay 24
3.3 Flow chart of Folin-Ciocalteu reagent method 26
3.4 Flow Chart of Research Methodology 30
4.1 Graph of antioxidant activity of fraction (Fl, F2, F3, F4 and F5), BHT and ascorbic acid 38
4.2 Proposed reaction mechanism of ascorbic acid with DPPH. (radical) 41
4.3 Proposed reaction mechanism for BHT and DPPH • (radical) 42
4.4 Proposed reaction mechanism of phenolic with DPPH • (radical) 45
4.5 Graph of Gallic Acid Equivalent (GAE) 46
4.5 Identification of alkaloid using TLC Visualizer at Xnlax 254nm and Xax 366nm for fraction F4 48
4.6 Pre-Identification of phenolic using TLC Visualizer at Xmax 2 54n and Xmax366nm for fraction F3. 49
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47 Pre-Identification of phenolic using TLC Visualizer at X.max 254nm and Xmax 366nm for fraction F4. 50
xii
LIST OF SYMBOLS
XIII
Wavelength
Percentage
Uncertainty
Greater than
cm Centimeter
h Hour
Microliter
g Gram
Kg Kilogram
L Liter
min Minutes
mg Milligram
ML Milliliter
tg Microgram
nm Nanometer
Rf Retention factor
LIST OF ABBREVATIONS
BUT Butylated hydroxytoluene
DPPH 2,2-Diphenyl- 1 -picrylhydrazyl
DMSO Dimethyl Sulfoxide
EtOH Ethanol
MeOH Methanol
TLC Thin Layer Chromatography
UV-Vis Ultraviolet-Visible
xiv
LIST OF APPENDICES
Appendix No. Page
A Antioxidant activity of Tinospora crispa 61
Al DPPH Assay absorbance at 0 min 61
Percentage (%) of inhibition at 0 min 61
DPPH Assay absorbance at 15 min 62
A4 Percentage (%) of inhibition at 15 min 62
AS DPPH Assay absorbance at 30 min 63
A6 Percentage (%) of inhibition at 30 min 63
A7 DPPH Assay absorbance at 45 min 64
A8 Percentage (%) of inhibition at 45 min 64
A9 DPPH Assay absorbance at 1 h 65
AlO Percentage (%) of inhibition at 1 h 65
All DPPH Assay absorbance at 1 h 15 min 66
Al2 Percentage (%) of inhibition at 1 h 15 min 66
A13 DPPH Assay absorbance at 1 h 30 min 67
A14 Percentage (%) of inhibition at 1 h 30 nun 67
A15 DPPH Assay absorbance at 1 h 45 min 68
A16 Percentage (%) of inhibition at 1 h 45 min 68
A17 DPPH Assay absorbance at 2 h 69
A18 Percentage (%) of inhibition at 2 h 69
B Total content of phenolics in Tinospora crispa stems fractions 70
xv
xvi
BI Concentration of fraction Fl, F2, F3, F4 and F5 from gallic acid equivalent standard curve 70
B2 Total content of phenolic compounds in fraction Fl, F2, F3, F4 and F5 70
B3 Absorbance of Fl, F2, P3, F4, F5 and control DMSO at 750 nm 70
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Tinospora crispa is a woody and glabrous plant in the family of
Menispermaceae. It is found in primary rainforests or mixed deciduous forests
throughout a large part of Asia and Africa including all parts of Thailand, Malaysia and
Indonesia (Pathak et al., 1995).
Free radicals contribute to more than one hundred disorders in humans including
atherosclerosis, arthritis, ischemia and repercussion injury of many tissues, a central
nervous system injury, gastritis and cancer (Pourmorad et al., 2006). Due to
environmental pollutants, radiation, chemicals, toxins, deep fries and spicy foods as well
as physical stress, free radicals cause depletion of the immune system antioxidants, the
change in gene expression and induce abnormal proteins. The oxidation process is one
Of the most important routs for producing free radicals in food, drugs, and even living
systems (Dullard and German, 2000 and Turkoglu et al., 2007).
Antioxidant means "against oxidation". An antioxidant is any substance that
retards or prevents deterioration, damage or destruction by oxidation. In definition of the
term by Halliwell and Gutteridge (1999) stated that an antioxidant is 'any substance that,
when present at low concentrations compared with that of an oxidizable substrate, significantly delays or inhibits oxidation of that substrate'. On the other hand, a free
radical is a compound with one or more unpaired electrons in its outer orbital. Such
2
unpaired electron(s) make these species very unstable and therefore quite reactive with
other molecules due to the presence of unpaired electron(s) and they try to pair their
electron(s) and generate a more stable compound. Antioxidants can be classified taking
into account their mechanism of action, although there are other possible classifications.
Bearing in mind the mechanism of action, they can be divided into primary antioxidants,
synergistic and secondary antioxidants (Rajalakhmi et al., 1996). Some substances
considered as antioxidants are ascorbate, tocopherols, some enzymes, carotenoids and
bioactive plant phenols. The health benefits of fruits and vegetables are largely due to
the antioxidant vitamin content supported by a large number of phytochemicals, some
with greater antioxidant properties. Sources of tocopherols, carotenoids and ascorbic
acid are well recognized antioxidants.
Antioxidants are important species which possess the ability of protecting
organisms from damage caused by free radical-induced oxidative stress. The antioxidant
activity of phenolics is mainly due to their redox properties, which allow them to act as
reducing agents, hydrogen donors, singlet oxygen quenchers and metal chelators
(Canadanovic-Brunet et al., 2005). A number of synthetic antioxidants such as butylated
hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been extensively
added to foodstuffs, although their use has begun to be questioned because of their
toxicity (Ito et al., 1985), so there is considerable interest in preventive medicine and in
the food industry in the development of natural antioxidants obtained from botanical
sources, especially herbal plants (Djilas et al., 2003). Medicinal plants have been used
for centuries as remedies for human diseases because they contain components of
therapeutic value. Moreover, the increasing use of plant extracts in the food, cosmetic and pharmaceutical industries suggests that, in order to find active compounds, a
systematic study of medicinal plants is very important (Nostro et al., 2000).
22-Diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical which has an
Unpaired valence electron at one atom of nitrogen bridge (Eklund et al., 2005). DPPH is
one of the free radicals widely used for testing preliminary radical scavenging activity of a compound or plant extract. It is stable and it contains an odd electron in its structure
that is usually utilized for the detection of the radical scavenging activity in chemical
analysis.
1.2 PROBLEM STATEMENT
Antioxidant in general is important for the anti-ageing study. Besides, Tinospora
Crispa is reported to contain different phenolic compounds with probable free radical
scavenging property (Amom et al., 2009). The antioxidant activities may vary depending
on the structural factors, such as number of phenolic hydroxyl or methoxyl group,
flavone hydroxyl, keto groups, free carboxylic groups and other structural features.
Thus, this study is simply based on further study of on Tinospora Crispa fractions
extracted to know their variation in antioxidant properties and their total content of
phenolics. This will be done because the role of free radicals in many disease conditions
has been well established (Pathak et al., 1995; Quisumbing et al., 1978 and Rahman et
al., 1999). Several biochemical reactions in our human body can damage crucial bio-
molecules due to presence of free radical and increase the rate of ageing in human while
cause severe health risks. Hence, this study is done to extract different fractions from
Tinospora crispa stems and then to determine the antioxidant activity of the different
fractions extracted with other positive controls such as ascorbic acid and butylated
hydroxytoluene using DPPH Assay. Finally, the total content for phenolics in the
respective fractions extracted from Tinospora crispa stems is determined.
1.3 OBJECTIVES
i. To extract different fractions from Tinospora crispa stems extracts.
ii. To determine antioxidant activity of the different fractions extracted from the
Tinospora crispa stems using DPPH Assay.
lii. To determine total content of phenolics in the respective fractions extracted from
Tinospora crispa stems.
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1.4 SCOPE OF RESEARCH
As a way to accomplish the objective of this study, the scope of this research
focuses on extraction of different fractions from Tinospora CriSpa stems and the
comparison of the antioxidant activity of the different fractions extracted with other
established antioxidants such as ascorbic acid and butylated hydroxytoluene (BHT)
using DPPH Assay. Besides, it also focuses on determination of the total content of
phenolics in the respective fractions extracted from Tinospora crispa stems. Finally it
involves identification of the phenolics and alkaloids present in some selected fractions
of extract.
1.4.1 SIGNIFICANCE OF THE STUDY
The extracted fractions from Tinospora crispa stems are believed to have anti-
oxidative properties which can help in anti-ageing of cells. Ageing of cells can cause
severe health risks to humans. Therefore, through this test of free radical scavenger
using 2,2-diphenyl-1-picrylhydrazyl DPPH of the fractions extracted from Tinospora
Crispa, it is trusted to help reduce ageing of cells that may eventually decrease health
risks. If this study proves that the fractions extracted from Tinospora Crispa has anti-
oxidative properties, it may be of great use to the medicinal industry and help treat
various deceases such as cancer, atherosclerosis, senility, aging, and many more. Thus,
the fractions are further studied to determine the responsible antioxidants which can take
Part in maintenance of good health as do vitamins, minerals and enzymes. Antioxidants
can be added to foodstuffs to prevent free radical damage as they scavenge the free
radical (Botsoglou et al., 2002 and Shetty, 1997). Besides, the ready availability of the
plant source widely distributed in Asia is a major factor that influences the plant
selection in this study. Other than that, the plant source is also cheap and is viable for industrial applications.
CHAPTER 2
LITERATURE REVIEW
2.1 INTRODUCTION
In this chapter, the detailed facts on the plant material Tinospora crispa,
antioxidants, the various type of antioxidants, and 2,2-diphenyl- 1 -picryihydrazyl
(DPPH) reagent functionality were discussed.
2.2 TINOSPORA CRISPA PLANT MATERIAL
Tinospora crispa as in Figure 2.1 is an indigenous climber plant that commonly
grows wild in Asean countries including Malaysia. Known by various local names like
'akar patawali' and 'akar seruntum' (Noor and Ashcroft, 1989). Most species of the
genus Tinospora are among the most widely employed medicinal plants throughout a
large part of Asia and Africa (Pathak et al., 1995). In general folklore, the stem
decoction is considered antipyretic, useful as an antimalarial and a wash for skin ulcers.
Traditionally an infusion is used to treat fever due to malaria and also in cases of
jaundice and for use against intestinal worms. The antimalarial effect was confirmed in a
study (Ra1man et al., 1999).
An infusion of the stem is drunk as a vermiflige while a decoction of the stem is
used for washing sore eyes and syphilitic sores. Its stem has been used by traditional
folklore for various therapeutic purposes such as treatment for diabetes, hypertension, stimulation of appetite and protection from mosquito bites. Based on research done it
was also used as hypoglycemic drug an antipyretic agent in both man and domestic
animals (KongSaktrak00I et al., 1994; Noor et al., 1989 and Pathak et al., 1995). This
bitter tasting plant locally known as Makabuhai in Philippines is used for the treatment
of stomach troubles,, ulcers and fevers, as a tonic and a febrifuge for malaria and
smallpox, as a vulnerary for itches and wounds, and many other purposes (Quisumbing,
1978).
Figure 2.1: Tinospora crispa plant material
Source: Noor et al. (1989)
2.3 ANTIOXIDANTS
Antioxidant compounds play an important role as a health-protecting factor.
Antioxidants are substance that significantly prevents or delays the oxidation of other
molecules by inhibiting the initiation or propagation of oxidizing chain reactions
(HalliWell and Gutteridge, 1990). Oxygen-centered free radicals and other reactive
oxygen species (ROS) have been associated with the beginning of many diseases and
degenerative processes in ageing (Halliwell, 1994). Since oxidative stress is common in
chronic degenerative diseases, dietary antioxidants in plant sources may provide a
beneficial effect. Antioxidants are known to protect cells against the damaging effects of
reactive oxygen species (ROS) such as singlet oxygen, superoxide and hydroxyl
radicals, among others (Halvorsen et al., 2002 and Wu et al., 2004).
Almost all organisms are well protected against free radical damage by
oxidative enzymes such as superoxide dismutase and catalase or chemical compounds
such as a-tocopherol, vitamin C (ascorbic acid), carotenoids, polyphenol compounds and
glutathione (Betancur-Ancona et al., 2004). However, these systems are frequently
insufficient to totally prevent the damage, especially under the conditions of severe
oxidative stress, resulting in diseases and accelerated ageing (Ames et al., 1993). The
balance between antioxidation and oxidation is believed to be a critical concept for
maintaining a healthy biological system. Hyper physiological burden of free radicals
causes imbalance in homeostatic phenomena (mechanisms) between oxidants and
antioxidants in the body. This imbalance leads to oxidative stress involved in aging and
various human diseases like atherosclerosis, stroke, diabetes, cancer and
neurodegeneratjve diseases such as Alzheimer's disease and Parkinsonism (Halliwell,
1994).
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2.4 TYPES OF ANTIOXIDANTS.
2.4.1 Phenolics
Phenolic compounds are secondary metabolites which are synthesized in
plants. They possess biological properties such as antioxidant, anti-apoptotic, anti-aging,
anti-carcinogenic, anti-inflammatory, anti-atherosclerotic, cardiovascular protection,
improvement of the endothelial function, as well as inhibition of angiogenesis and cell
proliferation activity. Most of these biological actions have been attributed to their
intrinsic reducing capabilities (Han et al., 2007). Phenolic compounds are one of the
most important, numerous and ubiquitous groups of compounds in the vegetable
kingdom, being synthesised by plants during normal development and in response to
different situations such as stress and ultraviolet radiation (Naczk and Shahidi, 2004).
These substances are obtained from phenyipropanoid metabolism and there are more
than 8000 different known structures (Herrero et al., 2005). However, these large ranges
of structures possess a common structural feature: an aromatic ring with one or more
hydroxy substituents. The way to classify these components it is not clearly established;
a possible classification can be based on the number of constitutive carbon atoms in
conjunction with the structure of the basic phenolic skeleton, including for instance,
simple phenols, phenolic acids, coumarins, flavonoids and stilbenes (Antolovich et al.,
2000).
Plant polyphenols are aromatic hydroxylated compounds, commonly found in vegetables, fruits and many food sources that form a significant portion of our diet, and
which are among the most potent and therapeutically useful bioactive substances.
Phenolic derivatives represent the largest group known as 'secondary plant products'
synthesized by higher plants, probably as a result of antioxidative strategies adapted in evolution by respirative organisms starting from precursors of cyanobacteria. Many of
these phenolic compounds are essential to plant life, e.g., by providing defense against microbial attacks and by making food unpalatable to herbivorous predators (Bennick, 2002).
Although a precise chemical definition may be given for plant phenolics, it
would inevitably include other structurally similar compounds such as the terpenoid sex
hormones. Therefore, an operational definition of metabolic origin is preferable, and
thus the plant phenols being regarded as those substances derived from the shikimate
pathway and phenyipropanoid metabolism, following the phosphoenolpyruvate
phenylalaflhlle -p cmnamate - 4-coumarate course, leading to chalcone, flavanone,
dihydrOflavono l, and anthocyanin as in Figure 2.2 (Robards and Antolovich, 1997).
0 II CH,—CHNH1---C—OH Pnjatn*e mmoia4yese
NH3 CHCH—Lo.
Cinnamic. acid
Jr
HOQ
C14=CH—C—OH
CH=CH—C—DH
!IC
acid CInnamate-4-hYdroxrIasaj( .1
+3 HOOC—CH3CO—SCaA
Strlfran8 sthas9
HO •CH=CHO1°H2O
sprMase 0Rasvratrc,
OR
OH
HO
GH
_JJOr
o 7-Hydroxycoumann chatcope
1J3kQfl £Om$e
H0(01.JO1Cr
wi o Ftiranocouniaijn Ravanon.
Figure 2.2: Schematic biochemical pathway of the synthesis of flavanone, resveratrol, and furanocoumarin.
Source: Robards and Antolovich (1997)
10
Significant antioxidant, antitumor, antiviral and antibiotic activities are
frequently reported for plant phenols. They have often been identified as active
principles of numerous folk herbal medicines. In recent years, the regular intake of fruits
and vegetables has been highly recommended, because the plant phenols and
polvphenolS they contain are thought to play important roles in long term health and
reduction in the risk of chronic and degenerative diseases. Recognition of the benefits
brought by these natural products to human health has encouraged the inclusion in
everyday diets of some typical plant-derived food and beverages, among the most
preferred examples being olive and vegetable oils, citrus and other fruit juices,
chocolate, tea, coffee and wine.
Over eight thousand naturally occurring phenolic compounds are known
(Balasundram et al., 2006). These substances contain at least one aromatic ring with one
or more attached —OH groups, in addition to other substituents (Bennick, 2002), and can
be divided into 15 major structural classes (Harborne and Simmonds, 1964). Major
classes of plant phenolics with 'the type of carbon skeleton, class name (example)'
format include: C6, simple phenols (resorcinol); C6-C1, phenolic acids (p-
hydroxybenzoic acid); C6-C2, acetophenones and phenylacetic acids; C6-C3,
hydroxycinnamic acids (caffeic acid); C6-C4, hydroxyanthraquinones (physcion); C6-
C2-C6, stilbenes (resveratrol); C6-C3-C6, flavonoids (quercetin); (C6-C3) 2, lignans
(matairesjno 1); (C6-C3 -C6) 2, biflavonoids (agathisfiavone); (C6-C3), lignins; (C6-C3-
C6), and condensed tannins (procyanidin) (Balasundram et al., 2006).
Tannins are considered to be polyphenolic metabolites of plants with a molecular
weight larger than 500 and with the ability to precipitate gelatin and other proteins from solution (Mehansho et al., 1987) and to give typical phenol reactions such as forming a
blue colour with FeCl3 (Khanbabaee and Van Ree, 2001). Tannins may be subdivided into hydrolyzable and condensed tannins' ; the former are esters of gallic acid (gallo - and ellag i .. taflhljfl5) while the latter are polyhydroxyflavan-3-ols, also known as proanthocyaflidins (Porter et al., 1989). (Bernick, 2002) defines hydrolyzable tannins
consisting of a polyhydric alcohol, such as glucose, to which gallic acid or its dimer
I
he.xaliydrodiphenic acid is linked in ester linkages, whereas the condensed tannins
contain the monomeric unit of a flavan-3-ol such as catechin or epicatechin that is linked
through C-C bonds. Basic plant phenolic structures with examples are shown in Figure
2.3.
HO OH
HO OH
Simple phenols (e.g., resorcinol) Hydroxybenzoic acids (e.g., gallic acid)
OH
OH
a0H
OH
HO 0
Flavanols (flavan-3 -ols) (e.g., (+)-
catechin)
Hydroxyciimamjc acids (e.g., p-Coumaric acid)
Figure 2.3: Basic plant phenolic structures with examples
Source: Bennick (2002)