quality control studies on luffa … in the present paper attempts were made towards quality control...
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Full Proceeding Paper
QUALITY CONTROL STUDIES ON LUFFA ACUTANGULA L.
MANIKANDASELVI .S2, BRINDHA .P1*
1CARISM, SASTRA University, Thanjavur, 2Department of Biochemistry, S.T.E.T. Women’s College, Mannargudi, Tamilnadu, India. Email:[email protected]
Received: 04 Dec 2013, Revised and Accepted: 01 Jan 2014
ABSTRACT
Studies on herbal drug standardization and quality control parameters are massively wide and deep and are needed for the globalization of herbal products. Hence in the present paper attempts were made towards Quality control studies on Luffa acutangula L. Physicochemical parameters determined in the present work along with the HPTLC and GCMS profile can contribute towards the Quality control of selected plant source which possess high therapeutic potentials and nutraceutical values.. The phytochemical screening conducted on fruit extracts of Luffa acutangula L. revealed the presence of oils, steroids, saponins, alkaloids, glycosides, phenols, tannins, flavonoids and resins. Attempts were also made to determine the quality standards for this traditional source as per Ayurvedic pharmacopoeia.
Keywords: Luffa acutangula L., nutraceutical, Ayurvedic pharmacopoeia.
INTRODUCTION
From the estimated world market for plant and plant derived drugs which would cross more than 2000 billion during 2015 it was understood that a major percentage of the world population relies mainly on plants and plant extracts for health care. A poor Indian contribution of less than Rs. 2000 crores in the world market [1] is due to the major lacuna existing in the herbal industry. Fingerprinting of herbal medicines will be useful for the authenticity and quality control of herbal products. All herbal producers should abide by WHO guidelines [2] and stick to the combined qualitative & quantitative analysis, a novel and rational method of quality control of herbal products [3]. The development of analytical techniques will serve as a fast and exact tool to set a quality standards as well as to authenticate the product to satisfy the requirement of the controlling authorities for therapeutic efficiency, safety and storage time of herbal medicines [4]. In the present paper attempts were made to
develop standards for fruits of Luffa acutangula L. using advanced analytical techniques.
MATERIALS AND METHODS
Fresh fruits of Luffa acutangula L. were purchased from herbal market in Thanjavur in the month of may 2012. Identification of the species was done at the department of CARISM, SASTRA University, Thanjavur. The collected materials were cleaned, shade dried and coarsely powdered. These powder materials were used for further physicochemical, phytochemical and fluorescence analysis. The procedures recommended by India pharmacopoeia were followed.
RESULTS AND DISCUSSION
Table 1: Organoleptic features
Plant Name Taste Color Odour Luffa acutangula L. Astrigent Brown No Characteristic odour
Table 2: Physicochemical data
Plant Name Foreign matter
Loss on drying
Total ash
Acid insoluble Ash
Water soluble
Sulphated ash
Solubility Alcohol Water
Luffa acutangula L.(%) 1.48 2.56 6.36 0.68 3.77 8.05 17.2 30.2
Table 3: Extractive values
Plant Name Hexane Chloroform Ethyl acetate Ethanol Water Luffa acutangula L.(%) 4.38 1.23 1.02 5.43 17.0
Table 4: Preliminary phytochemical analysis of Fruits of Luffa acutangula L.
Test Reagents used Hexane Chloroform Ethyl Acetate
Ethanol Water
Alkaloids
Dragendroff’s + - - - + Mayer’s + - - - + Wagner’s + - - - + Hager’s + - - - +
Reducing sugar Fehling’s - - - + - Carbohydrates Molisch’s - - - - + Saponins Foam’s + - - - + Glycosides Anthrone + - - + - Steroids Liebermann burchard + - - - - Flavonoids Shinado’s - - + + + Phenolic compound Ferric chloride - - - + + Tannin Lead acetate - - + - +
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 6, Suppl 1, 2014
AAccaaddeemmiicc SScciieenncceess
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Quinone Sulphuric acid - - - - + Anthraquinone Aqueous ammonia - - - - - Lignin Phloroglucinol - + + - + Proteins Millon’s - - - - - Amino acids Ninhydrin - - - - -
Table 5: Quantitative analysis of Phytoconstituents
Plant Name Alkaloid Flavonoid Phenol Tannin Lignin Luffa acutangula L. (mg/kg) 0.19 0.45 0.62 1.84 58.7
Table 6: Fluorescence analysis
Powder+Reagent Visible UV visible at 254nm 366nm
Powder Brown Dark brown Brown Powder+H2So4 Dark brown Black Black Powder+HNO3 Orange Yellowish orange Orange Powder+CH3COOH Reddish brown Brown Brown Powder+NH4OH Brown Light brown Orange Powder+I2 Brown Maroon Brown Powder+Fecl3 Dark green Black Black Powder+Picric Acid Light brown Yellowish dark green Brown Powder+NaOH Brown Dark brown Brown
Table 7: Nutraceutical values
Plant Name Protein (mg/g)
Carbohydrate(mg/g) Crude fiber (mg/g)
Total fat(mg/g)
Energy value(Kcal)
Vitamin E(mg/g)
Vitamin C(mg/g)
Free fatty acid(mg/g)
Luffa acutangula L.
9.6 5.5 2.8 2.5 22.9 0.01 2.05 43.9
Table 8: Analysis of metals and minerals Using XRF
Plant Name P S Mo Mg Si Fe Al Zn Cu Luffa acutangula L. (%) 4.86 2.22 0.07 2.62 2.19 0.85 0.17 0.06 0.10
Table 9: Inorganic mineral analysis
Sample Sodium(ppm) Potassium (ppm) Calcium (ppm) Luffa acutangula L. 282.9 702 312
Table 10: Heavymetal analysis by Atomic Absorption Spectroscopy
Sample name Pb (ppm) Hg (ppm) Cd (ppm) Luffa acutangula L. 8.28 9.96 <0.5
Table 11: GC-MS Analysis (Chloroform extract)
S. No. Peak Name Retention time Peak area %Peak area 1. Name: 2,4-Heptadienal, (E,E)-
Formula: C7H10O MW: 110
5.72 2384814 0.0695
2. Name: 2-Octenal, (E)- Formula: C8H14O MW: 126
6.57 3706182 0.1080
3. Name: Octanoic Acid Formula: C8H16O2 MW: 144
8.41 8094290 0.2358
4. Name: Benzoic acid, 4-methyl-, 2-oxo-2-phenylethyl ester Formula: C16H14O3 MW: 254
8.69 786483 0.0229
5. Name: Dianhydromannitol Formula: C6H10O4 MW: 146
9.06 5264952 0.1534
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6. Name: Z,Z-7,11-Hexadecadien-1-ol Formula: C16H30O MW: 238
9.37 8779424 0.2558
7. Name: E-11,13-Tetradecadien-1-ol Formula: C14H26O MW: 210
9.56 5572185 0.1623
8. Name: 2,4-Nonadienal, (E,E)- Formula: C9H14O MW: 138
10.43 5298051 0.1544
9. Name: 2-Methoxy-4-vinylphenol Formula: C9H10O2 MW: 150
10.55 1849574 0.0539
10. Name: 2-Butanone, 4-butoxy-3-methyl- Formula: C9H18O2 MW: 158
10.84 4430231 0.1291
11. Name: n-Decanoic acid Formula: C10H20O2 MW: 172
11.12 8061940 0.2349
12. Name: Vanillin Formula: C8H8O3 MW: 152
11.86 5957217 0.1736
13. Name: 9-Oxononanoic acid Formula: C9H16O3 MW: 172
12.80 8982378 0.2617
14. Name: D-Allose Formula: C6H12O6 MW: 180
13.44 3577727 0.1042
15. Name: Dodecanoic acid Formula: C12H24O2 MW: 200
13.66 40295036 1.1740
16. Name: 2-Cyclohexyl-3-isopropyl-pent-4-en-2-ol Formula: C14H26O MW: 210
13.99 3063707 0.0893
17. Name: 2-Butanoyl-5-methylfuran Formula: C9H12O2 MW: 152
14.65 1325165 0.0386
18. Name: Eicosen-1-ol, cis-9- Formula: C20H40O MW: 296
15.11 3627929 0.1057
19. Name: Tetradecanoic acid Formula: C14H28O2 MW: 228
16.02 40630544 1.1837
20. Name: 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Formula: C20H40O MW: 296
16.75 197389424 5.7508
21 Name: 2-Cyclohexen-1-one, 4-hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl)- Formula: C13H18O3 MW: 222
16.89 1515217 0.0441
22 Name: Heptadecane Formula: C17H36 MW: 240
17.68 4066410 0.1185
23 Name: Hexadecanoic acid, methyl ester Formula: C17H34O2 MW: 270
18.21 55610412 1.6202
24 Name: n-Hexadecanoic acid Formula: C16H32O2 MW: 256
19.68 1882238336 54.8379
25 Name: 11,14,17-Eicosatrienoic acid, methyl ester Formula: C21H36O2 MW: 320
22.15 44116852 1.2853
26 Name: Phytol Formula: C20H40O MW: 296
22.48 12328265 0.3592
27 Name: Octadecanoic acid, methyl ester Formula: C19H38O2 MW: 298
22.67 12079494 0.3519
28 Name: 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- Formula: C19H32O2 MW: 292
23.91 540299520 15.7413
29 Name: Octadecanoic acid Formula: C18H36O2 MW: 284
24.22 63641316 1.8542
30 Name: Tetracosane Formula: C24H50
24.59 31228096 0.9098
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MW: 338 31 Name: Citronellyl tiglate
Formula: C15H26O2 MW: 238
27.49 107356192 3.1278
32 Name: 6-Hepten-2-one, 7-phenyl- Formula: C13H16O MW: 188
30.71 14583465 0.4249
33 Name: Hexatriacontane Formula: C36H74 MW: 506
33.38 41979592 1.2231
34 Name: Triacontane Formula: C30H62 MW: 422
35.69 104867736 3.0553
35 Name: Tetratriacontane Formula: C34H70 MW: 478
38.42 157376992 4.5851
Chromatogram (x-axis = Retention time; y-axis = % intensity/% abundane)
(Hexane extract)
S. No. Peak Name Retention time Peak area %Peak area 1. Name: Undecane, 2,2-dimethyl-
Formula: C13H28 MW: 184
9.48 528405 0.2621
2. Name: Octane, 2,4,6-trimethyl- Formula: C11H24 MW: 156
9.79 781687 0.3877
3. Name: Undecane, 4-methyl- Formula: C12H26 MW: 170
10.03 42331 0.0210
4. Name: Heptane, 5-ethyl-2,2,3-trimethyl- Formula: C12H26 MW: 170
10.36 2182276 1.0824
5. Name: Decane, 3,7-dimethyl- Formula: C12H26 MW: 170
10.82 893777 0.4433
6. Name: Decane, 3,7-dimethyl- Formula: C12H26 MW: 170
11.07 267789 0.1328
7. Name: 1-Pentanol, 4-methyl-2-propyl- Formula: C9H20O MW: 144
11.60 34070 0.0169
8. Name: Decane, 2,4-dimethyl- Formula: C12H26 MW: 170
12.14 300013 0.1488
9. Name: Dodecane, 2,6,10-trimethyl- Formula: C15H32 MW: 212
12.26 578118 0.2868
10. Name: Decane Formula: C10H22 MW: 142
13.02 1935092 0.9598
11. Name: Undecane, 2,6-dimethyl- Formula: C13H28 MW: 184
13.25 361681 0.1794
, 19-JUN-2012 + 14:35:05
6.80 11.80 16.80 21.80 26.80 31.80 36.80Time0
100
%
PC chcl3 19 06 12 Scan EI+ TIC
7.70e919.68;73
16.75
68
13.66
7316.02
73
17.42
81
23.91
79
22.15
79
24.22
55 27.49
8326.59
5732.07
43
31.11
5735.69
57
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12. Name: Octane Formula: C8H18 MW: 114
13.41 21745 0.0108
13. Name: Oxalic acid, allyl octyl ester Formula: C13H22O4 MW: 242
13.52 34298 0.0170
14. Name: Heptadecane, 2,6-dimethyl- Formula: C19H40 MW: 268
14.32 705667 0.3500
15. Name: Tridecane Formula: C13H28 MW: 184
14.85 2591044 1.2852
16. Name: Oxalic acid, allyl pentadecyl ester Formula: C20H36O4 MW: 340
15.13 214338 0.1063
17. Name: Decane, 2,3,5,8-tetramethyl- Formula: C14H30 MW: 198
15.85 18358 0.0091
18. Name: n-Decanoic acid Formula: C10H20O2 MW: 172
16.43 353785 0.1755
19. Name: Oxalic acid, isobutyl nonyl ester Formula: C15H28O4 MW: 272
16.57 1444682 0.7166
20. Name: 6,10-Dimethyl-4-undecanol Formula: C13H28O MW: 200
18.03 410004 0.2034
21. Name: Heptadecane, 2,6,10,14-tetramethyl- Formula: C21H44 MW: 296
18.19 434426 0.2155
22. Name: Benzene, (1-butylhexyl)- Formula: C16H26 MW: 218
18.87 699728 0.3471
23. Name: Benzene, (1-propylheptyl)- Formula: C16H26 MW: 218
19.04 549199 0.2724
24. Name: Benzene, (1-ethyloctyl)- Formula: C16H26 MW: 218
19.35 641565 0.3182
25. Name: Dodecanoic acid Formula: C12H24O2 MW: 200
19.56 2470177 1.2252
26. Name: Undecanoic acid, ethyl ester Formula: C13H26O2 MW: 214
19.68 1123139 0.5571
27. Name: Benzene, (1-methylnonyl)- Formula: C16H26 MW: 218
19..95 788412 0.3911
28. Name: Benzene, (1-butylheptyl)- Formula: C17H28 MW: 232
20.36 3222040 1.5982
29. Name: Benzene, (1-propyloctyl)- Formula: C17H28 MW: 232
20.52 1680238 0.8334
30. Name: Benzene, (1-ethylnonyl)- Formula: C17H28 MW: 232
20.88 1553912 0.7708
31. Name: Benzene, (1-methyldecyl)- Formula: C17H28 MW: 232
21.44 1557298 0.7724
32. Name: Benzene, (1-butyloctyl)- Formula: C18H30 MW: 246
21.78 4143396 2.0552
33. Name: Benzene, (1-ethyldecyl)- Formula: C18H30 MW: 246
22.30 1621785 0.8044
34. Name: Ethyl tridecanoate Formula: C15H30O2 MW: 242
22.53 1694088 0.8403
35. Name: 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Formula: C20H40O MW: 296
23.16 9743021 4.8327
36. Name: Pentadecanoic acid, ethyl ester Formula: C17H34O2 MW: 270
24.36 266854 0.1324
37. Name: Hexadecanoic acid, methyl ester 24.69 7061104 3.5024
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Formula: C17H34O2 MW: 270
38. Name: E-11-Hexadecenoic acid, ethyl ester Formula: C18H34O2 MW: 282
25.50 1970486 0.9774
39. Name: Hexadecanoic acid, ethyl ester Formula: C18H36O2 MW: 284
25.80 93853592 46.5528
40. Name: 7,10,13-Hexadecatrienoic acid, methyl ester Formula: C17H28O2 MW: 264
27.56 5983708 2.9680
41. Name: 9,12-Octadecadienoic acid, ethyl ester Formula: C20H36O2 MW: 308
28.41 7979019 3.9577
42. Name: 9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)- Formula: C20H34O2 MW: 306
28.53 21444672 10.6369
43. Name: Octadecanoic acid, ethyl ester Formula: C20H40O2 MW: 312
28.83 6951718 3.4482
44. Name: Eicosanoic acid, ethyl ester Formula: C22H44O2 MW: 340
31.53 3926295 1.9475
45. Name: Hentriacontane Formula: C31H64 MW: 436s
32.81 6547896 3.2479
Chromatogram (x-axis = Retention time; y-axis = % intensity/% abundane)
Table 12: HPTLC fingerprinting profile (Hexane ,Chloroform Extracts) Photo documentation under UV
AT 254nm AT 366nm
, 17-MAY-2012 + 12:09:39
8.85 10.85 12.85 14.85 16.85 18.85 20.85 22.85 24.85 26.85 28.85 30.85 32.85Time0
100
%
PD hexane run2 17 05 12 Scan EI+ TIC
1.45e925.80;88
23.16
4321.71
9120.36
91
10.36
57
14.85
57
24.69
74
28.53
79
28.41
67
27.56
41
28.83
55
30.23
43
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TLC DETAILS
Track 1,2-5µl of sample (Luffa acutangula Hexane Extract)
Track 3, 4-5µl of sample (Luffa acutangula Chloroform Extract)
Fig I Fig II
Proximate nutrient assay of edible fruit and vegetables establish its nutritional importance. There is a correlation between the health maintenance [5] and fruit, vegetable utilization. It seems to be not feasible to spot an exact component but assumed to be a combination of several constituents that may be responsible for these benefits [6].
Luffangulin, a novel N-terminal ribosome inactivating peptide isolated from fruit seeds of Luffa acutangula. [7]. Presence of Sapogenin, oleanolic acid and a bitter principle, Cucurbitacin B were also identified from the seeds of Luffa acutangula [8].
The oil characteristics of Luffa acutangula with Iodine value, saponification value and acid value as 99.5, 190.8 and 10.5 respectively with the melting point range -3°C and -10°C have been correlated with its nutrient value [9].
The data obtained in the present work revealed interesting chemical features which were tabulated in table 1. Among all the extracts, water extracts of Luffa acutangula was found to have maximum extractive yield following Ethanol, Hexane, Chloroform, Ethylactate
extracts. Loss on drying,acid insoluble ash value, water soluble and sulphated ash were determined for Luffa acutangula extracts. These tests of purity revealed that the sample satisfies purity standards, and suggested the drug is rich in high polar compounds.
Preliminary phytochemical analysis of Luffa acutangula extracts exhibits the presence of Alkaloids, Reducing sugar, Carbohydrates, saponins, Glycosides,Steroids, Flavonoids, Phenolic Compounds, Tannins, Quinones and Lignins.
Percentage yield of Lignin, Tannins, Phenol, Flavonoids and Alkaloids were more in the test drug. Currently plant flavonoids have attracted interest as significant nutritional cancer chemo-protective and antioxidant agents. Tannins are complex phenolic polymers, which can bind to proteins and carbohydrates resulting in decline the digestibility of these macromolecules and therefore inhibit microbial growth. The experimental results in case of congestive heart failure are to amplify the force of myocardial contraction. The plant drug exercise its hypotensive effect by restraining Na+,K+ ATPase. Besides, the drug directly act upon the smooth muscle of the vascular system and ultimately activate the
PEAK DISPLAY
(10µl of Sample- Luffa acutangula L. (Hexane Extract)
PEAK DISPLAY
(10µl of Sample- Luffa acutangula (chloroform Extract)
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mechanical and electrical behavior of the heart and alter vascular resistance and capacitance [10]. Hence the test drug could serve as a dietary supplement especially for the patients with both cancer and heart problems.
XRF, FPM data suggested that the plant is rich in minerals especially K, Ca,P and Mg. Si and S are present in moderate quantity. Rich in nutraceutical values of the test drug can be understood from the following identifications. Diet high in and low in favoured lowering of blood pressure. It is also possible that a low Na and high K diet would decrease the development of cardiovascular disease [11, 12]. The requirement from plant source is not much important because of its availability as NaCl salt. Deficiency of calcium and phosphorous leads to the classic bone symptoms associated with rickets, such as bowlegs, knock knees, curvature of the spine and pelvic and thoracic deformities. Magnesium plays important role in the structure and the function of the human body [13].
GC-MS analysis of hexane extract of Luffa acutangula revealed the presence of forty-five different class of compounds such as Hexadecanoic acid ethylester, 9, 12, 15-oc tadecatrienoic acid, 3, 7, 11, 15-Tetramethyl – 2 – hexadecenl-ol, Hentriacontane, 9, 12 octadecanoic acid ethyl ester, Hexadecanoic acid methyl ester. GC-MS of chloroform extract revealed the presence of 35 compounds such as n-Hexade canoic acid, 9, 12, 15-octadecatrienoic acid methyl ester, (z, z, z), Tetratriacontane, Triacontane, citronellyl tiglate. HPTLC profile under 366 nm shows number of spots in Luffa acutangula (Hexane and Chloroform extract) with different Rf values revealed the distinguishing feature of the drug.
In the present work fruits of Luffa acutangula were studied from standardization point of view. The standards determined in the present work could be useful in evaluating the quality of the drug. Incidentally the bio chemical standards determined in the present
work suggested this drug could be a good dietary supplement particularly for Heart & Cancer patients.
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