quality control studies on luffa … in the present paper attempts were made towards quality control...

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

Brindha et al. Int J Pharm Pharm Sci, Vol 6, Suppl 1, 55-62

Proceedings-International Conference on Natural Products in the Management of Cancer, Diabetes and Viral Infections, SASTRA University, Thanjavur, Tamil Nadu

56

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



57

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



58

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


11.07 267789 0.1328

7. Name: 1-Pentanol, 4-methyl-2-propyl- Formula: C9H20O MW: 144

11.60 34070 0.0169


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



59

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



60

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



61

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)



62

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.

REFERENCES

1. Jian Zhao, Nutraceuticals, Nutritional Therapy, Phytinutrients, and Phytotherapy for Improvement of Human Health: A Perspective on Plant Biotechnology Application. Journal of Recent Patents on Biotech.2007;1:75-97.

2. Raina M.K, Quality control of herbal and herbo-mineral formulations. Indian journal of natural products 2003;19: 11-15.

3. Javed Abbas Bangash et al., Proximate Composition, Minerals and Vitamins Content of Selected Vegetables Grown in Peshawar. J.Chem.Soc.Pak 2011;33(1):118- 126.

4. Rama C .S, Shirode A.R, Mundada A.S and Kadam V.J, Nutraceuticals an emerging era in the treatment and prevention of cardiovascular diseases. Curr Pharm Biotechnol 2009;(1):15‐23.

5. Willett W. C, Diet, Nutrition, and the Prevention of Cancer. In Modem Nutrition in Health and Disease, eds ME Shils, J. A. Olson, M. Shike,and C. Ross, Williams and Wilkins, Baltimore, MD. 1998;1243.

6. Wang H, Tzi Bun N.G, 2002;70(8): 899-906. 7. Pal S, Chakraborthi S.K, Banerjee A and Mukerji B,

Ind.Jour.Med.Res 1968;56(4):54-57. 8. Kamel B. S, Blackman B 1982;9(4): 277-282 9. Hertog M.G. L, Hollman P. C. H, Katan M. B and Klohout M, Nutr

cancer 1993;20, 21-29. 10. Houston M.C, Nutraceuticals, Vitamins, Antioxidants, and

Minerals in the Prevention and Treatment of Hypertension. Progress in Cardiovascular Diseases 2005;47: 396-449.

11. Kalaskar Mohan G and Surana Sanjay J. Pharmacognostic and Phytochemical Investigation of Luffa acutangula var. amara Fruits. International Journal of PharmTech Research 2010; 2(2): 1609-1614.

quality control studies on luffa … in the present paper attempts were made towards quality control...

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