PHYTOCHEMICAL ANALYSIS OF ETHANOLIC EXTRACT OF PROSOPIS CINERARIA PODS BY GC-MS

PHYTOCHEMICAL ANALYSIS OF ETHANOLIC EXTRACT OF PROSOPIS CINERARIA PODS BY GC-MS

Garima Sharma, Devki, Ruchi Vyas and Rashmi Sisodia ^*

Radiation Biology Laboratory, Department of Zoology, Centre for Advanced Studies, University of Rajasthan - 302004, Jaipur, India.

ABSTRACT: This study was aimed to identify the possible bioactive chemical constituents of the state tree of Rajasthan, i.e., Prosopis cineraria L. (locally called Khejri) by GC-MS analysis using the GC-MS equipment (Shimadzu GCMSQP2010). For this purpose, the ethanolic extract of P. cineraria pods were used. Experimental conditions of the GC-MS system were as follows: Rtx-5MS capillary standard non-polar column, dimension: 30m, ID: 0.25 mm, Film: 0.25 µm was used, and flow rate of mobile phase (carrier gas: He) was set at 1.0 ml/min. Crude samples that were dissolved in methanol were run fully at a range of 40-550 m/z, and the results were compared by using NIST Library. GC-MS analysis of an ethanolic extract of pods led to the identification of 32 compounds viz. Bicyclo[3.1.1]hept-3-ene, 4, 6, 6-trimethyl-2-vi (30.47%), 5-Isopropyl-2methylbicyclo [3.1.0] hexan-2-ol(19.38%), (-)-.beta. –Bourbonene (10.62%), 3-O-Methyl- d-glucose (5.04%), Kessane (4.01%), Thunbergol (3.65%), alfa.-Copaene (2.23%). There is not much information available on the phytochemical constituents of P. cineraria pods. This is the first attempt to investigate the GC-MS analysis of the ethanolic extract of this plant.

P. cineraria, Gas chromatography-mass spectrometry analysis, Phytochemical

INTRODUCTION: The P. cineraria (L) Druce belonging to the family Leguminosae is an important tree (Khejri- a local name in Rajasthan) of the Thar Desert with hard climatic adaptation and is considered as one of the lifelines in desert habitat as mentioned in ancient literature. This is a species representing all five F viz., Forest, Fiber, Fuel, Fodder, and Food. Pods of Khejri are eaten by cattle, sheep, horses, mules, donkeys, goats, camels, and other wildlife in the desert, especially blackbuck and chinkara in the western Rajasthan have survived by eating pods and leaves of this tree. This tree is also of mythological importance in local communities.

The high value of this species has led it to be recognized as a State symbol (the state tree of Rajasthan). P. cineraria (L.) tree is endemic to the hot deserts of India. Pods of the tree are locally called “Sangri” and are considered as dry fruit of desert. It is one of the main ingredients of the quintessential Rajasthani dish ‘‘The Panchkuta’ ¹.

Pods contain various phytoconstituents like tannins (gallic acid), steroids (campestral, stigmasterol, sitosterol, etc.), flavone derivatives (prosogerin A, B, C, D, and E), alkaloids (prosophylline, spicigerine) etc. which have been isolated from the sangria pod ². The fresh ripe pods contain 7-10% preformed water, and on a dry matter basis contain 9-17% crude protein, 1.2-4.3% ether extractives, 47-61% nitrogen-free extracts ,16-34% crude fibre, 28% acid detergent fibre, 8% acid detergent lignin, 4-5% ash, 0.14-0.29% silica, 0.3-0.5% calcium and 0.40-0.44% phosphorus.  The qualitative chemical analysis of extracts of Prosopis pods was found positive for alkaloids, proteins, carbohydrates, flavonoids, glycosides, saponins, and tannins in alcohol and aqueous solvent extracts. Also, 11 amino acids have been isolated from leaves and pods viz; aspartic acid, glutamic acid, serine, glycine, histidine, threonine, arginine, alanine, proline, valine, tyrosine, methionine, cysteine, isoleucine, leucine, phenylalanine, and lysine ³.

Prosopis has been found to contain 5-hydroxy-tryptamine, apigenin, isorhamnetin-3-diglucoside, l- arabinose, quercetin, tannin, and tryptamine. The isolation of a flavone glycoside Patulitrin 3, 5, 6, 3, 4- pentamethoxy-7-hydroxy flavone from flowers of P. cineraria has been reported. The fruits of P. juliflora D.C. (leguminosae) were found to contain the same compound ⁴. Many researchers found that patulitrin showed significant activity against the Lewis lung carcinoma in-vivo ⁵.

P. cineraria pods contain protein, iron, vitamins A and C, and other micro minerals. Unripe pods are also nutritious and are used to prepare curries and pickles. P. cineraria (L) Druce is also a highly valued plant in the indigenous system of medicine. Its bark is said to be a potent drug for several ailments such as leprosy, dysentery, bronchitis, asthma ⁶, leucoderma, piles, muscular tremors, asthma, rheumatism and inflammations. Pharmaco-logical activities like analgesic, antipyretic, anti-hyperglycemic, anti-oxidant, antihyperchole-sterolemic, anti-tumor, nootropic, anthelmintic, anti-bacterial, anti-fungal, anti-viral, and anticancer activities have also been reported from different plant extracts ^{3, 7-14}. The inhibitory activities of enzymes lipid peroxidase, cyclooxygenase -1 and-2 in sangri pods was also reported ¹⁵.

The chloroform fraction of stem bark of P. cineraria shows anti-diabetic activity in Stepto-zotocin induced diabetes in rats. The possible mechanisms of its anti-diabetic action include inhibition of carbohydrate hydrolytic enzymes, enhancement of glycogen regulatory enzymes expression in the liver and glucose uptake by tissues and adipocytes as well as stimulation of pancreatic insulin release ¹⁶. Thus, P. cineraria have potential in-vitro and in-vivo anti-diabetic activity, and this effect could be due to multi-target mode of action that includes anti-hyperglycemic, post-prandial hypoglycemic, hypolipidemic, and insulin secretory actions ¹⁷.

Phytoconstituents of an ethanolic pod extract of P. cineraria triggers the inhibition of HMG-CoA reductase, which is the main enzyme for cholesterol biosynthesis and the regression of atherosclerotic plaque in hyperchole-sterolemic rabbits ¹⁸. P. cineraria pod extract components can be significantly considered in neuropharmacology as it showed inhibition against DPP-4, AChE, and BuChE target enzymes ¹⁹.

After the GC-MS analysis of the methanolic extract of roots of P. cineraria showed the presence of significant phytoconstituents belonging to the type acids, esters, alcohols, ethers, etc. Significant anticancer activity was also reported. Squalene, which was present in P. cineraria may be responsible for anticancer activity ²⁰.

GC-MS analysis is a breakthrough in the analysis of phytoconstituents and structure elucidation of these compounds as they have a sensitivity of detecting compounds as low as 1 mg ²¹. Therefore, we have done a GC-MS analysis for the identification of significant compounds in P. cineraria pods in the present study.

MATERIALS AND METHODS:

Collection of Pods: The fruits of P. cineraria were collected from the Dausa district of Rajasthan, India, in the month of March 2017. The plant material was identified and authenticated by the Department of Botany, University of Rajasthan, Jaipur (RUBL 211667).

Preparation of Plant Extract: 25 gm of P. cineraria pods were air-dried in the shade at room temperature, and then dried pods were coarsely powdered. Plant material was extracted with 100ml ethanol by using a Soxhlet apparatus.

GC-MS Analysis: Five grams of sample was extracted successively with ethanol in Soxhlet extractor for 8 h. Brown colored residues were obtained after concentrating the extract under reduced pressure using a rotary evaporator.

The obtained extracts were stored in desiccators for further analysis. The dried sample was reconstituted in methanol to obtain 10 μgml^-1 concentrations. Methanol was used as a solvent. Finally, 2 ml of supernatant was taken and filtered through Axiva 0.2 μm nylon syringe filter and transferred to GC vial for analysis. The analysis was carried out on a Shimadzu GCMSQP2010 Ultra system.

GC-MS Conditions: The injector temperature was 280 °C.The samples were injected in the split mode with a split ratio of 1/60. The injection volume was 1 µL. A capillary column Rtx-5MS (5% Diphenyl-95% Dimethyl Polysiloxane), 30 m × 0.25 mm × 0.25 mm, was used. The carrier gas was helium with a constant flow of 1.00 mL/min. The oven temperature was as follows: initial temperature of 60 °C, held for 2 min, increased to 10 °C/min up to 260 °C and held for 10 min. The MS ionization potential was 70 eV, and the temperatures were as follows: interface 260 °C, Ion source 280 °C. Mass scan range 40-550 m/z. GC-MS analysis of P. cineraria pods extracts led to the identification of 32 compounds Table 1.

TABLE 1: PHYTOCOMPONENTS IDENTIFIED IN THE ETHANOLIC EXTRACT OF P. CINERARIA PODS

FIG. 1: GC-MS CHROMATOGRAM OF CONSTITUENTS OF ETHANOLIC EXTRACT OF PROSOPIS CINERARIA PODS

FIG. 2: LIST OF IDENTIFIED COMPOUNDS FROM P. CINERARIA PODS ARE ASCERTAINED WITH NIST14 LIBRARY

RESULTS AND DISCUSSION: The results pertaining to GC-MS analysis led to the identi-fication of a number of compounds from the GC fractions of ethanolic extract of P. cineraria. These compounds were identified through mass spectrometry attached with GC. The results of the GC-MS was interpreted by using a database of the National Institute of Standards and Technology (NIST) library having more than 2,00,000 patterns. The GC-MS spectrum of the unknown constituent was then compared with the known components stored in NIST-14 library. The results of present study are tabulated in Table 1.

P. cineraria Pods Extract: GC-MS analysis of ethanolic extract of P.cineraria pods revealed the presence of 32 different phytocompounds namely1, 3, 5-Cycloheptatriene, 3, 7, 7-trimethyl (0.31%), 3-Carene (0.40%), cis-4-methoxy thujane (1.18%), (1R, 4R, 5S) -1-Isopropyl-4- methoxy-4-methylb (2.67%), Butanedioic acid, mono (3-phenyl-2-propeny) (0.54%),p-Mentha-1,5-dien-8-ol (0.35%), Bicyclo [3.1.0] hexan-3-ol, 4-methylene-1- (1-methylethyl) -acetate(0.59%), 5-Isopropyl-2-methyl-bicyclo [3.1.0] hexan-2-ol (19.38%), Bicyclo [3.1.1] hept-3-ene, 4, 6, 6-trimethyl-2-vi (30.47%), trans-Verbenol (0.73%), (1,2,3-Tri-methyl- cyclo-pent-2-enyl)- methanol (0.66%), 2, 4-Pentadienoic acid, 3, 4-dimethyl-, isopropy (1.01%), Carvacrol (0.60%), (1R, 3S, 4R, 5S)-1-Isopropyl-4- methyl-bicyclo [3.1.0] hexan- 3-yl acetate -rel (0.60%), alpha.-ylangene (0.76 %), .alfa.- Copaene (2.23%), (-)-. beta.- Bourbonene (10.62%), beta.- Longipinene (0.60%), cis-.alpha.-Bergamotene (1.38%), Germa-crene D (0.72%), alfa.- Copaene (1.77%),. beta.- Longipinene (1.21%), Isoledene (1.84 %), Kessane (4.01%), Diethyl Phthalate (2.84%), 3-O-Methyl-d-glucose (5.04%),. alpha.- Phellandrene, dimer (1.15%), (R, 1E, 5E, 9E)-1, 5, 9-Trimethyl-12-(prop-1-en-2-yl)cyclotetradeca-1, 5, 9-triene (0.90%), Isocembrol (0.57%), 1, 3, 6, 10-Cyclotetra-deca-tetraene, 3, 7, 11-trimet (0.51%), (R, 1E, 5E, 9E)-1, 5, 9-Trimethyl-12- (prop-1-en-2-yl) cyclo-tetradeca-1, 5, 9-triene (0.68%), Thunbergol (3.65%).

TABLE 2: PHARMACEUTICAL / THERAPEUTIC POTENTIAL OF THE PHYTOCOMPONENTS IDENTIFIED IN THE ETHANOLIC EXTRACT OF P. CINERARIA PODS

The GC-MS spectrum confirmed that the presence of 32 major components with the retention time 6.673, 7.227, 7.857, 8.224, 8.571, 8.910, 9.016, 9.210, 9.436, 10.238,10.449, 10.604, 10.904, 11,582, 11.982, 12.067, 12.193, 12.450, 12.785, 12.991, 13.355, 13.499, 13.889, 14.100, 14.643, 15.623, 17.113, 18.801, 19.069, 19.566 respectively Fig. 1. The name, molecular weight, molecular formula and structure of the component of the test materials were ascertained. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. The identified phytoconstituents from pods of P. cineraria possess the following properties Table 2.

CONCLUSION: In the present study, the GC-MS analysis of an ethanolic extract of pods of P. cineraria revealed the presence of thirty-two compounds. Such pods are consumed as the dietary agent and may be further explored for their medicinal potential as the phytoconstituents identified has potent anticancer and antioxidant activities which can be used to reduce the damages caused by free radicals in the body and therefore contribute to the prevention of diseases related to the oxidative stress and also for the management of diabetes, inflammation and microbial infections.

Thus, this type of GC-MS analysis is the first step towards understanding the nature of active principle bioconstituents in this medicinal plant. The identification of phytoconstituents would be helpful for further detailed studies.

ACKNOWLEDGEMENT: The authors are thankful to the Department of Zoology, Centre for Advanced Studies, University of Rajasthan, Jaipur, for providing the necessary infrastructure & facilities. We are also grateful to DST-PURSE for financial support.

CONFLICTS OF INTEREST: The authors declare that this article content has no conflict of interest.

REFERENCES:

1. Rani B, Singh U, Sharma R, GUPTA AA, Dhawan NG, Sharma AK, Sharma S and Maheshwari RK: Prosopis cineraria (L) Druce: a desert tree to brace livelihood in Rajasthan. Asian Journal of Pharmaceutical Research and Health Care 2013; 5(2): 58-64.
2. Chaudhary KK, Kumar G, Varshney A, Meghvansi MK, Ali SF, Karthik K, Dhama K, Siddiqui S and Kaul RK: Ethnopharmacological and phytopharmaceutical evaluation of Prosopis cineraria: An overview and future prospects. Current drug metabolism 2018; 19(3): 192-14.
3. Islam MW, Bloukh SH, Edis Z and Bhandare RR: Emerging phytochemicals and bioactive compounds from a desert plant Prosopis cineraria (L.) druce and future prospects. In International Conference on Pure and Applied Chemistry Springer Cham 2018; 19-51.
4. Gupta A, Sharma G, Pandey S, Verma B, Pal V and Agrawal SS: Prosopis cineraria and its various therapeutic effects with special reference to diabetes: A novel approach. Int J Pharm Sci Rev Res 2014; 27(2): 328-33.
5. Sharma P and Sharma DK: Medicinal value of three common plants of Rajasthan, India. Journal of Medicinal Plants 2018; 6(1): 96-01.
6. Younis W, Asif H, Sharif A, Riaz H, Bukhari IA and Assiri AM: Traditional medicinal plants used for respiratory disorders in Pakistan: a review of the ethno-medicinal and pharmacological evidence. Chinese Medicine 2018; 13(1): 48.
7. Kumar A, Yadav SK, Singh S and Pandeya SN: Analgesic activity of ethanolic extract of roots of Prosopis cineraria (L.) Druce. Journal of Applied Pharmaceutical Science 2011; 1(8): 158.
8. Sharma N, Garg V and Paul A: Antihyperglycemic, anti-hyperlipidemic and antioxidative potential of Prosopis cineraria Indian Journal of Clinical Biochemistry 2010; 25(2): 193-200.
9. Robertson S, Narayanan N and Kapoor RB: Antitumour activity of Prosopis cineraria (L.) Druce against Ehrlich ascites carcinoma-induced mice. Natural Product Research 2011; 25(8): 857-62.
10. Bithu BS, Reddy NR, Prasad SK, Sairam K and Hemalatha S: Prosopis cineraria: a potential nootropic agent. Pharmaceutical Biology 2012; 50(10): 1241-7.
11. Preeti K, Avatar SR and Mala A: Pharmacology, phytochemistry and therapeutic application of Prosopis cineraria Linn: a review. Journal of Plant Sciences 2015; 3(1-1): 33-9.
12. Napar AA, Bux H, Zia MA, Ahmad MZ, Iqbal A, Roomi S, Muhammad I and Shah SH: Antimicrobial and antioxidant activities of Mimosaceae plants; Acacia modesta Wall (Phulai), Prosopis cineraria (Linn.) and Prosopis juliflora (Swartz). Journal of Medicinal Plants Research 2012; 6(15): 2962-70.
13. Pareek AK, Garg S and Kuma M: Prosopis cineraria: a gift of nature for pharmacy. Int J Pharma Sci Res 2015; 6(6): 958-64.
14. Sharifi-Rad J, Kobarfard F, Ata A, Ayatollahi SA, Khosravi-Dehaghi N, Jugran AK, Tomas M, Capanoglu E, Matthews KR, Popović-Djordjević J and Kostić A: Prosopis plant chemical composition and pharmacological attributes: Targeting clinical studies from preclinical evidence. Biomolecules 2019; 9(12): 777.
15. Liu Y, Singh D and Nair MG: Pods of Khejri (Prosopis cineraria) consumed as a vegetable showed functional food properties. Journal of Function Foods 2012; 4(1): 116-21.
16. Soni LK, Dobhal MP, Arya D, Bhagour K, Parasher P and Gupta RS: In-vitro and in-vivo antidiabetic activity of isolated fraction of Prosopis cineraria against strepto-zotocin-induced experimental diabetes: A mechanistic study. Biomedi & Pharmacotherapy 2018; 108: 1015-21.
17. Kumar M, Govindasamy J and Nyola NK: In-vitro and in-vivo anti-hyperglycemic potential of Prosopis cineraria pods extract and fractions. Journal of Biologically Active Products from Nature 2019; 9(2): 135-40.
18. Ram H, Jaipal N, Charan J, Kashyap P, Kumar S, Tripathi R, Singh BP, Siddaiah CN, Hashem A, Tabassum B and Allah AEF: Phytoconstituents of an ethanolic pod extract of Prosopis cineraria triggers the inhibition of HMG-CoA reductase and the regression of atherosclerotic plaque in hypercholesterolemic rabbits. Lipids in Health and Disease 2020; 19(1): 6.
19. Ram H, Jaipal N, Kumar P, Deka P, Kumar S, Kashyap P, Kumar S, Singh BP, Alqarawi AA, Hashem A and Tabassum B: Dual Inhibition of DPP-4 and cholinesterase enzymes by the phytoconstituents of the ethanolic extract of Prosopis cineraria pods: therapeutic implications for the treatment of diabetes-associated neurological impairments. Curr Alzheimer Res 2019; 16(13): 1230-44.
20. Khokar A and Menghani E: TLC and GC-MS analysis of Methanol extract of root of Prosopis cineraria from Jaipur, Rajasthan, India. International Journal of Scientific & Engineering Research 2016; 7(6): 537-43
21. Liebler DC, Burr JA, Philips L and Ham AJ: Gas chromatography–mass spectrometry analysis of vitamin E and its oxidation products. Anal Bio 1996; 236(1): 27-34.
22. Huong LT, Huong TT, Bich NT, Viet NT, Giwa-Ajeniya AO and Ogunwande I: Antimicrobial efficacy and chemical constituents of pseudo-stem essential oils from Zingiber castaneum. Trends in Phytochemical Research 2020; 4(2): 93-100.
23. Woo J, Yang H, Yoon M, Gadhe CG, Pae AN, Cho S and Lee CJ: 3-Carene, a phytoncide from pine tree has a sleep-enhancing effect by targeting the GABAA-benzodiazepine receptors. Experimental Neurobiology 2019; 28(5): 593.
24. Cavaleiro C, Pinto E, Gonçalves MJ and Salgueiro L: Antifungal activity of Juniperus essential oils against dermatophyte, Aspergillus and Candida strains. Journal of Applied Microbiology 2006; 100(6): 1333-8.
25. Premjanu N and Jaynthy C: Antimicrobial activity of diethyl phthalate: an in-silico Asian J Pharm Clin Res 2014; 7(4): 141-2.
26. Kachur K and Suntres Z: The antibacterial properties of phenolic isomers, carvacrol and thymol. Critical Reviews in Food Science and Nutrition 2019; 1-2.
27. Utegenova GA, Pallister KB, Kushnarenko SV, Özek G, Özek T, Abidkulova KT, Kirpotina LN, Schepetkin IA, Quinn MT and Voyich JM: Chemical composition and antibacterial activity of essential oils from Ferula L. species against methicillin-resistant Staphylococcus aureus. Molecules 2018; 23(7): 1679.
28. Salim M, Kabeer TA, Nair SA, Dan M, Sabu M and Baby S: Chemical profile, anti-proliferative and antioxidant activities of rhizome oil of Zingiber anamalayanum from Western Ghats in India. Nat Pro Res 2016; 30(17): 1965-8.
29. Mohammed GJ, Omran AM and Hussein HM: Anti-bacterial and phytochemical analysis of piper nigrum using gas chromatography-mass spectrum and fourier-transform infrared spectroscopy. International Journal of Pharma-cognosy and Phytochemical Research 2016; 8(6): 977-96.
30. Wang Z, Liu F, Yu JJ and Jin JZ: β-Bourbonene attenuates proliferation and induces apoptosis of prostate cancer cells. Oncology Letters 2018; 16(4): 4519-25.
31. Mughees M and Wajid S: Evaluation of cytotoxicity of different part extracts of Ipomoea turpethum against breast cancer cell lines. Journal of Environmental Pathology, Toxicology and Oncology. 2020; 39(1): 51-60.
32. Zhang PJ, Zhao C, Ye ZH and Yu XP: Trade‐off between defense priming by herbivore‐induced plant volatiles and constitutive defense in tomato. Pest Management Science 2020; 1893-1901.
33. Oliveira PF, Alves JM, Damasceno JL, Oliveira RA, Dias Júnior H, Crotti AE and Tavares DC: Cytotoxicity screening of essential oils in cancer cell lines. Revista Brasileira de Farmacognosia 2015; 25(2): 183-8.
34. Tan WN, Tan ZH, Zulkifli NI, Nik Mohamed Kamal NN, Rozman NA, Tong WY, Leong CR and Lim JW: Sesquiterpenes rich essential oil from Garcinia celebica and its cytotoxic and antimicrobial activities. Natural Product Research 2019; 3404-08.
35. Asif M, Shafaei A, Jafari SF, Mohamed SK, Ezzat MO, Majid AS, Oon CE, Petersen SH, Kono K and Majid AM: Isoledene from Mesua ferrea oleo-gum resin induces apoptosis in HCT 116 cells through ROS-mediated modulation of multiple proteins in the apoptotic pathways: A mechanistic study. Toxicology Letters 2016; 257: 84-96.
36. Parki A, Chaubey P, Prakash O, Kumar R and Pant AK: Seasonal variation in essential oil compositions and antioxidant properties of Acorus calamus accessions. Medicines 2017; 4(4): 81.
37. Massodi MLE, Mime LC, Fogang HPD, Djikeng FT, Karuna MSL and Womeni HM: Chemical Composition and Antioxidant Activity of Syzygium aromaticum and Monodora myristica Essential Oils from Cameroon. Journal of Food Stability 2018; 1(1): 1-13.
38. Abd‐ElGawad AM, Elshamy AI, El‐Nasser El Gendy A, Al‐Rowaily SL and Assaeed AM: Preponderance of oxygenated sesquiterpenes and diterpenes in the volatile oil constituents of Lactuca serriola revealed antioxidant and allelopathic activity. Chem & Biodiv 2019; 16(8): e1900278.
39. Fajdek-Bieda A, Wróblewska A, Miądlicki P, Szymańska A, Dzięcioł M, Booth AM and Michalkiewicz B: Influence of technological parameters on the isomerization of geraniol using sepiolite. Cata Lett 2020; 150(3): 901-11.
40. Mitić ZS, Jovanović B, Jovanović SČ, Stojanović-Radić ZZ, Mihajilov-Krstev T, Jovanović NM, Nikolić BM, Marin PD, Zlatković BK and Stojanović GS: Essential oils of Pinus halepensis and heldreichii: chemical composition, antimicrobial and insect larvicidal activity. Industrial Crops and Products 2019; 140: 111702.
    

    ---

    How to cite this article:

    Sharma G, Devki, Vyas R and Sisodia R: Phytochemical analysis of ethanolic extract of Prosopis cineraria pods by GC-MS. Int J Pharm Sci & Res 2021; 12(1): 418-31. doi: 10.13040/IJPSR.0975-8232.12(1).418-31.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---