WORS.LBorhade D JOURNAL OF PHAJournal RMAC AND PH ARPharmaceutical MACEUTICSciences AL SCIENCES Shobha World ofYPharmacy and Volume 3, Issue 4, 953-963.

Research Article

ISSN 2278 – 4357

SYNTHESIS, CHARACTERISATION & ANTIBACTERIAL ACTIVITY OF NEW FATTY ACID THIOSEMICARBAZIDE FROM CANNABIS SATIVA (HEMP) SEED OIL *Shobha S. Borhade Department of Drug Chemistry, S.M.B.S.T. College,Sangamner, Dist Ahmednagar, University of Pune, Maharashtra, INDIA.422605 ABSTRACT Article Received on 21 February 2014, Revised on 12 March2014, Accepted on 31 March 2014

Thiosemicarbazone derivative are of special importance because of their versatile biological and pharmacological activities. It is potential intermediate for the synthesis of pharmaceutical and bioactive materials and used in the field of medicinal chemistry. Hemp seed oil

*Correspondence for Author Dr. Shobha S. Borhade Department of Drug Chemistry, S.M.B.S.T. College, Sangamner, Dist Ahmednagar, University of Pune, Maharashtra.

is bold yellow coloured slightly viscous liquid. It has high iodine value indicate higher content of fatty acid. Fatty acids are important to every cell in the body for normal growth. Technically a nut, hempseed typically contains over 30% oil and about 25% protein, with considerable amounts of dietary fiber, vitamins and minerals. Hempseed oil is over 80% in polyunsaturated fatty acids (PUFAs), and

is an exceptionally rich source of the two essential fatty acids (EFAs) linoleic acid (18:2 omega-6) and alpha-linolenic acid (18:3 omega-3). The omega-6 to omega-3 ratio (n6/n3) in hempseed oil is normally between 2:1 and 3:1, which is considered to be optimal for human health. Hempseed is an excellent source of nutrition. Fatty acid thiosemicarbazide of Cannabis sativa ( Hemp) Seed oil was synthesized.The infrared spectra and X-ray diffraction of of fatty acidthiosemicarbazide was studied . The antibacterial activity of fatty acid thiosemicarbazide was evaluated. The bacteria B.cerus,E.coli and S.aureus was found to be more active. Keywords:- Cannabis sativa ( Hemp) Seed oil, Thiosemicarbazide Antimicrobial sample, IR,X-RD.

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INTRODUCTION Thiosemicarbazone derivatives are of special importance because of their versatile biological and pharmacological activities. Thiosemicarbazone derivatives have found application in drug development for the treatment of central nervous system disorders, of bacterial infection, as well as analgesic and antiallergic agent. Thiosemicarbazones are potent intermediates for the synthesis of pharmaceutical and bioactive materials and thus, they are used extensively in the field of medicinal chemistry. Moreover, thiosemicarbazones have found their way into almost every branch of chemistry of thiosemicarbazone derivatives have demonstrated wide range of biological activity viz antimicrobial

1-6

, antitumor

7,8

sodium channel blocker 9,

anticancer 10-11 , antitubercular 12, antiviral 13. Cannabis sativa L. has been an important source of food, fiber and medicine for thousands of years in the Old World14-15.The fatty acid composition of hemp seed oil from Cannabis sativa L show that it contains 70-80% polyunsaturated fatty acids (PUFA) with ~10% saturated fatty acids. Hemp seed oil is a rich source of polyunsaturated fatty acids which are present mainly as linoleic acid (18:2n-6; LA) and alpha-linolenic acid (18:3n-3ALA). Compared with other culinary oils it is low in saturated fatty acids. Current national and international dietary recommendations for the prevention of cardiovascular disease advocate decreasing the intake of saturated fatty acids in order to lower blood cholesterol concentrations. Replacing fats with a higher polyunsaturated fatty acid content will result in a lower serum cholesterol concentration and a reduction in the ratio of total/HDL cholesterol. A reduction in the ratio of total/HDL cholesterol is associated with a reduction in risk of cardiovascular disease. Hempseed oil is an excellent source of the two essential fatty acids required by humans. There are two families of essential fatty acids required by humans derived from linoleic acid (LA) and alpha-linoleicacid (ALA) respectively Harris WS. The omega-6/omega-3 ratio and cardiovascular disease risk: uses and abuses. Dietary intake of alpha-linolenic acid and risk of fatal ischemic heart disease among women16.Dietary fat intake and risk of coronary heart disease in women: 20 years of follow-up of the nurses' health study17 . Dietary hempseed reduces platelet aggregation18. The seed oil of Cannabis sativa L. is typically over 90% in unsaturated fats. Hempseed oil, pressed from nondrug varieties of the Cannabis seed, is an

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especially rich source of the two EFAs, linoleic acid (18:2 omega-6) and alpha-linolenic acid (18:3 omega-3), in addition to their respective biologic metabolites, gamma-linolenic acid (18:3 omega-6, ‘GLA’) and stearidonic acid (18:4 omega-3, ‘SDA’). the significance of a dietary ratio for the intakes of omega-6 and omega-3 fatty acids is important to consider in health19 and in the interpretation of results from clinical studies

20

. Earlier, an optimal

omega-6/omega-3 (n6/n3) ratio was considered to be somewhere between 5:1 and 10:121. The healing

properties

of

hempseed

can

be

attributed

to

high

levels

of

EFAs

Fatty acids, esterified to glycerol, are the main constituents of oils and fats. The industrial exploitation of oils and fats, both for food and oleochemical products, is based on chemical modification of both the carboxyl and unsaturated groups present in fatty acids. Although the most reactive sites in fatty acids are the carboxyl group and double bonds, methylenes adjacent to them are activated, increasing their reactivity. Potential new oil crops with unusual unsaturation or additional functionality are under development. Compilations of the fatty acid composition of oils and fats22-25 and less-common fatty acids are available.Omega-3 fatty acids play a crucial role in brain function, as well as normal growth and development. They have also become popular because they may reduce the risk of heart disease. omega-3 fatty acids reduce inflammation and may help lower risk of chronic diseases such as heart disease, cancer, and arthritis. Omega-3 fatty acids are highly concentrated in the brain and appear to be important for cognitive (brain memory and performance) and behavioral function. In fact, infants who do not get enough omega-3 fatty acids from their mothers during pregnancy are at risk for developing vision and nerve problems. Symptoms of omega-3 fatty acid deficiency include fatigue, poor memory, dry skin, heart problems, mood swings or depression, and poor circulation. the proper ratio of omega-3 and omega-6 (another essential fatty acid) in the diet. Omega-3 fatty acids help reduce inflammation, and most omega-6 fatty acids tend to promote inflammation. One of the best ways to help prevent heart disease is to eat a diet low in saturated fat and to eat foods that are rich in monounsaturated and polyunsaturated fats (including omega-3 fatty acids). omega-3 fatty acids from help prevent the development of prostate cancer.In terms of prevention, it is a well-known fact that nutrition in western countries is

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generally deficient in n–3 fatty acids, ·-linolenic acid (ALA) C18:3 n–3 in particular, and proficient in n–6 fatty acids, C18:2 n–6 in particular. Indeed, results from epidemiological 26-27

clinical28 and interventional 29studies have confirmed the increased mortality risk linked

to cardiovascular disease (CVD) and cancer, related to such unbalanced dietarybehaviour. Nutritional guidelines on lipid composition advocate reducing the C18:2 n–6/C18:3 n–3 ratio and increasing n–3 polyunsaturated fatty acid (PUFAs) intake30 . In addition, recent studies have suggested a beneficial effect of dietary supplementation of certain conjugated linoleic acids (CLA) of dairy origin (rumenic acid C18:2 cis 9 trans 11 in particular) towards preventing breast cancer 31and certain types of diabetes32. Cannabis sativa (Hemp) is an angiosperm belonging to the cannabaceae family and cannabis genus. MATERIALS AND METHODS Collection of Materials The dried Cannabis sativa (Hemp) seeds were obtained from local market in Pathardi, Ahmednagar District, Maharashtra, India. They are dried in room ,clean and stored in sealed vessel wrapped with a polyethylene bag at 40C.

Extraction of Oil After cleaning and removal of sand and other materials, the dried Cannabis sativa (Hemp) seeds were ground to a fine powder using a mixer. The oil was extracted with n-hexane ( 1:4 w/v) by continous extraction in a soxhlet apparatus for 12 hours. The solvent was evaporated at 400C in rotavapour. The extracted oil was stored in sealed and dark bottles. Their

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physic-chemical analysis was done by standard BIS methods33.All the other chemicals used in the study were of laboratory grade and were used without any modification. Preparation of Mixed Fatty Acids From Oil Mixed fatty acids from Cannabis sativa (Hemp) seed oil were obtained by saponification method in which 50 g oil was taken in 250 ml round bottom flask and 30 % alcoholic NaOH was added. The content were refluxed for 3 hrs. on stirring water bath. At the end of the reaction, the excess alcohol was distilled off and soap was dissolved in hot water. Then fatty acids were liberated by acidifying the soap solution with 1:1 H2SO4( added till development of red colour in methyl red), washed and dried over anhydrous sodium sulphate. Preparation of Fatty Acid Thiosemicarbazide ( FATSC) Fatty acid ( 2 gm) were dissolved in 4 ml of methanol and 1:1 H2SO4 to this solution of fatty acid thiosemicarbazide (4gm) in methanol was added with constant stirring at room temperature about 4 hrs and then reflux at 700C at 4 hrs.added NH4OH till alkaline stir about 15 min and kept it overnight. Crystals was filtered ,dried and recrystallized.

O S

H2 N N

N

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Infrared Spectra of FATSC The infrared spectra of fatty acid thiosemicarbazide (FATSC) was taken in the range of 4000 cm-1 to 750 cm-1 on perkin Elmer 221 IR Spectrophotometer using KBr pellet technique. The characteristic bands observed are as in Table 1. Fig 1. Shows IR spectra of fatty acid thiosemicarbazide. X-RD Spectra of FATSC X-Rd spectra of fatty acid thiosemicarbazide was taken on PW 3710 diffractometer using CuK2radiation (Y=1.54060) The X-RD diffraction of fatty acid thiosemicarbazide recorded at angle 2 from 18.5230 to 38.6561. The data of X-ray diffraction of fatty acid thiosemicarbazide were presented in Table 2. And X-ray spectrum in Fig 2. For the determination of structure Hesse-Lipson procedure is used33. Antibacterial Activity of FATSC Antibacterial activity of fatty acid thiosemicarbazide of Cannabis sativa (Hemp) seed oil was analyzed. Table 3. Two Gram-positive bacteria, Staphylococus aureus,Bacillus cereus and one Gram-positive bacteria Escherichia coli were used. Inoculum size was adjusted to 1 to 2 x 177CFU ( Colony FormingUnits) / ml by serial dilution with sterilized nutrients broth media . Nutrient agar ( PH 7.2) was used for routine susceptibility testing of nonfastidious bacteria. Stock solution of 1000mg / ml was prepared in 20 % v/v water in DMSO using the stock solution , 6000mg/ml, 4000mg/ml , 2000mg /ml and 1500 mg/ml solutions were prepared from which 100 ml solution was taken for assay. Ciprofloxacin was used as a standard, 20 % v/v WFI in DMSO was used as a control. Antibacterial assay was carried out by agar Well Diffusion Method. After 16 to 18 hours of incubation, each plate is examined. RESULTS AND DISCUSSION Cannabis sativa ( Hemp) seed oil is black in colour and its oil is bold yellow viscous liquid at room temperature. It has pleasant nutty odour and bland taste. It’s refractive index & Specific gravity is 1.4570 & 0.8927. The seed oil indicate high content of polysaturated fatty acids. The seed oil has Acid value is 2.15 mg KOH / g and Iodine value 163.5 g/100g of oil it indicate a high composition of poly unsaturated fatty acid is an assest in nutrition as high www.wjpps.com

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content of saturated fatty acids is implicated in cardiovascular diseases. Cannabis sativa (Hemp )seed oil contain fatty acids that helps to maintain healthy blood vessels. The Saponification number (mg KOH / g of oil) is 190.2. It’s peroxide number is 7.2 Meg /Kg. Experimentally it found that Cannabis sativa (Hemp) seed oil is used a medicinal important. Infrared spectra of fatty acid of thiosemicarbazide of Cannabis sativa (Hemp) seed oil shows that at 800 cm-1 ring containing three adjacent H atoms , at 900 cm-1 tetra substituted ring containing three H atoms, at 1025 cm-1 expected sulphur, at 1350 cm-1 O-H stretch, at 1450 cm-1 methylene C-H stretch, at 1650 cm-1 N-H, H-bond, at 2800 cm-1 R-OH, at 2950 cm-1 alkanes –CH3, at 3200 cm-1 C=O. X-RD spectra of fatty acid of thiosemicarbazide of Cannabis sativa (Hemp) seed oil indicate a = 7.9876 , b = 6.9847 & c = 8.9834 using Hesse-Lipson procedure shows that the structure orthorhombic . The antibacterial activity of fatty acid of thiosemicarbazide of Cannabis sativa (Hemp) seed oil was evaluated by diffusion method. It shows that antibacterial activity at varied level in E. coli , S. aureus & B. cereus bacteria. The bacteria E. coli , S. aureus & B. cereus was found to be more active in inhibition zone. The result calculated that the fatty acid of thiosemicarbazide of Cannabis sativa (H emp) seed oil posses good antibacterial activity. Table 1.

Infrared Spectra of Fatty acid thiosemicarbazide (FATSC) of Cannabis

sativa (Hemp) seed oil

Sr. No. 1 2 3 4 5 6 7 8 9

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Frequency Wavenumber 800 900 1025 1350 1450 1650 2800 2950 3200

Expected Element Ring containing three adjacent H atoms Tetra substituted ring containing three H atoms Expected Sulphur O-H stretch Methylene C-H stretch N-H, H- bond R-OH Alkanes –CH3 C=O

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Table 2.

World Journal of Pharmacy and Pharmaceutical Sciences

X-RD Spectra of Fatty acid thiosemicarbazide (FATSC) of Cannabis sativa

(Hemp) seed oil Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Table 3.

2

hkl

18.5230 20.1648 21.6652 24.5695 25.4859 27.4989 28.1913 28.9456 29.7094 30.4584 31.9077 32.1362 33.3775 34.7474 35.1742 35.4832 36.3404 36.8424 38.6561

100 110 110 111 111 111 111 111 200 200 200 220 220 220 222 222 222 222 222

Sin2 Observed 0.1102 0.0787 0.0945 0.1102 0.1574 0.1521 0.1417 0.1260 0.1102 0.1417 0.1260 0.0945 0.1889 0.1889 0.1260 0.1102 0.2204 0.1574 0.2834

Sin2 Calculated 0.1204 0.0879 0.1023 0.1198 0.1621 0.1489 0.1497 0.1300 0.0997 0.1380 0.1312 0.0972 0.1994 0.1911 0.131 0.1205 0.2190 0.1613 0.2912

d (A0) Observed 4.7902 4.4037 4.1020 3.6233 3.4951 3.2436 3.1655 3.0847 3.0071 2.9349 2.8048 2.7854 2.6846 2.5818 2.5515 2.5299 2.4722 2.4397 2.3293

d (A0) Calculated 4.1232 3.9876 3.2454 3.0021 3.1341 2.9834 2.8374 2.9870 2.6845 2.1092 2.7654 2.9567 1.9898 1.9345 2.0034 2.9345 2.0013 2.1034 2.0456

Antibacterial Activity of Fatty acid thiosemicarbazide (FATSC) of Cannabis

sativa (Hemp) seed oil Reference Substance Bacteria

Inhibition Zone FATSC 150  / well 200  / well

E.coli

35.60  0.53

29.78  0.43

24.34  0.34

S.aureus

39.10  0.95

34.92  0.67

29.00  049

B.cereus

36.67  0.61

35.32  0.47

27.68 0.58

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Figure-1. . Infrared Spectra of Fatty acid thiosemicarbazide (FATSC) of Cannabis sativa (Hemp) seed oil

Figure-2. X-RD Spectra of Fatty acid thiosemicarbazide (FATSC) of Cannabis sativa (Hemp) seed oil. REFERENCES 1. N.Kalyan Couglu ,S. Rollas , Yegenogly; Pharmazie, 1992,47(10), 796-97 2. A. M. Abdel-Halim, S.Fekria ,RM. Sayad, H.Abdel-Aziz,S. El-Dein; Indian J. Heterocyclic Chen., 1994, 3, 201-204 3. T.Siatra , A.Tsotinis , C.Sambari , H.Thomou ; Eur. J. Med. Chem.,1995, 30(2), 107-14. 4. Teoh-Siang Guan, Ang Show- Hing, Ongchiwi; J. Orgmet. Chem., 1999, 580(1), 17-21. 5. Jin Shuhui, Chen Li, Zhang Zhenye, Liang Xiaomei; Nongyaoxue Xuebao ,1999,1(3), 88-90. www.wjpps.com

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6. A.Rajasekaran, S.Murugesan : J. Indian Chem. Soc., 2002, 79(6), 544-45. 7. E. Silva Maria Joselice, Alves Antonio Jose,C. Silence : Farmaco, 1998 ,53(3), 241-243. 8. ER.Dulanyan

,TR.Ovsepyan,

GM.Stepanyan,FG.

Avsenyan:

Khim.

Farm.

Zh.,

1998,32(7), 14-15. 9. Wang Deteng, Wan Xinbo, Liu Cuiyibang, Zhao Quianquin; Huxai Yaoque Zohi, 1998,13(2), 75-76. 10. Krezel Izabella; Acta Pol. Pharma., 1998, 55(2), 125-28. 11. Magalhaes Nereide, Stela Santos, Alves Antonio Jose, Alencer et al.; Rev. Cienc, Farm., 1998, 19(1), 49-66. 12. OV.Fedorova , GG.Mordovskoi ,GL. Rusinov, Khim-farm Zh., 1998, 32(2), 11-12. 13. Alves Antonio Jose, Ramos Selma Veronica, E. Silva Maria Joselice; Rev. Farm. Bioqyim Uni. Sao Paulo, 1998, 34(2), 77-83. 14. J.Zias., H. Stark, J. Sellgman, R. Levy, E. Werker, A. Breuer, R. Mechoulam, Early medical use of cannabis. Nature : 1993. 363(6426): 215. 15. L.Xiaozhai, R.C. Clarke, The cultivation and use of hemp (Cannabis sativa L.) in ancient China. J Int Hemp Assoc : 1995. 2(1): 26–33. 16. K.Oh , FB.Hu , JE.Manson , MJ.Stampfer , WC.Willett , Am. J. Clin Nut: 1995. 69(5):890-897. 17. MN.Richard ,R. Ganguly , SN.Steigerwald , A.Al-Khalifa , GN.Pierce , Am. J. Epidemiol : 2005 ,161(7): 672-679. 18. GS.Thromb , Haemost : 2007, 5:424-5. 19. HT.Okuyama, P.Kobayashi,S.Watanabe: Dietary fatty acidsthe N-6/N-3 balance and chronic elderly diseases. Excess linoleic acid and relative N-3 deficiency syndrome seen in Japan. Prog Lipid Res 1995, 3: 409–457. 20. AP.Simopoulos, A. Leaf, N. Salem,Workshop statement on the essentiality of and recommended dietary intakes from omega- 6 and omega-3 fatty acids. Prostaglandins Leukot Essent Fatty Acids 2000, 63(3): 119–121. 21. WHO & FAO Joint Expert Consultation Report : Fats and oils in human nutrition. Nutr Rev. 1995, 53(7): 202–205.

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22. FD. Gunstone, FB. Padley, eds., Lipid Technologies and Applications, Marceln Dekker, Inc., New York, 1997. 23. F D. Gunstone, JL. Harwood, FB. Padley, eds., The Lipid Handbook, 2nd ed.,Chapman and Hall, London, U.K., 1994. 24. Official Methods and Recommended Practices of the American Oil Chemists’ Society, Section I, American Oil Chemists’ Society, Champaign, Illinois, 1997. 25. E. Ucciani, Dictionnaire des Huiles Ve´ge´tales, Lavoisier Publishing, Paris, 1995. 26. VA.Keys , A.Menotti , C.Aravanis , H.Blackburn , BS.Djordevic , R.Buzina , AS.Dontas, F. Fidanza , MJ.Karvonen , N.Kimura : The Seven Countries Study, 1984, 2,289 27. AF.Kardinaal ,KJ. Kok , J.Ringstad , J.Gomez- Aracena , VP.Mazaev , L.Kohlmeier ,BC Martin , A.Aro , M.Delgado-Rodriguez , Antioxidants in adipose tissue and risk in myocardial infarction: The Euramic study. Lancet 1993, 342:1379–1384. 28. TA.Dolecek ,Epidemiological evidence of relationships between dietary polyunsaturated fatty acids and mortality in the Multiple Risk Factor Intervention Trial. Proc Soc Exp Biol Med 1992,200:177–182. 29. M.De Lorgeril , S.Renaud , N.Mamelle , P.Salen , JL.Martin , I.Monjaud , J.Guidollet ,P. Touboul , J.Delaye , Mediterranean alpha-linolenic acidrich diet in the secondary prevention of coronary heart desease. Lancet 1994,343:1454–1459. 30. P.Legrand , JM.Bourre,B. Descomps, G.Durand, S.Renaud ,Lipids in Martin

A,

AFSSA, CNERNA-CNRS (eds): Apports nutritionnels conseillés pour la population française, ed 3. Tec et Doc Lavoisier, 2001, 63–82. 31. F.Lavillonière , P.Bougnoux ,Conjugated linoleic acid (CLA) and the risk of breast cancer, AOCS, 1999, 1( 20), 276– 282. 32. MA.Belury ,JP. Vanden Heuvel ,Modulation of diabetes by conjugated linoleic acid, AOCS, 1999,1(32),404–411. 33. LV.Azaroff ,MJ.Burger ,The powder method-Analytical method for indexing powder photographs,MC Graw Hill Book Co.Inc., N.Y. 1958,83.

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