ISSN: 0975-766X CODEN: IJPTFI Available Online through Research Article www.ijptonline.com ANTIMICROBIAL EUGENOL FROM CITRULLUS COLOCYNTHIS L.OF ARID KACHCHH REGION OF INDIA Taslimahemad T. Khatri 1 * and Viresh H. Shah 2 1 Department of Chemistry, KSKV Kachchh University, Bhuj-Kachchh 370 001, Gujarat, India. 2 Department of Chemistry, Saurashtra University, Rajkot 360005, Gujarat, India. Email: khatri_taslim@yahoo.com Received on 06-12-2012 Accepted on 18-12-2012 Abstract Citrullus colocynthis (L.) a member of Cucurbitaceae family, was collected from the different areas of the Kachchh region. Eugenol was isolated from the fruit pulp of Citrullus colocynthis (L.) by continuous and successive soxtherm extraction with petroleum ether (60-80 C), chloroform, ethyl acetate and methanol. The extract was isolated on preparative Thin Layer Chromatography (TLC); characterized mainly 1 H NMR spectroscopy. The antimicrobial activity was measured. The eugenol has been reported in the fruit pulp of this species for the first time. Keywords: Soxtherm extraction, preparative thin layer chromatography, 1 H NMR, antimicrobial screening. Introduction Citrullus colocynthis (L.) a member of Cucurbitaceae family, is a desert plant with a rich history as an important medicinal plant and as a source of valuable oil. It is commonly known as bitter apple, colosynth or wild gourd is used as an abortifacient, cathartic, purgative and vermifuse, and for the treatment of fever, cancer, amenorrhea, jaundice, leukemia, rheumatism, tumour and as an insect repellant. 1 It is interesting to note that one of the folk cancer remedy made from this plant contains three antitumor ingredients: cucurbitacin B (active against PS-134 and KB tumor systems), cucurbitacin E (active against LL and KB system) and the D-glucoside of β-sitosterol (active against CA, LL and WA tumors) 2 and the leaf extract of the plant (800mg/Kg) exhibited anti-inflammatory activity in the carrageenan-induced paw oedema test in rats 3 but was severely toxic (60% of animals died, the remainder had severe diarrhoea and hepatorenal damage). 4 IJPT Jan-2013 Vol. 4 Issue No.4 5004-5010 Page 5004
Previous chemical investigations of compounds from this plant showed that it mainly contains fatty acids, 5-8 cucurbitacins, 9-10 flavones 11-12 and flavanoid glycosides. 13 The search for bioactive metabolites and chemical constituents from natural sources has been an ongoing project in our laboratory. The C. colocynthis of the desert area of Kachchh has not been evaluated for the bioactive compounds. We therefore tested fractions obtained from a crude extract of the fruit pulp of C. colocynthis for antibacterial activity. Positive results prompted us to further evaluate the chemical constituents of the fruit pulp of C. colocynthis. Herein we report the structure elucidation of eugenol (structure 1) from C. colocynthis fruit pulp that has not been previously reported from the fruit pulp of C. colocynthis. Structure-1: Eugenol (4-allyl-2-methoxyphenol). Materials and Methods Instrumentation Soxtherm extraction system (Gerhardt, Germany), TLC was performed on silica gel (Kieselgel 60, F 254 ) of 0.25 mm layer thickness. Compounds were detected by observing under UV cabinate. The Elemental analysis was performed on Elemental Vario EL III Carlo Erba 1108 model. The 1 H NMR was performed on Bruker Ac 500MHz. Plant material The samples were collected from the various part of the Kachchh district of Gujarat (India), during August- September 2010. The collected plant material was cleaned with tap water and pulp was separated from epicarp and seeds. The pulp was dried under the shade until complete removal of water from it. Such dried pulp was powdered using blender and stored in air tight container for the further use. Soxtherm extraction In the soxtherm extraction the first step, the immersion of the thimble including the sample in boiling solvent (also called hot extraction) accelerates the extraction procedure considerably versus e.g. the traditional Soxhlet method where the extracting solvent is cold, because it s dripping back from the reflux IJPT Jan-2013 Vol. 4 Issue No.4 5004-5010 Page 5005
condenser. Following this initial extraction, the extract solution is concentrated by evaporation within defined limits and continues to be extracted in the rising vapor and in the cold condensate from the refluxing solvent (basically the traditional Twisselmann). The resulting extract is taken to dryness. The drying of the beakers is carried out in the traditional way. By using the technique of the hot extraction the extraction time is reduced considerably. 10 gm of powdered fruit pulp of C. colocynthis was taken in each filter thimble (33 x 80 mm, total 6 thimbles) and continuously extracted successively with petroleum ether (60-80 C), chloroform, ethyl acetate and methanol (each solvent was taken 140ml at a time) in Soxtherm extraction apparatus for 4 to 6 hours. 14 The criteria for the extraction are shown in table 1. The solvent from the extracted material was removed under reduced pressure and the residues were dried in vacuum to remove trace of solvents. The residue so obtain was weighed and percentage of extraction was recorded. Table-1: The criteria for the extraction a. Parameters Petroleum Chlorofor Ethyl Methanol ether (60-80 C) m acetate T-Classification 200 o C 200 o C 200 o C 200 o C Extraction temp. 80 o C 80 o C 80 o C 80 o C Reductioninterval 1min 30s 1min 30s 1min 30s 1min 30s Reduction pulse 3s 3s 3s 3s Hot Extraction 0h 30mins 0h 40mins 0h 30mins 0h 40mins Evaporation A 5 x interval 5 x interval 5 x interval 5 x interval Extraction time 2h 30min 3h 30min 2h 30min 3h 30min Evaporation B 5 x interval 5 x interval 5 x interval 5 x interval Evaporation C 10min 10min 10min 10min Program length 3h 25min 4h 35min 3h 25min 4h 35min a The air pressure for all the solvents was maintained 4.5 to 4.9 bar. IJPT Jan-2013 Vol. 4 Issue No.4 5004-5010 Page 5006
Isolation of eugenol using preparative TLC On the basis of literature survey various solvent systems were applied in order to get optimum separation on TLC plates. 10,15 The maximum separation of the extracted components was achieved, with solvent system Hexane: Ethyl acetate [17:3]. The R f value was found to be 0.25 for the separated single band. After getting optimum separation on small scale, the preparative TLC was performed. Melting point was measured for the isolated compound; the characterization was mainly done by elemental analysis and 1 H NMR analysis. Antimicrobial screening The isolated compound was tested for its antibacterial and antifungal activity (MIC) in vitro by broth dilution method 16-18 with two Gram-positive bacteria Staphylococcus aureus MTCC 96, Bacillus Cereus MTCC 10650, two Gram-negative bacteria Escherichia coli MTCC 442, Pseudomonas aeruginosa MTCC 441 and three fungal strains Candida albicans MTCC 227, Aspergillus niger MTCC 282, Aspergillus clavatus MTCC 1323 taking ampicillin, chloramphenicol, ciprofloxacin, norfloxacin, nystatin, and griseofulvin as standard drugs. Serial dilutions of the test compounds and reference drugs were prepared in Muellere-Hinton agar. Drugs (10 mg) were dissolved in dimethylsulfoxide (DMSO, 1 ml). Further progressive dilutions with melted Muellere-Hinton agar were performed to obtain the required concentrations of 1.56, 3.12, 6.25, 10, 12.5, 25, 50, 62.5, 100, 125, 250, 500 and 1000 µg ml -1. The tubes were inoculated with 10 8 cfu ml -1 (colony forming unit ml -1 ) and incubated at 37 ºC for 24 h. The MIC was the lowest concentration of the tested compound that yields no visible growth (turbidity) on the plate. To ensure that the solvent had no effect on the bacterial growth, a control was performed with the test medium supplemented with DMSO at the same dilutions as used in the experiments and it was observed that DMSO had no effect on the microorganisms in the concentrations studied. Result and Discussion The isolated material was a clear, pale yellow, oily liquid which was slightly soluble in water and soluble in organic solvents. The boiling point of compound was 252-254ºC. Eugenol (structure 1) was determined as C 10 H 12 O 2 on the basis of Elemental analysis Anal. Calcd. : C, 73.15; H, 7.37; O, 19.49 %. Found: C, 73.11; H, 7.32; O, 19.43 %. 1 H NMR (500 MHz, DMSO-d 6, δ / ppm ): 3.885 (3H, s, OCH 3 ), 6.681 (1H, d, aromatic, J =10 Hz), 6.764 (1H, s, IJPT Jan-2013 Vol. 4 Issue No.4 5004-5010 Page 5007
aromatic), 6.836 (1H, d, aromatic, J =10 Hz), 5.322 (1H, s, OH), 3.285 (2H, d, -CH 2 -, J =6.5 Hz), δ H 5.865 (1H, m, -CH=), δ H 5.002 (1H, d, =CH 2, J =7.5 Hz), 5.051 (1H, d, =CH 2, J =10.5 Hz)]. As shown in Table 2, eugenol is effective against B. Cereus and E. coli (minimum inhibitory concentration [MIC] = 20 µg ml -1 ). Eugenol also shows inhibition toward other microorganisms assayed (Table 2). Table-2: Antibacterial and antifungal activity of eugenol. Name of the compound/drugs Minimal inhibition concentration (µg ml -1 ) Gram-positive Gram-negative Fungal species S.au- B.ce- E.- P.aeru- C.alb- A.nig-er A.cla- reus reus coli ginosa icans vatus Eugenol 100 20 20 250 >1000 >1000 >1000 Ampicillin 250 100 100 100 - - - Chloramphenicol 50 50 50 50 - - - Ciprofloxacin 50 50 25 25 - - - Norfloxacin 10 10 10 10 - - - Nystatin - - - - 100 100 100 Greseofulvin - - - - 500 100 100 One can collect more information about the biological effects of eugenol, and can also find new areas of therapeutic applications. A detailed knowledge of eugenol pharmacology could be utilized to consider eugenol as a lead molecule for the development of new drugs with enhanced therapeutic efficacy. Conclusion Present study of Citrullus colocynthis fruit pulp indicated that it contains biologically active compound eugenol. The properties of eugenol probably contribute, at least to some extent, to the pharmacological and traditional medicinal uses of the Citrullus colosynthis. Moreover, the soxtherm extraction performed on the C. colocynthis fruit pulp shows that it reduces extraction time remarkably. IJPT Jan-2013 Vol. 4 Issue No.4 5004-5010 Page 5008
Acknowledgments Taslimahemad T. Khatri* et al. /International Journal Of Pharmacy & Technology We are thankful to Dr. B A Golakia - Head, Food Testing Laboratory -Junagadh Agriculture University, Junagadh for providing Soxtherm facility. We are also thankful to KSKV Kachchh University-Bhuj for providing basic facilities for the research. References 1. Duke. Dr. Duke s Phytochemical and Ethnobotanical Databases, Ethnobotanical uses of Citrullus colocynthis (Cucurbitaceae) 2006. 2. Z. Feng, H. W. Yeung, Journal of Biochemistr, 1991, Vol 23, pp561-567. 3. I. A. Wasfi, A. K. Bashir, A. A. Abdalla, N. R. Banna, M. Tanira, Antiinflammatory activity of some medicinal plants of the United Arab Emirates. International Journal of Pharmacognosy, 1995, Vol 33(2), pp124-128. 4. I. A. Wasfi, Some pharmacological studies on Citrullus colocynthis. Journal of Herbs, Spices and Medicinal Plants, 1994, Vol 2(2), pp65-79. 5. R. A. Flath, Journal of Agric. Food Chemistry, 1978, Vol 26, pp835-837. 6. X. J. Faug, Journal of Natural Produc, 1984, Vol 47, pp988-993. 7. R. Gawarikar, Indian Journal of Chemistry. Section-B, 1994, Vol 33, pp877. 8. Vandevoorde, J. Med. Chem., 2003, Vol 46, pp1440-1448. 9. E. Hardegger, Helv. Chem. Acta., 1963, Vol 46, pp1166-1171. 10. M. J. Fadimatou, S. F. Kimbu, B. L. Sondengam, H. K. Mahmud Tareq, C. M. Iqbal, Atta-Ur-Rahman, Acta Pharmaceutica Sciencia. 2010, Vol 52, pp328-334. 11. S. H. Piper, Biochem. Journal, 1934, Vol 28, pp2175-2188. 12. G. T. Maatooq. Phytochemistry, 1997, Vol 44, pp187-190. 13. D. Abbas, G. Simon, R. K. Ali, N. Hossein, M. Masoud, N. Lutfun, D. S. Satyajit, DARU. 2006, Vol 14(3), pp109-114. 14. Qian et al, United State Patents. US 6,429,021, 2002. 15. C. M. Mahesh, P. Vidya, Isolation and Identification of Flavonoid "Quercetin" from Citrullus colocynthis (Linn.) Schrad. Asian J. Exp. Sci. 2008, Vol 22(1), pp137-142. 16. D. H. Isenberg, Essential Procedure for Clinical Microbiology, American Society for Microbiology, IJPT Jan-2013 Vol. 4 Issue No.4 5004-5010 Page 5009
Washington, 1998. Taslimahemad T. Khatri* et al. /International Journal Of Pharmacy & Technology 17. National Committee for Clinical and Laboratory Standards (NCCLS), Method for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically Approved Standard, fourth ed. NCCLS, Villanova, Italy (1997) Document M 100-S7. S100-S157 18. J. R. Zgoda, J. R. Porter, Pharm. Biol, 2001, Vol 39, pp221-225. Corresponding Author: Taslimahemad T. Khatri 1 *, Email: khatri_taslim@yahoo.com IJPT Jan-2013 Vol. 4 Issue No.4 5004-5010 Page 5010