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- Short communication

Antimicrobial, Antioxidant and Cytotoxic Activities of Citrus Hystrix DC. Fruits
Akhtaruzzaman Chowdhury1, Md. Ashraful Alam1, Mohammad S. Rahman2, Md. Aslam Hossain2 and Mohammad A. Rashid2,3
Chemistry Research Laboratory, Department of Chemistry, Rajshahi University of Engineering and Technology, Rajshahi, Bangladesh 2 Phytochemical Research Laboratory, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh 3 Centre for Biomedical Research, University of Dhaka, Dhaka-1000, Bangladesh
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Citrus hystrix DC. (Bengali name- Satkara; Family-Rutaceae) is a small and bushy tree, about 3-5 m tall, which grows well in Jayantapur, Gowainghat and Moulobibazar of Bangladesh,1 Khasia hills of Assam, India2 and South East regions of Asia. It has folkloric reputation to be used in flu, fever, hypertension, abdominal pains and diarrhoea in infants.3 The fruits are used as pickle as well as in cooking. The fruit juice is rubbed onto the skin to soften or mixed with bath water to control body odor.4 It is known as medicinal lime for killing land leeches as an anti-leech rub.5 The fruits are also used in shampoo as an insecticide for washing the head as a hair shampoo.6 In addition to be used in cosmetic products, the fruits have been reported to have antiinflammatory and anti-fertility effects.7,8 The stem bark of C. hystrix showed mild to moderate antimicrobial activity9 while the methanolic extract of leaves is known to inhibit the herpes virus3 and also used as mosquito repellent.10 Previous phytochemical studies on the leaves of C. hystrix led to the isolation
Correspondence to: Mohammad A. Rashid Tel: 880-2-8612069, 9661900-73, Extn.4363, 4364, 8137; Fax: 880-2-8612069, 8615583; E-mail: rashidma@univdhaka.edu, rashid_phdu@yahoo.com , akhtarcy@yahoo.com

of antitumor glyceroglycolipids, 1,2-di-O-αlinolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (DLGG) and 1-O-α-linolenoyl-2-palmitoyl-3galactopyranosyl-sn glycerol ( LPGG)11 as well as αtocopherol.12 The fruit oil contained citronellal, geranial and d-limonene.7 In this investigation, we are reporting the antimicrobial, antioxidant and cytotoxic activites of the fruits of C. hystrix for the first time. The fruits of C. hystrix were collected from the Bandar bazar of Sylhet in the month of December, 2008. A voucher specimen (accession no.-34181) for this collection has been deposited in Bangladesh National Herbarium, Dhaka. The dried and powdered fruits of C. hystrix (700 g) were soaked in 2.0 L methanol for 7 days with occassional shaking and stirring and filtered through a cotton plug followed by Whatman filter paper number-1. The extract was then concentrated by using a rotary evaporator at reduced temperature and pressure. A portion (5.0 g) of the concentrated methanol extract (ME) was fractioned with n-hexane, carbon tetrachloride and dichloromethane by the modified Kupchan partitioning method.13 Evaporation of solvents yielded n-hexane (HX, 1.75 g), carbon tetrachloride (CT, 0.425 g), dichloromethane (DCM, 0.475 g) and aqueous soluble (AQ, 2.35 g) materials.

Dhaka Univ. J. Pharm. Sci. 8(2): 177-180, 2009 (December)

(ABSsample / ABScontrol)] x 100 .66 ± 1.33 ± 1.14 Solutions of known concentration (mg/ml) of the test samples were made by dissolving measured amount of the samples in calculated volume of solvents.00 ± 1.52 36. The antimicrobial activity of the test agents was determined by measuring the diameter of zone of inhibition expressed in mm.33 ± 1.178 The antimicrobial activity of the extractives was determined against the test organisms (Table 1) by the disc diffusion method.57 35.00 10.33 ± 0.33 ± 0.58 38.66 ± 1.00 ± 1.00 ± 1.66 ± 0. The test material having antimicrobial activity will show a clear.50.98 µg/ml were obtained by serial dilution technique.00 ± 1.52 11. 250.33 ± 1.66 ± 0. Solution of varying concentrations such as 500.57 20. These plates were kept at low temperature (4ºC) for 24 hours to allow maximum diffusion of the test materials and kanamycin.00 ± 1.66 ± 0.52 15.52 13. megaterium B.23 12.23 13.00 ± 1. distinct zone of inhibition was visualized surrounding the discs.00 ± 1.00 ± 1.33 ± 0.33 ± 0.23 13.57 34.33 ± 1. The absorbances were determined at 517 nm and from these values the corresponding percentage of inhibitions were calculated by using the following equation: % inhibition = [ 1.00 17.33 ± 0.15 In this experiment.00 17.00 ± 1. 1.66 ± 1.00 ± 1.66 ± 1. Test microorganisms Gram positive bacteria Bacillus cereus B.64 21.70 19.15 14.33 ± 1.66 ± 0..66 ± 0.66 ± 0.00 10.90 17.50 21.33 ± 0.00 10. Boydii Vibrio mimicus V.00 12.41 15. Dried and sterilized filter paper discs (6 mm diameter) were then impregnated with known amounts of the test substances using micropipettes and the residual solvents were completely evaporated.66 ± 1.00 9.D. ( n = 3).15 13.00 ± 1.66 ± 2.65 The diameters of zones of inhibition are expressed as mean ± S.00 ± 1.52 18.66 ± 1.00 ± 1.66 ± 1.64 35.00 ± 1.33 ± 0. subtilis Staphylococcus aureus Sarcina lutea Gram negative bacteria Escherichia coli Pseudomonas aeruginosa Salmonella paratyphi S.08 38.66 ± 1.57 15.00 17.08 13.62 17.23 11.0 mg of each of the extract was dissolved in methanol. 15.81. parahemolyticus Fungi Candida albicans Aspergillus niger Sacharomyces cerevaceae ME 21.66 ± 0.00 ± 1.52 37.33 ± 0.33 ± 3.57 23.66 ± 0. AQ: aqueous soluble of methanlic extract fraction.66 ± 0. The plates were then incubated at 37ºC for 24 hours to allow maximum growth of the organisms.00 ± 1. followed by evaporation) were used as positive and negative control.66 ± 1.00 15.52 Diameter of zone of inhibition (mm) CT DCM AQ 11.66 ± 0.66 ± 0.66 ± 0.08 16.00 35.57 11.15 9.33 ± 0.66 ± 1.1diphenyl-2-picrylhydrazyl (DPPH) was determined by the method developed by Brand-Williams et al.52 20.00 33.00 ± 1. 31.57 15.23 15.57 16. 2. hystrix fruits at 400 µg/disc.00 35.00 17.15 9.33 ± 1.15 20. 62.33 ± 0.00 9.00 ± 1.00 ± 1.41 10. respectively.23 18.33 ± 1.62.00 35.66 ± 3.00 11.33 ± 1. Standard discs of kanamycin (30 µg/disc) and blank discs (impregnated with solvents Chowdhury et al.25.57 20. Discs containing the test materials were placed onto nutrient agar medium uniformly seeded with the test microorganisms.33 ± 1.08 34.33 ± 1.00 18. Antimicrobial activity of extractives of C.95. 1995. An aliquot of two ml of the extract in methanol was mixed with 3 ml of a DPPH-methanol solution (20 µg/ml) and was allowed to stand for 20 minutes for the reaction to occur.66 ± 2.23 34.91. CT: carbon tetrachloride soluble fraction of the methanolic extract.23 16.33 ± 0. KAN: kanamycin (30 µg/disc) The antioxidant activity (free radical scavenging activity) of the extracts on the stable radical 1.35 11. ME: crude methanolic extract.66 ± 2.00 ± 1.66 ± 0. a diameter less than 7.00 ± 1.33 ± 1.35 KAN 35. Table 1.52 17.66 ± 0.00 10.23 15. 7. and 0.23 17.57 12.31 ± 1.00 10.52 15.00 14.66 ± 1.52 20. 125.57 16.00 ± 1. typhi Shigella dysenteriae Sh. The experiment was carried out in triplicate and the mean values were taken.61 33. CF: chloroform soluble fraction of the methanolic extract.33 ± 0.52 34.00 16.00 12. 3.15 13.00 mm was considered inactive.66 ± 0.57 15.33 ± 1.23 9.33 ± 1.33 ± 0.

00 73. Table 2 shows the antioxidant activity of the test samples. AQ: aqueous soluble fraction of methanolic extract.00 and 51. carbon tetrachloride.80 ± 0.50 32.32 ± 0.03 1. antioxidant and cytotoxicity screenings. hystrix fruits support the traditional uses of this plant in different diseases as well as its popular uses in foods and cosmetics.80 ± 0.66.50 µg/ml.00.92 ± 0. HX: n-hexane soluble fraction of methanolic extract.781 µg/ml) were obtained by serial dilution.84 ± 0. DCM: dichloromethane soluble fraction of methanolic extract. BHT: tert butyl-1-hydroxy toluene (Std). ME: crude methanolic extract. dichloromethane and aqueous soluble materials. 0.563.00 ± 2.33-21. Samples VS ME HX CT DCM AQ LC50 (µg/ml) 0.50±0.00 ± 3. However. carbon tetrachloride. This demonstrates the C. free radical scavenging activity of various fractions of the crude methanol extract was also evaluated. Antioxidant and Cytotoxic Activities Then % inhibitions were plotted against concentrations used and from the graph the IC50 was calculated by using tert-butyl-1-hydroxytoluene (BHT) as a positive control. Brine shrimp lethality bioassay of C.00.00 ± 7. hystrix extractives. 50. VS: vincristine sulphate (std).04 The values of LC50 are expressed as mean ± S. hystrix fruits.00 ± 4. ME: crude methanolic extract.07 3. 238. Following the procedure of Meyer.33-23.00 ± 7. By the way.00 The values of IC50 are expressed as mean ± S. respectively (Table 3).D. n-hexane.00 µg/ml. ( n = 3).50 ± 0.13 and 3.33-21. The LC50 were found to be 0.03.00 ± 2. 9. 6.00 30.05 0.09 1. In case of antimicrobial screening. 1. hystrix exhibited mild to strong antimicrobial activity.50.6616. hystrix as well as its Kupchan fractions demonstrated various degrees of bioactivities when subjected to antimicrobial. ( n = 3).92 ± 0. dichloromethane and aqueous soluble partitionates to brine shrimp was determined after 24 hours of exposure.00. The cytotoxicity exhibited by the methanol crude extract and its n-hexane and carbon tetrachloride soluble fractions was significant. . dichloromethane and aqueous soluble fractions were ranged from 9. The IC50 values for the methanolic crude extract and its nhexane. the lethality of methanolic crude extract and its n-hexane. The methanol extract of the fruits of C.05 µg/ml of the positive control (vincristine sulphate).84 ± 0. 100. dichloromethane and aqueous sluble partitionates were found to be 32. 12.D.04 µg/ml for methanol crude extract. DMSO solutions of the samples were applied against Artemia salina in a 1-day in vivo assay.66 and 9. 200. The experiment was carried out in triplicate and the mean values were taken. 3. the n-hexane soluble fraction was found to be insensitive to microbial growth (data not shown in Table 1).25. Antioxidant activity of extractives of C. Vincristine sulphate was used as positive control.66. The bioactivities exhibited by the extractives of C. 25.12 ± 0.00 51.00 ± 1. For this experiment.Antimicrobial. 73. The experiment was carried out in triplicate and the mean values were taken. CT: carbon tetrachloride soluble fraction of methanolic extract. dichloromethane and aqueous soluble fractions were dissolved in DMSO and solutions of varying concentrations (400. In the antioxidant assay.00 ± 3.07. 30. AQ: aqueous soluble fraction of methanolic extract.66 mm. Table 3.00 ± 1.20 ± 0. 1. In comparison with the LC50 0. n-hexane. DCM: dichloromethane soluble fraction of methanolic extract.00 ± 4. HX: nhexane soluble fraction of methanolic extract.09.12±0. respectively (Table 1). The zone of inhibition produced by the crude methanol extract and its carbon tetrachloride. respectively. carbon tetrachloride. 3. carbon tetrachloride. Samples BHT ME HX CT DCM AQ IC50 (µg/ml) 9.32 ± 0.20 ± 0. 1.00 238.13 3. Brine shrimp lethality bioassay16 technique was applied for the determination of cytotoxicity of the fruit extractives. hystrix fruits as a potential source of antioxidant.125. CT: carbon tetrachloride soluble fraction of methanolic extract. 9. 179 Table 2. the IC50 value exhibited by the standard ( BHT ) was 9. the fruit extractives of C. 4 mg of each of the methanol crude extract.

2001. Brine shrimp: A convenient general bioassay for active constituents.. Larsen. In vitro bioactivities of essential oils used for acne control. 2001.179. Dongjin and Hlaing. J. Scott. Int.V. Sing.. 15. 28. Dassanayake.L. Antibiotic susceptibility testing by standard single disc diffusion method. Z. Ulosantoin. 5. J. 31013105. Wratten. P. Br. 45.E. Murakami.O. J. Engl.W. p. and related Techno. 26. 2002. P..N. J. D. Fortin.. 377-392. 16. New Delhi. and Ong. C. Taweechaisupapong. D... Glyceroglycolipids from Citrus hystrix. Amerind Publishing Co Ltd. Food Chem. Nichols.C.N. 8. H. Agric Food Chem. 7. 2007. Chromat. C. p.. J. M. Van Wagenen. U. J. S. 43-49. 2779-2783. R.like materials from selected Myanmar Natural plants and Their antimicrobial activity. Dhaka-1000. Ling. Koshimizu. N. Dermatol. C. 140. Bauer. Chowdhury et al. 16. T. 43.1995. 426-493...D. J. Published by Rahman.. Lebnsm Will Technol. Fitoterapia. Fitoterapia. Lacobsen. 70. B. and Mohamed. N. 12. 30. Pathol.M. 502-513. Lidert. F. Use of free radical method to evaluate antioxidant activity. 2009. Ethnopharmacol. S. of Liq. S. M.. Mehedi.. 11. 1999.432-433. India.L. Cardellina. M. 73. Motijheel C/A. Daily Prothom Alo.M. 335-337.. 9. A. 1982.. and McLaughlin. Aromdee. Meyer. 1985.D. J. Piyachaturawat. Boustiea. 1995. Cuvelier.. Published by National Book Trust. J. 10. 31-32. 49. 1985. Agric. 1985. 58.C. 52. and Chanjarunee.C. Indian Fruits. W. Y. 1999. M. Kirby. Clini. 45. Pyo. Alpha-Tocopherol content in 62 edible tropical plants. 14. Repellency of volatile oils from plants against three mosquito vectors. U. a potent insecticide from the sponge Ulosa ruetzler.H.E. C. A. Aromather. 2. B. 737-738. Glinsukon. Malaysia. S.. 76-82. Planta Med.31. 6.E. K. a traditional herb in Thailand. 346-350. A. Am J.. and Arnoros. Le Bosse. Med. 13. L. 105-110. pp. Robina.promoting activity of 12-O-tetradecanoylphorpol 13-acetate in mouse skin. Phytophotodermatitis due to the application of Citrus hystrix as a folk remedy. A Revised Handbook to the Flora of Ceylon. M. .. Lertsatitthanakorn. and Techadamrongsin. Nakamura. Puam. Y. A-5 Green park. Malay ethno-medico botany in Machang. Vigora.B. Brand-Williams W.R. B. potently inhibit the tumor.. H. Kelantan. 05 June.. and Truck. Vol. and Swithenbank.. and Berset C. 25-30 4. New Delhi-110016. Ong.. 1993. Sherries.180 REFERENCES 1. D. Supercritical fluid extraction of drugs. H. and Ohigashi. A. V. 1966. Thavara. M. In vitro antiviral activity of thirty-six plants from La R eunion Island. 3. Ran dazzo. R. J. Ferringni. Antifertility effect of Citrus hystrix DC. Chem. R. Lohezic-Le.. and Khunkitti. J. J. H. Tawtsin. J. Koh. 2006. 13.C. and Nordiana.. Org.