Francis Shantha Margaret, Balasubrahmaniyan Ranjini, Arunachalam Ambethkar, Chockalingam Uma Maheswari and Natesan Selvaraj, In Vitro Antimicrobial Activity of Anguria L. () - The Ethnomedicinal , Int.J.Curr.Biotechnol., 2014, 2(8):1-6.

International Journal of Current Biotechnology ISSN: 2321 - 8371 Journal Homepage : http://ijcb.mainspringer.com

In Vitro Antimicrobial Activity of Cucumis Anguria L. (Cucurbitaceae) - The Ethnomedicinal Plant

Francis Shantha Margaret, Balasubrahmaniyan Ranjini, Arunachalam Ambethkar, Chockalingam Uma Maheswari and Natesan Selvaraj*

Plant Tissue Culture Laboratory, Department of Botany, Periyar E.V.R. College (Autonomous), Tiruchirappalli 620 023, Tamil Nadu.

ARTICLE INF O ABSTRACT

Article History: The in vitro antimicrobial activity of Cucumis anguria L. callus extract was studied Received 5 August 2014 against selected pathogenic fungi and bacteria, by well diffusion method. Callus was Received in revised form 9 August 2014 extracted using Chloroform, Acetone, Methanol, Benzene, and Petroleum ether. The Accepted 19 August 2014 callus extracted with methanol was most effective than other solvents used against Available online 30 August 2014 pathogenic bacteria Escherichia coli, Streptococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella paratyphi and fungi Fusarium oxysporum, Aspergillus fumigatus, Candida albicans and Aspergillus flavus, Aspergillus niger. Acetone extracts showed maximum inhibitory activity (34.9mm) against Streptococcus aureus , whereas for fungal methanol extract exhibiting highest zone of inhibition (8.6 Key words: mm) against Candida albicans. Chloroform extract showed moderate range of Antimicrobial activity, Cucumis anguria, Callus extract, Minimum Inhibitory Con- inhibitory activity against bacterial pathogens and fungal pathogens. Benzene and centration. Petroleum ether extracts were inefficient in inhibiting the bacterial and fungal growth, when compared to other extracts.

Introduction for discovering superior drugs against microbial Over the years have been used in medicine for infections (Srinivasan et al., 2001). both preventive and curative therapies. The records of native knowledge from various parts of the world Cucumis anguria is one of the under utilized traditional represent on age long tradition of plants being the major medicinal plants. Cucumis anguria is of African origin bio resource base for health care (Stepp and Moerman, arise in the wild of East and Southern . It can yield 2001). bitter or non-bitter . Immature fruits are utilized as fresh . Bitter forms of Cucumis anguria are Enteric bacteria include Escherichia coli. Shigella spp., used traditionally as a organic pesticide in crops in Klebsiella spp., Salmonella spp., Staphylococcus . The plant has diverse medicinal applications aureus, Vibrio cholera and Pseudomonas spp , which (Herin et al., 2012, Figueroa et al., 2009). In folk medicine are major etiologic factors of sporadic and epidemic the plant is used to cure stomach pain and to reduce diarrhea in both children and adults. W.H.O announced oedema. The is believed to treat jaundice if consumed that 80% populations depend mainly on traditional (Senthil Kumar and Kamaraj, 2011). Juice prepared from remedy, require the use of plant extracts and their active leaves are applied to freckles in . Kidney ingredients (Tambekar et al., 2007). complications are treated through preparing a decoction in Colombia. The previous investigation concluded that Fungal infections remain a significant cause of morbidity the antibacterial and antifungal activities the ethanolic and mortality despite of new antifungal agents (McNeil fruit extracts are having strong activity against all the et al., 2001). More than 25% of the world cereals are bacterial and fungal organisms. So Cucumis anguria contaminated with known mycotoxins and more than 300 callus extract with methanol can be used as antimicrobial fungal metabolites are reported to be toxic to man and agents. The present work aimed to find out the new animals (Galvano et al., 2001). Therefore, researchers therapeutic compound from callus extract of Cucumis are increasingly turning their attention to folk medicine, anguria. The result can be lending credence to the folkloric use of this plant for curing microbial infection.

*Corresponding author. Email address: [email protected] Mobile: 9442411555 1 Int.J.Curr.Biotechnol. Volume 2; Issue 8; August, 2014 Materials and Methods inhibitory concentration (MIC), for the most active Plant materials component was recorded after 48 hours. Seeds of West Indian Gherkin (Cucumis anguria L.) variety AJAY F1 were obtained from Nunhems India Pvt. Results and Discussion Ltd., Andhra Pradesh, India. Mature seeds were used as Traditionally leaf extracts of many plants has been used explants source. Surface sterilization of seeds by washing to treat fungal infection (Villasenor et al., 2002). The the seeds with 3-5 drops of “Teepol” brand (commercial present study revealed that the dried callus extract of bleach solution, 0.6% sodium hypo chloride, Rickitt and Cucumis anguria shown to possess both antibacterial Benckiser Ltd., Kolkatta, India) for 5 minutes and rinsed as well as antifungal properties. The methanolic extract with distilled water five times to remove the soap solution. was the most effective among the five crude extracts The fungal contaminations were eliminated by soaking tested against bacterial organisms as shown in Table 1. the seeds in 70% ethyl alcohol for 30 seconds and then The tested microbial organisms showed with the MIC of rinsed with sterile distilled water for three to five times. varying degrees of antimicrobial activities in the examined Then the seeds were surface sterilized with 0.1% (w/v) plant extracts. Methanol extract showed strong activity mercuric chloride solution for three minutes. Finally the of inhibition against E.coli (18.0 mm), Streptococcus seeds were rinsed with sterile distilled water for five times aureus (28.0 mm), Klebsiella pneumonia (10.0 mm), and left to air dry. The explants were treated with the Pseudomonas aeruginosa (8.1 mm) and Salmonella fungicide (Bavistin) for 15 minutes and rinsed again with paratyphi (6.4 mm). The acetone extract of callus extract sterile distilled water for 3-5 times. of C.anguria showed maximum zone of inhibition against E.coli (11.8 mm) followed by Streptococcus aureus (3.4 The surface sterilized seeds were aseptically inoculated mm), Klebsiella pneumonia (6.4 mm), Pseudomonas on MS basal medium (Murashige and Skoog, 1962) for aeruginosa (4.5 mm) and Salmonella paratyphi (6.7 germination. The inoculated seeds were kept in dark for mm).The petroleum ether extract showed moderate zone 2 days to render uniform germination and then in light of inhibition against E.coli (7.0 mm), Streptococcus (18/6 photoperiod) with the intensity of 50 µmol m-2 s-1 aureus (6.2mm), Klebsiella pneumonia (3.4 mm), for subsequent days provided by cool white light. The Pseudomonas aeruginosa (8.0 mm) and Salmonella in vitro leaves of Cucumis anguria L. were collected paratyphi (9.2 mm). Extracts of benzene and chloroform from 35 day - old plants. They were cultured on MS were ineffective in inhibiting the bacterial growth (data basal medium containing 2, 4-D (2.0 mg/l) for callus not shown). induction. Then the callus was air dried under sterile conditions in shade, powdered and used for the The methanol extract of C. anguria callus tissue has experimental work. shown good antifungal properties for all the fungal strains tested in this work. The growth of aflatoxin producing Test organisms toxigenic Aspergillus flavus, Aspergillus fumigates and Five bacterial and five fungal species were used as test Aspergillus niger were totally inhibited at 100 and 150 organisms. These include two gram positive bacteria such µg/ml concentration (Table 2). The total inhibition rate as Streptococcus aureus and Klebsiella pneumonia and for A. flavus can be comparable to Gentamycin used as three gram negative bacteria, Escherichia coli, control. For plant pathogenic fungi Fusarium oxysporum, Salmonella paratyphi and Pseudomonas aeruginosa. 75% total inhibition of growth was noticed at 500 µg/ml. Fungal organisms like Candida albicans, Fusarium This total inhibition was comparable to control. oxysporum, Aspergillus niger, A. fumigatus and A. flavus. The fresh pure cultures were collected from K.A.P The human pathogenic fungal strain, Candida albicans Vishwanathan College of Medical Science, was subjected to a 100 percent of inhibition of growth at Tiruchirappalli, India. 150 µg/ml concentrate of extract. Whereas 75 percent of inhibition was observed for 100 µg/ml concentration of Preparation of extracts extract and this was comparable with the control.Similar About 15g each of dried powdered callus of Cucumis results were also reported in methanol extract of C. anguria were separately extracted with five different anguria fruits at 500 µg/ml against Aspergillus, solvents namely Acetone, Benzene, Petroleum ether, Pencillium, Microsporum and Trichophyton (Senthil Methanol and Chloroform. The crude extract yields were Kumar and Kamaraj, 2011, Anusharaj et al., 2011, tested for antibacterial and antifungal activities. Abubacker et al., 2008, Britto ., and Steena, 2013). The leaf and fruit extracts of methanol has been reported to Antibacterial assay have antifungal activities against Candida albicans, Antibacterial activities of the extracts were tested using Proteus, E.coli, Fusarium oxysporium and Cryptococcus the Agar diffusion method (Collins et al., 1989). Varying neoformans (Jigna et al., 2007, Philip et al., 2009, Jigna et concentrations, from 100 µg/ml up to 500 µg/ml of the al., 2009). The present work proved that the callus extract were used and Muller Hinton Agar is used as test methanol extract at 150 µg/ml has good antifungal medium for antibacterial assay. The plates were incubated activities against C. albicans. overnight or 24 hours at 37º C and the inhibition zone were measured. The minimum inhibitory concentration The MIC values of the callus extract varied from 100 µg/ (MIC) for the most active component was recorded for ml to 500 µg/ml for the fungi tested. The activity of the each microbe after 24 hours. extract on these fungi at different concentrations decreased with increasing concentrations of the extract. Antifungal assay The possibility for developing antimicrobials from higher Antifungal activities of the extracts were tested using plants will lead to the development of a modern drug to the Agar dilution method (Collins et al., 1989). The extract act against microbes. Further research is necessary to were prepared at different concentrations (100 µg/ml to determine and identify the antibacterial compounds of 500 µg/ml) and tested in potato dextrose agar medium. C. anguria and also to determine its full spectrum of in Inhibition of growth was noted, after the plates were vitro antimicrobial evaluation of this species. incubated at 25º C for 24 to 48 hours. The minimum

Volume 2; Issue 8; August, 2014 Int.J.Curr.Biotechnol. 2 3 Int.J.Curr.Biotechnol. Volume 2; Issue 8; August, 2014 mm). L. against various bacterial strains. bacterial various against L. Cucumis anguria Cucumis nhibition Zones (ZI) representing sensitivity of extracts against bacterial cultures in millimeter ( millimeter in cultures bacterial against extracts of sensitivity representing (ZI) Zones nhibition I Table 1: Antibacterial activity of callus extracts of of extracts callus of activity Antibacterial 1: Table

Volume 2; Issue 8; August, 2014 Int.J.Curr.Biotechnol. 4 ). L Cucumis anguria Cucumis Table 2: Antifungal activity of callus extract of extract callus of activity Antifungal 2: Table nhibition Zones (ZI) representing sensitivity of extracts against fungal pathogens in millimeter (mm millimeter in pathogens fungal against extracts of sensitivity representing (ZI) Zones nhibition I

5 Int.J.Curr.Biotechnol. Volume 2; Issue 8; August, 2014 References Abubacker MN, Ramanathan R., and Senthil Kumar T., 2008. In vitro antifungal activity of Cassia alata Linn. flower extract. Natural Product Radiance 7(1): 6-9. Anusharaj Chandrashekar R., Prabhakar, Adake, Rao SN, and Santanusaha, 2013. Wrightia tinctoria: an overview. Journal of drug delivery and Therapeutics. 3(2):196-198. Britto A., and Steena RS., 2013. Phytochemical evaluation and in vitro antibacterial screening of Wrightiatinctoria (Roxb.) R. Br. Against enteric pathogens. Intern. Jour. Pharmac. Che. Biol. Sci. 3(2): 210-214. Collins CH., Lyne PM., and Grange JM., 1989. Microbiolagical methods. Sixth edition Butterworths and Company Ltd., London. Figueroa BM., Tittonell P., Giller KE., and Ohiokpehai O., 2009. The contribution of traditional to household food security in two communities of Vihiga and Migori Districts, Kenya. Acta Hort. (ISHS), 806: 57- 64. Galvano F., Piva A., Ritieni A., and Galvano G., 2001. Dietary strategies to counteract the effect of mycotoxins: A review. Jour. Food Protection 64: 120-131. Herin Sheeba Gracelin D., Selvin Samuel A., and Pauldurai Samraj A., 2012. Gherkin (Cucumis anguria L.) - a potential crop for bioprocessing. Journal of chemical,Biological and Physical Sciences. 2: 345-349. Jigna P., Sumitra V.,Chanda., 2007. In vitro antimicrobial activity and phytochemical analysis of some Indian Medicinal Plants. Tur. J. Biol. 31: 53-58. Jigna Parekh and Sumitra Chanda., 2008. In vitro antifungal activity of methanol extracts of some Indian Medicinal Plants against pathogenic yeast and moulds. Afr.J. Biotechnol. 7(23): 4349-4353. McNeil MM., Nash SL., Hajjeh RA., Phelan MA., Conn LA., Plikaytis BD., and Warnock DW., 2001.Trends in mortality due to invasive mycotic diseases in united states, 1980-1997. Clin. Infect. Dis. 33(5): 641-647. Murashige T. and Skoog F., 1962.A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiol. 15: 473-497. Philip K., Sri Nurestri Abd Malek., Sani Wirakarnain., Sim Kae Shin., and Saravana Kumar., Hong Sok Lai.,Lee Guan Serm and Syarifah., Rahman N.S.A., 2009. Antimicrobial activities of some medicinal plants from Malaysia. Am. J. Applied. Sci. 6(8): 1613-1617. Senthil Kumar S., and Kamaraj M., 2011.Antimicrobial activity of Cucumis anguria L. by agar well diffusion method. Botany Research International 4(2): 41-42. Srinivasan D., Sangeetha N., Suresh T., and Perumalsamy PL., 2001. Antimicrobial activity of certain Indian Medicinal plants used in folkloric medicine. J. Ethno Pharmacol. 74: 217-220. Stepp JR., and Moerman DE., 2001. The importance of weeds in ethno pharmacology J. Ethno pharmacol. 75: 19 – 23. Tambekar DH., Khante BS., Dahikar SB., Zarey VM., 2007. Antibacterial properties of contents of triphala: A traditional Indian Herbal Preparation. Continental J. Microbiology. 1(3): 8-12. Villasenor IM., Caulas AP., Pescua MP., Saband MN., and Soliven LA., 2002. Bioactivity studies on Cassia alata Linn. Leaf extracts. Phytother. Res. 6 (1): 893 – 896.

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