1 Int.J.Curr.Biotechnol. Volume 2; Issue 8; August, 2014

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

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

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

Plant Tissue Culture Laboratory, Department of Botany, Periyar E.V.R. College (Autonomous), Tiruchirappalli 620023, Tamil Nadu.

A R T I C L E I N F O A B S T R A C T

Article History:Received 5 August 2014Received in revised form 9 August 2014Accepted 19 August 2014Available online 30 August 2014

Key words:Antimicrobial activity, Cucumis anguria,Callus extract, Minimum Inhibitory Con-centration.

The in vitro antimicrobial activity of Cucumis anguria L. callus extract was studiedagainst selected pathogenic fungi and bacteria, by well diffusion method. Callus wasextracted using Chloroform, Acetone, Methanol, Benzene, and Petroleum ether. Thecallus extracted with methanol was most effective than other solvents used againstpathogenic 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 Streptococcusaureus , whereas for fungal methanol extract exhibiting highest zone of inhibition (8.6mm) against Candida albicans. Chloroform extract showed moderate range ofinhibitory activity against bacterial pathogens and fungal pathogens. Benzene andPetroleum ether extracts were inefficient in inhibiting the bacterial and fungal growth,when compared to other extracts.

*Corresponding author.Email address: selvasarayu@gmail.comMobile: 9442411555

IntroductionOver the years plants have been used in medicine forboth preventive and curative therapies. The records ofnative knowledge from various parts of the worldrepresent on age long tradition of plants being the majorbio resource base for health care (Stepp and Moerman,2001).

Enteric bacteria include Escherichia coli. Shigella spp.,Klebsiella spp., Salmonella spp., Staphylococcusaureus, Vibrio cholera and Pseudomonas spp , whichare major etiologic factors of sporadic and epidemicdiarrhea in both children and adults. W.H.O announcedthat 80% populations depend mainly on traditionalremedy, require the use of plant extracts and their activeingredients (Tambekar et al., 2007).

Fungal infections remain a significant cause of morbidityand mortality despite of new antifungal agents (McNeilet al., 2001). More than 25% of the world cereals arecontaminated with known mycotoxins and more than 300fungal metabolites are reported to be toxic to man andanimals (Galvano et al., 2001). Therefore, researchersare increasingly turning their attention to folk medicine,

ISSN: 2321 - 8371

for discovering superior drugs against microbialinfections (Srinivasan et al., 2001).

Cucumis anguria is one of the under utilized traditionalmedicinal plants. Cucumis anguria is of African originarise in the wild of East and Southern Africa. It can yieldbitter or non-bitter fruits. Immature fruits are utilized asfresh cucumbers. Bitter forms of Cucumis anguria areused traditionally as a organic pesticide in crops inZimbabwe. The plant has diverse medicinal applications(Herin et al., 2012, Figueroa et al., 2009). In folk medicinethe plant is used to cure stomach pain and to reduceoedema. The fruit is believed to treat jaundice if consumed(Senthil Kumar and Kamaraj, 2011). Juice prepared fromleaves are applied to freckles in Cuba. Kidneycomplications are treated through preparing a decoctionin Colombia. The previous investigation concluded thatthe antibacterial and antifungal activities the ethanolicfruit extracts are having strong activity against all thebacterial and fungal organisms. So Cucumis anguriacallus extract with methanol can be used as antimicrobialagents. The present work aimed to find out the newtherapeutic compound from callus extract of Cucumisanguria. The result can be lending credence to thefolkloric use of this plant for curing microbial infection.

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Volume 2; Issue 8; August, 2014 Int.J.Curr.Biotechnol. 2

Materials and MethodsPlant materialsSeeds of West Indian Gherkin (Cucumis anguria L.)variety AJAY F1 were obtained from Nunhems India Pvt.Ltd., Andhra Pradesh, India. Mature seeds were used asexplants source. Surface sterilization of seeds by washingthe seeds with 3-5 drops of “Teepol” brand (commercialbleach solution, 0.6% sodium hypo chloride, Rickitt andBenckiser Ltd., Kolkatta, India) for 5 minutes and rinsedwith distilled water five times to remove the soap solution.The fungal contaminations were eliminated by soakingthe seeds in 70% ethyl alcohol for 30 seconds and thenrinsed with sterile distilled water for three to five times.Then the seeds were surface sterilized with 0.1% (w/v)mercuric chloride solution for three minutes. Finally theseeds were rinsed with sterile distilled water for five timesand left to air dry. The explants were treated with thefungicide (Bavistin) for 15 minutes and rinsed again withsterile distilled water for 3-5 times.

The surface sterilized seeds were aseptically inoculatedon MS basal medium (Murashige and Skoog, 1962) forgermination. The inoculated seeds were kept in dark for2 days to render uniform germination and then in light(18/6 photoperiod) with the intensity of 50 µmol m-2 s-1

for subsequent days provided by cool white light. Thein vitro leaves of Cucumis anguria L. were collectedfrom 35 day - old plants. They were cultured on MSbasal medium containing 2, 4-D (2.0 mg/l) for callusinduction. Then the callus was air dried under sterileconditions in shade, powdered and used for theexperimental work.

Test organismsFive bacterial and five fungal species were used as testorganisms. These include two gram positive bacteria suchas Streptococcus aureus and Klebsiella pneumonia andthree gram negative bacteria, Escherichia coli,Salmonella paratyphi and Pseudomonas aeruginosa.Fungal organisms like Candida albicans, Fusariumoxysporum, Aspergillus niger, A. fumigatus and A. flavus.The fresh pure cultures were collected from K.A.PVishwanathan College of Medical Science,Tiruchirappalli, India.

Preparation of extractsAbout 15g each of dried powdered callus of Cucumisanguria were separately extracted with five differentsolvents namely Acetone, Benzene, Petroleum ether,Methanol and Chloroform. The crude extract yields weretested for antibacterial and antifungal activities.

Antibacterial assayAntibacterial activities of the extracts were tested usingthe Agar diffusion method (Collins et al., 1989). Varyingconcentrations, from 100 µg/ml up to 500 µg/ml of theextract were used and Muller Hinton Agar is used as testmedium for antibacterial assay. The plates were incubatedovernight or 24 hours at 37º C and the inhibition zonewere measured. The minimum inhibitory concentration(MIC) for the most active component was recorded foreach microbe after 24 hours.

Antifungal assayAntifungal activities of the extracts were tested usingthe Agar dilution method (Collins et al., 1989). The extractwere prepared at different concentrations (100 µg/ml to500 µg/ml) and tested in potato dextrose agar medium.Inhibition of growth was noted, after the plates wereincubated at 25º C for 24 to 48 hours. The minimum

inhibitory concentration (MIC), for the most activecomponent was recorded after 48 hours.

Results and DiscussionTraditionally leaf extracts of many plants has been usedto treat fungal infection (Villasenor et al., 2002). Thepresent study revealed that the dried callus extract ofCucumis anguria shown to possess both antibacterialas well as antifungal properties. The methanolic extractwas the most effective among the five crude extractstested against bacterial organisms as shown in Table 1.The tested microbial organisms showed with the MIC ofvarying degrees of antimicrobial activities in the examinedplant extracts. Methanol extract showed strong activityof inhibition against E.coli (18.0 mm), Streptococcusaureus (28.0 mm), Klebsiella pneumonia (10.0 mm),Pseudomonas aeruginosa (8.1 mm) and Salmonellaparatyphi (6.4 mm). The acetone extract of callus extractof C.anguria showed maximum zone of inhibition againstE.coli (11.8 mm) followed by Streptococcus aureus (3.4mm), Klebsiella pneumonia (6.4 mm), Pseudomonasaeruginosa (4.5 mm) and Salmonella paratyphi (6.7mm).The petroleum ether extract showed moderate zoneof inhibition against E.coli (7.0 mm), Streptococcusaureus (6.2mm), Klebsiella pneumonia (3.4 mm),Pseudomonas aeruginosa (8.0 mm) and Salmonellaparatyphi (9.2 mm). Extracts of benzene and chloroformwere ineffective in inhibiting the bacterial growth (datanot shown).

The methanol extract of C. anguria callus tissue hasshown good antifungal properties for all the fungal strainstested in this work. The growth of aflatoxin producingtoxigenic Aspergillus flavus, Aspergillus fumigates andAspergillus niger were totally inhibited at 100 and 150µg/ml concentration (Table 2). The total inhibition ratefor A. flavus can be comparable to Gentamycin used ascontrol. For plant pathogenic fungi Fusarium oxysporum,75% total inhibition of growth was noticed at 500 µg/ml.This total inhibition was comparable to control.

The human pathogenic fungal strain, Candida albicanswas subjected to a 100 percent of inhibition of growth at150 µg/ml concentrate of extract. Whereas 75 percent ofinhibition was observed for 100 µg/ml concentration ofextract and this was comparable with the control.Similarresults were also reported in methanol extract of C.anguria fruits at 500 µg/ml against Aspergillus,Pencillium, Microsporum and Trichophyton (SenthilKumar and Kamaraj, 2011, Anusharaj et al., 2011,Abubacker et al., 2008, Britto ., and Steena, 2013). Theleaf and fruit extracts of methanol has been reported tohave antifungal activities against Candida albicans,Proteus, E.coli, Fusarium oxysporium and Cryptococcusneoformans (Jigna et al., 2007, Philip et al., 2009, Jigna etal., 2009). The present work proved that the callusmethanol extract at 150 µg/ml has good antifungalactivities against C. albicans.

The MIC values of the callus extract varied from 100 µg/ml to 500 µg/ml for the fungi tested. The activity of theextract on these fungi at different concentrationsdecreased with increasing concentrations of the extract.The possibility for developing antimicrobials from higherplants will lead to the development of a modern drug toact against microbes. Further research is necessary todetermine and identify the antibacterial compounds ofC. anguria and also to determine its full spectrum of invitro antimicrobial evaluation of this species.

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Volume 2; Issue 8; August, 2014 Int.J.Curr.Biotechnol. 4

Tabl

e 1: A

ntib

acte

rial a

ctiv

ity o

f call

us ex

tract

s of C

ucum

is an

guria

L. a

gain

st va

rious

bact

erial

stra

ins.

Inhi

bitio

n Zon

es (Z

I) re

pres

entin

g sen

sitiv

ity o

f ext

ract

s aga

inst

bact

erial

cultu

res i

n mill

imet

er (m

m).

5 Int.J.Curr.Biotechnol. Volume 2; Issue 8; August, 2014

Tabl

e 2: A

ntifu

ngal

activ

ity o

f call

us ex

tract

of C

ucum

is an

guria

L

Inhi

bitio

n Zon

es (Z

I) re

pres

entin

g sen

sitivi

ty o

f ext

ract

s aga

inst

fung

al pa

thog

ens i

n milli

met

er (m

m).

Volume 2; Issue 8; August, 2014 Int.J.Curr.Biotechnol. 6

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