Anti Microbial Activity

8/19/2019 Anti Microbial Activity

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Introduction

The steadily increasing drug resistant microorganismssuch as bacteria, Candida species and dermatophytes arenow a therapeutic challenge. Bacterial and fungal patho-gens have evolved numerous defense mechanisms

against antimicrobial agents, and resistance to theexisting drugs is on the rise. A study on antibioticresistance in Staphylococcus aureus clinical isolates fromdiabetic patients showed that 80 of the isolates weremultidrug-resistance to more than eight antibiotics and!" isolates were methicillin resistant S. aureus #$%&A'#%a(u et al., )0*0'. +pportunistic fungal infections arecommon among immunocompromised patients#$artine-%ossi et al., )008 &traten et al., )00!'. Theindiscriminate use of antibiotics also contributes to theworsening of this picture #&traten et al., )00!'. +ppor-tunistic fungal infections caused by Candida species produce a wide spectrum of diseases, ranging from

superficial mucocutaneous disease to systemic candidia-sis, of which C. albicans was reported to be the mostpredominant species #&hoohi et al., )0*0 &harma andBorthaur )00/'.

n ndia the data on the burden of opportunistic

mycoses is not clear though the climate in this countryis well suited for of fungal infections. 1owever,increased incidence of invasive candidiasis wasreported in ndia #2harabarti et al., )008'. 3redo-minance of dermatophyte infections has also beenreported in many countries #&harma and Borthaur)00/ 4ain et al., )008 4aiswal )00)'. Thus the clinicalefficacy of many antimicrobial drugs is being threat-ened by the emergence of multidrug-resistant patho-gens. This has led to the search for new antimicrobialsubstances in natural products particularly medicinalplants, focusing on the basis of their ethno-medicinaluses, which enhance the probability of success in

A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2014; 9: 4-9 Journal homepage: www.banglaol.info! www.bdpharmacol.comAbstracted"inde#ed in Academic Search $omplete% Agroforestry Abstracts% Asia Journals &nline% Bangladesh Journals &nline% Biological Abstracts%B'&S'S Preiews% $AB Abstracts% $urrent Abstracts% Directory of &pen Access Journals% *BAS"#cerpta *edica% +oogle Scholar% ,'-A' (/,&)%'nternational Pharmaceutical Abstracts% &pen J0gate% Science $itation 'nde# #panded% S$&P1S and Social Sciences $itation 'nde#'SS-: 233204456 (Print)! 233204477 (&nline)! D&': 24.8893"bp.3i2.254

Abstract

Antimicrobial evaluation of methanol extract of Sarcochlamys pulcherrima leafand its hexane, ethyl acetate, n-butanol and water fractions against !* strainsof microorganisms, using agar well, agar disc diffusion, and broth microdilu-tion methods, revealed the activity of methanol extract against Trichophyton mentagrophytes, Candida albicans, Staphylococcus aureus and Escheracia coli #oneof inhibition5 )*-60 mm, )00 mg7m, and $25 9.)"-"0 mg7m'. All fractionsalso displayed antimicrobial activity #"-)0 mg7m', indeed ethyl acetate and n-butanol fractions showed better activity #$25 0.*"9 to )." mg7m'. C.albicans was most sensitive to n-butanol fraction #*" mm, )." mg7m'. :thylacetate and n-butanol fractions were more active against T. mentagrophytes #*)mm at *.)" mg7m' and S. aureus #ethyl acetate-*9 mm, n-butanol-*6 mm at

0.9)" mg7m'. E. coli was inhibited by n-butanol fraction #*! mm at )." mg7m'. ;urther, n-butanol fraction #600 <g7disc' exhibited promising activityagainst *6 bacteria, ) dermatophytes and ) yeasts strains.

Article Info

%eceived5 * =ovember )0*!Accepted5 *" >ecember )0*!Available +nline5 " 4anuary )0*6

Keywords:Antimicrobial activityn-Butanol fractionSarcochlamys pulcherrima

=umber of ;igure5 *=umber of Tables5 !=umber of %efs5 )?

Correspondence: A1$e-mail5 mdaf(al*)[email protected]

This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in themanner specified by the author or licensor.

Antimicrobial activity of methanol extract and fractions fromAntimicrobial activity of methanol extract and fractions fromAntimicrobial activity of methanol extract and fractions fromAntimicrobial activity of methanol extract and fractions fromSarcochlamys Sarcochlamys Sarcochlamys Sarcochlamys pulcherrima pulcherrima pulcherrima pulcherrima

Afjal Hussain Mazumder1, Jayshree Das2, Hemanta Kumar Gogoi2,Pronobesh hatto!adhyay 2 and "atya #husan Paul1

1Department of Chemistry, Assam University, Silchar, Assam 788 011, India; 2 Defence esearch !a"oratory,#e$p%r, &ost 'a( )o* 02, Assam 78+ 001, India*



finding new drugs #&veta et al., )0*0'.

Sarcochlamys pulcherrima #%oxb.' aud. #rticaceae', is awild medicinal plant and highly consumed by some

ethnic tribes and castes of Assam, ndia. The leaves areused in treatment of boils and fever blisters, eyecomplications #%ahman et al., )00/', diarrhea and dysen-tery #&harma and 3egu, )0**'. Coung shoots, leavesand fruits are eaten as vegetable #&awian et al., )00/&ingh et al., )0*)'. t is believed that eaten with porfacilitate the digestion of fats #Buragohain, )0**'. t isalso claimed that S. pulcherrima leaves damages tapeworm egg present in por when boiled with it #3aul etal., )0*0'. n spite of its numerous medicinal propertiesthere is no report on S. pulcherrima related to antimicro-bial activity. n our earlier study, we observed promi-sing antimicrobial and antioxidant activity of the metha-nol extract of S. pulcherrima leaves. :ncouraging withthe findings, the present study was undertaen to frac-tionate the methanol extract followed by antimicrobialtest in order to identify the fractions with better activity.

Materials and Methods

Microorganism and inoculums: The test microorganismsconsisted of ? dermatophytes strains, / yeasts, *0 ram#Dve' bacteria and " ram #-ve' bacteria #Table I'.=utrient agar #=A, 1imedia' was used for culturing thebacteria. ;or dermatophytes and yeasts, sabourauddextrose agar #&>A, 1imedia'' was used. Bacterial

inoculum #* x *08 2;7m' was prepared in nutrientbroth #1imedia' #Tee et al., )0**'. >ermatophytes andyeasts were subcultured in sabouraud dextrose broth#1imedia' to obtain the inoculum density of )." x *06

2;7m #1ammer et al., )00)' and * x *08 2;7m#3areh and 2handa, )008' respectively. The bacteriaand the yeasts were incubated at !/ E )F2 and )8 E )F2respectively for 68 hours, while the dermatophytecultures were incubated at )8 E )F2 for *0 to *" days ineach experiment.

Plant: ;resh leaves of S. pulcherrima were collected fromthe Arun 3un(ee #Punjee means Tribal Gillage' of$achhal, 2achar >istrict, Assam #ndia'. The plantwas authenticated at Botanical &urvey of ndia, :astern%egional 2entre, &hillong, ndia. The herbarium wasdeposited at the herbarium repository of >efence

%esearch aboratory, Tepur, Assam.Extraction and fractionation of plant material: &hade driedpowdered leaves were extracted with methanol #1ayetet al., )008' and dried at 60F2 under reduced pressureusing rotary evaporator #1eidolph nstruments mb1H 2o. I, ermany and lyophilised #The Benchtop;reeJone plus 2ascade 6." ;reee >ry &ystem,abconco, &A'. The methanol extract was seKuentiallyfractionated with hexane, ethyl acetate, n-butanol andwater as shown in Figure 1. +n subseKuent evaporationof solvents afforded hexane #/.99 g', ethyl acetate #/.06g', n-butanol #*6.*? g' and water #)*.!/ g' fractions. Test

Table I: Test microorganisms

Dermatophytes Gram (!e" bacteria

1 Epidermophyton floccosum var. =igricans #$T22 9*!' *

)

Bacillus subtilis #$T22 *)*'

B. subtilis #$T22 66*'

# Microsporum boullardii #$T22 90"?' ! B. subtilis #$T22 9*?'

$ M. fulum #$T22 86/8' 6 B. subtilis #$T22 /!9'

% M. gypseum #$T22 )8)?' " B. subtilis #$T22 )9*9'

& M. gypseum #$T22 )8!0' 9 B. subtilis #2inical isolate'

' M. gypseum #$T22 869?' / Micrococcus luteus #$T22 *09'

Trichophyton rubrum #$T22 86//' 8 Staphylococcus aureus #2inical isolate'

)

T. rubrum #$T22 )?9' ? S. aureus sub sp. aureus #$T22 /!/'

* T. mentagrophytes #$T22 86/9' *0 S. aureus sub sp. aureus #$T22 ?9'

+easts Gram (,!e" bacteria

1 Candida albicans #2inical isolate' * Enterobacter aerogenes #$T22 ***'

# C. albicans #$T22 *8!' ) Proteus mirabilis #$T22 /6!'

$ C. albicans #$T22 !0*8' ! !ersinia enterocolitica #$T22 6868'

% C. glabrata #$T22 !0*?' 6 Salmonella enteria ser. typhi #$T22 /!!'

& C. parapsilosis #$T22 6668' " Escheracia coli #2inical isolate'

' C. tropicalis #$T22 *000'

/ Trichosporon beigelii #2inical isolate'

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samples of extract and fractions were prepared in

dimethyl sulfoxide #>$&+, w7v' and filtered using0.)) mm $illipore filter #$:LM 3, reland'.

"ntimicrobial assay and determination of minimum inhibi#tory concentration $M%C&: Antimicrobial activity wastested by agar well diffusion #Iaushi and oyal, )008'and agar disc diffusion method #Traranrungsie et al.,)008'. The culture #&>A7 =A' plates were swabbedwith *"0 m of the inoculum and loaded with *"0 mof the test extract 7 fraction into the well of 8 mmdiameter, made on the agar plate. >$&+ was used asnegative control while clotrimaole and amphotericin Bwere used as positive control for fungi and bacteriarespectively. The one of inhibition was recorded to

evaluate the antimicrobial activity. n agar discdiffusion method, sterile paper discs #Nhatman =o. *paper, " mm diameter' were impregnated with testsample #600 <g7disc', clotrimaole #*0 <g7disc',amphotericin B #*0 <g7disc' and methanol separately,dried. The discs were placed on the surface of the agarplate, preswabbed with *"0 m of inoculum andrecorded the ones of inhibition. $2 was determinedby broth microdilution method, as described earlier#1ammer et al., )00* :diri et al., )0*)' with somemodifications. Test sample was serially diluted in ?9-well microtiter plate prepared with =B or &>B to obtaina concentration ranging from !? to ",000 <g7m.

noculum concentration in each well was ad(usted as

mentioned above. $2 was interpreted as the lowestconcentration of the test sample which showed novisible growth. ;ive replicates were maintained in eachexperiment.

-esults and Discussion

As per standard protocol a small set of referencemicroorganisms, which represent common pathogenicspecies of different classes may be used in primaryantimicrobial screening #3aul et al., )009'. 1ence, fourmicroorganisms namely, T. mentagrophytes, C. albicans,S. aureus and E. coli, which represented dermatophytes,

yeast, ram #Dve' and ram #-ve' bacteria respectivelywere used in the initial antimicrobial study by agar welldiffusion assay. The methanol extract exhibited anti-microbial activity against the test pathogens exhibitingone of inhibition in between )* and 60 mm at )00 mg7m and $2 values of 9.)"-"0 mg7m #Table II'. Theram #Dve' bacterium, S. aureus was found to be moresensitive than the ram #-ve' bacterium, E. coli, whichhas been well established earlier #3aul et al., )009 4ayaraman )00? Cagi et al., )0*)'. 2lotrimaole #0.*mg7m' produced one of inhibition of !* mm againstT. mentagrophytes and */ mm against C. albicans, whilethe one of inhibition caused by amphotericin B against

Figure 1. ;ractionation of methanol extract from S. pulcherrima #leaf'

Table II: /ntimicrobial acti!ity o0 methanol etract 0rom S. pulcherrma lea!es

$icroorganism Jone of inhibition #mm' $2#mg7m' $ethanol extract

)00 mg7m 2lotrimaole #0.* mg7m'

Amphotericin B #0.* mg7m'

>$&+

T. mentagrophytes )8 !* =o one =o one 9.)"

C. albicans )* */ =o one =o one *)."0

S. aureus 60 =o one )" =o one *)."0

E. coli )) =o one )) =o one "0.00

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S. aureus and E. coli were )" and )) mm respectively.Activity of clotrimaole and amphotericin B at very lowconcentration #0.*0 mg7m' as compared to the plantextracts may be attributed to its pure nature. Thereforehigher $2s of the crude methanol extract, observed inthe present study were considered as effective. >$&+did not inhibit the growth of test pathogens. Allfractions #"-)0 mg7m' revealed activity against T.mentagrophytes, C. albicans, S. aureus and E. coli #one ofinhibition5 ?-!0 mm', although ethyl acetate and n-butanol fractions showed better activity #Table III'. ;orC. albicans, n-butanol fraction was found to be the most

active #*" mm at )." mg7m'. :thyl acetate and n-butanol fractions were more active against T.mentagrophytes #*) mm at *.)" mg7m' and S. aureus #ethyl acetate-*9 mm, n-butanol-*6 mm at 0.9)" mg7m'. ram #Dve' bacterium, S. aureus was found to bemore sensitive than ram #-ve' bacterium E. coli towards the test fractions. +nly n-butanol fractionshowed a bit activity against E. coli #*! mm at )." mg7m'. &usceptibility difference between ram #Dve' andram #-ve' bacteria may be due to the cell wall struc-tural differences between them. +uter phospholipidsmembrane with the structural lipopolysaccharide

components in ram #-ve' bacterium, mae the cell wallimpenetrable to antimicrobial agents, while the ram#Dve' bacterium is more susceptible having only anouter peptidoglycan, which is not an effectivepermeability barrier # 4ayaraman )00? Cagi et al., )0*)'.+ver all, n-butanol and ethyl acetate fraction exhibitedbetter antimicrobial profile with $2 within the rangeof 0.*"9-)." mg7m related to the tested bacteria andfungi #Table III'. The activity was more pronouncedthan the hexane and water fractions and methanol ex-tract. The results indicated that the pattern of inhibitiondepends largely upon the solvent used for fractionation

of the plant material and the organisms tested. t can beinferred that antimicrobial compounds are presentmostly in ethyl acetate and n-butanol fractions, whichmight be medium polar to polar in nature. &imilar obser-vations were also reported earlier in other medicinalplants #Tee et al., )0** >as et al., )0*0 $aumder etal., )0*)'. :arlier in a study, higher range of antiderma-tophytic activity of hexane fraction of '. gratissimum leaves was reported #&ilva et al., )00"'. 2ontrary to this,in our study the hexane fraction was found to be lesseffective. This difference may be due to differences inplant constituents in different plant species, time of

Table III: /ntimicrobial acti!ity o0 di00erent sol!ent 0ractions o0 S. pulcherrima methanol etract

;raction $icroorganism Jone of inhibition #mm', 2onc.entration- mg7m $2 #mg7m'

)0 *0 " )." *.)" 0.9)"

1exane T. mentagrophytes *" *) *0 - - - *.)"

C. albicans )6 *? *6 - - - )."

S. aureus )* )0 *8 *9 *) - *.)"

E. coli *! ** ? - - - "

: acetate T. mentagrophytes )) )0 *8 *" *) - 0.9)"

C. albicans )6 )0 *8 - - - *.)"

S. aureus )8 )/ )9 )" *8 *9 0.9)"

E. coli

*0 ? ? - - - "

n-Butanol T. mentagrophytes )) *? *8 *9 *) - 0.*"9

C. albicans !0 )9 *9 *" - - 0.9)"

S. aureus )6 )! )0 *? *" *6 0.9)"

E. coli )* )0 *8 *! - - )."

Nater T. mentagrophytes ** ? ? - - - "

C. albicans )6 )0 *" - - - )."

S. aureus *8 *9 *" ** / - *.)"

E. coli ? - - - - - )0

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sample collection or other geographical factors.

+wing to higher extractive value and the promisingactivity, n-butanol fraction was further tested against ?

strains of dermatophytes, / yeasts, *0 ram #Dve' and "ram #-ve' bacteria employing agar disc diffusionmethod to ascertain its maximum potentiality incombating a wide range of pathogenic microbes. The n-butanol fraction #600 <g7disc' exhibited broadspectrum of activity against ma(ority of the test bacteria,except B. subtilis #$T22 66*' and S. enteria ser. typhi#$T22 /!!'. The most sensitive bacterium was B.subtilis #$T22 9*?' #one of inhibition5 )0 mm',followed by S. aureus sub sp. aureus #$T22 ?9' #one ofinhibition5 *) mm'. The remaining bacteria were moder-ately sensitive #one of inhibition5 9-*) mm'. Thedermatophytes and yeasts were observed to be

comparatively less sensitive showing inhibition #one ofinhibition5 0.9 mm' of only E. floccosum ar. (igricans#$T22 9*!' and M. boullardii #$T22 90"?' and twoyeasts namely C. albicans #$T22 *8!' and C. glabrata#$T22 !0*?' #one of inhibition5 0.8 mm'. 2lotrimaole#*0 mg7disc' was effective against all test dermato-phytes #one of inhibition5 *.0-*.* mm'. Among theyeasts, C. albicans #$T22 *8!', C. glabrata #$T22 !0*?',C. parapsilosis #$T22 6668', C. tropicalis #$T22 *000'were found to be susceptible to n- butanol fraction.Three bacteria namely, M. luteus #$T22 *09' E.aerogenes #$T22 ***' S. enteria ser.typhi #$T22 /!!'were not inhibited by amphotericin B #*0 mg7disc',whereas for the remaining bacteria were found to besusceptible #one of inhibition5 9-8 mm'. The strongestantibacterial activity and a wea antifungal activity ofcrude methanol extracts from aerial parts of Penstemon campanulatus was reported earlier #Ja(del et al., )0*)'.Ceasts and dermatophytes are euaryotic organismswith more complex structural organiation comparedto the simple proaryotic bacterial cells. This probablyexplains the difference in sensitivity of these twogroups of microorganisms.

The antimicrobial potential of S. pulcherrima #leaf',especially its n-butanol fraction suggested futureresearch on isolation of active molecule to serve either

as novel antimicrobial drug or lead compounds.

/c2nowledgements

The authors are thanful to >r. . &ingh, >irector, >irectorateof ife &ciences, >efence %esearch and >evelopment+rganisation #>%>+', ovt. of ndia and >r. G. Geer, >irector,>efence %esearch aboratory, Tepur, Assam for theirsupport. A. 1. $aumder greatly acnowledged >%>+, ovt.of ndia for the research fellowship.

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Anti Microbial Activity