Research Article Antimicrobial Activity of Spices and ...downloads.hindawi.com/journals/ijmicro/2016/9015802.pdfIn Vitro Antimicrobial Activity of Spices and Medicinal Herbs against

Research ArticleIn Vitro Antimicrobial Activity of Spices and Medicinal Herbsagainst Selected Microbes Associated with Juices

Romika Dhiman1 Neeraj Aggarwal1 Kamal Rai Aneja2 and Manpreet Kaur1

1Department of Microbiology Kurukshetra University Kurukshetra India2Vaidyanath Research Training and Diagnostic Centre Kurukshetra India

Correspondence should be addressed to Romika Dhiman romikadhimangmailcom

Received 18 September 2015 Revised 12 November 2015 Accepted 15 November 2015

Academic Editor Joseph Falkinham

Copyright copy 2016 Romika Dhiman et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In the present investigation comparison of antimicrobial activities of different spices Curcuma longa Zingiber officinale andMentha arvensis andmedicinal herbs such asWithania somnifera Rauvolfia serpentina Emblica officinalis Terminalia arjuna andCentella asiatica was evaluated Different extraction solvents (acetone methanol ethanol and water) were used and extracts wereexamined against Bacillus cereus Serratia sp Rhodotorula mucilaginosa Aspergillus flavus and Penicillium citrinum isolated fromjuices Extracts from themedicinal herb and spices have significant activity B cereuswas themost sensitive andRmucilaginosawasthe most resistant among the microorganisms tested Ethanolic and methanolic extract of C asiatica displayed maximum diameterof inhibition zone against bacteria and yeast and percentage mycelial inhibition against mouldsThis study confirmed the potentialof selected extracts of spices as effective natural food preservative in juices

1 Introduction

Emergence of new technologies in food preservation leadsto a reduction in the levels of preservatives and promotesthe use of ldquonaturally derived antimicrobials of animal plantsand microbial origin [1] Antimicrobial compounds derivedfrom plants were used for centuries in food preservationEgyptians Chinese and Indians used spices and essential oilssince ancient time Some of the spices such as mint garlicand ginger are still practiced in alternative health remediesin India [2 3] About 250 to 500 thousand plant species areestimated to exist on the planet and only between 1 and 10of them are used as food by humans and other animals [4]Spices and herbs used in foods as flavoring agents in additionto enhancing flavors were used as folk medicines and foodpreservatives Spices and herbs extend the shelf life of foodsby restricting rancidity through their antioxidant activity orthrough their bacteriostatic and bactericidal activity [5]

Spices herbs and their constituents are generally reco-gonised as safe (GRAS) and approved by several regulatoryagencies such as US Food and Drug Act the EuropeanUnion standards Codex Alimentarius and Food Safety and

Standards Authority of India [3] In India the trend ofconsumption of spices and herbs in food or using them asmedicine aims to maintain proper sanitation health andhygiene and to increase longevity of life Several spices such asajowan clove ginger black pepper cumin and asafetida arecommonly used in the Indian diet [3 6 7] Literature cited thework of several authors on the antimicrobial activity of plantsagainst wide range of bacteria yeasts and moulds [2 3 8ndash20] The general description of spices and herbs used in thepresent study is tabulated in Table 1

Spices and herbs owing to their natural origin attractmore attention of consumers that have doubt regarding thesafety of chemical preservatives Several plant extracts havegained momentum in recent years due to their bioactiveprincipals and formed the basis of pharmaceutical and foodprocessing industries [17 21]

Unpasteurized fruit juice consumption has increased inthe last decades which is attributed to the contents ofantioxidants vitamins and minerals Fresh fruit juices arehighly vulnerable to spoilage since fluid components are incontact with air and microorganisms form the environmentwhile handling [22] Fruit juices spoilage bacteria include acid

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2016 Article ID 9015802 9 pageshttpdxdoiorg10115520169015802

2 International Journal of Microbiology

Table1Ethn

obotanicaldescrip

tion

phytochemicalcompo

sition

regulatorystatusand

partof

plantsused

inantim

icrobialstu

dy

Scientificn

ame

Com

mon

name

Family

Plantp

arttested

Phytocon

stituentsof

partused

Tradition

aluses

References

CentellaasiaticaL

Jalbrahm

iman

dukparni

Apiaceae

Who

leplant

Sapo

ninsasia

ticosides

brahminosideandcentelloside

Recommendedforthe

treatmento

fvarious

skin

cond

ition

ssuchas

leprosylupu

svaric

oseu

lcerseczema

psoriasisdiarrho

eafeverand

amenorrhea

anddiseases

ofthefem

aleg

enito

urinarytract

[2627]

Curcum

alonga

Haldi

Zing

iberaceae

Rhizom

eCu

rcum

in(diferuloylmethane)

120572-phelland

renesabinenebo

rneol

zing

ibereneandsesquiterpenes

Usedto

treatgastr

ointestin

alup

setsandarthritispain

and

istonicfor

thed

igestiv

esystem

[28]

Emblica

officin

alis

Amla

Euph

orbiaceae

Leaves

Gallic

acidethylgallate

12346-penta-O

-galloylglucose

and

luteolin-41015840-O

-neohesperidoside

Source

ofVitamin

Cenhances

food

absorptio

nbalances

stomachacidsfortifies

theliversup

portsthe

heartand

prom

otes

healthierh

air

[29]

Menthaarvensis

Pudina

Lamiaceae

Leaves

Tann

insph

enolsste

roids

flavono

idsandvolatileo

ilsUsedto

treatliver

andspleen

diseasesA

sthmaand

Jaun

dice

[30]

Rauvolfia

serpentin

aBe

nth

Sarpagandh

aAp

ocyn

aceae

Leaves

Alkaloids

Therapeutic

actio

nsbeingmainlyeffectiv

einthe

treatmento

fhypertensionandpsycho

ticdisorderslike

schizoph

reniaanxietyinsomniaandinsanity

[31]

Term

inaliaarjuna

Arju

nCom

bretaceae

Leaves

Flavon

oid

Usedas

arem

edyforthe

treatmento

fear

ache

[32]

With

aniasomnifer

aAs

hwagandh

aSolanaceae

Leaves

Steroidalw

ithanolides

Claimed

tohave

potent

aphrod

isiac

rejuvenativ

eand

life-prolon

ging

prop

ertie

s[33]

Zingiberoffi

cinale

Saun

thdrie

dginger

Zing

iberaceae

Rhizom

eGingerol(5-hydroxy-1-(4

hydroxy-3-metho

xyph

enyl)

decan-3-on

e)

Com

mon

lyused

infood

prod

uctsandbeverages

carm

inativeantispasm

odicdigestiv

esto

machic

vasodilatorappetizerexp

ectorantbroncho

dilatortopical

andlocalstim

ulantanalgesic

antiflatulentaphrod

isiac

antitussiv

earthritis

rheumatism

sprainsm

usculara

ches

painsandlaxativ

e

[15]

International Journal of Microbiology 3

tolerant bacteria such as acetic acid bacteria lactic acid bac-teria Clostridium Bacillus members of Enterobacteriaceaefamily (Klebsiella sp Citrobacter sp and Serratia sp) andsome heat resistant bacteria such as Alicyclobacillus acidoter-restris and Propionibacterium cyclohexinicum Among yeastsPichia Candida Saccharomyces and Rhodotorula are com-monly encountered genera responsible for spoilage of juicesCertain common moulds such as Penicillium sp Aspergillussp Eurotium Alternaria Cladosporium Paecilomyces andBotrytis have also been reported in spoilage of fruit juicesThepresence of pathogenic bacteria and mycotoxin producingmould cannot be ruled out in fruit juice which has beenresponsible for the increase in food borne outbreaks with theconsumption of fresh fruit juices during the last two decades[14 23 24]

Therefore themain objective of this studywas to examinethe in vitro antimicrobial activity of different spices and herbsextracts and to compare the effect of different solvents in theextraction method for antimicrobial activity

2 Materials and Methods

21 PlantMaterials Three Indian spices pudina saunth andhaldi were procured from local market in YamunanagarHaryana India Four medicinal plants herbs amla brahmiashwgandha arjun and sarpagandha were collected fromCh Devi Lal Park at Khizrabad Yamunanagar HaryanaIndia The taxonomic identity of these plants was confirmedby Dr B D Vashishta plant taxonomist professor in theDepartment of Botany Kurukshetra University KurukshetraThe scientific name and tested parts of the 8 plants aredetailed in Table 1

22 Extraction of Plant Material Four different solventsnamely ethanol methanol acetone and aqueous (hot andcold) were used for extraction and plant extracts wereprepared according to the methods described by Sharma etal [25]

23 Test Microorganisms In the previous study [24] micro-biological analysis of fruit juices was done by serial dilutionagar plate techniqueOn the basis of percentage of occurrenceof microorganisms in juice samples one Gram-positivebacterium one Gram-negative bacterium one yeast and twomouldswere selected for examining the antimicrobial activityof spices Bacterial strains were identified on the basis ofgram staining and biochemical and molecular characteristics(16S rRNA sequencing) [23] Yeast was identified on thebasis of staining morphological cultural characteristics andmolecular characteristics (28S rRNA sequencing) Mouldswere identified on the basis of morphological and culturalcharacteristics and further identification was confirmed byCABI International Mycological Institute UK

Two bacteria namely Serratia (KC67407lowast) and BacilluscereusKRC1 (KC67408) one yeastRhodotorulamucilaginosa(KC67409) and two moulds Aspergillus flavus (504472lowastlowast)and Penicillium citrinum (504473) were identified Thebacterial isolates were subcultured on nutrient agar and R

mucilaginosa A flavus and P citrinum were subcultured onpotato dextrose agar and incubated aerobically at 37∘C and25∘C respectively The media were procured from Hi MediaLaboratory Pvt Ltd Bombay India (lowastnucleotide sequence ofall microorganisms has been submitted to GenBank databasewhich provided the GenBank accession number KC67407ndashKC67409 lowastlowastInternational Mycological Institute referencenumber)

24 Screening for Antimicrobial Activity against Bacteria andYeast The acetone methanol ethanol and hot and coldaqueous extracts of different plants were used for evaluationof antimicrobial activity by the agar well diffusionmethod Inthis method a pure isolate of bacteria and yeast was grownon NA and PDA plates and incubated at 37∘C and 25∘C for24 h and 72 h respectively One plate of each microorganismwas taken and colonies were transferred into normal saline(085) under aseptic conditions Density of each microbialsuspension was adjusted to be equal to that of 106 cfumL(standardized by 05 McFarland standard) and to be used asthe inoculum for performing an agar well diffusion assay100 120583L of the inoculum of each test organism was spreadonto the agar plates so as to achieve a confluent growth Theagar plates were allowed to dry and 8mm wells were madewith a sterile borer in the inoculated agar plates The lowerportion of each well was sealed with molten agar mediumThe dried extracts were reconstituted to 20 in dimethylsulfoxide (DMSO) to the final concentration of 100mgmLfor the bioassay analysis A 100 120583L volume of each extractwas propelled directly into the wells (in triplicate) of theinoculated agar plates for each test organism The plateswere allowed to stand for 1 h at room temperature (40∘C)for diffusion of the extract into agar and incubated at 37∘Cand 25∘C for 24 h and 72 h respectively Sodium benzoate(100mgmL) was used as positive reference standards todetermine the sensitivity of each microbial species testedSterile DMSO served as the negative control The antimi-crobial activity indicated by an inhibition zone surroundingthe well containing the extract was recorded if the zonewas greater than 8mm The experiments were performed intriplicate and the mean values of the diameter of inhibitionzones plusmn standard deviations were calculated [34]

25 Screening for Antimicrobial Activity against Moulds Theantimould activity of plant extracts in different solvent wasaccessed by poison food technique 100120583L of plant extractwith concentration of 100mgmL was poured into sterilePetri plate (90mm diameter) and 15mL of molten potatodextrose agar (PDA) was added to the Petri plate and swirledto achieve a uniform mixture and allowed them to solidify atroom temperature The solidified agar plates were inoculatedat the centre with fungal disc (6mm diameter) obtainedfrom the actively growing one-week-old colony of the testfungus placed with inoculums side down in the centre ofeach Petri plate aseptically and incubated at point 25∘C for7 days DMSO was used as negative control and chemicalpreservative sodium benzoate served as the positive controlThe antifungal activity of each extract was evaluated by


measuring the radial growth of fungus in terms of diameterand expressed as percentage mycelia inhibition determinedby applying the following formula [35ndash37]

inhibition of mycelia growth =(119889119888minus 119889119905)

119889119888

times 100 (1)

where 119889119888is average diameter of fungal colony in negative

control plates and 119889119905is average diameter of fungal colony in

extract added plates

26 Determination of Minimum Inhibitory ConcentrationMinimum inhibitory concentration (MIC) for each testorganism was determined by the broth macrodilutionmethod [38]

27 Statistical Analysis The experimental results wererepeated thrice in triplicate each time and expressed asmean plusmn SD and results were statistically evaluated usingSPSS software version 16 at 5 significant level Means werecompared using Tukeyrsquos simultaneous test set at 119901 lt 005

3 Results and Discussion

31 Antimicrobial Activity of Plant Extracts The dietary herband spices are used as food additives in foods not onlyto improve the sensory characteristics of food but also toincrease the shelf life by reducing or eliminating survival ofpathogenic bacteria [2]

In the present study the antimicrobial activity of thedifferent plant extracts in different solvents was examinedPerusal data in Table 2 revealed the mean diameters of theinhibition zones of all plant extracts against two bacteria andyeast and percentage mycelia inhibition against two mouldsThere was significant variation (119901 lt 005) observed betweenacetone methanol ethanol cold aqueous and hot aqueoussolvents for the antimicrobial activities of each of the testedplant extracts and microorganisms

For B cereus (KC67408) the DIZ values of 18 extracts(accounting for 45 of the 40 tested extracts) were between196mm and 293mm and those of 16 extracts (40) werebetween 123mm and 193mm However 6 extracts had noinhibitory activity Ethanolic extract of C asiatica exhibitedthe strongest antibacterial activity (DIZ = 293mm) followedby methanolic extract (DIZ = 263mm) and ethanolic extractof R serpentina (DIZ = 253mm) (Table 2)

For Serratia (KC67407) a total of 25 extracts (625)exhibited inhibitory activity with DIZ values ranging from113mm to 226mm and 47 extracts had low activity Theremaining 15 extracts had no inhibitory activity (Table 2)Thesample with strongest antimicrobial activity was methanolicextract of T arjuna (DIZ = 226mm) followed by acetonicextract of R serpentina (213mm)

For R mucilaginosa (KC67409) the DIZ values of 9extracts (225) ranged from 133mm to 253mm (Table 2)Thirty-one extracts (775) showed no inhibitory activ-ity Ethanolic extract of C asiatica showed the strongestinhibitory activity (DIZ = 206mm) followed by methanolicextract of T arjuna (193mm)

Of the two moulds tested for A flavus the percentagemycelial inhibition of 8 extracts (20) varied from 206mmto 263mm Nine extracts (225) had low inhibitory activity(percentage mycelial inhibition ranging between 116mmand 193mm) The other 23 extracts (575) showed noinhibitory activity Ethanolic extract of C asiatica exhib-ited the strongest antimould activity (percentage mycelialinhibition = 263mm) followed by methanolic extract of Casiatica and T arjuna (246mm) For P citrinum 8 extractsexhibited high inhibitory activity (percentage mycelial inhi-bition = 196mmndash243mm) and nine extracts possessed lessinhibitory activity (percentage mycelial inhibition rangingbetween 103mm and 193mm) while the other 23 extractshad no inhibitory activity Methanolic extract of C asi-atica (243mm) showed the strongest inhibitory activityfollowed by ethanolic extract of C asiatica andW somnifera(233mm)

Literature search revealed the in vitro and in vivo antimi-crobial activities of plant extracts and essential oils againstfood borne pathogens but they are difficult to compareowing to the use of different methods of extraction solventsmicrobial strains and antimicrobial test methods [3 5 1617 39 40] In the present analysis 4 plants C asiaticaE officinalis M arvensis and T arjuna exhibited broadspectrum activity against testedmicrobesThe present resultswere in line with the observation of earlier workers [5ndash7 15ndash17 41 42] Methanolic and ethanolic extract of C asiaticaexhibited maximum zone of inhibition against all testedmicroorganisms in the present investigation The antimicro-bial potential of C asiatica was observed by Arumugam et al[43] in a study to check the antimicrobial efficacy ofC asiaticaagainst B cereus S aureus and Pseudomonas aeruginosain four different solvents such as methanol chloroformwater and acetone and they found that methanolic extractof C asiatica leaves at the concentration of 50120583g per mLdisplayed activity (24ndash27mm) against all tested microorgan-isms The antimicrobial efficacy of C asiatica is due to thepresence of terpenoids [44] Acetonic extracts of T arjunadisplayed maximum zone of inhibition (16ndash28mm) againstS aureus Acinetobacter P aeruginosa and Proteus mirabilisin comparison to the other extracts (methanolic ethanolicand water extracts) however all the extracts lacked activityagainst Candida albicans [32] In the present investigationmethanolic and ethanolic extracts of T arjuna exhibitedantimicrobial activity against all tested microbes due to thepresence of flavonoids (luteolin) [45 46]

Ethanolic extract of E officinalis leaves showed moreactivity against P aeruginosa Proteus mirabilis S aureus andB cereus in comparison to ethanolic extract of M arvensisleaves at the concentration of 100mgmL [47] Howeverin the present investigation the ethanolic extracts of Marvensis displayed more activity in comparison to E offic-inalis ethanolic extract at the concentration of 100mgmLThe antimicrobial potential of E officinalis and M arvensisis attributed to the presence of hydrolysable tannins andmenthol respectively [48] Although in present investiga-tion R serpentina and W somnifera revealed antibacterialand antimould activity but lacked antiyeast activity several


Table 2 Antimicrobial activity of plant extracts in different solvents

Plant Solvent Diameter of inhibition zone Percentage mycelia inhibitionB cereus Serratia sp Rmucilaginosa A flavus P citrinum

Centella asiatica

Acetone 233lowastbx plusmn 152lowastlowast mdash mdash mdash mdash

Methanol 273cx plusmn 057 153cy plusmn 057 183cz plusmn 057 246bu plusmn 057 233bv plusmn 152Ethanol 293dx plusmn 057 123dy plusmn 152 206dz plusmn 057 263cu plusmn 152 243cv plusmn 057

Cold aqueous mdash mdash mdash mdash mdashHot aqueous mdash mdash mdash mdash mdash

Curcuma longa

Acetone 223bx plusmn 152 133by plusmn 152 mdash mdash mdashMethanol 203cx plusmn 152 mdash mdash mdash mdashEthanol 173dx plusmn 152 mdash mdash mdash mdash


Emblica officinalis

Acetone 223bx plusmn 057 193by plusmn 057 186bz plusmn 152 193bu plusmn 152 206bv plusmn 152Methanol 196cx plusmn 152 143cy plusmn 057 163cz plusmn 057 176cu plusmn 152 183cv plusmn 057Ethanol 173dx plusmn 057 153dy plusmn 057 143dz plusmn 057 126du plusmn 057 136dv plusmn 057

Cold aqueous 146ex plusmn 057 mdash mdash mdash mdashHot aqueous 126fx plusmn 057 mdash mdash mdash mdash

Mentha arvensis

Acetone 263bx plusmn 057 193by plusmn 152 mdash 223bu plusmn 057 206bv plusmn 057Methanol 233cx plusmn 057 143cy plusmn 057 mdash 176cu plusmn 152 153cv plusmn 152Ethanol 243dx plusmn 057 166dy plusmn 057 mdash 166du plusmn 152 143dv plusmn 057

Cold aqueous 193ex plusmn 057 113ey plusmn 057 mdash mdash mdashHot aqueous 156fx plusmn 152 mdash mdash mdash mdash

Rauvolfia serpentina

Acetone 223bx plusmn 057 213by plusmn 152 mdash 173bu plusmn 152 193bv plusmn 152Methanol 266cx plusmn 057 186cy plusmn 057 mdash 156cu plusmn 152 126cv plusmn 152Ethanol 256dx plusmn 152 173dy plusmn 057 mdash mdash mdash

Cold aqueous 193ex plusmn 057 146ey plusmn 152 mdash mdash mdashHot aqueous 176fx plusmn 057 126fy plusmn 057 mdash mdash mdash

Terminalia arjuna


Cold aqueous 156ex plusmn 057 146ey plusmn 057 133ez plusmn 057 116eu plusmn 152 103ev plusmn 057Hot aqueous 123fx plusmn 057 mdash mdash mdash

Withania somnifera



Zingiber officinale

Acetone 233bx plusmn 057 186by plusmn 057 mdash mdash mdashMethanol 213cx plusmn 152 136cy plusmn 057 mdash mdash mdashEthanol 193dx plusmn 057 113dz plusmn 057 mdash mdash mdash


Sodium benzoate 206ax plusmn 057 166ay plusmn 057 146az plusmn 057 306 plusmn 057 263 plusmn 057lowastValues including diameter of the well (8mm) are means of three replicates lowastlowastStandard deviation within five extracts and control of the same spice with threedifferent microorganisms tested different letters are significantly (119901 lt 005) different mdash no activity

authors confirmed antibacterial and antifungal potential ofW somnifera and R serpentina [31 33 49 50]

Sunilson et al [15] observed the antimicrobial activity ofC longa andZ officinalis in four different solvents (petroleumether chloroform methanol and water) against food bornepathogens The solvent extracts of C longa and Z officinalisdisplayed antibacterial and antiyeast activity however in

present investigation C longa and Z officinalis possessedantibacterial activity

The extraction of biologically active compound fromplant material is largely dependent on the type of thesolvent used in the extraction procedure The present studyrevealed that the organic extracts provided more powerfulantimicrobial activity compared to aqueous extracts Among


Table 3 MIC of plant extracts in different solvents

Plant Solvent MIC value of different plant extractsB cereus Serratia sp Rmucilaginosa A flavus P citrinum

Centella asiaticaAcetone 125 Nt Nt Nt NtMethanol 625 25 25 125 125Ethanol 312 Nt 125 625 125

Curcuma longaAcetone 125 50 Nt Nt NtMethanol 125 Nt Nt Nt NtEthanol 25 Nt Nt Nt Nt

Emblica officinalis

Acetone 125 25 25 25 125Methanol 125 50 25 25 25Ethanol 25 50 50 Nt 50

Cold aqueous 50 Nt Nt Nt Nt

Mentha arvensis

Acetone 625 25 Nt 125 125Methanol 125 50 Nt 25 25Ethanol 625 25 Nt 25 50

Cold aqueous 50 Nt Nt Nt NtHot aqueous 50 Nt Nt Nt Nt


Acetone 25 125 Nt 25 25Methanol 125 25 Nt 50 NtEthanol 625 25 Nt Nt Nt

Cold aqueous 25 50 Nt Nt NtHot aqueous 25 Nt Nt Nt Nt

Terminalia arjuna

Acetone 25 25 25 25 25Methanol 125 125 50 125 125Ethanol 125 25 25 125 50

Cold aqueous 25 50 50 Nt Nt

Withania somniferaAcetone 125 25 Nt 25 25Methanol 50 50 Nt 125 125Ethanol 50 Nt Nt 125 125

Zingiber officinale

Acetone 125 25 Nt Nt NtMethanol 125 50 Nt Nt NtEthanol 25 Nt Nt Nt Nt

Cold aqueous 25 Nt Nt Nt NtHot aqueous Nt Nt Nt Nt Nt

Sodium benzoate 125 25 50 125 125Nt not tested

the organic extracts alcoholic extracts displayed the bestantimicrobial activity in comparison to acetonic extracts(Table 2) thus substituting the findings of earlier workerswho rated methanol and ethanol as best solvent for theextraction of antimicrobial compounds from plants followedby acetone and water [5 15 16 25] Several compoundstannins flavonoids coumarins thiosulfinates glucosino-lates and saponins isolated from these plants are secondarymetabolites which are responsible for the antimicrobial andmedicinal properties of plants [2 3 8 17]

32 Sensitivity of Five Tested Microbes B cereus SerratiaR mucilaginosa A flavus and P citrinum revealed varyingsensitivities to the 40 tested extracts (Table 2) With respectto B cereus thirty-four extracts showed inhibitory activityand 16 extracts had no activity In case of Serratia 25extracts displayed inhibitory activity and 15 extracts showedno activity R mucilaginosa was the most resistant of thetested microorganisms Nine extracts exhibited inhibitory

activity and 31 extracts had no inhibitory activity Seventeenextracts showed inhibitory activity against two moulds (Aflavus and P citrinum) Based on the inhibitory activity ofplant extract Gram-positive bacteria B cereus demonstratedmore sensitivity to the extracts than Gram-negative bacteriaand fungiThe present findings are consistent with the resultsobtained by earlier workers who reported that plant extractsare more active against Gram-positive bacteria than againstGram-negative bacteria [3 5 16 17 48 51ndash53] This isattributed to the differences in the outer layers of Gram-negative andGram-positive bacteria Gram-negative bacteriapossess an outer membrane and a unique periplasmic spacenot found in Gram-positive bacteria [5 54 55]

33 Determination of Minimum Inhibitory ConcentrationThe minimum inhibitory concentration was determined for31 active plant extracts which show antimicrobial activityagainst microbes associated with juices (Table 3)TheMIC ofthe 31 active plant extracts showed that MIC ranged between


312mgmL and 50mgmL for the organic extracts andbetween 25 and 50mgmL for the aqueous extracts Whencomparing the MIC values of the tested plants the ethanolicextract of C asiatica appeared to be the most effective withthe lowest inhibitory concentration being 315mgmL againstB cereus Methanolic extracts of C asiaticaM arvensis andR serpentina were the second most effective plant extractswith MIC of 625mgmL Many plant extracts show MICin the range of 25ndash50mgmL and were thus shown to beless effective against microbes isolated from juices It hasbeen established that MIC results do not always correlatewell with the DIZ values but in present investigation theobservation of MIC related with the DIZ value might be dueto the adoption of disc diffusion assay for both antimicrobialactivity andMIC determination of plant extracts [16] Similarresults of MIC of different plants in different solvents havebeen observed by several authors against different microbes[3 5 9ndash13 16 56 57]

4 Conclusion

The results of present work established that all the testedplant extracts possess antimicrobial activity against selectedmicrobes associated with juices Alcoholic extracts of medic-inal herbs such as C asiatica T arjuna and R serpentinadisplayed better antimicrobial activity than chemical preser-vative sodium benzoate therefore these plant extracts havethe potential to extend the shelf life or they are used as naturalpreservatives in fruit juices

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are thankful for Kurukshetra University Kuruk-shetra for the financial assistance

References

[1] K R Aneja R Dhiman N K Aggarwal and A AnejaldquoEmerging preservation techniques for controlling spoilageand pathogenic microorganisms in fruit juicesrdquo InternationalJournal of Microbiology vol 2014 Article ID 758942 14 pages2014

[2] MM Tajkarimi S A Ibrahim andDO Cliver ldquoAntimicrobialherb and spice compounds in foodrdquo Food Control vol 21 no 9pp 1199ndash1218 2010

[3] R Dhiman N K Aggarwal and M Kaur ldquoComparativeevaluation of antimicrobial activities of commonly used indianspices againstmicrobes associated with juicesrdquoResearch Journalof Microbiology vol 10 no 4 pp 170ndash180 2015

[4] N C C Silva and A Fernandes Junior ldquoBiological propertiesof medicinal plants a review of their antimicrobial activityrdquoThe Journal of Venomous Animals and Toxins Including TropicalDiseases vol 16 no 3 pp 402ndash413 2010

[5] B Shan Y-Z Cai J D Brooks and H Corke ldquoThe invitro antibacterial activity of dietary spice and medicinal herbextractsrdquo International Journal of FoodMicrobiology vol 117 no1 pp 112ndash119 2007

[6] D S Arora and J Kaur ldquoAntimicrobial activity of spicesrdquoInternational Journal of Antimicrobial Agents vol 12 no 3 pp257ndash262 1999

[7] P K Sofia R Prasad V K Vijay and A K SrivastavaldquoEvaluation of antibacterial activity of Indian spices againstcommon foodborne pathogensrdquo International Journal of FoodScience and Technology vol 42 no 8 pp 910ndash915 2007

[8] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999

[9] L R Beuchat ldquoEcological factors influencing survival andgrowth of human pathogens on raw fruits and vegetablesrdquoMicrobes and Infection vol 4 no 4 pp 413ndash423 2002

[10] M Friedman P R Henika and R E Mandrell ldquoBactericidalactivities of plant essential oils and some of their isolatedconstituents against Campylobacter jejuni Escherichia coli Lis-teria monocytogenes and Salmonella entericardquo Journal of FoodProtection vol 65 no 10 pp 1545ndash1560 2002

[11] M Friedman P R Henika C E Levin and R E MandrellldquoAntibacterial activities of plant essential oils and their compo-nents against Escherichia coli O157H7 and Salmonella entericain apple juicerdquo Journal of Agricultural and Food Chemistry vol52 no 19 pp 6042ndash6048 2004

[12] S Burt ldquoEssential oils their antibacterial properties and poten-tial applications in foods a reviewrdquo International Journal of FoodMicrobiology vol 94 no 3 pp 223ndash253 2004

[13] J Mosqueda-Melgar R M Raybaudi-Massilia and O Martın-Belloso ldquoNon-thermal pasteurization of fruit juices by combin-ing high-intensity pulsed electric fields with natural antimicro-bialsrdquo Innovative Food Science and Emerging Technologies vol9 no 3 pp 328ndash340 2008

[14] R M Raybaudi-Massilia J Mosqueda-Melgar R Soliva-Fortuny and O Martın-Belloso ldquoControl of pathogenic andspoilage microorganisms in fresh-cut fruits and fruit juices bytraditional and alternative natural antimicrobialsrdquo Comprehen-sive Reviews in Food Science and Food Safety vol 8 no 3 pp157ndash180 2009

[15] J A J Sunilson R Suraj G Rejitha K Anandarajagopal A VA G Kumari and P Promwichit ldquoIn vitro antimicrobial evalu-ation of Zingiber officinale Curcuma longa and Alpinia galangaextracts as natural food preservativesrdquoAmerican Journal of FoodTechnology vol 4 no 5 pp 192ndash200 2009

[16] N S Weerakkody N Caffin M S Turner and G A DykesldquoIn vitro antimicrobial activity of less-utilized spice and herbextracts against selected food-borne bacteriardquoFoodControl vol21 no 10 pp 1408ndash1414 2010

[17] P S Negi ldquoPlant extracts for the control of bacterial growthefficacy stability and safety issues for food applicationrdquo Inter-national Journal of Food Microbiology vol 156 no 1 pp 7ndash172012

[18] A K Tyagi D Gottardi A Malik and M E Guerzoni ldquoAnti-yeast activity of mentha oil and vapours through in vitro and invivo (real fruit juices) assaysrdquo Food Chemistry vol 137 no 1ndash4pp 108ndash114 2013

[19] A K Tyagi D Gottardi A Malik and M E GuerzonildquoChemical composition in vitro anti-yeast activity and fruitjuice preservation potential of lemon grass oilrdquo LWTmdashFoodScience and Technology vol 57 no 2 pp 731ndash737 2014


[20] D Bukvicki D Gottardi A K Tyagi et al ldquoScapania nemorealiverwort extracts investigation on volatile compounds in vitroantimicrobial activity and control of Saccharomyces cerevisiaein fruit juicerdquo LWTmdashFood Science and Technology vol 55 no2 pp 452ndash458 2014

[21] A Lucera C Costa A Conte and M A Del Nobile ldquoFoodapplications of natural antimicrobial compoundsrdquo Frontiers inMicrobiology vol 3 article 287 2012

[22] J Mosqueda-Melgar R M Raybaudi-Massilia and O MartinBelloso ldquoMicrobiological shelf life and sensory evaluationof fruit juices treated by high intensity electric fields andantimicrobialsrdquo Food and Bioproducts Processing vol 10 pp123ndash142 2012

[23] K A Lawlor J D Schuman P G Simpson and P J TaorminaldquoMicrobiological spoilage of beveragesrdquo in Compendium of theMicrobiological Spoilage of Foods and Beverages W H Sperberand M P Doyle Eds Food Microbiology and Food SafetySpringer New York NY USA 2009

[24] K RAneja RDhimanNKAggarwal VKumar andMKaurldquoMicrobes associated with freshly prepared juices of citrus andcarrotsrdquo International Journal of Food Science vol 2014 ArticleID 408085 7 pages 2014

[25] C Sharma K R Aneja R Kasera and R Aneja ldquoAntimicrobialpotential of Terminalia chebula Retz Fruit extracts against earpathogensrdquoWorld Journal of Otorhinolaryngology vol 2 pp 8ndash13 2012

[26] K J Gohil J A Patel andA K Gajjar ldquoPharmacological reviewon Centella asiatica a potential herbal cure-allrdquo Indian Journalof Pharmaceutical Sciences vol 72 no 5 pp 546ndash556 2010

[27] B Dora and J Khatri ldquoCentella asiatica elexier of liferdquo Interna-tional Journal of Research in Ayurveda and Pharmacy vol 2 no2 pp 431ndash438 2011

[28] I Chattopadhyay K Biswas U Bandyopadhyay and R KBanerjee ldquoTurmeric and curcumin biological actions andmedicinal applicationsrdquoCurrent Science vol 87 no 1 pp 44ndash532004

[29] K R Aneja R Joshi and C Sharma ldquoIn vitro antimicrobialactivity of Sapindus mukorossi and Emblica officinalis againstdental caries pathogensrdquo Ethnobotanical Leaflets vol 14 pp402ndash412 2010

[30] M M Kumbalwar A B Ingle and M H Shende ldquoAntimi-crobial activity of Mentha arvensis (pudina) against on gramnegative bacteriardquo Indian Journal of Applied Research vol 4 no4 pp 488ndash489 2011

[31] M B Azmi and S A Qureshi ldquoMethanolic root extract ofRauwolfia serpentina benth improves the glycemic antiathero-genic and cardioprotective indices in alloxan-induced diabeticmicerdquo Advances in Pharmacological Sciences vol 2012 ArticleID 376429 11 pages 2012

[32] K R Aneja C Sharma and R Joshi ldquoAntimicrobial activityof Terminalia arjuna Wight amp Arn an ethnomedicinal plantagainst pathogens causing ear infectionrdquo Brazilian Journal ofOtorhinolaryngology vol 78 no 1 pp 68ndash74 2012

[33] S K Verma and A Kumar ldquoTherapeutic uses of Withaniasomnifera (ashwagandha) with a note on withanolides and itspharmacological actionsrdquo Asian Journal of Pharmaceutical andClinical Research vol 4 no 1 pp 1ndash4 2011

[34] K R Aneja R Joshi and C Sharma ldquoPotency of Barleriaprionitis L bark extracts against oral diseases causing strains ofbacteria and fungi of clinical originrdquo New York Science vol 3pp 5ndash12 2010

[35] S R Sridhar R V Rajagopal R Rajavel S Masilamani and SNarasimhan ldquoAntifungal activity of some essential oilsrdquo Journalof Agricultural and Food Chemistry vol 51 no 26 pp 7596ndash7599 2003

[36] S K S Al-Burtamani M O Fatope R G Marwah A K Oni-fade and S H Al-Saidi ldquoChemical composition antibacterialand antifungal activities of the essential oil of Haplophyllumtuberculatum from Omanrdquo Journal of Ethnopharmacology vol96 no 1-2 pp 107ndash112 2005

[37] T K Chattapadhyay and P Dureja ldquoAntifungal activity of 4-methyl-6-alkyl-2H-pyran-2-onesrdquo Journal of Agricultural andFood Chemistry vol 54 no 6 pp 2129ndash2133 2006

[38] K Das R K S Tiwari and D K Shrivastava ldquoTechniques forevaluation of medicinal plant products as antimicrobial agentcurrent methods and future trendsrdquo Journal of Medicinal PlantsResearch vol 4 no 2 pp 104ndash111 2010

[39] K A Hammer C F Carson and T V Riley ldquoAntimicrobialactivity of essential oils and other plant extractsrdquo Journal ofApplied Microbiology vol 86 no 6 pp 985ndash990 1999

[40] C Thongson P M Davidson W Mahakarnchanakul and JWeiss ldquoAntimicrobial activity of ultrasound-assisted solvent-extracted spicesrdquo Letters in Applied Microbiology vol 39 no 5pp 401ndash406 2004

[41] I Ahmad and A Z Beg ldquoAntimicrobial and phytochemicalstudies on 45 Indian medicinal plants against multi-drugresistant human pathogensrdquo Journal of Ethnopharmacology vol74 no 2 pp 113ndash123 2001

[42] B K Dash H M Faruquee S K Biswas M K Alam S MSisir and U K Prodhan ldquoAntibacterial and antifungal activitiesof several extracts of Centella asiatica L against some humanpathogenic microbesrdquo Life Sciences and Medicine Research vol2011 pp 1ndash5 2011

[43] T Arumugam M Ayyanar Y J Koil Pillai and T SekarldquoPhytochemical screening and antibacterial activity of leafand callus extracts of Centella asiaticardquo Bangladesh Journal ofPharmacology vol 6 no 1 pp 55ndash60 2011

[44] J T James and I A Dubery ldquoPentacyclic triterpenoids from themedicinal herb Centella asiatica (L) urbanrdquoMolecules vol 14no 10 pp 3922ndash3941 2009

[45] S Dwivedi ldquoTerminalia arjuna Wight amp ArnmdashA useful drugfor cardiovascular disordersrdquo Journal of Ethnopharmacologyvol 114 no 2 pp 114ndash129 2007

[46] S Jain P P Yadav V Gill N Vasudeva and N SinglaldquoTerminalia arjuna a sacred medicinal plant phytochemicaland pharmacological profilerdquo Phytochemistry Reviews vol 8no 2 pp 491ndash502 2009

[47] S Saeed and P Tariq ldquoAntimicrobial activities of Emblica offic-inalis and Coriandrum sativum against gram positive bacteriaand Candida albicansrdquo Pakistan Journal of Botany vol 39 no 3pp 913ndash917 2007

[48] B Mahesh and S Satish ldquoAntimicrobial activity of someimportantmedicinal plant against plant and human pathogensrdquoWorld Journal of Agricultural Sciences vol 4 pp 839ndash843 2008

[49] S Arora S Dhillon G Rani and A Nagpal ldquoThe invitro antibacterialsynergistic activities of Withania somniferaextractsrdquo Fitoterapia vol 75 no 3-4 pp 385ndash388 2004

[50] A Nostro M P Germano V DrsquoAngelo A Marino and MA Cannatelli ldquoExtraction methods and bioautography forevaluation of medicinal plant antimicrobial activityrdquo Letters inApplied Microbiology vol 30 no 5 pp 379ndash384 2000


[51] N A A Ali W-D Julich C Kusnick and U LindequistldquoScreening of Yemeni medicinal plants for antibacterial andcytotoxic activitiesrdquo Journal of Ethnopharmacology vol 74 no2 pp 173ndash179 2001

[52] E A Palombo and S J Semple ldquoAntibacterial activity oftraditional Australian medicinal plantsrdquo Journal of Ethnophar-macology vol 77 no 2-3 pp 151ndash157 2001

[53] R Nair and S Chanda ldquoActivity of some medicinal plantsagainst certain pathogenic bacterial strainsrdquo Indian Journal ofPharmacology vol 38 no 2 pp 142ndash144 2006

[54] E Ceylan and D Y C Fung ldquoAntimicrobial activity of spicesrdquoJournal of Rapid Methods and Automation in Microbiology vol12 no 1 pp 1ndash55 2004

[55] P Lopez C Sanchez R Batlle and C Nerın ldquoSolid- and vapor-phase antimicrobial activities of six essential oils susceptibilityof selected foodborne bacterial and fungal strainsrdquoThe Journalof Agricultural and Food Chemistry vol 53 no 17 pp 6939ndash6946 2005

[56] N Kapoor U Narain and K Misra ldquoBio-active conjugatesof curcumin having ester peptide thiol and disulfide linksrdquoJournal of Scientific and Industrial Research vol 66 no 8 pp647ndash650 2007

[57] K S Parvathy P S Negi and P Srinivas ldquoAntioxidant antimu-tagenic and antibacterial activities of curcumin-120573-diglucosiderdquoFood Chemistry vol 115 no 1 pp 265ndash271 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table1Ethn

obotanicaldescrip

tion

phytochemicalcompo

sition

regulatorystatusand

partof

plantsused

inantim

icrobialstu

dy

Scientificn

ame

Com

mon

name

Family

Plantp

arttested

Phytocon

stituentsof

partused

Tradition

aluses

References

CentellaasiaticaL

Jalbrahm

iman

dukparni

Apiaceae

Who

leplant

Sapo

ninsasia

ticosides

brahminosideandcentelloside

Recommendedforthe

treatmento

fvarious

skin

cond

ition

ssuchas

leprosylupu

svaric

oseu

lcerseczema

psoriasisdiarrho

eafeverand

amenorrhea

anddiseases

ofthefem

aleg

enito

urinarytract

[2627]

Curcum

alonga

Haldi

Zing

iberaceae

Rhizom

eCu

rcum

in(diferuloylmethane)

120572-phelland

renesabinenebo

rneol

zing

ibereneandsesquiterpenes

Usedto

treatgastr

ointestin

alup

setsandarthritispain

and

istonicfor

thed

igestiv

esystem

[28]

Emblica

officin

alis

Amla

Euph

orbiaceae

Leaves

Gallic

acidethylgallate

12346-penta-O

-galloylglucose

and

luteolin-41015840-O

-neohesperidoside

Source

ofVitamin

Cenhances

food

absorptio

nbalances

stomachacidsfortifies

theliversup

portsthe

heartand

prom

otes

healthierh

air

[29]

Menthaarvensis

Pudina

Lamiaceae

Leaves

Tann

insph

enolsste

roids

flavono

idsandvolatileo

ilsUsedto

treatliver

andspleen

diseasesA

sthmaand

Jaun

dice

[30]

Rauvolfia

serpentin

aBe

nth

Sarpagandh

aAp

ocyn

aceae

Leaves

Alkaloids

Therapeutic

actio

nsbeingmainlyeffectiv

einthe

treatmento

fhypertensionandpsycho

ticdisorderslike

schizoph

reniaanxietyinsomniaandinsanity

[31]

Term

inaliaarjuna

Arju

nCom

bretaceae

Leaves

Flavon

oid

Usedas

arem

edyforthe

treatmento

fear

ache

[32]

With

aniasomnifer

aAs

hwagandh

aSolanaceae

Leaves

Steroidalw

ithanolides

Claimed

tohave

potent

aphrod

isiac

rejuvenativ

eand

life-prolon

ging

prop

ertie

s[33]

Zingiberoffi

cinale

Saun

thdrie

dginger

Zing

iberaceae

Rhizom

eGingerol(5-hydroxy-1-(4

hydroxy-3-metho

xyph

enyl)

decan-3-on

e)

Com

mon

lyused

infood

prod

uctsandbeverages

carm

inativeantispasm

odicdigestiv

esto

machic

vasodilatorappetizerexp

ectorantbroncho

dilatortopical

andlocalstim

ulantanalgesic

antiflatulentaphrod

isiac

antitussiv

earthritis

rheumatism

sprainsm

usculara

ches

painsandlaxativ

e

[15]















119889119888

times 100 (1)


















Centella asiatica




Curcuma longa



Emblica officinalis



Mentha arvensis






Terminalia arjuna



Withania somnifera



Zingiber officinale













Emblica officinalis



Mentha arvensis






Terminalia arjuna




Zingiber officinale










4 Conclusion




Acknowledgment


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















119889119888

times 100 (1)


















Centella asiatica




Curcuma longa



Emblica officinalis



Mentha arvensis






Terminalia arjuna



Withania somnifera



Zingiber officinale













Emblica officinalis



Mentha arvensis






Terminalia arjuna




Zingiber officinale










4 Conclusion




Acknowledgment


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




119889119888

times 100 (1)


















Centella asiatica




Curcuma longa



Emblica officinalis



Mentha arvensis






Terminalia arjuna



Withania somnifera



Zingiber officinale













Emblica officinalis



Mentha arvensis






Terminalia arjuna




Zingiber officinale










4 Conclusion




Acknowledgment


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Centella asiatica




Curcuma longa



Emblica officinalis



Mentha arvensis






Terminalia arjuna



Withania somnifera



Zingiber officinale













Emblica officinalis



Mentha arvensis






Terminalia arjuna




Zingiber officinale










4 Conclusion




Acknowledgment


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Emblica officinalis



Mentha arvensis






Terminalia arjuna




Zingiber officinale










4 Conclusion




Acknowledgment


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Conclusion




Acknowledgment


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Antimicrobial Activity of Spices and ...downloads.hindawi.com/journals/ijmicro/2016/9015802.pdfIn Vitro Antimicrobial Activity of Spices and Medicinal Herbs against