Journal of Agr 2011 Vol.

7(1): 133-142
Available online http://www.ij
7(1):Technology133-142 ISSN
1686-9141

In vitroevaluation of antibacterialAnnona p squamosaL.aAnnonad

reticulataL.fromSimilipal B Reserve, Orissa, India

1* 1 2 3
Padhi,, LPanda,..Satapathy,.K.andDutta,S.N .S.K.
1
2
PG Department of Microbiology; M. P. C. (Auto) Co
3
School of Engineering, Amity University, NOIDA (U
PG Department of Zoology; North Orissa University

Padhi, L.P., Panda,andDutta,S.K. Satapathy,.InK.vitro(2011)evaluationS..N. of

antibacterialAnnonapotentisquL.amosalAndofnona reticulataL.from Similipal Biosphere Reserve, Orissa,
India. Journal Agricult

Three different solveAnnonat squamosaextractsL.AnnonadofreticulataLleaf.wereof studied for its

antibacterial activity. Agar cup antibacterial activityBacillususing subtilis,threeGramandStaphyloco-positive

Staphylococcus)andepidermidisfiveEscherichiaGram-negativeandPseudomonascoli( aeruginosa, Salmonella

typhi,) bacteriaVibrioalginolyticu.Thescree results showed that highest inhibition was obser petroleum ether
and AnnonaaqueousandquamosaextractsAnnonareticulataleaffor.boh

Bacillus,Staphylococcussubtilis,StaphylococcuepidermidandVisbrioaureusalginolyticu are the most sensitive

bacterial strains among a showed growth ofSalmonellainhibitiontyphiagainst.

Key words:similipal biosphere reserve, antimicrobial a analysis

Introduction

In India, from ancient times, different used to cure specific ailments. Today, t derived from
plants. This interest primar medicine is safe and dependable, compared have adverse effects.
Hence, there is ne promising biological activityAnnona,members.Plantsof theof Annonaceae
family, are native to South an

*
Corresponding author:sujogyapSujogyanda@hotmK.Pandail;.ecom-mail:

133
small trees, and produceAnnona squamosacompouL.,knowndfruitsas custard apple, is
commonly found in decid in various parts of India. Literatures of partsA.squamosaofpossess
medicinal property (Kirt Roots are employed internally in depressi is known to be a powerful
astringent. In good tonic, enrich blood, used as expect cooling, lessens burning sensation and
te and relieves vomiting (Patel and Kumar, property in curing of different ailments, Annona
reticulataL.isreferred as bullock's hear that becomes dull red when ripe. Less vo diterpenoids,
and acetogeninsal2006).,have(Ogunwanbeeniden far from oil of various parts of the plan
Annona reticulataleafisnot yet been explored pro present work was an attempt to perform th the
leafAnnonaextractreticulataandofAnnonasquamosa.

Matherials and methods

Study area

Similipal Biosphere Reserve is located

northern region 08of- 2Oriss71Nla,titudebetween04-1586ad2186

E longitudes, unique habitat of mixed tro enriched with more than 500 medicinal plan

Plant material

LeafAnnonaof reticulataandAnnonasquamosawerecollected in month of April, 2007 from

Similipal Biosp and their identity were confirmed at Dept Baripada. The shed dried healthy
leaves mechanical grinder and then were passed t size is maintained.

Preparation of extracts

500 g of each powdered plant material w flasks and soaked with 2 lit. of each so distilled
water) at room temperature for

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 Journal of Agricultural2011Vol.7(1):Technology133-142

Buchner funnel using Whatman filter paper to dryness under reduced pressure and th dried to
remove the solvent at -2 C till

Fi . Map showing the study area.

Phytochemical analysis

Qualitative phytochemical analysis was described by Trease and Evans Trease and
screened for presence of alkaloids with Dragendorffs reagents; flavonoids (NaCl Molischs,
Benedicts and Fehlings reage and Borntragers; protein and amino aci

Ninhydrin and Millons reagent;andtannin a

3
Gelatin); triterpenoid with thionylchlori Burchard and Salkowskis reagents and fat reagents.

135
Antimicrobial activity

Microbial strains

The antibacterial BacillusactvityMTCCsubtiliswas7164,tested a

StaphylococcusMTCC 1144,aureusStaphylococcusMTCCepidermidis3615,
EscherchiaMTCCcoli1098,Salmonella MTCCtyphimurium,3216, Pseudomonas
MTCCaeruginosa1034,Vibrio MTCCcholerae3904 and
Vibrio alginolyticusMTCC4439bacterial.These strains were o MTCC, Chandigarh,
(CustomerIndiano. 4853).

Agar cup method

The agar cup method was used to study extracts as detscribedal(2009)..byMuellerPanda-

Hinton ag Media, India) was used as bacteriological by pouring molten media into sterile Petr
solidify for 5 min. Wells were prepared i suspension was swabbed uniformly and the 5 min. The
extracts were diluted in 100% wells were punched into the agar and fill extracts, 20 l DMSO
(negative control) (Ciprofloxacin at concentration 10 g/ml) plates were incubated at 37 C for
24 h. of zones around the wells, confirms the a extracts. The same procedure was followed
experiment was carried out in triplicates was taken for evaluating the antibacterial

Determination of minimum inhibitory conce

In the present experiment, extracts wh further evaluated for determination of MI was

adopted using 96 well micro-titer p Triphenyltetrazolium Chloride (TTC) was c following the
methods with few modificati

the plate,towasHA the blank and consistedtowasH of MH

1 1 3 3
having the stock solutionto tillHoftotheAwereHtest ext
4 4 9 9
the wells in which the test extracts were
A12 to12 wereD control having12to1220lservedHof asDMSOcontrand
over control. All wells were dispensed wi

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 Journal of Agricultural2011Vol.7(1):Technology133-142

herbal extract was transferred from stock

A to containingH 100l of MH broth. 20l of
4 4
extract was then transferred to the next the 0.5 McFarland adjusted activated cult the wells
except the blank. 5l of 0.5% dilutions, blank, control and control ove wells was 205l. The
Microplate was seale 10l of the broth from each culture tube taken aseptically and were plated
on one inoculum and allowed to dry for 10 min un plate was sealed and incubated at 37C at
the microorganism.

Results and discussion

The result of antibacterial screening b indicates that highest zone of inhibition followed by
petroleum etherAnnonaand squamaqueoussa e andAnnona
reticulataleaf.AnnonaExtractsquamosainhibitedof the gro of all
testSalmonellastrains.excepttyphimAqueousriumextracts sho activity than methanol extracts
possibly were present in water extracts, but in lo were soluble in organic solvents and ther also
suggestedetal(2005)by.de. BoerThe antibacterial a is more pronounced on Gram -positive than
these findings correlate toofthemedicinalobservati plants for
antibaceterial2009).,Bacillusactivity.,subtili(Pand

Staphylococcus,StaphylococcuepidermidisandVibrioaureusalginolyti were the most sensitive

bacterial strains extractsA. reticofaqueouslata and methanol extracts
activityBacilluagains,tStaphylococcussubtilisandVibrioaureus alginolyticus.However, no
extractEscherchiashowedanycoliact andPseudomonas.
aeruginosaIncomparedAnnonatothereticulata,exracts

Annona squamosahadstrong antibacterial activity. There are several reasons that people

includes improvement of health after herb non availability of synthetic drugs part available were
either fake or expired dru more accustomed to and comfortable with tr study showed that
aqueous and methanol ex bacteria superior to Gram-negatives. Fin

137
scientist those reported Annonathatthesquamosa,petrole to be activeE.coli,againstP.
aeruginosa,andB.subtwhilSise. bothaureus the methanol and aqueousS.
extractspidermidis,were Sac. andB. subtilis.From agar cup method results obta differences
between the activities of the antibacterial drugs (ampicillin and cipro are normally present when
crude (unpurifi pure drugs that are already in clinical method is not always dependable for
accur is because of the high degree of interfer from drug diffusionet1981)alproblems.,. (Dickert

A more generally accurate method of as technique. In this study, therefore, the

determining the activities measured as MI B. subtilisandV.alginolyticuscorrelated well with the
re the agar cup method (Table 2). The MIC va Gram-positive bacteria are lower in compar that
the Gram-positive bacteria are more fromAnnona squamosawithrespect to its Gram-negat MBC
values were higher than the MIC valu tested bacteria. The lowest MICs exhibited eight time of
MIC, in corresponding mic interesting antimicrobial potency. From t most of test samples
exerted a lethal eff these MMC/MIC ratios lower than 4 was obt suggesting killing
effectsetal1987).come,. to mind (C

Phytochemical screening of both plants Evaluation of phytochemicals such as alk

glycosides, tannin and phenolic compoun triterpenoidsA. aresquamosawhilepresentalkaloids,in
flavono phenolic compounds and carbohydrates are A. reticulatasuchas methanol and aqueous
extrac extract (petroleum ether). APresence.squamosaof mo might be resulted betterA.
anreticulata.bacterialThe phytochemical constitute such as alkaloi compound have been
reported to be import medicinal plants (Burapedjoand Bunchoo, 1988.). Comparison of the data
obtained i result ofet Rahmanal(2005). is problematic. First, t

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Journal of Agricultural2011Vol.7(1):Technology133-142

139
TablePhytochemical3. analysis of different ex
Name of the Petrole Meth Aque
Name of the extracttest extractextract
phytochemicals A A A A A A
Mayers + + + + +
Dragen + +
Alkaloids
Hager + + +
Wagner'
Molisc + + + + +
CarbohydratFehlis + + + + +
Benedi
With Fer + + + +
Tannin and phenolic
With le
compound +
With gel
Ke Killi + +
Glycoside Lega
Borntra
Biur
Proteins andNinhydamino
acids Xanthop
Millo
Gum & m Molisc
With + + + + +
FlavonoidsWit S
Wi Mg/
Honeyco
Saponins Foam
Salkows
Steroids and sterol
Liberman + +
Trite Thionyl s + +
With fi
Oils and fats
With alk
With Ind
Vitamin C
Sod. ni
1+) Present; (-) Absent

extracts is known to vary according to conditionset 1987;(Janssenal.,Sivropoulouet1995)al.,.

Secondly the m used to assess antibacterial activity and varies et(Janssen1987)al.,. Most
frequently used met activity are agar diffusion techniques an obtained by each of these
methods may dif assays et(Janssen1987;al.,etHili1997)al.In,.vivostudies may be req to confirm
the values of the some of the

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 Journal of Agricultural2011Vol.7(1):Technology133-142

antibacterialAnnonaactivitysquamosaleafextractsof is partic considering the test human

pathogenic bac

Fig AntibacterAnnona leaf by ag

Acknowledgements

The present researchDepartmenthasbeen offundedScienceby theand T GovernmentofOrissa (Grant

No. 2818/28.06.2006). We ar North Orissa University for providing necessary fa

References

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(Received 4 March 2010; accepted 1 Octobe

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 7(1):Technology133-142

TableAntibacterial1. activity by agar cup method.

Bact Petrole Meth Aque Anti

A A A A A A A C
B 11.7 12.7 12.7 16.7 11.7 15.0 21.3 22.7
S 17.0 17.0 16.6 13.0 15.3 15.6 19.6
S 12.3 16.6 12.3 14.0 32.0 29.3
E 10.6 15.6 17.3 19.0 24.0
P 11.6 13.0 16.0 29.0
S 23.7
V 12.7 13.6 12.7 17.3 12.3 14.3 26.0 17.3
V 14.6 14.7 14.3 13.3 16.3 17.7
All values are mean zone of inhibition SD; (-) No zone of inhibition; Zo Extract concentrBsB-atcionllus,Sa(30Staphy-
subtimg/ml);isococcus,SeStaphylococcus-aureus,EcEscherich-epiderm,Pa-iadiscoli PseudomonasStaerugi-SalmonellaVaosa,Vibrio-
typhi,alginolytiVcVibrio-,ArcholerAnnonus-ae reticulAsAnnon-ata;squamosa

TableDetermination2. of MIC and MBC.

Na Annona r(concentrat Annona s(concentrat Amp li

of Petr MeO Aque Petr MeO Aque mg/ml)
bacteriaMIMB MI MB MI MB MI MB MI MB MI MB MI MB
B 5. <1 5. <1 5. <1 5. <1 1. 5. 2. 10 0. 0.
S 2. <1 5. <1 1. 2. 1. 5. 2. 10 0. 0.
S 5. <1 2. 5. 1. 5. 5. <1 0. 0.
E 5. <1 2. 10 2. 10 0. 0.
P 5. <1 5. <1 2. 10 *0. *0.
V 5. <1 5. <1 5. <1 5. <1 1. 10 5. <1 0. 0.
V 5. <1 5. <1 2. <1 5. <1 0. 0.
BsBacillus-,SaStaphylococcus-subtilis,SeStaphylococcus-aureus,EcEscherichia-epidermidis,Pa-PseudomonascoliStaeruginosa,-SalmonellaVaVibrio-
typhi,alginolyti,VcVibrio-,cCiprofloxacinusholerae*.