Antimicrobial activity of Ethanol extract of the leaf, stem, Pulp and seed of Bryonia laciniosa PRESENTED BY: EHSAN BONYADI RAD RAJIV GANDHI INSTITUTE OF IT AND BTBHARATI VIDYAPEETH UNIVERSITY

PUNE- 411046, INDIA

Introduction

Herbal medicine and its prominence in use

Herbal medicine are originated mostly from ancient countries such as Egypt ,Greece, Persia, India and China.For healing the ailments as a drugs or food additive or in form of traditional use.Over three-quarters of world population relies mainly on plants and plant extracts for health care.3000 Medicinal plants are reported India.It is prominenet because:Inadequate supply drugs, high cost of treatment, side effect & development of the resistanceIt is preferable because:Little side effect, renewable resources, compatibility, safety and more efficacy.

Bryonopsis laciniosaNames and classification Family : Cucurbitaceae Botanical name: Bryonopsis laciniosa (Bryonia laciniosa) Local name : ShivlingiEnglish Name : Lollipop plantSanskrit Name : Apastambhini, Chandra, Lingaja, Lingini, Shivavalli

Found in most parts of the herbal Old World especially India.

B. laciniosa specifications

Physical properties Medicinal propertiesA herbaceous, slender, monoecious climberLeaves - Palmate 3-7 lobed Flower - Creamy white, in axillary fasciclesPulps - spherical and smoothSeeds - Obovate, creamy-white or pale yellowFlowering season - August to October Potent medication Tonic and aperient Impotency treatment Treatment of fevers with flatulenceWhole plant Used to treat: adenopathy, ague, asthma, bronchitis, cholera, colic, consumption, convulsion, cough, fertility, headache, paralysis, phthisis, snake bite

Rationale of projectBryonopsis Consist of more than 100 speciesB.dioica, B.alba and B.laciniosa are medicinal.B.alba and B.dioicia are utilized in European countries in treatment of many diseases.

B.alba used: As Diuretics In Gravel (In Urinary Blocks) For relieving coughs and colds

B. dioica is a powerful cathartic and expectorant used in treatment of : Asthma Intestinal ulcers and Arthritis Vermifuge Intestinal ulcers, Hypertension

In some reference literatures, the medicinal properties of Shivlingi leaves and whole plant are described as:

The methanol extract of B. Laciniosa showed significant analgesic and antipyretic activity.

The chloroform extract of B.laciniosa exhibited significant anti-inflammatory effect.

Goniothalamine isolated from B. laciniosa has shown significant cytotoxicity, anti-cancer, apoptosis-inducing properties.

The antimicrobial activity of this plant hasnt been reported so far.

So the aim is to find out the antimicrobial activity of B.laciniosa

Collection and ExtractionCollectionDryingGrindingMixing with solventIncubation Filtration Evaporation Checking the activityCollected from Katraj dairy medicinal plant farmDrying at 37c for 48 hsGround the samples with hammer-milled methodMixing 10 gm of each powdered sample with 50 ml of 80 % ethanol (further extraction was repeated 3 times)Incubated in dark place for 3 to 5 days in RTMixtures are filtered through whatman NO.1 filter paper.Removing the ethanol from extracted liquid by rota- evaporation and waterbath evaporation method (temperature 70 c).Semisolid extracts air-dried, weighed and dissolved in known amount of 5 % DMSO to give concentration of 40 mg/ml.

Antimicrobial assay Disc diffusion method (Modified Kirby Bauer method )Test organisms are: S.aureus, M.leuteus, B.cereus, E.coli, S.typhimurium,P.aeruginosa Autoclaved paper discs (diameter 6 mm) were soaked in different concentration of the extract and allow to evaporate the solvent.100 l Standardized fresh inoculums (McFarland standard NO. 0.5) spread on agar plates3. After 15 minutes the discs are placed on the agar plates4. Incubation for 24h at 37C. 10l Ampicilin (100 mg/ml) - positive control.

5% DMSO control-blank.

Well diffusion method

After pour plating 4 mm in diameter wells are punched in the agar medium

Wells filled by 10l of different concentration of each extract ,+ve and -ve control.

The plates incubated for 24 h at 37C and the diameter of any resulting zone of inhibition are measured.

10l Ampicilin (100 mg/ml) - positive control.

5% DMSO control-blank.

Minimum inhibitory concentration(MIC) (de Paiva et al, 2003) 0.1 ml (100 l) of sterile distilled water was added in each well.0.1 ml (100 l) of extract (40 mg/ml) were added to the first well and diluted serially by two-fold dilution method.Then each well was inoculated with 100 l of 0.5 McFarland standard bacterial suspensions.Plates were incubated at 37C for 24 h.The MIC was defined as the lowest concentration of the extract inhibiting the visible growth of each micro-organism.

Result (disc diffusion method)M. leuteusE.coliP. aeruginosaB. Cereus

B. CereusM. leuteusS.aureusResult (Well diffusion method)leave extract stem extractS.aureusM. leuteus

Antimicrobial activity exhibited by leave extract through agar disk diffusion method.Test Organism Zone of inhibition (mm) Concentration (mg/ml) Ampicilin100 mg/ml 10 20 30E. coli---17P. aeruginosa-7918S. typhimurium---24S. aureus14161735M. leuteus12151622B. cereus15182023

Antimicrobial activity of various extracts through agar well diffusion methodTest OrganismZone of inhibition (mm)Concentration (mg/ml)Ampicilin(mg/ml)100Leave extractStem extractPulpextractSeed extract10 20 3010 20 3010 20 3010 20 30E. coli------------15P. aeruginosa-56-67------17S. typhimurium------------21S. aureus8101381114------30M. leuteus69126711------20B. cereus9111471013------22

MIC values of active extracts on test bacteriaTest OrganismLeave extract (mg/ml)Stem extract (mg/ml)Ampicilin(mg/ml)aureus

M. leuteus

P. aeruginosa

B. cereus 0.625

0.625

10

1.25 0.156 0.156

5

1.25 0.048

0.097 3.125 0.097

Conclusion The present study proved antimicrobial activity of B. laconioca Bacillus cereus was the most susceptible bacteria to this extract.It is evident that tested bacteria are more sensitive to stem extract than leave extract. The MIC values ranged between 0.156 and 5 mg/ml for stem extractMIC varied between 0.625 and 10 mg/ml for leave extract against tested microorganismsThis in vitro study demonstrated that folk medicine can be as effective as modern medicine to combat pathogenic microorganisms.It is hoped that this study would lead to the establishment of some compounds that could be used to formulate new and more potent antimicrobial drugs of natural origin.

Thank you

*********Bacillus cereus**