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Journal of Phytology 2010, 2(3): 0812 ISSN: 2075-6240

An Open Access Journal Available Online: www.journal-phytology.com

REGULAR ARTICLE

ANTIBACTERIAL EVALUATION OF SNAKEWEED(EUPHORBIA HIRTAL.)

O. Aniel Kumar*, L. Mutyala Naidu and K.G. Raja Rao

Department of Botany, Andhra University, Visakhapatnam (A.P) 530 003, India

SUMMARY

Ethanol, Methanol, Chloroform and Aqueous (water) extracts of leaf, stem, root and wholeplant of Euphorbia hirta L. (Euphorbiaceae) were used to evaluate antibacterial activity. Theagar-well diffusion assay was employed against several Gram-positive (Bacillus subtilis andStaphylococcus aureus) and Gram-negative (Escherichia coli, Klebsiella pneumoniae and Proteus

vulgaris) bacterial species. Aqueous and chloroform extracts of stem and root did not expressany activity. Antibacterial activity was recorded in the order of ethanol, methanol, aqueous andchloroform extracts. Among these extracts ethanol and methanol extracts of leaf and wholeplant were more effective and significant than aqueous and chloroform extracts in inhibitingthe growth of the pathogenic bacteria under study, but were less potent when compared to thatof tetracycline used as positive control. Phytochemical screening of the plant revealed thepresence of tannins, flavonoids, alkaloids, glycosides and saponins. The study scientificallyvalidates the use of plant in traditional system of medicine to treat various diseases.

Keywords:Euphorbia hirta (L.), antibacterial activity, micro-organisms.

O. Aniel Kumar et al. Antibacterial Evaluation of Snake Weed (Euphorbia hirta L.). J Phytol 2/3 (2010) 08-12

*Corresponding Author, Email: [email protected]

1. Introduction

In many parts of the world, there is a richtradition in the use of herbal medicine for thetreatment of many infectious diseases [1]. Indeveloping countries, it is estimated thatabout 80% of the population rely ontraditional medicine for their primary healthcare [2]. Many currently used drugs areexpensive or not readily available and amajor set back to their continued usage is the

development of resistance. This situationurgently forced scientists for searching newinexpensive drugs that will be able to act forlonger periods before resistance set in.Because of the side effects and the resistancethat pathogenic microorganisms buildresistant against the common antibiotics,much recent attention has been paid toextracts and biologically active compoundsisolated from plants used in herbal medicine[3].

Euphorbia hirta (family: Euphorbiaceae) isan herb found in many parts of the world

and its common names, Australian asthmaplant, Garden spurge and Snake weed. Thestem and leaves produce white or milky juicewhen cut [4]. The leaves of Euphorbia hirta arefound to contain flavonoids, polyphenols,tannins, sterols, alkaloids, glycosides andtriterpenoides [5]. The plant has a reputationfor increasing milk flow in women becauseof its milky latex and is used for other female

complaints as well as diseases like bronchitis,asthma, eczema, dysentery. It is used asantidiarrheal, antispasmodic, anti-inflammatory, antifungal, anticancer,antimalarial, antiamoebic, antibacterial andantihelmentic etc. The present investigationwas carried out on different plant parts ofEuphorbia hirta (L) (Root, stem, leaf andwhole plant ) to determine the antibacterialactivity of their aqueous, alcoholic andchloroform extracts against five bacterial

strains (both Gram-positive and Gram-negative).

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O.A. Kumar et al./J Phytol 2/3 (2010) 08-12

2. Materials and MethodsCollection of plant materials:

The different parts of Euphorbia hirta (L.)such as leaf, stem, root and whole plant werecollected from the field grown plants in and

around the Andhra University,Visakhapatnam, Andhra Pradesh and wereused for the following experiments.

Sample preparation and extraction procedure:Freshly collected plant materials such as

leaves, stem, root and whole plant ofEuphorbia hirta (L.) were dried in shade inwell ventilated enclosures and ground intofine powder using a mechanical grinder. 50gof the fine powder of each plant part ofEuphorbia hirta (L.) was extracted in Soxlet

apparatus separately [6]. Sequentialextraction was done with methanol, ethanol,chloroform and distilled water for about 18hours with each solvent. The extracts wereevaporated to dryness under vacuum using arotary evaporator. The extract was thenstored below ambient temperature.

Preparation of dilution of crude extract forantibacterial assay:

The methods of Akujobi et al [7] andEsimone et al [8] were adopted. The crude

extracts were dissolved in 30% dimethylsulphoxide (DMSO) and further diluted toobtain of each extract sample at 750 g/mlconcentration was used for the determinationof antibacterial activities by agar - welldiffusion method.

Test micro organismsThe organisms Escherichia coli, Klebsiella

pneumoniae, Proteus vulgaris, Bacillus subtilisand Staphylococcus aureus were obtained frommicrobial type culture collection (MTCC),

IMTECH, Chandigarh, India. They were re-isolated and the pure cultures subculturedon nutrient agar slants. They were stored at40C until required for the study.

Evaluation of anti bacterial activityThe agar-well diffusion method as

described by Esimone et al (1998) wasadopted for the study. fifteen ml of moltennutrient agar was seeded with 1.0 ml ofstandardized broth cultures of the bacteria(1.0x108 CFU/ml) by introducing the broth

cultures into sterile petridishes,incorporating the molten agar, rotating

slowly to ensure uniform distribution of themicroorganisms and then allowed to solidifyon a flat surface. Four wells (holes) weremade in the plates (about 6.0 mm diameter)using sterile cork borer and equal volumes of

the extracts were transferred into the wellsusing micropipette. Three pertidishescontaining a particular microorganism wereused for each concentration of the extract.The plates were allowed to stand for onehour for pre-diffusion of the extract to occurand were incubated at 370C for 24 hours. Atthe end of incubation the plates werecollected and zones of inhibition thatdeveloped were measured. By average of thezone of inhibition was calculated. Antibiotic

tetracycline at a concentration of 30g/ml aspositive control and 100% Dimethysulphoxide (DMSO) as negative control wereused.

3. ResultsThe antibacterial activity of crude

extracts of the different parts of Euphorbiahirta (L.) were assessed using the agar-welldiffusion method by measuring the diameterof growth inhibition zones with aqueous(water), methanol, ethanol and chloroform

extracts are depicted in Table- 1. In total ofsixteen extracts belongs to different parts ofEuphorbia hirta (L.) were tested against bothGram-positive (Bacillus subtilis andStaphylococcus aureus) and Gram-negative(Escherichia coli, Klebsiella pneumoniae andProteus vulgaris) bacteria in the presentinvestigation. The aqueous and chloroformextracts of stem and root exhibited nobacterial activity, whereas the extracts ofleaves and whole plant exhibited higherantibacterial activity against Gram-positivebacteria than Gram-negative bacteria.Ethanol and methanol extracts of leaves andwhole plant were found to be highlysignificant antibacterial activity againstGram-positive bacteria but moderate andsignificant activity against Gram-negativebacteria. Similarly the same extracts of thestem and root were found moderatelysignificant antibacterial activities againstGram-positive bacteria. Chloroform extractsof leaf and whole plant exhibited moderately

significant antibacterial activity againstGram-positive bacteria whereas stem and

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root extracts exhibited lower andinsignificant antibacterial activity against

both Gram-positive and Gram-negativebacteria (Table 1).

Table 1. Antibacterial activity of different parts of Euphorbia hirta extracts.

Plant part Plant extractswith differentconcentrations

Zone of inhibition(mm)Escherichiacoli

Klebsiellapneumoniae

Proteusvulgaris

Bacillussubtilis

Staphylococcusaureus

Leaves

Stem

Root

Whole plant

Tetracycline(30g/ml)

AqueousMethanolEthanolChloroform



AqueousMethanolEthanolchloroform

512144

--89--

--78--

814156

17

4.59102.5

--68--

--56--

510123

14

610123

--710--

--47--

712147

5

916198

--1315--

--1214--

11182410

26

814166

--1113--

--1012--

916217

24

--: No activity

From the results of antibacterialscreening of four solvents (ethanol, methanol,chloroform and water) of different parts ofEuphorbia hirta (L.) used in this study ethanoland methanol extracts of whole plant andleaf exhibited the best antibacterial activity.

4. DiscussionThe presence of antibacterial substances

in the higher plants is well established [9].Plants have provided a source of inspirationfor novel drug compounds as plants derivedmedicines have made significantcontribution towards human health.Phytomedicine can be used for the treatmentof diseases as is done in case of Unani andAyurvedic system of medicines or it can bethe base for the development of a medicine.Successive isolation of botanical compoundsfrom plant material is largely dependent onthe type of solvent used in the extractionprocedure. The traditional healers useprimarily water as the solvent but we found

in the study the plant extracts by ethanol andmethanol provides more consistent

antibacterial activity compared to thoseextracted by water. The results of theantibacterial activity of different plant partsof Euphorbia hirta (L.)against the investigatedbacterial strains are shown in the Table-1.

In the present study the results obtainedindicated that the ethanol, methanol,chloroform and water extracts of differentplant parts of Euphorbia hirta inhibited thegrowth of the tested microorganisms viz.Escherichia coli, Klebsiella pneumoniae and

Proteusvulgaris (Gram-negative) and Bacillussubtilis and Staphylococcus aureus (Gram-positive). However, its effects are low whencompared with standard antibacterial agenttetracycline was used in this study aspositive control (Table 1). The present resultsrevealed that the alcoholic extracts (ethanoland methanol) showed the maximum degreeof inhibition of zone as compared withaqueous and chloroform extracts. It is quitepossible that the stem and root extracts ofwater and chloroform were ineffective in thepresent study do not posses antibacterial

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properties. This may be due to the activechemical constituents were not soluble inwater and chloroform. Ethanolic andmethanolic extracts of whole plant and leafof Euphorbia hirta showed the maximum

degree of antibacterial activity properties.This may be due to the presence of alkaloids,tannins, saponins and flavonoids which areplant secondary metabolites known to possesantibacterial properties. Similar observationswere also reported in various plant extractswith different concentrations [7, 8,10,11,12].

The higher resistance of Gram-negativebacteria to plant extracts as previously beendocumented and related to thick mureinlayer in their outer membrane, which

prevents the entry of inhibitor substances[2,13,14,15]. Similarly our results indicatedthat the antibacterial activities of the extractsof Euphorbia hirta were more pronounced onGram-positive than on Gram-negativebacteria. Alternatively, the passage of theactive compound through the Gram-negativecell wall may be inhibited that observeddifferences may result from doses used in thestudy. In addition, microorganisms showvariable sensitivity to chemical substances torelated to different resistance levels between

strains.The results of this study have shown the

different plant parts of Euphorbia hirta havegreat potential as antimicrobial agents in thetreatment of infectious diseases caused byresistance microorganisms. Further detailedstudy is necessary regarding individualscreening of phyto constituents from thedifferent plant parts of Euphorbia hirta toconclusively identify the antimicrobialcompound. However, the present study

indicates that the plant contains potentialantimicrobial compounds which can befurther developed as phytomedicine forprevention of infection.

AcknowledgmentsThe authors are grateful to UGC-SAP,

Department of Botany, Andhra University,Visakhapatnam for providing financialassistance.

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