INTRODUCTION:

The wealth of medicinal plants and the traditional knowledge drastically recede in the wake of burgeoning population pressure, acculturation, rapid modernization, multivarious human activities and various developmental activities. Much of the current work in ethnobotany is concerned with the loss of traditional knowledge and the preservation of biological diversity in remote parts of the world where cultures and their ecosystems are being destroyed by development. (1) Careful ethnobotanical investigations may become invaluable to rescue knowledge in imminent danger of being lost and to find out new bioactive compounds in plant. The use of plant preparations as foodstuff, insecticides, CNS active, cardio active, antitumour and antimicrobial agents are some examples of immense chemical diversity in plants (2) As old as mankind with advances in phytochemical techniques several active principles of many medicinal plants have been isolated and introduced as valuable drugs in morden system of medicine.(3)

The world is now looking towards India for new drugs to manage various challenging diseases because of its rich biodiversity of medicinal plants and abundance of traditional know how such as Siddha, Ayurveda, to cure different diseases (4).From over 3, 00,000 species of higher plans to occur in nature, only about 2 percent have been screened The extract of plants from 157 families have been reported to be active against micro-organisms.(5)

materials and methods:

Sample Collection

The medicinal plant species namely Strychnos nux-vomica and Cassia angustifolia were collected from Pochampalli hills environment of Krishnagiri District. The collected samples were carefully stored in polythene bags and used for further investigation.

Sterilization of Plant Materials

About 1 gram of fresh and healthy leaves were taken for each solvent including aqueous the leaves of both plants Strychnos nux-vomica and Cassia angustifolia were sterilized with running tap water and soaked in 0.1% mercuric chloride solution for few seconds finally the leaves were washed with distilled water three times and shade dried.

Preparation of Plant Leaf Extract

About 1 gram of sterilized plant leaves were ground in mortar and pestle with 10ml of aqueous and organic solvents (Methanol and n-Butanol) it was filtered through what man no1 filter paper the supernatant was collected and stored for antimicrobial screening.

Microbial Strains

The three human pathogenic bacterial species Staphylococcus aureus, klebsiella pneumonia, Bacillus subtilis and three fungal species Aspergillus terreus A. flavus A. niger were collected from the Microbial Culture Collection Unit (MCCU) Sri Gowri Biotech Research Academy Nagai Road, Thanjavur, Tamil Nadu.

Media Preparation

The Nutrient agar Medium was used for culturing bacterial species.

Composition of nutrient agar medium

Beef Extract - 3g

Peptone - 5g

Sodium Chloride - 5g

Agar - 15g

Dissolved the contents in 1000ml of distilled and the pH was adjusted to 7.

Preparation

The above ingredients were weighed and put into the conical flask containing 1000ml distilled water. The flask was sterilized by an autoclave at 121^oC for 15 LBS pressure for 15 minutes and allowed to cool.

Screening of Antibacterial Activity by Well Diffusion Method (Levy, 1994)

The antibacterial activities of the leaves were tested against the selected bacterial strains. The petriplate’s were washed and placed in an autoclave for sterilization. After sterilization nutrient agar medium was poured into each sterile petriplate and allowed to solidify in a laminar air flow chamber.

Inoculation

After solidification using a sterile cotton swabs count was spread over the plate by spread plate technique. One well of 5 mm size made in the agar plates with the help of sterile cork borer, the well were loaded with 200ml of solvent’s extract (Methanol, n-Butanol and water) of these plant leaves’ and the solvent loaded.

Incubation

All the plates were incubated at 37^oC for 24 hours. After incubation, the plates were observed for formation of clear inhibition zone around the well indicated the presence of antibacterial activity, the zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well.

Media Preparation

The nutrient agar medium was used for culturing fungal species.

Composition of Potato Dextrose Agar Medium

Potato - 200g

Dextrose - 20g

Agar - 20g

Distilled water - 1000ml

pH - 5.0 to 6.0

Dissolved the content’s in 1000ml of distilled and the pH was adjusted to 7.

Preparation

The above ingredient’s were weighed and put into the conical flask containing 1000ml distilled water. The flask was sterilized by an autoclave at 121^oC 15 LbS pressure for 15 minutes and allowed to cool.

Screening of antimicrobial fungal activity by well diffusion method (Perez et al., 1990)

The antifungal activities of the leaves were tested against the selected fungal strains. The petriplates were washed and placed in an autoclave for sterilization. After sterilization nutrient agar medium was poured into each sterile petriplate and allowed to solidify in a laminar air flow chamber.

Inoculation

After solidification using a sterile cotton swabs count was spread over the plate by spread plate technique one well of 5 mm size made in the agar plates with the help of sterile cork borer, the well were loaded with 200ml of solvents extract (Methanol, n-Butenol an water) of these plant leaves .

Incubation

All the plates were incubated at 37^oC for 24 hours. After incubation the plates were observed for formation of clear inhibition zone around the well indicated the presence of antifungal activity. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well.

result AND Discussion:

In the present investigation, the antimicrobial activity of n-butanol methanol and distilled water of two medicinal plant’s Strychnos nux-vomica and Cassia angustifolia, were tested against three human pathogenic bacteria and fungi. The antimicrobial properties of the two extract’s of Strychnos nux-vomica and Cassia angustifolia comparatively analysed against standard antibiotics by antibiotic sensitivity test.

Antibacterial Activity of Strychnos nux-Vomica

The antibacterial activity of Strychnos nuxvomica extracted from n-butanol, methanol, distilled water were tested against Staphylococcus, aureus, Salmonella and Klebsiella pneumonia. The n-butanol extract of Strychnos nux vomica maximum zone of inhibition against staphylococcus aureus (15mm)and salmonella (13mm) was observed. The n-butanol extract of Strychnos nux-vomica showed maximum of zone of inhibition against staphylococcus aureas (15mm) and salmonella (13mm) was observed. The aqueous extract of Strychnos nux vomica does not showed any activity on both the isolates.

Antifungal Activity of Strychnos – Nux Vomica

The antifungal activity of n-butanol, methanol and water, of Strychnos – nux vomica were tested against Aspergillus terreus, and Aspergillus flavus. Maximum zone of inhibition, was observed when n-butanol extract of Strychnos-nux vomica was were used aginst the said fungal oraganism. The n-butanol extract of Enicostemma littorale, exhibited antifungal activity against Aspergillus terreus (20mm) and Aspergillus flavus (20mm) the aqueous extract of Strychnos – nux vomica, does not showed any activity against both isolates.

Antibacterial Activity of Cassia Angustifolia

The anti bacterial activity of n-butanol methanol and water extract of Cassia-angustifolia were tested against Staphylococcus and Salmonella. The n-butanol extract of Cassia angustifolia exhibited maximum zone of inhibition against staphylococcus (15mm) and Salmonella (13mm) The n-butanol extract of Cassia angustifolia showed maximum zone of inhibition against Staphylococcus (10mm) and Salmonella (13mm) The aqueous extract of Cassia angustifolia showed any activity against both the Isolated.

Antifungal Activity of Cassia Angustifolia

The antifungal activity of n-butanol methanol and water extract of Cassia angustifolia were tested against Aspergillus terreus and (12mm) Aspergillus flavus (15mm) and the result were presented(Table) The n-butanol extract of Cassia angustifolia showed a maximum zone of inhibition against Aspergillus terreus A. flavus. The n-butanol extract of Cassia angustifolia exhibited antifungal activity against A. terreus (5mm) and Aspergillus flavus (15mm). The aqueous extract of Cassia angustifolia does not showed any activity against both the isolates.

Antibiotic Sensitivity Test

The antibiotic sensitivity test has been using standard antibiotics. Viz. Tetracycline, Erythromycin Chloramphenical were tested against both bacteria and fungi studied and the result of antibiotic sensitivity represented. All the antibiotics used were exhibited higher anti bacterial activity the results confirmed that both the solvent extract Strychnos - nux vomica and Cassia angustifolia. Similarly, when compared to the standard antibiotics. The solvent extract of Strychnos - nux vomica and Cassia angustifolia showed higher antifungal activity against the fungi.

Antimicrobial Effect of Solvents

The result of antimicrobial effect of n-butanol, methanol and water solvent revealed no activity against bacteria and fungi studied.

table – 1 anti fungal activity of strychnos nux-vomica

*Sl. No.*	*Strychnos nux-vomica*	*n-butanol*	*Methanol*	*Distilled water*
*Sl. No.*	*Strychnos nux-vomica*	*(Zone of inhibition in mm)*
1.	Aspergillus terreus	20	-	-
2.	A. flavus	20	-	-
3.	A. niger	35	-	-

Table – 2 anti fungal activity of cassia angustifolia

*Sl. No.*	*cassia angustifolia*	*n-butanol*	*Methanol*	*Distilled water*
*Sl. No.*	*cassia angustifolia*	*(Zone of inhibition in mm)*
1.	Aspergillus terreus	12 mm	5 mm	-
2.	Aspergillus flavus	15 mm	5 mm	-
3.	Aspergillus niger	25 mm	10 mm	-

table – 3 anti bacterial activity of strychnos nux-vomica

*Sl. No.*	*Strychnos nux-vomica*	*n-butanol*	*Methanol*	*Distilled water*
*Sl. No.*	*Strychnos nux-vomica*	*(Zone of inhibition in mm)*
1.	Staphylococus sp aureus	15	-	-
2.	Salmonella sp typhi	13	-	-
3.	Klebsiella Pneumonia	10	-	-

table – 4 anti bacterial activity of cassia angustifolia

*Sl. No.*	*cassia angustifolia*	*n-butanol*	*Methanol*	*Distilled water*
*Sl. No.*	*cassia angustifolia*	(Zone of inhibition in mm)
1.	Staphylococus	17	-	-
2.	Salmonella	12	-	-
3.	Klebsiella Pneumonia	10	-	-

Discussion:

The present investigation was carried the antimicrobial activity of N-Butanol, Methanol and aqueous leaf extract of two medicinal plants followed Cassia agustifolia and Strychnosnux vomica were tested against the human pathogenic micro-organisms. Such as Klebsiella pneumonia, Bacillus subtilis, A niger, A terreus and A. flavusThe antimicrobial potential of plants was compared according to their zone of inhibition against the several pathogenic organisms. (Sasidharan et al., 1998).The antibacterial activity of the herbal extracts, indicated by the size of their zones of inhibition. Herbal extracts inhibited only the gram positive bacterium and aureus. The greatest antibacterial activity was detected from the ethanol extract. None of the herbal extracts examined showed antibacterial against E. Coli or P. aeruginose (gram negative bacteria) Herbal extracts have a greater activity against gram +ve bacteria than G-ve bacteria (Kabuki et al., 2000).The n-butanol extract of Cassia angustifolia exhibited maximum zone of inhibition against Staphylococcus sp. and Salmonella sp. The plant extracts were screened against human pathogenic bacteria to check antibacterial activities by agar well diffusion method, which showed valuable zone of inhibition. The specific zone of inhibition against various types of pathogenic bacteria (Alam Morshed et al., 2011).In the present study concluded that cassias angtifolia is best antimicrobial activity compared to Strychnos nux-vomica plant extracts of Cassia angustifolia possesses prospective broad spectrum antimicrobial potency against the test organisms.

ACKnowledgement:

We Are Thankful To Sri Gowri Biotech Research Acadamy, Thanjvur For Providing Lab Facilities.

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Received on 13.11.2011 Accepted on 10.03.2012

Asian J. Pharm. Tech. 2(1): Jan.-Mar. 2012; Page 08-11