INTRODUCTION:

A medicinal plant represents a rich source of anti microbial agents. Plants are used medicinally in different countries and are a source of many potent and powerful drugs. The use of medicinal plants as a source for relief from illness can be traced back to over five millennia to written documents of the early civilization in India, China and the near east and the art is as old as mankind.^[1] Many efforts have been made to discover new antimicrobial compounds from various kinds of sources such as microorganisms, animals, and plants. Although hundreds of plant species have been tested for antimicrobial properties, the vast majority of plants have not been adequately evaluated. Systematic screening of plants may result in the discovery of novel effective anti microbial compounds. The increasing prevalence of multi drug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the specter of untreatable bacterial infections and adds urgency to the search for new infection-fighting strategies. Contrary to the synthetic drugs, antimicrobials of plant origin are not associated with many side effects and have an enormous therapeutic potential to heal many infectious diseases.²

India throughout its long history has accumulated a rich body of empirical knowledge of the use of medicinal plants for the treatment of various diseases. Chemical studies of Indian medicinal plants provide a valuable material base for the discovery and development of new drugs of natural origin.

Considering the vast potential of plants as a source for antimicrobial drugs a systemic investigation of ethanolic extracts of different parts (roots, rhizomes, aerial parts, leaves and fruits) of five plants, which had been described in herbal books and folklore medicine of India, was carried out for their antimicrobial activity. The species tested were: Butea frondosa Roxb, Momordica charantia Linn, Rubia cardifolia Linn, Swertia chirayita Ham, Tribulus terrestris Linn.

The Butea frondosa Roxb belongs to the family Papilionaceae. The flowers are used in pruritus and skin diseases, as diuretic, astringent and aphrodisiac. Seed are used as anthelmintics and are useful in flatulence and piles.^[3] Momordica charantia Linn (Family- Cucurbitaceae) fruits are used as stimulant; blood purifier anthelmintics and shows profound antidiabetic (Hypoglycemic) activity.^[4] The fruits and roots of Rubia cardifolia Linn belonging to family Rubiaceae are used as alternative, astringent, diuretic and in hepatic obstruction and skin diseases.^[5] Swertia chirayita Ham belongs to the family Gentianaceae and the whole plant is used in bronchial asthma and liver disorders, cough, dropsy, skin diseases, as an antimalarial, anthelmintic, antidiarrrhoeal and as bitter stomachic and tonic.^[6]The fruits of Tribulus terrestris Linn (Zygophyllaceae) are used as diuretic, tonic, aphrodisiac and astringent. They are also used in the treatement of renal calculi, urinary incontinence, gonorrhea, impotence and genitourinary tract diseases.⁷

MATERIALS AND METHODS:

Collection of Plant Material: Fresh leaves, bark, roots and fruits of five different plants viz., Butea frondosa Roxb, Momordica charantia Linn, Rubia cardifolia Linn, Swertia chirayita Ham, Tribulus terrestris Linn free from disease were collected from the local dealer of Ayurvedic medicines and authenticated at the Botany Dept. D.N. College, Faizpur. Different parts were first wash thoroughly 2-3 times with running water and once with sterile distill water. The material was then air dried on sterile blotter under shade.

Solvent extraction: Thoroughly washed dried leaves of five plants of Butea frondosa Roxb, Momordica charantia, Rubia cardifolia, Swertia chirayita Ham, Tribulus terrestris, flowers of Butea frondosa Roxb, fruits of Momordica charantia and roots of Rubia cardifolia, Swertia chirayita Ham, Tribulus terrestris were dried in shade for five days and then powdered with the help of a mechanical grinder and passed through a 20-mesh sieve. 25g of shade dried powder was filled in the thimble and extracted successively with ethanol solvent in Soxhlet extractor for 48h. The solvent extracts were concentrated under reduced pressure and preserved at 5 ˚C in airtight bottle until further use.

Growth and Maintenance of Test Microorganism for Antimicrobial Studies: Bacterial cultures of Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa) and fungal cultures of Candida albicans (C. albicans) and Aspergillus flavus ( A. flavus) were obtained from the culture collection center, Department of Microbiology, D.N. College, Faizpur. The bacteria were maintained on nutrient broth (NB) at 37 ˚C and funguses were maintained on Potato dextrose agar (PDA) at 28 ˚C.

Preparation of Inoculum: The bacteria were pre cultured in nutrient broth over night in a rotary shaker at 37 ˚C, centrifuged at 10,000 rpm for 5 min, pellet was suspended in double distilled water and cell density was adjusted. The fungal inoculum was prepared from 5 to 10 days old culture grown on potato dextrose agar medium. The Petri dishes were flooded with 8 to 10 ml of distilled water and the conidia were scraped using sterile spatula and the spore density of each fungus was adjusted.

Anti-bacterial Activity: The ethanol leaf extracts of Butea frondosa Roxb, Momordica charantia, Rubia cardifolia, Swertia chirayita Ham, Tribulus terrestris, flowers of Butea frondosa Roxb, fruits of Momordica charantia and roots of Rubia cardifolia, Swertia chirayita Ham and Tribulus terrestris were tested by the disc diffusion method.^[8] Different concentrations of the extracts (100 µg/ml) were prepared by reconstituting with ethanol. The test microorganisms (10 µl) were seeded into respective medium by spread plate method with the 24h cultures of bacteria growth in nutrient broth. After solidification the filter paper discs (5mm in diameter) impregnated with the extract were placed on test organism seeded plates. Streptomycin Sulphate (10 µg/ml) was used as positive control while Ethanol solvent (100 µg/ml) was used as negative control. The antibacterial assay plates were incubated at 37˚C for 24h. The diameter of each zone of inhibition was measured in mm.

Antifungal Activity: The antifungal activity was tested by disc diffusion method.^[9] The potato dextrose agar plates were inoculated with each fungal culture (10 days old) by point inoculation. The filter paper discs (5mm in diameter) impregnated with 100-μg/ml concentrations of the extracts was placed on test organism seeded plates. A blank disc impregnated with solvent ethanol followed by drying off was used as negative control and Nystatin (10 µg disc) was used as positive control. The activity was determined after 72h of incubating at 28˚C. The diameter of the inhibition zones was measured in mm.

RESULTS:

Results obtained in the present study exhibits that the tested five plants extracts possess potential antibacterial and anti fungal activity against the microorganisms studied. (Table 1 and 2) When tested by the disc diffusion method, the ethanol leaf extract of Butea frondosa Roxb showed significant activity against B. subtilis, E. coli and S. aureus (around 18 mm) The highest activity of 20 mm in B. subtilis and least activity of 16 mm was found against P. aeruginosa. Flower extract of Butea frondosa Roxb exhibits highest activity against B. subtilis, and S. aureus and lowest activity against A. flavus. Leaf extract of Momordica charantia showed highest activity against S. aureus, B. subtilis and E. coli and least activity against P. aeruginosa and C. albicans. The fruit extract shows highest activity against E. coli and S. aureus and lowest activity against C. albicans and P. aeruginosa. The leaf and root extract of Rubia cardifolia, Swertia chirayita Ham and Tribulus terrestris showed almost similar zone of inhibition against all the tested microorganisms. The root extract of Swertia chirayita Ham and Tribulus terrestris showed varied in the zone of inhibition from 10-14 mm against all the tested microorganisms. The ethanolic extracts of all the plant parts showed almost similar or higher activity when compared with the streptomycin Sulphate and Nystatin for anti microbial and anti fungal activity respectively.

DISCUSSION:

Plants are important source for development of potentially useful newer chemotherapeutic agents. The in vitro anti bacterial activity assay may be the first step towards development of such agents. Many reports are available regarding the antibacterial^[10-13], anti fungal^{[14, 15]} and anthelmintic^[16] activity of plants. Some of these studies have helped in identifying and isolation of the active principal responsible for such activities. However not many reports are available on the exploitation of antifungal or anti bacterial property of plant for developing commercial formulations. In the present study five medicinal plant species viz Butea frondosa Roxb, Momordica charantia, Rubia cardifolia, Swertia chirayita Ham and Tribulus terrestris were screened to detect the presence or absence of anti microbial activity. The presence of antimicrobial substance in one form or other was confirmed in all the tested plants.

The ethanol extract exhibits maximum activity may be attributed to the fact that being an organic solvent it

Table 1: Antibacterial activity of some medicinal plant ethanol extracts (100µgml) and antibiotic (10µg ml) against bacterial species tested by disc diffusion assay.

Bacterial sp.	Zone of Inhibition (mm)
	*Butea* *frondosa*		*Momordica charantia*		*Rubia cardifolia*		*Swertia chirayita*		*Tribulus terrestris*		Streptomycin sulphate
	Leaves	Flower	Leaves	Fruit	Leaves	Root	Leaves	Root	Leaves	Root
Bacillus subtilis	15± 0.66	20±1.20	16± 0.33	15 ± 0.57	13 ± 0.70	17± 0.51	14± 0.57	15± 0.33	11± 0.33	15± 0.33	15± 0.33
Escherichia coli	14± 0.33	17 ± 0.88	12± 0.33	19± 0.88	10± 0.70	14± 0.30	15± 0.15	17± 0.57	13± 0.57	14± 0.57	14± 0.33
Staphylococcus aureus	13± 0.57	18 ± 0.33	14 ±1.20	17 ± 1.15	12 ± 0.75	16± 0.50	14± 0.33	15± 0.25	12± 0.50	15± 0.25	15± 0.25
Pseudomonas aeruginosa	15 ± 0.66	16 ± 0.88	16 ± 0.33	14± 1.20	14 ± 0.25	17± 0.51	13 ± 0.51	14± 0.33	14± 0.33	17± 0.33	14± 0.33

Values are mean inhibition zone (mm.) ± S.D. of three replicates

Table 2: Antifungal activity of some medicinal plant ethanol extracts (100µg ml) and antibiotic (10µg ml) against fungal species tested by disc diffusion assay

Fungal sp.	Zone of Inhibition (mm)
	*Butea frondosa*		*Momordica charantia*		*Rubia cardifolia*		*Swertia chirayita*		*Tribulus terrestris*		Nystatin
	Leaves	Flower	Leaves	Fruit	Leaves	Root	Leaves	Root	Leaves	Root
Candida albicans	12± 0.57	14 ± 0.33	8 ± 0.33	11 ± 0.25	10 ± 0.33	12 ± 0.25	10 ± 0.33	12± 0.25	11 ± 0.88	14 ± 0.51	12± 0.33
Aspergillus flavus	8 ± 0.33	10 ± 0.33	8 ± 0.33	11± 0.33	11 ± 0.33	13 ± 0.51	9 ± 0.33	11± 0.57	10 ± 0.25	13 ± 0.633	10± 0.57

Values are mean inhibition zone (mm.) ± S.D. of three replicates

dissolves organic compounds better than aqueous extract and also liberate the active component required for antibacterial activity.

In the present study the leaf, fruit and the root extract of all the plants have shown antimicrobial activity against B. subtilis, E. coli, S. aureus, P. aeruginosa, C. albicans and A. flavus. Butea frondosa Roxb, Rubia cardifolia and Tribulus terrestris have shown maximum activity against all the strains of microorganisms as depicted from the respective zone of inhibitions. The maximum activity was seen against E. coli and B. subtilis while C. albicans and A. flavus were found to be less inhibited.

Many substances may be anti microbial, but only a few of them will be potential therapeutic agents for the simple reason that mammalian cells are more sensitive to chemical inhibition than microbial cells. The need for toxicity testing of drugs derived from plants is also emphasized because the crude products obtained from such sources are often associated with a large number of compounds that have discomforting abilities. Hence the herbal drugs have to be subjected to extensive pharmacological, toxicological and clinical tests to conform the prescribed status.

The present study has shown a variety of antimicrobial activities, which provides a support to some traditional uses of these few medicinal plants. The present study emphasizes the need of identification, isolation and extensive scientific and pharmacological screening of the effective biomolecules from these plants responsible for antimicrobial activity.

ACKNOWLEDGEMENT:

The authors are grateful to the Principal and The Head, Department of Botany and Department of Microbiology D. N. College, Faizpur, Maharashtra, India for their valuable help and suggestions during the study period.

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Received on 11.03.2010 Modified on 26.03.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 924-926