Enumeration of Antibacterial Activity of Few Medicinal Plants by Bioassay Method

The present study was aimed to investigate the antibacterial activity of some common locally available plants, in order to estimate the biological potential of these herbs. The alcoholic extract of Tagetes erecta L (Asteraceae), Argemone mexicana L (Papavaraceae), Datura stramonium L. (Solanaceae) and Tylophora indica (Burm.f.) Merr. (Asclepiadaceae) were evaluated for antibacterial activity using broth dilution bioassay method. It is clear from the results that, the extracts of these plants acts as a good source of antibiotics against various bacterial pathogens tested and exhibited broad spectrum of antibacterial activity. These plant extracts were shown to be moderate to maximum inhibitory effect against different bacterial forms such as Salmonella typhii, Proteus vulgaris, Pseudomonas aeruginosa and Escherichia coli, where as, mild to moderate activity against Klebsiella pneumoniae and Staphylococcus aureus. The results of these studies revealed most valuable information and also support the continued sustainable use of these plants in traditional systems of medicine.


Introduction
It is evident that the use of plants for various diseases since from vedic period. documentation of the ayurvedic system recorded by Sushruta and Charaka dates from about 1000 BC 1 . Plants are a goldmine of novel chemicals; much impressive number of modern drugs has been developed from them. There are more than 2,70,000 higher plants existing in this planet. But so far less than 10% of recorded flora has been explored phytochemically as well as clinical evaluation for various biological activities 2 . While vast majority of the plant resource is waiting for discovery. So far there is no appropriate treatment in the modern system of medicine for many of the diseases such as, Cancer, AIDS, Jaundice, Hepatitis, SARS, Chikun-guniya and Diabetes. Some of the nucleotide analogues, the current antiviral drug use reverse transcriptase as target and are highly toxic compounds. Recently certain polyphenolic and bioflavonoids have been found to be the potential source of reverse transcription inhibitors. Anti-hepatotoxicity, immunomodulatory, anti-inflammatory, antimicrobial, antifeedent and antioxidant activities are amongst other important properties of flavonoids [3][4] .
The green revolution in the industrialized world has convinced many companies that plant might also prove a gold mine profit, an endless source which leads to new drugs and environmental friendly pesticides. By considering these above and other factors the present investigation was undertaken to evaluate the antibacterial activity by bioassay method using the crude ethanolic extracts of Tagetes erecta, Argemone mexicana, Datura stramonium and Tylophora indica.

Experimental
The leaves of Argemone mexicana, Tylophora indica, Tagetes erecta and Datura stramonium were collected from in and around Bellary city, Karnataka, India. All these plants were authenticated [5][6][7] and the voucher specimens were deposited in the Herbarium of Department of Botany, Gulbarga University, Gulbarga, India. Later leaves of these plants were subjected to surface sterilization using 50% alcohol and then shade dried for further analysis.

Preparation of the extracts
The 100 g leaves of A. mexicana, T. indica, T. erecta and D. stramonium were extracted with 300 mL of 95% alcohol at 60 -80 °C in a soxhlet apparatus. The obtained extracts were collected in a separate container and concentrated to dryness in a flash evaporator (Buchi type) under reduced pressure and controlled temperature (40 -50 °C).

Microorganisms
Clinical laboratory bacterial isolates of Klebsiella pneumoniae, Staphylococcus aureus, Salmonella typhii, Proteus vulgaris, Pseudomonas aeruginosa and Escherichia coli clinical isolates were collected from the standard stock cultures of Microbiology Laboratory, Vijayanagar Institute of Medical Sciences, Bellary, India. These cultures were maintained on nutrient agar medium and were stored at 4 o C for determining antimicrobial activity of these selected medicinal plants.

Culture media
The nutrient agar and nutrient broth were purchased from HiMedia, Laboratories Limited, Mumbai. Streptomycin (powdered form) another standard antibiotic from Nanjing Asian Chemicals Co., Ltd. The solvents and other chemicals used were analytical grade.

Antimicrobial-bioassay method
Nutrient broth media was prepared and distributed into fourteen different test tubes, then autoclaved and cooled. To these tubes, 0.1 mL of 24 h old cultures of test microorganisms, different concentrations (0.8 mg/mL, 1.2 mg/mL/ and 1.6 mg/mL) of the of four plants extracts and 0.2 mg/mL of streptomycin (standard positive control) were added separately into thirteen different tubes, only the fourteenth tube was not treated with any of the drug and used as a negative control. These tubes were incubated at 37 o C for 24 h in a BOD incubator. Subsequently after 24 h 0.1 mL of formaldehyde solution was added to each inoculated tube as a growth stopping agent. Then the optical density of each tube was measured at 530 nm. Same procedure was repeated to all six different bacterial species separately. Nutrient medium devoid of plant extract and microorganisms was served as Blank 8 . This experiment was repeated thrice and found out the average mean and standard error using SPSS software package.

Antibacterial assay
In the present investigation, all these plants furnished evidence in favor of the antimicrobial activity. The alcoholic extract of the leaves of Datura stramonium was found to be most efficient in controlling the growth of certain bacterial forms such as Salmonella typhii, Pseudomonas aeruginosa, Proteus vulgaris and moderately effective against Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli (Table 1). Similar observations were noticed with the extract of Argemone mexicana, but Pseudomonas aeruginosa was very much susceptible and highly sensitive to the extract of A. Mexicana (Table 2). Where as S. typhii, P. vulgaris, P. aeruginosa and E. coli were found to be highly sensitive, but K. pneumoniae and S. aureus were moderately inhibited to Tagetes erecta (Table 3). Where as, similar observations were noticed with Tylophora indica, except S. aureus was found to be less sensitive (Table 4).  The present investigations revealed that, the different concentrations of the alcoholic extracts of D. stramonium, T. indica, T. erecta and A. mexicana were proved to be effective and concentration dependent antimicrobial activity against both Gram positive and Gram negative bacteria tested. This is also evidenced by several research groups, and supporting the presence of antibacterial activity of D. stramonium and T. indica against Gram positive bacteria by dose dependent manner [9][10][11][12] . Where as, the inhibition of Gram positive and negative bacterial growth by Argemone mexicana and Tagetes erecta based on agar diffusion method 13 . So far there are no reports on the clinical antibacterial and antifungal activity on these plants.

Conclusion
In conclusion, the alcoholic extracts of these plants showed promising antimicrobial activity against all the selected pathogens. However, further experiments including detail evaluation of antimicrobial potentials are required to elucidate their mechanism of action at cellular and molecular levels.