**2.4 Preparation of plant extracts**

130 Antimicrobial Agents

Natural plant products and their analogues are an important source of new agricultural chemicals (Cardellina, 1988, Gulter, 1988). Medicinal plants as a group comprise approximately 8000 species and account for around 50% of all the higher flowering plant species of India. Over one and a half million practitioners of the Indian System of Medicine use medicinal plants in preventive, promotive and curative applications. In recent years, secondary plant metabolites (Phytochemicals), previously with unknown pharmacological activities, have been extensively investigated as a source of medicinal agents (Krisharaju et al, 2005). Plants have been formed the basis of natural pesticides, that make excellent leads for new pesticide development (Newman et al., 2000). The potential of higher plants as a source of new drugs is still largely unexplored. Hence, last decade witnessed an increase in the investigation on plants as a source of new biomolecules for human disease management (Grierson and Afolayan, 1999). Green plants are found to be an effective reservoir for the bioactive molecules and can provide valuable sources for the discovery of natural pesticides (Akhtar *et al.,* 1997). Therefore in recent years medicinal plant extracts are intensively

Fifty medicinal plants (Table-1) were selected in this study based on the information collected from literature (Warrier *et al.,* 1994-1996 and Pullaiah, 2002). All the plant materials were collected in and around Visakhapatnam over the course of the respective growth season during February to April in the year 2005 because of the extracts were generally rich in antibacterial agents after the flowering (sexual) stage and plants from stressful environments (Mitscher *et al.,* 1972). Plant materials were identified with the help of Gamble, "Flora of the Presidency of Madras" and later verified by comparison with the authentic specimens available in the herbariums of NBRI, Lucknow and the Department of Botany, Andhra University, Visakhapatnam. Voucher specimens were deposited in the

All chemicals were purchased from Qualigens fine Chemicals, Mumbai and SD fine chemicals, Mumbai. All culture media components and antibiotics used in this study were

Based on the disease index of Sorghum (Horne and Frederiksen., 1993) crops in which five phytopathogenic microorganisms were selected to screen the antimicrobial inhibition of the selected plant extracts listed in Table-2. The organisms used were procured from Microbial Type Culture Collection & Gene Bank (MTCC), Chandigarh. The lyophilized form of pure strain is reconstituted in sterile water and produced a suspension of the microbial cells. Inoculation was done with sterile inoculating loop to liquid broth medium. Liquid cultures are then incubated to allow cell replication and adequate growth of the culture, for use in bioassays. Following incubation, liquid cultures are refrigerated to store for further use. Typically, 24 hours will provide sufficient growth to allow visibly thick spread of the

herbarium of the Botany Department, Andhra University, Visakhapatnam.

analyzed with an aim of isolating novel bioactive compounds.

**2. Materials and methods** 

**2.2 Solvents and chemicals used** 

**2.3 Tested organisms** 

procured from Hi Media, Mumbai, India.

**2.1 Plant materials** 

The collected plant materials were chopped into small pieces shade dried and coarsely powdered in Willy mill. The coarsely powdered material weighed and extracted with hexane, chloroform, methanol and water in sequential order of polarity using a soxhlet extractor for five to six hours at temperature not exceeding the boiling point of the solvent. For each gram of dry material 2ml of solvent was used. The extracted solvents were filtered through Whatman no-1 filter paper and subsequently concentrated under reduced pressure (in vacuo at 40°c) using a rotary evaporator. The residue obtained were designated as crude extracts and stored in a freezer at -20°c until assayed.

The dried plant extract residues obtained were redissolved in 0.1% Dimethyl Sulfoxide (DMSO) to get different concentrations (100mg/ml, 300mg/ml and 500mg/ml) of crude extracts and filtration through a 0.45μm membrane filter and stored in sterile brown bottles in a freezer at 20°c until bioassay.

The prepared hexane, chloroform, methanol and water extracts samples were tested for antimicrobial activity against the test organism's the plant pathogens using the agar cup plate method. Streptomycin (5µg) was placed as a positive control in all plates and inoculated with bacteria and for the bacterial cultures used that was incubated at 37°C for 18-24 hours. Bavistin (5µg) was placed as a positive control in all plates inoculated with fungi and for the fungal cultures that were incubated at 26°C for 36-48 h. The microbes were plated in triplicates and average zone diameter was noted.
