Usually for extraction solvents from nonpolar to polar are used. But solvents such as methanol, ethanol, acetone, ethyl acetate, acetic acid, petroleum ether, chloroform, benzene (Fuleki et al., 1997; Jayaprakasha et al. 2003; Ozkan et al., 2004; Xu and Chang 2007) and their combinations have been used for the extraction of phenolics from plant materials, often with different proportions of water. Since nearly all of the identified antimicrobial compounds from plants are aromatic or saturated organic compounds, they are most often obtained through initial ethanol or methanol extraction (Serkedjieva and Manolova, 1992). In particular, methanol is generally found to be more efficient in extraction of lower molecular weight polyphenols while the higher molecular weight flavanols are better extracted with aqueous acetone (Metivier et al., 1980; Labarbe 1999; Guyot et al. 2001; Prior et al. 2001). On the other hand a study reported that extraction of tannins and other phenolics was better in aqueous acetone than in aqueous methanol. Ethanol is another good solvent for polyphenol extraction and is safe for human consumption (Brain and Turner 1975; vanWyk and Wink 2004; Shi et al., 2005). Addition of water to ethanol improves extraction rate, but too high water content can increase concomitant extraction of other compounds. The extraction of hydrophilic compounds uses polar solvents such as methanol, ethanol or ethyl-acetate. In some instances, extraction with hexane is used to remove chlorophyll (Gomez et al. 1996; Baydar et al. 2006; Lafka et al., 2007). Chloroform was found to be the best solvent for the extraction of non-polar biological active compounds (Harmala et al., 1992). For extraction of more lipophilic compounds, dichloromethane or a mixture of dichloromethane/methanol in ratio of 1:1 are used. In preparing anthocyanin-rich phenolic extracts from plant materials, an acidified organic solvent, most commonly methanol or ethanol, is used. This solvent system denatures the cell membranes, simultaneously dissolves the anthocyanins, and stabilizes them. However, care should be taken to avoid addition of excess acid which can hydrolyze labile, acyl, and sugar residues during concentration steps. In certain cases weak, organic acids are used for extraction of phenolics such as formic acid, acetic acid, citric acid, tartaric acid and phosphoric acid, and low concentrations of strong acids, such as 0.5-3.0% of trifluoroacetic acid and 1.0% of hydrochloric acid (Jackman 1987; Revilla 1998; Nicoue, 2007). In addition, sulfured water has also been used as extraction solvent to reduce the cost of extraction (Cacace, 2002). The properties of extracting solvents significantly affected the measured total phenolics content (±25% variation) and antioxidant capacity (up to 30%) in fruits and vegetables (Zhou and Yieu, 2004).
PHYTOCHEMICAL SCREENING
After extraction phytochemical screening assay can be done which helps us in preliminary detection of specific compound(s) present in the extract mixture or fraction. It is a simple, quick, and inexpensive procedure.
2.3 ANTIMICROBIAL TESTING
To test the biological activity of plant extracts is an integral part to assess the efficacy of a phytochemical as an antimicrobial agent. The antimicrobial susceptibility test (AST) is an essential technique in modern biological science. It is used to determine resistance of certain microbial strains to different antimicrobials and in pharmacology research it is used to determine the efficacy of novel antimicrobials from biological extract against different microorganisms.
AST methods are widely employed now–a-days to screen the plant extracts for antimicrobial activity and to determine Minimum Inhibitory Concentration (MIC) of the antimicrobial substance. Although current standard AST methods approved by various organizations like National Committee for Clinical Laboratory Science (NCCCLS), British Society for Antimicrobial Chemotherapy (BBSAC) and the European Committee for Antimicrobial
Susceptibility Testing (EUCAST) exist, for antimicrobial susceptibility testing but modifications still have to be made (Hammer et al. 1999). Since some factors (culture medium composition,
PHYTOCHEMICAL SCREENING METHODS
Test microorganisms, extractive method, pH, solubility of the sample in the culture medium, etc. can change results. AST standard tests are broadly classified into diffusion and dilution methods for convenience. Diffusion tests include agar well diffusion, agar disk diffusion, poison food technique, and bioautography while dilution methods include agar diffusion, broth microdilution and broth macrodilution techniques (Tenover et al., 1995). However some workers simultaneously make use of different techniques together for antimicrobial testing. For example Burns et al. (2000) used comparison of agar diffusion methodologies (disk diffusion and microdilution method) for antimicrobial susceptibility testing of Pseudomonas aeruginosa. Whereas, Serban et al., (2011) used well and disc diffusion techniques for screening of the antibacterial and antifungal activity of volatile essential oils.
