The extraction method also affects quantity and composition of secondary metabolites in an extract. These variations could be due to:
• type of extraction followed
• time of extraction
• temperature
• Nature of solvent
• solvent concentration and polarity
2.2. Plant material
Plants are the source of effective phytomedicines since times immemorial. Man is able to obtain from them a wondrous variety of industrial chemicals. Plant based natural constituents can be derived from the plant part, like bark, leaves, flowers, roots, fruits, seeds, etc i.e. any part of the plant may contain active components. Fresh or dried plant materials can be used as a source for the extraction of secondary plant components. Many authors had reported about plant extract preparation from the fresh plant tissues. The logic behind this came from the ethno medicinal uses of fresh plant materials among the traditional and tribal people. But as many plants are used in the dry form by conventional healers and due to differences in water content within different plant tissues, plants are generally air dried to a constant weight before extraction. Other researchers dry the plants in the oven at about 40°C for 72 h. In most of the reported works, underground parts (roots, tuber, rhizome, bulb etc.) of a plant were used extensively compared with other above ground parts in search for bioactive compounds possessing antimicrobial properties.
2.3. Choice of solvents
Properties of a good solvent in plant extractions include, low toxicity, ease of evaporation at low heat, promotion of rapid physiologic absorption of the extract, preservative action, inability of the extract to complex or dissociate. The factors affecting the choice of solvent are, quantity of phytochemicals to be extracted, rate of extraction, diversity of different compounds extracted, diversity of inhibitory compounds extracted, ease of subsequent handling of the extracts, toxicity of the solvent in the bioassay process, potential health hazard of the extractant. The various solvents that are used in the extraction procedures are:
2.3.1. Water
Water is universal solvent, used to extract plant products with antimicrobial activity. Though traditional healers use primarily water but plant extracts from organic solvents have been found to give more consistent antimicrobial activity compared to water extract. Reports indicates that water soluble flavonoids (mostly anthocyanins) have no antimicrobial significance and water soluble phenolics are important as only antioxidant compounds.
2.3.2. Acetone
Acetone dissolves many hydrophilic and lipophilic components from the plants and is miscible with water. It is volatile and has a low toxicity to the bioassay used. It is a very useful extractant, especially for antimicrobial studies where more phenolic compounds are required to be extracted. A study reported that extraction of tannins and other phenolics was better in aqueous acetone than in aqueous methanol.
2.3.3. Alcohol
The higher activity of the ethanolic extracts compared to the aqueous extract can be attributed to the presence of higher amounts of polyphenols. More useful explanation for the decrease in the activity of aqueous extract can be ascribed to the enzyme polyphenol oxidase, which degrade polyphenols in water extracts, whereas in methanol and ethanol they are inactive.
2.3.4. Chloroform
Terpene lactones have been obtained by following extractions of dried barks with hexane, chloroform and methanol with activity concentrating in chloroform fraction. Rarely tannins and terpenoids will be found in the aqueous phase, but they are more often obtained by treatment with less polar solvents.
2.3.5. Ether
Ether is commonly used for the selective extraction of coumarins and fatty acids.
2.3.6. Dichloromethane
It is another solvent used for carrying out the extraction procedures. It is specially used for the selective extraction of only terpenoids.
2.4. Extraction procedures
2.4.1. Plant tissue homogenization
Plant tissue homogenization in solvent has been generally used by researchers. Dried or wet, plant parts are grinded in a blender, with certain quantity of solvent, to fine particles, and shaken vigorously for 5-10 min or left overnight before filtering the extract. The filtrate then may be dried under reduced pressure and redissolved in small amount of solvent during quantification by HPLC.