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Preliminary investigation of the activity
The aim of this
investigation is to confirm rapidly the presumed pharmacological activity
of the harvested plant. This stage is an essential precursor to the later
stage of complete pharmacological investigation of pure isolated products.
It will establish whether presumed activity exists or not. In the latter
case it will save considerable time and money on further investigations.
Due
consideration must be given to traditional methods of preparation, use and size
of the dose relative to the weight of original dry material. Usually an
aqueous extract is suitable for a drug used in the form of an infusion, a
chloroform extract is preferable for drugs administered in powdered form
with oil, butter or milk, a water alcohol extract is suitable for drugs
which are ground in palm wine
Usually an aqueous extract is suitable as it is similar to an in vivo
state. Two most preferred solvents are ethanol and dimethylsulfoxide(DMSO)
as they are miscible with water and are non-toxic at concentration below 3%
v/v. Alternatively emulsion can be prepared using Tween® as emulsifying
agent.
Phytochemical
analysis of the plant
To achieve this
goal several extraction procedures may be considered. Selection of a
particular process depends upon the aims of the extraction. Generally the
extraction are carried out with the following two aims:
1 Preparation of solvent extracts
for screening program
Many research laboratories are involved in scientific evaluation of plant
extracts for therapeutic use. In many cases chemical nature and the
constituents responsible for the activity is not known. Thus the decisions
have to be taken about the solvent to be used for extraction. The choice of
the solvent depends upon various factors viz polarity, ionizability,
toxicity, thermostability and cost. One approach may be to extract the
plant material with a range of solvents of increasing polarity using a
Soxhlet apparatus. Thus four or more extracts may be prepared.
Alternatively, a simpler approach using only two solvents one polar e.g.
70% methanol and one nonpolar e.g. hexane or light petroleum may be used.
This will result in considerable savings in time and cost of the screening
program.
2. Extraction process for specific
phytochemical groups
If the aim is to extract specific phytochemical groups such as fixed oils.
Fats,and waxes,essential or volatile oils, alkaloids, glycosides,
carotenoids, phenolic compounds, polysaccharides, and proteins solvent
extraction using appropriate solvent as listed in Table may be used.
Table
List of Solvents appropriate for extraction of chemical groups
Solvents
Chemical groups extracted
Low Polar Solvents
Light Petoleum,Hexane
Fixed Oils, Fats, Waxes, Volatile Oils
Hexane,Cyclohexane
Chloroform
Alkaloids, Aglycones
Medium Polar
Solvents
Dichloromethane,Ether
Alkaloids, Aglycones
Acetone Alkaloids,
Aglycones, Glycosides
Methanol Sugars,
Amino acids, Glycosides
High Polar Solvents
Water Sugars,
Amino acids, Glycosides
Aqueous
acids
Sugars,
Amino acids, Bases
Aqueous
alkali Sugars,
Amino acids, Acids
Some General
Methods for Extraction of a particular Phytochemical group
Fixed Oils, Fats
and Waxes Fixed oils and fats are triglycerides of higher fatty
acids, namely palmitic, stearic, oleic, linoleic, and linolenic. Waxes are
esters of long chain fatty acids and long chain alcholos or sterols. Fixed
oils, fats and waxes are all non-polar in nature and can be extracted using
solvents such as light petroleum or hexane. They can also be extracted
using other solvents such as chloroform, ethanol or methanol but these
solvents lack specificity as they may dissolve other plant constituents
also. On an industrial scale, these substances may be obtained by the
process of expression rather than solvent extraction.
Volatile or
Essential oils Essential oils are the mixtures of mono and
sesquiterpenic hydrocarbons and their oxygenated compounds such as ethers,
alcohols, ketones, aldehydes, lactones, oxides, carboxylic acids. They are
usually extracted by steam distillation. Solvent extraction using light
petroleum, chloroform and dichlorometane, advanced phytonics method and
extraction into fixed oils or waxes (Enfluerage) are other options.
Glycosides These are the
combination of sugars with other non-sugar moiety known as aglycone. They
can be extracted with polar solvents such as acetone, ethanol, methanol,
water or mixture of these. To avoid breakdown of glycosides by the enzyme
glycosidase boiling water or significant proportion of alcohol or ammonium
sulphate may be added to the extract. Cardiac glycosides which contain
bulky steroidal agycones can be extrcted with chloroform.
Flavonoids
Flavonoids can be extracted from the plant tissue by following procedure –
§
A small amount of plant tissue (leaf or flower) after
immersing in 2 m HCl is heated in a test tube for 30-40 min at 100oC.
§
The cooled extract is then filtered and extracted with
ethylacetate.
§
If the solution is coloured then the aqueous extract
is again heated to remove last traces of ethyl acetate and reextracted with
a small volume of amyl alcohol.
§
The ethyl acetate is concentrated to dryness, taken up
in 1-2 drops of ethanol and aliquot is chromatographed in following
solvents. Forestal acetic acid conc. HCl water : 30:
3:10
, 50% aqueous acetic acid, Butanol :
acetic acid : water : 4:1:5.
§
The amyl alcohol extract, which should be coloured, is
concentrated to dryness, taken up in a few drops of 1% methanolic HCl and
aliquot chromatographed in Forestal and in formic acid : conc HCl : Water :
5:2:3.
Proteins Proteins usually contain free carboxylic, amino and phenolic
groups on amino-acid side chains and therefore exists in ionized form at
high or low pH. The pH at which protein remain unionized is known as
isoelectric point. Proteins can be extracted with water, buffers, dilute
acid or base or simple salt solutions. More lipophilic proteins can be
extracted woth 75%
alcohol.
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