Phytochemical Screening:
a) Preparation of extractb)Screening of phytochemical
INTRODUCTION:
Plant-derived substances have recently
become of great interest owing to their versatile applications. Medicinal
plants are the richest bio-resource of drugs of traditional systems of
medicine, modern medicines, nutraceuticals, food supplements, folk medicines,
pharmaceutical intermediates and chemical entities for synthetic drugs. Extraction
(as the term is pharmaceutically used) is the separation of medicinally active
portions of plant (and animal) tissues using selective solvents through
standard procedures. The products so obtained from plants are relatively
complex mixtures of metabolites, in liquid or semisolid state or (after
removing the solvent) in dry powder form, and are intended for oral or external
use.
These include classes of preparations known as
decoctions, infusions, fluid extracts, tinctures, pilular (semisolid) extracts
or powdered extracts. Such preparations have been popularly called galenicals,
named after Galen, the second century Greek physician .Extraction methods used
pharmaceutically involves the separation of medicinally active portions of
plant tissues from the inactive/inert components by using selective solvents.
During extraction, solvents diffuse into the solid plant material and
solubilize compounds with similar polarity. The purpose of standardized
extraction procedures for crude drugs (medicinal plant parts) is to attain the
therapeutically desired portions and to eliminate unwanted material by
treatment with a selective solvent known as menstrum. The extract thus
obtained, after standardization, may be used as medicinal agent as such in the
form of tinctures or fluid extracts or further processed to be incorporated in
any dosage form such as tablets and capsules.
These products contains complex
mixture of many medicinal plant metabolites, such as alkaloids, glycosides,
terpenoids, flavonoids and lignans . The general techniques of medicinal plant
extraction include maceration, infusion, percolation, digestion.
Plants are a source of large amount of
drugs comprising to different groups such as antispasmodics, emetics, anti-cancer,
antimicrobials etc. A large number of the plants are claimed to possess the
antibiotic properties in the traditional system and are also used extensively
by the tribal people worldwide. It is now believed that nature has given the
cure of every disease in one way or another. Plants have been known to relieve
various diseases in Ayurveda. Therefore, the researchers today are emphasizing
on evaluation and characterization of various plants and plant constituents
against a number of diseases based on their traditional claims of the plants
given in Ayurveda. Extraction of the bioactive plant constituents has always
been a challenging task for the researchers. In this present review, an attempt
has been made to give an overview of certain extractants and extraction
processes with their advantages and disadvantages: Medicinal plants,
phytochemicals, extraction, solvent, screening. decoction, hot continuous
extraction (Soxhlet), aqueous-alcoholic extraction by fermentation,
countercurrent extraction, microwave-assisted extraction, ultrasound extraction
(sonication), supercritical fluid extraction, and phytonic extraction (with
hydrofluorocarbon solvents).
For aromatic plants: hydrodistillation techniques (water
distillation, steam distillation, water and steam distillation), hydrolytic
maceration followed by distillation, expression and enfl eurage (cold fat
extraction) may be employed. Some of the latest extraction methods for aromatic
plants include headspace trapping, solid phase microextraction, protoplast
extraction, microdistillation, thermomicrodistillation and molecular distillation
. The basic parameters influencing the quality of an extract are : 1. Plant
part used as starting material
2. Solvent used for extraction
3. Extraction procedure Effect of
extracted plant phytochemicals depends on :
1. The nature of the plant material
2. Its origin
3. Degree of processing
4. Moisture content
5. Particle size
The variations in different extraction
methods that will affect quantity and secondary metabolite composition of an
extract depends upon [
1. Type of extraction 2. Time of
extraction 3. Temperature 4. Nature of solvent 5. Solvent concentration 6.
Polarity Plant material Plants are potent biochemists and have been components
of phytomedicine since times immemorial; man is able to obtain from them a
wondrous assortment of industrial chemicals. Plant based natural constituents
can be derived from any part of the plant like bark, leaves, flowers, roots,
fruits, seeds, etc i.e. any part of the plant may contain active components.
The systematic screening of plant species with the purpose of discovering new bioactive
compounds is a routine activity in many laboratories. Scientific analysis of
plant components follows a logical pathway. Plants are collected either
randomly or by following leads supplied by local healers in geographical areas
where the plants are found .
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 use of fresh plant materials
among the traditional and tribal people. But as many plants are used in the dry
form (or as an aqueous extract) by traditional healers and due to differences
in water content within different plant tissues, plants are usually air dried
to a constant weight before extraction.
Choice of solvents Successful
determination of biologically active compounds from plant material is largely
dependent on the type of solvent used in the extraction procedure. Properties
of a good solvent in plant extractions includes, low toxicity, ease of
evaporation at low heat, promotion of rapid physiologic absorption of the
extract, preservative action, inability to cause 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 extractants . The various solvents that
are used in the extraction procedures are:
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.
Also water soluble flavonoids (mostly anthocyanins) have no antimicrobial
significance and water soluble phenolics only important as antioxidant compound.
2. Acetone: Acetone dissolves many
hydrophilic and lipophilic components from the two plants used, is miscible
with water, 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 .Both acetone and methanol were found to extract saponins
which have antimicrobial activity
3. Alcohol: The higher activity of the
ethanolic extracts as compared to the aqueous extract can be attributed to the
presence of higher amounts of polyphenols as compared to aqueous extracts.
4. Chloroform: Terpenoid lactones have
been obtained by successive extractions of dried barks with hexane, chloroform
and methanol with activity concentrating in chloroform fraction. Occasionally
tannins and terpenoids will be found in the aqueous phase, but they are more
often obtained by treatment with less polar solvents.
5. Ether: Ether is commonly used
selectively for the extraction of coumarins and fatty acids [10].
Dichloromethanol: It is another solvent used for carrying out the extraction
procedures. It is specially used for the selective extraction of only
terpenoids.
Process:
a. Plant tissue homogenization: Plant
tissue homogenization in solvent has been widely used by researchers. Dried or
wet, fresh plant parts are grinded in a blender to fine particles, put in a
certain quantity of solvent and shaken vigorously for 5 - 10 min or left for 24
h after which the extract is filtered. The filtrate then may be dried under
reduced pressure and redissolved in the solvent to determine the concentration.
Some researchers however centrifuged the filtrate for clarification of the
extract.
b. Serial exhaustive extraction: It is
another common method of extraction which involves involves successive
extraction with solvents of increasing polarity from a non polar (hexane) to a
more polar solvent (methanol) to ensure that a wide polarity range of compound
could be extracted. Some researchers employ soxhlet extraction of dried plant
material using organic solvent. This method cannot be used for thermolabile
compounds as prolonged heating may lead to degradation of compounds.
c. Soxhlet extraction: Soxhlet
extraction is only required where the desired compound has a limited solubility
in a solvent, and the impurity is insoluble in that solvent. If the desired
compound has a high solubility in a solvent then a simple filtration can be
used to separate the compound from the insoluble substance. The advantage of
this system is that instead of many portions of warm solvent being passed
through the sample, just one batch of solvent is recycled. This method cannot
be used for thermolabile compounds as prolonged heating may lead to degradation
of compounds.
d. Maceration: In maceration (for fluid
extract), whole or coarsely powdered plantdrug is kept in contact with the
solvent in a stoppered container for a defined period with frequent agitation
until soluble matter is dissolved. This method is best suitable for use in case
of the thermolabile drugs
e. Decoction: this method is used for
the extraction of the water soluble and heat stable constituents from crude
drug by boiling it in water for 15 minutes, cooling, straining and passing sufficient cold water through the drug
to produce the required volume
f. Infusion: It is a dilute solution
of the readily soluble components of the crude drugs. Fresh infusions are
prepared by macerating the solids for a short period of time with either cold
or boiling water.
g. Digestion: This is a kind of
maceration in which gentle heat is applied during the maceration extraction
process. It is used when moderately elevated temperature is not objectionable
and the solvent efficiency of the menstrum is increased thereby.
h. Percolation: This is the procedure
used most frequently to extract active ingredients in the preparation of
tinctures and fluid extracts. A percolator (a narrow, cone-shaped vessel open
at both ends) is generally used. The solid ingredients are moistened with an
appropriate amount of the specified menstrum and allowed to stand for
approximately 4 h in a well closed container, after which the mass is packed
and the top of the percolator is closed. Additional menstrum is added to form a
shallow layer above the mass, and the mixture is allowed to macerate in the
closed percolator for 24 h. The outlet of the percolator then is opened and the
liquid contained therein is allowed to drip slowly. Additional menstrum is added
as required, until the percolate measures about threequarters of the required
volume of the finished product. The marc is then pressed and the expressed
liquid is added to the percolate. Sufficient menstrum is added to produce the
required volume, and the mixed liquid is clarified by filtration or by standing
followed by decanting i. Sonication: The procedure involves the use of
ultrasound with frequencies ranging from 20 kHz to 2000 kHz; this increases the
permeability of cell walls and produces cavitation. Although the process is
useful in some cases, like extraction of rauwolfia root, its large-scale
application is limited due to the higher costs. One disadvantage of the
procedure is the occasional but known deleterious effect of ultrasound energy
(more than 20 kHz) on the active constituents of medicinal plants through
formation of free radicals and consequently undesirable changes in the drug
molecules.
Phytochemical screening: Phytochemical examinations were carried out for all the extracts
as per the standard methods.
1. Detection of alkaloids: Extracts were
dissolved individually in dilute Hydrochloric acid and filtered.
a) Mayer’s Test: Filtrates were
treated with Mayer’s reagent (Potassium Mercuric Iodide). Formation of a yellow
coloured precipitate indicates the presence of alkaloids.
b) Wagner’s Test: Filtrates were
treated with Wagner’s reagent (Iodine in Potassium Iodide). Formation of
brown/reddish precipitate indicates the presence of alkaloids.
c) Dragendroff’s Test: Filtrates were
treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide).
Formation of red precipitate indicates the presence of alkaloids.
d) Hager’s Test: Filtrates were
treated with Hager’s reagent (saturated picric acid solution). Presence of alkaloids
confirmed by the formation of yellow coloured precipitate.
2. Detection of carbohydrates:
Extracts were dissolved individually in 5 ml distilled water and filtered. The
filtrates were used to test for the presence of carbohydrates.
a) Molisch’s Test: Filtrates were
treated with 2 drops of alcoholic α-naphthol solution in a test tube. Formation
of the violet ring at the junction indicates the presence of Carbohydrates.
b) Benedict’s Test: Filtrates were treated
with Benedict’s reagent and heated gently. Orange red precipitate indicates the
presence of reducing sugars.
c) Fehling’s Test: Filtrates were
hydrolysed with dil. HCl, neutralized with alkali and heated with Fehling’s A
& B solutions. Formation of red precipitate indicates the presence of
reducing sugars.
3. Detection of glycosides: Extracts
were hydrolysed with dil. HCl, and then subjected to test for glycosides.
Modified Borntrager’s Test: Extracts were
treated with Ferric Chloride solution and immersed in boiling water for about 5
minutes. The mixture was cooled and extracted with equal volumes of benzene.
The benzene layer was separated and treated with ammonia solution. Formation of
rose-pink colour in the ammonical layer indicates the presence of anthranol
glycosides.
4. Legal’s Test: Extracts were treated
with sodium nitropruside in pyridine and sodium hydroxide. Formation of pink to
blood red colour indicates the presence of cardiac glycosides.
5. Detection of saponins
a) Froth Test: Extracts were diluted
with distilled water to 20ml and this was shaken in a graduated cylinder for 15
minutes. Formation of 1 cm layer of foam indicates the presence of saponins.
b) Foam Test: 0.5 gm of extract was
shaken with 2 ml of water. If foam produced persists for ten minutes it
indicates the presence of saponins.
6. Detection of phytosterols
a) Salkowski’s Test: Extracts were
treated with chloroform and filtered. The filtrates were treated with few drops
of Conc. Sulphuric acid, shaken and allowed to stand. Appearance of golden
yellow colour indicates the presence of triterpenes.
b) Libermann Burchard’s test: Extracts were
treated with chloroform and filtered. The filtrates were treated with few drops
of acetic anhydride, boiled and cooled. Conc. Sulphuric acid was added.
Formation of brown ring at the junction indicates the presence of phytosterols.
7. Detection of phenols Ferric
Chloride Test: Extracts were treated with 3-4 drops of ferric chloride
solution. Formation of bluish black colour indicates the presence of phenols.
8. Detection of tannins Gelatin Test:
To the extract, 1% gelatin solution containing sodium chloride was added.
Formation of white precipitate indicates the presence of tannins.
9. Detection of flavonoids
a) Alkaline Reagent Test: Extracts
were treated with few drops of sodium hydroxide solution. Formation of intense
yellow colour, which becomes colourless on addition of dilute acid, indicates the
presence of flavonoids.
b) Lead acetate Test: Extracts were
treated with few drops of lead acetate solution. Formation of yellow colour
precipitate indicates the presence of flavonoids.
10. Detection of proteins and
aminoacids
a) Xanthoproteic Test: The extracts
were treated with few drops of conc. Nitric acid. Formation of yellow colour
indicates the presence of proteins.
b) Ninhydrin Test: To the extract,
0.25% w/v ninhydrin reagent was added and boiled for few minutes. Formation of
blue colour indicates the presence of amino acid. 11. Detection of diterpenes
Copper acetate Test: Extracts were dissolved in water and treated with 3-4
drops of copper acetate solution. Formation of emerald green colour indicates
the presence of diterpenes
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