INTERNATIONALE PHARMACEUTICA SCIENCIA
| Jan-March 2011 | Vol. 1 | Issue 1 |
Available online http://www.ipharmsciencia.com
©2011 IPS
REVIEW ARTICLE
| Jan-March 2011 | Vol. 1 | Issue 1 |
Available online http://www.ipharmsciencia.com
©2011 IPS
REVIEW ARTICLE
ABSTRACT
Plants are a source of large amount of drugs comprising to different groups such as Kaur, Harleen Kaur
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.
Keywords: Medicinal plants, phytochemicals, extraction, solvent, screening.
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 [1].
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 [2].
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 [1].
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 [3].
The general techniques of medicinal plant extraction
include maceration, infusion, percolation, digestion, 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 [3].
The basic parameters influencing the quality of an
extract are [1]:
1. Plant part used as starting material
2. Solvent used for extraction
3. Extraction procedure
Effect of extracted plant phytochemicals depends on
[1]:
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]:
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 [5].
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 ethano 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.
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 [1, 4].
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 [6].
The choice of solvent is influenced by what is intended
with the extract. Since the end product will contain traces of residual solvent, the solvent should be nontoxic and should not interfere with the bioassay. The
choice will also depend on the targeted compounds to
be extracted [1, 4].
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 [4].
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 [4, 6]. Both acetone and methanol
were found to extract saponins which have
antimicrobial activity [1].
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.
It means that they are more efficient in cell walls
and seeds degradation which have unpolar
character and cause polyphenols to be released
from cells. More useful explanation for the
decrease in 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. Moreover, water is a better medium for
the occurrence of the micro-organisms as compared to ethanol [7].
The higher
concentrations of more bioactive flavonoid
compounds were detected with ethanol 70% due
to its higher polarity than pure ethanol. By
adding water to the pure ethanol up to 30% for
preparing ethanol 70% the polarity of solvent
was increased [8].
Additionally, ethanol was
found easier to penetrate the cellular membrane
to extract the intracellular ingredients from the
plant material [9].
Since nearly all of the
identified components from plants active
against microorganisms are aromatic or
saturated organic compounds, they are most
often obtained through initial ethanol or
methanol extraction [10].
Methanol is more
polar than ethanol but due to its cytotoxic
nature, it is unsuitable for extraction in certain
kind of studies as it may lead to incorrect
results.
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 [10].
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
[10].
Table 1: Solvents used for active component extraction [10]
 |
| Table 2: Structural features and activities of various phytochemicals from plants [10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22] |
Methods of extraction
Variation in extraction methods usually depends upon:
1. Length of the extraction period,
2. Solvent used,
3. pH of the solvent,
4. Temperature,
5. Particle size of the plant tissues
6. The solvent-to-sample ratio [4].
The basic principle is to grind the plant material (dry or wet) finer, which increases the surface area for extraction
thereby increasing the rate of extraction. Earlier studies reported that solvent to sample ratio of 10:1 (v/w) solvent
to dry weight ratio has been used as ideal [4].
Table 3: Mechanism of action of some phytochemicals [10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23].
Extraction procedures
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 [4].
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 [4].
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 [24].
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 [1].
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 [2].
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 [2].
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 [2].
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 [3].
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 rauwolfi a 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 [3].
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.
a) 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 [25,
26, 27].
CONCLUSION
Non standardized procedures of extraction may lead
to the degradation of the phytochemicals present in
the plants and may lead to the variations thus leading to the lack of reproducibility. Efforts should be made
to produce batches with quality as consistent as
possible (within the narrowest possible range) and to
develop and follow the best extraction processes.
ACKNOWLEDGEMENT
The authors are thankful to Dr. Monica Gulati, Dean,
Department of Pharmaceutical Sciences, Lovely
Professional University, Phagwara (Punjab) for
providing necessary facilities and cooperation during
this research work.
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