INTERNATIONALE PHARMACEUTICA SCIENCIA

| Jan-March 2011 | Vol. 1 | Issue 1 |

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©2011 IPS

REVIEW ARTICLE

Phytochemical screening and Extraction: A Review

ABSTRACT Prashant Tiwari*, Bimlesh
Kumar, Mandeep Kaur, Gurpreet
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
Department of Pharmaceutical
claimed to possess the antibiotic properties in the traditional system and are also used
Sciences, Lovely School of
extensively by the tribal people worldwide. It is now believed that nature has given the cure of Pharmaceutical Sciences, Phagwara,
every disease in one way or another. Plants have been known to relieve various diseases in Punjab
Ayurveda. Therefore, the researchers today are emphasizing on evaluation and
Date of Submission: 12-01-2011
characterization of various plants and plant constituents against a number of diseases based Date of Acceptance: 22-02-2011
on their traditional claims of the plants given in Ayurveda. Extraction of the bioactive plant Conflict of interest: Nil
Source of support: None
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 extracts. Such preparations have been popularly called

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,
medicines, nutraceuticals, food supplements,
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
tinctures, pilular (semisolid) extracts or powdered
Address for correspondence
*Prashant Tiwari (Research Scholar)
Department of Pharmaceutical sciences
Lovely Professional University,
Ludhiana-Jalandhar G.T. Road
Phagwara (Punjab), 144402, India
*Mob: +919888418518 E.mail: parshant.tiwari@gmail.com
galenicals, named after Galen, the second century
Greek physician [2].
Extraction methods used pharmaceutically involves
modern the separation of medicinally active portions of plant
folk 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
extracts, 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,

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 Prashant Tiwari, et al: Phytochemical screening and Extraction: A Review

decoction, hot continuous extraction (Soxhlet), part of the plant like bark, leaves, flowers, roots, fruits,
aqueous-alcoholic extraction by fermentation, counter- seeds, etc i.e. any part of the plant may contain active
current extraction, microwave-assisted extraction, components. The systematic screening of plant species
ultrasound extraction (sonication), supercritical fluid with the purpose of discovering new bioactive
extraction, and phytonic extraction (with compounds is a routine activity in many laboratories.
hydrofluorocarbon solvents). For aromatic plants, Scientific analysis of plant components follows a
hydrodistillation techniques (water distillation, steam logical pathway. Plants are collected either randomly
distillation, water and steam distillation), hydrolytic or by following leads supplied by local healers in
maceration followed by distillation, expression and geographical areas where the plants are found [5].
enfl eurage (cold fat extraction) may be employed. Fresh or dried plant materials can be used as a source
Some of the latest extraction methods for aromatic for the extraction of secondary plant components.
plants include headspace trapping, solid phase micro- Many authors had reported about plant extract
extraction, protoplast extraction, microdistillation, preparation from the fresh plant tissues. The logic
thermomicrodistillation and molecular distillation [3]. behind this came from the ethno medicinal use of fresh
The basic parameters influencing the quality of an plant materials among the traditional and tribal
extract are [1]: people. But as many
1. Plant part used as starting material plants are used in the dry form (or as an aqueous
2. Solvent used for extraction extract) by traditional healers and due to differences in
3. Extraction procedure water content within different plant tissues, plants are
usually air dried to a constant weight before extraction.
Effect of extracted plant phytochemicals depends on Other researchers dry the plants in the oven at about
[1]: 40°C for 72 h. In most of the reported works,
1. The nature of the plant material underground parts (roots, tuber, rhizome, bulb etc.) of
2. Its origin a plant were used extensively compared with other
3. Degree of processing above ground parts in search for bioactive compounds
4. Moisture content possessing antimicrobial properties [1, 4].
5. Particle size
Choice of solvents
The variations in different extraction methods that will Successful determination of biologically active
affect quantity and secondary metabolite composition compounds from plant material is largely dependent
of an extract depends upon [1]: on the type of solvent used in the extraction procedure.
1. Type of extraction Properties of a good solvent in plant extractions
2. Time of extraction includes, low toxicity, ease of evaporation at low heat,
3. Temperature promotion of rapid physiologic absorption of the
4. Nature of solvent extract, preservative action, inability to cause the
5. Solvent concentration extract to complex or dissociate. The factors affecting
6. Polarity the choice of solvent are quantity of phytochemicals to
be extracted, rate of extraction, diversity of different
Plant material compounds extracted, diversity of inhibitory
Plants are potent biochemists and have been compounds extracted, ease of subsequent handling of
components of phytomedicine since times the extracts, toxicity of the solvent in the bioassay
immemorial; man is able to obtain from them a process, potential health hazard of the extractants [6].
wondrous assortment of industrial chemicals. Plant The choice of solvent is influenced by what is intended
based natural constituents can be derived from any with the extract. Since the end product will contain

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 Prashant Tiwari, et al: Phytochemical screening and Extraction: A Review
traces of residual solvent, the solvent should be non-
toxic 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
100 Internationale Pharmaceutica Sciencia
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].
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Table 1: Solvents used for active component extraction [10]

Water Ethanol Methanol Chloroform Ether Acetone

Anthocyanins Tannins Anthocyanins Terpenoids Alkaloids Phenol
Starches Polyphenols Terpenoids Flavonoids Terpenoids Flavonols
Tannins Polyacetylenes Saponins Coumarins
Saponins Flavonol Tannins Fatty acids
Terpenoids Terpenoids Xanthoxyllines
Polypeptides Sterols Totarol
Lectins Alkaloids Quassinoids
Lactones
Flavones
Phenones
Polyphenols

Table 2: Structural features and activities of various phytochemicals from plants [10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22]

Activities
Phytochemicals Structural features Example(s)
Phenols and Antimicrobial, Anthelmintic,
C3 side chain, - OH groups, phenol ring Catechol, Epicatechin, Cinnamic acid
Polyphenols Antidiarrhoeal
Quinones Aromatic rings, two ketone substitutions Hypericin Antimicrobial
Flavones
Abyssinone
Phenolic structure, one carbonyl group
Flavonoids
Hydroxylated phenols, C6-C3 unit linked to Chrysin, Quercetin, Rutin
Antimicrobial
an aromatic ring
Antidiarrhoeal
Flavones + 3-hydroxyl group
Totarol
Flavonols
Antimicrobial, Anthelmintic,
Tannins Polymeric phenols (Mol. Wt. 500-3000) Ellagitannin
Antidiarrhoeal
Phenols made of fused benzene and α-
Coumarins Warfarin
pyrone rings Antimicrobial
Antimicrobial
Terpenoids and Acetate units + fatty acids, extensive
Capsaicin Antidiarrhoeal
essential oils branching and cyclized
Berberine, Piperine, Palmatine, Antimicrobial, Anthelmintic,
Alkaloids Heterocyclic nitrogen compounds
Tetrahydropalmatine Antidiarrhoeal
Lectins and
Proteins Mannose-specific agglutinin, Fabatin Antimicrobial
Polypeptides
Glycosides Sugar + non carbohydrate moiety Amygdalin Antidiarrhoeal
Saponins Amphipathic glycosides Vina-ginsenosides-R5 and -R6 Antidiarrhoeal

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].

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 Prashant Tiwari, et al: Phytochemical screening and Extraction: A Review

Table 3: Mechanism of action of some phytochemicals [10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23].

Phytochemicals Activity Mechanism of action

Quinones Antimicrobial Binds to adhesins, complex with cell wall, inactivates enzymes
Antimicrobial Complex with cell wall, binds to adhesins
Antidiarrhoeal Inhibits release of autocoids and prostaglandins,
Inhibits contractions caused by spasmogens,
Stimulates normalization of the deranged water transport across the mucosal cells,
Flavonoids Inhibits GI release of acetylcholine
Antimicrobial Binds to adhesins, enzyme inhibition, substrate deprivation, complex with cell wall, membrane disruption, metal ion
complexation

Antidiarrhoeal Makes intestinal mucosa more resistant and reduces secretion, stimulates normalization of deranged water transport
Polyphenols and across the mucosal cells and reduction of the intestinal transit, blocks the binding of B subunit of heat-labile enterotoxin to
Tannins GM1, resulting in the suppression of heat-labile enterotoxin-induced diarrhea, astringent action

Anthelmintic Increases supply of digestible proteins by animals by forming protein complexes in rumen, interferes with energy
generation by uncoupling oxidative phosphorylation, causes a decrease in G.I. metabolism
Phytochemicals Activity Mechanism of action
Coumarins Antiviral Interaction with eucaryotic DNA
Antimicrobial Membrane disruption
Terpenoids and Antidiarrhoeal Inhibits release of autocoids and prostaglandins
essential oils
Antimicrobial Intercalates into cell wall and DNA of parasites
Inhibits release of autocoids and prostaglandins
Antidiarrhoeal Possess anti-oxidating effects, thus reduces nitrate generation which is useful for protein synthesis, suppresses transfer of
sucrose from stomach to small intestine, diminishing the support of glucose to the helminthes, acts on CNS causing
Alkaloids Anthelmintic paralysis
Lectins and Antiviral Blocks viral fusion or adsorption, forms disulfide bridges
Polypeptides
Glycosides Antidiarrhoeal Inhibits release of autocoids and prostaglandins
Antidiarrhoeal Inhibits histamine release in vitro
Anticancer Possesses membrane permeabilizing properties
Saponins Anthelmintic Leads to vacuolization and disintegration of teguments
Steroids Antidiarrhoeal Enhance intestinal absorption of Na+ and water

Extraction procedures c. Soxhlet extraction: Soxhlet extraction is

a. Plant tissue homogenization:
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
soxhlet extraction of dried plant material
using organic solvent. This method cannot be
used for thermolabile compounds
prolonged heating may lead to degradation of
compounds [4].
Plant 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 plant-
drug is kept in contact with the solvent in a
stoppered container for a defined period with
frequent agitation until soluble matter is
employ dissolved. This method is best suitable for use
in case of the thermolabile drugs [1].
e. Decoction: this method is used for the
as extraction of the water soluble and heat stable
constituents from crude drug by boiling it in
water for 15 minutes, cooling, straining and

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passing sufficient cold water through the drug its large-scale application is limited due to the
to produce the required volume [2]. higher costs. One disadvantage of the
f. Infusion: It is a dilute solution of the readily procedure is the occasional but known
soluble components of the crude drugs. Fresh deleterious effect of ultrasound energy (more
infusions are prepared by macerating the than 20 kHz) on the active constituents of
solids for a short period of time with either medicinal plants through formation of free
cold or boiling water [2]. radicals and consequently undesirable
g. Digestion: This is a kind of maceration in changes in the drug molecules [3].
which gentle heat is applied during the
maceration extraction process. It is used Phytochemical screening: Phytochemical
when moderately elevated temperature is not examinations were carried out for all the extracts as
objectionable and the solvent efficiency of the per the standard methods.
menstrum is increased thereby [2]. 1. Detection of alkaloids: Extracts were
h. Percolation: This is the procedure used dissolved individually in dilute Hydrochloric
most frequently to extract active ingredients acid and filtered.
in the preparation of tinctures and fluid a) Mayer’s Test: Filtrates were treated with
extracts. A percolator (a narrow, cone-shaped Mayer’s reagent (Potassium Mercuric Iodide).
vessel open at both ends) is generally used. Formation of a yellow coloured precipitate
The solid ingredients are moistened with an indicates the presence of alkaloids.
appropriate amount of the specified b) Wagner’s Test: Filtrates were treated with
menstrum and allowed to stand for Wagner’s reagent (Iodine in Potassium
approximately 4 h in a well closed container, Iodide). Formation of brown/reddish
after which the mass is packed and the top of precipitate indicates the presence of alkaloids.
the percolator is closed. Additional menstrum c) Dragendroff’s Test: Filtrates were treated
is added to form a shallow layer above the with Dragendroff’s reagent (solution of
mass, and the mixture is allowed to macerate Potassium Bismuth Iodide). Formation of red
in the closed percolator for 24 h. The outlet of precipitate indicates the presence of alkaloids.
the percolator then is opened and the liquid d) Hager’s Test: Filtrates were treated with
contained therein is allowed to drip slowly. Hager’s reagent (saturated picric acid
Additional menstrum is added as required, solution). Presence of alkaloids confirmed by
until the percolate measures about three- the formation of yellow coloured precipitate.
quarters of the required volume of the 2. Detection of carbohydrates: Extracts were
finished product. The marc is then pressed dissolved individually in 5 ml distilled water and
and the expressed liquid is added to the filtered. The filtrates were used to test for the
percolate. Sufficient menstrum is added to presence of carbohydrates.
produce the required volume, and the mixed a) Molisch’s Test: Filtrates were treated with 2
liquid is clarified by filtration or by standing drops of alcoholic α-naphthol solution in a
followed by decanting [3]. test tube. Formation of the violet ring at the
i. Sonication: The procedure involves the use junction indicates the presence of
of ultrasound with frequencies ranging from Carbohydrates.
20 kHz to 2000 kHz; this increases the b) Benedict’s Test: Filtrates were treated with
permeability of cell walls and produces Benedict’s reagent and heated gently. Orange
cavitation. Although the process is useful in red precipitate indicates the presence of
some cases, like extraction of rauwolfi a root, reducing sugars.

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 Prashant Tiwari, et al: Phytochemical screening and Extraction: A Review

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
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
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
were 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
and 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

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 Prashant Tiwari, et al: Phytochemical screening and Extraction: A Review

to the lack of reproducibility. Efforts should be made Fedde against Dactylogyrus intermedius in
to produce batches with quality as consistent as Carassius auratus. Veterinary Parasitology 2010;

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10. Cowan MM. Plant products as antimicrobial agents.
develop and follow the best extraction processes.
Clinical microbiology reviews 1999; 12(4): 564-582.
11. Kumar R, Sharma RJ, Bairwa K, Roy RK, Kumar A.
ACKNOWLEDGEMENT
Pharmacological review on natural antidiarrhoel
The authors are thankful to Dr. Monica Gulati, Dean, agents. Der Pharma Chemica 2010; 2(2): 66-93.
Department of Pharmaceutical Sciences, Lovely 12. Sutar N, Garai R, Sharma US, Sharma UK.
Professional University, Phagwara (Punjab) for Anthelmintic activity of Platycladus orientalis leaves
providing necessary facilities and cooperation during extract. International Journal of Parasitology
this research work. Research 2010; 2(2): 1-3.
13. Mute VM. Anthelmintic effect of Tamarind indica
linn leaves juice exract on Pheretima posthuma.
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106 Internationale Pharmaceutica Sciencia Jan-Mar 2011 Vol 1 Issue 1