Phytochemistry

T
Phytochemical Screening of plants
And their Biological Activity
Dr. Gurumeet C Wadhawa Karmaveer
Bhaurao Patil College,Vashi

Medicinal plants
Medicinal plants constitute an effective source of both traditional and
modern medicines
Herbal medicine has been shown to have genuine utility
About 80% of rural population depends on it as primary health care. [WHO,
(2005)]

Medicinal plants are the richest bio-resource
drugs of traditional systems of medicine
modern medicines
nutraceuticals
food supplements
folk medicines
pharmaceutical intermediates
chemical entities for synthetic drugs

Natural bioactive compounds found in different parts of plant (fruit, flower, stem,
leaf, root)
Provide definite physiological action on the human body
Bioactive substances include tannins, alkaloids, carbohydrates, terpenoids, steroids
and flavonoids
Widely used in the human therapy, veterinary, agriculture, scientific research
and countless other areas
Have inhibitory effects on all types of microorganisms in vitro
Phytochemicals

Extraction
……… is the separation of medicinally active portions of plant tissues using
selective solvents through standard procedures
The basic parameters influencing the quality of an extract
•Plant part used as starting material
•Solvent used for extraction
•Extraction procedure

Choice of solvents
Successful determination of biologically active compounds depends on
the type of solvent used in the extraction procedure
Property of a good solvent in plant extraction
•Low toxicity
•Ease of evaporation at low heat
•Promotion of rapid physiologic absorption of the extract
•Preservative action

The factors affecting the choice of solvent
•Quantity of phytochemicals to be extracted
•Rate of extraction
•Diversity of different inhibitory compounds extracted
•Ease of subsequent handling of the extracts
•Toxicity of the solvent in the bioassay process
•Potential health hazard of the extractants

Solvents used for active component extraction
Water
Anthocyanins
Starches Tannins
Saponins
Terpenoids
Polypeptides
Lectins
Ethanol
Tannins
Polyphenols
Polyacetylenes
Flavonols
Terpenoids Sterols
Alkaloids
Methanol
Anthocyanins
Terpenoids
Saponins Tannins
Xanthoxyllines
Totarol Quassinoids
Lactones Flavones
Phenones
Polyphenols
Chloroform
Terpenoids
Flavonoids
Ether
Alkaloids
Terpenoids
Coumarins
Fatty acids
Acetone
Phenol
Flavonols

General techniques of medicinal plant extraction
Plant tissue homogenization
Maceration
Infusion
Percolation
Digestion
Decoction
Soxhlet extraction (Hot continuous extraction)
Sonication (Ultrasound extraction)

Maceration
Whole//coarsely powdered crude drug is plalaced in a stoppered containiner with the solvent
Allow to stand @ room Temperature for a perioiod off at least 3 days with aggititatioion until the soluble
mattter gets disisssoolvlved
The mixture then is strainined, ,the marc (the damp solilid material )is presssseed
The companied liquid are clarified by
filtration or decantation after
standining

Digestion
• A form of maceration in which gentle heat is used during the
process of extraction
• Used when moderately elevated temperature is not objectionable
• The solvent efficiency of the menstruum is thereby increased
Microwave digestion system

Decoction
Suitable for extracting water-soluble, heat-stable constituents
Typically used in preparation of Ayurvedic extracts

Percolation
•
•
Used most frequently to extract active ingredients in the preparation of fluid extracts
The solid ingredients are moistened with an appropriate amount of the specified
menstruum
Allowed to stand for approximately 4 hours in a well closed container, After stand
time, the mass is packed & the top of the percolator is closed The mixture is allowed
to macerate in the closed percolator for 24 h

• Additional menstruum is added as required, until the
percolate measures about three-quarters of the required volume of the finished
product
• The marc is then pressed and the expressed liquid is added to the
percolate
• Sufficient menstruum is added to produce the required volume
•
The mixed liquid is clarified by filtration or by standing followed by
decanting

Soxhlet Extraction
(Hot Continuous Extraction)

Sonication (Ultrasound Extraction)
• Involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz
Increases the permeability of cell walls & produces cavitation
Disadvantage
Deleterious effect of ultrasound energy
medicinal plants through formation of
(>20 kHz) on the active constituents of
free
radicals and consequently undesirable changes in the drug
molecules

Effect of extracted plant phytochemicals depends on
• The nature & origin of the plant material
• Degree of processing
• Moisture content
• Particle size

Variation in extraction methods
• Length of the extraction period
• Solvent used
• pH of the solvent
• Temperature
• Particle size of the plant tissues
• Solvent-to-sample ratio

•Phytochemicals have two categories:
Primary & Secondary constituents.
• The phytochemical analysis  Commercially
value.
• Great interest in pharmaceutical companies for the
production of the new drugs for curing of various
diseases.

Qualitative Quantitative
• Steroids,
• Reducing sugars,
• Triterpenoids,
• Sugars,
• Alkaloids,
• Phenolic compounds,
• Flavonoids,
• Saponins,
• Tannins,
• Anthroquinones,
• Amino acids.
• Determination of total
alkaloids,
• Total flavonoids,
• Total phenolics,
• Total saponins,
• Total tannins,
• Total glycosides.

Standard procedures
• Sofowara (1993).
• Trease and Evans (1989).
• Harborne (1973).

Detection of alkaloids
• The individual extract is dissolved in dilute hydrochloric acid
and filter.
• The filtrate was further tested with following reagents for the
presence of alkaloids.

• Filtrate was treated with potassium bismuth iodide solution (Dragendroff’s reagent).
• Formation of orange red precipitate indicated the presence of alkaloids.
Dragendroff’s Test:
Hager’s Test:
• Filtrate was treated with saturated aqueous solution of picric acid (Hager’s reagent).
• Presence of alkaloids were confirmed by the formation of yellow coloured
precipitate.
Mayer’s Test:
• Filtrate was treated with potassium mercuric iodide solution (Mayer’s reagent).
• Formation of a whitish yellow or cream coloured precipitate indicated the presence
of alkaloids.

Detection of carbohydrates
Dissolve 2g
extract in 5
ml distilled
water & filter
it.
The filtrates
were used to
test for the
presence of
carbohydrates.
Molisch’s Test:
• Filtrate was treated with
2 drops of alcoholic α-
naphthol solution in a
test tube, shaken
• Add conc. sulphuric acid
from the side of the test
tube.
• Development of a violet
ring @ the junction of
two liquid confirmed the
presence of
carbohydrates

Detection of reducing sugars
Benedict’s test:
• Filtrate was treated with Benedict’s reagent & boil in a thermostatic water
bath for 5 minutes.
• Formation of an orange red precipitate indicated the presence of reducing
sugars.
Fehling’s Test
• Filtrate was acidified with dil. Hydrochloric acid, neutralized with
alkali & heated with Fehling’s A & B solutions.
• Formation of red precipitate indicated the presence of reducing
sugars.

Detection of saponins
Froth Test:
• Extract was diluted with distilled water to 20 ml & shaken in a
graduated test tube for 15 minutes.
• Formation of 1 cm layer of foam indicated the presence of
saponins.
Foam Test:
• Small quantity of the extract was shaken with 2 ml of water.
• Persistence of foam produced for ten minutes indicated the
presence of saponins.

Detection of phytosterols
Small quantity of
extract dissolved
in 5 ml of
chloroform
Salkowski’s Test:
On adding a few drops of
conc. Sulphuric acid.
Allow the solution to
stand
Formation of brown ring
indicated the presence of
phytosterols
LibermannBurchard’s test:
The chloroform extracted
solution was treated with
few drops of acetic
anhydride.
Boil & cool.
Add conc. sulphuric acid.
Formation of a bluish
green colour solution
confirmed the presence of
phytosterols.

Detection of phenolic compounds:
Ferric Chloride Test:
• Treat the extract with 3-4 drops of ferric chloride solution.
• Formation of bluish black colour indicated the presence of phenols.
Lead AcetateTest:
• Treat the extract with 3ml of 10% lead acetate solution.
• A bulky white precipitate indicated the presence of phenolic
compounds.

Detection of tannins:
Take 0.5 g of
the dried
powdered
plant
Boil 0.5g
sample in
20 ml of
water in a
test tube.
Filter the
above
mixture
Add few drops
of 0.1% ferric
chloride.
Development of a
brownish green or a
blue-black
colouration indicated
the presence of tannins

Detection of flavonoids:
• Treat the extract with few drops of sodium
hydroxide solution.
Formation of intense yellow colour, which
becomes colourless on further addition of dilute
acid, indicated the presence of flavonoids.
Alkaline Reagent
Test
• Treat the extract with few drops of lead acetate
solution. Formation of yellow precipitate
indicated the presence of flavonoids.
Lead acetate Test:
• Add a few drops of ferric chloride solution to
the extract solution. Development of intense
green colour indicates the presence of
flavonoids.
Ferric chloride
Test:

Detection of proteins and amino acids:
Millon’s Test:
• Treat the test solution with few drops of Millon’s reagents.
• when warmed , a white precipitate is formed which changes to a brick red
or disappears: indicates the presence of proteins &A.A.
Biuret Test:
• Treat the test solution with few drops of 2% of copper sulphate solution
• Add 1ml of ethanol followed by excess of potassium hydroxide pellets
• formation of pink colour in the extract layer indicates the presence of Pr.
Ninhydrin Test:
• Add Ninhydrin reagent to the test solution & boiled for few minutes.
• Formation of blue colour indicated the presence of amino acids.

Detection of terpenoids:
Salkowski test:
Mix 2 ml of chloroform
to extract solution
carefully added conc.
Sulphuric acid (3 ml) to
form a layer.
A reddish brown
colouration of the
interface indicated
the presence of
terpenoids.

Detection of cardiac glycosides
Keller-Killani test
Add 1ml of conc.
sulphuric acid,
Appearance of brown ring @
the interface indicate the
deoxysugar characteristic of
cardenolides
Appearance of a violet ring
below the brown ring & a
greenish ring in the acetic acid
layer confirmed the results.
Treat the extract with 2 ml of
glacial acetic acid containing
one drop of ferric chloride
solution.

Test for fixed oils and fats:
Spot Test:
• Place small quantity of the extract in between two filter papers.
• Oil stain produced with any extract showed the presence of fixed
oils and fats in the extracts.
Saponification test:
• Add few drops of 0.5N alcoholic potassium hydroxide extract
with few drops of phenolphthalein solution.
• Heat on a water bath for 1-2 hours.
• Formation of soap indicated the presence of fixed oils and fats in
the extracts.

Test for gums and mucilages
Dilute small
quantity of the
ethanolic extract
with water
Add ruthenium
red solution.
A pink colour
production
showed the
presence of gums
and mucilages.

Quantitative determination of phytochemicals
• Total phenols determination: Hagerman A., Muller I., Makkar
H. (2000).
• Total alkaloid determination: Harborne.J. (1973).
• Total flavonoids determination: Kumaran A, Karunakaran R.
(2006).
• Total tannins determination: Van-Burden T, Robinson W.
(1981).
• Total saponins determination: Obdoni B, Ochuko P. (2001).

Determination of total phenolic compounds(Hagerman A,
Muller I, Makkar H, 2000)
• Weigh accurately 100 mg of the extract of the sample &
dissolved in 100 ml of triple distilled water (TDW).
• Transfer 1 ml of this solution to a test tube & add 0.5 ml 2N of
the Folin-Ciocalteu reagent.
• Add 1.5 ml 20% of Na2CO3 solution & make volume up to 8 ml
with TDW followed by vigorous shaking.
• Finally allow to stand for 2 hours.
• Take the absorbance at 765 nm. (Spectroscopic determination).
• Data use: To estimate the total phenolic content using a standard
calibration curve obtained from various diluted concentrations of
gallic acid.

Determination of total alkaloids (Harborne J, 1973)
• Weigh 5 g of the sample & add 5g sample into a 250 ml beaker.
• Add 200 ml of 10% acetic acid in ethanol & cover the beaker with aluminum
foil Allow to stand for 4 hour.
• Filter the extract & concentrated on a water bath to one-quarter of the original
volume.
• Add concentrated ammonium hydroxide drop wise to the extract until the
precipitation was complete.
• The whole solution was allowed to settle
• Collect the precipitate & wash with dilute ammonium hydroxide and then filter.
• The residue is the alkaloid, which was dried and weighed

Determination of total flavonoids
(Kumaran A, Karunakaran R. 2006)
• The method is based on the formation of the flavonoids - aluminium
complex which has an absorptivity maximum at 415nm.
• Mix 100μl of the plant extracts in methanol (10 mg/ml) with 100 μl of
20 % aluminum trichloride in methanol
• Add a drop of acetic acid, and then diluted with methanol to 5ml.
• After 40 minutes read the absorption @ 415 nm.
• Blank samples were prepared from 100 ml of plant extracts and a
drop of acetic acid, and then diluted to 5ml with methanol.
• The absorption of standard rutin solution (0.5 mg/ml) in methanol was
measured under the same conditions.
• All determinations were carried out in triplicates.

Determination of total tannins
(Van-Burden T, Robinson W. 1981).
• Weigh 500 mg of the sample & transfer to a 50 ml plastic bottle.
• Add 50 ml of distilled water & shaken for 1 hour in a mechanical
shaker.
• Filter the above mixture into a 50 ml volumetric flask & make up to
the mark.
• Pipette out 5 ml of the filtrate into a test tube & mix with 2 ml of 0.1
M FeCl3 in 0.I N HCl & 0.008 M potassium ferrocyanide.
• Measure the absorbance @ 120 nm within 10 min.

Determination of total saponins
(Obdoni B, Ochuko P. 2001)
• Ground the samples & 20 g of each were put into a conical flask
• Add 100 cm3 of 20% aqueous ethanol.
• Heat the samples over a hot water bath for 4 hour with continuous
stirring @ about 55°C.
• Filter the mixture.
• Re-extract the residue with another 200 ml 20% ethanol.
• The combined extracts were reduced to 40 ml over water bath @
about 90°C.
• The concentrate was transferred into a 250 ml separatory funnel &
add 20 ml of diethyl ether; shake vigorously.

• The aqueous layer was recovered while the ether layer was discarded.
• Repeat the purification process. Add 60 ml of n-butanol & wash twice
with 10 ml of 5% aqueous sodium chloride. Heat the remaining solution
in a water bath. After evaporation the samples were dried in the oven to a
constant weight. The saponins content was calculated using standard
formulae.

DPPH scavenging test
Antioxidant activity measured in terms of free radicle scavenging Quantitative
measurement of radical scavenging properties was carried out in a universal
bottle. The reaction mixture contained sample under study 50 μL taken or 80%
MeOH as blank and 5 mL of a 0.004% (w/v) solution of DPPH in methanol
during the antioxidant activity. Different known antioxidants, vitamin E, and
butylated hydroxytoluene (BHT, Sigma) were used for comparison or as a
positive control. Discoloration was measured at 517 nm after incubation for 30
min. Measurements was taken at least in triplicate. DPPH or antioxidant activity
concentration was measured using the following formula :
DPPH scavenging effect (%) or antioxidant activityradical’s
= Ao – A1 / Ao X100
Where Ao was the absorbance of the control and A1 was Absorbance in presence
of sample

Alpha -Amylase inhibition
Alpha -Amylase inhibition.
The Alpha -amylase inhibitory activity was determined using a different methods but one of
the newmodified assayof that described in the Worthington Enzyme Manual is good method
reported. A total of 500 µL of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl)
containing 0.5 mg/mL of Alpha - amylase were pre-incubated at 25oC for 10 min. After the
preincubation, 500 μL of a 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9
with 0.006 M NaCl) was added to each tube at timed intervals with constant shaking. The
reaction was stopped using 1.0 mL of dinitrosalicylic (DNS) acid color reagent. The test
tubes were incubated in a boiling water bath for 5 min and then cooled to room temperature.
The Phoboo et al. was reported In Vitro Assays of Anti-diabetic and reaction mixture was
diluted by adding 5 to15 mL of distilled water, and the absorbance was measured at 540 nm
using the previously described UV-Visible light spectrophotometer. The absorbance
readings were compared with the controls that contained buffer instead of sample extract .

Alpha -Glycosidase Inhibitory activity
In-vitro method Alpha-Glycosidase Inhibitory activity
A better method to check activity of compounds for diabetes is Alpha-Glucosidase.
Inhibitory activity of Alpha - glycosidase was measured following the protocol described
various by McCue et al. for the development of new protocol during recent years The Alpha -
glycosidase was usually assayed using 50 µL of sample extracts and 100 µL of 0.1 M
phosphate buffer (pH 6.9) containing ,Alpha -glycosidase solution (1 mL), from various
source which was then incubated in 96-well plates at 25°C for 10 min shaking After the pre-
incubation period, 50 µL of 5 mM p-nitro phenyl-µ-d-glucopyranoside solution in0.1 M
phosphate buffer (pH 6.9) was added to each well at timed intervals. The reaction mixtures
were incubated at 25°C for 5 min. Before and after incubation, absorbance readings of the
samples were recorded at 405 nm using a micro plate reader and compared with a control that
had 50 µLof buffer solutions in place of the extract.

Assessment of invitro anti-inflammatory activity
Inhibition of albumin denaturation The anti-inflammatory activity of
Enicostemma axillare was studied by using inhibition of albumin
denaturation technique which was studied according to Mizushima et al
and Sakat et al followed with minor modifications.The reaction mixture
was consists of test extracts and 1% aqueous solution of bovine albumin
fraction, pH of the reaction mixture was adjusted using small amount of
1N HCl. The sample extracts were incubated at 37 ºC for 20 min and
then heated to 51 º C for 20 min, after cooling the samples the turbidity
was measured at 660nm.( UVVisible Spectrophotometer Model 371,
Elico India Ltd) . The experiment was performed in triplicate.
The Percentage inhibition of protein denaturation was calculated as
follows: Percentage inhibition = (Abs Control –Abs Sample) X 100/ Abs
control

Assay for in vitro cytotoxicity study
In vitro cytotoxicity assay of plant extract
was performed by using Vero (African green Monkey Kidney), A-
549 (Human lung) and Dalton’s Lymphoma Ascites (Tumour cells)
cell line. Briefly, 1×106 cells were suspended in 0.1 ml of phosphate
buffered saline (PBS, 0.2 M, pH 7.4) and mixed with 100μl of
various concentration (25,50,100,150,200 and 300μg/ml) of plant
and standard drug 5-flurouracil. The final volume was adjusted 1
ml with PBS and was incubated at 37°C for 3 h after the incubation
was over, the viability of the cells was determined using trypan
blue (0.4% in normal saline) method and the percentage of
cytotoxicity was determined by calculating percentage inhibition
and IC50 value

References
III.
I. HERIN SHEEBA D. GRACELIN A. JOHN DE BRITTO & P. BENJAMIN JEYA
RATHNA KUMAR.2013. QUALITATIVE AND QUANTITATIVE ANALYSIS OF
PHYTOCHEMICALS IN FIVE PTERIS SPECIES. Int J Pharm Pharm Sci, Vol 5(1):
105-107 .
II. Adarsh Krishna T.P., Ajeesh Krishna T.P., Sanyo Raj V.N., Juliet S., Nair S.N.,
Ravindran R. and Sujith S. 2013.Evaluation of phytochemical constituents and
proximate contents of the ethanolic leaf extract of Tetrastigma leucostaphylum
(Dennst.) Alstone (Vitaceae) found in Western Ghats of Kerala, India Res. J.
Pharmaceutical Sci. 2(10): 1-6.
Prashant Tiwari, Bimlesh Kumar, Mandeep Kaur, Gurpreet Kaur, Harleen Kaur.
2011. Phytochemical screening and Extraction: A Review Internationale
Pharmaceutica Sciencia 1(1): 98-106.
IV. Google images.

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