phytochemical investigation and characterization of …
TRANSCRIPT
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PHYTOCHEMICAL INVESTIGATION AND CHARACTERIZATION
OF ABRIN PROTEIN WITH GEL ELECTROPHORESIS FROM SEEDS
OF ABRUS PRECATORIUS
*Vikas Singh1, Gyanendra Kumar Saxena1, Himanshu Joshi1, Priyanka Gupta2,
Ekta Arya1
1Faculty of Pharmacy, Naraina vidya peeth group of Institutions, Panki, Kanpur, India. 2Indian Veterinary Research Institute, Bareilly, India.
ABSTRACT
The present study was undertaken with an objective to explore the hair
growth activity of ethanol extract of Abrus precatorius seeds on male
wistar albino rats. Abrus precatorius is commonly called as Ratti,
belongs to family Fabaceae. A.precatorius is mainly found all through
the plains of India, from Himalayas down to southern India and
Ceylon. Most of the active constituents were found to be present in its
seeds and on exhaustive literature survey it was found that sufficient
activities on this plant have been done on seed part. Therefore, the
present study was also planned to concentrate upon seed part which is
a rich source of active chemical constituents.
In the traditional medicine system, it has been mentioned that paste of
the seeds of Abrus precatorius is used to treat alopecia but on exhaustive literature survey no
scientific record was found for this activity; so the present study was undertaken to evaluate
the hair growth activity and to check the authenticity of traditional claims. Pharmacological
screening of ethanol extracts of seeds of Abrus precatorius showed significant hair growth
activity which was slightly less effective in comparison to standard minoxidil. Isolation,
characterization & study of mechanism of action of abrin in the treatment of alopecia could
be another step forward to prove the utility of this constituent of plant Abrus precatorius.
Key words: hair growth, Abrus precatorius, alopecia.
World Journal of Pharmaceutical research
Volume 2, Issue 4, 924-937. Research Article ISSN 2277 – 7105
Article Received on 10 April 2013, Revised on 01 May 2013,
Accepted on 19 June 2013
*Correspondence for Author:
Vikas Singh
Faculty of Pharmacy, Naraina
vidya peeth group of
Institutions, Panki, Kanpur,
India.
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INTRODUCTION
Abrus precatorius is commonly called as Ratti. It is mainly found all through the plains of
India, from Himalayas down to southern India and Ceylon.
Taxonomical classification1, 2
Kingdom : - Plantae
Division : - Magnoliophyta
Class : - Magnoliopsida
Order : - Fabales
Family : - Fabaceae
Subfamily : - Faboideae
Genus : - Abrus
Species : - precatorius
Figure: 1 seeds of Abrus precatorius
Hair is considered to be a major component of an individual’s general appearance.
Throughout history and in most of civilization, scalp hair has been associated with positive
signals such as beauty and power. Baldness or hair loss on the other hand has negative
attribute. Hairs or pili are present on most skin surface except the palms, palmar surfaces of
the fingers, the soles and plantar surfaces of the feet. In adults, hair usually is most heavily
distributed across the scalp, in the eyebrows, in the axillae and around the external genitalia3.
Alopecia or hair loss is the medical description of the loss of hair from the head or body,
sometimes to the extent of baldness. Around 2% of the world’s population suffers from a hair
loss problem known as alopecia areata. In the last decade much progress has been made in
terms of research on the causes and treatment of this disorder.
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Factors which contribute to hair loss or fall
Genetic predisposition.
Hormonal factors.
Use of chemotherapeutic agents also causes hair fall or loss.
Various diseased states such as typhoid, malaria and jaundice .
Compulsive hair pulling such as trichotillomania.
Fungal infection such as "black dot" tinea or tinea capitis.
Heat damage as from repeated hot comb use.
Telogen effluvium resulting from physical or psychological stress.
Radiation therapy or other radiation exposure4.
Chemical Constituents
Seeds contain the glycoprotein abrin which resembles ricin.
A number of indole alkaloids and other bases (precatorine, trigonelline, choline and
abrine) are present in the seeds.
Some quinones present in the roots of Abrus precatorius are abruquinones A, B, C, O, E,
F and G.
Two new saponins have also been isolated and the effects of these compounds on HIV-
protease and HIV-induced cytopathogenecity are now under investigation
Leaves contain precol, abrol, abrasine and precasine5.
MATERIALS AND METHODS
Collection, Identification and Authentication of Plant Material
Seeds of Abrus precatorius (Ratti) were collected in month of December from the local
market of Lucknow (Uttar Pradesh). The seeds were identified and authenticated as Abrus
precatorius from National Botanical Research Institute (NBRI), Lucknow under the Ref. No.:
NBRI/CIF/127/2009 dated 23-12-2009.
Preparation Of Ethanol Extracts Of Abrus Precatorius Seeds
The seeds of Abrus precatorius, were dried, powdered (1kg) and defatted with petroleum
ether and solvent free powder extracted with ethanol (1.2 liter) in the soxhlet apparatus at
40ºc for about 72 hours. After the completion of extraction, the extract was concentrated on
vacuum rotary evaporator to get a brown viscous gummy residue which was placed in
vacuum desiccator for 4-5 days for drying and then used for subsequent experiments.6
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Fractionation of Ethanol Extract 7, 8,9,10
The ethanol extract (10 gram) was taken in a separating funnel and shaked vigorously with
petroleum ether (10ml x 5) for 45 minutes then pet ether solvent was filtered and the residue
was put in the separating funnel for next addition of solvent. The petroleum ether soluble
portion was evaporated to dryness and then chloroform (10ml x 5) was placed in the
separating funnel and shaked for 45 minutes vigorously. Chloroform was filtered and the
residue was again put in the separating funnel for next addition of solvent. The chloroform
soluble portion was evaporated to dryness and then methanol (10ml x 5) was placed in the
separating funnel and shaked vigorously for 45 minutes. Methanol was filtered and the
residue was again put in the separating funnel for next addition of solvent. The methanol
soluble portion was evaporated to dryness and then water (10ml x 5) was placed in the
separating funnel and shaked for 45 minutes vigorously. Finally, the water soluble portion
was evaporated to dryness to get water fraction.
Percentage Yield of Various Extracts/Fractions of Abrus Precatorius Seeds:
S. No Name of
extracts/fractions
Weight of drug
powder(gm)
Weight of extract
(gm)
Percentage yield
(w/w)
1 Ethanol extract 220 15.9 7.2
2 Pet ether extract 10 1.8 18
3 Chloroform extract 10 0.3 3.0
4 Methanol extract 10 6.4 64
5 Water extract 10 2.3 23
Identification of Ethanol Extract by Phytochemical Tests
QUALITATIVE TESTS FOR VARIOUS PHYTOCONSTITUENTS 11,12,13,14
TEST FOR ALKALOIDS
Chemical tests Observation Inference
Dragendorff’s test
Few drops of potassium bismuth iodide +
extract
Reddish brown
colored ppt was
observed.
Presence of alkaloid.
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Mayer’s test
Few drops of mercuric iodide + extract.
Dull white
creamish ppt was
observed.
Presence of alkaloid.
Wagner’s test
Few drops of iodine solution in potassium
iodide + extract.
Reddish brown
colored ppt was
found.
Presence of alkaloid.
Hager’s test
Few drops of saturated solution of picric
acid + extract.
Yellow colored
ppt was found.
Presence of alkaloid.
TEST FOR CARBOHYDRATES
Chemical tests Observation Inference
Molisch’s test
Few drops of alcoholic solution of α-
naphthol + extract+ few drops of conc.
Sulphuric acid through sides of test tube.
Violet color ring
at the junction
was observed.
Presence of carbohydrate
Fehling's test
Equal amount of Fehling’s A and B solution
+ extract, heated at boiling water bath.
Brick red ppt
was observed.
Presence of carbohydrate
Benedict’s test
Benedict reagent + extract + heated at
boiling water bath.
Red ppt was
observed.
Presence of carbohydrate
TEST FOR STEROIDS
Chemical tests Observation Inference
Lieberman-Burchard test
Extract + 2 ml chloroform in dried test tube
+ 10 drops acetic anhydride + 2 drops conc.
sulphuric acid.
Changing of red
color to blue and
blue to bluish
green was
observed.
Presence of steroids
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Salkowski test
Extract + few drops of concentrated
sulphuric acid.
Bluish red to
cherry red color
in chloroform
layer was
observed.
Presence of steroids
TEST FOR GLYCOSIDES
Chemical tests Observation Inference
Legal test
Extract + pyridine + sodium nitroprusside.
Pink red color
was observed.
Presence of glycosides.
Baljet test
Extract + picric acid
Orange color
was observed.
Presence of glycosides.
TEST FOR FLAVONOIDS
Chemical tests Observation Inference
Shinoda test
Extract + magnesium turnings + 1-2 drops
of conc. HCl.
Green color was
observed instead
of red color.
Absence of flavonoids.
Zinc hydrochloride test
Extract+ zinc dust+ 1-2 drops of conc. HCl.
Green color was
observed
Absence of flavonoids.
TEST FOR PROTEINS
Chemical tests Observation Inference
Biuret test
Extract + 4 % sodium hydroxide + few
drops of 15% copper sulphate.
Pink color was
observed.
Presence of proteins.
Ninhydrin test
Solution of ninhydrin + extract, mixture was
heated.
Bluish violet
color was
observed.
Presence of proteins.
Heat test
Heat the test solution on a boiling water
bath.
Protein got
coagulated.
Presence of proteins.
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TEST FOR PHENOLIC COMPOUNDS
Chemical tests Observation Inference
Ferric chloride test
Extract + ferric chloride.
Dark blue color
was observed
instead of
greenish black
color.
Absence of phenolic group.
Potassium dichromate test
Extract + potassium dichromate solution.
No ppt was
observed instead
of Brown ppt.
Absence of phenolic group.
Gelatin test
Extract + 1% gelatin solution containing
10% NaCl.
No ppt was
observed.
Absence of phenolic group.
TEST FOR SAPONINS
Chemical tests Observation Inference
Foaming test
Extract was shaken with water.
Foam not
produced.
Absence of saponins.
PHYTOCHEMICAL ANALYSIS OF ETHANOL AND WATER EXTRACTS OF ABRUS
PRECATORIUS SEEDS
Table 3.2 Preliminary phytochemical studies of different extracts of Abrus precatorius
S. No Chemical Tests Ethanol Extract Water Extract
1
TESTS FOR ALKALOIDS:
Dragendorff‘s test
Mayer’s test
Wagner’s test
Hager’s test
+
+
+
+
+
+
−
+
2
TESTS FOR CARBOHYDRATES:
Molisch’s test
Fehling's test
Benedict’s test
+
+
+
+
−
+
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3
TESTS FOR STEROIDS:
Lieberman-Burchard test
Salkowski test
+
+
−
−
4
TESTS FOR GLYCOSIDES:
Legal test
Baljet test
+
+
+
−
5
TESTS FOR FLAVONOIDS:
Shinoda test
Zinc hydrochloride test
+
+
+
−
6
TESTS FOR PROTEINS:
Biuret test
Ninhydrin test
Heat test
+
+
+
+
+
+
7
TESTS FOR PHENOLIC COMPOUNS:
Ferric chloride test
Potassium dichromate test
Gelatin test
−
−
−
−
−
−
8
TESTS FOR SAPONINS:
Foaming test
−
−
IDENTIFICATION OF COMPOUND BY THIN LAYER CHROMATOGRAPHY (TLC) 15, 16 17
In 1958 Stahl demonstrated application of TLC in analysis. It is at present an important
analytical tool for qualitative analysis of a number of natural products. The plates were
visualized for spot identification under iodine chamber and sprayed with spray reagent of the
category given in table 3.3. The Rf value was calculated by using formula
Distance travelled by solute from the base line
Distance travelled by solvent front from the base line
Rf value =
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TLC PROFILE OF CRUDE ETHANOL EXTRACT OF ABRUS PRECATORIUS SEEDS
Table 3.3 TLC profile of crude ethanol extract of Abrus precatorius seeds
Category Solvent system Detecting reagent Color of spot Rf value
Glycoprotein
(Abrin)
water : butanol : acetic
acid (1.5: 6: 1.5)
Ninhydrin in ethanol Reddish brown 0.672
Figure.2 TLC of ethanol extract of Figure.3 TLC of ethanol extract of
Abrus precatorius seeds Abrus precatorius seeds
In iodine chamber. With ninhydrin reagent.
CHARACTERIZATION OF ETHANOL EXTRACT
PHYSICAL PROPERTIES
Colour : Dark brown.
State : Semi solid
Solubility : Soluble in water usually with turbidity, sparingly soluble in methanol.
Rf value : 0.672
CHARACTERIZATION OF PROTEIN (ABRIN) USING SODIUM DODECYL
SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS 18, 19, 20,21
Abrin is a potent plant toxin isolated from the seeds of Abrus precatorius. It is a type 2 ribosome
inactivating protein with ‘A’ and ‘B’ chains linked by disulfide bond. The abrin ‘A’ comprises
251 amino acid residues compared to 267 in ricin ‘A’chain, both having three folding structural
domains and molecular weight approximately 30 kD. Abrin ‘B’ chain has molecular weight
approximately 35 kD with 60% of its amino acid residues identical to those of ricin’s ‘B’ chain.
The abrin ‘A’chain has N-glycosidase activity while the abrin ‘B’ chain has galactose- binding
activity. Its smaller ‘A’ chain inhibits protein synthesis and causes cell death, the larger ‘B’
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chain binds to plasma cell membrane. The median lethal dose (LD50) of abrin determined by
intraperitonial injection in mice has been found to 20 µg/kg.
It is a yellowish white powder soluble in solutions of sodium chloride usually with turbidity.
The toxic portion is stable to incubation at 60ºc for 30 minutes and at 80ºc most of the toxicity is
lost in 30 minutes. Abrin can be separated and identified on the basis of its molecular weight by
Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE).
SIMILARITIES BETWEEN ABRIN AND RICIN
Abrin and ricin both are phytotoxins and are composed of two peptide chains (A, B) which
are linked by disulfide bond.
They both have 102 conserved amino acid homology.
Mechanism of action is same (both inhibit protein syntheses by inactivating ribosome’s).
The ‘A’ chain of both (abrin and ricin) contain the toxic activity while the ‘B’ chain gives
the toxin a cell recognition and binding function to facilitate transport across the cell
membrane.
The ‘A’ chain of both (abrin and ricin) inhibits protein syntheses whereas B-chain binds to
cell surface receptors containing terminal galactose and acts as an immunotoxin.
Abrin like ricin belongs to type-П RIPS.
GEL ELECTROPHORESIS (SDS-PAGE)
Electrophoresis refers to the electromotive force that is used to move the molecules through the
gel matrix. By placing the molecules in the well containing gel and applying an electric field, the
molecules will move through the matrix at different rates, determined largely by their mass
when charge to mass ratio (z) of all species is uniform, towards the anode if negatively charged
or towards cathode if positively charged. This method is used to separate substances on the basis
of their charge to mass ratios, using the effect of an electric field on the charges of the
substances to be separated. These techniques are widely used for charged colloidal particles or
macromolecular ions such as proteins, nucleic acids and polysaccharides.
PROTOCOL FOR SDS-PAGE
PREPARATION OF AQUEOUS EXTRACT SAMPLE
200 mg of aq. extract was taken in an eppendorff’s tube with 500µl of water and was shaked
vigorously for 10 minutes then the sample was mixed on vortex mixer, sonicated and finally
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centrifuged in microcentrifuge at 3000 rpm.
PREPARATION OF SEPARATING GEL
To a clean glass beaker following reagents were added to prepare separating gel:
Distilled water - 4.0 ml
30% Acrylamide - 3.3 ml
Separating buffer (Ph 8.8) - 2.5 ml
10% SDS - 100 µl
10% Ammonium per sulfate (APS) - 100 µl
TEMED - 5.0 µl
PREPARATION OF STACKING GEL
Distilled water - 2.7 ml
30% Acrylamide - 670 µl
Stacking buffer (Ph 6.8) - 500 µl
10% SDS - 40 µl
10% Ammonium per sulfate (APS) - 40 µl
TEMED - 4.0 µl
PROCEDURE
1) Glass plates, comb and spacers were washed with water and dried, then the glass plates were
sealed with the spacers.
2) Prepared separating gel was poured in the gap between the glass plates to ¾th of its length
and the gel was allowed to polymerize for 30 minutes.
3) Prepared stacking gel was then overlaid to the separating gel up to the rim of notched plate
and immediately a clean Teflon-comb was inserted into the stacking gel solution then
stacking gel was allowed to polymerize.
4) After polymerization comb was removed carefully and wells were washed immediately with
water then water was removed by using filter paper.
5) Spacers were then removed from the bottom and the glass plates were fixed in the
electrophoresis apparatus filled with running buffer in lower and upper buffer tanks.
6) After that three eppendorf’s were taken and marked as sample 1, sample 2, sample 3.
7) To the respective tubes 10 µl of sample was added followed by the addition of 10 µl of
sample solubilizing buffer.
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8) All the three tubes were mixed well and boiled at 100ºc for 4 minutes using water bath.
9) All the three samples were loaded to the first three lanes and 10 µl of protein molecular
weight marker was loaded to the next nearest lane.
10) After loading, power was switch on and voltage was set to 50 V. Running was continued
until the dye front reached the separating gel. After that the voltage was increased to 100 V
and running was continued until the dye front reached the bottom of the gel.
11) Gel plates were then removed from the tank using spatula and the stacking gel was removed
completely from the separating gel.
12) For the identification of abrin a mark was made on one side of the separating gel and the gel
was completely immersed with stainer in a petriplate and was stored for one day. After one
day stainer was removed and 25 ml of destainer was added.
13) Finally, protein was identified viewing against marker (at 29 KD), as abrin.
Figure.4 SDS-PAGE of abrin
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