PHYTOCHEMICAL INVESTIGATION AND CHARACTERIZATION OF …

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Vikas et al. World Journal of Pharmaceutical Research

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.

[email protected]

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925


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.


Wagner’s test

Few drops of iodine solution in potassium

iodide + extract.

Reddish brown

colored ppt was

found.


Hager’s test

Few drops of saturated solution of picric

acid + extract.

Yellow colored

ppt was found.


TEST FOR CARBOHYDRATES


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.


Benedict’s test

Benedict reagent + extract + heated at

boiling water bath.

Red ppt was

observed.


TEST FOR STEROIDS


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


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


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


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.


Heat test

Heat the test solution on a boiling water

bath.

Protein got

coagulated.


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930


TEST FOR PHENOLIC COMPOUNDS


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.


Gelatin test

Extract + 1% gelatin solution containing

10% NaCl.

No ppt was

observed.


TEST FOR SAPONINS


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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