MATERIALS AND METHODS

Plant:
The whole plants of Erythrina indica were collected from country drug and herbal shop. The plant
was authenticated by plant anatomy research centre, chennai, Tamil Nadu, India.
Preparation of the plant extract:
The whole plants of Erythrina indica were shade dried and coarsely powdered and was
extracted by using aqueous as a solvent in a soxhlet apparatus. The solvent was completely removed by
vaccum and semisolid mass was obtained (11% w/w with respect to the powdered material) the extract
was dried under reduced pressure using rotary flash evaporator and stored in refrigerator for further
studies.
Preliminary Phytochemical Screening:
100mg of aqueous extract was dissolved in distilled water and the following test was done
separately.
TEST FOR ALKALOIDS:
Mayer’s Test:
Reagent:
Solution A: 1.358g of mercuric chloride was dissolved in 60ml of distilled water.
Solution B: 5g of potassium iodide was dissolved in 10ml of distilled water.
Solution A and B were mixed and made up to 100ml with distilled water. 0.5ml of Mayer’s
reagent (potassium mercuric iodide) was added to the filtrate of Erythrina indica. The presence of
alkaloids was confirmed by the formation of cream precipitate.

TEST FOR GLYCOSIDES:

A portion of Erythrina indica was hydrolyzed by boiling with dilute hydrochloric acid in a
water bath for 30mins cooled and filtered. The hydrolysates were subjected to Legal’s, Borntrager’s
and modified anthraquinone test.RAQ
Legal’s Test:
To each 1ml of the hydrolysates of Erythrina indica, 1ml of pyridine and a few drops of
sodium nitro prusside solution were added and then it was made alkaline with sodium hydroxide
solution. Appearance of pink colour showed the presence of glycosides.
Borntrager’s Test:
1ml of each hydrolysates of Erythrina indica were shaken gently with equal volume of
chloroform and then the chloroform layer was separated. To this equal quantity of dilute ammonium
solution was added. If ammonia layer acquires pink colour it showed the presence of O-anthraquinone
glycosides.
Modified anthraquinone test:
To 1ml of the hydrolysates, equal volume of 5% ferric chloride solution and dilute
hydrochloric acid were added and heated on boiling water bath for 5mins, cooled and shaken gently
with benzene. Benzene layer separated and equal volume ammonia was added to the benzene layer.
Formation of pink colour indicates the presence of C-anthraquinone glycosides;
Test for phenol:
To the filtrate of extracts (1ml), two drops of ferric chloride solution was added.
Formation of blue colour indicated the presence of phenols.
Test for flavonoids:
To the filtrate of extracts (1ml), magnesium bit and 2 drops of concentrated hydrochloric acid were
added and gently heated. Formation of pink colour indicated the presence of flavonoids.
To the filtrate of extracts, 0.5ml of amyl alcohol was added followed by the addition of 3 drops
of sodium acetate and 2 drops of ferric chloride. Formation of blood red colour indicated the presence
of flavonoids.
Test for triterpenoids:
To 1ml of extracts, tin and 2 drops of thionyl chloride were added. Formation of pink colour indicated
the presence of triterpenoids.
Test for coumarins:
To the filtrate of extracts (1ml), 1ml of 2N sodium hydroxide was added. Formation of yellow colour
indicates the presence of coumarins. Further 1ml of 5N hydrochloric acid was added. If colourless
solution formed at the upper layer, it shows the presence of coumarins.
Test for saponins:
Foam test: The extracts were diluted with 1ml of water separately and shaken well. Stable froth
formation indicated the presence of saponins.
Test for tannins:
To the filtrate of extracts (1ml), 0.5 ml of 10% lead acetate was added. Appearance of white precipitate
indicated the presence of tannins.

Test for carbohydrates:

To the filtrate of the extracts (1ml), 2drops of concentrated Sulphuric acid was added slowly on the
sides of the test tube and then 0.5 ml of α-naphthol solution was added. Formation of purple ring at the
junction of two liquids showed the presence of carbohydrates.
Test for reducing sugars:
To the aliquot of extracts, 2ml of diluted Hydrochloric acid was added and boiled for 10mins and it was
neutralized with diluted sodium hydroxide then cooled for 5 min, after cooling equal volume of
Fehling’s A and B solutions were added and boiled in a water bath. Formation of reddish brown
precipitate indicated the presence of reducing sugar.
Test for phytosterol:
The extracts were refluxed with the solution of alcoholic potassium hydroxide till complete
saponification takes place. The mixture was diluted with distilled water and extracted with ether. The
ether layer was evaporated and the residue was tested for Libermann Burchard test.
Libermann Burchard test:
The extracts (10 mg) were dissolved in 3 ml of acetic anhydride. To this solution 2
drops of concentrated sulphuric acid were added slowly along the sides of the test tube. Appearance of
bluish green colour showed the presence of phytosterol.
Salkowski Test:
1ml of each filtrates were mixed with 1ml of chloroform separately. To these
solutions 1ml of concentrated sulphuric acid was added carefully along the sides of the test tube.
Formation of red colour indicated the presence of sterols.
Test for proteins:
Biuret Test: To 1ml of filtrate of extracts, 1ml of 5% copper sulphate and 1% sodium hydroxide
solution were added. Deep blue colour indicates the presence of proteins.

INVITRO STUDIES:
Free Radical Scavenging activity of Erythrina indica:
DPPH assay:
The free radical quenching capacity of aqueous extract of Erythrina indica was
determined by the method involving the bleaching of stable DPPH (Ursini et al, 1994). A reaction
mixture containing methanol, DPPH (10Mm, 30µl) and 100µl of various concentrations of Erythrina
indica (0.01, 0.1 and 1.0mg in dimethyl sulfoxide, DMSO) was allowed to stand at room temperature
for 30mins before mixing with redistilled distilled water (1ml) and toluene (3ml). The solution was
then centrifuged, and the absorbance of the upper phase was read at 517nm against a blank without
Erythrina indica prepared and processed as described above. Vitamin C served as standard and
compared with the extract. The free radical scavenging activity was calculated as given below.

Radical scavenging (%) = {CONTROL – SAMPLE} X 100

CONTROL
The results were expressed as % inhibited/mg of extract.

Nitric Oxide (NO-) Scavenging effect:

According to the method of Sreejayan and Rao (1997), sodium nitro prusside (SNP,
5Mm) was dissolved in phosphate buffered saline and this was mixed with different concentrations of
Erythrina indica (10, 20, 30, 40 and 50µg) dissolved in 50mM of phosphate

buffer, before being incubated at 25∘C for 150 mins. The amount of NO- produced by
sodium nitro prusside was measured by Griess reagent (1%) (Sulphanilamide in
5% phosphoric acid and 0.1% N- 15- naphtyl ethylenediamine dihydrochloride in
distilled water). 0.5ml of the incubation mixture was mixed with same volume of
Griess reagent. The absorbance of the mixture was read at 550nm. Vitamin C
served as standard and compared with the extract. The percentage of inhibition
per each concentration of the extract was calculated as given below.

Radical scavenging (%) = {CONTROL – SAMPLE} × 100

CONTROL
The results were expressed as % inhibited / mg of extract.

Superoxide Anion Scavenging Activity:

Measurement of superoxide anion scavenging activity of MME and MMU were based on
the method described by Liu et al., (1997) with slight modifications of Gulcin et al., (2003).
Reagent:
 1. Tris-HCl buffer: 16mM, pH 8.0
2. Nitro blue tetrazolium (NBT): 50 µM
3. Phenazine methosulphate (PMS): 10 µM
4. NADH: 78µM
Superoxide anions were chemically generated in a mixture of phenazine methosulphate
(PMS) and NADH. The reaction was quantified by coupling superoxide generation to the reduction of
nitroblue tetrazolium (NBT). In this experiment, the superoxide radicals were generated in 3 ml of Tris-
HCl buffer (16mM,pH 8.0) containing 1ml of NBT (50 µm) solution, 1 ml of NADH (78 µM) solution
and 1 ml of various concentrations (200, 400,600,800,1000 µg/ml) of aqueous and methanol extract of
Erythrina indica were mixed. The reaction was started by adding 1 ml of PMS solution (10 µM) to the
mixture. The reaction mixture was incubated at 25∘C for 5 min and the absorbance at 560 nm in a
spectrophotometer was measured.

Assay of DNA sugar damage:

The protective role of aqueous and methanol extract of Erythrina indica on the inhibition of
free radical mediated DNA-sugar damage was assayed by the method of Halliwell and Gutteridge
(1981).
Reagents:
1. Phosphate buffer: 0.1 M, pH 7.4
2. Ascorbic acid: 1Mm
3. Ferric chloride: 100µM
4. Thiobarbituric acid- 0.67%
5. Calf thymus DNA
6. Sodium chloride solution: 0.15 M
The reaction mixture in a total volume of 1.24 ml contained 0.5 ml calf thymus DNA (1
mg/ml of 0.15 M Nacl), 0.5 ml of phosphate buffer (0.1 M, pH 7.4), 0.2ml ascorbic acid (1mM) and
0.04 ml of ferric chloride (100µM). To the reaction mixture various concentrations
(200,400,600,800,1000 µg/ml) of aqueous and methanol extract of Erythrina indica were added. The
reaction mixture was incubated for 1 h at 37°C in a water bath shaker. After the incubation was over,
0.66% TBA (1ml) was added to the reaction mixture and then it was kept in boiling water bath for 15
min. The TBA reacting species so generated forms adduct which shows a characteristic absorption at
535 nm.
Estimation of Total Antioxidant Activity:
Ferric thiocyanate method (FTC):
It is based on the determination of amount of peroxide at primary stage of lipid peroxidation.
The peroxidation reacts with ferrous chloride to form reddish ferric chloride pigments, which is
measured at 500 mm. Methods of mitsuda et al.,(1967)and namiki were modified by kikuzaki and
nakatani was followed.
Reagents:

1. Linoleic acid 2.523%

2. Phosphate buffer 0.05m, pH 7.0
3. Ferrous chloride 0.02m
4. Ammonium thiocyanate 30%
5. Vitamin E
6. Absolute alcohol
7. Ethanol 75%
8. Hydrochloric acid 3.5%
Procedure:
4mg of the aqueous and methanol extract of Erythrina indica were dissolved separatively in
4ml of absolute ethanol. To these solutions 4.1ml of 2.52% linoleic acid in absolute ethanol, 8ml of
0.005M phosphate buffer (pH 7.8).
And .39ml of distilled water were added. These mixtures were taken in vials with a
screwcap and placed in an oven at 40c in the dark. To 0.1 ml of each ammonium thiocyanate was
added. Precisely 3 min after the addition of 0.1ml of 0.02M ferrous chloride in 3.5% hydrochloric acid
to reaction mixture, the absorbance was measured at 500 nm for every 24 hours until the absorbance of
the control reached maximum. The control and standard were subjected to the same procedure as the
sample except that for control. Only the solvent was added, and for the standard, 4 mg of the sample
was replaced by 4 mg of vitamin E.

Thio Barbituric acid method (TBA):

The method of ottolength(1959) was used to determine the TBA values of the samples.
The formation of the MDA is the basis for the known TBA method used for evaluating the lipid
peroxidation. At low pH and high temperature (100c), MDA binds with TBA to form a red complex
that can be measured at 532 nm. TBA method is used to measure the carbonyl compounds obtaind by
linoleic acid oxidation at the later stage of linoleic acid peroxidation.
Regents:
1. TBA 0.67%
2. TCA20%
3. Vitamin C
Procedure:
The sample prepared for FTC method were used for this assay. To 2ml of the sample solution
in a 10 ml centrifuge tube, 1 ml of 20% aqueous trichloroacetic acid was added. The mixture was
placed in boiling water bath For 10 min and cooled. The mixture was centrifuged at 3000 rpm for
30min absorbance of the supernatant was measured at 532 nm on the final day of the
assay.