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Short Communication

In-vitro antioxidant activity of different fractionof roots of Cassia auriculata Linn.

Supriya Deshpande, Shailesh M. Kewatkar*, Vivek V. Paithankar

Department of Pharmaceutical Sciences, Bhagwant University, Ajmer 305004, Rajasthan, India

a r t i c l e i n f o

Article history:

Received 7 February 2013

Accepted 28 May 2013

Keywords:

Antioxidant

Cassia auriculata Linn.

Flavonoids

DPPH

Indian traditional medicines

* Corresponding author. Tel.: þ91 9303649756E-mail address: kewatkar.shailesh@gmai

0975-7619/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.dit.2013.05.006

a b s t r a c t

The aim of this work was to estimate the total phenolic and flavonoid content, and to

evaluate in-vitro antioxidant activity of flavonoids rich extract of Cassia auriculata Linn.

(CAF) saponins rich extract of C. auriculata Linn. (CAS) which belongs to the family Cae-

salpiniaceae. Total phenolic content in CAF was found to be 67.32 mg/mg of extract

calculated as gallic acid equivalent (r2 ¼ 0.976) and total flavonoids compound was found to

be 31 mg/mg of extract calculated as rutin equivalent (r2 ¼ 0.985). The extract was screened

for its potential antioxidant activities using tests such as DPPH free radical scavenging

activity, reducing power activity, and hydrogen peroxide-scavenging activity. The in-vitro

antioxidant assay showed that both CAF as well as CAS posses antioxidant activity. So C.

auriculata Linn. could be useful for preparation of neutraceuticals as potent antioxidants, to

treat various human diseases and its complications.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction The healing potential of many plants have been utilized by

Antioxidant compounds in food play an important role as a

health protecting factor. Primary sources of naturally occur-

ring antioxidants are whole grains, fruits and vegetables. The

main characteristic of an antioxidant is its ability to trap free

radicals. Highly reactive free radicals and oxygen species can

initiate degenerative diseases. Antioxidant compounds like

phenolic acids, polyphenols and flavonoids are commonly

found in plants have been reported to havemultiple biological

effects, including antioxidant activity.1 Currently, the possible

toxicity of synthetic antioxidants has been criticized. Thus

interest in natural antioxidant, especially of plant origin has

greatly increased in recent years.2

.l.com (S.M. Kewatkar).2013, JPR Solutions; Publi

Indian traditional medicines like Siddha, Ayurvedha and

Unani. Being nontoxic and easily affordable, there has been

resurgence in the consumption and demand for medicinal

plants.3 Cassia auriculata Linn. commonly known as tanners

cassia, also known as “avaram” in Tamil language is a shrub

belongs to the Caesalpiniaceae family. The shrub is specially

famous for its attractive yellow flowers which are used in the

treatment of skin disorders and body odour. It is widely used

in traditional medicine for rheumatism, conjunctivitis and

diabetes. It has many medicinal properties. Its bark is used as

an astringent, leaves and fruits anthelminthic, seeds used to

treat in eye troubles and root employed in skin diseases.4 It is

also used for the treatment of ulcers, leprosy and liver

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

d ru g i n v e n t i o n t od a y 5 ( 2 0 1 3 ) 1 6 4e1 6 8 165

diseases.5 The antidiabetic, hypolipidemic6 and antioxidant7

and hepatoprotective8 effect of C. auriculata Linn. have been

reported. It was also observed that flower and leaf extract of C.

auriculata Linn. shown to have antipyretic activity.9 The roots

contain a flavones glycoside, 70,40-dihydroxyflavone-5-O-b-D-

galactopyranoside.10 C. auriculata Linn. contains several active

constituents such as flavonoids, b-sitosterol-a-d-glucoside,

polysaccharides, anthracene, dimeric procyanidins and myr-

istyl alcohol that are known to have scavenging activities

against free radicals.11 Roots of C. auriculata are reported to

contain flavonoids, polysaccharides, tannins, and saponins,

among other components which may contribute to its diverse

uses in folklore medicine.12

The aim of the present study was to determine the anti-

oxidant potential of two different fractions of the root of the

plant i.e. flavonoids rich extract of C. auriculata Linn. (CAF)

saponins rich extract of C. auriculata Linn. (CAS) which is

having traditional claims for several diseases.

2. Materials and methods

2.1. Procurement and authentication of plant

The root of the plant C. auriculata Linn. was collected from

fields of Walgaon Road, Amravati, Maharashtra and in-

teriors of Bhopal, Madhya Pradesh respectively. The plant

has been authenticated by Safia College of Science, Peer

Gate, Bhopal, (Madhya Pradesh), and were given the voucher

specimen number 159/Bot/Safia/2010. The authenticated

roots were dried under shade and then coarsely powdered

with the help of mechanical grinder. The powdered was

passed through sieve no. 40 and stored in an airtight conta-

iner for extraction.

2.2. Preparation of flavonoids rich extract of Cassiaauriculata Linn. (CAF)

Extraction of the powdered roots of the plant was carried out

with the help of water by decoction method.13 Special care

was taken while decoction process regarding temperature

that it should not exceeding more than 40 �C � 5 �C as it may

cause the precipitation or crystallization of some phytocon-

stituents which will never solubalised into any solvents in

further process. Then this aqueous extract was filtered and

alcohol (Ethanol) was added slowly into this aqueous liquid

extract to precipitate out polysaccharides which are present

into the roots of individual plant.

Then the solution was filtered and obtained filtrate was

evaporated to 1/4th of the total volume. After evaporating 1/

4th of the total volume of the solution, it was successively

extracted with equal amount of ethyl acetate with the help of

separating funnel to get separate fraction of ethyl acetate

fraction of the roots of the plant. Then the ethyl acetate

extract was acidified with 0.1 N HCL to increase the yield of

extract. Then this ethyl acetate fraction of root of the plant

was evaporated to get precipitate which is then dissolved in

methanol and evaporated slowly to get crystalline powder of

flavonoids.

2.3. Preparation of saponins rich extract of C. auriculataLinn. (CAS)

Same plant materials were used for getting saponins rich

fraction.14 Pulverized plant materials was treated with

ethanol: water (70:30) for maceration till seven days after

defattingwith petroleumether (40:60). Mixturewas agitated at

regular interval in this period. Obtained extract after filtration

with muslin cloth followed by filter paper was concentrated

using rotary vacuum evaporator (40 �C), precaution was kept

that extract do not get powdered. Concentrated extract was

further treated with n-butanol to get n-butanol soluble frac-

tion. n-Butanol soluble fraction was further treated with

chilled diethyl ether. After treating with chilled diethyl ether,

precipitates were formed. This mixture with precipitate was

kept at �20 �C for 24 h. Precipitates were further separated by

centrifugation. These precipitate were further dissolved in

methanol and methanol was evaporated slowly, to get crys-

talline powder.

Powder was further investigated for presence/absence of

various phytoconstituents. It was observed hat powder gave

positive results for froath test and haemolysis test, which

ascertained presence of saponins in powder.

2.4. Total phenolics content

The total phenols of all extracts were measured at 765 nm by

Folin Ciocalteu reagent.15 Each dilute fraction of the plant i.e.

CAF/CAS (0.5 ml of 1:10 g/ml) or gallic acid (standard phenolic

compound) wasmixed with Folin Ciocalteu reagent (5 ml, 1:10

diluted with distilled water) and aqueous sodium carbonate

(4 ml, 1 M). The mixture was allowed to stand for 15 min and

the total phenols were determined by spectrophotometer at

765 nm. The standard curve was prepared using 10, 20, 30, 40

and 50 mg/ml solutions of gallic acid inmethanol: water (50:50,

v/v). Total phenol values are expressed in terms of gallic acid

equivalent (mg/g of dry mass), which is a common reference

compound.

2.5. Total flavonoid content

An aliquot of diluted sample (1 mg/ml) or standard solution of

rutin was added to a 75 ml of NaNO2 solution, and mixed for

6 min, before adding 0.15 ml AlCl3 (100 g/L).16 After 5 min,

0.5 ml of NaOH was added. The final volume was adjusted to

2.5 ml with distilled water and thoroughly mixed. Absorbance

of the mixture was determined at 510 nm against the same

mixture, without the sample, as a blank. Total flavonoid

content was expressed as mg rutin/g dry weight, through the

calibration curve of rutin (10, 20, 30, 40 and 50 mg/ml). All

samples were analysed in three replications.

2.6. DPPH free radical scavenging activity

Different concentration of plant extract was prepared.17 Af-

terwards these dilutions were mixed with 0.5 ml DPPH solu-

tion (4 mg in 100 ml methanol) and incubated for 30 min at

room temperature in dark condition. After incubation the

absorbance was noted at 517 nm using methanol as a blank.

Percentage inhibition was calculated using following

Fig. 1 e Standard curve of gallic acid.

d r u g i n v e n t i o n t o d a y 5 ( 2 0 1 3 ) 1 6 4e1 6 8166

formulae Inhibition % ¼ (1 � A1/A0) � 100. Where A1 is the

absorbance in the presence of plant extract or positive con-

trols, while A0 is the absorbance in the absence of plant

extract and positive controls.

2.7. Hydrogen peroxide-scavenging assay

A solution of hydrogen peroxide (40 mM) was prepared in

phosphate buffer (pH 7.4).18 Hydrogen peroxide concentration

was determined spectrophotometrically from absorption at

230 nm in a spectrophotometer. Various concentration of plant

extract which were prepared in of 40 mM phosphate buffer sa-

line of (PH 7.4), which were added to 2 ml of hydrogen peroxide

solution. Absorbance of hydrogen peroxide at 230 nm was

determined after 10 min against a blank solution containing

phosphate buffer without hydrogen peroxide. Percentage inhi-

bition was calculated using following formulae Inhibition

%¼ (1�A1/A0)�100.Where,A1 is theabsorbance inthepresence

ofplantextractorpositive controls,whileA0 is theabsorbance in

the absence of plant extract and positive controls.

2.8. Reducing power assay

Extracts were prepared in different concentrations and 1ml of

each in distilled water were mixed with phosphate buffer

(2.5 ml, 2 M, pH 6.6) and potassium ferricyanide (2.5 ml, 1%);

the mixture was incubated at 50 �C for 20 min.19 A portion

(2.5 ml) of trichloroacetic acid (TCA, 10%) was added to the

mixture which was then centrifuged at 1500 RPM for 10 min.

The upper layer of solution (2.5 ml) was mixed with distilled

water (2.5ml) and FeCl3 (0.5ml, 0.1%), and the absorbancewas

measured at 700 nm. Increased absorbance of the reaction

mixture indicated increased reducing power.

Fig. 2 e Standard curve of rutin.

3. Results and discussion

Free radical is a molecule with an unpaired electron and is

involved in bacterial and parasitic infections, lung damage,

inflammation, reperfusion injury, cardiovascular disorders,

atherosclerosis, ageing and neoplastic diseases.20 They are

also involved in autoimmune disorder like rheumatoid

arthritis etc.21 Our results demonstrated that the both the

fractions of root of C. auriculata Linn. possess free radical

scavenging activity in-vitro models like DPPH�, Hydrogen

peroxide-scavenging and reducing power activity.

3.1. Total phenolics content

Phenolic compounds are known as powerful chain breaking

antioxidant22 and they are very important plant constituents

because of their scavenging ability, which is due to their hy-

droxyl groups.23 In CAF, the total phenolic content was found

to be 67.32 mg/mg in terms of gallic acid equivalent. Standard

curve of gallic acid is shown in Fig. 1.

3.2. Total flavonoids content

Flavonoids are a group of polyphenolic compounds, which

exhibit several biological effects such as anti-inflammatory,

anti-hepatotoxic, anti-ulcer, anti-allergic, anti-viral and anti-

cancer activities.24 They are capable of effectively scav-

enging the reactive O2 species because of their phenolic hy-

droxyl groups and so they are potent antioxidants also.25 The

total flavonoids content of CAFwas determined to be 31 mg/mg

in terms of rutin equivalent. Standard curve of rutin is shown

in Fig. 2

3.3. DPPH free radical scavenging activity

DPPH� is one of the free radicals widely used for testing pre-

liminary radical scavenging activity of the plant extract,

which is based on the ability of DPPH�, a stable free radical, to

decolorize in the presence of antioxidants, is a direct and

reliable method for determining radical scavenging action.26

The DPPH� assay has been largely used as a quick, reliable

and reproducible parameter to search the in-vitro general

antioxidant activity of pure compounds as well as plant ex-

tracts.27 The reducing capacity of compounds could serve as

indicator of potential antioxidant property.

In the present study, the percentage of scavenging effect

on the DPPH� radical was concomitantly increased with the

increase in the concentration of both CAF and CAS from 10 to

100 mg/ml and 100 to 1000 mg/ml. The percentage of inhibition

existed from 21.46 at 10 mg/ml to 72.5 at 100 mg/ml for CAF and

for CAS; they were 6.45 at 100 mg/ml and 69.37 at 1000 mg/ml

Table 1 e DPPH free radical scavenging activity of CAFand CAS.

CAF CAS

Sr.no.

Conc(mg/ml)

%Inhibition

IC50 Sr.no.

Conc(mg/ml)

%Inhibition

IC50

1 10 21.46 60.977 1 100 6.45 707.930

2 20 28.67 2 200 12.46

3 40 32.25 3 400 26.375

4 60 48.56 4 600 46

5 80 64.24 5 800 57.62

6 100 72.5 6 1000 69.37

Table 3 e Reducing power assay of CAF and CAS.

CAF CAS

S. no. Conc.(mg/ml)

Absorbance S. no. Conc.(mg/ml)

Absorbance

1 10 0.092 1 10 0.064

2 20 0.129 2 20 0.097

3 40 0.213 3 40 0.168

4 60 0.278 4 60 0.191

5 80 0.342 5 80 0.293

6 100 0.388 6 100 0.318

d ru g i n v e n t i o n t od a y 5 ( 2 0 1 3 ) 1 6 4e1 6 8 167

(Table 1). From the results it is known that the species, C.

auriculata Linn. possess hydrogen donating capabilities and

does scavenging free radicals. Furthermore, it was noticed

that the CAF has more pronounced scavenging activity than

that of the CAS.

3.4. Hydrogen peroxide-scavenging assay

Hydrogen peroxide is generated in vivo by several oxidase

enzymes. In this method, when an antioxidant is incubated

with hydrogen peroxide, the decay or loss of hydrogen

peroxide is measured spectrophotometrically.28 Hydrogen

peroxide is a weak oxidizing agent which inactivates enzymes

by oxidation of the essential thiol (SHe) groups. It rapidly

transverses cell membranes and once inside the cell interior,

interacts with Fe2þ and Cu2þ to form hydroxyl radicals, which

is harmful to the cell.29 The extracts showed good scavenging

effects with IC50 values 52.249 mg/ml and 956.584 mg/ml for CAF

and CAS respectively (Table 2). The composition of hydrogen

peroxide into water may occur according to the antioxidant

compounds, as the antioxidant component present in the

extract are good electron donors, they may accelerate the

conversion of H2O2 to H2O.

3.5. Reducing power assay

Reducing power activity is often used to evaluate the ability of

natural antioxidant to donate electron.30,31 Many reports have

revealed that there is a direct correlation between antioxidant

activities and reducing power of certain plant extracts.32,33

The reducing power activity of CAF and CAS increased

consistently with the increase in the volume of extract from

Table 2 e Hydrogen peroxide- scavenging assay of CAFand CAS.

CAF CAS

Sr.no.

Conc(mg/ml)

%Inhibition

IC50 Sr.no.

Conc(mg/ml)

%Inhibition

IC50

1 10 24.7 52.249 1 100 4.22 956.584

2 20 29.57 2 200 9.86

3 40 34.66 3 400 23.52

4 60 49.21 4 600 29.65

5 80 78.25 5 800 41.22

6 100 81.5 6 1000 54.28

10 mg/ml to 100 mg/ml. When compared with the CAS, CAF

showed higher absorbance. It is known further that the

reducing power activity of CAF was due to the presence of

phenolic compound such as flavonoids (Table 3).

4. Conclusion

Searching plant sources may bring new natural products into

pharmaceutical, cosmetic and food production. An in-vitro

antioxidant study provides scientific evidence to prove the

traditional claims to the member of cassia species, On the

basis of the results obtained in the present study, it was

concluded that the CAF and CAS possess significant antioxi-

dant activity. Presence of adequate amount of phenol, flavo-

noids and saponins compounds may account for this fact. So

these findings of present study suggest that this plant have a

potential source of natural antioxidant. Further studies are

warranted for the isolation and characterization of antioxi-

dant compounds, and also in vivo studies are needed for un-

derstanding their mechanism of action as antioxidants.

Conflicts of interest

All authors have none to declare.

Acknowledgements

Authors are thankful to Mr. Manvendra Singh Karchuli

(Research Associate) and Director, Safia College of Science,

Bhopal for their support and help for the completion of this

research work.

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