I J P B C S Research Article - International Journal of...

12
International Journal of Pharmaceutical Biological and Chemical Sciences International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS) | JAN-MAR 2014 | VOLUME 3 | ISSUE 1 | 06-17 www.ijpbcs.net or www.ijpbcs.com Research Article Page6 ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR AND SEASONAL AYURVEDIC MEDICINAL PLANTS ELEPHANTOPUS SCABER L. AND BIOPHYTUM SENSITIVUM DC. A. Subramanian 1 , S. Santhosh Kumar 2* , S. K. Suja 3 , M. Sudarshan 4 and A. Chakraborty 5 1 Department of Biochemistry, Raja Serafoji Government College, Thanjavur-613005, Tamil Nadu, India *2 Department of Physics, Avvaiyar Government College for Women, Karaikal 609 602, U.T of Puducherry, India 3 Department of Chemistry, Lady Doak College, Tallakulam, Madurai - 625002, Tamilnadu, India 4 Trace Element Sciences Laboratory, UGC-DAE Consortium for Scientific Research, Kolkata - 700 098, India 5 Radiation Biology Laboratory, UGC-DAE Consortium for Scientific Research, Kolkata 700 098, India *Corresponding Author Email: [email protected] INTRODUCTION Medicinal plants are widely used in the ancient system of medicines such as Ayurveda, Siddha, Unani, and in Tibetan medicine. 3/4 th of the population in developing countries relies on traditional medicine, according to the world health organisation (WHO) that too in the form of crude extracts, decoction, arishta, lehya, surna, and even in tablet form. Millions of rural households use medicinal plants in a self help mode. In recent years secondary plant metabolites phytochemical, pharmacological activities have been extensively investigated as a source of curative agent[1]. Recently there has been an upsurge of interest in the therapeutic potentials of plants, antioxidants in reducing free radical induced tissue injury, although several butylated hydroxy toluene (BHT), are commercially available, but are quite unsafe and their toxicity is a problem of concern. Hence strong restrictions have been placed on their application and which is to be substituted with naturally occurring antioxidants. Many plant species have been investigated in the search for novel antioxidants [2-5] but generally there is still a demand to find more information concerning the antioxidant potential of plant species as they are safe and also bioactive. The antioxidant detoxifies the reactive oxygen species (ROS), a potentially reactive derivative of oxygen, present in the body. However excess production of ROS or inadequate antioxidant defense causes oxidative damage to various bio-molecules including DNA, which is a critical etiological factor implicated in several chronic diseases like, diabetes mellitus, cancer, arthritis and also in ageing process[6]. Therefore, considerable attention has been directed, now a days, towards the identification of plants with antioxidant ability. Natural antioxidants especially phenolics and flavanoids from tea, wine, fruits, vegetables and spices are already exploited commercially either as antioxidant additives or as nutritional supplements [7]. Gulcin[8] reported the antioxidant and radical scavenging activity of black pepper seeds from both water extract and ethanol extract. Due to the bioactive nature and safe to use of naturally occurring antioxidants, many studies have been conducted in recent years, and most recently, Vinay at al. [6] have studied the antioxidant activity of commonly available plants and found Hibiscus is comparatively ABSTRACT: Traditionally used Ayurvedic medicinal plants, Elephantopus scaber and Biophytum sensitivum are investigated for their antioxidant potency. Preliminary phytochemical analysis of pet-ether fraction of these plant leaves show the presence of alkaloids, tannins, saponins, flavanoids and phenolic compounds. Both the pet-ether and ethanol fractions exhibited antioxidant activity significantly. The DPPH radical scavenging and the nitric oxide scavenging activity are high for ethanol fraction than pet-ether and ascorbic acid. The pet-ether fraction of B.sensitivum showed comparatively good reducing power even at low concentration and shows higher potentiality in antioxidant activity than other extracts, which is in good coincidence with the phytochemical constituents. The results validate the potential usage of these plants in the traditional Ayurvedic system of medicine, to a greater extent. KEYWORDS: Ayurvedic medicinal plant, phytochemical, antioxidant activity, DPPH, E.scaber, B.sensitivum.

Transcript of I J P B C S Research Article - International Journal of...

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International Journal of Pharmaceutical

Biological and Chemical Sciences

International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS)

| JAN-MAR 2014 | VOLUME 3 | ISSUE 1 | 06-17 www.ijpbcs.net or www.ijpbcs.com

Research Article

Pag

e6

ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR

AND SEASONAL AYURVEDIC MEDICINAL PLANTS ELEPHANTOPUS SCABER L.

AND BIOPHYTUM SENSITIVUM DC.

A. Subramanian1, S. Santhosh Kumar

2*, S. K. Suja

3, M. Sudarshan

4 and A. Chakraborty

5

1Department of Biochemistry, Raja Serafoji Government College, Thanjavur-613005, Tamil Nadu, India

*2Department of Physics, Avvaiyar Government College for Women, Karaikal – 609 602,

U.T of Puducherry, India 3 Department of Chemistry, Lady Doak College, Tallakulam, Madurai - 625002, Tamilnadu, India

4Trace Element Sciences Laboratory, UGC-DAE Consortium for Scientific Research, Kolkata - 700 098, India

5Radiation Biology Laboratory, UGC-DAE Consortium for Scientific Research, Kolkata 700 098, India

*Corresponding Author Email: [email protected]

INTRODUCTION

Medicinal plants are widely used in the ancient system

of medicines such as Ayurveda, Siddha, Unani, and in

Tibetan medicine. 3/4th

of the population in developing

countries relies on traditional medicine, according to the

world health organisation (WHO) that too in the form of

crude extracts, decoction, arishta, lehya, surna, and even

in tablet form. Millions of rural households use

medicinal plants in a self help mode. In recent years

secondary plant metabolites phytochemical,

pharmacological activities have been extensively

investigated as a source of curative agent[1]. Recently

there has been an upsurge of interest in the therapeutic

potentials of plants, antioxidants in reducing free radical

induced tissue injury, although several butylated

hydroxy toluene (BHT), are commercially available, but

are quite unsafe and their toxicity is a problem of

concern. Hence strong restrictions have been placed on

their application and which is to be substituted with

naturally occurring antioxidants.

Many plant species have been investigated in the search

for novel antioxidants [2-5] but generally there is still a

demand to find more information concerning the

antioxidant potential of plant species as they are safe and

also bioactive. The antioxidant detoxifies the reactive

oxygen species (ROS), a potentially reactive derivative

of oxygen, present in the body. However excess

production of ROS or inadequate antioxidant defense

causes oxidative damage to various bio-molecules

including DNA, which is a critical etiological factor

implicated in several chronic diseases like, diabetes

mellitus, cancer, arthritis and also in ageing process[6].

Therefore, considerable attention has been directed, now

a days, towards the identification of plants with

antioxidant ability.

Natural antioxidants especially phenolics and flavanoids

from tea, wine, fruits, vegetables and spices are already

exploited commercially either as antioxidant additives or

as nutritional supplements [7]. Gulcin[8] reported the

antioxidant and radical scavenging activity of black

pepper seeds from both water extract and ethanol extract.

Due to the bioactive nature and safe to use of naturally

occurring antioxidants, many studies have been

conducted in recent years, and most recently, Vinay at al.

[6] have studied the antioxidant activity of commonly

available plants and found Hibiscus is comparatively

ABSTRACT:

Traditionally used Ayurvedic medicinal plants, Elephantopus scaber and Biophytum sensitivum are investigated for their

antioxidant potency. Preliminary phytochemical analysis of pet-ether fraction of these plant leaves show the presence of

alkaloids, tannins, saponins, flavanoids and phenolic compounds. Both the pet-ether and ethanol fractions exhibited

antioxidant activity significantly. The DPPH radical scavenging and the nitric oxide scavenging activity are high for ethanol

fraction than pet-ether and ascorbic acid. The pet-ether fraction of B.sensitivum showed comparatively good reducing power

even at low concentration and shows higher potentiality in antioxidant activity than other extracts, which is in good

coincidence with the phytochemical constituents. The results validate the potential usage of these plants in the traditional

Ayurvedic system of medicine, to a greater extent.

KEYWORDS: Ayurvedic medicinal plant, phytochemical, antioxidant activity, DPPH, E.scaber, B.sensitivum.

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S. Santhosh Kumar *

et al; ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR ……………

International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS) | JAN-MAR 2014 | VOLUME 3 | ISSUE 1 | 06-17| www.ijpbcs.net

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rich in flavanoids, which is used in many ways by the

south Indian people, such as, oil, food supplement,

gulkand, lehya, shampoo, etc. Hence it is a necessity

now to identify potentially antioxidant plants, available

in common.

The oxidative stress, defined as “the imbalance between

oxidants and antioxidants in favour of the oxidants

potentially leading to damage” has been suggested to be

the cause of aging [9] and various diseases in humans

[10]. In modern western medicine, the balance between

antioxidation and oxidation is believed to be a critical

concept maintaining a healthy biological system [10-14].

Such concept exists in China since 2000 years, called

yin-yang (antioxidant-oxidation process). It was found

that yin-tonic herbs are mainly having flavanoids[15],

which are phenolic compounds with strong antioxidant

activity. Hence increasing the intake of foods rich in

antioxidant compounds, (e.g., polyphenols, cartenoids)

will enhance human health. As a consequence many

plants have been identified as having potentially active

antioxidant [10, 16, 17].

The majority of the antioxidant activity is due to the

flavones, isoflovones, flavonoids, anthocynin, coumarin

lignans, catechins and isocatechins [18]. Besides,

phenolic compounds and flavonoids are also

widely distributed in plants having multiple biological

effects, including antioxidant, free radical scavenging

abilities, anti-inflammatory, anti-carcinogenic

etc.[19,20].

The current focus is towards natural antioxidants, and it

is interesting to investigate the antioxidant properties of

plants, especially, which are in traditional medicinal

practice. Hence we have identified two important plants

such as Elephatopus scaber Linn, and Biophytum

sensitivum Dc., which are used in Ayurvedic practice for

several ailments.

Eventhogh many plants are having reported to be good

antioxidants the availability of plants is still a big

question. Even though the Biophytum sensitivum is

having good antioxidant and free radical scavenging

capacity, this plant is available only for around three to

four months after rainy season. And hence we selected

an annual/perennial plant Elephatopus scaber for such

study in comparison with Biophytum sensitivum.

Numerous applications of E. scaber in folk medicine as

well as the bioactivities shown by various extracts and

respective compounds of this plant are discussed earlier

[21,22]. Bioactivity screening of the extracts and their

compounds had shown that E. scaber possess wound

healing, anti-venom, anti-microbial, anti-inflammatory,

anti-diabetic, cytotoxic and anti-tumour activities.

The sacred plant B. sensitivum is one among the

Dashapushpam in the traditional culture of Kerala, is a

potent medicinal plant. It is having the radio-protective

activity, immune modulatory action, cardio protective,

antitumor activity, wound healing activity, etc. [23-25].

MATERIALS AND METHODS

The Plant Elephatopus scaber Linn, collected from the

southernmost part of India, the Kanyakumari District of

TamilNadu at coordinates 8°18'8"N 77°10'34"E, and

Biophytum sensitivum Dc, collected from Mahe region at

coordinates 11°42′4″N 75°32′12″E, located in Kerala

under U.T. of Puducherry, India, are washed well in pure

water and shadow dried. The dried leaves are separated

and powdered using mortar and pestle. The pet-ether

extract of the sample was obtained by adding the plant

powder with pet-ether in the ratio of 1gm for 10ml and

extracting for 48 hours using Soxhlet apparatus. The

solvent is concentrated at temperature below 40oC and

the resulting extracts are used for determination of

flavanoids, phenols and free radical scavenging activity.

Phytochemical screening

The Phytochemical studies are carried out using the

freshly prepared pet-ether extracts of these plant leaves

to detect the presence of stereoids, terpenoids, tannins,

flavonoids, saponins, glycosides, amino acids etc., using

standard procedures[26-30].

Test for Flavonoid

Alkaline reagent test: Extracts are treated with few drops

of sodium hydroxide solution. Formation of intense

yellow colour, which becomes colourless on addition of

dilute acid, indicates the presence of flavonoids.

Flavanoids are found to be useful in disease resistance

[31]. They are hydroxylated phenolic and they have been

found to be antimicrobial substances against wide array

of microorganisms in vitro. Their activity is probably

due to their ability to complex with extracellular and

soluble proteins and to complex with bacterial cell wall

[32]. They also are effective antioxidant and show strong

anticancer activities [33-35].

Test for carbohydrates

Molisch’s test: Filtrates are treated with 2 drops of

alcoholic α- naphthol solution in a test tube. Formation

of the violet ring at the junction indicates the presence of

carbohydrates.

Systematical reduction of the amount of dietary

carbohydrates could suppress, or at least delay the

emergence of cancer, and that proliferation of already

existing tumor cells could be slowed down[36], but the

cell surface carbohydrates are acting as tumor

suppressors.

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S. Santhosh Kumar *

et al; ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR ……………

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Test for alkaloids

Dragendroff’s test:

Filtrates are treated with Dragendorffs’ reagent (solution

of potassium bismuth iodide). Formation of red

precipitate indicates the presence of alkaloids.

Alkaloids are known to play some metabolic roles and

control development in living system [37]. These have

protective role in human and animal and it is used in

medicine especially the steroidal alkaloids which

constitute most of the valuable drugs. One of their

biological properties is cytotoxicity [38]. Alkaloids also

have analgesic [39, 40], antispasmodic and antibacterial

[41, 42] properties.

Test for saponin

Froth test: Extract aere diluted with distilled water to

20ml and this was shaken in a graduated cylinder for 15

minutes. Formation of 1cm layer of foam indicates the

presence of saponins.

Saponins, which are known to produce inhibitory effect

on inflammation [43] and has the property of

precipitating and coagulating red blood cells. Crude

saponins are having inhibition of glucose transport.

Glycosides are known to lower the blood pressure [44].

Steroids have been reported to have antibacterial

properties [45] and are important especially due to their

relationship with compounds such as sex hormones [46].

Plant sterols could act as a natural preventive dietary

product [47]. It helps in the lowering of plasma

cholesterol and LDL (low-density lipoprotein)

cholesterol. Hence, it will assist in reducing drastically

the morbidity and mortality caused by cardiovascular

disease.

Plant extracts containing alkaloids, flavanoids, phenolic

and/or terpenes showing active trypanocidal activity

[48]. As per Sofowora [26], these compounds are potent

bioactive compounds that could be used for therapeutic

purpose or which are precursors for the synthesis of

useful drugs. The phenolic compounds are one of the

largest and most ubiquitous groups of plant metabolites

[49]. They possess biological properties such as anti-

apoptosis, anti-aging, anti-carcinogen, anti-

inflammation, anti-atherosclerosis, cardiovascular

protection and improvement of endothelial function, as

well as inhibition of angiogenesis and cell proliferation

activities [18]. The phenols are reported to inhibit alpha-

amylace, sucrose, as well as the action of sodium

glucose-transporter 1 (SGLUT-1) of the intestinal brush

border. The polyphenol compound has antimicrobial and

antifungal effect and has been used in disinfections.

Determination of antioxidant activity

The pet-ether fractions of the two plants, E. scaber and

B. sensitivum, and the ethanolic fractions of B.

sensitivum were screened for free radical scavenging

activity against DPPH, reducing power and Nitric oxide

radical at different concentrations. All determinations

were performed in triplicate. The percentage inhibition

of the DPPH radical by the sample was calculated as

follows [2, 50],

Absorbance of Control- Absorbance of Sample

% DPPH. Scavenging = 100 x --------------------------------------------------------------

Absorbance of Control

Where Absorbance of Control is the absorbance in absence of standards or extracts and Absorbance of Sample is the

absorbance in presence of standards or extracts.

The DPPH radicals are widely used to investigate the

scavenging activity of some natural compounds. The

antioxidant activity of the plant extracts and the standard

was assessed on the basis of the radical scavenging

effect of the stable 1, 1,-diphenyl-2-picryl- hydrazyl

(DPPH) - free radical activity by modified method[51].

The diluted working solutions of the test extracts are

prepared in pet-ether. Ascorbic acid is used as standard

in 100-500 µg/ml solution. 0.002% of DPPH was

prepared in pet-ether and 1ml of this solution was mixed

with 1ml of sample solution and standard solution

separately. These solution mixtures were kept in dark for

30 min and optical density was measured at 517 nm

using Amile photo calorimeter.

In the DPPH assay, the antioxidants were able to reduce

the stable radical DPPH to the yellow coloured diphenyl-

picrylhydrazine, resulting a colour change from purple to

yellow. The absorbance decreased when the DPPH was

scavenged by an antioxidant through donation of

hydrogen to form a stable DPPH molecule. In the radical

form, the molecule had an absorbance at 517nm which

disappeared after acceptance of an electron or hydrogen

radical from an antioxidant compound to become a

stable diamagnetic molecule [52]. This method is based

on the reduction of alcoholic DPPH solution in the

presence of a hydrogenating antioxidant due to the

formation of the non- radical form (DPPH-H) in the

reaction [53]. The experiment was repeated for three

times. Ascorbic acid was used as standard control. IC50

values denote the concentration of sample, which is

required to scavenge 50% of DPPH free radicals.

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S. Santhosh Kumar *

et al; ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR ……………

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Reducing power determination Yen and Chen [54] method was followed to determine

the reducing power. Different amounts of extracts (100-

500 μg/ ml) in water were mixed with phosphate buffer

(2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide

[K3Fe (CN) 6] (2.5 ml, 1%). The mixture was incubated

at 50oC for 20 min. and the reaction was stopped by

adding 2.5 ml of trichloroacetic acid (10%) and then it

was centrifuged at 3000 rpm for 10 min. 2.5ml of the

upper layer solution was mixed with equal quantity of

distilled water and 0.5ml of FeCl3, and the absorbance

was measured at 700nm[55]. Increased absorbance of the

reaction mixture indicated increased reducing power.

Ascorbic acid is used as positive control.

Nitric oxide-scavenging activity This procedure is based on the principle that, sodium

nitro-prusside in aqueous solution at physiological pH

spontaneously generates nitric oxide which interacts with

oxygen to produce nitrite ions that can be estimated

using Griess reagent. Scavengers of nitric oxide compete

with oxygen, leading to reduced production of nitrite

ions. For the experiment, sodium nitroprusside (10 mM),

in phosphate-buffered saline, was mixed with different

concentrations of each extracts dissolved in water and

incubated at room temperature for 60 minutes. After the

incubation period, 0.5 ml of Griess reagent was added.

The absorbance of the chromophore formed was read at

540nm [55]. Ascorbic acid is used as positive control.

RESULTS Phytochemical analysis

The phytochemical constituents of E.scaber and

B.sensitivum are analysed using the pet-ether fraction of

the samples. Many studies have been conducted in

E.scaber leaf [56] with benzene, chloroform, and

methanol extracts, but pet-ether fraction shows the

presence of alkaloids, tannins, saponins, flavanoids,

phenolic copmpounds and carbohydrates. The

B.sensitivum also shows the presence of such compounds

(Table.1)

In vitro antioxidant activity

DPPH- radical scavenging activity

DPPH is a stable nitrogen-centered free radical, the color

of which changes to yellow upon reduction by either the

process of hydrogen- or electron- donation. Substances

which are able to perform this reaction can be considered

as antioxidants and therefore radical scavengers [57]. It

is found that the radical-scavenging activities of all the

extracts increased with increasing concentration (Fig.1).

Among the three extracts of two plants, ethanol extract

of E. scaber showed the best activity, i.e., IC50 = 517.56

g/ml. IC50 for DPPH radical scavenging activity is

reported in figures plotted separately (Figs. 2-5).

Reducing power Fe (III) reduction is often used as an indicator of electron

donating activity, which is an important mechanism of

phenolic antioxidant action [58]. In the reducing power

assay, the presence of antioxidants in the samples would

result in the reducing of Fe3+

to Fe2+

by donating an

electron. Increasing absorbance at 700 nm indicates an

increase in reductive ability. Fig. 6 shows dose-response

curves for the reducing powers of the samples. It is

found that the reducing powers of all the samples

increased with their concentrations. Ethanol extract of

B.sensitivum and pet-ether extract of E. scaber show

good reducing power at lower concentrations compared

to ascorbic acid.

Nitric oxide-scavenging activity The % inhibition was increased with increasing

concentration of the extracts (Fig.7). The ethanol extract

of B.sensitivum showed better reducing power than other

extracts. However, activity of ascorbic acid is very more

pronounced than that of the samples at the concentration

>300g/ml. In addition to reactive oxygen species, nitric

oxide is also implicated in inflammation, cancer and

other pathological conditions [58].

Antioxidants scavenge for free radicals and consequently

are a very special group of nutritional supplements. The

free radicals are all along present in the body, due to the

occurrences of biochemical process, but it is harmful if it

exceeds the normal level. Antioxidants are substances

with free radical chain breaking properties. Among the

numerous antioxidants available, flavonoids are known

to inhibit lipid peroxidation to scavenge free radicals and

active oxygen to chelate iron ion and to inactive

lipoxygenase [59].

Table.1 Phytochemical constituents of E.scaber, and B.sensitivum.

Compound E.Scaber B.Sensitivum

Benzene* Chloroform* Methanol* Pet-ether Pet-ether

Alkaloids -- -- -- + +

Tannins -- + + + +

Saponins -- -- -- + +

Flavonoids -- -- + + +

Phenolic

compound -- -- + + +

Carbohydrate -- -- -- + +

* From Ref.[56].

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S. Santhosh Kumar *

et al; ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR ……………

International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS) | JAN-MAR 2014 | VOLUME 3 | ISSUE 1 | 06-17| www.ijpbcs.net

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Fig.1. DPPH radical scavenging activity of the pet-ether extract of E.scaber leaves

and pet-ether & ethanol extract of leaves compared with standard, Ascorbic acid.

Fig.2. IC50 value of ethanol extract of B.sensitivum.

The IC50 value marked in figure is obtained through linear fitting of the curve.

100 200 300 400 500

0

10

20

30

40

50

60

70

80

90

100

% in

hib

itio

n

Concentration (gm)

B. Sensitivum

E. Scaber

B. Sensitivum (Ethanol)

Ascorbic acid

100 200 300 400 500

0

10

20

30

40

50Linear Regression

Y = A + B * X

Parameter Value Error

-----------------------------------------

A -4.849 4.37839

B 0.10605 0.0132

-----------------------------------------

R SD N P

-----------------------------------------

0.97754 4.17463 5 0.00403

-----------------------------------------

IC50

=517.560

% in

hib

itio

n

Concentration(gm/ml)

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S. Santhosh Kumar *

et al; ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR ……………

International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS) | JAN-MAR 2014 | VOLUME 3 | ISSUE 1 | 06-17| www.ijpbcs.net

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Fig.3. IC50 value of pet-ether extract of B.sensitivum.

The IC50 value obtained is marked in the figure.

Fig.4. IC50 value of pet-ether extract of E.scaber. Linear interpolation is used to obtain the IC50 value.

100 200 300 400 500

0

10

20

30

40

50

60

70Linear Regression

Y = A + B * X

Parameter Value Error

-------------------------------------------

A -7.457 5.24374

B 0.14727 0.01581

-------------------------------------------

R SD N P

-------------------------------------------

0.983 54.99971 5 0.00262

-------------------------------------------

IC50

=393.99

% in

hib

itio

n

concentration(gm/ml)

100 200 300 400 500

0

10

20

30

40

50

60

70

80Linear Regression

Y = A + B * X

Parameter Value Error

----------------------------------------

A -0.578 4.64138

B 0.14807 0.01399

----------------------------------------

R SD N P

----------------------------------------

0.98686 4.42538 5 0.0018

----------------------------------------

IC50

=339.316

% inh

ibitio

n

Concentration(gm/ml)

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S. Santhosh Kumar *

et al; ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR ……………

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Fig.5. IC50 value of the standard, Ascorbic acid.

Fig.6. Reducing power of B.sensitivum and E.scaber. Ascorbic acid is used as control.

100 200 300 400 500

30

40

50

60

70

80

90

100Linear Regression

Y = A + B * X

Parameter Value Error

-------------------------------------

A 16.61072 3.37404

B 0.15592 0.01017

------------------------------------

R SD N P

------------------------------------

0.99368 3.21702 5 6.032E-4

------------------------------------

IC50

= 217.5836

%in

hib

itio

n

Concentration(g/ml)

100 200 300 400 500

0

100

200

300

400

500

% inh

ibitio

n

Concentration(g/ml)

B.sensitivum (pet.ether)

B.sensitivum (ethanol)

E.scaber

Ascorbic acid

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S. Santhosh Kumar *

et al; ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR ……………

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Fig.7. Nitric oxide scavenging activity of pet-ether extract of E.scaber, pet-ether

& ethanol extract of B.sensitivum and standard (Ascorbic acid).

Fig.8. Comparision of IC50 vales of different extracts of the plants E.scaber and

B.sensitivum and ascorbic acid.

DISCUSSION

In the present paper we have evaluated the free radical

scavenging activity, of E.scaber and B.sensitivum. The

crude extracts of these plants showed antioxidant

activity, of which B.sensitivum Dc. showed a weak

value. It has been shown that the scavenging effects on

the DPPH radical increases sharply with the increasing

concentration of the samples and standards to a certain

100 200 300 400 500

0

20

40

60

80

100

120

140

160

180

200

B. Sensitivum

E. Scaber

B. Sensitivum (Ethanol)

Ascorbic acid

% in

hib

itio

n

Concentration (gm)/ml

E.scaber B.sens (P) B.sens(E) Ascorbic Acid

0

100

200

300

400

500

IC50

Samples

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S. Santhosh Kumar *

et al; ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR ……………

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extent [60] and hence are said to be strongly dependent

on the phenols and antioxidant activity. The radical

scavenging activities of the selected plant extracts are

still less effective than the commercial available

synthetic like BHT, as the plant extracts are quite safe

and their toxicity is a not a problem of concern unlike

those of BHT, they could be exploited as antioxidant

additives or as nutritional supplements.

The results of the present study show that the extract of

E.scaber contains the highest amount of flavonoids and

alkaloids, which corresponds to higher nitric oxide

activity. Therefore the plant can be further studied for

novel antioxidant compounds, which is very well

evidenced by the present work. However , the chemical

constituents present in the extract, which are responsible

for this activity, need to be investigated, and it is obvious

that the constituents like alkaloids, tannins, saponins, and

flavanoids present in the extract may be responsible for

such activity. Several of such compounds are known to

possess potent antioxidant activity [17]. Some of these

constituents have already been isolated from these

plants. Hence, the observed antioxidant activity may be

due to the presence of any of these constituents. The

plants exhibited strong anticancer, hepato protective,

antiviral and several other activities which may be due to

its antioxidant activity. The crude pet ether extracts

merits further experiments in vivo. The DPPH

scavenging activity IC50 values (Fig.2-5) show a higher

inhibition of ethanol extract (IC50 = 517.560; Fig.2) of

B.sensitivum than its pet-ether extract (IC50 = 393.999;

Fig.3) which are still higher than the pet-ether extract

(IC50 = 339.316; Fig.4) of E.scaber. The standard,

ascorbic acid shows only IC50=217.5836 (Fig.5).

The order of DPPH radical scavenging activity is,

ethanol fraction of B.sensitivum, pet.ether fraction of

B.sensitivum and E.scaber and then ascorbic acid

(Fig.8). Ganga Rao et al.[61] extensively studied the

antioxidant and radical scavenging activity of E.scaber

using hydro alcohol, methanol, ethyl acetate and hexane

extracts. In this paper we have taken the pet.ether extract

of E.scaber for the study of radical scavenging activity.

The DPPH radical scavenging activity of this plant

shows a higher value at 50% inhibition than the other

extracts, compared to Ganga Rao et al.[61].

Increasing absorption at 700nm indicates an increase in

reductive ability. Fig.6 shows the reducing powers of the

extracts. It is found that the reducing power increased

with the increase of their concentrations. The pet-ether

extract had shown a good reducing power compared to

ascorbic acid, but at concentrations above 300g/ml, all

the fractions showed good reducing powers. And the

ethanol fraction of B.sensitivum showed a smooth

increase with concentration.

The nitric oxide scavenging capacity (Fig.7) also shows

IC50 value is higher for ethanol extract (IC50 = 681.361)

of B.sensitivum than its pet-ether extract (IC50 = 216.666)

followed by the pet-ether extract (IC50 = 158.207) of

E.scaber.

In very recent articles [23, 62] on B. sensitivum reports

only the usage of aqueous and methanolic extracts for its

study, but pet-ether and ethanol extract give additional

information about the chemical constituents of this plant

and hence possess higher antioxidant activity.

CONCLUSION

In conclusion, even though different extracts exhibited

different levels of antioxidant activities in different

models studied, ethanol and pet-ether extracts, in general

show a good result of antioxidant activity than any other

fraction and hence this may be the first report of these

plants on pet-ether and ethanol extract. The B.Sensitivum

can be replaced with E.scaber only for its phytochemical

constituents and its availability throughout the year . But

in the context of antioxidant and free radical scavenging

capacity ethanol and pet-ether extracts of B.Sensitivum

values higher than E.scaber. But further studies are

needed to identify the unknown antioxidant components

to establish their pharmacological properties.

ACKNOWLEDGEMENT

This research was partially supported by the UGC-DAE

Consortium for Scientific Research, Kolkata Center,

through a facility utilization project.

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S. Santhosh Kumar *

et al; ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF ANNULAR ……………

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*Corresponding author address:

S. Santhosh Kumar

Department of Physics,

Avvaiyar Government College for Women,

Karaikal – 609 602, U.T of Puducherry, India,

Telephone: 094864-59600

E-mail: [email protected]