1. INTRODUCTION:

Free radicals represent a class of highly reactive intermediate chemical entities, containing one or more unpaired electrons due to which they are highly unstable and thus they cause damage to other molecules by extracting electrons from them in order to attain stability¹.

The examples of free radicals are superoxide anions, hydroxyl and hydrogen peroxide radicals. They are often generated as byproducts of oxidative damage to the DNA molecules, lipids and proteins. This damage could lead to several human diseases such as diabetes mellitus, cancer, atherosclerosis, arthritis, anemia, asthma, inflammation and neurodegenerative disorders².If free radicals are not inactivated, their chemical reactivity can damage all cellular macromolecules including proteins, carbohydrates, lipids and nucleic acids³.

Antioxidants are the substances that delays, prevents or removes oxidative damage to a target molecule. Physiological role of these compounds is to protect our body from diseases by reducing the oxidative damage to cellular components caused by free radicals⁴. The antioxidants can interfere with the oxidation process by reacting with free radicals, chelating free catalytic metals and also by acting as oxygen scavengers. Recent investigations have shown that plant based antioxidants have great therapeutic importance due to fewer side effects and could be correlated with oxidative stress defense⁵.

Actinodaphne madraspatana (Lauraceae) is endemic to India. It is occasional in forests in peninsular India. It is common on rocky hill slopes, at higher elevations. It is commonly known as Kovangutti (Telugu) and as Putta thali (Tamil). Trade name of this plant is Ray Laurel. It is a medium sized tree, branchlets are pubescent. Leaves are elliptic to lanceolate, coriaceous and shining above. Flowers are dioecious, brownish- yellowish, in axillary or lateral dense clusters. Male flowers occur in clusters and female flowers are umbellate. Fruits are ellipsoidal berries. Propagation is by seeds^{6- 8}.

Leaves, flowers and fruits constitute the drug. Leaves are useful for curing diabetes, clearing wounds and acts as a stimulant. It is a precursor of Vitamin A. Flower is reported to be used in curing mania and fickle minded behaviour^6-8.

MATERIALS AND METHODS:

Collection and Authentication of plant material:

The leaves of Actinodaphne madraspatana were collected from native species growing in deciduous forests of Tirumala region, A.P, India. The leaves have been identified taxonomically and authenticated by Dr. S. Madhava Chetty, Associate Professor, Department of Botany, Sri Venkateswara University, Tirupathi, A.P, India.

Preparation of extracts:

The freshly collected leaves of Actinodaphne madraspatana (AM) were shade dried and coarsely powdered and passed through sieve no. 40. The sieved powder was stored in airtight container and kept in room temperature. Dried plant material (500gm) was subjected to cold maceration with petroleum ether for about 48 hours. Cold maceration was carried out in a closed conical flask, after defatted, the extracted material was separated by using filter and the marc was dried in hot air oven at 50⁰C. Packed in Soxhlet apparatus and further extracted with different solvents like methanol (MEAM), ethyl acetate (EAEAM), chloroform (CEAM) and aqueous (AEAM) exhaustively. The solvents were removed from the extracts under reduced pressure by using rotary vacuum evaporator.

Preliminary phytochemical screening:

The preliminary phytochemical screening was carried out for the detection of phytoconstituents. Tests for the presence of common phytochemicals were carried out by the standard methods^9,10.

Chemicals and reagents:

Diphenyl 2- picryl hydrazine (DPPH), ascorbic acid, potassium ferricyanide, trichloro acetic acid (TCA), ferric chloride, sodium nitroprusside and all other chemicals including solvents used were of analytical grad and procured from Sigma- Aldrich pvt.ltd, India.

Experimental procedure:

In-vitro Antioxidant activity:

DPPH (Diphenyl 2-Picryl Hydrazine) free radical scavenging activity:

The DPPH free radical scavenging activity of MEAM, EAEAM, CEAM and AEAM was determined according to the method of Blois¹¹. One ml of each extract in different concentrations (5, 10, 15, 25, 50, 75, 100 and 125 µg/ml) was added to one ml of 0.1 mM solution of DPPH in methanol. Similarly one ml of ascorbic acid solution (reference compound) in different concentrations (5, 10, 15, 25, 50, 75, 100 and 125 µg/ml) was added to one ml of 0.1 mM solution of DPPH in methanol. After 30 minutes absorbance was measured at 517 nm using a spectrophotometer. A 0.01 mM solution of DPPH in methanol was used as control. All tests were performed in triplicate. Percent inhibition was calculated using the following equation:

Absorbance_control – Absorbance_test

% Inhibition = ------------------------------------------ X 100

Absorbance _control

The effective concentration of sample required to scavenge DPPH radical by 50% (IC₅₀ value) was obtained by linear regression analysis of dose-response curve plotting between % inhibition and concentrations.

Nitric Oxide (NO) free radical scavenging method:

Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacted with oxygen to produce nitrite ions which were measured using the Griess reaction¹². To 2 ml of each MEAM, EAEAM, CEAM and AEAM in various concentrations (5, 10, 15, 25, 50, 75, 100 and 125 µg/ml) 3 ml of 10 mM sodium nitroprusside in phosphate buffer solution was added. Similarly to 2 ml of ascorbic acid (reference compound) solution in different concentrations (5, 10, 15, 25, 50, 75, 100 and 125 µg/ml) 3 ml of 10 mM sodium nitroprusside in phosphate buffer solution was added. The resulting solutions were then incubated at 25⁰C for 60 minutes. A similar procedure was repeated with methanol as a blank, which served as control. To 5 ml of incubated sample, 5 ml of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene Diamine dihydrochloride in 2% H₃PO₄) was added. The absorbance of the chromophore formed was measured using a spectrophotometer at 546 nm. All tests were performed in triplicate. The IC₅₀and R² valuefor each test compound as well as that of standard preparation were calculated.

Percent inhibition was calculated using the following equation:

Absorbance_control – Absorbance_test

% Inhibition = --------------------------------------- X 100

Absorbance _control

Reducing power assay:

Principle: This method is based on the principle that substances which have reduction potential, react with potassium ferricyanide (Fe³⁺) to form potassium ferrocyanide (Fe²⁺), which then reacts with ferric chloride to form ferric ferrous complex that has an absorption maximum at 700 nm. Increase in the absorbance indicates an increase in the antioxidant activity. This method is described by Oyaizu (1986)¹³.

Procedure: 2.5 ml of 0.2M phosphate buffer (pH 6.6) and 2.5 ml of Potassium ferricyanide [K₃Fe (CN)₆] (1% w/v) are added to 1.0 ml of sample dissolved in distilled water. The resulting mixture is incubated at 50⁰C for 20 min, followed by the addition of 2.5 ml of Trichloro acetic acid (10% w/v). The mixture is centrifuged at 3000 rpm for 10 min to collect the upper layer of the solution (2.5 ml), mixed with distilled water (2.5 ml) and 0.5 ml of Ferric chloride (FeCl3) (0.1%, w/v). The absorbance is then measured at 700 nm against blank sample.

RESULTS:

The preliminary phytochemical investigations of methanolic, ethyl acetate, chloroform and aqueous extracts of leaves of Actinodaphne madraspatana have revealed the presence of various bioactive principles such as flavonoids, tannins, steroids, saponins, alkaloids, glycosides, carbohydrates and proteins.

DPPH free radical scavenging activity of MEAM, EAEAM, CEAM and AEAM and their comparison with that of Ascorbic acid is presented in the Figure-1. IC₅₀ and R² values of all the extracts and the standard in DPPH method were also calculated and presented in Table-1.

Nitric Oxide (NO) free radical scavenging activity of all the leaf extracts of AM and that of Ascorbic acid is presented in the Figure-2. Their IC₅₀ and R² values were also calculated and presented in Table-1.

Reducing power assay was performed for MEAM, EAEAM, CEAM, AEAM and ascorbic acid and their antioxidant activity is presented in Figure-3.

In the three in-vitro antioxidant methods, all the leaf extracts of Actinodaphne madraspatana and the standard (Ascorbic acid) exhibited a dose dependent inhibition i.e, as the concentration increased an increase in the % inhibition was also observed which is evident from the figure-1, 2 and 3. All the extracts and the ascorbic acid (standard) exhibited highest % inhibition at a concentration of 125µg/ml. Among all the extracts MEAM has greater antioxidant activity which was almost similar to that of the standard whereas EAEAM, CEAM and AEAM exhibited lesser antioxidant activity comparatively.

Figure-1: Comparison graph of AM extracts- DPPH method

Figure-2: Comparison graph of AM extracts- Nitric Oxide method

Figure-3: Comparison graph of AM extracts- Reducing Power Assay

Table-1: IC₅₀ and R² values- DPPH & Nitric Oxide methods

Extract	DPPH method		Nitric Oxide method
Extract	IC₅₀	R²	IC₅₀	R²
Ascorbic acid	14.190	0.9983	17.517	0.9989
MEAM	14.728	0.9976	15.404	0.9941
EAEAM	107.650	0.9794	19.312	0.9947
CEAM	128.451	0.9966	32.301	0.9956
AEAM	61.919	0.9855	19.724	0.9913

DISCUSSION:

DPPH is relatively stable nitrogen centered free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecule. DPPH radicals react with suitable reducing agents as a result of which the electrons become paired off forming the corresponding hydrazine. The solution therefore loses the colour stoichometrically depending on the number of electrons uptake. From the results of the present study it may be postulated that active principles in the leaf extracts of AM have hydrogen donors thus scavenging the free radicals.

Nitric oxide (NO) is an important chemical mediator generated by the endothelial cells, neurons involved in the regulation of various physiological processes. Oxygen reacts with the excess NO to generate nitrite and peroxynitrite anions, which acts as free radicals. In the present study, the nitrite produced by the incubation of solutions of sodium nitroprusside in standard phosphate buffer at 25⁰C was reduced by the leaf extracts of Actinodaphne madraspatana. This may be due to the antioxidant principles in the leaf extracts, which compete with oxygen to react with nitric oxide there by inhibiting the generation of nitrite. In the present study, the leaf extracts of Actinodaphne madraspatana exhibited free radical inhibition but less than ascorbic acid in scavenging NO.

In Reducing power assay, the leaf extracts of Actinodaphne madraspatana reacted with potassium ferricyanide (Fe³⁺) to form potassium ferrocyanide (Fe²⁺), which then reacted with ferric chloride to form ferric ferrous complex that has an absorption maximum at 700 nm. This may be due to the antioxidant principles in the leaf extracts. In the present study, thus the leaf extracts of Actinodaphne madraspatana have exhibited the antioxidant activity.

CONCLUSION:

From the results of the present study it can be concluded that the methanolic, ethyl acetate, chloroform and aqueous extracts of leaves of Actinodaphne madraspatana possess antioxidant activity. Among all the extracts MEAM exhibited greater antioxidant activity which was almost equal to that of the standard (Ascorbic acid). However, further pharmacological and biochemical investigations are to be undertaken to elucidate the mechanisms of antioxidant activity of Actinodaphne madraspatana.

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Received on 24.12.2013 Modified on 20.01.2014

Res. J. Pharmacognosy & Phytochem. 6(1): Jan.-Mar. 2014; Page 01-04