1. INTRODUCTION:

Cells under aerobic conditions are threatened to the insult of reactive oxygen metabolites (ROMs) that are efficiently taken care by the powerful antioxidant system in the human body. The term antioxidant can be defined as any substance that delays or inhibits oxidative damage to a target molecule. Antioxidant enzymes, together with the substances that are capable of either reducing ROMs or preventing their formation, forms buffers which have strong reducing properties which effects the capability of the cell to counteract the activation of ROM. All reducing agents thereby form protective mechanisms, which maintain the lowest possible levels of ROMs inside the cell. The first line of defense against O₂.- and H₂O₂ mediated injury are antioxidant enzymes like SOD and CAT¹. Reactive oxygen species (ROS), such as superoxide anions, hydrogen peroxide, and hydroxyl, nitric oxide and peroxynitrite radicals, play an important role in oxidative stress related to the pathogenesis of various important diseases^2,3.

In healthy individuals, the production of free radicals is balanced by the antioxidative defense system; however, oxidative stress is generated when equilibrium favors free radical generation as a result of a depletion of antioxidant levels. Cancer chemoprevention by using antioxidant approaches has been suggested to offer a good potential in providing important fundamental benefits to public health, and is now considered by many clinicians and researchers as a key strategy for inhibiting, delaying, or even reversal of the process of carcinogenesis^4,5. Moreover, knowledge and application of such potential antioxidant activities in reducing oxidative stresses in vivo has prompted many investigators to search for potent and cost-effective antioxidants from various plant sources. These research activities have contributed to new or renewed public interests worldwide in herbal medicines, health foods, and nutritional supplements. Botanicals have been used for treatment or prevention of various human diseases throughout history. The cancer chemopreventive activities of naturally occurring phytocompounds is of great interest. Many indigenous herbal plants of regional interest have been used popularly as folk medicines in Asian; however, their bioactivities or pharmacological effects are remained to be elucidated.

Sesbania grandiflora (family: Fabaceae) is known as agati or the hummingbird tree (or scarlet wisteria), a small tree believed to have originated either in India or Southeast Asia and grows primarily in hot and humid tropical areas in the world. A native to Asian countries such as India, Malasia, Indonesia and the Philippines where it is commonly seen growing on the dikes between rice paddies, along roadsides and in backyards vegetable gardens⁶. The whole plant contains Grandifloral, arginine, cystine, histidine, isolucine, phenylalanine, tryptophan, valine, threonine, alanine, aspargine, aspartic acid and a saponin yielding oleanolic acid, galactose, rhamnose and glucuronic acid^{7,8, 9} and it also contains flavonol glycoside, kaempfrol^7,10. The root-bark of the red-flowered variety is useful in vitiated condition of vata and arthralgia. The bark is astringent, cooling, bitter, tonic, anthelmintic and febrifuge. The pounded bark is externally applied to cure scabies. The juice of the bark is good for dyspepsia, diarrhea and gastralgia¹¹. The leaves are acrid, bitter, sweet, cooling, aperient, tonic and diuretic and contain a non-poisonous saponine like substance. The leaf juice is used is nasal catarrh¹², nyctalopia and cephalagia. Leaves are chewed to disinfect mouth and throat and are useful in stomatalgia⁸. The flowers are cooling, bitter, astringent, acrid and antipyretic. The juice of the flowers is applied to the eyes for nyctalopia and is used for intermittent fevers. The fruits are sweet, bitter, laxative and alexiteric and are useful in flatulent-colic, astringent, cooling, bitter, tonic, anthelmintic, febrifuge, cure scabies, dyspepsia, diarrhea and gastralgia, astringent, antipyretic, for nyctalopia naemia, emaciation and vitated conditions of tridosa⁶.Based on the above medicinal properties of Sesbania grandiflora, in this study, we investigated the presence of bioactive compounds and antioxidant activity of different extracts of plant leaves.

2. MATERIALS AND METHODS:

2.1. PLANT MATERIAL

The leaves of Sesbania grandiflora were collected from Tripura, India in the month of October 2012. The plant was authenticated by Dr. A.P. Singh, Principal Investigator, Weed Control, Dept. of Agronomy, College of Agriculture, Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhattisgarh, India. A voucher specimen of the plant was preserved in the herbarium for further reference (Agro/WC/13/252).

2.2. PREPARATION OF THE EXTRACT

Leaves of Sesbania grandiflora were washed under running tap water and dried in shade for seven days. Dried leaves were mechanically reduced to a coarse powder and then sieved and stored in an air tight container at room temperature. The extraction method was based on the presence of active constituents in the drug, using various solvents ranging from non-polar to polar. Dried powder (500 g) was extracted sequentially with hexane, dichloromethane, ethyl acetate, ethanol and distilled water by using soxhlation method. The extracts were concentrated to dryness by distilling the solvent at low temperature using rotary evaporator. The extracts were preserved in refrigerator at 4^OC.

2.3. PHYTOCHEMICAL SCREENING

All the extracts (hexane, dichloromethane, ethyl acetate, ethanol and aqueous) obtained by successive soxhlet extraction method from the powdered leaves of Sesbania grandiflora were subjected to various qualitative tests for the identification of presence of active phytoconstituents like alkaloids, glycosides, flavonoids, tannins and phenolic compounds, steroids, terpenoids, proteins, carbohydrates, saponins, fixed oil and fats^13,14.

2.4. IN VITRO ANTIOXIDANT ACTIVITY

2.4.1. DPPH Method (Hydrogen-Donating Activity)¹⁵

Chemicals: DPPH(2,2-Diphenyl-1-picryl-hydrazyl), DMSO (dimethyl sulphoxide), methanol.

Preparation of test and standard solutions

Each of the extract and the standards were dissolved in DMSO to prepare 1000 µg/ml solution. From the above stock solution further dilutions were made to get different dilutions such as 50 µg /ml, 100 µg /ml, 150 µg /ml, 200 µg /ml, 250 µg /ml.

Preparation of DPPH stock solution

Weighed 21 mg of DPPH, placed in 100 ml volumetric flask, added methanol, and made up to the mark.

Procedure

This assay was used in many studies for testing antioxidant activity. 2,2-diphenyl-1-picryl-hydrazil stable radical (DPPH) evidently offers a convenient and accurate method for titrating the oxidizable groups of natural and synthetic antioxidants. This assay was based on the reduction of a methanolic solution of the colored free radical DPPH by free radical scavenger. The degradation of DPPH was evaluated by comparison with a control sample without hydrogen-donating compounds. The decrease in absorbance of DPPH at its absorbance maximum of 517 nm was proportional to the concentration of free radical scavenger added to DPPH reagent solution. Lower absorbance of reaction mixture indicated higher antioxidant activity.

In this experiment methanolic solution of DPPH (100 mM, 2.95 ml), 0.05 ml of each extracts dissolved in methanol was added at different concentrations (50-250 µg/ml). Reaction mixture was shaken and after 30 min at room temperature, the absorbance values were measured at 517 nm and converted into percentage of antioxidant activity (% AA). Ascorbic acid was used as standard. The degree of discoloration indicates the scavenging efficacy of the extract, was calculated by the following equation:

% AA = 100 – {[(Abs_sample – Abs_blank) x 100] / Abs_DPPH}

IC₅₀ was calculated by plotting the graph, taking % inhibition on y, axis and concentration on x, axis.

2.5. ESTIMATION OF TOTAL PHENOLIC CONTENT

Total phenolic content present in the extractives was measured by Folin-Ciocalteau assay (FC) using Gallic acid as the calibration standard to express results in mol-1 or Gallic acid equivalents (GAE)¹⁶.

Procedure

400 µl of each of the extracts (1 mg/ml to 0.5 mg/ml) was separately mixed with 2 ml of Folin-Ciocalteu reagent and 1.6 ml of sodium carbonate. After shaking, it was kept for 2 h reaction time. The absorbance was measured at 738 nm in a spectrophotometer. Using Gallic acid monohydrate, standard curve was prepared. Total phenol content of the extracts was calculated from the standard curve and expressed in terms of Gallic acid equivalent in mg/gm or % w/w of the extracts.

Table1. Phytochemicals present in different extracts of Sesbania grandiflora(L.) leaves.

	Phytoconstituents	Chemical tests	Hexane Extract	Dichloro methane	Ethyl acetate	Ethanol	Aqueous
1	Alkaloids	Dragendorff’s test	-	+	+	+	+
		Mayer’s test	-	+	+	+	+
		Hager’s test	-	-	-	+	+
		Wagner’s test	-	-	-	+	+
2	Glycoside	Legal’s test	-	-	-	-	-
		Baljet’s test	-	-	-	-	-
		Keller-Killani test	-	-	-	-	-
		Borntrager’s test	-	-	-	-	-
		Modified Borntrager’s	-	+	+	-	-
3	Flavonoid	Shinoda’s Test	-	-	-	+	+
		Sulfuric acid test	-	-	-	+	+
		Alkaline Reagent	-	-	-	+	+
		Lead Acetate Test	-	-	-	+	+
4	Tannins and phenolic compounds	With lead acetate	-	-	-	+	+
		With FeCl₃	-	-	-	+	+
		With KMnO₄	-	-	-	+	+
		With dilute HNO₃	-	-	-	+	+
		Gelatin Test	-	-	-	+	+
5	Steroids	Salkowski test	+	-	+	+	+
5	Steroids	Liebermann-Burchard reaction	+	-	-	+	+
6	Terpenoids	With H2SO4	+	-	-	+	+
7	Proteins	Biuret test	-	-	+	+	+
		Ninhydrin test	-	-	+	+	+
		Millon’s test	-	-	-	+	+
		Xanthoproteic test	-	-	-	+	+
		Sulpher Test	-	-	-	+	+
		Precipitation test	-	-	-	+	+
8	Carbohydrates	Molisch’s test	-	+	-	+	+
		Fehling’s test	-	+	+	+	+
		Benedict’s test	-	-	-	+	+
		Barfoed’s test	-	-	-	+	+
9	Saponins	Foam test	+	+	+	+	+
9	Saponins	With lead acetate	+	+	+	+	+
10	Fixed Oils and Fats	Spot Test	+	+	+	-	-
10	Fixed Oils and Fats	Saponification test	+	+	+	-	-

+ : present, - : absent

3. RESULTS:

3.1. PHYTOCHEMICAL SCREENING

Phytochemical investigations of the successive leaf extracts of Sesbania grandiflora revealed the presence of steroids, terpenoids, tannins, alkaloids, saponins, phenolic compounds and flavonoids, proteins and carbohydrates in ethanolic and aqueous extracts. Whereas hexane extract revealed the presence of steroids, terpinoids, saponins and fixed oils. Alkaloids, glycoside, carbohydrates, saponins, fixed oils were found in dichloromethane extract and alkaloids, glycoside, steroids, proteins, carbohydrate, saponins, fixed oils were observed in ethyl acetate extract. The details are presented in Table1.

3.2 IN VITRO ANTIOXIDANT ACTIVIY

After phytochemical screening, it was observed that maximum number of phytoconstituents were present in ethanolic and aqueous extracts of Sesbania grandiflora leaves including flavonoids, tannins and phenolic compounds. Hence, these two extracts were selected for in vitro anti- oxidant activity using DPPH assay method followed by estimation of total phenolic contents to evaluate the free radicals scavenging capacity.

3.2.1 DPPH assay of Sesbania grandiflora leaves extracts

DPPH is stable nitrogen centered free radical that can accept an electron or hydrogen radical to become a stable diamagnetic molecule. DPPH radicals react with suitable reducing agents, then losing colour stoichometrically with the number of electrons consumed, which is measured spectrophotometricallty at 517 nm. The ethanolic and aqueous extracts of leaves strongly scavenged DPPH radical with the IC₅₀ being 119.40 and 109.30 respectively (Table2 and Fig.1 and 2).

3.3 ESTIMATION OF TOTAL PHENOLIC CONTENT

3.3.1. Total polyphenol content assay

The extract was prepared for examination of the total phenolic content concentrations. The results of the total phenolic content of the extracts examined, using Folin-Ciocalteu method, are depicted in Table 4. The total phenolic content in ethanolic and aqueous extracts, expressed as gallic acid equivalents ranged between 88.56 mg GA/gm and 90.12 mg GA/gm respectively.

Table4. Determination of total polyphenol content of leaves extract of S.grandiflora

Extracts

Total polyphenol content

(GAE mg/gm)

Ethanolic extract

88.56±0.09

Aqueous extract

90.12±0.52

Data expressed as gallic acid equivalent (GAE) mg/gm of the extract,

Values are mean ± SEM of triplicate determinations

4. DISCUSSION AND CONCLUSION:

In the present study, certain phytoconstituents such tannins, saponins, carbohydrates, steroids, flavonoids and phenolic compounds were detected during qualitative phytochemical screening of the successive leaves extracts of Sesbania grandiflora.

Recently herbal products and synthetic drugs as antioxidant agents have received much attention. Many plant extracts and herbal products have been reported to possess significant antioxidant activity^17,18. Sesbania grandiflora selected for the present study possess several ethnomedical uses related to its antioxidant activity. Plants containing important phytoconstituents like phenolic compounds, tannins, flavonoids and terpenoids are known to possess antioxidant properties.

Free radicals are implicated in various pathological conditions like tissue injury, inflammatory conditions, neurodegenerative diseases, cancer and aging. The ability of Free radical scavenging compounds to ameliorate diseased conditions is appreciated. Thus the human body is protected by antioxidants against damage by reactive oxygen species¹⁹.

The proton radical scavenging action is known to be one of the important mechanisms for measuring antioxidant activity. This assay determines the scavenging of stable radical species DPPH by antioxidants compounds present in the extracts. The rates of DPPH scavenging activity of extracts are probably due to the presence phenolic compounds. Our study clearly indicated that the extracts exhibited high content of phenolic compounds which was significantly correlated with the DPPH radical scavenging activity.

Now it may be concluded that among the two leaves extracts like ethanolic and aqueous of Sesbania grandiflora tested for its in vitro antioxidant activity, both the extracts exhibited potent antioxidant activity with significant IC₅₀values in the scavenging of DPPH radicals. The potent antioxidant activity of the ethanolic and aqueous extracts may be due to the high phenolic content such as tannins and flavonoids.

5. REFERENCES:

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16. Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic - phosphotungstic acid reagents. Am J Enolvitic 1965; 16:144-158.

17. Couladis M, Tzakou O, Verykokidou E, Harvala C. Screening of some greek aromatic plants for antioxidant activity. Phytother Res 2003; 17: 194-195.

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Received on 15.02.2014 Accepted on 31.03.2014

Asian J. Res. Pharm. Sci. 4(1): Jan.-Mar. 2014; Page 16-21

Concentration (µg/ml)	DPPH Scavenging %
Concentration (µg/ml)	Ethanol extract	Aqueous extract	Ascorbic Acid
50	25.32±0.62	28.54±0.48	94.53±0.73
100	42.14±0.75	46.17±0.91	-
150	63.78±0.09	68.32±0.72	-
200	78.17±0.28	79.61±0.36	-
250	93.14±0.19	95.24±0.58	-
IC₅₀	119.40	109.30	-