MATERIALS AND METHODS

Plant materials: The leaves of O. cochinchinense collected from local area of Villupuram District, Tamil Nadu, India during October-December-2012.

Preparation of plant extract: In green leaves were dried powdered using with a mechanical grinder and stored in a jib lack cover. Leaf powder (1.25 kg) was refluxed with different solvents, Dimethyl sulfoxamide (DMSO), Ethyl acetate (EtoAc), ethanol (EtOH), methanol (MeOH) and chloroform (CHCl₃₎ for five days. The total filtrate was concentrated to dryness, in hot air oven at 32°C to render five solvent extract for investigation.

Chemicals: All were purchased from SD fine chemical company Mumbai and all chemicals were of analytical grade.

Phytochemical screening: The phytochemical analysis of the (DMSO, EtoAc, EtOH, MeOH, CHCl₃ of O. cochinchinense leaf was carried out by standard methods as described in Evans (2000), Harborne (1998) and Ghani (2003). Specifically, the extract was screened for the presence of secondary metabolites (flavonoids, saponins, glycosides, steroids, alkaloids, resins, tannins, terpenoids and acidic compounds and macronutrient carbohydrate, reducing sugar).

Chemical group tests of the extract: Testing different chemical groups present in the extract were performed the through phytochemical studies (Evans, 2000). In each test 10% (w/v) solution of extract was taken unless otherwise mentioned in individual test.

Active-principle analysis
Test for flavonoids: Two hundred milligram each of the extract from different solvent was heated with 10 mL of ethyl acetate in boiling water for 3 min. The mixture was filtered and the filtrate was used for the following tests:

Test for glycosides: Dilute sulphuric acid (5 mL) was added to 0.1 g of the test extract in a test tube and boiled for 15 min in a water bath. It was then cooled and neutralized with 20% potassium hydroxide solution. A mixture, 10 mL of equal parts of Fehling’s solution A and B were added and boiled for 5 min. A more dense red precipitate indicated the presence of glycoside.

Test for steroids and terpenoids: Nine mililiter of ethanol was added to extract and refluxed for a few minute and filtered. Each of the filtrates was concentrated to 2.5 mL in a boiling water bath. Five mL distilled water was added to the concentrated solution, the mixture was allowed to stand for 1 h and the waxy matter was filtered off. The filtrate was extracted with 2.5 mL of chloroform using a separating funnel. To 0.5 mL of the chloroform extract in a test tube, 1 mL of concentrated H₂SO₄ was carefully added to form a lower layer. A reddish brown interface showed the presence of steroids.

A quantity, 0.5 mL of the chloroform extract was evaporated to dryness on a water bath and heated with 3 mL of concentrated H₂SO₄ for 10 min on a water bath. A grey colour indicated the presence of terpenoids.

Test for alkaloids: A quantity (0.2 g) of the sample solvent was boiled with 5 mL of 2% HCl on steam bath. The mixture was filtered and 1 mL filtrate was treated with 2 drops of the following reagents:

Test for saponins: A quantity of 500 mg of the extract solvent was boiled with 5 mL of distilled water for 5 min. The mixture was filtered while still hot and the filtrate was used for the following test:

Test for tannins: A quantity, 2 g of the extract solvent was boiled with 5 mL of 45% ethanol for 5 min. The mixture was cooled and filtered. The filtrate was subjected to the following tests:

Test for acidic compounds: A quantity, 0.1 g of the extract was placed in a clear dry test tube and sufficient water added. These were warmed differently in a hot water bath and cooled. A piece of water-wetted litmus paper was dipped into the filtrate and observed for colour change. Acidic compounds turn blue litmus paper red.

Test for resins: Two tests were carried out to detect the presence of resins in the plant part extract under investigation:

Macronutrients analyses: The test which would be shown subsequently, were carried out to determine the presence of macronutrients in the herb O. cochinchinense leaf.

Test for protein
Burette test: A quantity, 2 mL of the extract was put in a test tube and 5 drops of 1% hydrated copper sulphate and 2 mL of 40% sodium hydroxide were added and the test-tube was shaken vigorously to mix the contents. A purple coloration showed the presence of proteins (presence of two or more peptide bonds).

Test for carbohydrate: A quantity, 0.1 g of the extract was shaken vigorously with water and then filtered. To the aqueous filtrate, few drops of molisch reagent were added, followed by concentrated H₂SO₄ (1 mL) was carefully added to form a layer below the aqueous solution. A brown ring at the interface indicated the presence of carbohydrates.

Test for reducing sugar: A quantity, 0.1 g of the extract was shaken vigorously with 5 mL of distilled water and filtered. To the filtrate equal volumes of Fehling solution A and B were added and shaken vigorously. A brick red precipitate indicated the presence of reducing sugars.

In vitro test for antioxidant activity
Free radical scavenging activity by DPPH method: Quantitative Assay was performed on the basis of the modified method of Gupta et al. (2003). Stock solutions (10 mg mL^-1) of the plant extracts were prepared in different solvent from which serial dilutions were carried out to obtain concentrations of 10, 20, 30, 40, 50, 100 and 500 mg mL^-1. A quantity (2 mL) of diluted solutions were added to 2 mL of a 0.004% ethanol solution of DPPH, mixed and allowed to stand for 30 min for reaction to occur. The absorbance was determined at 517 nm and from these values corresponding percentage of inhibition were calculated. The experiment was performed in duplicate and average absorption was noted for each concentration. Ascorbic acid was used as positive control.

DPPH free radical scavenging activity was determined by the method described by Choi et al. (2007) and Desmarchelier et al. (1997). Plant extract (0.1 mL) was added to 3 mL of a 0.004% MeOH solution of DPPH. Absorbance at 517 nm was determined after 30 min and percentage inhibition was calculated:

where, A₀ is the absorbance of the control and A₁ is the absorbance of the extract/ standard. IC₅₀ value was calculated from the equation.

Statistical analysis: Statistical analysis of the results was performed by student’s t-test for independent samples. Values of p<0.001 were considered significant.

RESULTS

Phytochemical and macronutrient screening: The phytochemical screening of DMSO from the O. cochinchinense leaves indicated the presence of alkaloids, steroids, glycosides, saponins, tannins, acidic compounds, resins and coumerines but not flavanoids, acidic compounds, resins and coumerins. Quantitatively, alkaloids were more whereas resins were least among the detected secondary metabolites (Table 1). Furthermore, only carbohydrates (in moderate abundance) were found to be present in the DMSO of O. cochinchinense (Table 2).

Ethyl acetate (EtoAc) extract of O. cochichinense leaves indicated the presence of flavanoids, steroids, glycosides, saponins, tannins, acidic compounds, resins and coumerines but not detected alkaloids. Quantitatively, flavanoids were more whereas resins were least among the detected secondary metabolites (Table 1). Furthermore, only carbohydrates (in moderate abundance) were found to be present in the ethyl acetate of O. cochinchinense (Table 2).

Ethanol extract of O. cochichinense leaves indicated the presence of flavanoids, alkaloids, steroids, glycosides, saponins, tannins, acidic compounds, resins and coumerines but not fats and oils. Quantitatively, alkaloids were more whereas resins were least among the detected secondary metabolites (Table 1). Furthermore, only carbohydrates (in moderate abundance) were found to be present in the ethanol of O. cochinchinense (Table 2).

Phytochemical screening of methanol from the O. cochinchinense leaves indicates the presence of flavanoids, steroids, glycosides, saponins, tannins, acidic compounds, resins and coumerines but not detected alkaloids and acidic compounds. Quantitatively, flavanoids were more whereas resins were least among the detected secondary metabolites (Table 1). Furthermore, only carbohydrates (in moderate abundance) were found to be present in the methanol of O. cochinchinense (Table 2).

Table 1:	Results of phytochemical analyses on O. cochinchinense leaves using the different solvent extracts
-: Absent, +: Low in abundance, ++: Moderate in abundance, +++: High in abundance and Nd: Not detected

Table 2:	Results of macronutrient analyses on O. cochinchinense leaves using the test solvent extracts
-: Absent, +: Low in abundance, ++: Moderate in abundance, +++: High in abundance and Nd: Not detected

Chloroform extract of O. cochichinense leaves indicates the presence of alkaloids, steroids, glycosides, saponins, tannins, acidic compounds, but not detected resins, coumerines fats and oils. Quantitatively, alkaloids were more whereas resins were least among the detected secondary metabolites (Table 1). Furthermore, only carbohydrates (in moderate abundance) were found to be present in the ethanol of O. cochinchinense (Table 2).

In vitro antioxidant activity
DPPH free radical scavenging activity: Dimethyl sulfoxamide extract of O. cochichinense showed potential antioxidant activity where the IC₅₀ was 13.05±0.39 μg mL^-1 (p<0.001), as compared that of ascorbic acid (IC₅₀ 6.10±0.18 μg mL^-1) (p<0.001) which is well known antioxidant (Table 3). The extract caused an increase in DPPH free radical scavenging activity (% inhibition) as increasing dose (Table 4). This table showed most potent inhibitor with a (IC₅₀ 83.36±0.43 μg mL^-1) which was comparable to ascorbic acid (IC₅₀ 85.12±1.25 μg mL^-1).

Ethyl acetate (EtoAc) extract of O. cochichinense showed potential antioxidant activity where the IC₅₀ was 14.08±0.42 μg mL^-1 (p<0.001), as compared that of ascorbic acid (IC₅₀ 7.18±0.21 μg mL^-1) (p<0.001) which is well known antioxidant (Table 3). Ethyl acetate extract caused an increase in DPPH free radical scavenging activity (% inhibition) as increasing dose (Table 4). EtoAc extract most potent the hydroxyl radical inhibition tests (IC₅₀ 83.33±0.40 μg mL^-1) which was comparable to (IC₅₀ 84.68±1.20 μg mL^-1) ascorbic acid.

Ethanol extract of O. cochichinense showed potential antioxidant activity where the IC₅₀ was 15.02±0.45 μg mL^-1 (p<0.001), as compared that of ascorbic acid (IC₅₀ 8.15±0.24 μg mL^-1) (p<0.001) which is well known antioxidant (Table 3). The ethanol extract caused an increase in DPPH free radical scavenging activity (% inhibition) as increasing dose (Table 4). As the ethanol extract exhibited considerable free radical inhibition properties are (IC₅₀ 82.89±0.41 μg mL^-1) which was comparable to (IC₅₀ 84.18±1.31 μg mL^-1) ascorbic acid.

Table 3:	DPPH free radical scavenging activity of O. cochinchinense leaves using the different solvent extract

Table 4:	DPPH radical scavenging activity of O. cochinchinense leaves using the different solvent extract
Values represent the Mean±SED, No. of readings in each group = 3

Table 5:	DPPH radical scavenging activity of O. cochinchinense leaves using the different solvent extract
Values represent the Mean±SED, No. of readings in each group = 3

Table 4 and 5 indicated that hydroxyl radical was scavenged from ethanol extract in a dose dependent manner. The other solvent extracts appeared to have relatively higher or lower activities.

Methanol extract of O. cochichinense showed potential antioxidant activity where the IC₅₀ was 16.12±0.48 μg mL^-1 (p<0.001), as compared that of ascorbic acid (IC₅₀ 9.10±0.27 μg mL^-1) (p<0.001) which is well known antioxidant (Table 3). Methanol extract caused an increase in DPPH free radical scavenging activity (% inhibition) as increasing dose (Table 5). This table showed most potent the hydroxyl radical scavenge inhibition test (IC₅₀ 84.73±0.40 μg mL^-1) which was comparable to (IC₅₀ 85.76±1.32 μg mL^-1) ascorbic acid.

Chloroform extract of O. cochichinense showed potential antioxidant activity where the IC₅₀ was 14.88±0.44g mL^-1 (p<0.001), as compared that of ascorbic acid (IC₅₀ 8.18±0.23 μg mL^-1) (p<0.001) which is well known antioxidant (Table 3). The extract caused an increase in DPPH free radical scavenging activity (% inhibition) as increasing dose (Table 5). The CHCl₃ extract are free radical inhibition tests (IC₅₀ 83.13±0.42 μg mL^-1) which was comparable to (IC₅₀ 85.68±1.25 μg mL^-1) ascorbic acid.

DISCUSSION

The preliminary phytochemical screening of the chemical constituents of different solvents from the O. cochinchinense plants showed that the leaves generally contain the major secondary metabolites in moderate abundance. In higher plants the flavonoids becomes inseparable with the antioxidant potentials that could cure heart diseases and also cance (Noroozi et al., 1998; Al-Humaid et al., 2010). Vitamin A, C and E and flavonoids from plant sources are antioxidants in diet (Pietta, 2000). Thus, the absence of flavonoids in the DMSO extract of O. cochinchinense leaves might limit the solvent choice of DMSO in the extraction of active medicinal ingredients from O. cochinchinense leaves. Hence, the moderate abundance of alkaloids in the leaves of O. cochinchinense appears to support the efficacy of the use of the leaves in ethno-medicinal practice. The absence of alkaloids in the (EtoAc) extract of O. cochinchinense leave limit the solvent choice of (EtoAc) in the extraction of active medicinal ingredients from O. cochinchinense leaves.

The presence of flavonoids from ethanol extracts of O. cochinchinense leaves observed in the present study also attests to the possible efficacy of therapeutic use of O. cochinchinense leaves. They are related to sex hormones and could, by serving as potent starting material in the synthesis of sex hormones, ensure such hormonal balance (Okwu, 2001). This could in addition of the noted high carbohydrate content of O. cochinchinense leaves that could provide useful energy, be highlighting the possible reproductive benefits from the prospective use of O. cochinchinense leave as nutraceutical beverage. The absence of acidic compounds from methanol extract of O. cochinchinense leaves might limit the solvent choice of MeOH in the extraction of active medicinal ingredients from O. cochinchinense leaves. The absence of flavonoids, acidic compounds, resins, coumerins in the CHCl₃ extract of O. cochinchinense leaves might limit the CHCl₃ solvent choice of extraction and active medicinal ingredients from O. cochinchinense leaves. The phytochemical analysis of the extract showed the presence of flavonoids, alkaloids, saponins, sterols, terpenoids, resins and sugar. These constituents may be responsible for antioxidant potentiality of O. cochinchinense.

CONCLUSION

The present study reveals that different solvent extracts of O. cochinchinense leaves possesses significant of phytochemical screening and antioxidative activity. Now research is continued to isolate lead compound from these extracts and also to develop a potent formulation for treatment of bone healing activities.