Introduction

Ventilago maderspatana Gaertn. is a plant of the family Rhamnaceae, Red Creeper, Raktavalli, and Pitti are the other common names of this plant in different languages (Warrier, Nambiar, & Ramankutty, 2006). It is a climbing shrub, and one can easily identify it by its dark greyish branches of bark having brownish buddings and also by its flowers foul odour in winter season ( RDA-Gene Bank, 2020; Sharma et al., 2020). It is widely distributed in south Greece, Indonesia, Bhutan, Malaysia, Srilanka and India. (Kirtikar & Basu, 1987; Pullaiah, 2006). The root barks powder of Ventilago maderspatana Gaertn. have many properties and application like it is used as stomachic, a tonic, carminative, stimulant. It is also useful in the treatment of weakness and fever. Similarly, the powder of Ventilago maderspatana Gaertn, the bark is used externally to treat itching and other skin ailments in different regions of South India. (Hanumaiah et al., 1985). The pale yellow wood of this plant is extensively used as fuel, and fishermen's use the long climbing stems of this plant as a substitute for ropes (Basu, Ghosh, & Hazra, 2005; Hanumaiah et al., 1985). Till date five isofuranonapthoquinones, three naphthoquinones and two naphthalene derivatives are isolated from the root bark of Ventilago maderspatana Gaertn. And many properties like antibacterial, anti-denaturation and ex-vivo scavenging activities are reported (Basu & Hazra, 2006; Duganath, Kumar, Kumanan, & Jayaveera, 2010). Still, Till date, there is no literature supporting anti-oxidant properties on Root Bark extracts of Ventilago maderspatana Gaertn using the models that are explained in this study. Therefore the current study was done to evaluate the anti-oxidant activity of ethanolic and aqueous extracts of Root Bark of Ventilago maderspatana Gaertn (Chaudhary, 1996)

Materials and Methods

For this study, all the chemicals of analytical grade were used and purchased from Sigma Aldrich Germany.

Plant materials

Ventilago maderspatana Gaertn. The plant was collected from Tirupati district (AP), India in the July month and Confirmed by Dr K. Madhavachetty Assistant professor (Botany Department) SV University Tirupati (AP) (Voucher No: SVU/DC/10/26/10-11) and one specimen of the plant was deposited in Pharmacognosy Department of JSSCOP, Ooty and The materials of the plant were subjected for cleaning and garbling process to ensure the quality of plant (Torel, Cillard, & Cillard, 1986).

Extraction of selected plant material.

For extraction, 500 gm of Root bark (In coarsely powdered form) were subjected to cold maceration process in 3 litres Round bottom flask using ethanol and distilled water. Nature and yield were noted and stored at 4⁰C in the refrigerator until further use. Both the extracts were labelled as Ventilago maderspatana Gaertn Root Bark aqueous (VMRBA) and Ventilago maderspatana Gaertn Root Bark Ethanolic (VMRBE) for convenient identification.

In-Vitro Anti-oxidant Studies

(LPO) Lipid Peroxidation Assay

Principle

Oxidation of polyunsaturated fatty acids (PUFA) which are present in membranes of the cell is caused by both enzymatic and auto-oxidation, peroxidation and by a free radical chain reaction. LPO proceeds in 3 stages, which starts from initiation then propagation and in the last termination. In starting phase free radicals obstructs hydrogen from PUFA which is responsible for the formation of lipid radicals and in the propagation stage more free radicals are generated when lipid radicals are broken. In termination stage there is a reaction between free radical species anti-oxidants which results in the formation of inert products and Inhibitory Concentration (IC₅₀= Sample concentration required to scavenge 50% of Free radicals) is calculated by using the formula

$\mathbf{\text{\%}} \mathbf{\text{Inhibition=}}\frac{\mathbf{Control}\mathbf{ }\mathbf{\left(}\mathbf{OD}\mathbf{\right)}\mathbf{-}\mathbf{Sample}\mathbf{ }\mathbf{\left(}\mathbf{OD}\mathbf{\right)}\mathbf{ }}{\mathbf{Control}}\mathbf{\times }\mathbf{100}\mathbf{ }\mathbf{OD}$

Chemicals and reagents

Saline Phosphate Buffer (pH 7.4), Egg Lecithin (3 mg/ml), Ascorbic acid (200mM), Ferric Chloride (400mM), Trichloroacetic acid (15% w/v) and Thio barbituric acid (0.375% w/v) etc.

Procedure

To prepare test solution take 1 ml of egg lecithin mixture to add test samples of different concentration and for control preparation takes only 1 ml of egg lecithin mixture without sample addition and Induce the Lipid Peroxidation by the addition of 10 µL Ferric chloride (400mM) and 10 µL ascorbic acid (200mM) Solution and incubate for 1 hour at 37°C to start the reaction, and for stopping the reaction add 2 ml of HCL (0.25 N) containing Thiobarbituric acid (0.375% w/v) and boil it for 15 minutes, cool it, centrifuge it and measure the absorbance of the supernatant at 532 nm (Duh, Yen, Yen, & Chang, 2001).

Estimation of total anti-oxidant Capacity (TAC)

Chemicals and reagents

Mix H₂SO₄ (0.6M), Na₂SO₄ (28mM) and Ammonium Molybdate (4mM) to prepare total anti-oxidant capacity (TAC) Reagent for the study.

Procedure

Dissolve 100 µL of extract in 1 ml of total anti-oxidant capacity reagent, use Distilled water as blank by replacing total anti-oxidant capacity reagent and measure the absorbance of both at 695 nm (Sánchez-Moreno, 2002).

Hydroxyl radical scavenging activity by p-Nitroso dimethyl aniline method

Principle

Fenton reaction is the basic principle behind this where H₂O₂ produces highly active species ^ºOH, in the presence of Fe²⁺ ions.

${\mathbf{\text{Fe}}}^{\mathbf{2}}\mathbf{+}{\mathbf{H}}_{\mathbf{2}}{\mathbf{O}}_{\mathbf{2}}\mathbf{\to }{\mathbf{Fe}}^{\mathbf{3}\mathbf{+}}\mathbf{+}{\mathbf{OH}}^{\mathbf{o}}\mathbf{+}{\mathbf{OH}}^{\mathbf{-}}$

This is the most common reaction in biological systems and source of various deleterious lipid peroxidation products in which there is an iron-catalyzed decomposition of hydrogen peroxide, and inhibition of p-NDA bleaching is measured to determine Hydroxyl Radical Scavenging.

Chemicals and reagents

p-NDA (dissolve 0.901 mg in 100 ml of phosphate buffer), FeCl₃ (dissolve 9.73 mg in 100 ml of phosphate buffer), Disodium EDTA (dissolve 22.3 mg in 100 ml of phosphate buffer), Ascorbic acid (dissolve 10.5 mg in 100 ml of phosphate buffer), Hydrogen Peroxide (dissolve 0.136 ml in 100 ml of Phosphate buffer).

Preparation of test and standard solution

Dissolve 30 mg of both extract and rutin (Standard) in 5 ml of DMSO Separately and dilute all the solutions serially to obtain further dilutions.

Procedure

To prepare 3 ml of sample solution take 0.5 ml of different concentrations of extracts or standards and add it to the reaction mixture containing 0.5 ml of FeCl₃ (0.1 mM), 0.1 ml of EDTA (0.1 mM), 0.5 ml of Ascorbic acid (0.1 mM), 0.5 ml of H₂O₂ (2 mM) and 0.5 ml of p-NDA (0.01mM) in phosphate buffer (pH 7.4 & 20 mM) and the blank was prepared by adding 0.5 mL sample and 2.5 ml Phosphate buffer (pH 7.4) and measure the absorbance of the solution at 440 nm and calculate the percentage of scavenging (Cook & Samman, 1996).

Nitric oxide radical inhibition activity

Principle

At physiological pH, Sodium Nitroprusside is in an aqueous form. It produces nitric oxide immediately, which reacts with oxygen to form nitrite ions which are estimated by Griess Illosvoy reaction. In current investigation Griess, Illosvoy reagent is modified by use of naphthyl ethylene diamine dihydrochloride (NEDD) (0.1% w/v) rather using 5% Napthylamine.

In this, nitric oxide competes with oxygen and causes reduction of Nitric oxide production.

Chemicals and reagents

Sodium Nitroprusside (dissolve 0.2998 gm in 100 ml of Distilled water), Napthyl ethylene diamine dihydrochloride (0.1% w/v), Sulphanilic acid (dissolve 0.33 gm in 20% glacial acetic acid and make up the volume up to 100 ml), Saline Phosphate buffer (pH 7.4) and Dimethyl sulphoxide.

Preparation of standard solution

Separately weigh 42 mg of each ascorbic acid and rutin and dissolve in 2 mL of Dimethyl sulphoxide to get a solution of 21 mg/mL Concentration and dilute with Dimethyl sulphoxide to get lower concentrations.

Procedure

Incubate the 6 ml of reaction mixture comprised of 4 ml of Sodium Nitroprusside (10 mM), 1 mL Saline Phosphate Buffer and 1 mL of extract in Dimethyl Sulphoxide at 25^ºC for 150 Minutes and after Incubation remove 0.5 mL of reaction mixture containing nitrate and add 1 mL of Sulphanilic acid reagent, mix it and allow to stand for 5 minutes to complete the diazotization then add 1 mL of Napthyl ethylenediamine dihydrochloride and allow to stand for 30 minutes In diffused sunlight at room temperature and measure the absorbance at 540 nm against blank (Garrat, 1964; Kunchandy & Rao, 1990).

ABTS radical scavenging method

Principle

ABTS on oxidation with potassium persulfate forms radical mono-cation 2, 2' -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and it is reduced in the presence of hydrogen donating anti-oxidants.

Preparation of ABTS solution

Prepare two mM of ABTS solution using distilled water and 17 mM of potassium persulfate solution using distilled water and mix 0.3 mL of 17 mM potassium persulfate solution with 50 mL of 2 mM ABTS solution and reaction mixture is allowed to stand overnight in the dark at room temperature.

Preparation of test solution

Separately weigh 13.5 mg of both extracts and standards (ascorbic acid and rutin) and dissolve in 2 mL of Dimethyl sulphoxide and dilute serially with Dimethyl sulphoxide to obtain further dilutions.

Procedure

Take 0.2 mL of extracts of different concentrations of standards and add 1 mL of Dimethyl sulphoxide and 0.16 mL of ABTS solution and make up the volume to 1.36 ml and measure the absorbance after 20 minutes at 734 nm against blank using spectrophotometer (Jayaprakasha, Rao, & Sakariah, 2004).

Results and Discussion

Lipid peroxidation of Root Bark extracts of Ventilago maderspatana Gaertn.

Ethanolic extract of Root Bark of Ventilago maderspatana Gaertn. has shown potent anti-oxidant activity with an IC50 value of 23.18 ± 0.07 as compared to aqueous extract of Root Bark of Ventilago maderspatana Gaertn. i.e. 224.0 ± 0.03. The details of the results are given in Table 1.

The total anti-oxidant capacity of Root Bark extracts of Ventilago maderspatana Gaertn.

Ethanolic extract of Root Bark of Ventilago maderspatana Gaertn, has shown potent anti-oxidant activity with 0.34 ± 0.06 mM equivalent to ascorbic acid as compared to aqueous extract of Root Bark of Ventilago maderspatana Gaertn.i.e. 0.67 ± 0.02. The details of the results are shown in Table 1.

Hydroxyl radical scavenging activity (p-NDA Method)

Ethanolic extract of Root Bark of Ventilago maderspatana Gaertn. has shown potent anti-oxidant activity with an IC50 value of 46.22 ± 0.04 as compared to aqueous extract of Root Bark of Ventilago maderspatana Gaertn. i.e. 427.0 ± 0.02. The details of the results are shown in Table 2.

Nitric oxide radical inhibition activity

Ethanolic extract of Root Bark of Ventilago maderspatana Gaertn. has shown potent anti-oxidant activity with an IC50 value of 31.14 ± 0.15 as compared to aqueous extract of Root Bark of Ventilago maderspatana Gaertn. i.e. 578.0 ± 0.06. The details of the results are shown in Table 3.

ABTS radical Scavenging method

Ethanolic extract of Root Bark of Ventilago maderspatana Gaertn. has shown potent anti-oxidant activity with an IC50 value of 0.79 ± 0.04 as compared to aqueous extract of Root Bark of Ventilago maderspatana Gaertn. i.e. 6.08 ± 0.07. The details of the results are shown in Table 4.

Conclusions

Results of all the standard methods used for evaluation of the in-vitro anti-oxidant activity of Root Bark extracts of Ventilago maderspatana Gaertn showed that Ethanolic Root Bark extract of Ventilago maderspatana Gaertn has more potent anti-oxidant activity than Aqueous Root Bark extract of Ventilago maderspatana Gaertn.