INTRODUCTION:

Oxidative stress is initiated due to disparity amid formation of free radicals and detoxification potential of a biological system [1,2]. Both enzymatic and non-enzymatic reactions in the cell can incessantly produce the free radicals instigating wide-ranging impairment to tissues and also in several medical ailments [3]. However, the cells are shielded from the injury produced by uninhibited radicals by antioxidants [4]. Phenolic acids, polyphenols and flavonoids have the potential antioxidants properties and aid in scavenging free radicals [5]. Inflammation is a host defense mechanism to combat the tissues injury, infection or irritation, which encompasses the activation of several enzymes, tissue breakdown and repair [6]. Some metabolites of plants and herbs are identified for retaining anti-inflammatory along with antioxidant activities [7].

The Vitex negundo L. (Verbenaceae), which also generally identified as the Chinese chaste tree. It is a large aromatic shrub with quadrangular tomentose [8]. This plant is grown all over India, Mediterranean countries and Central Asia. Vitex negundo possess numerous biological properties such as antioxidant, hepatoprotective, anti-fungal, anti-inflammatory, actions. The extracts of this plant have been utilized for treating various disorders [9]. V. negundo leaves decoction is given as a treatment to catarrhal fever. Further V. negundo leaves stuffed in a pillow is used to relief the headache. The juices of the leaves are used to remove worms from ulcers. The V. negundo blossoms have been found to beneficial for treating pyrexia, bleeding, liver disorders, diarrhea, cholera, also heart related conditions [10]. Leaves and bark are used as emollient in scorpion stings, tincture of root bark is used to treat irritable bladder and of rheumatism [11]. The dried fruit acts as anti-helminthic and is used as vermifuge. The literature survey revealed that V. negundo are having anti-inflammatory, antimicrobial, antioxidant activities [12]. Here in this current research work have assessed antioxidant and anti-inflammatory activity of Vitex negundo methanolic extract.

MATERIALS AND METHODS:

Plant material collection and authentication:

The plant Vitex negundo, erect aromatic shrub of Verbenaceae clan which is well distributed throughout India. The plant material was identified and authenticated by Dr. S.B. Padal, Associate professor, Department of Botany, Andhra University, Visakhapatnam. The voucher specimen was 22227.

Preparation of plant extracts:

Water cleansed leaves of Vitex negundo were shade dried and powdered by grinding in mechanical grinder. Crudely powdered plant materials of 75g, (75g/250ml) was obtained in a soxhlet extractor over a time period of 10 - 12 hours, consecutively with methanol, ethyl acetate, chloroform, petroleum ether. Finally, the derived extracts were, then concentrated. Lastly, they were dried to a constant weight. The dried extracts were maintained at 20°C until further usage.

DPPH radical scavenging activity:

The DPPH assay was done for the methanolic extract in accordance to Braca et al, [13] with some modifications methodology. In this study 0.1 ml of extract and standard compound Vitamin C (Ascorbic acid) of variable concentration (50-2000 μg/ml) was added to 3 ml of 0.004% DPPH (in methanol). The preparation of control was proceeded in similar manner excluding standard and extract. Control. Methanol is used as blank. The samples were shaken vigorously. Then incubation in dark was proceeded for half an hour. Following this the absorbance for the samples were assessed at 517nm using spectrophotometer. The standard utilized in this study was ascorbic acid i.e., Vitamin C was used as standard. Increased levels of free radicals are indicated by decrease in the absorbance. All experiments were performed in triplicate manner and DPPH percentage inhibition was observed and analyzed accordingly.

Nitric oxide scavenging activity:

Nitric oxide scavenging activity can be estimated by the use of Griess IlIosvoy reaction [14]. Briefly, Various concentrations of methanolic extract were added to 10 mM sodium nitroprusside and incubated for 2.5 h. After incubation, Griess reagent was added to the tubes and the absorbance of the chromophore formed was read at 590 nm. The blank solution contained a mixture of 0.5 mL PBS and 0.5 mL extract. The IC50 values and percent inhibition by various concentrations of extract were calculated by comparing the absorbance values of the control and methanolic extracts.

Total Anti-inflammatory activity:

The anti-inflammatory activity of the methanolic extract of Vitex negundo has been tested by means of two methods: protein denaturation inhibition [15,16]. About 100µl methanolic extract was then combined with 500µl BSA 1%. Then incubation of the same was carried out for 10 minutes, temperature of 37ºC. The contents were heated using water bath at 51ºC, with a time of 20 minutes and then dampened down to the room temperature before checking absorbance at 660nm against the blank. In this experiment Acetyl Salicylic acid has been utilized as the positive control and the action was assessed.

Determination of reducing power:

Reductive property was observed, where transformation of Fe³⁺-Fe²⁺has been evaluated in accordance to method described elsewhere [17]. In the presence of 2.5 ml of phosphate buffer (pH 6.6), 2.5 ml of 1% potassium ferric cyanide with extracts along with ascorbic acid using different concentrations (25, 50, 100, 200, 250, 500, 1000, 2000 μg/0.05ml) were incubated at 50˚C for 20 minutes. To the mixture, 2.5 ml of 10% TCA were added and centrifuged at 3000 rpm for 10 mins. After centrifugation, 2.5 ml of the supernatant was diluted with 2.5 ml of water and added 0.5 ml freshly prepared 0.1% ferric chloride. The absorbance was measured at 700 nm. The control solution consists of water in the place of sample. The increase in absorbance indicates higher reductive ability.

STATISTICAL ANALYSIS:

Experiments were done in triplicates. The results were expressed as the mean ±S. D deviation.

RESULTS:

The antioxidant, DPPH assay for Vitex negundo methanolic extract in various strengths compared to ascorbic acid as depicted in Figure 1. From this, Vitex negundo methanolic extract displayed radical scavenging action starting from 200μg/0.1 ml concentration, with highest percent inhibition of 81.87±1.53% at 2000μg/0.1 ml concentration (Figure 1). Nitric oxide free radical dousing action of the methanolic extract of Vitex negundo was then identified, assessed with Vitamin C (Figure 2). Compared to ascorbic acid Vitex negundo methanolic extract exhibited nitric oxide radical quenching activity at all concentrations. The methanolic extract of Vitex negundo displayed utmost inhibition percentage which is 75.29±1.33% at 2000μg/ml (Figure 2). The Vitex negundo methanolic extract exhibited reducing (antioxidant) ability in concentration-dependent pattern (Figure 3). The highest reducing power (83.75±0.57%) of Vitex negundo methanolic extract was observed at 2000μg/0.05 ml (Figure 3). The Vitex negundo methanolic extract showed strong anti-inflammatory activity by exhibiting 79.81% inhibition at 500ug concentration (Figure 4).

Figure 1: The percentage of DPPH scavenging activity of Vitex negundo methanolic extract.

Figure 2: The percentage of nitric oxide scavenging activity of Vitex negundo methanolic extract.

Figure 3. Inhibitory effect of Vitex negundo methanolic extract on reductive ability

Figure 4. Anti-inflammatory activity of Vitex negundo methanolic extract.

DISCUSSION:

This current research work findings uncovered convincing antioxidant activity for the methanolic extracts of leaves from Vitex negundo. Several lines of evidences showed presence of different bioactive metabolites such as alkaloid, carotene, benzoic acid, β-sitosterol, flavonoids, casticin, vitamin-C and C-glycoside [18,19,20,21].

Free radical reducing capacity of Vitex negundo extract has also been identified in diverse vivo assays such as Superoxide, Hydroxyl radical scavenging, iron chelation, 2, 2’-azino-bis- 3-ethyl benzothiazioline-6-sulfuric acid and Lipid peroxides [22,23]. Vitex negundo plant derived compounds such as Vitedoin A, Vitedoin B and some of the lignans were also assessed for their free radical lowering potential [24,25,26]. Studies in various animal models have identified the anti-inflammatory activity of Vitex negundo leaves, fruits, roots and seeds [27, 28, 29, 30]. Further, V. negundo leaf oil exhibited anti-inflammatory activity through blocking of COX-2 but not hindering COX-1 pathways [31]. In addition, Vitex negundo methanolic extract could able to act as neuroprotective agent by produce long neurites [32].

In summary, the methanolic extract of Vitex negundo showed potential antioxidant action and also anti-inflammatory activity. Hence further purification of the methanolic extract is warranted to identify and confirm the exact active compound responsible for its antioxidant and anti-inflammatory activities.

CONFLICT OF INTEREST:

I Dr. Anusha and the other co-authors of this article declare no conflict of interest regarding the content, authorship or publication of this article. This study is completely self funded.

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Received on 25.02.2019 Modified on 17.03.2019

Research J. Pharm. and Tech. 2019; 12(6): 2824-2827.

DOI: 10.5958/0974-360X.2019.00475.X