IN- VITRO ANTIOXIDANT ACTIVITY OF HYDROETHANOLIC EXTRACT OF STEM BARK OF OUGEINIA OOJEINENSIS (ROXB.) HOCHR (LEGUMINOSAE)

IN- VITRO ANTIOXIDANT ACTIVITY OF HYDROETHANOLIC EXTRACT OF STEM BARK OF OUGEINIA OOJEINENSIS (ROXB.) HOCHR (LEGUMINOSAE)

1. P. Shinde*, Y. M. Joshi and V. J. Kadam

Department of Pharmacology, Bharati Vidyapeeth’s College of Pharmacy, Sector 8, CBD, Belapur, Navi Mumbai - 400614, Maharashtra, India

ABSTRACT

Safer antioxidants suitable for long term use are needed to prevent or stop the progression of free radical mediated disorders. In this study, the antioxidant activity of hydroethanolic extract of stem bark of Ougeinia oojeinensis (Roxb.) (HEBO) was studied using various in vitro assays. The antioxidant activity of the extract was evaluated by using the free radical scavenging activity assay (DPPH method), reducing power assay. The findings indicated promising antioxidant activity of the hydroethanolic extract of stem bark of Ougeinia oojeinensis (Roxb.) and needs further exploration for its effective use in both modern and traditional system of medicines.

Nitric oxide

INTRODUCTION: Plants have become the basis of traditional medicine system throughout the world for thousands of years and continue to provide mankind with new remedies. In addition, plant based drugs remain an imp source of therapeutic agents because of their availability, relatively cheaper cost and non-toxic nature, when compared to modern medicine ¹. Many medicinal plants contain active chemical constituents with high antioxidant property which plays an important role in the prevention of various degenerative diseases ².

Antioxidants are vital substances which possess the ability to protect the body from damage caused by free radical induced oxidative stress. In fact, oxidative stress results from an imbalance between the generation of reactive oxygen species and endogenous antioxidant systems.ROS are major sources of primary catalysts that initiate oxidation in- vivo and in- vitro and create oxidative stress which results in numerous diseases and disorders ^{3, 4} such as cancer ⁵, cardiovascular disease ⁶, neural disorders ⁷, Alzheimer’s disease ⁸, mild cognitive impairment ⁹, Parkinsons disease ¹⁰, alcohol induced liver disease ¹¹, ulcerative colitis ¹², ageing ¹³.

Thus, there is a need for more effective, less toxic and cost effective antioxidant. Medicinal plants appear to have these desired comparative advantages; hence the growing interest in natural antioxidants from plants ¹⁴.

The plant, Ougeinia oojeinensis (Roxb.) Hochr (Leguminosae) is known as Tinsa in Hindi and Rathadru in Sanskrit ¹⁵. According to the Ayurvedic text, bark and gum are astringent, haemostatic, antidiuretic, antidermatoses and rejuvenating. Therefore useful in oedema, leucoderma and other skin diseases. Internally, it is used in diarrheoa, blood disorders, skin diseases, and obesity ^{15, 16}. The extract of the whole plant has shown anti-inflammatory activity ¹⁵. Preliminary phytochemical testing of 50% hydroethanolic extract of the stem bark of Ougeinia oojeinensis revealed the presence of flavonoids, saponins.

The aim of this study was to investigate the antioxidant properties of hydroethanolic extract of stem bark of Ougeinia oojeinensis (Roxb.) against the free radicals.

MATERIALS AND METHODS:

Plant Material: The stem bark of Ougeinia oojeinensis was collected from Belapur, Maharashtra, India. The plant was identified and authenticated by Dr. Ganesh Iyer, Department of Life Sciences, Ramnarain Ruia College, Matunga, Mumbai, Maharashtra, India. The sample specimen was preserved in our laboratory.

Preparation of Extract: The stem bark of the plant was shade dried and reduced to fine powder. This powdered bark was extracted with alcohol: water (1:1) in a Soxhlet extractor for 40 hours. The solvent was removed from the extract under vacuum rotator dryer. The dried extract was stored in dessicator and was used for preliminary phytochemical as well as pharmacological evaluation.

Antioxidant Activity Assessment:

The Free Radical Scavenging Activity Assay (DPPH method): The antioxidant activity of the plant extracts and standard were assessed on the basis of the radical scavenging effect of the stable DPPH free radical¹⁷. About 10-100 μl of each extract or standard was added to 2 ml of DPPH (HiMedia Laboratories Pvt. Ltd., Mumbai) in methanol (0.33%) in a test tube. After incubation at 37^oC for 30 minutes the absorbance of each solution was determined at 517 nm using spectrophotometer ¹¹.

The corresponding blank readings were also taken and the remaining DPPH was calculated by using the following formula:

DPPH radical scavenging activity (%) = [Abs (control) – Abs (standard)] ×100.

Where, Abs (control): Absorbance of DPPH radical + methanol,

Abs (standard): Absorbance of DPPH radical + extract/standard.

IC₅₀ value is the concentration of the sample required to scavenge 50% DPPH free radical.

Reducing Power Assay: The reducing power of the extract of the stem bark of Ougeinia oojeinensis was determined according to the method of Oyaizu (1986) ¹⁸. Different concentrations of the HEBO (10–100 μg/ml) in 1.0 ml of deionised water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferrocyanide (2.5 ml, 1%). The mixture was incubated at 50^oC for 20 min.

A portion of trichloroacetic acid (2.5 ml, 10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%) and the absorbance was measured at 700 nm and compared with standards. Increased absorbance of the reaction mixture indicated increased reducing power.

Statistical Analysis: Results are expressed as mean ± S.E.M. of three determinants. Comparisons among the groups were tested by two-way ANOVA using Graph Pad Prism, Version 5.0 (Graph Pad Software, San Diego, CA, USA). P-values < 0.005 were considered significant.

RESULTS AND DISCUSSION:

Free Radical Scavenging Activity: Figure 1 shows the dose-response curve of DPPH radical scavenging activity of HEBO, compared with ascorbic acid, as standard. At a concentration of 100μg/ml, the scavenging activity of methanol extract of stem bark was 81.07%, while at the same concentration, that of the standard ascorbic acid was 89.35%. IC₅₀ values of extract and standard were 53.50μg/ml and 25.00μg/ml, respectively.

The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability ¹⁹. Though the DPPH radical scavenging abilities of the extract was less than those of ascorbic acid at 100μg/ml, the study showed that the extract has the proton-donating ability and could serve as free radical inhibitor or scavenger, acting possibly as primary antioxidant.

FIG. 1: FREE RADICAL SCAVENGING ACTIVITY OF HEBO (n=3)

Reducing Power Assay: Figure 2 shows the reductive capabilities of the plant extract compared to ascorbic acid. The reducing power of the HEBO was very potent and the power of the extract was increased with quality of sample. The plant extract could reduce the most Fe³⁺ ions, which had a lesser reductive activity than the standard of ascorbic acid. Increased absorbance of the reaction indicated increased reducing power.

FIG. 2: REDUCING POWER ASSAY OF HEBO (n=3)

CONCLUSION: On the basis of the results obtained in the present study, it is concluded that the hydroethanolic extract of the stembark of Ougeinia oojeinensi, exhibits significant antioxidant and free radical scavenging activities. It also chelates iron and has reducing power. These in-vitro assays indicate that this plant extract is a significant source of natural antioxidants, which might be helpful in preventing the progress of various oxidative stress induced disorders. However, the components responsible for the antioxidant activity are currently unclear. Therefore, further investigations need to be carried out to isolate and identify the antioxidant compounds present in the plant extract.

Furthermore, the in vivo antioxidant activity of this extract needs to be assessed prior to clinical use.

ACKNOWLEDGEMENT: Authors are thankful to Dr. Ganesh Iyer, Department of Life Sciences, Ramnarain Ruia College, Matunga for providing the plant samples and authenticating the same.

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