IN VITRO ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF STEM OF MUSSAENDA ERYTHROPHYLLA

IN VITRO ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF STEM OF MUSSAENDA ERYTHROPHYLLA

M Chinna Eswaraiah* and T Satyanarayana

Anurag Pharmacy College, Kodad, Nalgonda (Dt), University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India

ABSTRACT

Mussaenda erythrophylla is native to western tropical Africa, occasionally seen in gardens and parks as ornamental plant in India and belongs to Rubiaceae family. The present study was concentrated on the in vitro antioxidant methods like superoxide radical, hydroxyl radical, lipid peroxidation and DPPH radical methods. The ethylacetate and methanolic extracts of Mussaenda erythrophylla were subjected for the above methods.  The results of anti oxidant activity revealed that, the ethylacetate extract has lower IC­₅₀ values than the methanolic extract of Mussaenda erythrophylla. The lower IC­₅₀ value indicates the higher free radical scavenging ability. So, the ethylacetate extract has better antioxidant activity than methanolic extract. The results were compared with the standard ascorbic acid. The plant contains phytosterols, triterpenoids, flavonoids, glycosides, saponins and tannins. These active constituents alone or in combination may be responsible for the observed antioxidant activity.

INTRODUCTION: Mussaenda erythrophylla (Rubiaceae) is native to western tropical Africa, occasionally seen in gardens and parks as ornamental plant in India and is commonly known as mussenda (telugu), nagavalli (Sanskrit) and red flag bush (English) ¹. It is a perennial, evergreen shrub with branched tap root system. The roots are useful for cough, jaundice and when chewed acts as an appetizer ². A number of triterpenoids and glycosides were reported. Mussaenda genus viz., contains mussaendosides U(1) and V(2) ³, mussaendosides G(1) and K(2) are two new triterpenoid saponins ⁴, mussaendosides A-C, M and N with cyclolanostene type aglycone ^5-6  and aureusidin⁷, iridoid glycosides ⁸.

The pharmacological activities reported from Mussaenda species were diuretic, antiphlogistic, antipyretic and effective in laryngopharyngitis, acute gastroenteritis and dysentery ⁹ and also anti-fertility activity ¹⁰. It is established that plants which have anti oxidant property exert hepatoprotective ^11-14 and anti diabetic actions ^15-17. It is in the light of this fact that antioxidant and free radical scavenging potential of stem of Mussaenda erythrophylla (M.E) was investigated.

MATERIAL AND METHODS:  All the chemicals used were of analytical grade obtained from S.D. Fine Chemicals Pvt. Ltd., Mumbai, Sigma chemical company, USA and Loba chemicals, Mumbai.

Plant Material: The stem of Mussaenda erythrophylla were procured from M.V.P colony, Visakhapatnam.The authentification of the plant was done by Prof. M. Venkaiah, Dept. of Botany, Andhra University, Visakhapatnam. The Voucher specimen was deposited in the herbarium of our department.

Preparation of Extract: Freshly collected plant material was shade dried at room temperature and coarsely powdered in Wiely mill. The powdered stem (1kg) was extracted successively with hexane, ethylacetate and methanol using soxhlet apparatus. The crude extract was evaporated to dryness in a rotary film evaporator (Roteava,Equitron, Medica instrument, India) and found to be 2.5, 30 and 25gms respectively. Preliminary phytochemical screening of ethylacetate extract of Mussaenda erythrophylla stem revealed the presence of   steroids, triterpenoids and flavonoids; methanol extract tested positive for glycosides, tannins and saponins.The constituents present in the ethylacetate extract and methanolic extracts of M. erythrophylla stem initiated to carry out the anti oxidant activity for the above said extract.

In vitro Anti Oxidant Study:             The ethylacetate extract and methanolic extract of M. erythrophylla stem tested for its free radical scavenging property using different in vitro models. All experiments were performed thrice and the results were averaged.

Superoxide Radical Scavenging Activity: Superoxide radical scavenging activity of the plant extract was measured according to the method of Mc Cord and Fridovich ¹⁸, which depends on light induced superoxide generation by riboflavin and the corresponding reduction of nitroblue tetrazolium. All the solutions were prepared in phosphate buffer (pH 7.8). The optical density was measured at 560nm. The percentage inhibition was calculated from formula ¹⁹.

Hydroxyl Radical Scavenging Activity: Hydroxyl radical scavenging activity was measured according to the method of Elizabeth and Rao²⁰, by studying the competition between deoxyribose and test extracts for hydroxyl radicals generated by Fenton’s reaction. The damage imposed on deoxyribose due to the free radicals was determined calorimetrically by measuring the thiobarbituric acid reactive substances (TBARS) at 532 nm. Percentage of inhibition was calculated using the formula.

Lipid Peroxidation Inhibition Activity: The inhibition of lipid peroxidation was performed as per the method described by Ohkawa et al., ²¹. Rat liver homogenate was used as the source of polyunsaturated fatty acids for determining the extent of lipid peroxidation. The absorbance was measured at 532nm. Percentage of inhibition was calculated using the formula.

DPPH Radical Scavenging Activity: DPPH radical scavenging activity was measured according to the method of Braca et al., ²². An aliquot of 3ml of 0.004% DPPH solution in ethanol and 0.1ml of plant extract at various concentrations were mixed and incubated at 37˚c for 30 min. and absorbance of the test mixture was read at 517nm.The percentage of inhibition of DPPH radical was calculated by comparing the results of the test with those of the control (not treated with extract) using the formula;

Percentage inhibition = (A_{o­­­­ - ­­}A­_{1)/ Ao X 100}                                             

Where A_o = Absorbance of the control; A₁ = Absorbance of the plant extract/ standard.

Statistical Analysis: Linear regression analysis was used to calculate IC₅₀ values ²³.

RESULTS AND DISCUSSION: Superoxides are produced from molecular oxygen due to oxidative enzymes of body as well as via non enzymatic reactions such as auto oxidation by catecholamines ²⁴. In the present study ethylacetate extract and methanol extract of M. erythrophylla stem was found to scavenge the superoxides generated by photo reduction of riboflavin. The ethylacetate extract and methanol extract of M. erythrophylla stem produced dose dependent inhibition of superoxide radicals. The IC₅₀ values for superoxide radical with ethylacetate extract and methanol extract of M. erythrophylla stem were found to be 216.63µg, 271.38µg with ascorbic acid were found to be 140.76 µg respectively. The ethylacetate extract of M. erythrophylla stem was found to have better superoxide radical scavenging activity than methanol extract of M. erythrophylla, as shown in Table 1, Fig. 1.

TABLE 1: PERCENTAGE INHIBITION OF SUPEROXIDE RADICAL SCAVENGING ACTIVITY IN VITRO BY ETHYLACETATE AND METHANOL EXTRACTS OF MUSSAENDA ERYTHROPHYLLA STEM

AA – Ascorbic acid, EAEME-Ethyl acetate extract of M erythrophylla,MEME-Methanol extract of M. erythrophylla

A single hydroxyl radical can result in formation of many molecules of lipid hydroperoxides in the cell membrane, which may severely disrupt its function and lead to cell death. The ethylacetate extract and methanol extract of M. erythrophylla stem showed concentration dependent activity and the ascorbic acid at various concentrations produced dose dependent inhibition of hydroxyl radicals. The IC₅₀ values for hydroxyl radical with ethylacetate extract and methanol extract of M. erythrophylla stem were found to be 250.82µg, 321.42µg, with ascorbic acid was found to be 231.96 µg respectively. The ethylacetate extract of M. erythrophylla stem was found to have better hydroxyl radical scavenging activity when compared to methanol extract of M. erythrophylla, as shown in Table 2, Fig. 2.

TABLE 2: PERCENTAGE INHIBITION AND IC₅₀ VALUES OF HYDROXYL RADICAL SCAVENGING ACTIVITY IN VITRO BY ETHYL ACETATE AND METHANOL EXTRACTS OF MUSSAENDA ERYTHROPHYLLA STEM

AA – Ascorbic acid, EAEME-Ethyl acetate extract of M erythrophylla, MEME-Methanol extract of M. erythrophylla

Free radicals induce lipid peroxidation in polyunsaturated lipid rich areas like brain and liver ²⁵. In this study, in vitro lipid peroxidation was induced in rat liver by using ammonium ferrous sulphate and ascorbic acid. The extract showed concentration dependent prevention towards generation of lipid peroxides. The IC₅₀ values for the lipid peroxidation inhibiting activity with ethyl acetate extract and methanol extract of M. erythrophylla stem were found to be 321.32µg, 355.13µg; with ascorbic acid was found to be 183.51 µg respectively. The lower the IC₅₀, the higher the free radical scavenging ability. The ethylacetate extract of M. erythrophylla stem was found to have higher lipid peroxidation inhibition than the methanol extract of M. erythrophylla as shown in Table 3, Fig. 3.

TABLE 3: PERCENTAGE INHIBITION AND IC₅₀ VALUES OF LIPID PEROXIDATION IN VITRO BY ETHYL ACETATE AND METHANOL EXTRACTS OF MUSSAENDA ERYTHROPHYLLA STEM

AA – Ascorbic acid, EAEME-Ethyl acetate extract of M erythrophylla,MEME-Methanol extract of M. erythrophylla

DPPH assay has been extensively used for screening antioxidant activity because it can accommodate many samples in a short period and is sensitive enough to detect active ingredients at low concentration ²⁶. When DPPH radicals encounter a proton donating substance such as an antioxidant, it would be scavenged and the absorbance is reduced. Thus, the DPPH radicals were widely used to investigate the scavenging activity of some natural compounds. The mean IC₅₀ values for DPPH radical with ethylacetate extract of M. erythrophylla stem and methanol extract of M. erythrophylla stem were found to be 159.86 µg, 174.35 µg; with ascorbic acid were found to be 75.22 µg respectively. The lower the IC₅₀, the higher the freeradical scavenging ability. The ethylacetate extract of M. erythrophylla stem was found to have better DPPH radical scavenging activity when compared to methanol extract of M. erythrophylla, as shown in Table 4, Fig. 4.

TABLE 4: PERCENTAGE INHIBITION AND IC₅₀ VALUES OF DPPH RADICAL SCAVENGING ACTIVITY IN VITRO BY ETHYL ACETATE AND METHANOL EXTRACTS OF MUSSAENDA ERYTHROPHYLLA STEM

AA – Ascorbic acid, EAEME-Ethyl acetate extract of M erythrophylla,MEME-Methanol extract of M. erythrophylla

FIG. 1: IN VITRO CONCENTRATION DEPENDENT PERCENTAGE INHIBITION OF SUPEROXIDE RADICAL SCAVENGING ACTIVITY BY ETHYL ACETATE AND METHANOL EXTRACTS OF MUSSAENDA ERYTHROPHYLLA STEM AND ASCORBIC ACID

FIG. 2: IN VITRO CONCENTRATION DEPENDENT PERCENTAGE INHIBITION OF HYDROXYL RADICAL SCAVENGING ACTIVITY BY ETHYLACETATE AND METHANOL EXTRACTS OF MUSSAENDA ERYTHROPHYLLA STEM AND ASCORBIC ACID

FIG. 3: IN VITRO CONCENTRATION DEPENDENT PERCENTAGE INHIBITION OF LIPID PEROXIDATION BY ETHYL ACETATE AND METHANOL EXTRACTS OF MUSSAENDA ERYTHROPHYLLA STEM AND ASCORBIC ACID

FIG. 4: IN VITRO CONCENTRATION DEPENDENT PERCENTAGE INHIBITION OF DPPH RADICAL SCAVENGING ACTIVITY BY ETHYLACETATE AND METHANOL EXTRACTS OF MUSSAENDA ERYTHROPHYLLA STEM AND ASCORBIC ACID

CONCLUSION: Natural antioxidants such as phenolic acids, flavonoids and tannins possess potent antioxidant activity ²⁷.Sterols like β-sitosterol have been reported for antioxidant activity ²⁸.Terpenoids are also reported to possess antioxidant activity ²⁹.Phytochemical analysis reveal that Mussaenda erythrophylla stem contains phytosterols, triterpenoids, flavonoids, saponins, glycosides and tannins; hence, the observed activity may be due to the presence of any of these constituents. The extracts merits further experiments in vivo.

ACKNOWLEDGMENT: The authors acknowledge UGC for financial support to M.Chinna Eswaraiah to   carry out this   research work.

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