Antioxidant potential and total phenolic content of methanolic bark extract of Madhuca indica (koenig) Gmelin.

This study was carried out to investigate the antioxidant and free radical scavenging activity of methanolic extract of Madhuca indica bark in varios systems. DPPH radical, superoxide anion radical, nitric oxide radical, hydroxyl radical, lipid peroxidation, and total phenolic content assays were carried out to evaluate the antioxidant potential of the extract. The percentage inhibition of 40 μg/ml concentration of MMI in DPPH radical scavenging model was found as 74.1%. The scavenging of nitric oxide by the plant extract was concentration dependent and IC(50) value of rutin was found to be 161.7 μg/ml. MMI elicited significant and concentration-dependent superoxide radical scavenging effect with MMI as well as standard curcumin, which exhibited IC(50) values of 38.1 and 5.84 μg/ml, respectively. MMI demonstrated significant scavenging activity of OH(-) radical generated from Fe(2+)-ascorbate-EDTA-H(2)O(2) in a concentration-dependent manner. The extract showed a significant dose-dependent free radical scavenging activity in all the models. The extract showed the presence of high phenolic content corresponding to 98.48 μg equivalent of gallic acid and the antioxidant activity could be attributed to this.

INTRODUCTION

It has been established that oxidative stress is among the major causative factors in induction of many chronic and degenerative diseases including atherosclerosis, diabetes mellitus, cancer, Parkinson's disease and immune dysfunction and is involved in aging.[1-3] Antioxidants, both exogenous and endogenous, whether synthetic or natural, can be effective in prevention of the free radical formation by scavenging or promotion of their decomposition and suppression of such disorders.[14] There is growing interest towards natural antioxidants from herbal sources.[5-7]

Madhuca indica Syn. Madhuca latifolia (Sapotaceae), commonly known as “mahua” in India, is an important economic plant growing throughout the subtropical region of the Indo-Pak subcontinent.[8] Different parts of this plant are used as stimulants, demulcents, emollients, heating, and astringents.[9] The bark is a good remedy for itching, swellings, fractures, and snake bites, as well as for diabetes mellitus.[10] Mahua oil is used for the treatment of skin diseases, rheumatism, headache, and as a laxative. Fruits are astringent and largely used as a lotion for chronic ulcers, in acute and chronic tonsillitis, and in pharyngitis. The constituents reported from M. Indica include fatty acids,[1112] sapogenins,[13] sugars,[10] triterpenoids, steroids,[14-16] saponins,[1718] flavonoids, and glycosides.[151619]

Keeping this in view, this study has been undertaken to investigate antioxidant potential of methanolic extract of M. Indica. The scavenging effect of M. indica bark extract was evaluated against DPPH (1,1-diphenyl,2-picryl hydrazyl), nitric oxide, super oxide, hydroxy radical, and inhibition of lipid peroxidation, and total phenolic content was also determined.

MATERIALS AND METHODS

Plant material

Madhuca indica (Sapotaceae) was purchased from a local market and identified by Dr. K. Madhava Chetty, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India. A voucher specimen (BIT/ Anu/2009/MI-520) was deposited in the herbarium of School of Pharmacy, Bharat Institute of Technology, Meerut, UP, India.

Preparation of plant extract

Madhuca indica bark sample was dried under shade and then powdered with a mechanical grinder to obtain a coarse powder. Equal quantity of powder was passed through 40 mesh sieve and extracted with methanol in Soxhlet apparatus at 60°C. The solvent was completely removed by rotary vacuum evaporator. The extract obtained with methanol was 0.39% w/w. The extract was freeze-dried and stored in vacuum desiccators.

Chemicals

All chemicals used were of analytical grade. Rutin was obtained from Acros organics, New Jersey, USA. DPPH (1,1-diphenyl,2-picryl hydrazyl), NBT (nitro blue tetrazolium), NADH (nicotinamide adenine dinucleotide phosphate reduced), PMS (phenazine methosulphate), TCA (trichloro acetic acid), BHT (butyl hydroxy toluene), and quercetin were obtained from Sigma Chemical Co., USA. Ascorbic acid and vitamin E were obtained from SD Fine Chemicals Ltd, Biosar, India. TBA (thiobarbituric acid) and pyridine were obtained from Loba Chemie, Mumbai, India. Acetic acid, EDTA (ethylene diamine tetra acetic acid disodium salt), and hydrogen peroxide (H2O2) were obtained from Qualigens Fine chemicals, Mumbai, India. Naphthyl ethylene diamine dihydrochloride was obtained from Roch-light ltd, Suffolk, England. Sodium nitro prusside was obtained from Ranbaxy Lab, Mohali, India. Potassium ferric cyanide was obtained from May and Bakers, Dagenham, UK. 2-Deoxy-2-ribose was obtained from Fluka (Buchs, Switzerland) and Folin-ciocalteu's phenol reagent was obtained from SISCO Research Laboratories Pvt. Ltd, Mumbai, India.

In vitro antioxidant activity

Methanolic extract of M. indica bark was tested for its free radical scavenging activity using different in vitro models. All experiments were performed in triplicate and the results averaged.

DPPH radical scavenging activity

The free radical scavenging capacity of the extracts was determined using DPPH. Briefly, 0.15% solution of DPPH in ethanol was prepared and 1 ml of this solution was added to 3 ml of MMI in methanol at different concentrations (1-40μg/ml). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min in dark. Then the absorbance was measured at 515 nm using a spectrophotometer (Perkin-Elmer Lambda 20 UV-visible spectrophotometer). The inhibition curve was plotted and IC50 values were obtained.[20]

Nitric oxide radical scavenging activity

Nitric oxide radical inhibition was estimated by the use of Griess Illosvoy reaction.[21] In this investigation, Griess Illosvoy reagent was modified by using naphthyl ethylene diamine dihydrochloride (0.1% w/v) instead of 1-napthylamine (5%). The reaction mixture (3 ml) containing sodium nitroprusside (10 mM, 2 ml), phosphate buffer saline (0.5 ml) and MMI (10-320 μg) or standard solution (rutin, 0.5 ml) was incubated at 25°C for 150 min. A control without test compound but equivalent amount of methanol was taken. After incubation, 0.5 ml of the reaction mixture was mixed with 1 ml of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Then, 1 ml of naphthyl ethylene diamine dihydrochloride was added, mixed, and allowed to stand for 30 min at 25°C. The concentration of nitrite was assayed at 540 nm and calculated with reference to the absorbance of the standard nitrite solutions.

Superoxide anion radical scavenging activity

The superoxide scavenging activity was determined by the PMS-NADH superoxide generating system[22] and slightly modified. About 1 ml of nitroblue tetrazolium (NBT) solution (156 μM NBT in 100 mM phosphate buffer, pH 7.4) 1 ml NADH solution (468 μM in 100 mM phosphate buffer, pH 7.4) and 0.1 ml of sample solution of MMI (5-80 μg) in water were mixed. The reaction was initiated by adding 100 μl of phenazine methosulphate (PMS) solution (60 μM PMS in 100 mM phosphate buffer, pH 7.4) to the mixture. The reaction mixture was incubated at 25°C for 5 min, and the absorbance at 560 nm was measured against blank samples. Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity.

Inhibition of lipid peroxidation

The effect of crude extract on Fe2SO4-H2O2- induced lipid peroxidation in rat liver homogenate was determined by malondialdehyde (MDA)–TBA adducts formation.[23] The reaction mixture containing 0.5 ml of liver homogenate, 0.05 ml of potassium phosphate buffer (pH 7.4), 0.025 ml of 5nm FeSO4, 0.025 ml of 0.3% H2O2, and different concentration of MMI (50-200 μg) were incubated for 1 h at 37° C. After incubation, TBA (0.4% in 0.2 M HCl) and BHT (0.2% in 95% ethanol) at a ratio of 1:2:0.3 were added, and the mixture was heated at 90°C for 30 min. After cooling, 5 ml of n-butanol was added, and the mixture was separated by centrifugation at 1000 × g for 10 min, and MDA production was measured at 532nm. Using the external standard tetramethoxy propane, the ability to inhibit MDA formation by test extracts was calculated using the following formula:

%Inhibition = MDA in liver homogenate without test - MDA in liver homogenate with test/MDA in liver homogenate without test

Inhibition of hydroxyl radical

The assay was performed as described by Halliwell[24] with minor changes. All solutions were prepared freshly. The reaction mixture (1 ml) contained 100 μl of 28 mM 2-deoxy-2-ribose (dissolved in KH2PO4-K2HPO4 buffer pH 7.4), 500 μl solution of various concentrations of MMI (10-80 μg), 200 μl of 200 μM FeCl3 and 1.04 mM EDTA (1:1 v/v), 100 μl H2O2 (1.0 mM), and 100 μl ascorbic acid (1.0 mM). After an incubation period of 1 h at 37°C, the extent of deoxyribose degradation was measured by the TBA reaction.[2526] The absorbance was measured at 532 nm against the blank solution. Vitamin E was used as a positive control.

Determination of total phenolic content

The contents of total phenolic compounds present in the MMI and MRI were determined using the FolinCiocalteu's phenol reagent and the absorbance was measured at 760 nm according to the method of Slinkard and Singleton.[27] All the tests were performed in triplicate and the results averaged. The concentration of total phenolic compounds in MMI was expressed as gallic acid (μg) equivalent by using the following equation, which was obtained from the standard gallic acid graph.[28]

Absorbance = 0.001 × gallic acid (μg) + 0.0033

RESULTS AND DISCUSSION

The extract showed significant antioxidant activity in vitro by free radical scavenging models. In all models MMI showed dose-dependent results. The percentage of inhibition in various models, viz., DPPH, nitric oxide, super oxide radical, hydroxy radical, and lipid peroxidation are shown in Tables 1–5, respectively. Determination of total phenolic compounds showed that 1 mg of MMI contains 98.48 μg equivalent of gallic acid.

Table 1

Scavenging effect of MMI on the DPPH radical

Table 5

The effects of different concentrations of MRM and MMI on the scavenging activities of hydroxyl radical

MMI as well as ascorbic acid produced significant quenching of DPPH radical due to its scavenging ability [Table 1]. Free radical scavenging activity increased with an increase in concentration. MMI had strong hydrogen donating ability with an IC50 value of 21.7 μg/ml and the value was found to be less than the standard vitamin C (IC50 value of 3.1 μg/ml). The percentage inhibition of 40 mg/ml concentration of MMI in DPPH radical scavenging model was found as 74.1%.

The scavenging of nitric oxide by the plant extract was concentration dependent as illustrated in Table 2. The IC50 value of MMI was found to be 192.8 μg/ml. The IC50 value of rutin was 161.7 μg/ml.

Table 2

Nitric oxide scavenging activity of different concentrations of MMI

MMI elicited significant and concentration-dependent superoxide radical scavenging effect in PMS-NADH-NBT system [Table 3]. MMI and standard curcumin exhibited IC50 values 38.1 and 5.84 μg/ml, respectively.

Table 3

The effects of different concentrations of MMI on the scavenging activities of superoxide anion

MMI inhibited the OH radical-mediated lipid peroxidation by the FeSO4-H2O2 system in a concentration-dependent manner that was determined by the amount of MDA formed in liver homogenate as given in Table 4. The percentage inhibition of MDA formation by 200 mg/ml of MMI was found to be 58.8%. IC50 of MMI was 178.9 μg/ml and that of vitamin E was 119.2 μg/ml.

Table 4

The effect of different concentrations of MMI on FeSO4-H2O2-induced lipid peroxidation in rat liver homogenate in vitro

MMI demonstrated significant scavenging activity of OH- radical generated from Fe2+-ascorbate-EDTA-H2O2 system [Table 5]. The IC50 values of MMI and vitamin E were 71.4 and 32.5 μg/ml, respectively.

The high content of phenolic components in the extract may be a contributing factor toward antioxidant activity because the phenolic compounds are known to have direct antioxidant property due to the presence of hydroxyl groups, which can function as hydrogen donor.[29-31]

CONCLUSION

This study shows that the methanolic extract of M. indica possesses significant antioxidant effects. It may be assumed that the antioxidant property of the methanolic extract of M. indica could be due to the presence of various phenolic compounds. These preliminary results lend to support the use of this plant in folk medicine as an antioxidant.

Thus, the radical scavenging activity, reductive capability, and anti-lipoperoxidant activity strongly suggests that MMI has antioxidant potential. Further studies are needed to evaluate the in vivo antioxidant potential of this extract in various animal models and to isolate the active component.