Introduction

Active oxygen species and free radicals play an important role in the initiation and evolution of numerous diseases. The use of compounds with antioxidant activity is expected to be useful for the treatment of these diseases.Therefore, there has been a growing interest in finding novel antioxidants in order to meet the requirements of pharmaceutical industries^1.Antioxidants are emerging as prophylactic and therapeutic agents for various diseases like cancer, diabetes, cardiovascular disorders, brain dysfunction, inflammation and other degenerative diseases owing to our sedentary way of life and stressful existence. Thus Antioxidants are the substances that delay or prevent the oxidation of cellular substrates.

Mimosa catechu Wild belonging to the family mimosacae  is an important medicinal plant used in ayurveda for many diseases. It was reported that the heartwood of the plant is widely used in conjunctivitis, haemoptysis, catarrh, cough pruritis, leprosy, leucoderma, skin diseases, helmenthiasis, diarrhea, and dysentery, foul ulcers, wounds, fever, anaemia, diabetes, hemorrhages, chest diseases, asthma etc. A mixture of catechu and myrrh is usually prescribed as a galactogogue to women after confinement. It is also used as a hepatoprotective agent. The important chemical constituents ^[2] reported in the heartwood are catechin (2-12%), epicatechin ^[3], catechutannic acid (25-33%), catechin tetramer, dicatechin, gallocatechin, acacatechin (10-12%), catechuic acid, catechu red, kaempferol, taxifolin^[4,5], isorhamnetin, (+) afzelechinn, L-arabinose, D-rhamnose, D-galactose, aldobiuronic acid, quercetin, phlebotannin (25-33%), quercetrin, quercitin. Very scanty work has been reported on Mimosa catechu bark ,hence we have selected the bark of this plant for the present study to explore its antioxidant properties.

The purpose of the present study was to carry out the phytochemical screening and to evaluate the in vitro antioxidant activity of bark of  Ethanolic extract of Mimosa catechu  .

Materials and Methods

Plant material 

The plant material (bark) was collected from a village named Punyakshethram, which is about 20km away from Rajahmundry. The plant was identified and authenticated by the Botanist, Dr.T.U.Raghuram.

Preparation of the plant extract

The fresh bark of the plant was collected, dried under shade and powdered. Finally the dried powder was repeatedly extracted with absolute alcohol (maceration followed by hot percolation).

Chemicals and equipment

All the chemicals used are of analytical grade. DPPH (2,2-diphenyl-1-picryl hydrazyl) has been procured from RESEARCH-LAB FINE CHEM INDUSTRIES, MUMBAI. Gallic acid was a gifted sample from GLAXOSMITHKLINE. Potassium ferricyanide, Ferric chloride, Trichloro acetic acid, Sodium phosphate buffer has been procured from SIGMA CHEMICALS.

The different glassware used for the experimental purpose are of standard quality. UV-Visible double beam (ELICO SL 210), centrifuge machine and shimadzu electronic balance were used for the analysis.

Phytochemical Screening  ^[6-7]

The Ethanolic extract of the bark was subjected to different qualitative tests for the identification of various possible constituents present in it. Thin layer chromatography of the alcoholic extract was also performed by using the solvent system of hexane and ethyl acetate in the ratio of 70:30. Concentrated sulphuric acid in methanol is used as the chromatographic spots detecting agent.

Antioxidant Activity 

The antioxidant potential of the ethanolic bark extract of Mimosa catechu wild was investigated by employing the following methods.

Dpph Method ^[8]

The free radical scavenging activity was followed by  preparing 0.002% DPPH solution in methanol. Gallic acid was taken as the reference standard. Different concentrations of the extract (25, 50 and 100 µg/ml) and standard drug (1 and 2.5 µg/ml) were prepared using methanol. 1ml of 0.002% DPPH solution is mixed with 1ml of all the concentrations of both extract and standard separately. These mixtures are kept in dark for about 30min and the optical density was measured at 517nm. 0.002% DPPH and methanol mixture is the blank. Finally the         % inhibition of the DPPH activity is calculated using the formula.

DPPH Scavenged (%) = [ (A_control– A_test ) / A_control ] × 100

Where A_control  is the absorbance of the control reaction and A_test is the absorbance in the presence of the sample of the extracts. The antioxidant activity of the Ethanolic bark extract was expressed as IC50 and compared with standard. The IC50 value was defined as the concentration (in μg/ml) of extracts that scavenges the DPPH radicals by 50%.

Reducing Power Assay ^[9]

Different concentrations of the extract (25, 50 and 100 µg/ml) and standard drug (1 and 2.5 µg/ml) were prepared using distilled water.1% potassium ferricyanide, 10% trichloro acetic acid, 0.1% ferric chloride and 0.2M phosphate buffer were prepared using distilled water. Gallic acid was taken as the reference standard. Then 1ml of each concentration of both extract and standard were taken separately and mixed with 1ml of 0.2M phosphate buffer (p^H 6.6) and 1ml of potassium ferricyanide. Incubate all these samples at 50⁰C for 20min. Then add 1ml of 10% trichloro acetic acid and centrifuge at 2000rpm for 10min. Now separate the upper layer (2.5ml) and then add (2.5ml) distilled water, 0.5ml of freshly prepared ferric chloride. Finally measure the absorbance at 700nm.

Results and Discussion

Phytochemical Screening

Qualitative screening of the ethanolic bark extract showed positive result for constituents which were displayed in the table1. In the TLC study, six major spots were observed in pink, purple and yellow colors indicating the presence of steroids, glycosides and flavonoids respectively. The results are displayed  in the fig1.

Figure 1 : TLC of Mimosa catechu.   Click here to View figure

Dpph Method 

The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants. Table 2 shows the percentage of DPPH radical scavenged by Gallic acid  and Ethanolic extract of bark at various concentrations (μg/ml). Figure 2,3  illustrates a decrease in the concentration of DPPH radical due to the scavenging ability of the soluble constituents in the Ethanolic extract of leaves of Mimosa catechu and the standard Gallic acid , as a reference compound, presented the highest activity at all concentrations. The IC50 values were found to be 8.6 µg/ml and 0.81 µg/ml for Ethanolic bark extract of Mimosa catechu and Gallic acid respectively.

Figure 2 : free radical scavenging activity of the Mimosa catechu extract.   Click here to View figure

Figure 3: free radical scavenging activity of  GALLIC ACID.   Click here to View figure

Table 2: Results of DPPH radical scavenging activity of ethanolic bark extract of Mimosa catechu

Values are the mean ± SEM., n=3

Reducing Power Assay 

Reducing power assay is based on the principle that substances, which have reduction potential, react with potassium ferricyanide (Fe+3) to form potassium ferrocyanide (Fe+2),which then reacts with ferric chloride to form ferrous complex that has an absorption maximum at 700nm.The reducing capacity of a compound may serve as a significant indicator of it’s potential antioxidant activity^[10]. Table 3  shows the reducing power of ethanolic bark extract of Mimosa catechu From figure 4,5 it was found that the absorbance of the extract increased with the increase in concentrations. Reducing power capabilities of extract was found to be closer to Gallic acid

Table 3: Results of Absorbance in Reducing power assay

Values are the mean ± SEM., n=3

Figure 4: Reducing power Mimosa catechu extract.                        Click here to View figure

Figure 5: Reducing power of  Gallic acid.   Click here to View figure

Conclusion

This research provides information which could trigger further research in the direction of partial or full isolation and characterization of the constituents of bark extract of Mimosa catechu in order to decipher the specific phytochemical constituent(s) responsible for the free radical scavenging activity of the plant. When this work is explored , Mimosa catechu could acquire important therapeutic application in phytotherapy. Experimental pharmacology confirming  the Biological activities of Mimosa catechu extract in vivo system would be helpful to demonstrate the Rational use of this Plant .

Acknowledgement

The authors are thankful to the Vice Chairman Sri.N.Satish Reddy , for providing necessary facilities and constant encouragement through out the research work carried out in the Institution.

References 

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