1. INTRODUCTION:

In recent years, there has been a great deal of attention toward the study of free radicals. Free radicals, reactive oxygen species and reactive nitrogen species are introduced in our body by various endogenous systems, exposure to different physiochemical conditions or some pathological states.^1,2,3 Free radicals are exposed to body more than the body's ability to regulate them, the condition is known as oxidative stress. These free radicals adversely alter lipids, proteins, and DNA and trigger a number of human diseases or disorders^4,5,6,7. A balance between free radicals and antioxidants is necessary for proper physiological functioning^8,9. Hence use of external source of antioxidants can be used to control or overcome oxidative stress due to free radicals^10,11.

The search for effective, nontoxic compounds with antioxidant activity has been intensified in recent years^12,13,14. There are various reports which evaluate antioxidant activity of many natural or herbal and synthetic antioxidant substances. N –Acetyl-D-Glucosamine is a well known compound used for various conditions like arthritis or diabetes to name some^15,16,17. Antioxidant activity can be one of the reasons for its effectiveness and need to be evaluated. The present study comparatively evaluates the antioxidant potential of N –Acetyl-D-Glucosamine with that of standard antioxidant ascorbic acid using DPPH and Hydrogen peroxide scavenging activity^18,19,20.

2. MATERIAL AND METHODS:

2.1 Materials

N-Acetyl-D-Glusosamine, Ascorbic acid, hydrogen peroxide and DPPH were purchased from SD Fine Chem, Mumbai, India. All other chemicals used were of AR grade and used as received.

2.2. Evaluation of In Vitro Antioxidant Activity

2.2.1.: Determination of 1,1- Diphenyl-2-Picrylhydrazyl (DPPH) Radical Scavenging Activity

Solutions of N Acetyl Glucosamine, ascorbic acid were prepared by dissolving/dispersing in distilled water and preparing dilutions ranging from 5 mcg/ml to 250 mcg /ml. Total seven dilutions were prepared for evaluation of antioxidant activity in this study. DPPH (1,1- diphenyl-2-picrylhydrazyl) 0.004% w/v solution was prepared by dissolving 4 mg of DPPH in 100 ml of ethanol. The solution was kept overnight in dark place for the generation of DPPH radical.

An aliquot of 3 ml of 0.004% DPPH solution in ethanol and 0.1 ml of test sample at various concentrations were mixed. The mixture was shaken vigorously and allowed to reach a steady state at room temperature for 30 min. De-colorization of DPPH was determined by measuring the absorbance at 517 nm. A control was prepared using 0.1 ml of distilled water in the place of test sample

The percentage inhibition of free radical by the test sample was calculated using the formula:

Inhibitory ratio =

(A₀ –A₁)

X 100

A₀

Where, A0 is the absorbance of control; A1 is the absorbance of test sample.

2.2.2: Determination of Hydrogen Peroxide Scavenging Activity

Same dilutions of test and standard were used for evaluating antioxidant activity of N-Acetyl-D-Glucosamine by Hydrogen Peroxide Scavenging Activity.

Hydrogen peroxide (0.002%v/v) solution was prepared by transferring 100 μl of hydrogen peroxide (30%) to a volumetric flask and the volume was made up to 15 ml with distilled water. From this, 1 ml of solution was transferred to a volumetric flask and was made to 100 ml with distilled water.

Phosphate buffer (100 mM, pH 7.4) solution was freshly prepared during the study. Solution A: 276 mg of NaH2PO4 was weighed, transferred to a volumetric flask and the volume was made up to 100 ml with distilled water.

Solution B: 568 mg of Na2HPO4 was weighed, transferred to a volumetric flask and the volume was made up to 100 ml with distilled water.

From the above solutions, 12 ml of solution A and 88 ml of solution B were mixed and pH was adjusted to 7.4. Phenol red (0.2 mg/ml) containing Peroxidase (0.1 mg/ml) solution was also freshly prepared during the free radical study. 2 mg of phenol red and 1 mg of horse radish peroxidase were dissolved in 10 ml of 100 mM phosphate buffer solution.

Each 100 μl of the standard and test solutions with different concentrations were mixed with 100 μl of 0.002% H2O2 and 0.8 ml of phosphate buffer and were pre-incubated for 10 min at 37⁰C. 1 ml of phenol red dye containing horseradish peroxidase solution was added to this reaction mixture. After 15 min 50 μl of 1 M NaOH was added and absorbance was measured at 610 nm immediately. A control was prepared using 0.1 ml of distilled water in the place of test or standard solution.

The percentage inhibition of radical by the test sample was calculated using the same formula as that of DPPH scavenging evaluation previously mentioned.

3. RESULTS AND DISCUSSION:

3.1.: DPPH free Radical Scavenging Activity

The results of evaluation of antioxidant activity using DPPH scavenging method indicated significant antioxidant activity for N- Acetyl-D-Glucosamine. Moreover the antioxidant activity of N-Acetyl-D-Glucosamine was found to be dose dependant and comparable to antioxidant activity of standard antioxidant ascorbic acid. N-Acetyl-D-Glucosamine showed about 90 % of antioxidant activity as that of ascorbic acid at same doses. The results of DPPH scavenging activity are depicted in Fig. No. 1.

Fig No. 1: Antioxidant activity of Nacetyl-D-Glucosamine by using DPPH scavenging activity

3.2.: H₂O₂ free Radical Scavenging Activity:

To consolidate the findings of antioxidant activity of N-Acetyl-D-Glucosamine by DPPH scavenging , the antioxidant potential of N-Acetyl-D-Glucosamine was further evaluated Hydrogen peroxide scavenging method. In this method also ascorbic acid was used as standard antioxidant agent. The results are shown in Fig. No. 2. The results confirms that N-Acetyl-D-Glucosamine has significant antioxidant activity. N-Acetyl-D-Glucosamine show more than 80 % of antioxidant activity as compared to ascorbic acid even in hydrogen peroxide scavenging study.

Fig No. 2: Antioxidant activity of Nacetyl d glucosamine using H₂O₂ scavenging activity

4. CONCLUSION:

The results of the study confirm that N-Acetyl-D-Glucosamine have significant antioxidant activity. Moreover the activity is comparable with that of ascorbic acid (more than 80%). This antioxidant potential may be one of the reasons of the therapeutic benefits of N-Acetyl-D-Glucosamine. The results indicate that there is a need for N-Acetyl-D-Glucosamine to be evaluated for more activities which are affected by antioxidant potential like haematinic, osteoarthritis, anti-inflammatory and analgesic effects.

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ABSTRACT: