ASSESSMENT OF ANTI-OXIDANT POTENCY OF SMALLER CHAIN TRIPEPTIDES USING DPPH FREE RADICAL SCAVENGING ASSAY

Transcription

1 ASSESSMENT OF ANTI-OXIDANT POTENCY OF SMALLER CHAIN TRIPEPTIDES USING DPPH FREE RADICAL SCAVENGING ASSAY *K. NAGARAJAN 1, SALIL TIWARI 2, ABHAY BHARDWAJ 3, U. K. BAJAJ 4 ABSTRACT 1, 1-Diphenyl-2-picryl-hydrazyl (DPPH) in-vitro assay was employed to determine the antioxidant potency of test compounds I, II, III, IV and V [Phe-Arg-Asn (FRN); Thr-Arg-Asn (TRN); Glu-His-Arg- (EHR); Glu-Trp-Lys (EWK); Thr-Phe-Arg (TFR)] using ascorbic acid as the standard drug. The percentage scavenging activity of different concentrations of the test drugs were determined and the IC 50 value of the test compounds were subsequently compared with that of standard, ascorbic acid. Among the compounds tested, Compund IV (EWK) have shown maximum potency with an IC 50 value of 14.5 g/ml; whereas compound I (FRN) and Compound II (TRN) gave an IC 50 value of g/ml and 16.1 g/ml correspondingly when compared with standard ascorbic acid (IC 50 = 15.8 g/ml). Based on the above results, Glu-Trp- Lys could be considered for various formulations of antioxidant effect suitable for prevention of human disease. KEY WORDS 1. 1-Diphenyl-2-picryl-hydrazyl, Antioxidant, Tripeptides, Ascorbic acid. AFFILIATION 1. Dr. K. Nagarajan, Professor & Head, Research Laboratory of Central Instrumentation Frontiers, Department of Pharmaceutical Chemistry, KIET School of Pharmacy, 13 Km. Stone, Ghaziabad Meerut Road, Ghaziabad , India, nagarajan_mph@yahoo.co.in. 2. Mr. Salil Tiwari, PG Research Scholar, M.Pharm Research Lab-II, Department of Pharmaceutical Chemistry, KIET School of Pharmacy, 13 Km. Stone, Ghaziabad Meerut Road, Ghaziabad , India. 3. Mr. Abhay Bhardwaj, Assistant Professor, Department of Pharmaceutical Chemistry, KIET School of Pharmacy, 13 Km. Stone, Ghaziabad Meerut Road, Ghaziabad , India. 4. Dr. U. K. Bajaj, Professor & Principal, KIET School of Pharmacy, 13 Km. Stone, Ghaziabad Meerut Road, Ghaziabad , India. *Main author for correspondence 66

2 INTRODUCTION Free radicals easily react with macro-molecules of crucial biological significance (DNA, lipids, protein) and destroy their structure and function what accelerates ageing and might lead to degenerative diseases, including cancer. [1,2] Certain portion of reactive oxygen species (ROS) is generated in normal human metabolism and the production rate is precisely controlled by specialized system of antioxidant defense. [3] This well-balanced ROS synthesis is impaired by inflammatory events, where activated macrophages and neutrophils upon contact with pro-inflammatory stimuli; release substantial amounts of aggressive oxygen and nitrogen-centered radicals. [4] Natural antioxidant defense system involves enzymes (Superoxide dismutase, Catalase, glutathione peroxidase), other proteins (Albumin, ferritin, ceruloplasmin) and numerous smaller molecules (e.g. reduced glutathione, -tocopherol, -carotene, bilirubin, uric acid) of various modes of action. Antioxidant molecules counteract ROS and diminish their deleterious effects. [5,6] This protective barrier can be enhanced by the use of antioxidant micronutrient (vitamins C, E, -carotene) and non-nutrient ingredients of edible plants, like polyphenols. Polyphenol subgroup of chemicals, flavonoids, is the extensively examined group of antioxidants. [7,8] Lunasin is a 43-amino acid peptide identified in soybean which has been found to exert antioxidant properties that could contribute to its chemopreventive effects [9]. Vasoactive intestinal peptide (VIP) was used to evaluate the renoprotective activity against hydrogen peroxide induced oxidative damage in a proximal tubule kidney cell line (human, non-tumor, HK2 cells) and found that VIP decreased the intracellular ROS levels reached by hydrogen peroxide induced oxidative stress, thereby exert a renoprotective effect by the suppression of oxidative stress [10]. Recently, there has been a particular focus on milk-derived peptides as a source of antioxidants, these peptides are inactive within the sequence of the parent protein but can be released during enzyme hydrolysis. Once released, the peptides have been shown to possess radical scavenging, metal ion chelation properties and the ability to inhibit lipid peroxidation [11]. The selenium analogues of captopril represent a novel class of ACE inhibitors exhibiting significant antioxidant activity with the observation that the introduction of L-Phe to Sec-Pro and Cys-Pro peptides significantly increases the ACE inhibitory activity [12]. A study was designed to investigate the in-vitro antioxidant activity of five tripeptide test compounds and to establish the most potent antioxidant drug having therapeutic value. 67

3 MATERIALS & METHODS Chemicals All chemicals were analytical grade and all chemicals required for biochemical assay were obtained from Sigma Chemicals Co., USA, DPPH Radical Scavenging Assay The free radical scavenging activity of the test compounds was measured in-vitro by 1, 1- diphenyl-2-picryl-hydrazyl (DPPH) assay. [9,10] About 0.3 mm solution of DPPH in 100% ethanol was prepared and 1ml of this solution was added to 3ml of test drug dissolved in ethanol at different concentrations (1-32 g/ml). The mixture was shaken and allowed to stand at room temperature for 30 minutes and the absorbance was measured at 517 nm using Uv-visible double beam spectrophotometer (Shimadzu-1800). The % scavenging activity at different concentrations of test drug were determined and the IC 50 value of the test drugs was compared with that of ascorbic acid, which was used as the standard. RESULTS & DISCUSSION The results of DPPH radical scavenging activity of the subjected tripeptide leads are shown in Table 1. Table 1: DPPH Radical Scavenging Activiy of the Tripeptide leads. Compound % Inhibition *IC 50 ( g/ml) 1 g/ml 2 g/ml 4 g/ml 8 g/ml 16 g/ml 32 g/ml Test Compound 1 (FRN) Test Compound 2 (TRN) Test Compound 3 (EHR) Test Compound 4 (EWK) Test Compound 5 (TFR) Standard Drug (Ascorbic Acid)

4 *Values obtained from regression lines with 95% of confidence level. IC 50 is defined as the concentration sufficient to obtain 50% of a maximum effect estimate in 100%. All values given are mean of triplicate experiments at S.D. (5%) for the above table. The graphs of IC50 Value prediction in uv-visible spectrophotometric determination of most potent test drugs and the standard with their linear regression and correlation coefficient are very well shown in figures 1-3. Figure 1: Graphical representation of Linearity for the Standard Drug Concentration with their Corresponding IC 50 Value 69

5 Figure 2: Graphical representation of Linearity for the Test Drug IV (EWK) 70

6 Figure 3: Graphical representation of Linearity for the Test Drug I (FRN). All the test drugs I-V demonstrated H-donor activity. The highest DPPH radical scavenging activity was detected in test drug-iv (Glu-Trp-Lys; EWK) with the IC 50 value of 14.5 g/ml (Table 1), which showed that antioxidant activities are greater than that of standard ascorbic acid (IC 50 = 15.8 g/ml). 71

7 Similarly test drug-i (Phe-Arg-Asn; FRN) and test drug-ii (Thr-Arg-Asn; TRN) have shown almost equal antioxidant capacity with that of the standard drug ascorbic acid by their corresponding IC 50 values as 16.0 g/ml, 16.1 g/ml and 15.8 g/ml respectively. Antioxidants are the compounds that protect cells against the damaging effects of reactive oxygen species, such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite. An imbalance between antioxidants and reactive oxygen species results in oxidative stress, leading to cellular damage. Oxidative stress has been linked to cancer, ageing, atherosclerosis, ischemic injury and inflammation and neuro degenerative diseases. As a comparision with other research works in the similar area, the antioxidant activities of the hydrolysates of walnut proteins was measured with DPPH assay, in which peptide hydrolysate obtained by three hours, especially, the tetra peptide having the sequence Ala-Asp-Ala-Phe exhibited the highest antioxidant activities, which could also quench the hydroxyl radical, chelate ferrous ion, exhibit reducing power and inhibit the lipid peroxidation [15]. In an another study, the antioxidant activities of 28 synthetic peptides, which were designed based on an antioxidative peptide (Leu-Leu-Pro-His-His) derived from proteolytic digests of a soybean protein, against the peroxidation of linoleic acid in an aqueous system measured with ferric thiocyanate method indicated that Pro-His-His was the most antioxidative tripeptide [16]. Earlier research studies investigated by Hernandez-Ledesmaa et al., have also shown that the antioxidant activity was exhibited by the larger aminoacid chain peptide lunasin (43 aminoacids) in concordance with the results obtained for recombinant sporamin and its synthesized peptides. The obtained peptides from later one, viz., GTEKC, SYCQ and VRL have shown corresponding IC 50 values of , and 4.23 mm respectively, when scavenging activity of DPPH radicals (%) was measured [17]. Our research studies suggest that test drugs IV, I and II (EWK, FRN, TRN) with the minimum chain possible amino acid tripeptide combination having better stability than any of the earlier reported comparatively larger chain tetra and pentapeptide and hence possess a remarkable significant antioxidant property that may maintain good health by boosting the immune system and reducing inflammation and allergies. It can be concluded that test drug IV (Glu-Trp-Lys; EWK) could be considered for the preparation of nutraceuticals as made of bio-friendly peptide moieties with potent antioxidant activity suitable for the prevention of human disease. ACKNOWLEDGEMENT The authors are very much thankful to the Director, Dr. S. Narendra Kumar, KIET Group of Institutions, Ghaziabad, India. Also, we remain thankful to Mrs. M. Uma Maheswari, Professor, Department of Pharmacology, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, India for her valuable technical suggestions. 72

8 REFERENCES 1. Ishizaki T, Kishi T, Sasaki F, et al. Effect of probucol, an oral hypocholesterolemic agent, on acute tobacco smoke inhalation in rats. Clin. Sci. 1996; 90: Kehrar JP. Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol. 1993; 23: Ignatowicz E, Rybczynska M. Some biochemical and pharmacological aspects of free radical-mediated tissue damage. Pol. J. Pharmacol. 1994; 46: Halliwell B. Oxygen and nitrogen are pro-carcinogens. Damage to DNA by reactive oxygen, chlorine and nitrogen species: measurement, mechanism and effects of nutrition. Mutat Res. 1999; 443: Halliwell B, Gutteridge JMC. The antioxidants of human extra cellular fluids. Arch Biochem Biophys. 1990; 280: Yu B.P. Cellular defenses against damage from reactive oxygen species. Physiol Rev. 1994; 74: Cotelle N. Role of flavonoids in oxidative stress. Curr Top Med Chem. 2001; 1: Rice-Evans C. Flavonoid antioxidants. Curr Med Chem. 2001; 8: Hernandez-Ledesmaa B, Hsiehb CC, De Lumenb BO. Lunasin, a seed peptide with promising cancer preventive properties. Protein & Peptide Letters. 2012; Sep 13. (Epub ahead of print). 10. Vacas E, Bajo AM, Schally AV, Sanchez-Chapado M, Prieto JC, Carmena MJ. Antioxidant activity of vasoactive intestinal peptide in HK2 human renal cells. Peptides. 2012; Sep 19. Pii: S (12) (Epub ahead of print). 11. Power O, Jakeman P, Fitzgerald RJ. Antioxidative peptides: enzymatic production, invitro and invivo antioxidant activity and potential applications of milk-derived antioxidative peptides. Amino Acids. 2012; Sep 12. (Epub ahead of print). 12. Bhuyan, Bhaskar J, Mugesh, Govindasamy. Antioxidant activity of peptide-based angiotensin converting enzyme inhibitors. Organic and Biomolecular Chemistry. 2012; 10 (11): Mensor LL, Menezes FS, Leitao GG, et al. Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytother. Res. 2001; 15: Nagarajan K, Mazumder A, Ghosh LK, In-vitro antioxidant activity of alcoholic extracts of Wrightia tomentosa. Pharmacologyonline. 2008; 1:

9 15. Chen N, Yang H, Sun Y, Niu J, Liu S. Purification and identification of antioxidant peptides from Walnut (Juglans regia L.) protein hydrolysates. Peptides. 2012; Sep 26. Pii: S (12) (Epub ahead of print). 16. Hua-Ming C, Koji M, Fumio Y, Kiyoshi N. Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. Journal of Agricultural and Food Chemistry. 1996; 44 (9): Huang GJ, Chen HJ, Chang YS, Sheu MJ, Lin YH. Recombinant sporamin and its synthesized peptides with antioxidant activities invitro. Botanical Studies. 2007; 48: