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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article

A COMPARATIVE STUDY OF ANTI OXIDANT PROPERTIES IN COMMON INDIAN SPICES S. Asha Devi*, M. E. Umasankar, S. Babu.

Division of Biomolecules and Genetics, School of Biosciences and Technology, VIT University, Vellore-632014, India

Article Received on: 16/02/12 Revised on: 27/03/12 Approved for publication: 18/04/12 *Email:devi_asha@reediffmail.com ABSTRCT The present study reports the anti-oxidant potential of the methanolic extract of three spices Syzygium aromaticum, Elettaria cardamom, Piper nigrum. The total phenolic content was high 36.63 mg GAE/g in the Syzygium aromaticum extract where as flavanoids, proanthocynidins contents was high in Piper nigrum 9.16 mg QE/g, and 3.13 mg CE/g respectively. The Syzygium aromaticum extract exhibited stronger 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, ability of chelating ferrous ions and reducing power, whereas Elettaria cardamom extract displayed the striking superoxide anion-scavenging activity. These activities of spices extracts were comparable to the standard antioxidants used. Syzygium aromaticum might be considered as potential antioxidant compared to Elettaria cardamom and Piper nigrum. Keywords: DPPH, Flavonoids, Phenolic, Free radicals, Proanthocyanidin, Scavenger INTRODUCTION Several studies have shown that aromatic and medicinal plants are sources of diverse nutrient and non nutrient molecules, of which many display antioxidant and antimicrobial properties. These can protect the human body against cellular oxidation reactions and pathogens respectively. Since ages several cultures have been using spices and herbs to enhance the flavor and aroma of foods. Scientific experiments since the late 19th century have documented the antioxidant properties of spices, herbs, and their components1. The purpose of this study was to investigate the antioxidant potential of Syzygium aromaticum, Elettaria cardamom, and Piper nigrum. These spices are very integral part of Indian cooking both in vegetarian as well as non-vegetarian cooking. They are common food adjuncts that impart flavor and aroma. The three spices are typical to the hot and humid tropical climates especially in southern India. Studies on the above have demonstrated impressive antioxidant and antibacterial effects. Many of the constituent compounds such as quercetin in pepper is a much more effective antioxidant than many fruit and vegetables, similarly antioxidant properties in cardamom and can increase levels of glutathione, a natural antioxidant in the body. Phenolics, proanthocyanidins and flavonoids are secondary metabolites produced in plants. These compounds exhibit many biological activities namely antiallergenic, antiviral, anti inflammatory, and vasodilating actions. Flavonoids and proanthocyanidin are compounds most commonly known for their antioxidant activity. Additionally, the antioxidant abilities of these metabolites are greater than those of vitamin C and E. Phenolics, flavonoids and phenylopropanoids are oxidized by the enzyme peroxidase, and act in H2O2-scavenging. Their antioxidant action resides mainly in their chemical structure. A rapid and simple method to measure antioxidant capacity of plants can be carried out by using the free radical, 2, 2-Diphenyl-1-picrylhydrazyl (DPPH). DPPH is widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors, and to evaluate antioxidant activity. Further, reducing power, ferrous ion

chelating ability, superoxide anion radical-scavenging activity was tested in this study2. MATERIALS AND METHODS Extract Preparation Fresh spices (Syzygium aromaticum, Piper nigrum, and Elettaria cardamom) were collected and 100g of each spice was powdered in a mixer-grinder. 4g of each of the three spices was extracted separately with methanol for 24 h. Extracts obtained this way were filtered through a Whattmann filter paper. The residue was extracted twice with the solvent. The extracts were dried at 45OC and weighed and stored at 4OC.100 mg of this was weighed and dissolved in 100 ml methanol to prepare the methanolic extract. Determination of Total Phenolic, Flavonoid, and Proanthocyanidin Content Total phenolics content was determined by the Folin-ciocalteu method3. 0.5mL of Folin- ciocalteu reagent was gently mixed with 0.5 mL of methanol extract, the concentration being 1 mg/mL of methanol. 0.5 mL of sodium carbonate (100 mg/mL) was added after 2 minutes. The contents were mixed and allowed to stand for 2 h. After 2 hours the optical density was measured at 765 nm, and total phenolic contents were expressed as mg gallic acid equivalent (GAE)/g dry weight. Flavonoid content was determined as per the procedure of Jia et al4. To 1.25 ml of distilled water, 250 μl of methanol extract solution 75μl of sodium nitrate solution (5%) was added. After 5 minutes 150 μl of 10% ammonium chloride was added. To this 500μl of 1M sodium hydroxide was added, left for 6 minutes and absorbance was measured at 510 nm by diluting the contents with 275µl of distilled water. The standard curve was prepared using quercetin. Total flavonoid contents were expressed as milligrams of quercetin equivalent (QE)/g dry weight. Proanthocyanidin content was determined according to Sun et al procedure5. 0.5 mL of extract solution was mixed with 3 mL of 4% vanillin methanol solution and 1.5 mL of hydrochloric acid. The content was mixed well and left in room temperature for 15 min, and absorbance was measured at 500 nm. Total proanthocyanidins content was expressed as milligrams of catechin equivalent (CE)/g dry weight.

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Determination of DPPH Radical-Scavenging Activity To get different methanolic concentrations (200, 100, 80, 60, 40, 20, 10 and 5 μg/ml) additional methanol was mixed to the extracts. 2 mL of extract from each concentration was mixed with 1 mL of methanolic solution containing DPPH radicals, with final concentration made up to 0.2 mM DPPH. The contents were shaken vigorously and kept in dark for 30 min. Absorbance was measured at 517 nm. Absorbance of control was determined by replacing the sample with methanol. The scavenging activity was calculated using the formula of scavenging Activity (%) = [(A517 of control - A517 of sample)/A562 of control] × 100. Quercetin and BHA were used as standard6. Determination of Reducing Power Methanol extract (2ml) was mixed with 2 mL of 0.2M (pH6.6) phosphate buffer and 2 mL of potassium ferricyanide (10 mg/mL) and incubated at 50⁰C for 20 min. Thereafter, 2 mL of trichloroacetic acid (100 mg/mL) was added. From this mixture, 2 mL was diluted with 2 mL of distilled water and 0.4 mL of 0.1% ferric chloride. The absorbance was measured at 700 nm after 10 minutes. Increased absorbance of the reaction mixture is in direct proportion to the high reducing ability which was duly indicated7. Determination of Superoxide Anion Radical-Scavenging Activity Riboflavin–light NBT system is used to determine superoxide anion scavenging activity8. 0.5 mL of phosphate buffer (50 mM pH 7.6), 0.3 mL riboflavin (50 μM), 0.25 mL PMS (20 mM), and 0.1 mL NBT (0.5 mM) was mixed and to this, 1 mL of methanol extract solution at different concentration was added. And reaction was initiated by illuminating fluorescence lamp. The absorbance was measured at 560 nm after 20 min of incubation. The percent of radical scavenging activity was calculated by the following method: scavenging activity (%) = [(A 560 of control – A560 of sample)/A560 of control] × 100. Quercetin and BHA were used as standard reference. Ability of Chelating Ferrous Ions The Fe2+ chelating ability of the extract was measured by the ferrous iron– ferrozine complex method9. Various dilutions of 10, 8, 6, 4, and 2 mg/mL were prepared and the test extracts were dissolved with methanol; then 0.8 mL extract was mixed with 50 μl of 2 mMFeCl2, 200μl of 5mM ferrozine, and then incubated for 10 min at 25 ± 2⁰C. Absorbance was measured at 562 nm against methanol as blank. Calculation was elucidated using the formula of chelating ferrous ion (%) = [(A562 of control–A 562 of sample/A517 of control] × 100. RESULTS Total Phenolic, Flavanoid, and Proanthocyanidin content Total phenolics, flavonoids and proanthocyanidins of methanolic extracts of Syzygium aromaticum, Piper nigrum, and Elettaria cardamom are presented in Table 1.The amount of these compounds varied in the spices under study. There is significant difference in the phenolic content of Syzygium aromaticum, Piper nigrum, Elettaria cardamom with values 36.63, 5.04 and 15.67 mg GAE/g dry weight respectively. The flavanoid content in Syzygium aromaticum and Elettaria cardamom with values 3.97 and 3.59 mg QE/g respectively. The flavanoid content in Piper nigrum is almost triple (9.16 mg QE/g) of the other two spices. The proanthocyanidin content of Syzygium aromaticum, Piper nigrum and Elettaria

cardamom is quite similar with values 1.51, 3.31 and 2.84 mg CE/g dry weight respectively. DPPH Radical-Scavenging Activity The Syzygium aromaticum, Piper nigrum and Elettaria cardamom extracts displayed DPPH radical scavenging activities but of varying order. (Figure1). Maximum activity was shown by Syzygium aromaticum and minimum by Elettaria cardamom. The radical scavenging potential of the spices used here were dose dependent that increased on increasing the concentration of spice extracts (5-200 μg/mL). The highest antioxidant activity shown by Syzygium aromaticum was 87.53% at 200μg/mL where as highest antioxidant activity shown by Elettaria cardamom and Piper nigrum were 11.77% and 12.61% respectively, very low as compared to Syzygium aromaticum. Reducing Power In this study, the Syzygium aromaticum, Piper nigrum and Elettaria cardamom extracts displayed varying reducing power. The Syzygium aromaticum displayed high reducing power than Elettaria cardamom and Piper nigrum (Figure 2). Superoxide Anion Radical-Scavenging Activity The Syzygium aromaticum, Piper nigrum and Elettaria cardamom extracts displayed difference in their superoxide anion radical-scavenging activities (Figure 3). The Elettaria cardamom and Syzygium aromaticum extracts showed quite similar superoxide anion radical-scavenging activity that is comparatively stronger than Piper nigrum extracts. The superoxide anion radical-scavenging potential of these spices increased with increased amount of dose (i.e.10-100µg/ml). Ability of Chelating Ferrous Ion Metal-chelating of the spices extracts was determined at the concentration range from 2 to 8 mg/ml. As shown in figure 4 the three different spices extracts Syzygium aromaticum, Piper nigrum and Elettaria cardamom demonstrated striking , yet comparable ability of chelating ferrous ion with record values 81.04% , 10.3% , 42.4% respectively at 8 mg/ml. The ability of the spices extracts chelating ferrous ion was also dose dependent that increased with increased amount of dose (i.e. 2-8 mg/ml). DISCUSSION Syzygium aromaticum was found to possess highest phenolic content and lowest proanthocyanidin content. Therefore Syzygium aromaticum proves to have maximum antioxidant activity. Piper nigrum showed highest flavonoids content. Elettaria cardamom shows the lowest amounts of phenolic, flavonoids and proanthocyanidin contents, signifying that it has the least radical scavenging10,11. DPPH is a stable nitrogen centered free radical, and their color change from violet to yellow when is reduced by either the process of hydrogen- or electrondonation12. Substances performing such radical scavenging activity can be considered as antioxidants; and from our study, when comparatively analysed Syzygium aromaticum showed the highest radical scavenging activity compared to Elettaria cardamom and Piper nigrum. One of the important parameter to be considered for determining the antioxidant property is the reducing power.The Syzygium aromaticum displayed high reducing power than Elettaria cardamom and Piper nigrum. Superoxide anion gives rise to the generation of powerful and dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to the oxidative stress13,14. The superoxide anion radical-scavenging potential of these spices is dose dependent. Elettaria cardamom and Syzygium aromaticum extracts showed quite

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similar superoxide anion radical-scavenging activity, but Elettaria cardamom comparatively stronger than Syzygium aromaticum extracts. Ferrous ion is well known for its effective pro-oxidant activity and is widely prevalent in the food systems15. Polyphenols chelate pro-oxidant metal ions, thus prevents free radical formation from the pro-oxidants.16 The Syzygium aromaticum extracts had the highest chelating ferrous ion ability. REFERENCES 1. Sujata RV, Rathod VS, Yesane DP . Screening of three wild edible fruits for their antioxidant potential. Current Botany 2011; 2:48-52. 2. Asha DS, Deepak G. Antioxidant Activities of Methanolic Extracts of Sweet-Flag (Acorus calamus) Leaves and Rhizomes. Journal of Herbs, Spices & Medicinal Plants 2011; 17: 1–11. 3. Liu X, Zhao M, Wang J, Yang B, JiangY. Antioxidant activity of methanolic extract of emblica fruit (Phyllanthus emblica L.) from six regions in China. J. Food Compt. Anal 2008; 21: 219–228. 4. Jia Z, Tang M,Wu J. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry 1999; 64: 555–599. 5. Sun BS, Ricardo-Da-Silva JM, Spranger MI. Critical factors of vanillin assay for catechins and proanthocyanidins. Journal of Agricultural and Food Chemistry 1998; 46: 4267–4274. 6. Shimada K, Fujikawa K, Yahara K, Nakamura T. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. Journal of Agriculture and Food Chemistry 1992; 40: 945–948. 7. Oyaizu M. Antioxidantive activities of browning products of glucosamine fractionated by organic solvent and thin-layer chromatography. Nippon Shokuhin Kogyo Gakkaishi 1998; 35: 771–775.

8. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry 1971; 44: 276–277. 9. Decker EA, Welch B. Role of ferritin as a lipid oxidation catalyst in muscle food. Journal of Agricultural and Food Chemistry 1990; 38: 674–677. 10. Manikandan S, Srikumar R, Parthasarathy NJ, Devi RS. Protective effect of Acorus calamus Linn on free radical scavengers and lipid peroxidation in discrete regions of brain against noise stress exposed rat. Biol. Pharm. Bull. 2005; 28: 2327–2330 11. Manikandan S, Devi RS. 2005. Antioxidant property of α-asarone against noise-stress-induced changes in different regions of rat brain. Pharmacol. Res 2005; 52: 467–474. 12. Brand-Williams W, Cuvelier M, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaftund-Technologie 1995; 28: 25–30. 13.Dahl MK, Richardson T. Photogeneration of superoxide anion in serum of bovine milk and in model systems containing riboflavin and amino acids. Journal of Dairy Science 1978; 61: 400–407. 14. Meyer AS, Isaksen A. Application of enzymes as food antioxidants. Trends Food Science Technology 1995; 6: 300–304 15. Hsu CL, Chen W, Weng YM, Tseng CY. Chemical composition, physical properties, and antioxidant activities of yam flours as affected by different drying methods. Food chemistry 2003; 83: 85–92. 16. Kris-Etherton PM, Hecker KD, Bonanome A, Coval SM, Binkoski AE, Hilpert KF et al.Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. American Journal of Medicine 2002; 113: 71–88.

TABLE 1: Amounts of Phenolics, Flavonoids, and Proanthocyanidins in Methanolic Extracts of Syzygium aromaticum, Piper nigrum, Elettaria

cardamom Compounds Syzygium aromaticum Piper nigrum Elettaria cardamom ____________________________________________________________________________________ Total phenolics (mg GAE/g) 36.63( ±0.15) 5.04(±0.17) 15.67(±0.08) Flavonoids (mg QE/g) 3.97 (±0.15) 9.16(±0.06) 3.59(±0.10) Proanthocyanidins (mg CE/g) 1.51(±0.08) 3.31(±0.11) 2.84(±.09)

Figure1: DPPH radical scavenging activities of methanolic extracts of Syzygium aromaticum, Piper nigrum and Elettaria cardamom.

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Figure 2: Reducing power of methanolic extracts of Syzygium aromaticum, Piper nigrum and Elettaria cardamom

Figure 3: Superoxide anion radical scavenging activity of methanolic extracts of Syzygium aromaticum, Piper nigrum and Elettaria cardamom.

Figure 4: Ferrous ion chelating ability of methanolic extracts of Syzygium aromaticum, Piper nigrum and Elettaria cardamom

Source of support: Nil, Conflict of interest: None Declared