In vitro antioxidant property of siddha formulation, Irunelli karpam

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1 Research Article In vitro antioxidant property of siddha formulation, Irunelli karpam S. Kumar 1, K. S. Maanickha Chelvi 2 *, A. Faridha 3, Gayathri Gunalan 4 ABSTRACT Introduction: Siddha system of medicine is a renowned holistic system of traditional medicine emphasizing curative and preventive measures. The medicines used in siddha are of plant origin, metals, minerals and animal products. Kaya-karpam (Elixir science) is a treasure for the siddha system as they improvise the longevity of life through their anti-oxidant activities. Irunelli karpam is one of the karpa medicine, is a herbo-mineral drug, widely used in siddha for the treatment of inflammatory skin diseases like psoriasis, eczema, urticaria, etc. Mateials and Methods: The aim of the present study was to evaluate the in vitro free radical scavenging activity of Irunelli karpam. DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, nitric oxide radical scavenging activity and total reducing power assay was determined as per standard procedures. Results and Discussion: The results of the present study, it was observed that the IC 50 of the tested drug was found to be 29.73±0.87 µg/ml for DPPH, 61.22±6.75 µg/ml for hydroxyl radical, ± 4.75µg/ml for superoxide radical and 37.94±3.44 for nitric oxide radical respectively. Hence it may be concluded that Irunelli karpam is a potent antioxidant candidate which can be used for the treatment of various non communicable diseases like Cancer, Diabetes, Arthritis and other inflammatory diseases which involve oxidative stress in its pathogenesis. This result is for the world at large from Siddha system of medicine KEY WORDS: Antioxidant, Irunelli karpam, Siddha system, Tamil medicine, Traditional medicine INTRODUCTION Free radicals or highly reactive oxygen species are formed by exogenous chemicals or endogenous metabolic processes in the human body. These are capable of oxidizing bio-molecules, viz., nucleic acids, proteins, lipids, and DNA and can initiate different degenerative diseases such as neurological disorders, cancer, emphysema, cirrhosis, atherosclerosis, and arthritis. [1,2] Antioxidants are the compounds which terminate the attack of free radicals and thus reduce the risk of these disorders. [3] Almost all organisms are protected up to some extent by free radical damage with the help of enzymes such as superoxide dismutase, catalase, and antioxidant compounds, viz., ascorbic acid, tocopherol, phenolic acids, polyphenols, flavonoids, and glutathione. Prior and Cao (1999) Access this article online Website: jprsolutions.info ISSN: reported that antioxidant supplements or dietary antioxidants protect against the damaging effects of free radicals. [4] Plants have a longest history of use as a medicine, food source, and for a variety of daily needs. [5] Of the 250,000 known plant species on the Earth, more than 80,000 are utilized for medicinal purposes. India is one of the world s 12 biodiversity centres with the presence of over 45,000 different plant species. Of these, about 15,000-20,000 plants have a potent medicinal value. However, only species are utilized in routine by traditional communities for their medicinal value. [6] In India, drugs of herbal origin have been used by Ayurveda, Siddha, and Unani systems of medicines since ancient times. Siddha system of medicine is one of the oldest one from Dravidian culture. This system is mainly focused on food as medicine. Kayakarpam is also called as elixir science is unique and treasure of the siddha system. Kayam means body karpam means stone also 1 Department of Siddha, Siddha Central Research Institute (CCRS), Arumbakkam, Chennai, Tamil Nadu, India, 2 Department of Siddha, Siddha Regional Research Institute (CCRS), Poojappura, Thiruvananthapuram, Kerala, India, 3 Department of Siddha, Siddha Regional Research Institute (CCRS), Kuyavarpalayam, Puducherry, India, 4 Department of Biochemistry, Siddha Regional Research Institute (CCRS), Kuyavarpalayam, Puducherry, India *Corresponding author: K. S. Maanickha Chelvi, Department of Siddha, Siddha Regional Research Institute (CCRS), Thiruvananthapuram , Kerala, India. manickaselvi@hotmail.com Received on: ; Revised on: ; Accepted on: Journal of Pharmacy Research Vol 11 Issue

2 known as life span of Brahma according to Hindu mythology. Hence, this medicine is one which makes human body as stone and not affected by any diseases or aging. These kind of medicines are available from herbal preparation, metals and from animal products also. Many of siddhars such as sage Agathiyar and Bohar are written about in various literature. These medicines are preventive as well as cure the disease. The kayakarpam prevent the aging process as one of the actions is antioxidant property. The Irunelli karpam (INK) is made up of Indian gooseberry (nellikai) and sulfur (nelikkai ghanthakam) in equal quantity and prepared by grinding then drying the finished product. This is one of the kayakarpam medicines used for various types of skin diseases such as psoriasis, eczema, and urticaria. The aim of the present study is to evaluate the in vitro free radical scavenging activity of INK and for this 1,1-diphenyl phenyl hydrazyl (DPPH) radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, nitric oxide (NO) radical scavenging activity, and total reducing power assay was determined as per standard procedures. MATERIALS AND METHODS Chemicals DPPH and curcumin were purchased from Sigma, St. Louis, MO, USA. All other reagents used, including solvents were of analytical grade and obtained from Himedia, India. Recordings were made in a UV-vis spectrophotometer (Shimadzu UV-2200). Siddha Drug Commercially available INK was purchased from IMPCOPS depot, Puducherry, was used for the analysis. Drug Preparation The decoction of the Siddha drugs can be prepared, and then aliquots can be used for the study. Prepare the decoction by dissolving 10 g of drug in 100 ml of sterile distilled water and boil for 20 min. Filter and then the filtrate (decoction) should be used for the analysis. 100 µl, 250 µl, 500 µl, 1000 µl, and 2000 µl can be used as aliquots for the antioxidant study of the selected Siddha drug. Radical Scavenging Activity of Epstein Barr Virus (EBV) DPPH radical scavenging activity The free radical scavenging activity was measured using DPPH by the method of Blois. [7] A 0.1 mm solution of DPPH in ethanol was prepared, and 2.96 ml of this solution was added to 0.4 ml of various quantities and the reference compound, after 30 min, absorbance was measured at 517 nm. Butylated hydroxyanisole was used as a reference material. Percent inhibition was calculated by comparing the absorbance values of control and samples. % inhibition = A control /A control Hydroxy radical scavenging activity The hydroxyl radical scavenging activity was determined according to the method reported by Klein and coworkers. Various concentrations of extracts were taken in different test tubes and evaporated to dryness. One milliliter of iron-ethylenediaminetetraacetic acid (EDTA) solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 ml of EDTA (0.018%), and 1 ml of dimethyl sulfoxide (DMSO) (0.85% v/v in 0.1 M phosphate buffer, ph 7.4) were added to these tubes, and the reaction was initiated by adding 0.5 ml of 0.22% ascorbic acid. Test tubes were capped tightly and heated on a water bath at C for 15 min. The reaction was terminated by the addition of 1 ml of ice-cold trichloroacetic acid (TCA) (17.5% w/v). 3 ml of Nash reagent (75.0 g of ammonium acetate, 3 ml of glacial acetic acid, and 2 ml of acetylacetone were mixed and made up to 1 L with distilled water) was added to all of the tubes and left at room temperature for 15 min for color development. The intensity of the yellow color formed was measured spectrophotometrically at 412 nm against reagent blank. [8] Butylated hydroxytoluene (BHT) and catechin were used as reference compound. Percent inhibition was calculated by comparing the results of control and test samples. Percent inhibition = (1 (Control OD/test OD))*100 Superoxide anion radical scavenging activity The superoxide anion scavenging activity was determined by the method described by Nishikimi et al. [9] slightly modified. About 1 ml nitroblue tetrazolium (NBT) solution containing 156 µm NBT dissolved in 1.0 ml 100 mm phosphate buffer, ph 7.4, 1 ml NADH solution containing 468 µm NADH dissolved in 1 ml 100 mm phosphate buffer, ph 7.4, and 0.1 ml various concentration of EBV and reference compounds (20, 40, 60, 80, and 100 µg) were mixwell and the reaction was started by adding 100 µl phenazine methosulfate solution containing 60 µm phenazine methosulfate in 100 mm phosphate buffer, ph 7.4. The reaction mixture was incubated at 25 C for 5 min, and the absorbance at 560 nm was measured against control samples. BHT and quercetin were used as reference compounds. Percent inhibition was calculated by comparing the results of control and test samples. NO radical scavenging activity NO generated from sodium nitroprusside in aqueous solution at physiological ph interacts with oxygen 1136 Journal of Pharmacy Research Vol 11 Issue

3 to produce nitrite ions, which were measured by the Griess reaction. [10] The reaction mixture (3 ml) containing 10 mm sodium nitroprusside in phosphate buffered saline and the reference compound at different concentrations were incubated at 25 C for 150 min. A 0.5 ml aliquot of the incubated sample was removed at 30 min intervals, and 0.5 ml Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride in 2% H 3 PO 4 ) was added. The absorbance of the chromophore formed was measured at 546nm. All tests were performed in triplicate. Percent inhibition of the NO generated was measured by comparing the absorbance values of control and test preparations. Curcumin was used as a positive control. NO scavenged (%)=(A cont )/A cont Where, Acont is the absorbance of the control reaction and Atest is the absorbance in the presence of the sample. Ferric reducing antioxidant potential (FRAP) assay This method is based on the reduction of colorless ferric complex (Fe 3+ tripyridyltriazine) to bluecolored ferrous complex (Fe 2+ tripyridyltriazine) by the action of electron donating antioxidants at low ph. The reduction was monitored by measuring the change of absorbance at 593 nm. The working FRAP reagent was prepared by mixing 10 volumes of 300 mm acetate buffer, ph 3.6, with 1 volume of 10 mm TPTZ (2,4,6-tri(2- pyridyl)-s-triazine) in 40mM HCl and with 1 volume of 20mM ferric chloride. All the required solutions were freshly prepared before their uses. 100 μl of samples were added to 3mL of prepared FRAP reagent. The reaction mixture was incubated in a water bath for 30 min at 37 C. Then, the absorbance of the samples was measured at 593 nm. The difference between absorbance of sample and the absorbance of blank was calculated and used to calculate the FRAP value. % Reduction=A control /A control Total reducing power assay The total reducing power was determined by the method of Oyaizu (1986). [11] Ten milligrams of EBV in 1 ml distilled water milligrams of EBV in 1 ml 200 µm potassium phosphate buffer, ph 6.6, and 2.5 ml 1% potassium ferricyanide (K 3 Fe [CN] 6 ). The mixture was incubated at 50 C for 20 min. A 2.5-ml aliquot of 10% TCA was added to the mixture, which was then centrifuged at 3000 g for 10 min. The upper layer of the solution (2.5 ml) was mixed with 2.5 ml distilled water and 0.5 ml 0.1% FeCl 3 and absorbance was measured at 700 nm. BHT was used as a reference material. All tests were performed in triplicate, and the graph was plotted with the average of the three determinations. RESULTS AND DISCUSSION Several concentrations ranging from 20 to 100 µg/ml of EBV were tested for their antioxidant activity using different in vitro models. It was observed that free radicals were scavenged by the test compounds in a dose-dependent manner in the various methods. DPPH Radical Scavenging Activity DPPH is a free radical, stable at room temperature, which produces a violet solution in ethanol. It is reduced in the presence of an antioxidant molecule, giving rise to uncolored ethanol solutions. [12] The DPPH radical is considered to be a model of lipophilic radical. A chain reaction in lipophilic radicals was initiated by lipid autoxidation. [13] The radical scavenging activity of INK was determined from the reduction in absorbance at 517 nm due to scavenging of stable DPPH free radical. The positive DPPH test suggests that the samples are free radical scavengers. The scavenging effects of INK and ascorbic acid on the DPPH radical are illustrated and compared in Figure 1. INK had significant scavenging effects on the DPPH radical which increased with increasing concentration in the µg/ml range; the scavenging effect of EBV was similar to that of ascorbic acid. The IC 50 value of EBT in the DPPH radical scavenging assay was µg/ml, a statistically significant result (P < 0.05). Hydroxy Radical Scavenging Activity Hydroxyl radical is an extremely reactive species formed in biological systems and has been implicated as highly damaging in free radical pathology, capable of damaging almost every molecule found in living cells. [14] This radical has the capacity to join nucleotides in DNA and cause strand breakage, which contributes to carcinogenesis, mutagenesis, and cytotoxicity. In addition, this species is considered to be one of the quick initiators of the lipid peroxidation process, abstracting hydrogen atoms from unsaturated fatty acids. Hydroxyl radical scavenging activity was estimated by generating the hydroxyl radicals using ascorbic acid iron EDTA. The hydroxyl radicals Figure 1: 1,1-diphenyl phenyl hydrazyl radical scavenging activity of Irunelli value for INK = µg/ml; ascorbic acid = µg/ml Journal of Pharmacy Research Vol 11 Issue

4 formed by the oxidation react with DMSO to yield formaldehyde, which provides a convenient method for their detection by treatment with Nash reagent. In this study, the crude ethanol leaf extract was checked for its inhibitory effect on hydroxyl radical production. Figure 2 illustrates the percent inhibition of hydroxyl radical by INK. BHT (35.99 µg/ml) was used as a reference compounds. The concentration of INK needed for 50% inhibition was 67.93µg/ml. The results were not significant (P < 0.05) as compared to reference compounds. Superoxide Radical Scavenging Activity Superoxide anion is produced from molecular oxygen due to oxidative enzymes [15] of body by non-enzymatic reaction such as auto-oxidation by catecholamines. [16] The scavenging activity toward the superoxide radical is measured in terms of inhibition of generation of O 2. In the present study superoxide radical reduces NBT to blue colored formazan that is measured at 560 nm. [17] Superoxide anions indirectly initiate lipid oxidation as a result of superoxide and hydrogen peroxide, serving as precursors of singlet oxygen and hydroxyl radicals (Figure 3). [18] NO Radical Scavenging Activity NO is an important chemical mediator generated by endothelial cells, macrophages, neurons and is involved in the regulation of various physiological processes. Excess concentration of NO is associated with several diseases. [19] Oxygen reacts with NO to generate nitrite and peroxynitrite anions which act as free radicals. [20] NO can react rapidly in the intracellular environment to form nitrate, nitrite and S-nitrosothiols. These metabolites play a key role in mediating many xenotoxic effects such as DNA damage. NO causes DNA damage through peroxynitrite. In this study, the ethanol leaf extract was checked for its inhibitory effect on NO production. Figure 2 illustrates the percent inhibition of NO generation by INK. Curcumin was used as a reference compound. The concentration of INK needed for 50% inhibition was µg/ml, whereas 20.4 µg/ml was needed for an equal weight of curcumin. The results were statistically significant (P < 0.05) (Figure 4). FRAP Assay The antioxidant potential of formulations was estimated from their ability to reduce TPRZ-Fe (III) complex to TPTZ-Fe (II). Antioxidant activities are known to increase directly proportional to the polyphenol content. This activity is believed to be mainly due to their redox properties, [21,22] which plays an important role in (a) adsorbing and neutralizing free radicals, (b) quenching singlet and triplet oxygen, and (c) decomposing peroxides. Figure 5 depicts the results of FRAP assay. Total Reducing Power In the reducing power assay, the presence of antioxidants in the sample would result in the reduction of Fe3+ to Fe2+ by donating an electron. The amount Figure 2: Hydroxy radical scavenging activity of Irunelli value for INK = µg/ml; butylated hydroxytoluene = µg/ml Figure 4: Nitric oxide radical scavenging activity of Irunelli value for INK = µg/ml; curcumin = µg/ml Figure 3: Superoxide radical scavenging activity of Irunelli value for INK = µg/ml; butylated hydroxytoluene = µg/ml Figure 5: Ferric reducing antioxidant potential of Irunelli value for INK = µg/ml; ethylenediaminetetraacetic acid = µg/ml 1138 Journal of Pharmacy Research Vol 11 Issue

5 Figure 6: Total reducing power of Irunelli karpam of Fe2+ complex can then be monitored by measuring the formation of Perl s blue at 700 nm. Increasing absorbance indicates an increase in reductive ability. [23] Since reducing power of a compound serves as a significant indicator of its antioxidant activity, INK was assayed for the reducing power activity. It has shown in vitro ferric reducing potential. The OD at 700 nm increased in a dose-dependent manner (Figure 6). CONCLUSION The Siddha formulation INK exhibits potent antioxidant property in various methods when compare to standards. INK is a potent antioxidant candidate which can be used for the treatment of various non-communicable diseases such as cancer, diabetes, and arthritis and other inflammatory diseases which involve oxidative stress in its pathogenesis. This result is for the world at large from Siddha system of medicine. REFERENCES 1. Halliwell B, Gutteridge JM. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J. 1984;219(1): Maxwell SR. Prospect for the use of antioxidant therapies. Drugs. 1995;49: Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med. 1996;20(7): Prior RL, Cao G. Variability in dietary antioxidant related natural product supplements: The need for methods of standardization. J Am Nutraceutical Assoc. 1999;2: Dewick PM. Drugs from nature medicinal natural products: A biosynthetic approach endogenous nitric oxide. Br J Pharmacogn. 1997;110: Joy PP, Thomas J, Mathew S, Skaria BP. Medicinal Plants. Kerala, India: Kerala Agricultural University, Aromatic and Medicinal Plants Research Station; Blois MS. Antioxidant determinations by the use of a stable free radical. Nature. 1958;29: Thabrew MI, Hughes RD, McFarlane IG. Antioxidant activity of Osbeckia aspera, Phytother Res. 1998;12: Nishikimi M, Appaji N, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun. 1972;46(2): Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982;126(1): Oyaizu M. Studies on product of browning reaction prepared from glucose amine. Jpn J Nutr. 1986;44: Silva CG, Herdeiro RS, Mathias CJ, Panek AD, Silveira CS, Rodrigues VP, et al. Evaluation of antioxidant activity of Brazilian plants. Pharmacol Res. 2005;52(3): Kumar RS, Sivakumar T, Sunderam RS, Gupta M, Mazumdar UK, Gomathi P, et al. Antioxidant and antimicrobial activities of Bauhinia racemosa L. stem bark. Braz J Med Biol Res. 2005;38(7): Shimada KK, Fujikawa KY, Nakamura T. Anti-oxidative properties of xanthan on auto-oxidation of soybean oil in cyclodextrin. J Agric Food Chem. 1992;40: Sainani GS, Manika JS, Sainani RG. Oxidative stress: A key factor in pathogenesis of chronic diseases. Med Update. 1997;1: Hemmani T, Parihar MS. Reactive oxygen species and oxidative DNA damage. India J Physiol Pharmacol. 1998;42: Khanam S, Shivprasad HN, Kshama D. In vitro antioxidant screening models: A review. Indian J Pharm Educ. 2004;38: Okuda T, Kimura Y, Yoshida T, Hatano T, Okuda H, Arichi S. Studies on the activities of tannins and related compounds from medicinal plants and drugs. I. Inhibitory effects on lipid peroxidation in mitochondria and microsomes of liver. Chem Pharm Bull (Tokyo). 1983;31(5): Lata H, Ahuja G. Role of free radicals in health and disease. Indian J Physiol Allied Sci. 2003;57: Ialenti A, Moncada S, Di Rosa M. Modulation of adjuvant arthritis by endogenous nitric oxide. Br J Pharmacogn. 1993;110: Adedapo AA, Jimoh FO, Afolayan AJ, Masika PJ. Antioxidant activities and phenolic contents of the methanol extracts of the stems of Acokanthera oppositifolia and Adenia gummifera. BMC Complement Altern Med. 2008;8: Bhaumik UK, Kumar AD, Selvan VT, Saha P, Gupta M, Mazumder UK. Antioxidant and free radical scavenging property of methanol extract of Blumea lanceolaria leaf in different in vitro models. Pharmacol Online. 2008;2: Olayinka AA, Anthony IO. Preliminary phytochemical screening and in vitro antioxidant activities of the aqueous extract of Helichrysum longifolium DC. BMC Complement Altern Med. 2010;10:21. Journal of Pharmacy Research Vol 11 Issue