Dominic Sophia et al. / Journal of Pharmacy Research 2012,5(2),1162-1164 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Antioxidant properties of sonchifolia (L.) :An in vitro study

Dominic Sophia, Paramasivam Ragavendran, Chinthamony Arul Raj, Velliyur Kanniapan Gopalakrishnan* Department of Biochemistry, Karpagam University, Coimbatore- 641 021 (T.N.), Received on:10-11-2011; Revised on: 15-12-2011; Accepted on:12-01-2012

ABSTRACT The present study was examined to assess the enzymatic and non-enzymatic antioxidant levels in the whole of Emila sonchifolia. It was found that the plant possess predominant amount of enzymatic antioxidants namely superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GPx), ascorbate oxidase (AO), glutathione-S-transferase (GST), peroxidase and non-enzymatic antioxidants like total reduced glutathione (TRG), vitamin C. Thus the study infers that Emila sonchifolia is a significant source of natural antioxidants which can scavenge free radicals and in turn can prevent oxidative stress.

Key words: Emila sonchifolia, antioxidants, free radicals, superoxide dismutase, catalase.

INTRODUCTION Natural products are rich in antioxidants that could act against highly reac- and the tubes were exposed for 10 minutes to 200 W-philips fluorescent tive free radicals. These free radicals react with biological macromolecules lamps. After the exposure time, 1ml of Greiss reagent (mixture of equal such as proteins, lipids and DNA and results in the induction of diseases volume of 1% sulphanilamide in 5% phosphoric acid) was added and the [1]. Current research into free radicals has confirmed that foods rich in absorbance of the color formed was measured at 543 nm. One unit of en- antioxidants play an essential role in the prevention of various ailments [2]. zyme activity was measured as the amount of SOD capable of inhibiting The balance between free radical generation and free radical defense deter- 50% of nitrite formation under assay conditions [6]. mines the survival of the system [3]. Emilia sonchifolia () is a well-known herbaceous plant seen in most tropical and subtropical regions Assay of CAT worldwide. It is an edible plant used in the Ayurvedic system of medicine The enzyme extract (0.5 ml) was added to the reaction mixture containing for the treatment of tumor, inflammation, cough, rheumatism and wounds 1ml of 0.01 M phosphate buffer (pH 7.0), 0.5 ml of 0.2 M H2O2, 0.4 ml [4]. Fresh juice and methanolic extract of Emilia sonchifolia leaves were H2O and incubated for different time period. The reaction was terminated found to be potent inhibitors of hydroxyl radical formation and superoxide by the addition of 2 ml of acid reagent (dichromate/acetic acid mixture) radical generation in vitro [5]. The main aim of this study was to assess the which was prepared by mixing 5% potassium dichromate with glacial acetic antioxidant levels of Emilia sonchifolia. acid (1:3 by volume). To the control, the enzyme was added after the addition of acid reagent. All the tubes were heated for 10 minutes and the MATERIALS AND METHODS absorbance was read at 610 nm. Catalase activity was expressed in terms of

Emilia sonchifolia was collected from Thrissur, Kerala, India. The plant µmoles of H2O2 consumed/min/mg protein [7]. was authenticated by Dr. G.V.S Moorthy, Botanical survey of India, TNAU Campus, Coimbatore. The voucher number is BSI/SRC/5/23/09-10/Tech/ Assay of GPx 782. Glutathione peroxidase was assayed according to the method of Rotruck [8] with some modifications. The reaction mixture consisting of 0.4 ml of Sample preparation for antioxidant assays 0.4 M sodium phosphate buffer (pH 7.0), 0.1 ml of 10mM sodium azide,

The samples were prepared by grinding one gram of the whole plant of 0.2 ml of 4 mM reduced glutathione, 0.1 ml of 2.5 mM H2O2, 0.2 ml of Emilia sonchifolia in 2 ml of 50% ethanol in a pre-chilled mortar and pestle. water and 0.5 ml of enzyme was incubated at 0, 30, 60, 90 seconds respec- The extracts were centrifuged at 10,000 g at 4ºC for 10 minutes. The super- tively. The reaction was terminated with 0.5 ml of 10% TCA and after natant was collected and used within 4h for various enzymatic and non- centrifugation, 2 ml of the supernatant was added to 3 ml of phosphate enzymatic antioxidants assays. buffer and 1ml of DTNB reagent (0.04% DTNB in 1% sodium citrate). The color developed was read at 412 nm and the enzyme activity is expressed in Assay of SOD terms of µg of glutathione utilized/min/mg protein. 1.4 ml aliquots of the reaction mixture (comprising 1.11 ml of 50 mM phosphate buffer of pH 7.4, 0.075 ml of 20 mM L-Methionine, 0.04ml of Assay of GST 1% (v/v) Triton X-100, 0.075 ml of 10 mM Hydroxylamine hydrochloride The cuvettes (final volume of 3.0 ml) contained 0.1M phosphate buffer and 0.1ml of 50 mM EDTA) was added to 100µl of the sample extract and (pH 6.5), 1 mM GSH and 1 mM of chlorodinitrobenzene and 20 µl of incubated at 30ºC for 5 minutes. 80 µl of 50 µM riboflavin was then added appropriately diluted enzyme from the different sources. Change in absor- bance at 340 nm was followed against a blank containing all reactants ex- cepting enzyme protein, Specific activity was expressed as µmol conjugate *Corresponding author. formed/min/mg protein [9]. Velliyur Kanniapan Gopalakrishnan Department of Biochemistry, Assay of peroxidase Karpagam University, The reaction mixture consisted of 3ml of buffered pyrogallol (0.05 M pyro- Coimbatore- 641 021 gallol in 0.1 M phosphate buffer (pH 7.0) and 0.5 ml of 1% H2O2. To this (T.N.), India added 0.1 ml enzyme extract and OD change was measured at 430 nm for every 30 seconds for 2 minutes. The peroxidase activity was calculated using an extinction coefficient of oxidized pyrogallol (4.5 litres/mol) [10].

Journal of Pharmacy Research Vol.5 Issue 2.February 2012 1162-1164 Dominic Sophia et al. / Journal of Pharmacy Research 2012,5(2),1162-1164 Assay of AO The sample was homogenized [1 : 5 (w/v)] with phosphate buffer (0.1 M/ pH 6.5) and centrifuged at 3000 g for 15 minutes at 50oC. The supernatant 120 obtained was used as enzyme source. To 3.0 ml of the substrate solution 100 (8.8 mg ascorbic acid in 300 ml phosphate buffer, pH 5.6), 0.1 ml of the 80 enzyme extract was added and the absorbance change at 265 nm was mea- 60 sured for every 30 seconds for a period of 5 minutes. One enzyme unit is 40 equivalent to 0.01 OD change per min [11]. 20 Concentration Estimation of vitamin C 0 The assay mixture for vitamin C consisted of 0.1 ml of brominated sample SOD CAT AO extract, 2.9 ml of distilled water, 1 ml of 2% DNPH reagent and 1-2 drops of thiourea. After incubation at 37ºC for 3 h, the orange-red osazone crys- tals formed were dissolved by the addition of 7 ml of 80% sulphuric acid Values are expressed as mean±SD (n=3). and absorbance was read at 540 nm after 30 minutes. Vitamin C concentra- SOD,CAT,AO = Unit/mg protein tion was expressed in terms of mg/g tissue [12]. 1unit of SOD= inhibition of 50% of nitrite formation

1 unit of CAT=1 µmole of H2O2 consumed/min/mg protein. Estimation of TRG 1 unit of AO=0.01 O.D. change/ min. 1ml of the sample extracts were treated with 4.0 ml of metaphosphoric acid precipitating solution (1.67 g of glacial metaphosphoric acid, 0.2 g EDTA Figure 1: SOD, CAT and AO activity in the whole plant of Emilia and 30 g NaCl dissolved in 100ml water). After centrifugation, 2.0 ml of the sonchifolia protein-free supernatant was mixed with 0.2 ml of 0.4 M Na2HPO4 and 1.0 ml of DTNB reagent (40 mg DTNB in 100 ml of aqueous 1% tri sodium citrate). Absorbance was read at 412 nm within 2 minutes. GSH concentra- tion was expressed as nmol/mg protein [13]. 1000 The values are expressed as mean ± SD (n = 3) and the data were statisti- 800 cally analyzed by using SPSS. 10.0. 600 400 RESULTS AND DISCUSSION Recently, research on phytochemicals and their effects on human health 200 Concentration have been intensively studied. In particular, research has been focused on a 0 search for antioxidants that are widely used as ingredients in dietary supple- GST Gpx Peroxidase ments and are exploited to maintain health and prevent oxidative stress- mediated diseases such as cancer, atherosclerosis, diabetes, inflammation Values are expressed as mean±SD (n=3). and ageing [14]. GST, GPx, Peroxidase = Unit/mg protein 1 unit of GST = nmol CDNB/min/mg protein The inevitable generation of reactive oxygen species (ROS) in biological 1 unit of GPx = µg of GSH utilized/min/mg protein. system and the oxidative damage is counterpoised by the antioxidant de- 1 unit of peroxidase = 1 µmole of pyrogallol oxidized/min fense mechanism. While the production of ROS can be important in several cellular processes (e.g. defense against infection, cellular signaling), the pres- Figure 2: GST, GPX and peroxidase activity in the whole plant of ence of ROS is more often associated with damage to cellular components such as proteins, lipids and nucleic acids [15]. Humans have evolved highly Emilia sonchifolia complex antioxidant systems (enzymatic and nonenzymatic), which work synergistically, and in combination with each other to protect the cells and 300 organ systems of the body against free radical damage. The antioxidants can 250 be endogenous or obtained exogenously eg, as a part of a diet or as dietary supplements. Some dietary compounds that do not neutralize free radicals, 200 but enhance endogenous activity may also be classified as antioxidants. The 150 most efficient enzymatic antioxidants involve SOD, CAT and GPx. Non- enzymatic antioxidants include vitamin E and C, thiol antioxidants (glu- 100 tathione, thioredoxin and lipoic acid), melatonin, carotenoids, natural fla- 50 vonoids, and other compounds. Concentration 0 In this study we have attempted to evaluate the levels of antioxidant in the whole plant of Emilia sonchifolia that is important for its scavenging prop- Vit C TRG erties. In previous study, it was reported that the n-hexane extract of Emilia sonchifolia possess a potential free radical scavenging activity and also Values are expressed as mean±SD (n=3). confirmed the presence of terpenoid, an important medicinal constituent Units: Vitamin C = mg/g plant tissue [16]. The levels of antioxidant enzymes SOD, CAT, AO, GST, GPx and TRG= mM/mg protein peroxidase assessed in Emilia sonchifolia is given in Figure 1 and 2.

Plants have developed several systems to protect themselves against oxida- Figure 3: Vitamin C and Total reduced glutathione activity in the tive stress. Antioxidative enzymes play an important role in this type of protection. The most frequently studied are SOD, which participates in the whole plant of Emilia sonchifolia first line of defence [17]. The most important enzymes protecting

Journal of Pharmacy Research Vol.5 Issue 2.February 2012 1162-1164 Dominic Sophia et al. / Journal of Pharmacy Research 2012,5(2),1162-1164 from oxidative stress belong to a family of several SODs [18]. SOD is apoptosis and protection from Dalton’s Lymphoma challenge in mice by an active fraction from Emilia sonchifolia, Indian J located in various cell compartments and catalyzes the disproportionation Pharmacol, 37, 2005, 232-37. _ of two O2 radicals to H2O2 and O2 [3]. CAT is a major H2O2 scavenging 5 Shylesh BS, Padikkala J, Antioxidant and anti-inflammatory activ- enzyme and is mainly associated with removal of hydrogen peroxide in ity of Emilia sonchifolia, Fitoterapia, 70, 1999, 275-78. peroxisomes, thus protecting cells against oxidative stress [9]. 6 Das K, Samanta L, Chainy GBN, A modified spectrophotometric assay of superoxide dismutase using nitrite formation by superox- AO is primarily located in chloroplast and cytosol and it is the key en- ide radicals, Indian J Biochem Biophys, 37, 2000, 201-204. 7 Sinha AK, Colorimetric assay of catalase, Anal Biochem, 47, 1972, zymes of the ascorbate-glutathione cycle that uses ascorbate as reducing 389-394. substrate for H2O2 detoxification. AO uses two molecules of ascorbate to 8 Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, reduce H2O2 to H2O, with the concomitant generation of two molecule of Hoekstra WG, Selenium: Biochemical role as a component of dehydroascorbate. glutathione peroxidase, Science, 179, 1973, 588-590. 9 Habig WH, Pabst MJ, Jakoby WB, Glutathione-S-Transferases: The GST play an important role in detoxification of xenobiotics and utilizes first enzymatic step in mercapturic acid formation, J Biol Chem, 249, 1974, 7130. glutathione to scavenge potentially the toxic compounds including those 10 Addy SK, Goodman RN. Polyphenol oxidase and peroxidase in produced as a result of oxidative stress and is part of the defense mecha- apple leaves inoculated with a virulent or an avirulent strain for nism against the mutagenic, carcinogenic and toxic effects of such com- Ervinia amylovora, Ind Phytopath, 25, 1972, 575-579. pounds. It has been well established that GPx, a selenium enzyme plays a 11 Vines HM, Oberbacher MF, Response of oxidation and phosphory- major role in regulating the concentration of H O and a wide variety of lation in citrus mitochondria to arsenate, Nature, 206, 1965, 319- 2 2 320. organic peroxides [20]. GPx activity generates glutathione disulfide (GSSG) 12 Boyne AF, Ellman GL, A methodology for analysis of tissue sulfhy- that is reduced back to GSH by GR, using NADPH as electron donor [21]. dryl components, Anal Biochem, 46, 1972, 639-53. Peroxidase catalyses the oxidation of various organic substrates in presence 13 Sadasivam S, Manickam A, Vitamins. In: Biochemical methods, of hydrogen peroxides [3]. Peroxidase participates in the lignin biosynthe- Eds. Sadasivam S and Manickam A, 2, New Age International (P) sis which may build up a physical barrier against poisoning of heavy metals Limited, New Delhi, 1996, 185-186. [22]. Besides enzymatic antioxidant reactions, active free radicals can be 14 Kalim MD, Bhattacharyya D, Banerjee A, Chattopadhyay S, Oxi- dative DNA damage preventive activity and antioxidant potential scavenged by small molecules such as alkaloids, flavonoids, carotenoids, of plants used in Unani system of medicine, BMC Complement ascorbate and thiol compounds. Altern Med, 10: 2010, 1-11. 15 Pospisil P, Production of reactive oxygen species by photosystem Like enzymatic antioxidants, non-enzymatic antioxidants like reduced glu- II, Biochim Biophys Acta, 1787, 2009, 1151–1160. tathione, vitamin C was also increased in Emilia sonchifolia (Figure 3). 16 Sophia D, Ragavendran P, Arulraj C, Gopalakrishnan VK, In vitro Glutathione is the major non-protein thiol in plants with many functions in antioxidant activity and HPTLC determination of n-hexane ex- tract of Emilia sonchifolia (L.)DC, J Basic Clin Pharm, 2, 2011, plant metabolism [23]. It can act as an antioxidant, by scavenging radicals, 179-183. resulting in the oxidation of GSH to glutathione disulfide (GSSG) [24]. Glutathione is related to ascorbic acid through the ascorbic acid- glutathione 17 Miszalski Z, Libik M, Surowka E, Niewiadomsk E, Cu/Zn superox- ide dismutase and catalase activities in Pinus mugo needles growing cycle as follows: The oxidized ascorbic acid (dehydroascorbic acid) is en- at elevated stands in the mountains, and their photochemical effi- zymatically reduced to ascorbic acid using electrons coming from GSH ciency of PSII, J Plant Physiol, 162, 2005, 895-902. [25]. 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In Oxidative Stress: Oxi- dants and Antioxidants, Academic Press, London, 1991, 15-22. On the basis of the above data, we can conclude that Emilia sonchifolia has 21 Mitozo PA, De Souza LF, Loch-Necke G, Flesch S, Maris AF, Figueiredo CP, Dos Santos ARS, Farina M, Dafre AL, A study of the a very potential antioxidant network system. relative importance of the peroxiredoxin, catalase, and glutathione- dependent systems in neural peroxide metabolism, Free Radical ACKNOWLEDGEMENT Biol Med, 51, 2011, 69–77. 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Journal of Pharmacy Research Vol.5 Issue 2.February 2012 1162-1164