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INTRODUCTION

Free radicals are highly reactive atom having one or more unpaired electron produced from the oxidation reaction that takes place in the transfer of electrons capable of attacking the stable biomolecules. These free radicals initiate cascades of events which ultimately lead to cellular damage [1]. However, physiologically these free radical preserve homeostatic action by signal transduction process [2]. Formation of these highly reactive free radical occur, which has a tendency to break the stable bonds present in the molecules by the pair of electrons [3]. Oxidative stress occurs due to the alteration in the antioxidant level which leads to several disorders such as neurodegenerative diseases, cancer, diabetes mellitus, coronary heart ailments and aging process [4, 5]. The potent role of many antioxidant compounds possesses anti-cancerous, anti-inflammatory, anti-carcinogenic, anti-bacterial or anti-viral activities [6-9].

The sequential event of metabolic processes is carried out in the aid of enzymes. Some of antioxidant enzymes systems catalyse reaction to neutralize the free radicals and reactive oxygen species (ROS) which includes superoxide dismutase, catalase, peroxidase, glutathione transferase and ascorbate oxidase [10]. These form the body’s endogenous defense mechanism to protect against free radical induced oxidative damage. In our system of naturally antioxidants are present such as vitamins which balance the redox reaction in the biological molecule [11]. Today’s and tomorrow’s pharma industry depends on the naturally derived pharmaceutical products for the development of active molecules against chronic diseases.

Lichens are symbiotic partner of photobiont (algal partner) and mycobiont (fungal partner) which exhibits a wide range of biological activities. Lichens occur in different forms such as crustose, foliose and fruticose. Traditionally lichens have been used to treat various ailments viz, diabetes, bronchitis, leprosy, tuberculosis, dyspepsia etc. The metabolites produced by the lichen possess antimicrobial, antioxidant, anthelmintic, anticaries, cytotoxic, insecticidal, larvicidal, wormicidal etc. [12-17]. The objectives of the present study aimed to evaluate different purified extracts of Leptogium papillosum.

MATERIALS AND METHODS

Collection and identification of lichens

The lichen sample was obtained from the Yercaud (Shevaroy hills), Tamil Nadu state, India. These foliose lichen belong to collemataceae family according to the study done by Nayaka et al. [18]. The collected was identified based on its anatomical, morphological and chemical tests. Colour tests were performed in the cortex and the medulla region using three reagents namely 10% potassium hydroxide (K), Steiner’s stable paraphenylenediamine solution (P) and calcium hypochlorite solution (C). The Species confirmation was done by performing thin layer chromatography (TLC) to identify the presence of appropriate secondary metabolites.

Preparation of Homogenate

The lichen thalli was dried and grounded with a mortar and pestle using liquid nitrogen containing 100 mM sodium phosphate buffer (pH 7.4), 0.05% PVPP for extraction. The samples were centrifuged at 10,000 g at 4ºC for 15 min and the supernatant was collected for further use [19].

Ammonium Sulphate Precipitation and Dialysis

The crude extract was subjected to ammonium sulphate precipitation which was done in an ice bath using finely powdered ammonium sulphate. The powder was added slowly to the extract by continuous stirring for complete solubilisation and the solution was centrifuged at 15 000g for 20 min. After discarding the supernatant, the pellet (precipitate) was suspended in 100 mM sodium phosphate buffer (pH 7.4). Then the precipitate was dialyzed in the magnetic stirring condition using dialysis membrane for 24 hrs at 4ºC against the above buffer mix.

Protein Concentration

Protein concentration for the above three purified extracts was determined by Lowry et. al. method [20], by using bovine serum albumin (BSA) as a standard protein.

Superoxide dismutase (SOD) activity

The assay of superoxide dismutase had to be carried out according to the method of Das et al [21]. To the 1.4 ml of aliquot (comprised of 1.11 ml of 50 mM phosphate buffer of pH 7.4, 0.075 ml of 20 mM L-Methionine, 0.04ml of 1% (v/v) Triton X-100, 0.075 ml of 10 mM Hydroxylamine hydrochloride and 0.1ml of 50 mM EDTA) add 100 µl of enzyme extract and incubated at 30ºC for 5 min. After incubation, add 80 µl of 50µM of riboflavin and the tubes were exposed to 200 W-philips fluorescent lamps for 10 min. After the exposure time, add 1 ml of Greiss reagent (mixture of equal volume of 1% sulphanilamide in 5% phosphoric acid) was added. The absorbance was read at 543 nm One unit of enzyme activity was measured as the amount of SOD capable of inhibiting 50% of nitrite formation under assay conditions.

Catalase (CAT) activity

Catalase activity was assayed according to the method of Sinha [22]. 100 µl of enzyme extract was added to the reaction mixture containing 1ml of 0.01 M phosphate buffer (pH 7.0), 0.5 ml of 0.2 M H₂O₂, 0.4 ml of H₂O and incubate at a different time interval. The reaction was terminated by adding 2 ml of the acid reagent (mixing 5% potassium dichromate with glacial acetic 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 absorbance was read at 610 nm. Catalase activity was expressed in terms of µ moles of H₂O₂ consumed/min/mg protein.

Peroxidase (POD) activity

The assay was done by the method of Addy and Goodman [23]. To 0.1 ml of the extract, add the reaction mix consisting of 3ml of buffered pyrogallol [0.05 M pyrogallol in 0.1 M phosphate buffer (pH 7.0)] and 0.5 ml of 1% H₂O₂. Change in the absorbance was measured at 430 nm for every 30 sec for 2 min. The peroxidase activity was expressed in terms of mole of pyrogallol oxidised/ min.

Polyphenol Oxidase (PPO) activity

Assay of Polyphenol oxidase activity was carried out in accordance with the procedure of Sadasivam and Manickam [24]. 0.1 ml of extract was mixed with 3.0ml of distilled water 1.0ml of catecol solution (0.4mg/ml) reactants were quickly mixed. The enzyme activity was measured as change in absorbance/min at 490nm.

Glutathione S Transferase (GST) activity

Glutathione transferase activity using 2, 4 dichloronitrobenzene as substrates was assayed spectrophotometrically by Habig et al., [25]. 3 ml reaction mix consists of 0.1 Μ phosphate buffer (pH 6.5), 1 mM GSH and 1 mM of chlorodinitrobenzene (CDNB) and add 100 µl of enzyme extract. Change in the absorbance at 340 nm was read against blank containing all reagents except the enzyme. Specific activity was expressed as µmol conjugate formed/min/mg protein.

Glutathione Peroxidase (GPx) activity

Glutathione Peroxidase activity was assayed according to the procedure of Rotruck et al. [26]. The reaction mixture consisting of 0.4 ml of 0.4 M sodium phosphate buffer (pH 7.0), 0.1 ml of 10mM sodium azide, 0.2 ml of 4 mM reduced glutathione, 0.1 ml of 2.5 mM H₂O_2, 0.2 ml of water and 0.1 ml of enzyme was incubated at 0, 30, 60, 90 seconds respectively. The reaction was terminated by adding 0.5 ml of 10% TCA and after centrifugation, 2 ml of the supernatant was in addition to 3 ml of phosphate buffer and 1ml of DTNB reagent (0.04% DTNB in 1% sodium citrate). The absorbance was read at 412 nm and the enzyme activity is expressed in terms of µg of glutathione utilized/min/mg protein.

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