Materials and Methods

Transcription

1 Materials and Methods

2 3. MATERIALS AND METHODS The objectives of the research investigation, Volvariella volvacea (Bulliard ex Fries) Singer: A therapeutic fungal biofactory were executed as follows: 3.1 Mushroom sample collection and extraction Sample collection The fruiting bodies and mycelial cultures of Volvariella volvacea were collected from Tamil Nadu Agricultural University (TNAU), Coimbatore, Tamil Nadu, India Inoculum preparation and mycelial growth V. volvacea cultures were maintained in potato dextrose agar (PDA) medium and sub cultured at regular intervals. Large scale production of mycelia was carried out using improved PDA liquid media under static culture (20 g of glucose, 5 g of protease peptone, 1.5 g of MgSO 4.7H 2 O, 0.2 g of K 2 HPO 4, and 0.4 g of KH 2 PO 4 in 1 L of water with 200 g of boiled potatoes) and were kept in temperature controlled incubator at 25 C for days Sample preparation for analysis of antioxidants The mycelia was separated from the filtrate and washed with deionized water. Fresh mycelium and fruiting bodies weighing, 1.0 g was homogenized with 2.0 ml of 0.1 M Tris-HCl buffer (ph 7.4). The samples were centrifuged at 4 C and the supernatant was collected and was used for the estimation of antioxidant constituents. 3.2 Quantitative analysis of enzymic and non-enzymic antioxidant constituents in the sample The enzymic antioxidants viz., superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-s-transferase, glucose-6-phosphate dehydrogenase, polyphenol oxidase and the non-enzymic antioxidants viz., total reduced glutathione and vitamin C were determined Determination of superoxide dismutase (SOD) activity (Das et al., 2000) This method involves the generation of superoxide radicals of riboflavin and its detection by nitrite formation from hydroxylamine hydrochloride. The formed nitrite reacts with sulphanilic acid and produces diazonium compound which subsequently reacts with naphthylamine to produce a red azo compound whose absorbance is measured at 543 nm.

3 50 mm Phosphate buffer (ph 7.4) 20 mm L-Methionine 1 % (v/v) Triton X mm Hydroxylamine hydrochloride (NH 2 OH. HCl) 50 µm Ethylenediaminetetraacetic acid (EDTA) 50 µm Riboflavin Griess reagent: Mixture of 1 % sulphanilamide, 2 % phosphoric acid and 0.1 % naphthylethylene diamine dihydrochloride. Pipetted 1.4 ml aliquot of the reaction mixture in a test tube containing 1.1 ml phosphate buffer, 75 µl L-methionine, 40 µl triton X-100, 75 µl hydroxylamine hydrochloride and 100 µl EDTA. To the tube, 100 µl of the sample was added followed by preincubation at 37 C for 5 min. 80 µl of riboflavin was added and the tubes were exposed to 200 W Philips fluorescent lamps for 10 min. The control tube contained equal amount of buffer instead of sample. The sample and its respective control were run together. At the end of exposure, 1.0 ml of Greiss reagent was added to each tube and absorbance was measured at 543 nm. One unit of enzyme activity was defined as the amount of SOD capable of inhibiting 50 % of nitrite formation under assay condition Determination of catalase (CAT) activity (Sinha, 1972) Catalase causes rapid decomposition of hydrogen peroxide to water. H 2 O 2 H 2 O + O 2 Dichromate in acetic acid is converted to perchloric acid and then to chromic acetate when heated in presence of H 2 O 2. The chromic acetate thus produced is measured spectrophotometrically at 610 nm. The catalase preparation was allowed to split H 2 O 2 for different periods of time. The reaction is stopped at specific time interval by the addition of dichromate acetic acid mixture and the remaining H 2 O 2 is determined by measuring chromic acetate M Phosphate buffer (ph 7.0)

4 0.2 M Hydrogen peroxide (H 2 O 2 ) 5 % Potassium dichromate Dichromate-acetic acid solution: 1:3 ratio of potassium dichromate mixed with glacial acetic acid. This was again diluted to 1:5 with acetic acid. Standard: 0.1 ml of 0.2 M H 2 O 2 was diluted to 100 ml using water. To 0.9 ml of phosphate buffer, 0.1 ml of sample extract and 0.4 ml of H 2 O 2 was added. At 0 and after 60 sec, 2.0 ml of dichromate-acetic acid solution was added and tubes were kept in boiling water bath for 10 min, and the colour developed was read at 620 nm. Standards in the range of µm were taken and processed as test and blank containing reagent alone. The activity of catalase was expressed as µmoles of H 2 O 2 decomposed/min/mg protein or ml of serum Determination of glutathione peroxidase (GPx) activity (Ellman, 1959) Glutathione peroxidase catalyses the following reaction: 2GSH + H 2 O 2 Se-GPxGSSH +2H 2 O Glutathione was measured by its reaction with DTNB (5,5 -dithionitrobis-2- benzoic acid) to give a compound that absorbs at 412 nm. 0.4 M Phosphate buffer (ph 7.0) 10 mm Sodium azide (NaN 3 ) 2.5 mm Hydrogen peroxide (H 2 O 2 ) 4 mm reduced glutathione 10 % Trichloroactetic acid (TCA) 0.3 M disodium hydrogen phosphate solution 4 mm EDTA: mg/10 ml water. Ellman s reagent: 19.8 mg DTNB (dithionitrobis benzoic acid) in 1 % sodium citrate. To 0.4 ml of buffer, 0.2 ml of EDTA, 0.1 ml of NaN 3, 0.2 ml of reduced glutathione and 0.1 ml of H 2 O 2 were added to two test tubes labeled as test (T) and control (C). To the test added, 0.2 ml of sample and to the control added 0.2 ml of

5 water. The contents were mixed well and incubated at 37 C for 10 min. The reaction was arrested by adding 0.5 ml of 10 % TCA and the contents were centrifuged. To 1.0 ml of the supernatant, 3.0 ml of buffer and 0.5 ml of Ellman s reagent was added and the colour developed was read at 412 nm. Standards in the range of µg of reduced glutathione was taken and treated similarly. The activity was expressed in terms of µmoles of glutathione consumed/min/mg protein or ml of serum Determination of glutathione reductase (GR) activity (Beutler, 1984) Glutathione reductase catalyses the reduction of oxidised glutathione (GSSG) and is assayed by measuring the decrease in absorbance at 340 nm. NADPH (NADH) + H + + GSSG 0.3 M Phosphate buffer (ph 6.8) GR NADP + (NAD) + + 2GSH 25 mm EDTA 12.5 mm oxidised glutathione (GSSG) 3 mm NADPH To 0.2 ml of sample, 1.5 ml of buffer, 0.5 ml EDTA, 0.2 ml GSSG and 0.1 ml NADPH were added. The decrease in optical density was measured against that of the blank at 340 nm. The enzyme activity was calculated in terms of µmoles of NADPH oxidised/min/mg protein or ml of serum Determination of glutathione-s-transferase (GST) activity (Habig et al., 1973) The reaction of 1-chloro 2,4 dinitrobenzene (CDNB) with the sulphydryl group of glutathione is catalysed by glutathione-s-transferase CDNB + GSH GST CDNB - S - Glutathione The conjugate, CDNB - glutathione absorbs light at 340 nm and the activity of the enzyme can therefore be estimated by measuring the increase in absorbance.

6 0.5 M Phosphate buffer (ph 6.5) 30 mm 1-chloro 2,4 dinitrobenzene (CDNB) in 95 % ethanol 30 mm reduced glutathione To 0.1 ml of sample, 1.0 ml of buffer, 1.7 ml of water and 0.1 ml of CDNB were added and incubated at 37 C for 5 min. After incubation, 0.1 ml of reduced glutathione was added. The increase in optical density of the enzyme was measured against blank at 340 nm. The enzyme activity was calculated in terms of µmoles of CDNB conjugate formed/min/mg protein or ml of serum Determination of glucose-6-phosphate dehydrogenase (G6PDH) activity (Balinsky and Bernstein, 1963) Glucose-6-phosphate dehydrogenase is assayed by measuring the increase in absorbance at 340 nm when NADP reduces to NADPH. This reaction takes place when electrons are transferred from glucose-6-phosphate to NADP. 0.1 M Tris HCl buffer (ph 8.2) 0.2 mm NADP 0.1 M Magnesium chloride (MgCl 2 ) 6.0 mm Glucose-6-phosphate The reaction was initiated by adding 0.2 ml of glucose-6-phosphate to the reaction mixture containing 0.4 ml of Tris-HCl buffer, 0.2 ml of NADP, 0.2 ml of MgCl 2, 1.0 ml water and 0.2 ml of sample in a cuvette and the increase in absorbance was measured at 340 nm. The activity was expressed in terms of units/mg protein, where one unit is equal to the amount of enzyme that brought about a change in OD of 0.01/min/mg protein or ml of serum.

7 3.2.7 Determination of polyphenol oxidase (PPO) activity (Rocha and Morais, 2001; Yemenicioglu, 2002) Polyphenol oxidase activity was determined by measuring the increase in absorbance at 412 nm. Substrate solution: 0.05 mm catechol in 0.2 M sodium phosphate and 0.1M citric acid buffer (ph 6.8). The reaction mixture contained 2.9 ml of substrate solution and 0.1 ml of sample. The reduced change in absorbance was recorded at every 30 sec for upto 3 min at 412 nm. One unit of PPO activity was defined as the enzyme that reduced the absorbance by 0.01 per min. The enzyme activity was calculated in terms of 0.01 optical density change/min/mg of protein Estimation of protein (Lowry et al., 1957) Blue colour developed by the reduction of the phosphomolybdicphosphotungstic components in the Folin-Ciocalteau s reagent by the amino acids tyrosine and tryptophan present in the protein, plus the colour developed by the Biuret reaction of the protein with alkaline cupric tartarate are measured at 660 nm. 2 % Sodium carbonate in 0.1 N sodium hydroxide (Reagent A) 0.5 % Copper sulphate in 1 % potassium sodium tartarate (Reagent B) Alkaline copper reagent: Mixed 50 ml of A and 1.0 ml of B prior to use Folin-Ciocalteau s reagent: Mixed 1 part of reagent with 2 parts of water Stock standard: 50 mg of bovine serum albumin/50 ml saline. Diluted 10 ml of the stock to 100 ml with distilled water. Working standard solution of 0.2 to 0.1 ml ( µg/ml) was pipetted into test tubes and 0.1 ml of the sample was also taken and their volumes were made up to 1.0 ml with distilled water. 5.0 ml of alkaline copper reagent was added to each tube, mixed well and were allowed to stand for 10 min. Folin-Ciocalteau s reagent of

8 0.5 ml volume was added, mixed well and incubated at room temperature for 30 min. Reagent blank was also run simultaneously. After 30 min, the blue colour developed was read at 660 nm. The results were expressed as mg/g tissue or g/l of serum Determination of total reduced glutathione (GSH) (Moron et al., 1979) Ellman s method for estimation of total reduced glutathione is based on the development of yellow colour, when dithionitrobis benzoic acid (DTNB) is added to the compounds containing sulphydryl groups. 0.2 M Phosphate buffer (ph 8.0) 5 % Trichloroacetic acid (TCA) Ellman s reagent: 19.8 mg of DTNB in 100 ml of 1 % sodium citrate solution 0.3 M Disodium hydrogen phosphate. Standard glutathione solution: 20 mg of reduced glutathione was dissolved in 100 ml of distilled water. Standard glutathione solution of 0.2 to 1.0 ml ( µg/ml) was used. Volume in all the tubes was made up to 1.0 ml with distilled water. 0.5 ml of sample was precipitated with 2.0 ml of 5 % TCA. 1.0 ml of supernatant was taken after centrifugation. 0.5 ml of Ellman s reagent and 3.0 ml of phosphate buffer was added to all the tubes. The absorbance was read at 412 nm within 2 min against the reagent blank. The amount of glutathione was expressed as μg/g tissue or ml of serum Estimation of vitamin C (Sadasivam and Manickam, 1997) Ascorbic acid is first dehydrogenated by bromination. The dehydro ascorbic acid reacts with 2,4 dinitro phenyl hydrazine to form osazone and dissolves in sulphuric acid to give an orange red coloured solution which is measured at 540 nm. 4 % Oxalic acid 0.5 N Sulphuric acid (H 2 SO 4 ) 2 % 2,4 dinitro phenyl hydrazine (DNPH) in 0.5 N sulphuric acid. 10 % Thiourea

9 80 % Sulphuric acid (H 2 SO 4 ) Stock standard: 100 mg of ascorbic acid/100 ml of 4 % oxalic acid. Diluted 10 ml of stock standard solution to 100 ml with 4 % oxalic acid to form the working standard. Sample extraction: 1.0 g of sample was grounded either mechanically or using a mortar and pestle in 10 ml of 4 % oxalic acid solution. Centrifuged at 1000 rpm for 20 min or filtered and the liquid was collected. An aliquot of the filtrate was transferred to a conical flask and bromine water was added drop wise with constant mixing. The enolic hydrogen atoms in ascorbic acid are removed by bromine. When the extract turned orange yellow due to excess bromine, it was expelled by blowing in air. The volume was made up to 10 ml with 4 % oxalic acid solution. Brominated sample extract of 0.1 ml was taken and was made to 3.0 ml by addition of distilled water. To that 1.0 ml of DNPH reagent followed by 1 to 2 drops of thiourea was added into each tube. A blank was set as above, but with water in place of ascorbic acid solution. The contents in the tube were thoroughly mixed and incubated at 37 C for 3 h and after incubation, tubes were kept in ice bath. Formed orange red ozazone crystals were dissolved by addition of 7.0 ml of 80 % H 2 SO 4 drop wise while the tubes were still in the ice bath. The tubes were removed and allowed to stand at room temperature for 30 min and its absorbance was read at 540 nm. The amount of vitamin C is expressed as µg/g tissue. 3.3 Determination of total carbohydrates by phenol sulphuric acid method (Dubois et al., 1956) In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This forms a green coloured product with phenol and has absorption maximum at 490 nm. 5 % Phenol Sulphuric acid (H 2 SO 4 ) Stock standard glucose: 100 mg/100 ml water. Working standard solution of 0.2 to 1.0 ml ( µg/ml) was pipetted out in test tubes and the volumes were made to 1.0 ml with distilled water. Sample of

10 1.0 ml volume and 1.0 ml distilled water as blank was taken in other tubes. 1.0 ml of phenol solution and 5.0 ml of 96 % H 2 SO 4 was added to all the tubes and after 10 min the tubes were heated in water bath at C for 20 min. The developed colour was read at 490 nm. 3.4 Mineral content determination in fruiting bodies and mycelium of V. volvacea Determination of ash content (Raghuramulu et al., 2003) About 5.0 g of the sample was weighed accurately into platinum (or porcelain) crucible (which has been previously heated to about 600 C and cooled). The crucible was placed over a low flame till all the material got completely charred followed by heating in a muffle furnace for about 3.5 h at about 600 C. It was cooled in a desiccator and weighed. To ensure completion of ashing, the crucible was again heated in the muffle furnace for an hour, cooled and weighed. This was repeated till two consecutive weights were same and the ash was almost white or grayish white in colour. Weight of the ash Ash content (mg/g sample) = Weight of the sample taken Ash Solution The ash that has been obtained as said above was moistened with small amount of glass distilled water ( ml) and 5.0 ml of diluted HCl was added to it. The mixture was evaporated to dryness on a boiling water bath and another 5.0 ml of HCl was added again and the solution was evaporated to dryness as before. 4.0 ml of HCl and a few ml of water were added and the solution was warmed over a boiling water bath and filtered into a 100 ml volumetric flask using Whatman No. 40 filter paper. After cooling, the volume was made to 100 ml and suitable aliquots were used for the estimation of phosphorus, iron, magnesium and calcium Estimation of calcium (Raghuramulu et al., 2003) Calcium is precipitated from sample as oxalate. The precipitate is dissolved in acid and the oxalate is determined titrimetrically with potassium permanganate. 4 % Ammonium oxalate Ammonia 2 % (v/v solution)

11 0.01 N Potassium permanganate (KMnO 4 ) (Prepared freshly) 1 N Sulphuric acid (H 2 SO 4 ) 0.01 N oxalic acid: 630 mg of oxalic acid was dissolved in redistilled water and 5.0 ml of concentrated sulphuric acid was added and final volume was made up to 1 L. To 2.0 ml of the sample, 2.0 ml of distilled water and 1.0 ml of 4 % ammonium oxalate were added, mixed well and allowed to stand overnight. Precipitated calcium was collected and 3.0 ml of 2 % ammonia was added along the sides of the tube. Precipitate was mixed well and centrifuged again and the supernatant was separated. This step was repeated until the supernatant gave no precipitate with calcium chloride solution. 2.0 ml of 1.0 N H 2 SO 4 was added and the precipitate was mixed well with the acid, placed in a beaker containing almost boiling water to complete the solution of oxalate. Removed and titrated against 0.01 N KMnO 4, keeping the mixture at C to a faint pink colour, which persisted for about a minute. 2.0 ml of 1.0 N H 2 SO 4 is titrated as blank. The difference between the two titration values gives the volume of 0.01 N KMnO 4. The results were expressed as mg/g sample Estimation of iron (Raghuramulu et al., 2003) Iron is determined colorimetrically with ferric iron which gives a blood red colour with potassium thiocyanate. 30 % Sulphuric acid (H 2 SO 4 ) 7 % Potassium per sulphate solution 30 % Potassium thio cyanate solution (KCNS): 40 g KCNS is dissolved in 90 ml glass distilled water, 4 ml acetone is added and the volume is made upto 100 ml. Standard iron solution: mg ferrous ammonium sulphate is dissolved in 100 ml glass distilled water and after addition of 5.0 ml of 1:1 HCl the solution is made upto 1 L and mixed thoroughly (0.1 mg Fe/mL). The standard solution is prepared fresh once in 6 months. Working standard (10 µg Fe/mL) is prepared by diluting the stock 10 fold.

12 : To an aliquot (6.5 ml or less) of mineral solution, enough water was added (if necessary) to make up the volume 6.5 ml. Into a series of test tubes pipetted out ml of standard iron solution, corresponding to 10 to 50 µg. 1.0 ml of 30 % H 2 SO 4, 1.0 ml of potassium per sulphate solution and 1.5 ml of 30 % KCNS solution were added. The developed red colour was measured within 20 min at 540 nm Estimation of magnesium (Raghuramulu et al., 2003) Magnesium that is present in the sample complexes with titan yellow in an alkaline medium resulting in red colouration, which is read at 540 nm. Titan yellow: 75 mg titan yellow was dissolved in 100 ml of double distilled water. 1 volume of solution was diluted with 4 volumes of double distilled water before use. 10 % Sodium hydroxide (NaOH) Magnesium standard - 5 mg/dl 5 % Trichloroacetic acid (TCA) The standard magnesium solution of 0.2 to 1.0 ml was pipetted into a series of test tubes and a blank was set up with 3.0 ml of water. Volume in all the tubes was made to 3.0 ml with water. Sample of 1.0 ml was mixed with 4.0 ml of 5 % TCA. The solutions were centrifuged and 3.0 ml of clear supernatant was taken. To all the tubes 1.0 ml of titan yellow and 1.0 ml of NaOH was added. The colour developed was read immediately at 540 nm and the results were expressed as mg/g sample Estimation of phosphorus (Fiske and Subbaraow, 1925) Phosphorus reacts with molybdic acid to form phosphor molybdic acid. On treatment with 1-amino-2-napthol-4 sulphonic acid (ANSA), phospho molybdic acid is reduced to produce a deep colour (molybdenum blue) which is a mixture of lower acids of molybdenum. The blue colour is measured spectrophotometrically at 660 nm.

13 2.5 % ammonium molybdate: 25 g of ammonium molybdate was dissolved in 200 ml of water and transferred to 1 L flask, containing 300 ml 10 N H 2 SO 4. The solution was made up to 1 L with water. 1-amino-2-napthol-4 sulphonic acid (ANSA) reagent: Added 0.5 g of ANSA to 195 ml of 15 % sodium bisulphate solution in a conical flask and 5 ml of 20 % sodium sulphite was also added and the contents were mixed well and dissolved. This reagent should be prepared freshly. Stock standard phosphate solution: 35.1 mg of potassium dihydrogen phosphate (KH 2 PO 4 ) was weighed and dissolved in water with addition of 1.0 ml of 10 N sulphuric acid and their volume was made up to 100 ml with water such that 1.0 ml of solution contains 80 μg of phosphorus. Working standard: 10 ml of the stock standard was diluted to 100 ml with distilled water. Working standard solution ( ml) corresponding to 8-40 μg of phosphorous and 1.0 ml of the sample solution was pipetted in a series of tubes. The volumes in all the tubes were made to 8.6 ml by addition of distilled water. A blank was set up with 8.6 ml of distilled water. 1.0 ml of 2.5 % ammonium molybdate and 0.4 ml of ANSA was added to all the tubes and were mixed well and allowed to stand for 10 min until blue colour developed, which was read at 660 nm. 3.5 Preparation of sample extracts Preparation of the extracts from V. volvacea fruiting bodies, mycelia and culture filtrate The fruiting bodies were dried and powdered. The propagated mycelia were separated from the filtrate and washed with deionized water, dried overnight at 90 C and powdered. One litre of mycelial extracellular medium was filtered and concentrated to 10 ml by a rotary evaporator and then freeze-dried to a powder form. The dried fruiting bodies, mycelia and culture filtrate (10 g) were extracted by stirring with 100 ml of boiling water at 100 C for 4 h, centrifuged at 5000 rpm for 15 min and filtered through Whatman No. 1 filter paper. The residue was then extracted twice with 100 ml portions of boiling water, as described above. The combined extracts were freeze dried. The dried extracts were stored at 4 C until further use.

14 3.5.2 Extraction of polysaccharides The water soluble polysaccharide fractions were prepared as described by Cheng et al. (2008) with few modifications. Polysaccharides were extracted from dried fruiting bodies and mycelium (~1.5 g) with boiling water (50 ml) for 4 h under agitation. The residues were further extracted with two portions of boiling water over a total of 6 h. The resulting suspension was then centrifuged (4000 rpm for 10 min). It was then concentrated in a rotary evaporator under reduced pressure at 50 C. The concentrated supernatants were then precipitated with three volumes of absolute ethanol (95 %) and polysaccharides were precipitated overnight at 4 C. The precipitated polysaccharides were collected after centrifugation at 3000 rpm for 40 min followed by filtration. 3.6 Qualitative analysis of phytochemicals of V. volvacea The different solvent extracts of fruiting bodies and mycelia were analysed for phytochemicals. The studies were performed as described by Paech and Tracey (1955) and Raaman (2006) Alkaloids Dragendroff s test Dragendroff s reagent: Dissolved 8 g of bismuth nitrate [Bi(NO 3 ) 3 ] in 20 ml of nitric acid and 2.72 g of potassium iodide in 50 ml of water. They were mixed and allowed to stand, where potassium nitrate crystals out. The supernatant was decanted off and made up to 100 ml with distilled water. To 0.5 ml of sample extract, 2.0 ml HCl was added and to that acidic medium 1.0 ml of the reagent was added. Formation of an orange red precipitate immediately indicates the presence of alkaloids Wagner s test Wagner s reagent: Iodine and potassium iodide weighing 1.2 g and 2.0 g each were dissolved in 5 ml of sulphuric acid and the solution was diluted to 100 ml. 10 ml of sample extract was acidified by addition of 15 % v/v HCl and few drops of Wagner s reagent. Formation of yellow or brown precipitate confirmed the presence of alkaloids Meyer s Test Mercuric chloride weighing 1.36 g was dissolved in 60 ml of distilled water and 5 g of potassium iodide in 10 ml of water. Both the solutions were mixed and diluted to

15 100 ml with distilled water. To 1.0 ml of sample extract, few drops of mercuric chloride reagent was added and the formation of white or pale precipitate showed the presence of alkaloids Flavonoids In a test tube containing 0.5 ml of sample extract, 5-10 drops of dilute HCl and small piece of zinc or magnesium was added and the solution was boiled for few min. Reddish pink or dirty brown colour production indicates the presence of flavonoids Saponins In a test tube containing 5.0 ml of sample extract, a pinch of sodium bicarbonate was added. The mixture were shaken vigorously and kept for 3 min. A honey comb like forth was formed, indicating the presence of saponins Carbohydrates Fehling s test Solution A: g of copper sulphate was dissolved in water and made upto 500 ml. Solution B: 125 g of potassium hydroxide and 173 g of Rochelle s salt (Sodium potassium tartarate) were dissolved in water and made upto 500 ml. To 2.0 ml sample extract, 1.0 ml of a mixture of equal parts of Fehling s solution A and B were added. The contents were boiled for a few minutes. Formation of red or brick red precipitate indicates the presence of carbohydrates Benedict s test Benedict s reagent: 173 g of sodium citrate and 100 mg of sodium carbonate were dissolved in 500 ml of water. To this solution 17.3 g of copper sulphate dissolved in 100 ml of water was added. To 0.5 ml of the sample extract, 5.0 ml of Benedict s reagent was added and boiled for 5 min. Formation of a bluish green colour showed the presence of carbohydrates Molisch s test In a test tube containing 2.0 ml of sample extract, 2 drops of freshly prepared 20 % alcoholic solution of α-naphthol was added and mixed. To this solution 2.0 ml of concentrated H 2 SO 4 was added so as to form a layer below the mixture. Formation

16 of the red violet ring at the junction of the solutions and its disappearance on the addition of an excess alkali solution indicates the presence of carbohydrates Proteins Millon s test One part of mercury was digested with two parts of nitric acid and the resulting solution was diluted with 2 volumes of water. To a small quantity of sample extract, 5-6 drops of Millon s reagent was added. A white precipitate which turns red on heating was formed, indicating the presence of proteins Biuret s test To 1.0 ml of sample extract, 5-8 drops of 10 % sodium hydroxide (NaOH) solution was added, followed by one or two drops of 3 % copper sulphate. Formation of red or violet colour confirmed the presence of proteins Phenols Ferric chloride test To 1.0 ml of sample extract, 2 ml of distilled water was added followed by a few drops of 10 % aqueous ferric chloride solution. Formation of blue or green indicates the presence of phenols Lead acetate test Sample extract of 0.1 ml was diluted to 5 ml with distilled water and to this few drops of 1 % aqueous solution of lead acetate was added. A yellow precipitate was formed to indicate the presence of phenols Liebermann s test A small quantity of sample extract was dissolved in 0.5 ml of 20 % sulphuric acid followed by the addition of a few drops of aqueous sodium nitrate solution. A red colour was obtained on dilution and turned blue when made alkaline with aqueous NaOH solution Steroids Libermann-Burchard s test To 1.0 ml of sample extract, 1.0 ml of concentrated sulphuric acid was added followed by the addition of 2.0 ml acetic anhydride solution. A greenish colour developed and it turned blue, indicating the presence of steroids.

17 Salkowski reaction To 2.0 ml of sample extract, 1.0 ml of concentrated sulphuric acid was added carefully along the sides of the tube. A red colour was produced in the chloroform layers Glycosides A small amount of sample extract was dissolved in 1.0 ml of water and then aqueous sodium hydroxide solution was added. Formation of a yellow colour indicates the presence of glycosides Resins To 2.0 ml of sample extract, 5-10 ml of acetic anhydride solution was added, dissolved by gently heating, cooled and then 0.5 ml of sulphuric was added. A bright purple colour rapidly changing into violet, indicates the presence of resins Tannins Ferric chloride test To 2.0 ml of sample extract few drops of 5 % aqueous ferric chloride solution was added. The formed bluish black colour disappears on addition of few ml of dilute sulphuric acid followed by the formation of a yellowish brown precipitate, indicating the presence of tannins Lead acetate test In a test tube containing about 5.0 ml of an aqueous extract, few drops of 1 % lead acetate solution was added. A yellow or red precipitate was formed indicating the presence of tannins Thiols To about 0.5 ml of sample extract, enough ammonium sulphate was added to saturate the solution. 2-4 drops of 5 % sodium nitroprusside was then added followed by one or more drops concentrated nitric acid. A transient magenta colour develops in the presence of thiols.

18 3.7 Estimation of bioactive components Estimation of total phenols (Singleton and Rossi, 1965) The phenolic compounds are oxidized to phenolates by Folin-Ciocalteau s reagent at alkaline ph in a saturated solution of sodium carbonate, resulting in a blue molybdenum-tungstate complex. The colour developed is measured at 650 nm. 35 % Sodium carbonate (Na 2 CO 3 ) Folin-Ciocalteau s reagent Stock standard ( mm): Gallic acid; Catechin; Catechol Mushroom extracts of 1.0 ml volume was mixed with 1.0 ml of Folin- Ciocalteau s phenol reagent, and after 3 min, 1.0 ml of saturated Na 2 CO 3 was added to the mixture and the volume was made up to 10 ml by adding distilled water. The reaction mixture was kept in dark for 90 min, after which its absorbance was read at 725 nm. A calibration curve was constructed with different concentrations of gallic acid, catechol and catechin. The results were expressed as mg of gallic acid (GAE), catechin (CAE), and catechol equivalents (CE) per g of extract Estimation of total flavonoids (Jia et al., 1999) The sample was mixed with a reagent containing aluminium chloride and sodium nitrite. Pink coloured flavonoid aluminium complex was formed in alkaline medium. The intensity of the colour is read at 510 nm. 5 % Sodium nitrite (NaNO 2 ) 10 % Aluminium chloride (AlCl 3 ) 1 M Sodium hydroxide (NaOH) Stock standard (1 mg/ml): Catechol, rutin and quercetin were used Mushroom extracts (0.5 ml) were added into test tubes containing 1.25 ml distilled water and ml of 5 % NaNO 2 solution. The tubes were allowed to stand

19 for 5 min ml of 10 % AlCl 3 was added and after 6 min 0.5 ml of 1.0 M NaOH was also added and the mixtures were diluted with ml of distilled water. The absorbance was measured immediately at 510 nm. The flavonoid content was expressed as milligram of catechol (CE), quercetin (QE) and rutin (RE) equivalents/g extracts. 3.8 In vitro scavenging assays The aqueous extracts of fruiting bodies (FBAE), mycelia (MAE) and culture filtrate (CFAE); polysaccharides from the fruiting bodies (FBPS) and mycelia (IPS) were taken up for analysis of antioxidant capacity by in-vitro scavenging assay methods DPPH radical scavenging activity (Shimada et al., 1992) DPPH (2,2 diphenyl-1-picryl hydrazyl) radical is scavenged by the antioxidants through donation of a proton forming the reduced DPPH. The colour change from purple to yellow after reduction can be quantified by its decrease in absorbance at 517 nm. 0.2 mm 2,2 diphenyl-1-picryl hydrazyl (DPPH) 80 % Methanol Various concentrations of mushroom extracts (4.0 ml) were mixed with 1.0 ml of methanolic solution containing DPPH, where the final concentration of DPPH is 0.2 mm. The mixture was shaken vigorously and left for 30 min at room temperature and its absorbance was measured at 517 nm. Ascorbic acid, trolox, BHA and quercetin ( mg/ml) were used as positive controls. The DPPH radical scavenging activity was calculated as follows: Scavenging activity = [(A 0 -A 1 /A 0 )] x 100, Where A 0 is the absorbance of the control (blank, without extract) and A 1 is the absorbance in the presence of the extract. EC 50 value (mg extract/ml) is the effective concentration at which 50 % of DPPH radicals were scavenged and was obtained by interpolation from linear regression analysis ABTS radical scavenging activity (Re et al., 1999) 2,2 -azinobis(3-ethylbenzothiazidine-6-sulphonicacid) (ABTS) decolourisation assay, involves the generation of ABTS + chromophores by oxidation of ABTS with

20 ammonium persulphate. It is applicable for both hydrophilic and lipophilic compounds. The scavenging activity of the mushroom and mycelium extracts on ABTS radical cation were measured at 734 nm. 7 mm 2,2 -azinobis(3-ethylbenzothiazidine-6-sulphonic acid) (ABTS) 2.45 mm Ammonium per sulphate ABTS solution: 7 mm of ABTS was mixed with 2.45 mm ammonium per sulphate and the mixture was allowed to stand in dark at room temperature for h before use. ABTS.+ solution was diluted to an absorbance of 0.7 ± 0.05 with ethanol at 734 nm. Ethanol The reaction was initiated by the addition of 1.0 ml of diluted ABTS.+ to 10 µl of different concentration of various mushroom extracts and also to 10 µl of methanol, acting as control. The absorbance was read at 734 nm after 6 min and the percentage inhibition was calculated. Ascorbic acid, trolox, BHA and quercetin ( mg/ml) were used as positive controls. The inhibition was calculated according to the equation I = A 0 -A 1 /A 0 x 100, where A 0 is the absorbance of control reaction, A 1 is the absorbance of test compound. EC 50 value (mg extract/ml) is the effective concentration at which 50 % ABTS radicals were scavenged, and was determined by interpolation from linear regression analysis DMPD scavenging activity (Fogliano et al., 1999; Gulcin, 2010) In the presence of Fe 3+ a coloured DMPD (N,N-dimethyl p-phenylenediamine) radical cation is generated; antioxidant compounds transfers a hydrogen atom to DMPD + causing decolouration of the solution that is measured by the decrease in absorbance at 505 nm. 0.1 M Acetate buffer (ph 5.3) 0.05 M Ferric chloride 100 mm DMPD + solution : 20.9 mg of DMPD + was dissolved in 1.0 ml of deionised water and 1.0 ml of this solution was added to 100 ml of 0.1M

21 acetate buffer (ph 5.3), and the coloured radical cation (DMPD + ) was obtained by adding 0.2 ml of 0.05 M ferric chloride. Different concentrations of mushroom extracts were added in test tubes and the total volume was adjusted with distilled water to 0.5 ml. 10 min later, the absorbance was measured at 505 nm. 1.0 ml of DMPD + solution was directly added to the reaction mixture and its absorbance was measured. Ascorbic acid, trolox, BHA and quercetin ( mg/ml) were used as positive controls. The buffer solution was used as a blank sample. The DMPD + scavenging activity was calculated using the following equation: scavenging activity (%) = (A 0 - A 1 /A 0 ) x 100, where A 0 is the absorbance of the initial concentration of DMPD +, A 1 is the absorbance of the remaining concentration of DMPD + in the presence of the extract. EC 50 value (mg extract/ml) is the effective concentration at which 50 % DMPD radicals were scavenged. The value was obtained by interpolation from linear regression analysis Hydroxyl radical scavenging assay (Smirnoff and Cumbes, 1989) Hydroxyl radicals were generated from FeSO 4 and hydrogen peroxide and detected by their ability to hydroxylate salicylate and the hydroxylated salicylate complex is measured at 562 nm. 1.5 mm Ferrous sulphate (FeSO 4 ) 6 mm Hydrogen peroxide (H 2 O 2 ) 20 mm Sodium salicylate Reaction mixture of 3.0 ml volume contained, 1.0 ml of 1.5 mm FeSO 4, 0.7 ml 6 mm H 2 O 2, 0.3 ml of 20 mm sodium salicylate and different concentrations of mushroom extracts. After incubation for an hour at 37 C, the absorbance of the hydroxylated salicylate complex was measured at 562 nm. Ascorbic acid, trolox, BHT and quercetin ( mg/ml) were used as positive controls. The percentage scavenging effect was calculated as, scavenging activity = [1-(A 1 -A 2 ) / A 0 ] x 100, where A 0 is absorbance of the control (without extract) and A 1 is the absorbance in the presence of the extract, A 2 is the absorbance without sodium salicylate.

22 EC 50 value (mg extract/ml) is the effective concentration at which 50 % hydroxyl radicals were scavenged and this value was obtained by interpolation from linear regression analysis Reducing power assay (Oyaizu, 1986) The reducing power was measured by direct electron donation in the reduction of Fe 3+ (CN - ) 6 to Fe 2+ (CN - ) 6. The product was visualized by the addition of free Fe 3+ ions after the reduction reaction, forming an intense prussian blue colour complex, Fe 3+ 4 [Fe 2+ (CN - ) 6 ] 3 and was quantified by measuring its absorbance at 700 nm. 0.2 M Sodium phosphate buffer (ph 6.6) 1 % Potassium ferricyanide 10 % Trichloroacetic acid (TCA) 0.1 % Ferric chloride The reaction mixture contained 2.5 ml of different concentrations of mushroom extracts, and also 2.5 ml of 1 % potassium ferricyanide and 2.5 ml of 0.2 M sodium phosphate buffer. The control contained all the reagents except the sample. The mixture was incubated at 50 C for 20 min and the reaction was terminated by the addition of 2.5 ml of 10 % (w/v) of TCA, followed by centrifugation at 3000 rpm for 10 min. 5.0 ml of the supernatant was mixed with 5.0 ml of deionized water and 1.0 ml of 0.1 % ferric chloride. The absorbance was measured at 700 nm against blank that contained distilled water and sodium phosphate buffer. Increase in absorbance indicates increased reducing power of the sample. Ascorbic acid, trolox, BHA and quercetin ( mg/ml) were used as positive controls. EC 50 value (mg extract/ml) is the effective concentration at which 50 % of reduction has occurred showing the reducing power at an absorbance of 0.5 and the values were obtained by interpolation from linear regression analysis FRAP assay (Benzie and Strain, 1996) The total antioxidant potential of sample is determined using ferric reducing ability as a measure of antioxidant power. FRAP (Ferric ion reducing antioxidant power) assay measures the change in absorbance at 593 nm owing to the formation

23 of blue coloured Fe II-tripyridyl triazine compound from, colourless oxidized Fe III form by the action of electron donating antioxidants. 10 mm 2,4,6-tripyridyl-s-triazine (TPTZ) 40 mm Hydrochloric acid (HCl) 20 mm Ferric chloride (FeCl 3.6H 2 O) 0.3 M Acetate buffer (ph 3.6) FRAP reagent: Reagent contains 2.5 ml TPTZ solution, 2.5 ml ferric chloride solution and 25 ml acetate buffer. The reagent was freshly prepared and warmed at 37 C. The stock solution of 10 mm TPTZ was prepared in 40 mm HCl, 20 mm FeCl 3.6H 2 O and 0.3 M acetate buffer. FRAP reagent of 900 µl was mixed with 90 µl water and with 30 µl different concentrations of mushroom and standard antioxidant solution. The reaction mixture was then incubated at 37 C for 30 min and its absorbance was recorded at 595 nm. An intense blue colour complex was formed when ferric tripyridyl triazine (Fe 3+ -TPTZ) complex got reduced to ferrous (Fe 2+ ) form. The absorption at 540 nm was recorded. Ascorbic acid, trolox, BHA and quercetin ( mg/ml) were used as positive controls. EC 50 value (mg extract/ml) is that effective concentration at reduction of ferrous ions are observed with an absorbance of 0.5 as reducing power and was obtained by interpolation from linear regression analysis CUPRAC assay (Apak et al., 2006; Karman et al., 2010) CUPRAC (Cupric ion reducing antioxidant capacity) method has also been used to determine the reducing power of antioxidant compounds. This method is based on the reduction of Cu 2+ to Cu + by antioxidants in the presence of neocuproine. 10 mm Cupric chloride (CuCl 2 ) 7.5 mm Ethanolic neocuproine 1 M Ammonium acetate buffer (CH 3 COONH 4 ) (ph 7.0)

24 CuCl 2 solution measuring 0.25 ml was added to 0.25 ml of ethanolic neocuproine solution and 0.25 ml CH 3 COONH 4 buffer solution to a test tube, followed by mixing with different concentrations of mushroom extracts. Then, total volume was adjusted to 2.0 ml with distilled water and mixed thoroughly. The tubes were stoppered and kept at room temperature. Absorbance was measured at 450 nm against a reagent blank after 30 min. Ascorbic acid, trolox, BHA and quercetin ( mg/ml) were used as positive controls. Increased absorbance of the reaction mixture indicates increased reduction capability. EC 50 value (mg extract/ml) is that effective concentration at which 50 % of the cupric ions are reduced corresponding to an optical density value of 0.5 and that was obtained by interpolation from linear regression analysis carotene bleaching assay (Yae et al., 2003) The antioxidant activity of the extract was measured by the bleaching of -carotene linoleate system and the absorbance is measured at 470 nm. β-carotene Tween 80 Linoleic acid The -carotene solution was prepared by dissolving 2 mg of -carotene in 10 ml chloroform and 2 ml of this solution was pipetted into a 100 ml round bottom flask. After the chloroform was removed at 40 C under vacuum, 40 mg of linoleic acid, 400 mg of Tween 80 emulsifier and 100 ml of distilled water were added to the flask with vigorous shaking. 4.8 ml of that emulsion was transferred to different test tubes containing 0.2 ml of different concentrations of the mushroom extracts. The tubes were shaken and incubated at 50 C in a water bath. As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm. A blank, devoid of - carotene was prepared for background subtraction. -carotene bleaching inhibition was calculated using the following equation: (-carotene content after 2 h of assay/initial - carotene content) x 100. Ascorbic acid, α-tocopherol, BHA and quercetin ( mg/ml) were used as positive controls.

25 EC 50 value (mg extract/ml) is that effective concentration at which 50 % inhibition of -carotene bleaching was observed and was obtained by interpolation from linear regression analysis Chelating ability on ferrous ions (Dinis et al., 1994) Iron (II) chelating activity was measured by the inhibition of iron (II) - ferrozine complex formation after preincubation of the sample. The Fe 2+ was monitored by measuring the formation of ferrous iron- ferrozine complex against methanol blank at 562 nm. 2 mm Ferric chloride (FeCl 2 ) Methanol 5 mm Ferrozine The reaction mixture contained 1.0 ml of different concentrations of the mushroom extracts, 0.1 ml of 2 mm FeCl 2 and 3.7 ml methanol. The control contained all the reaction reagents except sample. The reaction was initiated by addition of 0.2 ml of 5 mm ferrozine. After 10 min at room temperature, the absorbance of the mixture was determined at 562 nm. A lower absorbance of the reaction mixture indicated a higher Fe 2+ chelating ability. EDTA ( mg/ml) was used as a positive control. The capacity to chelate the ferrous ion was calculated as % chelation = [(A 0 - A 1 )/A 0 ] x 100 EC 50 value (mg extract/ml) is the effective concentration at which 50 % chelating ability is seen and value was obtained by interpolation from linear regression analysis Inhibition of lipid peroxidation in rat liver homogenate (Yen and Hsieh, 1998; Banerjee et al., 2005) Malondialdehyde has been identified as the product of lipid peroxidation that reacts with thiobarbituric acid to give a red colour absorbing at 535 nm. 0.5 mm FeCl 2

26 0.5 mm H 2 O 2 20 % TCA 0.8 % TBA Different concentrations of mushroom extracts of 1.0 ml ( mg/ml) was mixed with 1.0 ml of 10 % liver homogenate, then 0.05 ml of 0.5 mm FeCl 2 and 0.5 mm H 2 O 2 were added to initiate lipid peroxidation. After incubation at 37 C for 60 min, 1.5 ml of 20 % TCA and 1.5 ml of 0.8 % TBA solution (0.8 %, w/v) were added to quench the reaction. The resulting mixture was heated at 100 C for 15 min and then centrifuged at 4000 rpm for 10 min. The absorbance of the upper layer was measured at 532 nm. Ascorbic acid, trolox, BHA and quercetin ( mg/ml) were used as positive controls. The inhibition effect on lipid peroxidation was calculated as follows: Inhibition effect (%) = [1 - (A 1 -A 2 )/A 0 ] x 100, where A 0 was the absorbance of the control (water instead of sample), A 1 is the absorbance of the sample and homogenate, and A 2 was the absorbance of the sample only (water instead of liver homogenate). EC 50 value (mg extract/ml) is the effective concentration at which 50 % LPO of liver was inhibited and its values were obtained by interpolation from linear regression analysis Lipid peroxidation assay in rat liver mitochondria (Visavadiya et al., 2009) Malondialdehyde has been identified as the product of lipid peroxidation that reacts with thiobarbituric acid to give a red colour absorbing at 535 nm. Homogenization buffer (0.25 M sucrose in 5 mm Tris HCl buffer ph 7.4) 0.15 M KCl 20 mm Tris HCl buffer (ph 7.4) 0.2 mm Ascorbic acid 10 M FeSO 4 20 % TCA 0.4 % TBA n-butanol

27 Liver was washed with ice cold normal saline and a 10 % homogenate was prepared. The homogenate was centrifuged at 800 rpm for 5 min at 4 C to separate the nuclear debris and the supernatant was recentrifuged at 10,500 g for 20 min at 4 C. The resulting pellet was washed three times with homogenization buffer, and the final pellet was resuspended in 0.15 M KCl in 20 mm Tris HCl buffer (ph 7.4) to get the mitochondrial fractions (MF). The protein concentration in mitochondrial fraction was determined according to the method of Lowry et al. (1957). A reaction mixture containing 200 µl of mitochondrial fraction (equivalent to 0.5 mg of protein), 200 µl of water (control) or standard/ different concentrations of various mushroom and mycelial extracts ( mg/ml), 200 µl of 10 M FeSO 4 and 200 µl of 0.2 mm ascorbic acid were added to start peroxidation. At the end of this incubation period, 10 µl of 5 % BHT in ethanol was added followed by addition of 0.8 ml 20 % TCA and 0.4 % TBA each. The mixtures were heated in a water bath at 80 C for 20 min. The chromogen was extracted in 2 ml n-butanol and the extent of lipid peroxidation (TBARS) was measured in the organic layer at 532 nm. Ascorbic acid, gallic acid and BHT ( mg/ml) were used as positive controls. EC 50 value (mg extract/ml) is the effective concentration at which 50 % LPO of liver mitochondria was inhibited and its values were obtained by interpolation from linear regression analysis Inhibition of lipid peroxidation in egg homogenate (Ruberto et al., 2000) A modified thiobarbituric acid reactive species (TBARS) assay was used to measure the lipid peroxide formed, using egg yolk homogenates as lipid rich media. Malondialdehyde (MDA), a secondary end product of polyunsaturated fatty acid oxidation reacts with two molecules of thiobarbituric acid (TBA) yielding a pinkish red chromogen with an absorbance maximum at 532 nm. 0.8 % TBA 0.07 M FeSO 4 20 % Acetic acid (ph adjusted to 3.5 with NaOH) 20 % TCA n-butanol

28 10 % Egg homogenate 10 % egg homogenate of 0.5 ml volume and different concentrations of mushroom extracts of 0.1 ml ( mg/ml) were mixed in a test tube and their final volume was made to 1.0 ml by addition of distilled water. Finally 0.05 ml FeSO 4 was added to the above mixture and incubated for 30 min to induce lipid peroxidation. Thereafter, 1.5 ml of 20 % acetic acid and 1.5 ml of 0.8 % TBA (prepared in 1.1% sodium dodecyl sulphate) and 0.05 ml 20 % TCA were added, vortexed and heated in boiling water bath for 60 min. After cooling, 5.0 ml n-butanol was added to each tube and centrifuged at 3000 rpm for 10 min. The absorbance of the organic upper layer was measured at 532 nm. Ascorbic acid, trolox, BHT and quercetin ( mg/ml) were used as positive controls. EC 50 value (mg extract/ml) is the effective concentration at which 50 % LPO of egg homogenate was inhibited and its values were obtained by interpolation from linear regression analysis Inhibition of erythrocyte hemolysis mediated by peroxyl free radicals (Barros et al., 2007) The antioxidant activity of the mushroom and mycelium extracts was measured as the inhibition of erythrocyte hemolysis. 10 mm PBS (ph 7.4): Prepared by mixing 10 mm of NaH 2 PO 4 and Na 2 HPO 4 and 125 mm of NaCl in 1 L of distilled water. 200 mm 2,2 -azobis(2-amidinopropane)dihydrochloride (AAPH) Blood was collected from the rats and erythrocytes were separated from the plasma and the buffy coat was washed thrice with 10 ml 10 mm of PBS and centrifuged at 1500 rpm for 5 min. A suspension of erythrocytes in PBS (20 %, 0.1 ml) was added to 0.2 ml of 200 mm AAPH solution in PBS, mushroom and mycelial extracts of different concentrations (0.1 ml). The reaction mixture was shaken gently (3000 rpm) while being incubated at 37 C for 3 h. The reaction mixture was diluted with PBS (8 ml) and centrifuged at 3000 rpm for 10 min; the absorbance of its supernatant was then read at 540 nm. The percentage hemolysis inhibition = [(A AAPH - A S )/ A AAPH ] x 100, where A S is the absorbance of the sample containing the mushroom and mycelial extracts (

29 mg/ml) and A AAPH is the absorbance of the control sample containing no mushroom and mycelial extract. Ascorbic acid, gallic acid and rutin ( mg/ml) were used as positive controls. The extract concentration providing 50 % inhibition (EC 50 ) was calculated from the graph of hemolysis inhibition percentage against extract concentration Determination of total antioxidant capacity (Prieto et al., 1999) The assay is based on the reduction of Mo(VI)-Mo(V) by the extract and subsequent formation of green phosphate/mo(v) complex at acidic ph. The absorbance of the solution was measured at 695 nm. 0.6 M Sulphuric acid 28 mm Sodium phosphate 4 mm Ammonium molybdate The reagent solution (0.6 M sulphuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate) of 1.0 ml was taken in screw capped tubes to which 0.1 ml of mushroom and mycelial extracts were added and dissolved. The tubes were capped and incubated in a thermal block at 95 C for 90 min. After cooling to room temperature, the absorbance of the aqueous solution in each tube was measured at 695 nm against a blank. Ascorbic acid and gallic acid were used as standards and the total antioxidant capacity is expressed as equivalents of ascorbic acid (AAE) or gallic acid (GAE). 3.9 Procurement of experimental animals The laboratory animals (both male and female Sprague Dawley rats, Swiss albino mice) that were used throughout this study for experimental purpose were procured from a small Animal Breeding Station, Thrissur, Kerala, India. These animals were maintained under standard conditions of humidity, temperature (25 ± 2 ºC) and light (12 h light/dark). They were acclimatized to animal house conditions and were fed on a commercial pelleted rat chow (AVM Cattle Feeds, Coimbatore, Tamil Nadu) and water at ad libitum. Experimental animals were handled according to the University and Institutional Legislation, regulated by the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.