phytochemical analysis

3.1 Phytochemical analysis

3.1.1 Plant collection

Plants of Sonchus asper and Launaea procumbens at maturity were collected from Wah cantt and Bannu (Pakistan) during the month of June 2006 and July 2007 respectively. Plants were identified and a specimen was submitted vide voucher no R-153 and R-147 at Herbarium of Pakistan, Quaid-i-Azam University Islamabad, Pakistan. All parts of both plants (leaves, stem, flowers, seeds and roots) were shade dried at room temperature for two weeks, chopped, grinded mechanically of mesh size 1 mm.

3.1.2 Preparation of plant extract

5 kg powder of Sonchus asper and Launaea procumbens was socked in 10 L methanol, after a week of socking the extract was filtered through whatmann filter paper no 45, and the filtrate was evaporated through rotary vacuum evaporator to get 407/362 g methanolic crude extract. This crude extract was further extracted with solvents of increasing polarity; starting from n-hexane (28/23g), ethyl acetate (35/43g), chloroform (89/67g), butanol (100/105g), and remaining water extract (115/89g). The reflux time for each solvent was 5 h. The extract was cooled at room temperature, filtered and evaporated under reduced pressure in rotary evaporator. All the fractions were stored at 4 C for further phytochemical, in vitro and in vivo investigations.

3.1.3 Phytochemical screening

Chemical tests were carried for preliminary phytochemical screening of various fractions of both plants extract using standard procedures to identify the constituents as described by Sofowara (1993), Trease and Evans (1989) and Harborne (1973).

3.1.3.1 Test for tannins

50 mg of each fraction was boiled in 20 ml of distilled water in a test tube and was filtered. A few drops of 0.1% ferric chloride was added in each fraction and observed for colure change, the presence of brownish green or a blue-black coloration shows the presence of tannins.

3.1.3.2 Identification of phlobatinins

For of Identification of phlobatinins 80 mg of each plant extract was boiled in 1% aqueous hydrochloric acid, the deposition of a red precipitate indicates the presence of phlobatinins.

3.1.3.3 Saponin test

20 mg of the each sample was boiled in 20 ml of distilled water in a water bath for five minutes and filtered. 10ml of the each filtrate was mixed with 5 ml of distilled water and shaken vigorously for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously, then observed for the formation of emulsion.

3.1.3.4 Determination of flavonoids

Two methods were used to determine the presence of flavonoids in the plant sample (Sofowara, 1993; Harbrone, 1973).

  • 50 mg of each plant extract dissolved in 100 ml distal water to form aqueous solution. 5 ml of dilute ammonia solution were added to 10 ml of aqueous filtrate of each plant extract followed by few drops addition of
    concentrated H2S04. A yellow coloration observed in each fraction indicated the presence of flavonoids.
  • 50 mg portion of each plant sample was heated with 10 ml of ethyl acetate over a steam bath for 3 min. The mixture was filtered and 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution. A yellow coloration
    was observed indicating the presence of flavonoids.

3.1.3.5 Test for terpenoids (Salkowski test)

Five ml (1 mg/ml) of each extract was mixed in 2 ml of chloroform, and then 3 ml concentrated H2S04 was carefully added to form a layer. A reddish brown coloration of the inter face was formed which showed positive results for the presence of terpenoids.

3.1.3.6 Cardiac glycosides determination (Keller-Killani test)

Five ml of each extracts was treated with 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This was under layered with 1 ml of concentrated sulphuric acid. A brown ring of the interface indicates a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout thin layer.

3.1.3.7 Cumarine identification

0.3 g of each plant extract was taken in a small test tube and covered it with filter paper moistened with 1 N NaOH. The test tube was placed, for few minutes, in boiling water bath. Then the filter paper was removed and examined in UV light for yellow florescence to indicate the presence of Cumarine.

3.1.3.8 Test for Alkaloids

0.4 g extract of each plant was mixed with 8 ml of 1% HCl, warmed and filtered. 2 ml of each filtrate was titrated separately with (a) Mayer's reagent and (b) Dragendroff's reagent. Turbidity of precipitation was observed to indicate the presence of alkaloids.

3.1.3.9 Anthraquinone detection

200 mg of each plant fractions was boiled with 6 ml of 1% HCl and filtered. The filtrate was shaken with 5 ml of benzene. The layer was removed and then 10% NH4OH was added. Formation of pink, violet or red color in the alkaline phase was observed for the presence of anthraquinone.

3.2. Quantitative determination of the chemical constituents

3.2.1 Alkaloid determination using Harborne (1973) method

50 mg of the sample was weighed into a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was added, covered and allowed to stand for 4 h. This was then filtered and the extract was concentrated on a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. The whole solution was allowed to settle and the precipitated was collected and washed with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and weighed.

3.2.2 Tannin determination by Van-Burden and Robinson (1981) method

50 mg of each fraction was weighed into a 50 ml plastic bottle. 50 ml of distilled water was added and shaken for 1 h in a mechanical shaker. This was filtered into a 50 ml volumetric flask and made up to the mark. 5 ml of each filtrate was pipetted into a test tube and mixed with 2 ml of 0.1 M FeCl3 in 0.I N HCl and 0.008 M potassium ferro-cyanide. The absorbance was measured at 120 nm within 10 min.

3.2.3 Saponin determination (Obadoni and Ochuko 2001)

50 mg of each extract was put into a conical flask and 100 cm3 of 20% aqueous ethanol were added. The samples were heated over a hot water bath for 4 h with continuous stirring at about 550C. The mixture was filtered and the residue re-extracted with another 200 ml 20% ethanol. The combined extracts were reduced to 40 ml over water bath at about 900C. The concentrated filtrate was transferred into a 250 ml separatory funnel and 20 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated. 60 ml of n-butanol was added. The combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the samples were dried in the oven to a constant weight; the saponin content was calculated as percentage.

3.2.4 Determination of flavonoids (Bohm and Kocipai-Abyazan 1974)

50 mg of both plant samples were extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature. The whole solution was filtered through whatmann filter paper No 42 (125 mm). The filtrate was later transferred into a crucible and evaporated into dryness over a water bath and weighed to a constant weight.

3.2.5 Determination of the total phenolic Contents

Total phenolic Contents (TPC) was estimated using the method of Singleton & Rossi, 1965. 200 l (1-5 mg/ml) of each plant fraction and each concentration standard (50-200 mg/L) was added in ten milliliter of 1:10 Folin-Ciocalteau reagent. The mixture was mixed and incubated for 5 min before the addition of 7 ml of 0.115 mg/ml Na2CO3.The resulting solution was incubated a further 2 h before absorbance readings were taken at 765 nm. Gallic acid used for the calibration curve and a different concentration of gallic acid was used for calibration curve ranging from 50, 75, 100, 125, 150, 175, 200 mg/L. Results were expressed as mg gallic acid (GAE)/g dried plant material. All fractions were done in triplicate.

3.2.6 Determination of the total flavonoids content

Total flavonoids content was determined by using a method described by Sakanaka, Tachibana, and Okada (2005). Briefly, 0.25 ml of each extracts (1-5 mg/ml) and rutin standard solution (15-250 ug/ml) was mixed with 1.25 ml of distilled water in a test tube, followed by addition of 75 ul of a 5% (w/v) sodium nitrite solution. After 6 min, 150 ul of 10% (w/v) aluminum chloride solution was added and the mixture was allowed to stand for a further 5 min before 0.5 ml of 1M NaOH was added. The mixture was made up to 2.5 ml with distilled water and mixed well. The absorbance was measured immediately at 510 nm. The results of samples were expressed as mg of rutin equivalents of total extractable compounds. All samples were run in triplicate.

3.2.7 Elemental and nutritional analysis

100 ppm stock solution of the K, Mg, Ca, Na, Fe, Co, Mn, Cu, Cr, Zn, Ni, Li, Pb, Cd, were prepared by dissolving required amount of salts in distilled water for elemental analysis of Sonchus asper and Launaea procumbens plant powder. Powder of both plants were digested according to the of perchloric-acid digestion method (Allen, 1974). 0.25 g of each plant powder was taken in 50 ml flask, added 6.5 ml of mixed acid solution i.e. Nitric acid, Sulfuric acid, perchloric acid (5:1:0.1) in it and boiled it in fume hood on hot plate till the digestion has been completed which was indicated by white fumes coming out from the flasks. Digested samples were allowed to cool and transferred in 50 ml volumetric flask, by rising volume with distilled water. Filter the extract with whatmann No 42 filter paper and filtrate were collected in labeled plastic bottles. Concentration of these elements in the entire sample was determined by Shimadzu AA-670 Atomic Absorption Spectrophotometer and calculated by using formula

Nutrient cation in plants= (ppm in extract - blank) A/W dilution factor

A=Total volume of extract (ml)

W=Weight of dry plant

3.2.8 LEAF PROTEIN CONTENTS

Protein content of leaves was determined following the method of Lowery et al., (1951) using BSA as standard.

Phosphate Buffer (Stock Solution)

  • Monobasic sodium phosphate: 27.6g was dissolved in distill water (1000ml)
  • Dibasic sodium phosphate: 53.6g was dissolved in 1000ml

Monobasic sodium phosphate (16ml) and dibasic sodium phosphate (84ml) was mix together to get the desire pH (7.5) of phosphate buffer.

  • Reagent A: 2g sodium carbonate (Na2CO3) 0.4g NaOH (0.1 N) and 1g Na-K tartrate was dissolved in100 ml of distilled water.
  • Reagent B: CuSO4.5H2O (0.5g) was dissolved in 100ml of distill water.
  • Reagent C: Solution A (50ml) and Solution B (1ml) were mixed.
  • Reagent D: Folin phenol reagent was diluted with distill water in 1:1 ratio.

Fresh leaves 0.1g were ground with the help of mortar and pestle in 1ml of phosphate buffer pH 7.5 and, centrifuged for 10 min at 3000rpm. The supernatant (0.1ml) of given sample containing unknown amount of protein was poured in the test tubes and total volume of 1ml was made by distilled water. 1ml of reagent C was added. After shaking for 10min, 0.1ml of reagent D was added. The absorbance of each sample was recorded at 650nm after 30 min incubation. The concentration of protein contents was determined with the reference to standard curve made by using standard BSA (Bovine Serum Albumen). The BSA of different concentration viz 20,40,60,80,320, 640 mg and 2 g was prepared. The absorbance of BSA was recorded at 650 nm.

3.2.9 SUGAR ESTIMATION

Sugar estimation of fresh leaves was done following method of Dube et al., (1956).

Fresh plant material (0.5g) was homogenized with 10ml of distilled water in a clean mortar and then centrifuged at 3000 rpm for 5 min. In 0.1ml of supernatant 1ml of 80% (w/v) phenol was added, after incubation at room temperature, 5ml concentrated sulphuric acid was added. The sample is incubated for 4 hrs and ten absorbance of each sample was recorded at 420 nm. The concentration of unknown sample was calculated with reference to standard curve made by using glucose.

3.2.10 CHLOROPHYLL CONTENT OF LEAVES

Chlorophyll content of leaves was determined by the method of Aron (1968). Crude preparation 1ml was mixed with 4ml of 80% (w/v) acetone and allowed to stand in dark at room temperature. It was centrifuged at 2000 rpm for 5 min to clear the suspension. Supernatant was used for chlorophyll determination. Absorbance of solution was read at 645 nm (chlorophyll a) and at 663nm (chlorophyll b) on spectrophotometer against 80% (v/v) acetone blank.

Total chlorophyll (mg/l) = (20.2 x A645) + (8.02 x B663)

3.2.11Thin layer chromatography of fractions;

0.1 g of each fraction of Sonchus asper and Launaea procumbens were dissolved in 1 ml of methanol (HPLC grade). Silica gel TLC plates (20x20 cm) were marked 1cm from each side and heated at 110 0C for 40 minutes for silica activation. 5-10 ul of each samples was spotted by jet pointed capillary tube on the line marked at one corner of the plate. 150 ml of mobile phase solution composed of a mixture of butanol, acetic acid and water (4?1?5) was poured in tank and covered with a lid for vapors saturation. After 20 minutes, plates were kept and allowed to develop. When mobile phase reached at upper end (Just below 1 cm), plates were removed out. Solvent front was marked, air dried, sprayed with 1% ethanolic 2-Aminoethyle diphenyl borinate solution. Flavonoids have been detected through its characteristic colors under UV - 365 nm and retention factors and their RF values were calculated as;

RF value = Distance covered by visualized spot / Distance covered by solvent front

3.2.12 High Performance Liquid Chromatogrhy (HPLC)

3.2.12.1 Sample preparation for HPLC

100 mg of each fraction of both plants were extracted with 6 ml of 25% hydrochloric acid and 20 ml methanol for 1 h. The obtained extract was filtered to a volumetric flask. The residue was then heated twice with 20 ml of methanol for 20 min. The combined extracts were diluted with methanol to 100 ml. 5 ml portion of the solution was filtered and then was transferred to a volumetric flask and diluted with 10 ml of methanol. The filtrate (10 l) was injected into the HPLC apparatus.

3.2.12.2 HPLC determination of flavonoids and Phenolic compounds

Samples were analyzed on agilant HPLC system. Separation was carried out through column 20RBAX ECLIPSE, XDB-C18, (5 m; 4.6150 mm, Agilent USA) with UV-VIS Spectra-Focus detector, injector-auto sampler. Solvent A (0.05% trifluoroacetic acid) and solvent B (0.038% trifluoroacetic acid in 83% acetonitrile v/v) with the following gradient: 0-5 min, 15% B in A, 5-10 min, 70% B in A, 10-15 min, 70% B in A. The flow rate was 1 ml/min and injection volume was 10 l. The calibration curves were defined for each compound in the range of sample quantity 0.02-0.5 g. All samples were assayed in triplicate.

3.3 In vitro Screening of fractions

3.3.1 DPPH radical scavenging activity

The free radical scavenging activity was measured by using 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) assay. DPPH assay was performed according to the procedure as reported by Gyamfi et al., (1999). DPPH solution was prepared by dissolving 3.2 mg in 100ml of 82% methanol. 2800l of DPPH solution was added to glass vial followed by the addition of 200l of test sample solution, in methanol, leading to the final concentration of 1g/ml, 5g/ml, 10g/ml, 25g/ml, 50g/ml and 100g/ml. Mixture of DPPH and each fraction was shaken well and kept in dark at room controlled room temperature (25C- 28 C) for one hour. Absorbance was measured at 517 nm. Mixture of 2800l of 82% methanol and 200l of Methanol was used as blank while 200l of methanol and 2800l of DPPH solution was taken as control. The test of each fraction was performed in triplicate. Percentage inhibition was measured according to following formula and IC50 value was calculated by graph pad prism software.

% scavenging = absorbance of control- absorbance of test sample x 100

Absorbance of control

3.3.2. Phosphomolybdenum assay

The antioxidant activity of samples was evaluated by the Phosphomolybdenum method according to the procedure of Prieto, Pineda, and Aguilar (1999). An aliquot of 0.1 ml of sample solution was combined in a 4 ml vial with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The vials were capped and incubated in a water bath at 95 0C for 90 min. After the incubation samples were cooled to room temperature, the absorbance of the mixture was measured at 765 nm against a blank. ,

3.3.3. ABTS radical cation assay

ABTS radical cation assay was carried out using the protocol of Re et al., 1999. According to this protocol ABTS (2, 20-azinobis-(3-ethylbenzothiazoneline-6-sulphonic acid)) used as the free radical provider was treated with (7.4 mM) potassium persulphate (2.45 mM) to generate free radicals. The solution was diluted to obtain an absorbance of between 1.5 and 2.5 at 414 nm with 98% of ethanol, before used. Reagents (3 ml) were transferred to the glass cuvettes with one of them containing 3 ml ethanol as blank. The initial absorbance of the reagents in the glass cuvettes at 414 nm was recorded. The samples (100 ul, 1.0 mg/ml) were transferred into the cuvettes containing the reagent and the mixtures were shaken thoroughly. The mixture in the Cuvettes was examined after 90 min using a UV-vis spectrophotometer. The capability to scavenging the ABTS radical cation was calculated using the following equation:

% ABTS radical cation scavenging capability = (A1-A2/A1) x100

where A1 is the absorbance of the control (ABTS solution without test sample) and A2 is the absorbance in the presence of the test sample. Also, the antioxidant capacity of the extract was obtained by comparing the change of absorbance at 414 in a test reaction mixture containing extracts with that containing Rutin. The results reported are mean values expressed as milligrams of Rutin equivalents per gram sample.

3.3.4. Antioxidant activity by beta carotene bleaching method

The antioxidant activity of each fraction was evaluated using beta carotene-linoleate model system, as described by Sun and Ho (2005). Two milligrams of beta carotene were dissolved in 10 ml chloroform and 1 ml beta carotene solution was mixed with 20 mg of purified linoleic acid and 200 mg of Tween 40 emulsifier. Chloroform was then evaporated under a gentle stream of nitrogen and the resulting mixture was immediately diluted with 50 ml of distilled water. To an aliquot of 5 ml of this emulsion, 0.2 ml of each extract (0.625-5 mg/ml) or the reference antioxidants (gallic acid) were added and mixed well. The absorbance at 470 nm, which was regarded as t0, was measured, immediately, against a blank consisting of the emulsion without beta carotene. The capped tubes were placed in a water bath at 50 0C and the absorbance was measured every 15 min up to 120 min. For the positive control, sample was replaced with gallic acid or BHT. A negative control consisted of 0.2 ml distilled water or solvent instead of extract or reference antioxidants. All samples were assayed in triplicate. The antioxidant activity (AA) was measured in terms of successful bleaching of b-carotene by using the following equation: AA = ((1- (A0 - At / A00 - A0t)) x 100 where A0 and A00 are the absorbance values measured at zero time of the incubation for each fraction and control, respectively. At and A0t were the absorbance values measured in each fraction and control, respectively, after incubation for 120 min. The results were expressed on a % basis of preventing bleaching of beta carotene (Abdille, Singh, Jayaprakasha, & Jena, 2005).

3.3.5. Superoxide radical scavenging activity

Superoxide radical scavenging activity of each fraction was determined by the nitroblue tetrazolium reduction method (Nishikimi, Rao, & Yagi, 1972). One milliliter of nitroblue tetrazolium (NBT) solution (l M NBT in 100 mM phosphate buffer, pH 7.4), 1 ml NADH solution (l M NADH in 100 mM phosphate buffer, pH 7.4) and 0.1 ml of the extracts (0.125-5 mg/ml), gallic acid or quercitine (0.016-2.5 mg/ml) were mixed. The reaction was started by adding 100 ul of PMS solution (60 uM PMS in 100 mM phosphate buffer, pH 7.4) to the mixture. The reaction mixture was incubated at 25 0C for 5 min, and the absorbance at 560 nm was measured against blank samples, containing all the reagents except the PMS. The positive and negative controls were subjected to the same procedures as the sample, except that for the negative control, only the solvent was added, and for the positive control, sample was replaced with gallic acid and quercitine. All measurements were made in triplicate and averaged. The abilities to scavenge the superoxide radical were calculated using the following equation:

% Superoxide radical scavenging activity = (1- absorbance of sample / absorbance of control) x 100

3.3.6. Hydroxyl radical scavenging activity

The effect of extracts on hydroxyl radicals was assayed by using the deoxyribose method (Nagai, Myoda, & Nagashima, 2005). 2-Deoxyribose is degraded on exposure to hydroxyl radicals generated by Fenton reaction. Each fraction and BHA were prepared in ethanol/water, and ethyl acetate extract in ethyl acetate/water mixture (3:7; v/v). The reaction mixture contained 450 ul of 0.2 M sodium phosphate buffer (pH 7.0), 150 ul of 10mM 2-deoxyribose, 150 ul of 10 mM FeSO4-EDTA, 150 ul of 10 mM H2O2, 525 ul of H2O, and 75 ul of sample solution (2.5-20 mg/ml water or infusion extracts; 0.01-5 mg/ml ethanol or ethyl acetate extracts). The reaction was started by the addition of H2O2. After incubation at 37 0C for 4 h, the reaction was stopped by adding 750 ul of 2.8% trichloroacetic acid and 750 ll of 1% TBA in 50 mM NaOH, the solution was boiled for 10 min, and then cooled in water. The absorbance of the solution was measured at 520 nm. Gallic acid (2.5-10 mg/ml) and BHT (0.01-5 mg/ml) were used as positive controls. The ability to scavenge the hydroxyl radicals was calculated using the following equation:

% Superoxide radical scavenging activity = (1- absorbance of sample / absorbance of control) x 100

3.3.7. Hydrogen peroxide-scavenging activity

The ability of the extracts to scavenge hydrogen peroxide was determined according to the method of Ruch, Cheng, and Klaunig (1989). A solution of hydrogen peroxide (2mM) was prepared in 50 mM phosphate buffer (pH 7.4). Hydrogen peroxide concentration was determined spectrophotometrically at 230 nm absorption using the molar extinction coefficient for H2O2 of 81 mol-1 cm-1. Samples of various fractions (0.05-250 mg/ml) of extracts, gallic acid, ascorbic acid or rutin (0.05-250 mg/ml) were transferred into the test tubes and their volumes were made up to 0.4 ml with 50 mM phosphate buffer (pH 7.4) or solvents. After addition of 0.6 ml hydrogen peroxide solution, tubes were vortexed and absorbance of the hydrogen peroxide at 230 nm was determined after 10 min, against a blank. 50 mM phosphate buffer without hydrogen peroxide was used as blank. Hydrogen per oxide scavenging ability was calculated by following equation:

Hydrogen peroxide scavenging activity = (1- absorbance of sample / absorbance of control) x 100

3.3.8. Reducing power

The reducing powers of the extracts were determined according to the method described by Chung et al. (2005). 0.1 ml of each extract, ascorbic acid, rutin or gallic acid (0.05-250 mg/ml) were mixed with an equal volume of 0.2 M phosphate buffer (pH 6.6) and 1% potassium ferricyanide, and then incubated at 50 0C for 20 min. 0.25 ml of 1% trichloroacetic acid was added to the mixture to stop the reaction, and then the mixture was centrifuged at 2790g for 10 min. The supernatant (0.25 ml) was mixed with 0.25 ml distilled water and 0.1% FeCl3 (0.5 ml) and then the absorbance was measured at 700 nm. The reducing powers of the tested samples increased with the absorbance values.

3.3.9. Chelating activity on Fe2+

The extracts were assessed for their ability to compete with ferrozine for iron (II) ions in free solution. The chelating ability of ferrous ions by various fractions of Sonchus asper and Launea procumbens leaves was estimated by the method of Dinis, Madeira, and Almeida (1994). Extracts (0.05-250 mg/ml), 2.5 ml were added to a solution of 2 mM FeCl2.4H2O (0.05 ml). The reaction was initiated by the addition of 5 mM ferrozine (0.2 ml), the mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance of the solution was then measured at 562 nm against the blank performed in the same way using FeCl2 and water. EDTA (0.625-5 lg/ml) served as the positive control, and a sample without extract or EDTA served as the negative control. All tests were run in triplicate and averaged. The percentage of inhibition of ferrozine-Fe2+complex formation was calculated using the formula:

Chelating activity % = (1- absorbance of sample / absorbance of control) x 100

3.3.11 Antibacterial assay

3.3.11.1. Preparation of samples

All samples were prepared for assay by dissolving 100 mg of each sample in 10 ml of DMSO. The stock solution was further diluted as shown in Table (1.1).

3.3.11.2. Media for bacteria

Nutrient Broth medium (MERCK) was used to grow bacteria for inoculums preparation. It was composed of peptone (5g/l) and meat extract (3g/l) while Nutrient agar medium (MERCK) was composed of peptone (5g/l) Meat extract (3g/l) and Agar-Agar (12g/l). Nutrient broth medium was prepared by dissolving 0.8gm/100ml nutrient broth in distilled water while Nutrient agar medium was prepared by dissolving 2gm of nutrient agar in 100ml distilled water, pH 7.0 and was autoclaved.

3.3.11.3. McFarland 0.5 BaSO4 turbidity standard

To compare the turbidity of bacterial culture, McFarland 0.5 BaSO4 is used. The standard was prepared by adding 0.5ml of 0.048M BaCl2 to 99.5ml 0.36N H2SO4. Barium sulfate turbidity standard (4 to 6ml) was taken in screw capped test tube and was used to compare the turbidity.

3.3.12.4. Microorganisms

Four strains of bacteria were used; three were Gram positive, which were Staphylococcus aurous (ATCC 6538) and Bacillus subtitles (ATCC 6633), Micrococcus lutes and three were Gram negative, which were Escherichia coli (ATCC 15224), Klepsila and Enterobacter aerogenes (ATCC 13048). The organisms were maintained on nutrient agar medium at 4C. Twenty-four hours old culture in nutrient broth (MERCK) of selected bacterial strain was mixed with physiological saline (0.9% NaCl w/v.) and turbidity was corrected by adding sterile physiological saline until a McFarland 0.5 BaSO4 turbidity standard [106 colony forming unit (CFU) per ml density was obtained. Then this inoculum was used for seeding the nutrient agar.

3.3.13.5. Assay procedure (agar diffusion method)

Nutrient agar medium was prepared by suspending nutrient agar (MERCK) 2gm in 100 ml distilled water; pH 7.0 and was autoclaved. It was allowed to cool up to 45C. Then it was seeded with 1ml of prepared inoculum to have 106 CFU per ml. Petri plates (14 cm) were prepared by pouring 75 ml of seeded nutrient agar and allowed to solidify. Eleven wells per plate were made with sterile cork borer (8mm). Using micropipette, 100l of test solutions was poured in respective well. Eight samples, two solutions for positive control (Roxithromycin 1 mg/ml and Cefixime-USP 1mg/ml, one for each) and one for negative control (DMSO) was applied to each Petri plate. Finally, the Petri plates were incubated at 37 ?C for 24 h. Then clear (inhibition) zones were detected around each hole. DMSO alone (0.1 ml) was used as a control under the same condition for each organism, and, by subtracting the diameter of inhibition zone resulting with DMSO from that obtained in each case, both antibacterial activities were calculated as a mean of 3 replicates (Mohamed et al., 2006). Diameter of the clear zones, showing no bacterial growth, around each well was measured with the help of vernier caliper. Triplicate plates were prepared for each sample.

3.3.14. Antifungal assay

The agar tube dilution method was used for antifungal activity of plant extracts as reported by Choudhary et al. (1995).

3.3.14.1. Microorganisms used

  • F.Solani (0300)
  • Aspergillus niger (0198)
  • Aspergillus flavus (0064)
  • Aspergillus fumigatus (66)

3.3.14.2. Media for antifungal assay

Sabouraud dextrose agar (MERCK) was used to grow fungus for inoculums preparation. It was composed of peptone complex 10 g/l, glucose 40g/l and agar 15g/l.

3.3.14.3. Preparation of samples

The samples for antifungal assay were prepared from initial stock of 12mg/ml to get final concentration of 200g/ml. Solutions of terbinafine 12mg/mL in DMSO were prepared for positive control. Pure DMSO was used as negative control.

3.3.14.4. Assay Procedure

Media for fungus was prepared by dissolving 6.5gm/100mL in distilled water pH was adjusted at pH 5.6. Test tubes were marked to 10 cm mark. The Sabouraud dextrose agar (MERCK) dispensed as 4ml volume into screw capped tubes or cotton plugged test tubes and were autoclaved at 121C for 21 minutes. Tubes were allowed to cool to 50 C and non-solidified SDA was loaded with 67 l of sample pipette from the stock solution. This would give the final concentration of 200 g /ml of the sample. Tubes were then allowed to solidify in slanting position at room temperature. Tubes were prepared in triplicate for each fungus species. The tubes containing solidified media and test compound were inoculated with 4mm diameter piece of inoculum, taken from a seven days old culture of fungus. Positive and negative control test tubes with DMSO and terbinafine were also inoculated. The test tubes were incubated at 28C for 7 days. Cultures were examined twice weekly during the incubation. Reading was taken by measuring the linear length of fungus in slant by measuring growth (mm) and growth inhibition was calculated with reference to negative control. Percentage inhibition of fungal growth for each concentration of compound was determined by the following formula.

Percentage inhibition of fungal growth;

= (100 - linear growth in test (mm) / linear growth in control (mm) x 100

3.3.15. Antitumour Potato Disc Assay

The potato disc method was used for Antitumour activity of plant extracts as reported by (Ferrigini et al., 1985). All the samples were assayed at three different concentrations, i.e. 10000, 1000ppm, 100ppm and 10ppm.

3.3.15.1. Microorganisms used

The strain of Agrobacterium tumefaciens used was At10.

3.3.15.2. Preparation of bacterial culture

2.5% LB (Luria broth) was prepared by dissolving 2.5g of LB in 100ml of distilled water and was autoclaved. 20l of 50mg/mL rifampicin stock solution was added to it. In the final volume concentration of rifampicin was 10l /ml. Single colony from culture plate of Agrobacterium tumefaciens (AT10) was inoculated in it. It was allowed to grow for 48 hours at 28oC in shaking incubator.

3.3.15.3. Preparation of stock solutions

10,000 ppm stock solution of each test fractions was prepared by dissolving 10mg of test samples in 1ml of DMSO. Two more stock solutions of 1000ppm and 100ppm were prepared in DMSO from already prepared stock solution of 10,000 ppm. Final volume was 1ml.

3.3.15.4. Preparation of Innoculum

In order to attain test sample final concentrations of10ppm, 100ppm and 1000ppm in the innoculum, 1500l of innoculum was prepared as follows.150l of each stock solution was added in three autoclaved appendorfs. Then 750l of autoclaved distilled water and 600l of bacterial culture was added to it, so that final volume of innoculum was1500l.

3.3.15.5. Preparation of control solutions

Three controls were used in the assay.

  • Positive Control 1
  • Positive control was prepared by taking 150l of DMSO in autoclaved appendorfs and adding 1350l of autoclaved distilled water in it.
  • Negative Control 2
  • Negative control was prepared by taking 150l of DMSO in autoclaved appendorfs and adding 750l of autoclaved distilled water and 600l of bacterial culture in it.
  • Control 3
  • Blank potato discs

All the solutions were prepared in laminar flow hood by considering all precautionary measures to avoid contamination.

3.3.15.6. Preparation of Agar solution

1.5% Agar solution was prepared by dissolving agar in distilled water and was autoclaved. 25ml of agar solution was needed for each Petri plate

3.3.18.5. Preparation of Agar plates

Three petri plates were prepared for each concentration of test sample and control.

Agar solution was poured in autoclaved Petri plates and allowed to solidify.

3.3.15.7. Preparation of potato discs

Red skinned potatoes were soaked in 0.1% mercuric chloride solution for 10 minutes.

With the help of a large size sterilized forceps potatoes were taken out of mercuric chloride solution in large Petri plates (autoclaved). Cylinders were made with the help of a sterilized borer (8mm). Cylinders were washed in autoclaved distilled water and 1cm on both ends was cut with the help of a sterilized blade. Cut ends were thrown out. 5mm thick discs of these cylinders were cut in autoclaved Petri plates. Discs were washed with autoclaved distilled water and were placed on solidified Agar plates (10discs per plate). 50l of innoculum was added on the surface of each disc of respective concentration as well as controls. Innoculum was allowed to diffuse for 10-20 minutes. The edge of each Petri plates was sealed with Para film strips to make air tight and to prevent moisture loss during incubation period. Dish level was kept all the time to keep the innoculum on the tops of the discs. Petri plates were placed in dark at 28oC for 21 days.

3.3.15.8. Staining procedure

Lugol's solution was prepared in distilled water (10%KI, 5%I).

Discs were covered with Lugol's solution for staining purpose and after 30 min discs were observed under dissecting microscope with side illumination of light. Distained portions of the discs were tumors. Number of tumors per disc was counted %age inhibition for each concentration was determined as follows:

%age inhibition = 100- average number of tumors of sample____ X 100

Average number of tumors of -ve control

20% tumor inhibition was considered significant

3.3.16. Cytotoxic brine shrimp assay

3.3.16.1 Requirements

Various fractions of Sonchus asper and Launaea procumbens, sea salt (28 g/l of dH2O), Artemia salina (Shrimps eggs), hatching try, magnetic stirrer, magnifying glass, lamp, micropipette, glass vials, vials tray, solvents and dH2O according to (Meyer et al., 1992).

3.3.16.2 Sample preparation

Samples were prepared by dissolving 10 mg of each fraction in respective solvents to form stock solution of 1000 ppm, further dilution were made as in Table (1.2)

3.3.16.3 Saline preparation

28 g sea commercial sea salt (sigma) was dissolved in one liter of dH2O with continuous stirring for two hours.

3.3.16.4 Hatching of Shrimps

Brine shrimps were hatched in two compartment rectangular tray containing sea salt saline. Eggs were sprinkled in dark compartment of tray and after 24 hrs of shrimps hatching larvae was collected by pipette from the lightened side.

3.3.16.4 Bioassay

Two dram vials were used in this bioassay. 0.5 ml of each solution (1000 ppm, 100 ppm, 10 ppm) was taken in vials and evaporated the solvents. Residues was redisolved in saline of 2 ml. 10 shrimps were transferred to each vial and raised the volume up to 5 ml and incubate at 25-280C. After 24 hrs of incubation survivors was counted with help of 3x magnifying glass and calculation was done using Abbot's formula;

% Death = Sample-control/controlx100

LD50 was determined through prism graph pad software.

3.3.17 Phototoxic (Allelopathy) radish root inhibition assay

3.3.16.2 Sample preparation

Samples were prepared by dissolving 100 mg of each fraction in respective solvents to form stock solution of 10000 ppm, further dilution were made as in Table (1.2)

3.3.16.2 Bioassay

Radish seed was washed with dH2O and then with 1 % mercuric chloride. Filter paper was put in each autoclaved patri plates. Pour 5 ml of each fraction in each plate and evaporated the respective solvent. Put 10 seed in each plate and incubate in growth room for five days. After 3 days root and shoot inhibition was noted and again after 5 days. Fresh weight, dry weight, % inhibition and % germination velocity was recorded and IC50 was calculated using Graph prism pad software.

3.4. In vivo screening of fractions

3.4.1. Animals

Two hundred ninety two (292) male albino rats (190-200 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25 3 C with a 12 h dark/light cycle. They were allowed to standard laboratory feed and water. The study protocol was approved by Ethical committee of Quaid-i-Azam University Islamabad. All these rats were equally divided into four experiments for in vivo screening of the fraction against CCl4 and KBrO3 induced toxicity in various tissues.

3.4.1.1. Experimental design for Sonchus asper fractions in vivo screening

The first experiment was designed to evaluate the antioxidant effects of Sonchus asper, 96 male albino rats were equally divided into 16 groups (6 rats). Group 1 received only raw water and free access to food materials. Group II received olive oil intraparetoneally (Monday and Thursday) and DMSO orally (Wednesday and Saturday) at a dose of 3 ml/kg body weight. Group III received CCl4 3 ml/kg intraparetoneally in olive oil (Monday and Thursday). Group IV received 30 mg/kg b.w. rutin as reference chemical after 48 h of CCl4 (Wednesday and Saturday). Group V and VI were given orally 100; 200 mg/kg b.w. respectively, Sonchus asper methanolic extracts (SAME) after 48 h of CCl4 treatment as above. Groups VII received only SAME at a dose of 200 mg/kg b.w (Wednesday and Saturday). Group VIII and IX received 100; 200 mg/kg Sonchus asper chloroform extract (SACE) after 48 h of CCl4 treatment, while group X received only 200 mg/kg SACE. Group XI received XII received 100; 200 mg/kg b.w. Sonchus asper ethyl acetate extract (SAEE) after 48 h of CCl4 treatment, however, group XIII received only 200 mg/kg b.w. SAEE. Group XIV and XV was treated with 100; 200 mg/kg b.w. Sonchus asper n-hexane extract (SAHE) after 48 h of CCl4 treatment, while group XVI received only 200 mg/kg b.w. SAHE. All these treatments were given twice a week for four weeks.

3.4.1. 2. Experimental design for Launaea procumbens fractions in vivo screening

The 2nd experiment was designed to evaluate the antioxidant effects of Launaea procumbens, 96 male albino rats were equally divided into 16 groups (6 rats). Group 1 received only raw water and free access to food materials. Group II received olive oil intraparetoneally (Monday and Thursday) and DMSO orally (Wednesday and Saturday) at a dose of 3 ml/kg body weight. Group III received CCl4 3 ml/kg intraparetoneally in olive oil (Monday and Thursday). Group IV received 30 mg/kg b.w. rutin as reference chemical after 48 h of CCl4 (Wednesday and Saturday). Group V and VI were given orally 100; 200 mg/kg b.w. respectively, Launaea procumbens methanolic extracts (LPME) after 48 h of CCl4 treatment as above. Groups VII received only LPME at a dose of 200 mg/kg b.w (Wednesday and Saturday). Group VIII and IX received 100; 200 mg/kg Launaea procumbens chloroform extract (LPCE) after 48 h of CCl4 treatment, while group X received only 200 mg/kg LPCE. Group XI received XII received 100; 200 mg/kg b.w Launaea procumbens ethyl acetate extract (LPEE) after 48 h of CCl4 treatment, however, group XIII received only 200 mg/kg b.w. SAEE. Group XIV and XV was treated with 100; 200 mg/kg b.w. Launaea procumbens n-hexane extract (LPHE) after 48 h of CCl4 treatment, while group XVI received only 200 mg/kg b.w. LPHE. All these treatments were given twice a week for four weeks.

toxicity in various tissues.

3.4.1.3. Experimental approach for Sonchus asper fractions against KBrO3 induced toxicity

The first experiment was designed to evaluate the antioxidant effects of Sonchus asper, 96 male albino rats were equally divided into 16 groups (6 rats). Group 1 received only raw water and free access to food materials. Group II received olive oil intraparetoneally (Monday and Thursday) and DMSO orally (Wednesday and Saturday) at a dose of 3 ml/kg body weight. Group III received KBrO3 1 ml (20 mg/kg b.w.) intraparetoneally in olive oil (Monday and Thursday). Group IV received 30 mg/kg b.w. rutin as reference chemical after 48 h of KBrO3 (Wednesday and Saturday). Group V and VI were given orally 100; 200 mg/kg b.w. respectively, Sonchus asper methanolic extracts (SAME) after 48 h of KBrO3 treatment as above. Groups VII received only SAME at a dose of 200 mg/kg b.w (Wednesday and Saturday). Group VIII and IX received 100; 200 mg/kg Sonchus asper chloroform extract (SACE) after 48 h of CCl4 treatment, while group X received only 200 mg/kg SACE. Group XI received XII received 100; 200 mg/kg b.w. Sonchus asper ethyl acetate extract (SAEE) after 48 h of KBrO3 treatment, however, group XIII received only 200 mg/kg b.w. SAEE. Group XIV and XV was treated with 100; 200 mg/kg b.w. Sonchus asper n-hexane extract (SAHE) after 48 h of KBrO3 treatment, while group XVI received only 200 mg/kg b.w. SAHE. All these treatments were given twice a week for four weeks.

3.4.1.4. Experiment for Launaea procumbens fractions against KBrO3 induced toxicity

The 4th experiment was designed to evaluate the antioxidant effects of Launaea procumbens, 96 male albino rats were equally divided into 16 groups (6 rats). Group 1 received only raw water and free access to food materials. Group II received olive oil intraparetoneally (Monday and Thursday) and DMSO orally (Wednesday and Saturday) at a dose of 1 ml (20 mg/kg) body weight. Group III received KBrO3 20 mg/kg intraparetoneally in olive oil (Monday and Thursday). Group IV received 30 mg/kg b.w. rutin as reference chemical after 48 h of KBrO3 (Wednesday and Saturday). Group V and VI were given orally 100; 200 mg/kg b. w KBrO3 respectively, Launaea procumbens methanolic extracts (LPME) after 48 h of CCl4 treatment as above. Groups VII received only LPME at a dose of 200 mg/kg b.w (Wednesday and Saturday). Group VIII and IX received 100; 200 mg/kg Launaea procumbens chloroform extract (LPCE) after 48 h of KBrO3 treatment, while group X received only 200 mg/kg LPCE. Group XI received XII received 100; 200 mg/kg b.w Launaea procumbens ethyl acetate extract (LPEE) after 48 h of KBrO3 treatment, however, group XIII received only 200 mg/kg b.w. SAEE. Group XIV and XV was treated with 100; 200 mg/kg b.w. Launaea procumbens n-hexane extract (LPHE) after 48 h of KBrO3 treatment, while group XVI received only 200 mg/kg b.w. LPHE. All these treatments were given twice a week for four weeks.

3.4.1.5. Dissection procedure of animals

After completion of 4 weeks duration during each experiment all the animals were kept on normal feed without any treatment for at least 24 hrs before the dissection of animals. Urine was collected and stored at - 70 0C for further analysis, and then animals were given chloroform anesthesia and dissected from ventral side. First of all the blood was collected by cardiac puncture, put in the falcon tube and centrifuged for serum. The serum was stored in refrigerator. After taking blood the tissues were removed and washed in ice cold saline, dried with blotting paper and weighted. After weight the tissues were divided into two portions.

  • The part from all tissue were cut off and stored in fixative sera (Absolute alcohol 60%, Formaldehyde 30%, Glacial acetic acid 10%), for histology.
  • Second portion of the tissue was treated with liquid Nitrogen and stored at -700C for further biochemical and molecular studies.

3.4.2. Histological procedure of tissue

After weighting the portion specifies for histology is undergoes the process of fixation.

3.4.2.1. Tissue fixation process

A solution used for fixation purpose is known as fixative sera. .A small portion of each tissue was taken from the different sides and were fixed in freshly prepared fixative sera for 3-4 hrs. The composition of the fixative sera was:

  • Absolute alcohol 60ml
  • Formaldehyde 30ml
  • Glacial acetic acid 10ml

3.4.2.2. Dehydration

Fixation is followed by dehydration in following ascending grades of alcohol

  • 80% Alcohol Overnight at room temperature
  • 90% Alcohol 2-4 hrs at room temperature
  • 100% Alcohol 2-4 hrs at room temperature

Following the dehydration fixed liver tissue were transferred to cedar wood oil until they become clear and transparent at room temperature.

3.4.2.3. Embedding

Following steps were under taken.

  • Benzol 1 10 minutes at room temperature
  • Benzol 2 10 minutes at room temperature
  • Benzol + paraplast (1:1) 20 minutes at 60C
  • Paraplast 1 12 hrs at 60C
  • Paraplast 2 12 hrs at 60C
  • Paraplast 3 12 hrs at 60C

Embedded tissues were then transferred into melted wax in a boat. Bubbles were removed and wax was allowed to solidify. A block of wax containing tissue was prepared. Paraffin wax of blocks were trimmed with the help of sterilized knife or scalpel and then mounted on wooden block for sectioning.

3.4.2.4. Microtomy

Paraffin embedded tissue mounted on plastic cassettes and cut thin slices of 3um-5m using microtome. The thin section embedded in wax strip is firstly shift to cold water where the long ribbon of wax is divided into portion equal to the size of glass slide. The sections along with wax stripe is than shift to the water bath temperature 60co.In water bath the wax stripe becomes smooth and then shift on clean glass slide. Slides were incubated at 65co in oven to melt the extra wax and fix the tissue on slide. These glass slides were now placed in incubator for overnight.

3.4.2.5. Staining Procedure

After removing wax from tissue they were stained by Eosin hematoxylin staining and silver nitrate. Eosin and Hematoxylin stain were prepared by following procedure.

Hematoxylin

Following chemicals were used in hematoxylin preparation.

  • Hematoxylin 2 grams
  • Ethanol absolute 100ml
  • Ammonium alum 3 grams
  • Distilled water 100 ml
  • Glycerol 100ml
  • Sodium iodate 0.24grams
  • Acetic acid 10ml

In ethanol hematoxylin was added. Thoroughly prepare ammonium alum solution in distilled water and boiled. Add hematoxylin solution to ammonium alum solution and carefully added glycerol and sodium iodate and then added acetic acid one by one in given amount, mix thoroughly.

Eosin

It is prepared by dissolving 1gm of eosin in 100ml of 70% of ethanol.

3.4.2.6. Staining Methodology

Hematoxylin and eosin method was used for staining.

Hydration

These slides were deparafinised in xylene as follows

  • Xylene 1 2-5 minutes at room temperature
  • Xylene 2 2-5 minutes at room temperature

Paraplast removed thyroid tissues were hydrated in graded solutions of alcohol in descending order.

  • 100% Alcohol 2-5 minutes at room temperature
  • 90% Alcohol 2-5 minutes at room temperature
  • 70% Alcohol 2-5 minutes at room temperature
  • 50% Alcohol 2-5 minutes at room temperature
  • 30% Alcohol 2-5 minutes at room temperature
  • Hematoxylin 1-2 dips
  • Wash in tap water 5-10 minutes until the tissues were blue in colour.

Dehydration

Now cross sections were dehydrated in ascending order in alcoholic grades for 3-5 minutes.

  • 30% Alcohol 2-5 minutes at room temperature.
  • 50% Alcohol 2-5 minutes at room temperature.
  • 70% Alcohol 2-5 minutes at room temperature.
  • 90% Alcohol 2-5 minutes at room temperature.
  • Eosin 1-2 dip.
  • 90% Alcohol 1 dip.
  • 100% Alcohol 2-5 minutes at room temperature.
  • Xylene 5-10 minutes.

After staining the slides were mounted with Canada balsam. Cover slips were placed on the slides and they were placed in incubator overnight. Extra Canada balsam was removed by xylene.

3.4.2.7. Light microscopic study

3-5m thick sections were studied under compound microscope (DIALUX 20 EB) at 40x magnifications was photographed.

3.4.2.8. Silver staining technique, (AgNORs) Assay

Silver staining technique was used according to the Trere et al., (1996) and Rosana et al., (2005). The AgNORs technique was performed on dried slides as follows; First of all the unstained fixed slides were dewaxed by dipping for 3 minutes in xylene. After complete dewaxation the slides were hydrated in decrease ethanol concentration (90, 70 and 50%) and washed in distilled water for 10 minutes and dried in oven. After drying slides were treated with one drop of colloidal solution (2% gelatin and 1% formic acid) and two drops of 50% AgNO3 solution on the slide and incubated at 35C for about 8-12 minutes for each tissue. The progressive staining was followed under microscope to get golden colored nuclei and brown/black NORs. Then, the slide was washed in distilled water, treated for 1 minute with 1% sodium thiosulfate at room temperature to stop the reaction, and washed in tap water. The cells were examined under light microscope at 100x magnifications. Composition and preparation of solution/reagents used in AgNOR assay are given below.

  • 2% gelatin
  • Gelatin 2g
  • Distilled water 100ml
  • Formic acid 1%
  • Formic acid 1ml
  • Distilled water 100ml
  • 50% Silver Nitrate solution
  • Silver nitrate 2g
  • Distilled water 2ml
  • 1% Sodium thiosulfate solution
  • Sodium thiosulfate 1g
  • Distil water 100ml
  • 3.4.3. Biochemical investigations

    Physical analysis of urine

    Urine samples were assayed for red blood cells (RBCs) count, white blood cells (WBCs) count and specific gravity by using standard diagnostic kits (MediScreen Urine Strips, Orgenics, France). However, pH was determined by using pH meter.

    3.4.3.1. Urine and serum analysis

    After completion of 4 weeks duration all the animals were kept on normal feed without any treatment for at least 24 hrs and their urine was collected, stored in - 70 0C for further analysis, while blood was collected by cardiac puncture, put in the falcon tube and centrifuged at -40C and 4000 rpm for serum extraction. The serum was stored in refrigerator for assessment of further serum maker enzymes and hormonal analysis.

    3.4.4.1. Assessment of total protein

    Principle

    Total protein in serum and urine was assessed from a colored complex in the presence of copper salt in alkaline solution through AMP DIAGNOSTIC KIT (biuret method).

    Assay procedure

    Mix 1 ml reagent (potassium iodide, potassium sodium tartrate, copper sulphate and sodium hydroxide) with 10 ul sample or standard (albumin), 10 ul distilled water for blank and take OD (550nm) after 10 minutes incubation 37 0C.

    Total protein were calculated through formula; (OD of sample / OD of standard) x n

    Where n is standard concentration.

    3.4.4.2. Assessment of Albumin

    Principle

    Colorimetric determination of serum albumin using bromocresol green (BCG) at pH 4.20 through AMP DIAGNOSTIC KIT.

    Assay procedure

    Mix 1 ml reagent (succinate buffer, bromocresol green, Brij 35) with 10 ul sample or standard (bovine albumin), 10 u distilled water for blank and take OD (628nm) after 5 minutes incubation at 37 0C.

    Serum and urine albumin were calculated through formula; (OD of sample / OD of standard) x n

    Where n is standard concentration

    3.4.4.3. Assessment of globulin

    Globulin was assessed by using formula as; Total protein-albumin

    3.4.4.4. Determination of urea

    Principle

    Urea was determined using Kit purchased from AMP diagnostics company.

    Urea +H2O urease 2NH3 + CO2

    2NH4+ + 2 alpha Ketoglutarate + 2 NADH __ GIDH 2L-glutamate + 2NAD+ + 2H2O

    Assay procedure

    Mix 1 ml reagent (Tris buffer, alpha Ketoglutarate, urease, GIDH and ADP) with 10ul sample or standard and read the variation of OD (340nm) between 30 seconds and 90 seconds. Urea was calculated through formula; (OD of sample / OD of standard) xn

    Where n is standard concentration.

    3.4.4.5. Determination of Serum Bilirubin

    Principle

    Sulfanilic acid reacts with sodium nitrate to form diazotized sulfanilic acid. In the presence of DMSO total Bilirubin reacts with diazotized sulfanilic acid to form azobilirubin. In the presence of DMSO only direct Bilirubin reacts to give azobilirubin using AMP DIAGNOSTIC KIT.

    Assay procedure

    • Mix 1.5 ml working reagent (R1) (sulfanilic acid, DMSO, HCl) , 0.5 ml working reagent (R2) with 100 ul sample or calibrator, 100 ul sample without R2 for sample blank and take OD (555nm) after 5 minutes incubation at 37 0C.
    • Serum total bilirubin and direct Bilirubin were calculated through formula;
    • (OD of sample - OD of sample blank) x F
    • Where F is standard factor concentration.

    3.4.4.6. Determination of Cretinine and Cretinine clearance

    Principle

    The rat of formation of a colored complex between Creatinine and alkaline pirate was measured. Creatinine was determined using AMP DIAGNOSTIC KIT.

    Assay procedure

  • Mix 1 ml working reagent (R) (picric acid) with 100 ul sample or standard, read absorbance at 500nm 25 second after the sample or standard addition. Exactly 2 minutes after the first reading (A1), take 2nd reading (A2).
  • Creatinine calculated through formula;
  • (A2-A1sample / A2-A1standarad) x n
  • Where n is standard concentration.
  • Creatinine clearance was assessed by using formula Ccr = (Cu/Cs) x V
  • Where Cs is the concentration of Creatinine in serum, Where Cu is the concentration of Creatinine in urine and V is the urine flow rate in ml/min.
  • 3.4.4.7. Determination of serum cholesterol

    The quantitative determination of cholesterol was done by using AMP DIAGNOSTIC KIT.

    Principle

    The cholesterol present in the sample originates a colored complex, according to the following reactions:

    • Cholesterol esters + H2O cholesterol esterase_______ Cholesterol + fatty acids
    • Cholesterol + O2 _ cholesterol oxidase_________ 4-Cholestenona + H2O2
    • 2H2O2 + Phenol + 4-Aminoantipyrine ___ Peroxidase ____ Quinonimine +4 H2O2
    • The intensity of the color formed is proportional to the cholesterol concentration in the sample.

    Assay Procedure

    • The assay was done at 37 C temperature with wavelength of 505 nm in cm optical path. 10l of sample or standard was mixed with I ml reagent and incubated for 5 min. The absorbance of the sample was read against the
      blank within 60 min.
    • Serum Cholesterol was assessed by using formula;
    • (OD of sample / OD of standard) xn
    • Where n is standard concentration

    3.4.4.8. HDL Cholesterol

    The in vitro determination of HDL was done by the AMP DIAGNOSTIC KIT.

    Antihuman beta-lipoprotein antibody in R1 binds to lipoproteins. The antigen-antibody complexes formed block enzyme reactions when R2 is added. Cholesterol esterase (CHE) and cholesterol oxidase (CO) in R2 only react with HDL-C. Hydrogen peroxide produced by the enzyme reactions with HDL yields a blue color complex. By measuring the optimum wavelength of 593 nm, the HDL concentration in the sample can be calculated when compared with the absorbance of the HDL-C calibrator.

    Assay Procedure

    • The whole procedure was done at the temperature of 37C with 1 cm optical path at the wavelength of 600 nm against the reagent blank. 2.5L of sample was mixed with the 2.5 l calibrator and after 5 min incubation
      absorbance was measured then R2 of 60l was added and mixed. Then it was incubated for 5 min and absorbance was checked.
    • Calculation
    • (OD2-OD1) sample / (OD2-OD1) Calibrator x n
    • n=Calibrator concentration
    • Calibration:
    • AMP HDL-Calibrator (BR9812) used for calibration.

    3.4.4.9. LDL-Cholesterol

    • LDL- Cholesterol can be calculated by the formula:
    • LDL= TG/5+ HDL - Cholesterol

    3.4.4.9.10. Aspartate aminotransaminase (AST)

    The assay was performed according to the standard procedure available with kit used for in vitro determination of AST according to the method by Vitalab selectra Routine chemistry analyzer (Merck) and other photometric systems.

    Priciple of Reaction

    • L-Aspartate + 2-Oxoglutarate AST L-Glutamate + Oxalacetate
    • Oxalacetate + NADH + H+ MDH L-Maltate + NAD+

    The rate of NADH consumption consumption is measured photo metrically and is directly proportional to the AST activity in the sample

    Reagents (R)

    R1:

    • Tris HCl (pH 7.65) 110mM
    • L-aspartate 320mM
    • Malate dehydrogenase (MDH) 800u/l
    • Lactate dehydrogenase LDH 1200u/l

    R2:

    • 2- Oxoglutarate 65mM/l
    • NADH 1mM/l

    Pyridoxal-5-phosphate:

    • 100mM Good,s Buffer (pH 9.6)
    • 13.8mM pyrodoxal -5-phasphate

    Reagents preparation

    Mix four parts of reagent 1(R1) and one part of reagent 2 (R2) to form a mono reagent with out the mixing of pyridoxal-5-phasphate. The reagents were highly light sensitive so these were protected from light.

    Assay procedure

    The reaction was automatically performed by the Slectra Routine Chemistry analyzer under the feed program in computer. The temperature for the reaction was 370C at 340nm with 1cm optical path against air blank.

    3.4.4.9.11. Alanine amino transferase

    For the determination of ALT the photometric method was used based on the reference method of the international federation of clinical chemistry

    Principle

    • 2-Oxoglutarate + L-alanine ALT Glutamate + Pyruvate
    • Pyruvate + NADH + H+ LDH Lactate + NAD+

    The rate of NADH consumption consumption is measured photometrically and is directly proportional to the ALT activity in the sample.

    Reagents

    • Reagent a, 5x 80ml reagent solution
    • Reagent b, 1x 100ml start reagent

    Reaction solution

    Mix reagent a and reagent b at a ratio of 4 + 1 , e. g, 20ml of reaction solution plus 5ml start reagent.

    Reagents concentration

    • Tris buffer, pH 7.5 100mM/l
    • L-alanine 500mM/l
    • 2-Oxoglutarate 15mM/l
    • NADH 0.18mM/l
    • LDH > 1.2 ku/l

    Procedure

    The reaction was automatically performed by the Slectra Routine Chemistry analyzer under the feed program in computer. The temperature for the reaction was 370C at 340nm with 1cm optical path against air blank.

    Calculations for the enzyme activity

    • The activity of enzyme was calculated as unite per litter (U/l) by using the following formula.
    • Enzyme activity = (?A/min) x F
    • Where (?A/min) is the average absorbance of samples per min while F is a factor specific for each weave length. For 340nm it is 1746.

    3.4.4.12. Alkaline phosphatase (ALP)

    Alkaline phosphatase (ALP) enzyme was determined according to the recommendation of German society of clinical chemistry (DGKC) by vita lab selectra and other photometric systems.

    Principle

    Para - Nitro phenyl phosphate + H2 O ALP Phosphate + para-nitrophenol

    Reagent (R)

    R1:

    • Diethanolamine (pH 9.8) 1.25mM/l
    • Magnesium chloride 0.625mM/l

    R2:

    • p-Nitrophenylphosphate 50mM/l

    Reaction solution

    Mix reagent 1 (R1) and reagent 2 (R2) at a ratio of 4 + 1 , e. g, 20ml of R1and 5ml R2 reagent to form a mono reagent. Mono reagent was protected from light.

    Assay procedure

    The reaction was automatically performed by the Slectra Routine Chemistry analyzer under the feed program in computer. The temperature for the reaction was 370C at 405nm (400nm-420nm) with 1cm optical path against air blank. Mix 20ul of sample with 1000ul

    Mono reagent and read the absorbance after 1min and start stopwatch read the absorbance after 1, 2 and 3 minutes.

    Calculation

    • ALP activity (U/l) ?= (?A/min x F)
    • ?A/min = average of absorbance per min
    • F = factor (for ALP it is 2757 at 405nm)

    3.4.4.13. Gamma glutamyltransferase (GGT)

    This was determined by the method of Orlowski and Meister (1973) using glutamyl p-nitroanilide as substrate. The reaction mixture in a total volume of 1.0 ml contained 0.2 ml 10% homogenate which was incubated with 0.8 ml substrate mixture (containing 4 mM glutamyl p-nitroanilide, 40 mM glycylglycine and 11 mM MgCl2 in 185 mM Tris-HCl buffer, pH 8.25) at 37 C. Ten minutes after initiation of the reaction, 1.0 ml of 25% TCA was added and mixed to terminate the reaction. The solution was centrifuged and the supernatant fraction was read at 405 nm. Enzyme activity was calculated as nM p-nitroaniline formed/min/mg protein using a molar extinction coefficient of 1.74 x 103/M cm.

    3.4.5. RADIOIMMUNO ASSAYS

    3.4.5.1. Determination of Testosterone

    Testosterone in rat serum was calculated through 13276 - Marseille Cedex 9 France (IM1119) Kit purchased from IMMUNOTECH Company.

    Principal of Assay

    The radioimmunoassay of testosterone is a competition assay. Samples and calibrators are incubated with I125-labled testosterone in antibody-coated tubes. After incubation the liquid content of tubes was aspirated and the bound reactivity was determined in a gamma counter, a standard curve was prepared. Unknown valves were determined from the curve by interpolation

    Reagent

    • 100 Antibody-coated anti-testosterone tubes
    • Iodine label 55 ml testosterone tracer
    • Six calibrators
    • Control

    Assay procedure

    • 50 ul of calibrator, control or sample was added to antibody coated tubes
    • 500 ul of tracer was added in all tubes and mix
    • 500 ul of tracer was added to two blank tubes for total count
    • Incubated all the tubes in water bath at 37 centigrade for 3 hours
    • After incubation all the content of tubes were aspirated except the two tubes of total cpm
    • Count bound cpm (B) and total cpm (T) for I min in gamma counter
    • Results were calculated by making standard curve

    3.4.5.2. Cortisol determination

    Cortisol in rat serum was calculated through 10227-Czch Republic (IM1841) Kit purchased from IMMUNOTECH Company.

    Principal of Assay

    The radioimmunoassay of testosterone is a competition assay. Samples and calibrators are incubated with I125-labled testosterone in antibody-coated tubes. After incubation the liquid content of tubes was aspirated and the bound reactivity were determined in a gamma counter, a standard curve were prepared. Unknown valves were determined from the curve by interpolation

    Reagent

    • Anti-Cortisol monoclonal antibodies-coated tubes
    • Iodine label 55 ml Cortisol tracer
    • 5 calibrators
    • Control

    Assay procedure

    • 50 ul of calibrator, control or sample was added to antibody coated tubes
    • 500 ul of tracer was added in all tubes and mix
    • 500 ul of tracer was added to two blank tubes for total count
    • Incubated all the tubes at 18-25 centigrade for 1 hours with shaking at 400rpm
    • After incubation all the content of tubes were aspirated except the two tubes of total cpm
    • Count bound cpm (B) and total cpm (T) for I min in gamma counter
    • Results were calculated by making standard curve

    3.4.5.3. Determination of Prolactin

    Prolactin Hormones were calculated in rat serum through 10227-Czch Republic (IM2121-IM3303) Kit purchased from IMMUNOTECH Company.

    Principal of Assay

    The radioimmunoassay of testosterone is a sandwich type assay. The kit utilizes mouse monoclonal antibodies directed against two different epitopes of Prolactin and hence not competing. Samples and calibrators are incubated with I125-labled testosterone in antibody-coated tubes. After incubation the liquid content of tubes was aspirated and rinsed with wash buffer. The bound reactivity was determined in a gamma counter, a standard curve was prepared. Unknown valves were determined from the curve by interpolation

    Reagent

    • Anti-Prolactin monoclonal antibodies-coated tubes
    • Iodine label 55 ml Cortisol tracer
    • 5 calibrators + one 5 ml zero calibrator ready for use
    • Two control vials
    • 20x wash solution

    Assay procedure

    • 50 ul of calibrator, control or sample was added to antibody coated tubes
    • 500 ul of tracer was added in all tubes and mix
    • 500 ul of tracer was added to two blank tubes for total count
    • Incubated all the tubes at 18-25 centigrade for 1 hours with shaking (>350 rpm)
    • After incubation all the content of tubes were aspirated except the two tubes of total cpm
    • Wash twice all the tubes with wash solution
    • Count bound cpm (B) and total cpm (T) for I min in gamma counter
    • Results were calculated by making standard curve

    3.4.5.4. Determination of Estradiol

    Estradiol in serum was calculated through 13276 - Marseille Cedex 9 France (A21854) Kit purchased from IMMUNOTECH Company.

    Principal of Assay

    The radioimmunoassay of Estradiol is a competition assay. Samples and calibrators are incubated for 3 hours with I125-labled Estradiol in antibody-coated tubes. After incubation the liquid content of tubes was aspirated and the bound reactivity were determined in a gamma counter, a standard curve were prepared. Unknown valves were determined from the curve by interpolation

    Reagent

    • Anti- Estradiol monoclonal antibodies-coated tubes
    • Iodine label 54 ml Estradiol tracer
    • 7 calibrators
    • Control

    Assay procedure

    • 100 ul of calibrator, control or sample was added to antibody coated tubes
    • 500 ul of tracer was added in all tubes and mix
    • 500 ul of tracer was added to two blank tubes for total count
    • Incubated all the tubes at 18-25 centigrade for 3 hours with shaking at 350rpm
    • After incubation all the content of tubes were aspirated except the two tubes of total cpm
    • Count bound cpm (B) and total cpm (T) for I min in gamma counter
    • Results were calculated by making standard curve

    3.4.5.5. Determination of FSH

    FSH Hormones were calculated in rat serum through 10227-Czch Republic (IM2125-IM3301) Kit purchased from IMMUNOTECH Company.

    Principal of Assay

    The radioimmunoassay of FSH is a sandwich type assay. The kit utilizes mouse monoclonal antibodies directed against two different epitopes of Prolactin and hence not competing. Samples and calibrators are incubated with I125-labled testosterone in antibody-coated tubes. After incubation the liquid content of tubes was aspirated and rinsed with wash buffer. The bound reactivity was determined in a gamma counter, a standard curve was prepared. Unknown valves were determined from the curve by interpolation

    Reagent

    • Anti-Prolactin monoclonal antibodies-coated tubes
    • Iodine label 55 ml Cortisol tracer
    • 6 calibrators
    • Two control vials
    • 20x wash solution

    Assay procedure

    • 100 ul of calibrator, control or sample was added to antibody coated tubes
    • 50 ul of tracer was added in all tubes and mix
    • 50 ul of tracer was added to two blank tubes for total count
    • Incubated all the tubes at 18-25 centigrade for 90 min with shaking (>350 rpm)
    • After incubation all the content of tubes were aspirated except the two tubes of total cpm
    • Wash twice all the tubes with wash solution
    • Count bound cpm (B) and total cpm (T) for I min in gamma counter
    • Results were calculated by making standard curve

    3.4.5.6. Determination of LH

    LH Hormones were calculated in rat serum through 1057-Czch Republic (IM2325-IM3302) Kit purchased from IMMUNOTECH Company.

    PRINCIPLE OF THE ASSAY

    The immunoradiometric assay of luteinizing hormone (LH) is a sandwich-type assay. The kit utilizes mouse monoclonal antibodies directed against two different epitopes of LH and hence not competing. Samples or calibrators are incubated in tubes, coated with the first monoclonal antibody in the presence of the second 125I-labeled monoclonal antibody. The content of tubes is aspirated and rinsed after incubation and bound radioactivity is measured. Values are calculated by interpolation from the standard curve. The radioactivity bound is directly proportional to the concentration of LH in the sample.

    REAGENTS

    • All reagents of the kit are stable until the expiry date indicated on the kit label, if stored
    • at 2-8C. Storage conditions for reagents after reconstitution or dilution are indicated
    • in paragraph Assay Procedure.
    • 100 Anti-LH monoclonal antibody coated tubes, One 125I-labeled monoclonal anti-LH antibody, six vials of Calibrators, one vial (lyophilized) of Control serum, wash solution.

    Assay procedure

    • 100 ul of calibrator, control or sample was added to antibody coated tubes
    • 50 ul of tracer was added in all tubes and mix
    • 50 ul of tracer was added to two blank tubes for total count
    • Incubated all the tubes at 18-25 centigrade for 90 min with shaking (>350 rpm)
    • After incubation all the content of tubes were aspirated except the two tubes of total cpm
    • Wash twice all the tubes with wash solution
    • Count bound cpm (B) and total cpm (T) for I min in gamma counter
    • Results were calculated by making standard curve

    3.4.5.7. Determination of Total T4

    T4 Hormones were calculated in rat serum through 10227-Czch Republic (IM1447-IM3286) Kit purchased from IMMUNOTECH Company.

    Principal of Assay

    The radioimmunoassay of T4 is a competition assay. Samples and calibrators are incubated with I125-labled testosterone in antibody-coated tubes along with tracer. After incubation the liquid content of tubes was aspirated and the bound reactivity were determined in a gamma counter, a standard curve were prepared. Unknown valves were determined from the curve by interpolation.

    Reagent

    • Anti-T4 monoclonal antibodies-coated tubes
    • Iodine label 55 ml T4 tracer
    • 6 calibrators
    • Two control vials

    Assay procedure

    • 20 ul of calibrator, control or sample was added to antibody coated tubes
    • 500 ul of tracer was added in all tubes and mix
    • 500 ul of tracer was added to two blank tubes for total count
    • Incubated all the tubes at 18-25 centigrade for 1 hour with shaking (>280 rpm)
    • After incubation all the content of tubes were aspirated except the two tubes of total cpm
    • Count bound cpm (B) and total cpm (T) for I min in gamma counter
    • Results were calculated by making standard curve

    3.4.5.8. Determination of Total T3

    T3 Hormones were calculated in rat serum through 10227-Czch Republic (IM1699-IM3287) Kit purchased from IMMUNOTECH Company.

    Principal of Assay

    The radioimmunoassay of T3 is a competition assay. Samples and calibrators are incubated with I125-labled testosterone in antibody-coated tubes along with tracer. After incubation the liquid content of tubes was aspirated and the bound reactivity was determined in a gamma counter, a standard curve was prepared. Unknown valves were determined from the curve by interpolation.

    Reagent

    • Anti-T3 monoclonal antibodies-coated tubes
    • Iodine label 55 ml T3 tracer
    • 6 calibrators
    • Two control vials

    Assay procedure

    • 50 ul of calibrator, control or sample was added to antibody coated tubes
    • 500 ul of tracer was added in all tubes and mix
    • 500 ul of tracer was added to two blank tubes for total count
    • Incubated all the tubes at 18-25 centigrade for 1 hour with shaking (>280 rpm)
    • After incubation all the content of tubes were aspirated except the two tubes of total cpm
    • Count bound cpm (B) and total cpm (T) for I min in gamma counter
    • Results were calculated by making standard curve

    3.4.5.9. Determination of TSH

    TSH Hormones were calculated in rat serum through 10227-Czch Republic (IM3712-IM3713) Kit purchased from IMMUNOTECH Company.

    Principal of Assay

    The radioimmunoassay of TSH is a competition assay. Samples and calibrators are incubated with I125-labled testosterone in antibody-coated tubes along with tracer

    Reagent

    • Anti- TSH monoclonal antibodies-coated tubes
    • Iodine label 11 ml TSH tracer
    • 7 calibrators
    • Two control vials
    • 20x wash solution

    Assay procedure

    • 100 ul of calibrator, control or sample was added to antibody coated tubes
    • 100 ul of tracer was added in all tubes and mix
    • 100 ul of tracer was added to two blank tubes for total count
    • Incubated all the tubes at 18-25 centigrade for 1 hour with shaking (>280 rpm)
    • After incubation all the content of tubes were aspirated except the two tubes of total cpm
    • Wash twice with 2 ml of wash solution
    • Count bound cpm (B) and total cpm (T) for I min in gamma counter
    • Results were calculated by making standard curve

    3.4.5.10. Estimation of Insulin

    PRINCIPLE OF THE ASSAY

    The immunoradiometric assay of insulin is a "sandwich" type assay. In the kit, mouse monoclonal antibodies directed against two different epitopes of insulin and hence not

    competing is used. This kit may be employed for the measurement of: - immunoreactive insulin (free insulin + insulin bound to anti-insulin antibodies) directly in serum or plasma, - free insulin after pre-treatment of samples with precipitation reagent (Serum and plasma samples (pre-treated or not with precipitation reagent), controls (pre-treated or not with precipitation reagent) and calibrators are incubated in tubes coated with the first monoclonal antibody in the presence of the second monoclonal antibody which is labeled with iodine 125. After incubation, the content of tubes is rinsed so as to remove unbound 125I-labeled antibody. The bound radioactivity is then determined in a gamma counter. The insulin concentrations in the samples are obtained by interpolation from the standard curve. The concentration of insulin in the samples is directly proportional to the radioactivity.

    Reagents

    • Anti-insulin monoclonal antibody coated tubes
    • 125I-labeled monoclonal anti-insulin antibody
    • 5 1 ml Calibrators
    • Two control sera
    • One 50 ml 20x Wash solution

    Assay procedure

    • 50 ul 0f calibrator, control or sample was added to antibody coated tubes
    • 100 ul of tracer was added in all tubes and mix
    • Incubated all the tubes at 18-25 centigrade for 2 hour with shaking (>280 rpm)
    • After incubation all the content of tubes were aspirated except the two tubes of total cpm
    • Wash twice with 2 ml of wash solution
    • Count bound cpm (B) and total cpm (T) for I min in gamma counter
    • Results were calculated by making standard curve

    3.4.6 Assessment of tissue biochemical studies

    3.4.6.1. Protein estimation.

    The total soluble protein of homogenate and supernatant of tissues were determined by the method of Lowry et al. (1951)

    REAGENTS

    Phosphate buffer

    • Potassium dihydrogen phosphate (0.1M) 80ml
    • Disodium hydrogen phosphate (0.1M) 420ml

    Standard protein

    • Bovine serum albumin 1mg/ml

    Alkaline copper solution

    In this solution (50:1) fifty volume of solution (A) and one volume of solution (B) was added.

    Solution A

    • Sodium carbonates 2gm
    • Sodium potassium tartrate 1gm
    • Sodium hydroxide 0.4gm
    • Distilled water 100ml

    Solution B

    • Copper sulphate 0.5gm
    • Distilled water 100ml

    Folin ciocalteu phenol reagent

    Folin ciocalteu phenol+dH2O (1:1) were prepared immediately before use.

    Procedure

    • 50-80mg of tissues mixed in 4 volume of phosphate buffer and homogenized in ice bath throughout.
    • The homogenate is then centrifuged at 10,000 rpm for 20 minutes at 4co
    • Supernatant is decanted and saved for protein assay.
    • The saved supernatant is diluted in 5 folds if required in 0.1M phosphate buffer (pH, 7.4).
    • 1ml alkaline copper solution was then added to each sample tube and mix thoroughly.
    • After 10 minutes 0.1ml of 1:1 folin ciocalteu phenol reagent added to each tube.
    • Thoroughly mixed on vortex and incubated for 30 minutes at room temperature.
    • Then optical density was noted at 500, 595 and 650nm at smart specTM spectrophotometer.
    • For standard curve bovine serum albumin was dissolved 10mg/ml in phosphate buffer of same concentration used for samples and then its further serial dilutions were used and make slandered curve from it O D values.
    • The concentration of soluble protein was calculating in mg/ml by comparing the standard O. D values with sample valve.
    • The amount of protein was expressed as mg/ml of tissue homogenate in phosphate buffer.
      • 3.4.6.2. Extraction of antioxidant enzymes

        Tissues of 80-100mg weighted and were grinded in 1.5ml of 50mM phosphate buffer (pH 7.8) and centrifuged at 15,000xg for 20 minutes at 4C. The supernatant was stored at 4co and was used for the determination of activities of enzymes 70 mg of lung tissue were homogenized in 10 volume of 100 mM NaH2PO4 buffer containing 1 mM EDTA, pH 7.4 and centrifuged at 12,000 xg for 30 min at 4oC. The supernatant was collected and used for the following experiments as described below. Protein concentration of the supernatant of kidney tissue was determined by the method of Lowry et al, 1951 using crystalline BSA as standard.

        3.4.6.2.1. Catalase assay (CAT)

        CAT activities were determined by the method of Chance and Maehly (1955) with some modification. The reaction solution of CAT activities contained 2.5 ml of 50 mM phosphate buffer (pH 5.0), 0.4 ml of 5.9 mM H2O2 and 0.1 ml enzyme extract. Changes in absorbance of the reaction solution at 240 nm were determined after one minute. One unit of CAT activity was defined as an absorbance change of 0.01as units/min

        3.4.6.2.2. Peroxidase assay (POD)

        Activities of POD were determined by the method of Chance and Maehly (1955) with some modification. The POD reaction solution contained 2.5 ml 50 mM phosphate buffer (pH 5.0), 0.1 ml of 20 mM guaiacol, 0.3 ml of 40 mM H2O2 and 0.1 ml enzyme extract. Changes in absorbance of the reaction solution at 470 nm were determined after one minute. One unit of POD activity was defined as an absorbance change of 0.01units/min.

        3.4.6.2.3 Super oxide dismutase assay (SOD)

        SOD activity of lungs was estimated by the method of Kakkar et al., (1984). Reaction mixture of this method contained 0.1 ml of phenazine methosulphate (186 M), 1.2 ml of sodium pyrophosphate buffer (0.052 mM, pH 7.0), 0.3 ml of supernatant after centrifugation (1500 xg, 10 min followed by 10000 xg, 15 min) of 10% lung homogenate was added to the reaction mixture. Enzyme reaction was initiated by adding 0.2 ml of NADH (780 M) and stopped after 1 min by adding 1 ml of glacial acetic acid. Amount of chromogen formed was measured by recording color intensity at 560 nm. Results are expressed in units/mg protein.

        3.4.6.2.4. ?-glutamyl transpeptidase assay (?-GT)

        This was determined by the method of Orlowski and Meister (1973) using glutamyl p-nitroanilide as substrate. The reaction mixture in a total volume of 1.0 ml contained 0.2 ml 10% homogenate which was incubated with 0.8 ml substrate mixture (containing 4 mM glutamyl p-nitroanilide, 40 mM glycylglycine and 11 mM MgCl2 in 185 mM Tris-HCl buffer, pH 8.25) at 37 C. Ten minutes after initiation of the reaction, 1.0 ml of 25% TCA was added and mixed to terminate the reaction. The solution was centrifuged and the supernatant fraction was read at 405 nm. Enzyme activity was calculated as nM p-nitroaniline formed/min/mg protein using a molar extinction coefficient of 1.74 x 103/M cm.

        3.4.6.2.5. Reduced glutathione assay (GSH)

        Reduced glutathione was estimated by the method of Jollow et al., (1974). 1.0 ml sample of 10% homogenate was precipitated with 1.0 ml of (4%) sulfosalicylic acid. The samples were kept at 4 C for 1 h and then centrifuged at 1200 xg for 20 min at 4 C. The total volume of 3.0 ml assay mixture contained 0.1 ml filtered aliquot, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB (5,5-dithiobis-2-nitrobenzoic acid), (100 mM) . The yellow color developed was read immediately at 412 nm on a SmartSpecTM plus Spectrophotometer. It was expressed as M GSH/g tissue.

        3.4.6.2.6. Estimation of lipid peroxidation assay (MDA)

        The assay for lipid peroxidation was carried out following the method of Wright et al., (1981) as modified by Iqbal et al., (1996). The reaction mixture in a total volume of 1.0 ml contained 0.58 ml phosphate buffer (0.1 M, pH 7.4), 0.2 ml homogenate sample, 0.2 ml ascorbic acid (100 mM), and 0.02 ml ferric chloride (100 mM). The reaction mixture was incubated at 37 C in a shaking water bath for 1 h. The reaction was stopped by addition of 1.0 ml 10% trichloroacetic acid. Following addition of 1.0 ml 0.67% thiobarbituric acid, all the tubes were placed in boiling water-bath for 20 min and then shifted to crushed ice-bath before centrifuging at 2500 xg for 10 min. The amount of malonaldehyde formed in each of the samples was assessed by measuring optical density of the supernatant at 535 nm using spectrophotometer against a reagent blank. The results were expressed as nM MDA/min/mg tissue at 37 C using molar extinction coefficient of 1.56x105/M cm.

        3.4.6.2.7. Glutathione peroxidase assay (GSH-Px)

        Glutathione peroxidase activity was assayed by the method of Mohandas et al.,1984). The reaction mixture consisted of 1.49 ml phosphate buffer (0.1 M, pH 7.4), 0.1 ml EDTA (1 mM), 0.1 ml sodium azide (1 mM), 0.05 ml glutathione reductase (1 IU/ml), 0.05 ml GSH (1 mM), 0.1 ml NADPH (0.2 mM), 0.01 ml H2O2 (0.25 mM) and 0.1 ml 10% homogenate in a total volume of 2 ml. The disappearance of NADPH at 340 nm was recorded at 25 C. Enzyme activity was calculated as nM NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22103/M cm.

        3.4.6.2.8. Glutathione reductase assay (GSR)

        Glutathione reductase activity was determined by method of Carlberg and Mannervik (1975). The reaction mixture consisted of 1.65 ml phosphate buffer: (0.1 M, pH 7.6), 0.1 ml EDTA (0.5 mM), 0.05 ml oxidized glutathione (1 mM), 0.1 ml NADPH (0.1 mM) and 0.1 ml 10% homogenate in a total volume of 2 ml. Enzyme activity was quantitated at 25 C by measuring disappearance of NADPH at 340 nm and was calculated as nM NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 x103/M cm.

        3.4.6.2.9. Glutathione-S-transferase assay (GST)

        Glutathione-S-transferase activity was assayed by the method of Habig et al., 1974).

        The reaction mixture consisted of 1.475 ml phosphate buffer (0.1 M, pH 6.5), 0.2 ml reduced glutathione (1 mM), 0.025 ml (CDNB) (1 mM) and 0.3 ml of 10% homogenate in a total volume of 2.0 ml. The changes in the absorbance were recorded at 340 nm and enzymes activity was calculated as nM CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6103/M cm.

        3.4.6.2.10. Hydrogen peroxide assay (H2O2)

        Hydrogen peroxide (H2O2) was assayed by H2O2-mediated horseradish peroxidase-dependent oxidation of phenol red by the method of Pick and Keisari (1981). 2.0 ml of homogenate sample was suspended in 1.0 ml of solution containing phenol red (0.28 nM), horse radish peroxidase (8.5 units), dextrose (5.5 nM) and phosphate buffer (0.05 M, pH 7.0) and were incubated at 37 C for 60 min. The reaction was stopped by the addition of 0.01 ml of NaOH (10 N) and then centrifuged at 800 xg for 5 min. The absorbance of the supernatant was recorded at 610 nm against a reagent blank. The quantity of H2O2 produced was expressed as nM H2O2/min/mg tissue based on the standard curve of H2O2 oxidized phenol red.

        3.4.6.2.11. Quinone reductase assay

        The activity of quinone reductase was determined by the method of Benson et al., 1980). The 3.0 ml reaction mixture consisted of 2.13 ml Tris-HCl buffer (25 mM, pH 7.4), 0.7 ml BSA, 0.1 ml FAD, 0.02 ml NADPH (0.1 mM), and 0.l ml 10% homogenate. The reduction of dichlorophenolindophenol (DCPIP) was recorded at 600 nm and enzyme activity was calculated as nM of DCPIP reduced/min/mg protein using molar extinction coefficient of 2.1 x104/M cm

        3.4.6.2.12. Nitrite assay

        Nitrite assay was conducted by using Griess reagent. Tissue samples were deproteinized by equal volumes of 0.3 M NaOH and 5% ZnSO4 and centrifuged at 6400 xg for 20 min and supernatant was collected. 1.0 ml of Griess reagent was added into the cuvette and blanks the spectrophotometer at 540 nm. Then 20l supernatant was added in cuvette containing Griess Reagent. Nitrite concentration was calculated using a standard curve for sodium nitrite.

        3.4.7. Molecular study

        3.4.7. 1.DNA Isolation

        DNA was isolated by using the methods of Bin Wu et al. (2005), Barone et al. (2006) and Gilbert et al. (2007).Tissues used for DNA isolation were first treated with Liquid Nitrogen and stored at -70C up to start the DNA extraction. The reagents used in DNA isolation are given below.

        • Chloroform
        • EDTA, 0.5 M
        • Ethanol, absolute
        • Isoamyl alcohol
        • Phenol
        • Phosphate Buffered Saline (PBS), 1X
        • Proteinase K
        • RNase A
        • Sodium dodecyl sulfate (SDS) solution, 10%

        Preparation of Required Solution

        DNA buffer (Tris-EDTA)

        • 1 M Tris pH 8.0 20 ml
        • 0.5 M EDTA 20 ml
        • Sterile water 100 ml

        Proteinase K (10mg/ml)

        • Dissolve 100 mg Proteinase K in 10 ml TE for 30 min at room temperature (RT)
        • Aliquot and store at -20C

        RNase A (20 mg/ml)

        Dissolve 200 mg RNase A in 10 ml sterile water, boil for 15 min, and cool to room temperature.Aliquot and store at -20C

        10xTBE (Tris Borate EDTA)

        • Tris, 109gm
        • Boric acid, 55gm
        • EDTA, 9.3gm
        • Distilled Water, 1000ml

        Loading dye

        • 0.25% Bromophenol Blue
        • 4g sucrose in water
        • dH2O to 10mL

        Ethidum Bromide

        • 10mg/ml water

        Agrose gel

        • 1.5% agrose in 1xTBE

        Composition of DNA Lysis buffer

        • 10 mM Tris HCl (PH.8)
        • 25 mM EDTA
        • 100 mM NaCl
        • 0.5% SDS
        • 0.1 mg/ml protein kinase

        Procedure

        1. Take 0.15gms of tissue in a patri dish and washed with DNA Buffer.
        2. When the tissue becomes air dry it was grinded in aluminum foil by using pestle and Mortar.
        3. Put the grinded tissue into number of sterile 15 ml plastic falcon tubes.
        4. Put the l ml lysis buffer of given composition to each tube and regrind the tissue in the presence of lysis buffer with the help of glass rod.
        5. 5. Add 100 l proteinase K (10 mg/ml) and 240 l 10% SDS, shake gently, and
          incubate overnight at 45C in a water bath.
        6. 6. If there are still some tissue pieces visible, add proteinase K again, shake
          gently, and incubate for another 5 hr at 45C.
        7. 7. Add 2.4 ml of phenol, shake by hand for 5-10 min, and centrifuge at 3000 rpm
          for 5 min at 10C.
        8. 8. Pipette the supernatant into a new tube; add 1.2 ml of phenol, and 1.2 ml
          Chloroform/isoamyl alcohol (24:1); shake by hand for 5-10 min, and centrifuge
          at 3000 rpm for 5 min at 10C.
        9. 9. Pipette the supernatant into a new tube; add 2.4 of ml chloroform/isoamyl
          alcohol (24:1), shake by hand for 5-10 min, and centrifuge at 3000 rpm for 5
          min at 10C.
        10. 10. Pipette the supernatant into a new tube; add 25 l of 3 M sodium acetate (pH
          5.2) and 5 ml ethanol, shake gently until the DNA precipitates.
        11. 11. Pipit out the solution gently and the DNA thread is not disturbed.
        12. 12. Wash the DNA in 70% ethanol to dissolve all the impurities and dry it in the laminar flow.
        13. 13. Dissolve the DNA in T E buffer and put the RNAs to each tube having concentration
          at 4C on a rotating shaker.
        14. 14. Measure the DNA concentration in a spectrophotometer at 260 and 280nm

        3.4.7. 2. DNA ladder assay

        DNA Ladder was determined using the procedure of Wu, et al., (2005) and Baron et al. (2006)

        • Total DNA was extracted by from tissue was extracted sequentially using a Phenol-chloroform isoamyl alcohol mixture (25:24:1 v/v/v).
        • Proteins were removed and DNA was purified by using 70% alcohol.
        • Agarose gel Electrophoresis was performed using a 1.5% gel containing 1.0ug/ml ethidium bromide.
        • Depending upon experiment 5ug of total DNA per well was loaded.
        • DNA standards (0.5 g per well) were included to identify the size of the DNA fragments.
        • Electrophoresis was performed for 45minuts 100 V, and DNA was observed under ultraviolet fluorescent lighting.

        3.4.7. 3. DNA fragmentation assay with diphenylamine reaction

        DNA fragmentation from tissue extract was determined using the procedure of He et al., (2001) and Wu et al., (2005).

        Materials

        • Liver tissue 100mgs
        • TTE solution TE buffer pH 7.4 (AI) with 0.2% tritonX-100 (4C)
        • Trichloroacetic acid (TCA) at 5% and 25%

        Diphenylamine (DPA) solution preparation

        • 2g solid Diphenylamine (Recrystalyze in 100% ethanol)
        • 90ml glacial acetic acid
        • 10ml con H2SO4
        • Refrigerated cell centrifuge
        • Heating Plate
        • Spectro photometer (Smart spacTM Plus)
        • Water bath (20oC-100oC)

        Methodology

        • Grind 0.1g of tissue and mixed it in TTE solution.
        • Deliver1.0ml of cell suspension in tubes labeled B
        • Cells centrifuged at 200xg at 4C for 10 min
        • Supernatant transferred carefully in new tubes labeled S
        • Pellet added in tube B 1.0 ml TTE solution and were vortex vigorously
        • To separate fragmented DNA from intact chromatin, tubes were centrifuged at 20,000xg for 10 min at 4C
        • Supernatant transferred carefully in new tubes labeled T
        • Small pellet was added in tube B and 1.0 ml TTE solution
        • 1.0 ml of 25% TCA added to all tubes T,B and S and vortexes vigorously
        • Then allowed precipitation to proceed overnight at 4C
        • After incubation precipitated DNA recovered by pelleting for 10 min at 18,000xg at 4C
        • Supernatants was discard by aspiration
        • DNA was hydrolyzed by adding 160l of 5% TCA to each pellet and heating 15 min at 90C in a heating block.
        • To each tube 320l of freshly prepared DPA solution was added, then vortexes. Allowed to develop color for about 4 h at 37 C.
        • Optical density was read at 600 nm with a spectrophotometer (Smart spacTM Plus, catalog # 170-2525).

        3.4.7. 4. Telomeric repeats amplification protocol (TRAP assay)

        The method was used for measuring the Telomerase activity in tissue was according to the (Wen et al., 1998 and Torre et al., 2002).

        Reagents used in the TRAP assay are following:

        Washing solution

        • 10 mM Hepes-KOH (pH 7.5) (Adjust the pH of KOH solution (pH, 12) by Hepes solution (pH, 5-6.5).
        • 1.5mM MgCl2
        • 10mM KCl
        • 1mM Dithiothreitol (DTT)
        • 1ul RNAs inhibitor (40u/ul)

        Lysis Buffer

        • 10mM Tris-HCl (pH 7.5)
        • 1mM MgCl2
        • 1 mM EGTA
        • 0.1 mM AEBSF [4-(2-aminoethyl)-benzenesulphonyl-fluoride-hydrochlorine]
        • 5 mM mercaptoethanol
        • 0.5% CHAPS (3-cholamidopropyl-dimethylammonio-1-propane-sulphonate)
        • 10% glycerol
        • 1ul RNAs inhibitor (40u/ul)

        10xTRAP reaction solution

        • 200 mM Tris-HCl (pH 8.3)
        • 15 mM MgCl2
        • 630 mM KCl
        • 0.05% Tween-20
        • 10 mM EGTA
        • 1 mg/ml bovine serum albumin
        • 20U of RNAs inhibitors

        Composition of PCR mixture (50l)

        • 36.6 l DEPC treated water
        • 2 l (6 g protein) extract
        • 5 l 10x TRAP reaction solution
        • 2 l (50 M) each dNTP
        • 0.4 l (2 U) Taq DNA polymerase
        • 2 l (0.1 g), TS primer sequence (5'-AATCCGTCGAGCAGAGTT-3').
        • 2 l (0.1 g), CX primer sequence (5'-CCCTTACCCTTACCCTTACCCTAA-3')
        • 1ul RNAs inhibitor enzyme (40U/ul)

        Composition of 12% Polyacrylamide non denaturing gel

        • 40.5 ml 0f 30% polyacrylamide
        • 10.0 ml 10x TBE
        • 800ul Ammonium per sulphate
        • 44ul TEMED
        • 48.656ul dH2O

        Composition of 30% polyacrylamide solution

        • 29g acrylamide
        • 1g NN, Methylenebisacrylamide
        • Volume of dH2O up to 100ml

        Composition of fixing solution

        • 0.5% acitic acid
        • 10% ethanol
        • 0.2% AgNO3

        Composition of developind solution

        • 0.1% formaldehyde
        • 3% NaOH

        Composition of loading dye

        • Xylene cynole 0.25%
        • Bromophenol blue 0.25%
        • Glycerol 50%

        Procedure

        • Take 100 mg of liver tissue and were washed once in ice cold washing buffer.
        • Grind the tissue in ice clod pestle and mortar.
        • Homogenized the grinded tissue in 200 l ice cold lysis buffer
        • The homogenate was incubated on ice for 30 minutes and then centrifuged at
          10 000 g for 30 minutes at 4C.
        • The supernatant was collected and the protein content determined by Coomassie brilliant blue assay.
        • PCR reaction mixture (total 48 l) consisted of 36.6 l DEPC treated water, 2 l (6 g protein)extract, 5 l of 10x TRAP reaction solution, 2 l (50 M) each dNTP, 0.4 l (2 U) Taq DNA polymerase, and 2 l (0.1 g)
          of TS primer sequence.
        • The PCR reaction mixture was incubated at 25C in a thermal cycler for 30 minutes for extension of TS primer.
        • CX primer sequence 2 l (0.1 g)) was added after the completion of incubation period.
        • The reaction mixture (total 50 l) was subjected to 36 PCR cycles at 90oC for 90sec and than at 94C for 30 seconds, 55C for 30 seconds, and 72C for 90 seconds (then 10 minutes for the final step).
        • TRAP reaction product (25 l) was analyzed by electrophoresis in 0.5 Tris-borate EDTA buffer on 15% polyacrylamide non-denaturing gels.
        • Twenty-five microliters of each sample and controls were loaded onto a 15% non-denaturing polyacrylamide gel in 0.5X TBE buffer (1.5 mm spacer and 15 x 17 cm vertical gel).
        • Electrophoresis was carried out at 140 V in 0.5X TBE buffer at room temperature until the bromophenol blue just ran off the gel.
        • The gel was transferred to a fixing solution containing 0.5% acetic acid and 10% ethanol with gentle shaking for 15 min.
        • After addition of 0.2% AgNO3, the gel was stained for 10 min followed by washing twice in distilled water.
        • Then incubated in developing solution (1% formaldehyde and 3%NaOH) for about 10 min.
        • The gel was handled after staining, maintained wet and photographed on 677 Polaroid film.

        3.4.8. Statistical analysis

        To determine the treatment effects one way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% level of probability.

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