Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), CCl4, flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6-dichlorophenolindophenol (DCPIP), thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) were purchased from Sigma Chemicals Co. St. Louis, USA.
The plant was collected in March 2009 from the campus of Quaid-i-Azam University Islamabad, identified by its vernacular name and later validated by Dr. Mir Ajab Khan, Department of Plant Sciences, Quaid-i-Azam University, Islamabad. A voucher specimen was deposited at the Herbarium of Pakistan, Museum of Natural History, Islamabad. Leaves were shade dried for two weeks and powdered in a Willy Mill to 60-mesh size. Briefly, 1 kg powder was extracted with 6 litres of methanol (95%) at 25°C for 48 h. After extraction the mixture was filtered and the methanol solution was evaporated in a rotary evaporator (Panchun Scientific Co., Kaohsiung, Taiwan) at 40°C and stored at 4°C for further in vivo investigations.
Estimation of MCL dose
Male Sprague-Dawley rats (3; six week old) were kept fasting for overnight providing only water, after which the extract was administered intragastrically at the dose of 300 mg/kg bw and rats were remained under observation for 14 days to observe the mortality. Toxicity was not observed, and the procedure was repeated for next higher doses, i.e., 600, 1000, 1500 and 2000 mg/kg bw. One-tenth (200 mg/kg bw) of the maximum dose of the extract tested (2000 mg/kg bw), did not indicate mortality was selected for evaluation of hepatoprotective activity .
Animals and treatment
Six-week-old male Sprague-Dawley rats weighing 180 ± 10 g were kept at 20-22°C on a 12-h light-dark cycle during which time they had free access to standard laboratory rat chow containing protein, 21.05%; fat 4.33%; fiber, 3.07%; ash, 8.3%; sand (silica), 1.65%; carbohydrate, 52.06%; calcium, 0.9%, phosphorous, 0.5%, moisture, 10% (w/w) and fresh water ad libitum. We used only male rats because of their constant metabolism compared to the variation in the female physiology. All experimental procedures involving animals were conducted in accordance with the guidelines of National Institute of Health (NIH guidelines Islamabad, Pakistan). The study protocols were approved by Ethical Committee of Quaid-i-Azam University, Islamabad. The rats were acclimatized to laboratory conditions for 7 days before commencement of experiment.
For subchronic toxicity studies, 8 week experiment was designed with some modifications . 30 male rats of Sprague-Dawley strain were randomly divided into five groups with six animals in each. Group (I) the control received only vehicles; olive oil (0.5 ml/kg bw) and DMSO (0.5 ml/kg bw) and fed with a normal diet. Group (II) the CCl4 group received intraperitoneal administration of 0.5 ml CCl4/kg bw (20% CCl4/olive oil) twice a week for 8 weeks to cause subchronic reversible cirrhosis. Group (III) MCL group (200 mg/kg bw) and Group (IV) the silymarin group (50 mg/kg bw) was given intragastrically through a feeding tube twice a week for 8 weeks. These groups (III and IV) also received intraperitoneal injection of 0.5 ml CCl4/kg bw (20% CCl4/olive oil) twice a week for 8 weeks. Group (V) received only MCL (200 mg/kg bw) twice a week for 8 weeks. At the end of 8 weeks, 24 h of the last treatment, all the animals were anesthesized in an ether chamber. The liver was removed after perfusion with ice cold saline at 4°C. Blood was collected by cardiac puncture and serum obtained by blood centrifugation at 1500 × g for 10 min, at 4°C.
Assessment of serum marker enzymes
Serum analysis of various liver marker enzymes such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and gamma glutamyltransferase (γ-GT) were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria).
Assessment of liver antioxidant enzymes
10% homogenate of liver tissue was prepared in 100 mM KH2PO4 buffer containing 1 mM EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was collected and used for the following experiments as described below. Concentration of protein in supernatant was estimated by using crystalline BSA as standard .
Catalase assay (CAT)
CAT activity was determined by spectrophotometric method . Briefly, a reaction mixture of 3 ml containing 2.5 ml of 50 mM phosphate buffer (pH 5.0), 0.4 ml of 5.9 mM H2O2 and 0.1 ml liver supernatant was allowed to react for one min and change in absorbance of the reaction solution was noted at 240 nm. CAT activity was defined an absorbance change of 0.01 as unit/min.
Peroxidase assay (POD)
POD activity in the liver supernatant was determined by spectrophotometric method . Briefly, 0.1 ml liver supernatant was added to a reaction mixture containing 2.5 ml of 50 mM phosphate buffer (pH 5.0), 0.1 ml of 20 mM guaiacol and 0.3 ml of 40 mM H2O2. Change in absorbance of the reaction solution at 470 nm was determined after one min and POD activity was defined an absorbance change of 0.01 as unit/min .
Superoxide dismutase assay (SOD)
SOD activity was estimated by using NADH as substrate . Briefly, 0.3 ml of supernatant after centrifugation (1500 × g for 10 min followed by 10000 × g for 15 min) was added to the reaction mixture containing 0.1 ml of phenazine methosulphate (186 μM), and 1.2 ml of sodium pyrophosphate buffer (0.052 mM; pH 7.0). Enzyme reaction was initiated by adding 0.2 ml of NADH (780 μM) 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.
Glutathione-S-transferase assay (GST)
The assay mixture was made using 1.475 ml phosphate buffer (0.1 M, pH 6.5), 0.2 ml reduced glutathione (1 mM), 0.025 ml 1-chloro-2,4-dinitrobenzene (CDNB, 1 mM) and 0.3 ml of liver supernatant. Change in the absorbance was recorded at 340 nm and enzyme activity was calculated as nM CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 103M-1cm-1.
Glutathione reductase assay (GSR)
Glutathione reductase assay was carried out by using NADPH as the substrate . Briefly, 0.1 ml of liver supernatant was mixed with 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) and 0.1 ml of NADPH (0.1 mM) and the mixture was measured at 340 nm. Enzyme activity was calculated as nM NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 × 103 M-1cm-1.
Glutathione peroxidase assay (GSH-Px)
This procedure was based on a previously reported method . The reaction assay was done in a final volume of 2 ml solution constituted by: 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 of liver supernatant. 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 6.22 × 103 M-1cm-1.
Quinone reductase assay (QR)
This procedure was based on the reduction of dichlorophenolindophenol (DCPIP). Briefly, 3.0 ml reaction assay was constituted 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 0.1 ml of liver supernatant. The absorbance was determined at 600 nm and enzyme activity was calculated as nM of DCPIP reduced/min/mg protein using molar extinction coefficient of 2.1 × 104 M-1cm-1.
Reduced glutathione assay (GSH)
Reduced glutathione in liver homogenate was determined by reaction with 1,2-dithio-bis nitro benzoic acid (DTNB). Briefly, 1.0 ml of supernatant 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 × g 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 of 1,2-dithio-bis nitro benzoic acid (DTNB, 100 mM). The yellow color developed was read immediately at 412 nm on a SmartSpecTM plus Spectrophotometer. It was expressed as μM GSH/g tissue .
Estimation of lipid peroxidation assay (TBARS)
Malondialdehyde in liver homogenate was determined by reaction with thiobarbituric acid (TBA). Briefly, 1.0 ml reaction assay was consisted of 0.58 ml phosphate buffer (0.1 M; pH 7.4), 0.2 ml liver supernatant, 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 of trichloroacetic acid (10%). 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 × g for 10 min. The amount of TBARS 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 TBARS/min/mg tissue at 37°C using molar extinction coefficient of 1.56 ×105 M-1cm-1.
Hydrogen peroxide assay (H2O2)
Hydrogen peroxide (H2O2) was assayed by H2O2-mediated horseradish peroxidase-dependent oxidation of phenol red . 2.0 ml of liver supernatant 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 the resultant mixture was 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 × g 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.
For microscopic evaluation liver tissues were fixed in a fixative (absolute alcohol 60%, formaldehyde 30%, glacial acetic acid 10%) and embedded in paraffin, sectioned at 4 μm and subsequently stained with hematoxylin/eosin. First the slides were deparaffinized in xylene, and rehydrated in descending order of ethanol. Slides were dipped in hematoxylin, washed in tap water and dehydrated in ascending order of ethanol. Sildes were then stained with eosin and washed with absolute ethanol and xylene. Sections were studied under light microscope (DIALUX 20 EB) at 40× magnifications.
Qualitative tests of the MCL for the presence of alkaloids, anthraquinones, cardiac glycosides, coumarins, flavonoids, saponins, phlobatannins, tannins and terpenoids were carried out.
Test for alkaloids
0.4 g of MCL was stirred with 8 ml of 1% HCl, mixture was warmed and filtered. 2 ml of filtrate was treated separately with (a) with few drops of potassium mercuric iodide (Mayer's reagent) and (b) potassium bismuth (Dragendroff's reagent). Turbidity or precipitation with either of these reagents was taken as evidence for existence of alkaloids .
Test for saponins
The ability of saponins to produce emulsion with oil was used for the screening test. 20 mg of MCL was boiled in 20 ml of distilled water in a water bath for five min and filtered. 10 ml of the filtrate was mixed with 5 ml of distilled water and shaken vigorously for froth formation. 3 drops of olive oil were mixed with froth, shaken vigorously and observed for emulsion development .
Test for terpenoids
Presence of terpenoids in MCL was carried out by taking 5 ml (1 mg/ml) of MCL and mixed with 2 ml of chloroform, followed by 3 ml of concentrated H2SO4. A reddish brown coloration of the interface confirmed the presence of terpenoids .
Test for anthraquinones
200 mg of MCL was boiled with 6 ml of 1% HCl and filtered. The filtrate was shaken with 5 ml of benzene, filtered and 2 ml of 10% ammonia solution was added to the filtrate. The mixture was shaken and the presence of a pink, violet or red color in the ammoniacal phase indicated the presence of free hydroxy anthraquinones .
Cardiac glycosides determination
5 ml (10 mg/ml in methanol) of MCL was mixed with 2 ml of glacial acetic acid having one drop of FeCl3 solution. To the mixture obtained 1 ml of concentrated H2SO4 was added to form a layer. The presence of brown ring of the interface indicated deoxy sugar characteristic of cardiac glycosides .
Test for coumarins
In a small test tube, 300 mg of MCL was covered with filter paper moistened with 1 N NaOH. The test tube was placed for few minutes in a boiling water bath. After removing the filter paper it was examined under UV light, yellow florescence indicated the presence of coumarins .
Test for phlobatannins
80 mg of MCL was boiled in 1% aqueous hydrochloric acid; the deposition of a red precipitate indicated the presence of phlobatannins .
Test for flavonoids
Test for : 50 mg of MCL was suspended in 100 ml of distilled water to get the filtrate. 5 ml of dilute ammonia solution was added to 10 ml of filtrate followed by few drops of concentrated H2SO4. Presence of flavonoids was confirmed by yellow coloration .
Test for tannins
50 mg of MCL was boiled in 20 ml of distilled water and filtered. A few drops of 0.1% FeCl3 was added in filtrate and observed for color change; brownish green or a blue-black coloration was taken as evidence for the presence of tannins .
High Performance Liquid Chromatography (HPLC) of MCL
Sample preparation: 50 mg of MCL was 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 heated twice with 20 ml of methanol for 20 min to obtain the extract. The combined extract was diluted with methanol to 100 ml. 5 ml portion of the solution was filtered and transferred to a volumetric flask and diluted with 10 ml of methanol. The sample (10 μl) was injected into the HPLC apparatus.
HPLC determination: Samples were analyzed on Agilent HPLC system. Separation was carried out through column 20RBAX ECLIPSE, XDB-C18, (5 μm; 4.6 × 150 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, 50% B in A, 10-15 min, 70% B in A. The flow rate was 1 ml/min and injection volume was 10 μl. Eleven standard compounds including rutin, myricetin, vitexin, orientin, hyperoside, isovitexin, isoquercetin, luteolin, apigenin, kaempherol, and luteolin-7-glucoside were run for comparative detection and were optimized. The calibration curves were defined for each compound in the range of sample quantity 0.02-0.5 μg. All samples were assayed in triplicate.
The values are expressed as means ± standard deviation (SD) of six observations in each group. One-way analysis of variance (ANOVA) was carried out to determine the significant difference between parameters. Duncan's multiple range test was used to determine the significant difference between the groups at P < 0.05 by using SPSS ver. 14.0.