Fruit collection of M. buxifolia
The fruit was collected in April 2010 from Khyber Pakhtunkhwa province of Pakistan and the plant was identified by its local name and later identified by Dr. Mir Ajab Khan, Department of Plant Sciences, Quaid-i-Azam University, Islamabad. A specimen ((#43729)) was kept at the Herbarium of the Pakistan Museum of Natural History, Islamabad. The fruits (5.0 kg fresh) of uniform size at maturity were collected and dried under shade to obtain 1.0 kg dry powder excluding the seeds. The powder was exhaustively extracted with methanol and after evaporation of the solvent in a rotary evaporator at 40 °C the crude methanol extract (MBM) was obtained. MBM was stored at 4 °C and used for various studies.
Thin layer chromatography of MBM
An amount of 50 mg of MBM was dissolved in 1 ml of methanol (HPLC grade) Analysis for TLC was performed with silica coated aluminium plates (20 × 20 cm). For activation the silica plates were heated at 110 °C for 40 min. A volume of 10 μl of MBM and reference compounds including apigenin, isoquercetin, orientin, luteolin, rutin, hyperoside, myricetin, luteolin-7-glucoside, kaempherol, vitexin and isovitexin were dropped out to one end of the TLC plate, using a pointed capillary tube. Solvent for the spotted material (mobile phase solution, 150 ml) was prepared by mixing together water, acetic acid and butanol in a ratio of 5:4:1 and stored in a container with a covering to allow vapour circulation. After a gap of 20 min, plates were held vertically in this developing tank. Plates were taken out when mobile phase rose to the upper end, just below 1 cm from the end. Solvent front was marked immediately then the plate was subjected to drying and later sprayed with 2-APB 1 % solution. Detection of flavanoids was done by observing their characteristic colours under UV light at 365 nm. Retention factor values (RF values) have been taken using the formula;
$$ \mathrm{R}\mathrm{F} = \mathrm{Distance}\ \mathrm{the}\ \mathrm{spot}\ \mathrm{moved}/\mathrm{Distance}\ \mathrm{the}\ \mathrm{solvent}\ \mathrm{moved} $$
High performance liquid chromatography (HPLC) analysis
Presence of polyphenolic components of the MBM were detected by the HPLC-DAD analysis. The apparatus used was of Agilent Germany and analytical column was of Sorbex RXC8 (Agilent USA) with 5 μm particle size and 25 ml capacity. Mobile phase was consisted of eluent A, (acetonitrile-methanol-water-acetic acid /5: 10: 85: 1) and eluent B (acetonitrile-methanol-acetic acid/40: 60: 1). The gradient (A: B) utilized was the following: 0–20 min (0 to 50 % B), 20–25 min (50 to 100 % B), and then isocratic 100 % B (25–40 min) at flow rate of 1 ml/min. The injection volume of the sample was 20 μl. Before the injection samples were filtered through 0.45 μm membrane filter. Among the standards gallic acid was analyzed at 230, rutin at 257 nm, catechin at 279 nm, caffeic acid at 325 nm and quercetin, myricetin, kampferol were analyzed at 368 nm [15]. Each time the column was reconditioned for 10 min before the next analysis. All samples were assayed in triplicates. Quantification was carried out by the integration of the peak using the external standard method. All chromatographic operations were carried out at an ambient temperature.
Brine shrimp lethality assay for LD50 estimation
Survival assay against the MBM was carried out on the nauplii of brine shrimp (Artemia salina). For this purpose brine shrimp eggs were hatched in artificial sea water by dissolving 38 g/l of sea salt in distilled water [16]. A lamp was placed above the open side of the tank to attract the hatched shrimps towards wall of the tank. After 24 h the hatched shrimps matured as nauplii were used in the experiment. Stock solution of MBM (20 mg/ml) was prepared in 1 ml of propyleneglycol/Tween 80/water (4:1:4). The solution obtained was used for the two fold diluted serial solutions in salt water in the range of 0.08 – 10 mg/ml. A suspension of larvae (0.1 ml), containing 20 larvae, was added in to each vial and incubated for 24 h. The test tubes were examined after 6, 12 and 24 h and the dead larvae in each vial were counted. The death percentage was calculated for three independent experiments.
$$ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{death}=\left[\frac{Total\ nauplii- Alive\ nauplii}{Total\ nauplii}\right]\times 100 $$
Animal treatment
Acute toxicity studies
In order to investigate the acute toxicity of the methanol extract of the M. buxifolia fruit male and female Sprague-Dawley rats (150–200 g) were assorted in to eight groups having three rats in each. Administration of various dosages of the extract (50, 250, 500, 1000, 2000, 3000, 4000 mg/kg, p.o.) and saline (10 ml/kg) were given to different groups. The experiment was conducted according to the guidelines 425 advocated by the Organization for Economic Cooperation and Development (OECD, 2001) [17]. Abnormal behavior of rats as an effect of the different doses was recorded up to 6 h and the mortality rate was assessed for 14 days after each treatment. According to the results (mortality and abnormal behavior was not detected at the highest dose), 1/10 of the highest dose (400 mg/kg) along with 200 mg/kg was used in this experiment to evaluate the protective potential against the CCl4 induced renal toxicity in rat.
CCl4 induced toxicity studies
Sprague Dawley rats, each weighing about 150–200 g, were maintained in regular rat cages at 25–30 °C with normal 12 h light and dark cycles. The experimental proposal was endorsed (Bch#0231) by a committee of ethical issues, Quaid-i-Azam University Islamabad. Thirty six rats were equally divided into six groups with six rats in each group. Animals of Group I were treated as control. Rats of Group II–V were treated with CCl4 (1 ml/kg body weight i.p. 30 % v/v in olive oil) intraperitoneally. Animals of Group III were also treated with silymarin (50 mg/kg) whereas Group IV and V were additionally treated with MBM (200; 400 mg/kg body weight) orally. Group VI received only MBM at 400 mg/kg orally. The duration of the experiment was 30 days and animals received 15 dosages on alternate days. Urine samples were collected 24 h after the last treatment and stored at −70 °C for urine analysis. Under mild anesthesia of chloroform, dissection was performed ventrally. Blood samples were collected through cardiac puncture and were centrifuged at 500 × g for 15 min at 4 °C to collect the sera for estimation of biochemical parameters. The kidneys were also removed, washed (with ice cold saline to remove debris) and stored in liquid nitrogen at −70 °C for tissue homogenate tests. For performance of histopathological studies, small part of kidneys was stored with 10 % phosphate buffered formalin.
Urine analysis
Urine samples were analyzed for pH, specific gravity, albumin, pus cells, red blood cells and epithelial cells by using standard diagnostic kits (Krenngasse 12, 8010 Graz, Australia). The urine samples were also analyzed for urobilinogen, creatinine, urea, albumin and protein with AMP diagnostics company kits (Krenngasse 12, 8010 Graz, Australia) according to the manufacturers’ instruction.
Serum analysis
Level of urobilinogen, creatinine, urea, blood urea nitrogen (BUN), total bilirubin, albumin and total protein was assessed by the use of AMP diagnostics company kits (Krenngasse 12, 8010 Graz, Australia) according to the manufacturers’ instruction.
Assessment of antioxidant enzymes in renal tissues
Renal tissues were homogenized and 10× homogenates was prepared by the addition of potassium phosphate buffer (pH 7.4). Further, the homogenate was centrifuged at 1500 × g for 10 min at 4 °C. The supernatant obtained was used for estimation of various antioxidant enzymes.
Protein estimation
The estimation of soluble protein in renal tissues was carried out by a method reported earlier [18]. Briefly, 0.1 ml of the supernatant was added in 1 ml of alkaline solution and mixed thoroughly. After an incubation of 30 min the optical density of the mixture was recorded at 595 nm. The concentration of the soluble protein in renal tissue was calculated by using the standard currve of bovine serum albumin.
Catalase (CAT) activity
For the determination of catalase activity in renal tissues, 25 μl of the supernatant was added to a mixture prepared by mixing of 100 μl of 5.9 mM H2O2 and 625 μl of 50 mM potassium phosphate buffer (pH 5.0). Disintegrated the H2O2 occurred by the presence of catalase in the supernatant and its concentration began to decline in the reaction mixture. Activity level of catalase was monitored by a decline in absorbance at 240 nm for 1 min. One unit of catalase activity stated the change in absorbance of 0.01 as units/min [19].
Peroxidase (POD) activity
The method of Chance and Maehly [19] was followed to assess the peroxidase activity in supernatant of renal tissues. Reaction mixture was prepared by mixing of 25 μl of 20 mM guaiacol, 75 μl of 40 mM H2O2 and 625 μl of 50 mM potassium phosphate buffer (pH 5.0). The reaction was initiated by the addition of 25 μl of supernatant to the mixture. Change in absorbance of the reaction mixture was recorded at 470 nm for 1 min. One unit POD activity was deemed equivalent to change in absorbance of 0.01 as units/min.
Superoxide dismutase (SOD) activity
In order to assess the SOD activity in renal samples method of Kakkar et al. [20] was followed. For this purpose homogenate was centrifuged at 1500 × g for 10 min and then it was centrifuged at 10,000 × g for 15 min. To a previously prepared reaction mixture (50 μl of 186 μM phenazine methosulphate and 600 μl of 0.052 mM sodium pyrophosphate buffer (pH 7.0); 150 μl of renal supernatant was added. The reaction was initiated by the addition of 100 μl of 780 μM NADH while to stop the reaction after 1 min 500 μl of glacial acetic acid was added. The quantity of chromogen formed was assessed by recording optical density at 560 nm spectrophotometrically. The enzyme activity was calculated as the concentration of enzyme required to inhibit the 50 % chromogen formation in 1 min. The results were expressed as units/mg protein.
Quinone reductase (QR) activity
Method developed by Benson et al. [21] was adopted to assess the quinone reductase activity in renal tissues. Quinone reductase activity relies on reduction of 2,6-dichlorophenol indophenol (DCPIP). The assay system was composed of 233 μl of bovine serum albumin, 33.3 μl of 50 mM FAD, 6.6 μl of 0.1 mM NADPH, 710 μl of 25 mM Tris-HCl buffers (pH 7.4) and 33.3 μl of renal tissue supernatant. The QR activity was assessed by measuring the disappearance of DCPIP at 600 nm for 3 min at 30 s interval. Using molar extinction coefficient of 2.11 × 104/M/cm, QR activity was interpreted as nM of DCPIP reduced/min/mg protein.
Glutathione peroxidase (GSH-Px) activity
The glutathione peroxidase activity was estimated by following the protocol of Mohandas et al. [22]. GSH-Px activity was based on the oxidation of NADPH and decline in absorbance was recorded at 340 nm at 25 °C. An aliquot of 50 μl of renal supernatant was added to a mixture consisting of 50 μl of 1 mM sodium azide, 50 μl of 1 mM EDTA, 25 μl of glutathione reductase (1 unit/ml), 25 μl of 1 mM GSH, 5 μl of 0.25 mM H2O2 and 740 μl of 0.1 M sodium phosphate buffer (pH 7.4). To start the reaction 50 μl of 0.2 mM NADPH was added to the reaction mixture and optical density was recorded at 340 nm at 25 °C for 20 min. Distilled water was used as blank. By using molar coefficient of 6.23 × 103/M/cm, GSH-Px activity was estimated as amount of NADPH oxidized/min/mg protein.
Glutathione reductase (GSR) activity
The glutathione reductase activity in the renal tissues was assessed by the method of Carlberg and Mannervik [23]. GSR activity was based on the conversion of oxidized glutathione (GSSG) into reduced glutathione (GSH) at the expense of NADPH. Briefly, 50 μl of renal supernatant was added to a reaction mixture consisting of 25 μl of 1 mM oxidized glutathione (GSSG), 50 μl of 0.5 mM EDTA and 825 μl of 0.1 M sodium phosphate buffer (pH 7.6). Then an aliquot of 50 μl of 0.1 mM NADPH was added to the reaction mixture to initiate the process and decline in optical density was recorded at 340 nm at 25 °C for 20 min. Using molar extinction coefficient of 6.22 × 103/M/cm, GSR activity was assessed as amount of NADPH oxidized/min/mg protein.
Glutathione-S-transferase (GST) activity
The glutathione-S-transferase activity was based on the formation of conjugate between reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) [24]. Briefly, reaction mixture was prepared by mixing of 12.5 μl of 1 mM CDNB and 720 μl of sodium phosphate buffer (pH 6.5) and allowed to incubate at 37 °C for 10 min. Later on 150 μl of the renal tissue supernatant was added to the reaction mixture. The reaction was initiated by the addition of 100 μl of 1 mM reduced glutathione and increase in absorbance was recorded 340 nm for 10 min. The blank used in this assay was composed of all the ingredients except the renal tissue supernatant and was replaced by distilled water. With the help of the molar extinction coefficient (9.6 × 103/M/cm), enzymatic activity (GST) was determined and expressed as nM CDNB conjugate formed/min/mg protein.
γ-Glutamyl transpeptidase (γ-GT) activity
The protocol of Orlowski and Meister [25] was followed to estimate the γ-glutamyl transpeptidase activity in renal tissues. The reaction assay was prepared by mixing of 250 μl of 4 mM γ-glutamyl p-nitroanilide, 250 μl of 40 mM glycyl glycine and 250 μl of 11 mM MgCl2 (prepared in 185 mM Tris HCl buffer). An aliquot of 50 μl of renal tissue supernatant was added to the reaction mixture. And after incubation for 10 min at room temperature 250 μl of trichloro acetic acid (25 %) was added to stop the reaction. Optical density of the supernatant after centrifugation at 2500 × g for 10 min was recorded at 405 nm. Using a molar extinction coefficient of 1.75 × 103/M/cm, γ-GT activity was estimated as nM nitroaniline formed/min/mg protein.
Estimation of biomolecules in renal tissues
Reduced glutathione (GSH) assay
The method of Jollow et al. [26] was followed for estimation of GSH concentration in the renal tissues. This method involves oxidation of GSH by sulfhydral reagent 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5′-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. Briefly, 500 μl of renal supernatant was mixed with 500 μl of sulfosalicylic acid (4 %) to carry out precipitation. After an hour of incubation at 4 °C, samples were centrifuged at 1200 × g for 20 min. Then 33 μl of supernatant obtained after centrifugation was mixed with 900 μl of 0.1 M potassium phosphate buffer (pH 7.4) and 66 μl of 100 mM DTNB. The optical density of the yellow colored complex was recorded at 412 nm and GSH activity was determined as μM GSH/g tissue.
Lipid peroxidation (TBARS) assay
The method developed by Iqbal et al. [27] was adopted to measure TBARS (thiobarbituric acid reactive substances) in the renal tissues. The assay system was prepared by mixing of 100 μl of renal tissue homogenate, 10 μl of 100 mM FeCl3, 100 μl of 100 mM ascorbic acid and 290 μl of sodium phosphate buffer (pH 7.4) and incubated for 1 h at 37 °C. The reaction was ceased by the addition of 500 μl of 10 % TCA and after the addition of 500 μl of 0.67 % TBA the tubes were placed in boiling water bath for 15 min. Then shifted on crushed ice for 5 min and allowed to centrifuge at 2500 × g for 10 min. In order to determine the amount of TBARS formed, the absorbance of the supernatant was recorded at 535 nm. With the help of the molar extinction coefficient (1.560 × 105/M/cm), lipid peroxidation activity (TBARS) was expressed as TBARS formed/min/mg tissue.
Hydrogen peroxide (H2O2) assay
Pick and Keisari [28] protocol was followed to perform hydrogen peroxide (H2O2) assay. The oxidation of phenol red was carried out by H2O2-mediated horseradish peroxidase enzyme. Briefly, the reaction mixture was prepared by the addition of 1 ml of phenol red (0.28 nM) solution, 2.0 ml of lung tissue homogenate, 5.5 nM dextrose, 0.05 M phosphate buffer (pH 7.0). the reaction was initiated by the addition of horseradish peroxidase (8.5 units) and incubated for 60 min at 37 °C. To stop the reaction 0.01 ml of 10 N NaOH was added and centrifugation was done at 800 × g for 5–10 min. The absorbance of the sample was noted at 610 nm by using the reagent as a blank. The concentration of H2O2 was given as nM H2O2/min/mg tissue based on the standard curve of H2O2 oxidized phenol red.
Nitrite assay
The concentration of nitrite in renal tissue was assessed by the method of Green et al. [29] by using Griess reagent. The homogenate was treated with equal volume of ZnSO4 (5 %) and NaOH (0.3 M) and then centrifuged at 6400 × g for 15–20 min to obtain the protein free supernatant. A volume of 20 μl of supernatant was reacted with 1 ml of Griess reagent and the optical density of the assay mixture was recorded at 540 nm by using Griess reagent as blank.
DNA fragmentation assay
The DNA injuries in renal tissues were assessed by diphenylamine reaction method using the protocol of Wu et al. [30]. Kidney tissue about 100 mg was homogenized in Tris triton EDTA (TTE) solution. From homogenate 0.1 ml was taken in a tube and labelled B, was centrifuged at 200 × g at 48 °C for 10 min. The supernatant collected was labelled S and again centrifuged at 20,000 × g for 10 min at 48 °C. The intact chromatin obtained was labeled C. After addition of 1.0 ml of 25 % TCA to all tubes; B, S and C were incubated overnight at 48 °C. After centrifugation at 18,000 × g at 48 °C the precipitated DNA was recovered. To each tube 160 ml of 5 % TCA was added and heated for 15 min at 90 °C followed by the addition of 320 ml of freshly prepared DPA solution. Each tube was vortexed vigorously and incubated for 4 h at 37 °C. Optical density of the reaction assay was recorded at 600 nm. The results were presented as %fragmented DNA by using the formula:
$$ \mathrm{D}\mathrm{N}\mathrm{A}\ \mathrm{fragmentation}\ \left(\%\right)=\left[\frac{\mathrm{C}\times 100}{\mathrm{C}+\mathrm{B}}\right] $$
Histopathological study of kidneys
Small pieces of kidney tissues were fixed in fixative sera for 3–4 h and after dehydration in ascending order of alcohol (80 %, 90 %, 100 %) were shifted in cedar wood oil. The tissues after becoming clear were embedded in paraplast. Thin slices (3–4 μm) were prepared with the help of the microtome and then after removing wax, it was stained with hematoxylin-eosin stain and examined by light microscopy.
Statistical analysis
All parametric values were expressed as mean ± SE of six observations in each group. for each group. To determine the difference among various treatments for in vivo studies in rat one way analysis of variance was estimated by using the GraphPad Prism 5 software. Multiple comparisons among various treatments were determined using Boneferroni post hoc comparison test. A P value < 0.05 was considered significant.
