The plant was collected in April 2011 from Islamabad, Pakistan. The plant material was botanically identified by Dr. Mir Ajab Khan, Department of Plant Sciences, Quaid-i-Azam University Islamabad. A voucher specimen (accession #023541) was deposited at the Herbarium of Pakistan Museum of Natural History Islamabad.
The fresh, whole plant (3 kg) was collected and shade dried to obtain 500 g dry sample which was later coarsely powdered in a Willy Mill to 60-mesh size and used for solvent extraction. For sample preparation, 500 g of dried sample were extracted twice (2000 ml for each) with 95% methanol at 25°C for 48 h and concentrated using a rotary evaporator (Panchun Scientific Co., Kaohsiung, Taiwan) under reduced pressure at 40°C to yield the TLM (11.5%). The residue was suspended in water (50 ml) and partitioned successively with n-hexane, chloroform, ethyl acetate, n-butanol (a total of two aliquots of 100 ml each) and soluble residual aqueous fraction yielding respectively the TLH (5.4%), TLC (4.3%), TLE (6.1%), TLB (4.8%) and TLA (8.2%) .
Ascorbic acid; aluminum chloride, 2, 2′ - azino-bis-(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS); ferric chloride (FeCl3); Folin-Ciocalteu; bovine serum albumin (BSA); potassium persulphate; 2,2′-diphenyl-1-picrylhydrazyl (DPPH); nitro blue tetrazolium (NBT); phenazine methosulphate (PMS); reduced glutathione (GSH); 1,2-dithio-bis nitro benzoic acid (DTNB); sulphosalicylic acid; thiobarbituric acid (TBA) and trichloroacetic acid (TCA) were purchased from Sigma Co. (St. Louis, MO, USA). Sulphuric acid; 2-deoxyribose; riboflavin; sodium carbonate (Na2CO3); sodium hydroxide (NaOH); sodium nitrite (NaNO2); disodium hydrogen phosphate (Na2HPO4) and hydrogen peroxide (H2O2) were obtained from Wako Co. (Osaka, Japan). Potassium ferricyanide [K3Fe (CN)6]; triflouroacetic acid; sodium dihydrogen phosphate (NaH2PO4) and all solvents n-hexane (99.8%); chloroform (99.8%); ethyl acetate (99.8%) and n-butanol (99.8%) used were of analytical grade and purchased from Merck Co. (Darmstadt, Germany). Distilled deionized water (dd. H2O) was prepared by Ultrapure TM water purification system (Lotun Co., Ltd., Taipei, Taiwan).
In vitro studies
Each sample was dissolved in 95% methanol to make a concentration of 1 mg/ml and then diluted to prepare the series concentrations for antioxidant assays. Reference chemicals were used for comparison in all assays.
DPPH radical scavenging activity assay
The free radical scavenging activity of the fractions was measured in vitro by 2,2′- diphenyl-1-picrylhydrazyl (DPPH) assay according to the method described earlier [18, 19]. The stock solution was prepared by dissolving 24 mg DPPH with 100 ml methanol and stored at 20°C until required. The working solution was obtained by diluting DPPH solution with methanol to attain an absorbance of about 0.98±0.02 at 517 nm using the spectrophotometer. A 3 ml aliquot of this solution was mixed with 100 μl of the sample at various concentrations (10 - 500 μg/ml). The reaction mixture was shaken well and incubated in the dark for 15 min at room temperature. Then the absorbance was taken at 517 nm. The control was prepared as above without any sample. The scavenging activity was estimated based on the percentage of DPPH radical scavenged as the following equation:
Superoxide anion scavenging assay
The assay for superoxide anion radical scavenging activity was supported by riboflavin-light-NBT system . Briefly, 1 ml of sample was taken at different concentrations (25 to 500 μg/ml) and mixed with 0.5 ml of phosphate buffer (50 mM, pH 7.6), 0.3 ml riboflavin (50 mM), 0.25 ml PMS (20 mM), and 0.1 ml NBT (0.5 mM). Reaction was started by illuminating the reaction mixture using a fluorescent lamp. After 20 min of incubation, the absorbance was measured at 560 nm. Ascorbic acid was used as standard. The scavenging ability of the plant extract was determined by the following equation:
Phosphomolybdate assay (total antioxidant capacity)
The total antioxidant capacity of the fractions was determined by phosphomolybdate method using ascorbic acid as a standard . An aliquot of 0.1 ml of sample solution was mixed with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a water bath at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 765 nm against a blank. A typical blank contained 1 ml of the reagent solution and the appropriate volume of the solvent and incubated under the same conditions. Ascorbic acid was used as standard. The antioxidant capacity was estimated using following formula:
Hydroxyl radical scavenging assay
Hydroxyl radical scavenging activity was measured by the ability of the different fractions of T. leptophylla extract to scavenge the hydroxyl radicals generated by the Fe3+-ascorbate-EDTA-H2O2 system (Fenton reaction) . The reaction mixture contained; 500 μl of 2-deoxyribose (2.8 mM) in phosphate buffer (50 mM, pH 7.4), 200 μl of premixed ferric chloride (100 mM) and EDTA (100 mM) solution (1:1; v/v), 100 μl of H2O2 (200 mM) with or without the extract solution (100 μl). The reaction was triggered by adding 100 μl of 300 mM ascorbate and incubated for 1 h at 37°C. 0.5 ml of the reaction mixture was added to 1 ml of TCA (2.8%; w/v; aqueous solution), then 1 ml of 1% aqueous TBA were added to the reaction mixture. The mixture was heated for 15 min on a boiling water bath. After the mixture being cooled the absorbance at 532 nm was noted against a blank (the same solution but without reagent). The scavenging activity on hydroxyl radical was calculated as follows:
Hydrogen peroxide scavenging activity
Hydrogen peroxide solution (2 mM) was prepared in 50 mM phosphate buffer (pH 7.4). Aliquots (0.1 ml) of different fractions was transferred into the test tubes and their volumes were made up to 0.4 ml with 50 mM phosphate buffer (pH 7.4) 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 . The abilities to scavenge the hydrogen peroxide were calculated using the following equation:
ABTS radical scavenging activity
The 2,2′-azinobis (3-ethylbenzthiazoline-6-sulphonic acid), commonly called ABTS cation scavenging activity was performed . Briefly, ABTS solution (7 mM) was reacted with potassium persulfate (2.45 mM) solution and kept for overnight in the dark to yield a dark coloured solution containing ABTS radical cations. Prior to use in the assay, the ABTS radical cation was diluted with 50% methanol for an initial absorbance of about 0.70±0.02 at 745 nm, with temperature control set at 30°C. Free radical scavenging activity was assessed by mixing 300 μl of test sample with 3.0 ml of ABTS working standard in a microcuvette. The decrease in absorbance was measured exactly one minute after mixing the solution, then up to 6 min. The percentage inhibition was calculated according to the formula:
The antioxidant capacity of test samples was expressed as EC50 (anti-radical activity), the concentration necessary for 50% reduction of ABTS .
The reducing power was based on Fe (III) to Fe (II) transformation in the presence of the solvent fractions . The Fe (II) can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Various concentrations of the sample (2 ml) were mixed with 2 ml of phosphate buffer (0.2 M, pH 6.6) and 2 ml of potassium ferricyanide (10 mg/ml). The mixture was incubated at 50°C for 20 min followed by addition of 2 ml of trichloroacetic acid (100 mg/l). The mixture was centrifuged at 3000 rpm for 10 min to collect the upper layer of the solution. A volume of 2 ml from each of the mixture earlier mentioned was mixed with 2 ml of distilled water and 0.4 ml of 0.1% (w/v) fresh ferric chloride. After 10 min reaction, the absorbance was measured at 700 nm. Higher absorbance of the reaction mixture indicates a higher reducing power.
Estimation of total phenolic content
The total phenolic content was determined by the spectrophotometric method . In brief, a 1 ml of sample (1 mg/ml) was mixed with 1 ml of Folin-Ciocalteu’s phenol reagent. After 5 min, 10 ml of a 7% Na2CO3 solution was added to the mixture followed by the addition of 13 ml of deionized distilled water and mixed thoroughly. The mixture was kept in the dark for 90 min at 23°C, after which the absorbance was read at 750 nm. The TPC was determined from extrapolation of calibration curve which was made by preparing gallic acid solution. The estimation of the phenolic compounds was carried out in triplicate. The TPC was expressed as milligrams of gallic acid equivalents (GAE) per g of dried sample.
Estimation of total flavonoid content
Total flavonoid content was determined following a method by Park et al (2008) . In a 10 ml test tube, 0.3 ml of extracts, 3.4 ml of 30% methanol, 0.15 ml of NaNO2 (0.5 M) and 0.15 ml of AlCl3.6H2O (0.3 M) were mixed. After 5 min, 1 ml of NaOH (1 M) was added. The solution was mixed well and the absorbance was measured against the reagent blank at 506 nm. The standard curve for total flavonoids was made using rutin standard solution (0 to 100 mg/l) under the same procedure as earlier described. The total flavonoids were expressed as milligrams of rutin equivalents per g of dried fraction.
Phytochemical screening of TLM
Phytochemical screening of TLM for the presence of alkaloids, anthraquinones, cardiac glycosides, coumarins, flavonoids, saponins, phlobatannins, tannins and terpenoids was carried out.
Test for alkaloids
0.4 g of TLM was stirred with 8 ml of 1% HCl and the 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 TLM 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 TLM was carried out by taking 5 ml (1 mg/ml) of TLM and mixed with 2 ml of chloroform, followed by 3 ml of concentrated H2SO4. A reddish brown colouration of the interface confirmed the presence of terpenoids .
Test for anthraquinones
200 mg of TLM 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 colour in the ammoniacal phase indicated the presence of free hydroxyl anthraquinones .
Cardiac glycosides determination
5 ml (10 mg/ml in methanol) of TLM 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 TLM 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 TLM was boiled in 1% aqueous hydrochloric acid; the deposition of a red precipitate indicated the presence of phlobatannins .
Test for flavonoids
50 mg of TLM 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 colouration .
Test for tannins
50 mg of TLM was boiled in 20 ml of distilled water and filtered. A few drops of 0.1% FeCl3 was added in filtrate and observed for colour change; brownish green or a blue-black colouration was taken as evidence for the presence of tannins .
In vivo studies
Animals and treatment
Studies were carried out using male Sprague Dawley rats weighing 180±10 g. The animals were grouped and housed in polyacrylic cages with not more than six animals per cage and maintained under standard laboratory conditions. They had free access to standard diet and fresh water ad libitum. For acute toxicity studies, 30 rats were divided randomly into 5 groups, each comprising 6 animals. Group (I) the controls received only vehicles; olive oil (0.5 ml/kg bw) and DMSO (0.5 ml/kg bw) and fed with a normal diet for 7 days. Group II (induction controls) received single dose of CCl4 (CCl4 + Olive oil in 1:1 ratio; 2 ml/kg bw; i.p) on day 1 and day 7 of the experiment. Group ІІІ and IV received TLM (200 mg/kg bw; i.p) and silymarin (25 mg/kg, i.p) as the standard reference drug, once in a day for 7 days respectively along with the intraperitoneal administration of CCl4 on day 1 and 7. Group V received only TLM (200 mg/kg; ip) once in a day for 7 days. At the end of 7 days, 24 h of the last treatment, all the animals were anesthetized in an ether chamber. The liver was removed and placed at 4°C after perfusion with ice cold saline. Intraperitoneal route of administration is selected because it is more rapid and predictable absorption than oral administration. This route is also advantageous because the drug is not inactivated or destroyed as may happen in the gastrointestinal tract and usually smaller doses are required because large concentrations of drug is achieved at the affected site. All experimental procedures involving animals were conducted in accordance with the guidelines of National Institutes of Health (NIH guidelines Islamabad, Pakistan). The study protocol was approved (No. 0241) by Ethical Committee of Quaid-i-Azam University, Islamabad.
Determination of in vivo antioxidant activity
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.
Determination of thiobarbituric acid reactive substances (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 and 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 ˉ1cmˉ1.
Determination of reduced glutathione (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%) sulphosalicylic 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 colour developed was read immediately at 412 nm on a Smart- SpecTM plus Spectrophotometer. It was expressed as μM GSH/g tissue .
Data are expressed as mean ± SD from three separate observations. For in vitro antioxidant assays one way ANOVA test followed by Tukey’s test (P < 0.05) was used to analyze the differences among EC50 of various fractions for different antioxidant assays. The EC50 values were determined using the Graph Pad Prism 5 software. Data on biochemical investigations of in vivo experiments were analyzed by one-way (ANOVA) and the group means were compared by Dunnet’s Multiple Range Test. A probability of P < 0.05 was considered as significant.