Chemicals and reagents
DPPH (1,1-diphenyl-2-picrylhydrazyl) was purchased from Sigma- Aldrich Company. Quillaja saponin was purchased from Chendu Must Biotechnology CO., LTD. SOD, malonaldehyde (MDA), lactate dehydrogenase (LDH), and total antioxidant capacity (T-AOC) kit was obtained from Nanjing Institute of Jiancheng Biological Engineering. Mice were purchased from purchased from Xinxiang Medical University. All other agents were analytical of grade.
RT used in this study was collected from farmland of Henan Institute Science and Technology in autumn, 2012. RT was authenticated by Professor Li Meng from the Department of Botany, Henan Institute Science and Technology.
Preparation and extract of the crude saponins
Samples was dried at the room temperature and milled into dry power. Ethanol, n-butanol, and ethyl acetate (EtOAc) were used for the extraction. Briefly, 200 g of RT power was extracted three times with 10-fold ethanol. After ethanol solvent being removed by a rotary evaporator, the dried ethanol extract was then dissolved in hot water and partitioned successively with equal volumes of n-butanol and EtOAc respectively. The components of each fraction were subjected to rotary evaporator. All of samples were applied with a silica gel column, eluted with water, 80% ethanol and 100% ethanol in sequence, but only the fraction eluted with 80% ethanol was collected and evaporated. To be more specific, the dry extracts were: n-butanol fraction; EtOAc fraction; and the mixture of n-butanol and EtOAc fraction.
Determination of total saponins
The total saponins content of different extracts was determined by the vanillin-sulfuric acid method. All of the extracts were mixed with vanillin (8%, w/v) and sulfuric acid (72%, w/v), then incubated at 60°C for 10 min. Being cooled in an ice water bath for 15 min, the absorbance was measured at 538 nm. Quillaja saponin was used as a reference standard and the content of total saponins was expressed as Quillaja saponin equivalents (μg/mg extract).
DPPH radical scavenging assay
The radical scavenging activity of the plant extracts against DPPH was determined by measuring UV absorbance at 517 nm. The DPPH radical scavenging assay was performed as described by Nazari with slight modifications. In brief, each sample extract (1 ml) at different concentrations (1, 2, 3, 4, and 5 mg/ml) was added to 2 ml DPPH (10 mg/l in methanol). After a 30-min reaction, absorbance was determined. The scavenging ability on DPPH radicals was calculated as follows: Scavenging ability on DPPH radicals (%) = [(A1 - A2)/A1] × 100, where A1 is the absorbance of the control (containing all reagents except the sample extract), and A2 is the absorbance of the sample extract. Vitamin C was used as standard antioxidants.
Reducing power assay
The reducing power assay was conducted as previously described by Heo. In brief, each sample extract (1 ml) at different concentrations (1, 2, 3, 4, and 5 mg/ml) was first mixed with 0.2 M phosphate buffer (pH 6.6) (2.5 ml), and 1% K3Fe(CN)6 (w/v) (2.5 ml). After incubation at 50°C for 20 min, trichloroacetic acid (TCA, 10% w/v) (2.5 ml) were added to the mixture followed by centrifugation at 3000 × g for 10 min to stop the reaction. 2.5 ml of the collected upper layer of the mixture was mixed with 2.5 ml distilled water and 0.5 ml ferric chloride (FeCl3, 0.1% w/v), and the absorbance of the resulting solution was read at 700 nm against a blank. Vitamin C was used as positive controls.
Hydroxyl radical scavenging assay
The scavenging ability of the each fraction on hydroxyl radicals was determined according to the method described by Heo with some modifications. Briefly, individual sample extract (1 ml) at different concentrations (1, 2, 3, 4, and 5 mg/ml) was added to the reagent containing 1 ml FeSO4 (1.5 mM), 0.7 ml H2O2 (6 mM) and 0.3 ml sodium salicylate (20 mM). After incubation for 1 h at 37°C, absorbance of the reaction mixture was read at 562 nm. The scavenging ability of hydroxyl radicals was calculated using the following equation: Scavenging ability on hydroxyl radicals (%) = [(A1 - A2)/A1] × 100, where A1 is the absorbance of the control reaction (containing all reagents except the sample extract), and A2 is the absorbance of the sample extract. Again, vitamin C was used as positive controls.
Animals and treatment
The animal use and care protocols were approved by Institutional Animal Care and Use Committee (IACUC) of Xinxiang Medical University. Forty-eight adult male Kunming mice weighing from 18 to 20 g were purchased from Xinxiang Medical University. All animals were required to undergo institutional quarantine for 7 days prior to use. The environment for animal housing was equipped with controlled temperature (22 ± 3°C), humidity (40% – 70%), and a 12 h light/dark alternation. They were given standard pellet diet and water ad libitum. To study the antioxidant effects of saponins, mice were equally divided into eight groups (n = 6). Group I (control group) received saline (0.9%) intragastrically (2 ml/kg body weight) during the experiment. Group II (an oxidant control group) was given saline (0.9%) intragastrically (2 ml/kg body weight) for 15 days before CCl4 intoxication. Group III and IV (EtOAc fraction treatment group with a dose of 2 mg/kg/d and 3 mg/kg/d of crude drug, respectively), Group V and VI (n-butanol fraction treatment group with a dose of 2 mg/kg/d and 3 mg/kg/d of crude drug, respectively), and Group VII and VIII (the mixture of n-butanol and EtOAc fraction treatment group with a dose of 2 mg/kg/d and 3 mg/kg/d of crude drug, respectively) were administered intragastrically for 15 days before CCl4 injection. The mice received an intraperitoneal injection of CCl4 (0.2 mL/mouse of 0.1% CCl4 solution in olive oil) 13 h before the final administration in different groups, except the normal control groups (group I), which was intraperitoneally treated with an equal amount of olive oil.
After 24 h of CCl4 and olive injection, all the animals were sacrificed. For determination of oxidative stress, the fresh liver tissue was collected and homogenized in 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4). After centrifugation at 12,000 × g for 30 min at 4°C, the supernatant was collected and stored at −70°C for further studies. Protein concentration was determined by Bradford method.
The supernatant of all the samples was collected after homogenate and the LDH content was determined using an LDH assay kit according to the manufacturer’s instructions. LDH cytotoxicity was calculated using OD as LDH cytotoxicity (U/g protein) = (OD sample - OD blank)/(OD standard solution – OD blank standard solution) × standard solution concentration/sample protein concentration.
MDA level was measured by the thiobarbituric acid method with MDA assay kit according to the manufacturer’s instructions. Samples (10 μl) were suspended in 200 μl thiobarbituric acid (TBA) reagent and heated at 95°C for 40 min. After cooling, the reaction mixture was centrifuged at 6,000 × g for 10 min, and TBARS equivalent in supernatants monitored at 532 nm. MDA was calculated using OD as MDA level (nmol/mg protein) = (OD sample − OD blank)/(OD standard solution – OD blank standard solution) × standard solution concentration/sample protein concentration.
The SOD activity of liver tissue was estimated using SOD assay kit according to the manufacturer’s instructions. Briefly, samples (30 μl) were mixed with 3.3 ml of reaction mixture containing xanthine oxidase to oxidizing of NBT by O2
•− monitored at 550 nm. SOD was calculated using OD as SOD level (U/mg protein) = (OD blank − OD sample)/OD blank/50% × dilution of reaction system/sample protein concentration. One unit of SOD activity was defined as that producing 50% dismutation of O2
Ferric-reducing antioxidant power (FRAP) was measured by using total antioxidative capacity (T-AOC) commercial kit according to the manufacturer’s instructions. The stable color of the Fe2+ -o-phenanthroline complex was measured at 520 nm. T-AOC was expressed in U/mg protein where 1 U is defined as an increase in absorbance (A520) of 0.01/min/mg protein at 37°C. T-AOC was calculated using OD as T-AOC level (U/mg protein) = (OD sample − OD blank)/0.01/30 × dilution of reaction system/sample protein concentration.
All of the statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL) program. All data were reported as means ± SD of three independent experiments and were analyzed by oneway ANOVA followed by LSD multiple comparison post hoc analysis. For all comparisons, P < 0.05 was considered statistically significant.