Chemicals
Cyclophosphamide (Product NO. C0768) were purchased from Sigma-Aldrich (MO, USA.). All other chemicals were reagent grade or purer.
Preparation of water extract of G. hederacea
G. hederacea was planted in pots and the plants were harvested when the leaves grew up to 4–5 cm in diameter, which took about 2–3 weeks of growth during spring time (temperature around 20–25°C). The G. hederacea extracts were prepared in accordance with our previously reported procedures [11–12]. Briefly, fresh plants were extracted with distilled water at 100°C for 3 h using a heating mantle (NEW LAB MN-30000, Sunray Science Co., Ltd., Taipei, Taiwan). The decoctions were filtered, and then dried by a vacuum freeze-dryer. The extracts were sealed in plastic bottles and stored at -70°C until use.
Mouse Erythrocyte Micronucleus Assay
Weaned (six week old) and healthy (approval of the serology, parasitology and microbiology examinations) male and female mice (ICR strain, body weight: 25–35 g) obtained from Biolasco Taiwan Co., Ltd. (I-Lan, Taiwan) were housed in stainless steel cages (5 per cage, cage size: D29.5 × W18.8 × 13.0 cm) bedding with aspen chips (Nepco, U.S.A.) and provided regular diet (Fu-So pellet chow, Taichung, Taiwan) and water ad libitum. The stainless steel cages were kept at 25 ± 2℃, 65 ± 5% relative humidity and a 07:00–19:00 h lighting period. This study was approved by the animal research ethics committee at Providence University, Taichung, Taiwan (Approval No: 20111215-A03).
The micronucleus assay was conducted according to Krishna and Hayashi [13] and Organization for Economic Cooperation and Development (OECD) [14]. Five mice were allocated randomly to each group. The mice were given HWG at a limited dose of 1.25, 2.50 or 5.00 g/kg bw by oral gavage at 09:00–11:00 am daily. The administered volume was 0.2 ml. The CP group, as the positive control, was intraperitoneal injected with 0.04 g/kg bw of cyclophosphamide. After dosing, the animals were examined for mortality and clinical signs. The animals were anesthetized using CO2, and 100 µL of orbital peripheral blood was withdrawn at 48 and 72 h. Slides were prepared for staining with 0.1% acridine orange hemi. The reticulocytes (RETs), which stained orange, and the micronuclei (Mn) in the RETs, which stained yellow-green, on each slide were counted under a florescence microscope (Eclipse 50i, Nikon, Japan) with blue excitation (450∼490 nm) and a barrier filter (520 nm). In total, 1000 RETs per animal were analyzed for the existence of Mn. The ratio of RETs to normochromatic erythrocytes (NCEs) was determined based on 1000 NCEs. The Mn-to-NCE ratio was recorded while counting 1000 RETs per animal and the Mn-RETs/1000 RETs (‰) was calculated.
Acute And Subacute Toxicity Tests In Rats
Animals and diets
Weaned (five week old) SD male rats (body weight: 140–170 g) and female (body weight: 120–150 g) rats purchased from the Biolasco Taiwan Co., Ltd. (I-Lan, Taiwan) were used. The rats were acclimatized for 1 week prior to starting the experiment. Each rat was caged in a stainless steel cage (cage size: D47.3 × W25.5 × 21.5 cm) individually under controlled environmental conditions (22 ± 2℃, 65 ± 5% relative humidity, 07:00–19:00 h lighting period). The rats were allowed free access to commercial basic diets (Fu-So pellet chow Taichung, Taiwan) and water. The food intake was recorded every day, and the rats were weighed weekly. All the animals received humane care according to the guidelines of the Guidebook for the Care and Use of Laboratory Animals [15]. The study protocol was approved by the animal research ethics committee at Providence University, Taichung, Taiwan (Approval No: 20111215-A03).
Acute Oral Toxicity Study
A single dose of the test substance (1.00 and 5.00 g/kg bw) was gavaged to 10 male and 10 female rats. The administered volume was 1.0 ml. The water ad libitum was used as the vehicle for the acute and subacute studies. The animals were observed carefully for any signs of morbidity and mortality immediately after dosing, at 4 h and at 24 h and intervals, and twice daily for 14 days. The animals were sacrificed under CO2 after 14 days following an overnight fast, and a thorough necropsy was performed on all the animals.
Subacute Oral Toxicity Study
Ten rats were allocated with body weight-based randomization to four groups, including control (water) and HWG treated groups (0.25, 0.50 and 1.00 g/kg bw) for each sex. Each group consisted of 10 males and 10 females, and the rats were gavaged daily with HWG (1.0 mL) at 9:00–11:00 am for 28 days. The limited dose level of HWG at 1.00 g/kg bw/day was selected for the high dose group, and 0.25 g/kg bw/day was considered the low-dose group. Clinical signs and mortality were observed twice daily. The food intake of the rat was recorded every day. The rats were weighed weekly. At the end of the 28-day treatment period, the animals fasted overnight and were sacrificed under CO2. A 1-mL whole blood sample was taken from the abdominal aorta in an EDTA- containing tube (K3 EDTA syringes, product NO. 367835, Vacutainer, NJ, USA.) for the complete blood count (CBC) assay, and serum was obtained from 5 mL of whole blood in Vacutainer tubes (Product NO. 367955, Insepack, Japan) by centrifugation (Kubota 2010, Japan) at 775 xg for 10 min at 4℃.
Hematology Examination
Complete blood count (CBC) was examined by an automated hematology analyzer (Sysmex K-4500, Toa Medical Electronics Co., Ltd., Kobe, Japan) at a commercial analytical service center (Lian-Ming Co., Taichung, Taiwan). The white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelet (PLT) counts were measured.
Biochemical Examinations
The serum sample was used to determine the alkaline phosphatase (ALP), the total cholesterol, triglycerides, glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total bilirubin, glucose, blood urine nitrogen (BUN), and creatinine by enzymatic methods using an automatic analyzer (Synchron CX-7 systems, Beckman Coulter, Fullerton, Calif., U.S.A.) at a commercial analytical service center (Lian-Ming Co., Taichung, Taiwan).
Autopsy And Histology
All the animals, including those in the erythrocyte micronucleus assay and the acute / subacute toxicity tests, were subjected to necropsy, and the organ weights, histopathological examinations and biochemical analyses were evaluated. The morphologies of all the glands were examined visually, and all the organs were observed macroscopically. Selected vital organs (including the heart, liver, spleen, and kidney) were excised, blotted and weighted. Vital organs included brain, heart, liver and kidneys were examined grossly and weighed. Relative organ weight (%) is calculated as organ weight (g) / final body weight (g) x 100. The tissues were fixed in a 10% buffered formaldehyde solution and embedded in paraffin. The paraffin wax was cut into 2 µm sections, stained with hematoxylin and eosin, and examined under light microscopy (Opticphot-2, Nikon, Tokyo, Japan) for the histological examinations.
Antioxidant Activity In Rats
Tissue sampling and preparation
After 28 days of feeding, food was withheld overnight and the rats were sacrificed by decapitation the next morning. The livers, heart, and brains in male rates were subjected for assessment of the antioxidant status. One portion of this tissue was immediately homogenized (0.3 g/mL) in ice-cold 0.05 mole/L phosphate buffer (pH 7.4) using a Potter-Elvehjem-type homogenizer with a Teflon pestle and then centrifuged at 12,000 × g, 4 oC for 10 min (Himac CF 16 RX, Hettich, Tokyo, Japan). The supernatant was used for determining the activities of antioxidant-related enzymes including CAT, SOD, and GPx, and lipid peroxidation and the concentrations of ascorbic acid (vitamin C), vitamin E, GSH and MDA were assessed as well.
Lipid Peroxidation Measurement
The extent of tissue lipid peroxidation was determined by malonaldehyde- thiobarbituric acid (MDA-TBA) adduct according to the method described by Tatum et al [16]. MDA-TBA adduct standard was prepared by reacting 1,1,3,3-tetraethoxy propane (TEP) with TBA under acid condition.
Antioxidants And Plasma Of Total Antioxidant Status (tas) Measurement
The serum sample was used to determine TAS status. The TAS was measured by the colorimetric technique as described by Miller et al [17], using a commercialized kit (Randox Laboratoratories Ltd, UK), in which 2,2’-azinobis-(3-ethyl benzothiazoline-6-sulphonic acid (ABTS) is incubated with metmyoglobin and H2O2 to produce radical cations. The absorbance of 734 nm was monitored, and this absorbance was proportional inversely to the antioxidant capacity of the tested substance. The data of TAS was expressed as a trolox equivalent.
The levels of vitamin C and E in the tissues were measured by using HPLC with electrochemical detection according to the method of Mitton and Trevithick [18]. Reduced glutathione (GSH) in tissue homogenates were analyzed by HPLC with fluorimetric detection as previously described [19].
Determination of the catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities
CAT activity was determined spectrophotometricaly using H2O2 as the substrate [20]. The rate of H2O2 dismutated to H2O and O2 was proportional to the CAT activity. The decrease in H2O2 amount was monitored at 240 nm at every 15 s interval over 1 min period. One unit of CAT activity was defined as 1 mM H2O2 remaining / min. Specific activity of the enzyme was expressed as unit / mg protein.
The activity of SOD in tissue homogenate was assayed by the inhibition of autoxidation of pyrogallol as described by Marklund and Marklund [21]. Tissue supernatant was mixed with equal volume of 1% triton x-100 on ice for 30 min. After centrifuged at 9300 × g for 5 min (4°C), the supernatant was collected for SOD activity analysis. A final 3.017 mL volume of the reaction systems contained 10 µL sample, 3 mL of 50 mM sodium phosphate buffer containing 0.1 mM ethylenediamine tetraacetic acid (EDTA, pH 8.0) and 0.7 µL 50 mM pyrogallol, and the absorbance was recorded every 15 sec for 5 min at 420 nm. One unit of SOD activity was defined as the amount of enzyme required for producing half maximal inhibition of autoxidation.
GPx activity in the supernatant was measured as described by Paglia and Valentine [22]. Briefly, the assay mixture consisted of 0.62 mL of 250 mM phosphate buffer (pH 7.4), 50 µL of 40 mM GSH, 0.2 mL of 5 unit/mL GSH reductase, 10 µL of 20 mM NADPH and 0.1 mL of tissue supernatant. The reaction was started by the addition of 20 µL of 15 mM cumene hydroperoxide. Conversion of NADPH to NADP+ was monitored continuously at 340 nm for 4 min. One unit of GPx activity was defined as the amount of enzyme that catalyzed the oxidation of 1 µmole of NADPH / minute. Specific activity of the enzymes was as unit / mg of protein. Protein concentration of supernatant protein was determined by bicinchoninic acid (BCA) method using bovine serum albumin as the standard [23].
Statistical analysis
Data were expressed as the mean ± standard deviation in each group. Mean values were compared by student’s t test or analysis of variance (ANOVA) [24] using the SPSS 10.0 software (Spss Inc., Chicago, IL, USA). A significance level of 5% was adopted for all comparisons.