Hepatoprotection of Chlorella against Carbon Tetrachloride-induced Oxidative Damage in Rats

Materials and Methods

Materials. Ascorbic acid, bovine serum albumin (BSA), cumene hydroperoxide, α,α-diphenyl-β-picrylhydrazyl (DPPH), hydrogen peroxide (H₂O₂), GSH, glutathione reductase, pyrogallol, reduced β-nicotinamide adenine dinucleotide phosphate (β-NADPH), thiobarbituric acid (TBA), 1,1,3,3-tetraethoxypropane (TEP), meta-phosphoric acid (MPA), cysteine, butylated hydroxytoluene (BHT), methanol, vitamin E, dithiothrietol (DTT), monobromobimane (MbBr), silymarin and Triton® X-100 were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). The bicinchonic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL, USA). All reagents used were of analytical grade or purer.

Preparation of 80% ethanolic extract of chlorella (GPE). Dried, powdered chlorella (C. sorokiniana) was purchased from the Taiwan Chlorella Manufacturing Co. Ltd. (Taipei, Taiwan, ROC). Chlorella was extracted with 80% ethanol (50-fold) by stirring for 2 h. After filtering, the extracting solvent was evaporated at 40°C by a vacuum condenser and the remaining residue was dried by a vacuum freeze-dryer. The yield of the dried GPE was 14.0% . The extract was sealed in plastic bottles and stored at −70°C.

Measurement of the total phenolic content and DPPH radical-scavenging activity. The total phenol content was analyzed using Folin-Ciocalteu's reagent (22). Gallic acid was used as the standard for the calibration curve, and the total phenolic contents were expressed as mg gallic acid equivalents per gram of tested extracts. The DPPH radical-scavenging activity of the extract was measured according to the method of Chung et al. (23). The capability to scavenge DPPH radical was calculated by the following equation: scavenging effect (%)=(1-[absorbance of sample at 517 nm/absorbance of control at 517 nm]) ×100 % .

Treatment of animals. Fifty male weanling Sprague-Dawley rats were obtained and fed commercial chow diets (Fwusow Industry Co., LTD, Taiwan). They were randomly divided into five groups with ten animals in each group (A: control, B: GPE, C: CCl₄, D: GPE+CCl₄ and E: silymarin+CCl₄). All the animals were maintained in a controlled environment (12 h light/dark cycles at 22±2°C and 50±10% humidity). Food and tap water were supplied ad libitum. The body weight and food intake of each rat was recorded daily throughout the experiment. The control group (A) was gavaged with 0.5 ml of normal saline, 0.9% (w/v) NaCl, daily over four consecutive weeks. The GPE group (B) was gavaged with GPE, dissolved in 0.5 ml normal saline, at a dose of 0.5 g/kg body weight/day over four consecutive weeks. The CCl₄ group (C) was gavaged with 0.5 ml 20% CCl₄ in olive oil/rat, on Mondays and Thursdays and also administered 0.5 ml normal saline daily over four consecutive weeks. The GPE+CCl₄ group (D) and silymarin +CCl₄ group (E) were gavaged with GPE (0.5 g/kg body weight) or silymarin (0.2 g/kg body weight) daily over four consecutive weeks and received CCl₄ in the same manner as the C group. The A and B groups, rats not administered CCl₄, received 0.5 ml olive oil at the same time points.

Tissue sampling and preparation. After four weeks of feeding, food was withheld overnight and the rats were decapitated the next morning. The blood was collected directly into sterile tubes. The serum was prepared by centrifugation at 805 ×g for 10 min at 4°C and analyzed for aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The livers were excised, weighed and immediately homogenized in an ice-cold 0.05 mole/l phosphate buffer (pH 7.4) using a Potter-Elvehjem-type homogenizer with a teflon pestle. One portion of this tissue homogenate (0.3 g/ml) was used for assaying the levels of vitamin C, vitamin E and reduced GSH, while the remaining portion was centrifuged at 12,000 ×g, 4°C for 10 min (Universal 16R freeze Centrifuge; Hettich, Tuttlingen, Germany). The resulting supernatant was used to determine activities of SOD and GPX, protein contents and lipid peroxidation. This animal research and all the procedures were reviewed and approved by the Animal Research Ethics Committee at Providence University, Taichung, Taiwan.

Determination of biochemical markers of hepatic injury. The levels of AST and ALT in the serum samples were determined by enzymatic methods using an automatic analyzer (Synchron CX-7 Systems; Beckman, Fullerton, CA, USA) at a commercial analytical service center (Lian-Ming Co., Taiwan, ROC).

Lipid peroxidation measurement. Lipid peroxides are unstable and decompose to form MDA, thus MDA levels are widely used as an indicator of lipid peroxidation. The thiobarbituric acid-reactive substances (TBARS) method was used to estimate tissue MDA level spectrophotometrically at 535 nm (24). TEP was used as a standard and the results were calculated as nanomoles of MDA per mg of tissue protein.

Antioxidants measurement. The liver vitamin C content was stabilized by MPA and cysteine solution, then determined by high-performance liquid chromatography (HPLC) through a 5 μm Econosphere C-18 reversed-phase cartridge column (Merck, Darmstadt, Germany) with an electrochemical detector using a BSA PM-80 isocratic pump (Bioanalytical System, West Lafayette, USA) (25). The eluant was 40 mM sodium acetate/EDTA solution at a flow rate of 0.6 ml/min.

The vitamin E (α-tocopherol) standard and the tissue cytosols were diluted in 0.25 % BHT/0.2% ascorbate/methanol solution prior to HPLC analysis. The tissue vitamin E and GSH contents were measured by HPLC through a 5 μm Econosphere C-18 reversed-phase cartridge column (Merck) with a fluorimetric detector using the Hewlett Packard 1100 Series System (Agilent Technologies, Waldbronn, Germany) (26-27). For vitamin E separation, 10 mM sodium acetate/methanol (A solution) and 75% methanol (B solution) were used as the eluants at a flow rate of 1.2 ml/min. Thirty microliters of sample mixture were injected into the C-18 column. To achieve the separation, the gradient used was 100% A solution for 0-10 min, 100% B solution for 10-15 min and 100% A solution for 15-19 min. The tissue GSH was reduced by DTT/ phosphate solution and the MbBr derivative produced prior to HPLC analysis. A 30 mM TBA/ methanol solution was used as the eluant at a flow rate of 1 ml/min.

Determination of the antioxidative enzyme activities. GPX activity was determined spectrophotometrically at 340 nm by an enzyme-coupled method with glutathione reductase, utilizing cumene hydroperoxide as substrate at 30°C (28). The rate of decrease in the NADPH concentration was observed at 340 nm over a 3 min period at 30 s intervals. One unit of GPX activity was defined as the amount of enzyme that catalyzed the oxidation of 1 μmol of NADPH/min/ml.

SOD activity was determined spectrophotometrically at 325 nm based on the SOD-mediated decrease in the rate of pyrogallol autoxidation under alkali conditions (29). One unit of SOD activity was defined as half the rate of reduction of pyrogallol autoxidation over a 1 min period at 15 s intervals.

Catalase activity was determined spectrophotometrically utilizing H₂O₂ as substrate (30). The rate of H₂O₂ dismutated to H₂O and O₂ is proportional to the catalase activity. The decrease in the amount of H₂O₂ was observed at 240 nm over a 1 min period at 15 s intervals. One unit of catalase activity was defined as 1 mmol H₂O₂ remaining per minute. The protein content of the tissue cytosols was determined based on the Biuret reaction (31) of a BCA kit using BSA as standard. The specific activity of the enzyme was expressed as unit/mg protein.

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Figure 1.

Antioxidant activity of GPE determined by the DPPH method. Each value represents the mean±S.D (n=3).

Statistical analysis. All the results except those for AST and ALT activities, body weight and daily food intake were normalized to cellular protein content. The mean values were compared by analysis of variance (ANOVA) with the Scheffe method for comparing groups (32). A significance level of 5% was adopted for all the comparisons.