Article pubs.acs.org/JAFC
Characterization of Polysaccharides with Antioxidant and Hepatoprotective Activities from the Wild Edible Mushroom Russula vinosa Lindblad Qin Liu,† Guoting Tian,§ Hao Yan,‡ Xueran Geng,† Qingpeng Cao,† Hexiang Wang,*,† and Tzi Bun Ng*,# †
State Key Laboratory for Agrobiotechnology and Department of Microbiology and ‡State Key Laboratory for Agrobiotechnology and College of Biological Science, China Agricultural University, Beijing 100193, China § Institute of Biotechnology and Germplasmic Resource, Yunnan Academy of Agricultural Science, Kunming 650223, China # School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China ABSTRACT: The aim of the present study was to investigate the antioxidant and hepatoprotective effects of water-soluble polysaccharides (RVLWP) and alkali-soluble polysaccharides (RVLAP) from Russula vinosa on carbon tetrachloride (CCl4)induced acute liver damage in mice. For the in vitro antioxidant activities, RVLWP and RVLAP exhibited potent 1,1-diphenyl-2picrylhydrazyl (DPPH) radical scavenging activity (IC50 = 1.55 ± 0.04 and 3.37 ± 0.21 mg/mL, respectively), hydrogen peroxide scavenging activity (IC50 = 6.07 ± 0.24 and 9.23 ± 0.54 mg/mL, respectively), lipid peroxidation inhibitory effect (IC50 = 0.52 ± 0.095 and 0.86 ± 0.043 mg/mL, respectively), and moderate reducing power and Fe2+ chelating activity (IC50 = 1.86 ± 0.0036 and 0.22 ± 0.0057 mg/mL, respectively). Ascorbic acid was employed as the standard antioxidant in the present study. For the in vivo hepatoprotective activity, administration of RVLWP and RVLAP (200 mg/kg) significantly prevented the elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in acute liver damage induced by CCl4 and suppressed hepatic malondialdehyde (MDA) formation. Mice treated with RVLWP and RVLAP demonstrated a better profile of antioxidants with augmented activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver. The results suggest that RVLWP and RVLAP protect the liver from CCl4-induced hepatic damage via antioxidant mechanisms. KEYWORDS: Russula vinosa, polysaccharides, antioxidant activity, hepatoprotective activity
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INTRODUCTION In recent years, reactive oxygen species (ROS) have received considerable attention because of their role in several pathological conditions, such as heart diseases, diabetes, and cancer.1 Carbon tetrachloride (CCl 4) is a well-known hepatotoxic chemical. In the hepatic parenchyma cells, CCl4 is metabolized by cytochrome P450 to trichloromethyl radicals (CCl3•), which are highly reactive and attack molecules of the cellular membranes, causing lipid peroxidation and depletion of antioxidant enzymes that culminate in liver injury. Antioxidants can delay or reduce oxidation, thus protecting the body from damaging oxidation reactions. However, many studies indicated that some commercial synthetic antioxidants such as butylated hydroxytoluene (BHT), tertiary butylated hydroquinone, and gallic acid ester have potential adverse effects.2−4 Hence, there is increasing interest in searching for natural antioxidants that can replace synthetic antioxidants. Mushrooms can be defined as macrofungi with distinctive fruiting bodies, which are either epigeous or hypogeous and sufficiently conspicuous to the naked eye to be hand-picked.5 Many kinds of mushrooms are delicious and edible and at the same time possess valuable tonic and medicinal attributes.6 Medicinal mushrooms have a long history of application in traditional oriental therapies, and fungal metabolites are increasingly being employed to treat a wide range of diseases.7,8 According to previous reports, polysaccharides from mush© 2014 American Chemical Society
rooms manifest a diversity of useful biological properties, including antioxidant, antitumor, antibacterial, anti-inflammatory, immunomodulatory, antihyperglycemic, and antihypercholesterolemic activities.9−12 Lentinan and schizophyllan are efficacious in the treatment of gastric, breast, lung, cervical, and colorectal cancers when used in combination with conventional antineoplastic drugs.12 Polysaccharides from Coprinus comatus bring about immunomodulatory, hypoglycaemic, hypolipidemic, antitumor, and antibacterial effects.13,14 Recently, considerable interest has arisen in characterizing the polysaccharide components of mushrooms because they exhibited free radical scavenging activity that could counteract CCl4-induced liver damage.15,16 It has been reported that polysaccharides from Agaricus blazei, Macrocybe gigantea, Pholiota dinghuensis, and Phellinus rimosus exert a protective action against acute hepatotoxicity in rats.17−19 All of these activities confer mushrooms with a tremendous potential for promoting human health.19 The basidiomycete fungus Russula vinosa Lindblad is an edible wild mushroom with high medicinal value distributed in broad-leafed forests. Publications on this mushroom are Received: Revised: Accepted: Published: 8858
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was 30 °C. Sugar identification was done by comparison with reference sugars (D-glucose, D-galactose, L-arabinose, and L-fucose). Analysis of Antioxidant Activities in in Vitro Systems. DPPH Radical Scavenging Activity. The activity was measured by using the method reported by Cheng et al.27 with slight modifications. Briefly, 200 μL of different concentrations (0−5 mg/mL) of the sample were added to 600 μL of DPPH solution (0.004% in methanol solution). The mixture was shaken thoroughly and allowed to stand at room temperature in the dark for 30 min. The absorbance was measured at 517 nm. The scavenging percentage was calculated by using the equation
confined to its bionomics, nutritional and functional components, and cultivation.20 To our knowledge, there are as yet no reports about the hepatoprotective effect of polysaccharides from R. vinosa against CCl4-induced hepatotoxicity in rats. Hence, in this study, we used in vitro biochemical tests and an in vivo mouse model of CCl4 intoxication to investigate the activities of R. vinosa water-soluble and alkalisoluble polysaccharides. Our data demonstrate that they elicited pronounced antioxidant and hepatoprotective effects, suggesting that the mushroom can be used as a functional food additive for hepatoprotection.
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scavenging of DPPH radical (%) = (Acontrol − A sample)/Acontrol × 100
MATERIALS AND METHODS
Mushroom and Chemicals. Dried fruiting bodies of the mushroom R. vinosa Lindblad were collected from Fujian province in China. The diagnostic kits for assaying activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Ascorbic acid, linoleic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and monosodium salt of (2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′4″-disulfonic acid (ferrozine) were purchased from Sigma-Aldrich (Steinheim, Germany). All other chemicals were of analytical grade and purchased from Beijing Chemical Co. (Beijing, China). Extraction and Isolation of Polysaccharides. The polysaccharides of R. vinosa were isolated by employing a previously described procedure.21,22 Briefly, dried fruiting bodies of the mushroom (100 g) were crushed into powder by using a disintegrator. The powder was extracted twice with 95% ethanol (2 L) for 2 h to remove lipid, and the residue was extracted with 10 volumes of water (w/v) at 90 °C for 6 h. The supernatant was obtained after centrifugation (9000g, 4 °C, 10 min), and then the residue was extracted with 0.5 M NaOH solution at room temperature for 5 h. The extract was concentrated 10-fold and precipitated with ethanol (1:4, v/v) at 4 °C overnight. The precipitate was collected by centrifugation and deproteinated by employing the Sevage method.23 Finally, the deproteinated supernatant was extensively dialyzed against distilled water and lyophilized to give crude R. vinosa polysaccharides (cRVLWP/cRVLAP). cRVLWP/ cRVLAP was dissolved in distilled water, then applied to a 5 cm × 20 cm column of DEAE-cellulose (Sigma, USA), and eluted at a flow rate of 3.0 mL/min with distilled water. The eluent was concentrated, lyophilized, and then subjected to gel filtration on a Superdex 75 HR 10/30 column (GE Healthcare) in 0.15 M NH4HCO3 buffer (pH 8.5). The eluent was collected automatically, and carbohydrates were determined by the authrone−sulfuric acid colorimetric method.24 The main fraction was pooled and lyophilized to yield RVLWP/RVLAP, which were used for further study. Preliminary Characterization of RVLWP/RVLAP. Total carbohydrate content of the polysaccharide was determined by using the authrone−sulfuric acid colorimetric method, with glucose as the standard.24 The protein content in polysaccharide was measured by using Bradford’s method.25 UV absorption was determined with a UV−visible spectrophotometer (Thermo Scientific, USA) in the range of 200−400 cm−1.23,26 Gel filtration on an FPLC-Superdex 75 column, which had been calibrated with a molecular mass marker, was conducted to determine the molecular masses of RVLWP and RVLAP. The monosaccharide compositions of RVLWP/RVLAP were analyzed by ion exchange chromatography on a Dionex ICS-5000 HPIC system (Thermo Scientific, USA). RVLWP/RVLAP was hydrolyzed with 2 M trifluoroacetic acid (TFA) (2 mL) at 110 °C for 4 h. After removal of TFA with methanol, the hydrolyzed product was dissolved in distilled water. The supernatant was filtered through a 0.22 μm nylon membrane (MSI, Westborough, MA, USA), and 10 μL of the filtrate was injected into the Dionex CarboPac PA-200 anion exchange column (3 mm × 250 mm), eluted with 1 mM NaOH solution. The flow rate was 0.45 mL/min, and the column temperature
where Acontrol is the absorbance of control (water used instead of sample) and Asample is the absorbance in the presence of the sample. Inhibitory Effect on Lipid Peroxidation. The inhibitory effect on lipid peroxidation was determined in accordance with the conjugated diene method but with some modifications.28 Samples at different concentrations (0−5 mg/mL) were prepared and incubated with 500 μL of linoleic acid emulsion (10 mM) in 0.2 M sodium phosphate buffer (pH 6.5) for 15 h at 37 °C. Then 1.5 mL of 60% methanol in deionized water was added, and the absorbance of the mixture was measured at 234 nm. The inhibitory effect on lipid peroxidation was calculated according to the formula
inhibition of lipid peroxidation (%) = (Acontrol − A sample)/Acontrol × 100 where Acontrol is the absorbance of the control sample (water used instead of sample) and Acontrol is the absorbance in the presence of the tested sample. Ascorbic acid was used for comparison. Hydrogen Peroxide Scavenging Activity. The ability to scavenge hydrogen peroxide (H2O2) was determined according to the reported method,1 with minor modifications. The reaction mixture was composed of 200 μL of phosphate buffer (0.1 M, pH 7.4), 40 μL of H2O2 solution (0.3%), and 100 μL of RVLWP/RVLAP solution (0−5 mg/mL). Absorbance of the reaction mixture at 234 nm was determined after 10 min. Ascorbic acid was used as a reference compound. The scavenging of H2O2 was expressed as scavenging of H2O2 (%) = (Acontrol − A sample)/Acontrol × 100 where Acontrol and Asample are the absorbance readings of the control group (water used instead of sample) and sample group at 234 nm, respectively. Reducing Power. The reducing power was determined according to the method reported29 with some modifications. Different concentrations of 1 mL of sample (0−5 mg/mL) were mixed with 1 mL of phosphate buffer (0.2 M, pH 6.6) and 1 mL of potassium ferricyanide (0.1%). The mixtures were incubated at 50 °C for 20 min. Trichloroacetic acid solution (1 mL, 10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (1 mL) was mixed with 1 mL of distilled water and 0.2 mL of ferric chloride (0.3%), and the absorbance was measured at 700 nm. All assays were carried out in triplicate. Ascorbic acid was used as a positive control at the same concentration. Ferrous Ion Chelating Activity. For each sample, the chelating effect on Fe2+ ions was measured according to the reference method30 with some modifications. Briefly, the reaction mixture, containing 200 μL of sample (0−5 mg/mL), 10 μL of ferrous chloride (FeCl2), 40 μL of ferrozine solution (5 mM), and 550 μL of distilled water, was shaken well and incubated for 10 min at room temperature. The absorbance of the mixture was determined at 562 nm. A lower absorbance indicated stronger chelating activity. Ethylenediaminetetraacetic acid disodium salt (EDTA·2Na) was used as a positive control in the present study. The Fe2+ chelating activity was calculated according to the formula 8859
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Fe2 +chelating activity (%) = (Acontrol − A sample)/Acontrol × 100
Article
RESULTS Isolation and Characterization of Polysaccharides. RVLWP and RVLAP were isolated from R. vinosa through hot water extraction followed by 0.5 M NaOH extraction, ethanol precipitation, deproteinization, and lyophilization. As a result, the yields of RVLWP and RVLAP attained approximately 2.67 and 3.19% of dried R. vinosa fruiting bodies by weight, respectively. The extracts were further fractionated on a DEAEcellulose column and then on a Superdex 75 HR 10/30 column. The main eluted fraction named RVLWP/RVLAP was collected for further structural characterization and bioactivity assay. The chemical compositions of the purified polysaccharides were determined as shown in Table 1. The total carbohydrate
where Acontrol is the absorbance of the control sample (water used instead of sample) and Asample is the absorbance in the presence of tested sample. Animal Experiments. Animals. Male Kunming mice (weighing 18−22 g) were provided by Xinglong Experimental Animal Breeding Factory in Haidian District, Beijing, China. The animals were allowed to acclimatize for 7 days to conditions in the animal room and were maintained on standard pellet diet and water ad libitum at a temperature of 20−25 °C under a 12 h light/dark cycle throughout the experiment. All experiments were performed in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China. In Vivo Hepatoprotective Activity. The mice were randomly allocated into seven groups (I−VII) of eight animals each: I, control; II, CCl4; III, bifendate (200 mg/kg + CCl4); IV, RVLWP (100 mg/kg + CCl4); V, RVLWP (200 mg/kg + CCl4); VI, RVLAP (100 mg/kg + CCl4); and VII, RVLAP (200 mg/kg + CCl4). In the normal control group and CCl4-intoxicated group, animals were given a single dose of distilled water (0.2 mL, ig) once daily. The positive control group was treated with bifendate (200 mg/kg in 0.5% sodium carboxymethyl cellulose, ig) once daily for 10 days. In the four experimental groups, the mice were pretreated with RVLWP and RVLAP (100 and 200 mg/ kg, ig) once daily for 10 consecutive days. On the 11th day, all mice except those in the normal control group were given a CCl4/peanut oil mixture (1%, 5 mL/kg, ig) 2 h after the last administration, whereas the normal control group received peanut oil alone.31−33 All animals were sacrificed by anesthesia 16 h after CCl4 administration. The livers perfused with saline were dissected and processed immediately for further analysis. Biochemical Assays. After blood collection, serum was obtained by centrifugation at 5000 rpm for 10 min. The activities of the enzymes ALT and AST were measured with commercially available diagnostic kits, and the results were expressed as units per liter (IU/L). The liver was excised, weighed, and homogenized (10% w/v) in icecold physiological saline. The homogenate was centrifuged (8000g, 4 °C, 10 min), and the supernatant obtained was stored at −20 °C. The activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) and hepatic protein concentrations were assayed using commercially available diagnostic kits. The activities of SOD, GSH-Px, and MDA were normalized with reference to protein, and the results were expressed in units per milligram of protein, units per milligram of protein, and nanomoles per milliggram of protein, respectively. The hepatosomatic index (HI) was calculated according to the following formula: HI = liver weight/body weight × 1000%. Histopathological Study on the Liver. Tissue samples were taken immediately from the liver and fixed in 10% buffered formalin, dehydrated in alcohol, and embedded in paraffin. Sections of 4−5 μm thickness were cut and stained with hematoxylin-eosin. For evaluation of morphological changes, the slides were examined under the microscope. Finally, the images were examined (× 400 magnification) and evaluated for pathological changes. Acute Toxicity Study. Male Kunming mice were randomly divided into five groups of eight animals each. In the control group, mice were given distilled water by gavage. In the experimental group, mice were given RVLWP and RVLAP (500 and 1000 mg/kg, respectively). The animals were observed continuously in the first 24 h for any gross behavioral changes and toxic symptoms in the first 48 h for mortality. Statistical Analysis. All data are presented as the mean ± standard deviation (SD) from three independent experiments. Significant differences among the groups were determined by one-way ANOVA using the IBM SPSS Statistical software (version 20) package program. p < 0.05 was considered to be statistically significant.
Table 1. Compositions and Molecular Weights of RVLWP and RVLAP sample
RVLWP
RVLAP
carbohydrate (wt %) protein (wt %)
96.7 0
97.3 0
galactose (mol %) arabinose (mol %) glucose (mol %) molecular weight (Da)
2.51 0 41.69 8.71 × 104
1.3 0.18 63.93 10.72 × 104
contents of RVLWP and RVLAP were 96.7 and 97.3%, respectively. The negative results in Bradford’s test and the lack of absorption at either 280 or 260 nm in the UV−visible spectrum indicated that RVLWP/RVLAP contained no protein or nucleic acid.22 As can be seen from Table 1, RVLWP was mainly composed of glucose and galactose in a molar ratio of 41.69:2.51. RVLAP consisted of glucose, galactose, and fructose with a molar ratio of 63.93:1.3:0.18. The average molecular weights of RVLWP and RVLAP were 8.71 × 104 and 10.72 × 104 Da, respectively. In Vitro Antioxidant Activity. To analyze the antioxidant activities of RVLWP/RVLAP in vitro, five parameters were monitored, and the results are as follows. DPPH Radical Scavenging Activity. The DPPH radical scavenging activities of RVLWP, RVLAP, and ascorbic acid are presented in Figure 1A. It was observed that the DPPH radical scavenging effects of the samples increased dose-dependently. At the higher concentration (5 mg/mL) of RVLWP and RVLAP, respectively, 85.89 and 52.53% of DPPH radicals were scavenged. The antioxidant activity of the water-extracted polysaccharide, RVLWP (IC50 = 1.55 ± 0.04 mg/mL), was higher than that of RVLAP (IC50 = 3.37 ± 0.21 mg/mL) at the tested concentration as determined by the DPPH radical scavenging assay. Inhibitory Effect on Lipid Peroxidation. The abilities of RVLWP, RVLAP, and ascorbic acid to inhibit lipid peroxidation are presented in Figure 1B. Using the conjugated diene method, RVLWP, RVLAP, and ascorbic acid (IC50 = 0.12 ± 0.048 mg/mL), all when tested at 1 mg/mL, elicited respectively 64.39, 54.72, and 87.24% inhibition of lipid peroxidation. The results demonstrated that RVLWP (IC50 = 0.52 ± 0.095 mg/mL) and RVLAP (IC50 = 0.86 ± 0.043 mg/ mL) manifested potent activity in inhibiting linoleic acid peroxidation. Hydrogen Peroxide Scavenging Activity. The abilities of RVLWP and RVLAP to scavenge hydrogen peroxide are shown 8860
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Figure 1. Antioxidant activities of RVLWP and RVLAP: (A) scavenging of DPPH radicals; (B) inhibitory effect on lipid peroxidation; (C) hydrogen peroxide scavenging activity; (D) reducing power; (E) Fe2+ ion chelating activity. The values are representative of three separate experiments.
Table 2. Effects of RVLWP and RVLAP on Body Weight, Liver Weight, and Hepatosomatic Index (HI) in CCl4-Treated Micea treatment normal control CCl4 only CCl4 + bifendate CCl4 + RVLWP CCl4 + RVLWP CCl4 + RVLAP CCl4 + RVLAP
dose (RVLWP/RVLAP) (mg/kg)
200 100 200 100 200
body wt (g) 29.13 ± 3.22 29.07 ± 1.50 29.86 ± 27.37 ± 27.97 ± 28.17 ± 27.52 ±
1.10 1.44 2.32 2.71 1.67
liver wt (g) 1.23 ± 0.20 1.54 ± 0.09## 1.39 ± 0.12** 1.36 ± 0.09** 1.35 ± 0.11** 1.41 ± 0.24* 1.35 ± 0.18**
HI (%) 42.24 ± 2.87 53.25 ± 3.48### 46.66 ± 4.12*** 49.55 ± 2.27* 48.23 ± 1.83** 49.71 ± 4.68* 49.04 ± 4.38*
a
Mice were randomized into seven groups as follows: control group (normal control), CCl4 intoxication (CCl4 only group), pretreatment with bifendate at 200 mg/kg plus CCl4 intoxication (CCl4 + bifendate group), pretreatment with RVLWP at 100 mg/kg plus CCl4 intoxication (CCl4 + RVLWP group), pretreatment with RVLWP at 200 mg/kg plus CCl4 intoxication (CCl4 + RVLWP group), pretreatment with RVLAP at 100 mg/kg plus CCl4 intoxication (CCl4 + RVLAP group), pretreatment with RVLAP at 200 mg/kg plus CCl4 intoxication (CCl4 + RVLAP group). CCl4induced hepatotoxic mice were given a CCl4/peanut oil mixture (1%, 5 mL/kg, ig), whereas the normal control group received peanut oil alone. The values are reported as the mean ± SD of eight mice per group: ###, p < 0.001 compared with normal control; ***, p < 0.001, **, p < 0.01, and *, p < 0.05 compared with the CCl4 group.
in Figure 1C and compared with that of ascorbic acid. All samples demonstrated a dose-dependent H2O2 scavenging activity. When the concentration of the samples tested was raised from 1 to 5 mg/mL, the percentages of H2O2 scavenged
increased from 22.91 to 50.23% for RVLWP and from 8.97 to 35.03% for RVLAP. Ascorbic acid scavenged 52.28% H2O2 at 1 mg/mL. Compared with ascorbic acid (IC50 = 1.28 ± 0.04 mg/ mL), RVLWP (IC50 = 6.07 ± 0.24 mg/mL) and RVLAP (IC50 8861
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= 9.23 ± 0.54 mg/mL) exhibited moderate H2O2 scavenging activity. Reducing Power. As shown in Figure 1D, RVLWP and RVLAP brought about reduction of the Fe3+/K3Fe(CN)6 complex to Fe2+ ions and, consequently, the Fe2+ ions could be monitored by measurement of the enhanced formation of Perl’s Prussian blue at 700 nm.31 In the concentration range of 1−5 mg/mL, a concentration-dependent effect was discernible. At 5 mg/mL, RVLWP and RVLAP exhibited reducing powers of 0.67 and 0.54, respectively. The data presented here indicate that both RVLWP and RVLAP have potential to be exploited as strong antioxidants, albeit somewhat inferior to the reference ascorbic acid in antioxidant capacity. Ferrous Ion Chelating Activity. Figure 1E shows the Fe2+ ion chelating activity of RVLWP and RVLAP when compared to the standard, EDTA·2Na (IC50 < 0.1 mg/mL). The Fe2+ ion chelating activity of RVLWP and RVLAP increased with concentration. The percentage of Fe2+ ions chelated by RVLWP, RVLAP, and EDTA·2Na was respectively 69.08, 97.28, and 98.38% at a concentration of 5 mg/mL. The present results demonstrated that RVLWP (IC50 = 1.86 ± 0.0036 mg/ mL) and RVLAP (IC50 = 0.22 ± 0.0057 mg/mL) elicited strong activity in chelating Fe2+ ions. Animal Experiments. Effects of Polysaccharides on Body Weight, Liver Weight, and HI in Mice. Table 2 shows the changes in body weight, liver weight, and HI of different groups of experimental mice. The liver weight and HI of the mice increased significantly after CCl4 treatment when compared to the normal control (#, p < 0.01; #, p < 0.001). However, the CCl4-induced increases in the liver weight and HI could be mitigated by pretreatment with RVLWP and RVLAP at two different doses (100 and 200 mg/kg), similar to results of pretreatment with the positive control bifendate at a dose of 200 mg/kg (*, p < 0.01; *, p < 0.001, respectively). These results are in agreement with a previous study.31 The body weights of the experimental groups did not show statistically significant differences compared with the normal control group. Effects of Polysaccharides on AST, ALT, MDA, GSH-Px, and SOD Activities. Several enzymes in serum such as AST and ALT were used as biochemical markers for early acute hepatic damage. As displayed in Figure 2, CCl4 treatment significantly increased the activities of AST and ALT in serum. When compared with the normal control group, mice treated with CCl4 alone showed acute liver damage as evidenced by a significant rise in the serum activities of AST and ALT (both with #, p < 0.001), which are indicators of hepatocyte damage and loss of functional integrity. Interestingly, pretreatment with RVLWP and RVLAP significantly suppressed elevations in the serum levels of AST (∗, p < 0.05) and ALT (∗, p < 0.05), especially at the dose of 200 mg/kg (∗, p < 0.01). The results signified that supplementation with RVLWP and RVLAP could depress the serum activities of AST and ALT in CCl4intoxicated mice and appeared to be protective in undermining the deleterious effect of CCl4. Bifendate (positive control) at a dose of 200 mg/kg demonstrated the most significant hepatoprotective effect (∗, p < 0.001). In this study, the product of membrane lipid peroxidation (MDA) was also examined. As illustrated in Figure 3A, CCl4 treatment significantly raised the hepatic level of MDA (#, p < 0.001). RVLWP and RVLAP at 100 and 200 mg/kg significantly suppressed the level of MDA in a dose-dependent manner. Bifendate-treated mice also manifested a significant
Figure 2. Effects of RVLWP and RVLAP on the activities of serum AST (A) and ALT (B) in CCl4-treated mice. The values are reported as the mean ± SD of eight mice per group: (###)p < 0.001 compared with normal control; (∗∗∗) p < 0.001, (∗∗) p < 0.01, and (∗)p < 0.05 compared with the CCl4-treated group.
decline in hepatic MDA compared to CCl4-treated mice (∗, p < 0.001). The hepatic activities of GSH-Px and SOD in experimental animals are shown in Figure 3B, C. When compared to the normal control group, the acute administration of CCl4 to mice induced characteristic hepatotoxicity that affected the antioxidant parameters of liver tissue, as indicated by a significant decrease in SOD and GSH-Px levels (#, p < 0.001). The CCl4induced decrement was dose-dependently attenuated by pretreatment with RVLWP and RVLAP at 100 mg/kg (∗, p < 0.05) and 200 mg/kg (∗, p < 0.01). When tested at a dose of 200 mg/kg, bifendate as a positive control also effectively protected against the fall in hepatic activities of GSH-Px and SOD (∗, p < 0.001). Liver Histopathological Study. In the current study, histopathological observation of the liver was performed to corroborate the evidence from biochemical analyses. In comparison with the cellular architecture of hepatic tissue from the normal control group (Figure 4A), acute administration of CCl4 to mice induced extensive liver damage characterized by moderate to severe cellular degeneration, hepatocyte necrosis, and lipid droplet accumulation (Figure 4B). However, bifendate pretreatment effectively protected against CCl4-induced liver damage (Figure 4C). Likewise, RVLWP and RVLAP pretreatment ameliorated liver damage, as evidenced by a diminution of necrotic zones and attenuation of lipid droplet accumulation (Figure 4D−G). This finding was 8862
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Hence, it is possible to limit oxidative tissue injury and hence prevent disease progression by employing antioxidant defense supplements. Some of the natural polysaccharide-containing extracts from plants and fungi possess free radical scavenging activities.9,19,36 In our study, the antioxidant effects of watersoluble and alkali-soluble polysaccharides from R. vinosa Lindblad were determined using enzymatic and nonenzymatic methods under in vivo and in vitro conditions. The DPPH-based assay system is a rapid and efficient method for measuring the free radical scavenging activities of antioxidants. In this assay, the absorbance decreases due to a color change from deep violet to colorless as the radical is scavenged by RVLWP/RVLAP. DPPH radical is characterized as a stable free radical and can accept an electron or a hydrogen radical to become a stable diamagnetic molecule. To our knowledge, cysteine, glutathione, ascorbic acid, and aromatic amines could reduce and decolorize DPPH by virtue of their hydrogen-donating ability.37 Similarly, in this investigation, RVLWP and RVLAP exhibited significant dose-dependent DPPH radical scavenging activities. Thus, the results implied that RVLWP/RVLAP might also, like the antioxidants cysteine, glutathione, and ascorbic acid and aromatic amines, act as an electron or a hydrogen donor to scavenge DPPH radicals. Lipid peroxidation is a complicated process involving the interaction of oxygen-derived free radicals with polyunsaturated fatty acids, resulting in a variety of highly reactive electrophilic aldehydes.38 In this study, a well-known polyunsaturated fatty acid, linoleic acid, was oxidized in a water emulsion. Peroxyl (LOO•) and alkoxyl (LOS•) radicals, the products of linoleic acid oxidation, form pre-existing lipid peroxide (LOOH) to initiate lipid peroxidation.39,40 Antioxidants could inhibit lipid peroxidation by scavenging lipid-derived radicals (LOO• or LOS•).41,42 As observed in this investigation, RVLWP and RVLAP displayed significant activity in combatting the peroxidation of linoleic acid, indicating that these polysaccharides can serve as good antioxidants. The measurement of H2O2 scavenging activity is one of the useful methods for determining the ability of antioxidants to lower the level of pro-oxidants such as H2O2.43 Hydrogen peroxide causes toxicity to cells because it depletes antioxidants and produces strand breaks in DNA and oxidative degradation of most biological macromolecules such as lipids, proteins, carbohydrates, and nucleic acids.44 At the concentration of 5 mg/mL, the percentage inhibition of RVLWP and RVLAP on H2O2 observed was 50.23 and 35.03%, respectively. The present results disclosed that RVLWP and RVLAP evinced moderate dose-dependent H2O2 scavenging activity. The reducing power of a compound may serve as an indicator of its potential antioxidant activity.45 It has also been reported that the reducing properties were generally associated with the presence of reductones.46 Our results suggest that RVLWP and RVLAP have moderate but concentrationdependent reducing power. They might play a role as hydrogen donors and could react with free radicals to stabilize and terminate radical chain reactions. Fe2+ chelation might ensue in important antioxidative effects by retarding metal-catalyzed oxidation.47 Some transition metals could trigger free radical reactions to magnify the cellular damage. Among the transition metals, Fe is known as the most important pro-oxidant due to its high reactivity. The ferrous state of iron can stimulate lipid oxidation by generating reactive free radicals via the Fenton reaction.48,49 RVLAP showed stronger activity than RVLWP in chelating ferrous ions
Figure 3. Effects of RVLWP and RVLAP on (A) hepatic MDA content, (B) hepatic GSH-Px activity, and (C) hepatic SOD activity in CCl4-treated mice. The values are reported as the mean ± SD of eight mice per group: (###) p < 0.001 compared with normal control; (∗∗∗) p < 0.001, (∗∗) p < 0.01, and (∗) p < 0.05 compared with the CCl4-treated group.
consistent with the levels of serum and hepatic enzyme markers. Acute Toxicity Study. In the acute toxicity studies, RVLWP and RVLAP exhibited no toxicity in experimental mice. No mortality occurred within 48 h either in the control or in the treated group. No gross behavioral abnormalities were detected in the treated mice during the first 24 h.
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DISCUSSION Metabolism of chemicals takes place largely in the liver, which accounts for the organ’s susceptibility to metabolism-dependent injury.31,34,35 Many compounds, including clinically useful therapeutic drugs, can cause cellular damage in the liver through metabolic activation of chemicals to highly reactive compounds such as free radicals, ensuing in oxidative stress.3 8863
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Figure 4. Effects of RVLWP and RVLAP on liver as disclosed by morphological analysis (×400 H&E staining): (A) normal control group; (B) CCl4intoxicated group; (C) bifendate positive control group (200 mg/kg) + CCl4; (D) RVLWP (100 mg/kg) + CCl4; (E) RVLWP (200 mg/kg) + CCl4; (F) RVLAP (100 mg/kg) + CCl4; (G) RVLAP (200 mg/kg) + CCl4. Black arrows indicate necrotic zones; red arrows indicate lipid droplet accumulation.
and at 1 mg/mL concentration had a closer Fe2+ chelating activity to that of EDTA·2Na. The chelating effect of RVLWP and RVLAP might be partly due to the presence of Fe2+ chelating groups in their structure. Tsiapali et al. proposed a model for the antioxidant ability of carbohydrate polymers.42,50 They demonstrated that the antioxidant effect of glucans and nonglucan polymers, which are significantly better free radical scavengers than monosaccharides, does not correlate with the type of intrachain linkages, molecular weight, or degree of polymer branching, but rather with the monosaccharide composition of the polymer. The weak activity of monosaccharides is attributed to abstraction of the anomeric hydrogen and the enhanced activity of the polymers to the greater ease of abstraction of anomeric hydrogen from one of the internal monosaccharide units rather than from the reducing end. RVLWP and RVLAP were mainly composed of glucose and galactose. The higher antioxidant ability of RVLWP than of RVLAP is likely due to more facile anomeric hydrogen abstraction than RVLAP. Many studies have shown that the hepatoprotective effects may be associated with an antioxidant capacity to scavenge ROS.51 The present study was undertaken to evaluate the hepatoprotective effects of RVLWP/RVLAP on CCl4-induced oxidative stress in the liver of Kunming mice. Bifendate (biphenyldicarboxylate) is a synthetic hepatoprotective agent derived from Schisandrin C. It is active against a variety of hepatotoxins and has been used as a curative agent for the treatment of hepatitis with minimal observable side effects at the prescribed dosage. Many authors considered bifendate as a hepatoprotective agent against drug-induced liver injuries in animals.52 In this study, bifendate was utilized as a positive control medicine for exploring the hepatoprotective effect of water-soluble and alkali-soluble polysaccharides from R. vinosa (RVLWP and RVLAP).
The serum levels of AST and ALT have been used as biochemical markers for acute liver damage.53 When liver cells are damaged, these enzymes leak into the bloodstream from the liver tissue and produce markedly heightened serum levels.54 Our results demonstrate that CCl4 administration causes severe acute liver damage as evidenced by a dramatic rise of the serum levels of AST and ALT. It has been reported that the leakage of large quantities of enzymes into the bloodstream was associated with massive centrilobular necrosis, ballooning degeneration, and cellular infiltration of the liver.51,55 Our results demonstrate that CCl4 administration causes severe acute liver damage as evidenced by a dramatic rise of the serum levels of AST and ALT. The suppression of AST and ALT activities brought about by RVLWP/RVLAP indicates stabilization of plasma membrane as well as repair of hepatic tissue damage caused by CCl4. This hepatoprotective effect was also confirmed by the histopathological observations of diminished necrotic zones and lipid droplet accumulation. CCl4-mediated hepatotoxicity arises from the cytochrome P450-catalyzed biotransformation of CCl4 to the trichloromethyl free radicals (•CCl3), which is further converted to the highly reactive trichloromethylperoxy radicals (CCl3OO•) capable of bombarding biomolecules, thus engendering lipid peroxidation and depletion of the antioxidant enzymes and eventually liver damage.31 MDA is widely used as a marker of lipid peroxidation and oxidative stress.56 Enhanced hepatic MDA levels reflect a causal role of lipid peroxidation in CCl4induced liver damage. Our results revealed that pretreatment with RVLWP and RVLAP significantly reversed these deleterious changes. This effect might, at least in part, be derived from the capability of RVLWP/RVLAP to scavenge ROS. Free radical scavenging enzymes such as SOD and GSHPx are in the first line of defense against oxidative injury. The status of these antioxidant enzymes is an appropriate indirect 8864
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way to assess the pro-oxidant−antioxidant status in tissues.47 SOD and GSH-Px catalyze, respectively, reduction of the superoxide radical (O2−) into H2O2 and O2, and reduction of H2O2 to H2O and O2, thereby preventing the formation of hydroxyl radicals.27,47,53 The results of the present study showed that the activities of SOD and GSH-Px in the livers of CCl4-treated mice were suppressed compared with normal control mice, indicating escalated oxidative damage to the liver. However, pretreatment with RVLWP and RVLAP could notably restore the levels of SOD and GSH-Px in the livers of CCl4-treated mice, implying that RVLWP/RVLAP exerted a substantial protective effect against CCl4-induced acute hepatotoxicity in mice. A review of the recent literature revealed that the hepatoprotective activities of a diversity of extracts and compounds were related to antioxidant and free radical scavenging activities.53−56 Likewise, the hepatoprotective effect of water-soluble and alkali-soluble polysaccharides from R. vinosa might be a consequence of their free radical scavenging effect, antioxidant activity and inhibitory action on lipid peroxidation. The anticoagulant activities of different sea cucumber polysaccharides are probably related to monosaccharide composition and sulfate content.57 Hence, it is likely that the physicochemical differences between RVLWP and RVLAP may account for the observation that the former appeared to have higher activity. In conclusion, the wild edible mushroom R. vinosa has shown to be an excellent source of antioxidant and hepatoprotective polysaccharides, which could be developed as a natural functional food ingredient or a novel nutraceutical to enhance health.
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AUTHOR INFORMATION
Corresponding Authors
*(H.W.) Phone: 86-10-62732578. E-mail:
[email protected]. *(T.B.N.) Phone: 852-3943-6872. E-mail: b021770@mailserv. cuhk.edu.hk Funding
This work was financially supported by China Agriculture Research System (CARS24). Notes
The authors declare no competing financial interest.
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