Polysaccharides of Grifola frondosa ameliorate oxidative stress and hypercholesterolaemia in hamsters fed a high-fat, high-cholesterol diet

Abstract

Objectives

This study was to evaluate the antioxidant and anti-hypercholesterolaemia activities of Grifola frondosa in hamsters fed a high-fat, high-cholesterol (HFHC) diet.

Methods

G. frondosa, including fruiting bodies (FGF), fermented mycelia (MGF) and polysaccharides extracted from fruiting bodies (FPS), fermented mycelia (MIP) and fermented broth (BEP) were received intragastrically. Lipid profile and antioxidant status in the blood and liver of hamsters were assessed.

Key findings

FGF decreased weight gain, serum triglycerides and cholesterol and increased hepatic mRNA expression of cholesterol-7α-hydroxylase expression. FGF, MGF, FPS and MIP decreased the HFHC diet-increased area under the curve (AUC) of serum cholesterol. FGF and FPS further decreased AUC of serum triglycerides. When evaluating the redox status of erythrocytes, FPS and MIP increased non-protein sulfhydryl (NP-SH) groups, reduced glutathione (GSH) and catalase activity and FPS further increased GSH peroxidase activity. In the liver, MGF increased NP-SH groups and GSH and decreased triglycerides content. FPS, MIP and BEP decreased oxidized GSH and triglycerides content. Moreover, all treatments alleviated HFHC diet-increased LDL oxidation.

Conclusions

Fruiting bodies of G. frondosa may improve hypercholesterolaemia via increased bile acid synthesis. Additionally, fermented biomass and polysaccharides of G. frondosa may have the potential to prevent hepatic lipid accumulation.

Grifola frondosa, hyperlipidaemia, redox status, cholesterol-7α-hydroxylase, hepatic lipid accumulation

Introduction

Hyperlipidaemia has been identified as a critical step in the development of atherosclerosis and shown to be positively correlated with the risk of cardiovascular disease (CVD).^[1] People who chronically consumed excess calories, fat and cholesterol are accompanied by elevated circulating triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) and reduced circulating high-density lipoprotein cholesterol (HDL-C).^[2] It has been demonstrated that hyperlipidaemia is associated with abnormal hepatic cholesterol biosynthesis, mainly due to the imbalance between cholesterol synthesis and elimination. In clinical practice, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme in cholesterol synthesis, is the target of cholesterol-lowering drugs to lower serum cholesterol and reduce the risk of CVD.^[3] In addition, cholesterol-7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis that is exclusively expressed in hepatocytes, has drawn a lot of attention in drug development to increase the cholesterol catabolism for patients with hypercholesterolaemia.

Evidence showed that oxidative stress is initiated and augmented in the progression of hyperlipidaemia and its complications, such as non-alcohol fatty liver disease (NAFLD), atherosclerosis and CVD.^[4] Both animal and human studies have indicated that a poor antioxidant defence system was related to the increased lipid peroxidation in the presence of dyslipidaemia.^{[5, 6]} For example, animals fed with high-fat diets may induce hyperlipidaemia which results in an increased malondialdehyde (MDA) and decreased superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in the liver.^{[7, 8]} In patients with hyperlipidaemia, lower activities of SOD and GPx, diminished thiol concentration, and increased lipid peroxidation producing thiobarbituric acid reactive substances (TBARS) and LDL oxidation in the blood were characterized.^{[5, 6, 9, 10]} These results suggest that hyperlipidaemia is closely associated with increased oxidative stress. Therefore, dietary management to normalize lipid profile and enhance antioxidant ability are important strategies to prevent the complications of hyperlipidaemia.

Mushrooms, such as Phellinus pini, Ganoderma lucidum, Hericium erinaceus, Pleurotus ferulae, Lentinus edodes and Agaricus bisporus, have been recognized as functional foods for their anti-hyperlipidaemic activities.^[11–16]Grifola frondosa (Dicks.: Fr.) S.F. Gray (Meripilaceae, Polyporales, higher Basidiomycetes), known as maitake in Japanese, is an edible mushroom with both nutritional and medicinal properties.^[17] Evidence showed that the pharmacological effects of G. frondosa are associated with its bioactive compounds, such as polysaccharides, protein and peptides, fatty acids, polyphenolics, flavonoids, ascorbic acid, α-tocopherol and ergosterol peroxide.^[18] Our previous studies revealed that the fermented mycelia and broth of G. frondosa had beneficial bioactivities in improving serum TG level and glycaemic response, as well as had immunomodulatory effects in diabetic rats.^{[19, 20]} Evidence showed that polysaccharides from fruiting bodies of G. frondosa (FGF) are a promising agent in lipid-lowering, anti-hyperglycaemia and anti-oxidative stress.^{[21, 22]} Zhao et al. indicated that a heteropolysaccharide, possessed a 1,6-β-d-glucan backbone with a single 1,3-α-d-fucopyranosyl side-branching unit, from fermented mycelia of G. frondosa has antiviral activity.^[23] However, polysaccharides derived from various sources of G. frondosa may have different components and structure, as well as display diverse biological properties. In this study, we investigated the potentials of polysaccharides extracted from fruiting bodies, fermented mycelia and fermented broth of G. frondosa in improving hyperlipidaemia and oxidative stress in hamsters fed a high-fat high-cholesterol (HFHC) diet. The lipid profiles in the serum, lipoproteins and liver, and the activities and mRNA expression of HMGR and CYP7A1 in the liver were determined. To evaluate the effects of different products of G. frondosa on oxidative stress, the enzymatic and non-enzymatic antioxidant defence systems in the erythrocytes and the liver were measured.

Materials and Methods

Polysaccharides preparation

FGF were purchased from a local market in Changhua City, Taiwan. After 12 h of drying at 60°C, the dried FGF was ground into powder and added with 10-fold volume of deionized water (w/v) for 6-h incubation at 95°C. A 4-fold volume of 95% alcohol was added and incubated overnight. The precipitates were centrifuged and freeze-dried to obtain the crude polysaccharides of fruiting bodies (FPS). To produce fermented mycelia and broth, G. frondosa (BCRC 36434, the Bioresources Collection & Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan) was cultured with polysaccharide-free medium (2% glucose, 0.6% peptone, 2% sucrose and 0.1% soybean oil) for 7 days as our previous study.^[19] The culture supernatant (broth) and precipitates (mycelia, MGF) were harvested. The crude intracellular (MIP) and extracellular (BEP) polysaccharides were prepared from the mycelia and broth of G. frondosa, respectively, as described by Wang et al.^[24] In brief, dried MGF was mixed with a 10-fold volume of distilled water (w/v) and extracted at 121°C for 30 min in an autoclave. The autoclaved supernatant was filtered, mixed with a 4-fold volume of 95% ethanol and incubated at 4°C.

After an overnight extraction by ethanol and centrifugation, the precipitate was washed with distilled water three times and freeze-dried for 2 days to remove the residual ethanol. To obtain BEP, dried broth powder was mixed with a 4-fold volume of 95% ethanol, extracted overnight at 4°C, centrifuged and freeze-dried for 2 days. The amounts of crude polysaccharides in FPS, MIP and BEP were 58.27 mg/g fruiting body, 48.36 mg/g biomass and 1.68 mg/ml fermented broth, respectively, using the phenol-sulfuric acid assay.^[25] All test powders were re-dissolved in distilled water when they were given to the animals. The proximate compositions of G. frondosa, including crude protein, crude fat, crude carbohydrate, crude fibre and crude ash (Table 1), were determined according to the official methods of analysis of the Association of Official Analytical Chemists (AOAC).^[26]

Table 1.

The composition of fruiting bodies and fermented mycelium and broth powder of G. frondosa

Composition, %	Fruiting bodies	Mycelium	Broth powder
Crude protein	18.85 ± 0.74	18.51 ± 0.63	6.42 ± 0.04
Crude fat	2.39 ± 0.13	2.14 ± 0.16	1.12 ± 0.07
Crude carbohydrate	65.17 ± 5.37	63.94 ± 3.98	90.79 ± 5.37
Crude fibre	8.14 ± 0.64	8.29 ± 0.57	N.D.
Crude ash	5.45 ± 0.37	7.12 ± 0.58	1.67 ± 0.08

Abbreviation: N.D., non-detectable.

Values are means ± SEM, n = 3.

Analysis of monosaccharide composition in polysaccharides

The monosaccharide composition of crude polysaccharides was determined using HPLC as described by Liu et al. with some modifications.^[27] In brief, FPS, MIP and BEP were hydrolyzed with 4 M hydrochloric acid (HCl) at 90°C for 6 h. The cooled hydrolysates were mixed with methanol and evaporated to dryness under vacuum on a 60°C water bath. After repeated thrice to remove HCl, the dried hydrolysates were dissolved in 1 ml of water. To derivatize monosaccharides, 100 µl of hydrolyzed samples or monosaccharide standards (1 mg/ml), including d-mannose, d-glucose, d-galactose, d-rhamnose and d-arabinose, were mixed thoroughly with NaOH and reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP, 0.5 mol/L in methanol) at 70°C for 30 min. The cooled mixture was neutralized with HCl, diluted to 1 ml with distilled water and extracted by chloroform. After vigorous shaking and centrifugation at 4000 xg for 5 min, the organic phase was discarded. The procedure of chloroform extraction, shaking, centrifugation and organic phase removal was repeated thrice. The upper aqueous layer was filtered through a 0.45 µm syringe filter and used for HPLC analysis.

The PMP-derivatized monosaccharides were analysed by a JASCO HPLC system equipped with a quaternary pump and a UV detector (UV-2075 Plus, JASCO International Co., Ltd, Tokyo, Japan). The derivatized samples (10 µl) were separated using a C₁₈ Hypersil column (5 µm, 4.6 mm × 250 mm) with a guard column (5 µm, 4.0 mm × 10 mm, Thermo Fisher Scientific Inc., Branchburg, NJ, USA) at 30°C. The derivatized monosaccharides were separated by an eluent containing 0.05 M phosphate buffer (pH 6.8) and acetonitrile (83 : 17, v/v) at a flow rate of 1 ml/min and detected by a UV detector at 250 nm. The monosaccharides were identified and quantified by comparing their integration values of peak area with standards to calibration curves.

Animals

The 6-week-old male hamsters (n = 60) were purchased from National Laboratory Animal Center, Taipei, Taiwan and all animal procedures were approved by the Laboratory Animal Care and Use Committee at Changhua Christian Hospital, Changhua, Taiwan (License number: CCH-AE-94004, approved on 18/02/2015). Animals were free access to water and diet (AIN-76, MP Biomedicals, ICN Biomedicals Inc., Irvine, CA, USA). After 1-week acclimatization, the animals were randomized into seven groups. The control (CON) group was given a chow diet containing 5% fat (w/w, 11.7% of calories as fat and cholesterol-free) and six hyperlipidaemic groups were fed with a HFHC diet containing 15% fat (w/w, 32% of total calorie) and 0.5% cholesterol (w/w) for 8 weeks to induce hyperlipidaemia. In addition, the hyperlipidaemic groups received intragastrically cellulose (HTC group), 0.5 g of fruiting bodies (FGF group), fermented mycelia (MGF group), FPS, MIP or BEP. The amounts of crude polysaccharides in the FPS, MIP and BEP groups were equal to the FGF group. To control for the effects of fibre, the CON and HTC groups intragastrically received cellulose that was equal to the amount of FGF group.

The daily food intake and weekly body weight were recorded. Overnight fasting blood samples were collected from the retro-orbital sinus every 2 weeks. At the end of the experiment, hamsters were anaesthetized with intramuscular injections of 100 mg/kg ketamine and 10 mg/kg xylazine. Serum and whole blood were collected. The livers were immediately snap-frozen and stored at −80°C until use.

Serum biochemical parameters and hepatic lipid profiles

Serum albumin, glucose, blood urea nitrogen (BUN), creatinine, glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) were measured. Serum TG, TC and free cholesterol (FC) were determined using commercial assay kits (Fortress Diagnostics Limited, Antrim, UK). Serum VLDL, LDL and HDL were separated to measure the TG and TC.^{[28, 29]} The fractions with density <0.163, 1.006–1.0663 and 1.063–1.21 g/ml were collected as VLDL, LDL and HDL, respectively. The liver was extracted by chloroform/methanol (2 : 1) following the Folch method and TG and TC levels were determined.^[30]

Enzymatic activity and mRNA expression of HMGR and CYP7A1 in the liver

The liver was homogenized with 10-fold volume of 0.02 M Tris-HCl buffer, pH 7.5 on ice and centrifuged at 15250 xg for 30 min at 4°C to obtain the supernatants for the measurement of HMGR and CYP7A1 enzymatic activities.^{[31, 32]}

Total RNA was isolated from livers using the Trizol reagent (Invitrogen, Life Technologies, USA). Liver tissue (100 mg) was homogenized in Trizol reagent (1 ml) with Polytron PT-2100 (Kinematica, Switzerland), 0.2 ml of chloroform was added and vortexed vigorously for 15 s. The homogenate was incubated at room temperature for 2 min and centrifuged at 15250 xg for 15 min at 4°C. Adding 0.5 ml of isopropanol to the aqueous phase and mixed well, the mixture was incubated at room temperature for 10 min and centrifuged at 15250 xg for 15 min at 4°C. The RNA precipitate was recovered by decanting the supernatant, washed with 70% ethanol and centrifuged at 15250 xg for 15 min at 4°C. The RNA pellet was air-dried for 5 min and rehydrated with 20 µl DEPC-treated water. The RNA was reverse transcribed with M-MLV reverse transcriptase (Promega, USA) according to the manufacturer’s protocol. The real-time PCR was carried out to amplify the target sequence using SYBR Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA) with the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, USA). The primers of target genes were: HMGR, forward, 5ʹ-TGCTCATTAGCGTTGTCCAG-3ʹ, reverse, 5ʹ-CCCAAGACTGAACTCGAAGC-3ʹ; CYP7A1, forward, 5ʹ-GGAGTGCCATTTACTTGGA-3ʹ, reverse, 5ʹ-GACGGCAAAGAGTCTTCCAG-3ʹ; and β-actin (internal control), forward, 5ʹ-GTCGTACCACTGGCATTGTG-3ʹ, reverse, 5ʹ-CATCTCTTGCTCGAAGTCC-3ʹ. The relative gene expression was calculated as fold change, in relation to the CON group, according to the comparative Ct method.^[33]

Indices of redox status

Serum LDL oxidation, TBARS, non-enzymatic antioxidants, and the activities of antioxidant enzymes in the erythrocytes and liver were determined. Serum LDL was dialyzed in cold PBS for 15 h at 4°C in the dark. The kinetics of copper-induced LDL oxidation in terms of the lag time before oxidation were measured by using 75 µg of protein and 10 µM CuCl₂.^[34] The non-enzymatic antioxidants included protein-bound sulfhydryl (PB-SH) groups, non-protein sulfhydryl (NP-SH) groups, total sulfhydryl (TSH) groups, reduced glutathione (GSH), and oxidized glutathione (GSSG), and the activities of SOD, GPx and catalase were measured as described in our previous study.^[35]

Statistical analysis

Data are expressed as the mean ± standard error of the mean (SEM). The statistical significance of difference among groups of each parameter was analysed by one-way analysis of variance (ANOVA) using the SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA) followed by post hoc least significant difference (LSD) test. A P < 0.05 was considered statistically significant.

Results

Monosaccharide compositions of the crude polysaccharides

The results of HPLC analysis showed that glucose was the major monosaccharide and galactose, mannose, rhamnose and arabinose were the minor monosaccharides in FPS, MIP and BEP (Table 2). The proportion of glucose was less in BEP than in FPS and MIP, whereas that of galactose and mannose was greater in BEP than in FPS and MIP. MIP had the lowest proportion of galactose than FPS and BEP. In addition, FPS and BEP had less than 5% of monosaccharides as rhamnose and MIP had non-detectable rhamnose. MIP had approximately 13.6% of monosaccharides as arabinose and FPS and BEP had non-detectable arabinose. The monosaccharide compositions were different among the three crude polysaccharides.

Table 2.

Monosaccharide composition of the crude polysaccharide from G. frondosa

Composition, %	Glucose	Galactose	Mannose	Rhamnose	Arabinose
FPS	83.31 ± 5.12	8.24 ± 0.32	5.31 ± 0.89	1.32 ± 0.08	N.D.
MIP	76.47 ± 4.23	2.28 ± 0.13	5.14 ± 0.74	N.D.	13.58 ± 0.57
BEP	68.63 ± 3.58	12.37 ± 0.65	11.31 ± 0.62	4.53 ± 0.26	N.D.

Abbreviations: FPS, the crude polysaccharides extracted from fruiting bodies of G. frondosa; MIP, the crude intracellular polysaccharides extracted from fermented mycelia of G. frondosa; BEP, the crude extracellular polysaccharides from fermented broth of G. frondosa; N.D., non-detectable.

Values are means ± SEM, n = 3.

Body weight, food intake and serum biochemical parameters

The results of body weight during the experimental period were shown in Figure 1. There was no significant difference in body weight among groups in the first 3 weeks, whereas body weight was significantly greater in the HTC group than in the CON group from week 4 to week 8. In addition, the FGF group had significantly lower body weight than the HTC group from week 4 to week 8 and the CON group on weeks 7 and 8. The results of body weight change within 8 weeks showed that the FGF group had the lowest body weight gain when compared with the CON and HTC groups (P < 0.05, Figure 1, inset). Furthermore, the average daily food intake (Figure 2A) and feed efficiency (Figure 2B), that is, total weight gain divided by total caloric intake within 8 weeks, were significantly lower in the FGF group than in the HTC group.

Figure 1.

Effects of G. frondosa products on body weight and body weight change (inset) in the HFHC diet-fed hamsters. Data were expressed as means ± SEM. * indicates significantly different from the CON group and ^† indicates significantly different from the HTC group (P < 0.05, one-way ANOVA with LSD).

Figure 2.

Effects of G. frondosa products on (A) food intake and (B) feed efficiency in the HFHC diet-fed hamsters. Data were expressed as means ± SEM. * indicates significantly different from the CON group and ^† indicates significantly different from the HTC group (P < 0.05, one-way ANOVA with LSD).

The results of serum albumin, glucose, BUN, creatinine, GOT and GPT are shown in Table 3. Serum levels of albumin and GOT were not significantly different among groups. Hamsters in the HTC group had significantly increased serum glucose levels and this increase was significantly decreased by oral administration of FGF and BEP. Serum BUN was significantly decreased in hamsters with a HFHC diet, that is, the HTC, FGF, MGF, FPS, MIP and BEP groups, when compared with those fed with the regular chow diet, that is, the CON group. The FGF group had significantly decreased serum creatinine when compared with the CON and HTC groups. The HFHC diet-increased serum GPT was further increased by oral administration of FGF and decreased by that of BEP.

Table 3.

Serum substrate concentrations

Groups	CON	HTC	FGF	MGF	FPS	MIP	BEP
Albumin, mg/dL	3.56 ± 0.06	3.44 ± 0.06	3.15 ± 0.10	3.34 ± 0.08	3.41 ± 0.07	3.27 ± 0.23	3.42 ± 0.03
Glucose, mg/dL	182.2 ± 12.4	237.9 ± 24.3*	151.8 ± 10.0†	222.6 ± 27.0	196.4 ± 6.8	205.0 ± 11.5	183.3 ± 7.3†
BUN, mg/dL	24.4 ± 1.6	19.1 ± 1.1*	19.3 ± 0.9*	17.0 ± 0.7*	19.9 ± 1.2*	19.2 ± 1.0*	20.9 ± 0.8*
Creatinine, mg/dL	0.33 ± 0.03	0.38 ± 0.02	0.27 ± 0.02*†	0.36 ± 0.02	0.36 ± 0.03	0.36 ± 0.02	0.36 ± 0.02
GOT, U/L	79.6 ± 3.4	117.6 ± 22.8	124.0 ± 20.2	101.3 ± 9.3	98.8 ± 8.5	115.2 ± 12.3	91.0 ± 5.8
GPT, U/L	62.7 ± 3.1	100.8 ± 6.4*	150.3 ± 12.9*†	83.5 ± 3.8*	94.9 ± 7.3*	105.3 ± 3.7*	82.9 ± 3.1*†

Abbreviations: BUN, blood urea nitrogen; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; CON, control; HTC, hyperlipidaemia; FGF, hyperlipidaemia with G. frondosa fruiting bodies; MGF, hyperlipidaemia with fermented mycelia of G. frondosa; FPS, hyperlipidaemia with polysaccharides extracted from fruiting bodies of G. frondosa; MIP, hyperlipidaemia with polysaccharides extracted from fermented mycelia of G. frondosa; BEP, hyperlipidaemia with polysaccharides extracted from fermented broth of G. frondosa.

Values are means ± SEM, n = 6–10. * indicates significantly different from the CON group in all groups and † indicates significantly different from the HTC group in the hyperlipidaemic groups (P < 0.05, one-way ANOVA with least significant difference).

Serum lipid profiles

Serum levels of TG, TC and FC, from weeks 0 to 8 are shown in Figure 3. As shown in Figure 3A, serum TG was significantly increased in the HTC group in weeks 4 and 6 when compared with the CON group and this increase was alleviated in the FGF and FPS groups in weeks 4 and 6 and in the MIP group in week 6. Serum TC was significantly increased in the HTC group in weeks 2, 4, 6 and 8 (Figure 3B) and this HFHC diet-induced increase was significantly decreased in the FGF group in weeks 4, 6 and 8; in the MGF group in weeks 2 and 4; in the FPS group in weeks 2, 4 and 6; and in the MIP group in week 4. Serum FC were significantly increased in the HTC group from week 2 to week 8 (Figure 3C). The HFHC diet-increased serum FC was significantly decreased in the FGF and MGF groups in week 6.

Figure 3.

Effects of G. frondosa products on serum (A) triglycerides, (B) total cholesterol, (C) free cholesterol and (D) area under the curve (AUC) of lipid levels from week 0 to week 8 in the HFHC diet-fed hamsters. Data were expressed as means ± SEM. * indicates significantly different from the CON group and ^† indicates significantly different from the HTC group (P < 0.05, one-way ANOVA with LSD).

The area under the curve (AUC) of serum TG, TC and FC levels from week 0 to week 8 was calculated to assess the impact of the HFHC diets and G. frondosa products on the changes in serum lipids. The AUC of serum TG, TC and FC levels were significantly greater in the HTC group when compared with the CON group (Figure 3D). The HFHC diet-increased AUC of serum TG was significantly decreased in the FGF and FPS groups; that of TC was significantly decreased in the FGF, MGF, FPS and MIP groups; and that of FC was significantly decreased in the FGF and MGF groups.

To further evaluate the effects of G. frondosa on the lipid profile, TG and TC contents of VLDL, LDL and HDL were determined (Figure 4). The results showed that there were no significant differences in the TG contents of the VLDL, LDL and HDL, that is, the VLDL-TG, LDL-TG and HDL-TG, respectively, between the CON and HTC groups (Figure 4A). However, VLDL-TG was significantly greater in the FPS and MIP groups than in the CON and HTC groups, and HDL-TG was significantly greater in the FPS and MIP groups than in the HTC group. LDL-TG were not significantly different among groups.

Figure 4.

Effects of G. frondosa products on the contents of (A) triglycerides and (B) total cholesterol in serum VLDL, LDL and HDL of the HFHC diet-fed hamsters. Data were expressed as means ± SEM. * indicates significantly different from the CON group and ^† indicates significantly different from the HTC group in the hyperlipidaemic groups (P < 0.05, one-way ANOVA with LSD).

The TC content of VLDL, that is, VLDL-C, was significantly decreased in the FGF, FPS and MIP groups, and the TC content of LDL, that is, LDL-C, was significantly decreased in the FPS and MIP groups when compared with the HTC group (Figure 4B). The TC content of HDL, that is, HDL-C, was significantly increased in hamsters with a HFHC diet and this increase was significantly decreased by oral administration of FGF.

Liver weight and lipid content

As shown in Table 4, the liver weight, corrected by the body weight, was significantly increased by 66–78% in hamsters with a HFHC diet more than those with a regular chow diet. Oral administration of G. frondosa products did not have a significant impact on the HFHC diet-increased liver weights. The contents of TG and TC in the liver were significantly increased in the HTC group when compared with the CON group. The HFHC diet-increased hepatic TG content was significantly decreased in hamsters administered with MGF, FPS, MIP and BEP; and the increased hepatic cholesterol content was further increased in those with MIP.

Table 4.

Liver weights, lipid profile and the activities of enzymes in the liver

Groups	CON	HTC	FGF	MGF	FPS	MIP	BEP
Liver, g/kg body weight	29.5 ± 1.0	49.2 ± 0.9*	51.5 ± 2.0*	51.1 ± 1.0*	48.9 ± 1.2*	51.8 ± 1.6*	52.5 ± 1.7*
Triglycerides, mg/g liver	20.8 ± 0.2	28.3 ± 2.2*	29.4 ± 1.3*	18.7 ± 0.5†	19.4 ± 0.9†	19.1 ± 0.9†	21.6 ± 1.0†
Cholesterol, mg/g liver	15.7 ± 0.3	24.8 ± 1.4*	25.4 ± 0.9*	25.0 ± 1.3*	23.7 ± 2.3*	32.7 ± 2.6*†	24.4 ± 1.6*
HMGR, nmol/min/g protein	57.0 ± 3.9	84.1 ± 8.7*	111.2 ± 11.1*	84.7 ± 10.0*	98.4 ± 2.9*	82.5 ± 4.0*	91.1 ± 13.3*
CYP7A1, nmol/min/g protein	114.4 ± 13.4	119.5 ± 15.0	118.2 ± 5.2	114.3 ± 10.0	105.1 ± 11.9	99.3 ± 6.1	105.0 ± 9.4

Abbreviations: HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; CYP7A1, cholesterol 7α-hydroxylase.

Values are means ± SEM, n = 6–10. * indicates significantly different from the CON group in all groups and ^† indicates significantly different from the HTC group in the hyperlipidaemic groups (P < 0.05, one-way ANOVA with least significant difference). For name of groups, see Table 3.

Activities and mRNA expression of HMGR and CYP7A1 in the liver

To evaluate the cholesterol metabolism in the liver, the activities and mRNA expression of HMGR and CYP7A1 were determined. The activity of HMGR was significantly increased in hamsters fed a HFHC diet, that is, the HTC, FGF, MGF, FPS, MIP and BEP groups (Table 4). The HFHC diet did not have a significant impact on the mRNA expression of HMGR; however, the combination of a HFHC diet and MIP administration significantly increased mRNA expression of HMGR (Figure 5). The activity of CYP7A1 in the liver was not significantly different among groups (Table 4). However, the FGF group had approximately a 3-fold increase in CYP7A1 mRNA in the liver when compared with the CON and HTC groups.

Figure 5.

Effects of G. frondosa products on the mRNA levels of HMGR and CYP7A1 in the liver of the HFHC diet-fed hamsters. Data were expressed as means ± SEM. * indicates significantly different from the CON group and ^† indicates significantly different from the HTC group (P < 0.05, one-way ANOVA with LSD).

Redox status in the erythrocytes and liver

As shown in Table 5, the levels of TBARS, thiol groups, such as PB-SH, NP-SH, and TSH, GSH, GSSG, the ratio of GSH to GSSG (GSH/GSSG), and the activities of SOD, GPx and catalase in the erythrocytes were not significantly different among the CON, HTC, FGF and MGF groups. The lag time of copper-induced LDL oxidation in the serum was significantly decreased in the HTC group when compared with the CON group. Administration of FGF, MGF, FPS, MIP and BEP significantly eliminated the HFHC diet-induced decrease in the lag time of LDL oxidation. In addition, the FPS group had significantly increased NP-SH, GSH and GPx and catalase activities in the erythrocytes when compared with the CON and HTC groups; and had significantly decreased PB-SH and increased GSH/GSSH when compared with the CON and HTC groups, respectively. The MIP group had significantly lower levels of PB-SH and TSH and higher levels of NP-SH and GSH/GSSG than the CON and HTC groups and higher levels of GSH and catalase activity in the erythrocyte than the HTC group. The BEP group had a significantly higher level of NP-SH than the CON and HTC groups.

Table 5.

Redox status in the erythrocytes

Groups	CON	HTC	FGF	MGF	FPS	MIP	BEP
TBARS, µmol/ml	1.80 ± 0.04	1.79 ± 0.07	1.88 ± 0.09	1.73 ± 0.06	1.68 ± 0.10	1.76 ± 0.08	1.87 ± 0.04
LDL lag time, min	44.4 ± 0.8	37.2 ± 0.8*	46.7 ± 1.2†	42.9 ± 1.0†	42.5 ± 0.7†	41.5 ± 0.8*†	43.9 ± 0.7†
PB-SH, µmol/ml	14.5 ± 0.7	12.0 ± 1.1	14.3 ± 0.9	10.5 ± 0.7	10.1 ± 2.1*	4.5 ± 1.3*†	12.3 ± 1.7
NP-SH, µmol/ml	0.25 ± 0.04	0.26 ± 0.03	0.17 ± 0.04	0.21 ± 0.12	0.79 ± 0.06*†	0.77 ± 0.03*†	0.44 ± 0.06*†
TSH, µmol/ml	14.8 ± 0.7	12.2 ± 1.1	14.4 ± 0.9	10.7 ± 0.7	10.9 ± 2.1	5.3 ± 1.3*†	12.7 ± 1.7
GSH, µg/mg Hb	2.27 ± 0.26	2.02 ± 0.14	2.01 ± 0.14	2.21 ± 0.21	2.99 ± 0.24*†	2.72 ± 0.10†	2.27 ± 0.12
GSSG, µg/mg Hb	2.00 ± 0.24	2.14 ± 0.22	2.00 ± 0.21	2.41 ± 0.30	2.37 ± 0.22	1.83 ± 0.08	2.10 ± 0.23
GSH/GSSG ratio	1.15 ± 0.08	0.93 ± 0.07	1.07 ± 0.14	0.99 ± 0.14	1.30 ± 0.10†	1.53 ± 0.06*†	1.08 ± 0.09
SOD, U/mg Hb	27.1 ± 2.6	29.5 ± 2.5	27.6 ± 1.5	29.5 ± 5.2	34.4 ± 3.1	31.1 ± 1.6	28.8 ± 2.0
GPx, nmole NADPH/min/mg Hb	1.49 ± 0.08	1.38 ± 0.08	1.31 ± 0.04	1.58 ± 0.10	1.81 ± 0.09*†	1.54 ± 0.07	1.36 ± 0.06
Catalase, nmol H2O2/min/mg Hb	0.15 ± 0.02	0.14 ± 0.01	0.13 ± 0.01	0.20 ± 0.01	0.28 ± 0.03*†	0.20 ± 0.02†	0.16 ± 0.02

Abbreviations: PB-SH, protein-bound thiol group; NP-SH, non-protein thiol group; TSH, total thiol group; GSH, reduced glutathione; GSSG, oxidized glutathione.

Values are means ± SEM, n = 6–10. * indicates significantly different from the CON group in all groups and ^† indicates significantly different from the HTC group in the hyperlipidemic groups (P < 0.05, one-way ANOVA with least significant difference). For name of groups, see Table 3.

The indices of redox status in the liver are shown in Table 6. The contents of TBARS, PB-SH, TSH and GSH and the activities of SOD and GPx were significantly decreased in the HTC group when compared with the CON group. Hepatic TBARS content and SOD activity in the FGF group and NP-SH and GSH contents in the MGF group were significantly greater than those in the HTC group. In addition, hepatic GSSG in the FPS, MIP and BEP groups and hepatic TBARS in the MIP group were significantly lower than in the CON and HTC groups.

Table 6.

Redox status in the liver

Groups	CON	HTC	FGF	MGF	FPS	MIP	BEP
TBARS, nmol/g liver	281.6 ± 33.1	193.2 ± 13.6*	299.2 ± 27.1†	215.0 ± 24.5	146.5 ± 34.4*	108.6 ± 24.9*†	167.3 ± 24.2*
PB-SH, µmol/g liver	16.1 ± 0.6	12.8 ± 0.4*	11.6 ± 0.9*	11.5 ± 0.7*	13.3 ± 0.2*	12.3 ± 0.4*	12.7 ± 0.4*
NP-SH, µmol/g liver	4.80 ± 0.10	4.27 ± 0.32	4.09 ± 0.61	5.36 ± 0.10†	4.96 ± 0.27	4.59 ± 0.25	4.40 ± 0.29
TSH, µmol/g liver	20.9 ± 0.6	17.4 ± 0.6*	15.7 ± 1.3*	16.6 ± 0.9*	18.0 ± 0.4*	16.9 ± 0.6*	17.1 ± 0.5*
GSH, mg/g liver	1.98 ± 0.13	1.71 ± 0.07*	1.73 ± 0.04*	2.00 ± 0.11†	1.61 ± 0.08*	1.51 ± 0.04*	1.51 ± 0.07*
GSSG, mg/g liver	1.15 ± 0.06	1.13 ± 0.07	0.97 ± 0.02	1.20 ± 0.14	0.90 ± 0.03*†	0.89 ± 0.02*†	0.87 ± 0.03*†
GSH/GSSG ratio	1.74 ± 0.09	1.40 ± 0.11	1.80 ± 0.07	1.57 ± 0.36	1.73 ± 0.05	1.74 ± 0.07	1.67 ± 0.08
SOD, U/mg protein	0.65 ± 0.02	0.53 ± 0.01*	0.68 ± 0.03†	0.54 ± 0.01*	0.54 ± 0.02*	0.56 ± 0.01*	0.52 ± 0.03*
GPx, nmole NADPH/min/mg protein	146.6 ± 7.2	96.3 ± 6.7*	76.9 ± 4.1*	102.7 ± 9.0*	82.7 ± 7.5*	88.9 ± 6.3*	80.3 ± 6.8*
Catalase, nmol H2O2/min/mg protein	1.39 ± 0.16	1.43 ± 0.16	1.89 ± 0.35	1.08 ± 0.17	1.14 ± 0.21	1.54 ± 0.13	1.39 ± 0.06

Abbreviations: PB-SH, protein-bound thiol group; NP-SH, non-protein thiol group; TSH, total thiol group; GSH, reduced glutathione; GSSG, oxidized glutathione.

Values are means ± SEM, n = 6–10. * indicates significantly different from the CON group in all groups and ^† indicates significantly different from the HTC group in the hyperlipidaemic groups (P < 0.05, one-way ANOVA with least significant difference). For name of groups, see Table 3.

Discussion

Hyperlipidaemia and the accompanied oxidative stress are considered as the risk factors for CVD, the leading cause of death globally.^[2] Nutraceuticals and specific food components with antioxidant and lipid-lowering activities have drawn a significant amount of attention in the prevention of hyperlipidaemia.^{[11, 21]} In this study, we demonstrated that G. frondosa could ameliorate oxidative stress and hyperlipidaemia in HFHC-fed hamsters. We also found that the anti-hypercholesterolaemic activity of FGF is associated with the up-regulated mRNA expression of CYP7A1. In addition, the crude polysaccharides of G. frondosa were found to alleviate lipid accumulation in the liver of HFHC-fed hamsters. Moreover, the anti-hyperlipidaemic and antioxidant activities of natural and fermented G. frondosa may be closely related to the monosaccharide composition of polysaccharides.

By feeding a HFHC diet for 8 weeks, hamsters had obviously increased body weight (Figure 1) and abnormally lipid profiles in the serum (Figure 3) and liver (Table 4). These changes indicated that HFHC-fed hamsters were under the condition of hyperlipidaemia and had the potential to develop NAFLD, atherosclerosis and CVD. G. frondosa has been shown to control body weight and alleviate hyperlipidaemia and hepatic steatosis in mice fed with a high-fat diet.^[36] We found the similar results in the HFHC-fed hamsters administered with FGF (Figures 1 and 3). These beneficial effects of FGF were closely related to the reduced food intake and feed efficiency (Figure 2), suggesting the ability of G. frondosa fruiting bodies in regulating appetite and energy expenditure. The MGF showed the ability to decrease AUC of serum cholesterol (Figure 3D) but not in body weight and food intake (Figures 1 and 2A). These findings reveal that FGF may have superior abilities in improving diet-induced hyperlipidaemia. Since all the hamsters were administered with cellulose, we can be assured that FGF contain more bioactive compounds and nutrients, instead of dietary fibre, than mycelia in improving hyperlipidaemia.

The well-known bioactive components of mushrooms, including sterols, dietary fibres and polysaccharides have been shown to have anti-hypercholesterolaemia activity.^{[12, 13, 17, 18]} Administration of polysaccharides from G. frondosa fruiting bodies had been shown to alleviate dyslipidaemia in high-fat diet-fed rats.^[37] The fermented mycelia and broth of mushrooms are good sources of intracellular and extracellular polysaccharides.^[38] However, limited information is available regarding the biological activities of these polysaccharides from fermented G. frondosa. In this study, we found that FPS and MIP had decreased AUC of serum TC (Figure 3D) and serum VLDL-C and LDL-C (Figure 4B), with the increasing serum VLDL-TG and HDL-TG (Figure 4A). These changes implied that polysaccharides from fruiting bodies and fermented mycelia of G. frondosa may regulate cholesterol transport and have a positive against atherosclerosis. Therefore, we speculate that fruiting bodies, mycelia and polysaccharides of G. frondosa may result in different degrees of improvement in hypercholesterolaemia and these differences may be due to the various composition of G. frondosa, such as crude protein, crude fat, crude carbohydrate, crude fibre, crude ash (Table 1) and polysaccharides (Table 2). Further study is needed to investigate the relationship between the structural characterization of polysaccharides and the bioactivities of G. frondosa.

With habitual consumption of a high-fat diet, the accumulation of hepatic TG may result in oxidative stress, hepatocyte damage and NAFLD.^[39] Mushroom polysaccharides, especially β-glucan, have been reported to modulate cholesterol biosynthesis, bile acids excretion and cholesterol metabolism-associated enzymes.^[40] Administration of polysaccharides from G. frondosa fruiting bodies could ameliorate hepatic steatosis through increasing bile acid synthesis and excretion in rats with a high-fat diet.^[37] In this study, we found that the HFHC-fed hamsters had increased liver weights, TG and TC contents, and HMGR activity in the liver and those administered with mycelia and different G. frondosa polysaccharides had significantly decreased hepatic TG accumulation (Table 4). FGF had significantly increased hepatic mRNA expression of CYP7A1, suggesting the elevated conversion of cholesterol to bile acid (Figure 5). MIP had significantly increased HMGR mRNA expression and cholesterol content in the liver (Table 4) and decreased serum VLDL-C and LDL-C (Figure 4B). These changes revealed that MIP increased the hepatic cholesterol synthesis was independent of the circulating cholesterol. The changes in circulating and hepatic lipid profiles revealed that the lipid-lowering activity of G. frondosa is source-oriented.

Hyperlipidaemia-induced oxidative stress is considered to promote the development of NAFLD and atherosclerosis.^{[39, 41]} This study showed that the HFHC-induced hyperlipidaemia with decreased LDL oxidized lag time and hepatic antioxidant capacities in hamsters (Table 6), similar results were observed in the high-fat emulsion-induced hypertriglyceridaemia in mice.^[42] The polysaccharides from Ganoderma lucidum, Pleurotus eryngii var. tuoliensis and Pleurotus tuber-regium have been shown to prevent lipid peroxidation and restore activities of SOD and GPx in the livers of the high-fat diet-fed animals.^{[12, 42, 43]} This study observed that FGF had significantly increased the hepatic SOD activity, which may be a compensatory response to the elevated hepatic TBARS (Table 6). Furthermore, the beneficial effects of G. frondosa on alleviating hepatic oxidative stress were shown in increased NP-SH and GSH in the MGF group, decreased TBARS in the MIP group and decreased GSSG in the FPS, MIP and BEP groups. We assumed natural, fermented biomass and polysaccharides of G. frondosa play important and different roles in the prevention of hepatic oxidative stress in hyperlipidaemia. Our previous study demonstrated that extracellular polysaccharopeptides from fermented Trametes versicolor could mitigate oxidative stress, hyperglycaemia and hyperlipidaemia, as shown by the decreased TG and TBARS and increased GSH and SOD activity in the blood in diabetic rats with high-fat diet.^[44] In this study, G. frondosa products significantly decreased the LDL oxidized lag time, and FPS and MIP significantly increased NP-SH, GSH, GSH/GSSG ratio, and catalase activity in erythrocytes of the HFHC-fed hamsters (Table 5), implying the alleviated oxidative stress and risk for atherosclerosis. The erythrocytic NP-SH content and GPx activity were also increased by BEP and FPS, respectively. The elevated circulating antioxidant capacity of the FPS and MIP groups was parallel with the alteration of serum lipid profile (Figure 4). Taking together, the lipid-lowering activity of G. frondosa polysaccharides may contribute to mitigate oxidative stress.

It has been shown that monosaccharide composition profiles from fruiting bodies, mycelia and fermentation broth of mushrooms are different, mostly due to the sources and cultivating conditions.^[45] We also confirmed that monosaccharide composition profiles were different among FPS, MIP and BEP (Table 2). In this study, we showed a similar monosaccharide composition profiles of polysaccharides from G. frondosa fruiting bodies (Table 2) to those in the study of Zhao et al., that is, approximately 83% of glucose, 8–10% of galactose and 5–7% of mannose.^[46] Moreover, the proportions of glucose (68.6%) and galactose (12.4%) in BEP were different from that of exopolysaccharides from G. frondosa HB0071 (82.5% glucose and 9.8% galactose), suggesting different bioactive properties between these two exopolysaccharides.^[47] For example, exopolysaccharides with higher proportion of mannose (24.8%) had enhanced antioxidant activity in Inonotus obliquus.^[48] The enzymatic polysaccharides with high proportion of galactose (19.4%) had anti-hyperlipidaemic and antioxidant activities in Morehella esculenta.^[49] In this study, FPS, MIP and BEP extracted from G. frondosa had different monosaccharide composition profiles and alleviated hyperlipidaemia and oxidative stress differentially in the HFHC diet-fed hamsters.

Conclusions

This study suggests that fruiting bodies, fermented mycelia, and their polysaccharides of G. frondosa may improve hypercholesterolaemia and oxidative stress in hamsters fed a HFHC diet. In addition, FGF may ameliorate the diet-induced hyperlipidaemia via the increase in bile acid synthesis, as shown in the up-regulated expression of CYP7A1. Mycelia and crude polysaccharides of G. frondosa alleviated serum cholesterol, hepatic lipid accumulation and LDL oxidation, suggesting the abilities to prevent the development of diet-induced NAFLD, atherosclerosis and CVD. The increased thiol group molecules and antioxidant enzyme activities in the erythrocyte and the decreased oxidized GSH in the liver revealed the antioxidant activity of polysaccharides of G. frondosa. Collectively, these findings demonstrated that natural and fermented G. frondosa and their polysaccharides have potentials as functional foods to improve hypercholesterolaemia and oxidative stress, especially for people who have consumed high-fat diets.

Author Contributions

HCL and THH conceptualized the research; WTW, WLC, CKY and HCL carried out the experiments and analyses; THH provided the testing samples; WTW and HCL drafted the original manuscript; THH and HCL supervised the project; and all authors read, edited and approved the submission of the manuscript.

Funding

This work was supported by grants from Changhua Christian Hospital (94-CCH-NSC-13) and the Ministry of Science and Technology, Taiwan (NSC94-2320-B-309-001).

Conflict of Interest

The authors declare that there are no conflicts of interest.

Data Availability

The data that support the findings of this study are available from the corresponding author (H.-C.L.), upon reasonable request.