Background
High-fat diets provide excess energy intake and disrupt lipid metabolism, resulting in fat accumulation in many tissues, especially serum and liver [
1]. Hyperlipidaemia, always caused by high-fat diets, is mainly characterized by increased levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C), along with a decrease in high-density lipoprotein cholesterol (HDL-C). These changes are the major risk factors contributing to the development and progression of atherosclerosis, fatty liver and cerebrovascular disease [
2‐
5]. Many researchers have focused on the roles of oxidative damage and lipid peroxidation in the pathomechanism of hyperlipidaemia [
6]. Oxidative stress, always caused by superfluous reactive oxygen species (ROS), is an early event in the evolution of hyperlipidaemia. ROS, including hydroxyl (HO·), DPPH· and superoxide (O
−
2·) radicals, are potentially toxic to various biological molecules, resulting in oxidative damage that can accelerate the pathogenic progress of hyperlipidaemia and its complications [
6‐
8]. Under hyperlipidaemic conditions, enzymatic and non-enzymatic antioxidative defence systems such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) are altered, leading to ROS-mediated damage [
9]. Scientists have suggested that the appropriate support for enhancing the antioxidant supply in subjects with hyperlipidaemia can attenuate the course of the disease. Maladjusted lipid synthesis and lipid clearance also play roles in causing hyperlipidaemia, and methods to reduce blood lipid levels could be effective in treating this disease [
5]. Thus, effects on the antioxidant and hypolipidaemic properties of some bioactive compounds are particularly promising for improving human health [
10].
Recently, the treatment of hyperlipidaemia has been involved in diet control, exercise and pharmaceutical therapy. Since synthetic lipid-lowering drugs, including statins and fibrates, usually have side effects and contraindications with long-term use, the application of natural hypolipidaemic drugs seems to be urgent to prevent and treat hyperlipidaemia and its complications [
11].
Pleurotus eryngii (
P. eryngii), one type of common edible fungus in China, is rich in biologically active components, including polysaccharides, peptide, sterols and dietary fibre [
12]. As the most potent mushroom-derived substances, polysaccharides exhibit various biological activities, including antioxidant, anti-aging, antivirus and anti-lipid peroxidation properties. [
13]. Furthermore, modified polysaccharides have received more attention due to their superior physicochemical properties, including good water-solubility, high stability, and non-toxicity [
14]. Previous studies have shown that crude polysaccharides from the fruiting body of
P. eryngii have potential effects in reducing blood lipids [
15]. However, the hypolipidaemic effects of exopolysaccharides and their chemically modified forms have not been evaluated. In the present study, three kinds of novel polysaccharides – exopolysaccharides (EPS), enzymatic EPS (EEPS) and acidic EPS (AEPS) – were isolated, and their hypolipidaemic and hepatoprotective effects were investigated. EPS, EEPS and AEPS possessed hypolipidaemic and antioxidant activities, indicating that the polysaccharides could be developed as valuable functional foods/drugs for clinical hypolipidaemic and hepatoprotective treatments.
Methods
Strain and chemicals
The P. eryngii SI-04 strain was provided by the Fungi Institute of the Academy of Agricultural Sciences (Tai’an, China). The diagnostic kits for analysing SOD activities, GSH-Px activities, CAT activities, total antioxidant capacity (T-AOC) activities, lipid peroxidation (LPO) contents and malondialdehyde (MDA) contents were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The standard monosaccharide samples, including rhamnose (Rha), ribose (Rib), arabinose (Ara), xylose (Xyl), glucose (Glc), mannose (Man) and galactose (Gal) were provided by the Merck Company (Darmstadt, Germany) and Sigma Chemical Company (St. Louis, USA). Other reagents and chemicals used in the present work were analytical reagent grade and were supplied by local chemical suppliers.
Preparation of EPS
The liquid fermentation of
P. eryngii SI-04 was processed using the method from our present work [
16]. The EPS of
P. eryngii SI-04 was obtained by referencing the method of Ma et al. (2015) with slight modifications. After centrifugation (3000 rpm, 15 min), the supernatant fermentation broth was mixed with 3 volumes of 95% ethanol (
v/v), stirred thoroughly and stored at 4 °C for 24 h. The precipitate was deproteinized with Sevag reagent (chloroform/
n-butanol, 5:1,
v/v) and lyophilized by vacuum freeze-drying (Labconco, USA) to obtain EPS. The EPS was weighed, and the yield was 3.81 g/L.
Enzymatic and acidic hydrolysis of EPS
The enzymatic hydrolysis of EPS was processed according to the methods of Yang et al. [
17] and Li et al. [
18] with some modifications. The polysaccharide sample (0.5 g) and snailase (0.1 g) were dissolved in 100 mL of 1% acetic acid at pH 6 and 37 °C for 4 h. After quick pre-freezing, the enzymatic hydrolysis exopolysaccharides (EEPS) were lyophilized for further analyses.
The acidic hydrolysis of EPS was processed according to the method of Ma et al. [
19] with slight modifications. Briefly, EPS (0.5 g) was dissolved in 10 mL of 1 M H
2SO
4 solution, and the reaction was processed in a boiling water bath for 8 h. After centrifugation (6000 rpm, 10 min) and neutralization, the supernatant was concentrated and lyophilized to obtain acidic exopolysaccharides (AEPS).
Monosaccharide composition analysis
The monosaccharide compositions of EPS, EEPS and AEPS were calculated using gas chromatography (GC-2010, Shimadzu, Japan) equipped with a flame ionization detector (FID) and an Rtx-1 capillary column (30 m × 0.25 mm × 0.25 μm). The samples and standard monosaccharides were pre-processed using our previous method [
16]. The initial oven temperature of the column was maintained at 190 °C for 20 min and increased gradually to 200 °C at a rate of 3 °C /min. Nitrogen was used as the carrier gas at 0.8 mL/min of cavity flow and 19.8 mL/min of total flow. The samples (1.0 μL) were injected in the split model (1:20) at 260 °C. The monosaccharide content was expressed as the following formula:
$$ Monosaccharide content\ \left(\%\right)=\frac{A}{B}\times \frac{V}{M}\times C $$
(1)
where A and B were the peak areas of sample and standard monosaccharides, V was the sample constant volume (mL), M was the sample quality (g), and C was the monosaccharide concentration of the mixed standard (mg/mL).
Antioxidant effects in vitro
The reducing power was assayed according to our previous work [
16].
The scavenging capability on hydroxyl radicals was evaluated using the method of Koksal et al. [
20] with few modifications. The reaction mixture, including 1 mL of phenanthroline (7.5 mM), 1 mL of ferrous sulphate (0.75 mM), 5 mL of phosphate buffer (pH 7.4), 1 mL of sample (0–1000 mg/L) and 1 mL of hydrogen peroxide (3%,
v/v) was shaken sufficiently and incubated at 37 °C for 30 min. The absorbance was measured at 560 nm using distilled water as a blank, and the scavenging rate was calculated using the following formula:
$$ Scavenging rate\ \left(\%\right)=\frac{A-B}{B}\times 100 $$
(2)
where A was the absorbance of distilled water, and B was the absorbance of samples.
The scavenging capability on DPPH radicals was measured using the methods of Brand-Williams et al. [
21] and Kong et al. [
22] with some modifications. The reaction mixture, containing 2 mL of ethanol (95%,
w/
v), 0.1 mL of DPPH (l M) and 2 mL of sample (0–1000 mg/L), was incubated at room temperature and placed in the dark for 30 min. The absorbance of the solution was determined at 517 nm. The scavenging rate was evaluated using the following formula:
$$ Scavenging rate\ \left(\%\right)=\left(1-\frac{A}{B}\right)\times 100 $$
(3)
where A was the absorbance of the tested sample, and B was the absorbance of the blank.
The scavenging capability of superoxide anion radicals was measured using the method of Stewar and Beewley [
23] with slight modification. Briefly, 1.0 mL of sample (0–1000 mg/L) was added to the mixture containing phosphate-buffered saline (0.5 mL, 0.2 M, pH 7.8), riboflavin (0.3 mL, 10 mM) and methionine (0.25 mL, 13 mM), and the reaction was incubated at 25 °C for 30 min. The absorbance of the solution was determined at 560 nm, and the scavenging rate was calculated using the following formula:
$$ Scavenging rate\ \left(\%\right)=\left(1-\frac{A}{B}\right)\times 100 $$
(4)
where A was the absorbance of polysaccharide samples, and B was the absorbance of the blank.
Experimental design
Preparation of high-fat emulsion
The high-fat emulsion was prepared using the method of Zhao, Huang and Yuan [
11] with sight modifications. Briefly, the oil phase, including 25 g lard oil, 10 g cholesterol, 1 g methylthiouracil and 25 mL of Tween-80, was heated to the melting point on a magnetic stirring apparatus (Guohua Instrument Ltd. Co. Changzhou, China). Simultaneously, the water phase contained 30 mL distilled water, 20 mL propylene glycol and 2 g sodium deoxycholate. Subsequently, the water and oil phases were mixed thoroughly before animal administration.
Design of the animal experiment
Seventy-two Kunming strain mice (20 ± 2 g, male), purchased from Taibang Biological Products Ltd. Co. (Tai’an, China) were housed in polycarbonate cages and freely accessed food and water ad libitum at constant conditions of 22 ± 1 °C and constant humidity (50 ± 5%) under a 12-h light-dark cycle.
After adapting to the environment for 7 d, all mice were weighed and randomly distributed into nine groups (eight mice per group). In the hyperlipidaemia group (HL), mice were perfused with high-fat emulsion alternated with distilled water. Mice in the simvastatin group (ST) were perfused with high-fat emulsion alternated with simvastatin (200 mg/kg body weight). In the other six treatment groups (L-EPS, H-EPS, L-EEPS, H-EEPS, L-AEPS and H-AEPS), mice were perfused with high-fat emulsion alternated with EPS, EEPS and AEPS at 400 and 800 mg/kg body weight. Mice in the normal control group (NC) were given distilled water daily, and the entire experiment lasted 28 days. All the experiments were submitted to and approved by the ethics committee of the Shandong Agricultural University.
After overnight fasting, all mice were weighed and sacrificed under anaesthesia. The serum was obtained by centrifugation (10,000 rpm, 10 min) from blood in the retrobulbar vein. The livers were excised, weighed and homogenized (1:9, w/v, in normal saline and ethyl alcohol). After centrifugation (5000 rpm, 20 min, 4 °C), the supernatants were collected and stored at 0 °C for further biochemical analysis.
Biochemical and histopathological assays
Alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and TG, TC, HDL-C and LDL-C levels in serum were measured using an automatic biochemical analyser (ACE, USA). GSH-Px, SOD and CAT activities in serum/liver homogenate and the MDA, LPO, TC and TG contents in liver homogenate were analysed using commercial kits according to the instructions.
The liver tissue staining method followed a previously published study [
16].
Acute toxicity assay
The acute toxicity test in mice was performed on the basis of the reported method [
24] with some modifications. The mice were randomly divided into four groups, including one control group and three dose groups (eight mice per group). The mice in dose groups received intragastric administration with EPS, EEPS and AEPS at 5000 mg/kg body weight, while the control group received isometric saline solutions. All mice had free access to food and water ad libitum for 10 days under regular observation for any mortality or behavioural changes, including irritation, restlessness, respiratory distress, abnormal locomotion and catalepsy.
Statistical analysis
All data are presented as the means ± standard deviations (SD) of three independent experiments. Significant differences among groups were determined by one-way ANOVA (SPSS 16.0 software package, USA). P < 0.05 was considered statistically significant.
Discussion
The scientific literature has indicated that hyperlipidaemia plays a very important role in the developmental progress of non-alcoholic fatty liver disease, atherosclerosis and cardiovascular disease [
26,
27]. Several serum parameters, including elevated TC, TG and LDL-C levels and reduced HDL-C levels, are often considered indicators of hyperlipidaemia and are involved in increased risk of clinical diseases [
28], consistent with the present results (Table
2). As the main carrier of cholesterol, excess LDL-C can be deposited in blood vessel walls, directly inducing the formation of atherosclerosis. High levels of HDL-C had protective effects because HDL-C can transport cholesterol from peripheral tissues to the liver through the “reverse cholesterol transport” pathway for catabolism [
1,
29,
30]. In addition, TG levels play key roles in the regulation of lipoprotein interactions in maintaining normal lipid metabolism and have also been proposed as major determinants of cholesterol esterification, transfer and HDL remodelling in human plasma [
31]. However, the variation trends in lipid levels are significantly mitigated by treatment with these three polysaccharides (EPS, EEPS and AEPS), indicating that the polysaccharides extracted from the fermentation broth of
P. eryngii SI-04 showed positive antihyperlipidaemic effects on restoring high-fat emulsion-induced lipid metabolic disturbance. Ren et al. [
32] demonstrated that these polysaccharides might be combined with lipids in lipid metabolism, accelerating transport and excretion of serum lipids.
Previous literature has reported that oxidative stress, usually induced by ROS and motivationally accelerating the development of endothelial damage and atherosclerosis – owing to its oxidative roles for the destruction of the nucleic acids, proteins and lipids of endothelial cell membranes – may be regarded as a possible mechanism to induce hyperlipidaemia [
33,
34]. Lipid peroxidation could be a very sensitive biomarker for investigating the antioxidant effects, since lipid peroxidation could lead to hydroperoxide generation to toxic chemicals such as MDA. Excess MDA can oxygenate and modify LDL-C to form MDA-LDL-C, which can cause the degeneration and necrosis of endothelial cells, inflammatory reactions and disordered antioxidant systems [
35,
36]. Experimentally, the major antioxidant enzymes, such as SOD, GSH-Px and CAT, were commonly used as biomarkers reflecting the production of free radicals and can prevent oxidative damage cooperatively at different sites during ROS metabolic pathways [
37]. In the current study, serum GSH-Px, SOD and CAT activities decreased significantly (Fig.
2) after the perfusion of high-fat emulsion. The results were in accordance with those reported in previous articles [
38,
39]. The significant and dose-dependent increases in these enzyme activities after treatment with AEPS indicated that AEPS had superior activity in the treatment of hyperlipidaemia.
In addition, excessively accumulated lipids in the liver can damage hepatic biomembranes, leading to an imbalance in oxidative phosphorylation and accelerating ROS formation. The imbalance of oxidation and reduction can cause lipid peroxidation and produce significant toxic intermediate products in the liver, resulting in hepatic necrosis and apoptosis [
40]. Furthermore, oxidative stress can also produce an inflammatory reaction through cell injury, causing the infiltration of the liver parenchyma by inflammatory cells [
11], in accordance with the results of the hepatocyte morphological assay (Fig.
5). The hepatocytes showed obvious diffuse hepatic steatosis and inflammatory changes in the HL group, and treatment of the samples alleviated these symptoms.
Moreover, it is well known that the biological activities of polysaccharides are always associated with their monosaccharide compositions [
41]. The EPS consists of five monosaccharides, including Ara, Xyl, Man, Gal and Glc, in contrast to a previous conclusion for intracellular polysaccharides (IPS) from
P. eryngii SI-04 [
16]. Compared with the published literature, Chen et al. [
10] demonstrated that the polysaccharides of the
P. eryngii fruit body were mainly composed of Man, Glc and Gal. The difference in monosaccharide compositions may be related to the composition of the culture medium and the fermentation, extraction and purification conditions of polysaccharides [
42]. Additionally, Wu et al. [
14] demonstrated that the polysaccharides showed higher biological activities after hydrolysis with various glycosidases or acidic reagents. After enzymatic and acidic hydrolysis, the monosaccharide compositions and percentage compositions of EPS were altered. The results of in vitro antioxidant and antihyperlipidaemic assays indicated that AEPS with more abundant monosaccharide compositions than EEPS and higher Glc percentages than EPS performed better in these assays (Fig.
1).