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Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide
BIOMAC-10126; No of Pages 5 International Journal of Biological Macromolecules xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Review
Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide Shengjun Wu Jiangsu Key Laboratory of Marine Bioresources and Environment, Huaihai Institute of Technology, 59 Cangwu Road, Haizhou 222005, China Co-Innovation Center of Jiangsu Marine Bio-industry Technology, 59 Cangwu Road, Haizhou 222005, China Jiangsu Key Laboratory of Marine Biotechnology, 59 Cangwu Road, Haizhou 222005, China
a r t i c l e
i n f o
Article history: Received 28 May 2018 Received in revised form 5 July 2018 Accepted 12 July 2018 Available online xxxx Keywords: Ganoderma lucidum Hypolipidaemic Polysaccharide
a b s t r a c t Ganoderma lucidum polysaccharide (GLP) was prepared by hot water extraction and partly characterised by highperformance gel filtration chromatography and its monosaccharide composition. Male Sprague–Dawley rats were randomly divided into four groups: one group received a high-fat diet (control group) and the three other groups received a high-fat diet containing 100, 300 and 500 mg/kg of GLP. GLP administration reduced the body weight gain, food efficiency ratio, levels of plasma triacylglycerol (TG), plasma total cholesterol (TC) and low-density lipoprotein cholesterol and liver weight, TC and TG levels and malondialdehyde values, improved the levels of faecal fat, cholesterol and plasma high-density lipoprotein cholesterol and enhanced the activities of serum superoxide dismutase and glutathione peroxidase in rats compared with the control group. The appropriate dose of GLP was 300 mg/kg. Results indicate that GLP exhibits hypolipidaemic and lipid antioxidant activities and may be used as a drug for hyperlipidaemia treatment. © 2018 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Ethics statement . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. GLP preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. GLP characterisation . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Test animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Experimental design . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Serum lipids, faecal fat, faecal cholesterol, SOD, GPx and MDA of rats . . 2.8. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . 3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Product characterisation . . . . . . . . . . . . . . . . . . . . . . 3.2. Body weight gain (WG), food intake (FI) and food efficiency ratio (FER) . 3.3. Plasma lipid levels . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Faecal lipid and cholesterol excretion . . . . . . . . . . . . . . . . 3.5. Liver weight, TC and TG. . . . . . . . . . . . . . . . . . . . . . . 3.6. Serum SOD, GPx and MDA . . . . . . . . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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E-mail address: [email protected].
https://doi.org/10.1016/j.ijbiomac.2018.07.082 0141-8130/© 2018 Elsevier B.V. All rights reserved.
Please cite this article as: S. Wu, Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide, (2018), https://doi. org/10.1016/j.ijbiomac.2018.07.082
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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S. Wu / International Journal of Biological Macromolecules xxx (2018) xxx–xxx Table 1 The composition of the commercial diet. Constituent
Content
Protein (%, w/w) Fat (%, w/w) Fiber (%, w/w) Moisture (%, w/w) Ash (%, w/w) Nitrogen free extract (%, w/w) Calcium (%, w/w) Phosphate (%, w/w)
22.1 5.3 4.1 9.2 5.2 52 1.2 0.9 13.4
L-Lysine
(%, w/w)
L-Methionine
and L-cystine (%, w/w) Total calorie (kcal)
0.72 352
1. Introduction Hyperlipidaemia can result in atherosclerosis, which is a major risk factor for the development of numerous cardiovascular diseases, and is therefore a severe threat to human health [1]. Thus, effective therapeutic drugs must be developed for hyperlipidaemia. Chemosynthetic drugs can effectively decrease serum triacylglycerol (TG) and cholesterol levels and are thus widely used for hyperlipidaemia; however, these drugs exhibit numerous adverse effects [2]. Therefore, natural drugs that exhibit hypolipidaemic activity without adverse effects should be developed. Ganoderma lucidum, a rot fungus also known as ‘Lingzhi’ in Chinese, is a mushroom that has been widely used as a tonic for improving health and longevity in China for the past 2000 years [3]. A number of studies verified that the polysaccharides isolated from G. lucidum exhibit antioxidant, anti-inflammatory and anti-tumour activities and improve insulin sensitivity and immunomodulation [3–7]. At present, G. lucidum polysaccharides (GLPs) have been widely used in Food and pharmaceutical industries. However, data regarding the effects of GLPs on hypolipidaemic and anti-lipidperoxidant activities are limited. In the present study, a GLP was extracted using hot water and its hypolipidaemic and anti-lipidperoxidant activities was investigated in rats. 2. Materials and methods 2.1. Ethics statement This study was approved by the Ethics Committee of Huaihai Institute of Technology, Jiangsu, China. All procedures were performed in compliance with relevant laws and institutional guidelines. 2.2. Materials
distilled water under agitation at room temperature (approximately 20 °C) for 30 min to yield a suspension with a concentration of approximately 2%. The suspension was incubated under agitation in a water bath at 80 °C for 12 h and centrifuged at 5000 ×g for 10 min. The resulting supernatant was protein separated using Sevage method [8], filtered through a 1000 Da interception filter membrane, concentrated (approximately 10%) using a vacuum rotary evaporator (RE-2010; Shanghai Qiang Qiang Industrial Development Co., Ltd., Shanghai, China), precipitated with four volumes of absolute ethanol, filtered through a Whatman GF/A filter paper and freeze-dried. 2.4. GLP characterisation The total sugar, protein, lipid and moisture contents in the GLP sample were determined through phenol–sulphuric acid colorimetric method, Kjeldahl method, Soxhlet extraction and drying method, respectively, as described by Hou [9]. The GLP sample was redissolved in distilled water, loaded onto a DEAE-52 cellulose anion-exchange chromatography column (30 cm × 2.6 cm; GE Healthcare, Buckinghamshire, UK) and eluted successively with 0.05 mol L−1 phosphate-buffered saline and gradient solution of 0.05–1.0 mol L−1 NaCl at a flow rate of 1.0 mL min−1. The eluted polysaccharide fractions ware collected for subsequent analysis. The molecular weights (MWs) of GLP were determined by high-performance gel filtration chromatography (LC-10A; Shimadzu, Kyoto, Japan) in which an ultrahydrogel size exclusion column (LKB-Produkter AB, Bromma, Switzerland) and high-sensitivity refractive index detector (Model ERC-7515 A; ERC Inc., Kawaguchi, Japan) were used. The GLP was eluted with 0.1 N NaNO3 at a flow rate of 0.9 mL min−1. Pullulan standards (P20-P800; JM Science, Inc., Grand Island, NY, USA) were used as MW standard. The monosaccharide compositions of GLP were determined according to the methods described by Sheng et al. [10]. 2.5. Test animals Male Sprague–Dawley rats weighing 107 ± 9 g were housed individually in metabolic cages equipped with glass separators for urine and faeces collection in an air-conditioned room at 25 °C ± 1 °C on a 12 h light–dark cycle. Rats were allowed free access to food and water. A total of 40 rats were fed ad libitum with a commercial diet and water for 7 days. The rats were randomly divided into 4 groups (10 rats per group), namely, one control group and three GLP groups. The rats in all groups were fed with a high-fat diet containing 15% (w/w) egg yolk powder, 10% (w/w) lard, 1% (w/w) cholesterol and 76% (w/ w) basic diet with composition conforming to AIN 76 (Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China; Table 1). Rats in the GLP group were gavaged twice daily with 50, 100 and 150 mg/kg GLP, and the rats in the control group were gavaged twice daily with equal volumes of distilled water.
Dried fruit bodies of G. lucidum were purchased from Gansu Hall Nutritional Health Food Co. Ltd. (Gansu, China). Male Sprague–Dawley rats weighing 107 ± 9 g were used in the experiment. Commercial assay kits for low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), TG, plasma total cholesterol (TC), superoxide dismutase (SOD), glutathione peroxidase (GPx) and malondialdehyde (MDA) were obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Standard monosaccharides, such as glucose, fructose, galactose and mannose, were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of reagent grade.
2.6. Experimental design
2.3. GLP preparation
2.7. Serum lipids, faecal fat, faecal cholesterol, SOD, GPx and MDA of rats
Dried fruit bodies of G. lucidum were pulverised and sifted through a 100-mesh sieve to yield fine powder. The powder was soaked in
The levels of serum TG, TC and HDL-C were assayed using commercial assay kits. The LDL content in serum was calculated using the
The rats were deprived of food overnight, and blood samples were collected to analyse the initial plasma lipid level, SOD activity, GPx activity and MDA level before the experiments. At the sixth week, faeces were collected for three continuous days and dried at 100 °C for 2 h to analyse the cholesterol and fat contents. All rats were subjected to diethyl ether treatment after fasting for 18 h. Blood was collected and centrifuged at 5000 ×g for 10 min to yield plasma, which was stored in a freezer at −20 °C until use for further analysis.
Please cite this article as: S. Wu, Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide, (2018), https://doi. org/10.1016/j.ijbiomac.2018.07.082
S. Wu / International Journal of Biological Macromolecules xxx (2018) xxx–xxx
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Absorbance
Tube number Fig. 1. Elution curve of Ganoderma lucidum polysaccharide on a DEAE Sepharose Fast Flow ion exchange chromatography column.
following equation [11]:
2.8. Statistical analysis
LDL‐Cðmmol=LÞ ¼ ðTC−HDL‐C−TGÞ=2:2
All data are presented as the mean ± standard deviation. Analysis of variance was used to compare the means of two rat groups. Statistical significance at 95% and 99% probability levels were set at p b 0.05 and p b 0.01, respectively.
Faeces were dried at 105 °C for 2 h, weighed and pulverised. Faecal fat content was determined gravimetrically using the Soxhlet method [9]. Faecal cholesterol was extracted using a previously described method [12]. The cholesterol content in faeces was determined enzymatically using a commercial assay kit. Serum SOD activity, GPx activity and MDA level were analysed using enzyme-linked immunosorbent assay (ELISA) kits by following the methods of Gao et al. [13]. In brief, standards or samples were added to the appropriate micro-ELISA strip plate wells and combined with the specific antibody, followed by a horseradish-peroxidaseconjugated antibody specific for the target enzyme. The free components were washed away. Tetramethylbenzidine substrate solution was added to each well. Optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm. The OD was proportional to the activity of the target enzyme, which was calculated by comparing the OD of the samples with the standard curve. A unit of enzyme activity was defined as the amount of enzyme that decreased the absorbance by 0.001 min−1. All assays were performed in triplicate.
3. Results and discussion 3.1. Product characterisation The total sugar and moisture contents in the crude GLP products were 97.36% and 1.41%, respectively. GLP did not contain any proteins and lipids. The GLP samples were water-soluble powders. The elution curve of the GLP sample on a DEAE Sepharose Fast Flow ion-exchange chromatography column indicated that the sample comprised only one kind of polysaccharide, which was collected for subsequent experiments (Fig. 1). High-performance liquid chromatography spectrum analysis of the GLP products indicated one type of polysaccharide, with a MW of 13.7 kDa (Fig. 2). Monosaccharide composition analysis verified that GLP consisted of glucose and galactose at the molar ratio
13.7 kDa
MV
Time (min) Fig. 2. HPLC spectrum of Ganoderma lucidum polysaccharide.
Please cite this article as: S. Wu, Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide, (2018), https://doi. org/10.1016/j.ijbiomac.2018.07.082
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S. Wu / International Journal of Biological Macromolecules xxx (2018) xxx–xxx
Table 2 Effect of Ganoderma lucidum polysaccharide (GLP) on body weight gain of rats (WG), food intake (FI), food efficiency ratio (FER) of rats. Parameters
WG (g) FI (g) FER (%)
Group
Table 4 Effect of Ganoderma lucidum polysaccharide (GLP) on faecal fat and cholesterol excretion of rats. Parameters
Control
100 mg/kg GLP
300 mg/kg GLP
500 mg/kg GLP
13.6 ± 0.6a 131.4 ± 7.2a 10.4 ± 0.5a
12.1 ± 0.5b 130.8 ± 6.4a 9.3 ± 0.4b
10.8 ± 0.4c 129.6 ± 6.1a 8.3 ± 0.4c
10.7 ± 0.3c 129.3 ± 5.9a 8.3 ± 0.3c
Group
Faecal fat
Values are expressed as mean ± SD (n = 3). Different superscript letters in a row indicate significant difference (p b 0.05).
Cholesterol excretion
Control
100 mg/kg GLP
300 mg/kg GLP
500 mg/kg GLP
11.86 ± 0.59a 17.72 ± 0.81a
13.37 ± 0.62b 16.41 ± 0.74c
21.26 ± 0.86d
19.28 ± 0.86b 22.46 ± 0.85c
24.81 ± 0.91d
Values are expressed as mean ± SD (n = 3). Different superscript letters in a row indicate significant difference (p b 0.05).
of 3.72:1. The results obtained in this study were inconsistent with those reported by He et al. [14]. Differences between the monosaccharide compositions of GLP can be ascribed to the species of G. lucidum tested and the extraction methods used.
Table 5 Effect of Ganoderma lucidum polysaccharide (GLP) on liver weight, TG and TC levels in rats. Parameters
Group
3.2. Body weight gain (WG), food intake (FI) and food efficiency ratio (FER)
Control
100 mg/kg GLP
300 mg/kg GLP
500 mg/kg GLP
3.19 ± 0.24a 1.79 ± 0.19a 2.81 ± 0.22a
3.14 ± 0.22a
2.13 ± 0.16b
2.09 ± 0.15b
1.74 ± 0.16a
0.91 ± 0.14b
0.90 ± 0.12b
2.76 ± 0.19a
1.73 ± 0.13b
1.71 ± 0.11b
Table 2 shows the WG, FI and FER for the four groups after 6 weeks of feeding. Although no significant difference in FI was observed among the four groups (p N 0.05), WG and FER for the GLP group were lower than those of the control group (p b 0.05). This finding indicated that GLP can inhibit the increase in body weight of the rats fed with highfat diets. However, excessive administration of GLP (N300 mg/kg) did not decrease WG and FER further.
Values are expressed as mean ± SD (n = 3). Different superscript letters in a row indicate significant difference (p b 0.05).
3.3. Plasma lipid levels
3.5. Liver weight, TC and TG
The effect of GLP on the plasma lipid levels is shown in Table 3. After 6 weeks of gavage administration of GLP, the plasma TC, TG and LDL-C levels of rats were significantly lower than those of the control group and the HDL-C levels of the GLP group were significantly higher than those of the control group (p b 0.05). Similarly, the polysaccharides produced from submerged mycelial culture of G. lucidum reportedly decrease TC, TG and LDL-C levels and increase HDL-C level in rats [15].
The changes in the values of liver weight, TC and TG of rats fed with different diets are shown in Table 5. Although no differences in the liver weight, TC and TG of rats were observed between the control group and the 100 mg/kg GLP group (p N 0.05), the liver weight, TG and TC levels of the 300 and 500 mg/kg GLP groups were significantly lower than those of the control group (p b 0.05). The results indicated that GLP can effectively reduce the accumulation of lipids in the liver. Similarly, the polysaccharides produced from the submerged mycelial culture of G. lucidum reportedly decrease the liver weight, TC and TG of rats [15].
Liver weight (g) Liver TC (mmol/L) Liver TG (mmol/L)
3.4. Faecal lipid and cholesterol excretion The effect of GLP on the lipid and cholesterol contents in the dried faeces of rats collected for three successive days were determined, and the results are shown in Table 4. The faecal fat and cholesterol contents of all of the GLP groups were significantly higher than those of the control group in a dose-dependent manner (p b 0.05), indicating that GLP possessed considerable fat-binding capacities.
3.6. Serum SOD, GPx and MDA The changes in serum SOD and GPx activities and MDA level of rats fed with different diets are shown in Table 6. After 6 weeks of gavage administration of GLP, the serum SOD and GPx activities of rats were significantly higher than those of the control group and the MDA levels of the GLP group were significantly lower than those of the control group (p b 0.05). This finding can be attributed to the antioxidant activity of GLP [5].
Table 3 Effect of Ganoderma lucidum polysaccharide (GLP) on serum TG, TC, LDL-C and HDL-C levels in rats. Parameters
Group Control
Serum TG (mmol/L) Serum TC (mmol/L) Serum LDL-C (mmol/L) Serum HDL-C (mmol/L)
3.21 ± 0.32a 1.76 ± 0.16a 2.69 ± 0.18a 1.39 ± 0.17a
100 mg/kg GLP
300 mg/kg GLP
2.59 ± 0.26
b
1.43 ± 0.14
b
2.15 ± 0.13b 1.81 ± 0.23b
500 mg/kg GLP
1.73 ± 0.47
c
1.71 ± 0.43
0.79 ± 0.12
c
0.78 ± 0.10c
1.08 ± 0.11c
1.07 ± 0.11c
1.83 ± 0.24b
c
1.86 ± 0.25b
Values are expressed as mean ± SD (n = 3). Different superscript letters in a row indicate significant difference (p b 0.05).
Table 6 Effect of Ganoderma lucidum polysaccharide (GLP) on serum SOD, GPx and l malondialdehyde (MDA) levels in rats. Parameters
Group Control
SOD (U/ml) GPx (U/ml) MDA (ng/ml)
100 mg/kg GLP a
3.15 ± 0.15 21.13 ± 0.83a 10.78 ± 0.43a
b
3.85 ± 0.17 41.63 ± 1.17b 6.71 ± 0.36b
300 mg/kg GLP c
4.74 ± 0.21 50.94 ± 2.18c 4.62 ± 0.27c
500 mg/kg GLP 4.79 ± 0.22c 51.79 ± 2.19c 4.59 ± 0.26c
Values are expressed as mean ± SD (n = 3). Different superscript letters in a row indicate significant difference (p b 0.05).
Please cite this article as: S. Wu, Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide, (2018), https://doi. org/10.1016/j.ijbiomac.2018.07.082
S. Wu / International Journal of Biological Macromolecules xxx (2018) xxx–xxx
4. Conclusions This study verified the hypolipidaemic and lipid antioxidant activities of GLP in rats. GLP administration decreased the WG, FER, levels of TG, TC and LDL-C, liver weight, TC and TG levels and MDA values and increased the levels of faecal fat, cholesterol and HDL-C and serum SOD and GPx activities of rats relative to the control group. However, the mechanism of the hypolipidaemic and lipid antioxidant in the appropriate micro-ELISA strip plate wells, the activities of GLP needs further investigation. Acknowledgments This research was supported by A Project Funded by the Priority Academic Program Development （PAPD） of Jiangsu Higher Education Institutions. Conflict of interest statement The authors have declared that no competing interests exist. References [1] Z.W. Yang, K.H. Ouyang, J. Zhao, H. Chen, L. Xiong, W.J. Wang, Structural characterization and hypolipidemic effect of Cyclocarya paliurus polysaccharide in rat, Int. J. Biol. Macromol. 91 (2016) 1073–1080. [2] L.Y. Zhao, W. Huang, Q.X. Yuan, J. Cheng, Z.C. Huang, L.J. Ouyang, F.H. Zeng, Hypolipidaemic effects and mechanisms of the main component of Opuntia dillenii Haw. polysaccharides in high-fat emulsion-induced hyperlipidaemic rats, Food Chem. 134 (2012) 64–971. [3] L.X. Zhang, Y.J. Zhang, L.P. Zhang, Structure and immunological activity of a novel polysaccharide from the spores of Ganoderma lucidum, Afr. J. Biotechnol. 10 (2011) 10923–10929.
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Please cite this article as: S. Wu, Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide, (2018), https://doi. org/10.1016/j.ijbiomac.2018.07.082
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Review
Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide Shengjun Wu Jiangsu Key Laboratory of Marine Bioresources and Environment, Huaihai Institute of Technology, 59 Cangwu Road, Haizhou 222005, China Co-Innovation Center of Jiangsu Marine Bio-industry Technology, 59 Cangwu Road, Haizhou 222005, China Jiangsu Key Laboratory of Marine Biotechnology, 59 Cangwu Road, Haizhou 222005, China
a r t i c l e
i n f o
Article history: Received 28 May 2018 Received in revised form 5 July 2018 Accepted 12 July 2018 Available online xxxx Keywords: Ganoderma lucidum Hypolipidaemic Polysaccharide
a b s t r a c t Ganoderma lucidum polysaccharide (GLP) was prepared by hot water extraction and partly characterised by highperformance gel filtration chromatography and its monosaccharide composition. Male Sprague–Dawley rats were randomly divided into four groups: one group received a high-fat diet (control group) and the three other groups received a high-fat diet containing 100, 300 and 500 mg/kg of GLP. GLP administration reduced the body weight gain, food efficiency ratio, levels of plasma triacylglycerol (TG), plasma total cholesterol (TC) and low-density lipoprotein cholesterol and liver weight, TC and TG levels and malondialdehyde values, improved the levels of faecal fat, cholesterol and plasma high-density lipoprotein cholesterol and enhanced the activities of serum superoxide dismutase and glutathione peroxidase in rats compared with the control group. The appropriate dose of GLP was 300 mg/kg. Results indicate that GLP exhibits hypolipidaemic and lipid antioxidant activities and may be used as a drug for hyperlipidaemia treatment. © 2018 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Ethics statement . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. GLP preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. GLP characterisation . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Test animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Experimental design . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Serum lipids, faecal fat, faecal cholesterol, SOD, GPx and MDA of rats . . 2.8. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . 3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Product characterisation . . . . . . . . . . . . . . . . . . . . . . 3.2. Body weight gain (WG), food intake (FI) and food efficiency ratio (FER) . 3.3. Plasma lipid levels . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Faecal lipid and cholesterol excretion . . . . . . . . . . . . . . . . 3.5. Liver weight, TC and TG. . . . . . . . . . . . . . . . . . . . . . . 3.6. Serum SOD, GPx and MDA . . . . . . . . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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E-mail address: [email protected].
https://doi.org/10.1016/j.ijbiomac.2018.07.082 0141-8130/© 2018 Elsevier B.V. All rights reserved.
Please cite this article as: S. Wu, Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide, (2018), https://doi. org/10.1016/j.ijbiomac.2018.07.082
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S. Wu / International Journal of Biological Macromolecules xxx (2018) xxx–xxx Table 1 The composition of the commercial diet. Constituent
Content
Protein (%, w/w) Fat (%, w/w) Fiber (%, w/w) Moisture (%, w/w) Ash (%, w/w) Nitrogen free extract (%, w/w) Calcium (%, w/w) Phosphate (%, w/w)
22.1 5.3 4.1 9.2 5.2 52 1.2 0.9 13.4
L-Lysine
(%, w/w)
L-Methionine
and L-cystine (%, w/w) Total calorie (kcal)
0.72 352
1. Introduction Hyperlipidaemia can result in atherosclerosis, which is a major risk factor for the development of numerous cardiovascular diseases, and is therefore a severe threat to human health [1]. Thus, effective therapeutic drugs must be developed for hyperlipidaemia. Chemosynthetic drugs can effectively decrease serum triacylglycerol (TG) and cholesterol levels and are thus widely used for hyperlipidaemia; however, these drugs exhibit numerous adverse effects [2]. Therefore, natural drugs that exhibit hypolipidaemic activity without adverse effects should be developed. Ganoderma lucidum, a rot fungus also known as ‘Lingzhi’ in Chinese, is a mushroom that has been widely used as a tonic for improving health and longevity in China for the past 2000 years [3]. A number of studies verified that the polysaccharides isolated from G. lucidum exhibit antioxidant, anti-inflammatory and anti-tumour activities and improve insulin sensitivity and immunomodulation [3–7]. At present, G. lucidum polysaccharides (GLPs) have been widely used in Food and pharmaceutical industries. However, data regarding the effects of GLPs on hypolipidaemic and anti-lipidperoxidant activities are limited. In the present study, a GLP was extracted using hot water and its hypolipidaemic and anti-lipidperoxidant activities was investigated in rats. 2. Materials and methods 2.1. Ethics statement This study was approved by the Ethics Committee of Huaihai Institute of Technology, Jiangsu, China. All procedures were performed in compliance with relevant laws and institutional guidelines. 2.2. Materials
distilled water under agitation at room temperature (approximately 20 °C) for 30 min to yield a suspension with a concentration of approximately 2%. The suspension was incubated under agitation in a water bath at 80 °C for 12 h and centrifuged at 5000 ×g for 10 min. The resulting supernatant was protein separated using Sevage method [8], filtered through a 1000 Da interception filter membrane, concentrated (approximately 10%) using a vacuum rotary evaporator (RE-2010; Shanghai Qiang Qiang Industrial Development Co., Ltd., Shanghai, China), precipitated with four volumes of absolute ethanol, filtered through a Whatman GF/A filter paper and freeze-dried. 2.4. GLP characterisation The total sugar, protein, lipid and moisture contents in the GLP sample were determined through phenol–sulphuric acid colorimetric method, Kjeldahl method, Soxhlet extraction and drying method, respectively, as described by Hou [9]. The GLP sample was redissolved in distilled water, loaded onto a DEAE-52 cellulose anion-exchange chromatography column (30 cm × 2.6 cm; GE Healthcare, Buckinghamshire, UK) and eluted successively with 0.05 mol L−1 phosphate-buffered saline and gradient solution of 0.05–1.0 mol L−1 NaCl at a flow rate of 1.0 mL min−1. The eluted polysaccharide fractions ware collected for subsequent analysis. The molecular weights (MWs) of GLP were determined by high-performance gel filtration chromatography (LC-10A; Shimadzu, Kyoto, Japan) in which an ultrahydrogel size exclusion column (LKB-Produkter AB, Bromma, Switzerland) and high-sensitivity refractive index detector (Model ERC-7515 A; ERC Inc., Kawaguchi, Japan) were used. The GLP was eluted with 0.1 N NaNO3 at a flow rate of 0.9 mL min−1. Pullulan standards (P20-P800; JM Science, Inc., Grand Island, NY, USA) were used as MW standard. The monosaccharide compositions of GLP were determined according to the methods described by Sheng et al. [10]. 2.5. Test animals Male Sprague–Dawley rats weighing 107 ± 9 g were housed individually in metabolic cages equipped with glass separators for urine and faeces collection in an air-conditioned room at 25 °C ± 1 °C on a 12 h light–dark cycle. Rats were allowed free access to food and water. A total of 40 rats were fed ad libitum with a commercial diet and water for 7 days. The rats were randomly divided into 4 groups (10 rats per group), namely, one control group and three GLP groups. The rats in all groups were fed with a high-fat diet containing 15% (w/w) egg yolk powder, 10% (w/w) lard, 1% (w/w) cholesterol and 76% (w/ w) basic diet with composition conforming to AIN 76 (Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China; Table 1). Rats in the GLP group were gavaged twice daily with 50, 100 and 150 mg/kg GLP, and the rats in the control group were gavaged twice daily with equal volumes of distilled water.
Dried fruit bodies of G. lucidum were purchased from Gansu Hall Nutritional Health Food Co. Ltd. (Gansu, China). Male Sprague–Dawley rats weighing 107 ± 9 g were used in the experiment. Commercial assay kits for low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), TG, plasma total cholesterol (TC), superoxide dismutase (SOD), glutathione peroxidase (GPx) and malondialdehyde (MDA) were obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Standard monosaccharides, such as glucose, fructose, galactose and mannose, were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of reagent grade.
2.6. Experimental design
2.3. GLP preparation
2.7. Serum lipids, faecal fat, faecal cholesterol, SOD, GPx and MDA of rats
Dried fruit bodies of G. lucidum were pulverised and sifted through a 100-mesh sieve to yield fine powder. The powder was soaked in
The levels of serum TG, TC and HDL-C were assayed using commercial assay kits. The LDL content in serum was calculated using the
The rats were deprived of food overnight, and blood samples were collected to analyse the initial plasma lipid level, SOD activity, GPx activity and MDA level before the experiments. At the sixth week, faeces were collected for three continuous days and dried at 100 °C for 2 h to analyse the cholesterol and fat contents. All rats were subjected to diethyl ether treatment after fasting for 18 h. Blood was collected and centrifuged at 5000 ×g for 10 min to yield plasma, which was stored in a freezer at −20 °C until use for further analysis.
Please cite this article as: S. Wu, Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide, (2018), https://doi. org/10.1016/j.ijbiomac.2018.07.082
S. Wu / International Journal of Biological Macromolecules xxx (2018) xxx–xxx
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Absorbance
Tube number Fig. 1. Elution curve of Ganoderma lucidum polysaccharide on a DEAE Sepharose Fast Flow ion exchange chromatography column.
following equation [11]:
2.8. Statistical analysis
LDL‐Cðmmol=LÞ ¼ ðTC−HDL‐C−TGÞ=2:2
All data are presented as the mean ± standard deviation. Analysis of variance was used to compare the means of two rat groups. Statistical significance at 95% and 99% probability levels were set at p b 0.05 and p b 0.01, respectively.
Faeces were dried at 105 °C for 2 h, weighed and pulverised. Faecal fat content was determined gravimetrically using the Soxhlet method [9]. Faecal cholesterol was extracted using a previously described method [12]. The cholesterol content in faeces was determined enzymatically using a commercial assay kit. Serum SOD activity, GPx activity and MDA level were analysed using enzyme-linked immunosorbent assay (ELISA) kits by following the methods of Gao et al. [13]. In brief, standards or samples were added to the appropriate micro-ELISA strip plate wells and combined with the specific antibody, followed by a horseradish-peroxidaseconjugated antibody specific for the target enzyme. The free components were washed away. Tetramethylbenzidine substrate solution was added to each well. Optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm. The OD was proportional to the activity of the target enzyme, which was calculated by comparing the OD of the samples with the standard curve. A unit of enzyme activity was defined as the amount of enzyme that decreased the absorbance by 0.001 min−1. All assays were performed in triplicate.
3. Results and discussion 3.1. Product characterisation The total sugar and moisture contents in the crude GLP products were 97.36% and 1.41%, respectively. GLP did not contain any proteins and lipids. The GLP samples were water-soluble powders. The elution curve of the GLP sample on a DEAE Sepharose Fast Flow ion-exchange chromatography column indicated that the sample comprised only one kind of polysaccharide, which was collected for subsequent experiments (Fig. 1). High-performance liquid chromatography spectrum analysis of the GLP products indicated one type of polysaccharide, with a MW of 13.7 kDa (Fig. 2). Monosaccharide composition analysis verified that GLP consisted of glucose and galactose at the molar ratio
13.7 kDa
MV
Time (min) Fig. 2. HPLC spectrum of Ganoderma lucidum polysaccharide.
Please cite this article as: S. Wu, Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide, (2018), https://doi. org/10.1016/j.ijbiomac.2018.07.082
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S. Wu / International Journal of Biological Macromolecules xxx (2018) xxx–xxx
Table 2 Effect of Ganoderma lucidum polysaccharide (GLP) on body weight gain of rats (WG), food intake (FI), food efficiency ratio (FER) of rats. Parameters
WG (g) FI (g) FER (%)
Group
Table 4 Effect of Ganoderma lucidum polysaccharide (GLP) on faecal fat and cholesterol excretion of rats. Parameters
Control
100 mg/kg GLP
300 mg/kg GLP
500 mg/kg GLP
13.6 ± 0.6a 131.4 ± 7.2a 10.4 ± 0.5a
12.1 ± 0.5b 130.8 ± 6.4a 9.3 ± 0.4b
10.8 ± 0.4c 129.6 ± 6.1a 8.3 ± 0.4c
10.7 ± 0.3c 129.3 ± 5.9a 8.3 ± 0.3c
Group
Faecal fat
Values are expressed as mean ± SD (n = 3). Different superscript letters in a row indicate significant difference (p b 0.05).
Cholesterol excretion
Control
100 mg/kg GLP
300 mg/kg GLP
500 mg/kg GLP
11.86 ± 0.59a 17.72 ± 0.81a
13.37 ± 0.62b 16.41 ± 0.74c
21.26 ± 0.86d
19.28 ± 0.86b 22.46 ± 0.85c
24.81 ± 0.91d
Values are expressed as mean ± SD (n = 3). Different superscript letters in a row indicate significant difference (p b 0.05).
of 3.72:1. The results obtained in this study were inconsistent with those reported by He et al. [14]. Differences between the monosaccharide compositions of GLP can be ascribed to the species of G. lucidum tested and the extraction methods used.
Table 5 Effect of Ganoderma lucidum polysaccharide (GLP) on liver weight, TG and TC levels in rats. Parameters
Group
3.2. Body weight gain (WG), food intake (FI) and food efficiency ratio (FER)
Control
100 mg/kg GLP
300 mg/kg GLP
500 mg/kg GLP
3.19 ± 0.24a 1.79 ± 0.19a 2.81 ± 0.22a
3.14 ± 0.22a
2.13 ± 0.16b
2.09 ± 0.15b
1.74 ± 0.16a
0.91 ± 0.14b
0.90 ± 0.12b
2.76 ± 0.19a
1.73 ± 0.13b
1.71 ± 0.11b
Table 2 shows the WG, FI and FER for the four groups after 6 weeks of feeding. Although no significant difference in FI was observed among the four groups (p N 0.05), WG and FER for the GLP group were lower than those of the control group (p b 0.05). This finding indicated that GLP can inhibit the increase in body weight of the rats fed with highfat diets. However, excessive administration of GLP (N300 mg/kg) did not decrease WG and FER further.
Values are expressed as mean ± SD (n = 3). Different superscript letters in a row indicate significant difference (p b 0.05).
3.3. Plasma lipid levels
3.5. Liver weight, TC and TG
The effect of GLP on the plasma lipid levels is shown in Table 3. After 6 weeks of gavage administration of GLP, the plasma TC, TG and LDL-C levels of rats were significantly lower than those of the control group and the HDL-C levels of the GLP group were significantly higher than those of the control group (p b 0.05). Similarly, the polysaccharides produced from submerged mycelial culture of G. lucidum reportedly decrease TC, TG and LDL-C levels and increase HDL-C level in rats [15].
The changes in the values of liver weight, TC and TG of rats fed with different diets are shown in Table 5. Although no differences in the liver weight, TC and TG of rats were observed between the control group and the 100 mg/kg GLP group (p N 0.05), the liver weight, TG and TC levels of the 300 and 500 mg/kg GLP groups were significantly lower than those of the control group (p b 0.05). The results indicated that GLP can effectively reduce the accumulation of lipids in the liver. Similarly, the polysaccharides produced from the submerged mycelial culture of G. lucidum reportedly decrease the liver weight, TC and TG of rats [15].
Liver weight (g) Liver TC (mmol/L) Liver TG (mmol/L)
3.4. Faecal lipid and cholesterol excretion The effect of GLP on the lipid and cholesterol contents in the dried faeces of rats collected for three successive days were determined, and the results are shown in Table 4. The faecal fat and cholesterol contents of all of the GLP groups were significantly higher than those of the control group in a dose-dependent manner (p b 0.05), indicating that GLP possessed considerable fat-binding capacities.
3.6. Serum SOD, GPx and MDA The changes in serum SOD and GPx activities and MDA level of rats fed with different diets are shown in Table 6. After 6 weeks of gavage administration of GLP, the serum SOD and GPx activities of rats were significantly higher than those of the control group and the MDA levels of the GLP group were significantly lower than those of the control group (p b 0.05). This finding can be attributed to the antioxidant activity of GLP [5].
Table 3 Effect of Ganoderma lucidum polysaccharide (GLP) on serum TG, TC, LDL-C and HDL-C levels in rats. Parameters
Group Control
Serum TG (mmol/L) Serum TC (mmol/L) Serum LDL-C (mmol/L) Serum HDL-C (mmol/L)
3.21 ± 0.32a 1.76 ± 0.16a 2.69 ± 0.18a 1.39 ± 0.17a
100 mg/kg GLP
300 mg/kg GLP
2.59 ± 0.26
b
1.43 ± 0.14
b
2.15 ± 0.13b 1.81 ± 0.23b
500 mg/kg GLP
1.73 ± 0.47
c
1.71 ± 0.43
0.79 ± 0.12
c
0.78 ± 0.10c
1.08 ± 0.11c
1.07 ± 0.11c
1.83 ± 0.24b
c
1.86 ± 0.25b
Values are expressed as mean ± SD (n = 3). Different superscript letters in a row indicate significant difference (p b 0.05).
Table 6 Effect of Ganoderma lucidum polysaccharide (GLP) on serum SOD, GPx and l malondialdehyde (MDA) levels in rats. Parameters
Group Control
SOD (U/ml) GPx (U/ml) MDA (ng/ml)
100 mg/kg GLP a
3.15 ± 0.15 21.13 ± 0.83a 10.78 ± 0.43a
b
3.85 ± 0.17 41.63 ± 1.17b 6.71 ± 0.36b
300 mg/kg GLP c
4.74 ± 0.21 50.94 ± 2.18c 4.62 ± 0.27c
500 mg/kg GLP 4.79 ± 0.22c 51.79 ± 2.19c 4.59 ± 0.26c
Values are expressed as mean ± SD (n = 3). Different superscript letters in a row indicate significant difference (p b 0.05).
Please cite this article as: S. Wu, Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide, (2018), https://doi. org/10.1016/j.ijbiomac.2018.07.082
S. Wu / International Journal of Biological Macromolecules xxx (2018) xxx–xxx
4. Conclusions This study verified the hypolipidaemic and lipid antioxidant activities of GLP in rats. GLP administration decreased the WG, FER, levels of TG, TC and LDL-C, liver weight, TC and TG levels and MDA values and increased the levels of faecal fat, cholesterol and HDL-C and serum SOD and GPx activities of rats relative to the control group. However, the mechanism of the hypolipidaemic and lipid antioxidant in the appropriate micro-ELISA strip plate wells, the activities of GLP needs further investigation. Acknowledgments This research was supported by A Project Funded by the Priority Academic Program Development （PAPD） of Jiangsu Higher Education Institutions. Conflict of interest statement The authors have declared that no competing interests exist. References [1] Z.W. Yang, K.H. Ouyang, J. Zhao, H. Chen, L. Xiong, W.J. Wang, Structural characterization and hypolipidemic effect of Cyclocarya paliurus polysaccharide in rat, Int. J. Biol. Macromol. 91 (2016) 1073–1080. [2] L.Y. Zhao, W. Huang, Q.X. Yuan, J. Cheng, Z.C. Huang, L.J. Ouyang, F.H. Zeng, Hypolipidaemic effects and mechanisms of the main component of Opuntia dillenii Haw. polysaccharides in high-fat emulsion-induced hyperlipidaemic rats, Food Chem. 134 (2012) 64–971. [3] L.X. Zhang, Y.J. Zhang, L.P. Zhang, Structure and immunological activity of a novel polysaccharide from the spores of Ganoderma lucidum, Afr. J. Biotechnol. 10 (2011) 10923–10929.
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Please cite this article as: S. Wu, Hypolipidaemic and anti-lipidperoxidant activities of Ganoderma lucidum polysaccharide, (2018), https://doi. org/10.1016/j.ijbiomac.2018.07.082