Views: 4 Author: DU Yan1 HAN Yi‐Feng1 LU Zhen‐Ming2, 3 XU Guo‐Hua4 XU Zheng‐Hong2, 3 GENG Yan1 Publish Time: 2022-11-03 Origin: 22 May 2018, 37(5): 656‐666 Mycosystema ISSN1672‐6472 CN11‐5180/Q
Edible and medicinal mushrooms have been identified as remarkable therapeutic agents in traditional Chinese medicines and as culinary products all over the world. However, the ingredients responsible for their bioactive effects have not been fully investigated. In this study, we investigated glucose‐ and lipid‐lowering activities of n‐hexane, chloroform, ethyl acetate, and methanol extract of mycelia from six mycelium powder from Cephalosporium sinensis, Mortierella hepiali, Hericium erinaceus, Ganoderma lingzhi, Armillaria mellea and Antrodia cinnamomea. Their activities of lowering glucose and lipid were evaluated via insulin‐induced insulin resistance model and oleic acid (OA)‐induced triglycerides (TGs) deposition in human hepatoma cell line HepG‐2 cells. Of the 24 extract at nontoxic concentrations (<300μg/mL), n‐hexane, chloroform, ethyl acetate, and methanol extract from H. erinaceus and G. lingzhi and n‐hexane extract from C. sinensis dose‐dependently increased the consumption of glucose in HepG‐2 cells; n‐hexane, ethyl acetate, and methanol extract from C. sinensis, n‐hexane, ethyl acetate extract from G. lingzhi and n‐hexane extract from M. hepiali dose‐dependently inhibited OA‐induced TGs production. These results indicated that medicinal mushrooms could be applied as protective agents against glucose and lipid metabolism disorders.
Glucose and lipid metabolism disorders are risk factors for the metabolic syndrome (MS) and commonly found in several major diseases such as Type 2 diabetes mellitus (T2DM), nonalcoholic fatty liver disease (NAFLD), obesity, coronary heart disease, myocardial infarction, and chronic complications (Li et al. 2013). There is a strong association between NAFLD and T2DM. Approximately 70% of T2DM patients have a fatty liver (Park et al. 2013), and more than 90% of obese patients with T2DM have NAFLD (Silaghi et al. 2016). Insulin resistance (IR) is common in both conditions. The symptoms of T2DM are mainly for IR or insufficient insulin secretion (Guariguata et al. 2014). Most NAFLD patients also have hepatic IR, increasing the risk of T2DM (Perry et al. 2014). Liver is the major site of fatty acid synthesis. The formation of lipid droplets may be a protective response of preventing lipotoxicity from fatty acid‐induced oxidative stress in the liver (Choi & Diehl 2008). The storage of TG in the liver is a temporary strategy. If hepatic cells are unable to handle them appropriately through metabolic pathways, the stored triglycerides (TGs) could render the liver more susceptible to nonalcoholic steatohepatitis (NASH) which can ultimately lead to cirrhosis (Neuschwander‐Tetri 2010). The overload lipid could induce insulin resistance (Perry et al. 2014). Protein kinase‐Cε (PKCε) has been found to play a crucial role in mediating lipid‐induced hepatic insulin resistance, which binds and inhibits the activity of the intracellular kinase domain of the insulin receptor (Fruci et al. 2013). Insulin‐stimulated phosphorylation of IRS2, IRS2‐associated PI(3)K activity and phosphorylation of Akt2 were reduced, which impaired insulin activation of glycogen synthesis and increased gluconeogenesis (Heinrich et al. 2017). The first line of treatment for abnormal blood glucose/cholesterol is usually to eat a healthy diet which is low in saturated/trans fats and high in fruits and vegetables, and to increase exercise. But some patients still need medicine to bring down their blood glucose/cholesterol levels as close to normal as possible. However, none of the existing drugs ensures a complete glycemic/fat control without undesirable side‐effects (Suagee et al. 2011; Ou et al. 2016). It is still desirable to find new anti‐diabetic compounds that efficiently restore the glucose and fat levels in the body without significant adverse effects. Medicinal mushrooms have been known as a traditional source of natural bioactive compounds over many centuries and targeted as potential hypoglycemic and anti‐diabetic agents (Dong et al. 2016). Bioactive metabolites including DU Yan et al. /Glucose‐ and lipid‐lowering activities of mycelial extracts from medicinal mushrooms Research paper 658 polysaccharides, proteins, dietary fibers, and many other biomolecules were isolated from medicinal mushrooms (De Silva et al. 2012). Previous studies have indicated a lot of mushrooms species appear to be effective for both the control of blood glucose levels and the modification of the course of diabetic complications (De Mello et al. 2011), in addition, the consumption of mushrooms markedly decreased the lipid levels including as total cholesterol, total triglyceride, and low‐density lipoprotein cholesterol, and increased the level of high‐density lipoprotein cholesterol (Jeong et al. 2010). In this study, we established insulin resistance (IR) model and NAFLD model in vitro to investigate the activity of lowering glucose and/or lipid of 6 species of medicinal mushrooms, namely Cephalosporium sinensis, Mortierella hepiali, Hericium erinaceus, Ganoderma lingzhi, Armillaria mellea and Antrodia cinnamomea, which have a long history of use for disease treatment in folk medicines, especially in eastern countries such as China, India, Japan and Korea (Dai & Yang 2008; Cao et al. 2012; De Silva et al. 2012; Zhou et al. 2015; Dai et al. 2018).
To screen the anti‐diabetic activity of extracts from 6 species of medicinal mushrooms, C. sinensis, M. hepiali, H. erinaceus, G. lingzhi, A. mellea and A. cinnamomea, we established an IR cell model by incubating HepG2 cells with high concentration of insulin. Firstly, we found that there was a dose‐dependent relationship between insulin concentration and cell viability. The non‐toxic concentration range of insulin was determined to be 0.01–5μmol/L (Fig. 1A). Then the HepG2 cells were incubated with different concentrations of insulin for 36h. After incubation, culture medium was replaced with insulin free basal medium for 12h and then the supernatant was collected to detect glucose consumption. The glucose consumption was decreased in a concentration dependent manner (0.01–5μmol/L) (Fig. 1B). To determine the optimal induction duration of insulin, HepG2 cells were treated with 5μmol/L insulin for 24h, 36h and 48h respectively. It was revealed that treating for 36h was the best duration to establish HepG2/IR cells (Fig. 1C). In order to explore the stability of HepG2/IR cells, we treated HepG2 cells with 5μmol/L insulin for 36h, then culture medium was replaced with basal medium which is insulin free for 12h, 24h, 48h and 60h respectively. The supernatant was collected to detect glucose consumption. The result showed that the HepG2/IR cells could keep stable within 48h (Fig. 1D). Taking together, we found that HepG2 cells stimulated by 5μmol/L insulin for 36h were optimal for the IR model. These conditions were used for subsequent experiments.
Fig. 1 Induction of insulin resistance (IR) in HepG‐2 cells by insulin. A: Cell viability was assessed by MTT; B: Glucose consumption was detected by GO Assay Kit; C: HepG2 cells were treated with 5μmol/L insulin for 24h, 36h, 48h, then replaced the media without insulin for 12h to collect the supernatant to detect glucose consumption; D: The continued duration of insulin resistance in HepG‐2 cells (treatment of HepG2 cells with 5μmol/L insulin for 36h, then replaced the media without insulin for 12h, 24h, 48h, 60h to collect the supernatant to determine the glucose consumption). Data represent the mean ± standard deviation from 3 separate experiments. The one‐way ANOVA analysis (PASW Statistics 18.0, USA) was performed to assess data differences among various groups. Means followed by different letters are significantly different (Duncan′s test, P<0.05).
To explore whether the extract from medicinal mushrooms can alleviate non‐alcohol fatty liver, we established a screening model using oleic acid (OA) to induce hepatic steatosis. Firstly, the lipotoxicity of OA on HepG2 cells was determined. During exposure to OA (50–200μmol/L), cell viability did not alter as compared with the control (Fig. 2A). In these concentrations, OA induced TGs deposition significantly in HepG2 cells in a dose‐dependent manner (Fig. 2B). Lipid staining by Oil Red O was used in morphological observations (Fig. 2C) and after dissolving the dye, the quantitative results were obtained (Fig. 2D). The cells in the control group exhibited no red lipid droplets. OA induced the accumulation of lipid droplets in the HepG2 cells in a dose‐dependent manner (Fig. 2C, D). Considering reversibility and efficiency, TGs production stimulated by 100μmol/L OA in HepG2 cells was chosen as the optimal experimental condition for the subsequent investigation.
Glucose‐lowering activity of 6 different medicinal mushrooms The extraction rates of 4 organic (n‐hexane, chloroform, ethyl acetate, methanol) extract of these fungi were shown in preliminary studies (Geng et al. 2014a, 2014b). At first we examined the cytotoxicity of the 24 extract in HepG2 cells and calculated their half maximal inhibitory concentration (IC50) (Table 1). As compared with the normal cells, at the range of 50–300μg/mL, the majority of extract showed no significant effect on cell viability. IC50 values of n‐hexane extract from A. cinnamomea and chloroform extract from A. mellea and A. cinnamomea were less than 300μg/mL. To determine whether the extract of these fungi can play a role in the regulation of insulin resistance, glucose consumption was measured in the absence or presence of different concentration of the extract. As shown in Figure 3, except for M. hepiali, H. erinaceus and the ethyl acetate and methanol extract from C. sinensis (Fig. 3A–C), 10 other extract at nontoxic concentrations significantly increased glucose consumption in a dose‐dependent manner after stimulating by insulin (Fig. 3A, 3D, 3E). The results indicated that n‐hexane extract from C. sinensis, n‐hexane, chloroform, ethyl acetate, and methanol extract from G. lingzhi and the extract from A. cinnamomea increased glucose consumption more effective as compared with other organic extract. The improvement rate of glucose consumption reached a maximum value of approximately 50%.
Fig. 2 Induction of steatosis in HepG2 cells by OA. A, B: Effects of different concentrations of OA on cell viability and the production of TGs in HepG‐2 cells. C, D: OA‐induced steatosis in HepG2 cells determined by ORO staining (200×). Data represent the mean ± standard deviation from 3 separate experiments. The one‐way ANOVA analysis (PASW Statistics 18.0, USA) was performed to assess data differences among various groups. Means followed by different letters are significantly different (Duncan′s test, P<0.05).
Fig. 3 Different solvent extract of mycelia from 6 medicinal mushrooms—Cephalosporium sinensis, Mortierella hepiali, Hericium erinaceus, Ganoderma lingzhi, Armillaria mellea and Antrodia cinnamomea—ameliorate effects of insulin resistance on HepG‐2 cell. A–F: Cells cultured in 96‐well plates were treated with or without 5μmol/L insulin for 36h, then replaced the medium with increasing doses of mushroom extract without insulin for 24h, and the supernatant was used to measure glucose consumption by GO Assay Kit. Data represent the mean ± standard deviation from 3 separate experiments. The one‐way ANOVA analysis (PASW Statistics 18.0, USA) was performed to assess data differences among various groups. Means followed by different letters are significantly different (Duncan′s test, P<0.05). Hex, n‐hexane; Chl, chloroform; EtOAc, ethyl acetate; MeOH, methanol.
4 Lipid‐lowering activity of 6 different medicinal mushrooms To evaluate the inhibitory effect of different extract on OA induced lipid accumulation, HepG2 cells were treated with safe doses of the extract after treatment of OA for 24h. Then, the TG level was analyzed quantitatively. As illustrated in Figure 4, extract from H. erinaceus and A. mellea could not reduce lipid deposition in HepG2 cells (Fig. 4C, 4E). n‐Hexane, ethyl acetate and methanol extract from C. sinensis, n‐hexane extract from M. hepiali, n‐hexane and ethyl acetate extract from G. lingzhi and ethyl acetate extract from A. cinnamomea at nontoxic concentrations significantly attenuated TGs accumulation in a dose‐dependent manner (Fig. 4A, 4B, 4D, 4E). n‐Hexane and chloroform extract from A. cinnamomea could play a role in attenuating TGs accumulation at nontoxic concentrations (less than 100μg/mL), but had greater cytotoxicity at high concentrations (200–300μg/mL) (Fig. 4F). The results indicated that the n‐hexane and ethyl acetate extract from C. sinensis and G. lingzhi, and the n‐hexane, chloroform, and ethyl acetate extract from A. cinnamomea inhibited TGs production more effective as compared with other organic extract.
Fig. 4 Different solvent extract of mycelia from 6 medicinal mushrooms, Cephalosporium sinensis, Mortierella hepiali, Hericium erinaceus, Ganoderma lingzhi, Armillaria mellea and Antrodia cinnamomea, preventing OA‐induced TGs production in HepG‐2 cells. A–F: Cells cultured in 96‐well plates were treated with or without 100μmol/L OA for 24h, then replaced the medium with increasing doses of mushroom extract without OA, TGs deposition. Measurement was carried out after 24h. Data represent the mean ± standard deviation from 3 separate experiments. The one‐way ANOVA analysis (PASW Statistics 18.0, USA) was performed to assess data differences among various groups. Means followed by different letters are significantly different (Duncan′s test, P<0.05). Hex, n‐hexane; Chl, chloroform; EtOAc, ethyl acetate; MeOH, methanol.
DISCUSSIONS In our screening experiment of some promising mushrooms that are potential glucose‐ and lipid‐lowering agents, a glucose consumption in insulin induced HepG2/IR cells bioassay was used for the evaluation of anti‐diabetic activity and a TGs accumulation in OA induced HepG2 cells was used for the evaluation of lipid lowering activity. According to this study, n‐hexane extract from C. sinensis, n‐hexane and ethyl acetate extract from G. lingzhi and n‐hexane, and chloroform and ethyl acetate extract from A. cinnamomea showed activities in reducing lipid level and decreasing deposition of glucose. Paticularly, we found that n‐hexane extract from C. sinensis at nontoxic concentrations (<300μg/mL) has more higher glucose‐ and lipid‐ lowering activities than other extracts and up regulated glucose consumption (56.8%) as compared with HepG‐2/IR cell and down regulated the accumulation of TGs (63.5%) in oleic acid‐induced HepG‐2 cells. Several investigations have been conducted to develop advanced biomaterials and biologically active substances from Cordyceps sinensis (Lee et al. 2015). Modern experimental methods in biochemistry have proved that C. sinensis consists of active constituents such as mannitol, nucleosides, ergosterol, aminophenol and trace elements (Zhou et al. 2009). Fruiting bodies of Cordyceps attenuated diabetes induced weight loss, polydipsia and hyperglycemia, and these improvements suggest that the fruiting body of Cordyceps has potential to be a functional food for diabetes patients. In one randomized trial, 95% of patients treated with 3g/day of C. sinensis showed a decrease in their blood sugar levels, while the control group showed only 54% improvement with treatment by other methods (De Silva et al. 2012). G. lingzhi has an extensive variety of pharmacological activities responsible for health benefits such as antioxidant, anticancer, anti‐inflammatory, and immunomodulatory activities (Teng et al. 2011; Xuan et al. 2015). Moreover, G. lingzhi has been shown to be a rich source of biologically active metabolites, containing many bioactive components, including triterpenoids, polysaccharides, nucleotides, sterols, steroids, peptides and other bioactive ingredients (Wang et al. 2015). G. lingzhi consumption can provide beneficial effects in treating type 2 diabetes mellitus by lowering the serum glucose levels (De Silva et al. 2012). The fruiting body of A. cinnamomea is highly valued folk medicine in Taiwan. It is used as an antidote for diarrhea, abdominal pain, hypertension, itchy skin and liver cancer. Some polysaccharides, steroids, triterpenoids and sesquiterpene lactone have been isolated and characterized from the fruiting body of A. cinnamomea (Phuong et al. 2009). Biologically active metabolites and components derived from medicinal mushrooms have been demonstrated to have controlling effects on metabolic syndrome through the regulation of multi‐pathophysiological pathways including the glucose absorption, inflammation, islet β‐cell damage, insulin release, insulin signaling, AMP‐activated protein kinase (AMPK) signaling, phosphatidylinositol 3‐kinase (PI3K)‐protein kinase B (PKB/AKT) signaling and other signaling pathways (Lo & Wasser 2011; Li et al. 2012). A follow‐up study will be carried out in evaluating the active components/compounds of different extracts of medicinal mushrooms. As there are many complicated signaling pathways and the involvement of a number of systems in regulating glucose‐ and lipid‐metabolism disorders in the human body, the identification of the effect and activity of these metabolites is still uncertain. Studies are needed to explore this un‐tapped resource for the isolation and production of novel glucose‐ and lipid‐ lowering compounds having medicinal and biochemical potential with therapeutic importance.
