Anti-α-glucosidase, Anti-proliferative and Anti-enterovirus 71 Activity of Secondary Metabolites Identified from Grifola Frondosa

Grifola frondosa, an edible and medicinal resource, is widely used as functional foods worldwide. To explore bioactive compounds against α-glucosidase, human tumor cells and enterovirus 71 (EV71), eight compounds were isolated from G. frondosa by chromatographic column. Among the isolated compounds, heptadecanoic acid, uridine and adenosine exhibited potent inhibition activity against α-glucosidase, ergosterols and ergosterol-5,8-peroxide showed anti-proliferative activity on tumor cells, while ergosterol and methyl linoleate displayed inhibition against the replication of EV71. Also, to our knowledge, this is the first study to report that fatty acids isolated from G. frondosa show potent inhibition against α-glucosidase and EV71. Further molecular docking results revealed that the active compounds in G. frondosa form hydrogen bonding, hydrophobic interactive and π-stacking with the active sites on the surface of α-glucosidase, CASP3 and VP1 proteins, thus promoting the active compounds to combine with the target protein to form a stable complex, thus playing an antagonistic role. Our results could provide a new active compound and mode of action for G. frondosa to treat diabetes, cancer and EV71-infected patients.


Introduction
Grifola frondosa belongs to the family of Polyporaceae, also known as the king of mushrooms, and is popularly consumed as traditional medicines and healthy foods in China, Japan, Europe and North America.Numerous studies have found that G. frondosa has many pharmacological properties including immunomodulatory [1], antitumor [2], antiviral [3] and anti-diabetic activities [4].Previously, our group have launched a series of studies on polysaccharides from G. frondosa with hypoglycemic and antiviral effects.We have reported that polysaccharide components GFP1 from mycelia of G. frondosa could block EV71 viral replication and show apoptotic activity by suppressing EV71-induced caspase-3 cleavage and IκBα down-regulation [5].We also found that polysaccharides GFP-W from G. frondosa water extract exerted hypoglycemic effect through improving insulin resistance and promoting glucose consumption by IRS/PI3K signal pathway [6].Moreover, the novel polysaccharide GFP-A extracted from G. frondosa can increase the cytotoxic activity of CD4+ and CD8+ T cells and has significant anti-tumor activity.The secondary metabolites

EV71
enterovirus 71 HFMD hand-foot-and-mouth disease FBS fetal bovine serum DMEM dulbecco's modified eagle's medium OCFA odd-chain fatty acids TI therapeutic index isolated from G. frondosa was also reported to show different pharmacological effects.For example, pyrrole alkaloids showed potent inhibition against α-glucosidase and sterols exhibit promising antineoplastic potential [7].However, the information published to date about the antiviral, antitumor and hypoglycemic effects of the secondary metabolites from G. frondosa were individually evaluated and reported based on secondary metabolites of different origins or in different studies.Regarding the fact that those biological effects possibly could occur simultaneously in our body, once secondary metabolites from G. frondosa were consumed, it is necessary to conduct a comprehensive comparison of various pharmacological effects of the same compound from G. frondosa.
Cancer is a heterogeneous disease involving a complex set of signs and symptoms whereas inflammatory and infectious diseases caused by viruses play a significant role.Numerous studies indicate that inflammatory factors have the capacity to induce cell proliferation, and promote cell survival through the activation of oncogene-suppressor or inactivation of tumor-suppressor genes, thus resulting in genetic instability with increased cancer risk.Enterovirus 71 (EV71) is a major pathogenesis of hand foot and mouth disease (HFMD).After infection, it could induce immune cells to produce a large number of inflammatory factors, such as IL-6, IL-12 and TNF-α, which can lead to pathological changes in various cells and serious complications in the body [8].Additionally, as part of the metabolic syndrome, diabetes is believed to share many risk factors such as hyperinsulinemia, hyperglycemia, and chronic inflammation with a variety of cancers.The increased prevalence of non-alcoholic fatty liver disease in patients with diabetes will in turn lead to the development of cirrhosis and hepatocellular carcinoma [9].Therefore, the prevention and therapy of cancer, diabetes and HFMD has become a major health care focus.One possible approach is to explore secondary metabolite resources in natural fungi, due to their advantages of diverse chemical structures, high specificity and limited or nonexistent side effects.
In order to enrich the material basis of G. frondosa with hypoglycemic, antitumor and antiviral effects, we isolated and identified the compounds from G. frondosa, and analyzed their anti-α-glucosidase, anti-proliferative and anti-EV71 activities.Molecular docking was used to study the binding activity and detailed mode of action between active compounds and their receptors.The results of this study could provide a theoretical basis for the application of secondary metabolites from G. frondosa in functional food and medicine industry.

Materials and Methods
The material and methods section are presented as ESM_1.

Structure Identification of the Purified Compounds
Eight compounds (Fig. 1) including two ergosterol derivatives (1, 5), three fatty acids (2, 3, 8), one vitamin compound (4) and two nucleoside (6, 7), were isolated and identified from G. frondosa.Their chemical structures were deduced on the basis of spectroscopic analysis and comparison with the previously reported spectral data.Notably, three fatty acid compounds (2, 3, 8) were individually obtained from the G. frondosa with high purity for the first time to our knowledge.(ESM_2)

Anti-α-glucosidase Activity
High blood glucose level is the main feature of diabetic patients.Persistent hyperglycemia can cause many serious complications and endanger health.Controlling and lowering the blood glucose level has been regarded as the only way to treat diabetes.α-glucosidase promotes the conversion of carbohydrate into glucose, which leads to the increase of blood glucose level.Therefore, inhibiting the activity of α-glucosidase is an effective measure to treat diabetes [10].
The inhibitory activity of eight isolated compounds from G. frondosa against α-glucosidase is shown in Fig. 2. The IC 50 measuring results showed that the inhibitory activities of three compounds, adenosine, heptadecanoic acid and uridine against α-glucosidase were stronger (p < 0.05) than the positive control, acarbose.As for ergosterol (compound 1) and ergosterol-5,8-peroxide (compound 5), no obvious inhibition against α-glucosidase was found, which was consistent with previous report [11].Therefore, the results suggested that the bioactive material basis of the non-polar extract of G. frondosa, as for anti-α-glucosidase activity, would be fatty acid and nucleoside rather than steroids.
Compared with even-chain fatty acids, odd-chain fatty acids (OCFA) have positive promoting effects on human health, including antibacterial, anticancer, anti-inflammatory and antioxidant effects [12].OCFA can be metabolized into propionyl-CoA and then synthesized succinyl-CoA to participate in the TCA cycle, which may be related to the significant inhibitory effect of heptadecanoic acid on α-glucosidase.To our knowledge, this is the first study to report the potential hypoglycemic activity of heptadecanoic acid.Nucleosides can directly involve in synthesis and metabolism of nucleic acid in organisms, and also affect the metabolism regulation as the derivatives.They are often used as drugs to treat diseases related to central nervous system, urinary system and cardiovascular system [13].In this work, we found that nucleoside compounds (adenosine and uridine) may have anti-diabetic activity.Future work could be conducted focusing on the hypoglycemic activity of adenosine in vivo, as well as their action mechanisms.

Anti-proliferative Activity
In vitro, cytotoxicity test can be used as an economic and efficient method for screening anti-tumor drugs.The results of anti-proliferation activities of the compounds isolated from G. frondosa on SW480, HepG2 and A549 cells are shown in Table 1.Among the compounds examined, ergosterol and ergosterol-5,8-peroxide showed high anti-proliferation effect on three tumor cells, adenosine has weak inhibitory effects on cancer cells, other compounds have no anti-proliferation effect on cancer cells.Compared with the polysaccharide and glycoprotein in G. frondosa which have been found to inhibit the growth tumor cells, the results of this study showed that the secondary metabolites It was observed that except nicotinamide and uridine, the cell viability of the treatment groups were significantly higher (p < 0.05) than that of the EV71 virus-infection group (Fig. 3).Compared with the virus-infection group, the cell viability of ergosterol and methyl linoleate increased (p < 0.05) to 90% at low concentration (≤ 100 µg/mL), which indicated that the inhibitory effect of these two compounds on EV71 was stronger than that of other compounds.The inhibitory effect of methyl linoleate and ergosterol on EV71 was stronger than that of ribavirin based on IC 50 results (Table 2).The therapeutic index (TI = TC 50 /IC 50 ) also showed that methyl linoleate and ergosterol were stronger than ribavirin.Especially, the TC 50 values of all examined compounds were found to be higher than ribavirin, indicating lower cytotoxicity and higher safety.Thus, the results suggested that methyl linoleate and ergosterol have higher safety, stronger anti-EV71 effect and higher development value compared with ribavirin.
Ergosterol inhibits pro-inflammatory cytokines and activation of IκBα and NF-κB p65, and upregulates the expression of IFN-I, thus enhancing the immune regulation of the body, which may explain that ergosterol in our results has significant inhibitory effects on tumor cells and EV71 [19].Further work could be done to explore more links between ergosterol's anti-tumor and anti-EV71 effects.The EV71 virus shell consists of four coat proteins (VP1, VP2, VP3 and VP4), of which VP1 is the most important antigenic determinant [20].Therefore, the binding form between ergosterol and VP1 protein can be studied to reveal the anti-EV71 mechanism of ergosterol.

Molecular Modeling Docking
The binding mode and binding activity between bioactive compounds from G. frondosa and receptor proteins were analyzed by molecular docking, so as to predict whether the compounds have the potential to become candidate drugs.The value of binding affinity less than − 5 kcal/mol indicates that the compound has good binding activity with the in G. frondosa, such as ergosterol and adenosine, also have significant anti-proliferation effects on cancer cells, which increases the material basis of anti-tumor effects in G. frondosa.
Previous studies have shown that the accumulation of p53, the attenuation of ROS production, and the activation of caspase may all contribute to the cell protection of nucleoside compounds [14].Our research results showed that adenosine, which has a significant inhibitory effect on α-glucosidase, also has a slight inhibitory effect on tumor cells.These results indicate a connection between the compounds in G. frondosa and their anti-tumor and hypoglycemic effects.Further study could be conducted to provide more evidence to explain this specificity.Compared with ergosterol, peroxides on ergosterol-5,8-peroxide C-5 and C-8 can increase their antioxidant capacity, which may be the reason for their strong anti-proliferation activity.However, how ergosterol-5,8-peroxide can effectively inhibit the CASP3 protein of tumor cells needs further research [15].

Inhibition Effect of the Isolated Compounds on EV71-infected Vero Cells
In 2008, a major outbreak of HFMD occurred in Mainland China, and EV71 was the main pathogenesis causing this outbreak of the epidemic [16].Children under 5 years of age have been found to be particularly vulnerable to EV71 infection.The most effective way to prevent EV71 infection is to vaccinate children with EV71 vaccine [17].However, the immune efficacy and duration of the vaccine still need to be further verified.Since the research on anti-EV71 drug started late, the clinical effective and specific drugs is very limited.Ribavirin is widely used to treat viral infections, but its long-term use will lower leukocyte levels, resulting in bone marrow suppression and other adverse reactions [18].Therefore, screening highly effective and low-toxic antiviral drugs from natural products can provide ideas for new anti-EV71 drug development and HFMD treatment.

Conclusions
In this study, eight compounds were isolated from the G. frondosa, which included three compounds isolated from the G. frondosa for the first time.Among these eight bioactive compounds of G. frondosa, adenosine, uridine and heptadecanoic acid showed stronger inhibiting effect on α-glucosidase than acarbose, ergosterol and ergosterol-5,8-peroxide displayed high anti-proliferation effect on three tumor cells, the inhibitory effect of methyl linoleate receptor protein [21].As shown in Table 3, the results of our docking experiments suggested that adenosine has good binding activity with α-glucosidase, ergosterol-5,8-peroxide has high binding ability with CASP3, while ergosterol also could bind well to VP1. (ESM_2)  and ergosterol on EV71 was stronger than that of ribavirin.Molecular docking further verified the binding activity between compounds (adenosine, ergosterol-5,8-peroxide and ergosterol) and receptor proteins, suggesting that adenosine, ergosterol-5,8-peroxide and ergosterol have the potential to become natural hypoglycemic, antitumor and antiviral drugs.The results not only expand the bioactive compounds in G. frondosa used to treat patients with diabetes, cancer and EV71 infection, but also provide a good prerequisite for the differences and connections among the hypoglycemic, antiviral, and antitumor effects of bioactive compounds.