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 isolated from the G. frondosa 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[12].
The inhibitory activity of eight isolated compounds from G. frondosa against α-glucosidase is shown in Table 1. The IC50 measuring results showed that the inhibitory activities of three compounds, adenosine, heptadecanoic acid and uridine against α-glucosidase were stronger (p < 0.01) than the 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 anti-α-glucosidase 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[13]. OCFA can be metabolized into propionyl-CoA and then synthesized succinyl-CoA to participate in the TCA cycle[14], 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 involved 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[15]. 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 2. 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 of tumor cells, the results of this study show that the secondary metabolites 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.
Adenosine is an endogenous nucleoside distributed all over human cells, and it is a high-level metabolite in tumor environment. Adenosine A2A receptor is widely expressed in immune cells with high affinity, and tumor proliferation is closely related to human immune function[16], so blocking adenosine pathway may become an important part of cancer treatment. 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[17].
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[18]. 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[19]. 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[20]. Therefore, screening highly effective and low-toxic antiviral drugs from natural products can provide ideas for new anti-EV71 drug development and HFMD treatment.
It was observed that except nicotinamide and uridine, the cell viability of the treatment groups were significantly higher (p < 0.05 or p < 0.01) than that of the EV71 virus-infection group (Fig. 2). Compared with the virus-infection group, the cell viability of ergosterol and methyl linoleate increased (p < 0.01) 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 IC50 results (Table 3). The therapeutic index (TI) also showed that methyl linoleate and ergosterol were stronger than ribavirin. Especially, the TC50 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.
The active components in G. frondosa can regulate the immune function of the body by regulating the expression of immune-related factors, thus achieving the purpose of anti-virus. Methyl linoleate can improve immune function by lowering the levels of cytokines IL-1β and TNF-α[21], which may be related to its anti-EV71 effect. Ergosterol has been reported to enhance the immune activity of the body, but the study of ergosterol anti-EV71 has not been reported. The EV71 virus shell consists of four coat proteins (VP1, VP2, VP3 and VP4), of which VP1 is the most important antigenic determinant[22]. 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 receptor protein[23]. As shown in Table 4, 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)