Six new polyphenolic metabolites isolated from the Suillus granulatus and their cytotoxicity against HepG2 cells

Edible mushrooms are an important source of nutraceuticals and for the discovery of bioactive metabolites as pharmaceuticals. In this work, six new polyphenolic metabolites suillusol A-D (1–4), suillusinoic acid (5), ethyl suillusinoate (6), were isolated from the Suillus granulatus. The structures of new compounds were elucidated using high-resolution electrospray ionization mass spectroscopy, nuclear magnetic resonance data, and single-crystal X-ray diffraction analysis. As far as we know, compound 1 represents an unprecedented type of natural product and compound 3 represents a new type of polyphenol fungal pigment, which may be biosynthetically related to thelephoric acid. The cytotoxicity against HepG2 cells of the new compounds were also evaluated. Compound 2 demonstrate significant inhibitory activity against HepG2 cells with IC50 values of 10.85 μM, surpassing that of positive control cisplatin. Moreover, compound 1 and 3 also exhibited moderate cytotoxic activity with their IC50 values measured at 35.60 and 32.62 μM, respectively. Our results indicate that S. granulatus is a rich source of chemical constituents that may provide new lead compounds for the development of anticancer agents.


Introduction
The genus Suillus, a type of ectomycorrhizal fungi known for its high host specificity and commonly found in symbiosis with trees such as pines and firs, is classified within the Boletales order of the Basidiomycetes class (1).In some regions of China, it is commonly referred to as "pine mushrooms."The Suillus genus has attracted growing research interest due to its nutritional value, capacity to produce biologically active secondary metabolites, and potential applications.Since the 1960s, studies have been initiated on the extracts, chemical constituents, and biological activities of this genus (2,3).Studies have shown that the extracts derived from Suillus genus exhibit notable antioxidant, antineoplastic, antimicrobial properties and others (4)(5)(6)(7)(8)(9)(10)(11)(12)(13).This implies that they integrate culinary and medicinal properties, being a valuable forest resource with significant economic and ecological benefits (14).
Suillus granulatus, a species of edible fungus from the genus Suillus, is widely distributed around the world (15).Current investigations into the chemical composition of S. granulates have been primarily confined to the analysis of polyprenylphenols and fatty acids, with the notable absence of studies on the complex phenolic metabolites (16)(17)(18)(19)(20).
Liver cancer is a malignant tumor with a global distribution, and it is estimated that mortality rate attributed to liver cancer will surpass one million by the year 2030 (21-23).Chemotherapy is a pivotal method in the treatment of liver cancer, especially for patients who are not suitable candidates for surgical removal of the tumor.Nevertheless, given the protracted nature of such regimens, associated adverse reactions, and the propensity for chemoresistance development, the therapeutic effectiveness of chemotherapy often falls short of expectations (24,25).Therefore, natural products with a reliable anti-hepatocarcinoma effect that are less toxic and have fewer side effects have received increasing attention.
To identify new natural products with anti-hepatocarcinoma activity, we investigated chemical constituents of S. granulatus and isolated six novel polyphenolic compounds Subsequently, these compounds were evaluated for their cytostatic potential against hepatoma cell lines HepG2.Remarkably, all six compounds exhibited varying degrees of cytotoxic effects, with compound 2 exhibiting notably higher activity compared to the positive control cisplatin.The present study delineates the isolation, structural characterization, and antitumor activity against HepG2 cells of these novel polyphenolic compounds.

General experimental procedure
HR-ESI-MS spectra were acquired on a Waters Xevo G2 Q-TOF mass spectrometer (Waters Co., Milford, MA, USA).NMR spectra were recorded on n a Bruker AM-600 spectrometer with TMS as an internal standard (Bruker, Ettlingen, German).NMR spectra were recorded at 25 • C on a Bruker AM-600 spectrometer equipped with a cryoprobe, and deuterated solvents signal was used as an internal standard.The column chromatography (CC) was performed on YMC RP-18 gel (Fuji Silysia Chemical Ltd., Kasugai, Japan) and silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China).High-performance liquid chro-matography (HPLC) was performed on Waters 2,535 chromatography system (Waters, Milford, MA, USA) equipped with a Waters 2,489 UV/visible detector with a YMC-Pack ODS-A (250 × 10 mm, 5 µm) column (YMC Co., Ltd., Kyoto, Japan).

Mushroom material
The fresh fruiting bodies of S. granulatus were collected from the pine forests in Maojinba National Forest Park in Longhua Country, Chengde City, Hebei Province, China, in September 2022.It was morphologically identified by Prof. Li-an Wang (Hebei Normal University, China), and then verified by molecular analysis.
The n-butanol fraction (54 g) was separated by ODS reversedphase silica gel CC eluting with a methanol/water (1:9-8:2) gradient system, resulting in the isolation of six fractions designated as BA1-BA6.Compound 3 was separated as crystals from the BA6 fraction.

Cytotoxic activity
Cell viability assay was determined by the Cell Counting Kit-8 Assay Kit (CCK8, Bioss, China).The experiment was determined based on the method of Liu et al. (26).HepG2 cells (100 µL, 5 × 10 4 /mL) were seeded onto a 96-well plate overnight.The     3) in DMSO-d 6 of 3 exhibited four phenolic hydroxy signals at δ H 10.06 (1H, brs, 2-OH), 9.82 (1H, brs, 9-OH), 9.67 (1H, brs, 4′-OH), and 9.48 (1H, brs, 3′-OH), two sets of characteristic 1,3,4-trisubstituted benzene ring signals at δ H 8.75 (1H, d, J = 2.9 Hz, H-10), 7.32 (1H, d, J = 8.9 Hz, H-7), 7.01 (1H, dd, J = 8.9, 2.9 Hz, H-8), 7.72 (1H, d, J = 2.2 Hz, H-2′), 7.61 (1H, dd, J = 8.5, 2.2 Hz, H-6′), and 6.93 (1H, d, J = 8.5 Hz, H-5′).The 13 C NMR spectrum (Table 3) showed a total of 18 carbon resonances, and all were in the range of 111.6-172.0ppm, including six aromatic methine carbons and 12 quaternary ones.The aforementioned NMR features suggested that 3 should be a polyphenol containing two benzene rings.A detailed analysis of the HMBC correlations (Figure 2) allowed to establish the specific substitutions of two benzene ring moieties.However, the four carbon signals at δ C 172.0, 154.7, 142.6, and 142.5 were still unsolved, since no correlations were observed in the HMBC spectrum.Thereupon we had to resort to using single crystal X-ray diffraction method.Fortunately, its single crystals were eventually obtained through multiple attempts, and the structure was conclusively determined to be 3-(3,4-dihydroxyphenyl)-2,9dihydroxypyrano[2,3-c]chromene-1,5-dione by single crystal X-ray crystallographic analysis (Figure 3).Thus, the structure of 3 was established as shown in Figure 1 and named as suillusol C. As far as  4) in DMSO-d 6 of 4 showed a group of p-substituted benzene ring signals at δ H 7.92 (2H, d, J = 8.7 Hz, H-2′/6′) and 6.89 (2H, d, J = 8.7 Hz, H-3′/5′), as well as three aromatic or olefinic proton singlets at δ H 7.22 (1H, s, H-9), 7.03 (1H, s, H-5), and 7.00 (1H, s, H-8), which hinted the existence of three phenolic hydroxy protons.The 13 C NMR spectrum (Table 4) showed a total of 17 carbon resonances, including a conjugated lactone carbonyl carbon at δ C 166.6 (s, C-2), as well as 16 aromatic or olefinic carbons assignable to two benzene rings and two double bond groups.The analysis of the degrees of unsaturation hinted the existence of two additional rings.An analysis of the HMBC correlations (Figure 2) revealed the presence of a 4-hydroxyphenyl moiety, which was located at an up-field sp carbon at δ C 94.3 (s, C-3).According to the peak shape of the remaining proton signals, the other benzene ring was inferred as 1,2,4,5-tetrasubstituted and located on a chromene nucleus, which was supported by the observable HMBC correlations from H-5 [δ   7 (d,  C-8b).The analysis of the degrees of unsaturation was indicative of the presence of two aliphatic rings in 5.The aforementioned NMR features were very similar to those of suillusin, a unique cyclopenta[b]benzofuran derivative isolated from the same genus (19).Comparison of the NMR data of 5 with those of suillusin revealed that the signal differences were mainly from the carboxylic acid group at C-3a.The absence of the methoxy signals and the obvious downfield shift of the carboxylic carbon indicated that the methoxycarbonyl group was replaced by a carboxylic one in 5.The resulting structure was further verified by the careful HMBC analysis (Figure 2).While the relative configuration of two chiral centers was deduced to be the same as that of suillusin by comparison of their NMR data.Therefore, the structure of 5 was established as shown in Figure 1 and named as suillusinoic acid.
Compound 6, light yellow powder, possessed a molecular formula of C 20 H 16 O 8 by the negative HR-ESI-MS (m/z 383.0762 [M-H] − , calcd.383.0772).The 1 H and 13 C NMR spectra (Table 5) in CD 3 OD of 6 were very similar to those of suillusinoic acid (5).Comparison of the NMR data of 6 with those of 5 revealed that the signal differences were also from the carboxylic acid moiety at C-3a.The appearance of the ethoxy signals and the obvious upfield shift of the carboxylic carbon indicated that the carboxylic group was ethylated in 6, which was confirmed by the observable HMBC correlations from H 2 -1′' [δ H 4.16-4.22(2H, m)] and H-8b [δ H 4.12 (1H, brs)] to the ester carbonyl carbon [δ C 172.9 (s, C-9)].Similarly, the relative configuration was the same as that of suillusinoic acid by comparison of their NMR data.Thus, the structure of 6 was established as shown in Figure 1 and named as ethyl suillusinoate.

Cytotoxicity against HepG cells
We tested the antitumor activity against HepG2 human liver carcinoma cells of the isolated compounds 1-6.The results shown in Figure 4 indicated that compound 2 exhibit higher efficacy than cisplatin, a commonly used anticancer chemotherapeutic agent, and demonstrate exceptional antitumor activity, with the halfmaximal inhibitory concentrations (IC 50 ) measured at 10.85 µM.Additionally, compounds 1 and 3 also exhibit moderate antitumor activity, with IC 50 values of 35.60 and 32.62 µM.Compounds 5 and 6 exhibited the lowest inhibitory activity, with their IC 50 values showing no significant difference due to their structural similarity.But during the experimental process, compounds 5 and 6 displayed color-changing phenomena, suggesting that they may have a more pronounced effect in antioxidant activity.

Conclusion
Six new polyphenolic compounds, named suillusol A-D (1-4), suillusinoic acid (5), ethyl suillusinoate (6), were isolated from the macrofungus S. granulatus.All the isolated compounds exhibited some degree of cytotoxicity against HepG2 cells.Compound 2 showed improved inhibitory activities than positive control cisplatin.Moreover, compounds 1 and 3 also exhibited moderate cytotoxic activity against HepG2 cells.Those results suggested that the polyphenolic compounds isolated from the S. granulatus could be investigated as natural anticancer agent in the pharmaceutical and food industries.

FIGUREX
FIGURE X-ray crystal structure of compound .

TABLE The H
and C NMR data for in CD OD.
TABLE The H and C NMR data for in CD OD.

TABLE The H
and C NMR data for and in CD OD.The 13 C NMR spectrum (Table2) showed a total of 16 carbon resonances, including one carbonyl carbon at δ C 174.4 (s, C-4), 14 aromatic or olefinic carbons due to two benzene rings and a double bond group, as well as one aliphatic methylene carbon at δ C 35.1 (t, C-7′).One carbonyl, two benzene rings, and one double bond occupied 10 degrees of unsaturation, indicative of