Insulin resistance (IR) is defined as an impaired biological response to insulin stimulation of target tissues mainly including the liver, muscle, and adipose tissue. Insulin resistance syndrome, also known as metabolic syndrome, is associated with a broad spectrum of diseases such as obesity, hyperglycemia, hypertension, dyslipidemia, non-alcoholic fatty liver disease, cardiovascular disease, polycystic ovarian syndrome, and type 2 diabetes mellitus (Allahbadia and Merchant, 2008; Willard et al., 2016; Cortés-Rojo et al., 2020; García-Carretero et al., 2021). Insulin resistance is mainly related to excess body fat and also genetic causes, which affects as many as one in three Americans and becomes a tremendous burden for the healthcare system of the United States (Balasubramanyam, 2006; McCarthy, 2014; Sharma et al., 2019; Rajesh and Sarkar, 2021). Now, the incidence of insulin resistance is growing at an alarming speed, and it is actually becoming a non-ignorable public concern worldwide. Accumulating evidence revealed that the inactivation of Akt and the activation of Foxo1 via inhibiting insulin receptor substrate 1 (IRS1) and insulin receptor substrate 2 (IRS2) might act as the underlying mechanism of metabolic syndromes (Guo, 2014). Targeting the pivotal molecules such as IRS or Akt or Foxo1 in the signaling cascade will therefore be a precise strategy for the intervention of insulin resistance–related diseases (Guo, 2014).

Breast cancer is still the most common cancer among women (Chowdhury et al., 2021; Loibl et al., 2021). The incidence rate accounts for about 30%, and the mortality rate is as high as 15% (Loibl et al., 2021). Triple-negative breast cancer (TNBC) is one of the subgroups of breast cancer which has an incidence of 15–20% and many characteristics such as invasive, resistant, and rapid growth rate (Chen et al., 2021; Chowdhury et al., 2021). Due to its aggressive behavior, metastasis usually occurs in the liver, lungs, and brain (Chen et al., 2021; Chowdhury et al., 2021). Discovering some potential molecules which could inhibit cell migration of TNBC may provide some enlightenment for its treatment.

Ganoderma fungi, well-known mushrooms of numerous functional foods, have received long-term attention worldwide. For example, 28785 documents are related to Ganoderma when searched using SciFinder till August 29, 2021. The taste and functional components of the Ganoderma fungus lead to its applications as food additives, raw materials, dietary supplements and remedies, even in Western countries (Leskosek-Cukalovic et al., 2010; Veljović et al., 2019; Krobthonga and Yingchutrakul, 2020). Polysaccharides and triterpenoids have long been considered the major chemical compositional elements of Ganoderma, whereas the other chemical composition remains largely unknown. In recent years, a number of meroterpenoids in Ganoderma have been reported (Jiang et al., 2021), representing a new research trend for Ganoderma metabolites. Phenol and terpene moieties are considered biologically active, and Ganoderma meroterpenoids possess both groups (Lu et al., 2007; Tan et al., 2018). We have focused on the investigation of Ganoderma meroterpenoids since 2009. As a result, many structurally intriguing and biologically important meroterpenoids have been characterized by us (Yan et al., 2019; Dai et al., 2020; Qin et al., 2020; Zhang et al., 2020). Due to considerable economic values, Ganoderma species have been cultivated in several places of our country. The concept of “one strain many compounds (OSMAC)” has been widely accepted in the related field. Ganoderma belongs to a higher fungal family; hence, it is necessary to gain an insight into the chemical profiling of Ganoderma meroterpenoids of Ganoderma lucidum produced in different locations, despite that much work has been carried out on this fungal species. G. lucidum, cultivated at Dayao County of Yunnan Province, is locally dictated to have distinct effects on diabetes, which thus inspired our interest. G. lucidum was reported to have antitumor effects, and triterpenoids are generally regarded as its antitumor ingredients. Given the increasing number of Ganoderma meroterpenoids being discovered, it is worth being concerned whether they have a role in tumor treatment. As a consequence, this study afforded five pairs of meroterpenoid enantiomers, dayaolingzhiol I−M (1–5), with biological activities toward insulin resistance and metastasis of TNBC. Herein, we describe their isolation, structure characterization, and biological evaluation.

Dayaolingzhiol I (1) (Figure 1) was isolated as yellowish gums, and its molecular formula was assigned as C₂₁H₂₆O₆ (nine degrees of unsaturation) by the analysis of its HRESIMS, ¹³C NMR, and DEPT spectra. The ¹H NMR spectrum (Table 1) shows a typical ABX spin system [δ _H 7.27 (1H, d, J = 2.9 Hz, H-3), 7.00 (1H, dd, J = 8.9, 2.9 Hz, H-5), and 6.78 (1H, d, J = 8.9 Hz, H-6)], suggesting the presence of a 1,2,4-trisubstituted benzene ring. The ¹³C NMR and DEPT (Table 1) spectra show three methyl, five sp³ methylene, one oxygenated sp³ methine (δ _C 81.1), four sp² methine, eight non-protonated carbons (one ketonic carbonyl at δ _C 203.8, one ester carbonyl at δ _C 173.9, and four oxygenated quaternary carbons). The planar structure of 1 was mainly constructed by the results of 2D NMR experiments. The ¹H–¹H COSY spectrum (Figure 2) shows correlations of H₂-11/H₂-12/H-13 and H₂-16/H₂-17/H-18. The HMBC correlations of H-3, H₂-8/C-7 (δ _C 203.8) indicate that the ketonic carbonyl C-7 is connected with C-2; the HMBC correlations (Figure 2) of H₂-8, Ha-11, H-13 (δ _H 4.55)/C-10 (δ _C 173.9), and H₂-8/C-9 (δ _C 74.4), C-11 (δ _C 27.8) imply that C-9 is connected with C-8, C-10, and C-11 by carbon bonds, while C-10 and C-13 (δ _C 81.1) are connected via oxygen bridges. These data show that the six-membered lactone ring is formed in 1. In addition, the HMBC correlations of H₃-15 (δ _H 1.24)/C-13, C-14 (δ _C 78.6), C-16, and H₂-16 (δ _H 1.53)/C-13 imply that C-14 is connected with C-13, C-14, and C-15 through carbon bonds, H₂-17/C-19 (δ _C 133.0), H₃-20/C-18 (δ _C 124.8), C-19 (δ _C 133.0), C-21, and H₃-21/C-18 indicate that C-18 and C-19 are connected via a double bond, and two terminal methyl are attached to C-19. The ROESY correlations (in DMSO-d ₆) (Figure 2) of 1-OH (δ _H 11.06)/H-6 (δ _H 6.79) and 4-OH (δ _H 9.14)/H-3 (δ _H 7.16), H-5 (δ _H 6.96) indicate the presence of 1-OH and 4-OH groups. One benzene ring, one six-membered lactone ring, one ketone group, and one double bond account for eight degrees of unsaturation, and the remaining one degree of unsaturation, in consideration of the diagnostic chemical shift for C-9 (δ _C 74.4) and C-14 (δ _C 78.6), allows to conclude the presence of an ether bond formed by C-9 and C-14. Therefore, the planar structure of 1 was deduced. The relative configuration of 1 was assigned by the ROESY experiments. The ROESY correlation (Figure 2) of Ha-12/H₂-16 indicates the relative configuration of 1 as 9S*,13R*,14S*. To further confirm this structure, the density functional theory (DFT) at the B3LYP/6–311g(d,p) level was used to calculate chemical shifts of carbon atoms in the deduced structure with different relative configurations as 9S*,13R*,14S* (1–1) and 9R*,13S*,14S* (1–2), as well as the ether linkage broken structures of 1 with the relative configurations as 9R*,13S*,14R* (1–3); 9R*,13S*,14S* (1–4); 9R*,13R*,14R* (1–5); and 9R*,13R*,14S* (1–6) (Supplementary Figure S42). By the analysis of ¹³C NMR calculations, it is shown that the correlation coefficient (R ²) for calculated vs experimental chemical shifts in 1–1 to 1–6 were 0.99928, 0.99927, 0.99570, 0.99694, 0.99771, and 0.99776, respectively (Supplementary Figure S49), and the corrected mean absolute error (CMAE) values were 1.2, 1.2, 2.8, 2.5, 2.2, and 2.1 ppm (Supplementary Table S7–12). These values suggest that structural and stereochemical assignments to these substances are highly dependable. Moreover, the results of DP4+ probability analysis (Supplementary Figure S50) indicated a 100% matching degree between the experimental values and the calculated values of 1–1, which further confirmed the correctness of the deduced structure and its stereochemistry. To clarify the absolute configuration of 1, which is an enantiomeric isomer, ECD calculations at the PBE1PBE/def2SVP level and B3LYP/6–31(d,p) level were carried out. It was found that the ECD spectrum of (9S,13R,14S)-1 matches well with the experimental one of (–)-1 (Figure 3 and Supplementary Figure S51). Similarly, the calculated specific optical rotation for (9S,13R,14S)-1 was –4.14 (Supplementary Table S13), which is closer to the experimental one of (–)-1 ( [ α ] D 25 –6.67), supporting the absolute configuration of (–)-1. Thus, their absolute configurations were determined as 9S,13R,14S for (–)-1 and 9R,13S,14R for (+)-1. Therefore, compound 1 was deduced and named dayaolingzhiol I. Of note, compound 1 is characteristic of the presence of a dioxabicyclo[2.2.2]octan-3-one motif in the terpenoidal chain, which makes it a structurally intriguing compound.

Dayaolingzhiol J (2) was obtained as yellowish gums. It has a molecular formula C₂₂H₃₀O₅ deduced by its HRESIMS, ¹³C NMR, and DEPT spectra. The ¹H NMR spectrum of 2 (Table 1) shows a typical ABX spin system [(δ _H 6.92, d, J = 2.5 Hz, H-3; δ _H 6.72, dd, J = 8.8, 2.5 Hz, H-5; and δ _H 7.18, d, J = 8.8 Hz, H-6)]. The ¹³C NMR and DEPT spectra show four methyl (one oxygenated), four methylene, six methine (five sp² and one sp³), and eight non-protonated carbons (one carbonyl and three oxygenated quaternary carbons). These data are similar to those of ganofuran B (Adams et al., 2010). The only difference is that a methoxy group appears at C-7 in 2 supported by the HMBC correlations (Figure 2) of -OCH ₃ (δ _H 3.90)/C-7 (δ _C 141.1). The significant ROESY correlation of H₂-11 (δ _H 2.00)/H₃-20 (δ _H 1.52) indicates that the Δ ¹²⁽¹³⁾ double bond is E form. Compound 2 was isolated as a racemic mixture, and chiral HPLC was used to afford (+)-2 and (–)-2. In general, it is a great challenge to assign the absolute configuration at C-9 in the side chain. In this case, computational methods such as ECD calculations at the APFD/6–311+g(2d,p) and B3LYP/6–31(d,p) level were carried out (Figure 4 and Supplementary Figure S52). The results reveal that the ECD spectrum of (9S)-2 matches well with the experimental one of (–)-2 . Thus, the absolute configurations of the two enantiomers were assigned as 9R for (+)-2 and 9S for (–)-2, respectively. In this way, the structure of 2 was deduced and named dayaolingzhiol J.

Dayaolingzhiol K (3) has the molecular formula C₂₁H₂₈O₅ (eight degrees of unsaturation) on the basis of its HRESIMS, ¹³C NMR, and DEPT spectra. The ¹H NMR spectrum (Table 2) shows a typical ABX spin system [δ _H 6.46 (1H, d, J = 2.9 Hz, H-3), 6.61 (1H, dd, J = 8.7, 2.9 Hz, H-5), 6.68 (1H, d, J = 8.7 Hz, H-6)]. The ¹³C NMR and DEPT spectra show four methyl, five methylene, five sp² methine, one oxygenated sp³ methine, and seven non-protonated carbons (one ester carbonyl and three oxygenated quaternary carbons). These data resemble those of ganomycin K (Niedermeyer et al., 2013), differing in that the OH-18 in ganomycin K is reduced in compound 3. This alteration is supported by the ¹H–¹H COSY correlation of H₂-16/H₂-17/H₂-18 and the HMBC correlations of H₂-16 (δ _H 1.96)/C-18 (δ _C 44.3) and H₃-21 (δ _H 1.15)/C-18 (δ _C 44.3). Moreover, the significant ROESY correlations of H₂-12 (δ _H 2.30)/H₃-15 (δ _H 1.58) and H-13 (δ _H 5.14)/H₂-16 (δ _H 1.96) indicate that the Δ ¹³⁽¹⁴⁾ double bond is E form. Compound 3 was isolated as a racemic mixture, and chiral HPLC was used to afford (+)-3 and (–)-3. By carefully comparing the CD spectra of (+)-3 and (–)-3 with those of (+)- and (–)-zizhine A (Cao et al., 2016), the absolute configurations of the two enantiomers were assigned as R for (+)-3 and S for (–)-3, respectively. Thus, the structure of 3 was deduced and named dayaolingzhiol K.

Dayaolingzhiol L (4) was obtained as yellowish gums, and its molecular formula was assigned as C₂₂H₃₀O₅ by analysis of its HRESIMS, ¹³C NMR, and DEPT spectra. The ¹H NMR spectrum of 4 (Table 2) shows a typical ABX spin system (δ _H 7.25, d, J = 2.9 Hz, H-3; δ _H 7.01, dd, J = 8.9, 2.9 Hz, H-5; δ _H 6.79, d, J = 8.9 Hz, H-6), suggesting the presence of a 1,2,4-trisubstituted benzene ring. The ¹³C NMR and DEPT spectra show four methyl (one oxygenated), five methylene, six methine (five sp² and one sp³), and seven non-protonated carbons (one ketone and one carbonyl). These data are similar to those of fornicin C (Niu et al., 2006). The only difference is a methyl ester in 4, instead of the free carboxyl group in fornicin C, supported by the HMBC correlations (Figure 2) of -OCH ₃ (δ _H 3.68)/C-7 (δ _C 177.7). The ROESY correlation of H₂-12 (δ _H 2.08)/H₃-15 (δ _H 1.61) indicates that the Δ ¹³⁽¹⁴⁾ double bond is E form. Compound 4 is also a racemic mixture, and the absolute configurations of the two enantiomers after chiral separation by HPLC were assigned as R for (+)-4 and S for (–)-4, respectively, by comparison of their CD spectra with those of (+)− and (–)−applanatumol S (Luo et al., 2016). The structure of 4 was thus determined and named dayaolingzhiol L.

Dayaolingzhiol M (5) was isolated as yellow gums, and it bears the same carbon skeleton with that of 4 by careful analysis of their NMR spectra. The ¹H NMR spectrum of 5 (Table 3) shows a typical ABX spin system (δ _H 7.22, d, J = 2.9 Hz, H-3; δ _H 7.01, dd, J = 8.9, 2.9 Hz, H-5; δ _H 6.79, d, J = 8.9 Hz, H-6). The ¹³C NMR and DEPT spectra show three methyl, five methylene, five sp² methine, and eight non-protonated carbons (one ketone, one carbonyl, and three oxygenated quaternary carbons). These data also resemble those of fornicin C (Niu et al., 2006). Differently, a hydroxyl is attached to C-9 (δ _C 75.8) in 5, gaining support from the HMBC correlations of H₂-8 and H₂-11/C-9. The ROESY correlation of H₂-12 (δ _H 2.23)/H₃-15 (δ _H 1.62) indicates that the Δ ¹³⁽¹⁴⁾ double bond is E form. Compound 5 is also a racemate, which was further purified by chiral HPLC to afford (+)-5 and (–)-5. To clarify their absolute configurations, ECD calculation was used at the PBE1PBE/def2SVP level and B3LYP/6–31(d,p) level (Figure 5 and Supplementary Figure S53). It was found that the ECD spectrum of (9S)- 5 matches well with the experimental one of (+)-5 (Figure 5); thus, the absolute configurations of the two enantiomers were assigned as 9S for (+)-5 and 9R for (–)-5, respectively. Therefore, the structure of 5, named dayaolingzhiol M, was deduced.

In this experiment, we obtained five long-chain meroterpenoids dayaolingzhiols I−M (1–5). Among them, dayaolingzhiol I (1) is a relatively rare meroterpenoid with dioxabicyclo[2.2.2]octan-3-one motif of G. lucidum. At the same time, dayaolingzhiol J (2) belongs to the benzofuran type of meroterpenoid, and few benzofuran type of meroterpenoids have been reported from Ganoderma so far (Adams et al., 2010). These two pairs of meroterpenoids together with the remaining three pairs of new meroterpenoids reveal the structural diversity of Ganoderma meroterpenoids.

Ganoderma lucidum cultivated at Dayao County of Yunnan Province is locally used as a health-care product with a pronounced effect on diabetes. To explore the protective effect of Ganoderma meroterpenoids against IR, insulin exposed C2C12 myotubes were used. C2C12 cells were pretreated with different compounds for 24 h, and cell viability was not decreased at 20 μM. The CCK-8 assay showed no obvious cytotoxicity of compounds, except (+)-4 and (–)-4 (Supplementary Figure S54). When the cells differentiated into myotubes, they were pretreated with 100 nM insulin for 24 h to mimic the IR model and then exposed to 20 μM compounds for an additional 24 h. In the cellular model of IR, insulin-stimulated glucose uptake and the expression of phospho-adenosine monophosphate–activated protein kinase (p-AMPK) and phospho-AKT (p-AKT) were lower than those in control cells. The results showed that incubation with (+)-1, (–)-4, and (–)-5 caused an increase in the phosphorylation of AMPK in IR C2C12 cells after insulin stimulation. At the same time, compound (+)-5 treatment caused an increase in protein expression of p-AMPK and p-AKT in IR C2C12 cells (Figure 6A−C). Further glucose uptake experiments showed that compound (+)-5 improved the capacity of glucose uptake in IR cells in a concentration-dependent manner (Figure 6D). So far, some meroterpenoids were found to have beneficial effects to insulin sensitivity in PA-induced C2C12 cells, such as ganomycin C and ganodercin D (Qin et al., 2021). Ganomycin I exhibited potent insulin-sensitizing effects in KK-A^y mice, and its analog (R,E)-5-(4-(tert-butyl)phenyl)-3-(4,8-dimethylnona-3,7-dien-1-yl)furan-2(5H)-one ameliorates insulin resistance (Wang et al., 2017; Wang et al., 2018). These meroterpenoids with long side chains showed advantages in the amelioration of insulin resistance.

Our previous study showed that Ganoderma meroterpenoids with long side chains are active toward breast cancer cell migration (Cai et al., 2021). In this article, 10 optically active compounds, all possessing a long side chain, were tested for their suppressive activity in the triple-negative breast cancer cell line (MDA-MB-231) by using cell viability and cell migration assays. First, the cell viability assay was carried out in the MDA-MB-231 cell line. As shown in Figure 7A, compounds demonstrated weak effect on cell viability at the concentration of 20 μM. Wound healing assay was investigated in the MDA-MB-231 cell line at 20 μM. The results showed that compounds (+)-1, (+)-4, and (–)-4 exhibit a migration inhibitory effect compared to the DMSO group in the MDA-MB-231 cell line, of which (+)-4 showed stronger inhibitory effect (Figures 7B, D). Furthermore, the effect of compound (+)-4 was found to be dose-dependent at concentrations of 10 μM, 20 μM, and 30 μM (Figures 7C,E).

To conclude, triterpenoids and polysaccharides as chemical compositions of Ganoderma mushrooms have been traditional gnosia for decades. Our present study resulted in the isolation of five pairs of meroterpenoidal enantiomers, representing new chemical composition of G. lucidum and aiding an in-depth insight into Ganoderma metabolites. The activation of (+)-5 on Akt, AMPK, and glucose uptake in C2C12 cells indicates its role in diabetes prevention. In addition, the inhibitory effects of compounds (+)-1, (+)-4, and (–)-4 on cell mobility in MDA-MB-231 cells indicate their role in TNBC. These results shed light on the traditional uses of G. lucidum, implying G. lucidum–derived compounds’ potential in insulin-resistant diseases and cancer, and might inspire a consideration of Ganoderma meroterpenoids as dietary supplements.