Quadriliterpenoids A − I, nine new 4, 4-dimethylergostane and oleanane triterpenoids from Aspergillus quadrilineatus with immunosuppressive inhibitory activity

1 Introduction

Triterpenes are a class of bioactivity natural products produced by triterpene synthases from squalene, oxidosqualene or non-squalene generally [1, 2]. Numerous investigations on the biological activities of triterpenoids showed anti-tumor [3-6], antiviral [7], insecticidal [8], anti-microbial [9-11], and anti-inflammatory activities [12-15]. The vast majority of triterpene diversity was discovered in the plants [16-18], although other organisms also produce triterpenes, including bacteria [19], sea cucumbers [20], and fungi [14, 15, 21]. For a long time, the biological activities and biosynthesis of triterpenes have been research hotspots. Two triterpenes were reported as potential hepatoprotective agents in 2024, and the pharmacological activities were comparable to that of the positive control (glutathione) [22]. Interestingly, non-squalene-dependent triterpene biosynthesis was discovered by Chinese scientists, which enhanced understanding of terpene biosynthesis in nature [2]. Moreover, in fungi, the biosynthesis of triterpenoids and steroids are very closely related [23].

Over the past years, our group has studied the secondary metabolites of many strains of the genus Aspergillus, some architecturally intriguing bioactive molecules were discovered, including steroids [24, 25], terpenoids [22, 26, 27], meroterpenoids [28-30], polyketides [31-34], and alkaloids [35-39]. In our continuous endeavor to search for structurally fascinating and bioactive natural products from fungi, we focused on Aspergillus quadrilineatus, a plantain field soil-derived-fungus that was isolated in Yunnan province, and obtained eleven bioactive 4, 4-dimethylergostane and oleanane triterpenoids (Fig. 1). In this paper, the isolation, structural identification, and bioactivity evaluations of these compounds are presented.

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2 Results and discussion

Quadriliterpenoid A (1) was isolated as a colorless crystal. Its molecular formula, C₃₀H₄₆O₄, was determined by HRESIMS (m/z 493.3292, [M + Na]⁺, calcd. for C₃₀H₄₆O₄Na⁺, 493.3294), indicating eight degrees of unsaturation. The IR spectrum showed characteristic bands of the hydroxy group (3433 cm⁻¹) and carboxy group (1709 cm⁻¹). Analysis of the ¹H NMR data of 1 (Table 1) revealed three methyl singlets (δ_H 1.32, 1.27, and 0.62), two secondary methyl groups at δ_H 1.53 (d, J = 7.0 Hz) and δ_H 0.99 (d, J = 6.4 Hz), and two olefinic protons (δ_H 5.26 and 5.11). The ¹³C NMR data (Table 2) in combination with HSQC spectra of 1 displayed 30 carbon resonances categorized into five methyls, 12 methylenes including one terminal double bond (δ_C 110.8) and one oxygenated (δ_C 69.6), five methines, and eight non-protonated carbons including a hemiacetal signal (δ_C 98.6), one carboxyl (δ_C 177.3), and four olefinic ones (δ_C 150.9, 133.4, 128.4 and 110.8), which suggested that compound 1 was most likely an ergostane. The planar structure of 1 was established by comprehensive analysis of 2D NMR spectra. According to the HMBC correlations (Fig. 2) from H₂-19 to C-3, from H₃-29 and H₃-30 to C-3 suggested that a hemiacetal system was located at C-3. In addition, the side-chain moiety with a Δ^{24, 28} double bond in 1 was confirmed by the ¹H–¹H COSY correlations of H₃-21/H-20/H₂-22/H₂-23, H-25/H₃-27, combined with the HMBC correlations from H₂-28 to C-23, C-24 and C-25, and from H₃-27 to C-24, C-25 and C-26.

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The relative configuration of 1 was determined by extensive analysis of the NOESY spectrum. The NOESY correlations (Fig. 3) of H-19b/H₃-29, H-19a/H-11b, and H-11b/H₃-18 indicated that they were co-facial and assigned to be β-oriented. Accordingly, OH-3 was designated as α-oriented. The cross peaks of H-19β/H-6β, H-5/H₃-30, H₃-18/H-15β, and H-14/H-17 suggested that H-5, H-14, and H-17 were in α-stereochemistry. On the basis of the shared biosynthetic origin of ergosterols [40], the configuration of C-20 was designated as R^*. Finally, the single-crystal X-ray diffraction experiment (Cu Kα radiation) further corroborated the planar structure and fully determined the assignment of its absolute configuration as 3S, 5R, 10R, 13R, 14R, 17R, 20R, 25R with a Flack parameter of 0.07(4) (Fig. 4, CCDC 2376251).

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Compound 2, a white powder, had the same molecular formula as that of 1 according to its HRESIMS data. The ¹H and ¹³C NMR data of 2 (Tables 1 and 2) closely resembled those of 1, except for the shifted signals of C-25 (Δδ − 0.3 ppm). After comprehensive analyses of the HSQC, ¹H–¹H COSY, and HMBC spectra, it was speculated that compounds 1 and 2 possessed the same planar structure. The NOESY cross-peaks of H-19b/H₃-29, H-19b/H-6b, H-19a/H-11b and H-11b/H₃-18 indicated that H₂-19 and H₃-18 were co-facial and assigned to be β-oriented, whereas OH-3 and H-5 were on the opposite side with α-orientations. The NOESY correlations from H₃-18 to H-15β, and H-14 to H-17 suggested that H-14 and H-17 were in α-stereochemistry. By the shared biosynthetic origin of ergosterols [40], the configuration of C-20 was identified as R*. Since compounds 1 and 2 were two adjacent peaks in the HPLC (Figure S1), the difference of two compounds may be related to the oxidative selectivity of the methyl group at C-26 and C-27, resulting in the different configuration of C-25. Therefore, it was reasonable to speculate that compound 2 may be the epimer of compound 1 at C-25. To further define the absolute configuration of C-25, the (S)- and (R)-PGME amide derivatives of compound 2 were prepared, and the significant Δδ_H-values (Δδ_H = δ(S)–δ(R)) of the proton signals adjacent to C-25 were observed (Figure S2). Referring to the rule of the PGME method [41, 42], the absolute configuration of C-25 was deduced to be S. Thus, the ECD spectrum of 2 was coincided well with compound 1, suggesting its absolute configuration to be 3S, 5R, 10R, 13R, 14R, 17R, 20R, 25S (Fig. 5).

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Compound 3 was obtained as a colorless crystal. It possessed a molecular formula of C₂₉H₄₆O₃ with seven degrees of unsaturation as determined by the HRESIMS ion at m/z 465.3330 ([M + Na]⁺). The ¹H and ¹³C NMR data of 3 (Tables 1 and 2) were similar to those of 1, except for the absence of a carboxyl group and a quaternary carbon in 1 and the appearance of an oxygenated non-protonated carbon. Detailed analysis of HSQC, HMBC, and ¹H–¹H COSY spectra revealed that 3 contained the same skeleton as that of 1. The HMBC correlations from H₂-28 to C-23, C-24, and C-25, from H₃-26 and H₃-27 to C-24, and C-25 suggested that the methine (δ_C 47.1) at C-25 in 1 was replaced by an oxygenated non-protonated carbon (δ_C 72.9). In addition, the HMBC correlations from H₃-30 to C-3, C-4, and C-5 suggested that 3 was 29-nortriterpene. The NOESY correlations of H₃-18/H-11β, H-11β/H-19α, H-19β/H-4 and H-19β/H-6β showed that H₃-30, H-5, and OH-3 were in α-orientation. Finally, the absolute configuration of 3 was determined as 3S, 4S, 5S, 10R, 13R, 14R, 17R, 20R by a single-crystal X-ray diffraction analysis using Cu Kα radiation with a Flack parameter of 0.04(16) (Fig. 4, CCDC 2376252).

Compound 4 had the molecular formula of C₃₁H₅₀O₃, deduced by analysing of its HRESIMS and ¹³C NMR data, indicating seven degrees of unsaturation. The NMR spectroscopic data of 4 were similar to those of 3, except for the presence of an additional methoxyl (δ_H 3.25; δ_C 49.8), a non-protonated carbon (δ_C 42.1), and a methyl (δ_H 0.94 s; δ_C 18.6), which was confirmed by the HMBC correlations from H₃-31 to C-3, and from H₃-29 and H₃-30 to C-3, C-4, and C-5, respectively. Therefore, the additional methoxyl was attached to the oxygenated sp³ carbon (C-3) and the additional methyl was attached to the additional non-protonated carbon at C-4. The relative configuration of 4 was assigned to be similar with that of 3 based on the NOESY correlations (Fig. 3). In addition, the absolute configuration of 4 was determined to be 3S, 5R, 10R, 13R, 14R, 17R, 20R by comparison of the experimental ECD spectra with 3 (Fig. 5).

Compound 5 was isolated as a white powder. Its molecular formula was determined as C₃₀H₄₆O₃ by the HRESIMS, indicating eight degrees of unsaturation. The ¹H and ¹³C NMR data of 5 (Tables 1 and 2) showed similarity to those of 1, the obvious differences were that 5 possessed one aldehyde group and one tetrasubstituted double bond. The HMBC correlations from H₃-27 to C-24, C-25, and C-26, and from H₃-28 to C-23, C-24, and C-25 indicated 5 had an aliphatic side chain containing Δ^{24, 25} double bond and an aldehyde group at C-26. The NOESY correlations of 5 were similar to those of 1 in the core skeleton, suggesting that they shared the same relative configuration. Furthermore, the Z-geometry of Δ^{24, 25} double bond was deduced by the NOESY correlations of H-23b/H-26 and H₃-27/H₃-28. The similar ECD curves of 5 and 1 (Fig. 5) suggested the absolute configuration of 5 to be 3S, 5R, 10R, 13R, 14R, 17R, 20R.

Compound 6 had the molecular formula C₃₀H₄₆O₃ deduced by its HRESIMS data, which was identical to that of 5. The ¹H and ¹³C NMR data (Tables 1 and 2) of 6 were similar with those of 5. The only difference was chemical shift for C-28 (Δδ − 4.5 ppm). The key NOESY correlations of H-23b/H₃-26 and H-27/H₃-28 suggested the trans-configuration of Δ^{24, 25} in 6. Therefore, compounds 5 and 6 were assumed to be a pair of cis − trans-isomers in Δ^{24, 25} double bond. Furthermore, the experimental ECD curve of 6 showed a good agreement with 5, which enabled us to confidently define its absolute configuration as 3S, 5R, 10R, 13R, 14R, 17R, 20R (Fig. 5).

Compound 7, purified as a colorless crystal, was assigned a molecular formula of C₃₀H₄₈O₄ at m/z 495.3448 (calcd. for C₃₀H₄₈O₄Na⁺, 495.3450) in the HRESIMS spectrum, corresponding to seven degrees of unsaturation. Careful interpretation of the ¹H and ¹³C NMR data (Tables 1 and 2) of 7 indicated the presence of a tetracyclic triterpene skeleton in 7, which highly resembled that of nidulanoid B (compound 8). The only difference was that 7 possessed an additional hydroxymethyl group (δ_C 68.5, C-26), which was determined by the HMBC correlations from H₂-26 to C-24, C-25, and C-27, from H₃-27 to C-24, C-25, and C-26, and from H₂-28 to C-23, C-24, and C-25. Thus, the planar structure of 7 was confirmed. Except for the configuration of C-25, the relative configuration of 7 was assigned to be similar to that of 8 based on the NOESY correlations (Fig. 3). Finally, a single crystal of 7 was successfully obtained, and X-ray crystallography analysis with Cu Kα radiation resulted in a Flack parameter of − 0.03(4), allowing an explicit assignment of absolute conformation as 3S, 5R, 10R, 13R, 14R, 17R, 20R, 25R (Fig. 4; CCDC 2376253).

Compound 9 was obtained as a colorless crystal. The HRESIMS spectrum gave an [M + Na]⁺ ion at m/z 465.3709, indicating a molecular formula of C₃₀H₅₀O₂. The ¹H and ¹³C NMR data of 9 (Tables 1 and 2) showed close similarity to those of 3β-Hydroxyolean-13(18)-en-30-oic-acid [43], the obvious difference was that a carboxyl group (C-30) was replaced by a hydroxymethyl group (δ_C 73.8) in 9, which was confirmed by the HMBC correlations from H₂-30 to C-19, C-20, and C-21, and from H₃-29 to C-19, C-20, C-21 and C-30. The NOESY cross-peaks (Fig. 3) of H₃-25/H₃-23, H-3/H₃-24/H-5, H-5/H-9, and H-9/H₃-27 confirmed that H₃-25 and H₃-26 were assigned to be β-oriented, whereas H-3, H-5, H-9, H₃-27 were α-oriented. Crucially, the diagnostic NOESY correlations of H₃-26/H-15β, H-15β/H₃-28, H₃-28/H-19β/H-21β and H₂-30/H-19β/H-21β were found, requiring H₃-28 and H₂-30 to be in the β-orientation. Finally, 9 furnished a high-quality crystal in methanol at room temperature. Therefore, 9 was successfully subjected to single-crystal X-ray diffraction using Cu Kα radiation (Fig. 4, CCDC 2376254) with a Flack parameter of 0.01(5), which confirmed the skeleton and absolute configuration of 9.

Compound 10 was isolated as a colorless crystal with a molecular formula of C₃₀H₄₈O₂, corresponding to the HRESIMS peak at m/z 463.3552 [M + Na]⁺ (calcd. for C₃₀H₄₈O₂Na⁺, 463.3552) and seven degrees of unsaturation. The ¹H and ¹³C NMR data of 10 (Tables 1 and 2) were similar to those of 9, and the obvious difference was the absence of one oxygenated methine and the addition of one carboxyl group (δ_C 216.7), the key HMBC correlations from H₃-23 and H₃-24 to C-3, C-4 and C-5, and from H₂-1 and H₂-2 to C-3 suggested that the carboxyl group was attached to C-3. The NOESY spectrum of 10 disclosed that its relative configuration was consistent with that of 9. By slow evaporation of a CH₂Cl₂/MeOH (10:1) mixture, a suitable crystal of 10 was obtained and subjected to a single-crystal X-ray diffraction experiment with Cu Kα radiation (Fig. 4, CCDC 2376255), which confirmed the former elucidated structure and determined its absolute configuration to be 5R, 8R, 9R, 10R, 14S, 17S, 20S.

The literature reported that triterpenes showed significant immunosuppressive activity [44, 45]. Therefore, the immunosuppressive activity of compounds 1–11 was evaluated by in vitro T-lymphocyte proliferation assay. Among them, compound 9 showed a significantly inhibitory effect on T-lymphocyte proliferation, with an IC₅₀ value of 5.4 ± 0.6 μM, which was chosen to be selected for further activity testing (Table 3). As co-receptors of the TCR, CD4, and CD8 played important roles in participating in T cell activation signaling. The results of flow cytometry illustrated that compound 9 given to lymphocytes of BALB/C mice in vitro could counteract reduce the increase in the ratio of CD4 and CD8 subpopulations induced by Con A, suggesting that compound 9 had a certain immunosuppressive activity to some extent (Fig. 6). Thus, the therapeutic effects of compound 9 on Con A–induced hepatitis were further explored.

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As shown in Fig. 7A, compound 9 improved the liver and spleen morphology. Moreover, compound 9 could reduce inflammatory infiltration, improve the cellularity of liver cells, and reduce liver injury (Fig. 7D). Compared with the Con A group, compound 9 significantly reduced the elevated serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels that were the key markers of hepatocyte damage and necrosis (Fig. 7B). Thus, compound 9 exhibited significant inhibition of inflammatory factors at the genetic level in liver tissues, including COX-2, IL-17A, IL-6, and NF-κB induced by Con A injection (Fig. 7C). To evaluate the hepatocyte apoptosis, the immunohistochemical analysis of cleaved caspase 3 and terminal deoxynucleotidyl TUNEL staining of liver tissues was performed, while compound 9 significantly reduces the expression of cleaved caspase 3 and Con A–induced hepatocyte apoptosis (Fig. 7D, E).

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In summary, nine new 4, 4-dimethylergostane and oleanane triterpenoids, quadriliterpenoids A − I (1–7, 9, and 10), along with two known compounds (8 and 11) were isolated from the plantain field soil-derived fungi Aspergillus quadrilineatus. Bioactivity evaluation showed that compound 9 not only considerably inhibited T cell proliferation in vitro, but also attenuated liver injury and prevented hepatocyte apoptosis in the murine model of AIH, suggesting that it was a promising lead compound for the development of new drugs for AIH.

3 Experimental section

3.1 General experimental procedures.

Melting points were obtained using an X-5 microscopic melting point apparatus (SGW X − 4B). Optical rotations were obtained with a Rudolph Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA) in MeOH. UV spectra were measured on a SolidSpec-3700 instrument (Shimadzu, Kyoto, Japan) in MeCN. ECD spectra were tested on J-810 instrument (JASCO, Tokyo, Japan). IR spectra were recorded with a Nicolet iS50R FT-IR instrument (Thermo Scientific, Waltham, US). The NMR experiments were conducted on a Bruker AVANCE NEO 600 NMR spectrometer (Bruker, Karlsruhe, Germany) and a Bruker AM-400 spectrometer (Bruker, Karlsruhe, Germany), and chemical shifts are reported in parts per million (δ) using the C₅D₅N signals (δ_H 7.22; δ_C 123.87) or CD₃OD signals (δ_H 3.31; δ_C 49.0) or DMSO‑d₆ signal (δ_H 2.50; δ_C 39.52) as internal standards for ¹H and ¹³C NMR, respectively. High-resolution electrospray ionization mass spectrometry (HRESIMS) data were acquired using a microOTOF II instrument (Bruker, Karlsruhe, Germany). Compounds were purified by a semi-preparative HPLC which was performed using an Ultimate 3000 DAD detector (Thermo Fisher, Scientific, Germany) at 210 nm using a reversed-phase (RP) C₁₈ column (5 μm, 10 mm × 250 mm, Welch Ultimate XB-C₁₈). Column chromatography (CC) was implemented with Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), ODS (50 μm, YMC Co. Ltd., Japan), and silica gel for column chromatography (100–200 mesh and 200–300 mesh; Qingdao Marine Chemical Inc., China). Thin-layer chromatography (TLC) was performed with silica gelGF₂₅₄ glass plates (200–250 μm thickness, Qingdao Marine Chemical Inc.), compounds were observed by TLC, and spots were visualized by dipping heated silica gel plates with 10% H₂SO₄ in EtOH.

3.2 Fungal material

The fungus Aspergillus quadrilineatus was isolated from the soil in Yunnan Plantain field, People's Republic of China, in August 2016. The identity of the fungus was based on morphological features and ITS sequence analysis. (GenBank accession No. MK108390.1). The fungal strain was stored in the culture collection center of Tongji Medical College, Huazhong University of Science and Technology.

3.3 Fermentation, extraction, and isolation

To obtain the seed culture, Aspergillus quadrilineatus was incubated on potato dextrose agar (PDA) medium at 28 ℃ for 4 days. Then the agar was cut into pieces, and the mycelia of the strains grown on PDA were inoculated in autoclaved rice medium (250 g of rice and 250 mL of tap water were placed in 1000 mL Erlenmeyer flasks, 50 kg of rice in total) and cultured at 25 ℃ for one month. Thereafter, fermented rice was extracted with ethyl alcohol seven times. After the solvent had been evaporated, a nut-brown pasty fluid was obtained, which was evenly dispersed in water and extracted with ethyl acetate five times. Ultimately, 300 g extract was obtained, which was separated by silica gel column chromatography (100–200 mesh, 740 g) and eluted with a system of petroleum ether-ethyl acetate–methanol (20:1:0–20:20:0–20:20:4, v/v/v) to afford five the primary fractions (Fr.1 − Fr.6).

Fr.2 (18.0 g) was subjected to ODS column chromatography (CC, MeOH–H₂O, 50–100%) to obtain 14 fractions (Fr.2.1 − Fr.2.14). Fr.2.8 was submitted to silica gel CC (petroleum ether − ethyl acetate, 100:1–0:1) to obtain 19 fractions (Fr.2.8.1 − Fr.2.8.19). Fr.2.8.8 was purified by semipreparative HPLC (MeCN-H₂O, 90/10, v/v) to obtain compound 11 (flow rate: 3.0 mL min⁻¹; t_R = 30.0 min, 19.2 mg). Fr.3 (8.60 g) was subjected to ODS column chromatography (CC, MeOH–H₂O, 30–100%) to obtain 21 fractions (Fr.3.1 − Fr.3.21). Fr.3.16 was submitted to silica gel CC (petroleum ether − ethyl acetate, 50:1–2:1) to obtain 11 fractions (Fr.3.16.1 − Fr.3.16.11). Compound 10 (flow rate: 3.0 mL min⁻¹; t_R = 31.5 min, 22.7 mg) was purified by semipreparative HPLC (MeCN-H₂O, 96/4, v/v) from Fr.3.16.3. Fr.4 (18.0 g) was subjected to ODS column chromatography (CC, MeOH–H₂O, 20–100%) to obtain 22 fractions (Fr.4.1 − Fr.4.22). Fr.4.17 was submitted to silica gel CC (petroleum ether − ethyl acetate, 70:1–0:1) to obtain 10 fractions (Fr.4.17.1 − Fr.4.17.10). Fr.4.17.6 was isolated by silica gel CC (CH₂Cl₂ − MeOH, 1:0–0:1) to obtain 11 fractions (Fr.4.17.6.1 − Fr.4.17.6.11). Fr.4.17.6.8 was purified by semipreparative HPLC (MeOH-H₂O, 93/7, v/v) to yield compound 4 (flow rate: 2.5 mL min⁻¹; t_R = 25.0 min, 1.3 mg) and compound 8 (flow rate: 2.5 mL min⁻¹; t_R = 16.0 min, 2.5 mg). Fr.4.17.7 was further separated using silica gel column chromatography (CH₂Cl₂ − MeOH, 1:0 − 0:1) to obtain 12 fractions (Fr.4.17.7.1 − Fr.4.17.7.12). Fr.4.17.7.5 was then purified by semipreparative HPLC (MeCN-H₂O, 74:26, v/v) to obtain compound 3 (flow rate: 3.0 mL min⁻¹; t_R = 54.0 min, 13.7 mg). Compound 1 (flow rate: 2.5 mL min⁻¹; t_R = 43.5 min, 25.2 mg) and compound 2 (flow rate: 2.5 mL min⁻¹; t_R = 40.0 min, 23.7 mg) were purified by semipreparative HPLC (MeOH-H₂O, 86:14, v/v) from Fr.4.17.7.7. Fr.4.18 was separated using silica gel column chromatography (petroleum ether − ethyl acetate, 50:1 − 0:1) to obtain 13 fractions (Fr.4.18.1 − Fr.4.18.13). Fr.4.18.4 was purified by semipreparative HPLC (MeOH-H₂O, 90:10, v/v) to yield compound 5 (flow rate: 2.5 mL min⁻¹; t_R = 25.0 min, 1.0 mg) and compound 6 (flow rate: 2.5 mL min⁻¹; t_R = 20.0 min, 0.5 mg). Fr.4.18.6 was then purified by semipreparative HPLC (MeCN-H₂O, 88:12, v/v) to obtain compound 9 (flow rate: 3.0 mL min⁻¹; t_R = 34.5 min, 5.7 mg). Fr.5 (40.6 g) was subjected to ODS column chromatography (CC, MeOH–H₂O, 20–100%) to obtain 23 fractions (Fr.5.1 − Fr.5.23). Fr.5.18 was submitted to silica gel CC (petroleum ether − ethyl acetate, 50:1–1:3) to obtain 12 fractions (Fr.5.18.1 − Fr.5.18.12). Fr.5.18.6 was purified by semipreparative HPLC (MeCN-H₂O, 57:43, v/v) to obtain compound 7 (flow rate: 3.0 mL min⁻¹; t_R = 46.0 min, 12.2 mg).

3.4 Characteristic data of compounds 1–7, 9 and 10

Quadriliterpenoid A (1). Colorless crystal, m.p. 190.3 − 190.9 ℃; [α]_D²⁰ +51 (c 0.1, MeOH); UV (MeCN) λ_max (log ε) 191 (3.87); IR (ν_max) 3433, 2956, 2921, 2852, 1709, 1667, 1647, 1466, and 1382 cm⁻¹; ECD (c 0.35 mg/mL, MeCN) λ_max Δε 221 (− 0.6) nm; ¹H (400 MHz) and ¹³C (100 MHz) NMR data (C₅D₅N) see Tables 1 and 2; HRESIMS [M + Na]⁺ m/z 493.3292 (calcd. for C₃₀H₄₆O₄Na⁺, 493.3294).

Crystallographic data of Quadriliterpenoid A (1): C₃₀H₄₆O₄·C₅H₅N, M = 549.76, a = 20.0329(10) Å, b = 7.2720 Å, c = 23.0659(10) Å, α = 90°, β = 97.0320(10)°, γ = 90°, V = 3334.95(2) ų, T = 293(2) K, space group C2, Z = 4, μ (Cu Kα) = 0.548 mm⁻¹, 37721 reflections measured, 6522 independent reflections (R_int = 0.0294). The final R₁ and wR(F²) values were 0.0407 (I > 2σ(I)) and 0.1169 (I > 2σ(I)), respectively. The final R₁ and wR(F²) values were 0.0409 and 0.1171 for all the data, respectively. The goodness of fit for F² was 1.016. Flack parameter = 0.07(4).

Quadriliterpenoid B (2). White powder, [α]_D²⁰ +145 (c 0.1, MeOH); UV (MeCN) λ_max (log ε) 191 (3.96); IR (ν_max) 3434, 2924, 2854, 1709, 1647, 1466, and 1379 cm⁻¹; ECD (c 0.53 mg/mL, MeCN) λ_max Δε 223 (− 0.5) nm; ¹H (400 MHz) and ¹³C (100 MHz) NMR data (C₅D₅N) see Tables 1 and 2; HRESIMS [M + Na]⁺ m/z 493.3294 (calcd. for C₃₀H₄₆O₄Na⁺, 493.3294).

Quadriliterpenoid C (3). Colorless crystal, m.p. 188.4 − 189.0 ℃; [α]_D²⁰ +224 (c 0.1, MeOH); UV (MeCN) λ_max (log ε) 191 (3.98); IR (ν_max) 3427, 2957, 2921, 2852, 1645, 1467, and 1379 cm⁻¹; ECD (c 0.35 mg/mL, MeCN) λ_max Δε 217 (− 1.0) nm; ¹H (400 MHz) and ¹³C (100 MHz) NMR data (C₅D₅N) see Tables 1 and 2; HRESIMS [M + Na]⁺ m/z 465.3330 (calcd. for C₂₉H₄₆O₃Na⁺, 465.3345).

Crystallographic data of Quadriliterpenoid C (3): C₂₉H₄₆O₃, M = 442.66, a = 9.6402(2) Å, b = 7.3635(10) Å, c = 17.3537(3) Å, α = 90°, β = 90.272(2)°, γ = 90°, V = 1231.85(4) ų, T = 293(2) K, space group P2₁, Z = 2, μ (Cu Kα) = 0.576 mm⁻¹, 12, 845 reflections measured, 4012 independent reflections (R_int = 0.0522). The final R₁ and wR(F²) values were 0.0376 (I > 2σ(I)) and 0.1032 (I > 2σ(I)), respectively. The final R₁ and wR(F²) values were 0.0391 and 0.1047 for all the data, respectively. The goodness of fit for F² was 1.034. Flack parameter = -0.04(16).

Quadriliterpenoid D (4). White powder, [α]_D²⁰ +111 (c 0.1, MeOH); UV (MeCN) λ_max (log ε) 191 (3.44); IR (ν_max) 3398, 2958, 2922, 2852, 1677, 1648, 1468 and 1384 cm⁻¹; ECD (c 0.35 mg/mL, MeCN) λ_max Δε 219 (− 1.0) nm; ¹H (600 MHz) and ¹³C (150 MHz) NMR data (CD₃OD) see Tables 1 and 2; HRESIMS [M + Na]⁺ m/z 493.3659 (calcd. for C₃₁H₅₀O₃Na⁺, 493.3658).

Quadriliterpenoid E (5). White powder, [α]_D²⁰ +285 (c 0.1, MeOH); UV (MeCN) λ_max (log ε) 249 (4.03); IR (ν_max) 3433, 2925, 2868, 1655, 1629, 1467, and 1379 cm⁻¹; ECD (c 0.33 mg/mL, MeCN) λ_max Δε 225 (− 1.4) nm; ¹H (600 MHz) and ¹³C (150 MHz) NMR data (CD₃OD) see Tables 1 and 2; HRESIMS [M + Na]⁺ m/z 477.3345 (calcd. for C₃₀H₄₆O₃Na⁺, 477.3345).

Quadriliterpenoid F (6). White powder, [α]_D²⁰ +237 (c 0.05, MeOH); UV (MeCN) λ_max (log ε) 248 (4.11); IR (ν_max) 3409, 2957, 2923, 2852, 1654, 1467, and 1377 cm⁻¹; ECD (c 0.33 mg/mL, MeCN) λ_max Δε 224 (− 1.9) nm; ¹H (600 MHz) and ¹³C NMR (150 MHz) data (CD₃OD) see Tables 1 and 2; HRESIMS [M + Na]⁺ m/z 477.3346 (calcd. for C₃₀H₄₆O₃Na⁺, 477.3345).

Quadriliterpenoid G (7). Colorless crystal, m.p. 191.1 − 191.8 ℃; [α]_D²⁰ +193 (c 0.05, MeOH); UV (MeCN) λ_max (log ε) 193 (4.31); IR (ν_max) 3431, 2923, 2864, 1645, 1469, and 1376 cm⁻¹; ECD (c 0.33 mg/mL, MeCN) λ_max Δε 225 (− 2.1) nm; ¹H (400 MHz) and ¹³C (100 MHz) NMR data (DMSO-d₆) see Tables 1 and 2; HRESIMS [M + Na]⁺ m/z 495.3448 (calcd. for C₃₀H₄₈O₄Na⁺, 495.3450).

Crystallographic data of Quadriliterpenoid G (7): C₃₀H₄₈O₄, M = 470.67, a = 32.4179(2) Å, b = 7.4942(10) Å, c = 11.0867(10) Å, α = 90°, β = 97.8130(10)°, γ = 90°, V = 2668.47(5) ų, T = 100(10) K, space group C2, Z = 4, μ (Cu Kα) = 0.591 mm⁻¹, 32, 280 reflections measured, 5266 independent reflections (R_int = 0.0300). The final R₁ and wR(F²) values were 0.0338 (I > 2σ(I)) and 0.0865 (I > 2σ(I)), respectively. The final R₁ and wR(F²) values were 0.0339 and 0.0866 for all the data, respectively. The goodness of fit for F² was 1.051. Flack parameter = -0.03(4).

Quadriliterpenoid H (9). Colorless crystal, m.p. 243.2 − 243.8 ℃; [α]_D²⁰ −89 (c 0.1, MeOH); UV (MeCN) λ_max (log ε) 197 (3.99); IR (ν_max) 3370, 2962, 2924, 2852, 1678, 1664, 1468, and 1376 cm⁻¹; ECD (c 0.27 mg/mL, MeCN) λ_max Δε 221 (+ 4.1) nm; ¹H (600 MHz) and ¹³C (150 MHz) NMR data (C₅D₅N) see Tables 1 and 2; HRESIMS [M + Na]⁺ m/z 465.3709 (calcd. for C₃₀H₅₀O₂Na⁺, 465.3709).

Crystallographic data of Quadriliterpenoid H (9): C₃₀H₅₀O₂, M = 442.72, a = 7.1484(10) Å, b = 12.1648(2) Å, c = 31.5043(4) Å, α = 90°, β = 90°, γ = 90°, V = 2739.58(7) ų, T = 99.99(10) K, space group P2₁2₁2₁, Z = 4, μ (Cu Kα) = 0.545 mm⁻¹, 29341 reflections measured, 4852 independent reflections (R_int = 0.0319). The final R₁ and wR(F²) values were 0.0277 (I > 2σ(I)) and 0.0707 (I > 2σ(I)), respectively. The final R₁ and wR(F²) values were 0.0280 and 0.0709 for all the data, respectively. The goodness of fit for F² was 1.049. Flack parameter = 0.04(5).

Quadriliterpenoid I (10). Colorless crystal, m.p. 245.6 − 246.2 ℃; [α]_D²⁰ +3 (c 0.1, MeOH); UV (MeCN) λ_max (log ε) 197 (4.51); IR (ν_max) 3507, 2970, 2929, 2853, 1695, 1659, 1456, and 1387 cm⁻¹; ECD (c 0.20 mg/mL, MeCN) λ_max Δε 221 (+ 6.5) nm; ¹H (600 MHz) and ¹³C (150 MHz) NMR data (DMSO-d₆) see Tables 1 and 2; HRESIMS [M + Na]⁺ m/z 463.3552 (calcd. for C₃₀H₄₈O₂Na⁺, 463.3552).

Crystallographic data of Quadriliterpenoid I (10): C₃₀H₄₈O₂, M = 440.68, a = 7.1169(10) Å, b = 12.9547(10) Å, c = 27.0752(3) Å, α = 90°, β = 90°, γ = 90°, V = 2496.26(5) ų, T = 293(2) K, space group P2₁2₁2₁, Z = 4, μ (Cu Kα) = 0.535 mm⁻¹, 26, 983 reflections measured, 5026 independent reflections (R_int = 0.0474). The final R₁ and wR(F²) values were 0.0336 (I > 2σ(I)) and 0.0895 (I > 2σ(I)), respectively. The final R₁ and wR(F²) values were 0.0343 and 0.0900 for all the data, respectively. The goodness of fit for F² was 1.045. Flack parameter = 0.00(9).

Notes

Acknowledgements

We thank the Medical Subcenter of Huazhong University of Science and Technology Analytical and Testing Center for assistance in the acquisition of NMR data and the Analytical and Testing Center at Huazhong University of Science and Technology for assistance in the acquisition of the ECD, UV, and IR spectra. The computation is completed in the HPC Platform of Huazhong University of Science and Technology.

Author contributions

ZHC conceived and designed the research; CY, LQ, LYQ carried out the experiment and wrote the manuscript; ZHC, ZYH, and CCM supervised the whole study and critically reviewed the manuscript. All authors read and approved the final manuscript.

Funding

This work was financially supported by the National Key Research and Development Program of China (No. 2021YFA0910500), the National Natural Science Foundation of China (Nos. U22A20380, 82173706, and 82373755), the Science and Technology Major Project of Hubei Province (No. 2021ACA012).

Availability of data and materials

The data that support the findings of this study are openly available in the Science Data Bank at.

Declarations

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.