Qingqianliusus A-N, 3,4-seco-dammarane triterpenoids from the leaves of Cyclocarya paliurus and their biological activities

2.1 General and solvents

UV spectra were recorded with Hewlett-Packard 8452A, UV–vis spectrophotometer (L.P., Palo Alto, Ca, USA). Using a Frontier infrared spectrometer to record IR spectra (PerkinElmer, U.S.A). HR-ESI-MS spectra were performed on Xevo G2-S QTOF mass spectrometer (Waters Co., Milford, MA, USA), 6500 series Q-TOF mass spectrometer (Agilent Co., USA) and LTQ Orbitrap Velos Pro MS (Thermo Scientific, MA, USA). The single Crystal X-ray diffraction data were obtained using a SuperNova, Dual, Cu at zero, AtlasS2 diffractometer (Rigaku Co., Japan). 1D and 2D NMR spectra were acquired on a Bruker Avance DRX-600 MHz spectrometer (Bruker Co., Billerica, MA, USA) at room temperature with CD₃OD as solvent. The absolute configuration of the sugar moiety was detected by HPLC analysis at 210 nm using C18 columns (5 μm, 4.6 × 250 mm, Agilent, Palo Alto, CA, USA) on Agilent 1260 series HPLC instrument (Agilent, Palo Alto, CA, USA). Semi preparative HPLC was carried out on Agilent 1260 series semi preparative HPLC instrument (Agilent, Palo Alto, CA, USA) by using C18 columns (5 mm, 9.4 × 150 mm) with a flow rate of 2 mL/min. All analytical-grade solvents were bought from Merck KGaA (Darmstadt, Germany). Besides, chemical solvent were obtained from Shanghai Titan Scientific Co., ltd. P. R. China and silica gel (300–400 mesh) for column chromatographic were purchased from Qingdao Marine Chemical Inc. P. R. China. Similarly, Sephadex LH-20 gel were bought from GE Health care, Sweden and Lichrospher RP-18 gel (20–45 μm) from Merck KGaA (Darmstadt, Germany).

2.2 Plant material

The leaves of Cyclocarya paliurus (Batalin) Iljinskaja were purchased from Huaihua, Hunan Province, People’s Republic of China, in June 2015, and authenticated by Prof. Wei Wang of Hunan University of Chinese Medicine. The voucher specimen (No. 20150629) was preserved in the TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, People′s Republic of China.

2.3 Extraction and isolation

The crude extract (4 Kg) was extracted from Qingqianliu (20.0 Kg) using 95 % ethanol percolation for three times at room temperature. Afterwards, the ethanol extract was suspended in water and then extracted with petroleum ether (PE), chloroform (CHCl₃), and n-butanol (n-BuOH) respectively. The chloroform layer (980.0 g) was subjected to silica gel column (60 × 15.5 cm) chromatography eluted with CH₂Cl₂-CH₃OH (0:1–1:1) to gain nineteen fractions (Fr. E1-E19). Fraction E7 (5.2 g) was purified using silica gel column (60 × 15.5 cm) chromatography eluted with CYH-EA (0:1–1:1) and reverse-phase HPLC (2.0 mL/min, MeCN-H₂O, 7.0:3.0) afforded 10 (4.0 mg) and 14 (4.0 mg). Combined fractions E8 and E9 named fractions E8-9 (33.9 g), which through silica gel column with PE-EA (0:1–1:1) to give thirteen fractions (Fr. F1-F13). Sub fraction F9 (2.5 g) through silica gel column chromatography yield 1 (12.5 mg), 2 (7.0 mg), 3 (15.2 mg), 4 (5.8 mg), 5 (4.1 mg), 11 (4.4 mg), 12 (27.4 mg), and 13 (32.4 mg). Similarly, fraction F10 (4.6 g) was subjected using Sephadex LH-20 column (4 × 77 cm) chromatography eluted with CH₃OH and reverse-phase HPLC (2.0 mL/min, MeCN-0.01 % F₃CCOOH-H₂O, 6.5:3.5) to produce 6 (9.1 mg), 7 (4.4 mg), 8 (3.8 mg), 9 (17.9 mg), 15 (4.0 mg), and 18 (8.2 mg). Compounds 16 (5.2 mg) and 17 (4.2 mg) were separated from fraction E13 (10.0 g) via silica gel column (60 × 15.5 cm) chromatography eluted with CYH-EA (0:1–1:1) and purified by reverse-phase HPLC (2.0 mL/min, MeCN-H₂O, 7.0:3.0).

2.4 Determination of the absolute configurations of the Sugars

The acid hydrolysis was adopted from reported method (Liu et al., 2020; Shen et al., 2020). After the comparison of the retention times (t_R) of monosaccharide derivatives with those of standard products: l-Arabinose (19.75 min) and d-Arabinose (23.96 min), compounds 10, 11, and 14 were determined to be l-Arabinose. Detailed description of the procedure can be seen in the Supporting Information (Text S1.).

2.5 X-ray crystallographic Analysis

All data were obtained using a SuperNova, AtlasS2 diffractometer with graphite monochromated Cu Ka radiation (λ = 1.54184 Å). The structure was solved by direct method with the SHELXTL software package and was refined by full-matrix least-squares techniques. All the nonhydrogen atoms were refined anisotropically and H atoms were located in geometrical calculations. Crystal data for compounds 1 and 18 were listed below.

2.6 α-Glucosidase inhibitory activity Assay

The α-glucosidase inhibition assay was adopted from reported method (Perez et al., 2019; Smirnova et al., 2020; Zhou et al., 2021). The detailed protocol was described in the Supporting Information (Text 2).

2.7 Anti-inflammatory activity against COX-2

The COX-2 inhibition assay was detected with previous report (Chai et al., 2008; Shataer et al., 2021). The detailed procedures for this assay were discussed in the Supporting Information (Text 3).

2.8 Cytotoxic Bioassays

Using the previously described MTT method (Chen et al., 2018; Gao et al., 2016; Peng et al., 2019; Sun et al., 2021; Yan et al., 2014; Zhou et al., 2021), the cytotoxicities of new compounds against the human cancer cell lines (BGC-823, MCF-7, HCT-116, and HepG-2) were measured. Paclitaxel was used as positive control. The detailed process can be seen in the Supporting Information (Text 4).

3.1 Structure Determination

The chloroform extract of Qingqianliu was isolated and fractionated over Sephadex LH-20, silica gel, RP-18 (ODS), and semi-preparative HPLC to obtain fourteen undiscovered compounds named Qingqianliusus A-N (1–14), as well as four reported compounds, 20S,24S-dihydroxy-3,4-seco-dammara-4(28),25-dien,3-oic acid (15) (Aoki et al., 1988), cyclocariol B (16) (Chen et al., 2018), cyclocariol A (17) (Chen et al., 2018), and cyclocariol E (18) (Chen et al., 2018). In addition, compound 15 was reported from Qingqianliu for the first time.

Qingqianliusu A (1), an optical rotation value of [α]23 D = + 67.0 (c 0.19, MeOH), was obtained as cubic crystals (CH₃CN-CH₃OH) with mp 169 ∼ 171℃. The molecular formula C₃₀H₄₈O₄ was established by [M + Na]⁺ ion at m/z 495.3450 (calcd. for C₃₀H₄₈O₄Na, 495.3450) in HR-ESI-MS (Fig. S6). Its IR spectrum (Fig. S8) revealed characteristic absorption peaks for hydroxyl (3369 cm⁻¹), methyl (2950, 2930, 1457, 1387 cm⁻¹), double bond (1644 cm⁻¹), and carbonyl groups (1728 cm⁻¹). The ¹H NMR data (Table 1) exhibited resonances for four olefinic proton singlets at δ_H 5.67 (ddd, J = 15.6, 7.5, 6.2 Hz, H-23), 5.62 (d, J = 15.6 Hz, H-24), 4.86 (br s, H-28a), and 4.74 (br s, H-28b); one oxygenated methine at δ_H 4.78 (q-like, J = 8.8 Hz, H-11); as well as seven methyl singlets at δ_H 1.75 (s, H₃-29), 1.26 (s, H₃-27), 1.27 (s, H₃-26), 1.20 (s, H₃-19), 1.14 (s, H₃-21), 1.00 (s, H₃-18), and 0.97 (s, H₃-30). According to the ¹³C NMR (Table 2), DEPT-135°, and HSQC spectra (Fig. S12), 30 carbon signals including one carbonyl at δ_C 179.6 (C-3); four olefinic carbons at δ_C 148.3 (C-4), 142.2 (C-24), 123.7 (C-23), and 114.6 (C-28); one oxygenated sp³ methine at δ_C 78.5 (C-11); five sp³ quaternary carbons at δ_C 75.7 (C-20), 71.2 (C-25), 51.0 (C-14), 41.8 (C-8), and 40.7 (C-10); seven methyls at δ_C 29.9 (C-26), 30.0 (C-27), 26.1 (C-21), 23.9 (C-29), 19.3 (C-19), 17.4 (C-18), and 16.1 (C-30); as well as eight sp³ methylenes at δ_C 45.2 (C-22), 40.6 (C-1), 37.0 (C-12), 34.7 (C-7), 31.6 (C-15), 30.5 (C-2), 26.3 (C-6), and 25.8 (C-16) were determined. All the above data indicated that 1 possessed a 3,4-seco-dammarane triterpenoid skeleton.

The ¹H–¹H COSY correlations (Fig. 2) of 1 demonstrated the presence of four spin-coupling systems (C-1 to C-2, C-5 to C-7, C-9 to C-15, C-22 to C-24). The HMBC spectrum (Fig. S13) showed the correlations from δ_H 4.78 (H-11) to δ_C 179.6 (C-3)/40.7 (C-10), δ_H 2.81 (H-2a)/2.30 (H-2b) to δ_C 40.7 (C-10), as well as δ_H 1.78 (H-1a)/1.33 (H-1b) to δ_C 179.6 (C-3), suggesting the existence of a 3,11-heptacyclic lactone unit, which was supported by the down-field shift of the resonance with δ_H 4.78 (q-like, J = 8.8 Hz, H-11). The correlations from δ_H 5.67 (H-23) to δ_C 75.7 (C-20)/71.2 (C-25), δ_H 1.26 (H-26)/1.27 (H-27) to δ_C 142.2 (C-24), δ_H 1.14 (H-21) to δ_C 51.9 (C-17)/45.2 (C-22) and δ_H 1.89 (H-13)/1.80 (H-16a) to δ_C 75.7 (C-20) showed that an eight-carbon side chain containing a double bond was attached to C-17. The configuration of the Δ²³ double bond was E geometry due to the large coupling constant (³J_H-23/H-24 = 15.6 Hz). In addition, the correlations of δ_H 1.75 (H-29) to δ_C 114.6 (C-28)/58.7 (C-5), δ_H 4.86 (H-28a)/4.74 (H-28b) to δ_C 58.7 (C-5), δ_H 1.00 (H₃-18) to δ_C 34.7 (C-7)/53.5 (C-9) /51.0 (C-14), δ_H 1.00 (H₃-30) to δ_C 41.8 (C-8)/39.9 (C-13) /31.6 (C-15), implied the planar structure of 1 (Fig. 2). The relative configuration of 1 were determined by ROESY spectrum (Fig. S14), in which the correlations of δ_H 1.87 (H-5)/1.84 (H-9)/1.79 (H-17)/0.97 (H₃-30) revealed that H-5, H-9, H-17, and H₃-30 were cofacial and randomly allocated to be α-oriented. Consequently, the β-orientations of H-11, H-13, H₃-18, and H₃-19 were defined by the ROESY cross-peaks of δ_H 4.78 (H-11)/1.89 (H-13)/1.00 (H₃-18)/1.20 (H₃-19). Furthermore, a single crystal X-ray diffraction analysis was thus successfully performed to expressly assign the stereochemistry of 1 (Fig. 3). The Flack parameter of −0.3 (2) could only allow the relative configuration of 1. However, by analyzing several 3,4-seco-dammarane analogues (Aoki et al., 1988; Aoki et al., 1990; Chen et al., 2018; Phan et al., 2011; Takayuki and Toshifumi, 1979), as well as X-ray data (Fig. S4) of known analogue cyclocariol E (18) with a small Flack parameter of 0.05(6), it was found that the skeleton configurations of these compounds were stable. Therefore, based on the above evidence, the absolute structure of 1 was established as 23(E),11R,20S-11,20,25-trihydroxy-3,4-seco- dammara-4(28),23-dien-3 oic acid-3,11-heptacyclic lactone.

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Fig. 2

Key ¹H–¹H COSY (blue thick bonds) and HMBC (red solid arrows) correlations of compounds 1–4, and 6, together with NOESY (red dash arrows) correlations of compounds 1, 3, and 6.

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Fig. 3

Crystal structures of compounds 1, and 18.

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Qingqianliusu B (2) was obtained as a colorless amorphous powder with [α]23 D = + 76.0 (c 0.25, MeOH). The molecular formula, C₃₀H₄₈O₄, was based on its sodium adduct molecular ion [M + Na]⁺ at m/z 495.3451 (calcd. for C₃₀H₄₈O₄Na, 495.3450) in HR-ESI-MS spectrum (Fig. S15). Comparison of the NMR data (Tables 1 and 2) of 2 with compound 1 suggested that they share a similar skeleton with the same 3,11-heptacyclic lactone unit. The only difference between 1 and 2 were at the eight-carbon side chain. Terminal double bond with chemical shift at δ_H 4.91 (H-26a)/4.81 (H-26b), δ_C 148.8 (C-25) and 111.6 (C-26) in 2 replaced E-type disubstituted double bond in 1. Compound 2 also included one quaternary oxygen-bearing carbon at δ_C 75.4 (C-20), together with one oxygenated methine at δ_C 77.4 (C-24). The key HMBC correlations (Fig. S22) from δ_H 4.91 (H-26a)/4.81 (H-26b)/1.72 (H₃-27) to δ_C 77.4 (C-24), δ_H 1.65 (H-23a)/1.57 (H-23b) to δ_C 148.8 (C-25)/75.4 (C-20), and δ_H 1.14 (H-21) to δ_C 52.4 (C-17)/37.9 (C-22) revealed eight carbon side chain structure of 2. Notably, the absolute configuration of chiral center (C-24) on the branch chain was determined by summarizing NMR data characteristics of epimers with R/S configuration at C-24 and single crystal data of corresponding compound cyclocariol B (16) (Chen et al., 2018). Please see “3.3. Determination of the Absolute Configurations of Epimers” of this article for more details. Thus, the structure of 2 was assigned as 23(E),11R,20S,24S-11,20,24-trihydroxy-3,4-seco-dammara-4(28),25-dien-3 oic acid-3,11-heptacyclic lactone.

Qingqianliusu C (3), a colorless powder with [α]23 D = + 38.5 (c 0.29, MeOH), had a molecular formula C₃₁H₅₂O₅ as determined by HR-ESI-MS on its sodium adduct molecular ion [M + Na]⁺ at m/z 527.3712 (calcd. for C₃₁H₅₂O₅Na, 527.3712) (Fig. S24). In the ¹³C NMR spectrum (Fig. S28), singlets were exhibited for four oxygenated carbon singlets at δ 75.9 (C-20), 71.6 (C-11), 71.2 (C-25), and 51.9 (3-COOCH₃). The ¹H and ¹³C NMR data (Tables 1 and 2) of 3 were analogous to those of cyclocariol E (18) (Chen et al., 2018), illustrating that 3 had similar 3,4-seco-dammarane skeleton with cyclocariol E. Moreover, in the HMBC spectrum (Fig. 2 and Fig. S31), key correlations from δ_H 3.63 (s, 3-COOCH₃) to δ_C 177.3 (C-3) showed that a methoxy group was connected to the carbonyl. The correlations from δ_H 3.92 (td, J = 10.8, 5.0 Hz, H-11) to δ_C 41.7 (C-8) and 40.6 (C-10) verified the hydroxyl was located at C-11 in 3, while one hydroxyl located at C-12 in cyclocariol E (18). The α-orientations of H-5, H-9, H-17, and H₃-30 were further to confirm with the ROESY correlations (Fig. 2 and Fig. S32) of δ_H 2.07 (H-5)/1.73 (H-9)/1.79 (H₃-17)/0.97 (H₃-30). Then, key correlations of δ_H 3.92 (H-11)/1.87 (H-13) /1.00 (H₃-18)/1.20 (H₃-19) suggested that H-11, H-13, H₃-18, and H₃-19 are β-oriented. Thus, the configuration of C-11 in 3 was proved as S by comparing with the structure and ROESY correlation (Fig. 2) of 1. Accordingly, the compound 3 was determined as 23(E),11R,20S-11,20,25-trihydroxy-3,4-seco-dammara-4(28),23-dien-3-oic acid methyl ester.

Qingqianliusu D (4), colorless amorphous powder with [α]23 D = + 36.0 (c 0.25, MeOH). The HR-ESI-MS spectrum (Fig. S33) of 4 showed a quasi-molecular ion peak at m/z [M + Na]⁺ at m/z 527.3719 (calcd. for C₃₁H₅₂O₅Na, 527.3712), which illustrated a molecular formula C₃₁H₅₂O₅. The ¹H and ¹³C NMR data (Tables 1 and 2) of 4 were closely identical to 3 except for the side chain at C-17. A pair of terminal disubstitutd double bond carbon singlets at δ_C 148.8 (C-25) and δ_C 111.6 (C-26), one quaternary oxygen-bearing carbon at δ_C 75.6 (C-20), together with one oxygenated methine at δ_C 77.4 (C-24) suggested that compound 4 and 2 have the same side chain connected at C-17 (Table 2). Consequently, compound 4 was deduced as 11R,20S,24S-trihydroxy-3,4-seco-dammara-4(28),25-dien,3-oic acid methyl ester.

Qingqianliusu E (5) was isolated as a white amorphous powder with [α]23 D = + 39.4 (c 0.33, MeOH), which has the same molecular formula of C₃₁H₅₂O₅ as 4, as evidenced by the ¹³C spectrum (Fig. S46) and its sodium adduct molecular ion [M + Na]⁺ at m/z 527.3717 (calcd. for C₃₁H₅₂O₅Na, 527.3712) in the HR-ESI-MS spectrum (Fig. S42). The ¹H and ¹³C NMR (Tables 1 and 2) of 5 showed almost identical with 4 except for a slight difference in the chemical shifts [Δδ_C = δ_C (5) - δ_C (4)] at C-25 (Δδ_C + 0.35), C-26 (Δδ_C −0.30), and C-27 (Δδ_C + 0.31), which could be attributed to the difference configuration of the hydroxyl group (−OH) at C-24. Therefore, compound 5 was found to be epimer of 4 at C-24 according to spectroscopic data and characterized as 11R,20S,24R-trihydroxy-3,4-seco-dammara-4(28),25-dien,3-oic acid methyl ester.

Qingqianliusu F (6) was also obtained as white amorphous powder with [α]23 D = + 39.9 (c 0.28, MeOH). The molecular formula C₃₁H₅₂O₄ was confirmed by the HR-ESI-MS m/z 511.3764 [M + Na]⁺ (calcd. for C₃₁H₅₂O₄Na, 511.3763) (Fig. S51) and ¹³C NMR data (Table 2). Comprehensive analysis of the NMR data (Tables 1 and 2) of 5 and 6 revealed that compound 6 has one more methylene (δ_H 1.44/1.36, δ_C 23) while one less oxymethine (δ_H 3.91, δ_C 71.6). Moreover, the diagnostic ¹H–¹H COSY spectrum (Fig. S56) of δ_H 1.60 (m, H-9) and δ_H 1.44/1.36 (m, H₂-11) indicated that there was no hydroxyl group substitutied at C-11. Therefore, the structure of 6 was determined as 20S,24R-dihydroxy-3,4-seco-dammara-4(28),25-dien,3-oic acid methyl ester.

Qingqianliusu G (7) was separated as white amorphous powder with [α]23 D = + 36.0 (c 0.22, MeOH), which had a molecular formula of C₃₀H₅₀O₄ determined by its quasi-molecular ion peak [M + Na]⁺ at m/z 497.3589 (calcd. for C₃₀H₅₀O₄Na, 497.3607) in the HR-ESI-MS (Fig. S60), 14 mass units less than those of 6. The ¹H and ¹³C NMR spectral data (Tables 1 and 2) of 7 were also very similar to those of 6, except for the disappearance of one methoxyl group. As a result, compound 7 was deduced as 20S,24R-dihydroxy-3,4-seco-dammara-4(28),25-dien,3-oic acid.

Qingqianliusu H (8), a white amorphous powder with [α]23 D = + 14.3 (c 0.21, MeOH), had the molecular formula C₃₀H₄₈O₅ by the ¹³C NMR data (Fig. S73) and quasi-molecular ion peak [M + Na]⁺ at m/z 511.3382 (calcd. for C₃₀H₄₈O₅Na, 511.3399) in the HR-ESI-MS (Fig. S69). The IR absorption (Fig. S71) at 3432, 2868, 1708 and 1692 cm⁻¹ represented the presence of hydroxyl (—OH), aldehyde (—CHO) and carbonyl groups (—COOH), respectively. Analysis of the 1D and 2D NMR data (Tables 1 and 2) showed 8 was the high similarity with those of the know compound 20S,24S-dihydroxy-3,4-seco-dammara-4(28),25-dien, 3-oic acid (15). The only slight difference in the group observed was the methyl group (—CH₃) in 15 replaced by the aldehyde group (—CHO) in 8 at C-4. The correlations from δ_H 6.42 (H-28a)/6.22 (H-28b) to δ_C 196.6 (C-29)/40.2 (C-5) in the HMBC spectrum (Fig. S1 and Fig. S76) proved the linkage of aldehyde group located at C-4. These data generated the assignment of 8 as 20S,24S-dihydroxy-3,4-seco-dammara-4(28),25-dien,4-aldehyde group,3-oic acid.

Qingqianliusu I (9) was isolated as a colorless amorphous powder with [α]23 D = + 33.2 (c 0.21, MeOH). The molecular formula of 9 was determined to be C₃₂H₅₄O₅ by ¹³C NMR spectrum (Fig. S82) and positive-ion peak [M + Na]⁺ at m/z 541.3869 (calcd. for C₃₂H₅₄O₅Na, 541.3868) in the HR-ESI-MS (Fig. S78). The 1D and 2D NMR data (Tables 1 and 2) were nearly identical with cyclocariol E (18) (Chen et al., 2018), except for the replacement of the methoxy moiety (—OCH₃) by ethoxy unit (—OCH₂CH₃) at C-3 in 9. Furthermore, the crucial ¹H–¹H COSY (Fig. S83) correlation of 9 illustrated the presence of ethyl fragment δ_C 4.11 (H-3-COOCH₂CH₃) and 1.24 (H-3-COOCH₂CH₃). In its key HMBC correlation (Fig. S85) from δ_H 4.11 (H-3-COOCH₂CH₃) to δ_C 175.8 (C-3) suggested that ester ethyl unit (—OCH₂CH₃) at C-3. Therefore, the structure of 9 was determined as 23(E),12R,20S-11,20,25-trihydroxy-3,4-seco-dammara-4(28),23-dien-3-oic acid ethyl ester.

Qingqianliusu J (10), a white amorphous powder with [α]23 D = + 7.2 (c 0.29, MeOH), was assigned as molecular formula of C₃₉H₆₄O₁₀ determined on the basis of a positive-ion peak [M + Na]⁺ m/z 715.4400 (calcd. for C₃₉H₆₄O₁₀Na, 715.4397) in the HR-ESI-MS (Fig. S87). A detailed analysis of 1D NMR data (Table 1 and Table 2), compound 10 were almost similar to 9 except for the additional glycosyl derivatives unit, which was proven by presence seven carbon singlets including one carbonyl signal at δ_H 172.6 (C-5′–OCOCH₃); four oxygenated methines at δ_H 103.5 (C-1′), 83.7 (C-2′), 82.1 (C-4′), and 77.9 (C-3′); one oxygenated methylene at 65.2 (C-5′); and one methyl signal at δ_H 20.7 (C-5′–OCOCH₃). The glycosyl unit can be identified as α-arabinose by comparing the ¹³C NMR data with known compound cyclocarioside O (Liu et al., 2020). In addition, an acetoxy group (—OCOCH₃) attached to the C-5′ of α-arabinose based on the HMBC correlations (Fig. S1 and Fig. S94) of δ_H 4.26 (H-5′a)/4.13 (H-5′b) with δ_C 172.6 (C-5′–OCOCH₃). Then, the key HMBC correlations from anomeric hydrogen δ_H 5.27 (H-1′) with δ_C 83.8 (C-20) determined the position of glycosyl unit was located at C-20. Besides, the sugar was recognized with acid hydrolysis and HPLC analysis (Fig. S5) of l-arabinose standard. Therefore, the structure of 10 was deduced as 23(E),12R,20S-12,20,25-trihydroxy-3,4-seco-dammara-4(28),23-dien,3-oic acid ethyl ester-20-O-α-L-(5′-O-acetyl)-arabinofuranoside.

Qingqianliusu K (11) was deduced as a white amorphous powder with [α]23 D = + 12.0 (c 0.25, MeOH). The molecular formula of 11, C₄₁H₆₈O₁₀, yielded a positive sodium ion HR-ESI-MS peak (Fig. S96) at m/z 743.4718 [M + Na]⁺ (calcd. for C₄₁H₆₈O₁₀Na, 743.4710), which is 28 Da more than that of 10. Detailed comparison of the ¹H and ¹³C NMR data (Tables 1 and 2) showed that 11 was almost similar to 10, except for the hydroxyl group (–OH) at C-25 was replaced by the ethoxy (–OCH₂CH₃), which was proven by the key HMBC correlations (Fig. S103) from δ_H 3.39 (H-25-OCH₂CH₃) to δ_C 76.3 (C-25). In addition, the glycosyl unit was assigned as α-l-arabinofuranoside based on acid hydrolysis and HPLC analysis (Fig. S5). Therefore, compound 11 was a characteristic 3,4-seco-dammarane triterpenoid glycoside named as 23(E),12R,20S-12,20,25-trihydroxy-3,4-seco-dammara-4(28),23-dien,3-oic acid ethyl ester-25-O-ethyl,20-O-α-L-(5′-O-acetyl)-arabinofuranoside.

Qingqianliusu L (12) was obtained as a white amorphous powder and had a molecular formula of C₃₂H₅₄O₅, which was established by HR-ESI-MS (Fig. S105) from a positive sodium ion at m/z 541.3863 [M + Na]⁺ (calcd. for C₃₂H₅₄O₅Na, 541.3869). Comparing the ¹H and ¹³C NMR data (Tables 1 and 2) of 12 and cyclocariol B (16)(Chen et al., 2018), which demonstrated a considerable degree of similarity except for the replacement of the ester methoxy moiety (—OCH₃) by ethoxy unit (—OCH₂CH₃) at C-3 in 12. Accordingly, the structure of 12 was deduced as 12R,20S,24S-trihydroxy-3,4-seco-dammara-4(28),25-dien,3-oic acid ethyl ester.

Qingqianliusu M (13), a white amorphous powder with [α]23 D = + 40.8 (c 0.59, MeOH), had a molecular formula of C₃₂H₅₄O₅, the same as that 12, as determined by the [M + Na]⁺ ion peaks at m/z 517.3859 (calcd. for C₃₂H₅₄O₅Na, 541.3869) in the HR-ESI-MS (Fig. S114). Detailed analysis of 1D NMR spectroscopic (Tables 1 and 2), compound 13 contained exactly a 3,4-seco-dammarane triterpenoids skeleton as 12, except for a slight difference in the chemical shifts [Δδ_C = δ_C (13) - δ_C (12)] at C-25 (Δδ_C + 0.39), C-26 (Δδ_C −0.34), and C-27 (Δδ_C + 0.27), which suggested that 13 was a epimer at C-24 of 12. Therefore, compound 13 was elucidated as 12R,20S,24R-trihydroxy-3,4-seco-dammara-4(28),25-dien,3-oic acid ethyl ester.

Qingqianliusu N (14) was obtained as a white amorphous powder with [α]23 D = + 7.2 (c 0.28, MeOH). It provided the molecular formula C₃₉H₆₄O₁₀, which was determined by HRESI-MS ion [M + Na]⁺ at m/z 715.4382 (calcd. for C₃₉H₆₄O₁₀Na, 715.4397) (Fig. S123). The 1D NMR data (Tables 1 and 2) of 14 were highly analogous to those of 13 except for the additional glycosyl derivatives unit, which indicated that 14 also possesses a 3,4-seco-dammarane triterpenoids skeleton. The glycosyl unit of 14 was identified as α-(5′-O-acetyl)-arabinofuranoside by comparing the ¹³C data (Table 2) of compounds 10 and 11. Moreover, the acid hydrolysis results were also in agreement with the presence of l-arabinofuranoside standard (Fig. S5). Thus, 14 was identified as 12R,20S,24R-trihydroxy-3,4-seco-dammara-4(28),25-dien,3-oic acid methyl ester-20-O-α-L-(5′-O-acetyl)-arabinofuranoside.

3.2 Determination of the absolute configurations of isomers

To further clarify the absolute configuration of the hydroxyl group (—OH) substituted at the C-24, according to the ¹³C NMR date (Table 2) of C-25 to C-27 in the four pairs of epimers 24R/24S (17/16, 13/12, 7/15, and 5/4) were compared and analyzed. One pair of known isomers cyclocariol B (16) and cyclocariol A (17) has been isolated by our group. The X-ray crystallographic analysis of 17 demonstrated that the absolute configuration at C-24 was R. As shown in Table 3 and Fig. 4, the rules demonstrated that the slight variations between the epimers were the chemical shifts (24R-24S) at C-25 (Δδ_C + 0.20 to + 0.41), C-26 (Δδ_C −0.34 to −0.30), and C-27 (Δδ_C + 0.25 to + 0.32). Apparently, it could be basically inferred that compounds 5, 7, and 13 were found to be R configuration at C-24, while its corresponding epimers 4, 15, and 12 were in the S configuration.

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Fig. 4

Comparison of key ¹³C NMR data differences for the isomers 24R (represented in blue) and 24S (represented in red) (CD₃OD, 150 MHz).

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3.3 α-Glucosidase inhibitory activity assay

Diabetes mellitus is a complex chronic metabolic disorder with an increasing prevalence every year (Xiao et al., 2017; Zhu et al., 2015). The inhibitors of α-glucosidase had become one of the clinically effective drugs for the prevention and treatment of diabetes (Chen et al., 2021; Li et al., 2012). On this basis, in order to further explore the potential hypoglycemic effect of the active components in Qingqianliu, α-glucosidase inhibitory activity was tested on fourteen 3,4-seco-dammarane triterpenoids (1–14). The results (Table 4) showed that only three compounds 8, 11, and 14 exhibited strong α-glucosidase inhibitory activities with the IC₅₀ ranges from 3.76 ± 0.77 to 7.08 ± 0.53 μM, while compound 10 showed weak activity with IC₅₀ value of 19.62 ± 0.70 μM, as compared with positive control acarbose (IC₅₀ 1.07 ± 0.31 μM). The structure–activity relationship analysis indicated that the presences of the aldehyde group at C-4 and arabinose unit at C-20 could improve the α-glucosidase inhibitory activity.

3.4 Anti-inflammatory activity against COX-2

COX-2, known as Prostaglandin-endoperoxide synthase (PTGS), keeps closely related to the occurrence of inflammation and tumors (Jiang et al., 2022). The inhibitors of COX-2, which become a hot spot in drug research, could prevented inflammation and the formation of malignant tumors by blocking prostaglandin synthesis (Liu et al., 2020; Shataer et al., 2021). With celecoxib as positive control, the COX-2 inhibitory activity of compounds (1–14) were evaluated. The results were demonstrated in the Table 5. Only six compounds 1, 2, 4, 6, 10, and 11 showed COX-2 inhibitory activity with inhibition rates from 10.23 % to 35.83 % at the concentration of 25 µM. Among them, compound 11 had the highest inhibition rate and reached half of the inhibition rate of celecoxib. The results indicated that an additional sugar moiety at C-20 plays an important role in COX-2 inhibitory activity.

3.5 Cytotoxic bioassays

All unreported 3,4-seco-dammarane triterpenoids (1–14) were evaluated for their cytotoxicities against for human cancer cell lines (BGC-823, MCF-7, HCT-116, and HepG-2). As shown in Table 6, compounds 3, 7, 10, and 13 displayed moderate activities against BGC-823 with IC₅₀ values from 7.69 ± 0.21 to 9.04 ± 0.61. Moreover, compound 13 showed modest activity against MCF-7 and HCT-116 with IC₅₀ values of 9.19 ± 0.78 and 8.80 ± 0.36. respectively. Structurally, compounds 13 and 12 as a pair of isomers, which 13 exhibited stronger activities than 12. It can be inferred that the hydroxyl group at the C-24 was R configuration in the same type of 3,4-seco-dammarane triterpenoids skeleton possess potential cytotoxicity effect.