Cytotoxic diterpenoids from the roots of Euphorbia jolkinii Boiss against human pancreatic cancer SW1990 cells by regulating the expressions of Bcl-2, Bax, and Caspase-3 proteins

2.1 General experimental procedures

The high-resolution mass spectra were detected on LC-MS-IT-TOF (Shimaduz, Japan) and Agilent LC-MS-Q-TOF G6530 (Agilent Technologies, Santa Clara, USA). NMR data were conducted with a Brucker Advance III-600 spectrometer (Brucker, Massachusetts, USA) using tetramethylsilane (TMS) as an internal standard. The ultraviolet (UV) spectra were measured on a UV-2401 UV spectrometer (Shimadzu, Kyoto, Japan) and the electronic circular dichroism (ECD) spectra were recorded using a Chirascan instrument (Applied Photophysics, Surrey, UK). Infrared (IR) spectra were performed using KBr lithography on a NICOLET iS10 infrared spectrometer (Thermo Fisher Scientific Inc, Massachusetts, USA). Optical rotation data (ORD) were collected with Jascomodel 1020 polarimeter (Horiba, Tokyo, Japan). The LC-52 system (Beijing Saipuruisi Technology Co., Ltd., Beijing, China) was employed for high-performance liquid chromatography (HPLC) purifications with a ReproSil-Pur 120 CN column (10 × 250 mm, 5 μm). Silica gel (200–300 mesh, Qingdao Makall group Co. Ltd., Qingdao, China), Sephadex LH-20 (20–50 μm, Amersham Bioscience, Sweden), MCI gel CHP 20P (Mitsubishi Chemical Corporation, Tokyo, Japan), and Rp-C18 column (40–70 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), and thin-layer chromatography plate (GF254, 200 × 200 mm, Liyan Technology Co., Ltd., Kunming, China) were employed for the isolations. The thin layer chromatography (TLC) plate was sprayed with a 10 % H₂SO₄-EtOH (v/v) solution as a chromogenic agent. Microplate reader (HBS-1096B, DeTie, Nanjing, China) and thermostatic cell incubator (Thermo Forma 3310, Thermo Fisher Scientific Inc, Massachusetts, USA) were used. Fluorescein FITC-labelled Annexin-V, Propidine iodide (PI) and CCK-8 kit were obtained form BD Pharmingen, San Diego, USA. Paclitaxel (HPLC ≥ 98 %) was purchased from Liyan Technology Co., Ltd., Kunming, China. Primary antibodies against Bcl-2 (ab194583, abcam), Bax (ab32503, abcam), Caspase-3 (ab184787, abcam), β-actin (ab8226, abcam) were were purchased from Abcam Co. Ltd., Cambridge, UK. Human pancreatic cancer cells (SW1990) were obtained from the Kunming Cell Bank of the Typical Culture Preservation Committee of the Chinese Academy of Sciences.

2.2 Plant materials

The roots of Euphorbia jolkinii Boiss were collected in Shangri-la county, Yunnan province, in August 2020 and authenticated by Prof. Lu Lu (Kunming Medical University). A voucher specimen (No. 2020-0812) was deposited in Yunnan Key Laboratory of Southern Medicine Utilization.

2.3 Extraction and isolation

30.0 Kg of E. jolkinii roots were smashed and extracted with MeOH (3 × 150 L) for three times at room temperature. The combined methanol extracts were suspended in H₂O and extracted with ethyl acetate to obtain the EtOAc part (1.5 kg). Then the EtOAc fraction was fractionated by D101 macroporous resin column using an EtOH-H₂O gradient system (30:70, 50:50, 70:30, and 90:10, v/v) to afford four fractions (Fr.1–Fr.4).

Fr.3 (480 g) was subjected to MCI column chromatography (CC) and eluted with a gradient system of MeOH-H₂O (50:50–90:10, v/v) to give four fractions, Fr.3-1–Fr.3-4. Fr.3-2 (95.6 g) was subjected to silica gel CC eluting with a CH₂Cl₂-EtOAc gradient system (95:5, 80:20, and 70:30, v/v) to yield five fractions, Fr.3-2-1–Fr.3-2-5. Fr. 3-2-2 (15.3 g) was separated on RP-C18 CC and eluted with MeOH-H₂O (40:60–80:20, v/v) to afford four fractions, Fr.3-2-2-1–Fr.3-2-2-4. Fr.3-2-2-2 (2.5 g) was chromatographed on Sephadex LH-20 CC washing with MeOH and further separated by the semi-preparative HPLC (MeOH-H₂O, 60:40, v/v, λ = 254/210 nm, 3.0 mL/min) to afford compounds 11 (t_R = 15 min, 15 mg), 16 (t_R = 17 min, 17 mg), 13 (t_R = 18 min, 5 mg), and 12 (t_R = 21 min, 7 mg). Fr.3-2-2-3 (1.7 g) was subjected to silica gel CC eluting with CH₂Cl₂-MeOH (98:2 and 95:5, v/v) and then isolated by semi-preparative HPLC (MeCN-H₂O, 40:60, v/v, λ = 254/202 nm, 3.0 mL/min) to provide compounds 8 (t_R = 16 min, 25 mg), 10 (t_R = 18 min, 7 mg), 9 (t_R = 19 min, 49 mg), and 7 (t_R = 22 min, 8 mg). Fr. 3-2-3 (9.6 g) was isolated by RP-C8 CC eluted with MeOH-H₂O (30:70–70:30, v/v) to afford five fractions, Fr.3-2-3-1–Fr.3-2-3-5. Fr.3-2-3-2 (1.1 g) was repeatedly separated on Sephadex LH-20 CC (MeOH) and semi-preparative HPLC (MeCN-H₂O, 30:70 → 90:10, v/v, λ = 254/210 nm, 3.0 mL/min) to obtain compounds 5 (t_R = 19 min, 21 mg), 6 (t_R = 21 min, 28 mg), 14 (t_R = 22 min, 5 mg), and 15 (t_R = 25 min, 26 mg). Fr. 3-3-3-3 (3.3 g) was fractionated on a Rp-C18 CC (MeOH-H₂O, 30:70, 50:50, 70:30, and 90:10, v/v), silica gel CC (petroleum ether-EtOAc, 10:90–70:30, v/v), semi-preparative HPLC with YMC-Pack ODS-A column (MeCN-H₂O, 50:50, v/v, λ = 254/210 nm, 3.0 mL/min) to give out compounds 1 (t_R = 17 min, 41 mg), 2 (t_R = 18 min, 38 mg), 4 (t_R = 20 min, 7 mg), and 3 (t_R = 22 min, 11 mg).

Jolkiniiol A (1). White solids, C₂₀H₃₀O₃, (+)-HR-ESI-MS [M + H]⁺ m/z 319.2274 (calcd. 319.2268, +1.9 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{23.7}$  − 13.86 (c 0.07, MeOH); UV (MeOH) λ_max (log ε) 203 (4.15), 280 (3.84) nm; IR (KBr) ν_max 3372, 2926, 1615, 1458, 1386, 1277, 1057, 1022, 988, 902, 871, 615 cm⁻¹; ECD (c 0.35, MeOH) λ_max (Δε) 207 (+3.10), 212 (+2.96), 219 (+3.14), 242 (+3.73), 283 (−1.37), 306 (−0.76), 330 (−1.28) nm; ¹H and ¹³C NMR data: see Table 1.

Jolkiniiol B (2). White solids, C₂₀H₃₀O₃, (−)-HR-ESI-MS [M + Cl]⁻ m/z 353.1884 (calcd. 353.1889, −1.3 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{24.5}$  + 11.08 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 203 (3.75), 284 (3.43) nm; IR (KBr) ν_max 3419, 2924, 1639, 1454, 1379, 1278, 1152, 1094, 1055, 884, 721 cm⁻¹; ECD (c 0.50, MeOH) λ_max (Δε) 228 (+0.26), 247 (+1.27), 266 (+0.77), 287 (+1.14), 334 (−1.66) nm; ¹H and ¹³C NMR data: see Table 1.

Jolkiniiol C (3). White solids, C₂₂H₃₂O₄, (+)-HR-ESI-MS [M + H]⁺ m/z 361.2389 (calcd. 361.2373, +4.0 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25.9}$  − 46.80 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 228 (3.96) nm; IR (KBr) ν_max 3444, 2936, 1739, 1667, 1448, 1386, 1237, 1040, 918, 828, 585 cm⁻¹; ECD (c 0.33, MeOH), λ_max (Δε) 207 (+5.08), 236 (−9.30), 342 (+2.08) nm; ¹H and ¹³C NMR data: see Table 1.

Jolkiniiol D (4). White solids, C₂₂H₃₄O₅, (−)-HR-ESI-MS [M + HCOO]⁻ m/z 423.2378 (calcd. 423.2388, −2.2 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25.2}$  − 6.48 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 202 (2.97), 265 (2.04) nm; IR (KBr) ν_max 3432, 2931, 1702, 1449, 1384, 1250, 1051 cm⁻¹; ECD (c 0.50, MeOH) λ_max (Δε) 211 (+0.31) nm; ¹H and ¹³C NMR data: see Table 1.

Jolkiniiol E (5). White solids, C₂₀H₂₈O₃, (+)-HR-ESI-MS [M + H]⁺ m/z 317.2112 (calcd. 317.2111, +0.7 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{24.8}$  + 10.72 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 202 (3.60), 224 (3.52) nm; IR (KBr) ν_max 3431, 2928, 1671, 1382, 1273, 1004, 830, 580 cm⁻¹; ECD (c 0.50, MeOH) λ_max (Δε) 210 (+3.61), 242 (−0.28) nm, 355 (+0.34); ¹H and ¹³C NMR data: see Table 2.

Jolkiniiol F (6). White solids, C₂₀H₂₈O₃, HR-ESI-MS [M + H]⁺ m/z 317.2115 (calcd. 317.2111, +0.9 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{24.9}$  + 20.96 (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 203 (3.92), 267 (3.80) nm; IR (KBr) ν_max 3410, 2971, 1647, 1410, 1221, 1059, 915, 643 cm⁻¹; ECD (c 0.30, MeOH) λ_max (Δε) 202 (+5.92), 224 (+0.60), 245 (+0.90), 285(−0.12), 332 (+0.54) nm; ¹H and ¹³C NMR data: see Table 2.

Jolkiniiol G (7). White solids, C₂₂H₃₀O₄, (+)-HR-ESI-MS [M + H]⁺ m/z 359.2208 (calcd. 359.2217, −2.5 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{24.3}$  + 5.17 (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 202 (3.05), 215 (2.88) nm; IR (KBr) ν_max 3439, 2924, 1637, 1383, 1043, 668 cm⁻¹; ECD (c 0.60, MeOH) λ_max (Δε) 199 (+1.56), 233 (−0.24), 355 (+0.31) nm; ¹H and ¹³C NMR data: see Table 2.

Jolkiniiol H (8). White solids, C₂₂H₃₂O₃, (+)-HR-ESI-MS [M + Na]⁺ m/z 367.2248 (calcd. 367.2244, +1.0 ppm), ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25.2}$  − 32.00 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 205 (3.78), 224 (3.81) nm; IR (KBr) ν_max 3436, 2971, 1716, 1668, 1455, 1385, 1262, 1064, 830, 581 cm⁻¹; ECD (c 0.43, MeOH) λ_max (Δε) 204 (+0.37), 222 (−3.41), 256 (+0.63), 348 (+0.41) nm; ¹H and ¹³C NMR data: see Table 2.

Jolkiniiol I (9). White solids, C₂₂H₃₂O₃, (+)-HR-ESI-MS [M + H]⁺ m/z 345.2427 (calcd. 345.2424, +0.6 ppm), ${[\mathrm{\alpha }]}_{\mathrm{D}}^{24.4}$  − 25.73 (c 0.09, MeOH); UV (MeOH) λ_max (log ε) 203 (3.46), 225 (3.53) nm; IR (KBr) ν_max 3435, 2969, 1453, 1384, 1075, 831, 568 cm⁻¹; ECD (c 0.69, MeOH) λ_max (Δε) 207 (−1.38), 226 (−4.36), 263 (+0.19), 344 (+0.56) nm; ¹H and ¹³C NMR data: see Table 3.

Jolkiniiol J (10). White solids, C₂₂H₃₂O₃, (+)-HR-ESI-MS [M + H]⁺ m/z 345.2433 (calcd. 345.2424, +2.5 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25.3}$  − 10.00 (c 0.10, MeOH); UV (MeOH) λ_max(log ε) 201 (2.02) nm; IR (KBr) ν_max 3437, 2923, 1637, 1383, 1047, 373 cm⁻¹; ECD (c 1.00, MeOH) λ_max (Δε) 225 (−0.27), 355 (+0.39); ¹H and ¹³C NMR data: see Table 3.

Jolkiniiol K (11). White solids, C₂₀H₃₀O₃, (+)-HR-ESI-MS [M + H]⁺ m/z 319.2270 (calcd. 319.2268, +0.6 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{24.0}$  + 10.80 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 203 (3.65), 238 (2.83) nm; IR (KBr) ν_max 3434, 2970, 1716, 1636, 1390, 1111, 1058, 921, 587 cm⁻¹; ECD (c 0.33, MeOH) λ_max (Δε) 226 (+0.15), 248 (+0.42) nm; ¹H and ¹³C NMR data: see Table 3.

Jolkiniiol L (12). White solids, C₂₀H₃₀O₃, (+)-HR-ESI-MS [M + H]⁺ m/z 319.2270 (calcd. 319.2268, +0.6 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{24.8}$  − 56.32 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 204 (3.96), 239 (3.27) nm; IR (KBr) ν_max 3456, 2954, 1701, 1388, 1280, 1117, 1055, 926, 657 cm⁻¹; ECD (c 0.33, MeOH) λ_max (Δε) 229 (+0.65), 289 (−0.56) nm; ¹H and ¹³C NMR data: see Table 3.

Jolkiniiol M (13). White solids, C₂₀H₂₈O₃, (+)-HR-ESI-MS [M + Na]⁺ m/z m/z 339.1932 (calcd. 339.1931, +0.3 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{24.1}$  + 8.04 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 200 (3.20), 240 (3.47) nm; IR (KBr) ν_max 3432, 2968, 2927, 1714, 1634, 1386, 1064, 596 cm⁻¹; ECD (c 0.50, MeOH) λ_max (Δε) 205 (+0.21), 235 (+0.79) nm; ¹H and ¹³C NMR data: see Table 4.

Jolkiniiol N (14). White solids, C₂₀H₂₆O₄, (+)-HR-ESI-MS [M + H]⁺ m/z 331.1915 (calcd. 331.1904, +1.4 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25.2}$  + 3.84 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 202 (3.01), 215 (2.94), 238 (2.88) nm; IR (KBr) ν_max 3433, 2927, 1718, 1668, 1383, 1113, 585 cm⁻¹; ECD（c 0.50,MeOH）λ_max (Δε) 221 (+0.36), 249 (+0.10) nm; ¹H and ¹³C NMR data: see Table 4.

Jolkiniiol O (15), white solids, C₂₀H₂₆O₃, (+)-HR-ESI-MS [M + H]⁺ m/z 315.1960 (calcd. 315.1955, +1.3 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{25.4}$ + 98.40 (c 0.15, MeOH); UV (MeOH) λ_max (log ε) 202 (3.85), 229 (3.79), 290 (3.79) nm; IR (KBr) ν_max 3437, 2972, 1719, 1669, 1454, 1394, 1264, 1194, 1113, 1070, 928 cm⁻¹; ECD (c 0.45, MeOH) λ_max (Δε) 212 (−1.86), 288 (+5.47), 336 (−0.57) nm; ¹H and ¹³C NMR data: see Table 4.

Jolkiniiol P (16). white solids, C₂₀H₃₂O₂, (+)-HR-ESI-MS [M + H]⁺ m/z 305.2462. (calcd. 305.2475, −4.3 ppm); ${[\mathrm{\alpha }]}_{\mathrm{D}}^{24.1}$  − 21.6 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 204 (3.58) nm; IR (KBr) ν_max 3429, 2930, 1719, 1633, 1457, 1385, 1035, 919, 556 cm⁻¹; ECD (c 0.33, MeOH) λ_max (Δε) 248 (+0.23) nm; ¹H and ¹³C NMR data: see Table 4.

2.4 Cytotoxicity assay

The cytotoxicity of the compounds was detected by CKK-8 method. Pancreatic cancer cells were cultured in a 5 % carbon dioxide incubator containing at 37 °C. The culture mediums were composed of 89 % DMEM, 10 % fetal bovine serum, and 1 % penicillin–streptomycin solution. The cells were digested with 2 mL trypsin for 2 min when the density of cells on the petri dish was greater than 85 %. And then the cells were added with 4 mL mediums and transferred to a 15 mL centrifuge tube via trypsin for centrifugation. The cells were inoculated into a 96-well plate with 100 μL per well after centrifugation and inoculated continuously for 24 h. Then the cells were treated with the compounds and paclitaxel as positive control. The CCK-8 reagents were diluted ten-fold with DMEM medium 24 h later and the cells were cultivated for 2 h. Finally, the absorbances were measured by a microplate reader at 450 nm. Inhibition ratios were calculated as [A_control − A_sample]/A_control × 100 %. The IC₅₀ values were calculated according to Prism.8 software.

2.5 Flow cytometric analysis

Fluorescein FITC-labelled Annexin-V is applied to label the early apoptotic cells due to its high combining capacity with the phosphatidylserine in early apoptotic cells. Propidine Iodide (PI), a nucleic acid dye, is not permeable to the normal cell membranes. But it can through the membranes of apoptotic cells at middle and late-stage (Wei et al., 2023). Thus, Annexin-V and PI are utilized to detect apoptotic cells to determine the cell cycles. Firstly, the tumor cells were inoculated in 6-well plates at an inoculum density of 2 × 10⁶ cells/well. 24 h later, compounds 1 (13 μM, 26 μM), 9 (11 μM, 22 μM), and positive drug (paclitaxel 45 μM) were added. After 3 h, the cells were transferred to a 5 mL centrifuge tube via trypsin for centrifugation. Then FITC (5 μL) and PI (5 μL) were added sequentially into the cells for the incubation at room temperature of 15 min. Then 400 μL PBS were added to each culture tube, and the cells were subjected to the flow cytometer. The data were analyzed by FlowJo_v10.8.1 software.

2.6 Western blot assay

The Western blot assay was prepared according to our previous report (Chen et al., 2022). The cell culture procedures were similar with cytotoxicity assay. According to the IC₅₀ values, the concentrations of the samples were set as: 2.5 μM, 5 μM, and 10 μM for compound 1, 3 μM, 6 μM, and 12 μM for compound 9 and 20 μM for positive control. The mediums were removed 3 h later. Then the RIPA lysis buffer (protease inhibitor: EDTA = 98:1:1) was added to lyse the cells for 1 h. After the centrifugation at 13,200 rpm × 30 min, the supernatants were collected and 1/3 vol of protein buffer (β-hydrophobic ethanol: 4 × SDS concentrated gel buffer solution = 1:9) were added. The supernatants were subjected to heat denaturation at 95 °C for 5 min and stored at − 20 °C refrigerator. 30 μg proteins were added in SDS-PAGE and electroblotted onto PVDF membrane. The membranes were blocked with 5 % skim milk in TBS-T, and probed with desired antibodies. Super Signal West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA) was used to detect antibody-antigen complexes. The data were collated and analyzed by ChemiScope Analysis and Prism.8 software.

2.7 Molecular docking and druggability prediction

The molecular docking assay was prepared according to our previous report (Yang et al., 2023). The 3D structure of Bcl-2 protein was downloaded from Protein Data Bank (PDB, https://www.rcsb.org). Molecular docking was conducted using Discovery Studio 4.0 software. LibDockScores of compounds with target proteins were used to evaluate their affinity and the docking results were drawn using PyMol software. Discovery Studio 2016 Client software was used to predict the pharmacokinetics of the compounds.

2.8 Statistical analysis

All the data were statistically analyzed by using GraphPad prism 8.0 statistical software and expressed as mean ± standard deviations (SDs). Multiple comparisons between groups were performed using one-way ANOVA, and pairwise comparisons were conducted by independent t-test. In all cases, differences with P < 0.05 were considered statistically significant.

3.1 Structure elucidation

Compound 1 was obtained as white solids with the molecular formula as C₂₀H₃₀O₃ which was determined by the (+)-HR-ESI-MS at m/z 319.2274 ([M + H]⁺ calcd. 319.2268) indicating six unsaturation degrees. The ¹H NMR spectrum of 1 exhibited four olefinic protons at δ_H 5.22 (1H, t, J = 6.8 Hz), 6.26 (1H, d, J = 9.5 Hz), 6.42 (1H, d, J = 16.5 Hz), and 6.63 (1H, d, J = 16.5 Hz) and five methyl signals at δ_H 1.01 (3H, s), 1.18 (3H, s), 1.33 (3H, s), 1.61 (3H, s), and 1.90 (3H, s) (Table 1). The ¹³C NMR spectrum presented 20 carbons including five methyls, three methylenes, seven methines, and five quaternary carbons from which a carbonyl unit at δ_C 195.6 and three double bonds at δ_C 147.1 (CH), 143.8 (CH), 138.3 (C), 137.8 (C), 128.5 (CH), and 119.8 (CH) were deduced. Thus, the remaining two unsaturation degrees required by the molecular formula of 1 were attributed to two rings. The presence of a quaternary carbon at δ_C 26.0 (C-15) and a gem-dimethyl functionality at δ_C 29.0 (C-16)/16.1 (C-17), together with the ¹H–¹H-COSY correlations between two cyclopropyl protons of H-1 (δ_H 1.15, overlapped) and H-2 (δ_H 1.48, t, 8.6), indicated the presence of a substituted cyclopropyl ring (Fig. 2). Consequently, compound 1 was deduced to be a casbane diterpenoid with a 14-membered macrocycle. The ¹H–¹H-COSY correlations of H-2 and the olefinic proton at δ_H 6.26 suggested a double bond (δ_C 143.8 and 137.8) was located between C-3 and C-4. Both of H-3 and H₃-18 showed HMBC correlations to the carbonyl unit indicated that C-5 was oxygenated as a carbonyl group. Furthermore, the olefinic proton at δ_H 6.63 presented ¹H–¹H-COSY correlations with δ_H 6.42 and HMBC correlations with C-5 which suggested a disubstituted double bond was posited at C-6 and C-7. The double bond of Δ¹¹⁽¹²⁾ was determined by the HMBC cross peaks from H₃-20 to δ_C 119.8 (C-11) and 138.3 (C-12). An oxygenated quaternary carbon and an oxygenated methine were deduced as C-8 (δ_C 75.4) and C-9 (δ_C 78.7) respectively via the HMBC correlations of H-6/C-8 and H₃-19/C-9. Δ³⁽⁴⁾, Δ⁶⁽⁷⁾, and Δ¹¹⁽¹²⁾ were all E geometry according to the δ_C values of CH₃-18/CH₃-20 (<20 ppm) and the J values of H-6/H-7 (>16 Hz) (Li et al., 2010). The junction of the two rings between C-1 and C-2 was suggested to be cis on the basis of ¹³C NMR chemical shifts of C-16 (δ_C 29.0) and C-17 (δ_C 16.1), analogously with previous cis-fused casbane diterpenoids (Zhang et al., 2005; Li et al., 2010). Two hydroxyls were located at the same side verified by the ROESY correlations of H₃-19 and H-9 (Fig. 3). Furthermore, the similarity Cotton effect of the experimental electronic circular dichroism (ECD) spectrum of compound 1 between the calculated ECD spectrum of compounds 1a (1,2-cis-8α,9α-dihydroxyl) (Fig. 4) established compound 1 as (1S,2R,8R,9S)-8,9-diydroxyl-casbane-3,6,11-triene-5-one and named as jolkiniiol A.

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Fig. 2

The key 2D NMR of compounds 1–5 and 7–16.

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Fig. 3

The ROESY correlations of compounds 1–16.

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Fig. 4

The ECD calculations of compounds 1–16.

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Fig. 4

The ECD calculations of compounds 1–16.

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The molecular formula of compound 2 was C₂₀H₃₀O₃ according to the (−)-HR-ESI-MS at m/z 353.1884 ([M + Cl]⁻calcd. 353.1889). The NMR data of 2 were similar with those of 1 except for the chemical shifts of C-1 (δ_C 38.0), C-2 (δ_C 32.2), C-16 (δ_C 21.8), and C-17 (δ_C 23.5) (Table 1) which suggested the trans configuration of C-1 and C-2 on the basis of trans-fused casbane diterpenoids (Li et al., 2010). The two hydroxyls were located at the same side as well by the ROESY correlations of H₃-19 and H-9 (Fig. 3). 2a (1,2-trans-8α,9α-dihydroxyl) presented almost the same calculated ECD spectrum with the experimental ECD spectrum of 2 (Fig. 4). So, compound 2 was identified as (1R,2R,8R,9S)-8,9-dihydroxyl-casbane-3,6,11-triene-5-one and named as jolkiniiol B.

Compound 3 was white solids whose molecular formula was C₂₂H₃₂O₄ according to the (+)-HR-ESI-MS at m/z 361.2389 ([M + H]⁺ calcd. 361.2373). Two olefinic hydrogens at δ_H 6.92 (1H, d, J = 10.1 Hz) and 5.81 (1H, d, J = 10.1 Hz) were observed (Table 1) and were determined as a 1,2-disubstituted double bond by their ¹H–¹H-COSY correlations (Fig. 2). The ¹H NMR spectrum of 3 also afforded four methyl signals at δ_H 1.08 (3H, s), 1.14 (3H, s), 1.23 (3H, s), and 2.11 (3H, s), and two oxygenated protons at δ_H 3.97 (1H, d, J = 11.4 Hz) and 4.11 (1H, d, J = 11.4 Hz). In the ¹³C NMR spectrum, 22 carbons were deduced as four methyls, seven methylenes, five methines, and six quaternary carbons from which two carbonyl units at δ_C 205.2 and 171.1 were identified. Compared with the know compound, 17-acetoxyl-l6-hydroxyl-ent-atisane-3-one (Awasaki et al., 1987), there was one 1,2-disubstituted double bond in 3. The other NMR data of 3 were similar with those of 17-acetoxyl-16-hydroxyl-ent-atisane-3-one. The HMBC correlatios from δ_H 6.92 to C-5/C-9 and from δ_H 5.81 to C-4/C-10 suggested the double bond was located at C-1 and C-2 (Fig. 2). The oxygenated protons displayed HMBC correlations to a carbonyl unit at δ_C 205.2, an oxygenated quaternary carbon at δ_C 72.7 (C-16), a methylene at δ_C 52.0 (C-15), and a methine at δ_C 32.6 (C-12) which clarified the 17-acetoxyl-16-hydroxyl substituent group of 3. Most of the atisane-type diterpenoids isolated from Euphorbia species were ent-atisane diterpenoids on consideration of biosynthetic pathways (Zhao et al., 2014). Besides, the ROESY correlations of H-5/H-9/H₂-17 were found as well that indicated these H-atoms were all located at β configuration (Fig. 3). Finally, the calculated ECD spectrum of 3 exhibited similar Cotton effects with the experimental ECD spectrum of 3 (Fig. 4). So, compound 3 was identified as (16R)-17-acetoxyl-l6-hydroxyl-ent-atisane-1-ene-3-one and named as jolkiniiol C.

Compound 4 afforded the chemical formula of C₂₂H₃₄O₅ based on the (−)-HR-ESI-MS at m/z 423.2378 ([M + HCOO]⁻ calcd. 423.2388). Compared with the NMR data of 3, an oxygenated methine at δ_C 66.9 and δ_H 4.77 (1H, d, 10.3) (Table 1). At the same time, the absence of the 1,2-disubstituted double bond signal was deduced. On the contrary, two methylenes at δ_C 39.4 and 34.2 were observed and they were conjugated together due to the ¹H–¹H-COSY correlations of their protons (Fig. 2). The HMBC correlation of H₃-20 to the methylene at 39.4 was observed as well. So, the two methylenes were C-1 and C-2 respectively. The ¹H–¹H-COSY correlations from H-9 to δ_H 4.77 suggested that C-11 was an oxygenated methine. The β configuration of H-11 was determined by the ROESY correlations of H-11/H-9/H-5/H₂-17 (Fig. 3). The calculated ECD spectrum of 4 presented similar Cotton effects with the experimental ECD spectrum of 4 (Fig. 4). Thus, 4 was identified as (11S,16R)-17-acetoxyl-11,l6-dihydroxyl-ent-atisane-3-one and named as jolkiniiol D.

According to the (+)-HR-ESI-MS at m/z 317.2112 ([M + H]⁺ calcd. 317.2111), compound 5 afforded the chemical formula of C₂₀H₂₈O₃ as white solids. The ¹H NMR spectrum of 5 revealed the signals of four tertiary methyl units at δ_H 0.88, 1.08, 1.13, and 1.16 (each 3H, s), three vinyl protons for ABX system at δ_H 5.29 (1H, d, J = 17.8 Hz), 5.34 (1H, d, J = 11.0 Hz), and 6.30 (1H, dd, J = 17.8, 11.0 Hz), three other vinyl H-atoms at δ_H 5.88 (1H, br.s), 5.93 (1H, d, J = 10.2 Hz), and 6.92 (1H, d, J = 10.2 Hz), two oxygenated hydrogens at δ_H 3.98 (1H, br.s) and 3.96 (1H, s) (Table 2). Twenty carbons including four methyls, three methylenes, eight methines, and five quaternary carbons were observed from its NMR spectrum in which a carbonyl unit at δ_C 204.2, three pairs of double bonds at δ_C 154.0/142.7/136.8/127.7/125.7/114.4, and two oxygenated C-atoms at δ_C 81.7 and 74.5 were determined. The detailed analysis of the HSQC, ¹H–¹H-COSY, and HMBC (Fig. 2) spectra allowed the position assignment of all the C-atoms and H-atoms. The NMR data of 5 were similar to those of 2,12α-dihydroxyl-ent-isopimara-1,7,15-trien-3-one (Tian et al., 2016) except for the olefinic carbon at δ_C 125.7 (CH, δ_H 5.93) and the oxygenated C-atom at δ_C 81.7 (CH, δ_H 3.96). The ¹H–¹H COSY correlation of δ_H 5.9 and δ_H 6.92 (H-1) suggested that C-2 was a disubstituted olefinic carbon. H-7 and H-15 showed HMBC correlations to δ_C 81.7 which positioned C-14 linked with a hydroxyl group. The ROESY correlations of CH₃-17 with H-12 and H-14 indicated that these H-atoms were presented on the same side as α-configuration (Fig. 3). The (+) Cotton effect due to the n → π* transition at 355 nm according to the octant rule for α,β-unsaturated ketones suggested the trans junction of A and B rings (Mi et al., 1993). Thus, compound 5 was (12β,14β)-12,14-dihydroxyl-ent-pimara-1,7,15-trien-3-one whose absolute configurations was determined as 12R,14S by the calculated ECD method (Fig. 4), and named as jolkiniiol E.

The (+)-HR-ESI-MS at m/z 317.2115 ([M + H]⁺calcd. 317.2111) showed that the molecular formula of 6 was C₂₀H₃₈O₃. The ¹H NMR and ¹³C NMR data reveled that 6 was an ent-isopimara diterpenoid with a carbonyl group, three pairs of double bonds, and two hydroxyl units (Table 2). Huang et al. (Huang et al., 2014) and Tian et al. (Tian et al., 2016) reported almost the same NMR data of two compounds which were similar with those of 6. The former was identified as 2,12α-dihydroxy-ent-isopimara-1,7,15-trien-3-one and the later was identified as 2,12β-dihydroxy-isopimara-1,7,15-trien-3-one. However, H-12 was not exhibited ROESY correlations with H-5 and H-9 which verified the α-configuration of H-12 (Fig. 3). The ROESY correlations of H-12 to CH₃-20 and CH₃-17 also deduced the α-configuration of H-12. So, the hydroxyl group posited at C-12 was β-configuration. The trans junction of A and B rings was determined by the (+) Cotton effect at 355 nm according to the octant rule for α,β-unsaturated ketones (Mi et al., 1993). Compound 6 should be 2,12β-dihydroxy-ent-isopimara-1,7,15-trien-3-one and its absolute configurations was determined as 12R by comparing the experimental ECD and calculated ECD spectra of 6 (Fig. 4) which was named as jolkiniiol F.

Compound 7 was white solids with the molecular formula of C₂₂H₃₀O₄ determined by the (+)-HR-ESI-MS at m/z 359.2208 ([M + H]⁺calcd. 359.2217). In the ¹H NMR spectrum of 7 the most characteristic signals were five tertiary methyl units at δ_H 1.08, 1.09, 1.15, 1.18 and 2.03 (each 3H, s), three vinyl protons for ABX system at δ_H 5.00 (1H, d, J = 17.4 Hz), 5.14 (1H, d, J = 10.6 Hz), and 5.82 (1H, dd, J = 17.4, 10.6 Hz), three other vinyl H-atoms at δ_H 5.60 (1H, s), 5.95 (1H, d, J = 10.2 Hz), and 6.95 (1H, d, J = 10.2 Hz), two oxygenated hydrogens at δ_H 4.40 (1H, br.s) and 4.88 (1H, dd, J = 8.7, 3.4 Hz) (Table 2). Its ¹³C NMR spectrum afforded twenty-two carbons from which two carbonyl units at δ_C 204.2 and 170.8, three pairs of double bonds at δ_C 154.9/141.4/138.2/132.4/126.9/115.0, and two oxygenated C-atoms at δ_C 75.2 and 72.2 were identified. These NMR data were very similar with those of compound 5 except the carbonyl unit at δ_C 170.8 and a tertiary methyl group at δ_H 2.03/δ_C 21.2. They were linked together to form an acetoxyl unit according to the HMBC correlation from δ_H 2.03 to δ_C 170.8 (Fig. 2). Besides, the oxygenated H-atom at δ_H 4.88 showed HMBC correlation with δ_C 170.8 suggested that the acetoxyl unit was located at C-12 position. H-5, H₂-6 and the oxygenated H-atom at δ_H 4.40 were showed ¹H–¹H-COSY correlations together indicated there was a hydroxyl group posited at C-7 position. Both the HMBC correlations from the olefinic proton at δ_H 5.60 to C-7 and C-12 verified the structure of Δ⁸⁽¹⁴⁾. The junction of A and B rings was determined as trans according to the octant rule for α,β-unsaturated ketones (Mi et al., 1993) as well. The ROESY correlations of CH₃-20/H-7 and CH₃-17/H-12 showed that H-7 and H-12 were both located at α-configuration (Fig. 3). Thus, compound 7 was (7β,12β)-12-acetoxyl-7-hydroxyl-ent-isopimara-1,8(14),15-trien-3-one whose absolute configurations were determined as 7S,12R by the calculated ECD spectrum of 7 (Fig. 4) and named as jolkiniiol G.

Compounds 8–10 gave the same molecular formula of C₂₂H₃₂O₃ according to the (+)-HR-ESI-MS data. It found five methyl units, three vinyl protons for ABX system, two other vinyl H-atoms, and four oxygenated hydrogens in their ¹H NMR spectra respectively (Tables 2 and 3). Their ¹³C NMR spectra afforded twenty-two carbons including a carbonyl unit, three pairs of double bonds, and three oxygenated C-atoms as well. They gave out an oxygenated methylene at δ_C 64, a methyl group at δ_C 15, and a double bond at 135/128 without vinyl H-atom which were different from those of 5. The ¹H–¹H-COSY correlations from the protons of the oxygenated methylene at δ_C 64 and the methyl at δ_C 15 suggested they were linked together to make an ethoxyl group (Fig. 2). HMBC correlations from the H-atoms of the oxygenated methylene to C-7 indicated the ethoxyl group was located at C-7 position. The quaternary carbons of double bond were determined as C-8 and C-9 according to the HMBC correlations of H-7/C-9 and H₂-11/C-8. Compounds 8–10 exhibited the most same NMR data except the chemical shifts of the oxygenated carbons at C-7 and C-12. For compound 8, the chemical shifts of C-7 and C-12 were at δ_C 78.9 and 73.9. For 9, these were δ_C 74.8 and 74.4. And, these were δ_C 74.6 and 72.0 for 10. We speculated that the configurations of C-7 and C-12 in compounds 8–10 were different. They all showed (+) Cotton effect at 350 nm which suggested the trans junction of A and B rings according to the octant rule for α,β-unsaturated ketones (Mi et al., 1993). The observed ROESY correlations of H-5/H-7 and H-12/CH₃-17 in 8, H-7/CH₃-20 and H-12/CH₃-17 in 9, and H-7/CH₃-20 and H-12/H-15 in 10 indicated that the configurations of 8–10 were 7α-ethoxy-12β-hydroxyl, 7β-ethoxy-12β-hydroxyl, and 7β-ethoxy-12α-hydroxyl respectively (Fig. 3). Finally, their absolute configurations were determined according to their experimental ECD and calculated ECD spectra (Fig. 4). So, their structures were identified as shown in Fig. 1 and named as jolkiniiols H-J.

Compound 11 assigned the molecular formula of C₂₀H₃₀O₃ on the basis of (+)-HR-ESI-MS [M + H]⁺ at m/z 319.2270 ([M + H]⁺calcd. 319.2268). Its ¹H NMR spectrum afforded three vinyl protons for ABX system, one other vinyl H-atom, four methyl units, and two oxygenated H-atoms (Table 3). Its ¹³C NMR data gave twenty carbons including a carbonyl unit, two pairs of double bonds, and two oxygenated C-atoms. Just as that of compound 10, the ABX system was formed by H-15 and H₂-16. C-12 was ascribed to link with a hydroxy group by the HMBC correlations from CH₃-17 to the oxygenated C-atom at δ_C 73.7 (Fig. 2). The ¹H–¹H-COSY correlations of H-9/H₂-11/H-12 verified C(12)-hydroxyl as well. Furthermore, ¹H–¹H-COSY cross peaks of H-5/H₂-6/H-7 suggested that H-7 was a vinyl H-atom. The other oxygenated C-atom at C-3 position assigned by the HMBC correlation from CH₃-18 to C-3. H-3 presented HMBC correlation to the carbonyl unit which determined C-2 as a carbonyl C-atom. Both of the configurations of the hydroxyl groups at C-3 and C-12 positions were α-oriented according to the ROESY cross peaks of H-3/H-5/H-9/H-12 (Fig. 3). The calculated ECD spectrum of 11 was well matched with the experimental ECD spectrum of 11 (Fig. 4). Thus, 11 was identified as (3S,12S)-3,12-dihydroxyl-ent-isopimara-7,15-dien-2-one and named as jolkiniiol K.

Compound 12 afforded almost the same HR-ESI-MS and NMR data as those of 11. The difference between their NMR data was a double bond. For 11, the chemical shifts were at δ_C 133.2 and 122.3. For 12, the chemical shifts were at δ_C 136.3 and 128.6. The Δ⁸⁽¹⁴⁾ was established by the HMBC correlations from the olefinic H-atom at δ_H 5.12 to C-7, C-9, C-12, and C-17 (Fig. 2). The ROESY cross peaks of H-3/H-5/H-9/H-12 were observed as well which illuminated the α-oriented hydroxyl groups at C-3 and C-12 positions (Fig. 3). The structure of 12 was (3S,12S)-3,12-dihydroxyl-ent-isopimara-8(14),15-dien-2-one based on the calculated ECD spectrum of 12 (Fig. 4) and named as jolkiniiol L.

Compound 13 presented the molecular formula C₂₀H₂₈O₃, 2 mass units lower than that of 12, by (+)-HR-ESI-MS at m/z 339.1932 ([M + Na]⁺ calcd. 339.1931). Interpretation of the NMR spectrum established the ent-isopimara skeleton of 13 being identical with that of 12, except that there was a double bond in 13 at δ_H 6.12/5.68 and δ_C 130.7/126.1 (Table 4). The ¹H–¹H-COSY correlations of H-5/δ_H 6.12/5.68 indicated that the double bond was located at C-6 and C-7 positions (Fig. 2). This hypothesis was also corroborated by the observations of HMBC associations of H-7/C-5, H-7/C-9, H-14/C-7. The carbonyl unit, hydroxy groups, and the other two double bonds of 13 were same as those of 12 based on its 2D NMR data. The α-oriented hydroxyl groups at C-3 and C-12 positions were assigned by the ROESY correlations of H-3/H-5/H-9/H-12 (Fig. 3). And then, the absolute configurations of 13 were deduced as 3S,12S by the same Cotton effects of the calculated ECD spectrum of 13 and the experimental ECD spectrum of 13 (Fig. 4). So, 13 was (3S,12S)-3,12-dihydroxyl-ent-isopimara-6,8(14),15-trien-2-one and named as jolkiniiol M.

Compound 14 displayed the molecular formula of C₂₀H₂₆O₄ as derived from the (+)-HR-ESI-MS at m/z 331.1915 ([M + H]⁺ calcd. 331.1904). The ¹H NMR spectrum of 14 displayed the characteristic resonances for three vinyl protons for ABX system, one other vinyl H-atom at δ_H 5.93 (1H, s), four methyl units, and two oxygenated H-atoms at δ_H 3.91 (1H, d, J = 6.0 Hz) and 3.47 (1H, s) (Table 4). Two carbonyl units at δ_C 208.8 and 202.1, two pairs of double bonds, and three oxygenated C-atoms at δ_C 81.9/60.1/54.9 were observed in the ¹³C NMR spectrum of 14. The oxygenated C-atoms were divided into two methines and one quaternary carbon. C(2)-carbonyl group and C(3)-hydroxyl unit were established by the interpretation of the 2D NMR spectra as those of 11 (Fig. 2). Except for the vinyl protons for ABX system, the other vinyl H-atom at δ_H 5.93 was displayed HMBC correlations to C-10 and C-13 suggested that this double bond was located between C-9 and C-11. Besides, C-8 was illuminated as an oxygenated quaternary C-atom on the basis of the HMBC correlation of H-11 and the oxygenated quaternary carbon at δ_C 54.9. The other oxygenated C-atom was C-7 according the ¹H–¹H-COSY cross peaks of H-5/H₂-6/H-7. C-12 was a carbonyl unit assigned by the HMBC correlations from CH₃-17 to the carbonyl C-atom at δ_C 202.1. Thus, the remaining one unsaturation degree required by the molecular formula of 14 was attributed to one ring that might be located as C-7 and C-8 to form an epoxy ring. The configurations of H-3 and H-7 were β-oriented according to the ROESY cross peaks of H-3/H-5/H-7 (Fig. 3). The calculated ECD spectrum of 14 (Fig. 4) determined its absolute configurations were 3S,7R,8S. Thus, 14 was (3S,7R,8S)-7,8-epoxy-3-hydroxy-ent-isopimara-9(11),15-dien-2,12-dione and named as jolkiniiol N.

Compound 15 gave out the molecular formula of C₂₀H₂₆O₃ which came from the (+)-HR-ESI-MS at m/z 315.1960 ([M + H]⁺ calcd. 315.1955). Compared with the NMR data of 14, the absence of the epoxy ring signals and the presence of one double bond at δ_C 130.8 and 129.4 were observed (Table 4). The ¹H–¹H-COSY correlations of H-5/H₂-6/H-atom at δ_H 6.11 deduced the double bond was located between C-7 and C-8 positions which was determined by the HMBC correlations from H-7 to C-5 and C-14 as well (Fig. 2). The other NMR signals were similar with those of 14. H-3 was β-oriented according to the ROESY cross peaks of H-3/H-5 (Fig. 3). Thus, the absolute configuration of 15 was 3S based on the comparison of the calculated ECD spectrum of 15 and experimental ECD spectrum of 15 (Fig. 4), Finally, compound 15 was identified as (3S)-3-hydroxy-ent-isopimara-7,9(11),15-trien-2,12-dione and named as jolkiniiol O.

Compound 16 assigned the molecular formula of C₂₀H₃₀O₂ based on the (+)-HR-ESI-MS [M + H]⁺ at m/z 305.2462 ([M + H]⁺calcd. 305.2475). Just as those of compound 12, three vinyl protons for ABX system, one other vinyl H-atom, four methyl units, and two oxygenated H-atoms were found in its ¹H NMR spectrum (Table 4). Its ¹³C NMR data gave twenty carbons including two pairs of double bonds, and two oxygenated C-atoms. The ABX system was formed by H-15 and H₂-16 undoubtedly. The hydroxy group at C-12 position and Δ⁸⁽¹⁴⁾ were deduced by the HMBC correlations from CH₃-17 to the oxygenated C-atom at δ_C 72.8 and to the olefinic C-atom at δ_C 124.6 (Fig. 2). Furthermore, ¹H–¹H-COSY cross peaks of H-1/H-2/the oxygenated at δ_H 3.26 and the HMBC correlation from CH₃-19 to the oxygenated C-atom at δ_C 79.0 indicated that C-3 was an oxygenated C-atom. H-3 and H-12 were both β-oriented according to the ROESY cross peaks of H-3/H-5/H-9/H-12 (Fig. 3). The calculated ECD spectrum of 16 was well matched with the experimental ECD spectrum of 16 (Fig. 4). Therefore, 16 was identified as (3R,12S)-3,12-dihydroxyl-ent-isopimara-7,15-dien and named as jolkiniiol P.

3.2 Cytotoxicity on pancreatic cancer SW1990 cells

Compounds 1–16 were evaluated the cytotoxicity on pancreatic cancer SW1990 cells using CCK-8 method in vitro and paclitaxel was used as the positive control. The results showed that 1 and 9 displayed obvious cytotoxicity with the IC₅₀ values 26.50 ± 6.36 μM and 21.09 ± 5.98 μM respectively which were stronger than that of paclitaxel (IC₅₀ = 45.01 ± 7.65 μM) (Table 5). Other compounds did not present cytotoxicity on pancreatic cancer SW1990 cells with the IC₅₀ values > 50 μM.

3.3 Flow cytometric results

FITC Annexin-V combined with PI were used to detect the effects of compounds 1 and 9 on the apoptosis cycle of tumor cells. The experiments showed that the percentages of SW1990 cells in pre apoptosis cycle were increased with dose dependent after the administration of compounds 1 and 9 (Fig. 5). It suggested us that 1 and 9 could promote the pre-apoptosis of SW1990 cells to act a role in anti-tumor.

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Fig. 5

Compounds 1 (A) and 9 (B) induced the apoptosis rates of pancreatic cancer SW1990 cells (means ± SEM, n = 3; ***P < 0.001 vs control (DMSO)).

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3.4 Western blot assay results

Bcl-2 is an inhibitor of apoptosis in mitochondria, and Bax is a pro-apoptotic protein. Bcl-2 forms a heterodimer with the Bax and inhibits the pro-apoptotic activity of Bax to maintain the integrity of the mitochondrial outer membrane and block mitochondrial apoptosis (Hu et al., 2015; Zhang et al., 2018). Caspase-3 is an aspartate-specific protease with the ability of breaking down proteins which is recognized as an apoptosis executor as well (Hu et al., 2015; Zhang et al., 2018). Therefore, we investigated the levels of Bcl-2, Bax, and Caspase-3 proteins in pancreatic cancer SW1990 cells after the treatment of compounds 1 and 9. As a result, compound 1 could down-regulate the expressions of Bcl-2 and up-regulate the expressions of Bax and Caspase-3. The protein levels of Bcl-2 in SW1990 cells were decreased significantly with the administration of compound 9. But compound 9 only up-regulated the expressions of Bax and not affected the protein levels of Caspase-3 (Fig. 6). It reminded that compounds 1 and 9 played a role in inhibiting tumor cells by decreasing the expressions of intracellular anti-apoptotic protein Bcl-2 and increasing the levels of apoptotic proteins Bax and Caspase-3.

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Fig. 6

The effects of compounds 1 (A) and 9 (B) on Bcl-2, Bax, and Caspase-3 proteins in pancreatic cancer SW1990 cells (means ± SEM, n = 3; *P < 0.05, **P < 0.01 vs control (DMSO)).

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3.5 Molecular docking results and druggability prediction

The above findings suggested that Bcl-2 played an important role in the anti-tumor process of compounds 1 and 9. To reveal their binding sites with Bcl-2, we subjected 1 and 9 to molecular docking with Bcl-2 (Fig. 7). It found that compound 1 was able to inhibit Blc-2 with the binding energy of −6.2 kcal/mol by forming hydrogen bonds with aspartic acid (ASP), arginine (ARG), and asparagine (ASN) residues of Bcl-2 whose bond lengths were 3.0, 3.1, 2.6, and 2.3, respectively. Compound 9 also showed a good free energy binding ability with Bcl-2 with the molecular docking binding energy of −5.4 kcal/mol and formed hydrogen bonds with ASP, ARG, glutamic acid (GLU), and lysine acid (LYS) residues with the bond lengths of 2.3, 2.9, 2.2, 3.5, and 2.3, respectively (Table 6).

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Fig. 7

The molecular docking results of 1 and 9 with Bcl-2 protein.

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The druggability prediction showed that compounds 1 and 9 were lipophilic molecules with low solubility, low molecular polar surface area, and high Log P values. Thus, they were easily absorbed through the digestive system and displayed high plasma protein binding rates and blood–brain barrier permeabilities. Besides, the predictions also suggested that 1 and 9 could not inhibit the CYP2D6 to generated hepatotoxicity (Table 6). These results provided the basis for the later experimental study of their drug properties.

The spread of E. jolkinii caused serious damages to alpine meadows that prompted the research of E. jolkinii on bioactive compounds and allelochemicals (Huang et al., 2014; He et al., 2008; Duan et al., 2024; Niu et al., 2024). In this paper, we investigated the anti-tumor effects of diterpenoids from E. jolkinii roots that was helpful for the utilization of E. jolkinii and the development of anti-pancreatic cancer drugs. However, the allelochemicals of E. jolkinii were unclear and the prevention of E. jolkinii has not been solved yet. Diterpenoids, as the characteristic compounds isolated from E. jolkinii, they might present ecological significance, such as allelopathy or antifeedant activity. Therefore, we plant design experiments to study the allelopathy of these diterpenoids to reveal the allelochemicals of E. jolkinii in the future which will help to control E. jolkinii and protect the alpine meadow ecosystem.