FBS, RPMI1640 medium, glutamine, and DMEM medium were purchased from Biological Industries (Israel). Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1), Z-VAD-FMK (Zvad), and Necrostatin (Nec-1) were purchased from MedChemexpress (MCE) in the United States. AnnexinV/PI cell double staining apoptosis detection kit, TUNEL (ALEX647) apoptosis detection kit, cell cycle detection kit, and DAPI anti-fluorescence quenching mounting tablets were purchased from Shanghai Yisheng Biotechnology Co. Ltd. Sulfonyl Rhodamine B (SRB) was purchased from Shanghai Aladdin Reagent Co. Ltd. Tris (Tris) was purchased from Shanghai Shenggong Biological Engineering Co. Ltd. Trichloroacetic acid (TCA) was purchased from Shanghai Saen Chemical Technology Co. Ltd. Anti-Caspase3, anti-Caspase9, anti-Parp-1, anti-Cleaved-parp-1 were purchased from Shanghai Shenggong BBILIFE. Anti-Cleaved-caspase9 was purchased from Absin (Shanghai, China). Anti-active-Caspase3 was purchased from ProteinTech (Chicago, United States). All consumables are purchased in NEST Biotechnology (Wuxi, China).

In this experiment, Alstonia yunnanensis Diels was obtained from Changshan Mountain Yongde County, Lincang City, Yunnan Province. The plant samples (JGCC-2018-01) were collected and identified by Kunming Caizhi Biotechnology Co. Ltd. Plant specimens were preserved in the Key Laboratory of Chemistry in Ethnic Medicinal Resources.

IMARK multifunctional microplate reader (Bio-Rad, United States). 1658001 vertical electrophoresis belt transfer system (Bio-Rad, United States). Tanon 5200 chemiluminescence imager (Tianneng, China). DMIL-LED trinocular inverted microscope (Leica, Germany). TCS-SP8 laser confocal microscope (Leica, Germany). Thermo Scientific 3425 carbon dioxide cell incubator (Thermo Scientific World Er, United States). Thermo TSU-600V -80°C ultra-low temperature refrigerator (Thermo Fisher, United States). Bruker AV nuclear magnetic resonance instrument (Bruker, Germany). Waters AutoSpec Premier P776 high-resolution magnetic mass spectrometer (Waters, United States). The pipette and Eppendr of 5424R centrifuge (Eppendorf, Germany).

2.9 kg of dried Alstonia yunnanensis Diels root was extracted with methanol to obtain 150 g of extract. The extract was suspended in an appropriate amount of water, first extracted with an appropriate amount of petroleum ether (PE), then the aqueous phase was adjusted pH to 3-4 with glacial acetic acid, and then extracted with ethyl acetate (EA) to obtain 32 g of part A. Next, the aqueous phase of the EA extraction was adjusted to a pH of approximately 10 with ammonia, and the EA was re-extracted to obtain 7.2 g of part B. 60-100 mesh silica gels 150 g mixed with part A using column chromatography purification with PE/EA (50:1 to 10:1) to obtain 9 g Fr.3 elution segment. Then, these 9 g samples are separated by Sephadex LH-20 chromatography and detected by TLC. The components were combined with the same polarity to obtain a 30 mg component (Fr.3.2). This component was eluted by 300 mesh silica gel columns according to PE/EA (50:1 to 20:1) as the mobile phase, and TLC detection to combine the same components. Then, this part was recrystallized with methanol as the solvent to obtain 17.2 mg of compound Acetoxytabernosine (AC). Similarly, 60-100 mesh silica gels 50 g mixed with part B using column chromatography purification with PE/EA (30:1 to 10:1) to obtain 513 mg Fr.Ш elution segment. Fr.Ш was separated by PE/EA (50:1 to 5:1) to get 100 mg Fr.Ш.2. Thereafter, Fr.Ш.2 was separated by gel chromatography, and the same polarity was combined by TLC and then eluted by HPLC with MeOH/H₂O (100:1 to 20:1) to get 15.3 mg AC.

1 and 2-dimensional NMR experiments were recorded on a Bruker DRX-600 spectrometer operating at 400 MHz (¹H) and 100 MHz (¹³C) at 300 K (chemical shifts δ in ppm, coupling constants J in Hz) (Bruker, Germany). Mass spectra of AC were obtained on a Waters AutoSpec Premier P776 mass spectrometer (Waters Co., Milford, MA, United States). In this experiment, the ECD calculation is done by the research dog instrument testing platform. ECD Calculation The calculations for ECD spectra were conducted as published previously (Shi et al., 2019).

Cells BEL-7402, N87, AGS, CT26, HCT116, and SMMC7721 were provided by the National Collection of Authenticated Cell Cultures. After the cells were resuscitated at 37°C, they were cultured in RPMI1640/DMEM containing 10% FBS, in a 37°C, 5% CO2 incubator, and passaged every 2 days. Logarithmic growth phase cells were seeded into a 96-well culture plate (5000 cells/well) and medicine administration was conducted for 48 h. SRB (0.1%) was used to stain the cells, and the wavelength of 515 nm was used to detect the absorption peak.

SMMC7721 and BEL-7402 (5000 cells) grow on covering glass for 24 h. Then, according to AC 50 μM, and AC 50 μM with Zvad 10 μM co-administered for 24 h. After the cells were fixed with PBS with paraformaldehyde at 4°C for 25 min, aspirate the supernatant, and treat the sample with 2 μg mL⁻¹ of proteinase K for 5 min to make the cell permeable. Next, cells were fixed with 100 μL of 1×Equilibration Buffer for 10-30 min, aspirated the supernatant and incubated with Alexa Flour 647-12-dUTP Labeling Mix for 1 h at 37°C, washed away the background with PBS, and sealed the cell slide with DAPI-containing sealing agent. The pictures were observed and taken under a laser confocal microscope.

Flow cytometry was used to detect the total cell apoptosis rate induced by AC on SMMC7721 and BEL-7402, which were treated with AC and apoptosis inhibitors (Zvad) for 24 h, and the results were analyzed by prism 8.0. According to the experimental grouping, the cells were collected directly into 1.5 ml centrifuge tubes, the number of cells per sample was about 2×10⁶, centrifuged for 5 min, and the medium was discarded. The cells were washed once with PBS and centrifuged at 500–1000 r/min for 5 min. The cells were resuspended with 100 μL of the labeling solution and incubated for 10-15 min at room temperature in the dark. Next, the cells were centrifuged at 500–1000 r/min for 5 min and washed the cells with PBS once. Then, the incubation buffer solution was added and incubated for 20 min at 4°C, avoiding light and shaking softly. The excitation light wavelength of the flow cytometer was 488 nm, a band-pass filter with a wavelength of 515 nm was used to detect FITC fluorescence, and a filter with a wavelength greater than 560 nm was used to detect PI.

The cell cycle was detected by Flow cytometry, and cells were seeded into a 6-well plate. After the AC treatment, the cells were collected and fixed with 70% ethanol at 4°C for more than 2 h, centrifuged at 1500 RPM, and discarded the ethanol fixative. Then, the cells were washed with PBS and centrifuged to discard the PBS. At last, the cells were suspended with the fixative solution, and incubated with RNase and PI at 37°C in the dark for 30 min. Flow cytometry detects cell cycle distribution, and the final data of Flow cytometry were processed by Modfit TL 4.1 software.

Cells were collected and the final spreading density was 2×10⁵ cells/well in 6-well plates with 2 ml per well. The final concentration of AC was 50 μM and the final concentration of colchicine was 1.5 μM. The plates were gently shaken and incubated for 4 h at 37°C in a constant temperature incubator with 5% CO₂. Cells were collected for JC-1 staining (beyotime, China), incubated for 15 min Centrifuged and the supernatant discarded. Cells were resuspended again and flow cytometry was performed immediately.

Western blot was used to detect the expression of proliferation and metastasis-related proteins. SMMC7721 and BEL-7402 were treated with AC at three concentrations of 25, 50, and 100 μM. SMMC7721 and BEL-7402 were treated with AC for 24 h. The cells were digested and collected to lysis with ultrasound for 5 min. The protein concentration was determined by the coomassie brilliant blue method. Polyacrylamide gel electrophoresis was performed with a 50 μg protein mixture in each electrophoresis hole. Then the protein was transferred to the membrane. Subsequently, the membranes were incubated in TBST (Tween-20) containing 5% skim milk at 25°C. The membranes were rinsed with TBST and then incubated overnight with primary antibodies Parp-1, Cleaved-parp-1, Caspase9, Cleaved-caspase9, Caspase3, and Active-caspase3 using β-actin as the internal reference protein. Thereafter, the membranes were rinsed with TBST and incubated with a secondary antibody for 1 h. ECL reaction, darkroom development, exposure. ImageJ software analyzed the ratio of the protein content of each electrophoresis band of the protein content of β-actin.

Prism 8.0 was used to perform statistical analysis of the data. The results of each experiment were independently repeated 3 times. Data are shown as mean ± standard error of the mean (SEM). Student’s t-test and One-way ANVOA were used to calculate statistical differences between groups. p < 0.05 was considered significant.

AC was separated from the Alstonia yunnanensis Diels root, and the compound was structurally identified by modern spectral analysis using MS and NMR, and UV (Supplementary Figures S1–S8). As shown in Figure 1A, 17.2 mg AC was isolated from part A and 15.2 mg AC was obtained from part B, involving silica column chromatography, gel chromatography, recrystallization, and HPLC. In addition, modern wave spectroscopy to identify the chemical structure of AC, The ¹H NMR, and ¹³C-NMR data were consistent with Acetoxytabernosine. The mass spectrometry of AC [M + H]⁺ is 395 (m/z), [M + Na]⁺ is 419 (m/z), and AC was calcded for 394.19. In addition, compared with the detection result, 19(s), 20(s)-Acetoxytabernosine has a positive cotton effect at 270-300. The absolute stereochemistry calculated by ECD is 19(s), 20(s)-Acetoxytabernosine (Figure 1B).

To explore the toxicity of AC on GI tumors, liver cancer cells SMMC7721, BEL-7402, gastric cancer cells AGS, N87, intestinal cancer cells CT26, and HCT116 were selected for the study. The results of the SRB assay showed that AC could induce GI tumor death in a dose-dependent manner. AC has an inhibitory effect on the growth of SMMC7721 with IC₅₀ of 15.3 ± 2.5μM, and BEL-7402 with IC₅₀ of 19.3 ± 1.2 μM (Figures 2A, B). As for gastric cancer cells, the IC₅₀ of AC for AGS is 20.9 ± 2.5 μM and the IC₅₀ for N87 is 23.2 ± 4.87 μM (Figures 2C, D). As for intestinal cancer cells, AC’s IC₅₀ for CT26 is 35.8 ± 0.99μM, and for HCT116 IC₅₀ is 29.47 ± 0.73 μM (Figures 2E, F). In total, AC could inhibit the proliferation of GI tumors and exhibit smaller concentrations of IC₅₀ in hepatocellular carcinoma cells.

Earlier SRB cell activity assay showed that AC could inhibit SMMC7721 proliferation. And flow cytometry showed that AC could promote the death of SMMC7721 (Figures 3A, B). As shown in Figures 3C, D, AC could significantly promote BEL-7402 death with the strongest effect at 50 μM. The cell cycle of 0, 25, and 50 μM AC on SMMC7721 and BEL-7402 were detected by Flow cytometry. As for SMMC7721, 0 μM AC administered group G1 phase cells accounted for about 64% of the total cell number, and S phase cells about 30%. In the 25 μM administration group, G1 phase cells accounted for approximately 84% of the total cell number, and S phase cells approximately 10%. In the 50 μM administration group, G1 phase cells accounted for about 85% of the total cell number, and S phase cells accounted for 9% (Figures 3E, F). The effect of AC on the BEL-7402 cell cycle was similar to that of SMMC7721, with AC inducing a decrease in the BEL-7402 S phase and an increase in the G1 phase BEL-7402 (Figures 3G, H). Taken together, with the increase of the administration concentration, the number of cells in the G1 phase increased and the number of cells in the S phase decreased, indicating that the synthesis of genetic material is blocked in the G1 phase.

To further explore the cell death method of AC on hepatocellular carcinoma cells, the inhibitors Zvad, Fer-1, Lip-1, and Nec-1 were co-administered with AC. Figure 4A showed the full names, targets, and concentrations of the apoptosis inhibitor Zvad, the iron death inhibitors Fer-1 and Lip-1, and the necrosis inhibitor Nec-1. Besides, the SRB staining was used to detect the cell viability, and it was found that Zvad significantly inhibited the cell death of hepatocellular carcinoma cells caused by AC (Figures 4B, C), which advanced apoptosis of AC on hepatocellular carcinoma cells.

Flow cytometry was used to detect the distribution of apoptosis cells, SMMC7721 and BEL-7402 cells were treated with AC and Zvad for 24 h. In SMMC7721, the total apoptosis rate in the AC 50 μM administration group was 23.54%, the total apoptosis rate in the Zvad + AC 50 μM group was 7.59%, and the apoptosis inhibitor was significantly reduced the total cell apoptosis rate (Figures 5A, B). For BEL-7402 cells, the total apoptosis rate in the AC 50 μM administration group was 20.28%, and the total apoptosis rate in the Zvad + AC 50 μM group was 4.01%, and apoptosis was inhibited (Figures 5C, D). AC significantly reduced the total cell apoptosis rate. In addition, the TUNEL assay results showed that the red fluorescence of the AC 50 μM with Zvad 10 μM combined administration group was significantly weaker than the 50 μM administration group in SMMC7721(Figures 5E, F). As shown in Figures 5G, H, similar to the results of SMMC7721, the red fluorescence was stronger in the AC group compared with the AC 0 group. Compared with the AC group, the red fluorescence diminished when AC was combined with Zvad. Therefore, it is proved that AC can induce hepatocellular carcinoma cells to apoptosis, and this reaction can be reversed by the apoptosis inhibitor Zvad.

The flow cytometry results showed that the mitochondrial membrane potential, which was reduced due to the action of colchicine, was increased under the action of AC, indicating that AC can effectively increase the mitochondrial membrane potential and enhance the exchange of substances inside and outside the mitochondria (Figures 6A–D). Mitochondria are closely associated with apoptosis, which also suggests that AC can influence mitochondrial apoptotic mechanisms. Earlier experiments had shown that apoptosis inhibitor Zvad significantly inhibits AC, so we then examined the caspase-related pathway (Zvad targets) using a western blot. Hepatocellular carcinoma cells were treated with different concentrations of AC for 24 h, and the expression Parp-1, Cleaved-parp-1, Caspase-9, Cleaved-caspase9, Caspase3, and Active-caspase3 were analyzed. As for SMMC7721, we found that Parp-1, Caspase9, and Caspase-3 decreased with the increase of AC, while Cleaved-parp-1, Cleaved-caspase9, and Active-caspase3 increased (Figures 6E, F). AC on caspase pathway proteins in BEL-7402 showed similar results to SMMC7721, with caspase9, caspase3, and Parp-1 all cleaved and activated to subunits (Figures 6G, H). A general schematic of the isolation and activity testing of AC was shown in Figure 7. These experiments show that AC can cause the cascade activation of Caspase9-Caspase3, which further leads to the cleavage of parp-1 to prevent DNA damage repairment and lead to cell apoptosis.