3.1 General experimental procedures

SPR analysis were performed on Biacore T200. X-ray data were recorded using a Bruker APEX DUO instrument. NMR, HRESIMS, UV, and IR spectra were measured as described previously [13].

3.2 Plant material

H. erectum (whole herbs) was bought from Eastern medicinal materials market of Hebei Province in September 2023. The voucher specimen (No. KIBZY20230702) was identified by Dr. Chunlei Xiang and preserved at Kunming Institute of Botany.

3.3 Extraction and isolation

Crude alkaloids (800 g) were obtained from H. erectum whole herbs (40.0 kg) by following the published procedures [13], which were separated into five fractions (I-V) by silica gel CC eluted with CH2Cl2/MeOH (60:1 → 0:1, v/v). Fr. V (30.0 g) was eluted with CH2Cl2/MeOH (40:1 → 0:1, v/v) to give five subfractions (V1-5). Fr. V5 (6.0 g) was subjected to Sephadex LH-20 (MeOH), followed by semipreparative high-performance using Waters X-Bridge C18 (10 × 150 mm, 5 μm) column with CH3CN/H2O (32:68, 0.1%, v/v, formic acid) to afford compound 3 (8.5 mg, tR = 23.0 min). Fr. V4 (12.3 g) was subjected to gradient elution (CH2Cl2/MeOH: 30:1 → 0:1, v/v) and followed by semi-preparative high performance liquid chromatography (HPLC) separation using Waters X-Bridge C18 (10 × 150 mm, 5 μm) column, CH3CN/H2O (38:62, 0.1%, v/v, formic acid) to afford compound 4 (5.1 mg, tR = 42.0 min). Fr. IV (80.0 g) was eluted with CH2Cl2/MeOH (50:1 → 0:1, v/v) to give five subfractions (IV1-5). IV3 (15.3 g) was then subjected to gradient elution (CH2Cl2/MeOH: 30:1 → 0:1, v/v), purified using semi-preparative HPLC with Waters X-Bridge C18 (10 × 150 mm, 5 μm) column CH3CN/H2O (48:52, 0.1%, v/v, formic acid), which finally gave compounds 1 (6.2 mg, tR = 32.6 min), and 2 (26.8 mg, tR = 17.5 min).

3.4 Compound characterization

Hypecotumine A (1): white crystal; UV (MeOH) λmax (log ε) 210.0 (5.38), 241.5 (5.12), 312.0 (4.78), 357.5 (4.75) nm; IR (KBr) νmax 3432, 2912, 1630, 1588, 1504, 1485,1394 and 1366 cm−1; ESIMS m/z 382 [M + H]+; HRESIMS m/z 382.1283 [M + H]+ (calcd for C21H19NO6, 382.1285); 1H and 13C NMR data, see Tables 1 and 2, respectively.

Table 1 1H NMR (500 MHz) spectroscopic data for compounds 1–4 (δ in ppm, J in Hz)Table 2 13C NMR (125 MHz) spectroscopic data for compounds 1–4 (δ in ppm)

Hypecotumine B (2): white needle-like crystal, amorphous powder; UV (MeOH) λmax (log ε) 208.5 (5.23), 240.0 (4.95), 314.0 (4.69), 355.0 (4.59) nm; IR (KBr) νmax 3432, 2937, 1626, 1606, 1564, 1519, 1481 and 1461 cm−1; ESIMS m/z 398 [M + H]+; HRESIMS m/z 398.1594 [M + H]+ (calcd for C22H23NO6, 398.1598); 1H and 13C NMR data, see Tables 1 and 2, respectively.

Hypecotumine C (3): yellow oil; UV (MeOH) λmax (log ε) 204.5 (5.06), 242.5 (4.69), 304.5 (4.43), 372.0 (4.30) nm; IR (KBr) νmax 3432, 2932, 1709, 1604, 1564, 1519, 1444 and 1382 cm−1; ESIMS m/z 396 [M + H]+; HRESIMS m/z 396.1443 [M + H]+ (calcd for C22H21NO6, 396.1442); 1H and 13C NMR data, see Tables 1 and 2, respectively.

Hypecotumine D (4): yellow oil; UV (MeOH) λmax (log ε) 206.0 (5.49), 245.5 (5.12), 300.5 (4.84), 373.0 (4.66) nm; IR (KBr) νmax 3434, 2924, 1630, 1607, 1503, 1486 1465 and 1384 cm−1; ESIMS m/z 380 [M + H]+; HRESIMS m/z 380.1134 [M + H]+ (calcd for C21H17NO6, 380.1129); 1H and 13C NMR data, see Tables 1 and 2, respectively.

3.5 Crystal data for Hypecotumine A (1)

Hypecotumine A (1): C21H19NO6•3(H2O), M = 435.42, a = 40.812(3) Å, b = 13.2173(9) Å, c = 15.3426(9) Å, α = 90°, β = 90.934(7)°, γ = 90°, V = 8275.1(9) Å3, T = 150.(2) K, space group C12/c1, Z = 16, μ(Cu Kα) = 0.931 mm−1, 36,414 reflections measured, 7593 independent reflections (Rint = 0.3067). The final R1 values were 0.1195 (I > 2σ(I)). The final wR(F2) values were 0.3090 (I > 2σ(I)). The final R1 values were 0.1990 (all data). The final wR(F2) values were 0.3769 (all data). The goodness of fit on F2 was 0.996. Crystallographic data (excluding structure factor tables) for compound 1 have been deposited with the Cambridge Crystallographic Data Center as supplementary publication (deposit number CCDC 2348263). Copies of the data can be obtained free of charge by application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. + 44 (0) (1223) 336033; e-mail: deposit@ccdc.cam.ac.uk.

3.6 Experimental methods

Protein preparation: The protein concentration of PCSK9 was 40 µg/mL. Ligand pre-enrichment: ligand pre-enrichment was performed by manual injection to test the pre-enrichment of PCSK9 at a concentration of 40 µg/mL in 10 mM sodium acetate solution at pH 4.5 and 5.0 (pH 4.5 was selected for coupling). The proteins were immobilized in the Fc-4 channel of CM5 chip using amino coupling, respectively. And the Fc-1 channel was activated and closed as the reference channel of the Fc-4 channel, respectively [23].

3.7 Compounds interaction analysis with protein PCSK9

Biacore T200 was used to characterize interactions between small molecules and protein. The compounds 1–4 were formulated in a concentration gradient using running buffer to prepare a series of concentrations ranging from 500.0, 250.0, 125.0, 62.5, 31.25, 15.625, and 7.8125 µM. Flow through Fc1-Fc4 and determine the interaction between compounds and PCSK9 immobilized on the chip surface. Operation parameter setting: flow path 4–1 (contact time 80 s, flow rate 30 μL/min, dissociation time 80 s), 25 ℃.

3.8 Cell culture

Human hepatocellular liver cell, HepG2 was obtained from ATCC Cell Bank and cultured 12–24 h in advance in RMPI1640 medium using culture medium containing 10% fetal bovine serum. Positive drug and compounds were solubilized using DMSO and diluted to 12 µmol and 100 µmol individually. Then the compounds were added to the cells 6 well plates, incubated in a 37 °C cell culture incubator at 5% CO2 for 24 h.

3.9 Western blot assay

HepG2 cells were washed two times in pre-cooled PBS and lysed using RIPA buffer (Thermo fisher CN) on ice. The cells were centrifuged for 10 ~ 15 s per 10 min to fully lyse, continuing 60 min, and detected the protein concentration with BCA protein concentration assay kit. Proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) with electrophoresis and transferred to activated PVDF membrane (Millipore USA). Then, PVDF membrane were treated with 5% milk for 1.5 h. After that, the membrane was incubated with primary antibody (1:500) overnight at 4 °C and with secondary antibody (1:5000) for 60 min at room temperature. The band pictures were got by Gel imaging and chemiluminescence imaging system (UVITEC UK).

3.10 Statistical analysis

The experiment was repeated five times and the results were expressed by mean ± S.E.M. Significance level was fixed by one-way analysis of variance (ANOVA) and Dunnett’s t-test. P represented the degree of significance [20].

3.11 Molecular docking

Schrodinger 2012.1 soft was used for molecular docking. The docking results were processed by Pymol. The PDB of PCSK9 protein is 6U3X (resolution: 2.64 Å) (https://www.rcsb.org). The process of protein preparation includes addition of hydrogens, addition of missing side chains, removal of waters, assigning protonation states and energy minimization [24]. Ligands were prepared by generating 3D coordinates, using OPLS3e force field, assigning protonation states, and energy minimization formation. The position and size of the docking box was determined by the position and size of the original ligand. The SP docking mode was used to generate docking poses.