3.1 General experimental procedures

Optical rotations were determined using a Rudolph Autopol I automatic polarimeter, and X-ray crystal crystallographic data were collected on an Agilent Xcalibur Nova X-ray diffractometer. Melting points were measured on an X-4 melting instrument. UV and IR spectra were recorded using a Shimadzu UV-2600i spectrophotometer and a Bruker Tensor 37 infrared spectrophotometer, respectively. 1D and 2D NMR spectra acquired on a Bruker AM-500 spectrometer at 25 °C. HR-ESI–MS was performed using a Waters Micromass Q-TOF spectrometer. Semipreparative HPLC was performed on a Shimadzu LC-40B XR liquid chromatograph equipped with a YMC-Pack ODS-A column (250 mm × 10 mm, 5 μm). Silica gel (200 − 300 mesh, Qingdao Marine Chemical, Inc, Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden), and reversed-phase C18 (RP-C18) silica gel (50 μm, Quebec City Canada) were employed for column chromatography (CC).

3.2 Plant material

The roots of I. asprella were collected in April 2023 from Hezhou City, Guangxi Zhuang Autonomous Region, China. The plants were authenticated by one of the authors (W. Li) and a voucher specimen (No. GM202304) has been deposited in the Zhongshan Institute for Drug Discovery.

3.3 Extraction and isolation

The dried roots of I. asprella (20 kg) were crushed and subjected to reflux extraction using 65% EtOH for three times (3 × 100 L, 2 h each), producing a crude extract (1.6 kg). The extract was suspended in hot water (2.0 L) and successively partitioned with EtOAc (3 × 3.0 L) and n-BuOH (3 × 3.0 L), yielding EtOAc (105 g) and n-BuOH (303 g) fractions.

The n-BuOH extract was chromatographed on a silica gel column using a gradient of petroleum ether (PE)/EtOAc/MeOH solvent system (5:1:0 → 0:0:1), yielding six fractions (Frs. A − F). Fr. C (24.8 g) was processed on an ODS gel column (MeOH/H2O, 3:7 → 1:0) to afford four sub-fractions (Frs. C1 − C4). Fr. C2 (2.8 g) was separated on a Sephadex LH-20 column (MeOH) and further purified by semipreparative RP-HPLC with a YMC-park ODS-A column (CH3CN/H2O, 45:55, 3 mL/min) to obtain compounds 15 (17.5 mg, tR 38 min), 9 (16.6 mg, tR 41 min), 13 (2.7 mg, tR 43 min), and 17 (7.5 mg, tR 33 min). Fr. D (33.5 g) was subjected to repeated silica gel column chromatography with CH2Cl2/MeOH (100:1 → 0:1), affording five fractions (Frs. D1–D5). Fr. D4 (778.2 mg) was further separated using a Sephadex LH-20 column (MeOH), resulting in five subfractions (Frs. D4a–Fr. D4e). Fr. D4b (94.1 mg) was purified via HPLC (MeOH/H2O, 77:23, 2 mL/min) to yield 5 (1.9 mg, tR 28 min), 11 (7.2 mg, tR 34 min), and 12 (10.7 mg, tR 35 min). Similarly, Fr. D4c (39.1 mg) was processed using HPLC (CH3CN/H2O, 43:57, 2 mL/min) to yield 1 (19.7 mg, tR 50 min). Fr. D4e (88.5 mg) was also purified using HPLC (CH3CN/H2O, 52:48, 2 mL/min) to afford 3 (4.1 mg, tR 12 min), 14 (15.5 mg, tR 15 min), and 16 (15.7 mg, tR 17 min). Chromatography of Fr. D5 on an ODS gel column (MeOH/H2O, 1:1 → 1:0) produced six fractions (Fr. D5a–D5f). Fr. D5b (17.5 mg) was further refined using HPLC (CH3CN/H2O, 30:70, 2 mL/min) to isolate 7 (5.7 mg, tR 64 min) and 10 (9.6 mg, tR 74min). Compounds 8 (8.0 mg, tR 18 min), 4 (5.6 mg, tR 21 min), and 6 (12.7 mg, tR 22 min) were purified from Fr. D5c (50.5 mg) by HPLC (CH3CN/H2O, 45:55, 2 mL/min), and 2 (11.7 mg, tR 55 min) was obtained from Fr. D5f (24.2 mg) by HPLC (CH3CN/H2O, 38:62, 2 mL/min).

3.4 Spectroscopic data of compounds3.4.1 Asprecoside A (1)

Colorless needles; [α] 20D −16 (c 0.1, CH3CN); IR (KBr) νmax 3417, 2950, 1738, 1444, 1374, 1238, 1092, 1045, 1027, 982 cm−1; 1H and 13C NMR data (Tables 1 and 2); HRESIMS m/z 659.3803 [M − H]− (calcd for C37H55O10−, 659.3801).

3.4.2 Asprecoside B (2)

Colorless needles; [α] 20D −12 (c 0.1, CH3CN); IR (KBr) νmax 3424, 2925, 1738, 1458, 1376, 1168, 1051, 1025, 910 cm−1; 1H and 13C NMR data (Tables 1 and 2); HRESIMS m/z 659.3799 [M − H]− (calcd for C37H55O10−, 659.3801).

3.4.3 Asprecoside C (3)

White amorphous powder; [α] 20D −21 (c 0.1, MeOH); IR (KBr) νmax 3434, 2948, 1737, 1452, 1387, 1227, 1171, 1046 cm−1; 1H and 13C NMR data (Tables 1 and 2); HRESIMS m/z 659.3816 [M − H]− (calcd for C37H55O10−, 659.3801).

3.4.4 Asprecoside D (4)

White amorphous powder; [α] 20D −11 (c 0.1, MeOH); IR (KBr) νmax 3424, 2943, 1747, 1456, 1373, 1167, 1053 cm−1; 1H and 13C NMR data (Tables 1 and 2); HRESIMS m/z 689.3907 [M + HCOO]− (calcd for C38H57O11−, 689.3906).

3.4.5 Asprecoside E (5)

White amorphous powder; [α] 20D −14 (c 0.1, MeOH); IR (KBr) νmax 3441, 2925, 1743, 1456, 1376, 1049 cm−1; 1H and 13C NMR data (Tables 1 and 2); HRESIMS m/z 705.3857 [M + HCOO]− (calcd for C38H57O12− 705.3856).

3.4.6 Asprecoside F (6)

White amorphous powder; [α] 20D −25 (c 0.1, CH3CN); IR (KBr) νmax 3423, 2925, 1739, 1441,1374, 1244, 1164, 1046, 1025, 914 cm−1; 1H and 13C NMR data (Tables 2 and 3); HRESIMS m/z 659.3805 [M − H]− (calcd for C37H55O10−, 659.3801).

3.4.7 Asprecoside G (7)

White amorphous powder; [α] 20D −69 (c 0.1, CH3CN); IR (KBr) νmax 3423, 2925, 1738, 1443, 1375, 1249, 1166, 1048, 1026, 982 cm−1; 1H and 13C NMR data (Tables 2 and 3); HRESIMS m/z 677.3905 [M − H]− (calcd for C37H57O11−, 677.3906).

3.4.8 Asprecoside H (8)

White amorphous powder; [α] 20D +18 (c 0.1, CH3CN); UV (CH3CN) λmax (log ε) 225 (3.97) nm; IR (KBr) νmax 3417, 2924, 1738, 1442, 1372, 1238, 1166, 1084, 1044 cm−1; 1H and 13C NMR data (Tables 2 and 3); HRESIMS m/z 659.3817 [M − H]− (calcd for C37H55O10−, 659.3801).

3.4.9 Asprecoside I (9)

White amorphous powder; [α] 20D +14 (c 0.1, CH3CN); UV (CH3CN) λmax (log ε) 226 (3.99) nm; IR (KBr) νmax 3414, 2937, 2873, 1696, 1460, 1372, 1239, 1163, 1043 cm−1; 1H and 13C NMR data (Tables 2 and 3); HRESIMS m/z 585.3799 [M − H]− (calcd for C35H53O7−, 585.3797).

3.4.10 Asprecoside J (10)

White amorphous powder; [α] 20D −13 (c 0.1, CH3CN); IR (KBr) νmax 3418, 2924, 1729, 1443, 1375, 1251, 1168, 1045, 1023, 983 cm−1; 1H and 13C NMR data (Tables 2 and 3); HRESIMS m/z 677.3898 [M − H]− (calcd for C37H57O11−, 677.3906).

3.5 X-ray crystallographic data of 1 and 2

The X-ray crystallographic data of asprecosides A (1) and B (2) have been deposited at the Cambridge Crystallographic Data Centre, with the following CCDC deposition numbers: 2413613 (1) and 2,413,615 (2).

3.5.1 Crystallographic data of asprecoside A (1)

C37H56O10⋅4(H2O) (M = 732.88 g/mol): monoclinic, space group P21 (no. 4), a = 13.2729(3) Å, b = 7.0751(2) Å, c = 20.4063(5) Å, β = 96.8560(10)°, V = 1902.59(8) Å3, Z = 2, T = 170.00 K, μ(CuKα) = 0.801 mm−1, Dcalc = 1.279 g/cm3, 22,369 reflections measured (6.708° ≤ 2Θ ≤ 133.79°), 6424 unique (Rint = 0.0391, Rsigma = 0.0367) which were used in all calculations. The final R1 was 0.0387 (I > 2σ(I)) and wR2 was 0.1039 (all data). Flack parameter =  − 0.02(3).

3.5.2 Crystallographic data of asprecoside B (2)

C37H56O10 (M = 660.81 g/mol): monoclinic, space group P21 (no. 4), a = 7.0183(2) Å, b = 26.3891(6) Å, c = 23.1192(5) Å, β = 98.73°, V = 4232.22(18) Å3, Z = 4, T = 170.15 K, μ(CuKα) = 0.605 mm−1, Dcalc = 1.037 g/cm3, 12,902 reflections measured (5.116° ≤ 2Θ ≤ 133.192°), 12,902 unique (Rsigma = 0.0806) which were used in all calculations. The final R1 was 0.0631 (I > 2σ(I)) and wR2 was 0.1789 (all data). Flack parameter = 0.09(4).

3.6 Acid hydrolysis of asprecosides A − J (1−10)

Each compound (1 mg) was heated under refluxed in 1.5 mL of 2 M HCl (dioxane/H2O, 1:1) at 90 °C for 4 h. Following hydrolysis, 5 mL of water was added, followed by extraction with EtOAc. The aqueous layer was evaporated under vacuum and the residue was dissolved in anhydrous pyridine (400 μL), followed by the addition of 2 mg of L-cysteine methyl ester hydrochloride. After stirring the mixture at 60 °C for 1 h, 50 μL of o-tolyl isothiocyanate was added, and the reaction continued at the same temperature for an additional hour. The authentic samples D-xylose and D-glucuronic acid underwent identical treatment and were analyzed by reversed-phase HPLC. D-xylose (2 mL/min, MeCN/H2O, 25:75, tR = 51.7 min) was identified in 9, whereas D-glucuronic acid (2 mL/min, MeCN/H2O, 50:50, tR = 25.2 min) was observed in 1−8 and 10.

3.7 Cell culture

H1975 and HCC827 cells, sourced from the American Type Culture Collection, were cultured in RPMI 1640 medium (Gibco BRL, USA) containing 10% fetal bovine serum (FBS). These cells were grown under conditions of 5% CO2 and 37 °C.

3.8 Cell viability assay

Cell viability was evaluated through the Cell Counting Kit-8 (CCK-8) assay. In brief, Briefly, H1975 and HCC827 cells were seeded into 96-well plates (1× 103 cells/well) and incubated for 24 h. Then, the attached cells were treated with different concentrations of compounds. Following a 3-days incubation, CCK-8 reagent (Dojindo) was added, and luminescence was measured according to the manufacturer’s protocol.