Optical rotations were recorded on a JASCO P-1020 automatic digital polarimeter. UV spectra were measured on a Shimadzu UV-2401 PC ultraviolet spectrometer, and IR data were carried out on a BRUKER Tensor-27 Fourier transform infrared spectrometer, respectively. NMR spectra were obtained on a Bruker Avance III 600 MHz spectrometer. The chemical shifts are given in ppm with TMS (tetramethylsilane) as an internal reference, and coupling constants are reported in Hz. ESI-MS and HRESI-MS data were recorded on an Agilent G6230 time-of-flight mass spectrometer. Semipreparative HPLC was run on a LC-5510 analytical and semi-preparative high-performance liquid chromatographs with a Zorbax SB-C18 column (Agilent, 4.6 mm×250 mL, 5 µm). And preparative HPLC was run on a Waters 1525 High Performance Liquid Chromatograph with a Waters SunFire C18 column. TLC was run on precoated silica gel plates, using 10% ethanol-H2SO4 as a visualization reagent. ODS RP-18 (40–60 µm, Merck, Germany), Sephadex LH-20 (GE Healthcare Bio-Sciences Corp, Piscataway, USA), macroporous resin (D101 type, Mitsubishi Corporation, Japan), and silica gel (200 − 300 mesh, Qingdao Haiyang Chemical Co., Ltd., People’s Republic of China) were used for column chromatography.
Nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse liver microsomes (MLMs) were obtained from Research Institute for Liver Diseases (Shanghai) Co., Ltd. The inhibitor resveratrol and ɑ-Naphthoflavone were purchased from Shanghai Maclin Biochemical Technology Co., LTD or Shanghai yuanye Bio-Technology Co., Ltd. β-estradiol were also purchased from Shanghai Maclin Biochemical Technology Co., LTD. 4-hydroxyestradiol were purchased from Cayman Chemical Company. All solvents (methanol and acetonitrile) were UPLC-MS (ultraperformance liquid chromatography-mass spectrometry) grade and reagents were analytical grade.
The branches and leaves of M. ferrea were collected in Gengma, Yunnan Province, People’s Republic of China, in April 2018, and identified by Prof. Li-ping Tang of Kunming Medical University. And the voucher specimen (No. 20180430) was deposited at Kunming Medical University.
Extraction and isolation
The air-dried branches and leaves of M. ferrea (5 kg) were extracted exhaustively with 95% EtOH–H2O (24 h × 3) at room temperature, followed by filtration. The filtrates were combined and evaporated under reduced pressure to afford an extract (500 g). The dried extract was subjected to silicagel column chromatography and sequentially partitioned by eluting with petroleum ether, chloroform, EtOAc, acetone, and MeOH to afford five fractions (A-E).
Fraction B (61 g) was then separated by column chromatography over silica gel (200 − 300 mesh) with a gradient mixture of petroleum ethyl-EtOAc (200:1→0:1, v/v) to yield 20 fractions (Fr. 1–20). Then, Fr. 9 was applied to a silica gel column (200 − 300 mesh) and eluted with a gradient mixture of petroleum ethyl-EtOAc (80:1→1:1, v/v) and further purified by semi-preparative HPLC with acetonitrile-H2O-Acetic acid (85:15:0.5, v/v) to yield compound 8. Fr. 10 was chromatographed on a silica gel column with petroleum ethyl-chloroform (10:1→1:1, v/v) and then purified by semi-preparative HPLC, run isocratically using a mixture of MeOH-H2O (84:16, v/v), affording compound 13. Fr. 11 was subjected to passage over a Sephadex LH-20 column by elution with acetone to yield four subfractions (Fr. 11 − 1 − 11 − 4). The main fraction, Fr. 11 − 3 was then separated chromatographically on a preparative HPLC column with MeOH-H2O (90:10, v/v) to yield Fr. 11-3-1 − 11-3-4. Fr.11-3-1 was purified by semi-preparative HPLC with MeOH-H2O (90:10, v/v) to produce compound5. Fr.11-3-2 was subjected to separation using semi-preparative HPLC with acetonitrile-H2O-Acetic acid (90:10:0.5, v/v), affording compound 7. Fr. 11-3-3 was separated chromatographically by semi-preparative HPLC column with acetonitrile-H2O-Acetic acid (87:13:0.5, v/v) to yield compound 6. And Fr. 11-3-4 was purified by semi-preparative HPLC with acetonitrile-H2O-Acetic acid (85:15:0.5, v/v), affording compound 1. Fr. 12 was fractionated over Sephadex LH-20 using acetone to furnish eleven fractions (Fr. 12 − 1 − 12 − 11). The main part Fr. 12 − 9 was submitted to silicagel column chromatography and eluted with a gradient of petroleum ethyl-acetone (70:1→1:1, v/v) to afford ten fractions (Fr. 12-9-1 − 12-9-10). Fr. 12-9-6 was purified by semi-preparative HPLC with MeOH-H2O (85:15, v/v) to get compounds 10 and 12. Fr. 12-9-10 on preparative TLC over silica gel using petroleum ethyl-EtOAc (3:1, v/v) yielded compound 9. Fr. 13 was subjected to separation over a Sephadex LH-20 column with acetone as the eluent, to yield thirteen subfractions (Fr. 13 − 1 − 13–13). Fr. 13 − 6 was applied to a silica gel columnand eluted with a gradient mixture of petroleum ethyl-acetone (100:1→1:1, v/v) to yield thirteen subfractions (Fr. 13-6-1 − 13-6-13). Fr. 13-6-11 was subjected to separation using semi-preparative HPLC with acetonitrile-H2O-Acetic acid (85:15:0.5, v/v), affording compounds 3 and 4. Fr. 13-6-13 was purified by semi-preparative HPLC with MeOH-H2O (85:15, v/v) to produce compounds 2 and 11.
Mesuaferlinn A (1). Yellow gum. [α]D21 = + 4.95 (c = 0.124, MeOH). UV(MeOH)λmax (log ε): 363(3.73), 290(4.38), 237(4.29), 206(4.51) nm; IR(KBr): 3434, 3059, 3030, 2974, 2934, 2876, 1749, 1721, 1612, 1476, 1372, 1177, 1145, 945, 859, 767, 703 cm− 1. ESI-MS: 443 [M + Na]+ ; HR-ESI-MS: 443.1467 ([M + Na]+, C25H24O6Na; calc.443.1471). 1H-and 13C-NMR: see Table 1.
Mesuaferlinn B (2). Yellow gum. [α]D21 = + 6.91 (c = 0.118, MeOH); UV(MeOH)λmax (log ε): 360(3.72), 289(4.36), 232(4.28), 204(4.52) nm; IR (KBr): 3434, 3059, 3029, 2978, 2934, 2874, 1749, 1722, 1611, 1476, 1371, 1181, 1146, 951, 858, 766, 703cm− 1. ESI-MS: 429 [M + Na]+; HR-ESI-MS: 429.1307 ([M + Na]+, C24H22O6Na; calc.429.1314). 1H- and 13C-NMR: see Table 1.
Mesuaferlinn C (3). Yellow gum. [α]D22 = + 6.55 (c = 0.127, MeOH). UV(MeOH)λmax (log ε): 405(3.77), 372(3.83), 283(4.22), 229(4.16), 204(4.46) nm; IR (KBr): 3430, 3059, 3030, 2969, 2931, 2873, 1712. 1620, 1585,1428, 1379, 1130, 906, 853, 768, 700 cm− 1. ESI-MS: 391 [M + H]+; HR-ESI-MS: 391.1541 ([M + H]+, C24H23O5; calc.391.1546). 1H- and 13C-NMR: see Table 1.
Enzyme Activity Inhibition Assays of CYP1A1, CYP1A2 and CYP1B1
The methods for measuring CYP enzyme activity were described in previous literature  and were briefly described as follows: the incubation system was carried out in 96-well plates, and the reaction system consists of β-estradiol (20 µM), the chemical inhibitor or the compound, which the concentration was the same as that of the inhibitor, MLM (0.5mg/ml), and buffer solution (PBS, PH = 7.4). The chemical inhibitors of CYP1A1, CYP1A2 and CYP1B1 enzyme activity were α-naphthalenone (10.0 µM), α-naphthalenone (1.0 µM) and resveratrol (10 µM), respectively. After the reaction system was incubated, NADPH was added for reaction, the reaction was terminated and centrifuged, and the supernatant was taken for testing to determine the inhibitory effect of the compounds on CYP1s activity by UPLC-MS. Incubation system in triplicate. The groups were as follows: ①Positive group: the inhibitory, β-estradiol and NADPH. ②Negative group: no NADPH, replaced with equal volume PBS. ③Experimental group: β-estradiol, the compound and NADPH.
A series of compounds were added to the incubation system to test IC50, which was the same as that of CYPs inhibition assay (the final concentration in the system was 1, 5, 20, 50, 100 µM).
All samples were analyzed on a high-resolution Q Exactive Plus hybrid quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA). The drug metabolites were transported on an XDB-C18 column (2.1×100 mm, 1.8mm, Agilent, Santa Clara, CA). The injection volume was 5ul, and the liquid flow was 0.3 ml/min. Phase A was 0.01% formic acid aqueous solution, and phase B was acetonitrile containing 0.01% formic acid. The elution gradient was as follows: 0–12 min, 2–98%B; 12–14 min, 98%B; 14–16 min, 98%A. The column temperature was 45 ℃. The data were in positive ion mode. The flow rate of collision gas and dry gas was 9 L/min. Capillary voltage 3.5 kV, temperature 350 ℃, atomizer pressure 35 psi.
The incubation system and the samples analysis of the assay was the same as that of CYPs inhibition assay. The groups were as follows: ①Sample group: β-estradiol, the compound and NADPH. ②N-NADPH group: β-estradiol and the compound, no NADPH, with equal volume of PBS instead. ③Control group: β-estradiol and NADPH.
Cell Culture and Cell Activity Assay
The human hepatoma cell line HepG2 were purchased from Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and tested by STR. The cells were preserved in Dulbecco's modified Eagle medium (DMEM), supplemented with 1% penicillin-streptomycin solution and 10% fetal bovine serum in a humidified environment of 37℃ and 5% CO2. 2×104 cells/well were cultured on 96-well plates in 200 µL DMEM medium, and the cells were incubated with compounds (2.5, 12.5, 25 or 100 µM) for 24h, then MTT solution was added to detect the cell activity.
Compounds or diosmine, the AHR agonist, were added to cells to determine the effect of compounds on AHR signaling pathways. 2×104 cells/well were cultured on 12-well plates in 2 mL DMEM medium, and the cells were incubated with compounds (25 µM) or diosmin (25 µM) for 24h. RNA for the cells was extracted and QPCR was performed to determine the expressions of CYP1A1 mRNA.
Data and statistical analysis
Chromatographic and spectral data were analyzed using QE Plus data acquisition software. All values were expressed as mean ± SD, and Graphpad Prism software 9.0 (Inc., La Jolla, CA)) was used for statistical analysis to obtain IC50 value.