Ecx 500 spectrometer

Nuclear magnetic resonance (NMR) experiments were performed on a JEOL ECX-500 spectrometer (¹H, 500 MHz; ¹³C, 125 MHz; JEOL Ltd., Japan). All chemical shifts were referenced relative to the corresponding signals (δ_H 3.31/δ_C 49.15 for CD₃OD). Electron ionization mass spectrometer (EI-MS) data were obtained using micromass spectrum (AUTOSPEC, UK). High performance liquid chromatography (HPLC) was performed using Agilent 1200 series (Agilent Technologies, CA, USA). Open column chromatography (CC) was carried out over a silica gel 60 (70–230 mesh, 230–400 mesh ASTM, Merck, Darmstadt, Germany) and sephadex LH-20 gel (GE Healthcare, Uppsala, Sweden). Pre-coated silica gel 60 F₂₅₄ (Merck) were used for thin-layer chromatography (TLC).

The organic solvents, including methanol (MeOH), hexane (Hx), ethyl acetate (EtOAc), butanol (BuOH), and methylene chloride (MC) were purchased from Duksan Chemical Co. (Seoul, Korea). The column chromatography used was silica gel 60 (Merck 230–400 mesh, ASTM, Darmstadt, Germany). The preparative TLC was performed using 20 × 20 cm plates coated with 1 mm-thick F254 silica gel (Merck, Darmstadt, Germany). The NMR spectra were recorded on a JEOL ECX-500 spectrometer, operating at 500 MHz for ¹H and 125 MHz for ¹³C NMR spectra (JEOL Ltd., Tokyo, Japan). The determination of high-performance liquid chromatography (HPLC) spectra were recorded on an Agilent 1260 series system (Agilent Inc., Palo Alto, CA, USA) with a photodiode array (PDA) and an evaporative light scattering detector (ELSD).

Nuclear magnetic resonance (NMR) experiments were performed on a JEOL ECX-500 spectrometer (¹H, 500 MHz; ¹³C, 125 MHz; JEOL Ltd., Japan). All chemical shifts were referenced relative to the corresponding signals (δ_H 3.31/δ_C 49.15 for CD₃OD). EI-MS data were obtained using micromass spectrum (AUTOSPEC, UK). High-performance liquid chromatography (HPLC) was performed using the Agilent 1200 series (Agilent Technologies, CA, USA). Open column chromatography (CC) was carried out over a silica gel 60 (70–230 mesh, 230–400 mesh ASTM, Merck, Darmstadt, Germany) and sephadex LH-20 gel (GE Healthcare, Sweden). Pre-coated silica gel 60 F₂₅₄ (Merck) were used for thin-layer chromatography (TLC).

Artemisia capillaris Thunb was purchased from Korean medicine Omniherb (www.omnishop.co.kr). The P149 voucher specimen was deposited in the Natural Products Bank (NIKOM). Column chromatography (CC) was conducted using silica gel (Merck, Darmstadt, Germany). ¹³C Nuclear magnetic resonance (NMR) and ¹H NMR spectra were analysed using JEOL ECX‐500 spectrometer (JEOL Ltd.). Agilent 1260 series (Agilent Technologies) with a C18 column (Phenomenex, United States of America Synergi 10 μ Hydro‐RP 80A, 10 μm, 4.6 mm × 250 mm) was used for high‐performance liquid chromatography (HPLC).

All manipulations were carried out using standard Schlenk techniques or in a glovebox under an atmosphere of argon. Anhydrous hexane, pentane, and toluene were dried by passage through two columns of activated alumina and a Q-5 column, while anhydrous THF, Et₂O, and DME were dried by passage through two columns of activated alumina. Anhydrous deuterated benzene (benzene-d₆) was dried and degassed over a potassium mirror prior to use. NMR spectra were recorded on a JEOL ECX-500 spectrometer (JEOL Ltd., Tokyo, Japan). ¹H NMR spectra were reported with reference to solvent resonances of C₆D₆ residual protons (δ = 7.16 ppm). ¹³C NMR spectra were referenced to solvent (peaks δ = 128.06 (C₆D₆) ppm). ¹¹B chemical shifts were referenced to BF₃•OEt₂ (neat at 0.0 ppm) as an external standard. ¹⁵N chemical shifts were referenced to 90% formamide in dimethyl sulfoxide-d₆ (112.7 ppm with respect to NH₃ at 0.0 ppm) as an external standard. Complexes 2-K and 3-Na were prepared following the literature procedure [23 (link),24 (link)]. Elemental analyses (C, H, and N) were carried out on an Elementar vario MICRO Cube (Elementar, Frankfurt, Germany). IR spectra were recorded on a JASCO VIR-200 spectrometer (JASCO, Tokyo, Japan). Spectra and structures are available in Supplementary Materials.

Nuclear magnetic resonance (NMR) spectra were obtained using a JEOL ECX-500 spectrometer (JEOL Ltd., Tokyo, Japan) operating at ¹H-NMR (500 MHz) and ¹³C-NMR (125 MHz) with tetramethylsilane (TMS) as internal standard. High-performance liquid chromatography (HPLC) was performed using Agilent 1260 series (Agilent Technologies, Santa Clara, CA, USA). The MPLC system (puriFlash^TM430, interchim, Los Angeles, CA, USA) and prep-LC equipment (SpotⅡ, Armen, France) and Silica gel 60 (230–400 mesh ASTM, Merck, Darmstadt, Germany) were used to separate the active compound. The A. membranaceus were purchased at a commercial herbal medicine market. A voucher specimen (P357) has been deposited in the Natural Products Bank, National Institute for Korean Medicine Development (NIKOM).

The melting points of the products were determined using an XT-4 binocular microscope (Beijing Tech Instrument Co. Ltd., Beijing, China). ¹³C NMR were recorded using a JEOL-ECX500 spectrometer at 22°C, with tetramethylsilane as the internal standard and CDCl₃ as the solvent. Column chromatography was performed using silica gel (200–300 meshes) (Qingdao Marine Chemistry Co., Qingdao, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Sodium dodecyl sulfate (SDS) were purchased from Beijing Dingguo CO., Ltd; 2,2-diphenlyl-1-picrylhydrazyl (DPPH) and vitamin C (Vc) were purchased from Aladdin Reagent Inc; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and DMSO were purchased from Roche Molecular Biochemicals (1465–007); Adriamycin (ADM) was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd; α-amyrin, β-sitosterol, and stigmast-4-en-3-one had been prepared in previous work [22 (link),23 (link)]. β-amyrin was isolated from supercritical carbon dioxide extract of H. undatus peel, and its purification process and NMR data are presented in Additional file 1. All other chemicals were of analytical reagent grade and used without further purification.