General experimental procedures

1D and 2D NMR spectra were recorded on a Bruker AVANCE NEO 600 (1H: 600 MHz, 13C: 150 MHz) spectrometer and JEOL ECA-600 (1H: 600 MHz, 13C: 150 MHz), using TMS as an internal standard. Chemical Shifts (δ) are presented in ppm and coupling constants (J) in Hz. HR-ESI–MS experiments were acquired using an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific). Single-crystal X-ray diffraction data were acquired on a Rigaku XtaLAB Synergy-R diffractometer using Cu Ka radiation. Optical rotation value was recorded on a JASCO P-2200 polarimeter. UV spectra was obtained with a JASCO V-750 spectrophotometer. IR spectra was obtained with a JASCO FT/IR-4600 spectrophotometer. Circular dichroism was measured on JASCO J-1100 spectrometer. Powdered DIAION (Mitsubishi Chemical Co., Japan), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), MCI gel CHP20P (Mitsubishi Chemical Co., Japan), silica gel 60 (Merck, Darmstadt, Germany) and ODS-A-HG (YMC, Kyoto, Japan, 50 µm) were used for column chromatography (CC). Silica gel GF254 plates (Merck; 0.25 mm in thickness) were used for TLC analysis.

Plant material

The hook-bearing stems of Uncaria rhynchophylla (Miq.) Miq. were purchased from Santai Country Yuanhui Commerce and Trade Co., Ltd. in Sichuan, China. A voucher specimen (THS102306) was deposited in the herbarium of Tsumura Co., Ltd. in Japan.

Isolation of new compound 1

The dried hook-bearing stems of U. rhynchophylla (14.25 kg) were percolated with 60% MeOH in H2O (101.5 L). After concentration of MeOH, the crude extract in water was partitioned with CHCl3 to obtain the CHCl3-soluble extract and the water-soluble extract. After concentration of the aqueous phase, the crude extract was subjected to DIAION HP-20 eluting successively with 60% MeOH in H2O and MeOH to obtain two fractions (60 M, DM2).

60 M was fractionated by Sephadex LH-20 CC (10–100% MeOH in H2O) to afford fractions 60M1 (61.82 g), 60M2 (21.26 g), 60M3 (115.54 g). Further separation of 60M1 (61.82 g) by ODS CC (10–100% MeOH in H2O) to obtain five fractions 60M11, 60M12, 60M13, 60M14 and 60M15. 60M13 was chromatographed on silica gel CC (CHCl3/MeOH/H2O, 40:10:1–6:4:1, v/v, successively) to obtain fractions 60M131, 60M132 and 60M133. Purification of 60M133 on ODS CC (15–16% MeCN in 50 mM NH4Ac aq.) and MCI gel CHP-20P (H2O to MeOH) afford compound 1 (68 mg). The isolation scheme of other known compounds is summarized in supplementary Fig. S13.

Spectroscopic data of rhynchophylloside L 11-O-β-D-glucopyranoside (1)

Pale-brown powder; [α]D20 –99 (c = 0.1, MeOH); UV λmax (MeOH) nm (log ɛ): 247 (4.71), 253 (4.71), 310 (4.07), 321 (4.06); CD λmax (MeOH) nm (Δɛ): 224 (+ 10.47), 252 (–11.09), 294 (+ 1.02); IR (KBr) cm–1: 3398, 2923, 2877, 1635, 1604, 1574, 1454, 1250, 1076, 505; 1H and 13C NMR (600 and 150 MHz, respectively, DMSO-d6), see Table 1; positive ion HR-ESI–MS, m/z 875.2698 [M + Na]+ (calculated for C38H48N2O20Na, 875.2693).

SC-XRD analysis of 1, 2, 3, 4, 8 and 10

Compound 1 (50 µg) was placed into V-type vial and added 4 µL of water. After caped vial, the suspension was dissolved by heating. The suitable single crystals of compound 1 was crystalized within a day. (C38H48N2O20)·6(H2O), M = 960.88, crystal size 0.142 × 0.013 × 0.008 mm3, orthorhombic, space group P212121, a = 6.9177(1) Å, b = 21.6692(3) Å, c = 28.7263(5) Å, V = 4306.10(11) Å 3, Z = 4, α = β = γ = 90°, density (calcd) = 1.482 g·cm–3, F(000) = 2040.0, reflections collected/unique 64,432/8602 (Rint = 0.0467), final R indices (I > 2σ (I)) R1 = 0.0476, wR2 = 0.1043, goodness of fit = 1.149, Flack parameter = 0.01(5). Crystallographic data for compound 1 have been deposited with the Cambridge Crystallographic Data Center (CCDC 2258683).

Compound 2 (50 µg) was placed into V-type vial and added 10 µL of water/ethanol/acetonitrile. After caped vial, the suspension was dissolved by heating. The suitable single crystals of compound 2 was crystalized within a day. (C38H50N2O19)·2(H2O), M = 874.83, crystal size 0.19 × 0.04 × 0.03 mm3, orthorhombic, space group P212121, a = 5.8287(2) Å, b = 19.4840(4) Å, c = 38.4044(10) Å, V = 4361.4(2) Å 3, Z = 4, α = β = γ = 90°, density (calcd) = 1.332 g·cm–3, F(000) = 1856.0, reflections collected/unique 37,841/8418 (Rint = 0.0458), final R indices (I > 2σ (I)) R1 = 0.0557, wR2 = 0.1514, goodness of fit = 1.125, Flack parameter = 0.09(7). Crystallographic data for 2 have been deposited with the Cambridge Crystallographic Data Center (CCDC 2264009).

Compound 3 (50 µg) was placed into V-type vial and added 10 µL of water/acetonitrile. After caped vial, the suspension was dissolved by heating. The suitable single crystals of compound 3 was crystalized within a day. C32H40N2O14, M = 676.66, crystal size 0.05 × 0.03 × 0.01 mm3, monoclinic, space group P21, a = 16.8414(6) Å, b = 5.9860(2) Å, c = 18.8582(6) Å, V = 1881.77(11) Å 3, Z = 2, α = γ = 90°, β = 98.187(3)°, density (calcd) = 1.194 g·cm–3, F(000) = 716.0, reflections collected/unique 23,514/7326 (Rint = 0.0507), final R indices (I > 2σ (I)) R1 = 0.0607, wR2 = 0.1521, goodness of fit = 1.019, Flack parameter = − 0.2(2). Crystallographic data for 3 have been deposited with the Cambridge Crystallographic Data Center (CCDC 2263997).

Compound 4 (50 µg) was placed into V-type vial and added 10 µL of ethanol. After caped vial, the suspension was dissolved by heating. The suitable single crystals of compound 4 was crystalized within a day. (C26H30N2O8)·(C2H6O), M = 544.59, crystal size 0.25 × 0.02 × 0.014 mm3, monoclinic, space group P21, a = 8.2300(2) Å, b = 5.85790(10) Å, c = 27.3883(6) Å, V = 1307.99(5) Å 3, Z = 2, α = γ = 90°, β = 97.862(2), density (calcd) = 1.383 g·cm–3, F(000) = 580.0, reflections collected/unique 26,131/5286 (Rint = 0.0484), final R indices (I > 2σ (I)) R1 = 0.0469, wR2 = 0.1305, goodness of fit = 1.097, Flack parameter = 0.00(10). Crystallographic data for 4 have been deposited with the Cambridge Crystallographic Data Center (CCDC 2263990).

Compound 8 was recrystallized from methanol. 2(C27H32N2O10)·8(H2O), M = 1233.22, crystal size 0.12 × 0.05 × 0.03 mm3, orthorhombic, space group P212121, a = 12.7952(4) Å, b = 19.1742(9) Å, c = 24.2979(9) Å, V = 5961.2(4) Å 3, Z = 4, α = β = γ = 90°, density (calcd) = 1.374 g·cm–3, F(000) = 2624.0, reflections collected/unique 39,208/11453 (Rint = 0.0697), final R indices (I > 2σ (I)) R1 = 0.0790, wR2 = 0.1902, goodness of fit = 1.176, Flack parameter = 0.04(11). Crystallographic data for 8 have been deposited with the Cambridge Crystallographic Data Center (CCDC 2259276).

Compound 10 was recrystallized from acetone. C21H22N2O3, M = 350.40, crystal size 0.11 × 0.06 × 0.04 mm3, orthorhombic, space group P212121, a = 7.13640(10) Å, b = 9.8793(2) Å, c = 25.4299(5) Å, V = 1792.88(6) Å 3, Z = 4, α = β = γ = 90°, density (calcd) = 1.298 g·cm–3, F(000) = 744.0, reflections collected/unique 17,419/3672 (Rint = 0.0306), final R indices (I > 2σ (I)) R1 = 0.0339, wR2 = 0.0954, goodness of fit = 0.810, Flack parameter = 0.07(9). Crystallographic data for 10 have been deposited with the Cambridge Crystallographic Data Center (CCDC 2255637).

AChE Inhibitory Assay

The AChE inhibitory activities of isolated compounds were examined based on Ellman’s method [26] using Amplite® Colorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Inc.). Briefly, 10 µL of the tested compound solution dissolved in H2O with or without 4% DMSO was mixed with 30 µL of the Assay Buffer and 10 µL of acetylcholinesterase solution (200 mU/mL in the Assay Buffer) on a 96-well microplate. Neostigmine bromide was used as a positive control. After incubation at room temperature for 20 min to facilitate binding of compounds to AChE, 50 µL of the Working Solution containing 5,5′-dithiobis(2-nitrobenzoic acid) and acetylthiocholine iodide was added. After 30 min incubation, the absorbance of each well was recorded at a wavelength of 405 nm. Following the previous report [5], the AChE inhibitory activity was calculated as follows: inhibition % = (C − S)/ (C − B) × 100% (C, the absorbance of control; S, the absorbance of sample solution; and B, the absorbance of blank).