Introduction

Sophora japonica L. belongs to the Leguminosae family, and it is mainly distributed in Hebei, Henan, Jiangsu, Guangdong, and Guangxi provinces in China. As a Traditional Chinese Medicine (TCM), the flowers of S. japonica (Flos Sophorae) can be used for bloody diarrhea, bleeding, vomiting, epistaxis, headaches, dizziness, etc. [1]. Pharmacologic studies and clinical practice have demonstrated that it has anti-tumour, anti-fertility, and anti-oxidant activities [2, 3]. The main components in this plant were phenolic acids, flavonoids, and saponins [4]. During the course of our characterization studies on the constituents from Flos Sophorae, we have reported the isolation and structure elucidation of 15 constituents [5–7]. As a continuing study on this herbal medicine, three new maltol glycosides, designated soyamalosides A (1), B (2), and C (3), together with eight known ones were obtained from this TCM. In this paper, we describe the isolation and structure elucidation of the three new isolates. Furthermore, the protective effects of the isolated compounds against antimycin A-induced L6 cell injury were studied.

Results and discussion

The dried Flos Sophorae (8.0 kg) was extracted with 70 % ethanol–water 3 times to provide a 70 % ethanol–water extract (2.3 kg). The above-mentioned extract (670.0 g) was extracted with EtOAc–H2O (1:1) to afford an EtOAc layer (78.0 g), EtOAc precipitation (197.0 g), and H2O layer (385.0 g). The H2O layer (348.3 g) was subjected to D101 column chromatography (CC) (H2O → 95 % EtOH → acetone), and H2O (213.1 g), 95 % EtOH (122.1 g), and acetone (8.2 g) eluted fractions were obtained.

The EtOAc layer and 95 % EtOH-eluted fraction was subjected to normal- and reversed-phase silica gel and Sephadex LH-20 column chromatography, and finally HPLC to give three new compounds, designated soyamalosides A (1), B (2), and C (3), along with eight known ones, 3-O-[β-d-apiofuranosyl-(1→2)-β-d-glucopyranosyl] maltol (4) [8], N,N’-dicoumaroylputrescine (5) [9], kakkasaponin II (6) [10], kaikasaponin I (7) [11], kaikasaponin III (8) [12], soyasaponin I (9) [13], dehydrosoyasaponin I (10) [12], and phaseoside IV (11) [13] (Fig. 1). Of the known isolates, this is the first report of 4–6, 10 and 11 in the Sophora genus.

Fig. 1
figure 1

Structures of compounds 1–11 obtained from Flos Sophorae

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Soyamaloside A (1) was isolated as a white amorphous powder with negative optical rotation {[α] 25D −20.5° (MeOH)}. Its molecular formula was determined to be C21H22O10 by HRESI-TOF-MS (m/z 457.1109 [M + Na]+, calcd for C21H22O10Na, 457.1105). Its IR spectrum showed absorption bands ascribable to hydroxy (3446 cm−1) and unsaturated ketone (1642 cm−1) functions, aromatic ring (1618, 1514, 1446 cm−1), and O-glycosidic linkage (1081 cm−1). The 1H-NMR (C5D5N), 13C-NMR (Table 1) and various types of 2D-NMR experiments including 1H–1H COSY, HSQC, and HMBC spectra suggested the presence of a trans-p-coumaroyl [δ 6.57 (1H, d, J = 16.0 Hz, H-8′′), 7.17 (2H, d, J = 8.0 Hz, H-3′′,5′′), 7.54 (2H, d, J = 8.0 Hz, H-2′′,6′′), 7.94 (1H, d, J = 16.0 Hz, H-7′′)], a β-d-glucopyranosyl [δ 5.63 (1H, d, J = 8.0 Hz, H-1′)], and a maltol (3-hydroxy-2-methyl-4H-pyran-4-one) moiety [8] [δ 2.38 (3H, s, 2-CH3), 6.50 (1H, d, J = 5.0 Hz, H-5), 7.81 (1H, d, J = 5.0 Hz, H-6)]. The assignment of glycoside protons was made by proton–proton correlations observed from the 1H–1H COSY experiment (Fig. 2). Furthermore, the linkage positions of trans-p-coumaroyl and β-d-glucopyranosyl on the aglycone of 1 were determined by the HMBC experiment, which showed long-range correlations between δH 5.63 (H-1′) and δC 143.1 (C-3), δH 5.80 (H-4′) and δC 167.2 (C-9′′) (Fig. 2). Finally, on acid hydrolysis and identification with HPLC analysis, the presence of d-glucose in 1 was clarified [14, 15]. Consequently, the structure of soyamaloside A was elucidated as maltol 3-O-(4′-O-trans-p-coumaroyl)-β-d-glucopyranoside (1).

Table 1 13C-NMR data for 1–3
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Fig. 2
figure 2

The main 1H–1H COSY and HMBC correlations of compounds 1–3

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Soyamaloside B (2) was obtained as a white amorphous powder with negative optical rotation {[α] 25D –15.0° (MeOH)}. HRESI-TOF-MS analysis revealed its molecular formula to be C27H30O14 (m/z 577.1541 [M-H]−, calcd for C27H29O14, 577.1563). Treatment of 2 with 1 M HCl yielded d-glucose [14, 15]. The 1H-NMR (CD3OD) and 13C-NMR (Table 1), together with 1H–1H COSY, HSQC, and HMBC spectra of 2 indicated that there were the same moieties as 1, i.e. a maltol aglycone [δ 2.42 (3H, s, 2-CH3), 6.46 (1H, d, J = 4.5 Hz, H-5), 8.01 (1H, d, J = 4.5 Hz, H-6)], a β-D-glucopyranosyl [δ 5.00 (1H, d, J = 8.0 Hz, H-1′)], and a trans-p-coumaroyl [δ 6.41 (1H, d, J = 16.0 Hz, H-8′′), 6.81 (2H, d, J = 8.0 Hz, H-3′′,5′′), 7.48 (2H, d, J = 8.0 Hz, H-2′′,6′′), 7.68 (1H, d, J = 16.0 Hz, H-7′′)]. Additionally, the above-mentioned spectra further suggested there was a 3-hydroxy-3-methylglutaric acid moiety [4] [δH: 1.34 (3H, s, 3′′′-CH3), 2.60 (2H, m, H2-2′′′), 2.63, 2.69 (1H each, both d, J = 14.0 Hz, H2-4′′′)]. The long-range correlations between δH 5.00 (H-1′) and δC 143.3 (C-3), δH 5.11 (H-3′) and δC 168.9 (C-9′′), δH 4.24, 4.46 (H2-6′) and δC 172.3 (C-1′′′) elucidated the linkage positions of the maltol, trans-p-coumaroyl, β-d-glucopyranosyl, and 3-hydroxy-3-methylglutaric acid moieties (Fig. 2). This evidence led us to formulate the structure of soyamaloside B to be maltol 3-O-(3′-O-trans-p-coumaroyl-6′-O-(3-hydroxy-3-methylglutaroyl))-β-d-glucopyranoside (2).

Soyamaloside C (3) was isolated as a white amorphous powder, which exhibited negative optical rotation {[α] 25D −85.7° (MeOH)}. Its molecular formula was revealed to be C23H32O16 by positive HRESI-TOF-MS analysis (m/z 587.1589 [M + Na]+, calcd for C23H32O16 Na, 587.1583). The IR spectrum absorption bands indicated the presence of an ester carbonyl (1725 cm−1), an unsaturated ketone (1642 cm−1), and an O-glycosidic linkage (1062 cm−1). The 1H-NMR (DMSO-d 6), 13C-NMR (Table 1) and various 2D-NMR experiments, and references [8, 16], suggested that 3 possessed the same moiety as known compound 4, maltol 3-O-β-d-apiofuranosyl(1→2)-β-d-glucopyranosyl [δ 2.23 (3H, s, 2-CH3), 5.05 (1H, d, J = 7.0 Hz, H-1′), 5.31 (1H, br. s, H-1′′), 6.32 (1H, d, J = 5.5 Hz, H-5), 8.02 (1H, d, J = 5.5 Hz, H-6)]. In addition, the result of acid hydrolysis further validated the presence of d-glucose and d-apiose [14, 15]. Furthermore, there was a 3-hydroxy-3-methylglutaric acid functional group [δH: 1.15 (3H, s, 3′′′-CH3), 2.30, 2.35 (1H each, both d, J = 16.0 Hz, H2-4′′′), 2.41, 2.51 (1H each, both d, J = 14.0 Hz, H2-2′′′); δC: 27.2 (3′′′-CH3), 45.7 (C-2′′′), 45.8 (C-4′′′), 68.8 (C-3′′′), 170.2 (C-1′′′), 172.9 (C-5′′′)] in 3. In the HMBC experiment, a long-range correlation between δH [4.00 (1H, dd, J = 6.0, 11.5 Hz), 4.23 (1H, br. d, ca. J = 12 Hz), H2-6′] and δC 170.2 (C-1′′′) was observed. Consequently, the structure of soyamaloside C (3) was identified to be as shown in Fig. 1.

Antimycin A is known to bind to cytochrome C reductase, thereby inhibiting the oxidation of ubiquinol in the electron transport chain of oxidative phosphorylation, causing the inhibition of mitochondrial electron transport [17].

Compared with the normal group, 100 µg/mL antimycin A significantly induced an L6 cell survival rate of 73.5 %, while 10 µM probucol produced increased cell survival rate effects compared with the antimycin-treated group. Of the compounds 1–11, 2, 3, and 10 showed significant protective effects against antimycin A-induced L6 cell injury (Table 2). Further studies of the antioxidant mechanisms of compounds 2 and 3 are necessary.

Table 2 Effect of compounds 1–11 on cell survival rate of L6 cells treated with antimycin A
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Experimental

General

Optical rotations were recorded on a Rudolph Autopol® IV automatic polarimeter. IR spectra were measured on a Varian 640-IR FT-IR spectrophotometer. UV spectra were determined on a Varian Cary 50 UV–Vis spectrophotometer. NMR spectra were conducted on a Bruker 500-MHz NMR spectrometer at 500 MHz for 1H and 125 MHz for 13C-NMR, with TMS as an internal standard. Positive- and negative-ion HRESI-TOF-MS were recorded on an Agilent Technologies 6520 Accurate-Mass Q-Tof LC/MS spectrometer. Column chromatographies (CC) were performed on macroporous resin D101 (Haiguang Chemical Co., Ltd., Tianjin, China), Silica gel (74-149 μm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and ODS (50 μm, YMC Co., Ltd., Tokyo, Japan). Pre-coated TLC plates with Silica gel GF254 (Tianjin Silida Technology Co., Ltd., Tianjin, China) were used to detect the purity of isolates achieved by spraying with 10 % aqueous H2SO4–EtOH, followed by heating. Preparative HPLC (Prep-HPLC) column, Cosmosil 5C18-MS-II (20 mm i.d. × 250 mm, Nakalai Tesque, Inc., Tokyo, Japan), was used to purify the constituents.

Plant material

The Flos Sophorae were collected from Tangshan city, Hebei province, China and identified by Dr. Li Tianxiang. The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM (No. 20120909).

Extraction and isolation

The dried Flos Sophorae (8.0 kg) was extracted with 70 % ethanol–water 3 times. Evaporation of the solvent under reduced pressure provided a 70 % ethanol–water extract (2.3 kg, 29.4 %). The 70 % ethanol–water extract (670.0 g) was partitioned with EtOAc–H2O (1:1, v/v) to afford an EtOAc layer (SoE, 78.0 g, 3.4 %), EtOAc precipitation (197.0 g, 9.1 %), and H2O layer (385.0 g, 16.9 %). The H2O layer (348.3 g) was subjected to D101 CC (H2O → 95 % EtOH → acetone), and H2O (213.1 g, 10.9 %), 95 % EtOH (SoH, 122.1 g, 6.2 %), and acetone (8.2 g, 0.4 %) eluted fractions were obtained.

SoE (50.0 g) was isolated by silica gel CC [CHCl3 → CHCl3–MeOH (100:2 → 100:3 → 100:5, v/v) → CHCl3–MeOH–H2O (10:3:1, v/v/v, the lower layer) → MeOH], and seven fractions (SoE 1–7) were obtained. SoE 5 (7.0 g) was separated by ODS CC [MeOH–H2O (30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 100:0, v/v) to give 12 fractions (SoE 5-1–5-12). SoE 5-4 (136.1 mg) was purified by Prep-HPLC [CH3CN–(H2O + 1 % HAc) (20:80, v/v)] to afford soyamaloside A (1, 15.2 mg, 0.00066 %). SoE 6 (7.5 g) was subjected to ODS CC [MeOH–H2O (10:90 → 45:55 → 50:50 → 60:40 → 70:30 → 80:20 → 100:0, v/v)] to yield 12 fractions (SoE 6-1–6-12). SoE 6-3 (1462.0 mg) was isolated by Prep-HPLC [MeOH–H2O (45:55, v/v)], and 11 fractions (SoE 6-3-1–6-3-11) were obtained. SoE 6-3-9 (828.3 mg) was separated by Prep-HPLC [CH3CN–H2O (23:77, v/v)], yielding soyamaloside B (2, 132.0 mg, 0.0082 %). SoE 6-7 (200.3 mg) was isolated by Prep-HPLC [CH3CN–H2O (45:55, v/v)] to afford N,N’-dicoumaroylputrescine (5, 12.1 mg, 0.00098 %).

SoH (72 g) was subjected to silica gel CC [CHCl3 → CHCl3–MeOH (100:2 → 100:5 → 100:7, v/v) → CHCl3–MeOH–H2O (10:3:1 → 7:3:1 → 6:4:1, v/v/v, lower layer) → MeOH], and 19 fractions (SoH 1–19) were obtained. SoH 12 (3.2 g) was isolated by Prep-HPLC [MeOH–H2O (20:80 → 40:60 → 50:50 → 60:40 → 100:0, v/v)] to yield 21 fractions (SoH 12-1–12-21). SoH 12-5 (64.3 mg) was purified by Prep-HPLC [MeOH–(H2O + 1 % HAc) (10:90, v/v)] to give 3-O-[β-d-apiofuranosyl-(1→2)-β-d-glucopyranosyl] maltol (4, 6.8 mg, 0.00055 %). SoH 12-15 (238.7 mg) was further separated by Prep-HPLC [CH3CN–(H2O + 1 % HAc) (23:77, v/v)], and soyamaloside B (2, 31.3 mg, 0.0083 %) was obtained. SoH 13 (5.0 g) was subjected by ODS CC [MeOH–H2O (10:90 → 20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 100:0, v/v)] to give 15 fractions (SoH 13-1 → 13-15). SoH 13-5 (50.4 mg) was purified by Prep-HPLC [MeOH-(H2O + 1 % HAc) (12:88, v/v)] to yield 3-O-[β-d-apiofuranosyl-(1→2)-β-d-glucopyranosyl] maltol (4, 4.0 mg, 0.00055 %). SoH 13-8 (188.8 mg) was separated by MeOH–(H2O + 1 % HAc) (12:88, v/v) to obtain soyamaloside C (3, 16.1 mg, 0.00082 %). SoH 15 (6.0 g) was subjected to ODS CC [MeOH–H2O (10:90 → 20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 100:0, v/v)] to give 20 fractions (SoH 15-1–15-20). SoH 15-19 (272.8 mg) was centrifuged repeatedly to give 2 fractions (SoH 15-19-1 and 15-19-2). SoH 15-19-2 (103.2 mg) was subjected to silica gel CC [CHCl3–MeOH–H2O (15:3:1, v/v/v, lower layer)] to produce kaikasaponin I (7, 28.9 mg, 0.0010 %). SoH 16 (8.5 g) was subjected to Sephadex LH-20 CC [MeOH–H2O (1:1, v/v)] to give 4 fractions (SoH 16-1–16-4). SoH16-2 (1.54 g) was isolated by Prep-HPLC [MeOH–H2O (70:30 → 90:10 → 100:0)] to give 21 fractions (SoH 16-2-1–16-2-21). SoH 16-2-18 (300.5 mg) was isolated by Prep-HPLC [MeOH–H2O (72:28), v/v] and 10 fractions (SoH 16-2-18-1–16-2-18-10) were obtained. SoH 16-2-18-3 (123.4 mg) was further purified by silica gel CC [CHCl3–MeOH–H2O (7:3:1, v/v/v, lower layer)] to produce soyasaponin I (9, 70.1 mg, 0.0036 %). SoH 16-2-18-6 (89.1 mg) was pure and was identified as kaikasaponin III (8, 89.1 mg, 0.0046 %). SoH 16-2-19 (74.2 mg) was separated by silica gel CC [CHCl3–MeOH–H2O (10:3:1 → 7:3:1, v/v/v, lower layer)] to produce dehydrosoyasaponin I (10, 15.2 mg, 0.00078 %). SoH 16-2-20 (103.2 mg) was isolated by silica gel CC [CHCl3-MeOH-H2O (20:3:1 → 10:3:1, v/v/v, lower layer)] to produce kakkasaponin II (6, 6.8 mg, 0.00035 %) and phaseoside IV (9, 42.3 mg, 0.0022 %).

Soyamaloside A (1): white powder. [α] 25D –20.5° (c = 0.42, MeOH); IR νmax (KBr) cm−1: 3446, 2955, 2694, 2286, 1642, 1618, 1514, 1446, 1293, 1255, 1167, 1081, 994, 922, 834; UV λmax (MeOH) nm (log ε): 201 (4.50), 295 (3.99). 1H-NMR (500 MHz, C5D5N): δ 6.50 (1H, d, J = 5.0 Hz, H-5), 7.81 (1H, d, J = 5.0 Hz, H-6), 5.63 (1H, d, J = 8.0 Hz, H-1′), 4.27 (1H, dd, J = 8.0, 9.0 Hz, H-2′), 4.45 (1H, dd, J = 9.0, 9.5 Hz, H-3′), 5.80 (1H, dd, J = 9.5, 9.5 Hz, H-4′), 4.08 (1H, m, H-5′), [4.12 (1H, dd, J = 6.0, 11.0 Hz), 4.21 (1H, br. d, ca. J = 11 Hz), H2-6′], 7.54 (2H, d, J = 8.0 Hz, H-2′′,6′′), 7.17 (2H, d, J = 8.0 Hz, H-3′′,5′′), 7.94 (1H, d, J = 16.0 Hz, H-7′′), 6.57 (1H, d, J = 16.0 Hz, H-8′′), 2.38 (3H, s, 2-CH3); 1H NMR (500 MHz, CD3OD): δ 6.46 (1H, d, J = 5.5 Hz, H-5), 8.01 (1H, d, J = 5.5 Hz, H-6), 4.90 (1H, d, J = 8.0 Hz, H-1′), 3.53 (1H, dd, J = 8.0, 9.0 Hz, H-2′), 3.72 (1H, dd, J = 9.0, 9.0 Hz, H-3′), 4.93 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.53 (1H, m, H-5′), [3.54 (1H, dd, J = 5.0, 11.5 Hz), 3.85 (1H, br. d, ca. J = 12 Hz), H2-6′], 7.46 (2H, d, J = 8.5 Hz, H-2′′,6′′), 6.81 (2H, d, J = 8.5 Hz, H-3′′,5′′), 7.67 (1H, d, J = 16.0 Hz, H-7′′), 6.35 (1H, d, J = 16.0 Hz, H-8′′), 2.48 (3H, s, 2-CH3); 13C-NMR (125 MHz, C5D5N) and (125 MHz, CD3OD) spectroscopic data, see Table 1. HRESI-TOF-MS: positive-ion mode m/z 457.1109 [M + Na]+ (calcd for C21H22O10Na, 457.1105).

Soyamaloside B (2): white powder. [α] 25D –15.0° (c = 0.56, MeOH); IR νmax (KBr) cm−1: 3312, 2941, 1720, 1621, 1604, 1514, 1441, 1386, 1329, 1243, 1165, 1062, 1026, 926, 835; UV λmax (MeOH) nm (log ε): 208 (4.14), 259 (3.90), 309 (4.26). 1H-NMR (500 MHz, CD3OD): δ 6.46 (1H, d, J = 4.5 Hz, H-5), 8.01 (1H, d, J = 4.5 Hz, H-6), 2.42 (3H, s, 2-CH3), 5.00 (1H, d, J = 8.0 Hz, H-1′), 3.62 (1H, dd, J = 8.0, 9.0 Hz, H-2′), 5.11 (1H, dd, J = 9.0, 9.0 Hz, H-3′), 3.60 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.55 (1H, m, H-5′), [4.24 (1H, dd, J = 5.0, 12.0 Hz), 4.46 (1H, br. d, ca. J = 12 Hz), H2-6′], 7.48 (2H, d, J = 8.0 Hz, H-2′′,6′′), 6.81 (2H, d, J = 8.0 Hz, H-3′′,5′′), 7.68 (1H, d, J = 16.0 Hz, H-7′′), 6.41 (1H, d, J = 16.0 Hz, H-8′′), 2.60 (2H, m, H2-2′′′), 2.63, 2.69 (1H each, both d, J = 14.0 Hz, H2-4′′′), 1.34 (3H, s, 3′′′-CH3); 13C-NMR (125 MHz, CD3OD) spectroscopic data, see Table 1. HR-ESI-TOF-MS: negative-ion mode m/z 577.1541 [M-H]− (calcd for C27H29O14, 577.1563).

Soyamaloside C (3): white powder. [α] 25D –85.7° (c = 0.85, MeOH); IR νmax (KBr) cm−1: 3377, 2926, 1725, 1642, 1616, 1568, 1434, 1252, 1194, 1062, 1010, 927, 843; UV λmax (MeOH) nm (log ε): 207 (3.90), 255 (3.85). 1H-NMR (500 MHz, DMSO-d 6): δ 6.32 (1H, d, J = 5.5 Hz, H-5), 8.02 (1H, d, J = 5.5 Hz, H-6), 2.23 (3H, s, 2-CH3), 5.05 (1H, d, J = 7.0 Hz, H-1′), 3.37 (1H, m, overlapped, H-2′), 3.39 (1H, m, overlapped, H-3′), 3.17 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.28 (1H, m, H-5′), [4.00 (1H, dd, J = 6.0, 11.5 Hz), 4.23 (1H, br. d, ca. J = 12 Hz), H2-6′], 5.31 (1H, br. s, H-1′′), 3.79 (1H, br. s, H-2′′), [3.57 (1H, d, J = 9.0 Hz), 3.84 (1H, d, J = 9.0 Hz), H2-4′′], 3.37, 3.50 (1H each, both d, J = 9.5 Hz, H2-5′′), 2.41, 2.51 (1H each, both d, J = 14.0 Hz, H2-2′′′), 2.30, 2.35 (1H each, both d, J = 14.0 Hz, H2-4′′′), 1.15 (3H, s, 3′′′-CH3); 1H-NMR (500 MHz, CD3OD): δ 6.39 (1H, d, J = 5.5 Hz, H-5), 7.95 (1H, d, J = 5.5 Hz, H-6), 2.36 (3H, s, 2-CH3), 5.11 (1H, d, J = 7.5 Hz, H-1′), 3.57 (1H, dd, J = 7.5, 9.0 Hz, H-2′), 3.55 (1H, dd, J = 9.0, 9.0 Hz, H-3′), 3.36 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.38 (1H, m, H-5′), [4.20 (1H, dd, J = 5.5, 11.5 Hz), 4.37 (1H, dd, J = 1.5, 11.5 Hz), H2-6′], 5.43 (1H, br. s, H-1′′), 4.00 (1H, br. s, H-2′′), 3.70, 4.05 (1H each, both d, J = 9.5 Hz, H2-4′′], 3.64, 3.72 (1H each, both d, J = 11.5 Hz, H2-5′′), 2.59, 2.64 (1H each, both d, J = 14.0 Hz, H2-2′′′), 2.53, 2.59 (1H each, both d, J = 15.5 Hz, H2-4′′′), 1.30 (3H, s, 3′′′-CH3); 13C-NMR (125 MHz,DMSO-d 6) and (125 MHz, CD3OD) spectroscopic data, see Table 1. HR-ESI-TOF-MS: positive-ion mode m/z 587.1589 [M + Na]+ (calcd for C23H32O16Na, 587.1583).

Acid hydrolysis of 1–3

Solutions of soyamalosides A–C (1–3, each 1.5 mg) were dissolved in 1 M HCl (1.0 mL) and heated under reflux for 1 h. The reaction mixture was neutralized with Amberlite IRA-400 (OH− form) and removed by filtration. The aqueous layer was subjected to HPLC analysis under the following conditions: HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm i.d. × 250 mm (Tokyo Kasei Co. Ltd., Tokyo, Japan); detection, optical rotation [Chiralyser (IBZ Messtechnik GMBH, Mozartstrasse 14–16 D-30173 Hannover, Germany)]; mobile phase, CH3CN–H2O (75:25, v/v); flow rate 1.0 mL/min. Identification of d-apiose (i) from 3 and d-glucose (ii) from 1–3 present in the aqueous layer was carried out by comparison of their retention times and optical rotations with those of authentic samples: (i) t R 6.3 min (d-apiose, positive optical rotation), (ii) t R 13.5 min (d-glucose, positive optical rotation).

Mitochondrial oxidative stress protection assay

Antimycin A was used to induce mitochondrial oxidative stress [18]. Briefly, L6 cells (Cell Resource Center, IBMS, CAMS/PUMC, Beijing, China) were plated at a density of 5 × 104 cells/well in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific, UT, USA) supplemented with 10 % calf serum (Thermo Scientific) in a 96-well plate and were incubated at 37 °C for 24 h. Cells were treated with or without 10 μmol/L sample DMSO solution (final DMSO concentration was 0.5 %). One hour later, medium was removed and 100 μg/mL antimycin A (Sigma Co. Ltd, MO, USA) in 100 μL DMEM was added to each well, The MTT assay was performed 4 h later to detect the cell survival rate. Probucol (10 μmol/L) was used as positive control.