Original Papers

215

Authors

Yan Wang, Chun-Lei Zhang, Yan-Fei Liu, Dong Liang, Huan Luo, Zhi-You Hao, Ruo-Yun Chen, De-Quan Yu

Affiliation

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, Peopleʼs Republic of China

Key words " Araliaceae l " Schefflera kwangsiensis l " triterpenoid saponins l " hepatoprotective l " structure elucidation l

Abstract !

Seven new triterpenoid saponins, schekwangsiensides A–G (1–7), and a new triterpenoid, schekwangsienin (8), together with nine known triterpenoids and saponins (9–17), were isolated from the aerial parts of Schefflera kwangsiensis. The structures of these compounds were elucidated on the basis of spectroscopic data analysis

Introduction

July 26, 2013 Nov. 14, 2013 Nov. 22, 2013

Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1360179 Published online December 19, 2013 Planta Med 2014; 80: 215–222 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. De-Quan Yu Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College 2 Nan Wei Road Beijing, 100050 Peopleʼs Republic of China Phone: + 86 10 63 16 52 24 Fax: + 86 10 63 01 77 57 [email protected]

Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica

thermore, hepatoprotective activities of some compounds are reported herein.

!

received revised accepted

and chemical evidence. Furthermore, in in vitro assays, compounds 4, 8, 9, and 15 (10 µM) exhibited moderate hepatoprotective activities against D-galactosamine-induced HL-7702 cell damage.

The genus Schefflera (Araliaceae) consists of about 1100 species, 35 of which can be found in China [1]. Schefflera kwangsiensis Merr. ex Li is a traditional Chinese medicine, distributed in southern China. The aerial parts of this plant have been used for the treatment of trigeminal neuralgia, headache, sciatica, rheumatalgia, trauma, and other ailments [2]. Previous phytochemical studies on S. kwangsiensis have demonstrated the presence of triterpenoid saponins [3]. In the meridian tropism theory of traditional Chinese medicine, S. kwangsiensis has the pharmaceutical effect of activating liver and kidney meridians, which results in alleviating pain in patients. We hypothesized that S. kwangsiensis might be good for the liver and selected the 95 % EtOH extract of the aerial parts for investigation. We observed that the n-BuOH-soluble extract showed hepatoprotective activity against D-galactosamine-induced toxicity in HL-7702 cells at a concentration of 55 µg/mL. Fractionation of this extract led to the isolation of seven new triterpenoid saponins, schekwangsiensides A–G (1–7), and a new triterpenoid, schekwangsienin (8), together with nine known triterpenoids and saponins (9–17) " Fig. 1). This is the first report on the hepato(l protective activities of S. kwangsiensis. In this paper, we report the isolation and structural elucidation of the new triterpenoid and saponins. Fur-

Materials and Methods !

General experimental procedures Optical rotations were measured with a JASCO P2000 polarimeter. IR spectra were recorded on a Nicolet 5700 spectrometer by an FT‑IR microscope transmission method. NMR measurements were performed on VNS-600, INOVA-500, and Bruker AV500-III spectrometers. HRESIMS were obtained using an Agilent 1100 series LC/MSD ion trap mass spectrometer. Preparative HPLC was conducted using a Shimadazu LC-6AD instrument with an SPD-20A detector and a YMC-Pack ODS‑A column (250 × 20 mm, 5 µm). Silica gel (200–300 mesh; Qingdao Marine Chemical Factory), Sephadex LH-20 (GE), MCI gel (35–75 µm, CHP 20PY; Mitsubishi Chemical Corporation), and ODS (50 µm; YMC) were used for column chromatography. TLC was carried out with GF254 plates (Qingdao Marine Chemical Factory). Spots were visualized by spraying with 10 % H2SO4 in 95 % EtOH followed by heating.

Plant material The aerial parts of S. kwangsiensis were collected in Fusui, Guangxi Province, Peopleʼs Republic of China, in September 2011, and identified by Pro-

Wang Y et al. Hepatoprotective Triterpenoids and …

Planta Med 2014; 80: 215–222

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Hepatoprotective Triterpenoids and Saponins of Schefflera kwangsiensis

Original Papers

Fig. 1

fessor Lin Ma (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College). A voucher specimen (ID-22162) has been deposited at the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing.

Extraction and isolation Air-dried and powered aerial parts of S. kwangsiensis (15.4 kg) were exhaustively extracted with 95% aqueous EtOH (3 × 80 L, under reflux, each for 2.5 h). The combined extracts were concentrated under reduced pressure to dryness. The residue (940.0 g) was suspended in H2O (5 L) and partitioned with petroleum ether (3 × 5 L), EtOAc (3 × 5 L), and n-BuOH (3 × 5 L) successively. The n-BuOH-soluble residue (285.4 g) was applied to a Diaion HP20 column (i. d. 15 × 100 cm, 2.5 kg) that was eluted with 30 % (20 L), 50 % (20 L), 70 % (20 L), and then 95% aqueous EtOH (20 L). After removing the solvent, the 70 % aqueous EtOH eluate (67.3 g) was subjected to silica gel CC (100–200 mesh, i. d. 10 × 15 cm, 500 g) and eluted with a gradient of CH2Cl2-MeOH [30 : 1 (5 L); 20 : 1 (5 L); 10 : 1 (5 L); 5 : 1 (5 L); 3 : 1 (5 L); 2 : 1 (5 L); 1 : 1 (5 L); MeOH (5 L)] to afford 11 fractions. Fractions 2 (6.3 g), 3 (5.8 g), 5 (4.2 g), 6 (3.3 g), 8 (8.4 g), 10 (3.2 g), and 11 (3.9 g) were separated by MPLC (5.5 × 40 cm, ODS, 30 mL/min, 6 h) and eluted with 40%, 60%, 70 %, 80 %, 90 %, and 100 % MeOH‑H2O (each 3 L) to afford about 20 subfractions. The triterpenoid or saponin-containing fractions 2−17 (0.8 g), 3−17 (0.4 g), 5−16 (0.5 g), 6−16 (0.4 g), 8−15 (0.7 g), 10−14 (0.6 g), and 11−12 (2.1 g) were chromatographed over Sephadex LH-20 eluted with 70–80 % MeOH‑H2O (1 L) as the mobile phase to give two or three subfractions. The triterpenoid or saponin-containing fractions 2− 17–2 (513.4 mg), 3− 17–2 (208.7 mg), 5− 16–2 (312.2 mg), 6− 16–2 (258.5 mg), 8− 15–2 (202.6 mg), 10− 14–2 (247.1 mg), and 11− 12–2 (807.5 mg) were further separated via silica gel [EtOAc-EtOH‑H2O, 50 : 2 : 1 (0.5 L); Wang Y et al. Hepatoprotective Triterpenoids and …

Planta Med 2014; 80: 215–222

Chemical structures of compounds 1–17.

30 : 2 : 1 (0.5 L); 20 : 2 : 1 (0.5 L); 15 : 2 : 1 (0.5 L); 10 : 2 : 1 (0.5 L); 7 : 2 : 1 (0.5 L); 5 : 2 : 1 (0.5 L)], respectively. Fraction 2− 17–2–2 (367.5 mg) was purified by preparative HPLC using 92 % MeOH‑H2O (5 mL/min) as the mobile phase to yield 8 (8.2 mg, 47 min), 11 (103.0 mg, 38 min), and 12 (35.4 mg, 42 min). Fraction 3− 17–2–3 (147.2 mg) was purified by preparative HPLC using 90 % MeOH‑H2O (5 mL/min) as the mobile phase to yield 1 (12.2 mg, 43 min), 9 (7.9 mg, 35 min), 10 (6.3 mg, 49 min), and 17 (26.8 mg, 38 min). Fraction 5− 16–2–2 (158.3 mg) was purified by preparative HPLC using 87 % MeOH‑H2O (5 mL/min) as the mobile phase to yield 14 (23.2 mg, 48 min) and 16 (37.4 mg, 43 min). Fraction 6− 16–2–2 (120.7 mg) was purified by preparative HPLC using 85% MeOH‑H2O (5 mL/min) as the mobile phase to yield 2 (33.2 mg, 41 min) and 6 (12.4 mg, 36 min). Fraction 8− 15– 2–3 (26.1 mg) was purified by preparative HPLC using 83 % MeOH‑H2O (5 mL/min) as the mobile phase to yield 3 (2.1 mg, 42 min) and 7 (6.2 mg, 38 min). Fraction 8− 15–2–4 (32.9 mg) was purified by preparative HPLC using 82 % MeOH‑H2O (5 mL/min) as the mobile phase to yield 13 (19.3 mg, 32 min). Fraction 10–14–2–3 (48.5 mg) was purified by preparative HPLC using 73 % MeOH‑H2O (5 mL/min) as the mobile phase to yield 15 (22.0 mg, 44 min). Finally, fraction 11− 12–2–2 (402.4 mg) was further separated by MCI gel (35–75 µm, 60 g) to afford 11− 12–2–2–1 (93.2 mg) and 11− 12–2–2–2 (185.0 mg), eluting with a step gradient of MeOH‑H2O [40% (200 mL), 60% (200 mL), 70% (200 mL), 10 mL/ min], and then purified by preparative HPLC using 70 % MeOH‑H2O (5 mL/min) as the mobile phase to yield 4 (35.2 mg, 62 min) and 5 (70.3 mg, 55 min), respectively.

Acid hydrolysis and sugar analysis Compound 1 (2 mg) was dissolved in 2 M HCl-H2O (2 mL) and heated to 85 °C for 15 h. The reaction mixture was extracted with EtOAc. The aqueous layer was evaporated under vacuum, diluted

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216

repeatedly with H2O, and evaporated in vacuo to furnish a neutral residue. The residue was dissolved in anhydrous pyridine (1 mL), to which 2 mg of L-cysteine methyl ester hydrochloride was added. The mixture was stirred at 60 °C for 2 h, and after evaporation in vacuo to dryness, 0.2 mL of N-trimethylsilylimidazole was added; the mixture was kept at 60 °C for another 2 h. The reaction mixture was partitioned between n-hexane and H2O (2 mL each), and then the n-hexane extract was analyzed by GC under the following conditions: capillary column, HP-5 (30 m × 0.25 mm, with a 0.25 µm film; Dikma); detection, FID; detector temperature, 280 °C; injection temperature, 250 °C; initial temperature 160 °C, then raised to 280 at 5 °C/min, final temperature maintained for 10 min; carrier, N2 gas. From the acid hydrolysate of 1, D-glucofuranurono-6,3-lactone was confirmed by comparison of the retention time of its derivative with that of an authentic sugar derivatized in a similar way, which showed a retention time of 16.77 min. The constituent sugars of compounds 2–7 were identified by the same method as 1. Retention times of authentic samples were detected at 18.91 min (D-glucose) for 2 and 6, 15.56 min (D-xylose) for 3 and 7, and 15.98 min (D-glucuronic acid) and 16.63 min (L-rhamnose) for 4 and 5, respectively.

Schekwangsienside E (5): amorphous power, [α]20 D − 30.5 (c 0.11, MeOH); IR νmax 3378, 2938, 1731, 1651, 1365, 1078, 991 cm−1; 1H " Tables 1, 2, and 4; HRESIMS m/z NMR and 13C NMR, see l 1269.5842 [M + Na]+ (calcd. for C60H94O27Na 1269.5875). Schekwangsienside F (6): amorphous power, [α]20 D − 0.9 (c 0.21, MeOH); IR νmax 3436, 2940, 1728, 1399, 1059, 794 cm−1; 1H " Tables 1, 2, and 4; HRESIMS m/z NMR and 13C NMR, see l + 785.4457 [M + Na] (calcd. for C42H66O12Na 785.4446). Schekwangsienside G (7): amorphous power, [α]20 D − 3.1 (c 0.12, MeOH); IR νmax 3358, 2940, 1740, 1456, 1377, 1077, 886 cm−1; 1 " Tables 1, 2, and 4; HRESIMS m/z H NMR and 13C NMR, see l 773.4474 [M + Na]+ (calcd. for C41H66O12Na 773.4446). Schekwangsienin (8): amorphous power, [α]20 D + 85.8 (c 0.10, MeOH); IR νmax 3426, 2947, 1700, 1666, 1553, 1460, 1384, 1259, " Tables 1 and 2; HRE1043, 826 cm−1; 1H NMR and 13C NMR, see l SIMS m/z 467.3167 [M – H]− (calcd. for C30H43O4 467.3167).

Hepatoprotective effects assay

Results and Discussion

The hepatoprotective effects of compounds 1–17 were determined by a (MTT) colorimetric assay in HL-7702 cells (Cell Culture Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). Each cell suspension of 2 × 104 cells in 200 µL of RPMI 1640 containing fetal calf serum (10%), penicillin (100 U/mL), and streptomycin (100 µg/mL) was planted in a 96well microplate and precultured for 24 h at 37 °C under a 5 % CO2 atmosphere. Fresh medium (100 µL) containing bicyclol (purity, > 98%; Beijing Union Pharmaceutical Plant) and test samples were added, and the cells were cultured for 1 h. Next, the cultured cells were exposed to 25 mM D-galactosamine (Sigma) for 24 h. Then, 100 µL of 0.5 mg/mL MTT was added to each well after the withdrawal of the culture medium and incubated for an additional 4 h. The resulting formazan was dissolved in 150 µL of DMSO after aspiration of the culture medium. The optical density (OD) of the formazan solution was measured on a microplate reader at 492 nm. Inhibition (%) was obtained by the following formula:

!

Inhibition (%) = [(OD(sample) – OD(control)) /(OD(normal) – OD(control))] ×100 Schekwangsienside A (1): amorphous power, [α]20 D − 5.5 (c 0.11, MeOH); IR νmax 3425, 2940, 1785, 1694, 1462, 1073, 927 cm−1; 1 " Tables 1–3; HRESIMS m/z 637.3729 H NMR and 13C NMR, see l + [M + Na] (calcd. for C36H54O8Na 637.3711). Schekwangsienside B (2): amorphous power, [α]20 D + 20.7 (c 0.10, MeOH); IR νmax 3397, 2941, 1735, 1459, 1201, 1070, 1028, " Tables 1–3; HRESIMS m/z 973 cm−1; 1H NMR and 13C NMR, see l 761.4475 [M – H]− (calcd. for C42H65O12 761.4482). Schekwangsienside C (3): amorphous power, [α]20 D − 1.8 (c 0.05, MeOH); IR νmax 3359, 2931, 1739, 1662, 1462, 1388, 1076, " Tables 1–3; HRESIMS m/z 894 cm−1; 1H NMR and 13C NMR, see l 773.4438 [M + Na]+ (calcd. for C41H66O12Na 773.4446). Schekwangsienside D (4): amorphous power, [α]20 D − 29.6 (c 0.11, MeOH); IR νmax 3349, 2936, 1731, 1594, 1364, 1078, 993 cm−1; 1H " Tables 1–3; HRESIMS m/z 1125.5448 [M NMR and 13C NMR, see l + + Na] (calcd. for C54H86O23Na 1125.5452).

Supporting information IR, MS, and NMR spectra of compounds 1–8 are available as Supporting Information.

The 95% EtOH extract from the dried aerial parts of S. kwangsiensis was suspended in H2O and successively extracted with petroleum ether, EtOAc, and n-BuOH. The n-BuOH-soluble extract showed hepatoprotective activity against D-galactosamine-induced toxicity in HL-7702 cells at a concentration of 55 µg/mL. Therefore, it was subjected to multiple chromatographic steps, yielding compounds 1–17. Comparision of the NMR and MS data with reported values led to the identification of structures of the known compounds 9–16 as hederagenin (9) [4], maslinic acid (10) [5], 22α-hydroxyoleanolic acid (11) [6], 21β-hydroxy-3-oxo-olean-12-en-28-oic acid (12) [7], scheffleraside I (13) [8], oleanoic acid 28-O-β-D-glucopyranoside (14) [4], 3β-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl] oleanolic acid (15) [9], 28-O-β-D-glucopyranosyl-3β-hydroxy-lup-20(29)-en-28-oate (16) [10], and 2α,3β-dihydroxy-urs-12-en-28-oic acid (17) [11]. Schekwangsienside A (1) was obtained as a white, amorphous power. The molecular formula was determined to be C36H54O8 on the basis of HRESIMS (m/z 637.3729 [M + Na]+). The 1D NMR " Tables 1–3) revealed the presence of seven tertiary methdata (l yl groups at δH 1.23 (H3-27), 0.99 (H3-30), 0.95 (H3-23), 0.93 (H329), 0.92 (H3-26), 0.86 (H3-24), and 0.70 (H3-25), an olefinic proton at δH 5.40 (br s) with two typical olefinic carbon signals at δC 122.5 and 144.7, and a carboxylic carbon signal at δC 180.2, indicative of an olean-12-en-28-oic acid skeleton. An oxymethine proton signal assignable to H-3 of the aglycone moiety was observed at δH 3.14 (dd, J = 11.7, 4.3 Hz). A ROESY correlation between H-3 and H-5 (δH 0.67, d, J = 11.4 Hz) indicated the α-orientation of H-3. Thus, the aglycone was identified as 3β-hydroxyolean-12-en-28-oic acid (oleanolic acid) [4]. The 1H NMR spectrum of 1 showed a signal of a sugar at δH 5.69 (s), which gave a correlation in the HSQC spectrum with a carbon atom resonance at δC 113.3. After acid hydrolysis, the sugar unit was confirmed to be a D-glucofuranurono-6,3-lactone (Gluf L), which was identified by gas chromatographic (GC) analysis of its trimethylsilyl L-cysteine derivative. Furthermore, an HMBC correlation between Gluf L‑H‑1 (δH 5.69) and Gluf L–C‑4 (δC 79.9), and correlations between Gluf L‑H-3 (δH 5.17), H-4 (δH 5.34), H-5 (δH 4.96), Wang Y et al. Hepatoprotective Triterpenoids and …

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217

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Original Papers

218

Original Papers

H NMR spectroscopic data for the aglycones of compounds 1–7 and for 8. 1a

2a

3a

4b

5c

6d

7d

8a

1a

1.34 br d (13.0)

1.54 (o)

1.53 (o)

1.54 (o)

1.48 (o)

0.75 m 2.34 dd (14.0, 4.0) 1.75 d (14.0) 3.14 dd (11.7, 4.3)

0.97 (o) 1.89 (o) 1.78 (o) 3.41 dd (10.5, 4.5)

0.98 (o) 1.85 (o) 1.80 (o) 3.41 m

5 6a 6b

0.67 d (11.4) 1.40 m 1.23 (o)

0.83 d (10.5) 1.51 m 1.35 m

0.83 d (11.5) 1.51 (o) 1.33 (o)

0.89 (o) 1.78 (o) 1.64 (o) 3.09 dd (12.0, 4.5) 0.67 (o) 1.44 (o) 1.30 (o)

0.86 (o) 1.67 (o) 1.51 (o) 2.97, dd (10.5, 3.0) 0.67 (o) 1.45 (o) 1.26 (o)

1.62 br d (12.6) 0.93 (o) 1.82 br t 1.74 (o) 3.42 m

7.01 d (10.0)

1b 2a 2b 3

1.63 br d (12.6) 0.97 m 1.81 (o) 1.69 (o) 3.43 dd (15.9, 7.6) 0.79 d (9.6) 1.52 (o) 1.28 (o)

7a 7b 9

1.39 m 1.22 (o) 1.53 t (8.5)

1.46 m 1.34 (o) 1.64 br t

1.38 (o) 1.22 (o) 1.47 (o)

1.35 (o) 1.20 (o) 1.45 (o)

1.37 (o) 1.31 (o) 1.33 (o)

11a 11b 12a 12b 13

1.80 m

1.93 (o)

1.54 (o) 1.50 (o) 1.66 dd (10.0, 7.5) 1.93 (o)

0.77 (o) 1.44 m 1.28 dd (12.0, 2.4) 1.54 (o) 1.39 (o) 1.36 (o)

1.80 (o)

1.77 m

5.40 br s

5.43 br s

5.45 br s

5.15 br s

5.15 br s

15a

2.12 m

2.29 m

1.69 (o)

1.25 (o)

1.35 (o) 1.15 (o) 1.88 d (6.0) 1.16 (o) 2.71 td (11.4, 3.0) 2.04 (o)

15b 16a

1.14 (o) 2.09 m

1.13 (o) 2.03 m

2.30 td (13.5, 4.5) 1.25 (o) 2.10 (o)

0.98 (o) 1.94 br t (10.5)

0.95 (o) 1.93 (o)

16b 18

1.94 m 3.24 dd (13.5, 4.0)

1.92 (o) 3.18 (o)

19a

1.78 (o)

1.73 (o)

1.93 (o) 3.19 dd (13.5, 4.0) 1.80 (o)

1.61 (o) 2.75 dd (14.0, 4.0) 1.62 (o)

1.56 (o) 2.74 dd (14.0, 3.0) 1.62 (o)

1.36 (o) 1.15 (o) 1.86 (o) 1.18 (o) 2.67 br t (12.0) 1.98 br t (13.2) 1.43 (o) 2.61 br d (12.6) 1.48 (o) 1.73 (o)

1.60 d (10.0) 1.43 (o) 1.37 br d (12.7) 1.47 (o) 1.31 (o) 1.91 dd (11.3, 5.8) 2.14 (o) 2.03 d (14.0) 5.58 br s

3.39 td (9.6, 3.0)

3.37 td (11.4, 4.8)

19b

1.26 (o)

1.22 (o)

1.27 (o)

1.04 (o)

1.06 (o)

21a

1.43 m

1.32 m

1.29 (o)

1.32 (o)

2.22 (o)

2.06 (o)

21b 22a

1.18 (o) 2.02 m

1.09 (o) 1.92 m

1.11 (o) 1.63 (o)

1.14 (o) 1.56 (o)

1.43 (o) 2.24 (o)

22b 23 24 25 26 27 29a 29b 30

1.80 (o) 0.95 s 0.86 s 0.70 s 0.92 s 1.23 s 0.93 s

1.77 (o) 1.20 s 1.01 s 0.94 s 1.10 s 1.21 s 0.86 s

1.37 td (14.0, 3.5) 1.07 (o) 1.88 td (14.0, 4.0) 1.76 (o) 1.18 s 0.96 s 0.87 s 1.07 s 1.25 s 0.90 s

1.52 (o) 1.00 s 0.77 s 0.85 s 0.70 s 1.06 s 0.81 s

1.43 (o) 1.00 s 0.74 s 0.83 s 0.66 s 1.06 s 0.86 s

0.99 s

0.87 s

0.90 s

0.83 s

0.86 s

1.49 (o) 1.20 s 0.99 s 0.83 s 1.13 s 1.00 s 4.87 br s 4.71 br s 1.72 s

1.36 (o) 2.20 dd (12.6, 7.8) 1.49 m 1.18 s 0.95 s 0.75 s 1.11 s 1.04 s 4.90 br s 4.73 br s 1.73 s

No

a

1

1.38 (o) 2.89 br d (12.6) 1.55 (o) 1.75 t (11.4)

5.95 d (10.0)

2.12 (o) 1.15 m 2.20 (o) 2.13 (o) 3.48 dd (14.0, 4.0) 2.05 (o) 1.50 dd (14.5, 4.0) 3.97 t (8.0)

2.35 d (8.0)

1.20 s 1.09 s 1.01 s 1.01 s 1.25 s 1.28 s 1.29 s

Measured in pyridine-d5, at 500 MHz; b measured in methanol-d4, at 500 MHz; c measured in DMSO-d6, at 500 MHz; d measured in pyridine-d5, at 600 MHz

and δC 175.5 also confirmed the presence of a hexofuranurono6,3-lactone. The β-anomeric configuration for the sugar unit was judged from its small 3JH1,H2 coupling constant (J < 1.5 Hz) [12]. The glucofuranurono-6,3-lactone was located at C-3 of the aglycone by the HMBC signal between Gluf L‑H‑1 (δH 5.69) and C-3 (δC 88.6). Therefore, the structure of schekwangsienside A was determined as 3β-O-(β-D-glucofuranosylurono-6,3-lactone)-12en-28-oic acid (1). The molecular formula of schekwangsienside B (2), C42H66O12, was deduced by HRESIMS (m/z 761.4475 [M – H]−). The NMR data " Tables 1–3). The 1H of the aglycone of 2 resembled those of 1 (l NMR spectrum of 2 showed a signal of a sugar at δH 6.24 (d, J = 6.0 Hz), which correlated to the anomeric carbon at δC 95.4 in Wang Y et al. Hepatoprotective Triterpenoids and …

Planta Med 2014; 80: 215–222

the HSQC spectrum. After acid hydrolysis, the sugar unit was identified as a D-glucose by GC analysis. The 3JH1, H2 value (J = 6.0 Hz) suggested the β-glycosidic linkage of the glucose. An HMBC signal of Glc-H‑1 (δH 6.24) with C-28 (δC 176.4) was used to locate the glucose at C-28 of the aglycone. Moreover, the 13C NMR of 2 showed 42 carbon signals, 30 of which were assigned to the aglycone, 6 to the glucose, and the remaining 6 to carbons (δC 171.7, 46.7, 70.0, 46.4, 175.3, 28.3), and was determined to be a (3R)-3-hydroxy-3-methylglutaryl group (HMG) by comparison with the 1H NMR and 13C NMR data in the literature [13, 14]. This was also confirmed by the HMBC experiment. The location of this group was assigned at Glc-C‑6 by the correlations between δH 4.77 and 4.91 (Glc-H‑6) and δC 171.7 in the HMBC spectrum,

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Table 1

Original Papers

a

13

C NMR spectroscopic data for the aglycones of compounds 1–7 and for 8.

No

1a

2a

3a

4b

5c

6d

7d

8a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

38.5 25.5 88.6 39.0 55.7 18.5 33.2 39.7 48.0 36.9 23.7 122.5 144.7 42.0 28.3 23.7 46.6 42.1 46.5 30.9 34.2 33.1 28.3 16.9 15.3 17.3 26.1 180.2 33.3 23.8

39.0 28.1 78.1 39.3 55.8 18.8 33.2 39.9 48.1 37.4 23.8 122.9 144.1 41.7 28.2 23.4 47.0 42.1 46.2 30.7 34.0 32.5 28.8 16.5 15.7 17.5 26.1 176.4 33.1 23.6

39.0 28.1 78.1 39.4 55.9 18.8 33.2 40.0 48.2 37.4 23.9 122.7 144.4 42.1 28.7 23.1 47.1 41.8 46.3 30.8 34.1 32.4 28.8 16.5 15.6 17.5 26.1 176.5 33.2 23.7

39.8 26.9 92.4 40.5 56.9 19.3 33.9 40.7 49.0 37.9 24.5 123.8 144.8 42.9 28.9 24.0 48.0 42.6 47.2 31.5 34.9 33.1 28.7 16.8 16.0 17.7 26.3 178.1 33.5 24.0

38.0 25.4 89.1 38.7 55.0 17.7 32.3 38.8 47.0 36.3 23.0 121.8 143.4 41.2 27.1 22.4 46.0 40.7 45.6 30.3 33.2 31.6 27.7 16.1 15.1 16.7 25.5 175.2 32.8 23.4

39.3 28.3 78.1 39.5 55.9 18.8 34.7 41.2 50.9 37.5 21.1 26.1 38.4 42.8 30.1 32.2 57.0 49.8 47.5 150.9 30.9 36.9 28.7 16.3 16.4 16.3 14.9 175.0 110.1 19.4

39.3 28.3 78.1 39.5 55.9 18.8 34.8 41.3 51.0 37.5 21.1 26.1 38.2 42.8 30.7 31.8 57.1 49.8 47.3 151.0 30.8 37.0 28.7 16.3 16.4 16.5 14.9 174.9 110.0 19.5

159.2 125.1 204.3 44.6 53.8 19.1 32.9 40.4 42.2 39.7 23.6 122.2 144.5 42.5 28.4 25.1 48.8 42.0 47.2 36.7 72.6 41.8 27.9 21.9 18.7 17.8 25.8 179.8 29.9 17.8

Measured in pyridine-d5, at 125 MHz; b measured in methanol-d4, at 125 MHz; c measured in DMSO-d6, at 125 MHz; d measured in pyridine-d5, at 150 MHz

Table 3 No

1

H and 13C NMR spectroscopic data for the sugar units of compounds 1–4. 1 δH mult.

2 δc

(J in Hz) 1 2 3 4 5 6a 6b 1 2a 2b 3 4a 4b 5a 5b 6a 6b

Gluf L at C-3 5.69 s 4.88 s 5.17 d (4.0) 5.34 dd (6.0, 4.0) 4.96 d (6.0)

δH mult.

3 δc

(J in Hz) 113.3 78.6 83.9 79.9 70.6 175.7

Glc at C-28 6.24 d (6) 4.19 (o) 4.20 (o) 4.13 (o) 4.09 (o) 4.91 d (11.0) 4.77 d (11.0) HMG 3.10 d (14.0) 3.01 d (14.0) 3.14 d (14.0) 3.07 d (14.0)

1.71 s

4

δH mult.

δc

(J in Hz) 95.4 73.9 78.6 71.0 76.0 64.1

Glc at C-28 6.23 d (8.0) 4.34 m 3.92 m 4.30 (o)

δH mult.

δc

(J in Hz) 93.6 80.3 79.2 70.8

3.67 (o)

δc

(J in Hz) 105.7 77.9 78.1 73.5

Rha 5.09 br s 3.82 br s 3.65 (o) 3.32 (o)

76.5 172.7

4.02 m 1.15 d (6.0)

69.5 18.3

102.1 79.3

Glc II at C-28 5.28 d (8.0) 3.23 (o)

95.7 73.9

101.9 72.2 72.1 74.2

171.7 46.7

4.88 (o) 4.41 (o) 4.35 (o) Xyl 5.47 d (8.0) 4.07 t (8.5)

106.0 76.0

70.0 46.4

4.17 t (8.5) 4.27 (o)

78.5 71.2

3.37 (o) 2.96 t (9.0)

79.4 72.6

3.33 (o) 3.26 (o)

78.6 71.1

175.3

4.41 (o) 3.72 t (11.0)

67.4

3.14 t (9.0)

78.2

3.26 (o)

78.2

3.74 (o) 3.44 (o)

63.6

3.71 (o) 3.58 d (11.5)

62.3

28.3

78.8 62.1

Glc A at C-3 4.36 d (7.5) 3.64 (o) 3.50 t (9.0) 3.40 (o)

δH mult.

Glc I 4.77 (o) 3.27 (o)

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Table 2

219

Original Papers

Table 4 No

1

H and 13C NMR spectroscopic data for the sugar units of compounds 5–7. 5 δH mult.

6 δc

(J in Hz) 1 2 3 4 5 6a 6b

1 2a 2b 3 4a 4b 5a 5b 6a 6b

Glc A at C-3 4.21 d (6.0) 3.53 (o) 3.38 (o) 3.28 (o) 3.47 (o)

δH mult.

δc

(J in Hz) 105.5 76.1 77.0 71.7 75.2 170.6

Glc I 4.79 d (8.0) 3.16 (o)

99.7 76.9

3.26 (o) 2.88 t (9.0)

77.6 71.2

3.03 br t (6.5)

76.6

3.70 d (10.0) 3.35 (o)

61.9

Rha 5.02 br s 3.65 br s 3.50 (o) 3.15 (o) 3.93 m 1.08 d (6.0)

δH mult. (J in Hz)

99.9 70.6 70.6 72.3 68.0 18.1

Glc II at C-28 5.26 d (8.0) 3.12 (o) 3.22 (o) 3.13 (o) 3.41 (o) 4.23 d (11.5) 4.01 dd (11.5, 5.5) HMG 2.66 d (14.5) 2.47 (o) 2.50 (o) 2.46 (o)

δH mult.

1.24 s

Planta Med 2014; 80: 215–222

7 δc

(J in Hz) 93.8 69.6 76.3 72.2 74.4 63.1

170.4 45.5 69.0 45.3

Glc at C-28 6.34 d (7.2) 4.22 (o) 4.22 (o) 4.20 (o) 4.23 (o) 4.91 d (11.4) 4.78 dd (11.4, 3.6) HMG 3.08 (o) 3.00 (o) 3.12 (o) 3.02 (o)

172.5

which was also implied by the presence of a deshielded signal at Glc-C‑6 (by 2.0 ppm). Thus, the structure of schekwangsienside B was elucidated as 3β-O-hydroxyolean-12-en-28-oic acid 28-O[6-O-((3R)-3-hydroxyl-3-methylglutaryl)-β-D-glucopyranosyl] ester (2). Schekwangsienside C (3) was isolated as a white, amorphous power, and its molecular formula was established as C41H66O12 on the basis of HRESIMS (m/z 773.4438 [M + Na]+). Comparison of the spectroscopic data of 3 with those of 2 showed they were " Tables 1–3). almost superimposable on those of the aglycone (l 1 The H NMR spectrum of 3 showed signals of two sugars at δH 6.23 (d, J = 8.0 Hz) and 5.47 (d, J = 8.0 Hz), which gave correlations in the HSQC spectrum at δC 93.6 and 106.0. After acid hydrolysis, the sugar units were confirmed to be D-glucose and D-xylose in a ratio of 1 : 1, and were identified by GC analysis of their trimethylsilyl L-cysteine derivatives. The coupling constant confirmed the β-glycosidic linkages for the two sugar units. The glucose was connected to C-28 of the aglycone, which was deduced from the HMBC correlation between Glc-H‑1 (δH 6.23) and C-28 (δC 176.5), and the xylose was connected to Glc-C‑2, which was allowed by the HMBC correlation between Xyl-H‑1 (δH 5.47) and Glc-C‑2 (δC 80.3), and the ROESY correlation between Xyl-H‑1 (δH 5.47) and Glc-H‑2 (δH 4.34). Accordingly, the structure of schekwangsienside C was elucidated as 3β-O-hydroxyolean-12en-28-oic acid 28-O-[β-D-xylopyranosyl-(1 → 2)-β-D-glucopyranosyl] ester (3). Schekwangsienside D (4) was assigned a molecular formula of C54H86O23 from its HRESIMS (m/z 1125.5448 [M + Na]+). The 1H " Tables 1–3) were also and 13C NMR data of the aglycone of 4 (l in good agreement with those of oleanolic acid. Besides, the 1H NMR spectrum of 4 displayed signals of four sugars at δH 4.36 (d, J = 7.5 Hz), 4.77 (overlapped), 5.09 (br s), and 5.28 (d, J = 8.0 Hz), which correlated to the anomeric carbons at δC 105.7, 102.1, 101.9, and 93.6 in the HSQC spectrum. After acid hydrolysis, the sugar units were confirmed to be D-glucuronic acid, D-glucose, and L-rhamnegin in a ratio of 1 : 2 : 1. This was also confirmed by

Wang Y et al. Hepatoprotective Triterpenoids and …

δc

27.4

1.70 s

δH mult.

δc

(J in Hz) 95.2 74.0 78.5 71.0 76.1 64.1

Glc at C-28 6.32 d (7.8) 4.34 (o) 3.91 br d (7.5) 4.31 (o) 4.26 (o) 4.40 (o) 4.35 (o)

93.1 79.9 79.3 70.8 78.7 62.0

171.7 46.8

Xyl 5.49 d (7.8) 4.07 t (7.0)

105.7 76.0

70.1 46.7

4.16 t (7.5) 4.26 (o)

78.4 71.2

175.0

4.37 (o) 3.69 t (9.0)

67.3

28.4

GC analysis. The 3JH1, H2 value [J = 7.5, overlapped (about 7.5), and 8.0 Hz] suggested the β-glycosidic linkages for the two glycose units and the glucuronic acid, and the rhamnegin unit was determined to be α-anomer on the basis of the 3JH1, H2 value (J < 1.5 Hz). A sugar chain seemed to be composed of D-glucuronic acid, D-glucose, and L-rhamnegin, and located at C-3 of the aglycone, which was similar to 3β-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl] oleanolic acid in the literature [9]. This could also be deduced from the HMBC correlations between Glu A–H‑1 (δH 4.36) and C-3 (δC 92.4), Glc I‑H‑1 (δH 4.77) and Glu A–C‑2 (δC 77.9), Rha-H‑1 (δH 5.09) and Glc I– C‑2 (δC 79.3), and the ROESY correlations between Glc I‑H‑1 (δH 4.77) and Glu A–H‑2 (δH 3.64), Rha-H‑1 (δH 5.09) and Glc I‑H‑2 (δH 3.27). Furthermore, the other glucose was located at C-28 by the HMBC correlation between Glc II‑H‑1 (δH 5.28) and C-28 (δC 178.1). Thus, the structure of schekwangsienside D was elucidated as 3β-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl] olean-12-en-28-oic acid 28-O-(β-D-glucopyranosyl) ester (4). Schekwangsienside E (5) was assigned a molecular formula of C60H94O27 from its HRESIMS (m/z 1269.5842 [M + Na]+). Its NMR " Tables 1, 2, and 4) were closely comparable to those of 4, data (l except for the presence of a group composed of six carbons which was the same as the HMG of 2. The location of this group was assigned at Glc II–C‑6 by the correlations between δH 4.01, 4.23 (Glc II‑H‑6), and δC 170.4, respectively, in the HMBC sepctrum, and allowed by the presence of a deshielded signal at Glc II–C‑6 (by 2.0 ppm). Thus, the structure of schekwangsienside E was elucidated as 3β-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl] olean-12-en-28-oic acid 28-O-[6-O-((3R)-3-hydroxyl-3-methylglutaryl)-β-D-glucopyranosyl] ester (5). Schekwangsienside F (6) exhibited the same molecular formula, C42H66O12, as 2, as established by HRESIMS at m/z 785.4457 [M + " Tables 1, 2, and 4) revealed the presNa]+. The 1D NMR data (l ence of six tertiary methyl groups at δH 1.73 (H3-30), 1.18 (H3-

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220

Original Papers

Table 5 Hepatoprotective effects of compounds 4, 8, 9, and 15 (10 µM) against D-galactosamine-induced toxicity in HL-7702 cellsa. Compound Normal Control Bicyclol 4 8 9 15 a

Cell survival rate

Inhibition

(% of normal)

(% of control)

100 ± 2 64.6 ± 7.9 84.7 ± 5.7** 95.5 ± 1.2** 91.8 ± 5.5** 86.7 ± 7.9** 84.7 ± 9.9*

56.8 87.3 76.8 62.4 56.8

Results are expressed as means ± SD (n = 3; for normal and control, n = 6); bicyclol

was used as a positive control (10 µM). * P < 0.05, ** p < 0.01

were credible. Therefore, 4, 8, 9, and 15 might have some benefits in the prevention or treatment of hepatic disease. Further research should be conducted.

Acknowledgements !

The project was supported by the National Science and Technology Project of China (No. 2012ZX09301002–002) and the State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College.

Conflict of Interest !

The authors declare no conflict of interest.

References 1 Botany Editorial Board, The Chinese Academy of Sciences. Flora of China, Volume 13. Beijing: Science Press; 2007: 454 2 Chinese Pharmacopoeia Commission. Pharmacopoeia of the Peopleʼs Republic of China, Vol. 1. Beijing: China Medical Science Press; 1977: 193–195 3 Zhang Q, Shen J, Zhao YW, Wang ZZ, Xiao W. Study on glycosides from stems of Schefflera kwangsiensis. Chin Tradit Herb Drugs 2012; 43: 2141–2145 4 Srivastava SK, Jain DC. Triterpenoid saponins from plants of Araliaceae. Phytochemistry 1989; 28: 644–647 5 Yagi A, Okamura N, Haraguchi Y, Noda K, Nishioka I. Studies on the constituents of Zizyphi fructus II structure of new p-coumaroylates of maslinic acid. Chem Pharm Bull 1978; 26: 3075–3079 6 Yoshikawa M, Shimada H, Morikawa T, Yoshizumi S, Matsumura N, Murakami T, Matsuda H, Hori K, Yamahara J. Medicinal foodstuffs VII on the saponin constituents with glucose and alcohol absorption-inhibitory activity from a food garnish “tonburi”, the fruit of Japanese Kochia scoparia (L) Schrad: structures of scoparianosides A, B, and C. Chem Pharm Bull 1997; 45: 1300–1305 7 Capel CS, de Souza ACD, de Carvalho TC, de Sousa JPB, Ambrosio SR, Martins CHG, Cunha WR, Galan RH, Furtado NAJC. Biotransformation using Mucor rouxii for the production of oleanolic acid derivatives and their antimicrobial activity against oral pathogens. J Indian Microbiol Biotechnol 2011; 38: 1493–1498 8 Jiang QP, Xiao ZY. Study on saponins isolated from Schefflera delavayi Harms ex Diels. West China J Pharm Sci 1990; 5: 133–135 9 Pancharoen O, Tuntiwachwuttikul P, Taylor WC, Picker K. Triterpenoid glycosides from Schefflera lucantha. Phytochemistry 1994; 35: 987– 992 10 Chatterjee P, Peaauto JM, Kouzi SA. Glucosidation of betulinic acid by Cunninghamella species. J Nat Prod 1999; 62: 761–763

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23), 1.11 (H3-26), 1.04 (H3-27), 0.95 (H3-24), and 0.75 (H3-25), two olefinic protons at δH 4.71 (br s) and 4.87 (br s) with two typical olefinic carbon signals at δC 150.9 and 110.1, and a carboxylic carbon signal at δC 175.0, indicative of a lup-20(29)-en-28-oic acid skeleton. An oxymethine proton signal assignable to H-3 of the aglycone moiety was observed at δH 3.43 (dd, J = 15.9, 7.6 Hz). The ROESY correlation between H-3 and H-5 (δH 0.67, d, J = 11.4 Hz) indicated the α-orientation of H-3. Therefore, the aglycone was identified as 3β-hydroxylup-20(29)-en-28-oic acid (betulinic acid) [10]. Except for the aglycone, the remaining 1H NMR and 13C NMR data seemed to be the same as 2. The position of the 6-O‑HMG-β-D-glucose was determined to be at C-28 of the aglycone, based on the HMBC correlation between Glc-H‑1 (δH 6.34) and C-28 (δC 175.0). Thus, the structure of schekwangsienside F was elucidated as 3β-hydroxylup-20(29)-en-28-oic acid 28-O-[6-O-((3R)-3-hydroxyl-3-methylglutaryl)-β-D-glucopyranosyl] ester (6). Schekwangsienside G (7) was shown to have the same molecular formula, C41H66O12, as 3, as established by HRESIMS (m/z 773.4474 [M + Na]+). NMR data of the aglycones of 7 and 6 were " Tables 1, 2, and 4), and the NMR specalmost superimposable (l tra of the remaining sugar chain seemed to be closely comparable to those of 3. The positon of the sugar chain was confirmed by the HMBC correlation of Glc-H‑1 (δH 6.32) with C-28 (δC 174.9) of the aglycone. Accordingly, the structure of schekwangsienside G was elucidated as 3β-O-hydroxylup-20(29)-en-28-oic acid 28-O-[βD-xylopyranosyl-(1 → 2)-β-D-glucopyranosyl] ester (7). Schekwangsienin (8) exhibited an [M – H]− ion peak at m/z 467.3167 in its HRESIMS, corresponding to the molecular formu" Tables 1 and 2) revealed the la C30H44O4. The 1D NMR data (l presence of seven tertiary methyl groups at δH 1.29 (H3-30), 1.28 (H3-29), 1.25 (H3-27), 1.09 (H3-24), 1.20 (H3-23), 1.01 (H3-25), and 1.01 (H3-26), three olefinic protons at δH 7.01 (d, 10.0), δH 5.95 (d, 10.0), and δH 5.40 (br s), four olefinic carbon signals at δC 159.2, 144.7, 125.1, and 122.5, a carboxylic carbon signal at δC 180.2, and a carbonyl at δC 204.3. The signals of the seven methyl groups and δH 5.40 (br s), δC 144.7, 122.5, and 180.2 were typical characteristics of the olean-12-en-28-oic acid skeleton. Besides, the ketocarbonyl (δC 204.3) was assigned to C-3, because of the correlations of it with H3-23 (δH 1.09) and H3-24 (δH 1.20) in the HMBC spectrum, and the other double bond, composed by δC 159.2 and 125.1, which gave correlations in the HSQC spectrum with protons at δH 7.01 (d, 10.0) and δH 5.95 (d, 10.0), respectively, was determined to be the bond of C-1 and C-2 by the correlations of δH 7.01, δH 5.95 and δC 204.3 in the HMBC spectrum. An oxymethine proton signal observed at δH 3.97 (t, J = 8.0 Hz), which correlated to the carbon at δC 72.5 in the HSQC spectrum, was assigned to H-21 based on the correlation with H-22 (δH 2.35) in the 1H–1H COSY and signals of C-21 (δC 72.5) with H3-29 (δH 1.28) and H3-30 (δH 1.29) in the HMBC spectrum. Moreover, ROESY correlations between H-18 (β-orientation) and H-19a (δH 1.50) and between H-19b (δH 2.05, α-orientation) and H-21 (δH 3.97) indicated the α-orientation of H-21. Therefore, the structure of schekwangsienin H was elucidated as 3-oxo-21β-hydroxyolean-1(2), 12(13)-dien-28-oic acid (8). Triterpenoids and saponins 1–17 were evaluated for their hepatoprotective activities against D-galactosamine-induced toxicity in HL-7702 cells, using the hepatoprotective activity drug bicyclol " Table 5, compounds 4, as the positive control [15]. As shown in l 8, 9, and 15 exhibited pronounced hepatoprotective activity. According to the description in the literature [16, 17], activities of triterpenoids and saponins evaluated in vitro by a cellular assay

221

Original Papers 11 Mahato SB, Kundu AP. 13C NMR spectra of pentacyclic triterpenoids – a compilation and some salient features. Phytochemistry 1994; 37: 1517–1575 12 Liberek B, Tuwalska D, Santos-Zounon I, Konitz A, Sikorski A, Smiatacz Z. Conformations of methyl 2,5-di-O-acetyl-β-D-glucofuranosidurono6,3-lactone and 1,2,5-tri-O-acetyl-β-D-glucofuranurono-6,3-lactone in the crystal structure and in solution. Carbohydr Res 2006; 341: 2275–2285 13 Zhao J, Nakamura N, Hattori M, Yang XW, Komatsu K, Qiu MH. New triterpenoid saponins from the roots of Sinocrassula asclepiadea. Chem Pharm Bull 2004; 52: 230–237

14 Ma L, Gu YC, Luo JG, Wang JS, Huang XF, Kong LY. Triterpenoid saponins from Dianthus versicolor. J Nat Prod 2009; 72: 640–644 15 Liu GT. The anti-virus and hepatoprotective effect of bicyclol and its mechanism of action. Chin J New Drugs 2001; 10: 325–327 16 Masaharu T, Naoharu W, Mayumi S, Hideaki K, Sadafumi O, Zhang PL, Rao C, Chen WM. New hepatoprotective triterpenes from Canarium album. Planta Med 1989; 55: 44–47 17 Junei K, Masafumi O, Manabu U, Yoshimi S, Tomoki H, Yuichi S, Toshihiro N. Hepatoprotective and hepatotoxic actions of oleanolic acid-type triterpenoidal glucuronides on rat primary hepatocyte cultures. Chem Pharm Bull 1999; 47: 290–292

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Hepatoprotective triterpenoids and saponins of Schefflera kwangsiensis.

Seven new triterpenoid saponins, schekwangsiensides A-G (1-7), and a new triterpenoid, schekwangsienin (8), together with nine known triterpenoids and...
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