Polyacetylenic Oleanane-Type Triterpene Saponins from the Roots of

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Polyacetylenic Oleanane-Type Triterpene Saponins from the Roots of Panax japonicus Yang Liu,† Jianping Zhao,‡ Yang Chen,† Wei Li,† Bin Li,† Yuqing Jian,† Gulnar Sabir,§ Shaowu Cheng,† Qinhui Tuo,† Ikhlas Khan,‡ and Wei Wang*,† †

TCM and Ethnomedicine Innovation & Development Laboratory, Sino-Luxemburg TCM Research Center, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, 410208, People’s Republic of China ‡ National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, Mississippi 38677, United States § Xinjiang Institute of Chinese Material Medica and Ethnomedicine, Urumqi, 830002, People’s Republic of China S Supporting Information *

ABSTRACT: Three new polyacetylenic oleanane-type triterpenoids, baisanqisaponins A−C (1−3), and one new oleanane-type triterpenoid, chikusetsusaponin-V ethyl ester (4), together with 19 known compounds (5−23), were isolated from the roots of Panax japonicus. The structures were elucidated on the basis of spectroscopic analyses and chemical methods. Compounds 1−3 feature a rare panaxytriol group containing a polyacetylene on the saponin skeleton. Neuroprotective activity was evaluated for compounds 1−17, and angiotensin II-induced vascular smooth muscle cell proliferation inhibition was tested for compounds 5−7 and 10−12.

T

IVa butyl ester (7),12 chikusetsusaponin IVa (8),8 chikusetsusaponin IV methyl ester (9),8 taibaienoside I (10),8 taibaienoside II (11),8 28-desglucosylchikusetsusaponin IVa butyl ester (12),13 β-D-glucopyranosiduronic acid, (3β)-17-carboxy-28norolean-12-en-3-yl 2-O-β-D- glucopyranosyl 6-butyl ester (13),13 chikusetsusaponin V methyl ester (14),14 ginsenoside Ro 6′-O-butyl ester (15),14 (20 R)-ginsenoside Rh1 (16),15 ginsenoside Rh4 (17),16 oleanolic acid 28-O-β-D-glucopyranoside (18),8 pseudoginsenoside RT1 butyl ester (19),12 chikusetsusaponin V (20),14 chikusetsusaponin IV (21),8 daucosterol (22),17 and panaxytriol (23),18 by comparing their spectroscopic data with those reported in the literature. Compound 1 was obtained as a white, amorphous powder, and the molecular formula C59H90O16 was deduced from the HRESIMS at m/z 1089.5923 [M + Cl]− (calcd for 1089.5917) and its 13C NMR data. From the 1H NMR spectrum, seven methyl singlets at δH 0.87, 0.88, 0.92, 0.96, 1.10, 1.25, and 1.28 (each 3H, s), together with one methyl triplet at 0.81 (3H, t, J = 7.0 Hz) were observed. Further, one olefinic proton signal at δH 5.48 (1H, t, J = 3.7 Hz) and two anomeric proton signals at δH 6.32 (1H, d, J = 8.0 Hz) and 4.92 (1H, d, J = 8.0 Hz) were apparent. The 13C NMR spectrum showed signals at δC 176.2 and 169.6 (for two ester carbonyl groups), two olefinic carbons signals at δC 143.9, 138.5, 122.8, and 115.2 (for double bonds), and two anomeric carbons δC 107.4 and 95.5 (for sugar units).

ujia ethnomedicine is an important part of the folk medicine system in the People’s Republic of China. Panax japonicus C.A. Meyer (Araliaceae), which in the Tujia dialect is known as “Baisan Qi” and “Zhujie Shen”, is a perennial herb that grows in Northwestern Hunan, People’s Republic of China.1 This plant has been used to treat weakness and fatigue, bruises, sprains, traumatic hemorrhage, and cardiovascular disease by the Tujia people for thousands of years.2 Previous research has focused on the chemical constituents of the plant when collected in Japan and southwest mainland China. Dammarane-type triterpenoids have been reported to be the major components of the plant.3−7 In view of the clinical applications of this plant by the Tujia people, we have studied the constituents from the ethanol extract of P. japonicus. Herein, are reported the isolation and structure elucidation of triterpene saponins from the root of P. japonicus collected in northwestern Hunan, as well as their neuroprotective activity and inhibitory effects on vascular smooth muscle cell (VSMC) proliferation.

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RESULTS AND DISCUSSION The n-BuOH-soluble fraction from the ethanol extract of P. japonicus was subjected to MCI, normal-phase and reversedphase silica gel, and Sephadex LH-20 column chromatography to afford four new oleanane-type triterpene saponins, baisanqisaponins A−C (1−3) and chikusetsusaponin V ethyl ester (4), together with 19 known compounds (5−23), identified as chikusetsusaponin IVa methyl ester (5),8 chikusetsusaponin IVa ethyl ester (6),11 chikusetsusaponin © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 13, 2016

A

DOI: 10.1021/acs.jnatprod.6b00748 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Compound 2 was obtained as white, amorphous powder. The molecular formula was determined as C64H98O20 by HRESIMS [m/z 1221.6358 [M + Cl]− (calcd for 1221.6340)] and 13C NMR data analysis. The 1D NMR data (Table 1) were similar to those of compound 1, which suggested that 2 is also an oleanane-type triterpene saponin with a panaxytriol moiety. Furthermore, an additional anomeric proton H-1‴ (δH 5.98) signal and the corresponding carbon C-1‴ (δC 108.4) resonance were observed by comparing the 1D NMR data of 2 with those of 1. This implied that 2 possesses an additional αarabinofuranose unit, which was determined by the comparison of the chemical shifts of the carbons concerned (δC 108.4, 82.5, 78.5, 87.4, 62.3) with previously reported data.8−10 The HMBC correlations between H-1‴ (δH 5.98) of arabinose and C-4′ (δC 76.2) revealed that the arabinose moiety is located at C-4′. Furthermore, 2 showed similar NMR signals to chicususoponin IV (21) isolated from the plant except for the presence of a panaxytriol moiety. D-Glucose and L-arabinose were determined by acid hydrolysis. UHPLC-MS analysis also revealed 2 is a natural polyacetylenic saponin (Figure S42, Supporting Information). Thus, the structure of 2, baisanqisaponin B, was established as polyacetylenic chicususoponin IV. Compound 3 (baisanqisaponin C) was isolated as a white, amorphous powder. The molecular formula was established as C59H90O16 from the HRESIMS [m/z 1089.5909 [M + Cl]− (calcd for 1089.5917)] and 13C NMR data. Their same HRESIMS data and closely comparable 1D NMR data (Table 1) suggested that 3 possesses the same sapogenin, sugar units, and panaxytriol group as 1. The main difference found was the linkage site of the sugar residue, which was deduced from the HMBC correlations between H-1′ (δH 4.89) and C-3 (δC 89.0) and between H-1″ (δH 5.33) and C-2′ (δC 82.4), as shown in Figure 1. In addition, the triterpene saponin showed similar NMR data to those of 28-deglucosylchicususoponin V except for the presence of a panaxytriol moiety.23 Thus, the structure of 3 was assigned as polyacetylenic 28-deglucosylchicususoponin V. Compound 4 gave the molecular formula C50H80O19, according to its HRESIMS at m/z 1019.4960 [M + Cl]− (calcd for 1019.4982) and 13C NMR data. The NMR spectroscopic data of 4 were similar to those of 3 (Table 1), except for the signals due to the panaxytriol group at C-6′ in 3 being replaced by those for an ethyl group and additional signals due to a β-glucose unit (JH‑1″ = 7.7; δC 95.5, 73.9, 78.7, 70.9, 79.1, 62.0) in 4.12 D-Glucose was determined by acid hydrolysis. The connectivity sites of the ethyl group and β-Dglucose were confirmed by the HMBC correlations of H-1⁗ (δH 4.24) with C-6′ (δC 169.7) and H-1″ (δH 5.38) with C-28 (δC 176.2), respectively, as shown in Figure 1. Moreover, 4 showed similar NMR signals to chikusetsusaponin V (20), also isolated from the title plant, except for the presence of a panaxytriol moiety. Compound 4 is not an artifact due to being found in the methanol extract through UHPLC-MS analysis (Figure S42, Supporting Information). Therefore, the structure of 4 was established as chikusetsusaponin V ethyl ester. Compounds 1−17 were evaluated for neuroprotective activity to hydrogen peroxide-induced PC 12 cell injury by using the MTT assay.24 Compounds 3, 10, and 13 exhibited moderate protective effects compared with a positive control (Table S2, Supporting Information). Also, compounds 5−7 and 10−12 were evaluated for their inhibitory effects on angiotensin II (Ang II)-induced VSMC proliferation.25 Only

The above spectroscopic data indicated that 1 is an oleananetype triterpenoid saponin with both a glucose (δC 95.5, 73.9, 78.7, 70.9, 79.1, 62.0) and a glucuronic acid (δC 107.4, 75.1, 77.8, 72.5, 77.4, 169.6) substituent.19 The two sugar units were connected to C-3 and C-28, which was determined by the HMBC correlations between H-1‴ (δH 6.32) and C-28 (δC 176.2) and between H-1′ (δH 4.92) and C-3 (δC 88.9), respectively. The β configuration was confirmed by the coupling constants (J = 8.0 Hz), and D-glucose was determined by acid hydrolysis. The assignments of the 1H and 13C NMR signals of the triterpene saponin were based on the 1H−1H COSY, HSQC, and HMBC NMR data (Table 1), which were similar to those of chikusetsusaponin IVa (7) obtained from the same extract in this study. The only difference was that 1 exhibited the presence of an additional rare panaxytriol (23) moiety,20,22 including a methyl group (δC 14.0), seven methylenes (δC 31.9 30.7, 29.7, 29.4, 25.8, 24.9, 22.7), three oxygened methylenes (δC 76.7, 70.6, 62.8), a double bond (δC 138.5, 115.2), and two triple bonds (δC 79.8, 77.3, 70.4, 66.4), based on the 13C NMR and HSQC spectra. The connectivity between chikusetsusaponin IVa and the panaxytriol group was confirmed by the key correlation of H-10⁗ (δH 5.58) with C-6′ (δC 169.6) in the HMBC spectrum (Figure 1). The absolute configuration of the panaxytriol moiety was inferred as 3⁗R, 9⁗R, 10⁗R, owing to the measured optical rotation value of natural isolated panaxytriol (23) from P. japonicus being identical to the reported data.20,21 To date, saponins with a polyacetylene side chain have been reported only rarely.22 UHPLC-MS analysis was used to confirm the presence of 1 in the original plant methanol extract as a natural product (Figure S42, Supporting Information). Therefore, the structure of 1 was elucidated as polyacetylenic chicususaponin IVa and given the trivial name baisanqisaponin A. B

DOI: 10.1021/acs.jnatprod.6b00748 J. Nat. Prod. XXXX, XXX, XXX−XXX

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 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″

position

C

m; 1.08 m m; 1.74 m s s s s s

0.92 s 0.88 s 4.92b 4.02 m 4.24b 4.53b 4.60 d (9.8)

1.35 1.81 1.25 0.96 0.87 1.10 1.27

3.20 dd (14.1, 3.4) 1.77 m; 1.23 m

2.34 m; 1.18 m 2.09 m

1.97 m 5.48 brs

1.66 m

0.76 d (10.9) 1.45 m; 1.28 m 1.46 m; 1.34 m

1.62 m; 0.97 m 2.20 m; 1.89 m 3.26 dd (11.6, 4.3)

δH

1 38.6 CH2 26.6 CH2 88.9 CH 39.3 C 55.5 CH 18.3 CH2 32.9 CH2 39.7 C 47.8 CH 36.8 C 23.61 CH2 122.8 CH 143.9 C 41.9 C 28.0 CH2 23.2 CH2 46.8 C 41.5 CH 46.0 CH2 30.6 C 33.8 CH2 32.3 CH2 27.8 CH3 16.7 CH3 15.4 CH3 17.3 CH3 25.9 CH3 176.2 C 32.9 CH3 23.4 CH3 107.4 CH 75.1 CH 77.8 CH 72.5 CH 77.4 CH 169.6 C

δC

δH

s s d (7.8) m d (8.3) d (7.0) d (7.9)

m; 1.06 m m; 1.75 m s s s s s

5.98 brs 4.79b 4.76b 4.95 d (4.2) 4.24;b 4.13 dd (11.8, 4.4)

0.92 0.89 4.87 4.01 4.26 4.67 4.64

1.36 1.81 1.22 0.95 0.87 1.10 1.27

3.21 dd (13.8, 4.0) 1.78 m; 1.29 m

2.36 m; 1.18 m 2.09 m; 1.97 m

1.96 m 5.50 brs

1.66 m

0.74 d (11.8) 1.45 m; 1.26 m 1.46 m; 1.33 m

1.67 m; 1.01 m 2.18 m; 1.86 m 3.21 dd (11.8, 4.0)

Table 1. NMR Spectroscopic Data for Compounds 1−4a 2 38.6 26.6 89.1 39.3 55.5 18.3 32.9 39.7 47.9 36.8 23.6 122.8 143.8 41.9 28.0 23.2 46.8 41.5 46.0 30.6 33.8 32.3 27.8 16.7 15.4 17.3 25.9 1762 C 32.9 23.5 107.1 74.8 75.6 76.2 75.4 169.3 108.4 82.5 78.5 87.4 62.3

δC

CH3 CH3 CH CH CH CH CH C CH CH CH CH CH2

CH2 CH2 CH2 C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C CH CH2 C CH2 CH2 CH3 CH3 CH3 CH3 CH3 m; 1.21 m m s s s s s

5.33 d (7.5) 4.09 dd (9.1, 7.7) 4.22 d (8.9) 4.30b 3.91 m 4.47 m

0.96 s 1.00 s 4.89 d (7.6) 4.18 dd (9.2, 7.6) 4.27b 4.43 d (9.0) 4.52 d (9.7)

1.45 1.81 1.22 1.09 0.86 1.00 1.30

3.30 m 1.80 m; 1.30 m

2.15 m; 1.19 m 2.12 m, 1.96 m

1.96 m 5.52 t

1.65 m

0.71 d (11.6) 1.47 m; 1.23 m 1.43 m; 1.28 m

1.62 m; 0.96 m 2.17 m; 1.89 m 3.16 dd (11.8, 4.5)

δH

3 38.5 26.6 89.0 39.3 55.5 18.3 33.0 39.5 47.8 36.8 23.6 122.4 144.5 41.9 28.1 23.5 46.4 41.8 46.3 30.7 34.0 32.3 27.8 16.5 15.3 17.2 26.0 179.9 33.1 23.5 105.3 82.4 77.4 72.2 76.8 169.2 105.8 76.7 77.7 71.5 78.1 62.5

CH2 CH2 CH2 C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C CH CH2 C CH2 CH2 CH3 CH3 CH3 CH3 CH3 C CH3 CH3 CH CH CH CH CH C CH CH CH CH CH CH2

δC

4

5.38 d (7.7) 4.10 dd (9.1, 7.7) 4.23 m 4.31b 3.91 m 4.45b

0.90 s 0.87 s 4.94 d (7.2) 4.29b 4.30b 4.42b 4.48 d (9.9)

1.34 m; 1.07 m 1.82 m; 1.74 m 1.24 s 1.07 s 0.82 s 1.08s 1.24 s

3.21 dd (13.8, 4.0) 1.75 m; 1.24 m

2.32 m, 1.15 m 2.08 m; 1.94 m

1.86 m 5.40 brs

1.58 m

0.69 d (11.6) 1.43 m; 1.26 m 1.39 m; 1.30 m

1.39 m; 0.80 m 2.09 m; 1.83 m 3.22 dd (11.7, 4.3)

δH

CH2 CH2 CH2 C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C CH CH2 C CH2 CH2 CH3 CH3 CH3 CH3 CH3 C CH3 CH3 CH CH CH CH CH C CH CH CH CH CH CH2

δC 38.4 26.4 89.1 39.3 55.5 18.3 32.9 39.7 47.8 36.7 23.5 122.6 143.9 41.9 28.0 23.2 46.8 41.5 46.0 30.5 33.8 32.3 27.9 16.5 15.3 17.2 25.9 176.2 32.9 23.4 105.1 82.4 77.4 72.6 76.6 169.7 105.7 76.8 77.8 71.5 78.1 62.5

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DOI: 10.1021/acs.jnatprod.6b00748 J. Nat. Prod. XXXX, XXX, XXX−XXX

D

2.99 dd (17.2, 8.1) 2.94 dd (17.2, 4.9) 4.33 m 5.58 m 2.06 m; 1.97 m 1.21 m 1.24 m 1.59 m; 1.50 m 1.15 m 1.18 m 0.81 t (7.0) 70.6 76.7 30.6 29.7 29.4 25.7 31.9 22.7 14.0

138.5 62.8 77.3 70.4 66.4 79.8 24.9

95.5 73.9 78.7 70.9 79.1 62.0 115.2

CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH3

CH CH C C C C CH2

CH CH CH CH CH CH2 CH2

δC

2

2.99 dd (17.2, 7.8) 2.94 dd (17.2, 5.0) 4.35 m 5.53 m 2.02 m 1.26 m 1.24 m 1.49 m 1.16 m 1.20 m 0.82 t (7.2)

6.32 d (8.1) 4.20 t (8.6) 4.27b 4.34 d (9.2) 4.02 m 4.46 dd (11.9, 2.5); 4.39 dd (11.9, 4.4) 5.63 dt (17.0, 1.6) 5.22 dt (10.1, 1.6) 6.21 ddd (17.0, 10.2, 5.2) 5.31 d (5.4)

δH

70.4 77.3 30.6 29.7 29.3 25.7 31.9 22.7 14.1

138.4 62.8 77.4 70.3 66.5 79.6 25.0

95.5 73.9 78.7 70.9 79.1 62.0 115.2

δC

CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH3

CH CH2 C C C C CH2

CH CH CH CH CH CH2 CH2

3

2.97 dd (17.2, 8.0) 2.91 dd (17.2, 5.0) 4.32 m 5.57 m 1.97 m 1.26 m 1.21 m 1.50 m 1.17 m 1.19 m 0.82 t (6.9)

5.64 dt (17.0, 1.6) 5.22 dt (10.1, 1.6) 6.21 ddd (17.0, 10.2, 5.2) 5.31 m

δH

70.6 76.7 30.7 29.7 29.4 25.8 31.9 22.7 14.0

138.5 62.8 77.4 70.3 66.4 79.8 25.0 CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH3

CH CH2 C C C C CH2

115.2 CH2

δC

1.17 t (7.1)

6.32 d (8.1) 4.19 t (8.6) 4.27b 4.34 d (9.1) 4.02b 4.40 m 4.24b

δH

4

14.0 CH3

CH CH CH CH CH CH2 CH2

δC 95.5 73.9 78.7 70.9 79.0 62.0 61.0

Measured at 500 MHz for 1H and 125 MHz for 13C with reference to pyridine-d5. Assignments were established by 1H−1H COSY, HSQC, and HMBC spectra. Coupling constants (J) in Hz are given in parentheses. bSignal overlapped.

a

9⁗ 10⁗ 11⁗ 12⁗ 13⁗ 14⁗ 15⁗ 16⁗ 17⁗

2⁗ 3⁗ 4⁗ 5⁗ 6⁗ 7⁗ 8⁗

6.32 d (8.1) 4.19b 4.27b 4.34 d (9.2) 4.02 m 4.45 m; 4.40 m 5.63 dt (17.0, 1.5) 5.22 dt (10.1, 1.5) 6.21 ddd (17.0, 10.1, 5.2) 5.31 d (5.3)

1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 1⁗

1

δH

position

Table 1. continued

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DOI: 10.1021/acs.jnatprod.6b00748 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Key HMBC correlations (H−C) of 1−4. MeOH−H2O, to afford five pooled fractions (fractions A−E). Fraction D (40.3 g) was chromatographed on a silica gel column eluted with CH2Cl2−MeOH (100:1−0:1) gradients to give 10 further fractions. Fraction D-a was then separated by normal-phase silica gel column chromatography (CC) eluted with petroleum ether−EtOAc (60:1− 0:1) to offord 23 (18 mg). Fraction D-b was separated by silica gel CC with CHCl3−MeOH−H2O (7:3:0.4) and purified by passage over a C18 column eluted with 50−100% MeOH−H2O to yield 12 (15 mg) and 22 (20 mg). Fraction D-e was separated on silica gel CC with CH2Cl2−MeOH (20:1−1:1) to give five fractions. Fraction D-e-1 was separated by MCI CC with 50−100% MeOH−H2O to give 17 (11 mg) and 7 (25 mg). Fraction D-e-2 was separated by MCI CC eluted with 50−100% MeOH−H2O and purified by C18 CC eluted with 60− 100% MeOH−H2O to yield 1 (10 mg), 5 (67 mg), and 6 (12 mg). Fraction D-e-4 was separated by MCI CC with 50−100% MeOH− H2O to yield 10 (33 mg). Fraction D-f was separated on silica gel CC using CH2Cl2−MeOH (20:1−1:1) to give three fractions. Fr.D-f-2 was separated on silica gel CC with CH2Cl2−MeOH (15:1−1:1) and purified by C18 CC eluted with 60−100% MeOH−H2O to give 11 (29 mg) and 13 (22 mg) and then by semipreparative HPLC with the mobile phase CH3CN−H2O (37:63) to obtain 9 (25 mg, tR 37.9 min). In turn, semipreparative HPLC of this residue with the mobile phase CH3CN−H2O (35:65) produced 3 (6 mg, tR 46.7 min). Fraction D-g was separated by silica gel CC with CH2Cl2−MeOH (10:1−1:1) to give two fractions. Of these, fraction D-g-1 was separated over MCI eluted with 60−100% MeOH−H2O and purified by C18 CC eluted with 60−100% MeOH−H2O to give 4 (8 mg) and 14 (22 mg). Fraction D-h was separated by MCI CC eluted with 60−100% MeOH−H2O and purified by C18 CC eluted with 60−100% MeOH− H2O to give 2 (13 mg). Fraction C (131.1 g) was subjected to passage

compound 5 was found to exhibit a dose-dependent inhibitive effect (Table S3, Supporting Information).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were run on a Rudolph Research Analytical Autopol IV automatic polarimeter. UV data were measured in methanol on a Hewlett-Packard 8452A UV−vis spectrometer. IR spectra were obtained on an Agilent Technologies Cary 630 FTIR spectrometer. 1H, 13C, COSY, HSQC, and HMBC spectra were measured on an Agilent DD2-500 NMR spectrometer with a OneNMR probe at 500 MHz for 1H and 125 MHz for 13C, using the pulse programs provided in the Agilent Vnmrj 4.0 software. Tetramethylsilane was used as internal standard, and all chemical shifts are reported in parts per million (ppm, δ). HRESIMS were recorded on an Agilent Series 1100 SL mass spectrometer. Plant Material. The dried roots of P. japonicus were collected from Shimen, Hunnan Province, People’s Republic of China, in July 2012. The material was identified by one of us (W.W.). A voucher specimen (CEL 12045) has been deposited at TCM and Ethnomedicine Innovation and Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine. Extraction and Isolation. The dried roots of P. japonicus (6.7 kg) were treated with EtOH (95%, 3 × 15 L) by ultrasonic extraction. The filtrate was concentrated under reduced pressure to obtain a crude EtOH extract (2042 g), which was suspended in H2O and successively partitioned with EtOAc and n-BuOH to give an EtOAc-soluble fraction (19.5 g), a n-BuOH-soluble portion (366 g), and a H2O layer. The n-BuOH-soluble fraction was subjected to MCI column chromatography and eluted with 0%, 50%, 70%, 90%, and 100% E

DOI: 10.1021/acs.jnatprod.6b00748 J. Nat. Prod. XXXX, XXX, XXX−XXX

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over a silica gel column eluted with CHCl3−MeOH (2:1−0:1) to give 10 fractions. Compound 8 (17 mg, tR 5.2 min) was isolated from fraction C-a by semipreparative HPLC with the mobile phase CH3CN−H2O (55:45). In turn, compound 18 (6 mg, tR 15.7 min) was isolated from fraction C-a by semipreparative HPLC with the mobile phase CH3CN−H2O (45:55). Fraction C-b was isolated by C18 CC and purified by semipreparative HPLC to yield compound 16 (20 mg, tR 15.0 min). Compound 15 (18 mg, tR 11.3 min) was isolated from fraction C-d by semipreparative HPLC with the mobile phase CH3CN−H2O (44:56). Compound 19 (7 mg, tR 21.4 min) was obtained from fraction C-f by semipreparative HPLC with the mobile phase CH3CN−H2O (39:61). Compounds 20 (58 mg) and 21 (37 mg) were recrystallized from fraction C-g. Baisanqisaponin A (1): white, amorphous powder (MeOH); [α]25 D −12.4 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 201 (2.6) nm; IR (KBr) νmax 3369, 2929, 2252, 1737, 1644 cm−1; 1H NMR (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table 1; HRESIMS m/z 1089.5923 [M + Cl]− (calcd for C59H90O16Cl, 1089.5917). Baisanqisaponin B (2): white, amorphous powder (MeOH); [α]25 D −33.6 (c 0.24, MeOH); UV (MeOH) λmax (log ε) 203 (3.1) nm; IR −1 1 (KBr) νmax 3369, 2929, 2252, 1737, 1644 cm ; H NMR (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table 1; HRESIMS m/z 1221.6358 [M + Cl]− (calcd for C64H98O20Cl, 1221.6340). Baisanqisaponin C (3): white, amorphous powder (MeOH); [α]25 D −6.4 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 202 (3.4) nm; IR −1 1 (KBr) νmax 3369, 2927, 2359, 1735, 1697 cm ; H NMR (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table 1; HRESIMS m/z 1089.5909 [M + Cl]− (calcd for C59H90O16Cl, 1089.5917). Chikusetsusaponin V Ethyl Ester (4): white, amorphous powder (MeOH); [α]25 D +2.7 (c 0.24, MeOH); UV (MeOH) λmax (log ε) 201 (2.8) nm; IR (KBr) νmax 3378, 2939, 1736, 1598 cm−1; 1H NMR (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table 1; HRESIMS m/z 1019.4960 [M + Cl] − (calcd for C50H80O19Cl, 1019.4982). Acid Hydrolysis of Saponins. Compounds 1−4 (1 mg) were hydrolyzed by heating in 1.0 M HCl (1 mL). After drying in vacuo, the residue was dissolved in pyridine (0.1 mL) containing L-cysteine methyl ester hydrochloride (0.5 mg) and heated at 60 °C for 1 h. A 0.1 mL solution of o-tolylisothiocyanate (0.5 mg) in pyridine was added to the mixture, which was heated at 60 °C for 1 h. The reaction mixture was directly analyzed by reversed-phase HPLC.26 The peaks of 1, 3, and 4 at 20.58, 20.66, and 20.64 min coincided with derivatives of standard D-glucose with tR at 20.60 min. The peaks at 22.79 and 20.67 min in 2 coincided with those of derivatives of standard L-arabinose and D-glucose at 22.70 and 20.62 min, respectively. Neuroprotective Activity Bioassays. PC 12 cells were cultivated in DMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin, in a 5% CO2 humidified atmosphere at 37 °C. The cells were inoculated to 96-well plates (100 μL/plate) at a density of 1 × 106 cells/mL and treated with 3.125, 6.25, 12.5, 25, and 50 μM test sample and 12.5 μM edaravone, followed by coculture with 1 mM H2O2. The MTT reagent (5 mg/mL) was added to the cells and then incubated at 37 °C for 4 h. Then 100 μL of DMSO was added to dissolve the formazan crystal, and the absorbance was measured at 490 nm with a microplate reader (TECAN, Infinite F50, CH).24 VSMC Proliferation Inhibitory Bioassay. VSMCs were prepared from thoracic aortas of 2−3-month-old male Wistar rats using the explant method.26 This animal work was approved by Hunan University of Chinese Medicine (SYXK2013-0005). The cells were cultured in DMEM containing 10% fetal bovine serum. Cultured cells were then incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. VSMCs were grown in 96-well plates for 24 h and starved in serum-free medium for 24 h prior to experimental stimulation with Ang II. After incubating cells with compounds (0.1, 0.3, 1, 3, 10, and 30 μM) and curcumin (30 μM) for 48 h, cell viability was assayed by the MTT method.27

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00748. 1 H, 13C, 2D NMR, HRESIMS, and LC-MS spectra for compounds 1−4 and bioassay results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: 86-731-8845-8827. E-mail: wangwei402@hotmail. com (W. Wang). ORCID

Wei Wang: 0000-0003-0876-2205 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Ministry of Education of Overseas Returnees Start-up Fund (2013-693), Hunan Province Administration of TCM Scientific Research Key Project (201215), Changsha Science and Technology Bureau Key Project (K1205019-31), and Specialized Research Fund for the Doctoral Program of High Education (20134323110004).

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