Uralsaponins MâY, Antiviral Triterpenoid Saponins from the Roots of

Article pubs.acs.org/jnp

Uralsaponins M−Y, Antiviral Triterpenoid Saponins from the Roots of Glycyrrhiza uralensis Wei Song,† Longlong Si,† Shuai Ji,† Han Wang,† Xiao-mei Fang,‡ Li-yan Yu,‡ Ren-yong Li,§ Li-na Liang,§ Demin Zhou,† and Min Ye*,† †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China ‡ Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing 100050, People’s Republic of China § ThermoFisher Scientific Ltd., Beijing 100085, People’s Republic of China S Supporting Information *

ABSTRACT: Thirteen new oleanane-type triterpenoid saponins, uralsaponins M−Y (1−13), and 15 known analogues (14−28) were isolated from the roots of Glycyrrhiza uralensis Fisch. The structures of 1−13 were identified on the basis of extensive NMR and MS data analyses. The sugar residues were identified by gas chromatography and ion chromatography coupled with pulsed amperometric detection after hydrolysis. Saponins containing a galacturonic acid (1−3) or xylose (5) residue are reported from Glycyrrhiza species for the first time. Compounds 1, 7, 8, and 24 exhibited good inhibitory activities against the influenza virus A/ WSN/33 (H1N1) in MDCK cells with IC50 values of 48.0, 42.7, 39.6, and 49.1 μM, respectively, versus 45.6 μM of the positive control oseltamivir phosphate. In addition, compounds 24 and 28 showed anti-HIV activities with IC50 values of 29.5 and 41.7 μM, respectively.

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pulsed amperometric detection (IC-PAD) after hydrolysis. Furthermore, antiviral activities of the saponins were evaluated against the influenza virus strain A/WSN/33 (H1N1) and VSV-G pseudo-typed HIV-1 virus.

icorice (Gan-Cao in Chinese) is one of the most popular Chinese herbal medicines. It is derived from the dried roots and rhizomes of Glycyrrhiza uralensis Fisch., G. inf lata Bat., and G. glabra L.1 Among these species, G. uralensis is the major source for licorice in the herb market and is recorded in the pharmacopoeia of China, Japan, Europe, and the U.S.2 Licorice shows significant anti-inflammatory, antiaging, anticarcinogenesis, hepatoprotective, and antiviral activities.3−8 The major bioactive constituents of licorice include flavonoids and triterpenoid saponins.9−12 Thus far, 26 saponins have been isolated from G. uralensis, with glycyrrhizic acid (or glycyrrhizin) being the predominant constituent.13 In China, glycyrrhizic acid has been developed into a popular antihepatitis drug.14,15 As part of our continued study on the chemistry and biological activities of licorice, we report herein the isolation and structural elucidation of triterpenoid saponins from G. uralensis.16−18 A number of saponins with novel saccharide chains containing a galacturonic acid or xylose residue were obtained. The sugar residues were identified by gas chromatography (GC) and ion chromatography coupled with © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The dried roots of G. uralensis Fisch. were extracted with 70% EtOH, and the concentrated extract was suspended in water and partitioned successively with EtOAc and n-BuOH. The nBuOH extract was separated by macroporous resin, MCI, and ODS column chromatography and semipreparative RP-HPLC. A total of 28 triterpenoid saponins were obtained, including 13 new compounds (1−13, uralsaponins M−Y) and 15 known compounds (14−28). The structures of 1−13 were identified by physical data analyses, including 1D and 2D NMR and HRESIMS. The sugar residues were identified by GC and ICPAD analyses after hydrolysis. The known compounds were Received: March 19, 2014

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dx.doi.org/10.1021/np500253m | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

identified as uralsaponin C (14),19 uralsaponin F (15),19 licorice saponins A3 (16),20 G2 (17),21 E2 (18),21 H2 (19),22 B (20),22 B2 (21),20 and J2 (22),22 araboglycyrrhizin (23),23 22β-acetoxyglycyrrhizin (24),21 22β-acetoxyglycyrrhetaldehyde (25), 2 4 3β-O-[β- D -glucuronopyranosyl-(1→2)-β- D glucuronopyranosyl]olean-9,12-dien-30-oic-acid (26),25 glycyrrhizic acid (27), 26 and 3-O-β- D -glucuronopyranosylglycyrrhetinic acid (28).27 Compound 1 was obtained as a white, amorphous powder. Its molecular formula was determined to be C44H64O18 by HRESIMS analysis ([M − H]− m/z 879.3983, calcd for C44H63O18, 879.4009) and 13C NMR spectroscopic data. In the positive ESIMS/MS spectrum, the [M + H]+ ion at m/z 881 could successively lose two units of 176 Da to produce the m/z 705 and 529 ions, indicating that 1 was a saponin containing two residues of glucuronic acid or galacturonic acid. The 1H NMR spectrum exhibited eight methyl singlets at δH 0.90−1.44 and δH 2.03, together with one olefinic proton resonance at δH 6.02 (1H, s). The 13C NMR spectrum showed resonances for one carbonyl (δC 199.6, C-11), two carboxyl or ester groups (δC 179.2, C-30; 170.2, COCH3), two olefinic carbons (δC 129.0, C-12; 168.4, C-13), two oxygenated methines (δC 88.8, C-3; 77.4, C-22), and eight methyls (δC 16.8, 16.8, 18.8, 20.7, 21.9, 24.2, 28.2, 29.5) (Table 1). These data were consistent with the sapogenin of 22β-acetoxyglycyrrhizin (24).21 Thus, the sapogenin of 1 was determined to be 22β-acetoxy-3β-hydroxy11-oxo-olean-12-en-30-oic acid. This deduction was confirmed by enzymatic hydrolysis of 1 and 24 with β-glucuronidase, which yielded the same sapogenin (Figure S1). The NMR spectra for compound 1 also showed characteristic resonances for two sugar residues [δC 105.3 (C-1′), 172.0 (C-6′), δH 5.05 (H-1′); δC 106.7 (C-1″), 171.7 (C-6″), δH 5.27 (H-1″)]. Initially, we assigned the saccharide chain as two units of glucuronic acid (GluA), which is common for Glycyrrhiza

saponins. However, examination of the 1H NMR spectrum showed that H-4″ at δH 4.99 appeared as a broad singlet, which was different from GluA H-4′ (δH 4.57, t, J = 9.5 Hz) due to axial−axial couplings with H-3′ and H-5′. The broad H-4″ singlet indicated equatorial−axial couplings. Thus, 4″-OH should be β-oriented, and the terminal sugar residue was identified as galacturonic acid (GalA). In accordance, the resonances for GalA C-3″ and C-4″ appeared at δC 74.4 and 71.7, respectively, which were different from those of C-3′ (δC 77.4) and C-4′ (δC 73.3) of GluA. To further confirm the sugar residues, compound 1 was hydrolyzed by β-glucuronidase, and the water-soluble fraction was analyzed by IC-PAD. The chromatogram showed two peaks corresponding to GalA and GluA in a ratio of approximately 1:1 (Figure 1), which was consistent with our previous deduction. The GluA residue was linked to 3-OH of the sapogenin, according to the HMBC correlation of H-1′ with C-3 (δC 88.8). The terminal GalA was substituted at 2′-OH of GluA, according to the HMBC correlation of H-1″ with C-2′ (δC 83.4) (Figure 2). The βconfiguration of the GluA and GalA glycosidic bonds was established by the coupling constants of H-1′ (δH 5.05, d, J = 7.6 Hz) and H-1″ (δH 5.27, d, J = 7.6 Hz). On the basis of the above deductions, the structure of compound 1 was identified as 3β-O-[β-D-galacturonopyranosyl-(1→2)-β-D-glucuronopyranosyl]-22β-acetoxy-11-oxo-olean-12-en-30-oic acid and was named uralsaponin M. The molecular formula of compound 2 was determined to be C42H62O17 by HRESIMS analysis ([M − H]− m/z 837.3890, calcd for C42H61O17, 837.3903) and 13C NMR spectroscopic data. It contained the same sugar moiety (GluA-GalA) as 1, according to its NMR spectroscopic data. The 13C NMR spectrum exhibited an oxygen-bearing methylene resonance at δC 63.3, which showed HMBC correlation with CH3-23 (δH 1.42, s). This resonance was assigned to C-24.21 In addition, the B


C

CH3 CH3 CH3 CH3 CH3

16.8, 18.8, 24.2, 21.9, 29.5,

36.1, C 44.6, CH

17 18

25 26 27 28 29

25.7, CH2

16

28.2, CH3 16.8, CH3

45.8, C 62.2, CH 37.3, C 199.6, C 129.0,CH 168.4, C 43.7, C 26.8, CH2

8 9 10 11 12 13 14 15

23 24

32.8, CH2

7

77.4, CH

17.6, CH2

6

22

39.9, C 55.4, CH

4 5

40.7, C 35.2, CH2

88.8, CH2

3

20 21

26.7, CH2

2

40.1, CH2

39.6, CH2

1

19

δC, type

position

16.6, 18.4, 23.4, 28.5, 28.6,

1.22, 1.06, 1.43, 0.90, 1.30,

CH3 CH3 CH3 CH3 CH3

22.7, CH3 63.3, CH2

1.44, s 1.18, s

s s s s s

38.3, CH2

44.0, C 31.5, CH2

41.6, CH2

32.0, C 48.6, CH

26.7, CH2

43.3, C 61.7, CH 36.9, C 199.1, C 128.4, CH 169.6, C 45.3, C 26.5, CH2

33.0, CH2

17.7, CH2

44.0, C 55.5, CH2

1.76, m; 2.82, m 4.84, t (2.0)

3.07, dd (13.6, 3.6) 1.78, m; 2.19, br d (13.6)

1.10, m; 1.74, dd (13.4, 3.6) 1.02, m; 1.94, dd (13.4, 3.6)

6.02, s

2.44, s

0.73, br d (12.6) 1.12, m; 1.26, m 1.22, m; 1.52, m

89.5, CH

26.7, CH2

2.06, m; 2.29, dd (13.6, 3.6) 3.36, dd (11.6, 3.6)

δC, type

39.1, CH2

δH (J in Hz)

1.03, m; 3.03, m

1

1.42, s 3.40, d (11.6); 4.43, m 1.06, s 0.98, s 1.41, s 0.75, s 1.33, s

1.44, m; 2.26, m 1.43, m; 1.70, m

2.50, dd (13.6, 3.6) 1.71, m; 2.08, m

0.92, m; 2.06, m

1.06, m; 1.67, m

5.92, s

2.40, s

0.81, br d (12.0) 1.28, m; 1.56, m 1.23, m; 1.50, m

3.49, dd (11.6, 4.0)

0.95, m; 2.94, dd (13.2, 3.0) 2.06, m; 2.24, m

δH (J in Hz)

2

16.7, 18.7, 22.4, 23.9, 20.3,

CH3 CH3 CH3 CH3 CH3

28.2, CH3 16.7, CH3

83.9, CH

42.1, C 38.1, CH2

40.8, CH2

35.8, C 44.4, CH

25.9, CH2

44.9, C 62.0, CH 37.3, C 199.0, C 130.1, CH 164.4, C 45.1, C 25.3, CH2

33.1, CH2

17.6, CH2

39.5, C 55.4, CH

89.7, CH

26.7, CH2

39.8, CH2

δC, type

1.17, 0.97, 1.33, 0.90, 1.18,

s s s s s

1.42, s 1.20, s

1.95, m; 2.25, m 4.17, d (6.0)

1.45, m; 1.73, m

2.26, m

1.07, m; 1.82, m

1.06, m; 1.60, m

5.67, s

2.40, s

0.70, br d (12.6) 1.24, m; 1.48, m 1.20, m; 1.47, m

3.35, dd (11.6, 4.0)

1.05, m; 3.00, dd (13.2, 2.8) 1.95, m; 2.25, m

δH (J in Hz)

3

16.7, 18.8, 23.0, 29.0, 20.8,

CH3 CH3 CH3 CH3 CH3

28.0, CH3 16.8, CH3

43.0, CH2

48.1, C 71.9, CH

35.0, CH2

32.9, C 47.0, CH

26.8, CH2

45.3, C 61.9, CH 37.1, C 199.2, C 128.5, CH 169.6, C 43.8, C 26.6, CH2

32.9, CH2

17.5, CH2

39.9, C 55.2, CH

88.8, CH

26.6, CH2

39.0, CH2

δC, type

1.27, 1.12, 1.49, 0.92, 1.45,

s s s s s

1.81, br d (12.6); 1.85, br d (12.6) 1.32, s 1.15, s

4.50, br s

1.74, m; 3.16, m

2.36, m

0.91, m; 1.70, m

1.06, m; 1.70, m

5.91, s

2.45, s

1.25, m; 1.43, m

1.12, m; 1.26, m

0.70, br d (12.6)

3.36, dd (11.6, 4.0)

2.08, m; 2.53, m

1.09, m; 3.09, dd (13.2, 2.8)

δH (J in Hz)

4

16.7, 18.8, 23.0, 29.1, 20.9,

CH3 CH3 CH3 CH3 CH3

28.1, CH3 17.0, CH3

43.1, CH2

48.2, C 72.0, CH

35.0, CH2

33.0, C 47.1, CH

26.9, CH2

45.4, C 62.0, CH 37.3, C 199.6, C 128.8, CH 169.9, C 43.9, C 26.9, CH2

32.9, CH2

17.6, CH2

39.8, C 55.3, CH

88.9, CH

26.7, CH2

39.0, CH2

δC, type

1.24, 1.09, 1.48, 0.90, 1.43,

s s s s s

1.31, s 1.18, s

1.77, m; 1.85, m

4.50, br s

1.72, m; 3.17, m

2.36, dd (13.6, 3.2)

1.07, m; 1.70, m

1.04, m; 1.68, m

5.88, s

2.44, s

1.22, m; 1.49, m

1.30, m; 1.47, m

0.72, br d (12.6)

2.11, dd (13.2, 3.0); 2.32, dd (13.2, 3.0) 3.39, dd (11.6, 3.0)

1.08, m; 3.07, dd (13.2, 3.0)

δH (J in Hz)

5

Table 1. NMR Spectroscopic Data for Compounds 1−7 (400 MHz for 1−4 and 600 MHz for 5−7, Pyridine-d5)

16.7, 18.8, 22.9, 29.1, 20.9,

CH3 CH3 CH3 CH3 CH3

28.4, CH3 16.8, CH3

43.0, CH2

48.1, C 72.0, CH

34.9, CH2

32.9, C 47.0, CH

26.5, CH2

45.4, C 62.0, CH 37.3, C 199.6, C 128.8, CH 169.9, C 43.9, C 26.9, CH2

33.0, CH2

17.6, CH2

40.0, C 55.4, CH

89.6, CH

26.5, CH2

39.4, CH2

δC, type

1.22, 1.08, 1.44, 0.89, 1.43,

s s s s s

1.38, s 1.18, s

1.74, m; 1.83, dd (15.0, 2.4)

4.48, br s

1.70, m; 3.13, m

2.34, dd (13.6, 3.6)

1.06, m; 1.70, m

1.04, m; 1.68, m

5.86, s

2.45, s

1.24, m; 1.51, m

1.31, m; 1.49, m

0.73, br d (12.6)

2.03, dd (13.2, 2.8); 2.25, dd (13.2, 2.8) 3.33, dd (11.6, 2.8)

1.11, m; 3.05, dd (13.2, 2.8)

δH (J in Hz)

6

16.7, 18.8, 23.0, 29.1, 20.9,

CH3 CH3 CH3 CH3 CH3

28.3, CH3 16.8, CH3

43.0, CH2

48.2, C 72.0, CH

34.9, CH2

32.9, C 47.1, CH

26.5, CH2

45.4, C 62.0, CH 37.3, C 199.6, C 128.8, CH 169.9, C 43.9, C 26.9, CH2

33.0, CH2

17.6, CH2

40.0, C 55.4, CH

89.6, CH

26.5, CH2

39.4, CH2

δC, type

1.22, 1.09, 1.46, 0.90, 1.43,

s s s s s

1.76, dd (15.0, 2.4); 1.85, dd (15.0, 2.4) 1.35, s 1.13, s

4.48, br s

1.71, m; 3.14, m

2.35, dd (13.6, 3.2)

0.90, m; 1.85, m

1.05, m; 1.68, m

5.87, s

2.46, s

1.26, m; 1.54, m

1.31, m; 1.51, m

0.75, br d (12.6)

3.33, dd (11.6, 2.8)

2.00, m; 2.24, dd (13.2, 2.8)

1.10, m; 3.05, dd (13.2, 2.8)

δH (J in Hz)

7

Journal of Natural Products Article


179.2, C 105.3, CH 83.4, CH 77.4, CH 73.3, CH 77.5, CH 172.0, C 106.7, CH 74.5, CH 74.4, CH

71.7, CH 76.8, CH

171.7, C

30 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″

4″ 5″

6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ COCH3 COCH3

170.2, C 20.7, CH3

δC, type

position

Table 1. continued

2.03, s

5.27, d (7.6) 4.64, m 4.33, dd (10.4, 2.8) 4.99, br s 4.79, br s

d (7.6) t (7.6) m t (9.5) m

δH (J in Hz)

5.05, 4.38, 4.59, 4.57, 4.61,

1

171.4, C

71.9, CH 76.3, CH


δC, type d (7.6) t (7.6) m t (9.5) m

5.67, d (7.4) 4.59, m 4.24, dd (10.0, 2.8) 4.89, br s 4.71, br s

5.01, 4.44, 4.38, 4.55, 4.61,

δH (J in Hz)

2

171.5, C

71.6, CH 76.8, CH


δC, type d (7.6) t (7.6) m m m

5.27, d (7.6) 4.64, m 4.33, dd (10.0, 2.8) 4.99, br s 4.79, br s

5.05, 4.38, 4.59, 4.57, 4.61,

δH (J in Hz)

3

61.2, CH2

69.5, CH 76.9, CH


δC, type d (7.6) t (7.6) m m d (12.6)

4.44, m; 4.63, m

5.25, d (7.6) 4.60, m 4.20, dd (9.6, 2.4) 4.73, br s 4.08, m

5.02, 4.31, 4.61, 4.45, 4.38,

δH (J in Hz)

4

102.3, CH 72.4, CH 72.5, CH 73.9, CH 69.3, CH 18.8, CH3

66.5, CH 62.7, CH2


δC, type d (7.6) t (7.6) t (7.6) dd (7.6, 3.0) m

5.77, 4.60, 4.56, 4.33, 4.62, 1.76,

br s m dd (9.0, 3.0) t (9.0) m d (6.6)

4.43, t (9.0) 3.89, dd (9.0, 3.6); 4.64, m

5.94, d (7.6) 4.80, m 4.49, m

5.06, 4.20, 4.44, 4.56, 4.62,

δH (J in Hz)

5

62.0, CH2 102.0, CH 72.4, CH 72.7, CH 74.3, CH 69.4, CH 18.8, CH3

70.5, CH 76.6, CH


δC, type d (7.6) m m br s m

4.40, 6.30, 4.78, 4.73, 4.32, 5.05, 1.76,

m; 4.45, m br s d (2.8) dd (9.0, 2.8) t (9.0) m d (6.6)

4.46, m 3.90, t (6.6)

5.70, d (7.6) 4.64, m 4.16, dd (9.6, 2.4)

5.05, 4.50, 4.62, 4.48, 4.45,

δH (J in Hz)

6

63.3, CH2 102.0, CH 72.4, CH 72.7, CH 74.3, CH 69.5, CH 19.0, CH3

72.6, CH 77.4, CH


δC, type

d (7.6) m m dd (8.0, 2.4) m

4.32, 6.42, 4.77, 4.71, 4.35, 5.05, 1.79,

m; 4.51, m br s d (2.8) dd (9.2, 2.8) t (9.0) m d (6.6)

4.08, t (9.0) 3.85, m

5.87, d (7.6) 4.31, m 4.24, t (9.0)

5.02, 4.53, 4.61, 4.42, 4.60,

δH (J in Hz)

7


D



Article

Figure 1. IC-PAD analysis for sugar residues of licorice saponins.

Figure 2. Key HMBC correlations (H → C) for compounds 1 and 8.

−COOH resonance at δC 179.7 (C-30), and two olefinic resonances at δC 128.5 (C-12) and 169.6 (C-13), indicating the presence of an 11-oxo-olean-12-en-30-oic acid skeleton. An oxygen-bearing methine resonance appeared at δC 71.9, the corresponding proton (δH 4.50, br s) of which showed HMBC correlations with C-17 (δC 32.9) and C-19 (δC 35.0). Thus, a hydroxy group was present at C-21. H-21 appeared as a broad singlet due to its equatorial orientation, indicating the αorientation of 21-OH. In accordance, H-21 showed NOE enhancement with 29-CH3 (δH 1.45) (Figure 3). The sapogenin of 4 was thus identified as 3β,21α-dihydroxy-11-

acetoxy resonance for compound 1 was not observed in compound 2. Thus, the structure of 2 was identified as 3β-O[β-D-galacturonopyranosyl-(1→2)-β-D-glucuronopyranosyl]24-hydroxy-11-oxo-olean-12-en-30-oic acid and was named uralsaponin N. The molecular formula of compound 3 was determined to be C42H60O16 according to the HRESIMS spectrum ([M − H]− m/z 819.3772, calcd for C42H59O16, 819.3797) and 13C NMR spectroscopic data. The NMR spectra indicated that compound 3 contains the same sugar moiety as compounds 1 and 2. The IR spectrum of 3 exhibited an ester carbonyl absorption at 1753 cm−1, different from the 1725 cm−1 of compound 1. This information indicated the existence of a 22,30-lactone moiety. It was confirmed by the HMBC correlation of H-22 (δH 4.17) with C-30 (δC 179.6). These data were consistent with the known triterpenoid sapogenin glabrolide (3β,22β-dihydroxy11-oxo-olean-12-en-30-oic acid-22(30)-lactone).21 On the basis of the above deductions, the structure of compound 3 was established as 3β-O-[β-D-galacturonopyranosyl-(1→2)-β-Dglucuronopyranosyl]glabrolide and was named uralsaponin O. Compound 4 was obtained as a white, amorphous powder. Its molecular formula, C42H64O16, was established by the HRESIMS spectrum ([M − H]− m/z 823.4106, calcd 823.4111) and 13C NMR spectroscopic data. The 13C NMR spectrum showed a carbonyl resonance at δC 199.2 (C-11), a

Figure 3. Key NOE enhancements for compounds 4, 9, and 11. E



Article

Hz), 5.43 (1H, d, J = 7.6 Hz), and 5.35 (1H, s), suggesting that 8 was a triterpenoid saponin containing three sugar residues. The 13C NMR spectrum displayed a carbonyl resonance at δC 199.6 (C-11) and two olefinic resonances at δC 128.6 (C-12) and 168.9 (C-13), suggesting the presence of an 11-oxo-olean12-ene substructure. Unlike other Glycyrrhiza saponins, compound 8 contained eight instead of seven methyl groups and no carboxyl group. Both C-29 (δC 32.8) and C-30 (δC 27.4) were methyl groups, which was confirmed by the HMBC correlations of CH3-29 (δH 0.80, 3H, s) and CH3-30 (δH 0.92, 3H, s) with C-20 (δC 30.5) and C-21 (δC 35.4). Two oxygenated methine resonances were assigned to C-3 and C-22, according to the HMBC correlations from H-24 (δH 1.24) and H-1′ (δH 5.05) to C-3 (δC 89.2), from H-22 (δH 3.57) to C-20 (δC 30.5), and from H-28 (δH 0.91) to C-22 (δC 78.8) (Figure 2). The β-orientation of 22-OH was supported by the small coupling constant of H-22 (δH 3.57, br s).21 Ion chromatography analysis indicated that compound 8 contained two GluA units and one L-rhamonsyl unit (Rha) (Figure 1). Its 3-OH group was substituted with β-D-GluA-(1→2)-β-D-GluA, the same as glycyrrhizic acid (27).26 The Rha residue showed characteristic methyl resonances at δC 18.6 (C-6‴) and δH 1.70 (3H, d, J = 6.0 Hz). It was linked to 22-OH according to the HMBC correlation of H-1‴ (δH 5.35) with C-22 (δC 78.8). The glycosidic substitutions were confirmed by the NOE enhancements of GluA H-1′ (δH 5.05)/H-3 (δH 3.32), GluA H-1″ (δH 5.43)/GluA H-2′ (δH 4.38), and Rha H-1‴/H-22. The α-Rha glycosidic bond was established by the chemical shift of C-5 (δC 70.5). 28 Therefore, compound 8 was identified as a bisdesmosidic saponin, 3β-O-[β-D-glucuronopyranosyl-(1→2)β-D-glucuronopyranosyl]-22β-O-(α-L-rhamnopyranosyl)-11oxo-olean-12-ene, and was named uralsaponin T. Compound 9 was isolated as a white, amorphous powder. Its molecular formula was determined to be C42H62O17 by HRESIMS (m/z 837.3906 [M − H]−) and 13C NMR data. The NMR spectroscopic data of 9 were similar to those of licorice saponin G2 (17), except for the chemical shifts of C-29 and C-30.21 C-29 appeared at δC 28.8 in 17 and at δC 19.6 in 9. This resonance was consistent with triterpenoid saponins with a 29-carboxylic group.18 Hence, H-30 (δH 1.04) showed an NOE enhancement with H-18 (δH 2.22) (Figure 3). Therefore, the structure of compound 9 was identified as 3β-O-[β-Dglucuronopyranosyl-(1→2)-β-D-glucuronopyranosyl]-24-hydroxy-11-oxo-olean-12-en-29-oic acid and was named uralsaponin U. Compound 10 is an isomer of licorice saponin C2 with the molecular formula C42H64O16, according to its HRESIMS spectrum ([M − H]− m/z 823.4106, calcd 823.4111) and 13C NMR spectroscopic data. The UV spectrum showed characteristic absorption maxima at 241, 249, and 258 nm, indicating the presence of an 11,13-diene substructure.20 It was corroborated by the NMR resonances at δC 125.8 (C-11), 126.7 (C-12), 135.4 (C-13), 135.9 (C-18), and δH 5.57 (1H, d, J = 10.8 Hz) and 6.49 (1H, dd, J = 10.8, 2.8 Hz). Similar to compound 9, C30 resonated at δC 20.4, suggesting C-29 was a carboxylic group (δC 180.9). The saccharide moiety was determined to be GluA(2→1)GluA via the NMR data. Thus, the structure of 10 was established as 3β-O-[β-D-glucuronopyranosyl-(1→2)-β-Dglucuronopyranosyl]olean-11,13(18)-dien-29-oic acid and was named uralsaponin V. Compound 11 has the molecular formula C42H62O15, deduced from its HRESIMS spectrum ([M − H]− m/z 805.4008, calcd 805.4005) and 13C NMR spectroscopic data.

oxo-olean-12-en-30-oic acid (21α-hydroxyglycyrrhetinic acid), which is a new sapogenin among the Glycyrrhiza saponins. The NMR spectra indicated that compound 4 contained two sugar residues, which showed anomeric resonances at δH 5.02 (H-1′) and 5.25 (H-1″), and δC 105.2 (C-1′) and 107.1 (C-1″). These sugar residues were identified as glucuronic acid (GluA) and galactose (Gal) by ion chromatography after acid hydrolysis (Figure 1). GluA was linked to 3-OH of the sapogenin, according to the HMBC correlation of H-1′ with C-3 (δC 88.8). Gal was substituted at 2′-OH of GluA, according to the HMBC correlation of H-1″ with C-2′ (δC 83.4). For both GluA and Gal, the anomeric proton showed a coupling constant of 7.6 Hz, indicating a β-glycosidic bond. Therefore, compound 4 was identified as 3β-O-[β-D-galactopyranosyl-(1→2)-β-D-glucuronopyranosyl]-21α-hydroxy-11-oxo-olean-12-en-30-oic acid and was named uralsaponin P. The sapogenins of compounds 5, 6, and 7 were the same as compound 4, according to their NMR spectra. The molecular formula of compound 5 was determined to be C47H72O19 according to the HRESIMS spectrum (m/z 939.4595, calcd 939.4584) and 13C NMR spectroscopic data. Ion chromatography of the acid hydrolysis product suggested that compound 5 contained D-xylose (Xyl), L-rhamnose (Rha), and Dglucuronic acid (GluA) in a 1:1:1 ratio, by comparing with reference sugar standards (Figure 1). GluA was linked to 3-OH of the sapogenin, according to the HMBC correlation of H-1′ (δH 5.06) with C-3 (δC 88.9). Xyl was connected to 2′-OH of GluA according to the HMBC correlation of H-1″ (δH 5.94) with C-2′ (δC 79.1). Rha was substituted at 2″-OH of Xyl according to the HMBC correlation of H-1‴ (δH 5.77) with C2″ (δC 74.9). The β-glycosidic bonds for GluA and Xyl were confirmed by coupling constants (both 7.6 Hz) of the anomeric protons. The α-configuration of the L-rhamnosyl moiety was indicated by the chemical shift of C-5 at δC 69.3.28 Thus, compound 5 was identified as 3β-O-[α-L-rhamnopyranosyl(1→2)-β- D-xylopyranosyl-(1→2)-β-D-glucuronopyranosyl]21α-hydroxy-11-oxo-olean-12-en-30-oic acid and was named uralsaponin Q. Compound 6 has the molecular formula C48H74O20, determined by its HRESIMS spectrum ([M − H]− m/z 969.4699, calcd 969.4690) and 13C NMR spectroscopic data. The saccharide moiety of 6 was composed of D-GalA, D-Glu, and L-Rha, according to ion chromatography analysis (Figure 1). Similar to compound 5, linkage sites of the sugar residues of 6 were deduced from 2D NMR spectra. Thus, the structure of 6 was identified as 3β-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-β-D-galacturonopyranosyl]-21α-hydroxy11-oxo-olean-12-en-30-oic acid and was named uralsaponin R. Compound 7 was an isomer of 6, according to its HRESIMS ([M − H]−, m/z 969.4695) and 13C NMR data. Ion chromatography analysis indicated that the saccharide moiety of compound 7 was composed of D-GluA, D-Glu, and L-Rha (Figure 1). The structure of 7 was thus identified as 3β-O-[α-Lrhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]-21α-hydroxy-11-oxo-olean-12-en-30-oic acid and was named uralsaponin S. Compound 8 was obtained as a white, amorphous powder. Its molecular formula was determined to be C48H74O19 by HRESIMS analysis ([M − H]− m/z 953.4721, calcd for C48H73O19, 953.4741) and 13C NMR spectroscopic data. The 1 H NMR spectrum showed eight methyl resonances at δH 0.80, 0.91, 0.92, 1.07, 1.21, 1.24, 1.34, and 1.40 (each 3H, s) and three anomeric proton resonances at δH 5.05 (1H, d, J = 7.6 F


G

128.6, CH 168.9, C 43.7, C 26.5, CH2

26.8, CH2

37.3, C 45.3, CH 44.7, CH2

12 13 14 15

16

17 18 19

16.9, CH3 18.8, CH3 23.3, CH3 22.0, CH3 32.8, CH3 27.4, CH3 105.2, CH 84.5, CH

45.6, C 62.0, CH 37.2, C 199.5, C

8 9 10 11

25 26 27 28 29 30 1′ 2′

33.0, CH2

7

28.1, CH3 16.9, CH3

17.7, CH2

6

23 24

40.0, C 55.5, CH

4 5

78.8, CH

89.2, CH

3

22

26.8, CH2

2

30.5, C 35.4, CH2

39.6, CH2

1

20 21

δC, type

position

1.21, 1.07, 1.34, 0.91, 0.80, 0.92, 5.05, 4.38,

s s s s s s d (7.6) t (7.6)

1.40, s 1.24, s

3.57, br s

1.28, m; 1.55, m

2.26, dd (13.6, 3.4) 0.87, m; 1.71, m

1.03, m; 1.94, m

1.01, m; 1.64, m

5.69, s

2.41, s

1.22, m; 1.52, m

1.25, m; 1.47, m

0.71, br d (12.6)

2.06, m; 2.28, dd (13.6, 3.6) 3.32, dd (11.8, 3.6)

1.03, m; 3.03, m

δH (J in Hz)

8

2.40, s

1.23, m; 1.50, m

1.53, m; 1.68, m

0.81, br d (12.4)

3.47, dd (11.6, 4.0)

1.00, m; 3.00, dd (13.2, 3.0) 2.16, m; 2.28, m

δH (J in Hz)

9

1.46, s 3.71, d (11.6); 4.41, m 1.20, s 1.03, s 1.36, s 0.83, s

1.34, m; 1.50, m

1.71, m; 2.17, m

2.22, dd (13.6, 3.6) 1.59, m; 2.46, m

1.01, m; 2.10, m

16.5, CH3 18.4, CH3 23.4, CH3 28.4, CH3 180.8, C 19.6, CH3 1.04, s 104.6, CH 5.02, d (7.6) 81.3, CH 4.35, t (7.6)

22.8, CH3 63.3, CH2

35.7, CH2

42.4, C 29.6, CH2

32.4, C 46.5, CH 39.7, CH2

26.4, CH2

128.8, CH 5.77, s 169.1, C 45.4, C 26.5, CH2 1.01, m; 1.69, m

43.4, C 61.8, CH 36.9, C 199.1, C

33.0, CH2

18.4, CH2

44.3, C 55.8, CH2

89.5, CH

26.6, CH2

39.3, CH2

δC, type

0.73, br d (12.6) 0.80, m; 1.51, m 1.20, m; 1.41, m

0.82, m; 1.62, m 1.92, m; 2.27, m 3.31, dd (11.6, 4.0)

δH (J in Hz)

10

18.2, CH3 16.8, CH3 20.4, CH3 25.3, CH3 180.9, C 20.4, CH3 105.2, CH 84.5, CH

s s s s 1.07, s 5.07, d (7.6) 4.28, t (7.6)

0.78, 0.68, 1.07, 1.11,

40.6, C 54.5, CH 1.87, s 36.5, C 125.8, CH 6.49, dd (10.8, 2.8) 126.7, CH 5.57, d (10.8) 135.4, C 42.8, C 24.7, CH2 0.96, m; 1.60, m 36.2, CH2 1.37, m; 1.61, m 35.0, C 135.9, C 38.1, CH2 0.81, m; 1.48, m 44.3, C 35.2, CH2 1.76, m; 2.82, m 38.1, CH2 1.47, m 1.63, m 27.8, CH3 1.38, s 16.3, CH3 1.17, s

32.6, CH2

18.5, CH2

39.7, C 55.3, CH

89.4, CH

26.6, CH2

38.2, CH2

δC, type

Table 2. NMR Spectroscopic Data for Compounds 8−13 (400 MHz, Pyridine-d5)

16.7, CH3 17.6, CH3 23.6, CH3 28.1, CH3 23.7, CH3 206.1, CH 105.1, CH 84.4, CH

28.0, CH3 16.8, CH3

37.3, CH2

46.9, C 28.5, CH2

31.9, C 47.9, CH 38.5, CH2

26.8, CH2

128.7, CH 168.5, C 43.4, C 26.6, CH2

45.5, C 61.9, CH 37.2, C 199.5, C

32.8, CH2

17.5, CH2

39.9, C 55.4, CH

89.2, CH

26.7, CH2

39.5, CH2

δC, type

1.27, 0.98, 1.36, 0.69, 0.87, 9.48, 5.04, 4.29,

s s s s s s d (7.6) m

1.26, br d (12.6); 1.29, br d (12.6) 1.39, s 1.22, s

1.37, m; 1.28, m

2.06, m 1.01, m; 1.67, m

0.94, m; 1.80, m

1.03, m; 1.64, m

5.78, s

2.39, s

1.17, m; 1.50, m

1.29, m; 1.46, m

0.70, br d (12.6)

3.32, dd (12.0, 4.0)

1.00, m; 3.01, dd (13.6, 2.6) 2.05, m; 2.30, m

δH (J in Hz)

11

2.48, s

1.24, m; 1.62, m

1.34, m; 1.56, m

0.80, br d (12.6)

3.34, dd (12.0, 4.0)

1.08, m; 3.03, dd (13.0, 3.0) 1.97, m; 2.21, m

δH (J in Hz)

12

16.6, CH3 18.7, CH3 23.9, CH3 21.7, CH3 29.4, CH3 179.0, C 102.7, CH 78.9, CH

28.4, CH3 16.8, CH3

77.3, CH

39.9, C 35.0, CH2

35.9, C 44.5, CH 40.0, CH2

25.6, CH2

s s s s s 5.06, d (7.6) 4.52, t (7.6)

1.16, 1.05, 1.43, 0.90, 1.31,

1.45, s 1.25, s

1.78, br d (15.0); 2.70, br d (15.0) 4.84, t (2.4)

3.05, dd (13.6, 3.2) 1.75, m; 2.18, m

1.03, m; 1.93, m

128.8, CH 6.00, s 168.2, C 43.5, C 26.5, CH2 1.08, m; 1.71, m

45.7, C 62.1, CH 37.3, C 199.5, C

32.7, CH2

17.6, CH2

39.9, C 55.3, CH

89.8, CH

26.5, CH2

39.4, CH2

δC, type

16.6, CH3 18.7, CH3 22.2, CH3 23.9, CH3 20.3, CH3 179.7, C 105.0, CH 79.2, CH

28.4, CH3 16.7, CH3

84.1, CH

42.1, C 38.1, CH2

35.7, C 44.4, CH 40.8, CH2

25.9, CH2

130.0, CH 164.3, C 45.0, C 25.2, CH2

44.9, C 62.0, CH 37.3, C 199.1, C

33.1, CH2

17.6, CH2

39.4, C 55.4, CH

89.8, CH

26.5, CH2

40.0, CH2

δC, type

s s s s s 5.04, d (7.6) 4.49, m

1.13, 0.95, 1.33, 0.89, 1.17,

1.43, s 1.22, s

4.18, d (6.0)

1.95, m; 2.25, m

2.25, m 1.45, m; 1.71, m

1.07, m; 1.83, m

1.10, m; 1.64, m

5.65, s

2.42, s

1.26, m; 1.57, m

1.32, m; 1.54, m

0.77, br d (12.6)

3.32, dd (11.6, 3.6)

1.07, m; 2.95, dd (13.4, 2.8) 1.99, m; 2.23, m

δH (J in Hz)

13



δC, type

78.5, CH 73.3, CH 77.6, CH 172.5, C 106.9, CH 76.9, CH 77.8, CH 73.1, CH 77.5, CH 172.2, C 98.2, CH 72.6, CH 73.0, CH 73.9, CH 70.5, CH 18.6, CH3

position

3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ COCH3 COCH3

Table 2. continued

d (7.6) m m m m

s m m t (9.0) m d (6.0)

5.43, 4.62, 4.32, 4.62, 4.40,

5.35, 4.47, 4.57, 4.32, 4.25, 1.70,

4.59, m 4.57, t (9.5) 4.61, m

δH (J in Hz)

8 77.8, CH 73.1, CH 77.6, CH 172.2, C 105.2, CH 72.7, CH 77.6, CH 73.1, CH 76.7, CH 172.2, C

δC, type

5.70, 4.35, 4.30, 4.59, 4.58,

d (7.6) m m m m

4.38, m 4.55, t (9.5) 4.61, m

δH (J in Hz)


δC, type

5.41, 4.25, 4.31, 4.62, 4.59,

d (7.6) m m m m

4.59, m 4.57, m 4.63, m

δH (J in Hz)


δC, type

5.43, 4.26, 4.32, 4.63, 4.61,

d (7.6) m m m m

4.40, m 4.46, m 4.63, m

δH (J in Hz)

11 77.4, CH 73.5, CH 77.6, CH 172.1, C 105.0, CH 78.1, CH 78.7, CH 73.4, CH 78.9, CH 172.5, C 102.1, CH 72.4, CH 72.6, CH 74.3, CH 69.6, CH 19.0, CH3 170.2, C 20.7, CH3

δC, type

br s m dd (9.0, 3.0) t (9.0) m d (6.0)

d (7.6) m m m m

2.03, s

6.46, 4.79, 4.72, 4.37, 5.07, 1.82,

5.94, 4.46, 4.49, 4.47, 4.64,

4.60, m 4.46, m 4.62, m

δH (J in Hz)

12 77.3, CH 73.6, CH 77.5, CH 172.6, C 102.6, CH 78.1, CH 78.7, CH 73.5, CH 78.8, CH 172.2, C 102.1, CH 72.3, CH 72.6, CH 74.3, CH 69.6, CH 18.9, CH3

δC, type

6.42, 4.76, 4.71, 4.35, 5.05, 1.80,

5.92, 4.42, 4.46, 4.55, 4.44,

br s m dd (9.0, 3.0) t (9.0) m d (6.0)

d (7.6) m m m m

4.58, m 4.60, m 4.63, m

δH (J in Hz)

13


H



Article

Table 3. Antiviral Activities of Selected Licorice Saponins against H1N1 and HIV Virusesa anti-H1N1 (IC50 in μM) anti-HIV (IC50 in μM) a

1

7

8

24

48.0

42.7

39.6

49.1

Osv-P 45.6

18

21

23

24

25

28

GA

efavirenz

87.1

83.2

85.1

29.5

69.2

41.7

28.8

0.0015

Note: Osv-P and efavirenz are positive controls. GA, glycyrrhetinic acid; Osv-P, oseltamivir phosphate (Tamiflu).

Its NMR data were similar to those of glycyrrhizic acid (27).26 The only difference was that the C-30 carboxylic resonance disappeared, while a new carbonyl resonance emerged at δC 206.1. This resonance was correlated with a singlet at δH 9.48 in the HSQC spectrum, indicating C-30 as an formyl group in compound 11. This was confirmed by the HMBC correlation of CH3-29 (δH 0.87, 3H, s) with C-30 (δC 206.1). The βorientation of 30-CHO was supported by the NOE enhancement of H-29 (δH 0.87) with H-21 (δH 1.37) (Figure 3). The sapogenin should be glycyrrhetaldehyde.29,30 Therefore, compound 11 was identified as 3β-O-[β-D-glucuronopyranosyl-(1→ 2)-β-D-glucuronopyranosyl]glycyrrhetaldehyde and was named uralsaponin W. Compound 12 was obtained as a white, amorphous powder. Its molecular formula was determined as C50H74O22 according to HRESIMS analysis ([M − H]− m/z 1025.4600, calcd 1025.4588) and 13C NMR spectroscopic data. In the positive ESIMS/MS spectrum, the [M + H]+ ion at m/z 1027 produced a major fragment ion at m/z 881 by neutral loss of 146 Da, suggesting a terminal rhamnose residue of the saccharide moiety. The m/z 881 ion could further lose 352 Da, indicating the presence of two GluA residues. Comparing the NMR spectroscopic data with those of 22β-acetoxyglycyrrhizin (24) showed that compound 12 contained a 22β-acetoxy-3βhydroxy-11-oxo-olean-12-en-30-oic acid moiety substituted with a GluA-(2→1)-GluA sugar chain. The characteristic methyl resonances at δC 19.0 and δH 1.82 (3H, d, J = 6.0 Hz) confirmed the presence of a rhamnosyl residue. After acid hydrolysis, compound 12 showed a peak corresponding to LRha in GC analysis and a peak corresponding to 22βacetoxyglycyrrhizin (24) in LC/MS analysis (Figures S2 and S3). The Rha residue was linked to 2″-OH according to the HMBC correlation between H-1‴ (δH 6.46, 1H, s) and C-2″ (δC 78.1). The α-orientation of the Rha glycosidic bond was supported by the chemical shift of C-5 at δC 69.6.28 Thus, the structure of 12 was established as 3β-O-[α-L-rhamnopyranosyl(1→2)-β-D-glucuronopyranosyl-(1→2)-β-D-glucuronopyranosyl]-22β-acetoxy-11-oxo-olean-12-en-30-oic acid and was named uralsaponin X. Compound 13 has the molecular formula C48H70O20, according to HRESIMS analysis ([M − H]− m/z 965.4374, calcd 965.4377) and 13C NMR spectroscopic data. The MS and NMR data also indicated that 13 had the same sugar moiety as 12. The sapogenin of 13 was identified as glabrolide. The sugar moiety was linked to 3-OH of glabrolide according to the HMBC correlation of H-1′ (δH 5.04) with C-3 (δC 89.8). Therefore, the structure of 13 was identified as 3β-O-[α-Lrhamnopyranosyl-(1→2)-β-D-glucuronopyranosyl-(1→2)-β-Dglucuronopyranosyl]glabrolide and was named uralsaponin Y. Among the 13 new saponins, compounds 1−3 contain a galacturonic acid residue (GalA), and 5 contains a xylose residue (Xyl). Saponins containing these sugar residues are reported from Glycyrrhiza species for the first time. The sugar residues were identified by comparing with reference standards using GC or IC-PAD after hydrolysis. The GluA−GluA or

GluA−GalA bond was difficult to hydrolyze by acid treatment. Although this bond could be easily hydrolyzed by βglucuronidase, the derivatized sugar residues could not be detected by GC for unknown reasons. We also found that the reference standards for GluA and GalA both gave two peaks in GC analysis (Figure S4), which was different from other sugar residues. Finally, GluA and GalA were identified by IC-PAD, which proved to be convenient, fast, and reliable. The 28 saponins isolated from G. uralensis were evaluated for antiviral activities against influenza virus A/WSN/33 (H1N1) in MDCK cells.31 None of the compounds showed significant cytotoxicity against uninfected MDCK cells at 100 μM. Their abilities to inhibit H1N1 virus-induced MDCK cell death were evaluated using the CellTiter-Glo luminescent cell viability assay. All the saponins showed inhibitory activities against the H1N1 virus at 100 μM (Figure S5). The inhibition rates varied from 47.5% to 82.5%. The positive control oseltamivir phosphate (Osv-P, Tamiflu) showed an inhibition rate of 78% at the same dose.32 The IC50 values for significant inhibitors were measured (Table 3). Compounds 1, 7, 8, and 24 showed IC50 values of 48.0, 42.7, 39.6, and 49.1 μM, respectively, which were comparable to Osv-P (IC50 45.6 μM). Glycyrrhizic acid (27), the major saponin in licorice, showed an IC50 value of 158.0 μM. Compounds 1−28 were also evaluated for anti-HIV activities (Figure S6). Compounds 18, 21, 23, 24, 25, and 28 showed inhibition rates of above 50% at 100 μM, and their IC50 values were measured. Compounds 24 and 28 exhibited IC50 values of 29.5 and 41.7 μM, respectively. Glycyrrhetinic acid, the sapogenin of glycyrrhizic acid (27), inhibited the HIV-1 virus with an IC50 value of 28.8 μM. By comparing the activities of glycyrrhetinic acid (no GluA), 28 (GluA), and 27 (GluA− GluA), it appears as if GluA substitutions decrease the anti-HIV activities of licorice saponins.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter. UV spectra were measured on a Cary 300 Bio UV−visible spectrophotometer. IR spectra were recorded as KBr disks using a Nicolet NEXUS-470 FT-IR spectrophotometer. Unless otherwise specified, NMR spectra were recorded at 400 MHz for 1H and 100 MHz for 13C on a Bruker AVANCE III-400 spectrometer in pyridined5, using TMS as reference. NOE experiments were conducted using an Inova 600 MHz spectrometer. HRESIMS spectra were obtained on a Bruker APEX IV FT-MS spectrometer. Open column chromatography was performed using AB-8 macroporous resin (Cangzhou Bao’en Chemical Factory, China), SBC MCI gel (75−150 μm, Sci-Bio Chem Co. Ltd., Chengdu, China), and ODS C18 (75 μm, YMC Co. Ltd., Japan). Semipreparative HPLC was performed on an Agilent 1200 instrument equipped with a ZORBAX SB C18 column (250 mm × 9.4 mm, i.d. 5 μm, Agilent, USA) and a YMC Pack ODS-A column (250 mm × 10 mm, i.d. 5 μm, YMC Co. Ltd., Japan). Standard samples of L-rhamnose, L-arabinose, D-galactose, D-glucose, Dgalacturonic acid, and D-glucuronic acid were from Sigma-Aldrich with purities of above 97%. Anhydrous pyridine, L-cysteine methyl ester hydrochloride (TCI, >99%), and HMDS−TMCS−pyridine I



Article

Uralsaponin O (3): amorphous powder (MeCN); [α]25D +188 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 250 (2.82) nm; IR (KBr) νmax 3434, 2921, 1753, 1637, 1080 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 819.3772 [M − H]− (calcd for C42H59O16, 819.3798). Uralsaponin P (4): amorphous powder (MeCN); [α]25D +190 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 252 (2.80) nm; IR (KBr) νmax 3433, 2929, 1728, 1640, 1048 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 823.4106 [M − H]− (calcd for C42H63O16, 823.4110). Uralsaponin Q (5): white, amorphous powder (MeCN); [α]25D +190 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 255 (2.89) nm; IR (KBr) νmax 3434, 2919, 1661, 1048 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 939.4595 [M − H]− (calcd for C47H71O19, 939.4584). Uralsaponin R (6): white, amorphous powder (MeCN); [α]25D +189 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 255 (2.93) nm; IR (KBr) νmax 3340, 2928, 1661, 1037 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 969.4699 [M − H]− (calcd for C48H73O20, 969.4690). Uralsaponin S (7): white, amorphous powder (MeCN); [α]25D +189 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 251 (2.96) nm; IR (KBr) νmax 3422, 2922, 1675, 1205 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 969.4695 [M − H]− (calcd for C48H73O20, 969.4690). Uralsaponin T (8): white, amorphous powder (MeCN); [α]25D +199 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 253 (3.00) nm; IR (KBr) νmax 3429, 2930, 1668, 1205, 1045 cm−1; 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 953.4721 [M − H]− (calcd for C48H73O19, 953.4741). Uralsaponin U (9): white, amorphous powder (MeCN); [α]25D +192 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 252 (3.01) nm; IR (KBr) νmax 3435, 2948, 1667, 1206, 1046 cm−1; 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 837.3906 [M − H]− (calcd for C42H61O17, 837.3903). Uralsaponin V (10): white, amorphous powder (MeCN); [α]25D +187 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 241 (3.18), 249 (3.22), 258 (3.05) nm; IR (KBr) νmax 3435, 2943, 1731, 1382, 1045 cm−1; 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 805.4021 [M − H]− (calcd for C42H61O15, 805.4005). Uralsaponin W (11): white, amorphous powder (MeCN); [α]25D +170 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 248 (2.91) nm; IR (KBr) νmax 3429, 1681, 1208, 1142 cm−1; 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 805.4008 [M − H]− (calcd for C42H61O15, 805.4005). Uralsaponin X (12): white powder (MeCN); [α]25D +193 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 250 (2.71) nm; IR (KBr) νmax 3434, 1723, 1654, 1051 cm−1; 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 1025.4599 [M − H]− (calcd for C50H73O22, 1025.4588). Uralsaponin Y (13): white powder (MeCN); [α]25D +185 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 250 (2.98) nm; IR (KBr) νmax 3433, 2929, 1761, 1660, 1048 cm−1; 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 965.4374 [M − H]− (calcd for C48H69O20, 965.4377). Hydrolysis of Saponins. Acid hydrolysis was carried out to obtain the sugar residues of compounds 4, 5, 6, and 7. Compounds 4−7 (each 2.0 mg) were treated with 5 N TFA (trifluoroacetic acid, aqueous solution, 3 mL) at 90 °C for 6 h. After extraction with CH2Cl2 (3 mL × 3), the water-soluble layer was evaporated to dryness. Enzyme hydrolysis was carried out to obtain the sugar residues, GluA, and GalA in compounds 1 and 11. The compound (each 2.0 mg) was distributed in 1 mL of β-glucuronidase solution (containing 19.86 U/ μL, in NaOAc buffer, pH 5.5, Sigma-Aldrich) and incubated at 37 °C for 2 h. The mixture was treated with 5 mL of MeOH and centrifuged at 6000 rpm for 10 min to remove protein. The supernatant was dried under nitrogen flow. Compounds 8 and 9 were hydrolyzed with 5 N TFA and then treated with β-glucuronidase. Gas Chromatography Analysis. GC analysis was conducted on a Varian CP-3800 instrument (Varian Inc., USA). The hydrolyzed

(2:1:10) silylation reagent (Regis) were used for sugar derivatization before GC analysis. Plant Material. Dried roots of G. uralensis Fisch. were collected in September 2012 in Chifeng City, Inner Mongolia Autonomous Region, China. The plant was identified by the authors. A voucher specimen (No. GC-201209) was deposited at the School of Pharmaceutical Sciences, Peking University, Beijing, China. Extraction and Isolation. The dried material (35 kg) was powdered and extracted with 95% (90 L × 2 h × 2) and 70% EtOH (90 L × 2 h × 1) under reflux. After concentration in vacuo, the extract (10 L) was dispersed in H2O and successively extracted with EtOAc and n-BuOH. The n-BuOH extract (3 L) was separated on an AB-8 macroporous resin column eluted with EtOH−H2O (10 to 95%, v/v) to obtain fractions A−E. Fraction B (55 g) was subjected to MCI gel column chromatography and eluted with 20%, 30%, 35%, and 45% EtOH to afford fractions B-0 (23 g), B-1 (8 g), B-2 (5 g), and B-4 (12 g). Fraction B-1 was fractionated on an ODS C18 column eluted with MeCN−H2O−TFA (20:80:0.03−32:68:0.03, v/v/v) to obtain fractions B-11 (0.4 g), B-12 (1.5 g), and B-13 (5 g). Compounds 8 (7 mg, tR = 45.5 min), 14 (4.0 mg, tR = 25.0 min), and 15 (50.0 mg, tR = 33.0 min) were obtained from B-11 by semipreparative HPLC on an Agilent ZORBAX SB C18 column (5 μm, 250 × 9.4 mm; flow rate, 2 mL/min; MeCN−H2O−TFA, 22:78:0.03, v/v/v; 254 nm). Compounds 12 (12 mg, tR = 45.0 min) and 16 (220 mg, tR =38.0 min) were isolated from B-12 eluted with MeCN−H2O−TFA (26:74:0.03, v/v/v). Compounds 1 (15 mg, tR = 49.0 min), 6 (3 mg, tR = 42.0 min), and 24 (300 mg, tR = 45.0 min) were isolated from B-13 eluted with MeCN−H2O−TFA (27:73:0.03, v/v/v). Fraction B-2 was purified on an ODS C18 column and eluted with MeCN−H2O− TFA (32:68:0.03−38:62:0.03, v/v/v) to obtain fractions B-21 (0.2 g), B-22 (2.8 g), and B-23 (0.8 g). Compounds 4 (8 mg, tR = 52.0 min), 5 (4 mg, tR = 52.8 min), 7 (5 mg, tR = 54.0 min), and 13 (15 mg, tR = 56.5 min) were obtained from B-21 by semipreparative HPLC (MeCN−H2O−TFA, 28.5:71.5:0.02, v/v/v; 254 nm). B-22 was subjected to MCI column chromatography to yield compound 17 (950 mg), fraction B-22-1 (900 mg), and fraction B-22-2 (400 mg). B22-2 was then purified by semipreparative HPLC on a YMC Pack ODS-A column (5 μm, 250 × 10 mm; flow rate, 2 mL/min; MeCN− H2O−TFA, 28:72:0.03, v/v/v; 254 nm) to yield 2 (4 mg, tR = 56.0 min) and 18 (200 mg, tR = 53.5 min). Fraction B-23 was isolated by semipreparative HPLC on the same YMC column to afford 3 (5 mg, tR = 42.0 min), 9 (10 mg, tR = 44.6 min), and 25 (20 mg, tR = 47 min) with MeCN−H2O−TFA (30:70:0.05, v/v/v) as the mobile phase. Compound 27 (3 g) was precipitated from fraction B-3. Fraction D (12 g) was subjected to ODS column chromatography to afford fractions D-1 through D-4 eluted with MeCN−H 2 O−TFA (40:60:0.03−50:50:0.03, v/v/v). Compounds 22 (30 mg), 10 (80 mg), and 21 (65 mg) were precipitated from a 70% MeCN solution of fractions D-1, D-2, and D-3, respectively. Compounds 19 (5 mg, tR = 45.0 min), 20 (7 mg, tR = 47.0 min), and 23 (12 mg, tR = 55.0 min) were isolated from fraction D-1 by semipreparative HPLC (YMC Pack ODS-A column, 5 μm, 250 × 10 mm; flow rate, 2 mL/min; MeCN− H2O−TFA, 33:67:0.03, v/v/v; 254 nm). Similarly, compounds 11 (5 mg, tR = 38.0 min) and 26 (6 mg, tR = 41.0 min) were obtained from fraction D-2 by semipreparative HPLC eluted with MeCN−H2O− TFA (36:64:0.03, v/v/v). Compound 28 (11 mg, tR = 39.0 min) was obtained from fraction D-4 by semipreparative HPLC (YMC Pack ODS-A column, 5 μm, 250 × 10 mm; flow rate, 2 mL/min; MeCN− H2O−TFA, 48:52:0.03, v/v/v; 254 nm). Purities of compounds 1−28 were above 97% by HPLC/UV analysis. Uralsaponin M (1): white, amorphous powder (MeCN); [α]25D +204 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 251 (2.88) nm; IR (KBr) νmax 3434, 2923, 1725, 1653, 1048 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 879.3983 [M − H]− (calcd for C48H73O19, 879.4009). Uralsaponin N (2): white, amorphous powder (MeCN); [α]25D +193 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 252 (2.97) nm; IR (KBr) νmax 3435, 2930, 1731, 1659, 1046 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 837.3890 [M − H]− (calcd for C42H61O17, 837.3903). J



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sample of saponins or authentic monosaccharide was dissolved in 2 mL of anhydrous pyridine. L-Cysteine methyl ester hydrochloride (3 mg) was added before the mixture was stirred at 60 °C for 1.5 h. A 1 mL amount of HMDS−TMCS (hexamethyldisilazane−trimethylchlorosilane, 2:1) was added, and the mixture was kept at 60 °C for 2 h. A 1 μL portion of the supernatant was injected for analysis on a CP-Sil 8CB/MS GC column (30 m × 0.25 mm × 0.2 μm) at 120 to 260 °C at 6 °C min−1 using N2 (1 mL/min) as carrier gas.33 The injection and detector temperature was set at 250 °C, and the splitting ratio was 1/ 100. IC-PAD Analysis. IC-PAD analysis was carried out on an ICS3000 ion chromatography instrument (Thermo-Dionex Inc., USA) equipped with a DP-5 gradient pump, an AS-1 autosampler, and an ED-3000 electrochemical detector. The saponin hydrolysis product was dispersed in 5 mL of deionized H2O. The solution was purified by solid phase extraction through a preprocessing (5 mL of MeOH + 5 mL of H2O) OnGuard II RP column. After discarding the initial 3 mL, the eluent was collected and injected for analysis. Samples were separated on a Dionex CarboPac PA20 column (3 × 150 mm) protected with a CarboPacPA20 guard column (3 × 30 mm). An ED3000 electrochemical detector with a gold working electrode and a pH-Ag/AgCl reference electrode was used. The waveform was as follows: E1 = 0.10 V, 0−0.4 s; E2 = −2.00 V, 0.4−0.42 s; E3 = 0.60 V, 0.43 s; and E4 = −0.10 V, 0.44−0.50 s (E1 was the detection potential, and E2−E4 were potentials to clean and restore the electrode for subsequent detection). The linear gradient elution program was as follows: 0−13 min, 10 mM NaOH; 13.1−23 min, 10 mM NaOH + 120 mM NaOAc; 23.1−25 min, 200 mM NaOH; 25.1−32 min, 10 mM NaOH. The flow rate was 0.45 mL/min, and the temperature was 30 °C. The injection volume was 25 μL for each sample. The data were recorded by Chromeleon 2.2 software. Consequently, the sugar residues were identified by comparing the retention times with those of reference standards: L-rhamnose (tR, 6.75 min), L-arabinose (7.28 min), D-galactose (9.21 min), D-glucose (10.40 min), D-galacturonic acid (21.33 min), and D-glucuronic acid (23.53 min). Anti-H1N1 Assay. Madin-Darby canine kidney (MDCK) cells (ATCC) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. The influenza virus strain A/ WSN/33 (H1N1) was propagated in MDCK cells in the presence of 2 μg/mL TPCK-treated trypsin (from bovine pancreas, Sigma-Aldrich, lot #031M7358 V). After incubation at 37 °C for 2 days, the supernatant was centrifuged at 1000 rpm for 3 min, and the progeny virus was harvested and used for the infection. MDCK cells were seeded into 96-well plates at 1 × 105 cells per well 24 h prior to infection and incubated at 37 °C in 5% CO2. Compounds were mixed with the virus and incubated at room temperature for 15 min. The original medium in the 96-well plate was removed, and the mixed culture containing compound and virus was added to the cells. At 36 h postinfection, microscopy was performed to determine the antiviral activity, and the data were confirmed by the CellTiter-Glo luminescent cell viability assay (Promega, #G7570). In addition, cytotoxicity of the compounds was determined in uninfected MDCK cells, which were incubated with indicated concentrations of compounds for 36 h. Microscopy was performed to determine the cytopathic effect, and the data were confirmed by the CellTiter-Glo luminescent cell viability assay.31,34 The inhibition rate was calculated by the following formula: inhibition rate (%) = [1 − (luminescence with compounds − luminescence with compounds and virus)/(luminescence with DMSO − luminescence with DMSO and virus)] × 100%. Anti-HIV Activity Evaluation. 293T cells (2 × 105) were cotransfected with 0.6 mg of pNL-Luc-E and 0.4 mg of pHIT/G. The VSV-G pseudo-typed viral supernatant (HIV-1) was harvested by filtration through a 0.45 μm filter after 48 h, and the concentration of viral capsid protein was determined by p24 antigen capture ELISA (Biomerieux). SupT1 cells were exposed to VSV-G pseudo-typed HIV-1 (MOI = 1) at 37.8 °C for 48 h in the absence or presence of test compounds (with positive control efavirenz). A luciferase assay system (Promega) was used to determine the inhibition rate.35

Article

ASSOCIATED CONTENT

S Supporting Information *

Copies of 1D and 2D NMR, UV, IR, and HRESIMS spectra for compounds 1−13, flow chart for compound isolation, GC and LC/MS chromatograms for structural characterization, and biological evaluation data for compounds 1−28. This material can be accessed free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-8280-1516. Fax: +86-10-8280-2024. E-mail: [email protected] (M. Ye). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (No. 81173644, No. 81222054) and the Program for New Century Excellent Talents in University from China Ministry of Education (No. NCET-11-0019).

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REFERENCES

(1) Commission of Flora of China. Flora of China; Chinese Science Press: Beijing, 1998; Vol. 42, No. 2, p 168. (2) Zhang, Q. Y.; Ye, M. J. Chromatogr. A 2009, 1216, 1954−1969. (3) Fiore, C.; Eisenhut, M.; Ragazzi, E.; Zanchin, G.; Armanini, D. J. Ethnopharmacol. 2005, 99, 317−324. (4) Kim, J. K.; Oh, S. M.; Kwon, H. S.; Oh, Y. S.; Lim, S. S.; Shin, H. K. Biochem. Biophys. Res. Commun. 2006, 345, 1215−1223. (5) Oganesyan, R. Bull. Exp. Biol. Med. 2002, 134, 135−136. (6) Jo, E. H.; Kim, S. H.; Ra, J. C.; Kim, S. R.; Cho, S. D.; Jung, J. W.; Yang, S. R.; Park, J. S.; Hwang, J. W.; Aruoma, O. I.; Kim, T. Y.; Lee, Y. S.; Kang, K. S. Cancer Lett. 2005, 230−239. (7) Lee, J. R.; Park, S. J.; Lee, H. S.; Jee, S. Y.; Seo, J.; Kwon, Y. K.; Kwon, T. K.; Kim, S. C. Evid.-Based Complementary Altern. Med. 2009, 6, 195−201. (8) Eisenhut, F. M.; Krausse, R.; Ragazzi, E.; Pellati, D.; Armanini, D.; Bielenberg, J. Phytother. Res. 2008, 22, 141−148. (9) Kitagawa, I. Pure Appl. Chem. 2002, 74, 1189−1198. (10) Ashfaq, U. A.; Masoud, M. S.; Nawaz, Z.; Riazuddin, S. J. Transl. Med. 2011, 9, 112. (11) Li, X. H.; Qi, Y.; Cai, R. L.; Liu, B.; Song, Y.; Xie, C. Chin. J. Exp. Tradit. Med. Form. 2010, 16, 110−113. (12) Cherng, J. M.; Lin, H. J.; Hung, M. S.; Lin, Y. R.; Chan, M. H.; Lin, J. C. Eur. J. Pharmacol. 2006, 547, 10−21. (13) Zhou, S. J.; Cao, J. L.; Qiu, F.; Kong, W. J.; Yang, S. H.; Yang, M. H. Phytochem. Anal. 2013, 24, 527−533. (14) Shi, H. F.; Chen, Y. P.; Di, J. B.; Xu, Z. W. Chin. J. Contemp. Pediatr. 2010, 12, 272−274. (15) Wu, W. F.; Sun, W. W. China Pharm. 2004, 15, 558−559. (16) Wang, Q.; Ji, S.; Yu, S. W.; Wang, H. X.; Lin, X. H.; Ma, T. T.; Qiao, X.; Xiang, C.; Ye, M.; Guo, D. A. Fitoterapia 2013, 85, 35−40. (17) Wang, Q.; Miao, W. J.; Xiang, C.; Guo, D. A.; Ye, M. Chin. Tradit. Herbal Drugs 2012, 43, 1886−1890. (18) Ji, S.; Wang, Q.; Qiao, X.; Guo, H. C.; Yang, Y. F.; Bo, T.; Xiang, C.; Guo, D. A.; Ye, M. J. Pharm. Biomed. Anal. 2014, 90, 15−26. (19) Zheng, Y. F.; Qi, L. W.; Cui, X. B.; Peng, G. P.; Pen, Y. B.; Ren, M. T.; Cheng, X. L.; Li, P. Planta Med. 2010, 76, 1457−1463. (20) Kitagawa, I.; Hori, K.; Taniyama, T.; Zhou, J. L.; Yoshikawa, M. Chem. Pharm. Bull. 1993, 41, 43−49. (21) Li-Yang, J.; Nakajima, J. I.; Kimura, N.; Saitob, K.; Seo, S. Nat. Prod. Commun. 2007, 2, 243−248. (22) Kitagawa, I.; Zhou, J. L.; Sakagami, M.; Uchida, E.; Yoshikawa, M. Chem. Pharm. Bull. 1991, 39, 244−246.

K



Article

(23) Kitagawa, I.; Hori, K.; Sakagami, M.; Hashiuchi, F.; Yoshikawa, M.; Ren, J. L. Chem. Pharm. Bull. 1993, 41, 1350−1357. (24) Zhang, H.; Wang, S. S.; Li, W.; Wang, Y. Q.; Xue, X. Y.; Liang, X. M. Mod. Tradit. Chin. Med.-World Sci. Technol. 2009, 11, 253−256. (25) Stolyarova, O. V.; Baltina, L. A.; Gabbasov, T. M.; Kondratenko, R. M.; Il’ina, T. V.; Plyasunova, O. A. Russia. Patent RU 2,475,246, 2013. (26) Baltina, L. A.; Kunert, O.; Fatykhov, A. A.; Kondratenko, R. M.; Spirikhin, L. V.; Baltina, L. A., Jr.; Galin, F. Z.; Tolstikov, G. A.; Haslinger, E. Chem. Nat. Compd. 2005, 41, 432−435. (27) Tao, W. W.; Duan, J. A.; Yang, N. Y.; Li, J. P.; Tang, Y. P.; Yan, H. Chin. Tradit. Herbal Drugs 2013, 44, 1552−1557. (28) Nie, W.; Luo, J. G.; Kong, L. Y. Carbohydr. Res. 2010, 345, 68− 73. (29) Rozen, S.; Shahak, I.; Bergmann, E. D. Tetrahedron 1973, 29, 2327−2331. (30) Seki, H.; Sawai, S.; Ohyama, K.; Mizutani, M.; Ohnishi, T.; Sudo, H.; Fukushima, E. O.; Akashi, T.; Aoki, T.; Saito, K. Plant Cell 2011, 23, 4112−4123. (31) Jones, J. C.; Turpin, E. A.; Bultmann, H.; Brandt, C. R.; SchultzCherry, S. J. Virol. 2006, 12, 11960−11967. (32) Lew, W.; Chen, X.; Kim, C. U. Curr. Med. Chem. 2000, 7, 663− 672. (33) Pang, X.; Cong, Y.; Yu, H. S.; Kang, L. P.; Feng, B.; Han, B. X.; Zhao, Y.; Xiong, C. Q.; Tan, D. W.; Song, W.; Liu, B.; Cong, Y. W.; Ma, B. P. Planta Med. 2012, 78, 276−285. (34) Evelien, V.; Fusun, G.; Zafer, C.; Matheus, F.; Mark, L. R.; Charles, J. R.; Nesrin, C.; Lieve, N. J. Virol. 2010, 84, 4277−4288. (35) Zhang, Q.; Liu, Z.; Mi, Z.; Li, X.; Jia, P.; Zhou, J.; Yin, X.; You, X.; Yu, L.; Guo, F. Antiviral Res. 2011, 91, 321−329.

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