Dibenzoyl and Isoflavonoid Glycosides from Sophora flavescens

Article pubs.acs.org/jnp

Dibenzoyl and Isoflavonoid Glycosides from Sophora flavescens: Inhibition of the Cytotoxic Effect of D‑Galactosamine on Human Hepatocyte HL-7702 Yi Shen, Zi-Ming Feng, Jian-Shuang Jiang, Ya-Nan Yang, and Pei-Cheng Zhang* 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 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Twelve new dibenzoyl derivatives sophodibenzoside A−L (1−12) and five new isoflavone glycosides (13− 17) have been isolated from the roots of Sophora flavescens together with eight known compounds (18−25). Notably, the use of acetic acid-d4 was required to enable identification of the dibenzoyl glycoside structures. Compounds 1, 2, 13, 14, and 19 exhibited weak inhibition of the cytotoxic effect of Dgalactosamine on the human hepatic cell line HL-7702.

he perennial shrub Sophora flavescens Ait. (Leguminosae) is distributed widely throughout East Asia. In the People’s Republic of China, the roots of this plant are known as “ku shen”. It is commonly used in traditional Chinese medicine (TCM) as an antipyretic and diuretic agent and has been used for the treatment of skin and gynecological diseases. Pharmacological investigations indicated that S. flavescens can be beneficial for mental health, and that it has antiinflammatory, anthelmintic, free radical scavenging, and antimicrobial activity.1−4 Previous chemical studies on S. flavescens revealed that quinolizidine alkaloids and flavonoids are the main chemical components, and these are usually regarded as the active constituents.5−8 Recent reports suggested that S. flavescens possessed hepatoprotective properties and was active against the hepatitis B virus, with an alkaloid, oxymatrine, contributing to this activity.9−12 As a continuation of our efforts to explore new compounds with hepatoprotective activity, a series of new dibenzoyl derivatives (1−12) and isoflavonoid glycosides (13−17) were isolated from S. flavescens, together with eight known compounds (18−25). In the course of NMR experiments directed toward confirming the structures of the dibenzoyl glycosides, different deuterated solvents had to be examined to obtain the clearest NMR spectra. Ultimately, acetic acid-d4 was found the most appropriate deuterated solvent, and a possible mechanism underlying this result is discussed. Some compounds were assessed for inhibition of the cytotoxic effect of D-galactosamine on the human hepatic cell line HL-7702.

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© 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 was obtained as a yellow powder, and its molecular formula was established as C26H30O15 by HRESIMS {m/z 605.1479 [M + Na]+ (calcd for 605.1477)}. The IR spectrum of 1 revealed the presence of hydroxy (3355 cm−1) and carbonyl (1663 cm−1) groups. In the 1H NMR spectrum (Table 1), two sets of typical ABX systems were present at δH 7.45 (1H, d, J = 8.5 Hz), 6.79 (1H, d, J = 2.0 Hz), and 6.62 (1H, dd, J = 8.5, 2.0 Hz) and 7.02 (1H, d, J = 8.5 Hz), 7.51 (1H, d, J = 2.0 Hz), and 7.49 (1H, dd, J = 8.5, 2.0 Hz), Received: September 30, 2013 Published: December 2, 2013 2337

dx.doi.org/10.1021/np400784v | J. Nat. Prod. 2013, 76, 2337−2345

Journal of Natural Products

Article

Table 1. 1H NMR (500M) Spectroscopic Data (δ in ppm, J in Hz) for Compounds 1−6 in Acetic Acid-d4 position 3′ 5′ 6′ 2″ 3″ 5″ 6″ glc-1‴ 2‴ 3‴ 4‴ 5‴ 6‴a 6‴b 1′′′′ 2′′′′ 3′′′′ 4′′′′ 5′′′′ 4″-OCH3

1

2

3

4

6.79, 6.62, 7.45, 7.51,

d (2.0) dd (8.5, 2.0) d (8.5) d (2.0)

6.75, 6.60, 7.45, 7.51,

brs d (8.5) d (8.5) brs

6.70, 6.60, 7.45, 7.51,

d (2.0) dd (8.5, 2.0) d (8.5) d (2.0)

7.02, 7.49, 5.16, 3.72, 3.78, 3.67, 3.86, 4.16, 3.85, xyl 4.45, 3.46, 3.60, 3.76,

d (8.5) dd (8.5, 2.0) d (7.5) m m t (9.0) m d (9.5) m

7.01, 7.49, 5.15, 3.73, 3.78, 3.62, 3.81, 4.06, 3.70, api 5.09, 4.08,

d (8.0) overlapped d (7.5) m m t (9.0) m d (11.5) m

7.01, 7.48, 5.21, 3.72, 3.81, 3.69, 3.72, 4.01, 3.88,

d (8.5) dd (8.5, 2.0) d (7.5) m m t (9.0) m d (12.5) dd (12.5, 5.0)

d (7.5) dd (7.5, 9.0) t (9.0) m

4.00, dd (11.5, 5.0) 3.28, dd (11.5, 10.0) 3.95, s

brs brs

3.99, d (10.0) 3.88, d (10.0) 3.78, s 3.95, s

6.80, 6.62, 7.47, 7.96, 7.04, 7.04, 7.96, 5.15, 3.73, 3.79, 3.68, 3.86, 4.16, 3.85, xyl 4.44, 3.46, 3.59, 3.75,

5

d (2.5) dd (8.5, 2.5) d (8.5) d (8.5) d (8.5) d (8.5) d (8.5) d (7.5) m m m m d (10.0) m d (7.5) dd (7.5, 9.0) t (9.0) m

3.98, dd (11.5, 5.5) 3.28, t (11.5) 3.89, s

3.96, s

6.76, 6.61, 7.47, 7.95, 7.04, 7.04, 7.95, 5.15, 3.74, 3.76, 3.63, 3.80, 4.06, 3.70, api 5.10, 4.10,

d (2.0) dd (8.5, 2.0) d (8.5) d (8.5) d (8.5) d (8.5) d (8.5) d (7.5) m m m m d (11.5) m

6 6.72, 6.65, 7.47, 7.94, 7.07, 7.07, 7.94, 5.20, 3.72, 3.77, 3.64, 3.73, 4.00, 3.83,

d (2.0) d (8.5) d (8.5) d (8.5) d (8.5) d (8.5) d (8.5) d (7.5) m t (9.0) t (9.0) m d (12.0) dd (12.0, 5.5)

d (2.0) d (2.0)

3.99, d (10.5) 3.90, d (10.5) 3.77, m 3.89, s

3.90, s

Figure 1. Key HMBC correlations of compounds 1, 7, and 16.

characteristic of two trisubstituted benzene rings. A methoxy resonance (δH 3.96, 3H, s) and two anomeric proton resonances at δH 5.16, (1H, d, J = 7.5 Hz, glc-1-H) and 4.45 (1H, d, J = 7.5 Hz, xyl-1-H) were also observed. The remaining proton resonances corresponding to the sugar moieties appeared at δH 3.27−4.16. The 13C NMR spectrum of 1 showed 26 carbon resonances (Table 3). Of these carbon resonances, 12 were attributed to two benzene rings. Two carbonyl carbon resonances at δC 194.6 and 201.2 were observed in the downfield region. A methoxy resonance at δC 58.9 and two anomeric carbon resonances from sugar moieties at δC 102.5 and 106.8 were also observed. The HMBC experiment (Figure 1) showed correlations from H-6′ to C-2 and from H-2″ and H-6″ to C-1, which suggested two carbonyl carbons linked to two benzene rings. The methoxy group was assigned at C-4″ from the correlation of −OCH3 (δH 3.95, s) with C-4″(δC 156.8). The D-configurations of the xylopyranosyl and glucopyranosyl moieties were determined by comparison of the NMR data,13 and GC analysis after acidic hydrolysis and chiral derivatization of 1.14,15 The β-configurations were deduced based on the chemical shifts and coupling constants (glc: J = 7.5 Hz and xyl: J = 7.5 Hz) of the anomeric protons.13

The linkage positions of the two sugar moieties were established as β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl based on the 13C NMR shift values and the HMBC correlations showing correlation of xyl-H-1 with C-6‴. In addition, the key HMBC correlation of C-4′ with glc-H-1‴ confirmed that the glucopyranosyl moiety was located at C-4′ (Figure 1). Thus, 1 was determined to be 2′,3″-dihydroxy-4″-methoxydibenzoyl-4′O-β- D -xylopyranosyl-(l→6)-β- D -glucopyranoside and was named sophodibenzoside A. Compound 2 showed a quasi-molecular ion at m/z 605.1487 [M + Na] + in the HRESIMS (calcd for 605.1477), corresponding to the molecular formula C26H30O15. The 1H and 13C NMR spectra of 2 were similar to those of 1 with the exception of the sugar moiety. The 13C NMR spectrum of 2 (Table 3) showed the anomeric carbon at δC 112.4 (api-C-1) and the other four carbon resonances at δC 83.1, 80.5, 76.8, and 68.1. The 1H NMR spectrum (Table 1) also showed an anomeric proton at δH 5.09 (1H, br s) and the other proton resonances at δH 4.08−3.78. These implied the presence of an apiofuranosyl group in 2 instead of the xylopyranosyl moiety found in 1. The HMBC spectrum confirmed the linkage position at C-6‴, based on the observed correlation of C-6‴ 2338



Article

Table 2. 1H NMR (500M) Spectroscopic Data (δ in ppm, J in Hz) for Compounds 7−12 in Acetic Acid-d4 position 3′ 5′ 6′ 2″ 5″ 6″ glc-1‴ 2‴ 3‴ 4‴ 5‴ 6‴a 6‴b 1′′′′ 2′′′′ 3′′′′ 4′′′′ 5′′′′ 3″-OCH3 3″,4″-OCH2O-

7 6.79, 6.62, 7.45, 7.48, 6.92, 7.52, 5.16, 3.73, 3.80, 3.68, 3.85, 4.16, 3.86, xyl 4.45, 3.46, 3.60, 3.76,

d (2.0) dd (8.5, 2.0) d (8.5) d (2.0) d (8.5) dd (8.5, 2.0) d (8.0) m t (9.0) t (9.0) m d (10.5) m d (7.0) dd (7.0, 9.0) t (9.0) m

4.00, dd (11.5, 5.5) 3.28, t (11.5) 6.10, s

8 6.75, 6.60, 7.45, 7.48, 6.92, 7.52, 5.15, 3.73, 3.75, 3.63, 3.82, 4.05, 3.69, api 5.09, 4.07,

d (2.5) dd (9.0, 2.5) d (9.0) d (1.5) d (8.5) dd (8.5, 1.5) d (7.5) m m t (9.0) m d (12.5) m

9 6.70, 6.62, 7.45, 7.47, 6.92, 7.51, 5.21, 3.72, 3.81, 3.68, 3.72, 4.01, 3.87,

d (2.0) d (2.0)

4.00, d (10.0) 3.88, d (10.0) 3.75, s

6.10, s

10

d (1.5) dd (8.5, 1.5) d (8.5) d (2.0) d (8.0) d (8.0, 2.0) d (7.5) m t (9.0) t (9.0) m d (12.0) dd (12.0, 5.0)

6.79, 6.62, 7.47, 7.62, 6.96, 7.44, 5.16, 3.73, 3.79, 3.68, 3.86, 4.17, 3.85, xyl 4.45, 3.47, 3.60, 3.75,

11

d (2.5) dd (9.0, 2.5) d (9.0) d (2.0) d (8.0) dd (8.0, 2.0) d (7.5) m t (9.0) m m d (10.0) m

6.70, 6.60, 7.46, 7.62, 6.96, 7.44, 5.21, 3.71, 3.81, 3.67, 3.72, 4.00, 3.87,

d (2.5) dd (9.0, 2.5) d (9.0) d (1.5) d (7.5) dd (7.5, 1.5) d (8.0) m t (9.0) t (9.0) m dd (12.5, 2.0) dd (12.5, 5.0)

d (7.0) dd (9.0,7.0) t (9.0) m

4.00, dd (11.5, 5.0) 3.28, dd (11.5, 10.0) 3.95, s

12 6.78, 6.64, 7.46, 7.52, 6.97, 7.38, 5.17, 3.72, 3.77, 3.61, 3.85, 4.16, 3.85, xyl 4.43, 3.45, 3.55, 3.70,

d (2.0) dd (9.0, 2.0) d (9.0) d (2.0) d (8.0) dd (8.0, 2.0) d (7.0) m m m m d (10.0) m d (7.5) dd (9.0,7.5) m m

3.96, m 3.27, t (11.5) 3.95, s

6.10, s

Compound 7 was obtained as a yellow powder, and its HRESIMS showed a quasi-molecular ion at m/z 603.1332 [M + Na]+, corresponding to the molecular formula C26H28O15. The IR, UV, and NMR spectra permitted assignment of the structure as a dibenzoyl glycoside. Comparison of the NMR spectra of 7 and 1 showed that the differences between these two compounds were due to the substituents on ring B. The 3″-hydroxy-4″-methoxy arrangement in 1 was replaced in 7 with a methylenedioxy group with resonances at δH 6.10 (2H, s) and δC 105.9, suggesting the C-3″, C-4″dioxolane moiety. This was supported by the HMBC experiment (Figure 1), in which the correlations of the methylenedioxy protons with C3″ (δC 152.2) and C-4″ (δC 157.1) were present. With the exception of this dioxolane moiety, the remaining moieties of 7 were in accord with those of 1 based on the spectroscopic data (Tables 2 and 3) and chemical analysis. The structure of compound 7 was thus elucidated as 2′-hydroxy-3″,4″methylenedioxydibenzoyl-4′-O-β-D-xylopyranosyl-(l→6)-β-Dglucopyranoside and named sophodibenzoside G. With the use of similar identification methods, compounds 8 and 9 were readily defined as 2′-hydroxy-3″,4″-methylenedioxydibenzoyl4′-O-β-D-apiofuranosyl-(l→6)-β-D-glucopyranoside and 2′-hydroxy-3″,4″-methylenedioxydibenzoyl-4′-O-β-D-glucopyranoside, respectively. They were named sophodibenzoside H and I, respectively. The molecular formula of compound 10 was established as C26H30O15 by the HRESIMS ion at m/z 605.1461 [M + Na]+. Most of its spectroscopic data and chemical characteristics were in accord with 1, including UV, IR, MS, 1H NMR, and 13C NMR data. The ABX system at δH 7.62 (1H, d, J = 2.0 Hz), 6.96 (1H, d, J = 8.0 Hz), and 7.44 (1H, dd, J = 8.0, 2.0 Hz) on the B-ring was still present in the 1H NMR spectrum (Table 2). However, the HMBC spectrum led to assignment of the methoxy group at C-3″, according to the correlations of δH 3.95 (3H, s) and 6.96 with δC 151.2. The hydroxy group was unambiguously assigned at C-4″, according to the correlation of

(δC 70.4) with H-1′′′′. The configurations of the apiofuranosyl and glucopyranosyl groups were determined by the same methods as 1.14,15 Thus, the structure of compound 2 was elucidated as 2′,3″-dihydroxy-4″-methoxydibenzoyl-4′-O-β-Dapiofuranosyl-(l→6)-β-D-glucopyranoside, and it was named sophodibenzoside B. Compound 3 had the molecular formula C21H22O11 by analysis of its HRESIMS, which gave a quasi-molecular ion at m/z 473.1076 [M + Na]+. The 1H and 13C NMR resonances of the aglycone of 3 were similar to those of 1 (Tables 1 and 3), with the only differences arising due to the absence of a pentose moiety. This is supported by the presence of a glc-C-6‴ (δC 64.2) resonance in 3 instead of the corresponding glc-C-6‴ (δC 71.7) resonance found in 1. Thus, compound 3 was elucidated to be 2′,3″-dihydroxy-4″-methoxydibenzoyl-4′-O-β-D-glucopyranoside and was named sophodibenzoside C. The molecular formula of compound 4 was established as C26H30O14 by the HRESIMS {[M + Na]+ ion at m/z 589.1539}. Comparison of the NMR data of 4 with those of 1 (Tables 1 and 3) indicated the absence of an aromatic hydroxy resonance in 4. In the 1H NMR spectrum of 4 (Table 1), a set of AA′BB′ resonances at δH 7.96 (2H, d, J = 8.5 Hz) and 7.04 (2H, d, J = 8.5 Hz) replaced the corresponding ABX system arising from ring B of 1. Therefore, the structure of 4 was elucidated as 2′-hydroxy-4″-methoxydibenzoyl-4′-O-β-Dxylopyranosyl-(l→6)-β-D-glucopyranoside, and it was named sophodibenzoside D. Compounds 5 and 6 have the same aglycone moiety as 4, exhibiting a 2′-hydroxy-4″-methoxydibenzoyl-4′-O-substitution pattern, based on comparison of their NMR spectra (Tables 1 and 3). Furthermore, the sugar moieties were determined to be the same for 5 and 2 and for 6 and 3. Thus, these compounds were assigned the structures 2′-hydroxy-4″-methoxydibenzoyl4′-O-β-D-apiofuranosyl-(l→6)-β-D- glucopyranoside (5) and 2′hydroxy-4″-methoxydibenzoyl-4′-O-β-D-glucopyranoside (6). They were named sophodibenzoside E and F, respectively. 2339



Article

Table 3. 13C NMR (125 M) Spectroscopic Data (δ in ppm, J in Hz) for Compounds 1−12 in Acetic Acid-d4 position 1 2 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ glc-1‴ 2‴ 3‴ 4‴ 5‴ 6‴

1′′′′ 2′′′′ 3′′′′ 4′′′′ 5′′′′

1 194.6, 201.2, 115.7, 168.5, 107.6, CH 168.0, 112.8, CH 137.5, CH 129.6, 118.2, CH 149.8,

2 C C C C

C

C

C

156.8, C 114.1, CH 128.1, CH 102.5, CH 76.4, CH 79.4, CH 73.1, CH 78.5, CH 71.7, CH2 xyl 106.8, CH 76.4, CH 79.0, CH 72.7, CH 67.9, CH2

194.6, 201.2, 115.7, 168.6, 107.6, CH 168.1, 112.8, CH 137.5, CH 129.5, 118.2, CH 149.7,

3 C C C C

C

C

C

156.8, C 114.1, CH 128.1, CH 102.6, CH 76.5, CH 79.5, CH 73.2, CH 78.5, CH 70.4, CH2 api 112.4, CH 80.5, CH 83.1, C 76.8, CH2 68.1, CH2

194.5, 201.2, 115.7, 168.6, 107.4, CH 168.0, 112.5, CH 137.6, CH 129.6, 118.2, CH 149.8,

C C C C

C

C

C

156.8, C 114.1, CH 128.1, CH 102.4, CH 76.5, CH 79.2, CH 72.7, CH 79.2, CH 64.2, CH2

4

5

6

194.3, C 201.0, C 115.6, C 168.6, C 107.5, CH 168.0, C 112.8, CH 137.4, CH 129.0, C 135.7, CH 117.7, CH 168.5, C 117.7, CH 135.7, CH 102.4, CH 76.4, CH 79.4, CH 72.7, CH 78.4, CH 71.7, CH2 xyl 106.7, CH 76.4, CH 79.0, CH 73.0, CH

194.1, C 201.0, C 115.4, C 168.5, C 107.4, CH 167.9, C 112.2, CH 137.2, CH 128.9, C 135.6, CH 117.5, CH 168.5, C 117.5, CH 135.6, CH 102.4, CH 76.5, CH 79.2, CH 72.9, CH 78.3, CH 70.2, CH2 api 112.2, CH 80.5, CH 83.0, C 76.3, CH2 67.9, CH2

194.9, C 200.7, C 115.6, C 168.6, C 107.3, CH 167.9, C 112.6, CH 137.3, CH 128.5, C 135.7, CH 117.7, CH 168.6, C 117.7, CH 135.7, CH 102.2, CH 76.0, CH 78.8, CH 72.6, CH 79.1, CH 64.0, CH2

67.9, CH2

7 193.9, 200.7, 115.6, 168.6, 107.6, CH 168.0, 112.8, CH 137.4, CH 130.9, 111.1, CH 152.2,

8 C C C C

C

C

C

193.9, 200.8, 115.6, 168.7, 107.5, CH 168.2, 112.8, CH 137.4, CH 130.9, 111.1, CH 152.2,

9 C C C C

C

C

C



xyl 106.8, CH 76.5, CH 79.0, CH 72.7, CH

api 112.4, CH 80.5, CH 83.1, C 76.8, CH2

67.9, CH2

68.1, CH2

193.8, 200.8, 115.6, 168.6, 107.4, CH 168.1, 112.6, CH 137.5, CH 130.9, 111.6, CH 152.2,

58.9, CH3

58.9, CH3

58.9, CH3

58.3, CH3

58.2, CH3

C

C

C


194.4, 201.2, 115.7, 168.6, 107.6, CH 168.0, 112.8, CH 137.5, CH 130.3, 114.0, CH 151.2,

11 C C C C

C

C

C

156.4, C 118.2, CH 128.8, CH 102.5, CH 76.5, CH 79.5, CH 73.1, CH 78.5, CH 71.8, CH2 Xyl 106.8, CH 76.5, CH 79.0, CH 72.8, CH 68.0, CH2 58.8, CH3

3″-OCH3 4″-OCH3

10 C C C C

194.3, 201.2, 115.7, 168.6, 107.4, CH 168.0, 112.5, CH 137.5, CH 128.7, 113.9, CH 151.2,

12 C C C C

C

C

C


194.8, 201.1, 115.5, 168.0, 107.3, CH 167.8, 112.7, CH 137.3, CH 128.5, 118.6, CH 148.1,

C C C C

C

C

C

155.3, C 118.8, CH 128.3, CH 102.2, CH 76.4, CH 79.4, CH 71.3, CH 78.5, CH 71.7, CH2 Xyl 106.5, CH 76.5, CH 79.0, CH 72.7, CH 67.9, CH2

58.8, CH3

58.5, CH3

3″,4″-OCH2O-

105.9, CH2

δH 7.44 with δC 156.4. Thus, the structure of compound 10 was determined to be 2′,4″-dihydroxy-3″-methoxydibenzoyl-4′-O-βD-xylopyranosyl-(l→6)-β-D-glucopyranoside, and it was named sophodibenzoside J. Compounds 11 and 12 were identified as 2′,4″-dihydroxy-3″methoxydibenzoyl-4′-O-β-D-glucopyranoside (sophodibenzoside K) and 2′,3″,4″-trihydroxydibenzoyl-4′-O-β-D-xylopyranosyl-(l→6)-β-D-glucopyranoside (sophodibenzoside L), respectively. It is worth noting that dibenzoyl compounds are not only potential inhibitors of human carboxylesterases, but are also standard building blocks in the field of organic synthesis.16−19 With the exception of three dibenzoyl aglycones,20−22 no such glycosidic compounds have been isolated from natural sources. The structures of the three known dibenzoyl aglycones were identified based on their NMR spectra in common deuterated solvents. However, for the dibenzoyl glycosides with a C-2′ hydroxy group, standard NMR techniques became problematic. Typical deuterated solvents, such as the dipolar aprotic solvent

105.9, CH2

105.9, CH2

DMSO-d6, protic methanol-d4, aprotic pyridine-d5, acetone-d6, and a mixture of D2O and DMSO-d6, did not allow for the collection of quality NMR spectra: the proton splitting patterns were poorly resolved in the 1H NMR spectrum and some carbon resonances were absent in the 13C NMR spectra (Figures S103−S105 and S110, panels A and B, of the Supporting Information). Acid hydrolysis and methylation were used to clarify the chemical structures of these compounds. The aglycone 1a was thus obtained from 1, while 4a was derived from 4 by acid hydrolysis. NMR spectra of these compounds were then recorded in methanol-d4 and acetone-d6, respectively, but no improvement was observed in the quality of the spectra (Figures S106−S108 of the Supporting Information). Next, the tetra-O-methyl ether of aglycone (1b) was obtained from 1a through methylation with dimethyl sulfate in a solution of KOH and acetone. The NMR spectra of 1b recorded in DMSO-d6 were well-resolved and -integrated (Figure S110C of the Supporting Information). On the basis of its structural features, hydrogen bonding was presumed to be the main 2340



Article

Compound 13 gave a quasi-molecular ion [M + Na]+ at m/z 601.1550 in HRESIMS, responding to a molecular formula of C27H30O14. The UV spectrum showed absorption at 254 and 290 nm. In the 1H NMR spectrum of 13 (Table 4), one typical singlet was observed at δH 8.35 (1H, s, H-2), as was a set of protons in an ABX spin system at δH 7.25 (1H, d, J = 2.0 Hz), 8.07 (1H, d, J = 9.0 Hz), and 7.16 (1H, dd, J = 9.0, 2.0 Hz). Additional resonances were observed at δH 7.08 (1H, s) and 6.98 (2H, br s), along with two sets of sugar resonances with anomeric protons at δH 5.07 (1H, d, J = 7.5 Hz) and 4.82 (1H, d, J = 3.0 Hz). A methoxy group was also observed at δH 3.81 (3H, s). The 13C NMR spectrum of 13 showed 27 carbon resonances (Table 5). All of these resonances were in good agreement with an isoflavone glycoside structure.23 The types and configuration of the sugars were determined by NMR data comparison and GC analysis after acidic hydrolysis and chiral derivatization.14,15 The linkages for these moieties were established based on HMBC experiments, in which the correlations from glc-H-1″(δH 5.07) to C-7 (δC 161.8) suggested that the sugar moiety was located at C-7. The methoxy group was assigned at C-4′ from the correlations −OCH3 (δH 3.81, 3H, s) and H-6′ to C-4′ (δC 148.1). Thus, the structure of 13 was assigned as 3′-hydroxy-4′-methoxyisoflavone-7-O-β-D-apiofuranosyl-(l→6)-β-D-glucopyranoside. The HRESIMS of compound 14 gave a quasi-molecular ion [M + Na]+ at 601.1547, appropriate for a molecular formula of C27H30O14. The IR and UV spectra suggested that 14 was also an isoflavone glycoside. However, comparison between the NMR spectra of 14 and 13 showed two key differences between these compounds (Tables 4 and 5). These differences arose from the interchange of B-ring hydroxy and methoxy group and from the presence of a xylopyranosyl moiety instead

factors influencing the spectroscopic behavior. Thus, the influence of solvents facilitating the breaking of hydrogen bond between the C-2′ hydroxy group and proximal carbonyl groups were examined. In trifluoroacetic acid-d1, the resolution was greatly enhanced in the 1HNMR spectrum. However, the resonances from the xylopyranosyl moiety in 1 disappeared due to hydrolysis by the strong acid trifluoroacetic acid-d1 (Figure S109 of the Supporting Information). Finally, the NMR experiments were conducted in the weak acid, acetic acid-d4. As expected, all the NMR resonances were observed, including the split proton resonances and the aromatic and carbonyl carbon resonances (Figure S110D of the Supporting Information). It is possible that when deuterated acids, such as trifluoroacetic acid-d1 or acetic acid-d4 are employed as solvents, the hydrogen bonds are disrupted and the activated hydrogen of the C-2′ hydroxy group is exchangeable with deuterium. In other solvents, such as DMSO-d6, acetone-d6, methanol-d4, and D2O + DMSO-d6, slow rotation due to hydrogen bonds between the C-2′ hydroxy group and the C-1 and C-2 carbonyls (Figure 2) would give poorly resolved NMR spectra.

Figure 2. The possible solution structures of sophodibenzosides.

Table 4. 1H NMR (500M) Spectroscopic Data (δ in ppm, J in Hz) for Compounds 13−17 in DMSO-d6 position 2 5 6 8 2′ 3′ 5′ 6′ glc-1″ 2″ 3″ 4″ 5″ 6″a 6″b 1‴ 2‴ 3‴ 4‴ 5‴ 3′-OCH3 4′-OCH3 3′,4′-OCH2O-

13

14

15

8.35, 8.07, 7.16, 7.25, 7.08,

s d (9.0) dd (9.0, 2.0) d (2.0) brs

8.40, 8.08, 7.20, 7.29, 7.20,

s d (8.5) dd (8.5, 2.0) d (2.0) d (2.0)

8.39, 8.08, 7.16, 7.25, 7.20,

s d (9.0) dd (9.0, 2.0) d (2.0) d (2.0)

6.98, 6.98, 5.07, 3.32, 3.33, 3.14, 3.66, 3.90, 3.48, api 4.82, 3.80,

brs brs d (7.5) m m m m d (11.5) dd (11.5, 7.5)

6.84, 7.02, 5.05, 3.34, 3.31, 3.20, 3.65, 3.96, 3.61, xyl 4.19, 3.00, 3.09, 3.31,

d (8.5) dd (8.5, 2.0) d (7.0) m m m m d (11.0) dd (11.0, 6.5)

6.84, 7.00, 5.07, 3.31, 3.33, 3.14, 3.66, 3.90, 3.48, api 4.82, 3.80,

d (8.0) dd (8.0, 2.0) d (7.0) m m m m d (9.0) m

16 8.28, 7.91, 6.91, 6.82,

s d (9.0) dd (9.0, 2.0) d (2.0)

17 8.38, 8.04, 7.14, 7.22, 7.15,

s d (9.0) dd (9.0, 2.0) d (2.0) d (2.0)

6.95, s

d (3.0) m

3.95, d (9.5) 3.62, d (9.5) 3.38, s

d (7.5) m m m

6.76, 4.75, 3.10, 3.25, 3.07, 3.30, 3.71, 3.41,

s d (7.5) m t (9.0) t (9.0) m d (10.5) m

d (3.0) m

3.94, d (9.0) 3.62, d (9.0) 3.34, s

3.70, m 2.97, m 3.81, s

6.97, d (8.0) 7.05, dd (8.0, 2.0) 5.04, d (7.0) 3.29, m 3.30, m 3.11, m 3.63, m 3.87, d (10.0) 3.45 m api 4.80, d (3.0) 3.76, m 3.91, d (9.5) 3.58, d (9.5) 3.35, s

3.81, s

3.81, s

3.78, s 6.04, s 2341



Article

Table 5. 13C NMR (125M) Spectroscopic Data (δ in ppm, J in Hz) for Compounds 13−17 in DMSO-d6 position 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ glc-1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 3′-OCH3 4′-OCH3 3′,4′-OCH2O-

13

14

15

16

17

154.0, CH 125.0, C 175.1, C 127.5, CH 115.9, CH 161.8, C 104.1, CH 157.5, C 119.0, C 124.0, C 116.9, CH 146.5, C 148.1, C 112.5, CH 120.2, CH 100.5, CH 73.6, CH 77.0, CH 70.5, CH 76.1, CH 68.3, CH2 api 109.9, CH 76.4, CH 79.1, C 73.8, CH2 63.7, CH2

154.0, CH 124.2, C 175.2, C 127.6, CH 115.9, CH 161.8, C 104.1, CH 157.5, C 119.1, C 123.3, C 113.8, CH 147.7, C 147.1, C 115.7, CH 122.0, CH 100.6, CH 73.6, CH 76.9, CH 70.3, CH 76.3, CH 69.1, CH2 xyl 104.6, CH 73.9, CH 76.9, CH 70.0, CH 66.1, CH2 56.2, CH3

154.0, CH 124.2, C 175.2, C 127.5, CH 115.9, CH 161.8, C 104.1, CH 157.5, C 119.0, C 123.2, C 113.7, CH 147.7, C 147.1, C 115.7, CH 122.0, CH 100.5, CH 73.6, CH 77.0, CH 70.5, CH 76.1, CH 68.3, CH2 api 109.9, CH 76.4, CH 79.1, C 73.8, CH2 63.6, CH2 56.2, CH3

155.8, CH 120.5, C 175.5, C 127.5, CH 116.1, CH 164.4, C 102.6, CH 158.1, C 116.1, C 113.6, C 148.5, C 102.6, CH 148.2, C 141.3, C 118.5, CH 102.9, CH 73.9, CH 77.2, CH 70.5, CH 77.7, CH 61.4, CH2

154.4, CH 126.0, C 175.0, C 127.4, CH 115.9, CH 161.8, C 104.1, CH 157.4, C 119.0, C 123.8, C 109.6, CH 147.4, C 147.5, C 108.7, CH 122.9, CH 100.5, CH 73.6, CH 77.0, CH 70.5, CH 76.1, CH 68.3, CH2 api 109.8, CH 76.4, CH 79.1, C 73.8, CH2 63.7, CH2

56.2, CH3

55.6, CH3 101.5, CH2

glucopyranosyl moiety was confirmed to be at C-2′ by HMBC correlation between glc-H-1″ at δH 4.75 (1H, d, J = 7.5 Hz) and C-2′ (δC 148.5) (Figure 1). The position of the methoxy group was assigned at C-4′ by the correlation of −OCH3 and H-6′ with C-4′. The configuration of the glucopyranosyl group was determined on the basis of coupling constants (J = 7.5 Hz) and GC analysis after acid hydrolysis and chiral derivatization of 16.14,15 Thus, compound 16 was assigned as 5′-hydroxy-4′-methoxyisoflavone-2′-β-D-glucopyranoside. The spectroscopic data of compound 17 indicated that it differed from 13 only in that a methyl was absent, while a methylenedioxyl moiety appeared in 17. Analysis of the 2D NMR data, especially the HMBC correlations from the methylenedioxy to C-3′ (δC 147.4) and C-4′ (δC 147.5), confirmed that the dioxolane moiety was formed between C-3″ and 4″ on ring B. Thus, compound 17 was determined to be 3′,4′-methylenedioxyisoflavone-7-O-β-D-apiofuranosyl-(l→6)β-D-glucopyranoside. The structures of seven known compounds isolated from the roots of S. flavescens were also identified by comparison of their chromatographic and spectroscopic data to the literature data. The known compounds were 3′,4′-dihydroxyisoflavone-7-O-β24 D-glucopyranoside (18), 3′-hydroxy-4′-methoxyisoflavone-7O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside (19),25 4′hydroxyisoflavone-7-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside (20),26 4′-hydroxyisoflavone-7-O-β-D-apiofuranosyl(1→6)-β-D-glucopyranoside (21),27 4′-methoxyisoflavone-7O-β- D -xylopyranosyl-(1→6)-β- D -glucopyranoside (22), 25

of an apiofuranosyl group as the sugar. This assessment was supported by NMR data and GC analysis.14,15 The position of the methoxy group was assigned at C-3′ by the HMBC correlations of −OCH3 (δH 3.81, 3H, s) and H-5′ (δH 6.84, 1H, d, J = 8.5 Hz) with C-3′ (δC 147.7). Thus, compound 14 was assigned as 4′-hydroxy-3′-methoxyisoflavone-7-O-β-D-xylopyranosyl-(l→6)-β-D-glucopyranoside. Compound 15 gave a quasi-molecular ion [M + Na]+ at m/z 601.1546, appropriate for a molecular formula of C27H30O14. The 1H and 13C NMR spectra (Tables 4 and 5) were similar to those of 14. The most noticeable difference in 15 was the presence of an apiofuranosyl moiety and the absence of a xylopyranosyl group (Tables 4 and 5). This was confirmed by HMBC experiments and GC analysis after acid hydrolysis and chiral derivatization.14,15 Thus, 15 was determined to be 4′hydroxy-3′-methoxyisoflavone-7-O-β-D-apiofuranosyl-(l→6)-βD-glucopyranoside. Compound 16 gave a quasi-molecular ion [M + Na]+ at m/z 485.1064 in the HRESIMS, appropriate for a molecular formula of C22H22O11. The IR and UV spectra showed that 16 was an isoflavonoid glucoside. An ABX spin system [δH 6.82 (1H, d, J = 2.0 Hz), 7.91 (1H, d, J = 9.0 Hz), and 6.91 (1H, d, J = 9.0, 2.0 Hz)] present in the 1H NMR spectrum (Table 4), was characteristic of a trisubstituted ring. Aromatic protons at δH 6.95 (1H, s) and 6.76 (1H, s) implied the presence of a tetrasubstituted benzene B-ring. The 13C NMR spectrum showed 22 carbon resonances (Table 5). These data indicated that 16 was distinct from the aforementioned isoflavonoid glycosides in the position of its glucosidic moiety. The 2342



Article

(+)-oxysophocarpine (23),28 (+)-dehydromatrine (24),29 and 1, 2, 3, 4-tetrahydro-β-carboline-3-carboxylic acid (25).30 Some of the compounds were tested for inhibition of the cytotoxic effect of D-galactosamine on the human hepatic cell line HL-7702. Compounds 1, 2, 13, 14, and 19 showed comparable inhibition activities to the positive control bicyclol, a hepatoprotective drug. The results suggest that alkaloids of S. flavescens are not the only active components, but that dibenzoyl and isoflavonoid glycosides also contribute to its activity. Recent advances indicate that different compounds exert their hepatoprotective effects through scavenging oxidative damage31 and antioxidant properties.32

■

separated by preparative RP-HPLC, using MeOH−H2O (60:40) as the mobile phase, to yield 7 (9 mg), 9 (7 mg), 10 (16 mg), and 11 (8 mg). Fraction B was chromatographed over Sephadex LH-20, eluting with MeOH−H2O (1:2) and further purified by preparative RP-HPLC [MeOH−H2O (23:77)] to yield compounds 23 (18 mg), 24 (7 mg), and 25 (12 mg). Sophodibenzoside A (1): yellow powder; [α]20 D -78 (c 0.05, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3355, 2911, 1663, 1608, 1511, 1441, 1274, 1072, 1048, 1022, 764, 636 cm−1; 1H and 13C NMR data, see Tables 1 and 3; ESIMS m/z 605.2 [M + Na]+, 581.3 [M − H]−; HRESIMS m/z 605.1479 [M + Na]+ (calcd for C26H30O15Na, 605.1477). Sophodibenzoside B (2): yellow powder; [α]20 D -55 (c 0.05, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3391, 2933, 1661, 1620, 1511, 1441, 1274, 1070, 1018, 765, 637 cm−1; 1H and 13C NMR data, see Tables 1 and 3; ESIMS m/z 581.0 [M − H]−; HRESIMS m/ z 605.1487 [M + Na]+ (calcd for C26H30O15 Na, 605.1477). Sophodibenzoside C (3): yellow powder; [α]20 D -42 (c 0.05, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3498, 2911, 1659, 1607, 1509, 1445, 1347, 1258, 1189, 1079, 1047, 775, 638 cm−1; 1H and 13C NMR data, see Tables 1 and 3; ESIMS m/z 449.0 [M − H]−; HRESIMS m/z 473.1076 [M + Na]+ (calcd for C21H22O11Na, 473.1054). Sophodibenzoside D (4): yellow powder; [α]20 D -64 (c 0.1, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3506, 3438, 2918, 1667, 1603, 1510, 1369, 1232, 1082, 1045, 1023, 842, 763, 609 cm−1; 1 H and 13C NMR data, see Tables 1 and 3; ESIMS m/z 589.1 [M + Na]+, 565.3 [M − H]−; HRESIMS m/z 589.1539 [M + Na]+ (calcd for C26H30O14 Na, 589.1528). Sophodibenzoside E (5): yellow powder; [α]D20-102 (c 0.02, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax: 3400, 2934, 1658, 1598, 1513, 1426, 1268, 1220, 1170, 1070, 1019, 843, 613 cm−1; 1H and 13C NMR data, see Tables 1 and 3; ESIMS m/z 589.1 [M + Na]+, 565.1 [M − H]−; HRESIMS m/z 589.1546 [M + Na]+ (calcd for C26H30O14Na, 589.1528). Sophodibenzoside F (6): yellow powder; [α]20 D -56 (c 0.05, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3467, 3396, 3193, 1656, 1613, 1600, 1507, 1261, 1231, 1088, 1035, 997, 878, 839, 782, 663, 621 cm−1; 1H and 13C NMR data, see Tables 1 and 3; ESIMS m/ z 433.1 [M − H]−; HRESIMS m/z 457.1119 [M + Na]+ (calcd for C21H22O10 Na, 457.1105). Sophodibenzoside G (7): yellow powder; [α]20 D -62 (c 0.05, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3356, 2918, 1664, 1614, 1502, 1446, 1250, 1099, 1072, 1044, 926, 767, 633 cm−1; 1H and 13 C NMR data, see Tables 2 and 3; ESIMS m/z 603.1 [M + Na]+, 579.3 [M − H]−; HRESIMS m/z 603.1332 [M + Na]+ (calcd for C26H28O15Na, 603.1320). Sophodibenzoside H (8): yellow powder; [α]D20-25 (c 0.025, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3354, 2920, 1663, 1618, 1503, 1447, 1251, 1102, 1070, 1036, 927, 768, 635 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 603.1 [M + Na]+, 579.1[M − H]−; HRESIMS m/z 603.1347 [M + Na]+ (calcd for C26H28O15Na, 603.1320). Sophodibenzoside I (9): yellow powder; [α]D20-32 (c 0.025, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3355, 2917, 1664, 1603, 1504, 1446, 1251, 1000, 1075, 1030, 927, 767, 634 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 471.1 [M + Na]+; HRESIMS m/z 471.0912 [M + Na]+ (calcd for C21H20O11Na, 471.0898). Sophodibenzoside J (10): yellow powder; [α]D20-70 (c 0.05, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3388, 2917, 1626, 1589, 1513, 1430, 1252, 1072, 764, 636 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 581.3 [M − H]−; HRESIMS m/z 605.1461 [M + Na]+ (calcd for C26H30O15Na, 605.1477). Sophodibenzoside K (11): yellow powder; [α]D20-19 (c 0.05, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3356, 2919, 1624, 1589, 1513, 1429, 1253, 1075, 1024, 764, 635 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 473.1 [M + Na] +,

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-2000 polarimeter. UV spectra were obtained on a JASCO V-650 spectrophotometer. IR spectra were recorded on a Nicolet 5700 spectrometer using an FT-IR microscope transmission method. GC experiments were conducted on an Agilent 7890A instrument. NMR spectra were run on INOVA 500 spectrometers. HRESIMS were obtained using an Agilent 1100 series LC/MSD TOF from Agilent Technologies. ESI mass spectra were recorded on an Agilent 1100 series LC/MSD ion trap mass spectrometer. Column chromatography was performed with macroporous resin (Diaion HP-20, Mitsubishi Chemical Corp., Tokyo, Japan), Rp-18 (50 μm, YMC, Kyoto, Japan), Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden), and silica gel (200− 300 mesh, Qingdao Marine Chemical Inc. Qingdao, People’s Republic of China). Preparative HPLC was carried out on a Shimadzu LC-6AD instrument with an SPD-20A detector, using a YMC-Pack ODS-A column (250 × 20 mm, 5 μm, Japan). HPLC-DAD analysis was performed using an Agilent 1260 series system (Agilent Technologies, Waldbronn, Germany) with an Apollo C18 column (250 × 4.6 mm, 5 μm, Grace Davison). Plant Material. The roots of S. flavescens were collected in Weichang, Hebei Province, People’s Republic of China, in July 2010. The plant material was identified by Prof. Lin Ma. A voucher specimen (ID-5-2438) was deposited at the Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing 100050, People’s Republic of China. Extraction and Isolation. The air-dried powdered plant material (40 kg) was extracted twice with 70% EtOH under reflux for 2 h. The resulting crude extract of the plant was extracted sequentially with petroleum ether, EtOAc, and n-butanol. The n-butanol fraction (760 g) was subjected to chromatography on HP-20 macroporous resin, eluting with a step gradient, to give six fractions: A (eluted with H2O, 228 g), B (eluted with 15% EtOH-H2O, 150 g), C (eluted with 30% EtOH-H2O, 75 g), D (eluted with 50% EtOH-H2O, 194 g), E (eluted with 75% EtOH-H2O, 62 g), and F (eluted with 95% EtOH-H2O, 20 g). Fraction C was chromatographed over Sephadex LH-20 with a gradient of MeOH (5−100%) to give fractions C1−C22. Fraction C11 (1.5 g) was separated by reversed-phase (RP) preparative HPLC, using MeOH−H2O (35:65) as the mobile phase, to yield 1 (46 mg), 2 (20 mg), 13 (15 mg), 14 (9 mg), and 15 (7 mg). Fraction C12 (0.8 g) was separated by RP preparative HPLC, using MeOH−H2O (38:62) as the mobile phase, to yield 17 (6 mg), 19 (55 mg), 20 (13 mg), and 21 (9 mg). Fractions C14 (0.9 g), C15 (0.5 g), and C16 (0.7 g) were separated by RP preparative HPLC, using MeOH−H2O (30:70) as the mobile phase, to obtain 4 (15 mg), 5 (40 mg), 12 (8 mg), 16 (9 mg), and 18 (8 mg). Fraction D was subjected to a silica gel column (200− 300 mesh, 1.3 kg) and eluted sequentially with CHCl3 containing increasing amounts of MeOH (1:0, 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, and 0:1) to yield 54 fractions (D1−D54). Compound 22 (500 mg) was obtained by recrystallization in MeOH from fraction D19. Fractions D22−D28 were chromatographed over Sephadex LH-20, eluting with MeOH−H2O (45%) and then separated by preparative RP-HPLC, using MeOH−H2O (43:57) as the mobile phase, to yield 3 (12 mg), 6 (35 mg), and 8 (6 mg). Fractions D29−D33 were 2343



Article

449.2 [M − H]−; HRESIMS m/z 473.1074 [M + Na]+ (calcd for C21H22O11Na, 473.1054). Sophodibenzoside L (12): yellow powder; [α]D20-39 (c 0.05, MeOH); UV (MeOH) λmax 278, 330 nm; IR (KBr) νmax 3344, 2914, 1658, 1614, 1501, 1445, 1294, 1262, 1072, 1048, 1049, 1024, 825, 764, 636 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 591.1 [M + Na]+, 567.1[M − H]−; HRESIMS m/z 567.1347 [M − H]−(calcd for C25H27O15, 567.1355). 3′-Hydroxy-4′-methoxyisoflavone-7-O-β-D-apiofuranosyl-(1→6)β-D-glucopyranoside (13): yellow powder; [α]D20-120 (c 0.001, MeOH); UV (MeOH) λmax 254, 290 nm; IR (KBr) νmax 3351, 2922, 1623, 1512, 1443, 1266, 1199, 1071, 1024, 852, 825, 763, 661 cm−1; 1H and 13C NMR data, see Tables 4 and 5; ESIMS m/z 601.2 [M + Na]+, 577.1 [M − H]−; HRESIMS m/z 601.1550 [M + Na]+ (calcd for C27H30O14Na, 601.1528). 4′-Hydroxy-3′-methoxyisoflavone-7-O-β-D-xylopyranosyl-(1→6)β-D- glucopyranoside (14): yellow powder; [α]20 D -73 (c 0.03, MeOH); UV (MeOH) λmax 254, 290 nm; IR (KBr) νmax 3364, 2905, 1623, 1516, 1445, 1264, 1199, 1072, 1048, 1062, 850, 825, 764, 660 cm−1; 1 H and 13C NMR data, see Tables 4 and 5; ESIMS m/z 601.2 [M + Na]+; HRESIMS m/z 601.1547 [M + Na]+ (calcd for C27H30O14Na, 601.1528). 4′-Hydroxy-3′-methoxyisoflavone-7-O-β-D-apiofuranosyl-(1→6)β-D- glucopyranoside (15): yellow powder; [α]20 D -70 (c 0.03, MeOH); UV (MeOH) λmax 254, 290 nm; IR (KBr) νmax 3371, 2932, 1622, 1515, 1445, 1263, 1200, 1072, 1045, 851, 636, 610 cm−1; 1H and 13C NMR data, see Tables 4 and 5; ESIMS m/z 579.2 [M + H]+; HRESIMS m/z 601.1546 [M + Na]+ (calcd for C27H30O14Na, 601.1528). 5′-Hydroxy-4′-methoxyisoflavone-2′-β-D-glucopyranoside (16): yellow powder; [α]20 D -20 (c 0.03, MeOH); UV (MeOH) λmax 254, 290 nm; IR (KBr) νmax 3348, 2922, 1622, 1512, 1453, 1288, 1197, 1095, 1074, 1045, 836, 786, 634 cm−1; 1H and 13C NMR data, see Tables 4 and 5; ESIMS m/z 461.4 [M − H]−; HRESIMS m/z 485.1064 [M + Na]+ (calcd for C22H22O11Na, 485.1054). 3′,4′-Methylenedioxyisoflavone-7-O-β-D-apiofuranosyl-(1→6)-β20 D-glucopyranoside (17): yellow powder; [α]D -69 (c 0.02, MeOH); UV (MeOH) λmax 254, 290 nm; IR (KBr) νmax 3382, 2919, 2885, 1621, 1502, 1441, 1250, 1197, 1069, 1041, 926, 855, 815, 652 cm−1; 1 H and 13C NMR data, see Tables 4 and 5; ESIMS m/z 577.2 [M + H]+; HRESIMS m/z 599.1383 [M + Na]+ (calcd for C27H28O14Na, 599.1371). Determination of Absolute Configuration of Sugars. Compounds 1−17 were processed and analyzed based on the reported method.14,15 The products were subjected to GC analysis under the following conditions: capillary column, HP-5 (60 m × 0.25 mm, with a 0.25 μm film, Dikma); detection, FID; detector temperature, 280 °C; injection temperature, 260 °C; initial temperature 160 °C, raised to 280 °C at 5 °C/min and final temperature maintained for 10 min; and carrier N2 gas. From the acid hydrolysates of 1−17, the presence of D-glucose, D-xylose, and D-apiose was confirmed by comparing the retention times of their derivatives with those of authentic sugars. These standards showed the same retention times: 24.7, 15.0, and 15.8 min, respectively. Inhibition of the Cytotoxic Effect Assay. Inhibition of the cytotoxic effect of D-galactosamine on HL-7702 was measured using the procedure described previously,15 according to the standard MTT method33 in HL-7702 cells (Table 6). A hepatoprotective drug bicyclol was designed as positive control substance. Inhibition (%) was obtained by the following formula:

Table 6. Hepatoprotective Effects against D-Galactosamine Induced Toxicity in HL-7702 cellsa cell survival rate (% of normal)

inhibition (% of control)

± ± ± ± ± ± ± ±

29.4 27.1 52.4 36.6 45.8 46.3

normal control bicyclolb 1 2 13 14 19

100.0 46.8 62.5 61.3 74.7 66.3 71.2 71.5

2.9 8.7 6.9c 4.2d 6.5e 3.5e 3.4e 5.1e

The compounds were tested at 1 × 10−5 M. Results are expressed as means ± SD (n = 5). bPositive control substance (Beijing Union Pharmaceutical Factory, purity is 98% by HPLC). cp < 0.01. dp < 0.05. e p < 0.001. a

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ASSOCIATED CONTENT

S Supporting Information *

IR, HRESIMS, and NMR spectra for compounds 1−17. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-63165231. Fax: +86- 10-63017757. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research described in this publication was supported by the National Science and Technology Project of People’s Republic of China (Grant 2011ZX09307-002-01).

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REFERENCES

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Inhibition (%) = [(OD(sample) − OD(control)) /(OD(normal) − OD(control))] × 100 Statistical Analysis. Student’s t test for unpaired observations between normal or control and tested samples was carried out to identify significant differences; p values less than 0.05 were considered as significantly significant. 2344



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Dibenzoyl and Isoflavonoid Glycosides from Sophora flavescens

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