Hepatoprotective Iridoid Glycosides from the Roots of Rehmannia

Aug 23, 2012 - Min-Beom Kim , Chulwon Kim , Won-Seok Chung , Jae-Heung Cho , Dongwoo Nam , Sung-Hoon Kim , Kwang Seok Ahn. Phytotherapy ...
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Hepatoprotective Iridoid Glycosides from the Roots of Rehmannia glutinosa Yan-Fei Liu, Dong Liang, Huan Luo, Zhi-You Hao, Yan Wang, Chun-Lei Zhang, Qing-Jian Zhang, Ruo-Yun Chen, and De-Quan Yu* 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: Eleven new iridoid glycosides, rehmaglutosides A− K (1−11), together with 20 known analogues were isolated from the air-dried roots of Rehmannia glutinosa. The structures of these compounds were elucidated on the basis of spectroscopic data analysis and chemical evidence. Furthermore, in in vitro assays, compounds 3, 7, and 9−11 (10 μM) exhibited moderate hepatoprotective activities against D-galactosamine-induced HL7702 cell damage.

Rehmannia glutinosa Libosch (Scrophulariaceae) is indigenous to Mainland China, and the roots of this plant have been used in oriental medicine as an antianemic, an antipyretic, and a tonic.1 Due to the different processing methods, R. glutinosa is classified into three categories, namely, fresh roots, dried roots, and steamed roots, which are used in different ways in traditional Chinese medicine. Previous phytochemical studies on the dried and steamed roots of R. glutinosa have led to the isolation and identification of iridoid glycosides, ionone glycosides, phenethyl alcohol glycosides, and several other components.2 As part of our program to study traditional Chinese medicines, an ethanolic extract of the air-dried roots of R. glutinosa was investigated. Eleven new iridoid glycosides, rehmaglutosides A−K (1−11), together with 20 known analogues, were isolated from this extract. In this paper, we report the isolation and structural elucidation of these new iridoid glycosides. Furthermore, their cytotoxic and hepatoprotective activities are reported herein.

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additional cinnamoyl group. The C-5 and C-7 upfield shifts (δ 39.3 from δ 41.1; δ 47.8 from δ 49.9) and the C-6 downfield shift (δ 80.5 from δ 78.0) suggested the attachment of the cinnamoyl group to the C-6 carbon. The assumption was confirmed by the HMBC correlation of H-6 (δ 4.95) with the ester carbonyl (δ 168.4) of the cinnamoyl group. The relative configuration of 1 was established by analysis of the NOESY spectrum and coupling constants. The cinnamoyl group was assigned an E configuration from the large coupling constant between H-7″ and H-8″ (J = 16.0 Hz) in the 1H NMR spectrum. In the NOESY spectrum, correlations between H-9 and H-5/H-7β indicated that H-5 and H-9 are both β-oriented. Moreover, NOESY correlations between CH3-10 and H-7α/H6/H-1 confirmed that H-1, H-6, and CH3-10 are all α-oriented. Acid hydrolysis of 1 afforded D-glucose, which was identified by TLC comparison with an authentic sample, and the configuration was determined by measurement of the optical rotation value. The β-anomeric configuration for the glucosyl unit was judged from its large 3JH1,H2 coupling constant (J = 8.5 Hz). Analysis of the HMBC and HSQC spectra led to the complete assignments of the proton and carbon signals in compound 1 (Tables 1 and 3). Therefore, rehmaglutoside A was characterized as 1. The molecular formula of compound 2 was determined as C29H40O17 from the HRESIMS (m/z 683.2157 [M + Na]+). Comparison of the spectroscopic data of 2 with those of 1 showed they were almost superimposable on those of the iridoid skeleton, but the (E)-cinnamoyl group at C-6 in 1 was

RESULTS AND DISCUSSION

Compound 1 was obtained as an amorphous powder with a specific rotation of [α]20D −138.1. Its molecular formula, C24H30O10, was deduced by HRESIMS (m/z 501.1731 [M + Na]+). The IR spectrum showed absorption bands for hydroxy (3398 cm−1) and carbonyl (1705 cm−1) groups. The 13C NMR spectrum showed 24 carbon signals including six for a glucopyranosyl unit, nine for a cinnamoyl group, and the remaining nine for an iridoid skeleton. The 1H and 13C NMR data of 1 displayed signals characteristic of an iridoid glycoside, which were similar to those reported for ajugol.2c The only evident difference was that 1 showed resonances due to an © 2012 American Chemical Society and American Society of Pharmacognosy

Received: July 22, 2012 Published: August 23, 2012 1625

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together with HMBC correlations of H-8″ with C-9″/C-7″, H-7″ with C-9″/C-8″/C-3″, H-3″ with C-5″/C-7″, H-5″ with C-6″/C-3″, and H-6″ with C-2″/C-4″/C-5″ revealed the presence of a 3-(4-hydroxypyridin-2-yl)propanoic acid moiety. Furthermore, the HMBC correlation from H-6 to C-9″ verified the location of the ester group at C-6 of the ajugol moiety. From these data, compound 4 (rehmaglutoside D) was characterized as shown. Compound 5, a white powder, had the molecular formula C34H40O15 from its positive-mode HRESIMS (m/z 711.2268 [M + Na]+). The 13C NMR spectrum displayed 34 signals, of which 15 could be attributed to the ajugol moiety, and the other 19 to a lignan moiety (including a methoxy group). The 1 H NMR spectrum of the lignan moiety exhibited a large coupling constant between the protons at δ 7.59 (1H, d, J = 15.6 Hz) and 6.33 (1H, d, J = 15.6 Hz), showing the trans configuration for the double bond, a pair of meta-coupled aromatic protons [δ 7.05 (br s), 6.97 (d, J = 1.2 Hz)], and an ABX coupling system [δ 6.96 (1H, d, J = 1.8 Hz, H-2″), 6.83 (1H, dd, J = 7.8, 1.8 Hz, H-6″), and 6.76 (1H, d, J = 7.8 Hz, H5″)], indicating there were two benzene rings. The spectroscopic data of the lignan moiety were very similar to those of morindolin.3b In the HMBC spectrum, a correlation of H-6/C-9‴ revealed the presence of a lignan moiety attached to C-6 of the ajugol moiety as an ester, and a correlation of OMeC-3″/C-3″ indicated that the methoxy group is present at C-3″ of the lignan moiety. From the coupling constant of J7″,8″ = 6.6 Hz, the relative configuration of C-7″ and C-8″ in the furan ring was trans. On the basis of the reversed helicity rule of the 1Lb band CD for the 7-methoxy-2,3-dihydrobenzo[b]furan chromophore, a negative Cotton effect at 267 nm in the CD spectrum of 5 indicated that it had a 7R″, 8S″ configuration.3a From these data, compound 5 (rehmaglutoside E) was characterized as shown. Compound 6 gave a molecular formula of C30H46O12 as established by HRESIMS (m/z 621.2889 [M + Na]+). The 13C NMR spectrum of 6 showed 30 signals, of which 15 could be attributed to the ajugol moiety, and the other 15 to a norcarotenoid moiety.2f The 1H NMR spectrum of the norcarotenoid moiety of 6 showed an olefinic proton signal at δ 5.77 (1H, s), trans-olefinic proton signals at δ 6.37 and 6.67 (each 1H, d, J = 16.0 Hz), and an olefinic methyl proton signal at δ 2.27 (3H, s), which were in good agreement with those of aeginetic acid. The 1H NMR spectrum of the cyclohexyl end group of 6 exhibited three singlet methyl signals at δ 0.82, 1.09, and 1.22 (each 3H, s), and its 13C NMR spectrum showed two quaternary carbon signals carrying the hydroxy groups at δ 77.9 and 82.9. Detailed 2D-NMR analysis was used to assign the norcarotenoid moiety as aeginetic acid.2f In the HMBC spectrum, a correlation from H-6 (δ 4.80) to the ester carbonyl at δ 168.7 indicated that the norcarotenoid ester group is present at C-6 of the ajugol moiety. ROESY correlations of H4″β/H-3″β and Me-15″, and H-13″α/H-3″α and H-7″, indicated that the hydroxy groups at C-5″ and C-6″ are αand β-oriented, respectively. The CD spectrum of 6 exhibited similar Cotton effects (positive at 225 nm and negative at 266 nm, Supporting Information, Figure S50) to that of sechydroxyaeginetic acid,2f indicating that the asymmetric centers at C-5″ and C-6″ are both in the R configuration. On the basis of the above data, rehmaglutoside F was characterized as 6. Compound 7 gave a molecular formula of C30H46O13 by HRESIMS at m/z 637.2841 [M + Na]+, 16 mass units higher than that of 6, in accordance with the presence of an additional

replaced by a vanilloyl group in 2,2c and there was an additional glucopyranosyl unit [δ 5.01 (1H, d, J = 7.2 Hz)]. In the HMBC spectrum, a correlation from H-6 (δ 5.05) to C-7″ (δ 167.5) assigned the vanilloyl group to C-6 of the ajugol moiety. Furthermore, a HMBC correlation of H-1‴ (δ 5.01) with C-4″ (δ 152.2) was used to locate the additional glucopyranosyl unit at C-4″ of the vanilloyl group. Thus, rehmaglutoside B was characterized as 2. Compound 3 was assigned a molecular formula of C30H42O17 from its HRESIMS (m/z 697.2327 [M + Na]+). Its NMR data were very similar to those of 2 except for evidence of the presence of a syringic group, instead of a vanilloyl group at C-6 in 2, and the glucopyranosyl unit located at C-4″ was replaced by a rhamnopyranosyl unit [δ 5.34 (1H, d, J = 1.5 Hz), 1.19 (3H, d, J = 6.0 Hz)].2c The position of the syringic group was confirmed by HMBC correlation of H-6 (δ 5.06) with the carbonyl of the syringic group (δ 167.3) and that of the rhamnopyranosyl group by a cross-peak between H-1‴ (δ 5.34) and C-4″ (δ 140.0) of the syringic group. On the basis of the reported procedure,11 the absolute configurations of the glucose and rhamnose moiety were determined as D-glucose and L-rhamnose, respectively. Hence, rehmaglutoside C was characterized as 3. Compound 4 was isolated as an amorphous powder, and its molecular formula was established as C23H31NO11 on the basis of HRESIMS (m/z 520.1804 [M + Na]+). The 1H NMR and 13 C NMR spectra of 4 (Tables 1 and 3) resembled those of 1; however, the (E)-cinnamoyl group was replaced by a 3-(4hydroxypyridin-2-yl)propanoic acid moiety in 4. The 1D NMR data exhibited an ABX signal pattern [δ 6.33 (1H, br s, H-3″), 6.29 (1H, dd, J = 6.5, 1.5 Hz, H-5″), and 7.30 (1H, d, J = 6.5 Hz, H-6″)], a carbonyl group (δ 168.9), and two methylene signals at δ 2.78 (2H, t, J = 8.0 Hz, H-7″) and 2.62 (2H, t, J = 8.0 Hz, H-8″). 1H−1H COSY correlation of H-7″/H-8″ 1626

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Table 1. 1H NMR Spectroscopic Data of Compounds 1−5a position 1 3 4 5 6 7α 7β 9 10 1′ 2′ 3′ 4′ 5′ 6′a 6′b 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″a 9″b OMe 1‴ 2‴ 3‴ 4‴ 5‴ 6‴a 6‴b 7‴ 8‴

1 5.50, 6.22, 4.97, 2.93, 4.95, 2.24, 1.99, 2.58, 1.38, 4.67, 3.19, 3.37, 3.26, 3.30, 3.88, 3.66, 7.58, 7.38, 7.38, 7.38, 7.58, 7.68, 6.53,

d (2.5) dd (6.0, 2.5) m dd (9.0, 2.5) m dd (14.5, 7.0) dd (14.5, 4.0) dd (9.0, 2.5) s d (8.5) dd (9.0, 8.0) t (8.0) m m dd (12.0, 2.0) dd (12.0, 6.0) m m m m m d (16.0) d (16.0)

2 5.51, 6.23, 4.99, 2.99, 5.05, 2.28, 2.04, 2.63, 1.40, 4.65, 3.18, 3.36, 3.25, 3.30, 3.88, 3.64, 7.63,

d (2.4) dd (6.0, 2.4) dd (6.0, 3.0) dd (9.0, 1.8) m dd (14.4, 6.0) dd (14.4, 3.6) dd (9.6, 2.4) s d (8.0) dd (9.0, 7.5) t (9.0) br t (9.0) m m dd (12.0, 6.0) d (1.8)

3 5.51, 6.24, 4.99, 3.02, 5.06, 2.28, 2.05, 2.62, 1.41, 4.66, 3.18, 3.36, 3.27, 3.30, 3.89, 3.65, 7.36,

d (2.5) dd (6.5, 2.0) dd (6.5, 2.5) dd (9.0, 2.0) m dd (14.5, 6.0) dd (14.5, 3.5) dd (9.0, 2.5) s d (8.0) dd (9.5, 8.0) t (8.5) m m m dd (12.0, 6.0) s

4 5.44, 6.12, 4.82, 2.72, 4.76, 2.09, 1.82, 2.41, 1.29, 4.59, 3.15, 3.31, 3.22, 3.24, 3.82, 3.61,

d (2.5) dd (6.5, 2.0) m dd (9.0, 2.0) m dd (14.0, 6.5) dd (14.0, 4.0) dd (9.0, 2.0) s d (8.0) dd (9.0, 8.0) m m m dd (12.0, 1.5) dd (12.0, 5.5)

5 5.49, 6.20, 4.96, 2.92, 4.91, 2.23, 2.00, 2.57, 1.37, 4.66, 3.19, 3.37, 3.27, 3.30, 3.89, 3.66, 6.96,

d (2.4) dd (6.6, 2.4) dd (6.6, 3.0) br dd (9.0, 2.4) m dd (14.4, 6.0) dd (14.4, 3.6) dd (9.0, 2.4) s d (8.0) t (9.0) t (9.0) m m dd (12.0, 1.8) dd (12.0, 6.0) d (1.8)

6.76, 6.83, 5.56, 3.51, 3.83, 3.81, 3.80,

d (7.8) dd (7.8, 1.8) d (6.6) m m m s

6.33, s 7.19, d (8.4) 7.66, dd (8.4, 1.8)

3.88, 5.01, 3.52, 3.47, 3.39, 3.45, 3.86, 3.67,

s d (7.2) dd (9.0, 7.8) t (9.0) t (9.0) m m dd, (12.0, 6.0)

7.36, s

3.87, 5.34, 4.12, 3.90, 3.43, 4.23, 1.19,

s 6H d (1.5) m m t (9.5) dd (9.5, 6.0) d (6.0)

6.29, 7.30, 2.78, 2.62,

dd (6.5, 1.5) d (6.5) t (8.0) t (8.0)

6.97, d (1.2)

7.05, s 7.59, d (15.6) 6.33, d (15.6)

a1

H NMR data (δ) were measured in methanol-d4 at 500 MHz for 1, 3, 4, 6, and 8−11 and at 600 MHz for 2, 5, and 7. Coupling constants (J) in Hz are given in parentheses. The assignments were based on 1H−1H COSY, NOESY(ROESY), HSQC, and HMBC experiments.

confirmed by an HMBC correlation between H-4″ (δ 3.60) and C-15″ (δ 22.5) together with an 1H−1H COSY correlation between H-4″ and H-3″ (δ 1.51, 1.81). The HMBC correlation from H-6 (δ 4.80) to the ester carbonyl (δ 168.7) demonstrated that the norcarotenoid ester group is connected to C-6. ROESY correlations of H-4″/H-3″β and Me-15″, H2″β/H-14″ and H-3″β, and H-13″/H-2″α, H-3″α, and H-7″ suggested that the hydroxy groups at C-4″, C-5″, and C-6″ are α-, α-, and β-oriented, respectively. The CD spectrum of 8 exhibited similar Cotton effects (positive at 226 nm and negative at 268 nm, Supporting Information, Figure S68) to those seen for 6, indicating that the asymmetric centers of 8 at C-4″, C-5″, and C-6″ are all the R-configured. Thus, rehmaglutoside H was characterized as 8. Compound 9 was shown to have the molecular formula C36H56O17, by HRESIMS (m/z 783.3411 [M + Na]+). The spectroscopic data of 9 (Tables 2 and 3) were very similar to those of 6. The position of the norcarotenoid ester group was determined to be at C-6 of the ajugol moiety, based on the HMBC correlation of H-6/C-11″. Comparison of the NMR data of 9 and 6 indicated the presence of an additional glucopyranosyl unit. In addition, the 13C NMR shift of C-5″ of

hydroxy group. The spectroscopic data of 7 (Tables 2 and 3) were similar to those of 6, except for a secondary hydroxy group located at C-3″ of the norcarotenoid moiety. This was confirmed by HMBC correlations from H-3″ to C-1″and C-5″, together with 1H−1H COSY correlations between H-3″ and H4″/H-2″. The position of the norcarotenoid ester group was determined to be at C-6 of the ajugol moiety, based on a HMBC correlation for H-6/C-11″. ROESY correlations of H13″/H-2″α, H-3″, and H-7″, H-3″/H-2″α and H-4″α, and Me15″/H-4″β, H-7″, and H-8″ indicated that the hydroxy groups at C-3″, C-5″, and C-6″ are β-, α-, and β-oriented, respectively. The CD spectrum of 7 exhibited similar Cotton effects (positive at 228 nm and negative at 267 nm, Supporting Information, Figure S59) to those of 6, indicating that the asymmetric centers of 7 at C-3″, C-5″, and C-6″ are S-, R-, and R-configured, respectively. Consequently, rehmaglutoside G was characterized as 7. Compound 8 exhibited the same molecular formula C30H46O13 as 7, as established by HRESIMS at m/z 637.2834 [M + Na]+. The NMR spectroscopic data of 8 were very similar to those of 7 except that the position of the secondary hydroxy group was located at C-4″ instead of at C-3″. This was 1627

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Table 2. 1H NMR Spectroscopic Data of Compounds 6−11a position 1 3 4 5 6 7α 7β 9 10 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″α 2″β 3″α 3″β 4″α 4″β 5″ 6″ 7″α 7″β 8″ 9″ 10″ 12″ 13″ 14′ 15″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴a 6‴b

6

7

8

9

10

5.43, 6.15, 4.92, 2.81, 4.80, 2.16, 1.90, 2.48, 1.31, 4.60, 3.13, 3.31, 3.22, 3.24, 3.84, 3.60,

d (2.5) dd (6.5, 2.0) dd (6.0, 2.5) dd (9.0, 2.5) m dd (14.0, 6.5) dd (14.0, 4.5) dd (9.5, 2.0) s d (8.0) dd (9.0, 8.0) t (8.5) m m dd (12.0, 2.0) dd (12.0, 5.5)

5.47, d (2.4) 6.20, dd (6.5, 2.4) 4.98,dd (6.5, 3.0) 2.86,dd (9.0, 2.4) 4.85, m 2.21, dd (13.8, 6.0) 1.94, dd (13.8, 3.6) 2.52,dd (9.0, 1.8) 1.36, s 4.66, d (8.4) 3.19, dd (9.0, 7.8) 3.36, t (9.0) 3.27, m 3.29, m 3.87, dd (12.0, 1.8) 3.66, dd (12.0, 6.0)

5.43, 6.15, 4.92, 2.81, 4.80, 2.16, 1.90, 2.48, 1.31, 4.59, 3.13, 3.31, 3.20, 3.24, 3.84, 3.60,

d (2.5) dd (6.5, 2.0) dd (6.5, 2.5) dd (9.0, 2.5) m dd (14.0, 6.5) dd (14.0, 4.0) dd (9.0, 2.0) s d (8.0) dd (9.0, 8.0) t (8.5) m m dd (12.0, 1.5) dd (12.0, 5.5)

5.43, 6.15, 4.92, 2.81, 4.80, 2.16, 1.90, 2.48, 1.31, 4.60, 3.15, 3.31, 3.23, 3.24, 3.84, 3.60,

d (2.5) dd (6.5, 2.0) dd (6.5, 2.5) dd (9.0, 2.5) m dd (14.0, 6.5) dd (14.0, 4.5) dd (9.5, 2.0) s d (8.0) m t (8.5) m m dd (12.0, 1.5) dd (12.0, 5.5)

1.13, 1.63, 1.83, 1.31, 1.74, 1.41,

m m m m m m

1.65, m 1.47, m 4.08, m

1.74, 1.15, 1.81, 1.51, 3.60,

m m m m m

1.09, 1.63, 2.07, 1.22, 1.69, 1.59,

m m m m m m

1.78, m 1.74, m

6.67, d (16.0)

6.69, d (16.2)

6.67, d (16.0)

6.75, d (16.0)

6.37, d (16.0)

6.42, d (16.2)

6.33, d (16.0)

6.33, d (16.0)

5.77, 2.27, 1.15, 0.73, 1.00,

5.84, 2.31, 1.22, 0.82, 1.09,

5.79, 2.26, 1.13, 0.71, 1.08,

5.76, 2.26, 1.14, 0.76, 1.11, 4.40, 3.18, 3.29, 3.23, 3.16, 3.74, 3.55,

s s s s s

s d (1.2) s s s

s s s s s

s s s s s d (8.0) m t (9.0) m m dd (11.5, 2.0) dd (12.0, 6.0)

5.35, 6.11, 4.81, 2.68, 3.86, 1.97, 1.74, 2.51, 1.26, 4.59, 3.15, 3.31, 3.35, 3.38, 4.02, 3.74, 5.11,

d (2.0) dd (6.5, 2.0) m m m m m m s d (8.0) m m m m dd (11.5,4.0) d (11.5) d (5.0)

11 5.33, 6.11, 4.79, 2.68, 3.86, 1.96, 1.74, 2.50, 1.25, 4.56, 3.13, 3.31, 3.18, 3.39, 4.10, 3.60, 5.35,

d (2.0) dd (6.0, 2.0) m m m dd (13.5, 5.5) m m s d (8.0) m m m m dd (12.0, 1.5) m d (3.5)

4.85, m

5.00, m

1.96, 1.89, 2.54, 3.92, 1.97, 1.74,

1.35, 1.87, 2.43, 3.94, 2.11, 1.74,

m m m m m m

m m m m dd (14.0, 6.0) m

2.18, dd (9.0, 5.0) 1.26, s

2.22, dd (8.5, 3.0) 1.29, s

4.68, 3.17, 3.37, 3.20, 3.25, 3.85, 3.56,

4.65, 3.16, 3.32, 3.21, 3.29, 3.84, 3.59,

d (8.0) m m m m m m

d (8.0) m m m m m m

a1 H NMR data (δ) were measured in methanol-d4 at 500 MHz for 1, 3, 4, 6, and 8−11 and at 600 MHz for 2, 5, and 7. Coupling constants (J) in Hz are given in parentheses. The assignments were based on 1H−1H COSY, NOESY(ROESY), HSQC, and HMBC experiments.

and 3) of 10 exhibited signals for two sets of C9-iridoid glycosides; one unit was determined to be ajugol,2c and another an analogue of ajugol. The NMR data of the latter unit were very similar to those of ajugol except for another acetal carbon (δ 98.7, C-3″) and a methylene (δ 29.9, C-4″) instead of an olefinic carbon (C-3, 4). The NMR signal of C-6′ of 10 was deshielded significantly by comparison with those of ajugol. This indicated that the second unit is located at C-6′ through the connectivity of C-6′-O-C-3″, which was verified by correlations from H-6′ to C-3″ and from H-3″ to C-6′ in the HMBC spectrum of 10. In the ROESY spectrum, correlations between H-5″ and H-9″/H-4″β indicated that H-5″ and H-9″ are both β-oriented. In turn, ROESY correlations between CH3-10″ and H-7″α/H-6″/H-1″ confirmed that H-1″, H-6″, and CH3-10″ are all α-oriented. Regarding the configuration of H-3″, the ROESY correlation between H-3″ and H-1″ implied

9 was significantly deshielded by comparison with that of 6. This suggested that the glucopyranosyl unit is located at C-5″, which was verified by a correlation from H-1‴ to C-5″ in the HMBC spectrum. ROESY experiments indicated the relative stereochemistry for 9 to be the same as that of 6. The CD spectrum of 9 exhibited similar Cotton effects (positive at 228 nm and negative at 266 nm, Supporting Information, Figure S77) to those seen for 6, showing that the asymmetric centers of 9 at C-5″ and C-6″ are in the R configuration. The structure of this compound was confirmed by detailed analysis of the 2DNMR data including the HSQC, HMBC, 1H−1H COSY, and ROESY spectra. Accordingly, rehmaglutoside I was characterized as 9. Compound 10 exhibited a [M + Na]+ ion peak at m/z 719.2739 in its HRESIMS, corresponding to the molecular formula C30H48O18. The 1H and 13C NMR spectra (Tables 2 1628

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Table 3. 13C NMR Spectroscopic Data of Compounds 1−11a position

1

2

1 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″ 14″ 15″ OMe 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴

93.4 141.1 104.5 39.3 80.5 47.8 79.1 51.6 26.0 99.3 74.7 77.9 71.6 78.2 62.8 135.7 129.2 130.0 131.5 130.0 129.2 146.3 119.1 168.4

93.4 141.2 104.4 39.5 80.9 47.7 79.1 51.7 26.2 99.4 74.8 78.3 71.7 77.9 62.8 125.7 114.2 150.3 152.2 116.3 124.6 167.5

93.4 141.2 104.4 39.5 81.2 47.6 79.2 51.8 26.2 99.3 74.7 77.9 71.7 78.2 62.8 127.4 107.8 154.5 140.0 154.5 107.8 167.3

56.7 101.8 74.7 78.2 71.2 77.8 62.4

56.6 × 2 103.4 72.0 72.1 73.6 71.3 17.9

3

4

5

6

7

8

9

10

11

93.7 141.2 104.4 39.3 80.7 47.7 79.0 51.6 26.0 99.4 74.8 78.0 71.7 78.2 62.8

93.4 141.0 104.5 39.3 80.3 47.8 79.1 51.6 26.0 99.3 74.7 77.9 71.6 78.1 62.8 134.3 110.4 149.0 147.5 116.1 119.7 89.5 55.0 64.7

93.3 140.9 104.7 39.3 79.7 47.9 79.1 51.5 25.9 99.3 74.7 78.0 71.7 78.2 62.8 39.9 37.3 18.7 36.7 75.8 80.7 140.3 134.2 154.8 119.0 168.7 14.4 25.7 27.4 27.3

93.3 140.9 104.7 39.2 79.7 47.9 79.0 51.5 25.9 99.3 74.7 78.1 71.6 77.8 62.8 41.2 46.3 65.1 45.6 77.9 79.9 139.9 134.7 154.2 119.3 168.6 14.4 26.3 27.5 27.2

93.4 140.9 104.7 39.3 79.7 47.9 79.1 51.5 25.9 99.3 74.8 78.0 71.7 78.2 62.8 39.5 36.1 27.8 73.4 77.6 82.0 140.2 133.9 154.8 118.7 168.6 14.4 25.5 27.0 22.5

93.3 140.9 104.7 39.3 79.6 47.9 79.1 51.5 25.9 99.3 74.7 77.9 71.6 78.2 62.8 39.9 37.1 18.7 32.4 83.7 80.4 141.1 133.6 154.8 118.7 168.7 14.5 25.9 27.6 22.6

93.8 140.4 105.9 41.4 77.7 49.9 79.5 51.7 25.3 99.3 74.7 77.8 71.0 76.1 68.2 96.1

93.8 140.4 105.8 41.4 77.9 49.9 79.5 51.9 25.3 99.3 74.7 78.0 71.9 77.1 69.6 95.2

98.7 29.9 42.7 76.1 49.9 79.5 53.0 25.9

98.3 31.8 43.9 77.2 50.3 79.6 51.8 27.0

98.2 75.4 79.0 71.9 77.4 63.1

99.3 75.0 77.8 71.7 78.1 62.7

99.2 74.9 78.0 71.8 78.0 62.9

165.8 118.8 158.4 109.9 135.4 31.5 34.5 173.7

56.3 129.5 116.9 142.7 151.1 130.9 117.9 146.9 115.7 168.9

a13 C NMR data (δ) were measured in methanol-d4 at 125 MHz for 1, 3, 4, 6, and 8−11 and at 150 MHz for 2, 5, and 7. The assignments were based on 1H−1H COSY, NOESY(ROESY), HSQC, and HMBC experiments.

that H-3″ is α-oriented. The structure of this compound was confirmed by detailed analysis of the 2D-NMR data including its HSQC, HMBC, 1H−1H COSY, and ROESY spectra. Therefore, compound 10 (rehmaglutoside J) was characterized as shown. Compound 11 was found to have the same molecular formula, C30H48O18, as 10, as established by its HRESIMS (m/z 719.2728 [M + Na]+). The spectroscopic data of 11 (Tables 2 and 3) indicated that it is an epimer of 10. Regarding the configuration of H-3″, the ROESY correlation between H-3″ and H-5″ implied that H-3″ is β-oriented. The structure of 11 (rehmaglutoside K) was therefore established as shown. The known iridoid glycosides were identified as aucubin,8 myobontioside A,12 catalpol,2b ajugol,2c geniposide,2b 6-O-Eferuloyl ajugol,2c 8-epiloganin,5 gardoside methyl ester,5 geniposidic acid,6 6-O-p-hydroxybenzoyl ajugol,2c 6-O-vanillate ajugol,2c jioglutoside B,2b 8-epiloganic acid,2c 6-O-E-caffeoyl

ajugol,7 6-O-(4″-O-α-L-rhamnopyranosyl)vanilloyl ajugol,2c monomellittoside,2d 6-O-sec-hydroxyaeginetoyl ajugol,2e mussaenosidic acid,9 3′-O-β-D-glucopyranosyl catalpol,10 and genipin 1-O-α-L-rhamnopyranosyl-(1→6)-β-D- glucopyranoside,11 by NMR analysis and comparison with literature data. Compounds 6−9, together with 6-O-sec-hydroxyaeginetoyl ajugol, are interesting examples composed of iridoid glycoside and sesquiterpenoid or sesquiterpenoid glycoside units. Furthermore, compounds 10 and 11 represent the first example of iridoid glycoside dimers from R. glutinosa, although iridoid glycoside dimers were identified previously from other plants.13 Compounds 1−11 were tested for their cytotoxicity against five human tumor cell lines, HCT-8 (human ileocecal adenocarcinoma cell line), Bel-7402 (human hepatoma cell line), BGC-823 (human gastric cancer cell line), A549 (human lung epithelial cell line), and A2780 (human ovarian cancer cell line). However, all were inactive for all cell lines used (IC50 > 1629

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10 μM is defined as “inactive”). They were also bioassayed for their hepatoprotective activities against D-galactosamine-induced toxicity in HL-7702 cells, using the hepatoprotective activity drug bicyclol as the positive control.14 As shown in Table 4, compounds 3, 7, and 9−11 exhibited pronounced hepatoprotective activities.

mg), 8 (6 mg), 7 (10 mg), and 9 (11 mg). Eluting with a step gradient of 15−50% MeOH in H2O, fraction F6 (20 g) was chromatographed on a reversed-phase C18 silica gel column, to give subfractions F6‑1− F6‑5. Fraction F6‑5 was purified using Sephadex LH-20 (MeOH−H2O, 1:1) followed by semipreparative HPLC (19% MeCN in H2O) to afford 3 (12 mg). Fraction F7 (30 g) was chromatographed on a reversed-phase C18 silica gel column eluted with MeOH−H2O (15:85, 25:75, 35:65, 45:55, and 50:50 v/v) to give subfractions F7‑1−F7‑5. Fraction F7‑3 was purified using semipreparative HPLC (38% MeOH in H2O) to afford 2 (10 mg). Fraction F9 (120 g) was subjected to repeated silica gel column chromatography (CHCl3−MeOH−H2O, 3:1:0.1; 2:1:0.2; 1:1:0.25; 100% MeOH) to give subfractions F9‑1−F9‑4. Fraction F9‑2 (5.0 g) was further separated by a reversed-phase C18 silica gel column and eluted with MeOH−H2O (5:95, 10:90, 15:85, 20:80, and 30:70 v/v) to give subfractions F9‑2‑1−F9‑2‑5. Fractions F9‑2‑4 (20 mg) and F9‑2‑5 (13 mg) were subjected separately to separation over Sephadex LH-20 (MeOH−H2O, 1:1) to yield 10 (12 mg) from F9‑2‑4 and 11 (10 mg) from F9‑2‑5. Rehmaglutoside A (1): amorphous powder, [α]20D −138.1 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 203 (4.65), 216 (4.39), 278 (4.50) nm; IR νmax 3398, 2930, 1705, 1636, 1450, 1312, 1170, 1075 cm−1; 1H and 13C NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 501.1731 [M + Na]+ (calcd for C24H30O10Na, 501.1737). Rehmaglutoside B (2): amorphous powder, [α]20D −93.8 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 203 (4.78), 216 (4.52), 254 (4.19), 292 (3.92) nm; IR νmax 3383, 2921, 1700, 1658, 1600, 1512, 1419, 1274, 1075 cm−1; 1H and 13C NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 683.2157 [M + Na]+ (calcd for C29H40O17Na, 683.2158). Rehmaglutoside C (3): amorphous powder, [α]20D −127.8 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 203 (4.46), 212 (4.36), 266 (3.88) nm; IR νmax 3394, 2937, 1702, 1658, 1593, 1500, 1460, 1334, 1223, 1073 cm−1; 1H and 13C NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 697.2327 [M + Na]+ (calcd for C30H42O17Na, 697.2314). Rehmaglutoside D (4): amorphous powder, [α]20D −102.1 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 204 (4.79), 227 (4.23), 296 (3.90) nm; IR νmax 3349, 2921, 1728, 1653, 1611, 1425, 1077 cm−1; 1H and 13 C NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 520.1804 [M + Na]+ (calcd for C23H31NO11Na, 520.1789). Rehmaglutoside E (5): amorphous powder, [α]20D −94.0 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 204 (5.11), 224 (4.74), 330 (4.58) nm; CD (MeOH) 239.5 (Δε +0.85), 267 (Δε −0.24) nm; IR νmax 3402, 2935, 1687, 1606, 1516, 1276, 1137 cm−1; 1H and 13C NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 711.2268 [M + Na]+ (calcd for C34H40O15Na, 711.2259). Rehmaglutoside F (6): amorphous powder, [α]20D −125.3 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 270 (4.55) nm; CD (MeOH) 225.5 (Δε +1.89), 266 (Δε −4.25) nm; IR νmax 3419, 2929, 1693, 1660, 1610, 1370, 1237, 1154, 1075 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 621.2889 [M + Na]+ (calcd for C30H46O12Na, 621.2881). Rehmaglutoside G (7): amorphous powder, [α]20D −103.5 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 269 (4.68) nm; CD (MeOH) 228.5 (Δε +2.03), 267 (Δε −4.46) nm; IR νmax 3394, 2929, 1693, 1660, 1610, 1371, 1237, 1158, 1075 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 637.2841 [M + Na]+ (calcd for C30H46O13Na, 637.2831). Rehmaglutoside H (8): amorphous powder, [α]20D −99.7 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 270 (4.45) nm; CD (MeOH) 226.5 (Δε +2.33), 268 (Δε −4.38) nm; IR νmax 3402, 2923, 1694, 1659, 1610, 1370, 1237, 1154, 1075 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 637.2834 [M + Na]+ (calcd for C30H46O13Na, 637.2831). Rehmaglutoside I (9): amorphous powder, [α]20D −112.3 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 270 (4.22) nm; CD (MeOH) 228.5 (Δε +0.73), 266 (Δε −4.10) nm; IR νmax 3388, 2922, 1692, 1659, 1635, 1611, 1369, 1237, 1157, 1076 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 783.3411 [M + Na]+ (calcd for C36H56O17Na, 783.3410).

Table 4. Hepatoprotective Effects of Compounds 3, 7, and 9−11 (10 μM) against D-Galactosamine-Induced Toxicity in HL-7702 Cellsa compound normal control bicyclol 3 7 9 10 11

cell survival rate (% of normal)

inhibition (% of control)

± ± ± ± ± ± ± ±

16.7 33.6 38.7 28.2 25.2 35.5

100 56.3 63.6 71.0 73.2 68.6 67.3 71.8

2.4 2.1 3.4b 1.9c 5.0c 1.9c 5.3c 2.9d

Results are expressed as means ± SD (n = 3; for normal and control, n = 6); bicyclol was used as positive control (10 μM). bp < 0.05. cp < 0.01. dp < 0.001.

a

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-2000 polarimeter, and UV spectra with a JASCO V-650 spectrophotometer. IR spectra were recorded on a Nicolet 5700 spectrometer by an FT-IR microscope transmission method. NMR measurements were performed on VNS-600, INOVA500, and Bruker AV500-III spectrometers in methanol-d4. HRESIMS were obtained using an Agilent 1100 series LC/MSD ion trap mass spectrometer. Preparative HPLC was conducted using a Shimadazu LC-6AD instrument with an SPD-20A detector and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Silica gel (200−300 mesh, Qingdao Marine Chemical Factory, Qingdao, People’s Republic of China), Sephadex LH-20 (GE), and ODS (50 μm, YMC, Japan) were used for column chromatography. TLC was carried out with GF254 plates (Qingdao Marine Chemical Factory). Spots were visualized by spraying with 10% H2SO4 in 95% EtOH followed by heating. Plant Material. The roots of R. glutinosa were collected in Jiaozuo, Henan Province, People’s Republic of China, in March 2011, and identified by Professor Lin Ma (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College). A voucher specimen (ID-S-2427) has been deposited at the Herbarium of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (ID-S-2427), Beijing. Extraction and Isolation. The fresh roots (500 kg) of R. glutinosa were sliced, air-dried, and then exhaustively extracted with 95% aqueous EtOH (3 × 100 L) at reflux. The combined extracts were concentrated under reduced pressure to dryness. The residue was suspended in H2O and applied to a Diaion HP20 column that was eluted with H2O and then EtOH. After removing the solvent, the EtOH eluate (700 g) was subjected to passage over a silica gel column. Successive elution with a gradient of increasing methanol (0−100%) in chloroform afforded nine main fractions (F1−F9) based on TLC analysis. Fraction F3 (8.0 g) was chromatographed on a reversed-phase C18 silica gel column (4.5 × 42 cm) eluted with MeOH−H2O (20:80, 30:70, 40:60, 50:50, and 60:40 v/v) to give subfractions F3‑1−F3‑5. Separation of fraction F3‑3 (4.2 g) with preparative HPLC (26% MeOH in H2O) yielded 1 (12 mg). F5 (30 g) was chromatographed over Sephadex LH-20 eluted with MeOH as mobile phase to give subfractions F5‑1−F5‑3. Fraction F5‑2 (2.0 g) was further separated by repeated Sephadex LH-20 to afford 4 (10 mg) and 5 (8 mg). Fraction F5‑3 (10 g) was fractioned via silica gel (EtOAc−EtOH−H2O, 15:2:1; 10:2:1; 7:2:1) and Sephadex LH-20 (MeOH−H2O, 1:1) to yield 6 (7 1630

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Rehmaglutoside J (10): amorphous powder, [α]20D −112.4 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 201 (4.56), 225 (3.64) nm; IR νmax 3383, 2931, 1657, 1373, 1233, 1078, 1007, 949 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 719.2739 [M + Na]+ (calcd for C30H48O18Na, 719.2733). Rehmaglutoside K (11): amorphous powder, [α]20D −114.8 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 201 (4.55), 229 (3.60) nm; IR νmax 3376, 2932, 1657, 1373, 1233, 1077, 949 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 719.2728 [M + Na]+ (calcd for C30H48O18Na, 719.2733). Acid Hydrolysis of 1−3 and 9−11. Each compound (8 mg) was individually refluxed in 6% HCl (5.0 mL) at 80 °C for 2 h. Each reaction mixture was extracted with CHCl3 (3 × 6 mL), and the H2O phase was dried by using a N2 stream. The residues were separately subjected to column chromatography over silica gel with EtOAc− EtOH−H2O (7:4:1) as eluent to yield glucose (1.06 mg) from 1, [α]20D +44.3 (c 0.07, H2O), glucose (1.60 mg) from 2, [α]20D +72.5 (c 0.10, H2O), glucose (1.55 mg) and rhamnose (1.41 mg) from 3, [α]20D +73.4 (c 0.10, H2O) and [α]20D +13.5 (c 0.09, H2O), glucose (0.99 mg) from 9, [α]20D +52.1 (c 0.06, H2O), glucose (1.48 mg) from 10, [α]20D +68.6 (c 0.09, H2O), and glucose (0.73 mg) from 11, [α]20D +38.8 (c 0.05, H2O), respectively. The solvent system EtOAc−EtOH− H2O (6:4:1) was used for TLC identification of glucose and rhamnose. Cytotoxicity Assay. Compounds 1−11 were tested for cytotoxicity against HCT-8 (human colon carcinoma), Bel-7402 (human liver carcinoma), BGC-823 (human stomach carcinoma), A549 (human lung carcinoma), and A2780 (human ovarian carcinoma) by means of an MTT method described in the literature.15 Protective Effect on Cytotoxicity Induced by D-Galactosamine in WB-F344 Cells. The hepatoprotective effects of compounds 1−11 were determined by a (MTT) colorimetric assay16 in HL-7702 cells. Each cell suspension of 2 × 104 cells in 200 μL of RPMI 1640 containing fetal calf serum (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL) was placed in a 96-well microplate and precultured for 24 h at 37 °C under a 5% CO2 atmosphere. Fresh medium (100 μL) containing bicyclol and test samples was added, and the cells were cultured for 1 h. Then, the cultured cells were exposed to 25 mM D-galactosamine for 24 h. Then, 100 μL of 0.5 mg/mL MTT was added to each well after the withdrawal of the culture medium and incubated for an additional 4 h. The resulting formazan was dissolved in 150 μL of DMSO after aspiration of the culture medium. The optical density (OD) of the formazan solution was measured on a microplate reader at 492 nm. Inhibition (%) was obtained by the following formula:

Academy of Medical Sciences and Peking Union Medical College.

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(1) Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China; China Medical Science Press: Beijing, 2010; Vol. 2, pp 115−116. (2) (a) Sasaki, H.; Nishimura, H.; Chin, M.; Mitsuhashi, H. Phytochemistry 1989, 28, 875−879. (b) Morota, T.; Sasaki, H.; Nishimura, H.; Sugama, K.; Chin, M.; Mitsuhashi, H. Phytochemistry 1989, 28, 2149−2153. (c) Nishimura, H.; Sasaki, H.; Morota, T; Chin, M.; Mitsuhashi, H. Phytochemistry 1989, 28, 2705−2709. (d) Morota, T.; Sasaki, H.; Sugama, K.; Nishimura, H.; Chin, M.; Mitsuhashi, H. Phytochemistry 1990, 29, 523−526. (e) Sasaki, H.; Nishimura, H.; Morota, T.; Katsuhashi, T.; Chin, M.; Mitsuhashi, H. Phytochemistry 1991, 30, 1639−1644. (f) Sasaki, H.; Morota, T.; Nishimura, H.; Ogino, T.; Katsuhashi, T.; Sugama, K.; Chin, M.; Mitsuhashi, H. Phytochemistry 1991, 30, 1997−2001. (3) (a) Antus, S.; Kurtan, T.; Juhasz, L.; Kiss, L.; Hollosi, M.; Majer, Z. S. Chirality 2001, 13, 493−506. (b) Kamiya, K.; Tanaka, Y.; Endang, H.; Umar, M.; Satake, T. J. Agric. Food Chem. 2004, 52, 5843−5848. (4) Yamamoto, A.; Nitta, S.; Miyase, T.; Ueno, A.; Wu, L. J. Phytochemistry 1993, 32, 421−425. (5) Damtoft, S.; Hansen, S. B.; Jacobsen, B.; Jensen, S. R.; Nielsen, B. J. Phytochemistry 1984, 23, 2387−2389. (6) Toda, S.; Miyase, T.; Arichi, H.; Tanizawa, H.; Takino, Y. Chem. Pharm. Bull. 1985, 33, 1270−1273. (7) Gouda, Y. G.; Abdel-bakya, A. M.; Darwisha, F. M.; Mohameda, K. M.; Kasaib, R.; Yamasakib, K. Phytochemistry 2003, 63, 887−892. (8) Akdemir, Z.; Calis, I.; Junior, P. Phytochemistry 1991, 30, 2401− 2402. (9) Kobayashi, H.; Karasawa, H.; Miyase, T.; Fukushima, S. Chem. Pharm. Bull. 1985, 33, 3645−3650. (10) Kanchanapoom, T.; Ruchirawat, S.; Kasai, R.; Otsuka, H. Chem. Pharm. Bull. 2004, 52, 980−982. (11) Zhang, Y. L.; Gan, M. L.; Lin, S.; Liu, M. T.; Song, W. X.; Zi, J. C.; Wang, S. J.; Li, S.; Yang, Y. C.; Shi, J. G. J. Nat. Prod. 2008, 71, 905−909. (12) Kanemoto, M.; Matsunami, K.; Otsuka, H.; Shinzato, T.; Ishigaki, C.; Takeda, Y. Phytochemistry 2008, 69, 2517−2522. (13) Fan, Q. L.; Tan, C. H.; Liu, J.; Zhao, M. M.; Han, F. S.; Zhu, D. Y. Phytochemistry 2011, 72, 1927−1932. (14) Liu, G. T. Chin. J. New Drugs 2001, 10, 325−327. (15) Ni, G.; Zhang, Q. J.; Zheng, Z. F.; Chen, R. Y.; Yu, D. Q. J. Nat. Prod. 2009, 72, 966−968. (16) Xu, F.; Matsuda, H. J.; Ninomiya, K.; Yoshikawa, M. J. Nat. Prod. 2004, 67, 569−576.

inhibition (%) = [(OD(sample) − OD(control))

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REFERENCES

/(OD(normal) − OD(control))] × 100

ASSOCIATED CONTENT

S Supporting Information *

Copies of IR, MS, 1D and 2D NMR, and CD spectra for compounds 1−11. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-63165224. Fax: +86-10-63017757. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Science and Technology Project of China (No. 2011ZX09307-002-01) and the State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese 1631

dx.doi.org/10.1021/np300509z | J. Nat. Prod. 2012, 75, 1625−1631