Iridoid Glucosides and Diterpenoids from Caryopteris glutinosa

Article pubs.acs.org/jnp

Iridoid Glucosides and Diterpenoids from Caryopteris glutinosa Guoyong Luo,†,‡,§ Qi Ye,† Baowen Du,† Fei Wang,† Guo-lin Zhang,*,† and Yinggang Luo*,† †

Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of China University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § Guiyang College of Traditional Chinese Medicine, Guiyang 550025, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Five new iridoid glucoside derivatives (1−5), three new diterpenoids (7, 12, and 15), and 11 known compounds were isolated from the aqueous EtOH extract of Caryopteris glutinosa. Cell-based estrogen biosynthesis assays indicated that caryopteriside C (3) and caryopterisoid B (12) promote the biosynthesis of estrogen E2, with EC50 values of 11.1 and 8.0 μM, respectively, in human ovarian granulosa-like KGN cells via upregulating the expression of aromatase.

(1602 cm−1) groups. The UV spectrum suggested the presence of substituted phenyl and conjugated aromatic rings (233, 286, and 323 nm). An iridoid moiety was established from the 1 H−1H COSY cross signals for the H-3/H-4 and H-6/H-7 coupling systems and the following key HMBC correlations: H1/C-5, C-8; H-3/C-1, C-5; H-4/C-6, C-9; H-7/C-5, C-9, C-10; and H-10/C-9 (Figure 1A). The presences of a glucopyranosyl moiety was deduced from the 1H−1H COSY cross signals for the H-1′/H-2′/H-3′/H-4′/H-5′/H-6′ coupling systems and the key HMBC correlations H-1′/C-3′, C-5′ and H-6′/C-4′, C-5′ (Figure 1A). A trans-feruloyl group was established from the 1 H−1H COSY cross signals for the H-5″/H-6″ and H-7″/H-8″ coupling systems, the key HMBC correlations H-2″/C-4″, C6″; H-5″/C-1″, C-3″; H-6″/C-4″; H-7″/C-2″, C-6″, C-9″; H8″/C-1″; and 3″-OCH3/C-3″ (Figure 1A), and the coupling constant (15.9 Hz) of H-7″/H-8″ (Table 1). A guaiacylglyceryl, i.e., a 1-(4-hydroxy-3-methoxyphenyl)glyceryl unit, was established from the 1H−1H COSY cross signals for the H-5‴/H-6‴ and H-7‴/H-8‴/H-9‴ coupling systems and the key HMBC correlations H-2‴/C-4‴, C-6‴; H-5‴/C-1‴, C-3‴; H-6‴/C4‴; H-7‴/C-2‴, C-6‴, C-9‴; H-8‴/C-1‴; and 3‴-OCH3/C3‴ (Figure 1A). The crucial HMBC cross signals of H-1/C-1′ and H-1′/C-1 (Figure 1A) suggested the C-1−O−C-1′ linkage, indicating the presence of a harpagide moiety. Alkaline hydrolysis of 1 afforded harpagide, which was identified by an HPLC comparison of harpagide from the alkaline hydrolysis of 8-O-acetylharpagide (Figure S1, Supporting Information).6b The 6′-OH was acylated by the trans-feruloyl group in view of the HMBC correlation of H-6′/C-9″ (Figure 1A). The C-4″−

T

he plants of the genus Caryopteris are short shrubs distributed in the eastern and central Asian regions.1 Some of them have been used as folk medicine to treat cold, cough, and rheumatic pain.2 Phytochemical investigations on the plants of this genus led to the identification of several classes of natural products such as iridoid glucosides, diterpenoids, flavonoids, phenylethanoid glucosides, and pyridine-containing alkaloids. Many of them showed antioxidant, antimicrobial, antitumor, and insect-antifeeding bioactivities.2−4 Caryopteris glutinosa, an endemic species distributed in the Sichuan Province of China, is an ethnic herb used to treat inflammatory diseases.5 In the present work 19 compounds were isolated from the 95% EtOH extract of C. glutinosa. Five new iridoid glucoside derivatives (1−5) and three new diterpenoids (7, 12, and 15) were characterized by physical data. Cell-based estrogen biosynthesis assays indicated that compounds 3 and 12 promote the biosynthesis of estrogen E2 by enhancing the expression of aromatase that catalyzes the conversion of androgens to estrogens in vertebrates.

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RESULTS AND DISCUSSION The 95% EtOH extract of the whole plants of C. glutinosa was partitioned between H2O and EtOAc. Purification of the EtOAc-soluble fraction by a combination of column chromatographic methods afforded five new iridoid glucoside derivatives (1−5), three new diterpenoids (7, 12, and 15), and 11 known compounds. Compound 1 was isolated as a light yellow solid. Its molecular formula of C37H46O18 was determined from the 13C NMR (Table 1) and HRESIMS data (m/z 801.2566 [M + Na]+). The IR spectrum indicated the presence of hydroxy (3421 cm−1), conjugated carbonyl (1701 cm−1), and phenyl © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 23, 2015

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DOI: 10.1021/acs.jnatprod.5b00946 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. NMR Data for Compounds 1 and 2 in Methanol-d4a 1 position 1 3 4 5 6 7

8 9 10 8-OAc 1′ 2′ 3′ 4′ 5′ 6′

1″ 2″ 3″ 4″ 5″ 6″

O−C-8‴ linkage was concluded from the HMBC correlation of H-8‴/C-4″ (Figure 1A). An acetoxy group at C-8 defined the iridoid unit as a substituted 8-O-acetylharpagide moiety since the NMR data of this moiety were consistent with those of 8-Oacetylharpagide.6a The relative configuration of the 8-O-acetylharpagide moiety of 1 was established from the key NOESY correlations (Figure 1B). The chemical shift difference of C-8‴ and C-7‴ (Δ δC‑8‴ − δC‑7‴) was reported to be distinguishable for the different orientations of 7‴-OH and 8‴-OH of the guaiacylglyceryl moiety.7a−c A threo-configuration of C-7‴ and C-8‴ in 1 was determined by the Δ δC‑8‴ − δC‑7‴ value (12.3 ppm, Table 1). The 3JH‑7‴,H‑8‴ value of 4.6 Hz is characteristic for the threooriented hydroxy groups when the 1H NMR spectrum was recorded in DMSO-d6 (Figure S2A, Supporting Information).7d Therefore, the structure of compound 1, caryopteriside A, was determined as 6′-O-[(E)-4-O-(threo-β-guaiacylglyceryl)feruloyl]-8-O-acetylharpagide. The 13C NMR (Table 1) and HRESIMS data of 2 (m/z 801.2579 [M + Na]+) suggested that 2 has the same molecular formula as that of 1. Only slight differences between the IR, UV, and NMR data (Table 1) of 2 and 1 were observed, indicating that compound 2 is mostly like a diastereoisomer of 1. The structure of 2 (Figure 1A), similar to that of 1, was established from its NMR (Table 1) and alkaline hydrolysis data (Figure S1, Supporting Information). However, the chemical shifts of C-7‴, -8‴, and -9‴ of the guaiacylglyceryl moiety in 1 and 2 are different, indicating different relative configurations of C-7‴ and C-8‴. An erythro-guaiacylglyceryl moiety in 2 was established via the Δ δC‑8‴ − δC‑7‴ value (11.4 ppm, Table 1) and the coupling constant between H-7‴ and H8‴ (5.5 Hz, Figure S2B, Supporting Information). Thus, the structure of compound 2, caryopteriside B, was defined as 6′-O-

7″ 8″ 9″ 3″-OMe 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴

3‴OMe

δH (J in Hz) 6.01, d (1.4) 6.37, d (6.4) 4.91, m

3.70, dd (4.6, 2.0) 2.14, d (14.9) 1.96, dd (15.0 4.6) 2.84, s 1.39, s 1.93, s 4.61, d (8.0) 3.25, m 3.43, m 3.37, m 3.58, m 4.53, dd (11.9, 2.3) 4.38, dd (11.9, 6.4) 7.23, d (1.9)

7.04, d (8.3) 7.10, dd (8.5, 2.0) 7.67, d (15.9) 6.48, d (15.9) 3.89, s 7.03, br s

6.75, d (8.1) 6.85, dd (8.2, 2.0) 4.88, m 4.45, m 3.76, dd (12.0, 4.0) 3.52, dd (11.9, 5.7) 3.82, s

2 δC, type 94.3, CH 143.5, CH 107.3, CH 73.0, C 77.6, CH 46.0, CH2

87.8, 55.6, 22.5, 22.1, 172.5, 99.6, 74.4, 77.6, 71.9, 75.6, 64.8,

C CH CH3 CH3 C CH CH CH CH CH CH2

129.6, 112.2, 151.6, 152.0, 117.3, 123.7,

C CH C C CH CH

146.3, 116.8, 168.9, 56.6, 133.7, 111.6, 148.8, 147.2, 115.8, 120.6,

CH CH C CH3 C CH C C CH CH

δH (J in Hz) 6.01, d (1.4) 6.37, d (6.4) 4.92, dd (6.4, 1.6) 3.70, dd (4.7, 2.0) 2.14, d (14.7) 1.96, dd (15.1, 4.7) 2.84, s 1.39, s 1.92, s 4.61, d (7.9) 3.24, t (8.4) 3.42, m 3.36, m 3.58, m 4.53, dd (11.9, 2.1) 4.37, dd (11.8, 6.4) 7.15, d (2.0)

6.95, d (8.4) 7.06, dd (8.5, 2.0) 7.64, d (16.0) 6.45, d (16.0) 3.81, s 7.03, d (1.9)

73.8, CH 86.2, CH 62.0, CH2

6.71, d (8.1) 6.84, dd (8.1, 2.0) 4.82, d (5.9) 4.49, m 3.83, m

56.3, CH3

3.80, s

δC, type 94.3, CH 143.5, CH 107.3, CH 73.0, C 77.6, CH 46.0, CH2

87.8, 55.6, 22.5, 22.1, 172.5, 99.6, 74.4, 77.6, 71.9, 75.6, 64.8,


129.5, 112.3, 151.7, 151.8, 117.4, 123.6,

C CH C C CH CH

146.4, 116.7, 168.9, 56.6, 133.9, 111.9, 148.7, 147.0, 115.6, 121.1,

CH CH C CH3 C CH C C CH CH

74.0, CH 85.5, CH 62.4, CH2

56.3, CH3

a

Recorded at 400 MHz for 1H and 100 MHz for 13C. Chemical shifts and coupling constants (in parentheses) are given in ppm and Hz, respectively.

[(E)-4-O-(erythro-β-guaiacylglyceryl)feruloyl]-8-O-acetylharpagide. It is a C-7‴ epimer of caryopterisde A (1). Caryopterisides A (1) and B (2) feature a “mixed structure” of an iridoid glucoside and an oxyneolignan. Compound 3 was shown to have the molecular formula C27H34O14 from the 13C NMR data (Table 2) and the [M + Na]+ ion at m/z 605.1848 in its HRESIMS. The similarity of the IR, UV, and NMR data (Table 2) of 3 and 1 (Table 1) B



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of a guaiacylglyceryl group in 3 compared to the data of 1 and 2. A trans-sinapoyl group was established from the NMR data (Table 2, Figure S3, Supporting Information). It was linked to C-6′ via an ester bond (C-6′−O−C-9″ linkage) based on the key HMBC correlations of H-6′/C-9″ (Figure S3, Supporting Information). Thus, the structure of compound 3, caryopteriside C, was defined as 6′-O-[(E)-sinapoyl]-8-O-acetylharpagide. The 13C NMR data (Table 2) and the [M + Na]+ ion at m/z 635.1946 in the HRESIMS of compound 4 suggested the absence of a methoxy group in 4 compared with the structure of 3. The alkaline hydrolysis of 4 also afforded harpagide (Figure S1, Supporting Information). The NMR data (Table 2) showed the presence of a trans-feruloyl group in 4 (Figure S3, Supporting Information). The C-6′−O−C-9″ linkage was established from the key HMBC correlations of H-6′/C-9″ (Figure S3, Supporting Information). The structure of compound 4, caryopteriside D, was hence defined as 6′-O[(E)-feruloyl]-8-O-acetylharpagide. Compound 5 was isolated as a light yellow solid. Its molecular formula C25H32O14 was determined from the 13C NMR (Table 2) and the HRESIMS data (m/z 579.1684 [M + Na]+). The similarity of the IR, UV, and NMR data (Table 2) of 5 and 3 suggested that 5 was also a derivative of harpagide, which was supported by alkaline hydrolysis (Figure S1, Supporting Information). The NMR data (Table 2) suggested the presence of a vanilloyl group in 5 instead of a trans-sinapoyl

Figure 1. 1H−1H COSY, key HMBC, and selected NOESY correlations of compounds 1 and 2.

suggested that 3 was also a derivative of harpagide, which was confirmed by alkaline hydrolysis (Figure S1, Supporting Information). The NMR data (Table 2) indicated the absence Table 2. NMR Data for Compounds 3−5 in Methanol-d4a 3 position 1 3 4 5 6 7 8 9 10 8-OAc 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 3″-OMe 5″-OMe a

4

δH (J in Hz)

δC, type

δH (J in Hz)

6.01, d (1.0) 6.37, d (6.4) 4.92, dd (6.4, 1.5)

94.3, CH 143.5, CH 107.3, CH 73.0, C 77. 6, CH 45.9, CH2

6.02, d (1.3) 6.37, d (6.4) 4.92, dd (6.4, 1.6)

3.70, dd (4.4, 1.9) 2.14, d (14.5) 1.97, dd (15.0, 4.6) 2.84, s 1.41, s 1.93, s 4.61, 3.25, 3.43, 3.41, 3.58, 4.53, 4.38,

d (7.9) t (8.5) m m m dd (11.9, 2.1) dd (11.9, 6.3)

6.89, s

6.89, s 7.65, d (15.9) 6.45, d (15.9) 3.86, s 3.86, s

87.8, C 55., CH 22.5, CH3 22.1, CH3 172.4, C 99.6, CH 74.5, CH 77.6, CH 71.9, CH 75.6, CH 64.7, CH2 126.6, C 106.9, CH 149.5, C 139.7, C 149. 5, C 106.9, CH 147.1, CH 115.9, CH 169.0, C 56.9, CH3 56.9, CH3

3.70, dd (4.7, 1.9) 2.13, d (15.1) 1.95, dd (15.1, 4.6) 2.84, s 1.39, s 1.92, s 4.62, 3.27, 3.45, 3.39, 3.59, 4.53, 4.38,

d (7.9) t (8.4) t (8.9) t (9.1) m dd (11.9, 2.2) dd (11.9, 6.3)

7.16, d (1.9)

6.80, 7.04, 7.64, 6.41,

d (8.2) dd (8.2, 1.9) d (15.9) d (15.9)

3.87, s

5 δC, type 94.3, 143.5, 107.2, 73.0, 77.5, 45.9,

CH CH CH C CH CH2

87.8, 55.5, 22.5, 22.1, 172.5, 99.5, 74.4, 77.5, 71.9, 75.5, 64.7,


127.7, C 111.6, CH 149.2, C 150.5, C 116.4, CH 124.1, CH 146.9, CH 115.4, CH 169.1, C 56. 5, CH3

δH (J in Hz) 6.00, d (1.0) 6.36, d (6.4) 4.92, dd (6.4, 1.3) 3.70, dd (4.4, 1.8) 2.14, d (14.9), 1.96, dd (15.0, 4.6) 2.82, s 1.36, s 1.91, s 4.63, 3.26, 3.44, 3.41, 3.64, 4.67, 4.41,

d (7.9) t (8.2) m m t (7.1) dd (11.8, 1.9) dd (11.8, 6.7)

7.59, d (1.2)

6.80, d (8.3) 7.63, dd (8.3, 1.9)

3.89, s

δC, type 94.1, 143.5, 107.4, 72.9, 77.6, 45.9,

CH CH CH C CH CH2

87.8, 55.7, 22.4, 22.0, 172.4, 99.4, 74.5, 77.6, 72.1, 75.7, 65.1,


122.5, 113.8, 148.7, 153.0, 115.9, 125.4, 168.0,

C CH C C CH CH C

56.5, CH3

Recorded at 400 MHz for 1H and 100 MHz for 13C. Chemical shifts and coupling constants (in parentheses) are given in ppm and Hz, respectively. C



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Table 3. NMR Data for Compounds 7, 12, and 15 in CDCl3a 7b position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

a

δH (J in Hz) 1.65, 1.47, 2.31, 2.20, 6.82,

m m m m m

2.39, 1.14, 1.46, 1.40, 1.48,

d (12.9), m m, m overlap

1.30, 1.47, 1.39, 1.88, 1.75,

d (12.1) m m dt (12.8, 4.2) dt (13.0, 5.0)

5.39, t (6.7) 4.14, d (6.9)

16 17 18 19

1.67, s 0.80, d (5.9) 1.24, s

20

0.73, s

12c δC, type 17.6, CH2 27.6, CH2 140.1, 141.8, 37.7, 36.0,

CH C C CH2

27.4, CH2 36.4, 38.8, 46.8, 37.0,

CH C CH CH2

33.0, CH2

δH (J in Hz) 1.56, 1.49, 2.18, 2.06, 5.56,

m m m m br s

18.3, CH2 26.7, CH2

CH3 CH3 C CH3

18.5, CH3

122.3, 148.1, 38.0, 36.4,

1.37, m d (10.3) m, m ddd (16.2, 11.9, 4.4) m

140.8, C 123.1, CH 59.6, CH2 16.7, 16.1, 172.3, 20.7,

δC, type

1.75, dt (12.8, 2.9) 1.31, m 1.43, m

1.25, 1.66, 1.56, 2.33, 2.22,

15c

CH C C CH2

δH (J in Hz)

δC, type

3.09, m 1.89, m 1.94, m

29.4, CH2 35.3, CH2

1.86, m

72.7, 151.8, 167.6, 123.6,

6.22, s

27.3, CH2

189.8, C

36.6, 38.5, 46.7, 31.7,

109.5, 136.2, 42.5, 131.2,

CH C CH CH2

37.8, CH2

s d (6.4) d (5.1) s

30.2, 16.1, 63.1, 21.4,

0.76, s

CH3 CH3 CH2 CH3

18.3, CH3 b

C C C C

154.7, C

209.5, C

2.14, 0.80, 4.09, 1.07,

C C C CH

3.41, 2.88, 5.14, 1.53, 1.52, 5.28, 5.20, 1.50,

dd (15.3, 9.0), dd (15.3 7.3) m d (6.4) s s, s s

111.4, C 154.2, C 34.6, CH2 83.4, 22.1, 26.9, 112.4,

CH CH3 CH3 CH2

21.6, CH3

1

Chemical shifts and coupling constants (in parentheses) are given in ppm and Hz, respectively. Recorded at 400 MHz for H and 100 MHz for C. cRecorded at 600 MHz for 1H and 150 MHz for 13C.

13

confirmed by the NOESY correlation of H-15 and H-16 (Figure S4B, Supporting Information). Similar negative Cotton effects at ∼240 nm were observed in the electronic circular dichroism (ECD) spectra of 7, 9−11, and 13 with the same moieties (Figure S4C, Supporting Information),11 indicating identical absolute configurations. Thus, the structure of compound 7, caryopterisoid A, was determined as (13E)-15hydroxyneocleroda-3,13-dien-18-oic acid. It is a C-5 epimer of cis-ent-15-hydroxycleroda-3,13-dien-18-oic acid.9g Compound 12 was isolated as a colorless oil. Its molecular formula of C18H30O2 was determined from the [M + K]+ ion at m/z 317.1883 in the HRESIMS and the 13C NMR data (Table 3). The IR spectrum showed a characteristic absorption for a carbonyl group (1716 cm−1). The structure of 12 was established from the 1H−1H COSY, HSQC, and HMBC correlations (Figure S6A, Supporting Information). The relative configuration of 12 was established from the NOESY correlations (Figure S6B, Supporting Information) and the selective irradiation experiment (Figure S7, Supporting Information). Thus, the structure of compound 12, caryopterisoid B, was defined as 18-hydroxy-14,15-dinor-neocleroda-3en-13-one. Compound 15 was isolated as a yellow powder. Its molecular formula of C20H22O5 was determined from the HRESIMS (m/z 365.1348 [M + Na]+) and the 13C NMR data (Table 3). The IR and UV spectra indicated the presence of conjugated carbonyl (1642 cm−1), olefin (1635 cm−1), and phenyl (1602 cm−1) functionalities. The structure of 15 was established from

group in 3 (Figure S3, Supporting Information). Its location at C-6′ through an ester bond (C-6′−O−C-7″ linkage) was concluded from the key HMBC correlations of H-6′/C-7″ (Figure S3, Supporting Information). Thus, the structure of compound 5, caryopteriside E, was defined as 6′-O-vanilloyl-8O-acetylharpagide. Compound 7 was isolated as a white powder. Its molecular formula C20H32O3 was determined from the [M + Na]+ ion at m/z 343.2249 in the HRESIMS and the 13C NMR data (Table 3). The IR spectrum suggested the presence of α,β-unsaturated carboxylic (1688 and 1682 cm−1) and olefinic (1636 cm−1) groups. The similarity of the NMR data of 7 (Table 3) and those of kolavic acid (9) suggested that 7 is a neo-clerodane derivative.8 The structure of 7 was established from 2D NMR experiments (Figure S4A, Supporting Information). The presence of a C-14 hydroxymethyl group in 7 rather than a carboxylic group in 9 was established from the HMBC correlations of H-15/C-13, C-14 (Figure S4A, Supporting Information). The A- and B-rings of 7 are trans-fused in view of the characteristic chemical shift of C-19 (δC 20.7) in transclerodanes compared with the downfield shift in cis-clerodanes (Table S1, Supporting Information).9 The identical orientations of the C-5, -8, and -9 methyl groups were established from the NOESY correlations (Figure S4B, Supporting Information) and the enhancement of the 1H NMR signals for 5-CH3 and 8CH3 when 9-CH3 was selectively irradiated (Figure S5, Supporting Information). The chemical shifts of H3-16 and C-16 of 7 suggested an E-Δ13,14 double bond,10 which was D



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Figure 2. 1H−1H COSY, key HMBC, and selected NOESY correlations and ECD spectrum of compound 15.

Figure 3. Caryopteriside C (3) and caryopterisoid B (12) were shown to promote the biosynthesis of estrogen E2 via upregulating the expression of aromatase in human ovarian granulosa-like KGN cells. (A) Cell-based estrogen biosynthesis assays. (B) Concentration−response of compounds 3 and 12. (C) Cell lysates were immunoblotted with antiaromatase or anti-GAPDH antibodies and are shown on the left. The quantitative results are depicted on the right. DMSO, control; FSK, 10 μM forskolin; *p < 0.05; **p < 0.01; ***p < 0.001; compared with the control (n = 3).

2D NMR experiments (Figure 2A). The β-orientation of the C16 methyl group was inferred by a comparison of the coupling constants of H-15/H-16/H-17 and the chemical shifts of C-15, C-16, and C-17 (Table 3) with those of known compounds containing the same moieties and in view of the biosynthetic origin of the rearranged abietane diterpenoids.12 The 10Sconfiguration was concluded from the negative Cotton effect at 311 nm (n−π* transition of a carbonyl group), indicative of a 10S-configuration of this type of rearranged abietanes,12a,b,d in the ECD spectrum of 15 (Figure 2C). The configuration of C-3 should be R, in view of the relative configurations of C-3 and C10 from the NOESY correlations (Figure 2B). A positive Cotton effect at ∼260 nm (π−π* transition of the α,βunsaturated carbonyl moiety) was observed in the ECD spectra of 17(15→16)-abeo-abietanes and 17(15→16),18(4→3)-diabeo-abietanes.12a,b,d However, in the case of 15, a negative Cotton effect at 262 nm was observed (Figure 2C). It may be ascribed to the allylic C-3−C-4−C-19 moiety, which plays a dominant role in determining the sign of the Cotton effects of

olefins.13 The C-3 configuration was also concluded to be R, according to the symmetry rule for chiral olefins.13 Thus, the structure of compound 15, caryopterisoid C, was defined as (3R,10S,16S)-12,16-epoxy-3,11,14-trihydroxy-17(15→ 16),18(4→3)-diabeo-abieta-4(19),5,8,11,13-pentaen-7-one. The 11 known compounds were identified as 8-Oacetylharpagide (6),6a 15-oxocleroda-3,13E-dien-18-oic acid (8),14 kolavic acid (9),8 15-oxocleroda-3,13Z-dien-18-oic acid (10),14 glutinic acid (11),15 kolavonic acid (13),16 16-hydroxy18-acetoxykolavenic acid lactone (14),17 uncinatone (16),18 sugiol (17),19 11-hydroxysugiol (18),20 and 6-hydroxysalvinolone (19),21 respectively, on the basis of the spectroscopic and physicochemical data. The complete NMR spectroscopic and physiochemical data of 9 and 13 are included in the Supporting Information. Estrogens are steroidal hormones derived from cholesterol. They are popular in various tissues such as the reproductive tract, skeleton, and central nervous system.22a,b A number of diseases such as inflammation, cancer, osteoporosis, and E



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gel (160−200 mesh) CC with petroleum ether−acetone (10:1, v/v) to give nine subfractions. Fraction E6A (1.2 g) was separated by semipreparative HPLC with MeCN−H2O (61:39, v/v, 5 mL/min) to afford compounds 8 (tR 31 min, 10 mg) and 10 (tR 35 min, 5 mg). Fraction E6B (2 g) was separated with ODS gel CC with MeOH− H2O (65:35, v/v, 10 mL/min) to give four subfractions. Compound 13 (5 mg) was crystallized from the petroleum ether−acetone (10:1, v/v) solution of fraction E6B3B (60 mg) and collected by filtration. The filtrate was subjected to preparative TLC developed with petroleum ether−acetone (10:1, v/v) to afford compound 12 (2 mg). Fraction E6B3D (300 mg) was separated by semipreparative HPLC with MeCN−H2O (61:39, v/v, 4 mL/min) to give compound 14 (tR 28 min, 75 mg). Fraction E7 (18.5 g) was separated over a Sephadex LH-20 column eluted with CHCl3−MeOH (1:1, v/v) to afford five subfractions. Fraction E7D (400 mg) was separated on semipreparative HPLC with MeOH−H2O (70:30, v/v, 3 mL/min) to give compound 18 (tR 37 min, 17 mg) and an E7DA fraction with a retention time of 45 min. Compound 19 (9 mg) was obtained from fraction E7DA separated over a silica gel (200−300 mesh) column eluted with petroleum ether−acetone (5:1, v/v). Fraction E8 (53 g) was subjected to Sephadex LH-20 CC with CHCl3−MeOH (1:1, v/v) to give four subfractions. Fraction E8C (11 g) was subjected to an ODS gel column eluted with MeOH−H2O (70:30, v/v, 20 mL/min) to give 10 subfractions. Fraction E8C1 (800 mg) was separated by semipreparative HPLC eluted with MeOH−H2O (58:42, v/v, 3 mL/ min) to afford compound 15 (tR 27 min, 2 mg). Compound 9 (3 mg) was obtained from fraction E8C8 by silica gel CC with petroleum ether−acetone (4:1, v/v). Fraction E8C10C (50 mg) was separated by semipreparative HPLC eluted with MeOH−H2O (70:30, v/v, 1.5 mL/ min) to afford compounds 7 (tR 23 min, 9 mg) and 11 (tR = 31 min, 4 mg). Fraction E17 (80 g) was separated over Sephadex LH-20 CC with CHCl3−MeOH to give nine subfractions. Semipreparative HPLC separation of fraction E-17A6 (3 g) with MeCN−H2O (22:78, v/v, 15 mL/min) afforded compounds 5 (tR 23 min, 190 mg), 4 (tR 49 min, 116 mg), and 3 (tR 44 min, 82 mg). Fraction E17A7 (2 g) was subjected to semipreparative HPLC with MeCN−H2O (18:82, v/v, 15 mL/min) to give compound 6 (tR 8 min, 40 mg) and an E17A7A fraction with a retention time of 70 min. Semipreparative HPLC separation of fraction E17A7A with MeCN−H2O (20:80, v/v, 4 mL/ min) gave compounds 2 (tR 74 min, 4 mg) and 1 (tR 81 min, 9 mg). Caryopteriside A (1): light yellow solid; [α]20 D −28 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 204 (4.65), 233 (4.18), 286 (4.10), and 323 (4.16) nm; IR (KBr) νmax 3421, 2935, 1701, 1632, 1602, 1512, 1422, 1379, 1260, 1238, 1160, 1125, 1074, 1031, 848, 815, and 783 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 801.2566 (calcd for C37H46O18+Na+, 801.2576), error 1.3 ppm. Caryopteriside B (2): light yellow solid; [α]20 D −43 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (4.55), 233 (4.06), 287 (3.98), and 324 (4.05) nm; IR (KBr) νmax 3444, 2925, 1700, 1632, 1602, 1512, 1442, 1379, 1260, 1238, 1180, 1162, 1125, 1073, 1030, 848, 815, and 777 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 801.2579 (calcd for C37H46O18+Na+, 801.2576), error 0.4 ppm. Caryopteriside C (3): light yellow solid; [α]20 D −50 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (4.22), 224 (3.99), 238 (4.01), and 327 (4.09) nm; IR (KBr) νmax 3421, 2941, 2911, 1716, 1701, 1603, 1515, 1457, 1428, 1372, 1283, 1258, 1235, 1177, 1156, 1115, 1074, 1017, 989, 846, and 775 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 635.1946 (calcd for C28H36O15+Na+, 635.1946), error 0.0 ppm. Caryopteriside D (4): light yellow solid; [α]20 D −61 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (4.25), 219 (4.08), 236 (4.02), 296 (4.10), and 326 (4.26) nm; IR (KBr) νmax 3420, 2942, 2909, 1715, 1650, 1634, 1602, 1516, 1430, 1374, 1270, 1236, 1176, 1161, 1122, 1073, 1019, 989, 952, 847, 815, and 777 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 605.1848 (calcd for C27H34O14+Na+, 605.1841), error −1.1 ppm. Caryopteriside E (5): light yellow solid; [α]20 D −59 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (4.36), 219 (4.21), 262 (4.00), and 295 (3.72) nm; IR (KBr) νmax 3392, 2922, 1715, 1701, 1652, 1601, 1515, 1429, 1378, 1288, 1234, 1119, 1072, 1015, 848, and 765 cm−1; 1H and

cardiovascular diseases are associated with an abnormal estrogen level in vivo, indicating that screening estrogen biosynthesis modulators will be beneficial to the discovery of pharmaceutical leads.22c Cell-based estrogen biosynthesis assays showed that a ∼2-fold increase of the concentration of estrogen E2 (i.e., 17β-estradiol), the most active estrogen, in KGN cells was detected when the cells were administrated with caryopteriside C (3) or caryopterisoid B (12) at 25 μM (Figure 3A). Caryopteriside C (3) and caryopterisoid B (12) enhanced estrogen E2 biosynthesis in a dose-dependent manner (Figure 3B). The EC50 values of 3 and 12 were evaluated to be 11.1 and 8.0 μM, respectively (Figure 3B). Western blotting results indicated a ∼1.5-fold increase of the expression level of aromatase, the key enzyme that catalyzes the conversion of testosterone to estrogen, when the KGN cells were treated with caryopteriside C (3) or caryopterisoid B (12) (Figure 3C). It revealed that 3 or 12 enhanced estrogen biosynthesis via increasing the expression level of aromatase. The results implied caryopteriside C (3) and caryopterisoid B (12) are potential leads for the development of selective aromatase modulators for the diseases caused by estrogen deficiency, including osteoporosis, climacteric syndrome, and premature ovarian failure.23

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a PerkinElmer 341 automatic polarimeter. UV spectra were recorded on a PerkinElmer Lambda 35 UV/vis spectrometer with λmax given in nm. ECD spectra were acquired on an APP Chirascan CD spectrometer. IR spectra were recorded on a PerkinElmer Spectrum One spectrometer with νmax given in cm−1. NMR spectra were recorded on either a Bruker Avance 600 or an Ascend 400 spectrometer. HRESIMS was obtained on a Bruker Bio TOF IIIQ (quadrupole time-of-flight) mass spectrometer. Silica gel (Qingdao Haiyang Chemical Co., Ltd. (QHCC), China), ODS gel (YMC Co., Ltd., Kyoto, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography (CC). TLC analyses were conducted on plates precoated with 10−40 μm of silica gel GF254 from QHCC. Preparative HPLC was carried out on an LC3000 HPLC system (Beijing Chuangxin Tongheng Science and Technology Co., Ltd., China) with a YMC C18 column (20 × 250 mm, 10 μm, YMC Co., Ltd., Kyoto, Japan); semipreparative HPLC on a PerkinElmer 200 HPLC system (PerkinElmer Inc., Waltham, MA, USA) with a Welch C18 column (10 × 250 mm, 5 μm, Welch Materials Inc., Shanghai, China); and analytical HPLC on an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) with an Agilent C18 column (4.6 × 250 mm, 5 μm). All solvents were commercially purchased and distilled under normal atmospheric pressure prior to use. Plant Material. The whole plants of C. glutinosa were collected in July 2012 at Wenchuan County, Sichuan Province, China. The plant material was identified and authenticated by Prof. Fading Fu of the Chengdu Institute of Biology, Chinese Academy of Sciences (CIBCAS). A voucher specimen (2012-07) has been deposited in the herbarium of CIBCAS. Extraction and Isolation. The air-dried whole plants of C. glutinosa (25 kg) were powdered and extracted with 95% EtOH (3 × 80 L) at room temperature for 7 days. The EtOH was removed under reduced pressure to yield a dark brown residue (3.32 kg). One half of the residue was suspended in 1.7 L of H2O and partitioned with EtOAc (3 × 1.5 L) and n-BuOH (4 × 1.5 L). The EtOAc-soluble fraction (780 g) was separated over a silica gel (160−200 mesh) column eluted with petroleum ether and acetone (1:0, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, v/v, each 20 L) to give fractions E1−E17. Fraction E4 (15.29 g) was separated over a silica gel (200−300 mesh) column eluted with petroleum ether−acetone (10:1, v/v) to afford compounds 16 (42 mg) and 17 (4 mg). Fraction E6 (58 g) was subjected to silica F



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13

C NMR data, see Table 2; HRESIMS m/z 579.1684 (calcd for C27H34O14+Na+, 579.1684), error 0.0 ppm. Caryopterisoid A (7): white powder; [α]20 D −90 (c 0.4, EtOH); UV (EtOH) λmax (log ε) 203 (4.02) and 219 (3.43) nm; ECD (c 0.7 mM, MeOH) 215 (Δε = −1.52), 242 (Δε = −3.99); IR (KBr) νmax 2957, 2924, 2859, 1688, 1682, 1636, 1457, 1384, 1260, 1237, 992, 772, and 694 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 343.2249 (calcd for C20H32O3+Na+, 343.2244), error 1.7 ppm. Caryopterisoid B (12): colorless oil; [α]20 D −18 (c 0.2, CHCl3); UV (CHCl3) λmax (log ε) 243 (3.02) nm; IR (KBr) νmax 2955, 2925, 2856, 1716, 1463, 1457, 1385, 1169, 1127, 1003, and 798 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 317.1883 (calcd for C18H30O2+K+, 317.1887), error 1.9 ppm. Caryopterisoid C (15): yellow powder; [α]20 D −60 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 206 (4.57), 264 (4.05), 323 (3.76), and 384 (3.68) nm; ECD (c 0.8 mM, MeOH) 218 (Δε = +10.28), 262 (Δε = −4.95), 311 (Δε = −1.89); IR (KBr) νmax 2969, 2928, 1642, 1635, 1602, 1473, 1457, 1386, 1324, 1211, 1114, 1040, 1013, 976, 893, 817, and 557 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 365.1348 (calcd for C20H22O5+Na+, 365.1359), error 3.2 ppm. Alkaline Hydrolysis of Compounds 1−6. To a MeOH solution (2 mL) of each sample (1, 1 mg; 2, 0.5 mg; 3, 4, and 5, each 1 mg) was added 15 mg of Na2CO3. The reaction mixture was stirred under reflux for 3 h and filtered. The filtrate was subjected to an analytic HPLC analysis with isocratic MeOH−H2O (15:85), at a flow rate of 1 mL/min, monitored at 208 nm. For preparation of harpagide, 10 mg of 6 was hydrolyzed, and the hydrolysis product (tR 11 min) was separated by semipreparative HPLC using isocratic MeOH−H2O (20:80, v/v, 3 mL/min, and monitored at 208 nm). NMR spectroscopic data of the alkaline hydrolysis product from 6 were identical to those of harpagide.6b Cell-Based Estrogen Biosynthesis Assay. The assays were performed according to a published procedure.22d Briefly, the KGN cells were maintained in DMEM/F-12 medium supplemented with 5% fetal bovine serum (Gibco-Invitrogen), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The culture condition was set at 37 °C in a humidified atmosphere containing 5% CO2. The KGN cells were seeded in a 24-well plate and cultured overnight. The medium was replaced by a serum-free DMEM/F-12 medium, followed by addition of test samples, DMSO (control), or forskolin (positive control). The resulting mixture was incubated for 24 h. Testosterone (10 nM) was added to each well, and the resulting mixture was incubated for another 24 h. The medium and cell lysate were collected, and the protein content of the cell lysate was determined using the bicichoninic acid protein assay kit (Bestbio, Shanghai, China). The content of 17β-estradiol in the medium was determined by magnetic particle-based ELISA (Bio-Ekon Biotechnology). The results were normalized with total protein content of cell lysate and expressed as percentage 17β-estradiol production compared with the control. Western Blotting.22d The KGN cells were cultured in 60 mm dishes and treated with test samples for 24 h. The cells were lysed with RIPA lysis buffer (Beyotime, Haimen, China) containing protein inhibitor cocktail. A portion of the resulting cell lysate (40 μg protein) was mixed with loading buffer, boiled for 5 min, and subjected to a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins from the gel were blotted onto a nitrocellulose membrane. The resulting membrane was blocked with 5% bovine serum albumin and incubated at 4 °C overnight with anti-aromatase or anti-GAPDH antibody (Epitomics, Burlingame, CA, USA), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). An enhanced chemiluminescence detection method (Amersham Bioscience, Piscataway, NJ, USA) was developed, according to the instructions provided by the manufacturer. Quantity One (Bio-Rad, Hercules, CA, USA) was employed to calculate the chemiluminescence intensity of the protein bands.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00946. Figures S1−S7, Table S1, physiochemical and spectroscopic data of compounds 9 and 13, and spectra including 1H NMR, 13C NMR, 1H−1H COSY, HSQC, HMBC, NOESY, and HRESIMS for all new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-28-82890996. Fax: +86-28-82890288. E-mail: [email protected] (G.-L. Zhang). *Tel: +86-28-82890813. Fax: +86-28-82890288. E-mail: [email protected] (Y. Luo). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for financial support in part from the National Natural Sciences Foundation of China (21561142003, 21372213, 21372214), the Sichuan Youth Science & Technology Foundation (2014JQ0028), and Pillar Program of Science and Technology Department of Sichuan Province (2012SZ0219).

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REFERENCES

(1) Pei, J.; Chen, S. L. In Flora of China; Science Press: Beijing, 1982; Vol. 65, p 194. (2) (a) Dai, Y.; Zhang, B. B.; Liao, Z. X. Nat. Prod. Res. 2012, 26, 643−647. (b) Gao, J. J.; Han, G. Q. Phytochemistry 1997, 44, 759−761. (c) Zhang, Y. H.; Wang, Y. L.; Wei, Q. Y.; Cai, Y. J.; Wang, Q.; Liu, Z. L. Pharmazie 2005, 60, 551−553. (3) (a) Castro, A.; Coll, J. Nat. Prod. Commun. 2008, 3, 1021−1031. (b) Gao, J. J.; Igarashi, K.; Nukina, M. Chem. Pharm. Bull. 2000, 48, 1075−1078. (4) (a) Que, S.; Zhao, Y. Y.; Zhou, Y.; Zhang, Q. Y. China J. Chin. Mater. Med. 2009, 34, 1523−1526. (b) Zhao, D. P.; Matsunami, K.; Otsuka, H. J. Nat. Med. 2009, 63, 241−247. (5) Jia, M. R.; Li, X. W. In Records of Ethnodrugs in China; China Medical Science Press: Beijing, 2005; p 136. (6) (a) Li, G. Z.; Dumaa, M.; Pu, X.; Yi, J. H.; Zhang, G. L.; Luo, Y. G. Yingyong Yu Huanjing Shengwu Xuebao 2012, 18, 924−927. (b) Li, Y. M.; Jiang, S. H.; Gao, W. Y.; Zhu, D. Y. Phytochemistry 1999, 50, 101−104. (7) (a) Gan, M. L.; Zhang, Y. L.; Lin, S.; Liu, M. T.; Song, W. X.; Zi, J. C.; Yang, Y. C.; Fan, X. N.; Shi, J. G.; Hu, J. F.; Sun, J. D.; Chen, N. H. J. Nat. Prod. 2008, 71, 647−654. (b) Xiong, L.; Zhu, C. G.; Li, Y. R.; Tian, Y.; Lin, S.; Yuan, S. P.; Hu, J. F.; Hou, Q.; Chen, N. H.; Yang, Y. C.; Shi, J. G. J. Nat. Prod. 2011, 74, 1188−1200. (c) Kim, K. H.; Choi, S. U.; Ha, S. K.; Kim, S. Y.; Lee, K. R. J. Nat. Prod. 2009, 72, 2061−2064. (d) Gan, M. L.; Zhu, C. G.; Zhang, Y. L.; Zi, J. C.; Song, W. X.; Yang, Y. C.; Shi, J. G. China J. Chin. Mater. Med. 2010, 35, 456− 467. (8) Misra, R.; Pandey, R. C.; Dev, S. Tetrahedron Lett. 1964, 49, 3751−3759. (9) (a) Du, K.; Mieri, M. D.; Neuburger, M.; Zietsman, P. C.; Marston, A.; van Vuuren, S. F.; Ferreira, D.; Hamburger, M.; van der Westhuizen, J. H. J. Nat. Prod. 2015, 78, 2494−2504. (b) Pacheco, A. G.; de Oliveira, P. M.; Piló-Veloso, D.; de Carvalho Alcântara, A. F. Molecules 2009, 14, 1245−1262. (c) Puebla, P.; Correa, S. X.; Guerrero, M.; Carron, R.; Feliciano, A. S. Chem. Pharm. Bull. 2005, 53, 328−329. (d) Ahmad, V. U.; Farooq, U.; Hussain, J.; Ullah, F.; Nawaz, G



Article

S. A.; Choudhary, M. I. Chem. Pharm. Bull. 2004, 52, 441−443. (e) He, H. P.; Shen, Y. M.; Zuo, G. Y.; Yang, X. S.; Hao, X. J. Helv. Chim. Acta 2003, 86, 3187−3193. (f) Hara, N.; Asaki, H.; Fujimoto, Y.; Gupta, Y. K.; Singh, A. K.; Sahai, M. Phytochemistry 1995, 38, 189−194. (g) Zdero, C.; Bohlmann, F.; King, R. M. Phytochemistry 1991, 30, 2991−3000 and references therein. (h) Lopes, L. M. X.; Bolzani, V. D. S.; Trevisan, L. M. V. Phytochemistry 1987, 26, 2781−2784. (10) Toyota, M.; Nagashima, F.; Asakawa, Y. Phytochemistry 1989, 28, 3415−3419. (11) Bisio, A.; Schito, A. M.; Ebrahimi, S. N.; Hamburger, M.; Mele, G.; Piatti, G.; Romussi, G.; Piaz, F. D.; Tommasi, N. D. Phytochemistry 2015, 110, 120−132. (12) (a) Tian, X. D.; Min, Z. D.; Xie, N.; Lei, Y.; Tian, Z. Y.; Zheng, Q. T.; Xu, R. N.; Tanaka, T.; Iinuma, M.; Mizuno, M. Chem. Pharm. Bull. 1993, 41, 1415−1417. (b) Fan, T. P.; Min, Z. D.; Linuma, M.; Tanaka, T. J. Asian Nat. Prod. Res. 2000, 2, 237−243. (c) Rodríguez, B. Magn. Reson. Chem. 2002, 40, 752−754. (d) Wang, W. X.; Xiong, J.; Tang, Y.; Zhu, J. J.; Li, M.; Zhao, Y.; Yang, G. X.; Xia, G.; Hu, J. F. Phytochemistry 2013, 89, 89−95. (e) Liu, Q.; Hu, H. J.; Li, P. F.; Yang, Y. B.; Wu, L. H.; Chou, G. X.; Wang, Z. T. Phytochemistry 2014, 103, 196−202. (13) Scott, A. I.; Wrixon, A. D. Tetrahedron 1970, 26, 3695−3715. (14) Zdero, C.; Bohlmann, F.; Niemeyer, H. M. Phytochemistry 1991, 30, 3669−3677. (15) Pu, Z. L.; Miao, Z. C.; Zhao, H. Acta Pharm. Sin. 1992, 27, 908− 911. (16) Misra, R.; Pandey, R. C.; Dev, S. Tetrahedron 1979, 35, 979− 984. (17) Bohlmann, F.; Zdero, C.; Gupta, R. K.; King, R. M.; Robinson, H. Phytochemistry 1980, 19, 2695−2705. (18) Min, H.; Zhao, Z. M.; Guo, W. Y.; Yang, D. P.; Cheng, J. L. Chin. Tradit. Herb. Drugs 2012, 43, 1050−1056. (19) Zhuang, W. T.; Zhu, L. P.; Xiang, C.; He, J.; Li, P.; Li, B. C. Nat. Prod. Res. Dev. 2013, 25, 370−374. (20) Horvath, T.; Linden, A.; Yoshizaki, F.; Eugster, C. H.; Rüedi, P. Helv. Chim. Acta 2004, 87, 2346−2353. (21) Topcu, G.; Ulubelen, A. J. Nat. Prod. 1996, 59, 734−737. (22) (a) Heldring, N.; Pike, A.; Andersson, S.; Matthews, J.; Cheng, G.; Hartman, J.; Tujague, M.; Ström, A.; Treuter, E.; Warner, M.; Gustafsson, J. A. Physiol. Rev. 2007, 87, 905−931. (b) Prossnitz, E. R.; Sklar, L. A.; Oprea, T. I.; Arterburn, J. B. Trends Pharmacol. Sci. 2008, 29, 116−123. (c) Deroo, B. J.; Korach, K. S. J. Clin. Invest. 2006, 116, 561−570. (d) Lu, D. F.; Yang, L. J.; Li, Q. L.; Gao, X. P.; Wang, F.; Zhang, G. L. Eur. J. Pharmacol. 2012, 691, 275−282. (23) (a) Simpson, E. R. J. Steroid Biochem. Mol. Biol. 2003, 86, 225− 230. (b) Simpson, E. R.; Clyne, C.; Rubin, G.; Boon, W. C.; Robertson, K.; Britt, K.; Speed, C.; Jones, M. Annu. Rev. Physiol. 2002, 64, 93−127. (c) Simpson, E. R.; Davis, S. R. Endocrinology 2001, 142, 4589−4594. (d) Simpson, E.; Rubin, G.; Clyne, C.; Robertson, K.; O’Donnell, L.; Jones, M.; Davis, S. Trends Endocrinol. Metab. 2000, 11, 184−188.

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Iridoid Glucosides and Diterpenoids from Caryopteris glutinosa

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