Lignans from the Aerial Parts of Saururus chinensis - ACS Publications

Jan 3, 2014 - The 1H NMR data of 1 showed large coupling constants (J = 7.2 and 6.6 Hz) for the 9- and 9′-methyl protons as observed in saururin A (...
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Lignans from the Aerial Parts of Saururus chinensis: Isolation, Structural Characterization, and Their Effects on Platelet Aggregation Wei-Jern Tsai,† Chien-Chang Shen,† Tung-Hu Tsai,‡ and Lie-Chwen Lin*,†,§ †

National Research Institute of Chinese Medicine, Taipei, Taiwan Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan § Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan ‡

ABSTRACT: Five new diaryldimethylbutane lignans, saurulignans A−E (1−5), four new tetrahydrofuran lignans, saurufurins A−D (6−9), and one arylnaphthalene lignan, saurunarin (10), were isolated from Saururus chinensis, along with 18 known compounds. Lignan 5 showed significant inhibition of ADP-induced aggregation with an IC50 value of 9.8 μM and AA-induced aggregation with an IC50 value of 14.0 μM. Compound 19 showed significant activity to inhibit PAF-induced aggregation with an IC50 value of 9.1 μM. In addition, five isolated compounds could induce platelet aggregation. These results suggest that secondary metabolites in S. chinensis have bidirectional regulation on blood clotting and anticlotting effects.

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ities, while saucernetin-7 and saucernetin-8 displayed antiinflammatory15 and cytotoxic effects.16 However, pharmacological information on the blood aggregation effects of these compounds remained unclear. Thus, we re-examined the whole plant and isolated 10 new lignans, together with 18 known compounds. Some isolates were also studied for their effects on platelet aggregation. This paper deals with the isolation and structural elucidation of these new compounds. In addition, results of the antiaggregation activity and the prothrombotic activity of individual compounds are presented.

latelet aggregation plays a central role in hemostasis and thrombosis. Platelet-mediated thrombus formation in arteries is a primary factor in the development of thrombotic disorders such as unstable angina, myocardial infarction, stroke, and peripheral vascular diseases.1−3 Platelet aggregation is also caused by certain physiological substances, such as thrombin and prostaglandin endoperoxide, and can lead to arterial thrombosis.4 Platelet aggregation is induced by the action of endogenous agonists, such as collagen, thrombin, adenosine diphosphate, arachidonic acid, and platelet-activating factor.5 The inhibition of platelet function thus represents a promising approach for the treatment of thrombotic diseases. Many antiplatelet drugs have been used clinically, though they have certain disadvantages such as notable side effects and inefficient therapy.6,7 Therefore, the pursuit of new antiplatelet drugs with more effective, safer, and fewer side effects is important. Saururus chinensis (Lour.) Baill. (Saururaceae) is a perennial fetid herb distributed in the temperate regions of Asia.8 S. chinensis is used in the traditional Chinese medicine of Taiwan for a variety of diseases, including hepatitis, pneumonia, edema, jaundice, and gonorrhea.9 Recent advances in phytochemistry and pharmacology provided some scientific basis for these medicinal applications. Lignans,10,11 phenylpropanoids,11 and anthraquinones and alkaloids10 were reported as constituents of S. chinensis. Of these constituents, sauchinone showed neuroprotective,12 anti-inflammatory,13 and hepatoprotective14 activ© 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The aerial parts of S. chinensis were extracted with EtOH, and the extract was partitioned successively with n-hexane, EtOAc, and n-BuOH. The EtOAc fraction was chromatographed over silica gel, Sephadex LH-20, and reversed-phase C18 to afford 28 compounds. Compounds 1−4 (Table 1) were characteristic of 8,8′-dimethylbutane lignans since each exhibited two methyl doublets (H-9 and H-9′), two methine groups (H-8 and H-8′), two sets of benzylic methylene resonances (H2-7 and H2-7′), and aromatic proton resonances in their 1H NMR spectra. The differences observed in the NMR spectra of 1−4 were due to different substitution patterns in the aromatic rings. The Received: September 24, 2013 Published: January 3, 2014 125

dx.doi.org/10.1021/np400772h | J. Nat. Prod. 2014, 77, 125−131

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Table 1. 13C and 1H NMR Data of Compounds 1−5 (δ in ppm and J in Hz)a 1 position 1 2 3 4 5 6 7

8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′

8′ 9′ −OCH2O− 3′-OMe 4′-OMe 6′-OMe a

δC 121.1, 150.8, 98.4, 147.1, 141.5, 100.9, 36.2,

C C CH C C CH CH2

37.6, CH 14.3, CH3 134.6, 113.4, 148.7, 145.3, 115.8, 122.5,

C CH C C CH CH

42.2, CH2

39.1, CH 14.4, CH3 101.8, CH2 56.3, CH3

2 δH

6.31, s

6.41, s 2.30, dd (13.2, 7.8) 2.55, dd (13.2, 6.6) 1.78, m 0.80, d (7.2)

6.59, d (1.8)

6.64, d (7.8) 6.50, dd (7.8, 1.8) 2.34, dd (13.2, 7.8) 2.51, dd (13.2, 6.6) 1.75, m 0.82, d (6.6) 5.78, s 3.77, s

δC 119.3, 148.0, 98.2, 145.9, 141.0, 110.1, 35.1,

C C CH C C CH CH2

3 δH

6.36, s

6.49, s 2.32, m

δC 119.2, 147.9, 98.1, 146.0, 141.2, 110.1, 34.9,

C C CH C C CH CH2

2.59, m 37.1, CH 14.3, CH3 118.2, 113.6, 140.4, 144.2, 102.9, 148.0,

C CH C C CH C

35.4, CH2

6.40, s

37.1, CH 14.4, CH3 118.0, 114.4, 142.9, 147.8, 100.9, 147.3,

2.32, m

C CH C C CH C

35.3, CH2

2.59, m 37.3, CH 14.4, CH3 100.8, CH2 56.7, CH3

1.78, m 0.84, d (6.6) 5.84, s 3.78, s

6.34, s

6.49, s 2.33, m

δC 119.5, 148.1, 98.1, 145.7, 141.0, 110.0, 34.7,

C C CH C C CH CH2

2.58, m

1.78, m 0.83, d (6.6) 6.49, s

4 δH

1.78, m 0.84,d (6.6) 6.52, s

6.38, s

2.33, m

100.8, CH2 56.6, CH3 56.0, CH3

1.78, m 0.85, d (6.6) 5.85, s 3.77, s 3.81, s

6.35, s

6.49, s 2.28, m

δC 119.2, 148.2, 98.2, 145.9, 140.9, 110.1, 35.3,

C C CH C C CH CH2

2.56, m 36.9, CH 14.2, CH3 122.8, 116.5, 138.9, 144.6, 97.2, 151.1,

C CH C C CH C

35.1, CH2

2.58, m 37.3, CH 14.4, CH3

5 δH

1.75, m 0.80,d (6.6) 6.64, s

6.45, s

2.33, m

35.5, CH 14.0, CH3 149.7, 187.6, 107.7, 158.4, 182.3, 131.3,

C C CH C C CH

34.1, CH2

2.56, m 37.0, CH 14.3, CH3 100.8, CH2

1.75, m 0.81, d (6.6) 5.83, s

56.2, CH3 56.6, CH3

3.84, s 3.70, s

37.1, CH 14.3, CH3

δH

6.36, s

6.48, s 2.30, dd (14.5, 7.5) 2.54, dd (14.5, 6.0) 1.75, m 0.79, d (6.5)

5.87, s

6.39, s 3.33, dd (14.5, 7.5) 2.48, dd (14.5, 5.5) 1.77, m 0.83, d (6.5)

110.1, CH2

5.83, s

56.2, CH3

3.77, s

Compound 1 measured in methanol-d4; 2−5 measured in CDCl3.

molecular formula of 1 was established as C20H24O5 by HRESIMS at m/z 343.1535 [M − H]− (calcd for C20H23O5: 343.1545). In addition to signals of the 8,8′-dimethylbutane group, two aromatic singlets [δ 6.31 (H-3) and 6.41 (H-6)], an aromatic ABC spin system [δ 6.64 (d, J = 7.8 Hz, H-5′)/6.50 (dd, J = 7.8, 1.8 Hz, H-6′)/6.59 (d, J = 1.8 Hz, H-2′)], a methylenedioxy [δ 5.78 (s)], and a methoxy (δ 3.77) group were evident in the 1H NMR spectrum of 1. In its HMBC spectrum, the signal at δ 6.41 (H-6) correlating to C-7 (δ 36.2), C-2 (δ 150.8), and C-4 (δ 147.1), the signal at δ 6.31 (H-3) correlating to C-1 (δ 121.1) and C-5 (δ 141.5), and the signal at δ 5.78 correlating to C-4 and C-5 suggested that a 2-hydroxy4,5-methylenedioxyphenyl group was present. In addition, HMBC correlations of H-2′ and H-6′ to C-7′ (δ 42.2) and C-4′ (δ 145.3) and of −OCH3 (δ 3.77) and H-5′ to C-3′ confirmed the partial structure of a 4-hydroxy-3-methoxyphenyl group. The 1H NMR data of 1 showed large coupling constants (J = 7.2 and 6.6 Hz) for the 9- and 9′-methyl protons as observed in saururin A (11),17 which indicated they had the same relative configuration. Thus, 1 was identified as rel(8S,8′R)-7′-(4-hydroxy-3-methoxyphenyl)-7-(2-methoxy-4,5methylenedioxyphenyl)-8,8′-dimethylbutane and was named saurulignan A. HRESIMS analyses indicated that saurulignans B−D (2−4) had the molecular formulas C20H24O6, C21H26O6, and C21H26O6, respectively. Comparison of the 1H and 13C NMR data of 2−4 with those of 1 revealed that the 3,4-disubstituted phenyl group in 1 was replaced by a 2,4,5-trisubstituted phenyl

moiety in 2−4. In the latter aromatic ring of 2−4, two aromatic singlets and one methoxy singlet in 2 and two methoxy singlets in 3 and 4 were observed. The locations of these substituents were deduced by NOESY and HMBC experiments. The NOESY spectrum of 2, with the correlations of H-6′ with H-7′ and OMe-5′, together with the HMBC correlations of H-6′ to C-7′ and C-4′, H-3′ to C-1′ and C-5′, and MeO-5′ to C-5′, 126

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Table 2. 13C and 1H NMR Data of Compounds 6−10 (δ in ppm and J in Hz)a 6 position 1 2 3 4 5 6 7

8 9 1′ 2′ 3′ 4′ 5′ 6′

a

δC 136.5, 101.5, 150.6, 136.3, 145.0, 108.4, 88.9,

C CH C C C CH CH2

49.6, CH 14.9, CH3 135.1, 112.2, 150.2, 149.8, 112.6, 120.2,

C CH C* C* CH CH

7 δH 6.67, s

6.70, s 4.33, d (9.0)

1.73, m 1.05, d (6.6)

6.95, d (1.8)

7′

84.5, CH2

6.95, d (8.4) 6.82, dd (8.4, 1.8) 5.09, d (9.0)

8′ 9′

47.0, CH 15.3, CH3

2.26, m 0.63, d (7.2)

δC 138.2, 101.3, 150.5, 136.0, 144.9, 107.9, 88.9,

C CH C C C CH CH2

46.0, CH 12.9, CH3+ 135.9, 111.5, 150.5, 150.2, 112.8, 120.2,

C CH C# C# CH CH

8 δH 6.64, s

6.65, s 4.44, d (6.6)

2.28, m 1.04, d (6.6)

7.03, d (1.8)

88.8, CH2

6.93, d (8.4) 6.97, dd (8.4, 1.8) 4.46, d (6.6)

45.6, CH 13.2, CH3+

2.28, m 1.02, d (6.6)

OCH2O

102.7, CH2

5.96, s

102.6, CH2

5.91, s

OCH2O 5-OMe 3′-OMe 4′-OMe 5′-OMe

57.4, CH3 56.5, CH3 56.5, CH3

3.90, s 3.82, s 3.82, s

57.3, CH3 56.4, CH3 56.5, CH3

3.86, s 3.82, s 3.82, s

δC 137.9, 101.4, 150.6, 136.1, 144.9, 108.1, 88.8,

C CH CH C C CH CH2

45.6, CH 13.0, CH3 139.6, 104.6, 154.5, 138.4, 154.5, 104.6,

C C C C C CH

88.8, CH2 46.0, CH 13.2, CH3 102.6, CH2

57.3, 56.6, 61.1, 56.6,

9 δH

CH3 CH3 CH3 CH3

6.65, s 6.43, s

6.67, s 4.46, d (6.6)

2.28, m 1.01,d (6.6) 6.72, s

6.72, s 4.49, d (6.6) 2.28, m 1.04 (d, 6.6) 5.91, s CH2 3.87, 3.83, 3.87, 3.83,

δC 115.2, 150.8, 99.5, 147.7, 140.6, 106.7, 89.3,

C C CH C C CH CH2

48.1 CH 13.9 CH3 135.2, C 106.3CH 147.3, C 148.0, C 108.1, CH 119.6, CH

10 δH

6.43, s

6.44, s 4.69, d (9.6)

1.95, m 1.04, d (6.6)

6.85, d (1.8)

87.3, CH2

6.76, d (7.8) 6.78, dd (7.8, 1.8) 4.57, d (9.0)

50.4, CH 13.9, CH3

1.76, m 1.05 (d, 6.6)

δC 116.4, 146.5, 95.9, 145.3, 139.4, 123.1, 32.6,

C C CH C C C CH2

34.9, CH 20.0, CH3 139.9, C 108.9 CH 147.2, C 145.5, C 107.5, CH 122.0 CH

δH

6.28, s

2.20, dd (16.0, 3.5) 2.78, dd (16.0, 3.5) 1.48, m 1.07, d (6.0)

6.54, s

6.67, d (7.5) 6.60, d (7.5)

50.2 CH2

3.43, d (9.5)

44.7, CH 16.8, CH3

1.38, m 0.93, d (6.5)

101.0, CH2

5.87, s

100.4, CH2

101.1, CH2

5.94, s

100.7, CH2

5.51, s 5.60, s 5.89, s

s s s s

Compounds 6, 7, 8, and 10 measured in methanol-d4; 9 measured in CDCl3; *,#,+ the values may be exchanged with each other.

dd). The major NMR differences between 5 and 4 were found in one of the aromatic rings by comparison of their 1H and 13C NMR data. Two aromatic singlets at δ 5.87 (s, H-3′) and 6.39 (s, H-6′) and a methoxy singlet at δ 3.77 (OMe-4′) arising from one of the aromatic rings were observed. Two carbonyl resonances at δ 182.3 (s, C-5′) and 187.6 (s, C-2′) associated with the UV maxima absorption at 270 and 300 nm and IR absorptions at 1648 (CO) and 1603 (CC) cm−1 suggested the presence of a 1,4-benzoquinone system. HMBC cross-peaks of H-3′/C-1′ (δ 149.7) and C-5′ (δ 182.3), H-6′/C-2′ (δ 187.6), C-4′ (δ 158.4), and C-7′ (δ 34.1), and H-7′/C-2′ provided evidence of the 2-methoxy-1,4-benzoquinone moiety. Owing to the coupling constant (J = 6.5 Hz) and the chemical shifts of the C-9 and C-9′ carbons and protons, 5 had the same relative configuration as saururin A (11).17 Therefore, 5 was identified as rel-(8S,8′R)-7-(2-methoxy-4,5-methylenedioxyphenyl)-7′-(4-methoxy-1,4-benzoquinone)-8,8′-dimethylbutane. Saurufurin A (6) was isolated as a white, amorphous powder, [α]26D +18 (c 0.2, MeOH). Its HREIMS gave an [M]+ ion peak at m/z 386.1738, consistent with a molecular formula of C22H26O6. UV absorptions at 234 (sh.) and 279 nm suggested the presence of an aromatic moiety. The 1H NMR data (Table 2) showed two benzylic oxymethines (δ 4.33, d, J = 9.0 Hz; 5.09, d, J = 9.0 Hz), two methines (δ 1.73, m; 2.26, m), and two methyl groups (δ 1.05, d, J = 6.6 Hz; 0.63, d, J = 7.2 Hz),

confirmed a 2,4-dihydroxy-5-methoxyphenyl group. The NOESY spectrum of 3 showed correlations of H-6′ with H7′ and MeO-5′ and H-3′ with MeO-4′ and HMBC correlations of H-6′ to C-7′ and C-4′, H-7′ to C-2′ and C-6′, H-3′ to C-1′ and C-5′, MeO-4′ to C-4′, and MeO-5′ to C-5′, confirming the structure of the 2-hydroxy-4,5-dimethoxyphenyl group. The NOESY spectrum of 4 showed correlations of H-7′ with H-6′ and MeO-2′ and H-3′ with MeO-2′ and MeO-4′ and HMBC correlations of 4 of H-6′ to C-7′ and C-4′, H-7′ to C-2′ and C6′, H-3′ to C-1′ and C-5′, MeO-2′ to C-2′, and MeO-4′ to C-4′ to confirm the structure of the 5-hydroxy-2,4-dimethoxyphenyl group. Compounds 2−4 showed large coupling constants (J = 6.6 Hz) for the 9- and 9′-Me,17 and hence the structures were defined as rel-(8S,8′R)-7′-(2,4-dihydroxy-5-methoxyphenyl)-7(2-methoxy-4,5-methylenedioxyphenyl)-8,8′-dimethylbutane (2), rel-(8S,8′R)-7′-(2-hydroxy-4,5-dimethoxyphenyl)-7-(2-methoxy-4,5-methylenedioxyphenyl)-8,8′-dimethylbutane (3), and rel-(8S,8′R)-7′-(5-hydroxy-2,4-dimethoxyphenyl)-7-(2-methoxy-4,5-methylenedioxyphenyl)-8,8′-dimethylbutane (4). Saurulignan E (5) was obtained as an optically active oil with [α]26D −24 (c 0.5, MeOH). It possessed the molecular formula C20H22O6, as derived from the HRESIMS ion at m/z 357.1349 [M − H]− (calcd for C20H21O6: 357.1338). The 1H NMR spectrum of 5 showed characteristic signals for an 8,8′dimethylbutane lignan moiety (δ 0.79/0.83, each 3H, d, J = 6.5 Hz; 1.75/1.77, each 1H, m; 2.22/2.30, 2.48/3.33, each 1H, 127

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Table 3. Antiplatelet Aggregation Activitya of Isolated Compounds ADP-induced platelet aggregation

AA-induced platelet aggregation

compound

inhibition (%) at 100 μM

IC50 (μM)

2 3 4 5 6 7 10 14 17 19 20 21 23 25 26 27

68.2 ± 6.5 71.5 ± 0.7 85.2 ± 7.2 90.4 ± 5.7 19.1 ± 0.8 75.6 ± 3.8 84.0 ± 4.7 90.9 ± 3.9 92.1 ± 2.9 51.8 ± 2.2 not active 48.2 ± 19.0 86.7 ± 3.7 84.7 ± 5.9 65.2 ± 2.9 83.1 ± 0.8 80.2 ± 9.5 clopidogrel (30 μM)

N.D. N.D. 30.0 ± 2.7 9.8 ± 1.0 56.0 40.3 34.6 34.9

± ± ± ±

6.0 2.0 3.2 3.0

50.6 ± 8.0 28.1 ± 1.1

inhibition (%) at 100 μM 90.9 73.0 96.8 98.7 54.4 80.3 83.7 94.1 54.9 19.4 10.7 28.2 42.5 93.5 42.0 21.8 99.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7.3 4.4 1.3 0.2 5.6 8.2 6.2 3.2 4.0 3.0 4.9 2.2 5.0 3.0 5.1 8.7 0.5 aspirin (100 μM)

PAF-induced platelet aggregation

IC50 (μM) N.D. N.D. 29.4 ± 3.9 14.0 ± 5.0 85.0 ± 17.3 71.4 ± 31.6 25.9 ± 2.3

60.3 ± 0.7

inhibition (%) at 100 μM 11.7 7.0 26.7 92.0 42.0 98.9 0.6 3.9 18.4 94.4 11.8 23.6 5.3 68.1 27.3 6.8 94.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.0 8.9 9.0 4.6 5.4 0.9 0.5 1.5 4.6 3.0 0.9 5.9 5.3 10.3 6.0 3.3 4.3 CV3988 (10 μM)

IC50 (μM)

61.7 ± 2.9 37.2 ± 6.2

9.1 ± 3.8

79.4 ± 8.0

The antiplatelet aggregation activity was calculated by the following equation: Inhibition of platelet aggregation (%) = [1 − (platelet aggregation potency of sample/platelet aggregation potency of vehicle)] × 100%.

a

established as C20H20O6 from EIMS ([M]+ = m/z 356) and 13C NMR data, indicating that it contained one more oxygen atom than (−)-galbacin.25 The 1H and 13C NMR data of 9 (Table 2) are closely related to those of (−)-galbacin except for an aromatic system in which two singlets replaced the ABX signals in galbacin. The carbon resonance at δ 150.8 (s) in 9 was assigned to C-2, and the hydroxy group at C-2 caused the shift of C-1 to δ 115.2. HMBC cross-peaks of H-6 (δ 6.44)/C-2 and C-4 (δ 147.7) and H-3 (δ 6.43)/C-1 and C-5 (δ 140.6) confirmed the location of 2-OH. The 1H and 13C NMR data of 9 coincided with those of characteristic shifts for trans-oriented aryl/methyl and trans-oriented methyl/methyl substituents in the tetrahydrofuran ring.19,20 Therefore, the structure of 9 was deduced as rel-(7S,8S,7S,8′S)-2-hydroxy-4,5,3′,4′-dimethylenedioxy-7,7′-epoxylignan. Saurunarin (10), an optically active compound with [α]26D −9 (c 0.5, MeOH), had the molecular formula C20H20O5 as derived from the HRESIMS ion at m/z 341.1374 [M + H]+ (calcd for C20H21O5: 341.1389). The UV absorptions at 241 (sh.) and 289 nm suggested the presence of a benzenoid moiety. The 1H NMR data of 10 (Table 2) revealed the presence of a benzlic methine proton (δ 3.43, d, J = 9.5 Hz, H7′), two secondary methyl groups [δ 1.07 (d, Me-9′), 0.93 (d, Me-9′)], two methine protons [δ 1.48 (m, H-8), 1.38 (m, H8′)], and a methylene group (δ 2.20/2.78, each dd, H2-7) in the aliphatic region. The combination of one aromatic singlet (δ 6.28, H-3), a set of ABX signals (δ 6.54/6.60/6.67, H-2′/6′/5′), and two methylenedioxy signals (δ 5.51/5.60, 5.89) suggested that 10 is a phenyltetrahydronaphthalene-type lignan. The five oxygenated quaternary carbons at δ 139.4, 145.3, 145.5, 146.5, and 147.2 indicated that there should be one hydroxy group in the aromatic rings in addition to two methylenedioxy groups. The positions of methylenedioxy and hydroxy groups (C-2) were determined from HMBC data. HMBC correlations of H-7 to C-2 (δC 146.5)/C-6/C-8′ and H-7′ to C-1/-2′/-6′/-8′/-9′ were observed. The NOESY correlations of H-7′ with H3-9′ and H-8 revealed that the C-8 and C-8′ methyl groups were trans-oriented. Comparison of the spectral data and optical

suggesting 6 to be an asymmetric 8,8′-dimethyl-7,7′-disubstituted tetrahydrofuran lignan. Two singlets at δ 6.67 (H-2) and 6.70 (H-6), a methylenedioxy singlet at δ 5.96, and a methoxy singlet at δ 3.90 (OMe-3) revealed that one substituent was a 5-methoxy-3,4-methylenedioxyphenyl group. The remaining two methoxy groups at δ 3.82 (6H) and the protons of a 1,3,4-trisubstituted benzene ring at δ 6.95 (d, J = 8.4 Hz), 6.95 (d, J = 1.8 Hz), and 6.82 (dd, J = 8.4, 1.8 Hz) suggested that the other substituent was a 3,4-dimethoxyphenyl group. NOESY and HMBC experiments indicated a 3,4methylenedioxy-5,3′,4′-trimethoxy-7,7′-epoxylignan structure for 6. The relative configuration of 6 was identical to that of fragransin B318 on the basis of comparison of the specific rotation, coupling constants, and the chemical shifts of the aliphatic protons and carbons. Thus, the structure of 6 was defined as rel-(7S,8S,7S,8′R)-3,3′,4′-trimethoxy-4,5-methylenedioxy-7,7′-epoxylignan. Saurufurin B (7) was an oily substance, [α]26D −2 (c 0.2, MeOH), with the same UV spectrum as and identical molecular formula (m/z 386.1735 [M]+, calcd for C22H26O6, 386.1724) to 6. Comparison of 1H and 13C NMR data of 7 and 6 (Table 2) suggested that they differed in the configuration of the tetrahydrofuran ring. The proton and corresponding carbon shifts in the NMR spectra were characteristic for trans-oriented aryl/methyl and cis-oriented methyl/methyl substituents in the tetrahydrofuran ring.19,20 Therefore, the structure of 7 was deduced as rel-(7S,8S,7R,8′R)-5,3′,4′-trimethoxy-3,4-methylenedioxy-7,7′-epoxylignan. The 1H NMR spectrum of saurufurin C (8) (Table 2) was similar to that of 7 except for the presence of a methoxy singlet (δ 3.83) in place of an aromatic proton doublet, suggesting that the aromatic ring is a 3,4,5-trimethoxyl phenyl group. Compound 8 had the molecular formula C23H8O7 by analysis of the ESIMS and 13C NMR data, corresponding to the presence of an additional methoxy group compared to 7. Therefore, the structure of 8 was deduced as rel-(7S,8S,7R,8′R)5,3′,4′,5′-tetramethoxy-3,4-methylenedioxy-7,7′-epoxylignan. Saurufurin D (9) was isolated as an optically active yellowish oil, [α]26D −29 (c 0.2, MeOH). The molecular formula was 128

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measured on a JASCO P-2000 polarimeter. 1H, 13C, and 2D NMR spectra were measured with a Varian Unity Inova-500 spectrometer with deuterated solvents as internal standards. ESIMS and HRESIMS were recorded on Finnigan LCQ and Shimadzu LCMS-IT-TOF mass spectrometers, respectively. Column chromatography was performed on Sephadex LH-20 (Pharmacia) or silica gel 60 (70−230 or 230−400 mesh, Merck; or 12−26 μm, Eurochrom, Knauer). Silica gel 60F254 (Merck, Darmstadt, Germany) was used for TLC (0.25 mm). The preparative HPLC system consisted of a chromatographic pump (LC8A, Shimadzu, Kyoto, Japan) and a UV−visible detector (SPD-10A vp, Shimadzu, Kyoto, Japan). A Cosmosil 5C18-AR-II column (20 × 250 mm; particle size 5 μm; Nacalai tesque, Kyoto, Japan) or a LiChrosorb Si60 column (10 × 250 mm; particle size 7 μm; Merck, Darmstadt, Germany) was used for separation. Plant Material. Whole plants of Saururus chinensis were collected from the northeast coast of Taiwan during April 2010, and a voucher specimen was deposited in the National Research Institute of Chinese Medicine, Taipei, Taiwan. Extraction and Isolation. Aerial parts of S. chinensis (5.6 kg) were crushed and extracted with EtOH (80 L × 2) under reflux. The combined extracts were concentrated to about 1 L and extracted successively with n-hexane, EtOAc, and n-BuOH (each 1 L × 3). The EtOAc fraction (155 g) was subjected to column chromatography on silica gel (10 × 120 cm), with a gradient of EtOAc in n-hexane from 0 to100% and the last elution with 10% MeOH in EtOAc, to give 16 fractions (Fr. 1−16). Fr. 3 (2.0 g) was chromatographed on SephadexLH-20 with 5% CHCl3/MeOH as eluent to give 10 subfractions, 3A− 3J. Fr. 3J showed a homogeneous spot on TLC, and it was concentrated to give 26 (1.13 g). Fr. 3H and 3I were individually chromatographed by preparative HPLC (column: Cosmosil 5C18-ARII, 20 × 250 mm, 5 μm, solvent: 80% ACN/H2O, flow rate: 18.9 mL/ min, UV: 210 nm) to give 15 (5.7 mg) and 16 (4.8 mg) from Fr. 3H as well as 9 (7.8 mg), 17 (4.1 mg), and 23 (100.6 mg) from Fr. 3I. In addition, Fr. 5 (13.6 g) was chromatographed on silica gel with 50%, 70%, and 100% CHCl3/n-hexane (each 2 L) as eluents to give six subfractions, 5A−5F. The precipitate was separated from Fr. 5B and recrystallized from MeOH to give 20 (1.02 g). The combined filtrate of Fr. 5B and Fr. 5C (62.6 g) was chromatographed on a Sephadex LH-20 column (5% CHCl3/MeOH) to give 22 (1.3 g) and Fr. 5C2. Fr. 5C2 was further purified by preparative HPLC (column: LiChrosorb Si60, 10 × 250 mm, 7 μm, solvent: 5% EtOAc/n-hexane, flow rate: 7.0 mL/min, UV: 210 nm) to give 20 (36.6 mg) and 21 (22.2 mg). In addition, Fr. 5D (6.4 g) yielded 28 (405.2 mg) after purification on a Sephadex LH-20 column (EtOAc). Fr. 6 (25.6 g) was chromatographed on silica gel with a gradient solvent of CHCl3 in nhexane from 50% to 100% to give 10 subfractions, 6A−6J. Fr. 6C (2.7 g) was repeatedly chromatographed on Sephadex-LH-20 (10% EtOAc/MeOH) and preparative HPLC columns (column: Cosmosil 5C18-AR-II, 10 × 250 mm, 5 μm, solvent: 40−60% ACN/H2O, flow rate: 4.7 mL/min, UV: 254 nm) to give 6 (8.2 mg), 7 (39.8 mg), 12 (1.41 g), 13 (38.9 mg), 14 (1.3 mg), 18 (2.7 mg), and 24 (24.4 mg). Fr. 6D (10.1 g) was chromatographed on Sephadex-LH-20 (10% EtOAc/MeOH) to give eight subfractions, Fr. 6D1−6D8. Of these, Fr. 6D3, Fr. 6D4, and Fr. 6D5 were chromatographed on Sephadex LH20 (10% EtOAc/MeOH) to give 25 (26.4 mg from Fr. 6D3), 8 (36.7 mg), 19 (7.6 mg from Fr. 6D4), and 1 (9.0 mg from Fr. 6D5). Fr. 6H (6.4 g) gave 2 (19.1 mg), 3 (17.7 mg), 4 (101.1 mg), 5 (46.7 mg), 10 (7.6 mg), and 11 (2.5 g) after repeated chromatography on Sephadex LH-20 (acetone, MeOH) and silica gel columns (Europrep, 12−26 μm, eluted with a gradient solvent of 2−10% MeOH/CHCl3). Finally, Fr. 16 (1.5 g) was chromatographed on Sephadex LH-20 with MeOH as eluent to give 27 (83.3 mg). Saurulignan A (1): brown, amorphous powder; UV (MeOH) λmax (log ε) 231 sh. (3.88), 284 (3.56), 307 sh. (3.38) nm; [α]26D −7 (c 0.2, MeOH); IR (KBr) νmax 3475, 2956, 1612, 1511, 1445, 1376, 1269, 1155, 1037 cm−1; 1H, 13C NMR data, see Table 1; ESIMS m/z 343 [M − H]−; HRESIMS m/z 343.1535 [M − H]− (calcd for C20H23O5, 343.1545). Saurulignan B (2): brown, amorphous powder; UV (MeOH) λmax (log ε) 297 (2.88) nm; [α]26D −3 (c 0.5, MeOH); IR (KBr) νmax 3447,

rotations led to the conclusion that 10 had the same (7′S,8R,8′S) configuration as (−)-cagayanin.21 The other compounds were characterized as saururin A (11),17 (+)-dihydroguaiaretic acid (12),22 (−)-dihydroguaiaretic acid (13),22 saucerneol G (14),23 (8S,8′R)-3,4,3′,4′dimethylenedioxy-7-oxo-8,8′-neolignan (15),24 (−)-galbacin (16),25 d-epigalbacin (17),25 futokadsurin C (18),26 di-Omethyltetrahydrofuriguaiacin B (19),27 sauchinone (20), sauchinone A (21), (−)-licarin A (22),28 (−)-licarin B (23), bisnorneolignan aldehyde (24),29 (+)-7-acetylraphidecursinol B (25),30 saurufuran (26),31 quercetin 3-O-rhamnoside (27), and β-sitosterol (28). We evaluated 21 of the 28 isolated compounds for their inhibitory effects on the ADP-, AA-, and PAF-induced washed rabbit platelet aggregation (Table 3). Clopidogrel (30 μM), aspirin (100 μM), and CV3988 (10 μM) were used as positive controls, respectively. In addition to antiplatelet activity, five of the 21 compounds induced platelet aggregation without additional stimuli (Table 4). Table 4. Prothrombotic Activitya of Isolated Compounds compound

% increase in platelet aggregation at 100 μM

1 11 12 13 22

66.1 49.5 74.5 76.4 66.8

± ± ± ± ±

3.1 4.4 6.8 7.1 3.0

a

The prothrombotic activity was determined by the % increase in platelet aggregation, which was induced by compound alone without adding stimuli.

In the ADP-induced aggregation assay, eight compounds exhibited significant inhibitory effects. Of these, 5 showed inhibitory activity with IC50 values of 9.8 ± 1.0 μM; 25, 4, 14, and 17 showed moderate inhibitory activity with IC50 values of 28−35 μM; and 10, 23, and 7 exhibited weaker inhibitory activity with IC50 values ranging from 40 to 56 μM. In the AAinduced aggregation assay, six compounds exhibited significant inhibitory effects. Compound 5 showed potent inhibitory activity with IC50 values of 14.0 ± 5.0 μM; 4 and 14 showed moderate inhibitory activity with IC50 values of 26−30 μM; and 25, 10, and 7 exhibited weaker inhibitory activity with IC50 values of 60−85 μM. In the PAF-induced aggregation assay, four compounds exhibited significant inhibitory effects. Of these, 19 showed inhibitory activity with IC50 values of 9.1 ± 3.8 μM; 7 showed moderate inhibitory activity with IC50 values of 37.2 ± 6.2 μM; and 5 and 25 exhibited weaker inhibitory activity with IC50 values of 62−80 μM. Analysis of the specificity of antiplatelet aggregation activity showed that 17 and 23 specifically inhibited ADP-induced platelet aggregation, and 19 specifically and potently inhibited PAF-induced platelet aggregation. Compounds 4, 14, and 10 also inhibited ADP- and AA-induced platelet aggregations, while 5, 7, and 25 inhibited three stimuli-induced platelet aggregations. In addition, that compounds 1, 11, 12, 13, and 22 alone induced platelet aggregation, indicting that these five compounds could have prothrombotic activity.

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EXPERIMENTAL SECTION

General Experiment Procedures. IR spectra were obtained on a Nicolet Avatar 320 IR spectrometer. UV spectra were measured on a Hitachi U-3200 spectrophotometer in MeOH. Optical rotations were 129

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2924, 1621, 1516, 1448, 1382, 1202, 1174, 1082 cm−1; 1H, 13C NMR data, see Table 1; ESIMS m/z 361 [M + H]+; HRESIMS m/z 361.1650 [M + H]+ (calcd for C20H25O6, 361.1651). Saurulignan C (3): light yellow, amorphous powder; UV (MeOH) λmax (log ε) 233 (3.02), 276 (2.85), 297 (2.83) nm; [α]26D −2 (c 0.5, MeOH); 1H, 13C NMR data, see Table 1; ESIMS m/z 373 [M − H]−; HRESIMS m/z 373.1653 [M − H]− (calcd for C21H25O6, 373.1651). Saurulignan D (4): brown gum; UV (MeOH) λmax (log ε) 231 (3.88), 298 (3.84) nm; [α]26D −29 (c 1, MeOH); IR (KBr) νmax 3403, 2958, 1614, 1505, 1444, 1334, 1281, 1170, 1036 cm−1; 1H, 13C NMR data, see Table 1; ESIMS m/z 373 [M − H]−; HRESIMS m/z 375.1798 [M + H]+ (calcd for C21H27O6, 375.1808). Saurulignan E (5): brown, oily substance; UV (MeOH) λmax (log ε) 270 (3.91), 300 (3.58) nm; [α]26D −24 (c 0.5, MeOH); IR (KBr) νmax 3427, 2961, 1646, 1605, 1504, 1481, 1443, 1370, 1223, 1173, 1036 cm−1; 1H, 13C NMR data, see Table 1; ESIMS m/z 357 [M − H]−; HRESIMS m/z 357.1349 [M − H]− (calcd for C20H21O6, 357.1338). Saurufurin A (6): white, amorphous powder; UV (MeOH) λmax (log ε) 234 sh. (4.12), 279 (3.68) nm; [α]26D +18 (c 0.2, MeOH); IR (KBr) νmax 2960, 2871, 1633, 1513, 1456, 1434, 1321, 1248, 1134, 1093, 1028 cm−1; 1H, 13C NMR data, see Table 2; EIMS m/z 386 [M]+; HREIMS m/z 386.1738 [M]+ (calcd for C22H26O6, 386.1724). Saurufurin B (7): light yellow oil; UV (MeOH) λmax (log ε) 233 sh. (4.24), 278 (3.73) nm; [α]26D −2 (c 0.2, MeOH); IR (KBr) νmax 2962, 2908, 2871, 1635, 1512, 1453, 1322, 1264, 1235, 1133, 1093, 1031 cm−1; 1H, 13C NMR data, see Table 2; EIMS m/z 386 [M]+; HREIMS m/z 386.1735 [M]+ (calcd for C22H26O6, 386.1724). Saurufurin C (8): light yellow oil; UV (MeOH) λmax (log ε) 214 sh. (4.60), 240 sh. (4.26), 276 (3.47) nm; [α]26D +2 (c 1, MeOH); IR (KBr) νmax 2957, 1635, 1592, 1506, 1459, 1426, 1328, 1234, 1128, 1040 cm−1; 1H, 13C NMR data, see Table 2; EIMS m/z 439 [M + Na]+; HREIMS m/z 417.1897 [M + H]+ (calcd for C23H29O7, 417.1913). Saurufurin D (9): light yellow oil; UV (MeOH) λmax (log ε) 237 (3.32), 294 (3.25) nm; [α]26D −29 (c 0.2, MeOH); [α]26D −59 (c 0.5, CHCl3); 1H, 13C NMR data, see Table 2; EIMS m/z 356 [M]+; HREIMS m/z 355.1194 [M − H]− (calcd for C20H19O6, 355.1182). Saurunarin (10): brown, amorphous powder; UV (MeOH) λmax (log ε) 241 (3.0), 289 (2.9) nm; [α]26D −9 (c 0.5, MeOH); IR (KBr) νmax 3387, 2924, 1627, 1491, 1453, 1381, 1233, 1038 cm−1; 1H, 13C NMR data, see Table 2; ESIMS m/z 339 [M − H]−; HRESIMS m/z 341.1374 [M + H]+ (calcd for C20H21O5, 341.1389). Preparation of the Platelet Suspension. A washed platelet suspension was prepared as previously described with some modifications.32−34 Briefly, blood was collected from the marginal ear vein of New Zealand white rabbits into tubes containing one-sixth volume of acid-citrate dextrose as anticoagulant. The blood was centrifuged at 1000g for 8 min at room temperature. The upper portion was kept as platelet-rich plasma after mixing with EDTA to a final concentration of 5 mM and recentrifuged at 2000g for 12 min. The platelet pellet was suspended in modified Ca2+-free Tyrode’s buffer (137 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 0.33 mM NaH2PO4, 5 mM glucose, 10 mM HEPES) with 0.35% bovine serum albumin, heparin (50 unit/mL), and apyrase (1 unit/mL) and was incubated at 37 °C for 20 min. After centrifugation at 2000g for 6 min, the washed platelet pellet was resuspended in Tyrode’s buffer containing 1 mM Ca2+. For the aggregation test, the platelet numbers were counted by hemacytometer calculator and adjusted to 2.5 × 108 platelets/mL. Measurement of Platelet Aggregation. Platelet aggregation was measured turbidimetrically by the PACK4 platelet aggregation chromogenic kinetic system (Helena Laboratories, Beaumont, TX, USA) with some modifications.33,34 The platelet suspension was stirred at 900 rpm and incubated with an appropriate amount of vehicle, test compounds in DMSO, and positive control (30 μM clopidogrel, 100 μM aspirin, or 10 μM PAF receptor antagonist CV3988) at 37 °C for 2 min. Platelet aggregation was induced with ADP (20 μM), arachidonic acid (100 μM), or PAF (10 nM) for another 4 min. The light transmission of washed platelet suspension was assigned the value of 0% aggregation, while light transmission

through Tyrode’s buffer was assigned the value of 100% aggregation. The extent of platelet aggregation was measured as the maximal increase in light transmission within 4 min after the addition of an inducer. To eliminate or minimize any possible effects of the solvent, the final concentration of DMSO in the platelet suspension was fixed at 0.5%. The antiplatelet aggregation activity (%) was calculated by the following equation: Inhibition of platelet aggregation (%) = [1 − (platelet aggregation potency of sample /platelet aggregation potency of vehicle)] × 100% The prothrombotic activity was determined by the % increase in platelet aggregation, which was induced by compound alone without additional stimuli.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +886-2-28201999, ext. 7101. Fax: +886-2-28264276. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by the grants from National Science Council, ROC (NSC 100-2320-B-077-002), and from National Research Institute of Chinese Medicine, Taipei, Taiwan, ROC (NRICM 100-DHM-02).

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