Seven new sesquiterpenoids from the fruits of Schisandra sphenanthera.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)

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Seven New Sesquiterpenoids from the Fruits of Schisandra sphenanthera by Yao-Ching Tsai a ), Yuan-Bin Cheng a ) b ), I-Wen Lo a ), Ho-Hsi Cheng a ), Ching-Jie Lin a ), Tsong-Long Hwang c ), Yuh-Chi Kuo d ), Shorong-Shii Liou e ), Yi-Zsau Huang f ), Yao-Haur Kuo f ), and Ya-Ching Shen* a ) a

) School of Pharmacy, College of Medicine, National Taiwan University, Jen-Ai Rd. Sec. 1, Taipei 100, Taiwan (phone: þ 886-2-23123456, ext. 62226; fax: þ 886-2-23919098; e-mail: [email protected].) b ) Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan c ) Graduate Institute of Natural Products, Chang Gung University, Taoyuan, Taiwan d ) Department of Life Science, Fu-Jen University, Taipei Hsien, Taiwan e ) Department of Pharmacy, Tajen University, Ping-Tung, Taiwan f ) National Research Institute of Chinese Medicine, Taipei, Taiwan

Fractionation of the EtOH extract from the fruits of Schisandra sphenanthera resulted in the isolation of seven new sesquiterpenoids, 1 – 7, in addition to the known metabolites 8 – 23. Among them, schiscupatetralin A (1) possesses an unprecedented structure with a CC bond between cuparenol and tetralin. The isolated new compounds were evaluated for their anti-HSV-1 and anti-inflammatory activities. The results revealed that compound 4 exhibited anti-HSV-1 activity, while compound 6 showed a significant anti-inflammatory activity.

Introduction. – Schisandra sphenanthera Rehder et E.H.Wilson (Nan-Wuweizi, Schisandraceae) is a deciduous climber growing in western and southern China. It is different from S. chinensis (Bei-Wuweizi), which is widely grown and cultivated in northern China. Both plants had been described in ancient Chinese Material Medica as a superior drug (Wuweizi) more than a thousand years ago, implying that these fruits are edible and can be consumed for a long time without toxic side effects. In general, the commercial Bei-Wuweizi is prepared as a black product due to treatment with vinegar and wine. However, the commercial Nan-Wuweizi was simply dried without adding wine or vinegar, and heating. It was assumed that the character of Bei-Wuweizi is warmer and more useful than Nan-Wuweizi in kidney therapy in Chinese medicine. According to previous reports, the fruits of S. sphenanthera were used in traditional Chinese medicine for the treatment of chronic cough, diabetes, and insomnia, and as a tonic remedy [1]. Pharmacological studies revealed that the extracts of Nan-Wuweizi exhibited antiviral, antitumor, anti-inflammatory, and antioxidative activities [2] [3]. Previously, several lignans and triterpenoids were isolated from the stems and leaves [3 – 11]. Recent chemical investigations of this species led to the isolation of seven new lignans [12] and three new sesquiterpenes from the fruits of S. sphenanthera [13]. Some sesquiterpenes and lignans were found to possess immuno-modulatory and anti-liver fibrotic activities, respectively. Continued investigation of the constituents of S. sphenanthera has resulted in the isolation of seven new sesquiterpenoids (Fig. 1). Among them, schiscupatetralin A (1) 2014 Verlag Helvetica Chimica Acta AG, Zrich

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is a novel compound, a CC coupling product between a cuparenol skeleton and an arylated tetralin derivative. Others are ()-cuparenic acid methyl ester (2), ()-cuparenol methyl ether (3), ()-15-methoxy-3,6-peroxocupar-1-ene (4), (þ)-g-norcuparenol (5), schisansphenin C (6), and schisansphenin D (7). In addition, 16 known compounds were also isolated and characterized, i.e., methylgomisin O (8) [14], arisantetralone A (9) [15], arisantetralone C (10) [15], gomisin C (11) [16], methylgomisin R (12) [17], gomisin S (13) [18], interiotherin A (14) [4], macelignan (15) [19], deoxyschizandrin (16) [20], campherenol (17) [21], chamigrenal (18) [22], gcuparenal (19) [23], cuparenic acid (20) [24], d-cuparenol (21) [25], p-hydroxybenzoic acid (22) [26], and kadsuric acid (23) [27]. Their structures were elucidated on the basis of spectroscopic analysis such as 1D- and 2D-NMR (HMQC, HMBC, COSY, and NOESY) techniques, and physical methods such as IR, UV, CD, and HR-MS. These compounds were evaluated for their anti-HSV-1 and anti-inflammatory activities. Results and Discussion. – Extensive column and HPLC chromatographic separations of the EtOH extracts of the fruits of S. sphenanthera afforded seven new compounds. The structure elucidations, biological activities, and a plausible biogenetic pathway leading to 1 are discussed below. Compound 1 was isolated as pale amorphous powder. Its molecular formula, C35H42O4 , was deduced from a quasi-molecular-ion peak at m/z 527.3167 ([M þ H] þ ) and DEPT spectra. The IR absorptions at 3520, 1615, and 1590 cm 1, and UV bands at 216, 241, and 290 nm indicated that 1 contained OH and aromatic groups. The 1H-NMR spectrum of 1 (Table 1) exhibited three Me singlets (d(H) 0.53, 1.02, and 1.21), two Me doublets (d(H) 0.88 (J ¼ 6.9) and 0.89 (J ¼ 6.9)), a MeO singlet (d(H) 3.75), two aromatic doublets (d(H) 7.13 (J ¼ 8.1) and 7.22 (J ¼ 8.1, 4 H)), four aromatic singlets (d(H) 6.03, 6.25, 6.35, and 6.55), and a OCH2O singlet (d(H) 5.84). The 13C-NMR spectrum (Table 1) showed 16 aromatic C-atom signals (d(C) 104.4, 108.2, 109.6, 110.3, 112.9, 127.1, 127.4, 129.4, 130.3, 137.1, 145.1, 145.6, 145.8, 146.9, 154.2, and 158.1), suggesting that 1 possesses three aromatic rings. One of them is a 1,4-disubstituted benzene moiety due to symmetrical signals at d(C) 127.1 and 127.4. In addition, DEPT spectra disclosed five Me groups (d(C) 15.4, 16.3, 24.2, 24.3, and 26.4), a MeO group (d(C) 55.7) and a OCH2O group (d(C) 100.5), five additional CH2 groups (d(C) 19.7, 28.0, 35.2, 36.8, and 39.7), three aliphatic CH groups (d(C) 28.6, 40.3, and 51.8), and two quaternary C-atoms (d(C) 44.2 and 50.3). The COSY correlations of HC(10’’)/HC(9’’), HC(11’’), and HC(1’’,5’’)/ HC(2’’,4’’)), and HMBC correlations (Fig. 2) of HC(14’’)/C(7’’), C(8’’,9’’), C(13’’); HC(12’’)/C(7’’), C(8’’), C(11’’); and HC(9’’)/C(7’’) suggested that 1 contains a cyclopentane subunit, attached to the 4-disubstituted benzene ring. HMBC HC(12’’)/ C(6’’) and HC(1’’,5’’)/C(7’’) revealed that the two connecting C-atoms were C(6’’) and C(7’’), respectively. Thus, compound 1 has a partial structure involving a cuparanoid sesquiterpene. The HMBCs HC(15’’) (d(H) 3.94)/C(3’), C(4’), and C(5’); HC(2’)/C(3’), C(4’), and C(6); HC(6’)/C(1’), C(4’), and C(5’); and MeOC(5’)/C(5’) and OH/C(3’) revealed the presence of an aromatic ring encompassing C(1’) to C(6’). In addition, the HMBCs HC(7’)/C(2), C(1’), C(2’), and C(6’); HC(9’)/C(8), C(7’), C(8’); HC(3)/ C(7’); HC(9)/C(7), C(8), C(8’); and HC(7)/C(1), C(2), C(6), C(8’) indicated the


Fig. 1. Structures of compounds 1 – 23

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Table 1. 1H- and 13C-NMR Data of Compound 1. d in ppm, J in Hz. Atom numbering as indicated in Fig. 1. Position 1 2 3 4 5 6 7 8 9 10 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ a

d( H ) a )

6.35 (s)

6.55 (s) 2.42 (dd, J ¼ 16.5, 8.0, Hb ), 2.81 (dd, J ¼ 16.5, 5.3, Ha ) 2.00 – 2.03 (m) 0.88 (d, J ¼ 6.9) 5.84 (s) 6.03 (s)

6.25 (s) 3.60 (d, J ¼ 5.3) 1.92 – 1.98 (m) 0.89 (d, J ¼ 6.9)

d(C ) b ) 129.4 (s) 130.3 (s) 110.3 (d) 145.6 (s) c ) 145.8 (s) c ) 108.2 (d) 35.2 (t) 28.6 (d) 16.3 (q) 100.5 (t) 146.9 (s) 109.6 (d) 154.2 (s) 112.9 (s) 158.1 (s) 104.4 (d) 51.8 (d) 40.3 (d) 15.4 (d)

Position 1’’, 4’’ 2’’, 5’’ 3’’ 6’’ 7’’ 8’’ 9’’ 10’’ 11’’ 12’’ 13’’ 14’’ 15’’ 3’-OH 5’-MeO

d( H )

d(C )

7.22 (d, J ¼ 8.1) 7.13 (d, J ¼ 8.1)

127.1 (d) 127.4 (d) 137.1 (s) 145.1 (s) 50.3 (s) 44.2 (s) 39.7 (t)

1.50 – 1.55 (m), 1.62 – 1.68 (m) 1.71 – 1.77 (m) 1.60 – 1.66 (m, Ha ), 2.42 – 2.48 (m, Hb ) 1.21 (s) 0.53 (s) 1.02 (s) 3.94 (s) 4.60 (br. s) 3.75 (s)

19.7 (t) 36.8 (t) 24.3 (q) 26.4 (q) 24.2 (q) 28.0 (t) 55.7 (q)

) Recorded at 400 MHz. b ) Recorded at 100 MHz. c ) Assignments may be interchanged.

linkages between C(1)/C(7), C(2)/C(7’), and C(1’)/C(7’). The above observations determined the partial structure of the aryltetralin subunit. Finally, the key HMBC HC(15’’)/C(3’’), C(2’’,4’’) confirmed the connection of C(4’) of the aryltetraline with C(15’’) of the cuparane unit (Fig. 2). The relative configuration of 1 was determined by NOESY experiments and by comparison with the NMR data of known derivatives. The NOESY correlations

Fig. 2. 1H,1H-COSY (— —) and HMB (H ! C) correlations of compound 1


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(Fig. 3) HC(7’)/HC(9’), HC(9); HbC(7)/HC(9); HaC(7)/HC(8), HC(8’); HC(7’)/HC(2’), HC(6’), HC(9’), HC(9); HC(8’)/HC(2’), HC(6’); HC(6’)/ MeOC(5’), and HC(2’)/HOC(3’) suggested that the substituent at C(7’) favored aorientation, and MeC(9), MeC(9’), and HC(7’) were all on the b-face. Comparison of the NMR data of C(8), C(7’), and C(8’) in 1 with those of isogalbulin [28] also agreed with (R)-configurations at C(8), C(7’), and C(8’). On the other hand, NOESY correlations HC(1’’)/HC(12’’), HC(13’’), HC(14’’), HaC(11’’), HbC(11’’), HC(10’’); HC(13’’)/HbC(11’’); HC(15’’)/HC(2’’,4’’)), HOC(3’); and HC(2’’,4’’))/ HOC(3’), MeOC(5’) indicated the relative configuration of cuparane. Moreover, the CD spectrum of 1 displayed a negative Cotton effect at 294 nm confirming that C(7’) was (R)-configured [29]. The relative configuration at C(7’’) was tentatively assigned as (S) on the basis of biogenetic consideration and negative optical rotations of 1 – 3, whereas a positive value

Fig. 3. Key NOESY (H $ H) correlations and computer-generated perspective models of 1 using MM2 force-field calculation

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([a] ¼ þ 7.0) was found for cuparenic acid [24]. A molecular model for 1 was generated by CS Chem 3D version 9.0 using MM2 force-field calculation for energy minimization (28.6928 kcal/mol) as illustrated in Fig. 3. Therefore, the structure of compound 1 was established and a trivial name schiscupatetralin A was given. A plausible biogenetic pathway for compound 1 is proposed in Fig. 4. The intermediate c may be produced from nucleophilic attack of intermediate a at the benzylic position of b via Friedel Craft type alkylation. Compound 2 was isolated as pale-yellow oil and had a molecular formula C16H22O2 as deduced from a quasi-molecular-ion peak at m/z 269.1521 ([M þ Na] þ ) in the HREI-MS. The IR absorption bands (3094, 1723, and 1609 cm 1) and UV (lmax at 242 nm) suggested that 2 contained an aromatic ring and an ester function. In the 1H-NMR spectrum of 2 (Table 2), three Me singlets (d(H) 0.52, 1.06, and 1.26), a MeO singlet (d(H) 3.88), and two aromatic doublets (d(H) 7.39 (J ¼ 8.3), 7.91 (J ¼ 8.3)) were observed. Inspection of the 13C-NMR spectrum (Table 3) revealed that compound 2 contained an ester C¼O C-atom (d(C) 167.2) and four aromatic C-atoms (d(C) 127.0, 127.3, 128.8, and 153.3) attributed to a symmetrical 1,4-disubstituted benzene system. In addition, the presence of three Me groups (d(C) 24.2, 24.2, and 26.3), a MeO group (d(C) 51.9), three CH2 groups (d(C) 19.7, 36.7, and 39.7), and two quarternary C-atoms (d(C) 44.5 and 51.0) were deduced from DEPT. The COSY correlations CH2(9)/CH2(10), CH2(10)/CH2(11); HC(1,5)/HC(2,4), and HMBCs Me(14)/C(7), C(9), and C(13); Me(12)/C(8); CH2(9)/C(13); and CH2(11)/C(7) suggested that compound 2 possessed a five-membered carbocyclic ring as in the case of 1. HMBCs Me(12)/C(6) and HC(1)/C(7) indicated that the fivemembered ring and the aromatic ring are connected by a bond between C(6) and C(7)

Fig. 4. Biogenetic pathway for compound 1

7.35 (d, J ¼ 8.1) 7.26 (d, J ¼ 8.1)

7.39 (d, J ¼ 8.3) 7.91 (d, J ¼ 8.3)

1 2

3.49 (d, J ¼ 10.8), 3.54 (d, J ¼ 10.8) 3.38 (s)

4.44 (s) 3.41 (s)

15-MeO

3.88 (s)

1.41 – 1.47 (m) 2.21 – 2.26 (m) 1.01 (s) 1.03 (s) 1.04 (s)

1.38 – 1.44 (m, Ha ), 1.65 – 1.70 (m, Hb ) 1.53 – 1.59 (m)

1.68 – 1.74 (m) 2.50 – 2.55 (m) 1.28 (s) 0.57 (s) 1.08 (s)

1.70 – 1.76 (m) 2.46 – 2.52 (m) 1.26 (s) 0.52 (s) 1.06 (s)

1.77 – 1.83 (m)

1.68 – 1.73 (m, Hb )

1.38 – 1.44 (m), 2.03 – 2.08 (m) 1.50 – 1.56 (m), 2.23 – 2.29 (m)

6.82 (d, J ¼ 8.8) 6.48 (d, J ¼ 8.8)

4

11a 11b 12 13 14a 14b 15

10

1.50 – 1.56 (m, Ha ), 1.65 – 1.70 (m, Hb ) 1.77 – 1.82 (m)

7.35 (d, J ¼ 8.1)

7.39 (d, J ¼ 8.3)

5

6 7 8 9

7.26 (d, J ¼ 8.1)

7.91 (d, J ¼ 8.3)

4

3

3

2

Position

1.65 – 1.70 (m) 2.42 – 2.48 (m) 1.24 (s) 1.04 (s) 0.55 (s)

1.52 – 1.57 (m, Ha ), 1.65 – 1.70 (m, Hb ) 1.74 – 1.80 (m)

7.22 (d, J ¼ 8.5)

6.75 (d, J ¼ 8.5)

7.22 (d, J ¼ 8.5) 6.75 (d, J ¼ 8.5)

5

0.88 (d, J ¼ 6.3) 0.92 (d, J ¼ 6.3) 4.71 (s) 4.96 (s)

2.27 – 2.33 (m, Ha ), 2.53 – 2.58 (m, Hb ) 1.62 – 1.67 (m) 2.04 – 2.09 (m) 1.62 – 1.68 (m)

6.55 (d, J ¼ 10.1) 5.99 (d, J ¼ 10.1)

1.56 – 1.62 (m) 1.44 – 1.50 (m), 1.96 – 2.02 (m) 2.38 – 2.43 (m), 2.54 – 2.60 (m)

6

2.25 – 2.30 (m) 2.32 – 2.38 (m) 2.04 – 2.10 (m) 2.18 – 2.24 (m) 0.90 (s) 1.02 (s) 4.60 (s) 4.95 (s)

6.94 (d, J ¼ 10.5) 6.05 (d, J ¼ 10.5)

1.30 – 1.36 (m), 1.60 – 1.65 (m) 0.82 – 0.88 (m), 1.60 – 1.66 (m) 2.22 – 2.28 (m)

7

Table 2. 1H-NMR Data (400 MHz, CDCl3 ) of Compounds 2 – 7. d in ppm, J in Hz. Atom numbering as indicated in Fig. 1.

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Table 3.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014) 13

C-NMR Data (100 MHz, CDCl3 ) of Compounds 2 – 7 a ). Atom numbering as indicated in Fig. 1.

Position

2 b)

3 c)

4 b)

5 b)

6 b)

7 b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 15-MeO

127.0 (d, CH ) 128.8 (d, CH ) 127.3 (s) 128.8 (d) 127.0 (d) 153.3 (s) 51.0 (s) 44.5 (s) 39.7 (t) 19.7 (t) 36.7 (t) 24.2 (q) 26.3 (q) 24.2 (q) 167.2 (s) 51.9 (q)

127.0 (s) 127.0 (s) 135.0 (s) 127.0 (s) 127.0 (s) 147.1 (s) 50.4 (s) 44.2 (s) 39.7 (t) 19.7 (t) 36.8 (t) 24.3 (q) 26.3 (q) 24.2 (q) 74.5 (t) 58.0 (q)

134.9 (d, CH ) 131.5 (d) 76.3 (s) 25.1 (t) 25.2 (t) 82.6 (s) 49.9 (s) 44.9 (s) 42.0 (t) 19.3 (t) 34.1 (t) 21.4 (q) 25.9 (q) 28.0 (q) 73.9 (t) 59.9 (q)

128.1 (d) 114.2 (d) 153.1 (s) 114.2 (d) 128.1 (d) 139.9 (s) 49.9 (s) 44.2 (s) 39.6 (t) 19.7 (t) 36.9 (t) 24.4 (q) 24.2 (q) 26.4 (q)

58.8 (d) 27.5 (t) 29.4 (t) 152.2 (s) 51.0 (s) 158.8 (d) 127.9 (d) 200.6 (s) 33.3 (t) 24.9 (t) 29.4 (d) 22.3 (q) 22.4 (q) 108.8 (t)

37.4 (s) 35.8 (t) 22.7 (t) 32.0 (t) 147.9 (s) 50.0 (s) 154.8 (d) 130.5 (d) 200.2 (s) 34.5 (t) 26.2 (t) 26.1 (q) 24.0 (q) 112.9 (t)

a

) Assignments were accomplished by HMQC and HMBC techniques. b ) in CDCl3 .

(Fig. 5). Moreover, HMBCs MeOC(15) (d(H) 3.88)/C(15) and HC(2)/C(15) (d(C) 167.2) implied a MeO group at C(15). Thus, compound 2 had a planar structure similar to that of the methyl ester of cuparenic acid [24]. NOESY Correlations (Fig. 5) HC(1)/HbC(10), HbC(11), Me(12), Me(13), and Me(14); and HaC(11)/Me(12) and Me(14) were found similar to those of 1. It was noted that cuparenic acid had a positive specific rotation ([a] ¼ þ 7.0), while 2 had a negative one ([a] ¼ 8.0). The CD spectrum of 2 showed a positive Cotton effect at 223 nm and a negative one at 252 nm. Therefore, compound 2 was tentatively determined as ()-cuparenic acid methyl ester with (S)-configuration at C(7). Compound 3 was isolated as an oily substance, with the molecular formula C16H24O, as deduced from a quasi-molecular-ion peak at m/z 255.1719 ([M þ Na] þ ) in HR-EIMS and DEPT spectra. The UV (241 nm) and IR (3094 and 1609 cm 1) absorptions were similar to those of 2, suggesting a close analog. The 1H- and 13C-NMR spectra of 3 (Tables 2 and 3) resembled those of 2 indicating that compound 3 was also a cuparane-type sesquiterpene. The difference between them consisted in the nature of the substituent at C(3). The COSY correlations CH2(9)/

Fig. 5. 1H,1H-COSY (— —), HMB (H ! C), and key NOESY (H $ H) correlations of compound 2


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CH2(10); CH2(10)/CH2(11); HC(1,5)/HC(2,4), and key HMBCs (Fig. 6) indicated a MeO group at C(15) (d(H) 74.5). Detailed comparison of 1H- and 13C-NMR data of 3 with those of ()-g-cuparenol [13] led to the structure of 3 as ()-g-cuparenol methyl ether. NOESY Correlations (Fig. 6) and CD spectrum of 3 were similar to those of 2, suggesting the same sense of chirality. The b-disposition of Me(13) (d(H) 0.57) and aorientation of Me(14) (d(H) 1.08) were also assigned accordingly. Compound 4 had a molecular formula C16H26O3 as determined from a quasimolecular-ion peak at m/z 289.1778 ([M þ Na] þ ) in the HR-EI-MS. The 1H-NMR spectrum of 4 (Table 2) showed three Me singlets (d(H) 1.01, 1.03, and 1.04), a MeO singlet (d(H) 3.38), and signals of a CH2O group (d(H) 3.49 (d, J ¼ 10.8, HaC(15)) and 3.54 (d, J ¼ 10.8, HbC(15))), and of two olefinic H-atoms (d(H) 6.48 (d, J ¼ 8.8) and 6.82 (d, J ¼ 8.8)). The 13C-NMR and DEPT spectra (Table 3) exhibited signals of two olefinic C-atoms (d(C) 131.5 and 134.9), three Me groups (d(C) 21.4, 25.9, and 28.0), a MeO group (d(C) 59.9), six CH2 groups (d(C) 19.3, 25.1, 25.2, 34 .1, 42.0, and 73.9), and four quaternary C-atoms (d(C) 44.9, 49.9, 76.3, and 82.6). These data suggested that compound 4 is an analog of 2 and 3, but lacked an aromatic ring. The COSY correlations CH2(9)/CH2(10), CH2(10)/CH2(11), and CH2(4)/CH2(5), and HMBCs HC(1)/C(3), C(5), and C(6); HC(2)/C(3), C(4), and C(6); Me(12)/C(6); HC(1)/ C(7), MeOC(15)/C(15); HC(2)/C(15); and CH2(15)/C(3) (Fig. 7) further supported the structure of 4, in which C(3) and C(6) are linked with a peroxide ring. This was confirmed by an IR absorption at 1116 cm 1 and a quite low chemical shift of C(6) (d(C) 82.6). It was found that the planar structure of 4 was similar to that of 3,6peroxocupar-1-ene, a natural product previously isolated from Nardia spp. [30]. The relative configuration at C(7) was determined by NOESY (Fig. 7). Comparison of the specific rotation of 4 with that of 3,6-peroxocupar-1-ene led us to propose (S)configuration at C(7) in compound 4. Compound 5 was isolated as yellowish oil. It had the molecular formula C14H20O, as deduced from a quasi-molecular-ion peak at m/z 203.1444 ([M H] ) in the HR-EIMS. Its UV and IR spectra were similar to those of 3, suggesting a close analogy with cuparenol methyl ether (3). Detailed comparison of the 1H- and 13C-NMR spectra of 5 (Tables 2 and 3) with those of 3 suggested that the differences between 5 and 3 were in the signals of the MeOCH2 of 3 which were missing in the spectra of 5. The chemical shift of C(3) at d(C) 153.1 indicated that compound 5 contained a OH group at C(3). This finding was supported by COSY correlations CH2(9)/CH2(10), CH2(10)/CH2(11), and HC(1,5)/HC(2,4), and HMBCs correlations (Fig. 8) of 5. The planar structure of

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5 was thus established as a norcuparane-type sesquiterpene similar to 4-(1,2,2trimethylcyclopentyl)anisole [31]. The relative configuration was determined from NOESY correlations (Fig. 8) HC(1)/HaC(10), HaC(11), HbC(11), Me(12), Me(13), and Me(14); Me(13)/Me(12) and Me(14); and Me(14)/HaC(11) and HaC(9). The specific rotation of 5 was quite different from that of 2 and 3, suggesting that compound 5 had (R)-configuration at C(7). Therefore, 5 was a new compound and named (þ)-g-norcuparenol. Compound 6, isolated as pale-yellow oil, had a molecular formula C14H20O (doublebond equivalents, 5) as deduced from the quasi-molecular-ion peak at m/z 227.1405 ([M þ Na] þ ) in the HR-EI-MS. The IR absorptions at 3075 and 1677 cm 1, and UV absorption lmax at 224 nm suggested that compound 6 contained an a,b-unsaturated ketone function. The 1H-NMR spectrum (Table 2) revealed the presence of two Me groups (d(H) 0.88 (d, J ¼ 6.3) and 0.92 (d, J ¼ 6.3)), a pair of olefinic CH groups (d(H) 5.99 (d, J ¼ 10.1) and 6.55 (d, J ¼ 10.1)), and one terminal olefinic CH2 groups (d(H) 4.71 (s, HaC(14) and 4.96 (s, HbC(14)). The 13C-NMR spectrum of 6 (Table 3) exhibited signals of two sets of olefinic C-atoms (d(C) 108.8, 127.9, 152.2, and 158.8) and a C¼O C-atom (d(C) 200.6). The remaining two unsaturation degrees suggested that 6 should contain two cyclic ring systems. This interpretation was supported further from DEPT spectrum, which revealed the presence of two Me groups (d(C) 22.3 and 22.4), four CH2 (d(C) 24.9, 27.5, 29.4, and 33.3), and two CH groups (d(C) 29.4 and 58.8), and a quaternary C-atom (d(C) 51.0). The planar structure of 6 was deduced from COSY correlations and HMBCs (Fig. 9). COSY Correlations HC(11)/Me(12) and Me(13); HC(1)/CH2(2), HC(11);


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and CH2(3)/CH2(2), and HMBCs Me(12)/C(1), C(11), C(13); CH2(14)/C(3) and C(5); and CH2(2)/C(5) indicated the presence of a five-membered ring with an iPr group at C(1) and a terminal C¼C bond at C(4). On the other hand, COSY correlations HC(6)/HC(7) and CH2(9)/CH2(10), and HMBCs HC(6)/C(1), C(5), C(8), and C(10); HC(7)/C(5) and C(9); CH2(9)/C(5) and C(8); and CH2(10)/C(5) and C(8) evidenced an a,b-unsaturated keto group in the six-membered ring system. The spirostructure of the bicyclic sesquiterpene core of 6 was deduced from the signal of the quarternary C-atom at d(C) 51.0 (C(5)). The established structure is similar to that of schisansphenin B, [13] isolated from the same plant, but lacking of a COOH group. The relative configuration was determined from NOESY and CD spectra. The NOESY correlations (Fig. 9) HC(6)/HC(1), Me(12), and HaC(14), and HaC(14)/HbC(9) suggested that C(1) and C(5) had the same configuration. The absolute configuration was confirmed by CD spectroscopy. According to the helicity rule [32], the configuration of C(5) corresponded to the negative Cotton effect ([q]237 4583) of a,b-unsaturated keto group in 6 due to p ! p* transition (K-band) at 237 nm. Thus, 6 was a new natural product and named schisansphenin C. It was proposed that the noracorane structure of 6 may result by loss of C(15) during its biogenetic pathway. Compound 7 is an isomer of 6, because it showed a quasi-molecular ion at m/z 227.1406 ([M þ Na] þ ) in the HR-EI-MS, and had a formula C14H20O, as compound 6. The IR (3079 and 1682 cm 1) and UV (227 nm) bands were similar to those of 6, suggesting that 7 contained an a,b-unsaturated keto group as compound 6. The 1 H-NMR spectrum (Table 2) exhibited two Me singlets (d(H) 0.90 and 1.02), a couple of olefinic CH doublets (d(H) 6.05 (d, J ¼ 10.5), 6.94 (d, J ¼ 10.5)), terminal olefinic CH2 signals (d(H) 4.60 (s), 4.95 (s), CH2(14)). It was also supported from 13C-NMR spectrum (Table 3), which showed signals of two sets of olefinic C-atoms (d(C) 112.9, 130.5, 147.9, and 154.8) and of a conjugated C¼O group (d(C) 200.2). However, the DEPT spectra of 7 indicated the presence of five CH2 groups (d(C) 22.7, 26.2, 32.0, 34.5, and 35.8), and of not four as in 6, in addition to the signals of two Me groups (d(C) 24.0 and 26.1) and two quaternary C-atoms (d(C) 37.4 and 50.0). The COSY correlations CH2(3)/CH2(4); HC(7)/HC(8), and CH2(10)/CH2(11), and the HMBCs Me(12)/ C(1), C(2), C(6), and C(13); CH2(14)/C(4), C(5), C(6); CH2(4)/C(2) and C(5);

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CH2(2)/C(6); HC(7)/C(1), C(9), and C(11); HC(8)/C(6), C(10) and C(11); CH2(10)/C(9); and CH2(11)/C(5) and C(9) clearly evidenced the planar structure of 7 as illustrated in Fig. 10. Detailed comparison of the 13C-NMR data of 7 (Table 3) with those of chamigrenal [22] revealed that the CHO group of chamigrenal is missing in compound 7. The specific rotation of 7 ([a] ¼ þ 5.6 (MeOH)) is also different from that of chamigrenal ([a] ¼ 18.6 (MeOH)). The relative configuration was determined from NOESY and CD spectra. NOESY Correlations (Fig. 10) HC(7)/Me(12), Me(13), and HaC(14); and Me(12)/HbC(2), HaC(2), CH2(11) tentatively provided (R)-configuration at C(6), which was confirmed from positive Cotton effect ([q]215 þ 3822) at 215 nm due to the p ! p* transition of a,b-unsaturated keto moiety in the six-membered ring system. Thus, compound 7 was a new norchamigrane and named schisansphenin D. We propose that 7 may be produced from chamigrane by loss of C(15), followed by hydroxylation and further oxidation. The isolated new compounds 1 – 7 were tested for their in vitro inhibitory activities against the HSV-1 virus. Among them, compound 4 exhibited the most potent activity (Table 4). Acyclovir was used as a positive control. Also, inhibitory effects of compounds 1 – 7 were evaluated on superoxide-anion generation and elastase release by human neutrophils in response to FMLP/CB at a concentration of 10 mg/ml. As a result, compound 6 showed moderate anti-inflammatory effects (31.67 5.74 and 41.33 2.95 %, resp.) on superoxide anion and elastase release. Genistein was used as a standard compound (51.60 5.89%; Table 5). Table 4. Effects of Compounds 1 – 7 on HSV-1 Replication Compound (100 ml)

Inhibitory activity [%]

1 2 3 4 5 6 7 Acyclovir (2.5 mm)

6.94 6.66 21.97 12.17 8.96 6.56 43.93 5.13 12.14 6.81 17.92 1.15 9.54 4.51 96.96 1.01


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Table 5. Effects of Compounds 1 – 7 on Superoxide-Anion Generation and Elastase Release by Human Neutrophils in Response to FMLP/CB a ) Compound

1 2 3 4 5 6 7 Genistein a

Inhibition [%] Superoxide anion b )

Elastase release

15.1 3.9 22.0 6.5 14.4 5.4 23.6 6.4 17.8 1.5 31.7 5.7 10.6 3.0 65.0 5.7

4.3 2.0 0.7 3.3 4.9 2.4 13.6 3.4 10.0 3.8 41.3 2.9 3.2 4.8 51.6 5.9

) Results are presented as mean S.E.M. (n ¼ 3). b ) Percentage of inhibition at 10 mm concentration.

Experimental Part General. Silymarin and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). LiChrospher Si 60 (5 mm, 250 – 10; Merck) and LiChrospher 100 RP-18e (5 mm, 250 – 10; Merck) were used for NP-HPLC and RP-HPLC (Hitachi, L-6250; flow rate, 2 ml/min; UV detection at 254 nm), resp. Optical rotations: JASCO DIP-1000 polarimeter. CD Spectra: JASCO J-720 spectropolarimeter. UV Spectra: HITACHI U-2001 spectrophotometer; lmax (log e) in nm. IR Spectra: HORIBA FT-720 spectrophotometer; ˜n in cm 1. 1H- and 13C-NMR, and 2D-NMR spectra (COSY, HMQC, HMBC, and NOESY): Bruker AVX NMR spectrometer operating at 400 (1H) and 100 MHz (13C); in CDCl3 using the CDCl3 solvent peak as internal standard (d(H) 7.265, d(C) 77.0 ppm); d in ppm, J in Hz. Lowresolution EI-MS: VG Quattro 5022 mass spectrometer; in m/z. HR-ESI-MS: JEOL HX 110 mass spectrometer; in m/z. Plant Material. The fruits of S. sphenanthera were purchased from Miaoli County, Taiwan, and were identified by one of the authors (S.-S. L.). A voucher specimen was deposited with the School of Pharmacy, College of Medicine, National Taiwan University. Extraction and Isolation. The dried fruits of S. sphenanthera (28 kg) were extracted with EtOH at r.t., concentrated under reduced pressure to give a crude extract, and then partitioned between hexane, MeOH, and H2O (4 : 3 : 1) to give a MeOH/H2O layer (350 g). This residue was subjected to CC (flash column chromatography, eluted with mixtures of hexane, AcOEt, and MeOH, with increasing polarity), to yield ten fractions, Frs. SC9.1 – SC9.10. Fr. SC9.1 (17.6 g) was subjected to CC (Sephadex LH-20; MeOH) to give Fr. SC9.1.2 (14.2 g), which was further separated by CC (SiO2 ; hexane/AcOEt 85 : 15) to afford three fractions, Frs. SC9.1.2.1 – SC9.1.2.3. Fr. SC9.1.2.1 was subjected to CC (SiO2 ; hexane/acetone 97 : 3) to yield Frs. SC9.1.2.1.4 (162 mg) and SC9.1.2.1.5 (160 mg). The former was subjected to a normalphase HPLC (hexane/AcOEt 96 : 4) to furnish compound 3 (9.6 mg). The latter fraction was submitted to a normal-phase HPLC (hexane/AcOEt 95 : 5) to yield compound 2 (3.7 mg). Fr. SC9.1.2.2 was subjected to a normal-phase HPLC (hexane/CH2Cl2 7 : 3) to give a-cuparenol (21; 2.5 mg). Fr. SC9.1.2.3 (412 mg) was separated by CC (SiO2 ; hexane/acetone 9 : 1) to furnish three fractions, Frs. SC9.1.2.3.2 – SC9.1.2.3.4. Fr. SC9.1.2.3.2 (44 mg) was purified by a normal-phase HPLC (hexane/AcOEt 9 : 1) to yield chamigrenal (18; 4.9 mg). Fr. SC9.1.2.3.3 (159 mg) was separated by a normal-phase HPLC (hexane/AcOEt 9 : 1) to yield compounds 5 (5 mg) and 6 (3 mg), and a residue, which was purified by NP-HPLC (CH2Cl2/AcOEt 97 : 3) to furnish compound 4 (7.2 mg). Fr. SC9.1.2.3.4 (107 mg) was purified by a normal-phase HPLC (hexane/AcOEt; 85 : 15) to provide campherenol (17; 12.2 mg). Fr. SC9.3 (40.3 g) was subjected to CC (Sephadex LH-20; MeOH) to give Frs. SC9.3.2 (30 g) and SC9.3.3 (4.8 g). The former was purified by CC (SiO2 ; hexane/AcOEt 8 : 2) to give a residue, which was crystallized (MeOH) to yield deoxyschizandrin (16; 1.48 g). The latter fraction was separated by CC (SiO2 ; hexane/AcOEt 3 : 2) to provide two fractions, Frs. SC9.3.3.1 (126 mg) and SC9.3.3.4 (87 mg).

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Fr. SC9.3.3.1 was subjected to a normal-phase HPLC (hexane/AcOEt 9 : 1) to furnish compounds 1 (12.2 mg) and 7 (5 mg), and methylgomisin R (12; 3.3 mg), interiotherin A (14; 21.6 mg), macelignan (15; 33.1 mg), and g-cuparenal (19; 19.8 mg). Fr. SC9.3.3.4 was separated by a normal-phase HPLC (hexane/ AcOEt 7 : 3) to yield gomisin C (11; 5.9 mg), cuparenic acid (20; 4.8 mg), and kadsuric acid (23; 12.5 mg). Fr. SC9.5 (23 g) was subjected to CC (Sephadex LH-20; MeOH) to give Fr. SC9.5.3 (2.7 g), which was further separated by CC (SiO2 ) to afford Frs. SC9.5.3.2 (43 mg), SC9.5.3.7 (783 mg), and SC9.5.3.8 (90 mg). Methylgomisin O (8; 5.7 mg) was obtained from Fr. SC9.5.3.2 by NP-HPLC (hexane/AcOEt 85 : 15). Arisantetralone A (9; 321 mg) and arisantetralone C (10; 102 mg) were obtained from CC (SiO2 ; hexane/acetone 7 : 3), while gomisin S (13; 10 mg) and 4-hydroxybenzoic acid (22; 25 mg) were isolated from NP-HPLC (hexane/AcOEt 55 : 45). Schiscupatetralin A ( ¼ 5-[(5R,6R,7R)-5,6,7,8-Tetrahydro-6,7-dimethylnaphtho[2,3-d] [1,3]dioxol-5yl]-3-methoxy-2-{4-[(1S)-1,2,2-trimethylcyclopentyl]benzyl}phenol; 1). [a] 24 D ¼ 21.0 (c ¼ 1.22, MeOH). UV (MeOH): 290 (3.67), 241 (4.21), 216 (4.57). CD (c ¼ 0.1, MeOH): [q]215 þ 14006, [q]248 2951, [q]294 þ 5576. IR (neat): 3520, 3054, 1615, 1590. 1H- and 13C-NMR: see Table 1. HR-ESI-MS: 527.3167 ([M þ H] þ , C35H43O þ4 ; calc. 527.3161). Cuparenic Acid Methyl Ester ( ¼ Methyl 4-[(1S)-1,2,2-Trimethylcyclopentyl]benzoate; 2). [a] 24 D ¼ 8.0 (c ¼ 0.1, MeOH). UV (MeOH): 242 (4.0). CD (c ¼ 0.1, MeOH): [q]217 2958, [q]223 þ 4436, [q]252 3452. IR (neat): 3094, 1723, 1609. 1H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS: 269.1521 ([M þ Na] þ , C16H22NaO þ2 ; calc. 269.1517). ()-Cuparenic Methyl Ether ( ¼ (1-(Methoxymethyl)-4-[(1S)-1,2,2-trimethylcyclopentyl]benzene; 3). [a] 24 D ¼ 7.2 (c ¼ 0.34, MeOH). UV (MeOH): 241 (3.95). CD (c ¼ 0.01, MeOH): [q]216 1936, [q]227 þ 345, [q]255 223. IR (neat): 3094, 1609. 1H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS: 255.1719 ([M þ Na] þ , C16H24NaO þ ; calc. 255.1725). ()-15-Methoxy-3,6-peroxocupar-1-ene ( ¼ (1R,4S)-1-(Methoxymethyl)-4-[(1S)-1,2,2-trimethylcyclopentyl]-2,3-dioxabicyclo[2.2.2]oct-5-ene; 4). [a] 25 D ¼ 10.8 (c ¼ 0.72, MeOH). IR (neat): 3066, 1116. 1 H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS: 289.1778 ([M þ Na] þ , C16H26NaO þ3 ; calc. 289.1780). (þ)-rel-Norcuparenol ( ¼ 4-[(1R*)-1,2,2-Trimethylcyclopentyl]phenol; 5). [a] 25 D ¼ þ 2.2 (c ¼ 0.34, MeOH). UV (MeOH): 278 (2.93), 221 (3.66). IR (neat): 3415, 3067, 1614. 1H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS: 203.1444 ([M H] , C14H19O ; calc. 203.1430). Schisansphenin C ( ¼ (4S,5S)-1-Methylidene-4-(propan-2-yl)spiro[4.5]dec-6-en-8-one; 6). [a] 25 D ¼ 24.0 (c ¼ 0.3, MeOH). UV (MeOH) 224 (3.42). IR (neat): 3075, 1710, 1677. 1H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS: 227.1405 ([M þ Na] þ , C14H20NaO þ ; calc. 227.1412). Schisansphenin D ( ¼ (6R)-7,7-Dimethyl-11-methylidenespiro[5.5]undec-1-en-3-one; 7). [a] 24 D ¼ þ 5.6 (c ¼ 0.5, MeOH). UV (MeOH): 227 (3.43). CD (c ¼ 0.3, MeOH): [q]215 þ 3822. IR (neat): 3079, 1715, 1682. 1H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS: 227.1406 ([M þ Na] þ , C14H20NaO þ ; calc. 227.1412). Anti-HSV-1 Assay. Vero cells were cultured in minimal essential medium (MEM; GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT), 100 U/ml penicillin, and 100 mg/ml streptomycin, and incubated at 378 in a 5% CO2 incubator. To prepare a stock soln. of HSV-1 (KOS strain, VR-1493, ATCC), Vero cells were infected by HSV-1 at a multiplicity of infection of three plaque-forming units (PFU)/cell and harvested at 24 h post infection and centrifuged at 1500 g (centrifuge 5810 R, Eppendrof) at 48 for 20 min. The supernatant was collected and stored at 708 for use. The activities of various compounds and acyclovir for inhibition of plaque formation were calculated [33] [34]. Human Neutrophils Elastase Release Assay. Degranulation of azurophilic granules was determined by elastase release as described in [35]. Experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate. After supplementation with MeO-Suc-Ala-Ala-Pro-Val-pnitroanilide (100 mm), neutrophils (6 105 cell/ml) were equilibrated at 378 for 2 min and incubated with each test compound for 5 min. Cells were activated by fMLP (100 nm)/CB (0.5 ml), and changes in absorbance at 405 nm were monitored continuously for elastase release. The results are expressed as the percentage of the initial rate of elastase release in the fMLP/CB-activated, test compound-free (DMSO) control system.


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Human Neutrophil Superoxide Generation Assay. Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Superoxide-anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c [36]. In brief, after supplementation with 0.5 mg/ml ferricytochrome c and 1.0 mm Ca2 þ , neutrophils were equilibrated at 378 for 2 min and incubated with drugs for 5 min. Cells were activated with 100 nm fMLP for 10 min. When fMLP was used as a stimulant, CB (1 ml) was incubated for 3 min before activation by the peptide (fMLP/CB). Changes in absorbance with the reduction of ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell positioner spectrophotometer under constant stirring (Hitachi U3010, Tokyo, Japan). Calculations were based on differences in the reactions with and without SOD (100 U/ml) divided by the extinction coefficient for the reduction of ferricytochrome c. The authors thank the National Science Council, ROC (grant No. NSC-98-2320-B002-027-MY3) for providing financial support.

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New lignans from the roots of Schisandra sphenanthera.

Isolation, characterization and antioxidant activity of polysaccharide from Schisandra sphenanthera.

7,8-Secolignans from the fruits of Schisandra neglecta.

Seven new drimane-type sesquiterpenoids from cultures of fungus Laetiporus sulphureus.

Seven New Drimane-Type Sesquiterpenoids from Cultures of Fungus Phellinus tuberculosus.

Two new sesquiterpenoids from Artemisia annua.

Isolation and anti-hepatitis B virus activity of dibenzocyclooctadiene lignans from the fruits of Schisandra chinensis.

Predictions of potential geographical distribution and quality of Schisandra sphenanthera under climate change.

Two new sesquiterpenoids from cultures of the basidiomycete Tremella foliacea.

Five new sesquiterpenoids from Dalbergia odorifera.

Four New Sesquiterpenoids from Cultures of the Fungus Phellinidium sulphurascens.

Therapeutic efficacy of Wuzhi tablet (Schisandra sphenanthera Extract) on acetaminophen-induced hepatotoxicity through a mechanism distinct from N-acetylcysteine.

Two-steps extraction of essential oil, polysaccharides and biphenyl cyclooctene lignans from Schisandra chinensis Baill fruits.

Protective effect of Wuzhi tablet (Schisandra sphenanthera extract) against cisplatin-induced nephrotoxicity via Nrf2-mediated defense response.

Two New Compounds from Schisandra propinqua var. propinqua.

Four new neolignan glucosides from the fruits of Arctium lappa.

Sesquiterpenoids from the Rhizomes of Homalomena occulta.

Sesquiterpenoids from the roots of Homalomena aromatica.

Quantitative analysis of six lignans in fruits with different colours of Schisandra chinensis by HPLC.

Four new sesquiterpenoids from fruiting bodies of the fungus Inonotus rickii.

New eudesmane-type sesquiterpenoids from the processed rhizomes of Atractylodes macrocephala.

New free radical scavenging neolignans from fruits of Piper attenuatum.

New apoptosis-inducing sesquiterpenoids from the mycelial culture of Chinese edible fungus Pleurotus cystidiosus.

New Azulene-Type Sesquiterpenoids from the Fruiting Bodies of Lactarius deliciosus.