Abstract
A structurally unique hydrophobic compound, biyouyanagin A, was isolated from the MeOH extract of the leaves of Hypericum chinense L. var. salicifolium. The structure of biyouyanagin A was elucidated on the basis of spectroscopic evidence. Biyouyanagin A showed a significant activity against HIV and inhibited cytokine production.
The recent widespread interest in the antidepressant activity of Hypericum perforatum (St. John’s wort) has inspired the investigation of secondary metabolites from other Hypericum species.1 The genus Hypericum, which are distributed widely in temperate regions, have been used as traditional medicines in various parts of the world. In Japan, H. chinense L. var. salicifolium (Biyouyanagi in Japanese) is used as a folk medicine for treatment of female disorders.2
Antibacterial acylphloroglucinols and spirolactones were also isolated from this species.3 As a part of a program to discover new bioactive natural products from plants, we have examined the MeOH extract from the leaves of H. chinense and isolated a unique hydrophobic compound named biyouyanagin A, which contains sesquiterpene, cyclobutane, and spirolactone moieties. Biyouyanagin A showed a significant activity against HIV and inhibited cytokine production. In this paper, we report isolation, structural elucidation, and biological evaluation of biyouyanagin A.
Dried leaves of H. chinense L. var. salicifolium (1.48 kg) were extracted with MeOH. The MeOH extract (632.7 g) was partitioned with n-hexane and H2O, and the n-hexane fraction (92.6 g) was subjected to repeated column chromatography to give biyouyanagin A.
Biyouyanagin A (1) was obtained as a colorless oil, [α]D –240.0 (CHCl3, c 0.5). The IR spectrum of 1 showed absorption bands of two carbonyl groups (1792, 1743 cm−1). The 1H NMR showed the presence of a benzene ring [δH 7.26–7.37 (5H, m)], a 1-substituted ethylene moiety [δH 5.24 (1H, dd, J = 17.6, 11.2), 4.80 (1H, d, J = 11.2), 4.62 (1H, d, J = 17.6)], two olefinic protons [δH 5.46 (1H, m), 5.11 (1H, brt, J = 5.6)], one oxygenated methylene group [δH 4.71, 3.98 (each 1H, d, J = 8.8)], five methines, three methylenes, and five methyls. The HRFABMS gave a quasimolecular ion peak at m/z 475.2911 ([M + H]+, calcd 475.2848) suggesting the molecular formula of C31H38O4. The 13C NMR spectral data, including DEPT spectra, were in good agreement with the above analysis (Table 1).
Table 1.
NMR Data for 1a
| position | 13C (δC) | 1H (δH) | HMBC (13C no.) |
|---|---|---|---|
| 1 | 118.4 | 4.80 (1H, d, 11.2)
4.62 (1H, d, 17.6) |
3 |
| 2 | 134.5 | 5.24 (1H, dd, 17.6, 11.2) | 3, 4, 9, 10 |
| 3 | 49.0 | ||
| 4 | 93.1 | ||
| 5 | 209.6 | ||
| 6 | 51.9 | 3.16 (1H, dd, 6.0, 1.2) | 4, 5, 11, 17, 18, 22 |
| 7 | 89.7 | ||
| 8 | 171.6 | ||
| 9 | 73.7 | 4.71 (1H, d, 8.8)
3.98 (1H, d, 8.8) |
3, 4, 8, 10 |
| 10 | 20.1 | 1.31 (3H, s) | 2, 3, 4, 9 |
| 11 | 139.6 | ||
| 12 | 125.9 | 7.37–7.26 (1H, m) | 7 |
| 13 | 127.7 | 7.37–7.26 (1H, m) | |
| 14 | 127.8 | 7.37–7.26 (1H, m) | |
| 15 | 127.7 | 7.37–7.26 (1H, m) | |
| 16 | 125.9 | 7.37–7.26 (1H, m) | 7 |
| 17 | 35.9 | 3.01 (1H, ddd, 8.4, 6.6, 6.6) | 5, 6, 7, 18, 19, 21, 22, 24 |
| 18 | 50.3 | 3.49 (1H, d, 8.4) | 6, 7, 17, 19, 20, 23 |
| 19 | 131.4 | ||
| 20 | 123.9 | 5.46 (1H, m) | |
| 21 | 23.5 | 2.09 (1H, m)
1.99 (1H, m) |
|
| 22 | 38.8 | 1.73 (1H, m) | 6, |
| 23 | 21.7 | 1.02 (3H, d, 1.2) | 18, 19, 20 |
| 24 | 35.1 | 1.46 (1H, m) | |
| 25 | 16.8 | 0.83 (3H, d, 6.4) | 22, 24, 26 |
| 26 | 35.0 | 1.45 (1H, m)
1.20 (1H, m) |
24, 27, 28 |
| 27 | 25.9 | 2.02 (1H, m)
1.94 (1H, m) |
|
| 28 | 124.6 | 5.11 (1H, brt, 5.6) | 27, 30, 31 |
| 29 | 131.4 | ||
| 30 | 25.7 | 1.70 (3H, d, 1.2) | 28, 29, 31 |
| 31 | 17.7 | 1.61 (3H, s) | 28, 29, 30 |
Measured in CDCl3. Coupling constants given (J, Hz) in parentheses.
The 1H–1H COSY spectrum of 1 showed the following correlations: H3-25–H-24–H2-26–H2-27–H-28; H-20–H2-21–H-22–H-17–H-18. The structure of partial unit A (sesquiterpene unit, Figure 1) was indicated by the following long-range correlations in the HMBC spectrum: H3-30 and -31 with C-28, -29; H3-25 with C-22, -24, -26; H3-23 with C-18, -19, -20; H-17 with C-18, -19, -21, -22, -24; and H-18 with C-17, -19, -20, -23.
Figure 1.
Partial structures of 1.
The remaining 1H and 13C NMR signals of 1 were compared with those of hyperolactone C.4 These data showed good agreement except for the signals of H-6 [δH 3.16 (1H, dd, J = 6.0, 1.2) in 1 vs 5.99 (1H, s) in hyperolactone C, C-5 (δC 209.6 vs 196.6), C-6 (δC 51.9 vs 100.3), C-7 (δC 89.7 vs 187.3), and C-11 (δC 139.6 vs 127.7)]. In 1, the long-range correlations of H-6 with C-4, -5, -11 were observed in the HMBC spectrum. These results clearly indicated that 1 has a saturated C-6/C-7 bond (methine carbon and a quaternary carbon, respectively) rather than the double bond in hyperolactone C. Thus, the structure of partial unit B (spiro-lactone unit, Figure 1) was elucidated.
The connections of units A (sesquiterpene) and B (spiro-lactone) were established on the basis of the following key correlations: H-6 with H-17 (1H-1H COSY); H-6 with C-17, -18, -22, H-17 with C-5, -6, -7, H-18 with C-6, -7 (HMBC). Thus, the direct connections between C-6 and C-17, C-7 and C-18 formed a cyclobutane ring.
The relative configuration was established from the following NOE correlations: H-6 with H-17, -22, and aromatic protons; H-17 with H-18, -22; H3-10 with aromatic protons. Thus, the structure of 1 was elucidated (Figure 2).
Figure 2.
Biyouyanagin A (1).
Our postulated biosynthetic pathway of 1 from the related sesquiterpene and spirolactone is shown in Scheme 1.
Scheme 1.
In the search for anti-HIV natural products, various coumarins, terpenoids, and phloroglucinols5 have been reported to have anti-HIV activity. Accordingly, we evaluated anti-HIV activity of this novel compound. Compound 1 inhibited HIV replication in H9 lymphocytes with an EC50 value of 0.798 μg/mL and uninfected H9 cell growth with IC50 values of > 25 μg/mL, giving a calculated therapeutic index (TI) value of >31.3 (Table 2). Thus, 1 can be regarded as a promising new anti-HIV agent with a unique structure and merits further evaluation and analogue design.
Table 2.
Anti-HIV Activity of 1
| compd | IC50 (μg/mL) | EC50 (μg/mL) | TI |
|---|---|---|---|
| biyouyanagin A (1) | >25 | 0.798 | 31.3 |
| AZT | 500 | 0.0021 | 238, 738 |
Furthermore, we examined the effect of 1 in LPS-induced cytokine production, and it markedly inhibited the LPS-induced production of IL-10, IL-12, and TNF-α (Table 3). These data suggest that 1 is a strong inhibitor for cytokines and is worthy of further investigation.
Table 3.
Inhibitory Effects for Cytokine Release of 1a
| cytokine production ratio
|
|||
|---|---|---|---|
| compd | IL-10 | IL-12 | TNF-α |
| biyouyanagin A (1) | 0.03 | 0.02 | 0.48 |
| prednisolone | 0.14 | 0.24 | 0.48 |
PBMCs were treated with lipopolysaccharide (LPS) in the presence of 1 (10 μg/mL). Prednisolone (0.3 μg/mL) was used as a reference sample.
Data were expressed as ratios to cytokine production induced by LPS.
Acknowledgments
This investigation was supported in part by Grant No. AI-33066 from the National Institute of Allergy and Infectious Diseases (NIAID) awarded to K.H.L.
Footnotes
Supporting Information Available: Experimental section, plant material, extraction, isolation, and spectral data of biyouyanagin A (1). This material is available free of charge via the Internet at http://pubs.acs.org.
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