- Email: [email protected]
Anti-inflammatory abietane diterpenoids from the seeds of Podocarpus nagi
Phytochemistry Letters 21 (2017) 260–263
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Anti-inflammatory abietane diterpenoids from the seeds of Podocarpus nagi 1
MARK
1
Zhe-Ling Feng , Dan Li , Qian-Yu Liu, Jing-Xin Liu, Li Huang, Qing-Wen Zhang, Yi-Tao Wang, ⁎ Li-Gen Lin State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, 999078, China, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Podocarpus nagi Abietane diterpenoids NO production Anti-inflammation
In searching for naturally occurring anti-inflammatory agents, three new abietane-type diterpenoids, named 16hydroxylambertic acid (1), 7-oxo-18-hydroxyferruginol (2), and 5α,12-dihydroxy-6-oxa-abieta-8,11,13-trien-7one (3), were isolated from the seeds of Podocarpus nagi, together with three known compounds. The structures of the new compounds were elucidated by extensive analysis of NMR and HR-ESIMS data. All the new compounds were tested for nitric oxide (NO) inhibitory activities on lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Compound 1 significantly inhibited NO production with IC50 value of 5.38 ± 0.17 μM, and suppressed inducible NO synthase (iNOS) expression in a dose-dependent manner, which were mediated through inhibiting the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) activation.
1. Introduction Podocarpus nagi (Thunb.) Pilg (Podocarpaceae) is widely distributed in South of China, which has been used to treat rheumatism, arthritis and venereal diseases (Editorial Committee of Flora of China, 1978; Abdillahi et al., 2011). The seeds of this plant are rich in diterpenoids, including totarane-, abietane-, and ent-pimarane-types (Barrero et al., 2003; Zheng et al., 2016). These diterpenoids were reported with wide bioactivities, including anti-inflammation (Zheng et al., 2016), cytotoxicity (Hembree et al., 1979; Sato et al., 2009; Feng et al., 2017), and insecticidal activities (Zhang et al., 1992). In searching for naturally occurring anti-inflammatory agents, we identified an abietane- and an ent-pimarane-type diterpenoids from the twigs of P. nagi, with inhibitory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-treated macrophages (Zheng et al., 2016). In a continuing study, three new abietane diterpenoids, 16-hydroxylambertic acid (1), 7-oxo18-hydroxyferruginol (2), and 5α,12-dihydroxy-6-oxa-abieta-8,11,13trien-7-one (3) (Fig. 1), were isolated from the seeds of P. nagi, together with three known analogues. Herein, the isolation and structure elucidation of the new compounds, as well as their anti-inflammatory effects on LPS-stimulated RAW264.7 cells, are described. 2. Results and discussion Compound 1, a white amorphous powder, had a molecular formula of C20H28O4 based on the deprotonated ion peak in the HRESIMS (m/z
⁎
1
331.1912 for C20H27O4, calcd. m/z 331.1909), indicating seven degrees of unsaturation. The 1H NMR spectrum of 1 displayed resonances for two aromatic protons [δH 6.80 (1H, s) and 6.78 (1H, s)], one oxygenated methylene [δH 3.94 (1H, dd, J = 9.7, 3.6 Hz) and 3.68 (1H, m)], and three methyls [δH 1.32 (3H, s), 1.30 (3H, d, J = 7.2 Hz), and 1.11 (3H, s)] (Table 1). The 13C NMR spectrum of 1 (Table 1) exhibited 20 resonances attributed to one carbonyl carbon (δC 181.1), six aromatic carbons (δC 152.9, 147.7, 128.1, 127.3, 126.6, and 113.9), two quaternary carbons (δC 38.4 and 37.5), two methine carbons (δC 52.7 and 36.4), six methylene carbons (δC 69.7, 43.7, 39.2, 31.3, 21.1, and 19.9), and three methyl carbons (δC 28.8, 23.1, and 15.6). These data revealed that 1 possesses an abietane-type diterpenoid framework, and its 1H and 13C NMR data were closely related to those of lambertic acid (Cambie et al., 1983). The major difference was a methyl group in lambertic acid was replaced by an oxygenated methylene [δH 3.94 and 3.68] in 1. The structure of 1 was constructed by detailed analysis of the HMBC spectrum. The HMBC correlations between the oxygenated methylene protons and C-13 (δC 128.1), C-15 (δC 36.4), and C-17 (δC 15.6) assigned it as H2-16. The relative configuration of 1 was the same as abietane-type diterpenoids inferred by ROESY spectrum. Accordingly, the structure of 1 was established, and it was named 16-hydroxylambertic acid. Compound 2 was obtained as a white amorphous powder. Its molecular formula was assigned as C20H28O3 according to the ion peak at m/z 315.1970 [M − H]− (calcd. m/z 315.1960) in its HRESIMS. The 1H NMR spectrum exhibited signals for four methyl groups [δH 1.28 (3H, d,
Corresponding author. E-mail address: [email protected] (L.-G. Lin). Authors contributed equally.
http://dx.doi.org/10.1016/j.phytol.2017.07.011 Received 28 April 2017; Received in revised form 6 July 2017; Accepted 10 July 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 21 (2017) 260–263
Z.-L. Feng et al.
Fig. 1. Chemical structures of compounds 1–3.
Table 1 1 H (600 MHz, δ in ppm, J in Hz) and 1–3 in CDCl3. position
1
13
2
3
δC
δH
δC
δH
δC
δH
1α 1β 2α 2β 3α 3β 4 5 6α 6β 7α 7β
39.2
1.41 m 1.57 m 1.66 m 2.04 m 1.44 m 2.24 m
37.4
1.37 m 1.53 m 1.42 m 1.83 m 1.40 m 1.56 m
31.2
1.51 m 1.98 m 1.42 m 1.70 m 1.60 m 1.86 m
8 9 10 11 12 13 14 15 16
126.6 147.7 38.4 113.9 152.9 128.1 127.3 36.4 69.7
17
15.6
18
181.1
19 20
28.8 23.1
19.9 43.7 37.5 52.7 21.1 31.3
1.54 m 2.00 m 2.17 m 2.70 m 2.83 dd (16.2, 5.1)
6.80 s
6.78 s 3.18 m 3.94 dd (9.7, 3.6); 3.68 m 1.30 d (7.2)
1.32 s 1.11 s
group at C-11 in 5α,11,12-trihydroxy-6-oxa-abieta-8,11,13-trien-7-one. The HMBC correlations from the proton (δH 6.80, s) to C-8 (δC 133.6), C-9 (δC 146.5), C-10 (δC 42.0), C-12 (δC 159.2), C-13 (δC 133.7), and C15 (δC 26.7) suggested it was attached at C-11. Thus, the structure of 3 was characterized, named 5α,12-dihydroxy-6-oxa-abieta-8,11,13-trien7-one. Three known diterpenoids were identified as sugiol (Cheng and Lin, 1979), 6α-hydroxysugiol (Son et al., 2005), and lambertic acid (Cambie et al., 1983) by comparing their spectroscopic data with those reported in the literature. Compounds 1–3 were tested for their anti-inflammatory effects on LPS-stimulated RAW264.7 cells. NO is recognized as a mediator and regulator of inflammatory responses, which is mainly produced in macrophages by inducible nitric oxide synthase (iNOS). The results showed compound 1 significantly inhibited NO production with IC50 value of 5.38 ± 0.17 μM, and suppressed iNOS and COX2 expressions in a dose-dependent manner (Fig. 2A). The other two compounds didn’t show inhibitory effects up to 100 μM. The above evidences prompted us the anti-inflammatory effect of compound 1 might involve in the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways, which are the key regulators of inflammation. As expected, LPS treatment increased the phosphorylation of p38, c-Jun Nterminal kinase (JNK), and extracellular signal-regulated kinase (ERK); while, compound 1 significantly blocked the phosphorylation of p38, JNK, and ERK (Fig. 2B). Regarding to the NF-κB pathway, the phosphorylation of IKKα/β, IκBα, and p65 was increased remarkably by LPS stimulation, which was reversed by compound 1 (Fig. 2C). These results demonstrated that compound 1 suppressed the activation of MAPK and NF-κB signaling pathways.
C NMR (150 MHz, δ in ppm) data of compounds
18.2 34.7 37.7 42.6 35.7
2.23 m 2.62 2H m
18.2 36.7 39.2 107.6
198.3
165.7
124.5 156.2 37.4 109.9 158.3 132.7 126.6 26.8 22.5
7.91 s 3.17 m 1.28 d (6.9)
133.6 146.5 42.0 109.7 159.2 133.7 129.3 26.7 22.4
22.3
1.26 d (6.9)
22.5
70.9
3.46 d (11.4); 3.19 d (11.4) 0.95 s 1.27 s
22.8
7.90 s 3.18 m 1.24 d (6.3) 1.24 d (6.3) 1.25 s
25.5 17.9
1.24 s 1.12 s
17.3 23.8
6.73 s
6.80 s
3. Experimental 3.1. General experimental procedures
J = 6.9 Hz), 1.27 (3H, s), 1.26 (3H, d, J = 6.9 Hz), and 0.95 (3H, s)], one oxygenated methylene [δH 3.46 (1H, d, J = 11.4 Hz) and 3.19 (1H, d, J = 11.4 Hz)]; while, the 13C NMR spectrum of 2 exhibited 20 carbon resonances (Table 1). The 1H and 13C NMR data were quite similar to those of l8-hydroxyferruginol (Harrison and Asakawa, 1987). After careful comparison, the methylene at C-7 in l8-hydroxyferruginol was replaced by a carbonyl carbon in 2. In the HMBC spectrum, the correlations from H-5 (δH 2.23, m), H-6 (δH 2.62, 2H, m), H-11 (δH 6.73, s), and H-14 (δH 7.91, s) to the carbonyl carbon, assigned it as C-7. The relative configuration of 2 was assigned by ROESY spectrum. Therefore, the structure of 2 was established, named 7-oxo-18-hydroxyferruginol. Compound 3 was obtained as a yellow amorphous powder. Its molecular formula was assigned as C19H26O4 according to the HRESIMS ion peak at m/z 317.1761 [M − H]− (calcd. m/z 317.1753). The 1H and 13C NMR data of 3 (Table 1) closely resembled those of 5α,11,12trihydroxy-6-oxa-abieta-8,11,13-trien-7-one (Salae et al., 2012). The major difference was an aromatic proton in 3 instead of a hydroxyl
Optical rotation data were obtained using an Autopol VI polarimeter. UV data were recorded with a Varian CARY 50 spectrophotometer. IR spectra were recorded on a PerkinElmer spectrum-100 FTIR spectrometer using KBr disks. NMR spectra were recorded on a Bruker Avance-600 (Bruker, Switzerland, 600 MHz for 1H NMR, 150 MHz for 13C NMR) NMR spectrometer with the chemical shift values presented as δ values having TMS as an internal standard. The coupling constants (J) were given in Hz. CD spectra were measured on a Jasco J-180 spectrophotometer (Japan). HRESIMS spectra were recorded on an LTQ-Orbitrap XL spectrometer. All solvents were analytical grade (TianJing Chemical Plant, Tianjing, China). Silica gel used for column chromatography (CC) and precoated silica gel GF254 plates used for TLC were produced by Qingdao Haiyang Chemical Co., Ltd. TLC spots were viewed at 254 nm and visualized by spraying with 1% vanillin in H2SO4. MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical Industries Ltd.) and ODS C18 gel (50 μm, YMC) were used for CC.
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Fig. 2. Compound 1 inhibited LPS-induced inflammation in RAW264.7 macrophages through MAPK and NF-κB signaling pathways. (A) The protein expressions of iNOS and COX2 was analyzed by Western blotting. (B) The protein levels of phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, and JNK were detected by Western blotting analyses. (C) The protein levels of phospho-IKKα/β, IKKα, IKKβ, phospho-IκBα, IκBα, phospho-p65, and p65 were detected by Western blotting analyses. α-Tubulin was used as internal loading control.
successively. The EtOAc fraction (138.7 g) was subjected to CC over MCI gel eluted with H2O/MeOH (1:0 to 0:1), to yield eight major fractions (E1 to E8). The E6 fraction was subjected to CC over silica gel eluted with petroleum ether–acetone (15:1 to 1:1) to yield four subfractions (E6A to E6D), The E6D subfraction was subjected to CC over Sephadex LH-20, eluting with CHCl3/MeOH (1:1, v/v) to yield three fractions (E6D1 to E6D3), The E6D2 subfraction was further separated by preparative HPLC, eluting with H2O/MeCN (5:1, v/v), to obtain compounds 1 (2.6 mg), 2 (2.7 mg), and 6α-hydroxysugiol (3.9 mg). The E8 fraction was subjected to CC over ODS gel eluted with H2O/MeOH (1:1 to 0:1, v/v) to yield four subfractions (E8A to E8D). Sugiol (1.2 g) was crystallized from subfraction E8A. The remaining E8A subfraction was subjected to CC over Sephadex LH-20, eluting with CHCl3/MeOH (1:1, v/v), to yield five fractions (E8A1 to E8A4), The E8A2 subfraction was further separated by preparative HPLC, eluting with H2O/MeCN (1:1, v/v), to obtain compound 3 (4.7 mg), and lambertic acid (1.2 mg).
Preparative HPLC was performed on a Shimadzu LC-20AP instrument with a SPD-M20A PDA detector. Chromatographic separation was carried out on a C18 column (19 × 250 mm, 5 μm, Waters, SunFire™), using a gradient solvent system comprised of H2O (A) and MeCN (B) at a flow rate of 10 mL/min. 3.2. Plant material The seeds of P. nagi were collected from Lincang County, Yunnan Province, China, and identified by Professor Jingui Shen from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. A voucher was deposited at the herbarium of Institute of Chinese Medical Sciences, University of Macau (LL-20150901). 3.3. Extraction, isolation and characterization of compounds The air-dried seeds of P. nagi (15.0 kg) were ground into powder and extracted with 95% ethanol at room temperature (40 L × 3 times, each 2 days). After evaporation of the collected percolate, the crude extract (1.4 kg) was suspended in 4 L H2O and extracted with petroleum ether (2 L × 3), EtOAc (3 L × 3), and n-butanol (2 L × 3),
3.3.1. 16-Hydroxylambertic acid (1) White amorphous powder; [α]25 D +4.2 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 207.1 (0.22) nm; CD (MeOH, nm) λmax (Δε) 194.0 (+0.7), 202.0 (+3.5); IR νmax (KBr) 3457 (strong, broad), 1636, 1382, 1122,
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875 cm−1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 331.1912 [M − H]− (calcd for C20H27O4, 331.1909).
were isolated from the seeds of P. nagi. Moreover, compound 1 showed potent anti-inflammatory activity on macrophages through suppressing MAPK and NF-κB signaling pathways.
3.3.2. 7-Oxo-18-hydroxyferruginol (2) White amorphous powder; [α]25 D + 1.5 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 284.0 (0.5) nm; CD (MeOH, nm) λmax (Δε) 192.7 (–0.9), 198.7 (+0.3); IR νmax (KBr) 3426 (strong, broad), 2971, 1763, 1709, 1626, 1545, 1384, 1069, 994 cm−1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 315.1970 [M − H]− (calcd for C20H27O3, 315.1960).
Conflict of interest The author declare no conflict of interest. Acknowledgment Financial support by Science and Technology Development Fund, Macao S.A.R (FDCT 120/2013/A3) and the Research Fund of University of Macau (MYRG2015-00153-ICMS-QRCM and MYRG2017-00109ICMS) are gratefully acknowledged.
3.3.3. 5α,12-Dihydroxy-6-oxa-abieta-8,11,13-trien-7-one (3) Yellow amorphous powder; [α]25 D + 26.7 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 301.0 (0.17) nm; CD (MeOH, nm) λmax (Δε) 209.7 (–9.8), 230.0 (+23.2); IR νmax (KBr) 3430 (strong, broad), 2924, 2851, 1607, 1384, 1118, 965 cm−1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 317.1761 [M − H]− (calcd for C19H25O4, 317.1753).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.07.011.
3.4. Assay for NO production
References
RAW264.7 cells were maintained in DMEM supplemented with 10% FBS at 37 °C and 5% CO2. Cells were seeded into a 24-well plate (1 × 105 cells/well) and allowed to adhere for 24 h. Then, the cells were treated with different concentrations of compound for 1 h, followed by stimulation with 1 μg/mL LPS and incubated for another 18 h. DMSO was used as vehicle, where the final concentration of DMSO was maintained at up to 0.2% of all cultures. To evaluate the inhibitory activity of compounds, NO content in culture supernatant was assayed using Griess reagent as described previously (Liu et al., 2016).
Abdillahi, H.S., Finnie, J.F., van Staden, J., 2011. Anti-inflammatory, antioxidant, antityrosinase and phenolic contents of four Podocarpus species used in traditional medicine in South Africa. J. Ethnopharmacol. 136, 496–503. Barrero, A.F., Del Moral, J.F.Q., Herrador, M.M., 2003. Podolactones: a group of biologically active norditerpenoids. In: Rahman, Atta-ur (Ed.), Studies in Natural Products Chemistry. Elsevier, Amsterdam, pp. 453–516. Cambie, R.C., Cox, R.E., Croft, K.D., Sidwell, D., 1983. Phenolic diterpenoids of some Podocarps. Phytochemistry 22, 1163–1166. Cheng, Y.S., Lin, C.S., 1979. Study of the extractive constituents from the wood of Cuninghamia konishii Hayata. J. Chin. Chem. Soc. 26, 169–172. Editorial Committee of Flora of China, 1978. Flora of China. Science Press, Beijing, pp. 404. Feng, Z.L., Zhang, L.L., Zheng, Y.D., Liu, Q.Y., Liu, J.X., Feng, L., Huang, L., Zhang, Q.W., Lu, J.J., Lin, L.G., 2017. Norditerpenoids and dinorditerpenoids from the seeds of Podocarpus nagi as cytotoxic agents and autophagy inducers. J. Nat. Prod. http://dx. doi.org/10.1021/acs.jnatpro.7b00347. Harrison, L.J., Asakawa, Y., 1987. 18-Oxoferruginol from the leaf of Torreya nucifera. Phytochemistry 26, 1211–1212. Hembree, J.A., Chang, C.J., McLaughlin, J.L., Cassady, J.M., Watts, D.J., Wenkert, E., Fonseca, S.F., Campello, J.D.P., 1979. The cytotoxic norditerpene dilactones of Podocarpus milanjianus and Podocarpus sellowii. Phytochemistry 18, 1691–1694. Liu, Q.Y., Li, D., Wang, A.Q., Dong, Z., Yin, S., Zhang, Q.W., Ye, Y., Li, L.C., Lin, L.G., 2016. Nitric oxide inhibitory xanthones from the pericarps of Garcinia mangostana. Phytochemistry 131, 115–123. Salae, A.W., Rodjun, A., Karalai, C., Ponglimanont, C., Chantrapromma, S., Kanjana-Opas, A., Tewtrakul, S., Fun, H.K., 2012. Potential anti-inflammatory diterpenes from Premna obtusifolia. Tetrahedron 68, 819–829. Sato, K., Inaba, Y., Park, H.S., Akiyama, T., Koyama, T., Fukaya, H., Aoyagi, Y., Takeya, K., 2009. Cytotoxic bisnor- and norditerpene dilactones having 7α 8α-epoxy-9,11enolide substructure from Podocarpus macrophyllus D. Don. Chem. Pharm. Bull. 57, 668–679. Son, K.H., Oh, H.M., Choi, S.K., Han, D.C., Kwon, B.M., 2005. Anti-tumor abietane diterpenes from the cones of Sequoia sempervirens. Bioorg. Med. Chem. Lett. 15, 2019–2021. Zhang, M., Ying, B.P., Kubo, I., 1992. Nagilactones from Podocarpus nagi and their effects on the feeding and growth of tobacco budworm. J. Nat. Prod. 55, 1057–1062. Zheng, Y.D., Guan, X.C., Li, D., Wang, A.Q., Ke, C.Q., Tang, C.P., Lin, L.G., Ye, Y., Wang, Z.L., Yao, S., 2016. Novel diterpenoids from the twigs of Podocarpus nagi. Molecules 21, 1282.
3.5. Western blot analysis RAW264.7 cells were treated with or without different concentrations of compound 1 for 1 h, and then treated with or without LPS (1 μg/mL) for another 18 h, respectively. Cells were washed twice with cold PBS and then lysed with cold RIPA buffer containing freshly added phosphatase inhibitor cocktails and protease inhibitor on ice for 30 min. Whole cell lysate were centrifuged at 13,500 rpm for 20 min and the supernatants were transferred into new tubes. 15 μg protein extract was separated by 8% SDS-PAGE. Then extracted protein was transferred onto PVDF membrane (Bio-Rad Laboratories, Inc), blocked with 5% nonfat milk in TBST buffer for 1 h at room temperature and incubated with the specific primary antibodies overnight at 4 °C. After washing with TBST, the membrane was incubated with horseradish peroxidase conjugated secondary antibody for 2 h at room temperature. The protein-antibody complexes were detected by chemiluminescence (ECL System) and exposed by autoradiography. 4. Conclusion Three new abietane-type diterpenoids, 16-hydroxylambertic acid (1), 7-oxo-18-hydroxyferruginol (2), and 5α,12-dihydroxy-6-oxaabieta-8,11,13-trien-7-one (3), together with three known analogues,
263
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Anti-inflammatory abietane diterpenoids from the seeds of Podocarpus nagi 1
MARK
1
Zhe-Ling Feng , Dan Li , Qian-Yu Liu, Jing-Xin Liu, Li Huang, Qing-Wen Zhang, Yi-Tao Wang, ⁎ Li-Gen Lin State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, 999078, China, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Podocarpus nagi Abietane diterpenoids NO production Anti-inflammation
In searching for naturally occurring anti-inflammatory agents, three new abietane-type diterpenoids, named 16hydroxylambertic acid (1), 7-oxo-18-hydroxyferruginol (2), and 5α,12-dihydroxy-6-oxa-abieta-8,11,13-trien-7one (3), were isolated from the seeds of Podocarpus nagi, together with three known compounds. The structures of the new compounds were elucidated by extensive analysis of NMR and HR-ESIMS data. All the new compounds were tested for nitric oxide (NO) inhibitory activities on lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Compound 1 significantly inhibited NO production with IC50 value of 5.38 ± 0.17 μM, and suppressed inducible NO synthase (iNOS) expression in a dose-dependent manner, which were mediated through inhibiting the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) activation.
1. Introduction Podocarpus nagi (Thunb.) Pilg (Podocarpaceae) is widely distributed in South of China, which has been used to treat rheumatism, arthritis and venereal diseases (Editorial Committee of Flora of China, 1978; Abdillahi et al., 2011). The seeds of this plant are rich in diterpenoids, including totarane-, abietane-, and ent-pimarane-types (Barrero et al., 2003; Zheng et al., 2016). These diterpenoids were reported with wide bioactivities, including anti-inflammation (Zheng et al., 2016), cytotoxicity (Hembree et al., 1979; Sato et al., 2009; Feng et al., 2017), and insecticidal activities (Zhang et al., 1992). In searching for naturally occurring anti-inflammatory agents, we identified an abietane- and an ent-pimarane-type diterpenoids from the twigs of P. nagi, with inhibitory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-treated macrophages (Zheng et al., 2016). In a continuing study, three new abietane diterpenoids, 16-hydroxylambertic acid (1), 7-oxo18-hydroxyferruginol (2), and 5α,12-dihydroxy-6-oxa-abieta-8,11,13trien-7-one (3) (Fig. 1), were isolated from the seeds of P. nagi, together with three known analogues. Herein, the isolation and structure elucidation of the new compounds, as well as their anti-inflammatory effects on LPS-stimulated RAW264.7 cells, are described. 2. Results and discussion Compound 1, a white amorphous powder, had a molecular formula of C20H28O4 based on the deprotonated ion peak in the HRESIMS (m/z
⁎
1
331.1912 for C20H27O4, calcd. m/z 331.1909), indicating seven degrees of unsaturation. The 1H NMR spectrum of 1 displayed resonances for two aromatic protons [δH 6.80 (1H, s) and 6.78 (1H, s)], one oxygenated methylene [δH 3.94 (1H, dd, J = 9.7, 3.6 Hz) and 3.68 (1H, m)], and three methyls [δH 1.32 (3H, s), 1.30 (3H, d, J = 7.2 Hz), and 1.11 (3H, s)] (Table 1). The 13C NMR spectrum of 1 (Table 1) exhibited 20 resonances attributed to one carbonyl carbon (δC 181.1), six aromatic carbons (δC 152.9, 147.7, 128.1, 127.3, 126.6, and 113.9), two quaternary carbons (δC 38.4 and 37.5), two methine carbons (δC 52.7 and 36.4), six methylene carbons (δC 69.7, 43.7, 39.2, 31.3, 21.1, and 19.9), and three methyl carbons (δC 28.8, 23.1, and 15.6). These data revealed that 1 possesses an abietane-type diterpenoid framework, and its 1H and 13C NMR data were closely related to those of lambertic acid (Cambie et al., 1983). The major difference was a methyl group in lambertic acid was replaced by an oxygenated methylene [δH 3.94 and 3.68] in 1. The structure of 1 was constructed by detailed analysis of the HMBC spectrum. The HMBC correlations between the oxygenated methylene protons and C-13 (δC 128.1), C-15 (δC 36.4), and C-17 (δC 15.6) assigned it as H2-16. The relative configuration of 1 was the same as abietane-type diterpenoids inferred by ROESY spectrum. Accordingly, the structure of 1 was established, and it was named 16-hydroxylambertic acid. Compound 2 was obtained as a white amorphous powder. Its molecular formula was assigned as C20H28O3 according to the ion peak at m/z 315.1970 [M − H]− (calcd. m/z 315.1960) in its HRESIMS. The 1H NMR spectrum exhibited signals for four methyl groups [δH 1.28 (3H, d,
Corresponding author. E-mail address: [email protected] (L.-G. Lin). Authors contributed equally.
http://dx.doi.org/10.1016/j.phytol.2017.07.011 Received 28 April 2017; Received in revised form 6 July 2017; Accepted 10 July 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 21 (2017) 260–263
Z.-L. Feng et al.
Fig. 1. Chemical structures of compounds 1–3.
Table 1 1 H (600 MHz, δ in ppm, J in Hz) and 1–3 in CDCl3. position
1
13
2
3
δC
δH
δC
δH
δC
δH
1α 1β 2α 2β 3α 3β 4 5 6α 6β 7α 7β
39.2
1.41 m 1.57 m 1.66 m 2.04 m 1.44 m 2.24 m
37.4
1.37 m 1.53 m 1.42 m 1.83 m 1.40 m 1.56 m
31.2
1.51 m 1.98 m 1.42 m 1.70 m 1.60 m 1.86 m
8 9 10 11 12 13 14 15 16
126.6 147.7 38.4 113.9 152.9 128.1 127.3 36.4 69.7
17
15.6
18
181.1
19 20
28.8 23.1
19.9 43.7 37.5 52.7 21.1 31.3
1.54 m 2.00 m 2.17 m 2.70 m 2.83 dd (16.2, 5.1)
6.80 s
6.78 s 3.18 m 3.94 dd (9.7, 3.6); 3.68 m 1.30 d (7.2)
1.32 s 1.11 s
group at C-11 in 5α,11,12-trihydroxy-6-oxa-abieta-8,11,13-trien-7-one. The HMBC correlations from the proton (δH 6.80, s) to C-8 (δC 133.6), C-9 (δC 146.5), C-10 (δC 42.0), C-12 (δC 159.2), C-13 (δC 133.7), and C15 (δC 26.7) suggested it was attached at C-11. Thus, the structure of 3 was characterized, named 5α,12-dihydroxy-6-oxa-abieta-8,11,13-trien7-one. Three known diterpenoids were identified as sugiol (Cheng and Lin, 1979), 6α-hydroxysugiol (Son et al., 2005), and lambertic acid (Cambie et al., 1983) by comparing their spectroscopic data with those reported in the literature. Compounds 1–3 were tested for their anti-inflammatory effects on LPS-stimulated RAW264.7 cells. NO is recognized as a mediator and regulator of inflammatory responses, which is mainly produced in macrophages by inducible nitric oxide synthase (iNOS). The results showed compound 1 significantly inhibited NO production with IC50 value of 5.38 ± 0.17 μM, and suppressed iNOS and COX2 expressions in a dose-dependent manner (Fig. 2A). The other two compounds didn’t show inhibitory effects up to 100 μM. The above evidences prompted us the anti-inflammatory effect of compound 1 might involve in the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways, which are the key regulators of inflammation. As expected, LPS treatment increased the phosphorylation of p38, c-Jun Nterminal kinase (JNK), and extracellular signal-regulated kinase (ERK); while, compound 1 significantly blocked the phosphorylation of p38, JNK, and ERK (Fig. 2B). Regarding to the NF-κB pathway, the phosphorylation of IKKα/β, IκBα, and p65 was increased remarkably by LPS stimulation, which was reversed by compound 1 (Fig. 2C). These results demonstrated that compound 1 suppressed the activation of MAPK and NF-κB signaling pathways.
C NMR (150 MHz, δ in ppm) data of compounds
18.2 34.7 37.7 42.6 35.7
2.23 m 2.62 2H m
18.2 36.7 39.2 107.6
198.3
165.7
124.5 156.2 37.4 109.9 158.3 132.7 126.6 26.8 22.5
7.91 s 3.17 m 1.28 d (6.9)
133.6 146.5 42.0 109.7 159.2 133.7 129.3 26.7 22.4
22.3
1.26 d (6.9)
22.5
70.9
3.46 d (11.4); 3.19 d (11.4) 0.95 s 1.27 s
22.8
7.90 s 3.18 m 1.24 d (6.3) 1.24 d (6.3) 1.25 s
25.5 17.9
1.24 s 1.12 s
17.3 23.8
6.73 s
6.80 s
3. Experimental 3.1. General experimental procedures
J = 6.9 Hz), 1.27 (3H, s), 1.26 (3H, d, J = 6.9 Hz), and 0.95 (3H, s)], one oxygenated methylene [δH 3.46 (1H, d, J = 11.4 Hz) and 3.19 (1H, d, J = 11.4 Hz)]; while, the 13C NMR spectrum of 2 exhibited 20 carbon resonances (Table 1). The 1H and 13C NMR data were quite similar to those of l8-hydroxyferruginol (Harrison and Asakawa, 1987). After careful comparison, the methylene at C-7 in l8-hydroxyferruginol was replaced by a carbonyl carbon in 2. In the HMBC spectrum, the correlations from H-5 (δH 2.23, m), H-6 (δH 2.62, 2H, m), H-11 (δH 6.73, s), and H-14 (δH 7.91, s) to the carbonyl carbon, assigned it as C-7. The relative configuration of 2 was assigned by ROESY spectrum. Therefore, the structure of 2 was established, named 7-oxo-18-hydroxyferruginol. Compound 3 was obtained as a yellow amorphous powder. Its molecular formula was assigned as C19H26O4 according to the HRESIMS ion peak at m/z 317.1761 [M − H]− (calcd. m/z 317.1753). The 1H and 13C NMR data of 3 (Table 1) closely resembled those of 5α,11,12trihydroxy-6-oxa-abieta-8,11,13-trien-7-one (Salae et al., 2012). The major difference was an aromatic proton in 3 instead of a hydroxyl
Optical rotation data were obtained using an Autopol VI polarimeter. UV data were recorded with a Varian CARY 50 spectrophotometer. IR spectra were recorded on a PerkinElmer spectrum-100 FTIR spectrometer using KBr disks. NMR spectra were recorded on a Bruker Avance-600 (Bruker, Switzerland, 600 MHz for 1H NMR, 150 MHz for 13C NMR) NMR spectrometer with the chemical shift values presented as δ values having TMS as an internal standard. The coupling constants (J) were given in Hz. CD spectra were measured on a Jasco J-180 spectrophotometer (Japan). HRESIMS spectra were recorded on an LTQ-Orbitrap XL spectrometer. All solvents were analytical grade (TianJing Chemical Plant, Tianjing, China). Silica gel used for column chromatography (CC) and precoated silica gel GF254 plates used for TLC were produced by Qingdao Haiyang Chemical Co., Ltd. TLC spots were viewed at 254 nm and visualized by spraying with 1% vanillin in H2SO4. MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical Industries Ltd.) and ODS C18 gel (50 μm, YMC) were used for CC.
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Fig. 2. Compound 1 inhibited LPS-induced inflammation in RAW264.7 macrophages through MAPK and NF-κB signaling pathways. (A) The protein expressions of iNOS and COX2 was analyzed by Western blotting. (B) The protein levels of phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, and JNK were detected by Western blotting analyses. (C) The protein levels of phospho-IKKα/β, IKKα, IKKβ, phospho-IκBα, IκBα, phospho-p65, and p65 were detected by Western blotting analyses. α-Tubulin was used as internal loading control.
successively. The EtOAc fraction (138.7 g) was subjected to CC over MCI gel eluted with H2O/MeOH (1:0 to 0:1), to yield eight major fractions (E1 to E8). The E6 fraction was subjected to CC over silica gel eluted with petroleum ether–acetone (15:1 to 1:1) to yield four subfractions (E6A to E6D), The E6D subfraction was subjected to CC over Sephadex LH-20, eluting with CHCl3/MeOH (1:1, v/v) to yield three fractions (E6D1 to E6D3), The E6D2 subfraction was further separated by preparative HPLC, eluting with H2O/MeCN (5:1, v/v), to obtain compounds 1 (2.6 mg), 2 (2.7 mg), and 6α-hydroxysugiol (3.9 mg). The E8 fraction was subjected to CC over ODS gel eluted with H2O/MeOH (1:1 to 0:1, v/v) to yield four subfractions (E8A to E8D). Sugiol (1.2 g) was crystallized from subfraction E8A. The remaining E8A subfraction was subjected to CC over Sephadex LH-20, eluting with CHCl3/MeOH (1:1, v/v), to yield five fractions (E8A1 to E8A4), The E8A2 subfraction was further separated by preparative HPLC, eluting with H2O/MeCN (1:1, v/v), to obtain compound 3 (4.7 mg), and lambertic acid (1.2 mg).
Preparative HPLC was performed on a Shimadzu LC-20AP instrument with a SPD-M20A PDA detector. Chromatographic separation was carried out on a C18 column (19 × 250 mm, 5 μm, Waters, SunFire™), using a gradient solvent system comprised of H2O (A) and MeCN (B) at a flow rate of 10 mL/min. 3.2. Plant material The seeds of P. nagi were collected from Lincang County, Yunnan Province, China, and identified by Professor Jingui Shen from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. A voucher was deposited at the herbarium of Institute of Chinese Medical Sciences, University of Macau (LL-20150901). 3.3. Extraction, isolation and characterization of compounds The air-dried seeds of P. nagi (15.0 kg) were ground into powder and extracted with 95% ethanol at room temperature (40 L × 3 times, each 2 days). After evaporation of the collected percolate, the crude extract (1.4 kg) was suspended in 4 L H2O and extracted with petroleum ether (2 L × 3), EtOAc (3 L × 3), and n-butanol (2 L × 3),
3.3.1. 16-Hydroxylambertic acid (1) White amorphous powder; [α]25 D +4.2 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 207.1 (0.22) nm; CD (MeOH, nm) λmax (Δε) 194.0 (+0.7), 202.0 (+3.5); IR νmax (KBr) 3457 (strong, broad), 1636, 1382, 1122,
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875 cm−1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 331.1912 [M − H]− (calcd for C20H27O4, 331.1909).
were isolated from the seeds of P. nagi. Moreover, compound 1 showed potent anti-inflammatory activity on macrophages through suppressing MAPK and NF-κB signaling pathways.
3.3.2. 7-Oxo-18-hydroxyferruginol (2) White amorphous powder; [α]25 D + 1.5 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 284.0 (0.5) nm; CD (MeOH, nm) λmax (Δε) 192.7 (–0.9), 198.7 (+0.3); IR νmax (KBr) 3426 (strong, broad), 2971, 1763, 1709, 1626, 1545, 1384, 1069, 994 cm−1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 315.1970 [M − H]− (calcd for C20H27O3, 315.1960).
Conflict of interest The author declare no conflict of interest. Acknowledgment Financial support by Science and Technology Development Fund, Macao S.A.R (FDCT 120/2013/A3) and the Research Fund of University of Macau (MYRG2015-00153-ICMS-QRCM and MYRG2017-00109ICMS) are gratefully acknowledged.
3.3.3. 5α,12-Dihydroxy-6-oxa-abieta-8,11,13-trien-7-one (3) Yellow amorphous powder; [α]25 D + 26.7 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 301.0 (0.17) nm; CD (MeOH, nm) λmax (Δε) 209.7 (–9.8), 230.0 (+23.2); IR νmax (KBr) 3430 (strong, broad), 2924, 2851, 1607, 1384, 1118, 965 cm−1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 317.1761 [M − H]− (calcd for C19H25O4, 317.1753).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.07.011.
3.4. Assay for NO production
References
RAW264.7 cells were maintained in DMEM supplemented with 10% FBS at 37 °C and 5% CO2. Cells were seeded into a 24-well plate (1 × 105 cells/well) and allowed to adhere for 24 h. Then, the cells were treated with different concentrations of compound for 1 h, followed by stimulation with 1 μg/mL LPS and incubated for another 18 h. DMSO was used as vehicle, where the final concentration of DMSO was maintained at up to 0.2% of all cultures. To evaluate the inhibitory activity of compounds, NO content in culture supernatant was assayed using Griess reagent as described previously (Liu et al., 2016).
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3.5. Western blot analysis RAW264.7 cells were treated with or without different concentrations of compound 1 for 1 h, and then treated with or without LPS (1 μg/mL) for another 18 h, respectively. Cells were washed twice with cold PBS and then lysed with cold RIPA buffer containing freshly added phosphatase inhibitor cocktails and protease inhibitor on ice for 30 min. Whole cell lysate were centrifuged at 13,500 rpm for 20 min and the supernatants were transferred into new tubes. 15 μg protein extract was separated by 8% SDS-PAGE. Then extracted protein was transferred onto PVDF membrane (Bio-Rad Laboratories, Inc), blocked with 5% nonfat milk in TBST buffer for 1 h at room temperature and incubated with the specific primary antibodies overnight at 4 °C. After washing with TBST, the membrane was incubated with horseradish peroxidase conjugated secondary antibody for 2 h at room temperature. The protein-antibody complexes were detected by chemiluminescence (ECL System) and exposed by autoradiography. 4. Conclusion Three new abietane-type diterpenoids, 16-hydroxylambertic acid (1), 7-oxo-18-hydroxyferruginol (2), and 5α,12-dihydroxy-6-oxaabieta-8,11,13-trien-7-one (3), together with three known analogues,
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