Labdane diterpenes from Chloranthus serratus

Fitoterapia 91 (2013) 95–99

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Labdane diterpenes from Chloranthus serratus Mi Zhang, Junsong Wang, Jun Luo, Pengran Wang, Chao Guo, Lingyi Kong ⁎ State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China

a r t i c l e

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Article history: Received 27 June 2013 Accepted in revised form 17 August 2013 Available online 30 August 2013 Keywords: Labdane diterpene Chloranthus serratus Anti-inflammatory Induced circular dichroism

a b s t r a c t Five new labdane diterpenes (1–5), serralabdanes A–E, were isolated from the whole plant of Chloranthus serratus. Their structures were elucidated by spectroscopic methods, and the absolute configuration of the 12,13-diol moiety in serralabdane C (3) was determined by observing the induced circular dichroism (ICD) after addition of dimolybdenum teracetate in DMSO solution. Serralabdanes A–E (1–5) showed inhibitory effects on lipopolysaccharideinduced nitric oxide production in RAW264.7 cells. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Labdane diterpenes, possessing a variety of changeable skeletons, are mainly distributed widely in higher plants, such as Labiate, Asteraceae, Acanthaceae, Euhorbiaceae, Chloranthaceae, and Zingiberaceae, among others. Most of them have marked bioactivities, including anti-inflammatory, anti-bacterial, cardiotonic, and hypotensive activities [1]. Chloranthus serratus (Thnub.) Roem. et Schult., belonging in Chloranthaceae, is a perennial herbaceous plant usually distributed in eastern Asia. Its whole plant has been used for the treatment of bruises, bone fractures, rheumatoid arthritis, etc. in Chinese folk [2,3]. Previous investigations on the chemical constituents of this plant and their activities by our group [4] and other researchers [5–9] have discovered many terpenoids, mainly including sesquiterpene and sesquiterpenoid dimers, and some of which displayed significantly anti-inflammatory activities [4]. In our further study, five new labdane diterpenes (1–5), serralabdanes A–E, were isolated from the whole plant of C. serratus. Their structures and relative configurations were elucidated by spectroscopic methods (Fig. 1). And the absolute configuration of the 12,13-diol moiety in serralabdane C (3) was determined by observing the induced circular dichroism ⁎ Corresponding author. Tel./fax: +86 25 8327 1405. E-mail address: [email protected] (L. Kong). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.08.015

(ICD) after the addition of dimolybdenum teracetate in DMSO solution (Snatzke's method). Serralabdanes A–E (1–5) were evaluated for their inhibitory effects on lipopolysaccharideinduced nitric oxide production in RAW264.7 cells. 2. Experimental details 2.1. General Optical rotations were obtained on a JASCO P-1020 polarimeter. CD spectra were measured with a JASCO 810 spectropolarimeter. UV spectra were recorded on a Shimadzu UV-2450 spectropolarimeter. NMR spectra were obtained on Bruker ACF-500 with TMS as the internal standard. Highresolution mass spectra were obtained on an Agilent UPLCQ-TOF (6520B). Silica gel (200–400 mesh, Qingdao Haiyang Chem. Co.), MCI gel (75–150 μm, Mitsubishi) and RP-C18 (40–63 μm, Fuji) were used for column chromatography (CC). Recycling preparative HPLC was carried out using Agilent 1100 Series equipped with Shim-park RP-C18 column (5 μm, 20 × 200 mm) and 1100 Series Multiple Wavelength Detector. 2.2. Plant material Whole plants of C. serratus were collected in May 2010 from Tiantang Village, Anhui Province of China, and authenticated

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2

1

3

5

4 Fig. 1. Structures of compounds 1–5.

by Prof. Gan Yao of the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences. A voucher specimen (CS2010005) has been deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. 2.3. Extraction and isolation The whole plants of C. serratus (1.8 kg) were extracted with 95% EtOH (4 × 6 L) to give 163 g of residue. The residue was suspended in water and partitioned with EtOAc to afford the EtOAc fraction (101 g). The fraction extract was chromatographed over a silica gel column eluted with petroleum ether–EtOAc in a gradient (1:0 to 0:1) to yield twenty fractions (1–20), monitored by TLC. Fraction 12 (2.0 g) was chromatographed on MCI gel, eluted successively with MeOH– H2O (1:1 to 7:3) to give three subfractions (12a–12c). Fraction 12c (145.8 mg) was subjected to reversed-phase C18 silica gel, eluted with MeOH–H2O (1:1 to 7:3), to give five subfractions (12c1–12c5). Fraction 12c2 (15.3 mg) was separated by recycling preparative HPLC using MeOH–H2O (65:35) to give 1 (5.7 mg) and 2 (2.2 mg). Fraction 12c3 (25.6 mg) was separated by recycling preparative HPLC using MeOH–H2O (70:30) to give 3 (2.7 mg), 4 (7.5 mg), and 5 (4.8 mg). Serralabdane A (1): colorless oil; [α]25D − 3.8 (c = 0.22, MeOH); UV (MeOH) λmax (log ε) 203 (3.71) nm; 13C and 1 H NMR data, see Tables 1 and 2; HRESIMS m/z 303.2320 (calcd for C20H31O2, 303.2319). Serralabdane B (2): white powder; [α]25D + 5.2 (c = 0.26, MeOH); UV (MeOH) λmax (log ε) 206 (3.83) nm; CD (1.7 × 10− 4 MeOH) Δε216 nm − 16.95; 13C and 1H NMR data, see Tables 1 and 2; HRESIMS m/z 341.2084 (calcd for C20H30O3Na, 341.2087). Serralabdane C (3): white powder; [α]25D + 28.8 (c =0.18, MeOH); UV (MeOH) λmax (log ε) 204 (3.63) nm; 13C and 1H NMR data, see Tables 1 and 2; HRESIMS m/z 345.2402 (calcd for C20H34O3Na, 345.2400).

Serralabdane D (4): white powder; [α]25D − 2.9 (c =0.33, MeOH); UV (MeOH) λmax (log ε) 206 (3.71) nm; 13C and 1H NMR data, see Tables 1 and 2; HRESIMS m/z 373.2701 (calcd for C22H38O3Na, 373.2701). Serralabdane E (5): white powder; [α]25D + 2.6 (c = 0.21, MeOH); UV (MeOH) λmax (log ε) 220 (4.18) nm, 205 (4.15); 13 C and 1H NMR data, see Tables 1 and 2; HRESIMS m/z 277.1272 (calcd for C18H29O2, 277.1262). 2.4. Anti-inflammatory bioassays The protocol of the anti-inflammatory bioassays was provided in a previously published paper with dexamethasone as the reference [10]. Table 1 13 C NMR (125 MHz) spectral data of compounds 1–5 (in CDCl3). No.

1

2

3

4

5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 \OEt

37.2 28.2 79.0 39.4 54.8 24.1 38.1 148.4 54.4 39.5 21.9 150.9 113.6 113.1 139.6 10.3 107.4 28.6 15.7 14.5

36.7 28.0 78.5 39.1 52.2 24.0 37.9 148.1 54.3 39.1 27.8 83.4 169.2 116.6 173.1 13.9 106.5 28.3 15.4 14.5

37.1 27.9 78.8 39.0 54.6 24.0 38.2 148.4 52.1 39.2 26.3 75.9 75.8 141.0 114.5 24.5 106.9 28.3 15.4 14.7

37.0 28.6 78.8 39.2 54.7 24.0 38.1 148.4 52.9 39.2 28.0 77.2 140.3 124.8 66.7 10.7 107.1 28.3 15.4 14.6 65.6 15.3

38.6 27.6 78.8 39.0 53.7 22.9 36.4 147.9 60.5 39.3 145.8 133.7 197.9 27.3

109.0 28.3 15.1 15.6

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Table 2 1 H NMR (500 MHz) spectral data of compounds 1–5 (in CDCl3, J in Hz). No.

1

1α 1β 2α 2β 3 5 6α 6β 7α 7β 9 11α 11β 12 13 14 15

1.31 1.79 1.69 1.60 3.29 1.19 1.76 1.40 2.01 2.36 2.26 2.69 2.64

16 17 18 19 20 \OEt

2

3

1.29 (m) 1.61 (t, 3.0) 1.69 (dt, 12.5, 4.5) 1.58 (m) 3.27 (dd, 11.5, 4.5) 1.19 (dd, 12.5, 2.5) 1.79 (dt, 13.0, 2.5) 1.42 (dd, 13.0, 4.0) 2.06 (m) 2.44 (dt, 13.0, 4.0) 2.19 (dd, 12.5, 2.5) 1.94 (t, 12.5) 1.39 (m) 4.85 (d, 11.0)

1.23 1.75 1.70 1.56 3.26 1.18 1.78 1.40 2.02 2.41 1.99 1.63 1.31 3.47

(m) (m) (m) (m) (dd, 11.5, 4.0) (td, 12.5, 2.5) (m) (dd, 13.0, 4.0) (td, 12.5, 5.0) (dt, 12.5, 3.0) (m) (m) (m) (d, 10.5)

1.07 1.74 1.65 1.57 3.23 1.02 1.77 1.69 1.91 2.39 1.44 1.69 1.67 4.13

6.11 (d, 1.5) 7.17 (d, 1.5)

5.77 (s)

2.10 (s) 4.45 (s) 4.94 (s) 1.17 (s) 0.78 (s) 0.69 (s)

(dd, 17.5, 11.0) (d, 11.0) (d, 17.5) (s) (s) (s) (s) (s) (s)

5.47 (t, 6.5) 4.02 (m)

1.93 4.59 4.78 1.01 0.80 0.78

5.94 5.22 5.35 1.33 4.46 4.85 1.00 0.78 0.68

(m) (dt, 13.5, 3.0) (m) (m) (dd, 11.5, 4.5) (dd, 12.5, 4.3) (m) (dd, 12.5, 4.0) (td, 13.0, 5.0) (ddd, 13.0, 2.5, 2.5) (dd, 10.0, 2.5) (m) (m)

(s) (s) (s) (s) (s) (s)

3. Results and discussion Serralabdane A (1), obtained as a colorless oil, showed the molecular formula of C20H30O2 by positive-ion HRESIMS from the [M + H]+ signal at m/z 303.2320. Its 13C NMR spectrum displayed twenty signals (Table 1) and 1H NMR exhibited four methyl group proton signals at δH 1.93 (s), 1.01 (s), 0.80 (s), and 0.78 (s), one hydroxymethine proton at δH 3.29 (dd, 11.5, 4.5), and two trisubstituted olefinic proton signals at δH 6.11 (d, 1.5) and 7.17 (d, 1.5) (Table 2). Moreover, two protons at δH 4.59 (s) and 4.78 (s) characteristic of an exocyclic olefin in labdane diterpene [1] correlated with an olefinic methylene carbon at δC 107.4 in the HSQC spectrum which indicated that an exocyclic olefin was to be located at C-8 by the long-range HMBC correlations from the two protons to C-7 and C-9. Comparison of its NMR data with those of pumiloxide [11] revealed a difference in that a hydroxy was present in 1. This was supported by HMBC correlations of the two gem-dimethyl protons at δH 1.01 and 0.80 (each 3H, s) with the oxymethine carbon resonance δC 79.0, and a hydroxymethine proton at δH 3.29 (dd, 11.5, 4.5) correlated with the oxymethine carbon resonance δC 79.0 in the HSQC spectrum with C-18 and C-19. The relative configuration of 1 was established by a ROESY experiment in which correlations of Me-20/Me-19, Me-18/H-3, H-3/H-5 and H-5/H-9 revealed that 3-OH was β-oriented, and H-3, H-5 and H-9 were α-oriented (Fig. 2). Therefore, the structure of 1 was identified as 3β-hydroxy-15,12-epoxylabda-8(17),12,14triene, named serralabdane A. Serralabdane B (2) was isolated as a white powder and its molecular formula was determined to be C20H30O3 (HRESIMS). Twenty carbon signals were shown in the 13C NMR spectrum (Table 1) and four methyl group proton signals at δH 2.10 (s),

4

1.65 4.69 4.88 0.98 0.76 0.69 3.49 1.21

5 (m) (td, 10.0, 3.5) (m) (m) (dd, 11.5, 4.5) (m) (dt, 11.5, 3.5) (m) (td, 11.5, 5.0) (dt, 11.5, 3.0) (t, 6.5) (m) (m) (t, 7.0)

(s) (s) (s) (s) (s) (s) (dd, 7.0, 2.0) (t, 7.0)

1.43 1.60 1.64 1.55 3.26 1.09 1.75 1.47 2.08 2.47 2.43 6.85

(dt, 13.5, 3.5) (td, 13.5, 3.5) (m) (m) (dd, 11.5, 4.5) (td, 12.5, 4.0) (dt, 13.0, 2.5) (dd, 13.0, 4.5) (td, 13.5, 5.0) (dt, 13.5, 3.0) (d, 9.0) (dd, 15.5, 10.0)

6.10 (d, 16.0) 2.27 (s)

4.43 4.82 1.02 0.89 0.82

(s) (s) (s) (s) (s)

1.17 (s), 0.78 (s), and 0.69 (s), two hydroxymethine protons at δH 3.27 (dd, 11.5, 4.5) and 4.85 (d, 11.0), two characteristic exocyclic methylene proton signals at δH 4.45 (s) and 4.94 (s), and one trisubstituted olefinic proton signal at δH 5.77 (s) (Table 2). In its HMBC spectrum, cross-peaks from the proton at δH 3.27 (dd, 11.5, 4.5) to C-4, C-18 and C-19, from Me-16 to C-12, C-13, and C-14, from Me-18 and Me-19 to C-3, C-4, and C-5, and from Me-20 to C-1, C-5, C-9, and C-10 indicated that 2 possessed a 3-hydroxy labdane diterpene skeleton incorporating a 13-en-15,12-olide structural moiety. The relative configuration of 2 was found to be the same as 1 by a ROESY experiment. And the configuration of C-12 was determined as R because of the negative cotton effect curve at 216 nm [12]. Thus, the structure of 2 was identified as 3β-hydroxy-8(17),13labdadien-15,12R-olide, named serralabdane B.

Fig. 2. Selected 2D NMR correlations of serralabdane A (1).

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Serralabdane C (3), a white powder, had the molecular formula C20H34O3 as established by HRESIMS. Its 1H NMR spectrum showed four methyl group proton signals at δH 1.33 (s), 1.00 (s), 0.78 (s), and 0.68 (s), two hydroxymethine protons at δH 3.26 (dd, 11.5, 4.0) and 3.47 (d, 10.5), four characteristic exocyclic methylene proton signals at δH 4.45 (s), 4.94 (s), 5.22 (d, 11.0), and 5.35 (d, 17.5), and one trisubstituted olefinic proton signal at δH 5.94 (dd, 17.5, 11.0) (Table 2). Its 13C NMR spectrum also displayed twenty signals (Table 1). Comparison of the 1D NMR spectra of 3 with those of 12R,13Rdihydroxylabda-8(17),14-dien-19-oic acid [13] revealed the differences in that the carboxylic group was absent and a methyl group was instead found at δH 0.78 (s) in 3. And there was presence of a hydroxy at C-3 in 3, which was supported by HMBC correlations from the hydroxymethine protons at δH 3.26 (dd, 11.5, 4.0) to two gem-dimethyl carbons at δC 28.3 and 15.4. The relative configuration of 3-OH was established as β-oriented on the basis of the observed ROESY correlations from Me-18 to H-3 (Fig. 3). Moreover, the absolute configuration of the 12,13-diol moiety in serralabdane C (3), determined as 12R,13R, was also supported by the negative cotton effect curve at 313 nm in the induced circular dichroism (ICD) after the addition of dimolybdenum teracetate [Mo2(OAc)4] in DMSO solution (Snatzke's method) [14]. Hence, the structure of 3 was proposed as 3β,12R,13R-trihydroxy-8(17),14-dienlabdane, named serralabdane C. Serralabdane D (4) was obtained as a white powder. It showed the molecular formula C22H38O3 by positive-ion HRESIMS from the [M + Na]+ signal at m/z 373.2701. Detailed analysis of its 1D NMR spectra (Tables 1 and 2) suggested that the structure of 4 was similar to 12S,15dihydroxylabda-8(17),13E-dien-19-oic acid [13]. The main differences were that in the latter a carboxylic group was present instead of a methyl group at δH 0.76 (s), a hydroxy was present at C-3, and an ethyoxyl group at δH 3.49 (dd, 7.0, 2.0) and 1.21 (t, 7.0) was attached to C-15. Those were supported by HMBC correlations from the methyl group at δH 0.76 (s) to C-3, C-4, and C-5, the hydroxymethine protons at δH 3.23 (dd, 11.5, 4.5) to C-18 and C-19, and the oxymethylene protons at δH 3.49 (dd, 7.0, 2.0) to C-15. The relative configuration of 4 was established by a ROESY experiment, and 3-OH, H-5, and H-9 were found to be the same as those of 3. Consequently, the structure of 4 was

identified as 3β,12S-dihydroxy-15-ethyoxyl-8(17),13(E)-dienlabdane, named serralabdane D. Serralabdane E (5), isolated as a white powder, had the molecular formula C18H28O2. The 1D NMR data (Tables 1 and 2) suggested that 5 was structurally similar to 15,16-bisnor13-oxo-8(17),11E-labdandien-19-oic acid [15], and the significant differences were the absence of a carboxylic group and the presence of a hydroxy in 5. In the HMBC spectrum, the correlations between the hydroxymethine protons at δH 3.26 (dd, 11.5, 4.5) and C-1, C-18, and C-19 exhibited a hydroxy that was attached to C-3. And the relative configuration of 3-OH was established as β-oriented by a ROESY experiment in which key long-range correlation was observed between Me-18 and H-3. On the basis of the above data, 5 was determined as 11E-3β-hydroxy-15,16-bisnor-8(17),12-labdadien-13-one, named serralabdane E. Serralabdanes A–E (1–5) were evaluated for their inhibitory effects on the release of NO from macrophages using lipopolysaccharide (LPS)-induced RAW264.7 cells as a model system. Compared with IC50 values of the reference compound dexamethasone at 1.08 ± 0.15 μM, serralabdanes A–E (1–5) displayed weak activities with IC50 values of 38.45 ± 1.02, 29.78 ± 0.92, 44.37 ± 0.58, 53.68 ± 1.52, and 47.31 ± 1.26, respectively. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193), the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the National Natural Science Foundation of China (21072230). Appendix A. Supplementary data 1D and 2D NMR, and HRESIMS spectra of 1–5 are available as Supporting Information. Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j. fitote.2013.08.015. References

HO OH

HO

HMBC

ROESY

Fig. 3. Selected 2D NMR correlations of serralabdane C (3).

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