Fitoterapia 95 (2014) 229–233

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Two novel clerodane diterpenenes with NGF-potentiating activities from the twigs of Croton yanhuii Yihang Sun a,b,1, Meicheng Wang a,1, Quanhui Ren a, Shen Li a, Jing Xu a,⁎, Yasushi Ohizumi c, Chunfeng Xie a, Da-Qing Jin d, Yuanqiang Guo a,⁎ a b c d

College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China College of Pharmacy, Harbin University of Commerce, Harbin 150076, China Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan School of Medicine, Nankai University, Tianjin 300071, China

a r t i c l e

i n f o

Article history: Received 26 January 2014 Accepted in revised form 26 February 2014 Available online 29 March 2014 Keywords: Croton yanhuii Clerodane diterpene Nerve growth factor (NGF)-potentiating activity Alzheimer's disease

a b s t r a c t Nerve growth factor (NGF) and analog reagents to promote the neurite outgrowth of nerve cells against the neuron degeneration are expected to be potentially useful for the medical treatment of Alzheimer's disease. In our focus on the discovery of bioactive diterpenes, we investigated the chemical constituents of the plant Croton yanhuii. This investigation led to the isolation and identification of two novel clerodane diterpenes (1 and 2). Their structures were elucidated on the basis of extensive 1D and 2D NMR (COSY, HMQC, HMBC, and NOESY) and mass (ESIMS and HR-ESIMS) spectroscopic data analyses. Further biological screenings showed that both of the compounds enhanced NGF-mediated neurite outgrowth from PC12 cells. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The genus Croton, a member of the Euphorbiaceae family, contains about 800 species that are widely distributed in the subtropical and tropical regions [1]. About six species of the genus Croton, such as Croton tiglium, C. crassifolius, C. lachnocarpus, et al. were documented in Chinese Materia Medica for medical treatment of various diseases [2]. Previous phytochemical investigations on this genus Croton revealed the main presence of the diterpenes, especially clerodane-type, kaurane-type, and labdane-type diterpenes [3–10], which showed increasing effects of histamine-induced gastric acid secretion [3], cytotoxic and anti-inflammatory activities [7–9],

⁎ Corresponding authors. Tel./fax: +86 22 23502595. E-mail addresses: [email protected] (J. Xu), [email protected] (Y. Guo). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.fitote.2014.03.012 0367-326X/© 2014 Elsevier B.V. All rights reserved.

and stimulatory effects on osteoblast differentiation [10]. Though the chemistry and biological screenings have been investigated, neither the neurite outgrowth-promoting activity of the genus Croton, nor the chemical constituents of Croton yanhuii Y. T. Chang have been reported. In our search for pharmacologically active substances in plants [11–13], much attention has been given to the occurrence of compounds having NGF-potentiating activity, since these compounds are expected to be potentially useful for the medical treatment of dementia [14]. As a continuation of our search for bioactive substances with NGF-potentiating activities from plants [13], we investigated the chemical constituents of C. yanhuii, whose chemical constituents have never been reported. Our phytochemical investigation on C. yanhuii led to the isolation of two novel diterpenenes (1 and 2) (Fig. 1). On the basis of detailed spectroscopic and spectrometric analyses (IR, ESIMS, HR-ESIMS, and 1D and 2D NMR), two novel diterpenes were elucidated and named crotonpenes A and B (1 and 2). Further biological screenings showed that both of the compounds enhanced nerve

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Y. Sun et al. / Fitoterapia 95 (2014) 229–233

Fig. 1. Structures of compounds 1 and 2 from C. yanhuii.

growth factor (NGF)-mediated neurite outgrowth from PC12 cells. Herein, the isolation, structural elucidation and neurite outgrowth-promoting activity of two novel diterpenes are described.

2. Experimental 2.1. General The optical rotations were measured in CH2Cl2 using a Rudolph Autopol IV automatic polarimeter (Rudolph Research Analytical, America). The IR spectra were taken on a Bruker Tensor 27 FT-IR spectrometer with KBr discs (Bruker, Germany). The ESIMS spectra were obtained on a LCQ-Advantage mass spectrometer made by Finnigan Company (America). HR-ESIMS spectra were recorded by Agilent 6520 Q-TOF LC/MS (Agilent, Santa Clara, CA). 1D and 2D NMR spectra were recorded on a Bruker AV 400 instrument (400 MHz for 1H and 100 MHz for 13C) with TMS as an internal standard. HPLC separations were performed on a CXTH system (Beijing Chuangxintongheng instrument Co. Ltd., P. R. China), equipped with a UV3000 detector at 210 nm, and a YMC-pack ODS-AM column (20 × 250 mm, i.d.). Silica gel (200–300 mesh, Qingdao Marine Chemical Group Co. Ltd., P. R. China) was used for column chromatography. Chemical reagents for isolation were analytical grade and purchased from Tianjin Chemical Reagent Company, P. R. China. Biological reagents were from Sigma Company. The PC12 cell line was from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (China).

2.3. Extraction and isolation The air-dried twigs of C. yanhuii (5.2 kg) were cut into pieces and extracted with MeOH (3 × 31 L) under reflux. The solvent was evaporated to obtain a crude extract. The extract was suspended in H2O (0.4 L) and partitioned with EtOAc (3 × 0.4 L). The EtOAc soluble part (78 g) was subjected to a silica gel column chromatography, using a gradient of acetone in petroleum ether (1%–40%), to give four fractions (F1–F4) based on TLC analyses. F3 (10.3 g) was fractionated by middle pressure liquid chromatography (MPLC) over octadecylsilyl (ODS) eluting with a step gradient from 70% to 92% MeOH in H2O to give three subfractions (F3-1–F3-3). F3-2 was further purified by preparative HPLC (YMC-pack ODS-AM, 20 × 250 mm, 83% MeOH in H2O) to afford compounds 1 (tR = 33 min, 21.6 mg), and 2 (tR = 26 min, 35.2 mg). 2.3.1. Crotonpene A (1) Colorless oil; [α]12 D = + 175.4 (c = 0.13, CH2Cl2); IR (KBr) νmax cm−1: 2947, 2905, 1677, 1373, 1276, 1261, 1108, 1052, 975, 874; ESI-MS m/z 315 [M + H]+; HR-ESIMS m/z 315.1955 [M + H]+, calcd. for C20H27O3 315.1960; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data see Table 1. 2.3.2. Crotonpene B (2) Colorless oil; [α]12 D = − 210.6 (c = 0.32, CH2Cl2); IR (KBr) νmax cm−1: 2953, 2914, 1730, 1680, 1383, 1279, 1220, 1156, 983, 873. ESI-MS m/z 361 [M + H]+; HR-ESIMS m/z 361.2010 [M + H]+, calcd. for C21H29O5 361.2015; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data see Table 1.

2.2. Plant material 2.4. Bioassay for neurite outgrowth The twigs of C. yanhuii were collected in Xishuangbanna, Yunnan province, China in October, 2013. A voucher specimen (no. 20131001) was identified by Dr. Yuanqiang Guo (College of Pharmacy, Nankai University, China) and deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University, China.

PC12 cells were cultured at 37 °C in DMEM supplemented with 5% (v/v) inactivated fetal bovine serum (FBS), 5% (HS, v/v) inactivated horse serum (HS) and 100 U/mL penicillin/streptomycin under a water-saturated atmosphere of 95% air and 5% CO2. The cells were disassociated by

Y. Sun et al. / Fitoterapia 95 (2014) 229–233 Table 1 1 H and 13C NMR spectroscopic data of compounds 1 and 2 (in CDCl3, δ in ppm, J in Hz).a Position

1 δC

1

18.5

2

27.7

3 4 5 6

62.2 64.7 36.9 36.7

7

27.9

8 9 10 11

40.4 55.8 50.4 104.8

12 13 14 15 16 17 18 19 20

148.0 117.3 108.3 143.1 139.6 16.4 19.6 15.2 70.3

OMe

2 δH

δC

α 1.52 m β 1.45 m α 1.74 m β 2.12 m β 2.94 br. s

α 1.65 β 1.39 α 1.57 β 1.34 β 1.31

m m m m m

β 1.19 m 4.54 s

6.45 dd (1.8, 0.6) 7.35 t (1.8) 7.51 br. s 0.88 d (6.6) 1.17 s 0.87 s a 3.96 d (9.4) b 4.13 d (9.4)

18.3 28.0 62.2 65.5 37.4 37.3 28.2 38.9 50.9 49.5 43.2 192.9 128.7 108.6 144.3 146.9 17.8 19.8 13.8 174.6 50.9

δH α 1.58 m β 1.49 m α 2.08 m β 2.17 m β 2.94 br. s

α 1.69 β 1.47 α 1.70 β 1.46 β 1.82

m m m m m

β 1.84 m 3.11 d (16.9) 3.18 d (16.9)

6.74 dd (1.8, 0.7) 7.44 t (1.8) 8.03 br. s 1.10 d (6.9) 1.18 s 0.91 s

3.64 s

a

The assignments are based on DEPT, HMQC, HMBC, 1H–1H COSY, and NOESY experiments.

incubation with 1 mM of ethylene glycol-bis(2-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA) in phosphatebuffered saline (PBS) for 15 min and then seeded in 24-well culture plates (3 × 104 cells/well) coated with poly-L-lysine. After 24 h, the medium was changed to test medium containing various concentrations of NGF (100 ng/mL for positive control, 20 ng/mL for test samples and significant difference control), 1% FBS, 1% HS, and various concentrations of test compounds (1.5, 5, 15 μM). After a continuous incubation of 96 h, the neurite outgrowth was assessed under a phase

231

contrast microscope. Neurite processes with a length equal to or greater than the diameter of the neuron cell body were scored as neurite bearing cells. The ratio of the neurite-bearing cells to total cells (with at least 100 cells examined/viewing area; 3 viewing areas/well; 6 wells/sample) was determined and expressed as a percentage. 3. Results and discussion Compound 1 was isolated as a colorless oil. Its HR-ESIMS provided the molecular formula C20H26O3, through the presence of a peak at m/z 315.1955 [M + H]+ (calcd. for C20H27O3 315.1960), which was compatible with the NMR data. The 1H NMR spectrum of 1 exhibited three methyl groups at δH 1.17 (3H, s, H3-18), 0.88 (3H, d, J = 6.6 Hz, H3-17), and 0.87 (3H, s, H3-19), a set of oxygenated methylene protons at δH 3.96 and 4.13 (each 1H, d, J = 9.4 Hz, H2-20), and one olefinic proton at δH 4.54 (1H, s, H-11) (Table 1). In addition, three aromatic protons at δH 6.45 (1H, dd, J = 1.8, 0.6 Hz, H-14), 7.35 (1H, t, J = 1.8 Hz, H-15), and 7.51(1H, br. s, H-16) were also revealed in the 1H NMR spectrum. The 13C NMR spectrum of 1 showed 20 carbon resonances. From the 1H and 13C NMR spectra, a β-substituted furan ring moiety was obvious from the observation of the above three aromatic protons and the following carbon resonances [δC 117.3 (C-13), 108.3 (C-14), 143.1 (C-15), and 139.6 (C-16)]. Apart from the above four aromatic carbons for the furan ring moiety, the remaining 16 carbons displayed in the 13C NMR spectrum were classified into three methyl groups [δC 16.4 (C-17), 19.6 (C-18), and 15.2 (C-19)], five methylene groups [δC 18.5 (C-1), 27.7 (C-2), 36.7 (C-6), 27.9 (C-7), and 70.3 (C-20)], four methine group [δC 62.2 (C-3), 40.4 (C-8), 50.4 (C-10), and 104.8 (C-11)], four quaternary carbons [δC 64.7 (C-4), 36.9 (C-5), 55.8 (C-9), and 148.0 (C-12)] by the analyses of DEPT and HMQC spectra. Based on the above spectroscopic features, a comparison of the chemical shifts of compound 1 with those of clerodane diterpenes reported in the literature suggested that 1 should be a clerodane diterpene [3–5]. The following interpretation of HMQC and HMBC spectra confirmed the structure of clerodane diterpene for 1. In the HMBC spectrum, the long range correlations of protons H-10 to C-1, C-2, C-8, and C-9,

Fig. 2. Key 1H–1H COSY and HMBC correlations of compounds 1 and 2.

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Y. Sun et al. / Fitoterapia 95 (2014) 229–233

O O H

H H

H

H

H O H

H

H Hb CH3

CH3

Ha H

H

HH H3C O

Ha

H

H

HH H3C O

H

H

H CH3

1

Hb

H O

H CO2CH3 H

CH3

2 Fig. 3. Key NOESY correlations of compounds 1 and 2.

H3-19 to C-4, C-5, and C-6, H3-18 to C-3, C-4, and C-5, H3-17 to C-7, C-8, and C-9, and H-8 to C-6, C-7, C-9, C-10, and C-17 verified the presence of another structural moiety, two fused six-membered rings [3–5]. Consequently, the olefinic carbons at δC 104.8, and 148.0, and the oxygenated carbons at δC 70.3 were assigned to C-11, C-12, and C-20, respectively, by the corresponding HMBC correlations (Fig. 2). The long-range couplings of H2-20 with the carbon C-12 implied the presence of an unsaturated furan ring comprising C-9, C-11, C-12, and C-20. After further analysis of the HMQC, HMBC, and 1H–1H COSY spectra (Fig. 2), all of the proton and carbon signals were assigned unambiguously, which resulted in the establishment of the planar structure for compound 1. However, the molecular formula based on the deduced structure for compound 1 was not compatible with the HRESIMS data, which implied the presence of an additional ring based on the total unsaturation degrees. According to the NMR

120 Control

Neurite-bearing cells (%)

Compound 1 Compound 2 80

40

0 NGF Comp.

20

20

20

20

20

20

20

0

1.5

5

15

1.5

5

15

100 ng/mL 0

Fig. 4. Potentiating activities of compounds 1 and 2 on NGF-induced neurite outgrowth from PC12 cells. PC12 cells were treated with NGF alone or together with each compound at the concentrations indicated. After 96 h incubation, neurite processes with a length equal to or greater than the diameter of the neuron cell body were scored as neurite bearing cells. The proportion of neurite-bearing cells is expressed as a percentage against the maximum response to NGF (100 ng/ml, 100%). The experiment was performed three times, and the data are expressed as mean ± SD values. A statistically significant difference from the control (20 ng/mL NGF) in the absence of compounds is indicated in the figure: *p b 0.05.

data of compound 1, an oxygen bridge was further deduced, which could only be 3,4-epoxy. Thus, the planar structure of compound 1 was characterized. The relative configuration of 1 was elucidated by the NOESY spectrum. NOESY correlations observed for H-10/H-6β, H-10/ H-1β, H-10/H-11, H3-18/H-3, H-3/H-2β, H3-19/H-1α, H3-19/ H-6α, H3-19/H-7α, H3-17/H-7α, and H3-17/H-20a, but not for H-10/H-20a, H-10/H-20b, H-10/H3-17, and H-10/H3-19 Fig. 3, suggested that two six-membered rings were trans-fused and existed in a twist-chair conformation and a chair conformation. The H-10 proton was in a β-position with an axial orientation, the C-19 (methyl group) and the C-20 (methylene group) were both in α-positions with an axial orientation, and the C-17 (methyl group) was in an α-position with an equatorial orientation. All of the above evidence confirmed the structure of compound 1 as depicted in Fig. 1, which has been given the trivial name crotonpene A. Compound 2, colorless oil, possessed a molecular formula C21H28O5 as determined from the HR-ESIMS data (m/z 361.2010 [M + H]+, calcd. for C21H29O5 361.2015) and NMR data analyses. From the 1H NMR spectrum of 2, three methyl groups, one methoxy group, and three aromatic protons (Table 1) were apparent. The 13C NMR spectrum revealed that compound 2 possessed 21 carbon resonances. Based on the 1H and 13C NMR spectra, the same furan ring moiety in compound 2 as that in 1 and the methoxy group were deduced from the observation of the proton signals [δH 6.74 (1H, dd, J = 1.8, 0.7 Hz, H-14), 7.44 (1H, t, J = 1.8 Hz, H-15), 8.03(1H, br. s, H-16), and 3.64 (3H, s, OCH3-20)] and the corresponding carbons [δC 128.7 (C-13), 108.6 (C-14), 144.3 (C-15), 146.9 (C-16), and 50.9 (OCH3-20)]. In addition to the same furan ring moiety as that in 1, another structural moiety, two fused six-membered rings constituting of C-1–C-10 was deduced by comparing the 13C NMR spectrum of 2 with that of 1. In order to confirm the above deductions and elucidate the structure accurately, the following HMQC and HMBC experiments were performed. By the interpretation of 2D NMR spectra, the furan ring and the other structural moiety, two fused six-membered rings, were defined. Consequently, the methylene carbon at δC 43.2 and the two carbonyl carbons at δC 192.9 and 174.6 were assigned to C-11, C12, and C-20, respectively, by the HMBC correlations of H-11 to C-9, C-8, C-10, C-12, C-13, and C-20, H-10 to C-9, C-20, and C-11, and H-16(14) to C-12 (Fig. 2). The methoxy group was validated at C-20 by the HMBC correlation of OCH3/C-20. As in the case of 1, the

Y. Sun et al. / Fitoterapia 95 (2014) 229–233

relative configuration of 2 was determined based on the NOESY spectrum. The NOESY correlations of H-10/H-6β, H-10/H-11b, H-10/H-1β, H3-18/H-3, H-3/H-2β, H3-19/H-1α, H3-19/H-6α, H3-19/H-7α, and H3-17/H-7α, but not for H-10/H3-17, and H-10/H3-19, implied that two six-membered rings are transfused and existed in a twist-chair conformation and a chair conformation, H-10 and H-8 were β-axially oriented, C-19 (methyl group) and C-20 (methoxycarbonyl group) were α-axially oriented, and H-3 and C-18 (methyl group) were β-equatorially oriented, respectively. Therefore, compound 2 was elucidated completely and named crotonpene B. In order to explore the undiscovered and potential pharmacological activities of two novel clerodane diterpenes isolated from the twigs of C. yanhuii, compounds 1 and 2 were evaluated for their enhancing activities of NGF-induced neurite outgrowth from PC 12 cells according to the method previously reported [15,16]. NGF was used as inducers and the positive control. In control experiments, the percentages of neuritebearing cells were 17% and 100% following 96 h incubation with NGF 20 and 100 ng/mL, respectively. Compounds 1 and 2 (1.5, 5, 15 μM) had no effect on neurite outgrowth from PC12 cells in the absence of NGF, but at 15 μM markedly increased the NGF (20 ng/mL)-induced proportion of neurite bearing cells by 59%, and 47%, respectively (Fig. 4). In summary, the chemical constituents of C. yanhuii were investigated for the first time by us and two novel clerodane diterpenes (1 and 2) were isolated and characterized on the basis of extensive spectroscopic data analyses (IR, ESIMS, HR-ESIMS, and 1D and 2D NMR). Both compounds have the 3,4-epoxy moiety and compound 1 has an additional and rare unsaturated furan ring comprising C-9, C-11, C-12, and C-20. Based on the structures, two novel compounds should have the same biogenetic origin and compound 2 may probably be from compound 1 by oxidation or enzyme catalysis. Biological screenings disclosed that two compounds both exhibited potentiating activities of NGF-mediated neurite outgrowth from PC12 cells. The current biological data suggested that the two compounds (1 and 2) possessed the property to stimulate NGF-mediated neurite outgrowth from PC12 cells and may be potentially useful for the development of antineurodegenerative agents for Alzheimer's disease and other neurological disorders [17,18]. Further biological studies on two compounds are still underway by our group. Acknowledgments The project was supported by the Natural Science Foundation of China (no. 21372125).

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Appendix A. Supplementary data The 1D and 2D NMR and HR-ESIMS spectra of compounds 1 and 2 are available as Supplementary data. Supplementary data related to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2014.03.012.

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