Chemical components of Dendrobium crepidatum and their neurite outgrowth enhancing activities

Introduction

The stems of several Dendrobium species (Orchidaceae) are used as "Shi-Hu" in traditional Chinese medicine for a long time for the purpose of being beneficial to the stomach and promoting the production of body fluid, nourishing yin and clearing heat.^{1, 2} Inspired by this theory, "Shi-Hu" is mainly used for thrombotic diseases and health care especially for those older. D. crepidatum belongs to Sect. Dendrobium and the stems of this species used as a biological source of "ShiHu" and its flowers are very beautiful and get the name "Rosa Shi-Hu" in Chinese.³ Alkaloidal part of this species has been detected by Elander etc. and five octahydroindolizine alkaloids have been isolated and elucidated.⁴ To continue our research on "Shi-Hu"⁵ and provide chemical evidence for this traditional Chinese medicine, D. crepidatum was selected as our plant material and 15 compounds, including two new compounds, crepidatuols A (1) and B (2) (Figure 1) were isolated and elucidated herein, furthermore, 10 compounds were sent to assay their neurite outgrowth enhancing activities and the results indicated that three of them showed neuroprotective effects.

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Results and Discussion

Compound 1 was obtained as yellow oil. Its molecular formula was established as C₂₆H₂₈O₆ by the HREIMS showing a molecular ion peak at m/z 436.1882 (calcd. for C₂₆H₂₈O₆ 436.1886), indicating thirteen degrees of unsaturation. The UV spectra at λ_max (log ε): 282.5 (3.9) nm indicated an extended conjugated system. The ¹H and ¹³C NMR spectra data of 1 (Table 1) showed the existence of three methoxy groups, four aliphatic methylenes, one aliphatic methine and three aromatic rings. Two methylene carbons at δ_C 36.2 (C-7) and 35.0 (C-8) correlated with four benzylic protons at δ_H 2.82 (4H, m, H-7 and H-8) in the HSQC spectrum of 1 indicated a bibenzyl nucleus, 6 which was confirmed by the correlations [H-7, H-8/C-1, C-2, C-6, C-9, C-10, C-14] in the HMBC spectrum (Figure 2). Three aromatic hydrogen at δ_H 6.63 (¹H, d, 1.8), 6.85 (¹H, d, 8.1) and 6.71 (¹H, dd, 8.1, 1.8) indicated a 1, 3, 4-trisubstituted phenyl, moreover, two others at δ_H 6.42 (¹H, d, 2.5) and 6.39 (¹H, d, 2.5) showed another phenyl with two meta-coupling hydrogen for the bibenzyl nucleus. The correlations [H-16/H-15, H-17] in the COSY spectrum indicated a structural moiety of -CH₂-CH₂-CH-(C-15, C-16, C-17), furthermore, the correlations [H-15/C-14 and C-13, H 16/C-14 and C-18, H-17/C-18, C-19 and C-23] in the HMBC spectrum indicated that the partial structure and the related aromatic ring were linked with the bibenzyl nucleus as trigonopol B^5a except hydride reduction at C-16. Three methoxy groups could be observed that δ_H 3.92 (3H, s) belongs to δ_C 55.9, δ_H 3.76 (3H, s) belongs to δ_C 55.2, δ_H 3.86 (3H, s) belongs to δ_C 55.8 in the HSQC spectrum respectively, furthermore, cross peaks of δ_H 3.92 (3H, s) with δ_C 146.6, δ_H 3.86 (3H, s) with δ_C 146.2, δ_H 3.76 (3H, s) with δ_C 158.6 in the HMBC spectrum determined the position of three methoxy groups as shown in Figure 1. Thus, compound 1 was identified as 4-(5-(4-hydroxy-3-methoxyphenethyl)-7-methoxychroman-2-yl)-2-methoxyphenol, named as crepidatuol A.

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Compound 2 was obtained as yellow oil, its molecular formula was determined as C₂₆H₂₈O₇ by the HRESIMS (m/z 452.1832 [M]⁺, calcd. for 452.1835), with thirteen degrees of unsaturation. The ¹³C NMR and DEPT spectrum exhibited 26 carbon signals, including three methoxy, three aliphatic methylenes, two aliphatic methines and three aromatic rings. The ¹H and ¹³C NMR data of 2 (Table 1) were similar to those of trigonopol B^5a as compound 1, indicating a similar bibenzyl core for compound 2. The threo-isomer (6–8 Hz) is larger than in the erythro-isomer (2–4 Hz)5e, from the chemical shifts and the coupling constants (J = 8.7) of compound 2 and trigonopol B (J = 6.4), it could be predicated that relative stereochemistry of C-16 and C-17 were similar in two compounds. One more methoxy in 2 than trigonopol B and correlations [H-24/C-3, H-25/C-11, H-26/C-20] in the HMBC spectrum (Figure 2) indicated that compound 2 was 5-(4-hydroxy-3-methoxyphenethyl)-2-(4-hydroxy-3-methoxyphenyl)-7-methoxychroman-3-ol as shown in figure 1, named as crepidatuol B.

The 13 known compounds were elucidated as erianin, ⁷ crepidatin, ⁸ moscatilin, ⁸ hircinol, ⁹ confusarin, ¹⁰ syringaldazine, ¹¹ 3-(2-acetoxy-5-methoxy)phenylpropanol, ¹¹ kaempferol, ¹² 4, 6-dihydroxyisobenzofuran-1(3H)-one, ¹³ ergostan-7, 9, 22-trien-3, 5-diol, ¹⁴ ergosterol, ¹⁵ β-sitosterol and daucosterol by comparing with those from literature and TLC means.

For the purpose of evaluating the effect of neuroprotective effects of this species, the isolated 10 compounds including crepidatuol A (1), erianin, crepidatin, moscatilin, confusarin, 3-(2-acetoxy-5-methoxy)-phenylpropanol, kaempferol, 4, 6-dihydroxyisobenzofuran-1(3H)-one, ergostan-7, 9, 22-trien-3, 5-diol and ergosterol were evaluated for their enhancing activity on NGF-induced (nerve growth factor, R & D Systems Inc., 97%) neurite outgrowth in PC12 cells.16 The results indicated that the proportion of the NGF-induced (10 ng/mL) neurite-bearing cells were enhanced by crepidatuol A (1, 8.5%), confusarin (7.1%) and 3-(2-acetoxy-5-methoxy)-phenylpropanol (6.8%) at the concentration of 10 μM (Table 2). Others showed no activity on 10 ng/mL NGF-induced neurite outgrowth in PC12 cells at 10.0 μM.

Experimental Section

General Experimental Procedures

Optical rotations were measured using a JASCODIP-370 polarimeter. UV spectra were obtained with a Hitachi UV-3210. IR spectra were recorded on a Bio-Rad BRUKER TENSOR27 IR spectrophotometer as KBr disks. NMR spectra were run on Bruker AVANCE 400 NMR spectrometer (400 MHz for ¹H NMR, 100 MHz for ¹³C NMR) and DRX-500 NMR spectrometer (500 MHz for ¹H NMR, 125 MHz for ¹³C NMR) with TMS as internal standard. Mass spectra were performed on a AutoSpec Premier P776 mass spectrometer. Column chromatography was carried out on silica gel H60 (Qingdao Haiyang Chemical Group Corp., Qingdao, China), Sephadex LH-20 (Amersham Biosciences), and ODS (40–63 μm, Merck) as packing materials. Silica gel G was used for analytical TLC.

Plant Material

The fresh stems of D. crepidatum were collected in Yunnan Province in 2008 and identified by Professor Hong Yu of Yunnan University. A voucher specimen (Zsh-8) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Science, China.

Extraction and Isolation

The air-dried stems of D. crepidatum (15 kg) were refluxed three times with 95% EtOH. After evaporation of solvent in vacuo, the residue was suspended in H₂O and adjusted pH to 2 with hydrochloric acid. Then partitioned with chloroform and non-alkaloid fraction (1000 g) were obtained. Then the non-alkaloid fraction was subjected to column chromatography on silica gel (200–300 mesh, 6 kg) and eluted with petroleum ether/acetone (6:4→0:10) to afford 7 fractions. Fraction B (120g) was applied repeatedly to column chromatography over silica gel (petroleum ether/acetone, 10:1 to 1:1) and got 13 (267 mg), 14 (259 mg) and 16 (1.1 g), then residue were purified with Sephadex LH-20 (CHCl₃-MeOH, 1:1) to afford compounds 1 (29 mg), 4 (1.73 g), 9 (9 mg) and 10 (11 mg). Fraction C (62.7 g) was chromatographed on silica gel (petroleum ether-acetone, 5:1 to 1:1) to afford 7 fractions. Each fraction was subjected repeatedly to column chromatography over silica gel and then passed over Sephadex LH-20 (MeOH) respectively to afford compounds 5 (43 mg), 7 (11 mg) and 12 (17 mg). Fraction D was treated the same as Fraction C to afford compounds 6 (28 mg), 8 (14 mg) and 11 (300 mg). Fraction E was subjected to column chromatography over silica gel and got 15 (400 mg) and then applied to ODS column (MeOH-₂O, 3:7→5:5) and Sephadex LH-20 column (MeOH) to yield compounds 2 (5 mg) and 3 (4 mg).

Crepidatuol A (1)

yellow oil, [α] _D^16.8 – 11.89 (c 0.15, CHCl₃), UV (CHCl₃): λ_max (log ε) 282.5 (3.9) nm; IR (KBr): ν_max 3431, 2924, 2850, 1614, 1583, 1516, 1463, 1428, 1364, 1270, 1196, 1140, 1032 cm^–1; ¹H NMR (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz) data: see Table 1; EIMS m/z 436 (92), 299 (28), 285 (46), 137 (100); HREIMS m/z 436.1882 [M]⁺ (calcd for C₂₆H₂₈O₆, 436.1886).

Crepidatuol B (2)

yellow oil, [α] _D^11.9 + 0.24 (c 0.26, CHCl₃), UV (CHCl₃): λ_max (log ε) 282.0 (3.87) nm; IR (KBr): ν_max 3493, 3313, 2923, 2852, 1728, 1614, 1584, 1516, 1463, 1272, 1197, 1140, 1031 cm^–1; ¹H NMR (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz) data: see Table 1; EIMS m/z 452 (37), 287 (63), 137 (100); HREIMS m/z 452.1832 [M]⁺ (calcd for C26H28O7, 452.1835).

Cell Culture and Evaluation of Neurite OutgrowthPromoting Activity

The neurotrophic activities of the test compounds were examined according to an assay using PC12 cells as reported.¹⁷ Briefly, PC12 cells were maintained in F12 medium supplemented with 12.5% horse serum (HS), and 2.5% fetal bovine serum (FBS), and incubated at 5% CO₂ and 37 ℃. Test compounds were dissolved in DMSO. For the neurite outgrowth-promoting activity bioassay, PC12 cells were seeded at a density of 2 × 10⁴ cells/mL in 48-well plate coated with poly-L-lysine. After 24 h, the medium was changed to that containing 10 μM of each test compounds plus 10 ng/mL nerve growth factor (NGF), or various concentrations of NGF (50 ng/mL for the positive control, 10 ng/mL for the negative control). The final concentration of DMSO was 0.1%, and the same concentration of DMSO was added into the negative control and blank. After 72 h incubation, the neurite outgrowth was assessed under a phasecontrast 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/view area; 5 viewing area/well) was determined and expressed as a percentage.

Notes

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-012-0103-3 and is accessible for authorized users.

Acknowledgments

This work was financially supported by National Natural and Science Foundations of China (No. 30800090), "Xi-BuZhi-Guang" project (2009–2012) from Chinese Academy of Science and the Fund of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2010-ZZ012). The authors are grateful to the members of the analytical group of the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, for all the spectral measurements.