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Four new sesquiterpenes from Commiphora myrrha and their neuroprotective effects
Fitoterapia 83 (2012) 801–805
Contents lists available at SciVerse ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Four new sesquiterpenes from Commiphora myrrha and their neuroprotective effects Jing Xu a, Yuanqiang Guo a,⁎, Peng Zhao b, Ping Guo c, Yonggang Ma a, Chunfeng Xie a, Da-qing Jin d, Liping Gui a a College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China b Tianjin Children's Hospital, Tianjin 300074, China c Department of Analytical Chemistry, College of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China d School of Medicine, Nankai University, Tianjin 300071, China
a r t i c l e
i n f o
Article history: Received 8 February 2012 Accepted in revised form 7 March 2012 Available online 20 March 2012
a b s t r a c t Four new sesquiterpenes, myrrhterpenoids K−N (1− 4) have been isolated from the resin of Commiphora myrrha. Their structures and relative configurations were elucidated by spectroscopic methods (IR, ESIMS, HR-ESIMS, 1D and 2D NMR). Compounds 1 and 4 showed neuroprotective effects against MPP+-induced neuronal cell death in SH-SY5Y cells. © 2012 Elsevier B.V. All rights reserved.
Keywords: Commiphora myrrha Sesquiterpene Neuroprotective Burseraceae
1. Introduction The resin of Commiphora myrrha (Burseraceae) has been used for several centuries for the treatment of various diseases in the traditional medicines of India, China, Rome, Greece and Babylon. Previous phytochemical investigations on C. myrrha revealed that terpenoids were the characteristic constituents of C. myrrha [1–6]. Our previous search for new bioactive substances from C. myrrha led to the isolation and identification of some new sesquiterpenoids [7,8]. In our continuous investigation for neuroprotective compounds from C. myrrha, with focus on minor sesquiterpenes, four new sesquiterpenes, named myrrhterpenoids K−N (1−4) (Fig. 1) have been isolated from the resin of C. myrrha. Their structures and relative configurations were elucidated by spectroscopic methods (IR, ESIMS, HR-ESIMS, 1D and 2D NMR). Compounds 1 and 4 showed neuroprotective effects against MPP +induced neuronal cell death in SH-SY5Y cells. This paper ⁎ Corresponding author. Tel./fax: + 86 22 23502595. E-mail address: [email protected] (Y. Guo). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.03.016
herein describes the isolation and structure elucidation of these new sesquiterpenes and their neuroprotective effects against MPP +-induced neuronal cell death in SH-SY5Y cells. 2. Experimental 2.1. General The optical rotations were measured in CH2Cl2, using an Autopal IV automatic polarimeter made by Autopal Industries Limited Company. The IR spectra were taken on a Bio-Rad FTS 6000 Fourier transform infrared (FTIR) spectrometer with KBr discs. The ESIMS spectra were obtained on a LCQ-Advantage mass spectrometer made by Finnigan Company (America). HR-ESIMS spectra were taken by an Ionspec 7.0 T FTICR MS. 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
802
J. Xu et al. / Fitoterapia 83 (2012) 801–805
Fig. 1. Structures of 1−4 from C. myrrha.
(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 Yuanli Chemical Co. Ltd. P. R. China. Guanosine was purchased from Genview Company and other biological reagents were from Sigma Company. Human dopaminergic SH-SY5Y cells were obtained from the American Type Culture Collection (ATCC). 2.2. Plant material The resin of C. myrrha were imported from India in Aug. 2009 and identified by Mr. Zheyou Liu (Shennong Pharmaceuticals, Anhui Province, P. R. China). A voucher specimen (No. 20090801) was deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University, P. R. China. 2.3. Extraction and isolation The resin of C. myrrha (1.0 kg) were powdered and extracted with CH2Cl2 (4 L) at room temperature for three times (3 × 24 h). The organic solvent was evaporated and a crude extract (280 g) was obtained. The crude extract
(120 g) was subjected to a silica gel column chromatography, using a gradient of acetone in petroleum ether, to give eight fractions (F1–F8) based on TLC analyses. F8 was chromatographed by MPLC over ODS eluting with a step gradient from 50% to 85% MeOH in H2O to give five subfractions (F8-1–F8-5). Subfraction F8-4 was purified by preparative HPLC (YMC-pack ODS-AM, 20 × 250 mm, 74% MeOH in H2O) to afford compound 1 (12.3 mg). Compounds 2 (3.6 mg) and 3 (3.9 mg) were obtained from subfraction F8-2 by the above HPLC system (YMC-pack ODS-AM, 20 × 250 mm, 63% MeOH in H2O). The purification of subfraction F8-5 with the solvent system of 79% MeOH in H2O led to the isolation of compound 4 (14.5 mg). 2.3.1. Myrrhterpenoid K (1) Colorless oil; [α]D20 − 5.3 (c = 0.2, CH2Cl2); IR (KBr) νmax cm − 1: 3430, 2938, 1696, 1452, 1378, 1096; ESIMS: m/z 247 [M + H] +; HR-ESIMS m/z 269.1148 [M + Na] +, calcd. for C15H18NaO3 269.1154; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data see Table 1. 2.3.2. Myrrhterpenoid L (2) Colorless oil; [α]D20: − 9.1 (c = 0.1, CH2Cl2); IR (KBr) νmax cm − 1: 3452, 2930, 1698, 1459, 1378, 1094; ESIMS: m/z 303
Table 1 1 H and 13C NMR spectroscopic data of compounds 1−4 (in CDCl3, δ in ppm, J in Hz) a. No.
1
2
3
4
δC
δH
δC
δH
δC
δH
δC
δH
1 2
54.9 74.8
β 2.98 m α 4.23 dd (14.3,7.4)
58.5 83.0
α 2.03 m β 3.95 t (2.4)
77.7 26.1
69.4 31.0
3
42.6
39.1
37.9
34.5 64.0 198.0 122.1 159.2 36.1
α 1.95 m β 1.31 m α 2.49 m β 3.39 m
β 3.74 dd (10.0,4.2) α 1.62 m β 1.83 m α 2.31 m β 2.12 m
4 5 6 7 8 9
α 2.25 m β 1.36 m α 2.24 m β 2.92 m
β 3.58 dd (11.3,2.8) α 1.58 m β 1.89 m α 1.45 m β 1.82 m
10 11 12 13 14
141.2 121.4 138.3 9.2 19.8
15
116.0
α 3.72 d (17.4) β 3.57 d (17.4)
7.04 s 2.06 s 1.10 d (6.0) 5.05 s 5.06 s
32.2 56.5 198.3 121.9 160.7 73.2
α 4.68 s
69.9 59.4 198.0 118.3 165.3 38.7
71.7 121.3 139.3 9.5 19.6
7.13 s 2.17 s 1.15 d (6.4)
45.4 120.0 139.3 8.5 30.8
7.09 s 2.17 s 1.43 s
44.0 119.0 139.4 8.8 114.3
28.0
1.33 s
12.5
1.11 s
20.9
55.9
3.24 s
OMe a
30.2
1
1
The assignments are based on DEPT, HMQC, HMBC, H– H COSY, and NOESY experiments.
β 2.48 s
α 2.98 d (17.3) β 2.73 d (17.3)
142.5 62.1 194.2 118.9 165.1 32.1
α 3.02 br. s
α 3.31 d (17.7) β 2.55 d (17.7)
7.07 s 2.17 s 5.04 s 4.74 s 1.14 s
J. Xu et al. / Fitoterapia 83 (2012) 801–805
[M + Na] +; HR-ESIMS m/z 303.1203 [M + Na] +, calcd. for C15H20NaO5 303.1208; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data see Table 1. 2.3.3. Myrrhterpenoid M (3) Colorless oil; [α]D20: + 15.0 (c = 0.1, CH2Cl2); IR (KBr) νmax cm − 1: 3445, 2930, 1726, 1667, 1083; ESIMS: m/z 301 [M + Na] +; HR-ESIMS m/z 301.1410 [M + Na] +, calcd. for C16H22NaO4 301.1416; 1H NMR (400 MHz, CDCl3) and 13 C NMR (100 MHz, CDCl3) data see Table 1. 2.3.4. Myrrhterpenoid N (4) Colorless oil; [α]D20: + 30.7 (c = 0.3, CH2Cl2); IR (KBr) νmax −1 cm : 3435, 2928, 1725, 1699, 1457, 1066; ESIMS: m/z 269 [M + Na] +; HR-ESIMS m/z 269.1146 [M + Na] +, calcd. for C15H18NaO3 269.1154; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data see Table 1. 2.4. Bioassay for neuroprotective activity See Refs. [9-11]. 3. Results and discussion Compound 1 was isolated as a colorless oil. Its molecular formula was determined as C15H18O3 by HR-ESIMS (m/z 269.1148 [M + Na]+, calcd. for C15H18NaO3 269.1154). The 1H NMR spectrum of 1 exhibited two methyl groups at δ 2.06 (3H, s, H3-13) and 1.10 (3H, d, J = 6.0 Hz, H3-14), and three olefinic protons at δ 7.04 (1H, s, H-12), and 5.05 and 5.06 (each 1H, s, H-15) (Table 1). The 13C NMR spectrum of 1, in combination with the DEPT and HMQC spectra, showed 15 carbon signals that were classified into two methyls [δ 9.2 (C-13), and 19.8 (C-14)], three methylenes [δ 42.6 (C-3), 36.1 (C-9) and 116.0 (C-15)], five methines [δ 54.9 (C-1), 74.8 (C-2), 34.5 (C-4), 64.0 (C-5), and 138.3 (C-12)], and five quaternary carbons [δ 198.0 (C-6), 122.1 (C-7), 159.2 (C-8), 141.2 (C-10) and 121.4 (C-11)]. Based on the above spectroscopic data and the reported sesquiterpenoids form the genus Commiphora [6], compound 1 might be a guaiane-type sesquiterpene with a furan ring. This assumption was confirmed by an HMBC experiment. In the HMBC spectrum, the long-range correlations of H1 to C-9, C-10, C-15, C-2, C-3, C-4, C-5, and C-6, and H-5 to C-3, C-4, C-6, C-7, C-1, C-2, and C-10 (Fig. 2), revealed the fusion of a five-membered ring and a seven-membered ring. In addition, the HMBC correlations of H3-13 to C-7, C-11, and C-12, and H12 to C-11, C-7, C-8, and C-13 demonstrated the presence of a furan ring located at C-7, C-8, C-11, and C-12 with a methyl
803
attached at C-11. The remaining one terminal double bond was assigned to C-10 and C-15 based on the HMBC correlations of H2-15 to C-1, C-9, and C-10, and H-9 to C-7, C-8, C-1, C-10, and C-15. By further analyzing the HMQC, HMBC, and 1H–1H COSY NMR spectra (Fig. 2), all the proton and carbon signals were assigned unambiguously. Thus, the planar structure of a sesquiterpene with three rings for 1 was established. The relative configuration was deduced by the NOESY spectrum. The NOESY correlations observed for H-1/H-15, H-1/ H-5, H-5/H3-14, H-3β/H-1, H-3β/H3-14, H-2/H-9α, H-2/H-4, and H-9β/H-15 (Fig. 3), but not for H-2/H-5, suggested the cisfusion of the five-membered ring and the seven-membered ring, and the presence of a β-orientation for the C-1 hydroxy. Therefore, compound 1 was determined and named myrrhterpenoid K. Compound 2 had a molecular formula C15H20O5 as suggested by its HR-ESIMS (m/z 303.1203 [M+ Na] +, calcd. for C15H20NaO5 303.1208). Three methyl groups and one olefinic proton were evident from its 1H NMR spectrum. The 13C NMR spectrum of 2 revealed 15 resonances. The 1H and 13C NMR spectra of 2 were similar to those of compound 1, which suggested compound 2 was an analogue of 1. A careful comparison of the 1H and 13C NMR spectroscopic data of 2 with those of 1, revealed the disappearance of one methylene and the terminal double bond at C-10 and C-15. In order to assign all the proton and carbon signals accurately, the 2D NMR spectra were recorded. Similar to compound 1, the carbonyl carbon signal (δC 198.3) conjugated with the furan ring (C-7, C-8, C-11, and C-12) was assigned to C-6, which was demonstrated by the HMBC correlations (Fig. 2). The fusion of a five-membered ring and a seven-membered ring via C-1 and C-5 unit was revealed by the interpretation of 2D NMR spectra, which was also similar to that of compound 1. Three oxygenated carbon signals at δC 83.0, 73.2, and 71.7 were ascribed to C-2, C-9, and C-10, respectively by the interpretation of HMBC spectrum. The further analyses of 2D NMR spectra revealed the planar structure of 2. The NOESY spectrum showed the correlations of H-1/H-4, H-1/H-3α, H-3β/H3-14, H-2/H3-15, H-2/H-5, H-5/ H3-15, H-5/H3-14, H-5/H-2, and H-1/H-9α (Fig. 3). These correlations suggested a trans-fusion for two rings via C-1 and C-5 unit, and revealed the hydrxoy groups at C-1 and C-10 were α-oriented and the hydroxy at C-9 was β-oriented. Thus, compound 2 was determined and named myrrhterpenoid L. Compound 3, colorless oil, had a molecular formula C16H22O4 based on the HR-ESIMS (m/z 301.1410 [M+ Na]+, calcd. for C16H22NaO4 301.1416). The 1H NMR spectrum for 3 exhibited three methyl groups, an olefinic proton and one methoxy group (Table 1). The 13C NMR spectrum of 3 showed
Fig. 2. Selected 1H–1H COSY and HMBC correlations for compounds 1−3.
804
J. Xu et al. / Fitoterapia 83 (2012) 801–805
Fig. 3. Key NOESY correlations for compounds 1−3.
16 carbon signals. From the 1H and 13C NMR spectra, the same furan ring conjugated with a carbonyl moiety in 3 as those in compounds 1 and 2 was confirmative. Based on the spectroscopic data (Table 1) of 3 and the reported sesquiterpenoids form the genus Commiphora [6], the close similarity of the chemical shifts from C-1 to C-15 for 3 with the reported data for similar compounds implied that 3 might be a eudesmanetype sesquiterpene. A careful comparison of the 13C NMR spectroscopic data for 3 with those for zedoarofuran, a known eudesmane-type sesquiterpene [12], revealed compound 3 to be a eudesmane sesquiterpene related to the known compound structurally. The presence of the one methoxy group implied that one hydroxy in 3 was etherified to produce the methoxy, which was assigned to C-1 by the corresponding HMBC correlations (Fig. 2). By the further analyses of the HMQC, HMBC, and 1H–1H COSY NMR spectra (Fig. 2), all the proton and carbon signals were assigned unambiguously. According to the reported eudesmane-type sesquiterpenes and the NOESY correlations of H3-14/H-5, H3-14/H-3β, H-5/
Compound 4
Compound 1
120
Guanosine *
Cell viability (%)
90
*
*
*
*
*
*
# 60
30
0
MPP + Conc.
+
-
+ 30
+ 10
+ 3
+ 30
+ 10
+ 3
+ 30
-
H-1, H-1/H-9β, H-5/H-9β, H3-15/H-9α, H-1/H-9β, and H-2α/ OCH3 at C-1 (Fig. 3), the methyoxy at C-1 and the hydroxy at C-4 were both determined as α-orientations. Thus, compound 3 was characterized and named myrrhterpenoid M. Compound 4 possessed a molecular formula C15H18O3 suggested by the HR-ESIMS (m/z 269.1146 [M+ Na]+, calcd. for C15H18NaO3 269.1154). Similar to compounds 1−3, the same furan ring conjugated with the carbonyl moiety in 4 was evident from the 1H and 13C NMR spectra. In addition, one terminal double bond, one tertiary methyl (C-15) and one oxygenated carbon in 4 were also revealed by its 1H and 13 C NMR spectra. Based on the biosynthetic considerations and the above mentioned moieties, compound 4 should be a eudesmane-type sesquiterpene consisting of 15 carbons [6]. In order to determine the planar structure, the following HMQC and HMBC experiments were performed. The methylene carbon signal at δC 32.1 was assigned to C-9 by the HMBC correlations of H2-9 [3.31, 2.55 (each 1H, d, J = 17.7)] to C-7, C-8, C-10, C-15, C-5, and C-1. Consequently, the oxygenated carbon at C-1 and the terminal double bond at C-4 and C14 were also ascribed by the corresponding HMBC correlations. The further 2D NMR spectra analyses resulted in the establishment of the planar structure of eudesmane-type sesquiterpene for 4, which was an analogue of the known compound curcolone [13]. The NOESY correlations observed for H3-15 and H-5, but not for H3-15 and H-1, implied that two sixmembered rings were cis-fused and the C-1 hydroxy group was α-oriented. Compound 4 was therefore elucidated, which has been named myrrhterpenoid N. The neuroprotective effects of compounds 1 and 4 against MPP +-induced neuronal cell death in dopaminergic neuroblastoma SH-SY5Y cells were evaluated [9-11,14]. Guanosine was used as a positive control. The two compounds exhibited neuroprotective effects (Fig. 4). The above active compounds (3–30 μM) neither affected the cell viability nor showed any cytotoxicity with the absence of MPP + (data not shown). Compounds 2 and 3 were not investigated for the neuroprotective effects because of inadequate amount.
µM
References Fig. 4. Neuroprotective effects of compounds 1 and 4. The SH-SY5Y cells were exposed to MPP+and the cell viability was assessed by MTT assay. The cells were treated with 0.8 mM MPP+in the absence or presence of compounds 1 and 4. Data are expressed as the percentage of values in untreated control cultures. Each value indicates a mean ± SEM (n = 3). # P b 0.01, compared with control group. *P b 0.05, compared with the MPP+-treated group. Guanosine, positive control.
[1] Zhu N, Kikuzaki H, Sheng S, Sang S, Rafi MM, Wang M, et al. J Nat Prod 2001;64:1460. [2] Zhu N, Sheng S, Sang S, Rosen RT, Ho CT. Flavour Fragance J 2003;18: 282. [3] Shen T, Wan WZ, Wang XN, Yuan HQ, Ji M, Lou HX. Helv Chim Acta 2009;92:645.
J. Xu et al. / Fitoterapia 83 (2012) 801–805 [4] Su SL, Duan JA, Tang YP, Zhang X, Yu L, Jiang FR, et al. Planta Med 2009;75:351. [5] Ubillas RP, Mendez CD, Jolad SD, Luo J, King SR, Carlson TJ, et al. Planta Med 1999;65:778. [6] Shen T, Lou HX. Chem Biodivers 2008;5:540. [7] Xu J, Guo Y, Li Y, Zhao P, Liu C, Ma Y, et al. Planta Med 2011;77:2023. [8] Xu J, Guo Y, Li Y, Zhao P, Xie C, Jin DQ, et al. Fitoterapia 2011;82:1198.
[9] [10] [11] [12]
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Xu J, Zhao P, Guo Y, Xie C, Jin DQ, Ma Y, et al. Fitoterapia 2011;82:1133. Xu J, Liu C, Guo P, Guo Y, Jin DQ, Song X, et al. Fitoterapia 2011;82:772. Xu J, Guo Y, Xie C, Li Y, Gao J, Zhang T, et al. J Nat Prod 2011;74:2224. Matsuda H, Morikawa T, Toguchida I, Ninomiya K, Yoshikawa M. Chem Pharm Bull 2001;49:1558. [13] Hikino H, Sakurai Y, Takemoto T. Chem Pharm Bull 1967;15:1065. [14] Guo P, Li Y, Xu J, Guo Y, Jin DQ, Gao Jie, et al. Fitoterapia 2011;82:1123.
Contents lists available at SciVerse ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Four new sesquiterpenes from Commiphora myrrha and their neuroprotective effects Jing Xu a, Yuanqiang Guo a,⁎, Peng Zhao b, Ping Guo c, Yonggang Ma a, Chunfeng Xie a, Da-qing Jin d, Liping Gui a a College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China b Tianjin Children's Hospital, Tianjin 300074, China c Department of Analytical Chemistry, College of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China d School of Medicine, Nankai University, Tianjin 300071, China
a r t i c l e
i n f o
Article history: Received 8 February 2012 Accepted in revised form 7 March 2012 Available online 20 March 2012
a b s t r a c t Four new sesquiterpenes, myrrhterpenoids K−N (1− 4) have been isolated from the resin of Commiphora myrrha. Their structures and relative configurations were elucidated by spectroscopic methods (IR, ESIMS, HR-ESIMS, 1D and 2D NMR). Compounds 1 and 4 showed neuroprotective effects against MPP+-induced neuronal cell death in SH-SY5Y cells. © 2012 Elsevier B.V. All rights reserved.
Keywords: Commiphora myrrha Sesquiterpene Neuroprotective Burseraceae
1. Introduction The resin of Commiphora myrrha (Burseraceae) has been used for several centuries for the treatment of various diseases in the traditional medicines of India, China, Rome, Greece and Babylon. Previous phytochemical investigations on C. myrrha revealed that terpenoids were the characteristic constituents of C. myrrha [1–6]. Our previous search for new bioactive substances from C. myrrha led to the isolation and identification of some new sesquiterpenoids [7,8]. In our continuous investigation for neuroprotective compounds from C. myrrha, with focus on minor sesquiterpenes, four new sesquiterpenes, named myrrhterpenoids K−N (1−4) (Fig. 1) have been isolated from the resin of C. myrrha. Their structures and relative configurations were elucidated by spectroscopic methods (IR, ESIMS, HR-ESIMS, 1D and 2D NMR). Compounds 1 and 4 showed neuroprotective effects against MPP +induced neuronal cell death in SH-SY5Y cells. This paper ⁎ Corresponding author. Tel./fax: + 86 22 23502595. E-mail address: [email protected] (Y. Guo). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.03.016
herein describes the isolation and structure elucidation of these new sesquiterpenes and their neuroprotective effects against MPP +-induced neuronal cell death in SH-SY5Y cells. 2. Experimental 2.1. General The optical rotations were measured in CH2Cl2, using an Autopal IV automatic polarimeter made by Autopal Industries Limited Company. The IR spectra were taken on a Bio-Rad FTS 6000 Fourier transform infrared (FTIR) spectrometer with KBr discs. The ESIMS spectra were obtained on a LCQ-Advantage mass spectrometer made by Finnigan Company (America). HR-ESIMS spectra were taken by an Ionspec 7.0 T FTICR MS. 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
802
J. Xu et al. / Fitoterapia 83 (2012) 801–805
Fig. 1. Structures of 1−4 from C. myrrha.
(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 Yuanli Chemical Co. Ltd. P. R. China. Guanosine was purchased from Genview Company and other biological reagents were from Sigma Company. Human dopaminergic SH-SY5Y cells were obtained from the American Type Culture Collection (ATCC). 2.2. Plant material The resin of C. myrrha were imported from India in Aug. 2009 and identified by Mr. Zheyou Liu (Shennong Pharmaceuticals, Anhui Province, P. R. China). A voucher specimen (No. 20090801) was deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University, P. R. China. 2.3. Extraction and isolation The resin of C. myrrha (1.0 kg) were powdered and extracted with CH2Cl2 (4 L) at room temperature for three times (3 × 24 h). The organic solvent was evaporated and a crude extract (280 g) was obtained. The crude extract
(120 g) was subjected to a silica gel column chromatography, using a gradient of acetone in petroleum ether, to give eight fractions (F1–F8) based on TLC analyses. F8 was chromatographed by MPLC over ODS eluting with a step gradient from 50% to 85% MeOH in H2O to give five subfractions (F8-1–F8-5). Subfraction F8-4 was purified by preparative HPLC (YMC-pack ODS-AM, 20 × 250 mm, 74% MeOH in H2O) to afford compound 1 (12.3 mg). Compounds 2 (3.6 mg) and 3 (3.9 mg) were obtained from subfraction F8-2 by the above HPLC system (YMC-pack ODS-AM, 20 × 250 mm, 63% MeOH in H2O). The purification of subfraction F8-5 with the solvent system of 79% MeOH in H2O led to the isolation of compound 4 (14.5 mg). 2.3.1. Myrrhterpenoid K (1) Colorless oil; [α]D20 − 5.3 (c = 0.2, CH2Cl2); IR (KBr) νmax cm − 1: 3430, 2938, 1696, 1452, 1378, 1096; ESIMS: m/z 247 [M + H] +; HR-ESIMS m/z 269.1148 [M + Na] +, calcd. for C15H18NaO3 269.1154; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data see Table 1. 2.3.2. Myrrhterpenoid L (2) Colorless oil; [α]D20: − 9.1 (c = 0.1, CH2Cl2); IR (KBr) νmax cm − 1: 3452, 2930, 1698, 1459, 1378, 1094; ESIMS: m/z 303
Table 1 1 H and 13C NMR spectroscopic data of compounds 1−4 (in CDCl3, δ in ppm, J in Hz) a. No.
1
2
3
4
δC
δH
δC
δH
δC
δH
δC
δH
1 2
54.9 74.8
β 2.98 m α 4.23 dd (14.3,7.4)
58.5 83.0
α 2.03 m β 3.95 t (2.4)
77.7 26.1
69.4 31.0
3
42.6
39.1
37.9
34.5 64.0 198.0 122.1 159.2 36.1
α 1.95 m β 1.31 m α 2.49 m β 3.39 m
β 3.74 dd (10.0,4.2) α 1.62 m β 1.83 m α 2.31 m β 2.12 m
4 5 6 7 8 9
α 2.25 m β 1.36 m α 2.24 m β 2.92 m
β 3.58 dd (11.3,2.8) α 1.58 m β 1.89 m α 1.45 m β 1.82 m
10 11 12 13 14
141.2 121.4 138.3 9.2 19.8
15
116.0
α 3.72 d (17.4) β 3.57 d (17.4)
7.04 s 2.06 s 1.10 d (6.0) 5.05 s 5.06 s
32.2 56.5 198.3 121.9 160.7 73.2
α 4.68 s
69.9 59.4 198.0 118.3 165.3 38.7
71.7 121.3 139.3 9.5 19.6
7.13 s 2.17 s 1.15 d (6.4)
45.4 120.0 139.3 8.5 30.8
7.09 s 2.17 s 1.43 s
44.0 119.0 139.4 8.8 114.3
28.0
1.33 s
12.5
1.11 s
20.9
55.9
3.24 s
OMe a
30.2
1
1
The assignments are based on DEPT, HMQC, HMBC, H– H COSY, and NOESY experiments.
β 2.48 s
α 2.98 d (17.3) β 2.73 d (17.3)
142.5 62.1 194.2 118.9 165.1 32.1
α 3.02 br. s
α 3.31 d (17.7) β 2.55 d (17.7)
7.07 s 2.17 s 5.04 s 4.74 s 1.14 s
J. Xu et al. / Fitoterapia 83 (2012) 801–805
[M + Na] +; HR-ESIMS m/z 303.1203 [M + Na] +, calcd. for C15H20NaO5 303.1208; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data see Table 1. 2.3.3. Myrrhterpenoid M (3) Colorless oil; [α]D20: + 15.0 (c = 0.1, CH2Cl2); IR (KBr) νmax cm − 1: 3445, 2930, 1726, 1667, 1083; ESIMS: m/z 301 [M + Na] +; HR-ESIMS m/z 301.1410 [M + Na] +, calcd. for C16H22NaO4 301.1416; 1H NMR (400 MHz, CDCl3) and 13 C NMR (100 MHz, CDCl3) data see Table 1. 2.3.4. Myrrhterpenoid N (4) Colorless oil; [α]D20: + 30.7 (c = 0.3, CH2Cl2); IR (KBr) νmax −1 cm : 3435, 2928, 1725, 1699, 1457, 1066; ESIMS: m/z 269 [M + Na] +; HR-ESIMS m/z 269.1146 [M + Na] +, calcd. for C15H18NaO3 269.1154; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data see Table 1. 2.4. Bioassay for neuroprotective activity See Refs. [9-11]. 3. Results and discussion Compound 1 was isolated as a colorless oil. Its molecular formula was determined as C15H18O3 by HR-ESIMS (m/z 269.1148 [M + Na]+, calcd. for C15H18NaO3 269.1154). The 1H NMR spectrum of 1 exhibited two methyl groups at δ 2.06 (3H, s, H3-13) and 1.10 (3H, d, J = 6.0 Hz, H3-14), and three olefinic protons at δ 7.04 (1H, s, H-12), and 5.05 and 5.06 (each 1H, s, H-15) (Table 1). The 13C NMR spectrum of 1, in combination with the DEPT and HMQC spectra, showed 15 carbon signals that were classified into two methyls [δ 9.2 (C-13), and 19.8 (C-14)], three methylenes [δ 42.6 (C-3), 36.1 (C-9) and 116.0 (C-15)], five methines [δ 54.9 (C-1), 74.8 (C-2), 34.5 (C-4), 64.0 (C-5), and 138.3 (C-12)], and five quaternary carbons [δ 198.0 (C-6), 122.1 (C-7), 159.2 (C-8), 141.2 (C-10) and 121.4 (C-11)]. Based on the above spectroscopic data and the reported sesquiterpenoids form the genus Commiphora [6], compound 1 might be a guaiane-type sesquiterpene with a furan ring. This assumption was confirmed by an HMBC experiment. In the HMBC spectrum, the long-range correlations of H1 to C-9, C-10, C-15, C-2, C-3, C-4, C-5, and C-6, and H-5 to C-3, C-4, C-6, C-7, C-1, C-2, and C-10 (Fig. 2), revealed the fusion of a five-membered ring and a seven-membered ring. In addition, the HMBC correlations of H3-13 to C-7, C-11, and C-12, and H12 to C-11, C-7, C-8, and C-13 demonstrated the presence of a furan ring located at C-7, C-8, C-11, and C-12 with a methyl
803
attached at C-11. The remaining one terminal double bond was assigned to C-10 and C-15 based on the HMBC correlations of H2-15 to C-1, C-9, and C-10, and H-9 to C-7, C-8, C-1, C-10, and C-15. By further analyzing the HMQC, HMBC, and 1H–1H COSY NMR spectra (Fig. 2), all the proton and carbon signals were assigned unambiguously. Thus, the planar structure of a sesquiterpene with three rings for 1 was established. The relative configuration was deduced by the NOESY spectrum. The NOESY correlations observed for H-1/H-15, H-1/ H-5, H-5/H3-14, H-3β/H-1, H-3β/H3-14, H-2/H-9α, H-2/H-4, and H-9β/H-15 (Fig. 3), but not for H-2/H-5, suggested the cisfusion of the five-membered ring and the seven-membered ring, and the presence of a β-orientation for the C-1 hydroxy. Therefore, compound 1 was determined and named myrrhterpenoid K. Compound 2 had a molecular formula C15H20O5 as suggested by its HR-ESIMS (m/z 303.1203 [M+ Na] +, calcd. for C15H20NaO5 303.1208). Three methyl groups and one olefinic proton were evident from its 1H NMR spectrum. The 13C NMR spectrum of 2 revealed 15 resonances. The 1H and 13C NMR spectra of 2 were similar to those of compound 1, which suggested compound 2 was an analogue of 1. A careful comparison of the 1H and 13C NMR spectroscopic data of 2 with those of 1, revealed the disappearance of one methylene and the terminal double bond at C-10 and C-15. In order to assign all the proton and carbon signals accurately, the 2D NMR spectra were recorded. Similar to compound 1, the carbonyl carbon signal (δC 198.3) conjugated with the furan ring (C-7, C-8, C-11, and C-12) was assigned to C-6, which was demonstrated by the HMBC correlations (Fig. 2). The fusion of a five-membered ring and a seven-membered ring via C-1 and C-5 unit was revealed by the interpretation of 2D NMR spectra, which was also similar to that of compound 1. Three oxygenated carbon signals at δC 83.0, 73.2, and 71.7 were ascribed to C-2, C-9, and C-10, respectively by the interpretation of HMBC spectrum. The further analyses of 2D NMR spectra revealed the planar structure of 2. The NOESY spectrum showed the correlations of H-1/H-4, H-1/H-3α, H-3β/H3-14, H-2/H3-15, H-2/H-5, H-5/ H3-15, H-5/H3-14, H-5/H-2, and H-1/H-9α (Fig. 3). These correlations suggested a trans-fusion for two rings via C-1 and C-5 unit, and revealed the hydrxoy groups at C-1 and C-10 were α-oriented and the hydroxy at C-9 was β-oriented. Thus, compound 2 was determined and named myrrhterpenoid L. Compound 3, colorless oil, had a molecular formula C16H22O4 based on the HR-ESIMS (m/z 301.1410 [M+ Na]+, calcd. for C16H22NaO4 301.1416). The 1H NMR spectrum for 3 exhibited three methyl groups, an olefinic proton and one methoxy group (Table 1). The 13C NMR spectrum of 3 showed
Fig. 2. Selected 1H–1H COSY and HMBC correlations for compounds 1−3.
804
J. Xu et al. / Fitoterapia 83 (2012) 801–805
Fig. 3. Key NOESY correlations for compounds 1−3.
16 carbon signals. From the 1H and 13C NMR spectra, the same furan ring conjugated with a carbonyl moiety in 3 as those in compounds 1 and 2 was confirmative. Based on the spectroscopic data (Table 1) of 3 and the reported sesquiterpenoids form the genus Commiphora [6], the close similarity of the chemical shifts from C-1 to C-15 for 3 with the reported data for similar compounds implied that 3 might be a eudesmanetype sesquiterpene. A careful comparison of the 13C NMR spectroscopic data for 3 with those for zedoarofuran, a known eudesmane-type sesquiterpene [12], revealed compound 3 to be a eudesmane sesquiterpene related to the known compound structurally. The presence of the one methoxy group implied that one hydroxy in 3 was etherified to produce the methoxy, which was assigned to C-1 by the corresponding HMBC correlations (Fig. 2). By the further analyses of the HMQC, HMBC, and 1H–1H COSY NMR spectra (Fig. 2), all the proton and carbon signals were assigned unambiguously. According to the reported eudesmane-type sesquiterpenes and the NOESY correlations of H3-14/H-5, H3-14/H-3β, H-5/
Compound 4
Compound 1
120
Guanosine *
Cell viability (%)
90
*
*
*
*
*
*
# 60
30
0
MPP + Conc.
+
-
+ 30
+ 10
+ 3
+ 30
+ 10
+ 3
+ 30
-
H-1, H-1/H-9β, H-5/H-9β, H3-15/H-9α, H-1/H-9β, and H-2α/ OCH3 at C-1 (Fig. 3), the methyoxy at C-1 and the hydroxy at C-4 were both determined as α-orientations. Thus, compound 3 was characterized and named myrrhterpenoid M. Compound 4 possessed a molecular formula C15H18O3 suggested by the HR-ESIMS (m/z 269.1146 [M+ Na]+, calcd. for C15H18NaO3 269.1154). Similar to compounds 1−3, the same furan ring conjugated with the carbonyl moiety in 4 was evident from the 1H and 13C NMR spectra. In addition, one terminal double bond, one tertiary methyl (C-15) and one oxygenated carbon in 4 were also revealed by its 1H and 13 C NMR spectra. Based on the biosynthetic considerations and the above mentioned moieties, compound 4 should be a eudesmane-type sesquiterpene consisting of 15 carbons [6]. In order to determine the planar structure, the following HMQC and HMBC experiments were performed. The methylene carbon signal at δC 32.1 was assigned to C-9 by the HMBC correlations of H2-9 [3.31, 2.55 (each 1H, d, J = 17.7)] to C-7, C-8, C-10, C-15, C-5, and C-1. Consequently, the oxygenated carbon at C-1 and the terminal double bond at C-4 and C14 were also ascribed by the corresponding HMBC correlations. The further 2D NMR spectra analyses resulted in the establishment of the planar structure of eudesmane-type sesquiterpene for 4, which was an analogue of the known compound curcolone [13]. The NOESY correlations observed for H3-15 and H-5, but not for H3-15 and H-1, implied that two sixmembered rings were cis-fused and the C-1 hydroxy group was α-oriented. Compound 4 was therefore elucidated, which has been named myrrhterpenoid N. The neuroprotective effects of compounds 1 and 4 against MPP +-induced neuronal cell death in dopaminergic neuroblastoma SH-SY5Y cells were evaluated [9-11,14]. Guanosine was used as a positive control. The two compounds exhibited neuroprotective effects (Fig. 4). The above active compounds (3–30 μM) neither affected the cell viability nor showed any cytotoxicity with the absence of MPP + (data not shown). Compounds 2 and 3 were not investigated for the neuroprotective effects because of inadequate amount.
µM
References Fig. 4. Neuroprotective effects of compounds 1 and 4. The SH-SY5Y cells were exposed to MPP+and the cell viability was assessed by MTT assay. The cells were treated with 0.8 mM MPP+in the absence or presence of compounds 1 and 4. Data are expressed as the percentage of values in untreated control cultures. Each value indicates a mean ± SEM (n = 3). # P b 0.01, compared with control group. *P b 0.05, compared with the MPP+-treated group. Guanosine, positive control.
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