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Phenolic constituents from the roots of Phyllodium pulchellum a
b
c
a
Yang Zong , Ming Zhong , Dong-Mei Li , Bao-Jing Zhang , Zhena
a
a
a
Peng Mai , Xiao-Kui Huo , Shan-Shan Huang , Hou-Li Zhang , Chao a
a
b
Wang , Xiao-Chi Ma , Sheng-Min Yu & Dong-Ai Yang
b
a
College of Pharmacy, Dalian Medical University, Dalian 116044, China b
Institute of Nationality Medicine in Guangxi, Nanning 530001, China
Click for updates
c
Merro Pharmaceutical Corporation, Dalian 116036, China Published online: 23 Apr 2014.
To cite this article: Yang Zong, Ming Zhong, Dong-Mei Li, Bao-Jing Zhang, Zhen-Peng Mai, Xiao-Kui Huo, Shan-Shan Huang, Hou-Li Zhang, Chao Wang, Xiao-Chi Ma, Sheng-Min Yu & Dong-Ai Yang (2014) Phenolic constituents from the roots of Phyllodium pulchellum, Journal of Asian Natural Products Research, 16:7, 741-746, DOI: 10.1080/10286020.2014.910197 To link to this article: http://dx.doi.org/10.1080/10286020.2014.910197
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 7, 741–746, http://dx.doi.org/10.1080/10286020.2014.910197
Phenolic constituents from the roots of Phyllodium pulchellum Yang Zonga, Ming Zhongb*, Dong-Mei Lic, Bao-Jing Zhanga, Zhen-Peng Maia, Xiao-Kui Huoa, Shan-Shan Huanga, Hou-Li Zhanga, Chao Wanga*, Xiao-Chi Maa, Sheng-Min Yub and Dong-Ai Yangb a
College of Pharmacy, Dalian Medical University, Dalian 116044, China; bInstitute of Nationality Medicine in Guangxi, Nanning 530001, China; cMerro Pharmaceutical Corporation, Dalian 116036, China
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(Received 18 November 2013; final version received 27 March 2014) Three new phenolic constituents 1 –3 were obtained from the 95% ethanol extract of the roots of Phyllodium pulchellum (Leguminosae). Their structures were elucidated on the basis of spectroscopic analyses, such as NMR, UV, IR, HR-ESI-MS, and CD. Furthermore, in an in vitro bioassay, all compounds were tested for inhibitory effects against the proliferation of acetaldehyde-stimulated HSC-T6 cells, and compound 3 exhibited potent inhibitory activity with the IC50 value of 7.6 mM. Keywords: Phyllodium pulchellum; phenolic; anti-liver fibrosis
1. Introduction Phyllodium pulchellum (L.) Desv. (Leguminosae) is a folk medicine distributed in the southern parts of China, which is widely used for the treatment of fevers and liver fibrosis. Recently, many investigations exhibited that the ethanol extract of P. pulchellum had significant bioactivity in vivo system of liver fibrosis [1,2]. As a result, the investigation of bioactive substances of P. pulchellum indicated the presence of amines, flavonoids, phenols, and coumarins [3,4]. In the course of our phytochemical studies of P. pulchellum, we investigated the phenolic compounds of the EtOAc portion in an ethanol extract. Three new phenolic compounds were isolated and purified by the combination of several chromatographic techniques, such as PTLC, column chromatography (CC), and preparative HPLC. The structures of 1 –3 were elucidated using various spectroscopic data, including 1D, 2D NMR, HR-ESI-MS, IR, and CD. We report herein the isolation and structural
elucidation of compounds 1 –3 (Figure 1). The inhibitory effects of new compounds against the proliferation of acetaldehydeinduced HSC-T6 cells were also evaluated.
2. Results and discussion Compound 1 was obtained as a yellow amorphous powder. Its molecular formula was established as C21H22O4 by HR-ESIMS (339.1597 [M þ H] þ, calcd for 339.1591), implying 11 degrees of unsaturation. The UV spectrum displayed the presence of aromatic moiety (240, 283 nm). The 1H NMR spectroscopic data of 1 indicated the presence of one olefinic bond [dH 7.17 (1H, dd, J ¼ 9.6, 3.0 Hz), 6.19 (1H, d, J ¼ 9.6 Hz)], one aromatic proton at dH 6.25 (1H, s) from a penta-substituted aromatic ring, and one methoxyl group [dH 3.77 (3H, s)]. The 1H NMR spectrum also displayed the characteristic ABX2-type signals for the cisCHA(CHBCH2 moiety [dH 6.80 (1H,
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
742
Y. Zong et al. 5′
O 1′
13
3″
3′ 5 15
HO HO
11 9
3
5′
OH
4″
OH 8
O
O 2
6
1
10
7
OCH3
OH
1
4
3′
1′
HO
OH
5
OCH3 11
H3CO
OH
3 9 1
13
O
7
15
2
3
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Figure 1. Compounds 1 – 3 isolated from P. pulchellum.
J ¼ 10.2 Hz), 5.90 (1H, m), 2.42 (2H, m)], in which the cis configuration was deduced by the coupling constant of the olefinic protons (J AB ¼ 10.2 Hz). One prenyl group could also be established for 1 by the protons at dH 5.16 (1H, t, J ¼ 7.2 Hz), 2.98 (2H, d, J ¼ 7.2 Hz), 1.71 (3H, s) and 1.62 (3H, s). The 13C NMR spectrum of 1 confirmed the above-mentioned moieties, as well as exhibited one carbonyl carbon (dC 186.0). One a-substituted a,b,a0 ,b0 unsaturated carbonyl system could be deduced from the carbons at dC 186.0, 153.9, 149.1, 136.0, 126.0 and the protons at dH 7.17, 6.87, 6.19. The spectroscopic data indicated that 1 was a phenol, similar to pulchelstyrene D [4], which was also isolated from P. pulchellum. The differences were the substituent model and substituent moieties, such as one extra isopentenyl group of 1. In the HMBC spectrum, the long-range correlations of H-10 [dH 2.98 (2H, d, J ¼ 7.2 Hz)] of the prenyl group with C-11 (dC 149.1), C-12 (dC 136.0), and C-13 (dC 186.0) determined that the prenyl group
was located at C-12 (Figure 2). The longrange correlations of H-7 (dH 6.80) with C-2 (dC 143.3), and OCH3 (dH 3.77) with C-2 (dC 143.3) indicated that the methoxyl group was attached to C-2. The correlations of H-5/C-6, C-10, and H-5/C-3, C4 indicated that the C-5 site was unsubstituted, as well as confirmed the locations of two hydroxyl groups (C-3, C-4, respectively). It was difficult to determine the configuration of 1 for the limit of the mass of 1, which could be used by us. Therefore, the structure of 1 was elucidated as shown in Figure 1, named pulchelstyrene E. Compound 2, a yellow amorphous powder, gave the molecular formula C20H18O7, as established by HR-ESI-MS at m/z 371.1137 [M þ H]þ. The 1H NMR spectrum of 2 revealed the presence of a 1,2,3,4,5-penta-substituted benzene ring [dH 5.92 (1H, s)], a 1,3,4-tri-substituted benzene ring [dH 6.98 (1H, brs), 6.86 (1H, brd, J ¼ 7.2 Hz), 6.81 (1H, d, J ¼ 7.2 Hz)], and two oxygenated methines [dH 4.97 (1H, d, J ¼ 11.4 Hz), 4.53
O OH O HO
O A OH
HO
OH
C OH
HO
OH
B
OCH3
H3CO
O
OCH3 1
2
Figure 2. The key HMBC correlations of compounds 1– 3.
3
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Journal of Asian Natural Products Research (1H, d, J ¼ 11.4 Hz)]. In addition, two tertiary-methyl moieties [dH 1.41 (3H, s), 1.39 (3H, s); dC 26.4, 26.9], a pair of ciscoupled olefinic doublets [dH 6.45, 5.54 (each 1H, d, J ¼ 9.6 Hz); dC 125.9, 114.2], and an oxygen-bearing quaternary sp3 carbon (dC 77.4) were observed in the 1H and 13C NMR spectra of 2. The spectroscopic data indicated that 2 was a flavanonol derivative with a 2,2dimethyl-2H-pyran ring either at C-6, C-7 or C-7, C-8 [5 – 7]. In the HMBC spectrum, H-20 and H-60 of B-ring displayed long-range correlations with C-2 respectively, implying the hydroxyl groups of B-ring were assigned to be at C-30 and C-40 . The 2,2dimethyl-2H-pyran ring was attached to C7 and C-8 of A-ring on the basis of the HMBC correlations of H-400 with C-7, C-8, and C-9. The 2,3-trans configuration was assigned from the J value of H-2/H-3 (11.4 Hz). Analysis of the CD spectrum of 2 indicated positive Cotton effect at 336.5 nm, and negative Cotton effect at 299.5 nm, which were similar to those of the related compound [6]. Thus, the absolute configuration of 2 was determined to be 2R, 3R. Based on the observations, the structure of compound 2 was elucidated as (2R,3R)-5,30 ,40 -trihydroxy-200 ,200 -dimethylpyrano[b-7,8]-flavanone and named pulcheloid B (Figure 1). Compound 3, a yellow amorphous powder, had the molecular formula C17H18O4, calculated from the [M þ H]þ ion peak at m/z 287.1277 in its HR-ESIMS. Six aromatic protons derived from two aromatic rings were observed in the 1 H NMR spectrum of 3. Analyses of the chemical shifts and coupling constants of these aromatic protons indicated that one aromatic ring was para-disubstituted [A 2B 2 system, d H 7.19 (2H, d, J ¼ 8.4 Hz), 6.77 (2H, d, J ¼ 8.4)], and the other ring was 1,2,3,4-tetra-substituted [AB system, dH 6.68 (1H, d, J ¼ 8.4 Hz), 6.65 (1H, d, J ¼ 8.4 Hz)]. The 1H NMR data also exhibited the presence of one
743
allyl moiety [ d H 6.44 (1H, brd, J ¼ 11.4 Hz), 5.65 (1H, m), 3.58 (2H, d, J ¼ 7.2 Hz)] with the cis double bond, determined by the JAB (11.4 Hz). In addition, two methoxyl [dH 3.84 (3H, s), 3.76 (3H, s)] and two hydroxyl groups were also observed from the 1H NMR and HR-ESI-MS data. The 13C NMR spectroscopic data confirmed the above-mentioned moieties, and indicated a C6C3C6 skeleton of the structure. The attachments of these substituents could be determined by the long-range correlations of carbons and protons in the HMBC spectrum. The HMBC correlations of H-7 with C-2, 6 indicated that the olefinic carbon of the allyl moiety was attached at the para-disubstituted aromatic ring. The correlations of OCH3 (dH 3.76)/C-11 (dC 145.3), OCH3 (dH 3.84)/C-13 (dC 146.7), H-9 (dH 3.58)/C-10 (dC 126.3), C-11 (dC 145.3), C-15 (dC 117.8), H-14/C-13 (dC 146.7), C-12 (dC 138.7), H-15 (dH 6.65)/C13 (dC 146.7) determined the positions of substituents in the tetra-substituted aromatic ring. Thus, the structure of 3 was determined as shown in Figure 1, similar to pulchelstyrenes A – C [4], named pulchelstyrene F. Three new phenolic compounds (1 –3) were isolated from the roots of P. pulchellum, which was a folk medicine distributed in the southern part of China. Their structures were elucidated by MS, NMR, and CD spectroscopic data. The anti-liver fibrosis activities of compounds 1 –3 were also evaluated in an in vitro bioassay [8]. As a result, compound 3 inhibited the proliferation of acetaldehydestimulated hepatic stellate (HSC-T6) cells with an IC50 of 7.6 mM, while 1 and 2 inhibited with an IC50 . 100 mM. 3.
Experimental
3.1 General experimental procedures Optical rotations were measured on a JASCO P-2000 automatic digital polarimeter (JASCO, Hachioji-shi, Japan).
b
a
1
1.71 s 1.62 s 3.77 s
6.19 d (9.6) 7.17 dd (9.6, 3.0) 2.98 d (7.2) 5.16 t (7.2)
6.87 brs
6.80 d (10.2) 5.90 m 2.42 m
6.25 s
dHa
1
119.9 143.3 139.1 147.4 108.1 113.1 120.9 119.7 33.1 43.5 149.1 136.0 186.0 126.0 153.9 26.6 117.9 133.0 15.8 23.9 59.7
dCb 1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 200 300 400 500 600
Position
H and 13C NMR spectroscopic data of compounds 1 – 3.
Measured in methanol-d4, recorded at 600 MHz, dH in ppm, J in Hz. Recorded in methanol-d4, 150 MHz.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50 2-OCH3
Position
Table 1.
5.54 d (9.6) 6.45 d (9.6) 1.41 s 1.39 s
6.86 brd (7.2) 6.81 d (7.2)
6.98 brs
5.92 s
4.97 d (11.4) 4.53 d (11.4)
dHa
2
83.2 71.7 196.9 162.5 96.2 161.7 101.2 156.2 100.4 127.7 113.9 144.4 145.3 114.1 118.9 77.4 125.9 114.2 26.4 26.9
dCb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 11-OCH3 13-OCH3
Position
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6.68 d (8.4) 6.65 d (8.4) 3.76 s 3.84 s
6.77 d (8.4) 7.19 d (8.4) 6.44 d (11.4) 5.65 m 3.58 d (7.2)
7.19 d (8.4) 6.77 d (8.4)
dHa
3
128.3 129.1 114.0 155.4 114.0 129.1 128.2 128.0 27.6 126.3 145.3 138.7 146.7 106.4 117.8 58.7 54.7
dCb
744 Y. Zong et al.
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Journal of Asian Natural Products Research UV spectra were recorded on a JASCO V650 spectrophotometer (JASCO), and IR spectra were recorded on a Nicolet 5700 spectrometer using an FT-IR microscope transmission method (Thermo Fisher Scientific, Inc., Waltham, MA, USA). CD spectra were measured on a JASCO J-815 spectropolarimeter (JASCO). NMR spectra were acquired with BRUKER-600, (Bruker, Billerica, MA, USA). HR-ESIMS were collected on an Agilent 1100 series LC/MSD ion trap mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Preparative HPLC was conducted using a Shimadzu LC-6AD instrument with an SPD-20A detector (Shimadzu, Kyoto, Japan) and a YMC-Pack ODS-A column (250 mm £ 20 mm, 5 mm). CC was performed with silica gel (200 – 300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China) and ODS (50 mm, YMC, Kyoto, Japan). TLC was carried out on glass precoated silica gel GF254 plates (Qingdao Marine Chemical, Inc.). Spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating. 3.2 Plant material The roots of P. pulchellum were collected in Gongcheng, Guangxi, China, in August 2012, and identified by Chief physician Bin Dai from the Institute of Nationality Medicine in Guangxi. A voucher specimen has been deposited at the Herbarium of Institute of Nationality Medicine in Guangxi (S-1082). 3.3
Extraction and isolation
The air-dried and powdered roots (10 kg) of P. pulchellum were ultrasonically extracted with water, and 95% ethanol successively (30 l £ 1 h £ 3). After evaporation of ethanol in vacuo, the aqueous residue of the ethanolic extract was diluted with water and then partitioned with EtOAc. The EtOAc extract (20 g) was
745
subjected to CC on silica gel with petroleum ether– acetone (100:1 –2:1) to afford 11 fractions (F1 –F11). Sub-fraction F9 (1.2 g) was further isolated on RP-18 column and finally purified by preparative HPLC (5 ml/min, 210 nm, 20 – 40% CH3CN – H2O (90 min)) to yield compounds 1 (3.4 mg, Rf 73 min), 2 (8.5 mg, Rf 32 min), and 3 (6.0 mg, Rf 56 min).
3.3.1
Compound 1
A yellow amorphous powder. ½a20 D þ 31.7 (c ¼ 0.08, CH3OH). UV (MeOH) lmax: 240, 283 nm. IR (microscope) nmax: 3394, 2939, 1688, 1468, 1436, 1204, 1140 cm – 1. CD (CH3OH) De 216 nm 2 0.18, De 236 nm þ 0.36. For 1H and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS: m/z 339.1597 [M þ H]þ (calcd for C21H23O4, 339.1591).
3.3.2 Compound 2 A yellow amorphous powder. þ 10.6 (c ¼ 0.1, CH3OH). UV (MeOH) lmax: 229, 272, 295, 314 nm. IR (microscope) nmax: 3338, 2954, 2926, 1655, 1457, 1377 cm – 1. For 1H and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS: m/z 371.1137 [M þ H]þ (calcd for C20H19O7, 371.1139).
3.3.3 Compound 3 A yellow amorphous powder. UV (MeOH) lmax: 202, 255 nm. IR (microscope) nmax: 3399, 2943, 1612, 1511, 1465, 1210 cm – 1. For 1H and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS: m/z 287.1277 [M þ H] þ (calcd for C17 H19O 4, 287.1278).
Acknowledgements This project was financially supported by the National Natural Science Foundation of China (No. 81160559).
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References
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[1] C.N. Luo and S. Wang, Res. Pract. Chin. Med. 22, 74 (2008). [2] M. Zhong, M.S. Yu, L.Y. Huang, and Z.Y. Yang, Chin. Hepatol. 6, 168 (2001). [3] S. Ghosal, S.K. Banerjee, S.K. Bhattacharya, and A.K. Sanyal, Planta Med. 21, 398 (1972). [4] C.C. Shen, S.T. Wang, S.Y. Tsai, H.C. Yang, B.J. Shieh, and C.C. Chen, J. Nat. Prod. 68, 791 (2005).
[5] P. Innok, T. Rukachaisirkul, and A. Suksamrarn, Chem. Pharm. Bull. 57, 993 (2009). [6] H. Sasaki, Y. Kashiwada, H. Shibata, and Y. Takaishi, Phytochemistry 82, 136 (2012). [7] P. Khaomek, C. Ichino, A. Ishiyama, H. Sekiguchi, M. Namatame, N. Ruangrungsi, E. Saifah, H. Kiyohara, K. Otoguro, S. Omura, and H. Yamada, J. Nat. Med. 62, 217 (2008). [8] C. Li, L.C. Alyssa, L. Andrew, W.F. Samuel, and R.B. David, J. Hepatol. 55, 399 (2011).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Phenolic constituents from the roots of Phyllodium pulchellum a
b
c
a
Yang Zong , Ming Zhong , Dong-Mei Li , Bao-Jing Zhang , Zhena
a
a
a
Peng Mai , Xiao-Kui Huo , Shan-Shan Huang , Hou-Li Zhang , Chao a
a
b
Wang , Xiao-Chi Ma , Sheng-Min Yu & Dong-Ai Yang
b
a
College of Pharmacy, Dalian Medical University, Dalian 116044, China b
Institute of Nationality Medicine in Guangxi, Nanning 530001, China
Click for updates
c
Merro Pharmaceutical Corporation, Dalian 116036, China Published online: 23 Apr 2014.
To cite this article: Yang Zong, Ming Zhong, Dong-Mei Li, Bao-Jing Zhang, Zhen-Peng Mai, Xiao-Kui Huo, Shan-Shan Huang, Hou-Li Zhang, Chao Wang, Xiao-Chi Ma, Sheng-Min Yu & Dong-Ai Yang (2014) Phenolic constituents from the roots of Phyllodium pulchellum, Journal of Asian Natural Products Research, 16:7, 741-746, DOI: 10.1080/10286020.2014.910197 To link to this article: http://dx.doi.org/10.1080/10286020.2014.910197
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Baskent Universitesi] at 15:30 20 December 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2014 Vol. 16, No. 7, 741–746, http://dx.doi.org/10.1080/10286020.2014.910197
Phenolic constituents from the roots of Phyllodium pulchellum Yang Zonga, Ming Zhongb*, Dong-Mei Lic, Bao-Jing Zhanga, Zhen-Peng Maia, Xiao-Kui Huoa, Shan-Shan Huanga, Hou-Li Zhanga, Chao Wanga*, Xiao-Chi Maa, Sheng-Min Yub and Dong-Ai Yangb a
College of Pharmacy, Dalian Medical University, Dalian 116044, China; bInstitute of Nationality Medicine in Guangxi, Nanning 530001, China; cMerro Pharmaceutical Corporation, Dalian 116036, China
Downloaded by [Baskent Universitesi] at 15:30 20 December 2014
(Received 18 November 2013; final version received 27 March 2014) Three new phenolic constituents 1 –3 were obtained from the 95% ethanol extract of the roots of Phyllodium pulchellum (Leguminosae). Their structures were elucidated on the basis of spectroscopic analyses, such as NMR, UV, IR, HR-ESI-MS, and CD. Furthermore, in an in vitro bioassay, all compounds were tested for inhibitory effects against the proliferation of acetaldehyde-stimulated HSC-T6 cells, and compound 3 exhibited potent inhibitory activity with the IC50 value of 7.6 mM. Keywords: Phyllodium pulchellum; phenolic; anti-liver fibrosis
1. Introduction Phyllodium pulchellum (L.) Desv. (Leguminosae) is a folk medicine distributed in the southern parts of China, which is widely used for the treatment of fevers and liver fibrosis. Recently, many investigations exhibited that the ethanol extract of P. pulchellum had significant bioactivity in vivo system of liver fibrosis [1,2]. As a result, the investigation of bioactive substances of P. pulchellum indicated the presence of amines, flavonoids, phenols, and coumarins [3,4]. In the course of our phytochemical studies of P. pulchellum, we investigated the phenolic compounds of the EtOAc portion in an ethanol extract. Three new phenolic compounds were isolated and purified by the combination of several chromatographic techniques, such as PTLC, column chromatography (CC), and preparative HPLC. The structures of 1 –3 were elucidated using various spectroscopic data, including 1D, 2D NMR, HR-ESI-MS, IR, and CD. We report herein the isolation and structural
elucidation of compounds 1 –3 (Figure 1). The inhibitory effects of new compounds against the proliferation of acetaldehydeinduced HSC-T6 cells were also evaluated.
2. Results and discussion Compound 1 was obtained as a yellow amorphous powder. Its molecular formula was established as C21H22O4 by HR-ESIMS (339.1597 [M þ H] þ, calcd for 339.1591), implying 11 degrees of unsaturation. The UV spectrum displayed the presence of aromatic moiety (240, 283 nm). The 1H NMR spectroscopic data of 1 indicated the presence of one olefinic bond [dH 7.17 (1H, dd, J ¼ 9.6, 3.0 Hz), 6.19 (1H, d, J ¼ 9.6 Hz)], one aromatic proton at dH 6.25 (1H, s) from a penta-substituted aromatic ring, and one methoxyl group [dH 3.77 (3H, s)]. The 1H NMR spectrum also displayed the characteristic ABX2-type signals for the cisCHA(CHBCH2 moiety [dH 6.80 (1H,
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
742
Y. Zong et al. 5′
O 1′
13
3″
3′ 5 15
HO HO
11 9
3
5′
OH
4″
OH 8
O
O 2
6
1
10
7
OCH3
OH
1
4
3′
1′
HO
OH
5
OCH3 11
H3CO
OH
3 9 1
13
O
7
15
2
3
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Figure 1. Compounds 1 – 3 isolated from P. pulchellum.
J ¼ 10.2 Hz), 5.90 (1H, m), 2.42 (2H, m)], in which the cis configuration was deduced by the coupling constant of the olefinic protons (J AB ¼ 10.2 Hz). One prenyl group could also be established for 1 by the protons at dH 5.16 (1H, t, J ¼ 7.2 Hz), 2.98 (2H, d, J ¼ 7.2 Hz), 1.71 (3H, s) and 1.62 (3H, s). The 13C NMR spectrum of 1 confirmed the above-mentioned moieties, as well as exhibited one carbonyl carbon (dC 186.0). One a-substituted a,b,a0 ,b0 unsaturated carbonyl system could be deduced from the carbons at dC 186.0, 153.9, 149.1, 136.0, 126.0 and the protons at dH 7.17, 6.87, 6.19. The spectroscopic data indicated that 1 was a phenol, similar to pulchelstyrene D [4], which was also isolated from P. pulchellum. The differences were the substituent model and substituent moieties, such as one extra isopentenyl group of 1. In the HMBC spectrum, the long-range correlations of H-10 [dH 2.98 (2H, d, J ¼ 7.2 Hz)] of the prenyl group with C-11 (dC 149.1), C-12 (dC 136.0), and C-13 (dC 186.0) determined that the prenyl group
was located at C-12 (Figure 2). The longrange correlations of H-7 (dH 6.80) with C-2 (dC 143.3), and OCH3 (dH 3.77) with C-2 (dC 143.3) indicated that the methoxyl group was attached to C-2. The correlations of H-5/C-6, C-10, and H-5/C-3, C4 indicated that the C-5 site was unsubstituted, as well as confirmed the locations of two hydroxyl groups (C-3, C-4, respectively). It was difficult to determine the configuration of 1 for the limit of the mass of 1, which could be used by us. Therefore, the structure of 1 was elucidated as shown in Figure 1, named pulchelstyrene E. Compound 2, a yellow amorphous powder, gave the molecular formula C20H18O7, as established by HR-ESI-MS at m/z 371.1137 [M þ H]þ. The 1H NMR spectrum of 2 revealed the presence of a 1,2,3,4,5-penta-substituted benzene ring [dH 5.92 (1H, s)], a 1,3,4-tri-substituted benzene ring [dH 6.98 (1H, brs), 6.86 (1H, brd, J ¼ 7.2 Hz), 6.81 (1H, d, J ¼ 7.2 Hz)], and two oxygenated methines [dH 4.97 (1H, d, J ¼ 11.4 Hz), 4.53
O OH O HO
O A OH
HO
OH
C OH
HO
OH
B
OCH3
H3CO
O
OCH3 1
2
Figure 2. The key HMBC correlations of compounds 1– 3.
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Journal of Asian Natural Products Research (1H, d, J ¼ 11.4 Hz)]. In addition, two tertiary-methyl moieties [dH 1.41 (3H, s), 1.39 (3H, s); dC 26.4, 26.9], a pair of ciscoupled olefinic doublets [dH 6.45, 5.54 (each 1H, d, J ¼ 9.6 Hz); dC 125.9, 114.2], and an oxygen-bearing quaternary sp3 carbon (dC 77.4) were observed in the 1H and 13C NMR spectra of 2. The spectroscopic data indicated that 2 was a flavanonol derivative with a 2,2dimethyl-2H-pyran ring either at C-6, C-7 or C-7, C-8 [5 – 7]. In the HMBC spectrum, H-20 and H-60 of B-ring displayed long-range correlations with C-2 respectively, implying the hydroxyl groups of B-ring were assigned to be at C-30 and C-40 . The 2,2dimethyl-2H-pyran ring was attached to C7 and C-8 of A-ring on the basis of the HMBC correlations of H-400 with C-7, C-8, and C-9. The 2,3-trans configuration was assigned from the J value of H-2/H-3 (11.4 Hz). Analysis of the CD spectrum of 2 indicated positive Cotton effect at 336.5 nm, and negative Cotton effect at 299.5 nm, which were similar to those of the related compound [6]. Thus, the absolute configuration of 2 was determined to be 2R, 3R. Based on the observations, the structure of compound 2 was elucidated as (2R,3R)-5,30 ,40 -trihydroxy-200 ,200 -dimethylpyrano[b-7,8]-flavanone and named pulcheloid B (Figure 1). Compound 3, a yellow amorphous powder, had the molecular formula C17H18O4, calculated from the [M þ H]þ ion peak at m/z 287.1277 in its HR-ESIMS. Six aromatic protons derived from two aromatic rings were observed in the 1 H NMR spectrum of 3. Analyses of the chemical shifts and coupling constants of these aromatic protons indicated that one aromatic ring was para-disubstituted [A 2B 2 system, d H 7.19 (2H, d, J ¼ 8.4 Hz), 6.77 (2H, d, J ¼ 8.4)], and the other ring was 1,2,3,4-tetra-substituted [AB system, dH 6.68 (1H, d, J ¼ 8.4 Hz), 6.65 (1H, d, J ¼ 8.4 Hz)]. The 1H NMR data also exhibited the presence of one
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allyl moiety [ d H 6.44 (1H, brd, J ¼ 11.4 Hz), 5.65 (1H, m), 3.58 (2H, d, J ¼ 7.2 Hz)] with the cis double bond, determined by the JAB (11.4 Hz). In addition, two methoxyl [dH 3.84 (3H, s), 3.76 (3H, s)] and two hydroxyl groups were also observed from the 1H NMR and HR-ESI-MS data. The 13C NMR spectroscopic data confirmed the above-mentioned moieties, and indicated a C6C3C6 skeleton of the structure. The attachments of these substituents could be determined by the long-range correlations of carbons and protons in the HMBC spectrum. The HMBC correlations of H-7 with C-2, 6 indicated that the olefinic carbon of the allyl moiety was attached at the para-disubstituted aromatic ring. The correlations of OCH3 (dH 3.76)/C-11 (dC 145.3), OCH3 (dH 3.84)/C-13 (dC 146.7), H-9 (dH 3.58)/C-10 (dC 126.3), C-11 (dC 145.3), C-15 (dC 117.8), H-14/C-13 (dC 146.7), C-12 (dC 138.7), H-15 (dH 6.65)/C13 (dC 146.7) determined the positions of substituents in the tetra-substituted aromatic ring. Thus, the structure of 3 was determined as shown in Figure 1, similar to pulchelstyrenes A – C [4], named pulchelstyrene F. Three new phenolic compounds (1 –3) were isolated from the roots of P. pulchellum, which was a folk medicine distributed in the southern part of China. Their structures were elucidated by MS, NMR, and CD spectroscopic data. The anti-liver fibrosis activities of compounds 1 –3 were also evaluated in an in vitro bioassay [8]. As a result, compound 3 inhibited the proliferation of acetaldehydestimulated hepatic stellate (HSC-T6) cells with an IC50 of 7.6 mM, while 1 and 2 inhibited with an IC50 . 100 mM. 3.
Experimental
3.1 General experimental procedures Optical rotations were measured on a JASCO P-2000 automatic digital polarimeter (JASCO, Hachioji-shi, Japan).
b
a
1
1.71 s 1.62 s 3.77 s
6.19 d (9.6) 7.17 dd (9.6, 3.0) 2.98 d (7.2) 5.16 t (7.2)
6.87 brs
6.80 d (10.2) 5.90 m 2.42 m
6.25 s
dHa
1
119.9 143.3 139.1 147.4 108.1 113.1 120.9 119.7 33.1 43.5 149.1 136.0 186.0 126.0 153.9 26.6 117.9 133.0 15.8 23.9 59.7
dCb 1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 200 300 400 500 600
Position
H and 13C NMR spectroscopic data of compounds 1 – 3.
Measured in methanol-d4, recorded at 600 MHz, dH in ppm, J in Hz. Recorded in methanol-d4, 150 MHz.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50 2-OCH3
Position
Table 1.
5.54 d (9.6) 6.45 d (9.6) 1.41 s 1.39 s
6.86 brd (7.2) 6.81 d (7.2)
6.98 brs
5.92 s
4.97 d (11.4) 4.53 d (11.4)
dHa
2
83.2 71.7 196.9 162.5 96.2 161.7 101.2 156.2 100.4 127.7 113.9 144.4 145.3 114.1 118.9 77.4 125.9 114.2 26.4 26.9
dCb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 11-OCH3 13-OCH3
Position
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6.68 d (8.4) 6.65 d (8.4) 3.76 s 3.84 s
6.77 d (8.4) 7.19 d (8.4) 6.44 d (11.4) 5.65 m 3.58 d (7.2)
7.19 d (8.4) 6.77 d (8.4)
dHa
3
128.3 129.1 114.0 155.4 114.0 129.1 128.2 128.0 27.6 126.3 145.3 138.7 146.7 106.4 117.8 58.7 54.7
dCb
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Journal of Asian Natural Products Research UV spectra were recorded on a JASCO V650 spectrophotometer (JASCO), and IR spectra were recorded on a Nicolet 5700 spectrometer using an FT-IR microscope transmission method (Thermo Fisher Scientific, Inc., Waltham, MA, USA). CD spectra were measured on a JASCO J-815 spectropolarimeter (JASCO). NMR spectra were acquired with BRUKER-600, (Bruker, Billerica, MA, USA). HR-ESIMS were collected on an Agilent 1100 series LC/MSD ion trap mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Preparative HPLC was conducted using a Shimadzu LC-6AD instrument with an SPD-20A detector (Shimadzu, Kyoto, Japan) and a YMC-Pack ODS-A column (250 mm £ 20 mm, 5 mm). CC was performed with silica gel (200 – 300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China) and ODS (50 mm, YMC, Kyoto, Japan). TLC was carried out on glass precoated silica gel GF254 plates (Qingdao Marine Chemical, Inc.). Spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating. 3.2 Plant material The roots of P. pulchellum were collected in Gongcheng, Guangxi, China, in August 2012, and identified by Chief physician Bin Dai from the Institute of Nationality Medicine in Guangxi. A voucher specimen has been deposited at the Herbarium of Institute of Nationality Medicine in Guangxi (S-1082). 3.3
Extraction and isolation
The air-dried and powdered roots (10 kg) of P. pulchellum were ultrasonically extracted with water, and 95% ethanol successively (30 l £ 1 h £ 3). After evaporation of ethanol in vacuo, the aqueous residue of the ethanolic extract was diluted with water and then partitioned with EtOAc. The EtOAc extract (20 g) was
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subjected to CC on silica gel with petroleum ether– acetone (100:1 –2:1) to afford 11 fractions (F1 –F11). Sub-fraction F9 (1.2 g) was further isolated on RP-18 column and finally purified by preparative HPLC (5 ml/min, 210 nm, 20 – 40% CH3CN – H2O (90 min)) to yield compounds 1 (3.4 mg, Rf 73 min), 2 (8.5 mg, Rf 32 min), and 3 (6.0 mg, Rf 56 min).
3.3.1
Compound 1
A yellow amorphous powder. ½a20 D þ 31.7 (c ¼ 0.08, CH3OH). UV (MeOH) lmax: 240, 283 nm. IR (microscope) nmax: 3394, 2939, 1688, 1468, 1436, 1204, 1140 cm – 1. CD (CH3OH) De 216 nm 2 0.18, De 236 nm þ 0.36. For 1H and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS: m/z 339.1597 [M þ H]þ (calcd for C21H23O4, 339.1591).
3.3.2 Compound 2 A yellow amorphous powder. þ 10.6 (c ¼ 0.1, CH3OH). UV (MeOH) lmax: 229, 272, 295, 314 nm. IR (microscope) nmax: 3338, 2954, 2926, 1655, 1457, 1377 cm – 1. For 1H and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS: m/z 371.1137 [M þ H]þ (calcd for C20H19O7, 371.1139).
3.3.3 Compound 3 A yellow amorphous powder. UV (MeOH) lmax: 202, 255 nm. IR (microscope) nmax: 3399, 2943, 1612, 1511, 1465, 1210 cm – 1. For 1H and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS: m/z 287.1277 [M þ H] þ (calcd for C17 H19O 4, 287.1278).
Acknowledgements This project was financially supported by the National Natural Science Foundation of China (No. 81160559).
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References
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[5] P. Innok, T. Rukachaisirkul, and A. Suksamrarn, Chem. Pharm. Bull. 57, 993 (2009). [6] H. Sasaki, Y. Kashiwada, H. Shibata, and Y. Takaishi, Phytochemistry 82, 136 (2012). [7] P. Khaomek, C. Ichino, A. Ishiyama, H. Sekiguchi, M. Namatame, N. Ruangrungsi, E. Saifah, H. Kiyohara, K. Otoguro, S. Omura, and H. Yamada, J. Nat. Med. 62, 217 (2008). [8] C. Li, L.C. Alyssa, L. Andrew, W.F. Samuel, and R.B. David, J. Hepatol. 55, 399 (2011).
