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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Three new compounds isolated from the seeds of Vaccaria segetalis ab
b
c
b
Yan-Ling Zhang , Li-Long Jiang , Tong-Shu Xiao , Shou-Qian Chen ac
& You-Bin Li a
School of Pharmacy, Hainan Medical University, Haikou 571101, China b
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046, China c
Click for updates
Department of Phytochemistry, Jiangsu Provincial Institute of Traditional Chinese Medicine, Nanjing 210028, China Published online: 03 Jan 2015.
To cite this article: Yan-Ling Zhang, Li-Long Jiang, Tong-Shu Xiao, Shou-Qian Chen & You-Bin Li (2015): Three new compounds isolated from the seeds of Vaccaria segetalis, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.992887 To link to this article: http://dx.doi.org/10.1080/10286020.2014.992887
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 http://dx.doi.org/10.1080/10286020.2014.992887
Three new compounds isolated from the seeds of Vaccaria segetalis Yan-Ling Zhanga,b, Li-Long Jiangb, Tong-Shu Xiaoc, Shou-Qian Chenb and You-Bin Lia,c* a School of Pharmacy, Hainan Medical University, Haikou 571101, China; bSchool of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046, China; cDepartment of Phytochemistry, Jiangsu Provincial Institute of Traditional Chinese Medicine, Nanjing 210028, China
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(Received 27 July 2014; final version received 25 November 2014) Two new capsaicin analogs, N-(3-methoxy-4-hydroxyphenethyl)-tetracosanamide (1) and N-(3,4-dihydroxyphenethyl)-tetracosanamide (2), along with one new flavonoidal glycoside pinnatifin E (3) were isolated from the ethanolic extract of the seeds of Vaccaria segetalis. Their structures were elucidated on the basis of spectroscopic methods including 1D, 2D NMR, MS, and other spectroscopic techniques, as well as by comparison with the relevant literatures. All compounds were evaluated for their coagulation Factor Xa inhibition activities. Keywords: Vaccaria segetalis; N-(3-methoxy-4-hydroxyphenethyl)-tetracosanamide; N-(3,4-dihydroxyphenethyl)-tetracosanamide; pinnatifin E; FXa inhibition activity
1. Introduction Vaccaria segetalis (Neck.) Garcke (Caryophyllaceae) is an annual herb that is distributed all over China, except southern China [1]. The seeds of V. segetalis, which are known as Wang-Bu-Liu-Xing, have been used as a common folk medicine in China for the treatment of amenorrhea, dysmenorrhea, and breast milk stoppage [2]. The extract of V. segetalis demonstrates various bioactivities, such as the promotion of dieresis and milk secretion, activation of blood circulation, and the relief of carbuncle [3]. Previous phytochemical studies revealed the presence of triterpenoid saponins [4 –9] and cyclic peptides [10,11] in the seeds of V. segetalis. In the previous study, we screened the potential inhibitory activity of 24 species of those plants that were used as traditional Chinese medicine for the treatment of thrombosis. We demonstrated that the ethanol extract of the seeds of V. segetalis possessed inhibitory activity
against the Factor Xa (FXa) [12]. During the course of our search for bioactive ingredients from the traditional medicine, two new capsaicin analogs (1 and 2) and one new flavonoidal glycoside (3) (Figure 1) were isolated from the ethanolic extract of the seeds of V. segetalis. In this paper, we report the structure elucidation of the new compounds and their inhibitory activities against FXa.
2.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Results and discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula C33H59NO3 was deduced from the pseudomolecular ion peak at m/z 516.4456 [M – H] – in the negative HR-ESI-MS. The IR spectrum showed the characteristic absorptions of amidogen (3310 cm21), hydroxy (3519, 1029 cm21), and methyl (2919, 2850 cm21) groups. The 1H NMR spectrum (Table 1) displayed signals for a 1,3,4-trisubstituted aromatic ring at d 6.84
2
Y.-L. Zhang et al. OH
24'
RO
3
HO
4
2
1 6
7
8
H N
6'' 1'
HO 2'
O
5
1 R = CH3 2 R=H
5''
4''
HO
O O
O
7
2'' 3''
1''
OH
6
5
OH O
3
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Figure 1. Structures of compounds 1 –3.
(1H, d, J ¼ 7.8 Hz), 6.69 (1H, s), and 6.67 (1H, d, J ¼ 7.8 Hz); a methoxy signal at d 3.87 (3H, s); and aliphatic group signals at d 1.59 (2H, m), 1.20 – 1.40 (40H), and 0.89 (3H, t, J ¼ 6.9 Hz). In the 13C NMR spectrum (Table 1), a set of typical carbon signals at d 146.7 (C-3), 144.3 (C-4), 130.7 (C-1), 121.4 (C-6), 114.4 (C-5), and 111.2 (C-2) revealed the presence of a benzene ring. Signal at d 55.9 revealed the presence of a methoxy group. In addition, signals at d 173.1, 36.9, 29.7 (multiplet), 25.8, and 14.1 were assigned as a long-chain fattyacyl group. These NMR data clearly indicated that compound 1 was an analog of capsaicinoids. Comparing the NMR data of 1 with those of N-vanillylhexadecanamide [13], two main differences were unambiguously observed: (1) Rather than a 3-methoxy-4-hydroxybenzyl amine substituent in N-vanillyl-hexadecanamide, 1 contained a 3-methoxy-4hydroxy-phenethyl amine substituent. It was further supported by the HMBC correlations from H-7 at dH 2.74 (2H, t, J ¼ 6.9 Hz) to C-1 at dC 130.7, C-6 at dC 121.4, C-2 at dC 111.2, and C-8 at dC 40.6; and H-8 at dH 3.48 (2H, m) to C-10 at dC 173.1, C-1 at dC 130.7, and C-7 at dC 35.4. (2) Combined with mass spectrometry, the differences just from the chain length, the molecule of 1 was 126 larger than that of N-vanillyl-hexadecanamide, which weighed nine CH2 units. Except one more CH2 in the 3-methoxy-4-hydroxyphenethyl amine substituent in 1, the left eight CH2 were determined to be the fatty acid part that was elucidated as lignoceric acid and were confirmed by the detailed
analyses of the HMBC correlations (Table 1). Thus, the structure of 1 was elucidated as N-(3-methoxy-4-hydroxyphenethyl)-tetracosanamide and shown in Figure 1. Compound 2 was isolated as a yellow powder. Its molecular formula was established as C32H57NO3 deduced from the negative HR-ESI-MS at m/z 502.4308 [M– H] – . The IR spectrum showed the characteristic absorptions of amidogen (3303 cm21), hydroxy group (3091 cm21), and carboxyl group (1717 cm21). It was found to have a similar structure to compound 1 by comparison of their 1H and 13C NMR spectral data (Table 1), the main difference observed was the absence of a methyl signal at d 3.87 (3H, s) in the 1H NMR spectrum and a carbon signal at d 55.9 in the 13C NMR spectrum of 1. After careful analysis of the data, the result showed that there was a hydroxy group in 2 instead of a methoxy group in 1, which was confirmed by the obvious down-fielded shift of C-2 and C-4. Therefore, compound 2 was established to be N-(3,4-dihydroxyphenethyl)-tetracosanamide. Compound 3 was also obtained as a yellow powder with ½a20 2 25.43 D (c ¼ 0.07, DMSO) and positive to Mg – HCl color reaction. Its molecular formula, C21H18O9, was determined by negative HR-TOF-MS at m/z 413.0875. The IR spectrum of 3 showed the characteristic absorptions attributable to hydroxy groups (3384 cm21), conjugated carbonyl group (1667 cm21), and aromatic rings (1619 and 1546 cm21). These data suggested that compound 3 may be a flavone. In the 1H
130.7 (C) 111.2 (CH) 146.7 (C) 144.3 (C) 114.4 (CH) 121.4 (CH) 35.4 (CH2) 40.6 (CH2) 173.1 (C) 36.9 (CH2) 25.8 (CH2) 29.5 (CH2) , 29.7 (CH2) 31.9 (CH2) 22.7 (CH2) 14.1 (CH3) 55.9 (CH3)
1 2 3 4 5 6 7 8 10 20 30 40 50 – 210 220 230 240 OCH3
– 6.69 (1H, s) – – 6.84 (1H, d, 7.8) 6.67 (1H, d, 7.8) 2.74 (2H, t, 6.9) 3.45– 3.52 (2H, m) – 2.11 (2H, t, 7.5) 1.58– 1.60 (2H, m) 1.20– 1.40 1.20– 1.40 1.20– 1.40 1.20– 1.40 0.89 (3H, t, 6.9) 3.87 (3H, s)
dH
Compound 1 (CDCl3)
– C-3, C-4, C-6 – – C-1, C-3, C-4 C-2, C-4 C-1, C-2, C-6, C-8 C-1, C-7, C-10 – C-10 , C-30 – – – – – C-220 , C-230 C-3
HMBC 131.5 (C) 117.2 (CH) 147.2 (C) 145.6 (C) 116.5 (CH) 120.3 (CH) 35.9 (CH2) 41.7 (CH2) 173.1 (C) 36.7 (CH2) 26.3 (CH2) 30.0 (CH2) , 29.8 (CH2) 32.1 (CH2) 22.9 (CH2) 14.2 (CH3) –
dC (DEPT)
C NMR (75 MHz) and 1H NMR (300 MHz) spectral data of 1 and 2 (d in ppm, J in Hz).
dC (DEPT)
13
Position
Table 1.
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– 7.18 (1H, s) – – 7.14 (1H, d, 7.8) 6.76 (1H, d, 7.8) 2.90 (2H, t, 6.9) 3.68 – 3.75 (2H, m) – 2.33 (2H, t, 7.5) 1.75 – 1.79 (2H, m) 1.10 – 1.40 1.10 – 1.40 1.10 – 1.40 1.10 – 1.40 0.83 (3H, t, 6.3) –
dH
Compound 2 (C5D5N)
C-220 , C-230
C-10 , C-30
C-1, C-3, C-4 C-2, C-4, C-7 C-1, C-2, C-6, C-8 C-1, C-7, C-10
C-3, C-4, C-6
HMBC
Journal of Asian Natural Products Research 3
4 Table 2. in Hz).
Y.-L. Zhang et al. 13
C NMR (75 MHz) and 1H NMR (300 MHz) spectral data of 3 in DMSO-d6 (d in ppm, J
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Compound 3 Position
dC (DEPT)
dH
HMBC
2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100
164.1 (C) 102.9 (CH) 181.1 (C) 155.4 (C) 107.2 (C) 163.9 (C) 89.3 (CH) 157.2 (C) 104.7 (C) 121.1 (C) 128.5 (CH) 116.0 (CH) 161.2 (C) 116.0 (CH) 128.5 (CH) 32.7 (CH2)
200 300 400 500 600
117.9 (C) 79.7 (CH) 73.7 (CH) 83.8 (CH) 63.3 (CH2)
– 6.82 (1H, s) – – – – 6.64 (1H, s) – – – 7.93 (1H, d, 8.7) 6.93 (1H, d, 8.7) – 6.93 (1H, d, 8.7) 7.93 (1H, d, 8.7) 3.05 (1H, d, 16.8) 3.27 (1H, d, 16.8) – 3.99 (1H, d, 8.1) 3.90 (1H, m) 3.80 (1H, m) 3.62 (1H, m) 3.47 (1H, m) 13.23 (s) 10.37 (s)
– C-2, C-4, C-10, C-10 – – – – C-6, C-7, C-9, C-10 – – – C-2, C-30 , C-40 , C-60 C-10 , C-40 , C-50 – C-10 , C-40 , C-30 C-2, C-20 , C-50 , C-40 C-6, C-300 , C-5, C-7 C-6, C-200 , C-5, C-7 – C-400 C-300 , C-600 C-400 C-500 C-500 C-5, C-6, C-10 –
5-OH 40 -OH
– –
NMR spectrum (Table 2), a characteristic downfield singlet at d 13.23 confirmed the presence of 5-OH, and two sets of doublets at d 7.93 (2H, d, J ¼ 8.7 Hz), and 6.93 (2H, d, J ¼ 8.7 Hz) displayed AA0 BB0 system signals of flavonoid ring B and were assigned to H-20 /60 and H-30 /50 , respectively. In addition, two singlets at d 6.82 and 6.64 were due to the protons in rings A and C. In the 13C NMR spectrum (Table 2), 21 carbon signals were observed, which included 15 carbon signals from the aglycone moiety and a set of carbon signals of the sugar moiety resonating at d 117.9 (C), 83.8 (CH), 79.7 (CH), 73.7 (CH), 63.3 (CH2), and 32.7 (CH2). These data showed that compound 3 was closely related to the known compound pinnatifin C [14]. The significant difference was the NMR spectral data
of flavonoid ring A. Careful analysis of the NMR data suggested that 3 should have different substitution pattern compared with pinnatifin C. In the HMBC spectrum (Table 2), the correlations from H-8 at dH 6.64 (1H, s) to C-7 at dC 163.9, C-9 at dC 157.2, C-6 at dC 107.2, and C-10 at dC 104.7; and from H-100 a at dH 3.05 (1H, d, J ¼ 16.8 Hz), H-100 b at dH 3.27 (1H, d, J ¼ 16.8 Hz) to C-6 at dC 107.2, C-200 at dC 117.9, and C-300 at dC 79.7 confirmed the link position between sugar moiety and flavonoid skeleton was at C-6 in 3 rather than C-8 in pinnatifin C. Additionally, the configuration of sugar moiety was the same as that of pinnatifin C from the NOESY spectrum and its optical rotation. In the NOESY spectrum, the cross-peaks between protons at d 3.99 (1H, d, J ¼ 8.1 Hz, H-300 ) and 3.80 (1H, m, H-500 ),
Journal of Asian Natural Products Research Table 3. Inhibitory enzymatic activity of compounds 1 – 3 against FXa. Compounds
263 ^ 3.36 368 ^ 4.91 165 ^ 8.37
1 2 3
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IC50 (mmol/l)
and at d 3.90 (1H, m, H-400 ) and 3.62 (1H, m, H-600 a) as well as the optical rotation of ½a20 D 2 25.43 (c ¼ 0.07, DMSO) allowed the configuration of sugar moiety as b-D frucofurariose. In conclusion, the structure of 3 was characterized as depicted in Figure 1 and named pinnatifin E. In this study, the isolated compounds were tested for their FXa inhibition activities. The result is presented in Table 3, and new compounds showed weak inhibition activity to FXa with the IC50 of 263 ^ 3.36, 368 ^ 4.91, and 165 ^ 8.37 mmol/l (all P , 0.01), respectively.
macia Biotech AB, Uppsala, Sweden), and octadecyl silica (ODS; Merck, Darmstadt, Germany) were used for column chromatography (CC), while precoated silica gel GF254 plates were used for thin layer chromatography (Qingdao Marine Chemical Co., Ltd, Qingdao, China). All solvents used were of analytical grade (Jiangsu Hanbang Science and Technology, Co., Ltd, China). 3.2 Plant material The seeds of V. segetalis were collected in Bozhou Country of Anhui Province, China, in March 2011 and identified by Prof. Shi-Hui Qian (Jiangsu Province Academy of Traditional Chinese Medicine). A voucher specimen (No. S-1300002) was deposited in the Department of Traditional Chinese Medicine Materials, Jiangsu Province Institute of Traditional Chinese Medicine, Jiangsu, China. 3.3
3. 3.1
Experimental General experimental procedures
Optical rotation was obtained on an Autopl IV polarimeter (Rudolph Research Analytical, Flanders, NJ, USA). UV spectra were measured on a Shimadzu UV-210 spectrometer (Shimadzu, Kyoto, Japan). IR spectra were recorded on a Bruker Tensor 27 spectrometer (Bruker Co., 1 Ettlingen, Germany). H NMR 13 (300 MHz), C NMR (75 MHz) spectra and 2D NMR (HSQC and HMBC) were recorded on a Bruker AV-300 spectrometer with tetramethylsilane as an internal standard (Bruker Co.). Electrospray ionization mass spectra (ESI-MS) were performed with Waters 2695-Micromass Qauttro micro and HR-ESI-MS were measured with LCESI-TOF-MS (Agilent Technologies, Palo Alto, CA, USA). Silica gel (100 – 200 mesh, 200 – 300 mesh, Qingdao Marine Chemical Co., Ltd, Qingdao, China), Sephadex LH-20 (Phar-
5
Extraction and isolation
The seeds of V. segetalis (5.0 kg) were extracted three times with 80% ethanol (3 £ 40 l) under reflux for 2 h each time. All the extracts were pooled together and concentrated by a rotary evaporator under reduced pressure to yield a crude extract (999.3 g), which was dissolved and suspended in H2O and then partitioned with petroleum ether, EtOAc, and n-BuOH sequentially. The EtOAc extract (180 g) was separated by silica gel CC eluting with a step gradient of CHCl3/MeOH (100:1 to 2:1) to obtain eight fractions (Fr.1 –Fr.8). Fraction 1 (3.5 g) was chromatographed on a silica gel column eluting with petroleum ether –EtOAc (40:1, 10:1, 6.5:1) to get three subfractions 1a – 1c. Subfraction 1c (0.3 g) was purified by silica gel CC eluting with petroleum ether –EtOAc (8:1) to yield compound 1 (15.4 mg). Fraction 2 (11.2 g) was purified by silica gel CC eluting with petroleum ether – acetone (2:1) to yield compound 2 (28.7 mg).
6
Y.-L. Zhang et al.
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Fraction 5 (20.9 g) was decolorized on an ODS column chromatography eluting with MeOH – H2O (50:50, 40:60, 30:70, 20:80) to afford four subfractions (Fr.5a– 5d). Subfraction Fr.5b (4.3 g) was purified by silica gel CC eluting with CHCl3 – MeOH (14:1) to yield compound 3 (10.3 mg). 3.3.1 Compound 1 A white amorphous powder; UV (MeOH) lmax 284 nm; IR (KBr) nmax (cm21): 3519, 3310, 2955, 2919, 2850, 1639, 1550, 1521, 1463, 1273, 1236, 1158, 1097, 1056, 1123, 1029, 858, 823, 798, 725, 635. For 1H NMR (CDCl3, 300 MHz) and 13C NMR (CDCl 3, 75 MHz) spectral data, see Table 1. HR-ESI-MS: m/z 516.4456 [M – H] – (calcd for C33H58NO3, 516.4417). 3.3.2
Compound 2
A yellow amorphous powder; UV (MeOH) lmax 295 nm; IR (KBr) nmax (cm21): 3303, 3091, 2918, 2849, 1717, 1644, 1555, 1528, 1463, 1279, 1250, 1195, 1115, 957, 719. For 1H NMR (C5D5N, 300 MHz) and 13C NMR (C5D5N, 75 MHz) spectral data, see Table 1. HR-TOF-MS: m/z 502.4308 [M – H] – (calcd for C32H56NO3, 502.4260). 3.3.3 Compound 3 A yellow amorphous powder; ½a20 D 2 25.43 (c ¼ 0.07, DMSO); UV (MeOH) lmax 325, 271 nm; IR (KBr) nmax (cm21): 3448, 3384, 3083, 3012, 2918, 2821, 2616, 2512, 1667, 1619, 1597, 1573, 1546, 1462, 1365, 1246, 1171, 1031, 974, 841. For 1H NMR (DMSO-d6, 300 MHz) and 13C NMR (DMSO-d6, 75 MHz) spectral data, see Table 2. HR-TOF-MS: m/z 413.0875 [M – H] – (calcd for C21H17O9, 413.0873). 3.4 Assay of FXa inhibition activity of the isolated compounds In this paper, the FXa inhibitory activity was measured in a phosphate buffer solution at pH 8.34 using the method of
chromogenic substrate [15]. Each compound was diluted in dimethyl sulfoxide (DMSO). Every compound dilution (0.2 ml) and 20 ml of 2.5 mg/ml enzyme solution (FXa) were added to the prepared buffer and preincubated for 30 min. The reactions were initiated by the addition of 20 ml of 2.5 mg/ml substrate at 378C, and the color was monitored continuously at 405 nm using a Nanodrop 1000 Spectrophotometer (Thermo, Florida, USA), for 5 min (once every 30 s). The absorbancetime curve and the slope of curve (Vi) which reflect the activity of enzyme were observed. Inhibitory effect was calculated according to the equation (V0 – Vi)/V0. IC50 values were calculated from the regression line between inhibitory effect and concentration.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding The work was financially supported by The Jiangsu Provincial Cultivate Project “the Grant of the Peak of Six Major Talent” [grant number 2012-yy-011] and National Natural Science Foundation of China [grant number 81460591].
References [1] Jiangsu New Medical College, Zhongyao-da-ci-dian (Shanghai Science and Technology Publisher, Shanghai, 1986). [2] Pharmacopoeia Commission of People’s Republic of China, Pharmacopoeia of the People’s Republic of China (Chemical Industry Press, Beijing, 2010), Vol. 1. [3] L. Feng, X.P. Zhang, H. Hua, L.Y. Qiu, L.F. Zhang, and Z.W. Lv, Asian Biomed. 6, 683 (2012). [4] H. Morita, Y.S. Yun, K. Takeya, H. Itokawa, K. Yamada, and O. Shirota, Bioorg. Med. Chem. Lett. 7, 1095 (1997). [5] K. Koike, Z.H. Jia, and T. Nikaido, Phytochemistry 47, 1343 (1998). [6] Z.H. Jia, K. Koike, M. Kudo, H.Y. Li, and T. Nikaido, Phytochemistry 48, 529 (1998).
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[7] Y.S. Yun, K. Shimizu, H. Morita, K. Takeya, H. Itokawa, and O. Shirota, Phytochemistry 47, 143 (1998). [8] S.M. Sang, A.N. Lao, Y. Leng, Z.P. Gu, Z.L. Chen, J. Uzawa, and Y. Fujimoto, Tetrahedron Lett. 41, 9205 (2000). [9] H. Morita, Y.S. Yun, K. Takeya, H. Itokawa, and O. Shirota, Phytochemistry 42, 439 (1996). [10] H.X. Zhang, L.Z. Min, and M.S. Sheng, Chin. Chem. Lett. 15, 55 (2004). [11] H. Morita, M. Eda, T. Iizuka, Y. Hirasawa, M. Sekiguchi, Y.S. Yun, H. Itokawa, and
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K. Takeya, Bioorg. Med. Chem. Lett. 16, 4458 (2006). S. Shen, Q. Wang, and Y.B. Li, Inf. Tradit. Chin. Med. 29, 20 (2012). K. Kenji, S. Kazumi, T. Hitomi, N. Aki, O. Hiromi, T. Ikue, and M. Ken, J. Agric. Food Chem. 58, 3627 (2010). P.C. Zhang, Y.J. Zhou, and S.X. Xu, J. Asian Nat. Prod. Res. 3, 77 (2001). L.L. Jiang, Q. Wang, S. Shen, T.S. Xiao, and Y.B. Li, Thromb. Res. 3, 501 (2014).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Three new compounds isolated from the seeds of Vaccaria segetalis ab
b
c
b
Yan-Ling Zhang , Li-Long Jiang , Tong-Shu Xiao , Shou-Qian Chen ac
& You-Bin Li a
School of Pharmacy, Hainan Medical University, Haikou 571101, China b
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046, China c
Click for updates
Department of Phytochemistry, Jiangsu Provincial Institute of Traditional Chinese Medicine, Nanjing 210028, China Published online: 03 Jan 2015.
To cite this article: Yan-Ling Zhang, Li-Long Jiang, Tong-Shu Xiao, Shou-Qian Chen & You-Bin Li (2015): Three new compounds isolated from the seeds of Vaccaria segetalis, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.992887 To link to this article: http://dx.doi.org/10.1080/10286020.2014.992887
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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Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2014 http://dx.doi.org/10.1080/10286020.2014.992887
Three new compounds isolated from the seeds of Vaccaria segetalis Yan-Ling Zhanga,b, Li-Long Jiangb, Tong-Shu Xiaoc, Shou-Qian Chenb and You-Bin Lia,c* a School of Pharmacy, Hainan Medical University, Haikou 571101, China; bSchool of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046, China; cDepartment of Phytochemistry, Jiangsu Provincial Institute of Traditional Chinese Medicine, Nanjing 210028, China
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(Received 27 July 2014; final version received 25 November 2014) Two new capsaicin analogs, N-(3-methoxy-4-hydroxyphenethyl)-tetracosanamide (1) and N-(3,4-dihydroxyphenethyl)-tetracosanamide (2), along with one new flavonoidal glycoside pinnatifin E (3) were isolated from the ethanolic extract of the seeds of Vaccaria segetalis. Their structures were elucidated on the basis of spectroscopic methods including 1D, 2D NMR, MS, and other spectroscopic techniques, as well as by comparison with the relevant literatures. All compounds were evaluated for their coagulation Factor Xa inhibition activities. Keywords: Vaccaria segetalis; N-(3-methoxy-4-hydroxyphenethyl)-tetracosanamide; N-(3,4-dihydroxyphenethyl)-tetracosanamide; pinnatifin E; FXa inhibition activity
1. Introduction Vaccaria segetalis (Neck.) Garcke (Caryophyllaceae) is an annual herb that is distributed all over China, except southern China [1]. The seeds of V. segetalis, which are known as Wang-Bu-Liu-Xing, have been used as a common folk medicine in China for the treatment of amenorrhea, dysmenorrhea, and breast milk stoppage [2]. The extract of V. segetalis demonstrates various bioactivities, such as the promotion of dieresis and milk secretion, activation of blood circulation, and the relief of carbuncle [3]. Previous phytochemical studies revealed the presence of triterpenoid saponins [4 –9] and cyclic peptides [10,11] in the seeds of V. segetalis. In the previous study, we screened the potential inhibitory activity of 24 species of those plants that were used as traditional Chinese medicine for the treatment of thrombosis. We demonstrated that the ethanol extract of the seeds of V. segetalis possessed inhibitory activity
against the Factor Xa (FXa) [12]. During the course of our search for bioactive ingredients from the traditional medicine, two new capsaicin analogs (1 and 2) and one new flavonoidal glycoside (3) (Figure 1) were isolated from the ethanolic extract of the seeds of V. segetalis. In this paper, we report the structure elucidation of the new compounds and their inhibitory activities against FXa.
2.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Results and discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula C33H59NO3 was deduced from the pseudomolecular ion peak at m/z 516.4456 [M – H] – in the negative HR-ESI-MS. The IR spectrum showed the characteristic absorptions of amidogen (3310 cm21), hydroxy (3519, 1029 cm21), and methyl (2919, 2850 cm21) groups. The 1H NMR spectrum (Table 1) displayed signals for a 1,3,4-trisubstituted aromatic ring at d 6.84
2
Y.-L. Zhang et al. OH
24'
RO
3
HO
4
2
1 6
7
8
H N
6'' 1'
HO 2'
O
5
1 R = CH3 2 R=H
5''
4''
HO
O O
O
7
2'' 3''
1''
OH
6
5
OH O
3
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Figure 1. Structures of compounds 1 –3.
(1H, d, J ¼ 7.8 Hz), 6.69 (1H, s), and 6.67 (1H, d, J ¼ 7.8 Hz); a methoxy signal at d 3.87 (3H, s); and aliphatic group signals at d 1.59 (2H, m), 1.20 – 1.40 (40H), and 0.89 (3H, t, J ¼ 6.9 Hz). In the 13C NMR spectrum (Table 1), a set of typical carbon signals at d 146.7 (C-3), 144.3 (C-4), 130.7 (C-1), 121.4 (C-6), 114.4 (C-5), and 111.2 (C-2) revealed the presence of a benzene ring. Signal at d 55.9 revealed the presence of a methoxy group. In addition, signals at d 173.1, 36.9, 29.7 (multiplet), 25.8, and 14.1 were assigned as a long-chain fattyacyl group. These NMR data clearly indicated that compound 1 was an analog of capsaicinoids. Comparing the NMR data of 1 with those of N-vanillylhexadecanamide [13], two main differences were unambiguously observed: (1) Rather than a 3-methoxy-4-hydroxybenzyl amine substituent in N-vanillyl-hexadecanamide, 1 contained a 3-methoxy-4hydroxy-phenethyl amine substituent. It was further supported by the HMBC correlations from H-7 at dH 2.74 (2H, t, J ¼ 6.9 Hz) to C-1 at dC 130.7, C-6 at dC 121.4, C-2 at dC 111.2, and C-8 at dC 40.6; and H-8 at dH 3.48 (2H, m) to C-10 at dC 173.1, C-1 at dC 130.7, and C-7 at dC 35.4. (2) Combined with mass spectrometry, the differences just from the chain length, the molecule of 1 was 126 larger than that of N-vanillyl-hexadecanamide, which weighed nine CH2 units. Except one more CH2 in the 3-methoxy-4-hydroxyphenethyl amine substituent in 1, the left eight CH2 were determined to be the fatty acid part that was elucidated as lignoceric acid and were confirmed by the detailed
analyses of the HMBC correlations (Table 1). Thus, the structure of 1 was elucidated as N-(3-methoxy-4-hydroxyphenethyl)-tetracosanamide and shown in Figure 1. Compound 2 was isolated as a yellow powder. Its molecular formula was established as C32H57NO3 deduced from the negative HR-ESI-MS at m/z 502.4308 [M– H] – . The IR spectrum showed the characteristic absorptions of amidogen (3303 cm21), hydroxy group (3091 cm21), and carboxyl group (1717 cm21). It was found to have a similar structure to compound 1 by comparison of their 1H and 13C NMR spectral data (Table 1), the main difference observed was the absence of a methyl signal at d 3.87 (3H, s) in the 1H NMR spectrum and a carbon signal at d 55.9 in the 13C NMR spectrum of 1. After careful analysis of the data, the result showed that there was a hydroxy group in 2 instead of a methoxy group in 1, which was confirmed by the obvious down-fielded shift of C-2 and C-4. Therefore, compound 2 was established to be N-(3,4-dihydroxyphenethyl)-tetracosanamide. Compound 3 was also obtained as a yellow powder with ½a20 2 25.43 D (c ¼ 0.07, DMSO) and positive to Mg – HCl color reaction. Its molecular formula, C21H18O9, was determined by negative HR-TOF-MS at m/z 413.0875. The IR spectrum of 3 showed the characteristic absorptions attributable to hydroxy groups (3384 cm21), conjugated carbonyl group (1667 cm21), and aromatic rings (1619 and 1546 cm21). These data suggested that compound 3 may be a flavone. In the 1H
130.7 (C) 111.2 (CH) 146.7 (C) 144.3 (C) 114.4 (CH) 121.4 (CH) 35.4 (CH2) 40.6 (CH2) 173.1 (C) 36.9 (CH2) 25.8 (CH2) 29.5 (CH2) , 29.7 (CH2) 31.9 (CH2) 22.7 (CH2) 14.1 (CH3) 55.9 (CH3)
1 2 3 4 5 6 7 8 10 20 30 40 50 – 210 220 230 240 OCH3
– 6.69 (1H, s) – – 6.84 (1H, d, 7.8) 6.67 (1H, d, 7.8) 2.74 (2H, t, 6.9) 3.45– 3.52 (2H, m) – 2.11 (2H, t, 7.5) 1.58– 1.60 (2H, m) 1.20– 1.40 1.20– 1.40 1.20– 1.40 1.20– 1.40 0.89 (3H, t, 6.9) 3.87 (3H, s)
dH
Compound 1 (CDCl3)
– C-3, C-4, C-6 – – C-1, C-3, C-4 C-2, C-4 C-1, C-2, C-6, C-8 C-1, C-7, C-10 – C-10 , C-30 – – – – – C-220 , C-230 C-3
HMBC 131.5 (C) 117.2 (CH) 147.2 (C) 145.6 (C) 116.5 (CH) 120.3 (CH) 35.9 (CH2) 41.7 (CH2) 173.1 (C) 36.7 (CH2) 26.3 (CH2) 30.0 (CH2) , 29.8 (CH2) 32.1 (CH2) 22.9 (CH2) 14.2 (CH3) –
dC (DEPT)
C NMR (75 MHz) and 1H NMR (300 MHz) spectral data of 1 and 2 (d in ppm, J in Hz).
dC (DEPT)
13
Position
Table 1.
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– 7.18 (1H, s) – – 7.14 (1H, d, 7.8) 6.76 (1H, d, 7.8) 2.90 (2H, t, 6.9) 3.68 – 3.75 (2H, m) – 2.33 (2H, t, 7.5) 1.75 – 1.79 (2H, m) 1.10 – 1.40 1.10 – 1.40 1.10 – 1.40 1.10 – 1.40 0.83 (3H, t, 6.3) –
dH
Compound 2 (C5D5N)
C-220 , C-230
C-10 , C-30
C-1, C-3, C-4 C-2, C-4, C-7 C-1, C-2, C-6, C-8 C-1, C-7, C-10
C-3, C-4, C-6
HMBC
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4 Table 2. in Hz).
Y.-L. Zhang et al. 13
C NMR (75 MHz) and 1H NMR (300 MHz) spectral data of 3 in DMSO-d6 (d in ppm, J
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Compound 3 Position
dC (DEPT)
dH
HMBC
2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100
164.1 (C) 102.9 (CH) 181.1 (C) 155.4 (C) 107.2 (C) 163.9 (C) 89.3 (CH) 157.2 (C) 104.7 (C) 121.1 (C) 128.5 (CH) 116.0 (CH) 161.2 (C) 116.0 (CH) 128.5 (CH) 32.7 (CH2)
200 300 400 500 600
117.9 (C) 79.7 (CH) 73.7 (CH) 83.8 (CH) 63.3 (CH2)
– 6.82 (1H, s) – – – – 6.64 (1H, s) – – – 7.93 (1H, d, 8.7) 6.93 (1H, d, 8.7) – 6.93 (1H, d, 8.7) 7.93 (1H, d, 8.7) 3.05 (1H, d, 16.8) 3.27 (1H, d, 16.8) – 3.99 (1H, d, 8.1) 3.90 (1H, m) 3.80 (1H, m) 3.62 (1H, m) 3.47 (1H, m) 13.23 (s) 10.37 (s)
– C-2, C-4, C-10, C-10 – – – – C-6, C-7, C-9, C-10 – – – C-2, C-30 , C-40 , C-60 C-10 , C-40 , C-50 – C-10 , C-40 , C-30 C-2, C-20 , C-50 , C-40 C-6, C-300 , C-5, C-7 C-6, C-200 , C-5, C-7 – C-400 C-300 , C-600 C-400 C-500 C-500 C-5, C-6, C-10 –
5-OH 40 -OH
– –
NMR spectrum (Table 2), a characteristic downfield singlet at d 13.23 confirmed the presence of 5-OH, and two sets of doublets at d 7.93 (2H, d, J ¼ 8.7 Hz), and 6.93 (2H, d, J ¼ 8.7 Hz) displayed AA0 BB0 system signals of flavonoid ring B and were assigned to H-20 /60 and H-30 /50 , respectively. In addition, two singlets at d 6.82 and 6.64 were due to the protons in rings A and C. In the 13C NMR spectrum (Table 2), 21 carbon signals were observed, which included 15 carbon signals from the aglycone moiety and a set of carbon signals of the sugar moiety resonating at d 117.9 (C), 83.8 (CH), 79.7 (CH), 73.7 (CH), 63.3 (CH2), and 32.7 (CH2). These data showed that compound 3 was closely related to the known compound pinnatifin C [14]. The significant difference was the NMR spectral data
of flavonoid ring A. Careful analysis of the NMR data suggested that 3 should have different substitution pattern compared with pinnatifin C. In the HMBC spectrum (Table 2), the correlations from H-8 at dH 6.64 (1H, s) to C-7 at dC 163.9, C-9 at dC 157.2, C-6 at dC 107.2, and C-10 at dC 104.7; and from H-100 a at dH 3.05 (1H, d, J ¼ 16.8 Hz), H-100 b at dH 3.27 (1H, d, J ¼ 16.8 Hz) to C-6 at dC 107.2, C-200 at dC 117.9, and C-300 at dC 79.7 confirmed the link position between sugar moiety and flavonoid skeleton was at C-6 in 3 rather than C-8 in pinnatifin C. Additionally, the configuration of sugar moiety was the same as that of pinnatifin C from the NOESY spectrum and its optical rotation. In the NOESY spectrum, the cross-peaks between protons at d 3.99 (1H, d, J ¼ 8.1 Hz, H-300 ) and 3.80 (1H, m, H-500 ),
Journal of Asian Natural Products Research Table 3. Inhibitory enzymatic activity of compounds 1 – 3 against FXa. Compounds
263 ^ 3.36 368 ^ 4.91 165 ^ 8.37
1 2 3
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IC50 (mmol/l)
and at d 3.90 (1H, m, H-400 ) and 3.62 (1H, m, H-600 a) as well as the optical rotation of ½a20 D 2 25.43 (c ¼ 0.07, DMSO) allowed the configuration of sugar moiety as b-D frucofurariose. In conclusion, the structure of 3 was characterized as depicted in Figure 1 and named pinnatifin E. In this study, the isolated compounds were tested for their FXa inhibition activities. The result is presented in Table 3, and new compounds showed weak inhibition activity to FXa with the IC50 of 263 ^ 3.36, 368 ^ 4.91, and 165 ^ 8.37 mmol/l (all P , 0.01), respectively.
macia Biotech AB, Uppsala, Sweden), and octadecyl silica (ODS; Merck, Darmstadt, Germany) were used for column chromatography (CC), while precoated silica gel GF254 plates were used for thin layer chromatography (Qingdao Marine Chemical Co., Ltd, Qingdao, China). All solvents used were of analytical grade (Jiangsu Hanbang Science and Technology, Co., Ltd, China). 3.2 Plant material The seeds of V. segetalis were collected in Bozhou Country of Anhui Province, China, in March 2011 and identified by Prof. Shi-Hui Qian (Jiangsu Province Academy of Traditional Chinese Medicine). A voucher specimen (No. S-1300002) was deposited in the Department of Traditional Chinese Medicine Materials, Jiangsu Province Institute of Traditional Chinese Medicine, Jiangsu, China. 3.3
3. 3.1
Experimental General experimental procedures
Optical rotation was obtained on an Autopl IV polarimeter (Rudolph Research Analytical, Flanders, NJ, USA). UV spectra were measured on a Shimadzu UV-210 spectrometer (Shimadzu, Kyoto, Japan). IR spectra were recorded on a Bruker Tensor 27 spectrometer (Bruker Co., 1 Ettlingen, Germany). H NMR 13 (300 MHz), C NMR (75 MHz) spectra and 2D NMR (HSQC and HMBC) were recorded on a Bruker AV-300 spectrometer with tetramethylsilane as an internal standard (Bruker Co.). Electrospray ionization mass spectra (ESI-MS) were performed with Waters 2695-Micromass Qauttro micro and HR-ESI-MS were measured with LCESI-TOF-MS (Agilent Technologies, Palo Alto, CA, USA). Silica gel (100 – 200 mesh, 200 – 300 mesh, Qingdao Marine Chemical Co., Ltd, Qingdao, China), Sephadex LH-20 (Phar-
5
Extraction and isolation
The seeds of V. segetalis (5.0 kg) were extracted three times with 80% ethanol (3 £ 40 l) under reflux for 2 h each time. All the extracts were pooled together and concentrated by a rotary evaporator under reduced pressure to yield a crude extract (999.3 g), which was dissolved and suspended in H2O and then partitioned with petroleum ether, EtOAc, and n-BuOH sequentially. The EtOAc extract (180 g) was separated by silica gel CC eluting with a step gradient of CHCl3/MeOH (100:1 to 2:1) to obtain eight fractions (Fr.1 –Fr.8). Fraction 1 (3.5 g) was chromatographed on a silica gel column eluting with petroleum ether –EtOAc (40:1, 10:1, 6.5:1) to get three subfractions 1a – 1c. Subfraction 1c (0.3 g) was purified by silica gel CC eluting with petroleum ether –EtOAc (8:1) to yield compound 1 (15.4 mg). Fraction 2 (11.2 g) was purified by silica gel CC eluting with petroleum ether – acetone (2:1) to yield compound 2 (28.7 mg).
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Fraction 5 (20.9 g) was decolorized on an ODS column chromatography eluting with MeOH – H2O (50:50, 40:60, 30:70, 20:80) to afford four subfractions (Fr.5a– 5d). Subfraction Fr.5b (4.3 g) was purified by silica gel CC eluting with CHCl3 – MeOH (14:1) to yield compound 3 (10.3 mg). 3.3.1 Compound 1 A white amorphous powder; UV (MeOH) lmax 284 nm; IR (KBr) nmax (cm21): 3519, 3310, 2955, 2919, 2850, 1639, 1550, 1521, 1463, 1273, 1236, 1158, 1097, 1056, 1123, 1029, 858, 823, 798, 725, 635. For 1H NMR (CDCl3, 300 MHz) and 13C NMR (CDCl 3, 75 MHz) spectral data, see Table 1. HR-ESI-MS: m/z 516.4456 [M – H] – (calcd for C33H58NO3, 516.4417). 3.3.2
Compound 2
A yellow amorphous powder; UV (MeOH) lmax 295 nm; IR (KBr) nmax (cm21): 3303, 3091, 2918, 2849, 1717, 1644, 1555, 1528, 1463, 1279, 1250, 1195, 1115, 957, 719. For 1H NMR (C5D5N, 300 MHz) and 13C NMR (C5D5N, 75 MHz) spectral data, see Table 1. HR-TOF-MS: m/z 502.4308 [M – H] – (calcd for C32H56NO3, 502.4260). 3.3.3 Compound 3 A yellow amorphous powder; ½a20 D 2 25.43 (c ¼ 0.07, DMSO); UV (MeOH) lmax 325, 271 nm; IR (KBr) nmax (cm21): 3448, 3384, 3083, 3012, 2918, 2821, 2616, 2512, 1667, 1619, 1597, 1573, 1546, 1462, 1365, 1246, 1171, 1031, 974, 841. For 1H NMR (DMSO-d6, 300 MHz) and 13C NMR (DMSO-d6, 75 MHz) spectral data, see Table 2. HR-TOF-MS: m/z 413.0875 [M – H] – (calcd for C21H17O9, 413.0873). 3.4 Assay of FXa inhibition activity of the isolated compounds In this paper, the FXa inhibitory activity was measured in a phosphate buffer solution at pH 8.34 using the method of
chromogenic substrate [15]. Each compound was diluted in dimethyl sulfoxide (DMSO). Every compound dilution (0.2 ml) and 20 ml of 2.5 mg/ml enzyme solution (FXa) were added to the prepared buffer and preincubated for 30 min. The reactions were initiated by the addition of 20 ml of 2.5 mg/ml substrate at 378C, and the color was monitored continuously at 405 nm using a Nanodrop 1000 Spectrophotometer (Thermo, Florida, USA), for 5 min (once every 30 s). The absorbancetime curve and the slope of curve (Vi) which reflect the activity of enzyme were observed. Inhibitory effect was calculated according to the equation (V0 – Vi)/V0. IC50 values were calculated from the regression line between inhibitory effect and concentration.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding The work was financially supported by The Jiangsu Provincial Cultivate Project “the Grant of the Peak of Six Major Talent” [grant number 2012-yy-011] and National Natural Science Foundation of China [grant number 81460591].
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