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New coumarin derivative from Euphorbia wallichii a
b
a
a
Wen-Hui Xu , Yun-Heng Shen , Qian Liang & Ping Zhao a
Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming 650224, P.R. China b
Department of Phytochemistry, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, P.R. China Published online: 23 Jan 2015.
Click for updates To cite this article: Wen-Hui Xu, Yun-Heng Shen, Qian Liang & Ping Zhao (2015): New coumarin derivative from Euphorbia wallichii, Natural Product Research: Formerly Natural Product Letters To link to this article: http://dx.doi.org/10.1080/14786419.2014.1003930
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2014.1003930
SHORT COMMUNICATION New coumarin derivative from Euphorbia wallichii Wen-Hui Xua, Yun-Heng Shenb, Qian Lianga* and Ping Zhaoa* a Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming 650224, P.R. China; bDepartment of Phytochemistry, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, P.R. China
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(Received 12 November 2014; final version received 30 December 2014)
One new coumarin derivative (1) and two known compounds, quercetin (2) and glyceraldehyde (3) have been isolated from the whole plants of Euphorbia wallichii. Their structures were elucidated by means of extensive spectroscopic analysis (NMR and ESI-MS) and by comparison with data reported in the literature. This is the first isolation of dihydrocoumarin (1) from the genus of Euphorbia. Keywords: Euphorbia wallichii; coumarin derivative
1. Introduction Euphorbia is the largest genus of the plant family Euphorbiaceae, with approximately 2000 species (Shi et al. 2008). Several species within the genus Euphorbia have been studied chemically (Duarte and Ferreira 2007; Liu et al. 2012; Zhao et al. 2014). The characteristic compounds include diterpenoid, and triterpenoid derivatives. There is a very rare report of coumarin compounds from the genus Euphorbia (Shi et al. 2008). Euphorbia wallichii is a perennial shrub distributed in China, India, Nepal and Kashmir regions. The plant is used as a Chinese folk medicine for the treatment of oedema and skin disease (Pan et al. 2006). Previous phytochemical studies on E. wallichii led to the isolation of a number of structurally diverse diterpenoids (Wang et al. 2004; Zhang et al. 2006) and triterpenoids (Ali et al. 2008). In this study, one new coumarin derivative (1) and two known compounds (2 and 3) (Figure 1) have been isolated. Herein we report the isolation and structure elucidation of the new compound 1.
2. Results and discussion Compound 1, an amorphous colourless powder, possesses the molecular formula C20H22O11, corresponding to 10 degrees of unsaturation, which was established by high-resolution ESI-MS (m/z 461.1046 calcd. for [M þ Na]þ, 461.1054), and 20 carbon signals were observed in the 13C
*Corresponding authors. Email: [email protected]; [email protected] q 2015 Taylor & Francis
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Figure 1. Structures of isolated compounds 1 –3 from E. wallichii.
NMR spectrum. A DEPT NMR experiment permitted differentiation of the 20 resonances into 3 methyls, 3 methylenes, 4 methines and 10 quaternary carbons. The 1H NMR spectrum of 1 in acetone-d6 indicated that the three methyl groups resonate at dH 1.15, 1.16 and 1.25 (3H each), which are correlated with the carbons at dC 14.1 (C-60 ), 14.3 (C-40 ) and 14.4 (C-20 ), respectively, in the HMQC spectrum. The 13C NMR spectrum of 1 showed that the three methyleneoxy carbons at dC 61.8 (C-10 ), 62.3 (C-30 ) and 62.4 (C-50 ) are correlated with the protons at 4.05 (2H, m), 4.08 (2H, m) and 4.13 (2H, m), respectively, in the HMQC spectrum. The presence of three ethyl carboxylate moieties in 1 was evident from the 1H – 1H correlations of 1.12 (3H, m, Me-40 )/ 4.08 (2H, m, H-30 ), 1.15 (3H, t, Me-60 )/4.13 (2H, m, H-50 ) and 1.25 (3H, m, Me-20 )/4.05 (2H, m, H-10 ) in the COSY spectrum. This was further supported by the long range H – C correlations of 4.05 (H-10 )/165.8 (C-12), 4.08 (H-30 )/166.6 (C-13) and 4.13 (H-50 )/170.0 (C-14), respectively, in the HMBC spectrum. The 13C NMR spectrum contains six aromatic carbons at dC 108.5 (CH, C1), 116.1 (qC, C-6), 129.8 (CH, C-4), 138.7 (qC, C-2), 143.1 (qC, C-5) and 145.8 (qC, C-3), which indicates the presence of a benzene ring system. This was further supported by HMBC correlations between aromatic protons at dH 6.80 (s, H-4) with dC 143.1 (C-5), dH 7.13 (s, H-1) with dC 116.1 (C-6), 138.7 (C-2) and 145.8 (C-3). The methane proton at 5.26 (1H, d, H-8) was correlated to two aromatic carbons at 116.1 (C-6), 143.1 (C-5), ester carbonyl carbon at 163.8 (C-7) and methine carbon at 35.3 (C-9) in the HMBC spectrum, which gave a dihydrocoumarinlike skeleton (Chen et al. 2008). The presence of an olefinic bond moiety in 1 was evident from the residual two resonances at d 118.6 (qC, C-10) and 143.7 (qC, C-11). On the basis of above analysis, we believed initially compound 1 to be an dihydrocoumarin-like skeleton. The 2D NMR spectra were in turn used to determine the linkage of the olefinic bond and three ethyl carboxylate moieties to dihydrocoumarin skeleton. The double bond was located at 35.3 (C-9) of the dihydrocoumarin skeleton by the long range H – C correlation between the methine proton at 5.45 (1H, d, H-9) and two additional olefinic carbons at 118.6 (C-10) and 143.7 (C-11). The methine protons at d 5.26 (H-8) and 5.45 (H-9) showed key correlations with carbonyl at d 170.0 (C-14) in the HMBC spectrum, which confirmed that the ethyl carboxylate moiety was attached at 79.3 (C-8) of the dihydrocoumarin skeleton. The critical long-range correlation between the methine proton of the dihydrocoumarin skeleton at 5.45 (H-9) and the carbonyl at d 166.6 (C-13) indicated that another ethyl carboxylate unit was linked to olefinic carbon at 118.6 (C-10). Additional ethyl carboxylate moiety was placed at the oxygenated olefinic carbon at 143.7 (C11) according to published literature (Chen et al. 2008). The ROESY spectrum was employed to determine the relative configuration of 1. The NOE correlations from 5.45 (H-9a) to 1.25 (Me20 ) confirmed the cis orientation double bond between 118.6 (C-10) and 143.7 (C-11). The strong NOE correlation between 5.45 (H-9a) and 5.26 (H-8) and a relatively small coupling constant (J ¼ 1.5 Hz) between H-9a and H-8 indicate a-configuration of H-8 (Lee et al. 2007; Chen et al.
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2008). The full 1H and 13C NMR assignments were established by means of a combination of COSY, HMQC, HMBC and ROESY spectral measurements (Supporting Information Table S1 and Figure S10). Thus, the structure of 1 was established as 8-ethyl carboxylate, 9-di-ethyl carboxylate-10-ol-10,11-Z-2,3-dihydroxy dihydrocoumarin. It is notable that compound 1 is the first dihydrocoumarin example from Euphorbia genus, while coumarin compounds are rare and only isolated from E. lunulata, E. quinquecostata (Shi et al. 2008), E. Lagascae (Duarte et al. 2008) and E. portlandica (Madureira et al. 2004). The known compounds were identified by comparison of their ESI-MS and NMR spectroscopic data with those reported in the literature as quercetin (2) (Maria et al. 1984), D -glyceraldehyde (3) (Tu 2012). All of them are isolated from E. wallichii for the first time.
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3. Experimental 3.1. General The 1D and 2D NMR (COSY, HMQC, HMBC, ROESY) spectra using standard pulse programmes were recorded at room temperature on a Bruker Avance DRX 500 FT spectrometer operating at 500 (1H) or 125 (13C) MHz. The chemical shift values are relative to the internal standard TMS. HR-ESI-MS data were obtained on an Agilent Series 1100 SL mass spectrometer. Column chromatography was performed using normal phase silica gel (200 2 300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China) and reversed-phase silica gel (RP-18, YMC, 40 –60 mm, Fuji Silysia Chemical Ltd., Kasugai, Japan). TLC was carried out on silica gel sheets (Qingdao Marine Chemical, Inc.) and reversed-phase plates (RP-18 F254S, Merck, Darmstadt, Germany). Visualisation: UV at 254 nm or 10% H2SO4 followed by heating. 3.2. Plant material The whole plants of E. wallichii were collected in Lijiang, Yunnan Province, China, in August 2011, and identified by Prof. Fan Du. A voucher specimen (No. 110801) is deposited in the Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Southwest Forestry University, Kunming City, Yunnan Province, China. 3.3. Extraction and isolation Powdered, air-dried whole plants of E. wallichii (15.0 kg) were exhaustively extracted with 95% EtOH (20 L £ 3) at room temperature for 72 h. The extraction was evaporated to dryness in vacuo. The residue (710 g) was suspended in H2O and then partitioned with petroleum ether and EtOAc, respectively. The EtOAc layer was evaporated to dryness and the resultant residue (390.0 g) was subjected to silica gel liquid chromatography (9 cm £ 70 cm) using stepwise gradient elution CHCl3/MeOH at 100:0 (7600 mL), 200:1 (3100 mL), 100:1 (3000 mL), 50:1 (5900 mL), 25:1 (3200 mL), 10:1 (5300 mL), 5:1 (3000 mL), 2:1 (1600 mL) and finally with MeOH (2000 mL) to afford 17 pooled fractions A –Q according to TLC. Fraction L (61.0 g) was purified on silica gel column chromatography (5 cm £ 65 cm) eluting with petroleum ether/acetone at 8:1 (3700 mL), 6:1 (3000 mL), 4:1 (4000 mL), 3:1 (3000 mL), 2:1 (4700 mL) and 1:1 (3900 mL) to give 10 pooled fractions (1 –10). Fraction 8 was further purified on a C18 reversed-phase column (4 cm £ 60 cm) eluting with MeOH/H2O at 60:40 (1500 mL), 70:30 (1200 mL), 80:20 (1200 mL), 90:10 (1500 mL) and 95:5 (500 mL) to give 1 (49.7 mg, eluted with MeOH/H2O 60:40). Compound 2 (68.0 mg) was got by recrystallisation from Fraction M in CHCl3/Acetone (1:9). Fraction O (20.0 g) was further purified on silica gel column chromatography (4.5 cm £ 65 cm) eluting with CHCl3/acetone at 5:1 (1300 mL), 3:1 (1000 mL), 1:1 (3900 mL) and 0:1 (800 mL) to afford 3 (10.0 mg, eluted with CHCl3/acetone 1:1).
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3.4. Spectroscopic data Compound 1: a colourless amorphous powder, Rf ¼ 0.64 (silica gel TLC with petroleum ether/acetone, 1:1), ESI-MS at 439 for [M þ H]þ, 461 for [M þ Na] þ, 477 for [M þ K]þ, HRESI-MS found at m/z 461.1046 calcd. for [M þ Na] þ, 461.1054, 1H (500 MHz) and 13C (125 MHz) NMR data for compound 1 in acetone-d6 (d ppm, J in Hz). 1H NMR: 7.13 (s, 1H, H1), 6.80 (s, 1H, H-4), 5.26 (d, 1H, J ¼ 1.5 Hz, H-8), 5.45 (d, 1H, J ¼ 1.5 Hz, H-9), 4.05 (m, 2H, H-10 ), 1.25 (t, 3H, J ¼ 7.0 Hz, H-20 ), 4.08 (m, 2H, H-30 ), 1.12 (m, 3H, H-40 ), 4.13 (m, 2H, H-50 ), 1.15 (m, 3H, H-60 ); 13C NMR: 108.5 (d, C-1), 138.7 (s, C-2), 145.8 (s, C-3), 129.8 (d, C-4), 143.1 (s, C-5), 116.1 (s, C-6), 163.8 (s, C-7), 79.3 (d, C-8), 35.3 (d, C-9), 118.6 (s, C-10), 143.7 (s, C11), 165.8 (s, C-12), 166.6 (s, C-13), 170.0 (s, C-14), 61.8 (t, C-10 ), 14.4 (q, C-20 ), 62.3 (t, C-30 ), 14.3 (q, C-40 ), 62.4 (t, C-50 ), 14.1 (q, C-60 ). 4. Conclusion We studied the chemical constituents of the E. wallichii and reported the isolation and structural elucidation of one new coumarin derivative (1), together with two known compounds, quercetin (2) and glyceraldehyde (3). This is the first isolation of dihydrocoumarin (1) from the genus of Euphorbia. Supplementary material Supplementary materials relating to this article are available online, alongside Figures S1 – S14 and Table S1. Acknowledgements The authors thank Prof. Fan Du for providing the plant material.
Funding This work was supported by the Natural Science Foundation of China (NSFC) [grant number 31160075], [grant number 21362035].
References Ali MS, Ahmed S, Saleem M. 2008. Spirowallichiione: a rearranged multiflorane from Euphorbia wallichii Hook F. (Euphorbiaceae). Molecules. 13:405–411. Chen R, Liang JY, Lu HY, Yang Y, Liu R. 2008. Study on chemical constituents of the leaves of Canarium album (Lour.) Raeusch. Chem Ind Forest Prod. 27:45–48. Duarte N, Ferreira MJU. 2007. Lagaspholones A and B: two new jatropholane-type diterpenes from Euphorbia lagascae. Org Lett. 9:489–492. Duarte N, Kayser O, Abreu P, Ferreira MU. 2008. Antileishmanial activity of piceatannol isolated from Euphorbia lagascae seeds. Phytother Res. 22:455–457. Lee HS, Jung SH, Yun BS, Lee KW. 2007. Isolation of chebulic acid from Terminalia chebula Retz. and its antioxidant eVect in isolated rat hepatocytes. Arch Toxicol. 81:211–218. Liu JH, Latif A, Ali M, Zhang GP, Xiang WJ, Ma L, Arfan M, Hu LH. 2012. Diterpenoids from Euphorbia neriifolia. Phytochemistry. 75:153–158. Madureira AM, Molnar A, Abreu PM, Molnar J, Ferreira MJU. 2004. A new sesquiterpene-coumarin ether and a new abietane diterpene and their effects as inhibitors of p-glycoprotein. Planta Med. 70:828–833. Maria TP, Eliseo S, Amparo T. 1984. Flavones, sesquiterpene lactones and glycosides isolated from Centaurea aspera var. Stenophylla. Phytochemistry. 23:1995– 1998. Pan L, Zhou P, Zhang XF, Peng SL, Ding LS, Qiu SX. 2006. Skeleton-rearranged pentacyclic diterpenoids possessing a cyclobutane ring from Euphorbia wallichii. Org Lett. 8:2775–2778.
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Shi QW, Su XH, Kiyota H. 2008. Chemical and pharmacological research of the plants in genus Euphorbia. Chem Rev. 108:4295–4327. Tu PF. 2012. Natural sugar chemistry. Beijing: Editorial Committee of Natural Products Chemistry. Wang H, Zhang XF, Cai XH, Ma YB, Luo XD. 2004. Three new diterpenoids from Euphorbia wallichii. Chin J Chem. 22:199–202. Zhang XF, Wang H, Sheng JW, Luo XD. 2006. A new guaiane diterpenoid from Euphorbia wallichii. Nat Prod Res. 20:89–92. Zhao JX, Liu CP, Qi WY, Han ML, Han YS, Wainberg MA, Yue JM. 2014. Eurifoloids A-R, structurally diverse diterpenoids from Euphorbia neriifolia. J Nat Prod. 77:2224–2233.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
New coumarin derivative from Euphorbia wallichii a
b
a
a
Wen-Hui Xu , Yun-Heng Shen , Qian Liang & Ping Zhao a
Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming 650224, P.R. China b
Department of Phytochemistry, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, P.R. China Published online: 23 Jan 2015.
Click for updates To cite this article: Wen-Hui Xu, Yun-Heng Shen, Qian Liang & Ping Zhao (2015): New coumarin derivative from Euphorbia wallichii, Natural Product Research: Formerly Natural Product Letters To link to this article: http://dx.doi.org/10.1080/14786419.2014.1003930
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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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2014.1003930
SHORT COMMUNICATION New coumarin derivative from Euphorbia wallichii Wen-Hui Xua, Yun-Heng Shenb, Qian Lianga* and Ping Zhaoa* a Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming 650224, P.R. China; bDepartment of Phytochemistry, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, P.R. China
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(Received 12 November 2014; final version received 30 December 2014)
One new coumarin derivative (1) and two known compounds, quercetin (2) and glyceraldehyde (3) have been isolated from the whole plants of Euphorbia wallichii. Their structures were elucidated by means of extensive spectroscopic analysis (NMR and ESI-MS) and by comparison with data reported in the literature. This is the first isolation of dihydrocoumarin (1) from the genus of Euphorbia. Keywords: Euphorbia wallichii; coumarin derivative
1. Introduction Euphorbia is the largest genus of the plant family Euphorbiaceae, with approximately 2000 species (Shi et al. 2008). Several species within the genus Euphorbia have been studied chemically (Duarte and Ferreira 2007; Liu et al. 2012; Zhao et al. 2014). The characteristic compounds include diterpenoid, and triterpenoid derivatives. There is a very rare report of coumarin compounds from the genus Euphorbia (Shi et al. 2008). Euphorbia wallichii is a perennial shrub distributed in China, India, Nepal and Kashmir regions. The plant is used as a Chinese folk medicine for the treatment of oedema and skin disease (Pan et al. 2006). Previous phytochemical studies on E. wallichii led to the isolation of a number of structurally diverse diterpenoids (Wang et al. 2004; Zhang et al. 2006) and triterpenoids (Ali et al. 2008). In this study, one new coumarin derivative (1) and two known compounds (2 and 3) (Figure 1) have been isolated. Herein we report the isolation and structure elucidation of the new compound 1.
2. Results and discussion Compound 1, an amorphous colourless powder, possesses the molecular formula C20H22O11, corresponding to 10 degrees of unsaturation, which was established by high-resolution ESI-MS (m/z 461.1046 calcd. for [M þ Na]þ, 461.1054), and 20 carbon signals were observed in the 13C
*Corresponding authors. Email: [email protected]; [email protected] q 2015 Taylor & Francis
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Figure 1. Structures of isolated compounds 1 –3 from E. wallichii.
NMR spectrum. A DEPT NMR experiment permitted differentiation of the 20 resonances into 3 methyls, 3 methylenes, 4 methines and 10 quaternary carbons. The 1H NMR spectrum of 1 in acetone-d6 indicated that the three methyl groups resonate at dH 1.15, 1.16 and 1.25 (3H each), which are correlated with the carbons at dC 14.1 (C-60 ), 14.3 (C-40 ) and 14.4 (C-20 ), respectively, in the HMQC spectrum. The 13C NMR spectrum of 1 showed that the three methyleneoxy carbons at dC 61.8 (C-10 ), 62.3 (C-30 ) and 62.4 (C-50 ) are correlated with the protons at 4.05 (2H, m), 4.08 (2H, m) and 4.13 (2H, m), respectively, in the HMQC spectrum. The presence of three ethyl carboxylate moieties in 1 was evident from the 1H – 1H correlations of 1.12 (3H, m, Me-40 )/ 4.08 (2H, m, H-30 ), 1.15 (3H, t, Me-60 )/4.13 (2H, m, H-50 ) and 1.25 (3H, m, Me-20 )/4.05 (2H, m, H-10 ) in the COSY spectrum. This was further supported by the long range H – C correlations of 4.05 (H-10 )/165.8 (C-12), 4.08 (H-30 )/166.6 (C-13) and 4.13 (H-50 )/170.0 (C-14), respectively, in the HMBC spectrum. The 13C NMR spectrum contains six aromatic carbons at dC 108.5 (CH, C1), 116.1 (qC, C-6), 129.8 (CH, C-4), 138.7 (qC, C-2), 143.1 (qC, C-5) and 145.8 (qC, C-3), which indicates the presence of a benzene ring system. This was further supported by HMBC correlations between aromatic protons at dH 6.80 (s, H-4) with dC 143.1 (C-5), dH 7.13 (s, H-1) with dC 116.1 (C-6), 138.7 (C-2) and 145.8 (C-3). The methane proton at 5.26 (1H, d, H-8) was correlated to two aromatic carbons at 116.1 (C-6), 143.1 (C-5), ester carbonyl carbon at 163.8 (C-7) and methine carbon at 35.3 (C-9) in the HMBC spectrum, which gave a dihydrocoumarinlike skeleton (Chen et al. 2008). The presence of an olefinic bond moiety in 1 was evident from the residual two resonances at d 118.6 (qC, C-10) and 143.7 (qC, C-11). On the basis of above analysis, we believed initially compound 1 to be an dihydrocoumarin-like skeleton. The 2D NMR spectra were in turn used to determine the linkage of the olefinic bond and three ethyl carboxylate moieties to dihydrocoumarin skeleton. The double bond was located at 35.3 (C-9) of the dihydrocoumarin skeleton by the long range H – C correlation between the methine proton at 5.45 (1H, d, H-9) and two additional olefinic carbons at 118.6 (C-10) and 143.7 (C-11). The methine protons at d 5.26 (H-8) and 5.45 (H-9) showed key correlations with carbonyl at d 170.0 (C-14) in the HMBC spectrum, which confirmed that the ethyl carboxylate moiety was attached at 79.3 (C-8) of the dihydrocoumarin skeleton. The critical long-range correlation between the methine proton of the dihydrocoumarin skeleton at 5.45 (H-9) and the carbonyl at d 166.6 (C-13) indicated that another ethyl carboxylate unit was linked to olefinic carbon at 118.6 (C-10). Additional ethyl carboxylate moiety was placed at the oxygenated olefinic carbon at 143.7 (C11) according to published literature (Chen et al. 2008). The ROESY spectrum was employed to determine the relative configuration of 1. The NOE correlations from 5.45 (H-9a) to 1.25 (Me20 ) confirmed the cis orientation double bond between 118.6 (C-10) and 143.7 (C-11). The strong NOE correlation between 5.45 (H-9a) and 5.26 (H-8) and a relatively small coupling constant (J ¼ 1.5 Hz) between H-9a and H-8 indicate a-configuration of H-8 (Lee et al. 2007; Chen et al.
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2008). The full 1H and 13C NMR assignments were established by means of a combination of COSY, HMQC, HMBC and ROESY spectral measurements (Supporting Information Table S1 and Figure S10). Thus, the structure of 1 was established as 8-ethyl carboxylate, 9-di-ethyl carboxylate-10-ol-10,11-Z-2,3-dihydroxy dihydrocoumarin. It is notable that compound 1 is the first dihydrocoumarin example from Euphorbia genus, while coumarin compounds are rare and only isolated from E. lunulata, E. quinquecostata (Shi et al. 2008), E. Lagascae (Duarte et al. 2008) and E. portlandica (Madureira et al. 2004). The known compounds were identified by comparison of their ESI-MS and NMR spectroscopic data with those reported in the literature as quercetin (2) (Maria et al. 1984), D -glyceraldehyde (3) (Tu 2012). All of them are isolated from E. wallichii for the first time.
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3. Experimental 3.1. General The 1D and 2D NMR (COSY, HMQC, HMBC, ROESY) spectra using standard pulse programmes were recorded at room temperature on a Bruker Avance DRX 500 FT spectrometer operating at 500 (1H) or 125 (13C) MHz. The chemical shift values are relative to the internal standard TMS. HR-ESI-MS data were obtained on an Agilent Series 1100 SL mass spectrometer. Column chromatography was performed using normal phase silica gel (200 2 300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China) and reversed-phase silica gel (RP-18, YMC, 40 –60 mm, Fuji Silysia Chemical Ltd., Kasugai, Japan). TLC was carried out on silica gel sheets (Qingdao Marine Chemical, Inc.) and reversed-phase plates (RP-18 F254S, Merck, Darmstadt, Germany). Visualisation: UV at 254 nm or 10% H2SO4 followed by heating. 3.2. Plant material The whole plants of E. wallichii were collected in Lijiang, Yunnan Province, China, in August 2011, and identified by Prof. Fan Du. A voucher specimen (No. 110801) is deposited in the Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Southwest Forestry University, Kunming City, Yunnan Province, China. 3.3. Extraction and isolation Powdered, air-dried whole plants of E. wallichii (15.0 kg) were exhaustively extracted with 95% EtOH (20 L £ 3) at room temperature for 72 h. The extraction was evaporated to dryness in vacuo. The residue (710 g) was suspended in H2O and then partitioned with petroleum ether and EtOAc, respectively. The EtOAc layer was evaporated to dryness and the resultant residue (390.0 g) was subjected to silica gel liquid chromatography (9 cm £ 70 cm) using stepwise gradient elution CHCl3/MeOH at 100:0 (7600 mL), 200:1 (3100 mL), 100:1 (3000 mL), 50:1 (5900 mL), 25:1 (3200 mL), 10:1 (5300 mL), 5:1 (3000 mL), 2:1 (1600 mL) and finally with MeOH (2000 mL) to afford 17 pooled fractions A –Q according to TLC. Fraction L (61.0 g) was purified on silica gel column chromatography (5 cm £ 65 cm) eluting with petroleum ether/acetone at 8:1 (3700 mL), 6:1 (3000 mL), 4:1 (4000 mL), 3:1 (3000 mL), 2:1 (4700 mL) and 1:1 (3900 mL) to give 10 pooled fractions (1 –10). Fraction 8 was further purified on a C18 reversed-phase column (4 cm £ 60 cm) eluting with MeOH/H2O at 60:40 (1500 mL), 70:30 (1200 mL), 80:20 (1200 mL), 90:10 (1500 mL) and 95:5 (500 mL) to give 1 (49.7 mg, eluted with MeOH/H2O 60:40). Compound 2 (68.0 mg) was got by recrystallisation from Fraction M in CHCl3/Acetone (1:9). Fraction O (20.0 g) was further purified on silica gel column chromatography (4.5 cm £ 65 cm) eluting with CHCl3/acetone at 5:1 (1300 mL), 3:1 (1000 mL), 1:1 (3900 mL) and 0:1 (800 mL) to afford 3 (10.0 mg, eluted with CHCl3/acetone 1:1).
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3.4. Spectroscopic data Compound 1: a colourless amorphous powder, Rf ¼ 0.64 (silica gel TLC with petroleum ether/acetone, 1:1), ESI-MS at 439 for [M þ H]þ, 461 for [M þ Na] þ, 477 for [M þ K]þ, HRESI-MS found at m/z 461.1046 calcd. for [M þ Na] þ, 461.1054, 1H (500 MHz) and 13C (125 MHz) NMR data for compound 1 in acetone-d6 (d ppm, J in Hz). 1H NMR: 7.13 (s, 1H, H1), 6.80 (s, 1H, H-4), 5.26 (d, 1H, J ¼ 1.5 Hz, H-8), 5.45 (d, 1H, J ¼ 1.5 Hz, H-9), 4.05 (m, 2H, H-10 ), 1.25 (t, 3H, J ¼ 7.0 Hz, H-20 ), 4.08 (m, 2H, H-30 ), 1.12 (m, 3H, H-40 ), 4.13 (m, 2H, H-50 ), 1.15 (m, 3H, H-60 ); 13C NMR: 108.5 (d, C-1), 138.7 (s, C-2), 145.8 (s, C-3), 129.8 (d, C-4), 143.1 (s, C-5), 116.1 (s, C-6), 163.8 (s, C-7), 79.3 (d, C-8), 35.3 (d, C-9), 118.6 (s, C-10), 143.7 (s, C11), 165.8 (s, C-12), 166.6 (s, C-13), 170.0 (s, C-14), 61.8 (t, C-10 ), 14.4 (q, C-20 ), 62.3 (t, C-30 ), 14.3 (q, C-40 ), 62.4 (t, C-50 ), 14.1 (q, C-60 ). 4. Conclusion We studied the chemical constituents of the E. wallichii and reported the isolation and structural elucidation of one new coumarin derivative (1), together with two known compounds, quercetin (2) and glyceraldehyde (3). This is the first isolation of dihydrocoumarin (1) from the genus of Euphorbia. Supplementary material Supplementary materials relating to this article are available online, alongside Figures S1 – S14 and Table S1. Acknowledgements The authors thank Prof. Fan Du for providing the plant material.
Funding This work was supported by the Natural Science Foundation of China (NSFC) [grant number 31160075], [grant number 21362035].
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