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A new modified sesquiterpene glycoside from Cupressus chengiana a
a
a
a
Yan Lin , Rong-Jiao Li , Yan Li , Brian D. McGarvey , Hai-Feng Wu a
& Xiao-Ling Wang a
Ethnic Pharmaceutical Institute, Southwest University for Nationalities, Sichuan, Chengdu 610041, P.R. China b
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, P.R. China Published online: 28 Feb 2014.
Click for updates To cite this article: Yan Lin, Rong-Jiao Li, Yan Li, Brian D. McGarvey, Hai-Feng Wu & Xiao-Ling Wang (2014) A new modified sesquiterpene glycoside from Cupressus chengiana, Natural Product Research: Formerly Natural Product Letters, 28:9, 611-615, DOI: 10.1080/14786419.2014.886213 To link to this article: http://dx.doi.org/10.1080/14786419.2014.886213
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Natural Product Research, 2014 Vol. 28, No. 9, 611–615, http://dx.doi.org/10.1080/14786419.2014.886213
A new modified sesquiterpene glycoside from Cupressus chengiana Yan Lina, Rong-Jiao Lia, Yan Lia, Brian D. McGarveya, Hai-Feng Wub* and Xiao-Ling Wanga* a Ethnic Pharmaceutical Institute, Southwest University for Nationalities, Sichuan, Chengdu 610041, P.R. China; bInstitute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, P.R. China
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(Received 6 December 2013; final version received 19 January 2014) A new sesquiterpene glycoside, cupressusoside (1), and five known compounds were isolated from the 70% aqueous ethanol extract of the branches and leaves of Cupressus chengiana. Their structures were elucidated by using spectroscopic methods. All the isolates expressed no remarkable cytotoxic activity against colon carcinoma (HCT-8) and breast cancer (MCF-7) cell lines, with IC50 . 40 mg/mL. Keywords: Cupressus chengiana; sesquiterpene; cytotoxic activities
1. Introduction Cupressus chengiana S.Y. Hu. (Cupressaceae) is widely distributed in northwest Sichuan, China (Zhao et al. 2008) and has been traditionally used as Chinese folk medicine for the treatment of rheumatoid arthritis, pruritus, rheumatism and pertussis. Previous phytochemical investigation on the genus Cupressus resulted in the isolation of flavonoids, terpenes, phenols and organic acids (Lin et al. 1987, 1990), exhibiting some biological activities such as antineoplastic, antioxidation, antitussive, expectorant and inhibition of nucleoside operation effects (Lin 2000; Banerjee & Ziboh 2002). In our ongoing search for bioactive constituents, a new sesquiterpene glucoside, with an unusual modified sesquiterpene skeleton, named cupressusoside (1), as well as five known compounds, 10-O-b-D -glucopyranoside-oplopanone (2), pimaric acid (3), 13S15-hydroxylabd-8(17)-en-18-oic acid (4), 13S-15-acetoxylabd-8(17)-en-18-oic acid (5) and methyl 15-methyl imbricataloate (6) (Figure 1), were obtained from the branches and leaves of C. chengiana. Herein, we report the structural elucidation of the new compound and evaluation of the cytotoxic activity of all the isolated compounds.
2. Results and discussion Compound 1 was isolated as a white amorphous powder with ½a20 D 2 14.0 (c ¼ 0.132 MeOH). HR-ESI-MS revealed a quasi-molecular ion peak at m/z 439.2292 [M þ Na]þ (calcd for C21H36O8Na 439.2307) in the positive-ion mode. In conjunction with the analysis of 1H and 13C NMR spectra (Figure S1, Supplementary material online), the formula of compound 1 was deduced as C21H36O8, consistent with four degrees of unsaturation. The IR spectrum indicated the presence of hydroxyl (3385 cm21) and carbonyl groups (1717 cm21). The UV absorption at 280 nm along with the IR absorption band at 1717 cm21 attested to the presence of a ketone group.
*Corresponding authors. Email: [email protected], [email protected] q 2014 Taylor & Francis
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Y. Lin et al. OH O
HO HO
OH
10 O 9
HO HO
1 6
O 1
H
13 14 CH2OH 15
H O 2
OR
H
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O
OH
4 11
H
H COOH
OH O
O H
HOOC
HOOC
H 6
3 4R=H 5 R = Ac
Figure 1. The structures of compounds 1 – 6.
The 1H NMR spectrum revealed three methyl signals at dH 0.87, 0.91 and 0.92, hydroxymethylene signals at dH 4.37 and 4.27, one oxymethine proton signal at dH 3.68 (m, H-9) and four methine proton signals at dH 1.78 (m, H-4), 2.00 (m, H-5), 2.54 (q, H-6) and 1.67 (m, H11). The 13C NMR spectrum revealed the presence of three methyl carbon signals at dC 16.1 (C12), 20.4 (C-10) and 21.4 (C-13), a carbonyl carbon signal at dC 213.3 (C-14), a hydroxymethyl carbon signal at dC 66.4 (C-15), a glucoside anomeric carbon signal at dC 100.4 (C-100 ), an oxygenated methine carbon signal at dC 77.3 (C-9), a quaternary carbon signal at dC 45.8 (C-1), four methine carbon signals at dC 49.3 (C-4), 49.6 (C-5), 44.8 (C-6) and 29.9 (C-11), four methylene carbon signals at dC 22.4 (C-2), 36.4 (C-3), 21.1 (C-7) and 23.3 (C-8) and resonances of a glucosyl group at dC 100.4, 73.6, 76.8, 70.4, 76.2 and 61.5. The 1H and 13C NMR data of 1 were consistent with a modified sesquiterpene glucoside skeleton as deduced by 1H – 1H COSY (Figure S2). All protonated carbons were assigned from the HSQC spectrum and the locations of the substituents were determined from the HMBC spectrum. One methyl was located at C-1, on the basis of the long-range correlations of the quaternary carbon at dC 45.8 (C-1) with the protons at dH 0.92 (s, 3H, H-15). Two methyls were located at C-11, on the basis of the long-range correlation of the methine carbon at dC 29.9 (C-11) with the methyl protons at dH 0.87 (d, 3H, H12), dH 0.91 (d, 3H, H-13); C-12 and C-13 correlate with each other on the basis of 3J 13C – 1H coupling. This isopropyl group was located at C-4 on the basis of the long-range correlations of the methine carbon at dC 49.3 (C-4) with the protons at dH 0.91 (d, 3H, H-13) and dH 0.87 (d, 3H, H-12). The carbonyl group was located at C-5, on the basis of the long-range correlation of the carbonyl carbon at dC 213.3 (C-14) with the methine proton at dH 2.00 (m, H-5). The hydroxymethyl carbon at dC 66.4 (C-15) was located at the carbonyl carbon (C-14), on the basis of the long-range correlations of the proton signals at dH 4.27 (d, H-15) and 4.37 (d, H-15) with the carbonyl carbon at dC 213.3 (C-14). The glucosidic bond was located at C-9, on the basis of the long-range correlation of the methine carbon at dC 77.4 (C-9) with the anomeric proton of the glucoside at dH 4.26 (d, H-10 ). The key HMBC and HSQC correlations are shown in Figure S2. In the NOESY spectrum, the methine proton signal at dH 3.68 (H-9) revealed NOE enhancements with dH 2.54 (H-6) and 2.00 (H-5), which indicated that the carbonyl group at C-5 and the glucosidic bond were a-oriented. Similarly, NOE enhancements of the isopropyl protons (H-12 and H-13) with 5-H (dH 2.00), 6-H (dH 2.54) and 9-H (dH 3.68) indicated that they are
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b-oriented (Figure S3). Therefore, the structure of compound 1 was established as b-methyl-15hydroxymethyl-faurinone-9-a-O-b-D -glucoside and named cupressusoside. Bos et al. (1983) reported the structure of a related non-glycosidic compound without a hydroxyl group on C-15, a correction of a structure previously reported by Hikino et al. (1968). The structures of the known compounds were identified as those of 10-O-b-D -glucopyranoside-oplopanone (2) (Gan & Zhan 2006), pimaric acid (3) (Zhang 2005), 13S-15-hydroxylabd-8 (17)-en-18-oic acid (4) (Yueh & Ming 1996), 13S-15-acetoxylabd-8(17)-en-18-oic acid (5) (Yueh & Ming 1996) and methyl 15-methyl imbricataloate (6) (Shimizu et al. 1988), on the basis of their spectral data and by comparison of their spectral data with those of the previously reported compounds. This is the first report of the isolation of compounds 2– 6 from the genus Cupressus. The cytotoxicity of compounds 1 – 6 was assayed against two human cancer cell lines, HCT8 and MCF-7, using the MTT method as previously reported. The results demonstrated that compounds 1 –6 expressed no remarkable cytotoxic activity against HCT-8 and MCF-7 cancer cell lines with IC50 . 40 mg/mL. 3. Experimental 3.1. General experimental procedures UV and IR spectra were recorded on Beijing spectrum analysis Shimadzu TU-1901 (Beijing Purkinje General Instrument Co., Ltd., Beijing) and Thermo IR 200 spectrometers (Beijing Tektronix Instrument Co., Ltd., Beijing), respectively. NMR spectra were obtained with a Bruker Avance-600 NMR spectrometer (Bruker company, Germany; chemical shift values are presented as d values with TMS as the internal standard). HR-ESI-MS spectra were performed on an LTQ-Orbitrap XL spectrometer (Thermo Electric Finnigan Scientific Instruments Company, America). C18 reversed-phase silica gel (40 – 63 mm, Merck, Darmstadt, Germany, Nacalai Tesque, NACALAI TESQUE Company, Japan), Sephadex LH-20 (Pharmacia Company, America), MCI gel (CHP 20P, 75 –150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan) and silica gel (100 –200 and 300– 400 mesh, Qingdao Marine Chemical plant, Qingdao, People’s Republic of China) were used for column chromatography. All the solvents used were of analytical grade (Chengdu Chemical Works, Constant Ltd. Chengdu, China). 3.2. Plant material The branches and leaves of C. chengiana were collected in August 2010 from Xiaojin County, Sichuan Province, China, and identified by Prof. Yuan Liu, Ethnic Pharmaceutical Institute, Southwest University for Nationalities (No. XJX1008). 3.3. Extraction and isolation The air-dried and powdered leaves of C. chengiana (2.0 kg) were extracted three times with 95% EtOH (3 £ 2 L) at room temperature. Removal of the ethanol under reduced pressure yielded ethanol extract (120 g). The extract was suspended in distilled water and then the suspension was partitioned with petroleum ether, ethyl acetate and n-BuOH successively. The n-BuOH fraction (22.0 g) was subjected to silica gel (100 –200 mesh) column chromatography (L/D ratio ¼ 7:1) using a chloroform and methanol gradient (from 80:1 to 1:1) as eluent to yield three fractions (Frs A – C). Fr. A (5 g) was subjected to chromatography on a silica gel column (L/D ratio ¼ 7:1) (100 – 200 mesh) using a CHCl3 – MeOH solvent system of increasing polarity (20:1 ! 0:1, v/v), followed by Sephadex LH-20 (MeOH) and C18 reversed-phase silica gel column (L/D ratio ¼ 5:1) (30% MeOH) to yield 1 (65 mg). Fr. B (2.0 g) was subjected to chromatography on a silica gel column (L/D ratio ¼ 8:1) (100 – 200 mesh) with CHCl3 – MeOH gradient (20:1 ! 0:1,
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v/v) and was subjected to Sephadex LH-20 (L/D ratio ¼ 10:1) with MeOH as eluent to yield 2 (20 mg,). Fr. C was subjected to chromatography on a silica gel column (L/D ratio ¼ 7:1) (100 – 200 mesh) eluted with CHCl3 –MeOH gradient (20:1 ! 0:1, v/v) and was subjected to Sephadex LH-20 (L/D ratio ¼ 10:1) with MeOH as eluent and purified using C18 reversed-phase silica gel column(L/D ratio ¼ 5:1) (50% MeOH) to yield 3 (20 mg). The ethyl acetate fraction (30.0 g) was subjected to chromatography on a silica gel column (L/D ratio ¼ 7:1) (100 – 200, 300– 400 mesh) using a petroleum ether and acetone gradient (from 80:1 to 1:1) as eluent to yield three fractions (Frs E– G). Fr. E (8 g) was subjected to chromatography on a silica gel column (L/ D ratio ¼ 6:1) (100 –200 mesh) eluted with a petroleum ether and acetone gradient (20:1 ! 0:1, v/v) and was subjected to Sephadex LH-20 (L/D ratio ¼ 10:1) (MeOH) to yield 4 (10 mg) and 5 (15 mg). Fr. F (5 g) was subjected to chromatography on a silica gel column (L/D ratio ¼ 7:1) (100 – 200 mesh) eluted with petroleum ether and acetone gradient (20:1 ! 0:1, v/v), and was subjected to Sephadex LH-20 (L/D ratio ¼ 10:1) (MeOH) to yield 6 (50 mg). 3.3.1. Spectral data 21 Compound 1. White amorphous powder; ½a20 D 2 14.0 (c ¼ 0.132 MeOH). IR (KBr) cm : 3385 (– OH), 1717 (CvO). UV lmax (MeOH): 280 (nm). HR-ESI-MS m/z 439.2292 [M þ Na]þ (calcd for C21H36O8Na 439.2307). 1H NMR (CD3OD, 600 MHz) dH 1.44 (m, H-2a), 1.78 (m, H-2b), 2.08 (m, H-3a), 1.13 (m, H-3b),1.78 (m, H-4), 2.00 (m, H-5), 2.54 (q, H-6), 2.01 (m, H-7a), 1.59 (m, H7b), 1.74 (ddd, 2H, H-8), 3.68 (m, H-9), 0.92 (s, 3H, H3-10), 1.67 (m, H-11), 0.87 (d, J ¼ 6.7 Hz, 3H, H-12), 0.91 (d, J ¼ 6.8 Hz, 3H, H-13), 4.27 (d, J ¼ 18.2 Hz, H-15a), 4.37 (d, J ¼ 18.1 Hz, H15b), 4.26 (d, J ¼ 7.7 Hz, H-100 ), 3.14 (q, H-200 ), 3.34 (t, H-300 ), 3.28 (d, J ¼ 8.9 Hz, H-400 ), 3.23 (m, H-500 ), 3.85 (dd, J ¼ 9.4,11.6 Hz, H-600 a), 3.66 (m, H-600 b). 13C NMR (CD3OD, 150 MHz) dC 45.8 (C-1), 22.4 (C-2), 36.4 (C-3), 49.3(C-4), 49.6 (C-5), 44.8 (C-6), 21.1 (C-7), 23.3 (C-8), 77.3 (C-9), 20.4 (C-10), 29.9 (C-11), 16.1 (C-12), 21.4 (C-13), 213.3 (C-14), 66.4 (C-15), 100.4 (C-100 ), 73.6 (C-200 ), 76.8 (C-300 ), 70.4 (C-400 ), 76.2 (C-500 ), 61.5 (C-600 ). 3.4. Cytotoxicity bioassays Compounds 1 –6 were assessed by the MTT method using colon carcinoma (HCT-8) and breast cancer (MCF-7) human cancer cell lines. Each cell was seeded on to 96-well microtitre plates at a density of 6 £ 104 cells/mL (100 mL) per well. Cells were pre-incubated for 24 h at 378C in 5% CO2 for 24 h. Then, five different concentrations of each compound dissolved in dimethyl sulfoxide (DMSO) were added to each well. Each concentration was tested in triplicate. After 48 h, 10 mL of MTT (4 mg/mL) was added to each well and incubated for another 4 h. The liquid in each well was then removed and DMSO (200 mL) was added to each well. The absorbance was recorded on a microplate reader at a wavelength of 570 nm. 4. Conclusions In summary, a novel sesquiterpene glucoside with an unusual modified skeleton, cupressusoside (1), together with five known compounds, was isolated from C. chengiana. None of the compounds expressed remarkable cytotoxic activity against HCT-8 and MCF-7 cancer cell lines. Supplementary material Supplementary material relating to this article is available online, alongside and Figures S1 – S3. Acknowledgements This work was supported by the Project of Postgraduate Degree Construction, Southwest University for Nationalities (No. 2013XWD-S0703) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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References Banerjee T, Ziboh VA. 2002. Downregulation of COX-2 and iNOS by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line. Prostaglandins Lenkot Essent Fatty Acids. 66:485–492. Bos R, Hendricks H, Kloosterman J, Simpa G. 1983. A structure of faurinone, a sesquiterpene ketone isolated from Valeriana officinalis. Phytochemistry. 22:1505– 1506. Gan LS, Zhan ZJ. 2006. Two new terpenoid glucosides from Aster flaccidus. J Asian Nat Prod Res. 8:589– 594. Hikino H, Hikino Y, Agatsuma K, Takemoto T. 1968. Structure and absolute configuration of faurinone. Chem Pharm Bull. 16:1779– 1783. Lin LC. 2000. Cytotocix biflavonoids from Sleaginella delicatula. J Nat Prod. 63:627–630. Lin RC, Seguin E, Skaltsounis AL, Tillequin F, Koch M. 1990. Skaltsounis synthesis of the glycoalkaloids of Selaginella doederleinii and structure revision of one of them. J Nat Prod. 53:882–893. Lin RC, Seguin E, Tillequin F, Koch M. 1987. New alkaloid glycosides from Selaginella doederleinii. J Nat Prod. 50:422–426. Shimizu M, Tsuji H, Shogawa H, Fukumura H, Tanaami S, Hayashi T, Arisawa M, Morita N. 1988. Anti-inflammatory constituents of topically applied crude drugs. II. Constituents and anti-inflammatory effect of Cryptomeria japonica D. DON. Chem Pharm Bull. 36:3967–3973. Yueh HK, Ming TY. 1996. Three labdane type diterpenes from the bark of Juniperus formosana Hay. var. concolor HAY. Chem Pharm Bull. 44:1242 –1244. Zhang R. 2005. Study on the bioactive constituents of the needles of Pinus sylvestris L.[D]. Tianjin: Tianjin University. Zhao J, Yan M, Huang Y, He W-y, Zhao Y. 2008. Flavonoids from the leaves of Sabina vulgaris Antoine. Chem Indus Forest Prod. 2:33–37.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new modified sesquiterpene glycoside from Cupressus chengiana a
a
a
a
Yan Lin , Rong-Jiao Li , Yan Li , Brian D. McGarvey , Hai-Feng Wu a
& Xiao-Ling Wang a
Ethnic Pharmaceutical Institute, Southwest University for Nationalities, Sichuan, Chengdu 610041, P.R. China b
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, P.R. China Published online: 28 Feb 2014.
Click for updates To cite this article: Yan Lin, Rong-Jiao Li, Yan Li, Brian D. McGarvey, Hai-Feng Wu & Xiao-Ling Wang (2014) A new modified sesquiterpene glycoside from Cupressus chengiana, Natural Product Research: Formerly Natural Product Letters, 28:9, 611-615, DOI: 10.1080/14786419.2014.886213 To link to this article: http://dx.doi.org/10.1080/14786419.2014.886213
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
Natural Product Research, 2014 Vol. 28, No. 9, 611–615, http://dx.doi.org/10.1080/14786419.2014.886213
A new modified sesquiterpene glycoside from Cupressus chengiana Yan Lina, Rong-Jiao Lia, Yan Lia, Brian D. McGarveya, Hai-Feng Wub* and Xiao-Ling Wanga* a Ethnic Pharmaceutical Institute, Southwest University for Nationalities, Sichuan, Chengdu 610041, P.R. China; bInstitute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, P.R. China
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(Received 6 December 2013; final version received 19 January 2014) A new sesquiterpene glycoside, cupressusoside (1), and five known compounds were isolated from the 70% aqueous ethanol extract of the branches and leaves of Cupressus chengiana. Their structures were elucidated by using spectroscopic methods. All the isolates expressed no remarkable cytotoxic activity against colon carcinoma (HCT-8) and breast cancer (MCF-7) cell lines, with IC50 . 40 mg/mL. Keywords: Cupressus chengiana; sesquiterpene; cytotoxic activities
1. Introduction Cupressus chengiana S.Y. Hu. (Cupressaceae) is widely distributed in northwest Sichuan, China (Zhao et al. 2008) and has been traditionally used as Chinese folk medicine for the treatment of rheumatoid arthritis, pruritus, rheumatism and pertussis. Previous phytochemical investigation on the genus Cupressus resulted in the isolation of flavonoids, terpenes, phenols and organic acids (Lin et al. 1987, 1990), exhibiting some biological activities such as antineoplastic, antioxidation, antitussive, expectorant and inhibition of nucleoside operation effects (Lin 2000; Banerjee & Ziboh 2002). In our ongoing search for bioactive constituents, a new sesquiterpene glucoside, with an unusual modified sesquiterpene skeleton, named cupressusoside (1), as well as five known compounds, 10-O-b-D -glucopyranoside-oplopanone (2), pimaric acid (3), 13S15-hydroxylabd-8(17)-en-18-oic acid (4), 13S-15-acetoxylabd-8(17)-en-18-oic acid (5) and methyl 15-methyl imbricataloate (6) (Figure 1), were obtained from the branches and leaves of C. chengiana. Herein, we report the structural elucidation of the new compound and evaluation of the cytotoxic activity of all the isolated compounds.
2. Results and discussion Compound 1 was isolated as a white amorphous powder with ½a20 D 2 14.0 (c ¼ 0.132 MeOH). HR-ESI-MS revealed a quasi-molecular ion peak at m/z 439.2292 [M þ Na]þ (calcd for C21H36O8Na 439.2307) in the positive-ion mode. In conjunction with the analysis of 1H and 13C NMR spectra (Figure S1, Supplementary material online), the formula of compound 1 was deduced as C21H36O8, consistent with four degrees of unsaturation. The IR spectrum indicated the presence of hydroxyl (3385 cm21) and carbonyl groups (1717 cm21). The UV absorption at 280 nm along with the IR absorption band at 1717 cm21 attested to the presence of a ketone group.
*Corresponding authors. Email: [email protected], [email protected] q 2014 Taylor & Francis
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Y. Lin et al. OH O
HO HO
OH
10 O 9
HO HO
1 6
O 1
H
13 14 CH2OH 15
H O 2
OR
H
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O
OH
4 11
H
H COOH
OH O
O H
HOOC
HOOC
H 6
3 4R=H 5 R = Ac
Figure 1. The structures of compounds 1 – 6.
The 1H NMR spectrum revealed three methyl signals at dH 0.87, 0.91 and 0.92, hydroxymethylene signals at dH 4.37 and 4.27, one oxymethine proton signal at dH 3.68 (m, H-9) and four methine proton signals at dH 1.78 (m, H-4), 2.00 (m, H-5), 2.54 (q, H-6) and 1.67 (m, H11). The 13C NMR spectrum revealed the presence of three methyl carbon signals at dC 16.1 (C12), 20.4 (C-10) and 21.4 (C-13), a carbonyl carbon signal at dC 213.3 (C-14), a hydroxymethyl carbon signal at dC 66.4 (C-15), a glucoside anomeric carbon signal at dC 100.4 (C-100 ), an oxygenated methine carbon signal at dC 77.3 (C-9), a quaternary carbon signal at dC 45.8 (C-1), four methine carbon signals at dC 49.3 (C-4), 49.6 (C-5), 44.8 (C-6) and 29.9 (C-11), four methylene carbon signals at dC 22.4 (C-2), 36.4 (C-3), 21.1 (C-7) and 23.3 (C-8) and resonances of a glucosyl group at dC 100.4, 73.6, 76.8, 70.4, 76.2 and 61.5. The 1H and 13C NMR data of 1 were consistent with a modified sesquiterpene glucoside skeleton as deduced by 1H – 1H COSY (Figure S2). All protonated carbons were assigned from the HSQC spectrum and the locations of the substituents were determined from the HMBC spectrum. One methyl was located at C-1, on the basis of the long-range correlations of the quaternary carbon at dC 45.8 (C-1) with the protons at dH 0.92 (s, 3H, H-15). Two methyls were located at C-11, on the basis of the long-range correlation of the methine carbon at dC 29.9 (C-11) with the methyl protons at dH 0.87 (d, 3H, H12), dH 0.91 (d, 3H, H-13); C-12 and C-13 correlate with each other on the basis of 3J 13C – 1H coupling. This isopropyl group was located at C-4 on the basis of the long-range correlations of the methine carbon at dC 49.3 (C-4) with the protons at dH 0.91 (d, 3H, H-13) and dH 0.87 (d, 3H, H-12). The carbonyl group was located at C-5, on the basis of the long-range correlation of the carbonyl carbon at dC 213.3 (C-14) with the methine proton at dH 2.00 (m, H-5). The hydroxymethyl carbon at dC 66.4 (C-15) was located at the carbonyl carbon (C-14), on the basis of the long-range correlations of the proton signals at dH 4.27 (d, H-15) and 4.37 (d, H-15) with the carbonyl carbon at dC 213.3 (C-14). The glucosidic bond was located at C-9, on the basis of the long-range correlation of the methine carbon at dC 77.4 (C-9) with the anomeric proton of the glucoside at dH 4.26 (d, H-10 ). The key HMBC and HSQC correlations are shown in Figure S2. In the NOESY spectrum, the methine proton signal at dH 3.68 (H-9) revealed NOE enhancements with dH 2.54 (H-6) and 2.00 (H-5), which indicated that the carbonyl group at C-5 and the glucosidic bond were a-oriented. Similarly, NOE enhancements of the isopropyl protons (H-12 and H-13) with 5-H (dH 2.00), 6-H (dH 2.54) and 9-H (dH 3.68) indicated that they are
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b-oriented (Figure S3). Therefore, the structure of compound 1 was established as b-methyl-15hydroxymethyl-faurinone-9-a-O-b-D -glucoside and named cupressusoside. Bos et al. (1983) reported the structure of a related non-glycosidic compound without a hydroxyl group on C-15, a correction of a structure previously reported by Hikino et al. (1968). The structures of the known compounds were identified as those of 10-O-b-D -glucopyranoside-oplopanone (2) (Gan & Zhan 2006), pimaric acid (3) (Zhang 2005), 13S-15-hydroxylabd-8 (17)-en-18-oic acid (4) (Yueh & Ming 1996), 13S-15-acetoxylabd-8(17)-en-18-oic acid (5) (Yueh & Ming 1996) and methyl 15-methyl imbricataloate (6) (Shimizu et al. 1988), on the basis of their spectral data and by comparison of their spectral data with those of the previously reported compounds. This is the first report of the isolation of compounds 2– 6 from the genus Cupressus. The cytotoxicity of compounds 1 – 6 was assayed against two human cancer cell lines, HCT8 and MCF-7, using the MTT method as previously reported. The results demonstrated that compounds 1 –6 expressed no remarkable cytotoxic activity against HCT-8 and MCF-7 cancer cell lines with IC50 . 40 mg/mL. 3. Experimental 3.1. General experimental procedures UV and IR spectra were recorded on Beijing spectrum analysis Shimadzu TU-1901 (Beijing Purkinje General Instrument Co., Ltd., Beijing) and Thermo IR 200 spectrometers (Beijing Tektronix Instrument Co., Ltd., Beijing), respectively. NMR spectra were obtained with a Bruker Avance-600 NMR spectrometer (Bruker company, Germany; chemical shift values are presented as d values with TMS as the internal standard). HR-ESI-MS spectra were performed on an LTQ-Orbitrap XL spectrometer (Thermo Electric Finnigan Scientific Instruments Company, America). C18 reversed-phase silica gel (40 – 63 mm, Merck, Darmstadt, Germany, Nacalai Tesque, NACALAI TESQUE Company, Japan), Sephadex LH-20 (Pharmacia Company, America), MCI gel (CHP 20P, 75 –150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan) and silica gel (100 –200 and 300– 400 mesh, Qingdao Marine Chemical plant, Qingdao, People’s Republic of China) were used for column chromatography. All the solvents used were of analytical grade (Chengdu Chemical Works, Constant Ltd. Chengdu, China). 3.2. Plant material The branches and leaves of C. chengiana were collected in August 2010 from Xiaojin County, Sichuan Province, China, and identified by Prof. Yuan Liu, Ethnic Pharmaceutical Institute, Southwest University for Nationalities (No. XJX1008). 3.3. Extraction and isolation The air-dried and powdered leaves of C. chengiana (2.0 kg) were extracted three times with 95% EtOH (3 £ 2 L) at room temperature. Removal of the ethanol under reduced pressure yielded ethanol extract (120 g). The extract was suspended in distilled water and then the suspension was partitioned with petroleum ether, ethyl acetate and n-BuOH successively. The n-BuOH fraction (22.0 g) was subjected to silica gel (100 –200 mesh) column chromatography (L/D ratio ¼ 7:1) using a chloroform and methanol gradient (from 80:1 to 1:1) as eluent to yield three fractions (Frs A – C). Fr. A (5 g) was subjected to chromatography on a silica gel column (L/D ratio ¼ 7:1) (100 – 200 mesh) using a CHCl3 – MeOH solvent system of increasing polarity (20:1 ! 0:1, v/v), followed by Sephadex LH-20 (MeOH) and C18 reversed-phase silica gel column (L/D ratio ¼ 5:1) (30% MeOH) to yield 1 (65 mg). Fr. B (2.0 g) was subjected to chromatography on a silica gel column (L/D ratio ¼ 8:1) (100 – 200 mesh) with CHCl3 – MeOH gradient (20:1 ! 0:1,
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v/v) and was subjected to Sephadex LH-20 (L/D ratio ¼ 10:1) with MeOH as eluent to yield 2 (20 mg,). Fr. C was subjected to chromatography on a silica gel column (L/D ratio ¼ 7:1) (100 – 200 mesh) eluted with CHCl3 –MeOH gradient (20:1 ! 0:1, v/v) and was subjected to Sephadex LH-20 (L/D ratio ¼ 10:1) with MeOH as eluent and purified using C18 reversed-phase silica gel column(L/D ratio ¼ 5:1) (50% MeOH) to yield 3 (20 mg). The ethyl acetate fraction (30.0 g) was subjected to chromatography on a silica gel column (L/D ratio ¼ 7:1) (100 – 200, 300– 400 mesh) using a petroleum ether and acetone gradient (from 80:1 to 1:1) as eluent to yield three fractions (Frs E– G). Fr. E (8 g) was subjected to chromatography on a silica gel column (L/ D ratio ¼ 6:1) (100 –200 mesh) eluted with a petroleum ether and acetone gradient (20:1 ! 0:1, v/v) and was subjected to Sephadex LH-20 (L/D ratio ¼ 10:1) (MeOH) to yield 4 (10 mg) and 5 (15 mg). Fr. F (5 g) was subjected to chromatography on a silica gel column (L/D ratio ¼ 7:1) (100 – 200 mesh) eluted with petroleum ether and acetone gradient (20:1 ! 0:1, v/v), and was subjected to Sephadex LH-20 (L/D ratio ¼ 10:1) (MeOH) to yield 6 (50 mg). 3.3.1. Spectral data 21 Compound 1. White amorphous powder; ½a20 D 2 14.0 (c ¼ 0.132 MeOH). IR (KBr) cm : 3385 (– OH), 1717 (CvO). UV lmax (MeOH): 280 (nm). HR-ESI-MS m/z 439.2292 [M þ Na]þ (calcd for C21H36O8Na 439.2307). 1H NMR (CD3OD, 600 MHz) dH 1.44 (m, H-2a), 1.78 (m, H-2b), 2.08 (m, H-3a), 1.13 (m, H-3b),1.78 (m, H-4), 2.00 (m, H-5), 2.54 (q, H-6), 2.01 (m, H-7a), 1.59 (m, H7b), 1.74 (ddd, 2H, H-8), 3.68 (m, H-9), 0.92 (s, 3H, H3-10), 1.67 (m, H-11), 0.87 (d, J ¼ 6.7 Hz, 3H, H-12), 0.91 (d, J ¼ 6.8 Hz, 3H, H-13), 4.27 (d, J ¼ 18.2 Hz, H-15a), 4.37 (d, J ¼ 18.1 Hz, H15b), 4.26 (d, J ¼ 7.7 Hz, H-100 ), 3.14 (q, H-200 ), 3.34 (t, H-300 ), 3.28 (d, J ¼ 8.9 Hz, H-400 ), 3.23 (m, H-500 ), 3.85 (dd, J ¼ 9.4,11.6 Hz, H-600 a), 3.66 (m, H-600 b). 13C NMR (CD3OD, 150 MHz) dC 45.8 (C-1), 22.4 (C-2), 36.4 (C-3), 49.3(C-4), 49.6 (C-5), 44.8 (C-6), 21.1 (C-7), 23.3 (C-8), 77.3 (C-9), 20.4 (C-10), 29.9 (C-11), 16.1 (C-12), 21.4 (C-13), 213.3 (C-14), 66.4 (C-15), 100.4 (C-100 ), 73.6 (C-200 ), 76.8 (C-300 ), 70.4 (C-400 ), 76.2 (C-500 ), 61.5 (C-600 ). 3.4. Cytotoxicity bioassays Compounds 1 –6 were assessed by the MTT method using colon carcinoma (HCT-8) and breast cancer (MCF-7) human cancer cell lines. Each cell was seeded on to 96-well microtitre plates at a density of 6 £ 104 cells/mL (100 mL) per well. Cells were pre-incubated for 24 h at 378C in 5% CO2 for 24 h. Then, five different concentrations of each compound dissolved in dimethyl sulfoxide (DMSO) were added to each well. Each concentration was tested in triplicate. After 48 h, 10 mL of MTT (4 mg/mL) was added to each well and incubated for another 4 h. The liquid in each well was then removed and DMSO (200 mL) was added to each well. The absorbance was recorded on a microplate reader at a wavelength of 570 nm. 4. Conclusions In summary, a novel sesquiterpene glucoside with an unusual modified skeleton, cupressusoside (1), together with five known compounds, was isolated from C. chengiana. None of the compounds expressed remarkable cytotoxic activity against HCT-8 and MCF-7 cancer cell lines. Supplementary material Supplementary material relating to this article is available online, alongside and Figures S1 – S3. Acknowledgements This work was supported by the Project of Postgraduate Degree Construction, Southwest University for Nationalities (No. 2013XWD-S0703) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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