Natural Product Research Formerly Natural Product Letters
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A new diterpene from Clinopodium chinense Mingliang Zhong, Haifeng Wu, Xiaopo Zhang, Guangli Sun, Guibo Sun, Shichun Yu & Xudong Xu To cite this article: Mingliang Zhong, Haifeng Wu, Xiaopo Zhang, Guangli Sun, Guibo Sun, Shichun Yu & Xudong Xu (2014) A new diterpene from Clinopodium chinense, Natural Product Research, 28:7, 467-472, DOI: 10.1080/14786419.2013.879132 To link to this article: http://dx.doi.org/10.1080/14786419.2013.879132
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Date: 02 December 2016, At: 05:19
Natural Product Research, 2014 Vol. 28, No. 7, 467–472, http://dx.doi.org/10.1080/14786419.2013.879132
A new diterpene from Clinopodium chinense Mingliang Zhonga1, Haifeng Wua1, Xiaopo Zhanga, Guangli Sunb, Guibo Suna, Shichun Yuc and Xudong Xua* a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Peking Union Medical College, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing 100193, P.R. China; bDepartment of Pharmacy, School of Pharmacy, Hebei United University, Tangshan, Hebei 063000, P.R. China; cBeijing Hong Tai Chi Chung Medical Technology Co., Ltd., Beijing 102600, P.R. China
(Received 12 September 2013; final version received 23 December 2013) A new abietane diterpene, named as 3b-hydroxy-12-O-b-D -glucopyranosyl-8,11,13abietatrien-7-one (1), together with four known flavonoids, was isolated from the hot water extract of the aerial parts of Clinopodium chinense. Their structures were determined by analysing the spectroscopic data including 1D, 2D NMR and HR-ESI-MS. Compound 1 tested against HepG-2 and A549 cancer cell lines expressed weak cytotoxicity. Cardioprotective effects of compounds 2–5 against H2O2-induced apoptosis in H9c2 cells were also evaluated; compounds 2 and 3 exhibited moderate cardioprotective effect. Keywords: Clinopodium chinense; 3b-hydroxy-12-O-b-D -glucopyranosyl-8,11,13abietatrien-7-one; cytotoxicity; cardioprotective activity
1. Introduction Clinopodium chinense (Benth.) O. Kuntze (Labiatae), known as ‘Duanxueliu’ in Chinese Pharmacopoeia, has long been used as a traditional Chinese medicine for the treatment of influenza, heliosis, allergic dermatitis, dysentery, haematuria, trauma and haemorrhage (Chi & Lu 2007). Previous phytochemical investigation indicated the presence of flavonoids, triterpenoid saponins and volatile oil, responsible for haemostatic, anti-inflammatory, antioxidant, antibiotic and antihyperglycaemic activities (Zhong et al. 2012). Cardioprotective effects of some flavonoids on H9c2 cells were noteworthy (Sun et al. 2011; Sun, Sun, et al. 2012). In our ongoing research for bioactive constituents, a new abietane diterpene, 3b-hydroxy12-O-b-D -glucopyranosyl-8,11,13-abietatrien-7-one (1), as well as four known flavonoids, prunin (2), homoeriodictyol (3), hesperitin-7-O-b-D -glucopyranoside (4) and kaempferol-3-Orhamnopyranoside (5) were obtained from the ethanolic extract of the aerial parts of C. chinense. Herein, we present the structural elucidation and cytotoxicity of compound 1 as well as the cardioprotective effects of known compounds against H2O2-induced apoptosis in H9c2 cells. 2. Results and discussion Compound 1 (Figure 1) was obtained as a white amorphous powder with ½a25 D ¼ 2 16.4 (c ¼ 0.14, MeOH). It exhibited the molecular formula of C26H38O8 as deduced from its quasimolecular ion peak of [M –H]2 at m/z 477.2491 (calcd 477.2488) in an HR-ESI-MS experiment, consistent with eight degrees of unsaturation. The IR spectrum showed the presence of hydroxyl
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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M. Zhong et al. HO
6' 5'
O
O
4' OH 3'
R1 1' 2'
OH OH
12 11
20 2 3 HO
1 4
10 5
ORha O
OH OH
O 5
2 R1 = R3 = HR2 = OH R4 = Glu
O
3 R1 = OCH3 R2 = OH R3 = R4 = H
18
19
R3
17
8 7
O
HO O
R4O
14
9 6
13 15
OH R2
16
4 R1 = H R2 = OCH3 R3 = OH R4 = Glu
1
Figure 1. Chemical structures of compounds 1 –5.
(3390 cm21), carbonyl (1661 cm21) and phenyl (1601 cm21) groups. The 1H NMR spectrum of 1 showed signals of two aromatic protons [dH 7.83 (1H, s, H-14), 7.13 (1H, s, H-11)], an isopropyl group [dH 1.19 (3H, d, J ¼ 7.2 Hz, H-16), 1.24 (3H, d, J ¼ 7.2 Hz, H-17), 3.41 (1H, m, J ¼ 7.2 Hz, H-15)], three methyl groups bounded to quaternary carbon atoms [dH 1.26 (3H, s, H-20), 1.04 (3H, s, H-18), 0.96 (3H, s, H-19)] and an anomeric proton (dH 5.02). In the 13C NMR spectrum, 26 carbon signals were observed, which were resolved through the APT experiment into 5 methyl, 4 methylene, 10 methine and 7 quaternary carbons, including the resonance of a glucosyl group [dC 102.0 (C-10 ), 75.2 (C-20 ), 78.7 (C-30 ), 71.8 (C-40 ), 78.6 (C-50 ), 62.9 (C-60 )]. All carbon-bound protons were assigned from the HSQC spectrum and the locations of the substituents were determined by the HMBC spectrum. The overall 1H and 13C NMR spectroscopic data for 1 confirmed that the compound was an abietane-type diterpene skeleton (Niu et al. 2006). Comparison of the NMR spectra of 1 with that of abietatrien-12-O-bglucopyranoside (Carlos et al. 2007) reveals the appearance of hydroxyl and carbonyl groups in 1. The cross-peaks of dH 1.83 (H-5), 2.63 (H-6a), 2.71(H-6b), 7.13 (H-11) and 7.83 (H-14) with dC 201.1 (C-7) in the HMBC spectrum demonstrated that the carbonyl group was attached at C-7. The HMBC correlations of dH 1.04 (s, H3-18) and 0.96 (s, H3-19) with dC 79.0 (C-3) suggested that the hydroxyl group was located at C-3. The HMBC correlations from dH 5.02 (H-10 ) to dC 161.5 (C-12) indicated that the glucosyl group was connected with C-12, whereas correlations from dH 3.41 (H-15) to dC 161.5 (C-12)/126.5 (C-14), dH 1.19 (H3-16)/1.24 (H3-17) to dC 137.5 (C-13) suggested that the isopropyl group was linked to C-13 (Figure 2).
HMBC
HO O
O
NOESY
OH OH
OH HO H
HO
O
Figure 2. Key HMBC and NOESY correlations of compound 1.
H
O
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Table 1. In vitro cytotoxicity of compound 1. IC50 (mg/mL)
Compounds HepG-2 1 Taxolb a b
A549
76.8 ^ 1.7 5.6 ^ 0.13 a
68.1 ^ 2.1 4.8 ^ 0.24
Values present mean ^ SD of triplicate experiments. Positive control substance.
The relative stereochemistry of 1 was resolved by analysis of NOESY spectrum and H –H coupling constant value (J). The evidence from the large J value of the axial proton at C-3 (dH 3.28, dd, J ¼ 10.2, 5.4 Hz) suggested that the hydroxyl group at C-3 is positioned in b-configuration (Sun, Zhang, et al. 2012). The NOESY correlations between H-3 and H-5/H-18, H-19 and H-20 (Figure 2) indicated that methyl groups at C-19 and C-20 were b-oriented, whereas methyl at C-18 and H-5 were a-oriented. The J value of the anomeric proton (J ¼ 7.2 Hz) confirmed a b-configuration of the glucosyl group (Carlos et al. 2007). Based on the above-mentioned analysis, compound 1 was assigned as 3b-hydroxy-12-O-b-D -glucopyranosyl8,11,13-abietatrien-7-one. In addition, four known flavonoids, namely prunin (2), homoeriodictyol (3), hesperitin-7-Ob-D -glucopyranoside (4) and kaempferol-3-O-rhamnopyranoside (5) (Lewinsohn et al. 1986; Gohar et al. 2009; Nakano et al. 2011; Yousuf et al. 2013), were obtained and identified by comparing their spectroscopic data with those reported in the literature, and 3 – 5 were first isolated from the genus Clinopodium. In the cytotoxic assay, compound 1 exhibited weak antineoplastic activity against HepG-2 (IC50 76.8 mg/mL) and A549 (IC50 68.1 mg/mL) cell lines, compared with the positive control taxol (Table 1). It was previously reported that some abietane diterpenes with similar structures expressed moderate cytotoxicity against several cancer cell lines (Carlos et al. 2007; Lin et al. 2010). Moreover, cardioprotective effects of 2 –5 against H2O2-induced apoptosis in H9c2 cells were tested; compounds 2 and 3 were active in cardioprotection. The cell viability was improved from 85.7% to 91.6% (2) and 82.3% to 91.9% (3) at concentrations of 5 and 25 mg/mL, respectively, compared with those of exposure to H2O2. In conclusion, we described a new abietane diterpene, 3b-hydroxy-12-O-b-D -glucopyranosyl8,11,13-abietatrien-7-one (1) and four known compounds, prunin (2), homoeriodictyol (3), hesperitin-7-O-b-D -glucopyranoside (4) and kaempferol-3-O-rhamnopyranoside (5). Compound 1 expressed weak cytotoxicity against HepG-2 and A549 cancer cell lines, while compounds 2 and 3 exhibited moderate cardioprotective effect against H2O2-induced apoptosis in H9c2 cell line. 3. Experimental 3.1. General experimental procedures UV spectra were recorded on a Shimadzu UV-2550 UV/vis spectrophotometer (Shimadzu Corporation, Tokyo, Japan). IR spectrum was acquired using a Shimadzu FTIR-8400S spectrophotometer (Shimadzu Corporation, Tokyo, Japan). Optical rotations were measured by a Perkin-EImer 241 polarimeter (PerkinElmer, Inc., Waltham, US). The NMR data (1D and 2D NMR) were recorded on a Bruker AV 600 instrument (Bruker BioSpin Corporation, Billerica, US) in CD3OD or DMSO-d6 with TMS as internal standard (600 MHz for 1H and 150 MHz for 13 C). The HR-ESI-MS data were obtained by LTQ-Orbitrap mass spectrophotometer (ThermoFisher Scientific, Bremen, Germany). Chromatography was performed on AB-8 macroporous resin (Chemical Plant of NanKai University, Tianjin, China), silica gel (200 –300 mesh,
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Qingdao Haiyang Chemical Factory, Qingdao, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and ODS gel (40 – 60 mm, Daiso Co., Ltd, Osaka, Japan). Semipreparative HPLC was performed on a CXTH LC-3000 HPLC system with a CXTH LC-3000 UV spectrophotometric detector (Beijing Chuangxintongheng Science and Technology Co., Ltd, Beijing, China). 3.2. Plant material The plants were collected from Anhui Province in 2011, and identified by Dr Jing Quan Yuan at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College and a voucher specimen (no. 20101132) has been deposited in the same institute. 3.3. Extraction and isolation The dried aerial parts of C. chinense (5 kg) were decocted two times with boiled water (50 L, 2 h each time), and the combined solution was concentrated under reduced pressure to yield an extract (400 g). The extract was subjected to AB-8 macroporous resin column chromatography using ethanol–H2O (0:100 to 95:5, v/v) and the 50–95% fraction yielded the total flavonoids of C. chinense (CCF). The CCF (103 g) was subjected to column chromatography on silica gel with CHCl3 –MeOH (20:1 to 0:1, v/v), and fractionated into eight fractions (Frs 1–8). Fr. 4 (3.1 g) was subjected to silica gel column chromatography eluting with chloroform–acetone (0:1 to 1:0, v/v) to yield five subfractions (SubFrs 1–5). SubFr. 4 (0.3 g) was purified by Sephadex LH-20 column chromatography eluting with CH3OH and compound 1 was obtained after semi-preparative HPLC (5 mg, 96.8%). Compound 5 was obtained after semi-preparative HPLC (7 mg, 98.7%) from SubFr. 2 (0.2 g). SubFr. 3 (0.6 g) was fractionated into two parts by Sephadex LH-20 column chromatography eluting with CH3OH, and the second part yielded compounds 2 (23 mg, 97.3%) and 4 (5 mg, 96.4%). Fr. 1 (1.8 g) was subjected to Sephadex LH-20 column chromatography eluting with CH3OH to give A1–A3. A3 (1.1 g) was further separated by ODS column chromatography eluting with CH3OH–H2O (3:7 to 1:0, v/v) to give four fractions (Frs A1–A4). Fr. A4 (0.4 g) was applied to a semi-preparative HPLC to yield compound 3 (13 mg, 95.9%). The purity of compounds 1–5 was determined by the HPLC peak area method. 3.3.1. 3b-hydroxy-12-O-b-D -glucopyranosyl-8,11,13-abietatrien-7-one Compound 1, purity . 95% by HPLC: amorphous powder, ½a25 D ¼ 216.4 (c ¼ 0.14, MeOH); UV (MeOH) lmax: 201 nm. IR nmax (KBr): 3390, 2960, 1661, 1601 cm21. HR-ESI-MS m/z: 477.2491 [M–H]2 (calcd for C26H37O8, 477.2488). 1H NMR (600 MHz, CD3OD): d 1.72 (1H, m, H-1a), 2.41 (1H, dt, J ¼ 13.2, 3.0 Hz, H-1b), 1.85 (2H, m, H-2), 3.28 (1H, dd, J ¼ 10.2, 5.4 Hz, H-3), 1.83 (1H, dd, J ¼ 13.8, 4.2 Hz, H-5), 2.63 (1H, dd, J ¼ 18.0, 4.2 Hz, H-6a), 2.71(1H, dd, J ¼ 18.0, 13.8 Hz, H-6b), 7.13 (1H, s, H-11), 7.83 (1H, s, H-14), 3.41 (1H, m, J ¼ 7.2 Hz, H-15), 1.19 (3H, d, J ¼ 7.2 Hz, H-16), 1.24 (3H, d, J ¼ 7.2 Hz, H-17), 1.04 (3H, s, H-18), 0.96 (3H, s, H-19), 1.26 (3H, s, H-20), 5.02 (1H, d, J ¼ 7.2 Hz, H-10 ), 3.92 (1H, dd, J ¼ 12.0, 2.4 Hz, H-60 b), 3.69 (1H, dd, J ¼ 12.0, 6.6 Hz, H-60 a); 13C NMR (150 MHz, CD3OD): d 37.5 (C-1), 28.5 (C-2), 79.0 (C-3), 40.2 (C-4), 50.7 (C-5), 36.8 (C-6), 201.1 (C-7), 126.2 (C-8), 157.6 (C-9), 39.4 (C-10), 111.0 (C-11), 161.5 (C-12), 137.5 (C-13), 126.5 (C-14), 27.7 (C-15), 23.4 (C-16), 123.1 (C-17), 28.2 (C-18), 15.8 (C-19), 23.7 (C-20), 102.0 (C-10 ), 75.2 (C-20 ), 78.7 (C-30 ), 71.8 (C-40 ), 78.6 (C-50 ), 62.9 (C-60 ). 3.4. Bioactivity The cytotoxicity of compound 1 was assessed with MTT method using HepG-2 and A549 cancer cell lines supplied by the American Type Culture Collection. Taxol (purity . 99%,
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no. P106869) was purchased from ALADDIN (Xi’an Aladdin Biological Technology Co., Ltd, Xi’an, China). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum and were seeded at 6 £ 104 cells/mL per well in 96well plates. Compound 1 was dissolved in dimethyl sulfoxide (DMSO) and added at five different concentrations to the corresponding wells. After incubation in 5% CO2 at 378C for 48 h, 10 mL of MTT (4 mg/mL) was added to each well and incubated for another 4 h. Then, the supernates were aspirated, and 200 mL of DMSO was added. The absorbance was recorded at wavelength of 570 nm on a microplate reader (Ma et al. 2012). Taxol was used as positive control. The cell viability against H2O2-induced apoptosis in H9c2 cells treated with compounds 2– 5 was evaluated by MTT assay. The cells were cultured in DMEM supplemented with foetal bovine serum, 2 mM L -glutamine, 100 U/mL of penicillin, 100 mg/mL of streptomycin and maintained in a humidified incubator of 5% CO2 at 378C. Cells were seeded at 1 £ 104 cells/well in 96-well plates. After 4 h of treatment with different concentrations of compounds 2– 5 followed by incubation with 150 mM H2O2 for 6 h, 50 mL of 5 mg/mL MTT solution was added to each well (0.1 mg/well), and incubated for another 4 h. The supernates were aspirated, and the formazan crystals in each well were dissolved in 100 mL of DMSO. The absorbance was measured at 570 nm on a microplate reader (Sun et al. 2011). Supplementary material Supplementary material relating to this article is available online. Acknowledgements This work was financially supported by the National Nature Science Foundation of China (no. 81173511), the technological large platform for comprehensive research and development of new drugs in the Twelfth Five-Year ‘Significant New Drugs Created’ Science and Technology Major Project (no. 2012ZX09301002-001-026), and the chemical composition of the digital library of traditional Chinese medicine of drug discovery in the Twelfth Five-Year ‘Significant New Drugs Created’ (no. 2011ZX09307-002-01).
Note 1. These authors contributed equally to this work.
References Carlos A, Jaime AR, Ivan R, Tania Y, Cristina T, Guillemo SH. 2007. Gastroprotective and cytotoxic effect of semisynthetic ferruginol derivatives. J Pharm Pharmacol. 59:289–300. Chi HD, Lu JC. 2007. Advances in studies on medicinal plants in Clinopodium. J Shenyang Pharm Univ. 6:100–110. Gohar A, Gedara SR, Baraka HN. 2009. New acylated flavonol glycoside from Ceratonia siliqua L. seeds. J Med Plants Res. 3:424–428. Lewinsohn E, Berman E, Mazury Y, Gressel J. 1986. Glucosylation of exogenous flavanones by grapefruit (Citrus paradisi) cell cultures. Phytochemistry. 25:2531–2535. Lin S, Zhang YL, Liu MT, Yang S, Gan ML, Zi JC, Song WX, Fan XN, Wang SJ, Liu Y, et al., 2010. Abietane and C20-norabietane diterpenes from the stem bark of Fraxinus sieboldiana and their biological activities. J Nat Prod. 73:1914–1921. Ma GX, Xu XD, Cao L, Yuan JQ, Yang JS, Ma LY. 2012. Cassane-type diterpenes from the seeds of Caesalpinia minax with their antineoplastic activity. Planta Med. 78:1363–1369. Nakano H, Kawada N, Yoshida M, Ono H, Iwaura R, Tonooka T. 2011. Isolation and identification of flavonoids from accumulated in proanthocyanidin-free barely. J Agric Food Chem. 59:9581 –9587. Niu F, Cui Z, Li Q, Chang HT, Jiang Y, Qiao L, Tu PF. 2006. Complete assignments of 1H and 13C NMR spectral data for a novel diterpenoid from Semiaquilegia adoxoides. Magn Reson Chem. 44:724–726. Sun GB, Sun X, Wang M, Ye JX, Si JY, Xu HB, Meng XB, Qin M, Sun Y, Wang HW, Sun XB. 2012. Oxidative stress suppression by luteolin-induced heme oxygenase-1 expression. Toxicol Appl Pharmacol. 265:229–240.
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Sun GL, Zhang XP, Xu XD, Yang JS, Zhong ML, Yuan JQ. 2012. A new triterpene from Uncaria macrophylla and its antitumor activity. Molecules. 17:1883–1889. Sun X, Sun GB, Wang M, Xiao J, Sun XB. 2011. Protective effects of cynaroside against H2O2-induced apoptosis in H9c2 cardiomyoblasts. J Cell Biochem. 112:2019–2029. Yousuf S, Sudha N, Murugesan G, Enoch IVMV. 2013. Isolation of pruning from the fruit shell of Bixa orellana and the effect of b-cyclodextrin on its binding with calf thymus DNA. Carbohydr Res. 365:46–51. Zhong ML, Xu XD, Yu SC, Sun GL. 2012. Advances in studies on medicinal plants in Clinopodium Linn. Chin Tradit Herb Drugs. 43:820–828.
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A new diterpene from Clinopodium chinense Mingliang Zhong, Haifeng Wu, Xiaopo Zhang, Guangli Sun, Guibo Sun, Shichun Yu & Xudong Xu To cite this article: Mingliang Zhong, Haifeng Wu, Xiaopo Zhang, Guangli Sun, Guibo Sun, Shichun Yu & Xudong Xu (2014) A new diterpene from Clinopodium chinense, Natural Product Research, 28:7, 467-472, DOI: 10.1080/14786419.2013.879132 To link to this article: http://dx.doi.org/10.1080/14786419.2013.879132
View supplementary material
Published online: 31 Jan 2014.
Submit your article to this journal
Article views: 97
View related articles
View Crossmark data
Citing articles: 5 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20 Download by: [Athabasca University]
Date: 02 December 2016, At: 05:19
Natural Product Research, 2014 Vol. 28, No. 7, 467–472, http://dx.doi.org/10.1080/14786419.2013.879132
A new diterpene from Clinopodium chinense Mingliang Zhonga1, Haifeng Wua1, Xiaopo Zhanga, Guangli Sunb, Guibo Suna, Shichun Yuc and Xudong Xua* a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Peking Union Medical College, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing 100193, P.R. China; bDepartment of Pharmacy, School of Pharmacy, Hebei United University, Tangshan, Hebei 063000, P.R. China; cBeijing Hong Tai Chi Chung Medical Technology Co., Ltd., Beijing 102600, P.R. China
(Received 12 September 2013; final version received 23 December 2013) A new abietane diterpene, named as 3b-hydroxy-12-O-b-D -glucopyranosyl-8,11,13abietatrien-7-one (1), together with four known flavonoids, was isolated from the hot water extract of the aerial parts of Clinopodium chinense. Their structures were determined by analysing the spectroscopic data including 1D, 2D NMR and HR-ESI-MS. Compound 1 tested against HepG-2 and A549 cancer cell lines expressed weak cytotoxicity. Cardioprotective effects of compounds 2–5 against H2O2-induced apoptosis in H9c2 cells were also evaluated; compounds 2 and 3 exhibited moderate cardioprotective effect. Keywords: Clinopodium chinense; 3b-hydroxy-12-O-b-D -glucopyranosyl-8,11,13abietatrien-7-one; cytotoxicity; cardioprotective activity
1. Introduction Clinopodium chinense (Benth.) O. Kuntze (Labiatae), known as ‘Duanxueliu’ in Chinese Pharmacopoeia, has long been used as a traditional Chinese medicine for the treatment of influenza, heliosis, allergic dermatitis, dysentery, haematuria, trauma and haemorrhage (Chi & Lu 2007). Previous phytochemical investigation indicated the presence of flavonoids, triterpenoid saponins and volatile oil, responsible for haemostatic, anti-inflammatory, antioxidant, antibiotic and antihyperglycaemic activities (Zhong et al. 2012). Cardioprotective effects of some flavonoids on H9c2 cells were noteworthy (Sun et al. 2011; Sun, Sun, et al. 2012). In our ongoing research for bioactive constituents, a new abietane diterpene, 3b-hydroxy12-O-b-D -glucopyranosyl-8,11,13-abietatrien-7-one (1), as well as four known flavonoids, prunin (2), homoeriodictyol (3), hesperitin-7-O-b-D -glucopyranoside (4) and kaempferol-3-Orhamnopyranoside (5) were obtained from the ethanolic extract of the aerial parts of C. chinense. Herein, we present the structural elucidation and cytotoxicity of compound 1 as well as the cardioprotective effects of known compounds against H2O2-induced apoptosis in H9c2 cells. 2. Results and discussion Compound 1 (Figure 1) was obtained as a white amorphous powder with ½a25 D ¼ 2 16.4 (c ¼ 0.14, MeOH). It exhibited the molecular formula of C26H38O8 as deduced from its quasimolecular ion peak of [M –H]2 at m/z 477.2491 (calcd 477.2488) in an HR-ESI-MS experiment, consistent with eight degrees of unsaturation. The IR spectrum showed the presence of hydroxyl
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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M. Zhong et al. HO
6' 5'
O
O
4' OH 3'
R1 1' 2'
OH OH
12 11
20 2 3 HO
1 4
10 5
ORha O
OH OH
O 5
2 R1 = R3 = HR2 = OH R4 = Glu
O
3 R1 = OCH3 R2 = OH R3 = R4 = H
18
19
R3
17
8 7
O
HO O
R4O
14
9 6
13 15
OH R2
16
4 R1 = H R2 = OCH3 R3 = OH R4 = Glu
1
Figure 1. Chemical structures of compounds 1 –5.
(3390 cm21), carbonyl (1661 cm21) and phenyl (1601 cm21) groups. The 1H NMR spectrum of 1 showed signals of two aromatic protons [dH 7.83 (1H, s, H-14), 7.13 (1H, s, H-11)], an isopropyl group [dH 1.19 (3H, d, J ¼ 7.2 Hz, H-16), 1.24 (3H, d, J ¼ 7.2 Hz, H-17), 3.41 (1H, m, J ¼ 7.2 Hz, H-15)], three methyl groups bounded to quaternary carbon atoms [dH 1.26 (3H, s, H-20), 1.04 (3H, s, H-18), 0.96 (3H, s, H-19)] and an anomeric proton (dH 5.02). In the 13C NMR spectrum, 26 carbon signals were observed, which were resolved through the APT experiment into 5 methyl, 4 methylene, 10 methine and 7 quaternary carbons, including the resonance of a glucosyl group [dC 102.0 (C-10 ), 75.2 (C-20 ), 78.7 (C-30 ), 71.8 (C-40 ), 78.6 (C-50 ), 62.9 (C-60 )]. All carbon-bound protons were assigned from the HSQC spectrum and the locations of the substituents were determined by the HMBC spectrum. The overall 1H and 13C NMR spectroscopic data for 1 confirmed that the compound was an abietane-type diterpene skeleton (Niu et al. 2006). Comparison of the NMR spectra of 1 with that of abietatrien-12-O-bglucopyranoside (Carlos et al. 2007) reveals the appearance of hydroxyl and carbonyl groups in 1. The cross-peaks of dH 1.83 (H-5), 2.63 (H-6a), 2.71(H-6b), 7.13 (H-11) and 7.83 (H-14) with dC 201.1 (C-7) in the HMBC spectrum demonstrated that the carbonyl group was attached at C-7. The HMBC correlations of dH 1.04 (s, H3-18) and 0.96 (s, H3-19) with dC 79.0 (C-3) suggested that the hydroxyl group was located at C-3. The HMBC correlations from dH 5.02 (H-10 ) to dC 161.5 (C-12) indicated that the glucosyl group was connected with C-12, whereas correlations from dH 3.41 (H-15) to dC 161.5 (C-12)/126.5 (C-14), dH 1.19 (H3-16)/1.24 (H3-17) to dC 137.5 (C-13) suggested that the isopropyl group was linked to C-13 (Figure 2).
HMBC
HO O
O
NOESY
OH OH
OH HO H
HO
O
Figure 2. Key HMBC and NOESY correlations of compound 1.
H
O
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Table 1. In vitro cytotoxicity of compound 1. IC50 (mg/mL)
Compounds HepG-2 1 Taxolb a b
A549
76.8 ^ 1.7 5.6 ^ 0.13 a
68.1 ^ 2.1 4.8 ^ 0.24
Values present mean ^ SD of triplicate experiments. Positive control substance.
The relative stereochemistry of 1 was resolved by analysis of NOESY spectrum and H –H coupling constant value (J). The evidence from the large J value of the axial proton at C-3 (dH 3.28, dd, J ¼ 10.2, 5.4 Hz) suggested that the hydroxyl group at C-3 is positioned in b-configuration (Sun, Zhang, et al. 2012). The NOESY correlations between H-3 and H-5/H-18, H-19 and H-20 (Figure 2) indicated that methyl groups at C-19 and C-20 were b-oriented, whereas methyl at C-18 and H-5 were a-oriented. The J value of the anomeric proton (J ¼ 7.2 Hz) confirmed a b-configuration of the glucosyl group (Carlos et al. 2007). Based on the above-mentioned analysis, compound 1 was assigned as 3b-hydroxy-12-O-b-D -glucopyranosyl8,11,13-abietatrien-7-one. In addition, four known flavonoids, namely prunin (2), homoeriodictyol (3), hesperitin-7-Ob-D -glucopyranoside (4) and kaempferol-3-O-rhamnopyranoside (5) (Lewinsohn et al. 1986; Gohar et al. 2009; Nakano et al. 2011; Yousuf et al. 2013), were obtained and identified by comparing their spectroscopic data with those reported in the literature, and 3 – 5 were first isolated from the genus Clinopodium. In the cytotoxic assay, compound 1 exhibited weak antineoplastic activity against HepG-2 (IC50 76.8 mg/mL) and A549 (IC50 68.1 mg/mL) cell lines, compared with the positive control taxol (Table 1). It was previously reported that some abietane diterpenes with similar structures expressed moderate cytotoxicity against several cancer cell lines (Carlos et al. 2007; Lin et al. 2010). Moreover, cardioprotective effects of 2 –5 against H2O2-induced apoptosis in H9c2 cells were tested; compounds 2 and 3 were active in cardioprotection. The cell viability was improved from 85.7% to 91.6% (2) and 82.3% to 91.9% (3) at concentrations of 5 and 25 mg/mL, respectively, compared with those of exposure to H2O2. In conclusion, we described a new abietane diterpene, 3b-hydroxy-12-O-b-D -glucopyranosyl8,11,13-abietatrien-7-one (1) and four known compounds, prunin (2), homoeriodictyol (3), hesperitin-7-O-b-D -glucopyranoside (4) and kaempferol-3-O-rhamnopyranoside (5). Compound 1 expressed weak cytotoxicity against HepG-2 and A549 cancer cell lines, while compounds 2 and 3 exhibited moderate cardioprotective effect against H2O2-induced apoptosis in H9c2 cell line. 3. Experimental 3.1. General experimental procedures UV spectra were recorded on a Shimadzu UV-2550 UV/vis spectrophotometer (Shimadzu Corporation, Tokyo, Japan). IR spectrum was acquired using a Shimadzu FTIR-8400S spectrophotometer (Shimadzu Corporation, Tokyo, Japan). Optical rotations were measured by a Perkin-EImer 241 polarimeter (PerkinElmer, Inc., Waltham, US). The NMR data (1D and 2D NMR) were recorded on a Bruker AV 600 instrument (Bruker BioSpin Corporation, Billerica, US) in CD3OD or DMSO-d6 with TMS as internal standard (600 MHz for 1H and 150 MHz for 13 C). The HR-ESI-MS data were obtained by LTQ-Orbitrap mass spectrophotometer (ThermoFisher Scientific, Bremen, Germany). Chromatography was performed on AB-8 macroporous resin (Chemical Plant of NanKai University, Tianjin, China), silica gel (200 –300 mesh,
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Qingdao Haiyang Chemical Factory, Qingdao, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and ODS gel (40 – 60 mm, Daiso Co., Ltd, Osaka, Japan). Semipreparative HPLC was performed on a CXTH LC-3000 HPLC system with a CXTH LC-3000 UV spectrophotometric detector (Beijing Chuangxintongheng Science and Technology Co., Ltd, Beijing, China). 3.2. Plant material The plants were collected from Anhui Province in 2011, and identified by Dr Jing Quan Yuan at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College and a voucher specimen (no. 20101132) has been deposited in the same institute. 3.3. Extraction and isolation The dried aerial parts of C. chinense (5 kg) were decocted two times with boiled water (50 L, 2 h each time), and the combined solution was concentrated under reduced pressure to yield an extract (400 g). The extract was subjected to AB-8 macroporous resin column chromatography using ethanol–H2O (0:100 to 95:5, v/v) and the 50–95% fraction yielded the total flavonoids of C. chinense (CCF). The CCF (103 g) was subjected to column chromatography on silica gel with CHCl3 –MeOH (20:1 to 0:1, v/v), and fractionated into eight fractions (Frs 1–8). Fr. 4 (3.1 g) was subjected to silica gel column chromatography eluting with chloroform–acetone (0:1 to 1:0, v/v) to yield five subfractions (SubFrs 1–5). SubFr. 4 (0.3 g) was purified by Sephadex LH-20 column chromatography eluting with CH3OH and compound 1 was obtained after semi-preparative HPLC (5 mg, 96.8%). Compound 5 was obtained after semi-preparative HPLC (7 mg, 98.7%) from SubFr. 2 (0.2 g). SubFr. 3 (0.6 g) was fractionated into two parts by Sephadex LH-20 column chromatography eluting with CH3OH, and the second part yielded compounds 2 (23 mg, 97.3%) and 4 (5 mg, 96.4%). Fr. 1 (1.8 g) was subjected to Sephadex LH-20 column chromatography eluting with CH3OH to give A1–A3. A3 (1.1 g) was further separated by ODS column chromatography eluting with CH3OH–H2O (3:7 to 1:0, v/v) to give four fractions (Frs A1–A4). Fr. A4 (0.4 g) was applied to a semi-preparative HPLC to yield compound 3 (13 mg, 95.9%). The purity of compounds 1–5 was determined by the HPLC peak area method. 3.3.1. 3b-hydroxy-12-O-b-D -glucopyranosyl-8,11,13-abietatrien-7-one Compound 1, purity . 95% by HPLC: amorphous powder, ½a25 D ¼ 216.4 (c ¼ 0.14, MeOH); UV (MeOH) lmax: 201 nm. IR nmax (KBr): 3390, 2960, 1661, 1601 cm21. HR-ESI-MS m/z: 477.2491 [M–H]2 (calcd for C26H37O8, 477.2488). 1H NMR (600 MHz, CD3OD): d 1.72 (1H, m, H-1a), 2.41 (1H, dt, J ¼ 13.2, 3.0 Hz, H-1b), 1.85 (2H, m, H-2), 3.28 (1H, dd, J ¼ 10.2, 5.4 Hz, H-3), 1.83 (1H, dd, J ¼ 13.8, 4.2 Hz, H-5), 2.63 (1H, dd, J ¼ 18.0, 4.2 Hz, H-6a), 2.71(1H, dd, J ¼ 18.0, 13.8 Hz, H-6b), 7.13 (1H, s, H-11), 7.83 (1H, s, H-14), 3.41 (1H, m, J ¼ 7.2 Hz, H-15), 1.19 (3H, d, J ¼ 7.2 Hz, H-16), 1.24 (3H, d, J ¼ 7.2 Hz, H-17), 1.04 (3H, s, H-18), 0.96 (3H, s, H-19), 1.26 (3H, s, H-20), 5.02 (1H, d, J ¼ 7.2 Hz, H-10 ), 3.92 (1H, dd, J ¼ 12.0, 2.4 Hz, H-60 b), 3.69 (1H, dd, J ¼ 12.0, 6.6 Hz, H-60 a); 13C NMR (150 MHz, CD3OD): d 37.5 (C-1), 28.5 (C-2), 79.0 (C-3), 40.2 (C-4), 50.7 (C-5), 36.8 (C-6), 201.1 (C-7), 126.2 (C-8), 157.6 (C-9), 39.4 (C-10), 111.0 (C-11), 161.5 (C-12), 137.5 (C-13), 126.5 (C-14), 27.7 (C-15), 23.4 (C-16), 123.1 (C-17), 28.2 (C-18), 15.8 (C-19), 23.7 (C-20), 102.0 (C-10 ), 75.2 (C-20 ), 78.7 (C-30 ), 71.8 (C-40 ), 78.6 (C-50 ), 62.9 (C-60 ). 3.4. Bioactivity The cytotoxicity of compound 1 was assessed with MTT method using HepG-2 and A549 cancer cell lines supplied by the American Type Culture Collection. Taxol (purity . 99%,
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no. P106869) was purchased from ALADDIN (Xi’an Aladdin Biological Technology Co., Ltd, Xi’an, China). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum and were seeded at 6 £ 104 cells/mL per well in 96well plates. Compound 1 was dissolved in dimethyl sulfoxide (DMSO) and added at five different concentrations to the corresponding wells. After incubation in 5% CO2 at 378C for 48 h, 10 mL of MTT (4 mg/mL) was added to each well and incubated for another 4 h. Then, the supernates were aspirated, and 200 mL of DMSO was added. The absorbance was recorded at wavelength of 570 nm on a microplate reader (Ma et al. 2012). Taxol was used as positive control. The cell viability against H2O2-induced apoptosis in H9c2 cells treated with compounds 2– 5 was evaluated by MTT assay. The cells were cultured in DMEM supplemented with foetal bovine serum, 2 mM L -glutamine, 100 U/mL of penicillin, 100 mg/mL of streptomycin and maintained in a humidified incubator of 5% CO2 at 378C. Cells were seeded at 1 £ 104 cells/well in 96-well plates. After 4 h of treatment with different concentrations of compounds 2– 5 followed by incubation with 150 mM H2O2 for 6 h, 50 mL of 5 mg/mL MTT solution was added to each well (0.1 mg/well), and incubated for another 4 h. The supernates were aspirated, and the formazan crystals in each well were dissolved in 100 mL of DMSO. The absorbance was measured at 570 nm on a microplate reader (Sun et al. 2011). Supplementary material Supplementary material relating to this article is available online. Acknowledgements This work was financially supported by the National Nature Science Foundation of China (no. 81173511), the technological large platform for comprehensive research and development of new drugs in the Twelfth Five-Year ‘Significant New Drugs Created’ Science and Technology Major Project (no. 2012ZX09301002-001-026), and the chemical composition of the digital library of traditional Chinese medicine of drug discovery in the Twelfth Five-Year ‘Significant New Drugs Created’ (no. 2011ZX09307-002-01).
Note 1. These authors contributed equally to this work.
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