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A new neo-clerodane diterpene from Ajuga decumbens a
a
a
Huawei Lv , Jianguang Luo & Lingyi Kong a
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P.R. China Published online: 27 Jan 2014.
To cite this article: Huawei Lv, Jianguang Luo & Lingyi Kong (2014) A new neo-clerodane diterpene from Ajuga decumbens, Natural Product Research: Formerly Natural Product Letters, 28:3, 196-200, DOI: 10.1080/14786419.2013.866114 To link to this article: http://dx.doi.org/10.1080/14786419.2013.866114
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 & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 3, 196–200, http://dx.doi.org/10.1080/14786419.2013.866114
A new neo-clerodane diterpene from Ajuga decumbens Huawei Lv, Jianguang Luo and Lingyi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P.R. China
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(Received 21 August 2013; final version received 31 October 2013) A new neo-clerodane diterpene, named ajugacumbin J (1), together with 13 known compounds (2 – 14) was isolated from Ajuga decumbens. The structure of ajugacumbin J (1) was elucidated by 1D and 2D NMR spectra and MS. Ajugacumbin J (1) and ajugacumbin D (5) exhibited inhibition of lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages with an IC50 value of 46.2 and 35.9 mM, respectively. Keywords: Ajuga decumbens; neo-clerodane diterpene; anti-inflammatory activity
1. Introduction The genus Ajuga (Labiatae) is distributed over the temperate parts of Asia and Europe. Several species of this genus have been reported to be a rich source of diterpenes (Coll & Tandrno´n 2008; Guo et al. 2011). Ajuga decumbens Thunb is widely distributed in China, Korea and Japan. The whole plant of A. decumbens is used as a traditional Chinese medicine for its anti-inflammatory, antitussive and expectorant effects (Chinese Pharmacopoeia Commission 2010). Previous phytochemical investigations of A. decumbens resulted in the isolation of a series of diterpenoids (Min et al. 1990; Takasaki et al. 1998; Huang et al. 2008; Sun et al. 2012, Wang et al. 2012), ecdysteroids (Takasaki et al. 1999) and iridoids (Takeda et al. 1987; Konoshima et al. 2000). In the course of our search for bioactive metabolites, a new neo-clerodane diterpene, named ajugacumbin J (1) (Figure 1), together with 12 known diterpenes (2– 13) and 1 dipeptide (14), was isolated from the whole plant of A. decumbens. Herein, we report on the isolation and structural elucidation of ajugacumbin J (1) as well as on the inhibitory activities of lipopolysaccharide (LPS)-induced nitric oxide (NO) production of compounds 1–6 and 8–14 in RAW 264.7 macrophages.
2. Results and discussion Ajugacumbin J (1) was obtained as colourless oil. The molecular formula was established as C30H42O10 by HRESI-MS at m/z 597.2468 [M þ Cl]2. The IR spectrum showed absorption bands due to ester carbonyl groups (1739 cm21), aldehyde group (1690 cm21) and double bond group (1644 cm21). The 1H NMR spectrum of 1 showed one secondary methyl group at dH 0.81 (3H, d, J ¼ 6.5 Hz, H-17), one tertiary methyl group at dH 0.91 (3H, s, H-20) and downfield methylene protons at dH 4.89 and 4.43 (each 1H, d, J ¼ 12.5 Hz, H-19a and H-19b), which were characteristic chemical shifts and multiplicities for a clerodin-like diterpene previously isolated from the genus Ajuga (Min et al. 1989). The 13C NMR spectrum showed 30 carbon signals, including 5 carbonyl carbons, 4 olefinic carbons, 6 oxygenated carbons and 15 aliphatic carbons,
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Figure 1. The structures of compounds 1 and 1a.
of them, the following 10 carbon resonances (dC 170.5 and 21.4; 170.1 and 21.3; 166.8, 128.8, 138.8, 14.6 and 12.2; 52.2) as well as the presence of the proton signals [dH 2.16 (3H, s); 1.96 (3H, s); 6.65 (1H, dq, J ¼ 0.5, 7.0 Hz), 1.71 (3H, d, J ¼ 7.0 Hz) and 1.76 (3H, s); 3.63 (3H, s)] revealed the presence of two acetoxy groups, one tigloyloxy group and one methoxy group. The 1 H and 13C NMR of compound 1 exhibited a number of similarities to those of 1a (Sun et al. 2012). The main differences between them were the presence of an epoxide ring [dH 3.12 (1H, brs) and 2.37 (1H, d, J ¼ 4.0 Hz); dC 49.8] in 1, instead of the chloromethyl group [dH 4.07, 3.99 (each 1H, ABq, J ¼ 11.3 Hz); dC 49.5] in 1a. The HMBC correlations of H-18 with C-3 and C-4 supported the location of the epoxide ring at C-4 of 1. In addition, the HMBC correlations of H-11 with C-12, C-13 and C-20, H-14 with C-12, C-15 and C-16 as well as the ROESY crosspeak between H-11 and H-14 were observed, confirming the structure of the side chain at C-9 as shown in Figure 1. The full assignments of the proton and carbon signals were made unambiguously by HSQC and HMBC data. The relative configuration of compound 1 was elucidated as follows. The ROESY spectrum showed correlations of H-3a with H-1, H-19; H-10 with H-18 in ring A and H-10 with H-6, H-8; H-7a with H-20 and H-19 with H-20 in ring B, suggesting that the two six-membered rings were trans-fused and both existed in chair configuration, H-6, H-10 and H-18 were b oriented while H-1, H-17, H-19 and H-20 were a oriented. Therefore, the structure of compound 1 was determined as 6a,19-diacetoxy-1btigloyloxy-4a,18-epoxy-neo-clerod-12-en-15-oic acid methyl ester-16-aldehyde, a new natural product and named as ajugacumbin J. The structures of known compounds were identified as ajugacumbins A – D (2 –5) (Min et al. 1989), ajugarins I (6), II (7) (Coll 2002), ajugalides B (8), D (9), ajugapantin A (10) (Chan 2005), ajugamarin (11) (Shimomura et al. 1983), (12S)-1b,6a,19-triacetoxy-12-[(2S)-2-methylbutanoyloxy]-4,18-epoxyneo-clerod-13(14)-en-15,16-olide (12) (Shimomura et al. 1989), ajuganipponin A (13) (Coll & Tandrno´n 2006) and aurantiamide acetate (14) (Wahidulla et al. 1991) by comparison of their spectroscopic data with previously reported values. Compounds 1 –6 and 8 –14 were tested for their abilities to inhibit LPS-induced NO production in RAW 264.7 macrophages. The results showed that compound 1 and ajugacumbin D (5) expressed inhibitory effects with an IC50 value of 46.2 and 35.9 mM, respectively. N-Monomethyl-L -arginine was used as the positive control, with an IC50 value of 39.2 mM. 3. Experiments 3.1. General experimental procedures Optical rotation was measured with a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan). UV spectrum was recorded using a UV-2450 UV – visible spectrophotometer (Shimadzu, Tokyo,
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Japan). IR spectrum (KBr discs) was recorded on a Bruker Tensor 27 spectrometer (Bruker, Karlsruhe, Germany). NMR spectra were recorded on a Bruker AV-500 NMR instrument (1H: 500 MHz, 13C: 125 MHz, Bruker, Karlsruhe, Germany), with TMS as an internal standard. HRESIMS was measured with an Agilent 6520B Q-TOF mass instrument (Agilent Technologies, Santa Clara, CA, USA). Extracts were chromatographed on silica gel (Qingdao Marine Chemical Co., Ltd, Qingdao, China), ODS (40 –63 mm, FuJi, Japan), microporous resin (MCI) gel CHP-20 (Mitsubishi, Tokyo, Japan), Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and MPLC (Lisui, Suzhou, China), and purified on an Agilent 1100 Series prep-HPLC system (Agilent Technologies, Santa Clara, CA, USA) and a Shimadzu CBM-20A recyclingpreparative HPLC system (Shimadzu, Tokyo, Japan). 3.2. Plant material The whole plant of A. decumbens Thunb was collected from Fujian Province, China, in August 2011, and was authenticated by Professor Mian Zhang, Department of Medicinal Plants, China Pharmaceutical University. A voucher specimen (No. 110813AD) is deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. 3.3. Extraction and isolation The air-dried whole plant of A. decumbens (800 g) was ultrasonically extracted with 4 l MeOH (2 h £ 3). The crude extract (102 g) was suspended in water and successively partitioned with petroleum ether, CH2Cl2 and EtOAc. The CH2Cl2 fraction (28 g) was subjected to an MCI column with MeOH –H2O (3:7, 8:2, 10:0, v/v) to yield three fractions (A– C). Fraction B afforded 2 (1.2 g) after repeated crystallisation from MeOH. The mother liquor was performed on a silica gel column using a gradient of petroleum ether – Me2CO (7:3, 1:1, 3:7, 0:1, v/v) to yield four fractions (B1 –B4). Fraction B2 was subsequently separated by MPLC with a continuous gradient of MeOH – H2O (1:1 to 1:0, v/v, 10 ml/min) to yield 35 fractions. Fractions 13 –15 were combined and subjected to Sephadex LH-20 eluting with MeOH, subsequently separated by recycling-preparative HPLC using CH3CN – H2O (40:60, v/v, 10 ml/min) to afford 7 (2.0 mg), 8 (3.0 mg) and 10 (3.0 mg). Fractions 18– 23 were purified by prep-HPLC (MeOH – H2O, 60:40, v/v, 10 ml/min) to obtain 6 (8.0 mg), 9 (8.0 mg), 11 (9.0 mg) and 13 (3.0 mg). Fractions 24– 26 were purified by prep-HPLC (MeOH – H2O, 65:35, v/v, 10 ml/min) to afford 3 (12.0 mg), 4 (20.0 mg) and 12 (5.0 mg). Fraction 28 was performed on prep-HPLC (CH3CN – H2O, 60:40, v/v, 10 ml/min) to yield 1 (3.0 mg), 5 (14.0 mg) and 14 (6.0 mg). 3.4. Ajugacumbin J (1) Colourless oil; ½a25 d þ 29.8 (c 0.11, MeOH); UV (MeOH) lmax (log 1) 221 (3.78); IR (KBr) nmax 2959, 2926, 2852, 1739, 1690, 1644, 1436, 1397, 1251, 1134, 1081, 1026, 903 and 735 cm21; 1H NMR (500 MHz, CDCl3): d 5.77 (1H, dt, J ¼ 5.0, 10.5 Hz, H-1), 2.25 (1H, m, H-2a), 1.64 (1H, m, H-2b), 2.40 (1H, m, H-3a), 1.17 (1H, m, H-3b), 4.72 (1H, dd, J ¼ 11.5, 4.5 Hz, H-6), 1.53 (1H, m, H-7a), 1.62 (1H, m, H-7b), 1.53 (1H, m, H-8), 2.14 (1H, d, J ¼ 11.0 Hz, H-10), 2.46 (2H, d, J ¼ 8.5 Hz, H-11), 6.83 (1H, dd, J ¼ 8.5, 4.5 Hz, H-12), 3.23 (2H, s, H-14),d 9.51 (1H, s, H-16), 0.81 (3H, d, J ¼ 6.5 Hz, H-17), 3.12 (1H, brs, H-18a), 2.37 (1H, d, J ¼ 4.0 Hz, H-18b), 4.89 (1H, d, J ¼ 12.5 Hz, H-19a), 4.43 (1H, d, J ¼ 12.5 Hz, H-19b), 0.91 (3H, s, H-20), 6.65 (1H, dq, J ¼ 0.5, 7.0 Hz, H-30 ), 1.71 (3H, d, J ¼ 7.0 Hz, H-40 ), 1.76 (3H, s, H-50 ), 3.63 (3H, s, 15-OCH3), 2.16 [3H, s, methyl of acetate (at C-19)], 1.96 [3H, s, methyl of acetate (at C-6)]; 13C NMR (125 MHz, CDCl3):d 70.3 (C-1), 31.8 (C-2), 30.4 (C-3), 64.5 (C-4), 45.7 (C-5), 71.8 (C-6), 33.3 (C-7), 36.8 (C-8), 40.1 (C-9), 51.0 (C-10), 38.4 (C-11), 153.0 (C-12),
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138.4 (C-13), 29.7 (C-14), 170.0 (C-15), 193.1 (C-16), 15.7 (C-17), 49.8 (C-18), 62.8 (C-19), 17.5 (C-20), 166.8 (C-10 ), 128.8 (C-20 ), 138.8 (C-30 ), 14.6 (C-40 ), 12.2 (C-50 ), 170.1 [carbonyl of acetate (at C-6)], 21.3 [methyl of acetate (at C-6)], 170.5 [carbonyl of acetate (at C-19)], 21.4 [methyl of acetate (at C-19)], 52.2 (15-OMe); ESI-MS: m/z 580 [M þ NH4]þ, 597 [M þ Cl]2; HRESI-MS: m/z 597.2468 [M þ Cl]2 (calcd for C30H42O10Cl, 597.2472). 3.5. Nitric oxide production bioassay
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The protocol for NO production bioassays was provided in previously published papers (Yin et al. 2013). N-Monomethyl-L -arginine was used as the positive control. All experiments were conducted in three replicates. 4. Conclusions In this paper, 13 diterpenes and 1 dipeptide, including a new neo-clerodane diterpene, named ajugacumbin J, were isolated from the whole plant of A. decumbens. The structure of ajugacumbin J (1) was identified as 6a,19-diacetoxy-1b-tigloyloxy-4a,18-epoxy-neo-clerod12-en-15-oic acid methyl ester-16-aldehyde (1) by 1D and 2D NMR spectra and MS. Bioassay results showed that compound 1 and ajugacumbin D (5) exhibited the inhibitory activities of LPS-induced NO production in RAW 264.7 macrophages. Supplementary material Supplementary material relating to this article is available online: 1D NMR and 2D NMR spectra and HRESIMS of 1 (Figures S1 –S8). Acknowledgements This research was supported by the National Key Scientific and Technological Special Projects (2012ZX09103-101-007), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193).
References Chan YY. 2005. Neoclerodane diterpenoids from Ajuga taiwanensis. Chem Pharm Bull. 53:164–167. Chinese Pharmacopoeia Commission. 2010. Chinese pharmacopoeia. Vol. 1. Beijing: The Medicine Science and Technology Press of China. p. 325. Coll J. 2002. NMR shift data of neo-clerodane diterpenes from the genus Ajuga. Phytochem Anal. 13:372–380. Coll J, Tandrno´n YA. 2006. Isolation and identification of neo-clerodane diterpenes from Ajuga nipponensis Makino. Nat Prod Commun. 1:183–189. Coll J, Tandrno´n YA. 2008. Neo-clerodane diterpenoids from Ajuga: structural elucidation and biological activity. Phytochem Rev. 7:25–49. Guo P, Li Y, Xu J, Liu C, Ma Y, Guo Y. 2011. Bioactive neo-clerodane diterpenoids from the whole plants of Ajuga ciliata Bunge. J Nat Prod. 74:1575–1583. Huang XC, Qin S, Guo YW, Krohn K. 2008. Four new neoclerodane diterpenoids from Ajuga decumbens. Helv Chim Acta. 91:628–634. Konoshima T, Takasaki M, Tokuda H, Nishino H. 2000. Cancer chemopreventive activity of an iridoid glycoside, 8-acetylharpagide, from Ajuga decumbens. Cancer Lett. 157:87–92. Min Z, Mizuo M, Wang S, Munekazu I, Toshiyuki T. 1990. Two new neo-clerodane diterpenes in Ajuga decumbens. Chem Pharm Bull. 38:3167–3168. Min ZD, Wang SQ, Zheng QT, Wu B, Mizuo M, Toshiyuki T, Munekazu I. 1989. Four new insect antifeedant neoclerodane diterpenoids, Ajugacumbins A, B, C and D, from Ajuga decumbens. Chem Pharm Bull. 37:2505– 2508.
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Shimomura H, Sashida Y, Ogawa K. 1989. Neo-clerodane diterpenes from Ajuga ciliata var. Villosior. Chem Pharm Bull. 37:988–992. Shimomura H, Sashida Y, Ogawa K, Iitaka Y. 1983. The chemical constituents of Ajuga plants. I. neo-cleroanes from the leaves of Ajuga nipponensis Makino. Chem Pharm Bull. 31:2192– 2199. Sun Z, Li Y, Jin D, Guo P, Xu J, Guo Y, Zhang L. 2012. Structure elucidation and inhibitory effects on NO production of clerodane diterpenes from Ajuga decumbens. Planta Med. 78:1579–1583. Takasaki M, Tokuda H, Nishino H, Konoshima T. 1999. Cancer chemopreventive agents (antitumor-promoters) from Ajuga decumbens. J Nat Prod. 62:972–975. Takasaki M, Yamauchi I, Haruna M, Konoshima T. 1998. New glycosides from Ajuga decumbens. J Nat Prod. 61:1105–1109. Takeda Y, Tsuchida S, Fujita T. 1987. Four new iridoid glucoside p-coumaroyl esters from Ajuga decumbens. Phytochemistry. 26:2303–2306. Wang B, Wang XN, Tao S, Wang SQ, Guo DX, Lou HX. 2012. Rearranged abietane diterpenoid hydroquinones from aerial parts of Ajuga decumbens Thunb. Phytochem Lett. 5:271–275. Wahidulla S, D’Souza L, Kamat SY. 1991. Dipeptides from the red alga Acantophora spicifera. Phytochemistry. 30:3323–3325. Yin H, Luo JG, Shan SM, Wang XB, Luo J, Yang MH, Kong LY. 2013. Amomaxins A and B, two unprecedented rearranged labdane norditerpenoids with a nine-membered ring from Amomum maximum. Org Lett. 15:1572–1575.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new neo-clerodane diterpene from Ajuga decumbens a
a
a
Huawei Lv , Jianguang Luo & Lingyi Kong a
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P.R. China Published online: 27 Jan 2014.
To cite this article: Huawei Lv, Jianguang Luo & Lingyi Kong (2014) A new neo-clerodane diterpene from Ajuga decumbens, Natural Product Research: Formerly Natural Product Letters, 28:3, 196-200, DOI: 10.1080/14786419.2013.866114 To link to this article: http://dx.doi.org/10.1080/14786419.2013.866114
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 & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 3, 196–200, http://dx.doi.org/10.1080/14786419.2013.866114
A new neo-clerodane diterpene from Ajuga decumbens Huawei Lv, Jianguang Luo and Lingyi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P.R. China
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(Received 21 August 2013; final version received 31 October 2013) A new neo-clerodane diterpene, named ajugacumbin J (1), together with 13 known compounds (2 – 14) was isolated from Ajuga decumbens. The structure of ajugacumbin J (1) was elucidated by 1D and 2D NMR spectra and MS. Ajugacumbin J (1) and ajugacumbin D (5) exhibited inhibition of lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages with an IC50 value of 46.2 and 35.9 mM, respectively. Keywords: Ajuga decumbens; neo-clerodane diterpene; anti-inflammatory activity
1. Introduction The genus Ajuga (Labiatae) is distributed over the temperate parts of Asia and Europe. Several species of this genus have been reported to be a rich source of diterpenes (Coll & Tandrno´n 2008; Guo et al. 2011). Ajuga decumbens Thunb is widely distributed in China, Korea and Japan. The whole plant of A. decumbens is used as a traditional Chinese medicine for its anti-inflammatory, antitussive and expectorant effects (Chinese Pharmacopoeia Commission 2010). Previous phytochemical investigations of A. decumbens resulted in the isolation of a series of diterpenoids (Min et al. 1990; Takasaki et al. 1998; Huang et al. 2008; Sun et al. 2012, Wang et al. 2012), ecdysteroids (Takasaki et al. 1999) and iridoids (Takeda et al. 1987; Konoshima et al. 2000). In the course of our search for bioactive metabolites, a new neo-clerodane diterpene, named ajugacumbin J (1) (Figure 1), together with 12 known diterpenes (2– 13) and 1 dipeptide (14), was isolated from the whole plant of A. decumbens. Herein, we report on the isolation and structural elucidation of ajugacumbin J (1) as well as on the inhibitory activities of lipopolysaccharide (LPS)-induced nitric oxide (NO) production of compounds 1–6 and 8–14 in RAW 264.7 macrophages.
2. Results and discussion Ajugacumbin J (1) was obtained as colourless oil. The molecular formula was established as C30H42O10 by HRESI-MS at m/z 597.2468 [M þ Cl]2. The IR spectrum showed absorption bands due to ester carbonyl groups (1739 cm21), aldehyde group (1690 cm21) and double bond group (1644 cm21). The 1H NMR spectrum of 1 showed one secondary methyl group at dH 0.81 (3H, d, J ¼ 6.5 Hz, H-17), one tertiary methyl group at dH 0.91 (3H, s, H-20) and downfield methylene protons at dH 4.89 and 4.43 (each 1H, d, J ¼ 12.5 Hz, H-19a and H-19b), which were characteristic chemical shifts and multiplicities for a clerodin-like diterpene previously isolated from the genus Ajuga (Min et al. 1989). The 13C NMR spectrum showed 30 carbon signals, including 5 carbonyl carbons, 4 olefinic carbons, 6 oxygenated carbons and 15 aliphatic carbons,
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Figure 1. The structures of compounds 1 and 1a.
of them, the following 10 carbon resonances (dC 170.5 and 21.4; 170.1 and 21.3; 166.8, 128.8, 138.8, 14.6 and 12.2; 52.2) as well as the presence of the proton signals [dH 2.16 (3H, s); 1.96 (3H, s); 6.65 (1H, dq, J ¼ 0.5, 7.0 Hz), 1.71 (3H, d, J ¼ 7.0 Hz) and 1.76 (3H, s); 3.63 (3H, s)] revealed the presence of two acetoxy groups, one tigloyloxy group and one methoxy group. The 1 H and 13C NMR of compound 1 exhibited a number of similarities to those of 1a (Sun et al. 2012). The main differences between them were the presence of an epoxide ring [dH 3.12 (1H, brs) and 2.37 (1H, d, J ¼ 4.0 Hz); dC 49.8] in 1, instead of the chloromethyl group [dH 4.07, 3.99 (each 1H, ABq, J ¼ 11.3 Hz); dC 49.5] in 1a. The HMBC correlations of H-18 with C-3 and C-4 supported the location of the epoxide ring at C-4 of 1. In addition, the HMBC correlations of H-11 with C-12, C-13 and C-20, H-14 with C-12, C-15 and C-16 as well as the ROESY crosspeak between H-11 and H-14 were observed, confirming the structure of the side chain at C-9 as shown in Figure 1. The full assignments of the proton and carbon signals were made unambiguously by HSQC and HMBC data. The relative configuration of compound 1 was elucidated as follows. The ROESY spectrum showed correlations of H-3a with H-1, H-19; H-10 with H-18 in ring A and H-10 with H-6, H-8; H-7a with H-20 and H-19 with H-20 in ring B, suggesting that the two six-membered rings were trans-fused and both existed in chair configuration, H-6, H-10 and H-18 were b oriented while H-1, H-17, H-19 and H-20 were a oriented. Therefore, the structure of compound 1 was determined as 6a,19-diacetoxy-1btigloyloxy-4a,18-epoxy-neo-clerod-12-en-15-oic acid methyl ester-16-aldehyde, a new natural product and named as ajugacumbin J. The structures of known compounds were identified as ajugacumbins A – D (2 –5) (Min et al. 1989), ajugarins I (6), II (7) (Coll 2002), ajugalides B (8), D (9), ajugapantin A (10) (Chan 2005), ajugamarin (11) (Shimomura et al. 1983), (12S)-1b,6a,19-triacetoxy-12-[(2S)-2-methylbutanoyloxy]-4,18-epoxyneo-clerod-13(14)-en-15,16-olide (12) (Shimomura et al. 1989), ajuganipponin A (13) (Coll & Tandrno´n 2006) and aurantiamide acetate (14) (Wahidulla et al. 1991) by comparison of their spectroscopic data with previously reported values. Compounds 1 –6 and 8 –14 were tested for their abilities to inhibit LPS-induced NO production in RAW 264.7 macrophages. The results showed that compound 1 and ajugacumbin D (5) expressed inhibitory effects with an IC50 value of 46.2 and 35.9 mM, respectively. N-Monomethyl-L -arginine was used as the positive control, with an IC50 value of 39.2 mM. 3. Experiments 3.1. General experimental procedures Optical rotation was measured with a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan). UV spectrum was recorded using a UV-2450 UV – visible spectrophotometer (Shimadzu, Tokyo,
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Japan). IR spectrum (KBr discs) was recorded on a Bruker Tensor 27 spectrometer (Bruker, Karlsruhe, Germany). NMR spectra were recorded on a Bruker AV-500 NMR instrument (1H: 500 MHz, 13C: 125 MHz, Bruker, Karlsruhe, Germany), with TMS as an internal standard. HRESIMS was measured with an Agilent 6520B Q-TOF mass instrument (Agilent Technologies, Santa Clara, CA, USA). Extracts were chromatographed on silica gel (Qingdao Marine Chemical Co., Ltd, Qingdao, China), ODS (40 –63 mm, FuJi, Japan), microporous resin (MCI) gel CHP-20 (Mitsubishi, Tokyo, Japan), Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and MPLC (Lisui, Suzhou, China), and purified on an Agilent 1100 Series prep-HPLC system (Agilent Technologies, Santa Clara, CA, USA) and a Shimadzu CBM-20A recyclingpreparative HPLC system (Shimadzu, Tokyo, Japan). 3.2. Plant material The whole plant of A. decumbens Thunb was collected from Fujian Province, China, in August 2011, and was authenticated by Professor Mian Zhang, Department of Medicinal Plants, China Pharmaceutical University. A voucher specimen (No. 110813AD) is deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. 3.3. Extraction and isolation The air-dried whole plant of A. decumbens (800 g) was ultrasonically extracted with 4 l MeOH (2 h £ 3). The crude extract (102 g) was suspended in water and successively partitioned with petroleum ether, CH2Cl2 and EtOAc. The CH2Cl2 fraction (28 g) was subjected to an MCI column with MeOH –H2O (3:7, 8:2, 10:0, v/v) to yield three fractions (A– C). Fraction B afforded 2 (1.2 g) after repeated crystallisation from MeOH. The mother liquor was performed on a silica gel column using a gradient of petroleum ether – Me2CO (7:3, 1:1, 3:7, 0:1, v/v) to yield four fractions (B1 –B4). Fraction B2 was subsequently separated by MPLC with a continuous gradient of MeOH – H2O (1:1 to 1:0, v/v, 10 ml/min) to yield 35 fractions. Fractions 13 –15 were combined and subjected to Sephadex LH-20 eluting with MeOH, subsequently separated by recycling-preparative HPLC using CH3CN – H2O (40:60, v/v, 10 ml/min) to afford 7 (2.0 mg), 8 (3.0 mg) and 10 (3.0 mg). Fractions 18– 23 were purified by prep-HPLC (MeOH – H2O, 60:40, v/v, 10 ml/min) to obtain 6 (8.0 mg), 9 (8.0 mg), 11 (9.0 mg) and 13 (3.0 mg). Fractions 24– 26 were purified by prep-HPLC (MeOH – H2O, 65:35, v/v, 10 ml/min) to afford 3 (12.0 mg), 4 (20.0 mg) and 12 (5.0 mg). Fraction 28 was performed on prep-HPLC (CH3CN – H2O, 60:40, v/v, 10 ml/min) to yield 1 (3.0 mg), 5 (14.0 mg) and 14 (6.0 mg). 3.4. Ajugacumbin J (1) Colourless oil; ½a25 d þ 29.8 (c 0.11, MeOH); UV (MeOH) lmax (log 1) 221 (3.78); IR (KBr) nmax 2959, 2926, 2852, 1739, 1690, 1644, 1436, 1397, 1251, 1134, 1081, 1026, 903 and 735 cm21; 1H NMR (500 MHz, CDCl3): d 5.77 (1H, dt, J ¼ 5.0, 10.5 Hz, H-1), 2.25 (1H, m, H-2a), 1.64 (1H, m, H-2b), 2.40 (1H, m, H-3a), 1.17 (1H, m, H-3b), 4.72 (1H, dd, J ¼ 11.5, 4.5 Hz, H-6), 1.53 (1H, m, H-7a), 1.62 (1H, m, H-7b), 1.53 (1H, m, H-8), 2.14 (1H, d, J ¼ 11.0 Hz, H-10), 2.46 (2H, d, J ¼ 8.5 Hz, H-11), 6.83 (1H, dd, J ¼ 8.5, 4.5 Hz, H-12), 3.23 (2H, s, H-14),d 9.51 (1H, s, H-16), 0.81 (3H, d, J ¼ 6.5 Hz, H-17), 3.12 (1H, brs, H-18a), 2.37 (1H, d, J ¼ 4.0 Hz, H-18b), 4.89 (1H, d, J ¼ 12.5 Hz, H-19a), 4.43 (1H, d, J ¼ 12.5 Hz, H-19b), 0.91 (3H, s, H-20), 6.65 (1H, dq, J ¼ 0.5, 7.0 Hz, H-30 ), 1.71 (3H, d, J ¼ 7.0 Hz, H-40 ), 1.76 (3H, s, H-50 ), 3.63 (3H, s, 15-OCH3), 2.16 [3H, s, methyl of acetate (at C-19)], 1.96 [3H, s, methyl of acetate (at C-6)]; 13C NMR (125 MHz, CDCl3):d 70.3 (C-1), 31.8 (C-2), 30.4 (C-3), 64.5 (C-4), 45.7 (C-5), 71.8 (C-6), 33.3 (C-7), 36.8 (C-8), 40.1 (C-9), 51.0 (C-10), 38.4 (C-11), 153.0 (C-12),
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138.4 (C-13), 29.7 (C-14), 170.0 (C-15), 193.1 (C-16), 15.7 (C-17), 49.8 (C-18), 62.8 (C-19), 17.5 (C-20), 166.8 (C-10 ), 128.8 (C-20 ), 138.8 (C-30 ), 14.6 (C-40 ), 12.2 (C-50 ), 170.1 [carbonyl of acetate (at C-6)], 21.3 [methyl of acetate (at C-6)], 170.5 [carbonyl of acetate (at C-19)], 21.4 [methyl of acetate (at C-19)], 52.2 (15-OMe); ESI-MS: m/z 580 [M þ NH4]þ, 597 [M þ Cl]2; HRESI-MS: m/z 597.2468 [M þ Cl]2 (calcd for C30H42O10Cl, 597.2472). 3.5. Nitric oxide production bioassay
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The protocol for NO production bioassays was provided in previously published papers (Yin et al. 2013). N-Monomethyl-L -arginine was used as the positive control. All experiments were conducted in three replicates. 4. Conclusions In this paper, 13 diterpenes and 1 dipeptide, including a new neo-clerodane diterpene, named ajugacumbin J, were isolated from the whole plant of A. decumbens. The structure of ajugacumbin J (1) was identified as 6a,19-diacetoxy-1b-tigloyloxy-4a,18-epoxy-neo-clerod12-en-15-oic acid methyl ester-16-aldehyde (1) by 1D and 2D NMR spectra and MS. Bioassay results showed that compound 1 and ajugacumbin D (5) exhibited the inhibitory activities of LPS-induced NO production in RAW 264.7 macrophages. Supplementary material Supplementary material relating to this article is available online: 1D NMR and 2D NMR spectra and HRESIMS of 1 (Figures S1 –S8). Acknowledgements This research was supported by the National Key Scientific and Technological Special Projects (2012ZX09103-101-007), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193).
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