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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new acorane sesquiterpene from the aerial parts of Psychotria yunnanensis a
a
a
a
Qinpei Lu , Junsong Wang , Jun Luo , Xiaobing Wang , Siming a
Shan & Lingyi Kong
a
a
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing210009, People's Republic of China Published online: 10 Jul 2014.
To cite this article: Qinpei Lu, Junsong Wang, Jun Luo, Xiaobing Wang, Siming Shan & Lingyi Kong (2014) A new acorane sesquiterpene from the aerial parts of Psychotria yunnanensis, Natural Product Research: Formerly Natural Product Letters, 28:20, 1659-1663, DOI: 10.1080/14786419.2014.934234 To link to this article: http://dx.doi.org/10.1080/14786419.2014.934234
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Natural Product Research, 2014 Vol. 28, No. 20, 1659–1663, http://dx.doi.org/10.1080/14786419.2014.934234
A new acorane sesquiterpene from the aerial parts of Psychotria yunnanensis
Downloaded by [University of Newcastle (Australia)] at 06:34 14 September 2014
Qinpei Lu, Junsong Wang, Jun Luo, Xiaobing Wang, Siming Shan and Lingyi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China (Received 25 March 2014; final version received 4 June 2014) Psycacoraone A (1), a new acorane-type sesquiterpene possessing rare spirobicyclic carbon skeleton, was isolated from the aerial parts of Psychotria yunnanensis. Its structure was elucidated on the basis of spectroscopic methods including HR-ESI-MS, 1D and 2D NMR. The absolute configuration of 1 was determined by calculation of electronic circular dichroism using density functional theory. Keywords: Psychotria yunnanensis; Rubiaceae; acorane sesquiterpene; psycacoraone A; calculated ECD
1. Introduction Psychotria yunnanensis Hutch. (Rubiaceae) is a species of the genus Psychotria one of the largest genera in the Rubiaceae with more than 1800 species worldwide (Davis et al. 2009). Eighteen species of this genus were distributed in China, and some of them were used as folk medicines for swelling and relieving muscles, activating collaterals and strengthening bones and muscles (Editorial Board of National Chinese Medicine Assembly 1975; State Administration of Traditional Chinese Medicine of the People’ Republic of China 1999; Chen & Taylor 2011). Indoline alkaloids were the characteristic chemical compositions of some species in this genus, which led to the isolation of monoterpene indole (Paul et al. 2003; Kerber et al. 2008) and polyindoline alkaloids (Takayama et al. 2004; Zhou et al. 2010; Li et al. 2011; Liu et al. 2013) and other compounds (Solis et al. 1995; Zhang et al. 2013; Lu et al. 2014). The previous investigation on P. yunnanensis led to the isolation of 16 compounds, including norisoprenoids, monoterpenoid and phenolic compounds (Lu et al. 2014). In our continuing study, a new spirobicyclic acorane-type sesquiterpene featured a pentaheterocyclic ring with an a,bunsaturated carbonyl group (Figure 1), psycacoraone A (1), was isolated from the aerial parts of P. yunnanensis. Its structure was established on the basis of spectroscopic methods, and the absolute configuration was determined by calculation of electronic circular dichroism (ECD) using density functional theory (DFT). This was the first report of acorane-type sesquiterpene in the family Rubiaceae. Herein, the isolation and structural elucidation of this new compound were described. 2. Results and discussion Psycacoraone A (1) was isolated as pale yellow oil. Its molecular formula was determined as C15H22O3 on the basis of positive-ion HR-ESI-MS at m/z 273.1462 [M þ Na]þ (calcd for
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Figure 1. The structure, key HMBC (H ! C) and ROESY (H $ H) correlations of 1.
C15H22O3Na 273.1461). The IR absorption bands at 3437 and 1687 cm21 implied the presence of hydroxyl and conjugated carbonyl groups, respectively. The UV spectrum exhibited the absorption maxima at 243 nm, typical of an a,b-unsaturated carbonyl group (Nawamaki & Kuroyanagi 1996). The 1H NMR data of 1 showed the presence of two trisubstituted olefinic protons [dH 5.91 (s, 1H, H-2) and 5.49 (br s, 1H, H-9)], a hydroxymethyl group [dH 3.79 (br s, 2H, H-15)], an isopropyl group [dH 2.60 (m, 1H, H-11), 1.12 (d, J ¼ 6.5 Hz, 3H, H-13) and 1.08 (d, J ¼ 6.5 Hz, 3H, H-12)] and a singlet methyl [dH 1.14 (s, 3H, H-14)]. The 13C NMR spectrum displayed 15 carbon resonances, assigned to three methyls, four methylenes (one oxygenated) three methines (two olefinic) and five quaternary carbons (two olefinic and one carbonyl). There were five indices of hydrogen deficiency evidence in the molecule of 1, three of which were represented by one carbonyl and two double bonds, and the remaining two degrees of unsaturation required a bicyclic ring system in 1. The aforementioned 1D NMR spectroscopic data revealed that 1 was an acorane-type sesquiterpene (Figure 1) (Castro et al. 1984; Wen et al. 2013). The connection of the structure was determined by the HMBC long-range correlations (Figure 1). The crucial HMBC correlations from H-2 (dH 5.91) to C-4 (dC 81.4) and C-5 (dC 51.6), from H3-14 (dH 1.14) to C-3 (dC 209.7) and C-5, from OH-4 (dH 4.86) to C-14 (dC 22.1) C3 and C-5, and from gem-dimethyls H3-12 (dH 1.08) and H3-13 (dH 1.12) to C-1 (dC 192.1) constructed the cyclopentenone group containing an a,b-unsaturated carbonyl (ring A). In addition, long-range correlations from H-6a (dH 1.72) to C-7 (dC 22.6), C-8 (dC 136.9) and C-10 (dC 32.8), and from the hydroxylmethyl H-15 (dH 3.79) to C-7 and C-9 (dC 119.6) in the HMBC spectrum suggested a cyclohexene group in ring B. The key HMBC correlations from H-10b (dH 1.82) and H-6a to C-4 and C-5 indicated that those two rings A and B were linked by a spiro carbon C-5. Therefore, the planar structure of 1 was established as shown. The relative configuration of 1 was determined by the ROESY spectrum (Figure 1). The strong ROESY cross-peak between OH-4 (dH 4.86) and H-10b (dH 1.82) indicated that they were in the same orientation. In addition, the ROESY correlation between H3-14 (dH 1.14) and H-7b (dH 2.11) revealed that they were in the reversed face. The two possible isomers (4S,5S)-1 and (4R,5R)-1 were then concluded and ECD spectra of them were calculated by using timedependent density functional theory. The experimental ECD spectrum of 1 was in good
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agreement with the calculated ECD spectrum for (4R,5R)-1 (Figure 2), which established the assignment of the absolute configuration of 1 as depicted. Compound 1 was evaluated for inhibitory effect on nitric oxide (NO) production in lipopolysaccaride-activated RAW264.7 macrophages. Unfortunately, it did not show potent activity (IC50 value more than 50 mM).
Downloaded by [University of Newcastle (Australia)] at 06:34 14 September 2014
3. Experimental 3.1. General experimental procedures Optical rotations were measured with a JASCO P-1020 polarimeter (JASCO, Tokyo, Japan). ECD spectra were obtained on a JASCO J 810 spectropolarimeter (JASCO). UV spectra were recorded on a UV-2450 UV/vis spectrophotometer (Shimadzu, Tokyo, Japan). IR (KBr disks) spectra were recorded by Bruker Tensor 27 spectrometer (Bruker, Karlsruhe, Germany). NMR spectra were recorded on a Bruker AV III-500 NMR instrument (1H: 500 MHz, 13C: 125 MHz) with TMS as internal standard (Bruker). HR-ESI-MS was obtained on an Agilent 6520B Q-TOF spectrometer (Agilent, Santa Clara, CA, USA). Column chromatography (CC) was carried out on silica gel (200 – 300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China), MCI gel (Mitsubishi Chemical Corp., Tokyo, Japan) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Preparative HPLC was carried out using a Shimadzu LC-6A instrument with a SPD10A detector using a shim-pack RP-C18 column (20 mm £ 200 mm). 3.2. Plant material The aerial parts of P. yunnanensis were collected from Xishuangbanna, Yunnan Province, China in April 2012, and authenticated by Professor Mian Zhang, Department of Pharmacognosy, China Pharmaceutical University. A voucher specimen (No. PY-2012-04-LQP) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
Figure 2. Calculated ECD spectrum and experimental ECD spectrum of 1.
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3.3. Extraction and isolation The air-dried, powdered aerial parts of P. yunnanensis (5 kg) were extracted with 95% ethanol under reflux three times (3 £ 20 L). The solvent was removed under reduced pressure to give a residue (512 g), which was suspended in H2O and partitioned with petroleum ether, CH2Cl2, EtOAc and n-BuOH, respectively. The CH2Cl2 extract (60.0 g) was subjected to CC on silica gel eluted with a gradient of petroleum ether-EtOAc (10:1, 5:1, 2:1, 1:2, v/v) to give six fractions, Fr.C1 – C6 according to TLC monitor. Fraction C6 (2 g) was separated by MCI gel eluted with MeOH – H2O (30:70 to 90:10, v/v) to obtain seven subfractions, Fr. C6A-C6G. C6F (124 mg) was subjected to Sephadex LH-20 (MeOH), and further separated by preparative HPLC (MeOHH2O, 55:45, v/v, 10.0 mL/min) to yield compound 1 (5 mg).
3.4. Psycacoraone A (1) Pale yellow oil; ½a25 D þ 34 (c ¼ 0.08 CHCl3); IR (KBr) nmax 3437, 3364, 2918, 1687, 1594, 1386, 1162, 1027 cm21; UV (CHCl3) lmax (log 1) 243 (2.92) nm; 1H NMR (500 MHz, DMSOd6): dH 5.91 (1H, s, H-2), 1.72 (1H, ddd, J ¼ 13.5, 11.0 and 5.5 Hz, H-6a), 1.86 (1H, overlapped, H-6b), 2.01 (1H, overlapped, H-7a), 2.11 (1H, overlapped, H-7b), 5.49 (1H, br s, H-9), 2.03 (1H, overlapped, H-10a), 1.82 (1H, overlapped, H-10b), 2.60 (1H, m, H-11), 1.08 (3H, d, J ¼ 6.5 Hz, H-12), 1.12 (3H, d, J ¼ 6.5 Hz, H-13), 1.14 (3H, s, H-14), 3.79 (2H, br s, H-15), 4.86 (1H, s, OH-4), 4.60 (1H, t, J ¼ 5.5 Hz, OH-15); 13C NMR (125 MHz, DMSO-d6): dC 192.1 (C1), 121.9 (C-2), 209.7 (C-3), 81.4 (C-4), 51.6 (C-5), 25.4 (C-6), 22.6 (C-7), 136.9 (C-8), 119.6 (C-9), 32.8 (C-10), 27.6 (C-11), 23.4 (C-12), 23.1 (C-13), 22.1 (C-14), 65.1 (C-15); ECD (MeOH, c 0.25) lmax (D1) ¼ 215 (þ 6.8), 262 ( 2 0.08), 327 (þ 1.22) nm; HR-ESI-MS m/z: 273.1462 [M þ Na]þ (calcd for C15H22O3Na, 273.1461).
3.5. NO production bioassay The protocol for NO production bioassays was provided in the previously published paper (Yin et al. 2013). N-Monomethyl-L -arginine was used as the positive control. All the experiments were performed in three replicates.
4. Conclusions In this paper, a rare spirobicyclic acorane-type sesquiterpene was isolated from the aerial parts of P. yunnanensis. The absolute configuration of 1 was determined by calculation of ECD using DFT. It was the first report that such type of sesquiterpene had been found in the family Rubiaceae. Unfortunately, compound 1 did not show potent activity for inhibitory effect on NO production in lipopolysaccaride-activated RAW264.7 macrophages.
Supplementary material Supplementary material relating to this article is available online: 1D NMR, 2D NMR and HRESI-MS spectra of 1 (Figures S1 –S8).
Acknowledgements This research work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193), the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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References Castro V, Jakupovic J, Bohlmann F. 1984. A new type of sesquiterpene and acorane derivative from Calea prunifolia. J Nat Prod. 47:802–808. Chen T, Taylor CM. 2011. Psychotria Linnaeus. Fl China. 19:294– 301. Davis AP, Govaerts R, Bridson DM, Ruhsam M, Moat J, Brummitt NA. 2009. A global assessment of distribution, diversity, endemism, and taxonomic effort in the Rubiaceae. Ann Missouri Bot Gard. 96:68–78. Editorial Board of National Chinese Medicine Assembly. 1975. National medicine assembly. Beijing: People’s Medical Publishing House. Kerber VA, Passos CS, Verli H, Fett-Neto AG, Quirion JP, Henriques AT. 2008. Psychollatine, a glucosidic monoterpene indole alkaloid from Psychotria umbellate. J Nat Prod. 71:697–700. Li XN, Zhang Y, Cai XH, Feng T, Liu YP, Li Y, Ren J, Zhu HJ, Luo XD. 2011. Psychotripine: a new trimeric pyrroloindoline derivative from Psychotria pilifera. Org Lett. 13:5896– 5899. Liu Y, Wang JS, Wang XB, Kong LY. 2013. Two novel dimeric indole alkaloids from the leaves and twigs of Psychotria henryi. Fitoterapia. 85:178–182. Lu QP, Wang JS, Kong LY. 2014. Chemical constituents from Psychotria yunnanensis and its chemotaxonomic study. Biochem Syst Ecol. 52:20–22. Nawamaki K, Kuroyanagi M. 1996. Sesquiterpenoids from Acorus calamus as germination inhibitors. Phytochemistry. 43:1175–1182. Paul JHA, Maxwell AR, Reynolds WF. 2003. Novel bis(monoterpenoid) indole alkaloids from Psychotria bahiensis. J Nat Prod. 66:752–754. Solis PN, Langat C, Gupta MP, Kirby GC, Warhurst DC, Phillipson JD. 1995. Bioactive compounds from Psychotria camponutans. Planta Med. 61:62–65. State Administration of Traditional Chinese Medicine of the People’s Republic of China. 1999. Zhonghua Bencao. Shanghai: Shanghai Science and Technology Publishing House. Takayama H, Mori I, Kitajima M, Aimi N, Lajis NH. 2004. New type of trimeric and pentameric indole alkaloids from Psychotria rostrata. Org Lett. 6:2945–2948. Wen QF, Zhong YJ, Su XH, Li CY, Liu J, Li YF, Liang B. 2013. A new acorane sesquiterpene from Lysionotus pauciflorus. Chin J Nat Med. 11:185–187. 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. Zhang CX, Zhang DM, Chen MF, Guan SY, Yao JH, He XX, Lei LF, Zhong Y, Wang ZF, Ye WC. 2013. Antiproliferative triterpenoid saponins from the stem of Psychotria sp. Plant Med. 79:978–986. Zhou H, He HP, Wang YH, Hao XJ. 2010. A new dimeric alkaloid from the leaf of Psychotria calocarpa. Helv Chim Acta. 93:1650–1652.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new acorane sesquiterpene from the aerial parts of Psychotria yunnanensis a
a
a
a
Qinpei Lu , Junsong Wang , Jun Luo , Xiaobing Wang , Siming a
Shan & Lingyi Kong
a
a
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing210009, People's Republic of China Published online: 10 Jul 2014.
To cite this article: Qinpei Lu, Junsong Wang, Jun Luo, Xiaobing Wang, Siming Shan & Lingyi Kong (2014) A new acorane sesquiterpene from the aerial parts of Psychotria yunnanensis, Natural Product Research: Formerly Natural Product Letters, 28:20, 1659-1663, DOI: 10.1080/14786419.2014.934234 To link to this article: http://dx.doi.org/10.1080/14786419.2014.934234
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 &
Downloaded by [University of Newcastle (Australia)] at 06:34 14 September 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 20, 1659–1663, http://dx.doi.org/10.1080/14786419.2014.934234
A new acorane sesquiterpene from the aerial parts of Psychotria yunnanensis
Downloaded by [University of Newcastle (Australia)] at 06:34 14 September 2014
Qinpei Lu, Junsong Wang, Jun Luo, Xiaobing Wang, Siming Shan and Lingyi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China (Received 25 March 2014; final version received 4 June 2014) Psycacoraone A (1), a new acorane-type sesquiterpene possessing rare spirobicyclic carbon skeleton, was isolated from the aerial parts of Psychotria yunnanensis. Its structure was elucidated on the basis of spectroscopic methods including HR-ESI-MS, 1D and 2D NMR. The absolute configuration of 1 was determined by calculation of electronic circular dichroism using density functional theory. Keywords: Psychotria yunnanensis; Rubiaceae; acorane sesquiterpene; psycacoraone A; calculated ECD
1. Introduction Psychotria yunnanensis Hutch. (Rubiaceae) is a species of the genus Psychotria one of the largest genera in the Rubiaceae with more than 1800 species worldwide (Davis et al. 2009). Eighteen species of this genus were distributed in China, and some of them were used as folk medicines for swelling and relieving muscles, activating collaterals and strengthening bones and muscles (Editorial Board of National Chinese Medicine Assembly 1975; State Administration of Traditional Chinese Medicine of the People’ Republic of China 1999; Chen & Taylor 2011). Indoline alkaloids were the characteristic chemical compositions of some species in this genus, which led to the isolation of monoterpene indole (Paul et al. 2003; Kerber et al. 2008) and polyindoline alkaloids (Takayama et al. 2004; Zhou et al. 2010; Li et al. 2011; Liu et al. 2013) and other compounds (Solis et al. 1995; Zhang et al. 2013; Lu et al. 2014). The previous investigation on P. yunnanensis led to the isolation of 16 compounds, including norisoprenoids, monoterpenoid and phenolic compounds (Lu et al. 2014). In our continuing study, a new spirobicyclic acorane-type sesquiterpene featured a pentaheterocyclic ring with an a,bunsaturated carbonyl group (Figure 1), psycacoraone A (1), was isolated from the aerial parts of P. yunnanensis. Its structure was established on the basis of spectroscopic methods, and the absolute configuration was determined by calculation of electronic circular dichroism (ECD) using density functional theory (DFT). This was the first report of acorane-type sesquiterpene in the family Rubiaceae. Herein, the isolation and structural elucidation of this new compound were described. 2. Results and discussion Psycacoraone A (1) was isolated as pale yellow oil. Its molecular formula was determined as C15H22O3 on the basis of positive-ion HR-ESI-MS at m/z 273.1462 [M þ Na]þ (calcd for
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Figure 1. The structure, key HMBC (H ! C) and ROESY (H $ H) correlations of 1.
C15H22O3Na 273.1461). The IR absorption bands at 3437 and 1687 cm21 implied the presence of hydroxyl and conjugated carbonyl groups, respectively. The UV spectrum exhibited the absorption maxima at 243 nm, typical of an a,b-unsaturated carbonyl group (Nawamaki & Kuroyanagi 1996). The 1H NMR data of 1 showed the presence of two trisubstituted olefinic protons [dH 5.91 (s, 1H, H-2) and 5.49 (br s, 1H, H-9)], a hydroxymethyl group [dH 3.79 (br s, 2H, H-15)], an isopropyl group [dH 2.60 (m, 1H, H-11), 1.12 (d, J ¼ 6.5 Hz, 3H, H-13) and 1.08 (d, J ¼ 6.5 Hz, 3H, H-12)] and a singlet methyl [dH 1.14 (s, 3H, H-14)]. The 13C NMR spectrum displayed 15 carbon resonances, assigned to three methyls, four methylenes (one oxygenated) three methines (two olefinic) and five quaternary carbons (two olefinic and one carbonyl). There were five indices of hydrogen deficiency evidence in the molecule of 1, three of which were represented by one carbonyl and two double bonds, and the remaining two degrees of unsaturation required a bicyclic ring system in 1. The aforementioned 1D NMR spectroscopic data revealed that 1 was an acorane-type sesquiterpene (Figure 1) (Castro et al. 1984; Wen et al. 2013). The connection of the structure was determined by the HMBC long-range correlations (Figure 1). The crucial HMBC correlations from H-2 (dH 5.91) to C-4 (dC 81.4) and C-5 (dC 51.6), from H3-14 (dH 1.14) to C-3 (dC 209.7) and C-5, from OH-4 (dH 4.86) to C-14 (dC 22.1) C3 and C-5, and from gem-dimethyls H3-12 (dH 1.08) and H3-13 (dH 1.12) to C-1 (dC 192.1) constructed the cyclopentenone group containing an a,b-unsaturated carbonyl (ring A). In addition, long-range correlations from H-6a (dH 1.72) to C-7 (dC 22.6), C-8 (dC 136.9) and C-10 (dC 32.8), and from the hydroxylmethyl H-15 (dH 3.79) to C-7 and C-9 (dC 119.6) in the HMBC spectrum suggested a cyclohexene group in ring B. The key HMBC correlations from H-10b (dH 1.82) and H-6a to C-4 and C-5 indicated that those two rings A and B were linked by a spiro carbon C-5. Therefore, the planar structure of 1 was established as shown. The relative configuration of 1 was determined by the ROESY spectrum (Figure 1). The strong ROESY cross-peak between OH-4 (dH 4.86) and H-10b (dH 1.82) indicated that they were in the same orientation. In addition, the ROESY correlation between H3-14 (dH 1.14) and H-7b (dH 2.11) revealed that they were in the reversed face. The two possible isomers (4S,5S)-1 and (4R,5R)-1 were then concluded and ECD spectra of them were calculated by using timedependent density functional theory. The experimental ECD spectrum of 1 was in good
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agreement with the calculated ECD spectrum for (4R,5R)-1 (Figure 2), which established the assignment of the absolute configuration of 1 as depicted. Compound 1 was evaluated for inhibitory effect on nitric oxide (NO) production in lipopolysaccaride-activated RAW264.7 macrophages. Unfortunately, it did not show potent activity (IC50 value more than 50 mM).
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3. Experimental 3.1. General experimental procedures Optical rotations were measured with a JASCO P-1020 polarimeter (JASCO, Tokyo, Japan). ECD spectra were obtained on a JASCO J 810 spectropolarimeter (JASCO). UV spectra were recorded on a UV-2450 UV/vis spectrophotometer (Shimadzu, Tokyo, Japan). IR (KBr disks) spectra were recorded by Bruker Tensor 27 spectrometer (Bruker, Karlsruhe, Germany). NMR spectra were recorded on a Bruker AV III-500 NMR instrument (1H: 500 MHz, 13C: 125 MHz) with TMS as internal standard (Bruker). HR-ESI-MS was obtained on an Agilent 6520B Q-TOF spectrometer (Agilent, Santa Clara, CA, USA). Column chromatography (CC) was carried out on silica gel (200 – 300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China), MCI gel (Mitsubishi Chemical Corp., Tokyo, Japan) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Preparative HPLC was carried out using a Shimadzu LC-6A instrument with a SPD10A detector using a shim-pack RP-C18 column (20 mm £ 200 mm). 3.2. Plant material The aerial parts of P. yunnanensis were collected from Xishuangbanna, Yunnan Province, China in April 2012, and authenticated by Professor Mian Zhang, Department of Pharmacognosy, China Pharmaceutical University. A voucher specimen (No. PY-2012-04-LQP) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
Figure 2. Calculated ECD spectrum and experimental ECD spectrum of 1.
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3.3. Extraction and isolation The air-dried, powdered aerial parts of P. yunnanensis (5 kg) were extracted with 95% ethanol under reflux three times (3 £ 20 L). The solvent was removed under reduced pressure to give a residue (512 g), which was suspended in H2O and partitioned with petroleum ether, CH2Cl2, EtOAc and n-BuOH, respectively. The CH2Cl2 extract (60.0 g) was subjected to CC on silica gel eluted with a gradient of petroleum ether-EtOAc (10:1, 5:1, 2:1, 1:2, v/v) to give six fractions, Fr.C1 – C6 according to TLC monitor. Fraction C6 (2 g) was separated by MCI gel eluted with MeOH – H2O (30:70 to 90:10, v/v) to obtain seven subfractions, Fr. C6A-C6G. C6F (124 mg) was subjected to Sephadex LH-20 (MeOH), and further separated by preparative HPLC (MeOHH2O, 55:45, v/v, 10.0 mL/min) to yield compound 1 (5 mg).
3.4. Psycacoraone A (1) Pale yellow oil; ½a25 D þ 34 (c ¼ 0.08 CHCl3); IR (KBr) nmax 3437, 3364, 2918, 1687, 1594, 1386, 1162, 1027 cm21; UV (CHCl3) lmax (log 1) 243 (2.92) nm; 1H NMR (500 MHz, DMSOd6): dH 5.91 (1H, s, H-2), 1.72 (1H, ddd, J ¼ 13.5, 11.0 and 5.5 Hz, H-6a), 1.86 (1H, overlapped, H-6b), 2.01 (1H, overlapped, H-7a), 2.11 (1H, overlapped, H-7b), 5.49 (1H, br s, H-9), 2.03 (1H, overlapped, H-10a), 1.82 (1H, overlapped, H-10b), 2.60 (1H, m, H-11), 1.08 (3H, d, J ¼ 6.5 Hz, H-12), 1.12 (3H, d, J ¼ 6.5 Hz, H-13), 1.14 (3H, s, H-14), 3.79 (2H, br s, H-15), 4.86 (1H, s, OH-4), 4.60 (1H, t, J ¼ 5.5 Hz, OH-15); 13C NMR (125 MHz, DMSO-d6): dC 192.1 (C1), 121.9 (C-2), 209.7 (C-3), 81.4 (C-4), 51.6 (C-5), 25.4 (C-6), 22.6 (C-7), 136.9 (C-8), 119.6 (C-9), 32.8 (C-10), 27.6 (C-11), 23.4 (C-12), 23.1 (C-13), 22.1 (C-14), 65.1 (C-15); ECD (MeOH, c 0.25) lmax (D1) ¼ 215 (þ 6.8), 262 ( 2 0.08), 327 (þ 1.22) nm; HR-ESI-MS m/z: 273.1462 [M þ Na]þ (calcd for C15H22O3Na, 273.1461).
3.5. NO production bioassay The protocol for NO production bioassays was provided in the previously published paper (Yin et al. 2013). N-Monomethyl-L -arginine was used as the positive control. All the experiments were performed in three replicates.
4. Conclusions In this paper, a rare spirobicyclic acorane-type sesquiterpene was isolated from the aerial parts of P. yunnanensis. The absolute configuration of 1 was determined by calculation of ECD using DFT. It was the first report that such type of sesquiterpene had been found in the family Rubiaceae. Unfortunately, compound 1 did not show potent activity for inhibitory effect on NO production in lipopolysaccaride-activated RAW264.7 macrophages.
Supplementary material Supplementary material relating to this article is available online: 1D NMR, 2D NMR and HRESI-MS spectra of 1 (Figures S1 –S8).
Acknowledgements This research work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193), the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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