This article was downloaded by: [Bibliothèques de l'Université de Montréal] On: 02 December 2014, At: 04:02 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new benzopyran derivative from Pseuduvaria indochinensis Merr. a
a
a
a
Cai-Gui Zhao , Ming-Jun Yao , Jin-Wei Yang , Ya-Lin Chai , Xiaoa
a
Dong Sun & Cheng-Shan Yuan a
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P.R. China Published online: 04 Dec 2013.
To cite this article: Cai-Gui Zhao, Ming-Jun Yao, Jin-Wei Yang, Ya-Lin Chai, Xiao-Dong Sun & Cheng-Shan Yuan (2014) A new benzopyran derivative from Pseuduvaria indochinensis Merr., Natural Product Research: Formerly Natural Product Letters, 28:3, 169-173, DOI: 10.1080/14786419.2013.863197 To link to this article: http://dx.doi.org/10.1080/14786419.2013.863197
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 [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 3, 169–173, http://dx.doi.org/10.1080/14786419.2013.863197
A new benzopyran derivative from Pseuduvaria indochinensis Merr.
Downloaded by [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
Cai-Gui Zhao1, Ming-Jun Yao1, Jin-Wei Yang, Ya-Lin Chai, Xiao-Dong Sun and Cheng-Shan Yuan* State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P.R. China (Received 12 June 2013; final version received 30 October 2013) From the twigs and leaves of Pseuduvaria indochinensis Merr., a new benzopyran derivative, pseudindochin (1), was isolated together with three known compounds, oligandrol (2), (6E,10E)-isopolycerasoidol (3) and polycerasoidol (4). The structure of compound 1 was elucidated on the basis of extensive spectroscopic data interpretation, including 1D, 2D NMR, HR-ESI-MS, UV and IR. Moreover, compounds 1 – 4 were evaluated in vitro for their cytotoxic activities against HL-60 and SMMC-7721 cell lines, but these compounds were essentially non-cytotoxic (IC50 . 30 mg/mL). Keywords: Pseuduvaria indochinensis Merr; benzopyran derivatives; spectroscopic data; cytotoxic activities
1. Introduction The genus Pseuduvaria (family Annonaceae) consists of about 17 species, of which only one species is distributed in the Yunnan Province of China. In prior reports, alkaloids (Zhong et al. 1988; Taha et al. 2011) and diterpenes (Xie et al. 1989) were isolated from this genus. Pseuduvaria indochinensis Merr. is a species of trees, mainly distributed in Southeast Asia. In this work, a new benzopyran derivative, pseudindochin (1), and three known compounds, oligandrol (2) (Banfield et al. 1994), (6E,10E)-isopolycerasoidol (3) (Gonzalez et al. 1996) and polycerasoidol (4) (Gonzalez et al. 1995), were isolated from the 95% EtOH extracts of the twigs and leaves of P. indochinensis, which were collected from the Yunnan Province of China. In this paper, we describe the isolation, structural elucidation and cytotoxic activities of the compounds isolated from P. indochinensis.
2. Results and discussion 2.1. Structural elucidation of compound 1 Compound 1 was obtained as yellow oil. Its molecular formula was established as C23H30O5 from the [M þ Na]þ peak at m/z 409.1991 (calcd 409.1985) in the positive HR-ESI-MS, indicating nine degrees of unsaturation. The IR absorptions suggested the absorption bands for hydroxyl (3378 cm21), olefin (1649 cm21), the ester carbonyl (1704 cm21) and aromatic ring (1465 and 1439 cm21) functionalities. The 1H NMR spectroscopic data showed three tertiary methyls at dH 1.36, 1.57, 2.13, a methoxyl at dH 3.78 (s, 3H), a pair of cis-conjugated olefinic protons at dH 6.25 (d, J ¼ 9.8 Hz, 1H) and 5.65 (d, J ¼ 9.8 Hz, 1H), and two olefinic methines at dH 5.14 (t, J ¼ 7.0 Hz, 1H) and *Corresponding author. Email: [email protected] q 2013 Taylor & Francis
Downloaded by [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
170
C.-G. Zhao et al.
6.21 (t, J ¼ 7.4 Hz, 1H). It also revealed two aromatic proton signals in meta position at dH 6.32 (d, J ¼ 2.9 Hz, 1H) and d 6.47 (d, J ¼ 2.9 Hz, 1H). Combined analysis of 13C NMR and DEPT spectroscopic data revealed 23 carbon signals attributed to four methyls, five methylenes, six methines and eight quaternary carbons (including four aromatic ring quaternary carbons at dC 144.80, 121.30, 148.66 and 126.29, one ester carboxyl carbon at dC 167.49, one oxygenated quaternary carbon at dC 77.76 and two olefinic quaternary carbon at dC 133.98 and 130.41). The structural feature was further corroborated by 1 H – 1H COSY and HSQC data analysis, which showed connectivity of two structural moieties: CH2(4)ZCH2(5)ZCH(6) moiety and CH2(8)ZCH2(9)ZCH(10) moiety. Careful comparison of the NMR data of 1 with those of 4 indicated that the signals were basically similar to each other. However, the signals for two methylenes and one Me observed at dC 22.43, 31.44 and 20.48 were not present in 1; instead, the signals of a pair of cis-conjugated double bond at dH 6.25 (d, J ¼ 9.8 Hz, 1H) and 5.65 (d, J ¼ 9.8 Hz, 1H) and dC 122.91, 130.62, a CH2OH group at dH 4.22 (2H, d, 5.3) and dC 65.30, and a methoxyl at dH 3.78 (s, 3 H) and dC 51.49 were detected in the spectra of 1. Compound 1 was completely characterised by HMBC experiments. The HMBC correlations from H-14 to C-6, C-7 and C-8, from H-6 to C-8 and C-14, and from H-8 to C-6, C-7 and C-14 revealed that both C-6 and C-8 connected with C-7 and the C-14 was attached to C-7. The correlations from H-10 to C-12, from OCH3-13 to C-13, from H-12 to C-10, C-11 and C-13 indicated the presence of an a,b-unsaturated ester and the C-12 located at C-11. The correlations from H-30 to C-1, from H-2 to C-20 , C-3, C-4 and C-15, from H-4 to C-2 and C-15, and from H-15 to C-2, C-3 and C-4 were in accord with the presence of (C-30 )ZC(20 )Z CH(1)vCH(2)ZC(3)ZC(4) moiety, the C-15 was attached to C-3 and C-50 was attached to C-10 , C-30 and C-40 , a hydroxyl group must locate at C-40 . The structure was confirmed except two Obearing carbons C-10 and C-3. By means of formula C23H30O5, a remaining O-atom must be connected to both C-10 and C-3 to form a six-membered ring composed of C(10 )ZOZC(3)Z CH(2)ZCH(1)ZC(20 ). Thus, the planer structure of compound 1 was confirmed. The relative configuration of 1 was derived by NOE difference spectra. The resonance of H-6 was enhanced by irradiation of H-8, and H-10 was enhanced by irradiation of H-12, indicating the 6E, and 10Z geometry for D6,7 and D10,11, respectively. On the basis of these data, compound 1 was established and named pseudindochin (1) (Figure 1). The structures of the known compounds 2– 4 were determined by comparing their physical and spectral data with those published in the literatures. They were identified as oligandrol (2) (Banfield et al. 1994), (6E,10E)-isopolycerasoidol (3) (Gonzalez et al. 1996) and polycerasoidol (4) (Gonzalez et al. 1995).
6' 1' O 5' HO 4'
3' 2' 1
15 3
13 COOCH3
14 5
24
9 6
7
8
10
11 CH2OH 12
O HO 2
1
COOH
O O COOH
HO
HO 3
Figure 1. Chemical structures of compounds 1 –4.
4
Natural Product Research
171
Downloaded by [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
2.2. In vitro cytotoxic activity In order to investigate the in vitro cytotoxicity of compounds 1 –4, they were tested for their inhibitory activity against the human promyelocytic leukaemia cell line HL-60 and human hepatocellular carcinoma cell line SMMC-7721 by sulforhodamine B (SRB) colorimetric assay (Skehan et al. 1990). It was found that compounds 1– 4 were inactive towards all the tested cell lines (IC50 . 30 mg/mL).
3. Experimental 3.1. General Optical rotations were measured on a PerkinElmer Model 341 polarimeter with a 1dm cell. UV spectra were measured on a Varian 3542 UV –visible spectrometer (Varian Inc., California, USA). IR spectra were recorded on a Nicolet NEXUS 670 FT-IR spectrometer over the range of 400 –4000 cm21. HR-ESI-MS determinations were run on a Bruker APEX II mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). NMR spectra were conducted on a Bruker AM400BB (400 MHz) spectrometer (d in ppm rel. to TMS, J in Hz). Sephadex LH-20 (Amersham Pharmacia Biotech, Piscataway, NJ, USA), RP-C18 silica gel (150 – 200 mesh, Merck, Darmstadt, Germany) and silica gel (200 –300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) were used for column chromatography (CC). Thin-layer chromatography (TLC): silica gel GF254 (10 –40 mm; Qingdao Marine Chemical Factory); detection under UV light and visualised by spraying with 5% H2SO4 in C2H5OH (v/v), followed by heating. Analytical TLC was provided to follow the separation and check the purity of isolated compounds.
3.2. Plant material The leaves and twigs of P. indochinensis Merr. were collected from Yunnan Province, P. R. China, in December 2009, and authenticated by Prof. Guo-Da Tao of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (No. 200912PI) was deposited in the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences.
3.3. Extraction and isolation The air-dried powdered leaves and twigs of P. indochinensis (15.0 kg) were extracted with EtOH (95%, v/v) three times (each for 3 h) at 408C and concentrated in vacuo to give a crude extract (1500 g). The crude extract was suspended in H2O and then extracted with petroleum ether, chloroform and EtOAc, successively. The petroleum ether phase and CHCl3-soluble fractions were combined due to similar constituents on TLC. The combined extract (600 g) was subjected to silica gel CC (3200 g) eluting with a petroleum ether/acetone (100:0 to 0:100, CH3OH) gradient system to afford fractions Fr1 –Fr9 under the aid of TLC examination. Fr3 was applied to chromatographed over Sephadex LH-20 eluting with CHCl3/MeOH (v/v ¼ 2:1) to afford fractions Fr3.1 – Fr3.4. Fr3.3 was subjected to silica gel CC with a gradient elution of petroleum ether/AcOEt (30:1 ! 3:1) to afford 3 (70 mg) and 1 (10 mg). Fr6 was separated by CC over silica gel (petroleum ether/acetone, 10:1 ! 1:1) to provide fractions Fr6.1 –Fr6.5. Purification of subfraction Fr6.3 by preparative TLC using CHCl3/EtOAc (v/v ¼ 5:1) as a developing solvent system afforded 2 (130 mg). Fr6.4 was purified on a column of RP-C18 silica gel (H2O/ CH3OH 1:4, v/v) to afford 4 (18 mg).
Downloaded by [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
172
C.-G. Zhao et al.
3.3.1. Pseudindochin (1) Pseudindochin (1): Yellow oil; C23H30O5; ½a20 D : þ 4.4 (c 0.23 in CHCl3); HR-ESI-MS: m/z ¼ 409.1991 [M þ Na]þ (calcd 409.1985); IR (KBr) nmax: 3379, 1704, 1465, 1440, 1214 and 1001 cm21; 1H NMR (400 MHz, CDCl3) dH 1.36 (3H, s, H-15), 1.57 (3H, s, H-14), 1.66 (2H, m, H-4), 2.06 (2H, m, H-8), 2.10 (2H, m, H-5), 2.13 (3H, s, CH3-60 ), 2.61 (2H, q, J ¼ 7.4 Hz, H-9), 3.78 (3H, s, OCH3-13), 4.22 (2H, d, J ¼ 5.3 Hz, H-12), 4.80 (br s, HO-40 ), 5.14 (1H, t, J ¼ 7.0 Hz, H-6), 5.56 (1H, d, 9.8, H-2), 6.21 (1H, t, J ¼ 7.4 Hz, H-10), 6.24 (1H, d, 9.8, H-1), 6.32 (1H, d, J ¼ 2.9 Hz, H-30 ), 6.47 (1H, d, J ¼ 2.9 Hz, H-50 ); 13C NMR data (100 MHz, CDCl3) dC15.47 (CH3-60 ), 15.72 (C-14), 22.59 (C-5), 25.89 (C-15), 27.79 (C-9), 38.67 (C-8), 40.70 (C4), 51.49 (OCH3-13), 65.30 (C-12), 77.76 (C-3), 110.30 (C-30 ), 117.08 (C-50 ), 121.30 (C-20 ), 122.91 (C-1), 125.17 (C-6), 126.29 (C-60 ), 130.41 (C-11), 130.62 (C-2), 133.98 (C-7), 144.80 (C-10 ), 146.43 (C-10), 148.66 (C-40 ), 167.49 (C-13); CD [nm (D1)] (2.54 mg/100 mL MeOH): 211 (þ 29.52), 221 (2 31.79), 247 (þ 13.58).
3.4. Measurement of cytotoxicity 3.4.1. Cell culture HeLa cells HL-60 and human hepatoma cells SMMC-7721 were grown as a monolayer in RPMI1640 (Gibco, New York, USA) supplemented with 10% (v/v) foetal bovine serum (Sigma, Deisenhofen, Germany) and 1% penicillin –streptomycin solution (penicillin 10,000 IE/mL; streptomycin 10,000 mg/mL, Biochrom AG, Berlin, Germany). Cells were grown at 378C in 95% air humidity and 5% CO2, and sub-cultured twice weekly using trypsine/EDTA (0.05%/ 0.02%, Lonza, Shanghai, China). DMSO (0.1%) alone was used as a vehicle control.
3.4.2. SRB cytotoxicity assay For experimentation, exponentially growing cells were harvested and seeded in 96-well plates with the final volume 100 mL containing 4 £ 103 cells per well. After 24 h incubation, cells were treated with various concentrations of tested substances for 48 h. The tested substances were diluted in assay medium using a stock solution (40 mM vehicle DMSO), and the final vehicle concentration did not exceed 0.05%. Then, the cultures were fixed by the addition of 50% icecold trichloroacetic acid to give a final concentration of 10% and were placed at 48C for 1 h. The plates were flicked and washed five times with tap water to remove traces of medium, drug and serum, and were then air-dried. The air-dried plates were stained 10 min with 0.4% SRB 100 mL dissolved in 1% acetic acid. The wells were washed 5 times with 0.1% acetic acid and left to dry overnight. After dissolving the bound cells with 150 mL of 10 mM unbuffered aqueous Tris base (pH 10.5) using a gyratory shaker, the absorbance at 515 nm was determined. Binding of SRB dye to basic amino acids has a linear relationship with cell density, and SRB staining serves as an accurate method in measuring cell density through total protein staining. Cell viability was calculated as percentage of vehicle control after background reduction. All experiments were carried out thrice with five replicates for each concentration tested. Where applicable, IC50 values were calculated by linear regression.
4. Conclusions In conclusion, a new benzopyran derivative, pseudindochin (1), was isolated together with three known compounds from the twigs and leaves of Pseuduvaria indochinensis. However, these compounds were essentially noncytotoxic activities.
Natural Product Research
173
Supplementary material Supplementary Figures S1 –S9 relating to this article are available online. Acknowledgements
Downloaded by [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
Financial support from the National Basic Research Program Projects of China (2007CB108903), National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (J1103307) and the Fundamental Research Funds for the Central Universities (lzujbky-2012-62) is gratefully acknowledged.
Note 1. These authors contributed equally to this work.
References Banfield JE, Black DSC, Collins DJ, Hyland BPM, Lee JJ, Pranowo SR. 1994. Constituents of some species of Beilschmiedia and Endiandra (Lauraceae): new endiandric acid and benzopyran derivatives isolated from B. oligandra. Aust J Chem. 47:587–607. Gonzalez MC, Sentandreu MA, Rao KS, Zafra-Polo MC, Cortes D. 1996. Prenylated benzopyran derivatives from two Polyalthia species. Phytochemistry. 43:1361–1364. Gonzalez MC, Serrano A, Zafra-Polo MC, Cortes D. 1995. Polycerasoidin and polycerasoidol, two new prenylated benzopyran derivatives from Polyalthia cerasoides. J Nat Prod. 58:1278–1284. Skehan P, Storeng R, Scudiero D. 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. J Nat Cancer Inst. 82:1107–1112. Taha H, Hadi A, Hamid A, Nordin N, Najmuldeen IA, Mohamad K, Shirota O, Nugroho AE, Piow WC, Kaneda T, Morita H. 2011. Pseuduvarines A and B, two new cytotoxic dioxoaporphine alkaloids from Pseuduvaria rugosa. Chem Pharm Bull. 59:896–897. Xie N, Zhong SM, Zhao SX, Waterman PG, He CH, Zheng QT. 1989. Diterpenes from Pseuduvaria indochinensis. J China Pharm Univ. 20:203–207. Zhong SM, Zhao SS, Xie N. 1988. Alkaloids from Pseuduvaria indochinensis. Phytochemistry. 27:4004–4005.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new benzopyran derivative from Pseuduvaria indochinensis Merr. a
a
a
a
Cai-Gui Zhao , Ming-Jun Yao , Jin-Wei Yang , Ya-Lin Chai , Xiaoa
a
Dong Sun & Cheng-Shan Yuan a
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P.R. China Published online: 04 Dec 2013.
To cite this article: Cai-Gui Zhao, Ming-Jun Yao, Jin-Wei Yang, Ya-Lin Chai, Xiao-Dong Sun & Cheng-Shan Yuan (2014) A new benzopyran derivative from Pseuduvaria indochinensis Merr., Natural Product Research: Formerly Natural Product Letters, 28:3, 169-173, DOI: 10.1080/14786419.2013.863197 To link to this article: http://dx.doi.org/10.1080/14786419.2013.863197
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 [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 3, 169–173, http://dx.doi.org/10.1080/14786419.2013.863197
A new benzopyran derivative from Pseuduvaria indochinensis Merr.
Downloaded by [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
Cai-Gui Zhao1, Ming-Jun Yao1, Jin-Wei Yang, Ya-Lin Chai, Xiao-Dong Sun and Cheng-Shan Yuan* State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P.R. China (Received 12 June 2013; final version received 30 October 2013) From the twigs and leaves of Pseuduvaria indochinensis Merr., a new benzopyran derivative, pseudindochin (1), was isolated together with three known compounds, oligandrol (2), (6E,10E)-isopolycerasoidol (3) and polycerasoidol (4). The structure of compound 1 was elucidated on the basis of extensive spectroscopic data interpretation, including 1D, 2D NMR, HR-ESI-MS, UV and IR. Moreover, compounds 1 – 4 were evaluated in vitro for their cytotoxic activities against HL-60 and SMMC-7721 cell lines, but these compounds were essentially non-cytotoxic (IC50 . 30 mg/mL). Keywords: Pseuduvaria indochinensis Merr; benzopyran derivatives; spectroscopic data; cytotoxic activities
1. Introduction The genus Pseuduvaria (family Annonaceae) consists of about 17 species, of which only one species is distributed in the Yunnan Province of China. In prior reports, alkaloids (Zhong et al. 1988; Taha et al. 2011) and diterpenes (Xie et al. 1989) were isolated from this genus. Pseuduvaria indochinensis Merr. is a species of trees, mainly distributed in Southeast Asia. In this work, a new benzopyran derivative, pseudindochin (1), and three known compounds, oligandrol (2) (Banfield et al. 1994), (6E,10E)-isopolycerasoidol (3) (Gonzalez et al. 1996) and polycerasoidol (4) (Gonzalez et al. 1995), were isolated from the 95% EtOH extracts of the twigs and leaves of P. indochinensis, which were collected from the Yunnan Province of China. In this paper, we describe the isolation, structural elucidation and cytotoxic activities of the compounds isolated from P. indochinensis.
2. Results and discussion 2.1. Structural elucidation of compound 1 Compound 1 was obtained as yellow oil. Its molecular formula was established as C23H30O5 from the [M þ Na]þ peak at m/z 409.1991 (calcd 409.1985) in the positive HR-ESI-MS, indicating nine degrees of unsaturation. The IR absorptions suggested the absorption bands for hydroxyl (3378 cm21), olefin (1649 cm21), the ester carbonyl (1704 cm21) and aromatic ring (1465 and 1439 cm21) functionalities. The 1H NMR spectroscopic data showed three tertiary methyls at dH 1.36, 1.57, 2.13, a methoxyl at dH 3.78 (s, 3H), a pair of cis-conjugated olefinic protons at dH 6.25 (d, J ¼ 9.8 Hz, 1H) and 5.65 (d, J ¼ 9.8 Hz, 1H), and two olefinic methines at dH 5.14 (t, J ¼ 7.0 Hz, 1H) and *Corresponding author. Email: [email protected] q 2013 Taylor & Francis
Downloaded by [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
170
C.-G. Zhao et al.
6.21 (t, J ¼ 7.4 Hz, 1H). It also revealed two aromatic proton signals in meta position at dH 6.32 (d, J ¼ 2.9 Hz, 1H) and d 6.47 (d, J ¼ 2.9 Hz, 1H). Combined analysis of 13C NMR and DEPT spectroscopic data revealed 23 carbon signals attributed to four methyls, five methylenes, six methines and eight quaternary carbons (including four aromatic ring quaternary carbons at dC 144.80, 121.30, 148.66 and 126.29, one ester carboxyl carbon at dC 167.49, one oxygenated quaternary carbon at dC 77.76 and two olefinic quaternary carbon at dC 133.98 and 130.41). The structural feature was further corroborated by 1 H – 1H COSY and HSQC data analysis, which showed connectivity of two structural moieties: CH2(4)ZCH2(5)ZCH(6) moiety and CH2(8)ZCH2(9)ZCH(10) moiety. Careful comparison of the NMR data of 1 with those of 4 indicated that the signals were basically similar to each other. However, the signals for two methylenes and one Me observed at dC 22.43, 31.44 and 20.48 were not present in 1; instead, the signals of a pair of cis-conjugated double bond at dH 6.25 (d, J ¼ 9.8 Hz, 1H) and 5.65 (d, J ¼ 9.8 Hz, 1H) and dC 122.91, 130.62, a CH2OH group at dH 4.22 (2H, d, 5.3) and dC 65.30, and a methoxyl at dH 3.78 (s, 3 H) and dC 51.49 were detected in the spectra of 1. Compound 1 was completely characterised by HMBC experiments. The HMBC correlations from H-14 to C-6, C-7 and C-8, from H-6 to C-8 and C-14, and from H-8 to C-6, C-7 and C-14 revealed that both C-6 and C-8 connected with C-7 and the C-14 was attached to C-7. The correlations from H-10 to C-12, from OCH3-13 to C-13, from H-12 to C-10, C-11 and C-13 indicated the presence of an a,b-unsaturated ester and the C-12 located at C-11. The correlations from H-30 to C-1, from H-2 to C-20 , C-3, C-4 and C-15, from H-4 to C-2 and C-15, and from H-15 to C-2, C-3 and C-4 were in accord with the presence of (C-30 )ZC(20 )Z CH(1)vCH(2)ZC(3)ZC(4) moiety, the C-15 was attached to C-3 and C-50 was attached to C-10 , C-30 and C-40 , a hydroxyl group must locate at C-40 . The structure was confirmed except two Obearing carbons C-10 and C-3. By means of formula C23H30O5, a remaining O-atom must be connected to both C-10 and C-3 to form a six-membered ring composed of C(10 )ZOZC(3)Z CH(2)ZCH(1)ZC(20 ). Thus, the planer structure of compound 1 was confirmed. The relative configuration of 1 was derived by NOE difference spectra. The resonance of H-6 was enhanced by irradiation of H-8, and H-10 was enhanced by irradiation of H-12, indicating the 6E, and 10Z geometry for D6,7 and D10,11, respectively. On the basis of these data, compound 1 was established and named pseudindochin (1) (Figure 1). The structures of the known compounds 2– 4 were determined by comparing their physical and spectral data with those published in the literatures. They were identified as oligandrol (2) (Banfield et al. 1994), (6E,10E)-isopolycerasoidol (3) (Gonzalez et al. 1996) and polycerasoidol (4) (Gonzalez et al. 1995).
6' 1' O 5' HO 4'
3' 2' 1
15 3
13 COOCH3
14 5
24
9 6
7
8
10
11 CH2OH 12
O HO 2
1
COOH
O O COOH
HO
HO 3
Figure 1. Chemical structures of compounds 1 –4.
4
Natural Product Research
171
Downloaded by [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
2.2. In vitro cytotoxic activity In order to investigate the in vitro cytotoxicity of compounds 1 –4, they were tested for their inhibitory activity against the human promyelocytic leukaemia cell line HL-60 and human hepatocellular carcinoma cell line SMMC-7721 by sulforhodamine B (SRB) colorimetric assay (Skehan et al. 1990). It was found that compounds 1– 4 were inactive towards all the tested cell lines (IC50 . 30 mg/mL).
3. Experimental 3.1. General Optical rotations were measured on a PerkinElmer Model 341 polarimeter with a 1dm cell. UV spectra were measured on a Varian 3542 UV –visible spectrometer (Varian Inc., California, USA). IR spectra were recorded on a Nicolet NEXUS 670 FT-IR spectrometer over the range of 400 –4000 cm21. HR-ESI-MS determinations were run on a Bruker APEX II mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). NMR spectra were conducted on a Bruker AM400BB (400 MHz) spectrometer (d in ppm rel. to TMS, J in Hz). Sephadex LH-20 (Amersham Pharmacia Biotech, Piscataway, NJ, USA), RP-C18 silica gel (150 – 200 mesh, Merck, Darmstadt, Germany) and silica gel (200 –300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) were used for column chromatography (CC). Thin-layer chromatography (TLC): silica gel GF254 (10 –40 mm; Qingdao Marine Chemical Factory); detection under UV light and visualised by spraying with 5% H2SO4 in C2H5OH (v/v), followed by heating. Analytical TLC was provided to follow the separation and check the purity of isolated compounds.
3.2. Plant material The leaves and twigs of P. indochinensis Merr. were collected from Yunnan Province, P. R. China, in December 2009, and authenticated by Prof. Guo-Da Tao of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (No. 200912PI) was deposited in the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences.
3.3. Extraction and isolation The air-dried powdered leaves and twigs of P. indochinensis (15.0 kg) were extracted with EtOH (95%, v/v) three times (each for 3 h) at 408C and concentrated in vacuo to give a crude extract (1500 g). The crude extract was suspended in H2O and then extracted with petroleum ether, chloroform and EtOAc, successively. The petroleum ether phase and CHCl3-soluble fractions were combined due to similar constituents on TLC. The combined extract (600 g) was subjected to silica gel CC (3200 g) eluting with a petroleum ether/acetone (100:0 to 0:100, CH3OH) gradient system to afford fractions Fr1 –Fr9 under the aid of TLC examination. Fr3 was applied to chromatographed over Sephadex LH-20 eluting with CHCl3/MeOH (v/v ¼ 2:1) to afford fractions Fr3.1 – Fr3.4. Fr3.3 was subjected to silica gel CC with a gradient elution of petroleum ether/AcOEt (30:1 ! 3:1) to afford 3 (70 mg) and 1 (10 mg). Fr6 was separated by CC over silica gel (petroleum ether/acetone, 10:1 ! 1:1) to provide fractions Fr6.1 –Fr6.5. Purification of subfraction Fr6.3 by preparative TLC using CHCl3/EtOAc (v/v ¼ 5:1) as a developing solvent system afforded 2 (130 mg). Fr6.4 was purified on a column of RP-C18 silica gel (H2O/ CH3OH 1:4, v/v) to afford 4 (18 mg).
Downloaded by [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
172
C.-G. Zhao et al.
3.3.1. Pseudindochin (1) Pseudindochin (1): Yellow oil; C23H30O5; ½a20 D : þ 4.4 (c 0.23 in CHCl3); HR-ESI-MS: m/z ¼ 409.1991 [M þ Na]þ (calcd 409.1985); IR (KBr) nmax: 3379, 1704, 1465, 1440, 1214 and 1001 cm21; 1H NMR (400 MHz, CDCl3) dH 1.36 (3H, s, H-15), 1.57 (3H, s, H-14), 1.66 (2H, m, H-4), 2.06 (2H, m, H-8), 2.10 (2H, m, H-5), 2.13 (3H, s, CH3-60 ), 2.61 (2H, q, J ¼ 7.4 Hz, H-9), 3.78 (3H, s, OCH3-13), 4.22 (2H, d, J ¼ 5.3 Hz, H-12), 4.80 (br s, HO-40 ), 5.14 (1H, t, J ¼ 7.0 Hz, H-6), 5.56 (1H, d, 9.8, H-2), 6.21 (1H, t, J ¼ 7.4 Hz, H-10), 6.24 (1H, d, 9.8, H-1), 6.32 (1H, d, J ¼ 2.9 Hz, H-30 ), 6.47 (1H, d, J ¼ 2.9 Hz, H-50 ); 13C NMR data (100 MHz, CDCl3) dC15.47 (CH3-60 ), 15.72 (C-14), 22.59 (C-5), 25.89 (C-15), 27.79 (C-9), 38.67 (C-8), 40.70 (C4), 51.49 (OCH3-13), 65.30 (C-12), 77.76 (C-3), 110.30 (C-30 ), 117.08 (C-50 ), 121.30 (C-20 ), 122.91 (C-1), 125.17 (C-6), 126.29 (C-60 ), 130.41 (C-11), 130.62 (C-2), 133.98 (C-7), 144.80 (C-10 ), 146.43 (C-10), 148.66 (C-40 ), 167.49 (C-13); CD [nm (D1)] (2.54 mg/100 mL MeOH): 211 (þ 29.52), 221 (2 31.79), 247 (þ 13.58).
3.4. Measurement of cytotoxicity 3.4.1. Cell culture HeLa cells HL-60 and human hepatoma cells SMMC-7721 were grown as a monolayer in RPMI1640 (Gibco, New York, USA) supplemented with 10% (v/v) foetal bovine serum (Sigma, Deisenhofen, Germany) and 1% penicillin –streptomycin solution (penicillin 10,000 IE/mL; streptomycin 10,000 mg/mL, Biochrom AG, Berlin, Germany). Cells were grown at 378C in 95% air humidity and 5% CO2, and sub-cultured twice weekly using trypsine/EDTA (0.05%/ 0.02%, Lonza, Shanghai, China). DMSO (0.1%) alone was used as a vehicle control.
3.4.2. SRB cytotoxicity assay For experimentation, exponentially growing cells were harvested and seeded in 96-well plates with the final volume 100 mL containing 4 £ 103 cells per well. After 24 h incubation, cells were treated with various concentrations of tested substances for 48 h. The tested substances were diluted in assay medium using a stock solution (40 mM vehicle DMSO), and the final vehicle concentration did not exceed 0.05%. Then, the cultures were fixed by the addition of 50% icecold trichloroacetic acid to give a final concentration of 10% and were placed at 48C for 1 h. The plates were flicked and washed five times with tap water to remove traces of medium, drug and serum, and were then air-dried. The air-dried plates were stained 10 min with 0.4% SRB 100 mL dissolved in 1% acetic acid. The wells were washed 5 times with 0.1% acetic acid and left to dry overnight. After dissolving the bound cells with 150 mL of 10 mM unbuffered aqueous Tris base (pH 10.5) using a gyratory shaker, the absorbance at 515 nm was determined. Binding of SRB dye to basic amino acids has a linear relationship with cell density, and SRB staining serves as an accurate method in measuring cell density through total protein staining. Cell viability was calculated as percentage of vehicle control after background reduction. All experiments were carried out thrice with five replicates for each concentration tested. Where applicable, IC50 values were calculated by linear regression.
4. Conclusions In conclusion, a new benzopyran derivative, pseudindochin (1), was isolated together with three known compounds from the twigs and leaves of Pseuduvaria indochinensis. However, these compounds were essentially noncytotoxic activities.
Natural Product Research
173
Supplementary material Supplementary Figures S1 –S9 relating to this article are available online. Acknowledgements
Downloaded by [Bibliothèques de l'Université de Montréal] at 04:02 02 December 2014
Financial support from the National Basic Research Program Projects of China (2007CB108903), National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (J1103307) and the Fundamental Research Funds for the Central Universities (lzujbky-2012-62) is gratefully acknowledged.
Note 1. These authors contributed equally to this work.
References Banfield JE, Black DSC, Collins DJ, Hyland BPM, Lee JJ, Pranowo SR. 1994. Constituents of some species of Beilschmiedia and Endiandra (Lauraceae): new endiandric acid and benzopyran derivatives isolated from B. oligandra. Aust J Chem. 47:587–607. Gonzalez MC, Sentandreu MA, Rao KS, Zafra-Polo MC, Cortes D. 1996. Prenylated benzopyran derivatives from two Polyalthia species. Phytochemistry. 43:1361–1364. Gonzalez MC, Serrano A, Zafra-Polo MC, Cortes D. 1995. Polycerasoidin and polycerasoidol, two new prenylated benzopyran derivatives from Polyalthia cerasoides. J Nat Prod. 58:1278–1284. Skehan P, Storeng R, Scudiero D. 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. J Nat Cancer Inst. 82:1107–1112. Taha H, Hadi A, Hamid A, Nordin N, Najmuldeen IA, Mohamad K, Shirota O, Nugroho AE, Piow WC, Kaneda T, Morita H. 2011. Pseuduvarines A and B, two new cytotoxic dioxoaporphine alkaloids from Pseuduvaria rugosa. Chem Pharm Bull. 59:896–897. Xie N, Zhong SM, Zhao SX, Waterman PG, He CH, Zheng QT. 1989. Diterpenes from Pseuduvaria indochinensis. J China Pharm Univ. 20:203–207. Zhong SM, Zhao SS, Xie N. 1988. Alkaloids from Pseuduvaria indochinensis. Phytochemistry. 27:4004–4005.
