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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 phenolic glycoside from Juglans mandshurica a
ab
a
a
b
Dalei Yao , Mei Jin , Changhao Zhang , Jie Luo , Ren Li , a
a
a
Mingshan Zheng , Jiongmo Cui & Gao Li a
Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, Yanbian University College of Pharmacy, Yanji 133002, China b
Yanbian University Hospital, Yanji 133000, China Published online: 04 Apr 2014.
To cite this article: Dalei Yao, Mei Jin, Changhao Zhang, Jie Luo, Ren Li, Mingshan Zheng, Jiongmo Cui & Gao Li (2014) A new phenolic glycoside from Juglans mandshurica, Natural Product Research: Formerly Natural Product Letters, 28:13, 998-1002, DOI: 10.1080/14786419.2014.902946 To link to this article: http://dx.doi.org/10.1080/14786419.2014.902946
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Natural Product Research, 2014 Vol. 28, No. 13, 998–1002, http://dx.doi.org/10.1080/14786419.2014.902946
A new phenolic glycoside from Juglans mandshurica Dalei Yaoa,1, Mei Jinab,1, Changhao Zhanga, Jie Luoa, Ren Lib, Mingshan Zhenga, Jiongmo Cuia and Gao Lia* a Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, Yanbian University College of Pharmacy, Yanji 133002, China; bYanbian University Hospital, Yanji 133000, China
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(Received 5 December 2013; final version received 5 March 2014) A new phenolic glycoside, 6-O-(40 -hydroxy-30 ,50 -dimethoxybenzoyl)-D -glucopyranose (4), and nine known compounds (1 – 3 and 5 – 10) were isolated from Juglans mandshurica Maxim. Compound structures were elucidated by NMR, HR-ESI-MS and acid hydrolysis. Compounds 5 and 6 are reported from this genus for the first time. Among compounds 1 – 10, only 1 exhibited cytotoxicity against MGC-803, A549, K562, JAR, HeLa, CaSKi and SiHa cell lines (IC50: 2.0, 5.3, 2.3, 6.9, 4.0, 6.6 and 2.7 mM, respectively). Keywords: Juglans mandshurica; Juglandaceae; phenolic glycoside
1. Introduction Juglans mandshurica Maxim. (Juglandaceae), a type of walnut tree, is mainly distributed throughout north-eastern Asia. The roots, stem bark, leaves and fruits of this plant are used as a folk medicine for cancer treatment in China and Korea (Xu et al. 2013). Several naphthalenyl glucosides (Joe et al. 1996), quinones (Li et al. 2007), flavonoids (Min et al. 2002), triterpenes (Li, Lee, et al. 2003) and diarylheptanoids (Lee et al. 2002; Li, Xu, et al. 2003; Li et al. 2004, 2005) have been isolated from J. mandshurica. These compounds have shown cytotoxic activity (Joe et al. 1996; Lee et al. 2002; Li, Xu, et al. 2003; Li et al. 2004), topoisomerase I and II inhibitory activity (Li, Lee, et al. 2003) and inhibitory effects on DNA polymerase and the RNase H activity of HIV-1 reverse transcriptase (Min et al. 2002). In the continuation of our studies on this plant (Lee et al. 2002; Li, Lee, et al. 2003; Li, Xu, et al. 2003; Li et al. 2004, 2005), a new phenolic glycoside (4) and nine known compounds 1–3 and 5–10 (Figure 1) were isolated from the EtOAc solvent fraction of the MeOH extract of J. mandshurica stem bark. Herein, we report the isolation, structural elucidation and cytotoxic activity of these new compounds and the other constituents from the stem bark of J. mandshurica. 2. Results and discussion Compound 4 was obtained as white amorphous solid (MeOH). The HR-ESI-MS (negative-ion mode) exhibited a quasi-molecular ion peak at m/z 359.0967 [M – H] – (calcd 359.0978), corresponding to the molecular formula C15H20O10. The 1H NMR spectrum of 4 showed signals for a galloyl group (d 7.34, s, 2H, H-20 , 60 ), two methoxyl groups (d 3.89, s, 6H, H-30 , 50 -OCH3) and a glucopyranose moiety (d 3.16 –5.12, m, 7H). The a- and b-anomeric configuration for glucopyranose was determined from JH1,H2 coupling constant value (7.3 Hz and 3.6 Hz).
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Figure 1. Structures of compounds 1 – 10 isolated from J. mandshurica.
The 13C NMR and distortionless enhanced polarization transfer spectra of 4 also showed the presence of a glucopyranose moiety and the chemical shifts of anomeric carbon signals at d 94.0 and 98.3, corresponding to a- and b-anomeric configuration (Hsu et al. 1994), respectively. This difference is the change in the equilibrium between two epimers of D -glucopyranose, when the corresponding stereocentres interconvert. In the HMBC spectrum, the connectivity of the galloyl group with glucopyranose was indicated by the cross-peak between the carbonyl group and H-6 of glucopyranose. The position of the two methoxyl groups on the aromatic ring was determined by the HMBC correlation of C-30 with H-30 -OCH3, and that of C-50 with H-50 -OCH3. After acid hydrolysis of 4, the aqueous layer was separated by HPLC to give D -glucose, and then optical rotation of the purified glucose was determined (Kiem et al. 2003). Consequently, the structure of 4 was identified as 6-O-(40 -hydroxy-30 ,50 -dimethoxybenzoyl)-D -glucopyranose. The known compounds 1 –3 and 5 – 10 were identified as, 5-hydroxy-2-methoxy-1,4naphthoquinone (1) (Khanna et al. 1989), 4,8-dihydroxy-1-tetralone (2) (Lal et al. 2011), 3b,24dihydroxyurs-12-en-28-oic acid (3) (Sashida et al. 1994), 6-O-galloyl-D -glucopyranose (5) (Hsu et al. 1994), 1-O-galloyl-b-D -glucopyranose (6) (Yue et al. 1994), 1,2,6-trigalloylglucopyranose (7) (Min et al. 2000), 1,2,3,6-tetragalloylglucopyranose (8) (Min et al. 2000; Duan et al. 2004), 40 a,50 ,80 -trihydroxy-a-tetralone-50 -O-b-D -[6-O-(400 -hydroxy-300 ,500 -dimethoxybenzoyl)] glucopyranose (9) (Kim et al. 1998) and 1,4,8-trihydroxynaphthalene-1-O-b-D -glucopyranoside (10) (Son 1995). Compounds 5 and 6 are reported here for the first time from the genus Juglans. Primary bioassays showed that only 1 exhibited cytotoxicity against MGC-803, A549, K562, JAR, HeLa, CaSKi and SiHa cell lines (Table 1). 3. Experimental 3.1. General experimental procedures Optical rotations were measured using a Rudolph Autopol I (Rudolph Research Analytical, Hackettstown, NJ, USA) automatic polarimeter. IR spectra were recorded on a Shimadzu
1000
D. Yao et al.
Table 1. Cytotoxicity of compoundsa isolated from J. mandshurica on tumour cell growth. IC50 (mM) Sample 1
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a
MGC-803
A549
K562
JAR
HeLa
CaSKi
SiHa
2.0
5.3
2.3
6.9
4.0
6.6
2.7
Compounds 2– 10 were inactive (IC50 . 100 mM).
IRPrestige-21 (Shimadzu Ltd, Tokyo, Japan) FT-IR spectrophotometer. NMR spectra were recorded on a Bruker 600 MHz (AV-600) for 4 and on a Bruker 300 MHz (AV-300) for 1 –3 and 5 –10. HR-ESI-MS spectra were recorded on a Bruker microTOF QII (Bruker Daltonics, Fremont, CA, USA) mass spectrometer. Preparative HPLC was carried out on a Bonna-Agela FL-LC050 series chromatograph (Bonna-Agela Technologies, Tianjin, China), equipped with a HP-Q-P050 binary pump, a Rheodyne 7725i rheodyne injector and a HP-Q-UV100 multiple wave detector. Separations were performed on a YMC-Pack ODS-A C18 column (10 mm, 250 mm £ 10 mm, YMC, Kyoto, Japan) and a Venusil ASB C18 column (5 mm, 150 mm £ 4.6 mm, Bonna-Agela Technologies, Tianjin, China). High-speed countercurrent chromatography (HSCCC) was performed using a OptiChrome-360 HSCCC system manufactured by Counter Current Technology Co., Ltd (Jiangyin, China), equipped with a 366 mL coil column made of polytetrafluoroethylene tubing (i.d. 1.6 mm). Column chromatography was performed using silica gel (200 –300 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd, Qingdao, China). Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB (GE Healthcare, Uppsala, Sweden). TLC was performed with precoated Silica gel GF254 glass plates (Branch of Qingdao Haiyang Chemical Co., Ltd, Qingdao, China). 3.2. Plant material The stem bark of J. mandshurica was collected in August 2006 in a mountainous area of Yanji City, Jilin Province, P.R. China. Plant samples were identified by Prof. Huizi Lv (College of Pharmacy, Yanbian University). A voucher specimen (voucher number: YB-HT-0112) has been deposited at College of Pharmacy, Yanbian University. 3.3. Extraction and isolation The dried stem bark of J. mandshurica (10 kg) was extracted with 70% MeOH (3 £ 15 L) at room temperature. The MeOH extract was dissolved in water and partitioned successively with CHCl3, EtOAc and n-BuOH (each 3 £ 1 L) saturated with H2O. The EtOAc fraction (200 g) was separated by silica gel column chromatography with a gradient of CHCl3 – MeOH (100% CHCl3 to 100% MeOH) to give 15 major fractions (A– O). Fraction A (600 mg) was further applied to a silica gel column with a gradient of CHCl3 – MeOH (100:1 to 50:50), followed by reparative HPLC with MeOH – H2O (60:40 to 90:10) to afford compounds 1 (5.5 mg), 2 (20 mg) and 3 (10.2 mg). Fractions I (500 mg) and M (630 mg) were separately purified by HSCCC using a single solvent system composed of CHCl3 –MeOH –H2O (4:3:2) to afford 4 (26 mg), 5 (10.5 mg) and 6 (8.5 mg) from I and 9 (10.3 mg) and 10 (7.6 mg) from M. Fraction O (700 mg) was purified by Sephadex LH-20 (MeOH –H2O, 4:1) and reparative HPLC with MeOH – H2O (20:80 to 90:10) to yield 7 (23.7 mg) and 8 (12.8 mg). 6-O-(40 -Hydroxy-30 ,50 -dimethoxybenzoyl)-D -glucopyranose (4): White amorphous solid; 25 ½aD 2 40.38 (c ¼ 0.03, MeOH); IR (KBr) vmax: 3407, 2924, 2852, 1714, 1564, 1464, 1342, 1222, 1114 cm – 1; UV (MeOH) lmax (log 1): 277 (1.81), 223 (2.49) nm; HR-ESI-MS m/z 359.0967 [M – H] – (Calcd for C15H19O10, 359.0978); 1H NMR (CD3OD, 600 MHz) d 7.34
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(2H, s, H-20 , 60 ), 5.12 (1H, d, J ¼ 3.6 Hz, a form H-1), 4.59 (1H, dd, J ¼ 2.0, 11.5 Hz, a and b form H-6a), 4.53 (1H,d, J ¼ 7.3 Hz, b form H-1), 4.42 (1H, dd, J ¼ 5.5, 11.5 Hz, a and b form H-6b), 4.10 (1/2H, m, a form H-4), 3.89 (6H, s, H-30 , 50 -OCH3), 3.72 (1/2H, m, a form H-3), 3.62 (1/2H, m, b form H-2), 3.48 (2H, m, a form H-2, 5 and b form H-3, 4), 3.16 (1/2H, m, b form H-5); 13C NMR (CD3OD, 150 MHz) d 168.0 (C-70 ), 148.9 (C-30 , 50 ), 142.0 (C-40 ), 121.2 (C10 ), 108.2 (C-20 , 60 ), 98.3 (b form C-1), 94.0 (a form C-1), 77.9 (b form C-3), 76.2 (b form C-5), 75.5 (b form C-2), 74.7 (a form C-3), 73.8 (a form C-5), 72.1 (a form C-2), 71.8 (b form C-4), 70.9 (a form C-4), 65.3 (b form C-6), 65.2 (a form C-6), 56.8 (C-30 , 50 -OCH3).
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3.4. Acid hydrolysis of compound 4 A solution of compound 4 (2.0 mg) in 2 mL of 5% HCl was heated for 3 h. The reaction mixture was concentrated under reduced pressure and diluted with 15 mL H2O. The solution was neutralised with Ag2CO3 and filtered. The aqueous layer was concentrated, filtered and passed through a Venusil ASB C18 column (150 mm £ 4.6 mm) and then repeatedly separated by HPLC (MeCN – H2O, 4:1) to afford the D -glucose fraction. The optical rotation value {½a25 D þ 36.8 (c ¼ 0.05, H2O)} was in agreement with that of D -glucose (Crublet et al. 2003; Kiem et al. 2003).
3.5. Cytotoxicity The tetrazolium-based colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT assay) (Rubinstein et al. 1990) was used for the in vitro assay of cytotoxicity against human gastric cancer cell line (MGC-803), human lung cancer cell line (A549), human leukaemic cell line (K562), human placental choriocarcinoma cell line (JAR) and human cervical cancer cell lines (HeLa, CaSKi and SiHa).
4. Conclusion J. mandshurica is one of rare species of trees for medicinal resources. Ten compounds were isolated from the stem bark of J. mandshurica, including one new phenolic glycoside (4) and 9 known compounds (1 –3 and 5 –10). 1 exhibited cytotoxicity against MGC-803, A549, K562, JAR, HeLa, CaSKi and SiHa cell lines. The present study provides an important basis for further research and utilisation from endangered plant species, it could help to rationalise the conservation efforts of such plants considering that they may serve as drug leads to benefit human health and create socioeconomic value.
Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S15.
Acknowledgements This work was supported by the National Natural Science Foundation of China under grant numbers 30760291, 81160386 and 30911140276.
Note 1. These authors contributed equally to the work.
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References Crublet ML, Long C, Se´venet T, Hadi HA, Lavaud C. 2003. Acylated flavonol glycosides from leaves of Planchonia grandis. Phytochemistry. 64:589–594. Duan DL, Li ZQ, Luo HP, Zhang W, Chen LR, Xu XJ. 2004. Antiviral compounds from traditional Chinese medicines Galla Chinese as inhibitors of HCV NS3 protease. Bioorg Med Chem Lett. 14:6041–6044. Hsu FL, Lee YY, Cheng JT. 1994. Antihypertensive activity of 6-O-galloyl-D-glucose, a phenolic glycoside from Sapium sebiferum. J Nat Prod. 57:308–312. Joe YK, Son JK, Park SH, Lee IJ, Moon DC. 1996. New naphthalenyl glucosides from the roots of Juglans mandshurica. J Nat Prod. 59:159–160. Khanna RN, Singh KP, Yadav SK, Srinivas S. 1989. Iodine catalysed alkyloxylation of naphthoquinones. Synth Commun. 19:3151–3157. Kiem PV, Cai XF, Minh CV, Lee JJ, Kim YH. 2003. Lupane-triterpene carboxylic acids from the leaves of Acanthopanax trifoliatus. Chem Pharm Bull. 51:1432–1435. Kim SH, Lee KS, Son JK, Je GH, Lee JS, Lee CH, Cheong CJ. 1998. Cytotoxic compounds from the roots of Juglans mandshurica. J Nat Prod. 61:643–645. Lal C, Raja ASM, Pareek PK, Shakyawar DB, Sharma KK, Sharma MC. 2011. Juglans nigra: chemical constitution and its application on Pashmina (Cashmere) fabric as a dye. J Nat Prod Plant Resour. 1:13–19. Lee KS, Li G, Kim SH, Lee CS, Woo MH, Lee SH, Jhang YD, Son JK. 2002. Cytotoxic diarylheptanoids from the roots of Juglans mandshurica. J Nat Prod. 65:1707–1708. Li G, Cui JM, Kwon YJ, Seo CS, Lee CS, Woo MH, Lee ES, Jahng YD, Chang HW, Lee SH, Son JK. 2005. Two new diarylheptanoids from Juglans mandshurica. Bull Korean Chem Soc. 26:1878–1880. Li G, Lee SY, Lee KS, Lee SW, Kim SH, Lee SH, Lee CS, Woo MH, Son JK. 2003. DNA topoisomerases I and II inhibitory activity of constituents isolated from Juglans mandshurica. Arch Pharm Res. 26:466–470. Li G, Seo CS, Lee SH, Jahng YD, Chang HW, Lee CS, Woo MH, Son JK. 2004. Diarylheptanoids from the roots of Juglans mandshurica. Bull Korean Chem Soc. 25:397–399. Li G, Xu ML, Choi HG, Lee SH, Jahng YD, Lee CS, Moon DC, Woo MH, Son JK. 2003. Four new diarylheptanoids from the roots of Juglans mandshurica. Chem Pharm Bull. 51:262–264. Li ZB, Wang JY, Jiang B, Zhang XL, An LJ, Bao YM. 2007. Benzobijuglone, a novel cytotoxic compound from Juglans mandshurica, induced apoptosis in HeLa cervical cancer cells. Phytomedicine. 14:846–852. Min BS, Lee HK, Lee SM, Kim YH, Bae KH, Otake T, Nakamura N, Hattori M. 2002. Anti-human immunodeficiency virus-type 1 activity of constituents from Juglans mandshurica. Arch Pharm Res. 25:441–445. Min BS, Nakamura N, Miyashiro H, Kim YH, Hattori M. 2000. Inhibition of human immunodeficiency virus type 1 reverse transcriptase and ribonuclease H activities by constituents of Juglans mandshurica. Chem Pharm Bull. 48:194–200. Rubinstein LV, Shoemaker RH, Paull KD, Simon RM, Tosini S, Skehan P, Scudiero DA, Monks A, Boyd MR. 1990. Comparison of in vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines. J Natl Cancer Inst. 82:1113– 1118. Sashida Y, Ogawa K, Yamanouchi T, Tanaka H, Shoyama Y, Nishioka I. 1994. Triterpenoids from callus tissue of Actinidia polygama. Phytochemistry. 35:377–380. Son JK. 1995. Isolation and structure determination of a new tetralone glucoside from the roots of Juglans mandshurica. Arch Pharm Res. 18:203– 205. Xu HL, Yu XF, Qu SC, Sui DY. 2013. Juglone, isolated from Juglans mandshurica Maxim, induces apoptosis via downregulation of AR expression in human prostate cancer LNCaP cells. Bioorg Med Chem Lett. 23:3631– 3634. Yue J, Lin Z, Wang D, Sun H. 1994. A sesquiterpene and other constituents from Erigeron breviscapus. Phytochemistry. 36:717–719.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new phenolic glycoside from Juglans mandshurica a
ab
a
a
b
Dalei Yao , Mei Jin , Changhao Zhang , Jie Luo , Ren Li , a
a
a
Mingshan Zheng , Jiongmo Cui & Gao Li a
Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, Yanbian University College of Pharmacy, Yanji 133002, China b
Yanbian University Hospital, Yanji 133000, China Published online: 04 Apr 2014.
To cite this article: Dalei Yao, Mei Jin, Changhao Zhang, Jie Luo, Ren Li, Mingshan Zheng, Jiongmo Cui & Gao Li (2014) A new phenolic glycoside from Juglans mandshurica, Natural Product Research: Formerly Natural Product Letters, 28:13, 998-1002, DOI: 10.1080/14786419.2014.902946 To link to this article: http://dx.doi.org/10.1080/14786419.2014.902946
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 13, 998–1002, http://dx.doi.org/10.1080/14786419.2014.902946
A new phenolic glycoside from Juglans mandshurica Dalei Yaoa,1, Mei Jinab,1, Changhao Zhanga, Jie Luoa, Ren Lib, Mingshan Zhenga, Jiongmo Cuia and Gao Lia* a Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, Yanbian University College of Pharmacy, Yanji 133002, China; bYanbian University Hospital, Yanji 133000, China
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(Received 5 December 2013; final version received 5 March 2014) A new phenolic glycoside, 6-O-(40 -hydroxy-30 ,50 -dimethoxybenzoyl)-D -glucopyranose (4), and nine known compounds (1 – 3 and 5 – 10) were isolated from Juglans mandshurica Maxim. Compound structures were elucidated by NMR, HR-ESI-MS and acid hydrolysis. Compounds 5 and 6 are reported from this genus for the first time. Among compounds 1 – 10, only 1 exhibited cytotoxicity against MGC-803, A549, K562, JAR, HeLa, CaSKi and SiHa cell lines (IC50: 2.0, 5.3, 2.3, 6.9, 4.0, 6.6 and 2.7 mM, respectively). Keywords: Juglans mandshurica; Juglandaceae; phenolic glycoside
1. Introduction Juglans mandshurica Maxim. (Juglandaceae), a type of walnut tree, is mainly distributed throughout north-eastern Asia. The roots, stem bark, leaves and fruits of this plant are used as a folk medicine for cancer treatment in China and Korea (Xu et al. 2013). Several naphthalenyl glucosides (Joe et al. 1996), quinones (Li et al. 2007), flavonoids (Min et al. 2002), triterpenes (Li, Lee, et al. 2003) and diarylheptanoids (Lee et al. 2002; Li, Xu, et al. 2003; Li et al. 2004, 2005) have been isolated from J. mandshurica. These compounds have shown cytotoxic activity (Joe et al. 1996; Lee et al. 2002; Li, Xu, et al. 2003; Li et al. 2004), topoisomerase I and II inhibitory activity (Li, Lee, et al. 2003) and inhibitory effects on DNA polymerase and the RNase H activity of HIV-1 reverse transcriptase (Min et al. 2002). In the continuation of our studies on this plant (Lee et al. 2002; Li, Lee, et al. 2003; Li, Xu, et al. 2003; Li et al. 2004, 2005), a new phenolic glycoside (4) and nine known compounds 1–3 and 5–10 (Figure 1) were isolated from the EtOAc solvent fraction of the MeOH extract of J. mandshurica stem bark. Herein, we report the isolation, structural elucidation and cytotoxic activity of these new compounds and the other constituents from the stem bark of J. mandshurica. 2. Results and discussion Compound 4 was obtained as white amorphous solid (MeOH). The HR-ESI-MS (negative-ion mode) exhibited a quasi-molecular ion peak at m/z 359.0967 [M – H] – (calcd 359.0978), corresponding to the molecular formula C15H20O10. The 1H NMR spectrum of 4 showed signals for a galloyl group (d 7.34, s, 2H, H-20 , 60 ), two methoxyl groups (d 3.89, s, 6H, H-30 , 50 -OCH3) and a glucopyranose moiety (d 3.16 –5.12, m, 7H). The a- and b-anomeric configuration for glucopyranose was determined from JH1,H2 coupling constant value (7.3 Hz and 3.6 Hz).
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Figure 1. Structures of compounds 1 – 10 isolated from J. mandshurica.
The 13C NMR and distortionless enhanced polarization transfer spectra of 4 also showed the presence of a glucopyranose moiety and the chemical shifts of anomeric carbon signals at d 94.0 and 98.3, corresponding to a- and b-anomeric configuration (Hsu et al. 1994), respectively. This difference is the change in the equilibrium between two epimers of D -glucopyranose, when the corresponding stereocentres interconvert. In the HMBC spectrum, the connectivity of the galloyl group with glucopyranose was indicated by the cross-peak between the carbonyl group and H-6 of glucopyranose. The position of the two methoxyl groups on the aromatic ring was determined by the HMBC correlation of C-30 with H-30 -OCH3, and that of C-50 with H-50 -OCH3. After acid hydrolysis of 4, the aqueous layer was separated by HPLC to give D -glucose, and then optical rotation of the purified glucose was determined (Kiem et al. 2003). Consequently, the structure of 4 was identified as 6-O-(40 -hydroxy-30 ,50 -dimethoxybenzoyl)-D -glucopyranose. The known compounds 1 –3 and 5 – 10 were identified as, 5-hydroxy-2-methoxy-1,4naphthoquinone (1) (Khanna et al. 1989), 4,8-dihydroxy-1-tetralone (2) (Lal et al. 2011), 3b,24dihydroxyurs-12-en-28-oic acid (3) (Sashida et al. 1994), 6-O-galloyl-D -glucopyranose (5) (Hsu et al. 1994), 1-O-galloyl-b-D -glucopyranose (6) (Yue et al. 1994), 1,2,6-trigalloylglucopyranose (7) (Min et al. 2000), 1,2,3,6-tetragalloylglucopyranose (8) (Min et al. 2000; Duan et al. 2004), 40 a,50 ,80 -trihydroxy-a-tetralone-50 -O-b-D -[6-O-(400 -hydroxy-300 ,500 -dimethoxybenzoyl)] glucopyranose (9) (Kim et al. 1998) and 1,4,8-trihydroxynaphthalene-1-O-b-D -glucopyranoside (10) (Son 1995). Compounds 5 and 6 are reported here for the first time from the genus Juglans. Primary bioassays showed that only 1 exhibited cytotoxicity against MGC-803, A549, K562, JAR, HeLa, CaSKi and SiHa cell lines (Table 1). 3. Experimental 3.1. General experimental procedures Optical rotations were measured using a Rudolph Autopol I (Rudolph Research Analytical, Hackettstown, NJ, USA) automatic polarimeter. IR spectra were recorded on a Shimadzu
1000
D. Yao et al.
Table 1. Cytotoxicity of compoundsa isolated from J. mandshurica on tumour cell growth. IC50 (mM) Sample 1
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a
MGC-803
A549
K562
JAR
HeLa
CaSKi
SiHa
2.0
5.3
2.3
6.9
4.0
6.6
2.7
Compounds 2– 10 were inactive (IC50 . 100 mM).
IRPrestige-21 (Shimadzu Ltd, Tokyo, Japan) FT-IR spectrophotometer. NMR spectra were recorded on a Bruker 600 MHz (AV-600) for 4 and on a Bruker 300 MHz (AV-300) for 1 –3 and 5 –10. HR-ESI-MS spectra were recorded on a Bruker microTOF QII (Bruker Daltonics, Fremont, CA, USA) mass spectrometer. Preparative HPLC was carried out on a Bonna-Agela FL-LC050 series chromatograph (Bonna-Agela Technologies, Tianjin, China), equipped with a HP-Q-P050 binary pump, a Rheodyne 7725i rheodyne injector and a HP-Q-UV100 multiple wave detector. Separations were performed on a YMC-Pack ODS-A C18 column (10 mm, 250 mm £ 10 mm, YMC, Kyoto, Japan) and a Venusil ASB C18 column (5 mm, 150 mm £ 4.6 mm, Bonna-Agela Technologies, Tianjin, China). High-speed countercurrent chromatography (HSCCC) was performed using a OptiChrome-360 HSCCC system manufactured by Counter Current Technology Co., Ltd (Jiangyin, China), equipped with a 366 mL coil column made of polytetrafluoroethylene tubing (i.d. 1.6 mm). Column chromatography was performed using silica gel (200 –300 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd, Qingdao, China). Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB (GE Healthcare, Uppsala, Sweden). TLC was performed with precoated Silica gel GF254 glass plates (Branch of Qingdao Haiyang Chemical Co., Ltd, Qingdao, China). 3.2. Plant material The stem bark of J. mandshurica was collected in August 2006 in a mountainous area of Yanji City, Jilin Province, P.R. China. Plant samples were identified by Prof. Huizi Lv (College of Pharmacy, Yanbian University). A voucher specimen (voucher number: YB-HT-0112) has been deposited at College of Pharmacy, Yanbian University. 3.3. Extraction and isolation The dried stem bark of J. mandshurica (10 kg) was extracted with 70% MeOH (3 £ 15 L) at room temperature. The MeOH extract was dissolved in water and partitioned successively with CHCl3, EtOAc and n-BuOH (each 3 £ 1 L) saturated with H2O. The EtOAc fraction (200 g) was separated by silica gel column chromatography with a gradient of CHCl3 – MeOH (100% CHCl3 to 100% MeOH) to give 15 major fractions (A– O). Fraction A (600 mg) was further applied to a silica gel column with a gradient of CHCl3 – MeOH (100:1 to 50:50), followed by reparative HPLC with MeOH – H2O (60:40 to 90:10) to afford compounds 1 (5.5 mg), 2 (20 mg) and 3 (10.2 mg). Fractions I (500 mg) and M (630 mg) were separately purified by HSCCC using a single solvent system composed of CHCl3 –MeOH –H2O (4:3:2) to afford 4 (26 mg), 5 (10.5 mg) and 6 (8.5 mg) from I and 9 (10.3 mg) and 10 (7.6 mg) from M. Fraction O (700 mg) was purified by Sephadex LH-20 (MeOH –H2O, 4:1) and reparative HPLC with MeOH – H2O (20:80 to 90:10) to yield 7 (23.7 mg) and 8 (12.8 mg). 6-O-(40 -Hydroxy-30 ,50 -dimethoxybenzoyl)-D -glucopyranose (4): White amorphous solid; 25 ½aD 2 40.38 (c ¼ 0.03, MeOH); IR (KBr) vmax: 3407, 2924, 2852, 1714, 1564, 1464, 1342, 1222, 1114 cm – 1; UV (MeOH) lmax (log 1): 277 (1.81), 223 (2.49) nm; HR-ESI-MS m/z 359.0967 [M – H] – (Calcd for C15H19O10, 359.0978); 1H NMR (CD3OD, 600 MHz) d 7.34
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(2H, s, H-20 , 60 ), 5.12 (1H, d, J ¼ 3.6 Hz, a form H-1), 4.59 (1H, dd, J ¼ 2.0, 11.5 Hz, a and b form H-6a), 4.53 (1H,d, J ¼ 7.3 Hz, b form H-1), 4.42 (1H, dd, J ¼ 5.5, 11.5 Hz, a and b form H-6b), 4.10 (1/2H, m, a form H-4), 3.89 (6H, s, H-30 , 50 -OCH3), 3.72 (1/2H, m, a form H-3), 3.62 (1/2H, m, b form H-2), 3.48 (2H, m, a form H-2, 5 and b form H-3, 4), 3.16 (1/2H, m, b form H-5); 13C NMR (CD3OD, 150 MHz) d 168.0 (C-70 ), 148.9 (C-30 , 50 ), 142.0 (C-40 ), 121.2 (C10 ), 108.2 (C-20 , 60 ), 98.3 (b form C-1), 94.0 (a form C-1), 77.9 (b form C-3), 76.2 (b form C-5), 75.5 (b form C-2), 74.7 (a form C-3), 73.8 (a form C-5), 72.1 (a form C-2), 71.8 (b form C-4), 70.9 (a form C-4), 65.3 (b form C-6), 65.2 (a form C-6), 56.8 (C-30 , 50 -OCH3).
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3.4. Acid hydrolysis of compound 4 A solution of compound 4 (2.0 mg) in 2 mL of 5% HCl was heated for 3 h. The reaction mixture was concentrated under reduced pressure and diluted with 15 mL H2O. The solution was neutralised with Ag2CO3 and filtered. The aqueous layer was concentrated, filtered and passed through a Venusil ASB C18 column (150 mm £ 4.6 mm) and then repeatedly separated by HPLC (MeCN – H2O, 4:1) to afford the D -glucose fraction. The optical rotation value {½a25 D þ 36.8 (c ¼ 0.05, H2O)} was in agreement with that of D -glucose (Crublet et al. 2003; Kiem et al. 2003).
3.5. Cytotoxicity The tetrazolium-based colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT assay) (Rubinstein et al. 1990) was used for the in vitro assay of cytotoxicity against human gastric cancer cell line (MGC-803), human lung cancer cell line (A549), human leukaemic cell line (K562), human placental choriocarcinoma cell line (JAR) and human cervical cancer cell lines (HeLa, CaSKi and SiHa).
4. Conclusion J. mandshurica is one of rare species of trees for medicinal resources. Ten compounds were isolated from the stem bark of J. mandshurica, including one new phenolic glycoside (4) and 9 known compounds (1 –3 and 5 –10). 1 exhibited cytotoxicity against MGC-803, A549, K562, JAR, HeLa, CaSKi and SiHa cell lines. The present study provides an important basis for further research and utilisation from endangered plant species, it could help to rationalise the conservation efforts of such plants considering that they may serve as drug leads to benefit human health and create socioeconomic value.
Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S15.
Acknowledgements This work was supported by the National Natural Science Foundation of China under grant numbers 30760291, 81160386 and 30911140276.
Note 1. These authors contributed equally to the work.
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