- Email: [email protected]
A cytotoxic compound from the leaves of Juglans mandshurica
Chinese Chemical Letters 18 (2007) 846–848 www.elsevier.com/locate/cclet
A cytotoxic compound from the leaves of Juglans mandshurica Zhi Bo Li a, Yong Ming Bao a, Hong Bo Chen b, Jing Yun Wang a, Jie Han Hu c, Li Jia An a,* a
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China b School of Chemical Engineering, Dalian University of Technology, Dalian 116024, China c Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116011, China Received 12 March 2007
Abstract From Juglans mandshurica leaves, a new quinone compound was isolated through bioassay-guided fractionation. The structure elucidation of the compound was established based on spectroscopic studies, notably of the 2D NMR spectra. The compound exhibited moderate cytotoxic activities against Hela, MCF-7, BGC823 and 3T3-L1cell lines with IC50 ranges from 7.5 to 26.8 mmol/L. # 2007 Li Jia An. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Juglans mandshurica; p-Hydroxymethoxybenzobijuglone; Cytotoxicity
Juglans mandshurica’s leaves, roots and seeds have been used as a traditional medicine for treatment of cancer in China and Korea [1,2]. Up to now, the phytochemical investigations of J. mandshurica mainly involved diaryheptanoids [3], flavonoids [4], volatile oil [5], naphthoquinones [6,7]. Most of them have been isolated from the seeds and roots of J. mandshurica. The reports on the chemical or biological activity research of J. mandshurica leaves are rare. Our previous work showed that the ethanol extract of J. mandshurica leaves had anticancer activity in vivo and in vitro [8,9]. Subsequently, the fractionation and purification of the ethanol extract of J. mandshurica leaves with cytotoxicity assay using the Hela cell line, led to the isolation of a new quinone compound, p-hydroxymethoxybenzobijuglone (HMBBJ) (Fig. 1). Quinones are widespread among plants, which show biological activities including antitumour, wound healing, anti-inflammatory, antiparasitic, particularly cytotoxic activities. Commonly, the researches of quiniones focused on naphthoquinones and anthraquinones [10], however, few reports on benzobinaphthoquinone compound are available up to date. HMBBJ was isolated as red needles and its UV spectrum (lmax 275, 380, 480 nm) suggested it has a quinone skeleton. Twenty-three carbon signals showed in inverted gated decoupling 13C NMR and [M-H] ion peak at m/z 415.0462 in the negative ion HREIMS spectrum indicated that the molecular formula of HMBBJ was C23H12O8, accounting for eighteen degrees of unsaturation. The IR absorption at 3579, 3513 cm1 and 1721 cm1 showed the existence of hydroxyl and carbonyl groups in HMBBJ. The 13C NMR (Table 1), inverted gated decoupling 13C NMR
* Corresponding author. E-mail address: [email protected] (L.J. An). 1001-8417/$ – see front matter # 2007 Li Jia An. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2007.05.043
Z.B. Li et al. / Chinese Chemical Letters 18 (2007) 846–848
847
Fig. 1. The structure of p-hydroxymethoxybenzobijuglone (HMBBJ).
Table 1 NMR spectroscopic data of compound (500 MHz for 1H NMR, 125 MHz for Position
dH
0
1,1 2,20 3,30 4,40 5,50 6,60 7,70 8,80 9,90 10, 100 100 200 OCH3 OH
7.16 (dd, 8.0, 1.1) 7.57 (t, 8.0) 7.47 (dd, 8.0, 1.1)
3.81 (s) 13.76 (s)
13
C NMR, DMSO-d6, d ppm, J Hz) dC
HMBC (C ! H)
179.5 122.9 127.7 184.9 161.8 123.0 134.7 117.2 134.4 117.7 167.8 123.7 51.6
H8
OH, H7 OH, H8 H6 H7 OH, H6, H8 OCH3
and DEPT spectroscopic data disclosed the presence of four carbonyl carbons at d179.5(C-1, C-10 ), 184.9(C-4, C-40 ), 12 sp2 quaternary carbons at d167.8(C-100 ), 123.7(C-200 ), 122.9(C-2, C-20 ), 127.7(C-3, C-30 ), 161.8(C-5, C-50 ), 134.4(C-9, C-90 ), 117.7(C-10, C-100 ), 6 sp2 methines at d123.0(C-6, C-60 ), 134.7(C-7, C-70 ), 117.2(C-8, C-80 ), and 1 sp3methyl for OCH3 at d51.6, suggesting that the structure of HMBBJ is symmetrical with two naphthoquinones connected with benzene. In the1HNMR spectrum (Table 1), six signals with dH between 7.16 and 7.57 were attributed to the protons on the two aromatic rings and one methoxyl signal at dH 3.81. With the spectrum of 1H–1H COSY, it was further confirmed that dH 7.16 was for H-6, H-60 , dH 7.57 was for H-7, H-70 and dH 7.47 for H-8,H-80 , the coupling constant between H-6(60 ) and H-7(70 ) or H-7(70 )and H-8(80 ) was 8.0 Hz. All direct 1J connectivities between carbons Table 2 Cytotoxicity of compounds against tumor cell lines and normal cell line Compounds
Cell lines Hela IC50 (mmol/L)
MCF-7 IC50 (mmol/L)
BGC823 IC50 (mmol/L)
3T3-L1 IC50 (mmol/L)
HMBBJ 24 h 48 h 72 h
15.9 0.20 12.2 0.15 10.7 0.24
16.7 0.21 13.6 0.17 10.4 0.25
10.6 0.21 8.2 0.23 7.5 0.19
26.8 0.17 18.2 0.24 14.1 0.33
HCPT 24 h 48 h 72 h
13.2 0.18 10.1 0.21 5.4 0.32
24.7 0.26 11.9 0.33 5.1 0.14
14.2 0.25 9.1 0.27 5.3 0.33
25.5 0.16 17.1 0.32 14.5 0.27
848
Z.B. Li et al. / Chinese Chemical Letters 18 (2007) 846–848
and protons were determined in the HMQC spectrum. With the HMBC experiment, the position of methoxyl group was determined by the cross-peak between dC 167.8 (C-100 ) and dH 3.81(C-100 -OMe) protons and H-7(70 ) at dH 7.57 showed a correlation with C-9, C-90 . In addition, other correlations among OH and C-5, C-50 also were established by HMBC experiments, which showed the hydroxyl groups and H-7(70 ) at dH 7.57 were correlated with C-5, C-50 . The downfield shift of OH-5, 50 (dH 13.76) is caused by hydrogen-bonding with the oxygen atom of neighboring C O. Therefore, the compound was identified as p-hydroxymethoxybenzobijuglone. To study the cytotoxicity of the p-hydroxymethoxybenzobijuglone (HMBBJ), MTT assay was employed to evaluate its effects on the viability of four cell lines including three human tumor cells (Hela cervical carcinoma, MCF7 breast adenocarcinoma, BGC823 gastric carcinoma cell) and one normal cell (3T3-L1 mouse preadipocytes). From the results of MTT assay, it was found that HMBBJ showed different degrees of cytotoxicity on these cells, with the 50% inhibitory concentrations (IC50) values ranging from 7.5 to 26.8 mmol/L (Table 2). The inhibitory activity of HMBBJ to the three human tumor cells approached to Hydroxycamptothecin (HCPT) as positive control. Furthermore, the cytotoxic activities of HMBBJ and HCPT were both less pronounced on the normal cell (3T3-L1). Among the cancer cell lines tested, BGC823 cells were the most vulnerable to HMBBJ. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
M.L. Sun, J. Northeast For. Univ. 32 (85) (2004) (in Chinese). S.H. Kim, K.S. Lee, et al. J. Nat. Prod. 61 (1998) 643. K.S. Lee, G. Li, S.H. Kim, et al. J. Nat. Prod. 65 (2002) 1707. B.S. Min, H.Y. Lee, S.M. Lee, et al. Arch. Pharm. Res. 25 (2002) 441. J.L. Zhang, F.Y. He, T.C. Li, J. Liaoning Univ. 25 (135) (1998) (in Chinese). K. Hirakawa, E. Ogiue, J. Motoyoshiya, M. Yajima, Phytochemistry 25 (1986) 1494. S.W. Lee, K.S. Lee, J.K. Son, Planta Med. 66 (2000) 184. H. Song, Y.M. Bao, et al. J. Dalian Inst. Light Ind. 20 (109) (2001) (in Chinese). Ch.L. Wang, Y.M. Bao, et al. Chinese Traditional Patent Medicine 25 (2003) 643 (in Chinese). B. Onegi, C. Kraft, et al. Phytochemistry 60 (2002) 39.
A cytotoxic compound from the leaves of Juglans mandshurica Zhi Bo Li a, Yong Ming Bao a, Hong Bo Chen b, Jing Yun Wang a, Jie Han Hu c, Li Jia An a,* a
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China b School of Chemical Engineering, Dalian University of Technology, Dalian 116024, China c Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116011, China Received 12 March 2007
Abstract From Juglans mandshurica leaves, a new quinone compound was isolated through bioassay-guided fractionation. The structure elucidation of the compound was established based on spectroscopic studies, notably of the 2D NMR spectra. The compound exhibited moderate cytotoxic activities against Hela, MCF-7, BGC823 and 3T3-L1cell lines with IC50 ranges from 7.5 to 26.8 mmol/L. # 2007 Li Jia An. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Juglans mandshurica; p-Hydroxymethoxybenzobijuglone; Cytotoxicity
Juglans mandshurica’s leaves, roots and seeds have been used as a traditional medicine for treatment of cancer in China and Korea [1,2]. Up to now, the phytochemical investigations of J. mandshurica mainly involved diaryheptanoids [3], flavonoids [4], volatile oil [5], naphthoquinones [6,7]. Most of them have been isolated from the seeds and roots of J. mandshurica. The reports on the chemical or biological activity research of J. mandshurica leaves are rare. Our previous work showed that the ethanol extract of J. mandshurica leaves had anticancer activity in vivo and in vitro [8,9]. Subsequently, the fractionation and purification of the ethanol extract of J. mandshurica leaves with cytotoxicity assay using the Hela cell line, led to the isolation of a new quinone compound, p-hydroxymethoxybenzobijuglone (HMBBJ) (Fig. 1). Quinones are widespread among plants, which show biological activities including antitumour, wound healing, anti-inflammatory, antiparasitic, particularly cytotoxic activities. Commonly, the researches of quiniones focused on naphthoquinones and anthraquinones [10], however, few reports on benzobinaphthoquinone compound are available up to date. HMBBJ was isolated as red needles and its UV spectrum (lmax 275, 380, 480 nm) suggested it has a quinone skeleton. Twenty-three carbon signals showed in inverted gated decoupling 13C NMR and [M-H] ion peak at m/z 415.0462 in the negative ion HREIMS spectrum indicated that the molecular formula of HMBBJ was C23H12O8, accounting for eighteen degrees of unsaturation. The IR absorption at 3579, 3513 cm1 and 1721 cm1 showed the existence of hydroxyl and carbonyl groups in HMBBJ. The 13C NMR (Table 1), inverted gated decoupling 13C NMR
* Corresponding author. E-mail address: [email protected] (L.J. An). 1001-8417/$ – see front matter # 2007 Li Jia An. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2007.05.043
Z.B. Li et al. / Chinese Chemical Letters 18 (2007) 846–848
847
Fig. 1. The structure of p-hydroxymethoxybenzobijuglone (HMBBJ).
Table 1 NMR spectroscopic data of compound (500 MHz for 1H NMR, 125 MHz for Position
dH
0
1,1 2,20 3,30 4,40 5,50 6,60 7,70 8,80 9,90 10, 100 100 200 OCH3 OH
7.16 (dd, 8.0, 1.1) 7.57 (t, 8.0) 7.47 (dd, 8.0, 1.1)
3.81 (s) 13.76 (s)
13
C NMR, DMSO-d6, d ppm, J Hz) dC
HMBC (C ! H)
179.5 122.9 127.7 184.9 161.8 123.0 134.7 117.2 134.4 117.7 167.8 123.7 51.6
H8
OH, H7 OH, H8 H6 H7 OH, H6, H8 OCH3
and DEPT spectroscopic data disclosed the presence of four carbonyl carbons at d179.5(C-1, C-10 ), 184.9(C-4, C-40 ), 12 sp2 quaternary carbons at d167.8(C-100 ), 123.7(C-200 ), 122.9(C-2, C-20 ), 127.7(C-3, C-30 ), 161.8(C-5, C-50 ), 134.4(C-9, C-90 ), 117.7(C-10, C-100 ), 6 sp2 methines at d123.0(C-6, C-60 ), 134.7(C-7, C-70 ), 117.2(C-8, C-80 ), and 1 sp3methyl for OCH3 at d51.6, suggesting that the structure of HMBBJ is symmetrical with two naphthoquinones connected with benzene. In the1HNMR spectrum (Table 1), six signals with dH between 7.16 and 7.57 were attributed to the protons on the two aromatic rings and one methoxyl signal at dH 3.81. With the spectrum of 1H–1H COSY, it was further confirmed that dH 7.16 was for H-6, H-60 , dH 7.57 was for H-7, H-70 and dH 7.47 for H-8,H-80 , the coupling constant between H-6(60 ) and H-7(70 ) or H-7(70 )and H-8(80 ) was 8.0 Hz. All direct 1J connectivities between carbons Table 2 Cytotoxicity of compounds against tumor cell lines and normal cell line Compounds
Cell lines Hela IC50 (mmol/L)
MCF-7 IC50 (mmol/L)
BGC823 IC50 (mmol/L)
3T3-L1 IC50 (mmol/L)
HMBBJ 24 h 48 h 72 h
15.9 0.20 12.2 0.15 10.7 0.24
16.7 0.21 13.6 0.17 10.4 0.25
10.6 0.21 8.2 0.23 7.5 0.19
26.8 0.17 18.2 0.24 14.1 0.33
HCPT 24 h 48 h 72 h
13.2 0.18 10.1 0.21 5.4 0.32
24.7 0.26 11.9 0.33 5.1 0.14
14.2 0.25 9.1 0.27 5.3 0.33
25.5 0.16 17.1 0.32 14.5 0.27
848
Z.B. Li et al. / Chinese Chemical Letters 18 (2007) 846–848
and protons were determined in the HMQC spectrum. With the HMBC experiment, the position of methoxyl group was determined by the cross-peak between dC 167.8 (C-100 ) and dH 3.81(C-100 -OMe) protons and H-7(70 ) at dH 7.57 showed a correlation with C-9, C-90 . In addition, other correlations among OH and C-5, C-50 also were established by HMBC experiments, which showed the hydroxyl groups and H-7(70 ) at dH 7.57 were correlated with C-5, C-50 . The downfield shift of OH-5, 50 (dH 13.76) is caused by hydrogen-bonding with the oxygen atom of neighboring C O. Therefore, the compound was identified as p-hydroxymethoxybenzobijuglone. To study the cytotoxicity of the p-hydroxymethoxybenzobijuglone (HMBBJ), MTT assay was employed to evaluate its effects on the viability of four cell lines including three human tumor cells (Hela cervical carcinoma, MCF7 breast adenocarcinoma, BGC823 gastric carcinoma cell) and one normal cell (3T3-L1 mouse preadipocytes). From the results of MTT assay, it was found that HMBBJ showed different degrees of cytotoxicity on these cells, with the 50% inhibitory concentrations (IC50) values ranging from 7.5 to 26.8 mmol/L (Table 2). The inhibitory activity of HMBBJ to the three human tumor cells approached to Hydroxycamptothecin (HCPT) as positive control. Furthermore, the cytotoxic activities of HMBBJ and HCPT were both less pronounced on the normal cell (3T3-L1). Among the cancer cell lines tested, BGC823 cells were the most vulnerable to HMBBJ. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
M.L. Sun, J. Northeast For. Univ. 32 (85) (2004) (in Chinese). S.H. Kim, K.S. Lee, et al. J. Nat. Prod. 61 (1998) 643. K.S. Lee, G. Li, S.H. Kim, et al. J. Nat. Prod. 65 (2002) 1707. B.S. Min, H.Y. Lee, S.M. Lee, et al. Arch. Pharm. Res. 25 (2002) 441. J.L. Zhang, F.Y. He, T.C. Li, J. Liaoning Univ. 25 (135) (1998) (in Chinese). K. Hirakawa, E. Ogiue, J. Motoyoshiya, M. Yajima, Phytochemistry 25 (1986) 1494. S.W. Lee, K.S. Lee, J.K. Son, Planta Med. 66 (2000) 184. H. Song, Y.M. Bao, et al. J. Dalian Inst. Light Ind. 20 (109) (2001) (in Chinese). Ch.L. Wang, Y.M. Bao, et al. Chinese Traditional Patent Medicine 25 (2003) 643 (in Chinese). B. Onegi, C. Kraft, et al. Phytochemistry 60 (2002) 39.