Arch. Pharm. Res. DOI 10.1007/s12272-014-0398-1
RESEARCH ARTICLE
Chemical constituents from the leaves of Juglans mandshurica Da Lei Yao • Chang Hao Zhang • Jie Luo • Mei Jin • Ming Shan Zheng • Jiong Mo Cui Jong Keun Son • Gao Li
•
Received: 18 January 2014 / Accepted: 8 April 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Two new (1 and 3) and two known diarylheptanoids (2 and 4), along with two tetralones (5 and 6), one naphthoquinone (7), four phenylpropanoids (8–11), and one phenol (12) were isolated from the leaves of Juglans mandshurica. Their structures were elucidated on the basis of spectral and chemical data. Compounds 2 and 10 are firstly isolated from this plant and 8 and 12 were isolated from the Juglans genus for the first time. Among these compounds, only 7 exhibited moderate cytotoxicities against cultured MGC-803, A549, K562, and HeLa tumor cell lines with IC50 values of 25.90, 28.60, 39.06, 44.90 lM, respectively. Keywords Juglans mandshurica Juglandaceae Diarylheptanoid Cytotoxicity Tetralone Phenylpropanoid
Da Lei Yao and Chang Hao Zhang contributed equally to this study.
Electronic supplementary material The online version of this article (doi:10.1007/s12272-014-0398-1) contains supplementary material, which is available to authorized users. D. L. Yao C. H. Zhang J. Luo M. Jin M. S. Zheng J. M. Cui G. Li (&) Key Laboratory of Natural Resources of the Changbai Mountain and Functional Molecules, Ministry of Education, Yanbian University College of Pharmacy, Yanji 133002, People’s Republic of China e-mail: [email protected] M. Jin Yanbian University Hospital, Yanji 133000, China J. K. Son College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Korea
Introduction Juglans mandshurica Maxim. (Juglandaceae) is a fastgrowing deciduous tree that is widely distributed in China and Korea. The roots, stem barks, leaves and fruits of this plant had been used as a folk medicine for the treatment of cancer, gastritis, diarrhea and leucorrhoea (Park et al. 2012; Xu et al. 2013). J. mandshurica is known to contain polyphenols, naphthoquinones, naphthalenyl glucoside, tetralones, flavonoids, terpenoids, diarylheptanoids and galloyl glycosides (Li et al. 2005; Son 1995; Joe et al. 1996; Kim et al. 1998; Lee et al. 2000; Min et al. 2000; Lee et al. 2002; Li et al. 2003a, b, 2004; Liu et al. 2004; Machida et al. 2005; Li et al. 2007; Li et al. 2009; Liu et al. 2010; Yao et al. 2012; Lin et al. 2013). Additionally J. mandshurica and these constituents have been shown to have antioxidative activity, antitumor activity (Xu et al. 2013; Joe et al. 1996; Kim et al. 1998; Li et al. 2007, 2009; Yao et al. 2012; Xu et al. 2010; Xin et al. 2014), anti-human immunodeficiency virus-type 1 acivity (Min et al. 2002), topoisomerases I and II inhibitory activity (Li et al. 2003a), anti-inflammatory activity (Ju et al. 2009), anti-complement activity (Min et al. 2003), inhibitory effect on allergic dermatitis-like skin lesions (Park and Oh 2014), inhibitory effect on DNA polymerase and on the RNase H activity of HIV-1 reverse transcriptase (Min et al. 2000). In the continuation of our studies on this plant (Lee et al. 2002; Li et al. 2003a, b, 2004, 2005), we isolated two new diarylheptanoids (1 and 3) and two known ones (2 and 4), along with two tetralones (5 and 6), one naphthoquinone (7), five phenylpropanoids (8–11) and one phenol (12) (Fig. 1) from the CH2Cl2 fraction of MeOH extract of the leaves of this plant. This paper describes the isolation, structural elucidation, and cytotoxic activity of the new diarylheptanoids (1 and 3) and other constituents (2, 4–12) on the basis of their spectroscopic data.
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Materials and methods General procedures The NMR spectra were recorded on a Bruker 500 MHz (AV 500, Berne, Switzerland) instrument for compounds 1–4 and on a Bruker 300 MHz (AV 300) for 5–12, in CDCl3 and CD3OD as solvents. Chemical shifts are expressed in d (ppm), and are referenced to the residual solvent signals. Optical rotations were measured using a Rudolph Autopol I (Rudolph Research Analytical, New Jersey, USA) automatic polarimeter. Circular dichroism (CD) spectra were recorded in MeOH with a JASCO J-810 spectropolarimeter (JASCO Analytical Instruments, Easton, USA). HR-ESI–MS spectra were recorded on a Bruker microTOF QII (Bruker Daltonics, California, USA) mass spectrometer. MALDI-TOF–MS was performed on a Perceptive Biosystems Voyager-DE STR using 2-cyano-3-(4-hydroxyphenyl) acrylic acid as the matrix. Middle Pressure Liquid Chromatography (MPLC) was performed on an automatic flash chromatography device (CHEETAH MP-200, Bonna-Agela Technologies Co., Ltd., Tianjin, China). Preparative HPLC was carried out on a Bonna-Agela FL-LC050 series chromatograph (BonnaAgela), equipped with a HP-Q-P050 binary pump, a rheodyne 7725i injector, and a HP-Q-UV100 multiple wave detector. Separations were performed on a YMC ODS-A column (10 lm, 250 9 10 mm, YMC, Kyoto, Japan) and a Venusil ASB C18 column (5 lm, 150 9 4.6 mm, BonnaAgela). Column chromatography was performed using silica gel (200–300 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd., Qindao, China). Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ODS silica gel (40–63 lm, Merck, Darmstad, Germany) were used for column chromatography. TLC was performed with precoated Silica gel GF254 glass plates (Branch of Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Spots were visualized using UV light (254 and/or 365 nm) and by spraying with 10 % (v/v) H2SO4-H2O followed by heating to 120 °C. Plant material The leaves of J. mandshurica were collected in August 2006 in a mountainous area of Yanji City, Jilin Province, China. Plant samples were identified by Prof. Hui Zi Lv (College of Pharmacy, Yanbian University). A voucher specimen (voucher number: YB-HT-0113) was deposited at the College of Pharmacy, Yanbian University. Extraction and isolation The dried leaves of J. mandshurica (3 kg) were extracted three times with 70 % MeOH (3 9 5 L) at room
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Table 1 1H- (500 MHz) and 13C- (125 MHz) NMR data for 1 and 3 Position
1
3
dH
dC
dH
dC
1
–
124.8
–
146.0
2
–
132.5
–
140.5
3
–
142.7
–
148.1
4
–
152.6
–
126.8
5
6.73 d (2.0)
112.1
6.90 d (8.4)
126.0
6
–
136.1
6.66 d (8.4)
123.0
7
2.54 m
30.9
3.12 m 2.36 m
28.9
1.98 m
26.8
1.80 m
29.2
8
1.90 m 9
1.69 m
1.51 m 23.3
1.12 m
22.6
39.7
1.24 m
38.7
1.55 m 10
1.93 m 1.60 m
1.01 m
11
4.11 m
69.0
3.09 m
72.0
12
2.33 m
35.2
1.50 m
36.5
27.2
2.62 m
28.5
1.75 m 13
2.92 m 2.00 m
2.55 m
14
–
130.7
–
15
7.12 dd (8.2, 2.4)
130.6
5.65 d (2.0)
134.6 113.5
16 17
6.92 d (8.2) –
117.3 152.1
– –
147.5 142.6
18
7.23 d (2.4)
133.7
6.85 d (8.1)
115.5
19
6.84 d (2.0)
126.5
6.65 dd (8.1, 2.0)
115.8
20
–
–
–
–
3-OCH3
3.85 s
61.5
–
–
4-OCH3
3.91 s
56.4
–
–
2-OCH3
–
–
3.95
62.0
d in ppm, J value in Hz within parentheses
temperature. The combined extract was concentrated under reduced pressure to furnish a dark brown residue (500 g), which was suspended in H2O and partitioned in turn with petroleum ether (PE), CH2Cl2, EtOAc and n-BuOH. The CH2Cl2 extract was evaporated under reduced pressure to yield a residue (5 g). The latter was separated chromatographically by a MPLC using a Bonna-Agela FCH120-S cartridge (120 g, CH2Cl2–MeOH, 100 % CH2Cl2 to 100 % MeOH, v/v) to obtain 13 subfractions A–M. Fraction F (1.19 g) was applied to a silica gel column eluted with a PE– EtOAc gradient (100 % PE to 1:9, v/v) to afford six subfractions (F1–F7). Fractions F2 (300 mg) and F4 (310 mg) were individually subjected to a series of purification steps using Sephadex LH-20 column chromatography (MeOH), ODS silica gel column chromatography (MeOH–H2O, 2:8 to 9:1, v/v), and finally purified by preparative HPLC (MeOH–
Chemical constituents from Juglans mandshurica
H2O, 5:5 to 9:1, v/v) to afford 1 (11.6 mg), 2 (7.1 mg), 4 (17.3 mg), 5 (17.6 mg), 6 (10.2 mg), and 8 (20.5 mg). Fraction G (700 mg) was purified by a MPLC (SO230012-0, C18, 12 g) with MeOH–H2O (3:7 to 9:1, v/v), followed by preparative HPLC with MeOH–H2O gradients (5:5 to 9:1, v/v) to afford 3 (7.3 mg), 9 (10.5 mg), 12 (27.2 mg). Fraction H (280 mg) was applied to a Sephadex LH-20 column eluted with MeOH to give a major fraction, which was purified by preparative HPLC (MeOH–H2O, 4:1, v/v) to yield 7 (9.1 mg), 10 (11.0 mg), 11 (11.2 mg). (11S)-11, 17-dihydroxy-3,4-dimethoxy-[7,0]metacyclophane (1) Brown amorphous powder, [a]20 D ? 84.2° (c = 0.17, MeOH); HRFABMS m/z 343.1904 [M ? H]? (calcd. for C21H27O4, 343.1909); UV (MeOH) kmax (log e) 292.0, 250.0, 207.0. IR (KBr) vmax 3410, 2926, 2859, 2360, 1731, 1457, 1265, 1063 cm-1; CD [MeOH, nm (De)]: 215 (-6.2), 283 (-2.9), 319 (-2.9); 1H- (CDCl3, 500 MHz) and 13CNMR (CDCl3, 125 MHz) see Table 1. (11R)-3,11,17-trihydroxy-2-methoxy-1,16-oxo-7,13diphenyl-11-heptanol (3) Brown amorphous powder: [a]20 D -97.7°(c = 0.07, MeOH); HRFABMS m/z 343.1551 [M - H]- (calcd. for C20H23O5, 343.1545); UV (MeOH) kmax (log e) 280.5, 209.0; IR (KBr) vmax 3426, 2933, 2847, 2363, 1602, 1511, 1468, 1434, 1261, 1027 cm-1; CD [MeOH, nm (De)]: 215 (-6.7), 280 (-2.8), 297 (-2.6); 1H- (CDCl3, 500 MHz) and 13C-NMR (CDCl3, 125 MHz) see Table 1. Cytotoxicity bioassay The tetrazolum-based colorimetric assay (MTT assay) was used to quantify the in vitro cytotoxicity against cultured human carcinoma cell lines (Tian et al. 2007), including MGC-803 (human gastric cancer), A549 (human lung adenocarcinoma), K562 (human myeloid leukemia), and HeLa (human cervical cancer) cells.
Results and discussion Two new diarylheptanoids (1 and 3) were isolated from the MeOH extract of J. mandshurica leaves together with ten known compounds, (11R)-11,17-dihydroxy-3,4-methylenedioxy-[7,0]-metacyclophane (2) (Yang et al. 2011), myricatomentogenin (4) (Morihara et al. 1997), (–)-regiolone (5) (Lal et al. 2011), 4-hydroxy-1-tetralone (6) (Rebelo et al. 2005), juglone (7) (Rebelo et al. 2005), caffeic
acid methyl ester (8), trans-coumaric acid methyl ester (9) (Wan et al. 2012), ferulic acid (10) (Yoshioka et al. 2004), cinnamic acid (11) (Yang et al. 2000), and 4-hydroxybenzoic acid methyl ester (12) (Yoshioka et al. 2004). Their structures were elucidated on the basis of spectral and chemical data. Compounds 2 and 10 are isolated for the first time from this plant, 8 and 12 were isolated from the Juglans genus for the first time. Compound 1 was obtained as a brown, amorphous powder, and assigned the molecular formula C21H26O4 from HR-ESI– MS, 13C-NMR and DEPT spectral data. In the 1H-NMR (Table 1) and HMQC spectrum, there were two sets aromatic signals, such as an 1,2,3,5-tetrasubstituted [3JH5H19 (2.0 Hz)] benzene ring, attributed to the benzene ring A, an 1,3,4-trisubstituted benzene ring [2JH15H16 (8.2 Hz), 3JH15H18 (2.4 Hz)] attributed to the benzene ring B, and two methoxyl groups: 3-OCH3 (d 3.85, s), and 4-OCH3 (d 3.91, s). The 13CNMR (Table 1) and DEPT spectra showed two methyl, six methylene, six methine, and seven quaternary carbon signals including the characteristic peaks of two methoxyl groups and twelve aromatic carbons. A 1H–1H COSY experiment (Fig. 2) disclosed the presence of H-7 to H-13. In the HMBC spectrum (Fig. 2) of 1, the linkages of two benzene rings on the aliphatic chain were established by the cross peaks between C-13 and H-15, H-18, and those between C-7 and H-5, H-19. The correlation of H-18 with C-2, C-14, C-17, and H-19 with C-1, C-3, C-5 suggested the attachments between the two benzene rings while the location of the two methoxyl groups on the aromatic ring were identified by the correlations of C-3 with 3-OCH3 and C-4 with 4-OCH3. Taken together, the structure of 1 was similar to (11R)-11,17-dihydroxy-3,4-methylenedioxy-[7,0]-metacyclophane (2) previously reported from Juglans sinensis (Yang et al. 2011), except for signals caused by two additional methoxy groups on benzene ring B instead of a methylenedioxy group. The CD spectrum of 1 showed negative cotton effects at 215 (-6.2) nm, which together with the specific rotation data ([a]20 D ? 84.2°, MeOH) suggesting 11S configuration by comparison with compound 2. From this analysis, the structure of compound 1 was elucidated as (11S)11,17-dihydroxy-3,4-dimethoxy-[7,0]-metacyclophane. Compound 3 had the molecular formula C20H24O5, determined from its HR-ESI–MS, 13C-NMR and DEPT spectral data. The 13C-NMR (Table 1) and DEPT spectra exhibited one methyl, six methylene, five methine, and seven quaternary carbon signals, including the characteristic peaks of twelve aromatic carbons, suggesting that compound 3 was a diarylheptanoid derivative. In the 1H-NMR spectrum (Table 1), signals on the aromatic region showed coupling patterns caused by 1,3,4-trisubstituted [2JH18H19 (8.2 Hz), 3JH15H19 (2.0 Hz)] and 1,2,3,4-tetrasubstituted 2[JH5H6 (8.4 Hz)] benzene rings, respectively. The H-15 signal (d, d 5.65) appeared abnormally upfield from the other proton signals of the two
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Fig. 1 Structures of compounds 1–12 Fig. 2 Key 1H-1H COSY and HMBC correlations of compounds 1 and 3
H3CO OH
H3CO HO
O HO 1
benzene rings, and this shielding effect is characteristic of diphenylether-type diarylheptanoids that have an ether linkage between C-16 and C-1 (Li et al. 2003b). The correlations in the 1H-1H COSY (Fig. 2) spectrum displayed the connectivities from H-7 to H-13 on the aliphatic chain. In the HMBC (Fig. 2) spectrum of 3, the linkages of two benzene rings on the aliphatic chain were established by the cross peaks between H-7 and C-3, C-4, C-5, and those between H-13 and C-14, C-15, C-19. The position of the methoxyl group on the aromatic ring was determined by a correlation of C-2 with 2OCH3. The absolute configuration of a hydroxyl group at C-11 was determined to be 11R by comparison of specific rotation ([a]20 D -97.7°, MeOH) of compound 3 with the one of (11R)11,17-dihydroxy-2-methoxy-1,16-oxo-7,13-diphenyl-11heptanol (Li et al. 2003b). Based on these results, the structure of compound 3 was assigned to (11R)-3,11,17-trihydroxy-2methoxy-1,16-oxo-7,13-diphenyl-11-heptanol. Among these compounds, only 7 was moderately cytotoxic toward cultured MGC-803, A549, K562, and HeLa tumor cell
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OH
HMBC 1H-1H
OH
COSY
OCH3 3
lines with IC50 values of 25.90, 28.60, 39.06, 44.90 lM, respectively. Acknowledgments This work was supported by the National Natural Science Foundation of China under Grant Numbers 30760291, 81160386, and 30911140276.
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RESEARCH ARTICLE
Chemical constituents from the leaves of Juglans mandshurica Da Lei Yao • Chang Hao Zhang • Jie Luo • Mei Jin • Ming Shan Zheng • Jiong Mo Cui Jong Keun Son • Gao Li
•
Received: 18 January 2014 / Accepted: 8 April 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Two new (1 and 3) and two known diarylheptanoids (2 and 4), along with two tetralones (5 and 6), one naphthoquinone (7), four phenylpropanoids (8–11), and one phenol (12) were isolated from the leaves of Juglans mandshurica. Their structures were elucidated on the basis of spectral and chemical data. Compounds 2 and 10 are firstly isolated from this plant and 8 and 12 were isolated from the Juglans genus for the first time. Among these compounds, only 7 exhibited moderate cytotoxicities against cultured MGC-803, A549, K562, and HeLa tumor cell lines with IC50 values of 25.90, 28.60, 39.06, 44.90 lM, respectively. Keywords Juglans mandshurica Juglandaceae Diarylheptanoid Cytotoxicity Tetralone Phenylpropanoid
Da Lei Yao and Chang Hao Zhang contributed equally to this study.
Electronic supplementary material The online version of this article (doi:10.1007/s12272-014-0398-1) contains supplementary material, which is available to authorized users. D. L. Yao C. H. Zhang J. Luo M. Jin M. S. Zheng J. M. Cui G. Li (&) Key Laboratory of Natural Resources of the Changbai Mountain and Functional Molecules, Ministry of Education, Yanbian University College of Pharmacy, Yanji 133002, People’s Republic of China e-mail: [email protected] M. Jin Yanbian University Hospital, Yanji 133000, China J. K. Son College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Korea
Introduction Juglans mandshurica Maxim. (Juglandaceae) is a fastgrowing deciduous tree that is widely distributed in China and Korea. The roots, stem barks, leaves and fruits of this plant had been used as a folk medicine for the treatment of cancer, gastritis, diarrhea and leucorrhoea (Park et al. 2012; Xu et al. 2013). J. mandshurica is known to contain polyphenols, naphthoquinones, naphthalenyl glucoside, tetralones, flavonoids, terpenoids, diarylheptanoids and galloyl glycosides (Li et al. 2005; Son 1995; Joe et al. 1996; Kim et al. 1998; Lee et al. 2000; Min et al. 2000; Lee et al. 2002; Li et al. 2003a, b, 2004; Liu et al. 2004; Machida et al. 2005; Li et al. 2007; Li et al. 2009; Liu et al. 2010; Yao et al. 2012; Lin et al. 2013). Additionally J. mandshurica and these constituents have been shown to have antioxidative activity, antitumor activity (Xu et al. 2013; Joe et al. 1996; Kim et al. 1998; Li et al. 2007, 2009; Yao et al. 2012; Xu et al. 2010; Xin et al. 2014), anti-human immunodeficiency virus-type 1 acivity (Min et al. 2002), topoisomerases I and II inhibitory activity (Li et al. 2003a), anti-inflammatory activity (Ju et al. 2009), anti-complement activity (Min et al. 2003), inhibitory effect on allergic dermatitis-like skin lesions (Park and Oh 2014), inhibitory effect on DNA polymerase and on the RNase H activity of HIV-1 reverse transcriptase (Min et al. 2000). In the continuation of our studies on this plant (Lee et al. 2002; Li et al. 2003a, b, 2004, 2005), we isolated two new diarylheptanoids (1 and 3) and two known ones (2 and 4), along with two tetralones (5 and 6), one naphthoquinone (7), five phenylpropanoids (8–11) and one phenol (12) (Fig. 1) from the CH2Cl2 fraction of MeOH extract of the leaves of this plant. This paper describes the isolation, structural elucidation, and cytotoxic activity of the new diarylheptanoids (1 and 3) and other constituents (2, 4–12) on the basis of their spectroscopic data.
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Materials and methods General procedures The NMR spectra were recorded on a Bruker 500 MHz (AV 500, Berne, Switzerland) instrument for compounds 1–4 and on a Bruker 300 MHz (AV 300) for 5–12, in CDCl3 and CD3OD as solvents. Chemical shifts are expressed in d (ppm), and are referenced to the residual solvent signals. Optical rotations were measured using a Rudolph Autopol I (Rudolph Research Analytical, New Jersey, USA) automatic polarimeter. Circular dichroism (CD) spectra were recorded in MeOH with a JASCO J-810 spectropolarimeter (JASCO Analytical Instruments, Easton, USA). HR-ESI–MS spectra were recorded on a Bruker microTOF QII (Bruker Daltonics, California, USA) mass spectrometer. MALDI-TOF–MS was performed on a Perceptive Biosystems Voyager-DE STR using 2-cyano-3-(4-hydroxyphenyl) acrylic acid as the matrix. Middle Pressure Liquid Chromatography (MPLC) was performed on an automatic flash chromatography device (CHEETAH MP-200, Bonna-Agela Technologies Co., Ltd., Tianjin, China). Preparative HPLC was carried out on a Bonna-Agela FL-LC050 series chromatograph (BonnaAgela), equipped with a HP-Q-P050 binary pump, a rheodyne 7725i injector, and a HP-Q-UV100 multiple wave detector. Separations were performed on a YMC ODS-A column (10 lm, 250 9 10 mm, YMC, Kyoto, Japan) and a Venusil ASB C18 column (5 lm, 150 9 4.6 mm, BonnaAgela). Column chromatography was performed using silica gel (200–300 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd., Qindao, China). Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ODS silica gel (40–63 lm, Merck, Darmstad, Germany) were used for column chromatography. TLC was performed with precoated Silica gel GF254 glass plates (Branch of Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Spots were visualized using UV light (254 and/or 365 nm) and by spraying with 10 % (v/v) H2SO4-H2O followed by heating to 120 °C. Plant material The leaves of J. mandshurica were collected in August 2006 in a mountainous area of Yanji City, Jilin Province, China. Plant samples were identified by Prof. Hui Zi Lv (College of Pharmacy, Yanbian University). A voucher specimen (voucher number: YB-HT-0113) was deposited at the College of Pharmacy, Yanbian University. Extraction and isolation The dried leaves of J. mandshurica (3 kg) were extracted three times with 70 % MeOH (3 9 5 L) at room
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Table 1 1H- (500 MHz) and 13C- (125 MHz) NMR data for 1 and 3 Position
1
3
dH
dC
dH
dC
1
–
124.8
–
146.0
2
–
132.5
–
140.5
3
–
142.7
–
148.1
4
–
152.6
–
126.8
5
6.73 d (2.0)
112.1
6.90 d (8.4)
126.0
6
–
136.1
6.66 d (8.4)
123.0
7
2.54 m
30.9
3.12 m 2.36 m
28.9
1.98 m
26.8
1.80 m
29.2
8
1.90 m 9
1.69 m
1.51 m 23.3
1.12 m
22.6
39.7
1.24 m
38.7
1.55 m 10
1.93 m 1.60 m
1.01 m
11
4.11 m
69.0
3.09 m
72.0
12
2.33 m
35.2
1.50 m
36.5
27.2
2.62 m
28.5
1.75 m 13
2.92 m 2.00 m
2.55 m
14
–
130.7
–
15
7.12 dd (8.2, 2.4)
130.6
5.65 d (2.0)
134.6 113.5
16 17
6.92 d (8.2) –
117.3 152.1
– –
147.5 142.6
18
7.23 d (2.4)
133.7
6.85 d (8.1)
115.5
19
6.84 d (2.0)
126.5
6.65 dd (8.1, 2.0)
115.8
20
–
–
–
–
3-OCH3
3.85 s
61.5
–
–
4-OCH3
3.91 s
56.4
–
–
2-OCH3
–
–
3.95
62.0
d in ppm, J value in Hz within parentheses
temperature. The combined extract was concentrated under reduced pressure to furnish a dark brown residue (500 g), which was suspended in H2O and partitioned in turn with petroleum ether (PE), CH2Cl2, EtOAc and n-BuOH. The CH2Cl2 extract was evaporated under reduced pressure to yield a residue (5 g). The latter was separated chromatographically by a MPLC using a Bonna-Agela FCH120-S cartridge (120 g, CH2Cl2–MeOH, 100 % CH2Cl2 to 100 % MeOH, v/v) to obtain 13 subfractions A–M. Fraction F (1.19 g) was applied to a silica gel column eluted with a PE– EtOAc gradient (100 % PE to 1:9, v/v) to afford six subfractions (F1–F7). Fractions F2 (300 mg) and F4 (310 mg) were individually subjected to a series of purification steps using Sephadex LH-20 column chromatography (MeOH), ODS silica gel column chromatography (MeOH–H2O, 2:8 to 9:1, v/v), and finally purified by preparative HPLC (MeOH–
Chemical constituents from Juglans mandshurica
H2O, 5:5 to 9:1, v/v) to afford 1 (11.6 mg), 2 (7.1 mg), 4 (17.3 mg), 5 (17.6 mg), 6 (10.2 mg), and 8 (20.5 mg). Fraction G (700 mg) was purified by a MPLC (SO230012-0, C18, 12 g) with MeOH–H2O (3:7 to 9:1, v/v), followed by preparative HPLC with MeOH–H2O gradients (5:5 to 9:1, v/v) to afford 3 (7.3 mg), 9 (10.5 mg), 12 (27.2 mg). Fraction H (280 mg) was applied to a Sephadex LH-20 column eluted with MeOH to give a major fraction, which was purified by preparative HPLC (MeOH–H2O, 4:1, v/v) to yield 7 (9.1 mg), 10 (11.0 mg), 11 (11.2 mg). (11S)-11, 17-dihydroxy-3,4-dimethoxy-[7,0]metacyclophane (1) Brown amorphous powder, [a]20 D ? 84.2° (c = 0.17, MeOH); HRFABMS m/z 343.1904 [M ? H]? (calcd. for C21H27O4, 343.1909); UV (MeOH) kmax (log e) 292.0, 250.0, 207.0. IR (KBr) vmax 3410, 2926, 2859, 2360, 1731, 1457, 1265, 1063 cm-1; CD [MeOH, nm (De)]: 215 (-6.2), 283 (-2.9), 319 (-2.9); 1H- (CDCl3, 500 MHz) and 13CNMR (CDCl3, 125 MHz) see Table 1. (11R)-3,11,17-trihydroxy-2-methoxy-1,16-oxo-7,13diphenyl-11-heptanol (3) Brown amorphous powder: [a]20 D -97.7°(c = 0.07, MeOH); HRFABMS m/z 343.1551 [M - H]- (calcd. for C20H23O5, 343.1545); UV (MeOH) kmax (log e) 280.5, 209.0; IR (KBr) vmax 3426, 2933, 2847, 2363, 1602, 1511, 1468, 1434, 1261, 1027 cm-1; CD [MeOH, nm (De)]: 215 (-6.7), 280 (-2.8), 297 (-2.6); 1H- (CDCl3, 500 MHz) and 13C-NMR (CDCl3, 125 MHz) see Table 1. Cytotoxicity bioassay The tetrazolum-based colorimetric assay (MTT assay) was used to quantify the in vitro cytotoxicity against cultured human carcinoma cell lines (Tian et al. 2007), including MGC-803 (human gastric cancer), A549 (human lung adenocarcinoma), K562 (human myeloid leukemia), and HeLa (human cervical cancer) cells.
Results and discussion Two new diarylheptanoids (1 and 3) were isolated from the MeOH extract of J. mandshurica leaves together with ten known compounds, (11R)-11,17-dihydroxy-3,4-methylenedioxy-[7,0]-metacyclophane (2) (Yang et al. 2011), myricatomentogenin (4) (Morihara et al. 1997), (–)-regiolone (5) (Lal et al. 2011), 4-hydroxy-1-tetralone (6) (Rebelo et al. 2005), juglone (7) (Rebelo et al. 2005), caffeic
acid methyl ester (8), trans-coumaric acid methyl ester (9) (Wan et al. 2012), ferulic acid (10) (Yoshioka et al. 2004), cinnamic acid (11) (Yang et al. 2000), and 4-hydroxybenzoic acid methyl ester (12) (Yoshioka et al. 2004). Their structures were elucidated on the basis of spectral and chemical data. Compounds 2 and 10 are isolated for the first time from this plant, 8 and 12 were isolated from the Juglans genus for the first time. Compound 1 was obtained as a brown, amorphous powder, and assigned the molecular formula C21H26O4 from HR-ESI– MS, 13C-NMR and DEPT spectral data. In the 1H-NMR (Table 1) and HMQC spectrum, there were two sets aromatic signals, such as an 1,2,3,5-tetrasubstituted [3JH5H19 (2.0 Hz)] benzene ring, attributed to the benzene ring A, an 1,3,4-trisubstituted benzene ring [2JH15H16 (8.2 Hz), 3JH15H18 (2.4 Hz)] attributed to the benzene ring B, and two methoxyl groups: 3-OCH3 (d 3.85, s), and 4-OCH3 (d 3.91, s). The 13CNMR (Table 1) and DEPT spectra showed two methyl, six methylene, six methine, and seven quaternary carbon signals including the characteristic peaks of two methoxyl groups and twelve aromatic carbons. A 1H–1H COSY experiment (Fig. 2) disclosed the presence of H-7 to H-13. In the HMBC spectrum (Fig. 2) of 1, the linkages of two benzene rings on the aliphatic chain were established by the cross peaks between C-13 and H-15, H-18, and those between C-7 and H-5, H-19. The correlation of H-18 with C-2, C-14, C-17, and H-19 with C-1, C-3, C-5 suggested the attachments between the two benzene rings while the location of the two methoxyl groups on the aromatic ring were identified by the correlations of C-3 with 3-OCH3 and C-4 with 4-OCH3. Taken together, the structure of 1 was similar to (11R)-11,17-dihydroxy-3,4-methylenedioxy-[7,0]-metacyclophane (2) previously reported from Juglans sinensis (Yang et al. 2011), except for signals caused by two additional methoxy groups on benzene ring B instead of a methylenedioxy group. The CD spectrum of 1 showed negative cotton effects at 215 (-6.2) nm, which together with the specific rotation data ([a]20 D ? 84.2°, MeOH) suggesting 11S configuration by comparison with compound 2. From this analysis, the structure of compound 1 was elucidated as (11S)11,17-dihydroxy-3,4-dimethoxy-[7,0]-metacyclophane. Compound 3 had the molecular formula C20H24O5, determined from its HR-ESI–MS, 13C-NMR and DEPT spectral data. The 13C-NMR (Table 1) and DEPT spectra exhibited one methyl, six methylene, five methine, and seven quaternary carbon signals, including the characteristic peaks of twelve aromatic carbons, suggesting that compound 3 was a diarylheptanoid derivative. In the 1H-NMR spectrum (Table 1), signals on the aromatic region showed coupling patterns caused by 1,3,4-trisubstituted [2JH18H19 (8.2 Hz), 3JH15H19 (2.0 Hz)] and 1,2,3,4-tetrasubstituted 2[JH5H6 (8.4 Hz)] benzene rings, respectively. The H-15 signal (d, d 5.65) appeared abnormally upfield from the other proton signals of the two
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Fig. 1 Structures of compounds 1–12 Fig. 2 Key 1H-1H COSY and HMBC correlations of compounds 1 and 3
H3CO OH
H3CO HO
O HO 1
benzene rings, and this shielding effect is characteristic of diphenylether-type diarylheptanoids that have an ether linkage between C-16 and C-1 (Li et al. 2003b). The correlations in the 1H-1H COSY (Fig. 2) spectrum displayed the connectivities from H-7 to H-13 on the aliphatic chain. In the HMBC (Fig. 2) spectrum of 3, the linkages of two benzene rings on the aliphatic chain were established by the cross peaks between H-7 and C-3, C-4, C-5, and those between H-13 and C-14, C-15, C-19. The position of the methoxyl group on the aromatic ring was determined by a correlation of C-2 with 2OCH3. The absolute configuration of a hydroxyl group at C-11 was determined to be 11R by comparison of specific rotation ([a]20 D -97.7°, MeOH) of compound 3 with the one of (11R)11,17-dihydroxy-2-methoxy-1,16-oxo-7,13-diphenyl-11heptanol (Li et al. 2003b). Based on these results, the structure of compound 3 was assigned to (11R)-3,11,17-trihydroxy-2methoxy-1,16-oxo-7,13-diphenyl-11-heptanol. Among these compounds, only 7 was moderately cytotoxic toward cultured MGC-803, A549, K562, and HeLa tumor cell
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OH
HMBC 1H-1H
OH
COSY
OCH3 3
lines with IC50 values of 25.90, 28.60, 39.06, 44.90 lM, respectively. Acknowledgments This work was supported by the National Natural Science Foundation of China under Grant Numbers 30760291, 81160386, and 30911140276.
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