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A secoiridoid with quinone reductase inducing activity from Cortex fraxini
Fitoterapia 81 (2010) 834–837
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
A secoiridoid with quinone reductase inducing activity from Cortex fraxini Lijun Wang a, Fang Sun a, Xiaoyu Zhang a, Zhongjun Ma a,⁎, Lin Cheng b,⁎ a Institute of Pharmaceutical Informatics, School of Pharmaceutical Sciences, Zhejiang University, Zijingang Campus, No. 388 Yuhangtang Road, Hangzhou 310058, PR China b Zhejiang Academy of Traditional Chinese Medicine, No. 132 Tianmushan Road, Hangzhou 310058, PR China
a r t i c l e
i n f o
Article history: Received 29 March 2010 Accepted in revised form 1 May 2010 Available online 21 May 2010 Keywords: Cortex fraxni Secoiridoid Quinone reductase
a b s t r a c t A new secoiridoid named chinensisol (1) along with twenty known compounds were isolated from the 95% ethanol extract of Cortex fraxini. Their structures were elucidated on the basis of NMR, MS, IR and UV spectral evidences. The quinone reductase (QR) inducing activities of the compounds were evaluated and the results showed that compounds 1, 9 and 14 had moderate QR inducing activities with CD values (concentration required to double the specific activity of QR) of 72.4 ± 7.7, 34.3 ± 3.3 and 42.0 ± 0.4 μM respectively. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cortex fraxini (Chinese name Qin-pi) is the dried bark of Fraxinus rhynchophylla, F. chinensis, F. szaboana and F. stylosa, and is mainly distributed throughout the north of China. The leaves of Fraxinus species are odd-pinnate, opposite or rarely whorled at branch apices and the petiole and petiolule are often basally thickened. Their flowers are small, bisexual, unisexual or polygamous while the fruit is a samara with elongated wing. C. fraxini is officially listed in the Chinese Pharmacopoeia and has been widely used in the treatment of stomatitis, toothache, pyrexia, haemostatic and urinary organ infection, arthritis and dysentery in China for over 2000 years [1–4]. Pharmacological researches demonstrated that it possessed anticancer, anticoagulant, antioxidant, antiallergic, antibacterial, diuretic and central nervous system protecting activities [5–11]. The studies of the constituents in C. fraxini have been reported previously. Fraxin, fraxetin esculin and esculetin are main coumarin derivatives and the most important bioactive components. Other kinds of compounds such as lignans, secoiridoid glycosides, triterpenes, flavonoids, saponins, sterols and phenolic acids are also reported [12]. ⁎ Corresponding authors. Tel.: + 86 571 88208427; fax: + 86 571 88208428. E-mail address: [email protected] (Z. Ma). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.05.003
Quinone reductase (QR) is a flavoprotein that catalyzes two-electron reduction and detoxification of quinones and its derivatives. It has been demonstrated that QR could protect mammalian cells by converting toxic quinones to hydroquinones and reducing oxidative cycling [13]. Thus reduction of quinones by QR is an important detoxification pathway in the life processes. In the present study, we reported the isolation, structural elucidation and QR inducing activities of the compounds from the 95% ethanol extract of C. fraxini. 2. Experimental 2.1. Generals Preparative HPLC: Agilent-1200 system, photodiode array detector, Zorbax-C18 column (ODS, 21.2 mm × 250 mm, 7 μm). Optical rotations: Jasco P-1030 polarimeter. IR Spectrum: Jasco FTIR-4100. 1H, 13C, 1H–1H COSY and HMBC NMR: Bruker Ultrashield Plus 500 MHz spectrometer. HR-ESI-MS: Bruker APEX III spectrometer. 2.2. Plant material C. fraxini was purchased from Hangzhou traditional Chinese medicine factory (the plants were collected from Anhui province and identified as F. chinensis).
L. Wang et al. / Fitoterapia 81 (2010) 834–837
2.3. Extraction and isolation The air-dried C. fraxini (5 kg) was extracted with 95% ethanol (×4) at 90 °C for 2 h to obtain the crude ethanol extract. The residue was dissolved in H2O (1 L) and then extracted successively with petroleum ether (1 L × 3), dichloromethane (1 L × 3), ethyl acetate (1 L × 3) and nbutanol (1 L × 3). The dichloromethane part (13.4 g) was subjected to silica gel column chromatography and eluted with petroleum ether-ethyl acetate and dichloromethane– methanol system. Twenty four fractions were collected and their compositions were monitored by TLC. Those fractions showing similar TLC profiles were grouped into 6 major fractions. Fraction 3 was separated by column chromatography (silica gel) and eluted with petroleum ether–ethyl acetate system to afford 5 fractions. The sub-fraction 3–4 was further separated by preparative HPLC using methanolH2O as the mobile phase (a 20 min gradient was used from 25% methanol to 40% methanol and maintained 45% methanol during the next 20 min, flow rate 10 mL/min) to obtain compound 1 (5 mg, tR = 12.5 min). The other known compounds were all separated using the same method combining of silica gel column chromatography and preparative HPLC. Chinensisol (1): white powder; [α]25D: 91.0° (c = 0.10, MeOH); UV, λmax (MeOH) nm: 238; IR (KBr), νmax cm− 1: 3442, 2951, 1748, 1701, 1634, 1443; HR-ESI-MS m/z: 1 257.1166 [M–H]− (calcd. for C11H13O− 7 , 257.0667); H and 13 C NMR data: see Table 1. 2.4. QR induction assay QR inducing activity was assessed using Hepa 1c1c7 cells as described previously [14]. The cells were maintained in αminimum essential medium supplemented with 0.1% penicillin-streptomycin, 10% fetal bovine serum and incubated in 5% CO2 at 37 °C. Hepa lclc7 cells were seeded in 96-well plate at a density of 1 × 104 cells/mL in 190 μL of media. After incubation for 24 h, different concentrations of the compounds were added into each well and the cells were incubated for an additional 24 h. Then the cell culture medium was removed and 50 μL of 0.8% digitonin and 2 mM EDTA (pH 7.8) was added to each well. After incubation for 10 min at 37 °C, 200 μL of a solution containing bovine
Table 1 1 H and 13C NMR data for compound 1 (in CD3OD). Position
δH
1 3 4 5 6 7 8 9 10
5.89 7.75 / 3.42 2.77 / 4.50 3.26 4.42 4.30 / 3.74
11 11-OMe
δc (1 H, d, J = 5.5 Hz) (1 H, s) (1 H, m) (2 H, m) (1 (1 (1 (1
H, t, J = 4.5 Hz) H, m) H, d, J = 10.5 Hz), H, dd, J = 4.5, 10.5 Hz)
(3 H,s)
103.6 156.9 108.5 22.7 33.9 171.5 74.5 42.3 75.6
(CH) (C) (C) (CH) (CH2) (C) (CH) (CH) (CH2)
166.6 (C) 50.5 (CH3)
835
serum albumin (0.67 mg/mL), 0.01% Tween 20, FAD (5 μM), 3-(4, 5-dimethylthiazo-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 25 mM), Tris–HCl (0.5 M), NADP (30 μM), glucose-6-phosphate (1 mM), menadione (50 μM) and glucose-6-phosphate dehydrogenase (2 U/mL) was added to each well. After incubation for 5 min, the absorbance of each well was scanned at 550 nm. Induction of QR activity was determined by comparing the specific activities of QR of sample-treated with DMSO-treated cells (CD value). The 4′-bromoflavone was tested as the positive control. Experiments were carried out three times on separate occasions. 3. Results and discussion Dichloromethane fraction yielded a new secoiridoid (1) and twenty known compounds (Fig. 1): medioresinol (2) [15], pinoresinol (3) [16], syringaresinol (4) [17], 8-hydroxypinoresinol (5) [18], balanophonin (6) [19], dehydrodiconiferyl alcohol (7) [19], lariciresinol (8) [20], 2-(4-hydroxy-3methoxyphenyl)-3-(2-hydroxy-5-methoxyphenyl)-3-oxo-1propanol (9) [21], ficusal (10) [22], 4-n-butoxyphenol (11) [23], 4-hydroxy-3-methoxy-phenyl ethanol (12) [24], 2-(4hydroxyphenyl)ethanol (13) [25], coniferyl aldehyde (14) [26], coniferyl alcohol (15) [27], soscopoletin (16) [28], esculetin (17) [29], fraxetin (18) [29], daphnetin 7-methyl ether (19) [30], esculin (20) [31] and linolenic acid (21) [32]. Compound 1 was isolated as white powder. Its molecular formula was supposed to be C11H14O7 according to the NMR data (Table 1) and the deprotonated molecular ion peak in HR-ESI-MS at m/z 257.1166 ([M–H]−, calcd. for C11H13O− 7 , 257.0667). The UV and IR spectra of compound 1 suggested the presence of an enol–ether system conjugated to a carbonyl group (238 nm in UV spectrum and 1701, 1630 cm− 1 in IR spectrum), which is typical for secoiridoid nuclei. 13C NMR spectrum showed eleven carbon signals including one methoxyl, two methylene, two carbonyl and six methine carbon signals. 1H NMR spectrum showed signals for one olefinic proton at δ 7.75 (1 H, s) and three methine protons at δ3.42 (1 H, m), 4.50 (1 H, t, J = 4.5 Hz) and 3.26 (1H, m), indicating the presence of a dihydropyran ring. The methoxy group (δH 3.74 and δC 55.5) was assigned at C-11 (δC 166.6) based on the HMBC correlation between methoxy proton signal (δH 3.74) and C-11 (δC 166.6) (Fig. 2). The carboxyl group (δC 171.5) was assigned at C-6 (δC 33.9, δH 2.77) based on the HMBC correlation between methylene proton signals (δH 2.77) and C-7 (δC 171.5) while the methylene was assigned at C-5 (δC 22.7, δH 3.42) based on the COSY correlation between H-6 (δH 2.77) and H-5 (δH 3.42) (Fig. 2). The remaining four carbons were assigned by a series of COSY, HMQC and HMBC experiments and assumed to make up a five membered hemiacetal ring. The NOESY spectra of compound 1 showed correlations between H-9 and H-5, H-9 and H-1, H-9 and H-8 (Fig. 2). The 1H and 13C NMR spectra of compound 1 were similar with those of ligustrohemiacetals A [33]. Comparing with ligustrohemiacetals A, compound 1 lacked a methoxy group at C-7. Thus the structure of compound 1 was established as chinensisol. To investigate whether the semi-acetal of compound 1 was stabile, we tested the 1H NMR spectrum of compound 1 in different times. The result showed that the 1H NMR spectra
836
L. Wang et al. / Fitoterapia 81 (2010) 834–837
Fig. 1. Structures of compounds 1-21.
were not changed, indicating the configuration at the semiacetalic carbon was stabile. It might be attributed to the location of the semi-acetal in furan ring. Previous studies have demonstrated that the ring cleavage needs some strong reducing agents such as LiAlH4 [34] or NaBH4 [35]. Therefore, the semiacetalic carbon of compound 1 is relatively stabile. Then, the QR inducing activities of 21 isolated compounds were investigated (Table 2). The results indicated that compounds 1, 9 and 14 possessed moderate QR inducing activities with CD values of 72.4 ± 7.7, 34.3 ± 3.3 and 42.0 ± 0.4 μM respectively and certainly merited continued and comparative study for the future.
Fig. 2. The key HMBC, 1H–1H COSY and NOESY correlations of compound 1.
Table 2 The QR inducing activities of isolated compounds. Compound
Concentration (μg/mL) a
QR induction activities (fold increase from control)
1 2 3 4 5 6 7 8 9 10 14 15 17 18 19 20 21 4′-Bromoflavone c
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
2.17 ± 0.32 b1 b 1.02 ± 0.14 b1 b1 b1 b1 b1 2.01 ± 0.21 b1 3.02 ± 0.17 b1 b1 b1 1.16 ± 0.08 b1 b1 3.18 ± 0.29
a All the compounds showed no cytotoxicities at the concentration of 20 μg/mL (the cell viabilities were all larger than 55%). b It is no sense if the fold increase from control of the compound is less than 1. c 4′-Bromoflavone was tested as the positive control.
L. Wang et al. / Fitoterapia 81 (2010) 834–837
Acknowledgements The work was financially supported by Zhejiang Key Science & Technological Program (2009C13028). References [1] The Pharmacopoeia Committee of the Health Ministry of People's Republic of China. Pharmacopoeia of People's Republic of China, part 1. Beijing: Chemical Technologic Publisher; 2005. p. 191. [2] Koju W, Akira I, Takashi S, Tsuyoshi S, Haruhisa H, Yoshiaki N. Eur J Pharmacol 1999;370:297. [3] Duncan SH, Flint HJ, Stewart CS. FEMS Microbiol Lett 1998;164:283. [4] He SD, Ueda S, Inoue K, Akaji M, Fujita T, Yang CR. Phytochemistry 1994;35:177. [5] Chu CY, Tsai YY, Wang CJ, Lin WL, Tseng TH. Eur J Pharmacol 2001;416: 25. [6] Park C, Jin CY, Kim GY, Choi IW, Kwon TK, Choi BT, et al. Toxicol Appl Pharmacol 2008;227:219. [7] Pan SL, Hang YW, Guh JH, Chang YL, Peng CY, Teng CM. Biochem Pharmacol 2003;65:1897. [8] Wu CR, Huang MY, Lin YT, Ju HY, Ching H. Food Chem 2007;104:1464. [9] Wie MB, Koh JY, Won MH, Lee JC, Shin TK, Moon CJ, et al. Prog NeuroPsychopharmacol Biol Psychiatry 2001;25:1641. [10] Tsai JC, Tsai SL, Chang WC. J Pharmacol Sci 2004;94:60. [11] Yang TM, Xin G, Wang XN. Med J NDFNC 2003;24:387. [12] Kostova I, Iossifova T. Fitoterapia 2007;78:85. [13] Yang J, Liu RH. Food Chem 2009;114:898. [14] Prochaska HJ, Santamaria AB. Anal Biochem 1988;169:328.
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[15] Zhuang L, Seligmann O, Jurcic K, Wagner H. Planta Med 1982;45:172. [16] Pelter A, Ward ES, Watson DJ, Collins P, Kai IT. J Chem Soc Perkin Trans 1982;1:175. [17] Ratnayake S, Fang X, Anderson JE, McLaughlin JL, Evert DE. J Nat Prod 1992;55:1462. [18] Gu JQ, Wang YH, Franzblau SG, Montenegro G, Yang DZ, Timmermann BN. Planta Med 2004;70:509. [19] Yuen MSM, Xue F, Mak TCW, Wong HNC. Tetrahedron 1998;54:12429. [20] Katayama T, Davin LB, Lewis NG. Phytochemistry 1992;31:3875. [21] Rahman MAA, Moon SS. Bull Korean Chem Soc 2007;28:1261. [22] Li YC, Kuo YH. Chem Pharm Bull 2000;48:1862. [23] Said K, Stephane B, Sophie B, Nicolas C, Jean-Marc P, Selim AA. Rapid Commun Mass Spectrom 2008;22:3651. [24] LaLonde RT, Wong C, Tsai AIM. J Am Chem Soc 1976;98:3007. [25] Jong HK, Min WK, Jong HR, Sang UC, Ok PZ. Arch Pharm Res 2009;32: 1681. [26] Li Y, Wang SF, Zhao YL, Liu KC, Wang XM, Yang YP, et al. Molecules 2009;14:4433. [27] Ralph J, Zhang YS. Tetrahedron 1998;54:1349. [28] Yu HW, Li BG, Chen XZ, Li CS, Zhang GL. Chin J Appl Environ Biol 2010;16:72. [29] Takaaki Y, Mai F, Takahiro N, Ayumi H, Keisuke O. J Nat Prod 2006;69: 755. [30] Guha PK. Indian J Chem 1991;30B:714. [31] Koch H, Steinegger E. Pharm Acta Helv 1981;56:244. [32] Song HR, Hong Y. J Shenyang Pharm Univ 1990;7:202. [33] Tanahashi T, Takenaka Y, Okazaki N, Koge M, Nagakura N, Nishi T. Phytochemistry 2009;70:2072. [34] Nagib DA, Scott ME, MacMillan DWC. J Am Chem Soc 2009;131:10875. [35] Kumamoto H, Takahashi N, Shimamura T, Tanaka H, Nakamura KT, Hamasaki T, et al. Tetrahedron 2008;64:1494.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
A secoiridoid with quinone reductase inducing activity from Cortex fraxini Lijun Wang a, Fang Sun a, Xiaoyu Zhang a, Zhongjun Ma a,⁎, Lin Cheng b,⁎ a Institute of Pharmaceutical Informatics, School of Pharmaceutical Sciences, Zhejiang University, Zijingang Campus, No. 388 Yuhangtang Road, Hangzhou 310058, PR China b Zhejiang Academy of Traditional Chinese Medicine, No. 132 Tianmushan Road, Hangzhou 310058, PR China
a r t i c l e
i n f o
Article history: Received 29 March 2010 Accepted in revised form 1 May 2010 Available online 21 May 2010 Keywords: Cortex fraxni Secoiridoid Quinone reductase
a b s t r a c t A new secoiridoid named chinensisol (1) along with twenty known compounds were isolated from the 95% ethanol extract of Cortex fraxini. Their structures were elucidated on the basis of NMR, MS, IR and UV spectral evidences. The quinone reductase (QR) inducing activities of the compounds were evaluated and the results showed that compounds 1, 9 and 14 had moderate QR inducing activities with CD values (concentration required to double the specific activity of QR) of 72.4 ± 7.7, 34.3 ± 3.3 and 42.0 ± 0.4 μM respectively. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cortex fraxini (Chinese name Qin-pi) is the dried bark of Fraxinus rhynchophylla, F. chinensis, F. szaboana and F. stylosa, and is mainly distributed throughout the north of China. The leaves of Fraxinus species are odd-pinnate, opposite or rarely whorled at branch apices and the petiole and petiolule are often basally thickened. Their flowers are small, bisexual, unisexual or polygamous while the fruit is a samara with elongated wing. C. fraxini is officially listed in the Chinese Pharmacopoeia and has been widely used in the treatment of stomatitis, toothache, pyrexia, haemostatic and urinary organ infection, arthritis and dysentery in China for over 2000 years [1–4]. Pharmacological researches demonstrated that it possessed anticancer, anticoagulant, antioxidant, antiallergic, antibacterial, diuretic and central nervous system protecting activities [5–11]. The studies of the constituents in C. fraxini have been reported previously. Fraxin, fraxetin esculin and esculetin are main coumarin derivatives and the most important bioactive components. Other kinds of compounds such as lignans, secoiridoid glycosides, triterpenes, flavonoids, saponins, sterols and phenolic acids are also reported [12]. ⁎ Corresponding authors. Tel.: + 86 571 88208427; fax: + 86 571 88208428. E-mail address: [email protected] (Z. Ma). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.05.003
Quinone reductase (QR) is a flavoprotein that catalyzes two-electron reduction and detoxification of quinones and its derivatives. It has been demonstrated that QR could protect mammalian cells by converting toxic quinones to hydroquinones and reducing oxidative cycling [13]. Thus reduction of quinones by QR is an important detoxification pathway in the life processes. In the present study, we reported the isolation, structural elucidation and QR inducing activities of the compounds from the 95% ethanol extract of C. fraxini. 2. Experimental 2.1. Generals Preparative HPLC: Agilent-1200 system, photodiode array detector, Zorbax-C18 column (ODS, 21.2 mm × 250 mm, 7 μm). Optical rotations: Jasco P-1030 polarimeter. IR Spectrum: Jasco FTIR-4100. 1H, 13C, 1H–1H COSY and HMBC NMR: Bruker Ultrashield Plus 500 MHz spectrometer. HR-ESI-MS: Bruker APEX III spectrometer. 2.2. Plant material C. fraxini was purchased from Hangzhou traditional Chinese medicine factory (the plants were collected from Anhui province and identified as F. chinensis).
L. Wang et al. / Fitoterapia 81 (2010) 834–837
2.3. Extraction and isolation The air-dried C. fraxini (5 kg) was extracted with 95% ethanol (×4) at 90 °C for 2 h to obtain the crude ethanol extract. The residue was dissolved in H2O (1 L) and then extracted successively with petroleum ether (1 L × 3), dichloromethane (1 L × 3), ethyl acetate (1 L × 3) and nbutanol (1 L × 3). The dichloromethane part (13.4 g) was subjected to silica gel column chromatography and eluted with petroleum ether-ethyl acetate and dichloromethane– methanol system. Twenty four fractions were collected and their compositions were monitored by TLC. Those fractions showing similar TLC profiles were grouped into 6 major fractions. Fraction 3 was separated by column chromatography (silica gel) and eluted with petroleum ether–ethyl acetate system to afford 5 fractions. The sub-fraction 3–4 was further separated by preparative HPLC using methanolH2O as the mobile phase (a 20 min gradient was used from 25% methanol to 40% methanol and maintained 45% methanol during the next 20 min, flow rate 10 mL/min) to obtain compound 1 (5 mg, tR = 12.5 min). The other known compounds were all separated using the same method combining of silica gel column chromatography and preparative HPLC. Chinensisol (1): white powder; [α]25D: 91.0° (c = 0.10, MeOH); UV, λmax (MeOH) nm: 238; IR (KBr), νmax cm− 1: 3442, 2951, 1748, 1701, 1634, 1443; HR-ESI-MS m/z: 1 257.1166 [M–H]− (calcd. for C11H13O− 7 , 257.0667); H and 13 C NMR data: see Table 1. 2.4. QR induction assay QR inducing activity was assessed using Hepa 1c1c7 cells as described previously [14]. The cells were maintained in αminimum essential medium supplemented with 0.1% penicillin-streptomycin, 10% fetal bovine serum and incubated in 5% CO2 at 37 °C. Hepa lclc7 cells were seeded in 96-well plate at a density of 1 × 104 cells/mL in 190 μL of media. After incubation for 24 h, different concentrations of the compounds were added into each well and the cells were incubated for an additional 24 h. Then the cell culture medium was removed and 50 μL of 0.8% digitonin and 2 mM EDTA (pH 7.8) was added to each well. After incubation for 10 min at 37 °C, 200 μL of a solution containing bovine
Table 1 1 H and 13C NMR data for compound 1 (in CD3OD). Position
δH
1 3 4 5 6 7 8 9 10
5.89 7.75 / 3.42 2.77 / 4.50 3.26 4.42 4.30 / 3.74
11 11-OMe
δc (1 H, d, J = 5.5 Hz) (1 H, s) (1 H, m) (2 H, m) (1 (1 (1 (1
H, t, J = 4.5 Hz) H, m) H, d, J = 10.5 Hz), H, dd, J = 4.5, 10.5 Hz)
(3 H,s)
103.6 156.9 108.5 22.7 33.9 171.5 74.5 42.3 75.6
(CH) (C) (C) (CH) (CH2) (C) (CH) (CH) (CH2)
166.6 (C) 50.5 (CH3)
835
serum albumin (0.67 mg/mL), 0.01% Tween 20, FAD (5 μM), 3-(4, 5-dimethylthiazo-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 25 mM), Tris–HCl (0.5 M), NADP (30 μM), glucose-6-phosphate (1 mM), menadione (50 μM) and glucose-6-phosphate dehydrogenase (2 U/mL) was added to each well. After incubation for 5 min, the absorbance of each well was scanned at 550 nm. Induction of QR activity was determined by comparing the specific activities of QR of sample-treated with DMSO-treated cells (CD value). The 4′-bromoflavone was tested as the positive control. Experiments were carried out three times on separate occasions. 3. Results and discussion Dichloromethane fraction yielded a new secoiridoid (1) and twenty known compounds (Fig. 1): medioresinol (2) [15], pinoresinol (3) [16], syringaresinol (4) [17], 8-hydroxypinoresinol (5) [18], balanophonin (6) [19], dehydrodiconiferyl alcohol (7) [19], lariciresinol (8) [20], 2-(4-hydroxy-3methoxyphenyl)-3-(2-hydroxy-5-methoxyphenyl)-3-oxo-1propanol (9) [21], ficusal (10) [22], 4-n-butoxyphenol (11) [23], 4-hydroxy-3-methoxy-phenyl ethanol (12) [24], 2-(4hydroxyphenyl)ethanol (13) [25], coniferyl aldehyde (14) [26], coniferyl alcohol (15) [27], soscopoletin (16) [28], esculetin (17) [29], fraxetin (18) [29], daphnetin 7-methyl ether (19) [30], esculin (20) [31] and linolenic acid (21) [32]. Compound 1 was isolated as white powder. Its molecular formula was supposed to be C11H14O7 according to the NMR data (Table 1) and the deprotonated molecular ion peak in HR-ESI-MS at m/z 257.1166 ([M–H]−, calcd. for C11H13O− 7 , 257.0667). The UV and IR spectra of compound 1 suggested the presence of an enol–ether system conjugated to a carbonyl group (238 nm in UV spectrum and 1701, 1630 cm− 1 in IR spectrum), which is typical for secoiridoid nuclei. 13C NMR spectrum showed eleven carbon signals including one methoxyl, two methylene, two carbonyl and six methine carbon signals. 1H NMR spectrum showed signals for one olefinic proton at δ 7.75 (1 H, s) and three methine protons at δ3.42 (1 H, m), 4.50 (1 H, t, J = 4.5 Hz) and 3.26 (1H, m), indicating the presence of a dihydropyran ring. The methoxy group (δH 3.74 and δC 55.5) was assigned at C-11 (δC 166.6) based on the HMBC correlation between methoxy proton signal (δH 3.74) and C-11 (δC 166.6) (Fig. 2). The carboxyl group (δC 171.5) was assigned at C-6 (δC 33.9, δH 2.77) based on the HMBC correlation between methylene proton signals (δH 2.77) and C-7 (δC 171.5) while the methylene was assigned at C-5 (δC 22.7, δH 3.42) based on the COSY correlation between H-6 (δH 2.77) and H-5 (δH 3.42) (Fig. 2). The remaining four carbons were assigned by a series of COSY, HMQC and HMBC experiments and assumed to make up a five membered hemiacetal ring. The NOESY spectra of compound 1 showed correlations between H-9 and H-5, H-9 and H-1, H-9 and H-8 (Fig. 2). The 1H and 13C NMR spectra of compound 1 were similar with those of ligustrohemiacetals A [33]. Comparing with ligustrohemiacetals A, compound 1 lacked a methoxy group at C-7. Thus the structure of compound 1 was established as chinensisol. To investigate whether the semi-acetal of compound 1 was stabile, we tested the 1H NMR spectrum of compound 1 in different times. The result showed that the 1H NMR spectra
836
L. Wang et al. / Fitoterapia 81 (2010) 834–837
Fig. 1. Structures of compounds 1-21.
were not changed, indicating the configuration at the semiacetalic carbon was stabile. It might be attributed to the location of the semi-acetal in furan ring. Previous studies have demonstrated that the ring cleavage needs some strong reducing agents such as LiAlH4 [34] or NaBH4 [35]. Therefore, the semiacetalic carbon of compound 1 is relatively stabile. Then, the QR inducing activities of 21 isolated compounds were investigated (Table 2). The results indicated that compounds 1, 9 and 14 possessed moderate QR inducing activities with CD values of 72.4 ± 7.7, 34.3 ± 3.3 and 42.0 ± 0.4 μM respectively and certainly merited continued and comparative study for the future.
Fig. 2. The key HMBC, 1H–1H COSY and NOESY correlations of compound 1.
Table 2 The QR inducing activities of isolated compounds. Compound
Concentration (μg/mL) a
QR induction activities (fold increase from control)
1 2 3 4 5 6 7 8 9 10 14 15 17 18 19 20 21 4′-Bromoflavone c
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
2.17 ± 0.32 b1 b 1.02 ± 0.14 b1 b1 b1 b1 b1 2.01 ± 0.21 b1 3.02 ± 0.17 b1 b1 b1 1.16 ± 0.08 b1 b1 3.18 ± 0.29
a All the compounds showed no cytotoxicities at the concentration of 20 μg/mL (the cell viabilities were all larger than 55%). b It is no sense if the fold increase from control of the compound is less than 1. c 4′-Bromoflavone was tested as the positive control.
L. Wang et al. / Fitoterapia 81 (2010) 834–837
Acknowledgements The work was financially supported by Zhejiang Key Science & Technological Program (2009C13028). References [1] The Pharmacopoeia Committee of the Health Ministry of People's Republic of China. Pharmacopoeia of People's Republic of China, part 1. Beijing: Chemical Technologic Publisher; 2005. p. 191. [2] Koju W, Akira I, Takashi S, Tsuyoshi S, Haruhisa H, Yoshiaki N. Eur J Pharmacol 1999;370:297. [3] Duncan SH, Flint HJ, Stewart CS. FEMS Microbiol Lett 1998;164:283. [4] He SD, Ueda S, Inoue K, Akaji M, Fujita T, Yang CR. Phytochemistry 1994;35:177. [5] Chu CY, Tsai YY, Wang CJ, Lin WL, Tseng TH. Eur J Pharmacol 2001;416: 25. [6] Park C, Jin CY, Kim GY, Choi IW, Kwon TK, Choi BT, et al. Toxicol Appl Pharmacol 2008;227:219. [7] Pan SL, Hang YW, Guh JH, Chang YL, Peng CY, Teng CM. Biochem Pharmacol 2003;65:1897. [8] Wu CR, Huang MY, Lin YT, Ju HY, Ching H. Food Chem 2007;104:1464. [9] Wie MB, Koh JY, Won MH, Lee JC, Shin TK, Moon CJ, et al. Prog NeuroPsychopharmacol Biol Psychiatry 2001;25:1641. [10] Tsai JC, Tsai SL, Chang WC. J Pharmacol Sci 2004;94:60. [11] Yang TM, Xin G, Wang XN. Med J NDFNC 2003;24:387. [12] Kostova I, Iossifova T. Fitoterapia 2007;78:85. [13] Yang J, Liu RH. Food Chem 2009;114:898. [14] Prochaska HJ, Santamaria AB. Anal Biochem 1988;169:328.
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[15] Zhuang L, Seligmann O, Jurcic K, Wagner H. Planta Med 1982;45:172. [16] Pelter A, Ward ES, Watson DJ, Collins P, Kai IT. J Chem Soc Perkin Trans 1982;1:175. [17] Ratnayake S, Fang X, Anderson JE, McLaughlin JL, Evert DE. J Nat Prod 1992;55:1462. [18] Gu JQ, Wang YH, Franzblau SG, Montenegro G, Yang DZ, Timmermann BN. Planta Med 2004;70:509. [19] Yuen MSM, Xue F, Mak TCW, Wong HNC. Tetrahedron 1998;54:12429. [20] Katayama T, Davin LB, Lewis NG. Phytochemistry 1992;31:3875. [21] Rahman MAA, Moon SS. Bull Korean Chem Soc 2007;28:1261. [22] Li YC, Kuo YH. Chem Pharm Bull 2000;48:1862. [23] Said K, Stephane B, Sophie B, Nicolas C, Jean-Marc P, Selim AA. Rapid Commun Mass Spectrom 2008;22:3651. [24] LaLonde RT, Wong C, Tsai AIM. J Am Chem Soc 1976;98:3007. [25] Jong HK, Min WK, Jong HR, Sang UC, Ok PZ. Arch Pharm Res 2009;32: 1681. [26] Li Y, Wang SF, Zhao YL, Liu KC, Wang XM, Yang YP, et al. Molecules 2009;14:4433. [27] Ralph J, Zhang YS. Tetrahedron 1998;54:1349. [28] Yu HW, Li BG, Chen XZ, Li CS, Zhang GL. Chin J Appl Environ Biol 2010;16:72. [29] Takaaki Y, Mai F, Takahiro N, Ayumi H, Keisuke O. J Nat Prod 2006;69: 755. [30] Guha PK. Indian J Chem 1991;30B:714. [31] Koch H, Steinegger E. Pharm Acta Helv 1981;56:244. [32] Song HR, Hong Y. J Shenyang Pharm Univ 1990;7:202. [33] Tanahashi T, Takenaka Y, Okazaki N, Koge M, Nagakura N, Nishi T. Phytochemistry 2009;70:2072. [34] Nagib DA, Scott ME, MacMillan DWC. J Am Chem Soc 2009;131:10875. [35] Kumamoto H, Takahashi N, Shimamura T, Tanaka H, Nakamura KT, Hamasaki T, et al. Tetrahedron 2008;64:1494.