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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Lipoxygenase-inhibiting phenolic glycosides and monoterpene glycosides from Paeonia lactiflora a
b
a
a
Liang Zou , Lin-Feng Hu , Yi-Dong Guo , Yu Song & Qiang Fu
a
a
College of Biological Industry, Chengdu University, Chengdu 610106, China b
Pharmacy Department, Chongqing Medical and Pharmaceutical College, Chongqing 401331, China Published online: 23 Mar 2015.
Click for updates To cite this article: Liang Zou, Lin-Feng Hu, Yi-Dong Guo, Yu Song & Qiang Fu (2015): Lipoxygenaseinhibiting phenolic glycosides and monoterpene glycosides from Paeonia lactiflora, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1007960 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1007960
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Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2015.1007960
Lipoxygenase-inhibiting phenolic glycosides and monoterpene glycosides from Paeonia lactiflora Liang Zoua, Lin-Feng Hub, Yi-Dong Guoa, Yu Songa and Qiang Fua* a
College of Biological Industry, Chengdu University, Chengdu 610106, China; bPharmacy Department, Chongqing Medical and Pharmaceutical College, Chongqing 401331, China
Downloaded by [University of Nebraska, Lincoln] at 17:31 04 April 2015
(Received 12 November 2014; final version received 11 January 2015) The EtOH extract of the roots of Paeonia lactiflora afforded a new phenolic glycoside paenoside A (1) and a new monoterpene glycoside paeonin D (2), and five known monoterpene glycosides. Their structures were elucidated on the basis of spectroscopic means and hydrolysis products. All compounds displayed inhibitory potential against enzyme lipoxygenase. Keywords: Paeonia lactiflora; phenolic glycosides; monoterpene glycosides; lipoxygenase inhibitory activity
1.
Introduction
The roots of Paeonia lactiflora Pall. (Paeoniaceae) are one of the most important Chinese crude drugs, and used in many formulas for the treatment of muscular spasm, chest pains, diarrhea, blood, and liver disorders [1]. Extensive chemical studies have been conducted and have led to the isolation of “cage-like” monoterpene glycosides and phenolic glycosides, and some of those compounds showed anticoagulant and anti-inflammatory activities [2,3]. However, the activity of compounds from this plant has not been satisfactorily examined. In the course of a search for biologically active compounds from Chinese medicinal plants, we have investigated the dried roots of P. lactiflora. Two phenolic glycosides and five monoterpene glycosides were isolated and identified. Two of these were identified as new compounds. All compounds were investigated for inhibitory activity against lipoxygenase. *Corresponding author. Email: [email protected] q 2015 Taylor & Francis
2.
Results and discussion
The EtOH extract of roots of P. lactiflora was partitioned between H2O and nBuOH. The n-BuOH extract was separated by repeated column chromatography using silica gel, C18 silica gel, and high performance liquid chromatography (HPLC) to give two new compounds 1 and 2, and five known structures 3 – 7. The known compounds were identified as 2-O-[a-L -arabinopyranosyl-(1 ! 6)-b-D glucopyranosyl]-benzaldehyde (3) [4], paeonidanin A (4) [5], 4-O-methyl-paeoniflorin (5) [6], 40 -O-galloylpaeoniflorin (6) [7], and 4-O-galloylalbiflorin (7) [8]. Compound 1 was obtained as a colorless amorphous solid. The molecular formula of 1 was found to be C33H34O17 by HR-ESI-MS at m/z 725.1682 [M þ Na]þ. The 13C NMR spectrum of 1 showed 33 carbon signals, including 1 aromatic aldehyde, 2 ester carbons, 18 aromatic carbons, and 12 sugar carbons. The 1H and 13C NMR spectra of 1 (Table 1) were similar to those of 2-O-
2
L. Zou et al.
Table 1. 1H and 13C NMR spectral data of compound 1 (500 MHz, in DMSO-d6 ). 1
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Position 1 2 3 4 5 6 CHO 10 20 30 40 50 60 a 60 b 100 200 300 400 500 600 a 600 b 1000 2000 , 6000 3000 , 5000 4000 7000 10000 20000 , 60000 30000 , 50000 40000 70000
dH (J in Hz)
7.41 d (8.0) 7.66 dd (8.0, 7.5) 7.12 dd (7.5, 7.5) 7.66 d (7.5) 10.49 s 4.86 d (8.0) 3.37– 3.39 m 3.27– 3.29 m 3.12– 3.14 m 3.59 dd (7.0, 4.0) 4.01 dd (11.0, 4.0) 4.12 d (11.0) 4.89 d (7.5) 4.98 dd (9.5, 8.0) 3.60 dd (9.5, 8.0) 3.24– 3.26 m 3.62 dd (8.0, 5.5) 4.52 dd (12.0, 5.5) 4.61 dd (12.0) 7.90 d (7.5) 2H 7.36 t (7.5) 2H 7.48 t (7.5) 7.10 s, 2H
dC 124.6 159.6 116.7 136.5 122.2 126.4 189.7 101.2 70.6 76.2 69.7 75.8 67.6 98.3 78.5 76.7 75.8 75.4 65.3 131.2 129.9 129.8 134.5 167.3 121.0 110.6 147.6 141.2 167.4
[a-L -arabinopyranosyl-(1 ! 6)-b-D -glucopyranosyl]-benzaldehyde (3) [4], with same signals due to aldehyde proton signal at dH 10.49 (1H, s) and four aromatic protons signals at dH 7.41 (1H, br d, J ¼ 8.0 Hz), 7.66 (1H, dd, J ¼ 8.0, 7.5 Hz). 7.12 (1H, dd, J ¼ 7.5, 7.5 Hz), and 7.66 (1H, br d, J ¼ 7.5 Hz), suggesting a typical 1,2-substituted benzene pattern. However, the sugar signals of 1 were different from those of 3. The sugar obtained after aqueous acid hydrolysis of 1 was identified as D -glucose through comparison of the retention time of its trimethylsilyl ether with that of the standard
in gas chromatography. The large coupling constants 8.0 and 7.5 Hz for the anomeric protons in the 1H NMR spectrum of 1 suggested that the glucopyranosyl moieties have a b-configuration. Analysis of the 1 H– 1H COSY spectrum resulted in sequential assignment of all proton resonances of two monosaccharide units. In the HSQC experiment, proton resonances were correlated with those of the corresponding carbons, and associated anomeric protons were correlated with their respective carbon atoms from HSQC–TOCSY data, leading to unambiguous assignments of the carbons in each monosaccharide unit. The 1H NMR data (dH 7.10, 2H, s) and 13C NMR data (dC 110.6, 110.6, 121.0, 141.2, 147.6, 147.6, and 167.4), and signals at dC 129.8, 129.9, 131.2, 134.5, and 167.3 disclosed the presence of a galloyl moiety and a benzoyl unit in 1. In the HMBC spectrum, the correlations H-2000 , 6000 / C-7000 , H-3000 , 5000 /C-1000 , C-4000 , and H-20000 /C10000 , C-30000 , C-40000 , C-60000 and C-70000 confirmed their presence. The connections of C10 /C-2 via an oxygen atom and C-100 /C-60 via an oxygen atom were revealed by HMBC correlations of H-10 /C-2 and H-100 /C-60 . The connections of galloyl moiety and C-600 , and benzoyl moiety and C-200 were revealed by HMBC correlations of H-600 /C-70000 and H200 /C-7000 , respectively. From the above evidences, the structure of 1 was determined as shown in Figure 1, and named as paenoside A. Compound 2 was obtained as a yellowbrown amorphous solid. The molecular formula of 2 was found to be C31H34O15 by HR-ESI-MS at m/z 669.1792 [M þ Na]þ. The 1H and 13C NMR spectra of 2 (Table 2) displayed signals similar to those of 4-O-methylgalloylpaeoniflorin [5], showing the presence of common signals of a monoterpene system and a galloyl moiety. Differences were found in the signals due to sugar moiety. The sugar obtained after aqueous acid hydrolysis of 2 was identified as D -galactose through comparison of the retention time of its trimethylsilyl ether with that of the
Journal of Asian Natural Products Research HO
HO HO
O
4''''
O 1''''
HO
3
OH
1'' OH
OH 7''' 1'''
O 1' OH
OO
O 2
3
4'''
4
OH H
5
1
HO
O 10 1''' O O O 1 OH 7''' OH 6 1' OH
6
O
O
HO
4''
O
7
9 O
5
1'' 7'' O
OCH3
8
O
1
O 3
2
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Figure 1. The structures of compounds 1 and 2.
standard in gas chromatography. The b-configuration of the glycosidic linkage was determined from the coupling constant (J ¼ 7.5 Hz) of the anomeric proton. The connection of the galactose moiety and C-1 via an oxygen atom was Table 2. 1H and 13C NMR spectral data of compound 2 (500 MHz, in CD3OD). 2 Position 1 2 3a 3b 4 5 6 7a 7b 8 9 10 10 20 30 40 50 60 a 60 b 100 200 , 600 300 , 500 400 700 1000 2000 , 6000 3000 , 5000 4000 7000 OCH3
dH (J in Hz)
1.67 d (12.0) 1.77 d (12.0) 2.61 d (6.0) 1.81 d (10.5) 2.40 dd (10.5, 6.0) 4.63 s 2H 5.41 s 1.22 s 3H 4.54 d (7.5) 3.07 t (7.5) 3.18– 3.20 m 3.14– 3.15 m 3.53 t (6.0) 4.46 dd (12.0, 6.0) 4.67 d (12.0) 7.96 d (7.5) 2H 7.45 t (7.5) 2H 7.55 t (7.5) 7.82 s, 2H
3.39
dC 89.4 86.8 42.0 109.6 41.9 72.0 22.6 61.6 102.8 19.8 97.9 76.6 73.5 73.1 70.6 64.8 131.4 131.1 129.9 134.7 167.8 121.1 110.7 146.9 141.0 167.6 51.2
confirmed by HMBC correlation of H-10 / C-1. The stereochemistry at various stereocenters of the monoterpene unis was assigned on the basis of similarity of spectral data with related compounds and further confirmed by NOESY correlations [5], which showed close agreement with those of 4-O-methylgalloylpaeoniflorin. Therefore, the structure of 2 was determined as shown in Figure 1, and named as paeonin D. Compounds 1– 7 were investigated for inhibition of lipoxygenase activity (Table 3). All of those compounds showed potent inhibitory activity.
3. Experimental 3.1. General experimental procedures Optical rotations were measured on a JASCO P-1020 digital polarimeter (Jasco, Tokyo, Japan). UV spectra were obtained on a SHIMADZU UV-2201 UV/VIS recording spectrophotometer (Shimadzu, Table 3. In vitro quantitative inhibition of lipoxygenase by compounds 1 – 7. Compounds 1 2 3 4 5 6 7 Baicalein
IC50 ^ SEM (mM) 8.4 ^ 0.3 24.4 ^ 2.1 12.6 ^ 2.5 28.1 ^ 1.7 37.4 ^ 0.9 21.6 ^ 1.3 20.4 ^ 1.1 21.5 ^ 1.2
Note: Baicalein was used as a positive control.
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4
L. Zou et al.
Kyoto, Japan). IR spectra were recorded on a Bruker IFS-55 plus spectrometer (Bruker, Ettlingen, German). NMR spectra were recorded on an Inova 500 spectrometer (Bruker, Waltham, MA, USA). ESI-MS were measured on an Agilent 6320 ion trap mass spectrometer (Agilent, Santa Clara, CA, USA). HR-ESI/MS measurements were performed on a Bruker-Daltonics APES-III 7.0 TESLA FTMS spectrometer (Bruker, Billerica, MA, USA), HPLC separation was carried out on an octadecylsilanized column (YMC-pack ODS-A, 250 £ 10 mm, i.d. 5 mm, YMC, Kyoto, Japan) with a photodiode array detector (Waters, Millford, MA, USA). GC was performed with a SHIMADZU GC-14D. Column chromatography was performed with silica gel (Merck, Darmstadt, Germany) and C18 silica gel (Merck). 3.2.
Plant material
The roots of P. lactiflora were collected in May 2014 in Chengdu, Sichuan Province, China. The identification of the plant was performed by the author (Qiang Fu). A voucher specimen (PL 201403) is maintained in the herbarium of the College of Biological Industry, Chengdu University. 3.3.
Extraction and isolation
The dried roots of P. lactiflora (2.6 kg) were extracted with EtOH. After removing the solvent, the residue (321 g) was suspended in H2O and extracted with nBuOH. The n-BuOH-soluble fraction (105 g) was subjected to silica gel column chromatography and eluted in a gradient of CHCl3 – MeOH (100:1 ! 10:90) to afford fractions 1 – 5. Fraction 1 (5.3 g) was subjected to C18 silica gel chromatography and eluted in a gradient of MeOH – H2O (10:90 ! 95:5) to afford sub-fractions 1– 6 (0.3, 0.8, 0.1, 0.4, 1.7, and 0.6 g, respectively) and compound 2 (18 mg). Sub-fraction 2 (0.8 g) was separated by
HPLC (MeOH – H2O, 52:48, 2.0 ml/min) to yield compound 1 (31 mg, 21.3 min). Sub-fraction 6 (0.6 g) was separated by HPLC (MeOH – H2O, 54:46, 2.0 ml/min) to yield compound 5 (21 mg, 19.2 min). Fraction 3 (10.2 g) was subjected to C18 silica gel chromatography and eluted in a gradient of MeOH – H2O (10:90 ! 95:5) to afford compounds 3 (31 mg, 16.3 min) and 4 (39 mg, 19.2 min). Fraction 4 (10.2 g) was subjected to C18 silica gel chromatography and eluted in a gradient of MeOH – H2O (10:90 ! 95:5) to afford compounds 6 (66 13.2 min) and 7 (27 mg, 18.3 min). 3.3.1. Paenoside A (1) Colorless amorphous solid; ½a20 D 2 32.2 (c ¼ 0.5, MeOH); UV (MeOH) lmax (log 1) 210 (3.17), 220 (2.71), 250 (3.81), 308 (3.41) nm; IR (KBr) vmax (cm21): 3453, 1702, 1654, 1201, and 1071; for 1H and 13C NMR spectral data, see Table 1. m/z 725.1682 [M þ Na] þ, calcd for C33H34O17Naþ, 725.1688] 3.3.2.
Paeonin D(2)
Yellow-brown amorphous solid, ½a20 D 2 48.3 (c ¼ 0.5, MeOH); UV (MeOH) lmax (log 1) 229 (2.81), 273 (3.71) nm; IR (KBr) vmax (cm21): 3452, 1702, 1653, and 1602; for 1H and 13C NMR spectral data, see Table 2. C31H34O15 [(M þ Na)þ, m/z 669.1792, calcd for C31H34O15Na, 669.1790] 3.4. Acid hydrolysis Compounds 1 and 2 (5 mg, respectively) were hydrolyzed separately with 2 M HCl (0.5 ml) for 10 h at 958C. After filtration of the reaction mixture, the filtrate was evaporated under vacuum. After addition of H2O, the acidic solution was evaporated again to remove HCl. This procedure was repeated until a neutral solution was obtained, which was finally evaporated
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Journal of Asian Natural Products Research and dried in vacuo to furnish a monosaccharide residue. The residue was dissolved in pyridine (1 ml), to which 2 mg of L -cysteine methyl ester hydrochloride was added. The mixture was kept at 608C for 2 h and evaporated under N2 stream and dried in vacuo. The residue was trimethylsilylated with N-trimethylsilylimidazole (0.2 ml) for 2 h. The mixture was partitioned between n-hexane and H2O (2 ml each), and the n-hexane extract was analyzed by GC-MS under the following conditions: capillary column, DB-5 (30 m £ 0.25 mm £ 0.25 mm); detection, Flame Ionization Detector; detector temperature, 2808C; injection temperature, 2508C; initial temperature was maintained at 1008C for 2 min and then raised to 2808C at the rate of 108C/min, and final temperature was maintained for 5 min; carrier, N2. The retention times of the D glucose and D -galactose derivatives were 12.33 and 15.21 min, respectively. 3.5. In vitro lipoxygenase inhibition assay Lipoxygenase-inhibiting activity was conveniently measured by slightly modifying the spectrometric method developed by Tappel [9]. Lipoxygenase type I-B and linoleic acid were purchased from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade. Sodium phosphate (160 ml, 100 mM) buffer (pH 8.0), 10 ml of test-compound solution, and 20 ml of lipoxygenase solution were mixed and incubated for 10 min at 258C. The reaction was then initiated by the addition of 10 ml linoleic acid (substrate) solution, with the formation of (9Z,11E)-(13S)-13hydroperoxyoctadeca-9,11-dienoate, and
5
the change of absorbance at 234 nm was followed for 10 min. Test compounds and the control were dissolved in MeOH. All the reactions were performed in triplicate in 96-well micro-plate in SpectraMax 340. Baicalein was used as a positive control. The IC50 values were then calculated using the EZ-Fit Enzyme kinetics program. The data are expressed as mean ^ SEM of three assays. Disclosure statement No potential conflict of interest was reported by the authors.
References [1] R.R. Baumgartner, D. Steinmann, E.H. Heiss, A.G. Atanasov, M. Gznzera, H. Stuppner, H. Stuppner, and V.M. Dirsch, J. Nat. Prod. 73, 1578 (2010). [2] K. Washida, Y. Itoh, T. Iwashita, and K. Nomoto, Chem. Pharm. Bull. 57, 971 (2009). [3] Q. Fu, S.B. Wang, S.H. Zhao, X.J. Chen, and P.F. Tu, J. Asian Nat. Prod. Res. 15, 697 (2013). [4] R. Wang, G.X. Chou, E.Y. Zhu, Z.T. Wang, and K.S. Bi, J. Asian Nat. Prod. Res. 8, 277 (2006). [5] W.J. Duan, J.Y. Yang, L.X. Chen, L.J. Zhang, Z.H. Jiang, X.D. Cai, X. Zhang, and F. Qiu, J. Nat. Prod. 72, 1579 (2009). [6] A. Braca, P.V. Kiem, P.H. Yen, N.X. Nhiem, T.H. Quang, N.X. Cuong, and C.V. Minh, Fitoterapia 79, 117 (2008). [7] K. Washida, T. Yamagaki, T. Iwashita, and K. Nomoto, Chem. Pharm. Bull. 57, 1150 (2009). [8] M.L. Ren, X. Zhang, R. Ding, Y. Dai, F.J. Tu, Y.Y. Cheng, and X.S. Yao, J. Asian Nat. Prod. Res. 11, 670 (2009). [9] A.L. Tappel, Methods of Enzymology (Academic Press, New York, 1962), Vol. 5, p. 539.
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Lipoxygenase-inhibiting phenolic glycosides and monoterpene glycosides from Paeonia lactiflora a
b
a
a
Liang Zou , Lin-Feng Hu , Yi-Dong Guo , Yu Song & Qiang Fu
a
a
College of Biological Industry, Chengdu University, Chengdu 610106, China b
Pharmacy Department, Chongqing Medical and Pharmaceutical College, Chongqing 401331, China Published online: 23 Mar 2015.
Click for updates To cite this article: Liang Zou, Lin-Feng Hu, Yi-Dong Guo, Yu Song & Qiang Fu (2015): Lipoxygenaseinhibiting phenolic glycosides and monoterpene glycosides from Paeonia lactiflora, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1007960 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1007960
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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Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2015.1007960
Lipoxygenase-inhibiting phenolic glycosides and monoterpene glycosides from Paeonia lactiflora Liang Zoua, Lin-Feng Hub, Yi-Dong Guoa, Yu Songa and Qiang Fua* a
College of Biological Industry, Chengdu University, Chengdu 610106, China; bPharmacy Department, Chongqing Medical and Pharmaceutical College, Chongqing 401331, China
Downloaded by [University of Nebraska, Lincoln] at 17:31 04 April 2015
(Received 12 November 2014; final version received 11 January 2015) The EtOH extract of the roots of Paeonia lactiflora afforded a new phenolic glycoside paenoside A (1) and a new monoterpene glycoside paeonin D (2), and five known monoterpene glycosides. Their structures were elucidated on the basis of spectroscopic means and hydrolysis products. All compounds displayed inhibitory potential against enzyme lipoxygenase. Keywords: Paeonia lactiflora; phenolic glycosides; monoterpene glycosides; lipoxygenase inhibitory activity
1.
Introduction
The roots of Paeonia lactiflora Pall. (Paeoniaceae) are one of the most important Chinese crude drugs, and used in many formulas for the treatment of muscular spasm, chest pains, diarrhea, blood, and liver disorders [1]. Extensive chemical studies have been conducted and have led to the isolation of “cage-like” monoterpene glycosides and phenolic glycosides, and some of those compounds showed anticoagulant and anti-inflammatory activities [2,3]. However, the activity of compounds from this plant has not been satisfactorily examined. In the course of a search for biologically active compounds from Chinese medicinal plants, we have investigated the dried roots of P. lactiflora. Two phenolic glycosides and five monoterpene glycosides were isolated and identified. Two of these were identified as new compounds. All compounds were investigated for inhibitory activity against lipoxygenase. *Corresponding author. Email: [email protected] q 2015 Taylor & Francis
2.
Results and discussion
The EtOH extract of roots of P. lactiflora was partitioned between H2O and nBuOH. The n-BuOH extract was separated by repeated column chromatography using silica gel, C18 silica gel, and high performance liquid chromatography (HPLC) to give two new compounds 1 and 2, and five known structures 3 – 7. The known compounds were identified as 2-O-[a-L -arabinopyranosyl-(1 ! 6)-b-D glucopyranosyl]-benzaldehyde (3) [4], paeonidanin A (4) [5], 4-O-methyl-paeoniflorin (5) [6], 40 -O-galloylpaeoniflorin (6) [7], and 4-O-galloylalbiflorin (7) [8]. Compound 1 was obtained as a colorless amorphous solid. The molecular formula of 1 was found to be C33H34O17 by HR-ESI-MS at m/z 725.1682 [M þ Na]þ. The 13C NMR spectrum of 1 showed 33 carbon signals, including 1 aromatic aldehyde, 2 ester carbons, 18 aromatic carbons, and 12 sugar carbons. The 1H and 13C NMR spectra of 1 (Table 1) were similar to those of 2-O-
2
L. Zou et al.
Table 1. 1H and 13C NMR spectral data of compound 1 (500 MHz, in DMSO-d6 ). 1
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Position 1 2 3 4 5 6 CHO 10 20 30 40 50 60 a 60 b 100 200 300 400 500 600 a 600 b 1000 2000 , 6000 3000 , 5000 4000 7000 10000 20000 , 60000 30000 , 50000 40000 70000
dH (J in Hz)
7.41 d (8.0) 7.66 dd (8.0, 7.5) 7.12 dd (7.5, 7.5) 7.66 d (7.5) 10.49 s 4.86 d (8.0) 3.37– 3.39 m 3.27– 3.29 m 3.12– 3.14 m 3.59 dd (7.0, 4.0) 4.01 dd (11.0, 4.0) 4.12 d (11.0) 4.89 d (7.5) 4.98 dd (9.5, 8.0) 3.60 dd (9.5, 8.0) 3.24– 3.26 m 3.62 dd (8.0, 5.5) 4.52 dd (12.0, 5.5) 4.61 dd (12.0) 7.90 d (7.5) 2H 7.36 t (7.5) 2H 7.48 t (7.5) 7.10 s, 2H
dC 124.6 159.6 116.7 136.5 122.2 126.4 189.7 101.2 70.6 76.2 69.7 75.8 67.6 98.3 78.5 76.7 75.8 75.4 65.3 131.2 129.9 129.8 134.5 167.3 121.0 110.6 147.6 141.2 167.4
[a-L -arabinopyranosyl-(1 ! 6)-b-D -glucopyranosyl]-benzaldehyde (3) [4], with same signals due to aldehyde proton signal at dH 10.49 (1H, s) and four aromatic protons signals at dH 7.41 (1H, br d, J ¼ 8.0 Hz), 7.66 (1H, dd, J ¼ 8.0, 7.5 Hz). 7.12 (1H, dd, J ¼ 7.5, 7.5 Hz), and 7.66 (1H, br d, J ¼ 7.5 Hz), suggesting a typical 1,2-substituted benzene pattern. However, the sugar signals of 1 were different from those of 3. The sugar obtained after aqueous acid hydrolysis of 1 was identified as D -glucose through comparison of the retention time of its trimethylsilyl ether with that of the standard
in gas chromatography. The large coupling constants 8.0 and 7.5 Hz for the anomeric protons in the 1H NMR spectrum of 1 suggested that the glucopyranosyl moieties have a b-configuration. Analysis of the 1 H– 1H COSY spectrum resulted in sequential assignment of all proton resonances of two monosaccharide units. In the HSQC experiment, proton resonances were correlated with those of the corresponding carbons, and associated anomeric protons were correlated with their respective carbon atoms from HSQC–TOCSY data, leading to unambiguous assignments of the carbons in each monosaccharide unit. The 1H NMR data (dH 7.10, 2H, s) and 13C NMR data (dC 110.6, 110.6, 121.0, 141.2, 147.6, 147.6, and 167.4), and signals at dC 129.8, 129.9, 131.2, 134.5, and 167.3 disclosed the presence of a galloyl moiety and a benzoyl unit in 1. In the HMBC spectrum, the correlations H-2000 , 6000 / C-7000 , H-3000 , 5000 /C-1000 , C-4000 , and H-20000 /C10000 , C-30000 , C-40000 , C-60000 and C-70000 confirmed their presence. The connections of C10 /C-2 via an oxygen atom and C-100 /C-60 via an oxygen atom were revealed by HMBC correlations of H-10 /C-2 and H-100 /C-60 . The connections of galloyl moiety and C-600 , and benzoyl moiety and C-200 were revealed by HMBC correlations of H-600 /C-70000 and H200 /C-7000 , respectively. From the above evidences, the structure of 1 was determined as shown in Figure 1, and named as paenoside A. Compound 2 was obtained as a yellowbrown amorphous solid. The molecular formula of 2 was found to be C31H34O15 by HR-ESI-MS at m/z 669.1792 [M þ Na]þ. The 1H and 13C NMR spectra of 2 (Table 2) displayed signals similar to those of 4-O-methylgalloylpaeoniflorin [5], showing the presence of common signals of a monoterpene system and a galloyl moiety. Differences were found in the signals due to sugar moiety. The sugar obtained after aqueous acid hydrolysis of 2 was identified as D -galactose through comparison of the retention time of its trimethylsilyl ether with that of the
Journal of Asian Natural Products Research HO
HO HO
O
4''''
O 1''''
HO
3
OH
1'' OH
OH 7''' 1'''
O 1' OH
OO
O 2
3
4'''
4
OH H
5
1
HO
O 10 1''' O O O 1 OH 7''' OH 6 1' OH
6
O
O
HO
4''
O
7
9 O
5
1'' 7'' O
OCH3
8
O
1
O 3
2
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Figure 1. The structures of compounds 1 and 2.
standard in gas chromatography. The b-configuration of the glycosidic linkage was determined from the coupling constant (J ¼ 7.5 Hz) of the anomeric proton. The connection of the galactose moiety and C-1 via an oxygen atom was Table 2. 1H and 13C NMR spectral data of compound 2 (500 MHz, in CD3OD). 2 Position 1 2 3a 3b 4 5 6 7a 7b 8 9 10 10 20 30 40 50 60 a 60 b 100 200 , 600 300 , 500 400 700 1000 2000 , 6000 3000 , 5000 4000 7000 OCH3
dH (J in Hz)
1.67 d (12.0) 1.77 d (12.0) 2.61 d (6.0) 1.81 d (10.5) 2.40 dd (10.5, 6.0) 4.63 s 2H 5.41 s 1.22 s 3H 4.54 d (7.5) 3.07 t (7.5) 3.18– 3.20 m 3.14– 3.15 m 3.53 t (6.0) 4.46 dd (12.0, 6.0) 4.67 d (12.0) 7.96 d (7.5) 2H 7.45 t (7.5) 2H 7.55 t (7.5) 7.82 s, 2H
3.39
dC 89.4 86.8 42.0 109.6 41.9 72.0 22.6 61.6 102.8 19.8 97.9 76.6 73.5 73.1 70.6 64.8 131.4 131.1 129.9 134.7 167.8 121.1 110.7 146.9 141.0 167.6 51.2
confirmed by HMBC correlation of H-10 / C-1. The stereochemistry at various stereocenters of the monoterpene unis was assigned on the basis of similarity of spectral data with related compounds and further confirmed by NOESY correlations [5], which showed close agreement with those of 4-O-methylgalloylpaeoniflorin. Therefore, the structure of 2 was determined as shown in Figure 1, and named as paeonin D. Compounds 1– 7 were investigated for inhibition of lipoxygenase activity (Table 3). All of those compounds showed potent inhibitory activity.
3. Experimental 3.1. General experimental procedures Optical rotations were measured on a JASCO P-1020 digital polarimeter (Jasco, Tokyo, Japan). UV spectra were obtained on a SHIMADZU UV-2201 UV/VIS recording spectrophotometer (Shimadzu, Table 3. In vitro quantitative inhibition of lipoxygenase by compounds 1 – 7. Compounds 1 2 3 4 5 6 7 Baicalein
IC50 ^ SEM (mM) 8.4 ^ 0.3 24.4 ^ 2.1 12.6 ^ 2.5 28.1 ^ 1.7 37.4 ^ 0.9 21.6 ^ 1.3 20.4 ^ 1.1 21.5 ^ 1.2
Note: Baicalein was used as a positive control.
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4
L. Zou et al.
Kyoto, Japan). IR spectra were recorded on a Bruker IFS-55 plus spectrometer (Bruker, Ettlingen, German). NMR spectra were recorded on an Inova 500 spectrometer (Bruker, Waltham, MA, USA). ESI-MS were measured on an Agilent 6320 ion trap mass spectrometer (Agilent, Santa Clara, CA, USA). HR-ESI/MS measurements were performed on a Bruker-Daltonics APES-III 7.0 TESLA FTMS spectrometer (Bruker, Billerica, MA, USA), HPLC separation was carried out on an octadecylsilanized column (YMC-pack ODS-A, 250 £ 10 mm, i.d. 5 mm, YMC, Kyoto, Japan) with a photodiode array detector (Waters, Millford, MA, USA). GC was performed with a SHIMADZU GC-14D. Column chromatography was performed with silica gel (Merck, Darmstadt, Germany) and C18 silica gel (Merck). 3.2.
Plant material
The roots of P. lactiflora were collected in May 2014 in Chengdu, Sichuan Province, China. The identification of the plant was performed by the author (Qiang Fu). A voucher specimen (PL 201403) is maintained in the herbarium of the College of Biological Industry, Chengdu University. 3.3.
Extraction and isolation
The dried roots of P. lactiflora (2.6 kg) were extracted with EtOH. After removing the solvent, the residue (321 g) was suspended in H2O and extracted with nBuOH. The n-BuOH-soluble fraction (105 g) was subjected to silica gel column chromatography and eluted in a gradient of CHCl3 – MeOH (100:1 ! 10:90) to afford fractions 1 – 5. Fraction 1 (5.3 g) was subjected to C18 silica gel chromatography and eluted in a gradient of MeOH – H2O (10:90 ! 95:5) to afford sub-fractions 1– 6 (0.3, 0.8, 0.1, 0.4, 1.7, and 0.6 g, respectively) and compound 2 (18 mg). Sub-fraction 2 (0.8 g) was separated by
HPLC (MeOH – H2O, 52:48, 2.0 ml/min) to yield compound 1 (31 mg, 21.3 min). Sub-fraction 6 (0.6 g) was separated by HPLC (MeOH – H2O, 54:46, 2.0 ml/min) to yield compound 5 (21 mg, 19.2 min). Fraction 3 (10.2 g) was subjected to C18 silica gel chromatography and eluted in a gradient of MeOH – H2O (10:90 ! 95:5) to afford compounds 3 (31 mg, 16.3 min) and 4 (39 mg, 19.2 min). Fraction 4 (10.2 g) was subjected to C18 silica gel chromatography and eluted in a gradient of MeOH – H2O (10:90 ! 95:5) to afford compounds 6 (66 13.2 min) and 7 (27 mg, 18.3 min). 3.3.1. Paenoside A (1) Colorless amorphous solid; ½a20 D 2 32.2 (c ¼ 0.5, MeOH); UV (MeOH) lmax (log 1) 210 (3.17), 220 (2.71), 250 (3.81), 308 (3.41) nm; IR (KBr) vmax (cm21): 3453, 1702, 1654, 1201, and 1071; for 1H and 13C NMR spectral data, see Table 1. m/z 725.1682 [M þ Na] þ, calcd for C33H34O17Naþ, 725.1688] 3.3.2.
Paeonin D(2)
Yellow-brown amorphous solid, ½a20 D 2 48.3 (c ¼ 0.5, MeOH); UV (MeOH) lmax (log 1) 229 (2.81), 273 (3.71) nm; IR (KBr) vmax (cm21): 3452, 1702, 1653, and 1602; for 1H and 13C NMR spectral data, see Table 2. C31H34O15 [(M þ Na)þ, m/z 669.1792, calcd for C31H34O15Na, 669.1790] 3.4. Acid hydrolysis Compounds 1 and 2 (5 mg, respectively) were hydrolyzed separately with 2 M HCl (0.5 ml) for 10 h at 958C. After filtration of the reaction mixture, the filtrate was evaporated under vacuum. After addition of H2O, the acidic solution was evaporated again to remove HCl. This procedure was repeated until a neutral solution was obtained, which was finally evaporated
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Journal of Asian Natural Products Research and dried in vacuo to furnish a monosaccharide residue. The residue was dissolved in pyridine (1 ml), to which 2 mg of L -cysteine methyl ester hydrochloride was added. The mixture was kept at 608C for 2 h and evaporated under N2 stream and dried in vacuo. The residue was trimethylsilylated with N-trimethylsilylimidazole (0.2 ml) for 2 h. The mixture was partitioned between n-hexane and H2O (2 ml each), and the n-hexane extract was analyzed by GC-MS under the following conditions: capillary column, DB-5 (30 m £ 0.25 mm £ 0.25 mm); detection, Flame Ionization Detector; detector temperature, 2808C; injection temperature, 2508C; initial temperature was maintained at 1008C for 2 min and then raised to 2808C at the rate of 108C/min, and final temperature was maintained for 5 min; carrier, N2. The retention times of the D glucose and D -galactose derivatives were 12.33 and 15.21 min, respectively. 3.5. In vitro lipoxygenase inhibition assay Lipoxygenase-inhibiting activity was conveniently measured by slightly modifying the spectrometric method developed by Tappel [9]. Lipoxygenase type I-B and linoleic acid were purchased from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade. Sodium phosphate (160 ml, 100 mM) buffer (pH 8.0), 10 ml of test-compound solution, and 20 ml of lipoxygenase solution were mixed and incubated for 10 min at 258C. The reaction was then initiated by the addition of 10 ml linoleic acid (substrate) solution, with the formation of (9Z,11E)-(13S)-13hydroperoxyoctadeca-9,11-dienoate, and
5
the change of absorbance at 234 nm was followed for 10 min. Test compounds and the control were dissolved in MeOH. All the reactions were performed in triplicate in 96-well micro-plate in SpectraMax 340. Baicalein was used as a positive control. The IC50 values were then calculated using the EZ-Fit Enzyme kinetics program. The data are expressed as mean ^ SEM of three assays. Disclosure statement No potential conflict of interest was reported by the authors.
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