Acylated flavonol glycosides and δ-truxinate derivative from the aerial parts of Lysimachia clethroides

Phytochemistry Letters 11 (2015) 116–119

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Acylated flavonol glycosides and d-truxinate derivative from the aerial parts of Lysimachia clethroides Dong Liang a,b, Yan-Fei Liu a, Zhi-You Hao a, Huan Luo a, Yan Wang a, Chun-Lei Zhang a, Ruo-Yun Chen a, De-Quan Yu a,* a

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, PR China

b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 October 2014 Received in revised form 19 November 2014 Accepted 2 December 2014 Available online 12 December 2014

Phytochemical investigation of the aerial parts of Lysimachia clethroides led to the isolation of a new acylated flavonol glycoside (1) and a new d-truxinate derivative (2), together with three known acylated flavonol glycosides. The structures of the new compounds were determined by spectroscopic methods and chemical evidence as quercetin-3-O-b-D-(6-O-Z-p-coumaroyl)glucopyranoside (1) and monomethyl 3,30 ,4,40 -tetrahydroxy-d-truxinate (2), respectively. All of the isolates were evaluated for their in vitro inhibitory activity against aldose reductase. ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Lysimachia clethroides Acylated flavonol glycoside d-Truxinate derivative Aldose reductase

1. Introduction Lysimachia clethroides Duby (Primulaceae) is a traditional Chinese folk medicine, distributed widely in many provinces of China. The aerial parts of this plant have been used for the treatment of throat ache, edema, and menoschesis, etc. (Jiangsu Botanic Institute, 1991). Previous phytochemical studies on L. clethroides have led to the isolation and identification of triterpenoid saponins, flavonoids, and several other components (Ding et al., 2010; Kitagawa et al., 1972; Wu et al., 2011; Zou and Tu, 2009). Fourteen cytotoxic triterpenoid saponins (Liang et al., 2011) and several other constituents (Liang et al., 2012, 2013a,b) have been obtained from the aerial parts of L. clethroides in our previous work. A new acylated flavonol glycoside (1, Fig. 1) and a new d-truxinate derivative (2, Fig. 1), together with three known acylated flavonol glycosides, were isolated from its ethanolic extract in our continuing search for other biologically active natural products from this plant. Herein, the isolation and structural elucidation of the two new compounds (1–2), along with the evaluation of the in vitro inhibitory activity of all the isolates against aldose reductase were described.

* Corresponding author. Tel.: +86 10 63165224; fax: +86 10 63017757. E-mail addresses: [email protected], [email protected] (D.-Q. Yu).

The known acylated flavonol glycosides were identified as quercetin-3-O-b-D-(6-O-E-p-coumaroyl)glucopyranoside (3) (Chen et al., 2013), kaempferol-3-O-b-D-(6-O-E-p-coumaroyl)glucopyranoside (4) (Tsukamoto et al., 2004), kaempferol-3-O-b-D(6-O-Z-p-coumaroyl)glucopyranoside (5) (Tsukamoto et al., 2004), by NMR analysis and comparison with literature data. 2. Results and discussion Compound 1 was isolated as a yellow amorphous powder. The molecular formula C30H26O14 was established on the basis of the positive-ion HR-ESIMS (633.1220 [M + Na]+, calcd 633.1215) and was identical with that of 3. The IR spectrum showed characteristic absorption bands for hydroxyl (3249 cm1) and conjugated carbonyl (1655 cm1) groups. The 1H and 13C NMR spectroscopic data of 1 displayed signals characteristic of acylated flavonol glycosides (Chen et al., 2013; Tsukamoto et al., 2004). The 1H NMR spectrum showed two doublets at dH 6.31 (1H, d, J = 2.0 Hz, H-8), 6.18 (1H, d, J = 2.0 Hz, H-6), an ABX spin system due to the aromatic ring at dH 7.50 (1H, d, J = 2.0 Hz, H-20 ), 7.49 (1H, dd, J = 9.0, 2.0 Hz, H-60 ), 6.81 (1H, d, J = 9.0 Hz, H-50 ), and an anomeric signal at dH 5.43 (1H, d, J = 7.5 Hz, H-100 ). In addition, the 1H NMR spectrum showed the signals for a p-coumaroyl moiety, including an AA0 XX0 system at dH 7.52 (2H, d, J = 8.5 Hz, H-5000 , 9000 ), 6.69 (2H, d, J = 8.5 Hz, H-6000 , 8000 ), and an AX system at dH 6.64 (1H, d, J = 13.0 Hz, H-3000 ),

http://dx.doi.org/10.1016/j.phytol.2014.12.006 1874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

D. Liang et al. / Phytochemistry Letters 11 (2015) 116–119

OH HO

O

OH O

OH HO

1'

OH

2

O HO

1''

2

4

O 1''' O OH O OH

1' 2'

HO 4' 1

9 7 8 COOR

1

OH

OH

7' 8' COOMe 9'

2 R=H 2a R=Me

Fig. 1. Chemical structures of compounds 1–2.

5.45 (1H, d, J = 13.0 Hz, H-2000 ). These data were closely related to those of 3 except for the chemical shifts and magnitude of the coupling constants for H-2000 and H-3000 (dH 7.34, 6.13, each 1H, d, J = 16.0 Hz for 3) (Chen et al., 2013), indicating that 1 bears a Z-pcoumaroyl moiety instead of the E-p-coumaroyl moiety in 3, which was further confirmed by the NOESY correlation between two olefinic protons. Acid hydrolysis of 1 afforded D-glucose, which was identified by the positive optical rotation ([a]20D + 37.5), and the b form was judged from its large 3JH1,H2 coupling constant (J = 7.5 Hz). Therefore, the structure of 1 was determined to be quercetin-3-O-b-D-(6-O-Z-p-coumaroyl)glucopyranoside, which was unambiguously confirmed by 1H–1H COSY, HSQC, HMBC, and NOESY data. Compound 2 was obtained as a white solid. Its molecular formula was determined as C19H18O8 from the HR-ESIMS (397.0895 [M + Na]+, calcd 397.0894) and supported by the NMR spectroscopic data, with a calculated unsaturation number of 11. The IR spectrum displayed the presence of hydroxyl (3188 cm1) and carbonyl (1723, 1678 cm1) groups. The 1H NMR spectrum in pyridine-d5 showed six aromatic proton signals at dH 7.51 (1H, d, J = 1.8 Hz, H-2), 7.46 (1H, d, J = 1.8 Hz, H-20 ), 7.18 (2H, overlapped by solvent peaks, H-5, 50 ), 7.09 (1H, dd, J = 7.8, 1.8 Hz, H-6), and 7.02 (1H, dd, J = 7.8, 1.8 Hz, H-60 ) revealing the presence of two 1,3,4-trisubstituted aromatic rings. In addition, there were four upfield proton signals at dH 4.10 (1H, m, H-7), 4.00 (1H, m, H-70 ), 3.95 (1H, m, H-80 ), 3.92 (1H, m, H-8), together with a methoxy at dH 3.57 (3H, s). The 13C NMR spectrum displayed 19 carbon signals, including two carbonyl (dC 175.5, 173.9), 12 aromatic carbons for two aromatic rings, one methoxy (dC 51.8), and four methine carbons [dC 48.5 (C-7), 48.3 (C-70 ), 46.1 (C-80 ), 45.6 (C-8)]. It was evident from the unsaturation number that a

117

cyclobutane ring was present in the molecule of 2. The key correlations of H-7 with C-9 (dC 175.5), H-70 with C-90 (dC 173.9), H8 with C-9/C-90 , and H-80 with C-9/C-90 in the HMBC spectrum (Supplementary data, Fig. S19) suggested the molecule to be a modified lignan with two phenylpropenoid units coupled at the C7 (C-70 ) and C-8 (C-80 ) positions, which was a head-to-head dimer. HMBC correlation of OMe (dH 3.57) with C-90 indicated that the methoxy group was located at C-90 . The relative configuration of the cyclobutane ring was determined to be the same as that of dimethyl 3,30 ,4,40 -tetrahydroxy-d-truxinate (2a, Fig. 1), a dtruxinate derivative with an additional methoxy group (Deng et al., 2011), because of their almost superimposable NMR data both measured in acetone-d6 (see Table 1). This conclusion was confirmed by a NOESY correlation between H-7 and H-80 in the NOESY spectrum (Supplementary data, Fig. S20), and the lower field resonated methoxy group (dH 3.67 in acetone-d6) accounted for the adjacent trans-oriented phenyl group on the cyclobutane ring (Ben-Efraim and Green, 1974; Kuroyanagi et al., 1982). Therefore, the structure of compound 2 was determined as monomethyl 3,30 ,4,40 -tetrahydroxy-d-truxinate. Inhibition of aldose reductase by acylated flavonols was reported previously (Lee et al., 2010; Matsumoto et al., 2014). As shown in Table 2, compounds 1–5 showed moderate inhibitory activity with IC50 values of 2.04–8.54 mM. 3. Experimental 3.1. General experimental procedures Optical rotations were measured with a JASCO P-2000 polarimeter, and UV spectra with a JASCO V-650 spectrophotometer. IR spectra were recorded on a Nicolet 5700 spectrometer by an FT-IR microscope transmission method. NMR measurements were performed on BRUKER-600 or INOVA-500 in DMSO-d6, pyridine-d5 or acetone-d6 with solvent peaks as references. HR-ESIMS were obtained using an Agilent 1100 series LC/MSD ion trap mass spectrometer. Preparative HPLC was performed on a Lumtech instrument equipped with a Lumtech K-2501 UV detector, using a YMC-Pack ODS-A column (250  20 mm, 5 mm). Silica gel (200– 300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (GE), and ODS (50 mm, YMC, Japan) were used for column chromatography. TLC was carried out with GF254 plates

Table 1 1 H and 13C NMR data of compounds 2 and 2a. No.

2 (pyridine-d5)

dH 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 OMe a

7.51 d (1.8)

7.18a 7.09 dd (7.8, 1.8) 4.10 m 3.92 m

7.46 d (1.8)

7.18a 7.02 dd (7.8, 1.8) 4.00 m 3.95 m 3.57 s (3H)

Signal overlapped by solvent peaks. b–j May be interchangeable.

2 (acetone-d6)

dC 134.2b 115.4 147.30c 146.3d 116.7e 118.52f 48.5 45.6 175.5 134.0b 115.4 147.27c 146.2d 116.6e 118.46f 48.3 46.1 173.9 51.8

dH

dC

6.82 d (2.0)g

6.76 6.67 3.46 3.28

d (8.0) dd (8.0, 2.0)h dd like dd like

6.81 d (2.0)g

6.76 6.65 3.46 3.28

2a (acetone-d6)

d (8.0) dd (8.0, 2.0)h dd like dd like

3.67 s (3H)

134.3i 114.8 145.9 145.0 116.1 119.0 48.5j 45.6 174.1 134.2i 114.8 145.9 145.0 116.1 119.0 48.3j 45.6 173.6 52.1

dH

dC

6.80 d (2.1)

6.77 6.65 3.46 3.30

d (8.1) dd (8.1, 2.1) dd like dd like

6.80 d (2.1)

6.77 6.65 3.46 3.30

d (8.1) dd (8.1, 2.1) dd like dd like

3.68 s (6H)

134.2 114.8 145.9 145.0 116.1 119.1 48.5 45.6 173.6 134.2 114.8 145.9 145.0 116.1 119.1 48.5 45.6 173.6 52.3 (2C)

D. Liang et al. / Phytochemistry Letters 11 (2015) 116–119

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Table 2 Inhibitory activity of compounds 1–5 against aldose reductasea. Compounds

Inhibitory rate (%)

IC50 (mM)

1 2 3 4 5 Epalrestatb

86.9 68.9 92.3 65.8 81.3 98.5

3.91 7.93 2.04 8.54 5.12 1.12  102

a b

The inhibitory rate of each compound was tested at a concentration of 10 mM. Positive control.

3.3.2. Monomethyl 3,30 ,4,40 -tetrahydroxy-d-truxinate (2) White solid; [a]–82.8 (c 0.11, MeOH); UV (MeOH) lmax (log e) 203 (4.68), 285 (3.72) nm; IR nmax 3188, 2956, 1723, 1678, 1602, 1526, 1441 cm1; Positive-ion HR-ESIMS m/z 375.1081 [M + H]+ (calcd for C19H19O8, 375.1074), 397.0895 [M + Na]+ (calcd for C19H18O8Na, 397.0894). 1H NMR (pyridine-d5, 600 MHz), 13C NMR (pyridine-d5, 150 MHz), 1H NMR (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) see Table 1. 3.4. Acid hydrolysis and sugar analysis

(Qingdao Marine Chemical Factory). Spots were visualized by spraying with 10% H2SO4 acid in EtOH followed by heating.

The determination of the absolute configuration of the sugar in compound 1 was conducted as described previously by our group (Liu et al., 2012).

3.2. Plant material

3.5. In vitro aldose reductase assay

The aerial parts of L. clethroides was collected in Mount Lushan, Jiangxi province, People’s Republic of China, in September 2009, and was identified by Professor Ce-Ming Tan (Jiujiang Institute of Forestry). A voucher specimen (No. 21787) was deposited at the Herbarium of the Department of Medicinal Plants, the Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing.

Aldose reductase activity was assayed by the method described previously (Nishimura et al., 1991). Enzyme activity was spectrophotometrically measured by monitoring the decay in absorbance at 340 nm, which accompanies the oxidation of NADPH catalysed by aldose reductase. The reaction mixture contained 100 mM sodium phosphate buffer, 0.16 mM NADPH, 10 mM DL-glyceraldehyde, with or without Epal/Sample solution (purity of Epal > 98%), in a total volume of 0.1 mL. Reactions were initiated by the addition of enzyme.

3.3. Extraction and isolation Air-dried and powdered aerial parts of L. clethroides (10 kg) were exhaustively extracted with 70% aqueous EtOH (50 L  3) at reflux. The combined extracts were concentrated under reduced pressure to dryness. The residue was suspended in H2O and partitioned with petroleum ether, EtOAc, and n-BuOH, successively. The EtOAc-soluble residue (300 g) was subjected to silica gel CC and eluted with a gradient of petroleum ether-acetone (1:0 to 1:1, V:V) and pure MeOH. The fractions were combined according to TLC profiles into fourteen main fractions (A–N). Fraction N (10 g) was separated by MPLC (ODS, 50 mm, YMC), eluting with MeOHH2O (1:9 to 1:0, V:V) to afford thirty subfractions. Fraction N-8 (370 mg) was subjected to preparative HPLC to give compound 2 (5 mg, tR 37.7 min) by 18% MeCN-H2O (7 mL/min). Fraction N-24 (336 mg) was purified by preparative HPLC using 25% MeCN-H2O (7 mL/min) to yield compounds 3 (16 mg, tR 36.2 min), and 1 (29 mg, tR 42.6 min). Fraction N-26 (242 mg) was purified by preparative HPLC using 27% MeCN-H2O (7 mL/min) to yield compounds 4 (15 mg, tR 38.5 min), and 5 (11 mg, tR 46.5 min). 3.3.1. Quercetin-3-O-b-D-(6-O-Z-p-coumaroyl)glucopyranoside (1) Yellow amorphous powder; [a]–17.7 (c 0.11, MeOH); UV (MeOH) lmax (log e) 205 (4.49), 258 (4.09), 267 (4.08), 310 (4.04), 360 (3.94) nm; IR nmax 3249, 1655, 1605, 1512, 1444, 1360 cm1; ESIMS (positive) m/z 633 [M + Na]+; ESIMS (negative) m/z 609 [M  H]; positive-ion HR-ESIMS m/z 611.1401 [M + H]+ (calcd for C30H27O14, 611.1395), 633.1220 [M + Na]+ (calcd for C30H26O14Na, 633.1215). 1 H NMR (DMSO-d6, 500 MHz) d: 7.52 (2H, d, J = 8.5 Hz, H-5000 , 9000 ), 7.50 (1H, d, J = 2.0 Hz, H-20 ), 7.49 (1H, dd, J = 9.0, 2.0 Hz, H-60 ), 6.81 (1H, d, J = 9.0 Hz, H-50 ), 6.69 (2H, d, J = 8.5 Hz, H-6000 , 8000 ), 6.64 (1H, d, J = 13.0 Hz, H-3000 ), 6.31 (1H, d, J = 2.0 Hz, H-8), 6.18 (1H, d, J = 2.0 Hz, H-6), 5.45 (1H, d, J = 13.0 Hz, H-2000 ), 5.43 (1H, d, J = 7.5 Hz, H-100 ), 4.16 (1H, dd, J = 12.0, 2.0 Hz, H-600 a), 4.07 (1H, dd, J = 12.0, 6.5 Hz, H-600 b), 3.14–3.33 (4H, H-200 , 300 , 400 , 500 ); 13C NMR (DMSO-d6, 125 MHz) d: 156.4 (C-2), 133.1 (C-3), 177.3 (C-4), 161.2 (C-5), 98.6 (C-6), 164.1 (C-7), 93.5 (C-8), 156.3 (C-9), 103.9 (C-10), 121.5 (C-10 ), 115.1 (C-20 ), 144.8 (C-30 ), 148.5 (C-40 ), 116.1 (C-50 ), 121.0 (C-60 ), 100.8 (C-100 ), 74.1 (C-200 ), 76.3 (C-300 ), 69.9 (C-400 ), 74.0 (C-500 ), 62.8 (C-600 ), 165.4 (C-1000 ), 114.7 (C-2000 ), 143.6 (C-3000 ), 125.3 (C-4000 ), 132.6 (C-5000 , 9000 ), 114.8 (C-6000 , 8000 ), 158.8 (C-7000 ).

Acknowledgements The research work was supported by the National Natural Science Foundation of China (21132009), the Guangxi Natural Science Foundation of China (2014GXNSFBA118042). We thank the Department of Medicinal Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, for the measurements of IR, NMR and HR-ESIMS spectra. We are also grateful to Ms. Quan Liu and Zhu-Fang Shen (Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College) for the in vitro aldose reductase assay.

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