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Conjugates of a secoiridoid glucoside with a phenolic glucoside from the flower buds of Lonicera japonica Thunb
Phytochemistry 96 (2013) 423–429
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Conjugates of a secoiridoid glucoside with a phenolic glucoside from the flower buds of Lonicera japonica Thunb Yoshiki Kashiwada a,⇑, Yuka Omichi a, Shin-ichiro Kurimoto a, Hirofumi Shibata a, Yoshiyuki Miyake b, Tsukasa Kirimoto b, Yoshihisa Takaishi a a b
Graduate School of Pharmaceutical Sciences, University of Tokushima, Shomachi 1-78, Tokushima 770-8505, Japan Consumer Healthcare, S.H. Product Strategy Planning, Product Research, Taiho Pharmaceutical Co., Ltd., 224-2 Ebisuno, Kawauchicho, Tokushima 771-0194, Japan
a r t i c l e
i n f o
Article history: Received 25 June 2013 Received in revised form 13 September 2013 Available online 10 October 2013
a b s t r a c t Secoiridoid glucosides, including two conjugates with a phenolic and two conjugates with a nicotinic acid derivative (3 and 4), together with seven known secoiridoid derivatives, were isolated from flower buds of Lonicera japonica. The structures were elucidated by spectroscopic analyses. Anti-influenza activities of six isolated compounds were also evaluated by plaque assay and neuraminidase inhibitory assay. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Lonicera japonica Caprifoliaceae Secoiridoid Nicotonic acid Eugenol Anti-influenza
1. Introduction
2. Results and discussion
Lonicera japonica Thunb. (Caprifoliaceae), also known as Japanese Honeysuckle, is a perennial twining woody vine native to the East Asia. Its flower buds have been used since early times for treatment of arthritis, diabetes mellitus, fever, infections, sores, and swelling (Shanghai Science and Technology Press, 1977). Its flower buds are also one of the major ingredients in Yin Qiao San, a most popular prescription in traditional Chinese medicine, used for common colds, including fever, headache, cough, thirst, and sore throat, and for influenza infection (Kurokawa et al., 1998). In evaluation of the anti-influenza activity of Yin Qiao San in vitro, the H2O extract of Yin Qiao San inhibited all subtypes of influenza A, B, and C virus replication, and also exhibited inhibitory activity against an oseltamivir-resistant influenza A virus (Shibata et al., 2010). Based on this finding, the MeOH extract of flower buds of L. japonica were investigated. This resulted in isolation of four new secoiridoid glucoside derivatives including two novel conjugates with a phenolic glucoside (1 and 2), and two conjugates with a nicotinic acid derivative (3 and 4), together with seven known compounds. Described herein are the isolation and structure elucidation of these compounds, as well as evaluation of their inhibitory activity against influenza A virus replication and neuraminidase.
2.1. Structure elucidation and identification
⇑ Corresponding author. Tel./fax: +81 88 633 7276. E-mail address: [email protected] (Y. Kashiwada). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.09.021
Flower buds of L. japonica (10.0 kg) were extracted five times with MeOH at room temperature. After removal of the solvent, the MeOH extract was successively partitioned between EtOAc, n-BuOH and H2O. The EtOAc-soluble fraction was further partitioned between n-hexane and 90% aqueous MeOH giving a n-hexane-soluble fraction and 90% MeOH-soluble fraction. In contrast, the H2O-soluble fraction was subjected to Diaion HP-20 chromatography with H2O containing increasing amounts of MeOH to give nine fractions (W-F1–W-F9). The n-hexane-, 90% MeOH-, and n-BuOH-soluble fractions, as well as the fractions obtained from Diaion HP-20 chromatography, were then evaluated for their inhibitory effects on the growth of influenza A/PR/8/34 by plaque assay, as well as for influenza A neuraminidase inhibitory activity. Anti-influenza activities were found in both the n-hexane-soluble fraction and fractions W-F2–W-F9 (data not shown). Fractions obtained from the Diaion HP-20 column chromatography were further separated by repeated Sephadex LH-20, MCI gel CHP-20P, Toyopearl HW-40C, and YMC ODS-A, column chromatography, and purified by reversed-phase HPLC to yield 1 (3.5 mg), 2 (3.3 mg), 3 (9.2 mg), and 4 (0.9 mg), respectively, together with seven known compounds (5–11). The latter were identified as L-phenylalaninosecologanin B (5) (Zheng et al., 2012), vogeloside
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(6) (Boros and Stermitz, 1991), 7-epi-vogeloside (7) (Boros and Stermitz, 1991), secoxyloganin (8) (Calis and Sticher, 1984), dimethyl secologanoside (9) (Calis and Sticher, 1984), sweroside (10) (Takeda et al., 1999), and secologanin dimethylacetal (11) (Machida et al., 1994), respectively, by comparison of their spectroscopic data with those reported in the literature (Fig. 1). Lonicerjaponin A (1) was obtained as a white amorphous powder. Its molecular formula was determined as C35H46O17 by HRESIMS (m/z 737.2680 [MH], calcd for C35H45O17, 737.2657). The 1 H NMR spectrum of 1 had signals similar to those of secoxyloganin (8) in teams of the presence of a tri-substituted olefin [dH 7.52 (1H, s)], a vinyl group [dH 5.68 (1H, ddd, J = 17.1, 10.3, 8.5 Hz), 5.19 (1H, br d, J = 17.5 Hz) and 5.16 (1H, br d, J = 10.5 Hz)], an acetal proton [dH 5.47 (1H, d, J = 8.2 Hz)] and a methoxy group [dH 3.64 (3H, s)], together with an anomeric signal [dH 4.70 (1H, d, J = 8.0 Hz)]. It also showed resonances ascribable to a 1,2,4-trisubstituted aromatic group [dH 7.00 (1H, d, J = 8.2 Hz), 6.83 (1H, d, J = 1.8 Hz), 6.71 (1H, dd, J = 8.2, 1.8 Hz)], a trans-olefin [dH 5.53 (1H, brd, J = 15.4 Hz), 5.50 (1H, brd, J = 15.4 Hz)], a vinyl group [dH 5.94 (1H, ddd, J = 17.3, 10.2, 6.7 Hz), 5.07 (1H, brd, J = 17.3 Hz), 5.03 (1H, brd, J = 10.2 Hz)] and a methoxy group [dH 3.83 (3H, s)], along with an anomeric signal [dH 4.80 (1H, d, J = 7.5 Hz)]. The 13C NMR spectrum displayed 35 carbon resonances, due to two ester carbonyls (dC 173.2 and 168.8) and two hexosyl moieties assignable to two glucosyl moieties. The sugar moiety was identified as D-glucose by direct HPLC analysis of the acid hydrolysate of 1 using an optical rotation detector. The structure of 1 was elucidated by 1H–1H COSY and HMBC spectroscopic analyses (Fig. 2). The 1H–1H COSY examination established the occurrence of a partial structure corresponding to H-1–H-9–H-8–H2-10, in which H-9 had correlations of a H-9–H5–trans-olefin; thus, the trans-olefin was assigned to H-6–H-7. The HMBC correlations of H-1 with C-3 and C-5, H-3 with C-4 and C-11, and of OMe with C-11, together with the HMBC correlation of the anomeric proton signal with H-1, indicated the presence of a secoiridoid moiety. In addition, H-7 exhibited a 1H–1H COSY correlation with a methylene proton (H2-12), which further displayed an HMBC correlation with an ester carbonyl carbon (dC 173.2), providing a 2-(secoloan-6-en-7-yl)-acetate moiety as a partial structure. In contrast, HMBC correlations of aromatic signals [dH 6.83 (1H, d, J = 1.8 Hz), 6.71 (1H, dd, J = 8.2, 1.8 Hz)] with an sp3 methylene carbon (dC 40.8), whose protons further exhibited a 1H–1H COSY correlation with a vinyl group, suggesting the presence of a phenylpropanoid moiety. Analysis of the following HMBC
Fig. 2. Key COSY and HMBC correlations in lonicerjaponin A (1).
correlations resulted in deduction of a 4-O-glucosyl-eugenol moiety: H-50 with C-30 and C-10 ; H-20 with C-40 ; OMe with C-300 ; H-100 with C-400 . These two moieties were connected through an ester linkage between C-600 0 and C-13 from the HMBC correlation of H2-6000 with C-13, and thus the structure of 1 was elucidated. Its relative configuration was elucidated by analysis of its NOESY spectrum. Thus, NOESY correlations of H-5 with H-9, as well as H-1 with H-8, indicated the relative configuration of 1 to be identical with secologanin. The b-linkages of both glucosyl moieties were concluded from the J-value of the anomeric proton signals. On the basis of this examination, its structure was characterized as shown in Fig. 1. The molecular formula of lonicerjaponin B (2) was assigned as C34H44O17 by positive HRESIMS (m/z 747.2477 [M+Na]+, calcd for C34H44O17Na, 747.2476). The 1H and 13C NMR spectra of 2 were similar to those of 1 except for the absence of signals of a methoxy and a trans-olefin. It also instead showed resonances ascribable to a methylene [dH 2.65 (1H, ddd, J = 13.2, 13.2, 12.0 Hz), 1.94 (1H, brdd, J = 12.0, 3.5 Hz); dC 30.8] and an oxygen-bearing sp3 methine group [dH 4.84 (1H, m); dC 77.1]. These signals were shown to be coupled to each other and were assigned to be H-6 and H-7, respectively, from the 1H–1H COSY correlation of H-5 with this methylene signal. Furthermore, H-7 showed a 1H–1H COSY correlation with H2-12, which further displayed a HMBC correlation with
Fig. 1. Structures of secoiridoid derivatives isolated from L. japonica.
Y. Kashiwada et al. / Phytochemistry 96 (2013) 423–429
an ester carbonyl carbon (dC 171.1). The presence of a lactone ring between C-7 and C-11 was deduced, taking into account the chemical shift of H-7 (dH 4.84), as well as the degree of unsaturation of its molecular formula. Thus, the occurrence of a 7-carboxylmethylsweroside moiety was elucidated. This moiety was shown to be located at the glucosyl C-6 position of the 4-O-glucosyl-eugenol moiety from the HMBC correlation of H-6000 with C-13. The identification of D-glucose was carried out by direct HPLC analysis of its hydrolysate, and b-linkages were concluded from the J-values of the anomeric proton signals. The relative configuration of 2 was elucidated by analysis of its NOESY spectrum, in which NOESY correlations of H-5 with H-7 as well as H-9 indicated that the relative configuration of 2 was the same as that of sweroside (7). On the basis of these observations, the structure of 2 was assigned as shown in Fig. 1. Compound 3 was obtained as an off-white amorphous powder. Its molecular formula was determined as C24H29NO11 by HRESIMS (m/z 530.1616 [M+Na]+, calcd for C24H29NO11Na, 530.1638). The 1H and 13C NMR spectra of 3 were also correlated to those of 1, except for the absence of the signals due to the 4-O-glucosyl-eugenol moiety. 2D NMR examinations established that the presence of a secoloan-6-en-7-yl moiety (Fig. 3). In addition, the 1H NMR spectrum also showed three aromatic signals [dH 8.91 8.70, 8.33 (each 1H, br s)] and a methoxy resonance [dH 3.94 (3H, s)], while the 13C NMR spectrum displayed seven carbon signals, including one ester carbonyl (dC 166.8), three sp2 methines (dC 152.0, 149.3, and 135.0), two sp2 quaternary carbons (dC 135.2 and 127.8) and a methoxy carbon (dC 53.0), except for the resonances arising from the secoloan-6-en-7-yl moiety. The presence of a D-glucosyl moiety was confirmed by direct HPLC analysis of its hydrolysate. The HMBC correlations of H-400 with C-200 , C-600 and C-700 , H-200 with C-300 , and of OMe with C-700 indicated the presence of a C-5 substituted methyl nicotinate moiety. In addition, the secoloan-6-en-7-yl moiety was shown to be connected to C-5 of a methyl nicotinate moiety from the HMBC correlation of H-400 with C-7 and of H-7 with C-400 and C-600 . The following NOESY correlations indicated that the relative stereochemistry of 3 was identical with that of secologanin: H-1 with H-6 and H-8; H-5 with H-9; H-6 with H-8. From these observations, structure 3 was elucidated as shown in Fig. 1. The molecular formula of 4 was assigned as C27H35NO11 by positive HRESIMS (m/z 572.2100 [M+Na]+, calcd for C27H35NO11Na, 572.2108). The 1H and 13C NMR spectra of 4 were quite similar to those of 3 in showing signals for a secoiridoid moiety and a nicotinate moiety. However, resonances due to a terminal methyl [dH 1.00 (3H, t, J = 7.5 Hz); dC 14.2], two methylenes [dH 1.78 (2H, m), 1.49 (2H, m); dC 32.2, 20.3] and an oxygen-bearing methylene [dH 4.37 (2H, t, J = 6.5 Hz); dC 65.2] were observed instead of the methoxy signal. 1H–1H COSY analysis indicated the presence of a
425
n-butyloxy group, which was shown to be connected at C-700 from the HMBC correlation of H-100 0 with C-700 . The NOESY correlation of H-1 with H-6 and H-8, and of H-5 and H-9 indicated that the relative stereochemistry of 4 was identical with that of 3. Thus, structure 4 was assigned as shown in Fig. 1. 2.2. Biological evaluation Compounds 3 and 6–10 were evaluated for their inhibitory effect on the growth of influenza A/PR/8/34 by plaque assays at 100 lg/mL, while all tested compounds showed little cytotoxicity (IC50 > 100 lg/mL) against MDCK cells in the cytotoxicity assay. As shown in Table 3, secoxyloganin (8) had the most potent inhibitory activity against influenza A virus replication by 53%, while dimethylsocolologanoside (9) also showed a similar level of antiinfluenza activity (49.3% inhibition). Other secoiridoid derivatives, except for 3, showed weak to moderate inhibitory activity ranging from 53.1 to 28.4%. In contrast, no iridoid derivatives displayed influenza A neuraminidase inhibitory activity. The anti-influenza activity of Yin Qiao San (Kobayashi et al., 1999) as well as its constituents, including glycyrrhizin (Michaelis et al., 2010), arctiin and arctigenin (Hayashi et al., 2010), were reported previously, whereas there have been no reports on the antiinfluenza activity of the flower buds of L. japonica. The flower buds of L. japonica also contained rutin and caffeoyl quinic acids, whose inhibitory activity was also reported (Jeong et al., 2009; Kashiwada et al., 2012). Although the anti-influenza activity of the secoiriodoid glucosides was weak, they might also play, together with rutin and caffeoyl quinic acids of the flower buds of L. japonica, a role in the anti-influenza activity of Yin Qiao San. 3. Conclusions Four new secoiridoid derivatives, including two novel conjugates with a phenolic glucoside (1 and 2) and two conjugates with a nicotinic acid derivative (3 and 4), together with seven known secoiridoid derivatives were isolated from the flower buds of L. japonica. Their structures were elucidated by extensive spectroscopic analyses. Compounds 1 and 2 are structurally unique conjugates of a secoiridoid glucoside with a phenolic glucoside, in which a 60 -O-malonyl moiety of the phenolic glucoside was considered to be involved in conjugation. In contrast, pyridinium alkaloid coupled secoiridoids similar to 3 and 4 were previously isolated from the flower buds of L. japonica (Song et al., 2008; Yu et al., 2011). In an evaluation of inhibitory effect on the growth of influenza A/PR/8/34 by plaque assay, secoxyloganin (8) and dimethylsecolologanoside (9) showed inhibitory activity against influenza A virus replication by ca. 50% at non-cytotoxic concentration (100 lg/mL), while a weak inhibitory activity was found in the other evaluated secoiridoid derivatives, except for 3. However, none of the iridoid derivatives displayed influenza A neuraminidase inhibitory activity. 4. Experimental 4.1. General experimental procedures
Fig. 3. Key COSY and HMBC correlations in compound 3.
Optical rotations were measured on a JASCO P-2200 polarimeter. NMR spectra were recorded on a Bruker AVANCE-400 instrument (1H NMR: 400 MHz, 13C NMR: 100 MHz) and a Bruker AVANCE-500 instrument (1H NMR: 500 MHz, 13C NMR: 125 MHz) using TMS as an internal standard. HRESIMS were obtained on a Waters LCT Premier. CD spectra were acquired on a CD-J600 spectropolarimeter (JASCO). Column chromatography (CC) was performed using silica gel 60 N (63–210 lm, Kanto Kagaku),
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Sephadex LH-20 (25–100 lm, GE Healthcare Bioscience), YMCpack ODS-A (S-50 lm, YMC Co., Ltd), MCI gel CHP20P (75–150 lm, Mitsubishi Chemical Corporation), and Toyopearl HW-40C (TOSHO). HPLC was carried out using on a JASCO apparatus consisting of a PU-980 prep pump, UV-970UV/VIS (at wavelength 280 nm), and Mightysil RP-18 GP (Kanto Chemical, 5 lm, 20 i.d. 250 mm, nacalai tesque), or Cosmosil 5C18-AR-II (5 lm, 20 i.d. 250 mm, nacalai tesque), while analyses of sugar moieties were carried out on an HPLC consisting of a PU-2089 plus pump, RI-2031 plus, OR-2090 plus, CO-2065 plus, and Capcell Pak NH2 SG80 (4.6 mm i.d. 250 mm, 5 lm, Shiseido, Tokyo, Japan). TLC was conducted on precoated silica gel 60 F254 (0.20 mm, Merck) and spots were detected by UV illumination and by spraying with 10% sulfuric acid reagent followed by heating. 4.2. Extraction and Isolation of compounds Flower buds of L. japonica (10.0 kg), purchased from Tochimoto Tenkaido Co., Ltd. (Lot. 028909003), were extracted with MeOH (36 L, 5) at room temperature. After removal of solvent, the MeOH extract (2.8 kg) was successively partitioned between EtOAc, n-BuOH and H2O. The EtOAc-soluble fraction was further partitioned between n-hexane and 90% aqueous MeOH to give a n-hexane-soluble fraction and a 90% MeOH-soluble fraction. The H2O-soluble fraction (1.74 kg) was subjected to Diaion HP-20 chromatography with H2O containing increasing amounts of MeOH (1:0 ? 0:1) as eluent to give nine fractions (W-F1–W-F9). Half (24.7 g) of fraction W-F2 (48.2 g) was subjected to over YMCODS-A CC [H2O–MeOH (1:0 ? 0:1)] to afford seven fractions (W-F2.1–2.7). Toyopearl HW-40C CC [H2O-MeOH (1:0 ? 0:1)] of W-F2.3 (3.9 g) yielded four further fractions (W-F2.3.1–2.3.4). An aliquot of (215 mg) of W-F2.3.4 (1.42 g) was then separated by Mightysil RP-18 on HPLC [MeOH-2%AcOH (3:7)] and then purified by silica gel CC [CHCl3–MeOH–H2O (10:1:01 ? 8:2:0.2)] to furnish 6 (20 mg) and 7 (15 mg). W-F2.5 (6.0 g) was applied to a Toyo pearl HW-40C CC [H2O–MeOH (1:0 ? 0:1)] to give four fractions (W-F2.5.1–2.5.4). An aliquot of (470 mg) of W-F2.5.4 (2.26 g) was further separated by silica gel CC [CHCl3–MeOH–H2O (10:1:01 ? 8:2:0.2)] yielding 8 (22 mg), 9 (8 mg), and 10 (26 mg), respectively. W-F9 (6.7 g) was applied to a Sephadex LH-20 column with EtOH as eluant to furnish five fractions (W-F9.1–9.5). An aliquot of (2.5 g) of W-F9.2 (3.4 g) was applied to over Toyo pearl HW-40C column [H2O–MeOH (1:0 ? 0:1)] to give eight fractions (W-F9.2.1–9.2.8). W-F9.2.3 (339 mg) was applied to YMC-ODS-A CC [H2O–MeOH (1:0 ? 0:1)] giving twelve fractions (frs. 9.2.3.1–9.2.3.12). W-F9.2.3.8 (35 mg) and W-F9.2.3.10 (45.6 mg) were separately purified by COSMOSIL 5C18-AR-II on HPLC [MeOH-2%AcOH (45:55)] to furnish 3 (10.8 mg) and 5 (3.5 mg), respectively. Sephadex LH-20 CC [H2O-MeOH (4:6 ? 0:1)] of W-F9.2.3.11 (121 mg), followed by purification with COSMOSIL 5C18-AR-II [MeOH–2%AcOH (1:1)] on HPLC yielded 2 (4.5 mg), together with a fraction containing 1, which was further purified by silica gel CC [EtOAc–MeOH–H2O (30:1:0 ? 50:3:2)] to give 1 (3.3 mg). YMC-ODS-A chromatography [H2O–MeOH (1:0 ? 0:1)] of W-F9.2.4 (351 mg), followed by purification with COSMOSIL 5C18-AR-II [MeOH–2%AcOH (3:2)] on HPLC yielded 4 (0.9 mg). W-F9.2.2 (580 mg) was separated by YMC-ODS-A [H2OMeOH (1:0 ? 0:1)] chromatography giving eight fractions (W-F9.2.2.1–9.2.2.8). W-F9.2.2.2 and W-F9.2.2.5 were separately purified by Mightysil RP-18 on HPLC [MeOH–H2O (3:7)] to give 10 (5.0 mg) and 11 (4.7 mg), respectively. 4.3. Lonicerjaponin A (1) White amorphous powder; [a]D 72.3 (MeOH, c 0.31); For 1H (500 MHz, CD3OD) and 13C (125 MHz, CD3OD), NMR spectroscopy,
see Table 1; HRESIMS: m/z 737.2680 [MH] (calcd for C35H45O17, 737.2657). 4.4. Lonicerjaponin B (2) Off-white amorphous powder; [a]D 53.3 (MeOH, c 0.33); For H (500 MHz, CD3OD) and 13C (125 MHz, CD3OD) NMR spectroscopy, see Table 1; HRESIMS: m/z 747.2477 [M+Na]+ (calcd for C34H44O17Na, 747.2476). 1
4.5. Compound (3) Pale brown amorphous powder; [a]D 110.8 (MeOH, c 0.92); For 1H (500 MHz, CD3OD) and 13C (125 MHz, CD3OD) NMR spectroscopy, see Table 2; HRESIMS: m/z 530.1616 [M+Na]+ (calcd for C24H29NO11Na, 530.1638). 4.6. Compound (4) Pale brown amorphous powder; [a]D 25.0 (MeOH, c 0.08); 1H (500 MHz, CD3OD) and 13C (125 MHz, CD3OD) NMR spectroscopy, see Table 2; HRESIMS: m/z 572.2100 [M+Na]+ (calcd for C27H35NO11Na, 572.2108). 4.7. Acid hydrolysis of 1–3 A solution of each sample (1 mg each) in 5% H2SO4 (1 mL) was heated at 70C for 2 h. The reaction mixture was neutralized with 5% NaOH solution, and concentrated under reduced pressure. The residue was partitioned between EtOAc and H2O, and the H2O-soluble fraction was analyzed by HPLC [column, Capcell Pak NH2 SG80 (4.6 mm i.d.250 mm, 5 lm, Shiseido, Tokyo, Japan); solvent, CH3CN-H2O (17:3); flow rate, 0.75 mL/min; column temperature: 35 °C; detection, OR]. The sugar moiety was identified as D-glucose in each case by comparison of its retention time and sign of optical rotation with those of an authentic sample. [tR: 16 min, optical rotation (+)]. 4.8. Viruses and cells Madin-Darby canine kidney (MDCK) cells were routinely passaged in the DMED supplemented with 10% FBS and 100 lg/mL kanamycin. Virus-infected cells were maintained in DMEM supplemented 0.5% bovine serum albumin. Influenza viruses were propagated in MDCK cells. Influenza A/PR/8/34 (H1N1) was purchased from the American Type Culture Collection (ATCC; Manassas, Va). 4.9. Cell viability by MTT assay Cell viability was determined by a colorimetric assay using MTT (Hussain et al., 1993). In the mitochondria of living cells, yellow MTT undergoes a reductive conversion to formazan, giving a purple color. Confluent MDCK cells in 96-well plates were treated with various concentrations of each test compound, which were dissolved in small amounts of DMSO and diluted in the appropriate culture medium (final concentration of DMSO < 0.5%), for 24 h (Furuta et al., 2002). The MTT solution (dissolved in phosphate buffer) was then added (final concentration 0.5 mg/mL), and the cells were further incubated at 37 °C for 1 h. After removal of the culture medium, 200 lL acidified isopropanol was added to dissolve the formazan formed by the reduction of MTT. The absorbance at 570 nm was measured using microplate reader. IC50 values are defined as the concentration of each test samples that reduced absorbance to 50% of vehicle-treated controls.
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Y. Kashiwada et al. / Phytochemistry 96 (2013) 423–429 Table 1 H and 13C NMR spectroscopic data for Lonicerjaponins A (1) and B (2) in CD3OD.
1
Position
1
2
dHa 1 2 3 4 5 6 7 8 9 10
7.52 (1H, s) 3.35 (1H, m) 5.53 (1H, br d, 15.4) 5.50 5.68 2.56 5.19 5.16
(1H, (1H, (1H, (1H, (1H,
br d, 15.4) ddd, 17.1, 10.3, 8.5) ddd, 8.5, 8.5, 5.5) br d, 17.5) br d, 10.5)
5.56 (1H, d, 1.5)
98.0
154.2 109.4 39.5 133.7
7.60 (1H, d, 2.5)
154.4 105.2 28.1 30.8
126.4 135.8 46.3 118.9 168.8 38.3
13 11-OMe
3.64 (3H, s)
173.2 51.8
200 -OMe 100 0 200 0 300 0 400 0 500 0 600 0
4.70 3.18 3.29 3.27 3.35 3.88 3.66
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 8.0) dd, 9.0, 8.0) m) m) m) dd, 11.7, 1.4) m)
6.83 (1H, d, 1.8)
7.00 6.71 3.32 5.94 5.07 5.03 3.83
(1H, (1H, (2H, (1H, (1H, (1H, (3H,
d, 8.2) dd, 8.2, 1.8) m) ddd, 17.3, 10.2, 6.7) br d, 17.3) br d, 10.2) s)
4.80 3.49 3.45 3.34 3.56 4.38 4.24
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 7.5) dd, 9.0, 7.5) t, 9.0) m) m) dd, 11.8, 2.0) dd, 11.8, 6.7)
d Cb
97.5
3.05 (2H, m)
100 200 300 400 500 600 700 800 900
a
5.47 (1H, d, 8.2)
dHa
11 12
10 20 30 40 50 60
b
dC
b
100.3 74.7 78.3 71.5 78.0 62.8 136.6 114.2 150.9 146.1 118.5 121.9 40.8 139.0 116.0 56.7 103.0 74.9 77.7 71.7 75.4 64.9
3.22 2.65 1.94 4.84 5.50 2.71 5.25 5.24
(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,
m) ddd, 13.2, 13.2, 12.0) brdd, 12.0, 3.5) m) dt, 17.0, 10.0) m) d, 17.0) d, 10.0)
2.76 (1H, dd, 16.0, 7.3) 2.65 (1H, dd, 16.0, 5.9)
77.1 133.2 43.6 121.0 168.0 41.6 171.1
4.68 3.21 3.37 3.39 3.30 3.89 3.67
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 8.0) 9.0, 8.0) m) m) m) dd, 12.0, 2.0) dd, 12.0, 5.0)
6.83 (1H, d, 2.0)
7.01 6.70 3.27 5.94 5.07 5.03 3.83
(1H, (1H, (2H, (1H, (1H, (1H, (3H,
d, 8.0) dd, 8.0, 2.0) m) ddd, 17.0, 10.0, 6.5) br d, 17.0) br d, 10.0) s)
4.81 3.49 3.46 3.28 3.57 4.50 4.26
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 8.0) dd, 9.0, 8.0) t, 9.0) m) m) dd, 12.0, 2.0) dd, 12.0, 6.0)
99.7 74.6 78.4 71.5 77.9 62.7 136.7 114.2 150.9 146.1 118.6 121.9 40.8 139.0 116.0 56.7 103.0 74.9 77.8 71.5 75.3 64.5
dH ppm (mult., J in Hz), 500 MHz. dC ppm, 125 MHz.
4.10. In vitro antiviral assay Confluent MDCK cells grown in 6-well tissue culture plates were inoculated with 1 105 PFU of virus. After adsorption at 37 °C for 1 h, the inoculum was removed. The cells were washed twice with phosphate-buffered saline (pH 7.4) to overlay with DMEM containing 5 mg/mL trypsin (Wako Pure Chemical Industries, Co., Ltd.) and various concentrations of tested samples. The plates were incubated at 37 °C in 5% CO2 for 18 h, and the titer of virus harvested from the cultures was determined in MDCK cells by the plaque assay. 4.11. Plaque assay The plaque assay was performed essentially as previously reported (Yen et al., 2005). Confluent MDCK cells grown in 6-well tissue culture plates were inoculated with appropriate dilutions of virus. After adsorption at 37 °C for 1 h, the inoculum was removed. The cells were washed twice with phosphate-buffered saline to overlay with DMEM containing 1% agarose and 5 mg/mL trypsin.
The plaques were visualized after incubation at 37 °C in 5% CO2 for 3 days by staining 0.5% crystal violet solution containing 3.7% formaldehyde. Oseltamivir carboxylate, which was prepared by hydrolysis of oseltamivir phosphate (obtained from Tamiflu capsules 75, Chugai Pharmaceutical Co., Ltd., Tokyo, Japan) as previously described (Yamanaka et al., 2006), was used as a positive control. 4.12. Influenza A neuraminidase inhibition assay A modified fluorometric assay using the fluorogenic substrate 20 -(4-methylumbellyferyl)-a-D-N-acetylneuraminic acid (MUNANA, Sigma) was used to determine the NA activity on the type A (H1N1) viruses (Ullah et al., 1999). All compounds were dissolved in DMSO and diluted to the corresponding concentrations in PBS. A 96-well plate containing a mixture of the diluted virus suspension (1 105 PFU, 50 lL) and the different concentration of compound solution (50 lL) was incubated on ice for 1 h. MUNANA substrate solution (0.2 mM in 0.1 M acetate buffer (pH 4.6), 25 lL) was added and the mixture was incubated for 30 min at 37 °C. The
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Table 2 H and 13C NMR spectroscopic data for compounds 3 and 4 in CD3OD.
1
Position
3
4
dHa 1 2 3 4 5 6 7 8 9 10
5.61 (1H, d, 7.5) 7.62 (1H, s) 3.62 6.40 6.50 5.75 2.70 5.26 5.22
11 11-OMe
4.75 3.21 3.38 3.26 3.39 3.87 3.63
100 200 300 400 500 600 700
m) dd, 16.0, 8.5) d, 16.0) ddd, 17.5, 10.5, 1.5) dd, 8.5) d, 17.5) d, 10.5)
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 7.6) dd, 8.9, 7.6) t, 8.9) t, 8.9) m) dd, 12.0, 1.5) dd, 12.0, 6.0)
8.91 (1H, s) 8.33 (1H, br s) 8.70 (1H, br s)
100 0 200 0 300 0 400 0 a
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
3.67 (3H, s)
10 20 30 40 50 60
b
dC
3.94 (3H, s)
b
dHa
97.5 154.5 109.0 39.8 134.7 128.8 135.4 46.5 119.4 168.6 51.9
d Cb
5.61 (1H, d, 7.5) 7.64 (1H, s) 3.63 6.41 6.51 5.76 2.72 5.27 5.22
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
m) dd, 15.5, 8.0) d, 15.5) ddd, 17.0, 10.5, 2.0) m) d, 17.0) d, 10.5)
3.68 (3H, s)
100.3 74.7 78.4 71.6 78.0 62.7
4.74 3.20 3.37 3.32 3.31 3.87 3.62
149.3 127.8 135.0 135.2 152.0 166.8
8.92 (1H, br s)
53.0
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 8.0) dd, 9.0, 8.0) t, 9.0) t, 9.0) m) dd, 11.5, 2.0) dd, 12.0, 6.0)
8.33 (1H, br s) 8.72 (1H, s) 4.37 1.78 1.49 1.00
(1H, (1H, (1H, (1H,
t, 6.5) q, 7.0, 6.5) ddd, 7.5, 7.0) t, 7.5)
97.3 152.0 109.8 40.3 134.9 127.7 135.3 46.2 120.9 168.5 51.4 100.1 73.2 77.1 70.3 76.7 61.2
147.6 125.3 133.7 135.6 150.5 166.7 65.2 32.2 20.3 14.2
dH ppm (mult., J in Hz), 500 MHz. dC ppm, 125 MHz.
Table 3 Effect of compounds on influenza A/PR/8/34 replication.a
a b
Compound
Inhibition at 100 lg/mL (%)
3 6 7 8 9 10 Oseltamivir carboxylateb
14.3 ± 2.7 37.2 ± 2.1 44.0 ± 3.6 53.1 ± 3.9 49.3 ± 3.2 28.4 ± 2.4 45.5 ± 2.3
Data are mean ± SE from three experiments. Oseltamivir carboxylate was tested at 0.1 lg/mL.
enzymatic reaction was quenched by adding 100 lL of glycine– NaOH buffer solution (pH 10.7). The fluorescence intensity of the product (4-methylumbellyferone) was measured in a spectrophotometer with excitation and emission wavelengths of 360 and 440 nm, respectively. The drug concentrations required to inhibit 50% of the NA activity (IC50) were determined by plotting the percent inhibition of NA activity as a function of the drug concentrations (Shi et al., 2007). Oseltamivir carboxylate was used as a positive control, which inhibited influenza A neuraminidase with an IC50 value of 0.005 ± 0.001 lg/mL.
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Furuta, Y., Takahashi, K., Fukuda, Y., Kuno, M., Kamiyama, T., Kozaki, K., Nomura, N., Egawa, H., Minami, S., Watanabe, Y., Narita, H., Shiraki, K., 2002. In vitro and in vivo activities of anti-influenza virus compound T-705. Antimicrob. Agents Chemother. 16, 977–981. Hayashi, K., Narutaki, K., Nagaoka, Y., Hayashi, T., Uesato, S., 2010. Therapeutic effect of arctiin and arctigenin in immunocompetent and immunocompromised mice infected with influenza A virus. Biol. Pharm. Bull. 33, 1199–1205. Hussain, R.F., Nouri, A.M., Oliver, R.T., 1993. A new approach for measurement of cytotoxicity using colorimetric assay. J. Immunol. Methods 160, 89–96. Jeong, H.J., Ryu, Y.B., Park, S.-J., Kim, J.H., Kwon, H.-J., Kim, J.H., Park, H.H., Rho, M.-C., Lee, W.S., 2009. Neuraminidase inhibitory activities of flavonols isolated from Rhodiola rosea roots and their in vitro anti-influenza viral activities. Bioorg. Med. Chem. 17, 6816–6823. Kashiwada, Y., Ahmed, F.A., Kurimoto, S., Kim, S.Y., Shibata, H., Fujioka, T., Takaishi, Y., 2012. New a-glucosides of caffeoyl quinic acid from the leaves of Moringa oleifera Lam. J. Nat. Med. 66, 217–221. Kobayashi, M., Davis, S.M., Utsunomiya, T., Pollard, R.B., Suzuki, F., 1999. Antiviral effect of Gingyo-san, a traditional chinese herbal medicine, on influenza A2 virus infection in mice. Am. J. Chinese Med. 27, 53–62. Kurokawa, M., Yamamura, J., Li, Z., Sato, H., Hitomi, N., Tatsumi, Y., Shiraki, K., 1998. Antipyretic activity of Gingyo-san, a traditional medicine, in influenza virusinfected mice. Chem. Pharm. Bull. 46, 1444–1447. Machida, K., Asano, J., Kikuchi, M., 1994. Caeruleosides A and B, bis-iridoid glucosides from Lonicera caerulea. Phytochemistry 39, 111–114. Michaelis, M., Geiler, J., Naczk, P., Sithisarn, P., Ogbomo, H., Altenbrandt, B., Leutz, A., Doerr, H.W., Cinatl Jr., J., 2010. Glycyrrhizin inhibits highly pathogenic H5N1 influenza A virus-induced pro-inflammatory cytokine and chemokine expression in human macrophages. Med. Microbiol. Immun. 199, 291–297. Shanghai People’s Publishing House, 1977. In: Jiangsu New Medical College (Ed.), Dictionary of Traditional Chinese Medicine, Shanghai, pp. 1403–1405. Shi, S.Y., Zhou, Q., Peng, H., Zhou, C.X., Hu, M.H., Tao, Q.F., Hao, X.J., Stöckigt, J., Zhao, Y., 2007. Four new constituents from Taraxacum mongolicum. Chin. Chem. Lett. 18, 1367–1370. Shibata, H.; Higuti, T.; Kirimoto, T., 2010. Antiviral agent. Jpn. Kokai Tokkyo Koho, JP-2010-83758A. Song, W., Li, S., Wang, S., Wu, Y., Zi, J., Gan, M., Zhang, Y., Liu, M., Lin, S., Yang, Y., Shi, J., 2008. Pyridinium Alkaloid-Coupled Secoiridoids from the Flower Buds of Lonicera japonica. J. Nat. Prod. 71, 922–925. Takeda, Y., Masuda, T., Honda, G., Takaishi, Y., Ito, M., Ashurmetov, O.A., Khodzhimatov, O.K., Otsuka, H., 1999. Secoiridoid glycosides from Gentiana olivieri. Chem. Pharm. Bull. 47, 1338–1340.
Y. Kashiwada et al. / Phytochemistry 96 (2013) 423–429 Ullah, N., Ahmed, S., Muhammad, P., Ahmed, Z., Nawaz, H.R., Malik, A., 1999. Coumarinolignoid glycoside from Daphne oleoides. Phytochemistry 51, 103– 105. Yamanaka, T., Tsujimura, K., Kondo, T., Matsumura, T., 2006. In vitro efficacies of oseltamivir carboxylate and zanamivir against equine influenza A virus. J. Vet. Med. Sci. 68, 405–408. Yen, H.-L., Herlocher, L.M., Hoffmann, E., Matrosovich, M.N., Monto, A.S., Webstrer, R.G., Govorkova, E., 2005. A. Neuraminidase inhibitor-resistant influenza viruses
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may differ substantially in fitness and transmissibility. Antimicrob. Agents Chemother. 49, 4075–4080. Yu, Y., Song, W., Zhu, C., Lin, S., Zhao, F., Wu, X., Yue, Z., Liu, B., Wang, S., Yuan, S., Hou, Q., Shi, J., 2011. Homosecoiridoids from the Flower Buds of Lonicera japonica. J. Nat. Prod. 74, 2151–2160. Zheng, Z.F., Zhang, Q.J., Chen, R.Y., Yu, D.Q., 2012. Four new N-contained iridoido glycosides from flower buds of Lonicera japonica. J. Asian Nat. Prod. Res. 14, 729–737.
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Conjugates of a secoiridoid glucoside with a phenolic glucoside from the flower buds of Lonicera japonica Thunb Yoshiki Kashiwada a,⇑, Yuka Omichi a, Shin-ichiro Kurimoto a, Hirofumi Shibata a, Yoshiyuki Miyake b, Tsukasa Kirimoto b, Yoshihisa Takaishi a a b
Graduate School of Pharmaceutical Sciences, University of Tokushima, Shomachi 1-78, Tokushima 770-8505, Japan Consumer Healthcare, S.H. Product Strategy Planning, Product Research, Taiho Pharmaceutical Co., Ltd., 224-2 Ebisuno, Kawauchicho, Tokushima 771-0194, Japan
a r t i c l e
i n f o
Article history: Received 25 June 2013 Received in revised form 13 September 2013 Available online 10 October 2013
a b s t r a c t Secoiridoid glucosides, including two conjugates with a phenolic and two conjugates with a nicotinic acid derivative (3 and 4), together with seven known secoiridoid derivatives, were isolated from flower buds of Lonicera japonica. The structures were elucidated by spectroscopic analyses. Anti-influenza activities of six isolated compounds were also evaluated by plaque assay and neuraminidase inhibitory assay. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Lonicera japonica Caprifoliaceae Secoiridoid Nicotonic acid Eugenol Anti-influenza
1. Introduction
2. Results and discussion
Lonicera japonica Thunb. (Caprifoliaceae), also known as Japanese Honeysuckle, is a perennial twining woody vine native to the East Asia. Its flower buds have been used since early times for treatment of arthritis, diabetes mellitus, fever, infections, sores, and swelling (Shanghai Science and Technology Press, 1977). Its flower buds are also one of the major ingredients in Yin Qiao San, a most popular prescription in traditional Chinese medicine, used for common colds, including fever, headache, cough, thirst, and sore throat, and for influenza infection (Kurokawa et al., 1998). In evaluation of the anti-influenza activity of Yin Qiao San in vitro, the H2O extract of Yin Qiao San inhibited all subtypes of influenza A, B, and C virus replication, and also exhibited inhibitory activity against an oseltamivir-resistant influenza A virus (Shibata et al., 2010). Based on this finding, the MeOH extract of flower buds of L. japonica were investigated. This resulted in isolation of four new secoiridoid glucoside derivatives including two novel conjugates with a phenolic glucoside (1 and 2), and two conjugates with a nicotinic acid derivative (3 and 4), together with seven known compounds. Described herein are the isolation and structure elucidation of these compounds, as well as evaluation of their inhibitory activity against influenza A virus replication and neuraminidase.
2.1. Structure elucidation and identification
⇑ Corresponding author. Tel./fax: +81 88 633 7276. E-mail address: [email protected] (Y. Kashiwada). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.09.021
Flower buds of L. japonica (10.0 kg) were extracted five times with MeOH at room temperature. After removal of the solvent, the MeOH extract was successively partitioned between EtOAc, n-BuOH and H2O. The EtOAc-soluble fraction was further partitioned between n-hexane and 90% aqueous MeOH giving a n-hexane-soluble fraction and 90% MeOH-soluble fraction. In contrast, the H2O-soluble fraction was subjected to Diaion HP-20 chromatography with H2O containing increasing amounts of MeOH to give nine fractions (W-F1–W-F9). The n-hexane-, 90% MeOH-, and n-BuOH-soluble fractions, as well as the fractions obtained from Diaion HP-20 chromatography, were then evaluated for their inhibitory effects on the growth of influenza A/PR/8/34 by plaque assay, as well as for influenza A neuraminidase inhibitory activity. Anti-influenza activities were found in both the n-hexane-soluble fraction and fractions W-F2–W-F9 (data not shown). Fractions obtained from the Diaion HP-20 column chromatography were further separated by repeated Sephadex LH-20, MCI gel CHP-20P, Toyopearl HW-40C, and YMC ODS-A, column chromatography, and purified by reversed-phase HPLC to yield 1 (3.5 mg), 2 (3.3 mg), 3 (9.2 mg), and 4 (0.9 mg), respectively, together with seven known compounds (5–11). The latter were identified as L-phenylalaninosecologanin B (5) (Zheng et al., 2012), vogeloside
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(6) (Boros and Stermitz, 1991), 7-epi-vogeloside (7) (Boros and Stermitz, 1991), secoxyloganin (8) (Calis and Sticher, 1984), dimethyl secologanoside (9) (Calis and Sticher, 1984), sweroside (10) (Takeda et al., 1999), and secologanin dimethylacetal (11) (Machida et al., 1994), respectively, by comparison of their spectroscopic data with those reported in the literature (Fig. 1). Lonicerjaponin A (1) was obtained as a white amorphous powder. Its molecular formula was determined as C35H46O17 by HRESIMS (m/z 737.2680 [MH], calcd for C35H45O17, 737.2657). The 1 H NMR spectrum of 1 had signals similar to those of secoxyloganin (8) in teams of the presence of a tri-substituted olefin [dH 7.52 (1H, s)], a vinyl group [dH 5.68 (1H, ddd, J = 17.1, 10.3, 8.5 Hz), 5.19 (1H, br d, J = 17.5 Hz) and 5.16 (1H, br d, J = 10.5 Hz)], an acetal proton [dH 5.47 (1H, d, J = 8.2 Hz)] and a methoxy group [dH 3.64 (3H, s)], together with an anomeric signal [dH 4.70 (1H, d, J = 8.0 Hz)]. It also showed resonances ascribable to a 1,2,4-trisubstituted aromatic group [dH 7.00 (1H, d, J = 8.2 Hz), 6.83 (1H, d, J = 1.8 Hz), 6.71 (1H, dd, J = 8.2, 1.8 Hz)], a trans-olefin [dH 5.53 (1H, brd, J = 15.4 Hz), 5.50 (1H, brd, J = 15.4 Hz)], a vinyl group [dH 5.94 (1H, ddd, J = 17.3, 10.2, 6.7 Hz), 5.07 (1H, brd, J = 17.3 Hz), 5.03 (1H, brd, J = 10.2 Hz)] and a methoxy group [dH 3.83 (3H, s)], along with an anomeric signal [dH 4.80 (1H, d, J = 7.5 Hz)]. The 13C NMR spectrum displayed 35 carbon resonances, due to two ester carbonyls (dC 173.2 and 168.8) and two hexosyl moieties assignable to two glucosyl moieties. The sugar moiety was identified as D-glucose by direct HPLC analysis of the acid hydrolysate of 1 using an optical rotation detector. The structure of 1 was elucidated by 1H–1H COSY and HMBC spectroscopic analyses (Fig. 2). The 1H–1H COSY examination established the occurrence of a partial structure corresponding to H-1–H-9–H-8–H2-10, in which H-9 had correlations of a H-9–H5–trans-olefin; thus, the trans-olefin was assigned to H-6–H-7. The HMBC correlations of H-1 with C-3 and C-5, H-3 with C-4 and C-11, and of OMe with C-11, together with the HMBC correlation of the anomeric proton signal with H-1, indicated the presence of a secoiridoid moiety. In addition, H-7 exhibited a 1H–1H COSY correlation with a methylene proton (H2-12), which further displayed an HMBC correlation with an ester carbonyl carbon (dC 173.2), providing a 2-(secoloan-6-en-7-yl)-acetate moiety as a partial structure. In contrast, HMBC correlations of aromatic signals [dH 6.83 (1H, d, J = 1.8 Hz), 6.71 (1H, dd, J = 8.2, 1.8 Hz)] with an sp3 methylene carbon (dC 40.8), whose protons further exhibited a 1H–1H COSY correlation with a vinyl group, suggesting the presence of a phenylpropanoid moiety. Analysis of the following HMBC
Fig. 2. Key COSY and HMBC correlations in lonicerjaponin A (1).
correlations resulted in deduction of a 4-O-glucosyl-eugenol moiety: H-50 with C-30 and C-10 ; H-20 with C-40 ; OMe with C-300 ; H-100 with C-400 . These two moieties were connected through an ester linkage between C-600 0 and C-13 from the HMBC correlation of H2-6000 with C-13, and thus the structure of 1 was elucidated. Its relative configuration was elucidated by analysis of its NOESY spectrum. Thus, NOESY correlations of H-5 with H-9, as well as H-1 with H-8, indicated the relative configuration of 1 to be identical with secologanin. The b-linkages of both glucosyl moieties were concluded from the J-value of the anomeric proton signals. On the basis of this examination, its structure was characterized as shown in Fig. 1. The molecular formula of lonicerjaponin B (2) was assigned as C34H44O17 by positive HRESIMS (m/z 747.2477 [M+Na]+, calcd for C34H44O17Na, 747.2476). The 1H and 13C NMR spectra of 2 were similar to those of 1 except for the absence of signals of a methoxy and a trans-olefin. It also instead showed resonances ascribable to a methylene [dH 2.65 (1H, ddd, J = 13.2, 13.2, 12.0 Hz), 1.94 (1H, brdd, J = 12.0, 3.5 Hz); dC 30.8] and an oxygen-bearing sp3 methine group [dH 4.84 (1H, m); dC 77.1]. These signals were shown to be coupled to each other and were assigned to be H-6 and H-7, respectively, from the 1H–1H COSY correlation of H-5 with this methylene signal. Furthermore, H-7 showed a 1H–1H COSY correlation with H2-12, which further displayed a HMBC correlation with
Fig. 1. Structures of secoiridoid derivatives isolated from L. japonica.
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an ester carbonyl carbon (dC 171.1). The presence of a lactone ring between C-7 and C-11 was deduced, taking into account the chemical shift of H-7 (dH 4.84), as well as the degree of unsaturation of its molecular formula. Thus, the occurrence of a 7-carboxylmethylsweroside moiety was elucidated. This moiety was shown to be located at the glucosyl C-6 position of the 4-O-glucosyl-eugenol moiety from the HMBC correlation of H-6000 with C-13. The identification of D-glucose was carried out by direct HPLC analysis of its hydrolysate, and b-linkages were concluded from the J-values of the anomeric proton signals. The relative configuration of 2 was elucidated by analysis of its NOESY spectrum, in which NOESY correlations of H-5 with H-7 as well as H-9 indicated that the relative configuration of 2 was the same as that of sweroside (7). On the basis of these observations, the structure of 2 was assigned as shown in Fig. 1. Compound 3 was obtained as an off-white amorphous powder. Its molecular formula was determined as C24H29NO11 by HRESIMS (m/z 530.1616 [M+Na]+, calcd for C24H29NO11Na, 530.1638). The 1H and 13C NMR spectra of 3 were also correlated to those of 1, except for the absence of the signals due to the 4-O-glucosyl-eugenol moiety. 2D NMR examinations established that the presence of a secoloan-6-en-7-yl moiety (Fig. 3). In addition, the 1H NMR spectrum also showed three aromatic signals [dH 8.91 8.70, 8.33 (each 1H, br s)] and a methoxy resonance [dH 3.94 (3H, s)], while the 13C NMR spectrum displayed seven carbon signals, including one ester carbonyl (dC 166.8), three sp2 methines (dC 152.0, 149.3, and 135.0), two sp2 quaternary carbons (dC 135.2 and 127.8) and a methoxy carbon (dC 53.0), except for the resonances arising from the secoloan-6-en-7-yl moiety. The presence of a D-glucosyl moiety was confirmed by direct HPLC analysis of its hydrolysate. The HMBC correlations of H-400 with C-200 , C-600 and C-700 , H-200 with C-300 , and of OMe with C-700 indicated the presence of a C-5 substituted methyl nicotinate moiety. In addition, the secoloan-6-en-7-yl moiety was shown to be connected to C-5 of a methyl nicotinate moiety from the HMBC correlation of H-400 with C-7 and of H-7 with C-400 and C-600 . The following NOESY correlations indicated that the relative stereochemistry of 3 was identical with that of secologanin: H-1 with H-6 and H-8; H-5 with H-9; H-6 with H-8. From these observations, structure 3 was elucidated as shown in Fig. 1. The molecular formula of 4 was assigned as C27H35NO11 by positive HRESIMS (m/z 572.2100 [M+Na]+, calcd for C27H35NO11Na, 572.2108). The 1H and 13C NMR spectra of 4 were quite similar to those of 3 in showing signals for a secoiridoid moiety and a nicotinate moiety. However, resonances due to a terminal methyl [dH 1.00 (3H, t, J = 7.5 Hz); dC 14.2], two methylenes [dH 1.78 (2H, m), 1.49 (2H, m); dC 32.2, 20.3] and an oxygen-bearing methylene [dH 4.37 (2H, t, J = 6.5 Hz); dC 65.2] were observed instead of the methoxy signal. 1H–1H COSY analysis indicated the presence of a
425
n-butyloxy group, which was shown to be connected at C-700 from the HMBC correlation of H-100 0 with C-700 . The NOESY correlation of H-1 with H-6 and H-8, and of H-5 and H-9 indicated that the relative stereochemistry of 4 was identical with that of 3. Thus, structure 4 was assigned as shown in Fig. 1. 2.2. Biological evaluation Compounds 3 and 6–10 were evaluated for their inhibitory effect on the growth of influenza A/PR/8/34 by plaque assays at 100 lg/mL, while all tested compounds showed little cytotoxicity (IC50 > 100 lg/mL) against MDCK cells in the cytotoxicity assay. As shown in Table 3, secoxyloganin (8) had the most potent inhibitory activity against influenza A virus replication by 53%, while dimethylsocolologanoside (9) also showed a similar level of antiinfluenza activity (49.3% inhibition). Other secoiridoid derivatives, except for 3, showed weak to moderate inhibitory activity ranging from 53.1 to 28.4%. In contrast, no iridoid derivatives displayed influenza A neuraminidase inhibitory activity. The anti-influenza activity of Yin Qiao San (Kobayashi et al., 1999) as well as its constituents, including glycyrrhizin (Michaelis et al., 2010), arctiin and arctigenin (Hayashi et al., 2010), were reported previously, whereas there have been no reports on the antiinfluenza activity of the flower buds of L. japonica. The flower buds of L. japonica also contained rutin and caffeoyl quinic acids, whose inhibitory activity was also reported (Jeong et al., 2009; Kashiwada et al., 2012). Although the anti-influenza activity of the secoiriodoid glucosides was weak, they might also play, together with rutin and caffeoyl quinic acids of the flower buds of L. japonica, a role in the anti-influenza activity of Yin Qiao San. 3. Conclusions Four new secoiridoid derivatives, including two novel conjugates with a phenolic glucoside (1 and 2) and two conjugates with a nicotinic acid derivative (3 and 4), together with seven known secoiridoid derivatives were isolated from the flower buds of L. japonica. Their structures were elucidated by extensive spectroscopic analyses. Compounds 1 and 2 are structurally unique conjugates of a secoiridoid glucoside with a phenolic glucoside, in which a 60 -O-malonyl moiety of the phenolic glucoside was considered to be involved in conjugation. In contrast, pyridinium alkaloid coupled secoiridoids similar to 3 and 4 were previously isolated from the flower buds of L. japonica (Song et al., 2008; Yu et al., 2011). In an evaluation of inhibitory effect on the growth of influenza A/PR/8/34 by plaque assay, secoxyloganin (8) and dimethylsecolologanoside (9) showed inhibitory activity against influenza A virus replication by ca. 50% at non-cytotoxic concentration (100 lg/mL), while a weak inhibitory activity was found in the other evaluated secoiridoid derivatives, except for 3. However, none of the iridoid derivatives displayed influenza A neuraminidase inhibitory activity. 4. Experimental 4.1. General experimental procedures
Fig. 3. Key COSY and HMBC correlations in compound 3.
Optical rotations were measured on a JASCO P-2200 polarimeter. NMR spectra were recorded on a Bruker AVANCE-400 instrument (1H NMR: 400 MHz, 13C NMR: 100 MHz) and a Bruker AVANCE-500 instrument (1H NMR: 500 MHz, 13C NMR: 125 MHz) using TMS as an internal standard. HRESIMS were obtained on a Waters LCT Premier. CD spectra were acquired on a CD-J600 spectropolarimeter (JASCO). Column chromatography (CC) was performed using silica gel 60 N (63–210 lm, Kanto Kagaku),
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Sephadex LH-20 (25–100 lm, GE Healthcare Bioscience), YMCpack ODS-A (S-50 lm, YMC Co., Ltd), MCI gel CHP20P (75–150 lm, Mitsubishi Chemical Corporation), and Toyopearl HW-40C (TOSHO). HPLC was carried out using on a JASCO apparatus consisting of a PU-980 prep pump, UV-970UV/VIS (at wavelength 280 nm), and Mightysil RP-18 GP (Kanto Chemical, 5 lm, 20 i.d. 250 mm, nacalai tesque), or Cosmosil 5C18-AR-II (5 lm, 20 i.d. 250 mm, nacalai tesque), while analyses of sugar moieties were carried out on an HPLC consisting of a PU-2089 plus pump, RI-2031 plus, OR-2090 plus, CO-2065 plus, and Capcell Pak NH2 SG80 (4.6 mm i.d. 250 mm, 5 lm, Shiseido, Tokyo, Japan). TLC was conducted on precoated silica gel 60 F254 (0.20 mm, Merck) and spots were detected by UV illumination and by spraying with 10% sulfuric acid reagent followed by heating. 4.2. Extraction and Isolation of compounds Flower buds of L. japonica (10.0 kg), purchased from Tochimoto Tenkaido Co., Ltd. (Lot. 028909003), were extracted with MeOH (36 L, 5) at room temperature. After removal of solvent, the MeOH extract (2.8 kg) was successively partitioned between EtOAc, n-BuOH and H2O. The EtOAc-soluble fraction was further partitioned between n-hexane and 90% aqueous MeOH to give a n-hexane-soluble fraction and a 90% MeOH-soluble fraction. The H2O-soluble fraction (1.74 kg) was subjected to Diaion HP-20 chromatography with H2O containing increasing amounts of MeOH (1:0 ? 0:1) as eluent to give nine fractions (W-F1–W-F9). Half (24.7 g) of fraction W-F2 (48.2 g) was subjected to over YMCODS-A CC [H2O–MeOH (1:0 ? 0:1)] to afford seven fractions (W-F2.1–2.7). Toyopearl HW-40C CC [H2O-MeOH (1:0 ? 0:1)] of W-F2.3 (3.9 g) yielded four further fractions (W-F2.3.1–2.3.4). An aliquot of (215 mg) of W-F2.3.4 (1.42 g) was then separated by Mightysil RP-18 on HPLC [MeOH-2%AcOH (3:7)] and then purified by silica gel CC [CHCl3–MeOH–H2O (10:1:01 ? 8:2:0.2)] to furnish 6 (20 mg) and 7 (15 mg). W-F2.5 (6.0 g) was applied to a Toyo pearl HW-40C CC [H2O–MeOH (1:0 ? 0:1)] to give four fractions (W-F2.5.1–2.5.4). An aliquot of (470 mg) of W-F2.5.4 (2.26 g) was further separated by silica gel CC [CHCl3–MeOH–H2O (10:1:01 ? 8:2:0.2)] yielding 8 (22 mg), 9 (8 mg), and 10 (26 mg), respectively. W-F9 (6.7 g) was applied to a Sephadex LH-20 column with EtOH as eluant to furnish five fractions (W-F9.1–9.5). An aliquot of (2.5 g) of W-F9.2 (3.4 g) was applied to over Toyo pearl HW-40C column [H2O–MeOH (1:0 ? 0:1)] to give eight fractions (W-F9.2.1–9.2.8). W-F9.2.3 (339 mg) was applied to YMC-ODS-A CC [H2O–MeOH (1:0 ? 0:1)] giving twelve fractions (frs. 9.2.3.1–9.2.3.12). W-F9.2.3.8 (35 mg) and W-F9.2.3.10 (45.6 mg) were separately purified by COSMOSIL 5C18-AR-II on HPLC [MeOH-2%AcOH (45:55)] to furnish 3 (10.8 mg) and 5 (3.5 mg), respectively. Sephadex LH-20 CC [H2O-MeOH (4:6 ? 0:1)] of W-F9.2.3.11 (121 mg), followed by purification with COSMOSIL 5C18-AR-II [MeOH–2%AcOH (1:1)] on HPLC yielded 2 (4.5 mg), together with a fraction containing 1, which was further purified by silica gel CC [EtOAc–MeOH–H2O (30:1:0 ? 50:3:2)] to give 1 (3.3 mg). YMC-ODS-A chromatography [H2O–MeOH (1:0 ? 0:1)] of W-F9.2.4 (351 mg), followed by purification with COSMOSIL 5C18-AR-II [MeOH–2%AcOH (3:2)] on HPLC yielded 4 (0.9 mg). W-F9.2.2 (580 mg) was separated by YMC-ODS-A [H2OMeOH (1:0 ? 0:1)] chromatography giving eight fractions (W-F9.2.2.1–9.2.2.8). W-F9.2.2.2 and W-F9.2.2.5 were separately purified by Mightysil RP-18 on HPLC [MeOH–H2O (3:7)] to give 10 (5.0 mg) and 11 (4.7 mg), respectively. 4.3. Lonicerjaponin A (1) White amorphous powder; [a]D 72.3 (MeOH, c 0.31); For 1H (500 MHz, CD3OD) and 13C (125 MHz, CD3OD), NMR spectroscopy,
see Table 1; HRESIMS: m/z 737.2680 [MH] (calcd for C35H45O17, 737.2657). 4.4. Lonicerjaponin B (2) Off-white amorphous powder; [a]D 53.3 (MeOH, c 0.33); For H (500 MHz, CD3OD) and 13C (125 MHz, CD3OD) NMR spectroscopy, see Table 1; HRESIMS: m/z 747.2477 [M+Na]+ (calcd for C34H44O17Na, 747.2476). 1
4.5. Compound (3) Pale brown amorphous powder; [a]D 110.8 (MeOH, c 0.92); For 1H (500 MHz, CD3OD) and 13C (125 MHz, CD3OD) NMR spectroscopy, see Table 2; HRESIMS: m/z 530.1616 [M+Na]+ (calcd for C24H29NO11Na, 530.1638). 4.6. Compound (4) Pale brown amorphous powder; [a]D 25.0 (MeOH, c 0.08); 1H (500 MHz, CD3OD) and 13C (125 MHz, CD3OD) NMR spectroscopy, see Table 2; HRESIMS: m/z 572.2100 [M+Na]+ (calcd for C27H35NO11Na, 572.2108). 4.7. Acid hydrolysis of 1–3 A solution of each sample (1 mg each) in 5% H2SO4 (1 mL) was heated at 70C for 2 h. The reaction mixture was neutralized with 5% NaOH solution, and concentrated under reduced pressure. The residue was partitioned between EtOAc and H2O, and the H2O-soluble fraction was analyzed by HPLC [column, Capcell Pak NH2 SG80 (4.6 mm i.d.250 mm, 5 lm, Shiseido, Tokyo, Japan); solvent, CH3CN-H2O (17:3); flow rate, 0.75 mL/min; column temperature: 35 °C; detection, OR]. The sugar moiety was identified as D-glucose in each case by comparison of its retention time and sign of optical rotation with those of an authentic sample. [tR: 16 min, optical rotation (+)]. 4.8. Viruses and cells Madin-Darby canine kidney (MDCK) cells were routinely passaged in the DMED supplemented with 10% FBS and 100 lg/mL kanamycin. Virus-infected cells were maintained in DMEM supplemented 0.5% bovine serum albumin. Influenza viruses were propagated in MDCK cells. Influenza A/PR/8/34 (H1N1) was purchased from the American Type Culture Collection (ATCC; Manassas, Va). 4.9. Cell viability by MTT assay Cell viability was determined by a colorimetric assay using MTT (Hussain et al., 1993). In the mitochondria of living cells, yellow MTT undergoes a reductive conversion to formazan, giving a purple color. Confluent MDCK cells in 96-well plates were treated with various concentrations of each test compound, which were dissolved in small amounts of DMSO and diluted in the appropriate culture medium (final concentration of DMSO < 0.5%), for 24 h (Furuta et al., 2002). The MTT solution (dissolved in phosphate buffer) was then added (final concentration 0.5 mg/mL), and the cells were further incubated at 37 °C for 1 h. After removal of the culture medium, 200 lL acidified isopropanol was added to dissolve the formazan formed by the reduction of MTT. The absorbance at 570 nm was measured using microplate reader. IC50 values are defined as the concentration of each test samples that reduced absorbance to 50% of vehicle-treated controls.
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Y. Kashiwada et al. / Phytochemistry 96 (2013) 423–429 Table 1 H and 13C NMR spectroscopic data for Lonicerjaponins A (1) and B (2) in CD3OD.
1
Position
1
2
dHa 1 2 3 4 5 6 7 8 9 10
7.52 (1H, s) 3.35 (1H, m) 5.53 (1H, br d, 15.4) 5.50 5.68 2.56 5.19 5.16
(1H, (1H, (1H, (1H, (1H,
br d, 15.4) ddd, 17.1, 10.3, 8.5) ddd, 8.5, 8.5, 5.5) br d, 17.5) br d, 10.5)
5.56 (1H, d, 1.5)
98.0
154.2 109.4 39.5 133.7
7.60 (1H, d, 2.5)
154.4 105.2 28.1 30.8
126.4 135.8 46.3 118.9 168.8 38.3
13 11-OMe
3.64 (3H, s)
173.2 51.8
200 -OMe 100 0 200 0 300 0 400 0 500 0 600 0
4.70 3.18 3.29 3.27 3.35 3.88 3.66
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 8.0) dd, 9.0, 8.0) m) m) m) dd, 11.7, 1.4) m)
6.83 (1H, d, 1.8)
7.00 6.71 3.32 5.94 5.07 5.03 3.83
(1H, (1H, (2H, (1H, (1H, (1H, (3H,
d, 8.2) dd, 8.2, 1.8) m) ddd, 17.3, 10.2, 6.7) br d, 17.3) br d, 10.2) s)
4.80 3.49 3.45 3.34 3.56 4.38 4.24
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 7.5) dd, 9.0, 7.5) t, 9.0) m) m) dd, 11.8, 2.0) dd, 11.8, 6.7)
d Cb
97.5
3.05 (2H, m)
100 200 300 400 500 600 700 800 900
a
5.47 (1H, d, 8.2)
dHa
11 12
10 20 30 40 50 60
b
dC
b
100.3 74.7 78.3 71.5 78.0 62.8 136.6 114.2 150.9 146.1 118.5 121.9 40.8 139.0 116.0 56.7 103.0 74.9 77.7 71.7 75.4 64.9
3.22 2.65 1.94 4.84 5.50 2.71 5.25 5.24
(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,
m) ddd, 13.2, 13.2, 12.0) brdd, 12.0, 3.5) m) dt, 17.0, 10.0) m) d, 17.0) d, 10.0)
2.76 (1H, dd, 16.0, 7.3) 2.65 (1H, dd, 16.0, 5.9)
77.1 133.2 43.6 121.0 168.0 41.6 171.1
4.68 3.21 3.37 3.39 3.30 3.89 3.67
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 8.0) 9.0, 8.0) m) m) m) dd, 12.0, 2.0) dd, 12.0, 5.0)
6.83 (1H, d, 2.0)
7.01 6.70 3.27 5.94 5.07 5.03 3.83
(1H, (1H, (2H, (1H, (1H, (1H, (3H,
d, 8.0) dd, 8.0, 2.0) m) ddd, 17.0, 10.0, 6.5) br d, 17.0) br d, 10.0) s)
4.81 3.49 3.46 3.28 3.57 4.50 4.26
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 8.0) dd, 9.0, 8.0) t, 9.0) m) m) dd, 12.0, 2.0) dd, 12.0, 6.0)
99.7 74.6 78.4 71.5 77.9 62.7 136.7 114.2 150.9 146.1 118.6 121.9 40.8 139.0 116.0 56.7 103.0 74.9 77.8 71.5 75.3 64.5
dH ppm (mult., J in Hz), 500 MHz. dC ppm, 125 MHz.
4.10. In vitro antiviral assay Confluent MDCK cells grown in 6-well tissue culture plates were inoculated with 1 105 PFU of virus. After adsorption at 37 °C for 1 h, the inoculum was removed. The cells were washed twice with phosphate-buffered saline (pH 7.4) to overlay with DMEM containing 5 mg/mL trypsin (Wako Pure Chemical Industries, Co., Ltd.) and various concentrations of tested samples. The plates were incubated at 37 °C in 5% CO2 for 18 h, and the titer of virus harvested from the cultures was determined in MDCK cells by the plaque assay. 4.11. Plaque assay The plaque assay was performed essentially as previously reported (Yen et al., 2005). Confluent MDCK cells grown in 6-well tissue culture plates were inoculated with appropriate dilutions of virus. After adsorption at 37 °C for 1 h, the inoculum was removed. The cells were washed twice with phosphate-buffered saline to overlay with DMEM containing 1% agarose and 5 mg/mL trypsin.
The plaques were visualized after incubation at 37 °C in 5% CO2 for 3 days by staining 0.5% crystal violet solution containing 3.7% formaldehyde. Oseltamivir carboxylate, which was prepared by hydrolysis of oseltamivir phosphate (obtained from Tamiflu capsules 75, Chugai Pharmaceutical Co., Ltd., Tokyo, Japan) as previously described (Yamanaka et al., 2006), was used as a positive control. 4.12. Influenza A neuraminidase inhibition assay A modified fluorometric assay using the fluorogenic substrate 20 -(4-methylumbellyferyl)-a-D-N-acetylneuraminic acid (MUNANA, Sigma) was used to determine the NA activity on the type A (H1N1) viruses (Ullah et al., 1999). All compounds were dissolved in DMSO and diluted to the corresponding concentrations in PBS. A 96-well plate containing a mixture of the diluted virus suspension (1 105 PFU, 50 lL) and the different concentration of compound solution (50 lL) was incubated on ice for 1 h. MUNANA substrate solution (0.2 mM in 0.1 M acetate buffer (pH 4.6), 25 lL) was added and the mixture was incubated for 30 min at 37 °C. The
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Y. Kashiwada et al. / Phytochemistry 96 (2013) 423–429
Table 2 H and 13C NMR spectroscopic data for compounds 3 and 4 in CD3OD.
1
Position
3
4
dHa 1 2 3 4 5 6 7 8 9 10
5.61 (1H, d, 7.5) 7.62 (1H, s) 3.62 6.40 6.50 5.75 2.70 5.26 5.22
11 11-OMe
4.75 3.21 3.38 3.26 3.39 3.87 3.63
100 200 300 400 500 600 700
m) dd, 16.0, 8.5) d, 16.0) ddd, 17.5, 10.5, 1.5) dd, 8.5) d, 17.5) d, 10.5)
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 7.6) dd, 8.9, 7.6) t, 8.9) t, 8.9) m) dd, 12.0, 1.5) dd, 12.0, 6.0)
8.91 (1H, s) 8.33 (1H, br s) 8.70 (1H, br s)
100 0 200 0 300 0 400 0 a
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
3.67 (3H, s)
10 20 30 40 50 60
b
dC
3.94 (3H, s)
b
dHa
97.5 154.5 109.0 39.8 134.7 128.8 135.4 46.5 119.4 168.6 51.9
d Cb
5.61 (1H, d, 7.5) 7.64 (1H, s) 3.63 6.41 6.51 5.76 2.72 5.27 5.22
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
m) dd, 15.5, 8.0) d, 15.5) ddd, 17.0, 10.5, 2.0) m) d, 17.0) d, 10.5)
3.68 (3H, s)
100.3 74.7 78.4 71.6 78.0 62.7
4.74 3.20 3.37 3.32 3.31 3.87 3.62
149.3 127.8 135.0 135.2 152.0 166.8
8.92 (1H, br s)
53.0
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 8.0) dd, 9.0, 8.0) t, 9.0) t, 9.0) m) dd, 11.5, 2.0) dd, 12.0, 6.0)
8.33 (1H, br s) 8.72 (1H, s) 4.37 1.78 1.49 1.00
(1H, (1H, (1H, (1H,
t, 6.5) q, 7.0, 6.5) ddd, 7.5, 7.0) t, 7.5)
97.3 152.0 109.8 40.3 134.9 127.7 135.3 46.2 120.9 168.5 51.4 100.1 73.2 77.1 70.3 76.7 61.2
147.6 125.3 133.7 135.6 150.5 166.7 65.2 32.2 20.3 14.2
dH ppm (mult., J in Hz), 500 MHz. dC ppm, 125 MHz.
Table 3 Effect of compounds on influenza A/PR/8/34 replication.a
a b
Compound
Inhibition at 100 lg/mL (%)
3 6 7 8 9 10 Oseltamivir carboxylateb
14.3 ± 2.7 37.2 ± 2.1 44.0 ± 3.6 53.1 ± 3.9 49.3 ± 3.2 28.4 ± 2.4 45.5 ± 2.3
Data are mean ± SE from three experiments. Oseltamivir carboxylate was tested at 0.1 lg/mL.
enzymatic reaction was quenched by adding 100 lL of glycine– NaOH buffer solution (pH 10.7). The fluorescence intensity of the product (4-methylumbellyferone) was measured in a spectrophotometer with excitation and emission wavelengths of 360 and 440 nm, respectively. The drug concentrations required to inhibit 50% of the NA activity (IC50) were determined by plotting the percent inhibition of NA activity as a function of the drug concentrations (Shi et al., 2007). Oseltamivir carboxylate was used as a positive control, which inhibited influenza A neuraminidase with an IC50 value of 0.005 ± 0.001 lg/mL.
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