- Email: [email protected]
Chromone derivatives from Halenia elliptica and their anti-HBV activities
Phytochemistry 75 (2012) 169–176
Contents lists available at SciVerse ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Chromone derivatives from Halenia elliptica and their anti-HBV activities Yu-wei Sun a, Guang-ming Liu b, Hai Huang c, Pei-zhong Yu a,⇑ a
Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, PR China Department of Medicinal Chemistry, School of Pharmacy, Dali University, Dali, Yunnan 671000, PR China c Department of Biosynthetic Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, PR China b
a r t i c l e
i n f o
Article history: Received 13 January 2011 Received in revised form 28 July 2011 Accepted 20 September 2011 Available online 21 December 2011 Keywords: Halenia elliptica Gentianaceae Chromone-2-carboxylic acid 2-Methylchromone Acid-catalyzed isomerization Anti-HBV
a b s t r a c t Nine water-soluble chromone derivatives, including chromone-2-carboxylic acids, 2-methylchromones and their structural hybrids, were isolated from aerial tissues of Halenia elliptica (Gentianaceae), six of which were previously unknown. Their structures were elucidated by comprehensive mass, 1D and 2D NMR spectroscopic analyses and chemical derivatization. Two unstable structural hybrids of chromone-2-carboxylic acids and 2-methylchromones, viz. 3-acetyl-8-hydroxy-4H-1-benzopyran-4-one-2carboxylic acid (halenic acid C) and 2-(8-hydroxy-2-methyl-4H-1-benzopyran-4-one-3-yl)-2-oxoacetic acid (halenichromone A), were isomers and were interconvertible. The proposed mechanism of their acid-catalyzed isomerization in aqueous solvent is described. In addition, 2-methylchromones, 8hydroxy-2-methyl-4H-1-benzopyran-4-one, and 8-methoxy-2-methyl-4H-1-benzopyran-4-one, were found to exhibit a strong inhibitory effect towards hepatitis B virus (HBV) in vitro without showing significant cytotoxicity. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Halenia elliptica D. Don (Gentianaceae) is distributed across the Tibetan plateau and throughout western China. It has been used for centuries in traditional Tibetan and Mongolian medicine to cure hepatitis. Previous phytochemical investigations led to the isolation of a series of simple oxygenated xanthone aglycones and glycosides (Bennett and Lee, 1991; Dhasmana and Garg, 1989, 1990; Liu et al., 2009; Sun et al., 1983, 1987, 2011; Yu et al., 2008). These xanthone compounds are believed to contribute to the hepatoprotective (Gao et al., 2004b; Huang et al., 2010), vasorelaxant (Wang et al., 2007, 2008, 2009b) and antioxidant (Gao et al., 2004a; Harborne et al., 1999) activities ascribed to this particular species. In the current research, the water-soluble portion of the aerial tissues of H. elliptica was found to have potent anti-HBV activity in vitro (data not shown). This finding prompted us to characterize the polar constituents present in these aqueous extracts. Described herein is the isolation and structural elucidation of six chromone derivatives (1–6, halenic acid A–C, halenichromone A–C) and three known 2-methylchromones (7–9) from the water-soluble extract of H. elliptica (Fig. 1). Compounds 7–9 were isolated from natural sources for the first time. In vitro HBV inhibitory effects of compounds 1–4, 8 and 9 were also tested. Chromones are a class of plant secondary metabolites having a 4H-1-benzopyran-4-one skeleton and occur only in a relatively ⇑ Corresponding author. Tel./fax: +86 21 51980103. E-mail address: [email protected] (P.-z. Yu). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.09.015
small number of plant species (Harborne et al., 1999). To our knowledge, there have hitherto been no reports describing the presence of chromone derivatives in the Gentianaceae, which contains 87 genera and over 1600 angiosperm species (Struwe and Albert, 2002). The genus Halenia is one of the genera in this family rich in polyoxygenated xanthones. Up to now, only lipophilic xanthones have been reported as taxonomically informative compounds within the genus Halenia (Jensen and Schripsema, 2001). Chromone derivatives found in this research may be used as polar markers with chemotaxonomic significance. 2. Results and discussion 2.1. Structural elucidation Halenic acid A (1, Fig. 1) was obtained as an amorphous white powder with molecular formula C10H6O5 as determined by HRESI-MS. The IR spectrum of 1 showed a characteristic absorption band due to the presence of a conjugated carbonyl (1624 cm–1), and a strong broad band (3600–2400 cm–1) which clearly indicated the presence of a carboxyl group. An apparent increase in its HPLC retention time in low pH buffer (pH = 3–5) also indicated the acidic nature of 1 (Supplementary data, Fig. S74). The 1H NMR spectrum of 1 in DMSO-d6 had one olefinic proton (dH 6.69, H-3) assigned to a trisubstituted olefin and three aromatic protons (dH 7.39, H-5; dH 7.25, H-6; dH 7.18, H-7) attributed to a 1,2,3-trisubstituted aromatic ring (Table 1). Ten carbon signals were also observed in the 13C NMR spectrum, including four sp2 methines (one olefinic
170
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
Fig. 2. Key HMBC correlations of 1 and 5 (H ? C).
also showed a strong broad band representative of a carboxyl group (3600–2500 cm–1) which was responsible for the same HPLC chromatographic behavior as shown by 1. An additional signal of a methoxyl group (dH 3.97, dC 56.4) was observed in its 1H and 13C NMR spectra (Table 1), indicating that the 8-hydroxyl group in 1 was replaced by a methoxyl group. This conclusion was proved by the NOE correlation between H-7 and the methoxyl protons. The HMBC spectrum showed all the important long-range C–H correlations identical to those of 1. Therefore, compound 2 was established as 8-methoxy-4H-1-benzopyran-4-one-2-carboxylic acid, with a trivial name of halenic acid B. Halenic acid C (3) and halenichromone A (4) were obtained as a mixture. Although they could be separated by C18 reversed-phase silica gel column chromatography (CC), both purified compounds interconverted into an equilibrium during the removal of the solvent (MeOH–H2O) at 45 °C (Supplementary data, Fig. S75). The composition of the mixture also changed gradually in aqueous DMSO at room temperature over several days. The color of the sample was a good indicator of purity, since mixtures of 3 and 4 were yellow and upon purification they became colorless (Supplementary data, Fig. S76). The above facts suggested that 3 and 4 equilibrated in the solvent. Since these compounds could not be thoroughly separated by conventional chromatographic methods, fraction C1 (a mixture containing ca. 55% of 3 and ca. 35% 4, as calculated from HPLC peak area integral) was analyzed by LC–ESI-MS; Compounds 3 and 4 were distinguished by their retention times. A low pH buffer greatly increased their retention times, implying that 3 and 4 were also acidic compounds like 1 and 2. LC–ESI-MS analyses of 3 (peak A, tR = 10.5 min) and 4 (peak B, tR = 7.1 min) both gave quasimolecular ion peaks at m/z 247 [M H] . Furthermore, compound 3 (peak A, tR: 10.5 min) was prone to lose a moiety represented by a carboxyl group to give a peak at m/z 203 [M–H–COO] (Supplementary data, Fig. S77). The 1H and 13C NMR spectra of 3 and 4 could only be recorded using relatively purified mixtures. Analysis
Fig. 1. Compounds 1–9 and methyl ester derivatives 3a and 4a.
methine, three aromatic methines), four quaternary sp2 carbons (dC 124–161, showing no HSQC correlation with any protons), a conjugated carbonyl carbon (dC 179.1) and a carboxyl carbon (dC 161.7) (Table 1). The HMBC experiment connected the above-mentioned partial structures and functional groups though long-range C–H correlations to establish a chromone skeleton (Fig. 2). Three adjacent protons were assigned to H-5, H-6 and H-7 on the B ring of the chromone skeleton according to the HMBC correlations of H5/carbonyl carbon (C-4), C-7 and C-8a; H-6/C-8 and C-4a; H-7/C5 and C-8a. Other important HMBC correlations observed between the olefinic proton (H-3) and C-4a, C-2 and the carboxyl carbon suggested that the carboxyl group was at C-2. By considering the molecular formula and the downfield chemical shift of C-8 (dC 147.5), a hydroxyl group was located at C-8. Thus, the structure of compound 1 was established as 8-hydroxy-4H-1-benzopyran4-one-2-carboxylic acid and 1 was named halenic acid A. Halenic acid B (2) was deduced to be a congener of 1 on the basis of their similar 1H and 13C NMR spectra and its molecular formula C11H8O5 determined by HR-ESI-MS. The IR spectrum of 2 Table 1 1 H (400 MHz) and
13
C (100 MHz) NMR spectroscopic data for compounds 1–6.
Position 1 (in DMSO-d6)
2 3 4 4a 5 6 7 8 8a COOH-2 OCH3-8
a b c
dC
dH, mult (J in Hz)
160.4 110.2 179.1 124.7 113.7 125.5 119.4 147.5 145.1 161.7 –
– 6.69, – – 7.39, 7.25, 7.18, – – – –
2 (in DMSO-d6) dC
153.4 113.4 177.7 124.8 dd (7.8, 1.5) 115.3 t (7.8) 126.0 d (7.8) 116.1 149.0 145.8 161.4 56.4 s
Position 3 (in DMSO-d6)
dH, mult (J in Hz)
dC
dH, mult (J in Hz)
– 6.91, – – 7.56, 7.45, 7.50, – – – 3.97,
155.5 125.3 176.8 124.3 113.5 125.4 119.5 147.7 144.8 198.3 31.6 161.3
– – – – 7.41, 7.27, 7.27, – – – 2.33, –
2 3 4 4a d (7.6) 5 t (7.8) 6 d (6.8) 7 8 8a 3a s 3b 2a s
a
4 (in DMSO-d6)
a
Position 5 (in acetone-d6)
dC, mult dH, mult (J in Hz)
175.2 121.5 176.8 124.2 dd (7.0, 2.4) 114.0 c m 125.5 mc 119.5 146.6 144.8 N.D.b s 166.1 18.9
– – – – 7.39, 7.27, 7.22, – – – – 2.33,
2 3 4 4a d (7.7) 5 t (7.7) 6 d (7.6) 7 8 8a CH3-2 OCH3-6 s OH-5 OH-3
6 (in acetone-d6)
dC
dH, mult (J in Hz)
dC
dH, mult (J in Hz)
169.8 108.4 184.8 111.2 150.6 144.2 121.0 106.5 151.4 20.5 57.3 – –
– 6.16, s – – – – 7.40, d (9.0) 6.94, d (9.1) – 2.42, s 3.87, s 12.84, s –
137.4 152.8 177.5 111.0 149.7 143.2 121.7 106.9 150.6 15.1 57.6 – –
– – – – – – 7.41, d (9.2) 6.97, d (9.1) – 2.45, s 3.88, s 12.27, s 7.72, s
The assignments of 3 and 4 were based on HSQC and HMBC experiments, together with comparison with equivalent signals of compounds 3a and 4a. Signal undetermined. This signal was absent probably due to the instability of sample. 2H, overlapped signals.
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
of both 1H NMR spectra indicated presence of a methyl group at dH 2.33, and three aromatic protons (dH 7.2–7.4) (Table 1). This similarity, combined with the same molecular formulae C12H8O6 established by HR-ESI-MS, indicated that 3 and 4 were structurallyrelated isomers. The interconversion of 3 and 4 occurred during the 13C and 2D NMR spectroscopic measurements, which caused great difficulties for further structural elucidation. In their 13C NMR spectra, some quaternary carbons were indistinguishable or absent due to their instability. However, the main parts of the 13 C NMR spectra (C4-8, C-4a and C-8a) showed good agreement with those of 1, which suggested that the frameworks of 3 and 4 were also chromones (Table 1). In spite of all the difficulties due to the impurity of the sample, the 13C NMR spectra of compounds 3 and 4 did display a few notable differences between these molecules. The chemical shift of a methyl carbon in 3 was shifted significantly from 31.6 to 18.9 ppm in 4. In order to prevent their interconversion and allow the structural elucidation of 3 and 4, fraction C1 (mainly composed of 3 and 4) were refluxed with methanol, using the Fischer–Speier esterification method, to afford a mixture of methyl ester derivatives 3a and 4a. Compounds 3a and 4a were stable and could be readily separated using silica gel CC. The MS, IR, 1H, 13C and 2D NMR spectroscopic analyses of 3a and 4a finally led to the structural characterization of 3 and 4. Compounds 3a and 4a both showed the presence of a pseudomolecular ion peak at m/z 263 [M+H]+ in the ESI-MS and a characteristic absorption band consistent with an ester (1725 cm–1) in their IR spectra. Taken together with the evidence of additional signals indicating the presence of methoxyl groups (dH 3.97, dC 53.9 in 3a; dH 3.86, dC 52.4 in 4a) in their 1H and 13C NMR spectra, these were confirmed as methyl ester derivatives of 3 and 4. Thirteen carbon signals of 3a and 4a were clearly observed in both 13C NMR spectra. Compounds 3a and 4a shared the same chromone skeleton, especially their trisubstituted B ring, because of the similar 13C NMR spectroscopic data (C-4a, C5–8, C-8a, DdC < ±0.7). Three adjacent protons were assigned to H-5, H-6 and H-7 on the B ring in the same way as those of 1 and 2. The olefinic proton (H-3) in 1 and 2 was absent in 3a and 4a (also in 3 and 4). In 3a, H-3 was replaced by an acetyl group and a methyl-esterified carboxyl group was linked to C-2. The HMBC spectrum of 3a supported this conclusion by C–H correlation from methyl protons (dH 2.51) to a ketone carbonyl (dC 197.6) and further to the quaternary sp2 carbon C-3 (dC 128.5) (Fig. 3). In 4a, a methyl group located at C-2 was deduced from the HMBC correlation of methyl protons (dH 2.74)/oxygenated quaternary sp2 carbon C-2 (dC 175.1) and quaternary sp2 carbon C-3 (dC 117.7) (Fig. 3). Thus, it is possible to explain why the methyl carbons of 3 (also 3a) and 4 (also 4a) differ in their chemical shifts. The methyl group of 3 (3a) appeared in a ketone (RCOCH3), showing a representative 13C chemical shift of 31 ppm, while for 4 (4a), the methyl group was attached directly to the A ring of chromone at C-2 to establish a 2-methylchromone skeleton. According to reported data of known 2-methylchromones (Ito et al., 2004; Kuroda et al., 2009; Tanaka et al., 1995; Tsui and Brown, 1996; Tuntiwachwuttikul et al., 2006), the typical chemical shift of the 2-methyl carbon is around 20 ppm. On the basis of the above findings, the structures of methyl ester derivatives 3a and 4a
Fig. 3. Key HMBC correlations of 3a and 4a (H ? C).
171
were elucidated and their corresponding carboxylic acids were established as 3-acetyl-8-hydroxy-4H-1-benzopyran-4-one-2-carboxylic acid (3, halenic acid C), and 2-(8-hydroxy-2-methyl-4H1-benzopyran-4-one-3-yl)-2-oxoacetic acid (4, halenichromone A), respectively. Halenichromone B (5), a pale yellow amorphous solid, showed a pseudomolecular ion peak at m/z 207 [M+H]+ in its positive mode ESI-MS. Its molecular formula, C11H10O4, determined by HR-ESIMS, indicated seven degrees of unsaturation. The IR spectrum of 5 showed characteristic absorption bands due to conjugated carbonyl (1661 cm–1) and hydroxyl (3424 cm–1) groups. Furthermore, the hydroxyl group was confirmed by a hydrogen-bonded OH singlet observed at dH 12.84 in the 1H NMR spectrum. The 1H and 13C NMR spectra of 5 also had one broad singlet (dH 6.16, dC 108.4) assigned to a trisubstituted olefin, a pair of ortho-coupled doublets [dH 6.94 (d, J = 9.1 Hz), dC 106.5; dH 7.40 (d, J = 9.0 Hz), dC 121.0], indicating the presence of a 1,2,3,4-tetrasubstituted aromatic ring, along with one aromatic methyl group (dH 2.42, dC 20.5) and one methoxyl group (dH 3.87, dC 57.3) (Table 1). The remaining carbons included one quaternary sp2 carbon, four oxygenated quaternary sp2 carbons and a conjugated carbonyl carbon (C-4, dC 184.8) (Table 1). In the 1D-NOESY experiment, irradiation of the 2-methyl protons enhanced the signal intensity of the olefinic proton (H3). All the above analyses, in combination with the comparison of 13C NMR spectroscopic data with those of the reported 2-methylchromones, suggested the 2-methylchromone skeleton of 5 as previously described (Ito et al., 2004; Kuroda et al., 2009; Tanaka et al., 1995; Tsui and Brown, 1996; Tuntiwachwuttikul et al., 2006). Analysis of the HMBC spectrum then supported this by the long-range C–H correlations of the 2-methyl protons to C-2 (dC 169.8) and C-3, and of H-3 to C-2, methyl carbon and C-4a (dC 111.2) (Fig. 2). Also in the HMBC spectrum, the chelated OH-5 showed C–H correlations with C-5, C-4a and a methoxyl-bearing carbon (based on the HMBC correlation from methoxy protons to this carbon) at dC 144.2. This indicated that the methoxyl group was adjacent to OH-5 and located at C-6. The ortho-coupled aromatic protons were assigned to H-7 and H-8, on the basis of the HMBC correlations of H-7/C-5 and C-8a, H-8/C-6 and C-4a. Thus, compound 5 was characterized as 5-hydroxy-6-methoxy-2methyl-4H-1-benzopyran-4-one. Halenichromone C (6) was obtained as an amorphous yellow powder. Its molecular formula was established to be C11H10O5 by HR-ESI-MS, and was larger than that of 5 by an oxygen atom. The IR spectrum of 6 showed absorption bands consistent with conjugated carbonyl (1652 cm–1) and hydroxyl (3323 cm–1) groups. The 1H NMR spectroscopic data of 6 were superimposable on those of 5, except for the absence of an olefinic proton (H-3) (Table 1). Thus, 6 appeared to be the 3-hydroxylated derivative of 5. In the 13C NMR spectrum, carbon signals corresponding to the B ring of the chromone skeleton were almost identical with those of 5 (C4a, C5–8, C-8a, DdC < ±1.0) (Table 1), while resonances assigned to the A ring were notably different from those of 5. Due to the hydroxylation of H-3, the signal of C-3 was shifted downfield significantly from dC 108.4 in 5 to dC 152.8 in 6. In contrast, the neighboring C-2 was shifted upfield by about 30 ppm compared with the equivalent carbon in 5 (Table 1). The carbonyl (C-4, dC 177.5) and 2-methyl carbon (dC 15.1) were also shifted to some extent. Analysis of the HMBC spectrum established long-range C–H correlations of methyl protons/C-2 and C-3, methoxy protons/C-6 as expected. Therefore, compound 6 was established as 3,5-dihydroxy-6-methoxy-2-methyl-4H-1-benzopyran-4-one. Three known 2-methylchromones were identified as 5-hydroxy-8-methoxy-2-methyl-4H-1-benzopyran-4-one (7), 8-hydroxy-2-methyl-4H-1-benzopyran-4-one (8), and 8-methoxy-2-methyl-4H-1-benzopyran-4-one (9). They were all previously synthesized compounds. Compounds 7 (Rao and
172
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
Fig. 4. Proposed acid-catalyzed isomerization of 3 and 4.
Venkateswarlu, 1956) and 8 (Linke, 1969a, b) were synthesized during the 1950–1970s for UV or IR studies, however, no NMR spectroscopic data were reported. The 1H and 13C NMR spectroscopic data of 9 were reported though in DMSO-d6 (Jung et al., 2001). This is the first report of 7, 8 and 9 isolated from a natural source. Their structures were confirmed by MS, IR, 1D and 2D NMR spectroscopic techniques. In this study, the NMR spectroscopic data of 7, 8 and 9 are reported as a supplement to the known compounds. The amount of 7 was insufficient to obtain 13C NMR spectroscopic data. The difference between our NMR spectroscopic data and those reported for compound 9 may be due to the different solvents used. 2.2. Proposed mechanism of interconversion of 3 and 4 Among the chromones isolated from H. elliptica, halenic acid C (3) and halenichromone A (4) intrigued us by their isomerization equilibrium. This phenomenon was presumed to result from an intramolecular rearrangement, involving an acid-catalyzed opening of the A ring (pyrone ring) in aqueous solvent to form a triketone intermediate product 10, and then a ring closure with C-3a (carbonyl) to the phenolic hydroxyl at C-8a (Fig. 4). This intramo-
lecular rearrangement is similar to the classic Wessely–Moser rearrangement. In this rearrangement, a rotation takes place in the bond between C-4 and C-4a, resulting in the change of the B ring (Fig. 5), whereas for the isomerization of 3 and 4, it takes place in the bond between C-4 and C-3 (Fig. 4). Thus, in the Wessely– Moser rearrangement, an intermediate product 11 with two meta-phenolic hydroxyl groups at C-5 and C-8a are prerequisites. Wessely–Moser rearrangements have been reported in flavonoids and chromones, in both synthesized and naturally occurring compounds (Grayer and Veitch, 1998; Larget et al., 2000; Ma et al., 1996). In this research, compound 7 appears to be a classic Wessely–Moser rearrangement product of compound 5 (Fig. 5) and may be an artifact formed during the isolation procedure. In the rearrangement of 3 and 4, the non-regiospecific cyclization of triketone 10 is the key step. Triketone 10 contains more hydroxyls than 3 and 4. Since hydroxy in direct conjugation with benzene ring is an auxochrome, the mixture of 3 and 4 shows a darker color than both purified compounds. Compounds 1 and 2 are stable because they are unable to form triketone intermediates like 10 after the opening of their A rings. Compounds 3a and 4a, the methyl ester derivatives of 3 and 4, are also stable during their isolation and structural elucidation. This is because they are relatively lipophilic, and the use of an aprotic solvent during purification prevents opening of the A ring. This type of rearrangement was reported in synthesized compounds (Clarke et al., 2005; Klutchko et al., 1974). To our knowledge, 3 and 4 are the first examples of natural chromones that isomerize through this type of rearrangement. Since the chromone derivatives found in this research featured either a carboxyl (compounds 1–3) or a methyl group (compounds 4–9) at the C-2 position, they can be classified as a chromone-2carboxylic acid for 3 and a 2-methylchromone for 4. These two compounds were thus named as halenic acid C and halenichromone A, respectively, but they are actually structural hybrids of chromone-2-carboxylic acid and 2-methylchromone. They are presumed to be a pair of important transitional compounds in the biosynthetic pathway of chromone-2-carboxylic acids and 2-methylchromones present in this plant.
2.3. Anti-HBV activity Compounds 1–4, 8 and 9, in sufficient amounts, were tested for their potencies towards inhibiting the secretion of HBV antigens in
Fig. 5. Wessely–Moser rearrangement of 5 and 7.
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176 Table 2 HBV inhibitory activity of compounds 1–4, 8 and 9. Compound
Concentration (lg/mL)
Cell growth inhibition rate (%)a
HBsAg (inhibition rate %)
HBeAg (inhibition rate %)
1
100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 100
0 0 0 0 0 0 0 0 0 25 0 0 0 0 0 0
16.4 ± 0.8 0±0 0±0 12.0 ± 0.6 3.6 ± 1.0 1.9 ± 0.3 0±0 0±0 0±0 /b 36.8 ± 2.3 5.9 ± 2.3 70.9 ± 2.4 60.2 ± 2.2 55.6 ± 3.8 20.1 ± 1.2
0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 /b 41.5 ± 3.4 18.0 ± 2.5 14.1 ± 1.4 6.3 ± 0.8 0±0 19.7 ± 1.3
2
3/4
8
9
3TCc
a Cell damage was assessed by means of MTT assay, cell growth inhibition P10% was considered as cytotoxic. b / Means the compound was cytotoxic at this concentration. c Positive control (3TC = Lamivudine).
the human HBV-transfected liver cell line HepG2 2.2.15. Lamivudine (3TC) was used as a positive control that can suppress HBsAg secretion by 20.1% and HBeAg secretion by 19.7%, at 100 lg/mL (436 lM). Compounds 3 and 4 were tested using a mixture due to their instability in DMSO. The results are summarized in Table 2. 2-Methylchromone compounds 8 and 9 exhibited strong anti-HBV activities, inhibiting HBsAg secretion by 36.8% at a non-cytotoxic concentration of 50 lg/mL (284 lM) for 8 and by 70.9% at a noncytotoxic concentration of 100 lg/mL (526 lM) for 9. Compound 8 also suppressed HBeAg secretion by 41.5% at 50 lg/mL (284 lM). Acidic compounds 1–4, which possessed a carboxyl group, were slightly active or totally inactive at low concentration. Further investigations are warranted to explore the value of 2methylchromones as anti-HBV agents.
173
genus Halenia consists of approximately 100 species, among which only 4 have been studied (Yang et al., 2006). Species belonging to Halenia are found to produce similar xanthones, flavonoids and secoiridoids (Dhasmana and Garg, 1989, 1990; Gao et al., 2004a; Gendaramyn et al., 1998; Liu et al., 2009; Recio-Iglesias et al., 1992; Rodriguez et al., 1995, 1996; Stout and Fries, 1970; Sun et al., 1983, 1987, 2011). Our work is the first systematic study of H. elliptica that has focused on its water-soluble constituents, suggesting simple chromones are representatives of polar constituents in this species. Moreover, since chromone-2-carboxylic acids are quite rare in nature, these unique chromone compounds may be used as chemotaxonomic markers in the classification of Halenia species. 4. Experimental 4.1. General experimental procedures Melting points were determined on an X4 micromelting point apparatus. UV spectra were recorded on Hitachi U-2900 double beam spectrophotometer, whereas IR spectra (KBr) were obtained using an Avatar 360 ESP FT-IR spectrometer (v in cm–1). NMR spectra were recorded at 400 MHz for 1H and 100 MHz for 13C on a Varian Mercury Plus-400 spectrometer, in DMSO-d6 or acetone-d6, using residual solvent signals (dH/C 2.50/39.51 for DMSO-d6; dH/C 2.05/29.92 for acetone-d6) or tetramethylsilane (TMS) as internal standards. The chemical shifts (d) were given in ppm and the coupling constants (J) in Hz. ESI-MS and LC–ESI-MS data were measured with an Agilent 1100 chromatography system coupled with an Agilent G1946D mass detector. HR-ESI-MS data were measured with a YA019 Q-Tof micro mass spectrometer. Column chromatography (CC) was performed with silica gel H (10–40 lm, Qingdao Haiyang Chemical Co., Qingdao, China), Diaion HP-20 (Mitsubishi Chemicals Co., Tokyo, Japan), MCI gel CHP 20P (75– 150 lm, Mitsubishi Chemicals Co., Tokyo, Japan), Sephadex LH-20 (Amersham Pharmacia Biosciences, Inc., Piscataway, NJ, USA) and OUYA-RP18 silica gel (15–35 lm, Unicorn, Switzerland). 4.2. Plant material
3. Concluding remarks In this research, nine polar chromone derivatives were isolated from H. elliptica and can be categorized into chromone-2-carboxylic acids (1 and 2), 2-methylchromones (5–9) and their structural hybrids (3 and 4). Six major chromones (1–4, 8 and 9) possess a similar trisubstituted B ring (5,6,7-H; 8-OH or 8-OCH3). According to reports on the phytochemical composition of H. elliptica and from our previous work on its lipid soluble fractions, some of the most abundant xanthones isolated from this plant are 1-hydroxy-2,3,4,5-tetramethoxyxanthone, 1-hydroxy-2,3,5-trimethoxyxanthone, and 1,5-dihydroxy-2,3-dimethoxyxanthone (Wang et al., 2007, 2008; Yu et al., 2009). They also share a trisubstituted ring (B ring) with three adjacent protons (6,7,8-H; 5-OH or 5-OCH3) (Supplementary data, Fig. S78). Such structural similarity suggests that xanthone and chromone compounds presenting in this plant may be biosynthetically related. Chromone compounds are thought to be distributed in a limited number of plant families including the Ranunculaceae, Myrtaceae, Liliaceae, and Clusiaceae (another xanthone-rich family) (Harborne et al., 1999). To our knowledge, this is the first report describing the characterization of chromone derivatives in the Gentianaceae family. The Gentianaceae is, however, rich in natural xanthones (Peres and Nagem, 1997; Peres et al., 2000), and most polyoxygenated xanthones in the Gentianaceae come from the genera Swertia, Halenia, Veratrilla and Frasera (Jensen and Schripsema, 2001). The
H. elliptica was collected during April 2005 in Dali, Yunnan Province, China. The plant was identified by Professor Xiao-kuang Ma, Department of Pharmacognosy, School of Pharmacy, Dali University, PR China. A voucher specimen (No. HE0505) has been deposited at the Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai, PR China. 4.3. Extraction and isolation Air-dried aerial part of H. elliptica (5 kg) was extracted with EtOH–H2O (95:5, v/v, 50 L) at room temperature for 120 h. After evaporation of EtOH under reduced pressure, the residue (140 g) was suspended in H2O and partitioned sequentially with petroleum ether (b.p. 60–90 °C) and EtOAc to remove the lipophilic constituents. The aqueous phase was lyophilized to give a powder (45.2 g), the bulk of which (40.0 g) was dissolved in hot H2O and subjected to CC using a Diaion HP 20 resin column (EtOH–H2O, 0:100 ? 95:5, v/v) to give five fractions: Fr. A–E. Fr.B (2.7 g, eluted by EtOH–H2O, 10:90, v/v) was subjected to C18 reversed-phase silica gel CC (MeOH–H2O, 15:85, v/v) to afford compound 1 (120 mg). Fr.C (1.5 g, eluted by EtOH–H2O, 20:80, v/v) was subjected to MCIgel CHP 20P cc (MeOH–H2O, 0:100 ? 100:0, v/v) to give four subfractions: Fr. C1-C4. Fr.C1 (650 mg) was then purified repeatedly on Sephadex LH-20 (MeOH–H2O, 0:100 ? 10:90, v/v) and on a C18 reversed-phase silica gel column (MeOH–H2O, 15:85, v/v) to
174
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
yield compounds 2 (12 mg), 3 and 4 (mixture, 25 mg), and a small amount of compound 1 (5 mg). Fr.C1 was then analyzed by LC–ESIMS. Fr.C2 (100 mg) was purified on a C18 reversed-phase silica gel column (MeOH–H2O, 30:70, v/v) to yield compound 8 (45 mg). Fr.C4 (80 mg) was purified repeatedly on a C18 reversed-phase silica gel column (MeOH–H2O, 40:60, v/v) to yield compounds 5 (3.5 mg), 6 (2.5 mg) and 7 (0.8 mg). Fr.E (1005 mg, eluted by EtOH–H2O, 95:5, v/v) was subjected to Sephadex LH-20 cc (MeOH–H2O, 0:100 ? 100:0, v/v) to give three subfractions: Fr. E1–E3. Fr. E2 (200 mg) was then separated repeatedly on a C18 reversed-phase silica gel column (MeOH–H2O, 55:45, v/v) to afford compound 9 (38 mg). 4.4. LC–ESI-MS analysis of 3 and 4 Fraction C1 (2 mg) was dissolved in H2O and diluted to 0.5 lg/ lL. Separation was performed on a 5 lm Hypersil ODS2 column (4.6 i.d 250 mm, Dalian Elite Analytical Instruments Co. Ltd., Dalian, PR China). The mobile phase consisted of 0.1% HCOOH in MeOH–H2O (25:75, v/v). The flow rate was 1 mL/min and the temperature was 25 °C (detection: 280 nm). Analyses were conducted in negative ion mode using selected ion monitoring of m/z 247 [M H] ions and m/z 203 [M H–COO] ions. 4.5. 8-Hydroxy-4H-1-benzopyran-4-one-2-carboxylic acid (halenic acid A, 1) White amorphous solid; UV (H2O) kmax (log e): 324 (3.24), 259 (3.76), 241 (4.05) nm; IR vmax (KBr) cm–1: 3482 (COOH), 1695, 1624 (C@O), 1575, 1493, 1410, 1347, 1301, 1155, 1003, 848, 799, 783; for 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) spectroscopic data, see Table 1; ESI-MS (negative) m/z: 205 [M H] , 411 [2M H] ; HR-ESI-MS m/z: 205.0136 (calcd for C10H5O5, 205.0137). 4.6. 8-Methoxy-4H-1-benzopyran-4-one-2-carboxylic acid (halenic acid B, 2) Pale brown amorphous solid; UV (MeOH) kmax (log e): 316 (3.45), 268 (sh, 3.66), 235 (4.10) nm; IR vmax (KBr) cm–1: 3448 (COOH), 1635 (C@O), 1581, 1493, 1455, 1352, 1272, 1145, 1050, 877, 795, 740; for 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) spectroscopic data, see Table 1; ESI-MS (negative) m/z: 219 [M H] ; HR-ESI-MS m/z: 219.0295 (calcd for C11H7O5, 219.0293). 4.7. 3-Acetyl-8-hydroxy-4H-1-benzopyran-4-one-2-carboxylic acid (halenic acid C, 3) Pale yellow amorphous solid; UV (H2O) kmax (log e): 321 (3.17), 274 (sh, 3.61), 242 (3.98) nm; for 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) spectroscopic data, see Table 1; ESI-MS (negative) m/z: 247 [M H] , 203 [M H–COOH] ; HR-ESIMS m/z: 247.0240 (calcd for C12H7O6, 247.0243).
4.9. 5-Hydroxy-6-methoxy-2-methyl-4H-1-benzopyran-4-one (halenichromone B, 5) Pale yellow amorphous solid; UV (MeOH) kmax (log e): 347 (2.86), 262 (sh, 3.46), 237 (3.74); IR vmax (KBr) cm–1: 3424 (OH), 2924, 1661 (C@O), 1631, 1594, 1480, 1402, 1385, 1280, 1240, 1139, 1047, 965, 839, 767, 697 cm–1; for 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) spectroscopic data, see Table 1; ESI-MS (positive) m/z: 207 [M+H]+, 229 [M+Na]+, 435 [2M+Na]+; HR-ESI-MS m/z: 229.0478 (calcd for C11H10O4Na, 229.0477). 4.10. 3,5-Dihydroxy-6-methoxy-2-methyl-4H-1-benzopyran-4-one (halenichromone C, 6) Yellow amorphous solid; UV (MeOH) kmax (log e): 359 (3.45), 290 (sh, 3.31), 249 (4.05); IR vmax (KBr) cm–1: 3323 (OH), 2924, 1652 (C@O), 1630, 1594, 1566, 1479, 1384, 1320, 1299, 1233, 1039, 800, 751; for 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) spectroscopic data, see Table 1; ESI-MS (positive) m/z: 223 [M+H]+; HR-ESI-MS m/z: 245.0425 (calcd for C11H10O5Na, 245.0426). 4.11. 5-Hydroxy-8-methoxy-2-methyl-4H-1-benzopyran-4-one (7) Pale yellow amorphous solid; UV (MeOH) kmax (log e): 350 (2.81), 287 (2.64), 253 (3.45), 226 (3.63); 1H NMR (400 MHz, acetone-d6): d 12.06 (1H, s, OH-5), 7.33 (1H, d, J = 9.0 Hz, H-7), 6.67 (1H, d, J = 8.8 Hz, H-6), 6.23 (1H, s, H-3), 3.91 (3H, s, OCH3-8), 2.46 (3H, s, CH3-2); HR-ESI-MS m/z: 229.0479 (calcd for C11H10O4Na, 229.0477). 4.12. 8-Hydroxy-2-methyl-4H-1-benzopyran-4-one (8) White amorphous solid; UV (MeOH) kmax (log e): 310 (3.55), 252 (3.97), 231 (4.28); IR vmax (KBr) cm–1: 3447 (OH), 2736, 2655, 1634 (C@O), 1566, 1496, 1388, 1371, 1298, 1191, 1154, 1022, 991; 1H NMR (400 MHz, DMSO-d6): d 10.41 (1H, br, OH-8), 7.39 (1H, dd, J = 6.6, 2.6 Hz, H-5), 7.21 (2H, m, H-6 and H-7), 6.20 (1H, s, H-3), 2.39 (3H, s, CH3-2); 13C NMR (100 MHz, DMSO-d6): d 177.2 (C-4), 166.5 (C-2), 146.6 (C-8), 145.7 (C-8a), 125.0 (C-6), 124.4 (C-4a), 118.9 (C-7), 114.1 (C-5), 109.8 (C-3), 20.1 (CH3-2); ESI-MS (positive) m/z: 177 [M+H]+; HR-ESI-MS m/z: 175.0394 (calcd for C10H7O3, 175.0395). 4.13. 8-Methoxy-2-methyl-4H-1-benzopyran-4-one (9) White amorphous solid; UV (MeOH) kmax (log e): 307 (3.59), 252 (3.91), 230 (4.30); IR vmax (KBr) cm–1: 3451 (OH), 2926, 1646 (C@O), 1581, 1491, 1441, 1391, 1367, 1274, 1219, 1196, 1144, 1059, 748; 1H NMR (400 MHz, acetone-d6): d 7.59 (1H, dd, J = 6.8, 2.7 Hz, H-5), 7.34 (2H, m, H-6 and H-7), 6.16 (1H, s, H-3), 3.99 (3H, s, OCH3-8), 2.42 (3H, s, CH3-2); 13C NMR (100 MHz, acetoned6): d 177.6 (C-4), 167.0 (C-2), 149.8 (C-8), 147.7 (C-8a), 125.5 (C-6), 125.5 (C-4a), 116.6 (C-5), 115.4 (C-7), 110.9 (C-3), 56.7 (OCH3-8), 20.4 (CH3-2); ESI-MS (positive) m/z: 191 [M+H]+, 403 [2M+Na]+; HR-ESI-MS m/z: 213.0527 (calcd for C11H10O3Na, 213.0528).
4.8. 2-(8-Hydroxy-2-methyl-4H-1-benzopyran-4-one-3-yl)-2oxoacetic acid (halenichromone A, 4)
4.14. Esterification of 3 and 4
Pale yellow amorphous solid; UV (H2O) kmax (log e): 306 (3.67), 260 (sh, 4.00), 221 (4.22) nm; for 1H NMR (DMSO-d6, 400 MHz) and 13 C NMR (DMSO-d6, 100 MHz) spectroscopic data, see Table 1; ESIMS (negative) m/z: 247 [M H] ; HR-ESI-MS m/z: 247.0240 (calcd for C12H7O6, 247.0243).
Fraction C1 (80 mg) was dissolved in MeOH (20 mL) and 3 drops of concentrated H2SO4 were added. The reaction mixture was heated until reflux began these beams maintained for 2 h, then cooled, neutralized with saturated aqueous NaHCO3 to pH 5–6, and concentrated. The concentrated mixture was diluted with
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
H2O to 15 mL and extracted with CHCl3 for three times (15 mL, 10 mL 2). After evaporation of the combined organic solvent layers, a crude product (85 mg) was purified by silica gel CC, eluting with CHCl3 to afford 3a (25 mg) and 4a (18 mg). 4.15. Compound 3a Pale yellow needles (Me2CO); mp 198–200 °C; UV (MeOH) kmax (log e): 305 (sh, 3.61), 264 (4.01), 239 (4.20) nm; IR vmax (KBr) cm– 1 : 3295 (OH), 1725 (OC@O), 1639 (C@O), 1580, 1499, 1438, 1398, 1366, 1299, 1263, 1216, 1112, 1065, 1047, 990, 919, 859, 760, 644; 1H NMR (400 MHz, acetone-d6): d 7.58 (1H, dd, J = 7.1, 2.4 Hz, H-5), 7.39 (2H, m, H-6 and H-7), 3.97 (3H, s, OCH3), 2.51 (3H, s, COCH3); 13C NMR (100 MHz, acetone-d6): d 197.6 (C-3a), 176.6 (C-4), 161.9 (C-2a), 150.0 (C-2), 147.9 (C-8), 145.8 (C-8a), 128.5 (C-3), 127.1 (C-6), 126.0 (C-4a), 121.3 (C-7), 115.9 (C-5), 53.9 (OCH3), 31.2 (C-3b); ESI-MS (positive) m/z: 263 [M+H]+. 4.16. Compound 4a Pale yellow needles (Me2CO); mp 163–165 °C; UV (MeOH) kmax (log e): 310 (3.77), 259 (sh, 3.99), 222 (4.31) nm; IR vmax (KBr) cm– 1 : 3550 (OH), 2925, 1724 (OC@O), 1679, 1645 (C@O), 1592, 1540, 1384, 1364, 1299, 1206, 1127, 1039, 985, 778, 747, 726; 1H NMR (400 MHz, acetone-d6): d 7.55 (1H, m, H-5), 7.36 (2H, m, H-6 and H-7), 3.86 (3H, s, OCH3), 2.74 (3H, s, CH3-2); 13C NMR (100 MHz, acetone-d6): d 187.4 (C-3a), 176.4 (C-4), 175.1 (C-2), 164.7 (C3b), 147.0 (C-8), 145.5 (C-8a), 126.9 (C-6), 125.0 (C-4a), 121.1 (C7), 117.7 (C-3), 115.6 (C-5), 52.4 (OCH3), 19.8 (C-2a); ESI-MS (positive) m/z: 263 [M+H]+, 547 [2M+Na]+. 4.17. Anti-HBV tests The human HBV-infected cell line HepG2 2.2.15 was used in this study. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, 100 lg/mL streptomycin and 2 mmol/L L-glutamine (all from Invitrogen, USA). Cells were cultured at 37 °C in a humidified incubator at 5% CO2. Test reagents were dissolved in DMSO and then further diluted to 100, 50, and 25 lg/mL, respectively. Cells were seeded in 96-well tissue culture plates and incubated for 48 h, then serial dilutions of the test reagents were added into cultures and the medium was refreshed every 3 days. Nine days after incubation at 37 °C, the culture media were collected to measure their HBsAg and HBeAg concentration using enzyme-linked immunosorbent assay (ELISA) kits (Shanghai SIIC KEHUA Biotech Co. Ltd.). Each measurement was performed in triplicate. The inhibition rates (%) of the test reagents were calculated using the following formula: inhibition ratio% = [OD (control) OD (sample)]/OD (control) 100%. Cytotoxicity of the test reagents was assessed using the MTT assay (Wang et al., 2009a). Acknowledgments This work was financially supported by National Drug Innovative Program (Grant No. 2009ZX09301-011). We thank Mrs. Huimin Wang, Shanghai Institute of Pharmaceutical Industry, for acquiring the HR-ESI-MS data. We also thank Prof. Xiao-kuang Ma, Department of Pharmacognosy, School of Pharmacy, Dali University, PR China, for collection and identification of the plant. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2011.09.015.
175
References Bennett, G.J., Lee, H.H., 1991. Halenia elliptica xanthone – a structural revision. Phytochemistry 30, 1347–1348. Clarke, D.S., Gabbutt, C.D., Hepworth, J.D., Heron, B.M., 2005. Synthesis of 3-alkenyl2-arylchromones and 2,3-dialkenylchromones via acid-catalysed retro-Michael ring opening of 3-acylchroman-4-ones. Tetrahedron Lett. 46, 5515–5519. Dhasmana, H., Garg, H.S., 1989. Two xanthone glucosides from Halenia elliptica. Phytochemistry 28, 2819–2821. Dhasmana, H., Garg, H.S., 1990. Xanthones of Halenia elliptica. Phytochemistry 29, 961–964. Gao, J., Wang, S.J., Fang, F., Si, Y.K., Yang, Y.C., Liu, G.T., Shi, J.G., 2004a. Xanthones from Tibetan medicine Halenia elliptica and their antioxidant activity. Acta Acad. Med. Sin. 26, 364–367. Gao, J., Wang, S.J., Xu, R.M., Yang, Y.C., Zhang, S.R., Si, Y.K., Shi, J.G., 2004b. Studies on chemical constituents of Halenia elliptica guided by in vivo absorption and distribution of the constituents. Acta Pharm. Sin. 39, 198–203. Gendaramyn, O., Kojima, K., Rodriguez, S., Ondognii, P., Ogihara, Y., Hostettmann, K., 1998. Xanthones from Halenia corniculata. Chem. Pharm. Bull. 46, 1827–1828. Grayer, R.J., Veitch, N.C., 1998. An 8-hydroxylated external flavone and its 8-Oglucoside from Becium grandiflorum. Phytochemistry 47, 779–782. Harborne, J., Baxter, H., Moss, G., 1999. Phytochemical Dictionary: A Handbook of Bioactive Compounds from Plants. Taylor & Francis, London, pp. 381–389. Huang, B., Ban, X., He, J., Zeng, H., Zhang, P., Wang, Y., 2010. Hepatoprotective and antioxidant effects of the methanolic extract from Halenia elliptica. J. Ethnopharmacol. 131, 276–281. Ito, H., Kasajima, N., Tokuda, H., Nishino, H., Yoshida, T., 2004. Dimeric flavonol glycoside and galloylated C-glucosylchromones from Kunzea ambigua. J. Nat. Prod. 67, 411–415. Jensen, S.R., Schripsema, J., 2001. 6 Chemotaxonomy and pharmacology of Gentianaceae. In: Struwe, L., Albert, V.A. (Eds.), Gentianaceae: Systematics and Natural History. Cambridge University Press, Cambridge, pp. 573–631. Jung, J.C., Min, J.P., Park, O.S., 2001. A highly practical route to 2-methylchromones from 2-acetoxybenzoic acids. Synth. Commun. 31, 1837–1845. Klutchko, S., Shavel, J., von Strandmann, M., 1974. Base rearrangement of chromone-3-carboxylic esters to 3-acyl-4-hydroxycoumarins. J. Org. Chem. 39, 2436–2437. Kuroda, M., Uchida, S., Watanabe, K., Mimaki, Y., 2009. Chromones from the tubers of Eranthis cilicica and their antioxidant activity. Phytochemistry 70, 288–293. Larget, R., Lockhart, B., Renard, P., Largeron, M., 2000. A convenient extension of the Wessely–Moser rearrangement for the synthesis of substituted alkylaminoflavones as neuroprotective agents in vitro. Bioorg. Med. Chem. Lett. 10, 835–838. Linke, H.A.B., 1969a. Hydroxychromones – synthesis ultraviolet spectrophotometric and polarographic properties. Z. Naturforsch. B: Chem. Sci. 24, 225. Linke, H.A.B., 1969b. Infrarotspektren der Hydroxychromone: die absorption der carbonyl-valenzschwingung. Spectrochim. Acta A 25, 1067–1074. Liu, X., Liu, Y., Shi, Y., 2009. Xanthone glycosides from Tibetan herb Halenia elliptica. China J. Chin. Mater. Med. 34, 580–582. Ma, W.G., Fuzzati, N., Lu, S.L., Gu, D.S., Hostettmann, K., 1996. Further chromones from Eriosema tuberosum. Phytochemistry 43, 1339–1343. Peres, V., Nagem, T.J., 1997. Trioxygenated naturally occurring xanthones. Phytochemistry 44, 191–214. Peres, V., Nagem, T.J., de Oliveira, F.F., 2000. Tetraoxygenated naturally occurring xanthones. Phytochemistry 55, 683–710. Rao, C.R., Venkateswarlu, V., 1956. Synthesis of 5- and 5,8-dimethoxy-2-methyl chromones. Recl. Trav. Chim. Pay. B 75, 1321–1326. Recio-Iglesias, M.C., Marston, A., Hostettmann, K., 1992. Xanthones and secoiridoid glucosides of Halenia campanulata. Phytochemistry 31, 1387–1389. Rodriguez, S., Wolfender, J.L., Hostettmann, K., Odontuya, G., Purev, O., 1996. Corniculoside, a new biosidic ester secoiridoid from Halenia corniculata. Helv. Chim. Acta 79, 363–370. Rodriguez, S., Wolfender, J.L., Odontuya, G., Purev, O., Hostettmann, K., 1995. Xanthones, secoiridoids and flavonoids from Halenia corniculata. Phytochemistry 40, 1265–1272. Stout, G.H., Fries, J.L., 1970. The xanthones of a Halenia species. Phytochemistry 9, 235–236. Struwe, L., Albert, V.A., 2002. Gentianaceae: Systematics and Natural History. Cambridge University Press, Cambridge. Sun, H.F., Hu, B.L., Ding, J.Y., Fan, S.F., 1987. Three new glycosides from Halenia elliptica. Acta Bot. Sin. 29, 422–428. Sun, H.F., Hu, B.L., Fan, S.F., Ding, J.Y., 1983. Three new xanthones from Halenia elliptica D. Don. Acta Bot. Sin. 25, 460–467. Sun, Y.W., Sun, Z.H., Yu, P.Z., 2011. A new xanthone from Halenia elliptica D. Don. J. Asian Nat. Prod. Res. 13, 88–92. Tanaka, T., Koike, K., Mitsunaga, K., Narita, K., Takano, S., Kamioka, A., Sase, E., Ouyang, Y., Ohmoto, T., 1995. Chromones from Harrisonia perforata. Phytochemistry 40, 1787–1790. Tsui, W.Y., Brown, G.D., 1996. Chromones and chromanones from Baeckea frutescens. Phytochemistry 43, 871–876. Tuntiwachwuttikul, P., Phansa, P., Pootaeng-on, Y., Taylor, W.C., 2006. Chromones from the branches of Harrisonia perforata. Chem. Pharm. Bull. 54, 44–47. Wang, S.G., Li, J.Y., Huang, H., Gao, W., Zhuang, C.L., Li, B., Zhou, P., Kong, D.Y., 2009a. Anti-hepatitis B virus activities of astragaloside IV isolated from Radix Astragali. Biol. Pharm. Bull. 32, 132–135.
176
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
Wang, Y., Shi, J.G., Wang, M.Z., Che, C.T., Yeung, J.H.K., 2007. Mechanisms of the vasorelaxant effect of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery. Life Sci. 81, 1016–1023. Wang, Y., Shi, J.G., Wang, M.Z., Che, C.T., Yeung, J.H.K., 2008. Mechanisms of the vasorelaxant effect of 1,5-dihydroxy-2,3-dimethoxy-xanthone, an active metabolite of 1-hydroxy-2,3,5-trimethoxy-xanthone isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery. Life Sci. 82, 91–98. Wang, Y., Shi, J.G., Wang, M.Z., Che, C.T., Yeung, J.H.K., 2009b. Vasodilatory actions of xanthones isolated from a Tibetan herb, Halenia elliptica. Phytomedicine 16, 1144–1150.
Yang, H.P., Yue, M.E., Shi, Y.P., 2006. CZE Determination of flavonoids in Halenia elliptica. Chromatographia 63, 449–452. Yu, P.Z., Shen, X.J., Sun, Y.W., Hu, C.Q., 2009. HPLC simultaneous determination of four xanthones in Halenia elliptica D. Don. Chin. J. Pharm. Anal. 29, 540–543. Yu, P.Z., Shen, X.J., Hu, C.Q., Meehan, E.J., Chen, L.Q., 2008. 1,7-Dihydroxy-2,3,4trimethoxy-9H-xanthen-9-one monohydrate from Halenia elliptica. Acta Crystallogr. Sect. E: Struct. Rep. Online 64, O651–U823.
Contents lists available at SciVerse ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Chromone derivatives from Halenia elliptica and their anti-HBV activities Yu-wei Sun a, Guang-ming Liu b, Hai Huang c, Pei-zhong Yu a,⇑ a
Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, PR China Department of Medicinal Chemistry, School of Pharmacy, Dali University, Dali, Yunnan 671000, PR China c Department of Biosynthetic Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, PR China b
a r t i c l e
i n f o
Article history: Received 13 January 2011 Received in revised form 28 July 2011 Accepted 20 September 2011 Available online 21 December 2011 Keywords: Halenia elliptica Gentianaceae Chromone-2-carboxylic acid 2-Methylchromone Acid-catalyzed isomerization Anti-HBV
a b s t r a c t Nine water-soluble chromone derivatives, including chromone-2-carboxylic acids, 2-methylchromones and their structural hybrids, were isolated from aerial tissues of Halenia elliptica (Gentianaceae), six of which were previously unknown. Their structures were elucidated by comprehensive mass, 1D and 2D NMR spectroscopic analyses and chemical derivatization. Two unstable structural hybrids of chromone-2-carboxylic acids and 2-methylchromones, viz. 3-acetyl-8-hydroxy-4H-1-benzopyran-4-one-2carboxylic acid (halenic acid C) and 2-(8-hydroxy-2-methyl-4H-1-benzopyran-4-one-3-yl)-2-oxoacetic acid (halenichromone A), were isomers and were interconvertible. The proposed mechanism of their acid-catalyzed isomerization in aqueous solvent is described. In addition, 2-methylchromones, 8hydroxy-2-methyl-4H-1-benzopyran-4-one, and 8-methoxy-2-methyl-4H-1-benzopyran-4-one, were found to exhibit a strong inhibitory effect towards hepatitis B virus (HBV) in vitro without showing significant cytotoxicity. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Halenia elliptica D. Don (Gentianaceae) is distributed across the Tibetan plateau and throughout western China. It has been used for centuries in traditional Tibetan and Mongolian medicine to cure hepatitis. Previous phytochemical investigations led to the isolation of a series of simple oxygenated xanthone aglycones and glycosides (Bennett and Lee, 1991; Dhasmana and Garg, 1989, 1990; Liu et al., 2009; Sun et al., 1983, 1987, 2011; Yu et al., 2008). These xanthone compounds are believed to contribute to the hepatoprotective (Gao et al., 2004b; Huang et al., 2010), vasorelaxant (Wang et al., 2007, 2008, 2009b) and antioxidant (Gao et al., 2004a; Harborne et al., 1999) activities ascribed to this particular species. In the current research, the water-soluble portion of the aerial tissues of H. elliptica was found to have potent anti-HBV activity in vitro (data not shown). This finding prompted us to characterize the polar constituents present in these aqueous extracts. Described herein is the isolation and structural elucidation of six chromone derivatives (1–6, halenic acid A–C, halenichromone A–C) and three known 2-methylchromones (7–9) from the water-soluble extract of H. elliptica (Fig. 1). Compounds 7–9 were isolated from natural sources for the first time. In vitro HBV inhibitory effects of compounds 1–4, 8 and 9 were also tested. Chromones are a class of plant secondary metabolites having a 4H-1-benzopyran-4-one skeleton and occur only in a relatively ⇑ Corresponding author. Tel./fax: +86 21 51980103. E-mail address: [email protected] (P.-z. Yu). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.09.015
small number of plant species (Harborne et al., 1999). To our knowledge, there have hitherto been no reports describing the presence of chromone derivatives in the Gentianaceae, which contains 87 genera and over 1600 angiosperm species (Struwe and Albert, 2002). The genus Halenia is one of the genera in this family rich in polyoxygenated xanthones. Up to now, only lipophilic xanthones have been reported as taxonomically informative compounds within the genus Halenia (Jensen and Schripsema, 2001). Chromone derivatives found in this research may be used as polar markers with chemotaxonomic significance. 2. Results and discussion 2.1. Structural elucidation Halenic acid A (1, Fig. 1) was obtained as an amorphous white powder with molecular formula C10H6O5 as determined by HRESI-MS. The IR spectrum of 1 showed a characteristic absorption band due to the presence of a conjugated carbonyl (1624 cm–1), and a strong broad band (3600–2400 cm–1) which clearly indicated the presence of a carboxyl group. An apparent increase in its HPLC retention time in low pH buffer (pH = 3–5) also indicated the acidic nature of 1 (Supplementary data, Fig. S74). The 1H NMR spectrum of 1 in DMSO-d6 had one olefinic proton (dH 6.69, H-3) assigned to a trisubstituted olefin and three aromatic protons (dH 7.39, H-5; dH 7.25, H-6; dH 7.18, H-7) attributed to a 1,2,3-trisubstituted aromatic ring (Table 1). Ten carbon signals were also observed in the 13C NMR spectrum, including four sp2 methines (one olefinic
170
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
Fig. 2. Key HMBC correlations of 1 and 5 (H ? C).
also showed a strong broad band representative of a carboxyl group (3600–2500 cm–1) which was responsible for the same HPLC chromatographic behavior as shown by 1. An additional signal of a methoxyl group (dH 3.97, dC 56.4) was observed in its 1H and 13C NMR spectra (Table 1), indicating that the 8-hydroxyl group in 1 was replaced by a methoxyl group. This conclusion was proved by the NOE correlation between H-7 and the methoxyl protons. The HMBC spectrum showed all the important long-range C–H correlations identical to those of 1. Therefore, compound 2 was established as 8-methoxy-4H-1-benzopyran-4-one-2-carboxylic acid, with a trivial name of halenic acid B. Halenic acid C (3) and halenichromone A (4) were obtained as a mixture. Although they could be separated by C18 reversed-phase silica gel column chromatography (CC), both purified compounds interconverted into an equilibrium during the removal of the solvent (MeOH–H2O) at 45 °C (Supplementary data, Fig. S75). The composition of the mixture also changed gradually in aqueous DMSO at room temperature over several days. The color of the sample was a good indicator of purity, since mixtures of 3 and 4 were yellow and upon purification they became colorless (Supplementary data, Fig. S76). The above facts suggested that 3 and 4 equilibrated in the solvent. Since these compounds could not be thoroughly separated by conventional chromatographic methods, fraction C1 (a mixture containing ca. 55% of 3 and ca. 35% 4, as calculated from HPLC peak area integral) was analyzed by LC–ESI-MS; Compounds 3 and 4 were distinguished by their retention times. A low pH buffer greatly increased their retention times, implying that 3 and 4 were also acidic compounds like 1 and 2. LC–ESI-MS analyses of 3 (peak A, tR = 10.5 min) and 4 (peak B, tR = 7.1 min) both gave quasimolecular ion peaks at m/z 247 [M H] . Furthermore, compound 3 (peak A, tR: 10.5 min) was prone to lose a moiety represented by a carboxyl group to give a peak at m/z 203 [M–H–COO] (Supplementary data, Fig. S77). The 1H and 13C NMR spectra of 3 and 4 could only be recorded using relatively purified mixtures. Analysis
Fig. 1. Compounds 1–9 and methyl ester derivatives 3a and 4a.
methine, three aromatic methines), four quaternary sp2 carbons (dC 124–161, showing no HSQC correlation with any protons), a conjugated carbonyl carbon (dC 179.1) and a carboxyl carbon (dC 161.7) (Table 1). The HMBC experiment connected the above-mentioned partial structures and functional groups though long-range C–H correlations to establish a chromone skeleton (Fig. 2). Three adjacent protons were assigned to H-5, H-6 and H-7 on the B ring of the chromone skeleton according to the HMBC correlations of H5/carbonyl carbon (C-4), C-7 and C-8a; H-6/C-8 and C-4a; H-7/C5 and C-8a. Other important HMBC correlations observed between the olefinic proton (H-3) and C-4a, C-2 and the carboxyl carbon suggested that the carboxyl group was at C-2. By considering the molecular formula and the downfield chemical shift of C-8 (dC 147.5), a hydroxyl group was located at C-8. Thus, the structure of compound 1 was established as 8-hydroxy-4H-1-benzopyran4-one-2-carboxylic acid and 1 was named halenic acid A. Halenic acid B (2) was deduced to be a congener of 1 on the basis of their similar 1H and 13C NMR spectra and its molecular formula C11H8O5 determined by HR-ESI-MS. The IR spectrum of 2 Table 1 1 H (400 MHz) and
13
C (100 MHz) NMR spectroscopic data for compounds 1–6.
Position 1 (in DMSO-d6)
2 3 4 4a 5 6 7 8 8a COOH-2 OCH3-8
a b c
dC
dH, mult (J in Hz)
160.4 110.2 179.1 124.7 113.7 125.5 119.4 147.5 145.1 161.7 –
– 6.69, – – 7.39, 7.25, 7.18, – – – –
2 (in DMSO-d6) dC
153.4 113.4 177.7 124.8 dd (7.8, 1.5) 115.3 t (7.8) 126.0 d (7.8) 116.1 149.0 145.8 161.4 56.4 s
Position 3 (in DMSO-d6)
dH, mult (J in Hz)
dC
dH, mult (J in Hz)
– 6.91, – – 7.56, 7.45, 7.50, – – – 3.97,
155.5 125.3 176.8 124.3 113.5 125.4 119.5 147.7 144.8 198.3 31.6 161.3
– – – – 7.41, 7.27, 7.27, – – – 2.33, –
2 3 4 4a d (7.6) 5 t (7.8) 6 d (6.8) 7 8 8a 3a s 3b 2a s
a
4 (in DMSO-d6)
a
Position 5 (in acetone-d6)
dC, mult dH, mult (J in Hz)
175.2 121.5 176.8 124.2 dd (7.0, 2.4) 114.0 c m 125.5 mc 119.5 146.6 144.8 N.D.b s 166.1 18.9
– – – – 7.39, 7.27, 7.22, – – – – 2.33,
2 3 4 4a d (7.7) 5 t (7.7) 6 d (7.6) 7 8 8a CH3-2 OCH3-6 s OH-5 OH-3
6 (in acetone-d6)
dC
dH, mult (J in Hz)
dC
dH, mult (J in Hz)
169.8 108.4 184.8 111.2 150.6 144.2 121.0 106.5 151.4 20.5 57.3 – –
– 6.16, s – – – – 7.40, d (9.0) 6.94, d (9.1) – 2.42, s 3.87, s 12.84, s –
137.4 152.8 177.5 111.0 149.7 143.2 121.7 106.9 150.6 15.1 57.6 – –
– – – – – – 7.41, d (9.2) 6.97, d (9.1) – 2.45, s 3.88, s 12.27, s 7.72, s
The assignments of 3 and 4 were based on HSQC and HMBC experiments, together with comparison with equivalent signals of compounds 3a and 4a. Signal undetermined. This signal was absent probably due to the instability of sample. 2H, overlapped signals.
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
of both 1H NMR spectra indicated presence of a methyl group at dH 2.33, and three aromatic protons (dH 7.2–7.4) (Table 1). This similarity, combined with the same molecular formulae C12H8O6 established by HR-ESI-MS, indicated that 3 and 4 were structurallyrelated isomers. The interconversion of 3 and 4 occurred during the 13C and 2D NMR spectroscopic measurements, which caused great difficulties for further structural elucidation. In their 13C NMR spectra, some quaternary carbons were indistinguishable or absent due to their instability. However, the main parts of the 13 C NMR spectra (C4-8, C-4a and C-8a) showed good agreement with those of 1, which suggested that the frameworks of 3 and 4 were also chromones (Table 1). In spite of all the difficulties due to the impurity of the sample, the 13C NMR spectra of compounds 3 and 4 did display a few notable differences between these molecules. The chemical shift of a methyl carbon in 3 was shifted significantly from 31.6 to 18.9 ppm in 4. In order to prevent their interconversion and allow the structural elucidation of 3 and 4, fraction C1 (mainly composed of 3 and 4) were refluxed with methanol, using the Fischer–Speier esterification method, to afford a mixture of methyl ester derivatives 3a and 4a. Compounds 3a and 4a were stable and could be readily separated using silica gel CC. The MS, IR, 1H, 13C and 2D NMR spectroscopic analyses of 3a and 4a finally led to the structural characterization of 3 and 4. Compounds 3a and 4a both showed the presence of a pseudomolecular ion peak at m/z 263 [M+H]+ in the ESI-MS and a characteristic absorption band consistent with an ester (1725 cm–1) in their IR spectra. Taken together with the evidence of additional signals indicating the presence of methoxyl groups (dH 3.97, dC 53.9 in 3a; dH 3.86, dC 52.4 in 4a) in their 1H and 13C NMR spectra, these were confirmed as methyl ester derivatives of 3 and 4. Thirteen carbon signals of 3a and 4a were clearly observed in both 13C NMR spectra. Compounds 3a and 4a shared the same chromone skeleton, especially their trisubstituted B ring, because of the similar 13C NMR spectroscopic data (C-4a, C5–8, C-8a, DdC < ±0.7). Three adjacent protons were assigned to H-5, H-6 and H-7 on the B ring in the same way as those of 1 and 2. The olefinic proton (H-3) in 1 and 2 was absent in 3a and 4a (also in 3 and 4). In 3a, H-3 was replaced by an acetyl group and a methyl-esterified carboxyl group was linked to C-2. The HMBC spectrum of 3a supported this conclusion by C–H correlation from methyl protons (dH 2.51) to a ketone carbonyl (dC 197.6) and further to the quaternary sp2 carbon C-3 (dC 128.5) (Fig. 3). In 4a, a methyl group located at C-2 was deduced from the HMBC correlation of methyl protons (dH 2.74)/oxygenated quaternary sp2 carbon C-2 (dC 175.1) and quaternary sp2 carbon C-3 (dC 117.7) (Fig. 3). Thus, it is possible to explain why the methyl carbons of 3 (also 3a) and 4 (also 4a) differ in their chemical shifts. The methyl group of 3 (3a) appeared in a ketone (RCOCH3), showing a representative 13C chemical shift of 31 ppm, while for 4 (4a), the methyl group was attached directly to the A ring of chromone at C-2 to establish a 2-methylchromone skeleton. According to reported data of known 2-methylchromones (Ito et al., 2004; Kuroda et al., 2009; Tanaka et al., 1995; Tsui and Brown, 1996; Tuntiwachwuttikul et al., 2006), the typical chemical shift of the 2-methyl carbon is around 20 ppm. On the basis of the above findings, the structures of methyl ester derivatives 3a and 4a
Fig. 3. Key HMBC correlations of 3a and 4a (H ? C).
171
were elucidated and their corresponding carboxylic acids were established as 3-acetyl-8-hydroxy-4H-1-benzopyran-4-one-2-carboxylic acid (3, halenic acid C), and 2-(8-hydroxy-2-methyl-4H1-benzopyran-4-one-3-yl)-2-oxoacetic acid (4, halenichromone A), respectively. Halenichromone B (5), a pale yellow amorphous solid, showed a pseudomolecular ion peak at m/z 207 [M+H]+ in its positive mode ESI-MS. Its molecular formula, C11H10O4, determined by HR-ESIMS, indicated seven degrees of unsaturation. The IR spectrum of 5 showed characteristic absorption bands due to conjugated carbonyl (1661 cm–1) and hydroxyl (3424 cm–1) groups. Furthermore, the hydroxyl group was confirmed by a hydrogen-bonded OH singlet observed at dH 12.84 in the 1H NMR spectrum. The 1H and 13C NMR spectra of 5 also had one broad singlet (dH 6.16, dC 108.4) assigned to a trisubstituted olefin, a pair of ortho-coupled doublets [dH 6.94 (d, J = 9.1 Hz), dC 106.5; dH 7.40 (d, J = 9.0 Hz), dC 121.0], indicating the presence of a 1,2,3,4-tetrasubstituted aromatic ring, along with one aromatic methyl group (dH 2.42, dC 20.5) and one methoxyl group (dH 3.87, dC 57.3) (Table 1). The remaining carbons included one quaternary sp2 carbon, four oxygenated quaternary sp2 carbons and a conjugated carbonyl carbon (C-4, dC 184.8) (Table 1). In the 1D-NOESY experiment, irradiation of the 2-methyl protons enhanced the signal intensity of the olefinic proton (H3). All the above analyses, in combination with the comparison of 13C NMR spectroscopic data with those of the reported 2-methylchromones, suggested the 2-methylchromone skeleton of 5 as previously described (Ito et al., 2004; Kuroda et al., 2009; Tanaka et al., 1995; Tsui and Brown, 1996; Tuntiwachwuttikul et al., 2006). Analysis of the HMBC spectrum then supported this by the long-range C–H correlations of the 2-methyl protons to C-2 (dC 169.8) and C-3, and of H-3 to C-2, methyl carbon and C-4a (dC 111.2) (Fig. 2). Also in the HMBC spectrum, the chelated OH-5 showed C–H correlations with C-5, C-4a and a methoxyl-bearing carbon (based on the HMBC correlation from methoxy protons to this carbon) at dC 144.2. This indicated that the methoxyl group was adjacent to OH-5 and located at C-6. The ortho-coupled aromatic protons were assigned to H-7 and H-8, on the basis of the HMBC correlations of H-7/C-5 and C-8a, H-8/C-6 and C-4a. Thus, compound 5 was characterized as 5-hydroxy-6-methoxy-2methyl-4H-1-benzopyran-4-one. Halenichromone C (6) was obtained as an amorphous yellow powder. Its molecular formula was established to be C11H10O5 by HR-ESI-MS, and was larger than that of 5 by an oxygen atom. The IR spectrum of 6 showed absorption bands consistent with conjugated carbonyl (1652 cm–1) and hydroxyl (3323 cm–1) groups. The 1H NMR spectroscopic data of 6 were superimposable on those of 5, except for the absence of an olefinic proton (H-3) (Table 1). Thus, 6 appeared to be the 3-hydroxylated derivative of 5. In the 13C NMR spectrum, carbon signals corresponding to the B ring of the chromone skeleton were almost identical with those of 5 (C4a, C5–8, C-8a, DdC < ±1.0) (Table 1), while resonances assigned to the A ring were notably different from those of 5. Due to the hydroxylation of H-3, the signal of C-3 was shifted downfield significantly from dC 108.4 in 5 to dC 152.8 in 6. In contrast, the neighboring C-2 was shifted upfield by about 30 ppm compared with the equivalent carbon in 5 (Table 1). The carbonyl (C-4, dC 177.5) and 2-methyl carbon (dC 15.1) were also shifted to some extent. Analysis of the HMBC spectrum established long-range C–H correlations of methyl protons/C-2 and C-3, methoxy protons/C-6 as expected. Therefore, compound 6 was established as 3,5-dihydroxy-6-methoxy-2-methyl-4H-1-benzopyran-4-one. Three known 2-methylchromones were identified as 5-hydroxy-8-methoxy-2-methyl-4H-1-benzopyran-4-one (7), 8-hydroxy-2-methyl-4H-1-benzopyran-4-one (8), and 8-methoxy-2-methyl-4H-1-benzopyran-4-one (9). They were all previously synthesized compounds. Compounds 7 (Rao and
172
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
Fig. 4. Proposed acid-catalyzed isomerization of 3 and 4.
Venkateswarlu, 1956) and 8 (Linke, 1969a, b) were synthesized during the 1950–1970s for UV or IR studies, however, no NMR spectroscopic data were reported. The 1H and 13C NMR spectroscopic data of 9 were reported though in DMSO-d6 (Jung et al., 2001). This is the first report of 7, 8 and 9 isolated from a natural source. Their structures were confirmed by MS, IR, 1D and 2D NMR spectroscopic techniques. In this study, the NMR spectroscopic data of 7, 8 and 9 are reported as a supplement to the known compounds. The amount of 7 was insufficient to obtain 13C NMR spectroscopic data. The difference between our NMR spectroscopic data and those reported for compound 9 may be due to the different solvents used. 2.2. Proposed mechanism of interconversion of 3 and 4 Among the chromones isolated from H. elliptica, halenic acid C (3) and halenichromone A (4) intrigued us by their isomerization equilibrium. This phenomenon was presumed to result from an intramolecular rearrangement, involving an acid-catalyzed opening of the A ring (pyrone ring) in aqueous solvent to form a triketone intermediate product 10, and then a ring closure with C-3a (carbonyl) to the phenolic hydroxyl at C-8a (Fig. 4). This intramo-
lecular rearrangement is similar to the classic Wessely–Moser rearrangement. In this rearrangement, a rotation takes place in the bond between C-4 and C-4a, resulting in the change of the B ring (Fig. 5), whereas for the isomerization of 3 and 4, it takes place in the bond between C-4 and C-3 (Fig. 4). Thus, in the Wessely– Moser rearrangement, an intermediate product 11 with two meta-phenolic hydroxyl groups at C-5 and C-8a are prerequisites. Wessely–Moser rearrangements have been reported in flavonoids and chromones, in both synthesized and naturally occurring compounds (Grayer and Veitch, 1998; Larget et al., 2000; Ma et al., 1996). In this research, compound 7 appears to be a classic Wessely–Moser rearrangement product of compound 5 (Fig. 5) and may be an artifact formed during the isolation procedure. In the rearrangement of 3 and 4, the non-regiospecific cyclization of triketone 10 is the key step. Triketone 10 contains more hydroxyls than 3 and 4. Since hydroxy in direct conjugation with benzene ring is an auxochrome, the mixture of 3 and 4 shows a darker color than both purified compounds. Compounds 1 and 2 are stable because they are unable to form triketone intermediates like 10 after the opening of their A rings. Compounds 3a and 4a, the methyl ester derivatives of 3 and 4, are also stable during their isolation and structural elucidation. This is because they are relatively lipophilic, and the use of an aprotic solvent during purification prevents opening of the A ring. This type of rearrangement was reported in synthesized compounds (Clarke et al., 2005; Klutchko et al., 1974). To our knowledge, 3 and 4 are the first examples of natural chromones that isomerize through this type of rearrangement. Since the chromone derivatives found in this research featured either a carboxyl (compounds 1–3) or a methyl group (compounds 4–9) at the C-2 position, they can be classified as a chromone-2carboxylic acid for 3 and a 2-methylchromone for 4. These two compounds were thus named as halenic acid C and halenichromone A, respectively, but they are actually structural hybrids of chromone-2-carboxylic acid and 2-methylchromone. They are presumed to be a pair of important transitional compounds in the biosynthetic pathway of chromone-2-carboxylic acids and 2-methylchromones present in this plant.
2.3. Anti-HBV activity Compounds 1–4, 8 and 9, in sufficient amounts, were tested for their potencies towards inhibiting the secretion of HBV antigens in
Fig. 5. Wessely–Moser rearrangement of 5 and 7.
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176 Table 2 HBV inhibitory activity of compounds 1–4, 8 and 9. Compound
Concentration (lg/mL)
Cell growth inhibition rate (%)a
HBsAg (inhibition rate %)
HBeAg (inhibition rate %)
1
100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 100
0 0 0 0 0 0 0 0 0 25 0 0 0 0 0 0
16.4 ± 0.8 0±0 0±0 12.0 ± 0.6 3.6 ± 1.0 1.9 ± 0.3 0±0 0±0 0±0 /b 36.8 ± 2.3 5.9 ± 2.3 70.9 ± 2.4 60.2 ± 2.2 55.6 ± 3.8 20.1 ± 1.2
0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 /b 41.5 ± 3.4 18.0 ± 2.5 14.1 ± 1.4 6.3 ± 0.8 0±0 19.7 ± 1.3
2
3/4
8
9
3TCc
a Cell damage was assessed by means of MTT assay, cell growth inhibition P10% was considered as cytotoxic. b / Means the compound was cytotoxic at this concentration. c Positive control (3TC = Lamivudine).
the human HBV-transfected liver cell line HepG2 2.2.15. Lamivudine (3TC) was used as a positive control that can suppress HBsAg secretion by 20.1% and HBeAg secretion by 19.7%, at 100 lg/mL (436 lM). Compounds 3 and 4 were tested using a mixture due to their instability in DMSO. The results are summarized in Table 2. 2-Methylchromone compounds 8 and 9 exhibited strong anti-HBV activities, inhibiting HBsAg secretion by 36.8% at a non-cytotoxic concentration of 50 lg/mL (284 lM) for 8 and by 70.9% at a noncytotoxic concentration of 100 lg/mL (526 lM) for 9. Compound 8 also suppressed HBeAg secretion by 41.5% at 50 lg/mL (284 lM). Acidic compounds 1–4, which possessed a carboxyl group, were slightly active or totally inactive at low concentration. Further investigations are warranted to explore the value of 2methylchromones as anti-HBV agents.
173
genus Halenia consists of approximately 100 species, among which only 4 have been studied (Yang et al., 2006). Species belonging to Halenia are found to produce similar xanthones, flavonoids and secoiridoids (Dhasmana and Garg, 1989, 1990; Gao et al., 2004a; Gendaramyn et al., 1998; Liu et al., 2009; Recio-Iglesias et al., 1992; Rodriguez et al., 1995, 1996; Stout and Fries, 1970; Sun et al., 1983, 1987, 2011). Our work is the first systematic study of H. elliptica that has focused on its water-soluble constituents, suggesting simple chromones are representatives of polar constituents in this species. Moreover, since chromone-2-carboxylic acids are quite rare in nature, these unique chromone compounds may be used as chemotaxonomic markers in the classification of Halenia species. 4. Experimental 4.1. General experimental procedures Melting points were determined on an X4 micromelting point apparatus. UV spectra were recorded on Hitachi U-2900 double beam spectrophotometer, whereas IR spectra (KBr) were obtained using an Avatar 360 ESP FT-IR spectrometer (v in cm–1). NMR spectra were recorded at 400 MHz for 1H and 100 MHz for 13C on a Varian Mercury Plus-400 spectrometer, in DMSO-d6 or acetone-d6, using residual solvent signals (dH/C 2.50/39.51 for DMSO-d6; dH/C 2.05/29.92 for acetone-d6) or tetramethylsilane (TMS) as internal standards. The chemical shifts (d) were given in ppm and the coupling constants (J) in Hz. ESI-MS and LC–ESI-MS data were measured with an Agilent 1100 chromatography system coupled with an Agilent G1946D mass detector. HR-ESI-MS data were measured with a YA019 Q-Tof micro mass spectrometer. Column chromatography (CC) was performed with silica gel H (10–40 lm, Qingdao Haiyang Chemical Co., Qingdao, China), Diaion HP-20 (Mitsubishi Chemicals Co., Tokyo, Japan), MCI gel CHP 20P (75– 150 lm, Mitsubishi Chemicals Co., Tokyo, Japan), Sephadex LH-20 (Amersham Pharmacia Biosciences, Inc., Piscataway, NJ, USA) and OUYA-RP18 silica gel (15–35 lm, Unicorn, Switzerland). 4.2. Plant material
3. Concluding remarks In this research, nine polar chromone derivatives were isolated from H. elliptica and can be categorized into chromone-2-carboxylic acids (1 and 2), 2-methylchromones (5–9) and their structural hybrids (3 and 4). Six major chromones (1–4, 8 and 9) possess a similar trisubstituted B ring (5,6,7-H; 8-OH or 8-OCH3). According to reports on the phytochemical composition of H. elliptica and from our previous work on its lipid soluble fractions, some of the most abundant xanthones isolated from this plant are 1-hydroxy-2,3,4,5-tetramethoxyxanthone, 1-hydroxy-2,3,5-trimethoxyxanthone, and 1,5-dihydroxy-2,3-dimethoxyxanthone (Wang et al., 2007, 2008; Yu et al., 2009). They also share a trisubstituted ring (B ring) with three adjacent protons (6,7,8-H; 5-OH or 5-OCH3) (Supplementary data, Fig. S78). Such structural similarity suggests that xanthone and chromone compounds presenting in this plant may be biosynthetically related. Chromone compounds are thought to be distributed in a limited number of plant families including the Ranunculaceae, Myrtaceae, Liliaceae, and Clusiaceae (another xanthone-rich family) (Harborne et al., 1999). To our knowledge, this is the first report describing the characterization of chromone derivatives in the Gentianaceae family. The Gentianaceae is, however, rich in natural xanthones (Peres and Nagem, 1997; Peres et al., 2000), and most polyoxygenated xanthones in the Gentianaceae come from the genera Swertia, Halenia, Veratrilla and Frasera (Jensen and Schripsema, 2001). The
H. elliptica was collected during April 2005 in Dali, Yunnan Province, China. The plant was identified by Professor Xiao-kuang Ma, Department of Pharmacognosy, School of Pharmacy, Dali University, PR China. A voucher specimen (No. HE0505) has been deposited at the Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai, PR China. 4.3. Extraction and isolation Air-dried aerial part of H. elliptica (5 kg) was extracted with EtOH–H2O (95:5, v/v, 50 L) at room temperature for 120 h. After evaporation of EtOH under reduced pressure, the residue (140 g) was suspended in H2O and partitioned sequentially with petroleum ether (b.p. 60–90 °C) and EtOAc to remove the lipophilic constituents. The aqueous phase was lyophilized to give a powder (45.2 g), the bulk of which (40.0 g) was dissolved in hot H2O and subjected to CC using a Diaion HP 20 resin column (EtOH–H2O, 0:100 ? 95:5, v/v) to give five fractions: Fr. A–E. Fr.B (2.7 g, eluted by EtOH–H2O, 10:90, v/v) was subjected to C18 reversed-phase silica gel CC (MeOH–H2O, 15:85, v/v) to afford compound 1 (120 mg). Fr.C (1.5 g, eluted by EtOH–H2O, 20:80, v/v) was subjected to MCIgel CHP 20P cc (MeOH–H2O, 0:100 ? 100:0, v/v) to give four subfractions: Fr. C1-C4. Fr.C1 (650 mg) was then purified repeatedly on Sephadex LH-20 (MeOH–H2O, 0:100 ? 10:90, v/v) and on a C18 reversed-phase silica gel column (MeOH–H2O, 15:85, v/v) to
174
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
yield compounds 2 (12 mg), 3 and 4 (mixture, 25 mg), and a small amount of compound 1 (5 mg). Fr.C1 was then analyzed by LC–ESIMS. Fr.C2 (100 mg) was purified on a C18 reversed-phase silica gel column (MeOH–H2O, 30:70, v/v) to yield compound 8 (45 mg). Fr.C4 (80 mg) was purified repeatedly on a C18 reversed-phase silica gel column (MeOH–H2O, 40:60, v/v) to yield compounds 5 (3.5 mg), 6 (2.5 mg) and 7 (0.8 mg). Fr.E (1005 mg, eluted by EtOH–H2O, 95:5, v/v) was subjected to Sephadex LH-20 cc (MeOH–H2O, 0:100 ? 100:0, v/v) to give three subfractions: Fr. E1–E3. Fr. E2 (200 mg) was then separated repeatedly on a C18 reversed-phase silica gel column (MeOH–H2O, 55:45, v/v) to afford compound 9 (38 mg). 4.4. LC–ESI-MS analysis of 3 and 4 Fraction C1 (2 mg) was dissolved in H2O and diluted to 0.5 lg/ lL. Separation was performed on a 5 lm Hypersil ODS2 column (4.6 i.d 250 mm, Dalian Elite Analytical Instruments Co. Ltd., Dalian, PR China). The mobile phase consisted of 0.1% HCOOH in MeOH–H2O (25:75, v/v). The flow rate was 1 mL/min and the temperature was 25 °C (detection: 280 nm). Analyses were conducted in negative ion mode using selected ion monitoring of m/z 247 [M H] ions and m/z 203 [M H–COO] ions. 4.5. 8-Hydroxy-4H-1-benzopyran-4-one-2-carboxylic acid (halenic acid A, 1) White amorphous solid; UV (H2O) kmax (log e): 324 (3.24), 259 (3.76), 241 (4.05) nm; IR vmax (KBr) cm–1: 3482 (COOH), 1695, 1624 (C@O), 1575, 1493, 1410, 1347, 1301, 1155, 1003, 848, 799, 783; for 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) spectroscopic data, see Table 1; ESI-MS (negative) m/z: 205 [M H] , 411 [2M H] ; HR-ESI-MS m/z: 205.0136 (calcd for C10H5O5, 205.0137). 4.6. 8-Methoxy-4H-1-benzopyran-4-one-2-carboxylic acid (halenic acid B, 2) Pale brown amorphous solid; UV (MeOH) kmax (log e): 316 (3.45), 268 (sh, 3.66), 235 (4.10) nm; IR vmax (KBr) cm–1: 3448 (COOH), 1635 (C@O), 1581, 1493, 1455, 1352, 1272, 1145, 1050, 877, 795, 740; for 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) spectroscopic data, see Table 1; ESI-MS (negative) m/z: 219 [M H] ; HR-ESI-MS m/z: 219.0295 (calcd for C11H7O5, 219.0293). 4.7. 3-Acetyl-8-hydroxy-4H-1-benzopyran-4-one-2-carboxylic acid (halenic acid C, 3) Pale yellow amorphous solid; UV (H2O) kmax (log e): 321 (3.17), 274 (sh, 3.61), 242 (3.98) nm; for 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) spectroscopic data, see Table 1; ESI-MS (negative) m/z: 247 [M H] , 203 [M H–COOH] ; HR-ESIMS m/z: 247.0240 (calcd for C12H7O6, 247.0243).
4.9. 5-Hydroxy-6-methoxy-2-methyl-4H-1-benzopyran-4-one (halenichromone B, 5) Pale yellow amorphous solid; UV (MeOH) kmax (log e): 347 (2.86), 262 (sh, 3.46), 237 (3.74); IR vmax (KBr) cm–1: 3424 (OH), 2924, 1661 (C@O), 1631, 1594, 1480, 1402, 1385, 1280, 1240, 1139, 1047, 965, 839, 767, 697 cm–1; for 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) spectroscopic data, see Table 1; ESI-MS (positive) m/z: 207 [M+H]+, 229 [M+Na]+, 435 [2M+Na]+; HR-ESI-MS m/z: 229.0478 (calcd for C11H10O4Na, 229.0477). 4.10. 3,5-Dihydroxy-6-methoxy-2-methyl-4H-1-benzopyran-4-one (halenichromone C, 6) Yellow amorphous solid; UV (MeOH) kmax (log e): 359 (3.45), 290 (sh, 3.31), 249 (4.05); IR vmax (KBr) cm–1: 3323 (OH), 2924, 1652 (C@O), 1630, 1594, 1566, 1479, 1384, 1320, 1299, 1233, 1039, 800, 751; for 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) spectroscopic data, see Table 1; ESI-MS (positive) m/z: 223 [M+H]+; HR-ESI-MS m/z: 245.0425 (calcd for C11H10O5Na, 245.0426). 4.11. 5-Hydroxy-8-methoxy-2-methyl-4H-1-benzopyran-4-one (7) Pale yellow amorphous solid; UV (MeOH) kmax (log e): 350 (2.81), 287 (2.64), 253 (3.45), 226 (3.63); 1H NMR (400 MHz, acetone-d6): d 12.06 (1H, s, OH-5), 7.33 (1H, d, J = 9.0 Hz, H-7), 6.67 (1H, d, J = 8.8 Hz, H-6), 6.23 (1H, s, H-3), 3.91 (3H, s, OCH3-8), 2.46 (3H, s, CH3-2); HR-ESI-MS m/z: 229.0479 (calcd for C11H10O4Na, 229.0477). 4.12. 8-Hydroxy-2-methyl-4H-1-benzopyran-4-one (8) White amorphous solid; UV (MeOH) kmax (log e): 310 (3.55), 252 (3.97), 231 (4.28); IR vmax (KBr) cm–1: 3447 (OH), 2736, 2655, 1634 (C@O), 1566, 1496, 1388, 1371, 1298, 1191, 1154, 1022, 991; 1H NMR (400 MHz, DMSO-d6): d 10.41 (1H, br, OH-8), 7.39 (1H, dd, J = 6.6, 2.6 Hz, H-5), 7.21 (2H, m, H-6 and H-7), 6.20 (1H, s, H-3), 2.39 (3H, s, CH3-2); 13C NMR (100 MHz, DMSO-d6): d 177.2 (C-4), 166.5 (C-2), 146.6 (C-8), 145.7 (C-8a), 125.0 (C-6), 124.4 (C-4a), 118.9 (C-7), 114.1 (C-5), 109.8 (C-3), 20.1 (CH3-2); ESI-MS (positive) m/z: 177 [M+H]+; HR-ESI-MS m/z: 175.0394 (calcd for C10H7O3, 175.0395). 4.13. 8-Methoxy-2-methyl-4H-1-benzopyran-4-one (9) White amorphous solid; UV (MeOH) kmax (log e): 307 (3.59), 252 (3.91), 230 (4.30); IR vmax (KBr) cm–1: 3451 (OH), 2926, 1646 (C@O), 1581, 1491, 1441, 1391, 1367, 1274, 1219, 1196, 1144, 1059, 748; 1H NMR (400 MHz, acetone-d6): d 7.59 (1H, dd, J = 6.8, 2.7 Hz, H-5), 7.34 (2H, m, H-6 and H-7), 6.16 (1H, s, H-3), 3.99 (3H, s, OCH3-8), 2.42 (3H, s, CH3-2); 13C NMR (100 MHz, acetoned6): d 177.6 (C-4), 167.0 (C-2), 149.8 (C-8), 147.7 (C-8a), 125.5 (C-6), 125.5 (C-4a), 116.6 (C-5), 115.4 (C-7), 110.9 (C-3), 56.7 (OCH3-8), 20.4 (CH3-2); ESI-MS (positive) m/z: 191 [M+H]+, 403 [2M+Na]+; HR-ESI-MS m/z: 213.0527 (calcd for C11H10O3Na, 213.0528).
4.8. 2-(8-Hydroxy-2-methyl-4H-1-benzopyran-4-one-3-yl)-2oxoacetic acid (halenichromone A, 4)
4.14. Esterification of 3 and 4
Pale yellow amorphous solid; UV (H2O) kmax (log e): 306 (3.67), 260 (sh, 4.00), 221 (4.22) nm; for 1H NMR (DMSO-d6, 400 MHz) and 13 C NMR (DMSO-d6, 100 MHz) spectroscopic data, see Table 1; ESIMS (negative) m/z: 247 [M H] ; HR-ESI-MS m/z: 247.0240 (calcd for C12H7O6, 247.0243).
Fraction C1 (80 mg) was dissolved in MeOH (20 mL) and 3 drops of concentrated H2SO4 were added. The reaction mixture was heated until reflux began these beams maintained for 2 h, then cooled, neutralized with saturated aqueous NaHCO3 to pH 5–6, and concentrated. The concentrated mixture was diluted with
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
H2O to 15 mL and extracted with CHCl3 for three times (15 mL, 10 mL 2). After evaporation of the combined organic solvent layers, a crude product (85 mg) was purified by silica gel CC, eluting with CHCl3 to afford 3a (25 mg) and 4a (18 mg). 4.15. Compound 3a Pale yellow needles (Me2CO); mp 198–200 °C; UV (MeOH) kmax (log e): 305 (sh, 3.61), 264 (4.01), 239 (4.20) nm; IR vmax (KBr) cm– 1 : 3295 (OH), 1725 (OC@O), 1639 (C@O), 1580, 1499, 1438, 1398, 1366, 1299, 1263, 1216, 1112, 1065, 1047, 990, 919, 859, 760, 644; 1H NMR (400 MHz, acetone-d6): d 7.58 (1H, dd, J = 7.1, 2.4 Hz, H-5), 7.39 (2H, m, H-6 and H-7), 3.97 (3H, s, OCH3), 2.51 (3H, s, COCH3); 13C NMR (100 MHz, acetone-d6): d 197.6 (C-3a), 176.6 (C-4), 161.9 (C-2a), 150.0 (C-2), 147.9 (C-8), 145.8 (C-8a), 128.5 (C-3), 127.1 (C-6), 126.0 (C-4a), 121.3 (C-7), 115.9 (C-5), 53.9 (OCH3), 31.2 (C-3b); ESI-MS (positive) m/z: 263 [M+H]+. 4.16. Compound 4a Pale yellow needles (Me2CO); mp 163–165 °C; UV (MeOH) kmax (log e): 310 (3.77), 259 (sh, 3.99), 222 (4.31) nm; IR vmax (KBr) cm– 1 : 3550 (OH), 2925, 1724 (OC@O), 1679, 1645 (C@O), 1592, 1540, 1384, 1364, 1299, 1206, 1127, 1039, 985, 778, 747, 726; 1H NMR (400 MHz, acetone-d6): d 7.55 (1H, m, H-5), 7.36 (2H, m, H-6 and H-7), 3.86 (3H, s, OCH3), 2.74 (3H, s, CH3-2); 13C NMR (100 MHz, acetone-d6): d 187.4 (C-3a), 176.4 (C-4), 175.1 (C-2), 164.7 (C3b), 147.0 (C-8), 145.5 (C-8a), 126.9 (C-6), 125.0 (C-4a), 121.1 (C7), 117.7 (C-3), 115.6 (C-5), 52.4 (OCH3), 19.8 (C-2a); ESI-MS (positive) m/z: 263 [M+H]+, 547 [2M+Na]+. 4.17. Anti-HBV tests The human HBV-infected cell line HepG2 2.2.15 was used in this study. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, 100 lg/mL streptomycin and 2 mmol/L L-glutamine (all from Invitrogen, USA). Cells were cultured at 37 °C in a humidified incubator at 5% CO2. Test reagents were dissolved in DMSO and then further diluted to 100, 50, and 25 lg/mL, respectively. Cells were seeded in 96-well tissue culture plates and incubated for 48 h, then serial dilutions of the test reagents were added into cultures and the medium was refreshed every 3 days. Nine days after incubation at 37 °C, the culture media were collected to measure their HBsAg and HBeAg concentration using enzyme-linked immunosorbent assay (ELISA) kits (Shanghai SIIC KEHUA Biotech Co. Ltd.). Each measurement was performed in triplicate. The inhibition rates (%) of the test reagents were calculated using the following formula: inhibition ratio% = [OD (control) OD (sample)]/OD (control) 100%. Cytotoxicity of the test reagents was assessed using the MTT assay (Wang et al., 2009a). Acknowledgments This work was financially supported by National Drug Innovative Program (Grant No. 2009ZX09301-011). We thank Mrs. Huimin Wang, Shanghai Institute of Pharmaceutical Industry, for acquiring the HR-ESI-MS data. We also thank Prof. Xiao-kuang Ma, Department of Pharmacognosy, School of Pharmacy, Dali University, PR China, for collection and identification of the plant. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2011.09.015.
175
References Bennett, G.J., Lee, H.H., 1991. Halenia elliptica xanthone – a structural revision. Phytochemistry 30, 1347–1348. Clarke, D.S., Gabbutt, C.D., Hepworth, J.D., Heron, B.M., 2005. Synthesis of 3-alkenyl2-arylchromones and 2,3-dialkenylchromones via acid-catalysed retro-Michael ring opening of 3-acylchroman-4-ones. Tetrahedron Lett. 46, 5515–5519. Dhasmana, H., Garg, H.S., 1989. Two xanthone glucosides from Halenia elliptica. Phytochemistry 28, 2819–2821. Dhasmana, H., Garg, H.S., 1990. Xanthones of Halenia elliptica. Phytochemistry 29, 961–964. Gao, J., Wang, S.J., Fang, F., Si, Y.K., Yang, Y.C., Liu, G.T., Shi, J.G., 2004a. Xanthones from Tibetan medicine Halenia elliptica and their antioxidant activity. Acta Acad. Med. Sin. 26, 364–367. Gao, J., Wang, S.J., Xu, R.M., Yang, Y.C., Zhang, S.R., Si, Y.K., Shi, J.G., 2004b. Studies on chemical constituents of Halenia elliptica guided by in vivo absorption and distribution of the constituents. Acta Pharm. Sin. 39, 198–203. Gendaramyn, O., Kojima, K., Rodriguez, S., Ondognii, P., Ogihara, Y., Hostettmann, K., 1998. Xanthones from Halenia corniculata. Chem. Pharm. Bull. 46, 1827–1828. Grayer, R.J., Veitch, N.C., 1998. An 8-hydroxylated external flavone and its 8-Oglucoside from Becium grandiflorum. Phytochemistry 47, 779–782. Harborne, J., Baxter, H., Moss, G., 1999. Phytochemical Dictionary: A Handbook of Bioactive Compounds from Plants. Taylor & Francis, London, pp. 381–389. Huang, B., Ban, X., He, J., Zeng, H., Zhang, P., Wang, Y., 2010. Hepatoprotective and antioxidant effects of the methanolic extract from Halenia elliptica. J. Ethnopharmacol. 131, 276–281. Ito, H., Kasajima, N., Tokuda, H., Nishino, H., Yoshida, T., 2004. Dimeric flavonol glycoside and galloylated C-glucosylchromones from Kunzea ambigua. J. Nat. Prod. 67, 411–415. Jensen, S.R., Schripsema, J., 2001. 6 Chemotaxonomy and pharmacology of Gentianaceae. In: Struwe, L., Albert, V.A. (Eds.), Gentianaceae: Systematics and Natural History. Cambridge University Press, Cambridge, pp. 573–631. Jung, J.C., Min, J.P., Park, O.S., 2001. A highly practical route to 2-methylchromones from 2-acetoxybenzoic acids. Synth. Commun. 31, 1837–1845. Klutchko, S., Shavel, J., von Strandmann, M., 1974. Base rearrangement of chromone-3-carboxylic esters to 3-acyl-4-hydroxycoumarins. J. Org. Chem. 39, 2436–2437. Kuroda, M., Uchida, S., Watanabe, K., Mimaki, Y., 2009. Chromones from the tubers of Eranthis cilicica and their antioxidant activity. Phytochemistry 70, 288–293. Larget, R., Lockhart, B., Renard, P., Largeron, M., 2000. A convenient extension of the Wessely–Moser rearrangement for the synthesis of substituted alkylaminoflavones as neuroprotective agents in vitro. Bioorg. Med. Chem. Lett. 10, 835–838. Linke, H.A.B., 1969a. Hydroxychromones – synthesis ultraviolet spectrophotometric and polarographic properties. Z. Naturforsch. B: Chem. Sci. 24, 225. Linke, H.A.B., 1969b. Infrarotspektren der Hydroxychromone: die absorption der carbonyl-valenzschwingung. Spectrochim. Acta A 25, 1067–1074. Liu, X., Liu, Y., Shi, Y., 2009. Xanthone glycosides from Tibetan herb Halenia elliptica. China J. Chin. Mater. Med. 34, 580–582. Ma, W.G., Fuzzati, N., Lu, S.L., Gu, D.S., Hostettmann, K., 1996. Further chromones from Eriosema tuberosum. Phytochemistry 43, 1339–1343. Peres, V., Nagem, T.J., 1997. Trioxygenated naturally occurring xanthones. Phytochemistry 44, 191–214. Peres, V., Nagem, T.J., de Oliveira, F.F., 2000. Tetraoxygenated naturally occurring xanthones. Phytochemistry 55, 683–710. Rao, C.R., Venkateswarlu, V., 1956. Synthesis of 5- and 5,8-dimethoxy-2-methyl chromones. Recl. Trav. Chim. Pay. B 75, 1321–1326. Recio-Iglesias, M.C., Marston, A., Hostettmann, K., 1992. Xanthones and secoiridoid glucosides of Halenia campanulata. Phytochemistry 31, 1387–1389. Rodriguez, S., Wolfender, J.L., Hostettmann, K., Odontuya, G., Purev, O., 1996. Corniculoside, a new biosidic ester secoiridoid from Halenia corniculata. Helv. Chim. Acta 79, 363–370. Rodriguez, S., Wolfender, J.L., Odontuya, G., Purev, O., Hostettmann, K., 1995. Xanthones, secoiridoids and flavonoids from Halenia corniculata. Phytochemistry 40, 1265–1272. Stout, G.H., Fries, J.L., 1970. The xanthones of a Halenia species. Phytochemistry 9, 235–236. Struwe, L., Albert, V.A., 2002. Gentianaceae: Systematics and Natural History. Cambridge University Press, Cambridge. Sun, H.F., Hu, B.L., Ding, J.Y., Fan, S.F., 1987. Three new glycosides from Halenia elliptica. Acta Bot. Sin. 29, 422–428. Sun, H.F., Hu, B.L., Fan, S.F., Ding, J.Y., 1983. Three new xanthones from Halenia elliptica D. Don. Acta Bot. Sin. 25, 460–467. Sun, Y.W., Sun, Z.H., Yu, P.Z., 2011. A new xanthone from Halenia elliptica D. Don. J. Asian Nat. Prod. Res. 13, 88–92. Tanaka, T., Koike, K., Mitsunaga, K., Narita, K., Takano, S., Kamioka, A., Sase, E., Ouyang, Y., Ohmoto, T., 1995. Chromones from Harrisonia perforata. Phytochemistry 40, 1787–1790. Tsui, W.Y., Brown, G.D., 1996. Chromones and chromanones from Baeckea frutescens. Phytochemistry 43, 871–876. Tuntiwachwuttikul, P., Phansa, P., Pootaeng-on, Y., Taylor, W.C., 2006. Chromones from the branches of Harrisonia perforata. Chem. Pharm. Bull. 54, 44–47. Wang, S.G., Li, J.Y., Huang, H., Gao, W., Zhuang, C.L., Li, B., Zhou, P., Kong, D.Y., 2009a. Anti-hepatitis B virus activities of astragaloside IV isolated from Radix Astragali. Biol. Pharm. Bull. 32, 132–135.
176
Y.-w. Sun et al. / Phytochemistry 75 (2012) 169–176
Wang, Y., Shi, J.G., Wang, M.Z., Che, C.T., Yeung, J.H.K., 2007. Mechanisms of the vasorelaxant effect of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery. Life Sci. 81, 1016–1023. Wang, Y., Shi, J.G., Wang, M.Z., Che, C.T., Yeung, J.H.K., 2008. Mechanisms of the vasorelaxant effect of 1,5-dihydroxy-2,3-dimethoxy-xanthone, an active metabolite of 1-hydroxy-2,3,5-trimethoxy-xanthone isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery. Life Sci. 82, 91–98. Wang, Y., Shi, J.G., Wang, M.Z., Che, C.T., Yeung, J.H.K., 2009b. Vasodilatory actions of xanthones isolated from a Tibetan herb, Halenia elliptica. Phytomedicine 16, 1144–1150.
Yang, H.P., Yue, M.E., Shi, Y.P., 2006. CZE Determination of flavonoids in Halenia elliptica. Chromatographia 63, 449–452. Yu, P.Z., Shen, X.J., Sun, Y.W., Hu, C.Q., 2009. HPLC simultaneous determination of four xanthones in Halenia elliptica D. Don. Chin. J. Pharm. Anal. 29, 540–543. Yu, P.Z., Shen, X.J., Hu, C.Q., Meehan, E.J., Chen, L.Q., 2008. 1,7-Dihydroxy-2,3,4trimethoxy-9H-xanthen-9-one monohydrate from Halenia elliptica. Acta Crystallogr. Sect. E: Struct. Rep. Online 64, O651–U823.