Original Papers
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Authors
Cui-Ting Luo 1, Huan-huan Zheng 1, Shuang-Shuang Mao 1, Mao-xun Yang 1, Cheng Luo 2, Heru Chen 1, 2, 3
Affiliations
The affiliations are listed at the end of the article
Key words " Gentianaceae l " Swertia mussotii l " xanthones l " α‑glucosidase inhibitor l " diabetes mellitus l
Abstract !
Two new xanthones, 1,8-dihydroxy-3-methoxyxanthone 7-O-[α-L-rhamnopyranosyl(1 → 2)-βD-glucopyranoside] (1) and 1,8- dihydroxy-3methoxyxanthone 7-O-[α-L-rhamnopyranosyl (1 → 3)-α-L-rhamno-pyranosyl (1 → 2)-β-D-xylopyranoside] (2), together with 26 known xanthones (3–28), were isolated from the aqueous ethanol extract of the traditional Chinese herb Swertia mussotii. Their structures were elucidated via spectroscopic analyses including 2D NMR. The
Introduction !
received revised accepted
June 9, 2013 Nov. 19, 2013 Nov. 20, 2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1360173 Published online December 19, 2013 Planta Med 2014; 80: 201–208 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Dr. Heru Chen Institute of Traditional Chinese Medicine and Natural Products College of Pharmacy Jinan University Huangpu Avenue West 601 Guangzhou 510632 P. R. China Phone: + 86 20 38 37 52 99 Fax: + 86 20 85 22 15 59 [email protected]
Xanthones are the secondary metabolites that commonly occur in a few higher plant families, fungi, and lichens. Their taxonomic values in such families and pharmacological properties have provoked great interest [1]. To date, the isolated xanthones are classified into five major groups: simple oxygenated xanthones, xanthone glycosides, prenylated and related xanthones, xanthonolignoids, and miscellaneous. The simple oxygenated xanthones can further be subdivided into six groups according to the degree of oxygenation [2, 3]. The study of xanthones is interesting not only from a chemosystematic viewpoint but also from a pharmacological viewpoint. It has been reported that xanthones possess various biological and pharmacological properties such as antidepressant, antileukemic, antitumor, antitubercular, choleretic, diuretic, antimicrobial, antifungal, anti-inflammatory, antiviral, cardiotonic, and hypoglycemic activities [4–18]. We are most interested in the antidiabetic activity of xanthones. Two tetraoxygenated xanthones, methylswertianin and bellidifolin, were found to be potent hypoglycemic agents in streptozotocin (STZ)-induced diabetic rats by both oral and intraperitoneal administration [16, 17].
inhibition of α-glucosidase by the isolated xanthones was evaluated by an in vitro high-throughput screening assay. Our results indicated that 1,3,5,8-tetrahydroxyxanthone is the best inhibitor with an IC50 value of 5.33 ± 0.09 µM, while the O-glycosylated xanthones were poor α-glycosidase inhibitors. Supporting information available online at http://www.thieme‑connect.de/ejournals/toc/ plantamedica
Many people have type II diabetes mellitus (TII‑DM), which is associated with low insulin production or insulin resistance because of genetic and/or epigenetic causes. This disease is regarded as one of the most important public health problems throughout the world in the 21st century. Although many effective therapies have been developed to treat type II diabetes, there are a number of new approaches identified that provide better treatment options [19]. α-Glucosidase inhibitors are one type of useful agent that prevent the progression of the disease and are used for treating prediabetic conditions [20]. Interestingly, some xanthones have already been identified as α-glucosidase inhibitors [8]. As part of our ongoing research into the identification of lead compounds as α-glucosidase inhibitors from traditional Chinese medicine, a systematic study was initiated to investigate the chemical constituents (with a focus on xanthones) isolated from the aqueous ethanol extract of Swertia mussotii Franch, which is a traditional Tibetan folk medicine belonging to the family Gentianaceae. " Figs. 1 and 2, we describe Herein, as shown in l the isolation and characterization of two new xanthones (1–2), together with 26 known ones (3–28). The α-glucosidase inhibitory activities
Luo C-T et al. Xanthones from Swertia …
Planta Med 2014; 80: 201–208
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Xanthones from Swertia mussotii and Their α-Glycosidase Inhibitory Activities
Original Papers
Fig. 1 Chemical structures of compounds 1 and 2 from Swertia mussotii. (Color figure available online only.)
Fig. 2
Chemical structures of compounds 3–28.
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and associated structure-activity relationships of these compounds are also included.
Results and Discussion !
The aqueous ethanol extract of the whole Swertia mussotii plant dry powder was partitioned successively with petroleum ether, dichloromethane (DCM), ethyl acetate, and n-butanol. Both the DCM and n-butanol fractions were subjected to repeated separation on silica gel column chromatography (CC) as well as re-
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versed-phase chromatography with an ODS column, Sephadex LH-20, and preparative HPLC to afford two new xanthones (1–2) " Fig. 1) and 26 known xanthones (3–28) (l " Fig. 2). Compounds (l 1 and 2 belong to the tetraoxygenated xanthone group and contain a disaccharide and a trisaccharide, respectively. To date, there are only four reports of xanthone disaccharides [21–24] and just one report of a xanthone trisaccharide [25]. Compound 1 was obtained as an amorphous yellow solid. Its molecular formula, C26H30O15, was deduced from its HRESIMS, which exhibited a quasi-molecular ion peak at m/z 605.14 610 ([M + Na]+). The UV spectrum displayed absorption bands at λmax
Original Papers
1 δH (J in Hz)
1 2 3 4 4a 4b 5 6 7 8 8a 8b 9 1-OH 8-OH 3-OCH3 Glc-1 Glc-2 Glc-3 Glc-4 Glc-5 Glc-6 Rha-1 Rha-2 Rha-3 Rha-4 Rha-5 Rha-6 Rha-1, Rha-2, Rha-3, Rha-4, Rha-5, Rha-6, Xyl-1 Xyl-2 Xyl-3 Xyl-4 Xyl-5
6.31, d (3) 6.54, d (3)
6.92, d (9) 7.56, d (9)
11.75, s 11.75, s 3.85, s 5.09, d (9)
5.22, s
1.12, d (6)
2 δC 161.9 97.4 167.2 92.9 157.6 149.9 105.7 125.1 139.6 149.8 107.3 101.8 184.2
56.3 98.8 76.2 76.9 70.6 77.7 60.6 100.3 69.9 70.6 72.1 68.4 18.1
236, 260, and 330 nm, which implied a xanthone derivative [26]. The IR (KBr) υmax at 3396, 1645, 1606, and 1505 cm−1 indicated the existence of a hydroxyl group, a carbonyl group, as well as a " Taconjugated benzene ring group. The 1H NMR spectrum of 1 (l ble 1) exhibited peaks indicating hydroxyl protons at δH 11.75 (2H, s, OH-1, and OH-8), which implied the existence of an intramolecular hydrogen bond, along with meta-coupled aromatic protons at δH 6.31 (1H, d, J = 3 Hz, H-2) and δH 6.54 (1H, d, J = 3 Hz, H-4), and ortho-coupled aromatic protons at δH 6.92 (1H, d, J = 9 Hz, H-5) and δH 7.56 (1H, d, J = 9 Hz, H-6). The signals for an anomeric proton at δH 5.09 (1H, d, J = 6 Hz) substantiated that 1 was a β-O-glucoside. In addition, the methyl group signal at δH 1.12 (3H, d, J = 6 Hz) combined with an anomeric proton at δH 5.22 (1H, s) confirmed that 1 had an α-rhamnopyranose moiety. The spectrum also showed signals for a methoxy group at δH " Table 1) indi3.88 (3H, s). Analysis of the 13C NMR spectrum (l cated 13 carbon signals at a low field, including one carbonyl at δC 184.2, suggesting that two hydroxyls are located at C-1 and C8. A set of glycosyl carbon signals at a high field, δc 98.8, 76.2, 76.9, 70.6, 77.7, and 60.6, suggested that there was a β-glucopyranosyloxy unit in the molecule, and a set of glycosyl carbon signals at δC 100.3, 69.9, 70.6, 72.1, 68.4, and 18.1 implied that there
δH (J in Hz) 6.23, d (1.8) 6.41, d (1.8)
6.86, d (9) 7.50, d (9)
δC 161.9 97.4 167.2 92.8 157.5 150.8 106.0 127.2 138.7 150.6 107.5 101.3 184.0
3.80, s
56.3
4.89, s
101.4 70.6 70.8 72.0 69.0 17.9 101.3 70.6 70.7 72.0 68.9 17.8 99.8 81.5 76.9 68.4 65.4
1.13, d (6) 4.83, s
1.05, d (6) 5.22, d (6)
Table 1 1H and 13C NMR spectroscopic data for compounds 1 and 2 (δ in ppm).
was an α-rhamnopyranosyl unit in the structure. The structure of compound 1 was further confirmed by HSQC and HMBC. HSQC results confirmed that Glc-C-1 is at δc 98.8, Rha-C-1 at δc 100.3, " Fig. 3) showed a cross-peak of and -OCH3 at δc 56.3. HMBC (l -OCH3 with C-3 implying that there is a substitution at C-3 by -OCH3. It was observed that the anomeric proton (δH 5.09) is correlated with the carbon at δC 139.6 (C-7). This is the direct evidence that the glucosyl moiety is linked to C-7. The meta-coupled aromatic proton at δH 6.31 (H-2) was correlated with δC at 184.2 (C=O), 167.2 (C-3), 161.9 (C-1), 101.8 (C-8b), and 92.9 (C-4). Another meta-coupled aromatic proton at δH 6.54 (H-4) was correlated with δC at 167.2 (C-3), 157.6 (C-4a), 101.8 (C-8b), 97.43 (C2), and 184.2 (C=O). The doublet at δH 6.92 (H-5) was correlated with δC at 184.2 (C=O), 149.9 (C-4b), 139.7 (C-7), and 107.3 (C8a); whereas another doublet at δH 7.56 (H-6) was found to be correlated with δC at 149.8 (C-8), 139.7 (C-7), 105.7 (C-5), and 149.9 (C-4b). Taken together, these data supported that the assignments of H-2, H-4, H-5, and H-6 were correct. Furthermore, it was observed that another anomeric proton (δH 5.22) was correlated with δC 76.2 (Glc-C-2), which moved to a lower field when compared with the reference glucopyranosyl. Therefore the α-rhamnopyranosyl was suggested to be linked to Glc-2. AcLuo C-T et al. Xanthones from Swertia …
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Position
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Original Papers
Fig. 3 Key HMBC correlations of compounds 1 and 2 (recorded on Bruker AV-400).
idic hydrolysis of compound 1 and derivation of the resulting compounds with o-tolylisothiocyanate were carried out. HPLC analysis of the derivatives confirmed the existence of one β-Dglucopyranose at a retention time (RT) of 23.8 min and one αrhamnopyranose at RT of 41.0 min. Hence, the structure of 1 was determined to be 1,8-dihydroxy-3-methoxyxanthone 7-O-[α-Lrhamnopyranosyl (1 → 2)-β-D-glucopyranoside]. Compound 2 was isolated as a yellow powder. Its HRESIMS spectrum exhibited a quasi-molecular ion peak at m/z 721.19 658 ([M + Na]+), which indicated a molecular formula of C31H38O18. The UV spectrum with absorption bands at λmax 236, 260, and 330 nm implied a xanthone derivative [26]. The 1H NMR spec" Table 1) exhibited peaks for hydroxyl protons at δ trum of 2 (l H 11.75 (2H, s, OH-1, and OH-8), which implied the existence of an intramolecular hydrogen bond along with a set of meta-coupled aromatic protons at δH 6.41 (1H, d, J = 1.8 Hz, H-4) and δH 6.23 (1H, d, J = 1.8 Hz, H-2) as well as two ortho-coupled aromatic protons at δH 6.86 (1H, d, J = 9 Hz, H-5) and δH 7.50 (1H, d, J = 9 Hz, H6). Three anomeric protons were found at δH 5.22, 4.89, and 4.83. The signal at δH 5.22 (1H, d, J = 9 Hz) suggested that 2 probably contained a β-O-xylopyranosyl. The signals at δH 4.89 (1H, s) and 4.83 (1H, s) together with two methyl signals at δH 1.13 and 1.05 (3H, d, J = 6 Hz) indicated that 2 may have two α-rhamnopyranosyl units. The spectrum also had a signal for a methoxyl group at " Table 1) δH 3.80 (3H, s). Analysis of the 13C NMR spectrum (l showed 13 carbon signals at a low field and one carbonyl signal at δC 184.0. These data further confirmed the existence of a xanthone. A set of glycosyl carbon signals at a high field, δc 99.8, 81.5, 76.9, 68.4, and 65.4, further supported a β-O-xylopyranose unit in the structure. Another two sets of glycosyl carbon signals were observed at δC 101.4, 72.0, 70.8, 70.6, and 69.0 and at δC 101.3, 71.9, 70.7, 70.6, and 68.9. This implied two α-rhamnopyranosyl units in the molecule. Comparison of the 1H NMR and 13C NMR spectra indicated that compound 2 had the same xanthone scaffold as 1. The only difference between them appeared to be the polysaccharide moiety. Therefore, the structural exploration of 2 focused on confirming the identity and spatial arrangement of the glycosyl components. Based on a polysaccharide analysis assay, it was confirmed that 2 contained one D-xylopyranosyl unit with an RT of 27.8 min and two α-rhamnopyranosyl units with an RT of 41.0 min. The glycosyl connection was confirmed from the " Fig. 3) and HSQC. It was found that the HMBC spectrum (l anomeric proton (δH 5.22) was correlated with δC 138.7 (C-7) in the HMBC and with the carbon signal at δc 99.8 (Xyl-1) in the HSQC spectrum. This fact suggested that the xylopyranosyl unit was linked to C-7. In addition, it was observed that the second anomeric proton (δH 4.89) was correlated with δC 76.9 (Xyl-3), which moved to a lower field when compared with the reference xylopyranose. This anomeric proton was correlated with δC 101.4 Luo C-T et al. Xanthones from Swertia …
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(Rha-1) in the HSQC. Therefore, one α-rhamnopyranose unit was suggested to be linked to Xyl-3. The third anomeric proton (δH 4.83) was correlated with δC 81.5 (Xyl-2), which moved to a lower field when compared with the reference xylopyranose. This third anomeric proton was correlated with δC 101.3 (Rha-1′) in the HSQC. Thus the second α-rhamnopyranose unit was suggested to be linked to Xyl-2, and compound 2 was identified as 1,8-dihydroxy-3-methoxyxanthone 7-O-[α-L-rhamnopyranosyl(1 → 3)α-L-rhamnopyranosyl (1 → 2)-β-D-xylopyranoside]. " Fig. 2) were identified as 1,3,5-trihydroxyCompounds 3–28 (l xanthone 8-O-[β-D-glucopyranoside] (3) [27], 3,7,8-trihydroxyxanthone 1-O-[β-D-glucopyranoside] (4) [28], 1,8-dihydroxy3,4-dimethoxyxanthone 7-O-[β-D-glucopyranoside] (5) [29], 1,8-dihydroxy-3-methoxyxanthone 7-O-[β-D-glucopyranoside] (6) [30], 1-hydroxy-3,4-dimethoxyxanthone 7-O-[β-D-glucopyranoside] (7) [31], 3,7,8-trimethoxyxanthone 1-O-[β-D-glucopyranoside] (8) [27], 1,8-dihydroxy-3-methoxyxanthone 7-O[α-L-rhamnopyranosyl(1 → 2)-β-D-xylopyranoside] (9) [27], mangiferin (10) [27], 2,6,8-trihydroxyxanthone 7C-(β-D-glucoside) (11) [27], 1,6-dihydroxy-3,4,8-trimethoxyxanthone (12) [32], 1,7-dihydroxy-3,8-dimethoxyxanthone (13) [27], 1,7,8-trihydroxy-3,4-dimethoxy-xanthone (14) [33], 1,8-dihydroxy-3,7dimethoxy xanthone (15) [27], 1-hydroxy-3,7,8-trimethoxyxanthone (16) [27], 1,7,8-trihydroxy-3-methoxyxanthone (17) [27], 1,3,5,8-tetrahydroxyxanthone (18) [30], 1,7-dihydroxy-3,4,8-trimethoxyxanthone (19) [33], 1-hydroxy-3,4,7,8-tetramethoxy xanthone (20) [33], 1,8-dihydroxy-7-methoxyxanthone (21) [34], 1,7-dihydroxy-3-methoxyxanthone (22) [35], 1,3,7-trihydroxyxanthone (23) [36], 1,3,7,8-tetrahydroxylxanthone (24) [37], 1,3-dihydroxy-7,8-dimethoxyxanthone (25) [38], 1,5,8-trihydroxy-3,4-dimethoxyxanthone (26) [39], 1-hydroxy-3,4,5,8tetramethoxyxanthone (27) [37], and 1-hydroxy-3,5,8-trimethoxy xanthone (28) [37] by comparison of their spectroscopic data with those reported in the literature. It needs to be pointed out that compounds 11 and 14 were first isolated from this plant family, while compound 4 was first isolated from this plant. Several xanthones such as swerchirin, methylswertianin, and bellidifolin have been confirmed as antidiabetic agents [16–18, 40, 41]. Some have been identified as α-glucosidase inhibitors [8, 42]. With 28 isolated xanthones (1–28) on hand, we systematically investigated their potential to inhibit α-glucosidase and to " Tadisclose their structure-activity relationship. As shown in l ble 2, glycosylated xanthones (1–11) are generally poor inhibitors except for mangiferin (10) and 9, where 10 is a C-glycoside with an IC50 value of 13.02 ± 0.23 µM and 9 is a xanthone glycoside without a glucosyl group. This suggests that O-glycosylated xanthones containing a glucosyl unit are substrates of α-glucosidase, while C-glycosylated xanthones and O-glycosylated xanthones without a glucosyl unit are not. The hydroxyl group at C-3 is very
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Original Papers
Acarbosea 1 2 3 4 5 6 7 8 9 10 11
IC50 (µM)±SEM 39.6 ± 0.1 75.8 ± 1.3 84.5 ± 1.2 > 500 > 500 133.7 ± 3.0 394.9 ± 2.0 115.0 ± 2.4 83.8 ± 1.2 31.1 ± 0.2 13.0 ± 0.2 140.4 ± 0.8
Compounds
IC50 (µM)±SEM
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
> 500 > 500 5.42 ± 0.07 107.8 ± 0.1 142.1 ± 0.2 30.6 ± 0.1 5.3 ± 0.1 77.4 ± 0.1 65.8 ± 0.1 71.2 ± 0.2 17.6 ± 0.1 31.8 ± 0.1 7.09 ± 0.08 18.0 ± 0.1 82.2 ± 0.2 40.1 ± 0.1 75.8 ± 0.1
Table 2 α-Glucosidase inhibition of all the isolated xantones.
IC50 values expressed as mean ± SEM (standard error of mean), where n = 3; a positive control
important for maintaining the activity, e.g., mangiferin is a good inhibitor, but compound 11 is not. Compound 18 is the most active inhibitor with an IC50 value of 5.33 ± 0.09 µM followed by 14 with an IC50 value of 5.42 ± 0.07 µM. It is of note that 18 is structurally similar to bellifolin (1,5,8-trihydroxy-3-methoxyxanthone) which was reported to be a small molecule anti-diabetes candidate. The only difference between them is that the 3-hydroxyl group in compound 18 is a 3-methoxy group in bellifolin. A comparison of the inhibitory activities of 18 with 3 and 24 with 4 indicated that O-glycosylation at either C-1 or C-8 results in a loss of almost all the α-glucosidase inhibitory activity. This is supported by the fact that the IC50 values of 3 and 4 are all greater than 500 µM. Methylation of a hydroxyl group in xanthone will generally decrease the inhibitory activity of the compound, e.g., methylations of hydroxyl groups at C3, C5, and C8 in 18, which convert it into compound 28, decreases its activity by 14-fold. A comparison of the structure-activity relationship among compounds 24, 25, and 13 allowed us to conclude that free hydroxyl groups at C-3 and C-8 are very important for maintaining inhibitory activity. Taken together, the current studies offer useful information for further structural modification of xanthones for the purposes of designing small molecule inhibitors, which may prove useful as therapeutics for a variety of disease areas.
Materials and Methods !
(> 99%), L-glucose (> 99 %), D-xylose (> 99%), L-xylose (> 99%), Drhamnose (> 99 %), and L-rhamnose (> 99 %) were purchased from Ada-Mas-Beta Company. The 96-well Fluotrac 200 black microplates (part # 655 076) were purchased from Greiner America, Inc., and the 48-well microplates (Falcon No. 3230) were purchased from VWR. Clear polystyrene 96-well plates (Nunc) were purchased from Fisher Scientific.
General experimental procedures UV spectra were obtained using a Jasco V-550 spectrometer, whereas FT‑IR spectra were acquired on a Jasco FI/IR‑480 Plus Fourier Transform (using KBr disk method). The ESI‑MS spectra were acquired on a Finnigan LCQ Advantage Max spectrometer. The HRESIMS spectra were obtained on an Agilent QTOF spectrometer. NMR spectra were recorded on a Bruker AV-300 or Bruker AV-400 NMR spectrometer using TMS as an internal standard. Silica gel (200–300 mesh) for CC was purchased from Yantai Chemical Factory, and ODS (50 µm) was purchased from YMC Co. Ltd. Precoated thin-layer chromatography (TLC) plates (Institute of Yantai Chemical Industry) were used for TLC. Spots on TLC plates were detected by either a ZF-7A portable UV detector or spraying a KMnO4 solution followed by subsequent heating. Sephadex LH-20 was purchased from GE Healthcare. The RP-HPLC analysis and preparation were conducted using a P680 pumping system equipped with a PDA-100 photodiode array detector and Cosmosil columns (C18; 4.6 × 250 mm for analysis and 20 × 250 mm for preparation).
Plant materials One batch of plant material (batch no. 106785) was collected from Zhongda Village, Yushu County, Qinghai Province (China) on October 20, 2010. It was identified as Swertia mussotii by Dr. Jing Sun from the Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China. A reference sample (voucher sample NIPB-1136) is stored at the Institute.
Chemicals and apparatus α-Glucosidase (maltase) was purchased from Oriental Yeast Co. Ltd. 4-Nitrophenyl-α-D-glucopyranoside (PNPG) was purchased from Aldrich. L-cysteine methyl ester and acarbose (purity > 99%) were purchased from Aladdin. O-Tolyl isothiocyanate was purchased from Aldrich. Standard sugars including D-glucose
Extraction and isolation Dried powder of Swertia mussotii (5.0 kg) was extracted three times (2 h, 2 h, and 1 h) with 75 % aqueous ethanol (10 L) under reflux. The extracts were concentrated in vacuo to yield a brown-black gum (1.1 kg). A total of 509 g of the gum was then suspended in H2O and partitioned successively with petroleum ether (PE, 1.5 L), dichloromethane (DCM, 1.5 L), ethyl acetate (EE, 1.5 L), and n-butanol (NB, 1.5 L) to yield PE (35 g), DCM (92 g), EE (42 g), NB (170 g), and water (171 g) fractions. The DCM fraction was then subjected to silica gel CC (695 × 130 mm) using a gradient system of petroleum ether-ethyl acetate (30 : 1–0 : 1) (25 L, flow 2.5–3.5 mL/ min). The collected fractions were combined based on their TLC
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Planta Med 2014; 80: 201–208
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Compounds
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Original Papers
characteristics to offer eight subfractions (F1-F8), whose amount was 9.2, 13.5, 11.6, 10.3, 9.8, 12.6, 10.5, and 10.1 g, respectively. The F1 subfraction was separated further by silica gel CC (550 × 65 mm) using a gradient system of petroleum ether-ethyl acetate (15 : 1–0 : 1) and by recrystallization to yield pure com" Fig. 2), whose amount was 21.3, 32.5, and pounds (14–16) (l 45.7 mg, respectively. F2 was subjected to Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (6 L, flow 1.5– 2.5 mL/min) to yield compounds 12 (46.5) and 13 (58.7). Compounds 16 (60.2 mg) and 17 (55.3 mg) were isolated from F3 by repeated silica gel CC (550 × 65 mm) with PE-acetone and further separated by HPLC on a preparative Cosmosil column (20 × 250 mm) applying MeOH‑H2O as the eluant with flow rate 3.5 mL/min. F4 was purified by Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) to produce compound 19 (27.8 mg). Compound 20 (38.9 mg) was isolated from F5 by Sephadex LH-20 CC eluted with CHCl3-MeOH (1 : 1) (6.5 L, flow 1.5– 2.5 mL/min) and further purified by preparative HPLC (Cosmosil column, 20 × 250 mm) using MeOH‑H2O as the eluant at a flow rate of 3.5 mL/min. Compounds 21 (39.6 mg) and 22 (61.8 mg) were isolated from F6 by repeated silica gel CC (550 × 65 mm) with PE-acetone and then further purified by preparative HPLC as described above. F7 was purified by Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (5 L, flow 1.5– 2.5 mL/min) to yield compounds 23 (19.3 mg) and 24 (36.3 mg). Compounds 26–28 were isolated from F8 by Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (7 L, flow 1.5– 2.5 mL/min) and then further purified by preparative HPLC as described above resulting in 26 (41.5 mg), 27 (53.2 mg), and 28 (33.7 mg). The n-butanol fraction was first separated by a silica gel CC (695 × 130 mm) using a gradient system of CHCl3-MeOH (15 : 1– 0 : 1) (25 L, flow 2.5–3.5 mL/min). The collected fractions were combined based on their TLC characteristics to yield ten subfractions (P1-P10), whose amount was 15.2, 13.5, 11.6, 17.3, 19.8, 12.6, 11.5, 13.1, 20.3, and 14.9 g, respectively. Compounds 5 (15.6 mg) and 8 (21.8 mg) were isolated from P3 by preparative HPLC as described above. Compounds 6 (20.8 mg), 7 (18.2 mg), and 9 (23.7 mg) were isolated from P4 by reversed-phase ODS CC (800 × 35 mm) eluted with an MeOH‑H2O (1 : 1) (4 L, flow 1.0– 1.5 mL/min) and further submitted to Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (5 L, flow 1.5– 2.5 mL/min). Compounds 10 (15.6 mg) and 11 (19.3 mg) were isolated from P5 by preparative HPLC as described above. Compounds 3 (11.5 mg) and 4 (16.3 mg) were isolated from P6 by Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (4 L, flow 1.5–2.5 mL/min) and then further purified by prepara" Fig. 1) were tive HPLC. Compounds 1 (9.8 mg) and 2 (13.2 mg) (l isolated from P7 by Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (4 L, flow 1.5–2.5 mL/min) and then further purified by preparative HPLC as described above. 1,8-Dihydroxy-3-methoxyxanthone 7-O-[α‑L-rhamnopyranosyl (1 → 2)-β‑D-glucopyranoside] (1): Yellow amorphous powder, purity: 98.5 %; [α]20 D −50.5 (c 0.10, DMSO); UV (MeOH) λmax (log ε): 236 (4.41), 260 (4.51), 330 (4.25) nm; IR νmax (KBr): 3396, 1645, 1606, 1505 cm−1; ESI‑MS (m/z): 605.4 [M + Na]+ (calcd. for C26H30O15 + Na, 605.1); HRESIMS (m/z): 605.14 610 [M + Na]+ (calcd. for C26H30O15 + Na, 605.14 824); for 1H and 13C NMR " Table 1. (300 MHz, DMSO‑d6) spectroscopic data, see l 1,8-Dihydroxy-3-methoxyxanthone 7-O-[α‑L-rhamnopyranosyl (1 → 3)-α‑L-rhamno-pyranosyl(1 → 2)-β‑D-xylopyranoside] (2): Yellow amorphous powder, purity: 99.2 %; [α]20 D −61.3 (c 0.10, Luo C-T et al. Xanthones from Swertia …
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DMSO); UV (MeOH) λmax (log ε): 236 (4.52), 260 (4.64), 330 (4.35) nm; IR νmax (KBr): 3395, 1636, 1606, 1499 cm−1; ESI‑MS (m/z): 697.2 [M – H]− (calcd. for C31H38O18-H: 697.3); HRESIMS (m/z): 721.19 658 [M + Na]+ (calcd. for C31H38O18 + Na, 721.19 558); for 1H and 13C NMR (300 MHz, DMSO‑d6) spectro" Table 1. scopic data, see l
Confirmation of glycosyl units Compounds 1 and 2 (1 mg each) were individually hydrolyzed with 3 mL of 1 M HCl for 2 h at 90 °C. The mixture was evaporated to dryness under vacuum. The residue was dissolved in pyridine (2–3 mL) containing L-cysteine methyl ester (1 mg) and heated at 60 °C for 1 h. Then, o-tolyl isothiocyanate (10–15 µL) was added to the mixture which was heated at 60 °C for another 1 h. The reaction mixture (2 µL) was analyzed directly by reversed-phase HPLC [43]. Analytical HPLC was performed on a 250 × 4.6 mm i. d. Cosmosil 5C18 column at 30 °C with isocratic elution of 22 % CH3CN for 50 min and subsequent washing of the column with 90 % CH3CN at a flow rate of 1.0 mL/min. Peaks were detected using a UV detector at 250 nm. The standard sugars including Dglucose, L-glucose, D-xylose, L-xylose, D-rhamnose, and L-rhamnose were subjected to the same method. The peaks of the standard sugar derivatives were recorded at retention times (RT) of 23.5 (D‑Glc), 21.6 (L‑Glc), 27.3 (D‑Xyl), 25.3 (L‑Xyl), 42.5 (D‑Rha), and 40.6 (L‑Rha) min. Following the chemical reaction, the derivatives of 1 gave peaks at an RT of 23.8 min (D‑Glc) and 41.0 min (L‑Rha) with a ratio of peak area of 1 : 1. This supports the idea that compound 1 has one β-D-glucopyranosyl unit and one αrhamnopyranosyl unit. The derivatives of 2 gave peaks at 27.8 min (D‑Xyl) and 41.1 min (L‑Rha) with a ratio of peak area of 1 : 2. This supports the conclusion that compound 2 has one D-xylosyl unit and two α-rhamnopyranosyl units.
In vitro α-glucosidase inhibition assay The α-glucosidase inhibition was carried out by either assay (A) or (B). IC50 values were determined by using EZ‑FIT, an enzyme kinetics software by Perrella Scientific Inc. Results are presented as mean ± SEM (standard error of mean) from three experiments. (A) α-Glucosidase inhibitory activity was assayed in 0.1 M sodium phosphate buffer (pH 6.8) with p-nitrophenyl-α-D-glucopyranoside (PNPG) as the substrate. The concentration of α-glucosidase was 0.2 U/mL in each experiment. The enzyme (20 mL) along with 100 mL of phosphate buffered saline was incubated with various concentrations of test compounds at 37 °C. After a preincubation time of 15 min, 20 mL of substrate (0.7 mM) was added, and the reaction was carried out at 37 °C for 30 min. The enzymatic activity was quantified by measuring the absorbance of pnitrophenol at 400 nm on a microtiter plate spectrophotometer (Spectra Max; Molecular Devices). One unit of α-glucosidase was defined as the amount of enzyme capable of liberating 1.0 mmol of p-nitrophenol per minute under the conditions specified [44]. Acarbose was used as the positive control (IC50 = 39.75 ± 0.06 µM) " Table 2). The percent inhibition of p-nitrophenol formation in (l the test sample versus the reference (without the addition of sample) was calculated for each compound by using the following formula: % inhibition ¼ ð1
ODsample Þ 100% ODreference
(1)
(B) The α-glucosidase inhibition was determined spectrophotometrically in a 96-well plate using PNPG as the substrate [45].
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All the test samples were prepared as stock solutions of 1000, 500, 200, 100, and 50 µM, and then added to the wells as 10 µL aliquots. This provided a final concentration of 62.5, 31.25, 12.5, 6.25, and 3.125 µM for each sample. Each sample was tested in triplicate at each concentration. Aside from the 10 µL of sample solution, the other components of the well included 110 µL of phosphate buffer (pH 6.8) and 20 µL of enzyme solution (0.2 U · ml−1). The reaction was incubated at 37 °C for 15 min, then 20 µL of substrate solution (4.5 mmol · L−1 PNPG) was added, and the reaction was monitored at 400 nm by following the enzyme catalyzed release of p-nitrophenol from the substrate. The reference was prepared by adding an equivalent volume of phosphate buffer instead of sample solution. Acarbose was used as the positive control. The initial velocity was calculated from the slope of the progress curve of the enzymatic reaction. Based on the calculations of the velocities of the reference (Vr), the positive control, and each sample (Vs), the inhibitory rates (%) of the positive control and each sample were calculated according to the following formula: inhibition ð%Þ ¼ð1
Vs Þ 100% Vr
(2)
Supporting information Original spectra for compounds 1 and 2 as well as physicochemical data of compounds 3–28 are available as Supporting Information.
Acknowledgements !
This reasearch was financially supported by the National Natural Science Foundation of China (No. 81172982), Guangdong Provincial Project of Science and Technology (No. 2010A030100006), and the State Key Laboratory of Drug Research (SIMM1302KF12).
Conflict of Interest !
The authors state that they have no conflict of interest.
Affiliations 1
2
3
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou, P. R. China State key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, P. R. China Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou, P. R. China
References 1 Cardona ML, Fernández MI, Pedro JR, Serrano A. Xanthones from Hypericum reflexum. Phytochemistry 1990; 29: 3003–3006 2 Peres V, Nagem TJ. Trioxygenated naturally occurring xanthones. Phytochemistry 1997; 44: 191–214 3 Peres V, Nagem TJ, de Oliveira FF. Tetraoxygenated naturally occurring xanthones. Phytochemistry 2000; 44: 683–710 4 Hostettmann K, Wagner H. Xanthone glycosides. Phytochemistry 1977; 16: 821–829 5 Louh NG, Lannang AM, Mbazoa DC, Tangmouo JG, Komguem J, Castillo P, Mofo NF, Naz Q, Lontsi D, Iqbal MC, Sondengam BL. Polyanxanthone A, B and C, three xanthones from the wood trunk of Garcinia polyantha Oliv. Phytochemistry 2008; 69: 1013–1017 6 Pant N, Jain DC, Bhakuni RS. Phytochemicals from genus Swertia and their biological activities. Indian J Chem B 2000; 39: 565–586
7 Chin Y, Jung H, Chai H, Keller WJ, Kinghorn AD. Xanthones with quinone reductase-inducing activity from the fruits of Garcinia mangostana (Mangosteen). Phytochemistry 2008; 69: 754–758 8 Fouotsa H, Lannang AM, Mbazoa CD, Rasheed S, Marasini BP, Ali Z, Devkota KP, Kengfack AE, Shaheen F, Choudhary MI, Sewald N. Xanthones inhibitors of α-glucosidase and glycation from Garcinia nobilis. Phytochem Lett 2012; 5: 236–239 9 Jantan I, Saputri FC. Benzophenones and xanthones from Garcinia cantleyana var. cantleyana and their inhibitory activities on human lowdensity lipoprotein oxidation and platelet aggregation. Phytochemistry 2012; 80: 58–63 10 Zhang JS, Wang XM, Gao YT, Li GP. Studies on antioxidant activities of chemical constituents of Swertia mussotii Franch. Asian J Chem 2011; 23: 1966–1968 11 Uvarani C, Chandraprakash K, Sankaran M, Ata A, Mohan PS. Antioxidant and structure–activity relationships of five tetraoxygenated xanthones from Swertia minor (Griscb.) Knobl. Nat Prod Res 2012; 26: 1265–1270 12 Neerja P, Jain DC, Bhakuni RS. Phytochemicals from genus Swertia and their biological activities. Indian J Chem B 2000; 39: 565–586 13 Rukachaisirikul V, Kamkaew M, Sukavisit D, Phongpaichit S, Sawangchote P, Taylor WC. Antibacterial xanthones from the leaves of Garcinia nigrolineata. J Nat Prod 2003, 66: 1531–1535 14 Ito C, Itoigawa M, Takakura T, Ruangrungsi N, Enjo F, Tokuda H, Nishino H, Furukawa H. Chemical constituents of Garcinia fusca: structure elucidation of eight new xanthones and their cancer chemopreventive activity. J Nat Prod 2003; 66: 200–205 15 Matsumoto K, Akao Y, Kobayashi E, Ohguchi K, Ito T, Tanaka T, Iinuma M, Nozawa Y. Induction of apoptosis by xanthones from mangosteen in human leukemia cell lines. J Nat Prod 2003; 66: 1124–1127 16 Basnet P, Kadota S, Shimizu M, Takata Y, Kobayashi M, Namba T. Bellidifolin stimulates glucose uptake in rat 1 fibroblasts and ameliorates hyperglycemia in streptozotocin (STZ)-induced diabetic rats. Planta Med 1995; 61: 402–405 17 Tian LY, Bai X, Chen XH, Fang JB, Liu SH, Chen JC. Anti-diabetic effect of methylswertianin and bellidifolin from Swertia punicea Hemsl. and its potential mechanism. Phytomedicine 2010; 17: 533–539 18 Chhetri DR, Parajuli P, Subba GC. Antidiabetic plants used by Sikkim and Darjeeling Himalayan tribes, India. J Ethnopharmacol 2005; 99: 199– 202 19 Holst JJ, McGill MA. Potential new approaches to modifying intestinal GLP‑1 secretion in patients with type 2 diabetes mellitus: focus on bile acid sequestrants. Clin Drug Invest 2012; 32: 1–14 20 Israili ZH. Advances in the treatment of type 2 diabetes mellitus. Am J Ther 2011; 18: 117–152 21 Ghosal S, Sharma PV, Chaudhuri RK. Chemical constituents of Gentianaceae. 18. Xanthones of Swertia bimaculata. Phytochemistry 1975; 14: 2671–2675 22 Rana VS, Rawat MSM. A new xanthone glycoside and antioxidant constituents from the rhizomes of Swertia speciosa. Chem Biodivers 2005; 2: 1310–1315 23 Rezanka T, Jachymova J, Dembitsky VM. Prenylated xanthone glucosides from Uralʼs lichen Umbilicaria proboscidea. Phytochemistry 2003; 62: 607–612 24 Ikeya Y, Sugama K, Maruno M. Xanthone C-glycoside and acylated sugar from Polygala tenuifolia. Chem Pharm Bull 1994; 42: 2305–2308 25 Qiao Y, Okazaki T, Ando T, Mizoue K, Kondo K, Eguchi T, Kakinuma K. Isolation and characterization of a new pyrano[4′,3′:6, 7]naphtho[1, 2-b] xanthene antibiotic FD‑594. J Antibiot 1998; 51: 282–287 26 Meli AL, Komguem J, Ngounou FN, Tangmouo JG, Lontsi D, Ajaz A, Iqbal MC, Ranjit R, Devkota KP, Sondengam BL. Bangangxanthone A and B, two xanthones from the stem bark of Garcinia polyantha Oliv. Phytochemistry 2005; 66: 2351–2355 27 Wang S, Liang X. Isolation and chemical characterization of bioactive principles from Swertia franchetiana. Dalian: Dalian Institute of Chemical Physics, Chinese Academy of Sciences; 2004 28 Bian QY, Luo CN, Xiao PG. Four xanthone glycosides from Swertia calycina Franch. Pharm Pharmacol Commun 1998; 4: 597–598 29 Otsuka H. Triptexanthosides A–E: xanthone glycosides from aerial parts of Tripterospermum japonicum. Chem Pharm Bull 1999; 47: 962–965 30 Qing Z, Liu YW. Studies on the constituents of Swertia kauitchensis Franch. Hubei: Hubei College of Traditional Chinese Medicine; 2003 31 Yao S, Tang CP, Ye Y. Secoiridoids and xanthones from Tylophora secamonoides Tsiang. J Asian Nat Prod Res 2008; 10: 591–596
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Original Papers
Original Papers
32 Takashi H, Takeo N, Yuichi H, Keiichi F, Yukiteru K. 5,6-Dimethoxysterigmatocystin, a new metabolite from Aspergillus multicolor. Tetrahedron Lett 1997; 32: 2765–2766 33 Silveira ER, Falcão MJC, Menezes jr. A, Kingston DGI, Glass TE. Pentaoxygenated xanthones from Bredemeyera floribunda. Phytochemistry 1995; 39: 1433–1436 34 Bennett GJ, Lee HH, Lee LP. Synthesis of minor xanthones from Garcinia mangostana. J Nat Prod 1990; 53: 1463–1470 35 Hidehiro A, Yasuaki H, Mikio F, Yumiko H, Motonori F, Yujiro N, Yoshijiro N, Toshiro S, Kazuo T, Yoshiteru I. The chemical constituents of fresh Gentian root. J Nat Med 2007; 61: 269–279 36 Wang H, Ye G, Ma CH, Tang YH, Fan MS, Li ZX, Huang CG. Identification and determination of four metabolites of mangiferin in rat urine. J Pharm Biomed Anal 2007; 45: 793–798 37 Li S, Shi Y, Shang XY, Cui BS, Yuan Y, Chen XG, Yang YC, Shi JG. Triterpenoids from the roots of Pterospermum heterophyllum Hance. J Asian Nat Prod Res 2009; 11: 652–657 38 Cortez DAG, Young MCM, Marston A, Wolfender JL, Hostettmann K. Xanthones, triterpenes and a biphenyl from Kielmeyera coriacea. Phytochemistry 1998; 47: 1367–1374
Luo C-T et al. Xanthones from Swertia …
Planta Med 2014; 80: 201–208
39 Xu KP, Li FS, Liu JF, Tan JB, Zhang LH, Zeng GY, Tan GS. Studies on chemical constituents of Swertia nervosa (G. Don) Wall. Chin Pharm 2008; 43: 1612–1614 40 Chhetri DR, Parajuli P, Subba GC. Antidiabetic plants used by Sikkim and Darjeeling Himalayan tribes. Indian J Ethnopharmacol 2005; 99: 199– 202 41 Tian LY, Bai X, Chen XH, Fang JB, Liu SH, Chen JC. Anti-diabetic effect of methylswertianin and bellidifolin from Swertia punicea Hemsl. and its potential mechanism. Phytomedicine 2010; 17: 533–539 42 Masand VH, Mahajan DT, Patil KN, Chinchkhede KD, Jawarkar RD, Hadda TB, Alafeefy AA, Shibi IG. k-NN, quantum mechanical and field similarity based analysis of xanthone derivatives as α-glucosidase inhibitors. Med Chem Res 2012; 21: 4523–4534 43 Tanaka T, Nakashima T, Ueda T, Tomii K, Kouno I. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem Pharm Bull 2007; 55: 899–901 44 Choudhary MI, Shah SA, Atta-ur-Rahman Khan SN, Khan MT. Alpha-glucosidase and tyrosinase inhibitors from fungal hydroxylation of tibolone and hydroxytibolones. Steroids 2010; 75: 956–966 45 Wang YF, Ma L, Li Z, Du ZY, Liu Z, Qin JK, Wang XD, Huang ZS, Gu LQ, Chen ASC. Synergetic inhibition of metal ions and genistein on α-glucosidase. FEBS Lett 2004; 576: 46–50
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Authors
Cui-Ting Luo 1, Huan-huan Zheng 1, Shuang-Shuang Mao 1, Mao-xun Yang 1, Cheng Luo 2, Heru Chen 1, 2, 3
Affiliations
The affiliations are listed at the end of the article
Key words " Gentianaceae l " Swertia mussotii l " xanthones l " α‑glucosidase inhibitor l " diabetes mellitus l
Abstract !
Two new xanthones, 1,8-dihydroxy-3-methoxyxanthone 7-O-[α-L-rhamnopyranosyl(1 → 2)-βD-glucopyranoside] (1) and 1,8- dihydroxy-3methoxyxanthone 7-O-[α-L-rhamnopyranosyl (1 → 3)-α-L-rhamno-pyranosyl (1 → 2)-β-D-xylopyranoside] (2), together with 26 known xanthones (3–28), were isolated from the aqueous ethanol extract of the traditional Chinese herb Swertia mussotii. Their structures were elucidated via spectroscopic analyses including 2D NMR. The
Introduction !
received revised accepted
June 9, 2013 Nov. 19, 2013 Nov. 20, 2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1360173 Published online December 19, 2013 Planta Med 2014; 80: 201–208 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Dr. Heru Chen Institute of Traditional Chinese Medicine and Natural Products College of Pharmacy Jinan University Huangpu Avenue West 601 Guangzhou 510632 P. R. China Phone: + 86 20 38 37 52 99 Fax: + 86 20 85 22 15 59 [email protected]
Xanthones are the secondary metabolites that commonly occur in a few higher plant families, fungi, and lichens. Their taxonomic values in such families and pharmacological properties have provoked great interest [1]. To date, the isolated xanthones are classified into five major groups: simple oxygenated xanthones, xanthone glycosides, prenylated and related xanthones, xanthonolignoids, and miscellaneous. The simple oxygenated xanthones can further be subdivided into six groups according to the degree of oxygenation [2, 3]. The study of xanthones is interesting not only from a chemosystematic viewpoint but also from a pharmacological viewpoint. It has been reported that xanthones possess various biological and pharmacological properties such as antidepressant, antileukemic, antitumor, antitubercular, choleretic, diuretic, antimicrobial, antifungal, anti-inflammatory, antiviral, cardiotonic, and hypoglycemic activities [4–18]. We are most interested in the antidiabetic activity of xanthones. Two tetraoxygenated xanthones, methylswertianin and bellidifolin, were found to be potent hypoglycemic agents in streptozotocin (STZ)-induced diabetic rats by both oral and intraperitoneal administration [16, 17].
inhibition of α-glucosidase by the isolated xanthones was evaluated by an in vitro high-throughput screening assay. Our results indicated that 1,3,5,8-tetrahydroxyxanthone is the best inhibitor with an IC50 value of 5.33 ± 0.09 µM, while the O-glycosylated xanthones were poor α-glycosidase inhibitors. Supporting information available online at http://www.thieme‑connect.de/ejournals/toc/ plantamedica
Many people have type II diabetes mellitus (TII‑DM), which is associated with low insulin production or insulin resistance because of genetic and/or epigenetic causes. This disease is regarded as one of the most important public health problems throughout the world in the 21st century. Although many effective therapies have been developed to treat type II diabetes, there are a number of new approaches identified that provide better treatment options [19]. α-Glucosidase inhibitors are one type of useful agent that prevent the progression of the disease and are used for treating prediabetic conditions [20]. Interestingly, some xanthones have already been identified as α-glucosidase inhibitors [8]. As part of our ongoing research into the identification of lead compounds as α-glucosidase inhibitors from traditional Chinese medicine, a systematic study was initiated to investigate the chemical constituents (with a focus on xanthones) isolated from the aqueous ethanol extract of Swertia mussotii Franch, which is a traditional Tibetan folk medicine belonging to the family Gentianaceae. " Figs. 1 and 2, we describe Herein, as shown in l the isolation and characterization of two new xanthones (1–2), together with 26 known ones (3–28). The α-glucosidase inhibitory activities
Luo C-T et al. Xanthones from Swertia …
Planta Med 2014; 80: 201–208
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Xanthones from Swertia mussotii and Their α-Glycosidase Inhibitory Activities
Original Papers
Fig. 1 Chemical structures of compounds 1 and 2 from Swertia mussotii. (Color figure available online only.)
Fig. 2
Chemical structures of compounds 3–28.
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202
and associated structure-activity relationships of these compounds are also included.
Results and Discussion !
The aqueous ethanol extract of the whole Swertia mussotii plant dry powder was partitioned successively with petroleum ether, dichloromethane (DCM), ethyl acetate, and n-butanol. Both the DCM and n-butanol fractions were subjected to repeated separation on silica gel column chromatography (CC) as well as re-
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versed-phase chromatography with an ODS column, Sephadex LH-20, and preparative HPLC to afford two new xanthones (1–2) " Fig. 1) and 26 known xanthones (3–28) (l " Fig. 2). Compounds (l 1 and 2 belong to the tetraoxygenated xanthone group and contain a disaccharide and a trisaccharide, respectively. To date, there are only four reports of xanthone disaccharides [21–24] and just one report of a xanthone trisaccharide [25]. Compound 1 was obtained as an amorphous yellow solid. Its molecular formula, C26H30O15, was deduced from its HRESIMS, which exhibited a quasi-molecular ion peak at m/z 605.14 610 ([M + Na]+). The UV spectrum displayed absorption bands at λmax
Original Papers
1 δH (J in Hz)
1 2 3 4 4a 4b 5 6 7 8 8a 8b 9 1-OH 8-OH 3-OCH3 Glc-1 Glc-2 Glc-3 Glc-4 Glc-5 Glc-6 Rha-1 Rha-2 Rha-3 Rha-4 Rha-5 Rha-6 Rha-1, Rha-2, Rha-3, Rha-4, Rha-5, Rha-6, Xyl-1 Xyl-2 Xyl-3 Xyl-4 Xyl-5
6.31, d (3) 6.54, d (3)
6.92, d (9) 7.56, d (9)
11.75, s 11.75, s 3.85, s 5.09, d (9)
5.22, s
1.12, d (6)
2 δC 161.9 97.4 167.2 92.9 157.6 149.9 105.7 125.1 139.6 149.8 107.3 101.8 184.2
56.3 98.8 76.2 76.9 70.6 77.7 60.6 100.3 69.9 70.6 72.1 68.4 18.1
236, 260, and 330 nm, which implied a xanthone derivative [26]. The IR (KBr) υmax at 3396, 1645, 1606, and 1505 cm−1 indicated the existence of a hydroxyl group, a carbonyl group, as well as a " Taconjugated benzene ring group. The 1H NMR spectrum of 1 (l ble 1) exhibited peaks indicating hydroxyl protons at δH 11.75 (2H, s, OH-1, and OH-8), which implied the existence of an intramolecular hydrogen bond, along with meta-coupled aromatic protons at δH 6.31 (1H, d, J = 3 Hz, H-2) and δH 6.54 (1H, d, J = 3 Hz, H-4), and ortho-coupled aromatic protons at δH 6.92 (1H, d, J = 9 Hz, H-5) and δH 7.56 (1H, d, J = 9 Hz, H-6). The signals for an anomeric proton at δH 5.09 (1H, d, J = 6 Hz) substantiated that 1 was a β-O-glucoside. In addition, the methyl group signal at δH 1.12 (3H, d, J = 6 Hz) combined with an anomeric proton at δH 5.22 (1H, s) confirmed that 1 had an α-rhamnopyranose moiety. The spectrum also showed signals for a methoxy group at δH " Table 1) indi3.88 (3H, s). Analysis of the 13C NMR spectrum (l cated 13 carbon signals at a low field, including one carbonyl at δC 184.2, suggesting that two hydroxyls are located at C-1 and C8. A set of glycosyl carbon signals at a high field, δc 98.8, 76.2, 76.9, 70.6, 77.7, and 60.6, suggested that there was a β-glucopyranosyloxy unit in the molecule, and a set of glycosyl carbon signals at δC 100.3, 69.9, 70.6, 72.1, 68.4, and 18.1 implied that there
δH (J in Hz) 6.23, d (1.8) 6.41, d (1.8)
6.86, d (9) 7.50, d (9)
δC 161.9 97.4 167.2 92.8 157.5 150.8 106.0 127.2 138.7 150.6 107.5 101.3 184.0
3.80, s
56.3
4.89, s
101.4 70.6 70.8 72.0 69.0 17.9 101.3 70.6 70.7 72.0 68.9 17.8 99.8 81.5 76.9 68.4 65.4
1.13, d (6) 4.83, s
1.05, d (6) 5.22, d (6)
Table 1 1H and 13C NMR spectroscopic data for compounds 1 and 2 (δ in ppm).
was an α-rhamnopyranosyl unit in the structure. The structure of compound 1 was further confirmed by HSQC and HMBC. HSQC results confirmed that Glc-C-1 is at δc 98.8, Rha-C-1 at δc 100.3, " Fig. 3) showed a cross-peak of and -OCH3 at δc 56.3. HMBC (l -OCH3 with C-3 implying that there is a substitution at C-3 by -OCH3. It was observed that the anomeric proton (δH 5.09) is correlated with the carbon at δC 139.6 (C-7). This is the direct evidence that the glucosyl moiety is linked to C-7. The meta-coupled aromatic proton at δH 6.31 (H-2) was correlated with δC at 184.2 (C=O), 167.2 (C-3), 161.9 (C-1), 101.8 (C-8b), and 92.9 (C-4). Another meta-coupled aromatic proton at δH 6.54 (H-4) was correlated with δC at 167.2 (C-3), 157.6 (C-4a), 101.8 (C-8b), 97.43 (C2), and 184.2 (C=O). The doublet at δH 6.92 (H-5) was correlated with δC at 184.2 (C=O), 149.9 (C-4b), 139.7 (C-7), and 107.3 (C8a); whereas another doublet at δH 7.56 (H-6) was found to be correlated with δC at 149.8 (C-8), 139.7 (C-7), 105.7 (C-5), and 149.9 (C-4b). Taken together, these data supported that the assignments of H-2, H-4, H-5, and H-6 were correct. Furthermore, it was observed that another anomeric proton (δH 5.22) was correlated with δC 76.2 (Glc-C-2), which moved to a lower field when compared with the reference glucopyranosyl. Therefore the α-rhamnopyranosyl was suggested to be linked to Glc-2. AcLuo C-T et al. Xanthones from Swertia …
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Fig. 3 Key HMBC correlations of compounds 1 and 2 (recorded on Bruker AV-400).
idic hydrolysis of compound 1 and derivation of the resulting compounds with o-tolylisothiocyanate were carried out. HPLC analysis of the derivatives confirmed the existence of one β-Dglucopyranose at a retention time (RT) of 23.8 min and one αrhamnopyranose at RT of 41.0 min. Hence, the structure of 1 was determined to be 1,8-dihydroxy-3-methoxyxanthone 7-O-[α-Lrhamnopyranosyl (1 → 2)-β-D-glucopyranoside]. Compound 2 was isolated as a yellow powder. Its HRESIMS spectrum exhibited a quasi-molecular ion peak at m/z 721.19 658 ([M + Na]+), which indicated a molecular formula of C31H38O18. The UV spectrum with absorption bands at λmax 236, 260, and 330 nm implied a xanthone derivative [26]. The 1H NMR spec" Table 1) exhibited peaks for hydroxyl protons at δ trum of 2 (l H 11.75 (2H, s, OH-1, and OH-8), which implied the existence of an intramolecular hydrogen bond along with a set of meta-coupled aromatic protons at δH 6.41 (1H, d, J = 1.8 Hz, H-4) and δH 6.23 (1H, d, J = 1.8 Hz, H-2) as well as two ortho-coupled aromatic protons at δH 6.86 (1H, d, J = 9 Hz, H-5) and δH 7.50 (1H, d, J = 9 Hz, H6). Three anomeric protons were found at δH 5.22, 4.89, and 4.83. The signal at δH 5.22 (1H, d, J = 9 Hz) suggested that 2 probably contained a β-O-xylopyranosyl. The signals at δH 4.89 (1H, s) and 4.83 (1H, s) together with two methyl signals at δH 1.13 and 1.05 (3H, d, J = 6 Hz) indicated that 2 may have two α-rhamnopyranosyl units. The spectrum also had a signal for a methoxyl group at " Table 1) δH 3.80 (3H, s). Analysis of the 13C NMR spectrum (l showed 13 carbon signals at a low field and one carbonyl signal at δC 184.0. These data further confirmed the existence of a xanthone. A set of glycosyl carbon signals at a high field, δc 99.8, 81.5, 76.9, 68.4, and 65.4, further supported a β-O-xylopyranose unit in the structure. Another two sets of glycosyl carbon signals were observed at δC 101.4, 72.0, 70.8, 70.6, and 69.0 and at δC 101.3, 71.9, 70.7, 70.6, and 68.9. This implied two α-rhamnopyranosyl units in the molecule. Comparison of the 1H NMR and 13C NMR spectra indicated that compound 2 had the same xanthone scaffold as 1. The only difference between them appeared to be the polysaccharide moiety. Therefore, the structural exploration of 2 focused on confirming the identity and spatial arrangement of the glycosyl components. Based on a polysaccharide analysis assay, it was confirmed that 2 contained one D-xylopyranosyl unit with an RT of 27.8 min and two α-rhamnopyranosyl units with an RT of 41.0 min. The glycosyl connection was confirmed from the " Fig. 3) and HSQC. It was found that the HMBC spectrum (l anomeric proton (δH 5.22) was correlated with δC 138.7 (C-7) in the HMBC and with the carbon signal at δc 99.8 (Xyl-1) in the HSQC spectrum. This fact suggested that the xylopyranosyl unit was linked to C-7. In addition, it was observed that the second anomeric proton (δH 4.89) was correlated with δC 76.9 (Xyl-3), which moved to a lower field when compared with the reference xylopyranose. This anomeric proton was correlated with δC 101.4 Luo C-T et al. Xanthones from Swertia …
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(Rha-1) in the HSQC. Therefore, one α-rhamnopyranose unit was suggested to be linked to Xyl-3. The third anomeric proton (δH 4.83) was correlated with δC 81.5 (Xyl-2), which moved to a lower field when compared with the reference xylopyranose. This third anomeric proton was correlated with δC 101.3 (Rha-1′) in the HSQC. Thus the second α-rhamnopyranose unit was suggested to be linked to Xyl-2, and compound 2 was identified as 1,8-dihydroxy-3-methoxyxanthone 7-O-[α-L-rhamnopyranosyl(1 → 3)α-L-rhamnopyranosyl (1 → 2)-β-D-xylopyranoside]. " Fig. 2) were identified as 1,3,5-trihydroxyCompounds 3–28 (l xanthone 8-O-[β-D-glucopyranoside] (3) [27], 3,7,8-trihydroxyxanthone 1-O-[β-D-glucopyranoside] (4) [28], 1,8-dihydroxy3,4-dimethoxyxanthone 7-O-[β-D-glucopyranoside] (5) [29], 1,8-dihydroxy-3-methoxyxanthone 7-O-[β-D-glucopyranoside] (6) [30], 1-hydroxy-3,4-dimethoxyxanthone 7-O-[β-D-glucopyranoside] (7) [31], 3,7,8-trimethoxyxanthone 1-O-[β-D-glucopyranoside] (8) [27], 1,8-dihydroxy-3-methoxyxanthone 7-O[α-L-rhamnopyranosyl(1 → 2)-β-D-xylopyranoside] (9) [27], mangiferin (10) [27], 2,6,8-trihydroxyxanthone 7C-(β-D-glucoside) (11) [27], 1,6-dihydroxy-3,4,8-trimethoxyxanthone (12) [32], 1,7-dihydroxy-3,8-dimethoxyxanthone (13) [27], 1,7,8-trihydroxy-3,4-dimethoxy-xanthone (14) [33], 1,8-dihydroxy-3,7dimethoxy xanthone (15) [27], 1-hydroxy-3,7,8-trimethoxyxanthone (16) [27], 1,7,8-trihydroxy-3-methoxyxanthone (17) [27], 1,3,5,8-tetrahydroxyxanthone (18) [30], 1,7-dihydroxy-3,4,8-trimethoxyxanthone (19) [33], 1-hydroxy-3,4,7,8-tetramethoxy xanthone (20) [33], 1,8-dihydroxy-7-methoxyxanthone (21) [34], 1,7-dihydroxy-3-methoxyxanthone (22) [35], 1,3,7-trihydroxyxanthone (23) [36], 1,3,7,8-tetrahydroxylxanthone (24) [37], 1,3-dihydroxy-7,8-dimethoxyxanthone (25) [38], 1,5,8-trihydroxy-3,4-dimethoxyxanthone (26) [39], 1-hydroxy-3,4,5,8tetramethoxyxanthone (27) [37], and 1-hydroxy-3,5,8-trimethoxy xanthone (28) [37] by comparison of their spectroscopic data with those reported in the literature. It needs to be pointed out that compounds 11 and 14 were first isolated from this plant family, while compound 4 was first isolated from this plant. Several xanthones such as swerchirin, methylswertianin, and bellidifolin have been confirmed as antidiabetic agents [16–18, 40, 41]. Some have been identified as α-glucosidase inhibitors [8, 42]. With 28 isolated xanthones (1–28) on hand, we systematically investigated their potential to inhibit α-glucosidase and to " Tadisclose their structure-activity relationship. As shown in l ble 2, glycosylated xanthones (1–11) are generally poor inhibitors except for mangiferin (10) and 9, where 10 is a C-glycoside with an IC50 value of 13.02 ± 0.23 µM and 9 is a xanthone glycoside without a glucosyl group. This suggests that O-glycosylated xanthones containing a glucosyl unit are substrates of α-glucosidase, while C-glycosylated xanthones and O-glycosylated xanthones without a glucosyl unit are not. The hydroxyl group at C-3 is very
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Acarbosea 1 2 3 4 5 6 7 8 9 10 11
IC50 (µM)±SEM 39.6 ± 0.1 75.8 ± 1.3 84.5 ± 1.2 > 500 > 500 133.7 ± 3.0 394.9 ± 2.0 115.0 ± 2.4 83.8 ± 1.2 31.1 ± 0.2 13.0 ± 0.2 140.4 ± 0.8
Compounds
IC50 (µM)±SEM
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
> 500 > 500 5.42 ± 0.07 107.8 ± 0.1 142.1 ± 0.2 30.6 ± 0.1 5.3 ± 0.1 77.4 ± 0.1 65.8 ± 0.1 71.2 ± 0.2 17.6 ± 0.1 31.8 ± 0.1 7.09 ± 0.08 18.0 ± 0.1 82.2 ± 0.2 40.1 ± 0.1 75.8 ± 0.1
Table 2 α-Glucosidase inhibition of all the isolated xantones.
IC50 values expressed as mean ± SEM (standard error of mean), where n = 3; a positive control
important for maintaining the activity, e.g., mangiferin is a good inhibitor, but compound 11 is not. Compound 18 is the most active inhibitor with an IC50 value of 5.33 ± 0.09 µM followed by 14 with an IC50 value of 5.42 ± 0.07 µM. It is of note that 18 is structurally similar to bellifolin (1,5,8-trihydroxy-3-methoxyxanthone) which was reported to be a small molecule anti-diabetes candidate. The only difference between them is that the 3-hydroxyl group in compound 18 is a 3-methoxy group in bellifolin. A comparison of the inhibitory activities of 18 with 3 and 24 with 4 indicated that O-glycosylation at either C-1 or C-8 results in a loss of almost all the α-glucosidase inhibitory activity. This is supported by the fact that the IC50 values of 3 and 4 are all greater than 500 µM. Methylation of a hydroxyl group in xanthone will generally decrease the inhibitory activity of the compound, e.g., methylations of hydroxyl groups at C3, C5, and C8 in 18, which convert it into compound 28, decreases its activity by 14-fold. A comparison of the structure-activity relationship among compounds 24, 25, and 13 allowed us to conclude that free hydroxyl groups at C-3 and C-8 are very important for maintaining inhibitory activity. Taken together, the current studies offer useful information for further structural modification of xanthones for the purposes of designing small molecule inhibitors, which may prove useful as therapeutics for a variety of disease areas.
Materials and Methods !
(> 99%), L-glucose (> 99 %), D-xylose (> 99%), L-xylose (> 99%), Drhamnose (> 99 %), and L-rhamnose (> 99 %) were purchased from Ada-Mas-Beta Company. The 96-well Fluotrac 200 black microplates (part # 655 076) were purchased from Greiner America, Inc., and the 48-well microplates (Falcon No. 3230) were purchased from VWR. Clear polystyrene 96-well plates (Nunc) were purchased from Fisher Scientific.
General experimental procedures UV spectra were obtained using a Jasco V-550 spectrometer, whereas FT‑IR spectra were acquired on a Jasco FI/IR‑480 Plus Fourier Transform (using KBr disk method). The ESI‑MS spectra were acquired on a Finnigan LCQ Advantage Max spectrometer. The HRESIMS spectra were obtained on an Agilent QTOF spectrometer. NMR spectra were recorded on a Bruker AV-300 or Bruker AV-400 NMR spectrometer using TMS as an internal standard. Silica gel (200–300 mesh) for CC was purchased from Yantai Chemical Factory, and ODS (50 µm) was purchased from YMC Co. Ltd. Precoated thin-layer chromatography (TLC) plates (Institute of Yantai Chemical Industry) were used for TLC. Spots on TLC plates were detected by either a ZF-7A portable UV detector or spraying a KMnO4 solution followed by subsequent heating. Sephadex LH-20 was purchased from GE Healthcare. The RP-HPLC analysis and preparation were conducted using a P680 pumping system equipped with a PDA-100 photodiode array detector and Cosmosil columns (C18; 4.6 × 250 mm for analysis and 20 × 250 mm for preparation).
Plant materials One batch of plant material (batch no. 106785) was collected from Zhongda Village, Yushu County, Qinghai Province (China) on October 20, 2010. It was identified as Swertia mussotii by Dr. Jing Sun from the Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China. A reference sample (voucher sample NIPB-1136) is stored at the Institute.
Chemicals and apparatus α-Glucosidase (maltase) was purchased from Oriental Yeast Co. Ltd. 4-Nitrophenyl-α-D-glucopyranoside (PNPG) was purchased from Aldrich. L-cysteine methyl ester and acarbose (purity > 99%) were purchased from Aladdin. O-Tolyl isothiocyanate was purchased from Aldrich. Standard sugars including D-glucose
Extraction and isolation Dried powder of Swertia mussotii (5.0 kg) was extracted three times (2 h, 2 h, and 1 h) with 75 % aqueous ethanol (10 L) under reflux. The extracts were concentrated in vacuo to yield a brown-black gum (1.1 kg). A total of 509 g of the gum was then suspended in H2O and partitioned successively with petroleum ether (PE, 1.5 L), dichloromethane (DCM, 1.5 L), ethyl acetate (EE, 1.5 L), and n-butanol (NB, 1.5 L) to yield PE (35 g), DCM (92 g), EE (42 g), NB (170 g), and water (171 g) fractions. The DCM fraction was then subjected to silica gel CC (695 × 130 mm) using a gradient system of petroleum ether-ethyl acetate (30 : 1–0 : 1) (25 L, flow 2.5–3.5 mL/ min). The collected fractions were combined based on their TLC
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Planta Med 2014; 80: 201–208
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characteristics to offer eight subfractions (F1-F8), whose amount was 9.2, 13.5, 11.6, 10.3, 9.8, 12.6, 10.5, and 10.1 g, respectively. The F1 subfraction was separated further by silica gel CC (550 × 65 mm) using a gradient system of petroleum ether-ethyl acetate (15 : 1–0 : 1) and by recrystallization to yield pure com" Fig. 2), whose amount was 21.3, 32.5, and pounds (14–16) (l 45.7 mg, respectively. F2 was subjected to Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (6 L, flow 1.5– 2.5 mL/min) to yield compounds 12 (46.5) and 13 (58.7). Compounds 16 (60.2 mg) and 17 (55.3 mg) were isolated from F3 by repeated silica gel CC (550 × 65 mm) with PE-acetone and further separated by HPLC on a preparative Cosmosil column (20 × 250 mm) applying MeOH‑H2O as the eluant with flow rate 3.5 mL/min. F4 was purified by Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) to produce compound 19 (27.8 mg). Compound 20 (38.9 mg) was isolated from F5 by Sephadex LH-20 CC eluted with CHCl3-MeOH (1 : 1) (6.5 L, flow 1.5– 2.5 mL/min) and further purified by preparative HPLC (Cosmosil column, 20 × 250 mm) using MeOH‑H2O as the eluant at a flow rate of 3.5 mL/min. Compounds 21 (39.6 mg) and 22 (61.8 mg) were isolated from F6 by repeated silica gel CC (550 × 65 mm) with PE-acetone and then further purified by preparative HPLC as described above. F7 was purified by Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (5 L, flow 1.5– 2.5 mL/min) to yield compounds 23 (19.3 mg) and 24 (36.3 mg). Compounds 26–28 were isolated from F8 by Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (7 L, flow 1.5– 2.5 mL/min) and then further purified by preparative HPLC as described above resulting in 26 (41.5 mg), 27 (53.2 mg), and 28 (33.7 mg). The n-butanol fraction was first separated by a silica gel CC (695 × 130 mm) using a gradient system of CHCl3-MeOH (15 : 1– 0 : 1) (25 L, flow 2.5–3.5 mL/min). The collected fractions were combined based on their TLC characteristics to yield ten subfractions (P1-P10), whose amount was 15.2, 13.5, 11.6, 17.3, 19.8, 12.6, 11.5, 13.1, 20.3, and 14.9 g, respectively. Compounds 5 (15.6 mg) and 8 (21.8 mg) were isolated from P3 by preparative HPLC as described above. Compounds 6 (20.8 mg), 7 (18.2 mg), and 9 (23.7 mg) were isolated from P4 by reversed-phase ODS CC (800 × 35 mm) eluted with an MeOH‑H2O (1 : 1) (4 L, flow 1.0– 1.5 mL/min) and further submitted to Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (5 L, flow 1.5– 2.5 mL/min). Compounds 10 (15.6 mg) and 11 (19.3 mg) were isolated from P5 by preparative HPLC as described above. Compounds 3 (11.5 mg) and 4 (16.3 mg) were isolated from P6 by Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (4 L, flow 1.5–2.5 mL/min) and then further purified by prepara" Fig. 1) were tive HPLC. Compounds 1 (9.8 mg) and 2 (13.2 mg) (l isolated from P7 by Sephadex LH-20 CC (700 × 35 mm) eluted with CHCl3-MeOH (1 : 1) (4 L, flow 1.5–2.5 mL/min) and then further purified by preparative HPLC as described above. 1,8-Dihydroxy-3-methoxyxanthone 7-O-[α‑L-rhamnopyranosyl (1 → 2)-β‑D-glucopyranoside] (1): Yellow amorphous powder, purity: 98.5 %; [α]20 D −50.5 (c 0.10, DMSO); UV (MeOH) λmax (log ε): 236 (4.41), 260 (4.51), 330 (4.25) nm; IR νmax (KBr): 3396, 1645, 1606, 1505 cm−1; ESI‑MS (m/z): 605.4 [M + Na]+ (calcd. for C26H30O15 + Na, 605.1); HRESIMS (m/z): 605.14 610 [M + Na]+ (calcd. for C26H30O15 + Na, 605.14 824); for 1H and 13C NMR " Table 1. (300 MHz, DMSO‑d6) spectroscopic data, see l 1,8-Dihydroxy-3-methoxyxanthone 7-O-[α‑L-rhamnopyranosyl (1 → 3)-α‑L-rhamno-pyranosyl(1 → 2)-β‑D-xylopyranoside] (2): Yellow amorphous powder, purity: 99.2 %; [α]20 D −61.3 (c 0.10, Luo C-T et al. Xanthones from Swertia …
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DMSO); UV (MeOH) λmax (log ε): 236 (4.52), 260 (4.64), 330 (4.35) nm; IR νmax (KBr): 3395, 1636, 1606, 1499 cm−1; ESI‑MS (m/z): 697.2 [M – H]− (calcd. for C31H38O18-H: 697.3); HRESIMS (m/z): 721.19 658 [M + Na]+ (calcd. for C31H38O18 + Na, 721.19 558); for 1H and 13C NMR (300 MHz, DMSO‑d6) spectro" Table 1. scopic data, see l
Confirmation of glycosyl units Compounds 1 and 2 (1 mg each) were individually hydrolyzed with 3 mL of 1 M HCl for 2 h at 90 °C. The mixture was evaporated to dryness under vacuum. The residue was dissolved in pyridine (2–3 mL) containing L-cysteine methyl ester (1 mg) and heated at 60 °C for 1 h. Then, o-tolyl isothiocyanate (10–15 µL) was added to the mixture which was heated at 60 °C for another 1 h. The reaction mixture (2 µL) was analyzed directly by reversed-phase HPLC [43]. Analytical HPLC was performed on a 250 × 4.6 mm i. d. Cosmosil 5C18 column at 30 °C with isocratic elution of 22 % CH3CN for 50 min and subsequent washing of the column with 90 % CH3CN at a flow rate of 1.0 mL/min. Peaks were detected using a UV detector at 250 nm. The standard sugars including Dglucose, L-glucose, D-xylose, L-xylose, D-rhamnose, and L-rhamnose were subjected to the same method. The peaks of the standard sugar derivatives were recorded at retention times (RT) of 23.5 (D‑Glc), 21.6 (L‑Glc), 27.3 (D‑Xyl), 25.3 (L‑Xyl), 42.5 (D‑Rha), and 40.6 (L‑Rha) min. Following the chemical reaction, the derivatives of 1 gave peaks at an RT of 23.8 min (D‑Glc) and 41.0 min (L‑Rha) with a ratio of peak area of 1 : 1. This supports the idea that compound 1 has one β-D-glucopyranosyl unit and one αrhamnopyranosyl unit. The derivatives of 2 gave peaks at 27.8 min (D‑Xyl) and 41.1 min (L‑Rha) with a ratio of peak area of 1 : 2. This supports the conclusion that compound 2 has one D-xylosyl unit and two α-rhamnopyranosyl units.
In vitro α-glucosidase inhibition assay The α-glucosidase inhibition was carried out by either assay (A) or (B). IC50 values were determined by using EZ‑FIT, an enzyme kinetics software by Perrella Scientific Inc. Results are presented as mean ± SEM (standard error of mean) from three experiments. (A) α-Glucosidase inhibitory activity was assayed in 0.1 M sodium phosphate buffer (pH 6.8) with p-nitrophenyl-α-D-glucopyranoside (PNPG) as the substrate. The concentration of α-glucosidase was 0.2 U/mL in each experiment. The enzyme (20 mL) along with 100 mL of phosphate buffered saline was incubated with various concentrations of test compounds at 37 °C. After a preincubation time of 15 min, 20 mL of substrate (0.7 mM) was added, and the reaction was carried out at 37 °C for 30 min. The enzymatic activity was quantified by measuring the absorbance of pnitrophenol at 400 nm on a microtiter plate spectrophotometer (Spectra Max; Molecular Devices). One unit of α-glucosidase was defined as the amount of enzyme capable of liberating 1.0 mmol of p-nitrophenol per minute under the conditions specified [44]. Acarbose was used as the positive control (IC50 = 39.75 ± 0.06 µM) " Table 2). The percent inhibition of p-nitrophenol formation in (l the test sample versus the reference (without the addition of sample) was calculated for each compound by using the following formula: % inhibition ¼ ð1
ODsample Þ 100% ODreference
(1)
(B) The α-glucosidase inhibition was determined spectrophotometrically in a 96-well plate using PNPG as the substrate [45].
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All the test samples were prepared as stock solutions of 1000, 500, 200, 100, and 50 µM, and then added to the wells as 10 µL aliquots. This provided a final concentration of 62.5, 31.25, 12.5, 6.25, and 3.125 µM for each sample. Each sample was tested in triplicate at each concentration. Aside from the 10 µL of sample solution, the other components of the well included 110 µL of phosphate buffer (pH 6.8) and 20 µL of enzyme solution (0.2 U · ml−1). The reaction was incubated at 37 °C for 15 min, then 20 µL of substrate solution (4.5 mmol · L−1 PNPG) was added, and the reaction was monitored at 400 nm by following the enzyme catalyzed release of p-nitrophenol from the substrate. The reference was prepared by adding an equivalent volume of phosphate buffer instead of sample solution. Acarbose was used as the positive control. The initial velocity was calculated from the slope of the progress curve of the enzymatic reaction. Based on the calculations of the velocities of the reference (Vr), the positive control, and each sample (Vs), the inhibitory rates (%) of the positive control and each sample were calculated according to the following formula: inhibition ð%Þ ¼ð1
Vs Þ 100% Vr
(2)
Supporting information Original spectra for compounds 1 and 2 as well as physicochemical data of compounds 3–28 are available as Supporting Information.
Acknowledgements !
This reasearch was financially supported by the National Natural Science Foundation of China (No. 81172982), Guangdong Provincial Project of Science and Technology (No. 2010A030100006), and the State Key Laboratory of Drug Research (SIMM1302KF12).
Conflict of Interest !
The authors state that they have no conflict of interest.
Affiliations 1
2
3
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou, P. R. China State key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, P. R. China Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou, P. R. China
References 1 Cardona ML, Fernández MI, Pedro JR, Serrano A. Xanthones from Hypericum reflexum. Phytochemistry 1990; 29: 3003–3006 2 Peres V, Nagem TJ. Trioxygenated naturally occurring xanthones. Phytochemistry 1997; 44: 191–214 3 Peres V, Nagem TJ, de Oliveira FF. Tetraoxygenated naturally occurring xanthones. Phytochemistry 2000; 44: 683–710 4 Hostettmann K, Wagner H. Xanthone glycosides. Phytochemistry 1977; 16: 821–829 5 Louh NG, Lannang AM, Mbazoa DC, Tangmouo JG, Komguem J, Castillo P, Mofo NF, Naz Q, Lontsi D, Iqbal MC, Sondengam BL. Polyanxanthone A, B and C, three xanthones from the wood trunk of Garcinia polyantha Oliv. Phytochemistry 2008; 69: 1013–1017 6 Pant N, Jain DC, Bhakuni RS. Phytochemicals from genus Swertia and their biological activities. Indian J Chem B 2000; 39: 565–586
7 Chin Y, Jung H, Chai H, Keller WJ, Kinghorn AD. Xanthones with quinone reductase-inducing activity from the fruits of Garcinia mangostana (Mangosteen). Phytochemistry 2008; 69: 754–758 8 Fouotsa H, Lannang AM, Mbazoa CD, Rasheed S, Marasini BP, Ali Z, Devkota KP, Kengfack AE, Shaheen F, Choudhary MI, Sewald N. Xanthones inhibitors of α-glucosidase and glycation from Garcinia nobilis. Phytochem Lett 2012; 5: 236–239 9 Jantan I, Saputri FC. Benzophenones and xanthones from Garcinia cantleyana var. cantleyana and their inhibitory activities on human lowdensity lipoprotein oxidation and platelet aggregation. Phytochemistry 2012; 80: 58–63 10 Zhang JS, Wang XM, Gao YT, Li GP. Studies on antioxidant activities of chemical constituents of Swertia mussotii Franch. Asian J Chem 2011; 23: 1966–1968 11 Uvarani C, Chandraprakash K, Sankaran M, Ata A, Mohan PS. Antioxidant and structure–activity relationships of five tetraoxygenated xanthones from Swertia minor (Griscb.) Knobl. Nat Prod Res 2012; 26: 1265–1270 12 Neerja P, Jain DC, Bhakuni RS. Phytochemicals from genus Swertia and their biological activities. Indian J Chem B 2000; 39: 565–586 13 Rukachaisirikul V, Kamkaew M, Sukavisit D, Phongpaichit S, Sawangchote P, Taylor WC. Antibacterial xanthones from the leaves of Garcinia nigrolineata. J Nat Prod 2003, 66: 1531–1535 14 Ito C, Itoigawa M, Takakura T, Ruangrungsi N, Enjo F, Tokuda H, Nishino H, Furukawa H. Chemical constituents of Garcinia fusca: structure elucidation of eight new xanthones and their cancer chemopreventive activity. J Nat Prod 2003; 66: 200–205 15 Matsumoto K, Akao Y, Kobayashi E, Ohguchi K, Ito T, Tanaka T, Iinuma M, Nozawa Y. Induction of apoptosis by xanthones from mangosteen in human leukemia cell lines. J Nat Prod 2003; 66: 1124–1127 16 Basnet P, Kadota S, Shimizu M, Takata Y, Kobayashi M, Namba T. Bellidifolin stimulates glucose uptake in rat 1 fibroblasts and ameliorates hyperglycemia in streptozotocin (STZ)-induced diabetic rats. Planta Med 1995; 61: 402–405 17 Tian LY, Bai X, Chen XH, Fang JB, Liu SH, Chen JC. Anti-diabetic effect of methylswertianin and bellidifolin from Swertia punicea Hemsl. and its potential mechanism. Phytomedicine 2010; 17: 533–539 18 Chhetri DR, Parajuli P, Subba GC. Antidiabetic plants used by Sikkim and Darjeeling Himalayan tribes, India. J Ethnopharmacol 2005; 99: 199– 202 19 Holst JJ, McGill MA. Potential new approaches to modifying intestinal GLP‑1 secretion in patients with type 2 diabetes mellitus: focus on bile acid sequestrants. Clin Drug Invest 2012; 32: 1–14 20 Israili ZH. Advances in the treatment of type 2 diabetes mellitus. Am J Ther 2011; 18: 117–152 21 Ghosal S, Sharma PV, Chaudhuri RK. Chemical constituents of Gentianaceae. 18. Xanthones of Swertia bimaculata. Phytochemistry 1975; 14: 2671–2675 22 Rana VS, Rawat MSM. A new xanthone glycoside and antioxidant constituents from the rhizomes of Swertia speciosa. Chem Biodivers 2005; 2: 1310–1315 23 Rezanka T, Jachymova J, Dembitsky VM. Prenylated xanthone glucosides from Uralʼs lichen Umbilicaria proboscidea. Phytochemistry 2003; 62: 607–612 24 Ikeya Y, Sugama K, Maruno M. Xanthone C-glycoside and acylated sugar from Polygala tenuifolia. Chem Pharm Bull 1994; 42: 2305–2308 25 Qiao Y, Okazaki T, Ando T, Mizoue K, Kondo K, Eguchi T, Kakinuma K. Isolation and characterization of a new pyrano[4′,3′:6, 7]naphtho[1, 2-b] xanthene antibiotic FD‑594. J Antibiot 1998; 51: 282–287 26 Meli AL, Komguem J, Ngounou FN, Tangmouo JG, Lontsi D, Ajaz A, Iqbal MC, Ranjit R, Devkota KP, Sondengam BL. Bangangxanthone A and B, two xanthones from the stem bark of Garcinia polyantha Oliv. Phytochemistry 2005; 66: 2351–2355 27 Wang S, Liang X. Isolation and chemical characterization of bioactive principles from Swertia franchetiana. Dalian: Dalian Institute of Chemical Physics, Chinese Academy of Sciences; 2004 28 Bian QY, Luo CN, Xiao PG. Four xanthone glycosides from Swertia calycina Franch. Pharm Pharmacol Commun 1998; 4: 597–598 29 Otsuka H. Triptexanthosides A–E: xanthone glycosides from aerial parts of Tripterospermum japonicum. Chem Pharm Bull 1999; 47: 962–965 30 Qing Z, Liu YW. Studies on the constituents of Swertia kauitchensis Franch. Hubei: Hubei College of Traditional Chinese Medicine; 2003 31 Yao S, Tang CP, Ye Y. Secoiridoids and xanthones from Tylophora secamonoides Tsiang. J Asian Nat Prod Res 2008; 10: 591–596
Luo C-T et al. Xanthones from Swertia …
Planta Med 2014; 80: 201–208
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Original Papers
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32 Takashi H, Takeo N, Yuichi H, Keiichi F, Yukiteru K. 5,6-Dimethoxysterigmatocystin, a new metabolite from Aspergillus multicolor. Tetrahedron Lett 1997; 32: 2765–2766 33 Silveira ER, Falcão MJC, Menezes jr. A, Kingston DGI, Glass TE. Pentaoxygenated xanthones from Bredemeyera floribunda. Phytochemistry 1995; 39: 1433–1436 34 Bennett GJ, Lee HH, Lee LP. Synthesis of minor xanthones from Garcinia mangostana. J Nat Prod 1990; 53: 1463–1470 35 Hidehiro A, Yasuaki H, Mikio F, Yumiko H, Motonori F, Yujiro N, Yoshijiro N, Toshiro S, Kazuo T, Yoshiteru I. The chemical constituents of fresh Gentian root. J Nat Med 2007; 61: 269–279 36 Wang H, Ye G, Ma CH, Tang YH, Fan MS, Li ZX, Huang CG. Identification and determination of four metabolites of mangiferin in rat urine. J Pharm Biomed Anal 2007; 45: 793–798 37 Li S, Shi Y, Shang XY, Cui BS, Yuan Y, Chen XG, Yang YC, Shi JG. Triterpenoids from the roots of Pterospermum heterophyllum Hance. J Asian Nat Prod Res 2009; 11: 652–657 38 Cortez DAG, Young MCM, Marston A, Wolfender JL, Hostettmann K. Xanthones, triterpenes and a biphenyl from Kielmeyera coriacea. Phytochemistry 1998; 47: 1367–1374
Luo C-T et al. Xanthones from Swertia …
Planta Med 2014; 80: 201–208
39 Xu KP, Li FS, Liu JF, Tan JB, Zhang LH, Zeng GY, Tan GS. Studies on chemical constituents of Swertia nervosa (G. Don) Wall. Chin Pharm 2008; 43: 1612–1614 40 Chhetri DR, Parajuli P, Subba GC. Antidiabetic plants used by Sikkim and Darjeeling Himalayan tribes. Indian J Ethnopharmacol 2005; 99: 199– 202 41 Tian LY, Bai X, Chen XH, Fang JB, Liu SH, Chen JC. Anti-diabetic effect of methylswertianin and bellidifolin from Swertia punicea Hemsl. and its potential mechanism. Phytomedicine 2010; 17: 533–539 42 Masand VH, Mahajan DT, Patil KN, Chinchkhede KD, Jawarkar RD, Hadda TB, Alafeefy AA, Shibi IG. k-NN, quantum mechanical and field similarity based analysis of xanthone derivatives as α-glucosidase inhibitors. Med Chem Res 2012; 21: 4523–4534 43 Tanaka T, Nakashima T, Ueda T, Tomii K, Kouno I. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem Pharm Bull 2007; 55: 899–901 44 Choudhary MI, Shah SA, Atta-ur-Rahman Khan SN, Khan MT. Alpha-glucosidase and tyrosinase inhibitors from fungal hydroxylation of tibolone and hydroxytibolones. Steroids 2010; 75: 956–966 45 Wang YF, Ma L, Li Z, Du ZY, Liu Z, Qin JK, Wang XD, Huang ZS, Gu LQ, Chen ASC. Synergetic inhibition of metal ions and genistein on α-glucosidase. FEBS Lett 2004; 576: 46–50
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