Dipeptidyl peptidase-IV inhibitory activity of dimeric dihydrochalcone glycosides from flowers of Helichrysum arenarium

A methanol extract of everlasting flowers of Helichrysum arenarium L. Moench (Asteraceae) was found to inhibit the increase in blood glucose elevation in sucrose-loaded mice at 500 mg/kg p.o. The methanol extract also inhibited the enzymatic activity against dipeptidyl peptidase-IV (DPP-IV, IC50 = 41.2 μg/ml), but did not show intestinal α-glucosidase inhibitory activities. From the extract, three new dimeric dihydrochalcone glycosides, arenariumosides V–VII (2–4), were isolated, and the stereostructures were elucidated based on their spectroscopic properties and chemical evidence. Of the constituents, several flavonoid constituents, including 2–4, were isolated, and these isolated constituents were investigated for their DPP-IV inhibitory effects. Among them, chalconaringenin 2′-O-β-D-glucopyranoside (16, IC50 = 23.1 μM) and aureusidin 6-O-β-D-glucopyranoside (35, 24.3 μM) showed relatively strong inhibitory activities.


Introduction
An Asteraceae plant, Helichrysum arenarium L. Moench (common names ''dwarf everlasting'' or ''immortelle''), has an extensive distribution throughout Europe. The choleretic, hepatoprotective, and detoxifying activities of the inflorescence of this plant are known to European folk medicine [1]. The extract from the inflorescence of this plant has been characterized as showing free-radical scavenging actions [1] and antibacterial activities against lower respiratory tract pathogens [2]. Several flavonoids, megastigmanes, a-pyrones, and phthalides have been isolated from the flowers [3][4][5][6][7][8][9], achenes [10], roots [11,12], capitula, and leafy stems [13] of H. arenarium. In the course of our studies on bioactive constituents from the flowers of H. arenarium [4][5][6], we have reported that the methanol extract and several flavonoids showed inhibitory effects on tumor necrosis factor-a (TNFa)-induced cytotoxicity in L929 cells [4]. We further evaluated the extract and its constituents and found that the methanol extract inhibited blood glucose elevation in sucrose-loaded mice. A serine protease dipeptidyl peptidase-IV (DPP-IV) is widely expressed in the endothelial cells throughout the body and is found in a circulating soluble form [14]. It has been shown that the incretin hormone glucagon-like peptide-1 (GLP-1) is released from the intestinal L-cells into the circulation in response to the ingestion of food and stimulates both insulin biosynthesis and secretion [15,16]. Apart from several other beneficial effects, GLP-1 regulates insulin in a strictly glucose-dependent manner, and inhibition of the enzyme DPP-IV, which rapidly inactivates GLP-1, has been shown to increase the half-life of GLP-1 and to prolong the effects of this incretin hormone. The human recombinant DPP-IV inhibitory activity was revealed to regulate the antidiabetic effect of the extract. Further separation of the active constituents in the extract allowed us to isolate a known and novel new dimeric dihydrochalcone glycosides, tomoroside A (1) [17] and arenarumosides V-VII (2)(3)(4), respecitvely. Here, we describe the isolation and elucidation of the structure of 2-4 as well as the DPP-IV inhibitory activity of the flavonoid constituents .

Results and discussion
Effects of the methanol extract on plasma glucose levels in sucrose-loaded mice Dried flowers of H. arenarium were extracted with methanol under reflux to yield a methanol extract (19.8 % from the dried material). As shown in Table 1, the methanol extract showed an inhibitory effect against the increase in blood glucose levels in sucrose-loaded mice at a dose of 500 mg/kg, p.o.

Effects of the methanol extract and its fractions on human recombinant DPP-IV activity
To characterize the mode of action of the antihyperglycemic effect of the methanol extract, in vitro inhibitory activities on enzymes such as rat small intestinal a-glucosidases, maltase, and sucrose, and human recombinant DPP-IV inhibitory activities were examined. As shown in Table 2

Isolation
In the present study, we additionally isolated four dimeric dihydrochalcone glycosides, tomoriside A (1, 1.09 %) [17] and arenariumosides V (2, 0.0030 %), VI (3, 0.010 %), and VII (4, 0.018 %) from the MeOH-eluted fraction ( Structural identification and determination of dimeric dihydrochalcone glycosides (1)(2)(3)(4) In the positive-ion fast atom bombardment (FAB) MS spectrum of 1, a quasimolecular ion peak was observed at m/z 891 [M?Na] ? , and HRFABMS analysis revealed the molecular formula to be C 42 H 44 O 20 . The 1 H and 13 C NMR (pyridine-d 5 at 40°C with TMS as an internal standard) spectroscopic properties of 1 were in accordance with those of reported data [17] except for the deviations of the chemical shift, as shown in   (Fig. 2). Thus, the configuration of the cyclobutane moiety in 1 was determined to be an a-truxillic type structure [18][19][20]. Based on these findings, the structure of 1 was identified to be tomoroside A.
Arenariumoside V (2) was obtained as a yellow powder with positive optical rotation ([a] D 27 ?32.1 in MeOH). The IR spectrum showed absorption bands at 1619 cm -1 (carbonyl), 1509 and 1458 cm -1 (aromatic ring) and broad bands at 3568 and 1074 cm -1 , suggestive of a glycosyl moiety. In the positive ion FABMS, a quasimolecular ion  Table 3 1 H (500 MHz) and 13 C NMR (125 MHz) data of tomoroside A (1)   Table 3 continued   (Fig. 2). The ROE correlations in the ROESY experiments of 3 and 4 were observed as shown in Fig. 2; the stereochemistry of the cyclobutane moieties in 3 and 4 were elucidated to be dand b-truxinic type structures, respectively. To our knowledge, dimeric dihydrocalcones isolated from natural resources are rare, and the first report of a dimeric dihydrochalcone, brackenin, which was isolated from Brackenridgea zanguebarica, was established in 1983 [23]. A few other dimeric dihydrochalcones have been reported [24][25][26][27][28][29][30]. Recently, a dimeric dihydrochalcone glycoside, tomoroside A (1), was isolated from the same genus Helichysum zivojinii [17]. This article is the second report on the isolation and structural determination of dimeric dihydrochalcone glycosides from the genus Helichrysum.

Effects of the flavonoid constituents on human recombinant DPP-IV activity
In order to specify the principal active constituents, the inhibitory effects of the isolates from the active fractions described above (the EtOAc-soluble and the MeOH-eluted  Measured in a pyridine-d 5 or b DMSO-d 6 at 40°C (tetramethylsilane as an internal standard) fraction) on human recombinant DPP-IV activity were tested. In our previous study, we isolated 30 flavonoid constituents from the active MeOH-eluted fraction, including arenariumosides I (5, 0.0045 %), II (6, 0.0059 %), III (7, 0.0046 %), and IV (8, [4][5][6]. As shown in Table 5 strong inhibitory activity. In order to examine the inhibition type of DPP-IV by 16 and 35, the kinetic analysis was examined. Analysis of the Lineweaver-Burk plots indicated that the DPP-IV inhibition by 16 and 35 was of mixed type (Fig. 4).

Plant material
This item was described in a previous report [4].

Extraction and isolation
The dried flowers of H. arenarium (3.0 kg) were extracted under reflux with methanol three times for 3 h. Evaporation of the solvent under reduced pressure provided a methanolic extract (593. 8 [21,22].

Animals
Male ddY mice (6 weeks old) were purchased from Kiwa Laboratory Animal Co., Ltd., Wakayama, Japan. The animals were housed at a constant temperature of 23 ± 2°C and were then fed a standard laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). The animals were fasted for 20-24 h prior to the beginning of the experiment, but were allowed free access to tap water. All of the experiments were performed on conscious mice unless otherwise noted. The experimental protocol was approved by the Experimental Animal Research Committee at Kinki University.

Effects on plasma glucose levels in sucrose-loaded mice
The experiments were performed according to the method as described in our previous reports with a slight modification [31,32]. Thus, a mixture of each test sample and sucrose (2 g/kg) suspended in 5 % (w/v) acacia solution (20 ml/kg) was administered orally to fasted mice (body weight 24-27 g). Blood samples (ca. 0.1 ml) were collected from the infraorbital venous plexus under ether anesthesia 0.5, 1, and 2 h after the oral administration. The collected blood was immediately mixed with heparin sodium (5 units/tube). After centrifugation of the blood samples, the plasma glucose level was determined enzymatically by the Wako Glucose CII test (Wako Pure Chemical Industries Ltd., Osaka, Japan) according to the manufacturer's instructions. The intestinal a-glucosidase inhibitor acarbose was used as a reference compound [31].

Inhibitory effects on rat intestinal a-glucosidases
The experiments were performed as described previously [33][34][35]. Briefly, the assay was performed in 96-well microplates. Rat small intestinal brush border membrane vesicles were prepared, and a suspension of these in 0.1 M maleate buffer (pH 6.0) was used to determine the activities of maltase and sucrase. A test sample was dissolved in dimethyl sulfoxide (DMSO), and the resulting solution was diluted with 0.1 M maleate buffer to prepare the test sample solution (concentration of DMSO: 10 %). A substrate solution in the maleate buffer (maltose or sucrose: 74 mM, 50 ll), the test sample solution (25 ll), and the enzyme solution (25 ll) were mixed at 37°C for 30 min and then immediately heated by boiling water for 2 min to stop the reaction. The glucose concentrations were determined by a glucose-oxidase method. The final concentration of DMSO in the test solution was 2.5 %, and no influence of DMSO on the inhibitory activity was detected. The IC 50 was determined graphically (N = 2-4). The intestinal a-glucosidase inhibitors acarbose (IC 50 = 1.7 and 1.5 lM against maltase and sucrase, respectively) and salacinol (IC 50 = 6.0 and 1.3 lM) were used as reference compounds [33][34][35].

Inhibitory effect on human recombinant DPP-IV
The experiment was performed according to the method described in our previous reports with a slight modification [16]. Briefly, the assay was performed in 96-well half area white microplates (flat bottom). Pre-incubation of 15 ll of DPP-IV enzyme (from human recombinant; 17.3 mU/ml) or a blank buffer (50 mM tris HCl buffer, pH 7.5) with 35 ll of sample or control was incubated at room temperature (25°C) for 10 min. Fifty microliters of H-Gly-Pro-7-amino-4-methylcoumarin (Gly-Pro-AMC) substrate (4 mg/ml) was added, and the mixture was incubated at 25°C for 30 min. During the incubation, the fluorescence was measured using a fluorescence microplate reader (SH-9000, CORONA) at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The final concentration of DMSO in the test solution was 1.0 %, and no influence of DMSO on the inhibitory activity was detected. The IC 50 was determined graphically (N = 2-4). The DPP-IV inhibitors alogliptin (IC 50 = 18 nM) and diprotin A (Ile-Pro-Ile, IC 50 = 2.3 -lM) were used as reference compounds.

Statistical analysis
Values were expressed as mean ± SEM. For statistical analysis, one-way analysis of variance followed by Dunnett's test was used.