Glucose Tolerance-Improving Activity of Helichrysoside in Mice and Its Structural Requirements for Promoting Glucose and Lipid Metabolism

An acylated flavonol glycoside, helichrysoside, at a dose of 10 mg/kg/day per os for 14 days, improved the glucose tolerance in mice without affecting the food intake, visceral fat weight, liver weight, and other plasma parameters. In this study, using hepatoblastoma-derived HepG2 cells, helichrysoside, trans-tiliroside, and kaempferol 3-O-β-d-glucopyranoside enhanced glucose consumption from the medium, but their aglycones and p-coumaric acid did not show this activity. In addition, several acylated flavonol glycosides were synthesized to clarify the structural requirements for lipid metabolism using HepG2 cells. The results showed that helichrysoside and related analogs significantly inhibited triglyceride (TG) accumulation in these cells. The inhibition by helichrysoside was more potent than that by other acylated flavonol glycosides, related flavonol glycosides, and organic acids. As for the TG metabolism-promoting activity in high glucose-pretreated HepG2 cells, helichrysoside, related analogs, and their aglycones were found to significantly reduce the TG contents in HepG2 cells. However, the desacyl flavonol glycosides and organic acids derived from the acyl groups did not exhibit an inhibitory impact on the TG contents in HepG2 cells. These results suggest that the existence of the acyl moiety at the 6′′ position in the D-glucopyranosyl part is essential for glucose and lipid metabolism-promoting activities.


Introduction
Flavonoids are one of the most abundant classes of secondary plant metabolites. Flavonoids are biosynthesized by the shikimate and acetate-malonate pathways and are comprised of compounds that possess a common C 6 -C 3 -C 6 skeleton, where two aromatic rings (named ring A and B) are linked via a heterocyclic 4H-pyrane ring (ring C). Modification of the 15-carbon skeleton through different oxidation levels and substituents to ring C gives rise to different classes of flavonoids, such as flavones, flavonols, flavanones, chalcones, dihydroflavonols (flavanonols), isoflavones, aurones, anthocyanidins,

Synthesis of Acylated Flavonol Glycosides (1-15)
Rutin (19 = quercetin 3-O-α-l-rhamnopyraniosyl(1→6)-β-d-glucopyranoside), constructed with quercetin (20) as an aglycone, is one of the most widely distributed naturally occurring flavonoids and has been reported to have several pharmacological activities, such as anti-oxidant, anti-inflammatory, anti-diabetic, anti-adipogenic, and neuroprotective effects, and has been used in hormone therapy [27][28][29][30]. In order to achieve the practical synthesis of 1 from 19, the most inexpensive and commercially available flavonoid, the optimal conditions for enzymatic hydrolysis of the terminal rhamnosyl part were investigated. Therefore, the practical derivation from 19 to 17 was carried out using naringinase (from Penicillium decumbens) under an optimal pH and temperature (pH 7 and 50 • C), and the time course of the reaction mixture was monitored by high performance liquid chromatography (HPLC) analysis ( Figure S1 and Table S1). As shown in Table 1, the highest content of 17 in the reaction mixture of 2 h was observed. By applying these conditions, a large-scale derivation of 17 (6.50 g and 14.0 mmol, 56.9%) from 19 (15.0 g and 24.6 mmol) was achieved. Table 1. Peak area ratio of rutin (19), quercetin 3-O-β-d-glucopyranoside (17), and quercetin (20) in the reaction mixture.

Effect of Helichrysoside (1) on the Liver Triglyceride (TG) Content and Glucose Tolerance Test after 14 Days of Administration in Mice
Diabetes is characterized by a high incidence of cardiovascular disease and poor control of hyperglycemia caused by insulin resistance (IR). IR can be defined as the inability of insulin to stimulate glucose uptake into the liver, skeletal muscle, or adipose tissue. Hyperglycemia is an important factor contributing to the development of atherosclerosis, and is relevant to the pathophysiology of late diabetic complications. Therefore, improving IR may form part of the strategy for the prevention and management of cardiovascular disease in diabetes [37,38]. We have reported that several anti-diabetogenic therapeutic candidates obtained from natural resources, such as acylated flavonol glycosides from Sinocrassula indica [39]; saponins from Borassus flabellifer [40]; and thiosugars from Salacia reticulata, S. oblonga, and S. chinensis [41][42][43][44][45][46], showed the inhibition of postprandial hyperglycemia and/or improvement of glucose tolerance in sugar-loaded animal models. As mentioned above, the structure of 16 isolated from R. canina [26] is quite similar to 1, so we presumed that 1 also exhibits similar anti-diabetogenic activity to 16 in an in vivo study. To continue our search for new candidates of the anti-diabetogenic and/or anti-diabetic principles and to evaluate the anti-diabetogenic effect of 1, the effect of 14 days of the continuous administration of 1 on glucose tolerance was performed in mice. Following this continuous administration, 1 was found to significantly suppress the increase in blood glucose levels at doses of 1 and 10 mg/kg/day per os (p.o.), at 60 min post glucose loading ( Figure 2 and Table S3). The area under the curve (AUC) of blood glucose levels was significantly reduced at the dose of 10 mg/kg/day (p.o.). As indicated in Table S4, the continuous administration of 1 tended to reduce the weights of visceral fat and the liver and the liver TG content, without affecting the food intake and other plasma parameters, including plasma TG, total cholesterol, and free fatty acids (FFA).

Effects on Glucose Consumption in HepG2 Cells
The liver is one of the tissues important for maintaining blood glucose homeostasis and greatly affects the formation of abnormal glucose tolerance [47]. Since the improving activity of helichrysoside (1) in terms of glucose tolerance in mice was observed in the previous section, we investigated the effects of 1 and its related compounds (16-18 and 20-22), to clarify the structural requirement of glucose consumption using human hepatoblastoma-derived HepG2 cells. As shown in Table 2, the glucose concentration in the medium was found to be significantly reduced at 6 days pretreatment with 1, trans-tiliroside (16), kaempferol 3-O-β-d-glucopyranoside (18), and metformin. On the other hand, the desacyl derivative of 1, quercetin 3-O-β-d-glucopyranoside (17); the aglycones of 1 and 16, quercetin (20) and kaempferol (21); and trans-p-coumaric acid (22) did not result in changes in the glucose concentration in the medium. These results suggested that the p-coumaroyl moiety at the 6 position in the D-glucopyranosyl part was essential for promoting glucose consumption. Recent related studies have reported that compounds 17, 20, and 21 promoted glucose uptake into muscle and hepatocytes [48][49][50]. Due to the long-term treatment of test samples of cells in our study, the treatment with compounds 17, 20, and 21 showed cytotoxicity at the concentration of 30-100 µM.

Effects on Lipid Metabolism in HepG2 Cells
A fatty liver is recognized as a significant risk factor for serious liver diseases [51,52]. A strong causal link has been identified between fatty liver diseases and hyperinsulinemia, caused by insulin resistance [53,54]. Therefore, a fatty liver is considered to be closely associated with obesity and type 2 diabetes [54]. In previous studies on the identification of anti-fatty liver principles from natural medicines, several flavonoids [55][56][57][58] were revealed to inhibit lipid accumulation in HepG2 cells. Similarly, we also reported that several megastigmanes [59], diterpenes [60], and limonoids [61] inhibited lipid metabolism in high glucose-pretreated HepG2 cells.

Enzymatic Hydrolysis of Rutin (19) Monitored by HPLC
A suspension of rutin (19, 100.0 mg) in H 2 O (50 mL) was mixed and stirred at 50 • C in a water bath for a few minutes. Then, naringinase (5.0 mg) was added to the suspension to start the reaction. Aliquots (1 mL) of the reaction mixture after 0, 5, and 30 min and 1, 1.5, 2, 3, 4.5, 8, and 24 h were transferred into a 10 mL volumetric flask and methanol was added to make up the volume, respectively. Each solution was filtered through a syringe filter (0.45 µm), and an aliquot of 1 µL was subjected to the following HPLC analytical conditions.
A series LC-20A Prominence HPLC system (version 3.40, Shimadzu Co., Kyoto, Japan) was equipped with a UV-VIS detector, a binary pump, a degasser, an autosampler, a thermostatic column compartment, and a control module. The chromatographic separation was performed on a Cosmosil The flow rate was 0.2 mL/min with UV detection at 254 nm and the injection volume was 1 µL. The standard curves were prepared with five concentration levels in the range of 25-400 µg/mL (25,50,100,200, and 400 µg/mL, respectively). Linearity for each compound, such as rutin (19), quercetin 3-O-β-d-glucopyranoside (17), and quercetin (20), was plotted using linear regression of the peak area versus concentration. The coefficient of correlation (R 2 ) was used to judge the linearity ( Figure S1 and Table S1).

Synthesis of Helichrysoside (1)
Under an argon atmosphere, imidazole (1.50 g, 22.0 mmol, 3.2 eq) and TBDPSCl (4.54 g, 16.5 mmol, 2.4 eq) were added to a solution of trans-p-coumaric acid (22, 1.30 g, 6.88 mmol) in dry-DMF (12.0 mL), and the mixture was stirred at 40 • C for 16 h. The reaction mixture was poured into ice-water and extracted with EtOAc, before being washed with brine. The extract was condensed under a reduced pressure to give a white solid, which was dissolved in CHCl 3 /MeOH (10:7, v/v, 17 mL) and acidified by 1 M HCl until pH 3.0. After stirring at room temperature for 1.5 h, the reaction mixture was condensed under a reduced pressure to give a pale yellow oil, which was crystallized in n-hexane/EtOAc (9:1, v/v, 20 mL) to give 22a (2.12 g, 76.5%).

Animals
Male ddY mice were purchased from Kiwa Laboratory Animal Co., Ltd., (Wakayama, Japan). The animals were housed at a constant temperature of 23 ± 2 • C and fed a standard laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). All experiments were performed with conscious mice, unless otherwise noted. The experimental protocol was approved by Kindai University's Committee for the Care and Use of Laboratory Animals (KAPR-26-004, 1 April 2014).

Effects on the Glucose Tolerance Test in Mice
Effects on the glucose tolerance test after 14 days of administration of 1 in mice were determined according to the previously described protocol [26]. A test sample was administrated orally to male ddY mice (11 weeks old and fed a standard laboratory chow) once a day (10:00-12:00) for 14 days. Body weight and food intake were measured every day before administration of the test sample. Fasting for 20 h was carried out after the final administration, and 10% (w/v) glucose solution was intraperitoneally (i.p.) administrated to mice at 10 mL/kg. Blood samples (ca. 0.2 mL) were collected in tubes containing 10 units of heparin sodium from the infraorbital venous plexus before (0 h) and 0.5, 1, and 2 h after the loading of glucose. Mice were then killed by cervical dislocation, and the epididymal, mesenteric, and paranephric fat pads were removed and weighed. Plasma glucose, TG, total cholesterol, and FFA levels were determined using commercial kits (Glucose CII-test Wako, Triglyceride E-test Wako, Cholesterol E-test Wako, and NEFA C-test Wako, respectively, FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan). After removing the liver, ca. 300 mg of liver tissue was cut and homogenized with 9 mL of distilled water. An aliquot of the homogenate (500 µL) was diluted with distilled water (1 mL) and the TG concentration in the suspension was determined using Triglyceride E-test Wako.

Effects on Glucose Consumption in HepG2 Cells
HepG2 cells were inoculated in a 48-well tissue culture plate (10 5 cells/well in 150 µL/well in MEM). After 20 h, the medium was replaced with 150 µL/well of Dulbecco's Modified Eagle's Medium (DMEM) containing low-glucose (1000 mg/L) and a test sample. Cells were cultured for 6 days, and the medium was replaced every 2 days. The medium was then transferred to 200 µL/well of DMEM containing high-glucose (4500 mg/L) and the cells were cultured. After 20 h, the glucose content in the medium was determined using commercial kits (Glucose CII-test Wako, FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan). Medium was removed, and the cells were homogenized in distilled water (105 µL/well) by sonication. The protein content in the homogenate was determined using the BCA protein Assay Kit (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan). Each test compound was dissolved in DMSO and added to the medium (final DMSO concentration was 0.5%). An anti-diabetic agent, metformin, was used as a reference compound.

Effects on High Glucose-Induced TG Accumulation in HepG2 Cells
HepG2 cells were inoculated in a 48-well tissue culture plate (10 5 cells/well in 150 µL/well in MEM). After 20 h, the medium was replaced with 150 µL/well of DMEM containing high-glucose and a test sample, which was cultured for 4 days, with medium containing a test sample being replaced every 2 days. Medium was then removed, and the cells were homogenized in distilled water (105 µL/well) by sonication. The TG and protein content in the homogenate were determined using commercial kits (Triglyceride E-test Wako and BCA protein Assay Kit, respectively, FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan). Data were expressed as the % of control of TG/protein (µg/mg). Each test compound was dissolved in DMSO and was added to the medium (final DMSO concentration was 0.5%). An anti-diabetic agent, metformin, was used as a reference compound.

Effects on TG contents in High Glucose-Pretreated HepG2 Cells
Effects on TG metabolism-promoting activity in high glucose-pretreated HepG2 cells were evaluated according to the method described previously [61], with slight modifications. HepG2 cells were inoculated in a 48-well tissue culture plate (10 5 cells/well in 150 µL/well in MEM). After 20 h, the medium was replaced with 150 µL/well of DMEM containing high-glucose and cultured for 6 days, with the medium being replaced every 2 days. After accumulation of the lipid, the medium was transferred to 150 µL/well of DMEM containing low-glucose and a test sample, and the cells were cultured. After 20 h, the TG and protein content in the cells were determined by the same manner as described above. Data were expressed as the % of control of TG/protein (µg/mg). Each test compound was dissolved in DMSO and added to the medium (final DMSO concentration was 0.5%). An anti-diabetic agent, metformin, was used as a reference compound.

Statistics
Values are expressed as means ± S.E. One-way analysis of variance (ANOVA) followed by Dunnett's test was used for statistical analysis. Probability (p) values of less than 0.05 were considered significant.

Conclusions
The present study demonstrated that helichrysoside (1), an acylated flavonol glycoside, improved glucose tolerance in ddY mice. In the study, using HepG2 cells, helichrysoside (1) was shown to significantly enhance glucose consumption in the medium, inhibit high glucose-induced TG accumulation in cells, and promote the effect of TG metabolism in high glucose-pretreated cells. The results from various acylated flavonol glycosides, flavonol glycosides, flavonols, and organic acids indicated that the acyl group at the 6 position in the D-glucopyranosyl part was essential for the improved glucose tolerance activities. Previous evidence, along with this study, suggests that helichrysoside (1) might be considered as a possible candidate for the prevention of glucose and lipid metabolism-related disorders.