Glucose tolerance-improving activity of helichrysoside in mice and its structural requirements for promoting glucose and lipid metabolism

Article abstract of DOI:10.3390/ijms20246322

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.

Full text of DOI:10.3390/ijms20246322

International Journal of
Molecular Sciences
Article
Glucose Tolerance-Improving Activity of
Helichrysoside in Mice and Its Structural
Requirements for Promoting Glucose and
Lipid Metabolism
Toshio Morikawa 1
,2, ,†
*
, Akifumi Nagatomo , Takahiro Oka , Yoshinobu Miki ,
1,†
1
1
1
1
1
1,2
Norihisa Taira , Megumi Shibano-Kitahara , Yuichiro Hori , Osamu Muraoka
and
Kiyofumi Ninomiya 1
,2
1
Pharmaceutical Research and Technology Institute, Kindai University, 3-4-1 Kowakae, Higashi-osaka,
Osaka 577-8502, Japan; a-nagatomo@jintan.co.jp (A.N.); tmykoka0325@gmail.com (T.O.);
sanmokuhoushin@hokuriku.me (Y.M.); Taira_Norihisa@seiwakasei.co.jp (N.T.);
kabazakura2@yahoo.co.jp (M.S.-K.); hori.yuichiro.0208@gmail.com (Y.H.);
muraoka@phar.kindai.ac.jp (O.M.); ninomiya@phar.kindai.ac.jp (K.N.)
Antiaging Center, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan
Correspondence: morikawa@kindai.ac.jp; Tel.: +81-6-4307-4306; Fax: +81-6-6729-3577
These authors contributed equally to this work.
2
*
†
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 21 October 2019; Accepted: 13 December 2019; Published: 14 December 2019
Abstract: An acylated flavonol glycoside, helichrysoside, at a dose of 10 mg/kg/day per os for
4 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- -d-glucopyranoside enhanced
1
β
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.
0
0
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.
Keywords: helichrysoside; acylated flavonol glycoside; glucose tolerance-improving activity; lipid
metabolism-promoting activity
1
. 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 -C -C skeleton, where two aromatic rings (named ring A and B) are linked
6
3
6
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,
Int. J. Mol. Sci. 2019, 20, 6322; doi:10.3390/ijms20246322
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2019, 20, 6322
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leucoanthocyanidines (flavan-3,4-diols), and flavan-3-ols. They naturally occur in not only aglycone
forms, but also as glycosylated and/or acylated derivatives and oligomeric and polymeric structures,
such as the flavan-3-ol-derived condensed tannins and proanthocyanidins [1–5]. Flavonoid health
benefits are well-recognized, such as their antioxidant properties, properties for weight management,
cardiovascular disease protection, anti-allergic activity, vascular fragility, prevention of viral and
bacterial infections, anti-inflammatory activity, age-related neurodegenerative disease prevention,
anti-platelet aggregation effects, and cancer protection, etc. [
from medicinal and/or food resources have reported several bio-functional properties of flavonoids.
These included aldose reductase inhibitory [ 12], anti-platelet aggregation [ ], anti-allergic [12–14]
anti-inflammatory [12,15–18], aminopeptidase N inhibition [11 17 19], hepatoprotective [20],
gastroprotective [21], melanogenesis inhibition [22], and dipeptidyl peptidase-IV inhibitory [23
activities. This paper deals with the practical synthesis and glucose tolerance-improving
2–8]. Our studies on bioactive constituents
9–
9
,
,
,
]
0
0
activity of helichrysoside (1 = quercetin 3-O-(6 -O-trans-p-coumaroyl)-
from Helichrysum kraussii and H. stoechas [24 25] by other research groups.
synthetic studies of the related analogs of 1 ( 15, Figure 1) were also carried out, as well
β
-d-glucopyranoside), isolated
,
2
Furthermore,
–
as characterization of its structural requirements for glucose and lipid metabolism in HepG2
cells. In our previous report, an acylated flavonol glycoside, trans-tiliroside (16 = kaempferol
0
0
3
-O-(6 -O-trans-p-coumaroyl)-
to suppress visceral fat weight gain and improve glucose tolerance in mice [26]. The structures of
and 16 are similar: the former has a p-coumaroyl ester at the 6-position in the -D-glucopyranosyl
moiety of quercetin 3-O- -d-glucopyranoside (isoquercitrin, 17), while the latter has the common acyl
β-d-glucopyranoside), isolated from the fruit of Rosa canina, was found
1
β
β
group at the same position of kaempferol 3-O-β-d-glucopyranoside (18).
Figure 1. Structures of acylated flavonol glycosides (1–16) and related compounds (17–31).

Int. J. Mol. Sci. 2019, 20, 6322
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2
. Results and Discussion
2
.1. 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 [27–30]. 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
4.0 mmol, 56.9%) from 19 (15.0 g and 24.6 mmol) was achieved.
1
Table 1. Peak area ratio of rutin (19), quercetin 3-O-β–d-glucopyranoside (17), and quercetin (20) in the
reaction mixture.
Peak Area (%)
Reaction Time
Rutin (19)
Quercetin 3-O-Glc (17)
Quercetin (20)
0
5
min
min
99.1
96.6
60.8
29.7
16.4
6.3
0.0
0.0
0.0
0.0
0.0
1.6
0.0
0.7
7.5
22.9
32.6
43.2
57.7
72.5
89.4
95.5
3
0 min
30.7
46.3
50.1
49.6
40.8
25.9
9.0
1
h
1
.5 h
2
3
h
h
4
.5 h
8
h
2
4 h
1.5
Linearities for 19, 17, and 20 in the HPLC analytical condition were calculated as shown in Table S1.
Synthesis of the acylated flavonol glycosides, including helichrysoside (1–15) from 17, using
the corresponding acylation reactions, was carried out. Therefore, protection of a phenol group in
p-coumaric acid (22) with tert-butyldiphenylsilyl chloride (TBDPSCl) yielded the corresponding silyl
ether, 22a. Acylation of 17 with 22a in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC
deprotection with tetrabutylammonium fluoride (TBAF), provided 1 with a 36.9% yield (Scheme 1).
In a similar procedure to that of 31 32], compounds 33], 34], 10 33], 12 33 35 36],
, and 15 were synthesized with 17 and the corresponding organic acids. The cis-isomer of , quercetin
), was derived under a UV lamp in a methanol
·
HCl) and 4-dimethylaminopyridine (4-DMAP) in pyridine, followed by
1
[
,
3,
4,
5,
6,
7
[
8
[
[
[
,
,
1
4
1
0
0
3
-O-(6 -O-cis-p-coumaroyl)-
solution of . Using a similar procedure, quercetin 3-O-(6 -O-cis-caffeoyl)-
), quercetin
-O-(6 -O-cis-cinnamoyl)-
respectively. Among these synthetic products, known compounds (
by a comparison of their physicochemical data with those of authentic samples or with reported
values. The structural determination of new compounds ( 11, and 13 15) was elucidated on
their spectroscopic properties, including the 13C-NMR data, as shown in Table S2.
β-d-glucopyranoside (2
0
0
1
β
-d-glucopyranoside
and quercetin
-d-glucopyranoside (13) were also isomerized from 10, and 12
10, and 12) were identified
0
0
(9
3-O-(6 -O-cis-feruloyl)-
β-d-glucopyranoside
(11),
0
0
3
β
8
,
,
1, 7, 8,
2
–
6
,
9
,
–

Int. J. Mol. Sci. 2019, 20, 6322
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Scheme 1.
Reagents and conditions:
(a) tert-butyldiphenylsilyl chloride (TBDPSCl),
◦
◦
) naringinase/H O, 50 C, 2 h,
2
imidazole/N,N-dimethylformamide (DMF), 40 C, 16 h, 76.5%; (
b
5
4
6.9%; (
c
)
22a (1.2 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC
·
HCl),
◦
-dimethylaminopyridine (4-DMAP)/pyridine, 50 C, 12 h; (
d) tetrabutylammonium fluoride
(TBAF)/tetrahydrofuran (THF), r.t., 1 h, 36.9% (two steps from 17).
2
.2. 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
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 , so we presumed that 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
the effect of 14 days of the continuous administration of on glucose tolerance was performed in mice.
Following this continuous administration, was found to significantly suppress the increase in blood
–46], showed the inhibition of postprandial hyperglycemia
1
1
1
,
1
1
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).

Int. J. Mol. Sci. 2019, 20, 6322
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Figure 2. Effect of helichrysoside (1) on the glucose tolerance test after 14 days of administration
in mice.
2
.3. 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
) in terms of glucose tolerance in mice was observed in the previous section, we investigated the
effects of and its related compounds (16 18 and 20 22), to clarify the structural requirement of
(1
1
–
–
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 and 16,
1
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
0
0
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 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.
,
–
Table 2. Effects of helichrysoside (1) and related compounds (16–18 and 20–22) on glucose consumption
in HepG2 cells.
Glucose in the Medium (% of Control)(Protein (% of Control))
Treatment
0
µM
00.0 ± 2.6
100.0 ± 5.4)
3 µM
10 µM
30 µM
100 µM
1
94.7 ± 1.1 **
86.7 ± 1.2 **
89.3 ± 1.1 **
82.9 ± 1.5 **
Helichrysoside (1)
(
(
(96.6 ± 2.0)
(98.3 ± 2.0)
(102.9 ± 1.5)
(105.2 ± 1.4)
1
00.0 ± 6.4
84.6 ± 0.9 **
(105.4 ± 1.5)
90.1 ± 0.6 **
(102.3 ± 2.7)
96.8 ± 1.7
79.8 ± 1.0 **
(110.0 ± 1.0)
Trans-tiliroside (16)
Quercetin 3-O-Glc
100.0 ± 4.7)
(103.4 ± 1.9)
100.0 ± 1.1
100.9± 1.4
100.6 ± 1.3
241.7 ± 3.8 **
269.4 ± 5.2 **
(
17)
Kaempferol
-O-Glc (18)
(100.0 ± 1.3)
(104.0 ± 1.0)
(104.1 ± 0.9)
(47.2 ± 0.6 **)
(23.1± 1.4**)
100.0 ± 3.0
95.9 ± 0.7
98.9 ± 2.7
93.4 ± 0.4 **
(100.1 ± 0.4)
92.8 ± 1.1 **
(98.7 ± 1.4)
3
(100.0 ± 3.5)
(96.4 ± 2.0)
(94.2 ± 1.4 *)
1
00.0 ± 2.0
98.6 ± 0.4
97.2 ± 1.7
105.6 ± 2.0
249.2 ± 3.9 **
Quercetin (20)
(
(
100.0 ± 0.5)
(102.0 ± 0.9)
(103.4 ± 0.8 **)
(98.9 ± 0.6)
(41.7 ± 0.4 **)
1
00.0 ± 3.1
98.4 ± 2.2
95.2 ± 1.5
98.1 ± 0.9
224.4 ± 3.1 **
(55.4 ± 0.5 **)
Kaempferol (21)
100.0 ± 0.7)
(101.0 ± 0.8)
(100.7 ± 0.5)
(100.3 ± 1.2)
Trans-p-coumaric
acid (22)
100.0 ± 3.6
96.9 ± 1.5
96.5 ± 0.5
96.8 ± 1.7
97.7 ± 1.8
(100.0 ± 3.7)
(99.7 ± 0.6)
(98.2 ± 1.7)
(98.3 ± 0.4)
(105.8 ± 1.8 *)
0
µM
00.0 ± 1.5
100.0 ± 1.2)
62.5 µM
125 µM
250 µM
500 µM
1
98.9 ± 1.9
95.1 ± 0.7
97.7 ± 1.8
87.5 ± 4.8 *
Metformin
(
(102.0 ± 1.8)
(100.9 ± 1.7)
(100.5 ± 2.1)
(100.6 ± 2.3)
Each value represents the mean
where * p < 0.05 and ** p < 0.01.
±
S.E. (n = 4 or 8); asterisks denote significant differences from the control group,

Int. J. Mol. Sci. 2019, 20, 6322
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2
.4. 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
diabetes [54]. In previous studies on the identification of anti-fatty liver principles from natural
medicines, several flavonoids [55 58] were revealed to inhibit lipid accumulation in HepG2 cells.
,
2
–
Similarly, we also reported that several megastigmanes [59], diterpenes [60], and limonoids [61
]
inhibited lipid metabolism in high glucose-pretreated HepG2 cells.
Intracellular TG accumulated in HepG2 cells via increasing the expression of lipogenesis-related
proteins, such as sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase
(
FAS), when cultured in high glucose-containing medium [55
we examined the inhibitory effects of the acylated flavonol glycosides (
17 22 24 26, and 28 31) on (i) high glucose-induced TG accumulation in HepG2 cells and (ii) TG
contents in high glucose-pretreated HepG2 cells.
,
62]. To characterize this phenomenon,
1
–16) and related compounds
(
–
,
,
–
As
shown
helichrysoside
quercetin
in
Table
3,
quercetin
3-O-(6 -O-trans-p-methylcoumaroyl)-
-d-glucopyranoside (15), and trans-tiliroside (16), significantly inhibited high
M: (80.4 1.2%),
76.9 ± 2.3%),
3.0%)). In contrast, the other acylated flavonol glycosides
19), and organic acids, which related to the corresponding acyl
31), did not show significant inhibitory activity up to a concentration of 100 M. As for the
inhibitory effects of the corresponding aglycones, quercetin (20, 45.7 0.4% at 100 M) and kaempferol
21, 25.5 1.4% at 100 M) showed stronger activity than that of the acylated flavonol glycosides, with
several
acylated
flavonol
3-O-(6 -O-cis-p-coumaroyl)-
-d-glucopyranoside
glycosides,
-d-glucopyranoside
), quercetin
such
0
0
as
(
1),
β
0
0
(2),
β
(3
0
0
3
-O-(6 -O-trimethylgalloyl)-
β
glucose-induced TG accumulation in HepG2 cells (% of control at 100
(58.2 7.5%), 15 (85.8 4.1%), and 16 (82.3
14), related flavonol glycosides (17
groups (22
µ
1
(
2
±
3
±
±
±
(4–
–
–
µ
±
µ
(
±
µ
cytotoxicity under the effective concentrations (data not shown). The structural requirements of the acylated
flavonol glycosides were assessed and showed that (1) the acylated flavonol glycosides with a p-coumaroyl,
0
0
p-methylcoumaryl, or trimethylgalloyl moiety as the acyl group in the 6 position of the D-glucopyranosyl
part are essential for the activity, and (2) the glycoside structure contributes to reducing the cytotoxicity.
On the other hand, all the tested acylated flavonol glycosides (
1–16) and their aglycones
(
20 and 21) were found to significantly inhibit the TG content in high glucose-pretreated HepG2
0
0
cells at a concentration of 100
µ
M, as shown in Table 4. Specifically, the 6 -O-acylated quercetin
M:
0.9%), trans-m-coumaroyl
2.0%), trans- and cis-cinnamoyl (12 (87.2 2.1%)
7.3%), showed potent activities. However, the
19) and organic acids (22 24 26, and 28 31) lacked this potency.
3
1
-O-
β
-d-glucopyranosides structure, having a trans- and cis-p-coumaroyl (% of control at 10
1.3) and (86.0 3.2%)), trans-p-methylcoumaroyl ( , 87.4
, 88.4
and 13 (88.9 ± 1.8%)), and vanilloyl moiety (14, 75.0
corresponding flavonol glycosides (17
µ
(82.9
±
2
±
3
±
(6, 87.5 ± 4.2%), trans-m-methylcoumaroyl (
7
±
±
±
–
,
,
–
Based on these results, the following structural requirements can be concluded: the acylated flavonol
glycosides with a p- or m-coumaroyl, p- or m-methylcoumaryl, cinnamoyl, or vanilloyl moiety with the
0
0
acyl group in the 6 position of the D-glucopyranosyl part are essential for the potent inhibition of TG
content in high glucose-pretreated HepG2 cells.

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Table 3. Effects of acylated flavonol glycosides (1–16) and related compounds (17–22, 24, 26, and 28–31) on high glucose-induced triglyceride accumulation in
HepG2 cells.
TG/Protein (% of Control)
10 µM
Treatment
0
µM
3 µM
30 µM
100 µM
76.9 ± 2.3 **
80.4 ± 1.2 **
58.2 ± 7.5 **
93.4 ± 1.2
125.9 ± 3.6
106.0 ± 4.8
92.2 ± 8.5
98.1 ± 1.7
93.8 ± 2.2
114.9 ± 3.6
92.9 ± 5.2
Helichrysoside (1)
100.0 ± 1.6
100.0 ± 0.7
100.0 ± 4.8
100.0 ± 2.6
100.0 ± 6.7
100.0 ± 3.7
100.0 ± 3.4
100.0 ± 0.7
100.0 ± 2.2
100.0 ± 3.2
100.0 ± 7.0
100.0 ± 1.3
100.0 ± 4.0
100.0 ± 3.0
100.0 ± 2.9
100.0 ± 2.0
100.0 ± 1.1
100.0 ± 3.5
100.0 ± 4.1
100.0 ± 0.9
100.0 ± 1.2
100.0 ± 0.8
100.0 ± 1.1
100.9 ± 7.1
102.0 ± 2.1
99.8 ± 10.2
104.6 ± 4.7
84.4 ± 11.7
97.4 ± 10.9
108.0 ± 4.5
108.4 ± 1.6 *
102.4 ± 1.8
104.7 ± 5.3
104.9 ± 3.3
106.8 ± 3.5
106.3 ± 4.1
96.3 ± 4.5
99.7 ± 0.7
99.1 ± 1.2
97.1 ± 1.9
96.8 ± 3.2
102.1 ± 7.6
99.8 ± 1.1
94.2 ± 1.2
98.4 ± 2.9
99.1 ± 0.7
96.0 ± 1.5
97.3 ± 2.1
107.4 ± 2.3
98.2 ± 3.0
73.5 ± 12.2
99.0 ± 2.7
80.5 ± 10.6
107.0 ± 1.7 *
102.2 ± 1.6
110.6 ± 2.4
101.6 ± 2.7
100.6 ± 2.0
100.2 ± 3.9
101.7 ± 2.7
96.4 ± 1.1
90.2 ± 2.2
93.2 ± 1.2 *
112.9 ± 2.6
98.3 ± 2.4
107.6 ± 13.8
96.4 ± 5.6
114.2 ± 8.5
103.6 ± 2.3
100.7 ± 1.7
108.4 ± 4.2
100.3 ± 2.5
102.8 ± 0.9
107.8 ± 3.2
105.9 ± 3.2
92.3 ± 3.4
109.6 ± 3.8
96.0 ± 3.1
99.8 ± 1.3
103.9 ± 2.5
82.6 ± 2.4 **
90.2 ± 2.4 **
99.6 ± 1.7
97.1 ± 1.5
0
0
Quercetin 3-O-(6 -O-cis-p-coumaroyl)-Glc (2)
0
0
Quercetin 3-O-(6 -O-trans-p-methylcoumaroyl)-Glc (3)
0
0
Quercetin 3-O-(6 -O-trans-o-coumaroyl)-Glc (4)
0
0
Quercetin 3-O-(6 -O-trans-o-methylcoumaroyl)-Glc (5)
0
0
Quercetin 3-O-(6 -O-trans-m-coumaroyl)-Glc (6)
0
0
Quercetin 3-O-(6 -O-trans-m-methylcoumaroyl)-Glc (
7
)
0
0
Quercetin 3-O-(6 -O-trans-caffeoyl)-Glc (8)
0
0
Quercetin 3-O-(6 -O-cis-caffeoyl)-Glc (9)
0
0
Quercetin 3-O-(6 -O-trans-feruloyl)-Glc (10)
0
0
Quercetin 3-O-(6 -O-cis-feruloyl)-Glc (11)
0
0
Quercetin 3-O-(6 -O-trans-cinnamoyl)-Glc (12)
115.6 ± 2.9 **
121.0 ± 3.1 **
104.5 ± 1.3
85.8 ± 4.1 **
82.3 ± 3.0 **
94.3 ± 5.2
0
0
Quercetin 3-O-(6 -O-cis-cinnamoyl)-Glc (13)
0
0
Quercetin 3-O-(6 -O-vanilloyl)-Glc (14)
0
0
Quercetin 3-O-(6 -O-trimethylgalloyl)-Glc (15)
Trans-tiliroside (16)
95.5 ± 2.0
124.5 ± 1.3 **
95.5 ± 2.0
Quercetin 3-O-Glc (17)
Kaempferol 3-O-Glc (18)
Rutin (19)
91.9 ± 2.1
108.4 ± 3.4
93.8 ± 0.4 **
100.0 ± 2.4
97.6 ± 3.4
103.1 ± 2.6
45.7 ± 0.4 **
25.5 ± 1.4 **
102.7 ± 2.1
96.2 ± 2.2
Quercetin (20)
Kaempferol (21)
Trans-p-coumaric acid (22)
Trans-o-coumaric acid (24)
97.4 ± 1.7

Int. J. Mol. Sci. 2019, 20, 6322
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Table 3. Cont.
TG/Protein (% of Control)
10 µM
Treatment
0
µM
3 µM
30 µM
97.6 ± 1.9
99.9 ± 3.3
101.9 ± 3.4
100.0 ± 2.5
99.3 ± 2.3
0.5 mM
100 µM
99.6 ± 1.9
89.5 ± 4.7
102.0 ± 2.6
103.2 ± 5.6
101.3 ± 1.4
1 mM
Trans-m-coumaric acid (26)
Trans-caffeic acid (28)
Trans-ferulic acid (29)
Trans-cinnamic acid (30)
Vanillic acid (31)
100.0 ± 1.1
100.0 ± 3.8
100.0 ± 3.4
100.0 ± 4.7
100.0 ± 2.4
99.2 ± 0.6
97.2 ± 1.9
95.0 ± 1.8
103.4 ± 2.7
97.2 ± 1.5
0.125 mM
86.8 ± 1.5 **
97.7 ± 2.1
105.1 ± 4.3
105.3 ± 4.5
107.1 ± 1.3
100.6 ± 0.7
0
mM
0.25 mM
Metformin
100.0 ± 0.4
75.6 ± 1.5 **
64.8 ± 1.0 **
61.1 ± 2.8 **
Each value represents the mean ± S.E. (n = 4); asterisks denote significant differences from the control group, where * p < 0.05 and ** p < 0.01.
Table 4. Effects of acylated flavonol glycosides (1–16) and related compounds (17–22, 24, 26, and 28–31) on the triglyceride content in high glucose-pretreated
HepG2 cells.
TG/Protein (% of Control)
10 µM
Treatment
0
µM
3 µM
30 µM
100 µM
Helichrysoside (1)
100.0 ± 3.5
100.0 ± 3.7
100.0 ± 2.2
100.0 ± 2.3
100.0 ± 3.0
100.0 ± 1.3
100.0 ± 2.0
100.0 ± 13.8
100.0 ± 2.9
100.0 ± 7.9
90.5 ± 0.6
86.0 ± 0.8 *
90.3 ± 1.0 **
93.3 ± 1.5
94.5 ± 2.7
91.3 ± 3.6
91.2 ± 1.9 *
91.1 ± 6.0
95.6 ± 1.4
89.2 ± 3.9
82.9 ± 1.3*
83.3 ± 4.6*
81.6 ± 1.4 **
83.3 ± 1.5 **
79.6 ± 1.6**
86.4 ± 1.2 **
83.7 ± 3.1 **
83.8 ± 1.5 **
84.2 ± 6.3
62.1 ± 3.6**
74.6 ± 2.3 **
66.4 ± 1.4 **
64.5 ± 1.8 **
64.6 ± 2.0 **
69.8 ± 3.2 **
76.2 ± 1.5 **
65.8 ± 5.4 **
87.4 ± 4.6 *
75.8 ± 3.7 **
0
0
Quercetin 3-O-(6 -O-cis-p-coumaroyl)-Glc (2)
86.0 ± 3.2 *
87.4 ± 0.9 **
89.5 ± 2.6**
93.4 ± 1.6
0
0
Quercetin 3-O-(6 -O-trans-p-methylcoumaroyl)-Glc (3)
0
0
Quercetin 3-O-(6 -O-trans-o-coumaroyl)-Glc (4)
0
0
Quercetin 3-O-(6 -O-trans-o-methylcoumaroyl)-Glc (5)
0
0
Quercetin 3-O-(6 -O-trans-m-coumaroyl)-Glc (6)
87.5 ± 4.2 *
88.4 ± 2.0 **
87.0 ± 5.2
91.8 ± 2.0
94.0 ± 4.3
0
0
Quercetin 3-O-(6 -O-trans-m-methylcoumaroyl)-Glc (7)
0
0
Quercetin 3-O-(6 -O-trans-caffeoyl)-Glc (8)
0
0
Quercetin 3-O-(6 -O-cis-caffeoyl)-Glc (9)
87.4 ± 4.2 *
74.3 ± 5.8 **
0
0
Quercetin 3-O-(6 -O-trans-feruloyl)-Glc (10)

Int. J. Mol. Sci. 2019, 20, 6322
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Table 4. Cont.
TG/Protein (% of Control)
10 µM
Treatment
0
µM
3 µM
30 µM
100 µM
77.3 ± 3.5 **
68.7 ± 1.3 **
82.5 ± 1.0 **
60.8 ± 6.1 **
81.7 ± 3.9 **
72.8 ± 3.1 **
108.0 ± 1.4
92.4 ± 5.1
0
0
Quercetin 3-O-(6 -O-cis-feruloyl)-Glc (11)
100.0 ± 10.6
100.0 ± 3.4
100.0 ± 2.1
100.0 ± 9.7
100.0 ± 5.7
100.0 ± 1.7
100.0 ± 2.1
100.0 ± 3.2
100.0 ± 9.2
100.0 ± 2.4
100.0 ± 2.4
100.0 ± 3.3
100.0 ± 2.2
100.0 ± 2.3
100.0 ± 13.2
100.0 ± 11.2
100.0 ± 3.0
100.0 ± 9.2
86.8 ± 4.7
91.4 ± 2.6
93.2 ± 0.8
102.5 ± 7.4
92.2 ± 2.0
98.9 ± 2.9
100.9 ± 2.6
98.6 ± 3.3
95.3 ± 4.0
92.5 ± 1.1
103.4 ± 2.8
95.1 ± 4.5
97.2 ± 0.9
93.7 ± 1.0
89.9 ± 6.6
88.6 ± 6.3
102.8 ± 2.7
92.9 ± 4.7
0.125 mM
92.1 ± 1.4 *
90.7 ± 2.2
82.4 ± 4.8 *
80.9 ± 2.1 **
89.9 ± 0.5 **
70.2 ± 5.2 **
83.7 ± 1.3 **
86.1 ± 3.1 **
108.3 ± 1.2
88.8 ± 3.3
0
0
Quercetin 3-O-(6 -O-trans-cinnamoyl)-Glc (12)
87.2 ± 2.1 *
88.9 ± 1.8 **
75.0 ± 7.3 **
93.1 ± 1.0
0
0
Quercetin 3-O-(6 -O-cis-cinnamoyl)-Glc (13)
0
0
Quercetin 3-O-(6 -O-vanilloyl)-Glc (14)
0
0
Quercetin 3-O-(6 -O-trimethylgalloyl)-Glc (15)
Trans-tiliroside (16)
96.2 ± 1.2
Quercetin 3-O-Glc (17)
Kaempferol 3-O-Glc (18)
Rutin (19)
100.5 ± 2.5
96.0 ± 2.3
91.8 ± 5.8
99.3 ± 2.7
94.9 ± 7.6
Quercetin (20)
92.1 ± 3.5
71.5 ± 3.3 **
82.8 ± 1.5 **
103.7 ± 3.3
97.5 ± 4.1
63.2 ± 0.7 **
56.1 ± 3.6 **
118.6 ± 6.2 *
102.4 ± 4.2
94.4 ± 1.7
Kaempferol (21)
95.0 ± 4.6
Trans-p-coumaric acid (22)
Trans-o-coumaric acid (24)
Trans-m-coumaric acid (26)
Trans-caffeic acid (28)
Trans-ferulic acid (29)
Trans-cinnamic acid (30)
Vanillic acid (31)
99.8 ± 4.9
93.8 ± 3.3
97.1 ± 3.4
92.1 ± 0.8
91.1 ± 2.6
100.5 ± 4.7
98.5 ± 2.9
95.1 ± 8.4
0.5 mM
89.6 ± 0.6
97.6 ± 1.4
97.4 ± 3.1
104.3 ± 6.6
102.0 ± 5.3
106.7 ± 2.5
1 mM
99.2 ± 7.0
98.4 ± 2.8
0
µM
0.25 mM
Metformin
100.0 ± 1.2
88.6 ± 1.5 **
87.8 ± 2.2 **
81.4 ± 1.4 **
Each value represents the mean ± S.E. (n = 4 or 8); asterisks denote significant differences from the control group, where * p < 0.05 and ** p < 0.01.

Int. J. Mol. Sci. 2019, 20, 6322
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0
Recently, it has been reported that derivatives of 16 activate adenosine 5 -monophosphate-activated
protein kinase (AMPK) in 3T3-L1 cells [63] and stimulate glucose transporter (GLUT) 4 translocation
in skeletal muscle cells [64]. AMPK is known as a key molecule involved in regulating glucose and
lipid metabolism in the liver. From the similarity of the structure, the activities exhibited by 1 and its
analogs in this study may have been caused via the same mechanism, but further investigations are
needed to clarify the details.
3
. Materials and Methods
3
.1. Chemicals and Reagents
Rutin and naringinase were purchased from Sigma-Aldrich Co. LLC., St. Louis, MO, USA. DMF,
tert-butyldiphenylsilyl chloride (TBDPSCl), EDC
were purchased from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan. Flavonols (17
organic acids (22 31), and other chemicals, unless otherwise indicated, were purchased from Nakalai
Tesque Inc., Kyoto, Japan.
·
HCl, 4-DMAP, TBAF, and tetrahydrofuran (THF)
,
18, and 20 21),
–
–
3
.2. General Experimental Procedures
The following instruments were used to obtain spectroscopic data: specific rotations, Horiba
SEPA-300 digital polarimeter (l = 5 cm); UV spectra, Shimadzu UV-1600 spectrometer; IR spectra,
Shimadzu FTIR-8100 spectrometer; FAB-MS and high-resolution MS, JEOL JMS-SX 102A mass
spectrometer; ESIMS and HRESIMS, Exactive Plus mass spectrometer (Thermo Fisher Scientific Inc.,
1
MA, USA); H-NMR spectra, JEOL JNM-ECA600 (600 MHz) and JNM-ESC400 (400 MHz) spectrometers;
13
C-NMR spectra, JEOL JNM-ECA600 (150 MHz) and JNM-ESC400 (100 MHz) spectrometers, with
tetramethylsilane as an internal standard; HPLC detector, Shimadzu SPD-10Avp UV-VIS detectors; and
HPLC column, Cosmosil 5C -MS-II (Nacalai Tesque Inc.). The following experimental conditions were
18
used for column chromatography (CC): ordinary-phase silica gel CC, silica gel 60N (Kanto Chemical
Co., Tokyo, Japan; 63–210 mesh, spherical, neutral), normal-phase thin-layer chromatography (TLC),
pre-coated TLC plates with silica gel 60F254 (Merck, Darmstadt, Germany; 0.25 mm), with detection
achieved by spraying with 1% Ce(SO ) –10% aqueous H SO , and followed by heating.
4
2
2
4
3
.3. Enzymatic Hydrolysis of Rutin (19) Monitored by HPLC
A suspension of rutin (19, 100.0 mg) in H O (50 mL) was mixed and stirred at 50 C in a water
◦
2
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
the following HPLC analytical conditions.
µm), and an aliquot of 1 µL was subjected to
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
◦
5
C -MS-II (3
µ
m particle size, 150
×
2.0 mm i.d., Nacalai Tesque Inc., Kyoto, Japan) operated at 40 C
1
8
with mobile phase A (acetonitrile) and B (H O containing 0.1% acetic acid). The gradient program
2
was as follows: 0–3 min (A:B = 20:80, v/v)
The flow rate was 0.2 mL/min with UV detection at 254 nm and the injection volume was 1
standard curves were prepared with five concentration levels in the range of 25–400 g/mL (25, 50,
00, 200, and 400 g/mL, respectively). Linearity for each compound, such as rutin (19), quercetin
→
10–15 min (90:10, v/v)
→
15–25 min (20:80, v/v, hold).
µL. The
µ
1
µ
3
-O-
β
-d-glucopyranoside (17), and quercetin (20), was plotted using linear regression of the peak area
2
versus concentration. The coefficient of correlation (R ) was used to judge the linearity (Figure S1 and
Table S1).

Int. J. Mol. Sci. 2019, 20, 6322
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3
.4. Practical Derivation from Rutin (19) to Quercetin 3-O-β-d-glucopyranoside (17) by Naringinase
Naringinase (750.0 mg) was added to a suspension of rutin (19, 15.0 g, 24.6 mmol) in H O (7.5 L),
2
◦
and the mixture was stirred at 50 C for 2 h. Removal of the solvent from the reaction mixture was
carried out under reduced pressure using EtOH as an azeotropic solvent to give a crude product,
which, on silica gel CC (500 g, CHCl /MeOH/H O (10:3:0.4, v/v/v)), gave a title compound (17, 6.50 g,
3
2
5
6.9%).
3
.5. 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 /MeOH (10:7, v/v, 17 mL) and acidified by
3
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,
0 mL) to give 22a (2.12 g, 76.5%).
-O-tert-Butyldiphenylsilyl ether of trans-p-coumaric acid (22a): H NMR (400 MHz, CDCl ):
2
1
4
δ
3
1
.10 (9H, s, tert-Bu), 6.23, 7.65 (1H each, both d, J = 16.0 Hz, H-8 and 7), 6.76, 7.30 (2H each, both d,
13
J = 8.7 Hz, H-3,5 and 2,6), [7.37 (4H, m), 7.43 (2H, m), 7.70 (4H, dd, J = 1.9, 8.2 Hz), arom.]. C NMR
(
100 MHz, CDCl3): δ 127.2 (C-1), 129.8 (C-2,6), 120.3 (C-3,5), 158.1 (C-4), 146.7 (C-7), 114.8 (C-8), 172.3
C
(
C-9), 132.3, 135.4, 127.9, 130.1 (arom. C-1, 2,6, 3,5, 4), 19.4 (CH C-), 26.4 (CH C-).
3
3
Compound 22a (0.40 g, 0.96 mmol, 1.2 eq), EDC HCl (0.31 g, 1.60 mmol, 2.0 eq), and 4- DMAP
·
(
0.15 g, 1.20 mmol, 1.5 eq) were added to a solution of 17 (0.37 g, 0.80 mmol) in pyridine (4.0 mL),
◦
and the mixture was stirred at 50 C for 12 h. The reaction mixture was condensed under a reduced
pressure to give a crude product. Then, a solution of the crude product in THF (5.0 mL) was added
to TBAF (ca. 1.0 mol/L in THF, 800
µL, 0.80 mmol, 1.0 eq) at room temperature for 1 h. The reaction
mixture was quenched in H O, and removal of the solvent under a reduced pressure then furnished a
2
residue, which, on silica gel CC (10 g, CHCl /MeOH/H O (10:3:0.4, v/v/v)), gave a title compound (1,
3
2
0
.18 g, 36.9%).
3
.6. Synthesis of 3–8, 10, 12, 14, and 15
In a manner similar to that used for 22a (vide supra), trans-o-coumaric acid (24), trans-m-coumaric
acid (26), trans-caffeic acid (28), trans-ferulic acid (29), and vanillic acid (31) were derived to yield
the corresponding silyl ether derivatives, 24a (95.1%), 26a (94.6%), 28a (393.0%), 29a (87.3%), and 31a
(
98.5%), respectively.
1
2
-O-tert-Butyldiphenylsilyl ether of trans-o-coumaric acid (24a): H NMR (400 MHz, CDCl ):
δ
3
1.15 (9H, s, tert-Bu), 6.49, 8.53 (1H each, both d, J = 16.0 Hz, H-8 and 7), 6.48 (1H, dd, J = 1.8, 8.7 Hz,
H-3), 6.88 (1H, br dd, J = ca. 8, 8 Hz, H-5), 6.96 (1H, ddd, J = 1.8, 8.3, 8.7 Hz, H-4), 7.59 (1H, dd, J = 1.8,
.8 Hz, H-6), (7.39 (4H, m), 7.44 (2H, m), 7.72 (4H, dd, J = 1.8, 8.3 Hz), arom.). 13C NMR (100 MHz,
CDCl3): 125.0 (C-1), 154.6 (C-2), 119.9 (C-3), 131.5 (C-4), 121.4 (C-5), 127.5 (C-6), 142.3 (C-7), 117.1
7
δ
C
(
C-8), 172.6 (C-9), 132.2, 135.4, 127.9, 130.1 (arom. C-1, 2,6, 3,5, 4), 19.6 (CH C-), 26.5 (CH C-).
3 3
1
3-O-tert-Butyldiphenylsilyl ether of trans-m-coumaric acid (26a): H NMR (400 MHz, CDCl ): δ
3
1.12 (9H, s, tert-Bu), 6.17, 7.59 (1H each, both d, J = 16.0 Hz, H-8 and 7), 6.78 (1H, br d, J = ca. 8 Hz,
H-4), 6.94 (1H, br s, H-2), 7.04 (br d, J = ca. 8 Hz, H-6), 7.10 (dd, J = 7.8, 7.8 Hz, H-5), (7.38 (4H, m), 7.44
(
2H, m), 7.71 (4H, m), arom.). 13C NMR (100 MHz, CDCl3):
δ
C
135.2 (C-1), 119.1 (C-2), 156.0 (C-3),
1
22.2 (C-4), 129.7 (C-5), 121.5 (C-6), 146.8 (C-7), 117.3 (C-8), 172.1 (C-9), 132.2, 135.5, 127.9, 130.1 (arom.
C-1, 2,6, 3,5, 4), 19.5 (CH C-), 26.5 (CH C-).
3
3
1
3
,4-Di-O-tert-butyldiphenylsilyl ether of trans-caffeic acid (28a): H NMR (400 MHz, CDCl ):
δ
3
1.14, 1.18 (9H each, both s, tert-Bu), 5.48, 7.17 (1H each, both d, J = 16.0 Hz, H-8 and 7), 6.39 (1H, d,

Int. J. Mol. Sci. 2019, 20, 6322
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J = 8.2 Hz, H-5), 6.53 (1H, dd, J = 1.8, 8.2 Hz, H-6), 6.59 (1H, d, J = 1.8 Hz, H-2), (7.40 (8H, m), 7.45 (4H,
13
m), 7.79 (8H, m), arom.). C NMR (100 MHz, CDCl3):
δ 126.8 (C-1), 119.4 (C-2), 146.5 (C-3), 148.9
C
(
C-4), 120.5 (C-5), 122.3 (C-6), 146.6 (C-7), 114.4 (C-8), 172.0 (C-9), 132.5/132.9, 135.4/135.6, 127.9/128.0,
1
30.0/130.2 (arom. C-1, 2,6, 3,5, 4), 19.5, 19.5 (CH C-), 26.6, 26.8 (CH C-).
3 3
1
4-O-tert-Butyldiphenylsilyl ether of trans-ferulic acid (29a): H NMR (400 MHz, CDCl ):
δ
1.11
3
(
9H, s, tert-Bu), 3.60 (3H, s, -OCH ), 6.23, 7.64 (1H each, both d, J = 16.0 Hz, H-8 and 7), 6.69 (1H, d,
3
J = 8.2 Hz, H-5), 6.86 (1H, dd, J = 1.8, 8.2 Hz, H-6), 6.95 (1H, d, J = 1.8 Hz, H-2), (7.35 (4H, m), 7.41 (2H,
1
3
m), 7.70 (4H, dd, J = 1.8, 7.8 Hz), arom.). C NMR (100 MHz, CDCl3):
δ 127.7 (C-1), 111.2 (C-2), 150.8
C
(
(
C-3), 147.9 (C-4), 120.4 (C-5), 122.5 (C-6), 147.1 (C-7), 114.7 (C-8), 172.3 (C-9), 133.1, 135.3, 127.6, 129.8
arom. C-1, 2,6, 3,5, 4), 19.8 (CH C-), 26.6 (CH C-).
3
3
1
3
-O-tert-Butyldiphenylsilyl ether of vanillic acid (31a): H NMR (400 MHz, CDCl ):
δ
1.12 (9H, s,
3
tert-Bu), 3.61 (3H, s, -OCH ), 6.37 (1H, d, J = 9.2 Hz, H-5), 7.47 (1H, dd, J = 1.8, 9.2 Hz, H-6), 7.48 (1H, d,
3
13
J = 1.8 Hz, H-2), (7.35 (4H, m), 7.41 (2H, m), 7.69 (4H, m), arom.). C NMR (100 MHz, CDCl3):
δ
C
1
1
22.4 (C-1), 113.5 (C-2), 150.4 (C-3), 150.3 (C-4), 119.8 (C-5), 124.0 (C-6), 171.7 (C-9), 132.9, 135.2, 127.6,
29.8 (arom. C-1, 2,6, 3,5, 4), 19.8 (CH C-), 26.5 (CH C-).
3
3
In a manner similar to that used for the preparation of
of 17 (500.0 mg, 1.08 mmol) and 24a 26a 28a 30a, and 31a (521.1, 521.1, 521.1, 849.6, 558.6, and
26.3 mg, respectively, 1.29 mmol, 1.2 eq) were conducted to yield the corresponding 6 -O-acylated
quercetin 3- -d-glucopyranosides, (134.6 mg, 31.0%), (147.7 mg, 34.0%), (248.5 mg, 35.1%), 10
176.9 mg, 38.0%), and 14 (130.7 mg, 29.8%), respectively. Syntheses of the 6 -O-acylated quercetin
1 (vide supra), dehydration condensation
,
,
,
0
0
5
β
4
6
8
0
0
(
3
3
-
β
-d-glucopyranosides,
3 (39.0 mg, 20.3%), 5 (63.2 mg, 32.9%), 7 (57.2 mg, 29.8%), 12 (53.2 mg,
3.3%), and 15 (75.2 mg, 32.9%) were also carried out by the dehydration condensation of 17 with
trans-p-methylcoumaric acid (23), trans-o-methylcoumaric acid (25), trans-m-methylcoumaric acid (27),
trans-cinnamic acid (30), and trimethylgallic acid, respectively, without the deprotection procedure of
the tert-butyldiphenylsilyl (TBDPS) ether group.
0
0
Quercetin 3-O-(6 -O-trans-p-methylcoumaroyl)-
β
-d-glucopyranoside (
3
): A yellow powder,
+
1
high-resolution positive-ion FABMS: Calcd for C H O Na (M+Na) : 647.1377. Found: 647.1373. H
NMR (600 MHz, DMSO-d6):
.31 (1H, dd, J = 7.4, 8.8 Hz, H-2 ), 3.40 (1H, ddd, J = 2.0, 6.9, 8.9 Hz, H-5 ), 3.82 (3H, s, -OCH ), (4.30
1H, dd, J = 2.0, 11.8 Hz), 4.66 (1H, dd, J = 6.9, 11.8 Hz), H -6 ), 5.49 (1H, d, J = 7.4 Hz, H-1 ), 6.13,
.33 (1H each, both d, J = 2.0 Hz, H-6 and 8), 6.17, 7.36 (1H each, both d, J = 16.0 Hz, H-8 and 7 ),
.83 (1H, d, J = 8.4 Hz, H-5 ), 6.95, 7.44 (2H each, both d, J = 8.8 Hz, H-3 ,5 and 2 ,6 ), 7.53 (1H,
dd, J = 2.2, 8.4 Hz, H-6 ), 7.55 (1H, d, J = 2.2 Hz, H-2 ), 12.60 (1H, br s, 5-OH). C NMR (150 MHz,
DMSO-d ): δ given in Table S2.
Quercetin 3-O-(6 -O-trans-o-coumaroyl)-
high-resolution positive-ion FABMS: Calcd for C H O Na (M+Na) : 633.1220. Found: 633.1226. H
NMR (600 MHz, DMSO-d6):
.30 (1H, dd, J = 7.3, 8.8 Hz, H-2 ), 3.40 (1H, ddd, J = 2.2, 6.4, 9.5 Hz, H-5 ), (4.07 (1H, dd, J = 6.4, 11.9
Hz), 4.29 (1H, dd, J = 2.2, 11.9 Hz), H -6 ), 5.47 (1H, d, J = 7.3 Hz, H-1 ), 6.15, 6.38 (1H each, both d,
31
28 14
00 00
δ 3.21 (1H, dd, J = 8.9, 9.0 Hz, H-4 ), 3.29 (1H, dd, J = 8.8, 9.0 Hz, H-3 ),
0
0
00
3
(
6
6
3
0
0
00
2
0
00
000
0
000 000
000 000
0
0
13
6
C
0
0
β-d-glucopyranoside
(
4):
A
yellow powder,
+
1
30
26 14
00 00
δ 3.22 (1H, dd, J = 8.8, 9.5 Hz, H-4 ), 3.28 (1H, dd, J = 8.8, 8.8 Hz, H-3 ),
0
0
00
3
0
0
00
2
0
00 000
J = 2.0 Hz, H-6 and 8), 6.34, 7.73 (1H each, both d, J = 16.1 Hz, H-8 , 7 ), 6.83 (1H, ddd, J = 1.7, 7.8,
000 00 000
8
8
.7 Hz, H-5 ), 6.84 (1H, d, J = 8.2 Hz, H-5 ), 6.93 (1H, dd, J = 1.7, 8.3 Hz, H-3 ), 7.22 (1H, ddd, J = 1.6,
0
00
000
00
.3, 8.7 Hz, H-4 ), 7.38 (1H, dd, J = 1.6, 7.8 Hz, H-6 ), 7.52 (1H, dd, J = 2.0, 8.2 Hz, H-6 ), 7.55 (1H, d,
0
0
13
J = 2.0 Hz, H-2 ), 12.58 (1H, br s, 5-OH). C NMR (150 MHz, DMSO-d ): δ given in Table S2.
6
C
0
0
Quercetin 3-O-(6 -O-trans-o-methylcoumaroyl)-β-d-glucopyranoside (5): A yellow powder,
+
high-resolution positive-ion FABMS: Calcd for C H O Na (M+Na) : 647.1377. Found: 647.1381.
H NMR (600 MHz, DMSO-d ):
.9, 9.5 Hz, H-5 ), 3.82 (3H, s, -OCH ), (4.11 (1H, dd, J = 6.9, 12.1 Hz), 4.30 (1H, dd, J = 2.3, 12.1 Hz),
H -6 ), 5.48 (1H, d, J = 7.5 Hz, H-1 ), 6.07, 6.30 (1H each, both d, J= 2.0 Hz, H-6 and 8), 6.32, 7.70 (1H
each, both d, J = 16.1 Hz, H-8 and 7 ), 6.83 (1H, d, J = 8.9 Hz, H-5 ), 6.98 (1H, br dd, J = ca. 8, 8 Hz,
H-5 ), 7.05 (1H, br d, J = ca. 8 Hz, H-3 ), 7.41 (1H, ddd, J = 1.7, 8.2, 8.3 Hz, H-4 ), 7.47 (1H, dd,
3
1
28 14
1
00
00 00
δ
3.21 (1H, m, H-4 ), 3.33 (2H, m, H-2 , 3 ), 3.41 (1H, ddd, J = 2.3,
6
0
0
6
3
0
0
00
2
0
00
000
00
0
00
000
000

Int. J. Mol. Sci. 2019, 20, 6322
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0
00
00
00
J = 1.7, 8.2 Hz, H-6 ), 7.52 (1H, dd, J = 2.3, 8.9 Hz, H-6 ), 7.53 (1H, d, J = 2.3 Hz, H-2 ), 12.57 (1H, br s,
-OH). 13C NMR (150 MHz, DMSO-d ): δ given in Table S2.
5
6
C
0
0
Quercetin 3-O-(6 -O-trans-m-coumaroyl)-
high-resolution positive-ion FABMS: Calcd for C H O Na (M+Na) : 633.1220. Found: 633.1226. H
NMR (600 MHz, DMSO-d6):
.31 (1H, dd, J = 8.8, 8.9 Hz, H-3 ), 3.40 (1H, ddd, J = 2.0, 6.6, 9.0 Hz, H-5 ), (4.08 (1H, dd, J = 6.6, 11.9
Hz), 4.30 (1H, dd, J = 2.0, 11.9 Hz), H -6 ), 5.46 (1H, d, J = 7.6 Hz, H-1 ), 6.12, 6.33 (1H each, both br
β
-d-glucopyranoside (
6
):
A yellow powder,
+
1
3
0
26 14
00 00
δ 3.22 (1H, dd, J = 8.8, 9.0 Hz, H-4 ), 3.28 (1H, dd, J = 7.6, 8.9 Hz, H-2 ),
0
0
00
3
0
0
00
2
0
00
000
0
s, H-6, 8), 6.24, 7.35 (1H each, both d, J = 16.0 Hz, H-8 and 7 ), 6.82 (1H, d, J = 8.4 Hz, H-5 ), 6.83
000 000 000
(
1H, br d, J = ca. 8 Hz, H-4 ), 6.91 (1H, br d, J = ca. 8 Hz, H-6 ), 6.94 (1H, br s, H-2 ), 7.20 (1H, dd,
0
00
0
0
J = 8.0, 8.0 Hz, H-5 ), 7.52 (1H, dd, J = 2.2, 8.4 Hz, H-6 ), 7.54 (1H, d, J = 2.2 Hz, H-2 ), 12.57 (1H, br s,
5
-OH). 13C NMR (150 MHz, DMSO-d ): δ given in Table S2.
6
C
0
0
Quercetin 3-O-(6 -O-vanilloyl)-
positive-ion FABMS: Calcd for C H O Na (M+Na) : 637.1169. Found: 637.1174. H NMR (600 MHz,
DMSO-d6): δ 3.24 (1H, dd, J = 8.9, 9.1 Hz, H-4 ), 3.30 (1H, dd, J = 8.8, 8.9 Hz, H-3 ), 3.33 (1H, dd,
J = 7.4, 8.8 Hz, H-2 ), 3.45 (1H, ddd, J = 2.1, 6.9, 9.1 Hz, H-5 ), 3.70 (3H, s, -OCH ), (4.13 (1H, dd,
J = 6.9, 11.9 Hz), 4.40 (1H, dd, J = 2.1, 11.9 Hz), H -6 ), 5.54 (1H, d, J = 7.4 Hz, H-1 ), 6.18, 6.35 (1H
each, both d, J = 1.9 Hz, H-6 and 8), 6.70 (1H, d, J = 8.2 Hz, H-5 ), 6.78 (1H, d, J = 8.4 Hz, H-5 ), 7.19
1H, dd, J = 2.0, 8.2 Hz, H-6 ), 7.27 (1H, d, J = 2.0 Hz, H-2 ), 7.51 (1H, d, J = 2.2 Hz, H-2 ), 7.53 (1H,
dd, J = 2.2, 8.4 Hz, H-6 ), 12.58 (1H, br s, 5-OH). C NMR (150 MHz, DMSO-d6):
β
-d-glucopyranoside (14): A yellow powder, high-resolution
+
1
29
26 15
00 00
0
0
00
3
00
0
0
2
0
00
0
000 000 0
(
0
13
δ
C
given in Table S2.
yellow powder,
0
0
Quercetin 3-O-(6 -O-trimethylgalloyl)-
high-resolution positive-ion FABMS: Calcd for C H O Na (M+Na) : 681.1432. Found: 681.1436.
β
-d-glucopyranoside
(
15):
A
+
31
30 16
UV (MeOH, nm (log
ε
)): 259 (3.99), 295 (3.62), 359 (3.77). IR (KBr): 3590, 1701, 1655, 1597, 1503, 1341,
-
1
1
00
1
202, 1075 cm . H NMR (600 MHz, DMSO-d ):
δ
3.23 (1H, dd, J = 9.1, 9.7 Hz, H-4 ), 3.30 (2H, m,
6
0
0
00
00
H-2 , 3 ), 3.50 (1H, ddd, J = 2.4, 7.3, 9.7 Hz, H-5 ), (3.67 (6H, s), 3.73 (3H, s), -OCH ), (4.26 (1H, dd,
3
0
0
00
0
J = 7.3, 11.9 Hz), 4.43 (1H, dd, J = 2.4, 11.9 Hz), H -6 ), 5.49 (1H, d, J = 7.6 Hz, H-1 ), 6.10, 6.30 (1H
2
0
each, both d, J = 2.1 Hz, H-6 and 8), 6.74 (1H, d, J = 8.6 Hz, H-5 ), 7.48 (1H, br s, H-2 ), 7.49 (1H, br d,
0
13
J = ca. 9 Hz, H-6 ), 12.48 (1H br s, 5-OH). C NMR (150 MHz, DMSO-d ): δ given in Table S2.
6
C
3
.7. Isomerization of 1, 8, 10, and 12
A methanol solution (20 mL) of
1 (100.0 mg, 0.17 mmol) in a Pyrex tube was left standing for 8 h
under a UV lamp (short wave) at room temperature. The reaction mixture was condensed under a
reduced pressure to give a crude product, which, on HPLC (Cosmosil 5C -MS-II, MeOH–1% aqueous
18
AcOH (55:45, v/v)), gave the cis-isomer
similar procedure, a methanol solution (10 mL) of
corresponding cis-isomer (12.2 mg (recovered , 25.8 mg)), 11 (10.8 mg (recovered 10, 26.9 mg)), or 13
14.2 mg (recovered 12, 30.1 mg)).
2
(25.1 mg) and a recovering compound (1, 69.8 mg). Using the
8,
10, or 12 (each 50.0 mg) was isomerized to the
9
8
(
0
0
Quercetin 3-O-(6 -O-cis-p-coumaroyl)-
β
-d-glucopyranoside (
2
): A yellow powder, high-resolution
+
1
positive-ion FABMS: Calcd for C H O Na (M+Na) : 633.1220. Found: 633.1226. H NMR (600 MHz,
δ 3.29 (1H, dd, J = 8.9, 9.0 Hz, H-4 ), 3.42 (2H, m, H-3 , 5 ), 3.49 (1H, dd, J = 7.3, 8.8 Hz,
H-2 ), 4.14–4.22 (2H, m, H -6 ), 5.19 (1H, d, J = 7.3 Hz, H-1 ), 5.51, 6.69 (1H each, both d, J = 12.8 Hz
H-8 and 7 ), 6.18, 6.30 (1H each, both d, J = 1.8 Hz, H-6 and 8), 6.67, 7.49 (2H each, both d, J = 8.6 Hz
H-3 ,5 and 2 ,6 ), 6.80 (1H, d, J = 8.7 Hz, H-5 ), 7.56 (2H, m, H-2 , 6 ). C NMR (150 MHz,
CD OD): δ given in Table S2.
3
0
26 14
00 00 00
CD OD):
3
0
0
00
00
,
,
2
0
0
00
000
00 000
000 000
0
0
0
13
3
C
0
0
Quercetin 3-O-(6 -O-cis-caffeoyl)-
β
-d-glucopyranoside (
9): A yellow powder, high-resolution
+
1
positive-ion FABMS: Calcd for C H O Na (M+Na) : 649.1169. Found: 649.1170. H NMR (600 MHz,
3.18–3.36 (4H, m, H-2 –5 ), 3.71(3H, s, -OCH ), (4.04 (1H, dd, J = 6.4, 11.9 Hz), 4.19 (1H,
dd, J = 2.2, 11.9 Hz), H -6 ), 5.42 (1H, d, J = 7.5 Hz, H-1 ), 5.42, 6.58 (1H each, both d, J = 12.8 Hz
30
26 15
00
0
0
DMSO-d6):
δ
3
00
0
0
,
2
0
00
000
000
H-8 and 7 ), 6.20, 6.36 (1H each, both d, J = 1.7 Hz, H-6 and 8), 6.28 (1H, d, J = 2.0 Hz, H-2 ), 6.67
000 0 000
(
1H, d, J = 8.5 Hz, H-5 ), 6.82 (1H, d, J = 8.6 Hz, H-5 ), 6.98 (1H, dd, J = 2.0, 8.5 Hz, H-6 ), 7.52 (2H,
0
0
13
m, H-2 , 6 ), 12.59 (1H, br s, 5-OH). C NMR (150 MHz, DMSO-d ): δ given in Table S2.
6
C

Int. J. Mol. Sci. 2019, 20, 6322
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0
0
Quercetin 3-O-(6 -O-cis-feruloyl)-
β
-d-glucopyranoside (11): A yellow powder, high-resolution
+
1
positive-ion FABMS: Calcd for C H O Na (M+Na) : 663.1326. Found: 663.1330. H NMR (600 MHz,
DMSO-d6): δ 3.19 (1H, m, H-4 ), 3.27 (2H, m, H-2 , 3 ), 3.36 (1H, ddd, J = 2.3, 6.0, 9.6 Hz, H-5 ),
.71 (3H, s, -OCH ), (4.18 (1H, dd, J = 6.4, 11.9 Hz), 4.19 (1H, dd, J = 1.8, 11.9 Hz), H -6 ), 5.46 (1H, d,
J = 7.4 Hz, H-1 ), 5.48, 6.65 (1H each, both d, J = 12.8 Hz, H-8 and 7 ), 6.19, 6.31 (1H each, both
d, J = 1.8 Hz, H-6 and 8), 6.72 (1H, d, J = 8.2 Hz, H-5 ), 6.81 (1H, d, J = 8.7 Hz, H-5 ), 7.08 (1H, dd,
J = 1.8, 8.2 Hz, H-6 ), 7.45 (1H, br d, J = ca. 9 Hz, H-6 ), 7.52 (1H, br s, H-2 ), 7.59 (1H, d, J = 1.8 Hz,
31
28 15
00 00 00 00
0
0
3
3
2
0
0
000
000
0
00
0
0
0
00
0
0
00
13
H-2 ), 12.57 (1H, br s, 5-OH). C NMR (150 MHz, DMSO-d ): δ given in Table S2.
6
C
0
0
Quercetin 3-O-(6 -O-cis-cinnamoyl)-
β-d-glucopyranoside (13): A yellow powder, high-resolution
+
1
positive-ion FABMS: Calcd for C H O Na (M+Na) : 617.1271. Found: 617.1277. H NMR (600 MHz,
DMSO-d6): δ 3.14 (1H, dd, J = 8.6, 9.5 Hz, H-4 ), 3.25 (1H, dd, J = 7.5, 8.9 Hz, H-2 ), 3.28 (1H, dd,
J = 8.6, 8.9 Hz, H-3 ), 3.33 (1H, ddd, J = 2.3, 6.6, 9.5 Hz, H-5 ), (4.07 (1H, dd, J = 6.6, 11.8 Hz), 4.18 (1H,
30
26 13
00 00
0
0
00
0
0
000
000
dd, J = 2.3, 11.8 Hz), H -6 ), 5.69, 6.81 (1H each, both d, J = 12.9 Hz, H-8 and 7 ), 6.19, 6.33 (1H
2
0
000 000 000 000
each, both d, J = 2.0 Hz, H-6 and 8), 6.82 (1H, d, J = 8.9 Hz, H-5 ), 7.27 (4H, m, H-2 ,6 , 3 ,5 ), 7.43
000 0 0
(
1H, m, H-4 ), 7.51 (1H, dd, J = 2.0, 8.9 Hz, H-6 ), 7.51 (1H, d, J = 2.0 Hz, H-2 ), 12.56 (1H, br s, 5-OH).
1
3
C NMR (150 MHz, DMSO-d ): δ given in Table S2.
6
C
3
.8. 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).
3
.9. 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
2
(
1
0 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
0 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.
3
.10. Cell Culture
HepG2 cells (RCB1648, Riken Cell Bank, Tsukuba, Japan) were maintained in Minimum Essential
Medium Eagle (MEM, Sigma-Aldrich Co. LLC., St. Louis, MO, USA) containing 10% fetal bovine
serum, 1% MEM non-essential amino acids (FUJIFILM Wako Pure Chemical Corporation, Tokyo,
◦
Japan), penicillin G (100 units/mL), and streptomycin (100
µ
g/mL) at 37 C under 5% CO atmosphere.
2
3
.11. Effects on Glucose Consumption in HepG2 Cells
5
HepG2 cells were inoculated in a 48-well tissue culture plate (10 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

Int. J. Mol. Sci. 2019, 20, 6322
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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.
3
.12. Effects on High Glucose-Induced TG Accumulation in HepG2 Cells
5
HepG2 cells were inoculated in a 48-well tissue culture plate (10 cells/well in 150
µL/well in
MEM). After 20 h, the medium was replaced with 150
test sample, which was cultured for 4 days, with medium containing a test sample being replaced every
days. Medium was then removed, and the cells were homogenized in distilled water (105 L/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 ( g/mg). Each test
compound was dissolved in DMSO and was added to the medium (final DMSO concentration was
µL/well of DMEM containing high-glucose and a
2
µ
(
µ
0
.5%). An anti-diabetic agent, metformin, was used as a reference compound.
3
.13. 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
5
were inoculated in a 48-well tissue culture plate (10 cells/well in 150
µL/well in MEM). After 20 h,
the medium was replaced with 150
days, with the medium being replaced every 2 days. After accumulation of the lipid, the medium
was transferred to 150 L/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 ( g/mg). Each test
µL/well of DMEM containing high-glucose and cultured for 6
µ
µ
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.
3
.14. Statistics
Values are expressed as means
test was used for statistical analysis. Probability (p) values of less than 0.05 were considered significant.
±
S.E. One-way analysis of variance (ANOVA) followed by Dunnett’s
4
. Conclusions
The present study demonstrated that helichrysoside (
glucose tolerance in ddY mice. In the study, using HepG2 cells, helichrysoside (
1
), an acylated flavonol glycoside, improved
) was shown
1
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
0
0
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.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/24/
6
322/s1.

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Author Contributions: T.M., A.N., O.M., and K.N. conceived and designed the experiments; T.M., A.N., T.O.,
Y.M., N.T., M.S.-K., Y.H., and K.N. performed the experiments; T.M., A.N., and N.K. analyzed the data; T.M. and
A.N. wrote the paper.
Funding: This work was supported by a Grant-in-aid for Scientific Research (KAKENHI), 18K06726 (T.M.),
1
6K08313 (O.M.), and 18K06739 (K.N.).
Acknowledgments: The authors gratefully thank Division of Joint Research Center, Kindai University for the
NMR and MS measurements. We would like to thank Editage (www.editage.com) for English language editing.
Conflicts of Interest: The authors declare no conflicts of interest.
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