Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : February 13, 2024
Accepted : March 6, 2024
Published online : March 31, 2024
doi:10.24659/gsr.11.1_33

Original article

Suppressive effects of O 4 coordination-type zinc complexes
on in vitro AGE formation
Yuki Naito 1), Masaki Kojima 1), Kana Hayashi 1), Katsuyuki Tsukamoto 1),
Masayuki Yagi 2), Yutaka Yoshikawa 3), Hiroyuki Yasui 1)
1) Laboratory of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan.
2) Anti-Aging Medical Research Center and Glycative Stress Research Center, Doshisha University, Kyoto 610-0394, Japan.
3) Department of Health, Sports, and Nutrition, Kobe Women’s University, Hyogo 650-0046, Japan

Abstract

Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia. Persistent hyperglycemia causes
various complications and vascular damage. Increased production of advanced glycation end products (AGEs) via
glycation is involved in DM onset. Previous reports have demonstrated the therapeutic effects of zinc (Zn) complexes on
diabetic model mice in vivo. In this study, we aimed to investigate the in vitro anti-glycation effects of the Zn(O 4) type Zncomplexes, [Zn(mal) 2], [Zn(emal) 2 ], [Zn(trp) 2 ], and [Zn(hkt) 2]. We determined the effects of the Zn(O 4) coordination
type complexes on glycation via screening tests of their suppressive effects on fluorescent AGE formation and pentosidine,a fluorescent AGE. The screening test was performed using a phosphate buffer (pH 7.4) at 60 °C for 40 h. Then, pentosidine
was extracted from the supernatant using MonoSpin AG and quantified using high-performance liquid chromatographyfluorescence analysis. The tested Zn(O 4)-type complexes significantly suppressed fluorescent AGE formation more thanthe positive control, aminoguanidine. Moreover, pentosidine levels were decreased by Zn(O 4) complexes with a 7-member
troponoide structure, [Zn(trp) 2 ] and [Zn(hkt) 2 ]. To the best of our knowledge, this study is the first to demonstrate thesuppressive effects of Zn complexes on glycation reactions in vitro.

KEY WORDS: Zn complex, AGEs (advanced glycation end products), pentosidine, anti-glycation
Introduction
Diabetes mellitus (DM) is a chronic metabolic diseasecharacterized by hyperglycemia. Approximately 537 million
DM cases were reported worldwide in 2021, and this number
is predicted to increase to 783 million by 2045 1). Approximately
20 million DM or pre-DM cases are observed in Japan 2). DM
is classified into two types based on its pathogenesis: type 1
and type 2 DM. Type 1 DM is caused by autoimmunity
induced by viral infections, damage of pancreatic β cells by
immune cells, and genetic factors. In contrast, type 2 DM is achronic metabolic disease caused by genetic factors, such as
insufficient secretion or action of insulin, and environmental
factors, such as overeating and lack of exercise. Persistent
hyperglycemia causes various complications, thereby
reducing the patient quality of life 2). To prevent thesecomplications, DM treatments involve lifestyle changes anddrugs to control the blood glucose levels. Complications

caused by chronic hyperglycemia in diabetes include diabetic
nephropathy, diabetic retinopathy, neuropathy, and vasculardisorders. Non-enzymatic glycation reactions are a major
cause of these complications, and advanced glycation end
products (AGEs) formed by glycation reactions are involvedin the pathogenesis of such complications 3 - 6). Glycation
reactions can be divided into early- and late-stage reactions.
In early-stage reactions, reducing sugars combine with theamino groups of proteins to yield Schiff bases and Amadori
rearrangement products. HbA1c is a compound producedin early- stage glycation reactions, and its level indicatesthe blood glucose status. In late-stage reactions, AGEs are
generated via the crosslinking Amadori rearrangement
products 7). Accumulation of AGEs reduces insulin secretionin patients with type 2 DM; therefore, suppression of AGE
production has attracted considerable attention for DM
treatment 8). In addition to DM, AGE-induced glycationstress causes various changes, including osteoporosis andCorresponding author: Yuki Naito
Laboratory of Analytical and Bioinorganic Chemistry,
Division of Analytical and Physical Sciences,
Kyoto Pharmaceutical University,
5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan.
TEL: +81-75-595-4630 Fax: +81- 75-595- 4753
e-mail: naito@mb.kyoto-phu.ac.jp

Glycative Stress Research 2024; 11 (1): 33-39(c) Society for Glycative Stress Research_ 33 _

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Zn Complexes Suppress Glycation

Alzheimer’s disease, leading to lifestyle-related diseases andaging 9, 10). Glycated insulin levels are increased in animals
and patients with DM, and the glycation of insulin reduces
its biological activity 11-15).
Pentosidine, a fluorescent AGE initially isolated fromthe human brain dural collagen in 1989 16), is produced fromthe amino-terminal ends of ribose, arginine, and lysine ( Fig.
1 ). Plasma pentosidine levels are elevated in patients with
DM and chronic renal disease and used as early clinicalmarkers of nephropathy. Recently, pentosidine has attracted
attention as a biological marker of bone aging and is used forthe diagnosis of osteoporosis 17).

N
Arg

HN

N

N
Lys

Fig. 1. Structure of pentosidine.
Zinc (Zn) is an essential trace element present at aconcentration of approximately 2 g in the body. Many studies
have reported the association of Zn with DM 18, 19). Previously,
we synthesized Zn complexes with improved bioavailabilityof Zn 2 + and anti-DM effects 20 -25). In 2000, bis (maltolato)
zinc (II ) complex ([Zn(mal) 2]) was reported to exhibit highinsulin-like activity 24). Our team has also focused on Zn
complexes to improve their absorption in the body. We
previously revealed that the enhanced activation of theinsulin signaling pathway by Zn complexes exerts anti-DM
effects in DM model mice 25). These studies indicate that Zncomplexes exert insulin-like and anti-DM effects, including
HbA1c-lowering effects; however, the specific anti-DM
mechanisms of Zn complexes remain unclear. Therefore, inthis study, we focused on AGEs, factors involved in the onset
of DM. Particularly, we investigated the inhibition of AGE
formation by the Zn(O4) coordination type Zn complexes,[Zn(mal) 2 ], [Zn(emal) 2], [Zn(trp) 2], and [Zn(hkt) 2 ]. All
these Zn complexes have been reported to exhibit insulinlike effects in vitro and blood glucose-lowering effects invivo. Therefore, we examined their inhibitory effects on
fluorescent AGE formation and determined the pentosidine
levels via high-performance liquid chromatography withfluorescence detection (HPLC-FL).

Methods
Chemicals

Zinc sulfate heptahydrate (ZnSO4 ·7H 2 O), di-potassiumhydrogen phosphate, sodium borohydride, sodium hydroxide(for volumetric analysis), tris(hydroxymethyl)aminomethane,acetonitrile, and acetic acid were obtained from Nacalai
Tesque, Inc. (Kyoto, Japan). Lithium hydroxide monohydrate,ethanol, methanol, zinc acetate dihydrate, hinokitiol, dimethyl
sulfoxide (DMSO), d-(+)-glucose, sulfuric acid, potassium
dihydrogen phosphate, formic acid, 6 N hydrochloric acid,

100 % w/v trichloroacetic acid solution, ammonium formate,and carboxymethyl cellulose sodium salt were purchased from
Fujifilm Wako Pure Chemical Corporation (Osaka, Japan).
Additionally, 3-hydroxy- 2 -methyl - 4 -pyrone (maltol),
2- ethyl-3-hydroxy- 4 -pyrone (ethylmaltol), tropolone, andaminoguanidine (AG) were obtained from Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan). Human serum albumin(HSA) was obtained from Sigma-Aldrich (St. Louis, MO,
USA). Pentosidine standard was obtained from GL Sciences(Tokyo, Japan).

Preparation of Zn complexes[Zn(mal) 2 ] complex was prepared in deionized waterby mixing ZnSO4 , LiOH ·H 2 O, and maltol in a 1: 2:2 molar
ratio. The solution was stirred for 12 h at 25 ºC. The resulting
white precipitate was collected via vacuum filtration,
washed several times with a small amount of water, anddried overnight in vacuo, as previously described 24). The
prepared complex was characterized via elemental analysis
and infrared (IR) absorption spectroscopy (Shimadzu FT-IR
8100A on KBr pellets; Shimadzu Co., Kyoto, Japan).
Anal. Found (%): C, 39.60; H, 4.11. Calcd. (%) for[ Zn(mal) 2 ] · 2.7H 2 O; C, 39. 57; H, 4. 26. I R spectra(complex/ligand); υ C = O; 1614 cm -1/1653 cm -1 (Fig. 2-a).
[Zn(emal) 2 ] complex was prepared in deionized waterby mixing ZnSO 4 , LiOH ·H 2 O, and ethylmaltol in a 1: 2: 2
molar ratio. The solution was stirred for 12 h at 25 ºC.
The resulting white precipitate was collected via vacuum
filtration, washed several times with a small amount of water,and dried overnight in vacuo, as previously described 24). The
prepared complex was characterized via elemental analysis
and IR absorption spectroscopy (Shimadzu F T-IR 8100A on
KBr pellets; Shimadzu Co.).
Anal. Found (%): C, 42.98; H, 4. 32. Calcd. (%) for[Zn(emal) 2 ]· 2.6H 2 O;C, 43.06; H, 4.96. IR (complex/ligand);υ C = O; 1595 cm -1/1647 cm -1 ( Fig. 2-b).
[Zn(trp) 2] and [Zn(hkt) 2 ] complexes were prepared aspreviously described 26). For the [Zn(trp) 2] complex, deionizedwater of (CH 3 COO) 2 Zn ·2H 2 O and tropolone liquid indeionized water were mixed in a 1: 2 ratio. For the [Zn(hkt) 2]
complex, deionized waster of (CH 3 COO) 2 Zn·2H 2 O andmethanol solution of hinokitiol were mixed in a 1: 2 ratio.
Both mixed solutions were stirred for 12 h at 25 ºC. The
resulting white-yellow precipitate was collected via vacuum
filtration, washed several times with a small amount of water,and dried overnight in vacuo 27).
Anal. Found (%): C, 54.82; H, 3.26. Calcd. (%) for[Zn(trp) 2]; C, 54.66; H, 3.28. IR spectra (complex/ligand); υ
C = O; 1595 cm -1/1607 cm -1 ( Fig. 2-c).
Anal. Found (%): C, 61. 31; H, 5. 59. Calcd. (%) for[Zn(hkt) 2 ]; C, 61.31; H, 5.66. IR spectra (complex/ligand);υ C = O; 1589 cm -1/1608 cm -1 (Fig. 2-d ).

Measurement of fluorescent AGE levels in the HSA–
glucose glycation model

Suppressive effects of Zn complexes on AGE formation
were evaluated using the HSA–glucose model, as previously
described 28, 29). AG, a glycation reaction inhibitor, was used asa positive control, and the sample buffer (50 mM phosphatebuffer [pH 7.4], 8 mg/mL HSA, and 200 mM glucose) was_ 34 _Glycative Stress Research

a)

b)
O
O

c)

O
Zn

O

O

O

O

O

O
O

d)

O
Zn

O

O

O

O
Zn

Zn
O

O

O

O

O

Fig. 2. Estimated structures of Zn complexes.
(a) [ Zn(mal) 2 ]. (b) [Zn(emal) 2 ]. (c) [Zn(trp) 2 ]. (d) [Zn(hkt) 2 ].
prepared. Zn complexes were dissolved in 100 % DMSO,
added as test samples to the sample buffer in 1% DMSO,
incubated at 60 ºC for 40 h, and centrifugated at 18,000 × g
and 25 ºC for 10 min. Then, the supernatants were collected
and the fluorescence intensity was measured to quantify thefluorescent AGEs (excitation wavelength 360 nm/fluorescentwavelength 450 nm) using a 96-well black bottom plate with
Tecan infinite F200PRO ( Tecan, Männedorf, Switzerland).
The obtained values were calculated as AGE inhibition rates,
and the fluorescence vales were adjusted using 5 μg/mL
quinine sulfate solution.
Inhibition rate (%) = {1 – (A – B) /(C – D)} × 100
A: Reaction solution (Glucose (+) + tested sample)
B: Reaction solution without glucose(Glucose (−) + tested sample)
C : Without sample (Glucose (+) + sample buffer)
D: Blank (Glucose (−) + sample buffer)

Measurement of pentosidine levels in the HSA–
glucose glycation samples via HPLC

Inert Sustain AG (3 μm, 100 mm × 4.6 mm ID; GL Sciences)and RF-20A (Shimadzu Co.) Fluorescence detector. The
measurement conditions of HPLC were as follows: column
temperature, 20 °C; eluent, A solution: 0.1% formic acid (v/v)
in H 2 O solution, B solution: 100 % acetonitrile; elution
conditions as step gradient: 0 % B (0–10 min), 50 % B (10–12min), 0 % B (12 –30 min) flow rate, 1.0 mL/min; detection,
fluorescence (excitation [Ex] wavelength 325 nm, emission[Em] wavelength 385 nm); injection volume, 20 μL. In thisgradient program, the latter two steps (with 50 % B [10 –12min] and 0 % B [12 –30 min]) were programmed for column
washing and conditioning, respectively.

Statistical analyses

Data are expressed as the mean ± standard deviation.
Multiple comparisons with the AG group were analyzed
using analysis of variance and Dunnett’s post-hoc tests with
Bell Curve for Excel (Social Survey Research Information
Co., Ltd., Tokyo, Japan).

Next, supernatants of the HSA– glucose glycation
samples were pretreated as previously described 30, 31). The
supernatants were reduced with 200 mM sodium borohydride
solution (pH 9.2) to prevent pentosidine formation duringhydrolysis for 30 min at 25 ºC. Proteins were removed
using 20 % and 5 % TCA solutions. After removing thesupernatants, ultrapure water was added, and the precipitate
was resolved. Then, 6 N iron-free hydrochloric acid was added,
and the mixture was heated at 105 ºC for 18 h for hydrolysis.
After hydrolysis, 1.5 M Tris was added to dilute hydrochloric
acid in the solution and centrifuged using Centricut Ultra
Mini (Kurabo, Osaka, Japan). The hydrolyzed samples werepretreated using the Monospin AG column (GL Sciences),and the eluates were obtained using a measurement sample.
Analytical HPLC was performed using Shimadzu LC-20AB(Shimadzu Co.) fitted with the reverse-phase silica gel column

Results

Suppressive effects of Zn complexes on fluorescent
AGE formation

In this study, we examined the suppressive effects ofZn complexes including Zn2+ on fluorescent AGE formation.
We found that all Zn complexes with ZnSO4 (half-maximal
inhibitory concentration [ IC 50] = 610 ± 93 μM), except[Zn(emal) 2] (IC 50 = 1,531 ± 257 μM), significantly suppressed
AGE formation better than AG ( IC 50 = 859 ± 70 μM ).
Moreover, Zn complexes exerted their suppressive effects
in a concentration-dependent manner. Especially, Zn(O4)complexes with a 7-member troponoide structure, [Zn(trp) 2 ]and [Zn(hkt) 2 ], exhibited approximately 4 0 –100- times
stronger anti-glycation effects than AG, with IC 50 value_ 35 _

Zn Complexes Suppress Glycationof 20 ± 1 and 9 ± 2 μM, respectively ( Table 1 ). We also
investigated the suppressive effects of their ligands.
Compared with AG, only Trp and Hkt exhibited significant
suppressive effects ( Table 1 ).
Table 1. IC50 values of Zn complexes for AGE formationin glycation samples.
SampleIC 50 (µM)
859

±

70

610

±

93**

[Zn(mal) 2]

468

±

33**

[Zn(emal) 2]

1,531

±

257

[Zn(trp) 2]

20

±

1**

[Zn(hkt) 2]

9

±

3**

1,093

±

26

emal

1,823

±

44

trp

38

±

1**

18

±

6**

AG
ZnSO4 · 7H 2 O

Measurement of pentosidine levels in glycatedresidues
Next, we set the gradient conditions for column washingand conditioning in the latter two steps: 50 % B (10 –12 min)and 0 % B (12 – 30 min). A standard pentosidine-derived
peak was detected with an average retention time of 9.1 min( Fig. 3-a). The peak area and concentration of pentosidine
showed good linearity ( y = 260,085 x + 1199, R = 1) in theconcentration range of 0.1– 2.0 ng/mL. The amount ofpentosidine was quantified based on the calibration curve
prepared for each HPLC-FL measurement.

Suppressive effects of Zn complexes on pentosidine
formation

Values are expressed as the mean ± standard deviation (SD). ** p < 0.01
vs. AG. IC 50, half-maximal inhibitory concentration; AGE, advanced
glycation end product; AG, aminoguanidine; SD, standard deviation.

Notably, all tested Zn complexes were found to suppress
pentosidine formation in a concentration-dependent manner.
Next, we determined the IC 50 values of these complexes
for pentosidine formation ( Table 2 ). All Zn(O 4 ) complexes
exhibited suppressive effects. Particularly, [ Zn(trp) 2 ] and[Zn(hkt) 2 ], Zn(O 4 ) complexes with a 7-member troponoide
structure, exhibited the strongest suppressive effects on
pentosidine formation among the tested Zn complexes.
Although [Zn(emal) 2 ] did not sufficiently inhibit fluorescent

a)

b)

mal

hkt

Pentosidine 2 ng/mL

40000
30000
20000
10000
0

0

5

10

-10000

20

25

30

40000
30000
20000
10000
0
-10000time (min)

c)

0

5

10

15

20

25

30

time (min)AG 5 mM

50000

Intensity (mV)

15

Ref (+)

50000

Intensity (mV)Intensity (mV)

50000

40000
30000
20000
10000
0
-10000

0

5

10

15

20

25

30

time (min)Fig. 3. Chromatograph of glycation samples using a spin column (Monospin AG).
(a) Pentosidine ( 2 ng) standard curve. ( b) Reference (+) sample. (c) AG (5 mM). Glycation sample (50 μL) was hydrolyzed and preparedusing a spin column (Monospin AG). Arrow indicates the pentosidine peak. High-performance liquid chromatography with fluorescence
detection ( HPLC-F L) was performed as follows: column, Inert Sustain AG ( 3 μm, 100 mm × 4.6 mm ID); column temperature, 20 °C;
flow rate, 1.0 mL/min; detection, Ex 325 nm /Em 385 nm; eluent, A solution: 0.1% formic acid (v/v) in H 2 O solution, B solution: 100 %acetonitrile; elution conditions as step gradient: 0 % B (0 –10 min), 50 % B (10 –12 min), 0 % B (12 – 30 min); injection volume, 20 μL.
In this gradient program, the latter two steps (50 % B [10 –12 min] and 0 % B [12 – 30 min]) were programmed for column washing andconditioning, respectively. AG, aminoguanidine.

_ 36 _Glycative Stress Research

Table 2. IC50 values of Zn complexes for pentosidine
formation in glycation samples.
SampleIC 50 (µM)
102 x 10 3±73 x 10 3

638

±

212**

[Zn(mal) 2]

304

±

84**

[Zn(emal) 2]

371

±

155**

[Zn(trp) 2]

19

±

6**

[Zn(hkt) 2]

15

±

8**

AG
ZnSO4 · 7H 2 O

Values are expressed as the mean ± SD. ** p < 0.01 vs. AG. IC 50,
half-maximal inhibitory concentration; AG, aminoguanidine; SD, standard
deviation.

AGE formation in HSA–glucose screening test, it exerted
stronger suppressive effect on pentosidine formation than AG
or ZnSO 4 .

Discussion

AGEs are associated with various DM complications
and aging by increasing and accelerating glycation reactionsin the body under hyperglycemic conditions. Pentosidine is
an AGE that accumulates in human tissues with aging, and its
accumulation associated with many diseases, including DM
complications 32). Anti-glycation effects of various substances,such as flavonoids and L-carnitine, have been evaluated 33-36).
Such pathological conditions need to be further investigatedto develop new treatments.
Zn complexes exert insulin-like and hypoglycemic
effects. In this study, we investigated the anti-glycation
effects of Zn complexes from a novel perspective. We
evaluated the effects of Zn(O 4) coordination type Zncomplexes on the inhibition of AGE formation. Determinationof the association between AGEs and Zn complexes, inaddition to the anti-DM effects of Zn complexes, will aid
in their long-term administration for the prevention andtreatment of various diseases, including DM complications.
Here, we examined the anti-glycation effects of Zn
complexes by determining their effects on glycation reactions
using the HSA–glucose glycation model. In this glycation
model, glucose was used as the reducing sugar, and the model
assumed a DM state. The influence of Zn complexes onprotein glycation was evaluated based on the inhibition rateof fluorescent AGEs. We found that Zn compounds, including
Zn 2 + and Zn complexes, suppressed fluorescent AGEs in thescreening test. These ligands exhibited suppressive effects inthe screening test ( Table 1 ). A previous study revealed thatbis(ascrobinato) Zn(II) complex ([Zn( Vc) 2 ]), with weak
coordinating strength between ascorbic acid and Zn (logβ= 1) 37), exists as a Zn complex in both 0.015 M HEPESphosphate buffer (pH 7.4) and 0.015 M HEPES buffer (pH7.4) using cyclic voltammograms data 38). As [Zn(Vc) 2 ]maintains its complex structure in both buffer solutions
with and without phosphoric acid, we hypothesized that
Zn complexes inhibit fluorescent AGE formation in thepresent study. Furthermore, for detailed evaluation of AGEs,
quantitative analysis of pentosidine against Zn complexes
was also conducted using HPLC-FL.

Pentosidine is a type of fluorescent AGE. Blood
pentosidine concentration is associated with various diseases,
such as DM and renal failure, and used as a biomarker. To
evaluate the HPLC-FL method, a calibration curve wasplotted using standard pentosidine, and the sample solution
was prepared from a previously glycated sample solution in
the HSA–glucose model and pentosidine extraction process.
A pentosidine peak was observed at approximately 9.1 min,and the calibration curve showed good linearity ( Fig. 3 ).
Here, samples were analyzed in two ways: one sample wassubjected to HPLC measurement after undergoing pentosidine
extraction without refrigeration, whereas the other was
subjected to HPLC measurement after refrigeration for oneweek post-pentosidine extraction. No significant differenceswere observed in the results of the two samples, suggestingthat the samples could be refrigerated for one week after
pentosidine extraction (data not shown). As AGEs are produced
in a non-enzymatic manner, their long-term storage underheat and light is not desirable.
In this study, we found that Zn(O4 ) type complexes,
especially [ Zn(trp) 2 ] and [Zn(hkt) 2 ] complexes with a
7-member troponoide structure, exhibited significantly
greater suppressive effects on pentosidine formation than AG.
Although [Zn(emal) 2 ] suppressed pentosidine formation, it
did not sufficiently suppress fluorescent AGE formation in
the screening test ( Table 2 ). [Zn(hkt) 2] exhibited the strongest
inhibitory effect on pentosidine formation among all tested
complexes. We previously reported that [Zn(hkt) 2 ] activatesthe insulin signaling pathway and ameliorates insulin
resistance in peripheral tissues via islet-protecting effects intype 2 DM model KK-Ay mice 23, 39).
To the best of our knowledge, this study is the first to
demonstrate the anti-glycation effects of Zn complexes invitro. Specifically, [ Zn(trp) 2 ] and [Zn(hkt) 2 ], Zn(O 4 ) typecomplexes with a 7-member troponoide structure, exerted
strong anti-glycation effects, suppressing both fluorescent
AGE and pentosidine formation. Furthermore, they inhibitedAGE formation and accumulation, thereby preventing DM
complications. Notably, the anti-glycation effects differeddepending on the type of Zn coordination. Therefore, further
studies are necessary to investigate the mechanism of the antiglycation effects based on the structure–activity relationshipsof Zn complexes. With regard to glycative stress and thedevelopment of type 2 DM, it has recently been suggested
that postprandial hyperglycemia is followed by a chain
reaction of the formation of multiple aldehydes 40), and thatthese aldehydes non-physiologically modify intravascularand intracellular proteins and are involved in both insulin
biosynthesis and insulin resistance in β cells 41). The aldehyde
scavenging activity of Zn complexes to prevent these seriesof reactions will also be investigated in the future.

Acknowledgements
The authors are grateful to the members of theAnalytical Center of Kyoto University for assistance withelemental analyses. This study was supported by the JSPS
Grant-in-Aid for Research Activity Start-up (grant number#21K20734).

_ 37 _

Zn Complexes Suppress Glycation

Conflict of Interest
The authors declare no conflicts of interest.

Funding
This study was supported by the JSPS Grant-in-Aid for
Research Activity Start-up (grant number # 21K20734).

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