Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : October 11, 2023
Accepted : November 2, 2023
Published online : December 31, 2023
doi:10.24659/gsr.10.4_171

Original article

Protein cross-linking inhibition effects induced by glycative stress of
black galangal, Kaempferia parviflora Wall. Ex. Baker (Zingiberaceae)
Masayuki Yagi, Kayoko Inoue, Yasuo Sato, Chieko Sakiyama, Yoshikazu Yonei
1) Anti-Aging Medical Research Center and Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan.
2) Rene Co. Ltd., Osaka, Japan

Abstract

Accumulation of advanced glycation endproducts (AGEs) due to glycative stress in the body leads to tissue stiffening andphysiological dysfunction due to protein cross-link formation, and is one of the factors contributing to aging and lifestylerelated diseases. Suppression of glycative stress includes suppression of postprandial hyperglycemia, suppression of glycation
reaction, and degradation and excretion of AGEs. Fluorescent AGEs include both cross-linked and non-cross-linked AGEs.
Inhibition of fluorescent AGE formation has been reported in various plant materials, one of which is the ginger plant black
galingal (Kaempferia parviflora Wall. Ex. Baker; KP), with hydrophilic components and polymethoxy flavonoids (PMF)
involved in this effect. On the other hand, there are several types of cross-linked structures of glycated proteins, α-diketone
being one of them. The cleavage action of α-diketones is called AGE cross-link cleavage, and phenylpropanedione (1-phenyl1, 2 -propanedione; PPD) has been used as a cross-linking model substance. The rate of AGE cross-link cleavage has been
evaluated from the rate of free benzoic acid formation. KP rhizome has also been reported to have AGE cross-link-cleaving
activity. There have been only a few reports on the effect of AGE formation inhibition and AGE cross-link cleavage on protein
cross-linking by glycation. In this study, we examined the inhibition of glycated-protein cross-linking (IGPC) and cleavageof glycated-protein cross-linking (CGPC) of KP extract, which previously has been shown to have effects of fluorescent AGE
formation inhibition and AGE cross-linking cleavage.

KEY WORDS: inhibition of AGE formation, AGE cross-linking, inhibition of glycated-protein cross-linking,		

glycated-protein cross-linking

Introduction
Accumulation of advanced glycation endproducts (AGEs)
due to glycative stress in the body leads to tissue stiffening
and physiological dysfunction through the formation ofprotein cross-links, and is one of the factors that contributeto aging and lifestyle-related diseases. Inhibition of glycative
stress in the body is called "anti-glycation" or "glycation care,"and includes suppression of postprandial hyperglycemia,
suppression of glycation reaction, and degradation andexcretion of AGEs 1, 2). Inhibition of fluorescent AGE formation,
one of the indicators of glycation care, has already beenreported in various plant materials such as vegetables andherbs 3, 4). This effect has also been observed in black galangal(Kaempferia parviflora Wall. Ex. Baker; KP), a member ofthe ginger family (Zingiberaceae) 5, 6), and the presence ofpolymethoxy flavonoid (PMF) and hydrophilic components

have been reported 7). Fluorescent AGEs include various
substances such as non-crosslinking argpyrimidine,crosslinking vesperlysine, and crossline 8).
As one of the indicators of AGE degradation action, thecleavage action of α-diketone, a kind of crosslink structureof glycated proteins, is evaluated 9-11). This is called AGE
crosslink cleavage effect. Phenylpropanedione (1-phenyl1, 2-propanedione; PPD) is used as a crosslinking model
substance to evaluate the AGE crosslink cleavage effect.
The AGE crosslink cleavage rate is calculated from theproduction rate of benzoic acid, which is liberated by thecleavage of α-diketones by the test substance. Substancesthat have the AGE crosslink cleavage effect include
N-phenacylthiazolium bromide (PTB) 9), rosmarinic acid 12),ellagitannins 10), and flavonoids 11) have been reported. Thiseffect has also been reported in the rhizome of KP 13).

Contact Address: Visiting Professor Masayuki Yagi, PhD
Anti-Aging Medical Research Center / Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1- 3, Tatara Miyakodani, Kyotanabe-shi, Kyoto, 610- 0394 Japan
TEL & FAX : + 81-774 - 65-6394 e -mail: myagi@mail.doshisha.ac.jp
Co-authors: Inoue K, tmkayoko@miracle.ocn.ne.jp; Sato Y, ceo.gran@gmail.com;
Sakiyama C, csakiyam@mail.doshisha.ac.jp; Yonei Y, yyonei@mail.doshisha.ac.jp_ 171 _

Glycative Stress Research 2023; 10 (4): 171-176(c) Society for Glycative Stress ResearchProtein cross-linking inhibition effects induced by glycative stress of black galangal

On the other hand, there are only a few reports that haveexamined the effects of components with AGE formation
inhibitory effects on protein cross-link formation and AGE
cross-link cleavage effects on glycated proteins 14, 15).
In this study, we aimed to evaluate the effects of
KP, which has the effect of inhibiting fluorescent AGE
formation and cleavage of AGE crosslinks, on the inhibitionof glycated-protein cross-linking (IGPC) and cleavage ofglycated-protein cross-linking (CGPC).

Materials and Methods(1) Reagents
Reagents used were purchased from the following
manufacturers: N-phenacylthiazolium bromide (PTB) from
Sigma-Aldrich Japan (Meguro-ku, Tokyo, Japan). Lysozymehydrochloride (lysozyme derived from egg white)aminoguanidine hydrochloride (AG) is from Fujifilm Wako
Pure Chemical Industries, Osaka, Japan. 4-20 % Mini-Protean
TGX Precast gel is from Bio-Rad (Hercules, California, USA).
Other reagents were purchased from Fujifilm Wako Pure
Chemical Industries or Nakalay Tesque (Kyoto, Japan).
(2) Kaempferia parviflora (KP) rhizome samples andextraction conditions
Dried powder of KP rhizome, which is sold as a dietary
supplement product in Japan, was used as the sample; KP
powder was provided by Rene Co. The dried powder sample
was extracted by mixing 40 mL purified water and 2 g of thepowder. The hot water extraction conditions were incubationof the mixture in a water bath set at 80 °C for 60 minutes.
The extract was then centrifuged at 2,500 rpm (800 x g) for
10 min and further filtered. The resulting solution was used
as KP extract (Kaempferia parviflora extract; KPE) for
verification. The solid concentration of KPE was calculated
by placing 5 mL of KPE in an aluminum tray, evaporating it
at 120 °C for 2 hours, and weighing the evaporated residue.
(3) Inhibition of glycated-protein cross-linking (IGPC)
The inhibition of glycated-protein cross-linking (IGPC)
was performed using the lysozyme-glucose glycated-protein
cross-linking model based on previous reports 16, 17). The
reaction solution was prepared by adding 1/10 of the sample
solution in 0.1 mol/L phosphate buffer (pH 7.4) containing
5 mg/mL lysozyme and 0.5 mol/L glucose. The following
four reaction solutions were prepared and incubated at 60 °C
for 40 hours; (A) solution with phosphate buffer, lysozyme
solution, glucose solution, and each of the sample solutions
added, (B) solution with purified water added in place ofthe glucose solution in A, (C) solution with sample solution
added in place of the sample solution in A, and (D) solution
with purified water added in place of the glucose solution in
C. After completion of the reaction, the reaction solution wascentrifugally filtered using a 3 kDa ultrafiltration membrane(Amicon Ultra-0.5 mL centrifugal filters Ultracel-3K; Merck,
Darmstadt, Germany) to remove low molecular weightsubstances. The reaction solution was then subjected to 4-20 %polyacrylamide gel electrophoresis (SDS-PAGE). The gel was
stained with CBB Stain One (Nacalai Tesque), and the gel

images were imaged with the Pharos FX System (Bio-Rad).
The intensities of the monomer (12.4 kDa), dimer (25.8 kDa),
and trimer (40.5 kDa) bands of the enzyme were analyzed
using ImageJ (NIH, Maryland, USA). AG was used as apositive control substance for inhibition of glycated protein
cross-link formation.
The inhibition rate of protein cross-link formation was
calculated based on the following formula, as previously
reported 14).
Percentage inhibition of protein cross-link formation (%)= {1 − (A - B) / (C − D) } × 100
A - D ; band intensity of lysozyme dimer or trimer in SDSPAGE gel stained images of each reaction solution(4) Cleavage of glycated-protein cross-linking (CGPC)
For the cleavage of glycated-protein cross-linking(CGPC), glycated lysozyme was prepared with reference toa previous report 14), and the degradation rate of lysozyme
dimer by the sample was measured. Glycated lysozyme wasprepared by reacting 1 mg/mL lysozyme with 0.1 mol/L
phosphate buffer (pH 7.4) containing 0.2 mol/L glucose at
37 °C and then removing low molecular weight substances
by ultrafiltration (3 kDa).
The following two reaction solutions were prepared;(A) A solution in which 1/2 volume of sample solution wasadded in 0.05 mol/L phosphate buffer containing 0.5 mg/mL
glycosylated lysozyme and (B) a solution in which sample
lysate was added in place of the sample solution in A. Afterincubation at 37 °C, the reaction solution was centrifugedthrough an ultrafiltration membrane (3 kDa) to remove low
molecular weight substances. The reaction solution was
subjected to SDS-PAGE using the same method as describedin the previous section, and the gel was stained after
electrophoresis, imaged, and analyzed for band intensities oflysozyme monomer and dimer in the reaction solution. PTB
was used as a positive control for glycated protein cross-linkcleavage.
The protein cross-link cleavage rate was calculated
based on the following formula as previously reported 18).
Percentage of glycated protein cross-link cleavage (%)= [1 − {( D S / MS) / (D R / M R)}] × 100
S ; Lysozyme band intensity at the time of sample addition
R; Band intensity of lysozyme when only sample solution is
added
M; monomer band of lysozyme
D; dimer band of lysozyme

Statistical Analysis

Measurements are expressed as mean ± standard
deviation. The 50 % inhibition concentration ( IC 50; mg/mL)
or 50 % effective concentration (EC 50; mg/mL) was calculated
from the results of the three sample concentrations 19, 20).
A smaller IC 50 or EC 50 value indicates a stronger effect.
Tukey's multiple comparison test was used to compare themeasured values. Statistical analysis results were consideredsignificant at a risk rate of less than 5%.

_ 172 _Glycative Stress Research

Results

monomer band of the glucose-sensitive sample was slightly
larger than that of the glucose-free sample in the cathodic
side (Fig. 1).

IGPC showed concentration-dependent inhibition of
dimer and trimer formation in both KPE (0.097 – 0.87 mg/mL)
and AG (0.10 – 1.00 mg/mL) (Table 1). The IC 50 of IGPC
was 3.7-fold smaller for AG than for KPE in dimer and30.9-fold smaller in trimer, indicating a stronger effect of
AG. Comparing IGPCs of trimer and dimer, both KPE andAG showed a stronger inhibitory effect on trimer formationcompared to dimer. SDS-PAGE images showed that the

Glycated protein cross-link cleavage (CGPC)

Inhibition of glycoprotein cross-link formation(IGPC)CGPC was concentration dependent in KPE (0.1 – 1.0
mg/mL) and PTB (1.4 – 5.7 mg/mL) (Table 2). The EC 50 of
CGPC was 1.6 - fold smaller in KPE (1.65 mg/mL) than in
PTB (2.65 mg/mL), indicating a strong effect. The dimer
formation rate of Ref (with water) ranged from 10.3 to 11.9 %,and no trimer formation was observed (Fig. 2).

Table 1. Inhibitory effects of KPE and AG on cross-linking formation in the lysozyme-glucose reaction model.
Sample

KPE

AG

Conc.
(mg/mL)

Inhibition ratio on cross-linking formation (%)
IC50 (mg/mL)

Dimer

Trimer

0.097- 3. 4 ± 4.7

0.29

9.7 ± 3. 1

0.87

45.6 ± 3. 9

76. 8 ± 1. 6

0.10

30.1 ± 4.3

72. 9 ± 3. 8

0.30

49.2 ± 3.6

1.00

65.9 ± 1.6

IC50 (mg/mL)- 31.7 ± 10. 8*

1. 26 1)*

0.34

21. 8 ± 0.6

88.7 ± 2. 8

*

0. 51

*

0. 011

98. 1 ± 3. 3

1) Extrapolated value, mean ± SD, n = 3; * p < 0.05, Tukey's multiple comparison test; KPE, incubation with Kaempferia parviflora
rhizome hot water extract; AG, incubation with aminoguanidine; IC 50, 50 % inhibition concentration; SD, standard deviation.

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Inhibitory effects of KPE and AG on cross-linking formation in the lysozyme-glucose reaction model.

5 mg/mL lysozyme were incubated at 60 ℃ for 40 hours. SDS-PAGE was conducted using 4 - 20 % acrylamide gels. M, molecular$03.>0=%:<$?2.3%&/%"2-,.*&=.7%$(
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Protein cross-linking inhibition effects induced by glycative stress of black galangal

Table 2. Cleavage effects of KPE and PTB on lysozyme dimer in the glycated lysozyme.
Sample

KPE

PTB

Conc.
(mg/mL)

Cleavage(%)

0.1

22.6 ± 7.9

0.3

34. 5 ± 6.7

1

4 4.8 ± 4. 0

1.4

42.8 ± 6. 5

2.8

51.1 ± 9. 1

5.7

58. 4 ± 5. 6EC50(mg/mL)*

1. 65 1)*

2.65

1) Extrapolated value, mean ± SD, n = 3, * p < 0.05, Tukey's multiple comparison test; KPE, incubation with
Kaempferia parviflora rhizome hot water extract; P TB, incubation with N-phenacylthiazolium bromide;
EC 50, 50 % effective concentration; SD, standard deviation.

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Fig. 2. Cleavage effects of KPE and PTB on lysozyme dimer in the glycated lysozyme.

0.5 mg/mL glycated lysozyme were incubated at 37 ℃ for 16 hours. SDS-PAGE was conducted using 4 - 20 % acrylamide gels.
D,<(&@(&A$%"B"<%&%11%2-3&.1&456&"*7&5!C&.*&$03.>0=%&7,=%/&,*&-+%&<$02"-%7&$03.>0=%(M, molecular weight markers; Ref, incubation with water; KPE, incubation with Kaempferia parviflora rhizome hot water extract;!#1MQ1R#M+?C(&,-#+?A*J?1,#G,',#B2C/F(&,-#(&#6^T#I*'#!S#H*/'A;#343VEKL$#G(A#PTB, incubation with N-phenacylthiazolium bromide.
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Discussion

IGPC of KPE

KPE has been shown to inhibit the formation of
AGEs (fluorescent AGEs, pentosidine, N ε-(carboxymethyl)
lysine; CML) and glycation reaction intermediates(3-deoxyglucosone; 3DG, glyoxal; GO, methylglyoxal;
MGO) 13). Hydrophilic components of KPE are involved inthese effects 7). AGEs with crosslinking properties include
argpyrimidine, vesperlysine, and crossline 8). These aremeasured as fluorescent AGEs (excitation wavelength: 370
nm, fluorescence wavelength: 440 nm). Pentosidine is also
fluorescent (excitation wavelength: 335 nm, fluorescencewavelength: 385 nm) and crosslinkable 21). Furthermore,glycation intermediates such as 3DG, GO, and MGO are atype of aldehyde and are involved in the formation of cross-linkable AGEs 22, 23). From these facts, it is presumed that
the IGPC of KPE, a hot water extract of KP, is related tothe inhibitory effect of KPE on the formation of AGEs andglycation reaction intermediates. Protein cross-linking byglycation is different from physiological cross-linking by
disulfide bridges and lysyl oxidase, and is called pathologicalcross-linking because it forms disordered cross-links. The
formation of pathological cross-links by glycation of skinand bone collagen is considered to be a factor in the loss ofskin elasticity and bone fractures due to the deteriorationof bone quality 24, 25). IGPC of KPE may function to prevent
hardening and functional deterioration caused by protein
cross-linking and to inhibit aging in the body.
In the SDS-PAGE image of IGPC, the band width ofthe monomer was larger in the cathode side in the glucoseadded (+) sample than in the glucose-free (−) sample (Fig. 1).

_ 174 _Glycative Stress Research

The native-PAGE image of aldehyde-induced AGE-modified
BSA is shifted toward the positive side, presumably due toa relative negative change in the protein surface charge 26).
In SDS-PAGE, SDS neutralized the protein charge, and theshift of the band was on the cathodic side, suggesting thatthe difference in the glucose (+) monomer band was due
to a different factor than the protein charge change. In theglycation reaction of lysozyme and glucose, not only crosslinked AGEs but also non-cross-linked AGEs such as CML
are generated. Therefore, the band shift of monomer may be
due to the formation of non-crosslinking AGEs of lysozymes.

CGPC of KPE

The involvement of α-diketones, glucospan, and lysinedihydropyridinium-lysine 27, 28) in the formation of cross-links
by protein glycation has been reported. A hydrophilic version
of PTB, agebrium (3-phenacyl- 4, 5-dimethylthiazolium
chloride), has been called an AGE breaker because of its
ability to cleave α-diketones 29, 30). KPE has been reported
to cleave α-diketones. However, when KPE and PTB werecompared in terms of α-diketone cleavage at 1.42 mg/mL,
KPE was only about one-fifth as potent as P TB 13). The
CGPC of KPE was 1.6 times stronger than that of PTB,
which was different from the evaluation results of α-diketone
cleavage. The reason for this difference may be that theprotein cross-links of glycated lysozyme contain many crosslinks other than α-diketones, and the protein cross-linkcleaving effect of KPE may have been stronger in cleaving
cross-links other than α-diketones.
Proteins cross-linked by glycation are not easily
degraded by proteases and are not easily metabolized 31).

Therefore, CGPC may be involved in the reparative action ofproteins cross-linked by glycation. On the other hand, thecross-linking structure in which CGPC acts is not clear.
In particular, the effect on physiological cross-linking ofproteins needs to be verified.

Conclusion
The effect of KPE on the formation of protein crosslinks by glycation was verified, and both IGPC and CGPC
were observed. In IGPC, the involvement of KPE in theinhibition of crosslinkable AGEs and aldehyde formation
was inferred. In CGPC, KPE may be involved in crosslink
cleavage action other than α-diketone cleavage as well as inthe α-diketone cleaving action of KPE. KPE may function
to inhibit glycation-induced protein cross-link formation,
prevent stiffening and functional decline, and inhibit the
aging process in the body.

Declaration of Conflict of Interest
Research funding was received from Rene Corporation
for conducting this study.

Acknowledgments
Outline of this study were presented at the 27 th Meetingof Society for Glycative Stress Research (September 9, 2023,
Kyoto, Japan).

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