Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : January 3, 2021
Accepted : January 26, 2021
Published online : March 31, 2021
doi:10.24659/gsr.8.1_1

Original article

Antiglycative effect of black galangal, Kaempferia parviflora Wall. Ex. Baker(Zingiberaceae)
Masayuki Yagi 1), Kayoko Inoue 2), Yasuo Sato 2), Kaori Ishizaki 1), Chieko Sakiyama 1), Yoshikazu Yonei 1)
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 end products (AGEs) in the body due to glycative stress is a factor in the onset of aging
and lifestyle-related diseases. Suppression of glycative stress in the body is called anti-glycation, and includes suppression of
postprandial hyperglycemia, suppression of glycative reaction, and decomposition and excretion of AGEs. It is already known
that plant materials, i.e., vegetables and herbs, have an anti-glycative effect. Of these, the rhizome of black galangal (Kaempferia
parviflora Wall. Ex. Baker; KP), which is a type of ginger family plant (Zingiberaceae), has an action of suppressing theformation of fluorescent AGEs, and polymethoxy flavonoids (PMF); and hydrophilic components are thought to be involved
as the active components. In this study, for the purpose of further verifying the possibility of KP as an anti-glycation material,
we focused on the hydrophilic component in the KP rhizome and evaluated the anti-glycative effect and AGE degradation
action. As a sample, a hot water (80 °C) extract of dry KP rhizome was used. The anti-glycative effect of the KP extract was
examined for actions of inhibiting the formation of AGEs and intermediates in the human serum albumin (HSA)-glucose
glycation model. Regarding the degrading action of AGEs, the cleaving action of AGE cross-links and the enhancing actionof oxidized protein hydrolase (OPH) activity. In the results, the KP extracts inhibited the formation of fluorescent AGEs,
pentosidine, CML (N ε-carboxymethyllysine) and intermediates, 3-deoxyglucosone (3DG), glyoxal (GO) and methylglyoxal(MGO). For the degradation effect, the KP extract enhanced actions of both cross-link cleaving of AGEs and OPH activity.
In conclusion, it is indicated that KP, as an anti-glycative material, can suppress the production of many types of AGEs withdifferent pathways and acts on the degradation of AGEs, thus reducing the amount of accumulated AGEs in the body.

KEY WORDS: advanced glycation endproducts (AGEs), anti-glycation, pentosidine, N ε-carboxymethyllysine (CML),		

AGE-crosslink cleavage, oxidized protein hydrolase (OPH)

Introduction
Non-enzymatic binding of protein to glucose produces
advanced glycation end products (AGEs). The accumulationof AGEs in vivo is a factor in the onset of aging and lifestylerelated diseases. These are called glycative stress, which is
a comprehensive combination of the stress reaction on theliving body caused by reducing sugar and aldehyde loading
and subsequent reactions. On the other hand, suppression ofglycative stress in the living body is called anti-glycation.
Anti-glycation includes suppression of postprandial
hyperglycemia, suppression of glycative reaction, anddegradation and excretion of AGEs 1, 2). Aminoguanidine(AG) has already been reported to have an AGEs production
inhibitory effect, thus showing the effects of preventing andinhibiting the progress on nephropathy, retinopathy, andContact Address: 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; Ishizaki K, ko-sei12@mail.doshisha.ac.jp;
Sakiyama C, csakiyam@mail.doshisha.ac.jp; Yonei Y, yyonei@mail.doshisha.ac.jp_ 1 _Glycative Stress Research 2021; 8 (1) : 1- 7(c) Society for Glycative Stress ResearchAnti-glycative Effect of Black Galangal

neuropathy 3, 4). However, AG has side effects, i. e., anemia,
liver damage, and vitamin B6 deficiency, and has not been
put into practical use. It has already been reported that foodmaterials such as tea/herbal tea 5), ​​vegetables/herbs 6), fruits 7),
and yogurt 8) have inhibitory effects on glycative reaction.
In addition, N-phenacylthiazolium bromide (PTB) has beenreported as a substance that may act on the degradation of
AGEs 9). PTB cleaves the α-diketone structure, which is a
type of cross-linked structure of AGEs-modified proteins.
Similar effects have been observed with rosmarinic acid 10),ellagitannin 11), and flavonoids 12). It has also been reported
that oxidized protein hydrolase (OPH), which is a typeof in vivo enzyme, degrades AGEs-degrading proteins 13).
Furthermore, many kinds of herbal extracts have an OPH
activity enhancing effect 14).
It has already been reported that ginger (Zingiber
officinale Roscoe) has an inhibitory effect on glycative
reaction 15, 16). Furthermore, the rhizome of black galangal(Kaempferia parviflora Wall. Ex. Baker; KP), which is a kindof ginger family plant (Zingiberaceae), has an anti-glycative
effect via inhibiting postprandial hyperglycemia 17) and afluorescent AGEs production 18, 19). The inhibitory effect on
the production of fluorescent AGEs in KP rhizomes isreported to involve polymethoxyflavonoids (PMF), which
are characteristic components of this plant species, andhydrophilic components 20).
In this study, for the purpose of verifying the potential
of KP as an anti-glycation material, we focused on thehydrophilic components in the KP rhizome and evaluated theanti-glycative effect and AGEs degradation action of a hotwater extract of KP rhizome powder.

provided by Rene Co., Ltd (Osaka, Japan). The dry powder
sample was extracted by mixing 40 mL of distilled water and
2 g of powder. The condition of hot water extraction was thatthe mixture was incubated for 60 minutes in a water bath set
at 80 °C. The obtained extract was centrifuged at 2,500 rpm(800 × g) for 10 minutes, and then further filtered to obtain asample solution. The solid content concentration of the sample
solution was calculated by placing 5 mL in an aluminum tray,
drying and evaporating it at 120 °C for one hour, and thenweighing the evaporation residue.

3) HSA-glucose glycation model
The HSA-glucose glycation model was used to verify
the inhibitory effect on glycative reaction 21). Solution (A), asolution mixed sample solution, 0.1 mol/L phosphate buffer(pH 7.4), 40 mg/mL HSA, 2.0 mol/L glucose aqueous
solution, and distilled water at a ratio of 1: 5: 2: 1: 1; Solution(B), a solution in which purified water is added instead of the
glucose solution of (A); Solution (C), a solution prepared andincubated with purified water instead of the sample solutionof (A); Solution (D), purified water instead of the glucose
solution of (A) was prepared, followed by incubation at 60 °C
for 40 hours. The amounts of AGEs and intermediates, i.e.,
3DG, GO, MGO, in the reaction solution after incubation
were measured. AG and EGCg were used for the positive
control of inhibitory actions on glycative reaction.

4) Measurement of AGEs
For the evaluation of fluorescent AGEs, 200 µL of theglycative reaction solution was placed in a black microplate
and the fluorescence derived from AGEs (excitation
wavelength 370 nm/fluorescence wavelength 440 nm) was
measured according to the previous report 21). Pentosidine
was measured by HPLC after mixing 50 µL of the reaction
solution and 6 N hydrochloric acid and hydrolyzing at 110 °C
for 18 hours according to the previous report 22). CML wasmeasured using 30 µL of reaction solution using CircuLex
CML /N ε - (Carboxymethyl) lysine ELISA Kit.

Materials and methods
1) Reagent

The reagents used in the experiment were purchased fromthe following manufacturers; human serum albumins (HSA,
lyophilized powder, ≥ 96 %, agarose gel electrophoresis),
40 % methylglyoxal solution (MGO), N-phenacylthiazolium
bromide; PPD) from Sigma-Aldrich Japan (Meguro-ku, Tokyo,
Japan); aminoguanidine hydrochloride (AG), epigallocatechingallate (EGCg), 2,3-diaminonaphthalene (DAN), 4 0 %glyoxal solution (GO), 1-phenyl -1, 2-propanedione (PPD) andrubusoside (Ru) from Fuji Film Wako Pure Chemical
Industries, Ltd. (Osaka,Japan); 3-deoxyglucosone (3DG) from
Dojin Chemical Research Institute (Kamimashiki-gun,
Kumamoto, Japan); acylamino-acid releasing enzyme(AARE) from Takara Bio (Kusatsu, Shiga, Japan); N-acetylL-alanine p -nitroanilide (AAPA) from Bachem (Bubendorf,
Switzerland); CircuLex CML/ N ε- (Carboxymethyl) Lysine
ELISA Kit from MBL (Nagoya, Aichi, Japan). Other reagentsof special grade or HPLC grade were purchased from Fuji
Film Wako Pure Chemical Industries or Nacalai Tesque(Kyoto, Japan).

5) Measurement of glycative reaction intermediates
The intermediates, 3DG, GO, and MGO, were measuredby HPLC after deproteinizing 200 µL of the reaction solution
with perchloric acid, adding DAN under alkaline conditions,and labeling according to the previous report 23, 24).

6) Calculation of anti-glycative effect
As for the anti-glycative effect, the production inhibition
rate (%) of AGEs and intermediates were calculated by thefollowing formula according to the previous report 21).
Formation inhibitory rate (%) = {1 − (A − B) / (C − D)} × 100
Then, a 50 % inhibitory concentration (IC 50; mg/mL) was
calculated from the formation inhibitory rate of the sample
with three different concentrations 21, 25). The smaller the IC 50
value, the stronger the glycative reaction inhibitory effect.

2) Rhizome sample and extraction conditions of
Kaempferia parviflora (KP)

As a sample, dry powder of KP rhizome sold as asupplement product in Japan was used. The KP powder was_ 2 _Glycative Stress Research

negative control. The OPH activation rate (%) was calculatedby the following formula, assuming that the amount of pNA
generated in 60 minutes immediately after the start of thereaction (0 minutes) in the reference reaction was 100 %.

7) AGE crosslink cleavage action
According to previous report 9, 12), the AGEs cross-linking
and cleavage action was carried out by mixing the sample
solution, 10 mmol/L PPD, and 0.2 mol/L phosphate buffer(pH 7.4) at a ratio of 5: 1: 4 at 37 °C for 8 hours, thenstopping the reaction by adding 0.7 N hydrochloric acid,followed by measuring the amount of benzoic acid cleaved
from PPD by HPLC.

OPH activity enhancing rate (%) ={ (S 60 − S 0 ) / ( R60 − R 0 ) } x 100
S; pNA concentration of sample solution addition reaction
solution, R; pNA concentration of reference reaction solution,
60; 60 minutes later, 0; immediately after reaction (0 minutes).

AGE Cleavage rate (%) = { ( A − B ) / C } × 100
A; amount of benzoic acid in the reaction solution, B; amountof benzoic acid in the sample, C; amount of PPD used in thereaction.

Statistical analysis
The measured values ​​are shown as mean ± standard
deviation. Tukey's test was used to compare the measured
values. As a result of the statistical analysis, a significancelevel of less than 5 % was considered significant.

8) OPH activity enhancing effectAccording to the previous report 14), the OPH activity
enhancing action was made via mixing AARE as OPH, asample solution, 0.025 U/mL OPH, 0.025 mol/L AAPA, and0.12 mol/L Tris-hydrochloric acid buffer (pH 7.4) at a ratioof 1: 1: 2: 21, then incubated at 37 °C for one hour, followedby measuring the amount of p-nitroaniline ( pNA) released
from AAPA using absorbance meter at 405 nm; (S). As areference for measuring OPH activity, distilled water wasadded, instead of the sample solution added to the mixed
solution; (R). For the OPH activity enhancing action, Ru
was used for the positive control and EGCg was used for the

Result

AGE formation inhibitory effect
The formation inhibitory effects on fluorescent AGEs
and CML by the KP extract were found to be concentrationdependent ( Fig. 1 ). The formation inhibitory rate by KP at asolid content concentration of 0.79 mg/mL was 79.9 % forfluorescent AGEs, 52.0 % for pentosidine, and 98.8 % for

a)

b)
100

Fluorescent AGEs

*

80

*

60

*

40
20
0

Inhibition ratio (%)

Inhibition ratio (%)

120*
0.0079

0.079

0.79

KP

Inhibition ratio (%)

120
100

100

Pentosidine

*

80
60
40
20
0

0.1

0.0079

0.079

0.79

KP

AG

Concentration (mg/mL)

c)

120

0.1
EGCg

Concentration (mg/mL)

CM L

80
60
40
20
0

*
0.0079

0.079

0.79

KP

0.1
AG

Concentration (mg/mL)

Fig. 1. Inhibitory activity of KP extract on AGE formation.
a) Fluorescent AGEs, b) Pentosidine, c) CML. Results are expressed as mean ± standard deviation, n = 3, * p < 0.05 by Tukey’ s test. KP,
Kaempferia parviflora rhizome extract; AG, aminoguanidine; AGEs, advanced glycation end products; CML, N ε -(carboxymethyl)lysine.

_ 3 _Anti-glycative Effect of Black Galangal

CML. The IC 50 values ​​were 0.078 mg/mL for fluorescent
AGEs, 0.292 mg/mL for pentosidine, and 0.031 mg/mL for
CML ( Table 1 ). The IC 50 value of the KP extract was 0.65
times higher for fluorescent AGEs and 0.28 times higher for
CML than AG. The formation inhibitory rate of pentosidine
by KP was 42.7 % to 52.0 % at an extract concentration of
0.0079 to 0.79 mg/mL, in which concentration-dependence
was not observed. The IC 50 value of pentosidine was 21.3
times higher in KP extract than in EGCg.

Formation inhibitory actions on glycative reaction
intermediates
The formation inhibitory effects on 3DG and GO by
the KP extract were found to be concentration-dependent( Fig. 2 ). The formation inhibitory rate by KP with a solid
content concentration of 0.79 mg/mL was 89.4 % for 3DG
and 88.2 % for GO. The IC 50 values ​​were 0.028 mg/mL for
3DG and 0.038 mg/mL for GO ( Table 1 ). The IC 50 value of

b)
120

3DG

100

Inhibition ratio (%)

Inhibition ratio (%)

a)

*

80

*

60

*

40
20
0

0.0079

0.079

0.79

GO

100

*

80

*

60
40

*

20
0

0.1

KP

120

0.0079Inhibition ratio (%)

120

M GO

0.79

KP

Concentration (mg/mL)

c)

0.079

AG

0.1
AG

Concentration (mg/mL)

*

100
80
60
40
20
0

0.0079

0.079

0.79

0.1

KP

AG

Concentration (mg/mL)

Fig. 2. Inhibitory activity of KP extract on intermediate of AGE formation.
a) 3DG, b) GO, c) MGO. Results are expressed as mean ± standard deviation, n = 3, *; p < 0.05 by Tukey’ s test. KP, Kaempferia

parviflora rhizome extract; AG, aminoguanidine; EGCg, epigallocatechin gallate; AGEs, advanced glycation end products; 3DG,
3-deoxyglucosone; GO, glyoxal; MGO, methylglyoxal.

Table 1. Inhibitory activity of KP extract on AGEs and intermediate formation.
Index foranti-glycative effect

KP
IC 50 (mg/mL)

Aminoguanidine
IC 50 (mg/mL)

EGCg
IC 50 (mg/mL)

Fluorencet AGEs

0.078

0.120

-

0.292

-

0.014

0.031

0.114

-

0.028

0.126

-

0.038

0.117

-

Pentosidine
CML
3DG
GO

MGO

< 0.010< 0.010-

KP, Kaempferia parviflora rhizome extract; EGCg, epigallocatechin gallate; AGEs, advanced glycation end products; CML, N ε(carboxymethyl)lysine; 3DG, 3-deoxyglucosone; GO, glyoxal; MGO, methylglyoxal.

_ 4 _Glycative Stress Research

the KP extract was 0.22 times that of 3DG and 0.38 times
that of GO compared to AG. The MGO formation inhibitory
rate by KP was strong, 99 % or more in each of the extract
concentrations of 0.0079 to 0.79 mg/mL.

water extract of KP rhizome powder. As a result, the antiglycative effect of the KP extract was observed in all themeasurement items. IC 50 values for the fluorescent AGEs,
CML, 3DG showed equal to, or higher effect of the KP
extract than those of AG, a positive control. AG suppresses
the formation of AGEs by blocking carbonyl groups suchas intermediates 3). In a clinical trial in which patients withdiabetic nephropathy were administered with AG at 150 mg
or 300 mg per day for 42 consecutive months, their urinary
protein excretion was reduced by inhibiting AGE formationfor 21 months. However, after that, the usefulness was notrecognized in the 300 mg intake group 4). The reason may
be that the glycative reaction in vivo has multiple pathways,
and there are many types of AGEs and intermediates, which
are produced during the reaction process 26). Therefore, inorder to inhibit the glycative reaction in vivo, it is necessary
to suppress the formation of various types of AGEs andintermediates. It has already been verified in vitro andin human clinical trials that the mixed herb extract of
Houttuynia cordata, chamomile, hawthorn, and grape leaves
inhibits the glycative reaction in multiple pathways withvarious components 27- 30). The anti-glycative effect of the KP
extract may have the same effect as the mixed herb extract
because it suppressed the formation of many types of AGEs
with different pathways.
The hydrous ethanol extract of KP has an inhibitory
effect on the formation of fluorescent AGEs, and PMF has
been reported as an active ingredient 18). For the purpose ofanalyzing the functional components responsible for the anti-

AGE crosslink cleavage action
The AGE crosslink cleavage action was found to beconcentration-dependent at KP extract concentrations of 0.13
to 3.94 mg/mL ( Fig. 3 ). The cleavage rate at the KP extract
concentration of 3.94 mg/mL was 6.81%, which was 0.27
times that of the 5 mmol/mL PTB (cleavage rate 25. 5 %), a
positive control.

OPH activity enhancing effect
The OPH activity enhancing effect was 203.3 % at a KP
extract concentration of 0.315 mg/mL ( Table 2 ). This effect
was 0.97 times that of 0.04 mg/mL Ru (acting rate 210.2 %),a positive control, and 11. 5 times that of 0.04 mg/mL EGCg
(acting rate 17.7 %), a negative control.

Discussion

Anti-glycative effect by KP
The formation inhibitory rate of fluorescent AGEs,
pentosidine, CML, 3DG, GO, and MGO were measured forthe purpose of verifying the anti-glycative effect of a hot

Percent breakage (%)

30

*

25
20
15
10

*

5
0

0.13

0.39

1.31

*

3.94

PTB

KP

Concentration (mg/mL)

Fig. 3. AGE cross-link breaking activity.

Results are expressed as mean ± standard deviation, n = 3, * p < 0.05 by Tukey’ s test. KP,
Kaempferia parviflora rhizome extract; PTB, 5 mmol/L N-phenacylthiazolium bromide.

Table 2. OPH enhance activity.
SampleKP

Rubusoside (positive control)
EGCg (negative control)
Ref (water)

Concentration(mg/mL)

Percent enhancement(%)

0. 315203.3 ± 19. 4 *0.040

210.2 ± 27. 2 *0.040

17.7 ± 3. 2 *-100

Results are expressed as mean ± standard deviation, n = 3, * p < 0.05 by Tukey’ s test. KP, Kaempferia parviflora
rhizome extract; EGCg, epigallocatechin gallate; OPH, oxidized protein hydrolase; Ref, reference,_ 5 _Anti-glycative Effect of Black Galangal

glycative effect, we compared formation inhibitory actions
on fluorescent AGEs from ten types of rhizome samples of
KP and verified the relationship with PMF contained in KP 20).
As a result, the formation inhibitory effect on fluorescent
AGEs was stronger in the hot water extract having a low
PMF content than in the 70 % ethanol extract containing alarge amount of PMF. In this study, the formation inhibitory
effects by the KP hot water extract showed not only on
fluorescent AGEs but also on pentosidine, CML, 3DG, GO,
and MGO. The anti-glycative effect of KP may involve morewater-soluble components than hydrophobic components
such as PMF.

on the site where the material is used and is stronger inroots and seeds than in leaves. The KP used in this study isrhizome and may be associated with the site of action of theherb. OPH is an in vivo enzyme present in the liver, brain,
and blood. From these facts, KP may be involved in the
degradation of aged proteins including glycation in the body.

Conclusion
For the purpose of verifying the potential of KP as ananti-glycative material, the anti-glycative effect and AGE
decomposition action of an 80 °C hot water extract of KP
rhizome dry powder were verified. The KP extract has
been shown to inhibit the formation of fluorescent AGEs,
pentosidine, CML and intermediates, 3DG, GO and MGO.
In addition, both an AGE crosslink cleavage action andan OPH activity enhancing action were found in the AGE
degrading process by the KP extract. It is suggested that
KP inhibited the formation of many types of AGEs withdifferent pathways, and decomposed AGEs, thus acting as
an anti-glycative material to reduce the amount of AGEs
accumulated in the body.

AGE degradation action by KP
The AGE crosslink cleavage rate and the OPH activity
enhancing rate were measured for the purpose of verifying
the AGE degradation action by a KP rhizome powder hot
water extract. As a result, the KP extract was found to have
an AGE crosslink cleavage action and an OPH activity
enhancing action.
It has been shown that the PTB used for the positive
control in this study degradate PPD to produce benzoic
acid, which may cleave AGE-modified protein crosslinks 9, 31).
PTB has also been shown to reduce AGEs in rat arterial
walls 32). AGE crosslink cleavage actions have been reported
in various plant components 11-13). KP extracts that act similarly
to these may contain similar agents. On the other hand, in areport using the PTB-administered rat model, a reduction in
the AGE crosslink of tail collagen has not been observed 33).
Further verification is required for the possibility of AGE
decomposition by the components verified by the AGE
crosslink cleavage model.
OPH has long been known as an in vivo enzyme having
the action of degrading and releasing the N- terminal
modifying group of modified proteins such as acyl, formyl,
and acetylation 34). Furthermore, OPH has shown to degrade
AGE-modified proteins 13). It is also speculated that OPH andthe proteasome act in concert with the degradation of aging
proteins 35). Many types of herbal extracts have been reported
as active ingredients that enhance the OPH activity 14). The
OPH activity enhancing effect by herbs differs depending

Conflict of interest declaration
In carrying out this research, we received support from
Rene Co. Ltd.

Acknowledgment
Part of this study was presented at the 16 th Meeting of
the Japanese Society of Anti-aging Medicine on June 10th,
2016, Yokohama, Japan and the 18th Meeting of the Society
for Glycative Stress Research on August 31st, 2019, Kyoto,
Japan.

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