Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : December 21, 2022
Accepted : January 16, 2023
Published online : March 31, 2023
doi:10.24659/gsr.10.1_6

Original article

Antiglycative effect of plant extract complex
Masayuki Yagi 1), Chieko Sakiyama 1), Kyoko Mori 2), Masato Ohta 2),
Kiyoshi Amami 2), 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) Cocochicosme Co., Ltd., Tokyo, Japan

Abstract

Accumulation of advanced glycation end products (AGEs) in skin tissues due to glycative stress is one of the factors that
accelerate skin aging, including decreased elasticity and loss of texture. Suppression of glycative stress is called antiglycation or glycation care. For anti-glycation of the skin, there are treatments from the inside of the body such as diet andapproaches from the outside of the body such as skin care preparations. Furthermore, anti-glycation includes the suppressionof postprandial hyperglycemia, suppression of glycation reactions, and decomposition and excretion of AGEs. Various
plant materials have been shown to have anti-glycation effects. However, the glycation reaction in the body that leads to theformation of AGEs is a complex multi-pathway reaction, and it is thought that a multi-component approach may be useful.
This study was conducted to evaluate the anti-glycation effects of five plant extract complexes (sea buckthorn fruit, Chinese
blackberry tea, tea plant leaves, loquat leaves, and rosemary leaves) that can be used in skin care formulations. To evaluate
the anti-glycation effects of plant extract complex (PEC) on skin, we examined its inhibitory effects on glycation reaction,
AGE cross-link cleavage, inhibitory formation of glycated protein cross-link, glycated protein cross-link cleavage, andantioxidant activity. As results, PEC inhibited the production of fluorescent AGEs in protein glycation models of humanserum albumin (HSA), collagen, and keratin. PEC also inhibited the production of pentosidine, N ε-(carboxymethyl)lysine(CML), 3-deoxyglucosone (3DG), and glyoxal (GO) in a keratin-glucose glycation model. Furthermore, PEC has AGE crosslink-cleavage effect, inhibits protein cross-link formation in a lysozyme-glucose glycation cross-linking model, and degrades
proteins dimerized by glycation. PECs may be useful in preventing glycation of skin by suppressing AGE accumulation inskin proteins and preventing functional deterioration caused by the formation of glycated protein cross-links.

KEY WORDS: inhibition of glycation reaction, protein cross-linking degradation, plant extract complex, skin aging

Introduction

Anti-glycation includes suppression of postprandial
hyperglycemia, suppression of glycation reaction, anddegradation and excretion of AGEs 2). Postprandial
hyperglycemia can be suppressed by taking glycolytic enzyme
inhibitors with meals or by improving dietary methods 3).
Aminoguanidine (AG) is a compound known to inhibit
glycation reaction 4, 5). However, AG cannot be added in foods
and cosmetics due to side effects such as decreased liverfunction and vitamin B6 deficiency. On the other hand, natural

Accumulation of advanced glycation end products(AGEs) in skin tissues due to glycative stress is one of
the factors that contribute to skin aging, including loss ofelasticity and texture. In addition, oxidation is a factor that
promotes glycative stress 1). Suppression of glycative stress iscalled anti-glycation or glycation care. Anti-glycation for skinincludes treatments from the inside of the body, such as dietaryintake, and treatments s from the outside, such as applicationof skin care products.

Contact 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; Sakiyama C, csakiyam@mail.doshisha.ac.jp;
Ohta M, masato.ohta@cocochicosme.com; Mori K, kyoko.mori@cocochicosme.com;
Amami K, yj@cocochi.cn; Yonei Y, yyonei@mail.doshisha.ac.jp_ 6 _Glycative Stress Research 2023; 10 (1): 6- 15(c) Society for Glycative Stress ResearchGlycative Stress Research

products including many plant materials such as tea 6),
vegetables and herbs 7), and fruits 8) have been reported toinhibit glycation reactions.
N-phenacylthiazolium bromide (PTB) is known as a
substance that cleaves AGE cross-links by glycation 9). PTB
cleaves α-diketones, a type of cross-linked structure ofglycated proteins 10). Natural products with similar effects
to PTB include extracts of such as pomegranate (Punica
granatum) 11), water chestnuts (Trapa bicornis) 12), fennel(Trigonella foenum-graecum), fenugreek (Foeniculum vulgare),hibiscus (Hibiscus sabdariffa) 13). These AGE cross-linkcleavage agents may be involved in the degradation andexcretion of AGEs. In addition, edible purple chrysanthemum(Chrysanthemum morifolium) extract has been shown to
inhibit the formation of glycated protein cross-links andcleave glycated protein cross-links as well as AGE cross-linkcleaving action 14). Plants with glycative stress inhibitory
effects include flavonoids 15) and tannins 16). These components
have antioxidant properties.
Glycation reactions leading to the formation of AGEs
in vivo are complex and multi-pathway 17). For this reason,
it is considered useful to suppress the formation of AGEs,
which have different formation pathways, by using multiple
components 18). In vitro studies of four herbal mixtures
showed that they inhibited the formation of various AGEs 19).
In addition, intake studies of herbal mixtures showed that they
inhibited the accumulation of AGEs in blood and skin 20 -22)and improved skin aging indices 23). A skin care formulation
containing mugwort (Artemisia indica) extract, which has
AGE cross-link-cleaving activity, has been shown to improveskin elasticity in clinical studies 24, 25). Thus, materials that
inhibit AGE formation and degrade AGE crosslink structures
in in vitro anti-glycation studies have the potential to reduce
the effects of glycative stress in humans as foods or skin carecosmetics.
In this study, we evaluated the anti-glycation effectsof five plant extract complexes (PECs) that can be used inskin care formulations by examining their inhibitory effects
on glycation reactions, AGE cross-link cleavage, glycatedprotein cross-link formation, glycated protein cross-linkcleavage, and antioxidant effects.

Japan), CircuLex CML/Nε -(carboxymethyl) lysine) ELISA
Kit is manufactured by MBL (Aichi, Japan). Pentosidine(pentosidine-TFA salt) was from Peptide Institute (Osaka,
Japan). 1,1-diphenyl-2-picrylhydrazyl (DPPH) was from
Cayman Chemical (Ann Arbor, MI, USA). Other reagentswere purchased from Fujifilm Wako Pure Chemical
Industries or Nakalai Tesque (Kyoto, Japan) in special or
HPLC grade.

2) Samples

The plant extract complex (PEC) was made bymixing 1, 3 butylene glycol (BG) extracts of sea buckthorn(Hippophae rhamnoides) fruit, Chinese blackberry (Rubus
suavissimus) tea, tea plants (Camellia sinensis) leaves,loquat (Eriobotrya japonica) leaves, and rosemary (Salvia
rosmarinus) leaves. The BG extract of mugwort leaves wascompared as a working control solution (RF) for known antiglycation components 24, 25). PEC and RF were diluted with
BG solution (dissolving solution) as appropriate and used as
sample solutions. The sample concentration was 100 % of
the extracted solution concentration of each sample stock
solution. These samples were provided by Cosmo Beauty(Osaka, Japan).

3) Protein-glucose glycation model

A protein-glucose glycation reaction model was used toverify the inhibitory effect of the glycation reaction, basedon a previous report 26). The composition of the reaction
solution was prepared by adding 1/10 of the sample solution
to 0.1 mol/L phosphate buffer (pH 7.4) containing protein
and glucose. The protein and glucose concentrations were
HSA 8 mg/mL and glucose 0.2 mol/L, collagen 1.2 mg/mL
and glucose 0.4 mol/L, and keratin 5 mg/mL and glucose
0.2 mol/L. For the glycation reaction, phosphate buffer,protein solution, glucose solution, and sample solution were
all added (solution A), purified water was added in place ofthe glucose solution in A (solution B), dissolving solution
was added in place of the sample solution in A (solutionC), and purified water was added in place of the glucose
solution in C (solution D) were prepared and incubated at
60 °C. Incubation time was 40 hours for HSA and 10 days forcollagen and keratin.

Materials and methods
1) Reagents

4) Measurement of AGEs

The reagents used were purchased from thefollowing manufacturers and used. Human serum
albumin (HSA, lyophilized powder, ≥ 96 %, agarose gelelectrophoresis), methylglyoxal solution (40 % in H2O; MGO),
N-phenacylthiazolium bromide (PTB) was obtained from
Sigma-Aldrich Japan (Tokyo, Japan). Collagen Type I (bovine
skin, Pepsin degradation) is supplied by Nippi (Tokyo,
Japan). Wool-derived keratin (keratin, partially sulfonated,
5 % in water) is supplied by Tokyo Chemical Industry (Tokyo,
Japan). Aminoguanidine hydrochloride (AG), glyoxal (40 %glyoxal solution; G O), 2, 3-diaminonaphthalene (DAN ),1-phenyl-1, 2 (±)- 6 -Hydroxy-2, 5,7,8-tetramethylchroman2-carboxylic acid (Trolox) from Fujifilm Wako Pure
Chemical Industries (Osaka, Japan), 3-deoxyglucosone(3DG) from Dojin Chemical Laboratory (Kumamoto,

Fluorescent AGEs (F-AGEs) were measured, according
to a previous report 26), AGE-derived fluorescence (excitation
wavelength 370 nm / fluorescence wavelength 440 nm) in
200 μL of the glycation reaction solution after incubationby using a black microplate. Pentosidine was measured by
HPLC after hydrolysis of 50 μL of the glycation reaction
mixture with 6 N HCl at 110 °C for 18 h according to aprevious report 27). CML was measured using the CircuLex
CML / Nε- (Carboxymethyl) lysine ELISA Kit after
appropriate dilution of the reaction mixture.

5) Measurement of glycation reaction intermediates

The glycation reaction intermediates were measured for
3DG, GO, and MGO according to previous reports 28, 29). After_ 7 _Antiglycative Effect of Plant Extract Complex

deproteinizing 100 μL of the glycation reaction solution withperchloric acid, DAN was added under alkaline conditions
for labeling, followed by HPLC measurement.

After completion of the reaction, the reaction solution
was centrifuged and the supernatant was subjected to
4-20 % SDS-PAGE. The gel was stained with CBB Stain
One (Nakalai Tesque, Kyoto, Japan), the electrophoretic gel
images were imaged, and the intensities of the lysozyme
dimer (28.6 kDa) and trimer (42.9 kDa) bands were analyzed
with Image J. AG was used as a positive control substancefor inhibition of glycated protein cross-link formation. The
inhibition rate of glycated protein cross-link formation was
calculated based on the following formula.

6) Evaluation of the inhibitory effect on
glycation reaction

As an evaluation of the inhibitory effect on glycation
reaction, the inhibition rate (%) of production of AGEs
and glycation reaction intermediates was calculated by thefollowing equation according to a previous report 26),. The
positive control for inhibition of glycation reaction was AG.

Percentage inhibition of glycated protein cross-linkformation (%) = {1 − (A − B) / (C − D)} × 100

Inhibition (%) = {1 − (A − B) / (C − D) } × 100

A-D; band intensity of lysozyme dimer or trimer in SDSPAGE gel stained images of each reaction solution

The half maximal inhibitory concentration ( IC 50 ;mg/mL) was calculated from the inhibition rates of thethree concentrations for each sample 26, 30). A smaller IC 50
value indicates a stronger inhibitory effect on the glycation
reaction.

9) Evaluation of cleavage of glycated-protein
cross-link

The cleavage of glycated-protein cross-link (CGPC) was
measured by referring to previous reports 14, 33), glycatedlysozyme was prepared, and the degradation rate of lysozyme
dimer by the sample was measured. Glycated lysozyme wasprepared by reacting lysozyme with 0.1 mol/L phosphatebuffer (pH 7.4) containing glucose at 60 °C for 40 hours,
followed by ultrafiltration to remove low molecular weightsubstances. Reaction solutions were prepared by adding
sample solution in 0.05 mol/L phosphate buffer containing
glycated lysozyme (solution S), and dissolving solution inplace of sample solution in S (solution R), and incubated at
37 °C for 16 hours to react. The reaction solution was thencentrifuged and the supernatant was subjected to 4-20 %SDS-PAGE. After gel electrophoresis, gels were stainedwith CBB Stain One and imaged with a scanner GT- X830
(Epson, Suwa, Nagano, Japan), and the intensity of lysozyme
monomer (M) and dimer (D) bands were analyzed with
Image J. P TB was used as a positive control substance for
CGPC. The glycated protein cross-link cleavage rate wascalculated based on the following equation.

7) Evaluation of cleavage of AGE cross-linkings

The cleavage of AGE cross-link was measured according
to a previous report 31-33). PPD was used as a model substancefor AGE cross-linking, and the cleavage rate of α-diketoneof PPD by the sample was measured. The mixture solutionof sample solution, PPD, and phosphate buffer (pH 7.4) was
reacted at 37 °C for 8 hours, and after the reaction was stopped
by adding hydrochloric acid. The α-diketone of PPD was
cleaved by the sample, and the amount of free benzoic acidwas measured by HPLC under the same conditions aspreviously reported 32). The AGE cross-link cleavage rate wascalculated using the equation shown below, based on theprinciple that "when the α-diketone of a PPD molecule iscleaved, 1 mol of PPD yields 1 mol of benzoic acid".
AGE cross-link cleavage rate (%) = { (A − B − R) / C} × 100
A; Amount of benzoic acid in the reaction solution,
B; Amount of benzoic acid in the sample,
R; Amount of benzoic acid in the reaction solution when
only dissolving solution was added,
C; amount of PPD used in the reaction

Glycated-protein cross-link cleavage rate (%)= [1 − {(D S / MS ) / (DR / MR )}] × 100
S ; Band intensity of lysozyme in sample solution
R ; Band intensity of lysozyme in sample dissolving solution
M; Monomer band of lysozyme
D ; Dimer band of lysozyme8) Inhibitory actions on glycation-induced
protein cross-linking

The glycation-induced lysozyme-glucose cross-linking
model was used for evaluation of the inhibitory actions ofglycation-induced protein cross-link formation according toa previous report 14, 34, 35). The reaction solution was preparedby adding the sample solution to 0.1 mol/L phosphate buffer(pH 7.4) containing lysozyme and glucose. Reaction solutions
were prepared by adding all of the phosphate buffer, lysozyme
solution, glucose solution, and sample solution (solution A),
solution with purified water added in place of the glucose
solution in A (solution B), solution with dissolving solution
added in place of the sample solution in A (solution C),
solution with purified water added in place of the glucose
solution in C (solution D) were prepared and incubated at
60 °C for 40 hours.

10) Evaluation of antioxidant activity

The DPPH radical scavenging activity corresponding
to the sample solution was measured using the slope of theregression line generated by Trolox, referring to previous
reports 36, 37). DPPH radical scavenging activity was measured,by adding sample extracts, MES buffer ( pH 6.0), DPPH
solution, and 50 % ethanol solution into the wells of a
microplate, followed by a 20-minute reaction at room
temperature, absorbance at 520 nm.
The slope of the linear regression line between theamount of sample extract added and absorbance was thendetermined. The DPPH radical scavenging activity value_ 8 _Glycative Stress Research

was calculated as the Trolox equivalent (TE) of the sample
extract by dividing the slope of the linear regression lineof the absorbance, obtained from the reaction solution of
Trolox (a water-soluble analog of vitamin E) that was reacted
concurrently with the sample, by the slope of the regression
line of the sample extract.

collagen (0.175 %), followed by HSA (0.290 %) and keratin(2.92 %, Table 1 ). Similarly, the IC 50 for RF was lowest for
HSA (0.826 %), followed by collagen (1. 557 %) and keratin(greater than 2.0%). The IC 50 of PEC inhibition of F-AGE
formation was 0.4-fold for HSA, 0. 5-fold for collagen, andless than 0.4 - fold for keratin compared to RF. The IC 50
values of PEC were lower than those of RF for all model
proteins.

Statistical analysis

Measurements are expressed as mean ± standard
deviation (SD). Tukey's test or t-test was used to compare
measurements. Statistical analysis results were consideredsignificant at a risk rate of less than 5%.

Inhibition of AGEs and glycation intermediates
formation in the keratin-glucose glycation
reaction model

A keratin-glucose glycation reaction model was used toverify the inhibitory effects of PEC and RF on pentosidine,
CML, 3DG, GO, and MGO formation ( Table 2 ). The IC 50
for PEC was lowest for CML and GO (both <1%), followedby MGO (1.9 %), 3D G (4.7 %), and pentosidine (9.6 %).
Similarly, IC 50 values for RF were lowest for CML (0.9 %)and highest for GO (1.2 %), MGO (4.1%), and pentosidine(8.8 %), in that order. IC 50 values could not be calculated
due to little inhibition rate of formation of MGO for PEC
and 3DG for RF. IC 50 values for AG could not be calculated
due to small effect except for GO (0.1%).

Results

Inhibition of fluorescent AGE formation inprotein-glucose glycated reaction models

PEC and RF inhibition rate of F-AGE formation increased
with sample concentration in all three reaction models using
HSA, collagen, and keratin as model proteins (Fig. 1 ). The
IC 50 of PEC inhibition of F-AGE formation was lowest for

100

80

80

inhibition ratio (%)

inhibition ratio (%)

A
100

60
40
20
0

0.1

0.3

1.0

PEC

RF
concentration (%)

0.2

0.7

inhibition ratio (%)

100

2.0

60
40
20
0

0.01

B

0.1

AG

0.3

1.0

PEC

RF
concentration (%)

0.2

0.7

2.0

0.01

AG

C

80
60
40
20
0

1.0

3.0

PEC

10.0

0.2

0.7

RF
concentration (%)

2.0

0.01

AG

Fig. 1. Inhibitory activity of F-AGE formation.

A, HSA-glucose model; B, collagen-glucose model; C, keratin-glucose model. Results are expressed as mean ± standard deviation, n = 3, * p <0.05 vs different concentration samples by Tukey’s test; PEC, plant extract complex; RF, Artemisia indica Willd. var. maximowiczii leaf extract,
AG, aminoguanidine; F-AGE, fluorescent advanced glycation end product; HSA, human serum albumin.

_ 9 _Antiglycative Effect of Plant Extract Complex

Table 1. Half maximal inhibitory concentration (IC50) on the protein-glucose glycation model.
Model protein

PEC 1) (%)

RF 2) (%)

AG 3) (%)

HSA

0.290

0.826

0.006

keratin

2.92

2.0 <

collagen

0.175

1.557

0.049
0.0 071) plant extract complex, 2) Artemisia indica Willd. var. maximowiczii leaf extract, 3) aminoguanidine; positive control ofglycation inhibitor. HSA, human serum albumin.

Table 2. Half maximal inhibitory concentration (IC50) on the keratin-glucose glycation model.
AGE compound

PEC 1) (%)

RF 2) (%)

AG 3) (%)

pentosidine

9.6

8. 8

NC 8)

4.7

NC

NC

1. 9

4.1

CML
3DG

4)

1.0 >

5)

GO 6)

MGO

1. 0 >

7)

NC

0. 9

0.1

1. 2

NC

1) plant extract complex, 2) Artemisia indica Willd. var. maximowiczii leaf extract, 3) aminoguanidine; positive control ofglycation inhibitor, 4) N ε - carboxymethyl lysine, 5) 3-deoxyglucosone, 6) glyoxal, 7) methylglyoxal, 8) can't calculation.
70*

cleavage ratio (%)

60

*

50
40
30
20
10
0

0.15

0.5

1.5

5.0

1.5

PEC

5.0

13.5

RF

45.0

0.14

PTB

concentration (%)

Fig. 2. AGE cross-link cleavage activity.

Results are expressed as mean ± standard deviation, n = 3; * p < 0.05 vs different concentration samples by
Tukey’s test; PEC, plant extract complex; AGE, advanced glycation end product; RF, Artemisia indica Willd.
var. maximowiczii leaf extract, PTB, N-phenacylthiazolium bromide.

AGE cross-link cleavage

The AGE cross-link cleavage rates of PEC and
RF increased with sample concentration. At a sample
concentration of 1.5 %, the AGE cross-linking cleavage
rate of PEC (16.3 ± 0.4 %) was 7.8-fold higher than thatof RF ( 2.1 ± 0.2 %, p < 0.05, Fig. 2 ).

Inhibition of glycated protein cross-link formation

The inhibition rates of lysozyme trimer and dimer
formation by PEC, RF, and AG increased with sample
concentration ( Table 3 ). The IC 50 values for inhibition ofglycated protein cross-link formation were lowest for AG

for both the dimer and trimer, followed by PEC and RF.
The inhibition rate for dimer formation at a sample
concentration of 0. 3 % was 2.8-fold higher for PEC (54.9 ±3. 5 %) than for RF (19.7 ± 4.3 %, p < 0.05).

Cleavage of Glycated Protein Cross-link

The glycated proteins cross-link cleavage rateincreased with the sample concentration. However, there
was no difference in the cross-link cleavage rate of RF at
sample concentrations between 0.2 ~ 2 % ( Fig. 3, 4 ). The
cross-link cleavage rate of 0. 5 % PEC was 30. 9 ± 7.8 %,and 1.7 times higher than that of 0.5 % RF (18.6 ± 3. 5 %, p
< 0.05).

_ 10 _Glycative Stress Research

Table 3. Inhibitory effect of PEC, RF and aminoguanidine on cross-linking formation in the lysozyme-glucose reaction model.
sample

PEC

1)

RF 2)

AG 3)

dimer

conc.
(%)

inhibition ratio (%)

0.1

43.3 ± 4.1

0.3

5 4.9 ± 3.5

1.0

6 8.7 ± 2.0

0.1-1.6 ± 3.7

0.3

19.7 ± 4.3

1.0

41.2 ± 3.7

0.01

25.7 ± 6.8

0.03

42.4 ± 3.8

0.1

58.9 ± 8.3

trimerinhibition ratio (%)

IC 50 (%)

IC 50 (%)

88.9 ± 0.8

0.1 >89.2 ± 3.3

0.19

*

86.3 ± 1.1
19.5 ± 2.8
66.8 ± 3.1

1.6*

84.6 ± 4.0
79.5 ± 3.8
92.9 ± 1.4

0.053

*

0. 24

*

*

98.6 ± 1.2*

0.01 >

Results are expressed as mean ± standard deviation, n = 3; * p < 0.05 vs different concentration samples by Tukey’s test; IC 50, half maximal inhibitory
concentration. 1) plant extract complex, 2) Artemisia indica Willd. var. maximowiczii leaf extract, 3) aminoguanidine; positive control of glycation inhibitor.
IC 50, half maximal inhibitory concentration.

cleavage ratio (%)

100

*

*

80
60
40
20
0

0.5

1.5

5

0.5

PEC

1.5

5

RF

0.14

PTB

concentration (%)

Fig. 3. Cleavage of glycated-protein cross-link activity.

Results are expressed as mean ± standard deviation, n = 3; * p < 0.05 vs different concentration samples by
Tukey’s test; PEC, plant extract complex; RF, Artemisia indica Willd. var. maximowiczii leaf extract, P TB,
N-phenacylthiazolium bromide.

MW 0

PEC

0.5

1.5

5 (%)

MW 0

RF

0.5

1.5

5 (%)

lysozyme dimer

25 kDa15 kDalysozyme monomer

Fig. 4. CGPC of PEC and RF on lysozyme dimer in the glycated lysozyme.

Sample and 0.5 mg/mL glycated lysozyme were incubated at 37℃ for 16 hours; SDS-PAGE was conducted using 2-40 % acrylamide gels.
Stained with CBB stain one; CGPC, cleavage of glycated-protein cross-linking; MW, molecular weight markers; BG, butylene glycol; PEC,
plant extract complex; RF, Artemisia indica Willd. var. maximowiczii leaf extract; 0 %, incubation without sample (with 2.5% BG).

_ 11 _

Antiglycative Effect of Plant Extract Complex

Antioxidant activity
The DPPH radical scavenging activity was 13.0-foldhigher in PEC (26.0 ± 3.8 mmol-TE/L) than in RF (2.0 ±0.3 mmol-TE/L, p < 0.05, Table 4 ).
Table 4. Antioxidative activity of PEC and RF.
sampleDPPH radical scavenging activity(mmol-TE/L)PEC 1)

26.0 ± 3.8RF2)2.0 ± 0.3*

Results are expressed as mean ± standard deviation, n = 3; * p < 0.05 byt test; IC 50 , half maximal inhibitory concentration. 1) plant extract
complex, 2) Artemisia indica Willd. var. maximowiczii leaf extract.
DPPH, 1,1-diphenyl-2-picrylhydrazyl.

Discussion

Inhibitory actions of AGE formation andprotein-crosslinking

PEC, RF, and AG were found to inhibit glycation
reactions. PEC is a plant extract complex of sea buckthorn
fruit, Chinese blackberry tea, tea plants leaves, loquat leaves,and rosemary leaves. The 70 % ethanol extract of the leaves
and fruits of sea buckthorn has an inhibitory effect on
AGE formation in a bovine serum albumin (BSA)-fructose
glycation reaction model 38). In addition, the fruits of sea
buckthorn contain carotenoids, tocopherols, flavonoids, andtannins, which have strong antioxidant effects 39).
Hot water extract of Chinese blackberry has an
inhibitory effect on AGE formation in a collagen-glucose
glycation reaction model 40). In addition, Chinese blackberry
tea contains ellagitannins 41) and has anti-allergic 42) andantibacterial 43) effects. Tea leaves contain catechins 45).
Catechins have been shown to have antioxidant effects 44) andinhibitory effects on glycation reactions 40, 46). loquat leaves
have been shown to inhibit glycation 40). Furthermore, corosolic
acid contained in loquat leaves has antioxidant 47) and antiinflammatory 48) effects. Rosemary contains rosmarinic acid,
which has antioxidant 49) and anti-inflammatory 50) effects.
Since PEC is a complex, the action of each plant component
may act integrally. The taxonomic families of the extract
plants that make up the PEC are diverse, with sea buckthorns
belonging to the Elaeagnaceae family, Chinese black tea andloquats belonging to the Rosaceae family, tea plants belonging
to the Theaceae family, and rosemary belonging to theLamiaceae family. In plants, the characteristic componentsof each family are included 51).
Therefore, PECs may contain multiple phytoconstituentsthat are not present in a single plant. The glycation reaction
system leading to the AGE formation is a complex multipathway 17). Therefore, multi-component suppression isconsidered useful for suppressing the formation of AGEs withdifferent pathways 18). In the present study, PEC showedstronger inhibition of AGE formation, especially of fluorescent
AGEs (Table 2), CML, 3DG, GO, and MGO (Table 3 ), in the

keratin-glucose glycation model than RF from a single plant.
PEC, RF, and AG all showed inhibitory effects on
glycated protein cross-link formation. AGEs include
pentosidine, crossline, and glucosepane, which have crosslinking properties. When they accumulate in proteins, they
form cross-linked proteins. Some AGEs have fluorescent
properties, such as pentosidine 52), crossline 53), andpyrropyridine 54). The fluorescent and crosslinking properties
of AGEs are independent characteristics, with each AGE
having both or either. In this study, the inhibitory effectof samples on the formation of dimers and trimers in thelysozyme-glucose glycation reaction model was verified.
The inhibitory effect of PEC on glycated protein cross-linkformation was stronger than that of RF. However, the IC 50
value of pentosidine, a type of cross-linking AGEs, was 1.1
times higher with PEC than with RF, which was almost samelevel. In addition, PEC was 2.8 times stronger than RF in
dimer formation inhibition. The difference inhibition effectof dimer formation between PEC and RF suggests that PEC
may inhibit the formation of cross-linked AGEs other thanpentosidine.
Mugwort, used as a control (RF ) for the anti-glycation
component, is a plant of the Asteraceae family. An 80 %ethanol extract of mugwort has been reported to remove
AGEs generated in collagen gel and to improve skinelasticity by application of a formulation containing mugwort
extract 25). Although the evaluation of the anti-glycation
effect in this study was only an in vitro study, PEC generally
showed stronger effect than RF. The anti-glycation effect of
mixed herbal extracts consisting of Dokudami (Houttuynia
cordata) aerial parts, hawthorn (Crataegus laevigata) fruit,
Roman chamomile (Chamaemelum nobile) flowers and grape(Vitis vinifera) leaves was verified both in vitro and in clinical
trials 19). Ingestion studies of mixed herbal extracts haveshown to inhibit the accumulation of AGEs in the blood 20),
improve skin elasticity 21), inhibit the accumulation of stratum
corneum AGEs 22), and improve dullness 23). Mixed herbal
extracts are thought to inhibit the formation of a varietyof AGEs, with four taxonomically distinct plant species
inhibiting multiple AGE formation pathways 19). PEC
complexes are mixed from the same perspective as mixed
herbal extracts and may have similar effects when verified inclinical trials on human subjects.

Cleavage actions against AGE cross-linking andglycated protein cross-linking

PEC and RF showed cleavage effects of AGE crosslinking and glycated protein cross-linking. However, theglycated protein cross-link-cleaving effect of RF showedcleavage rates of less than 20 % in the range of 0. 5 ~ 5 %sample concentration, and the cleavage rate was independentof sample concentration. The cleavage activity of AGE
cross-linking indicates the cleavage against α-diketone
among protein cross-links caused by glycation 10). On theother hand, the mechanism of glycated protein cross-linkcleavage remains to be elucidated. In addition to α-diketones,
glucospan and lysine-dihydropyridinium-lysine have beenreported to be involved in the formation of protein crosslinks during glycation 10). In a study of the AGE cross-linkcleaving and glycated protein cross-link-cleaving effectsof 12 substances contained in herbs, no correlation was_ 12 _Glycative Stress Researchobserved between the two cleavage effects 33). The threeflavonoids with AGE cross-link- cleaving activity did notshow any protein cross-link- cleaving activity 33). These results
indicate that there is no single cross-link structure in theprotein molecule cross-linked by glycation. In addition, asingle substance involved in cross-link cleavage may not
sufficiently cleave the glycated protein cross-links. Based onthese results, we speculate that the difference in the action of
PEC and RF is due to the action of multiple components inthe complex, PEC, other than α-diketones.
The number and intramolecular position of gallate
groups attached to flavonoid and tannin molecules are
involved in the AGE cross-link cleavage activity of plantcomponents, and the AGE cross-link cleavage activity of
substances with trihydroxybenzene structure, the backboneof ellagitannins, is particularly strong 32).
In this study, glycated lysozyme, which was preparedby the lysozyme-glucose glycated reaction model, was used
for the glycated protein cross-link cleavage action. The
dimer formation rate of the glycated lysozyme was about
12 % (results not shown), but the type and number of crosslinked structures were unknown. In order to elucidate themechanism of the cross-link- cleavage action of glycatedproteins, it is necessary to identify the cross-link structuresin the glycated proteins and to verify the molecular structurescommon to substances having the cross-link-cleavage action.

Research limitations

Potential for prevention of skin aging

Declaration of Conflict of Interest

Proteins in living organisms are constantly exposed
to glucose, which exists as a source of cellular energy, andare therefore at risk of glycation 1). In addition, the AGE
formation proceeds due to the progress of glycation reactions
and the formation of aldehydes such as 3DG, GO, and MGO
associated with the oxidation of glucose 55). Furthermore,skin tissues are affected by radical formation due toultraviolet rays in sunlight and oxidation by substances thatcome in contact with the skin. Oxidation is an accelerator of
AGE formation through glycation. Therefore, inhibition ofglycation, degradation of AGE cross-links, and suppressionof oxidation of skin tissues are important to prevent theaccumulation of AGEs.
AGE accumulation in skin tissues has been observedin the dermis 56), epidermis 57, 58), and stratum corneum 59).
Glycation of the dermis forms cross -linked structures incollagen and elastin, reducing skin elasticity 58). Glycation of
the stratum corneum disrupts skin texture and accelerates
the appearance of aging 60). The main protein of the stratum
corneum is keratin. In this study, PEC was found to inhibit
the production of various AGEs on the glycation of keratin
and the formation of AGE cross-links. These effects indicate
the possibility of suppressing skin aging by applying PEC. In
addition, the AGE cross-linking and glycated protein crosslinking cleavage effects of PEC have shown the possibilityof being involved in the prevention and improvement of skintissue elasticity loss. Furthermore, the antioxidant effectinhibits the progression of glycation reactions. These results
suggest that the use of skin care formulations containing
PEC on skin has potential to act prevention of aging andimprovement the functionality of skin tissue.

The plant extracts comprising PEC have already beenused as raw materials for cosmetics and are considered tobe safe for use in skin care products. On the other hand, theeffects of PEC obtained in this study were verified in in
vitro studies, and the usefulness of PEC for the prevention of
aging in skin tissues needs to be verified in clinical studies
on humans.

Conclusion
Plant extract complex (PEC) of sea buckthorn fruit,
Chinese blackberry tea, tea plant leaves, loquat leaves, androsemary leaves inhibited the formation of fluorescent AGEs
in protein glycation models of HSA, collagen, and keratin.
PEC also inhibited the formation of pentosidine, CML,
3DG, GO, and MGO in a keratin-glucose glycation model.
Furthermore, PEC inhibited glycated protein cross-linking,cleaved AGE cross-links, and cleaved glycated protein crosslinks, suggesting that PEC may be useful in preventing skinaging by suppressing AGE accumulation in skin proteins andpreventing protein cross-linking due to glycation.

Cocochicosme Co. Ltd. provided research funding
support for this study and Cosmo Beauty Co. Ltd. provided
samples.

Acknowledgments
Support for the publication of this study was provided
by the Isyoku-Dogen Syoyaku Research Foundation.

_ 13 _

Antiglycative Effect of Plant Extract Complex

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