Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : September 16, 2016
Accepted : November 1, 2016
Published online : December 31, 2016

Original Article

Anti-glycative effect of palladium and platinum nanoparticle solution
Wakako Takabe 1), Masayuki Yagi 1), Masamitsu Ichihashi 1, 2), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center and Glycation Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan2) Saisei Mirai Clinic Kobe, Kobe, Japan

Abstract

Aim: Glycation, the non-enzymatic reaction between proteins and reducing sugars, generates advanced glycation end products(AGEs), and accumulation of AGEs is exceeded in diabetic patients. Oxidation is one of the critical steps in the process of
glycation. In the present study, we investigated the effect of PAPLAL (palladium and platinum nanoparticles), which is known
to be a potent antioxidant on glycation.
Methods: AGEs were formed by using three different combinations of proteins and sugars. Human serum albumin (HSA)
and glucose were incubated at 60 °C for 40 hours. Both type I collagen and type II collagen were mixed with fructose andwere incubated at 60 °C for 24 hours. Fluorescent AGEs were measured by their typical fluorescence of 370/440 nm. Nonfluorescent AGEs, N ε -(carboxymethyl)lysine (CML) and intermediates of AGEs: 3-deoxyglucosone (3-DG), glyoxal (GO)and methylglyoxal (MGO) were determined using ELISA and HPLC-UV analyses, respectively.
Results: Palladium and platinum nanoparticles inhibited the formation of fluorescent AGEs in all three protein-sugarmodels. In the HSA-glucose model, PAPLAL inhibited CML, 3-DG, GO and MGO formation in a dose dependent manner.
Conclusions: These results suggest that the palladium and platinum nanoparticle eliminated the formation of several different
kinds of AGEs and their intermediates in vitro.

KEY WORDS: Glycation, palladium and platinum nanoparticle, fluorescent AGEs, N ε -(carboxymethyl)lysine (CML),
intermediates of AGEs

Introduction

Advanced glycation end products (AGEs) are generated
and accumulated in the body due to non-enzymatic reactions
between proteins and excess substances which have reduction
abilities, such as reducing sugar and aldehydes. Thisphenomenon is named glycation and several lines of evidence
indicated that accumulation of AGEs is associated withage-related diseases such as cancer 1), diabetes mellitus 2),
osteoporosis 3), Alzheimer’s disease 4, 5) and cardiovasculardisease 6). If not a deadly disease, when AGEs are formed ina long-lived protein such as collagen and elastin, modified
protein may cause dysfunction in the body.
N ε-(carboxymethyl)lysine (CML) is one of the wellstudied AGEs. CML is accumulated in cardiovascular tissue
in diabetic patients 7). Hwang et al. also reported that serum
levels of CML associated with diabetic neuropathy 8). In
human skin, CML is predominantly accumulated in epidermis
in aged skin, and cytokeratin 10 in stratum corneum is oneof the proteins modified by CML 9). CML also accumulated

in elastic fibers in dermis, especially sun-exposed areas 10).
Iwai et al. reported that the amount of protein carboxylationin stratum corneum correlates to loss of skin clarity 11). Thus,
prevention of AGEs formation is a beneficial for not only
lifestyle-disease prevention, but also anti-aging and healthpromotion.
In the process of AGE formation, proteins and sugars
firstly form a schiff base, they then rearrange amadori
products and intermediates of AGEs 12). Once their structures
were cleaved by oxidation, various kinds of AGEs are
generated, such as pentosidine, CML, N ω -(carboxymethyl)
arginine (CMA), N ε-(carboxyethyl)lysine (CEL), pyrralin,crossline and so on. CML is also directly formed throughautoxidation of glucose 13). Therefore, oxidation is one of thecritical steps in glycation.
Platinum (Pt) nanoparticles are recently reported asstrong and broad antioxidants which scavenge superoxides
and peroxides 14, 15). However, the surface of Pt nanoparticles
is easy to oxidize, even in the air, at room temperature

Corresponding author: Wakako Takabe, PhD
Anti-Aging Medical Research Center and Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1-3 Tatara Miyakodani, Kyotanabe City, Kyoto 610-0394, Japan
TEL& FAX: 0774-65-6382 Email: wtakabe@mail.doshisha.ac.jp
Contact for Co-authors: Yagi M, myagi@mail.doshisha.ac.jp;
Ichihashi M, mm_ichihashi@hotmail.com; Yonei Y, yyonei@mail.doshisha.ac.jp_ 222 _

Glycative Stress Research 2016; 3 (4): 222- 228(c) Society for Glycative Stress ResearchGlycative Stress Researchand that causes loss of anti-oxidative effects. Okamoto
et al. demonstrated that co-treatment of palladium (Pd)nanoparticles suppressed oxidative stress-induced deteriorationof Pt nanoparticles 16). PAPLAL, the mixture of Pt (0.2 mg/mL) and Pd (0.3 mg/mL) nanoparticles, has been used forvarious diseases associated with inflammation for many
years in Japan. However, to our knowledge, there is no reportabout its anti-glycative effect.
In the present study, we investigated the effect of
PAPLAL, which is known to be a potent antioxidant on
glycation.

Materials and Methods
Materials

Human serum albumin (HSA) was purchased from
Sigma-Aldrich (St. Louis, MO), type I collagen was obtained
from Nippi (Tokyo, Japan) and type II collagen was provided
from Elastin Products Company (Owensville, MO). CML
ELISA kit was purchased from MBL (Nagoya, Japan). Allother chemicals were obtained from Wako (Osaka, Japan) or
Dojindo (Kumamoto, Japan) for analytical grade.

Nanoparticles

Palladium and platinum nanoparticle solution, namely
PAPLAL, was supplied from Musashino Pharmaceutical Co.
(Tokyo Japan). PAPLAL is contained with 0.3 mg/mL (2.82mM) of Palladium nanoparticles and 0.2 mg/mL (1.03 mM)
of Platinum nanoparticles 17).

Preparation of glycated proteins

Three glycation models, i) HSA with glucose, ii) type
I collagen with fructose and iii) type II collagen withfructose were used. Briefly, i) 8 mg/mL HSA was mixed
with 0.2 M glucose in 50 mM phosphate buffer (PB, pH
7.4) and incubated at 60 °C for 4 0 hours, ii) and iii) 0.6
mg/mL collagen with 0.4 M fructose in 50 mM PB (pH7.4) were incubated at 60 °C for 24 hours (named “solutionA”). As a background, heated proteins without sugars werealso prepared (solution B). To determine the effects of
PAPLAL on glycation, indicated amounts of PAPLAL wereintroduced into the reaction mixture with (solution C) or
without sugars (solution D).

Measurement of AGEs-derived fluorescence

AGEs-derived fluorescence was measured as previously
described 18). Briefly, 200 μL of the reaction mixture was used
to measure fluorescence at an excitation wavelength of 370
nm and an emission wavelength of 440 nm by a Varioscan®
Flash (Thermo scientific, Waltham, MA) microplate reader.
The value was calculated using the equation below.
Ratios of AGEs-derived fluorescence [%]= {fluorescence of (solution C – solution D) /fluorescence of(solution A-solution B)} x 100

CML measurement

CircuLex CML/N ε- (Carboxymethyl)lysine ELISA kits
were used according to manufacturer protocol to determine

the amount of CML in glycated proteins.

Measurement of intermediates of AGEs

Three kinds of intermediates of AGEs, 3-deoxyglucosone(3-DG), glyoxal (GO) and methylglyoxal (MGO), were
measured using a Shimadzu high-performance liquid
chromatography ultraviolet (HPLC-UV) system (Shimadzu
Corporation, Kyoto, Japan). Samples were prepared aspreviously described 19 ) with slight modification. Briefly,
reaction mixture were deproteinized using 6 % perchrolicacid. After centrifugation, supernatant was immediately
neutralized by excess amounts of sodium bicarbonate.
Then, 3-DG, GO and MGO were labeled with 2, 3 diaminonaphthalene (Dojindo) for 24 hours at 4 °C. The
HPLC conditions were as follows; Column, UnisonUK Phenyl, 75 mm x 3 mm I.D. column (Imtakt Corp, Kyoto,
Japan); eluent, 50 mM phosphoric acid and acetonitrile = 89
: 11. The flow rate and detection wavelengths were 1. 0 mL/
min and 268 nm.

Statistics

Data were expressed as mean ± standard deviation (SD)of at least three independent experiments. The statisticalanalyses were performed by the Student’s t-test and ananalysis of variance (ANOVA) was taken using TukeyKramer test for multiple comparisons. Differences wereconsidered significant at p values less than 0.05.

Results

PAPLAL inhibited fluorescent AGEs formation invarious glycation models.

First, we investigated that whether or not PAPLAL
affected glycation. Ten percent volume content of PAPLAL
was introduced into three different kinds of protein-sugarmodels: HSA-glucose model, type I collagen-fructose modeland type II collagen-fructose model as described in “Materials
and Methods”. Fluorescencent AGEs were measured at
370/440 nm which is the characteristic wavelength offluorescent AGEs. As shown in Fig. 1, 10 % PAPLAL
inhibited fluorescent AGEs formation significantly in all
three models. Especially, in the HSA-glucose model, ten %PAPLAL decreased glycation-derived fluorescent AGEs byover 70% ( Fig. 1A).

PAPLAL inhibited fluorescent AGEs formation ina dose-dependent manner.

As shown in Fig. 1A, 10 % PAPLAL has a drastic effectagainst fluorescent AGEs formation in the HSA-glucose
model. We further evaluated the effects of PAPLAL atlower concentrations. PAPLAL inhibited fluorescent AGEs
formation dose-dependently in the HSA-glucose model;
even 0.1% PAPLAL suppressed it significantly ( Fig. 2 ).

Effect of PAPLAL on CML formation in the HSAglucose model.

Even though 370/440 nm is a characteristic fluorescentwavelength of AGEs, there are several AGEs which do nothave fluorescence. In this experiment, we assessed the effects_ 223 _

0water

10% PAPLAL

Anti-Glycative Effect of Palladium and Platinum Nanoparticles

C. Collagen, type II with fructose

120

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100

Fluorescent AGEs[% control]

Fluorescent AGEs[% control]

A. HSA with glucose

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water

10% PAPLAL

10% PAPLAL

B. Collagen, type I with fructose

Fluorescent AGEs[% control]

120
100

Fig. 1. Effect of PAPLAL on fluorescent AGEs formationin various glycation models.

80
60

To determine the inhibitory effect of PAPLAL on fluorescent
AGEs formation, 10 % volume content of PAPLAL was used.
Fluorescent AGEs were measured at 370/440 nm. (A) HSAglucose model, (B) type I collagen-fructose model and (C) type
II collagen-fructose model. All data were shown as the mean
± SD (n = 3) of the ratios against water. ** p < 0. 01 vs. water.
AGEs, advanced glycation end products; HSA, human serum
albumin; SD, standard deviation.

＊＊

40
20
0

water

10% PAPLAL

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C. Collagen, type II with fructose
Fluorescent AGEs
% ratios of fluorescent[% control]AGEs

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[%]

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10

Fig. 2. Dose-dependent study of PAPLAL on fluorescent AGEs formation in HSA-glucose model.

Indicated percentages of PAPLAL were used to investigate the inhibitory effect of PAPLAL on fluorescent AGEs formation in HSA-glucose
model. Fluorescent AGEs were measured at 370/440 nm. All data were shown as the mean ± SD (n = 3) of the ratios against 0 % PAPLAL. **p < 0.01 vs. 0 % PAPLAL. AGEs, advanced glycation end products; HSA, human serum albumin; SD, standard deviation.

_ 22 4 _Glycative Stress Researchfunctions. Also accumulated AGEs induce inflammation intissues, leading to dysfunctions. In bone collagen, glycation
causes loss of bone stiffness and resilience and is involvedin age-related diseases such as osteoarthritis, rheumatoid
arthritis and osteoporosis 3, 20-22). Gomi reported that glycationof long-lived protein in skin tissue, such as type I collagenand elastic fibers, mediates loss of skin elasticity 23).
Accumulation of AGEs in skin are evaluated using anoninvasive autofluorescence reader. In diabetic patients,skin autofluorescence is strongly associated with cardiac
mortality 24). It also has a strong correlation between age 25)
and short sleeping hours 26) in healthy subjects. These findings
suggested that AGEs in skin tissues are not only a diagnostic
indicator but also may be a serious cause of skin aging.
In our previous study, we investigated over 500 kinds offood materials such as tea leaves, vegetables, fruits and spices
against glycation 27-31). Also, we demonstrated that the antilycative effect in herbs has a strong correlation with theirpolyphenol content 32). Because oxidation plays a critical rolein the early steps of glycation, antioxidants such as polyphenol
may affect glycation. PAPLAL, the mixture of Pt and Pd
nanoparticles is known to be a strong antioxidant and has beenused for a many years in Japan 33). In clinical trials andexperimental studies on skin disorders, topical use of
PAPLAL cream improved vitiligo subjects 34) and transdermal
PAPLAL treatment mitigated skin atrophy associated withincreased lipid peroxidation in Sod1-/- mice 17).
In the present study, we investigated the efficacy of
PAPLAL on anti-glycation by using three different proteinssuch as HSA, type I collagen and type II collagen. Serum
albumin is the most abundant protein in blood and exposedof PAPLAL on the formation of CML, one of the nonfluorescent AGEs, by using ELISA. As well as fluorescent
AGEs, PAPLAL decreased CML formation in a dose
dependent manner, while PAPLAL did not alter basal levelsof CML ( Fig. 3 ).

Effect of PAPLAL on formation of intermediate of
AGEs in the HSA-glucose model.

In the process of AGE formation, various kinds ofintermediates are produced and accumulated in the body.
In this experiment, we investigated the effects of the serial
dilution of PAPLAL on the formation of intermediates ofAGEs in the HSA-glucose model. We performed HPLC-UV
analyses to measure three different kinds of intermediates,
3-DG, GO and MGO. GO and MGO were reduced by
PAPLAL in a dose-dependent manner ( Figs. 4A, B ), while
3-DG was slightly, but significantly increased to over a 0.5% content in the reaction mixture ( Fig. 4C ). None of themwere detectable when HSA was heated without glucose.

Discussion

Glycation, the non-enzymatic reaction between reducing
sugars and proteins, forms various kinds of AGEs in humantissues and organs. Mostly, AGEs-modified proteins aredecomposed or metabolized, thus, the body can maintainphysiological homeostasis. However, when long-lived proteinsare glycated, it may disturb their metabolism and affect their

200

CML

[ng/mg-protein]

180
160
140
120
100
＊

80
60
40
20
0

PAPLAL [ % ]glucose

0

0.2

1

5

−

0

0.2

1

5

＋

Fig. 3. PAPLAL inhibited CML formation in a dose-dependent manner in HSA-glucose model.

Indicated percentage of PAPLAL were introduced into HSA-glucose model and heated at 60 °C for 4 0 hours. CML formations were determinedby ELISA. All data were shown as the mean ± SD (n = 3). * p < 0.05 vs. glucose with 0 % PAPLAL. CML, N ε- (carboxymethyl)lysine; HSA,
human serum albumin; SD, standard deviation.

_ 225 _

Anti-Glycative Effect of Palladium and Platinum NanoparticlesA% ratios of GO formation

120
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20
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0.2

0.5

5

PAPLAL [ % ]Fig. 4. PAPLAL inhibited formation of intermediate of AGEs in HSA-glucose model.

Indicated percentages of PAPLAL were introduced into HSA-glucose model and heated at 60 °C for 4 0 hours. Intermediates of AGEs weredetected by HPLC-UV. (A) GO, (B) MGO and (C) 3-DG. All data were shown as the mean ± SD (n = 3) of the ratios against 0 % PAPLAL.
** p < 0.01 vs. 0 % PAPLAL. AGEs, advanced glycation end products; HSA, human serum albumin; GO, glyoxal; MGO, methylglyoxal; 3-DG,
3-deoxyglucosone; SD, standard deviation.

_ 22 6 _Glycative Stress Research

to blood glucose constitutively, even though its half life iswithin a couple of weeks. Collagen is one of the long-life
proteins; type I collagen reside in bone and dermis and type
II collagen exist in cartilage. We demonstrated that 10 %PAPLAL inhibited fluorescent AGEs formation in all threeproteins ( Fig. 1 ). Even 0.1 % PAPLAL showed significant
suppression against HSA-glucose model ( Fig. 2 ). We
previously demonstrated that the pattern of formed AGEs
were different in each protein 30). Therefore, these results
suggest that PAPLAL is an extraordinary effective productagainst glycation-related protein disorders.
Accumulation of CML in the dermis is one of the causesof dullness 35) and loss of skin clarity 11). Even though CML
is a non-fluorescent AGEs, its amount has a correlation withskin autofluorescence AGEs 36). Also, accumulation of skin
AGEs has a correlation with glycated proteins in blood, suchas hemoglobin A1c (HbA1c) 24). In the present study, wedemonstrated that PAPLAL inhibited CML formation in
HSA-glucose model ( Fig. 3 ). This result indicates that orally
administrated PAPLAL may also be potentially effective forskin problems caused by blood glycation. CML was detectablein heated HSA in the absence of glucose, and PAPLAL
had no effect on CML amount in this reaction ( Fig. 3 ).
We considered that HSA which isolate from human blood
originally contained certain amount of CML, and PAPLAL
could not decompose already formed CML. However, further
studies are needed to discuss the effect of PAPLAL on
breakdown of AGEs.
Intermediates of AGEs such as GO, MGO and 3-DG
are highly reactive compounds to modulate proteins to form
various kinds of AGEs. In our data, PAPLAL inhibited GO(Fig. 4A) and MGO (Fig. 4B) formation. Because GO is anintermediate of CML, PAPLAL inhibited CML formation
through inhibition of GO formation. MGO is known to be anintermediate of CEL, PAPLAL may affect CEL formation,

as well. In skin collagen, Monnier et al. demonstrated that
accumulation of AGEs including CML and CEL increased
with age 37). These findings suggest that PAPLAL may havethe potential to reduce AGEs formation in skin, leading tobetter maintenance of skin health, while 3-DG was elevatedslightly, but significantly, by PAPLAL ( Fig. 4C ). Niwa et al.
reported that imidazolone, metabolite of 3-DG accumulated
in glomeruli and in the renal artery wall in an advanced stageof diabetic nephropathy in diabetes patients 38). 3-DG inhibitedmesangial cell adhesion, leading to the loss of critical cellmatrix interactions in the kidney 39). From this point of view,
an increase in 3-DG is undesirable side effect. The mechanism
by which PAPLAL induce 3-DG is still unknown, however,
elevated levels of 3-DG by PAPLAL was very little, as while
GO and MGO, other critical intermediated against diabetes 40)
were drastically inhibited. Further study is required todetermine the relevant concentration of PAPLAL fortreatment.
In conclusion, our study shows that PAPLAL, the colloidsof Pt, and Pd nanoparticles are potentially effective in themaintenance of healthy and young tissues and organs, which
are easily glycated. Even though the AGEs we investigatedin this study are limited, our present results strongly indicatethat the production of CML, reported to be strongly related
to dullness in skin, may be inhibited in human skin by
topical PAPLAL application. Further, our findings provide anovel approach for anti-aging of the skin.

Conflict of Interest Statement

The authors state that the performance of this study
entailed no issues representing a conflict of interest.

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