Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : August 15, 2023
Accepted : October 30, 2023
Published online : December 31, 2023
doi:10.24659/gsr.10.4_159

Original article

Anti-atherosclerotic and anti-glycation effects of collagen peptides
Michiya Igase 1), Seiko Koizumi 2), Ai Himeno 2), Keiji Igase 3),
Yoko Okada 4), Masayuki Ochi 4), Yasumasa Ohyagi 4)1) Department of Antiaging Medicine Ehime University Graduate School of Medicine, Ehime Japan.
2) Research and Development Center, Nitta Gelatin Inc., Osaka, Japan.
3) Department of Advanced Brain Therapy, Ehime University Graduate School of Medicine, Ehime, Japan.
4) Department of Neurology and Geriatric Medicine, Ehime University Graduate School of Medicine, Ehime, Japan.

Abstract

Collagen peptides derived from fish scales and pig skin have a molecular weight of about 1,000, which is a low molecularcollagen peptide (CP). We have conducted two RCTs and reported that CP has anti-atherosclerotic effects and improves
glucose tolerance. However, the recent finding that CP activates fibroblasts has opened the door to discussing the mechanismof action of CP from a new perspective. This paper introduces the involvement of glycative stress in the mechanism of actionof CP. In the first RCT, pulse wave velocity (baPWV) was shown to improve. The results of multivariate analysis of factors
involved in the improvement of baPWV indicated that the involvement of improvement in arterial patency, independent of
the effect on blood pressure improvement, was important. The 2nd RCT showed that CP intake improved insulin resistanceand reduced cutaneous autofluorescence (SAF), a measure of terminal glycation products (AGEs) accumulation in the skinincluding toxic AGEs (TAGEs), which are highly toxic. One possible mechanism of action is the fibroblast activating andproliferative effects of the CP-derived dipeptides Pro-Hyp (PO) and Hyp-Gly (OG). The production of matrix proteins byfibroblasts is important for maintaining vascular wall homeostasis. In particular, elastic fibers (elastin) are strongly involvedin the elasticity of arteries. The second mechanism is aldehyde trapping by CP-derived dipeptides and amino acids. Amongthe many short-chain aldehydes induced by postprandial hyperglycemia (blood glucose spikes), glyceraldehyde is the mostdangerous, generating TAGE. Aldehydes also adversely affect insulin biosynthesis in pancreatic beta cells because theyreadily cross cell membranes. From the above, it can be inferred that aldehyde trapping is of great significance. In conclusion,
since CP has anti-atherosclerotic and anti-glycation effects, it is expected to contribute to the extension of healthy life spanthrough the prevention of cerebro-cardiovascular diseases.

KEY WORDS: collagen peptide, pulse wave propagation velocity, anti-atherosclerotic effect,		advanced glycation endproducts (AGEs), AGE Reader

Introduction
Collagen constitutes about 30 % of the proteins in ourbodies. Collagen is characterized by its triple-helical, fibrousform and its molecular weight of approximately 300,000,
making it a very large protein. Since it is said that the humanbody is composed of about 20 % protein, it is calculated
that a person weighing 50 kg has 10 kg of protein, whichcontains about 3 kg of collagen. When collagen is heated
and dissolved, the triple-helical structure is loosened to form
gelatin, and when gelatin is further hydrolyzed using special
enzymes, its molecular weight is further reduced, and it isgenerally called collagen peptide. The molecular weightof collagen peptides derived from fish scales and pig skin,which have recently been used in research, is about 1000,
and these are called low molecular collagen peptides (CP). It
is also easily absorbed. It has been shown that orally ingested

CP is transferred into the blood, not only as an amino acidbut also as a dipeptide or tripeptide, which is a combinationof two or three amino acids, and that it remains in the bloodfor a relatively long time 1). CP has been reported to containhigh concentrations of dipeptides such as Pro-Hyp and
Hyp-Gly. In the field of cosmetic dermatology, they havebeen reported to be highly effective for skin beautification.
Recent studies have also reported that CP supplements have asignificant effect on the reduction of "blemishes," which havea significant impact on the "appearance" of the skin after one
month 2). Our laboratory has conducted three RCTs with CP
and reported its anti-atherosclerotic effects and improvementof skin conditions 3-5). In this paper, we introduce the novel
functional properties of CP that support these clinical resultsfrom the viewpoints of fibroblast activation and glycative
stress.

Corresponding to: Michiya Igase MD, PhD.
Department of Antiaging Medicine, Ehime University Graduate School of Medicine
454 Shitsukawa, Toon, Ehime 791- 0295, Japan.
TEL: +81-89-960-5851 e-mail: migase@m.ehime-u.ac.jp
Co-authors: Koizumi S, se-koizumi@nitta-gelatin.co.jp; Himeno A, a-himeno@nitta-gelatin.co.jp;
Igase K, kigcam0911@gmail.com; Okada Y, yokokada@m.ehime-u.ac.jp;
Ochi M, g4 01025@mail.goo.ne.jp; Ohyagi Y, ohyagiy@m.ehime-u.ac.jp_ 159 _

Glycative Stress Research 2023; 10 (4): 159-163(c) Society for Glycative Stress ResearchAnti-Atherosclerotic and Anti-Glycation Effects of Collagen Peptides

Results and Discussion

Anti-atherosclerotic effects of CP

Former RCT on the anti-arteriosclerotic effect of CP,
chicken-derived CP was administered to 58 normotensive tomildly hypertensive subjects (mean age 52.8 ± 8.6 years, 30
males) 3).
The results showed that, over the 12-week study period,
arterial progressivity, one of the indicators of arterial
stiffness as measured by pulse wave velocity of propagation(baPWV) testing, was significantly improved. An important
finding of this study is that there was a significant reduction
in systolic blood pressure and a trend toward improvement
in serum NOx, one of the indicators of vascular endothelial
function, only in the CP group after 12 weeks compared tobaseline (p < 0.1). In other words, the angiotensin-converting
enzyme inhibitory effect of chicken-derived CP, which
has been previously reported in basic research, and theaccompanying increase in NO via an increase in bradykinin 6),
may have been proven in human clinical trials. Tomosugi etal. 7) also reported a similar single-arm study in which 30
subjects (mean age 53.7 ± 7.2 years, 15 males) were treatedwith CP derived from porcine skin for 24 weeks and foundthat it improved arterial patency. The index used in this study
was the Cardio Ankle Vascular Index (CAVI) 8), which is lesssensitive to blood pressure than the aforementioned baPWV.
In this study, CAVI improved compared to baseline, andthere was a significant reduction in the L/H ratio in the highrisk group for atherosclerosis, where the initial value of theratio of low-density lipoprotein cholesterol (LDL-C) to highdensity lipoprotein cholesterol (HDL-C) (L/H ratio) was
greater than 2.5 (p = 0.025).
In the first RCT, the CP administration test was
conducted on 70 examinees (mean age 72 ± 8 years, 18 males)at the "Anti-Aging Dock" to evaluate arterial stiffness 4). The
subjects were randomly assigned to either (A) the actual
drug group: 2.5 g of CP (high dipeptide content, Pro-Hyp
and Hyp-Gly > 3000 ppm) derived from pork skin, or (B)the placebo group: 2.5 g of whey protein, using a random

number table, and 36 in the placebo (P) group. During
the observation period, one participant dropped out of thestudy after experiencing a mild diarrhea adverse event(which resolved quickly after participation in the study was
discontinued). Another participant was lost to follow-up due
to relocation, and no data were available. An additional four
participants dropped out of the study due to taste intolerance
and refusal of sustained intake. Data from 64 subjects (30 in
the CP group and 34 in the P group) were available for the
final analysis. Blood pressure measurements and baPWV
tests were performed on the subjects before and afteradministration, in addition to general blood sampling, todetermine differences between the two groups.
The results showed that neither the CP nor the placebogroup had side effects that caused problems with blood tests(Table 1). As shown in Table 2, baPWV at 12 weeks in the
CP group (30 subjects, 6 males) was significantly lower
than baseline data (p = < 0.01). In contrast, baPWV at 12
weeks in the placebo group (34 subjects, 8 males) was not
significantly different from baseline values. There was no
significant difference between the two groups with respect
to systolic blood pressure. baPWV improvement rate related
factors were examined by multiple regression analysis, andthe results showed that being in the actual drug group wasselected as a significant explanatory factor independentof age, gender, systolic blood pressure before starting, andpercentage change in systolic blood pressure, suggesting thatpork skin-derived CP has a function that resembles chickenderived CP and that it has an ameliorative effect on arterial
patency independent of its blood pressure ameliorative effect(Table 3).
One of the reasons why CP has an arteriosclerosis
ameliorative effect independent of its blood pressure
ameliorative effect may be the effects of CP-derived
dipeptides, Pro-Hyp (PO) and Hyp-Gly (OG), on fibroblasts.
The CPs used in the aforementioned studies all contain large
amounts of PO and OG. The PO and OG have been reported
to promote fibroblast proliferation 9, 10). Elastic fiber (elastin)
is strongly involved in the elasticity of arteries, and it isTable 1. Blood examination in the first RCT on CP.
CP (n = 30)
Base line
12 weeks

Placebo (n = 34)
Base line
12 weeks

0.76 ± 0.20
15.1 ± 4.0

0.78 ± 0.19
14.9 ± 4.1

0.77 ± 0.16
16.5 ± 4.3

0.79 ± 0.18
16.5 ± 4.6

66.8 ± 16.3
98 ± 5064.2 ± 14.3
101 ± 47

63.0 ± 10.4
107 ± 35

61.6 ± 11.8
105 ± 2765 ± 12113 ± 63

65 ± 11102 ± 49

59 ± 11112 ± 55

59 ± 13108 ± 53

25 ± 622 ± 922 ± 420 ± 723 ± 621 ± 1025 ± 923 ± 16g/dL

34 ± 35
13.6 ± 1.0

31 ± 32
13.5 ± 1.1

27 ± 1713.5 ± 1.3

28 ± 19
13.8 ± 1.4%

41.1 ± 2. 5

41.0 ± 3.1

40.7 ± 3.6

41.0 ± 3.9

sC r

mg/dL

BUN

mg/dL

eGFR

mL/min/1.73 m2

LDL-C

mg/dL

HDL-C

mg/dL

TG

mg/dL

GOT

mg/dL

GP T

mg/dL

γ - GTP

cm/s

Hb
Ht

Values are expressed as the mean ± standard deviation. sCr, serum creatinine; BUN, blood urea nitrogen; eGFR, estimated glomerular filtration rate;
LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride; GOT, glutamic oxaloacetic transaminase;
GPT, glutamic pyruvic transaminase; γ-GTP, γ-glutamyl trans peptidase; Hb hemoglobin; Ht, hematocrit value.

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Table 2. BP, pulse rate and baPWV in the first RCT on CP.

Systolic BP (mmHg)
CP
Placebo
Diastolic BP (mmHg)
CP
Placebo
Pulse rate (bpm)
CP
PlacebobaPWV (cm/s)
CP
PlaceboBase line

12 weeks

Change ratio (%)

129 ± 15130 ± 17125 ± 16131 ± 17

− 3.1 ± 9. 6
1.1 ± 11. 0

74 ± 1076 ± 1172 ± 1176 ± 11− 2. 4 ± 11. 7
0.6 ± 11. 9

69 ± 770 ± 869 ± 1071 ± 90. 3 ± 11. 8
1. 3 ± 7. 81709 ± 248
1670 ± 266

1611 ± 221
1646 ± 211− 5.4 ± 6. 5 *− 0.7 ± 8. 7

Values are expressed as the mean ± standard deviation. CP group, n = 30; Placebo group, n = 34, *p <0.01, comparison between
groups. BP, blood pressure; baPWV, brachial-ankle pulse wave velocity; CP, collagen peptide.

Table 3. Correlation analysis in the first RCT on CP (n = 64).
Univariate

MultivariaterP− 0.048

0. 7070.099

0. 437

Systolic BP (base line)− 0.166

baPWV (base line)− 0.462

0. 191< 0. 001

baPWV (12 weeks)

0.071

0. 578CP ingestion (+)

5.682

0.007

Age
Sex (male)βP− 0.428< 0. 001

0.279

0.013

BP, blood pressure; baPWV, brachial-ankle pulse wave velocity; CP, collagen peptide.

possible that the proliferative effect of fibroblasts induced
to proliferate by PO and OG also contributes to elastin
synthesis, thereby improving arterial elasticity.
Another possibility is the action of CP-derived dipeptides
and amino acids to trap blood aldehydes. Although 99 % of
the glucose in the blood presents a saccharide form with acyclic structure, some of it presents an open ring structurewith aldehyde groups exposed. During hyperglycemia,
especially postprandial hyperglycemia (glucose spike), thealdehyde-type glucose triggers the formation of short-chain
aldehydes such as 3-deoxyglucosone, glyoxal, methylglyoxal
and glyceraldehyde 11). Aldehydes readily cross cell membranes
and induce carbonylative modifications of peptides during
insulin biosynthesis in pancreatic beta cells, resulting in
the generation of glycated insulin and increased insulin
resistance 12). These aldehydes are highly reactive and easily
injure vascular endothelial cells, resulting in an increased
susceptibility to induce thrombus formation and an increased
risk of cardiovascular events. These aldehyde traps are
attracting attention as a strategy to protect the vascular wallfrom injury.

Anti-glycation effects of CP

Fish-derived CP has been reported to improve fastingblood glucose, HbA1c levels, and insulin resistance index(HOMA-R) in patients with type 2 diabetes 13, 14).
As mentioned earlier, postprandial hyperglycemia(glucose spike) produces carbohydrate-derived aldehydes,
which react with proteins to cause carbonylative
modifications and undergo several additional reactions to
produce advanced glycation endproducts (AGEs). Aldehydes
readily cross cellular and nuclear membranes, resulting in
the formation and accumulation of AGEs in blood vessels,
tissues, and cells. Aldehydes also promote damage not only
to proteins but also to lipids and nucleotides in the body.
Since intracellular AGEs induce ER stress and impair
cellular functions, inhibition of AGE formation may be a newmolecular target for organ protection in diabetes with respect
to prevention of aging-related and chronic diseases 15).
To date, it has not been fully clarified whether CP
affects AGEs. The second RCT aimed to confirm theefficacy of daily CP intake on AGE levels in skin andsubcutaneous blood vessels and on glucose metabolism inblood 5). A total of 30 subjects (male 8, female 22), aged 4787 (69 ± 11) years without overt diabetes, were randomly
assigned to the test food or placebo for 12 weeks. The test
food was 5 g of fish-derived CP (TYPE-S, average molecularweight = approximately 500 -1200 Da, containing 3 g/kg of
Pro-Hyp and Hyp-Gly; Nitta Gelatin Co., Osaka, Japan) andthe placebo was maltodextrin (Paindex # 2 AG; Matsutani
Chemical Industry Co, Hyogo, Japan). AGE levels were

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Anti-Atherosclerotic and Anti-Glycation Effects of Collagen Peptides

measured at the beginning and end of the study; AGEs were
evaluated from skin via skin autofluorescence using an
AGE reader (Diagno Optics Technologies BV, Groningen,
Netherlands). The results showed significantly lower AGE
levels in the CP group compared to the placebo group (Fig. 1)
and a lower insulin resistance index; HOMA-R = fasting
insulin level (μU/mL) x fasting blood glucose level (mg/dL) /405). Furthermore, a strong positive correlation was observed
between AGEs and the rate of change in HOMA-R levels inboth groups (Fig. 2). These results suggest that fish-derived

CP may reduce AGE levels and improve insulin resistance.
The aforementioned report by Tomosugi et al. 7)
also measured toxic AGEs (TAGEs) derived fromglyceraldehyde, a sugar metabolism intermediate, which isparticularly toxic among AGEs in conjunction with the antiatherosclerotic effect of CP. A significant decrease in TAGE
was observed in all subjects (p = 0.031). One of the reasonsfor the reduced TAGE formation may involve glyceraldehyde
trapping by CP-derived dipeptides and amino acids.

40.0

p < 0.05

Delta AGEs (A.U.)

30.0
20.0
10.0
0.0
- 10.0- 20.0- 30.0CP

PlaceboFig. 1. Comparison of percent change in AGEs in each group.

CP group, n = 16; Placebo group, n = 14. The two groups were compared by one-way ANOVA.
AGEs: advanced glycation endproducts, evaluated by using AGE Reader; CP: collagen peptides.

100.0
80.0

CP
Placebo

Delta HOMA-R (A.U.)

60.0
40.0
20.0
0.0
- 20.0- 40.0r = 0.542
r = 0.564- 60.0- 80.0- 100.0- 30.0- 20.0- 10.0

0.0

10.0

20.0

30.0

40.0

Delta AGEs (A.U.)

Fig. 2. Correlation between percent change in AGEs and percent change in HOMA-R in the collagen peptide (CP)and placebo (P) groups.

Black circles indicate the CP group (n = 16) and white circles the placebo group (n = 14). Spearman's rank correlation coefficients with thesolid line indicating the CP group (r = 0.542) and the dotted line indicating the placebo group (r = 0.564). Both groups showed a strong
correlation between the rate of change in AGEs and the rate of change in HOMA-R. HOMA-R: homeostasis model assessment ratio; AGEs:
advanced glycation endproducts, evaluated by using AGE Reader; CP: collagen peptide.

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Conclusion
In this paper, we introduced the possibility thatfibroblast activation and antiglycation are involved in themechanism of the anti-atherosclerotic effect of CP intake.
The detailed mechanism of the anti-glycation effect, inparticular, is still unclear in many respects. For example,
the variability in the data suggests that there are individual
differences in the production of AGEs. This may be due toindividual differences in the degree of carbohydrate-derived
aldehyde production even when blood glucose spikes of thesame magnitude occur. The trapping effect of CP-derived
amino acids is expected to be particularly effective for thosewith high levels of aldehyde production. AGEs, which were
evaluated as an anti-glycation agent, are found to increase
not only in hyperglycemia-induced glycative stress but also
in dyslipidemia as fatty acid-derived aldehydes including
methylglyoxal, as described earlier 16). The formation ofshort-chain aldehydes by fatty acid oxidation may reaffirm
the significance of antioxidants, and it will be important toanswer these questions as we search for additional benefits
from CP intake.
As Japan's population ages, the extension of healthy life
expectancy is a major theme. The anti-arteriosclerosis andanti-glycation effects of CP are expected to contribute to theextension of healthy life expectancy through the preventionof cerebrovascular diseases. In the future, the intake of CP asa food may become one of the options for extending healthy
life span. Currently, CP is generally consumed dissolvedin tea, coffee, black tea, etc. In the future, it will also benecessary to search for the optimal combination to maximize
the functional effects of CP.

Declaration of Conflict of Interest
This work was supported by Nitta Gelatin Inc., Osaka,
Japan.

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