International Journal of
Molecular Sciences
Article
Effect of a Topical Collagen Tripeptide on Antiaging and
Inhibition of Glycation of the Skin: A Pilot Study
Young In Lee 1,2,† , Sang Gyu Lee 1,† , Inhee Jung 3 , Jangmi Suk 3 , Mun-Hoe Lee 4 , Do-Un Kim 4
and Ju Hee Lee 1,2, *






Citation: Lee, Y.I.; Lee, S.G.; Jung, I.;
Suk, J.; Lee, M.-H.; Kim, D.-U.; Lee,
J.H. Effect of a Topical Collagen
Tripeptide on Antiaging and
Inhibition of Glycation of the Skin: A
Pilot Study. Int. J. Mol. Sci. 2022, 23,
1101. https://doi.org/10.3390/
ijms23031101
Academic Editors: Johann Bauer and
Ulrich Koller
Received: 21 December 2021
Accepted: 18 January 2022
Published: 20 January 2022
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Copyright: © 2022 by the authors.
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This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2022, 23, 1101. https://doi.org/10.3390/ijms23031101
1 Department of Dermatology, Cutaneous Biology Research Institute, Yonsei University College of Medicine,
Seoul 03722, Korea; ylee1124@yuhs.ac (Y.I.L.); dltkdrb5658@yuhs.ac (S.G.L.)
2 Scar Laser and Plastic Surgery Center, Yonsei Cancer Hospital, Seoul 03722, Korea
3 Global Medical Research Center, Seoul 06526, Korea; ihjung@gmrc.co.kr (I.J.); rose@gmrc.co.kr (J.S.)
4 Health Food Research and Development, NEWTREE Co., Ltd., Seoul 05604, Korea;
mhlee@inewtree.com (M.-H.L.); dkim@inewtree.com (D.-U.K.)
* Correspondence: juhee@yuhs.ac; Tel.: +82-2-2228-2080
† These authors contributed equally to this work.
Abstract: The glycation process has been recognized as one of the critical parameters that accelerate
signs of skin aging, especially in skin exposed to environment factors, such as ultraviolet radiation.
Although previous studies showed the anti-inflammatory and antiaging properties of the hydrolyzed
collagen tripeptide (CTP), its exact mechanism is not fully understood. Therefore, in this study,
we sought to investigate the effect of a topical CTP on facial skin. Our group designed a 4 week
prospective, single-arm study of 22 Asian women who applied topical CTP. We observed significant
improvements in skin wrinkles, elasticity, and density with a reduction in skin accumulation of
advanced glycated end products (AGEs) at week 4 without any adverse effects. The in vitro study
revealed a preventive effect of the topical CTP on the accumulation of AGEs, denatured collagen
production, and reactive oxygen species in dermal fibroblasts. Moreover, treatment with the CTP
decreased induction of matrix metalloproteinases while increasing the collagen 1 level. These results
suggest that the application of a topical CTP might improve clinical aging phenotypes via the
inhibition of glycation and oxidative stress, leading to a delay in cellular aging.
Keywords: hydrolyzed collagen; tripeptide; glycation; advanced glycated end products; antiaging
1. Introduction
Skin aging is the combined result of the effects from intrinsic factors and external
factors. In particular, external factors, such as ultraviolet radiation (UVR) exposure, have
shown significant impacts on skin cell aging through DNA damage, increased oxidative
stress, and the subsequent release of inflammatory mediators [1]. The consequent skin
aging process results in aging skin phenotypes, such as wrinkles, irregular pigmentation,
skin dryness, and decreased dermal and epidermal thickness [2]. As the average human
life expectancy has increased, the general population worldwide has become increasingly
interested in these skin aging outcomes, leading to the establishment of the aging-related
nutraceutical market, which is one of the largest consumer markets today [3].
The hydrolyzed collagen tripeptide (CTP) is one of the popular ingredients considered
to be an antioxidant that provides skin antiaging effects. The CTP is an enzymatically
hydrolyzed form of collagen, enabling it to be used as a food supplement and for cos-
meceutical skincare with better solubility and bioavailability along with lower allergenic
properties compared to the traditional collagen formulations [4]. Previous studies on
patients with atopic dermatitis (AD) and dry skin showed that oral and topical formu-
lations of CTPs showed anti-inflammatory properties, resulting in improvement of AD,
including skin hydration and itching [5]. Collagen hydrolysate has also been identified
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022, 23, 1101 2 of 16

as a safe cosmetic ingredient for topical formulations with good moisturizing properties
at the stratum corneum layer of the skin, thus reducing the effect of skin aging (dryness,
laxity, and wrinkles) [6]. Although those previous studies showed the anti-inflammatory
and antiaging properties of the CTP, its exact mechanism is not fully understood.
The aging process of skin is caused by DNA damage in nuclei and mitochondria,
inflammation, glycation, decreased function of keratinocytes and fibroblasts, and the break-
down of heparan sulfate, hyaluronic acid, collagen, and elastin [7]. Among these multiple
factors, glycation has been recognized as one of the critical parameters that accelerate signs
of skin aging, especially in skin exposed to environmental factors, such as UVR. Glycation
is the binding process of sugar to molecular structures, such as proteins, lipids, and nucleic
acids, during which advanced glycated end products (AGEs) are formed. During the skin
aging process, collagens, which are major components of the connective tissue that account
for 30% of the human protein component, are modified by mineralization, the accumulation
of AGEs, and depletion of glycosaminoglycans; these processes affect fiber stability and the
skin’s susceptibility to matrix metalloproteinase (MMP)-mediated degradation [8].
AGEs, through the promotion of oxidative stress, activate several stress-induced
transcription factors, with the production of proinflammatory mediators [9]. Moreover,
AGEs alter the skin’s mechanical properties and, therefore, can be closely associated with
the clinical manifestation of skin aging. Therefore, in this study, we aimed to investigate
the effects of a topical CTP on antiaging of the skin via its antioxidant behavior and the
inhibition of glycation process.

2. Results
2.1. Patient Characteristics
The clinical study included 22 Asian women with noticeable periorbital and glabellar
wrinkles. Participants’ mean age was 47.1 years (range, 30–54 years). No participant
dropped out because of noncompliance. In the final analysis after 4 weeks, data from
22 participants were collected and analyzed.

2.2. Clinical Efficacy and Safety of a Topical CTP on Skin Aging and AGE Accumulation
All participants applied the Ever Collagen Corrector Collagen Tripeptide Ampoule
(CTP ampoule; NEWTREE Co., Ltd., Seoul, Korea), the commercialized cosmeceutical
product used in the clinical study, twice daily onto their facial skin for 4 weeks. The total
ingredient included in the test ampoule is listed in Table S3. After 4 weeks, the mean
periorbital skin roughness (Ra) measured by PRIMOS was significantly reduced from
20.77 ± 3.51 µm to 19.24 ± 3.52 µm (Figure 1A, p < 0.001). The mean glabellar Ra was also
significantly reduced from 21.40 ± 3.09 µm to 20.21 ± 2.83 µm (Figure 1B, p < 0.001). In
addition, the maximum periorbital wrinkle roughness (Rmax) significantly decreased from
191.11 ± 32.58 µm to 182.01 ± 33.63 µm (Figure 1C, p < 0.001). The maximum glabellar
Rmax also significantly decreased from 169.92 ± 21.57 µm to 159.76 ± 21.58 µm (Figure 1D,
p < 0.001).
Meanwhile, clinical improvement of wrinkles was also visualized by a three-dimensional
(3D) skin analysis camera system (Antera 3D® , Miravex, Dublin, Ireland). The mean
depressed depth of periorbital wrinkles at baseline was 0.06 ± 0.01 mm, which significantly
improved to 0.05 ± 0.01 mm at week 4 (Figure 2A,B, p < 0.001).
2022, 23, x FOR PEER REVIEW 3 of 16
Int. J. Mol. Sci. 2022, 23, 1101 3 of 16

Figure 1. Comparison of skin

Figure roughnessofand
1. Comparison skinmaximum
roughnessskin
and roughness
maximum skin before and after
roughness 4 weeks
before of 4 weeks
and after
applying the CTP of ampoule. Skin roughness and maximum skin roughness were reduced
applying the CTP ampoule. Skin roughness and maximum skin roughness were reduced in in re-
sponse to application of thetoCTP
response ampoule
application over
of the CTP4 weeks
ampoule (A–D);
over 4 #weeks
p < 0.001,
(A–D);Wilcoxon signed-rank
# p < 0.001, Wilcoxon signed-rank
test; * p < 0.001, paired samples t-test). The 4 week follow-up images taken with PRIMOS
test; * p < 0.001, paired samples t-test). The 4 week follow-up images taken with (PRIMOS
PRIMOS (PRIMOS
CR, SnT Lab, Seoul, CR,Korea) areSeoul,
SnT Lab, shown in (E,F).
Korea) CTP ampoule,
are shown Ever
in (E,F). CTP Collagen
ampoule, Corrector
Ever CollagenCollagen
Corrector Collagen
Tripeptide Ampoule. Tripeptide Ampoule.
Int.
Int. J.J.Mol.
Mol. Sci. 2022, 23,
Sci. 2022, 23, 1101
x FOR PEER REVIEW 44of
of16
16

Figure 2. Comparison of the depth of periorbital wrinkles before and after 4 weeks of applying the
CTP ampoule. The depth of periorbital wrinkles was reduced in response to application of the CTP
ampoule over 4 weeks (A); # p < 0.001, Wilcoxon signed-rank test). The 4 week follow-up images
taken with the Antera 3D® (Miravex, Dublin, Ireland) are shown in (B). CTP ampoule, Ever Collagen
Corrector Collagen Tripeptide Ampoule.

Within 4 weeks, the skin density measured by Ultrascan UC22 (Courage + Khazaka
electronic GmbH, Köhn, Germany) showed a significant increase from 55.66 ± 7.61 to 59.67
± 7.84 (Figure 3A,B, p < 0.001). Furthermore, the skin surface elasticity (total recovery/total
Figure2. 2. Comparison
Comparison of of the
the depth
depth of of periorbital
periorbital wrinkles
wrinkles before and and after
after 44 weeks
weeks ofofapplying
applying the the
Figure
elongation; gross elasticity (R2)) increased from 0.81before ± 0.03 to 0.83 ± 0.03 (Figure 3C, * p <
CTP ampoule. The depth of periorbital wrinkles was reduced in response
CTP ampoule. The depth of periorbital wrinkles was reduced in response to application of the CTP to application of the CTP
0.001).
ampoule Theovermaximum collagen strength (R4) after application of the topical CTP also sig-
ampoule over 44 weeks
weeks (A);
(A); ##pp<<0.001,
0.001, Wilcoxon
Wilcoxon signed-rank
signed-rank test). The
test). The 44 week
week follow-up
follow-up images
images
nificantly increased from 68.02 ± 5.48 to 70.24 ± 5.14 (Figure 3D,
taken with the Antera 3D (Miravex, Dublin, Ireland) are shown in (B). CTP ampoule, Ever Collagen
® p < 0.001). Meanwhile,
taken with the Antera 3D® (Miravex, Dublin, Ireland) are shown in (B). CTP ampoule, Ever Collagen
AGE accumulation
Corrector in the skin
Collagen Tripeptide was 2.26 ± 0.32 AU at baseline, and it was significantly
Ampoule.
Corrector Collagen Tripeptide Ampoule.
reduced to 2.16 ± 0.29 AU at week 4 (Figure 3E, p < 0.001). The summary of the overall
clinical studies
Within
Within data outcomes
44weeks,
weeks, theskin
the is listedmeasured
skindensity
density in Table S1.
measured byUltrascan
by UltrascanUC22 UC22(Courage
(Courage++ Khazaka
Khazaka
Overall, GmbH,the average
Köhn, percentage
Germany) of
showedsubjects
a who
electronic GmbH, Köhn, Germany) showed a significant increase from 55.66 ± to
electronic significant responded
increase to
from “very
55.66 satisfied”
± 7.61 7.61 and
59.67to
“satisfied”
± 7.84 (Figureto the 4
3A,B, week
p < use
0.001).of topical
Furthermore,CTP product
the skin and the
surface overall
elasticity
59.67 ± 7.84 (Figure 3A,B, p < 0.001). Furthermore, the skin surface elasticity (total re- improvement
(total in facial
recovery/total
skin problems
elongation;
covery/total grosscaused by gross
elasticity
elongation; aging atincreased
the end(R2))
(R2))elasticity of
fromthe study
0.81
increased was
± 0.03fromto95.5%
0.81±(“very
0.83 ±0.03 satisfied”:
0.03(Figure
to 0.833C,± 0.03four
*p<
subjects,
0.001). The “satisfied”:
maximum 17 subjects).
collagen One
strength subject
(R4) rated
after “slightly
application satisfied”
of
(Figure 3C, * p < 0.001). The maximum collagen strength (R4) after application of the topical the with
topical respect
CTP also to the
sig-
test product
nificantly and
increased the overall
from improvement
68.02 ± 5.48 to of
70.24 the± facial
5.14 skin.
(Figure
CTP also significantly increased from 68.02 ± 5.48 to 70.24 ± 5.14 (Figure 3D, p < 0.001).No3D,adverse
p < events
0.001). were
Meanwhile, ob-
served
Meanwhile, during
AGE accumulation AGE the accumulation
4 in
week the study
skin was period,
in the2.26 and
± 0.32
skin none
was AU of at
2.26 the participants
±baseline,
0.32 AU and dropped
it outitofwas
was significantly
at baseline, and the
study
reducedbecause
to 2.16 of±adverse
0.29 AU events,
at week suggesting
4 (Figure that
3E, the
p <test product
0.001).
significantly reduced to 2.16 ± 0.29 AU at week 4 (Figure 3E, p < 0.001). The summary of The formulation
summary of was
the safe
overall to
use.
clinical
the studies
overall data
clinical outcomes
studies data is listed in is
outcomes Table
listedS1.in Table S1.
Overall, the average percentage of subjects who responded to “very satisfied” and
“satisfied” to the 4 week use of topical CTP product and the overall improvement in facial
skin problems caused by aging at the end of the study was 95.5% (“very satisfied”: four
subjects, “satisfied”: 17 subjects). One subject rated “slightly satisfied” with respect to the
test product and the overall improvement of the facial skin. No adverse events were ob-
served during the 4 week study period, and none of the participants dropped out of the
study because of adverse events, suggesting that the test product formulation was safe to
use.

Figure 3. Comparison of skin density, skin surface elasticity, skin collagen strength, and the AGE
measurement before and after 4 weeks of applying the CTP ampoule. Skin density, skin surface
elasticity, skin collagen strength, and the AGE measurement were increased in response to applying
the CTP ampoule over 4 weeks (B–E); # p < 0.001, Wilcoxon signed-rank test; * p < 0.001, paired
samples t-test. The 4 week follow-up images taken with the Ultrascan UC22 (Courage + Khazaka
electronic GmbH, Köhn, Germany) are shown in (A). CTP ampoule, Ever Collagen Corrector Collagen
Tripeptide Ampoule; AGE, advanced glycated end product.
Int. J. Mol. Sci. 2022, 23, 1101 5 of 16

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 16

Overall, the average percentage of subjects who responded to “very satisfied” and “sat-
isfied” to the 4 week use of topical CTP product and the overall improvement in facial skin
Figure 3. Comparison of skin density, skin surface elasticity, skin collagen strength, and the AGE
problems
measurement caused byand
before aging
afterat4the endofofapplying
weeks the study thewas
CTP95.5% (“very
ampoule. Skinsatisfied”: four
density, skin subjects,
surface
“satisfied”: 17 subjects). One subject rated “slightly satisfied” with respect to the
elasticity, skin collagen strength, and the AGE measurement were increased in response to applying test product
and the overall
the CTP ampouleimprovement
over 4 weeks of the #facial
(B–E); skin. Wilcoxon
p < 0.001, No adverse events were
signed-rank test; *observed
p < 0.001,during
paired the
4samples
week study
t-test).period, and none
The 4 week of the
follow-up participants
images dropped
taken with out of the
the Ultrascan UC22 study because
(Courage of adverse
+ Khazaka
electronic GmbH, Köhn, Germany) are shown in (A). CTP ampoule,
events, suggesting that the test product formulation was safe to use. Ever Collagen Corrector Colla-
gen Tripeptide Ampoule; AGE, advanced glycated end product.
2.3. In Vitro Evaluation of the Effect of EverCTP TM on Cellular Aging
2.3. In Vitro Evaluation of the Effect of EverCTPTM on Cellular Aging TM ;
As the cell viability of 1000 µg/mL of the hydrolyzed fish skin extract (EverCTP
As the cell viability of 1000 μg/mL of the hydrolyzed fish skin extract (EverCTPTM; TM
NEWTREE Co., Ltd., Seoul, Korea) was 96.88%, the investigators concluded that EverCTP
NEWTREE Co., Ltd., Seoul, Korea) was 96.88%, the investigators concluded that
had no cell cytotoxicity with treatment up to 1000 µg/mL (Figure S1).
EverCTPTM had no cell cytotoxicity with treatment up to 1000 μg/mL (Figure S1).
To investigate the effect of EverCTPTMTM on cellular aging, we performed a quantitative
To investigate the effect of EverCTP on cellular aging, we performed a quantitative
real-time reverse transcription polymerase chain reaction (qRT-PCR) to measure collagen 1,
real-time reverse transcription polymerase chain reaction (qRT-PCR) to measure collagen
matrix metallopeptidase (MMP)1, MMP3, and MMP9 messenger (m)RNA expression levels.
1, matrix metallopeptidase (MMP)1, MMP3, and MMP9 messenger (m)RNA expression
UVR exposure
levels. UVR exposurewas used
wasin thisinexperiment
used for the
this experiment forgeneration of extrinsic
the generation cellular
of extrinsic aging
cellular
on HDFs.
aging Transforming
on HDFs. growth
Transforming factor-beta
growth (TGF-β;
factor-beta Peprotech,
(TGF-β; Peprotech, Rocky
Rocky Hill,
Hill,NJ,
NJ,USA)
USA)and
retinoic
and retinoic acid (RA; Sigma Aldrich, Saint Louis, MO, USA) were used as positive controls
acid (RA; Sigma Aldrich, Saint Louis, MO, USA) were used as positive con-
for collagen
trols 1 and1MMPs,
for collagen respectively.
and MMPs, When
respectively. compared
When to the
compared to control group,
the control cellscells
group, treated
with 500 µg/mL and 1000 µg/mL of EverCTP TM showed significantly higher expressions
treated with 500 μg/mL and 1000 μg/mL of EverCTP showed significantly higher ex-
TM

of collagenof1collagen
pressions (Figure 14A, p < 0.05
(Figure 4A, and p < and
p < 0.05 0.005,
p <respectively). The expression
0.005, respectively). levels of
The expression
MMP-1, -3, and -9 reduced significantly after the treatment with 250
levels of MMP-1, -3, and -9 reduced significantly after the treatment with 250 μg/mL,µg/mL,
µg/mL, 500 500
and 1000 of EverCTP TM compared to to
thethe
UVR
μg/mL, andµg/mL
1000 μg/mL of EverCTP TM compared UVRexposure
exposuregroup. (Figure4B4B–D;
group. (Figure –
pD;< p0.05, p <p0.01,
< 0.05, and
< 0.01, andp<
p <0.005,
0.005,respectively).
respectively).

Figure
Figure 4. Relativemessenger
4. Relative messenger RNA
RNA expression
expression levels
levels of Collagen
of Collagen 1, MMP1,
1, MMP1, MMP3,MMP3,
and MMP9 MMP9
and in the in
the
HDF HDF cells.
cells. The The collagen
collagen 1 gene
1 gene expression
expression levelincreased
level was was increased concentration
concentration dependently
dependently in the in
EverCTP
the EverCTP TM (NEWTREE
TM (NEWTREE Co., Ltd.,
Co., Seoul, Korea)Korea)
Ltd., Seoul, treatment group compared
treatment to the CON
group compared group
to the CON (A).
group
MMP1
(A). MMP1(B), MMP3 (C), and
(B), MMP3 (C),MMP9 (D) gene
and MMP9 (D)expressions were induced
gene expressions by 10 mJ/cm
were induced by 10ofmJ/cm
2 UVB irradi-
2 of UVB
ation, and induced
irradiation, MMP MMP
and induced familyfamily
gene expressions were decreased
gene expressions with EverCTP
were decreased
TM treatment; # p <
with EverCTP TM treatment;
0.005, independent samples t-test compared with the CON group; * p < 0.05, *** p < 0.005, independ-
# p < 0.005, independent samples t-test compared with the CON group; * p < 0.05, *** p < 0.005,
ent samples t-test compared with the CON group (A) or the UV irradiation group (B–D). TGF-β,
independent samples t-test compared with the CON group (A) or the UV irradiation group (B–D).
transforming growth factor-beta; RA, retinoic acid; MMP, matrix metalloproteinase; HDF, human
TGF-β, transforming growth factor-beta; RA, retinoic acid; MMP, matrix metalloproteinase; HDF,
dermal fibroblast; CON, control; UVB, ultraviolet-B; UV, ultraviolet.
human dermal fibroblast; CON, control; UVB, ultraviolet-B; UV, ultraviolet.
 Int. J. Mol. Sci. 2022, 23, 1101 6 of 16
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 16

The procollagen type I peptide (PIP1) concentration increased significantly when

The procollagen
participants type with
were treated I peptide (PIP1) and
250 µg/mL concentration
500 µg/mL increased significantly
of EverCTP whento
TM compared
participants were treated with 250 μg/mL and 500 μg/mL of EverCTP TM compared to the
the control (Figure 5A, p < 0.01 and p < 0.005, respectively). Treatment with 1000 µg/mL
control (Figure
of EverCTP TM 5A,
alsop increased
< 0.01 andthep <PIP1
0.005, respectively).(885.65
concentration Treatment withcompared
± 89.69) 1000 μg/mL of
to the
EverCTP TM also increased the PIP1 concentration (885.65 ± 89.69) compared to the control
control (824.65 ± 8.12), but this was not statistically significant. On the other hand, the
(824.65
MMP1±protein
8.12), but this was
secretion notultraviolet
after statistically
(UV)significant.
exposureOn the other
showed hand, thereduction
a significant MMP1
protein secretion after ultraviolet (UV) exposure showed
when treated with 250 µg/mL, 500 µg/mL, and 1000 µg/mL of EverCTP a significant reduction
TM when
(Figure 5B,
treated with
p < 0.005). 250 μg/mL, 500 μg/mL, and 1000 μg/mL of EverCTP TM (Figure 5B, p < 0.005).

Figure 5. Concentrations of PIP1 and MMP1 in HDF cells according to the ELISA assay. The
Figure 5. Concentrations of PIP1 and MMP1 in HDF cells according to the ELISA assay. The PIP1
PIP1 concentration was significantly increased after treatment with 250 µg/mL and 500 µg/mL
concentrationTMwas significantly increased after treatment with 250 μg/mL and 500 μg/mL of
of EverCTP (NEWTREE Co., Ltd., Seoul, Korea) and TGF-β compared to the CON (A). MMP1
EverCTPTM (NEWTREE Co., Ltd., Seoul, 2Korea) and TGF-β compared to the CON (A). MMP1 pro-
production
duction was induced
was induced by 10by 10 mJ/cm
mJ/cm 2 of UVB ofirradiation,
UVB irradiation, and induced
and induced MMP1 MMP1 production
production was
was dose-
dose-dependently decreased according TM treatment (B); # p < 0.005,
dependently decreased according to theto the concentration
concentration of EverCTP
of EverCTP TM treatment (B); # p < 0.005, in-
independent
dependent samples
samples t-test
t-test compared
compared withwith
thethe
CON; ** p**< p0.01,
CON; < 0.01, p < p0.005,
*** *** < 0.005, independent
independent samples
samples t-
test compared
t-test compared with thethe
with CONCON group
group(A) oror
(A) the UV
the UVirradiation
irradiationgroupgroup(B).
(B).TGF-β
TGF-βand
andRA RAwere
wereused
used
asasthe
thepositive
positivecontrols.
controls. TGF-β,
TGF-β, transforming
transforming growth factor-beta;
factor-beta; RA, RA,retinoic
retinoicacid;
acid;PIP1,
PIP1,procollagen
procolla-
gen type I; MMP, matrix metalloproteinase; ELISA, enzyme-linked immunosorbent
type I; MMP, matrix metalloproteinase; ELISA, enzyme-linked immunosorbent assay; CON, control; assay; CON,
control; UVB, ultraviolet-B; UV, ultraviolet; HDF, human
UVB, ultraviolet-B; UV, ultraviolet; HDF, human dermal fibroblast. dermal fibroblast.

ToTomeasure
measurethe theelastase
elastaseactivation
activationthat
thatincreases
increasesdue
duetotoUV
UVlight
lightand
andreactive
reactiveoxygen
oxygen
species
species(ROS)
(ROS) during
during the aging
aging process,
process,ananelastase
elastaseinhibition
inhibition assay
assay waswas performed.
performed. Ac-
Accord-
cordingly, 1 mM phosphoramidon (PR; Sigma Aldrich) was used as the positive
ingly, 1 mM phosphoramidon (PR; Sigma Aldrich) was used as the positive control. Elas- control.
Elastase inhibition
tase inhibition activity
activity waswas significantly
significantly increased
increased afterafter treatment
treatment with with TM dose-
EverCTP
EverCTP TM

dose-dependently.
dependently. Compared Compared
to thetonegative
the negative
control,control, 250 μg/mL,
250 µg/mL, 500 μg/mL,
500 µg/mL, andµg/mL
and 1000 1000
μg/mL
of EverCTP TM showed
of EverCTP TM showed
42.12%, 42.12%,
42.74%,42.74%, and 48.47%
and 48.47% increases
increases in elastase
in elastase inhibition
inhibition activity,
activity, respectively
respectively (Figure (Figure 6, p < 0.005).
6, p < 0.005).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 16

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Int. J. Mol. Sci. 2022, 23, 1101 7 of 16

Figure 6. Percentage of elastase inhibition activity according to PR (positive control) and 250, 500,
and 1000 μg/mL of EverCTPTM (NEWTREE Co., Ltd., Seoul, Korea) treatment. The treatment in-
Percentage
Figure 6.elastase
creased of elastase
inhibition inhibition
activity as muchactivity
as PR. according to independent
*** p < 0.005, PR (positive control)
samplesand 250,
t-test 500, and
compared
Figure 6. Percentage of elastase inhibition activity according to PR (positive control) and 250, 500,
TM (NEWTREE
1000
with µg/mL of EverCTP
the control. PR, phosphoramidon. Co., Ltd., Seoul, Korea) treatment. The treatment increased
and 1000 μg/mL of EverCTPTM (NEWTREE Co., Ltd., Seoul, Korea) treatment. The treatment in-
elastase inhibition activity as much as PR. *** p < 0.005, independent samples t-test compared with
creased elastase inhibition activity as much as PR. *** p < 0.005, independent samples t-test compared
2.4. In
theVitro
the control.
with Evaluation
PR,
control. of the Effect of EverCTPTM on Reactive Oxygen Species and the
phosphoramidon.
PR, phosphoramidon.
Glycation Process
2.4. In Vitro Evaluation of the Effect of EverCTPTM on Reactive Oxygen Species and the
2.4. InAfter
Vitro
Glycation Evaluationwith
treatment
Process of the
10Effect of EverCTP
μM hydrogen TM on Reactive
peroxide (H2O2Oxygen Species and
), the increased the oxygen
reactive
Glycation Processlevel was significantly reduced when cotreated with 1000 μg/mL of
species (ROS)
After treatment with 10 µM hydrogen peroxide (H2 O2 ), the increased reactive oxy-
EverCTP
gen After
TM (Figure 7, p < 0.05, p < 0.01, p < 0.005). Meanwhile, the effect of EverCTPTM on
speciestreatment
(ROS) levelwithwas10 μM hydrogen peroxide
significantly reduced (H when2O2), the increased
cotreated withreactive oxygen
1000 µg/mL of
the
speciesglycation
EverCTP(ROS)
TM process
level 7,was
(Figure in human dermal
significantly
p < 0.05, fibroblast
p < 0.01, preduced
< 0.005).when (HDF) cells
cotreated
Meanwhile, was investigated
with of1000
the effect μg/mL
EverCTP by
TMen-
of
on
zyme-linked
EverCTP
the glycation immunosorbent
TM (Figure
process 7, in 0.05, passay
p <human < 0.01,(ELISA)
dermal pfibroblastafter
< 0.005). UV cells
Meanwhile,
(HDF) exposure. Treatment
the investigated
was effect with
of EverCTP
by TM1000
enzyme-on
μg/mL
the
linked of EverCTP
glycation
immunosorbent
TM significantly decreased the productions of pentosidine (from 211.682
process inassay human dermal
(ELISA) afterfibroblast (HDF) Treatment
UV exposure. cells was withinvestigated by en-
1000 µg/mL of
±EverCTP
0.425 toTM
zyme-linked 156.975 ± 0.135),decreased
immunosorbent
significantly methylglyoxal
assay the
(ELISA) (from 5.586
after
productions UVof±pentosidine
0.014 to 5.307
exposure. ± 0.001),
Treatment
(from and±AGEs
with
211.682 1000
0.425
(from
μg/mL 16.688
to 156.975 ± 0.235
of EverCTP
± 0.135), to 10.762 ± 0.424),
TM significantly
methylglyoxal (fromwhile
decreased 5.586thesignificantly
±productions
0.014 to 5.307 increasing
± 0.001),the
of pentosidine andproduction
(from
AGEs211.682
(fromof
glyoxalase
±16.688
0.425 ± I
to0.235 compared
156.975 ± 0.135),
to 10.762 to the UV group
methylglyoxal
± 0.424), (from 0.008
(from 5.586
while significantly ± 0.001 to
± 0.014 to
increasing 0.777
the5.307 ± 0.003;
± 0.001),
production Figure
ofand AGEs<
8, p
glyoxalase
0.005).
(from 16.688to± the
I compared 0.235
UVtogroup
10.762(from
± 0.424), ± 0.001
0.008while to 0.777 ± 0.003;
significantly increasing
Figure the
8, production
p < 0.005). of
glyoxalase I compared to the UV group (from 0.008 ± 0.001 to 0.777 ± 0.003; Figure 8, p <
0.005).

Figure 7. Visualization
VisualizationofofROSROSproduction
productionininHDF HDFcells (A).
cells ROS
(A). ROS production
production was stimulated
was stimulatedby by
10
μMµM
10 of2O
of H H22treatment andand
O2 treatment significantly reduced
significantly by 1000
reduced μg/mL
by 1000 of EverCTP
µg/mL TM (NEWTREE
TM (NEWTREE
of EverCTP Co., Ltd.,
Co.,
Seoul, Korea)Korea)
Ltd., Seoul, treatment (B); * p < 0.05,
treatment *p< ** 0.05,
p < 0.01,
** p*** p < 0.005,pindependent samples t-test. Scale bar
t-test.
Figure 7. Visualization of ROS (B);
production in HDF <cells
0.01, *** ROS
(A). < 0.005, independent
production samples
was stimulated by 10
indicates
Scale 100 μm. ROS, reactive oxygen species; HDF, human dermal fibroblast; H 2O2, hydrogen
μM of bar
H2Oindicates
2 treatment100
andµm. ROS, reactive
significantly oxygen
reduced species;
by 1000 μg/mL HDF, human dermal
of EverCTP TM fibroblast;
(NEWTREE Co.,HLtd.,
2 O2 ,
peroxide.
Seoul, Korea)
hydrogen treatment (B); * p < 0.05, ** p < 0.01, *** p < 0.005, independent samples t-test. Scale bar
peroxide.
indicates 100 μm. ROS, reactive oxygen species; HDF, human dermal fibroblast; H2O2, hydrogen
peroxide.
Int.
Int.J.J.Mol.
Mol.Sci. 2022,23,
Sci.2022, 23,1101
x FOR PEER REVIEW 8 of816
of 16

Figure 8. Comparison
Comparisonof ofthe
thepentosidine,
pentosidine,methylglyoxal,
methylglyoxal, glyoxalase
glyoxalase 1 activity, and
1 activity, advanced
and advanced gly-gly-
cation end
cation end product
product (AGE)
(AGE) production
productionininHDF
HDFcells
cellsbefore
beforeandandafter
afterthe
thetreatment.
treatment. Pentosidine,
Pentosidine,
methylglyoxal, and
methylglyoxal, and AGE
AGE production
productionwaswasinduced
inducedbyby1010mJ/cm
mJ/cmof
2 UVB
2 of UVB irradiation (A,B,D).
irradiation (A,B,D).TheThe
induced pentosidine, methylglyoxal, and AGE concentrations were significantly reduced after treat-
induced pentosidine, methylglyoxal, and AGE concentrations were significantly reduced after treat-
ment with 1000 μg/mL of EverCTPTM (NEWTREE Co., Ltd., Seoul, Korea). Glyoxalase 1 activity was
TM (NEWTREE Co., Ltd., Seoul, Korea). Glyoxalase 1 activity was
ment with 1000 µg/mL of EverCTP
reduced by 10 mJ/cm of UVB irradiation, and the reduced glyoxalase 1 activity was significantly
2

reduced 2
increasedby 10 mJ/cm
with treatmentofofUVB1000irradiation, and theTMreduced
μg/mL of EverCTP (C); *** pglyoxalase 1 activity was
< 0.005, independent significantly
samples t-test.
increased with treatment of 1000 µg/mL of EverCTP TM (C); *** p < 0.005, independent samples t-test.
HDF, human dermal fibroblast; ultraviolet-B; UV, ultraviolet.
HDF, human dermal fibroblast; ultraviolet-B; UV, ultraviolet.
Additional investigations of the CTP ampoule were performed to assess its effect on
the productions investigations
Additional of the CTP
of glycated collagen andampoule
AGEs. The were performed to
fluorescence assess its
intensity ofeffect on the
glycated
productions of glycated collagen and AGEs. The fluorescence intensity of
collagen production of the control group was 0.999 ± 0.058, while that of the CTP ampouleglycated collagen
production
control group of without
the controlcollagen was 0.999 ±
groupcomponent 0.058,
(CTP whilewas
control) that0.09
of the CTP (Figure
± 0.027 ampoule control
9A, p<
group
0.005). without
Compared collagen component
to the control group,(CTP control)
the CTP was 0.09
ampoule group± 0.027
(with (Figure
collagen)9A, p < 0.005).
showed a
Compared to the control
significant decrease group,collagen
in glycated the CTPproduction
ampoule group (with
to 0.517 collagen)
± 0.114 (Figureshowed a signifi-
9A, p < 0.005).
cant decrease
Moreover, theinfluorescence
glycated collagen production
intensity to 0.517 ± of
of AGE production 0.114
the (Figure
control 9A, p <was
group 0.005). More-
2.476 ±
over,
0.005,the fluorescence
while that of CTP intensity
ampoule ofgroup
AGE production of the decreased
was significantly control group 0.002±(Fig-
was±2.476
to 2.296 0.005,
while
ure 9B,that
p <of CTP ampoule
0.005). Lastly, wegroup was significantly
measured the degree ofdecreased 2.296 ± production
denaturedtocollagen 0.002 (Figure in-9B,
pduced
< 0.005). Lastly,
by heat we measured
treatment the the
to observe degree ofof
effect denatured
CTP ampoulecollagen production
cotreatment. Theinduced
fluores-by
heat
cencetreatment
intensity toofobserve
denatured thecollagen
effect of CTP ampoule
production cotreatment.
after The fluorescence
heat treatment was 13.361 ± intensity
2.532,
of denatured collagen production after heat treatment was 13.361 ±
and the degree of intensity significantly decreased to 8.676 ± 0.623 after treatment2.532, and with
the degree
the
of
CTPintensity
ampoule significantly p < 0.05). to 8.676 ± 0.623 after treatment with the CTP ampoule
(Figure 9C,decreased
(Figure 9C, p < 0.05).
3, x FOR PEER REVIEW 9 of 16
Int. J. Mol. Sci. 2022, 23, 1101 9 of 16

Figure 9. Effect of the Figure

CTP ampoule
9. Effect ofon
theglycated collagen,
CTP ampoule AGE,collagen,
on glycated and denatured collagen production.
AGE, and denatured collagen production.
Glycated collagen andGlycated
AGE production
collagen and decreased in the
AGE production CTP ampoule
decreased in the CTPtreatment group compared
ampoule treatment group compared
to the control group (A,B). Denatured collagen production induced
to the control group (A,B). Denatured collagen production induced by heat treatment decreased by heat treatment decreased
with CTP ampoule treatment (C), as visually shown in (D); * p
with CTP ampoule treatment (C), as visually shown in (D); * p < 0.05, *** p < 0.005, independent< 0.05, *** p < 0.005, independent
samples t-test. Scale bar indicates 80 µm. CTP ampoule, Ever Collagen Corrector Collagen Tripeptide
samples t-test. Scale bar indicates 80 μm. CTP ampoule, Ever Collagen Corrector Collagen Tripep-
Ampoule; AGEs, advanced glycated end products.
tide Ampoule; AGEs, advanced glycated end products.
2.5. Assessment of Skin Absorption after Topical Application of EverCTPTM
2.5. Assessment of Skin Absorption
The efficacyafter Topical
of collagen Application
1 absorption afteroftopical
EverCTP TM
application of gelatin (nonhydrolyzed
TM
The efficacy ofcollagen)
collagen and EverCTP to the skin was evaluated in reconstructed human micro-tissue
1 absorption after topical application of gelatin (nonhydro-
(KeraSkin-FT, Bio Solution Co., Ltd., Seoul, Korea). The fluorescence intensity of collagen 1
lyzed collagen) andabsorbed
EverCTP TM to skin
into the the after
skin1000
wasµg/mL
evaluated in reconstructed
of EverCTP TM treatment was human
2.316 ±micro-
0.444, which
tissue (KeraSkin-FT, Bio
was Solution
increased Co., Ltd.,
compared to thatSeoul, Korea).
after 1000 µg/mLThe fluorescence
of gelatin treatment intensity of and
(2.051 ± 0.550)
the negative control (0.266 ±
collagen 1 absorbed into the skin after 1000 μg/mL of EverCTP treatment was 2.316 ±in the
0.111; Figure 10A,B, p <
TM0.005). Despite an increase
EverCTPTM
0.444, which was increased treatment group,
compared to thattheafter
difference
1000 in florescent
μg/mL intensitiestreatment
of gelatin compared to the gelatin
(2.051
group did not reach a statistical significance (p = 0.5520).
± 0.550) and the negative control (0.266 ± 0.111; Figure 10A,B, p < 0.005). Despite an in-
Additionally, the results of the additional quantitative evaluation on the rates of colla-
crease in the EverCTP TM treatment group, the difference in florescent intensities compared
gen absorption via high-performance liquid chromatography (HPLC) compared between
TM treatments are shown in Figure 10C. The rates of collagen ab-
to the gelatin groupthe
did not reach
gelatin a statistical
and EverCTP significance (p = 0.5520).
sorption in the gelatin treatment group and EverCTP TM treatment group were 0.425 ± 0.100
Additionally, the results of the additional quantitative evaluation on the rates of col-
and 12.221 ± 0.684, respectively, thus showing a significantly improved quantitative ab-
lagen absorption via high-performance liquid chromatography (HPLC) compared be-
sorption rate of collagen in the EverCTPTM group (p < 0.005).
tween the gelatin and EverCTP treatments are shown in Figure 10C. The rates of colla-
TM

gen absorption in the gelatin treatment group and EverCTPTM treatment group were 0.425
± 0.100 and 12.221 ± 0.684, respectively, thus showing a significantly improved quantita-
tive absorption rate of collagen in the EverCTPTM group (p < 0.005).
 Int. J.J. Mol.
Int. Mol. Sci. 2022, 23,
23, 1101
x FOR PEER REVIEW 10 of
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16

Figure Comparisonofofcollagen
Figure 10. Comparison collagen 1 expression
1 expression using
using immunofluorescence
immunofluorescence staining
staining (A). 1000
(A). The The
1000
μg/mL gelatin
gelatin
µg/mL (nonhydrolyzed
(nonhydrolyzed collagen)
collagen) treatment
treatment collagen
collagen inducedmore
induced morecollagen
collagen 11 expression
expression
than the
than the control
control(B).
(B).The
The1000 μg/mL EverCTPTM
1000µg/mL TM treatment induced more collagen expression than
treatment induced 1 expression than
the 1000 μg/mL gelatin treatment. Collagen absorption was considerably increased
the 1000 µg/mL gelatin treatment. Collagen absorption was considerably increased with with1000
1000µg/mL
μg/mL
of EverCTP
of EverCTP
TM compared to 1000 μg/mL of gelatin (C); *** p < 0.005, independent samples t-test. Scale
TM compared to 1000 µg/mL of gelatin (C); *** p < 0.005, independent samples t-test.
bar indicates 80 μm.
Scale bar indicates 80 µm.

3. Discussion
3.
Recently, both
Recently, both topical
topical and
and oral
oral collagen-containing
collagen-containing products
products have
have become
become popular
popular
for their antiaging
for antiaging properties
properties[3].[3].Although
Althoughthe theantioxidant
antioxidant ability of of
ability thethe
CTP CTPis due to the
is due to
presence
the presenceof hydrophobic
of hydrophobic amino acids
amino in the
acids in peptide [10],[10],
the peptide the exact molecular
the exact molecularmechanism
mecha-
nism underlying
underlying how howthe CTPthe CTP
acts acts
as anasantiaging
an antiaging product
product hashas
notnot
beenbeen fully
fully uncovered.
uncovered. A
A previous
previous placebo-controlledclinical
placebo-controlled clinicaltrial
trialdemonstrated
demonstratedthat thatoral
oral ingestion
ingestion of of the CTPCTP
helped
helped retain
retain skin
skin water contents [11]. Moreover, a recent study on the cutaneous hydra-
tion
tion effect
effect of
of CTP
CTP intake
intake in
in mice
mice showed
showed significant
significant reductions
reductions in in transepidermal
transepidermal water water
loss,
loss, scratching
scratching behavior,
behavior, andand levels
levels of
of hyaluronidase-1,
hyaluronidase-1, tumortumor necrosis
necrosis factor-alpha,
factor-alpha, and and
interleukin-6,
interleukin-6, while showing increased water content and hyaluronan synthase-2 levels;
these
these results
results suggested
suggested thatthat CTP
CTP products
products could
could enhance
enhance skin
skin hydration
hydration withwith potential
potential asas
aa skin
skin hydration agent [12]. The present study showed improvements in facial wrinkles,
hydration agent [12]. The present study showed improvements in facial wrinkles,
skin
skin elasticity,
elasticity, and
and density
density without
without anyany adverse
adverse effects
effects after
after use
use of
of the
the topical
topical CTP.
CTP. TheThe
participants’
participants’ satisfaction
satisfaction scores
scores also
also subjectively
subjectively showed
showed an an overall
overall improvement
improvement of of their
their
facial
facial skin.
skin. Moreover,
Moreover,theretherewas
wasaasignificant
significantreduction
reductionininAGE
AGEautofluorescence
autofluorescence at at
week
week 4,
implying that the topical CTP has both antiaging and anti-glycation effects
4, implying that the topical CTP has both antiaging and anti-glycation effects clinically. clinically.
To
To further
further investigate
investigate the the molecular
molecular mechanism
mechanism underlying
underlying thethe antiaging
antiaging effect
effect of
of
the topical CTP, we performed an in vitro study on HDF cells.
the topical CTP, we performed an in vitro study on HDF cells. After UV exposure of After UV exposure of fi-
fi-
broblasts
broblasts totoinduce
induce cellular senescence
cellular senescence and and
aging, expressions
aging, of Collagen1,
expressions MMP1, MMP1,
of Collagen1, MMP3,
and MMP9 mRNA with or without CTP treatment were measured.
MMP3, and MMP9 mRNA with or without CTP treatment were measured. The Collagen1 The Collagen1 gene
expression significantly
gene expression increased
significantly with CTP
increased withtreatment, whereas
CTP treatment, MMP1,MMP1,
whereas MMP3,MMP3,and MMP9 and
expressions significantly decreased dose-dependently. Moreover,
MMP9 expressions significantly decreased dose-dependently. Moreover, ELISA ELISA of PIP1 andof MMP1
PIP1
levels in UV-exposed HDF cells with CTP treatment showed an increased PIP concentration,
and MMP1 levels in UV-exposed HDF cells with CTP treatment showed an increased PIP
while MMP1 levels decreased with CTP treatment, suggesting that the CTP showed an
concentration, while MMP1 levels decreased with CTP treatment, suggesting that the CTP
antiaging effect on fibroblasts. The additional elastase inhibition assay showed signifi-
showed an antiaging effect on fibroblasts. The additional elastase inhibition assay showed
cantly increased elastase inhabitation activity after CTP treatment, further indicating its
significantly increased elastase inhabitation activity after CTP treatment, further indicat-
antiwrinkle effect.
ing its antiwrinkle effect.
Over recent years, various benefits of supplementation with the CTP have been
Over recent years, various benefits of supplementation with the CTP have been re-
reported including improvements in joint pain, wound healing, blood pressure, glucose
ported including improvements in joint pain, wound healing, blood pressure, glucose tol-
tolerance, elasticity and wrinkles of the skin, and epidermal barrier function [4,13,14].
erance, elasticity and wrinkles of the skin, and epidermal barrier function [4,13,14]. More-
Moreover, the CTP has been shown to stimulate cell proliferation in dermal fibroblasts,
over, the CTP has been shown to stimulate cell proliferation in dermal fibroblasts, while
while increasing synthesis of hyaluronic acid and acceleration of cell migration [15–17].
increasing synthesis of hyaluronic acid and acceleration of cell migration [15–17]. During
Int. J. Mol. Sci. 2022, 23, 1101 11 of 16

During the process of skin aging, a profound atrophy of dermal connective tissue including
collagen occurs, which impairs strength and resiliency of the skin. Glycation and production
of AGEs further debilitate the function of collagen, making it stiffer and more brittle [17].
The glycation process also leads to profound changes in the behavior of dermal fibroblasts,
reducing their proliferation and migration, while simultaneously disrupting collagen I
maturation and preventing collagen deposition in the extracellular matrix [18].
Herein, we investigated the relationship between the application of a CTP and re-
ductions in the glycation process. Previous studies have shown that the production of
AGEs results in the activation of nuclear factor-kappa beta (NF-κB) transcription factors
via the generation of oxygen radicals and MAP kinase signaling; the NF-κB activation
subsequently leads to the induction of MMPs and formation of various proinflammatory
cytokines, which leads to cellular aging [19]. Our in vitro study revealed that the ROS
expression level of H2 O2 -treated HDF cells was significantly reduced when cotreated
with the CTP, implying its antioxidant effect. Furthermore, the subsequent ELISA with
UV-exposed HDF cells showed a significant decrease in productions of pentosidine and
methylglyoxal, the two major AGE biomarkers, after treatment with the CTP. However, the
level of glyoxalase-1, a ubiquitous cellular enzyme that participates in the detoxification of
methylglyoxal, significantly increased upon treatment with the CTP. The in vitro study of
the CTP ampoule also showed significant reductions in the production of glycated collagen,
AGEs, and denatured collagen in the treatment group compared to the control group. These
results suggest that supplementation with the CTP not only provided an antioxidant effect,
but also inhibited AGE accumulation, which could lead to the reduction in MMPs and
proinflammatory cytokines, thus promoting overall antiaging of the skin.
The antioxidant properties of the CTP depend on the lower molecular weight of pep-
tides, as smaller molecules with average molecular weights of 5 kDa showed greater ability
to donate an electron or hydrogen to stabilize oxygen radicals than larger molecules [20].
Since the absorption capacity of a topical collagen product through the skin barrier is fre-
quently limited by its molecular size, manufacturing small (1–10 kDa) collagen hydrolysate
with excellent biocompatibility, easy biodegradability, and weak antigenicity is crucial
for utilization [6,21]. In our study, we compared the absorption rate of EverCTPTM , with
gelatin—a form of a heterogeneous mixture of peptides derived from the parent protein
collagen [21]. The EverCTPTM group showed a significantly improved collagen absorp-
tion rate compared to the gelatin group, suggesting the enhanced biocompatibility of the
CTP formulation.
In conclusion, our pilot study showed the effect of a topical CTP formulation on
antiaging and inhibition of the glycation process of skin. These results suggest that the
application of a topical CTP might improve clinical aging phenotypes via the inhibition
of glycation and oxidative stress, leading to a delay in cellular aging. Nevertheless, our
study had several limitations. First, it included a small number of samples and short
follow-up period in the clinical study. Hence, a further clinical study with a larger sample
size and longer follow-up period with a double-blinded, controlled design is required.
Secondly, an additional skin absorption study on collagen 1 between EverCTPTM and the
simple tripeptide, Gly–Pro–Hyp, would further show efficacy and biocompatibility of the
test product in its topical formulation. Lastly, although our in vitro study revealed not
only a reduction in ROS and AGE production but also an increase in collagen 1 and the
reciprocal reduction of MMPs, additional studies on the molecular pathogenesis are needed
to provide a complete understanding of the effect of a topical CTP on antiaging of the skin.

4. Materials and Methods

4.1. Study Design and Ethical Considerations
This prospective, single-arm clinical trial was approved by the Institutional Review
Board (IRB) of the Global Medical Research Center (IRB number: GIRB-21624-DX), and
informed consent was obtained from each participant. The investigation was performed
Int. J. Mol. Sci. 2022, 23, 1101 12 of 16

in full compliance with the principles of Good Clinical Practices and the Declaration
of Helsinki.
All participants were instructed to apply the test product, the CTP ampoule, to their
entire face twice daily for 4 weeks to evaluate its clinical efficacy and safety. To assess its
safety, all participants were asked to report any adverse events they experienced while
using the topical product.

4.2. Participants
Twenty-two healthy women aged between 30 and 50 years with noticeable periorbital
and glabellar wrinkles were considered eligible for inclusion in the study. The main exclu-
sion criteria were as follows: major internal disorders, major or active skin diseases, current
pregnancy or lactation, history of allergy or hypersensitivity reactions, history of under-
going dermatologic procedures (e.g., lasers, filler, botulinum toxin injection) in the past
3 months, use of topical medications with any skin reactions (e.g., glucocorticoids, retinoids,
and topical immunomodulators) in the past 3 months, use of systemic medications, such as
glucocorticoids and immunomodulators within the last 1 month, and application of skin
care products or topical medications with similar functions to topical collagen peptides
within the last 3 months before participating in the study.

4.3. Test Product

The CTP ampoule containing a hydrolyzed fish skin extract (EverCTPTM ) was used
in the clinical study. EverCTPTM is a form of collagen hydrolysate obtained from the skin
of Pangasius hypophthalmus that contains 4% Gly–Pro–Hyp with the tripeptide content
exceeding 25%; it was used as the test ingredient in our laboratory study. The preliminary
human skin ex vivo penetration study with analysis of Franz diffusion cells showed 6.74%
permeability of the Gly–Pro–Hyp tripeptide.

4.4. Clinical Efficacy Assessment

All participants were followed up at baseline and week 4 after the initiation of the
treatment. The clinical efficacy of EverCTPTM was investigated by assessing biophysical
parameters determined by an optical 3D measurement system (PRIMOS CR, SnT Lab, Seoul,
Korea), Cutometer Dual MPA580 (Courage Khazaka electronic GmbH, Köln, Germany),
and Ultrascan UC22 (Courage + Khazaka electronic GmbH). The wrinkle volume and skin
roughness of the periorbital area (crow’s feet) and glabellar area were measured by the
phase-shift rapid in-vivo measurement of skin (PRIMOS) at baseline and week 4. Among
the skin surface descriptors of PRIMOS, Ra and Rmax were chosen to measure the 3D
wrinkle changes during the study period. Antera 3D was used to perform a quantitative
assessment of the depressions caused by the wrinkles.
Changes in skin elasticity were measured using the Cutometer MPA580. Among the
various parameters of the Cutometer, we chose R2 and R4 to represent the efficacy of the
test product in improvement of skin elasticity. The Ultrascan UC22 was used to measure
changes in the density of the skin.
To noninvasively assess AGE accumulation in the skin, we used skin autofluorescence
(skin AF) measured by the AGE Reader (DiagnOptics Technologies B.V., Groningen, The
Netherlands). Skin AF, expressed as arbitrary units (AU), was measured in triplicate at the
skin site on participants’ inner forearm, which is a body part easily accessible and rarely
exposed to the sun, as previously described [22].
All subjects underwent a physical examination to assess safety outcomes at every visit.
Participants were also asked to report side-effects during the study period. Additionally,
the participants were questioned to score their rates of satisfaction on the use of topical
CTP product and the overall improvement in facial skin problems caused by aging using
the following scale: 1 = unsatisfied, 2 = no change, 3 = slightly satisfied, 4 = satisfied, and
5 = very satisfied.
Int. J. Mol. Sci. 2022, 23, 1101 13 of 16

4.5. Cell Culture

HDF cells purchased from ATCC® (Manassas, VA, USA) were cultured in Dulbecco’s
modified Eagle’s medium (Lonza, Walkersville, MD, USA) supplemented with 1% penicillin–
streptomycin (Gibco, Grand Island, NY, USA) and 10% fetal bovine serum (Gibco). HDF
cells were incubated in a humidified atmosphere containing 5% carbon dioxide (CO2 ) at
37 ◦ C. TGF-β, RA, and PR were used as appropriate positive controls. UV exposure and
the treatment with H2 O2 were performed for the generation of extrinsic cellular aging and
the induction of ROS stress on HDFs, respectively.

4.6. Cell Viability Measurement

To determine if EverCTPTM was cytotoxic, a cell counting kit-8 (CCK-8; Dojindo,
Mashiki, Japan) assay was used according to the manufacturer’s instructions to evaluate
HDF cells (ATCC® ). In brief, HDF cells (5 × 104 cells/well) were seeded onto a 96-well
plate. When cell confluence was >80%, the HDF cells were treated with EverCTPTM and
incubated for 24 h. The ELISA microplate reader (VersaMax; Molecular Devices, San Jose,
CA, USA) was used to measure the absorbance at 450 nm.

4.7. Total RNA Extraction and qRT-PCR

Total RNA was extracted from the HDF cells treated with EverCTPTM after no ir-
radiation or 10 mJ/cm2 of ultraviolet-B (UVB) irradiation using a UVB lamp (BLX26,
BIO-LINK® -Crosslinker, FRA). Using the TRIzol reagent (Invitrogen, Waltham, MA, USA)
according to the manufacturer’s instructions, total RNA extraction was performed. For
quantification and reverse transcription of total RNA, the NanoDrop 2000 spectrophotome-
ter (ThermoFisher Scientific, Carlsbad, CA, USA) and RNA to complementary DNA EcoDry
Premix Kit (Takara Sake, Berkley, CA, USA) were used. qRT-qPCR was conducted using the
SYBR Green Master MIX (4309155, Promega Co., Madison, WI, USA), specific primer pairs,
and the QuantStudio 3 Real-Time Polymerase Chain Reaction System (Applied Biosystems,
Waltham, MA, USA). Primer sequences used in the present study are shown in Table S2.
Relative mRNA expression levels were normalized to GAPDH and calculated using the
2−∆∆Ct method.

4.8. Enzyme-Linked Immunosorbent Assay

PIP and MMP1 protein concentrations were detected using ELISA kits (Takara, Kusatsu,
JPN; Amersham Life Science, Amersham, UK). MMP1 production was induced by 10 mJ/cm2
of UVB irradiation. The Human Pentosidine ELISA kit (MyBioSource, San Diego, CA, USA),
Methylglyoxal ELISA kit (Abcam, Cambridge, UK), Glyoxalase I assay kit (Abcam), and
AGE assay Kit (Abcam) were used to measure pentosidine, methylglyoxal, gyloxalase I, and
overall AGE production. All kits were used according to the manufacturers’ instructions.
The absorbance detection was performed using an ELISA microplate reader (VersaMax) at
450 nm.

4.9. Elastase Inhibitory Assay

To measure elastase inhibition activity, an elastase solution extracted by HDF cells
was prepared by freezing/thawing the cells more than three times. After the solution was
centrifuged (3000 rpm) at 4 ◦ C for 20 min, the supernatant was harvested and used as
the elastase solution. The elastase solution was placed in 100 µg of protein per well on a
96-well plate, and we added 0.2 M Tris–HCl buffer (pH 8.0) to make the total volume 88 µL.
We added 2 µL of N-succinyl-tri-alanyl-p-nitroanilide, a substrate of elastase, and 10 µL
of EverCTPTM of three concentrations or 10 µL of 1 mM PR (Sigma Aldrich, Saint Louis,
MO, USA) to each well containing the elastase solution and cultured the plate at 37 ◦ C for
90 min. We added 10 µL of 0.2 M Tris–HCl buffer as a control group. Finally, using an
ELISA microplate reader (VersaMax), the absorbance was measured at 405 nm at 37 ◦ C.
Int. J. Mol. Sci. 2022, 23, 1101 14 of 16

The elastase inhibition activity was expressed as a decrease (%) in absorbance of the test
group with and without the sample solution.

Elastase inhibition activity (%) = (A − B)/A × 100,

where A is the absorbance of control, and B is the absorbance of sample.

4.10. ROS Assessment

Intracellular ROS accumulation assessment was performed using the 20 70 -
dichlorofluorescin diacetate (DCFDA) cellular ROS Detection Assay Kit (Abcam). Briefly,
the HDF cells (5 × 104 cells/dish) were seeded onto a glass bottom dish, and, when the cell
confluence was >80%, we added 200 µL of 25 µM DCFDA to each dish and incubated the
cells at 37 ◦ C for 45 min. To induce ROS stress as the positive control, the treatment with
10 µM H2 O2 was performed. For the test group, 10 µM H2 O2 was added and incubated
for 24 h prior to DCFDA. After diffusion into the cells, DCFDA was deacetylated and
subsequently oxidized by ROS to form dichlorofluorescein, which is highly fluorescent, and
it was assessed by confocal microscopy. After discarding the DCFDA solution, each dish
was washed using phosphate-buffered saline (PBS). Fluorescence intensity was imaged
and measured using a confocal microscope (LSM 700, Carl Zeiss, Jena, Germany), and
compared between groups using ImageJ software (National Institutes of Health, Bethesda,
MD, USA).

4.11. Assessment of Glycated Collagen Production and AGEs

Glycated collagen production was estimated using the Collagen Glycation Assay Kit
(COSMO BIO, Tokyo, Japan), which was used according to the manufacturer’s instructions.
In brief, 50 µL of a collagen solution was placed onto a 96-well black plate and incubated at
37 ◦ C for 18 h. After it reacted for 18 h, the sample dilution buffer was added to the buffer
group. In order to check the glycation reaction of the test product itself, the topical CTP
ampoule without collagen was added to the CTP control group (without collagen). In the
control group, fructose was added, while both fructose and the topical CTP ampoule (with
collagen) were added to the CTP ampoule group. Using the microplate reader (VersaMax)
at 370 nm and 440 nm, the absorbance was immediately detected after 4 weeks of reaction at
37 ◦ C. Detected absorbance was used to compare the glycated collagen production between
the groups.
In the assessment of AGE production, a mixture of bovine serum albumin (BSA)
(5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA), glucose (25 mM), and PBS or the CTP
ampoule was placed in a 1:1:1 ratio and reacted at 37 ◦ C for 2 weeks. PBS only was added
to the buffer group, while BSA and glucose were added to the control group. The CTP
ampoule was additionally treated along with BSA and glucose in CTP ampoule group.
In order to verify the glycation reaction of the CTP ampoule itself, a control for the CTP
ampoule excluding BSA was tested as the CTP control group.

4.12. Immunofluorescence Staining for Evaluating Denatured Collagen Production

The HDF cells (6 × 104 cells/well) were seeded on four-well chamber slides and
incubated with the CTP ampoule for 24 h when cell confluence reached > 80%. The slides
were washed, treated with heated PBS (95 ◦ C) for 1 min, and fixed with 4% paraformalde-
hyde. To induce denatured collagen production by HDFs as the positive control, heat
stimulation was performed. After fixation, the slides were incubated with a denatured
collagen detection reagent (Funakoshi, Tokyo, Japan) and incubated with FITC-conjugated
streptavidin. Finally, the slides were fixed using the VECTASHIELD® antifade mounting
medium with DAPI (Vector Laboratories Inc., Burlingame, CA, USA) and evaluated using
a fluorescence microscope (M2, Carl Zeiss). The fluorescence intensity was calculated using
obtained fluorescence images and ImageJ software (National Institutes of Health).
Int. J. Mol. Sci. 2022, 23, 1101 15 of 16

4.13. Immunofluorescence Staining and HPLC to Assess Collagen 1 Absorption

The reconstructed human micro skin tissue model KeraSkin-FT was cultured in the
KeraSkin-FT growth medium (Bio Solution Co., Ltd.) and incubated for 24 h in a humidified
atmosphere containing 5% CO2 at 37 ◦ C after covering with 10 µL of 1000 µg/mL of
EverCTPTM or gelatin (non-hydrolyzed collagen) extracted from fish skin. Following
fixation with 4% paraformaldehyde, the samples were made into 6 µm thick frozen sections.
The frozen sections were incubated with primary anti-fish collagen 1 (Abcam) and a goat
anti-rabbit immunoglobulin G H&L secondary antibody (Abcam). Then, the frozen sections
were fixed using the VECTASHIELD® mounting medium with DAPI and evaluated using
a fluorescence microscope. The fluorescence intensity was calculated using obtained
fluorescence images and ImageJ software (National Institutes of Health).
Additionally, after incubation, the medium containing collagen absorbed through the
reconstructed human micro skin tissue model was analyzed to quantify hydroxyproline
using HPLC. The increased absorption rates were calculated as previously described.

4.14. Statistical Analyses

All data were expressed as the mean ± standard deviation. Datasets were assessed
for normality using the Kolmogorov–Smirnov test. Clinical parameters at baseline and
week 4 were compared between groups using the Wilcoxon signed-rank test or paired
samples t-test according to the results from the normality test. The data were statistically
analyzed using SPSS statistics version 25.0 software (IBM Corp., Armonk, NY, USA). Mean
differences were considered significant when p < 0.05, p < 0.01, and p < 0.005. All laboratory
experiments in the present study were performed at least three times (n ≥ 3); for statistical
data analyses, the obtained data were used.

Supplementary Materials: The following supporting information can be downloaded at https:

//www.mdpi.com/article/10.3390/ijms23031101/s1.
Author Contributions: Conceptualization, Y.I.L., S.G.L., M.-H.L., D.-U.K. and J.H.L.; methodology,
S.G.L., Y.I.L., I.J. and J.S.; formal analysis, I.J., M.-H.L., D.-U.K., Y.I.L., S.G.L. and J.S.; investigation,
S.G.L., M.-H.L., D.-U.K., I.J., J.S. and Y.I.L.; writing—original draft preparation, S.G.L. and Y.I.L.;
writing—review and editing, Y.I.L., S.G.L. and J.H.L.; supervision, Y.I.L. and J.H.L. All authors read
and agreed to the published version of the manuscript.
Funding: This research was supported by NEWTREE Co., Ltd.
Institutional Review Board Statement: The study was conducted in accordance with the guidelines
of the Declaration of Helsinki and approved by the Institutional Review Board (IRB) of the Global
Medical Research Center (IRB number: GIRB-21624-DX).
Informed Consent Statement: Informed consent was obtained from all participants involved in
the study.
Conflicts of Interest: The authors declare no conflict of interest.

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