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Supplementating with Dietary Astaxanthin Combined with Collagen Hydrolysate
Improves Facial Elasticity and Decreases Matrix Metalloproteinase-1 and -12
Expression: A Comparative Study with Placebo
Hyun-Sun Yoon,
1–3,
*Hyun Hee Cho,
1,
*Soyun Cho,
1–3
Se-Rah Lee,
1,2
Mi-Hee Shin,
1,2
and Jin Ho Chung
1,2,4
1
Department of Dermatology, Seoul National University College of Medicine, Seoul, Korea.
2
Institute of Human-Environment Interface Biology, Seoul National University, Seoul, Korea.
3
Department of Dermatology, Seoul National University Boramae Hospital, Seoul, Korea.
4
Institute on Aging, Seoul National University, Seoul, Korea.
ABSTRACT Photoaging accounts for most age-related changes in skin appearance. It has been suggested that both as-
taxanthin, a potent antioxidant, and collagen hydrolysate can be used as antiaging modalities in photoaged skin. However,
there is no clinical study using astaxanthin combined with collagen hydrolysate. We investigated the effects of using a
combination of dietary astaxanthin and collagen hydrolysate supplementation on moderately photoaged skin in humans. A
total of 44 healthy subjects were recruited and treated with astaxanthin (2 mg/day) combined with collagen hydrolysate (3 g/
day) or placebos, which were identical in appearance and taste to the active supplementation for 12 weeks. The elasticity and
hydration properties of facial skin were evaluated using noninvasive objective devices. In addition, we also evaluated the
expression of procollagen type I, fibrillin-1, matrix metalloproteinase-1 (MMP-1) and -12, and ultraviolet (UV)-induced
DNA damage in artificially UV-irradiated buttock skin before and after treatment. The supplement group showed significant
improvements in skin elasticity and transepidermal water loss in photoaged facial skin after 12 weeks compared with
the placebo group. In the supplement group, expression of procollagen type I mRNA increased and expression of MMP-1 and
-12 mRNA decreased compared with those in the placebo group. In contrast, there was no significant difference in
UV-induced DNA damage between groups. These results demonstrate that dietary astaxanthin combined with collagen
hydrolysate can improve elasticity and barrier integrity in photoaged human facial skin, and such treatment is well tolerated.
KEY WORDS: anti-aging astaxanthin collagen hydrolysate photoaging
INTRODUCTION
Photoaging is caused by the superpositioning of
chronic ultraviolet (UV)-induced damage on the intrin-
sic aging process and accounts for the majority of age-
associated changes in skin appearance. Aged skin, especially
photoaged skin, manifests as a decrease of skin thickness and
elasticity, skin dryness, epidermal barrier dysfunction, and
changes in pigmentation.
1
Reactive oxygen species (ROS)
likely contribute to this process.
2
UV radiation has numerous direct and indirect effects on
the skin.
2
The indirect damage induced by UV irradiation is
suggested to be initiated by ROS,
2
which are involved in
connective tissue alterations.
3
Numerous antioxidants have
been tested for their ability to prevent or reverse clinical
signs associated with photoaging secondary to ROS. Stra-
tegies utilizing endogenous skin antioxidants as well as
plant-derived or synthetic compounds have been examined.
2
Although both topical and systemic application of antiox-
idants can significantly increase antioxidant levels in skin,
systemically applied supplements and topical agents are often
combined to enhance efficacy through their synergistic af-
fects.
4
Furthermore, the systemic application of antioxidants
has produced more pronounced and sustained effects than
topical application.
5
Thus, antioxidants are one of the most
popular categories of nutraceutical ingredients used to im-
prove skin health. Among the various antioxidants, astax-
anthin has a long history of use as an antioxidant dietary
supplement
6
and its antioxidant properties can be 10-fold
greater compared with other carotenoids, such as lutein and
b-carotene, and 100-fold greater compared with a-tocopherol.
7
Collagen hydrolysate is also a popular nutraceutical, and
collagen polypeptides have exhibited numerous bioactivities,
including antioxidant activity, mineral binding capacity,
*These authors contributed equally to this work.
Manuscript received 10 September 2013. Revision accepted 1 May 2014.
Address correspondence to: Jin Ho Chung, MD, PhD, Department of Dermatology, Seoul
National University College of Medicine, 101 Daehak-ro Jongno-gu, Seoul 110-744,
Korea, E-mail: jhchung@snu.ac.kr
JOURNAL OF MEDICINAL FOOD
J Med Food 17 (7) 2014, 810–816
#Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2013.3060
810
antihypertensive activity, lipid-lowering effects, and immu-
nomodulatory activity.
8–10
Collagen hydrolysate has also
been shown to be involved in the synthesis of the extracellular
matrix and is used for improving pathological conditions
involving the joints, nails, and hair.
11
In the skin aging pro-
cess, collagen hydrolysate might be beneficial for slowing
chronological aging
11
and photoaging
8
in rats.
When used as nutraceuticals, antioxidants and collagen
hydrolysate have different mechanisms of action, but might
show additive or synergistic effects for preventing or re-
versing the skin aging process. Therefore, we investigated
the effects of a dietary antioxidant, astaxanthin, combined
with collagen hydrolysate on skin aging and UV-induced
damage in human skin in vivo.
MATERIALS AND METHODS
We conducted a 12-week, randomized, double-blind placebo-
controlled study to evaluate the effects of dietary astaxanthin
and collagen hydrolysate supplementation on cutaneous aging.
This study was approved by the Institutional Review Board
of Seoul National University Hospital, and written informed
consent was obtained from all subjects participating in the trial.
Study participants
A total of 44 healthy female volunteers, aged ‡40 years and
who had wrinkles ‡grade 2,
12
were enrolled in the study. The
subject exclusion criteria for the present trial were as follows:
(1) received medical or cosmetic treatment that interferes with
the general aging process within 3 months of the study; (2)
reported taking functional foods within 1 month of the study;
(3) history of acute or chronic disease such as severe liver or
kidney disease or uncontrolled diabetes; (4) history of allergies
against any component of trial foods; (5) any visible skin dis-
ease that might be confused with a skin reaction to the test
procedure or materials used, or interfere with clinical mea-
surements; and (6) abnormal blood test results (hemoglobin,
hematocrit, aspartate aminotransferase [AST], alanine trans-
ferase [ALT], or fasting glucose).
Dietary supplement
Two types of oral dosage forms (capsules containing as-
taxanthin and tablets containing fish collagen) were pre-
pared. The capsules were manufactured by Cerebos Pacific
Limited (Singapore), and each capsule included 480 mg of
medium chain triglycerides and 20 mg of dark red lipid ex-
tract of Haematococcus pluvialis microalgae, standardized
with safflower oil to yield a minimum 5% content of as-
taxanthin (Cyanotech Corporation, Kailua-Kona, HI, USA;
1 mg of astaxanthin per capsule). The tablets were manu-
factured by Cerebos Pacific Limited (Singapore), and each
tablet contained 0.75 g of enzymatic hydrolyzed fish colla-
gen (Rousselot SAS, Puteaux, France). Identical placebo
capsules with only medium chain triglycerides (500 mg per
capsule) and tablets with hydrolyzed casein (0.75 g per
tablet) instead of hydrolyzed collagen were prepared as
controls. All 44 study participants were randomly assigned
to either the placebo group or the supplement group. Each
participant took two capsules of astaxanthin and four tablets
of hydrolyzed collagen or the control capsules and tablets
per day for 12 weeks.
Noninvasive assessment
Clinical assessments were performed at baseline and at 4 and
12weeksoftreatment.Skinelasticityonthecheek(atpoint
3 cm inferior to the lower eyelid) was measured using a Cut-
ometer MPA580 (C +K Electronic, Cologne, Germany). Hy-
dration of facial skin was evaluated on the cheek (at point 5 cm
inferior to the outer corner of eye) measured using a Corne-
ometer and a Tewameter (C +K Electronic). All measurements
wereperformedinaroomwithaconstanttemperatureof
20–25C and a constant humidity of 45–55% at the Clinical
Research Institute, Seoul National University Hospital.
Compliance and safety
Adverse events related to the medications and clinical
protocol were evaluated at 4 and 12 weeks of treatment. The
subjects were instructed to return any remaining capsules or
tablets to the investigators, and subjects who failed to take
‡80% of the study medications were disqualified from fur-
ther participation. Blood samples were drawn at baseline and
12 weeks after the start of treatment, and AST, ALT, glucose,
hemoglobin levels, and hematocrits were measured.
UV irradiation and skin biopsy samples
Two 2-mm skin biopsy samples were obtained taken from
the buttock area at 24 h following UV irradiation at baseline
and after 12 weeks of supplementation in subjects who
agreed to biopsies (n=6/group). For these subjects, skin on
the buttock area was irradiated with two minimal erythema
doses at baseline and the same UV doses again at 12 weeks
of study participation. The UV source was a Waldmann UV-
800 (Waldmann, Villingen-Schwenningen, Germany; 285–
350 nm, peak at 310–315 nm) phototherapy device fitted
with Philips TL-20W/12 fluorescent lamps.
Specimens for immunohistochemical staining were im-
mediately oriented in a low-temperature embedding me-
dium (Tissue-Tek OCT compound; Miles, Naperville, IL,
USA), frozen in liquid nitrogen, and stored at -70C. Skin
samples saved for RT-PCR analysis were frozen in liquid
nitrogen.
Immunohistochemical staining
DNA damage induced by UV irradiation was evaluated
by immunohistochemical staining for the presence of thy-
mine dimers and 8-OHdG (8-hydroxy-2-deoxyguanosine),
as previously described.
13
Quantitative real-time RT-PCR
Expressions of procollagen type I, fibrillin-1, matrix
metalloproteinase (MMP)-1, and MMP-12 were evaluated
using quantitative real-time PCR. Total RNA was prepared
from skin samples and converted to complementary DNA
EFFECT OF DIETARY ASTAXANTHIN AND COLLAGEN ON PHOTOAGED SKIN 811
using the First Strand cDNA Synthesis Kit (MBI Fermentas,
Vilnius, Lithuania). Quantitation of procollagen type I,
MMP-1, and -12 cDNA, and endogenous reference 36B4
was performed using a 7500 Real-time PCR System (Ap-
plied Biosystems, Foster City, CA, USA) and SYBR Green
PCR Master Mix (Takara Bio, Inc., Shiga, Japan) using
the primers for human genes listed in Supplementary
Table S1 (Supplementary Data are available online at www
.liebertpub.com/jmf). The comparative C
T
method
14
was
used to quantify relative changes in gene expression.
Statistical analyses
The Mann–Whitney U test was used to identify differences
in values obtained by noninvasive measurements between
two groups (R2, R5, and R7 values; Corneometer values;
transepidermal water loss [TEWL] values). In addition, the
treatment effects at weeks 4 and 12 were further compared
using analysis of covariance (ANCOVA) to adjust for cor-
responding baseline values. The changes from baseline
values for expression of mRNAs for procollagen type I,
fibrillin-1, MMP-1, and MMP-12 were analyzed using the
Mann–Whitney U test. The SPSS version 20.0 (SPSS, Inc.,
Chicago, IL, USA) was used for all analyses. A Pvalue <.05
was considered statistically significant.
RESULTS
Subject demographics
This study enrolled 44 Korean women between the ages
of 41 and 60 years (mean 51.0 years, SD –5.2 years).
Baseline values for most parameters were not significantly
different between groups (Table 1). However, despite ran-
domization, baseline values for gross elasticity (R2 by
Cutometer) were significantly different between groups.
Skin elasticity as measured by Cutometer
An improvement in skin elasticity began to be noticeable
in the supplement group at 4 weeks and was more prominent
at 12 weeks. After 12 weeks of treatment, the mean im-
provement from baseline in elasticity was significantly
greater for the supplement group vs. the placebo group (R2,
0.0252 vs. -0.0294, P=.035; R5, 0.0602 vs. -0.0195,
P=.020; R7 0.0222 vs. -00185, P=.012, Fig. 1). No im-
provements in any parameters representing elasticity (R2,
R5, R7) were noted in the placebo group. Additionally, the
main parameters representing skin elasticity (R2, R5, R7)
significantly improved in the supplement group compared
with those in the placebo group when adjusted for the
baseline corresponding values using ANCOVA (Supple-
mentary Table S2).
Epidermal hydration by Corneometer and TEWL
(barrier integrity) by Tewameter
Figure 2 shows the changes in epidermal hydration pa-
rameters as measured by Corneometer after 4 and 12 weeks
Table 1. Baseline Demographics and Baseline Values
of Key Parameters
Control
group
(n=22)
Treatment
group
(n=22)
P
value
a
Age (years) 50.6 –5.3 51.5 –5.2 0.580
Weight (kg) 57.5 –6.5 55.3 –6.4 0.361
Epidermal hydration 44.5 –13.2 46.0 –14.0 0.573
TEWL (g/h/m
2
) 9.2 –2.8 10.5 –3.8 0.162
Gross elasticity, R2 0.6446 –0.0675 0.6097 –0.0556 0.044
Net elasticity, R5 0.5244 –0.0953 0.4799 –0.1076 0.146
Biological elasticity, R7 0.3028 –0.0502 0.2813 –0.0501 0.136
a
By Mann–Whitney Utest.
TEWL, transepidermal water loss.
FIG. 1. Results of skin elasticity measurements using Cutometer. Subjects receiving supplements showed significant improvements in the three
viscoelastic parameters compared with subjects in the control group. Pvalues by Mann–Whitney U test.
812 YOON ET AL.
of treatment. Epidermal hydration was not significantly
different between the two groups after 4 and 12 weeks. The
stratum corneum barrier in both groups was significantly
improved with TEWL decreasing from baseline values by
3.4 g/h/m
2
in the control group and 5.2 g/h/m
2
in the sup-
plement group at 12 weeks. This difference in changes be-
tween the two groups after 12 weeks of treatment was
statistically significant (P=.045 by Mann–Whitney U test,
Fig. 2) and remained significant after controlling for the
corresponding baseline TEWL values (P=.048 by ANCO-
VA, Supplementary Table S3).
Safety and compliance
Treatment was well tolerated and no subjective adverse
events were reported during the 12-week trial period. La-
boratory evaluations revealed no significant abnormalities
after 12 weeks of treatment. One subject who took only
64.3% of the supplements was dropped from the study, ac-
cording to the study protocol.
Real-time RT-PCR
Supplementing with astaxanthin combined with colla-
gen hydrolysate induced a 3.4-fold increase in procolla-
gen mRNA levels in UV-irradiated skin compared with
those in the placebo group (P=.038 by Mann–Whitney
Utest). Induction of fibrillin-1 mRNA in the supplement
group was greater than in the placebo group, but the
difference was not statistically significant. After UV ir-
radiation, expression of MMP-1 (collagenase) and MMP-
12 (elastase) mRNA was suppressed by 68% (P=.027)
and 77% (P=.050), respectively, in the supplement
group compared with those expressions in the placebo
group (Fig. 3).
UV-induced DNA damage
There were no differences between groups in immuno-
histochemical analyses of thymine dimers and 8-OHdG after
UV irradiation (Fig. 4).
FIG. 2. Results of epidermal hydra-
tion measured using Corneometer, and
skin barrier integrity measured with
Tewameter. TEWL from facial skin in
the supplement group was significantly
lower than those in the placebo group at
12 weeks. Pvalues by Mann–Whiney U
test. TEWL, transepidermal water loss.
FIG. 3. Results of procollagen type I, fibrillin-1, and matrix metalloproteinase-1 (MMP-1) and -12 mRNA induction after UV radiation. While
levels of procollagen type I mRNA in the supplement group increased, levels of MMP-1 and -12 mRNA in the supplement group decreased
significantly compared with those in the placebo group. Expression of mRNA was measured by real-time RT-PCR (n=6 for each group). Pvalues
by Mann–Whiney U test. UV, ultraviolet.
EFFECT OF DIETARY ASTAXANTHIN AND COLLAGEN ON PHOTOAGED SKIN 813
DISCUSSION
The latest trend in antiaging strategies for skin is to use a
combination of dietary and oral supplements to produce an
appearance benefit. These methods of treatment are thought
to work synergistically with topical agents to enhance effi-
cacy.
4
In the antiaging market, there are many nu-
traceuticals, which are products derived from food sources
and provide extra physiologic benefits in addition to their
basic nutritional values.
15
Recently, astaxanthin and colla-
gen hydrolysate have been described as beauty foods, which
have the potential to prevent skin aging.
4,16
We evaluated the effects of dietary supplementation with
the antioxidant astaxanthin combined with collagen hydro-
lysate on facial skin elasticity, hydration, and dermal matrix
homeostasis in photoaged skin. We found that 12 weeks
of oral dosing with astaxanthin plus collagen hydrolysate
improved elasticity and epidermal integrity in photoaged fa-
cial skin. Furthermore, the induction of procollagen type I
mRNA was observed after 12 weeks of treatment, along with
significant decreases in the expression of collagen-degrading
enzyme, MMP-1 mRNA and elastin-degrading enzyme,
MMP-12 mRNA after UV irradiation. The improvements in
facial skin elasticity might be related to these molecular
changes.
Astaxanthin (3,30-dihydroxy-b,b0-carotene-4,40-dione) is
one of the pigments that belongs to the xanthophyll subclass
of carotenoids and is widely distributed in marine organisms.
6
Astaxanthin has been used as a food supplement ever since
experimental studies revealed its antioxidant properties.
6
Further studies have suggested that astaxanthin has health-
promoting benefits for the treatment and prevention of vari-
ous diseases such as diabetes, cardiovascular diseases, and
inflammatory diseases.
17,18
In vitro, astaxanthin effectively
suppresses cell damage by free radicals and induction of
MMP-1 in skin after UV irradiation.
19,20
Additionally, topical
administration of astaxanthin prevented UV-induced skin
damage in mice.
21
These studies suggest that astaxanthin may
prevent tissue damage caused by UV irradiation.
Collagens are the most common family of proteins in the
human body and have been used as dietary supplements for
promoting articular function and for cosmetic purposes.
Ingestion of collagen peptide induces increased fibroblast
density and enhances formation of collagen fibrils in the
dermis in a protein-specific manner.
22
Collagen hydrolysate
has been demonstrated to have protective effects on chro-
nological skin aging by its influence on collagen matrix
homeostasis in rats.
11
The proposed mechanisms of action
for collagen polypeptide mainly involve enhancing immu-
nity, reducing the loss of moisture and lipids, promoting
antioxidative activity, and repairing endogenous collagen
and elastin protein fibers.
8
Consistent with these previous reports, oral dosing with
astaxanthin and collagen hydrolysate improved elasticity in
photoaged facial skin and suppressed UV-induced MMP-1
and -12 expression in human skin in vivo. However, despite
the known antioxidant properties of astaxanthin, DNA
damage caused by endogenous or exogenous ROS, as
measured by 8-OHdG staining, did not significantly differ
between groups in this study. Nevertheless, the suppression
of MMP expression in the supplement group suggests that
oral supplementation with astaxanthin and collagen hydro-
lysate partially protects skin from UV-induced damage.
In addition to the improved viscoelastic property of skin,
which is mainly due to the dermal extracellular matrix, we
also found improved barrier integrity as represented by de-
creased TEWL. Because collagen hydrolysate itself has
good moisture absorption and retention properties,
8
the
improvement of skin hydration might be related to the ef-
fects of collagen hydrolysate. In addition, astaxanthin might
contribute to the improvement in TEWL by protecting the
keratinocyte differentiation and cornification from oxidative
damages such as inflammation in epidermis.
23
This study has some limitations. First, we did not prove
the concentrations of individual ingredients in the skin.
FIG. 4. (A) Thymine dimer immunostaining before and after sup-
plementation. Nuclear staining of thymine dimer in UV-irradiated
buttock skin 24 h after UV irradiation. The figures are representative
images from six subjects (original magnification ·200). (B) 8-OHdG
immunostaining before and after supplementation. Nuclear staining
of 8-OHdG in UV-irradiated buttock skin 24 h after UV irradiation.
Figures are representative images from six subjects (original mag-
nification ·200). 8-OHdG, 8-hydroxy-2-deoxyguanosine.
814 YOON ET AL.
Previous studies only measured the serum levels of collagen
or astaxanthin. Ninety-five percent of enterally administered
collagen hydrolysate is absorbed within the first 12 h as 2.5–
15 kDa peptides.
24
Major collagen peptides in serum and
plasma were identified as proline-hydroxyproline; this was
identical to the abundance motif reported for collagen when
healthy human volunteers ingested several food-derived
collagen peptides.
25
In humans, the absorption of astax-
anthin after 4 h ranges from 6% to 34%.
26
It should be
elucidated whether the concentration of astaxanthin or col-
lagen-derived peptide in skin is great enough to exhibit
relevant biologic activity. Second, we did not examine
changes in glycosaminoglycans, including hyaluronic acid
in the skin. Ohara et al. reported that collagen-derived di-
peptide, which was detected in circulation after collagen
ingestion, stimulated proliferation and hyaluronic acid syn-
thesis in cultured dermal fibroblasts.
27
Because hyaluronic
acid is important for tissue elasticity and hydration,
28
the
improvement of elasticity might be related to hyaluronic
acid in the dermis.
Despite these limitations, the present study shows that a
combination of astaxanthin and collagen hydrolysate im-
proves elasticity and barrier integrity in human skin in vivo.
Skin elasticity especially began to improve after 4 weeks of
supplementation, and the effect was maintained with con-
tinued supplementation for 12 weeks. Further studies should
focus on the underlying mechanism that produces an im-
provement in skin conditions after dietary supplementation
with a combination of astaxanthin and collagen hydrolysate.
ACKNOWLEDGMENT
This study was funded by Cerebos Pacific Limited.
AUTHOR DISCLOSURE STATEMENT
The authors declare they have no conflicts of interest.
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