Skin Antiageing and Systemic Redox Effects of Supplementation with Marine Collagen Peptides and Plant-Derived Antioxidants: A Single-Blind Case-Control Clinical Study

Abstract

Recently, development and research of nutraceuticals based on marine collagen peptides (MCPs) have been growing due to their high homology with human collagens, safety, bioavailability through gut, and numerous bioactivities. The major concern regarding safety of MCPs intake relates to increased risk of oxidative stress connected with collagen synthesis (likewise in fibrosis) and to ROS production by MCPs-stimulated phagocytes. In this clinical-laboratory study, fish skin MCPs combined with plant-derived skin-targeting antioxidants (AO) (coenzyme Q
10
+ grape-skin extract + luteolin + selenium) were administered to volunteers (

n
=
41

). Skin properties (moisture, elasticity, sebum production, and biological age) and ultrasonic markers (epidermal/dermal thickness and acoustic density) were measured thrice (2 months before treatment and before and after cessation of 2-month oral intake). The supplementation remarkably improved skin elasticity, sebum production, and dermal ultrasonic markers. Metabolic data showed significant increase of plasma hydroxyproline and ATP storage in erythrocytes. Redox parameters, GSH/coenzyme Q
10
content, and GPx/GST activities were unchanged, while NO and MDA were moderately increased within, however, normal range of values.
Conclusions
. A combination of MCPs with skin-targeting AOs could be effective and safe supplement to improve skin properties without risk of oxidative damage.

Full text

Clinical Study
Skin Antiageing and Systemic Redox Effects of Supplementation
with Marine Collagen Peptides and Plant-Derived
Antioxidants: A Single-Blind Case-Control Clinical Study
Chiara De Luca,1Elena V. Mikhal’chik,2Maxim V. Suprun,2
Michael Papacharalambous,3Arseniy I. Truhanov,4and Liudmila G. Korkina4,5
1Evidence-Based Well-Being Ltd., 31 Alt-Stralau, 10245 Berlin, Germany
2Russian Institute of Physical Chemical Medicine, 1A Malaya Pirogovskaya Street, Moscow 117513, Russia
3Dermatology Clinic “Orthobiotiki Clinical”, 3-5 Sorou Street, 15125 Athens, Greece
4Active Longevity Clinic, Beauty Institute on Arbat, 8 Maliy Nikolopeskovskiy Lane, Moscow 119002, Russia
5Centre of Innovative Biotechnological Investigations (CIBI-Nanolab), 197 Vernadskogo Prospekt, Moscow 119571, Russia
Correspondence should be addressed to Liudmila G. Korkina; korkina@cibi-nanolab.com
Received  August ; Revised  December ; Accepted  December 
Academic Editor: Tiziana Persichini
Copyright ©  Chiara De Luca et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Recently, development and research of nutraceuticals based on marine collagen peptides (MCPs) have been growing due to their
high homology with human collagens, safety, bioavailability through gut, and numerous bioactivities. e major concern regarding
safety of MCPs intake relates to increased risk of oxidative stress connected with collagen synthesis (likewise in brosis) and to ROS
production by MCPs-stimulated phagocytes. In this clinical-laboratory study, sh skin MCPs combined with plant-derived skin-
targeting antioxidants (AO) (coenzyme Q10 + grape-skin extract + luteolin + selenium) were administered to volunteers (𝑛=41).
Skin properties (moisture, elasticity, sebum production, and biological age) and ultrasonic markers (epidermal/dermal thickness
and acoustic density) were measured thrice ( months before treatment and before and aer cessation of -month oral intake). e
supplementation remarkably improved skin elasticity, sebum production, and dermal ultrasonic markers. Metabolic data showed
signicant increase of plasma hydroxyproline and ATP storage in erythrocytes. Redox parameters, GSH/coenzyme Q10 content,
and GPx/GST activities were unchanged, while NO and MDA were moderately increased within, however, normal range of values.
Conclusions. A combination of MCPs with skin-targeting AOs could be eective and safe supplement to improve skin properties
without risk of oxidative damage.
1. Introduction
Dietary vitamins, plant-derived polyphenols, fatty acids, pro-
teins, essential amino acids, and trace elements have demon-
strated benecial eects on skin health and appearance [–
]; hence, the use of nutraceuticals targeting skin is stead-
ily growing. For example, photoprotection by intake of dieta-
ry antioxidants has been a subject of numerous in vitro,
animal, and human studies [, ]. Within this direction, the
search for reliable and eective antiageing remedies for
both topical and systemic administration has become a “hot
spot” for cosmetic, food, and biomedical companies. One
of the strongest limitations for skin-targeting plant-derived
dietary substances with potential antiageing ecacy is their
low bioavailability due to limited and selective penetration
through the intestinal barrier, destruction by the intestinal
microorganisms, high rate of metabolism, and preferential
distribution between tissues and organs [, ].
Marine sh collagen and collagen peptides have been
widely used as functional foods or dietary supplements due to
their homology to human collagen structure [], safety prole
[], stability, biocompatibility, high bioavailability through
gastrointestinal barrier [], and potent bioactivities [].
Marine collagen peptides (MCPs) obtained by enzymatic
digestion of sh skin have been shown to exert several health
eects mainly in two directions: metabolic disorders and
Hindawi Publishing Corporation
Oxidative Medicine and Cellular Longevity
Volume 2016, Article ID 4389410, 14 pages
http://dx.doi.org/10.1155/2016/4389410

Oxidative Medicine and Cellular Longevity
skin/bone repair. us, they positively aected glucose and
lipid metabolism in patients with type II diabetes mellitus
[],improvedlipidmetabolisminobesepeople[]andge-
netically modied mice [], ameliorated early alcoholic liver
injury [], and possessed hypotensive and lipid normalising
action in patients with primary hypertension []. A great
majority of publications demonstrated signicant wound
healing ecacy of orally administered MCPs in animal
models of excision and full-thickness skin wounds [, , ].
Recently, collagen peptides isolated by enzymatic digestion
from sh, bovine, and porcine skin as well as from chicken
and bovine cartilage have drawn particular interest for the
treatment of patients with osteoarthritis. Several clinical trials
showed that MCPs were safe and provided an improvement
in terms of pain and functions in such patients []. From
mechanistic point of view, the oral intake of MCPs stim-
ulated the synthesis of extracellular matrix (ECM) macro-
molecules such as endogenous collagen, by upregulating gene
expression of several collagen-modifying enzymes involved
in posttranslational collagen modication and cross-linking
[]. Several in vitro studies have shown antioxidant prop-
erties of very-low-molecular-weight (– Da) MCPs [, ]
containing proline, which is a scavenger of hydroxyl radicals.
Of importance for the present study, MCPs are considered
antiageing compounds because they seem to increase life
span in rats by inhibiting spontaneous tumour incidence [],
possess photoprotective and immunomodulating properties
[–], and improve/eliminate signs of premature senes-
cence of human skin [, ].
e major concern regarding safety and clinical feasibility
ofregularintakeofMCPshasbeenraisedfromthewellestab-
lished fact that the induction of collagen synthesis, mainly
assessed by the increased hydroxyproline levels, is oen
associated with oxidative stress [–]. Moreover, MCPs of
dierent origin have been shown to activate innate immune
response of macrophages and neutrophils through Toll-like
receptor , which leads to NADPH-oxidase (NOX) activa-
tion and reactive oxygen species overproduction [, ]. A
newly developed composition of MCPs with a complex of
essential skin-targeting antioxidants, that is, coenzyme Q10 +
selenium + luteolin + grape-skin extract, demonstrated UVA-
protective eects in the preliminary in vitro experiments on
humanskinbiopsies[].However,thecompositionunder
commercial name of CELERGENⓇhas never been evaluated
clinically when administered as a food supplement.
e goal of the present clinical-laboratory study was to
elucidate the eects of the oral administration of CELERGEN
on skin physiology and dermal collagen deposition in the
group of healthy middle-aged subjects with clinical signs
of skin ageing. e cutaneous clinical-instrumental data
were compared with the systemic metabolic parameters of
collagen synthesis, redox balance, and energy storage. For the
rst time, we demonstrated (i) remarkable improvement of
ageing skin physiology and structure, which corresponded
to enhanced systemic markers of collagen synthesis; (ii)
systemic redox balance, sustained by the antioxidant com-
plex; and (iii) increased systemic energy storage. We also
hypothesised that moderately increased plasmatic levels of
nitric oxide (NO) and malonyl dialdehyde (MDA) may play
positive roles of mediators in the MCPs-induced collagen
and ATP synthesis/storage, as well as in sebum production.
On these grounds, we suggested that selected antioxidants
targeting the distinct organs/tissues should be essential
components of MCPs-containing nutraceuticals for more
eective, individualised, and safe supplementation.
2. Materials and Methods
2.1. Patients. e study enrolled a group of  adult healthy
Caucasian volunteers of both sexes recruited from the Beauty
Institute on Arbat (Moscow, Russia) sta (age – years;
mean age . ±. years;  males and  females) following
the exclusion and inclusion criteria for an open single-blind
clinical study. e inclusion criteria were as follows: (i)
healthywhiteadultsubjectsofbothsexes,–yearsofage,
(ii) subjects with visible symptoms of aged facial skin, (iii)
subjects who agreed to interrupt any intake of antioxidant
nutraceuticals/drugs for at least  week before and during
the entire duration of the trial, and (iv) subjects without any
diculty to understand and follow the clinical investigator
instructions. Pregnant and breastfeeding women, subjects
with allergic/intolerance reactions to any component of the
tested product, subjects on any other nutraceutical interven-
tions or/and therapies, and subjects simultaneously engaged
in other clinical trials were excluded from the study. e
participants were informed that they could interrupt clinical
trial at any moment, without any explanation of causative
reason for their action, or in case they noticed any adverse
reaction to the tested product or had any sensation that the
product intake aected their appearance negatively.
e protocol of the clinical trial was duly analysed and
approved by the Ethical Committee of the Beauty Insti-
tute on Arbat, Moscow, Russia (number /EK-). All
recruited subjects gave their informed consent to personal
and anamnestic data collection and biological material sam-
pling. e guidelines of Helsinki Declaration for human
experimentation were strictly followed during the conduct of
the clinical trial.
2.2. Food Supplement under Investigation. Food supplement
containing marine collagen peptides derived from skin of
deep sea sh (MCPs,  mg), grape-skin extract ( mg),
coenzyme Q10 of plant origin ( mg), luteolin ( mg), and
selenium (. mg) of plant origin was formulated in so
gelatine capsules. As inactive solvents, rened and partly
hydrogenated soybean oil as well as small admixture of pure
soybean lecithin were used. e product, under the commer-
cial name of CELERGEN (manufacturer: Laboratories-Dom,
Carouge, Switzerland), was kindly provided by Suisse Ueli
Corporation. According to the manufacturer’s information,
thedeepseashsources,thatis,Pollachius virens,Hippoglos-
sus hippoglossus,andPleuronectes platessa,originatedfrom
the French coast of the North Sea.
Fish skin was homogenised in distilled water, with
addition of complex proteases. e enzymatic proteolytic
process was carried out at ∘C and pH . for  h, aer
which the proteases were inactivated by short-term heating
(∘Cformin).eliquidwassterilisedbyMillipore

Oxidative Medicine and Cellular Longevity 
ltration (pore size . mm) and spray-dried to prepare
MCP powder, as described in detail previously [, ].
Chemical analysis by Kjeldahl assay of the powder conrmed
a>% content of collagen peptides, with moisture and ash
content <%. According to previous publications [, ,
], the molecular weight distribution of MCPs aer the
described enzymatic digestion process was within the range
of–Da,andMCPswereenrichedinglycine,glutamine,
proline, hydroxyproline, asparagine, alanine, and arginine.
e aqueous extract of grape skin was obtained from Vitis
vinifera Linn. fruit and contained at least % of polyphenols
and % of procyanidins as per UV/Vis spectrophotometry
data.ecoenzymeQ
10 component of plant origin was
of highest purity (100 ± 3%), conrmed by both IR spec-
trophotometry and high performance liquid chromatography
(HPLC) methods. Food-quality luteolin was extracted from
Marigold plant petals, and the extract contained % of
luteolin and % of zeaxanthin evaluated by HPLC analysis.
Selenium in the form of selenite (according to gravimetric
method) was extracted from plant bulbs and leaves. Acute
and chronic toxicity data and documents of Certicates of
Analyses, Security, and Registration in Switzerland were duly
provided by the manufacturer.
2.3. Clinical Study Design. e entire trial duration was 
months(May–December),thatis,monthsofpretreat-
ment period, followed by  months of treatment with the
test nutraceutical administration. e facial skin parameters
of recruited volunteers were analysed three times: at the
rst visit (enrollment), at the second visit  months aer
the pretreatment period, and at the third visit immediately
aer the treatment period. Each assessment session com-
prised instrumental methods for measuring skin physiology
parameters and ultrasound properties of the skin layers. is
design allowed us to use the same subject as a control and
an experiment. During the treatment period, the volunteers
were recommended to take  capsules of CELERGEN a
day (at breakfast and dinner time) for  consecutive days.
At the second and third visits, the participants donated
 mL of venous blood aer overnight fasting and test tubes
were coded by the principal clinical investigator. Blood
samples were routinely processed for general haematology
(haemoglobin content, dierential cell count, and the rate of
erythrocyte sedimentation) and biochemistry (glucose levels,
plasma protein and lipid proles, transaminases activities,
and C-reactive protein content). Laboratory operators carried
out analytical determinations blindly, and statistician was not
informed which set of analyses was done in the control or
experimental periods, hence ranking this study of clinical
ecacy of the nutraceutical as “a single-blind” clinical inves-
tigation.
2.4. Assessment of Skin Physiology Parameters. Several phys-
iological parameters, mainly barrier properties, of the facial
skin were assessed by appropriate SOFT PLUS TOP probes,
with microcamera visual analysis and patented comput-
erised programs (Callegari, Parma, Italy). Skin elasticity was
determined by the elastometric approach used in the SOFT
PLUS technique. e transepidermal water loss (TEWL), an
index of skin moisture, was assessed with Tewameter, which
measures the water evaporation through cutaneous levels.
When the skin is aged or damaged, the barrier properties of
the skin are aected, with increased water evaporation and
reduced skin hydration. Sebum content was measured by the
SOFT PLUS sebometric probe.
2.5. Assessment of Ultrasound Properties of the Skin. Assess-
ment of ultrasound properties of the skin was performed by
a digital ultrasound imaging system DUB CUTIS (Digital
Ultraschall Bildsystem, Germany), which allowed determin-
ing four parameters simultaneously: epidermal and dermal
thickness and epidermal and dermal ultrasonic density. e
rst two parameters are indirect markers of collagen (dermis)
and lipid (epidermis) synthesis and retention while the
second pair of parameters characterises the evenness and
order in the epidermal and dermal structures, respectively.
e elastic properties of the skin were additionally analysed
by a TPM system containing elastometric sensor ( MHz)
which combines digital ultrasound examination with an
imaging record (DUB CUTIS, Germany). A computerised
multifunctional diagnostic tool integrating dierent morpho-
metric parameters (epidermal thickness, tone, wrinkles, and
elasticity) for face skin biological age determination was used
(SOFT PLUS TOP, Callegari, Parma, Italy).
2.6. Reagents and Assay Kits. e majority of chemical
reagents, HPLC standards, mediums, solvents, and luciferin-
luciferase for ATP assay were from Sigma Chemical Co. (St.
Louis, MO, USA); kits for enzyme activity assays and Griess
reagent for nitrites/nitrates determination were from Cay-
man Chemical Company (Ann Arbor, MI, USA). Manufac-
turers of other reagents are mentioned within the respective
methods.
2.7. Redox and Oxidation Markers’ Studies. Complete dier-
ential blood cell counts and metabolic analyses were per-
formed on fresh ethylenediaminetetraacetic acid- (EDTA-)
anticoagulated venous blood of  hrs fasting subjects. Bio-
chemical assays were performed on peripheral blood plasma
or red blood cells (RBC), either immediately (ATP, glu-
tathione, and coenzyme Q10)orwithinhrs,onsample
aliquots stored at −∘C under argon. Plasma levels of
nitrites/nitrates (NO2−/NO3−, expressed as 𝜇moles/L) were
measured spectrophotometrically by Griess reagent []. Pro-
tein content was measured according to Bradford [], using
a microplate assay kit (Bio-Rad, Hercules, CA, USA). Total
glutathione (reduced + oxidized glutathione, GSH + GSSG,
mg/g Hb) levels in erythrocytes were measured by HPLC
(Shimadzu Scientic Instruments, Columbia, MD, USA)
accordingtoReedetal.[].TotalcoenzymeQ
10 (CoQ10H2+
CoQ10, mg/L) levels in plasma were quantied by HPLC
as described previously []. In brief,  mL plasma sample,
with adequate amount of coenzyme Q9(internal standard)
and  𝜇L acetic acid (% solution), was extracted twice,
rst with . mL and then with . mL of ethanol/hexane
mixture ( :  vol/vol), with homogenisation and subsequent

Oxidative Medicine and Cellular Longevity
T : Subjective evaluation of the -month food supplement administration eects, by participants (𝑛=41).
Parameter Number (%) of participants
Improvement No eect Aggravation
General health conditions  (%)  (%)  (%)
Stamina/muscle strength/joint motility  (%)  (%)  (%)
Digestive system  (%)  (%)  (%)
Skin conditions  (%)  (%)  (%)
centrifugation. e upper phase containing hexane extract
was evaporated under nitrogen ux and then resuspended in
an adjusted amount of a methanol/isopropanol ( :  vol/vol)
mixture for HPLC analysis. Reduced and oxidized forms of
coenzyme Q10 (CoQ10H2and CoQ10)werequantiedsimul-
taneously with HPLC equipped with analytical Supelcosil LP-
 column ( cm ×. mm,  𝜇m, Supelco, Bellefonte, PA,
USA)plusitsguardcolumn,andinlinephotodiodearray
and electrochemical detector (ESA CoulArray, Bedford, MA,
USA) in accord with previously published methods [, ].
e clinical normality range was extrapolated from the above
publications.
Plasmatic Cu,Zn-superoxide dismutase  (Cu,Zn-SOD,
U/g protein) activity was measured spectrophotometrically
at  nm using appropriate kit from Cayman Chemical
Company (Ann Arbor, MI, USA) [, ]. RBC were lysed
in hypotonic solution and the postspin cell lysates were anal-
ysed. Total RBC glutathione-S-transferase (GST, U/mg Hb)
activity was measured spectrophotometrically by the meth-
ods described previously, using chloro-,-dinitrobenzene
as substrate []. RBC glutathione peroxidase (GPx, U/g
Hb) activity was determined using Cayman Chemical kit,
according to the method [].
Plasma levels of MDA were determined by slightly
modied spectrophotometric analysis of thiobarbituric acid-
reactive substances (TBARS) described elsewhere []. Aer
a  min treatment of plasma ( 𝜇L) with trichloroacetic
(. M) and hydrochloric (. M) acids, alkaline solution
of TBA was added and the mixture was boiled for  min.
TBARS were extracted with butanol and analysed spec-
trophotometrically at  nm. e results were expressed in
𝜇M of MDA using the appropriate calibration curve.
2.8. ATP Measurement in Erythrocytes.  𝜇Loferythrocyte
pellet was stored on ice until analysis. Ice-cold water ( 𝜇L)
was added to  𝜇L of the erythrocytes pellet and mixed
andthelysederythrocyteswerekeptonice.eprinciple
of ATP assay is based on the quantitative bioluminescent
determination of adenosine 5󸀠-12 triphosphate (ATP), assessed
by the Bioluminescence Assay Kit.Intheassay,ATPis
consumed when rey luciferase catalyses the oxidation of
D-luciferin to adenyl-luciferin which, in the presence of
oxygen, is converted to oxyluciferin with light emission. is
second reaction is essentially irreversible. When ATP is the
limiting reagent, the light emitted is proportional to the ATP
present. e measurements of luciferin-luciferase chemilu-
minescence were performed on a Victor  multilabel
counter, equipped with Wallac  Soware (Perkin Elmer,
MA, USA). Results were expressed as mmoles/L.
2.9. Hydroxyproline Assay. e plasma levels of free hydrox-
yproline (Hyp) and hydroxyproline in the form of oligopep-
tides, mainly proline-hydroxyproline, were determined by
a chemical colorimetric method using a commercial kit
(Hydroxyproline Detection Kit) in accord with the manufac-
turer’s instructions. Hyp concentrations were quantied in
the linear range of its calibration curve using an array reader
(Bio-Rad, Hercules, CA, USA) and expressed in 𝜇g/mL of
plasma.
2.10. Statistical Analysis. Statistical analysis of clinical data
was carried out using WINSTAT programs for personal
computers (Statistics for Windows , Microso, USA).
All biochemical and molecular measurements were done in
triplicate and data were statistically evaluated. Values were
presented as mean, standard error of the mean, and . ×
standard error of triplicate analyses. When several datasets
were compared, data were analysed by Student’s 𝑡-test for
unpaired data. Dierences between initial/nal data for a
single participant were analysed by paired 𝑡-test and by
Mann-Whitney test for changes from baseline. All reported
𝑝values are from two-tailed tests, and 𝑝values of less than
. were considered to indicate statistical signicance.
3. Results
3.1. Subjective Evaluation by Participants and Clinical Inves-
tigators. All healthy volunteers (𝑛=41) recruited in the
trial duly completed it. ere were no drop-os due to
low compliance or adverse eects of the supplementation.
Routine haematological and biochemical analyses, which
were carried out aer blood donation in the beginning and
aer the cessation of the study, did not show statistically
signicant changes possibly reecting adverse consequences
of the test nutraceutical in the prescribed dosages (data not
shown).esubjectiveevaluationoftheproducteectson
selected general health parameters is shown in Table . e
participants were predominantly satised with the eects
obtained on general health conditions and skin properties
and partly also by enhanced muscle strength and stamina. No
eect whatsoever on digestion was registered.
3.2. Eects on Facial Skin Properties. Comparison of digital
photos taken before and aer clinical trial showed visible

Oxidative Medicine and Cellular Longevity 
(a) (b)
(c)
F : Digital images of facial skin ultrasound examinations (patient number , e.g.), made  months before the beginning of the trial
(a), at the day of the trial beginning (b), and immediately aer the trial cessation (c).
qualitative improvement of aesthetic aspect of face with
pronounced liing eect (data not shown).
Characteristic digital images of ultrasound examinations
made at trial beginning ( months before the beginning of
supplementation), at the rst day of nutraceutical adminis-
tration, and immediately aer the trial cessation are shown,
respectively, in Figures (a), (b), and (c). e individual
ultrasonic characteristics were rather stable and were not
subjected to statistically signicant changes during the 
months of pretreatment period (Tables  and ; compare
columns  and ). e analysis of individual data showed
that highly enhanced dermal thickness and homogenous
distribution of collagen bers in dermis were detectable in
% (𝑛=11) of the participants aer the trial cessa-
tion. Statistical evaluation of dermal thickness and acoustic
density revealed signicant changes exclusively at the third
visit (Table ), while the ultrasonic properties of epidermis
remained unchanged (Table ).
Analyses of the main physiological parameters of the
skin relevant to ageing, such as elasticity, moisture, and
sebum content, demonstrated their comparative stability in
the pretreatment period, as there were no signicant changes
between the rst and the second sets of measurements
(Table , columns  and ). Conversely, CELERGEN adminis-
tration statistically signicantly enhanced skin elasticity and
sebum production (𝑝 < 0.0001), whilst not inuencing
cutaneous moisture (Table , columns  and ). Biological
T : Eects of the -month food supplement administration on
the ultrasonic properties of the dermis (𝑛=41).
Parameter
Dermis
Pretreatment
period
Before
treatment
Aer
treatment
ickness, 𝜇m ±  ±  ±∗
Acoustic density . ±. . ±. . ±.∗
∗𝑝 < 0.05 versus “before treatment.”
T : Eects of the -month food supplement administration on
the ultrasonic properties of the epidermis (𝑛=41).
Parameter
Epidermis
Pretreatment
period
Before
treatment
Aer
treatment
ickness, 𝜇m. ±. . ±. . ±.
Acoustic density . ±. . ±. . ±.
age, calculated on the basis of ultrasound and cutaneous
physiology measurements, tended to decrease aer the trial;
however, the dierence did not reach statistical signicance.
It should be noticed that all tested parameters of skin
physiology and structure were not subjected to temporal
uctuations during the -month pretreatment period, and

Oxidative Medicine and Cellular Longevity
T : Eects of the -month food supplement administration on the parameters of skin physiology (𝑛=41).
Parameter Pretreatment period Before treatment Aer treatment
Elasticity . ±. . ±. . ±.∗∗∗
Moisture . ±. . ±. . ±.
Sebum . ±. . ±. . ±.∗∗∗
Skin biological age . ±. . ±. . ±.
∗∗∗𝑝 < 0.0001 versus “before treatment.”
therefore changes observed can be viewed as a result of
CELERGEN administration.
3.3. Plasmatic Oxidation Markers and Antioxidants. Sur-
prisingly, CELERGEN administration did not aect several
markers of glutathione metabolism such as total glutathione
levels (normality range: .–. mg/g Hb) and glutathione-S-
transferase and glutathione peroxidase activities (normality
ranges: .–. U/mg Hb and .–. U/g Hb, resp.) (Fig-
ures (a), (b), and (c)). At the same time, nitrite/nitrate and
MDA levels in plasma (normality ranges: .–. 𝜇Mand
.–. 𝜇M, resp.) were statistically signicantly increased
(𝑝 < 0.05 and 𝑝 < 0.0001, resp.), although they remained
within normal physiological range established in our lab-
oratory (Figures (a) and (b)). Extracellular Cu,Zn-SOD
activity was slightly suppressed (𝑝 < 0.001) but did not drop
below the normality border (.–. U/mL) (Figure (c)).
3.4. Parameters of Collagen and ATP Metabolism. Plasma
contentofhydroxyprolinewasfoundhighlyelevated(𝑝<
0.01) (Figure (a)), the same with ATP content in erythro-
cytes (𝑝 < 0.001) (Figure (c)), although total content of
coenzyme Q10 was not changed aer supplementation with
thecoenzymeQ
10-containing nutraceutical (Figure (b)).
4. Discussion
In a preliminary ex vivo study of CELERGEN components
against UVA-induced damage in human skin biopsies and
broblasts [], marine collagen peptides but not the complex
of plant-derived antioxidants inhibited transcriptional and
posttranscriptional matrix metalloproteinase- and elastase
upregulation, leading the authors to hypothesise clinical
feasibility for the prevention of skin photoaging. In contrast,
another publication demonstrated that the bioavonoid lute-
olin, a component of the CELERGEN antioxidant complex,
eectively attenuated UVB-induced DNA damage, inam-
mation, and ROS overproduction in skin cells in vitro and in
vivo [].
In the present study, we obtained convincing clinical
data on the ecacy of the marine collagen peptide and
plant antioxidant formulation CELERGEN in improving
dermal collagen deposition and structure (Table ), as well
as skin elasticity (Table ). ese eects were consistent
with enhanced plasma levels of hydroxyproline, a systemic
metabolic marker of collagen synthesis (Figure (a)). Nearly
% of human Hyp is in fact found in collagen []. Hyp
being an oxidative derivative of proline, both amino acids
are essential for collagen biosynthesis, maturation, mode
of deposition, and collagen ber structure. Dietary proline
intake promotes tissue repair in humans and animals [].
Recently, Wang et al. [] reported the experimental evidence
that MCPs might improve collagen synthesis and maturation
by inducing the expression of transforming growth factor
beta- (TGF-𝛽) and basic broblast growth factor (bFGF).
Our data (Figure (a)) are consistent with previously pub-
lished ones on rats fed with MCPs from salmon or trout
skin [], showing that plasma levels of free and dipeptide
(Pro-Hyp) forms of hydroxyproline were highly increased
aer single intake of MCPs in soybean oil. Similar data on
the blood levels of Hyp and Hyp-containing peptides were
obtained on healthy human volunteers [].
Numerous animal studies on the eects of oral adminis-
tration of natural or synthetic antioxidants towards collagen
deposition, reactive species levels, and antioxidant defences
generated highly conicting data, depending on the exper-
imental system. us, with various wound healing models,
it was repeatedly demonstrated that either complex plant
extracts containing active secondary metabolites (triterpenes,
polyphenols, alkaloids, etc.) [, ] or a composition of
collagen inducing polysaccharides like chitosan and antiox-
idants such as curcumin [] or resveratrol [] ameliorated
wound healing increasing skin collagen deposition, while
suppressing proinammatory iNOS and myeloperoxidase,
decreasing pathologically elevated levels of MDA and hydro-
gen peroxide, and improving enzymatic antioxidant defence.
Recent studies showed that collagen peptides from sh skin
remarkablypromotedbothwoundhealingandangiogenesis
in dierent experimental settings [, ]. Of importance,
excessive NO produced during the inammatory phase
of wound healing process impaired collagen accumulation
[], while moderate NO levels accelerated the granulation
phaseofwoundclosure[,].Moreover,woundhealing
acceleration by moderate levels of H2O2through induction
of vascular endothelial growth factor in keratinocytes and
macrophages was proved in a number of experimental and
clinical studies [, ]. Here, we found that, along with Hyp
accumulation, plasma levels of nitrites and nitrates, related
to NO production in the bloodstream, were moderately
increased aer CELERGEN treatment, though remaining
within the range of normal values (Figure (a)). Similar
results were obtained with plasmatic MDA (Figure (b)). is
allowedustosuggestthatredoxregulationofcutaneous
collagen synthesis process or/and broblast proliferation acti-
vation could have occurred due to physiologically relevant
NO and/or MDA amounts generated following supplement

Oxidative Medicine and Cellular Longevity 
Glutathione (GSH + GSSG, RBC, mg/g Hb)
0.96
1.00
1.04
1.08
1.12
1.16
1.20
Before Aer
Mean
Mean ±1.96 ∗ ES
Mean ±ES
(a)
GST activity (RBC, U/mg Hb)
Before Aer
Mean
Mean ±1.96 ∗ ES
Mean ±ES
0.36
0.38
0.40
0.42
0.44
0.46
0.48
(b)
GPx activity (RBC, U/g Hb)
10
20
30
40
50
60
70
Before Aer
Mean
Mean ±1.96 ∗ ES
Mean ±ES
(c)
F : Glutathione cycle parameters: erythrocyte levels of total glutathione (reduced and oxidized forms, GSH + GSSG) (a) and erythrocyte
enzymatic activities of glutathione-S-transferase (b) and of glutathione peroxidase (c) in the study group of patients (𝑛=41), before and aer
food supplement administration period. Values are represented as mean (◻), standard error of the mean (upper and lower limits of the box),
and . ×standard error (upper and lower whiskers). GSH: reduced glutathione; GSSG: oxidized glutathione; RBC: red blood cells; Hb:
haemoglobin; GST: glutathione S-transferase; GPx: glutathione peroxidase. Reference normality range: RBC total glutathione (.–.mg/g
Hb); RBC GST activity (.–. U/mg Hb); RBC GPx activity (.–. U/g Hb).
intake. However, the suggestion deserves further mechanistic
in vitro and clinical research.
On the other hand, in the models of cardiac brosis [,
],thesignicantdecreaseofthemodel-relatedoxidative
stress obtained by the use of Momordica charantia fruit
extract [] or Fructose Chorpondiatis total avonoids was
indeed associated with simultaneous attenuation of collagen
deposition, as assessed by Hyp levels. Similar results were
obtained in other tissue models of brosis [, –],
including skin brosis []. It seems that complex mixtures
of fruit extracts contained both collagen synthesis aecting
agents and antioxidants.
UV irradiation could cause skin photodamage causing
the symptoms of premature photoageing. Evaluating the
photoprotective eects of dietary MCPs isolated from jel-
lysh umbrella [] or from sh scale [] in the model

Oxidative Medicine and Cellular Longevity
Before Aer
105
120
135
150
165
180
195
Mean
Mean ±1.96 ∗ ES
Mean ±ES
Nitrites/nitrates (NO2+NO3, plasma, 𝜇M)
p < 0.02706
(a)
Before Aer
Mean
Mean ±1.96 ∗ ES
Mean ±ES
MDA (plasma, 𝜇M)
p < 0.000000001
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
(b)
Cu,Zn-SOD3 (plasma, U/mL)
Before Aer
Mean
Mean ±1.96 ∗ ES
Mean ±ES
p < 0.00035
6
7
8
9
10
11
12
13
(c)
F : Systemic oxidative stress markers: plasma levels of nitrites/nitrates (NO2+NO
3) (a), of malonyl dialdehyde (MDA) (b), and of
Cu,Zn-superoxide dismutase  (Cu,Zn-SOD) (c) in the study group of patients (𝑛=41), before and aer food supplement administration
period. Values are represented as mean (◻), standard error of the mean (upper and lower limits of the box), and . ×standard error
(upper and lower whiskers). Intergroup signicant dierences (𝑝) are indicated in the relative panels. NO2+NO
3: nitrites + nitrates; MDA:
malonyl dialdehyde. Reference normality range: plasma NO2+NO
3(.–. 𝜇M); plasma MDA (.–. 𝜇M); plasma Cu,Zn-SOD (.–
. U/mL).
of chronic UVA + UVB irradiation of mice, the authors
concluded that MCPs enhanced skin immunity, reduced
water loss, restored cutaneous collagen and elastin levels and
structure, and maintained type III to I collagen ratio. Under
similar experimental design, Zhuang et al. [] showed the
protective action of MCPs on antioxidant enzymes activities
and glutathione, lipid, and Hyp contents of murine skin. In
this connection, we found a signicant reduction (within
the range of normality) of plasmatic SOD activity follow-
ing CELERGEN supplementation (Figure (c)). Extracellu-
lar plasmatic Cu,Zn-SOD, a glycoprotein with a heparin-
binding domain, is predominantly expressed in tissue ECM,
where it is bound to heparin sulfate proteoglycan [].
Physiologically, SOD maintains redox balance and tissue

Oxidative Medicine and Cellular Longevity 
Hydroxyproline (plasma, 𝜇g/mL)
0.8
0.9
1.0
1.1
1.2
1.3
1.4
Before Aer
Mean
Mean ±1.96 ∗ ES
Mean ±ES
p<0.0068
(a)
Coenzyme Q10 (CoQ10H2+CoQ10, plasma, mg/L)
Before Aer
Mean
Mean ±1.96 ∗ ES
Mean ±ES
0.44
0.48
0.52
0.56
0.60
0.64
0.68
(b)
ATP (RBC, mM)
Before Aer
Mean
Mean ±1.96 ∗ ES
Mean ±ES
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
p<0.00125
(c)
F : Metabolic parameters related to collagen and ATP synthesis: levels of plasma hydroxyproline (a) and of the lipophilic antioxidant
total coenzyme Q10 (reduced and oxidized forms, CoQ10H2+CoQ
10) (b) and erythrocyte ATP (c) in the study group of patients (𝑛=41),
before and aer food supplement administration period. Values are represented as mean (◻), standard error of the mean (upper and lower
limits of the box), and . ×standard error (upper and lower whiskers). Intergroup signicant dierences (𝑝) are indicated in the relative
panels. CoQ10H2: reduced coenzyme Q10;CoQ
10: oxidized coenzyme Q10; ATP: adenosine triphosphate; RBC: red blood cells. Reference
normality range: total coenzyme Q10 (.–. mg/L); ATP (.–. mM).
homeostasis and modulates innate and adaptive immune
responses. Cutaneous homeostasis strongly depends on the
ECM microenvironment; therefore, an elevated SOD activ-
ity may be a marker of adaptive response against intrinsic age-
associated and external hazardous factors inducing immune
suppression in the skin [].
Since the supplementation of compounds with a direct
antioxidant eect has failed so far to show clinical ecacy and
sometimes even aggravated clinical picture [], the search
for drugs/therapeutic strategies to modulate oxidative stress
has been drastically redirected nowadays towards () indirect
AOs inducing endogenous enzymatic system of antiox-
idative defence, mainly, through Nrf-connected pathway;
() selective inhibitors of ROS/RNS-producing enzymes,
for example, dierent isoforms of NADPH-oxidase, having
shown denite clinical eects; () recognising essential and
multiple physiological roles of redox balancing agents rather
than mere inhibitors of free radical processes. Plant-derived

 Oxidative Medicine and Cellular Longevity
MCPs
AOs
GI Circulation Skin
AOs
RNS NO
ROS
MCPs
TLR4
TLR4
AOs
Hyp
Hyp-ox
Hyp
Pro-Hyp
E
M
GROS, Sq-ox, PUFA-ox,
?
Sebum production ↑
Elasticity ↑
Collagen deposition ↑
SOD3↑
GST ↑
GPx ↑
GSH ↓
MDA, 4-HNE, and so forth
↗
OH∙
Pro-Hyp
F : Scheme of the hypothesised redox-dependent mechanisms of CELERGEN physiological eects. Marine collagen peptides (MCPs)
easily penetrate gastrointestinal wall (GI, three arrows) and through blood circulation are mainly deposited in the skin. Antioxidant
component of the nutraceutical is partly metabolised in GI thus possessing low bioavailability (one arrow); however, skin-targeting
antioxidants and their metabolites reach dierent skin layers. While in the circulation, MCPs stimulate blood phagocytes (granulocytes
and monocytes) and endotheliocytes (E) to produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) by activating
Toll-like receptors  (TLR). Hydroxyproline (HYP) and prolyl-hydroxyproline (Pro-HYP) dipeptides as major components of MCPs are
metabolised by corresponding oxidases and hydroxyl radicals are formed as by-products. Antioxidants prevent systemic oxidative stress
blocking GSH oxidation, GPx, GST, and SOD activation. In the skin, collagen synthesis and deposition as well as elasticity areincreased while
(hypothetically) low levels of oxidised forms of skin lipids such as unsaturated fatty acids (PUFA-ox), squalene (Sq-ox), malonyl dialdehyde
(MDA), and -hydroxy--nonenal (-HNE) may facilitate cell signalling for ATP synthesis and sebum production.
polyphenols, quercetin, resveratrol, luteolin, and many others
appear to possess all these multipotent capabilities []. e
presence of quercetin and resveratrol from grape-skin extract
and of luteolin in the antioxidant combination of CELERGEN
maythenwellaccountfortheobservedredoxbalancing
eects during the upregulation of MCPs-induced collagen
synthesis (Figures (a), (b), and (c)). e majority of
publications have in fact demonstrated a drop of GSH content
and an increase of protective GPx activity when Hyp content
was raised [, , ]. Of great importance, the presence
of antioxidants in the tested formulation, whilst possibly
protecting the redox balance from harmful side eects of
collagen metabolism, did not negatively aect the desired
process of dermal collagen synthesis/deposition. In fact, the
observed elevation of plasmatic Hyp was comparable with
that found previously [], with pure sh MCPs in much
higher dosages.
In the last decade, endogenously produced systemic and
cutaneous redox-active substances (superoxide, hydrogen
peroxide, NO, lipid peroxides, stable end products of lipid
peroxidation, oxidative metabolites of cholesterol and squa-
lene, etc.), previously recognised exclusively as undesirable
metabolic by-products and markers of oxidative damage,
have shown essential functions in cellular signalling and reg-
ulation of cell proliferation, dierentiation, migration, innate
immunity, energy production, ECM dynamics, vascular tone,
stress responses and adaptation, and inammation [, –
]. In this frame, the moderate plasmatic elevation of MDA
(Figure (b)) and NO (Figure (a)) observed in this study
may reect the regulatory functions of these mediators both
in MCPs-induced collagen synthesis (Figure (a)) [] and in
the process of mitochondrial ATP production (Figure (c)).
Only excessive amounts are damaging as they initiate cell
senescence and death. It seems that, notwithstanding coen-
zyme Q10 supplementation during CELERGEN course, its
plasmatic levels were not increased (Figure (b)), due to
elevated consumption of the coenzyme in the mitochon-
drial cycle of energy production enhancing ATP storage in
erythrocytes (Figure (c)) and in cell redox balance control
(Figures (a)–(c)).
e antiageing eects of CELERGEN supplementation
were evidenced also by the highly increased sebum pro-
duction (Table ). It is established that the production of
sebaceous lipids is strongly age dependent, being low in
the prepubertal period, rising with sexual maturation, and
gradually declining in the aging populations (starting from
– years) [] or in UV-induced premature skin age-
ing [, ]. Cutaneous lipid-soluble antioxidants such as
vitaminEandsqualenedecayaccordingly[].Sinceskin
surface lipids (SSL) play multiple essential roles in skin
barrier properties, skin smoothness, elasticity, and moisture,
they are regarded as natural guards of normal cutaneous
ecology. Moreover, moderate concentrations of specic SSL
unsaturated components (squalene, cholesterol, and free fatty
acids) are able to generate oxidised lipid by-products (MDA,
-hydroxynonenal, oxidised cholesterol, and others), since
being long recognised as key signalling molecules for skin
immune and metabolic responses to environmental insults
andmicrobialinvaders[,,].Ontheotherhand,
excessive levels of microbially or photooxidised derivatives
of unsaturated fatty acids and other sebum lipids could
induce a vicious cycle of sebum overproduction followed by

Oxidative Medicine and Cellular Longevity 
oxidation, thus maintaining inammation characteristic for
acne disease []. As shown by our clinical data (Table ), no
complaints about skin conditions were registered during the
trial. Conversely, the marked improvement of skin elasticity
could be attributed not only to collagen deposition in derma,
but also to a moderate physiological increase of SSL content.
On the grounds of the results obtained and existing
literature data, we hypothesised redox-dependent pathways
(Figure ) which may lead to clinical and generalised health
eects of CELERGEN supplementation. Obviously, more
profound basic research and further clinical studies are
needed to prove this hypothesis and to evaluate the under-
lying mechanisms.
5. Conclusions
e addition of dietary plant-derived antioxidants with
known skin tropism and health eects towards human skin
did not impair denite induction of collagen synthesis and its
deposition as compact organised bres in the dermal layer by
marine sh skin-derived collagen peptides. Additional ben-
ecial eects of antioxidants were observed systemically, as
normal balance of systemic endogenous antioxidant defence
was maintained, and protection of energy storage occurred.
Abbreviations
MCPs: Marine collagen peptides
ECM: Extracellular matrix
NOX: NADPH-oxidase
NO: Nitric oxide
MDA: Malonyl dialdehyde
HPLC: High performance liquid chromatography
TEWL: Transepidermal water loss
EDTA: Ethylenediaminetetraacetic acid
RBC: Red blood cells
Cu,Zn-SOD: Cu,Zn-superoxide dismutase 
GST: Glutathione-S-transferase
GPx: Glutathione peroxidase
TBARS: iobarbituric acid-reactive substances
Hyp: Hydroxyproline
TGF-𝛽: Transforming growth factor beta-
bFGF: Basic broblast growth factor
ROS: Reactive oxygen species
RNS: Reactive nitrogen species.
Conflict of Interests
e authors declare that they have no conict of interests.
Acknowledgments
e authors gratefully acknowledge Suisse Ueli Corporation
for providing the product for the clinical study free of charge
andforcoveringthecostsofreagentsandanalyses.ey
are also grateful to Elena Schukina for excellent technical
assistance and to Valeriy Chertushkin for organisation of the
trial logistics.
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