nutrients

Article
A Collagen Supplement Improves Skin Hydration,
Elasticity, Roughness, and Density: Results of a
Randomized, Placebo-Controlled, Blind Study
Liane Bolke 1 , Gerrit Schlippe 1 , Joachim Gerß 2 and Werner Voss 1, *
1 Dermatest GmbH, Engelstraße 37, D-48143 Münster, Germany; dr.bolke@dermatest.de (L.B.);
dr.schlippe@dermatest.de (G.S.)
2 Institut für Biometrie und klinische Forschung (IBKF) der Westfälischen Wilhelms-Universität Münster,
Schmedding Straße 56, D-48149 Münster, Germany; joachim.gerss@ukmuenster.de
* Correspondence: dr.voss@dermatest.de; Tel.: +49-251-481637-20




Received: 7 August 2019; Accepted: 8 October 2019; Published: 17 October 2019


Abstract: The purpose of this randomized, placebo-controlled, blind study was to investigate the
effects of the drinkable nutraceutical ELASTEN® (QUIRIS Healthcare, Gütersloh, Germany) on skin
aging and skin health. Drinking ampoules provides a blend of 2.5 g of collagen peptides, acerola
fruit extract, vitamin C, zinc, biotin, and a native vitamin E complex. This controlled interventional
trial was performed on 72 healthy women aged 35 years or older. They received either the food
supplement (n = 36) or a placebo (n = 36) for twelve weeks. A skin assessment was carried out and
based on objective validated methods, including corneometry (skin hydration), cutometry (elasticity),
the use of silicon skin replicas with optical 3D phase-shift rapid in-vivo measurements (PRIMOS)
(roughness), and skin sonography (density). The verum group was followed for an additional four
weeks (without intake of the test product) to evaluate the sustainability of the changes induced by
the intake of the test product. The test product significantly improved skin hydration, elasticity,
roughness, and density. The differences between the verum group and the placebo group were
statistically significant for all test parameters. These positive effects were substantially retained
during the follow-up. The measured effects were fully consistent with the subjective assessments of
the study participants. The nutraceutical was well tolerated.

Keywords: aging; beauty; bioavailability; collagen peptides; cutometry; corneometry; wrinkles; high
coverage [HC] collagen complex

1. Introduction
Healthy skin provides an active interface between the internal and external environments of
the body and enables permanent adaptation and acclimatization of an organism during its lifetime.
Many different factors exacerbate the aging process of the skin, including intrinsic ageing, irradiation,
consumption of a non-balanced diet, and stress-related deficiencies in micronutrients [1–3], leading to
an age-dependent collagen loss in the skin.
Collagen, the most abundant component of the extracellular matrix, is the decisive protein that
determines skin physiology, by maintaining the skin structure and enabling its numerous functions to
take place [4–6]. The extracellular matrix retains water and supports a smooth, firm, and strong skin.
The structure of collagen is reminiscent of a rope. Three chains wind around each other forming a
collagen triple helix. These building blocks combine to form collagen fibrils of enormous strength and
tensile force [4–6].
Studies have shown that age-dependent reduction in collagen synthesis can be reversed by oral
administration of specific bioactive collagen peptides [4,7–11]. These oligopeptides are obtained by

Nutrients 2019, 11, 2494; doi:10.3390/nu11102494 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 2494 2 of 14

enzymatic hydrolysis of natural collagen. After ingestion, they are further metabolized to bioactive
di- and tri-peptides in the gastrointestinal tract, which are then released into the blood stream and
accumulated in the skin to form the collagen biomatrix [4,7,9]. Typical collagen hydrolysates are
composed of peptides of different lengths and, depending on the collagen source, are characterized by
a special amino acid composition. The unique (high coverage—[HC]) collagen complex (ELASTEN® )
tested here contains short chain oligopeptides composed of 5 to 8, 9 to 15, and 16 to 26 amino acids and
have a high coverage with amino acids sequences found in human skin collagen proteins.
External and internal factors drive the physiological process of skin aging associated with a decline
in collagen formation [12,13]. The collagen content of young and healthy skin has been demonstrated
to exceed 75% [4–6,12,13]. Collagen fibers are synthesized primarily by the fibroblasts in the deeper
layers of the skin. As such, rejuvenation of the biomatrix can be effectively improved only through a
supply of sufficient nutrients via the blood stream [2]. Collagen formation is diminished in mature
skin and the biomatrix of the skin begins to collapse when the collagen scaffold loses its strength and
stability [5,7,9,14]. Factors such as sunlight, smoking, environmental pollution, alcohol abuse, and
nutrient deficiency can accelerate this process [12,13]. The elasticity is then diminished, and lines and
wrinkles emerge. Due to the loss of collagen, the skin becomes increasingly thinner and drier.
A previous pilot study demonstrated that the oral intake of special bioactive collagen peptides,
also tested in the present trial, induce beneficial effects on human skin structure and function [8]. In
this former non-controlled pilot trial, which was conducted in 16 healthy women, ELASTEN® was
orally applied for 3 months. The study showed significant and sustainable improvements of skin
hydration, elasticity, and roughness. The present study was performed to confirm the results obtained
in the pilot trial under randomized, placebo-controlled conditions. The peptides provided by the food
supplement ELASTEN® are characterized by a high contribution of amino acid sequences from human
collagen, and are combined with specific synergistically acting dermonutrients.

2. Materials and Methods

2.1. Study Design and Ethical Aspects

This clinical study employed a randomized, placebo-controlled, single-blind design. It was
conducted according to the applicable principles of good clinical practice (GCP) [15]. The clinical study
was registered in the German Clinical Trial Register and at the International Clinical Trials Registry
Platform (no. DRKS00015664). The investigation was in full compliance with the principles outlined in
the Declaration of Helsinki and with national regulations of Germany. A written informed consent
was received from all volunteers. The trial protocol was approved by the Freiburg Ethics Commission
International, Freiburg, Germany on 11 September 2017.

2.2. Study Participants

A total of 72 female subjects aged 35 years or more with healthy skin of any type participated in
this study. The main exclusion criteria were as follows: Major or chronic skin diseases, major internal or
chronic diseases, intake of drugs with any impact on skin reactions (e.g., glucocorticoids, antiallergics,
and topical immunomodulators), application of preparations and care products containing active
substances 7–10 days before participating in the study, heavy allergies or the occurrence of severe
adverse events due to cosmetics in the past, sunbathing or solarium visits during the study, known
cancer, pregnancy, and lactation.
Participants were instructed to maintain their living habits 9 and to not begin or change any
estrogen or progesterone therapies. However, none of the participants were under estrogen or
progesterone treatment during the study. Any intake of other products similar to the test product
during the study was not allowed.
The recruited subjects were randomized to receive either ELASTEN® (verum group) or a placebo.
Randomization was performed with alternating allocation of subjects.
Nutrients 2019, 11, 2494 3 of 14

2.3. Study Schedule

The study duration was twelve weeks. After obtaining informed consent from the subjects, their
suitability for participation in the study was checked. Before the first intake of the study product,
after twelve weeks of intake, and after a follow-up period (without intake) of 4 weeks (T16), subjects
were dermatologically examined, and tolerability and efficacy data were collected. At the end of the
follow-up period, the sustainability of the previously observed effects was evaluated in the test group
of subjects who had taken the test product for the twelve-week period.

2.4. Test Product and Placebo

The test product is classified as a food supplement. Both the test product and placebo were
delivered in identical drinking ampoules. The test product ELASTEN® (QUIRIS Healthcare, Gütersloh,
Germany) contained a specially developed blend of 2.5 g collagen peptides, 666 mg acerola fruit
extract, 80 mg vitamin C, 3 mg zinc, 2.3 mg vitamin E, and 50 µg biotin. Other ingredients, which
were also contained in the placebo, were potassium sorbate, sodium benzoate, carboxymethylcellulose,
citric acid, natural aroma, and water. The placebo (QUIRIS Healthcare, Gütersloh, Germany) did not
contain any nutrients. The test product and placebo were taken daily before or together with a meal.
The specific collagen peptides were produced following GMP (good manufacturing practice) [16]
guidelines in an IFS (international featured standards)—and ISO (International Organization for
Standardization)—certified plant by water extraction of the endogenous collagen from bovine skin,
and subsequent enzymatic hydrolysis and sterilization.

2.5. Assessment of Safety and Tolerability

Safety and tolerability of the test material was assessed by three methods: (1) Evaluation of
tolerability by dermatological examinations before, during, and after the application phase of the
study; (2) monitoring of adverse events during the study through information collected in interviews
and questionnaires; and (3) the questionnaire at the end of the study.

2.6. Dermatological Examinations

Before, during, and after the study, the skin of subjects was examined according to established
clinical and dermatological assessment criteria. Each finding and type of reaction before and after the
study was documented and graded on a four-point scale: 0 = no pathological finding, 1 = mild skin
reaction, 2 = moderate skin reaction, and 3 = severe skin reaction.

2.7. Measurements
All measurements were done under rested conditions of the subjects and under stable physical
environmental conditions (room temperature 20 ◦ C and humidity 40%–60%).

2.7.1. Skin Hydration

Hydration of the external layer of the epidermis (stratum corneum) was measured on the forearm
capacitively with a Corneometer CM 825 (Courage and Khazaka, Cologne, Germany). For each
measurement time, at least three measurements at different locations in the test area on the forearm
were performed [17].

2.7.2. Skin Elasticity

For the assessment of skin elasticity, a Cutometer MPA 580 (Courage and Khazaka, Cologne,
Germany) was used. The functional principle is based on suction of the skin using a probe with
negative pressure, which causes the test area to be drawn into the aperture of the probe [18]. For each
measurement, skin elasticity (“R2”) at three different places of the test area (forearm) was assessed and
the values were averaged.
Nutrients 2019, 11, 2494 4 of 14

2.7.3. Skin Roughness

Skin roughness was measured on the face of the subject, on the basis of silicone imprints (skin
replicas). The structure of the skin surface was determined by means of the optical 3D in-vivo measuring
method PRIMOS (phase-shift rapid in-vivo measurement of skin; PRIMOS Compact, GFMesstechnik
GmbH, Teltow, Germany). The “arithmetic average roughness” and the “maximum roughness” were
used as common skin surface descriptors. A replica was prepared for each measurement in order
to determine skin roughness Rz, which is defined as arithmetic mean of the individual depths of
five contiguous individual measuring sections of the (digitally) filtered profile of equal lengths. The
technology reproducibly determines skin roughness [19].

2.7.4. Skin Density

Sonographic measurements were used to determine skin density [14]. The SkinScanner DUB®
Simple (tpm® taberna pro medicum, Lüneburg, Germany) enables the visualization of structures up to
a maximal depth of 1 cm. Measurements were performed on the thigh.

2.8. Questionnaire
After 12 and 16 weeks, participants filled out questionnaires regarding their subjective assessments
of different parameters concerning the characteristics and performance of the product such as efficacy,
odor, taste, consistency, and skin appearance.

2.9. Statistical Analysis

Statistical analyses were performed according to the principles of ICH (International Conference
on Harmonization) guideline E9 “Statistical Principles for Clinical Trials” [20] using SAS software
(Version 9.4 for Windows, SAS Institute Inc., Cary, NC, USA).
The objective skin parameters—hydration, elasticity, roughness, and density—were evaluated
by descriptive analyses at T0 (day 0), T12 (day 84), and T16 (day 112). Efficacy was determined by
relative changes of these parameters, which were determined by the following differences of the
means: T12–T0, T16–T0, and T16–T12. The location and scale statistics of all the parameters were
calculated, including the arithmetical mean, standard deviation, minimum, and maximum. From the
calculated values of skewness and kurtosis it was concluded that all the parameters could be regarded
as (approximately) normally distributed.
The results are presented graphically by means of box-and-whisker plots. The box ranges from
the 25th percentile up to the 75th percentile. Whiskers are drawn from the ends of the boxes to the
largest and smallest values that did not represent outliers. Outliers are defined as being values more
than 1.5 times the interquartile range away from the box. Outliers are represented by symbols beyond
the whiskers.
Inferential statistical analyses were performed using Student’s t test [21]. Intra-individual mean
changes of skin parameters (comparing T12 versus T0, T16 versus T0, and T16 versus T12) were
evaluated using paired t tests. The mean outcomes in the verum and placebo groups were compared
using the t test for independent samples. The primary outcomes and test hypotheses of the trial
were based on the comparison of the verum and placebo groups with respect to the relative change
(T12–T0) of: (i) Skin hydration, (ii) skin elasticity, (iii) skin roughness, and (iv) skin density. The
multiple significance level across the four primary tests was set to 5% (two-sided). In order to adjust for
multiple testing, the Bonferroni method [22] was applied. The primary results provide confirmatory
statistical evidence. Beyond the primary statistical analyses, all other statistical analyses are considered
exploratory. The p-values are regarded noticeable (“significant”) for p ≤ 0.05.
Nutrients 2019, 11, 2494 5 of 14

3. Results
Nutrients FOR PEER REVIEW 5 of 14
3.1. CONSORT (Consolidated Standards of Reporting Trials) Flow Diagram of the Controlled
Interventional Trial
3.1. CONSORT (Consolidated Standards of Reporting Trials) Flow Diagram of the Controlled Interventional
Trial
Seventy-two subjects aged 35 to 73 years were statistically analyzed. The mean age in the verum
= 36) was 50.6
group (nSeventy-two ± 11.2
subjects years
aged 35 and
to 73inyears
the placebo = 36), 52.4The
group (n analyzed.
were statistically ± 8.3 years.
mean age No subjects
in the
had verum
to be excluded
group (n =during
36) wasscreening
50.6 ± 11.2 and
yearsthroughout the study.
and in the placebo groupNo(n protocol
= 36), 52.4violations
± 8.3 years.occurred.
No
subjects had
All subjects to be for
assessed excluded during
eligibility screening
could be analyzed (n = 72).theHence,
and throughout study. the
No intention-to-treat
protocol violations (ITT)
occurred. All subjects assessed for eligibility could be analyzed (n = 72).
population and the per-protocol (PP) population were identical. The safety population Hence, the intention-to-
(SP) also
treat (ITT) population and the per-protocol (PP) population were identical. The
included all 72 enrolled subjects. The flow of subjects through the controlled interventional safety populationtrial is
(SP) also included all 72 enrolled subjects. The flow of subjects through the controlled
depicted in a CONSORT conform diagram [23] (Figure 1).
interventional trial is depicted in a CONSORT conform diagram [23] (Figure 1).

Enrollment Assessed for eligibility (n = 72)

Excluded (n = 0)

Randomized (n = 72)

Allocation
Allocated to ACTIVE intervention Allocated to PLACEBO intervention
(n = 36) (n = 36)

Follow-Up
Lost to follow-up (n = 0) Lost to follow-up (n = 0)
Discontinued intervention (n = 0) Discontinued intervention (n = 0)

Analysis
Analyzed (n = 36) Analyzed (n = 36)
—Excluded from analysis (n = 0) —Excluded from analysis (n = 0)

Figure
Figure 1. Recruitmentofofthe
1. Recruitment theeligible
eligible subjects
subjects with
withthe
theintervention protocol
intervention and and
protocol assessment.
assessment.

The The clinical

clinical study
study waswasconducted
conducted as
as aa two-arm
two-arm clinical
clinicalinterventional
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of the
of test
the product
test product
against placebo for twelve weeks with a follow-up period of four weeks without intake in the
against placebo for twelve weeks with a follow-up period of four weeks without intake in the subjects
subjects that received the test product. At the end of the follow-up period, the sustainability of the
that previously
received the test product. At the end of the follow-up period, the sustainability of the previously
observed effects was demonstrated in the test group that was no longer treated with the
observed effects All
test product. wasenrolled
demonstrated
subjects in theexamined
were test group as that was before
intended no longer
first treated
intake ofwith theproduct
the test test product.
All enrolled
or placebo (T0), after twelve weeks of intake of test product or placebo (T12), and after a follow-up (T0),
subjects were examined as intended before first intake of the test product or placebo
afterperiod
twelve weeksintake
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in subjects placebo (T12), andtaken
had previously afterthe
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intake of four weeks in subjects that had previously taken the test product (T16).
3.2. Tolerability
oughout the study
throughout theand follow-up
study period. period.
and follow-up

Skin3.3.
Hydration
Skin Hydration
Figure 2Figure
shows2 the descriptive
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of skin hydration beforeof studyofproducts
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study products (at
Nutrients FOR PEER REVIEW 6 of 14
, after twelve
T0), after weeks
twelve of intake
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2019, 11, T12),(atand
intake
2494 T12), at the
and end of the
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nificant differences
significant were found
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were the mean
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initial
tolerated. skin at T0 for
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revealed group and
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placebo group (35.0
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4.8
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± 4.8 33.7 vs.
irritancy
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or
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p =reactions.
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At skin
tolerability
T12, mean hydration
of testwas
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was was
nificantly increased,
significantly 3.2. Tolerability
rated
by 28.0%
increased, by subjects
by± 28.0% as
11.5% ±(44.5 follows:
11.5% ± 4.4 “very
AU± vs.
(44.5 good”
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AU vs.= 66%/56%,
± 5.7 AU,
36.6 “good”
p < AU,
± 5.7 =
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in the verum
p < 0.0001), “neither
in the verumnor” = 0%/8%,
up. On the On
group. otherthehand, “bad”
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hand, 0%/3%,
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in the =placebo
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was togroup,
9.0%
limited one
to± subject
6.6%.
9.0% reported
±The
6.6%. nausea and
The
The products were well tolerated. Dermatological examinations revealed no pathological skin
one heartburn. Apart from these two cases, participants did not report any
erence betweenbetween
difference the groupsthe at
reactions, T12, as
groups
irritancy well
T12, as the
atreactions, well
ordifference betweenbetween
as the difference
allergic reactions. the
The relative changes
the relative
tolerability of in test
the in skin events was
adverse
skinproduct/placebo
changes
throughout the study and follow-up period.
dration of both groups
hydration ratedproves
of both groups to
by subjects be highly
proves asto besignificant
highly
follows: (p < 0.0004)
significant
“very good” < in favorin
(p66%/56%,
= 0.0004) offavor
the test
“good” of product.
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= 33%/33%,test product.
“neither nor” = 0%/8%,
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measurements
= 0%/3%,
3.3. T16 show
at T16 that
show such effects
that such persist
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persist time.
for
and “very bad” = 0%/0%. In the placebo group, one subject
Skin Hydration some After
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Afterweeks
four weeks
reported nausea and one
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further further
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taking test
of the product,
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from skin
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skin participants
cases, hydration significantly
did stillreport
not increased
significantly
any adverse inevents in
increased throughout the
Figure 2 shows the descriptive analysis of skin hydration before intake of study products (at
mparison to the initial
comparison thestate
tostudy (40.1
initial state± 4.9 AU).
(40.1
and follow-up period. ± 4.9 AU).
T0), after twelve weeks of intake (at T12), and at the end of the follow-up period (at T16). No
significant
3.3. Skin differences were found for the mean initial skin hydration at T0 for the verum group and
Hydration
the placebo group (35.0 ± 4.8 AU vs. 33.7 ± 5.1 AU, p = 0.2405). At T12, mean skin hydration was
Figure 2 shows the descriptive analysis of skin hydration before intake of study products (at T0),
significantly increased, by 28.0% ± 11.5% (44.5 ± 4.4 AU vs. 36.6 ± 5.7 AU, p < 0.0001), in the verum
after twelve
group. Onweeksthe of intake
other (at the
hand, T12), and atinthe
increase end
the of thegroup
placebo follow-up period to
was limited (at9.0%
T16).± No significant
6.6%. The
differences were found for the mean initial skin hydration at T0 for the verum group
difference between the groups at T12, as well as the difference between the relative changes in skinand the placebo
(35.0 ± 4.8
grouphydration AU groups
of both ± 5.1to
vs. 33.7proves AU, p = 0.2405).
be highly At T12,
significant mean in
(p < 0.0004) skin hydration
favor of the testwas significantly
product.
increased,Theby 28.0% ± 11.5% (44.5at±T16
final measurements 4.4 show
AU vs. 36.6
that ± 5.7
such effects p < 0.0001),
AU,persist for some in time.
the verum group.
After four On the
weeks
other without
hand, the further taking
increase in theof placebo
the test group
product,
was skin hydration
limited to 9.0%was still significantly
± 6.6%. increased
The difference in the
between
comparison to the initial state (40.1 ± 4.9 AU).
groups at T12, as well as the difference between the relative changes in skin hydration of both groups
proves to be highly significant (p < 0.0004) in favor of the test product.

Figure 2.Figure
Skin hydration before (T0)
2. Skin hydration and (T0)
before afterand
(T12) intake
after ofintake
(T12) study of
products, as well asasafter
study products, wellfollow-
as after follow-
up (at T16)
up for
(at the
T16)test
forproduct
the test (grey)
productand placebo
(grey) and (white)
placebogroups.
(white) The boxplot
groups. The shows
boxplotthe meanthe
shows (×),mean (×),

2. Skin
FigureFigure hydration
2. Skin before
hydration (T0)(T0)
before andand
after (T12)
after (T12)intake
intakeofofstudy
study products, as well
products, as wellasasafter
afterfollow-
follow-up
(at T16) for the test product (grey) and placebo (white) groups. The boxplot shows the mean
up (at T16) for the test product (grey) and placebo (white) groups. The boxplot shows the mean (×), (×), median
median ( max–min
),whiskers
and max–min
median (median ( (), and), max–min
), and
and max–min ( (whiskers
whiskers
whiskers (). AU( indicates
).).AU
AUindicates
indicates arbitrary arbitrary
units, units,
arbitrary
).arbitrary
AU indicates units,where
where absolute levels and
units,
absolute levels and changes
changes are significantly different for the test product and placebo with p ≤ 0.0001 (****).
are significantly different for the test product and placebo with p ≤ 0.0001 (****).
3.4. Skin Elasticity
The final measurements at T16 show that such effects persist for some time. After four weeks
At T0, taking
without further skin elasticity wasproduct,
of the test similar inskin
the verum group
hydration (R2
was = 0.69
still ± 0.05) andincreased
significantly placebo group (R2 =
in comparison
0.71 ± 0.06; p = 0.3240). After
to the initial state (40.1 ± 4.9 AU). intake of the test product and placebo, the elasticity (R2 values)
significantly increased in both groups (Figure 3). At T12, skin elasticity was increased in the verum
3.4. Skin Elasticity
group by 0.81 ± 0.04 AU and in the placebo group by 0.75 ± 0.06 AU. This difference proved to be
highly
At T0, statistically significant
skin elasticity in the verum group (R2 = 0.69 ± 0.05) and placebo group
(p < 0.0004).
was similar
(R2 = 0.71 ± 0.06; p = 0.3240). After intake of the test product and placebo, the elasticity (R2 values)
significantly increased in both groups (Figure 3). At T12, skin elasticity was increased in the verum
group by 0.81 ± 0.04 AU and in the placebo group by 0.75 ± 0.06 AU. This difference proved to be
highly statistically significant (p < 0.0004).
Furthermore, skin elasticity in the test group was only slightly reduced at the end of the follow-up
period (R2 = 0.76 ± 0.07), corresponding to a mean decline of −6.38% ± 7.01%, however it did not drop
to its initial level.
 Nutrients FOR PEER REVIEW 7 of 14
Nutrients 2019, 11, 2494 7 of 14
Nutrients FOR PEER REVIEW

ER REVIEW 6 of 14
The products were well tolerated. Dermatolo
ucts were well tolerated. Dermatological examinations revealed no pathological skin reactions, irritancy reactions, or allergic reactions.
rated
ancy reactions, or allergic reactions. The tolerability of the test product/placebo was by subjects as follows: “very good” = 66%/5
ects as follows: “very good” = 66%/56%, “good” = 33%/33%, “neither nor” = 0%/8%, “bad” = 0%/3%, and “very bad” = 0%/0%. In the
%, and “very bad” = 0%/0%. In the placebo group, one subject reported nausea and one heartburn. Apart from these two cases, p
n. Apart from these two cases, participants did not report any adverse events throughout the study and follow-up period.
e study and follow-up period.
3.3. Skin Hydration
ation Figure 2 shows the descriptive analysis of sk
Figure 2. Skin hydration
Figure before (T0)
2. Skin hydration and(T0)
before after (T12)
and intake
after (T12)ofintake
studyofproducts, as well as
study products, as after follow-
wellT0),
as after follow-
after twelve weeks of intake (at T12), and a
shows the descriptive analysis of skin hydration before intake of study products (at
up (at T16) forT16)
up (at the test
for product (grey) and
the test product placebo
(grey) (white) groups.
and placebo The boxplot
(white) groups. shows the
The boxplot mean
shows (×),
the mean (×),
significant differences were found for the mean ini
lve weeks of intake (at T12), and at the end of the follow-up period (at T16). No
ferences were found for the mean initial skin hydration at T0 for the verum group and the placebo group (35.0 ± 4.8 AU vs. 33.7 ± 5.1 A
roup (35.0 ± 4.8 AU vs. 33.7 ± 5.1 AU, p = 0.2405). At T12, mean skin hydration was significantly increased, by 28.0% ± 11.5% (44.5 ± 4
ncreased, by 28.0% ± 11.5% (44.5 ± 4.4 AU vs. 36.6 ± 5.7 AU, p < 0.0001), in the verum group. On the other hand, the increase in the p
e other hand, the increase in the placebo group was limited to 9.0% ± 6.6%. The difference between the groups at T12, as well as th
ween the groups at T12, Figure
as well3. Skin elasticity
as3.the beforebetween
(T0) and the afterrelative
(T12) intake of study hydration
products, andof both
after groups (at
follow-up proves to be highly signi
Figure Skindifference
elasticity before (T0) and after changes
(T12) intake in skin
of study products, and after follow-up (at
both groups proves to be T16) for the
highly test product (grey) and placebo (white) groups. The boxplot shows The the
final measurements
mean (×), median at T16 show that suc
T16)significant
for the test (p < 0.0004)
product (grey)inand
favor of the
placebo test product.
(white) groups. The boxplot shows the mean (×),
median median
(
measurements at ), and
( T16 (show max–min
), and
that such ),whiskers
( max–min
effects ( ( whiskers
whiskers
persist (). The
for some ).).AU indicates
time.).levels
AU arbitrary
indicates
After four without
units,
arbitrary
weeks units,further taking of the test product, skin
),median
and max–min andwhiskers
max–min ( absolute
The absolute levels
and and
changeschanges are significantly
are significantly different
different for the
er taking of the test test product,for theskin
product test product
and and
hydration
placebo placebo
was
groups, groups,
withstill with p (****).
p ≤significantly
0.0001 ≤ 0.0001 increased
(****). comparison
in to the initial state (40.1 ± 4.9 AU).
o the initial state (40.1 ± 4.9 AU).
Furthermore, skin elasticity in the test group was only slightly reduced at the end of the
3.5. Skin Roughness
Infollow-up
line withperiod (R2 = 0.76 ± 0.07), corresponding to a mean decline of −6.38% ± 7.01%, however it
improvements in skin hydration and elasticity, skin roughness decreased during
did not drop to its initial level.
intake of the test product. As shown in Figure 4, the depth of wrinkles measured by PRIMOS replicas
was reduced
3.5. Skin in the verum group after twelve weeks of intake. Starting from similar initial mean
Roughness
values of 161.6 ± 11.4 µm (test product) and 161.7 ± 13.0 µm (placebo; p = 0.9702), the wrinkle depth
In line with improvements in skin hydration and elasticity, skin roughness decreased during
decreased to 118 ± 16.4 µm in the verum group and to 151.4 ± 15.9 µm in the placebo group. The
intake of the test product. As shown in Figure 4, the depth of wrinkles measured by PRIMOS
relative difference
replicas
between
was PEER
reduced
T0 and T12 was four times higher in the verum group (−26.8% ± 8.1%)
in the verum group after twelve weeks of intake. Starting from similar initial
Nutrients FOR REVIEW 8 of 14
than inmean
the placebo group (−6.4%
values of 161.6 ± 11.4 µ m± (test
5.8%; p < 0.0004).
product) The±maximum
and 161.7 improvement
13.0 µ m (placebo; of skin
p = 0.9702), roughness
the wrinkle
was 41%.
depth decreased to 118 ± 16.4 µ m in the verum group and to 151.4 ± 15.9 µ m in the placebo group.
The relative difference between T0 and T12 was four times higher in the verum group (−26.8% ±
8.1%) than in the placebo group (−6.4% ± 5.8%; p < 0.0004). The maximum improvement of skin
roughness was 41%.
After the follow-up period, the depth of wrinkles increased between T12 and T16 from 118 ±
16.4 µm to 131.6 ± 21.9 µm, demonstrating a persistent, highly significant difference of −18.9% ±
9.9% in skin roughness in relation to the initial state (Figure 4; p < 0.0001).

Figure 2. Skin hydration before (T0) and after (T12

up (at T16) for the test product (grey) and placebo
Skin hydration before (T0) and after (T12) intake of study products, as well as after follow-
) for the test product (grey) and placebo (white) groups. The boxplot shows the mean (×),

4. Skin roughness
Figure Figure (wrinkle (wrinkle
4. Skin roughness depth determined from the phase-shift
depth determined rapid in-vivo
from the phase-shift measurement
rapid in-vivo
of skin measurement
(PRIMOS) skinof replica) before (T0)
skin (PRIMOS) skin and after
replica) (T12)(T0)
before intake
andofafter
study products
(T12) and
intake of afterproducts
study follow-up
and after follow-up (FU) with test product (grey) and placebo (white), boxplot showing the mean ), and max–min whiskers (
median (
(FU) with test product (grey) and placebo (white), boxplot showing the mean (×), median ( ), and
(×), median ( ), and max-min whiskers ( ), absolute levels and changes are significantly different
), and max–min whiskers
max-min ( product
forwhiskers
the test ( ). AU
and placebo
), absolute indicates
at p and
levels arbitrary
< 0.0001 (****).are
changes units,
O represent outlier.
significantly different for the test product and
placebo at p < 0.0001 (****). O represent outlier.
3.6. Skin Density
Both test product and placebo induced an improvement in the skin density, sonographically
measurable as the thickness of the epidermis. Starting from similar initial values (35.7 ± 7.2 µ m for
the test product and 36.9 ± 8.6 µ m for placebo), measurements revealed a highly significant increase
in the thickness of the epidermis and the corresponding skin density in the verum group by 24.8% ±
T0), after twelve weeks
T0), after of intake
twelve weeks(atofT12),intakeand (atatT12),
the end
and ofat the
the follow-up
end of theperiod (at T16).
follow-up period No(at T16). No
significant differences
significantwere found for
differences werethefound
meanfor initial skin hydration
the mean initial skinathydration
T0 for theat verum
T0 forgroup and group and
the verum
the placebo the
group (35.0 group
placebo ± 4.8 AU (35.0vs.± 33.7
4.8 AU± 5.1vs.AU,
33.7p ±= 5.1
0.2405).
AU, Atp = T12, mean
0.2405). Atskin
T12,hydration
mean skinwas hydration was
significantlysignificantly
increased, by 28.0% ± 11.5%
increased, by 28.0% (44.5± ±11.5%
4.4 AU(44.5vs.±36.6 ± 5.7vs.
4.4 AU AU, p <± 0.0001),
36.6 5.7 AU, in p <the verumin the verum
0.0001),
group.Nutrients
On the other2494
group.
2019, 11,On
hand, the increase
the other hand, the in increase
the placebo group
in the was group
placebo limitedwas to 9.0% ± 6.6%.
limited to 9.0%The±8 6.6%.
of 14 The
difference between the groups at T12, as well as the difference between the relative
difference between the groups at T12, as well as the difference between the relative changes in skinchanges in skin
hydration ofhydration
both groups proves
of both to beproves
groups highly to significant
be highly(psignificant
< 0.0004) in(p favor of the
< 0.0004) in test
favorproduct.
of the test product.
After
The final the final
The follow-up
measurements period,
at T16
measurements showthe depth
atthat
T16 such
show of that
wrinkles
effects persist
such increased
for some
effects between
time.
persist for AfterT12
some fourand
time. T16 four
weeks
After fromweeks
118 ± 16.4
without further µm
withouttakingto 131.6
of the
further ± 21.9
test of
taking µm,
product, demonstrating
the test skin hydration
product, a persistent,
skin was highly significant
still significantly
hydration difference
increased inincreased
was still significantly of in
−18.9%
comparison to±the9.9% in skin
initial
comparison roughness
state
to the (40.1 4.9inAU).
initial± state relation
(40.1 ± 4.9 the initial state (Figure 4; p < 0.0001).
to AU).

3.6. Skin Density

Both test product and placebo induced an improvement in the skin density, sonographically
measurable as the thickness of the epidermis. Starting from similar initial values (35.7 ± 7.2 µm for the
test product and 36.9 ± 8.6 µm for placebo), measurements revealed a highly significant increase in the
thickness of the epidermis and the corresponding skin density in the verum group by 24.8% ± 16.8%
44.0 ±FOR
(toNutrients 7.6 µm, p < 0.0001; Figure 5). Furthermore, a significant improvement was observed in9the
PEER REVIEW of 14
placebo group of 6.8% ± 14.8% (to 39.0 ± 8.7 µm, p < 0.0109). The difference between the relative
changes of both groups proved to be highly significant (p < 0.0004) in favor of the test product.

Figure 2. SkinFigure
hydration before
2. Skin (T0) and
hydration after(T0)
before (T12) intake
and afterof(T12)
study products,
intake as well
of study as after
products, asfollow-
well as after follow-
up (at T16) forup
the
(attest product
T16) for the(grey) and placebo
test product (grey)(white) groups.
and placebo The boxplot
(white) groups.shows the mean
The boxplot (×), the mean (×),
shows

Figure 5. Skin density (sonographically measured) before (T0) and after (T12) intake of study products,
andFigure 5. Skin density
after follow-up (at T16)(sonographically measured)
for the test product (grey) andbefore
placebo(T0) and groups.
(white) after (T12)
The intake
boxplotofshows
study
(products,
medianthe meanmedianand( after
), and
(×), median
follow-up
( max–min
(at
), and
), max–min
T16)
whiskersfor
( the
max–min
whiskers
test product
whiskers
( ). AU(grey) and). AU
indicates
), and( outliers
placebo
arbitrary
(o). Absolute
(white)
units,
indicates
levels
groups. The
arbitrary
and changesunits,
are
boxplot shows the mean (×), median ( ), max–min whiskers ( ), and outliers (o). Absolute levels
significantly different for the test product and placebo with p ≤ 0.0001 (****), p ≤ 0.001 (***), and p ≤ 0.05
and changes are significantly different for the test product and placebo with p ≤ 0.0001 (****), p ≤
(*). O represent outlier.
0.001 (***), and p ≤ 0.05 (*). O represent outlier.
The effect of the test product within twelve weeks did not disappear during the four weeks
of thePersistence
3.7. follow-upofperiod.
Skin Improvements
After discontinuing the application, the mean skin density decreased to
36.7 ± The
6.9 µm at T16. This value was still
efficacy and sustainability ofathe
highly significantly
effects increase
of the test productcompared to the initial
are summarized value at 6
in Figures
(p =7.0.0008).
T0and In Figure 6, the differences between the changes (T0–T12) in skin hydration (A), skin
elasticity (B), skin roughness (C), and skin density (D) of the test product and placebo are
3.7. Persistence of Skin Improvements
illustrated. The relative differences between T0 and T12 were significantly higher in the verum
group
Thethan in the
efficacy andplacebo group (p
sustainability of<the
0.0004).
effects of the test product are summarized in Figures 6 and 7.
In Figure 6, the differences between the changes (T0–T12) in skin hydration (A), skin elasticity (B),
skin roughness (C), andA. skin density (D) of the
B. test product andC. placebo are illustrated.
D. The relative
differences between T0 and T12
Skin Hydration were significantly higher
Skin Elasticity in the verum group
Skin Roughness than in the placebo group
Skin Density
Change from Baseline (%)

(p < 0.0004).
30
20
10
0
-10
-20
-30
 3.7. Persistence of Skin Improvements
The efficacy and sustainability of the effects of the test product are summarized in Figures 6
and 7. In Figure 6, the differences between the changes (T0–T12) in skin hydration (A), skin
elasticity (B), skin roughness (C), and skin density (D) of the test product and placebo are
illustrated.
Nutrients The relative differences between T0 and T12 were significantly higher in the verum
2019, 11, 2494 9 of 14
group than in the placebo group (p < 0.0004).

A. B. C. D.
Change from Baseline (%) Skin Hydration Skin Elasticity Skin Roughness Skin Density
30
20
10
0
-10
Nutrients FOR PEER REVIEW 10 of 14
-20
After-30discontinuation of administration of the test product, the effect on skin hydration was
reduced by −45.8% during the follow up compared to T12. A better persistence was demonstrated
for the improvement of skin roughness. This parameter decreased by only 30.6% compared to T12.
Test Product: T0–T12
Skin elasticity and skin density were reduced by 38.5% and 31.3%, respectively (Figure 7). For all
Placebo: T0–T12
skin parameters tested in this study, the percentage improvements in the verum group at the end of
the
FigureFigure
follow-up 6.period
Percentage
6. Percentage (T16,change
change Figure
of skinof7)
skin
wereparameters
much
parameters between
more
between the baseline
pronounced
the baseline than
and andthe
the theimprovements
end ofend
theof the twelve
twelve in the
week
placebo week interventional
group at period
T12 (Figure (T0–T12) in the test group (blue bars) and in the placebo group (red
interventional period (T0–T12)6).in the test group (blue bars) and in the placebo group (red bars). Error
bars). Error bars indicate the standard errors of the mean. Changes are significant at p < 0.0001 (all
bars indicate the standard errors of the mean. Changes are significant at p < 0.0001 (all parameters
3.8. parameters
Subjective except skin density at T16 with a significance level of p < 0.0008).
Rating
except skin density at T16 with a significance level of p < 0.0008).

A. B. C. D.
Skin Hydration Skin Elasticity Skin Roughness Skin Density
30
Change from Baseline (%)

20 12
12
12 16
10 16
16
0

-10
16
-20 12

-30 12 after 12 week Intervention: T0–T12

16 after 12 week Intervention and 4 week Follow up: T0–T16

Figure 7. Percentage changes of skin parameters in the test group between baseline and the end of the
Figure 7. Percentage changes of skin parameters in the test group between baseline and the end of
twelve week interventional period (T0–T12, solid blue bars) and between baseline and the end of the
the twelve week interventional period (T0–T12, solid blue bars) and between baseline and the end of
fourthe
week
fourfollow-up periodperiod
week follow-up (T0–T16, open open
(T0–T16, whitewhite
bars)bars)
for skin hydration
for skin (A), (A),
hydration skinskin
elasticity (B),(B),
elasticity skin
roughness (C), and skin density (D) in the test group. The error bars indicate standard errors
skin roughness (C), and skin density (D) in the test group. The error bars indicate standard errors of of the
mean. Changes are significant at p < 0.0001.
the mean. Changes are significant at p < 0.0001.

After discontinuation
In the of administration
beginning, about of the test
64% of all participants product, the
characterized effect
their skinonasskin
more hydration was
or less dry,
reduced by −45.8%
approximately during
22% the follow
as normal, up compared
and about to T12.
6% as fatty A better
(8% did persistence
not provide such was demonstrated
information). All
for prompted
the improvement of skin roughness. This parameter decreased by only 30.6% compared
effects were more positively assessed by the test group as compared to the placebo to T12.
Skin elasticity and skin density were reduced by 38.5% and 31.3%, respectively (Figure
group. Up to three quarters of subjects in the test group agreed with the positive statements. As7). For all skin
parameters
shown intested
Tablein1,this
the study, the percentage
differences of the testimprovements
group compared in the verum
to the group
placebo at the
group endmost
were of the
follow-up
apparentperiod (T16,scaly
regarding Figure
skin7) (47%
were more
muchagreements),
more pronounced than the(23%),
skin elasticity improvements in the (29%),
skin appearance placebo
andatskin
group T12dryness (13%). After discontinuation of taking the study products, experienced effects
(Figure 6).
largely persisted for at least four weeks in both study groups.

Table 1. Self-perception rating, agreement to statements concerning test product effects reported as
a percentage in relation to placebo effects.

After 12 Weeks of Taking the Product  (%) Test Group Relative to Placebo Group
my skin is less dry. +13.0
my skin is less scaly. +47.4
the elasticity of my skin is improved. +22.7
my skin appearance is improved. +28.6
Nutrients 2019, 11, 2494 10 of 14

3.8. Subjective Rating

In the beginning, about 64% of all participants characterized their skin as more or less dry,
approximately 22% as normal, and about 6% as fatty (8% did not provide such information). All
prompted effects were more positively assessed by the test group as compared to the placebo group.
Up to three quarters of subjects in the test group agreed with the positive statements. As shown in
Table 1, the differences of the test group compared to the placebo group were most apparent regarding
scaly skin (47% more agreements), skin elasticity (23%), skin appearance (29%), and skin dryness (13%).
After discontinuation of taking the study products, experienced effects largely persisted for at least
four weeks in both study groups.

Table 1. Self-perception rating, agreement to statements concerning test product effects reported as a
percentage in relation to placebo effects.

After 12 Weeks of Taking the Product ∆ (%) Test Group Relative to Placebo Group
my skin is less dry. +13.0
my skin is less scaly. +47.4
the elasticity of my skin is improved. +22.7
my skin appearance is improved. +28.6
my skin appears more wrinkle-free. +10.5
external skin care products can be used less frequently. +27.3
∆ = Delta, difference in percent after 12 weeks of intake.

4. Discussion
Topically applied skin care products such as creams, lotions, and sera often fail to reach the
deeper layers of the skin in order to causally and lastingly influence the skin aging processes. The
aim to reach the dermis, the most important skin layer for the restoration of collagen synthesis, has
been achieved by developing highly bioavailable and thus bioactive short chain nutritional collagen
peptides [4,5,10,24]. This was also confirmed by the present trial, particularly by the improvement of
the density and elasticity of the skin. It was clearly shown that the effects of the test product were not
restricted to the epidermis.
A systematic review of the dermatological applications of oral collagen supplementation has
recently demonstrated that collagen supplements can increase skin hydration, elasticity, and dermal
collagen density. Eleven studies with a total of 805 patients suggest that administration of collagen
peptides can positively impact various skin conditions and skin aging [9]. Collagen supplementation
was safe with no reported adverse effects. Asserin et al. demonstrated in a randomized controlled
trial that the intake of 10 g of collagen hydrolysate over at least 56 days leads to an increase in skin
moisture and collagen density compared to placebo [25]. Collagen dipeptides that were taken over
56 days in a study by Inoue et al. also resulted in significantly more improvement in skin moisture,
elasticity, wrinkles, and roughness for the subjects that received 10 mg per day in comparison to study
participants who received 0.5 mg per day or placebo [26].
Compared to placebo, a significant reduction in eye wrinkle volume along with an increase of
procollagen type I and elastin was reported by Proksch et al. after the intake of 2.5 g collagen per
day over 56 days [27,28]. In addition, it was shown by Schunck et al. the intake of 2.5 g collagen
peptides over a period of 180 days led to a statistically significant decrease in the degree of cellulite
and a reduced skin waviness on thighs [29].
The supplement tested here provides a drinkable blend of collagen peptides and acerola fruit extract
with endogenous antioxidants, such as vitamin C, zinc, biotin, and a native vitamin E complex. Studies
suggest that an age-related reduction in collagen synthesis can be addressed by the administration of
oral collagen peptides together with other specific skin nutrients [7,9,11].
Nutrients 2019, 11, 2494 11 of 14

A clinical pilot study has previously demonstrated significant effects of the test product on skin
hydration, elasticity, and roughness after three months of application of the supplement in 16 women
aged 45–60 years [8].
The present study was conducted as a clinical interventional study on 72 healthy women treated
either with the food supplement (n = 36) or a placebo (n = 36) for three months to confirm and
extend these observations. It was demonstrated that drinkable collagen peptides together with other
dermonutrients could induce long lasting visible improvements in the skin’s appearance. However, it
should also be considered that collagen peptides, depending on the source material and manufacturing
process, could differ with respect to molecular size and amino acid composition.
One reason for the significant improvements of skin parameters shown in the present study might
be the high similarity between the collagen peptides provided by the bovine [HC] collagen complex
and those of human collagen. Hydrolysis of the bovine collagen yields specific bioactive short chain
peptides that are characterized by a high coverage of their amino acid profile with the amino acid
sequence of human collagen I. The amino acid sequence coverage of the bovine collagen peptides
used in this study was determined by liquid chromatography–mass spectrometry (nanoLC/MS/MS
hardware [24]: Aquity® UPCL M-Class HSS T3, Waters, MA, USA) using Mascot software (Matrix
Science, London, UK). This analysis revealed a coverage of 31% for collagen type I alpha 1 chains, 18%
for collagen type I alpha 2 chains, and 13% for collagen type III alpha 1 chains. These values were
compared to 4% to 20%, 7% to 16%, and 6% to 11% ranges, respectively, for other collagen peptides,
obtained from pigs, chickens, or marine sources (see Reference [30] for a review).
Moreover, the amino acid composition of the peptides is reflected by a high content of specific
amino acids that are abundant building blocks of human collagen, such as hydroxyproline, proline,
glycine, glutamic acid, alanine, and arginine [7,31,32]. During digestion, the oligopeptides are further
metabolized to bioactive di- and tri-peptides in the gastrointestinal tract and are subsequently released
into the blood stream [11,33–38]. The concentration and type of collagen, as well as the content of
other skin-relevant nutrients can differ in mono and combination products. Therefore, it is crucial to
underline the importance of product-specific trials.
In the follow-up phase, the verum group was observed one month after cessation of the treatment
to evaluate the sustainability of the changes induced by the application. The results show that all the
effects were substantially retained during the follow-up period. Throughout the study, there were no
adverse effects, neither for the placebo nor for the test product, which has been on the market for many
years as a food supplement.
The placebo effects shown here were attributed to the unconscious lifestyle changes of the subjects
during the study and conclusively demonstrated the necessity of a placebo control to reliably assess
the effects of a test product. The differences between the verum group and the placebo group were
highly significant for all of the test parameters after three months of treatment. For all of the skin
parameters tested in this study, the percentage improvement in the verum group was still higher at the
end of the follow-up period, after four months, than at the beginning of the application.
The findings were relevant in terms of skin physiology and decisive for demonstrating the validity
of this specific approach. Moreover, the effects were not only fully confirmed in objective test methods
for assessing skin hydration, elasticity, roughness, and density, but also in the subjective assessment of
the subjects. Therefore, the oral application of dermonutrients allows for a long-lasting regeneration of
the skin that is clearly visible and cosmetically relevant.

5. Conclusions
This randomized, placebo-controlled clinical trial confirmed that skin aging could be addressed
using nutrients that are able to restore skin hydration, elasticity, and density. Objective dermatological
measurements, such as cutometry and corneometry, have proven that oral collagen peptides together
with other dermonutrients significantly improve skin hydration, elasticity, roughness, and density
after three months of intake. These tests thus verify the results obtained in previous trials. Moreover,
Nutrients 2019, 11, 2494 12 of 14

and in line with the objective measurements, the study participants, in their subjective assessments,
concluded that their skin appearance had significantly improved.
Finally, the collagen supplement did not cause any side effects and proved to be safe and well
tolerated during the entire period of application and thereafter.
Since the collagen peptides and the skin nutrients were taken orally, the effects reached the deeper
layers of the skin and sustainably improved skin physiology and appearance. In conclusion, the tested
dermonutrient allowed for a long-lasting and cosmetically relevant regeneration of the skin. Ongoing
studies are exploring the decisive nutritional mechanisms involved in improving skin physiology
and appearance.

Author Contributions: Conceptualization, L.B., G.S., and W.V.; methodology, L.B., G.S., and J.G.; validation, L.B.
and J.G.; formal analysis, J.G.; investigation, G.S. and L.B.; data curation, J.G.; draft writing—review and editing,
L.B., G.S., J.G., and W.V.; visualization, J.G.; supervision, W.V.; and project administration, L.B.
Funding: This research was funded by Quiris Healthcare, Germany.
Conflicts of Interest: The authors declare no conflict of interest. The sponsor had no influence on execution,
analysis and interpretation of the data.

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