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Effects of collagen-derived bioactive peptides and natural antioxidant compounds on proliferation and matrix protein synthesis by cultured normal human dermal fibroblasts

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Nutraceuticals containing collagen peptides, vitamins, minerals and antioxidants are innovative functional food supplements that have been clinically shown to have positive effects on skin hydration and elasticity in vivo. In this study, we investigated the interactions between collagen peptides (0.3-8 kDa) and other constituents present in liquid collagen-based nutraceuticals on normal primary dermal fibroblast function in a novel, physiologically relevant, cell culture model crowded with macromolecular dextran sulphate. Collagen peptides significantly increased fibroblast elastin synthesis, while significantly inhibiting release of MMP-1 and MMP-3 and elastin degradation. The positive effects of the collagen peptides on these responses and on fibroblast proliferation were enhanced in the presence of the antioxidant constituents of the products. These data provide a scientific, cell-based, rationale for the positive effects of these collagen-based nutraceutical supplements on skin properties, suggesting that enhanced formation of stable dermal fibroblast-derived extracellular matrices may follow their oral consumption.
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Scientific RepoRts | (2018) 8:10474 | DOI:10.1038/s41598-018-28492-w
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Eects of collagen-derived
bioactive peptides and natural
antioxidant compounds on
proliferation and matrix protein
synthesis by cultured normal
human dermal broblasts
Suzanne Edgar1, Blake Hopley1, Licia Genovese2, Sara Sibilla2, David Laight1 & Janis Shute1
Nutraceuticals containing collagen peptides, vitamins, minerals and antioxidants are innovative
functional food supplements that have been clinically shown to have positive eects on skin hydration
and elasticity in vivo. In this study, we investigated the interactions between collagen peptides
(0.3–8 kDa) and other constituents present in liquid collagen-based nutraceuticals on normal primary
dermal broblast function in a novel, physiologically relevant, cell culture model crowded with
macromolecular dextran sulphate. Collagen peptides signicantly increased broblast elastin synthesis,
while signicantly inhibiting release of MMP-1 and MMP-3 and elastin degradation. The positive eects
of the collagen peptides on these responses and on broblast proliferation were enhanced in the
presence of the antioxidant constituents of the products. These data provide a scientic, cell-based,
rationale for the positive eects of these collagen-based nutraceutical supplements on skin properties,
suggesting that enhanced formation of stable dermal broblast-derived extracellular matrices may
follow their oral consumption.
e biophysical properties of the skin are determined by the interactions between cells, cytokines and growth fac-
tors within a network of extracellular matrix (ECM) proteins1. e bril-forming collagen type I is the predomi-
nant collagen in the skin where it accounts for 90% of the total and plays a major role in structural organisation,
integrity and strength2. A complex network of interlaced collagen brils in the dermis provides support to the
epidermis, and together with elastin and microbrils gives the skin its elasticity and resilience1. In addition, pro-
teoglycans and polymeric oligosaccharides, including abundant hyaluronic acid, play a key role in skin hydration.
Collagen I, elastin and proteoglycans, the three major groups of dermal ECM proteins, are secreted mainly
by dermal broblasts activated by TGFβ, a multifunctional growth factor regulating the expression, deposition
and turnover of skin extracellular matrix proteins1. Production of collagen and of the other components of the
extracellular matrix is high when there is a sucient level of mechanical tension on broblasts. When this ten-
sion is reduced, for example with age, the production of the matrix proteins falls and there is an increase of
matrix-degrading enzymes3. e mature interstitial collagen brils are resistant to most proteolytic enzymes, but
are susceptible to degradation by the collagenolytic matrix metalloproteinases MMP-1, MMP-8 and MMP-134.
Elastolytic MMPs include the macrophage metalloelastase MMP-12 and the weakly elastolytic MMP-3 which is
expressed by broblasts.
e skin is subject to intrinsic (chronological) and extrinsic (environmental and lifestyle factors including
UV radiation and smoking) ageing, which are both associated with histopathological and immunohistochem-
ical changes5. Intrinsic ageing is characterised by cell senescence6, and altered levels of collagen7, elastin8 and
1Portsmouth, University of Portsmouth, Portsmouth, PO1 2DT, UK. 2Minerva Research Labs, 1-6 Yarmouth Place,
London, W1J 7BU, UK. Correspondence and requests for materials should be addressed to S.S. (email: ssibilla@
minervalabs.com)
Received: 29 December 2017
Accepted: 19 June 2018
Published: xx xx xxxx
OPEN
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glycosaminoglycans, including hyaluronic acid9. In extrinsic ageing, there is loss of reticular collagen and an
accumulation of disorganised elastic bres and glycosaminoglycans. Photo-aged skin (UV-irradiated) displays
alterations of the extracellular matrix, with an increase in the expression of matrix metalloproteinases and colla-
genases10. Increased expression and activity of MMPs, notably MMP-1, MMP-3 and MMP-911,12, has been associ-
ated with photo-ageing, and a direct eect of UV on the integrity of elastic microbril associated proteins and on
the elastin network has been suggested8,13.
A major cause of ageing-related skin damage is thought to be a consequence of decreased antioxidant defences
leading to increased levels of intracellular reactive oxygen species (ROS). ese form through aerobic metabolism
and stimulate signal transduction resulting in the increased expression of MMPs and decreased collagen I syn-
thesis14. e generation of ROS has been directly associated with protein damage, and with up-regulation in the
expression and activity of MMPs in intrinsically and extrinsically aged skin15,16. Moreover, the ECM proteins can
work as skin photo-sensitizers, enhancing the genotoxicity of a given dose of UV irradiation, contributing there-
fore to skin photo-ageing17. With age, the imbalance between synthesis and degradation of the ECM proteins leads
to glycation and the consequent formation and accumulation of AGEs (advanced glycation end-products), which
are a hallmark of age-related diseases18. Further, with age, the ability to replenish collagen naturally decreases by
about 1% per year19. us, the administration of antioxidants might help in counteracting ROS-induced signs of
ageing20. Along this line, it was shown that the administration of antioxidants can decrease oxidative stress in a
model of prematurely ageing mice21. Moreover, clinical studies have shown that the oral administration of antiox-
idants can help improve skin condition in photo-aged skin22,23 and UV-induced erythema24.
In addition, evidence from placebo-controlled clinical studies supports the notion that daily oral consumption
of collagen peptides derived by hydrolysis of native porcine and piscine collagen improves the density and integ-
rity of the collagen network, hydration and elastic properties of normal skin25,26. Further, dietary supplements
combining piscine collagen peptides with other active ingredients including hyaluronic acid, antioxidants, vita-
mins and minerals improve the appearance of the ageing skin2729. However, the cellular mechanisms underpin-
ning these observations remain to be elucidated.
e aim of this in vitro study is to investigate the eects on normal human dermal broblast synthesis of col-
lagen I and elastin, release of transforming growth factor-β (TGF-β), plasminogen activator inhibitor-1 (PAI-1),
matrix metalloproteinases (MMP), MMP-1 and MMP-3, and elastin degradation of collagen bioactive peptides,
alone and in combination with other bioactive compounds found in two dierent collagen-based nutraceutical
supplements previously reported to increase skin elasticity in vivo28,29.
Our hypothesis is that theantioxidant activity associated with the additives within these nutraceutical prod-
ucts interacts with the eects of the collagen peptides to enhance the stability of matrix proteins by inhibiting
release of MMP-1 and MMP-3 in dermal broblast culture.
Materials and Methods
Test products. e collagen peptides, natural antioxidants and other bioactive molecules tested in this study
are those found in the collagen-based nutraceutical supplements ACTIVE GOLD COLLAGEN® (ACTIVE) and
GOLD COLLAGEN® FORTE (FORTE), which are manufactured by Minerva Research Labs (London, UK). e
collagen peptides component (0.3–8 kDa; Peptan® by Rousselot) was tested on normal primary human dermal
broblasts in culture in the absence and presence of a full combination of other active ingredients at the con-
centrations shown in Table1. ese data arepresented in the main results section. e collagen peptides were
also tested following addition of individual active ingredients in the combinations shown in Table1 to investi-
gate possible additive eects of individual components. ese data aredescribed in the main results section and
arepresented graphically in Supplementary FigsS3–S8.
Collagen Peptides Production. Peptan® collagen peptides are produced by hot water extraction of the
endogenous collagen from sh skin, ltration, concentration, subsequent standardized and controlled enzymatic
hydrolysis, sterilization and spray-drying. e production follows GMP guidelines and is HAACP-controlled in
a IFS and ISO certied plant.
Molecular Weight distribution. e molecular weight of Peptan® collagen peptides is between 0.3 and
8 KDa. e molecular weight distribution of these collagen peptides is determined by high performance size
exclusion chromatography (HPSEC) using an Agilent HPLC, 1260 Innity series (G1316A, G1329B, G1311C,
G1315D) with a TSKgel SWXL precolumn und a G2000SWXL column (Tosoh Bioscience). Analysis is performed
with the WinGPC soware (PSS). Samples are eluted from the column with 170 mM phosphate buer contain-
ing 15% acetonitril and monitored with UV detection. Calibration is performed with the Narrow Calibration
Standard (Low FILK).
Amino Acids composition. e samples were hydrolysed in 9 M HCl at 110 C for 20 h, and subsequently
derivatized and stabilized by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Fluor reagent kit
WAT052880). These fluorescent derivatives were separated by RP-HPLC on a Waters 2695 Alliance HPLC
Separation Module and detected by a uorescent detector. Quantication is performed by the Soware Waters
– Empower 3 using standards for each amino acid (Amino Acid Standard H WAT088122). Peptan® collagen pep-
tides are characterised by a high content of Glycine, Hydroxyproline/Proline and Glutamic Acid which represent
56% of the total amino acids. ese collagen peptides comprise also Arginine (8%), Alanine (8%), essential amino
acids (16%) and other amino acids (12%).
Cell Culture. Normal adult human dermal broblasts (NHDF) were purchased from Lonza (Walkersville,
MD, USA) and grown in broblast growth medium-2 (FGM), containing 2% FBS (Lonza), at 37 °C and 5% CO2.
Cells at passage 3–7 were seeded into 24 and 96 well plates at a density of 5 × 104 cells/well, and 5 × 103 cells/well
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Scientific RepoRts | (2018) 8:10474 | DOI:10.1038/s41598-018-28492-w
for protein analysis and proliferation assays respectively. e broblasts were grown for 16 hours, quiesced for
24 h in medium containing 0.3% FBS and then treated for 48 hours with combinations of the bioactive constitu-
ents of ACTIVE or FORTE (Table1) at a concentration found in a 1:50 dilution of the whole product. e ration-
ale for testing components at this dilution is based on the absorption and distribution of avian collagen peptides
of similar composition in an animal model, as detailed in the Supplementary Material.
ese treatments were added in FGM containing 0.3% FBS, supplemented with 100 µM L-ascorbic acid and
100 µg/ml > 500 kDa dextran sulphate (Sigma-Aldrich, St. Louis, USA), which will be referred to hereaer as
‘crowded medium30,31. Under these conditions cells remained viable, as conrmed by their adherence to cell
culture plates and proliferation over 48 hours, as described in section 2.7. In all experiments, cell culture super-
natants were collected, cleared by centrifugation at 1000 g for 10 minutes (min) at 4 °C and stored at 80 °C until
assayed.
Collagen I. Cells were washed twice with phosphate buered saline. Cell-associated collagen was solubilised
by the addition of 0.5 M acetic acid (250 µl/well) and gentle agitation at 4 °C for 32 hours, before adding 0.1 mg/ml
pepsin from porcine mucosa (Sigma-Aldrich) and continuing agitation for a further 16 hours. Pepsin digestion
was then inhibited with 2 µg/ml pepstatin A (Sigma-Aldrich). Cell samples were stored at -80 °C until assayed. A
novel quantitative immuno-blot assay for native collagen I was developed and validated by Western blot analysis
of TGFβ-activated NHDF cells and supernatants (Supplementary Fig.S1). Standards (0.625–10 ng calf skin col-
lagen I, Sigma Aldrich) and samples (100 µl) diluted with PBS (acid-soluble cell fraction, 1:10, and supernatants,
1:20), were loaded onto nitrocellulose membrane using a vacuum manifold to create protein dots. Blots were
dried, blocked with 5% dried skimmed milk powder and 2% Tween 20 in PBS and incubated overnight at 4 °C
with rabbit anti collagen I antibody (Abcam, Cambridge, UK) at 200 ng/ml in block buer. e blots were then
incubated with 50 ng/ml goat anti rabbit-HRP antibody (Dako, Glostrup, Denmark) for 1 hour at room tempera-
ture. Blots were visualised using chemiluminescence (ermo-Fisher, Waltham, USA).
Elastin. Cells were lied with trypsin-EDTA (Sigma-Aldrich) and diluted 3:1 with 1 M oxalic acid to 0.25 M
oxalic acid. Cell samples were incubated at 95–100 °C for 2 hours, with intermittent mixing, and the entire cell and
supernatant samples from each well were assayed separately for solubilised elastin using the Fastin elastin assay
kit (Biocolor, Carrickfergus, UK), following manufacturer’s instructions.
AP-1 analysis. NHDF were lysed in 20 mM TRIS-HCl, pH 7.6, containing 150 mM NaCl plus 1% Triton
X-100, lysis buer and 2 × protease inhibitors, protease cocktail I (Calbiochem), and Complete® protease inhibi-
tor (Roche), PhosSTOP® phosphatase inhibitor (Roche), 4 mM MgCl2 and benzonase (Sigma-Aldrich) at 1:1000
dilution in sample buer (35 ul per well of 24-well plate).
Samples were mixed with sample buer and separated on 10% SDS-PAGE and proteins transferred to nitro-
cellulose using semi-dry electrophoresis. Membranes were blocked with 3% dried skimmed milk powder in
ACTIVE GOLD COLLAGEN® constituents Antioxidant
activity Combinations tested
Code Constituent µg/ml TE (µM) No. Addition
CHydrolysed piscine collagen 2000 76 1 C
HA Hyaluronic acid 0.8 nd 2 C + HA
GGlucosamine hydrochloride 400 nd 3 C + HA + G
CA L-carnitine 400 10 4 C + HA + G + CA
MDried Maca root extract 8 93 5 C + HA + G + CA + M
PPiperine (Piper Nigrum seed extract) 0.6 41 6 C + HA + G + CA + M + P
GOLD COLLAGEN® FORTE constituents
Code Constituent µg/ml TE (µM) No.Addition
CHydrolysed piscine collagen 2000 76 1 C
CS Carnosine 24 44 2 C + CS
Q10 CoEnzyme Q10 10 19 3 C + CS + Q10
RResveratrol (red wine powder extract) 1 338 4 C + CS + Q10 + R
OBorage seed oil/Primrose oil 10 95 5 C + CS + Q10 + R + O
HA Hyaluronic acid 16 6 6 C + CS + Q10 + R + O + HA
PPiperine (Piper Nigrum seed extract) 0.6 41 7 C + CS + Q10 + R + O + HA + P
LLycopene (tomato pulp extract) 0.04 64 8 C + CS + Q10 + R + O + HA + P + L
AAcai berry extract 12 115 9 C + CS + Q10 + R + O + HA + P + L + A
PO Pomegranate juice (concentrated) 8 95 10 C + CS + Q10 + R + O + HA + P + L + A + PO
Table 1. e concentrations at which collagen peptides alone and in combination with other individual
bioactive constituents of the nutraceuticals, ACTIVE and FORTE, were tested in normal dermal broblast
cultures. e antioxidant activity measured as Trolox equivalents (TE) by peroxyl scavenging is shown for the
individual constituents (mean, n = 4); nd = not detected. Combination number 6 for the ACTIVE constituents
and combination number 10 for the FORTE constituents correspond to ‘All’.
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TRIS (20 mM) buered saline (150 mM) plus 0.1% Tween-20, and stained with rabbit anti-p-c-jun (Ser 63/73)
at 200 ng/ml, and secondary goat-anti-rabbit-HRP (Sigma-Aldrich) at 1:2000 dilution. Chemiluminescence
(Promega) was used to detect bands following 30 mins exposure in the ChemiDoc imager (BioRad).
ELISAs. Cells were lysed using hypotonic lysis buer (1% Triton X-100 in 10 mM Tris-HCl buer, pH 7.4)
containing 2 × protease cocktail I inhibitors (Merck (Calbiochem), Darmstadt, Germany) and stored at 80 °C
until assayed. Supernatants were analysed neat for TGF-β, PAI-1 and desmosine, and at a 1:25 dilution for MMPs
-1 and-3. Total TGF-β, total MMP-1, total MMP-3, TIMP-1 and PAI-1 ELISA kits from R&D systems (Abingdon,
UK) were used according to the manufacturer’s instructions.
A desmosine ELISA was performed using an in-house competitive ELISA as previously described32. In brief,
the wells of a 96-well Nunc maxisorp microtitre plate were coated with 250 ng desmosine-egg-albumin complex
(Elastin Products Company (EPC), Missouri, USA) in 100 µl bicarbonate buer, pH 9.6 (Sigma-Aldrich), over-
night at 4 °C. Desmosine standards (EPC) were prepared in the range 0–2000 ng/ml in 100 mM Tris-HCl pH 7.2,
containing 0.1%Tween-20. Standards and samples (100 µl) were added to 200 µl of a 1:3000 rabbit anti-desmosine
serum (EPC) and incubated at 37 °C for 30 min. Plates were washed, and standards and samples (100 µl in dupli-
cate) were added to the wells and incubated for 2 h at 4 °C. Biotinylated swine anti-rabbit (Dako) antibody, 100 µl
at 1:1000 dilution, was added to each well and incubated for 1 h at room temperature. Streptavidin-HRP com-
plexes (Vector Labs), 100 µl at 1:1000 dilution, were added for 30 min at room temperature, followed by substrate
(5.5 mM o-phenylene-diamine solution in TRIS-citrate buer pH 6). Reactions were stopped by the addition of
100 ul 2 M H2SO4 and the plate read at 490 nm.
Cell Proliferation. e proliferation assay was performed aer 48 hours of cell culture, as described in sec-
tion 2.2, using the CyQuant NF assay kit (ermo Fisher) according to the manufacturer’s instructions. A stand-
ard curve to calculate cell number was prepared with 100–50,000 cells per well, in triplicate, which were allowed
to adhere for 4 h, then stained with CyQuant dye binding solution for 40 min, and uorescence wasmeasured at
excitation/emission wavelengths 485/530.
Antioxidant activity. e antioxidant activity of the collagen peptides and the other bioactive ingredients
tested was measured using a pyranine-based procedure to evaluate the total peroxyl scavenging capacity33,34. e
method is based on the ability of antioxidants to prevent the bleaching of pyranine (200 μM) by peroxyl radicals
generated from AAPH (2,2-azo-bis- (2-amidinopropane) hydrochloride (200 mM). Trolox, a water-soluble ana-
logue of vitamin E was used as a standard and values wereexpressed as Trolox equivalents (TE). Samples (25 μl)
were mixed with 25 μl pyranine and incubated for 3 min at 37 °C. AAPH (50 μl) was added and the reaction
monitored at 454 nm every minute for 80 min. e lag phase to bleaching was determined for samples and Trolox
standards in the range 0 to 0.5 mM. Reagents were all from Sigma-Aldrich.
Statistical analysis. Fibroblast responses are presented as mean ± sem and were analysed by one-way
ANOVA followed by Fisher’s LSD post-hoc test using GraphPad Prism, version 7, soware. Correlation between
the cumulative antioxidant activity of individual bioactives and elastin, MMP-1 and MMP-3 levels in NHDF cul-
tures was analysed by one-tailed Pearson correlation coecient test. Dierences where p < 0.05 were considered
to be statistically signicant.
Results
It has previously been reported that TGF-β at 5 ng/ml strongly stimulates collagen synthesis by embryonic pulmo-
nary broblasts in crowded culture30. erefore, because TGF-β controls expression, deposition and turnover of
collagens and other extracellular matrix proteins in the skin1, and primary dermal broblast responses to TGF-β
as a positive control in cultures crowded with high molecular weight dextran sulphate have not previously been
reported, we initially tested the eect of TGF-β (5 ng/ml) on NHDF protein synthesis and proliferation aer
48 hour incubation under similarly crowded conditions, compared to media alone (Fig.1). e values for baseline
concentrations of all proteins in quiescent cultures are presented in the legend to Fig.1. Signicantly more colla-
gen I was found in the supernatant (2.5 ± 0.6 ng/well) compared to the cell layer (0.6 ± 0.2 ng/well). ese values
were normalised to 100% to test the relative eect of TGF-β. TGF-β stimulated a signicant increase in collagen
I in the cell lysate (942.0 ± 268.3%; Fig.1a) and supernatant (154.6 ± 20.7%; Fig.1b) compared to media alone
(100%). Unlike collagen I, signicantly more elastin was found, in quiescent cultures, associated with cell layers
(11.4 ± 1.9 µg/well) compared to supernatants (6.5 ± 0.8 µg/well). Further, TGF-β signicantly increased elastin
in the cell lysate (165.1 ± 35.1%; Fig.1d) and supernatant (151.8 ± 5.5%; Fig.1e). TGF-β signicantly decreased
total MMP-1 (42 ± 7.4%; Fig.1c), total MMP-3 (58.1 ± 9.9%; Fig.1f) and desmosine (75.4 ± 4.1%; Fig.1h) in the
culture supernatants, signicantly increased PAI-1 (721.2 ± 96.5%; Fig.1g) in the supernatant and signicantly
increased the proliferation of NHDF (121.0 ± 5.4%; Fig.1i) compared to the eect of media alone, normalised
to 100%.
e eects of the addition of collagen peptides alone (C) and in combination with all the other bioactive and
antioxidant constituents (All) present within the two nutritional supplements, ACTIVE and FORTE, at the con-
centrations described in Table1, were tested and compared to the eect of media alone (designated as the 100%
value in all experiments). In parallel, the eect of adding individual constituents to the collagen peptides, in the
combinations described in Table1, were tested and these results are presented in Supplementary FigsS3–S8.
Collagen I, elastin and TGF-β synthesis by NHDF was measured in both the cell lysate and supernatant. PAI-
1, MMP-1, MMP-3, TIMP-1 and desmosine were measured in the supernatant alone where they were most
abundant.
Collagen I was found predominantly in soluble form when cells were grown in media alone (Fig.2, legend).
Although collagen peptides alone had no signicant eect on collagen I in the supernatant, soluble collagen I was
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Scientific RepoRts | (2018) 8:10474 | DOI:10.1038/s41598-018-28492-w
signicantly increased in response to the combination of collagen peptides with the other ve ACTIVE constit-
uents tested (All; 265.8 ± 169% of media control; Fig.2b). Similarly, although cell-associated collagen in the cell
lysate was not signicantly increased in response to collagen peptides alone (C; 142.5 ± 32.2%), in combination
with all other ACTIVE constituents cell lysate collagen was signicantly increased (All; 196.2 ± 16.2%; Fig.2a).
Considering the individual combinations of ACTIVE constituents (Table1, Fig. Supplementary Fig.S3a) all those
including glucosamine in combination with hyaluronic acid and collagen peptides, but not hyaluronic acid or col-
lagen peptides alone, signicantly increased cell-associated collagen I (Supplementary Fig.S3a,c). In contrast, the
combination of collagen peptides with the other nine constituents of FORTE (All) did not increase cell-associated
or soluble collagen I (Fig.2c,d).
Unlike collagen, elastin was found predominantly in cell lysates in unstimulated cultures (Fig.3, legend). e
addition of collagen peptides alone (C) signicantly increased the amount of soluble elastin (Fig.3b,d). Moreover,
in combination with the nine other bioactive constituents of FORTE (All) the eect was further signicantly
enhanced (Fig.3d).
Although all combinations (Table1, Supplementary Fig.S4c,d) of the FORTE and ACTIVE constituents
increased soluble elastin in the supernatant compared to media alone, only FORTE constituents stimulated a
further signicant increase in soluble elastin compared to the collagen peptides alone (Supplementary Fig.S4d).
However, there was no signicant increase in the cell-associated elastin in cell lysates on addition of collagen
peptides toany combination with other bioactives (Fig.3a,c, Supplementary Fig.S4a,b).
Addition to dermal broblast cultures of the whole ACTIVE and FORTE products (including acidity regula-
tors, stabilisers, natural sweeteners, avourings and vitamins) at 1:50 dilution, to match the tested concentrations
of individual components, signicantly increased soluble elastin to 173 ± 27.9% and 187 ± 40%, respectively, of
the media control value.
MediaTGF1
0
500
1000
1500
CellLysate C o llagen (% )
**
MediaTGF1
0
50
100
150
200
SolubleCollagen(%)
**
MediaTGF1
0
50
100
150
MMP -1 ( %)
****
MediaTGF1
0
50
100
150
200
250
Cell Ly sate Elastin(% )
**
MediaTGF1
0
50
100
150
200
SolubleElastin (%)
****
MediaTGF1
0
50
100
150
MMP -3 ( %)
***
MediaTGF1
0
200
400
600
800
1000
P A I-1(% )
****
MediaTGF1
0
50
100
150
Desmosine (%)
****
MediaTGF1
0
50
100
150
Proliferation(%)
**
(a)(c)
(d
)
(g
)(h)
(e
)(
f)
(i)
(b)
Figure 1. e eect of TGF-β1 on protein levels in normal human dermal broblasts (NHDF) cultures.
Proteins were measured in cell lysates and/or supernatants of NHDF grown in 24 well plates and activated with
TGF-β1 (5 ng/ml) for 48 hours. Data was expressed as % of media control which was normalised to 100% in
each experiment. Collagen I and elastin were measured in cell lysates (a,d) and supernatants (b,e), MMP-1 (c),
MMP-3 (f), PAI-1 (g) and desmosine (h) were measured in supernatants. Proliferation was measured using the
CyQuant assay (i). e data is presented as mean ± SEM. **indicates P < 0.01, ***P < 0.001, ****P < 0.0001.
Media control values in (a) = 0.6 ± 0.2 ng/well, n = 11 (b) = 2.5 ± 0.6 ng/well, n = 11 (c) = 22.7 ± 3.5 ng/ml, n = 7
(d) = 11.4 ± 1.9 µg/well, n = 7 (e) = 6.5 ± 0.8 µg/well, n = 7 (f) = 23.7 ± 6.4 ng/ml, n = 6 (g) = 1618.7 ± 126.3
pg/ml, n = 8 (h) = 1655 ± 109.2 ng/ml, n = 8 (i) = 25564.5 ± 5758.9 uorescence units, n = 4.
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Scientific RepoRts | (2018) 8:10474 | DOI:10.1038/s41598-018-28492-w
We rst considered whether the observed increases in collagen and elastin concentrations were due to an
autocrine eect of TGF-β released by dermal broblasts under specic culture conditions. However, TGF-β was
detected at low (pg/ml) levels (Fig.4, legend) and found in an inactive form (i.e. requiring acid activation for
detection). TGF-β was signicantly increased in cell lysates (All; 184.2 ± 32%; Fig.4a) and in supernatants (All;
119.6 ± 7%; Fig.4b) in response to collagen peptides in combination with all ACTIVE constituents. However,
there was no signicant increase in TGF-β in either the supernatant (Fig.4d) or cell lysate (Fig.4c) in response to
addition of the constituents of FORTE.
Active TGF-β is a potent inducer of PAI-1 in NHDF culture supernatants (Fig.1g). However, PAI-1 was not
increased under any culture condition (data not shown), conrming that TGF-β was present only at low lev-
els and in an inactive form. Further, while TGF-β strongly induced AP-1 activation, detected as an increase in
phospho-c-jun in cell lysates (Supplementary Fig.S2) the collagen peptides and other bioactives under investiga-
tion did not (data not shown).
Because the measured increases in collagen and elastin potentially reect reduced degradation by MMP-1
and MMP-3, respectively, we measured levels of these proteases and their cognate inhibitor, TIMP-1, in cul-
ture supernatants. A highly signicant decrease in MMP-1 protein levels in supernatants (Fig.5a,b) was seen
in response to collagen peptides alone and the combination of all six ACTIVE constituents (All; 33.8 ± 3% of
media control; Fig.5a) and all ten FORTE constituents (All; 47.4 ± 7% of media control; Fig.5b). e eect of the
collagen peptides was further signicantly increased on addition of the other constituents of FORTE (Fig.5b).
In addition, all combinations of ACTIVE and FORTE constituents signicantly decreased MMP-1 levels in the
supernatants (Supplementary Fig.S6a,b), but an additive eect of the FORTE constituents stimulated further
signicant decrease in MMP-1 protein levels compared to collagen peptides alone (Supplementary Fig.S6b).
Similarly, MMP-3 (Fig.5c,d) was signicantly decreased in supernatants in response to collagen peptides
alone and in the presence of the six ACTIVE ingredients (All; 59.6 ± 18% of media control) and ten FORTE
ingredients (All; 52 ± 14.8% of media control). e antioxidants and bioactive constituents of FORTE further
signicantly enhanced the eect of the collagen peptides (Fig.5d). In fact, collagen peptides and all combina-
tions of ACTIVE and FORTE constituents signicantly reduced MMP-3 levels in supernatants (Supplementary
Fig.S6c,d), but an additive eect of the FORTE constituents stimulated further signicant decrease in MMP-3
protein levels compared to collagen peptides alone (Supplementary Fig.S6d).
Figure 2. e eect of collagen peptides alone and in combination with all other bioactives on collagen I levels
in NHDF cultures. Full length native collagen I was measured in cell lysates (a,c) and supernatants (b,d) of
NHDF grown in 24 well plates and incubated in media plus collagen peptides alone (C) and in combination
with the other constituents listed in Table1 (All) of ACTIVE (a,b) or FORTE (c,d) for 48 hours. Data was
expressed as % of media control, normalised to 100% in each experiment and is presented as mean ± SEM of 3
independent experiments. *Indicates P < 0.05. Media control in (a) = 0.7 ± 0.3 ng/well, (b) = 1.3 ± 0.4 ng/well,
(c) = 0.4 ± 0.1 ng/well and (d) = 2.9 ± 0.9 ng/well.
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In parallel with the significant decrease in MMP-1 and MMP-3 expression, a small but non-significant
increase in TIMP-1 levels were observed (Fig.5e,f) with collagen peptides alone that was not altered by addition
of other constituents of ACTIVE or FORTE supplements.
In parallel with the decrease in MMP-3 concentrations, desmosine as a marker of elastin breakdown
(Fig.5g,h) was signicantly decreased in response to collagen peptides in combination with all six ACTIVE con-
stituents (All; 72 ± 12.6%; Fig .5g), and all ten FORTE constituents (All; 64.3 ± 8.2%; Fig.5h). All combinations
(Supplementary Fig.S7a,b) of ACTIVE constituents, but only some combinations of FORTE constituents caused
a signicant decrease in the breakdown of elastin.
In view of the lack of evidence for autocrine eects of endogenous TGF-β in the cultures, we considered
whether the observed inhibition of MMP-1 and MMP-3 expressionwas related to the total antioxidant activity
of the added bioactives. In particular, the individual constituents of FORTE appeared to have an additive eect
(Supplementary Fig.S6), and when added all together theysignicantly enhanced the eect of the collagen pep-
tides (Fig.5,b,d and h). e antioxidant activity of the collagen peptides and each of the bioactives under inves-
tigation was measured as peroxyl scavenging activity, and expressed as Trolox equivalents (Table1). e data
in Fig.6 show the strong, signicant, negative correlations between the cumulative antioxidant activity of the
added FORTE constituents and MMP-1 and MMP-3 expression levels (Fig.6c,d). Further, there is a signicant
negative correlation of soluble elastin with MMP-3 activity (Fig.6a) and a signicant positive correlation with
antioxidant activity (Fig.6b), indicating that basal expression and release of MMPs is driven by reactive oxygen
species and that antioxidant activity added to the cultures was responsible for the further signicant inhibition of
MMP-3 release (Fig.5d) and increase in soluble elastin (Fig.3d, Supplementary Fig.S6d) beyond that seen with
the collagen peptides alone.
Although there was no signicant eect on proliferation (Fig.7) whenadding the collagen peptides alone, the
combination of all ten constituents of FORTE signicantly stimulated NHDF proliferation over 48 hours (All;
123.8 ± 6.5%; Fig.7b). In addition, incubation of NHDF with HA, a constituent of ACTIVE (Supplementary
Fig.S8a), and carnosine, a constituent of FORTE (Supplementary Fig.S8b) resulted in signicantly increased cell
proliferation that was not further enhanced by the addition of other bioactives.
Figure 3. e eect of collagen peptides alone and in combination with all other bioactives on elastin levels
in NHDF cultures. Elastin was measured in cell lysates (a,c) and supernatants (b,d) of NHDF grown in 24 well
plates and incubated in media plus collagen peptides alone (C) and in combination with the other constituents
listed in Table1 (All) of ACTIVE (a,b) or FORTE (c,d) for 48 hours. Data was expressed as % of media control,
normalised to 100% in each experiment and is presented as mean ± SEM of 4 independent experiments. **/††
indicates P < 0.01, ***P < 0.001, ****P < 0.0001. Media control in (a) = 15.2 ± 2.7 µg/well, (b) = 8.3 ± 1.7 µg/well,
(c) = 10.8 ± 3.4 µg/well and (d) = 6.1 ± 1.7 µg/well. *Indicates groups compared to 0 (media alone), indicates
groups compared to 1 (collagen peptides).
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Discussion
TGF-β is a well-recognised pro-brotic growth factor that promotes the deposition of ECM proteins, whilst lim-
iting their degradation. e pro-brotic eects of TGF-β were conrmed in our model in which normal human
dermal broblasts were cultured under conditions of macromolecular crowding to mimic the extracellular matrix
environment of the skin. Exogenously added TGF-β1 signicantly increased broblast proliferation, collagen and
elastin synthesis and release of PAI-1, while inhibiting release and MMP-1 and MMP-3 and the breakdown of
elastin. ese eects are mediated by SMAD and AP-1 dependent signalling pathways that stimulate synthesis
of collagen-I via Smad3 and PAI-1 via AP-135 and interact to repress MMP-1 and MMP-3 gene expression36,37.
Redox signalling also plays an important role in the pro-brotic eects of TGF-β38.
Media crowded with macromolecules such as high molecular weight dextran sulphate (0.01% w/v) was
reported to increase collagen synthesis by embryonic pulmonary broblasts 20–30 fold, compared to normal
media31 and soluble procollagen was completely processed into insoluble collagen by dermal fibroblasts39,
although the collagen was deposited as aggregates and not brils30. Using a sensitive in-house immunoblot
method to detect collagen I, the presence of soluble collagen I indicated processing was incomplete in our study.
However, it is possible that the high assay sensitivity detected more soluble collagen I than previous studies.
Collagen peptides signicantly increased cell-associated collagen, but only in the presence of glucosamine
(Supplementary Fig.S3). Positive eects on collagen levels may be associated with the ability of glucosamine to
stabilise collagen matrices by reducing MMP synthesis, as demonstrated in synovial broblasts40. Although we
found no peroxyl scavenging activity associated with glucosamine (data not shown), superoxide/hydroxyl-radical
scavenging antioxidant activity of glucosamine hydrochloride has previously been reported, and it was suggested
that glucosamine hydrochloride could be eectively employed as an ingredient in functional food, to alleviate
oxidative stress41. However, the lack of any signicant eect of adding antioxidants such as resveratrol, CoQ10,
acai berry, lycopene and pomegranate on collagen expression, despite signicant inhibition of MMP-1 expression,
indicates an eect of glucosamine on collagen synthesis that is not mediated through antioxidant activity.
In contrast, soluble elastin was signicantly increased by collagen peptides alone and with all combinations
of antioxidants and other bioactives tested. Each of the constituent antioxidants found in FORTE appeared to
have an additive eect on total soluble elastin synthesis. Moreover, when added to broblast cultures as the whole
product (diluted 1:50 to give the same concentrations of the tested individual components), both collagen-based
nutraceuticals signicantly (p < 0.05) increased soluble elastin almost two-fold. is increase in elastin expression
Figure 4. e eect of collagen peptides alone and in combination with all other bioactives on TGF-β1
levels in NHDF cultures. TGF-β1 was measured in cell lysates (a,c) and supernatants (b,d) of NHDF grown
in 24 well plates and incubated in media plus collagen peptides alone (C) and in combination with the other
constituents listed in Table1 (All) of ACTIVE (a,b) or FORTE (c,d) for 48 hours. Data was expressed as %
of media control, normalised to 100% in each experiment and is presented as mean ± SEM of 3 independent
experiments for ACTIVE and 3 independent experiments for FORTE. *Indicates P < 0.05. Media control in
(a) = 29.72 ± 12.2 pg/ml, (b) = 49.1 ± 10.3 pg/ml, (c) = 3.6 ± 1.2 pg/ml and (d) = 25 ± 7.5 pg/ml.
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Figure 5. e eect of collagen peptides alone and in combination with all other bioactives on MMP-1, MMP-3
and TIMP-1 levels and elastin breakdown in NHDF cultures. MMP-1, MMP-3, TIMP-1 and desmosine were
measured in supernatants of NHDF grown in 24 well plates and incubated in media plus collagen peptides
alone (C) and in combination with the other constituents listed in Table1 (All) of ACTIVE (a,c,e,g) or FORTE
(b,d,f,h) for 48 hours. Data was expressed as % of media control, normalised to 100% in each experiment and
is presented as mean ± SEM of 3 independent experiments. */†Indicates P < 0.05, **/††P < 0.01, ***P < 0.001,
****P < 0.0001. Media control in (a) = 26.4 ± 8.2 ng/ml, (b) = 5.6 ± 1.7 ng/ml, (c) = 25.5 ± 8.6 ng/ml,
(d) = 0.6 ± 0.3 ng/ml, (e) and (f) = 314.1 ± 34.6 ng/ml, (g) = 1.6 ± 0.5 µg/ml and (h) = 1.7 ± 0.5 µg/ml. *Indicates
groups compared to 0 (media alone), indicates groups compared to 1 (collagen peptides).
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reects what has already been observed in vivo. A signicant increase in skin rmness (Young’s elasticity) was
demonstrated in vivo aer 90 days of supplementation with both whole collagen-based supplements investigated
in this study28,29. Low molecular weight avian collagen peptides of similar composition, when orally administered
in rats, were shown to accumulate preferentially in the skin42, at concentrations which could be scaled to the con-
centration of peptides consumed daily in the test products (100 mg/ml) and used in our in vitro study (2 mg/ml).
0200 400600
0
5
10
15
20
MMP-3 (pg/ml)
SolubleElastin g/well)
r=-0.8911
r
2
=0.7940
P=***
0.00.2 0.40.6 0.
81
.0
0
5
10
15
20
25
Trolox Equivalence
S o lu b l eElastin (µg/well)
r=0.85
23
r
2
=0.726
4
P=***
0.00.2 0.40.6 0.81.0
0
1000
2000
3000
4000
5000
Trolox Equivalence
MMP-1 (pg/ml)
r=-0.8575
r
2
=0.7353
P=***
0.00.2 0.40.6 0.
81
.0
0
200
400
600
Trolox Equivalence
MMP-3 (pg/ml)
r=-0.8 714
r
2
=0.7593
P=***
(a)
(b)
(c)(d)
Figure 6. Correlation between MMP-3 and soluble elastin (a) andthe cumulative antioxidant activity of
individual bioactives in FORTE and solubleelastin (b), MMP-1 (c) and MMP-3 (d) levels in NHDF cultures. A
one-tailed Pearson correlation coecient test was used to analyse signicance of the data. **Indicates P < 0.01,
***P < 0.001, ns = no signicance.
Figure 7. e eect of collagen peptides alone and in combination with other constituents of ACTIVE and
FORTE on proliferation of NHDF. Proliferation was measured in NHDF grown in 96 well plates and incubated
in media plus collagen peptides alone (C) and in combination with the other constituents listed in Table1 (All)
of ACTIVE (a) or FORTE (b) for 48 hours. Data was expressed is presented as mean ± SEM of 4 independent
experiments. Media control value is 19,848 ± 2,476 cells. Indicates P < 0.05, ***P < 0.001. *Indicates groups
compared to 0 (media alone), indicates groups compared to 1 (collagen peptides).
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us our in vitro observations support those from our previous in vivo studies, validating the model we have used
to investigate dermal broblast responses to individual components of the products.
e very low levels of TGF-β released (25–50 pg/ml) by dermal broblasts in this model, and the lack of eect
of any of the additives on thelevels of active TGF-β, indicated that an autocrine eect of TGF-β was unlikely, and
this is evidenced by the lack of eect on AP-1 activation and PAI-1 expression.
Collagen peptides alone and in combination with antioxidants and other bioactives signicantly reduced
MMP-1 and MMP-3 expression, in the absence of any change in TIMP-1 levels. Notably, the constituents of
FORTE appeared to have an additive eect on inhibition of MMPs expression, as was also seen for the increase
in soluble elastin in culture supernatants. A major cause of ageing-related skin damage is thought to be due to
increased levels of ROS and oxidative stress14 which directly and indirectly, through increased expression of
MMPs, damage structural proteins43. MMPs -1, -2, -3 and -9 are mainly responsible for ECM damage and degra-
dation, and can fully degrade collagen together. However, the only MMP that can damage intact collagen I bres
is MMP-1, while MMP-3 is capable of degrading elastin43. Protective eects of the collagen peptides and other
constituents on elastin integrity are indicated by a decrease in desmosine, a cross-linked amino acid and spe-
cic marker of elastin breakdown, in the cultures. e negative correlation of soluble elastin concentration with
MMP-3 supports the notion that MMP-3 is responsible for elastin breakdown and the generation of desmosine
in the cultures.
e signicant positive association between total peroxyl scavenging activity of the additives and soluble elas-
tin content in the cultures may now suggest a mechanistic role for antioxidant activity in the observed increase in
soluble elastin. Potentially the eect is mediated by increased elastin transcription in the presence of the antioxi-
dants, as previously shown for the eect of the antioxidant N-acetylcysteine44 or decreased expression of MMP-1
and MMP-3, which was previously reported for the eect of the antioxidant Tiron45 and mitochondria-targeted
vitamin E46 on dermal broblasts. e signicant negative correlation of the MMPs with antioxidant activity
indicates that the antioxidants are protecting the cells from constitutive intracellular ROS, activation of AP-1 sig-
nalling and MMP expression14. ese eects transcend any potential activities associated with the physiological
(100 µM) concentration of ascorbic acid added to the culture medium as a co-factor for prolyl and lysyl hydroxy-
lases involved in collagen synthesis, since 88–98% of ascorbate disappears from culture medium within 24 h47 and
we found the peroxyl scavenging activity of 100 µM ascorbic acid was low (6 µM TE).
Finally, collagen peptides in combination with antioxidants and other bioactives under investigation, but not
alone, stimulated broblast proliferation, and the magnitude of the eect was similar to that seen with TGF-β.
A strong eect of HA on dermal broblast proliferation was not further increased by other additions. While HA
has not been shown to be directly mitogenic48, through facilitating proliferation in response to other mitogenic
factors such as TGF-β49 and, in this instance, collagen peptides, HA may have an important but indirect role in
cell proliferation. Similarly, the stimulating eect due to addition of carnosine on broblast proliferation was not
further enhanced by the other additions. McFarland and Holliday (1999) showed in their classical experiments
that carnosine enhances the proliferative potential of broblasts by protecting the cells from telomere shorten-
ing50. Together with stimulating eects on dermal broblast proliferation51 carnosine may play an important role
in skin regeneration.
In conclusion, 48 hour incubation of dermal broblasts with collagen-derived peptides and other nutraceu-
tical constituents increases structural ECM protein synthesis, particularly elastin, and decreases synthesis of
MMPs -1 and -3 and the elastin degradation product desmosine. e eect of these collagen-based supplements
and their constituents therefore support normal adult human dermal broblast function in terms of potentially
increasing matrix stability in the skin. is eect is not likely to be mediated through TGF-β signalling pathways
but is more likely correlated with antioxidant eects on dermal broblast MMP expression.
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Acknowledgements
We gratefully acknowledge Dr Anna Czajka, Dr Ewa Kania and Dr Martin Godfrey for critical reading of the
manuscript. e study was funded by Minerva Research Lab (London, UK) which also provided the supplements
used in this study, GOLD COLLAGEN® FORTE and ACTIVE GOLD COLLAGEN®, and the derived
compounds.
Author Contributions
All authors contributed to the experimental design of the research, which was carried out at the University of
Portsmouth by SE, BH, DL and JS, who analysed the data alleviating any perceived conict of interest. All authors
contributed to the writing and preparation of the manuscript. SE and DL were responsible for conception of the
study, analysis and interpretation of the data and writing of the manuscript; BH was responsible for analysis and
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
13
Scientific RepoRts | (2018) 8:10474 | DOI:10.1038/s41598-018-28492-w
interpretation of the data and writing of the manuscript; LG and SS were responsible for conception of the study
and writing of the manuscript; JS was responsible for conception of the study, analysis and interpretation of the
data, writing of the manuscript and nal approval.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-28492-w.
Competing Interests: e authors declare no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
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... Although elastin is structurally stable, it readily undergoes proteolytic degradation, producing elastin-derived peptides (EDPs) [261][262][263]. Utilizing in vivo and in vitro models, researchers have found that EDPs enhance Aβ formation, which could contribute to subsequent AD development [257,264]. Additionally, EDPs released from elastin due to proteolytic degradation gradually develop into amyloid-like structures [265,266]. Interestingly, like tau, described above, a recent review by Szychowski and Skóra examined the reciprocal relationship between the production of ROS and EDPs [268]. ...
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Dermal fibroblasts play a key role in maintaining skin homeostasis by synthesizing and degrading extracellular matrix components. During aging, they are subjected to changes, such as the loss of type I collagen expression and an increased synthesis of metalloproteinase I, leading to fragmentation of collagen fibrils with consequent reduction of the mechanical tension and defects of skin wound healing. Most information about fibroblast aging was obtained from experiments performed on replicative senescent dermal fibroblasts in vitro. However, the senescence status of fibroblasts isolated from intrinsically aged skins and its consequences on functionality need to be deeper investigated. Herein, we studied age-related phenotypic and functional alteration of fibroblasts from "young" (<35 years) and "old" (>50 years) donors. Our results brought evidence of the senescent status of "old" fibroblasts by senescence associated β-galactosidase (SA-βgal) positive staining and p16 expression. A PCR array focusing on senescence highlighted a subset of down-regulated genes including cell cycle progression and ECM genes in "old" fibroblasts as well as a subset of up-regulated genes involved in senescence features. In "old" fibroblasts, we measured a down-regulation of proliferative and contractile capacities, of migratory potential under PDGF stimulation and activation into myofibroblasts under TGFβ. Old fibroblasts were also more sensitive to oxidative stress than "young" ones. Of interest, downregulation of p16 expression partially reversed the senescent phenotype of "old" fibroblasts but failed to restore their functional properties. In conclusion, our data brought evidence of phenotypic and functional differences between fibroblasts from young and intrinsically aged skin that may contribute to the alterations observed with aging. This article is protected by copyright. All rights reserved.
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