Recently, development and research of nutraceuticals based on marine collagen peptides (MCPs) have been growing due to their high homology with human collagens, safety, bioavailability through gut, and numerous bioactivities. The major concern regarding safety of MCPs intake relates to increased risk of oxidative stress connected with collagen synthesis (likewise in fibrosis) and to ROS production by MCPs-stimulated phagocytes. In this clinical-laboratory study, fish skin MCPs combined with plant-derived skin-targeting antioxidants (AO) (coenzyme Q10 + grape-skin extract + luteolin + selenium) were administered to volunteers (
Dietary vitamins, plant-derived polyphenols, fatty acids, proteins, essential amino acids, and trace elements have demonstrated beneficial effects on skin health and appearance [
Marine fish collagen and collagen peptides have been widely used as functional foods or dietary supplements due to their homology to human collagen structure [
The major concern regarding safety and clinical feasibility of regular intake of MCPs has been raised from the well established fact that the induction of collagen synthesis, mainly assessed by the increased hydroxyproline levels, is often associated with oxidative stress [
The goal of the present clinical-laboratory study was to elucidate the effects of the oral administration of CELERGEN on skin physiology and dermal collagen deposition in the group of healthy middle-aged subjects with clinical signs of skin ageing. The cutaneous clinical-instrumental data were compared with the systemic metabolic parameters of collagen synthesis, redox balance, and energy storage. For the first time, we demonstrated (i) remarkable improvement of ageing skin physiology and structure, which corresponded to enhanced systemic markers of collagen synthesis; (ii) systemic redox balance, sustained by the antioxidant complex; and (iii) increased systemic energy storage. We also hypothesised that moderately increased plasmatic levels of nitric oxide (NO) and malonyl dialdehyde (MDA) may play positive roles of mediators in the MCPs-induced collagen and ATP synthesis/storage, as well as in sebum production. On these grounds, we suggested that selected antioxidants targeting the distinct organs/tissues should be essential components of MCPs-containing nutraceuticals for more effective, individualised, and safe supplementation.
The study enrolled a group of 41 adult healthy Caucasian volunteers of both sexes recruited from the Beauty Institute on Arbat (Moscow, Russia) staff (age 37–72 years; mean age 50.6 ± 10.4 years; 5 males and 36 females) following the exclusion and inclusion criteria for an open single-blind clinical study. The inclusion criteria were as follows: (i) healthy white adult subjects of both sexes, 35–75 years of age, (ii) subjects with visible symptoms of aged facial skin, (iii) subjects who agreed to interrupt any intake of antioxidant nutraceuticals/drugs for at least 1 week before and during the entire duration of the trial, and (iv) subjects without any difficulty to understand and follow the clinical investigator instructions. Pregnant and breastfeeding women, subjects with allergic/intolerance reactions to any component of the tested product, subjects on any other nutraceutical interventions or/and therapies, and subjects simultaneously engaged in other clinical trials were excluded from the study. The participants were informed that they could interrupt clinical trial at any moment, without any explanation of causative reason for their action, or in case they noticed any adverse reaction to the tested product or had any sensation that the product intake affected their appearance negatively.
The protocol of the clinical trial was duly analysed and approved by the Ethical Committee of the Beauty Institute on Arbat, Moscow, Russia (number 11/EK-2014). All recruited subjects gave their informed consent to personal and anamnestic data collection and biological material sampling. The guidelines of Helsinki Declaration for human experimentation were strictly followed during the conduct of the clinical trial.
Food supplement containing marine collagen peptides derived from skin of deep sea fish (MCPs, 570 mg), grape-skin extract (10 mg), coenzyme Q10 of plant origin (10 mg), luteolin (10 mg), and selenium (0.05 mg) of plant origin was formulated in soft gelatine capsules. As inactive solvents, refined and partly hydrogenated soybean oil as well as small admixture of pure soybean lecithin were used. The product, under the commercial name of CELERGEN (manufacturer: Laboratories-Dom, Carouge, Switzerland), was kindly provided by Suisse Ueli Corporation. According to the manufacturer’s information, the deep sea fish sources, that is,
Fish skin was homogenised in distilled water, with addition of complex proteases. The enzymatic proteolytic process was carried out at 40°C and pH 8.0 for 3 h, after which the proteases were inactivated by short-term heating (56°C for 10 min). The liquid was sterilised by Millipore filtration (pore size 0.02 mm) and spray-dried to prepare MCP powder, as described in detail previously [
The aqueous extract of grape skin was obtained from
The entire trial duration was 4 months (May–December 2014), that is, 2 months of pretreatment period, followed by 2 months of treatment with the test nutraceutical administration. The facial skin parameters of recruited volunteers were analysed three times: at the first visit (enrollment), at the second visit 2 months after the pretreatment period, and at the third visit immediately after the treatment period. Each assessment session comprised instrumental methods for measuring skin physiology parameters and ultrasound properties of the skin layers. This design allowed us to use the same subject as a control and an experiment. During the treatment period, the volunteers were recommended to take 2 capsules of CELERGEN a day (at breakfast and dinner time) for 60 consecutive days. At the second and third visits, the participants donated 20 mL of venous blood after overnight fasting and test tubes were coded by the principal clinical investigator. Blood samples were routinely processed for general haematology (haemoglobin content, differential cell count, and the rate of erythrocyte sedimentation) and biochemistry (glucose levels, plasma protein and lipid profiles, transaminases activities, and C-reactive protein content). Laboratory operators carried out analytical determinations blindly, and statistician was not informed which set of analyses was done in the control or experimental periods, hence ranking this study of clinical efficacy of the nutraceutical as “a single-blind” clinical investigation.
Several physiological parameters, mainly barrier properties, of the facial skin were assessed by appropriate SOFT PLUS TOP probes, with microcamera visual analysis and patented computerised programs (Callegari, Parma, Italy). Skin elasticity was determined by the elastometric approach used in the SOFT PLUS technique. The transepidermal water loss (TEWL), an index of skin moisture, was assessed with Tewameter, which measures the water evaporation through cutaneous levels. When the skin is aged or damaged, the barrier properties of the skin are affected, with increased water evaporation and reduced skin hydration. Sebum content was measured by the SOFT PLUS sebometric probe.
Assessment of ultrasound properties of the skin was performed by a digital ultrasound imaging system DUB CUTIS (Digital Ultraschall Bildsystem, Germany), which allowed determining four parameters simultaneously: epidermal and dermal thickness and epidermal and dermal ultrasonic density. The first two parameters are indirect markers of collagen (dermis) and lipid (epidermis) synthesis and retention while the second pair of parameters characterises the evenness and order in the epidermal and dermal structures, respectively. The elastic properties of the skin were additionally analysed by a TPM system containing elastometric sensor (22 MHz) which combines digital ultrasound examination with an imaging record (DUB CUTIS, Germany). A computerised multifunctional diagnostic tool integrating different morphometric parameters (epidermal thickness, tone, wrinkles, and elasticity) for face skin biological age determination was used (SOFT PLUS TOP, Callegari, Parma, Italy).
The majority of chemical reagents, HPLC standards, mediums, solvents, and luciferin-luciferase for ATP assay were from Sigma Chemical Co. (St. Louis, MO, USA); kits for enzyme activity assays and Griess reagent for nitrites/nitrates determination were from Cayman Chemical Company (Ann Arbor, MI, USA). Manufacturers of other reagents are mentioned within the respective methods.
Complete differential blood cell counts and metabolic analyses were performed on fresh ethylenediaminetetraacetic acid- (EDTA-) anticoagulated venous blood of 12 hrs fasting subjects. Biochemical assays were performed on peripheral blood plasma or red blood cells (RBC), either immediately (ATP, glutathione, and coenzyme Q10) or within 72 hrs, on sample aliquots stored at −80°C under argon. Plasma levels of nitrites/nitrates (
Plasmatic Cu,Zn-superoxide dismutase 3 (Cu,Zn-SOD3, U/g protein) activity was measured spectrophotometrically at 505 nm using appropriate kit from Cayman Chemical Company (Ann Arbor, MI, USA) [
Plasma levels of MDA were determined by slightly modified spectrophotometric analysis of thiobarbituric acid-reactive substances (TBARS) described elsewhere [
100
The plasma levels of free hydroxyproline (Hyp) and hydroxyproline in the form of oligopeptides, mainly proline-hydroxyproline, were determined by a chemical colorimetric method using a commercial kit (Hydroxyproline Detection Kit) in accord with the manufacturer’s instructions. Hyp concentrations were quantified in the linear range of its calibration curve using an array reader (Bio-Rad, Hercules, CA, USA) and expressed in
Statistical analysis of clinical data was carried out using WINSTAT programs for personal computers (Statistics for Windows 2007, Microsoft, USA). All biochemical and molecular measurements were done in triplicate and data were statistically evaluated. Values were presented as mean, standard error of the mean, and 1.96 × standard error of triplicate analyses. When several datasets were compared, data were analysed by Student’s
All healthy volunteers (
Subjective evaluation of the 2-month food supplement administration effects, by participants (
Parameter | Number (%) of participants | ||
---|---|---|---|
Improvement | No effect | Aggravation | |
General health conditions | 21 (51%) | 20 (49%) | 0 (0%) |
Stamina/muscle strength/joint motility | 15 (36%) | 26 (64%) | 0 (0%) |
Digestive system | 0 (0%) | 41 (100%) | 0 (0%) |
Skin conditions | 25 (61%) | 16 (39%) | 0 (0%) |
Comparison of digital photos taken before and after clinical trial showed visible qualitative improvement of aesthetic aspect of face with pronounced lifting effect (
Characteristic digital images of ultrasound examinations made at trial beginning (2 months before the beginning of supplementation), at the first day of nutraceutical administration, and immediately after the trial cessation are shown, respectively, in Figures
Effects of the 2-month food supplement administration on the ultrasonic properties of the dermis (
Parameter | Dermis | ||
---|---|---|---|
Pretreatment period | Before treatment | After treatment | |
Thickness, |
3884 ± 30 | 3900 ± 31 | 4133 ± 28 |
Acoustic density | 5.2 ± 0.2 | 5.1 ± 0.2 | 6.3 ± 0.1 |
Effects of the 2-month food supplement administration on the ultrasonic properties of the epidermis (
Parameter | Epidermis | ||
---|---|---|---|
Pretreatment period | Before treatment | After treatment | |
Thickness, |
76.9 ± 1.0 | 77.0 ± 0.8 | 77.6 ± 0.9 |
Acoustic density | 35.6 ± 2.4 | 35.2 ± 2.2 | 35.4 ± 2.0 |
Digital images of facial skin ultrasound examinations (patient number 23, e.g.), made 2 months before the beginning of the trial (a), at the day of the trial beginning (b), and immediately after the trial cessation (c).
Analyses of the main physiological parameters of the skin relevant to ageing, such as elasticity, moisture, and sebum content, demonstrated their comparative stability in the pretreatment period, as there were no significant changes between the first and the second sets of measurements (Table
Effects of the 2-month food supplement administration on the parameters of skin physiology (
Parameter | Pretreatment period | Before treatment | After treatment |
---|---|---|---|
Elasticity | 34.06 ± 1.54 | 33.66 ± 1.21 | 40.26 ± 0.87 |
Moisture | 48.83 ± 3.02 | 49.03 ± 3.52 | 46.54 ± 3.02 |
Sebum | 29.89 ± 4.16 | 29.37 ± 4.76 | 56.86 ± 4.04 |
Skin biological age | 50.11 ± 1.91 | 49.51 ± 1.68 | 48.09 ± 1.74 |
It should be noticed that all tested parameters of skin physiology and structure were not subjected to temporal fluctuations during the 2-month pretreatment period, and therefore changes observed can be viewed as a result of CELERGEN administration.
Surprisingly, CELERGEN administration did not affect several markers of glutathione metabolism such as total glutathione levels (normality range: 0.5–1.6 mg/g Hb) and glutathione-S-transferase and glutathione peroxidase activities (normality ranges: 0.2–0.7 U/mg Hb and 18.0–54.0 U/g Hb, resp.) (Figures
Glutathione cycle parameters: erythrocyte levels of total glutathione (reduced and oxidized forms, GSH + GSSG) (a) and erythrocyte enzymatic activities of glutathione-S-transferase (b) and of glutathione peroxidase (c) in the study group of patients (
Systemic oxidative stress markers: plasma levels of nitrites/nitrates (NO2 + NO3) (a), of malonyl dialdehyde (MDA) (b), and of Cu,Zn-superoxide dismutase 3 (Cu,Zn-SOD3) (c) in the study group of patients (
Plasma content of hydroxyproline was found highly elevated (
Metabolic parameters related to collagen and ATP synthesis: levels of plasma hydroxyproline (a) and of the lipophilic antioxidant total coenzyme Q10 (reduced and oxidized forms, CoQ10H2 + CoQ10) (b) and erythrocyte ATP (c) in the study group of patients (
In a preliminary
In the present study, we obtained convincing clinical data on the efficacy of the marine collagen peptide and plant antioxidant formulation CELERGEN in improving dermal collagen deposition and structure (Table
Numerous animal studies on the effects of oral administration of natural or synthetic antioxidants towards collagen deposition, reactive species levels, and antioxidant defences generated highly conflicting data, depending on the experimental system. Thus, with various wound healing models, it was repeatedly demonstrated that either complex plant extracts containing active secondary metabolites (triterpenes, polyphenols, alkaloids, etc.) [
On the other hand, in the models of cardiac fibrosis [
UV irradiation could cause skin photodamage causing the symptoms of premature photoageing. Evaluating the photoprotective effects of dietary MCPs isolated from jellyfish umbrella [
Since the supplementation of compounds with a direct antioxidant effect has failed so far to show clinical efficacy and sometimes even aggravated clinical picture [
In the last decade, endogenously produced systemic and cutaneous redox-active substances (superoxide, hydrogen peroxide, NO, lipid peroxides, stable end products of lipid peroxidation, oxidative metabolites of cholesterol and squalene, etc.), previously recognised exclusively as undesirable metabolic by-products and markers of oxidative damage, have shown essential functions in cellular signalling and regulation of cell proliferation, differentiation, migration, innate immunity, energy production, ECM dynamics, vascular tone, stress responses and adaptation, and inflammation [
The antiageing effects of CELERGEN supplementation were evidenced also by the highly increased sebum production (Table
On the grounds of the results obtained and existing literature data, we hypothesised redox-dependent pathways (Figure
Scheme of the hypothesised redox-dependent mechanisms of CELERGEN physiological effects. Marine collagen peptides (MCPs) easily penetrate gastrointestinal wall (GI, three arrows) and through blood circulation are mainly deposited in the skin. Antioxidant component of the nutraceutical is partly metabolised in GI thus possessing low bioavailability (one arrow); however, skin-targeting antioxidants and their metabolites reach different skin layers. While in the circulation, MCPs stimulate blood phagocytes (granulocytes and monocytes) and endotheliocytes (E) to produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) by activating Toll-like receptors 4 (TLR4). Hydroxyproline (HYP) and prolyl-hydroxyproline (Pro-HYP) dipeptides as major components of MCPs are metabolised by corresponding oxidases and hydroxyl radicals are formed as by-products. Antioxidants prevent systemic oxidative stress blocking GSH oxidation, GPx, GST, and SOD3 activation. In the skin, collagen synthesis and deposition as well as elasticity are increased while (hypothetically) low levels of oxidised forms of skin lipids such as unsaturated fatty acids (PUFA-ox), squalene (Sq-ox), malonyl dialdehyde (MDA), and 4-hydroxy-2-nonenal (4-HNE) may facilitate cell signalling for ATP synthesis and sebum production.
The addition of dietary plant-derived antioxidants with known skin tropism and health effects towards human skin did not impair definite induction of collagen synthesis and its deposition as compact organised fibres in the dermal layer by marine fish skin-derived collagen peptides. Additional beneficial effects of antioxidants were observed systemically, as normal balance of systemic endogenous antioxidant defence was maintained, and protection of energy storage occurred.
Marine collagen peptides
Extracellular matrix
NADPH-oxidase
Nitric oxide
Malonyl dialdehyde
High performance liquid chromatography
Transepidermal water loss
Ethylenediaminetetraacetic acid
Red blood cells
Cu,Zn-superoxide dismutase 3
Glutathione-S-transferase
Glutathione peroxidase
Thiobarbituric acid-reactive substances
Hydroxyproline
Transforming growth factor beta-1
Basic fibroblast growth factor
Reactive oxygen species
Reactive nitrogen species.
The authors declare that they have no conflict of interests.
The authors gratefully acknowledge Suisse Ueli Corporation for providing the product for the clinical study free of charge and for covering the costs of reagents and analyses. They are also grateful to Elena Schukina for excellent technical assistance and to Valeriy Chertushkin for organisation of the trial logistics.