Preventive remedies for skin photoaging are required because of the inevitability of exposure to sunlight. Collagen has been consumed as functional dietary supplements because of its efficacy for skin health. Collagen hydrolysates or collagen peptides (CPs) are also receiving attention, with studies in vitro and in vivo investigating the properties of various CPs, including fish-collagen hydrolysates derived from type I collagen from fish skin. In hairless mice, oral administration of Low-molecular-weight Collagen peptide (LMWCP), which is a fish-derived collagen hydrolysate, promotes recovery of collagen fibers and normal elastic fibers in the skin from degraded collagen and abnormal elastic fibers caused by UVB irradiation [ 8 ]. The study showed that this treatment leads to reductions in levels of collagenases (matrix metalloproteinase (MMP)-3 and MMP-13) expression and activities of gelatinases (MMP-2 and MMP-9), thus inhibits the breakdown of dermal collagen and results in significant reductions in skin wrinkling and trans-epidermal water loss (TEWL), and increases in skin elasticity and hydration [ 8 ]. These results suggest that LMWCP affects the regeneration of collagen and elastic fibers, thereby improving skin health (barrier function, wrinkling, hydration, and elasticity). LMWCP contains 15% tripeptide which makes it differ from other CPs which rarely contain tripeptides.

In the skin, collagen, elastic fibers, and HA are major structural constituents of dermal extracellular matrix (ECM) [ 10 12 ]. Collagen may constitute >70% of the dry weight of the normal human skin dermis [ 13 14 ]. The dermis is composed of two morphologically different layers: the adventitial dermis of thin collagenous fibers and the reticular dermis of thick, coarse collagen bundles [ 15 ]. Skin elastic fibers are composed of an inner core of crosslinked elastin with outer layers of fibril in micro fibrils [ 4 16 ]. The HA content of the dermis is much higher than that of the epidermis, and papillary dermis has significantly greater levels of HA than reticular dermis [ 12 ].

Various factors contribute to the aging of human skin. Photoaging of the skin is induced by chronic sunlight exposure, and is a form of extrinsic aging, whereas intrinsic aging arises mainly from the decline of biological function and the action of reactive oxygen species derived from cellular metabolism [ 1 ]. Although both intrinsic and extrinsic processes cause age-dependent skin alterations, changes are more prominent in photoaged skin, as evidenced by comparisons between the facial skin which has been exposed to the sun and the buttock skin which has been protected from the sun [ 2 3 ]. In the skin severely photoaged, elastic fibers are highly disorganized and deposited abundantly throughout the dermis, and in the skin mildly photoaged, loss of fibril in micro fibrils from the dermis is shown, whereas the elastic-fiber network is gradually fragmented in intrinsic aging [ 4 7 ]. Characteristics of photoaged skin include coarse wrinkles, dryness, loss of elasticity, pigmentation [ 1 ]. In the process of photoaging resulting from solar UV radiation, skin may experience loss of collagen and elastic fibers, which, along with a reduction in the synthesis of hyaluronic acid (HA), eventually leads to wrinkle formation, dryness, and loss of elasticity [ 1 9 ].

Statistical analysis was performed in two ways: through an intention-to-treat (ITT) analysis and a per-protocol (PP) analysis, with significance level of 0.05 unless otherwise noted. Missing data were dealt with by the last-observation-carried-forward method for ITT analysis. PP analysis was used for the efficacy evaluation, and ITT analysis was used for the safety evaluation. All results in the efficacy test, blood test, urine test, and vital-signs test were analyzed using descriptive statistics and compared between before and after treatment by paired t -tests and between groups by independent t -tests. In urinalysis, for the parameters except for specific gravity and pH, McNemar’s tests and Mann–Whitney U tests were used after results were classified as either normal or abnormal. The incidence rate of adverse reactions in each group was compared by Chi-square tests or Fisher’s exact tests.

A Cutometer MPA 580 (Courage and Khazaka) was used for assessment of skin elasticity of the designated crow’s-feet area, based on suction of the skin using a probe with negative pressure of 450 mbar, which makes the test area drawn into the aperture of the probe. Measurement was repeated three times with 2 s of suction time followed by 2 s of relaxation time for each measurement. Curves of skin deformation obtained were analyzed with Win Cutometer MPA software to obtain the values of skin-elasticity parameters: R2 (overall elasticity of the skin), R5 (net elasticity), and R7 (the ratio of elastic recovery to total deformation) [ 22 ].

Skin wrinkling was assessed by two methods: visual assessment by dermatologists and instrumental analysis of skin-replica images. For visual assessment, cutaneous examinations of the crow’s-feet area were conducted by two dermatologists with a double-blind method based on a global photodamage scoring system [ 18 ]. The average score from the two dermatologists was used. For instrumental analysis of skin-replica images, replicas made with a silicone-based solution and a catalyst (Courage and Khazaka) were taken from the designated crow’s-feet area and analyzed with a Visiometer (SkinVisiometer SV 600, Courage and Khazaka) that evaluates the topography of the skin surface by light transmission of a very thin silicone replica based on skin-wrinkling parameters: R1 (skin roughness), R2 (maximum roughness), R3 (average roughness), R4 (smoothness depth), and R5 (arithmetic average roughness) [ 19 21 ].

All participants took one bottle (orally) of their assigned study formulation once daily. To minimize any weakness of the study, all participants were required to refrain from intake of any similar dietary supplements, and from the use of any skincare treatments such as face masks or packs and massages. They were also not permitted to apply topical cosmetics except those provided by Ellead for a 2-week wash-out period before the study starts and for the 12-week study period, to maintain constant skin conditions. Each participant visited the research center for assessment four times in total: prior to intake of the study formulation at baseline (0 W), at 6 weeks (6 W) and 12 weeks (12 W) after intake of the study formulation, for efficacy measurements, and at 2 days after completion of intake at 12 weeks (12 W + 2 D) for safety evaluation. Prior to visiting the test facility, participants were banned from use of cosmetic products for 12 h. At each visit, participants shaved the crow’s-feet area, washed the entire face with foam cleansers, and rested for 30 min under the constant relative humidity (40–60%) and temperature (22–24 °C) prior to assessment. The same part of the face was examined at each visit, according to the same methods used in the examination conducted at 0 W. Prior to the visit for safety evaluation, participants were prohibited from eating and drinking for ≥8 h.

Women aged 40–60 years (= 70) who volunteered and met specified inclusion and exclusion criteria were recruited for the study ( Table 2 ). Inclusion criteria included crow’s-feet scores between 2 and 6, as determined by dermatologists according to the global photodamage scoring system [ 18 ]. Before proceeding to the clinical study, the participants were informed clearly and precisely of the objective and the protocol of the study, and of foreseeable risks involved in the trial. Participants signed an informed-consent form. Six individuals withdrew consent, and the remaining 64 commenced the study. Eleven participants dropped out of the study for personal reasons, and 53 completed the study ( Figure 1 ).

In the previous animal study using hairless mice, LMWCP exerted its effects on skin wrinkling, skin hydration, TEWL, skin elasticity, collagen formation in the dermis, and the regulation of expression of MMP-3 and MMP-13 and the activities of MMP-2 and MMP-9 at the dose of 167 mg/kg b.w. and 333 mg/kg b.w. [ 8 ]. Based on the doses of efficacy in the UVB-irradiated hairless mice, human dose was calculated using the body surface area normalization method [ 17 ]: 167 mg/kg b.w. of mice was translated to 13.5 mg/kg b.w. of human; 333 mg/kg b.w. of mice to 27.0 mg/kg b.w. of human. Thus 1000 mg/day for human was chosen as a dose for human study from the translated values applied to the average adult with 60 kg body weight: between 810 mg/day and 1620 mg/day.

The LMWCP used herein (obtained from Newtree, Seongnam, Korea) was a collagen hydrolysate obtained from the sutchi catfish’s skin (), with >15% tripeptide content including 3% Gly-Pro-Hyp. A 50 mL test bottle contained 1000 mg of LMWCP along with vehicle material ( Table 1 ). The placebo had the same formulation with the identical flavor and taste as the test material, except that LMWCP was replaced by water.

The results of blood-chemical tests, urine tests, and vital-sign measurements showed that all the measured parameters of the test group and the placebo group were in normal ranges before and after intake. Statistical analysis of all the data showed no significant differences from the normal range. No adverse reactions were observed in any of the participants during the course of the study.

The values of the skin-elasticity parameters of overall elasticity of the skin (R2) and net elasticity of the skin (R5) were significantly higher in the test group than in the placebo group at 12 weeks (0.025 and= 0.027, respectively) ( Figure 4 A,B, Table 7 ). In the test group, however, from the baseline, only the value of R5 was significantly improved at 12 weeks (0.002) ( Figure 4 B, Table 7 ). In the placebo group, none of the skin-elasticity parameters was shown to significantly improve from baseline values during the test period ( Figure 4 A,B, Table 7 ).

The instrumentally measured wrinkling parameters R1, R3, and R4 were significantly improved in the test group compared with the placebo group at 12 weeks (0.043,= 0.025, and= 0.004, respectively) ( Figure 3 B–D, Table 6 ). In the test group, R1, R3, R4, and R5 (data not shown) were significantly improved from baseline at 12 weeks (= 0.004,= 0.001,= 0.008, and= 0.035, respectively), whereas none of these parameters was significantly improved in the placebo group ( Figure 3 B–D, Table 6 ). In the test group, R3 was also significantly improved at 6 weeks compared with baseline (0.044) ( Figure 3 C, Table 6 ).

In the test group, the visual grade related to wrinkle formation was significantly improved from the baseline value at 12 weeks (0.000) ( Figure 3 A, Table 5 ). The test group also showed a significantly different visual grade to the placebo group at 12 weeks (0.013) ( Figure 3 A, Table 5 ). The increment of wrinkle improvement in visual grade in the test group was 10.5-fold greater than in the placebo group at 12 weeks ( Figure 3 A).

Skin hydration in the test group was significantly greater at 6 weeks (0.000) and 12 weeks (0.000) than at baseline, whereas in the placebo group there was no difference from baseline at 6 weeks (0.273), but hydration was significantly increased at 12 weeks (= 0.001) ( Figure 2 Table 4 ). In comparisons between groups, skin-hydration values were significantly higher in the test group than in the placebo group at 6 weeks and 12 weeks (0.000 and= 0.003, respectively) ( Figure 2 Table 4 ). The increment of skin hydration in the test group was 7.23-fold greater than in the placebo group at 6 weeks, and 2.9-fold greater at 12 weeks.

4. Discussion

The use of collagen or collagen hydrolysates as food or dietary supplements for the improvement of skin properties is increasing. LMWCP is a fish-derived type I collagen hydrolysate that is prepared by enzymatic hydrolysis and has a >15% content of tripeptides (Gly-X-Y, where X and Y are different amino acid residues that are often proline, hydroxyproline, or alanine) including 3% Gly-Pro-Hyp. The result of previous study in animal model suggested that oral intake of LMWCP may improve human skin function [ 8 ]. In this clinical trial, we investigated the efficacy of oral intake of LMWCP on human skin function. The results demonstrated that oral intake of 1 g of LMWCP once daily for 12 weeks significantly improved skin hydration, wrinkling, and elasticity in human participants.

24, Upon oral intake of collagen hydrolysates, small peptides derived from collagen (such as the tripeptide Gly-Pro-Hyp) can be detected in human blood, with peak concentrations observed 1–2 h after intake [ 23 25 ]. Gly-Pro-Hyp is readily absorbed intact across the intestine into the blood in rats [ 26 ] and humans [ 24 ]. In rats and mice, absorbed Gly-Pro-Hyp remains in the plasma for several hours, with a peak concentration ~2–4 h after administration, and can be detected in various tissues, especially in the skin, where it remains for longer than in other tissues [ 26 ]. In humans, the dipeptide Pro-Hyp (which is not digestible by peptidase) has been detected in the blood along with Gly-Pro-Hyp, suggesting that Pro-Hyp is originated from Gly-Pro-Hyp [ 24 25 ]. Transfer of biologically functional Pro-Hyp to the skin may stimulate migration and growth of fibroblasts [ 25 27 ], thereby exerting favorable effects on skin function, because dermal fibroblasts enable the synthesis of collagen, elastic fibers, and dermal HA [ 28 29 ]. Our observation of beneficial effects on the skin resulting from oral intake of LMWCP suggests that the tripeptides contained in LMWCP are efficiently absorbed and biologically active.

HAS2 transcription in human dermal fibroblasts in vitro [ In this study, both the test group and the placebo group showed significantly improved skin hydration from the baseline value at 12 weeks. However, only the test group showed significant improvement earlier at 6 weeks whereas no significant improvement was seen in the placebo group. And both at 6 weeks and 12 weeks, skin hydration values were significantly higher in the test group compared with the placebo group. The improvement form baseline values in the placebo group at 12 weeks might have been due to the effect of the vehicle material and/or cosmetics supplied by investigators. However, it was proved that the skin hydrating efficacy of LMWCP alone is statistically significant after zeroing out that of the vehicle material and/or cosmetics used. Skin hydration is affected by water bounded to HA in the dermis and the epidermis [ 12 ]. The collagen-derived Hyp-containing peptide Pro-Hyp has been shown to stimulate HA synthesis by activation oftranscription in human dermal fibroblasts in vitro [ 30 ]. It may be hypothesized that the improvements in skin hydration that occurred in the present study might therefore have resulted from increased production of HA via Pro-Hyp derived from LMWCP. However, this needs to be proved with the further studies in the future.

The improvements in skin wrinkling that we observed in this study in response to intake of LMWCP coincide well with the results of the previous animal study where expression and activation of MMPs (MMP-1(MMP-13 in rodents), MMP-2, MMP-3, and MMP-9) are down-regulated by oral administration of LMWCP in UVB-irradiated mice, thereby increasing collagen content in the skin and reducing wrinkle formation [ 8 ].

The mechanical properties of human skin: elasticity, resilience, and toughness, are mainly affected by collagen and elastic-fiber networks in the ECM in the dermis, and the elastic-fiber network is known to be contributing to the skin elasticity most [ 31 ]. MMP-2 and MMP-9 degrade insoluble elastin to soluble fragments [ 32 33 ], and MMP-2, MMP-9, and MMP-13 catabolize fibrillin microfibrils [ 34 35 ], so down-regulation regarding these proteins is likely to affect levels of elastic fibers in the skin [ 4 16 ]. Therefore, the oral intake of LMWCP might have favorably affected the content of both collagen and elastic fibers in the skin via down-regulation of MMPs, thereby improving skin wrinkling and skin elasticity. The skin-elasticity parameter R5 (net elasticity) represents only elastic characteristics of the skin, and so may be solely affected by elastic fibers [ 22 ]. In this study, R5 in the test group significantly improved from the baseline level at 12 weeks, while both R2 (overall elasticity) and R5 were higher with significance compared with the placebo group, suggesting that LMWCP directly exerts its effect on skin elasticity of the photoaged skin because one of the inclusion criteria for recruiting participants was to specify the eligible photodamage score of the UV-exposed site, crow’s feet. Skin hydration also strongly affects skin elasticity, because conformational change in elastin occurs only in hydrated protein [ 36 ]. The increased level of skin hydration that we observed following intake of LMWCP may therefore have contributed to the increase in skin elasticity. No adverse reactions to LMWCP were observed, and the results of laboratory tests and vital-sign tests in the LMWCP group were in normal ranges.

Our results demonstrated that oral intake of LMWCP (1000 mg once daily for 12 weeks) efficiently improved the health of photoaged skin in a cohort of women aged 40–60 years, by significantly improving skin hydration as early as after 6 weeks of intake, and skin wrinkling and elasticity after 12 weeks of intake. The LMWCP preparation was well tolerated by participants in the study. Although this study was done only for female individuals at the age between 40 to 60 years old, the results may still be valid for the male and other age groups. However, the further studies using both male and female at different age group are recommended to generalize the anti-photoaging efficacy of LMWCP. Furthermore, it would be also desirable if the larger number of participants is used hence the more robust results can be expected in the future studies. Interest in the development of anti-skin aging agents has been increasing considerably, and LMWCP may have potential as a safe and effective ingredient for health-functional food or dietary supplements, to improve skin wrinkling, hydration, and elasticity.