Oral Intake of Low-Molecular-Weight Collagen Peptide Improves Hydration, Elasticity, and Wrinkling in Human Skin: A Randomized, Double-Blind, Placebo-Controlled Study

The LMWCP used herein (obtained from Newtree, Seongnam, Korea) was a collagen hydrolysate obtained from the sutchi catfish’s skin (Pangasius hypophthalmus), 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.

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.

This clinical study had a randomized, double-blind, placebo-controlled design. It was conducted according to the applicable Good Clinical Practice and the Standard Operating Procedures of Ellead Skin and Bio Research (Ellead, Seongnam, Gyeonggi-do, Korea) from 27 February 2012, to 15 June 2012. The study protocol was reviewed and approved by the institutional review board of Ellead (Project number: EL-120208047A003).

Women aged 40–60 years (n = 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).

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.

Hydration of the skin of the cheek area of the designated side of the face is measured with a Corneometer CM 825 (Courage and Khazaka, Cologne, Germany).

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) [19,20,21].

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].

To retain the test area for instrumental measurements to a fixed position, a face mask made of acetate film was attached to the face after baseline measurements. The crow’s-feet area and cheek area selected as the test region were marked on the acetate film with a pen.

After 6 weeks and 12 weeks, participants filled out questionnaires regarding their subjective assessments of the efficacy of their assigned formulations and their appropriateness as a health food, safety (adverse reactions), and preference for the product.

Laboratory tests conducted at 0 W and 12 W + 2 D were hematological blood-chemical tests, urine tests, and vital-sign measurements. Blood-chemical test items were total protein, albumin, aspartate transaminase, alanine transaminase, γ-GTP, blood urea nitrogen, creatinine, glucose, total cholesterol, hemoglobin, hematocrit, white blood cell count, red blood cell count, platelet count, average red blood cell size (MCV), hemoglobin per red blood cell (MCH), and hemoglobin concentration per red blood cell (MCHC). Urine-evaluation parameters were urine pH, specific gravity, protein, glucose, ketone, urobilinogen, bilirubin, and nitrite. Vital signs measured were systolic and diastolic blood pressure and bodyweight.

Safety of the test material was also evaluated by monitoring adverse reactions during the study, through information collected in interviews and questionnaires.

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.

Participants (n = 64) were randomized at baseline and allocated to the test group (n = 33) or the placebo group (n = 31). Values for age, bodyweight, systolic and diastolic blood pressures, and crow’s-feet visual grade (global photodamage score) did not show any significant difference between the test group and the placebo group at baseline (Table 3).

Skin hydration in the test group was significantly greater at 6 weeks (p = 0.000) and 12 weeks (p = 0.000) than at baseline, whereas in the placebo group there was no difference from baseline at 6 weeks (p = 0.273), but hydration was significantly increased at 12 weeks (p = 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 (p = 0.000 and p = 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.

In the test group, the visual grade related to wrinkle formation was significantly improved from the baseline value at 12 weeks (p = 0.000) (Figure 3A, Table 5). The test group also showed a significantly different visual grade to the placebo group at 12 weeks (p = 0.013) (Figure 3A, 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 3A).

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

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 (p = 0.025 and p = 0.027, respectively) (Figure 4A,B, Table 7). In the test group, however, from the baseline, only the value of R5 was significantly improved at 12 weeks (p = 0.002) (Figure 4B, 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 4A,B, Table 7).

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.