Low‐molecular‐weight collagen peptides supplement promotes a healthy skin: A randomized, double‐blinded, placebo‐controlled study

Oral collagen peptides supplementation was reported to improve skin integrity and counteract skin aging.


| INTRODUC TI ON
As humans long for youth and beauty without aging, cosmetic supplements to improve biophysical properties of the skin have gained popularity. 1 In particular, collagen supplements became a research hotspot owing to their potential to improve skin integrity and counteract skin aging.During intrinsic and extrinsic aging, skin changes involve both epidermal and dermal thickness, which are mainly attributed to the loss of collagen and elasticity, as new extracellular matrix component synthesis by dermal fibroblasts diminishes and matrix metalloproteinase (MMP)-mediated collagen degradation is accelerated. 2Therefore, the dermal collagen network undergoes the accumulation of shorter and fragmented collagen fibers, resulting in wrinkled and loosen skin. 2,3Further, the capacity to contain moisture and the amount of hyaluronic acid in the skin decrease with age, therefore leading to dry skin of the elderly. 2 Regarding antiaging strategies, collagen peptides are promising for enhancing skin moisture, elasticity, and density without significant adverse effects. 4Collagen hydrolysates generated by enzymatic modulation of collagen molecules are composed of peptides with varying lengths.6][7][8] Moreover, in vivo studies demonstrated that collagen-derived bioactive peptides can reach the skin and are retained in the tissue for up to 2 weeks, 9 where they can attenuate ultraviolet B-induced aging. 10w-molecular-weight collagen peptides are a form of collagen hydrolysates rich in hydroxyproline, glycine, and proline amino acids.
It was suggested that these peptides can stimulate human fibroblasts to synthesize extracellular matrix molecules and strengthen the skin tissue. 72][13] The aim of this study is to evaluate the clinical efficacy of oral low-molecular-weight collagen peptides supplementation for preventing wrinkles as well as promoting skin whitening via biophysical and skin imaging techniques.

| Test product
Test product is a food supplement that contains low-molecularweight collagen peptides (GPVGPS Collagen®, Daehan Chemtech Co., Ltd.) obtained from fish scales of Nile tilapia (Oreochromis niloticus), along with maltodextrin, silicon dioxide, and sucralose.
The placebo contained all these ingredients but no nutrients.The test product and placebo were provided as a 2.5 g white powder blend, which was administered with water daily, in the morning before a meal.Detailed percentage of each ingredient is shown in Table 1.

| Study design
In our randomized, double-blinded, placebo-controlled clinical trial, all participants were randomly distributed to either the test product or placebo at a 1:1 ratio.Each participant took a packet of powder, which was either a study product or a placebo, once a day for 12 weeks.The number of assessments included four visits: a baseline visit at Week 0 and three more visits determined at Weeks 4, 8, and 12.

| Study participants
A total of 100 adults with their age in 35-60 years, who had dry skin and periorbital wrinkles, were enrolled in the study.Written informed consent of all participants was obtained.The eligibility criteria of the healthy volunteers were as follows: a score of three or more on the 10-grade crow's feet photo scale 14 and skin hydration less than 50 arbitrary units measured using a Corneometer (Courage & Khazaka Electronic) on both cheeks.The study had several exclusion criteria, which were carefully selected to ensure the safety and integrity of the results.These criteria included current pregnancy or lactation, major visceral organ disease, diffuse or active cutaneous disorders, past history of hypersensitivity reactions, recent cosmetic procedures within the past 6 months, applying retinoids, systemic steroids, and topical immunomodulators within the past 3 months, and intake of oral bioactive substances within 14 days before the participation.

| Measurements
Under a controlled environment with 40%-60% humidity and 20°C-24°C room temperature, the clinical efficacy of each parameter was measured.All participants were instructed to maintain rested conditions and restrict water intake 1 h before the assessment.Skin wrinkles were evaluated on the periorbital area using PRIMOS CR (GFMesstechnik, Teltow, Germany), an optical threedimensional non-contact measuring device.Among the skin surface descriptors in PRIMOS, Ra, Rmax, Rp, Rv, and Rz were assessed to represent skin wrinkles and roughness.In addition, the eye wrinkle volume was assessed to determine three-dimensional volumetric changes during the period of this study.

| Safety assessments
After being randomly assigned to the study, the participants were included for safety analysis if the test product or placebo was consumed at least once.A comprehensive physical examination at each visit was conducted to assess the safety.Furthermore, blood samples from all participants were collected at baseline and after the end of study to measure the complete blood cell count, liver and renal function, and glucose and cholesterol levels.Urine samples were also collected and analyzed for abnormalities in the same manner.

| Baseline characteristics of the participants
The randomized participants at baseline were allocated to the test (n = 51) or placebo (n = 49) groups.Six participants of the test group were removed from the analysis: four with consent to withdrawal and two due to protocol violation.In the placebo group, seven participants withdrew from the study.Hence, the per-protocol population included a total of 87 participants, including 45 and 42 participants in the test and placebo groups, respectively.The safety population included 100 patients who consumed the test product or placebo at least once.
The baseline clinical characteristics of the participants are listed in Table 2.No statistically significant differences were found between the groups regarding age, sex, height, and weight.Moreover, no statistically significant differences in lifestyles (e.g., smoking, alcohol, outdoor activity time, daily sleeping hours, and frequency of using sunblock) were observed between the groups.

TA B L E 2
Baseline clinical characteristics of the per-protocol study population.

| Effect on skin wrinkling
The skin wrinkling parameters-average skin roughness (Ra), maximum of all peak-to-valley values (Rmax), the maximum peak height of the wrinkle (Rp), and average maximum height of the wrinkle (Rz)measured using the PRIMOS optical system were found to be considerably improved in the test group compared with the placebo group at 12 weeks (p < 0.0001 for all parameters; Figure 1, Table 3).The starting level of skin wrinkling was similar between the two groups.
In the test group, Ra, Rmax, Rp, and Rz considerably improved from baseline to 12 weeks (p < 0.0001 for all parameters), whereas they remained unchanged or tended to worsen in the placebo group during the same period.In the test group, the maximum valley depth of the wrinkle (Rv) also tended to be reduced at 12 weeks compared with the baseline value (p = 0.6527), whereas in the placebo group tended to increase (p = 0.4625; Table 3).
After 12 weeks, the eye wrinkle volume in the test group was reduced remarkably compared to the baseline (from 19.01 ± 7.23 to 16.40 ± 6.66 mm 3 , p < 0.0001), whereas the parameter in the placebo group showed an increment (from 17.19 ± 6.58 to 17.67 ± 6.94 mm 3 , p = 0.3449).Furthermore, comparison of the changes from baseline in the eye wrinkle volume between the two groups showed statistically significant differences (p < 0.0001; Figure 1, Table 3).

| Effect on skin hydration
In

| Effect on skin elasticity
The skin overall (R2), net (R5), and biological (R7) elasticities showed greater improvements in the test group compared with the placebo group at 12 weeks (p < 0.0001 for all parameters; Figure 3, Table 4).
However, the change from the baseline of the R5 value was statistically significant only at 12 weeks (p = 0.0032), whereas that of R2 and R7 was statistically significant from Weeks 4 (p < 0.0001) and 8 (p = 0.018), respectively.

| Effect on skin whitening
Skin-whitening parameters in the test group showed much greater changes during the treatment compared with those of the placebo group (Figure 4).No significant differences in skin-whitening levels were observed between the groups at baseline.Comparison of the changes from baseline in the erythema index between the two groups revealed statistically significant differences (p = 0.0002).

| Adverse effects and laboratory analyses
The blood and urine analyses conducted on both the test and placebo groups revealed results within the normal ranges.Furthermore, when all the biochemical data were statistically analyzed, no significant variances were found between the two groups.Additionally, none of the participants experienced any adverse effects during the treatment period.

| DISCUSS ION
Low-molecular-weight collagen peptides are highly absorbable and favorably affect the quality and quantity of dermal component of the skin. 12,13Several in vivo studies revealed the underlying mechanisms of oral collagen supplementation.In particular, this therapeutic approach reduces the expression and activation TA B L E 3 Skin wrinkling parameters of the per-protocol study population.6][17][18] Further, bioactive collagen peptides, mainly consisting of prolyl-hydroxyproline, stimulate chemotaxis and proliferation of dermal fibroblasts, thereby enabling the production of dermal components: hyaluronic acids, elastic fibers, and collagen.
Skin surface hydration was measured using Corneometer CM 825 (Courage & Khazaka Electronic), which detects the hydration level below the stratum corneum by electrical capacitance.Skin surface hydration was assessed on the forehead (2 cm above the glabella), forearm (10 cm apart from the inner side of the wrist), cheeks (right angle intersection between lateral canthus and the tip of the nose), and 1 cm below the eyes.The average value of three stabilized measurements was used.Skin elasticity was evaluated using Cutometer MPA 580 (Courage & Khazaka Electronic).The study evaluated the skin's extension when subjected to a suction vacuum applied above the cheeks using a 450-mbar vacuum.This involved a pattern of 2-s exposure followed by a 2-s non-exposure period, repeated three times.Skin overall (R2), net (R5), and biological (R7) elasticities were determined.Skin whitening was assessed using Mexameter MX 18 (Courage & Khazaka Electronic) on the cheek, representing melanin and erythema indexes.The average value of three stabilized measurements was used.

F I G U R E 1
Normalized changes in skin wrinkle parameters of participants in the per-protocol study population.(A) Average skin roughness (Ra), (B) maximum of all peak-to-valley values (Rmax), (C) maximum peak height of the wrinkle (Rp), and (D) average maximum height of the wrinkle (Rz).Changes in parameter values from baseline are shown in micrometers (μm).(E) Eye wrinkle volume shown in cubic millimeters (mm 3 ).Data are expressed as mean ± standard deviation.*p < 0.05, **p < 0.001, and ***p < 0.0001 between groups as determined by two-sample t or Wilcoxon rank sum tests.

F I G U R E 2
Normalized changes in skin hydration in the per-protocol study population.Hydration parameters were determined in the (A) forehead, (B) both forearms (average values are shown), (C) both cheeks (average values are shown), and (D) 1 cm below both eyes (average values are shown).Changes in parameter values from baseline are shown in arbitrary units.Data are expressed as the mean ± standard deviation.*p < 0.05, **p < 0.001, and ***p < 0.0001 between groups as determined by two-sample t or Wilcoxon rank sum tests.F I G U R E 3 Normalized changes in skin elasticity parameters of the per-protocol study population.(A) Overall elasticity (R2), (B) net elasticity (R5), and (C) biological elasticity (R7).Changes in parameter values from baseline are shown in arbitrary units.Data are expressed as mean ± standard deviation.*p < 0.05, **p < 0.001, and ***p < 0.0001 between groups as determined by two-sample t or Wilcoxon rank sum tests. of MMPs (specifically MMP-1, MMP-2, MMP-3, MMP-9, and MMP-12) following ultraviolet B radiation exposure, which subsequently leads to increased synthesis of collagen and elastic fib-

F I G U R E 4
Normalized changes in skin-whitening parameters in the per-protocol study population.(A) Melanin index and (B) erythema index.Changes in parameter values from baseline are shown in arbitrary units.Data are expressed as mean ± standard deviation.*p < 0.05, **p < 0.001, and ***p < 0.0001 between groups as determined by two-sample t or Wilcoxon rank sum tests.
Ingredient composition of the test product and placebo product.

Parameter Time of visit Test group Placebo group Intergroup comparison p-value b Mean (SD) p-value a Mean (SD) p-value a
Skin wrinkle parameters: Ra, average skin roughness; Rmax, maximum of all peak-to-valley values; Rp, the maximum peak height of the wrinkle; Rv, maximum valley depth of the wrinkle; Rz, the average maximum height of the wrinkle.
a Intra-group comparisons; p-values were determined by paired t or Wilcoxon signed-rank tests.b Intergroup comparisons; p-values were determined by two-sample t or Wilcoxon rank sum tests.

μm) Time of visit Test group Placebo group Intergroup comparison p-value b Mean (SD) p-value a Mean (SD) p-value a
Skin elasticity parameters of the per-protocol study population.
22[19][20][21]The findings of the present study agree with these previous reports.Overall, oral consumption with low-molecularweight collagen peptides for 12 weeks improved skin elasticity and reduced skin wrinkles compared with a placebo.Nonetheless, impoints (from Weeks 4, 8, and 12, respectively).The R5 parameter is commonly preferred for quantifying skin aging since it reflects the ability of the skin to recover after deformation and skin roughness without being affected by skin thickness, whereas R2 and R7 represent the elasticity of the skin and its ability to recover its original position, which is highly correlated with skin aging.22Therefore, TA B  E 4 Parameter (Note: Skin elasticity parameters: R2, overall elasticity; R5, ratio of elastic recovery to immediate deformation, representative of net elasticity; and R7, ratio of elastic recovery to total deformation, representative of biological elasticity.a Intra-group comparisons; p-values were determined by paired t or Wilcoxon signed-rank tests.b Intergroup comparisons; p-values were determined by two-sample t or Wilcoxon rank sum tests.