A Cosmetic Formulation Containing Hydrolyzed Fish Skin Extract Enhances Procollagen Production and Improves Wrinkle Appearance: A Randomized, Double-Blind, Split-Face Clinical Trial

Abstract

Skin aging is characterized by decreased collagen synthesis and increased extracellular matrix degradation, leading to wrinkle formation and reduced skin elasticity. This study evaluated the anti-aging potential of hydrolyzed fish skin (HFS) extract through complementary in vitro and clinical investigations. In human dermal fibroblasts, treatment with HFS extract enhanced type I procollagen production and suppressed UVB-induced matrix-degrading enzymes, including matrix metalloproteinase-1 (MMP-1) and elastase, suggesting a mechanism that supports dermal matrix homeostasis. A randomized, double-blind, split-face clinical trial was conducted in 20 female participants over 12 weeks. A formulation containing 0.5% HFS extract was applied to one side of the face, while an identical vehicle control formulation without HFS extract was applied to the contralateral side. Wrinkle parameters were assessed using a three-dimensional imaging system. After 12 weeks, the test group showed significant improvements compared to baseline, with reductions of 12.75% in arithmetic mean roughness (Ra), 12.46% in root mean square roughness (Rq), and 11.32% in maximum wrinkle height (Rmax) (p < 0.05). No adverse events were observed. These findings demonstrate that HFS extract improves wrinkle-related skin parameters, potentially through promoting collagen synthesis while inhibiting matrix degradation. The combined molecular and clinical evidence supports its application as a functional cosmetic ingredient in anti-aging formulations.

1. Introduction

Skin aging is a complex biological process influenced by both intrinsic and extrinsic factors, resulting in visible changes such as wrinkle formation, skin sagging, and loss of elasticity. Among these age-related alterations, the degradation of collagen and elastin fibers within the dermal matrix plays a central role. Collagen, the primary structural protein in the skin, provides tensile strength, whereas elastin is responsible for maintaining skin elasticity. The preservation of these extracellular matrix components is therefore essential for maintaining youthful and resilient skin. However, aging disrupts the balance between their synthesis and degradation, a process that is further accelerated by environmental factors such as ultraviolet (UV) radiation. This imbalance is mediated by proteolytic enzymes, including matrix metalloproteinase-1 (MMP-1), which degrades collagen, and elastase, which breaks down elastin [1,2,3].

To counteract these age-related changes, various strategies aimed at enhancing collagen synthesis and inhibiting MMP-1 and elastase activity have been investigated. In this context, natural bioactive compounds have attracted considerable attention due to their favorable safety and efficacy profiles.

Hydrolyzed fish skin (HFS) extract is widely used as a functional ingredient in cosmetics, food, and pharmaceutical applications due to its anti-aging and antioxidant effects [4]. It is produced by breaking down large collagen molecules into smaller peptides, which are characterized by low molecular weight and potential biological activity [5]. Low-molecular-weight collagen peptides from HFS are typically composed of specific tripeptide sequences such as Glycine–Proline–Hydroxyproline (Gly-Pro-Hyp; GPH) and Glycine–Proline–Alanine (Gly-Pro-Ala; GPA) [6]. These tripeptides have been identified as key functional components contributing to skin health. Among these, GPH has been reported to stimulate collagen synthesis, thereby enhancing skin hydration and elasticity [7].

Accordingly, HFS extract has emerged as a promising candidate for anti-aging applications. Previous studies have suggested that collagen-derived peptides may promote collagen production while suppressing MMP-1 and elastase activity, thereby mitigating the structural degradation associated with skin aging [8,9]. In addition, several clinical studies have demonstrated that oral supplementation with collagen tripeptides can improve skin moisture and elasticity in female subjects [10,11].

Although hydrolyzed collagen has been widely used as an oral supplement, recent studies suggest that low-molecular-weight collagen-derived peptides may also exert biological effects on dermal fibroblasts by acting as signaling molecules [12]. In particular, tripeptides such as GPH have been reported to stimulate collagen synthesis and modulate extracellular matrix homeostasis. In addition, several cosmetic formulations containing hydrolyzed collagen have demonstrated potential efficacy when applied topically, although the available clinical evidence remains limited and often lacks rigorous study designs [13].

Despite these findings, there is still a lack of well-controlled clinical studies evaluating the topical application of hydrolyzed fish skin-derived peptides, particularly those enriched in specific bioactive sequences such as GPH. Furthermore, the relationship between in vitro biological activity and clinically observable skin improvements remains insufficiently characterized.

The HFS extract used in this study is a commercially available, food-grade material with a well-defined peptide composition and established safety profile. Its low-molecular-weight peptide content suggests potential bioactivity relevant to skin physiology; however, its efficacy as a topical cosmetic ingredient has not been fully investigated in a controlled clinical setting.

In this context, the present study extends previous findings by integrating mechanistic in vitro assays with a randomized, double-blind, split-face clinical study conducted over a 12-week period, combined with quantitative three-dimensional skin imaging. This approach enables a more robust and comprehensive evaluation of both biological activity and clinical efficacy.

We hypothesized that topical application of a cosmetic formulation containing HFS extract would result in measurable improvements in wrinkle parameters compared with those of a vehicle control, as assessed by quantitative three-dimensional imaging over a 12-week period.

2. Materials and Methods

2.1. Preparation of Hydrolyzed Fish Skin (HFS) Extract

The HFS extract used in this study was supplied by NEWTREE Co., Ltd. (Seoul, Republic of Korea). This material is a food-grade collagen product originally developed for oral consumption as a functional ingredient in the food and nutraceutical industries. It is derived from the skin of Pangasius hypophthalmus and produced through enzymatic hydrolysis to generate low-molecular-weight collagen peptides enriched with bioactive sequences such as GPH [14,15]. The manufacturing process involved enzymatic hydrolysis of fish skin-derived gelatin, followed by filtration, ultrafiltration, sterilization, and drying to obtain a purified powder enriched in low-molecular-weight collagen peptides with over 4% of GPH.

For the in vitro experiments, the HFS extract was dissolved in culture medium and serially diluted to the desired concentrations after confirming its solubility. Based on a preliminary cytotoxicity evaluation, treatment concentrations ranging from 0.13 to 4 mg/mL were selected.

2.2. In Vitro Assay

2.2.1. Cell Culture

Human dermal fibroblasts (HDFs) used in this study were primary cells obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; Catalog No. PCS-201-012). The cell line information is registered in the Cellosaurus database (Accession No. CVCL_0046). All cells were authenticated and confirmed to be free of contamination, including Mycoplasma, bacteria, yeast, fungi, hepatitis B virus, hepatitis C virus, and HIV-1. The HDFs were seeded in culture dishes and cultured in a DMEM/F12 3:1 mixed medium (Gibco, Grand Island, NY, USA) containing a 1% antibiotic–antimycotic solution (Gibco) and 10% FBS (Gibco). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. All in vitro experiments were performed with at least three independent biological replicates, with each condition tested in triplicate. Statistical analyses were conducted based on independent experiments. The concentration range used in subsequent assays was determined based on preliminary cytotoxicity evaluation and solubility assessment, ensuring that the selected concentrations were within a non-cytotoxic and physiologically relevant range.

2.2.2. Cell Viability Assay

Cell viability was analyzed using the MTT assay. HDFs were seeded in a 24-well plate at a density of 5 × 10⁴ cells/well and cultured for 24 h. After this incubation, the medium was replaced with FBS-free medium containing serial dilutions of the test substance, and the cells were incubated for another 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) solution was then added to each well to achieve a final concentration of 0.05%, and the cells were incubated for an additional 4 h. After removing the culture medium, 1000 µL of DMSO was added to each well, followed by shaking for over an hour. The absorbance was measured at 540 nm using an Epoch2 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

2.2.3. Measurement of Type I Procollagen Production

HDFs were seeded in a 24-well plate at a density of 5 × 10⁴ cells/well and cultured for 24 h. The medium was then replaced with FBS-free medium containing serial dilutions of the test substance. After 24 h of treatment, culture supernatants were collected, and type I procollagen synthesis was quantified by measuring procollagen type I C-peptide (PIP) levels using an ELISA kit (Takara Bio Inc., Kusatu, Japan). TGF-β1 (final treatment concentration 0.00001 mg/mL) was used as the positive control for comparison. Collagen production was assessed by normalizing to total protein content, and protein content was quantified using the Bradford method, with BSA (Sigma-Aldrich, St. Louis, MO, USA) as the standard.

2.2.4. Measurement of Matrix Metalloproteinase-1 (MMP-1) Production in UVB-Irradiated HDFs

HDFs were seeded in a well plate and cultured for 24 h and then irradiated with UVB at 10 mJ/cm². The medium was then replaced with FBS-free medium containing serial dilutions of the test substance, and the cells were cultured for another 24 h. After this period, the culture supernatant from each well was used to measure absorbance at 450 nm using an MMP-1 Human ELISA Kit (Invitrogen, Waltham, MA, USA). Retinoic acid (final treatment concentration 0.003 mg/mL) was used as the positive control for comparison. MMP-1 production was assessed by normalizing to total protein content, and protein content was quantified using the Bradford method, with BSA (Sigma-Aldrich) as the standard.

2.2.5. Measurement of Elastase Activity Inhibition

HDFs were seeded in a culture dish and harvested by adding a 0.1% Triton X-100 (Yakuri Pure Chemicals Co., Ltd., Uji, Japan) and 0.2 M Tris-HCl (pH 8.0) solution. The harvested cells were frozen and thawed three times, centrifuged at 10,000 rpm for 20 min at 4 °C, and then the supernatant was used as the elastase enzyme solution. The protein concentration of the elastase enzyme solution was determined, and a specific protein amount was added to a 96-well plate with 0.2 M Tris-HCl (pH 8.0) buffer to reach a final volume of 80 µL. The test substance was diluted in buffer and added in 10 µL increments, followed by 10 µL of 10 mM STANA (Sigma-Aldrich) solution (the substrate for elastase) to each well. The reaction was carried out at 37 °C in an oven (JSOF-150; JSR, Gongju, Republic of Korea) for 90 min. After the reaction, absorbance was measured at 405 nm to assess elastase activity. Phosphoramidon (final treatment concentration 0.0059 mg/mL) was used as the positive control for comparison.

2.3. Clinical Study

2.3.1. Study Design

A randomized, double-blind, split-face clinical study was conducted to evaluate the efficacy and safety of the test product (Evercollagen Collagen Corrector GPH, EVERCCG; NEWTREE Co., Ltd., Seoul, Republic of Korea) for the improvement of skin wrinkles.

The study was conducted at the Dermapro Skin Research Center (Seoul, Republic of Korea), a dedicated clinical research facility specializing in dermatological and cosmetic evaluation.

The study protocol was reviewed and approved by the Institutional Review Board of DERMAPRO Co., Ltd. (Seoul, Republic of Korea) on 9 February 2023 (IRB No. 1-220777-A-N-01-DICN23021). The study was conducted from February to May 2023 in accordance with the ethical principles of the Declaration of Helsinki. The study was registered with the Clinical Research Information Service (CRIS), Republic of Korea (Registration No. KCT0011965, Registration Date: 12 May 2026).

Healthy female volunteers who met the predefined inclusion and exclusion criteria were enrolled in the study. Prior to study initiation, all participants received a detailed explanation of the study objectives and procedures and provided written informed consent.

The test and control formulations were identical in composition and shared the same vehicle base, with the only difference being the inclusion of 0.5% HFS extract in the test formulation. The control formulation contained no additional active ingredients, making HFS extract the primary variable distinguishing the two formulations.

Both products were applied twice daily (morning and evening) to the designated areas. Approximately 300 mg of product was applied per application to the crow’s feet region. Participants were instructed to apply the products using gentle circular motions and to avoid cross-contamination between the two sides.

Formal power calculation was not performed; instead, the sample size was determined based on the functional cosmetic evaluation guidelines issued by the MFDS, which recommend a minimum of 20 subjects for wrinkle efficacy studies.

Participants were randomized using a predefined block randomization scheme, with allocation determining which side of the face (left or right) received the test product or the control product in a 1:1 ratio. The block size was not disclosed to participants or investigators prior to study completion in order to maintain allocation concealment.

The random allocation sequence was generated by an independent investigator. Participant enrolment was conducted by the clinical research staff, and assignment to interventions was performed by a designated study coordinator who was not involved in outcome assessment. Treatment assignment was concealed from both investigators and participants until completion of the study.

Blinding was maintained by providing the test and control products in identical formulations and containers, with labeling performed by an independent product manager. Both investigators and participants remained blinded to treatment allocation throughout the study.

Clinical assessments were performed at baseline and after 4, 8, and 12 weeks of product use. The split-face design is a well-established approach in cosmetic clinical research, as it allows each participant to serve as their own control, thereby minimizing inter-individual variability and reducing potential confounding factors [16]. This design facilitates a more accurate assessment of the effects of the active ingredient. No changes to the study methods or eligibility criteria were made after trial commencement.

2.3.2. Participants (Inclusion and Exclusion Criteria)

Inclusion Criteria

Participants were eligible for inclusion if they met all of the following criteria:

(1)

Female subjects aged 45–58 years with wrinkles of grade ≥3 at the test site, as determined using standardized photographic grading according to the judgment of the principal investigator;

(2)

Absence of acute or chronic systemic diseases, including dermatological disorders;

(3)

Provision of written informed consent after receiving sufficient explanation of the study objectives and procedures;

(4)

Ability and willingness to comply with all study visits and follow-up assessments throughout the study period.

Exclusion Criteria

Participants were excluded if they met any of the following criteria:

(1)

Pregnancy, breastfeeding, or the possibility of becoming pregnant;

(2)

Use of topical formulations containing steroids for the treatment of skin diseases for more than 1 month;

(3)

Participation in the same or a similar clinical study within 6 months prior to enrollment;

(4)

Presence of sensitive or hypersensitive skin;

(5)

Abnormal skin conditions at the test site, including visible pigmentation, acne, erythema, or telangiectasia;

(6)

Use of identical or similar cosmetic products or pharmaceutical agents on the test site within 3 months prior to study initiation;

(7)

History of dermatological procedures at the test site, such as skin resurfacing, botulinum toxin injections, wrinkle removal procedures, or other skin treatments, or plans to undergo such procedures within 6 months;

(8)

Presence of chronic systemic diseases, including asthma, diabetes mellitus, or hypertension;

(9)

Any condition considered by the principal investigator to interfere with safe participation or reliable evaluation of the study outcomes.

2.3.3. Instrumental Assessment of Wrinkles

Wrinkle assessments were conducted to evaluate the efficacy of the test product. Wrinkles were quantified using skin surface roughness parameters measured at the crow’s feet region. The arithmetic mean roughness (Ra), root mean square roughness (Rq), and maximum wrinkle height (Rmax) were assessed using the PRIMOS CR Small Field system (Canfield Scientific, Parsippany, NJ, USA), where Ra reflects the overall average surface roughness, Rq represents the variability in surface height deviations, and Rmax indicates the maximum wrinkle depth or height within the measured area. These parameters have been widely adopted in cosmetic clinical research for quantitative wrinkle evaluation [17,18]. All parameters were measured at the same facial region before and after product application.

2.3.4. Visual Assessment of Wrinkles

Two independent investigators performed visual assessments of wrinkles before and after product use. Wrinkle severity was graded on a 10-point scale. The inter-rater reliability between the two evaluators was examined using the intraclass correlation coefficient (ICC), and an ICC value of ≥0.8 confirmed a high level of agreement, consistent with established guidelines for ICC interpretation [19].

2.3.5. Outcomes

The primary outcome was the change from baseline in skin wrinkle parameters (Ra, Rq, and Rmax), measured using a three-dimensional imaging system at 4, 8, and 12 weeks. Secondary outcomes included visual assessment scores evaluated by trained investigators.

2.4. Statistical Analysis

Statistical analyses were performed using SPSS^® software Version 26.0 (IBM Corp., Armonk, NY 10504, USA). Data are presented as mean ± standard deviation (SD). For the in vitro experiments, statistical significance was evaluated using an unpaired two-tailed Student’s t-test for comparisons between two groups. For the clinical data, normality was assessed using the Shapiro–Wilk test. Changes over time and differences between test and control conditions in the split-face design were analyzed using repeated-measures analysis of variance (RM-ANOVA) with paired comparisons, with baseline values considered where appropriate. Inter-rater reliability for visual grading was evaluated using the intraclass correlation coefficient (ICC). Statistical significance was defined as p < 0.05. For in vitro experiments, percentage changes were calculated relative to the solvent control. For clinical data, percentage changes were calculated relative to the control group using the following formula: ((treated value − control value)/control value) × 100.

3. Results

3.1. Quantitative Determination of Gly–Pro–Hyp (GPH) Content in HFS Extract

The GPH (Glycine–Proline–Hydroxyproline; ≥95.2%, Bachem, H-3630.0250) content in the HFS extract was quantitatively determined using high-performance liquid chromatography (HPLC) with UV/PDA detection at 214 nm.

Quantitative analysis was performed using three independent batches, each analyzed in triplicate. The mean GPH content across all measurements was 4.0446%. Batch-specific mean values were 4.0884%, 4.0524%, and 3.9930% for sample 1, sample 2, and sample 3, respectively.

3.2. In Vitro Effects of HFS Extract on Wrinkle Improvement

3.2.1. Cell Viability

As shown in Figure 1A, HFS extract did not exhibit cytotoxicity in human dermal fibroblasts at concentrations up to 1 mg/mL. Cell viability remained comparable to that of the solvent control, with a slight but significant increase observed at 0.13 mg/mL (p < 0.05), whereas a significant reduction in cell viability was observed at concentrations ≥ 2 mg/mL. Based on these results, subsequent in vitro experiments were conducted at concentrations of 1 mg/mL or lower.

3.2.2. Type I Procollagen Production in Human Dermal Fibroblasts

HFS extract significantly increased type I procollagen production in human dermal fibroblasts, as determined by PIP levels. PIP production increased significantly by 5.85% to 27.89% compared to the solvent control at concentrations ranging from 0.13 to 1 mg/mL (p < 0.05, Figure 1B). TGF-β1 was used as a positive control.

3.2.3. Inhibition of MMP-1 Production in UVB-Irradiated HDFs

HFS extract significantly reduced UVB-induced MMP-1 levels in culture supernatants. UVB irradiation (10 mJ/cm²) markedly increased MMP-1 levels compared to the non-irradiated control, whereas treatment with HFS extract significantly decreased MMP-1 contents by 10.31% to 26.68% at concentrations ranging from 0.25 to 1 mg/mL compared to the UVB-irradiated solvent control (p < 0.05, Figure 1C).

3.2.4. Inhibition of Elastase Activity

HFS extract significantly reduced elastase activity. Elastase activity decreased by 1.36% to 17.86% at concentrations ranging from 0.06 to 1 mg/mL compared to the solvent control (p < 0.05, Figure 1D). Phenylmethylsulphonyl fluoride (PMSF) was used as a positive control.

3.3. Clinical Efficacy and Safety of the Test Product on Wrinkle Improvement

3.3.1. Participant Characteristics

A total of 23 female participants who met the inclusion and exclusion criteria were enrolled in the study. During the study period, three participants discontinued the study due to loss to follow-up related to COVID-19 (n = 1) or voluntary withdrawal (n = 2). As a result, 20 participants aged 45 to 58 years (mean age: 52.40 ± 3.79 years) completed the study and were included in the final analysis. Efficacy analysis was conducted on the per-protocol (PP) population comprising participants who completed all assessments. Intention-to-treat (ITT) analysis was not performed, as the split-face design precluded independent group assignment, and the PP population represented all randomized participants who completed the study (Figure 2).

Baseline demographic and lifestyle characteristics of the 20 participants are summarized in

Table 1. Baseline Demographic and Lifestyle Characteristics of Study Participants (n = 20).

Characteristic	Category	Frequency (n)	Percentage (%)
Age (years)	Range	45–58
	Mean ± SD	52.40 ± 3.79
Skin Type	Dry	14	70.00
	Normal	5	25.00
	Oily	1	5.00
	Combination	0	0.00
	Problem skin	0	0.00
Skin Surface Texture	Smooth	3	15.00
	Moderate	14	70.00
	Rough	3	15.00
Daily UV Exposure	<1 h	7	35.00
	1–3 h	12	60.00
	>3 h	1	5.00
Average Sleep Duration	≤5 h	0	0.00
	5–8 h	18	90.00
	>8 h	2	10.00
Smoking Status	Non-smoker	20	100.00
	<10 cigarettes/day	0	0.00
	≥10 cigarettes/day	0	0.00
Skin Irritation Sensitivity	Yes	0	0.00
	No	20	100.00
Stinging Sensitivity	Yes	0	0.00
	No	20	100.00
History of Adverse Skin Reactions	Yes	0	0.00
	No	20	100.00

Data are presented as frequency (n) and percentage (%) unless otherwise indicated. Age is presented as mean ± SD.

. All participants were female and aged 45–58 years (mean age: 52.40 ± 3.79 years). The majority had dry skin (70.0%) with moderate surface texture (70.0%). Most participants reported daily UV exposure of 1–3 h (60.0%) and an average sleep duration of 5–8 h per night (90.0%). All participants were non-smokers (100%) and reported no history of skin irritation sensitivity, stinging sensitivity, or adverse skin reactions, confirming a homogeneous study population at baseline. The trial was completed as planned over the predefined study period without early termination. No interim stopping rules were applied, and no external factors led to premature discontinuation of the study.

3.3.2. Analysis of Wrinkle Parameters Using a 3D Imaging System

The analysis of wrinkle parameters using a 3D imaging system demonstrated significant improvements in the test group over time. In the test group, both Ra and Rq values showed significant reductions after 8 and 12 weeks of product use compared to baseline. Specifically, Ra decreased by 4.87% at 8 weeks and 12.75% at 12 weeks (Figure 3A), while Rq decreased by 4.89% at 8 weeks and 12.46% at 12 weeks (Figure 3B). Rmax exhibited a significant reduction only at 12 weeks, with an 11.32% decrease compared to baseline (Figure 3C).

In contrast, the control group did not show significant changes in Ra, Rq, or Rmax at any time point. When the test and control groups were compared, significantly greater reductions in Ra and Rq were observed in the test group at both 8 and 12 weeks, indicating superior wrinkle improvement efficacy of the test product (Figure 3A,B). Representative three-dimensional skin images illustrating wrinkle improvement over time are shown in Figure 3D.

3.3.3. Visual Grading Results of Wrinkles

Visual grading of wrinkles by trained investigators showed significant improvement in the test group after 12 weeks of product use. The wrinkle score in the test group decreased by 3.51% at 12 weeks compared to baseline (p < 0.05, Figure 4). In contrast, no significant changes were observed in the control group throughout the study period. When comparing the two groups, a significantly greater reduction in wrinkle score was observed in the test group than in the control group at 12 weeks (Figure 4). The inter-rater reliability between the two evaluators was high, with an intraclass correlation coefficient (ICC) ≥ 0.8. These visual grading results are consistent with the improvements observed in the instrumental wrinkle parameters.

3.3.4. Subjective Evaluations by Participants

Subjective self-assessments indicated perceived improvements in skin appearance among participants in the test group after 8 and 12 weeks of product use. At both time points, 70% of participants in the test group reported an improvement in eye wrinkle appearance. In contrast, no notable changes were reported in the control group.

3.3.5. Compliance and Safety

Product adherence was high throughout the study period, with compliance rates of 100% at 4 and 8 weeks and ranging from 98% to 100% at 12 weeks. No adverse events or skin reactions were observed in any of the participants throughout the study period.

4. Discussion

Skin aging is characterized by progressive alterations in the dermal extracellular matrix, particularly the degradation of collagen and elastin fibers, which leads to wrinkle formation and a loss of skin elasticity. These processes are strongly influenced by both intrinsic aging and extrinsic factors such as ultraviolet (UV) radiation, which induces the upregulation of matrix metalloproteinases, including MMP-1, thereby accelerating collagen breakdown [1,2,3].

In this study, we observed that a cosmetic formulation containing HFS extract enhanced type I collagen synthesis, reduced MMP-1 and elastase activity, and improved multiple wrinkle parameters in a human clinical study.

Marine- and fish-derived collagen peptides are known to modulate fibroblast activity through growth factor-related signaling. Several studies suggest that bioactive di- and tri-peptides derived from collagen can stimulate the TGF-β/Smad pathway, a central regulator of extracellular matrix (ECM) homeostasis. These peptides have been shown to increase TGF-β1 expression and enhance Smad2/3 phosphorylation, thereby promoting type I collagen synthesis [4,8,11]. The significant increase in type I collagen production observed in our study is consistent with previous reports on the biological activity of collagen-derived peptides. However, the involvement of specific signaling pathways such as TGF-β/Smad was not directly investigated in this study, and thus the involvement of specific signaling pathways remains to be clarified.

The observed effects on procollagen production and MMP-1 expression are consistent with previously reported mechanisms of collagen-derived peptides, which have been suggested to influence pathways such as TGF-β/Smad and MAPK/AP-1 signaling. UV exposure activates ERK, JNK, and p38 MAPK signaling, which subsequently increases AP-1 activity and MMP-1 transcription [1,2]. Prior studies have shown that marine collagen peptides can attenuate MAPK activation and suppress AP-1, leading to reduced MMP-1 expression [7,20,21]. While these findings provide a plausible biological context for our results, the present study did not include pathway-specific analyses; therefore, the proposed mechanisms should be interpreted with caution.

The elastase inhibition observed in this study was based on a cell-free enzymatic assay, indicating a direct inhibitory effect of the HFS extract on elastase activity rather than a cellular regulatory mechanism. This finding suggests that HFS extract may contribute to the preservation of extracellular matrix components through direct enzyme inhibition. Since this assay did not involve living cells, no conclusions can be drawn regarding intracellular signaling or gene regulation.

Objective three-dimensional imaging revealed significant reductions in skin surface roughness parameters (Ra and Rq) after 8 and 12 weeks of product application, as well as a decrease in maximum wrinkle height (Rmax) at 12 weeks. These changes indicate a gradual and sustained improvement in wrinkle morphology, which may reflect gradual improvements in skin surface characteristics associated with overall skin condition. Similar improvements in wrinkle parameters and dermal structural integrity have been reported in previous studies evaluating collagen-containing cosmetic or nutraceutical interventions [22,23,24], supporting the consistency of our findings with established evidence. Systematic review and meta-analysis have further supported the role of collagen-derived peptides in improving skin wrinkle parameters [25,26]. A previous study demonstrated that hydrolyzed collagen tripeptides exhibited enhanced skin absorption compared to non-hydrolyzed collagen, with significantly higher quantitative absorption rates confirmed by high-performance liquid chromatography [27].

In addition to the instrumental assessments, visual grading by trained investigators demonstrated a significant reduction in wrinkle severity at 12 weeks, and subjective evaluations by participants further supported these findings, with most users reporting improvements in skin smoothness and elasticity. The concordance between instrumental measurements, visual assessments, and subjective perceptions has also been observed in prior clinical studies of collagen-containing cosmetic or nutraceutical products, highlighting the clinical relevance of such multimodal evaluation approaches [22,23,24].

The test product containing 0.5% HFS extract was well tolerated throughout the study period, with no adverse events reported. This favorable safety profile is consistent with previous reports on the use of collagen-based materials and marine-derived collagen peptides in cosmetic and dermatological applications [5,10,23,28].

An important consideration in the topical application of collagen-derived ingredients is their ability to interact with skin cells despite their relatively large molecular size. Previous studies have suggested that collagen hydrolysates contain low-molecular-weight peptides, including dipeptides and tripeptides such as Pro-Hyp and Gly-Pro-Hyp, which can act as bioactive signaling molecules in dermal fibroblasts. These peptides have been reported to be associated with cellular pathways related to collagen synthesis and extracellular matrix regulation. In addition, several studies evaluating cosmetic formulations containing hydrolyzed collagen have demonstrated improvements in skin elasticity and wrinkle parameters following topical application. Although the precise mechanisms underlying the dermal activity of collagen peptides remain to be fully clarified, the biological and clinical findings of the present study are consistent with previous reports suggesting that collagen-derived peptides may be associated with improvements in skin-related parameters reported in previous studies [8,22,23].

The HFS extract used in this study has been primarily utilized as a functional food ingredient, with several studies reporting its beneficial effects on skin, joint, and bone health following oral administration [29]. While these effects are generally attributed to systemic absorption and distribution of bioactive peptides, emerging evidence suggests that certain low-molecular-weight peptides may also directly influence dermal fibroblast activity.

In the present study, we explored its potential as a topical cosmetic ingredient, focusing on its ability to modulate collagen synthesis and matrix-degrading enzymes at the cellular level. The observed in vitro findings and clinical outcomes suggest that collagen-derived peptides may have potential relevance in skin-related applications beyond their traditional use as oral supplements. A previous study using a similar hydrolyzed fish collagen material reported improvements in skin parameters following 4 weeks of topical application in a single-arm design [13,27]. In contrast, the present study extends these findings by employing a longer treatment duration and a more rigorous split-face, double-blind design, along with objective 3D imaging analysis. These methodological improvements strengthen the clinical evidence supporting the efficacy of HFS extract.

Despite these promising results, the present study has certain limitations. The clinical sample size in this study (n = 20) was determined based on the regulatory guidelines for functional cosmetic evaluation provided by MFDS of Korea, which recommend a minimum of 20 subjects and a 12-week treatment period. However, the relatively small sample size may limit the statistical power and generalizability of the findings. Therefore, the present results should be interpreted as moderate evidence. Future studies with larger and more diverse populations, as well as extended treatment durations, are warranted. In addition, although the inhibitory effects on MMP-1 and elastase were clearly demonstrated, the precise upstream molecular pathways responsible for these changes remain to be fully elucidated. Advanced approaches, such as transcriptomic, proteomic, or phosphoproteomic profiling, may provide deeper insights into the bioactive components of HFS extract and identify their specific molecular targets within the dermal extracellular matrix. The proposed mechanisms involving collagen synthesis and matrix regulation should be interpreted with caution, as no direct pathway-level analyses were performed in the present study.

In contrast to many previous studies that have primarily examined the oral ingestion of collagen peptides, the present study provides evidence supporting the potential efficacy of topical application based on both in vitro cellular observations and clinically observed wrinkle improvements.

However, the relationship between the in vitro findings and clinical outcomes should be interpreted with caution. In the present study, relatively higher concentrations of HFS extract were used in vitro to evaluate biological activity under controlled conditions. These concentrations do not directly correspond to the levels used in the clinical formulation (0.5%), as in vitro systems lack the structural and physiological barriers present in human skin. Therefore, direct quantitative comparison between the in vitro and clinical concentrations is not feasible. The in vitro experiments were designed to assess the intrinsic bioactivity of the extract, whereas the clinical study evaluated its efficacy under real-use conditions. This difference should be considered when interpreting the relationship between the in vitro findings and clinical outcomes.

5. Conclusions

In this study, HFS extract demonstrated significant biological activity in vitro, including increased type I procollagen production and reduced UVB-induced MMP-1 expression and elastase activity in human dermal fibroblasts. In addition, topical application of an HFS-containing formulation resulted in measurable improvements in wrinkle-related parameters in a 12-week randomized, double-blind, split-face clinical study.

These findings suggest that HFS extract has potential as a functional cosmetic ingredient for improving visible signs of skin aging. The combined in vitro and clinical results provide complementary evidence supporting its biological activity and cosmetic efficacy, although the direct mechanistic link between cellular responses and clinical outcomes remains to be further elucidated.

Nevertheless, this study has several limitations, including the relatively small sample size and the lack of an active comparator. Therefore, future studies are warranted to investigate the underlying molecular mechanisms and confirm efficacy in larger and more diverse populations.

Overall, this study provides clinically relevant evidence supporting the potential use of HFS extract as a topical anti-aging ingredient in cosmetic formulations.