The effect of a serum containing acetyl hexapeptide‐8, dipeptide diaminobutyroyl benzylamide diacetate and gluconolactone on skin biomarkers, wrinkles and skin texture: Ex vivo and clinical studies

Abstract

Objective

This study aimed to explore the anti‐skin aging efficacy of a novel serum containing acetyl hexapeptide‐8, dipeptide diaminobutyroyl benzylamide diacetate, gluconolactone, niacinamide and laminaria extract through one ex‐vivo study and two clinical studies.

Methods

In ex‐vivo study, skin samples were exposed to the serum (Formula 1) and its active ingredient combinations (Formula 2). Biomarkers including matrix metalloproteinase‐1, elastic fibres and collagen types I, III, IV and XVII content were analyzed to evaluate the efficacy. In clinical study 1 (n = 50), the Formula 1's efficacy in reducing static wrinkles and improving skin quality was examined, while study 2 (n = 42) focused on its efficacy against dynamic wrinkles.

Results

Formula 1 significantly increased levels of anti‐aging biomarkers including elastic fibres, collagen fibres, etc. Meanwhile, Formula 1 significantly improved static wrinkle appearance within the first week, with mean clinical scoring from 35% to 69% for various types of wrinkles after 12 weeks of use (all p < 0.001). The clinical scoring for dynamic wrinkles improved by 10%–13% (all p < 0.001). Additionally, significant improvements were observed in skin quality, indicated by clinical scoring changes of smoothness (30%), radiance (27%), skin pore (43%), elasticity (33%) and firmness (36%) (all p < 0.001).

Conclusion

The serum demonstrates significant anti‐aging effects, effectively improving static and dynamic wrinkles and skin quality.

The novel serum demonstrates significant anti‐skin aging effects, effectively improving static and dynamic wrinkles and skin texture. Therefore, this formulation holds promise as a safe and effective product in the field of anti‐wrinkle skincare products.

INTRODUCTION

Skin aging is a multifaceted process influenced by physiological, environmental, lifestyle and genetic factors [1, 2]. Intrinsic aging (or chronological aging) is predominantly influenced by genetic predispositions and hormonal variations [3, 4]. As individuals age, the metabolic rate of the skin cells decelerates, accompanied by a reduction in collagen and elastin fibres. These alterations result in reduced skin elasticity [2, 5, 6]. On the other hand, extrinsic aging involves environmental factors, especially environmental pollutants, ultraviolet (UV) radiation and tobacco use [5, 7, 8]. Such factors accelerate the skin aging process, resulting in wrinkles, sagging and pigmentation [2, 8].

Two primary types of wrinkles develop in human skin: dynamic wrinkles and static wrinkles. Dynamic wrinkles are initially formed due to facial expressions, resulting from the contraction of underlying skeletal muscles [9]. Over time, with repeated muscle contractions, these dynamic wrinkles can transition into static wrinkles, which persist even in the absence of muscle activity. This progression is further exacerbated by the natural decline of collagen and elastin within the dermal layer that follows the aging process [9, 10]. Currently, topical skincare products, including topical peptides, are widely utilized interventions due to their targeted efficacy such as promoting collagen synthesis and enhancing skin rejuvenation, as well as their affordability and accessibility [6, 11]. Nevertheless, considering the ongoing demand for more effective anti‐wrinkle products, it is essential to develop and introduce new products that reduce signs of aging by targeting both static and dynamic wrinkles.

The present studies examined a new anti‐aging skin care product containing several ingredients with known anti‐wrinkle properties. These include acetyl hexapeptide‐8, dipeptide diaminobutyroyl benzylamide diacetate, gluconolactone, niacinamide and laminaria extract. Acetyl hexapeptide‐8 has been increasingly used in anti‐aging skincare products [12]. It has a mechanism similar to botulinum toxin (BoNT), which is commonly used to reduce both static and dynamic wrinkles [13, 14]. By inhibiting the assembly of the SNARE complex—a group of proteins pivotal in the formation of wrinkles—acetyl hexapeptide‐8 prevents the repetitive contraction of facial expression muscles [15]. Due to the underlying mechanisms that target both static and dynamic wrinkles, acetyl hexapeptide‐8 serves as an ideal active ingredient for comprehensive wrinkle reduction. A study from 2013 has demonstrated that 48.9% participants using 10% synthetic hexapeptide experienced significant wrinkle reduction compared with 0% in the placebo group, with a generally manageable safety profile [16]. Dipeptide diaminobutyroyl benzylamide diacetate is a snake venom‐mimicking peptide that also works similarly to BoNT by blocking signal transduction and relaxing the muscles in the treated area. While there is limited published research on its anti‐wrinkle effects, this ingredient has begun to be incorporated into skin care products [17]. Considering the similar mechanism with BoNT, this compound has been incorporated as an active ingredient. Gluconolactone is a mild polyhydroxy acid (PHA) that can help exfoliate the skin, leading to improved smoothness and radiance while minimizing potential irritation such as stinging or burning sensations [18, 19]. Niacinamide, a water‐soluble vitamin, has been utilized in skincare for decades. It is widely used to help reduce signs of skin aging and brighten skin tone [20]. Research has indicated that niacinamide is well‐tolerated by the skin and can help reduce hyper‐pigmentation and improve overall skin texture [21]. Additionally, preclinical studies have suggested that laminaria extracts may accelerate collagen deposition and delay collagen breakdown, suggesting their potential in improving wrinkles [22, 23].

Although these ingredients are recognized for improving skin texture, their combined impact has not been previously studied. Furthermore, existing anti‐wrinkle research predominantly focuses on static wrinkles, with less attention given to dynamic wrinkles, which also play a significant role in skin aging [24]. Therefore, to thoroughly explore the anti‐wrinkle efficacy of the serum, an ex vivo study was conducted to investigate specific biomarkers associated with wrinkles, thereby providing a scientific evidence base for its anti‐wrinkle properties. Subsequently, clinical studies were conducted to validate the serum's effects on both static and dynamic wrinkles, as well as overall skin texture in human subjects. This approach aims to address the clinical needs of anti‐wrinkle interventions in a comprehensive manner.

METHODS

Test product

Both the ex vivo and clinical studies evaluated the effects of a novel serum formulation (Formula 1) containing five active ingredients: acetyl hexapeptide‐8, dipeptide diaminobutyroyl benzylamide diacetate, gluconolactone, niacinamide and laminaria extract. In addition, the ex vivo study assessed the effects of an active ingredient combination (Formula 2) composed of the same five actives prepared in phosphate‐buffered saline (PBS). Formula 1 (the serum) includes auxiliary ingredients such as thickeners and stabilizers, whereas Formula 2 contains only the active ingredients. Details of all experimental conditions and ingredient compositions are summarized in Table S1.

Ex vivo study

The ex vivo study used skin explants to evaluate the anti‐aging effects of the Formulas 1 and 2. The changes in biomarkers, such as elastic fibres and collagen, were measured after applying the Formulas 1 and 2 to the skin samples.

The fresh skin tissues, the skin samples were washed in 75% alcohol for 30 s and cleaned three times with sterile PBS buffer. Subsequently, disposable sterile skin samplers were used to cut the skin tissues into fixed samples of 24 ± 2 mm², ensuring that the epidermis faced up and the dermis faced down. These samples were placed in culture inserts and transferred to six‐well plates, with each well containing 3.7 mL of culture medium. The samples were cultured at 37°C and under 5% CO₂, with fresh culture medium changed daily.

The skin tissues were divided into different test groups: Formula 1 group, Formula 2 group, positive control group (vitamins C + E), negative control group and blank control group, each containing three skin samples. The study also examined other combinations of active ingredients. The information and results of other combinations are listed in Materials S1.

After 2 days of skin tissue culture, the samples were exposed to UV radiation (30 J/cm² UVA and 50 m J/cm² UVB) for 4 days consecutively. After each irradiation, the fresh culture medium was replaced, then the sample was applied on the skin surface. The positive control group was administered under the liquid, and the sample group was administered on the surface. After 4 consecutive days of irradiation, the Ex vivo skin tissue was cultured for a further 3 days during which no irradiation was performed and only sample administration was performed.

Various biomarkers were used to evaluate the anti‐aging effects, including (1) Masson's trichrome and Victoria blue staining to respectively detect collagen fibres and elastic fibres, with image capture and analysis using Image‐Pro® Plus software, (2) enzyme‐linked immunosorbent assay (ELISA) to assess matrix metalloproteinase‐1 (MMP‐1) expression, (3) immunofluorescence to detect collagen I, collagen III, collagen IV and collagen XVII, with microscopic image capture, and (4) immunohistochemistry to evaluate elastin and Ki67 expression, with microscopic image capture.

The changes in elastic fibres and elastin content were used to assess firmness, while changes in MMP‐1, collagen fibres, collagen types I, III, IV and XVII content were used to evaluate anti‐wrinkle effects. Changes in Ki67 content were assessed to evaluate epidermal cell renewal capability. The improvement and inhibition rates were calculated using the following formulas:

$$\begin{array}{c}\text{Improvement rate}\left(\%\right)\hfill \\ =\frac{\text{Sample group}-\text{Negative control group}}{\text{Negative control group}}\times 100\%\hfill \end{array}$$

$$\begin{array}{c}\text{Inhibition rate}\left(\%\right)\hfill \\ =\frac{\text{Negative control group}-\text{Sample group}}{\text{Negative control group}}\times 100\%\hfill \end{array}$$

Clinical studies

The clinical studies included two prospective, single‐arm, single‐centre trials involving Chinese female participants. Participants applied the Formula 1 twice a day (morning and evening) on their entire face, including the eye area, following standard cleansing, moisturizing and sunscreen application. The dermatologist used the Aging Skin Atlas and modified Griffith's 10‐point scale (from 0 to 9) [25] to assess participants for signs of skin aging and evaluate the anti‐aging effects of the Formula 1. The dermatologist was not allowed to reference previous scores at post‐baseline assessments.

The complete inclusion and exclusion criteria are listed in Material S1. Before commencing the studies, the study protocols and informed consent forms were reviewed and approved by the ethics committees at each centre. Participants provided written informed consent prior to any study procedures.

Efficacy against static wrinkles

In Study 1, clinical scorings were assessed by dermatologists, including evaluations of various facial wrinkles and fine lines (T0, T1W, T2W, T4W, T8W and T12W). Trained technicians used a VISIA 7 camera to capture pictures of the participants' faces at different time points. Primos‐lite and Ultrascan UC22 devices were used to conduct detailed measurements of skin characteristics, such as the number, length and area of forehead wrinkles and crow's feet, as well as skin thickness (T0, T4W, T8W and T12W).

Efficacy against dynamic wrinkles

In addition to evaluating the efficacy against static wrinkles, Study 2 assessed the effects against dynamic wrinkles in 42 participants, including crow's feet wrinkles, nasolabial folds and underneath eye wrinkles in photos captured by high‐speed cameras (T0, T4W and T8W). During the shooting process, participants wrapped black scarves around their collars and covered their heads with black cloth to ensure that their faces and necks were not obstructed. Once located through a locator, the participants completed the smile actions under command. The shooting direction was from the front, the left and the right, with a right angle of 45°. Ten photos at each time point from no expression to max expression were selected. Referring to the Atlas, 10 photos of the underneath eye wrinkles and the nasolabial folds under the smile expression with the front side, crow's feet wrinkles under the smile expression with the right side were graded by a dermatologist.

Improvement in skin texture

The clinical scorings of skin texture improvements were assessed by the dermatologist, including evaluations of skin smoothness, skin pores, radiance, elasticity and firmness (T0, Timm, T1W, T2W, T4W, T8W and T12W).

Self‐assessment

The participants' experiences when using the Formula 1 at home were evaluated at various time points (Timm, T1W, T2W, T4W, T8W and T12W). The questionnaire covered the perceptions related to the anti‐aging effects, improvement in skin quality, radiance, hydration and freshness, skin comfort, and cosmeticity. Participants completed a self‐assessment questionnaire using a five‐point Likert scale (strongly agree, somewhat agree, neither agree nor disagree, somewhat disagree and strongly disagree). The reported percentages represent the proportion of participants who selected strongly agree or somewhat agree at week 12.

Statistical analysis

In the ex vivo study, GraphPad Prism was used for data visualization, presenting results as mean ± standard deviation. Statistical analyses were performed using t‐tests. Across all studies, a significance level of p < 0.05 was considered statistically significant, while p < 0.01 indicated highly significant differences. In the clinical studies, the continuous data were tested for normality using the Kolmogorov–Smirnov test. If the data were normally distributed, paired t‐tests or analysis of variance (ANOVA) were used for comparisons. For non‐normally distributed data, the Wilcoxon signed‐rank test was applied.

RESULTS

Ex vivo study

Anti‐wrinkle efficacy

In the ex vivo study, the Formula 1 group showed a significant reduction in MMP‐1 content compared with the negative control group (53%). Furthermore, there were increases in collagen fibres (86%), collagen type I (289%), collagen type III (71%), collagen type IV (109%) and collagen type XVII (37%) (Table 1). In the Formula 2 group, the MMP‐1 inhibition rate was 58%, and collagen fibres, collagen type I, collagen type III, collagen type IV and collagen type XVII were increased by 131%, 167%, 125%, 229% and 300%, respectively. Except for collagen type XVII in the Formula 1 group with a p‐value of <0.005, all other results had p < 0.001 (Table 1), demonstrating significant anti‐wrinkle effects of the Formulas 1 and 2 on ex vivo skin tissue. Moreover, the combination of the five active ingredients not only showed significant anti‐wrinkle effects compared with the negative control, but also demonstrated superior effects in increasing collagen type I and IV compared with each peptide and the combination of two peptides (acetyl hexapeptide‐8 and dipeptide diaminobutyroyl benzylamide diacetate) (Table S2). Specifically, compared with the combination of two peptides, the five‐ingredient combination exhibited more significant anti‐wrinkle properties (collagen type I: 167% vs. 85%; collagen type IV: 229% vs. 162%) (all p < 0.05). This not only proves the efficacy of the combination itself but also shows superior anti‐wrinkle effects compared with the single individual active ingredients.

TABLE 1.

Summary from the ex vivo study results.

Parameters	BC	NC	PC		Formula 1		Formula 2
			Value	Improvement rate (vs. NC)	Value	Improvement rate (vs. NC)	Value	Improvement rate (vs. NC)
Tightening efficacy
Elastic Fibre (relative area average)	1.0 ± 0.04	0.4 ± 0.02##	1.3 ± 0.19**	215%	0.9 ± 0.10**	122%	1.2 ± 0.16**	195%
Elastin (relative IOD average)	1.0 ± 0.04	0.5 ± 0.07##	1.2 ± 0.07**	135%	1.8 ± 0.24**	252%	1.9 ± 0.12**	260%
MMP‐1 (Average concentration, pg/mL)	8119.2 ± 413.88	24 504.5 ± 434.65##	16 878.8 ± 90.71**	31%	11 552.6 ± 372.14**	53%	10 274.9 ± 196.60**	58%
Anti‐wrinkle efficacy
Collagen fibres (relative area average)	1.0 ± 0.02	0.4 ± 0.02##	1.0 ± 0.12**	126%	0.8 ± 0.02**	86%	1.0 ± 0.07**	131%
Collagen I (relative IOD average)	1.0 ± 0.08	0.3 ± 0.02##	1.0 ± 0.07**	278%	1.1 ± 0.14**	289%	0.7 ± 0.05**	167%
Collagen III (relative IOD average)	1.0 ± 0.15	0.5 ± 0.04##	1.2 ± 0.17**	156%	0.8 ± 0.05**	71%	1.1 ± 0.1**	125%
Collagen IV (relative IOD/area average)	1.0 ± 0.10	0.3 ± 0.02##	1.0 ± 0.05**	179%	0.7 ± 0.03**	109%	1.1 ± 0.04**	229%
Collagen XVII (relative IOD average)	1.0 ± 0.08	0.3 ± 0.04##	0.6 ± 0.04**	107%	0.4 ± 0.03*	37%	1.2 ± 0.18**	300%
Epidermal cell renewal capacity
Ki67 (mean value of positive cell rate)	57.7% ± 1.86%	21.7% ± 2.90%##	57.6% ± 4.63%**	165%	51.2% ± 8.82%**	136%	59.2% ± 5.17%**	172%

Note: Values in table are presented as the mean ± standard deviation. #p < 0.05 and ##p < 0.01 versus the BC group. *p < 0.05 and **p < 0.01 versus the NC group.

Abbreviations: BC, blank control; IOD, integrated optical density; MMP‐1, matrix metalloproteinase 1; NC, negative control; PC, positive control.

Skin firmness

Compared with the negative control group, the Formula 1‐treated group showed a significant increase in skin elasticity fibres by 122% and elastin by 252%. In the Formula 2 group, there was also a significant increase in elasticity fibres by 195% and elastin by 260% (all p < 0.001) (Table 1). The results indicate that the Formulas 1 and 2 can increase the content of elasticity fibres and elastin in ex vivo skin tissue, thereby significantly improving skin firmness.

Epidermal cell renewal capacity

In the Formula 1 group, the percentage of Ki67‐positive cells was significantly higher than in the negative control group (136%). In the Formula 2 group, this increase was 172% (all p < 0.001) (Table 1). Hence, the Formulas 1 and 2 could significantly enhance the Ki67‐positive cell rate in ex vivo skin tissue, suggesting their ability to promote epidermal cell renewal capacity.

Clinical studies

Patients

Study 1 enrolled 50 participants for clinical evaluation and image capture, all of whom completed the study. In addition, 24 participants were included in the questionnaire surveys only. Among these, one participant withdrew for personal reason, resulting in 73 participants who completed the survey. Study 2 enrolled 49 participants, but seven were lost to follow‐up, leaving 42 participants who completed the study. The baseline characteristics of the participants are presented in Table 2.

TABLE 2.

Characteristics of the participants.

Characteristics	Study 1 (anti‐static wrinkles efficacy)		Study 2 (anti‐dynamic wrinkles efficacy)
	Total	Efficacy	Total
No. of subjects	73	50	42
Mean age ± SD, years (min‐max)	47.6 ± 5.44 (25–55)	48.3 ± 5.55 (25–55)	45.6 ± 3.83 (38–55)
Combined	28 (38%)	15 (30%)	12 (29%)
Dry	30 (41%)	25 (50%)	18 (43%)
Normal	11 (15%)	7 (14%)	8 (19%)
Oily	4 (6%)	3 (6%)	4 (10%)
Sensitive	36 (49%)	24 (48%)	21 (50%)

Abbreviation: SD, standard deviation.

Anti‐static wrinkle efficacy

In Study 1, significant efficacy of the Formula 1 on static wrinkles was observed as early as the first week of use (Figure 1). After 12 weeks of treatment, compared with baseline, the mean score improvements were 58% for forehead wrinkles, 69% for fine lines on the forehead, 47% for glabellar wrinkles, 35% for inter‐ocular wrinkles, 39% for crow's feet wrinkles, 55% for underneath eye wrinkles, 55% for cheek folds, 44% for nasolabial folds and 44% for wrinkles around the corner of the lip. All these changes were statistically significant (all p < 0.001) (Table 3). According to the Primos‐lite measurements, the Formula 1 showed significant anti‐wrinkle efficacy, resulting in a 29% decrease in the count of forehead wrinkles, a 9% reduction in their length, and an 11% reduction in their area. In addition, the number of crow's feet count was reduced by 34%, accompanied by an 11% reduction in the length and an 11% reduction in the area (all p < 0.001) (Figure 2a and Table S3). The Ultrascan UC22 results indicated that after 12 weeks of Formula 1 application, the participants experienced a decrease in average epidermal thickness by 60.4 μm (57%) (p < 0.001), while dermal thickness increased by 149.9 μm (12%) (p < 0.001). However, there were no significant differences in the combined thickness of both layers compared with the baseline (Figure 2b and Table S3).

FIGURE 1.

Representative Primos 3D images showing improvement in static crow's feet wrinkles after 12 weeks of use (average case).

TABLE 3.

Efficacy on wrinkles and skin texture.

Attributes (N = 50)	T0	T1W	Evolution over time (%)	T2W	Evolution over time (%)	T4W	Evolution over time (%)	T8W	Evolution over time (%)	T12W	Evolution over time (%)
Wrinkles and fine lines
Forehead wrinkles	2.9 ± 0.12	2.3 ± 0.13	−20%**	1.9 ± 0.13	−34%**	1.5 ± 0.13	−47%**	1.4 ± 0.12	−53%**	1.2 ± 0.12	−58%**
Fine lines on forehead	1.7 ± 0.04	1.5 ± 0.04	−14%**	1.2 ± 0.04	−29%**	0.9 ± 0.05	−45%**	0.7 ± 0.05	−58%**	0.5 ± 0.04	−69%**
Glabellar wrinkles	2.3 ± 0.10	1.9 ± 0.11	−15%**	1.7 ± 0.11	−27%**	1.5 ± 0.12	−35%**	1.4 ± 0.12	−41%**	1.2 ± 0.12	−47%**
Inter‐ocular wrinkles	2.5 ± 0.10	2.4 ± 0.10	−7%**	2.1 ± 0.11	−16%**	1.9 ± 0.12	−25%**	1.8 ± 0.12	−29%**	1.6 ± 0.13	−35%**
Crow's feet wrinkle	3.2 ± 0.11	2.8 ± 0.11	−12%**	2.5 ± 0.11	−22%**	2.2 ± 0.11	−29%**	2.1 ± 0.11	−34%**	1.9 ± 0.11	−39%**
Underneath eye wrinkle	3.2 ± 0.09	2.6 ± 0.10	−18%**	2.1 ± 0.10	−34%**	1.8 ± 0.10	−44%**	1.7 ± 0.09	−48%**	1.5 ± 0.10	−55%**
Cheek folds	2.5 ± 0.04	2.2 ± 0.04	−13%**	2.0 ± 0.05	−20%**	1.7 ± 0.06	−32%**	1.4 ± 0.05	−43%**	1.1 ± 0.06	−55%**
Nasolabial fold	2.8 ± 0.08	2.5 ± 0.08	−12%**	2.2 ± 0.09	−23%**	2.0 ± 0.09	−30%**	1.8 ± 0.10	−38%**	1.6 ± 0.10	−44%**
Wrinkles at the corner of the lips	2.5 ± 0.10	2.2 ± 0.11	−12%**	1.9 ± 0.10	−23%**	1.7 ± 0.10	−31%**	1.5 ± 0.10	−38%**	1.4 ± 0.09	−44%**
Skin quality
Skin smoothness (visual)	5.1 ± 0.07	4.6 ± 0.07	−9%**	4.3 ± 0.07	−16%**	4.0 ± 0.08	−22%**	3.8 ± 0.08	−26%**	3.6 ± 0.09	−30%**
Skin radiance (visual)	4.8 ± 0.04	4.3 ± 0.05	−11%**	3.9 ± 0.06	−18%**	3.7 ± 0.07	−22%**	3.6 ± 0.07	−25%**	3.5 ± 0.07	−27%**
Skin pore	5.0 ± 0.09	4.5 ± 0.09	−10%**	4.1 ± 0.09	−18%**	3.8 ± 0.10	−24%**	3.3 ± 0.10	−33%**	2.9 ± 0.11	−43%**
Skin elasticity (tactile)	4.5 ± 0.06	4.1 ± 0.07	−9%**	3.7 ± 0.07	−17%**	3.4 ± 0.06	−23%**	3.3 ± 0.06	−27%**	3.0 ± 0.07	−33%**
Skin firmness (visual)	5.0 ± 0.09	4.5 ± 0.09	−10%**	4.1 ± 0.09	−19%**	3.8 ± 0.08	−25%**	3.5 ± 0.08	−30%**	3.2 ± 0.09	−36%**

Attributes (N = 42)	T0	T1W	Evolution over time (%)	T2W	Evolution over time (%)	T4W	Evolution over time (%)	T8W	Evolution over time (%)	T12W	Evolution over time (%)
Dynamic wrinkles (mean grading of 10 pcs)
Underneath eye wrinkles (Front side, smile expression)	4.3 ± 0.11	—	—	—	—	4.0 ± 0.11	−6%**	3.8 ± 0.11	−10%**	—	—
Nasolabial fold (Front side, smile expression)	3.3 ± 0.10	—	—	—	—	3.1 ± 0.10	−8%**	2.9 ± 0.10	−13%**	—	—
Crow's feet wrinkles (right side, smile expression)	4.3 ± 0.13	—	—	—	—	4.0 ± 0.13	−6%**	3.8 ± 0.13	−11%**	—	—

Note: Values are presented as mean ± SD.

^** p < 0.01.

FIGURE 2.

Changes in instrumental measurements over 12 weeks. (a) Wrinkle attributes by Primos. (b) Skin thickness by UC22. *p < 0.05.

Anti‐dynamic wrinkle effect

In addition to significant improvements in static wrinkles, the Formula 1 also demonstrated a significant improvement in dynamic wrinkles (Figure 3). Under smile actions, the clinical scoring of underneath eye wrinkles decreased by 10%, the nasolabial folds by 13%, and the crow's feet wrinkles by 11% (all p < 0.001) (Table 3). Furthermore, across different facial expressions (from no expression to maximum expression, comprising 10 levels), the Formula 1 exhibited significant improvements in dynamic wrinkles consistently (Table 4).

FIGURE 3.

Representative photographs showing improvement in dynamic crow's feet wrinkles under smiling expression after 8 weeks of use (average case).

TABLE 4.

Anti‐dynamic wrinkles efficacy under smile expression (n = 50).

Attributes (N = 42)	P1‐no expression	P2	P3	P4	P5	P6	P7	P8	P9	P10‐max expression
Underneath eye wrinkle
T0	3.5 ± 0.09	3.6 ± 0.10	3.8 ± 0.11	4.0 ± 0.11	4.2 ± 0.12	4.4 ± 0.12	4.5 ± 0.12	4.7 ± 0.13	4.9 ± 0.13	5.0 ± 0.13
T4W	3.3 ± 0.10	3.4 ± 0.11	3.6 ± 0.11	3.8 ± 0.11	3.9 ± 0.11	4.1 ± 0.11	4.3 ± 0.12	4.4 ± 0.12	4.6 ± 0.13	4.7 ± 0.12
Evolution Over Time (%**)	−6%**	−7%**	−7%**	−6%**	−6%**	−7%**	−5%**	−6%**	−6%**	−5%**
T8W	3.1 ± 0.10	3.2 ± 0.10	3.4 ± 0.11	3.6 ± 0.12	3.7 ± 0.12	3.9 ± 0.12	4.1 ± 0.12	4.3 ± 0.12	4.4 ± 0.13	4.6 ± 0.13
Evolution Over Time (%**)	−12%**	−12%**	−12%**	−12%**	−11%**	−10%**	−9%**	−9%**	−9%**	−7%**
Nasolabial folds
T0	2.4 ± 0.09	2.6 ± 0.09	2.8 ± 0.10	3.0 ± 0.10	3.2 ± 0.11	3.4 ± 0.11	3.6 ± 0.12	3.9 ± 0.13	4.1 ± 0.14	4.2 ± 0.14
T4W	2.2 ± 0.10	2.3 ± 0.10	2.5 ± 0.10	2.8 ± 0.11	3.0 ± 0.10	3.2 ± 0.11	3.4 ± 0.11	3.5 ± 0.11	3.8 ± 0.12	3.9 ± 0.14
Evolution Over Time (%**)	−10%**	−9%**	−8%**	−8%**	−9%**	−7%**	−8%**	−9%**	−8%**	−7%**
T8W	2.0 ± 0.10	2.2 ± 0.10	2.3 ± 0.10	2.6 ± 0.10	2.8 ± 0.10	3.0 ± 0.11	3.2 ± 0.11	3.4 ± 0.12	3.6 ± 0.13	3.8 ± 0.14
Evolution Over Time (%**)	−18%**	−16%**	−16%**	−15%**	−14%**	−13%**	−12%**	−12%**	−11%**	−10%**
Crow's feet wrinkles
T0	3.7 ± 0.12	3.8 ± 0.13	3.9 ± 0.12	4.0 ± 0.13	4.1 ± 0.13	4.3 ± 0.14	4.5 ± 0.14	4.7 ± 0.15	4.9 ± 0.15	5.0 ± 0.15
T4W	3.5 ± 0.12	3.5 ± 0.12	3.6 ± 0.12	3.8 ± 0.13	3.9 ± 0.13	4.1 ± 0.13	4.2 ± 0.14	4.4 ± 0.14	4.6 ± 0.15	4.8 ± 0.15
Evolution Over Time (%**)	−6%**	−7%**	−7%**	−7%**	−6%**	−5%**	−6%**	−6%**	−6%**	−5%**
T8W	3.2 ± 0.12	3.3 ± 0.12	3.4 ± 0.12	3.5 ± 0.13	3.7 ± 0.13	3.8 ± 0.14	4.1 ± 0.14	4.3 ± 0.15	4.5 ± 0.16	4.7 ± 0.16
Evolution over time (%**)	−14%**	−14%**	−14%**	−14%**	−11%**	−10%**	−10%**	−8%**	−7%**	−7%**

Note: Values are presented as mean ± standard deviation.

^** p < 0.01.

Skin quality improvement effect

Significant improvements in various skin quality indicators were observed as early as after the first week of Formula 1 use, with increasingly pronounced anti‐aging effects over time. After 12 weeks of treatment, compared with baseline, improvements were observed for smoothness (30%), radiance (27%), skin pore (43%), elasticity (33%) and firmness (36%) (all p < 0.001) (Table 3). These results demonstrate the significant anti‐aging efficacy of Formula 1.

Self‐assessment

After 12 weeks of use, the participants acknowledged the anti‐aging effects of the Formula 1. Specifically, they observed an obvious reduction in fine lines and wrinkles, an increase in skin firmness, an improvement in face contour clarity, and a noticeable enhancement in skin rejuvenation and vitality. The participants also recognized improvements in skin texture and complexion, including pore tightening, increased smoothness, softness, elasticity, refinement, fullness and evenness, as well as moisturizing effects and enhanced skin radiance. In addition, the participants found that the Formula 1 provided a pleasant user experience, noting it was easy to apply, quickly absorbed, comfortable, relaxing, fresh, mild, non‐sticky, non‐greasy, lightweight, odourless, makeup‐friendly, with a pleasant texture and scent, and without any noticeable drawbacks (Table S4).

Safety

No adverse events were reported in any of the clinical studies.

DISCUSSION

This study revealed promising anti‐wrinkle outcomes of a novel serum, as evidenced by notable enhancements in collagen and elastic fibre content. The efficacy of Formula 1 was assessed using a multitude of methods, encompassing ex vivo testing, clinical scoring by dermatologists and instrumental measurements. These comprehensive assessments consistently revealed substantial anti‐wrinkle efficacy of Formula 1, providing robust scientific support for its extensive use in skincare regimens.

Significant increases in collagen and elastic fibre content, which are crucial for improving skin wrinkles and maintaining skin firmness [26], were observed in the ex vivo skin tissues treated with the positive control, Formula 1, and Formula 2. Moreover, the synergistic combination of the five active ingredients in this study not only exhibits substantial efficacy on its own but also significantly elevated the levels of collagen types I and IV compared with the single ingredients. Collagen type I constitutes 85%–90% of total skin collagen, providing structural support and strength to the skin [27, 28]. Collagen type IV is primarily found in the basement membrane, serving as a critical component linking the epidermis and dermis. There is a reduction in type IV collagen at the dermal‐epidermal junction with skin aging, potentially linked to a decreased expression in aging dermal fibroblasts [28, 29]. Furthermore, aging and environmental factors contribute to the degradation of elastic fibres and elastin, compromising skin structural integrity and leading to laxity and sagging [26, 30]. While numerous methods exist for enhancing collagen, fewer skin care products effectively target elastic fibre enhancement due to the complexity of elastic fibre synthesis involving the crosslinking of tropoelastin monomers and microfibrillar proteins [26]. Of note, the positive control and Formula 2 yielded more favourable outcomes in the ex vivo model relative to Formula 1. This is likely attributable to the composition of the Formula 1 vehicle. As a finished cosmetic product, Formula 1 contains preservative ingredients, including phytic acid, salicylic acid, chlorphenesin and alcohol. In the ex vivo model, these components may interact with the tissue sample, potentially altering tissue viability and thereby attenuating the efficacy of improving biomarkers [31]. However, the precise mechanism of this interaction warrants further investigation. Despite this observed attenuation in the model, Formula 1 significantly enhanced the synthesis of both collagen and elastic fibres. These findings underscore the critical roles of collagen and elastic fibres in wrinkles formation, highlighting the importance of using collagen biomarkers in anti‐wrinkle product development.

Currently, peptides are commonly used as anti‐wrinkle ingredients in clinical practice. A study showed that applying a peptide and vitamin C‐based serum product twice daily for 28 consecutive days resulted in significant improvements in dermatologist clinical scoring for crow's feet wrinkles, forehead wrinkles and the nasolabial folds, with reductions of 9%, 11% and 5.2%, respectively [32]. Similarly, the present study indicates that the Formula 1 containing five active ingredients has shown significant improvements in wrinkles over time. Notably, the mean clinical scoring for crow's feet wrinkles, forehead wrinkles and the nasolabial folds exhibited significant reductions of 29%, 47% and 30%, respectively, at week 4, which further improved to 39%, 58% and 44% at week 12. These results indicated that the improvement gradually increased with prolonged application, and these improvements appear to be numerically better than those in previous research. Moreover, this study innovatively explored the anti‐dynamic wrinkles as an anti‐aging parameter and revealed significant improvements. Previous research has established a significant positive correlation between age and wrinkle severity, regardless of whether they are dynamic or static, and the severity of dynamic wrinkles may reflect the severity of static wrinkles [24]. In the present study, the Formula 1 significantly reduced both static and dynamic wrinkles. Such findings suggest a synergistic effect between anti‐static and anti‐dynamic wrinkle treatments, offering valuable insights into the mechanisms underlying wrinkle reduction.

The present findings are consistent with and extend previous studies that have investigated the efficacy of individual peptide components used in our formulations. A randomized, placebo‐controlled clinical study conducted in Chinese subjects demonstrated that topical application of a synthetic hexapeptide (acetyl hexapeptide‐8, Argireline) produced significant wrinkle reduction, with a 48.9% total anti‐wrinkle efficiency after 4 weeks of treatment, accompanied by histological evidence of increased type I collagen deposition in murine skin models [33]. Similarly, a separate clinical trial evaluating a formulation containing 4% dipeptide diaminobutyroyl benzylamide diacetate reported a significant decrease in wrinkle area and length on the forehead compared with placebo after 4 weeks of use, confirming the peptide's ability to attenuate expression‐induced wrinkles through neuromodulatory mechanisms [34]. In our current study, both peptides were included at lower concentrations, 2% each, yet the formulation achieved comparable or greater improvements in static and dynamic wrinkle parameters over a longer evaluation period (8–12 weeks), without any adverse events. These results suggest a possible synergistic interaction among the five active ingredients, enhancing collagen synthesis, improving elasticity and contributing to wrinkle reduction while maintaining an excellent safety profile.

Although the results indicated significant anti‐wrinkle effects, this research has certain limitations. The absence of a control group may have influenced the interpretation of the results, as it may not adequately account for potential confounders such as the routine use of standard skincare products. Additionally, the study population was limited to Chinese patients, which may restrict the generalizability of the findings to other ethnic groups. To address these limitations, future research should incorporate a control group and include a more diverse patient population to provide a more robust comparison.

CONCLUSIONS

The novel serum demonstrates significant anti‐skin aging effects, effectively improving static and dynamic wrinkles and skin texture. Therefore, this formulation holds promise as a safe and effective product in the field of anti‐wrinkle skincare products.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ETHICS STATEMENT

Before commencing the studies, the study protocols and informed consent forms were reviewed and approved by the ethics committees at each centre. Participants provided written informed consent prior to any study procedures.

Supporting information

Data S1.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethics restrictions.