An Optimized Naturally Derived Formulation Extract Alleviates UV-Induced Skin Photoaging and Supports Topical Lotion and Cream Development

Abstract

This study investigated the protective effect of an optimized naturally derived formulation extract against ultraviolet-induced skin photoaging and its preliminary potential for topical formulation development. A mouse model was established by combined UVA + UVB irradiation and D-galactose administration. Skin phenotype, histopathology, oxidative stress, and inflammation-related indicators were evaluated, and representative constituents were identified by HPLC. The loading level of the active extract was screened using a DPPH radical-scavenging assay, and lotion and cream formulations were optimized through emulsification-condition screening and response surface methodology. The final products were further evaluated for appearance, pH, short-term physical stability, moisture-retention performance, and DPPH radical-scavenging capacity. The extract significantly alleviated skin roughness, wrinkle deepening, epidermal thickening, and collagen fiber disorganization in mice, increased SOD, GSH-Px, and CAT activities, and reduced MDA, ROS, 8-oxoG, TNF-α, IL-6, and MMP-3 levels. HPLC identified representative constituents including 6-gingerol, ferulic acid, senkyunolide, ligustilide, atractylenolide, cinnamaldehyde, quercetin, amygdalin, and sarsasapogenin. The optimal loading level was 1.6 μg/mL. The optimized lotion and cream exhibited acceptable appearance, suitable pH, and short-term physical stability under the tested conditions, while retaining measurable DPPH radical-scavenging capacity. These findings indicate that the naturally derived formulation extract exerts anti-photoaging effects by alleviating oxidative damage, suppressing inflammatory responses, and improving extracellular matrix abnormalities, and that it has preliminary potential for topical formulation development.

1. Introduction

The skin serves as an important barrier protecting the body from external stimuli. Long-term exposure to sunlight, especially continuous ultraviolet (UV) irradiation, can induce skin photoaging, which is mainly characterized by skin roughness, laxity, deepened wrinkles, hyperpigmentation, and reduced elasticity, accompanied by impaired barrier function and diminished repair capacity [1]. Therefore, clarifying the mechanisms underlying skin photoaging and identifying safe and effective intervention strategies are of considerable importance.

UV radiation is the major exogenous factor responsible for skin photoaging. Persistent UV exposure can lead to the accumulation of reactive oxygen species (ROS), resulting in oxidative stress and DNA damage, while simultaneously activating inflammatory responses and upregulating matrix metalloproteinases (MMPs), thereby promoting the degradation of collagen and elastic fibers and ultimately causing typical photoaging changes such as wrinkling and skin laxity [2,3]. Accordingly, oxidative stress, inflammation, and extracellular matrix imbalance are considered key events in the development of skin photoaging. Current preventive and therapeutic strategies mainly focus on photoprotection, antioxidant application, and the development of natural bioactive substances. Although photoprotection remains a basic strategy, it has limited ability to reverse already established tissue damage. Antioxidants have shown certain benefits in alleviating skin photoaging by reducing oxidative stress-related injury [4]. For example, Lithospermum-derived constituents such as shikonin have been reported to support skin repair and wound healing [5], while Aloe vera polysaccharides exhibit antioxidant, anti-inflammatory, and tissue-regenerative potential in skin-related applications [6]. Other plant-derived phenolics, such as ferulic acid and quercetin, have also been highlighted for their antioxidant, anti-inflammatory, and skin-protective activities [7,8]. However, the anti-photoaging potential of naturally derived ingredients cannot be explained by antioxidant activity alone. Certain naturally derived constituents, especially polyphenols and other aromatic compounds with conjugated structures, may provide partial UV absorption or light-screening effects, whereas many natural ingredients more commonly exert protective effects by attenuating oxidative stress, suppressing inflammatory signaling, limiting MMP-mediated extracellular matrix degradation, supporting barrier repair, and helping preserve collagen and elastic fiber homeostasis [9,10,11]. However, most existing studies still focus on single compounds or individual pathways, and their ability to comprehensively regulate inflammatory responses and extracellular matrix damage remains limited.

In recent years, naturally derived active ingredients have attracted increasing attention in the management of skin photoaging. Previous studies have shown that ginsenosides and Panax ginseng-derived preparations can alleviate photoinduced skin damage [12,13], while Ophiopogon japonicus polysaccharides also exhibit antioxidant and protective activities relevant to stress injury and tissue protection [14]. These findings indicate that naturally derived bioactive substances have a solid research basis in the anti-photoaging field. In addition to plant-derived ingredients, some bee-derived natural materials such as propolis have also been reported to contain abundant phenolic constituents and exhibit antioxidant potential [15]. Taken together, these findings suggest that natural cosmetic ingredients from different sources may provide diverse functional benefits; however, a major challenge lies not only in demonstrating biological activity, but also in translating such materials into stable and cosmetically acceptable topical formulations [16,17]. In semisolid systems such as lotions and creams, problems involving solubility, compatibility, consistency, physical stability, and retention of activity can substantially limit practical applicability [18,19,20]. Nevertheless, current studies on natural anti-photoaging products still tend to focus either on the activity of individual bioactive ingredients or on formulation performance alone, whereas reports integrating efficacy validation, representative constituent characterization, and activity-guided optimization of semisolid formulations within a single study remain relatively limited. Therefore, the novelty of the present study lies not only in evaluating the anti-photoaging potential of a naturally derived formulation extract in vivo, but also in further translating it into lotion and cream formulations through activity-guided optimization, thereby linking biological efficacy with formulation feasibility.

Lujiaosan has been documented in traditional literature and has long been regarded as a preparation associated with skin beautification and anti-aging effects. Modern studies have shown that multiple botanical ingredients and representative constituents related to its original composition, such as Asparagus cochinchinensis, Atractylodes macrocephala, Angelica dahurica, ferulic acid, and quercetin, possess antioxidant, anti-inflammatory, or collagen-regulating activities [21,22,23,24,25,26,27]. These activities are particularly relevant to photoaging because collagen loss and extracellular matrix disorganization are major structural bases of wrinkle formation and skin laxity [10,28]. However, the original Lujiaosan formula was not directly suitable for topical cosmetic development because some of its components are restricted under the current cosmetic regulatory framework, particularly the banned raw-material lists [29] (NMPA, 2021). Therefore, in the present study, Lujiaosan was used as a source formula and further optimized for compliance-oriented topical application while retaining its skin-related functional orientation. Angelica sinensis, Cinnamomum cassia twig, and dried ginger were introduced because their representative constituents have been reported to exhibit antioxidant, anti-inflammatory, and skin-protective activities, thereby improving topical translational feasibility while preserving the anti-photoaging relevance of the original formula [15,30,31].

Based on this background, the present study used an optimized naturally derived formulation extract as the research material to systematically evaluate its protective effects against UV-induced skin photoaging and, on this basis, further developed topical formulations. By screening base components capable of forming stable lotion and cream systems, optimizing the proportions of the oil phase, aqueous phase, and emulsifier, and further evaluating the physicochemical properties, stability, moisturizing performance, and antioxidant activity of the final products, this study explored the potential of the extract as an active ingredient for topical anti-photoaging applications and as a skincare formulation, with the aim of providing experimental evidence for the development of naturally derived anti-photoaging products.

2. Materials and Methods

2.1. Preparation of Extracts

2.1.1. Preparation of the Original Lujiaosan Extract

The original Lujiaosan formula consisted of Lujiaoshuang, Chuanxiong, Tianmendong, Baizhu, Bailian, Xingren, Xixin, Baifuzi, and Baizhi. All crude materials were pulverized and passed through a 60-mesh sieve. Lujiaoshuang and Tianmendong were each weighed at 60 g, whereas Chuanxiong, Baizhu, Bailian, Xingren, Xixin, Baifuzi, and Baizhi were each weighed at 30 g. The mixed powder was extracted with 10 volumes of absolute ethanol, soaked at room temperature for 12 h, and then refluxed for 45 min. After filtration, the residue was re-extracted twice under the same conditions. The combined extracts were concentrated under reduced pressure at 45 °C and freeze-dried to obtain the original Lujiaosan extract, with a yield of 19.83%. The extract was stored at −20 °C until use.

2.1.2. Preparation of the Modified Lujiaosan Extract

The modified Lujiaosan formula consisted of Lujiaoshuang, Danggui, and Tiandong (80 g each), and Baizhu, Bailian, Chuanxiong, Xingren, Guizhi, and Ganjiang (50 g each). All crude materials were pulverized, passed through a 60-mesh sieve, and extracted with 10 volumes of absolute ethanol. After soaking at room temperature for 12 h, the mixture was refluxed for 45 min and filtered. The residue was then extracted twice again with 10 volumes of absolute ethanol. The combined extracts were concentrated under reduced pressure at 45 °C and freeze-dried to obtain the modified Lujiaosan ethanolic extract (LJSMF), with a yield of 25.55%. The extract was stored at −20 °C until use.

2.2. Animal Experiments

SPF-grade female KM mice (11 months old, 34–36 g) were purchased from the Hubei Center for Disease Control and Prevention (Wuhan, China). Animals were housed in the SPF animal facility of South-Central Minzu University (Wuhan, China) under controlled conditions (22 ± 2 °C, 12 h light/dark cycle) with free access to food and water. All experimental procedures were approved by the Research Ethics and Science Safety Committee of South-Central Minzu University (Approval No. 2023-scuec-034; approved on 3 March 2023). After one week of acclimatization, mice were assigned to experimental groups according to a random number table. Group allocation was recorded using coded animal numbers and cage labels. Because different interventions were administered among groups, blinding of the operator during treatment was not feasible. However, group allocation was concealed from outcome assessors by the use of coded identifiers whenever feasible.

A skin photoaging model was established by combined UVA + UVB irradiation and D-galactose administration. The UV irradiation device consisted of two UVB lamps and four UVA lamps mounted in a custom-made irradiation chamber. Before each exposure, the lamps were preheated for 10 min, and the irradiation intensity was measured using a UV radiometer. The peak wavelengths of UVA and UVB were 365 nm and 313 nm, respectively, and the total UV irradiance was approximately 1400 μW/cm². The minimum erythema dose (MED) was determined in a preliminary experiment. The irradiation schedule was set as follows: 1.0 MED in week 1 (504 mJ/cm²), 1.2 MED in week 2 (604.8 mJ/cm²), 1.4 MED in week 3 (705.6 mJ/cm²), and 1.6 MED in week 4 (806.4 mJ/cm²). The UV irradiation setup is shown in Figure 1. During ultraviolet irradiation, mice were handled gently and placed in a fixed position to ensure uniform dorsal exposure under the same irradiation conditions.

2.2.1. Original LJS Experiment

After one week of acclimatization, mice were randomly assigned to five groups: Blank, Model, positive control (VE), low-dose LJS (LJS-L), and high-dose LJS (LJS-H). The detailed grouping and dosing regimen are shown in

Table 1. Grouping and dosing regimen of the original LJS experiment.

Group	LJS-H	LJS-L	Model	Positive Control (VE)	Blank
Administration route	Topical application	Topical application	Topical application	Topical application	Topical application
Treatment composition	Lujiaosan extract (50 mg/mL) + vehicle	Lujiaosan extract (25 mg/mL) + vehicle	Blank vehicle (anhydrous ethanol:propylene glycol = 3:7)	Vitamin E solution + blank vehicle	—
Dose	0.02 g/cm2	0.02 g/cm2	0.02 g/cm2	0.02 g/cm2	—
Number of mice	9	9	9	9	9

. Before modeling, a 3 cm × 4 cm area on the dorsal skin was depilated, and newly grown hair was removed before each UV exposure. The Blank group received depilation only without UV irradiation or drug treatment. All other groups were exposed to UV and then subcutaneously injected with 6% D-galactose in the dorsal neck region. The VE, LJS-L, and LJS-H groups were topically treated with the corresponding preparations, whereas the Model group received vehicle alone. Treatment was administered once daily for 6 consecutive days followed by 1 day without treatment, for a total of 4 weeks.

2.2.2. Modified LJSMF Experiment

A separate animal experiment was conducted to evaluate the anti-photoaging effect of the modified Lujiaosan formulation (LJSMF). After one week of acclimatization, mice were randomly divided into four groups: Blank, Model, low-dose LJSMF (LJSMF-L), and high-dose LJSMF (LJSMF-H). The detailed grouping and dosing regimen are shown in

Table 2. Grouping and dosing regimen of the modified LJSMF experiment.

Group	LJSMF-H	LJSMF-L	Model	Blank
Administration route	Topical application	Topical application	Topical application	Topical application
Treatment composition	LJSMF (50 mg/mL) + vehicle	LJSMF (25 mg/mL) + vehicle	Blank vehicle (anhydrous ethanol:propylene glycol = 3:7)	—
Dose	0.02 g/cm2	0.02 g/cm2	0.02 g/cm2	—
Number of mice	9	9	9	9

. Before modeling, a 3 cm × 4 cm area on the dorsal skin was depilated, and newly grown hair was removed before each UV exposure. The Blank group received depilation only without UV irradiation or treatment. All other groups were irradiated with UV and then subcutaneously injected with 6% D-galactose in the dorsal neck region. The LJSMF-L and LJSMF-H groups were topically administered different concentrations of LJSMF, whereas the Model group received vehicle alone. Treatment was given once daily for 6 consecutive days followed by 1 day without treatment, for a total of 4 weeks.

2.2.3. Skin Observation, Scoring, and Tissue Collection

During the experiments, changes in dorsal skin appearance, including erythema, roughness, laxity, scaling, and wrinkling, were recorded, and skin thickness was measured at designated time points. Skin injury was further evaluated using a scoring system, and the detailed scoring criteria are provided in Supplementary Table S1. Skin scores were independently assigned by three evaluators according to predefined criteria, and the mean score was used for analysis. Histological evaluation of H&E- and Masson-stained sections was performed under blinded conditions using coded slides/images. All topical treatments, routine observations, and scoring procedures were performed under standardized environmental conditions by the same trained personnel following the same handling procedure to minimize animal stress and inter-operator variation. On the day after the last treatment, mice were sacrificed and dorsal skin tissues were collected. One portion was fixed in 4% paraformaldehyde for histological staining, and another portion was used for biochemical and ELISA assays.

Approximately 0.1 g of skin tissue was cut into small pieces, homogenized in prechilled normal saline, and centrifuged at 12,000× g for 15 min at 4 °C. The supernatant was collected for subsequent analysis. The activities of SOD, GSH-Px, and CAT, as well as the levels of MDA, were measured using commercial assay kits. ELISA kits were used to determine the levels of MMP-3, TNF-α, IL-6, and HA in skin tissue homogenates. All biochemical assays and ELISA measurements were performed according to the manufacturers’ instructions. Each sample was analyzed in duplicate; ELISA concentrations were calculated from standard curves, and biochemical assay results were normalized to total protein concentration.

Total RNA was extracted from mouse skin using the AFTSpin Tissue/Cell Fast RNA Extraction Kit for Animals (ABclonal, Wuhan, China), and cDNA was synthesized with the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed using Hieff UNICON^® Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China) in a 20 μL reaction system.The amplification program was as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The primers utilized in these experiments are detailed in Supplementary Table S2. The relative abundance of each transcript was calculated using 2^−ΔΔCT and normalized to endogenous β-actin expression. For RT-qPCR, equal amounts of total RNA were used for reverse transcription, each sample was analyzed in technical triplicate, and amplification specificity was confirmed by melting-curve analysis.

2.3. HPLC Analysis

HPLC analysis was performed using an Agilent 1200 liquid chromatographic system equipped with a UV detector and an autosampler. Separation was achieved on a COSMOSIL C18 column (4.6 mm × 250 mm, 5 μm) at 25 °C with a flow rate of 1.0 mL/min. The detection wavelength was 205 nm, and the injection volume was 10 μL. The mobile phase consisted of acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B). The gradient elution program was as follows: 0–10 min, 19% A/81% B; 10–18 min, 21% A/79% B; 18–34 min, 24% A/76% B; 34–40 min, 26% A/74% B; 40–50 min, 29% A/71% B; 50–60 min, 36% A/64% B; 60–70 min, 44% A/56% B; 70–80 min, 52% A/48% B; 80–81 min, 99% A/1% B; 81–91 min, 99% A/1% B; 91–92 min, 19% A/81% B; and 92–102 min, 19% A/81% B.

The sample solution was prepared by dissolving LJSMF in methanol at a concentration of 0.1 g/mL and filtering it through a 0.22 μm membrane before analysis. Reference standards included 6-gingerol, ferulic acid, senkyunolide, ligustilide, atractylenolide, cinnamaldehyde, quercetin, amygdalin, and sarsasapogenin. Qualitative and quantitative analyses were performed according to retention times and standard curves.

2.4. Preparation and Evaluation of the Naturally Derived Topical Formulations

2.4.1. Preparation of Test Solutions

Based on the preceding animal experiments, the LJSMF-H extract showed a more pronounced anti-photoaging effect and was therefore selected for subsequent topical formulation studies. The LJSMF-H extract was dissolved in methanol to prepare test solutions at different concentrations (0.4, 0.8, 1.2, 1.6, and 2.0 μg/mL) for antioxidant evaluation and loading-level screening.

2.4.2. Screening of the Extract Loading Level

The suitable loading level of the active extract was screened using the DPPH radical scavenging assay. DPPH (0.4 mg) was dissolved in 10 mL methanol under light-protected conditions to prepare a 40 mg/L DPPH solution, which was stored at 4 °C. For the assay, A_i consisted of 100 μL DPPH solution mixed with 100 μL sample solution, A_j consisted of 100 μL sample solution mixed with 100 μL methanol, and A₀ consisted of 100 μL DPPH solution mixed with 100 μL methanol. After incubation at room temperature for 60 min in the dark, absorbance was measured at 517 nm. The DPPH radical scavenging rate was calculated as:

Scavenging rate (%) = [1 − (A_i − A_j)/A₀] × 100%

The concentration corresponding to the highest scavenging rate was selected for subsequent formulation studies.

2.4.3. Screening of Emulsification Conditions

Based on preliminary trials, lotion and cream base formulations were established separately. For the lotion system, the oil phase consisted of dimethicone and caprylic/capric triglyceride; the aqueous phase consisted of purified water, glycerol, 1,3-butanediol, and caprylyl glycol; and the emulsifier phase consisted of glyceryl stearate, self-emulsifying glyceryl stearate, and lecithin, with phenoxyethanol used as preservative. For the cream system, the oil phase consisted of cetearyl alcohol and caprylic/capric triglyceride; the aqueous phase consisted of purified water, glycerol, Carbomer 940, and methylparaben; and the emulsifier phase consisted of steareth-2 and steareth-21, with phenoxyethanol used as preservative. The active extract screened in Section 2.4.2 was incorporated into both formulation systems.

Emulsification time and temperature were then optimized using appearance uniformity, texture, and system stability as evaluation criteria. Different emulsification times (15, 20, and 25 min) were first compared at 75 °C. Subsequently, with the emulsification time fixed at 20 min, different emulsification temperatures (70, 75, and 80 °C) were evaluated. Conditions yielding formulations with uniform appearance, fine texture, and good stability were selected for subsequent optimization.

2.4.4. Sensory Evaluation

Sensory evaluation was performed with reference to the method reported by Montenegro et al. [32], with minor modifications according to the characteristics of the present formulations. A total of 20 volunteer panelists, aged 20–30 years, were recruited for this study. All panelists had no apparent skin disease, olfactory dysfunction, or known history of cosmetic allergy. Before the formal evaluation, all participants received standardized training to familiarize them with the evaluation procedure, sensory attributes, and scoring criteria. The panelists were instructed to avoid the use of perfume, hand cream, and other topical products that might affect the evaluation results within 12 h before testing. The study was conducted in accordance with ethical principles and was approved by the Ethics Committee of Gezhouba Central Hospital of Sinopharm (Approval No. GYGZBZXYY20260013; approved on 5 January 2026). Written informed consent was obtained from all participants prior to testing.

The sensory evaluation was carried out in a laboratory under controlled temperature, humidity, and lighting conditions, with the temperature maintained at 22 ± 2 °C and the relative humidity at 50% ± 5%. Lotion and cream samples were evaluated in two separate sessions to minimize panel fatigue and order bias. A total of 17 lotion samples and 17 cream samples were coded using randomized three-digit numbers and presented in randomized order. To reduce subjectivity and bias, all panelists scored the samples independently without communication, and they were blinded to the formulation composition and group identity throughout the evaluation process. Approximately 0.05 g of each sample was evenly applied to a designated area on the dorsal hand or inner forearm, with sufficient spacing between adjacent test areas. A 3 min interval was allowed between consecutive sample evaluations to minimize carryover effects.

The evaluated sensory attributes included overall uniformity, gloss, moisturization, spreadability, smooth feel, absorbability, and freshness, giving a total of seven parameters. Each attribute was scored on a 0–5 scale, with a total possible score of 35. The detailed scoring criteria are provided in Supplementary Table S3 [33]. The overall sensory scores of the lotion and cream formulations were then used for subsequent response surface analysis, and the complete experimental design matrices together with the corresponding scoring results are shown in Supplementary Tables S4 and S5, respectively.

2.4.5. Response Surface Optimization of the Formulations

On the basis of the single-factor experiments, response surface methodology was used to optimize the lotion and cream formulations. Oil phase content (A), aqueous phase content (B), and emulsifier content (C) were used as independent variables, while the overall sensory score was used as the response variable. The factor levels used for the response surface optimization of the lotion and cream formulations are summarized in

Table 3. Factors and levels used for response surface optimization of the lotion and cream formulations.

Formulation	Factor	Symbol	−1	0	1
Lotion	Oil phase content (%)	A	3	5	7
Lotion	Aqueous phase content (%)	B	86.1	90.2	94.3
Lotion	Emulsifier content (%)	C	1.1	2	2.9
Cream	Oil phase content (%)	A	4	7	10
Cream	Aqueous phase content (%)	B	82.3	85.4	88.5
Cream	Emulsifier content (%)	C	3	5	7

. The complete design matrices together with the corresponding sensory scores are provided in Supplementary Tables S4 and S5. Design-Expert software (version 13.0; Stat-Ease Inc., Minneapolis, MN, USA) was used for model fitting and optimization to obtain the optimal formulation composition.

2.4.6. Physicochemical Properties and Stability Evaluation

The optimized lotion and cream formulations were prepared and evaluated for appearance, pH, and stability. Appearance evaluation included color, physical state, uniformity, and odor. For pH determination, 1 mL of sample was mixed with 9 mL purified water at 40 °C, cooled to room temperature, and then measured using a pH meter.

Stability evaluation included room-temperature, low-temperature, high-temperature, and centrifugation tests. For room-temperature stability, samples were kept at room temperature for 24 h before observation. For low-temperature stability, samples were stored at −20 °C for 24 h and then equilibrated to room temperature. For high-temperature stability, samples were placed at 60 °C for 24 h. For centrifugation stability, 10 g of sample was centrifuged at 3000 rpm for 30 min. Changes such as phase separation, water release, or discoloration were recorded.

2.4.7. Moisture Retention and Antioxidant Activity of the Final Products

Moisture retention was evaluated at 25 °C and 50% relative humidity. Lotion and cream samples were evenly applied onto the same substrate, and the mass change before and after water evaporation was recorded. Water loss was then calculated to assess moisturizing performance.

The antioxidant activity of the final products was also evaluated using the same DPPH radical scavenging procedure described in Section 2.4.2, except that the lotion and cream samples were first diluted 10-fold with purified water before analysis. The DPPH scavenging rate was calculated to evaluate the antioxidant retention of the extract after formulation.

2.5. Statistical Analysis

Data are presented as the mean ± SD. For single-endpoint outcomes, comparisons among multiple groups were performed using one-way ANOVA followed by Dunnett’s multiple-comparisons test. For longitudinal parameters repeatedly measured in the same animals, such as skin thickness and skin score, repeated-measures two-way ANOVA was used; when appropriate, a mixed-effects model was applied. For key comparisons, effect sizes and 95% confidence intervals were additionally estimated and are summarized in Supplementary Table S6. Statistical analyses were performed using GraphPad Prism 9.0 and SPSS 26.0. A value of p < 0.05 was considered statistically significant.

3. Results

Guided by historical records describing Lujiao Powder as an anti-aging and skin-nourishing natural-origin formulation, we first evaluated whether the original formulation could protect against ultraviolet-induced skin photoaging in vivo. The results showed that LJS alleviated ultraviolet-induced skin damage at the levels of gross phenotype, histopathological structure, and oxidative stress/inflammation-related indicators.

3.1. Protective Effect of LJS Against Ultraviolet-Induced Skin Photoaging in Mice

As shown in Figure 2a, the dorsal skin of mice in the Blank group remained smooth and intact throughout the experiment, whereas the Model group developed typical photoaging features after repeated ultraviolet exposure, including roughness, dryness, scaling, and deepened wrinkles. Compared with the Model group, the VE group as well as the low- and high-dose LJS groups (LJS-L and LJS-H) showed visible improvement in skin appearance, with reduced scaling, roughness, and wrinkle formation, indicating that LJS mitigated ultraviolet-induced skin surface damage.

The skin thickness growth rate is shown in Figure 2b. Repeated-measures analysis showed significant effects of group, time, and their interaction on skin thickness. Mice in the Model group exhibited a marked increase in skin thickness during the modeling period, whereas VE and both LJS-treated groups showed lower thickness growth overall. Similarly, repeated-measures analysis of skin score (Figure 2c) indicated significant effects of group and time, with the Model group showing the highest scores across the observation period and the VE, LJS-L, and LJS-H groups showing reduced scores. These findings suggest that LJS attenuated ultraviolet-induced skin thickening and wrinkle progression. Effect sizes and 95% confidence intervals for the key comparisons at the final observation time point are provided in Supplementary Table S6.

Histopathological observations further supported these findings. As shown in Figure 2d, H&E staining revealed obvious epidermal thickening, disorganized epidermal and dermal structures, and inflammatory cell infiltration in the Model group. These pathological changes were attenuated in the VE and LJS-treated groups. Masson staining showed that collagen fibers in the Model group were sparse, loose, and disorganized, whereas VE and LJS treatment preserved a denser and more regular collagen arrangement. Quantitative analysis further indicated that the collagen volume ratio was increased in the VE and LJS-L groups and showed a recovery trend in the LJS-H group compared with the Model group (Figure 2e). Relative to the Model group, the collagen volume ratio increased by approximately 14.9% in the VE group, 17.5% in the LJS-L group, and 7.8% in the LJS-H group, indicating partial restoration of collagen homeostasis after LJS treatment. Together, these results suggest that LJS reduced ultraviolet-induced skin structural injury and partially restored collagen homeostasis.

To further define the protective effects of LJS against skin photoaging, oxidative stress-, inflammation-, and extracellular matrix-related indicators were evaluated. As shown in Figure 3, compared with the Model group, SOD activity was elevated in the VE, LJS-L, and LJS-H groups, with a more pronounced increase in the LJS-treated groups (Figure 3a). Specifically, SOD increased by approximately 49.1% in the VE group, 60.1% in the LJS-L group, and 80.1% in the LJS-H group relative to the Model group. GSH-Px activity was also elevated, with increases of about 24.2%, 31.7%, and 43.7% in the VE, LJS-L, and LJS-H groups, respectively (Figure 3b). Meanwhile, MDA levels were lower in all intervention groups than in the Model group, showing reductions of approximately 26.4% (VE), 20.0% (LJS-L), and 30.3% (LJS-H) (Figure 3c). The level of 8-oxoG was also decreased, with reductions of about 59.3% in the VE group, 26.5% in the LJS-L group, and 49.9% in the LJS-H group, indicating that LJS alleviated ultraviolet-induced oxidative DNA damage (Figure 3d).

For inflammatory and extracellular matrix-related markers, the Model group showed markedly elevated levels of MMP-3, TNF-α, and IL-6. Relative to the Model group, TNF-α decreased by approximately 36.4% in the VE group, 25.9% in the LJS-L group, and 37.0% in the LJS-H group. MMP-3 showed a more pronounced reduction, decreasing by about 64.2% in the VE group, 19.3% in the LJS-L group, and 32.7% in the LJS-H group (Figure 3e,f). IL-6 was reduced more moderately, by approximately 22.5% in the VE group, 12.7% in the LJS-L group, and 27.1% in the LJS-H group (Figure 3g). In contrast, HA content increased in the intervention groups, with increases of about 149.0% in the VE group, 114.0% in the LJS-L group, and 134.8% in the LJS-H group relative to the Model group (Figure 3h). TIMP1 levels were also higher in the VE and LJS groups than in the Model group, showing increases of approximately 57.8%, 39.8%, and 53.5% in the VE, LJS-L, and LJS-H groups (Figure 3i), respectively. Together, these findings indicate that the original LJS formulation improved ultraviolet-induced oxidative stress, inflammatory responses, and extracellular matrix imbalance to different extents across endpoints.

Taken together, the original formulation of Lujiao Powder not only improved ultraviolet-induced skin appearance and histopathological abnormalities, but also exerted protective effects by enhancing antioxidant enzyme activities, reducing oxidative damage, and suppressing inflammatory responses during skin photoaging.

3.2. Protective Effect of LJSMF Against Ultraviolet-Induced Skin Photoaging in Mice

Based on the protective effect observed for the original formulation LJS, the modified formulation LJSMF was further evaluated in a mouse model of ultraviolet-induced skin photoaging. The results showed that LJSMF alleviated ultraviolet-induced skin damage at multiple levels, including gross phenotype, histopathological changes, oxidative stress, inflammation, and extracellular matrix-related indicators.

As shown in Figure 4a, the dorsal skin of mice in the Blank group remained smooth and intact, whereas the Model group developed typical photoaging features after continuous ultraviolet exposure, including skin thickening, erythema, scaling, and coarse wrinkles. Compared with the Model group, both the low- and high-dose LJSMF groups (LJSMF-L and LJSMF-H) showed obvious improvement in skin appearance, with reduced erythema and scaling, lower roughness, and fewer wrinkles. The skin thickness growth rate is shown in Figure 4b. Repeated-measures analysis showed significant effects of group, time, and their interaction on skin thickness. Skin thickness increased continuously in the Model group during the modeling period, whereas LJSMF treatment reduced the overall thickness growth rate. Consistently, repeated-measures analysis of skin score (Figure 4c) showed that the LJSMF-treated groups had lower scores than the Model group over the observation period. These findings indicate that LJSMF effectively attenuated ultraviolet-induced skin thickening and surface damage. Effect sizes and 95% confidence intervals for the key comparisons at the final observation time point are provided in Supplementary Table S6.

Histopathological examination further supported these observations. As shown in Figure 4d, H&E staining revealed marked epidermal thickening, disorganized epidermal and dermal structures, and inflammatory cell infiltration in the Model group, whereas these pathological changes were alleviated after LJSMF treatment. Masson staining showed that collagen fibers in the Model group were sparse, loose, and disorganized, while the LJSMF-treated groups displayed denser and more orderly collagen distribution. Quantitative analysis further demonstrated that the collagen volume ratio was increased in both LJSMF-treated groups compared with the Model group, with a more pronounced increase in the LJSMF-L group (Figure 4e). These findings indicate that LJSMF reduced ultraviolet-induced histological damage and improved collagen homeostasis.

To further clarify the protective effect of LJSMF, oxidative stress-, inflammation-, and extracellular matrix-related indicators were measured. As shown in Figure 5, compared with the Model group, both LJSMF-L and LJSMF-H increased the activities of SOD, GSH-Px, and CAT (Figure 5a–c), while decreasing the levels of MDA, ROS, and 8-oxoG (Figure 5d–f), suggesting that LJSMF enhanced antioxidant capacity and alleviated lipid peroxidation and oxidative DNA damage. SOD increased by approximately 20% in the LJSMF-L group and 15% in the LJSMF-H group; GSH-Px increased by about 90% and 50%, respectively; and CAT increased by approximately 50% and 25%, respectively. Meanwhile, MDA decreased by about 40% in the LJSMF-L group and 30% in the LJSMF-H group, while ROS decreased by about 40 and 30%, respectively. The level of 8-oxoG was also lower, showing an approximate reduction of 40% in the LJSMF-H group, whereas the change in the LJSMF-L group was less obvious on visual inspection of the bar graph. For inflammation- and extracellular matrix-related markers, MMP-3 was reduced by roughly 40% in the LJSMF-L group and about 10% in the LJSMF-H group; TNF-α decreased by approximately 60% and 25%, respectively; and IL-6 decreased by about 30% and 20%, respectively. In contrast, HA increased by approximately 15% in the LJSMF-L group and 30% in the LJSMF-H group, while TIMP1 increased by roughly 35% in both treatment groups. Overall, these results suggest that LJSMF alleviated ultraviolet-induced photoaging by improving antioxidant defense, attenuating oxidative injury, suppressing inflammatory responses, and partially restoring extracellular matrix homeostasis.

3.3. Determination of Representative Chemical Constituents in LJSMF

Since LJSMF showed clear protective effects against skin photoaging in the animal experiments, HPLC analysis was further performed to characterize its representative chemical constituents and preliminarily clarify its material basis. As shown in Figure 6, both the reference standards and the LJSMF exhibited distinct chromatographic peaks, and the corresponding peaks in the sample showed retention times consistent with those of the standards. Specifically, Figure 6A shows that the nine reference compounds had good peak shape and separation, while Figure 6B demonstrates that corresponding peaks for 6-gingerol, ferulic acid, senkyunolide, ligustilide, atractylenolide, cinnamaldehyde, quercetin, amygdalin, and sarsasapogenin could be detected in the LJSMF, indicating good specificity of the established method.

The contents of these representative constituents were further quantified, and the results are shown in

Table 4. Determination of main components in LJSMF.

Source Medicinal Material	Compound	Peak Area (μV·s)	Content (mg/g)
Dried ginger	6-Gingerol	62,866	0.013
Angelica sinensis, Ligusticum chuanxiong	Ferulic acid	598,146	0.014
Angelica sinensis, Ligusticum chuanxiong	Senkyunolide	510,382	0.054
Angelica sinensis, Ligusticum chuanxiong	Ligustilide	2,514,803	0.128
Atractylodes macrocephala	Atractylenolide	140,336	0.023
Cinnamon twig	Cinnamaldehyde	777,124	0.101
Ampelopsis japonica	Quercetin	930,559	0.021
Bitter apricot kernel	Amygdalin	2,311,763	0.244
Asparagus cochinchinensis	Sarsasapogenin	94,700	0.109

. The levels of individual compounds varied considerably in the extract. Among them, amygdalin (0.244 mg/g), ligustilide (0.128 mg/g), sarsasapogenin (0.109 mg/g), and cinnamaldehyde (0.101 mg/g) were relatively abundant, whereas 6-gingerol (0.013 mg/g) and ferulic acid (0.014 mg/g) were present at lower levels. Overall, LJSMF contains multiple representative bioactive constituents, suggesting that its anti-photoaging effect may be supported by a multi-component material basis.

3.4. Formulation Optimization and Quality Evaluation of the Natural-Origin Topical Preparations

Given that LJSMF exhibited clear protective effects in the ultraviolet-induced skin photoaging model and contained multiple representative bioactive constituents, it was further developed into natural-origin topical preparations. The formulation process, stability, and physicochemical properties were subsequently evaluated to assess its application potential in skin care products.

3.4.1. Screening of Active Extract Loading and Emulsification Conditions

As shown in Figure 7, the DPPH radical scavenging rate of the natural-origin active extract increased with increasing concentration and reached a maximum at 1.6 μg/mL (approximately 94%). A slight decline was observed when the concentration increased further to 2.0 μg/mL. Therefore, 1.6 μg/mL was selected as the optimal loading level for subsequent formulation studies (Figure 7a). Further comparison of emulsification time and temperature showed that the formulation prepared at 20 min and 80 °C exhibited a more homogeneous appearance and better texture stability than the other tested conditions (Figure 7b,c), and these parameters were therefore selected as the optimal processing conditions.

3.4.2. Response Surface Optimization of the Lotion and Cream Formulations

Based on the single-factor screening results, response surface methodology was further applied to optimize the lotion and cream formulations. The overall sensory score, integrating color, texture, spreadability, absorbability, and moisturizing sensation, was selected as the primary response variable because the aim of formulation optimization in this study was to obtain semisolid topical products with acceptable appearance and user-related application properties. This composite score reflects multiple practical attributes directly relevant to preliminary formulation acceptability. Importantly, sensory score was not used as the sole quality criterion, because the optimized formulations were further evaluated for pH, physical stability, moisture retention, and DPPH radical-scavenging capacity. The complete response surface design matrices together with the corresponding sensory scores for the lotion and cream are provided in Supplementary Tables S4 and S5, respectively. The results showed that different proportions of the oil phase, aqueous phase, and emulsifier markedly affected the overall sensory score of the formulations. Among all response surface experimental runs, the highest observed score for the lotion was 35, corresponding to a formulation containing 5.0% oil phase, 90.2% aqueous phase, and 2.0% emulsifier. The highest observed score for the cream was also 35, corresponding to a formulation containing 7.0% oil phase, 85.4% aqueous phase, and 5.0% emulsifier. These findings indicate that the selected factor ranges and levels were appropriate for subsequent optimization.

Further model-fitting analysis showed that the overall sensory scores of both the lotion and cream could be well described by quadratic polynomial models. Both models were highly significant (p < 0.0001), while the lack-of-fit terms were not significant, indicating good model fitness and suitability for formulation prediction and optimization. For the lotion system, the order of factor influence on the overall score was emulsifier > oil phase > aqueous phase, whereas for the cream system the order was emulsifier > aqueous phase > oil phase. The detailed model analysis results are shown in

Table 5. Analysis results of the response surface models.

Formulation	Model P	Lack-of-Fit P	R2	Adjusted R2	Order of Factor Influence
Lotion	<0.0001	0.7158	0.9915	0.9806	Emulsifier > oil phase > aqueous phase
Cream	<0.0001	0.4553	0.9856	0.9670	Emulsifier > aqueous phase > oil phase

. The response surface and contour plots further demonstrated that interactions among the tested factors existed within the investigated ranges (Figure 8).

According to model prediction, the optimal lotion formulation consisted of 5.03% oil phase, 89.87% aqueous phase, and 1.10% emulsifier, with a predicted overall score of 34.82. For the cream system, considering the response surface optimization results together with the sensory evaluation outcomes, the cream formulation selected for subsequent quality evaluation consisted of 7.0% oil phase, 85.4% aqueous phase, and 5.0% emulsifier. These results indicate that response surface methodology effectively yielded topical formulations with desirable color, texture, and stability. The formulation composition and quality evaluation results of the final products are summarized in

Table 6. Optimal formulation composition and quality evaluation results of the final products.

Formulation	Oil Phase (%)	Aqueous Phase (%)	Emulsifier (%)	Overall Score	pH	Mean Water Loss (%)	DPPH Scavenging Rate (%)	Stability
Lotion	5.03	89.87	1.1	34.82	5.44 ± 0.20	1.41	83.43	Good
Cream	7.00	85.40	5.00	35.00	5.63 ± 0.20	1.08	75.64	Good

Note: The lotion composition was obtained from model prediction, whereas the cream composition was selected for subsequent quality evaluation based on the response surface optimization results together with the sensory evaluation outcomes.

.

3.4.3. Physicochemical Properties, Stability, and Functional Evaluation of the Final Formulations

Temperature-stability testing showed that no obvious phase separation, water release, or discoloration was observed after storage at room temperature, −20 °C, or 60 °C for 24 h, indicating acceptable short-term physical stability under the tested temperature conditions. Centrifugal stability testing further showed that both the lotion and cream remained macroscopically homogeneous after centrifugation at 3000 rpm for 30 min, with no obvious phase separation (Figure 9c).

Further evaluation showed that the pH values of the lotion and cream were 5.44 ± 0.20 and 5.63 ± 0.20, respectively, both of which fall within the acceptable range for cosmetic products. Moisture-retention testing revealed mean water-loss rates of approximately 1.41% for the lotion and 1.08% for the cream. In addition, the DPPH radical scavenging rates of the final lotion and cream were 83.43% and 75.64%, respectively. The formulation composition and quality evaluation results of the final products are summarized in Table 5. Overall, the optimized natural-origin topical preparations demonstrated acceptable appearance, suitable pH, short-term physical stability under the tested conditions, and retained DPPH radical-scavenging capacity, supporting their preliminary feasibility for topical formulation development.

4. Discussion

4.1. Anti-Photoaging Potential and Material Basis of the Natural-Derived Active Extract

Skin photoaging is a complex biological process induced by long-term ultraviolet exposure, and its progression is closely associated with oxidative stress accumulation, persistent inflammatory activation, and extracellular matrix degradation. Recent studies have shown that oxidative stress not only directly causes damage to lipids, proteins, and DNA, but also amplifies inflammatory signaling and promotes tissue remodeling, thereby driving the progression of skin photoaging [26,34,35,36,37]. Meanwhile, sustained elevation of inflammatory mediators and abnormal activation of matrix metalloproteinases (MMPs) accelerate the degradation of collagen and elastic fibers, thereby contributing to wrinkle formation, skin laxity, and structural deterioration of photoaged skin [34,35,36,38,39]. Therefore, natural-derived active ingredients capable of simultaneously modulating oxidative damage, inflammatory responses, and extracellular matrix homeostasis are considered promising candidates for anti-photoaging research and product development.

In the present study, HPLC analysis identified several representative constituents in the extract, including 6-gingerol, ferulic acid, senkyunolide, ligustilide, atractylenolide, cinnamaldehyde, quercetin, amygdalin, and sarsasapogenin. Among the identified constituents, several compounds have previously been reported to exert activities relevant to skin photoaging. Quercetin has been shown to suppress UV-induced MMP-1 and COX-2 expression and prevent collagen degradation in human skin, suggesting a plausible link to the reduced MMP-3 level and improved collagen-related outcomes observed here [40]. Ferulic acid has also been associated with antioxidant and photoprotective effects and with inhibition of UV-induced matrix-degrading processes [41]. In addition, ligustilide and 6-gingerol have been reported to exhibit anti-inflammatory and antioxidant activities, which may be relevant to the reductions in oxidative stress and inflammatory markers observed in the present study [42,43]. However, these links remain inferential, and the present study does not establish compound-specific causal mechanisms. Therefore, the identified constituents may provide only a preliminary material basis for the observed anti-photoaging activity, but their individual contributions require further verification.

4.2. Comprehensive Improvement of Oxidative Damage, Inflammatory Responses, and Extracellular Matrix Abnormalities

The present results showed that this natural-derived active extract significantly ameliorated typical photoaging phenotypes in UV-irradiated mice, including skin roughness, wrinkle deepening, epidermal thickening, and collagen fiber disorganization. In addition, the extract increased the activities of SOD, GSH-Px, and CAT, while reducing the levels of MDA, ROS, and 8-oxoG. These findings indicate that the extract enhanced the endogenous antioxidant defense system of the skin and attenuated lipid peroxidation as well as oxidative DNA damage. Previous studies have suggested that persistent ROS elevation after ultraviolet exposure is a major driving force in photoaging, and natural-derived antioxidant ingredients remain an important focus in anti-photoaging research because of their biological activity and development potential [34,36,37,39]. Therefore, the increased antioxidant enzyme activities and reduced oxidative damage markers observed in this study support a clear protective role of the extract against UV-induced oxidative stress.

Beyond its antioxidant effect, the extract also reduced the levels of TNF-α, IL-6, and MMP-3, while increasing the level of HA, suggesting that it also exerted beneficial effects on inflammatory responses and extracellular matrix dysregulation. Previous studies have shown that sustained elevation of inflammatory mediators is a hallmark of ultraviolet-mediated skin aging, whereas abnormal activation of MMPs directly contributes to extracellular matrix degradation and is closely associated with collagen loss and structural loosening of the skin [28,34,35,38]. In addition, persistent abnormalities in extracellular matrix structure and function are also considered an important basis for the worsening of visible skin aging [38,44]. Combined with the histological findings of the present study, these results suggest that the natural-derived active extract does not act through a single pathway alone, but more likely exerts an overall anti-photoaging effect by reducing oxidative damage, suppressing inflammatory mediator release, and attenuating extracellular matrix degradation. Although the high-dose treatment tended to show greater improvement than the low-dose treatment in several endpoints, this dose-related pattern was not uniformly observed across all measurements, suggesting variability in endpoint sensitivity.

These findings are also consistent with previously reported signaling pathways involved in skin photoaging. Recent studies have indicated that UV-induced oxidative stress and inflammatory activation are closely associated with MAPK/AP-1- and NF-κB-related signaling, which contributes to MMP upregulation and extracellular matrix degradation, while collagen loss and matrix disorganization are also linked to impaired collagen/ECM homeostasis-related regulation in photoaged skin [9,10,28,45,46]. However, the present study did not directly analyze specific signaling pathways, and therefore the mechanistic interpretation remains inferential rather than pathway-specific.

4.3. Significance of Formulation Translation from a Cosmetic Application Perspective

From the perspective of cosmetic research, demonstrating the biological activity of an active extract is only the first step; whether it can be further developed into a stable, mild, and user-friendly topical formulation is equally important for evaluating its application value. Recent studies have indicated that the development of topical anti-aging products has gradually shifted from focusing solely on active ingredients to a more integrated strategy that also considers formulation stability, skin compatibility, and consumer experience [16,18,47]. In addition, the advantages of plant- or natural-derived ingredients in cosmetics are not limited to antioxidant and anti-inflammatory activities, but also include moisturizing, reparative, and formulation-compatible properties [19,24,47].

In the present study, the natural-derived active extract was further formulated into a lotion and a cream, and its loading amount, emulsification conditions, response surface optimization results, physicochemical properties, and stability were systematically evaluated. The results showed that the optimized formulations exhibited a uniform appearance, fine texture, and pH values within the acceptable range for skin application. They also showed acceptable short-term physical stability under the tested high-/low-temperature and centrifugation conditions, while retaining measurable DPPH radical-scavenging capacity in the final products. These findings suggest that the antioxidant-related activity of the extract was not completely lost during formulation processing and that the designed formulations could preliminarily balance formulation stability with application-related performance [16,19].

Nevertheless, the present work still has important limitations. The formulation evaluation was mainly based on short-term physical stability testing and in vitro antioxidant screening, and the present study did not include long-term stability, microbial stability, preservative-efficacy, or shelf-life evaluation. More comprehensive physicochemical characterization, such as rheological behavior and droplet-size distribution, was also not included. In addition, although several measures were taken to reduce subjectivity and bias in the sensory evaluation, formal inter-rater reliability statistics were not assessed. The DPPH assay was used in the present study as a practical tool for extract-loading screening and for preliminary assessment of antioxidant-related activity retention after formulation. However, additional cellular, ex vivo, or in vivo antioxidant assays on the final formulations would be required to more comprehensively evaluate their biological antioxidant efficacy. Therefore, the current results support preliminary formulation feasibility rather than real-world applicability.

5. Conclusions

The present study demonstrated that the optimized naturally derived formulation extract alleviated UV-induced skin photoaging in mice, as evidenced by reduced skin roughness, wrinkle deepening, epidermal thickening, and collagen fiber disorganization. It also increased the activities of SOD, GSH-Px, and CAT, while decreasing the levels of MDA, ROS, 8-oxoG, TNF-α, IL-6, and MMP-3, indicating antioxidant, anti-inflammatory, and extracellular matrix-protective effects. HPLC analysis identified multiple representative constituents, providing a preliminary material basis for its anti-photoaging activity. Further formulation studies showed that the extract exhibited relatively high DPPH radical-scavenging capacity at 1.6 μg/mL, and that the optimized lotion and cream formulations possessed acceptable appearance, suitable pH, and short-term physical stability under the tested conditions, together with measurable moisture-retention performance and DPPH radical-scavenging capacity. Overall, this naturally derived formulation extract showed anti-photoaging activity in the present experimental setting and demonstrated preliminary potential for topical formulation development. However, further long-term stability, microbiological, preservative-efficacy, shelf-life, safety, and efficacy studies are still required before practical application can be established.