Normal human dermal fibroblasts (nHDFs; Lonza, Basel, Switzerland) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (Gibco Life Technologies) at 37°C in an atmosphere of 5% CO₂. Apigenin was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO).

The nHDFs (1×10⁶/well) were seeded into 6-well plates and cultured until 70–80% confluent. Prior to irradiation, the cells were washed twice with phosphate-buffered saline (PBS). Fresh PBS was then added, and the cells were irradiated with UVA light (25 J/cm² UVA; UVA lamp; UVP, Inc., Upland, CA, USA). The radiation intensity was monitored by a fiber optic spectrometer system USB2000 (Ocean Optics, Dunedin, FL, USA). The control cells were treated identically, except for the exposure to UV light. Following irradiation, various concentrations (0–100 µM) of the treatment agent (apigenin) in fresh medium were added to cells at 37°C for 24 h.

The nHDFs were seeded at a density of 3×10³ cells/well in 96-well plates and incubated for 24 h. The cells were irradiated with UVA (0–50 J/cm²) and incubated with various concentrations of apigenin (0–200 mM) for 24 h. nHDF cell toxicity due to apigenin was evaluated using the EZ-Cytox Cell Viability Assay kit (Itsbio, Seoul, Korea), a water-soluble tetrazolium salt (WST-1) assay. WST-1 solution was added to the cultured cells at a volume equal to 10% that of the culture medium, and the cells were then incubated at 37°C for 1 h. Cell viability was evaluated by measuring the absorbance at 450 nm using an iMark microplate reader (Bio-Rad, Hercules, CA, USA).

Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. The purity and concentration of the RNA were evaluated using a MaestroNano^®, a microvolume spectrophotometer (Maestrogen, Las Vegas, NV, USA), and cDNAs were synthesized using the miScript II RT kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. In order to evaluate the expression of MMP-1, quantitative PCR was performed using the following primers: forward, 5′-TCT GACGTTGATCCCAGAGAGCAG-3′ and reverse, 5′-CAGGG TGACACCAGTGACTGCAC-3′ using EvaGreen dye (Solis BioDyne, Tartu, Estonia) with Line-Gene K software (Bioer Technology Co., Ltd., Hangzhou, China). The Ct value for each gene was normalized to β-actin using the following primers: forward, 5′-GGATTCCTATGTGGGCGACGA-3′ and reverse, 5′-CGCTCGGTGAGGATCTTCATG-3′. The relative expression levels of each gene were calculated using the 2^−ΔΔCt method, as previously described (25).

The expression of galactosidase as a marker for senescent nHDFs was determined using the SA-β-galactosidase staining kit (BioVision, Inc., Milpitas, CA, USA) following the manufacturer's instructions. The nHDFs were seeded at a density of 2×10⁵ cells/well in 60 mm cell culture plates and incubated at 37°C until they were 90% confluent. The cells were then pre-treated with apigenin, irradiated with UVA, and incubated for 24 h. These cells were washed with PBS and fixed by treatment with 0.5 ml fixing solution/well (4% formaldehyde, 0.5% glutaraldehyde in PBS buffer, pH 7.2) for 1 h. The fixed cells were stained in staining solution mix (staining solution, 470 µl; staining supplement, 5 µl; 20 mg/ml X-Gal in dimethyl-formamide, 25 µl) for 24 h, at 37°C. After 1 day, 70% glycerol (1 ml/well) was added, and images were captured using an an Olympus IX51 microscope (Olympus, Tokyo, Japan).

All clinical evaluations were approved by the Ethics Committee of the Korea Institute for Skin and Clinical Sciences and performed in accordance with the Declaration of Helsinki Principles. We enrolled 40 women, aged over 30 years, in a randomized and double-blinded clinical trial. The subjects were selected based on age and were not pregnant or nursing. All subjects were informed about the objective of the study, signed an informed consent, and agreed to use only our products for skin care during the study duration. Factors for dropping out of the trial included itching, erythema, or hindrance to evaluation by excessive drinking or smoking. Th subjects were divided into the control and experimental groups, each containing 20 subjects (control group, 44.40±5.97 years; experimental group, 45.30±6.29 years). All subjects were subjected to the same conditions, apart from the experimental group which was administered the test treatment. The study duration was 4 weeks, and no participants dropped out. Biometric parameters were measured 3 times: before application, and then at 2 and 4 weeks after application. An investigator also questioned the subjects about their condition and performed visual evaluations for skin disorders, such as erythema, itching, scaling, edema, tingling and burning sensations, at each visitation.

To investigate the effects of apigenin on dermal density, skin elasticity, skin texture, moisture, transepidermal water loss (TEWL) and fine wrinkles around the eyes (also known as crow's feet), the subjects were instructed to apply 2 g of the test treatment to the face, including the eye rim, every morning and night for 4 weeks. The subjects and investigators were blinded to the test and control treatments. At each visit, all subjects washed with the cleanser provided and lay quietly in a room with a constant temperature (22±1°C) and humidity (45±5%), so that they would all be evaluated under the same conditions. The cream provided to the experimental group contained 1% (wt%) apigenin; whereas, the cream provided to the control group was prepared using the same volume of water in the place of apigenin.

To evaluate the improvement in skin elasticity, a DermaLab USB elasticity probe (Cortex Technology Inc., Hadsund, Denmark) was applied to the skin, and the results were analyzed using the associated application software, version 1.09. The measurement was performed by applying a single fixed elasticity probe on the left cheek of a subject. To analyze the measured value (in MPa), Young's modulus (E) was used, and the detected value is dependent on skin elasticity. To evaluate improvement, measurements were taken 3 times, before treatment and both at 2 and 4 weeks after application.

To evaluate dermal density, a DUB^® SkinScanner (taberna pro medicum, Luneburg, Germany) was utilized. Dermal density was measured (in µm) 3 cm beside the left eye, applying a couplant for ultrasonic examination. The analysis range was limited to the region between the dermis and the upper panniculus. To evaluate improvement, measurements were performed three times, before treatment and both 2 and 4 weeks after first application.

To evaluate the improvement of wrinkles, particularly crow's feet, a Robo skin analyzer CS50 (Inforward Inc., Tokyo, Japan) was used. All facial images were captured under the same position and with equal lighting. The capturing was performed 3 times at each evaluation, on the front, left and right sides of the face. To evaluate improvement, measurements were performed 3 times, before treatment and both at 2 and 4 weeks after application. We analyzed the captured images matching the facial feature points to reenact accurately, and the measurement unit was in mm.

To evaluate improvement in skin moisture, a DermaLab USB moisture probe (Cortex Technology Inc.) was applied to the skin, and the data were analyzed using the associated application software, version 1.09. All subjects were evaluated on the same region of the right cheek, 5 times consecutively, and we calculated the mean value, excluding the maximum and minimum values. To evaluate improvement, measurements were performed 3 times, before treatment and both at 2 and 4 weeks after application. The probe measures skin conductance in micro Siemens (µS), and the numerical value is dependent on skin moisture.

To evaluate improvements in TEWL, a DermaLab USB TEWL probe (Cortex Technology, Inc.) was applied to the skin, and the data were analyzed using the associated application software, version 1.09. The measurement was performed 5 times consecutively, on the right cheek of the subjects, and we calculated the mean value, excluding the maximum and minimum values. To evaluate improvement, measurements were performed 3 times, before treatment and both at 2 and 4 weeks after application.

To evaluate improvements in facial evenness, a PRIMOS Lite system (field of view 45×30; GFMesstechnik GmbH, Teltow, Germany) was used, and the captured clinical images were analyzed using the associated imaging software, PRIMOS Lite version 5.6E. The images were captured 3 times consecutively, on the left side of the forehead of the subjects. We analyzed facial evenness by calculating surface roughness, Ra (average of all heights and epths to the reference plane) value. The Ra value, which is the most well used measurement for facial evenness, is the arithmetic mean of the absolute values within the total measurement range. To evaluate improvement, measurements were performed 3 times, before treatment and both at 2 and 4 weeks after application.

For cellular efficacy tests, all results are presented as the mean percentage ± standard deviation (SD) of 3 independent experiments. Differences with a P-value <0.05, as determined by the Student's t-test, were considered statistically significant. For clinical efficacy tests, statistical analyses were conducted using SPSS software (SPSS, version 17.0 for Windows; IBM SPSS, Armonk, NY, USA). Paired Student's t-tests were performed in the cases of repeated measurements on the same subject. To analyze subject questionnaires, the mean values, standard deviations and percentages were calculated. The formula used to measure the percentage change for each skin parameter was 'Percentage change = [(A – B)/B] ×100', where A is defined as the individual value of any parameter at the 2-and 4-week visits, and B represents the zero hour of the assessed parameter.

To determine whether apigenin affects nHDF viability, the cells were exposed to apigenin at concentrations ranging from 0–100 µM for 24 h. As shown in Fig. 1, apigenin reduced cell viability by 1.23% at 1 µM, 3.63% at 5 µM, and 12.61% at 20 µM. The apigenin-induced cytotoxicity increased significantly at concentrations >50 µM. Therefore, we used the concentration of 20 µM as the maximum concentration in all the subsequent experiments.

To evaluate the effects of apigenin on the viability of damaged cells, the nHDFs were irradiated with 25 J/cm² UVA, and these cells were then treated with apigenin at various concentrations. As shown in Fig. 2, this dose of UVA reduced cell viability by 34.60%; however, treatment with 10 and 20 µM apigenin increased viability back to 90.00 and 98.92%, respectively, suggesting that apigenin protects cells from UVA-induced cytotoxicity.

We then investigated the ability of apigenin to inhibit senescence, using a SA-β-galactosidase assay. When the cells were irradiated with 25 J/cm² UVA, the percentage of senescent cells was found to be as high as 61.29%. This number decreased in a dose-dependent manner to 50.49, 32.03 and 17.34% when cells were post-treated with 5, 10 and 20 µM apigenin, respectively (Fig. 3). These results indicate that UVA acts as a stimulator of senescence, and that apigenin can inhibit UVA-induced cellular senescence.

UVA radiation corresponds to 90–95% of solar UV radiation (26) and is mainly responsible for the high production of reactive oxygen species (ROS) in skin, leading to oxidative stress (27). ROS are able to induce several disruptive cellular processes, such as senescence, DNA cleavage, lipid peroxidation and cell death (28). In addition, UVA induces the expression of MMP-1 in dermal fibroblasts in vivo and stimulates the expression of MMP-1, MMP-2 and MMP-3 in cell culture, all of which are induced during wrinkling and skin aging (29,30). MMP-1, when generated from fibroblasts, has been reported to ultimately promote a decrease in collagen (23,31,32). Collagen is the most abundant protein in the dermis, and type-1 collagen, in particular, provides structure to skin and composes >90% of collagen in the body (33). We found that the mRNA expression of MMP-1 in the UVA-irradiated nHDFs increased up to 2.91-fold as compared with the non-irradiated cells. However, treatment with 5, 10 and 20 µM apigenin reduced MMP-1 expression 2.43-, 1.85- and 1.31-fold, respectively (Fig. 4), indicating that apigenin inhibits the UVA-induced induction of MMP-1 expression.

To evaluate the effects of apigenin on skin aging in vivo, we measured the density of the dermis in subjects treated with a cream containing 1% apigenin. Using a DUB SkinScanner, we found that the subjects using the non-apigenin-containing control cream displayed a mean density of 50.41 µm before use, and densities of 50.02 and 50.05 µm after 2 and 4 weeks, respectively (Fig. 5). However, subjects using the apigenin-containing cream displayed a mean density of 49.96 µm before use, and densities of 55.31 and 62.32 after 2 and 4 weeks of application, respectively (Fig. 5). Dermal density measurement, represented as a mathematical value, is proportional to density, and these experimental data were statistically significant (P<0.001). To compare the results from the treatment and control groups, we calculated the improvement as a percentage based on the density values before and after application. Using this metric, the dermal density improvement was found to be −0.77 and −0.72% after 2 and 4 weeks of application, respectively, in the control group. Conversely, in the experimental group, the dermal density improvement was calculated as 10.70 and 24.75% after 2 and 4 weeks of application, respectively. These results suggest that the topical application of apigenin enhances dermal thickness.

We then measured the length of crow's feet in the subjects treated with the apigenin-containing cream and the controls. In the control group, the mean length was found to be 63.10 mm before application, and 63.95 and 64.25 mm after 2 and 4 weeks of application, respectively (Fig. 6). In the experimental group, the mean length was 65.05 mm prior to application, and 56.60 and 48.65 mm after 2 and 4 weeks of application, respectively (Fig. 6). The measured values for the experimental group were statistically significant (P<0.001). To compare the results from the control and experimental groups, we calculated the improvement as a percentage based on the values before and after application. For the control group, the percentage improvement was calculated as −1.35 and −1.82% after 2 and 4 weeks of application, respectively. Whereas for the experimental group, the percentage improvement was found to be 12.99 and 25.21% after 2 and 4 weeks of application, respectively. These data suggest that the topical application of apigenin can lead to a reduction in wrinkle length.

The dermis is composed of an extracellular matrix consisting of fibrous proteins, such as collagen and elastin, and is involved in the regulation of skin elasticity. Factors such as ROS, UV, or age can cause skin damage, wrinkle formation and a reduction in elasticity through the estructural denaturation of collagen and elastin (34). In order to examine the effects of apigenin on skin elasticity, we measured elasticity in subjects treated with apigenin-containing cream or the control cream using a DermaLab USB probe. In the control group, elasticity was found to be 7.38 MPa before application, and 7.33 and 7.27 MPA after 2 and 4 weeks of application, respectively. In the experimental group, elasticity was 7.40 MPa before application, and 9.14 and 10.60 MPa after 2 and 4 weeks of application, respectively (Fig. 7). The experimental group values were statistically significant (P<0.001). To compare the results from the control and experimental groups, we calculated the improvement as a percentage based on the values before and after application. For the control group, the percentage improvement was calculated to be −0.68 and −1.42% after 2 and 4 weeks of application, respectively. However, the experimental group showed an improvement of 23.60 and 43.34% after 2 and 4 weeks of treatment, respectively. These results suggest that the topical applicatino of apigenin increases skin elasticity.

Keratinocyte moisture content is pivotal for maintaining moisture in the skin. Normal keratinocytes maintain 10–30% moisture; however, when the moisture content drops below 10%, keratinocytes are unable to maintain the skin's barrier function, and skin becomes dry, acquires an uneven texture and produces wrinkles, accelerating senescence (4). To evaluate the effect of apigenin treatment on skin moisture, we analyzed the skin moisture content in our study subjects using the DermaLab USB moisture probe. We found that the moisture content in the control group was 391.25 µS before use, and 393.30 and 397.87 µS after 2 and 4 weeks of application, respectively (Fig. 8). By contrast, the moisture content in the experimental group, which used the apigenin-containing cream, was 399.48 µS before use, and 528.75 and 604.74 µS after 2 and 4 weeks of application, respectively (Fig. 8). To compare the results from the control and experimental groups, we calculated the degree of improvement as a percentage based on the values before and after application. For the control group, moisture was increased by 0.52 and 1.69% after 2 and 4 weeks, respectively. These changes were not statistically significant (P>0.05), indicating that the control cream had no measurable effect on moisture content. Conversely, the use of the apigenin-containing cream significantly improved skin moisture by 32.36 and 51.38% after 2 and 4 weeks, respectively (P<0.001). These data demonstrate that use of apigenin-containing cream results in an improved skin moisture content.

To determine the efficacy of apigenin as a skin moisturizer, we used the DermaLab USB TEWL probe to measure TEWL in the skin of subjects who used either the control or apigenin-containing cream. In the control subjects, the TEWL was found to be 7.50 g m⁻² h⁻¹ before use, and 7.49 and 7.28 g m⁻² h⁻¹ after 2 and 4 weeks of application, respectively (Fig. 9). By contrast, in the experimental group, the TEWL was 7.92 g m⁻² h⁻¹ before use, and 7.20 and 5.73 g m⁻² h⁻¹ after 2 and 4 weeks of application, respectively (Fig. 9). To compare the results from the control and experimental groups, we calculated the improvement in TEWL as a percentage based on the values before application. In the control group, TEWL was increased by 0.20 and 2.93% after 2 and 4 weeks of use, respectively. These changes were not statistically significant (P>0.05), indicating that the control cream had no measurable effect on TEWL. Conversely, the use of the apigenin-containing cream significantly improved the TEWL by 9.10 and 27.61% after 2 and 4 weeks, respectively (p<0.001). Through these experiments, we identified an improvement in TEWL as an outcome of using the apigenin-containing cream.

The thickness of the stratum corneum changes depending on its moisture content, and insufficient moisture in this layer gradually roughens skin texture (35). To investigate the effects of the apigenin-containing cream on skin texture, facial skin evenness was measured using a PRIMOS Lite system. Evenness in the control group was 19.71 Ra before use, and 19.43 and 19.30 Ra after 2 and 4 weeks of application, respectively (Fig. 10). By contrast, skin evenness in the experimental group was 20.05 Ra before use, and 18.41 and 16.11 Ra after 2 and 4 weeks of application, respectively (Fig. 10). To compare the results from the control and experimental groups, we calculated the improvement as a percentage based on the value before application. Consequently, we found that skin texture in the control group improved by 1.42 and 2.06%, after 2 and 4 weeks of use, respectively. These data were not statistically significant (P>0.05), indicating that the control cream had no measurable effect on skin texture. However, the use of the apigenin-containing cream significantly improved the evenness of skin texture by 8.20 and 19.65% after 2 and 4 weeks of application (P<0.001), respectively, suggesting that the use of the apigenin-containing cream can improve skin texture.

In this study, investigators questioned the subjects individually about the condition of their skin and performed a visual evaluation of skin reactions, including erythema, itching, scaling, tingling, tightness, prickling and burning sensations at each visit. No extraordinary reactions were reported based on either visual evaluation or the questionnaire (Table I).