Contributed equally
Apigenin (4′,5,7-trihydroxyflavone) is a flavone that has been reported to have anti-inflammatory, antioxidant and anti-carcinogenic properties. In this study, we investigated the protective effects of apigenin on skin and found that, in experiments using cells, apigenin restored the viability of normal human dermal fibroblasts (nHDFs), which had been decreased by exposure to ultraviolet (UV) radiation in the UVA range. Using a senescence-associated (SA)-β-gal assay, we also demonstrate that apigenin protects against the UVA-induced senescence of nHDFs. Furthermore, we found that apigenin decreased the expression of the collagenase, matrix metalloproteinase (MMP)-1, in UVA-irradiated nHDFs. UVA, which has been previously identified as a photoaging-inducing factor, has been shown to induce MMP-1 expression. The elevated expression of MMP-1 impairs the collagen matrix, leading to the loss of elasticity and skin dryness. Therefore, we examined the clinical efficacy of apigenin on aged skin, using an apigenin-containing cream for clinical application. Specifically, we measured dermal density, skin elasticity and the length of fine wrinkles in subjects treated with apigenin cream or the control cream without apigenin. Additionally, we investigated the effects of the apigenin-containing cream on skin texture, moisture and transepidermal water loss (TEWL). From these experiments, we found that the apigenin-containing cream increased dermal density and elasticity, and reduced fine wrinkle length. It also improved skin evenness, moisture content and TEWL. These results clearly demonstrate the biological effects of apigenin, demonstrating both its cellular and clinical efficacy, and suggest that this compound holds promise as an anti-aging cosmetic ingredient.
Aging is defined as the progressive accumulation of damage over time, leading to the disruption of functions at the cellular, tissue and organ levels. Eventually, disease and death are induced by a complex, multifactorial process, involving genetic, endogenous and environmental factors (
Apigenin (4′,5,7-trihydroxyflavone), a member of the flavone subclass of flavonoids, is widely found in herbs, fruits and vegetables, and thus, is a substantial component of the human diet. It has been shown to possess a variety of biological characteristics, including antioxidant (
Ultraviolet (UV) radiation that reaches the earth's surface is comprised of wavelength ranges referred to as UVB (280
Photoaging is characterized by the macro- and micro-structural deterioration of the skin, which includes damage to collagen fibers, the excessive deposition of abnormal elastic fibers and increased levels of glycosaminoglycans (18
In the present study, we examined the protective effects of apigenin on skin aging and demonstrated that apigenin induces anti-aging effects in skin by improving its barrier function and reducing UVA-induced damage.
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
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 (
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
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
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 (
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
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
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
UVA radiation corresponds to 90
To evaluate the effects of apigenin on skin aging
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 (
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 (
Keratinocyte moisture content is pivotal for maintaining moisture in the skin. Normal keratinocytes maintain 10
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 (
The thickness of the stratum corneum changes depending on its moisture content, and insufficient moisture in this layer gradually roughens 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 (
Apigenin has been reported to have various biological activities in various cell types, such as antioxidant, anti-inflammatory, anti-mutagenic and anti-tumorigenic properties (
We first evaluated the viability of nHDFs that were irradiated with 25 J/cm2 UVA and found that the post-treatment of UVA-irradiated nHDFs with apigenin significantly reduced cell cytotoxicity. This suggests that apigenin can reduce and/or mitigate UVA-induced cellular damage. We then evaluated the effects of apigenin on cellular senescence using a SA-β-galactosidase assay and found that while the percentage of senescent nHDFs increased in response to UVA irradiation, treatment with apigenin reduced the percentage of senescent cells in a dose-dependent manner. Furthermore, under the same conditions, the mRNA expression of MMP-1 collage-nase-1, which is a reported initiator for the degradation of type I and III fibrillary collagen and is induced in response to UVA irradiation, was reduced by apigenin in a dose-dependent manner (
UVA, a component of the UV spectrum, has been reported to have a greater average skin penetration than other UV types. Based on previous studies (
Based on the results of our cellular experiments demonstrating an apigenin-mediated protection from UVA-induced toxicity and an inhibition of MMP-1 upregulation in nHDFs, we constructed an apigenin-containing cream and a non-apigenin-containing control and enrolled 40 women (>30 years old) in a randomized and double-blinded clinical trial to examine the effects of apigenin on aging skin
The skin is an important organ that separates the human body from the external environment. It has been previously reported that both the barrier function and the water-holding capacity of human skin are decreased by solar UV exposure (
This study was supported by the KU Research Professor Program (H.-J. Cha) of Konkuk University. Support was also provided by grants from the Ministry of Science, ICT and Future Planning (grant no. 20110028646), the Korean Health Technology R&D Project, the Ministry of Health & Welfare (grant no. HN13C0075), and the Ministry of Oceans and Fisheries, Republic of Korea (grant no. OF123321).
Cytotoxicity of apigenin in normal human dermal fibroblasts (nHDFs). nHDFs were treated with apigenin at the indicated concentrations for 24 h. The results are representative of 3 independent experiments (means ± SD are shown). *P<0.05 and ***P<0.001, as determined by the Student's t-test.
Effect of apigenin on the viability of UVA-irradiated normal human dermal fibroblasts (nHDFs). nHDFs were irradiated with 25 J/cm2 UVA and then post-treated with apigenin for 24 h. The results are representative of 3 independent experiments (means ± SD are shown). **P<0.01, as determined by the Student's t-test.
Inhibitory effect of apigenin on cellular senescence in UVA-irradiated normal human dermal fibroblasts (nHDFs). nHDFs were irradiated with 25 J/cm2 UVA and then post-treated with apigenin for 24 h. Senescent nHDFs were detected using an optical microscope. The results are representative of 3 independent experiments (means ± SD are shown). **P<0.01 and **P<0.001, as determined by the Student's t-test.
Effect of apigenin on matrix metalloproteinase (MMP-1) mRNA expression in normal human dermal fibroblasts (nHDFs). Relative mRNA expression levels of MMP-1 in UVA- and apigenin-treated HDFs, as measured by quantitative PCR. The results are representative of 3 independent experiments (means ± SD are shown). **P<0.01 and ***P<0.001, as determined by the Student's t-test.
Dermal density of subjects treated with apigenin-containing cream or the control cream. The measurements were performed 3 times, namely, before application and after 2 and 4 weeks of use, using a DUB-Skin scanner, 3 cm beside the left eye, applying the couplant for ultrasonic examination. The unit of measurement is micro Meter (
Length of crow's feet in subjects treated with the apigenin-containing cream or the control cream. The measurements were performed 3 times, namely, before application and after 2 and 4 weeks of use, using a Robo skin analyzer CS50, on the front, left, and right side of face. ***P<0.001 as determined by the Student's t-test.
Skin elasticity in subjects treated with the apigenin-containing cream or the control cream. Skin elasticity measurements were performed 3 times, namely, before application and after 2 and 4 weeks of use, using a DermaLab USB elasticity probe. The detection was on the left cheek of subject, and the data were analyzed using application software, version 1.09. ***P<0.001 as determined by the Student's t-test.
Skin moisture content in subjects treated with the apigenin-containing cream or the control cream. Measurements were taken 3 times, namely, before application and after 2 and 4 weeks of use. A DermaLab USB moisture probe was applied, and the data were analyzed using the associated software application version 1.09. ***P<0.001 as determined by the Student's t-test.
Transepidermal water loss (TEWL) in subjects treated with the apigenin-containing cream or control cream. Measurements were performed 3 times, namely, before application and after 2 and 4 weeks of use, using a DermaLab USB TEWL probe, and the data were analyzed using the associated application software, version 1.09. ***P<0.001 as determined by the the Student's t-test.
Skin evenness in subjects treated with the apigenin-containing cream or the control cream. Evenness of the skin surface was measured using the PRIMOS Lite system. Measurements were taken 3 times, namely, before application and after 2 and 4 weeks of use. The captured images were analyzed using the associated imaging software PRIMOS Lite version 5.6E. ***P<0.001 as determined by the Student's t-test.
Adverse skin reactions reported by the subjects.
Abnormal reaction | Severity | Abnormal reaction | Severity |
---|---|---|---|
Erythema | 0 |
Tingling | 0 |
Swelling (edema) | 0 | Burning | 0 |
Scaling (epidermis) | 0 | Tightness | 0 |
Itching | 0 | Prickling | 0 |
0, none; 1, mild; 2, severe; 3, very severe.