Contributed equally
Rutin, a quercetin glycoside is a member of the bioflavonoid family which is known to possess antioxidant properties. In the present study, we aimed to confirm the anti-aging effects of rutin on human dermal fibroblasts (HDFs) and human skin. We examined the effects of rutin using a cell viability assay, senescence-associated-β-galactosidase assay, reverse transcription-quantitative polymerase chain reaction, and by measuring reactive oxygen species (ROS) scavenging activity
The clinical manifestations of aging are fine wrinkles, thin and transparent skin, loss of underlying fat leading to hollowed cheeks and eye sockets, dry and itchy skin, lack of sufficient perspiration, hair graying, hair loss or hirsutism, and thinning of the nail plates (
ROS play an important role in skin aging. In the skin, about 1.5–5% of the consumed oxygen is converted into ROS by intrinsic processes (
Rutin, a quercetin glycoside (
The present study confirms that rutin reduces skin aging by strengthening dermal density and elasticity through the regulation of enzymes in the extracellular matrix (ECM).
Human dermal fibroblasts, (HDFs; Lonza, Basel, Switzerland) were cultured in Dulbecco's modified Eagle medium (Gibco/Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (Gibco/Life Technologies) at 37°C in an atmosphere of 5% CO2. Rutin was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide.
HDFs were seeded at a density of 3×103 cells/well in a 96-well plate and incubated for 24 h. Rutin (0–200
The expression of lysomal galactosidase as a marker for senescent HDFs was determined using the senescence-associated (SA)-β-galactosidase staining kit (BioVision, Milpitas, CA, USA) according to the manufacturer's instructions. HDFs were seeded at a density of 2×105 cells/well in a 60-mm cell culture plate and incubated until they reached 90% confluence. Subsequently, the cells were treated with rutin and H2O2 for 24 h, the medium was removed, and the cells were washed once with phosphate-buffered saline (PBS). A fixing solution (0.5 ml; 4% formaldehyde and 0.5% glutaraldehyde in PBS buffer, pH 7.2) was added to the culture plate for 1 h in order to fix the cells, and 0.5 ml of a staining solution [staining solution 470
Intracellular ROS formation was measured by adding 2′,7′-dichlorofluorescein diacetate (DCF-DA; Sigma-Aldrich). DCF-DA is non-fluorescent until it is hydrolyzed by intracellular esterases and oxidized into the highly fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of ROS. The cells were seeded in 60-mm cell culture plates at a density of 2×105 cells/well and incubated for 24 h. Following incubation, the cells were pre-treated with rutin (0–50
Total RNA was isolated using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. The purity and concentration of the RNA were evaluated using MaestroNano®, a microspectrophotometer (Maestrogen, Las Vegas, NV, USA). All cDNAs for sensitive and specific miRNA detection were synthesized using the miScript II RT kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The following primers were used: collagen, type I, alpha 1 (
The study protocols were approved by the Institutional Review Board of GeneCellPharm Incorporated (Cheongju-si, Korea). All subjects were informed of the objective of the study and provided informed consent and agreed to use skin care products during the study. Forty women, aged 30–50 years, were selected for a randomized and double-blind clinical trial (control group, 45.50±5.79 years; experimental group, 45.70±5.34 years). The selection of subjects was based on age, signs of skin aging, and being neither pregnant nor nursing. Subjects who presented with symptoms of itching or erythema, or those who hindered the evaluation process due to excessive drinking or smoking were excluded from the experiment. The subjects were divided into control and experimental groups, each containing 20 subjects. All conditions were the same except for the test material used on the experimental group. The study was conducted for 4 weeks. Biometric parameters were measured three times: prior to application, and 2 and 4 weeks after application. During this study, each subject performed a self-evaluation, and using a scale of severity (0, none; 1, mild; 2, severe; 3, very severe), completed a questionnaire to indicate to what extent they exhibited any skin disorders, such as erythema, itching, scaling, edema and tingling and burning sensations, at each visit. The subjects who presented with skin disorders withdrew from the clinical evaluation process.
In order to determine whether the rutin-containing cream had any adverse effects, the subjects were asked individually about the condition of their skin, and a visual evaluation of skin reactions, such as erythema, itching, scaling, tingling, tightness and prickling or burning sensations was performed at each visit. No adverse effects were reported based on either a visual evaluation or the questionnaire (
The cream was prepared by incorporating the ingredients in the 3 phases (A, B, C). The ingredients in the A phase (distilled water, glycerin, 1,3-butylene glycol) were combined and heated until all the components were melted, and the ingredients in the B phase (distilled water, dipotassium phosphate, sodium hydroxylate, rutin) were combined and heated to the same temperature, to ensure homogeneity. The A and B phase ingredients were combined and emulsified using a homo mixer (Tokushu Kika Kogyo Co., Ltd., Japan) at 5,000 rpm for 10 min. The mixture was cooled to 60°C and blended with the homogenized phase C (emulium delta, sepipuls 400) at 5,000 rpm for 10 min. By then, the temperature of the mixture had dropped to 45°C. The mixture was combined and homogenized, while maintaining the pH at 6.2. The cream provided to the experimental group contained 2% (wt %) rutin and the cream provided to the control group was prepared using the same volume of water in place of rutin.
To evaluate the improvement in skin elasticity, a DermaLab USB elasticity probe (Cortex Technology ApS, Hadsund, Denmark) was applied and the results were analyzed using the associated application software (version 1.09). Measurements were obtained using a fixed elasticity probe on the left cheek of each subject. To analyze the elasticity measurements, Young's modulus (E) was calculated as a dose-dependent representation of skin elasticity. The measurements of elasticity were taken three times: prior to application, and 2 and 4 weeks after application. The measurement unit is MPa.
To evaluate dermal density, a DUB SkinScanner (tpm taberna pro medicum GmbH, Lüeneburg, Germany) was used. Dermal density was measured 3 cm from the left eye, applying a couplant for ultrasonic examination. The analysis range was limited to the area between the dermis and upper panniculus. The measurements were taken three times: prior to application, and 2 and 4 weeks after application.
To evaluate the extent of improvement in wrinkles, particularly at the eye rim, a Robo skin analyzer CS50 (Inforward Inc., Tokyo, Japan) was used. The facial images were captured from each subject placed in identical positions with equal lighting: on the front, left and right sides of the face. To evaluate improvement, measurements were taken three times: prior to application, and 2 and 4 weeks after application. We analyzed the captured images, matching the facial feature points accurately. The measurement unit of crow's feet length is mm and area is mm2.
In cellular efficacy tests, all results are presented as the mean percentages ± standard deviation (SD) of three independent experiments. A P-value <0.05 as determined by the Student's t-test, was considered statistically significant. In clinical efficacy tests, statistical analyses were conducted using SPSS software (version 17.0 for Windows; IBM, Armonk, NY, USA). Paired t-tests were performed in cases of repeated measurements on the same subject. To analyze subject questionnaires, mean values, SDs, and percentages were used. 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 rutin affects the viability of HDFs, cultured HDFs were exposed to concentrations of rutin ranging from 0–200
Next, we examined the inhibition of senescence by rutin using the SA-β-galactosidase assay. The percentage of senescent cells was 59.80% in the cells exposed to H2O2 only. This ratio decreased in a dose-dependent manner to 49.28, 35.30 and 19.26% with the administration of 1, 10 and 50
To determine cellular ROS levels, we performed the DCF-DA assay in H2O2- exposed HDFs. The exposure of HDFs to 0.2 mM H2O2 increased ROS levels >three times that of the control cells. The cells exposed to 0.2 mM H2O2 and treated with rutin exhibited decreased ROS levels in a dose-dependent manner to a relative intensity of 2.6-, 1.9- and 1.6-fold following treatment with 1, 10 and 50
ROS are known to be inducers of MMPs, which degrade intracellular substances (
As aging occurs, the expression of MMPs is increased which results in the degradation of skin substrate proteins (
Wrinkles, a representative aging phenomenon, occur in response to structural alterations of cells and tissues. Wrinkles arising due to intrinsic aging are formed by decreases in collagen and elasticity, denaturalization of elastic fibers and the stratum corneum, and a loss of skin moisture (
The length of crow's feet were 63.50 mm prior to application, 64.25 mm after 2 weeks, and 64.95 mm after 4 weeks in the control group (
In the control group, the area affected by crow's feet was 70.95 mm2 prior to application, and 69.65 mm2 after 2 weeks and 69.75 mm2 after 4 weeks of use (
The use of rutin-containing cream significantly reduced the areas affected by crow's feet over time. In the control group, the improvement rates were −1.83% after 2 weeks and 1.69% after 4 weeks. The improvement rates in the experimental group were 14.66% after 2 weeks and 27.19% after 4 weeks. From these results, we again confirmed the effects of rutin on wrinkle improvement.
In the control group, the average number of under-eye wrinkles was 4.15 prior to application, 4.20 after 2 weeks, and 4.30 after 4 weeks (
We then assessed the improvement in under-eye wrinkles over time. In the control group, improvements were −1.20% after 2 weeks and −3.61% after 4 weeks application. Improvement rates in the experimental group were 31.96% after 2 weeks and 49.48% after 4 weeks. From these results, we confirmed the effects of rutin on wrinkle improvement.
The dermis is composed of ECM that contains fibrous proteins such as collagen and elastin. Dermal fibroblasts regulate skin elasticity (
The present study examined the antioxidant and anti-aging effects as well as the beneficial effects of rutin on skin barrier function through cellular and clinical efficacy experiments. The results of cellular experiments on HDFs indicated the antioxidant and anti-aging effects of rutin. In HDFs, rutin protects cells against damage induced by H2O2. Furthermore, we identified a ROS scavenging effect of rutin by DCF-DA assay and reduced H2O2-induced cellular senescence. After analyzing the expression of
We first manufactured rutin-containing cream. The clinical efficacy tests were conducted with 40 subjects, women between 30–50 years of age, selected for a randomized and double-blind study. The subjects were divided into two groups, experimental and control, and 1% rutin-containing or basic cream were applied, respectively. Dermal density improved 10.73% after 2 weeks and 20.16% after 4 weeks of application of the rutin-containing cream in the experimental group. We also showed that rutin-containing cream improved skin elasticity 25.34% after 2 weeks and 40.50% after 4 weeks of application. Reduced skin elasticity contributes to damaged skin structure and aging, particularly wrinkle formation (
The length of crow's feet improved by 13.45% after 2 weeks and 23.90% after 4 weeks. Additionally, the area of crow's feet improved by 14.66% after 2 weeks and 27.19% after 4 weeks. After analyzing the average number of under-eye wrinkles, we demonstrated the effects of rutin on facial wrinkles. The number of under-eye wrinkles improved by 31.96% after 2 weeks and 49.48% after 4 weeks. These results indicate that rutin improves skin dermal density, reduces fine winkles, and enhances elasticity. We suggest that rutin may be used as a major ingredient in anti-aging cosmetics in order to improve skin elasticity and reduce wrinkles.
The present study was supported by the KU Research Professor Program (H.-J.C.) of Konkuk University. This study was also supported by grants from the Ministry of Science, ICT and Future Planning (no. 20110028646), the Korean Health Technology R&D Project, Ministry of Health and Welfare (grant no. HN13C0075), and the Ministry of Oceans and Fisheries (no. OF123321) of Republic of Korea.
Effects of rutin on cell viability in rutin- and H2O2-treated human dermal fibroblasts (HDFs). (A) Measurement of cell cytotoxicity of rutin in HDFs. (B) Inhibitory effects of rutin on H2O2-induced cellular damage response. *P<0.05, **P<0.01 and ***P<0.001 as determined by the Student's t-test.
Inhibitory effects of rutin on H2O2-induced cellular senescence of human dermal fibroblasts (HDFs). **P<0.01 and ***P<0.001 as determined by the Student's t-test. SA-β-gal, SA-β-galactosidase.
Reactive oxygen species (ROS) scavenging effects of rutin on H2O2-treated human dermal fibroblasts (HDFs). **P<0.01 as determined by the Student's t-test.
Effects of rutin on the mRNA expression of collagen, type I, alpha 1 (
Dermal density of skin to which rutin-containing or control cream was applied. **P<0.05 and ***P<0.001 versus the control group at the same time point as determined by the Student's t-test.
Beneficial effects of rutin-containing cream on eye wrinkles. The measurements of the (A) length and (B) area of crow's feet. (C) Measurements of wrinkle number under the eye rim. **P<0.05 and ***P<0.001 versus the control group at the same time point as determined by the Student's t-test.
Effects on Young's modulus of skin elasticity of rutin-containing cream applied over time. ***P<0.001 versus the control group at the same time point as determined by the Student's t-test.
Abnormal skin reactions reported by 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 |
Scale of severity, 0, none; 1, mild; 2, severe; 3, very severe.