Biological Activity and Component Analyses of Chamaecyparis obtusa Leaf Extract: Evaluation of Antiwrinkle and Cell Protection Effects in UVA-Irradiated Cells

Abstract

Background and Objectives: Chamaecyparis obtusa (C. obtuse) extract has been used as a folk medicinal remedy in East Asian countries to alleviate inflammation and prevent allergies. Active oxygen causes skin aging and leads to skin cell and tissue damage. Extensive research has been conducted to control active oxygen generation to prevent skin aging. We evaluated the antioxidant activity and antiwrinkle effect of C. obtusa extract to determine its potential as a cosmetic material. Materials and Methods: The antioxidant activity of a 70% ethanol extract of C. obtusa (COE 70) and a water extract of C. obtusa (COW) was determined using 2,2-diphenyl-1-picrylhydrazy (DPPH) scavenging, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) scavenging, superoxide dismutase-like activity, xanthine oxidase inhibition, and ferric-reducing antioxidant power assays. The effective concentration of the extracts was determined using the methyl thiazolyl tetrazolium assay to evaluate their toxicity. The effects of COE 70 on the production of matrix metalloproteinases (MMPs) and procollagen, and expression of activated cytokines, interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), in UVA-irradiated fibroblasts were determined using quantitative real-time PCR. Additionally, quercitrin, amentoflavone, hinokiflavone, and myricetin concentrations in COE 70 were determined using high-pressure high-performance liquid chromatography. Results: COE 70 had higher polyphenol and flavonoid concentrations than COW and exhibited an excellent antioxidant effect. COE 70 suppressed UVA-induced fibroblast death by 21.3% at 25 µg/mL. It also increased MMP-1, MMP-3, TNF-α, and IL-6 mRNA levels at 5-25 µg/mL compared with those in control UVA-irradiated fibroblasts. Moreover, mRNA levels of collagen type I and superoxide dismutase significantly increased, indicating the antiwrinkle and anti-inflammatory effects of the extract. Among the COE 70 components, quercitrin concentration was the highest; hence, quercitrin could be an active ingredient. Conclusions: COE 70 could be used as a natural antioxidant and antiwrinkle agent.

Keywords: Chamaecyparis obtusa; antioxidant; antiwrinkle; fibroblast; flavonoid; quercitrin.

Figure 1

Antioxidant effects of C. obtusa leaf extract. Antioxidant activity was evaluated by (a) electron-donating ability, (b) ABTS⁺ radical scavenging assay, (c) SOD-like activity, (d) xanthine oxidase assay, and (e) FRAP. AA: ascorbic acid. The values are expressed as mean ± SD (n = 3). All experiments were independently conducted three times. Values with different characters (a–g) are significantly different from each other, as determined by ANOVA and Duncan’s multiple range test (p < 0.05).

Figure 2

Viability of CCD-986sk fibroblasts treated with C. obtusa leaf extract. (a) The cells were cultured in 96-well plates and treated with the final extract at concentrations of 0, 12.5, 25, 50, and 100 µg/mL for 24 h. Survival rates of cells treated with the extracts were calculated using the MTT assay. (b) The cells were irradiated with UVA (10 mJ/cm²) in the absence or presence of C. obtusa extract in the medium. All experiments were independently conducted three times. Statistical analysis was performed using ANOVA and the results were compared with those of the experimental controls with Bonferroni’s test; * p < 0.05 compared to the control (a) and UVA (10 mJ/cm²)-irradiated (b) groups.

Figure 3

Anti-aging effect of C. obtusa leaf extract. Antioxidant activity was measured by (a) elastase inhibition activity and (b) collagenase inhibition activity. The mRNA level of (c) MMP-1, (d) MMP-3, (e) TNF-α, (f) IL-6, (g) collagen type I, and (h) SOD 1 in UVA-irradiated fibroblasts using qRT-PCR. Each value is expressed as the mean ± SD (n = 3). All experiments were independently conducted three times. Values with different characters (a–h) are significantly different from each other, as determined using ANOVA and Duncan’s multiple range test (p < 0.05). Nor: non-UVA-irradiated cells; Con: UVA-irradiated cells without C. obtusa treatment (c–h). Statistical analysis was performed using ANOVA and the results were compared with those of the experimental controls with Bonferroni’s test; * p < 0.05 and ** p < 0.01 compared to the control (a) and UVA (10 mJ/cm²)-irradiated (b) groups.

Figure 4

High-performance liquid chromatography. Chromatogram of the ethanol extract of (a) C. obtusa and (b) the quercitrin, myricetin, amentoflavone, and hinokiflavone standard compounds.