Skin-Whitening, Antiwrinkle, and Moisturizing Effects of Astilboides tabularis (Hemsl.) Engl. Root Extracts in Cell-Based Assays and Three-Dimensional Artificial Skin Models

Abstract

This study investigated the potential cosmetic properties of the ethyl acetate (EtOAc) fraction obtained from the roots of Astilboides tabularis (Hemsl.) Engl., focusing on skin-whitening, antiwrinkle, and moisturizing effects using cell-based assays and three-dimensional (3D) artificial skin models (Neoderm-ED and Neoderm-ME). The EtOAc fraction showed significant dose-dependent inhibitory activity against tyrosinase (TYR) (72.0% inhibition at 50 µg/mL), comparable to that of kojic acid. In α-melanocyte-stimulating hormone (α-MSH)-stimulated Neoderm-ME artificial skin containing melanocytes, the EtOAc fraction reduced melanin synthesis at concentrations of 50 and 75 µg/mL and decreased melanogenesis-related gene expression, including TYR, microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1) and TRP-2. In the antiwrinkle assays, the EtOAc fraction effectively inhibited elastase activity (41.5% inhibition at 10 µg/mL), exceeding the efficacy of ursolic acid. In the Neoderm-ED artificial skin model, the EtOAc fraction reversed structural damage induced by particulate matter (PM10), restoring epidermal thickness and dermal density. This improvement was supported by the increased expression of skin barrier and antiwrinkle genes, including filaggrin, hyaluronic acid synthase-1 (HAS-1), HAS-2, aquaporin-3 (AQP-3), collagen type I alpha 1 chain (COL1A1), elastin, tissue inhibitor of metalloproteinases-1 (TIMP-1), and TIMP-2, as well as decreased expression of matrix metalloproteinases (MMP-1, MMP-3, and MMP-9). Our results indicate that the EtOAc fraction from A. tabularis root has considerable potential as a multifunctional cosmetic.

Keywords: Astilboides tabularis (Hemsl.) Engl.; antiwrinkle; ethyl acetate; moisturizing; skin whitening.

Figure 1

Tyrosinase inhibition assay of the EtOAc fraction from A. tabularis root extract. Treatment groups include the EtOAc fraction at concentrations of 10, 50, and 100 µg/mL and kojic acid (KA) as a positive control. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using a two-tailed unpaired Student’s t-test. *** p < 0.001, compared with the untreated control.

Figure 2

Elastase inhibition assay of the EtOAc fraction from A. tabularis root extract. Treatment groups include the EtOAc fraction at concentrations of 10, 50, and 100 µg/mL and ursolic acid (UA) as a positive control. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using a two-tailed unpaired Student’s t-test. *** p < 0.001, compared with the untreated control.

Figure 3

Inhibitory effect of the EtOAc fraction from A. tabularis root extract on cytotoxicity in three cell lines: (A) B16F10 (murine metastatic melanoma), (B) HaCaT (human immortalized keratinocyte), and (C) Detroit 551 (human skin fibroblasts). The treatment groups include the EtOAc fraction at concentrations of 10, 25, 50, 75, 100, and 200 µg/mL. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using a two-tailed unpaired Student’s t-test. ** p < 0.01, *** p < 0.001, compared with the untreated control.

Figure 4

Effect of the EtOAc fraction from A. tabularis root extract on melanin content in B16F10 cells. Treatment groups include the EtOAc fraction at concentrations of 10, 25, 50, and 75 µg/mL. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using a two-tailed unpaired Student’s t-test. ** p < 0.01, *** p < 0.001, compared with the untreated control.

Figure 5

mRNA expression of EtOAc fraction from A. tabularis root extract in α-MSH-stimulated B16F10 cells. (A) MITF, (B) TYR, (C) TRP-1, and (D) TRP-2. Each data point is presented as the mean ± standard deviation of three replicate experiments.

Figure 6

Correlation analysis in mRNA expression of the EtOAc fraction from A. tabularis root extract in α-MSH-stimulated B16F10 cells. The correlation between the respective genes was analyzed. Superscripts mean significant differences between the experimental groups, as determined by an independent sample t-test (* p < 0.05).

Figure 7

mRNA expression of EtOAc fraction from A. tabularis root extract in fine dust-stimulated HaCaT cells. (A) Filaggrin, (B) AQP-3, (C) HAS-1, and (D) HAS-2. Each data point is presented as the mean ± standard deviation of three replicate experiments.

Figure 8

Correlation analysis in mRNA expression of EtOAc fraction from A. tabularis root extract in fine dust-stimulated HaCaT cells. The correlation between the respective genes was analyzed. Superscripts mean significant differences between the experimental groups, as determined by an independent sample t-test (* p < 0.05, ** p < 0.01).

Figure 9

mRNA expression of EtOAc fraction from A. tabularis root extract in fine dust-stimulated Detroit 551 cells. (A) COL1A1, (B) elastin, (C) TIMP-1, (D) TIMP-2, (E) MMP-1, (F) MMP-3, and (G) MMP-9. Each data is presented as the means ± standard deviation of three replicate experiment.

Figure 10

Correlation analysis of mRNA expression of the EtOAc fraction from A. tabularis root extract in fine dust-stimulated Detroit 551 cells. The correlation was analyzed between the respective genes. Superscripts mean significant difference between the experimental groups, as determined by an independent sample t-test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 11

Inhibitory effect of the EtOAc fraction from A. tabularis root extract on cytotoxicity in artificial skin tissues: Neoderm-ED ((A): epidermis + dermis) and Neoderm-ME ((B): epidermis + melanocytes). Treatment groups include the EtOAc fraction at concentrations of 10, 25, 50, and 75 µg/mL. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using a two-tailed unpaired Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the untreated control.

Figure 12

Histological analysis of the EtOAc fraction from A. tabularis root extract in fine dust-stimulated 3D artificial skin tissue (Neoderm-ED). Hematoxylin and eosin (H&E) staining images were acquired at 1000× magnification. (A) Untreated control (negative control), (B) fine dust-stimulated only (positive control), (C) fine dust + EtOAc 10 µg/mL, (D) fine dust + EtOAc 25 µg/mL, (E) fine dust + EtOAc 50 µg/mL, (F) fine dust + EtOAc 75 µg/mL. Data represent one of three independent experiments (n = 3). Morphological improvement in tissue structure was evaluated qualitatively.

Figure 13

mRNA expression of the EtOAc fraction from A. tabularis root extract in fine dust-stimulated artificial skin tissue Neoderm-ED. (A) Filaggrin, (B) AQP-3, (C) HAS-1, (D) HAS-2, (E) COL1A1, (F) elastin, (G) TIMP-1, (H) TIMP-2, (I) MMP-1, (J) MMP-3, and (K) MMP-9. Each data point is presented as the mean ± standard deviation of three replicate experiments.

Figure 14

Correlation analysis in mRNA expression of the EtOAc fraction from A. tabularis root extract in fine dust-stimulated artificial skin tissue Neoderm-ED. The correlation between the respective genes was analyzed. Superscripts mean significant differences between the experimental groups, as determined by an independent sample t-test (* p < 0.05, ** p < 0.01).

Figure 15

Effect of the EtOAc fraction from A. tabularis root extract on melanin content in the artificial skin tissue model Neoderm-ME. Treatment groups include the EtOAc fraction at concentrations of 10, 25, 50, and 75 µg/mL. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using two-tailed unpaired Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the untreated control.

Figure 16

mRNA expression of the EtOAc fraction from A. tabularis root extract in α-MSH-stimulated artificial skin tissue Neoderm-ME. (A) MITF, (B) TYR, (C) TRP-1, and (D) TRP-2. Each data point is presented as the mean ± standard deviation of three replicate experiments.

Figure 17

Correlation analysis in mRNA expression of the EtOAc fraction from A. tabularis root extract in fine dust-stimulated artificial skin tissue Neoderm-ME. The correlation between the respective genes was analyzed. Superscripts mean significant difference between the experimental groups, as indicated by an independent sample t-test ** p < 0.01, *** p < 0.001).

Figure 18

Proposed mechanisms of skin-whitening, antiwrinkle, and moisturizing effects of the EtOAc fraction from A. tabularis root extract.