Chemical Composition of a Novel Distillate from Fermented Mixture of Nine Anti-Inflammatory Herbs and Its UVB-Protective Efficacy in Mouse Dorsal Skin via Attenuating Collagen Disruption and Inflammation

Abstract

Since ancient times, various herbs have been used in Asia, including Korea, China, and Japan, for wound healing and antiaging of the skin. In this study, we manufactured and chemically analyzed a novel distillate obtained from a fermented mixture of nine anti-inflammatory herbs (Angelica gigas, Lonicera japonica, Dictamnus dasycarpus Turcz., D. opposita Thunb., Ulmus davidiana var. japonica, Hordeum vulgare var. hexastichon Aschers., Xanthium strumarium L., Cnidium officinale, and Houttuynia cordata Thunb.). The fermentation of natural plants possesses beneficial effects in living systems. These activities are attributed to the chemical conversion of the parent plants to functional constituents which show more potent biological activities. In our current study, the distillate has been manufactured after fermenting the nine oriental medical plants with Lactobacillus fermentum, followed by distilling. We analyzed the chemical ingredients involved in the distillate and evaluated the effects of topical application of the distillate on ultraviolet B (UVB)-induced skin damage in Institute of Cancer Research (ICR) mice. Topical application of the distillate significantly ameliorated the macroscopic and microscopic morphology of the dorsal skin against photodamage induced by UVB radiation. Additionally, our current results showed that topical application of the distillate alleviated collagen disruption and reduced levels of proinflammatory cytokines (tumor necrosis factor alpha and interleukin 1 β expressions) in the dorsal skin against UVB radiation. Taken together, our current findings suggest that the distillate has a potential to be used as a material to develop a photoprotective adjuvant.

Keywords: UVB; distillation; fermentation; natural herb complex; skin protection.

Figure 1

Representative gas chromatography (GC) chromatogram of the distillate. 2, 6, 10-trimethyldodecane (A; C₁₅H₃₂; retention time, 17.65 min; peak area, 3.69%), 2, 6, 11, 15-tetramethylhexadecane (B; C₂₀H₄₂; retention time, 20.58 min; peak area, 2.45%), n-heptadecane (C; C₁₇H₃₆; retention time, 18.25 min; peak area, 2.02%), and n-docosane (D; C₂₂H₄₆; rettention time, 19.31 min; peak area, 1.64%) are identified as important ingredients. a–f: siloxane compounds (impurities derived from the column).

Figure 2

(A) The clinical skin severity score observed in the control, UVB + vehicle, and UVB + distillate groups at 1 day, 3 days, and 5 days after UVB exposure. The clinical skin severity score in the UVB + vehicle group was significantly increased from 1 day after UVB irradiation, however, the score in the UVB + distillate group was significantly decreased from 3 days to 5 days after UVB exposure compared to that in the UVB + vehicle group. The data represent mean ± standard errors of means (n = 10/group, * p < 0.05 vs. control group; ^# p < 0.05 vs. UVB + vehicle group). (B) Photos of the dorsal skin of the control, UVB + vehicle, and UVB + distillate groups at 1 day, 3 days, 5 days, and 7 days after UVB exposure.

Figure 3

H and E staining of mouse dorsal skin in the control (A), UVB + vehicle (B), and UVB + distillate (C) groups at 5 days after UVB exposure. In the UVB + vehicle group, the thickness of the epidermis was significantly increased (arrows) at 5 days after UVB exposure. However, in the UVB + distillate group, the epithermal thickness was significantly reduced. Ep, epidermis; HF, hair follicle. Scale bar = 100 μm. (D) The mean epidermal thickness in the control, UVB + vehicle, and UVB + groups at 5 days after UVB exposure. The data represent mean ± standard errors of means (n = 10/group, * p < 0.05 vs. control group; ^# p < 0.05 vs. UVB+vehicle group).

Figure 4

Collagen I (A–C) and III (D–F) immunohistochemistry in the dorsal skin of the control (A,D), UVB + vehicle (B,E), and UVB + distillate (C,F) groups at 5 days after UVB exposure. In the UVB + vehicle group, collagen I and III immunoreactivity in the dermis was significantly decreased compared with that in the control group. However, collagen I and III immunoreactivity in the UVB + distillate group was markedly increased compared with that in the UVB + vehicle group. Scale bar = 100 μm. (G,H) Relative optical density (ROD) of collagen I (G) and III (H) immunoreactivity in the dorsal skin as percentage of the control group. The data represent mean ± standard errors of means (n = 10/group, * p < 0.05 vs. control group; ^# p < 0.05 vs. UVB + vehicle group).

Figure 5

Tumor necrosis factor alpha (TNF-α) (A–C) and interleukin (IL-1β) (D–F) immunohistochemistry in mouse dorsal skin in the control (A,D), UVB + vehicle (B,E), and UVB + distillate (C,F) groups at 5 days after UVB exposure. TNF-α and IL-1β immunoreactivity in the UVB + vehicle group was significantly increased in the epidermis (asterisk) and hair follicles (arrows). However TNF-α and IL-1β immunoreactivity in the UVB + distillate group was significantly reduced compared to that in the UVB + vehicle group. Scale bar = 100 μm. (G,H) ROD of TNF-α (G) and IL-1β (H) immunoreactivity in the dorsal skin as percentage of the control group. The data represent mean ± standard errors of means (n = 10/group, * p < 0.05 vs. control group; ^# p < 0.05 vs. UVB + vehicle group).