Improvement in Yield of Extracellular Vesicles Derived from Edelweiss Callus Treated with LED Light and Enhancement of Skin Anti-Aging Indicators

Abstract

The process of skin aging is currently recognized as a disease, and extracellular vesicles (EVs) are being used to care for it. While various EVs are present in the market, there is a growing need for research on improving skin conditions through microbial and plant-derived EVs. Edelweiss is a medicinal plant and is currently an endangered species. Callus culture is a method used to protect rare medicinal plants, and recently, research on EVs using callus culture has been underway. In this study, the researchers used LED light to increase the productivity of Edelweiss EVs and confirmed that productivity was enhanced by LED exposure. Additionally, improvements in skin anti-aging indicators were observed. Notably, M-LED significantly elevated callus fresh and dry weight, with a DW/FW ratio of 4.11%, indicating enhanced proliferation. Furthermore, M-LED boosted secondary metabolite production, including a 20% increase in total flavonoids and phenolics. The study explores the influence of M-LED on EV production, revealing a 2.6-fold increase in concentration compared to darkness. This effect is consistent across different plant species (Centella asiatica, Panax ginseng), demonstrating the universality of the phenomenon. M-LED-treated EVs exhibit a concentration-dependent inhibition of reactive oxygen species (ROS) production, surpassing dark-cultured EVs. Extracellular melanin content analysis reveals M-LED-cultured EVs' efficacy in reducing melanin production. Additionally, the expression of key skin proteins (FLG, AQP3, COL1) is significantly higher in fibroblasts treated with M-LED-cultured EVs. These results are expected to provide valuable insights into research on improving the productivity of plant-derived EVs and enhancing skin treatment using plant-derived EVs.

Keywords: LED; Leontopodium alpinum L.; extracellular vesicles; magenta; plant-derived EVs.

Figure 1

Flowchart for the Edelweiss callus induction process and illumination conditions. (A) Sourcing Edelweiss flowers and seeds. (B) Cultivating Edelweiss plants in vitro from sterilized seeds. (C) Preparing explants from Edelweiss leaves for callus induction. (D) Initiating callus formation from leaf explants. (E) Promoting callus induction and ensuring the uniformity of callus through subculturing. (F) Achieving homogeneous Edelweiss callus. (G) Administering LED light treatment to the uniform callus cultures.

Figure 2

Following a 4-week cultivation period, we scrutinized the structure of Edelweiss callus cultures under four distinct lighting conditions. Specifically, selected Edelweiss callus lines were exposed to the following lighting environments: R-Edel-Callus (R)—red LED treatment (24 h, 635 nm), B-Edel-Callus (B)—blue LED treatment (24 h, 448 nm), M-Edel-Callus (M)—magenta LED treatment (24 h, comprising a 50% blend of red and blue), and D-Edel-Callus (D)—the control condition maintained in darkness for 24 h.

Figure 3

Analysis of non-volatile secondary metabolites in Edelweiss callus extracts cultured under varying LED light conditions. The Edelweiss callus extracts from different growth conditions were assessed for their total flavonoid content (TFC) and total phenolic content (TPC).

Figure 4

The protein profile of Edelweiss callus cultivated under diverse light sources was examined using SDS/PAGE gel. The soluble total protein from the Edelweiss callus was loaded and separated accordingly. D: dark condition, R: red light condition, B: blue light condition, M: magenta light condition.

Figure 5

NTA analysis comparing the size (A) and image (B) of EVs derived from D-Edel-Callus (D-Edel-Callus-EV), as well as the size (C) and image (D) of EVs derived from M-Edel-Callus (M-Edel-Callus-EV). D-Edel-Callus-EV exhibits a particle size of 141.3 nm with a concentration of 1.3 × 10¹¹ particles/mL, while M-Edel-Callus-EV, slightly smaller at 139.2 nm, demonstrates a higher concentration of 3.4 × 10¹¹ particles/mL. This indicates a 2.3-fold increase in concentration when exposed to magenta light.

Figure 6

Transmission electron microscopy (TEM) image of EVs derived from Edelweiss (M-Edel-Callus-EV), Centella asiatica (M-Cica-Callus-EV), and Panax ginseng (M-Ginseng-Callus-EV) under magenta light conditions.

Figure 7

Intracellular antioxidant activity was assessed using EVs derived from Edelweiss callus, specifically designated as D-Edel-Callus-EV and M-Edel-Callus-EV. These EVs were subjected to varying concentrations in the analysis. A 2 μM Trolox solution served as the positive control. Statistical significance concerning the UV-treated control (Con) was determined (* p < 0.05 and ** p < 0.01).

Figure 8

The assessment of extracellular melanin production in the mouse-derived melanoma cell line (B16F10) involved treating the cells with EVs derived from Edelweiss callus, specifically designated as D-Edel-Callus-EV and M-Edel-Callus-EV. Varying concentrations of these EVs were employed in the evaluation. Following a 24 h culture period, the cells were stimulated with α-MSH to induce melanin production. Significance in comparison to the control (Con) was determined (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 9

Western blot analysis images depicting the protein expression of Filaggrin, AQP3, and Collagen I are presented. Detroit cells (fibroblasts) underwent treatment with 5 × 10⁸ Particles/mL of D-Edel-Callus-EV and M-Edel-Callus-EV for 24 h. Notably, the particle numbers of EVs treated with magenta LED (M-Edel-Callus-EV) and those under dark conditions (D-Edel-Callus-EV) were identical. The proteins examined in this analysis encompass Filaggrin (filament-aggregating protein), AQP3 (aquaporin 3), and Col1 (collagen I), with GADPH (glyceraldehyde 3-phosphate dehydrogenase) employed as a control.