Cistanche Lacerata Mycelium Culture Medium As A Novel Anti-Aging Microbial Material For Cosmeceutical Application Ⅱ
Mar 27, 2023
3. Results and Discussion
3.1. Measurement of Antioxidant Activity
The antioxidant activity of the solutions was evaluated by the DPPH and ABTS assays. Given the fact that most natural antioxidants hold reactive hydrogens, which serve as reductants, both DPPH and ABTS assays are a useful measure of standard antioxidant profile. As shown in Figure 2, CLEPS solution (5 mg/mL) exhibited the ability to scavenge ~81% DPPH (a) and ~85% ABTS (b) free radicals (active oxygen) as equivalent to ascorbic acid (AA, 1 mg/mL).
3.2. Cell Viability Assay
As presented in Figure 3, no toxicity on the viability of NHDF (a) and B16 melanoma cells (b) was observed when CLEPS was tested at a wide range of concentrations (0.05–7 mg/mL). Moreover, it was determined that the CLEPS treatment could induce fibroblast cell proliferation in a dose-dependent manner at 1–7 mg/mL. This finding was in good agreement with that of another study, which reported on the cell proliferation effect of a bioactive polysaccharide extract from Cistanche Tubulosa [21].
3.3. Expression Level of Filaggrin
Filaggrins are adhesives to hold keratin together, gradually flattening the shape of keratinocytes, increasing the strength of the skin barrier, and strengthening the cohesion between cells on the skin surface [22]. As presented in Figure 3c, when HaCaT cells were treated with CLEPS (30–300 µg/ml), the level of filaggrin expression at 300 µg/ml was signifificantly increased by 36% in contrast to the untreated group, confirming CLEPS could enhance or preserve the capability of the skin barrier and moisturize through upregulation of filaggrin. To evaluate effective anti-dry skin function via filaggrin upregulation, plant extracts derived from Artemisia princeps [23], Rhodiola crenulate [24], or a mixture of Actinidia arguta and Perilla frutescens [25] were studied, while a fungal metabolite such as GFF [26] were investigated. In this regard, CLEPS would be a new filaggrin-enhancing ingredient based on a fungal metabolite if further developed.
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3.4. Melanogenesis Inhibition Test of B16 Melanoma Cells
When CLEPS (0.5, 1.5 mg/mL) was added to the α-MSH-induced melanocytes, melanin synthesis was effectively inhibited by 43%, 54%, and 57%, respectively, in a concentration-dependent manner (Figure 3d). It has been confirmed that CLEPS can downregulate melanin synthesis more efficiently than 50 µg/ml of arbutin (41%). As an active compound extracted from the bearberry plant in Arctostaphylos, arbutin is a well-known melanin suppressor in cultured human melanocytes or B16 murine melanocytes [27]. Although it is, therefore, being widely used as a skin-lightening agent in the cosmetic market, many uncertainties remain, i.e., arbutin is a glycosylated hydroquinone that may cause cancer risks [28]. Considering arbutin is a recognized whitening single compound, it is very encouraging that CLEPS not only exhibited a skin-whitening effect by inhibiting the synthesis of melanin but also showed superior whitening function over arbutin, even though it is an unidentified complex compound.
3.5. Anti-Inflflammation Assay of NO, iNOS, COX2, and TNFα
When CLEPS 500 µg/mL was treated, the levels of inflammation-related factors, NO (a), iNOS (b), COX2 (c), and TNFα (d) were signifificantly reduced by ~75%, ~85%, 62%, and 55%, respectively (Figure 4). It was confirmed that CLEPS exerts an anti-inflflammatory effect by regulating the inflflammatory signaling pathway via suppressing the production of NO and the expression of inflammation-related genes. The potent inhibition of NO and TNF-α activity was comparable to those of commercially available edible mushrooms, such as Enoki, Shiitake, and Oyster, as the most potent anti-inflflammatory species among mushrooms (IC50 < 0.1 mg/mL) [29].
3.6. Synthesis of Collagen and Inhibition of Collagenase
Collagen is not only a key component supporting skin structure, but it can also directly control the wound microenvironment, serve as a scaffold for cell adhesion and function, or deliver biologically active ingredients to aid wound healing [1]. When damaged or deficient, the skin can wrinkle, deform, or cause skin disease. To confirm the anti-wrinkle efficacy of the CLEPS, synthesis of the collagen and inhibition of collagenase assays were performed as shown in Figure 5. Compared with the control groups, the groups treated with 10 ng/mL of TGF-β as positive control showed a substantial increase in collagen synthesis by ~52%. The collagen synthesis was increased to 27.4%, 31.9%, and 65.4% in the groups treated with CLEPS at concentrations of 0.5, 1, and 5 mg/mL, accordingly in a concentration-dependent manner. PMA is a major ROS-generating substance and has been routinely used as an endogenous inducer of superoxide production, which can increase MMP activity in dermal fibroblasts, resulting in extracellular matrix (ECM) protein degradation and premature skin aging [30]. In this work, the MMP-1 proteins were substantially increased after being treated with 50 nM of PMA, while it substantially dropped to 93.4% in the presence of CLEPS (5 mg/mL) in a concentration-dependent manner, indicating that CLEPS suppressed the collagenase activity by inhibiting PMA-induced oxidative stress to make NHDF cell death. Thus, it was confirmed that CLEPS could be effective in wrinkle improvement through collagen synthesis and collagenase reduction in skin ECM.
Figure 5. Effect of CLEPS on the synthesis of collagen (a) (p > 0.05 vs. control) and inhibition of collagenase (b) (p > 0.05 vs. no PMA).
3.7. In Vitro Wound Healing Assay
Figure 6. performed from 0 h to 48 h to analyze the effects of CLEPS (0-1 mg/mL) in HaCaT cellsScale bar = 100 um (a). Wound recovery by CLEPS treatment was quantified compared to each zero dose group (* p < 0.05) (b).
Thus, CLEPS may contain bioactive ingredients in related to anti-aging, anti-oxidation, and anti-inflflammatory properties. In the previous initial study, a submerged culture condition for the mycelium biomass and EPS production by C. lacerate were studied [33]. As a result of EPS analyses using HPLC, FT-IR, and NMR, it was found to be composed of polysaccharides, including mannose (83.36%), galactose (12.54%), and glucose (4.10%). Some flavonoids (e.g., dimethyl chalcone) and tremulant sesquiterpenoids (e.g., triphenyl) have been also isolated as potent bioactive components [34,35]. However, further research is needed to clarify these active compounds.
Taken together, our findings indicated that CLEPS as a new skincare solution exerts a potential cosmeceutical effect on human skin cell aging through anti-oxidation, anti-inflammation, anti-breakdown of the skin barrier, and wound healing. Especially, CLEPS not only signifificantly increased collagen and filaggrin synthesis but also markedly inhibited the activity of collagenase, possibly indicating that CLEPS could prevent skin barrier damage or skin wrinkles. Although the exact compounds producing these effects are unknown, CLEPS as a novel natural anti-aging ingredient could be used as innovative functional cosmetics. Furthermore, the CLEPS manufacturing process is not an extraction process that mainly uses chemical organic solvents or conducts animal testing, but a bio-, eco-friendly, green microbial culture process, which can be well fit with the latest beauty trends in the “vegan” cosmetics market.
Author Contributions: Conceptualization, J.-H.K., and Y.S.K.; data curation and analysis, C.A. and J.-H.K.; project administration, J.-H.K., and Y.S.K.; funding acquisition, Y.S.K.; writing—original draft, J.-H.K.; writing—review and editing, J.-H.K., C.A., and S.D.H. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded in part by Korea National Food Cluster (FOODPOLIS) Promotion Agency under the Korean Ministry of Agriculture, Food, and Rural Affairs, Grant name: “Our Food-To the World (Safety and Efficacy Evaluation)”.
Institutional Review Board Statement: Not Applicable.
Informed Consent Statement: Not Applicable.
Conflicts of Interest: There are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Abbreviations
UV Ultraviolet
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