Introduction
The skin, the human body's largest organ, is a crucial barrier against various external stressors, including ultraviolet (UV) radiation, pollutants, and pathogens. Comprising multiple specialized cell types such as keratinocytes, fibroblasts, and melanocytes, the skin maintains its integrity and functionality through a complex interplay of cellular and molecular mechanisms. However, chronic exposure to oxidative stress and inflammatory agents can impair these mechanisms, leading to conditions like inflammation—an age-associated chronic inflammatory state. This review evaluates the recent clinical study on Chaga mushroom extracts (International Journal of Molecular Sciences, Volume 24, Issue 16, 2023), focusing on their efficacy in mitigating oxidative stress and inflammation using experimental models.
Overview of Chaga Mushroom and Inotodiol
Chaga mushroom (
Inonotus obliquus) has been a staple in traditional medicine, particularly in Northern Europe, Russia, and Asia, for its therapeutic benefits. The mushroom is rich in bioactive compounds, particularly lanostane-type triterpenoids like inotodiol, which have been identified as key agents in its medicinal properties. Previous studies have highlighted inotodiol’s potential anti-tumor, anti-inflammatory, anti-allergic, antiviral, anti-aging, and antioxidant properties. Yet, the specific anti-inflammatory and anti-aging mechanisms of inotodiol, particularly under oxidative stress conditions, had not been comprehensively studied until the present research.
Objectives of the Study
The study aimed to investigate two primary objectives:
- Evaluate the impact of inotodiol on pro-inflammatory cytokine production in keratinocytes stimulated with UV or TNF-α.
- Assess the effect of inotodiol on collagen and hyaluronic acid synthesis in HaCaT cells, elucidating its potential role in preventing skin aging.
Methodology
Preparation and Characterization of Inotodiol Concentrate
The study began by preparing an inotodiol-rich concentrate from Chaga mushroom extracts, containing 10% inotodiol. The concentrate was analyzed using Liquid Chromatography-Mass Spectrometry (LC/MS) to determine its triterpene composition. The primary compounds identified included inotodiol, betulin, inonotsuoxide A, and (3β)-3-hydroxylanosta-8,24-dien-21-al.
Cytotoxicity Assessment
To ensure the safety and effectiveness of the inotodiol concentrate, its cytotoxicity was evaluated using the MTT assay on HaCaT cells (human keratinocyte cell line). The study determined that concentrations up to 20 µg/mL for inotodiol concentrate and 5 µg/mL for pure inotodiol were non-toxic, maintaining more than 70% cell viability.
Anti-inflammatory and Anti-aging Evaluations
The anti-inflammatory efficacy of inotodiol was assessed by measuring the mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) in HaCaT cells treated with TNF-α. The anti-inflammatory response of cells exposed to UV radiation was also examined by evaluating the expression levels of IL-6 and TNF-α cytokines. Furthermore, the study investigated the impact of inotodiol on synthesizing collagen (COL1A2) and hyaluronic acid (HAS2/3), crucial components for maintaining skin structure and hydration.
Results
Triterpene Composition of Inotodiol Concentrate
The LC/MS analysis revealed that inotodiol was the concentration's predominant triterpene, accounting for approximately 69.1% of the total peak area. Betulin was the second most abundant (12%), with other compounds comprising less than 10% each. This high concentration of inotodiol underscores its potential significance in the observed biological effects.
Cytotoxicity Evaluation
The MTT assay results indicated pure inotodiol and inotodiol concentrate were non-toxic to HaCaT cells at concentrations below 20 μg/mL. Specifically, inotodiol showed more than 90% cell viability up to 5 μg/mL, gradually decreasing at higher concentrations. Conversely, inotodiol concentrate maintained cell viability above 70% across all tested concentrations, indicating its potential safety for therapeutic applications.
Anti-inflammatory Effects
TNF-α Induced Inflammation:
In HaCaT cells treated with TNF-α, the mRNA expression levels of IL-1β, IL-6, and IL-8 were significantly elevated. However, adding inotodiol and inotodiol concentrate resulted in a dose-dependent reduction of these pro-inflammatory cytokines, particularly at higher concentrations (Figure 3). This suggests that inotodiol effectively mitigates TNF-α induced inflammation in keratinocytes, highlighting its potential as an anti-inflammatory agent.
UV Radiation-Induced Inflammation:
Exposure to UV radiation induces acute inflammation by promoting the release of pro-inflammatory mediators such as IL-6 and TNF-α. The study demonstrated that adding inotodiol and its concentrate considerably reduced the mRNA expression levels of these cytokines. At a concentration of 20 μg/mL, the inotodiol concentrate brought IL-6 expression levels almost back to baseline (Figure 5), suggesting its potential utility in combating photoaging and UV-induced inflammatory responses.
Effects on Collagen and Hyaluronic Acid Synthesis
Maintaining the integrity of the extracellular matrix (ECM) is essential for skin health, particularly in aging and chronic inflammation. The study revealed that inotodiol and inotodiol concentrate significantly increased the mRNA expression of COL1A2, a major collagen component, indicating enhanced collagen synthesis (Figure 6).
Moreover, inotodiol also upregulated the expression of HAS2 and HAS3, key enzymes involved in hyaluronic acid synthesis. Hyaluronic acid plays a crucial role in skin hydration and elasticity. In TNF-α-stimulated HaCaT cells, inotodiol effectively downregulated the expression of HYAL-3, an enzyme responsible for hyaluronic acid degradation, further contributing to the maintenance of hyaluronic acid levels in the skin (Figure 7).
Discussion
The findings from this study point to significant anti-inflammatory and anti-aging effects of inotodiol derived from Chaga mushrooms. The capacity of inotodiol to mitigate inflammation induced by TNF-α and UV radiation can be attributed to its ability to downregulate the expression of key pro-inflammatory cytokines. This regulation is crucial for reducing acute inflammatory responses and preventing chronic inflammatory conditions such as inflammation, which are linked to skin aging and various dermatological diseases.
Mechanisms of Action
Anti-inflammatory Action:
The anti-inflammatory effects of inotodiol can be explained by its potential interference with key inflammatory pathways like the NF-κB pathway. NF-κB is a transcription factor that plays a central role in regulating immune responses, including the expression of pro-inflammatory cytokines. By inhibiting NF-κB activation, inotodiol likely reduces cytokine production, thereby alleviating inflammation.
Anti-aging Action:
The study also suggests that inotodiol enhances the synthesis of crucial ECM components like collagen and hyaluronic acid. Collagen provides structural integrity, while hyaluronic acid maintains moisture and elasticity in the skin. By promoting the expression of COL1A2, HAS2, and HAS3, and simultaneously downregulating HYAL-3, inotodiol helps preserve the ECM structure and function, thereby exhibiting anti-aging properties.
Clinical Implications and Future Research
The promising results from this study pave the way for inotodiol to be considered a potential therapeutic agent for conditions characterized by oxidative stress and chronic inflammation. Its dual action in reducing inflammation and promoting skin health makes it a valuable candidate for treating various skin disorders and preventing early skin aging.
Potential Applications
Dermatology:
Inotodiol can be formulated into topical creams or ointments to reduce inflammation and promote skin regeneration in conditions like eczema, psoriasis, and photoaging.
Cosmetics:
Given its anti-aging properties, inotodiol can be incorporated into cosmetic products designed to enhance skin hydration, texture, and elasticity, targeting the growing anti-aging skincare market.
Systemic Therapy:
Beyond dermatological applications, the anti-inflammatory properties of inotodiol suggest potential benefits in systemic inflammatory conditions, warranting further investigation into its efficacy and safety profile in systemic formulations.
Future Directions
In vivo Studies:
While the current study provides robust in vitro data, in vivo studies are crucial for understanding the pharmacokinetics, bioavailability, and long-term effects of inotodiol. Animal models and clinical trials in humans will help validate these findings and determine appropriate dosages for therapeutic use.
Mechanistic Studies:
Further research is needed to elucidate the precise molecular mechanisms by which inotodiol exerts anti-inflammatory and anti-aging effects. Identifying specific signaling pathways and molecular targets will provide deeper insights into its therapeutic potential.
Safety and Toxicity:
Comprehensive studies on the safety and potential toxicity of inotodiol, including its effects on different cell types and organs, are essential before it can be recommended for widespread clinical use.
Conclusion
The study under review highlights the significant potential of inotodiol, a lanostane triterpenoid from Chaga mushrooms, in reducing oxidative stress and inflammation while promoting skin health. By modulating the expression of pro-inflammatory cytokines and enhancing the synthesis of collagen and hyaluronic acid, inotodiol shows promise as a natural bioactive compound for therapeutic and cosmetic applications. Continued research and clinical validation will be key to unlocking its full potential and integrating it into effective inflammatory and aging-related skin conditions treatments.
For further reading and access to the full article, visit the
International Journal of Molecular Sciences.
