Dihydromyricetin: A Natural DNA Methylation Inhibitor Identified with Human Skin Rejuvenation Activity

1 Introduction

Aging is a process characterized by the gradual decline of physiological functions and adaptability over time, among which abnormal epigenetic regulation is regarded as one of the core hallmarks of aging. DNA methylation is an important epigenetic modification closely associated with human health and diseases, catalyzed by DNA methyltransferases (DNMTs). DNMT3A and DNMT3B establish methylation patterns during early embryonic development, while DNMT1 is responsible for copying and maintaining these patterns during cell division, serving as a key enzyme for sustaining genomic methylation. As the largest human organ exposed to the external environment, skin aging is highly correlated with alterations in DNA methylation, particularly hypermethylation. Previous studies have established epidermal DNA methylation clocks that can accurately predict an individual’s chronological age based on methylation status, fully demonstrating the central role of methylation changes in skin aging. Healthy lifestyles—such as sun protection, smoking cessation, and intake of antioxidants—can improve skin condition through epigenetic regulation. Therefore, moderately inhibiting DNMT1, reducing aging-related hypermethylation, and restoring the basal epigenetic pattern represent an ideal strategy for screening anti-aging ingredients. Dihydromyricetin (DHM), a flavonoid abundantly found in traditional medicinal plants (e.g., Ampelopsis grossedentata), is known for its diverse biological activities including antioxidant, anti-inflammatory, and anticancer effects. This study is the first to discover that DHM inhibits DNMT1, modulates DNA methylation in skin cells, reduces cellular methylation age, reverses aging-silenced genes, and exerts distinct rejuvenation effects in skin models, indicating high potential for cosmetic applications.

2 Results

2.1 Dihydromyricetin is Identified as a Natural Small-Molecule DNMT Inhibitor

A high-throughput 384-well screening system based on recombinant human DNMT1 (using scintillation proximity assay) was established. Two replicate screens were performed on 1,800 natural products and 640 FDA-approved drugs, with a correlation coefficient of 0.78 between the two runs. The screening revealed that both myricetin and dihydromyricetin (DHM) exhibited significant DNMT1 inhibitory activity. Subsequently, three highly pure (>95%) DHM extracts (derived from Ampelopsis grossedentata and Myrica cerifera bark) significantly inhibited DNMT1 in independent biochemical assays, with no significant difference in inhibitory efficacy. Considering availability, physicochemical properties, and human safety, DHM was selected for further investigation. This study is the first to confirm DHM as a DNMT1 inhibitor.

Figure 1 Identification of dihydromyricetin (DHM) as a DNMT inhibitor.

2.2 DHM Induces DNA Hypomethylation in Primary Human Keratinocytes

Genomic DNA methylation was analyzed using the 850K methylation array in 13 independent primary human keratinocyte lines treated with the highest tolerable concentration of DHM. A total of 101,067 differentially methylated probes were detected. Principal component analysis showed clear separation between the DHM-treated group and the control group.DHM induced moderate yet statistically highly significant global DNA hypomethylation, which was significant in all genomic regions except CpG islands and their shores. These findings confirm that DHM regulates DNA methylation in human cells, providing direct evidence for its mechanism of action.

Figure 2 DHM inhibits DNA methylation in primary human keratinocytes.

2.3 DHM Has No Significant Impact on Epigenetic and Genetic Integrity in Keratinocytes

Excessive loss of DNA methylation may cause genomic instability and induce apoptosis. To evaluate the safety of DHM, the following indicators were assessed: DHM treatment did not alter the methylation level of LINE-1 retrotransposons (a common marker for genomic stability); did not increase the proportion of γ-H2AX-positive cells (a marker for DNA double-strand breaks); and did not activate caspase-3/7 (a key executioner of apoptosis). In summary, DHM induces mild regulation of DNA methylation without genotoxicity or pro-apoptotic risk.

Figure 3 No evidence of critical epigenetic changes in keratinocytes after DHM treatment.

2.4 DHM Treatment Does Not Elicit Key Epigenetic Alterations in Keratinocytes

Epidermal DNA methylation age is positively correlated with visually assessed skin age and negatively correlated with skin elasticity, independently of chronological age, indicating that methylation age accurately reflects the skin aging phenotype. Three methylation models were used to analyze DHM-treated keratinocytes: The Skin & Blood Clock (Horvath model) showed a 1.86-year reduction in biological age; The self-established human epidermal clock showed a 2.03-year reduction; The wrinkle prediction model showed a decrease in predicted wrinkle grade, equivalent to delaying wrinkle accumulation by approximately 3.7 years.

Figure 4 DHM reduces DNA methylation age and predicted wrinkle grade.

2.5 In Vivo Effects of DHM on Skin Aging Phenotypes and Function

In vitro toxicological assays demonstrated that DHM was non-cytotoxic at the applied concentration. With a molecular weight of 320.25 and a logP value of 1.23, DHM meets the basic requirements for skin penetration. A comprehensive assessment confirmed that topical application of 0.15% DHM on human skin is safe. For in vivo methylation regulation: After 2 weeks of DHM treatment on the back skin of 30 female volunteers followed by UV-induced aging stimulation, epidermal methylation analysis showed that more than 95% of 12,444 aging-related methylation probes reverted to a youthful epidermal pattern. For in vivo gene expression activation: After 8 weeks of topical DHM treatment on the forearm skin of 19 female volunteers aged 50–65 years, all 34 aging-silenced genes were reactivated, many of which are directly linked to wrinkle formation. For functional improvement in 3D skin models: Skin models derived from elderly donors exhibited typical aging phenotypes with notably thinner epidermis. After DHM treatment, epidermal thickness increased significantly, with a structure closer to that of youthful skin. Additionally, supernatant from DHM-pretreated keratinocytes significantly promoted fibroblast proliferation, ameliorating the aging-related decline in fibroblast proliferative capacity. This study confirms that DHM exerts clear skin rejuvenation effects at both the molecular regulatory and functional improvement levels.

Figure 5 In vivo effects of DHM on skin aging phenotypes and function.

3 Conclusion

Natural products are important sources for screening anti-aging active ingredients, and epigenetic regulation has been validated as a key strategy to slow or even reverse aging. This study screened dihydromyricetin (DHM) from natural products and revealed its epigenetic anti-aging mechanism for the first time: DHM directly inhibits DNMT1 activity and mildly regulates DNA methylation levels; It reduces the methylation age of skin cells to achieve cellular rejuvenation; It reverses aging-related methylation alterations and gene silencing in vivo; Functionally, it improves epidermal atrophy and promotes fibroblast proliferation. Compared with clinically used demethylating drugs (e.g., azacytidine), DHM exerts milder methylation regulation with higher safety and good human tolerance (e.g., DHM is abundant in Ampelopsis grossedentata, and long-term consumption is safe). Thus, DHM is more suitable for cosmetic and health food applications. This study provides high-quality preclinical evidence for the application of DHM in anti-aging skin care products and oral anti-aging formulations. Future research may further explore its potential in systemic anti-aging and combination therapy. In conclusion, this study is the first to confirm that DHM is a natural DNMT1 inhibitor. By mildly regulating DNA methylation patterns, DHM reduces the biological age of skin cells, reverses aging-related gene silencing, and improves epidermal atrophy, thereby achieving human skin rejuvenation. With a clear mechanism of action and favorable safety profile, DHM is a highly promising anti-aging active ingredient for skin care.

Reference: Falckenhayn C, Bienkowska A, Söhle J, et al. Identification of dihydromyricetin as a natural DNA methylation inhibitor with rejuvenating activity in human skin[J]. Front Aging, 2024, 4: 1258184.