Researchers at a South Korean institution have identified a specific fraction of Aruncus dioicus leaf extract that protects differentiated muscle cells from dexamethasone-induced atrophy in a laboratory setting. Published in Applied Biological Chemistry, the study demonstrates that a fraction designated F2, derived from the plant’s leaves, significantly attenuates the degradation of muscle cells caused by the synthetic glucocorticoid dexamethasone. The findings provide foundational cell-based evidence for the anti-atrophic potential of this polyphenol-rich fraction, highlighting its capacity to preserve muscle cell morphology and suppress key molecular markers of muscle wasting.

Skeletal muscle atrophy is a clinical challenge often associated with long-term glucocorticoid therapy, characterized by excessive protein degradation and increased oxidative stress. The condition involves the activation of the ubiquitin-proteasome system, a cellular machinery responsible for breaking down proteins. Central to this process are muscle-specific E3 ubiquitin ligases, specifically muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). These proteins are largely regulated by forkhead box O (FoxO) transcription factors, which promote the expression of atrophy-related genes when the anabolic signaling pathways are suppressed.

The study focused on Aruncus dioicus, a traditional wild edible plant native to East Asia and belonging to the Rosaceae family. While previous research has noted its antioxidant and metabolic regulatory activities, its potential role in glucocorticoid-induced muscle atrophy remained largely unexplored. To investigate this, the research team prepared a 70% ethanolic extract from freeze-dried leaves and subjected it to preparative high-performance liquid chromatography (HPLC). This process yielded two distinct fractions, F1 and F2, based on their UV chromatographic profiles, with F2 exhibiting a distinct yellow coloration and a yield of 28.53%.

Phytochemical analysis revealed that F2 possessed the highest total phenolic content and total flavonoid content among all tested samples. The fraction also demonstrated the strongest antioxidant capacity, as determined by both DPPH radical-scavenging and oxygen radical absorbance capacity (ORAC) assays. In the DPPH assay, F2 showed the lowest half-maximal inhibitory concentration, indicating superior radical-scavenging activity compared to the crude extract and fraction F1. These chemical properties suggested that F2 was enriched in bioactive polyphenols, specifically flavonol glycosides and phenolic acids, which were later confirmed through metabolite profiling.

To evaluate the biological effects, the researchers used C2C12 myoblasts, a mouse cell line that differentiates into multinucleated myotubes in culture. These myotubes were treated with dexamethasone to induce a model of muscle atrophy. Dexamethasone is a synthetic glucocorticoid widely used in experimental models because it activates catabolic programs that disrupt anabolic signaling and promote oxidative stress. The study assessed the protective effects of the extract fractions by measuring cell viability, intracellular reactive oxygen species (ROS) levels, myotube diameter, and the expression of the atrogenes MAFbx and MuRF1.

Pretreatment with F2 at a concentration of 50 micrograms per milliliter significantly reduced dexamethasone-induced ROS accumulation by 33%. This reduction brought oxidative stress levels closer to those of the untreated control group, suggesting that the chemical antioxidant capacity of F2 is effectively translated into the cellular environment. In contrast, the crude extract and fraction F1 exhibited comparatively weaker effects on ROS suppression. The ability of F2 to mitigate oxidative stress is considered a key mechanism in its protective action, as oxidative stress is implicated in activating multiple catabolic pathways that contribute to muscle wasting.

Morphological analysis using immunofluorescence staining showed that dexamethasone treatment caused a marked reduction in myotube diameter, a characteristic feature of atrophy. The control group exhibited a mean myotube diameter of 23.01 micrometers, which decreased to 17.70 micrometers in the dexamethasone-treated group. Pretreatment with F2 restored the myotube diameter to 22.52 micrometers, a level not significantly different from the untreated control. This morphological preservation indicates that F2 effectively attenuated the physical signs of atrophy, maintaining the structural integrity of the muscle cells.

At the molecular level, western blot analysis demonstrated that dexamethasone treatment markedly upregulated the protein expression of MAFbx and MuRF1. F2 pretreatment attenuated the expression of both atrogenes in a fraction-dependent manner. For MAFbx, F2 restored protein expression to levels comparable to the untreated control, representing significantly greater suppression than that achieved by the crude extract or F1. Similarly, F2 exhibited the strongest inhibitory effect on MuRF1 expression, with levels significantly lower than those observed in the dexamethasone-treated group. These findings suggest that F2 suppresses the ubiquitin-proteasome mediated proteolysis that drives muscle protein degradation.

Metabolite profiling using UHPLC–Orbitrap MS/MS identified several compounds enriched in F2, including isoquercetin, kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, tiliroside, and dicaffeoylquinic acid. Isoquercetin, a flavonoid with documented antioxidant properties, has been reported to suppress ROS-mediated oxidative stress and downregulate atrogenes through the Nrf2/HO-1 signaling pathway. Kaempferol glycosides have also been shown to protect against dexamethasone-induced atrophy by activating the PI3K/Akt/mTOR signaling pathway and inhibiting FoxO transcription factors. The combined presence of these bioactive polyphenols likely constitutes the molecular basis for the potent antioxidant and anti-atrophic effects observed in F2.

The study acknowledges that while the findings are promising, they are limited to an in vitro cell-based model. The C2C12 myotube system does not fully recapitulate the physiological complexity of skeletal muscle in vivo, and a direct causal relationship between ROS suppression and atrogene downregulation has not yet been definitively established. The authors note that further studies using specific pathway inhibitors or genetic modulation approaches are necessary to clarify these mechanistic links. Additionally, the potential involvement of endogenous antioxidant defense systems, such as the Nrf2/HO-1 pathway, requires further investigation to fully understand the protective mechanisms of F2.

The research highlights the potential of natural polyphenols as therapeutic agents for muscle atrophy, particularly in the context of glucocorticoid therapy. The ability of F2 to preserve myotube morphology and suppress atrogenes provides a strong rationale for further investigation. However, the authors emphasize that in vivo validation and targeted analysis of marker compounds are needed to confirm the physiological relevance of these findings. The study serves as a foundational step in exploring Aruncus dioicus as a source of anti-atrophic compounds, with the F2 fraction emerging as a candidate for further development.

In conclusion, the F2 fraction of Aruncus dioicus leaf extract effectively mitigates dexamethasone-induced muscle atrophy in C2C12 myotubes by suppressing oxidative stress and the expression of key atrogenes. The enrichment of flavonol glycosides and phenolic acids in F2 underpins its superior antioxidant and anti-atrophic activities. These findings contribute to the growing body of evidence supporting the role of dietary polyphenols in muscle health and provide a basis for future research into the development of natural anti-atrophic therapies.

Detailed extraction protocols involved two grams of freeze-dried leaf powder mixed with seventy percent ethanol at a one-to-fifty ratio and sonicated for thirty minutes. Following centrifugation at 12,000 times gravity and filtration, rotary vacuum evaporation at 36 degrees Celsius yielded 32.10 percent crude extract by dry weight. Preparative chromatography on a YMC Triart C18-S column separated the material into two distinct fractions monitored at 240, 280, and 320 nanometers. Fraction F1 eluted between 2 and 12 minutes with a reddish appearance and a 45.77 percent yield, whereas fraction F2 eluted between 12 and 25 minutes, yielding 28.53 percent yellow residue.

Phytochemical quantification confirmed distinct separation across preparations. Crude leaf extract contained 220.48 milligrams gallic acid equivalents per gram of total phenolics and 154.73 milligrams quercetin equivalents per gram of total flavonoids. Fraction F1 exhibited lower values at 108.84 milligrams gallic acid equivalents and 79.51 milligrams quercetin equivalents per gram. In contrast, F2 concentrated both compound classes, yielding 437.79 milligrams gallic acid equivalents and 324.48 milligrams quercetin equivalents per gram. In DPPH radical scavenging assays, F2 achieved a half-maximal inhibitory concentration of 18.01 micrograms per milliliter, outperforming the crude extract at 23.60 and F1 at 59.86 micrograms per milliliter. The ORAC assay likewise showed superior antioxidant capacity for F2 at 4841.46 micromoles of Trolox equivalents per milligram, substantially exceeding the crude extract at 3125.80 and F1 at 2059.52 micromoles per milligram.

Cell differentiation and viability assays established precise operational boundaries in C2C12 mouse myotubes. Myoblasts were expanded in medium containing ten percent fetal bovine serum and differentiated into multinucleated myotubes across 72 hours using medium containing two percent horse serum. In cytotoxicity screening, the crude extract and F1 maintained myotube viability above eighty percent at 30, 50, and 100 micrograms per milliliter. Fraction F2 preserved viability above ninety percent at 30 and 50 micrograms per milliliter, but viability dropped below eighty percent at 100 micrograms per milliliter, prompting researchers to select 50 micrograms per milliliter or less for subsequent experiments. Morphological evaluation across 150 measured myotubes per condition demonstrated that dexamethasone exposure reduced myotube diameter to 17.70 micrometers compared to 23.01 micrometers in untreated controls. Pretreatment with F1 produced only limited recovery at 19.89 micrometers, while the crude extract yielded 21.56 micrometers and F2 restored diameter to 22.52 micrometers.

High-resolution metabolomic profiling clarified the discriminatory chemical signatures separating the preparations. Separation on a Hypersil GOLD aQ C18 column using acidified water and acetonitrile mobile phases generated principal component analysis models explaining 78.8 percent and 8.3 percent of the total variance across the first two axes. Orthogonal partial least squares-discriminant analysis identified 56 features exceeding variable importance in projection thresholds of 1.5, with nineteen distinct features enriched in fraction F2. Tentative compound identifications resolved specific mass transitions, including kaempferol 3-O-(6”-malonylglucoside) at mass-to-charge ratio 489.1044 with a neutral loss of 205 daltons, alongside characteristic aglycone fragmentation for kaempferol and quercetin glycosides.

Subject of Research: Protective effects of Aruncus dioicus leaf extract against dexamethasone-induced muscle atrophy in C2C12 myotubes

Article Title: Protective effects of Aruncus dioicus leaf extract against dexamethasone-induced muscle atrophy in C2C12 myotubes

Article References: Kim, K. B., & Auh, J.-H. (2026). Protective effects of Aruncus dioicus leaf extract against dexamethasone-induced muscle atrophy in C2C12 myotubes. Applied Biological Chemistry, 69(1), Article 37. https://doi.org/10.1186/s13765-026-01107-z

Image Credits: AI Generated

DOI: 10.1186/s13765-026-01107-z

Keywords: Protective, effects, Aruncus, dioicus, leaf, extract, against, dexamethasone-induced, muscle, atrophy, C2C12, myotubes