Obesity is a growing global health concern, with a significant increase in prevalence. Current pharmacological treatments often have side effects. Vinegar has shown promise in reducing body fat and hyperglycemia in previous studies. This study focuses on *Cudrania tricuspidata* fruit vinegar (CTFV), exploring its anti-obesity efficacy, molecular mechanism, and effects on obesity-related metabolizing enzymatic activities. *Cudrania tricuspidata*, common in East Asia, is used as a food and medicinal plant, with its fruits containing bioactive substances including flavones, isoflavonoids, and phenolic compounds. While anti-obesity effects of *C. tricuspidata* fruits have been reported, information regarding CTFV's mechanism and efficacy is limited. This study aimed to analyze the anti-obesity effects of CTFV and its polyphenolics, comparing its efficacy to pomegranate fruit vinegar (PFV) and fenofibrate in HFD-fed obese mice. The rising prevalence of obesity in South Korea, coupled with the increasing commercialization of vinegar as a fat-reduction aid, further underscores the importance of this research.

Previous research indicates that vinegar intake can contribute to reduced body fat and hyperglycemia. Studies have demonstrated vinegar's role in reducing risk factors for high cholesterol and atherosclerosis in rabbits, and lowering food intake, body weight, and lipid levels in obese mice models. Acetic acid in vinegar has been shown to influence blood lipid levels and reduce body weight. Clinical studies have shown vinegar supplementation reducing glucose levels and postprandial hyperglycemia. Vinegar's biological effects are diverse, including antimicrobial, anti-tumor, antioxidant, anti-hepatic fibrosis, and anti-kidney stone recurrence activities. Fruit vinegars, incorporating fruits with health benefits, are increasingly studied and produced, with various fruits like black raspberries, *Schizandra chinensis*, *Vitis coignetiae*, apples, and blueberries being utilized.

The study involved several key steps: **1. CTFV and PFV Preparation:** *Cudrania tricuspidata* fruits were collected, processed, and fermented to produce CTFV. A traditional starter vinegar was used for the fermentation process. PFV was obtained commercially. **2. HPLC Analysis:** High-performance liquid chromatography (HPLC) was used to analyze the phenolic compounds and parishin derivatives in CTFV. Twenty phenolic compound standards were used for calibration curves. **3. Enzyme Assays:** In vitro enzyme activity assays were conducted for pancreatic lipase, lipoprotein lipase, β-glucosidase, α-amylase, phosphodiesterase IV, citrate synthase, and alkaline phosphatase, assessing the inhibitory effects of CTFV and its major compounds. **4. Cell Culture and Viability Assay:** HepG2, 3T3-L1, and Raw264.7 cells were cultured, and the cytotoxicity of CTFV was assessed using an MTT assay. **5. Animal Model and Treatment:** Male ICR mice were fed an HFD for 50 days. Mice were divided into five groups: control, DIO (high-fat diet-induced obese), DIO + fenofibrate, DIO + PFV, and DIO + CTFV. Food intake and body weight were monitored. At the study's end, tissues and blood samples were collected for analysis. **6. Histology:** Liver and fat tissue histology was performed using hematoxylin and eosin and Oil Red O staining. **7. Oxidative Stress Parameters:** Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), and nitric oxide (NOx) levels were measured in serum and liver. **8. Biochemical Analysis:** Serum and liver biochemical parameters were determined, including lipid profiles, glucose, insulin, proteins, leptin, adiponectin, and cardiovascular risk indices. **9. Immunoblotting:** Immunoblotting was used to analyze the protein expression levels of various proteins involved in metabolic pathways. **10. Statistical Analysis:** Data were analyzed using one-way ANOVA followed by a post-hoc Tukey's test.

CTFV production yielded a product with specific physicochemical properties, including pH and acidity levels, which changed during the fermentation process. HPLC analysis identified and quantified various polyphenolic compounds and parishin derivatives in CTFV. In vitro assays showed that CTFV and its major components reduced the activities of several obesity-related metabolizing enzymes. CTFV treatment didn't show cytotoxicity at lower concentrations in cell lines. In vivo studies using HFD-fed obese mice demonstrated that CTFV administration significantly reduced body weight gain, feed efficiency, liver mass, fat tissue mass, and adipocyte size compared to the DIO group. CTFV also effectively reduced hepatic fat accumulation. Furthermore, CTFV improved serum oxidative stress parameters, reducing MDA and NOx while increasing CAT and SOD activities. Similar improvements were seen in liver oxidative stress parameters. CTFV also modulated serum and liver metabolizing enzyme activities. Serum biochemical analysis showed CTFV significantly lowered lipid levels (TC, TG, LDL, VLDL), increased HDL, reduced insulin levels and insulin resistance, and improved cardiovascular risk indices. Immunoblotting revealed that CTFV modulated the expression and phosphorylation of proteins involved in AdipoR1, OBR, IRS1, and PTP1B signaling pathways, as well as PI3K/AKT/MAPKs and AMPK pathways, impacting glucose transport, lipid synthesis, and fatty acid oxidation.

The findings demonstrate that CTFV effectively attenuates obesity in HFD-fed mice. The reduction in body weight gain, fat accumulation, and improved metabolic parameters are consistent with previous studies on the anti-obesity effects of vinegar and its components. The observed effects of CTFV on metabolizing enzymes suggest a mechanism involving reduced nutrient absorption and enhanced energy expenditure. The modulation of signaling pathways related to adipogenesis, fatty acid oxidation, and glucose transport supports the multifaceted actions of CTFV in combating obesity. The superior efficacy of CTFV compared to PFV and fenofibrate suggests the presence of unique bioactive compounds in CTFV contributing to its anti-obesity effects. The study highlights the potential of CTFV as a functional food ingredient for obesity management.

This study demonstrates that CTFV effectively reduces HFD-induced obesity in mice through multiple mechanisms, including the inhibition of obesity-related enzymes, modulation of key signaling pathways, and improvement of oxidative stress parameters. The results suggest CTFV holds significant promise as a functional food ingredient for combating obesity. Future research could focus on identifying the specific bioactive compounds responsible for the observed effects, exploring the long-term effects of CTFV administration, and conducting human clinical trials to validate the findings.

The study used a mouse model, and the results may not be directly translatable to humans. The study duration was relatively short (50 days), limiting the assessment of long-term effects. Further research is needed to elucidate the precise mechanisms of action of CTFV's bioactive components and to determine optimal dosages for human consumption.