I. Introduction
Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver disorders globally, closely associated with obesity, insulin resistance, dyslipidemia, and systemic inflammation (Pouwels et al. 2022). The accumulation of advanced glycation end products (AGEs), formed through non-enzymatic glycation and oxidation of proteins and lipids under hyperglycemic and oxidative conditions, has emerged as a key driver of metabolic dysfunction (Gu et al. 2024). AGEs contribute to the development and progression of NAFLD by promoting oxidative stress, inflammation, and hepatocellular injury through the activation of their receptor (RAGE), which activates NF-κB and MAPK pathways thereby amplifying reactive oxygen species (ROS) production and inflammatory cytokine release (Lee et al. 2021). Nε- (carboxymethyl)lysine (CML), a major AGE, accumulates in metabolic tissues including the liver and contributes to hepatic steatosis, insulin resistance, and systemic metabolic disturbances, particularly when combined with a high-fat diet (Lee et al. 2024a). Pyridoxamine (PM), a derivative of vitamin B6, has been reported to exert anti-glycation effects, however long-term use may be associated with potential neuronal toxicity (Muhamad et al. 2023;Rivas Navarrete et al. 2025).
Oenothera biennis (evening primrose) seeds (OBS) are rich in omega-6 polyunsaturated fatty acids primarily linoleic acid and gamma-linolenic acid (GLA, 8-10%) alongside phenolics, flavonoids, tocopherols, sterols, and triterpenes with known antioxidant and anti-inflammatory capacities (Farag et al. 2023). OBS oil has chemotherapeutic properties in human pancreatic ductal adenocarcinoma cell lines (Zeppa et al. 2022). Evening primrose seed oil has demonstrated efficacy in reducing inflammatory markers and lipid peroxidation in various inflammatory conditions (Sharifi et al. 2024). The defatted OBS contained a high amount of dietary fiber and a low sugar content, suggesting its potential as a functional food for diabetes management (Wang et al. 2021). Despite these reported benefits, the role of OBS extract in mitigating AGE-induced metabolic dysfunction has not been reported.
Therefore, we investigated the protective effects of OBS in a CML-high fat diet (HFCML) mouse model. We assessed body weight, organ hypertrophy, serum liver enzyme activities, lipid profile, and glucose tolerance following OBS administration. Understanding these effects may elucidate the potential of OBS as a functional food ingredient to prevent AGEassociated metabolic diseases such as NAFLD and insulin resistance.
II. Materials and Methods
1. Preparation of Oenothera biennis seed (OBS) extract
The OBS were obtained from a commercial market (Jayeonhanjae Agricultural Cooperative, Nonsan-si, Republic of Korea) and finely ground into powder. The OBS powder was then added to 50% ethanol (1:10 w/v) at 50°C for 3 h, followed by reflux extraction. The extract was filtered through Whatman filter paper and concentrated using a rotary evaporator (R205; Buchi, Fostfach, Switzerland). The concentrate was then freeze-dried, and the samples were preserved at -20°C for experimental use.
2. Animal model
Six weeks old C57BL/6J male mice were purchased from the Orient Bio (Seongnam, Republic of Korea). The mice were randomly divided into the following five groups (n=5- 6 per group): (1) normal diet (ND), mice fed 7% kcal fat ND (AIN-93G diet; Research Diet, Inc., New Brunswick, NJ, USA); (2) HFD+CML (HFCML), mice fed 45% kcal HFD (D12492; Research Diet, Inc., New Brunswick, NJ, USA)+ CML (Sundia Meditech Company, Ltd., Shanghai, China) 10 mg/kg/day; (3) HFCML+OBS extract 100 mg/kg/day (OBS100); (4) HFCML+OBS extract 200 mg/kg/day (OBS200); and (5) HFCML+PM 100 mg/kg/day (positive control; pyridoxamine). PM was obtained from Sigma- Aldrich (P9380; St. Louis, Mo, USA) and used as an AGEs inhibitor. CML (200 μL) and OBS extracts (200 μL) were administrated once daily by gavage for 12 weeks. CML, OBS, and PM were dissolved in distilled water (DW) and orally administered at the described dose. The ND group received an equivalent volume of DW orally for 12 weeks. Body weight was measured once weekly during the 12 weeks of the experiment. At the end of the experiment, the mice were fasted for 12 h, euthanised by isoflurane exposure, and their blood was collected from the postcaval vein. Serum samples were centrifuged at 1500×g for 15 min. All animal experiments followed the guidelines of the Animal Care and Use Committee of the Korea Food Research Institute (KFRIM- 22019). The mice were housed under consistent conditions of 23±1°C, humidity 55±5%, and a 12 h light/dark cycle. The animals were provided food and water ad libitum.
3. Oral glucose tolerance test (OGTT)
OGTT was performed at 12 weeks of feeding. Following a 12 h fast, fasting blood glucose was collected from the tail and quantified using a glucometer (Accu-Chek Performa; Roche, Switzerland). Subsequently, the mice were orally administered glucose solution (2 g/kg body weight). Blood glucose levels were checked every 30 min for 2 h. To determine overall glucose tolerance, the area under the curve (AUC) was derived from the glucose levels measured throughout the duration.
4. Biochemical analysis
Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (T-Chol), low- and high-density lipoprotein cholesterol (LDL-Chol, and HDL-Chol) were analyzed by automatic blood biochemical test analyzer (AU680, Beckman Coulter, Tokyo, Japan).
5. Statistical Analysis
The experimental data are presented as means±standard error of the mean (SEM). A one-way analysis of variance was conducted, followed by dunnett’s post hoc test, using GraphPad Prism software (version 10.0; San Diego, CA, USA). Statistical significance is indicated as #p<0.05, ##p<0.01, and ###p<0.001 in the comparison between the ND and HFCML groups, and *p<0.05, **p<0.01, and ***p<0.001 in the comparison between the HFCML and treated groups.
III. Results and Discussion
1. Effects of OBS extract on body weight in HFCML diet mice
Previous study showed that CML-supplemental HFD significantly induced body weight, organ weight, and fat mass, which were suppressed by natural product administration (Lee et al. 2024). To investigate the effect of OBS extract on body weight and organ weight in HFCML diet- induced mice, changes in body weight were monitored for 12 weeks. As shown in <Figure 1A>, mice fed the HFCML diet exhibited a marked increase in body weight compared with the ND group. Treatment with OBS (100 or 200 mg/kg) attenuated HFCML-induced body weight <Figure 1A>.
CML group than in the ND group (p<0.001), whereas both OBS treated groups exhibited lower weight gain compared with the HFCML group <Figure 1B>. Liver weight did not change significantly between the ND and HFCML groups <Figure 1C>. In contrast, kidney weight was significantly increased in the HFCML group compared with the ND group (p<0.05), and this increase was markedly suppressed by OBS extract supplement. (p<0.001) <Figure 1D>. Furthermore, the weight of epididymal white adipose tissue (EWAT) was substantially elevated in the HFCML group compared to the ND group (p<0.001), whereas OBS administration showed a tendency to lower EWAT weight, the effect did not observe statistical significance <Figure 1E>. These results suggest that OBS extract may mitigate body weight gain and abnormal kidney hypertrophy induced by HFCML feeding.
2. Effects of OBS extract on serum liver biomarkers in HFCML diet mice
ALT and AST are key aminotransferases involved in amino acid metabolism and elevated serum levels reflect hepatocellular injury, serving as sensitive biochemical indicators of liver damage (Vogli et al. 2023). To evaluate the hepatoprotective effects of OBS extract, serum liver injury markers were measured in HFCML diet-induced mice. As shown in <Figure 2A>, ALT activity was markedly elevated in the HFCML group compared with the ND group (p<0.001), indicating liver injury. Treatment with OBS at both 100 and 200 mg/kg significantly reduced ALT levels (p<0.01). Similarly, AST activity was significantly increased in the HFCML group relative to the ND group (p<0.001) <Figure 2B>. Administration of OBS extract attenuated this elevation in a dose dependently, with significant decreases observed at both 100 mg/kg (p<0.05) and 200 mg/kg (p<0.001). These findings suggest that OBS extract effectively ameliorates HFCML-induced hepatic injury, as evidenced by reductions in serum ALT and AST activities.
3. Effects of OBS extract on serum lipid profile in HFCML diet mice
Elevated serum AST and ALT levels are closely associated with dysregulated lipid metabolism, leading to increased TG, T-Chol, and LDL-Chol levels, and decreased HDL-Chol levels (Xuan et al. 2024). To assess the effects of OBS extract on serum lipid metabolism, lipid profiles were analyzed in HFCML diet-induced mice. As shown in <Figure 3A>, serum TG levels were markedly increased in the HFCML group compared with the ND group (p<0.001). Administration of OBS at both 100 and 200 mg/kg significantly reduced TG concentrations (p<0.001). T-Chol levels were significantly elevated in the HFCML group relative to the ND group (p<0.001) <Figure 3B>. While OBS treatment tended to lower T-Chol levels, the effect was not statistically significant. Similarly, LDL-Chol was substantially higher in the HFCML group than in the ND group (p<0.001) <Figure 3C>. Both doses of OBS significantly decreased LDL-Chol levels (p<0.01), with the PM group showing the strongest reduction (p<0.001). In contrast, serum HDL-Chol was increased in the HFCML group compared with the ND group (p<0.01) <Figure 3D>. OBS administration did not significantly alter HDL-Chol levels. Collectively, these findings indicate that OBS extract effectively improves dyslipidemia in HFCML-fed mice by lowering TG and LDL-Chol levels, thereby exerting a protective effect against abnormal lipid metabolism.
4. Effects of OBS extract on oral glucose tolerance in HFCML diet mice
AGE accumulation disrupts glucose homeostasis by inducing insulin resistance and hepatic gluconeogenesis, thereby exacerbating metabolic dysfunction associated with NAFLD progression (Wang et al. 2022). The effects of OBS extract on glucose homeostasis were evaluated using OGTT in HFCML-induced mice. As shown in <Figure 4A>, fasting blood glucose levels were slightly elevated in the HFCML group compared with the ND group; however, no significant differences were observed among the OBS or PM treated groups. During the OGTT, blood glucose levels in the HFCML group were markedly increased after glucose loading and remained higher throughout the test compared with the ND group (p<0.01-0.001) <Figure 4B>. OBS supplementation attenuated the glucose excursion in a dose dependently, with the 200 mg/kg dose showing a more pronounced effect. Consistently, the AUC for blood glucose was significantly greater in the HFCML group compared with the ND group (p<0.001) and OBS administration groups exhibited reduced AUC values, reaching statistical significance at 200 mg/kg (p<0.05), while PM treatment markedly decreased the AUC (p<0.001) <Figure 4C>. These results indicate that OBS extract improves glucose tolerance and mitigates glucose intolerance induced by HFCML feeding, thereby exerting beneficial effects on systemic glucose metabolism.
IV. Summary and Conclusion
In the study, we demonstrated that OBS extract exerts protective effects against metabolic disturbances induced by HFCML feeding in mice. CML promotes hepatic lipid accumulation by enhancing de novo lipogenesis and suppressing fatty acid oxidation through RAGE-mediated oxidative and inflammatory signaling (Gaens et al. 2012;Lee et al. 2024b). Previous study showed that OBS contained phenolic compounds, lignin, and amino acids which contributed to health benefits (Timoszuk et al. 2018). OBS Specifically, OBS supplementation attenuated body weight g ain, reduced kidney hypertrophy, improved liver function, ameliorated dyslipidemia, and enhanced glucose tolerance showing effects comparable to PM positive control. These findings suggest that OBS extract may represent a promising dietary intervention for the prevention of AGEs-related metabolic disorders, NAFLD and insulin resistance.
HFCML feeding resulted in significant weight gain and adipose tissue accumulation, consistent with previous reports that AGEs exacerbate obesity and metabolic dysfunction through activation of the AGE-RAGE axis and downstream inflammatory signaling (Lee et al. 2023). OBS treatment reduced body weight gain and improved kidney and adipose tissue indices, indicating potential benefits in modulating energy balance and organ hypertrophy.
HFD feeding induces hepatic steatosis and liver injury, which is commonly reflected by elevated ALT and AST levels (Ou et al. 2024). Serum biochemical markers further supported the hepatoprotective role of OBS. The significant reductions in ALT and AST levels suggest that OBS can alleviate hepatic injury triggered by AGE and lipid overload. This hepatoprotective effect may be attributed to the antioxidant and anti-inflammatory properties of bioactive compounds in OBS, including polyphenols and flavonoids, which have been reported to suppress oxidative stress and inflammatory cascades (Timoszuk et al. 2018). Dyslipidemia is a hallmark of metabolic syndrome and a critical driver of NAFLD progression (Rong et al. 2022). Lipid profile analysis revealed that OBS markedly decreased serum TG and LDL-Chol levels while maintaining HDL-Chol within the normal range. Therefore, the lipid-lowering effect of OBS highlights its potential to improve systemic lipid metabolism and reduce cardiovascular risk factors associated with AGE accumulation.
Accumulating evidence reported that natural plant extracts rich in antioxidants improve insulin sensitivity by reducing oxidative stress and modulating signaling pathways related to glucose uptake (Esfahani et al. 2023;Gao et al. 2025;Zhang et al. 2025). OBS significantly improved glucose tolerance as demonstrated by a reduction in glucose excursion and AUC during OGTT. These findings indicate that OBS enhances glucose homeostasis and may ameliorates insulin resistance.
In Summary, present study has several limitations. The underlying molecular mechanisms the AGE-RAGE axis and associated pathway such as NF-κB and MAPK were not fully elucidated. However, our results suggest that OBS extract mitigates HFCML-induced metabolic dysfunction through multiple mechanisms involving hepatoprotection, lipid lowering, and improvement of glucose tolerance. These effects might mediate the attenuation of AGE-induced oxidative and inflammatory stress, thereby suppressing downstream metabolic dysregulation.
