1. INTRODUCTION
Hyperlipidemia, characterized by elevated levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL), coupled with decreased high-density lipoprotein (HDL), constitutes a major global health concern [1]. This condition can precipitate serious difficulties in disease management and foster the development of severe health conditions, such as stroke, degenerative joint disorders, and cardiovascular conditions (CVDs), thereby elevating mortality risk. The global prevalence of hyperlipidemia among adults is estimated to range from 20% to 80%, depending on population characteristics and diagnostic criteria [2]. In Thailand, the burden is particularly severe: a nationwide survey reported that 88.9% of adults with type 2 diabetes had dyslipidemia, while 79.6% of those with coexisting atherosclerotic CVD continued to exhibit uncontrolled lipid levels despite treatment [3]. Given its widespread impact, hyperlipidemia is directly related to SDG 3: Good Health and Well-being, particularly Target 3.4, which seeks to lower premature deaths from non-communicable diseases through preventive measures and effective treatments.
Chronic high-fat diet (HFD) intake is strongly linked to various detrimental health consequences, including dyslipidemia, elevated liver enzymes, hepatic cell damage, and heightened oxidative stress [4]. Excessive dietary fat promotes lipid accumulation in the liver, leading to hepatic steatosis and subsequent inflammation. Consequently, this inflammation can prompt the release of liver enzymes, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), into the bloodstream, resulting in their elevated systemic levels [5]. Beyond dyslipidemia, long-term intake of an HFD markedly elevates reactive oxygen species generation while impairing the body’s major antioxidant defense mechanisms. This imbalance results in oxidative stress, which damages cells, particularly hepatocytes, through mechanisms such as lipid peroxidation and protein oxidation [6]. Addressing these HFD-induced health issues is paramount for achieving SDG 3.
The conversion of agro-industrial waste into high-value health products is gaining considerable traction due to their promising bio-pharmacological activities, especially their antioxidant potential [7]. This strategy aligns with SDG 12, specifically Target 12.5, by promoting a substantial reduction in waste through measures such as prevention, reduction, recycling, and reuse. Complementing this trend, growing evidence shows that various parts of medicinal plants, including peels, seeds, and leaves, possess diverse pharmacological properties and strong antioxidant capabilities, making them effective and well-tolerated options for treating hyperlipidemia [8–10].
Cleistocalyx nervosum var. paniala (C. nervosum), commonly referred to as Makiang in northern Thailand, is a member of the Myrtaceae family and plays a key role in the Royal Project initiated by Her Royal Highness Princess Maha Chakri Sirindhorn, aimed at conserving Makiang genetic resources [11,12]. Cleistocalyx nervosum, a plant with recognized medicinal properties, has garnered attention for its various biological activities, including anticancer [13], antimutagenic [14], neuroprotective [15], and anti-neuroinflammatory effects [16], with a particular emphasis on its potent antioxidant capabilities [17]. While studies have explored the general pharmacological effects of C. nervosum seeds, there is a notable gap in research specifically examining their impact on hyperlipidemia. As mentioned earlier, hyperlipidemia, characterized by elevated lipid profiles and often accompanied by liver damage, inflammation, and oxidative stress, presents a significant health challenge. Given the seeds’ known antioxidant and anti-inflammatory properties, it is plausible that an extract from C. nervosum seeds could offer a therapeutic solution. Based on the known antioxidant and anti-inflammatory effects of C. nervosum seeds, we hypothesize that the administration of a C. nervosum seed extract to an HFD-fed rat model will attenuate the increase in lipid profiles, decrease hepatic enzyme levels, and downregulate pro-inflammatory markers, such as interleukin-6 (IL-6) expression. Furthermore, we predict that the extract will improve the antioxidant status in the liver of the rats, thereby demonstrating its potential as an anti-hyperlipidemic and hepatoprotective agent. This study will also highlight a potential link between sustainable waste management (utilizing the seeds) and global health initiatives.
2. MATERIALS AND METHODS
2.1. In vitro study on antioxidant activity of C. nervosum seed extract
2.1.1. C. nervosum seed extract preparation
In mid-November 2024, mature C. nervosum fruits were harvested from the Plant Genetic Conservation Project farm, Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn, at Maejo University–Phrae Campus, Thailand. Seeds were separated from mature fruits and thoroughly washed with tap water three times. The seed samples were arranged on stainless steel trays and subjected to drying in a hot-air oven at 50°C for 48 hours. After drying, the seeds were pulverized and homogenized using an electric blender, followed by maceration in 70% ethanol for 72 hours. The resulting mixture was subjected to vacuum filtration, and the obtained filtrate was concentrated at 40°C using a rotary evaporator before being lyophilized, as illustrated in Figure 1. The final dry yield of the seed extract was calculated as a percentage using the equation:
 | Figure 1. Process of C. nervosum seed extraction. [Click here to view] |
% yield = (weight of dried extract / weight of dried plant sample) × 100
The dried extract was preserved in an amber bottle at –20°C for subsequent animal studies. A preliminary determination of the total phenolic content (TPC) of the C. nervosum seed extract was also conducted.
2.1.2. Phytochemical analysis and antioxidant potential of C. nervosum seed extract
Qualitative phytochemical screening of the ethanolic C. nervosum seed extract was carried out using the following standard methods: tannins (bromine water test), alkaloids (Wagner’s reagent test), terpenoids (Salkowski test), flavonoids (dilute sodium hydroxide test), phenols (10% ferric chloride test), and reducing sugars (Fehling’s test) [18].
Quantitative analysis of TPC in the C. nervosum seed extract was performed using the Folin–Ciocalteu method, with slight modifications from Phachonpai et al. [19]. An ethanolic solution of the extract was prepared at a concentration of 1 mg/ml and used for the assay. For the reaction, 0.1 ml of the extract solution or 0.1 ml of gallic acid standard solution (20–100 mg/l) was mixed with 7.9 ml of distilled water, 0.5 ml of Folin–Ciocalteu reagent, and 1.5 ml of 20% Na2CO3. A blank contained distilled water instead of the extract. After 2 hours of incubation at room temperature, absorbance was detected at 760 nm using a UV-Vis spectrophotometer. TPC values were expressed as milligrams of gallic acid equivalents per gram of dry seed extract (mg GAE/g DW).
The antioxidant activity of C. nervosum seed extract was also evaluated using a modified 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, based on the method of Kordali et al. [20]. Briefly, 100 mg of dried extract was dissolved in 95% ethanol to prepare concentrations ranging from 1.25 to 50 mg/ml. Each extract solution was mixed with 200 μl of DPPH ethanolic solution and incubated in the dark at room temperature for 30 minutes. Using Trolox (TE) as the standard, absorbance was detected at 517 nm with a microplate reader, and all analyses were carried out in triplicate (n = 3), and mean values were used to calculate percentage inhibition and determine the radical scavenging potential (IC50) using the following equation:
% Inhibition = [(Absorbance of the control − Absorbance of the sample) / Absorbance of the control] * 100
2.2. In vivo study on biological and pharmacological activities of C. nervosum seed extract
2.2.1. HFD formulation
An HFD was prepared in our laboratory based on Srinivasan et al. [21], with minor modifications using locally available ingredients. The diet consisted of 20% carbohydrates (starch), 20% protein (casein), and 60% fat (primarily lard and soybean oil), providing 594 kcal per 100 g of dry weight. The macronutrient proportions were maintained as described, while ingredient sources were adapted to those available in our laboratory.
2.2.2. Selection of the test doses of C. nervosum seed extract
Based on a preliminary acute toxicity study following OECD guidelines [22], rats received single oral doses of 1,250, 2,500, or 5,000 mg/kg BW of C. nervosum seed extract and were observed for clinical signs, behavioral changes, and mortality during the first 30 minutes, followed by additional observations at 4, 24, and 48 hours post-administration. No mortality or significant adverse effects were observed. For the present study, one-tenth of these doses (125, 250, and 500 mg/kg BW) were selected to evaluate the effects on lipid profiles, liver enzyme activity, IL-6 levels, and oxidative stress in a hyperlipidemic rat model.
2.2.3. Experimental design and animal study
Forty-eight male Sprague–Dawley rats (7 weeks old, 240–270 g) were supplied by Nomura Siam International Co., Ltd. (Thailand) and housed in groups of three per cage under controlled environmental conditions (humidity 55% ± 5%, temperature 22°C ± 2°C, 12 hours light/12 hours dark). A one-week acclimatization period was provided, during which the rats received a standard commercial pellet diet (No. 082, C.P. Company, Bangkok, Thailand) and drinking water ad libitum prior to the commencement of dietary treatments. As depicted in Figure 2, after the 7-day acclimation period, all rats were randomly assigned to six groups (eight rats per group) as follows:
 | Figure 2. Workflow diagram of experimental design. ALP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate aminotransferase; C. nervosum = Cleistocalyx nervosum var. paniala; HFD = high-fat diet; IL-6 = interleukin-6. [Click here to view] |
- Group 1 (Control): Standard pellet diet (20% energy from fat) + 1 ml distilled water.
- Group 2 (HFD): High-fat diet.
- Group 3 (Simvastatin; SIM 10): HFD + simvastatin 10 mg/kg BW.
- Group 4 (C. nervosum 125): HFD + C. nervosum seed extract 125 mg/kg BW.
- Group 5 (C. nervosum 250): HFD + C. nervosum seed extract 250 mg/kg BW.
- Group 6 (C. nervosum 500): HFD + C. nervosum seed extract 500 mg/kg BW.
Simvastatin was prepared as a suspension in distilled water, vortexed thoroughly before administration, and delivered by oral gavage immediately after preparation to ensure uniform dosing, as described in previous studies [23]. Distilled water was also used as the vehicle for dissolving the C. nervosum seed extract. The total oral administration volume per rat did not exceed 1 ml and was delivered once daily via gavage for four consecutive weeks at the same time each day (8:00–9:00 a.m.). Body weight, as well as food and water intake, were measured on a weekly basis.
On day 29, rats were fasted overnight (16 hours) with restricted water access and anesthetized with thiobutabarbital sodium (100 mg/kg, intraperitoneally). Blood was collected via the abdominal vein, allowed to clot at room temperature, and serum was separated by centrifugation at 3,000 rpm for 20 minutes at 4°C. Serum was used to analyze lipid profiles, IL-6, and liver enzymes (AST, ALT, ALP). Livers were immediately removed, rinsed with ice-cold 0.9% NSS solution, weighed, and homogenized in ice-cold phosphate-buffered saline (PBS, 5 mM, pH 7.4). Homogenates were centrifuged at 16,000 rpm for 20 minutes at 4°C, and the supernatants were analyzed for malondialdehyde (MDA), an end product of lipid peroxidation, antioxidant enzyme activities [superoxide dismutase (SOD) and glutathione peroxidase (GPx)], and protein content
2.3. Biochemical parameters determination
2.3.1. Evaluation of lipid profiles
Serum levels of TG, TC, and HDL were analyzed using an automatic dry-chemistry analyzer (Fuji Dri-Chem 3500, Fujifilm, Tokyo, Japan). LDL cholesterol was derived using the Friedewald equation (LDL = TC − HDL − TG/5; all values expressed in mg/dl) [24]. The TG concentrations ranged from X to Y mg/dl, within the acceptable range for this calculation. Although originally developed for humans, the Friedewald formula is widely employed in rat studies to estimate LDL [25,26].
2.3.2. Evaluation of liver enzyme levels
Serum activities of AST, ALT, and ALP, indicators of liver-protective properties of C. nervosum seed extract, were analyzed using an automated clinical chemistry analyzer based on the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) methods [27].
2.3.3. Evaluation of IL-6 concentrations in serum
Serum IL-6 levels were quantified using a rat IL-6 ELISA kit (No. ER0042, Fine Biotech, China) in accordance with the manufacturer’s guidelines. The detection limit of this assay was 37.5 pg/ml.
2.3.4. Evaluation of liver lipid peroxidation
Hepatic content of MDA was quantified using the thiobarbituric acid reactive substances assay, adapted from the method of Buege and Aust [28]. Briefly, 100 μl of liver supernatant was reacted with 2 ml of TBA-TCA-HCl solution (0.37% thiobarbituric acid, 0.25 N hydrochloric acid, and 10% trichloroacetic acid) and incubated for 60 minutes at 95°C in a water bath. After incubation, the mixture was cooled in an ice bath and centrifuged at 12,000 rpm for 10 minutes at 4°C. The absorbance of the resulting supernatant and standard solutions was read at a wavelength of 535 nm with the aid of the spectrophotometer, with 1,1,3,3-tetraethoxypropane as the standard, and presented as nmol/mg protein.
2.3.5. Evaluation of liver antioxidant enzyme activities
The impact of crude C. nervosum seed extract on hepatic SOD activity was assessed using a commercially available SOD assay kit (No.0544-MBS036924, MyBioSource, Inc., San Diego, USA). Rat liver SOD activity was determined spectrophotometrically at 450 nm, and results are presented as units per milligram of protein (U/mg protein).
Liver GPx activity was quantified using a commercial rat GPx assay kit (No. MBS727547, MyBioSource, Inc., San Diego, USA), which was detected spectrophotometrically at 450 nm and reported as U/mg of protein.
2.3.6. Evaluation of protein concentrations assay
Protein concentrations in the samples were quantified using the Bradford method [29], with values derived from a standard curve established using bovine serum albumin.
2.4. Statistical analysis
All numerical data are expressed as mean ± SD. Normality of data distribution was assessed using the Shapiro–Wilk test, and homogeneity of variances was evaluated using Levene’s test. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was applied for multiple group comparisons. Exact p-values are reported in the Results section. Additionally, 95% confidence intervals and effect sizes (Cohen’s d) were calculated for key comparisons to provide further information on the magnitude and precision of the effects, even if not shown in all tables and figures. Data analysis was performed using GraphPad Prism software, version 11.5.
3. RESULTS
3.1. Percentage yield, antioxidant activity, and phytochemical screening of crude C. nervosum seed extract
Quantitative analysis of the crude ethanolic C. nervosum seed extract contained the concentrations of TPC (284.437 ± 0.113 mg GAE/g DW). In the DPPH assay, the seed extract exhibited an IC50 value of 3,173 ± 1.07 μg/ml, which was substantially higher than that of the Trolox standard (IC50 = 85.17 ± 1.02 μg/ml), indicating lower in vitro free radical scavenging potency (Table 1). Qualitative phytochemical screening confirmed the presence of tannins, alkaloids, terpenoids, flavonoids, and phenols. The extraction yield was 7.5%. A summary of these findings is provided in Table 2.
Table 1. Total phenolic content and DPPH radical scavenging potential of crude C. nervosum seed extract.
| Sample | Total phenolic content (mg GAE/g DW) | DPPH assay IC50 (μg/ml) |
|---|---|---|
| A 70% ethanolic extract of C. nervosum seed | 284.437 ± 0.113 | 3,173 ± 1.07 |
| Trolox | - | 85.17 ± 1.02 |
C. nervosum = Cleistocalyx nervosum var. paniala; DPPH = 2,2-diphenyl-1-picrylhydrazyl; GAE/g DW = gallic acid equivalent per gram of dry weight of the extract.
Table 2. Percentage yield and phytochemical composition screening of crude C. nervosum seed extract.
| Sample | Solvent | Phytochemical compounds | Qualitative and quantitative assessment |
|---|---|---|---|
| C. nervosum seed extract | 70% ethanol | Tannins | + |
| Alkaloids | + | ||
| Terpenoids | + | ||
| Phenols | + | ||
| Flavonoids | + | ||
| Reducing sugars | − | ||
| Percentage yield | 7.5% |
+ = presence; − = absent; C. nervosum = Cleistocalyx nervosum var. paniala.
3.2. Effect of crude C. nervosum seed extract on body weight, weight gain, and food consumption
Figure 3A shows the weekly average body weight of each experimental rat group over the 4-week study period. At baseline (week 0), there were no significant differences in mean body weight among the groups. From week 2 onward, body weight increased significantly (p < 0.05) in the HFD-only group compared to the control group, and this trend persisted throughout the study. In contrast, rats receiving simvastatin or any dose of C. nervosum seed extract did not show significant differences in mean body weight compared to either the control or HFD groups. This pattern was reflected in weight gain data: HFD fed rats exhibited a significant increase in body weight gain relative to controls (p < 0.01), whereas simvastatin and extract treated groups showed no significant differences from either the control or HFD groups (Fig. 3B). Additionally, as illustrated in Figure 3C, food consumption did not differ significantly among any of the experimental groups throughout the study
 | Figure 3. Effect of crude C. nervosum seed extract on: A) average body weight (g), B) body weight gain (g), and C) food consumption (g/24 h) in hyperlipidemic rats. Data are presented as mean ± SD, with n = 8 per group. Asterisks indicate significant differences: ***p < 0.001, **p < 0.01, and *p < 0.05 versus the control group. [Click here to view] |
3.3. Effect of crude C. nervosum seed extract on lipid profiles
As shown in Figure 4A–D, HFD induced hyperlipidemia in rats, evidenced by significantly elevated serum levels of TC, TG, and LDL (p < 0.0001 for all), along with a significant reduction in HDL (p < 0.001) compared to controls. Treatment with simvastatin (p < 0.05) and all doses of C. nervosum seed extract (p < 0.01 for all) significantly reduced TC, TG, and LDL levels relative to the hyperlipidemic group. Additionally, these treatments significantly increased serum HDL concentrations (p < 0.001 for all) compared to HFD-fed rats.
 | Figure 4. Effect of crude C. nervosum seed extract on: A) TC, B) TG, C) HDL, and D) LDL in hyperlipidemic rats. All data are expressed as mean ± SD, with n = 8 per group. Statistical significance versus the control group is indicated by asterisks (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05), while ampersands (&&&p < 0.001, &&p < 0.01, &p < 0.05) denote significance versus the hyperlipidemic group. [Click here to view] |
3.4. Effect of crude C. nervosum seed extract on liver enzyme levels
As depicted in Figure 5A–C, HFD consumption induced liver inflammation or damage, indicated by significantly elevated AST, ALT, and ALP levels in hyperlipidemic rats compared to control (p < 0.001 for all). Supplementation with all doses of crude C. nervosum seed extract significantly attenuated these elevations (p < 0.05 for all) relative to the hyperlipidemic group, without causing significant changes compared to the control group. Notably, simvastatin treatment significantly increased AST, ALT, and ALP levels (p < 0.05 for all) compared to controls.
 | Figure 5. Effect of crude C. nervosum seed extract on: A) AST, B) ALT, and C) ALP in hyperlipidemic rats. All data are expressed as mean ± SD, with n = 8 per group. Statistical significance versus the control group is indicated by asterisks (***p < 0.001, *p < 0.05), while ampersands (&p < 0.05) denote significance versus the hyperlipidemic group. [Click here to view] |
3.5. Effect of crude C. nervosum seed extract on liver antioxidant enzyme activities, lipid peroxidation, and serum pro-inflammatory cytokine IL-6 level
As presented in Figure 6A–C, rats treated with an HFD alone exhibited a significant reduction in hepatic antioxidant enzymes, specifically SOD (p < 0.01) and GPx (p < 0.05), alongside a significant induction in MDA (p < 0.001) compared to controls. Conversely, hyperlipidemic rats treated with all doses of C. nervosum seed extract exhibited significantly higher SOD and GPx activities (p < 0.05 for all doses) and lower MDA concentrations relative to the HFD group. Hyperlipidemic rats treated with simvastatin, however, showed no significant changes in these parameters compared to HFD alone.
 | Figure 6. Effect of crude C. nervosum seed extract on: A) SOD, B) GPx, C) MDA, and D) IL-6 in hyperlipidemic rats. All data are expressed as mean ± SD, with n = 8 per group. Statistical significance versus the control group is indicated by asterisks (***p < 0.001, **p < 0.01, *p < 0.05), while ampersands (&&p < 0.01, &p < 0.05) denote significance versus the hyperlipidemic group. [Click here to view] |
Furthermore, our results also revealed that HFD intake significantly induced pro-inflammatory cytokine IL-6 levels (p < 0.05) compared to control. Nevertheless, all doses of crude C. nervosum seed extract and simvastatin significantly reduced IL-6 levels (all p < 0.05 for all, except 250 mg/kg, p < 0.01 relative to the HFD group, as illustrated in Figure 6D.
4. DISCUSSION
Herbal by-products are rich in bioactive compounds with therapeutic potential against chronic diseases, including dyslipidemia [30]. In this study, oral supplementation with crude C. nervosum seed extract improved lipid metabolism, enhanced liver function, and reduced inflammation and oxidative stress in HFD-fed rats. These effects were mediated by upregulation of antioxidant enzymes (SOD, GPx), suppression of lipid peroxidation (reduced hepatic MDA), and a marked decrease in the pro-inflammatory cytokine IL-6, without mortality or observable side effects.
In our study, 70% ethanol was chosen as the extraction solvent because it efficiently recovers both polar and non-polar phytochemicals, thereby maximizing yield and preserving the bioactivity of compounds such as phenolics, flavonoids, tannins, alkaloids, and terpenoids [31]. Previous studies reported that dried C. nervosum seed extract contains phenolics, flavonoids, chalcones, phytosterols, alkaloids, terpenoids, and anthocyanins [13,32]. Our phytochemical screening confirmed the presence of alkaloids, tannins, terpenoids, phenols, and flavonoids, with tannins identified as particularly prominent compounds not previously documented. These differences may be attributed to variations in plant species, extraction method, solvent polarity, harvest period, or environmental factors.
Chronic HFD consumption has been shown to induce hyperlipidemia and oxidative stress, manifested by elevated TC, TG, LDL, and MDA, along with reduced HDL and antioxidant enzyme activities [33,34]. Elevated oxidative stress, in turn, damages various cells, particularly hepatocytes, leading to increased liver enzymes ALT, AST, and ALP, which serve as indicators of liver injury [35]. Dyslipidemia and oxidative stress also promote chronic low-grade inflammation, evidenced by increased circulating IL-6 levels [36]. This establishes a vicious cycle: HFD induces dyslipidemia and oxidative stress, which in turn exacerbates inflammation and liver dysfunction, perpetuating systemic metabolic imbalance [37]. Consistent with these findings, rats fed an HFD for 4 weeks in our study exhibited significant hyperlipidemia, elevated hepatic MDA, reduced SOD and GPx activities, increased IL-6 levels, and higher liver enzyme concentrations. Simvastatin, a widely prescribed HMG-CoA reductase inhibitor, lowers cholesterol and cardiovascular risk and exerts pleiotropic effects, including anti-inflammatory, vascular protective, antioxidant, and immunomodulatory actions, while improving plaque stability and endothelial function [38]. In this study, however, SOD and GPx activities in the simvastatin group were lower than controls, though not significantly, possibly due to dosage or treatment duration affecting antioxidant responses.
Nutraceuticals derived from agricultural by-products are increasingly recognized as promising agents for managing hyperlipidemia due to their antioxidant, hypolipidemic, and hepatoprotective properties [39]. Although several studies have reported potent antioxidant activity of C. nervosum extracts, particularly from fruits or when extracted with organic solvents [11,12], our aqueous seed extract exhibited weak radical scavenging potency in the DPPH assay, with an IC50 value substantially higher than that of Trolox. This discrepancy may be explained by differences in the extraction method, plant part, and phytochemical profile. Organic solvents typically yield higher concentrations of phenolic and flavonoid compounds, while aqueous preparations often show lower activity. In addition, seeds and fruits differ in their phytoconstituent composition, and the DPPH assay itself may underestimate antioxidant potential when activity is mediated through indirect mechanisms, such as activation of endogenous defenses, rather than direct free radical neutralization.
Despite its weak in vitro activity, in vivo administration of the seed extract attenuated oxidative stress in hyperlipidemic rats by reducing hepatic MDA levels, enhancing endogenous antioxidant enzymes (SOD, GPx), and improving lipid profiles. These effects are likely mediated by phytochemicals such as phenols, flavonoids, tannins, terpenoids, and alkaloids, which can neutralize reactive oxygen species, chelate transition metals, and activate the Nrf2 pathway to upregulate endogenous antioxidant defenses [40]. Our phytochemical analysis confirmed the presence of tannins, which may contribute to reduced MDA through free radical scavenging, metal chelation, and induction of antioxidant enzymes, consistent with previous reports [41,42]. Thus, the extract’s protective actions are more plausibly attributed to indirect mechanisms, including enhancement of endogenous antioxidant defenses, modulation of pro-inflammatory pathways, and improvements in lipid metabolism rather than strong direct radical scavenging. Importantly, limited in vitro activity does not preclude meaningful biological effects in vivo, and the observed antioxidant and anti-lipid peroxidation effects are likely the result of synergistic interactions among multiple bioactive compounds rather than the action of a single constituent.
Furthermore, C. nervosum seed extract demonstrated significant hypolipidemic effects. Treatment lowered TC, TG, and LDL concentrations while elevating HDL levels. The reduction in TC was accompanied by a marked decrease in LDL, suggesting enhanced LDL clearance, possibly via upregulation of hepatic LDL receptors that promote cholesterol elimination through conversion to bile acids [43]. The elevation in HDL implies a stimulatory effect on lecithin–cholesterol acyltransferase, facilitating reverse cholesterol transport [44,45]. The observed TG-lowering effect may be due to enhanced activity of lipoprotein lipase (LPL), which hydrolyzes TG-rich lipoproteins such as VLDL and chylomicrons, promoting their clearance and uptake by peripheral tissues [46]. Another possible mechanism involves modulation of intestinal lipid absorption through the inhibition of pancreatic lipase, thereby reducing dietary TG assimilation [47].
In addition to improving lipid metabolism, C. nervosum seed extract demonstrated anti-inflammatory and hepatoprotective properties. Treatment significantly reduced IL-6 levels, thereby attenuating hepatic inflammation and protecting against liver injury. These benefits, together with enhanced endogenous antioxidant enzyme activities and reduced MDA, contributed to improved liver function, as reflected by lower ALT, AST, and ALP levels. Since IL-6 is closely linked to hepatic inflammation, altered lipid metabolism, and hepatocellular injury [48,49], its reduction highlights the extract’s therapeutic relevance.
In contrast, liver enzymes were elevated in the simvastatin-treated group, which may indicate hepatocellular stress. Although simvastatin undergoes hepatic metabolism and has been associated with enzyme elevations in both experimental and clinical studies [50,51], such effects are generally uncommon and dose dependent. In the absence of histological evidence, these findings should be interpreted with caution and may reflect transient hepatic stress, formulation-related variability, or small sample effects rather than definitive hepatotoxicity.
Despite this, the hypolipidemic effect of C. nervosum seed extract was comparable to that of simvastatin, but without adverse changes in food intake, body weight, or weight gain, suggesting a targeted effect on lipid metabolism rather than overall energy balance. While these findings are promising, further studies are required to isolate and characterize the active constituents and to clarify the molecular mechanisms underlying the extract’s lipid-lowering and anti-inflammatory effects. Such insights will be essential for advancing this extract toward potential clinical applications
Finally, this research aligns with global health priorities. By exploring the therapeutic use of an underutilized plant resource, it contributes to SDG 3: Good Health and Well-being by offering a natural, accessible strategy to combat non-communicable diseases such as dyslipidemia, and to SDG 12: Responsible Consumption and Production through the sustainable valorization of agricultural by-products.
5. CONCLUSION
This study provides compelling evidence that oral administration of crude C. nervosum seed extract can effectively modulate lipid metabolism, improve liver function, and alleviate both inflammation and oxidative stress in HFD-induced hyperlipidemic rats. These protective effects appear to be mediated through the upregulation of endogenous antioxidant defenses and the suppression of pro-inflammatory pathways. Beyond its biomedical potential, the use of C. nervosum seed extract also supports the sustainable valorization of agro-industrial by-products, aligning with public health and environmental objectives. Collectively, our findings highlight C. nervosum seed extract as a promising candidate for development into a novel nutraceutical for the prevention and management of hyperlipidemia and associated metabolic disorders. Nevertheless, the specific bioactive compounds and the precise molecular mechanisms responsible for its hypolipidemic effects remain unclear, representing an important avenue for future research.
6. ACKNOWLEDGMENTS
The authors gratefully acknowledge financial support from the University of Phayao and Thailand Science Research and Innovation (TSRI) through the Fundamental Fund 2025 (Grant No. 5067/2567).
7. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
8. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
9. ETHICAL APPROVALS
The study protocol was approved by the Animal Ethics Committee of School of Medical Sciences, University of Phayao, Thailand (Approval No. 1-035-67). All experimental procedures complied with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1985).
10. DATA AVAILABILITY
All data generated and analyzed are included in this research article.
11. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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