Marmelosin Protects Against Metabolic Disturbances in High-Fat Diet an

Introduction

Type 2 Diabetes Mellitus (T2DM) belongs to a chronic metabolic disorder with a rapidly escalating global incidence, primarily attributed to contemporary lifestyle factors such as excessive consumption of energy-rich diets and diminished physical activity. An array of symptoms, including polyuria, polydipsia, unintended weight loss, fatigue, and visual impairments, characterizes it.1,2 T2DM pathogenesis involves a multifaceted etiology encompassing both impaired insulin secretion and abnormal insulin sensitivity in peripheral tissues. This intricate pathophysiology leads to elevated blood glucose levels, a hallmark of this debilitating condition.3 The pathogenesis of T2DM is associated with an insulin resistance condition distinguished by the abnormal biological response of several target tissues to insulin stimulation. This reduced insulin sensitivity is exacerbated by factors including obesity, physical inactivity, and aging. Pancreatic islets enlarge and increase β-cell mass to produce more insulin in response to insulin resistance. However, when this compensatory mechanism proves insufficient to maintain euglycemia, hyperglycemia ensues, marking the onset of T2DM.4,5 Diabetic conditions are characterized by hyperglycemia, hyperlipidemia, and inflammation, which converge to impair pancreatic β-cell function.6 These metabolic disturbances primarily induce endoplasmic reticulum (ER) stress, oxidative deficit, and mitochondrial dysfunction, leading to β-cell damage and dedifferentiation. While ER stress as well as mitochondrial dysfunction independently contribute to β-cell dysfunction and apoptosis, their synergistic role in exacerbating oxidative stress has emerged as a crucial factor.7 The β-cells are more susceptible to oxidative stress resulting from high endogenous ROS generation and low antioxidant capabilities, highlighting the pivotal role of oxidative stress in β-cell failure.6 Reduced expression of key antioxidant enzymes in these cells exacerbates their sensitivity to diabetogenic agents and promotes cytotoxicity.8

Streptozotocin (STZ), a glucose analog derived from Streptomyces achromogenes, selectively targets and destroys pancreatic beta cells in rodent models, serving as a widely utilized tool for inducing experimental diabetes. This specificity arises from its uptake via the GLUT2 glucose transporter, which is highly expressed in these cells. STZ-induced DNA damage leads to β-cell destruction, which subsequently results in hyperglycemia, serving as a widely employed rodent model for experimental diabetes.9 The consumption of high-fat diets (HFD) can cause insulin resistance and hyperinsulinemia, which are characteristic features of T2DM and often fail to elicit hyperglycemia. This suggests limitations in their ability to recapitulate the complex pathophysiology of human T2DM. Conversely, STZ, particularly at low doses, can induce a more gradual impairment of insulin secretion, resembling the progressive decline observed in late-stage T2DM. This makes it a valuable tool for assessing the mechanisms underlying β-cell dysfunction in this condition.10,11

Numerous conventional therapeutic interventions exist for managing the complications associated with diabetes. However, the potential for adverse side effects associated with pharmacological agents has increased interest in exploring herbal remedies. These natural alternatives offer maximal preventative benefits while minimizing the risk of undesirable side effects.12 Marmelosin, a naturally occurring coumarin derivative, is predominantly isolated from Aegle marmelos (Bael), a plant widely used in traditional medicine systems. It has been reported to exhibit a broad spectrum of pharmacological activities, including anti-inflammatory and antioxidant effects.13,14 Several studies have demonstrated the hypoglycemic potential of Aegle marmelos in experimental models of diabetes, where it effectively reduced blood glucose levels and improved lipid profiles.15,16 These beneficial effects are attributed to the compound’s phytoconstituent properties, although detailed mechanistic pathways remain to be fully elucidated.

Existing studies highlight Aegle marmelos as a promising natural remedy for managing T2DM, but there is no evidence yet on marmelosin effects on HFD and STZ-induced T2DM rats. This research investigated the effects of marmelosin, a natural compound, in rats with T2DM induced by HFD and STZ. The study assessed marmelosin potential to protect pancreatic and liver tissues, its impact on oxidative stress biomarkers, and its ability to reduce diabetes-related complications.

Materials and Methodology
Drugs and Chemicals

Marmelosin (HPLC purity: 99.32%), STZ, and Enzyme-linked immunosorbent assays (ELISA) kits were procured from MSW Pharma, M.S., India. ELISA was used to quantify biochemical parameters, including tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The glucose was quantified utilizing a commercially available glucometer kit (Accu-Check Active, Roche Diagnostics, India).

Animal

This experimental study utilized 24 adult male Wistar rats (10–12 weeks old, 180 ± 20 g) housed under standard conditions in a controlled 12-hour light/dark cycle. Rats were given laboratory chow ad libitum and had unrestricted access to water. All experimental procedures were performed in strict accordance with the ethical use of laboratory animals, following the guidelines and recommended procedures of the National Committee of Bioethics (NCBE). Study rats were obtained from Batterjee Medical College, Jeddah, Saudi Arabia. The experimental protocol received approval from the Institutional Research Board of Batterjee Medical College, Jeddah (RES-2025-0017).

Experimental Design
Induction of DM

To induce T2DM, the experimental protocol outlined by Gaballah et al17 was adopted. Rats underwent a one-week acclimation phase prior to receiving unrestricted access to HFD. After two weeks of dietary intervention, rats on the HFD were given a single injection of STZ [35 mg/kg/intraperitoneal (i.p.)]. After 72 hrs of STZ administration, FBG levels were measured. Animals with blood glucose levels exceeding 250 mg/dL were considered to have T2DM and were subsequently used for further experimental investigations.

Experimental Protocol

Following the establishment of the experimental groups, rats (n=6 per group) were randomly selected using a simple method for the treatment protocol for the duration of twenty-eight days.

• Group I: Served as the control and received 0.5 mL of normal saline.
• Group II: Served as diabetic control (T2DM control) and received STZ and HFD.
• Group III: Served as the treatment group, and administered 10 mg/kg orally Marmelosin.
• Group IV: Served as the treatment group, and administered 20 mg/kg orally Marmelosin.

Following completion of the treatment protocol, blood was obtained from the retro-orbital plexus of the rats. Before the collection of blood, rats were euthanized utilizing ketamine (75 mg/kg/i.p.) and xylazine (10 mg/kg/i.p.). Following anesthesia, blood samples were subsequently subjected to biochemical analyses. The pancreas was subsequently isolated for further biochemical and histopathological investigations. Histopathological assessments were conducted by an investigator blinded to the treatment groups to ensure unbiased evaluation.18–20 The experimental protocol design is illustrated in Figure 1.

Figure 1 Design of the experiment.

Body Weight

Body weight was determined at baseline and at intervals following the induction of diabetes in experimental rats. This parameter serves as a crucial indicator of the rat’s overall health and well-being within the experimental paradigm.

Biochemical Parameters
Estimation of Biochemical Parameters

The liver was isolated, rinsed with ice-cold saline, and homogenized in a buffer containing 0.1 M Tris–HCl at pH 7.4 to measure antioxidant enzyme activity.

Determination of Blood Glucose and HbA1c

To evaluate diabetic status, blood glucose levels were monitored in fasted rats (6 h) via a non-invasive tail-prick method utilizing a commercial glucometer. Blood samples were collected at different intervals throughout the study. Additionally, glycated hemoglobin (HbA1c) levels were quantified using commercially available assay kits (MSW Pharma, India).

Determination of Insulin and Insulin Resistance

The concentration of serum insulin was determined using an ELISA as per the manufacturer’s standard protocol. The HOMA-IR index was used to estimate insulin sensitivity using values of glucose and insulin, respectively. Samples were analyzed in triplicate, with insulin levels reported as μU/mL. The HOMA-IR was determined based on the following formula, thereafter.21

Determination of Lipid Profile

The whole blood was centrifuged at room temperature for 10 minutes to separate the serum from the remaining cellular components within the supernatant. The isolated serum was then stored at temperatures below 50°C to ensure sample stability. Commercially available kits from MSW Pharma, India, were used for the accurate quantification of total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL-C) concentrations.

Determination of Biomarkers

Serum biomarkers indicative of pancreatic function were quantified. This included the assessment of ALT, ALP, and AST utilizing a bioanalyzer and adhering to the manufacturer’s protocols. Enzyme concentrations were subsequently expressed as units per liter (U/L).

Determination of Antioxidant Enzymes

Superoxide dismutase (SOD) was quantified utilizing the Misra and Frodvich method. A 0.2 mL aliquot of pancreatic homogenate supernatant was combined with 0.8 mL of 50 mM glycine buffer (pH 10.4) to initiate the reaction. After a 5-minute incubation, epinephrine (0.02 mL) was added, and the absorbance was quantified spectrophotometrically at 480 nm.22

Reduced glutathione (GSH) levels were determined using the Ellman method with minor modifications.23 Tissue homogenates were deproteinized with 10% TCA, and the supernatant was reacted with DTNB in phosphate buffer (0.2 M, pH 8.0). The absorbance of the sample was determined at 412 nm.

Catalase (CAT) enzyme was quantified using a modified colorimetric method adapted from Sinha et al.24 This involved measuring the absorbance at 570 nm following the reaction of the enzyme with hydrogen peroxide(H2O2) in the presence of glacial acetic acid.

Determination of Inflammatory Markers

The pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) were evaluated utilizing commercially available ELISA kits. These kits, utilizing antibody-based capture within microplates, allowed the precise quantification of each cytokine. Data were expressed as picograms of cytokine per milliliter (pg/mL) of the sample.

Apoptotic Marker

The quantification of apoptotic markers, specifically Caspase 3, was determined through the utilization of commercially available ELISA kits. Experimental procedures adhered to the manufacturer’s recommended protocol.

Histopathology of the Pancreas

Pancreatic tissues from experimental rats were collected and subjected to standard histological processing. Briefly, Pancreatic tissues were fixed in 10% formalin, dehydrated in a graded ethanol series, and embedded in paraffin. 5-μm-thick sections were cut on a rotary microtome, deparaffinized, and rehydrated. Tissue sections were stained with hematoxylin and eosin (H&E) and subsequently visualized under light microscopic examination at a magnification of 40x.

Statistical Analysis

Statistical analysis of the experimental data was performed using GraphPad Prism software (Version 8.02), and the data are represented as the mean ± SEM. The normality of the result data was quantified by using the Shapiro–Wilk test before applying parametric tests. Since the data followed a normal distribution, one-way ANOVA was used to compare group means, with a significance level set at P ≤ 0.05 (n = 6 animals per group). When ANOVA indicated significant differences, Tukey’s post hoc test was employed to identify which groups differed.

Result

The present study directly evaluated the anti-diabetic effects of isolated marmelosin using standardized biochemical methods and repeated administration in diabetic animal models. Previous research has primarily assessed whole plant extracts or composite phytoconstituents. In contrast, this study focuses on marmelosin as an active compound, evaluates its hypoglycemic activity, and compares its performance with that of synthesized derivatives and standard controls. These findings provide new insights into the pharmacological potential of marmelosin, substantiating its role as a unique and promising agent in diabetes management compared to conventional treatments.

Outcome of Marmelosin on Body Weight

Figure 2 illustrates the outcome of marmelosin on body weight in HFD/STZ-induced T2DM. A marked elevation in body weight was found in rats fed with HFD compared with the control group prior to STZ administration or marmelosin treatment. Following STZ injection, a significant weight loss was evident in the diabetic groups. Conversely, T2DM rats treated with both doses of marmelosin exhibited a marked increase in body weight [F(3, 40) = 2.934, P = 0.0449].

Figure 2 Outcome of Marmelosin on body weight in HFD/STZ-induced T2DM rats. #P < 0.001 vs normal.

Outcome of Marmelosin on Blood Glucose and HbA1C

Figure 3A and B represents the outcome of marmelosin on FBG and HbA1c levels in rats subjected to HFD and STZ injection to induce T2DM. A marked elevation in both FBG and HbA1c was found in the HFD/STZ-induced T2DM rats compared with the control group, confirming the establishment of a diabetic state. Notably, these elevated levels were markedly attenuated by marmelosin treatment. Administration of both doses (10 and 20 mg/kg) of marmelosin demonstrated a marked reduction in FBG [F (3, 24) = 110.6, P<0.0001] and HbA1c [F (3, 20) = 10.36, P=0.0003].

Figure 3 (A and B) Outcome of marmelosin on (A) FBG and (B) HbA1c levels in HFD/STZ-induced T2DM rats. #P < 0.001 vs normal, *P < 0.05, **P< 0.01 vs HFD/STZ.

Outcome of Marmelosin on Insulin and Insulin Resistance

Figure 4A and B depicts the outcome of marmelosin on insulin and insulin resistance, as assessed by HOMA-IR, in HFD/STZ-induced T2DM rats. In contrast to the control group, the HFD/STZ-induced T2DM rats exhibited a marked reduction in plasma insulin levels, accompanied by a concomitant increase in HOMA-IR, indicative of insulin resistance. Notably, marmelosin treatment effectively ameliorated these metabolic abnormalities. Both doses of marmelosin (10 and 20 mg/kg) show significant elevation of plasma insulin [F (3, 20) = 7.565, P=0.0014] and a concomitant decrease in HOMA-IR [F (3, 20) = 6.798, P=0.0024].

Figure 4 (A and B) Outcome of marmelosin on (A) insulin and (B) HOMA-IR in HFD/STZ-induced T2DM rats. #P < 0.001 vs normal, *P < 0.05, **P< 0.01 vs HFD/STZ.

Outcome of Marmelosin on Lipid Profile

Figure 5A–C illustrates the outcome of marmelosin on the lipid profile of rats subjected to HFD and STZ administration to evoke T2DM. A marked elevation in serum concentrations of TC and TG was observed in the STZ/HFD-induced T2DM group compared to the normal control group, while HDL-C levels were significantly decreased. Conversely, marmelosin treatment demonstrated a significant reduction of TC and TG levels, concurrently with an elevation of HDL-C levels. Notably, both tested doses of marmelosin (10 and 20 mg/kg) exhibited a significant ameliorative effect on the serum lipid profile such as TC [F (3, 20) = 30.81, P<0.0001], TG [F (3, 20) = 90.11, P<0.0001], HDL-C [F (3, 20) = 14.98, P<0.0001] in diabetic rats, suggesting a potential protective role against dyslipidemia compared with T2DM.

Figure 5 (A–C) Outcome of marmelosin on the lipid profile: (A) TC, (B) TG, and (C) HDL-C in HFD/STZ-induced T2DM rats. #P < 0.001 vs normal, *P < 0.05, **P< 0.01 vs HFD/STZ.

Outcome of Marmelosin on Biomarkers

Figure 6A–C illustrates the outcome of marmelosin on several biomarkers, including ALT, AST, and ALP, of rats subjected to HFD/STZ administration to induce T2DM. The concentration of these markers was significantly higher in the STZ/HFD-induced T2DM control rats compared to the normal control rats. However, administration of marmelosin lowered these elevated levels of ALT [F (3, 20) = 29.09, P<0.0001], AST [F (3, 20) = 25.00, P<0.0001], and ALP [F (3, 20) = 16.10, P<0.0001]in HFD/STZ-induced T2DM rats.

Figure 6 (A–C) Outcome of marmelosin on several biomarkers: (A) ALT, (B) AST, and (C) ALP in HFD/STZ-induced T2DM rats. #P < 0.001 vs normal, *P < 0.05, **P< 0.01 vs HFD/STZ.

Outcome of Marmelosin on Antioxidant Enzymes

Figure 7A–C depicts the outcome of marmelosin on key antioxidant enzymes, namely SOD, GSH, and CAT, in HFD/STZ-induced T2DM rats. The diabetic rats demonstrated lower concentrations of GSH, SOD, and CAT compared to the normoglycemic control group. This reduction in antioxidant enzyme activity strongly suggests the presence of oxidative stress damage in diabetic rats. Notably, administration of marmelosin at both doses effectively reversed these alterations in the antioxidant enzymes such as SOD [F (3, 20) = 8.194, P=0.0009], GSH [F (3, 20) = 6.103, P=0.0040], and CAT [F (3, 20) = 10.56, P=0.0002] levels observed in diabetic rats.

Figure 7 (A–C) Outcome of marmelosin on antioxidant enzymes: (A) SOD, (B) GSH, and (C) CAT in HFD/STZ-induced T2DM rats. #P < 0.001 vs normal, *P < 0.05, **P< 0.01 vs HFD/STZ.

Outcome of Marmelosin on Inflammatory Markers

Figure 8A–C represents the outcome of marmelosin on inflammatory cytokines (IL-1β, IL-6, and TNF-α) in rats with T2DM induced by HFD/STZ injection. Compared to normoglycemic controls, rats with HFD/STZ-induced T2DM exhibited significantly higher levels of these pro-inflammatory cytokines. Notably, the administration of both doses of marmelosin effectively attenuated the augmented expression of IL-1β [F (3, 20) = 59.76, P<0.0001], IL-6 [F (3, 20) = 39.24, P<0.0001], and TNF-α [F (3, 20) = 83.15, P<0.0001].

Figure 8 (A–C) Outcome of marmelosin on the expression of pro-inflammatory cytokines (A) IL-1β, (B) IL-6, and (C) TNF-α in HFD/STZ-induced T2DM rats. #P < 0.001 vs normal, *P < 0.05, **P< 0.01 vs HFD/STZ.

Outcome of Marmelosin on Apoptotic Marker

Figure 9 illustrates the impact of marmelosin on caspase-3 expression, an established marker of apoptosis, in a rat with T2DM induced by HFD/STZ. Compared to normoglycemic control rats, the HFD/STZ-treated group demonstrated a significant upregulation of caspase-3 levels. However, treatment with both doses of marmelosin resulted in a significant attenuation of this elevated caspase-3 [F (3, 20) = 12.50, P<0.0001] expression.

Figure 9 Outcome of marmelosin on caspase-3 expression in HFD/STZ-induced T2DM rats. #P < 0.001 vs normal, *P < 0.05, **P< 0.01 vs HFD/STZ.

Histopathology

Histological examination of pancreatic tissue samples, as depicted in Figure 10, revealed a spectrum of pathological alterations in diabetic rats. Control rats exhibited no discernible histological abnormalities. In contrast, untreated diabetic rats displayed the most pronounced pancreatic pathology, characterized by cellular degeneration, interstitial lymphocytic infiltration, and progressive necrosis. This was further evidenced by the significant disruption of islet architecture, including pyknotic nuclei, homogeneous cytoplasmic masses within islet cells, and the expansion of halofollicular cells with minor cytoplasmic vacuolation. Furthermore, pyknotic nuclei within the pancreatic parenchyma were observed alongside lymphocytic infiltration. Importantly, treatment with marmelosin at both doses demonstrated a significant attenuation of these pathological changes, encompassing a reduction in interstitial inflammation, amelioration of pancreatic follicle cell swelling and necrosis, and a notable improvement in the organization of islet architecture (Figure 10A–D).

Figure 10 (A–D) Pancreatic tissue sections were stained with H & E at X 100 to compare experimental groups. (A) Control rats show normal Langerhans islets and intralobular ducts. (B) HFD/STZ rats with abnormal Langerhans islet structure and degenerative changes in their nuclei (black arrow). (C) Marmelosin-10 rats with moderate hyperplasia of the islets (green arrow). (D) Marmelosin-20 rats with improved histopathological architecture (blue arrow).

Discussion

Numerous studies have documented that HFD in rats induces insulin resistance, while low-dose STZ administration selectively impairs insulin secretion, replicating the progressive nature of T2DM.25 The synergistic effect of HFD and low-dose STZ in inducing T2DM in rats is well-established. These pathological conditions contribute to pancreatic β-cell dysfunction and aberrant insulin signaling.26,27 This investigation aimed to evaluate the therapeutic potential of marmelosin, a bioactive compound derived from Aegle marmelos, in a rat model of Type 2 T2DM induced by a HFD and STZ. The study focused on assessing the effects of marmelosin on metabolic parameters, including fasting blood glucose, HbA1c, plasma insulin, insulin resistance (HOMA-IR), lipid profile, liver function enzymes, body weight, and pancreatic histopathology. These outcomes were evaluated to determine the overall protective efficacy of marmelosin against HFD/STZ-induced diabetic alterations.

Numerous investigations have demonstrated a positive correlation between HFD and increased body mass in rats. The weight gain in HFD-fed rats is likely attributed to the consumption of diets enriched in saturated fats, such as lard, resulting in a significant increase in dietary energy intake. Obesity, characterized by excessive fat accumulation primarily within white adipose tissue, arises from a persistent disequilibrium between caloric intake and energy expenditure. Conversely, the administration of low doses of STZ has been observed to alleviate body weight.10 Our study demonstrated a markedly reduced body weight among rats subjected to HFD/STZ administration to induce T2DM. Notably, treatment with marmelosin at doses of 10 and 20 mg/kg resulted in a marked increase in body weight. This finding suggests that marmelosin may possess the ability to restore body weight in T2DM rats, indicating its potential protective effect against HFD/STZ-induced weight loss. These results are consistent with previous research findings.28

Several studies demonstrated a synergistic approach to inducing a rat model of T2DM. HFD was initially employed to cause insulin resistance, followed by the administration of a low dose of STZ to selectively destroy pancreatic β-cells, which are the primary source of insulin within the islets of Langerhans. This combination effectively recapitulated key metabolic characteristics of human type 2 diabetes. The destruction of β-cells resulted in impaired glucose-stimulated insulin secretion, a hallmark of this disease.29,30 Insulin, an essential hormone, regulates metabolism by facilitating the uptake of glucose into tissues such as fat, skeletal muscle, and the liver. However, insulin resistance, often associated with obesity, disrupts the insulin signaling pathway, hindering the metabolic effects of insulin. This reduced insulin sensitivity, a prominent feature of T2DM, contributes to this chronic metabolic disorder’s elevated blood glucose levels.31

HbA1c quantifies the concentration of circulating hemoglobin molecules that have undergone glycation. This non-enzymatic process, where glucose irreversibly binds to hemoglobin, serves as a biological indicator of average blood glucose concentrations over a preceding period, typically spanning weeks to months. As a robust biomarker, HbA1c plays a crucial role in both the diagnosis and therapeutic management of diabetes mellitus. Notably, elevated HbA1c levels are strongly compared with the presence of T2DM.32

Diabetes and prediabetes pathophysiology primarily involve dysregulated glucose homeostasis. These factors exhibit varying degrees of influence across different racial and ethnic subgroups within the prediabetic population. Insulin resistance (HOMA-IR) has emerged as a more commonly employed surrogate marker in large-scale epidemiological investigations.33

The current investigation observed a significant elevation in both FBG and HbA1c levels in HFD/STZ-induced T2DM rats compared to controls, confirming the establishment of a diabetic state. Concurrently, these diabetic rats exhibited a reduction in plasma insulin and a concomitant increase in the HOMA-IR, indicative of insulin resistance. Notably, treatment with marmelosin significantly attenuated these elevated levels of FBG and HbA1c. Administration of both doses (10 and 20 mg/kg) of marmelosin demonstrated a marked reduction in FBG and HbA1c levels. Furthermore, marmelosin treatment effectively ameliorated these metabolic abnormalities by significantly elevating plasma insulin levels and concomitantly decreasing HOMA-IR. These findings collectively support the protective effects of marmelosin in ameliorating HFD/STZ-induced diabetes in rats.34,35

Studies demonstrated that HFD/STZ induces hyperglycemia, which stimulates insulin secretion, consequently activating hepatic lipogenesis and cholesterol excretion, thereby disrupting lipid metabolism, manifested as hyperlipidemia. The state of hyperlipidemia is characterized by elevated levels of TC, TG, and LDL-C, often accompanied by reduced HDL-C, hyperlipidemia arises from dysregulated lipid metabolism or transport. Chronic hyperlipidemia can lead to severe complications, including fatty liver and atherosclerosis. Both elevated lipids (lipotoxicity) and glucose (glucotoxicity) have been shown to impair β-cell function in both healthy and diabetic individuals.36 Studies have shown a high prevalence of lipid abnormalities in T2DM, with up to 92% exhibiting dyslipidemia.37

The present investigation demonstrated a hyperlipidemic state in the STZ/HFD-induced diabetic rat, characterized by elevated serum levels of TC and TG, and a concomitant decrease in HDL-C compared to the normoglycemic control group. Marmelosin, at both 10 and 20 mg/kg doses, significantly ameliorated this dyslipidemia, resulting in a notable reduction in TC and TG levels, and a concomitant increase in HDL-C. These outcomes strongly suggest that marmelosin exerts a beneficial effect on the lipid profile in the context of T2DM, aligning with previous research observations.38

The liver serves a crucial role in glucose homeostasis within the diabetic milieu, encompassing insulin clearance, glucose production, and the synthesis of inflammatory cytokines. Hepatic enzymes, including ALP, ALT, and AST, play a crucial role in these physiological processes. Deviations from normal serum levels of these enzymes can serve as sensitive indicators of hepatocellular or biliary tract injury. Moreover, elevated hepatic enzyme activity can signify underlying inflammation, which can disrupt insulin signaling pathways.39,40 Clinical and experimental evidence support the notion that an excessive influx of free fatty acids from visceral adipose tissue can precipitate hepatic steatosis and contribute to the development of insulin resistance. Furthermore, research findings consistently demonstrate a strong association between elevated ALT levels and an increased risk of developing T2DM in the future. Hepatic enzymes, notably ALT and AST, are routinely employed as surrogate markers of liver health. Elevated levels of these enzymes reflect hepatocellular damage and are closely associated with T2DM and insulin resistance. These findings collectively underscore the potential utility of hepatic enzymes as valuable biomarkers for diabetes and its related complications.39 The present investigation observed significantly increased serum concentrations of AST, ALT, and ALP in rats with T2DM induced by STZ and HFD compared to control rats. Notably, treatment with both dosage regimens of marmelosin demonstrated a significant reduction in the elevated levels of these hepatic enzymes (ALT, AST, ALP) in the HFD/STZ-induced T2DM rats. These findings align with previous reports where Aegle marmelos (L.) methanolic leaf extract reduced elevated hepatic enzymes and improved liver function in diabetic and hepatotoxic models.41,42

Hyperglycemia promotes oxidative stress by stimulating the excessive generation of reactive oxygen species (ROS), thereby surpassing endogenous antioxidant defenses. This oxidative burden disrupts cellular homeostasis and induces oxidative damage to vital biomolecules, including lipids, proteins, and DNA.25 Lipid peroxidation is particularly exacerbated. Consequences include membrane dysfunction, mitochondrial impairment, and increased susceptibility to cellular injury. Oxidative stress is crucial in the pathogenesis of diabetic complications, contributing to a myriad of other diseases. Cellular defense mechanisms, primarily involving the SOD, CAT, and GSH, counteract ROS-mediated damage.43

The present investigation demonstrated a marked reduction in GSH, SOD, and CAT in T2DM rats induced by HFD/STZ compared to the non-diabetic control group. This significant reduction in the activity of these crucial antioxidant enzymes strongly indicates oxidative stress-mediated damage in diabetic rats. Notably, treatment with marmelosin at both doses effectively reversed these alterations in the antioxidant enzyme levels observed in the diabetic group. This amelioration of antioxidant enzyme activity strongly supports the hypothesis that marmelosin possesses potent antioxidant properties capable of mitigating the oxidative stress compared with HFD/STZ-induced T2DM in rats, findings that are consistent with previously reported studies.25

Cytokines, particularly IL-1β, IL-6, and TNF-α, exert a significant influence on the pathogenesis of diabetes. In T2DM, a chronic, low-grade inflammatory state prevails, characterized by an imbalance that favors the production of pro-inflammatory cytokines. This inflammatory milieu contributes to insulin resistance and hyperglycemia. IL-1β, a key player in innate immunity, is produced by activated macrophages and orchestrates the inflammatory response through interactions with pattern recognition receptors. IL-6, a pleiotropic cytokine, influences adaptive immunity by driving the differentiation of naive CD4+ T cells.44,45 Dysregulated production of IL-1β and IL-6 disrupts immunological tolerance, contributing to the pathogenesis of autoimmune and chronic inflammatory diseases, including T2DM. While elevated IL-6 levels are generally associated with inflammation, it has also been shown to exert protective effects on pancreatic β-cells by modulating autophagy and enhancing antioxidant responses, thereby mitigating oxidative stress. Conversely, TNF-α, when elevated within adipose tissue, promotes insulin resistance. Conversely, inhibition of TNF-α improved glucose control and insulin sensitivity.45

The current investigation observed a significant elevation of IL-1β, IL-6, and TNF-α in rats with T2DM induced by HFD/STZ compared to healthy controls. Notably, the administration of marmelosin at both tested doses effectively suppressed the increased expression of these pro-inflammatory cytokines. These results strongly suggest that marmelosin exerts anti-inflammatory effects by attenuating the inflammatory response, which likely contributes significantly to its observed amelioration of pancreatic dysfunction in rats with T2DM. These findings corroborate the conclusions of previous studies.34

Apoptosis, a fundamental and evolutionarily conserved cellular process, orchestrates programmed cell death, which is crucial for development, tissue maintenance, and the suppression of tumors. It is triggered by various stimuli, including DNA damage induced by therapies like chemotherapy and radiotherapy.46 The cysteine protease family, caspases, orchestrate apoptotic cell death. Initiator caspases leading to cellular inhibition. Suppression of apoptosis proteins (IAPs) regulates caspase activity. While primarily associated with apoptosis, caspases also participate in other regulated cell death, such as necroptosis, pyroptosis, and autophagy. Dysregulation of caspase activity is implicated in numerous human pathologies, including diabetes.46,47 In the current study, T2DM rats induced by HFD/STZ showed a marked elevation in caspase-3 protein levels compared to healthy controls. This elevation in caspase-3 expression, a key marker of apoptosis, indicates increased cell death in diabetic animals. However, treatment with marmelosin, a natural compound, resulted in a marked reduction in caspase-3 levels. This finding strongly suggests that marmelosin exerts protective effects in T2DM by inhibiting apoptotic pathways, specifically by suppressing the activation of caspase-3. These results are consistent with previous research findings on the anti-apoptotic properties of marmelosin.48

Histological analysis of pancreatic tissue from diabetic rats revealed severe pathological changes, including cellular degeneration, lymphocytic infiltration, and necrosis. Islet architecture was significantly disrupted, characterized by pyknotic nuclei, homogenous cytoplasmic masses within islet cells, and expanded halofollicular cells with vacuoles. Treatment with marmelosin at both doses demonstrated a significant attenuation of these pathological changes, including reduced interstitial inflammation, ameliorated follicle cell injury, and improved islet architecture organization, corroborating previous findings.25

Aegle marmelos extracts have shown evidence of upregulating PPAR-γ expression, improving insulin sensitivity and β-cell function, and reducing inflammatory cytokines in models of T2DM.49 The efficacy of Aegle marmelos is comparable to established PPAR-γ agonists like rosiglitazone, but with a broader safety profile due to its natural origin and additional anti-inflammatory and cytoprotective effects.50 Marmelosin normalizes blood glucose and lipid parameters, effects which parallel those of GLP-1 analogues in improving metabolic profiles and reducing inflammation, but again with the added advantage of direct anticancer properties and modulation of multiple disease-relevant pathways. The broad metabolic effects and cytoprotective action of mermelosin position it as a multi-target adjunct in metabolic diseases and related cancer risks, supplementing or potentially replacing monotherapy with PPAR-γ agonists or GLP-1 analogues for select patient populations.

Based on our findings, marmelosin exerts its antidiabetic effects through a multifaceted mechanism involving antioxidant, anti-inflammatory, metabolic regulatory, and anti-apoptotic pathways. It enhances insulin sensitivity by increasing plasma insulin levels and decreasing HOMA-IR, likely through improved insulin signaling in target tissues such as the liver and muscle. Marmelosin also mitigates dyslipidemia by lowering triglycerides and total cholesterol while increasing HDL cholesterol, which may contribute to improved metabolic control and reduced cardiovascular risk. Furthermore, marmelosin exhibits potent antioxidant properties by enhancing endogenous antioxidant enzymes, including SOD, GSH, and CAT, thereby reducing oxidative stress, a key factor in the development of insulin resistance and β-cell dysfunction. Its anti-inflammatory effects are evidenced by the reduction of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which help alleviate inflammation-associated complications in T2DM. Additionally, marmelosin shows anti-apoptotic activity by downregulating caspase-3 expression, suggesting a protective effect against pancreatic β-cell apoptosis. These combined actions support the therapeutic potential of marmelosin in managing T2DM by preserving β-cell function, reducing inflammation and oxidative stress, and improving overall metabolic health. The multifactorial properties of marmelosin make it promising for translational use in metabolic diseases and cancer, rivaling single-target drugs like PPAR-γ modulators and GLP-1 analogues by providing broad-spectrum metabolic and cytoprotective benefits.

Limitations

The present study is limited by the absence of gene and protein expression analysis using advanced molecular techniques. The quantification of pro-inflammatory cytokines (IL‑1β, IL‑6, and TNF‑α) and apoptotic markers was performed using ELISA kits; however, methods such as RT-PCR for gene expression and Western blotting for protein expression were not employed. Furthermore, the evaluation of apoptosis was limited to caspase-3, without considering other key apoptotic markers, such as caspase-9, p53, and Bax. Additionally, no signaling pathway analysis was conducted, which limits mechanistic insights into the observed pharmacological effects of marmelosin.

Conclusion

This study investigated the therapeutic potential of Marmelosin, a naturally occurring compound, in ameliorating the metabolic disturbances compared with T2DM. The results demonstrated that marmelosin effectively improved glycemic control, enhanced insulin sensitivity, and suppressed dyslipidemia in rats with T2DM. Furthermore, marmelosin exhibited potent antioxidant and anti-inflammatory properties, while also attenuating pancreatic cell apoptosis. These findings collectively suggest that marmelosin exerts multifaceted protective effects, highlighting its potential as a promising source for the management of T2DM and its related complications.

Data Sharing Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics Approval and Consent to Participate

The experimental protocol received approval from the Institutional Research Board of Batterjee Medical College, Jeddah (RES-2025-0017). The authors adhered to the ARRIVE guidelines.

Acknowledgment

The researchers would like to thank the Deanship of Graduate Studies and Scientific Research at Qassim University for financial support (2025-QU-APC).

Author Contributions

Conceptualization: Sattam Khulaif Alenezi, Muhammad Afzal; Data curation and Formal analysis: Khalid Saad Alharbi, Tariq Alsahli, Reem ALQahtani; Funding acquisition: Khalid Saad Alharbi; Investigation: Tariq Alsahli, Muhammad Afzal; Methodology: Sattam Khulaif Alenezi, Reem ALQahtani, Krishna Kumar Sharma, Nadeem Sayyed; Project administration: Tariq Alsahli, Muhammad Afzal; Resources and Software: Nadeem Sayyed; Supervision, Validation and Visualization: Tariq Alsahli, Muhammad Afzal; Writing – original draft: Nadeem Sayyed & Tariq Alsahli; Writing – review and editing: Tariq Alsahli, Khalid Saad Alharbi, Sattam Khulaif Alenezi, Reem ALQahtani, Krishna Kumar Sharma and Muhammad Afzal.

All authors have agreed on the journal to which the article will be submitted; reviewed and agreed on all versions of the article before submission, during revision, the final version accepted for publication, and any significant changes introduced at the proofing stage and agree to take responsibility and be accountable for the contents of the article.

Funding

Deanship of Graduate Studies and Scientific Research at Qassim University for financial support (2025-QU-APC).

Disclosure

The authors declare that they have no competing interests.

References

1. Brady V, Whisenant M, Wang X, et al. Characterization of symptoms and symptom clusters for type 2 diabetes using a large Nationwide Electronic Health Record Database. Diabetes Spectrum. 2022;35(2):159–170. doi:10.2337/ds21-0064

2. Cloete L. Diabetes mellitus: an overview of the types, symptoms, complications and management. Nurs Standard. 2022;37(1):61–66. doi:10.7748/ns.2021.e11709

3. Galicia-Garcia U, Benito-Vicente A, Jebari S, et al. Pathophysiology of type 2 diabetes mellitus. Int J Mol Sci. 2020;21(17):6275. doi:10.3390/ijms21176275

4. Berbudi A, Rahmadika N, Tjahjadi AI, Ruslami R. Type 2 diabetes and its impact on the immune system. Curr Diabetes Rev. 2020;16(5):442–449. doi:10.2174/1573399815666191024085838

5. Freeman AM, Acevedo LA, Pennings N. Insulin resistance. In: StatPearls. Treasure Island (FL): StatPearls Publishing LLC; 2024.

6. Eguchi N, Vaziri ND, Dafoe DC, Ichii H. The role of oxidative stress in pancreatic β cell dysfunction in diabetes. Int J Mol Sci. 2021;22(4):1509. doi:10.3390/ijms22041509

7. Ježek P, Jabůrek M, Plecitá-Hlavatá L. Contribution of oxidative stress and impaired biogenesis of pancreatic β-cells to type 2 diabetes. Antioxidants Redox Signal. 2018;31(10):722–751. doi:10.1089/ars.2018.7656

8. Oguntibeju OO. Type 2 diabetes mellitus, oxidative stress and inflammation: examining the links. Int J Physiol Pathophysiol Pharmacol. 2019;11(3):45–63.

9. Nahdi AMA, John A, Raza H. Elucidation of molecular mechanisms of streptozotocin‐induced oxidative stress, apoptosis, and mitochondrial dysfunction in Rin‐5F pancreatic β‐cells. Oxidative Med Cell Longev. 2017;2017(1):7054272. doi:10.1155/2017/7054272

10. Magalhães DA, Kume WT, Correia FS, et al. High-fat diet and streptozotocin in the induction of type 2 diabetes mellitus: a new proposal. Anais da Academia Brasileira de Ciências. 2019;91:e20180314. doi:10.1590/0001-3765201920180314

11. Guo -X-X, Wang Y, Wang K, Ji B-P, Zhou F. Stability of a type 2 diabetes rat model induced by high-fat diet feeding with low-dose streptozotocin injection. J Zhejiang Univ Sci B. 2018;19(7):559. doi:10.1631/jzus.B1700254

12. Tran N, Pham B, Le L. Bioactive compounds in anti-diabetic plants: from herbal medicine to modern drug discovery. Biology. 2020;9(9):252. doi:10.3390/biology9090252

13. Pynam H, Dharmesh SM. Antioxidant and anti-inflammatory properties of marmelosin from Bael (Aegle marmelos L.); Inhibition of TNF-α mediated inflammatory/tumor markers. Biomed Pharmacother. 2018;106:98–108. doi:10.1016/j.biopha.2018.06.053

14. Choudhary S, Chaudhary G, Kaurav H. Aegle marmelos (bael patra): an ayurvedic plant with ethnomedicinal value. Int J Res Ayurveda Pharm. 2021;12(3):147–156. doi:10.7897/2277-4343.120392

15. Kesari AN, Gupta RK, Singh SK, Diwakar S, Watal G. Hypoglycemic and antihyperglycemic activity of Aegle marmelos seed extract in normal and diabetic rats. J Ethnopharmacol. 2006;107(3):374–379. doi:10.1016/j.jep.2006.03.042

16. Narender T, Shweta S, Tiwari P, et al. Antihyperglycemic and antidyslipidemic agent from Aegle marmelos. Bioorgan Med Chem Lett. 2007;17(6):1808–1811. doi:10.1016/j.bmcl.2006.12.037

17. Gaballah HH, Zakaria SS, Mwafy SE, Tahoon NM, Ebeid AM. Mechanistic insights into the effects of quercetin and/or GLP-1 analogue liraglutide on high-fat diet/streptozotocin-induced type 2 diabetes in rats. Biomed Pharmacother. 2017;92:331–339. doi:10.1016/j.biopha.2017.05.086

18. Bukhari HA, Afzal M, Al-Abbasi FA, et al. In vivo and computational investigation of butin against alloxan-induced diabetes via biochemical, histopathological, and molecular interactions. Sci Rep. 2024;14(1):20633. doi:10.1038/s41598-024-71577-y

19. Omer AB, Altayb HN, Al-Abbasi FA, et al. Acemannan ameliorates STZ-activated diabetes by attenuating high glucose via inhibiting inflammatory cytokines and apoptosis pathway. Int J Biol Macromol. 2023;253:127127. doi:10.1016/j.ijbiomac.2023.127127

20. Almalki NAR, Al-Abbasi FA, Moglad E, et al. Protective activity of hirsutidin in high-fat intake and streptozotocin-induced diabetic rats: in silico and in vivo study. Heliyon. 2024;10(19):e38625. doi:10.1016/j.heliyon.2024.e38625

21. Assadi S, Shafiee SM, Erfani M, Akmali M. Antioxidative and antidiabetic effects of Capparis spinosa fruit extract on high-fat diet and low-dose streptozotocin-induced type 2 diabetic rats. Biomed Pharmacother. 2021;138:111391. doi:10.1016/j.biopha.2021.111391

22. Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972;247(10):3170–3175. doi:10.1016/S0021-9258(19)45228-9

23. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82(1):70–77. doi:10.1016/0003-9861(59)90090-6

24. Sinha AK. Colorimetric assay of catalase. Analyt Biochem. 1972;47(2):389–394. doi:10.1016/0003-2697(72)90132-7

25. Gothandam K, Ganesan VS, Ayyasamy T, Ramalingam S. Antioxidant potential of theaflavin ameliorates the activities of key enzymes of glucose metabolism in high fat diet and streptozotocin–induced diabetic rats. Redox Rep. 2019;24(1):41–50. doi:10.1080/13510002.2019.1624085

26. Zhuo J, Zeng Q, Cai D, et al. Evaluation of type 2 diabetic mellitus animal models via interactions between insulin and mitogen‑activated protein kinase signaling pathways induced by a high fat and sugar diet and streptozotocin. Mol Med Rep. 2018;17(4):5132–5142. doi:10.3892/mmr.2018.8504

27. Zhao Y, Wang Q-Y, Zeng L-T, et al. Long-term high-fat high-fructose diet induces type 2 diabetes in rats through oxidative stress. Nutrients. 2022;14(11):2181. doi:10.3390/nu14112181

28. Kamalakkannan N, Prince PSM. The effect of Aegle marmelos fruit extract in streptozotocin diabetes: a histopathological study. J Herbal Pharmacother. 2005;5(3):87–96. doi:10.1080/J157v05n03_08

29. Li W, Chang M, Qiu M, et al. Exogenous obestatin decreases beta-cell apoptosis and alfa-cell proliferation in high fat diet and streptozotocin induced type 2 diabetic rats. Eur J Pharmacol. 2019;851:36–42. doi:10.1016/j.ejphar.2019.02.028

30. Ramalingam S, Karuppiah M, Thiruppathi M, Palanivelu S, Panchanatham S. Antioxidant potential of biflavonoid attenuates hyperglycemia by modulating the carbohydrate metabolic enzymes in high fat diet/streptozotocin induced diabetic rats. Redox Rep. 2020;25(1):1–10. doi:10.1080/13510002.2020.1722914

31. Wondmkun YT. Obesity, insulin resistance, and type 2 diabetes: associations and therapeutic implications. Diabetes Metabol Syndrome Obesity. 2020;13:3611–3616. doi:10.2147/DMSO.S275898

32. Chehregosha H, Khamseh ME, Malek M, Hosseinpanah F, Ismail-Beigi F. A view beyond HbA1c: role of continuous glucose monitoring. Diabetes Ther. 2019;10:853–863. doi:10.1007/s13300-019-0619-1

33. Khalili D, Khayamzadeh M, Kohansal K, et al. Are HOMA-IR and HOMA-B good predictors for diabetes and pre-diabetes subtypes? BMC Endocrine Disorders. 2023;23(1):39. doi:10.1186/s12902-023-01291-9

34. Sharma AK, Bharti S, Goyal S, et al. Upregulation of PPARγ by Aegle marmelos ameliorates insulin resistance and β-cell dysfunction in high fat diet fed-streptozotocin induced type 2 diabetic rats. Phytother Res. 2011;25(10):1457–1465. doi:10.1002/ptr.3442

35. Hafizur RM, Momin S, Fatima N. Prevention of advanced glycation end-products formation in diabetic rats through beta-cell modulation by Aegle marmelos. BMC Complement Altern Med. 2017;17(1):227. doi:10.1186/s12906-017-1743-y

36. Jasim O, Mahmood M, Ad’hiah A. Significance of lipid profile parameters in predicting pre-diabetes. Arch Razi Instit. 2022;77(1):277. doi:10.22092/ARI.2021.356465.1846

37. Artha IMJR, Bhargah A, Dharmawan NK, et al. High level of individual lipid profile and lipid ratio as a predictive marker of poor glycemic control in type-2 diabetes mellitus. Vasc Health Risk Manage. 2019;15:149–157. doi:10.2147/VHRM.S209830

38. Kamalakkannan N, Stanely Mainzen Prince P. Antihyperlipidaemic effect of Aegle marmelos fruit extract in streptozotocin‐induced diabetes in rats. J Sci Food Agri. 2005;85(4):569–573. doi:10.1002/jsfa.1978

39. Rafaqat S, Sattar A, Khalid A, Rafaqat S. Role of liver parameters in diabetes mellitus–a narrative review. Endocrin Regulations. 2023;57(1):200–220. doi:10.2478/enr-2023-0024

40. Lala V, Zubair M, Minter D. Liver function tests. StatPearls. 2023.

41. Narendhirakannan RT, Subramanian S. Biochemical evaluation of the protective effect of Aegle marmelos (L.), Corr. leaf extract on tissue antioxidant defense system and histological changes of pancreatic beta-cells in streptozotocin-induced diabetic rats. Drug Chem Toxicol. 2010;33(2):120–130. doi:10.3109/01480540903203984

42. Birudu RB, Pamulapati P, Manoharan SK. Evaluation of biochemical changes in diabetic rats treated with Aegle marmelos (L.) methanolic leaf extract. Pharmacogn Res. 2020;12(2).

43. Yaribeygi H, Sathyapalan T, Atkin SL, Sahebkar A. Molecular mechanisms linking oxidative stress and diabetes mellitus. Oxidat Med Cell Longev. 2020;2020:1–13. doi:10.1155/2020/8609213

44. Chen L-H, Chien Y-W, Chang M-L, et al. Taiwanese green propolis ethanol extract delays the progression of type 2 diabetes mellitus in rats treated with streptozotocin/high-fat diet. Nutrients. 2018;10(4):503. doi:10.3390/nu10040503

45. Dludla PV, Mabhida SE, Ziqubu K, et al. Pancreatic β-cell dysfunction in type 2 diabetes: implications of inflammation and oxidative stress. World J Diabetes. 2023;14(3):130–146. doi:10.4239/wjd.v14.i3.130

46. Asadi M, Taghizadeh S, Kaviani E, et al. Caspase‐3: structure, function, and biotechnological aspects. Biotechnol Appl Biochem. 2022;69(4):1633–1645. doi:10.1002/bab.2233

47. El Azab EF, Mostafa HS. Geraniol ameliorates the progression of high fat‐diet/streptozotocin‐induced type 2 diabetes mellitus in rats via regulation of caspase‐3, Bcl‐2, and Bax expression. J Food Biochem. 2022;46(7):e14142. doi:10.1111/jfbc.14142

48. Katram N, Garlapati PK, Yadavalli C, Methal RE, Rajappa SBG, Raghavan AK. Aegle marmelos extract rich in marmelosin exacted ameliorative effect against chromium‐induced oxidative stress and apoptosis through regulation of Gadd45 in HepG2 cell line. J Food Biochem. 2021;45(4):e13704. doi:10.1111/jfbc.13704

49. Mudi SR, Akhter M, Biswas SK, et al. Effect of aqueous extract of Aegle marmelos fruit and leaf on glycemic, insulinemic and lipidemic status of type 2 diabetic model rats. J Complement Integrat Med. 2017;14(2):20160111. doi:10.1515/jcim-2016-0111

50. Panda C, Sharma P, Dixit US, Pandey LM. Potential and prospective of traditional Indian medicinal plants for the treatment of diabetes. J Biol Active Products Nat. 2023;13(4):316–360.