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Article

Insulinotropic and Beta-Cell Proliferative Effects of Unripe Artocarpus heterophyllus Extract Ameliorate Glucose Dysregulation in High-Fat-Fed Diet-Induced Obese Mice

by
Prawej Ansari
1,2,3,*,
Sara S. Islam
2,4,
Asif Ali
3,
Md. Samim R. Masud
2,
Alexa D. Reberio
2,
Joyeeta T. Khan
2,5,
J. M. A. Hannan
2,
Peter R. Flatt
3 and
Yasser H. A. Abdel-Wahab
3
1
Department of Pharmacology, National Medical College and Teaching Hospital, Parsa, Birgunj 44300, Nepal
2
Department of Pharmacy, School of Pharmacy and Public Health, Independent University, Bangladesh (IUB), Dhaka 1229, Bangladesh
3
Centre for Diabetes Research, School of Biomedical Sciences, Ulster University, Coleraine BT52 1SA, UK
4
Faculty of Science, Medicine and Health, University of Wollongong, Northfields Ave., Wollongong, NSW 2522, Australia
5
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences (UAMS), Little Rock, AR 72205, USA
*
Author to whom correspondence should be addressed.
Diabetology 2025, 6(8), 83; https://doi.org/10.3390/diabetology6080083
Submission received: 10 June 2025 / Revised: 29 July 2025 / Accepted: 5 August 2025 / Published: 13 August 2025
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Abstract

Background: Artocarpus heterophyllus, familiar as jackfruit, is a tropical fruit highly valued not only for its nutritional content but also for its medicinal properties, including potential antidiabetic effects. Objectives: This study aimed to evaluate the insulinotropic, β-cell proliferative and anti-hyperlipidaemic properties of the ethanol extract of unripe Artocarpus heterophyllus (EEAH) in high-fat-fed (HFF) diet-induced obese mice. Method: We evaluated acute insulin secretion and β-cell proliferation in BRIN-BD11 cells, and assessed in vitro glucose diffusion and starch digestion. In vivo, acute and chronic studies in HFF induced obese mice measured glucose tolerance, body weight, food and fluid intake, and lipid profiles. A preliminary phytochemical screening was also performed. Results: In this study, EEAH exhibited significant antidiabetic activity through multiple mechanisms. EEAH enhanced glucose-stimulated insulin secretion in BRIN-BD11 β-cells via KATP channel modulation and cAMP-mediated pathways, with partial dependence on extracellular calcium, and it also promoted β-cell proliferation. In vitro assays revealed its ability to inhibit starch digestion and glucose diffusion, indicating delayed carbohydrate digestion and absorption. In high-fat-fed (HFF) obese mice, the acute and chronic oral administration of EEAH improved oral glucose tolerance, reduced fasting blood glucose, decreased body weight, and normalized food and fluid intake. Lipid profile analysis showed increased HDL and reduced total cholesterol, LDL, and triglycerides, while higher doses of EEAH also enhanced gut motility. Phytochemical screening revealed the presence of bioactive compounds such as alkaloids, tannins, flavonoids, saponins, steroids, and terpenoids, which are likely responsible for these therapeutic effects. Conclusion: These findings highlight EEAH as a promising natural candidate for adjunctive therapy in managing type 2 diabetes and associated metabolic disorders and emphasize the importance of future multi-omics studies to elucidate its molecular targets and pathways.

1. Introduction

Diabetes mellitus (DM) is a complicated, multifactorial metabolic disease, characterized by excessive glucose in the circulation resulting from impaired insulin sensitivity, a deficient secretion of insulin, an anabolic hormone, or both [1]. Globally, DM presents a significant public health challenge; the International Diabetes Federation (IDF) estimated that approximately 537 million people had DM in 2021, and this number is projected to rise to 783 million by 2045 [2]. Nearly 10% of adults worldwide are diagnosed with diabetes mellitus (DM), being a major contributor to heightened morbidity and mortality [3].
The development of diabetes fundamentally stems from inadequate insulin production or impaired insulin action, both of which are critical for maintaining metabolic balance. Insulin is an essential peptide hormone produced by β-cells and plays a crucial role in regulating metabolic processes [4,5]. The synthesis begins with the transcription of the insulin gene into mRNA, followed by translation into pre-proinsulin. This precursor is subsequently processed, first into proinsulin and then further into mature insulin and C-peptide [5,6]. Insulin promotes glucose uptake in skeletal muscle by facilitating its absorption and conversion to glycogen, and it also stimulates glycogen storage and inhibits excessive glucose production in the liver [7].
Diabetes can be seen in diverse phenotypes. Type 1 diabetes (T1DM) is an autoimmune disorder marked by the destruction of pancreatic β-cells, causing absolute insulin deficiency and dysregulated α-cell activity followed by excessive glucagon secretion [1,8,9]. In contrast, type 2 diabetes mellitus (T2DM) accounts for 90% of diabetes cases and is a heterogeneous condition primarily driven by impaired insulin secretion from pancreatic β-cells and reduced insulin function due to insulin resistance [10]. Acute complications of diabetes include life-threatening conditions such as diabetic ketoacidosis (DKA) and severe hypoglycemia. Furthermore, chronic hyperglycemia leads to both microvascular damage (retinopathy, nephropathy, and neuropathy) and macrovascular complication, thereby increasing the risk of diabetic foot ulcers and premature mortality [11].
Research indicates that obesity is a major risk factor for the development of type 2 diabetes mellitus (T2DM), with approximately 90% of individuals with the disease being overweight or obese [12]. While diet and exercise aid weight loss in T2DM, they often fall short in sustaining optimal glycemic control [13]. Therefore, a range of antidiabetic medications, including metformin, sulfonylureas, meglitinides, thiazolidinediones, SGLT2 inhibitors, GLP-1 mimetics, and DPP-IV inhibitors, are essential for effective blood glucose management. However, these medications are often expensive and inaccessible for individuals in underprivileged communities and can also have undesirable side effects [14]. Consequently, affordable and effective alternatives are required to mitigate the detrimental effects of uncontrolled diabetes [13,15].
Traditionally, people worldwide rely on various herbs and medicinal plants to manage and treat diabetes mellitus [13,15]. Artocarpus heterophyllus, generally known as jackfruit, belongs to member of the Moraceae family and is cultivated in tropical regions, particularly in countries such as India, Bangladesh, Sri Lanka, Thailand, Indonesia, the Philippines, Brazil, and parts of East and West Africa [16,17,18,19,20,21,22]. Indigenous people have been using it to treat illness, such as diabetes, inflammation, infection, cancer, and CVD, for centuries, due to its innumerable medicinal properties [17,18]. A. heterophyllus is reported to be abundant in dietary fiber, carbohydrates, minerals, carboxylic acids, and vitamins such as thiamine and ascorbic acid [16,19]. Previous studies have shown that A. heterophyllus possesses antioxidant, anti-inflammatory, anti-fungal, antidiabetic, antibacterial, immunomodulatory, and wound-healing properties [18,20]. Recent studies also suggested that A. heterophyllus can lower blood glucose and improve oral glucose tolerance and body weight [21,22]. Therefore, our present aim was to investigate the insulinotropic and antidiabetic properties of A. heterophyllus, as well as the underlying mechanisms of action on pancreatic β-cells, using in vitro and in vivo models.

2. Materials and Methods

2.1. Preparation and Extraction of EEAH

Unripe jackfruit (Artocarpus heterophyllus) was collected from Jahangirnagar University, Savar, Dhaka, Bangladesh (botanical accession no. 87,070), and authenticated by a taxonomist from the Bangladesh National Herbarium, Mirpur, Dhaka. The collected fruits were thoroughly washed, air-dried for several hours, and then oven-dried at 45–50 °C to prepare for ethanolic extraction. The dried material was further ground into a fine powder using a grinder. Two hundred grams of this powdered material were mixed with 1 L of 80% (v/v) ethanol and shaken at 900 rpm for 48–72 h on an orbital shaker. After extraction, the mixture was filtered through Whatman No. 1 filter paper, and the filtrate was concentrated under reduced pressure using a rotary evaporator (BibbyRE-200, Sterilin Ltd., Newport, UK). The resulting semi-solid crude extract of A. heterophyllus was kept at 4 °C until used in experiments. Prior to administration, the 250 mg/kg and 500 mg/kg doses of the semisolid EEAH extract were each dissolved in 5 mL of 6% DMSO prepared in normal saline [23].

2.2. Studies of Insulin Release In Vitro

The ability of EEAH to stimulate insulin secretion was tested using BRIN-BD11 pancreatic β-cells [24]. Cells were treated with increasing concentrations of EEAH (8–5000 μg/mL) and incubated at 37 °C for 20 min in a medium containing 5.6 mM glucose [25]. Supernatants were collected and stored at −20 °C for insulin quantification by radioimmunoassay [26]. Additionally, the pathways responsible for EEAH-induced insulin secretion were investigated by using known insulin secretagogues and inhibitors. These included tolbutamide (a sulfonylurea and KATP channel blocker; Sigma-Aldrich, Gillingham, UK), IBMX (Isobutyl methylxanthine; a cAMP-mediated insulin modulator and phosphodiesterase inhibitor; Cayman Chemical, via Cambridge Bioscience, Bar Hill, UK), 30 mM KCl (Thermo Fisher Scientific, Loughborough, UK), 10 mM alanine (Sigma-Aldrich, Gillingham, UK), diazoxide (a KATP channel opener; Tocris Bioscience, Bristol, UK), and verapamil (a voltage-dependent Ca2+ channel blocker; Sigma-Aldrich, Gillingham, UK). Both KCl and alanine induce plasma membrane depolarization and Ca2+ influx, with alanine primarily achieving this through Na+ co-transport and ATP-generating metabolism [15,22].

2.3. β-Cell Proliferation In Vitro

To assess whether EEAH promotes β-cell proliferation, BRIN-BD11 cells were treated with EEAH at concentrations of 40 and 200 μg/mL. Glucagon-like peptide-1 (GLP-1; 10−6 M) was used as a positive control. Cells (40,000 per coverslip) were incubated for 18 h at 37 °C. After washing with PBS, cells were fixed with 4% paraformaldehyde. Antigen retrieval was performed using citrate buffer (90 °C, 20 min), and blocking was performed with 1.1% bovine serum albumin (BSA) for 30 min. The cells were then incubated with a primary antibody against Ki-67 (1:500; Abcam, Cambridge, UK) followed by an Alexa Fluor® 488-conjugated secondary antibody (1:400; Invitrogen, Waltham, MA, USA). Finally, coverslips were washed, mounted, and observed under a fluorescence microscope (Olympus System, Tokyo, Japan) equipped with a DP70 camera adapter. The number of Ki-67–positive cells was counted to determine proliferation [27].

2.4. In Vitro Starch Digestion

The effect of EEAH on starch digestion was tested by mixing 100 mg/50 mL starch solution (Sigma-Aldrich, St. Louis, MO, USA) with or without EEAH and acarbose (a reference α-glucosidase inhibitor). The reaction mixtures were incubated with 0.01% heat-stable α-amylase from Bacillus licheniformis and 0.1% amyloglucosidase from Rhizopus sp. (Sigma-Aldrich, St. Louis, MO, USA) at 80 °C and 60 °C for 20 and 30 min, respectively. After incubation, samples were collected, and glucose levels were measured using the glucose oxidase-phenol-4-aminophenazone (GOD-PAP) method (Randox GL 2623) [25,26]. An alpha glucosidase inhibitor, Acarbose, was used as a reference drug.

2.5. In Vitro Glucose Diffusion

To evaluate the impact of EEAH on glucose permeability in vitro, cellulose ester dialysis tubing (CEDT) was employed (dimensions: 20 cm × 7.5 mm, Spectra/Por®CE layer, MWCO: 2000, Spectrum, Amsterdam, The Netherlands). The experimental setup involved adding 2 mL of 0.9% NaCl solution containing 220 mM glucose, either with or without EEAH, into dialysis tubes. These sealed tubes were then submerged in 50 mL centrifuge tubes (Orange Scientific, Orange, CA, USA) containing 45 mL of 0.9% NaCl solution. The system was maintained at 37 °C with continuous agitation using an orbital shaker for 24 h. Post-incubation, samples were analyzed to determine the quantity of glucose that had diffused across the membrane into the external medium using the GOD/PAP methodology (Randox GL 2623) [28,29].

2.6. Animals

Two-week-old male Swiss albino mice weighing between 20 and 25 g were sourced from the Animal Resources Facility at ICDDRB, Dhaka, Bangladesh. Housing conditions were maintained at 22 ± 3 °C with 55–65% humidity, providing ad libitum access to food and water under an automated 12 h light/dark cycle at the Animal House of Independent University, Bangladesh. After a one-week acclimation period, mice were categorized into two primary groups based on body weight: a normal control cohort (NC) receiving standard chow (comprising 10% fat, 30% protein, and 60% carbohydrates) and a high-fat-fed (HFF) cohort consuming a high-fat diet (containing 19.4% powdered normal animal food, 17.5% sugar, 21% beef fat, 39.5% condense milk, 1.1% vitamin-B complex, and 1.5% salt) over 6–8 weeks. Subsequently, the HFF cohort was further stratified into four subgroups: a high-fat-fed control group (HFF) (Group 2), two experimental treatment groups (Groups 3 and 4), and a standard treatment group (Group 5). HFF diet-induced obese type 2 diabetic mice were characterized by fasting glucose concentrations above the normal threshold (>6.0 mmol/L) and body weights ranging from 40 to 45 g. Control groups (normal and high-fat-fed) received saline solution (5 mL/kg), while treatment groups were administered 250 and 500 mg/5 mL/kg of EEAH, respectively. The standard treatment group received glibenclamide (5 mg/5 mL/kg) twice daily (BID) for 60 days, purchased from Incepta Pharmaceuticals, Bangladesh. Each experimental group consisted of n = 6 mice. This research protocol received approval from the Institutional Review Board (IRB) at Independent University, Bangladesh (IUB) on 19 December, 2019, for animal experimentation. All procedures adhered to the animal Welfare Act 2019 of Bangladesh, UK Animals (Scientific Procedures) Act 1986, and EU Directive 2010/63/EU. Research activities were conducted under UK Home Office Animal license PIL1822, ensuring ethical treatment and minimizing animal distress. The experimental groups were organized as follows:
GroupTreatment Description
Group 1Lean control (saline; 5 mL/kg)
Group 2HFF diet-induced diabetic control (saline; 5 mL/kg)
Group 3HFF diet + EEAH (250 mg/5 mL/kg)
Group 4HFF diet + EEAH (500 mg/5 mL/kg)
Group 5HFF diet + glibenclamide (5 mg/5 mL/kg)

2.7. Acute and Chronic Oral Glucose Tolerance Test

Oral glucose tolerance tests (OGTTs) were conducted on HFF diet-induced obese mice at days 0, 15, 30, 45, and 60 during the 60-day experimental period to evaluate glucose metabolism over time. The baseline OGTT on day 0 was performed before treatment initiation to establish initial glucose tolerance, while follow-up assessments monitored treatment progression and therapeutic efficacy. HFF diet mice underwent a 12 h fasting period before testing. Blood glucose measurements were taken via tail vein sampling at specific time points before (0 min) and following (30, 60, 120, and 180 min) oral glucose administration (2.5 gm/5 mL/kg body weight, control) with/without EEAH (250 and 500 mg/5 mL/kg body weight) or standard treatment with glibenclamide (5 mg/5 mL/kg body weight). Glucose concentrations were assessed using an Ascencia Contour glucose meter (Bayer, Newbury, UK), with glibenclamide serving as the reference insulin secretagogue [25].

2.8. Chronic Study of Blood Glucose, Body Weight, Food and Fluid Intake

A 60-day long-term investigation was performed on high-fat-fed diet-induced obese mice. Multiple parameters, including fasting glucose levels, body weight, and food and fluid consumption, were monitored at three-day intervals. All experimental groups, including both the high-fat diet-induced obese mice and the normal control group, were subjected to 12 h fasting periods before these assessments. Treatment groups received EEAH at dosages of 250 and 500 mg/5 mL/kg, respectively. The positive control group was treated with glibenclamide (5 mg/5 mL/kg), while both the high-fat-diet control group and the normal control group received saline (5 mL/kg). Blood glucose measurements were obtained using an Ascensia Contour Blood Glucose Meter (Bayer, Newbury, UK) [22,25,30].

2.9. Lipid Profile Test

During the 60-day investigation, high-fat-fed diet-induced obese mice received oral treatments twice daily with either EEAH (250 and 500 mg/5 mL/kg) or glibenclamide (5 mg/5 mL/kg). Lipid profile analysis was performed following previously established protocols [15]. Upon study completion, mice were euthanized, and cardiac blood collection was performed using 5 mL syringes into heparinized microcentrifuge tubes (Sarstedt, Nümbrecht, Germany) to prevent blood coagulation. Blood samples underwent centrifugation at 12,000 rpm for 5 min, and the separated plasma serum was collected. High-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride (TG), and total cholesterol (TC) concentrations were subsequently analyzed using CHOD-PAP (Biolabo SAS, Maizy, France), COD-PAP, and GPO-PAP (Elabscience Biotechnology Co., Ltd., Houston, TX, USA) reagent systems with automated analyzer technology [31].

2.10. Gut Motility In Vivo

Following the 60-day chronic studies, mice were euthanized, and gastrointestinal motility was evaluated by measuring the transit distance of barium sulfate (BaSO4) milk solution in mice after a 16 h fasting period. One hour prior to BaSO4 solution administration, treatment groups received EEAH (250 and 500 mg/5 mL/kg), bisacodyl (10 mg/5 mL/kg), or loperamide (5 mg/5 mL/kg). The high-fat-fed (HFF) control and normal control groups received only the BaSO4 solution. Fifteen minutes following BaSO4 milk solution administration, mice were euthanized, and the entire small intestine was extracted. The BaSO4 transit distance was subsequently quantified and reported as a percentage of total small intestine length (measured from pylorus to ileocecal junction) [32,33].

2.11. Phytochemical Screening

To confirm the presence of alkaloids, tannins, flavonoids, saponins, steroids, terpenoids, glycosides, and reducing sugar, a phytochemical analysis of EEAH was performed. All the chemicals were purchased from Labtexbd, Dhaka, Bangladesh. Phytochemical screening of EEAH was conducted using standard qualitative methods to identify major secondary metabolites. Alkaloids were detected by acidifying the extract with dilute HCl followed by the addition of Dragendorff’s reagent, where a reddish-brown precipitate indicated the presence of alkaloids, which form colored complexes due to their nitrogenous structure. Tannins were identified by the formation of a white precipitate upon mixing the extract with 10% lead acetate, as tannins form insoluble complexes with metal ions. Flavonoids were confirmed by heating the extract with methanol, then adding magnesium and concentrated HCl, which produced a pink color due to the formation of flavilium salts under acidic conditions. Saponins were detected by vigorous shaking with distilled water, resulting in stable foam formation, a characteristic property due to their surfactant-like nature. Steroids were identified by treating the extract with chloroform, acetic anhydride, and concentrated sulfuric acid, producing a bluish-green color from sterol-acetic acid reactions. Terpenoids were confirmed by dissolving the extract in chloroform and layering with concentrated sulfuric acid, which formed a reddish-brown interface indicating terpenoid backbone reactivity. Glycosides were identified by adding glacial acetic acid and ferric chloride followed by sulfuric acid, where the appearance of a bluish-green color indicated the presence of glycosidic aglycones forming colored complexes. Reducing sugars were confirmed by heating the extract with Fehling’s reagent, where the formation of a red-brick precipitate indicated the reduction of Cu2+ ions to Cu+ by the sugar moieties [28,34,35].

2.12. Statistical Analysis

GraphPad Prism version 10 was used for statistical analysis. Data were assessed using a two-tailed unpaired Student’s t-test or one-way or two-way analysis of variance (ANOVA) with a Bonferroni post hoc test. Results were presented as mean ± SEM, with statistical significance defined as p < 0.05.

3. Results

3.1. Insulin Release with EEAH

At 5.6 mM glucose, EEAH significantly stimulated insulin secretion from BRIN-BD11 β-cells in a concentration-dependent manner across a range of 40–5000 µg/mL (p < 0.05 to p < 0.001; Figure 1A). Alanine (10 mM), served as a positive control, remarkably increased insulin secretion (p < 0.001; Figure 1A).

3.2. Insulin Release with EEAH, Known Modulators and Absence of Extracellular Ca2+

To elucidate the insulinotropic mechanisms of non-cytotoxic concentrations (200 µg/mL) of EEAH, further investigations were conducted (Figure 1B). As depicted in Figure 1B, EEAH-stimulated insulin secretion exhibited potentiation in the presence of key modulators: high glucose (16.7 mM, p < 0.01), the non-selective phosphodiesterase inhibitor IBMX (p < 0.001), and the sulfonylurea receptor 1 (SUR1) agonist tolbutamide (p < 0.001). The synergistic action of IBMX, leading to elevated intracellular cyclic AMP, and tolbutamide, promoting SUR1 closure and subsequent membrane depolarization, amplified both cAMP production and insulin secretion. Conversely, the KATP channel opener diazoxide and the L-type voltage-gated calcium channel (VGCC) antagonist verapamil attenuated EEAH-induced insulin release (p < 0.05–0.01; Figure 1B), as evidenced by reduced insulin secretion when incubated with Ca2+ free buffer (p < 0.05; Figure 1C). Notably, EEAH retained its capacity to stimulate insulin secretion from cells depolarized with 30 mM KCl (p < 0.001; Figure 1B), indicating a mechanism downstream of membrane potential. However, the absence of extracellular Ca2+ diminished, though did not completely abrogate, EEAH’s insulin-releasing activity (p < 0.001; Figure 1C), underscoring the partial dependence on extracellular calcium for its full insulinotropic effect.

3.3. Proliferation In Vitro with EEAH

Figure 2A–D displays BRIN BD11 β-cell proliferation images for three conditions: (A) Control, (B) GLP-1 (10−6 M), and (C,D) EEAH (at concentrations of 40 and 200 μg/mL). Quantitative analysis (Figure 2E) revealed that EEAH significantly enhanced BRIN-BD11 β-cell proliferation at both 40 μg/mL (p < 0.01) and 200 μg/mL (p < 0.001) compared to the control. The positive control, GLP-1 (10−6 M), demonstrated the most potent effect on BRIN-BD11 β-cell growth (p < 0.001; Figure 2E).

3.4. Starch Digestion In Vitro with EEAH

Figure 1D illustrates the impact of Acarbose and EEAH on starch digestion. The positive control, Acarbose (62.5–1000 µg/mL), demonstrated a concentration-dependent reduction in starch digestion ranging from 20% to 80%. EEAH also significantly inhibited starch digestion (11–33%, p < 0.05–0.001, Figure 1D) at higher concentrations (250–1000 µg/mL).

3.5. Glucose Diffusion In Vitro with EEAH

EEAH demonstrated significant (p < 0.01–0.001; Figure 1E) inhibitory effects on glucose diffusion during a 24 h incubation period when compared to the control condition. As illustrated in Figure 1E, the extract decreased total glucose diffusion by 16–26%, with maximum inhibition (26%) occurring at a concentration of 5000 μg/mL.

3.6. Oral Glucose Tolerance Test with EEAH

Oral administration of EEAH (250 and 500 mg/5 mL/kg) in combination with glucose (2.5 gm/5 mL/kg) significantly (p < 0.05–0.001) enhanced oral glucose tolerance at 30, 60, and 120 min on days 0, 15, 30, and 60 of the studies (Figure 3A–D) in HFF diet-induced obese mice when compared to high-fat-fed diet-induced obese control mice. EEAH treatment at doses of 250 and 500 mg/5 mL/kg improved oral glucose tolerance by 1.2-fold and 1.4-fold at 30 and 60 min, respectively, consistently throughout the studies. In comparison to the HFF diet-induced obese control mice, the standard drug glibenclamide (5 mg/5 mL/kg) also significantly (p < 0.05–0.001) reduced blood glucose levels at 30, 60, and 120 min on days 0, 15, 30, and 60 (Figure 3A–D).

3.7. Chronic Study of Fasting Blood Glucose, Body Weight, Food and Fluid Intake with EEAH

The oral administration of EEAH (250 and 500 mg/5 mL/kg) and glibenclamide (5 mg/5 mL/kg) twice daily demonstrated notable reductions in fasting blood glucose levels in HFF diet-induced obese mice during the study period of 60 days (Figure 4A). Compared to HFF alone, EEAH at doses of 250 and 500 mg/5 mL/kg considerably (p < 0.05–0.001) reduced blood glucose levels from day 27 onwards, with a 500 mg/5 mL/kg dose exhibiting greater potency than a 250 mg/5 mL/kg dose. Additionally, the standard drug glibenclamide (5 mg/5 mL/kg) consistently decreased (p < 0.05–0.001; Figure 4A) blood glucose throughout the study period.
The oral administration of EEAH (250 and 500 mg/5 mL/kg) and glibenclamide (5 mg/5 mL/kg) twice daily improved body weight in HFF mice during 60-day study period (Figure 4B). Compared to the HFF group alone, EEAH at doses of 250 and 500 mg/5 mL/kg progressively (p < 0.05–0.001) reduced body weight from day 6 onwards. However, a 500 mg/5 mL/kg dose showed a more consistent effect from day 18 onwards. In addition, glibenclamide (5 mg/5 mL/kg) showed a remarkable (p < 0.05–0.001; Figure 4B) improvement in the body weight of HFF diet-induced obese mice from day 36 onwards.
The oral administration of EEAH (250 and 500 mg/5 mL/kg) twice daily for 60 days significantly (p < 0.05–0.001; Figure 4C,D) improved food and fluid intake, and this improvement was consistent from day 18 compared to HFF diet-induced obese control mice. Similarly, glibenclamide (5 mg/5 mL/kg) noticeably (p < 0.01–0.001; Figure 4C,D) decreased food and fluid intake consistently throughout the studies.

3.8. Lipid Profiling with EEAH

Long-term treatment (twice daily for 60 days) with EEAH (250 and 500 mg/5 mL/kg) or glibenclamide (5 mg/5 mL/kg) effectively (p < 0.05–0.01; Figure 5A) elevated HDL cholesterol while significantly (p < 0.05–0.001; Figure 5B–D) reducing total cholesterol, LDL, and triglyceride levels, respectively, in high-fat-fed diet-induced obese mice. A high dose of EEAH at 500 mg/5 mL/kg showed the most potent antihyperlipidemic effect. A sulfonylurea drug, glibenclamide (5 mg/5 mL/kg), similarly increased (p < 0.05, Figure 5A) HDL concentrations and decreased (p < 0.05–0.01; Figure 5B–D) total cholesterol, LDL and triglyceride levels compared to high-fat-fed diet-induced obese mice alone.

3.9. Gut Motility with EEAH

Following 60 days of treatment with EEAH at 250 mg/5 mL/kg, no significant changes were observed in gastrointestinal motility (Figure 5E). However, EEAH at 500 mg/5 mL/kg significantly (p < 0.001; Figure 5E) ameliorated gut motility. Additionally, treatment with the stimulant laxative bisacodyl (10 mg/5 mL/kg) considerably (p < 0.001; Figure 5E) stimulated gut motility, whereas the antidiarrheal agent loperamide (5 mg/5 mL/kg) was associated with decreased (p < 0.01, Figure 5E) intestinal motility.

3.10. Phytochemical Screening with EEAH

To identify potential antidiabetic phytochemicals present in unripe A. heterophyllus ethanolic extract, additional phytochemical screening was performed. Phytochemical analysis of EEAH confirmed the presence of alkaloids, tannins, flavonoids, saponins, terpenoids, and steroids (Table 1).

4. Discussion

Medicinal plants exhibit natural antioxidant properties that serve as an effective source of herbal glucose-lowering medicine, particularly due to their bioactive phytochemicals such as flavonoids, saponins, tannins, phenolics, and alkaloids. These compounds can help in enhancing endocrine pancreatic function through the stimulation of insulin secretion or modulation of intestinal glucose absorption [36]. This study investigated the antidiabetic potential of unripe Artocarpus heterophyllus (EEAH) extract via comprehensive in vitro and in vivo analyses to elucidate its mechanisms in the regulation of blood glucose homeostasis.
The ethanolic extract of EEAH demonstrated a significant insulinotropic effect by enhancing insulin secretion from pancreatic β-cells under basal glucose conditions in a dose-dependent manner. This stimulatory activity was comparable to that of known insulin secretagogues, which indicates the potent ability of EEAH to modulate β-cell function. Importantly, EEAH maintained its insulin-releasing capacity in the presence of tolbutamide and high extracellular potassium, suggesting that its mechanism of action may involve both KATP channel-dependent and -independent pathways. The suppression of EEAH-induced insulin secretion by diazoxide supports the involvement of KATP channel closure in its effect [37]. Furthermore, the reduction of insulin release by verapamil implicates voltage-dependent Ca2+ influx as a crucial mediator of EEAH action. The synergistic enhancement of insulin secretion with IBMX co-treatment implies the potential involvement of cAMP-mediated pathways [38]. Collectively, these findings demonstrate that EEAH stimulates insulin secretion through a multimodal mechanism, including closure of KATP channels, activation of voltage-dependent Ca2+ channels, and modulation of the cAMP signaling pathway.
The significant proliferative effect of EEAH on BRIN-BD11 β-cells at both lower and higher concentrations compared to control also represents a potentially therapeutically relevant finding for diabetes management. This proliferative response mirrored the effect of GLP-1, a positive control that exhibits its action through established cAMP/PKA pathway activation [39]. This differential response indicates that bioactive compounds in EEAH, likely including polyphenols and flavonoids, may stimulate this action via this pathway [40]. EEAH may stimulate the proliferation of insulin-producing β-cells by activating key pathways such as PI3K/Akt, Wnt/β-catenin, or JAK-STAT, which are known to be involved in this regenerative process [41,42].
The pathogenesis of diabetes mellitus is generally multifactorial, with key contributions often coming from enzymatic starch hydrolysis mediated by α-amylase and α-glucosidase as well as subsequent glucose absorption and diffusion across the intestinal epithelium. In this context, the ethanol extract of unripe Artocarpus heterophyllus (EEAH) exhibited a significant concentration-dependent inhibitory effect on enzymatic starch degradation, suggesting it may have the potential to attenuate postprandial hyperglycemia. This inhibitory activity may be attributed to the presence of bioactive phytoconstituents such as flavonoids and phenolic compounds known for their capacity to suppress α-amylase activity [43,44,45]. Additionally, EEAH significantly also impeded glucose diffusion and intestinal absorption in a concentration-dependent manner, indicating its possibility in limiting glucose bioavailability in the gastrointestinal tract. These findings align with the previous literature documenting the antidiabetic potential of plant-based extracts through enzymatic and absorptive interference mechanisms [46].
Obesity, known as a critical predisposing factor in cases of T2DM, is often linked to elevated levels of non-esterified fatty acids (NEFAs) being released from adipose tissue and is notorious in impairing insulin sensitivity and disrupting pancreatic β-cell function [47]. In the present investigation, EEAH resulted in a significant improvement in the oral glucose tolerance of high-fat-fed (HFF) obese mice. These findings are in concordance with earlier plant-based antidiabetic studies, demonstrating that A. heterophyllus and other phytochemical-rich plant extracts can enhance glycemic control and preserve β-cell functionality in HFF-induced rodent models [48].
In the present study, chronic oral administration of the EEAH also demonstrated a significant antihyperglycemic effect in high-fat-fed (HFF) diet-induced obese mice during the 60-day treatment period. Twice-daily administration of EEAH effectively led to a time-dependent reduction in blood glucose levels, with the higher dose exerting an earlier and more sustained glycemic control. These findings indicate a dose-responsive antihyperglycemic potential of EEAH, comparable to the standard antidiabetic agent, glibenclamide, which consistently reduced blood glucose throughout the study duration. The glycemic-lowering effect of EEAH may be attributed to its capacity to enhance pancreatic β-cell responsiveness, potentiate insulin secretion, and possibly modulate insulin signaling pathways [49]. These observations align with a previous study of A. heterophyllus, where a similar blood glucose-lowering trend was observed [50]. In both studies, glibenclamide was used as a positive control that lowers blood glucose by binding to SUR1 on pancreatic β-cells and closing KATP channels, leading to calcium influx and stimulating insulin secretion [51]. Therefore, the bioactive compounds present in A. heterophyllus might be modulating through this pathway, as it showed a comparable glucose-lowering trend compared to the control.
EEAH administration was also associated with a gradual and sustained reduction in body weight in obese mice, indicating its potential role in ameliorating obesity-linked metabolic disturbances. Excessive caloric intake combined with inadequate energy expenditure disrupts lipid metabolism, leading to obesity and associated metabolic disorders like diabetes and non-alcoholic fatty liver disease (NAFLD). The effect of EEAH on body weight may reflect improved energy homeostasis and reduced adiposity, potentially through the modulation of lipid metabolism and appetite regulation [52,53]. The presence of polyphenol also contributes to this appetite suppression and weight loss [54]. In line with this, EEAH also significantly improved both food and fluid intake patterns, which are typically disrupted in diabetic and obese states. These observations are consistent with earlier reports on A. heterophyllus [55].
Both tested doses of EEAH showed a marked increase in HDL-cholesterol levels while displaying a concurrent decrease in total cholesterol, LDL-cholesterol, and triglyceride levels. This lipid-modulating potential may be attributed to the extract’s ability to enhance lipoprotein catabolism and promote reverse cholesterol transport, aligning with previous findings on plant-derived compounds with antihyperlipidemic activity [56]. The observed increase in HDL may further be supported by improved hormonal regulation, potentially involving thyroid-related pathways that facilitate the hepatic clearance of LDL [57].
EEAH treatment over 60 days resulted in a significant improvement in gastrointestinal motility at higher doses. This suggests that EEAH may be helpful in reducing glucose absorption by accelerating transit time through the gastrointestinal tract. The increased motility could limit the duration available for nutrient absorption, contributing to the extract’s antihyperglycemic activity [58]. Disaccharides like sucrose require breakdown into monosaccharides for absorption due to the absence of specific carriers in the GI tract; thus, the increased sucrose content following EEAH treatment likely reflects reduced sucrose digestion [59]. Such effects are likely linked to the presence of dietary fibers or bioactive compounds within EEAH that modulate gut function, similar to other plant-based treatments known to influence glucose regulation via digestive mechanisms [60,61,62].
Phytochemical analysis of the ethanol extract of unripe A. heterophyllus (EEAH) revealed the presence of several bioactive constituents, including alkaloids, tannins, flavonoids, saponins, steroids, and terpenoids. Tannins are known to improve glucose uptake, block adipocyte differentiation, inhibit inflammation and oxidative stress, and improve insulin sensitivity [62,63]. Saponins, particularly steroidal types, have been reported to mimic insulin action and improve glucose metabolism and weight management by modulating the activity of adipokines [64,65]. Flavonoids, including well-studied compounds such as catechin and epicatechin, exert antioxidant effects and improve glycemic control by targeting pathways involved in insulin resistance and glucose homeostasis [66,67]. Alkaloids and terpenoids also play significant roles in modulating carbohydrate metabolism, enhancing GLUT-4 translocation and increasing pancreatic β-cell function [68,69]. The presence of these diverse phytochemicals in EEAH suggests a multifaceted mechanism underlying its antidiabetic potential, warranting further investigation into their individual and combined therapeutic contributions.

5. Conclusions

This study highlights the therapeutic potential of the ethanol extract of unripe A. heterophyllus (EEAH) as a promising candidate for the management of type 2 diabetes mellitus. EEAH demonstrated significant insulinotropic, antihyperglycemic, and hypolipidemic effects and improved gastrointestinal motility, and it contained a diverse array of phytochemicals known to modulate glucose metabolism, insulin sensitivity, and lipid profiles. These findings suggest a multimodal mechanism of action, including the enhancement of insulin secretion, the modulation of nutrient absorption, and possible interaction with signaling pathways involved in metabolic regulation. Given the complex interplay of phytochemicals within EEAH and their broad biological targets, future research should prioritize the isolation and characterization of the bioactive phytomolecules responsible for the observed antidiabetic activity and the elucidation of their detailed mechanisms of action, ultimately paving the way for the development of targeted and effective therapeutic strategies. To further unravel these complex interactions, subsequent investigations using multi-omics approaches are essential. These integrative studies will provide a comprehensive understanding of the molecular mechanisms underpinning EEAH’s antidiabetic action and aid in identifying specific biomarkers of efficacy and safety, ultimately supporting the development of EEAH as a standardized, evidence-based nutraceutical or adjunct therapy for diabetes management in humans.

Author Contributions

The conceptualization and design of this study were carried out by P.R.F., P.A. and Y.H.A.A.-W., who also jointly supervised its implementation. P.A., S.S.I., M.S.R.M. and A.A. performed the experimental work and analyzed the data. P.A., A.D.R., J.T.K., A.A. and J.M.A.H. interpreted the results, prepared the figures, and drafted the initial manuscript. The revised manuscript was edited by P.R.F., P.A. and Y.H.A.A.-W. All authors have read and agreed to the published version of the manuscript.

Funding

Financial support for this research was provided by the Independent University, Bangladesh (IUB) sponsored research project fund (2019-SESM-09).

Institutional Review Board Statement

Animal experiments for this study were ethically approved by the Institutional Review Board (IRB) at Independent University, Bangladesh (IUB) on 19 December 2019. All procedures adhered strictly to national and international animal welfare guidelines, including the Animal Welfare Act 2019 of Bangladesh, the UK Animals (Scientific Procedures) Act 1986, and EU Directive 2010/63/EU. Experiments were conducted under UK Home Office Animal license PIL1822, ensuring high standards of animal care and minimizing distress.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data generated and analyzed during this study are not publicly accessible due to ethical or legal constraints. Requests for access to the data can be directed to the corresponding author and will be considered on a reasonable basis.

Acknowledgments

The authors thank Ulster University for Strategic Research Funding and Independent University, Bangladesh (IUB), Dhaka, for providing laboratory resources that facilitated this research.

Conflicts of Interest

The authors have no conflicts of interest to disclose.

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Diabetology 06 00083 g001
Figure 1. Effects of ethanol extract of unripe A. heterophyllus (EEAH) on insulin secretion from (A) clonal pancreatic BRIN BD11 β-cells alone, (B) with known modulators or inhibitors, (C) with or without extracellular calcium, (D) starch digestion, and (E) glucose diffusion. Percentage of glucose liberation from starch digestion and glucose diffusion in vitro are represented in the scatter-dot plot. Values are mean ± SEM; n = 8 for insulin secretion and n = 4 for starch digestion and glucose diffusion. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control. φ p < 0.05, φφ p < 0.01 and φφφ p < 0.001 compared to 5.6 mM glucose with EEAH. ΔΔ p < 0.01, ΔΔΔ p < 0.001 compared to respective incubation without EEAH. EEAH, ethanol extract of A. heterophyllus (unripe fruit).
Figure 1. Effects of ethanol extract of unripe A. heterophyllus (EEAH) on insulin secretion from (A) clonal pancreatic BRIN BD11 β-cells alone, (B) with known modulators or inhibitors, (C) with or without extracellular calcium, (D) starch digestion, and (E) glucose diffusion. Percentage of glucose liberation from starch digestion and glucose diffusion in vitro are represented in the scatter-dot plot. Values are mean ± SEM; n = 8 for insulin secretion and n = 4 for starch digestion and glucose diffusion. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control. φ p < 0.05, φφ p < 0.01 and φφφ p < 0.001 compared to 5.6 mM glucose with EEAH. ΔΔ p < 0.01, ΔΔΔ p < 0.001 compared to respective incubation without EEAH. EEAH, ethanol extract of A. heterophyllus (unripe fruit).
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Figure 2. Effects of ethanol extract of unripe A. heterophyllus (EEAH) on (A–E) BRIN-BD11 β-cells proliferation. BRIN-BD11 cells were cultured (18 h) with EEAH (40 and 200 μg/mL) or GLP-1 (10−6 M) and proliferation assessed by Ki-67 staining. Representative images depict cells stained with DAPI (blue) and Ki-67 (red), with arrows indicating Ki-67-positive (proliferating) cells. Figure 2A–D shows BRIN BD11 β-cell proliferation images under four conditions: (A) Control, (B) GLP-1 (10−6 M), (C) EEAH at 40 μg/mL, and (D) EEAH at 200 μg/mL. Percentage of Ki-67+ve cells are represented in the scatter-dot plot. All values are mean ± SEM with n = 6. **, *** p < 0.01–0.001 compared with control. ΔΔ, ΔΔΔ p < 0.01–0.001 compared with GLP-1 (10−6 M).
Figure 2. Effects of ethanol extract of unripe A. heterophyllus (EEAH) on (A–E) BRIN-BD11 β-cells proliferation. BRIN-BD11 cells were cultured (18 h) with EEAH (40 and 200 μg/mL) or GLP-1 (10−6 M) and proliferation assessed by Ki-67 staining. Representative images depict cells stained with DAPI (blue) and Ki-67 (red), with arrows indicating Ki-67-positive (proliferating) cells. Figure 2A–D shows BRIN BD11 β-cell proliferation images under four conditions: (A) Control, (B) GLP-1 (10−6 M), (C) EEAH at 40 μg/mL, and (D) EEAH at 200 μg/mL. Percentage of Ki-67+ve cells are represented in the scatter-dot plot. All values are mean ± SEM with n = 6. **, *** p < 0.01–0.001 compared with control. ΔΔ, ΔΔΔ p < 0.01–0.001 compared with GLP-1 (10−6 M).
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Figure 3. Acute and chronic effects of ethanol extract of unripe A. heterophyllus (EEAH) in high-fat-fed mice on oral glucose tolerance at (A) 0, (B) 15, (C) 30 and (D) 60 days respectively expressed as line graphs. High-fat-fed mice fasted for overnight were used to measure the blood glucose from the tail tips prior to and after receiving glucose (2.5 gm/5 mL/kg, body weight, control) with or without EEAH (250 and 500 mg/5 mL/kg, body weight) or glibenclamide (5 mg/5 mL/kg). Values are the mean ± SEM for n = 6 mice. *, ** p < 0.05–0.01 compared to lean control (saline), Δ, ΔΔ, ΔΔΔ p < 0.05–0.001 compared to high-fat-fed diet control mice. Glibenclamide was used as a positive control.
Figure 3. Acute and chronic effects of ethanol extract of unripe A. heterophyllus (EEAH) in high-fat-fed mice on oral glucose tolerance at (A) 0, (B) 15, (C) 30 and (D) 60 days respectively expressed as line graphs. High-fat-fed mice fasted for overnight were used to measure the blood glucose from the tail tips prior to and after receiving glucose (2.5 gm/5 mL/kg, body weight, control) with or without EEAH (250 and 500 mg/5 mL/kg, body weight) or glibenclamide (5 mg/5 mL/kg). Values are the mean ± SEM for n = 6 mice. *, ** p < 0.05–0.01 compared to lean control (saline), Δ, ΔΔ, ΔΔΔ p < 0.05–0.001 compared to high-fat-fed diet control mice. Glibenclamide was used as a positive control.
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Figure 4. Effects of 60-days chronic treatment (twice-daily) with ethanol extract of unripe A. heterophyllus (EEAH) on (A) body weight, (B) fasting blood glucose, (C) food intake, (D) fluid intake in high-fat-fed mice represented as line graphs. Parameters were measured at 3-day intervals prior to and after oral administration of either EEAH (250 and 500 mg/5 mL/kg, body weight) or glibenclamide (5 mg/5 mL/kg, body weight) twice daily. Values are the mean ± SEM for n = 6 mice. *, **, *** p < 0.05–0.001 compared to control (saline). Δ, ΔΔ, ΔΔΔ p < 0.05–0.001 compared to high-fat-fed diet control mice. As a positive control, glibenclamide was used.
Figure 4. Effects of 60-days chronic treatment (twice-daily) with ethanol extract of unripe A. heterophyllus (EEAH) on (A) body weight, (B) fasting blood glucose, (C) food intake, (D) fluid intake in high-fat-fed mice represented as line graphs. Parameters were measured at 3-day intervals prior to and after oral administration of either EEAH (250 and 500 mg/5 mL/kg, body weight) or glibenclamide (5 mg/5 mL/kg, body weight) twice daily. Values are the mean ± SEM for n = 6 mice. *, **, *** p < 0.05–0.001 compared to control (saline). Δ, ΔΔ, ΔΔΔ p < 0.05–0.001 compared to high-fat-fed diet control mice. As a positive control, glibenclamide was used.
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Figure 5. Effects of 60-day (twice-daily) oral administration of ethanol extract of unripe A. heterophyllus (EEAH) on (A) HDL cholesterol, (B) total cholesterol, (C) triglycerides, (D) LDL cholesterol, and (E) gut motility in lean mice and high-fat-fed mice expressed as scatter-dot plot. Parameters were measured on day 60 following twice-daily treatment with/without EEAH (250 and 500 mg/5 mL/kg, body weight) or glibenclamide (5 mg/5 mL/kg, body weight) or loperamide (5 mg/5 mL/kg) or bisacodyl (10 mg/5 mL/kg). Gastrointestinal motility was assessed by measuring BaSO4 transit length in 20 h fasted mice after ingestion of BaSO4 solution. Values are expressed as mean ± SEM with n = 6 mice. *, **, *** p < 0.05–0.001 compared to control (saline) and Δ, ΔΔ, ΔΔΔ p < 0.05–0.001 compared to high-fat-fed diet control mice. As a positive control, glibenclamide, Loperamide and bisacodyl were used.
Figure 5. Effects of 60-day (twice-daily) oral administration of ethanol extract of unripe A. heterophyllus (EEAH) on (A) HDL cholesterol, (B) total cholesterol, (C) triglycerides, (D) LDL cholesterol, and (E) gut motility in lean mice and high-fat-fed mice expressed as scatter-dot plot. Parameters were measured on day 60 following twice-daily treatment with/without EEAH (250 and 500 mg/5 mL/kg, body weight) or glibenclamide (5 mg/5 mL/kg, body weight) or loperamide (5 mg/5 mL/kg) or bisacodyl (10 mg/5 mL/kg). Gastrointestinal motility was assessed by measuring BaSO4 transit length in 20 h fasted mice after ingestion of BaSO4 solution. Values are expressed as mean ± SEM with n = 6 mice. *, **, *** p < 0.05–0.001 compared to control (saline) and Δ, ΔΔ, ΔΔΔ p < 0.05–0.001 compared to high-fat-fed diet control mice. As a positive control, glibenclamide, Loperamide and bisacodyl were used.
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Table 1. Phytochemical screening of ethanol extract of A. heterophyllus (EEAH) unripe fruit.
Table 1. Phytochemical screening of ethanol extract of A. heterophyllus (EEAH) unripe fruit.
Group TestObservation
Alkaloids+
Tannins+
Flavonoids+
Saponins+
Steroids+
Terpenoids+
Glycoside−
Reducing Sugar−
‘+’ indicates present; ‘−’ indicates absent.
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Ansari, P.; Islam, S.S.; Ali, A.; Masud, M.S.R.; Reberio, A.D.; Khan, J.T.; Hannan, J.M.A.; Flatt, P.R.; Abdel-Wahab, Y.H.A. Insulinotropic and Beta-Cell Proliferative Effects of Unripe Artocarpus heterophyllus Extract Ameliorate Glucose Dysregulation in High-Fat-Fed Diet-Induced Obese Mice. Diabetology 2025, 6, 83. https://doi.org/10.3390/diabetology6080083

AMA Style

Ansari P, Islam SS, Ali A, Masud MSR, Reberio AD, Khan JT, Hannan JMA, Flatt PR, Abdel-Wahab YHA. Insulinotropic and Beta-Cell Proliferative Effects of Unripe Artocarpus heterophyllus Extract Ameliorate Glucose Dysregulation in High-Fat-Fed Diet-Induced Obese Mice. Diabetology. 2025; 6(8):83. https://doi.org/10.3390/diabetology6080083

Chicago/Turabian Style

Ansari, Prawej, Sara S. Islam, Asif Ali, Md. Samim R. Masud, Alexa D. Reberio, Joyeeta T. Khan, J. M. A. Hannan, Peter R. Flatt, and Yasser H. A. Abdel-Wahab. 2025. "Insulinotropic and Beta-Cell Proliferative Effects of Unripe Artocarpus heterophyllus Extract Ameliorate Glucose Dysregulation in High-Fat-Fed Diet-Induced Obese Mice" Diabetology 6, no. 8: 83. https://doi.org/10.3390/diabetology6080083

APA Style

Ansari, P., Islam, S. S., Ali, A., Masud, M. S. R., Reberio, A. D., Khan, J. T., Hannan, J. M. A., Flatt, P. R., & Abdel-Wahab, Y. H. A. (2025). Insulinotropic and Beta-Cell Proliferative Effects of Unripe Artocarpus heterophyllus Extract Ameliorate Glucose Dysregulation in High-Fat-Fed Diet-Induced Obese Mice. Diabetology, 6(8), 83. https://doi.org/10.3390/diabetology6080083

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