Abstract

Introduction: Ensete glaucum seeds have been shown to possess antidiabetic properties. However, the specific hypoglycemic activities of the compounds they contain remain poorly understood. This study aimed to elucidate the hypoglycemic effects of E. glaucum seed compounds and to explore their mechanisms of action, particularly their interaction with pancreatic islets.

Methods: We employed spectroscopic techniques to determine the chemical structures of the isolated compounds, which we then compared with structures reported in the literature. In a streptozotocin (STZ)-induced diabetic mouse model, we evaluated the hypoglycemic response to an ethanol extract of E. glaucum seeds. Further, we assessed the islet-protective effects and the capacity to stimulate insulin release of both the crude extract and its individual compounds in STZ-challenged pancreatic islets.

Results: We isolated two novel compounds from E. glaucum seeds: afzelechin (a flavan-3-ol) and coniferaldehyde (a phenolic aldehyde), marking the first such report. Administration of the extract in doses of 25 and 50 mg/kg/day led to a significant reduction in blood glucose levels over a period of seven days in STZ-induced hyperglycemic mice. Notably, the extract and the isolated compounds afzelechin and coniferaldehyde exhibited a protective effect on islet β cells, mitigating STZ-induced cytotoxicity. Both the extract, at concentrations of 50 and 100 µg/mL, and the individual compounds, at 100 µM, demonstrated an ability to counteract STZ's inhibition of glucose-stimulated insulin secretion in isolated pancreatic islets. Importantly, molecular docking studies suggest that these compounds may stimulate insulin release from β cells by inhibiting the K_ATP channel, a hypothesis that warrants further experimental investigation.

Conclusion: Our findings suggest that E. glaucum seeds, along with their bioactive compounds, show promise in reducing blood sugar levels and enhancing insulin secretion, offering potential therapeutic avenues for diabetes management.

Introduction

Diabetes represents a significant global health issue, with its prevalence escalating rapidly. Type 2 diabetes, which accounts for the majority of cases, is marked by impaired pancreatic β-cell function and persistent insulin resistance. Pancreatic β cells are essential, as they regulate blood glucose by producing insulin. When these cells malfunction or die, insulin scarcity ensues, disrupting glucose homeostasis and causing blood glucose levels to rise1. It's therefore vital to maintain β cells' ability to secrete insulin. For treating diabetes, it's necessary to increase insulin levels to facilitate glucose uptake into cells, particularly for insulin-sensitive individuals, to decrease circulating blood glucose levels. Insulin and sulfonylurea treatments are typical examples of therapies that amplify this process. However, these treatments can have significant adverse effects. Insulin therapy can lead to various complications, including hypoglycemia, the Somogyi effect (a form of reactive hyperglycemia), insulin allergy, adipose tissue dystrophy, weight gain, and immune resistance to insulin2. Similarly, sulfonylureas may cause hypoglycemia, weight gain, and gastrointestinal issues, such as abdominal pain, nausea, and diarrhea. Particular caution is advised when prescribing these medications to patients with kidney failure, as drugs like glibenclamide and gliclazide are contraindicated in severe cases3. Hence, identifying substances that can protect and improve β cell functionality for more effective and safe insulin production and release is of paramount importance.

Plant-based sources are abundant in nutraceuticals and bioactive compounds with therapeutic potential. An estimated 80% of the world's population depends on herbal remedies for primary healthcare4. The application of medicinal plants and natural substances in diabetes treatment is garnering attention, supported by findings from preclinical and clinical research. For instance, a 2024 study demonstrated that a 70% ethanol extract of Ardisia elliptica Thunb., at a dosage of 250 mg/kg, was as effective in lowering serum glucose, creatinine, and urea levels as metformin, a leading antidiabetic drug5. A 2019 clinical trial found that consuming raspberries (Rubus idaeus) daily significantly decreased serum glucose levels after meals and reduced pro-inflammatory markers such as IL-6 and TNF-α four hours after consumption, compared to a control group6. Reports from 2023 have further underscored the importance of herbal medicines and plant secondary metabolites in diabetes management, particularly highlighting the elucidation of their action mechanisms7, 8, 9. Various plants, such as Momordica charantia L., Moringa oleifera Lam., Andrographis paniculata (Burm.f.) Nees, Clitoria ternatea L., and Gymnema sylvestre R.8, 10, 11, along with natural compounds like rutin, quercetin, hesperidin, kaempferol, cinnamic acid, resveratrol, paeoniflorin, and berberine7, 12, have been recognized for their antioxidant and hypoglycemic properties, as well as their role in enhancing the viability and insulin secretion of pancreatic β cells. The snow banana, scientifically designated as Ensete glaucum (Roxb.) Cheesman and a member of the Musaceae family13, is not just nutritionally rich in carbohydrates, fiber, vitamins, and minerals, but also in healing and health-promoting phytochemicals14. Traditionally, E. glaucum seeds have been extensively utilized by indigenous communities to treat a variety of ailments, including digestive disorders, diabetes, kidney stones, edema, stomach ulcers, and allergic reactions14, 15. Previous studies have indicated that E. glaucum seed extract can protect pancreatic cells in vivo and stimulate insulin secretion16; however, the responsible compounds and their mechanisms of action are yet to be identified.

Overall, this study aims to assess the hypoglycemic potential of E. glaucum seed extract and its isolated compounds in vivo, as well as their protective and stimulatory effects on insulin secretion from isolated mouse pancreatic islets in vitro, against streptozotocin toxicity, which is commonly used to induce diabetes by damaging pancreatic β cells. Moreover, this study anticipates determining the mechanism by which E. glaucum seed-derived compounds stimulate insulin, with a predictive in silico approach targeting the pancreatic ATP-sensitive potassium (K_ATP) channel.

Methods

Plant Material and Extraction Ensete glaucum bunches were harvested in Bac Ai District, Ninh Thuan Province, Viet Nam, in April 2020. An identification form (TNDL-CCD-2020) was kept at the Department of Natural Resources and Medicinal Materials Development, Research Center of Ginseng and Medicinal Materials in Ho Chi Minh City. The plant's scientific name has also been verified with the World Flora Online Plant List (https://wfoplantlist.org/plant-list). Seeds were separated from mature and ripe fruits and thoroughly rinsed with running water. After drying, the seeds were ground into a powder using a grinder. They were then extracted with 98% ethanol by hot extraction at 70°C for 60 minutes. The resultant liquid extract was filtered, and the extraction was repeated to achieve a weight/volume ratio of 1/20. The liquid extract was subsequently concentrated using a rotary evaporator at 70°C under reduced pressure to obtain the E. glaucum seed extract (EGE). The extract was preserved at 2-8°C and dissolved in an appropriate solvent to create a stock solution.Fractionation and Isolation The fractionation and isolation of compounds from E. glaucum seeds are summarized in Figure 1 and described in the Supplementary Information (SI).Animals The Institute of Vaccines and Medical Biologicals (IVAC) in Nha Trang City, Vietnam, supplied healthy Swiss albino mice (male, 4-5 weeks old, 18-22 g). The mice were cared for in an animal house with 50-60% relative humidity, a temperature of 25°C, and 12h light-dark cycles. They were acclimatized for 7 days before the experiment and housed in cages made of PP plastic. They had unlimited access to tap water and standard food supplied by IVAC. The administered volume (oral or injection) was 10 mL/kg body weight in the morning (8-9 a.m.).Experimental Design This study was a single-center, double-blind, controlled preclinical trial. The experiments were carried out at the Research Center of Ginseng and Medicinal Materials in Ho Chi Minh City. Streptozotocin (Sigma-Aldrich) (STZ, 170 mg/kg, i.p., single dose)17 was used after an overnight fast of 16 hours to induce a hyperglycemic mouse model. The streptozotocin was weighed and dissolved in 0.1 M sodium citrate pH 4.5 immediately before injection into mice. After 7 days, the fasting plasma glucose (FPG) was determined, and those with FPG ≥ 200 mg/dL18 were classified as hyperglycemic and chosen for the experiment.

The STZ-induced hyperglycemic mice were divided into 5 groups (n = 9). Group I received distilled water as the pathological control; Group II and III were administered EGE daily (25 and 50 mg/kg, respectively), and Group IV was administered glibenclamide daily (Medical Import Export Joint Stock Company DOMESCO, Vietnam) (GLI, 5 mg/kg). A group of normoglycemic mice injected with sodium citrate was also included in the study as a control. The animals were treated orally with/without EGE or glibenclamide for 7 days. The dosage and administration time of the extract and glibenclamide were selected based on previous reports16, 17. EGE or glibenclamide was mixed with distilled water for oral administration to the mice. The weight of the mice was measured daily.

Blood Biochemical Assessment

At baseline and at the end of day 7, mouse tail blood was collected to measure FPG concentration after one hour of treatment. Ten microliters of blood were combined with an equal volume of 2% EDTA (Ethylenediaminetetraacetic acid). The mixture was then centrifuged at 5,000 rpm for 5 minutes. Subsequently, 5 µL of plasma was gently mixed with 250 µL of glucose indicator agent at 37°C for 10 minutes. The glucose concentration was calculated according to the manufacturer’s guidelines for the GOD-PAP Kit (Glucose oxidase-phenol amino phenazone) from Erba, based on absorbance determined at 500 nm using a Biotek Microplate Reader (Synergy™ HTX Multi-Mode, USA)16.

Isolation and Culturing of Pancreatic Islets

Pancreatic islet isolation was performed in accordance with the previously described methods with some slight alterations19, 20. Mice were euthanized with carbon dioxide inhalation, and the pancreas was collected and treated with 3 mL of collagenase II (Gibco, Thermo Fisher Scientific, US) at 200 U/mL in sterile Hank's Balanced Salt Solution (HBSS, Gibco, Thermo Fisher Scientific, US) at 37°C for 30 minutes to achieve complete digestion. The islets were placed in a 50 mL falcon tube, rapidly cooled on ice, and then 25 mL of HBSS/CaCl2 at 3 mM was added with vigorous shaking for 10 seconds 40 times. The samples were centrifuged at 290 g for 30 seconds at 4°C. The precipitate was resuspended in 20 mL of HBSS/CaCl2 at 3 mM and filtered through a 0.419 mm sieve. Continuous centrifugation at 290 g for 30 seconds at 4°C was performed. The precipitate was then suspended in 10 mL of Ficoll 1077 and 10 mL of RPMI 1640 and centrifuged at 900 g for 20 minutes at 20°C. Pancreatic islets from the middle layer of Ficoll/medium were collected in RPMI 1640 containing 10% FBS, penicillin-streptomycin (100 U/mL-100 µg/mL), and centrifuged at 290 g for 5 minutes at 4°C. The pancreatic islets were filtered through a 70 nm filter into the culture medium. Individual islets, which had smooth perimeters and lacked a dark core, were selected using a 200-µL micropipette under a microscope and grown in a 6-well plate with fresh RPMI 1640 medium. After 24 hours, they were incubated at 37°C with 5% CO2. These islets were checked for their specificity and functionality.

Assessment of Islet Specificity and Functionality

Islet specificity was tested by Dithizone staining. Briefly, 10 µL of Dithizone (Sigma-Aldrich) solution (10 mg/mL) was added to 1 mL of Krebs-Ringer bicarbonate buffer (KRB, pH 7.4) containing suspended islets and incubated for 5-10 minutes at 37°C. Islets that stained bright red were observed under an inverted microscope (Nikon Eclipse Ts2R-FL, China), whereas non-islet tissues remained unstained19.

Islet functionality was assessed by measuring the level of insulin secreted. Isolated islets were washed and placed in KRB buffer. Then, 10 islets were exposed to 2.8 mM glucose in the buffer at 37°C for 60 minutes. Following this, the islets were stimulated with 16.8 mM glucose for another 60 minutes at 37°C. The insulin released into the medium under both conditions was determined using an ELISA insulin kit (Abcam) according to the manufacturer’s instructions21. The total cell protein content was also estimated using the Bradford reagent with BSA (Bovine Serum Albumin) as the standard for calculating insulin levels.

Assessment of Islet Mitochondrial Metabolism

An MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma-Aldrich) was used to evaluate the effect of test samples on cell viability. Ten islets were treated with test samples at various concentrations at 37 °C in 5% CO2 for 24 hours. These islets were then collected and exposed to 100 μL of MTT (0.5 mg/mL) at 37 °C for 4 hours. Subsequently, 100 μL of dimethyl sulfoxide (Sigma-Aldrich) was added and the mixture was incubated for 15 minutes to solubilize the formazan crystals. Absorbance was recorded at 570 and 630 nm using a BioTek Microplate Reader. The viability of islet cells treated with and without test samples was presented as the percentage of cell viability (%) by comparing the mean absorbance of cells from different groups using the formula: Cell viability (%) = (Mean absorbance of treated cells – Mean absorbance of blank) / (Mean absorbance of untreated cells – Mean absorbance of blank) × 10022.

Experimental Design for Assessing Islet Protective Effects Glucose-Stimulated Insulin Secretion (GSIS) Assay

Ten islets per group were treated for 1 hour at 37 °C in 500 μL KRB buffer containing either 2.8 or 16.8 mM glucose, along with either extracts or compounds from E. glaucum seeds at various concentrations23. Glimepiride (Stellapharm J.V. Co., Ltd.) was used as a positive control. Aliquots of supernatant were collected for insulin assays using an ELISA insulin kit (Abcam), following the manufacturer’s guidelines, and islets were gathered for protein assays using the Bradford reagent.

Effect of Extract and Compounds on Insulin Secretion in STZ-Treated Cells

The insulin-secreting effect of the test samples was evaluated in STZ-treated cells. Ten islets were treated with or without the extract (50 and 100 μg/mL) or compounds (50 and 100 μM) in the presence of STZ (5 mM)24. After 24 hours, the treated islets were incubated in KRB solution containing 16.8 mM glucose for 1 hour at 37 °C. Subsequently, the supernatant was collected, and the insulin content was estimated by an ELISA insulin kit (Mercodia), according to the manufacturer’s guidelines. The islets were then collected for protein assays using the Bradford reagent. Glimepiride served as a positive control. These experiments were repeated to confirm the protective effects of the extract and compounds from E. glaucum seeds on islet cells using the MTT assay.

Molecular Docking Simulation

The interaction between the compounds and the protein was evaluated through molecular docking. The 3D structures of the compounds were retrieved from PubChem25. The crystal structure of the recombinant pancreatic ATP-sensitive potassium channel (KATP channel), accession no. 6JB1, was obtained from the RCSB Protein Data Bank. The channel is a fusion construct of four inward-rectifier potassium channel 6 (Kir6.2) subunits from Mus musculus, surrounded by four sulfonylurea receptors 1 (SUR1) from Mesocricetus auratus (Figure S1-A in the Supplementary Information). The structure was processed with PyMOL 2.5 (https://pymol.org), to remove heteromolecules and reduce the size of the structure. Due to its symmetric nature, the KATP structure was trimmed to one SUR1 and two adjacent KIR6 subunits (Figure S1-B in the Supplementary Information), to lower the computational cost for docking. The trimmed structure and the compounds were processed as macromolecules and ligands using PyRx 1.9.2 with the default settings26. Docking was performed using the AutoDock Vina 1.2.0 protocol27 with the Vinardo scoring function, covering the entire protein with the grid, an exhaustiveness of 1000, and 15 output poses. The docking poses were evaluated based on the minimized affinity as calculated by the scoring function. The pose with the best affinity (lowest score) for each compound was visualized in PyMOL 2.5, and the protein-ligand interactions were analyzed using BIOVIA Discovery Studio Visualizer.

Statistical Analysis

Data were statistically analyzed using GraphPad Prism software (version 8.0.2, Inc., La Jolla, CA, USA) and reported as Mean ± SD. The data were initially tested for normality using the Anderson-Darling test and the Shapiro-Wilk test. One-way analysis of variance (ANOVA) was employed for statistical analysis, followed by Tukey’s test for homogeneity of variance and Dunnett’s test for heterogeneity of variance, assuming a normal distribution. Alternatively, the Kruskal-Wallis test was used in the case of a non-normal distribution, followed by Dunn’s post hoc test. Results were considered statistically significant when p

× Figure 1 . Schematic representation of fractionation of E. glaucum seed extract . Figure 1 . Schematic representation of fractionation of E. glaucum seed extract .

Table 1.

1 H-NMR and 13C -NMR Data of the compound EG1

Position C/H DEPT 1H-NMR ( δ H ppm) (No. H, mult., J Hz) 13 C-NMR ( δ C ppm) HMBC correlations ( 1 H → 13 C) 2 CH 4.80 (1H, s ) 78.0 C-7, C-1’ 3 CH 4.02 (1H, dd , 4.8, 9.6) 64.8 C-10, C-1’ 4 CH 2 H a : 2.48 (1H, dd , 3.0, 16.2) H b : 2.68 (1H, dd , 4.8, 16.2) 28.1 C-2, C-3, C-5, C-6, C-8, C-9, C-10 5 C 156.5 6 CH 5.71 (1H, d , 2.4) 94.1 C-5, C-7, C-8, C-10 7 C 155.7 8 CH 5.89 (1H, d