Phytochemical Profiling and Mechanistic Investigation of the Antidiabetic Properties of Parquetina Nigrescens

Abstract

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder marked by elevated blood glucose levels, primarily caused by insulin resistance and impaired insulin secretion. Despite significant pharmacological advancements, managing this condition remains challenging, particularly in low- and middle-income countries, where access to standard medications is often limited. Consequently, many individuals in these regions rely on traditional plant-based remedies to help manage their condition. Parquetina nigrescens (PN), a plant known for its antidiabetic properties, has attracted attention due to its biological activities. However, the specific bioactive metabolites responsible for these effects, as well as the mechanisms behind its insulin-releasing properties are not fully understood. This study aimed to identify the phytochemical composition of PN and explore its insulin-releasing effects, providing a deeper understanding of its therapeutic potential in management of T2DM. PN dried leaves were extracted and fractionated using solvents of varying polarities. Preliminary phytochemical screening was conducted using standard reagents, and the phytochemical composition was analysed via liquid chromatography-mass spectrometry (LC-MS). Biological activity was assessed by measuring cell viability in INS-1 β-cells using a methylthiazolyl diphenyl-tetrazolium bromide (MTT) assay, and enzyme inhibitory effect was tested against α-amylase, α-glucosidase, and pancreatic lipase. Cytoprotective effects were evaluated under glucotoxic, lipotoxic, lipo-glucotoxic, and high cytokine-induced stress conditions. Additionally, glucose-stimulated insulin secretion (GSIS) assay was performed to examine PN’s impact on insulin secretion at basal (1.1 mM) and stimulatory (16.7 mM) glucose concentrations in INS-1 cells and mouse pancreatic islets. Gene and protein expressions were analysed by qPCR and Western blotting. Intracellular calcium modulation was assessed in FURA-2AM-loaded cells under different treatment conditions, and specific pathway targeted by PN was further examined through β2-adrenergic receptor knockdown, followed by GSIS and intracellular calcium modulation. LC-MS identified over 6,000 compounds in PN, with key bioactive metabolites being apigenin, luteolin, and rutin, known for their antioxidant, anti-inflammatory, and insulinotropic properties. Flavonoids were the most abundant compounds in the ethanol (EtOHF) and ethyl acetate fractions (EAF), suggesting they are likely responsible for PN’s antidiabetic effects. Most extracts were non-cytotoxic up to 1 mg/mL during 24–72 h exposure, except n-Hexane fraction (n-HF) at 48 h and EtOHF at 72 h, which showed reduced viability at 1 mg/mL. EAF and EtOHF offered significant cytoprotection to INS-1 β-cells under stress conditions induced with high glucose, free fatty acid, and cytokines. Both crude extract (CE) and Parquetina nigrescens fractions improved cell viability, indicating protection of pancreatic β-cells from hyperglycaemia and elevated lipid levels, which are key contributors to β-cell dysfunction in T2DM. This protection was mediated through the upregulation of antioxidant pathway; Nuclear Factor Erythroid 2–Related Factor 2 – Kelch-like ECH-Associated Protein 1 (NRF2-KEAP1), and subsequent upregulation of antioxidant response elements, including Superoxide Dismutase 1 and 2, Catalase,Glutathione Peroxidase 1, and Heme Oxygenase 1 (SOD1, SOD2, CAT, GPX1, and HO1). Additionally, PN extracts significantly enhanced insulin secretion in INS-1 β-cells and mouse pancreatic islets at stimulatory glucose and, to a lesser extent, under basal glucose condition, suggesting PN acts as an insulin secretagogue. PN also upregulated the expression of genes involved in insulin biosynthesis and secretion, namely Insulin 1 and Musculoaponeurotic Fibrosarcoma Oncogene Homolog A (INS1 and MAFA); insulin maturation, Proprotein Convertase Subtilisin/Kexin Type 1 (PCSK1); and glucose metabolism, Glucokinase (GCK). The expression of HO1, SOD1, NRF2, GCK, and Pancreatic and Duodenal Homeobox 1 (PDX1) proteins were also upregulated, whereas KEAP1 expression was downregulated. Calcium imaging revealed that PN extracts enhanced intracellular calcium mobilisation, a crucial step in insulin exocytosis. This effect was completely blocked by verapamil (an L-type voltage-gated calcium channel blocker) and partially blocked by propranolol (a non-selective β-blocker) and SN6 (a selective inhibitor of the Na⁺/Ca²⁺ exchanger). Further studies showed that the modulatory effect of PN on intracellular calcium was mediated via the β2-adrenergic receptor (β2AR) signalling pathway, as confirmed by the inhibition of intracellular calcium mobilisation by ICI118551 (a selective β2AR blockers). Additionally, selective knockdown of β2AR led to blockade of PN-induced intracellular calcium modulation and insulin release, suggesting that PN increases insulin secretion by activating β2AR, leading to increased calcium influx through the Na+/Ca2+ exchanger. In addition to its insulinotropic effects, PN extracts demonstrated dose-dependent inhibition of key metabolic enzymes, including alpha-amylase, alpha-glucosidase, and pancreatic lipase. These enzymes are essential for carbohydrate and lipid digestion, and by inhibiting them, PN may reduce postprandial glucose spikes and improve metabolic control, making it a promising therapeutic agent for T2DM management. These data provide compelling evidence that PN has significant therapeutic potential for managing T2DM as it demonstrated a multifaceted pharmacological profile, including cytoprotection, antioxidant, insulinotropic, and enzyme inhibitory effects. PN’s ability to protect pancreatic β-cells, enhance insulin secretion, and modulate key signalling pathways via β2AR and NCX1, underlines its promise as a novel therapeutic option for T2DM management. In conclusion, PN holds great promise as a natural product-based therapeutic for T2DM management. Further research is needed to validate these data in primary cells and in vivo studies, explore other pathways, optimize its formulation for clinical application, and ensure its safety and efficacy as a complementary or alternative therapy for T2D.