| Summary: | Due to the numerous adverse effects of synthetic drugs, researchers are currently studying traditional medicinal plants to find alternatives for diabetes treatment. <i>Eucalyptus citriodora</i> is known to be used as a remedy for various illnesses, including diabetes. This study aimed to explore the effects of ethanol extract of <i>Eucalyptus citriodora</i> (EEEC) on in vitro and in vivo systems, including the mechanism/s of action. The methodology used involved the measurement of insulin secretion from clonal pancreatic β-cells, BRIN BD11, and mouse islets. Other in vitro systems further examined EEEC’s glucose-lowering properties. Obese rats fed a high-fat-fed diet (HFF) were selected for in vivo evaluation, and phytoconstituents were detected via RP-HPLC followed by LC-MS. EEEC induced insulin secretion in a concentration-dependent manner with modulatory effects, similar to 1 µM glucagon-like peptide 1 (GLP-1), which were partly declined in the presence of Ca<sup>2+-</sup>channel blocker (Verapamil), K<sub>ATP</sub>-channel opener (Diazoxide), and Ca<sup>2+</sup> chelation. The insulin secretory effects of EEEC were augmented by isobutyl methylxanthine (IBMX), which persisted in the context of tolbutamide or a depolarizing concentration of KCl. EEEC enhanced insulin action in 3T3-L1 cells and reduced glucose absorption, and protein glycation in vitro. In HFF rats, it improved glucose tolerance and plasma insulin, attenuated plasma DPP-IV, and induced active GLP-1 (7-36) levels in circulation. Rhodomyrtosone B, Quercetin-3-O-β-D-glucopyranoside, rhodomyrtosone E, and quercitroside were identified as possible phytoconstituents that may be responsible for EEEC effects. Thus, these findings revealed that <i>E. citriodora</i> could be used as an adjunct nutritional supplement to manage type 2 diabetes.
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