Insulin Secretory Actions of Ethanol Extract of Eucalyptus citriodora Leaf, including Plasma DPP-IV and GLP-1 Levels in High-Fat-Fed Rats, as Well as Characterization of Biologically Effective Phytoconstituents

Abstract

Due to the numerous adverse effects of synthetic drugs, researchers are currently studying traditional medicinal plants to find alternatives for diabetes treatment. Eucalyptus citriodora is known to be used as a remedy for various illnesses, including diabetes. This study aimed to explore the effects of ethanol extract of Eucalyptus citriodora (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^2+-channel blocker (Verapamil), K_ATP-channel opener (Diazoxide), and Ca²⁺ 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 E. citriodora could be used as an adjunct nutritional supplement to manage type 2 diabetes.

Keywords: diabetes; glucose; insulin; medicinal plants; phytoconstituents.

Figure 1

Schematic diagram represents the experimental design for animal studies.

Figure 2

Effects of EEEC on insulin secretion from (A,B) clonal pancreatic β-cells (BRIN-BD11) and (C) islets of Langerhans, (D) glycation of protein, (E) secretion of insulin with known stimulators or inhibitors and (F) plus or minus extracellular calcium from clonal β-cells. Values n = 8 and 4 for insulin secretion and n = 3 for glycation of protein are mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to control. ^ϕ p < 0.05 and ^ϕϕϕ p < 0.001 compared to 5.6 mM glucose with EEEC. ^Δ p < 0.05, ^ΔΔ p < 0.01 and ^ΔΔΔ p < 0.001 compared to respective incubation without EEEC. EEEC, Ethanol extract of E. citriodora.

Figure 3

Effects of EEEC on (A) membrane potential and (B) intracellular calcium in clonal pancreatic β cell (BRIN BD11) and, (C–G) glucose uptake, (H) starch digestion and (I) glucose diffusion in vitro. Changes of fluorescence intensity in differentiated 3T3L1 adipocyte incubated with EEEC (E) minus or (F) plus 100 nM insulin. The ×10 magnification was used to take the images. Values n = 6 for membrane potential and intracellular calcium, n = 4 for uptake of glucose, digestion of starch and diffusion of glucose are mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to control. ^Δ p < 0.05 compared to insulin alone.

Figure 4

Effects of EEEC on (A) DPP-IV enzyme in vitro, (B) glucose tolerance, (C) plasma insulin, (D) DPP-IV and (E) plasma active GLP-1 (7–36) in high-fat-fed rats. In vivo parameters were evaluated before and after oral gavage of glucose alone (18 mmol/kg body weight, control) or with EEEC (250 mg/5 mL/kg body weight), sitagliptin and vidagliptin (both at 10 μmol /5 mL/kg, body weight). Plasma active GLP-1 (7–36) levels was assayed at 30 min after treatments. Values n = 4 for in vitro DPP-IV enzyme activity and n = 6 for in vivo parameters are mean ± SEM. * p <0.05, ** p <0.01 and *** p < 0.001 compared to lean control and ^Δ p < 0.05, ^ΔΔ p < 0.01 and ^ΔΔΔ p < 0.001 compared to high-fat-fed diet control rats.

Figure 5

Representative (A) HPLC profile and (B) insulin-releasing effects of peak fractions (1,2,7-9) of EEEC. Crude extract was chromatographed at a flow rate of 1.0 mL/min on a (10 × 250 mm) semi-preparative 5 μm C-18 column (Phenomenex, UK). Using linear gradients of acetonitrile (0–20% up to 10 min, 20–70% up to 40 min), the concentration of the eluting solvent was increased. Compounds were detected by measurement of absorbance at 254 nm. Peak fractions 1, 2 and 7–9 were collected and insulin-releasing activity assessed using BRIN-BD11 cells. Values n = 8 for insulin release are mean ± SEM. *** p < 0.001 compared to control.

Figure 6

Chemical structure of possible phytoconstituents extracted from the EEEC. Chemical structures of (A) Rhodomyrtosone B, (B) Quercetin-3-O-β-D-glucopyranoside, (C) Quercitroside and (D) Rhodomyrtosone E with their corresponding molecular formulae: C₂₆H₃₄O_6, C₂₁H₂₀O₁₂, C₂₁H₂₀O₁₁, and C₃₀H₃₄O_6.