Hypoglycaemic activity of Bauhinia holophylla through GSK3-β inhibition and glycogenesis activation

Abstract

Context: Bauhinia L. species, including Bauhinia holophylla (Bong.) Steud. (Fabaceae), have traditionally been used to treat diabetes. Bauhinia is a complex botanical genus, and the indiscriminate use of the diverse Bauhinia species is reflected in the experimental divergence of their medicinal potential.

Objective: The hypoglycaemic and hypolipidaemic effects, molecular mechanism of action and phytochemical properties of an authentic extract of B. holophylla leaves were evaluated.

Materials and methods: A phytochemical study of a 70% EtOH extract was performed using FIA-ESI-IT-MS/MSⁿ and HPLC-PAD-ESI-IT-MS. The extract (200 or 400 mg/kg b.w.) was administered for 14 days to streptozotocin-induced diabetic Swiss mice. Glucose tolerance and insulin sensitivity, blood parameters, gene and protein expression, and the in vivo and in vitro inhibition of intestinal glucosidases were assessed.

Results: HPLC-PAD-ESI-IT-MS analysis identified flavonoid derivatives of quercetin, myricetin, luteolin and kaempferol. Treatment with 400 mg/kg of the extract reduced blood glucose (269.0 ± 32.4 mg/dL vs. 468.0 ± 32.2 mg/dL for diabetic animals), improved glucose tolerance, decreased cholesterol and triglyceride levels, and increased the mRNA expression of proteins involved in glucogenesis in the liver and muscle, such as PI3-K/Akt, GS, GSK3-β (ser-9), AMPK and Glut4. The activity of intestinal maltase was inhibited in vitro (IC₅₀: 43.0 µg/mL for the extract compared to 516.4 µg/mL for acarbose) and in vivo.

Discussion and conclusions: Treatment with B. holophylla was associated with a marked hypoglycaemic effect through the stimulation of glycogenesis and inhibition of gluconeogenesis and intestinal glucose absorption, without increasing basal insulinaemia.

Keywords: Antidiabetic; Fabaceae; flavonoid--glycosides; liver; plasma lipids.

Figure 1.

Effect of Bauhinia holophylla treatment on glycaemia, glucose tolerance and insulin sensitivity. (A) Average glycaemia during the treatment period. (B) Average glycaemia values during the intraperitoneal glucose tolerance test (ipGTT). (C) Areas under the curves (AUC) values obtained from ipGTT experiment. (D) Average insulinaemia at the end of the treatment period and (E) HOMA-IR index of the groups. Bauhinia holophylla treatment significantly decreased glycaemia and HOMA-IR index. Basal insulin values were not changed, suggesting an extra-pancreatic action of the extract. Different letters indicate significant differences (ANOVA followed by Tukeyʼs post-test, n = 8, p < 0.05).

Figure 2.

Liver glycogenesis stimulation by the treatment with Bauhinia holophylla extract. (A) The expression of PI3K, Akt, GSK3-β and GS genes analyzed by real-time PCR. (B) The expression of phosphorylated and total proteins by western blot. (C) Representative images of the proteins bands analyzed. Different letters indicate significant differences (ANOVA followed by Tukeyʼs post-test, n = 8, p < 0.05).

Figure 3.

Liver gluconeogenesis inhibition by the treatment with Bauhinia holophylla. (A) G6Pase and PEPCK genes expression analyzed by real-time PCR. (B) Protein expression of G6Pase, PEPCK and AMPK analyzed by western blot. (C) Representative images of the bands. Different letters indicate significant differences (ANOVA followed by Tukeyʼs post-test, n = 8, p < 0.05).

Figure 4.

Effect of Bauhinia holophylla treatment on gene and protein expression in muscle. (A) Expression of PI3K, Akt and Glut-4 genes. (B) Expression of phosphorylated and total forms of proteins involved in glucose uptake in muscles. (C) Representative images of the proteins analyzed. Different letters indicate significant differences (ANOVA followed by Tukeyʼs post-test, n = 8, p < 0.05).

Figure 5.

The inhibitory effect of the Bauhinia holophylla treatment on intestinal enzymes. In vitro determination of the IC₅₀ for α-amylase (A) and α-glucosidase (B). In vivo inhibition of α-amylase after an oral starch load (C) with the respective areas under the curves (D) and of α-glucosidase (E) with the respective areas under the curves (F). Different letters indicate significant differences (ANOVA followed by Tukeyʼs post-test, n = 6, p < 0.05).