Bioactive fraction isolated from Curcuma angustifolia rhizome exerts anti-diabetic effects in vitro, in silico and in vivo by regulating AMPK/PKA signaling pathway

Abstract

Curcuma angustifolia Roxb. is a therapeutic herb and a member of the Zingiberaceae family. A potential bioactive fraction was isolated from the methanolic extract of Curcuma angustifolia rhizome using column chromatography, and it was characterised using ¹H-NMR, GCMS and FTIR analyses. The bioactive fraction showed no toxic effects on the HepG2 cell line and it demonstrated inhibition of α-amylase and α-glucosidase enzymes in vitro with IC₅₀ values of 2.75 ± 0.09 and 4.9 ± 0.07 µM, respectively. Molecular docking analysis also showed that nerolidol, the major constituent of the bioacive fraction inhibits α-amylase and α-glucosidase enzymes competitively, supporting in vitro antihyperglycemic activity. ADMET analysis showed that nerolidol has the necessary physicochemical parameters for drug-likeness. It also complies with Lipinski's rule, indicating that its chemical structure is appropriate for designing safe and bioavailable oral drug. The antidiabetic efficacy of the isolated bioactive fraction was validated in type 2 diabetic albino wistar rats induced with a high-fat diet and a low dose (35 mg/kg bw) of streptozotocin. After 28 days of intervention, the lower and higher doses of the bioactive fraction (100 and 200 mg/kg BW) substantially decreased fasting blood glucose levels and ameliorated hyperglycemia, glucose intolerance, insulin resistance, and hyperlipidemia. The higher dose of bioactive fraction significantly ameliorated liver, kidney, and lipid profiles compared to the standard drug metformin and exhibited lower toxicity in the liver, kidney, pancreas, and epididymal adipose tissue than the lower dose of the bioactive fraction. Gene expression studies revealed that the bioactive fraction upregulated AMPK through downregulating PKA, a mechanism similar to the action of metformin. The results indicate that the isolated bioactive fraction could be a natural alternative to synthetic antidiabetic medications.

Keywords: AMPK; Curcuma angustifolia; PKA; bioactive fraction; molecular docking; type 2 diabetes mellitus.

FIGURE 1

(A) α-amylase inhibitory effect of bioactive fraction 8, (B) α-glucosidase inhibitory effect of bioactive fraction 8. Significant differences between the tested sample and the standard are indicated by different letters (p < 0.001).

FIGURE 2

Viability of the HepG2 cell line after treatment with various concentrations of bioactive fraction 8. Significant differences between the tested sample and control are indicated by different letters (p < 0.05).

FIGURE 3

¹H NMR spectra of bioactive fraction 8.

FIGURE 4

(A) GC-MS chromatogram of bioactive fraction 8, (B) Mass spectra of nerolidol.

FIGURE 5

Effect of bioactive fraction 8 on (A) body weight, (B) food intake, (C) water intake. The values are represented as the mean ± standard deviation (n = 6). Results were analysed using one-way ANOVA, and significant differences were denoted as ^###p < 0.001, ^##p < 0.01 in comparison with the NC group, whereas ***p < 0.001, **p < 0.01, *p < 0.05 in comparison with the DC group.

FIGURE 6

Effect of bioactive fraction 8 on (A) FBG level, (B) OGTT, (C) AUC of OGTT. The values are represented as the mean ± standard deviation (n = 6). Results were analysed using one-way ANOVA, and significant differences were denoted as ^###p < 0.001 in comparison with the NC group, whereas ***p < 0.001, **p < 0.01 in comparison with the DC group.

FIGURE 7

Effect of bioactive fraction 8 on FINS level and HOMA-IR. The values are represented as the mean ± standard deviation (n = 6). Results were analysed using one-way ANOVA, and significant differences were denoted as ^###p < 0.001, ^##p < 0.01 in comparison with the NC group, whereas ***p < 0.001, *p < 0.05 in comparison with the DC group.

FIGURE 8

Effect of bioactive fraction 8 on (A) Total cholesterol, Low-density lipoprotein, (B) Triglycerides, High-density lipoprotein, (C) Very low-density lipoprotein. The values are represented as the mean ± standard deviation (n = 6). Results were analysed using one-way ANOVA, and significant differences were denoted as ^##p < 0.01, ^#p < 0.05 in comparison with the NC group, whereas **p < 0.01, *p < 0.05 in comparison with the DC group.

FIGURE 9

Effects of bioactive fraction 8 on (A) AST, ALT, (B) ALP. The values are represented as the mean ± standard deviation (n = 6). Results were analysed using one-way ANOVA, and significant differences were denoted as ^###p < 0.001 compared to the NC group, whereas ***p < 0.001, **p < 0.01 compared to the DC group.

FIGURE 10

Effects of bioactive fraction 8 on (A) Urea, (B) Creatinine, (C) Total protein, (D) Albumin, Globulin. The values are represented as the mean ± standard deviation (n = 6). Results were analysed using one-way ANOVA, and significant differences were denoted as ^###p < 0.001, ^##p < 0.01, ^#p < 0.05 compared to the NC group, whereas ***p < 0.001, **p < 0.01, *p < 0.05 compared to the DC group.

FIGURE 11

Effects of bioactive fraction 8 on the histology of (A) liver, (B) kidney, (C) pancreas, and (D) epididymal adipose tissue (magnification, ×800)

FIGURE 12

Expression of AMPK and PKA genes in fold. The values are represented as the mean ± standard deviation (n = 3). Results were analysed using one-way ANOVA, and significant differences were denoted as ^{# #}p < 0.01 compared to the NC group, whereas **p < 0.01, *p < 0.05 compared to the DC group.

FIGURE 13

Proposed mechanisms underlying the antidiabetic effects of the bioactive fraction isolated from Curcuma angustifolia rhizome in type 2 diabetic rats.