Chemical and biological investigations of Limonium axillare reveal mechanistic evidence for its antidiabetic activity

Abstract

Root and bark of Limonium axillare (Forssk) Kuntze are used as antidiabetic remedies in parts of East Africa, but this activity has never been fully investigated. To validate its ethnobotanical use, we compared the chemical and pharmacological profiles of the ethanolic extracts of L. axillare root (REE) and aerial parts (AEE). Administration of REE (500 mg kg-1) reduced streptozotocin-induced hyperglycemia by 44%, restored serum insulin levels, reestablished Glut2 and Glut4 expression and ameliorated pancreatic tissue damage in diabetic rats. In vitro studies revealed a strong radical scavenging effect, α-glucosidase, and α-amylase inhibition activity of REE at IC50 at 25.2, 44.8 and 89.1μg/mL, respectively. HPLC analysis identified ten phenolic compounds in REE with umbelliferone as the major constituents at 10 ± 0.081 mg/g of extract. Additionally, six compounds were isolated from REE including, β-sitosterol-3-palmitate, β-sitosterol, myricetin and gallic acids with two new tetrahydrofuran monoterpenes; 2-isopropyl- 3,4,4, trimethyl-tetrahydrofuran (3), and 2-isopropyl-4-methyl-tetrahydrofuran-3,4 dicarboxylic acid (4), the latter was revealed by molecular docking to be a good ligand to glycerol-3-phosphate dehydrogenase a key enzyme in glycolysis.

Fig 1. Photomicrographs of rat pancreases isolated from different animal groups.

Photomicrographs of cross section of the pancreatic tissue of A: normal healthy animals showing normal pancreatic islets (PI) and pancreatic acini (arrow), B: diabetic group showing pancreatic duct inspissiated (arrow), pancreatic islet (pi) with sever reduction in size and hypocellularity. C: animals treated with REE 250 mg/kg showing: pancreatic islet (short arrow), inspissated pancreatic duct (long arrow) and blood vessel (double arrow) and pancreatic acini (arrow head). D: animals treated with REE 500 mg kg^-1 bwt showing pancreatic islet moderate in size and dilated congested blood vessel (arrow). E: animals treated with 250 mg kg^-1 bwt AEE showing pancreatic blood vessels with thickened walls (arrow) and perivascular fibrotic reaction (short arrow). F: animals treated with AEE (500 mg kg^-1 bwt) showing islets with normal size and vaccuolation between cells (arrowhead), pancreatic duct (arrow). (H & E). G: Animals treated with metformin showing: pancreatic islets with vacuolation between cells (arrow), dilated congested blood vessel (BV), and intact pancreatic acini (double arrow).

Fig 2. HPLC chromatograms for AEE and REE at λ = 280 and 360 nm.

A and B:HPLC analysis for REE showing eluted compounds with UV absorbance at λ = 280 and 360 nm, respectively. C and D: HPLC chromatogram of eluted compounds detected at λ = 280 and 360, respectively. Identified compounds are labeled as 1: gallic acid, 2:ellagic acid, 3: ferulic acid, 4: umbelliferone, 5: myricetin, 6:quercetin, 7:coumarin, 8:apigenin and 9:kaempferol.

Fig 3. Compounds isolated from the root extract of Limonium axillare.

Fig 4. Enzyme ligand interactions of Limonium axillare phytoconstituents in the active site glycerol phosphate dehydrogenase.

Diagrams representing 2D enzyme ligand interaction in the active site of GPDH with tested compounds A: 2’,4’ dihydroxychalcone, B: metformin, C: gallic acid, D: myricetin, E: compound 3, F: compound 4.