Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata

Abstract

Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15% SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution. Correspondence

The hypoglycemic effect of plant derived insulin like protein in comparison to the hypoglycemic effect of the human soluble insulin in diabetic mice

“…Bauhenina varigata alcoholic extracts have compounds with a retention times very close to that of soluble insulin, which means that both plant alcoholic extract have compound with chemical structure similarities to that of soluble insulin, excluding any factors affecting the retention time since all the compound were analyzed under the same chromatographic condition. ).this is in agreement with (19) which indicated that HPLC chromatographic analysis of Bauhinia varigata show insulin like properties. for Bauhenina varigata alcoholic extract, no deaths or toxicity signs and symptoms observed after 24h and 48 h in all concentration that was administered and therefore the LD50 is considered higher than 4000mg/kg (which is the crude or highest extract concentration) and according to (Loomis and Hayes, 1996) its considered slightly toxic.…”

Detection of Insulin-Like Protein and Some Active Compounds in Bauhinia Variegata Linn. Leaf Ethanolic Extracts and the Effect in Reducing Blood Glucose Levels in Mice

“…The effect of the active compounds such as flavonoids that found in B. variegata leaves on glucose levels in the serum of diabetic mice caused a significant decrease in blood glucose levels in diabetic mice similar to that reported on the hypoglycemic activity of Bauhinia variegate in diabetic patients by Azevedo et al, [20]. Several published study showed that streptozotocin-diabetic rats treated with a decoction of Bauhinia forficata leaves presented a significant reduction in serum and urinary glucose as compared to streptozotocindiabetic controls [21].…”

Detection of immunoactive insulin in Spirulina

“…We detected a protein band in the same position as a bovine insulin band with a molecular mass of approximately 6 kDa. Experimental insulin showed an identical electrophoretic mobility with human insulin (5.8 kDa), bovine insulin (5.7 kDa) and other plant insulins from Momordica charantia (11 kDa; Khanna et al 1976), Spinacea oleracea and L. gibba (6 kDa; Collier et al 1987), Vigna unguiculata (6 kDa; Venâncio et al 2003) and Bauhinia variegate (5.7 kDa; Azevedo et al 2000). During Western blotting, SDS-PAGE gels were treated with the anti-insulin antibody and the band observed at the same position as bovine insulin.…”