Characterization of bio-functional activities of glycoprotein purified from Undaria pinnatifida with its compositional analysis

Abstract

Seaweeds are nurtures of bioactive compounds that could be used as functional ingredients for human health. Macroalgae are known as good source of healthy food due to their low content of lipid, high concentration of polysaccharides, rich in minerals, polyunsaturated fatty acids (PUFA), vitamins and other bioactive molecules. Many efforts have been carried out to isolate and identify new compounds with potential medicinal and pharmaceuticals activities. However, very few compounds with real potency are reported. Till now, most of the researches related to seaweed have been focused on polysaccharides, lipid, protein, phlorotanins etc and reported to possess strong anti-viral, anticancer, antioxidant, anti-inflammatory and anti-diabetic activities. Apart from these compounds, glycoprotein from plants and other sources showed potent biofunctional activities. Thus, this study has been carried out to characterize the bio-functional activity of purified glycoprotein from Undaria pinnatifida with its compositional analysis. Firstly, I purified and determined the chemical composition of glycoprotein from Undaria pinnatifida (UPGP) and also evaluated antioxidant and DNA protection activity using in vitro digestion model. On SDS-PAGE, UPGP migrated as a single band with a molecular weight of approximately 10 kDa and confirmed by staining with Schiff’s reagent as glycoprotein. It consists of a carbohydrate (42.53%) and protein (57.47%) component. Amino acid profile, FT-IR spectrum and enzymatic glycosylation analysis suggested that protein is linked with carbohydrate by O- glycosylation. UPGP showed dose-dependent antioxidant activities as detected by different assays before and after in vitro digestion. The IC50 values of undigested UPGP were 0.25 ± 0.03, 0.08 ± 0.005, 0.69 ± 0.12, and 0.25 ± 0.08 mg/mL for DPPH, ABTS, FRAP, and NO, respectively. Following in vitro digestion, the antioxidant activities of UPGP were decreased during the gastric phase compared to those of undigested UPGP, with an increase occurring during the duodenal phase in all assays. However, the reducing power was unchanged after in vitro digestion. Furthermore, UPGP showed protective activity against oxidative DNA damage both undigested, after saliva and duodenal phase of digestion. These results indicate that the antioxidant and DNA protection activities of UPGP may be pH-dependent and assay specific. I next evaluated the hypoglycaemic effect of UPGP through monitoring α-glucosidase inhibition and glucose transport across yeast cell. Dose-dependent inhibitions of UPGP against yeast and rat intestinal α-glucosidase were observed with IC50 values of 0.11 and 0.29 mg/ml, respectively. UPGP showed stable inhibition following incubation at different temperatures and metal ions. Regarding bioaccessibility, the inhibition was decreased slightly during the gastric phase compared to undigested UPGP, with an increase during the duodenal phase. Kinetics and membrane dialysis revealed mixed and reversible inhibition, respectively. Furthermore, UPGP with acarbose showed synergistic inhibition against α-glucosidase, and UPGP increased the rate of glucose transport across the yeast cell. In conclusion, our study demonstrated that UPGP may be used as bioaccessible food additives for controlling postprandial hyperglycemia. Lastly, I examined the anti-Alzheimer’s and anti-inflammatory activities of UPGP. To evaluate the anti-Alzheimer’s potentiality of UPGP, the inhibitory activities of acetycholinesterase (AChE), butyrylcholinesterase (BChE), and β-secretase (BACE1) were assessed using standard protocols. Neuronal cell viability was determined with propidium iodide exclusion and lactate dehydrogenase assays using primary hippocampal cells. Anti-inflammatory activity was measured in two ways: first, by measuring nitric oxide (NO) production in lipopolysaccharides (LPS) activated RAW 264.7 macrophages, and second, with the cyclooxygenase (COX) inhibitory assay. The effect of UPGP on the enzymatic antioxidant activities of superoxide dismutase (SOD) and xanthine oxidase (Xox) was also studied. UPGP showed predominantly AChE, BChE, and BACE1 inhibitory activities with IC50 values of 63.56±1.86, 99.03±4.64, and 73.35±2.54 μg/mL, respectively. The addition of UPGP (5 μg/mL) to the culture medium demonstrated that it was not cytotoxic to cultured hippocampal cells, but rather, protected neurons from natural death. The ability of UPGP to inhibit enzymes COX-1 and COX-2 was determined by calculating the percent inhibition of prostaglandin production, as measured by enzymatic assays. UPGP inhibited COX-1 and COX-2 with IC50 values of 53.03±1.03 μg/mL for COX-1 and 193.35±3.08 μg/mL for COX-2. UPGP inhibited NO production by 94.2% at a concentration of 100 μg/mL; cell viability was 94.64% at this concentration. Moreover, UPGP improved SOD activity (53.45%) and inhibited Xox (82.05%) activity at a concentration of 5 mg/mL and 1 mg/mL, respectively. These results suggest that UPGP is a bioactive compound with the potential to control AD and inflammatory- and oxidative stress-related diseases.