Isolation and biological activity studies of some proteins from Cucurbitaceae family

Abstract

newlineObjective: The present research work was undertaken to evaluate the proteolytic, newlineantioxidant and antimicrobial activities followed by determination of amino acid newlinecomposition and functional properties of seed protein fractions of some indigenous newlineCucurbit plants such as Cucurbita moschata, Citrullus lanatus and Lagenaria siceraria. newlineMethods: A modified Osborne fractionation method was used to isolate albumin, globulin, newlineprolamin and glutelin successively from seeds of C. moschata, C. lanatus and L. siceraria. newlineThe total protein content of seeds was determined using a Bio-Rad protein assay method. newlineThe proteolytic activity study of the fractionated proteins was analyzed by using casein and newlinemilk-clotting activity was assessed by using skim milk. The effect of temperature (30 90 newline°C) and pH (3 11) on enzymatic activities were also evaluated. The antioxidant activities newlineof the all the protein fractions were evaluated using DPPH, ABTS, hydrogen peroxide, newlinenitric oxide radical scavenging, lipid peroxidation inhibition, phosphomolybdate and newlinereducing power assays. The further investigation was performed using enzymatic newlinehydrolysis of potent globulin fractions of seeds of all these plants by trypsin, and their newlineantioxidant properties were evaluated by using both in vivo and in vitro methods. The newlinefunctional and structural properties of globulin hydrolysates of these plant seeds were newlinedetermined by measuring various physicochemical parameters. The exploration of newlineantimicrobial activity of protein fractions of all these plants were carried out using two newlinedifferent in-vitro antimicrobial methods (viable cell count method and broth-based newlineturbidometric assay) against both Gram positive and Gram negative bacteria. The most newlineeffective protein fractions of all these tested plants were selected for hydrolysis using newlinetrypsin. Further, protein hydrolysates were evaluated by in vitro and in vivo methods. newlineResults: The total protein content of the seed flour of C. moschata, C. lanatus and L. newlinesiceraria was found to be 60%, 46% and 56%, respectively. Globulin fraction of L. newlinesiceraria contains highest amount of hydrophobic amino acid (138.1 mg/gm) as well as newlineessential amino acid (165.6 mg/g) as compared to other tested seed proteins. Among all newlinethese plants, C. moschata showed highest milk-clotting activity over a broad temperature newlinerange (50 70 °C) and pH range (6 7). In the present study, albumin fraction of C. newlinemoschata and C. lanatus seeds and prolamin fraction of L. siceraria showed more newlineproteolytic and milk-clotting activities than other protein fractions. The antioxidant activity newlinevi newlineof globulins fraction of all tested Cucurbitaceae seeds showed strongest antioxidant newlinecapacity while glutelin fractions showed weaker antioxidant potential. Globulin newlinehydrolysates of L. siceraria showed effective antioxidant properties in both in vivo and in newlinevitro models, whereas globulin hydrolysates of C. moschata and C. lanatus exhibited newlineremarkable antioxidant properties, as well. The functional property of globulin hydrolysates newlineof C. moschata, C. lanatus and L. siceraria was investigated. Globulin hydrolysates of L. newlinesiceraria was found to have good water absorption capacity (5 g/g), heat stability (89%), newlineemulsifying activity index (98.3m2/g) and emulsifying stability index (45.1 min) as newlinecompared to other globulin hydrolysates of seeds of C. moschata and C. lanatus. Among newlineall the tested plants, albumin fractions of C. moschata and L. siceraria were found to be newlineactive against A. baumanii while prolamin fraction of C. lanatus was found to be active newlineagainst P. aeruginosae. Similarly, albumin hydrolysates of C. moschata and L. siceraria newlinewere active against A. baumannii. The P. aeruginosa was found to be most susceptible to newlineprolamine hydrolysate of C. lanatus. The in vitro studies do not depend upon the newlinefluctuation of drug concentration within the body. Therefore, the vital potency of tested newlinedrug is determined by in vivo model. The albumin hydrolysates of C. moschata and L. newlinesiceraria showed significant antibacterial activities against A. baumannii whereas, newlineprolamin hydrolysate of C. lanatus exhibited remarkable antibacterial activity against P. newlineaeruginosa. newlineConclusion: It is concluded that the protein fractions of all these three plants like C. newlinemoschata, C. lanatus and L. siceraria seeds may be used in the formulation of nutritive newlineproducts. The resultant globulin hydrolysates could be utilized in food and pharmaceutical newlineindustries for the development of functional foods with potent antioxidant properties. The newlinepotent antimicrobial protein hydrolysates of these seeds might be used in development of newlinenovel antimicrobial agents in near future