Original ResearchLow-molecular-weight fractions of Alcalase hydrolyzed egg ovomucin extract exert anti-inflammatory activity in human dermal fibroblasts through the inhibition of tumor necrosis factor–mediated nuclear factor κB pathway
Introduction
Egg is a widely used, affordable, and high-quality source of protein. Egg protein–derived compounds have been extensively studied for their potential uses either as functional foods or as nutraceutical preparations [1]. Ovomucin is a mucin-like glycoprotein from egg white, accounting for 2% to 4% of total egg protein [2]. Each ovomucin molecule is composed of a carbohydrate-poor α-subunit and a carbohydrate-rich β-subunit, containing 11% to 15% and 50% to 57% (wt/wt) carbohydrate, respectively [3], [4]. It is well documented that ovomucin and its hydrolysates exert a number of beneficial bioactivities such as antitumor and cholesterol-lowering activities [5]. A structurally similar molecule, MUC1 mucin, a transmembrane glycoprotein expressed on the surface of mucosal epithelial cells, has been demonstrated to have anti-inflammatory property by suppressing the Toll-like receptor 5 signaling pathway [6]. In addition, ovomucin is rich in sialic acid, which can further contribute to anti-inflammatory effects [7]. Thus, ovomucin might have the potential to modulate inflammation, although its specific anti-inflammatory functions remain largely unknown.
Proteolytic digestion has been used to enhance both solubility and bioactivities of ovomucin [8]. For example, glycopeptides generated upon pronase digestion of ovomucin can exert antiadhesive activity against Escherichia coli O157:H7 as well as antitumor activity in a double-grafted tumor system [9], [10]. Ovomucin hydrolysates generated by microbial proteases such as Protamex, Flavourzyme, and Alcalase also possess antioxidant activity as determined by free radical scavenging assays [11].
However, not all protein hydrolysates are created equal. Indeed, depending on the enzymes used, the posthydrolysis treatments undertaken and the intrinsic properties (molecular weight [MW], solubility, hydrophobic vs hydrophilic nature) of the substrate protein, hydrolysates from the same protein source can differ widely in their observed biological functions [12]. For example, a recent publication by Schadow et al [13] has demonstrated the stark differences in biological effects of several commercially available collagen hydrolysates on articular cartilage function, whereas other studies suggested that lower-MW peptides were better suited for topical use and those rich in proline and/or hydroxyproline had higher bioavailability upon oral ingestion [14], [15]. Thus, there is a need to identify those enzymatic hydrolysates that might exert beneficial actions such as anti-inflammatory effects. Development of bioactive preparations from proteins like ovomucin can then lead to their use in the management of inflammatory conditions such as those affecting the skin, the largest organ in the body.
Inflammatory damage to the skin occurs from external injurious stimuli (excessive exposure to sun rays, trauma, radiation, infections) as well as from internal processes such as aging and the dermatologic manifestations of systemic illnesses [16]. Wound healing is also dependent on a low level of dermal inflammation, yet excessive and uncontrolled inflammatory changes may lead to widespread scarring and weaknesses of the healed skin [17]. Anti-inflammatory drugs and laser therapy are widely used for the treatment of skin diseases and aging, but are associated with their own complications and adverse effects, especially upon long-term usage [18], [19]. As such, there is increasing interest in using naturally derived compounds for maintenance and enhancement of skin health and reversal of skin aging effects. Natural compounds such as those derived from food sources are generally perceived as safer alternatives to pharmacologic agents and also likely to be better tolerated due to their preexisting use as dietary components [20]. Indeed, several enzymatic hydrolysates from food protein sources have shown promise in ameliorating pathological mechanisms associated with skin aging and dermal diseases [21], [22].
Egg white as a natural agent has been used in many parts of the world as an ingredient of folk cosmetics such as facial mask for improving skin health [23]. Given the implied anti-inflammatory potential in skin health and possible anti-inflammatory activity of ovomucin, we hypothesized that ovomucin-derived peptides might exert anti-inflammatory activity. The specific objectives were to test the anti-inflammatory activities of different ovomucin hydrolysates and its various fractions (enzymatically prepared and/or subjected to desalting or ultrafiltration) using human dermal fibroblasts (HDFs), a widely used model system for examining skin health [24], [25], and to understand the possible molecular mechanisms of the anti-inflammatory activity.
Section snippets
Materials and reagents
Fresh eggs from White Leghorn laid within 24 hours were obtained from the Poultry Research Centre of the University of Alberta (Edmonton, AB, Canada) and used same day for ovomucin extraction. Sodium dodecyl sulfate (SDS) and trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Coomassie Brilliant Blue (CBB) R-250 was purchased from Bio-Rad laboratories Inc (Hercules, CA, USA). Acetonitrile was bought from Fisher Scientific Inc (Ottawa, ON, Canada). Milli-Q water was
Ovomucin hydrolysates differentially regulate anti-inflammatory actions depending on enzymatic hydrolysis and desalting
Three different ovomucin hydrolysates were prepared as described under Methods and materials section, and these 3 ovomucin hydrolysates were further desalted to compare the anti-inflammatory activity to the original hydrolysates. Inflammatory changes were induced by stimulation with TNF, which causes up-regulation of the inflammatory leukocyte adhesion molecule, ICAM-1. Preincubation with different ovomucin hydrolysates showed that only the desalted Alcalase hydrolysate could reduce TNF-induced
Discussion
Natural products or natural product–derived compounds offer great opportunity to be the alternatives to drugs for the treatment of inflammatory diseases and provide mankind with new adjunctive therapy due to their great structural diversity and safety [32]. Various bioactive peptides from food sources have been proved their anti-inflammatory activity, especially those from food-derived collagen [22], [33], [34]. After oral ingestion of gelatin hydrolysates, the concentrations of
Acknowledgment
This study was funded by grants from Alberta Livestock and Meat Agency and the Natural Sciences and Engineering Research Council of Canada to J. Wu. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Both authors contributed equally.