Identification and characterization of health-promoting benefits and bioactive molecules in black walnuts (Juglans nigra l.) and waste materials from agroforestry systems
Authors:   , Ho Van Khanh 
Published: December 2023

Black walnut (Fagales: Juglandaceae; Juglans nigra L.), known as eastern black walnut or America walnut, is one of the most economically valuable hardwood species and a high value tree for edible nut production in the United States. This native plant species to North America that adapts to grow on a wide range of cultivation conditions is an ideal tree for agroforestry practices. Black walnut agroforestry systems provide landowners the opportunity to develop a solid long-term investment in future generations, both economically and spiritually while allowing establishing a balanced relationship with the environment. Remarkably, black walnut provides an excellent nutritious food material that contains a rich source of lipids and proteins. Consumption of black walnut has been linked to many health-promoting benefits such as decreased risk of cardiovascular disease, reduced levels of cholesterol, stimulated brain functions and prevention of certain cancers (e.g., prostate and breast cancers). However, the biological activities of black walnuts are remained unknown. We systematically explored multiple biological functions (antibacterial and antioxidant activities, and anti-inflammatory potential) of a numerous of black walnut cultivars that were selected and cultivated for nut production by the University of Missouri - Center for Agroforestry (Columbia, MO, USA). Additionally, it is likely that the bioactive activities of black walnut are correlated with its bioactive constituents including polyphenols. We further identified and characterized bioactive molecules that are driving the health-promoting benefits of black walnuts by modern analytical techniques combined with an array of in vitro bioassays. The exploration of health -promoting benefits in black walnut and its associated bioactive compounds would promote the development of novel applications of black walnut and its by-products such as new drug discoveries to mitigate human diseases (e.g., diabetes, cancer), cosmetic products (e.g., skin care products, personal care products), and functional foods (e.g., natural dietary supplements), and thereby potentially increase the sustainability of the black walnut agro-industry. We report the biological properties of black walnuts and their associated bioactive molecules. Black walnuts possessed antibacterial activities against a Gram-positive bacterium (Chapter 2). The antibacterial capacity of black walnuts was variable among different cultivars in which Mystry exhibited the strongest antibacterial capacity. Chapter 2 also describes a discovery of glansreginin A as a novel bacterial compound responsible for the antibacterial capacity of Mystry extract via a metabolomic approach coupled with bioassay-guided fractionation strategy. In Chapter 3, anti-inflammatory potential of black walnuts is described. Our results demonstrated for the first time that black walnut possessed compounds that exerted an inhibitory effect on the secretion of six measured cytokines [tumor necrosis factor alpha (TNF-[alpha]), interleukin (IL)-1[beta], IL-6, IL-8, IL-10, and monocyte chemoattractant protein (MCP)-1] induced by a human promonocytic cell line U937. Black walnut kernels contain a wealth of bioactive metabolites putatively identified through a metabolomics approach. Chapter 4 describes biological properties of glansreginin A. This compound was presented at moderate levels (6.8 - 47.0 mg/kg) in all 12 black cultivars examined. Glansreginin A possessed moderate antibacterial activities against Gram-positive bacteria including Staphyloccocus aureus and Bacillus anthracis. However, this polyphenolic compound exhibited no antioxidant activity, did not induced activity of antioxidant response element signaling pathways, and had no antiproliferative effects on the growth of both the tumorigenic alveolar epithelial cells and non-tumorigenic lung fibroblast cells. Furthermore, we identified and characterized antioxidant and anticancer capacities of 16 phenolic compounds that have been reported in black walnuts using high-throughput screening technologies (Chapter 5). Our findings suggested that penta-O-galloyl-[beta]-D-glucose, which is one of the most abundant phenolic compounds in Mystry, could be a potential bioactive agent for the cosmetic and pharmaceutical industries. Since several phenolic compounds had anticancer activities against growth of tumorigenic alveolar epithelial cells, revealing that black walnut extracts possibly possess anticancer activities. In addition to black walnuts, we investigated biological activities and associated bioactive compounds in waste materials [spent coffee grounds and switchgrass (Panicum virgatum)] from agroforestry systems. Chapter 6 describes the identification and quantification of bioactive molecules inhibiting pro-inflammatory cytokine production in spent coffee grounds. We found that methanolic extracts of spent coffee grounds obtained from 3 Arabica cultivars possess compounds that exerted inhibitory effects on the secretion of inflammatory mediators (TNF-[alpha], IL-6 and IL-10) induced by a human promonocytic cell line U-937. Spent coffee grounds contain a wealth of anti-inflammatory bioactive compounds with caffeine and 5-caffeoylquinic acid as the most abundant compounds identified via robust analytical metabolomic analyses. The health-promoting activities of the extracts from switchgrasses are assessed in Chapter 7. The extractives from switchgrass exhibited anti-inflammatory potentials, whereas no other biological activities (antibacterial and anticancer activities, tyrosinase and elastase inhibition) were observed on the switchgrass extracts. Switchgrass metabolic profile of anti-inflammatory compounds was putatively identified via untargeted metabolomics profiling and many of them were quantified using targeted metabolomics analyses.