Effect of osmotic conditions and germination time on the content and profile of glucosinolates, carotenoids and phenolic compounds in kale, and their antioxidant and anti-inflammatory properties

Abstract

In the past years, abiotic stresses such as saline soaking and the exogenous application of phytohormones such as methyl jasmonate (MeJA) have been studied as an effective tool to improve the accumulation of bioactive phytochemicals in fresh products. While the application of selenium (Se), sulfur (S), and methyl jasmonate (MeJA) has been studied in mature crops of Brassicaceae family such as kale, information about their effects on carotenoid and phenolic compound content in sprouts is scarce. Moreover, bioactivity studies of kale extracts treated with Se, S, and MeJA have not been performed. Therefore, the present thesis evaluated, the effect of Se, S, and MeJA on the accumulation of glucosinolates, carotenoids and phenolic compounds in kale sprouts. On the other hand, the individual and combined effects of Se, S, and MeJA stress over the anti-inflammatory and antioxidant activity in an in vitro model of Inflammatory bowel disease (IBD). The present study was divided into three phases. In the first phase, the effects of Se, S, and MeJA on lutein, glucosinolate, and phenolic accumulation were assessed in kale sprouts. Red Russian and Dwarf Green kale were chamber-grown using different treatment concentrations of Se (10, 20, 40 mg/L), S (30, 60, 120 mg/L), and MeJA (25, 50, 100 µM). Sprouts were harvested every 24 h for 7 days to identify and quantify phytochemicals. The highest lutein accumulation occurred 7 days after S 120 mg/L (178%) and Se 40 mg/L (199%) treatments in Red Russian and Dwarf Green kale sprouts, respectively. MeJA treatment decreased the level of most phenolic levels, except for kaempferol and quercetin, where increases were higher than 70% for both varieties when treated with MeJA 25 µM. The most effective treatment for glucosinolate accumulation was S 120 mg/L in the Red Russian kale variety at 7 days of germination, increasing glucoraphanin (262.4%), glucoerucin (510.8%), 4-methoxy-glucobrassicin (430.7%), and glucoiberin (1150%). Results show that kales treated with Se, S, and MeJA could be used as a functional food for fresh consumption or as raw materials for different industrial applications. In the second phase of this study, it was assessed the individual and combined effects of Se, S, and MeJA on the content of carotenoids, phenolic compounds, and glucosinolates present in undigested extracts and intestinal-digested fractions of 7-day-old Red Russian kale sprouts. Also, the effect of undigested extract and digested kale on cellular antioxidant activity (CAA) and nitric oxide (NOx) production was evaluated. Kale sprouts treated with Se showed higher levels of total glucosinolates (53.2%) and lutein (149.3%) in the ethanolic extract, whereas phenolic content remained without significant changes. After in vitro digestion, a significant liberation of lutein was observed in all samples of kale, mainly the Se-enriched fraction (210.3%). On the other hand, phenolic compounds and glucosinolates suffered significant losses. Abiotic stress did not cause a significant change in phenolic content compared to control sprouts; however, Se treatment significantly preserved the concentration of glucoraphanin (70.8%%), glucobrassicin (58.2%), 4-methoxy-glucobrassicin (41.6%) and total glucosinolate content (63.2%) in kale compared with control sprouts. In correlation, the maximum percentage of CAA and NOx was achieved in the undigested extract and intestinal digested fraction from kale sprouts treated with selenium. Due to the best results of phases 1 and 2 were obtained with Se (40 mg/L) and S (120 mg/L) treatments, they were selected for the third phase of this research. In this study, phytochemical extracts from 7-days-old Red Russian kale sprouts treated with Se and S were encapsulated with maltodextrin by spray-drying to evaluate its ability to protect phytochemicals from degradation during the digestion process. Particle morphology, encapsulation efficiency, and storage stability were characterized. Mouse macrophages (Raw 264.7) and human intestinal cells (Caco-2) were treated with the digested fraction to assess the cellular antioxidant capacity, the production of nitric oxide (NOx), and the levels of inflammatory biomarkers such as cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, IL-2, IL-6, and IL-10. Se (89.4%) and S (85.8%) kale sprouts showed the highest encapsulation efficiency and spherical morphology. Digestion affected the content of compounds in both encapsulated and non-encapsulated extracts. However, encapsulation promoted the control of oxidative processes during storage, and the kale extracts germinated with S and Se showed less degradation of lutein (35.6%, 28.2%), phenolic compounds (20.3%, 25.7%) and glucosinolates (15.4%, 18.9%) compared to non-encapsulated samples, respectively. The encapsulate treated with S exerted the highest antioxidant capacity (94.2%) and anti-inflammatory activity by stimulating IL-10 by 88.9% and inhibiting COX-2 and NO production by 84.1% and 92.2%, respectively. Therefore, kale sprouts with enhanced levels of phytochemicals could be used as feedstock to produce processed foods or undergo further processing for extraction and purification of high-value health-promoting biomolecules. Based on these results, general strategies to yield a high accumulation of specific phytochemicals are presented in this work. With this information, stressed kale with high concentrations of Se or S can be used as a plant model of high-value biomolecule for the treatment of inflammatory diseases.