Nanoencapsulation of antioxidant peptides from Lupinus mutabilis in chitosan nanoparticles obtained by ionic gelling and spray freeze drying intended for colonic delivery
Introduction
There is growing scientific evidence regarding the benefits of biopeptides from plant proteins (Wong et al., 2020). Soy peptides such as lunasin and soymorphins play an essential role in preventing multiple chronic diseases. Soybean glycinin tripeptide VPY effectively inhibits pro-inflammatory mediators in intestinal epithelial and immune cells, which are both involved in the pathogenesis of inflammatory bowel disease (IBD) (Fernández-Tomé et al., 2019; Montesano et al., 2020). IBD is a chronic disease that affects the middle or lower part of the gastrointestinal (GI) tract, where the two main subtypes are ulcerative colitis (UC) and Crohn's disease (CD). Both are debilitating conditions that carry an increased risk of colorectal cancer (from 0.5% to 20% per year), particularly in the case of UC (Moura et al., 2015). The aetiology of IBD remains unknown, but it is recognised that it is produced by a combination of genetic, immunological and environmental factors such as microbiota and diet. Oxidative stress has long been associated with the pathogenesis of IBD (Bourgonje et al., 2020). It has been suggested that the combined administration of antioxidants and anti-inflammatory agents may be helpful for its treatment (Fernández-Tomé et al., 2019; Shahinfar et al., 2021).
Lupinus mutabilis (tarwi) is an ancient legume cultivated throughout the South American Andes. It is of high industrial value due to its high content of proteins (32.0–52.6%) and lipids (13.0–24.6), which is similar to soybeans (glycine max), where the majority of the protein fraction (91–94%) that contains these seeds is comprised of globulins. Unfortunately, cultivation is currently poorly valued in Peru, and its cultivation is underutilised (Carvajal-Larenas et al., 2016) (Chirinos-Arias, 2015). In previous works, we optimized time parameters and enzyme/substrate ratio for the production of peptides with antioxidant capacity from a tarwi protein isolate using alcalase, enzyme of microbial origin (Intiquilla et al., 2018). Recent strategies suggest the previous use of digestive enzymes in combination with enzymes of microbial origin to generate small and stable antioxidant peptides (Palma-Albino et al., 2021; Vilcacundo et al., 2018), as well as protecting them in micro or nanocarriers (De Oliveira et al., 2021). Antioxidant peptides may help treat IBD; however, when administered orally, they can be prematurely degraded during their passage through the gastrointestinal tract, affecting their structural integrity, which is crucial for their bioavailability and pharmacological activity (Fernández-Tomé et al., 2019). According to the ABTS method, Zhao et al. (2020) identified five antioxidant peptides after hydrolyzing wheat protein with alcalase. However, the peptides CGFPGHC, RNF, and WF lost their biological activity (p < 0.05) after being subjected to an enzyme system that simulates the gastrointestinal tract.
Nanoencapsulation is an appropriate approach to protect the structure and, therefore, the biological activity of biopeptides in their passage through the gastrointestinal tract in an oral administration, improving their bioavailability compared to non-encapsulated peptides (Auwal et al., 2017). The colon is an appropriate site for peptide absorption. The colonic site has reduced proteolytic enzymatic activity and prolonged transit time, which can be increased by supplying biopeptides in mucoadhesive nanocarriers (chitosan, alginate, other gums), favouring their absorption, biological activity and controlled release for effective administration (De Oliveira et al., 2021). The intrinsic properties of biopeptides (solubility, molecular weight, hydrophilicity, and charge) are relevant to choosing the most appropriate nanocarrier to transport them.
Lipid nanoparticles (NP) have been developed as an alternative, however, their physicochemical instability and high sensitivity to oxidation can induce a premature release of the active ingredient, limiting its use at the colonic level (Ramezanzade et al., 2021). Chitosan-based NP are a practical and suitable alternative for the delivery of biopeptides to the colon since they are positively charged and favour their adhesion to mucins (Cone, 2009). In addition, the enzymatic activity of the resident microbiota in the colon triggers a controlled release (Gamboa et al., 2015). Despite, peptide fractions of size 10–30 kDa from fish (Hosseini et al., 2018) and peptides of 1–3 kDa and 3–10 kDa from egg (Du et al., 2019) have been encapsulated in chitosan nanoparticles by ionic gelation with tripolyphosphate, there is no description in the literature on the nanoencapsulation of biopeptides obtained from plant proteins with a size of less than 3 kDa in different chitosan NP. In previous work, prednisolone and inulin were nano encapsulated together in chitosan-tripolyphosphate (CTPP) obtained by ionic gelation and chitosan NP obtained by spray freeze-drying (SFDC). In both NPs, the encapsulation efficiency and loading capacity of inulin were lower than prednisolone due to its higher water solubility. In the case of prednisolone, due to its low solubility, it was necessary to use Pluronic F-127, which favoured its encapsulation in CTPP and SFDC. Although it is more challenging to obtain high-efficiency encapsulation for active ingredients with high water solubilities, such as inulin, in CTPP and SFDC, it is worth it because both systems allow colonic delivery of active ingredients. We proved that both systems, CTPP and SFDC, are degraded by Lysozyme, an enzyme which is presented in high concentration in IBD and E. coli, but only human faecal slurries degraded CTPP (Gamboa et al., 2015). Since patients with UC have diminished mucus-layer barrier properties due to the overall depletion of goblet cells and higher permeability of the intestinal epithelium, NPs can be easily transported into the mucosa, where they are phagocytized by macrophages and neutrophils at the sites of inflammation and can directly interact with the mucosal layer. Size (<500 nm) and positive charge are relevant for the ability of Chitosan NPs to passively diffuse into the inflamed intestinal mucosa and reach maximum retention times in the tissue (Cone, 2009; Ensign et al., 2012).
In this study, a protein isolate from Lupinus mutabilis seeds was sequentially hydrolysed in the enzyme system pepsin-pancreatin-alcalase to promote a higher release of antioxidant peptides. The peptide fraction with the highest antioxidant capacity was encapsulated in two nanosystems based on chitosan, CTPP and SFDC. Encapsulation parameters and physicochemical properties of both types of NPs were compared. The performance of both NPs was evaluated based on biopeptide release behaviour, antioxidant effect, and cytotoxicity on cell lines HT-29. This study contributes to the investigation of nanoparticles for colonic delivery of antioxidant peptides.
Section snippets
Materials
Tarwi seeds were collected in Huamachuco (Sánchez Carrión-La Libertad, Peru). Pepsin from porcine gastric mucosa (EC 3.4.23.1; 0.7 U/mg) and pancreatin from porcine pancreas (350 U/g of protease, 6000 U/g of lipase and 7500 U/g of amylase) were supplied by Merck Chemicals Co. (Darmstadt, Germany). Ortho-phthalaldehyde (OPA) and sodium tripolyphosphate (TPP) were purchased from Merck Millipore Corp, Darmstadt, Germany). Alcalase® 2.4 L from Bacillus licheniformis was purchased from Novozymes
Obtaining a tarwi protein isolate (TPI)
The tarwi seeds had an initial protein content of 46.37%, a value similar to that described by Carvajal-Larenas et al. (2016), who reported 32.0%–52.6% of Lupinus mutabilis proteins. The flour was defatted with ethanol, and tarwi protein isolate (TPI) was obtained at pH 10 with a protein content of 76.74%, which is similar to other legumes such as Phaseolus lunatus (71.8%) and Phaseolus vulgaris (63.8%) (Torruco-Uco et al., 2009). Fig. S1 shows the SDS–PAGE profile of TPI under reducing
Conclusion
This work made it possible to prepare chitosan nanoparticles by gelling with tripolyphosphate and spray freeze drying to encapsulate a peptide fraction with antioxidant capacity obtained from Lupinus mutabilis proteins. CTPP and SFDC presented suitable encapsulation parameters, achieving an LC of 5.35% and 6.03% and an EE of 63.80% and 71.75%, respectively. Both formulations showed a controlled release of the active compound, throughout 6 h of study, of 42.9 and 37.4%, respectively, maintaining
Author Statement
Arturo Intiquilla: Investigation, Validation, Formal analysis, writing. Karim Jiménez-Aliaga: Analysis. Amparo Iris Zavaleta: Analysis. Alexander Gamboa: Methodology. Nelson Caro: Methodology. Mario Diaz: Methodology. Martin Gotteland: Investigation. Lilian Abugoch: Analysis - Review & Editing, Resources, Project co-administration, Supervision. Cristian Tapia: Conceptualization, Formal analysis, Writing – review & editing, Resources, Supervision, Project administration
Declaration of competing interest
We do not have conflict of interest.
Acknowledgments
This work has received financial support from the “Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica” (CONCYTEC), Perú (Financial Agreement Number 007-2014-FONDECYT-Perú) and financing of research stays from the “Red de Macrouniversidades de América Latina y el Caribe” Banco Santander and the Doctorate Program in Nutrition and Food at the University of Chile.
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