Black rice anthocyanins embedded in self-assembled chitosan/chondroitin sulfate nanoparticles enhance apoptosis in HCT-116 cells
Introduction
Anthocyanins are plant pigments which are of wide interest due to their extensive range of colouring properties and their bioactivities (Zhan et al., 2016). The reported bioactivities of anthocyanins including antioxidant properties, cardioprotective capacity, anti-inflammatory activity, and prevention against oxidative damage (Krga and Milenkovic, 2019, Yang et al., 2019). For cancer cells, the anthocyanins can inhibit the cell proliferation, induce apoptosis, reduce oxidative stress and modulate the cell cycle (Bontempo et al., 2015, Vishnu et al., 2019). Anthocyanins are preferred over synthetic food colorants not only because of their nutritional value and health benefits, but due to the adverse side-effects of the synthetic food colorants.
Nevertheless, there are often challenges associated with incorporating these natural colorants into food and beverages due to their poor chemical stability. The stability of anthocyanins is influenced by environmental factors including light, enzymes, oxygen, pH, temperature, proteins, and metal ions (West & Mauer, 2013). Hence, it is essential to develop effective systems for improving the stability of anthocyanins in order to expand their further usage in the food industry.
The stability of anthocyanins can be increased by various methods including encapsulation, copigmentation, and the usage of metallic ions (Cavalcanti, Santos, & Meireles, 2011). Encapsulation is a technique that uses microspheres or liquid vesicles to incorporate and immobilize bioactive constituents to enhance the bioactive properties (Arroyo-Maya & McClements, 2015). The shelf life and stability of anthocyanins can be increased by microencapsulation which can protect molecules that are sensitive to environmental factors such as light and heat (Cortez, Luna-Vital, Margulis, & Elvira, 2017). Nanoparticles based on biopolymers have become one of the best methods to encapsulate and improve the stability of active substances due to advantages, such as controlled release, enhance absorption, reduce undesirable effects, and can easily penetrate cell membranes because of the small particle size (Liang et al., 2017a, Pan et al., 2018, Zhang et al., 2019). The nanoparticles prepared by biopolymers, such as chitosan (CH) and chondroitin sulfate (CS), were widely used in carrier systems due to advantages which include better biocompatibility and enhanced biodegradability (Nunes et al., 2017, Soe et al., 2019). Furthermore, CH/CS nanoparticles have been successfully explored as novel carriers for active substances delivery (Fan et al., 2017, Umerska et al., 2017). Hence, the CH/CS nanoparticles are promising carriers for ACN which may slow down ACNs degradation during exposure to adverse environments. However, the use of CH/CS nanoparticles for improving the stability and explored the biological activity of anthocyanins has been rarely reported and need further investigation.
Therefore, the aim of this present study was to evaluate CH/CS nanoparticles prepared by an ionic method, loaded with the black rice anthocyanins (ACN). Multiple microscopic (TEM, SEM, AFM) and spectroscopic approaches were used to explore the ACN embedded within CH/CS nanoparticles. This will help in understanding the bioactivity of anthocyanins in the embedded (nanoformulation) state. In addition, the effects of prepared ACN embedded nanoparticles on HCT-116 cells were investigated via cell viability, apoptosis and the structural changes in mitochondria.
Section snippets
Materials
Chitosan (Mw = 180,000–310,000 Da, 80%–95% degree of deacetylation, viscosity 50–800 cP) and Chondroitin sulfate (95% purity), acetic acid, HCl, and NaOH of analytical grade were purchased from Sinopharm Chemical Reagent Co., (Shanghai, China). The black rice-derived mixture of anthocyanins provided by Shanghai Purple Stone Biotech Co., (Shanghai, China) with ACN content of approximately 33.9% (w/w) by pH-differential method.
Preparation of CH/CS nanoparticles
The pH of polysaccharide solution (3–6.0), polysaccharide
Preparation of ACN/CH/CS nanoparticles
The influence of pH, CH/CS ratio and polysaccharide concentration on the particle size and zeta potential were investigated in the preparation of CH/CS nanoparticles. DLS results suggested that the zeta potential and particle size of nanoparticles increases as the concentration of polysaccharide increases (Fig. 1A). Our findings are in agreement with results on chitosan-gum arabic PECs (Coelho et al., 2011). The increase of CH concentration results in more –NH3+, which causes greater repulsion
Conclusions
The present findings indicated that the ACN/CH/CS nanoparticles were obtained at the polysaccharide concentrations of 1.5 mg/ml, CH:CS ratio 1:1, pH 3.0 and ACN: polysaccharide mass ratio of approximately 6% (w/w). The nanoparticles showed high loading efficiency (86.32 ± 0.15%), a relative lower particle size (350.1 ± 0.99 nm), and high Zeta potential (42.55 ± 0.54 mV). Morphological study and thermogravimetric analysis showed the prepared nanoparticles exhibits nearly spherical with
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We gratefully acknowledge financial support from the Key R&D Program of Guangdong Province (Grant No. 2018B020239001) and Shanghai Food Safety and Engineering Technology Research Center (16DZ2281400).
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