Green extraction and encapsulation of black rosehip polyphenols: İn vitro bioaccessibility, bioavailability, and biological activities

Abstract

Due to the increased prevalence of degenerative diseases, there has been a high consumer demand for functional foods with added health benefits. The term "functional food" generally refers to products providing an additional health benefit, beyond basic nutrition. Consuming foods fortified with functional ingredients (e.g., vitamins, probiotics, omega n-3 fatty acids, minerals, and antioxidants) could help to mitigate the risk of chronic diseases and contribute to the physical and mental well being. In that sense, (poly)phenols, phenolic compounds containing at least one phenolic hydroxyl group, are the most pronounced secondary metabolites found in plants possessing strong antioxidant power. However, to exploit the use of underutilized wild edible plant resources, convenient extraction methods need to be developed to separate and concentrate the (poly)phenols without using toxic hazardous chemical solvents. Hence, 'green' isolation of value-added ingredients for enrichment purposes from underutilized plant sources have importance for food industry. Moreover, (poly)phenols are susceptible to against harsh processing and environmental factors such as heat, oxygen, light, alkaline pH, or enzymes and therefore mostly have poor solubility and bioavailability. Moreover, their biological functions are significantly dependent on their bioavailability. In this regard, encapsulation applications enable increasing their stability by protecting from stresses mentioned above and/or improve their solubility, bioaccessibility and intestinal uptake which is especially desirable for manufacturing of functional foods/beverages and dietary supplements. Taking these facts into consideration, this thesis consist of the following objectives: (i) to characterize the (poly)phenols composition of black rosehip (Rosa pimpinellifolia L.) and its antioxidant as well as antiproliferative activity in comparison to common rosehip (Rosa canina L.); (ii) to optimize the extraction conditions of black rosehip (poly)phenols by means of green methods, in particular pressurized hot water extraction and ethanol modified supercritical carbon dioxide extraction; (iii) to evaluate the effects of extraction methods on bioaccessibility and bioavailability of (poly)phenols using combined in vitro gastrointestinal digestion/Caco-2 cell culture model; (iv) to improve processing and digestive stability of hydrophilic and lipophilic (poly)phenols with high antioxidant acitivity by converting them into edible and stable form for functional foods by means of encapsulation and to investigate the effects of liposomal encapsulation and spray drying processing on bioaccessibility and bioavailability of (poly)phenols using combined in vitro gastrointestinal digestion/Caco-2 cell culture model. The first chapter was designed to characterize Rosa pimpinellifolia fruits collected from Gümüşhane Province in the Black Sea region of Turkey by means of (poly)phenol composition, in vitro antioxidant activity and other biological activities in comparison to commonly consumed rosehip specie. The angiotensin-converting enzyme inhibition potential of black rosehip was also assessed by spectrophotometric assay. Since rosehip (Rosa canina) fruit has traditionally been widely consumed in the form of herbal tea, tea-type infusions of both Rosa species were prepared to reveal the consumption potential of black rosehip. In this context, tea-type infusions and aqueous methanol (80%, v/v) extracts were analyzed by LC-MS/MS in both rosehip species studying different parts of the fruits; whole fruit, flesh, and seed. Apart from other rosehip species, Rosa pimpinellifolia has its dark purplish-black color due to its rich anthocyanin content. Unsurprisingly, any anthocyanin was absent in Rosa canina fruit. Chromatographic analysis showed that (poly)phenol contents varied between rosehip species and drying method applied (freeze drying and air drying). Gallic acid, catechin, protocatechuic acid, procyanidin-B2, procyanidin oligomers and quercetin acid derivatives were principal in both rosehip species. For black rosehip, high level of anthocyanins, (poly)phenols content and high antiradical scavenging capacity were characteristic. Reactive oxygen species (ROS)-induced-DNA damage has been linked to the onset of many degenerative and cardiovascular diseases. Polyphenolic compounds protect cells against the harmful effects of ROS in various mechanisms. Flavonoids and phenolic acids fractions of Rosa canina have been reported to inhibit cell proliferation, however no study exist regarding the antiproliferative properties of black rosehip to the best of our knowlege. In that sense, cytotoxicity by MTT assay, ROS generation, and apoptotic effects by Acridine orange assay in breast cancer cells were evaluated in tea-type infusions and hydroalcoholic extracts from both species. Response surface methodology was adopted to achieve an effective extraction procedure of (poly)phenols from black rosehip (Rosa pimpinellifolia) fruits. Pressurized hot water extraction and ethanol modified supercritical carbon dioxide extraction techniques were investigated by screening parameters including temperature, pressure, solvent composition, and solvent-to-solid ratio. Ultrasound assisted solvent extraction was also assessed. Simultaneous maximization in terms of extraction yield, total antioxidant capacity, total (poly)phenol content, catechin content, total monomeric anthocyanin content, and cyanidin-3-O-glucoside content was performed. Antioxidant activity was evaluated using 2,2-diphenyl-1- picrylhydrazyl radical (DPPH) radical scavenging activity and cupric ion reducing antioxidant capacity (CUPRAC) assays. The experimental data was subjected to the regression analysis to obtain second-order polynomial equations and the fitted polynomial equations are presented as contour plots also showing the linear and quadratic effects of the tested dependent variables. The optimum conditions for extraction was compared to the predicted values of RSM using an independent sample t-test. The optimized conditions were as follows: 75 °C with 10 mL g-1 solvent-to solid ratio under 100 bar during 60 mins, and 60 °C with 25 % aqueous ethanol under 280 bar during 60 mins for pressurized hot water extraction and ethanol modified supercritical carbon dioxide extraction, respectively. When these extracts were examined in terms of bioaccessibility and epithelial cells (Caco-2) uptake upon gastric and intestinal digestion in vitro, all extracts exhibited lower levels of total phenolic content compared to their undigested counterparts (p < 0.05). Chapter 4 aimed to achieve the encapsulation of black rosehip extracts obtained via green techniques applied in previous chapter and to evaluate their antioxidant properties as well as processing and digestive stability. The black rosehip extract (BRE) which was obtained at optimized pressurized hot water extraction conditions, was further encapsulated in biopolymer-coated liposomes and spray dried using maltodextrin as carrier. Being readily available natural biopolymers, chitosan and whey protein were used in coating of liposomes via electrostatic deposition method. In the literature, these biopolymers have been widely used for encapsulation purposes of various polyphenolic extracts. However, in the present thesis, for the first time a comparison between these materials for their efficiency to fabricate stable antioxidant powders. Nanosized particle diameters were achieved by 5 cycle microfluidization of the liposomal dispersions. During spray drying process, total phenolic content (TPC) in extracts decreased due to oxygen and heat exposure. However, the retention efficiency of TPC in biopolymer coated liposomal powders was found significantly higher than spray dried BRE. In a similar pattern as observed with retention of phenolics, retention of antioxidant capacity of the powders was confirmed with CUPRAC assay. In addition, encapsulation provided remarkable protection of the phenolics under in vitro gastrointestinal digestion conditions, resulting in up to a 5.6- fold more phenolics in the bioaccessible fraction, which also had 2.9–8.6-fold higher antioxidant activity compared to the non-encapsulated BRE. Similar results were obtained for encapsulated RPFE using ethanol injection method in preparation of liposomes. Besides, as an alternative colloidal delivery system to liposomes, solid lipid nanoparticles (SLN) were prepared using plant saponin glycyrrhizin to ensure stabilization at low concentrations which has great importance for the food, nutraceutical and pharmaceutical industries. For the first time a series of glycyrrhizin emulsified tristearin SLNs were successfully fabricated and physical stability was determined during 21 days of storage. The influence of glycyrrhizin on the physical stability and crystallization behavior of SLNs were evaluated by dynamic and static lights scattering, electrophoretic light scattering, optical microscopy, visual observations, and differential scanning calorimetry. The SLN formulations containing lower amount of surfactant revealed poor stability against aggregation as clearly observed by optical microscopy. Beyond bioaccessibility, the absorption of (poly)phenols through the intestinal epithelial layer is an important factor in functional food development. Thus, the behavior of individual phenolic compounds loaded in dried liposomes should be investigated to gain insight regarding their bioavailability and resulting bioactivity. In the last chapter, the co-encapsulation of phenolic compounds into nanoliposomes in combination was explored in terms of particle characterization and bioavailability compared to their native (non-encapsulated) form. Phenolic compounds representing berry matrix (catechin, epicatechin, ferulic acid, and resveratrol) were loaded into chitosan- and whey protein-coated liposomal powders. For control purposes, the phenolic compounds were also spray dried without liposomes to observe the effect of liposomal encapsulation on protection of bioactive molecules against processing or digestive conditions. The digestive fate of the samples was determined using in vitro digestion coupled with Caco-2 cell monolayer model. The phenolic compounds, both in encapsulated or free form, decreased upon simulated gastrointestinal digestion, except cis-resveratrol possibly due to trans-to-cis isomerization that occurs during in vitro digestion. Significantly higher digestive stability, solubility, micellization efficiency, and bioaccessibility were found in encapsulated phenolic compounds (p < 0.05). On the contrary, whey protein coated liposomal formulation resulted in substantial increase in the cellular uptake of trans-resveratrol in comparison to other encapsulated formulations or in native form (p < 0.05). The epithelial permeation of the native and encapsulated micellar phenolics were further evaluated in Caco-2 transwell model system. Moreover, all digested formulations was analyzed in terms of fatty acid induced lipid accumulation in human hepatic cancer (HepG2) cells as an indicator of the effect of encapsulation on the potential biological activity.