Microwave hydrodiffusion and gravity (mhg) - extraction and characterization of compounds with antioxidant properties

Resumo

Natural resources can be utilized as raw material to obtain compounds with high-added value by the selection of suitable extraction procedures. Antioxidant bioactive compounds represent an important field in the development of these methods. This phytoconstituents has a relevant application in cosmetic and nutraceutical products due to their capacity to mitigate the generation of process-generated unwanted chemical as well as the health benefits that they provide, when compared with the potential health risks that the use of synthetic antioxidants could generate. The recovery of bioactive compounds should be carried out by extraction methodologies that combine the production of high quality bioactive extracts without jeopardizing the recovery of other compounds of interest, with efficient and economically viable methods. Conventional systems use organic solvents, which are difficult to remove from the final product. Moreover, these traditional methods provide low selectivity and recovery capacity in addition to promote the thermal degradation of the high valuable bioactive components. For these reasons, the development of alternative methodologies is necessary to obtain these products by economic and environmental friendly procedures. In this context, microwave hydrodiffusion and gravity (MHG) technology is a novel extraction method that does not employ added solvent. This system uses a non-ionizing of electromagnetic radiation that promote the electropermeabilization of the cellular tissues at atmospheric pressure in a unit operation, so that in situ water removes the high bioactive metabolites. This is a green technology that can lead to an enhancement in the obtained yields and extraction kinetics saving energy and time when compared with traditional extraction procedures. The use of sustainable and renewable sources with green extraction strategies allows recovery the bioactive compounds derived by secondary metabolites of vegetable materials and alleviate the current high market demand in this sector. This is also realized by the employment of underused raw materials as diverse agroforestry sources, by-products and surpluses of food and marine resources that were subjects of study in this PhD Thesis. Currently, a growing demand of cosmeceutical products and functional food enriched with bioactive compounds from natural resources responds to the increasing consumer´s awareness about their health care priority. In this context, products enriched with functional natural ingredients are designed by the cosmetic and food industries, since diverse natural extracts could promote positive effects. This PhD Thesis presents an approach to the utilization of the emerging MHG technology from different agroforestry, food by-products and seaweed sources. The optimization of the extraction procedures and their physicochemical and bioactive characterization were realized. Distinct applications of the derived liquid and solid phases were developed. In this study, personal care products and hydrogels were enriched with some aqueous MHG extracts from a wide range of feedstocks when compared with extracts obtained by distillation and solid-liquid extraction methods. The determination of their physicochemical and phytochemical features as well as the mechanical properties of the formulated functional matrices was critical to determine their potential food or non-food applications. Expanding the study horizon, MHG process was also studied as a dehydration method for edible brown seaweeds corroborating that this method could allow in parallel to recover their bioactive compounds, integrating these two above targets in an only operation. The first section of this PhD Thesis presented the obtained results for the extraction of antioxidant compounds from distinct agroforestry such as diverse wild flowers (Ulex europaeus L., Erica australis L., Acacia dealbata Link and Cytisus scoparius (L.) Link); fruit bodies of mushrooms of Pleurotus ostreatus (Jacq.) P. Kumm.; 1871; acorns without cupules of Quercus robur L. and leaves of Brassica rapa subsp. rapa and Urtica dioica L.; based on their healthy properties in combination with their wide distribution in the Galicia region and its economic availability. These raw materials were submitted to MHG and the optimization of the operation conditions was made. Steam distillation and solid-liquid extraction (SLE) were also used with comparative purposes. Selected extracts were employed in the development of different personal care products. In the first study, the environmentally friendly MHG extraction was used to extract compounds of interest from defrosted wild U. europaeus flowers. Different microwave irradiation powers were tested (ranged to 25 from 200 W) to find the most adequate extraction conditions. Steam distillation without cohobation system was studied also for comparative purposes. Extraction kinetics, jointly with the collected volume extraction yield of the tested extraction systems allowed defining the time and temperature conditions. Total phenolic content, antioxidant capacities and color features were determined for each gathered liquid fraction. The originated structural changes in flower tissues were also exhibited. The selection of optimum irradiation power and time extraction was realized based on the above results. MHG treatment at 100 W for around 80 min provided the highest collected volume by MHG (around 53 % of the initial moisture content) and the extraction time was noticeable considered in comparison with steam distillation (153 min). Both methodologies, exhibited comparable extraction yields and retaining the antioxidant properties. Specifically, U. europeaus extract collected by MHG at 100 W showed the highest reducing power capacity (approximately 0.102 mM FeSO4 * 7 H2O). Similar shrinkage effect was observed in wall cell of the flower tissues using both tested extraction systems. Also, the flower extracts from the both methods showed similar visual appearance (colorless) and olfactory features defined by floral and ripe fruity aroma. In the second work, similar above MHG work conditions were evaluated from defrosted E. australis flowers ranging the irradiation microwave powers between 25 and 200 W. This wild flower was also subjected to steam distillation for comparative purposes. Again, the total phenolic content, antioxidant capacity and color features of the gathered MHG and distillation extracts were analyzed. An evaluation of the effect of these extraction process on the flower tissues was realized by scanning electron microscopy. Selected MHG bioactive extracts were incorporated in the formulation of sunscreen creams with different thermal spring waters for their cosmetic application. Optimum U. europaeus MHG extract from the first study was also utilized in these creams for comparative purposes. The chemical, bioactive and rheological properties of these cosmetic emulsions were analyzed. An accelerated oxidation study of above sunscreen creams was also realized by the determination of the chemical and bioactive stability of these oil-in-water (O/W) model emulsions. Based on the results, E. australis flowers submitted by MHG at 50 W for 130 min allowed the optimum aqueous extracts with noticeable antioxidant properties (~ 0.27 mg Trolox eq/g flower dry weight and 0.01 mg ascorbic acid/g flower dry weight or 0.04 mg FeSO4 * 7 H2O/g flower dry weight). This method also led to a reduction of the required energy consumption and the produced environmental impact (about 8 times less) in comparison with steam distillation procedure. Similar color features were described by the extracts of both extraction technologies since small total color differences were detected between the gathered extracts. The incorporation of flower extracts produced similar effects (e.g. their total solid content, sun protection factor (SPF) or color characteristics) when compared with added commercial antioxidants. This tendency was also provided by the stability of their pH values and thiobarbituric acid reactive substances (TBARS) content. In all samples, the apparent viscosity decreased (about 4 decades) with increasing shear rate, displaying a pseudoplastic behavior. Specifically, the viscous properties of the sunscreen creams with added E. autralis extract and formulated with thermal spring water 3 displayed lower apparent viscosity profiles, which suggests a positive topical application effect to extend this cream across large body surface areas. In the third research, MHG extraction was used to extract antioxidant compounds from frozen wild A. dealbata flowers in comparison with steam distillation. The range of evaluated irradiation powers was from 50 to 125 W. The solid phases from MHG processes were treated by SLE with ethanolic solvent in order to evaluate the capacity of this method to obtain compounds with antioxidant capacity. Their total phenolic content, antioxidant profile, total carotenoid content and color features were determined as well as their pH and SPF values. Chosen extracts were incorporated to a sunscreen cosmetic model elaborated with three different thermal spring waters. Commercial antioxidants were also added to the cream with comparative purposes. Their chemical and rheological features were also tested jointly with their bioactive capacity by an acceleration oxidation analyze. Experimental analysis revealed that the optimum MHG extract was collected at 75 W for 180 min. Flower aqueous extracts obtained in this conditions exhibited the highest total phenolic content (about 0.15 mg GAE/g flower dry weight) and antioxidant capacities (e.g. around 0.39 mg Trolox eq/g flower dry weight and 0.36 µg β-carotene/g flower dry weight). Color parameters of MHG extracts and DPPH values exhibited a positive correlation whereas their pH and SPF data were ~ 5.4 and 0.24, respectively. Commercial antioxidants and selected extracts provided similar chemical and bioactive properties in the oil-in-water (O/W) model emulsions. Overall, this trend was also observed in the viscous behavior of the creams elaborated with the different thermal spring waters. Only the cosmetics formulated with thermal spring water 3 presented distinct viscous features since lower apparent viscosity data could indicate an improvement skin application effect. In the fourth study, diverse chosen vegetable sources were submitted to MHG method in comparison with ethanolic SLE. Their antioxidant capacity, color characteristics and proximate chemical characterization were analyzed. MHG extracts, commercial antioxidants and three distinct thermal spring waters were employed in the elaboration of a sunscreen cream cosmetic model. Their chemical and bioactive profile as well as the rheological features were assayed. Results indicated that MHG C. scoparius flower extract supplied the highest total phenolic content, antioxidant capacity and total carotenoid content (about 0.24 mg GAE/g raw material dry weight, 0.68 mg Trolox eq/g raw material dry weight, 0.33 mg ascorbic acid/g raw material dry weight or 0.71 mg FeSO4 * 7 H2O/g raw material dry weight and 3.1 µg β-carotene/g raw material dry weight). P. ostreatus MHG extract stood out for their highest total solid extraction yield (around 5.1 mg extract/g raw material dry weight). B. rapa MHG liquor sample exhibited the highest total protein content approximately 2.1 mg BSA/ g raw material dry weight and their monosaccharide and oligosaccharide concentration. Last studied resource, acorns of Q. robur divided into quarters provided MHG extracts with higher inhibition percentage (around 84.0 %), total lipid content (about 783.9 g lauric acid/g raw material dry weight) and the highest anti-elastase and anti-tyrosinase capacities (EC50 values of 606 mg extract/L and 496 mg extract/L, respectively) with the lowest energy requirements (~ 50 kJ). Differences between the chemical properties and bioactive stability of the sun creams with added selected MHG extracts and commercial antioxidants were not appreciable as well as the distinct thermal spring waters. Only the results of the viscous behavior of the spring waters 3 creams with added P. ostreatus and B. rapa MHG extracts could indicate a more suitable skin application due to their lower apparent viscosity value. In the fifth work, defrosted leaves of U. dioica were treated by MHG at low microwave irradiation powers to 50 from 125 W based on the results obtained in previous works. For comparative purposes, conventional steam distillation was also assayed as well as from solid phases of both extraction systems. Their total phenolic content, antioxidant capacity and color properties were researched. The effect of these extraction procedures on the nettle leaves tissues was also assessed. Selected extracts were used to supplemented distinct personal care products: these were added in a sunscreen cream made with three distinct spring waters and in a shampoo commercial sample. The chemical properties, bioactive capacities and mechanical features were determined. Based on the results, the optimum MHG extract was gathered at 100 W for 98 min. These extraction conditions provided the extract with the highest total phenolic content and antioxidant capacities (~ 0.04 mg GAE/g nettle leaves dry weight, 0.11 mg Trolox eq/g nettle leaves dry weight, 0.07 mg ascorbic acid/g nettle leaves dry weight or 0.20 mg FeSO4 * 7 H2O/g nettle leaves dry weight and 10 % inhibition percentage) by microwave extraction with the lowest required energy consumption (~ 788 kJ). Similar color features were obtained for all samples. This tendency was also disclosed for the chemical and bioactive stability properties of the sun creams with added nettle leave extracts and commercial antioxidants. The same trend was identified in the rheological analysis of these cosmetic emulsions except to the creams with thermal spring water 3 and added MHG extract since their lowest apparent viscosity at fixed shear rate suggested a more appropriate topical application of the studied product. One other hand, the enrichment with MHG extract of commerical shampoo caused a positive variation in their foaming ability and foam stability (Δ ~ 4 mL) jointly with an initial increase of the lightness on the volunteer’s natural hair (ΔL* ~ 2.6). The second section of this PhD Thesis supplies the treatment of natural and blanched processed Cynara scolymus L. and Brassica oleracea L. industrial by-products by MHG. Their valorization was done by means of the recovery of the water-soluble diffused bioactive compounds presents in these samples. The optimization of this extraction method was realized in combination to their use as solvent in the formulation of hydrogels. Chosen work conditions were also employed from these samples after a frozen storage of three years in order to evaluate the effect of this process relating to the collected aqueous extracts. In the sixth research, natural and blanched defrosted C. scolymus processed waste samples were subjected to MHG technology. Microwave irradiation powers between 70 and 500 W were utilized to recover bioactive compounds. The phytochemical and color parameters of the gathered aqueous phases were analyzed. Selected bioactive extracts were employed to prepare innovative functional gelling matrices. The textural and rheological properties of these hydrogels were also determined. The realized assays indicated that the extracts from blanched samples obtained at 500 W presented the highest bioactive features (e.g. around 0.70 mg GAE/g artichoke dry weight and 1.66 mg Trolox eq/g artichoke dry weight). The pre-treatment of the raw material facilitated the bioactive compound recovery: these aspect was especially noted in the color features of the samples. For both type of samples, an average about 443 kJ of the estimated energy requirements were defined. Alginate and gelatin hydrogels with bioactive artichoke liquors presented some typical gel spectra with softening effect. This fact was also observed in their textural features. Results of chemical and color characteristics of the bioactive extracts obtained after the frozen storage of the samples showed that this conservation method could reserve the antioxidant capacities of the samples for long time periods (3 years). In the seventh study, natural and blanched defrosted B. oleracea by-products were submitted to a similar work conditions that the last raw materials. Therefore, broccoli samples were submitted at irradiation powers from 70 to 500 W. The collected liquors were analyzed to determine their chemical, antioxidant capacity and color features. Functional alginate and gelatin hydrogels were prepared with the selected aqueous phases and their mechanical characteristics were evaluated. Based on the results, 300 W was the irradiation power more appropriate from natural and blanched samples. As in the previous case, blanched method of the broccoli samples promoted the recovery of bioactive compounds. Against this background, blanched broccoli at 300 W supplied the highest total solid extraction content (roughly 18 mg extract/g blanched sample dry weight) and bioactive content (close to 0.34 mg GAE/g broccoli dry weight and 19.4 % of inhibition percentage). Color characteristics were similar in all cases as well as the expected energy consumption (approximately 370 kJ). Hydrogels had typical gel response: the utilization of the selected broccoli extracts affected on to their rheological ant texture profile since these caused a gel softening behavior. The bioactive properties and color features of broccoli liquid phases were not influenced by their frozen storage for at least 3 years. The third section of this PhD Thesis exhibited the development of an interesting dehydration method for marine algae Laminaria ochroleuca and Undaria pinnatifida seaweeds. The exclusive characteristics of MHG technology also allowed the recovering of the water-soluble diffused scavenging compounds during this processing. In this section, sequential MHG treatments were proposed in order to achieve the best dehydration and bioactive extraction features. Moreover, selected extracts were used in the formulation of hydrogels matrices. In the eighth work, an innovative dehydration method by MHG was proposed to treat defrosted edible L. ochroleuca brown seaweed samples. Distinct microwave irradiations (50, 300 and 800 W) and a proposed combination of irradiation powers and time extraction in four steps (300 W (5 min), 100 W (10 min), 50 W (35 min) and 25 W (90 min)) were evaluated. The required energy consumption of these procedures were estimated. Resulting solid phases were analyzed in order to determine their drying kinetics and its modelling, shrinkage, microstructural and colorimetric characteristics. Their total phenolic content and antioxidant capacities were provided by ethanolic SLE. Results indicated that high final moisture contents were obtained by irradiation powers in an only step. However, proposed drying method led to solid phase with an adequate final moisture content (around 9.9 % dry basis). All drying kinetics were favorably (R2 > 0.95) adapted with the Page model. A suitable lineal dependence (R2 > 0.94) between moisture content and shrinkage was described. The energy requirements for MHG proposed method was around two decades lower than the data showed for conventional algae dried process of other brown algae. The selected method offered microstructural, color and antioxidant capacities features similar to the commercially dehydrated algae samples. In the ninth research, U. pinnatifida brow seaweed was treated by MHG for their dehydration. In this case, irradiation powers over the range of 50 and 800 W were tested as well as the above proposed drying method (300 W (5 min), 100 W (10 min), 50 W (35 min) and 25 W (90 min)). The rehydration of selected samples was also determined. The kinetics and modelling of these both procedures were realized as well as their volumetric shrinkage and microstructural changes and color features. Their bioactive antioxidant profile was also provided by ethanolic SLE. The expected energy requirements of MHG dehydration method were defined. Results displayed that lower time algae dehydration were produced at higher irradiation powers. Recommended drying procedure allowed a suitable final moisture content (< 10 % dry basis). The collected solid phase exhibited a uniform tissue surface and reduced total color differences. This sample display a suitable bioactive profile. Page and Peleg models fitted to experimental dehydration and rehydration kinetics. Expected energy consumptions were intensely lower than the requirements of conventional convective air drying method. In the last study, the characteristics of the liquid phase collected in the eighth and ninth works were compiled. For comparative purposes, conventional distillation technique was also realized. The total phenolic content, antioxidant capacities and color features of the water-soluble diffused bioactive compounds presents in these two edible algae were determined. Selected aqueous extracts were incorporated as solvent in functional alginate hydrogel formulations. Their mechanical profile was described. L. ochroleuca and U. pinnatifida liquid extracts at 300 W and suggested combination of microwave irradiation powers and times (300 W (5 min), 100 W (10 min), 50 W (35 min) and 25 W (90 min)) were chosen as the most interest extracts due their high phenolic content or antioxidant capacities. Distilled extract did not present better antioxidant properties. In all cases, color features of the liquors were comparable. Alginate hydrogel matrices showed typical gel rheological and textural profiles. Data indicated that selected extracts could allow softening behavior so that these solvents could be used for the formulation of diverse hydrogels with possible food and non-food functions. Overall, the works contained in this PhD Thesis showed that MHG technology is an emerging eco-friendly extraction method that allow the recovery of bioactive compounds from distinct underused agroforestry resources, by-products and surpluses of food and seaweed raw materials. It also highlights the importance of testing a wide range of raw materials to optimize the most adequate operation conditions. This treatment needed lower energy requirements and caused lower environmental impact than other conventional systems, as steam distillation or SLE. MHG procedure permits obtain aqueous antioxidant phases that could be incorporated in personal care products or hydrogels with positive effects in food and non-food applications. It is also necessary to emphasize that MHG could contribute not only to the adequate recovery of high valuable bioactive extracts, but also could simultaneously allow the dehydration of marine resources.