“EXTRACTION AND PURIFICATION OF ANTIOXIDANT FRACTIONS FROM FOREING SPECIES”

EUROPEAN DOCTORAL THESIS

María Parada Casas Ourense, julio 2017

International Doctoral School

María Parada Casas “EXTRACTION AND PURIFICATION OF ANTIOXIDANT FRACTIONS FROM FOREIGN SPECIES”

Dª Herminia Domínguez González, D. Andrés Moure Varela, Dª Enma Conde Piñeiro

2017

International Doctoral School

Dª Herminia Domínguez González, Dr. Andrés moure Varela and Dr. Enma Conde Piñeiro,

DECLARE that the present work, entitled “EXTRACTION AND PURIFICATION OF ANTIOXIDANT FRACTIONS FROM FOREIGN SPECIES” to obtain the title of Doctor, was carried out by Dª MARIA PARADA CASAS, under our supervision in the PhD programme “RD 1393/2007”

Ourense, July 2017

Dr. Herminia Domínguez González Dr. Andrés Moure Varela Dr. Enma Conde Piñeiro

AGRADECIMIENTOS‐ACKNOWLEDGMENTS

Me gustaría que estas líneas sirvieran para expresar mi más profundo y sincero agradecimiento a todas aquellas personas que me han ayudado de una u otra manera en la realización del presente trabajo el cual ha significado un reto tanto personal como profesional…

Quisiera agradecer a mis directores de tesis, Dra. Herminia Domínguez, Dr. Andrés y Dra. Enma Conde por haberme facilitado tanto la realización de este trabajo, muy especialmente a Herminia, por haberme aceptado primero como proyectista y después como estudiante de doctorado, por la confianza que me ha dado, por su ayuda infinita, y apoyo y palabras de ánimo constante y hasta el último momento.

Un punto y aparte merece Enma, de la cual he aprendido muchísimas cosas, tanto a nivel profesional como personal, por su capacidad de trabajo, templanza y paciencia conmigo (sobre todo al principio) por sus consejos, risas y charlas entre experimental y párrafos…te admiro.

Gracias al grupo de investigación de Ingeniería Química, muy especialmente a Jose Luis Alonso, por brindarme la oportunidad de formar parte de un gran equipo.

Gracias también al grupo de investigación da FRAU (free radicals and antioxidants unit) de Química Aplicada do Departamento de Ciências Químicas da Faculdade de Farmácia da Universidade do Porto, a la Dra. Eduarda Fernandes, por haberme permitido trabajar y aprender en su laboratorio, por su hospitalidad desde antes incluso de que llegara. Gracias a las Dras. Marisa Freitas y Daniela Ribeiro por haberme hecho sentir como en casa, por su ayuda en todos los ámbitos, por crear ese ambiente de trabajo tan genial. También a Carina y a Priscilla y al resto de doctorandas de Brasil sin todas vosotras no habría sido lo mismo.

Quiero dar las gracias a mis compañer@s del grupo de investigación de Ingeniería Química, a Carlos, Mª Jesús Susana, Belén, Sandra, Miguel, Elena, Noelia…a los que están y a los que ya se han ido, por su ayuda y colaboración y con los cuales también he compartido muy buenos momentos.

Gracias a todos mis amigos y amigas, a los de la infancia y a los que habéis llegado más tarde, por vuestro interés, porque de todos he recibido constantes palabras de ánimo, por vuestro empuje en los momentos más complicados y las palmaditas en el hombro tras cada pequeño logro. Gracias a Carmen por ayudarme siempre en todo y en todos los aspectos de mi vida sin importar día, hora o lugar.

Gracias a mi familia, empezando por mi abuelo, por sus sabios consejos, el que siempre me ha inculcado el estudio y la capacidad de trabajo, te siento siempre conmigo y sé que me sigues ayudando. A mi abuela por escucharme siempre, por ayudarme en todo, por su risa contagiosa… A mi tío, el cual siempre me lo ha dado todo y me lo sigue dando, por su preocupación, consejos y palabras de reconocimiento. A mi madre, por su amor incondicional, por su vida dedicada a nosotras, como no, por sus consejos, porque siempre acertaste y porque en el fondo nunca me dejaste. A mi hermana porque a pesar de su juventud es una de las personas más fuertes y con más fuerza de voluntad que conozco, un ejemplo para mi en muchas aspectos de la vida.

A mi madre

INDEX

INDEX

PREFACE ………………………………………………………………………………………………….…….…………….I

SUMMARY…………………………………………………………………………………………………….…………….II

RESUMEN………………………………………………………………………………………………..……….…………IX

1.INTRODUCTION

1.1 Antioxidants……………………………………………………………………………………………………………1

1.1.1. Definition of antioxidant………………………………………………………………….……..1

1.1.2. Natural and synthetic antioxidants………………………………………………………...2

1.1.3. Antioxidant activity…………………………………………………………………………..….…3

1.1.4. Measurement of antioxidant activity…………………………………………………...... 5

1.2 Natural phenolic compounds………………………………………………………………………………….7

1.2.1. Origin and formation of phenolic compounds………………………………….……..7

1.3 Natural compounds with antioxidant activity and major sources…………………………..7

1.3.1. Natural compounds with antioxidant activity……………………………..………..…7

1.3.2. Classification………………………………………………………………………………………..…9

1.3.3. Environmentally friendly extraction technologies…………………………………14

1.3.3.1. Hydrothermal processes for extraction…………………………………..14

1.3.3.2. Enzyme assisted extraction…………………………………………………….15

1.3.3.3. Microwave assisted extraction…………………………………………....…16

1.3.3.4. Supercritical fluids……………………………………………………….………….18

1.3.4. Recovery, concentration and purification of phenolic compounds…….….26

1.3.4.1. Adsorption-desorption processes………………………………….……….26

2. OBJECTIVES AND WORK PLAN

2.1. Objectives……………………………………………………………………………………………………………33

2.2. Work plan……………………………………………………………………………………..……………..……..33

3. MATERIALS AND METHODS

3.1. Raw materials…………………………………………………………………………………..…………………35

3.2. Experimental procedures……………………………………………………………………..……………..36

3.2.1. Extraction methodologies……………………………………………………………………..36

3.2.1.1. Hydrothermal treatments: autohydrolysis …………………………..…36

3.2.1.2. Enzyme-assisted extraction………………………………………………….…37

3.2.1.3. Solvent fractionation……………………………………………………...... 38

3.2.1.4. Supercritical fluid extraction…………………………………………………..38

3.2.1.5. Microwave hydrogravity extraction (MHG)………………………….…39

3.2.2. Recovery, fractionation and purification of extracts………………………………39

3.2.2.1. Static adsorption and desorption studies………………………………..40

3.2.2.2. Determination of adsorption‐desorption properties ………………41

3.2.2.3. Dynamic adsorption and desorption studies…………………………..45

3.3. Analytical methods……………………………………………………………………………………………..45

3.3.1. Moisture content and total solids determination …………...... …..45

3.3.2. Extraction yield determination………………………………………………..……………46

3.3.3. Total phenols determination…………………………………………………………..….…46

3.3.4. Floroglucinol equivalents………………………………………………………………………47

3.3.5. Chromatographic techniques……………………………………………………….……....48

3.3.5.1 High performance liquid chromatography (HPLC) ……………….....49

3.3.5.2. Gas chromatography (GC)……………………………………………………….50

3.3.6. In vitro antioxidant activity methods…………………………………………………….50

3.3.6.1. DPPH radical scavenging capacity assay ………………………………..51

3.3.6.2. Trolox equivalent antioxidant capacity assay or ABTS radical scavenging capacity assay ………………………………………………………………………………………….52

3.3.6.3. Ferric reducing antioxidant power assay ……………..……………..…54

3.3.6.4. Antitumoral activity…………………………………………………………..……55

3.3.6.5. Anti-inflammatory activity………………………………………………….…..56

3.3.6.6. COX-1 and COX-2 assays………………………………………………………...57

3.4. Scanning electron microscopy (SEM)…………………………………………………………………..58

3.5. Mathematical procedure…………………………………………………………………………………….58

3.5.1. Calculation of adsorption capacity and adsorption ratio………………...…….58

3.5.2. Adsorption kinetics……………………………………………………………………………....59

3.5.3. Adsorption isotherms……………………………………………………………………….…..60

3.5.4. Desorption process……………………………………………………………………….………62

3.5.4.1. Calculation of desorption ratio……………………………………………….62

3.5.4.2. Dynamic desorption studies…………………………………………………...62

3.5.4.3. Dynamic adsorption modeling………………………………………………..62

3.5.4.3.1. Modelo of Thomas……………………………………………………63

3.5.4.3.2. Modelo of Adams–Bohart ………………………………….……64

3.5.4.3.3. Modelo dof Yoon–Nelson…………………………….…………..65

4. RESULTS

4.1. Enzyme aided aqueous procesing of Sargassum muticum…………………………………..67

4.2. In vitro bioactive properties of phlorotannins recovered from hydrothermal treatment of Sargassum muticu………………………………………………………………………………...85

4.3. Solvent fractionation of ethanolic extracts from Acacia dealbata flowers…………119

4.4. Supercritical CO2 extracts from Acacia dealbata flowers……………………………………131

4.5. Sequential extraction of Hericium erinaceus using Green…………………………….……145

5. GENERAL CONCLUSIONS………………………………………………………………………………….….161

6. REFERENCES………………………………………………………………………………………………………...163

7. APPENDIX…………………………………………………………………………………………………………….179

PREFACE

This PhD Thesis has been conducted at the Department of Chemical Engineering at the University of Vigo (Faculty of Science, Ourense Campus) under the supervision of Dr. Herminia Domínguez González, Dr. Andrés Moure Varela and Dr. Enma Conde Piñeiro.

During my PhD Thesis period my research work has been based on the optimization of the conditions for the extraction and purification of bioactive products of three different types of plant biomass: Sargassum muticum macroalga, Acacia dealbata flowers and Hericium erinaceus, using clean extraction technologies such as enzyme assisted extraction, subcritical water extraction, supercritical CO2 extraction and microwave assisted extraction. Purification using resins and gels served to obtain high purity concentrates and the possible application of the fractions obtained in the cosmetic and pharmaceutical field was evaluated by in vitro cell cultures to assess the anti-inflammatory properties of the extracts. This study was developed during a brief stay at a foreign university in the Faculty of Farmacy of the University of Porto, Portugal.

The results of the studies performed during the PhD period have been published in a number of book chapters and papers in journals indexed in the Journal Citation Reports.

I

SUMMARY

The valuation of foreign plant species present in the local environment has been of special interest in recent years due to the growing demand for natural ingredients obtained with environmentally friendly technologies for food, pharmaceutical, cosmetic and biotechnological applications.The present work included the optimization of the conditions for the extraction and purification of bioactive products of three different types of plant biomass: the Sargassum muticum macroalga, the flower Acacia dealbata and the fungus Hericium erinaceus.To this end, experiments have been carried out with state-of-the-art technologies in different biomass matrices such as biological extraction, such as enzyme assisted extraction and physicochemical extraction, such as subcritical water extraction, supercritical CO2 extraction, microwave assisted extraction and solvent fractionation. Purification of the fractions with resins allowed them to obtain potentially high value added products with concentrated active compounds. The promising activities of the products are based on the results of the in vitro analytical methods for antioxidant and antiradical abilities and the biological methods of in vitro cell culture that developed during a short-term research at a foreign university in O Porto, Portugal.

Enzyme assisted extraction is an innovative technology useful to enhance the extraction yields and rates during aqueous extraction of bioactives. This technique has been applied to different brown seaweeds, but when this study was carried out, it has not been applied to Sargassum muticum. The hydrolytic enzyme assisted processing of carbohydrase and protease activities of Sargassum muticum was proposed to obtain soluble bioactive fractions. Commercial hydrolases have been applied to whole algae (Sm) and to the alginate free solids (AESm). It was intended to select the enzymes from an initial set of nine, to subsequently use those that showed the best extraction yields and increased the in vitro antioxidant activity in the extracts. The enzymatic hydrolysis was performed under experimental conditions similar to those reported in other previous studies with Ecklonia cava, Ishige okamurae, Sargassum fullvelum, S. horneri, S. coreanum, S. thunbergii or Scytosiphon lomentaria. The enzymes were used under the values of pH and T recommended by the manufaturers. The incubation for 3 h with 2-5% (wt%) of the enzyme were optimum conditions for the solubilization II

performance, up to 50% of the material could be solubilized, the most effective enzymes were Celluclast (cellulase), Viscozyme (a multi-enzyme complex containing a wide range of carbohydrases, including arabanase, cellulase, beta-glucanase, hemicellulase and xylanase) and Amylase (-amylase). A direct relationship between the amount of total phenols present in the enzymatic hydrolyzate of each enzyme and the antioxidant activity for the enzymes with proteolytic activity was observed. The ABTS radical cation scavenging capatity of one gram of extract was equivalent to 20 mg ascorbic acid and 100 mg Trolox. As expected, an increase in the monomeric and oligomeric sugar contents of the extracts after the enzymatic treatment was observed. The oligomeric sugar content was higher than the monomeric sugars irrespective of the enzyme and the substrate used. Therefore, this technology could be also suited for the preparation of polysaccharide soluble fractions. An additional experiment was performed using an enzyme treatment assisted by ultrasounds. This configuration increased by 50% the yield attained in control experiments, with a moderated increase of the phenolic content and radical scavenging capacity of the extracts. The extracts from cellulase digested samples and from control samples of Sm contained a similar ω3:ω6 ratio (close to one), regardless the use of ultrasound assisted extraction processes. However a lower ratio was found in the extracts from AESm samples. The selected extracts showed cytotoxic activity against murine melanoma B16F10 cells and human liposarcoma SW872 cells in the concentration range of 50-250 g/L, and on human foreskin fibloblasts HFF-1 more than 500 g/L were required. Since the autohydrolysis process of Sargassum muticum provided higher extraction yields than the enzymatic treatment, liquors produced from autohydrolysis were produced and used for the recovery, purification and concentration of polyphenols using adsorption-desorption processes. These separation techniques are relatively simple and represent promising alternatives to energy-consuming alternatives as it is an energy-efficient technology for the separation of thermosensitive compounds. The growing consumer demand for ingredients and additives derived from natural sources, and the growing market for functional foods and nutraceuticals, have favored the search for bioactive compounds and fractions.

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Phenolic compounds are one of the most numerous and widespread natural structures showing biological effects, such as antioxidants, antimicrobial, antiallergenic, cardioprotective, anticancer and anti-inflammatory. When the concentration of phenolic compounds in the extracts is low, further separations and concentrations should be performed. Since aqueous extraction is the first step in obtaining the target compounds from plant materials, the liquors from the autohydrolysis processes were directly processed with and without previous alginate removal with calcium chloride. Different adsorbents have been successfully proposed for the recovery and concentration of phenolic compounds from natural sources. The adsorbents used in this study for selective recovery are resins, these are hydrophilic or hydrophobic synthetic polymers can be polystyrene-divinylbenzene copolymers, polymethacrylate, divinylbenzene-ethylvinylbenzene and vinylpyridine copolymers. They are highly advantageous for their durability, chemical stability and high adsorption capacity, efficiency, selectivity and ease of regeneration, with relatively low cost and limited toxicity. For the present study it was based on an aqueous extract of Sargassum muticum obtained after the application of optimized conditions in previous works of the group. The food grade resins were Purosorb PAD428, PAD 500, PAD700, PAD900, PAD910 and PAD950 are macroporous, nonionic, designed to adsorb and separate the more hydrophobic organic compounds from aqueous solution. In summary the results of this study confirmed that commercial resins were more favorable for adsorption than organoclays. Adsorption kinetics followed a pseudo-second order and equilibrium was described by the Freundlich model. The phenolic content, the reducing and antiradical properties of the desorbed product increased in relation to those of the autohydrolysis liquors. The eluate showed promising antitumor and anti-inflammatory activities, through inhibition of the growth of cancer cell lines and inhibition of the production of ROS and PGE2, COX-1, COX-2, neutrophil inhibition and inhibition of the growth of carcinoma cells Lung and colon. The adsorption capacity (q, mg/g) achieves in batch adsorption experiments is a useful parameter to provide information about the selectivity and efficiency of the adsorbent used. However, this parameter is generally not useful in adsorption dynamic systems. Under this circumstance, the adsorption behavior of adsorbent in dymanic

IV

systems should be evaluated on the basis of the study and simulation of breakthrough curves obtained in the different possible combinations of the independent variables selected. The aim of this present work is to evaluate the performance of an adsorption dymanic process, where the Sargasum muticum phenolic compounds are recovered in a polymer matrix. Phloroglucinol standard solution has also been used as pattern. The effect of influent flow (1, 2 and 4 mL/min) and of resin bed height (10, 10 and 30 cm) on the phenolic adsorption is evaluated. Fixed bed column performance is usually evaluated using the breakthrough curves obtained by plotting the dimensionless concentration Ct/C0 versus time or volume of the effluent, where Ct and C0 are the outlet and inlet adsorbate concentration. The breakthrough time, tb, was determined when the Ct/C0 ratio reached 10 % of the influent value. The shape of the breakthrough curve was analyzed to correlate the compounds uploading and the time and volume of elution. Bohart-Adams, Yoon-Nelson and Thomas models were used to predict the column performance. The effect of bed depth on the breakthrough curves of Sm hydrolyzates and pholoroglucinol with the same phenolic concentration and flow rates of 1, 2 and 4 mL/min were evaluated. Samples were passed through the column obtaining higher breakthrough times for the highest fixed bed and lower column height. The mathematical description of the adsorption data from the fixed-bed column studies were analyzed using three simplified empirical models: Bohart-Adams, Yoon-Nelson and Thomas. The Thomas model allows the prediction of the maximum adsorption capacity of the column (qTh) and the Thomas rate constant (kTh); the Yoon- Nelson model simulates the time for 50% breakthrough () and the Yoon-Nelson rate constant (kYN); and the Adams-Bohart model predicts the saturation concentration

(N0). The Bohart-Adams model provides a good correlation between the initial part of the curve break calculated by the model and experimental data. This is to confirm the validity of this method only for the incipient area of breaking curve. The rate constant of the Yoon-Nelson model increases with increased flow rate and the fixed bed height. The type of feed used had influence in the shape of breakthrough curves. The slope of ploroglucinol breakthrough curves was higher than Sm solutions, which resulted in a broadened mass transfer zone and higher operational time. In general, these findings

V

confirmed the potential of adsorption systems continuous to retrieve phenolic compounds and their application on a larger scale. The utilization of different extraction technologies was also tried with some underutilized terrestrial materials. The leguminosa Acacia dealbata is native to Australia and in Europe became an invasive species. It is currently widely naturalized in Galicia, invading the lower and middle parts of the region, up to an altitude of 600 m. It prevents the regeneration of the altered natural vegetation due mainly to its facility to germinate and to regrow after fires and to its rapid growth. Its eradication is difficult and costly and the valorization of some fractions could be of interest. Solvent extraction is the conventional method for obtaining plant components, which can be concentrated. This strategy was proposed to obtain different fractions. Since conventional methods with organic solvents can lead to thermal degradation, oxidative transformations of the components and production of undesirable residues or traces of solvents in the final products, combination with emergent technologies can be proposed. Microwave hydrogravity was also proposed to obtain A. dealbata flower extracts from the solids after conventional solvent and aqueous extraction. The aim of the present study is to evaluate the potential applications of A. dealbata flower extracts based on their phenolic content, antiradical potency and other properties of interest in cosmetics and pharmaceuticals. The higher extraction yields were obtained in the first stage of ethanolic extraction, corresponding also to the higher phenolic content and TEAC values. The sequential extraction with solvents allowed to obtain fractions with phenolic content of up to 27% more. The extracts were not active as inducers of HFF-1 cell proliferation. Similarly, all extracts showed inhibition of melanin production at the maximum assay concentration of 1000 μg/mL but were cytotoxic at 500 and 1000 μg/mL and no inhibition was observed. All extracts showed similar cytotoxicity against the tumor cell lines tested. The IC50 values against A549 cells were 250-500 g/mL for acidified water extracts, the IC50 values against the colon tumoral cell line HCT-116 were 250-500 g/mL, observed for all tested extracts, except for the extract in butanol. Concentration of 500 g/mL and higher were needed for pancreatic adenocarcinoma (PSN1), and Caucasian human glioblastoma cells (T98G).

VI

The supercritical (sc) CO2extraction of Acacia dealbata flowers was optimized in a semi continuous flow lab equipment. Extraction conditions at pressures in the range from 100 to 350 bar and at temperatures from 35 to 55 °C were studied to assess their influence on the yield and antiradical properties. Using pure scCO2 at 30 MPa, 45 °C and 1.5 kg/h CO2, a maximum extraction yield of 1.1% of the initial material (d.b.) and 8.92% (measured as percentage of the total ethanol extractables) were obtained. The yield attained with 96% ethanol was 12.77% (d.b.). The kinetics of the process showed an initial linear period at constant rate during the first 30 minutes. This stage provided a quantitative extraction and was followed by a subsequent diffusion controlling mass transfer rate period. The typical behavior of extraction curves with three phases: a constant extraction rate, a falling extraction rate and the third stage where diffusion is the main extraction mechanism. The extraction yields were increased with an increase in the pressure due to the higher solvent density, since increasing solvent density leads to increased extraction yields and faster rate of extraction due to the positive effect of density on solvent power. An increase in the extraction pressure led to decreased ABTS radical scavenging properties of the extracts, but the effect of the operation temperature was less marked. Supercritical CO2 is a poor solvent for polar components and the extraction efficiency can be greatly improved by the addition of polar modifier such as methanol or ethanol. In the present work ethanol was selected for its non toxic and biorenewable character. When 10% ethanol was added as cosolvent, the yields increased up to 6% and the radical scavenging activity by 1.3. The extract obtained under optimal conditions was equivalent to 0.034 g Trolox in the ABTS radical scavenging assay. As an additional study, we proposed operation in a sequence of different conditions to obtain different fractions: a static stage, followed by a dynamic stage with pure CO2 and with ethanol-modified CO2. The application of a static stage allowed the extraction of 0.4% and 0.6% at 10 and 30 min, respectively. However, the active compounds were maximum at 20 min. The joint application of a static 30 min period followed by a dynamic one of 30 min allowed to increase the yield to 1.2%. Finally, the combination of conventional and alternative extraction techniques and adsorption-desorption onto resins was tried with a mushroom. The lion's mane (Hericium erinaceus) is an edible fungus, common during Summer and Autumn in

VII

hardwoods, especially American beech. It presents whitish color that becomes yellowish with time and finally brown at older age. It is commonly used for medicinal or nutritional purposes and has attracted recent attention because of its health effects. Carbohydrates are the most abundant fraction in H. erinaceus, most of the therapeutic polysaccharides are β-glucans, but fungi also contain other bioactive compounds, such as tocopherols, ascorbic acid and carotenoids. The objective of this work was to separate the phenolic fraction of Hericium erinaceus by a sequence of green solvent extraction steps (the limitation of toxic organic solvents and the integral fractionation of raw materials are desirable) including water extraction, microwave hydrogravity extraction (MHG), supercritical carbon dioxide, enzyme assisted extraction or autohydrolysis. The initial stage of the process, after water extraction overnight, was based on the application of microwave technology in an open system, applied to the biomass of fresh fungi using irradiation powers in the range 70-900 W. Due to the structural alterations produced after the treatment with MHG, it is facilitated the successive extraction of the solids. The mushroom processed by MHG was ground and extracted with CO2 using ethanol as modifier and the pressure and temperature conditions based on the literature. The irradiation power during MHG influenced the performance of the supercritical fluid extraction stage, with maximum yields after irradiation at 500-700 W, and a parallel increase in the ABTS radical cation scavenging capacity. The use of ethanol as cosolvent improved the extraction of active components, the yields increased by 20%, and the ABTS radical cation scavenging was doubled. Enzyme assisted extraction was performed on the remaining solid after MHG with a mixture of Viscozyme:Celluclast: Flavourzyme at an enzyme to substrate ratio of 5% (wt%). However, the hot pressurized water extraction under subcritical conditions (non isothermal autohydrolysis at 200 °C), also performed on the solids from MHG, resulted in higher solubilization yields and the phenolic content of the extracts. The aqueous extract obtained after overnight maceration of fresh H. erinaceus with distilled water was contacted by stirring with SP700 resins and after elution, the phenolic content and antiradical properties were enhanced, one gram of the extract obtained was equivalent to 0.6 g Trolox.

VIII

Resumen La valorización de las especies vegetales extranjeras presentes en el entorno local es de especial interés en los últimos años debido a la creciente demanda de ingredientes naturales obtenidos con tecnologías respetuosas con el medio ambiente para aplicaciones alimentarias, farmacéuticas, cosméticas y biotecnológicas. El presente trabajo incluyó la optimización de las condiciones para la extracción y purificación de productos bioactivos de tres tipos diferentes de biomasa vegetal: la macroalga Sargassum muticum, la flor Acacia dealbata y el hongo Hericium erinaceus. Para ello se han desarrollado experimentos con tecnologías de vanguardia en diferentes matrices de biomasa como extracción biológica, como extracción asistida por enzimas y extracción físico-química, como extracción subcrítica de agua, extracción supercrítica de CO2, extracción asistida por microondas y fraccionamiento de solventes. La purificación de las fracciones con resinas le permitió obtener productos potencialmente de alto valor añadido con compuestos activos concentrados. Las actividades prometedoras de los productos se basan en los resultados de los métodos analíticos in vitro para las capacidades antioxidantes y antiradicales y los métodos biológicos de cultivo celular in vitro que desarrolló durante una investigación a corto plazo en una universidad extranjera en O Porto, Portugal. La extracción asistida por enzimas es una tecnología innovadora eficaz para aumentar los rendimientos de extracción y la velocidad de extracción durante los procesos de extarcción acuosa de compuestos bioactivos. Esta técnica se aplicó a la extracción de diversas algas y cuando este estudio se llevó a cabo, todavía no se había aplicado a Sargassum muticum. El tratamiento hidrolítico asistido por enzimas de actividades carbohidrasa y proteasa se propuso para obtener fracciones solubles. Para ello se seleccionaron diversas hidrolasas comerciales que se aplicaron tanto a muestras del alga entera (Sm) como a los sólidos libres de alginato (AESm). Se planteó la selección de las enzimas más adecuadas a partir de un conjunton inicial de nueve formulaciones, para posteriormente emplear aquéllas que proporcionasen los mayores rendimientos extracción y un aumento de la actividad antioxidante de los extractos determinada por métodos in vitro. La hidrólisis enzimática se llevó a cabo en condiciones experimentales similares a las indicadas en estudios previos con Ecklonia

IX

cava, Ishige okamurae, Sargassum fullvelum, S. horneri, S. coreanum, S. thunbergii or Scytosiphon lomentaria. Los enzimas se emplearon siempre en condiciones de pH y T recomendadas por los fabricantes. Las condiciones óptimas de incubación fueron 3 h con 2-5% (p%) de enzimas para proporcionar los mayores rendimientos de solubilización, pues se logró solubilizar hasta el 50% del material de partida. Los enzimas más efectivos fueron Celluclast (celulasa), Viscozyme (un complejo multienzimático de carbohidrases, incluyendo arabanasa, celulasa, β-glucanasa, hemicelulasa y xilanasa) y Amylase (-amilasa). Se observó una relación directa entre la cantidad total de compuestos fenólicos en los extractos de cada enzima ensayada y la actividad antioxidante de los extractos obtenidos con enzimas de actividad proteolítica. La capacidad de captación del radical catión ABTS de un gramo de extracto fue la equivalente a 20 mg ácido ascórbico y a 100 mg Trolox. Se observó, como se esperaba, un incremento en el contenido de azúcares mono- y oligoméricos de los extractos tras el tratamiento enzimático. El contenido de oligómeros fue superior al de monómeros independientemente del enzima y tipo de muestra de alga empleados. Por lo tanto, esta tecnología podría ser adecuada para la preparación de fracciones solubles de polisacáridos de S. muticum. También se llevó a cabo un experimento con enzimas asistido por ultrasonidos. Esta configuración aumentó los rendimientos alcanzados en el experimento control, con un moderado incremento del contenido fenólico de los extractos y su capacidad de captación de radicales libres. Los extractos obtenidos con celulasas y en muestras control de Sm contienen una relación ω3:ω6 similar (cercana a uno), independientemente de que se hubiese empleado un proceso enzimático asistido por enzimas. Sin embargo, los extractos obtenidos a partir de las muestras de AESm mostraton valores menores de esta relación de ácidos grasos. Los extractos seleccionados mostraron actividad citotóxica frente a células B16F10 de melanoma y células SW872 de liposarcoma cells af 50-250 g/L, y sobre fibloblastos humanos HFF-1 se requerirían valores superiores a 500 g/L. Dado que los procesos de autohidrólisis proporcionan rendimientos de solubilización superiores a los obtenidos con tratamientos enzimáticos, los estudios de fraccionamiento y purificación se centraron en el empleo de la tecnología de autohidrólisis por ser más favorable y ser el plan de trabajo considerado en el proyecto

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de investigación que financió trabajo. Se procedió a la recuperación, purificación y concentración de polifenoles presentes en los licores de autohidrólisis mediante procesos de adsorción-desorción empleando resinas no iónicas comerciales. Estas técnicas de separación son relativamente simples y representan alternativas prometedoras a otras con más consumo de energía ya que son energéticamente eficientes para la separación de compuestos termosensibles. La creciente demanda de los consumidores de ingredientes y aditivos derivados de fuentes naturales, y el creciente mercado de alimentos funcionales y nutracéuticos, han favorecido la búsqueda de compuestos y fracciones bioactivas. Los compuestos fenólicos son una de las estructuras naturales más numerosas y extendidas que muestran efectos biológicos, como antioxidantes, antimicrobianos, antialergénicos, cardioprotectores, anticancerígenos y antiinflamatorios. Cuando la concentración de compuestos fenólicos en los extractos es baja, deben realizarse más separaciones y concentraciones. La extracción acuosa es el primer paso para obtener los compuestos de interés del material de partida. Se emplearon directamente los licores de autohidrólisis del alga y también se ensayaron cuando se había eliminado el alginato por precipitación con cloruro cálcico. Se ha propuesto diferentes adsorbentes que proporcionaron buenos resultados para la recuperación y concentración de compuestos fenólicos a partir de fuentes naturales. Los adsorbentes utilizados en este estudio para la recuperación selectiva son resinas, estas son polímeros sintéticos hidrófilos o hidrófobos pueden ser copolímeros de poliestireno-divinilbenceno, polimetacrilato, copolímeros de divinilbenceno-etilvinilbenceno y vinilpiridina. Entre sus ventajas destacan la durabilidad, estabilidad química y alta capacidad de adsorción, eficiencia, selectividad y facilidad de regeneración, con coste relativamente bajo y toxicidad limitada. Para el presente estudio se partió de un extracto acuoso de Sargassum muticum obtenido tras la aplicación de condiciones optimizadas en trabajos anteriores del grupo. Las resinas de grado alimentario fueron Purosorb PAD428, PAD 500, PAD700, PAD900, PAD910 Y PAD950, que son resinas macroporosas, no iónicas, diseñadas para adsorber y separar los compuestos orgánicos más hidrófobos presentes en disoluciones acuosas. En resumen los resultados de este estudio confirmaron que las resinas comerciales fueron más favorables para la adsorción que las organoarcillas.

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La cinética de adsorción siguió un modelo de pseudo-segundo orden y el equilibrio fue descrito por Freundlich. El contenido fenólico, las propiedades reductoras y antiradicales del producto desorbido se incrementó en relación con los de los licores de autohidrólisis. El eluato mostró actividades antitumorales y antiinflamatorias prometedoras, a través de la inhibición del crecimiento de líneas celulares de cáncer y la inhibición de la producción de especies reactivas de oxígeno, inhibición de prostaglandinas y ciclooxigenasas (PGE2, COX-1, COX-2), inhibición de neutrófilos e inhibición del crecimiento de células de carcinoma de pulmón y colon. El dato de capacidad o tasa de adsorción obtenida a partir de un experimento en discontinuo es útil para proporcionar información acerca de la eficacia del adsorbente empleado. Sin embargo, no suelen ser útiles en sistemas de tratamiento realizados en columna. Existe una necesidad real de realizar estudios dinámicos en lechos de relleno para evaluar el comportamiento de los sistemas de adsorción y la simulación de las curvas de rotura obtenidas en las diferentes combinaciones posibles de las variables independientes seleccionadas para el estudio del proceso. En este trabajo se realizan estudios de recuperación de compuestos fenólicos en una matriz polimérica con soluciones patrón de floroglucinol y Licores de autohidrólisis empleando tres caudales de operación diferentes de 1, 2 y 4 mL/min. Se determinaron las curvas de rotura para longitudes de lecho de resina de 10, 20 y 30 cm con diferentes flujos de alimentación. La eficiencia de la adsorción se determina mediante la evaluación de las curvas de rotura obtenidas de la representación gráfica de la relación entre la concentración de la muestra a cada momento respecto la concentración inicial (Ct/C0) frente al tiempo o volumen de efluente. El posterior ajuste y modelización de los datos experimentales mediante modelos matemáticos simplificados (Bohart-Adams, Yoon-Nelson, Thomas) permite establecer las capacidades de adsorción de los sistemas empleados en función de las variables operacionales elegidas y determinar el tiempo de operación de cada sistema en función del denominado punto de rotura. El modelo Bohart-Adams aporta una buena correlación entre la parte inicial de la curva de rotura calculada por el modelo y los datos experimentales. Esto viene a confirmar la validez de este método únicamente para la zona incipiente de la curva de rotura, encontrando que la constante cinética del

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modelo está influída por el flujo de la alimentación. En el modelo de Yoon-Nelson se encuentra que los valores de la constante cinética del modelo aumentaron con el incremento del flujo de alimentación y la longitud del relleno. Respecto a la influencia de tipo de alimentación se muestra como el proceso de adsorción de muestras modelo produce curvas de rotura con una forma más simétrica que las curvas de rotura obtenidas para los procesos de adsorción con alimentaciones de licores de Sargassum muticum. Estas curvas con una inclinación más suave obtenidas con Sm implica una zona más ancha de lecho donde se produce transferencia de materia separando una mayor cantidad de compuestos. Estos resultados ponen en evidencia que el empleo de efluentes reales frente a sistemas modelo conlleva a comportamientos del sistema que se alejan de la idealidad. Los aumentos de los lechos de columna implicaron un aumento en los tiempos de operación y por tanto en la cantidad tratada. En general, estos resultados confirmaron el potencial de los sistemas de adsorción en continuo para recuperar compuestos fenólicos y su aplicación a mayor escala. El empleo de tecnologías alternativas de extracción también se probó con otras fuentes terrestres, como las flores de mimosa. La leguminosa Acacia dealbata es originaria de Australia y en Europa se convirtió en una especie invasora. Actualmente se encuentra ampliamente naturalizada en Galicia, invadiendo las partes bajas y medias de la región, hasta una altitud de 600 m. Impide la regeneración de la vegetación natural alterada debido principalmente a su facilidad para germinar y rebrotar después de incendios y a su rápido crecimiento. Su erradicación es difícil y costosa y a valorización de algunas fracciones podría ser de interés comercial. La extracción con disolvente es el método convencional para obtener componentes de materiales vegetales, que pueden ser concentrados. Sin embargo, los métodos con disolventes orgánicos pueden conducir a degradación térmica, transformaciones oxidativas de los componentes y producción de residuos indeseables o trazas de disolventes en los productos finales y podría ser interesante plantear una combinación con otras tecnologías emergentes más eficaces. La hidrogravedad asistida por microondas es una técnica de extracción novedosa basada en la irradiación de material y drenaje por gravedad del extracto acuoso. Esta técnica se puede proponer para aprovechar los sólidos resultantes tras la extracción con disolventes orgánicos y

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acuosos. Este estudio tuvo como objetivo evaluar las aplicaciones potenciales de los extractos en disolventes convencionales de flores de A. dealbata, basados en su contenido fenólico y potencial antiradicalario y otras propiedades de interés en cosmético o para productos farmacéuticos. Los mayores rendimientos de extracción se obtuvieron en la primera etapa de extracción etanólica, al igual que el mayor contenido fenólico y los valores de TEAC. La extracción secuencial con disolventes permite conseguir fracciones con contenido fenólico de hasta el 27 % mayor. Los extractos no fueron activos como inductores de proliferación de fibroblastos HFF-1, un test empleado como ensayo antiarrugas. Todos los extractos mostraron inhibición de la producción de melanina en la concentración máxima ensayada de 1000 μg/mL pero fueron citotóxicos a 500 y 1000 μg/mL por lo que no se observó inhibición a concentractiones no tóxicas. Todos los extractos mostraron una citotoxicidad similar frente a las líneas celulares tumorales ensayadas. Los valores de EC50 frente a células A549 estuvieron en el intervalo 250-500 g/mL para extractos obtenidos con agua acidificada y también se obtuvieron valores de 250-500 g/mL frente a la línea HCT- 116 de células tumorales de colon para todos los extractos, excepto el de butanol. Sin embargo, frente a las células PSN1 y T98G los valores de EC50 fueron de 500 g/mL y superiores. En este estudio se optimizaron las condiciones de extracción con dióxido de carbono supercrítico CO2-sc a partir de flores de Acacia dealbata. Los experimentos se realizaron en un equipo de extracción sólido-líquido operando en semicontinuo a escala de laboratorio. Las condiciones de extracción estudiadas fueron presiones en el interval de 100 a 350 bar y temperaturas de 35 a 55 °C, y se evaluó su influencia sobre el rendimiento de extracción, propiedades antirradicalarias. Cuando se empleó dióxido de carbon puro a 30 MPa, 45 °C y con un caudal másico de 1.5 kg/h CO2, se obtuvo un rendimiento máximo de extracción de 1.1% del material de partida (base seca), que corresponde a un 8.92% (como porcentaje del total de extraíbles en etanol). El rendimiento en etanol del 96% fue de 12.77% (b.s.). La cinética del proceso de extracción presenta un periodo inicial de velocidad constante, apreciable durante los primeros 30 minutos. Esta etapa proporciona una extracción casi cuantitativa y a esta etapa sigue un periodo controlado por la difusión. La curva típica del proceso de

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extracción muestra tres fases, la del periodo de velocidad constante, una velocidad decreciente y la tercera, controlada por la difusión como mecanismo principal. El rendimiento de extracción se vio incrementado con el aumento de la presión de operación, debido al aumento de la densidad del disolvente, que aumenta los rendimientos y velocidad del proceso debido a la influencia positiva de la densidad sobre el poder solvatante. El aumento de la presión de operación llevó a menores valores de capacidad de captación del radical ABTS y los efectos de la temperatura de operación fueron menos evidentes. Dado que el dióxido de carbon supercrítico es un disolvente poco adecuado para sustancias polares, se planteó aumentar los rendimientos de extracción mediante la adición de un modificador polar como el etanol. Este disolvente se seleccionó por su carácter biorrenovable y no tóxico. Cuando se añadió etanol al 10% como cosolvente, los rendimientos se aumentaron hasta el 6% y la actividad antirradicalaria aumentó en un 30%. Los extractos obtenidos en condiciones óptimas mostraron una actividad equivalente a 0.034 g Trolox en el ensayo de captación del radical catión ABTS. Adicionalmente se propuso la operación en distintas condiciones de trabajo, con el objetivo de obtener distintas fracciones de modo independiente. Se consideró una etapa de extracción en condiciones estáticas (sin circulación continua de disolvente), seguida por una etapa dinámica empleando

CO2 puro y una última etapa, también con circulación continua de disolvente, empleando CO2 modificado con etanol. Durante la etapa de extracción en condiciones estáticas se alcanzaron rendimientos de 0.4% y 0.6% a 10 y 30 min, respectivamente. Sin embargo, los compuestos activos alcanzaron un máximos a 20 min y la aplicación conjunta de un periodo estático de 30 min y uno dinámico de otros 30 min, permitieron obtener un rendimiento de 1.2%. Finalmente se evaluó la combinación de tecnologías convencionales y emergentes y el empleo de resinas para la extracción de bioactivos de una seta. La melena de león (Hericium erinaceus) es un hongo comestible, común durante el verano y el otoño en las maderas duras, en especial en la de haya americana. Presenta color blanquecino que se hace amarillento con el tiempo y finalmente marrón a mayor edad. Se utiliza comúnmente para fines medicinales o alimenticios y ha atraído la atención reciente debido a sus efectos sobre la salud. Los hidratos de carbono son la

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fracción más abundante en H. erinaceus, la mayoría de los polisacáridos terapéuticos encontrados en H. erinaceus son β-glucanos, pero las seetas también contienen otros compuestos bioactivos, como tocoferoles, ácido ascórbico y carotenoides. El objetivo de este trabajo se centró en separar la fracción fenólica de Hericium erinaceus mediante una secuencia de etapas con disolventes verdes (es deseable la limitación de disolventes orgánicos tóxicos y el fraccionamiento integral de materias primas) incluyendo extracción con agua, hidrogravedad asistida por microondas (MHG) y extracción con dióxido de carbono supercrítico. También se aplicó un tratamiento enzimático o la extracción con agua presurizada en condiciones subcríticas, para la solubilización de los componentes del sólido remanente. La etapa inicial del proceso, tras la extracción acuosa se basó en la aplicación de microondas en un sistema abierto empleando una potencia entre 70 y 900 W. Debido a las alteraciones estructurales producidas por el tratamiento de MHG se pueden ver favorecidos los procesos de extracción empleados. El sólido procesado por MHG, una vez molido, se extrajo con

CO2 supercrítico con etanol como modificador empleando condiciones de presión y temperatura de la bibliografía. La potencia de irradiación durante el tratamiento de MHG influye en el proceso de extracción supercrítica, con valores máximos de rendimiento a valores de 500-700 W, y un aumento parallelo en la capacidad de captación del radical catión ABTS. El empleo de etanol como cosolvente mejoró los rendimiento de extracción de bioactivos en un 20%, y la actividad antirradicalaria due el doble. La extracción asistida por enzimas realizada sobre los sólidos tras MHG con una mezcla Viscozyme:Celluclast:Flavourzyme se realizó a una relación enzima:sustrato de 5% (wt%), pero los valores mayores de rendimiento y de contenido fenólico se alcanzaron cuando se realizó la extracción con agua caliente en condiciones subcríticas (autohidrólisis no isoterma a 200 °C), también a los sólidos tras MHG. El extracto acuoso obtenido después de la maceración durante la noche de H. erinaceus fresco con agua destilada se puso en contacto con resinas SP700 y tras la elución el contenido fenólico y las propiedades antirradicalarias del extracto se vieron mejoradas. El extracto desorbido presentó una capacidad de captación del radical catión ABTS de 0.6 g Trolox por gramos de extracto.

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1. INTRODUCTION

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1. INTRODUCTION 1.1. Antioxidants Antioxidants are often used in food and biological systems. Their main function is to inhibit lipid peroxidation, but also can protect proteins, and minimize deoxyribonucleic acid (DNA) degradation by reactive oxygen and nitrogen species (ROS and RNS, respectively). Free radicals are highly reactive and unstable molecules that in order to achieve their electronic stability try to pick up unpaired electrons or those that are easy to obtain from the nearest molecules. As a result of these reactions, when this process takes place in a living organism, a disorganization is generated in the cellular membranes, causing damage at different levels in the cell.

1.1.1. Definition of antioxidant There are different ways of defining antioxidants, since this depends on the author or the Regulations in the different states. The most used are: “Antioxidants: substances which extend the storage life of foodstuffs, protecting them from deterioration caused by oxidation, and changes in color”, of February, approving the positive list of additives other than coloring matter and sweeteners for use in the manufacture of foodstuffs, as well as their conditions of use). Another way to define it is: “the substance that, if present in as those substances which when present at low concentrations, compared to those of an oxidizable substrate, will significantly delay or inhibit oxidation of that substrate (lipids, carbohydrates), is capable of retarding or preventing the oxidation of said substrate (Halliwell and Gutteridge, 1999; Halliwell, 2002).. Food additives are added to foodstuffs in order to perform certain functions, such as conferring color, sweetening or preservation. Food additives are defined as: "Any substance which is not normally consumed as a food in itself and which is not normally used as a characteristic food ingredient, whether or not it has a nutritional value, the intentional addition of which to food for technological purposes in manufacture, processing, preparation, packaging, transport or storage of such

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foodstuffs, or may be reasonably expected to result in it or its by-products becoming directly or indirectly a component of such foodstuffs”. Antioxidants and stabilizers are included in the categories of food additives listed in Annex I to this Directive. According to the European Regulation No 1333/2008 of the european parliament and of the council of 16 December 2008 on food additives

1.1.2. Natural and synthetic antioxidants

There is now increased awareness and concern for the safety of synthetic antioxidants. In recent years, the global trend is to try to replace these compounds with natural antioxidants (in both nutraceuticals and functional foods, where they are used as ingredients). This change is due to the publication of numerous studies that have shown the harmful effects of synthetic antioxidants after a long period of ingestion. This fact has stimulated the search and identification of new substances of natural origin with antioxidant capacity, in order to reduce the consumption of synthetics. The worldwide use of synthetic antioxidants started because they were cheaper, with more consistent purity and had more uniform antioxidant properties. There have been some studies that have confirmed the therapeutic potential of the natural products, these include actions such as: protection against cellular or molecular damage caused by free radicals, reduction of radical-induced tissue damage and delayed progression of chronic diseases; although these effects can not be attributed exclusively to the antioxidant properties, but also influence other beneficial action complexes (Balboa et al., 2013). In spite of this, the health safety of natural compounds should not be taken for granted and it is necessary to study the toxic effects of natural substances isolated and added as pure substances. Employing synthetic antioxidants in food requires the consideration of several aspects: the technological need, the toxicity of lipid oxidation products and the toxicity of the antioxidants themselves. The most commonly used synthetic antioxidants are butylhydroxyanisol (BHA, E-320), butylhydroxytoluene (BHT, E-321), tert-butylhydroquinone (TBHQ, E-319), propyl (E310), octyl (E-311) and dodecyl (E-312) gallates. These antioxidants have many disadvantages, among which are: they are quite volatile substances; decompose

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easily at high temperatures; in refined vegetable oils do not prevent reversion, which produces an unpleasant taste; and in some foods such as noodles and noodles stabilized with TBHQ develop a strong stale smell. Several studies have found that high levels of BHT, BHA and TBHQ produce a significant increase in liver weight and marked proliferation of the endoplasmic reticulum. Other alterations noted have been a decreased growth and hair fall in rats, hyperplasia of epithelial cells in the folds of the stomach and a toxic effect on monkey cells by BHT. Another study reported the ineffectiveness of BHT in delaying oxidative deterioration in complex food systems, such as muscle foods where both lipoxygenase and hemoproteins are involved in the initiation of oxidation.

1.1.3. Antioxidant activity Although the activity and antioxidant capacity are used interchangeably, they do not mean the same. The activity of an antioxidant substance would be meaningless without the context of specific reaction conditions, such as pressure, temperature, reaction medium, reactants and reference points. Because the antioxidant activity measured by an individual assay reflects only the chemical reactivity under the specific conditions that are applied in that assay, it is inappropriate and misleading to generalize the data as indicators of total antioxidant activity. In contrast, the term antioxidant capacity is used to refer to the results obtained by the different tests, for example the amount (in moles) of free radicals captured by a sample. sample (Huang et al., 2005. Antioxidants may act as:

 Preventive antioxidants, which prevent the formation of ROS or the uptake of species responsible for the initiation of oxidation, and which prevent the decomposition of hydroperoxides.  Antioxidants that interrupt the propagation of the chain reaction of the autoxidation, which convert reactive free radicals to stable molecules, through a hydrogen atom or the transfer of a missing electron.  Suppressors of oxygen free radicals, which transform singlet oxygen (excited state of molecular oxygen).

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 Synergists are compounds that increase the activity of antioxidants, and can disrupt the chain reaction in a mixture.  Reducing agents, which reduce hydroperoxides to a non-radical form.  Metal chelants, responsible for stabilizing pro-oxidizing metals (iron or copper cations).  Oxidative enzyme inhibitors, which are very specific (especially lipoxygenases).

Antioxidant compounds, which are substances capable of counteracting the action of free radicals and certain oxidizing species, can be classified into three groups:

Primary antioxidants. They are those capable of capturing free radicals, so they can delay the initiation step or interrupt the propagation of autoxidation. These antioxidants react with lipids and peroxyl radicals, and then convert them into more stable products. The product obtained by this process is much less reactive than lipids and peroxyl radicals, and therefore less effective in promoting oxidation. These antioxidants have a higher affinity for peroxyl radicals than lipids, and tend to sequester the free radicals produced during the initiation and propagation stages. These antioxidants are also capable of terminating the lipid oxidation reaction by reacting with peroxyl and alkoxy radicals. In this group, the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione transferase (GST) and metal binding proteins such as ferritin and ceruloplasmin stand out. It can be said that they are destroyed during the period of induction.

Secondary antioxidants. They can act through a variety of mechanisms, such as metal ion bonding, oxygen uptake, conversion of hydroperoxides to non-radical species, absorbing UV radiation or deactivating singlet oxygen oxygen (Gordon, 2001; Zapata et al., 2007). Secondary antioxidants generally only act once the radical has formed, examples of which are citric acid (a sequestering agent which is only effective in the presence of metal ions) and ascorbic acid (a reducing agent, effective in the presence of tocopherols or other primary antioxidants). Some of these antioxidants are vitamin E, β carotenes, uric acid, bilirubin and albumin.

Tertiary antioxidants. They are in charge of the recovery of the chemicals structures, and these antioxidants include DNA repair enzymes, which are valuable because of the

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role they play in the conservation and transmission of hereditary characters from one generation to another. In general, antioxidants act by different mixed and cooperative mechanisms. An antioxidant may behave as a prooxidant depending on the structure, chemical environment and operating conditions Galati and O'Brien, 2004). There are situations where alterations occur in the delicate pro-antioxidant-antioxidant balance. When the former prevail, the organism is in a situation of oxidative stress. It is under these circumstances that damage can occur at the cellular level and in the organism, which can cause diseases such as arthritis, cataracts, cerebro-vascular diseases, various types of cancer, etc. One of the ways to control this imbalance is to increase the levels of antioxidants contributed by the diet (Halliwell, 2006).

1.1.4. Measurement of antioxidant activity

The antioxidant activity depends on a number of factors, including the type of substrate, the medium and the oxidizing agent. The oxidation conditions, the partitioning properties of the antioxidants between the lipids and the aqueous phases, and the experimental method used. Both low oxidation levels and possible modifying mechanisms in accelerated oxidation conditions must be taken into account (Frankel, 1993, Gordon, 2001, Yanishlieva 2001, Antolovich et al., 2002, Schlesier et al., 2002, Collins, 2005, Decker et al., 2005, Huang and Prior, 2005, Frankel and Finley, 2008). A list of in vitro tests used in the evaluation of antioxidant activity, including antiradical capacity, anti-lipoperoxidation, reduction and chelation of metals are shown in Table 1.1.

Table 1.1.In vitro methods frequently used to check antioxidant activity in extracts and in pure compounds.

Antioxidant activity tests

Capture of hydrogen peroxide

Capability of the hydroxyl radical

Uptake of hypochlorous acid

Absorption capacity of oxygen radicals

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Capture of peroxyl radical

Peroxynitrite uptake

Uptake of superoxide radical

Total capacity of oxidant uptake

Total Radical Capture Parameter

Antioxidant capacity equivalent to Trolox (TEAC)

Capability of the DFPH radical

Total antioxidant reactivity

Phosphomolybdenum method

Copper ion reducing capacity

Fremy radical reduction

Discoloration of β-carotene

1.2. Natural phenolic compounds 1.2.1. Origin and formation of phenolic compounds

Phenols can be formed by two metabolic pathways:

- The polyketide pathway (also known as the acetate-malonate or malonic acid route).

- The pathway of psychic acid, which can participate in the formation of complex phenols in a photodependent process. This pathway begins in the plastids of the plant cells by condensation of two photosynthetic products, erythrone 4-P and phosphoenolpyruvate (PEP), which after several reactions give rise to the shikonic acid. Next, the incorporation of a second molecule of PEP leads to the formation of phenylamine, which is converted into transcinnamic acid by the action of the enzyme phenylalanine ammonolase (PAL). Later this acid is transformed into coumaric acid, thanks to the incorporation of a hydroxyl group at the level of the aromatic ring and the action of a CoAligase, which transforms it into p-cumariolCoA, precursor of the most phenols of vegetal origin.

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The action performed by PAL in this process is a very important factor, since this enzyme is activated with light and its activity depends on the concentration of different plant hormones. PAL activity increases when vegetables are subjected to stress, lack of water, fungal or bacterial infections, low temperatures and UV radiation. 1.3. Natural compounds with antioxidant activity and major sources To avoid these negative effects, antioxidants are used in the food industry. The most commonly used are synthetic antioxidants such as butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA), but since the possible toxicity of these compounds to the human body has recently been described, the search for antioxidants from natural sources has increased). These include phenolic compounds such as tocopherol or vitamin E, carotenoids and catechins. These natural antioxidants have some disadvantages. Their antioxidant capacity is lower and most of them (carotenoids and phenolic compounds) are insoluble in water.

1.3.1. Natural compounds with antioxidant activity The most important groups of natural antioxidants include terpenes, vitamin E, small proteins and peptides, Maillard reaction products (MRPs), carbohydrates and phenolics. These are synthesized by plants, microorganisms, fungi and animals.

Terpenes

Terpenes are the main constituent of the essential oils of some plants and flowers. In plants the terpenes fulfill many primary functions: some carotenoid pigments are terpenes, they also form part of chlorophyll and hormones gibberellin and abscisic acid. The terpenes of plants are widely used for their aromatic qualities. They play an important role in traditional medicine and herbal remedies, and are investigating their possible antibacterial and other pharmaceutical uses.

Monomers are generally referred to as isoprene units because the heat decomposition of many terpenes results in that product, and because under suitable chemical conditions, isoprene can be induced to polymerize into multiples of 5 carbons, generating numerous terpene skeletons. Terpenes are classified as hemi-, mono- (C10), sesqui- (C15), di- (C20), sester- (C25), tri- (C30) and tetraterpenoids (carotenoids), which have eight isoprenoid C5 residues. - 7 -

The antioxidant activity of terpenes has been described (Escuder et al., 2002, Grassmann, 2005, Grassmann et al., 2002, Bourgou et al., 2010). They may also have synergistic effects with other compounds, such as phenolics (Milde et al., 2004). Carotenoids and xanthophylls (carotenoids) have been of particular interest because of their health implications and their provitamin and antioxidant actions (Bohm et al., 2002).

Vitamin E

Vitamin E includes four tocopherols, called alpha, beta, gamma and delta. Tocopherol is the name of several organic compounds formed by several methylated phenols, which form a class of chemical compounds called tocopherols, of which several act as Vitamin E.

Alpha-tocopherol is an antioxidant that has the property of protecting polyunsaturated fatty acids from membranes and other cellular structures of lipid peroxidation. Hence, it is the form of vitamin E which is preferably absorbed and accumulated in humans

Small proteins and peptides

Antioxidant peptides can be obtained from the digestion of animal or vegetable proteins, either using endogenous or exogenous enzymes, microbial fermentation, processing and during gastrointestinal digestion.

The therapeutic potential of a peptide is attributed to the presence of specific functional groups in the sequence. The mechanism is related to the composition, position in the peptide sequence, structure and hydrophobicity of each functional group. The studies performed so far, suggest different mechanisms for the physiological activities exhibited by the peptides, however, few of them have been confirmed.

The increased ability of peptides and larger proteins to decrease the reactivity of a species is related to the increased exposure of amino acids. In general the 20 amino acids present in proteins can interact with reactive species of oxygen and nitrogen, the most reactive include sulfur methionine and cysteine, histidine for its

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imidazole group and the aromatics tryptophan, tyrosine and phenylalanine, in addition to the imidazole ring such as histidine.

Maillard reaction products (MRPs)

The Maillard reaction, also known as non-enzymatic browning, is one of the most frequent reactions during food processing, mainly during heating but also during storage at room temperature or even, although much more moderate to low temperatures Mlotkiewicz, 1998; Somoza, 2005).

This reaction originates between the amino group of an amino acid, peptide or protein and the carbonyl group of a reducing sugar or an oxidative lipid that undergo a series of complex chemical reactions.

The most studied MRPs for their antioxidant properties are melanoidines. They show the antioxidant capacity of the hydroxyl scavenger radical, superoxide and hydrogen peroxide, as well as the chelating activity of the metal.

Phenolics

Phenolic compounds are one of the most important and ubiquitous groups of compounds in the plant kingdom. Currently, phenolic compounds are classified into two groups, based on the number of the basic phenolic skeleton present in the molecule: simple phenols and polyphenols, if only one or more aromatic groups are present, respectively.

Generally, soluble phenolic compounds are found within the vacuoles of plant cells and insoluble phenolics are present in cell walls. Some biochemical functions were described for these compounds in plants, such as: structural components, protein synthesis, enzymatic activity, photosynthesis and allelopathy.

These compounds play a wide variety of functions for plants, including defense against herbivores and pathogens, absorption of UV light, contributing to pigmentation of plants, attracting pollinators, reducing the growth of competitive plants and promoting symbiotic relationships with fixing bacteria Of nitrogen, and are implicated in numerous significant benefits to human health and nutrition due to their wide variety of bioactivities .

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1.3.2. Classification

The term "phenolics" encompasses about 8000 natural compounds, ranging from simple phenols, with low molecular weight, to large and complex polyphenols, but all have a common structural feature, a phenol (an aromatic ring bearing at least one hydroxyl substituent).

Some subclasses may be separated according to the modifications of the basic structure (C6-C3-C6), of which flavones and flavanols are the most widely present and structurally diverse. Since they absorb light energy UV-B (280 to 320 nm), they are believed to play a protective role in plants. Tannins are a high molecular weight (hydrolyzable and condensed) group with high and are commonly combined with alkaloids, polysaccharides and proteins.

Phenolic acids are found in a variety of plant-based foods; seeds, fruit skins and vegetable leaves contain the highest concentrations. Phenolic acids are easily absorbed through the walls of your intestinal tract and can be beneficial to your health as they function as antioxidants that prevent cell damage due to free radical oxidation reactions. They can also promote anti-inflammatory conditions in your body when you eat regularly.

Other types of polyphenols include flavonoids and stilbenes.

Stilbenes are an aromatic hydrocarbon of formula C14H12 formed by two aromatic rings (benzenes) separated from each other by an ethane or ethene bridge. Stilbenes may be free or form heterosides at times polymeric, to this family belong the natural polyphenols present in many families of higher plants (for example, trans-resveratrol of the grape). Resveratrol is the most representative stilbene, it has recently received a lot of attention for its specific biological properties and its potential therapeutic effects. Wine is the main dietary source of resveratrol. This compound is produced in the leaves and skin of grapes in response to an infection or stress induced by herbicides, fungicides or UV light. Resveratrol is found as aglycon (trans- and cis- resveratrol), or as glucoside (trans- and cis-piceid) and dimeric forms (viniferin and pallidol).

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Lignans the basic structure of these substances are two units C6-C3 joined by β, β bonds. Lignans are secondary metabolites of plants found in a wide variety of plants including flax seeds, pumpkin seeds, sesame seeds, rye, soybeans, broccoli, beans, and in some berries in very small quantities.

Flavonoids are low molecular weight compounds that share a common skeleton of diphenylpyrans (C6-C3-C6), composed of two phenyl rings (A and B) bound through a pyran (heterocyclic) C ring. This basic structure allows a multitude of substitution patterns and variations in the C ring (Figure 1.1). Depending on their structural characteristics they can be classified in:

1. Flavans, such as catechin, with an -OH group at position 3 of ring C.

2. Flavonols, represented by quercetin, which has a carbonyl group in the 4- position and a -OH group in the 3-position of the C-ring.

3. Flavones, such as diosmetine, which have a carbonyl group at the 4-position of the C-ring and lack the hydroxyl group at the C 3-position.

4. Anthocyanidins, which have the -OH group in the 3-position but also have a double bond between the carbons 3 and 4 of the C-ring.

Flavonoids are biosynthesized in all "terrestrial plants" or embryophytes and also in some algae. The play different roles in plants: they confer resistance to the photooxidation of the ultraviolet light of the sun, they take part in the transport of the hormone auxin, and are believed to function as a defense against herbivorism through the color or smell they give the plant or its flowers attract pollinating insects.

Flavonoid Flavanol Anthocyanidin

Flavone Flavonol- 11 -

Figure 1.1.Flavonoids, basic structure and types

Tannins are polyphenolic compounds, more or less complex, of vegetable origin and relatively high molecular mass. These are water-soluble compounds, sometimes giving colloidal solutions in water, soluble in alcohol and acetone and insoluble in non- organic solvents. Within the plants the tannins are usually found in cellular vacuoles, combined with alkaloids or proteins. Hydrolyzable tannins are derivatives of gallic acid (Figure 1.2). They present antioxidant properties behaving like free radical scavengers. Of the pharmacological activities of the tannins we can emphasize their astringent properties, both internally and topically. They also have vasoconstricting properties.

Figure 1.2.Molecular structure of gallic acid

Lignin is one of the most abundant biopolymers in plants and together with cellulose and hemicellulose it forms the cell wall of the same in a regulated arrangement at the nanostructural level, resulting in lignin-carbohydrate networks. Given their structural characteristics they may have antioxidant properties.

Phlorotannins are phenolic oligomeric structures of phloroglucinol (1,3,5- trihydroxybenzene), which can be linked in various ways and they are usually classified according to the type of linkage (ether, phenyl or dibenzodioxin). Phlorotannins are abundant in brown algae, and may account for 10-15 % of their dry weight. Some representative compounds are shown in Figure 1.3.

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HO OH CH3 O

HO OH HO OH

OH O CH3 O OH OH OCH3 3-(3,3-dimethylallyl)-5-(3-acetyl-2,4- O O dihydroxy-5-methyl-6-methoxybenzyl)- HO OH OH phlorobutyrophenone HO OH

OH O O O OH HO O O

HO O OH OH Dieckol Dioxinodehydroeckol

OH OH OH

OH O OH OH O OH Phloroglucinol HO OH O O OH OH

OH O HO O O OH OH

O O OH HO OH OH Phlorofucofuroeckol A

OH - 13 -

1-(3,5-dihydroxyphenoxy)-7-(2,4,6- trihydroxyphenoxy) 2,4,9- trihydroxydibenzo-1,4,-dioxin Figure 1.3.Some structures of algal derived phlorotannins

1.3.3. Environmentally friendly extraction technologies

The interest in more environmentally friendly extraction technologies biomass has favoured the research on alternatives that have economic, and environmental advantages over conventional extraction including selectivity, cleanliness and ease of product recovery, enabling cost savings and reduction in time and energy.

1.3.3.1. Hydrothermal processes for extraction Subcritical-water extraction (SWE) is an environmentally friendly technology for the selective extraction of bioactives and offers advantages derived from its simplicity, reduced extraction time, high quality of the extract, low cost of the extracting solvent and possibility of scaling-up. Water under pressure (temperature in the range 100–374 °C and pressure <22.1 MPa) is also known as superheated water extraction, high- temperature water extraction, pressurized hot water extraction, or hot water extraction. Water in subcritical state is environmentally safe, non-toxic, and presents unique solvent and transport properties, including decreased dielectric constant, surface tension, and viscosity with increasing temperature. It is a good solvent for ionic species at ambient pressure and temperature, and for nonpolar solutes near the critical temperature. As temperature increases the dielectric constant decreases to values similar to the ones of methanol or acetonitrile, improving the extractability of nonpolar compounds. The unique properties of subcritical water make it a suitable reaction medium for biomass conversion and can be scaled up. SWE was more efficient than conventional solvents for extracting flavanols from grape seeds or antioxidant phenolics from bitter melon, and was sucessfully employed to concentrate and isolate antioxidant compounds from oregano leaves. Temperature has a marked effect on yield and selectivity. The dielectric constant of water decreases with temperature,

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enabling the extraction of compounds having different polarities at different temperatures. High temperature also enhances diffusivity, facilitating the transport of solutes from the solid matrix, and a compromise to avoid thermal degradation must be reached. The conditions of SWE influence the degree of polymerization and structure of molecules, as reported for procyanidin extraction. Subcritical water extraction was successfully used for extracting various compounds from different materials including plants, algae and mushrooms (González- López et al., 2012b; Balboa et al., 2013). Compared to conventional extraction, SWE is faster and more efficient for the extraction and depolymerization of polysaccharides and phenolics phenolics (Garrote et al., 2003; Parajó et al., 2004; Moure et al., 2005 and 2014; García-Marino et al., 2006; Rodríguez-Meizoso et al., 2006; Budrat and Shotipruk, 2008; Conde et al., 2008; Balboa et al., 2013).

1.3.3.2. Enzyme assisted extraction The destruction of structural cell wall components facilitates the extraction of compounds with biological activities. Hydrolytic enzymes offer the possibility of degrading the cell walls and membranes and this is an alternative to conventional solvent-based methods. This technology provides extraction yields and selectivity, high reproducibility and operation in aqueous mild processing conditions that preserve the integrity of the products. Also has advantages such as the lower capital investment costs and energy requirements and increased safety (Jung and Mahfuz, 2009). This technique has been proposed for extraction of bioactives from plants (Puri et al., 2012a), and mushrooms, and from seaweeds (Wijesinghe and Jeon, 2012). Enzymes can aid in the extraction of polysaccharides, oils, carotenoids, phenolics, proteins. Enzymatic hydrolysis achieves cell wall disruption and breakdown of internal storage components for more effective release of intracellular bioactive compounds. In addition, hydrolysis of proteins produces peptides which enhance the bioactivity of the algal extract. Selection of appropriate hydrolytic activities or optimal mixtures is determinant because enzymes may constitute one of the major costs of treatment.

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The mechanical treatment has a large impact on the solvent extraction process, since it breaks plant cell walls, facilitating the accessibility of solvent to solutes. Grinding is proposed to cause cell disruption and to increase the surface area for an efficient enzyme treatment of a variety of materials. Enzyme assisted aqueous processing can be combined with thermal trearments and thermophysical techniques (extrusion, pressurized processing) for the simmultaneous extraction of oil and protein. The pH and temperature influence the activity to a large extent and the values at which maximum values are attained depend on the enzyme. The pH value, usually selected at the optimal for enzyme activity, is maintained by the buffer. The choice of enzyme depends on the cellular composition and structure of the vegetal material and the efficiency can be increased due to the presence of collateral activities of the formulation, which guarantees the disintegration of the cell walls and membranes, and due to the synergistic effects of mixtures. The interaction between time, temperature and enzyme to substrate ratio are important in most cases, thaerefore the joint optimization should be considered.

1.3.3.3. Microwave assisted extraction Microwaves (MW) are radiation of the electromagnetic spectrum ranging in frequency from 300 MHz (radio radiation) to 300 GHz (infrared radiation), which correspond to wavelengths from 1 m to 1 mm. Household microwave ovens operate at 2450 MHz with adjustable power output up to a maximum of 900-1000 W. Microwave heating is based on the direct effect of microwaves on molecules by dipole polarization and ionic conduction. Microwaves possess electric field and magnetic field components perpendicular. As the dipole re-orientates to align with the electric field, produces molecular friction and collisions and the liberation of thermal energy into the medium results in fast dielectric heating (Galema, 1997; Lidström et al., 2001). Conduction is responsible for the more rapid heating and higher temperature reached in a solution containing ions than in another without ions (Lidström et al., 2001).

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The ability of a material to interact with electromagnetic energy can be measured by the relative permittivity, ε=ε′−jε″, where j is the imaginary unit. The dielectric constant, ɛ′, is proportional to the amount of energy absorbed. The dielectric loss, ɛ″, or loss factor and indicates the ability of a medium to dissipate input dielectric energy as heat. The efficiency of microwave heating at a given frequency and temperature depends on the ability of the material to absorb electromagnetic energy and to dissipate heat, and can be measured by tan = ´´/ ´, which is the energy dissipation factor or dielectric loss tangent. Microwave assisted water extraction processes, either adding water as the only solvent or solvent free microwave assisted extraction, using the naturally present water in vegetal materials, is promising for the extraction of valuable components from renewable vegetal sources. The application of this technology on the extraction of natural compounds and secondary plant metabolites has been reviewed (Chan et al., 2011). The benefits of microwave in extraction processes are in relation to selective heating and thermal effects on the physicochemical and transport properties of solvent and solutes, as well as the thermal induced structural damage of the matrix. In conventional processes, heat is transferred by convection and conduction, whereas microwaves penetrate into the solid at the speed of light, causing direct generation of heat within the material and a simultaneous temperature rise. The application of microwave energy may be performed in pressurized equipments or in open-systems at atmospheric pressure (Chan et al., 2011). The extraction performed by a single stage involving the application of microwave irradiation and earth gravity is named microwave hydrodiffusion and gravity (MHG). The hydrodiffusion allows the extract to diffuse outside the plant material and the extract is dropped on a spiral condenser outside the microwave cavity; water and essential oil are collected and can be separated by density. MHG was successfully applied to the extraction of volatile compounds from fresh plant materials with a minimum 60% of initial moisture (Bousbia et al., 2009). Microwave assisted extraction was reported for the extraction of oils, polysaccharides, protein and phenolics. The MW extraction of vegetal compounds is

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influenced by the properties of the matrix, the solute, the solvent and the operational conditions, mainly the irradiation power and temperature. However, temperature should be controlled to avoid formation of by-products, decomposition and/or degradation. In closed systems, temperature can increase above the normal boiling point and significantly alter the properties of water. The increasing microwave irradiation energy can enhance the molecular interaction of the electromagnetic field and the materials, but must be chosen to maximize yields, selectivity and stability of the target solutes. Time is also an important factor in microwave assisted extraction processes, with an initial positive impact, but degradation of solutes and/or excessive breakage of the oligomeric fractions can occur at prolonged times. The water content in a plant sample has great influence on the extraction efficiency.

1.3.3.4. Supercritical fluids Supercritical fluid extraction (SFE) has been considered as a sustainable “green” technology for the selective isolation of plant compounds. The term “supercritical” refers to substance above its critical pressure and temperature (Figure 1.4). Supercritical fluids possess unusual characteristics as: high compressibility, low surface tension, liquid-like density, gas-like viscosity and diffusivity between those of liquids and gases. These properties confer supercritical fluids a greater ability to diffuse into solids than the conventional solvents and allow improved extraction yields of solutes from complex matrices. The density of supercritical fluids can be modified by adjusting pressure and temperature conditions; small changes in pressure provide significant changes in density and the solvating properties of solvent can be controlled by changing these variables. Carbon dioxide is the most widely used solvent for the supercritical fluid extraction of natural compounds. The main reasons are in the carbon dioxide properties. Supercritical carbon dioxide (SC-CO2) is cheap, nontoxic, nonflammable and environmentally safe. Its critical point (Pc = 7.38 MPa; Tc = 304.1 K) (Figure 1.4) allows operation at mild temperatures so that prevents thermal degradation of labile compounds. Furthermore, extracts obtained using SC-CO2 are solvent-free since CO2 is easily separated by depressurizing and the extract can be considered natural and

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would have GRAS (Generally Recognized As Safe) status. The major drawbacks are the high cost of the investment and the limited ability of SC-CO2 to dissolve hydrophilic compounds, requiring the addition of small amounts of a polar cosolvent (such as water or ethanol) to involve the extraction of polar compounds.

72.8

Solid

5.11 Pressure Pressure (bar)

216.8 304.2 Temperature (K)

Figure 1.4.Typical pressure-temperature phase diagram of pure CO2

A supercritical extraction process with fractionation from solid matrix is composed of an extraction vessel, which is heated to the extraction temperature, where the solid matrix is packed, and one or more separators where the extract could be fractionated (Figure 1.5). The supercritical fluid is subcooled prior to the pump, assuring a liquid phase to avoid cavitation issues. The pressurized solvent id heated above its critical temperature to the extraction temperature prior to the extraction vessel. The supercritical fluid flows uniformly distributed through the matrix bed into the extraction vessel and the solutes are extracted from the carrier material. Supercritical fluid and solubilized compounds leave the extraction vessel through a pressure reduction valve. The solvent power decreases with pressure reduction, so the compounds precipitate in the separators. The use of several separators in series,

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operating at different pressures and temperatures, allows the fractional precipitation of the solutes extracted.

Figure 1.5.General scheme of a supercritical fluid extractor

Adequate selection of the operation conditions defines the efficiency and selectivity of the supercritical CO2 extraction process. For this purpose, the most influent variables should be considered, including the previous preparation of the raw material, the extraction and separation conditions.

The performance of the SC extraction process depends on the solid matrix chosen, its physicochemical characteristics and the previous thermal and mechanical conditioning operations done. These conditioning operations can alter the main characteristics of the solid matrix as: moisture, particle size and structural characteristics. Particle reduction facilitates the diffusion of the solutes out of the solid particle because the grinding enhances the transfer area and reduces the diffusional resistance, but the presence of too fine particles can produce compaction bed increasing the internal mass transfer resistance and reducing the extraction

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effectiveness. The effect of particle size has been studied for several matrixes Bed compaction can be avoided by mixing with inert material as sea sand or glass beads. The moisture content is also an important variable that must be evaluated case by case. High content of moisture is usually not desirable because moisture acts as a mass transfer barrier. On the other hand, moisture expands the cell structure, facilitating the mass transfer of the solvent and the solute through the solid matrix. Other important parameters in the supercritical fluid extraction are the solvent- feed ratio and the solvent flow. The solvent-feed ratio depends on many factors, such as the concentration of the solute in the feed material, solubility in the solvent at supercritical conditions, type of matrix, and distribution of the solute in the initial material. As a general trend solvent feed ratios lower than 30 are used in industrial processes because higher solvent feed ratios imply higher operating costs and lower production capacity. A variable to optimize in the SFE is the flow rate because high solvent flow rates imply high operating cost. A high residence time (low solvent flow rates) implies a long batch time, while a short residence time may result in shorter contact time between the solvent and solute. In view of the above, the design and optimization of the SFE process will depend on an optimal selection of the most influential variables affecting the supercritical fluid extraction, for instance:

Pressure and temperature. The solubility of the target compounds is determined by both pressure and temperature and their optimal values should be evaluated case by case. At constant temperature, an increase in pressure results in increased solvent density and solvating power, however co-extraction of other compounds may occur, resulting in decreased selectivity of the process. At constant pressure, the solubility is controlled by two competing effects: increasing the temperature reduces the density of the supercritical fluid and thus its solvating power; but also, an increase in temperature increases the vapor pressure of the solute raising its solubility in the supercritical fluid. The effect of the density of the supercritical fluid over the solubility

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predominates at low pressures, while the effect of the vapor pressure is higher at high pressures. Solvent flow rate. Increasing flow rate may enhance extraction rates when the extraction is controlled by an external mass transfer resistance. This effect is more pronounced during the initial process stages when the easily accessible solutes are extracted. However, high solvent flow rates may result in an insufficient contact time between solute and solvent and increase operational and capital costs.

Modifier. The addition of a modifier may improve the extraction rate of the process by changing the polarity, density and viscosity of the supercritical fluid, and causing swelling of the matrix, allowing solute-fluid interactions and structural changes of the solid matrix (Díaz-Reinoso et al., 2006). The amount of modifier must be optimized case by case in order to maximize the extraction rate of the compounds of interest, but high modifier concentrations may result in decreased selectivity because the coextraction of other components (Moure et al., 2010).

Alcohols are the most frequently used modifier in SC-CO2 extraction, among them ethanol has been widely used in the extraction of bioactive compound due to its low toxicity (Pereira and Meireles, 2010). Water has been used as modifier in several cases such as in the extraction of caffeine or nicotine. It has the advantage of being a clean and non-toxic cosolvent but some problems may arise during the extraction including the formation of ice blockages during expansion or foam generation (Moure et al., 2010; Pereira and Meireles, 2010).

Table 1.2.Compounds extracted by SC-CO2 from vegetal matrixes

Plant Target compounds Extraction conditions Reference Agrimonia Phenolic compounds 25 / - / - / EtOH 1% / - Venskutonis et eupatoria al., 2008 Agrimonia procera Apium graveolens Phenolic compounds 20 / 45 / - / - / 1.1 kg/h Papamichail et seeds al., 2000 Arrabidaea chica Phenolic compounds 30 / 40 / St: 0.5 h / - / Paula et al., 2013 leaves 2.75·10-5 kg/s Arrabidaea chica Phenolic compounds 30-40 / 40-50 / - / - / 1.65 Paula et al., 2014 leaves g/min Artemisia Phenolic compounds, 18 / 40 / - / -/ 1.08 kg/h Martín et al., absinthium terpenes 2011 Camellia sinensis Phenolic compounds, 30 / 54 / - / EtOH 87.5% Bahar et al., 2013 Alkaloids (0.7 mL/min)/ - Cuscuta reflexa Phenolic compounds 25 / 55 / 2.5 h / 0.09 % Mitra et al., 2011 - 22 -

Acetone Echinacea Phenolic compounds 30 / 80 / - / 13% Co-S / - Yildiz et al., 2014 purpurea Echinacea No polyphenols, 30 / 45 / - / 12% EtOH/ - Catchpole et al., purpurea aerial Alkamides 2002 portions

Eucalyptus cinerea Phenolic compounds 9 / 40 / - / - / 20 g/min Herzi et al., 2013 and E. Fragances camaldulensis Eucalyptus Triterpenic acids 12–20 / 40-60 / - / 0-5% Domingues et al., globulus bark EtOH / 6-14 g /min 2012 Eucalyptus grandis Aromatic compounds, 20/ 60 / 10 h/ - / 6 g Patinha et al., x globulus bark sterols, triterpenoids CO2/min 2013 Garcinia Xanthones 30 / 50 / - / 0-3% EtOH / - Zarena & Sankar, mangostana 2009. Gracilaria Carotenoids, Phenolic 10-30 / 40-60 /-/ 2-8% Ospina et al., mammillaris compounds EtOH/- 2017 Hylocereus Triterpenoids, 30 / 40 / 1h / - / 30 L/h Luo et al., 2014 polyrhizus steroids Hylocereus undatus Hymenaea Phenolic compounds, 35 / 50 / St:10 min, Dt: Veggi et al., 2014 courbaril bark Terpenoids 40min / - / 1.11 kg CO2/s Hypericum Phenolic compounds 45 / 40 / - / - / 7 kg CO2/h Cossuta et al., perforatum 2012 Hypericum Phenolic compounds 30 / 40 / - / 10% EtOH/ - Catchpole et al., perforatum 2002 flowers Hypnea charoides Fatty acids 24-38/ 40-50 / 2 h / 1 Cheung, 1999 mL/min Ishige okamurae Fatty acids 40/ 40 /2 h / -/ - Ko et al., 2016 Laminaria japonica Carotenoids, fatty 30-40 / 40/ - / -/ - Lee and Chun, acid, phenolics 2013 compounds Lessonia vadosa Fucosterol 18 / 50/ -/20-30% cell/-/- Becerra et al., 2015 Levisticum Phenolic compounds 10-45 / 60 / 1.5 h (roots), Kemzūraitė et al., officinale Koch. 0.5 h (leaves) / - / - 2014 roots and leaves Mangifera indica Alkaloids, phenolic 10-40 / 40-50 / - / - / - Prado et al., 2013 leaves compounds, terpenoids Mangifera indica Phenolic compounds 10-40 / 35-55 / - / 0-20% / Fernández-Ponce leaves - et al., 2012 Mentha spicata Phenolic compounds 10-30 / 40-60 / - / 3-9 Bimakr et al.,

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leaves g/min / - 2012 Mikania laevigata Phenolic compounds > 17.5 / > 45 / - / - / - Oliveira et al., Mikania 2013 glomerata Myrtus communis Phenolic compounds 23 / 45 / - / 30% EtOH / Pereira et al., leaves 0.3 kg/h 2013

Origanum Phenolic compounds, 9 / 40/ - / - / - Mechergui et al., glandulosum Terpenes 2013 flowers, leaves Origanum vulgare Phenolic compounds 25 / 40 / - / 4% EtOH / - Cavero et al., 2006 Origanum vulgare Phenolic compounds, 30 / 40 / -/ - / 2.4 kg/h Fornari et al., leaves Terpenoids 2012 Panax Gingsenosides 15/ 50 / -/ TFA in MeOH Samimi et al., quinquefolius (0.05% v/v) /- 2014 Physalis peruviana Phenolic compounds 40 / 60 / - / - / - Wu et al., 2006 Piper regnellii Phenolic compounds 10.9-25 / 40-60 / - / - / Lemos et al., 3x10-6 m3/min 2012 Pipper Lactones 30 / 40 / - / - / - Catchpole et al., methysticum root 2002 and stems Prunus persica Phenolic compounds 15 / 60 / - / Co-S 6% / - Kazan et al., 2014 leaves Pteris semipinnata Terpenoids 30 / 55 / 4h / 10% EtOH / Lu et al., 2012 160 kg/h Quercus infectoria Fatty acids, sterols 30 / 40 / - / - / 2-4 mL/min Salleh et al., 2014 Quercus infectoria, Phenolic compounds 26.84 / 49.6 / 2 h / - / 2 Hasmida et al., mL/min 2014 Ridolfia segetum Terpenes 9 / 50 / - / -/ 1 kg/h Marongiu et al., Moris 2007 Rosmarinus Phenolic compounds 15/ - / 3h / CoS 5% / - Vicente et al., officinalis 2013 Rosmarinus Terpenoids 30 / 40 / - / - / - Pereira et al., officinalis 2007 Rosmarinus 30 / 40 / - / - / - García-Risco et officinalis al., 2011 Rosmarinus 30 / 40 / - / - / - Carvalho et al., officinalis 2005 Rosmarinus 22 / 40 / - / - / 7 g/min Zermane et al., officinalis 2010 Rosmarinus Terpenes 30 / 40 / - / - / 2.4 kg/h Fornari et al., officinalis leaves 2012 S. japonica Fatty acids 25 / 45/ 2 h / 1ml/min Sivagnanam et EtOH/ 27 g /min al., 2015

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Saccharina Carotenoids, fatty 45°C, 250 bar, 27 g Sivagnanam et japonica acids, phenolic CO2/min, 2 h al., 2016 compounds Saccharina Carotenoids, fatty 30 /49-51/ 4 h / 2% SO-W Saravana et al., japonica acids / 27 g CO2/min 2017 Salvia officinalis Phenolic compounds 12.8 / 50 / - / - / - Aleksovski & Sovova´, 2007 Salvia officinalis Phenolic compounds 35 / 100 / - / 1% EtOH / - Dauksas et al., 2001 Salvia officinalis Terpenoids 12.8 / 50 / - / - / - Aleksovski & Sovova´, 2007 Salvia officinalis Terpenoids 30 / 40 / - / - / 2.4 kg/h Fornari et al., leaves 2012 Sargassum horneri Carotenoids, fatty 25/ 45/2h/-/ 27 g CO2/min Sivagnanam et acids, phenolic al., 2016 compounds Sargassum Carotenoids, fatty 10 - 30 /30-50/ 1h/5-10% Conde et al., − muticum acids, Phenolic EtOH/ 25 g /min 2014 compounds Sargassum Carotenoids 10-35 / 45/1 h/-/ 25 g Balboa et al., muticum CO2/min 2015 Sargassum Lipids 29/45/-/-/10 kg/h Terme et al., muticum 2017 Satureja montana Phenolic compounds Silva et al., 2009 Scutellaria barbata Phenolic compounds 30 / 75 / 1.6 h / 15.6 % W/ Yang et al., 2013 2.2 mL/min Serenoa repens Fatty acids, sterols 28 / 40 / - / -/ 10 kg Catchpole et al., berries CO2/kg herb 2002 Solieria chordalis Lipids 29/45/-/-/10 kg/h Terme et al., 29/45/-/2-8% EtOH/ 2017 10kg/h Syzygium Phenolic compounds 25 / 60 / 1 h / - / 2 L/min Chatterjee & aromaticum Bhattacharjee, 2013 Syzygium Phenolic compounds 10 / 40 / - / - / - Ivanovic et al., aromaticum / 2013 Origanum vulgare Syzygium Phenolic compounds 10 / 40 / - / - / - Ivanovic et al., aromaticum / 2011 Origanum vulgare and Syzygium aromaticum / Origanum vulgare /Thymus vulgare mixtures Thymus vulgare Phenolic compounds 30 / 40 / - / - / 2.4 kg/h Fornari et al.,

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leaves Terpenoids 2012 Typhonium Fatty acids, sterols, 30 / 45 / - / - / 15 kg/h Li et al., 2011 giganteum Undaria Carotenoids, phenolic 8-30/30-60/-/ 3.0%EtOH Roh et al., 2008 pinnatifida compounds Undaria Carotenoids 20-40/25-60/ 3 h/-/ 1.0- Quitain et al., pinnatifida 4.0 mL /min 2013a and 2013 b (pretreated with microwaves) Urtica dioica Phenolic compounds, 28 / 40 / - / 9.4 % EtOH / - Sajfrtova´ et al., sterols 2005 Ziginber officinale Phenolic compounds 30 / 30 / - / - / 1,42·10-4 Justo et al., 2008 kg/s

1.3.4. Recovery, concentration and purification of phenolic compounds 1.3.4.1. Adsorption-desorption processes. Adsorption-desorption is an energy-efficient technology for separation and concentration of thermolabile compounds. These separation techniques are relatively simple and flexible, and represent promising alternatives to other more energy- consuming processes. On the other hand, the increasing comsumer demand for natural additives and ingredients and their low concentration in the extract obtained from several technologies involve the need of purification steps to produce enriched products with these biactive compounds. Adsorption is driven by attractive forces between the adsorbate (solute present in the solution) and the adsorbent (solid phase) and can involve physical adsorption (physisorption) or chemical adsorption (chemisorption), depending on the nature of the bonding between adsorbate and adsorbent. Thus, the interactions between the adsorbent and the molecules in the fluid phase, can be of different nature (van der Waals, acid-base interaction, electrical interaction, hydrogen bond, π-bond, covalent bond), but when the objective is to recover the solute, reversible adsorption is desirable. The adsorption-desorption principles are well established, and useful information can be found in texts dealing with theoretical and practical principles. The adsorption process is composed of a series of mechanisms and the knowledge of these mechanisms isimportant for the optimal design of and adsorption-desorption process.

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In general, sorption should be described by a series of resistances due to external mass transfer, intraparticle diffusion and reaction being described in the following steps: 1. Mass transfer from the fluid phase to the external surfaces of the sorbent particles (film diffusion resistance). 2. Pore diffusion in the fluid phase (pore diffusion resistance) 3. Adsorption reaction at the phase boundaries (surface reaction resistance) 4. Diffusion in the solid (particle diffusion resistance or solid diffusion resistance), which can occur in a pore-surface layer. The overall rate of the process can be controlled by one or morel of these steps. It is dertermined by the physico-chemical characteristics of the solute and adsorbent, particle size and affinities, as well as the conditions. In general terms, adsorbent particle size, adsorbate concentration, degree of mixing, affinity between adsorbate and adsorbent, and diffusion coefficients of the adsorbate (in the bulk solution and within the adsorbent) are important factors to be considered. In poorly mixed systems, the transport in the bulk phase may be of major importance, particularly in problems involving dilute adsorbates, small adsorbent particle sizes and high affinity adsorbent-adsorbate. In systems with good mixing, large adsorbent particle sizes, high concentration of adsorbate, and low affinity adsorbent- adsorbate, adsorption is controlled by intra-particle mass transfer (Soto et al., 2011). Besides the above mentioned factors, the usual complex composition of the systems employed in the adsorption processes difficults extrapolation between different systems. This behavior indicates the need for individual study of each case since the solute properties strongly influences over the affinity towards the adsorbent, and interactions between compounds of the system may alter adsorption affinities. The careful adjustment of the conditions requires that the influence of pH and temperature should be established for each adsorbent and solute solution to optimize rates, adsorption capacities and attainable desorption yields. In order to prevent oxidation and hydrolysis during the recovery procedure, the working temperature and the contact time have to be optimized (Fava et al., 2017). Selection of resins among those available is frequently performed in batch assays and further studies in column

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allow to define the breakthrough curves and the selection of the suited eluting agents and conditions (Soto et al., 2017) and further process scale up (Kammerer et al., 2010; Savarese et al., 2016). A number of materials can be suited for the selective adsorption of phenolic compounds. Zeolites and activated carbon are commonly used for removal of phenols from wastewaters and inhibitors from lignocellulosic biomass hydrolyzates and fermentation broth. The irreversible nature of the process and the need of regeneration at high temperature or with chemicals limit the recovery of thermosensitive compounds. Resins are the most studied adsorbents, based on their low cost, limited toxicity, chemical stability, high adsorption capacity, selectivity and ease of regeneration at moderated temperatures. Ion-exchange resins, proposed for the adsorption of pure phenolics and natural extracts. This technology has a big potential for the selective recovery, concentration and fractionation of phenolic compounds from aqueous extracts and from wastewaters as an only process or in combination with other green technologies. Adsorptios has been evaluated in addition to i) conventional extraction with water, or organic solvents (Zeng and Wang, 2016), ii) enzymatic aided extraction Dong et al., 2015; Xu et al., 2016), iii) pressurized hot water extraction (Casas et al., 2016; Conde et al., 2017), iv) microwave assisted extraction and v) ultrasound assisted extraction (Yi et al., 2015; Xu et al., 2016b; Zhou et al., 2016). Some of these applications are summarized in Table 1.3.

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Table 1.3.Examples of phenolic extracts purified by adsorption-desorption

Source of Adsorbent brand Operational conditions Eluent Reference extract or liquid stream

Artichoke Lewatit RSL 1:16.7; 2 mL/min E (70 %); 1 mL/min Condi et al., wastewaters 2015

Barley husks Sepabeads RSL3: 5; 175 rpm; 25 °C; E (>80 %) Conde et al., 20 min 2017

Barbat DM130; D101; RSL 1:10; 100 rpm; 45 E (95 %) Huang et al., scullcap herba HPD-5000; HPD- °C; 12 h 2017 E (10-95 %); 2 BV/h 100; AB-8; SP700 C (DM130, D101): 15 mL resin; 2 BV/h

Blackberries Polyamide resins 5: 50 ( mL R:mL E) E25 E (0.1 %v/v HCl, pH 3) Wen et al., °C; 2016 E (80% v/v); 2-10 C: 40 mL resin; 2-10 BV/h BV/h

Black AB-8 2.2 BV; pH2 E (80 %); 2.0 BV/h Zeng and chokeberry Wang, 2016

Black tea Amberlite: 25 mL E; 100 rpm; 240 E, 25 mL Monsanto et min; 20 °C al., 2015 Diaion

Blueberry D101 - E (40 %, 0.5 % CA); Xu et al., 1.5 mL/min 2016

Blueberry Amberlite 5-15 cm; 4 mL/min E (20-95 %, 1 % FA) Sandhu et al., 2017

Chinese AB-8; D101; X-5; RSL 0.5: 40; pH 5-9 ;150 E (15-100%); 50 mL; Wu et al., boxthorn NKA; ADS-7 rpm; 25-45 °C, 24 h 100 rpm; 24 h; 25 °C 2015 C (D101): 3 g A, 3 BV/h E (60 %); 3 BV/h

Chinese herb AB-8; NKA-9; RSL 0.3:20; 100 rpm; Wen et al., D3520; ADS-5; 30-50 °C; 150 min 2016 E (95 %); 0.5 mL/min ADS-17; D101; C (HPD-100): 1.5 g A; HPD-100; HPD-722 0.5 mL/min

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Chinese herb HPD-200A; HPD- RSL 2: 50; 120 rpm; 25- E (70 %); 50 mL Li et al., 2017 722; HPD-300; 45 °C; 12 h E (30-40 %); 2 BV/h HPD-750; HPD- C (HPD-300): 8 g A; 2 5000; HPD-826 BV/h

Chinese AB-8; D101; HPD- RSL 1: 50; 100 rpm; 8 h, E; 50 mL; 25 °C; 8 h Yue et al., magnoliavine series 0-40 °C 2016 E (90 % v/v); 1 BV/h C: 5 g A; 4 h; 3 BV/h

Eucalypt bark Amberlite; RSL 2:100; 200 rpm; 25 E (95 %); 100 mL; 200 Pinto et al., Sepabeads °C; 60 h rpm; 25 °C 2017

C (SP700): 25 °C; 5 E (95 %); 5 mL/min; mL/min; 2.5 h 2.5 h

Green tea Ionic liquid- -; 100 rpm; 25-45 °C; EA; E; M; M (2 % HCl); Zhang et al., modified silica gel pH 4-7; 3 h 150 rpm 2017

Japanese Sepabeads; RSL 3:5; 175 rpm; 25 °C, E (68%); 2.25 mL/min; Casas et al., wireweed PuroSorb pH 5; 30 min 25 °C 2016

Lignocellulosic PVPP; Amberlite; ; -; room T; -; 30 min; M (96%) Da Costa biomass Silica C18 López et al., 2016

Liquorice Amberlite: B: 5 g A; 100 mL E; 23- E (0-100 %); 25-55 °C; Dong et al., leaves Diaoin; 55 °C; 150 rpm 150 rpm; 120 min 2015 Sepabeads: C: 20 mg/mL feed; 4 °C W (10%)+E (30-100 %)

Mango peels Amberlite; B: -;350 rpm; 20-100 M (1 M HCl)+ NaCl Geerkens et Lewait: °C; -; 3 h (10 %) + M al., 2015

C (Lewait S6368 A): 5 NaOH (0.03 M) + mL A; 50 mL E NaOH (0.25 M) + M

Moldavian HPD-600 1:9 D/L; 0.32 g E/mL A; E (70 %); 4BV; 1.5 Yu et al., balm 1.5 BV/h; 12 h BV/h 2016 + W, 4 BV; 1.5 BV/h

Olive mill Wheat bran RSL: 1-6:100; W Achak et al., wastewaters 200rpm;30°C; pH 3-11; 2014 24 h

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Olive mill Amberlite 50 mL, 3 h W, E, Ac Zagklis et al., wastewaters 2015

Olive mill Amberlite 24-30 °C; 0.5-1.8 m; E (0.5 % v/v HCl 0.1 Pinelli et al., wastewaters 1.3-3.1 m/h N) 2016

Olive mill Amberlite: RSL:0.01: 10, 150 rpm; DW, DW (HCl pH 3); Kaleh et al., wastewater Lewatit; 21°C; -; - 20-60 °C; 150 rpm 2016 MonoPlus

Olive mill Macronet RSL 1:3 (L:L); 3 BV/h E ; 75 L; 1.5 BV/h Savarese et wastewaters al., 2016

Olive mill Amberlyst; 35 g A; pH 3-11; 10 L/h NaOH (2, 4 %) Victor-ortega wastewater Amberlite et al, 2016

Olive mill Amberlite 21, 30 °C; 0.40, 1.44 E (0.5 % v/v HCl 0.1 Frascari et al., wastewater m/h N) 2016

Olive waste Amberlite; RSL 1:5;120rpm; 25-40 E (40-80 %) Wang et al., Polyamide °C; -; 24 h 2017

Olive water Amberlite RSL 1:15; 150 rpm; 20 M; 20 mL Casado et al., °C;-; 2h 2014

Onion Amberlite RSL 1:100; 200 rpm;25- - Kühn et al., residues 65 °C; pH 2-6; 24 h 2014

Persimmon D101 - M (90 %) Zhou et al., 2016

Pine cones AB-8 RSL 1:10; 100 rpm; -; -; E (60 %); 5 g; 50 mL; Yi et al., 2015 12 h 100 rpm; 12 h

Pomegranate D101; AB-8; HPD- RSL 1:10;-; 25°C; -; 8 h E (60 %); 8 h, 120 Jiang et al., 100; HPD-450; rpm; 25 °C 2016 HPD-600 C: 20 mL A, 1-6 BV/h; 2- 4 mg/mL E(0-100 % v/v); 1-6 BV/h Pricklypear Amberlite 30 mg polyphenols/g A; E (96 %) Matias et al., cactus juice 4 h 2014

Purple-fleshed Amberlite; RSL 1:4;-; -;-; 15 h E (50 %); 1 h Heinonen et Amberlys 15 (H+ potato C (XAD-7HP): 1.7-10.2 al., 2016 form); CS16GC E (25-75 %, 0-7 % Ac) (Na+ form) BV/h

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Quinguan X-5; Nylon-6 1 BV/h E (30 %- 70 %) 56 apple

Radix salvia AB-8 C: 2 mL/min; 0.4 g E (40 %); 10 BV Sun et al., E/mL; 3 BV; 3 h 2014

Red raspberry AB-8; NKA-9; S-8; RSL 1:71.5; 100 rpm; E-W, 50 mL; pH 2; Yang et al., D(101;102, 103; 25-35 °C; -;10 h; 100 rpm; 2 h 2015 3520; 4006) C (AB-8): 4 g A; 25 °C E (60 %); pH 2

Saskatoon AB-8 RSL 1:5;-; 25 °C;-; 12 h E (80 %, 0.1/ HCl) Yang et al., berry 2016

Tea LX-68,5B,2000; RSL 1:40-100; 120 rpm; E (80 %) Wang et al., XDA-1; LSA-10; 30-50 °C;-; 4 h 2014

ADS-5C; ADS-21; C (PA): 1.5 BV/h PA; HP-20; AB-8 E (10-80 %)

Tian Shan AB-8 C: 10 g A; 10 mL E; 1 E (50 %); 4 BV; 1.5 Gu et al., rowan leaves BV/h BV/h 2016

Walnut de- AB-8; NAK-9; B: RSL 1:20; 120 rpm; E (80 %); 100 mL Zhu et al., pellicle DPD-100; D101; 25-45 °C; pH 7-9; 6-24 h 2017 wastewaters X-5 C (AB-8): 5 g resin E (80 %); 2 BV/h

Yellow poplar Amberlite; RSL 1:20; 150 rpm; 30 E Um et al., hydrolysates Activated carbon °C; -; 2 h 2017

C: 2.5 g A; 1.6 mL/min

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2. OBJECTIVES AND WORK PLAN

2. OBJECTIVES 2. 1. Objectives The main objective of this Thesis is to apply different green techniques to the extraction of bioactive compounds from different types of biomass (terrestrial and marine) and to evaluate different techniques for their fractionation and purification. To reach this goal, the following secondary objectives have been defined:  Objective 1: Production of crude extracts with green techniques: enzyme assisted extraction, subcritical water extraction, microwave hydrogravity and

supercritical CO2 extraction.  Objective 2: Purification by solvent fractionation and adsorption-desorption onto resins.  Objective 3: Combination of different stages in multiproduct processes.  Objective 4: Characterization of in vitro antioxidant properties and some biological properties.

2.2. Work plan In order to reach the planned objectives a list of tasks were proposed:

 Tasks to achieve Objective 1 1. Development of hydrothermal (SWE) production of extracts from Sargassum muticum under optimized conditions and evaluation of the effects of temperature for the hydrothermal processing of Hericium erinaceus on the solubilization yield, the phenolic extraction and antioxidant properties. 2. Evaluation of the enzyme dose effects on the release of phenolic compounds from alginate-free S. muticum, frosted S. muticum and exhausted H. erinaceus. 3. Evaluation of irradiation power in the solubilization of phenolic compounds from H. erinaceus by microwave hydrogravity extraction. 4. Evaluation of the effect of pressure, temperature, co-solvent concentration on the yield and phenolic solubilization from Acacia dealbata and exhausted H. erinaceus.

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 Tasks to achieve Objective 2 1. Assessment of a liquid-liquid extraction protocol to recover and concentrate phenolic compounds from H. erinaceus solubilized by conventional solvent extraction. 2. Evaluation of the sorption process (adsorption-desorption) of phenolic compounds solubilized previously. - Interpretation of kinetic, isotherm data in a static process (S. muticum). - Assessment of the effect of operational conditions as feed rate, feed concentration, fixed bed length on the adsorption capacities, breakthrough time and saturation time in the dynamic process of phenolic recovery from hydrothermal extracts of S. muticum.

 Tasks to achieve Objective 3 1. Development of a novel combination extraction process for the bioactives production from Hericium erinaceus. 2. Consideration of several options to produce bioactive products from whole and alginate free S. muticum.

 Tasks to achieve Objective 4 1. Evaluation of the antioxidant capacity of crude extracts obtained from the several feedstocks by all the methodologies assessed. 2. Evaluation of antiradical, anti-proliferative and anti inflammatory properties of purified phenolic fractions obtained from the different feedstocks.

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3. MATERIALS AND METHODS

3. MATERIALS AND METHODS 3.1. Raw materials There is a wide variety of natural sources available for use, exploitation and valorization. For this work, the following were selected: - Sargassum muticum, collected in playa da Mourisca (Vigo, Pontevedra, Spain). - Acacia dealbata, collected in Ourense (Spain). - Hericium erinaceus, provided by Hifas da Terra (Pontevedra, Spain). Each feedstock had its own conditioning protocol.

 Sargassum muticum. Sargassum muticum (Sm), were collected in Praia da Mourisca (Pontevedra, Spain) in June 2012. Depending upon the aim of treatment applied to Sargassum samples, two conditioning protocols were stablished. i) For the hydrothermal treatment the algae material were cleaned from epiphytes and sand, washed with tap water, frozen and stored in closed plastic bags at -4 C until use. ii) For the valorization of the solid residue remaining after the extraction of alginate, the alginate exhausted algal biomass (AESm) was also studied. In this option S. muticum was defrosted, ground and contacted with 1 % formaldehyde. The solids were afterwards extracted with 0.2 N sulphuric acid, washed with water and extracted with 1% sodium carbonate to convert the alginate salts to the sodium salt, which were then precipitated by ethanol. The residual solid phase or alginate-exhausted algae (AESm) was oven dried at 50 C and stored in sealed plastic bags until use.  Acacia dealbata. Acacia flowers were collected in Tibiás (Ourense, Spain) in March 2014 and in Pereiro de Aguiar (Ourense, Spain) in February 2016. The flowers were manually separated from the branches and air-dried at room temperature. The dried flowers were milled to pass a 1 mm, homogenized to avoid differences in composition, and stored in sealed plastic bags at -4 °C until use.  Hericium erinaceus. Mushroom samples were cultivated by Hifas da Terra (Pontevedra, Spain) in 2014. Strains were cultured on potato dextrose agar medium (PDA: potato 200 mg/mL glucose 20 mg/mL and agar 20 mg/mL) and incubated at 25 °C for 7–10 days (Fen et al., 2014). These cultures were used to seed Populus alba autoclaved wood pellets previously humidified. Mycelium was

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allowed to grow on wood pellets for 7-10 days at 25 °C, and fruit bodies developed after 20 days. Fresh fruit bodies were cut into pieces of aprox. 2 cm x 2 cm before use and afterwards freeze-dried and stored at -20°C until use.

3.2. Experimental procedures This section describes experimental procedures performed to i) extract antioxidant compounds by green technologies, ii) for recover, fractionate and characterize the solubilized compounds, and iii) to develop purification processes by adsorption-desorption techhnologies onto polymeric resins.

3.2.1. Extraction metodologies The proposed raw materials were subjected to different green extraction

technologies such as, supercritical CO2 extraction (SC-CO2), subcritical water extraction (SWE) also called in this thesis as autohydrolysis treatment, enzymatic-assisted extraction (EAE) and conventional processes as solvent and liquid-liquid extraction technologies.

3.2.1.1. Hydrothermal treatments: autohydrolysis Samples of Sargasum and mushrooms were treated with water in a batch reactor (Parr Instrument Company, Moline, Illinois, USA).

 Sargassum samples were treated under operational conditions optimized in a previous work (González-López et al., 2012). This operational conditions set consisted of a liquid:solid ratio (LSR) of 60: 1 (w: w), non-isothermal heating up to the temperature of 190 °C and continuous stirred at 160 rpm.  The ground wet mushroom or the exhausted solid after MHG were contacted with water (liquid:solid ratio 12:1, w:w) and heated in a 0.6 L Parr reactor (Parr Instr. Co., USA) under non isothermal operation, up to 200 °C. When the desired temperature was reached, the heating was stopped and cold water was circulated through the internal coil. By lowering the internal temperature to 45 °C, the reactor vessel was opened and emptied.

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The liquid and solid phases were separated by vacuum filtration and the liquid phase or the hydrolizate was separated from the solid phase by vacuum filtration as quickly as possible to avoid possible oxidation of phenolic compounds with antioxidant capacity present in the hydrolyzate, stored at a temperature of -18 °C until further analysis.

3.2.1.2. Enzyme-assisted extraction The study of enzyme-assisted extraction of Sargassum muticum (Sm) and mushrooms was carried out in this study. In the seaweed case, the process was performed in duplicate or triplicate independent assays in Erlenmeyer flasks, placed in an orbital shaker (Innova 4000, New Brunswich). The enzymes used including the proteases Alcalase and Protamex, the β-glucanases Celluclast and Viscozyme and the amylolytic formulation Amylase. The enzymes listed in Table 3.1 were added and mixed to wet Sm and samples and 1 g dry AESm samples with 0.5-15% enzyme/sample weight ratio. This mixture was at a liquid to solid ratio 50 (v/w), at pH fixed according to the manufacturer recommendations (Table 3.1) and commitment temperature 50 °C. In the mushroom’s case, the study was carried out in triplicate independent assays in Erlenmeyer flasks placed on an orbital shaker (INNOVA 4000, New Brunswick) at 50 °C for 3 h. In this study, the fresh biomass or the MHG residue were contacted with a commercial enzyme mixture Viscozyme: Celluclast: Flavourzyme (2:1.5:1.5) (Novozymes) at an enzyme to substrate ratio of 5% (weight) in sodium acetate-acetic acid buffer at pH 4.5 in a liquid to solid ratio of 25 (v:w). To inactivate the enzymes the suspension was heated to 100 ° C for 10 minutes. After the inactivation, the suspensions were centrifuged (3500 rpm, 10 min by Sargassum and 4500 rpm, 8 min for mushrooms) to remove the solid residue, the supernatant was filtered through 0.45 μm membranes and the filtrate was stored at - 20 ° C in the darkness until analysis.

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3.2.1.3. Solvent fractionation The dried solid or dry extract was extracted successively by Soxhlet extraction with solvents of increasing polarity. The crude extract was placed in a cellulose extraction thimble, and filled with fresh solvent from a distillation flask. The solute or solvent extract is left in the flask and the fresh solvent is passed back through the raw extract bed. The operation is repeated until extraction is complete (9 h). The partially extracted solid crude extract remaining in the thimble support was successively withdrawn with a solvent of greater polarity. The corresponding extract was obtained by vacuum evaporation of the solvent under consideration. The sample was then placed in a desiccator under vacuum and oven dried at 50 °C for 48 h. The extracts were analyzed for extraction yield, total phenols and antioxidant activity.

3.2.1.4. Supercritical fluid extraction The equipment used was a SFE-1000F-2-C10 (Thar Designs, Pittsburgh, USA), consisting of a 1 L extractor and two 500 mL separators. The pilot plant was also equipped with a refrigerated circulating baths (PolyScience, USA, model 9506) and a P-

200A piston pump (Thar Design Inc., Pittsburgh, PA, USA) to pump the CO2 and a HPLC pump (Scientific Systems, Inc., USA, model Series III) to pump the cosolvent. The extraction vessel was covered with polypropylene wool to avoid solid particles from entering the extraction line. The extract was collected in the first separator at room temperature and further recovered with 96 % ethanol. Ethanol was removed in rotavapor and the extracts were kept under N2, at -20 °C in the dark until analysis. Acacia dealbata dried flowers were placed with glass beads in the extractor vessel. The CO2 was precooled and pumped at a flow rate of 25 g CO2/min. The extract was collected in the first separator, operating at room temperature. Pressure and temperature effects on the extraction yield, phenolic compounds yield and antiradical activies were studied in the range 10-35 MPa and 35-55 °C, respectively. Freeze-dried Hericium erinaceus and MHG processed fungus were packed in 10 g bags with glass beads. The CO2 flow rate was 25 g CO2/min and the modifier was added in the concentration range 0.5-15% (wt basis). The extraction was carried out at

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a fixed pressure (20 MPa) and temperature (40 C) based on literature (Kitzberger et al., 2007; Mazzutti et al., 2012).

3.2.1.5. Microwave hydrogravity extraction (MHG) During the night, the fresh mushroom was immersed in distilled water and kept in a refrigerator at 4 C. After filtration with filter paper to separate the solid fraction from the liquid, were then processed by microwave hydrogravity extraction (MHG). The MHG procedure for the extraction of flowers was carried out in an open vessel NEOS-GR (Milestone Srl, Italy) microwave extractor with a 1.5 L Pyrex extraction vessel. During experiments samples were subjected to several irradiation powers (79- 900 W) and fractions of 5 mL were collected, cooled down and analyzed. The drained liquid phasewas analyzed for total solubles, total phenolics, and in vitro antioxidant properties. The temperature was measured by using an optic fiber temperature sensor inserted in the microwave cavity.

3.2.2. Recovery, fractionation and purification of extracts Diffent extracts/hydrolizates obtained by some of the processes proposed for Sargassum muticum and Hericium erinaceus were proposed to be enriched by means of adsorption–desorption processes in their phenolic fraction. In a adsoption–desorption process the choice of the adsorbent material is one of the most important factors, so 7 different resins, 6 provided by Purolite® (PAD428, PAD500, PAD700, PAD900, PAD910 and PAD950, belonging to its PurosorbTM line) and 1 by Mitsubishi Chemical (SP700, from DiaionTM, SepabeadsTM). Each of these resins has particular characteristics, which give rise to a different behavior with the same organic compound. Table 3.1 lists the main physico-chemical characteristics of the different resins used during the study.

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Table 3.1.Properties of the polymeric resins used in this study

Pore Area Porosity Partícle Density Resin Structure radius (m2/g) (mL/g) (Å) Size (µm) (g/mL)

Sepabeads SP700 Styrene-DVB 1200 90 2.2 >250 690

Brominated PuroSorb PAD428 720 400 0.7 350-1200 - polystyrene

PuroSorb PAD500 Polystyrene 800 500 1.0 300-500 -

PuroSorb PAD700 Polystyrene 550 700 1.2 350-1200 -

PuroSorb PAD900 Polystyrene 800 1000 1.5 350-1200 -

PuroSorb PAD910 Polystyrene 540 1100 1.6 350-1200 -

PuroSorb PAD950 Acrylic 535 960 1.3 350-1200 -

3.2.2.1. Static adsorption and desorption studies The operational mode for Sargassum muticum autohydrolysis liquors was the following: Dry 0.2 g dry resin was contacted with 5 mL of aqueous solutions of crude extract with a known concentration. The flasks were introduced into the stirring apparatus for a set period of 20 minutes, at 175 rpm and 25 °C as stipulated ambient temperature. The pre-desorption wash was carried out by shaking the resin used in the adsorption in a flask with water in a ratio of 1:3 (1 g of wet resin: 3 g of water). The stirring interval was 20 min, at room temperature and 175 rpm. A series of desorption stages have been carried out at different times, ranging from 5 min to 24 h. This process was carried out only with those resins that demonstrated better adsorbent capacity with the liquor of Sargassum muticum. The operational mode for H. erinaceus was the following: Only Sepabeads SP700 was used to recover and concentrate the phenolic fraction from the crude extract of H. erinaceus. The suspensions at a resin:liquid ratio

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1:25 (w:v) were placed in Erlenmeyer flasks at 175 rpm in an orbital shaker at 25 °C for 60 min. After solid liquid separation, the resin was washed (1:3 moist resin: distilled water ratio (w/v)) in an orbital shaker at 175 rpm and 25 °C during 20 min. After solid liquid separation, desorption was carried out contacting the saturated resins at a moist resin: 96% ethanol ratio 1:3 (w:v) at 175 rpm and 25 °C during 60 min.

3.2.2.2. Determination of adsorption‐desorption properties

Calculation of adsorption capacity and adsorption ratio

The adsorption capacity, qt (mg/g resin), quantity of solutes adsorbed at time t onto a mass unit of dry resin, was calculated according to equation:

[3.1]

C0 initial concentration of solutes in liquid phase (mg/mL) Ct concentration of solutes in liquid phase (mg/mL) at t time V volume of feed solution (mL) W weight of the dry resin (g)

The adsorption ratio, E, percentage of total solute being absorbed at adsorption equili brium, was calculated as indicated in equation:

[3.2]

C0 initial concentration of solutes in liquid phase (mg/mL) Ce equilibrium concentration of solutes in liquid phase (mg/mL)

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Adsorption kinetics

Adsorption kinetic is important from the point of view to control the process efficiency Various kinetic models have been used. The pseudo‐first order rate equation of Lagergren is generally described in equation:

[3.3] qe amount of solutes adsorbed (mg/g) at equilibrium qt amount of solutes adsorbed (mg/g) at contact time t (min) k1 pseudo‐first‐order rate constant (min‐1) t contact time (min)

The pseudo‐second order kinetic model may be represented by:

[3.4] qt amount of solutes adsorbed (mg/g) at contact time t (min) k2 second‐order rate constant (g/mg min) qe amount of solutes adsorbed (mg/g) at equilibrium t contact time (min)

Elovich equation can be expressed by equation:

[3.5]

qt amount of solutes adsorbed (mg/g) at contact time t (min) α initial adsorption rate (mg/g min)

- 42 -

β Elovich rate constant (g/mg) t contact time (min) The intra‐particle diffusion model, based i¡on the proposed by Weber and Morris (1962) is characterized by the relationship between specific adsorption and the square root of time, according to the following equation:

[3.6] qt amount of solutes adsorbed (mg/g) at contact time t (min) ki intra‐particle diffusion rate constant (mg/ g min0.5) I intercept (mg/g)

Adsorption isotherms

Adsorption isotherms can be studied in batch experiments and are then used to predict the behaviour of the adsorbents in dynamic systems like the fixed bed chromatographic process. Adsorption isotherms were carried out by mixing a known amount of resin with a fixed volumen of simple solution at vaious initial concentration in an orbital shaker kept at constant temperatura (25 °C). Two standard theoretical models were us used to describe the adsorption behaviour between adsorbate and adsorbent. Langmuir isotherm (Langmuir, 1918) assumes a monolayer adsorption, a homo geneous distribution of the sorption energy and no mutual interaction between adsorb ed molecules. The model of Langmuir,can be expressed by the following mathematical formula:

[3.7] qe amount of solute adsorbed per unit weight of adsorbent (mg/g) at equilibrium qm maximum adsorption capacity (mg/g) KL constant related to the free energy of adsorption (L/mg) Ce equilibrium concentration of solutes in liquid phase (mg/mL) - 43 -

The essential characteristics of Langmuir isotherm can be expressed by a dimensionles s constant called separation factor (or equilibrium parameter), RL. The parameter RL in dicates the characteristics of isotherm as follows:

[3.8] RL>1 unfavourable RL=1 linear 0

Freundlich isotherm (Freundlich, 1909) is an empirical equation, for non-ideal adsoption systems. This model describes the equilibrium conditions of adsorption for a heterogeneous surface and does not imply the formation of a monolayer. It is expressed by the formula below:

[3.9] qe amount of solute adsorbed per unit weight of adsorbent (mg/g) at equilibrium KF distribution coefficient (mg/g)(mg/L)n Ce equilibrium concentration of solutes in liquid phase (mg/mL) 1/n measurement of adsorption intensity

Calculation of desorption ratio The desorption ratio, D, percentage of total desorbed substrate with respect to the mass present in the loaded resin), was calculated as indicated in equation:

[3.10]

Cd concentration of the solutes of the desorption effluent (mg/mL) Vd volume of the desorption solution (mL) C0 initial concentration of solutes in liquid phase (mg/mL)

- 44 -

Ce equilibrium concentration of solutes in liquid phase (mg/mL) Vi volume of the feed solution (mL)

3.2.2.3. Dynamic adsorption and desorption studies The resin was satured with the florotannin fraction contained inthe autohidrolysis extract from the ground algae. Dinamic elution of the florotannin fraction was addressed at room temperature, using 68% ethanol at a flow rate 2.25 mL/min. The eluted samples were colleted and analyzed.

3.3. Analytical methods The methodologies used during the experimental procedures are presented in this section, including the analytical techniques and protocols performed.

3.3.1. Moisture content and total solids determination The moisture content was determined by placing in a stove at 105 °C to constant weight about 2 g of sample in a container of known weight previously dried. Subsequently it is poured into a desiccator before being weighed.

[3.11] Mb mass of the sample before drying

Ma mass of the sample after drying

[3.12] Ma mass of the sample after drying

Vi volumen of the process liquid

- 45 -

3.3.2. Extraction yield determination The extraction efficiency is a measure that indicates the effectiveness of the technique used to extract specific components from the source material. It can be calculated on the basis of the unhydrolyzed solid or the liquid phase obtained after the extraction. The corresponding extract was obtained by evaporation of the considered solve nt under reduced pressure (200 mm Hg) in a vacuum evaporator (60 °C). Then the sam ple was placed in a desiccator under vacuum and was oven dried at 50 °C during 48 h. The extraction yield was determined gravimetrically and calculated as the percentage of the weight of the extract respect to the raw material:

[3.13] Me weight of the extracts M rm weight of the raw material

The use of low temperatures for drying reduces the problems associated with the degradation of thermolabile substances or chemical changes of some components.

3.3.3. Total phenols determination The total phenol concentration of the extract and the fractions was determined with the Folin-Ciocalteu reagent by the method described by Singleton and Rossi (1965). The Folin-Ciocalteu reagent is mixed with water in a 1:1 ratio, a 10% solution of

Na2CO3, 0.5 mL of sample, 3.75 mL of H2O, 0.25 mL of Folin reagent (prepared above) and 0.5 mL of Na2CO3 are placed in a test tube. The whole is homogenised and left at room temperature for 1 h. The absorbance at 765 nm is then read against a blank which was prepared in the same manner but substituting the solvent for the sample with the phenolic solution. If necessary, some samples are diluted to fall within the ranges established by the standard straight line. The standard line was made with various dilutions of a gallic acid solution (Sigma-Aldrich Chemical S.A.) in distilled water of 0.01 to 0.1 g/L. The results

- 46 -

were expressed as g GAE/L, which are the equivalent grams of gallic acid per liter of sample.

0.9

0.8 y = 8.175x + 0.0018 0.7 R² = 0.9997

0.6

0.5

0.4

0.3 Absorbance (nm) Absorbance

0.2

0.1

0 0 0.02 0.04 0.06 0.08 0.1 0.12 Gallic acid concentration (mM)

Figure 3.1 Shows the calibration line for the Folin-Ciocalteu method, using gallic acid as the standard

3.3.4. Phloroglucinol equivalents The concentration of total phenols of the extract and of the fractions was determined with the Folin-Ciocalteu reagent by the method described by Rodríguez- Bernaldo de Quirós et al. (2010). The Folin-Ciocalteu reagent is mixed with water in a 1:1 (v/v) ratio, a 6% solution of Na2CO3 is prepared. 0.1 mL of sample, 2 mL of Na2CO3 and 0.1 mL of the Folin reagent (prepared above) are added to a test tube. The whole is homogenized and left at room temperature for 30 minutes in the dark. The absorbance at 720 nm is then read against a blank which was prepared in the same manner but substituting the solvent for the sample with the phenolic solution. If necessary, some samples are diluted so that they can be read at the intervals established by the standard straight line. The standard line was made with various dilutions of a solution with phloroglucinol (Sigma Chemical Co) in water-methanol 1:1 (v/v) of 0.05 to 0.3 g/L. The results were expressed as g PGE/L, which are the equivalent grams in phloroglucinol per liter of sample. - 47 -

1.20 y = 3.6648x - 0.0013 1.00 R² = 0.9992

0.80

0.60

0.40 Absorbance (nm)Absorbance

0.20

0.00 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Phloroglucinol concentration (g/L)

Figure 3.2.Shows the calibration line for the Folin-Ciocalteu method, using phloroglucinol as the reference standard

3.3.5. Chromatographic techniques Chromatography is a physical separation method for the characterization of complex mixtures, which has application in all branches of science; is a set of techniques based on the principle of selective retention, whose objective is to separate the different components of a mixture, allowing to identify and determine the quantities of said components. Subtle differences in the partition coefficient of the compounds result in a differential retention on the stationary phase and, therefore, an effective separation as a function of the retention times of each component of the mixture. Chromatography can fulfill two basic functions that are not mutually exclusive: -Separate the components of the mixture, to obtain them more pure and that can be used later (final stage of many syntheses). -Measure the proportion of the components of the mixture (analytical purpose). In this case, the quantities of material used are usually very small.

- 48 -

3.3.5.1 High performance liquid chromatography (HPLC)

The analytical HPLC system employed comprised an Agilent 1100 Series high‐performance liquid chromatograph equipped with an Agilent 1100 Series diode‐array detector (DAD) (Agilent). The HPLC pumps, autosampler, column oven, and diode‐array system were monitored and controlled using the HP Chem Station computer program (Agilent). Wavelengths used for the identification of phenolic and sugar derived compounds were 254, 270, 280, 329 and 360 nm. The injection volume was 2‐40 μL and a flow rate of 1 mL/min was used. The method used to separate the compounds consists of a combination of binary solvents using a polar solvent A (water) and a less polar B (methanol or acetonitrile) solvent, acid (acetic or formic or both) was added to maintain constant concentration of Acid during the gradient tests. These solvents were HPLC grade and were purchased from Scharlau. Analyses were carried out using an Agilent HPLC 1100 instrument equipped with a Supelcosil LC18 column (5 m, 250 mm x 4.6 mm; Supelco) in series with a C18 guard cartridge at 30 ± 1 °C. A nonlinear gradient of solvent A

(MeOH/H2O/CH3COOH, 10:89:1, v:v:v) and solvent B (MeOH/H2O/CH3COOH, 90:9:1, v:v:v) was used.

Table 3.2.Non‐linear gradients for method I and II used for HPLC analyses

Samples were analyzed directly, after saponification and after acid hydrolysis. S aponification of the phenolic compounds present in liquors was carried out with 0.75 M sodium hydroxide at 25 °C for 40 min under nitrogen atmosphere. Acid hydrolysis was carried out by adding sulfuric acid to the liquids to reach 4% (weight basis), and

- 49 -

were kept for 30 min at 121 ° C in stirred reactors to the liquids to reach 4% (weight basis), and the solutions were kept for 30 min at 121 ° C in stirred reactors. Samples were analysed by triplicate and all solutions were filtered through 0.45 µm me mbranebefore HPLC analysis. Identification of the eluted compounds was achieved by comparing their retentiontimes and UVVis spectra with those of the standards in the li brary that was built by using the inline DAD with a 3D feature. All standards were prepared as stock solutions at 1 mg/mL in methanol. Stock solutions of the standards were stored in darkness at -18 °C. Quantification was performed from calibration curves of standard compounds. Quantification was based on peak area at the corresponding wavelengths of má ximum absorption.

3.3.5.2. Gas chromatography (GC) Elution occurs by the flow of a mobile phase of inert gas. Unlike the other types of chromatography, the mobile phase does not interact with the analyte molecules; Its only function is to transport the analyte through the column. There are two types of gas chromatography (GC): gas-solid chromatography (GSC) and gas-liquid chromatography (GLC), the latter being the most widely used, and can be simply called gas chromatography (GC). The GLC uses as stationary phase liquid molecules immobilized on the surface of an inert solid. GC is performed on a gas chromatograph. It consists of various components such as the carrier gas, the sample injection system, the column (usually inside a furnace), and the detector.

3.3.6. In vitro antioxidant activity methods

The antioxidant activity was evaluated using in vitro assays: - 2,2‐Diphenyl‐1 picrylhydrazyl (DPPH.) scavenging capacity assay, Trolox equivalent antioxidant activity (TEAC) assay or 2,2´‐Azino‐bis(3‐ethylbenzothiazoline‐6 sulfonic acid) (ABTS.+) scavenging capacity assay, and oxygen radical absorbance ca acity ( RAC) assay. - Ferric reducing antioxidant power (FRAP) assay; reducing power assay.

- 50 -

3.3.6.1. DPPH radical scavenging capacity assay The technique employing 2,2-diphenyl-1-picrylhydrazyl as the radical was used. DPPH is a free radical which can be obtained directly by dissolving the reagent in an organic medium. The reduction of DPPH is monitored by the decrease in absorbance of 515 nm. In its free radical form, DPPH absorbs at 515 nm and when it is reduced by an antioxidant, the absorption lowers. Accordingly, the disappearance of the DPPH provides an index for estimating the ability of the test compound to trap free radicals. When mixed with a substance that can donate one or more hydrogen atoms, the concentration of the DPPH moiety decreases as the DPPH-H reduced form appears causing a change from purple to yellow color and the absorbance of the solution decreases.

Figure 3.3.DPPH assay mechanism

DPPH radical scavenging spectrophotometric assay was used to determine:

‐ The Inhibition Percentage, IP, which is described as the percentage of total DPPH rad cal which reacted with the antioxidant

‐ The Efficient Concentration, EC50, which is described as the amount of antioxidant re quired to lower the initial concentration of DPPH radical by 50%. The method was carried out as follows: ‐ A stock solution of DPPH (1.5 mM) in methanol was prepared and stored in darkness at ‐18 °C. Each day of analysis an aliquot of the stock solution of DPPH was diluted with m ethanol to reach a final concentration of 0.06 mM.

- 51 -

‐ 2 mL of a methanolic solution of DPPH (0.06 mM) were added to 50 µL of a solution o f the extract. ‐ The decrease in absorbance at 515 nm after 16 min was recorded using methanol as blank.

3.3.6.2. Trolox equivalent antioxidant capacity assay or ABTS radical scavenging capacity assay The TEAC (Trolox Equivalent Antioxidant Capacity) method is based on the suppression of the absorbance of the cationic radicals of ABTS (2,2'-azinobis (3- ethylbenzothiazolin-6-sulfonic acid), formed by the reaction of this compound with potassium persulfate, which is shown in Figure 3.2 (Wang et al., 2004). The ABTS compound is characterized by a blue-green color, an important water solubility and chemical stability. Its absorption maximum occurs at 342 nm. On the other hand, the radical derived from this compound shows a dramatic change in its properties, reaching its maximum absorption at 414, 645, 734 and 815 nm (Re et al., 1999). The nearness of the absorption maximum of 734 nm with the infrared region allows the ABTS radical to be the most suitable compound for testing with colored compounds as it significantly reduces the potential for interference. Another advantage to emphasize is that its versatility allows its use with both natural compounds and complex mixtures. It is a simple process, which due to its operation in a wide pH range, can be used to investigate the effects of this parameter on the antioxidant mechanisms. The characteristic solubility of the ABTS radical allows its application to be viable with both hydrophilic and lipophilic antioxidants. The determination of the activity is performed at 30 ° C by measuring the absorbance at 734 nm at 6 minutes. For this purpose the reagent (in PBS) is pre-diluted until it has a value of Absorbance of 0.7 and 20 μL of sample is added to 2 mL of diluted reagent. A control (with the medium in which the sample is diluted) is prepared. A calibration is performed with aqueous solutions of Trolox (water-soluble

- 52 -

derivative of vitamin E) in a range of 0.1 to 2 mM. The results were expressed as g Trolox /L which are the equivalent grams of Trolox per liter of sample.

Figura 3.4.Oxidation of ABTS by potassium persulfate to generate the radical ABTS.

0.50 0.45 y = 0.2248x - 0.0054 R² = 0.9986 0.40 0.35 0.30 0.25 0.20

Absorbance (nm) Absorbance 0.15 0.10 0.05 0.00 0.00 0.50 1.00 1.50 2.00 2.50 Antioxidant activity (mM Trolox)

Figure 3.5.Standard curve for TEAC assay

- 53 -

3.3.6.3. Ferric reducing antioxidant power assay FRAP (Ferric Reducing Antioxidant Power) is a method based on the reduction of the ferric compound Fe (+3) -TPTZ (2,4,6-tripyridyl-1,3,5-triazine) to a deep blue ferrous complex. This reaction allows the detection of compounds with redox potential, such as antioxidants. A differential factor in this technique is pH, which should be as close to 3.6 to maintain iron solubility. Of similar importance is the operating time, since it can cause significant variations in the results. Faster reactivity phenolic compounds are more efficiently analyzed in short times (around 4 minutes), while slower ones can take up to 30 minutes (Prior et al., 2005; Benzie and Strain, 1996).

Figure 3.6.Free radical TPTZ in the presence of an antioxidant

For the preparation of the mixture used in the FRAP procedure, it was necessary to prepare three different solutions:

- 300 mM acetate buffer at pH = 3.6: 0.78 g of sodium acetate trihydrate are dissolved in 4 mL of glacial acetic acid and brought to 250 mL with distilled water.

- 10 mM TPTZ (2,4,6-Tri(2-pyridyl)-s-triazine) in 40 mM HCl: 0.078 g of TPTZ are dissolved in 88 μL of hydrochloric acid and brought to 25 mL with distilled water.

- 20 mM iron (III) chloride hexahydrate: dissolving 0.135 g of this compound in 25 mL of distilled water.

After mixing the three solutions and homogenizing the resultant, the vessel is covered with aluminum foil in order to preserve the reagent from degradation caused by light.

- 54 -

From this point, the process is similar to that described in the TEAC, with certain modifications. The wavelength used will be 593 nm, at which the reagent should give an absorbance of 0.06, to verify that the proportions of its constituents are correct. Test tubes shall be provided with 0.1 mL of each sample (diluted, if necessary), and 3 mL of the reagent shall be added, leaving a 15 second interval between each tube and the next. After 6 minutes from the contact of the first sample with the reagent, proceed to the reading respecting the aforementioned intervals. The results can be expressed as grams of A.A. (ascorbic acid) per gram of extract.

1.20

y = 0.6535x - 0.0365 1.00 R² = 0.9938

0.80

0.60

Absorbance (nm) Absorbance 0.40

0.20

0.00 0.00 0.50 1.00 1.50 2.00 g AA/g extract

Figure 3.7.Standard curves for FRAP assay

3.3.6.4. Antitumoral activity The cytotoxic activity of the extracts was determined against four human cancer cell lines. The human A549 (lung adenocarcinoma epithelial cell line), HCT-116 (colon carcinoma), PSN1 (pancreatic adenocarcinoma), T98G (Caucasian human glioblastoma cells) were purchased from the ECACC (European Collection of Cell Cultures). A549 and HCT-116 cells were grown in RPMI-1640 medium supplemented with 5% FBS, 1% L-Glutamin (Sigma) and 1% Penicillin/Streptomycin (Hyclone, Thermo Scientific). PSN1 cells were grown in the same medium, supplemented with 10% FBS,

- 55 -

1% L-Glutamin and 1% Penicillin/Streptomycin. T98G cells were grown in RPMI-1640 medium supplemented with 10% FBS, 1% L-Glutamin and 1% Penicillin/Streptomycin, 1% sodium pyruvate and 1 % aminoacid solution (RPMI 1640, Sigma). The freeze-dried extracts (5 mg) of autohydrolysis from Sm and AESm and the porductos desorbed from SP700 with 68 % ethanol were dissolved in 500 µL methanol: DMSO (9:1) to prepare concentrations in the range of 25-500 µg/mL which were incubated during 48 h at 37 °C and 5 % CO2. Staurosporine (Biomar collection, AQUAe) was used as a positive control and two negative controls were used, one is the specific medium without cells and the other is the untreated cells suspensión. The cell viability was determined by a colorimetric assay based on the reduction of MTT to formazan by mitocondrial dehydrogenases in viable cells. To previously grown cells, 50 µL of MTT (1 mg/mL) solution were dispensed an incubated in a humidified atmosphere of 5 % CO2 during 2 h at 37 °C. Then, the medium was removed and 100 µL of pure DMSO were added to solubilize the colored compounds generated. Optical density measurements at 490 nm provided a measure of the proliferation and the cell survival. The IC50 has been calculated as the extract concentration, which inhibits 50 % of cellular growth in relation to non-treated control cells.

3.3.6.5. Anti-inflammatory activity Evaluation of neutrophils oxidative burst- oxidation of luminol. Venous blood has been collected from healty human voluteers, following informed consent, by antecubital venipunture into K3-EDTA vacuum tubes. The density gradient centrifugation method was used to isolate human neutrophils as previously described.

Tris-glucose (25 nM Tris, 1.26 nM CaCl2 2H2O, 5.37 nM KCL, 0.81 nM MgSO4 , 140 nM

NaCl, and 5.55 nM D -(+)- glucose) was the incubation media used. The measurement of neutrophils oxidative burst has undertaken by monitoring ROS-induced oxidation of luminol, as previously described. Luminol is a chemiluminescent probed and in is used to monitor the intra and extracelular production of reactive species (RS) by neutrophils , namely superoxide anion radical

(O2), hydrogen peroxide (H2O2) hydoxyl radical (HO) hypochlorous acid (HOCl), nitric oxide (NO) and peroxynitrite anion (ONOO). The reaction mixtures contained

- 56 -

neutrophils (1 x 106 cells/mL) and the following reagents at the indicared final concentrations (in a final volumen of 250 µL): the extracts desorbed under select optimal conditions at various concentrations (3.9-62.5 µg/mL), luminol (500 µM) and PMA (160 nM) cells were pre-incubated which luminol and the tested compounds for 5 min before the addiction of PMA. Kinetic readings were immediately initiated after cell simulation and carried out at 37 °C, under continuous soft shaking. Measurements were taken at the peak of the curve. This peak was observed at arround 10 min. Effects are expressed as the percent inibition of luminol oxidation. Quercetin has used as positive control.

3.3.6.6. COX-1 and COX-2 assays The assays were performed in human whole blood. The venous blood was collected into heparin-Li+ vacuum tubes, from healty human voluteers, as described above. Briefly, to detect the effect of the selected extracts in the production of prostagrandin E2 (PGE2), via COX-1, the collected blood (500 µL) was placed in microtubes and incubated in a water bath 37 °C with TXBSI (1 µM) and the extracts desorbed with 38 % ethanol both in static and in dynamic conditions from Sm and ASEm autohydrolysis liquids (10-200 µg/mL) in DMSO for 15 min. Then, the ionophore A23187 (12.2 µg/mL) was added and the mixture was for 15 min. Then the samples were placed on ice for 5 min to stop the reaction. Afterwards the samples were centrifuged at 1000 g for 20 min, at 4 °C. The supernatant has then collected and stored at -20 °C until used.

The evaluation of the effect of the selected extracts in the production of PGE2, via COX-2, undertaken by placing the collected blood (800 µL) in a six wells plate and incubation in a humidified incubator at 37 °C with TXBSI (1 µM), acetylsalicylic acid (10 µg/mL), the extracts desorbed with 68 % ethanol both in static and in dymanic conditions from Sm and AESm autohydrolysis extracts (10-200 µg/mL) in DMSO:chremophor in ethanol 1% (1:10) for 15 min. Then, LPS (10 µg/mL) was added and the mixture has incubated for 5 h, allowing the activation of COX-2. The reaction has been stoped by adding DPBS-gentamicin buffer (1 mL) to the samples and placing then on ice for 10 min. The samples were subsequently centrifuged at 1000 g for 15

- 57 -

min, at 4 °C to separeted the blood cells from the superntant, maintaining the low temperature. The supernatant was then collected and stored at -20 °C until use.

The determination of the amount of PGE2 in the samples was measured using the above mentioned commercial EIA kit, according in the manufacturer´s instructions, as an indicator of COX-1 and COX-2 activities. COX inhibitors, indomethacin (0.125-1 µM) and celecoxib (0.5-50 µM) were used as positive controls and the activities found were similar to those already reported. Results are expressed as the percent inhibition of control PGE2 production.

3.4. Scanning electron microscopy (SEM) Freeze-dried samples were fixed on aluminium stubs and sputter coatedwith gold in an Emitech K550X equipment and examined with a FEI Quanta 200 scanning electron microscope under vacuum conditions at an accelerating voltage of 12.5 kV.

3.5. Mathematical procedure

3.5.1. Calculation of adsorption capacity and adsorption ratio

The adsorption capacity, qt (mg/g resin), quantity of solutes adsorbed at time t onto a mass unit of dry resin, was calculated according to equation:

[3.14] Where, C0 and Ct (mg/mL) are initial concentration and concentration in specified time of solutes in liquid phase, V (mL) is volume of feed solution and W (g) is weight of the dry resin. The adsorption ratio, E, percentage of total solute being absorbed at adsorption equilibrium, was calculated as indicated in equation:

[3.15]

- 58 -

Where, C0 and Ce (mg/mL) are initial concentration and equilibrium concentration of solutes in liquid phase (mg/mL).

3.5.2. Adsorption kinetics Adsorption kinetic is important from the point of view to control the process efficiency. In this Thesis warious kinetic models have been tested.

 Pseudo‐first order model The pseudo‐first order rate equation of Lagergren is generally described in equation:

[3.16]

Where qe and qt (mg/g) are amount of solutes adsorbed at equilibrium and at ‐1 specified time t (min), k1 (min ) is pseudo‐first‐order rate constant and t (min) contact time.

 Pseudo-second order model The pseudo‐second order kinetic model may be represented by:

[3.17]

Where qe and qt (mg/g) are amount of solutes adsorbed at equilibrium and at

specified time t (min), k2 (g/(mg ·min)) is pseudo‐first‐order rate constant and t (min) is contact time.

 Elovich model Elovich equation can be expressed by equation:

[318]

- 59 -

Where qt (mg/g) are amount of solutes adsorbed at specified time t (min), α (mg/g--min‐) is initial adsorption rate, β (g/mg) is Elovich rate constant and t (min) is contact time.

 Intraparticle diffusion model. The intra‐particle diffusion model, based i¡on the proposed by Weber and Morris (1962) is characterized by the relationship between specific adsorption and the square root of time, according to the following equation:

[3.19]

Where qt (mg/g) are the amount of solutes adsorbed at specified time t (min), -0.5 ki (mg/g.min ) is intraparticle diffusion rate constant and (mg/g) is the intercept.

3.5.3. Adsorption isotherms

The study of asorption isotherms in static systems is necessary to know the interations between adsorbent and adsorbate. To evaluate the behaviour of the adsorbents and adsorbates a serial of batch experiments was carried out by mixing a known amount of resin with a fixed volumen of simple solution at various initial concentration in an orbital shaker kept at constant temperature (25 °C). The experimental data were fitted at the two theoretical models more employed in adsorption literature. Their results were used to describe the adsorption behaviour between adsorbate and adsorbent, and subsequently, were used for understanding the system behavior in dynamic processes.

 Langmuir isotherm (Langmuir, 1918) assumes a monolayer adsorption, a homogeneous distribution of the sorption energy and no mutual interaction between adsorbed molecules. The model of Langmuir, can be expressed by the following mathematical formula:

- 60 -

[3.20]

Wheres qe (mg/g) is amount of solute adsorbed per unit weight of adsorbent

(mg/g) at equilibrium, KL (L/mg) is constant related to the free energy of

adsorption and Ce (mg/mL) is the equilibrium concentration of solutes in liquid phase. The essential characteristics of Langmuir isotherm can be expressed by a

dimensionless constant called separation factor (or equilibrium parameter), RL.

The parameter RL indicates the characteristics of isotherm as follows:

[3.21]

RL >1 unfavourable; RL =1 linear; 0< RL <1 favourable; RL = 0 irreversible

 Freundlich isotherm (Freundlich, 1909) is an empirical equation, for non-ideal adsoption systems. This model describes the equilibrium conditions of adsorption for a heterogeneous surface and does not imply the formation of a monolayer. It is expressed by the formula below:

[3.22]

Wheres qe (mg/g) is amount of solute adsorbed per unit weight of adsorbent n (mg/g) at equilibrium, KF (mg/g)(mg/L) is the distribution coefficient, Ce (mg/mL) is the equilibrium concentration of solutes in liquid phase; and 1/n is measurement of adsorption intensity

- 61 -

3.5.4. Desorption process

3.5.4.1. Calculation of desorption ratio

The desorption ratio, D, percentage of total desorbed substrate with respect to the mass present in the loaded resin, was calculated as indicated in equation:

[3.23]

Where C0 and Ce (mg/mL) are initial and equilibrium concentration of solutes in liquid phase, Cd (mg/mL) is the concentration of the solutes in desorption effluent, Vi and Vd (mL) are feed solution and desorption solution volumes.

3.5.4.2. Dynamic desorption studies The resin was satured with the phlorotannin fraction contained inthe autohydrolysis extract from the ground algae. Dynamic elution of the florotannin fraction was addressed at room temperature, using 68 % ethanol at a flow rate 2.25 mL/min. The eluted samples were colleted and analyzed.

3.5.4.3. Dynamic adsorption modeling The performance of fixed packed beds is described through the concept of the breakthrough curve. The assessment of breakthrough time and the shape of the breakthrough curve are essential for characterizing a dynamic process into an adsorption column. The capacity of packaged adsorbent and its interaction with adsorbates with regard to the feed concentration and the flow rate will determine the breakthrough curve shape. The breakthrough curves provide information about the removing of adbsorbate from a flowing solution through a fixed bed. Usually, this behavior is expressed in term of normalized concentration, which is defined as the ratio of effluent adsorbate concentration to inlet absorbate concentration (C/Co) as a function of time or volume of effluent for a given bed height.

- 62 -

Successful design of a column adsorption process requires prediction of the concentration–time profile or breakthrough curve for the effluent. Developing a model to accurately describe the dynamic behavior of adsorption in a fixed bed system is inherently difficult. Since the concentration of the adsorbate as the feed moves through the bed, the process does not operate at steady state. The adsorption rate for a fixed bed column operation depends on the mechanism responsible for adsorption. This mechanism may be controlled by mass transfer from the bulk solution to the surface of the adsorbent. Alternatively, it may be controlled by diffusion and reaction within the adsorbent particles. So the mathematical description of adsorption rate must be added to these transport equations given for the fixed bed. The adsorption isotherm equation is the fourth and final key equation for the mathematical modeling of a fixed bed. Various simple mathematical models have been developed to predict the dynamic behavior of the column. In this study, the following models characterizing fixed bed were use to adjust the system performance: The Adams-Bohart model The Thomas model The Yoon and Nelson model

3.5.4.3.1. Model of Thomas The Thomas solution is one of the most general and widely used methods in column performance theory. The Thomas model assumes Langmuir kinetics of adsorption–desorption, and no axial dispersion is derived with the assumption that the rate driving force obeys second-order reversible reaction kinetics. Thomas’ solution also assumes a constant separation factor but it is applicable to either favorable or unfavorable isotherms. The primary weakness of the Thomas solution is that its derivation is based on second order reaction kinetics. Adsorption is usually not limited by chemical reaction kinetics but is often controlled by interphase mass transfer. Successful design of a column adsorption process requires prediction of the concentration–time profile or breakthrough curve for the effluent. The maximum adsorption capacity of an adsorbent is also needed in design. Traditionally, the Thomas

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model is used to fulfil the purpose. The linearized form of the Thomas model is as follows:

C0 kThq0 w ln( 1)   kThC0t [3.24] Ct  where kTh is the Thomas rate constant (mL/(min·mg)) and qo is the maximum solid- phase concentration of the solute (mg/g). C0 (mg/L) is the concentration of phenolic compounds in the influent; Ct (mg/L) is the concentration of phenolic compounds in the effluent; w (g) is the mass of resin and ν (mL/min) the flow rate of influent.

The value Ct/C0 is the ratio between the concentrations of phenolic compounds in the inlet and outlet streams, respectively. The linearization of ln[(C0/Ct)−1] against time (t) allows calulation of q0 and kTh.

3.5.4.3.2. Model of Adams–Bohart

This model assumes that the adsorption rate is proportional to both the residual capacity of the adsorbent and the concentration of the sorbing species. The Adams–Bohart model is used for the description of the initial part of the breakthrough curve. The mass transfer rates obeys the following equations:

∂q/∂t=− [3.25]

∂Cb/∂Z=−kABUoqCb [3.26] where kAB is the kinetic constant (L/(mg min)). Some assumptions are made for the solution of these differential equation systems: (i) the concentration field is considered to be low, e.g. effluent concentration C <

0.15 Co;

(ii) for t→∞, q→No, (where No is the saturation concentration (mg/L). When the differential equation systems are solved, the following equation is obtained with parameters kAB and No:

Ct Z ln = k ABC0 t k AB N0 [3.27] C0 F

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where Co and Ct are the inlet and effluent phenol concentrations (mg/L), respectively. From this equation values describing the characteristic operational parameters of the column can be determined from a plot of lnCt/Co against t at a given bed height and flow rate.

3.5.4.3.3. Model of Yoon–Nelson This model is a less complicated model which can be used to investigate the adsorption and breakthrough curves in fixed bed column. This model is based on the hypothesis that the probability of adsorption decreased rate of each adsorbate molecule is linearly related to the probability of amount of adsorbate adsorption and breakthrough on the adsorbent.

The linear form of Yoon-Nelson model, adapted from Aksu y Gonen (2004), is represented as follows,

C t ln  kYNt kYN [3.28] C0  Ct

-1 Where: kYN is the Yoon-Nelson rate constant (min ) and  (min) is the breakthrough time required for 50 % adsorbate breakthrough (min).

A simplification on the above equation permets determined Ct/(C0 - Ct) versus elution time (t) by means of the línea plot of ln( ) against t. From the mathematical adjustment of the experimental data the parameters of kYN and kYN can be obtained by linear regression from the value of the intercept and slope of the graph.

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4. RESULTS

4.1. Enzyme aided aqueous procesing of Sargassum muticum

4.1.1. SUMMARY The enzyme assisted processing of Sargassum muticum was proposed with the aim of obtaining soluble fractions with radical scavenging properties. Commercial hydrolytic enzymes of different activities have been applied under recommended operational conditions of pH and T. Both the whole algae (Sm) and the solid residue after alginate extraction (AESm) were used as substrate. Incubation during 3 h with 2-5 g enzyme solution/100 grams algae were selected as optimum conditions for total soluble yield, phenolic content and ABTS radical scavenging activity in the extracts. The enzyme treatment assisted by ultrasounds increased by 50% the yield attained in control, with a moderated increase of the phenolic content and radical scavenging capacity of the extracts. The selected soluble extracts contained 5% total phenolics and 20% protein, with most sugars present in the oligomeric form, the major components being glucose and fucose. The extracts from Sm showed slightly higher proportion of unsaturated fatty acids than those from AESm and the 3:6 ratio in the extracts from Sm samples was close to 1. One gram of extract was equivalent to 20 mg ascorbic acid and 100 mg Trolox and the cytotoxic activity against murine melanoma B16F10 cells and human liposarcoma SW872 cells was observed at 50-250 g/L, whereas on fibroblast occurred at more than 500 g/L.

4.1.2. STATE OF THE ART The degradation of cell wall polysaccharide facilitates the solid liquid extraction of solutes from the plant structures improving both the extraction efficiency and rate. The application of enzymatic technology has been proposed for the aqueous extraction of vegetal oils (Domínguez et al., 1994), proteins and bioactives (Puri et al., 2102) and more recently to marine macroalgae (Denis et al., 2009; Hahn et al., 2012; Wijesinghe and Jeon, 2012), although it had been suggested previously. Seaweed cell walls present a complex structure more heterogenous than those of land plants, since mixtures of sulfated and branched polysaccharides are associated with proteins and ions and limit the efficiency of extraction processes. Enzyme assisted

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extraction has been proposed for the maceration of seaweed tissues, to break down the cell walls and complex storage structures, facilitating the recovery of bioactive components from seaweeds. This relatively simple technology avoids the use of organic solvents and could be adapted to large-scale production processes (Wijesinghe and Jeon, 2012). The enzyme aided aqueous extraction has been successfully applied to the brown algae Ecklonia cava, Hizikia fusiformis, Ishige okamurae, Sargassum fulvellum, S. horneri, S. coreanum, S. thunbergii and Scytosiphon lomentaria (Heo et al., 2003; Ahn et al., 2004; Heo et al., 2005; Heo and Jeon, 2008; Ko et al., 2012; Wijesinghe and Jeon, 2012). Commercially available food-grade digestive enzymes with carbohydrase and protease action have been succesfully used for degrading the macroalgal structure rendering the intracellular materials more exposed for solvent extraction (Ahn et al., 2004; Ko et al., 2012; Siriwardhana et al., 2004 and 2008), whereas fucoidan-degrading enzymes are comparatively less studied (Urvantseva et al., 2006). Enzyme-assisted extraction resulted in high yields and high radical scavenging activity of extracts with improved water solubility (Je et al., 2009). The enzymatic extracts have shown ability to scavenge 1,1, diphenyl-2-picrylhydrazyl, alkyl, superoxide and hydroxyl radicals and to inhibit lipid peroxidation (Heo et al., 2003 and 2005; Ahn et al., 2004; Heo and Jeon, 2008). Phenolic compounds have been suggested as the major responsable for the antioxidant properties of extracts (Heo et al., 2003; Heo et al., 2005; Athukorala et al., 2006), although macroalgae contain different active components. The extracts produced by this technology showed heat stability at 100 °C (Heo et al., 2005) and some fractions of the enzymatic extracts proved to have a variety of bioactivities, including immunomodulatory (Ahn et al., 2008), antiinflammatory (Kang et al., 2001), inhibitory effect of DNA damage (Heo et al., 2005 BRT; Heo and Jeon, 2008), antiproliferative and apoptotic actions on cancer cells (Athukorala et al., 2006; Ko et al., 2012). Ultrasonic assisted extraction (UAE) is used as a low cost, efficient and scalable alternative method to improve the extraction of plant bioactives. Sonication simultaneously enhances the hydration and fragmentation process and facilitates the mass transfer of solutes to the solvent. Ultrasound effects on liquid systems are mainly related to cavitation, a phenomenon caused by the violent collapse of bubbles in the

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liquid, induced by alternating high pressure (compression) and low pressure (rarefraction) cycles. The mechanical effects associated to ultrasound can accelerate the eddy and internal diffusion and increase mass transfer, swelling and hydration, causing disruption of the cell walls and facilitating the release of cell contents into the bulk medium (Albu et al., 2004). Mild operating conditions usually employed in UAE do not cause significant changes in the structural properties and functionality of most bioactives, although sonication of water may produce free radicals within the cavitation bubbles (Kidak and Ince, 2006). Sonication has been proposed as a simple technique to enhance enzyme performance (Capelo et al., 2009). Sargassum muticum is an invasive seaweed in Atlantic waters and a hydrothermal based process with hot water (autohydrolyisis) has been useful for solubilisation of fractions with antiradical action (González-López et al., 2012), but no information is available on the enzyme digestion of this algae nor on the influence of ultrasound during this process.

4.1.2.1 Main Goal To develop a process for the extraction of soluble fractions with antioxidant properties from S. muticum (Sm) through enzymatic hydrolysis coupled with ultrasound, and to determine the potential the solid residue remaining after the extraction (AESm) of the alginate fraction as source of natural antioxidants.

4.1.3. METHODOLOGY 4.1.3.1. Raw material recollection, storage and characterization Sargassum muticum (Sm), collected in Praia da Mourisca (Pontevedra, Spain) in June 2012 was cleaned from epiphytes and sand, washed with tap water, frozen and stored in closed plastic bags at -4 C until use. The proximal composition of the alga, expressed as weight percent in dry basis, was 26% ash, 7% protein, 1% total phenolics, as gallic acid equivalents,10.2% alginate, 20.3% acid insoluble residue and 16.1% polysaccharide fraction (the monomers were present at the mass ratio Glu:Xyl:Gal:Man:Fuc= 1:0.10:0.30:0.04:0.47). 4.1.3.2. Alginate extraction

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A conventional stage of alginate extraction was performed to obtain the AESm. S. muticum was defrosted, ground and contacted with 1 % formaldehyde, and the resulting solids were subjected to extraction with 0.2 N sulphuric acid, washing with water and extraction with 1 % sodium carbonate to convert all the alginate salts to the sodium salt, which were precipitated by ethanol. The residual solid phase or alginate- exhausted algae (AESm) was oven dried at 50 °C and stored in sealed plastic bags until being used as substrate for the enzyme assisted aqueous extraction process. The proximal composition of the alginate exhausted solid residue in weight percent dry basis was 30.7% ash, 8.4% protein, 32.7% acid insoluble residue and 19.5% polysaccharide fraction (the monomers were present at a mass ratio Glu:Xyl:Gal:Man:Fuc= 1:0.06:0.25:0.05:0.48).

4.1.3.3. Enzymes Commercial enzymatic formulations succesfully employed for other brown algae (Heo et al., 2003 and 2005; Je et al., 2009) were incubated using either 0.1 M phosphate buffer (PB) or 0.1 M acetate buffer (AB). Different activities provided by Novozymes (Bagsvaerd, Denmark) were tested under the conditions in Figure 4.1.1, including the proteases Alcalase and Protamex, the β-glucanases Celluclast and Viscozyme and the amylolytic formulation Amylase. Also the mixture Amylase and Protamex was tested (PB pH 7, 50-60 °C). The proteolytic formulation RFT (DSM, Netherlands) was also used at condiciones pH y T recommended by the manufacturer.

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Figure 4.1.1.Effect of incubation time on the a) total solubles yield, b) phenolic content of the extracts expressed as gallic acid equivalents and as phloroglucinol equivalents and ABTS+·scavenging capacity of the extracts. Experiments were performed at the optimal pH and T for each enzyme, at liquid to solid (AESm) ratio 1:50, and at enzyme to AESm ratio of 5%

4.1.3.4. Enzyme assisted extraction Enzyme aided extraction was performed in duplicate independent assays in 250 mL Erlenmeyer flasks, placed in an orbital shaker (INNOVA 4000, New Brunswick) using 50 g of Sm samples and 1 g AESm at a liquid to solid ratio 50 (v/w), at pH and temperature fixed according to the manufacturer recommendations and to previous results. Enzyme to substrate ratios in the range of 0.5-15% (wt, db) were tested. The samples were heated at 100 °C for 10 min to inactivate the enzyme. After incubation, the - 71 -

extracts were centrifuged (3500 rpm, 10 min) and the filtrate was stored at -20 °C in the darkness until analysis.

4.1.3.5. Ultrasound assisted enzyme extraction The flasks with the suspension of the ground wet Sargassum muticum on the buffer with or without enzyme were sonicated in a Labsonic® P ultrasonic processor (Sartorius, Spain) (400 W, 24 kHz). Intermittent sonication of the algal suspension was applied inserting the titanium probe tip of the sonotrode horn (10 cm length, 1.9 cm diameter) 2 cm into the reaction solution with power discharges during 5 min and off periods of 25 min. The first stage was performed with 60 % amplitude and 1 cycle to allow the sample reached the temperature quickly, whereas in the next stages 50% amplitude and 1/2 cycle were used. During the process, temperature was measured using a thermocouple. To limit overheating in the area close to the tip of the ultrasonic probe, suspensions were cooled down in cold water or ice water bath. The extracts were centrifuged (3500 rpm, 10 min) to separate the solid residue, the supernatant was vacuum filtered and the filtrate stored at -20 °C in the absence of light until analysis. Each extraction was performed in duplicate using three different samples.

4.1.3.6. Analytical methods Ash was determined by calcination at 575 °C. The total phenolic content was measured by the Folin-Ciocalteu method (Singleton and Rossi, 1965), and expressed as gallic acid and phloroglucinol (Sigma) equivalents. The soluble protein was determined by the Lowry method, expressed as BSA (Sigma Aldrich) equivalents. Glucose, xylose, arabinose and fucose were determined by HPLC-RI using a 1100 series Hewlett-Packard chromatograph and a 300 × 7.8 mm Aminex HPX-87H column (BioRad, Hercules, CA) operating at 60 °C (mobile phase: 0.003 M H2SO4, flow rate: 0.6 mL/min). The concentration of oligosaccharides were determined from the concentrations of monosaccharides present in samples previously subjected to a quantitative posthydrolysis (4% sulfuric acid, 121 °C, 20 min).

Total lipids were gravimetrically determined after extraction with CHCl2:MeOH. Fatty acid methyl esters (FAME) were obtained by transmethylation of lipid samples (8–10

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mg) using 1 mL of 1% NaOH in MeOH, heating for 15 min at 55 °C, adding 2 mL of 5% methanolic HCl, heating again for 15 min at 55 °C and then adding 1 mL of H2O (Carreau and Dubacq, 1978). Fatty acid methyl esters were extracted by hexane and the organic phase was evaporated. FAMEs were analyzed in a GC–MS QP 2010 (Shimadzu, Japan) using 1 mL He/min as carrier gas and the column temperature was programmed by heating from 50 °C (2 min) at 10 °C/min to 240 °C; the final temperature was maintained for 27 min. FAMEs in the sample were identified by comparing their mass spectra with those of authentic standards. The results of two independent determinations are expressed as a percentage of total FAME.

4.1.3.6.1. TEAC (Trolox Equivalent Antioxidant Capacity) ABTS radical cation (ABTS•+) was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate. The ABTS•+ solution was diluted with PBS (pH 7.4) and equilibrated at 30 °C. After addition of 1 mL of diluted ABTS•+ solution to 10 μL of Trolox standards in PBS the absorbance reading was taken at 734 nm, 30°C and 6 min. Appropriate solvent blanks were run and the percentage inhibition was calculated as a function of the concentration of extracts and Trolox.

4.1.3.6.2. Ferric reducing antioxidant power (FRAP) The FRAP reagent was freshly prepared with 25 mL of 0.3 M acetate buffer (pH 3.6) (25 mL), 10 mmol/L of 2, 4, 6-tripyridyl-s-triazine (TPTZ) solution in 2.5 mL of 40 mmol/L HCl and 2.5 mL of 20 mmol/L FeCl3·6 H2O in distilled water. This solution was used as a blank, and a solution of 1 mMol ascorbic acid was used for calibration. Samples (100 μL) were mixed with 3 mL of FRAP reagent and the absorbance was monitored at 593 nm.

4.1.3.6.3. Cell viability Three different cell lines were selected for assessing the potential toxicity of extracts: murine melanoma B16F10 cells, human liposarcoma SW872 and human foreskin fibroblasts HFF-1.

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The cell viability was determined by a colorimetric assay based on the reduction of 3- (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT reagent, Sigma- Aldrich) to formazan by mitochondrial dehydrogenases in viable cells. To previously grown cells, 50 mL of 1 mg/mL MTT solution were dispensed and incubated in a humidified atmosphere of 5% of CO2 during 2 h at 37° C. Then, the medium was removed and 100 µL pure DMSO was added to solubilize the colored compounds generated. A negative control (DMEM without cells) and a positive control (untreated cells) provided minimum and maximum supervivence values, respectively. Five mg of extract were dissolved in 9:1 methanol:DMSO (Panreac) to prepare concentrations between 50 and 1000 g/mL. The amount of formazan formed can be measured spectrophotometrically at 490 nm and is proportional to the number of viable cells. Murine melanoma B16F10 cells (ATCC CRL-6475) were cultured in phenol red free Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma–Aldrich), supplemented with 10% fetal bovine serum (Sigma-Aldrich), 1% penicillin/streptomycin (Hyclone, Thermo Scientific) and 1% L-Glutamin (Sigma-Aldrich). Human liposarcoma SW872 cells (ATCC, HTB92) were cultivated in DMEM/Ham´s F12 (3/1 v/v) medium (Sigma-Aldrich) containing 10% fetal bovine serum (FBS, 1%, Sigma- Aldrich), 1% non essential aminoacids and 1% P/S (50 U/mL penicillin; 50 mg/mL streptomycin) (Hyclone, Thermo Scientific). Human foreskin fibloblasts HFF-1 (ATCC, SCRC-1041) were cultured in DMEM supplemented with FBS (1-10%), 1% P/S (50 U/mL penicillin; 50 mg/mL streptomycin) (Hyclone, Thermo Scientific) and 1% L-Glutamine.

4.1.4. RESULTS AND DISCUSSION 4.1.4.4. Enzyme assisted extraction 4.1.4.4.1. Effect of hydrolysis time The influence of time during the enzymatic hydrolysis of AESm was assessed during experiments carried out at optimal temperature and pH using a fixed enzyme to AESm solids ratio of 2.7% (wt basis). These initial experiments were carried out with AESm samples in order to select the most suited activities acting on the alginate free material. Figure 4.1.1 shows a higher extraction yield for both control (no enzyme

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added in the buffer) and enzyme treated samples operating at acidic pH, the optimum pH for amylases and β-glucanases. The highest phenolic liberation was also observed at acidic pH. Operating at the pH values used in the present study (4 and 8), other authors found no significant difference in the phenolic yield from H. fusiformis, although a significant increase in the total extraction yield was reported (Siriwardhana et al., 2008). Hardly any increase at more prolonged incubation times in the studied period was observed. In other works higher incubation times have been reported (Siriwardhana et al., 2008). Despite hardly any increase at more polonged incubation times of AESm was observed, in other works incubation times of 12 h or longer have been reported (Ahn et al., 2004; Athukorala et al., 2006; Heo et al., 2003; Siriwardhana et al., 2004), but also shorter times proved suitable (Heo et al., 2003). The mixture of amylolytic and proteolytic formulations offered solubles extraction yield lower than that attained with amylases and similar to that attained with proteases. However, the phenolic extraction yield was higher than that obtained with amylase and with protease. A similar performance was reported when a combination of proteases and carbohydrases increased the polyphenols yield but not the total solubles yield from H. fusiformis (Siriwardhana et al., 2004).

The extraction yields attained for different species of Sargassum (S. fulvellum, S. horneri, S. coreanum, S. thumbergii) were similar (21.5-31.5 g extract/100 g alga) (Heo et al., 2003). Also, yields in the range 27-45% of the initial material were obtained during treatment of Hizikia fusiformis with carbohydrases and proteases at 5% (Siriwardhana et al., 2004 and 2008). The phenolics extraction yield from AESm was in the range 2-4 g GAE/100 g S. muticum, which is slightly higher than the value reported from the whole algae collected in the same location using ethanol as solvent (1-2 g GAE/100 g S. muticum) and comparable to that reported for the algae collected in other Atlantic coasts (Connan et al., 2006). The phenolic content of the extract accounted for 4-10% (wt%, d.b.) of the extract, when this value is expressed as gallic acid equivalents and as phloroglucinol equivalents per gram of extract. Values were in the range of those reported for Sargassum sp (0.2-1 %) (Heo et al., 2003), for H. fusiformis hydrolyzates with protease

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(Siriwardhana et al., 2004) or with a mixture protease and carbohydrase (Siriwardhana et al., 2008), and for Sargassum coreanum protease hydrolyzates (Ko et al., 2012). One gram of the control alkaline extracts, Alcalase and Alcalase+Protease extracts were equivalent up to 0.3-0.4 g of the synthetic antioxidant Trolox and their radical scavenging capacity increased with the treatment time (Fig. 1.b). The most active were the proteolytic extracts, also favourable for other species, such as Ecklonia cava (Heo et al., 2003), Ishige okamurae (Heo and Jeon, 2008), Undaria pinnatifida (Je et al., 2009) or Sargasum coreanum (Ko et al., 2012). Despite the relatively low phenolic content, a correlation with TEAC values exists, and was also found for proteolytic extracts from brown algae (Heo et al., 2003, 2005; Athukorala et al., 2006), although other components in enzymatic extracts, including low molecular weight phenolics, polysaccharides, pigments such as fucoxanthin, proteins, and peptides, may also influence this activity.

4.1.4.4.2. Effect of enzyme to alga ratio Operating at incubation times of 3 h, the influence of the enzyme to the solid residue after alginate extraction (AESm) ratio is shown in Figure 4.1.2. Optimal doses in the range 2-5 genzyme protein per gram of AESm were found for Amylase and Cellulase extracts (Fig. 2.a). Optimal values in the range 10-20 g enzyme/100 g algae were reported (Ahn et al., 2004; Heo and Jeon, 2008; Je et al., 2009; Ko et al., 2012), also lower values (Athukorala et al., 2006; Siriwardhana et al., 2008). Up to 50% of the material could be solubilised, the most efficient enzymes were Cellulast, Viscozyme and Amylase. However, the extracts with higher phenolic and protein purity were those produced with proteolytic activities (Figs. 4.1.2.b and 4.1.2.c), which also showed higher reducing power and ABTS radical scavenging properties (Figs. 2.d and 2.e). Both the reducing and the radical scavenging capacity were lower than those of ascorbic acid and Trolox, respectively. Lower activity for algal enzyme extracts than for synthetic antioxidants as BHA and BHT (Athukorala et al., 2006), or vitamin C (Je et al., 2009), although comparable values for Hizikia fusiformis enzymatic extracts have been reported (Siriwardhana et al., 2008).

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The monomeric and oligomeric sugar content in the extracts increased with the enzyme dose during the treatment (Figs. 4.1.2.c and 4.1.2.d). The monomeric sugar content was higher than the oligomeric sugar content only at the highest enzyme loading tested. The carbohydrases, Viscozyme and Celluclast, provided higher glucose yields whereas the protease Alcalase provided the highest yields in xylose+galactose+mannose and fucose. The fucose proportion was lower in the monomeric fraction than in the oligomeric one.

a 60 b 0.7 a 0.6 50

a

0.5 40 0.4

30 0.3

20

0.2 Protein content(g BSAeq/g extract)

10 0.1 Extractionyield(g extract/ 100g AESm)

0 0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 enzyme concentration (%) enzyme concentration (%)

c e 0.09 a a 0.08

0.07

0.06

0.05 (1.0:0.7:0.0:0.08:0.06) 180000 0.04

160000 (1.0:0.7:0.0:0.06:0.08) 0.03

140000 (0.0:0.07:0.0:0.05:1.0) 0.02

120000 Phenolic content(g PGE/gextract)

0.01 (0.0:0.1:0.0:0.07:1.0) 100000 (1.0:0.7:0.0:0.08:0.06)

(1.0:0.8:0.0:0.1:0.1) 0.00 (1.0:0.8:0.0:0.2:0.2) 80000 (1.0:0.7:0.0:0.06:0.08) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

(0.0:0.2:0.0:0.07:1.0) enzyme concentration (%)

(0.9:1.0:0.0:0.1:0.1)

60000 1.0:0.8:0.0:0.1:0.1) (

Area of sugars

(0.9:1.0:0.0:0.1:0.1)

(0.0:0.2:0.0:0.1:1.0) (1.0:0.9:0.0:0.3:0.2)

40000 (1.0: 0.8:0.0:0.2:0.2)

(0.2:1.0:0.0:0.1:0.6)

(1.0:0.9:0.0:0.3:0.2)

(0.89:1.0:0.0:0.4:0.4)

(0.0:0.7:0.0:0.2:1.0)

(0.9:1.0:0.0:0.4:0.4)

(0.8:1.0:0.0:0.1:0.1) (0.7:1.0:0.0:0.2:0.3)

(0.0:0.9:0.0:0.2:1.0) g 0.05

(0.8:1.0:0.0:0.2:0.2)

(0.0:1.0:0.0:0.3:0.3) (0.0:1.0:0.0:0.2:0.2) 20000 (0.0:1.0:0.0:0.3:0.4) a 0.045 0 0.04 0.035 0.03 enzyme concentration (%) 0.025 0.02

(g 0.015AA/gextract) 0.01 d 0.005 Ce Vi Al Am a 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 % enzyme

(1.0:0.2:0.0:0.2:0.07) (1.0:0.2:0.0:0.2.:0.04)

120000 (1.0:0.3:0.0:0.2:0.06) f 0.30

(0.0:0.5:0.0:0.4:1.0) (0.0:1.0:0.0:0.6:0.1)

(0.0:0.4:0.0:0.1:1.0) a

100000 (1.0:0.3:0.0:0.2:0.07) 0.25

(0.0:0.7:0.0:0.4:1.0) (1.0:0.7:0.0:0.5:0.2) 80000

(1.0:0.9:0.0:0.4:0.1) 0.20

(0.8:1.0:0.0:0.5:0.1)

(1.0:0.9:0.0:0.5:0.2)

(0.8:1.0:0.0:0.6:0.1) (1.0:0.3:0.0:0.2:0.09)

60000 (0.6:1.0:0.0:0.7:0.2)

(0.8:1.0:0.0:0.6:0.2) (0.0:1.0:0.0:0.7:0.5)

(0.0:1.0:0.0:0.7:0.3) 0.15

(0.3:0.8:0.0:1.0:0.2)

(0.4:0.9:0.0:1.0:0.2)

(0.4:0.9:0.0:1.0:0.1)

(0.0:1.0:0.0:0.4:0.3)

(0.0:1.0:0.0:0.8:0.2)

(0.0:1.0:0.0:0.7:0.1)

(0.7:1.0:0.0:0.6:0.2) (0.0:1.0:0.0:0.8:0.2) 40000 (0.0:1.0:0.0:0.8:0.0) Area of sugars 0.10 20000

Antioxidantactivity Trolox/g(g extract) 0.05 0 0.00 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 enzyme concentration (%) enzyme concentration (%)

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Figure 4.1.2 Effect of enzyme to AESm ratio on the a) total solubles yield, b) protein content, c) monomeric and d) oligomer sugars, expressed as sum of the peak areas (Glu, Xyl+Gal+Man, Ara, Fuc), e) phenolic content of the extracts phenolic content expressed as gallic acid equivalents and as phloroglucinol equivalents, f) ABTS radical scavenging capacity and g) FRAP. Experiments were performed at the optimal pH and T for each enzyme during 3 h, at a liquid to AESm ratio of 50

4.1.4.5. Ultrasound and enzyme (USE) assisted extraction process In order to assess the potential of USAE enzyme treatment, initial experiments with a carbohydrase at 5% enzyme to alga ratio were performed using Sargassum muticum (Sm) as substrate, since this material is less processed and the effects could be better observed than in the AESm, subjected to a series of extraction and washing steps. Despite the total extraction yields were only slightly improved with US or with enzyme assisted processes, the phenolic extraction was more selective, and the phenolic content was improved over that in control. The performance of US assisted processes was better at prolonged times and a certain synergism with the enzyme treatment was observed on the solubles and phenolics extraction yields. A comparison of the performance of the different processes operating with the whole alga Sm and with the alginate exhausted solids, AESm, is shown in Figure 4.1.3. Two enzyme activities were selected, Cellulase, selected on the basis of the results in the previous section and the protease RTF from a previous screening study (unpublished results). The extraction yields were 3-4 times higher when AESm is used and also the protein content was slightly higher. Literature information on the protease treatment of other Sargassum species confirms that extraction yields of 40-48% can be attained (Ko et al., 2012). The solubles extraction yield attained with enzyme and US enzyme assisted extraction were comparable to the values reported for conventional solvent extraction yields with 80% ethanol, in the range 20-36% (Piao et al., 2011; Balboa et al., 2013). The beneficial effects of sonication has been reported in relation to: i) the activation of the enzymes due to the facilitated combination of substrate and enzyme, reducing processing times and increasing efficiency, and ii) the disruption of the cell structures and increased accessibility of the solvent to the internal structure, enhancing the intra-

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particle diffusivity and promoting the release of soluble compounds. Also unfavourable effects could occur, including: i) degradation of active compounds and ii) inactivation of enzymes. At prolonged times, probably the deactivating effect of US became more marked than the mechanical effects. The maximum temperature was determined by the optimal value for the enzyme, avoiding values higher than 50 °C, although in the present study thermostability concerns could also be associated to phenolics and components from the phlorotannin fraction. Phenolic compounds destruction can occur either directly by thermal decomposition, or indirectly by the production and/or enhancement of oxidative species (Kidak and Ince, 2006). Although the mechanisms of enzyme inactivation by ultrasound are not clear, most of them can be attributed to the mechanical and chemical effects of cavitation, which can produce free radicals and molecules such as OH· and H· radicals, susceptible of initiating radical chain reactions. Both the breakage of polysaccharide structures and decompositions of some phenolic components could occur induced by radical mediated reactions. Degradation of phenolic antioxidants and ascorbic acid in food products and modifications in the fatty acids profile with an increase in saturated ones in vegetal oil has been observed after sonication (Pingret et al., 2013). When US are applied in a continuous mode, the maximum extraction times for phenolic compounds have been established in several minutes (Jang et al., 2013), since additional reductions at prolonged times were observed (Liu et al., 2010).

50 30 0.06 0.16 45 0.14 ) 0.05 40 25

60000 160000 0.12 (1.0:0.4:0.0:0.5)

35 (1.0:0.3:0.0:0.3) (1.0:0.3:0.0:0.6) 0.04

140000 (1.0:0.3:0.0:0.6) (0.8:0.5:0.0:1.0) 20 (1.0:0.06:0.0:0.07) 0.10 Sm, AESm Sm, 30 50000 120000 (1.0:0.3:0.0:0.6)

(1.0:0.3:0.0:.4) 0.03 0.08

25 15 40000 100000 (1.0:0.2:0.7:0.07)

Extraction yield Extraction 20 80000 0.06 30000 0.02 10

(g extract/100g extract/100g (g 15 60000 0.04

20000 (0.0:1.0:0.0:0.9)

(1.0:0.8:0.0:0.3) 40000

(1.0:0.9:0.0:0.3) Oligomeric sugar area 10 (0.9:1.0:0.0:0.3)

Phenolic content (g GAE/ g extract) g GAE/ (g content Phenolic 0.01

(0.9:0:0.0:0.3) Monomeric Monomeric sugar area

Antioxidant activity (g Trolox/ g extract) g Trolox/ (g activity Antioxidant 0.02

5 (1.0:0.8:0.0:0.0)

10000 (1.0:0.9:0.0:0.0) 20000

5 Protein content ( g BSAE/ 100g extract) 100g BSAE/ g ( content Protein 0 0.00 0.00 0 0 0 Sm AESm Sm AESm Sm AESm Sm AESm Sm AESm Sm AESm a b e f c d 45 30 0.06 0.14

40 0.12 25 0.05 35 160000

120000 0.10

(1.0:0.6:0.0:0.6) (1.0:0.3:0.0:0.4) 30 140000 (1.0:0.5:0.0:0.9) 0.04

20 (1.0:0.3:0.0:0.5)

(1.0:0.1:0.1:0.03:0.0) (1.0:0.4:0.0:0.5) 100000 120000 25 (1.0:0.4:0.0:0.6) 0.08 0.03 15 100000 20 80000 0.06 80000 Extraction yield Extraction 15 60000 0.02 10

60000 0.04

(1.0:0.0:0.9:0.0)

(1.0:0.1:0.0:0.0:0.9) (g extract/100g Sm, AESm) Sm, extract/100g (g

10 40000 (0.0:0.8:0.0:0.1) 40000 Oligomeric sugar area 0.01

5 extract) g GAE/ (g content Phenolic

(1.0:0.3:0.0:0.0) Monomeric Monomeric sugar area

(1.0:0.6:0.0:0.2) 0.02

Antioxidant activity (g Trolox/ g extract) g Trolox/ (g activity Antioxidant Protein content (BSAE/ 100g extract) 100g (BSAE/ content Protein 5 (1.0:0.5:0.0:0.1)

20000 (0.8:0.8:0.0:0.0:0.3) 20000

(1.0:0.9:0.0:0.3) (1.0:0.9:0.0:0.2)

0 0 0.00 0 0 0.00 Sm AESm Sm AESm Sm AESm Sm AESm Sm AESm Sm AESm

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Figure 4.1.3.Effect of different treatments ( control,  enzyme assisted extraction, ultrasound assisted extraction and enzyme and ultrasound assisted extraction) on a) the extraction yield, b) protein content, c) phenolic content of the extracts, d) ABTS radical scavenging capacity, e) monomeric and f) oligomer sugars, expressed as sum of the peak areas (Retention times: 10.3, 10.9, 12.0, 12.4, 15.7), when the whole alga, Sm, and the alginate exhausted solids, AESm, were processed. Samples were incubated with Ce at 2% (upper row) and with RTF at 5% (lower row) during 3 h

4.1.4.6. Microstructural changes Microstructural features of the control and enzyme treated Sm tallus samples are shown in Figure 4.1.4. Despite no dramatic differences are observe, enzyme treated samples seem to present a more eroded surface.

Figure 4.1.4.Scanning electron images of control (upper row) and enzyme treated (lower row) Sm samples

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4.1.4.7. Characterization of extracts The phenolic content was under 5% of the extract (Figure 4.1.3), a value lower than the 11-12% reported for protease hydrolyzates from H. fusiformis (Siriwardhana et al., 2004), and from Sargassum coreanum protease hydrolyzates (Ko et al., 2012). The phenolic content and radical scavenging capacity was similar regardless the raw material and the extraction technologies, except US assisted processes. The protein content of the extracts was higher than that observed for a crude protease extract from S. coreanum (Ko et al., 2012). The oligomeric sugar content was higher than the monomeric sugars regardless the enzyme and substrate used. Fucose was mostly present in the oligomeric fractions and the sum of xylose, galactose and mannose in the monomeric. The influence of enzyme treatment was only significant on the AESm samples. As a general trend, non significant differences in composition were observed as a result of ultrasound application. Lipids are minor components in brown algae but they contain higher levels of essential polyunsaturated fatty acids than earth vegetables. The detailed percentual composition is shown in Figure 4.1.5. Palmitic acid (C16:0) was the most abundant saturated fatty acid accounting for 35-45%, as expected based on the composition of the lipids of this alga (data not published), and the content in extracts from AESm was significantly higher than from Sm. However palmitoleic acid (C16:1) showed an opposite trend, also different from other monousaturared fatty acids. The negative effect of ultrasounds was noticed on the saturated fatty acids C16:0 and C18:0. An average mass degradation of more than 20% was reported of palmitic and for stearic acids (Kwiatkowski et al., 2013), although no significant changes in oil composition have been reported on oil from seeds (Luque-García and Luque de Castro, 2004). Oleic acid (18:1, ω9) content was higher in extracts from AESm than in those from Sm. The linolenic acid (18:3, ω3) content was higher than linoleic acid (18:2, ω6) in extracts from Sm than in those from AESm samples. Arachidonic acid (C20:4), the most abundant among ω6 fatty acid accounted for 8-9% of the fatty acids, and eicosapentaenoic (20:5, ω3) contents in the extracts did not depend on the sample processing. Eicosatrienoic acid (20:3, ω3) was not found, although it was present in the

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initial alga and in the enzymatic extracts docosenoic acid (22:1, ω9) was found in low amounts. Nutritional standards recommend a ω3:ω6 ratio of 1:1.5 to 1:5. This desirable ratio was observed for S. muticum (1:1.2 to1: 2.95).

Figure 4.1.5.Effect of ultrasound (US) assisted enzyme (E) treatment of Sm and AESm on the fatty acid composition of the extracts

Extracts from cellulase digested samples and from control samples contained a similar ω3:ω6 ratio, close to 1:1, regardless the use of ultrasound assisted extraction processes, whereas extracts from AESm samples presented a significantly lower ratio. The IC50 values for cytotoxity with the MTT assays are shown in Table 4.1 for the extracts produced with the selected cellulose and protease formulations. No significant differences in the cytotoxicity values of the extracts obtained with the aid of ultrasounds were observed. The IC50 value on liposarcoma and melanoma cells for enzyme extracted products was 50-500 µg/mL, except for AESm extracts on BF10 cells, whereas toxicity on human foreskin fibroblasts HFF-1 was detected at more than 500 g/L. The cytotoxic effect of Sargassum polycystum ethyl acetate fractions in this range of concentrations on B6F10 cells was reported (Chan et al., 2011). Also the ethyl acetate extracts of Sargassum thunbergii showed in vitro cytotoxicity against B16F10 cells in the MTT assay (Kim et al., 2009). These values were only slightly lower than - 82 -

those showing cytotoxicity on human fibroblasts. However, lower IC50 values (< 30 μg/mL) are usually considered significant for the selection of crude extracts (Guedes et al., 2013).

Table 4.1.1 IC50 (g/mL) values obtained by MTT assays for the S. muticum extracts produced

with the aid of enzymes in different cell lines

Non US assisted US assisted

Sm AESm Sm AESm

Control Enzyme Control Enzyme Control Enzyme Control Enzyme

B6F10 100- 50-500 ≥ 1000 > 1000 500/1000 50/500 1000 1000 500

SW872 ≥250 >250 100-250 ≥250 >250 50-250 ≥250 >250

HFF-1 250- ≥ 500 >500 >500 250-500 >500 > 500 > 500 500

4.1.5. CONCLUSIONS Enzyme aided extraction of Sargassum muticum was feasible to provide soluble fractions with radical scavenging properties and an enzyme and ultrasound assisted process proved more efficient to enhance selectively the phenolic extraction than the enzyme aided process. Cellulolytic and proteolytic commercial formulations successfully dissolved up to 10-15 % of the crude alga and up to 50% of the algae without alginate. The dried extracts contained up to 70% oligomeric carbohydrates, 20% protein and 5% phenolics. The use of ultrasounds positively enhanced the solubilisation of both raw materials, but neither the antioxidant or antiproliferative properties were significantly influenced.

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4.2. In vitro bioactive properties of phlorotannins recovered from hydrothermal treatment of Sargassum muticum

4.2.1. SUMMARY The present work addresses the potential of different adsorbents for the recovery of the phlorotannin fraction present in the liquid phase from the autohydrolysis of Sargassum muticum. Operating with the extract generated during autohydrolysis of alginate exhausted alga, the kinetics followed pseudo-second order and the equilibrium corresponded to Freundlich model. In batch configuration, up to 70 % phlorotannins were adsorbed and up to 30 % were desorbed with ethanolic solutions. Comparable performance was observed with the autohydrolysis extracts from the whole algae. The phloroglucinol content, the reducing and ABTS antiradical capacities of the desorbed product increased by 2-4 times in relation to the values in the autohydrolysis extracts. Dynamic elution with 68 % ethanol allowed 80 % desorption and provided a concentrated extract with antiradical capacity equivalent to 1 g Trolox / g extract. This product showed anti-proliferative properties against lung adenocarcinoma A549 cells and against colon carcinoma HCT-116 cells. It also inhibited the production of reactive oxygen species by human neutrophils and inhibited prostaglandin E2 production via cyclooxygenase 2 in human whole blood.

4.2.2. STATE OF THE ART The valorization of the invasive brown seaweed Sargassum muticum (Yendo) Fensholt has been proposed, since its eradication by other means proved to be unfeasible (Critchley et al., 1986). The first stage of a biorefinery approach processing was based on the hydrothermal treatment (autohydrolysis) of the wet material using pressurized water to solubilize the alginate, fucoidan (Balboa et al., 2013) and phenolic (González- López et al., 2012) fractions, providing an optimal scenario for the integral utilization of this macroalga (Pérez-López et al., 2014). Antioxidant and antiproliferative actions have been reported for different fractions of Sargassum sp. (Namvar et al., 2013). Brown algae polyphenols, exclusively found in Phaeophyta, are termed phlorotannins and are polymers of phloroglucinol (1,3,5-trihydroxybenzene). The structural groups of

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phlorotannins differ by the type of linkage or by the presence of one or more additional phloroglucinol units (Singh & Bharate, 2006). They have different roles, including protection against UV radiation, herbivores or bacteria attacks, and metal sequestration. Phloroglucinol, diphlorethol, bifuhalol, trifuhalol A and trifuhalol B were isolated from S. muticum (Glombtiza et al., 1978), and more recently fuhalols, hydroxyfuhalols and phlorethols, with degrees of polymerization from 3 to 11, showing particularities depending on the geographical location, were identified in pressurized extracts (Montero et al., 2016). The phenolic content in the autohydrolysis extract phase is around 10 % of the dry extract and other components are oligomeric fucoidan fractions and minerals (González-López et al., 2012). The selective separation of phenolics could provide a more concentrated phenolic product and could be proposed to enhance the purity of fucoidan. Adsorption onto resins for the purification of solvent extracts from plants allows the selective recovery of phenolic components in a flexible scalable technique with simultaneous regeneration of the adsorbent. Nonionic polymeric resins have been successfully used for the recovery and concentration of phenolic fractions from industrial effluents, natural extracts and autohydrolysis of lignocellulosic materials of forestal and agroindustrial origin (Conde et al., 2008; D'Alessandro et al., 2013; Soto et al., 2011 and 2012; Víctor-Ortega et al., 2016). New organoclays prepared by loading the biocompatible surfactant Tween 20 onto the K10 montmorillonite proved to be suitable for the treatment of olive mill waste waters (Calabrese et al., 2013). Different resins have been proposed for the adsorption of phlorotannins, such as polyvinylpolypyrrolidone and macroporous resins (Kim et al., 2014; Toth, 2001). Several biological properties, such as anti-inflammatory and anti-proliferative activities have been found to correlate with the phenolic content of brown algae (Aravindan et al., 2013; Murugan et al., 2013 and 2014), although these seaweeds also contain a variety of other bioactive compounds, such as polysaccharides, sterols, carotenoids, etc. A phlorotannin-rich (18%) Ascophyllum nodosum extract showed inhibitory activity against oxidative stress, inflammation, and senescence. A. nodosum extract prevented tert-butyl hydroperoxide-induced production of reactive oxygen species (ROS) in epithelial cells, lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-α)

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and interleukin (IL)-6 release in macrophages, and also increased nuclear SIRT1 activity in epithelial cells (Dutot et al., 2012). Different phlorotannins isolated from the ethyl acetate fraction of Eisenia bicyclis dose-dependently inhibited LPS-induced nitric oxide (•NO) production. Treatment with 20 M of phlorofucofuroeckol A, isolated from Ecklonia stolonifera, significantly decreased the LPS- induced levels of inflammatory mediators, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and pro- inflammatory cytokines such as IL-1β, IL-6 and TNF-α in RAW 264.7 cells. Phlorofucofuroeckol A inhibited the promoter activities of transcriptional factors [nuclear factor-kappa B (NF-ĸB) and AP-1)] and inhibited activation of Akt and p38 MAPK in LPS-treated RAW 264.7 cells (Kim et al, 2011). The extracts from some species of the genus Sargassum were also found to be anti-inflammatory and were active against some human cancer cell lines growth (Kim et al., 2013; Stirk e al., 2003).

4.2.2. Main goal. To recover and concentrate the phlorortanin fraction from autohydrolysis liquors of S muticum by means of polymeric adsorbents and to select and evaluate polimeric adsorbents in basis os their adsorption capacities and mechanisms. Specific goals. To select the adsorbents with autohydrolysis extract from the alginate free alga to avoid the absorption compentece of some compounds present in the whole alga. To devolpment of a dynamic adsorption process to recover the fraction of phlorotanins from a concentrated extract and to evaluate it`s anti-proliferative and anti-inflammatory properties.

4.2.3. METHODOLOGY 4.2.3.1. Raw materials and standard solutions The samples of Sargassum muticum used in this study were collected in a local beach (Praia da Mourisca, Pontevedra, Spain) in the summer of 2012. Samples were brought to the researc centre were cleaned and washed with tap water. Drained sampled were frozen and stored in closed plastic bags at -4 °C until use. Main reactives and adsorbents used in this study. Adsorbents.

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According to Calabrese et al., 2013, Montmorillonite (MMT) at two different pH (2.5 and 5) and MMT organically modified by adsorption of saponin have been used as potential adsorbents Several food grade resins with different propertires from several suppliers were used. PuroSorb PAD428, PAD500, PAD700, PAD900, PAD910, PAD950 are macroporous, non-ionic, polymeric adsorbent designed to adsorb and separate more hydrophobic organic compounds from aqueous solutions and polar solvents, supplied by PuroSorb (Purolite Corporation, Barcelona, Spain). The non-ionic polymeric resin Sepabeads SP700 (PS-DVB copolymer), supplied by Resindion S.R.L. (Mitsubishi Chemical Corp.), has been selected based on previos results obtained in our research group (Conde et al., 2008, Soto et al., 2012) Physicochemical properties of the adsorbents selects in this work are summarized in Table 4.2.1. Table 4.2.1. Physicochemical characteristics of the resins used.

Surface Pore Porosity Particle Resin name Structure area radius (mL/g) size (µm) (m2/g) (Å) Sepabeads SP700 Styrene-DVB 1200 90 2.2 >250 PuroSorb PAD428 Polystyrenic-Br 720 400 0.7 350-1200 PuroSorb PAD500 Polystyrenic 800 500 1.0 300-500 PuroSorb PAD700 Polystyrenic 550 700 1.2 350-1200 PuroSorb PAD900 Polystyrenic 800 1000 1.5 350-1200 PuroSorb PAD910 Polystyrenic 540 1100 1.6 350-1200 PuroSorb PAD950 Acrylic 535 960 1.3 350-1200

Reactives The following reagents were purchased from Sigma- Aldrich Co. LLC (St. Louis, USA): phloroglucinol (PG), dimethylsulfoxide (DMSO), phorbol-12-myristate-13-acetate (PMA), trizma, D-(+)-glucose, acetylsalicylic acid, gentamicin sulfate, cremophor® EL, lipopolysaccharides from Escherichia coli 026:B6 (LPS), calcium ionophore (A23187), arachidonic acid (AA), RPMI 1640 medium, fetal bovine serum, L-glutamine, thiazolyl blue tetrazolium bromide (MTT). Penicillin and streptomycin were from Hyclone (Thermo Scientific). Staurosporine (Biomar collection, AQUAe) was used as reference

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compound. Absolute ethanol absolute was purchased from Fisher Chemical (Loughborough, Leic, UK) and 96 % ethanol from Scharlab. The thromboxane synthase inhibitor (TXBSI) was obtained by chemical synthesis as previously described (Kato et al., 1985). Luminol was purchased from Fluka Chemie GmbH (Steinheim, Germany). The “PGE2 Enzyme Immunoassay (EIA) Kit” was obtained from Enzo Life Sciences (Lausen, Switzerland).

4.2.3.2. Analitical, extraction and recovery methodolgies 4.2.3.2.1. Extraction and recovery protocols a) Preparation of hydrothermal extracts Whole S. muticum and alginate exhaustes S. muticum were treated by autohydrolysis to obtain the phlorotanin fraction present in this raw material. 1) Solubilization of phlorotanins from whole S. muticum (Sm) by non-isothermal treatment. According to conditions optimized previously in our research group (Balboa et al., 2013), the ground defrosted algae were contacted with water, in a Parr reactor, at a liquid:solid ratio 30:1 ( w:w). The mixture was heated under non isothermal operation up to 220 °C. Next, the reactor was quickly cooled with circulatin tap water up to 60°C, and then opened. The hydrolysate liquor and solids were separated by filtration. The hydrolysate liquor separated by filtration contained an average phenolic concentration of 0.72 g PGE/L. 2) Non-isotermal treatment of alginate exhausted Sargassum muticum (AESm). The alga solid residue from the traditional alginate extraction process algal biomass was treated in non-isotermal conditions according to the operational conditions optimized by González-López et al (2012). The liquid phase contained an average phenolic concentration of 0.47 g phloroglucinol equivalents (PGE)/L.The protocol of alginate extraction was the following: Sm were extracted at room temperature with consecutive additions of formaldehyde 1% (15 h), sulfuric acid 0.2 N (4 h) and sodium carbonate 1% (15 h) in a sequential process with intermediate filtrations and washings of solids using distilled water. b) Recovery of bioactive compounds solubilized: Adsorption and desorption studies. b.1 Static studies (Batch adsorption)

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Two kinds of adsorbents, several food grade resins and an organoclay composite, were evaluated to determine the interation between the target compounds solubilized in the hydrolyzates and the adsorbents. Previously their use, resins must be activated according to the following protocol: by the contact with 2 bed volumes (BV) of methanol at 175 rpm at 25 °C during 15 min. Resins were rinsed with 5 BV of deionized water at a liquid to solid ratio of 5 (g/g) before use. The batch adsorption experimente were conducted as follows: an aliquot of the sample solution (5 mL) containing a kwon amount of target compounds was added to 25 mL Erlenmeyer flasks containing 3 g of adsorbents. In order to ensure the adsorption to reach the equilibrium, the flasks were shaken in an orbital shaker 175 rpm at 25 °C, during 40 min, since no differences were found at more prolonged times. Atfer equilibrium was reached, tha samples were filtered through a 0.45 μm membrane filter and the liquid phase was analyzed. Organoclays were used directly or after activation, which consists on a shaking process at 25 °C for 4 h to allow hydration (80 g/L). Batch adsorption tests were performed by promoting the contact of 0.2 g of organoclay with 5 mL aqueous solutions of diluted autohydrolysis extract from AESm (0.42 g PGE/L) or phloroglucinol (2 g PGE/L) (pH 2.5, pH 5, pH 12.8) in Erlenmeyer flasks under shaking (175 rpm) at 25 °C for up to 900 min. Batch adsorption isotherms of phloroglucinol and phloroglucinol equivalents on the testes adsorbents were consequently obtained in the solutions evaluated at 25 °°C by mixing a known amount of resin with 5 mL of sample solutions at various initial concentrations. The amount of phloroglucinol or phloroflucinol equivalents adsorbed at any time (qt , mg phloroglucinol equivalents/g resin) was calculated according to eq. 4.2.1:

qt=((C0-Ct))/W V 4.2.1

where C0 and Ct are the concentrations of phloroglucinol equivalents in solution (mg/L) initially and at t time (min), respectively, V is the volume of the solution (L), and W is the weight of the wet adsorbent (g). Experiments were performed in triplicate.

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The adsorption efficacy at time t was calculated from eq. 4.2.2:

Ads (%) = ((C0-Ct))/C0 * 100 4.2.2

Batch desorption Washing was performed in two stages with deionized water at a water:resin ratio of 3 (g:g) in and orbital shaker (175 rpm) at 25 °C for 20 min. During elution, the resin, saturated at conditions previously selected, was in contact with the ethanol:water solution at a moist resin to ethanol ratio 1:3 (g:mL). The desorbed extract was freeze- dried, resuspended in deionized water and analyzed. The resin was regenerated by overnight contact with 1 M of NaOH and further washing with deionized water.

Dynamic adsorption Fixed-bed absorption runs were carried out in jacketed glass columns (internal diameter 10 mm) and three column lengths of 10, 20 and 30 cm. The solutions of standard phloroglucinol and aqueous S muticum extract at same concentrations mentioned above were fed to the top of the column at a desired flow rate using a F10 pump (Bio-Rad, CA, USA). The feed solution bottles with known contents of phenolic compounds and the column were jacketed for temperature control. The eluent from the column was collected at fixed time intervals using a fraction collector and analyzed spectrometrically to determine the phoroglucinol concentration. Fixed bed column performance is usually evaluated using the breakthrough curves obtained by plotting the dimensionless concentration Ct/C0 versus time or volume of the effluent, where Ct and C0 are the outlet and inlet adsorbate concentration, respectively (Arsuaga et al., 2014). The breakthrough time, tb, was determined when the Ct/C0 ratio reached 10 % of the influent value. The saturation time, often called exhaustion time, te, was estimated when the ratio Ct/C0 achieved 95% value of the normalized ratio. Breakthrough curves modeling. Three simplified models (Bohart-Adams, Yoon-Nelson and Thomas) were applied to reproduce the phenolic adsorption dynamics in the fixed bed column. Experiments were performed in duplicate.

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Dynamic desorption Sepabeads SP700 (packed into a 10 mm x 30 cm column) was saturated with the phlorotannin fraction contained in the autohydrolysis extract from the ground alga. Dynamic elution of the phlorotannin fraction was addressed at room temperature (25 5 °C), using 68 % ethanol at a flow rate of 2.25 mL/min. The eluted samples were collected and analyzed.

4.2.3.2.2. Analytical procedures And DETERMINATION OF BIOACTIVE PROPERTIES 4.2.3.2.2.1 Total phenolic content The total phenolic content was determined by the Folin-Ciocalteu colorimetric method (Singleton and Rossi, 1965) and expressed as gallic acid equivalents (GAE) and as phloroglucinol equivalents PGE (Rodríguez-Bernaldo de Quirós et al., 2010).

4.2.3.2.2.2. Antioxidant activity Ferric reducing antioxidant power (FRAP). The original FRAP method was adapted for analysis of no biological samples. The FRAP reagent was daily prepared by mixing of 25 mL of acetate buffer (0.3 M, pH 3.6), 2,4,6-tripyridyl-s-triazine solution (10 mmol/L) in

2.5 mL of HCl (40 mmol/L) and 2.5 mL of FeCl3·6 H2O (20 mmol/L) in distilled water. This solution was used as a blank. Calibration curves were made with a 1mM solution of ascorbic acid (AA). Samples (100 μL) were mixed with 3 mL of FRAP reagent and after 6 minutes the absorbance was monitored at 593 nm. Trolox equivalent antioxidant capacity (TEAC). ABTS radical cation (ABTS•+) was produced by the reaction of ABTS (7 mM) stock solution with potassium persulfate (2.45 mM). The ABTS•+ solution was diluted with PBS (pH 7.4) and equilibrated at 30 °C. After addition of 1 mL of diluted ABTS•+ solution to 10 μL of Trolox standards in PBS, the absorbance was read at 734 nm and after 6 min. The activity is expressed as Trolox equivalents, calculated from a standard curve with known amounts of this synthetic antioxidant leading to the same absorbance change as the sample.

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4.2.3.2.2.3. Biological activities Evaluation of neutrophils' oxidative burst - Oxidation of luminol (Anti-inflammatory activity) Venous blood was collected from healthy human volunteers, following informed consent, by antecubital venipuncture, into K3EDTA vacuum tubes. The density gradient centrifugation method was used to isolate human neutrophils as previously described by Ribeiro et al., 2013. Tris-glucose (25 mM Tris, 1.26 mM CaCl2·2 H2O, 5.37 mM KCl,

0.81 mM MgSO4, 140 mM NaCl, and 5.55 mM D-(+)-glucose) was the incubation media used. The measurement of neutrophils’ oxidative burst was undertaken by monitoring ROS- induced oxidation of luminol, as previously described by Ribeiro et al., 2013. Luminol is a chemiluminescent probe and it is used to monitor the intra and extracellular production of reactive species (RS) by neutrophils, namely superoxide anion radical •- • (O2 ), hydrogen peroxide (H2O2), hydroxyl radical (HO ), hypochlorous acid (HOCl), nitric oxide (•NO) and peroxynitrite anion (ONOO-). The reaction mixtures contained neutrophils (1 x 106 cells/mL) and the following reagents at the indicated final concentrations (in a final volume of 250 µL): the extracts desorbed under selected optimal conditions at various concentrations (3.9-62.5 µg/mL), luminol (500 µM) and PMA (160 nM). Cells were pre-incubated with luminol and the tested compounds for 5 min before the addition of PMA. Kinetic readings were immediately initiated after cell stimulation and carried out at 37 °C, under continuous soft shaking. Measurements were taken at the peak of the curve. This peak was observed at around 10 min. Effects are expressed as the percent inhibition of luminol oxidation. Quercetin was used as positive control and the IC50 value found was similar to those already reported by the research group (Ribeiro et al., 2013).

COX-1 and COX-2 assays The assays were performed in human whole blood. The venous blood was collected into heparin-Li+ vacuum tubes, from healthy human volunteers, as described above. Briefly, to detect the effect of the selected extracts in the production of prostaglandin E2 (PGE2), via COX-1, the collected blood (500 μL) was placed in microtubes and

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incubated in a water bath at 37 °C with TXBSI (1 μM) and the extracts desorbed with 68 % ethanol both in static and in dynamic conditions from Sm and AESm autohydrolysis liquids (10-200 μg/mL) in DMSO for 15 min. Then, the ionophore A23187 (12.2 μg/mL) was added and the mixture was incubated for 15 min. Then the samples were placed on ice for 5 min to stop the reaction. Afterwards the samples were centrifuged at 1,000 g for 20 min, at 4 °C. The supernatant was then collected and stored at -20 °C until use (Ribeiro et al., 2015). The evaluation of the effect of the selected extracts in the production of PGE2, via COX-2, was undertaken by placing the collected blood (800 μL) in a six-wells plate and incubating in a humidified incubator at 37 °C with TXBSI (1 μM), acetylsalicylic acid (10 μg/mL), the being extracts desorbed with 68% ethanol both in static and in dynamic conditions from Sm and AESm autohydrolysis extracts (10-200 μg/mL) in DMSO:chremophor in ethanol 1 % (1:10) for 15 min. Then, LPS (10 μg/mL) was added and the mixture was incubated for 5 h, allowing the activation of COX-2. The reaction was stopped by adding DPBS-gentamicin buffer (1 mL) to the samples and placing them on ice for 10 min. The samples were subsequently centrifuged at 1,000g for 15 min, at 4 °C, to separate the blood cells from the supernatant, maintaining the low temperature. The supernatant was then collected and stored at -20 °C until use. The determination of the amount of PGE2 in the samples (thawed plasma supernatants) was measured using the above mentioned commercial EIA kit, according to the manufacturer’s instructions, as an indicator of COX-1 and -2 activities. COX inhibitors, indomethacin (0.125–1 μM) and celecoxib (0.5–50 μM) were used as positive controls and the activities found were similar to those already reported (Ribeiro et al., 2013). Results are expressed as the percent inhibition of control PGE2 production.

4.2.3.2.2.4. Antitumoral activity The cytotoxic activity of the extracts was determined against four human cancer cell lines, A549 (lung adenocarcinoma epithelial cell line), HCT-116 (colon carcinoma), PSN1 (pancreatic adenocarcinoma), and T98G (Caucasian human glioblastoma cells), purchased from the ECACC (European Collection of Cell Cultures). A549 and HCT-116

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cells were grown in RPMI-1640 medium supplemented with 5% FBS, 1% L-glutamine and 1% penicillin/streptomycin. PSN1 cells were grown in the same medium, supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin. T98G cells were grown in RPMI-1640 medium supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin, 1% sodium pyruvate and 1% aminoacid solution. The freeze-dried extracts (5 mg) of autohydrolysis from Sm and AESm and the products desorbed from SP700 with 68 % ethanol were dissolved in 500 µL methanol: DMSO (9:1) to prepare concentrations in the range of 25 - 500 µg/mL, which were incubated during 48 h at 37 °C and 5 % CO2. Staurosporine was used as a positive control and two negative controls were used, one is the specific medium without cells and the other is the untreated cells suspension. The cell viability was determined by a colorimetric assay based on the reduction of (MTT) to formazan by mitochondrial dehydrogenases in viable cells. To previously grown cells, 50 µL of MTT (1 mg/mL) solution were dispensed and incubated in a humidified atmosphere of 5% of CO2 during 2 h at 37 °C. Then, the medium was removed and 100 µL of pure DMSO were added to solubilize the colored compounds generated. Optical density measurements at 490 nm provided a measure of the proliferation and cell survival. The IC50 was calculated as the extract concentration, which inhibits 50% of cellular growth, in relation to non-treated control cells.

4.2.4. RESULTS AND DISCUSSION 4.2.4.1. Recovery of phlorotannins from autohydrolysis liquid extracts In orde to determine the best adsorbent for the recovery of pproglucinol derived compound the batch adsorption in the above mentioned conditions were performed using standard phlorogucinol solutions and AESm autohydrolysis extract diluted with water. The main results on the adsorption efficiency for the assayed absorbents are shown in Figure 4.2.1 where the different adsorbent are compared in basis of their recovering capacity of phloroglucinol from standard solutions and phloroglucinol derived compounds present in autohydrolysis extracts from alginate exhausted Sargassum muticum.

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100 90 80 70 60 50 40

30 Adsorption (%) Adsorption 20 10 0 MMT-5 MMT-2.5 SAP-2.5 PAD428 PAD500 PAD700 PAD900 PAD910 PAD950 SP700 Adsorbents Figure 4.2.1.Comparative performance of organoclays and polymeric resins for the selective adsorption of the phlorotannin fraction from phloroglucinol standard solutions (, PG, 1.4-2.0 g PGE/L) and from alginate exhausted Sargassum muticum autohydrolysis extract (, AH, 0.42 g PGE/L)

According to data no shown in Figure 4.2.1, no difference in adsorption efficiency at pH 2.5 and 12 operating with SAP-2.5 was observed, however MMT performed better at pH 5. On other hand, to the best of our knowledge, the adsorption of phenolic compounds onto different adsorbents was favored at acidic pH, because the phenols are undissociated and the dispersion interactions predominate (Soto et al., 2012). Toht (2001) found that tannins were removed more effectively from solutions with a low pH since phenolics could also bind to the adsorbent molecules with hydrogen bonds. This behavior together with the acid pH of extracts causes the possibility of using these directly without the need to adjust the pH. This is advantageous and as far as we are aware, the adsoption has not been previously tried with liquid phases from autohydrolysis. Experiments with resins were performed at the unmodified pH of these hydrolyzates (pH=6.5). Based on the results, the resins were further used for the recovery of phlorotannins from the autohydrolysis extract from AESm. Adsorption percentages in the range 35-90% have been achieved with PVPP for extracts from A.

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nodosum (Toth, 2001), and 20-60% onto macroporous resins for E. cava extracts (Kim et al., 2014). To evaluate the influence of specific process parameters on adsorption, kinetic paramenters can be used, which are derived from concentration-time curves of adsorption. Figure 4.2.2 shows the percentage of phloroglucinol derived compounds adsorpbed-time curves on different adsorbents. PAD 950 and SP700 showed higher adsorption capacity than other resins at relatively short times. Those is according to contact times (10-30 min) reported by Toth (2001) with PVPP for removing phlorotannins from algal extracts. The adsorption capacity was lower for phloroglucinol (20-32 mg/g) than for the phlorotannins from S. muticum autohydrolysates (43-69 mg/g). Reported values for the phlorotannin fraction up to 100 mg/g were found for the E. cava ethanolic extract onto HP-20 resin (Kim et al., 2014)]. Values in these ranges were also reported for the adsorption of phenolics from terrestrial sources (Soto et al., 2012).

100 PAD 428 PAD 500 PAD 700 PAD 900 PAD 910 PAD 950 SP 700 90 80 70 60 50 40

Adsorption (%) Adsorption 30 20 10 0 0 5 10 15 20 25 30 35 40 45 Time (min) Figure 4.2.2.Adsorption kinetics of phloroglucinol standard solutions onto selected resins: () PAD 428, () PAD500, () PAD700, () PAD900, () PAD910, (●) PAD950, () SP700

Within this work, the pseudo-first order model, the pseudo-second order model according to Ho and McKay, the Elovich model and the intraparticle diffusion model of Weber and Morris were chosen to determine the kinetic parameters of adsorption for the phlorogucinol standard and the plorotanins fraction from the sargassum muticum

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hydrolysate. Experimental data were adjusted to the most frequently used theoretical models. The pseudo-first-order rate equation is described by Lagergren equation:

4.2.3 where, qt and qe are the amount of phenol adsorbed (mg/g) at contact time t (min) and -1 at equilibrium, k1 is the pseudo-first-order rate constant (min ). The pseudo-second order rate equation (Ho & McKay, 1999) is expressed as: tt 1 1  2  (t) 4.2.4 qt k2 qe qe where, t is the contact time (min), qt and qe are the concentrations of phenolics adsorbed (mg/g) at the considered time and at the equilibrium respectively, k2 is the pseudo-second order kinetic parameter. Elovich model (Ho, 2006; Özacar et al., 2008), useful in adsorption onto highly heterogeneous adsorbents or describing chemisorption, was also used:

1   1  4.2.5 qt    ln     ln t  β   β  where,  (mg/(g·min)) is the initial sorption rate and  (mg/g) is related to surface coverage and activation energy for chemisorption. The role of diffusion in the adsorption process was assessed with the intraparticle diffusion model of Weber and Morris (1963):

0.5 qt  ki t  I 4.2.6 where, t is the contact time (min), qt is the amount of phenolics adsorbed (as mg/g) at 0.5 the considered time, ki is the intra-particle diffusion rate constant [mg/(g min )] and I is the intercept (mg/g), which is in relation to the boundary layer thickness. Table 4.2.2 lists the results of the kinetic parameters of the four models. The correlation coefficient values, R, are not satisfied in all models assayed except those from the pseudo second-order model, indicating great deviation of the calculated values from the experimental values. So, the pseudo-second order model provides the best correlation for the adsorption process. When the intraparticle diffusion model was tested, multilinearity indicates that two or more steps took place, suggesting that the sorption process proceeds firstly by adsorption onto the exterior surface and then

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by intraparticle diffusion. With decreased solute concentration in the solution, the diffusion rate became lower and the diffusion processes reached the final equilibrium stage. A third stage where intraparticle diffusion starts to slow down due to extremely low adsorbate concentrations in the solution has also been observed (Özacar et al., 2008).

Table 4.2.2.Adsorption kinetic parameters of the selected resins for adsorption of ploroglucinol from alginate free S. muticum autohydrolysis extract. Pseudo-first Pseudo-second Elovich Intraparticle order order

2 2 2 2 k1 R k2 R α β R Ki I R

PAD 428 0.450 0.419 0.075 0.994 403 0.354 0.699 1.65 14.34 0.533

PAD 500 0.371 0.727 1.09 0.997 39727 0.495 0.908 1.29 19.87 0.829

PAD 700 0.399 0.654 0.69 0.989 1063 0.466 0.801 1.36 13.17 0.726

PAD 900 0.397 0.492 1.22 0.998 2893 0.402 0.815 1.47 17.78 0.634

PAD 950 0.352 0.460 1.18 0.998 2208 0.451 0.774 1.30 15.45 0.594

PAD 950 0.459 0.485 1.38 0.999 6407 0.348 0.799 1.70 22.35 0.629

SP 700 0.472 0.547 1.23 0.998 10386 0.364 0.782 1.70 22.58 0.675

In order to characterize the adsorption equilibrium of phloroglucinol derived compounds onto the resins, the effects of the initial phenolic concentration on its uptake were assessed. The initial concentration was evaluated in the range 300-11000 mg/L for standard solutions of phloroglucinol (Figure 4.2.3) and 100-3000 mg/L for the AESm autohydrolysis extract (Figure 4.2.4). Theoretical model of Freundlich was used to describe the adsoption behavior between target compounds and absorbent. Freundlich isotherm is an empirical equation based on the sorption onto heterogeneous surfaces or surfaces with sites of varied affinities. This model assumes that the stronger binding sites are occupied first and that the binding strength decreases with the increasing degree of site occupation. Adsorption capacity of freundlich isotherme is calculated according the next equation:

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1/N Qe = K Ce 4.2.7 where, qe is the amount of solute adsorbed per unit weight of adsorbent at equilibrium

(mg/g), Ce is the equilibrium concentration of the solute in the bulk solution (mg/L), 1/n is temperature dependent and indicates if the adsorption is favorable or not.

300

250

200

150 qe (mg/g) qe 100

50

0 0 2000 4000 6000 8000 10000 12000 Ce (mg/L)

Figure 4.2.3.Adsorption equilibrium onto polymeric resins of the phlorotannin fraction from the aqueous soluble phase generated during autohydrolysis of alginate exhausted Sargassum muticum. () PAD 428, () PAD500, () PAD700, () PAD900, () PAD910, (●) PAD950, () SP700

Table 4.2.3 lists the coefficients of Freundlich isotherm for the different resins (eq. 4.2.7). As a general trend, higher K values are obtained for the isotherms with the autohydrolysis extracts from AESm than for PG solutions, whereas the n values are similar for both extracts.

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Table 4.2.3. Freundlich parameters for the adsorption of phlorotannin from synthetic solutions and from the autohydrolysis extracts from alginate free Sargassum muticum.

Phloroglucinol standard solution S. muticum autohydrolysis liquor Resin K 1/n R2 K 1/n R2 ((mg/g)/(L/g)n) ((mg/g)/(L/g)n) PAD 428 0.025 1.002 0.9990 4.722 1.049 0.9251 PAD 500 0.028 0.9896 0.9987 5.450 0.997 0.9969 PAD 700 0.025 1.0015 1.000 5.180 0.9853 0.9972 PAD 900 0.025 1.0014 1.000 5.071 0.9969 0.9999 PAD 910 0.024 1.0058 0.9934 0.000 38.096 0.8265 PAD 950 0.028 0.9877 0.9988 1.423 1.0028 0.9991 SP700 0.030 0.9798 0.9956 5.316 0.9873 0.9996

Ethanol is a superior desorbent for macroporous resins because it can be effortlessly removed from the solution, easily recycled and has low cost and no toxicity to the phloroglucinol and phlorotanins fractions which are the target compounds in this work. The phenolic enrichment in desorbed fractions at different ethanol concentration is shown in Figure 4.2.5.

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30 18 A

25 15

) ) )

20 12

extract extract

15 9

6 g(g/100 PGE GAE (g/ 100g (g/ GAE 10

5 3

0 0 AH 40% 68% 96% EtOH

50 B SP 700 TEAC PAD 910 TEAC PAD 950 TEAC 45 SP 700 FRAP PAD 910 FRAP PAD 950 FRAP 40 35 30 25 20

15 TEAC (g/100 g extract) g (g/100TEAC

FRAP (g AA/100 g extract)) g AA/100(g FRAP 10 5 0 40% 68% 96% EtOH

Figure 4.2.5.Effect of ethanol concentration on A) the phenolic content of the desorbed products B) TEAC and FRAP of the desorbed fractions from resins saturated with alginate exhausted S. muticum hydrolyzates. AH refers to hydrolyzates

The ethanol concentration did not significantly influenced the desorption performance, whereas the resin was more determinant. The highest phenolic content in the desorbed products was found using SP700, with values up to 22 % of the extract (expressed as GAE/100 g dry extract) and a lower value when expressed as PGE. The TEAC value increased from 17 to values higher than 35 g Trolox equivalents/100 g extract, in the original autohydrolysis extract to the desorbed product, respectively. Maximum values were obtained when SP 700 was used and a similar behavior for the reducing capacity was observed. In order to propose an operationally simpler alternative, the direct autohydrolysis processing of the alga was considered. A comparative experiment between the adsorption-desorption onto SP700 of the liquid - 102 -

phase generated from autohydrolysis of i) AESm and ii) Sm was carried out. The overall performance of the adsorption stage onto SP700 resins and desorption stage with 68% ethanolic solutions was similar for both autohydrolysis liquids. Therefore, the direct utilization of liquors from autohydrolysis of the whole algae (Sm) could offer a simpler processing alternative (Balboa et al., 2013).

4.2.4.3 Dynamic adsorption 4.2.4.3.1 Adsorption of phloroglucinol standart solutions The adsorption capacity parameter obtained from a batch experiment is useful in providing information about the effectiveness of the adsorbent system. However, the data obtained under batch conditions are generally not applicable to other systems such as column operation, being the need to perform dynamic studies using fixed-bed columns. The breakthrough curves were obtained by plotting the effluent/influent concentration ratio versus time and or elution volume according to the model selected. Experimental data for model solutions of the breakthrough curves are presented in Figures 4.2.6.

Effect of phloroglucinol flow rate and different bed depths on breakthrough curve. In order to determine the effect of the target compounds concentration on the breakthrough curves, solutions of phloroglucinol at concentration of 0.7 g/L were passed through Sepabeads SP700 resin at a bed depth of 10, 20 and 30 cm and flow rates of 1, 2 and 4 mL/min. The effect of adsorbate concentration on the breakthrough curve was evaluated plotting the effluent/influent concentration ratio versus elution time (Figure 4.2.6.a-c). It can be observed that the higher flow rates not provided important diffenences in the breakthrough time (Figure 4.2.6.b-c), while significant differences in the breakthough time were observed for the higher depth columns operating al flow rate of 1 mL/min. Thus, breakthrough curves occurred faster with higher flow rate, so the efficiency of the sorption process was lower at higher flow rate. The lower flow rate used (1 mL/min) allowed a higher time contact between phenolic compounds and resin, and that involved a higher removal of these compounds. Increasing flow rate from 1 mL/min to 2 and 4 mL/min in a 10 cm depth

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column, involved a decreasing in the breakthrough time from 96 min to 41 and 16 min respectively, and besides, increasing the flow rates and decreasing the phenolic concentration in the feed led to steeper breakthrough curves (Figure 4.2.6.a-c). This lower breakthrough time at higher flow rate could be explained on the basis of the improved diffusion of phenolic compounds into the pores of resin.

a) b) A) b)

c)

Figure 4.2.6.Effect of height packed bed column on the breakthrough curve shapes at three differents flow rate

From the figure 4.2.6.a-c, it was seen that breakthrough time varied greatly with the bed height at the lowest flow rate. Increasing the bed depth the solution had more time to contact with the resin that resulted in higher removal efficiency of phloroglucinol. However the bed depth had not significant effect on the breakthrough time at higher flow rates. - 104 -

The slope of breakthrough curve maked more gentle with increasing bed height independently of the flow rate, which resulted in a narrowest mass transfer zone. Higher uptake was observed at the highest bed height due to an increase in the binding sites for the sorption due to a higher surface area available.

Dynamic adsoption of phlorotannin fraction from S. muticum extracts. Effects of flow rate and bed depths on breakthrough curve. Initially, the effect of flow rates on the phlorotannin fraction from S. muticum extracts adsorption of Sepabeads SP700 resin in the bed packed column was examined at natural pH pf the extract aqueous solution by varying the flow rate from 1–4.0 mL/min while the temperature and the phenolic concentration of feed expressed as phloroglucinol equivalents were held constant. Fig. 4.2.7 shows the breakthrough curve obtained by plotting C/C0 versus time at different flow rates. It was seen from Figure 4.2.4.a-b that breakthrough curves generally occurred faster with higher flow rate. Breakthrough time for reaching saturation was increased significantly with a decrease in the flow rate. When at a lower rate of influent, the compouds from the phloroglucion fraction had more time to contact with the sepabeads resin and resulted in higher adsorption efficiency of target compounds in the fixed bed. The variation in the slope of the breakthrough curve may be explained on the basis of mass transfer fundamentals. The reason is that at higher flow rate, the rate of mass transfer tends to increase. The amount of phloroglucinol derived compounds adsorbed onto the unit bed height (mass transfer zone) increased with increasing flow rate leading to faster saturation at a higher flow rate (Ko et al., 2000).

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Figure 4.2.7.Effect of height packed bed column and flow rate on the breakthrough curve shapes of phlorotanin adsorption from S muticum hydrolysate

The breakthrough curves at different bed depths were also shown in Fig. 4.2.7.a-b. For this cases, the influent flow rates were the mentioned above. It was seen that the breakthrough curves for higher ber showed a higher breaktrough time, which can be explained based on a more time to contact between the absorbent and the solutes.

As can be seen from these figures, the breakthrough time (elution time when C/Co =

0.1*C/Co initial) and the exhaustion time (elution time when C/Co = 0.95*C/Co initial) were found to increase with increasing bed depth. For the higher bed dept, more resin was used. So, there were more sites for holding target compounds, resulting in greater uptake capacity.

Testing of breakthrough curves by mathematical empirical models The empirical models by Bohart-Adams, Thomas and Yoon-Nelson have been used for a mathematical description of the experimental data previously showed for adsorption of phlorogluciinol and phlorotanin fractions from standard solutions and aqueous S. muticum extracs respectively. Calculations were carried out by linear regression analysis. The regression coefficient R2 was calculated as indicator of fitting of experimental points with mathematical equations of models given in Table 4.2.4 and 4.2.5. Slope and intercept were used for calculation of model parameters, and they are also shown.

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Each model allows the determination of different parameters on the fixed-bed adsorption of phloroglucinol and phlorotanins onto the Sepabeads SP700 resin. Thus,

the Adams-Bohart model predicts the saturation concentration (N0); the Yoon- Nelson model simulates the time for 50% breakthrough () and the Yoon-Nelson rate constant

(kYN); and The Thomas model allows the prediction of the maximum adsorption

capacity of the column (qTh) and the Thomas rate constant (kTh); Table 4.2.x lists the relevant estimated kinetic parameters as well as the regression coefficients. These parameters have been inserted into equations of breakthrough curves described in material and method section, and for the chosen values of time

and volume, the values of C/C0 have been calculated for each kinetic equation. The

calculated values of C/C0 versus t or V have been used to plot the breakthrough curves that were then compared with the experimental ones in Figs 4.2.8 to Fig 4.2.11. a) Phloroglucinols solutions Figures 4.2.8a, 4.2.9a, and Figure 4.2.10.a, shows the comparison between the experimental data and the predicted data by the Bohart-Adams and Yoon-Nelson models. The figures show a reasonably good agreement of the Bohart-Adams and Yoon-Nelson models with experimental points for the initial values on the x-axis in the simulation of breakthrough curves for bed depths of 10, 20 and 30 mm. The Adams-Bohart adsorption allows fitting the experimental data of the initial part of the breakthrough curve. This approach was focused on the estimation of maximum

adsorption capacity (N0) and kinetic constant (kAB), listed in Table4.2.4, at different flow rates and bed.

The kinetic constant of the Sepabeads SP700 (kAB) and the maximun adsorption capacity had not a regular behavior with increasing flow rates and bed depths. For the lower bed depth the kinetic constant increased with increasing flow rate while the adsorption capacity did follow the same behavior. The fact that the kinetic constant increased with increasing flow rate showed that the overall system kinetics is dominated by external mass transfer in the initial part of adsorption in the column (Lin et al., 2013) in the shortest column. The values of correlation coefficients ranged between 0.8246 and 0.9835.

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Figure 4.2.8.Experimental data and modeled breakthrough curves for the sorption of phlortanin standard solution on a 10 cm fixed bed column od Sepabeads SP700 at feed flow of 1, 2 and 4 mL/min

From a mathematical point of view, the Yoon-Nelson model is analogous to the Bohart-Adamds model, but its main advantage is that it is less complicated than other models, and also requires no detailed data concerning the characteristics of the kind of solute adsorbed, the type of the adsorbent, and the physical properties of the packed bed.

The rate constant KYN increased with increased flow rate and bed height except to in the 30cm bed depth, in this case there is not a regular behaviour. On the contrary to the rate constants behavior, the time required for 50% breakthrough decreased with increased flow rate and bed height.

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The values of correlation coefficients ranged between 0.9376 and 0.9975.

Figure 4.2.9.Experimental data and modeled breakthrough curves for the sorption of phlortanin standard solution on a 20 cm fixed bed column of Sepabeads SP700 at feed flow of 1, 2 and 4 mL/min

The Thomas model is based on the Langmuir isotherm for equilibrium systems and follows second-order reversible reaction kinetics. It describes the adsorption- desorption process where no axial dispersion is present. The coefficient of linear regression (r2) ranged between 0.9333 and 0.9973. A quite good agreement between the predicted and experimental data at experimental conditions assayed was observed for the initial part of the breakthrough curve (Figure 4.2.8-10.b). Thomas rate constant, kTh is dependent on flow rate and bed depth. On the same bed depth, the rate constant decreased with increasing the flow rate. This is because the residence time of solute in the bed was less. In the same way, on the same flow rate, the kTh decreased with increasing the bed height. According to these results showed above, and assuming that the best correlations between predicted model and experimental data have found in the application of Yoon-Nelson model, we can affirm that the phoroglucinol adsorption on packed bed of Sepabeads SP700 did follow a second – order rate equation and

The maximum adsorption capacity, qTh, increased with increase in flow rate but showed an irregular behabiour decreased with increase in bed depth. In general trend,

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at higher bed heights, more reactive sites were available and the maximun adsorption capacity would be increased.

Figure 4.2.10.Experimental data and modeled breakthrough curves for the sorption of phlortanin standard solution on a 30 cm fixed bed column od Sepabeads SP700 at feed flow of 1, 2 and 4 mL/min

Table 4.2.4.Values of the Bohart–Adams, Yoon-Nelson and Thomas models parameters for the sorption of phoroglucinol onto SP700 resin

Bohart-Adams model Yoon-Nelson model Thomas model

h Q kBA KTh qTh N K  cm mL/m L/ o r2 YN q r2 mL/mg· mg/g r2 g/L min-1 min in g·min min 10 1 0.1010 8.22 0.9835 0.0952 81.7 7.15 0.9854 0.1556 6.75 0.9475 2 0.1703 9.65 0.9754 0.1949 46.3 8.10 0.9679 0.1476 16.2 0.9679 4 0.2235 8.60 0.8246 0.2067 19.7 6.88 0.9975 0.0699 24.8 0.9973 20 1 0.0569 6.86 0.9902 0.0494 137.6 6.88 0.9676 0.0858 6.33 0.9457 2 0.1101 6.53 0.9833 0.1140 60.2 6.023 0.9837 0.0844 11.8 0.9768 4 0.1031 8.64 0.9746 0.1348 34.8 6.953 0.9686 0.0536 27.6 0.9648 30 1 0.0607 8.48 0.9500 0.0569 268.8 9.41 0.9938 0.0798 9.40 0.9856 2 0.0354 7.05 0.9418 0.0460 83.1 5.816 0.9376 0.03597 11.5 0.9333 4 0.0477 10.88 0.8977 0.0569 63.4 8.872 0.9197 0.02226 35.6 0.9391

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a) Aqueous Sargasum muticum extracts In the Figure 4.2.11.a-d shows the predicted curves obtained by fitting the experimental data with the mathematical model selected. The values of correlation coefficients for the Bohart-Adams model ranged between 0.7016 and 0.9930 (Table 4.2.4, and Figure 4.2.11.a and b). With the data found for the aqueous extract of Sargasum mutricum is not possible to stablish a behavior pattern for the kinetic constants calculated by this model whit increasing the flow rate. Nevertheless, the kinetic constant decrease with increasing the bed depth

a b , ,

c d , ,

Figure 4.2.11.Experimental data and modeled breakthrough curves for the sorption of Sargassum muticum extract on a 10 cm (a, b) and 20 cm (c-d) fixed bed column of Sepabeads SP700 at feed flow of 1, 2 and 4 mLmin

- 111 -

The rate constant KYN of Yoon-Nelson model decrease with increasing the bed height, but as in the Bohart-Adams model there was not a well-defined behavior of kinetic constant. On the contrary, the time required for 50% breakthrough always decreased with increased flow rate and bed height. The values of correlation coefficients ranged between 0.9399 and 0.9988.

Table 4.2.5.Values of the Bohart–Adams, Yoon-Nelson and Thomas models parameters for the sorption of phoroglucinol onto SP700 resin

Bohart-Adams model Yoon-Nelson model Thomas model

2 h Q kBA No r KTh qTh KYN  2 2 cm mL/min L/g·min g/L -1 q r mL/mg·min mg/g r min min 10 1 0.2112 4.79 0.7016 0.0141 206.4 18.6 0.9399 0.01889 18.5 0.9273 2 0.0362 33.3 0.9930 0.0156 205.1 36.9 0.9772 0.01298 73.7 0.9672 4 0.1176 13.8 0.8588 0.0377 53.9 19.4 0.9730 0.01284 77.5 0.9879 20 1 0.00956 87.2 0.9732 0.0083 93.97 93.9 0.988 0.0079 99.7 0.9735 2 0.01417 119.6 0.9655 0.01213 128.9 128.9 0.9785 0.0050 286 0.9258 4 0.0054 133.3 0.8919 0.0041 130.3 130.9 0.9677 0.0015 508 0.9474

4.2.4.4. Dynamic desorption The SP700 resins were previously compacted in a glass column and saturated with autohydrolysis extract prepared from Sargassum muticum samples. During elution with 68 % ethanol, fractions were collected and analyzed for total phenolics, expressed as phloroglucinol equivalents (Figure 4.2.12). The fractions collected during the first four minutes of dynamic elution were combined and freeze-dried to yield the desorbed product, which was further analyzed. The autohydrolysis extract once dried showed a phenolic content of 0.117 ± 0.002 g GAE/g or 0.053 ± 0.005 g PGE/g and the ABTS radical scavenging capacity was equivalent to 0.173 ± 0.014 g Trolox/g. The phenolic content of the desorbed product was 0.359 ± 0.018 g GAE/g extract or 0.168 ± 0.008 g PGE/g extract and the ABTS radical scavenging capacity was equivalent to 1.056 ± 0.053 g Trolox/g extract. - 112 -

12

10

8

6

4 Concentration (g PGE/L)(g Concentration 2

0 0 5 10 15 20 25 30 35 40 Time (min)

Figure 4.2.12.Desorption of the phlorotannin fraction from S. muticum hydrolyzates adsorbed onto SP700 resins and eluted with 68% ethanol

4.2.4.4. Biological properties 4.2.4.4.1. Antitumoral activity The effect of the different algal extracts (autohydrolysis extract and desorbed products) on the viability of four human cancer cell lines was determined using an MTT assay. None of the extracts significantly influenced the activity of pancreatic cancer and glioblastoma cells. However, the viability of lung adenocarcinoma cells was reduced by 30-40%, in the presence of the autohydrolysis extract both from the alginate free alga (AESm) and from the whole alga (Sm). Colon carcinoma cells were inhibited by more than 50% in the presence of all extracts at 250 and 500 g/mL, except by AESm autohydrolysis liquid (Figure 4.2.13). The inhibitory potential of the extracts was lower than those of commercial standards. The control IC50 values for staurosporine were 0.001 g/mL against A549, PSN1 and T98G cells, and 0.005 g/mL against HCT-116 cells.

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100 100

90 A 90 B

80 80

70 70

60 60

50 50

40 40

Cell viabity (%) viabity Cell Cell viabity (%) viabity Cell 30 30

20 20

10 10 1A5492 3 4 HTC1 -21163 4 0 0 0 100 200 300 400 500 600 0 100 200 300 400 500 600 Concentration (µg/mL) Concentration (µg/mL)

100 100 90 C 90 D 80 80

70 70

60 60

50 50

40 40

Cell viabity (%) viabity Cell Cell viabity (%) viabity Cell 30 30

20 20

10 10 1PSN12 3 4 1 T98G2 3 4 0 0 0 100 200 300 400 500 600 0 100 200 300 400 500 600 Concentration (µg/mL) Concentration (µg/mL)

Figure 4.2.13 Effect of the extract concentration on the cell viability of human tumor cell lines: A) A549 (lung adenocarcinoma epithelial), B) HCT-116 (colon carcinoma), C) PSN1 (pancreatic adenocarcinoma), and D) T98G (Caucasian human glioblastoma). () AESm autohydrolysis liquor, () Sm autohydrolysis liquor, (●) AESm autohydrolysis liquor, adsorbed onto SP700 and desorbed with 68 % ethanol in batch conditions, () Sm autohydrolysis liquor, adsorbed onto SP700 and desorbed with 68 % ethanol under dynamic operation

Literature reports on the correlation of the antitumoral properties with the phenolic content of macroalgal extracts. Antiproliferative activities of methanol, chloroform, ethyl acetate, and aqueous extracts from Caulerpa peltata, Gelidiella acerosa, Padina gymnospora, and Sargassum wightii at 100 g/mL was confirmed in human cancer cell lines. Chloroform and ethyl acetate extracts showed higher antiradical and growth inhibitory activities in lung adenocarcinoma A549 cells and colon adenocarcinoma HCT-15 cells (Murugan and Iyer, 2014). The higher growth inhibition of methanol, chloroform, ethyl acetate and aqueous extracts of Turbinaria ornata, Kappaphycus alvarezii, Acanthophora spicifera and Gracilaria corticata on lung adenocarcinoma

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A549 and osteosarcoma MG-63 cells was observed in extracts with higher phenolic content, reducing and antioxidant capacity (Murugan and Iyer, 2014). Both the anti- inflammatory and antiproliferative efficacies of the chloroform, ethyl acetate and methanol extracts from Cystoseira compressa correlated with their total phenol content (Dellai et al., 2012). The IC50 values for growth inhibition of lung carcinoma A549 cells, colon carcinoma HCT15 cells and breast adenocarcinoma MCF7 cells ranged from 78-82 mg/mL; 27-50 mg/mL and 110-130 mg/mL, respectively. Antioxidant-rich polyphenol fractions retard pancreatic cancer cell viability, although less efficiently than gallic acid (Aravindan et al., 2013). Ex vivo exposure of pancreatic cancer stem cells to the ethyl acetate fractions of Hormophysa triquerta, Spatoglossum asperum or Padina tetrastromatica, exhibited dose-dependent inhibition of cell viability at polyphenolcs concentrations of 10, 50, 100 g/mL (Aravindan et al., 2015). Our results suggest that increased phenolic concentration in the resin desorbed products led to higher antitumoral properties against HCT-116 cells, but not against A549 cells. Therefore other components also present in the autohydrolysis liquids, such as fucoidans could be responsible for this effect (Atashrazm et al., 2015; Balboa et al., 2013).

4.2.4.4.2. Anti-inflammatory activity In order to evaluate the anti-inflammatory effects of the selected extracts (S. muticum desorbed fractions from AESm autohydrolysis extract in batch conditions and the desorbed peak from Sm autohydrolysis extract in dynamic experiments), two models were selected: human neutrophils and human whole blood. Human neutrophils are the first cells arriving at the inflammation site and are the main producers of RS responsible for the resolution of the inflammatory process. However, in a chronic state, these RS are overproduced and become deleterious to the organism and their removal become vital to solve inflammation. So these cells may be considered one of the best models to study the modulation of RS production (Ribeiro et al., 2015b). The human neutrophils’ oxidative burst was effectively modulated by the two selected extracts. The S. muticum desorbed fractions from AESm autohydrolysis extract and the desorbed peak from Sm autohydrolysis extract in

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dynamic experiments presented an inhibition of luminol oxidation of 45.52  5.86 and 79.44  2.87 %, respectively, for the highest tested concentration, 62.5 µM (Figure 4.2.8b). Contrarily to the cytotoxic effects, a relationship between the inhibition of luminol oxidation and the phenolic content was observed. COXs are crucial enzymes in the initiation and progression of the inflammatory process. They are responsible for the production of prostaglandins (PGs) which levels are truly augmented due to COX-2 induction under inflammatory stimuli. PGs exert their effects on the cell of origin or nearby cellular structures. In this sense, to understand the effect of the selected extracts on PGE2 production, a matrix most approximate to the physiological conditions was selected, human whole blood (Ribeiro et al., 2015b). From all the tested extracts, only those containing the highest phenolic content, the fractions obtained during dynamic desorption from Sm autohydrolysis extract, showed inhibitory activity of PGE2 production in human whole blood. This result is even more interesting considering this extract is selective for COX-2. A steady increase in COX-2 inhibition was observed at increasing concentrations from 10 to 200 mg/mL (Figure 4.2.14.a). COX-1 is a constitutive enzyme with other physiological functions, and so its inhibition is more interesting in another kind of diseases, such as coagulation disturbances. On the contrary, COX-2 expression is induced under inflammatory stimuli and its activity is augmented, as already stated, so its inhibition is considered an excellent strategy to favor inflammation resolution. The phlorotannins, 6,6-bieckol, phloroeckol, dieckol and phlorofucofuroeckol, isolated from Ecklonia cava, were previously shown to significantl inhibit hyperglycemia- stimulated oxidative stress, reducing the induced production of ROS, and cell death in zebrafish. Dieckol also significantly reduced the overexpression of iNOS and COX-2 (Aravindan et al., 2013). Eckol, phlorofucofuroeckol A, dieckol and 8,8'-bieckol isolated from Eisenia bicyclis proved to interfere in the PGs production cascade by inhibiting the secretory phospholipase A2 from porcine pancreas. However, they only slightly inhibited COX-1 and COX-2, except for dieckol that inhibited COX-1 in 74.7% and for eckol that inhibited COX-2 in 43.2%, at 100 M (Shibata et al., 2003). Various studies in RAW 264.7 macrophage cell line have been published describing the inhibitory effects of the E. stolonifera extract, the ethanolic extract of Sargassum horneri on COX-2

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mRNA level, possibly through the interference in the NF-ĸB pathways (Kim et al- 2014; Yang et al., 2010).

100 90 80 70 60

50 Inhibitionof PGE

- 40 2 2

- 30 productioncontrol)(% COX 20 10 0 200 150 100 10 Conc. (mg/mL) 100 AH Sm AH AESm 90 80 70 60 50 40 30

neutrophils(%inhibition) 20 Luminoloxidation by activated 10 0 62.5 31.3 15.6 7.8 3.9 Conc. (µg/mL)

Figure 4.2.14 Inhibitory action of the desorbed extracts from the autohydrolysis liquid phase generated from AESm () and from Sm () on A PGE2 production - via COX-2 and B neutrophils oxidative burst

Stirk et al. [2003] have also proved that the ethanolic extracts from macroalgae collected from the KwaZulu-Natal coast and the Western Cape coast were very active in the COX-1 anti-inflammatory assay whereas the aqueous extracts were not active. The ethanolic extract of Sargassum sagamianum inhibited COX-2 and iNOS expression in LPS-induced RAW 264.7 cells without affecting cell viability. In addition, the expression of NF-ĸB p65 was suppressed by SA-E extract. Furthermore, the rate of - 117 -

formation of edema in the mouse ear was reduced by 46% at the highest dose tested (250 mg/kg) compared to that in the control (Kim et al., 2013).

4.2.5. CONCLUSIONS The phlorotannin fraction solubilized in autohydrolysis treatment of Sargassum muticum was retained in polymeric resins and desorbed with ethanolic solutions. Commercial resins were more efficient adsorbents than organoclays; the adsorption kinetics followed a pseudo-second order and the equilibrium was described by the Freundlich model. During batch desorption from SP700 resins neither the ethanol concentration nor the type of raw material used to prepare the autohydrolysis extract was significantly influencing the process performance. The phenolic content and reducing capacity of the desorbed product were four times higher than in the initial autohydrolysis extract, and the antiradical capacity was increased in relation to that of the autohydrolysis extract. Dynamic elution allowed the preparation of an extract which showed promising antitumoral and anti-inflammatory activities, through the inhibition of cancer cell lines growth and inhibition of RS and PGE2 production.

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4.3. Solvent fractionation of ethanolic extracts from Acacia dealbata flowers

4.3.1. ABSTRACT Acacia dealbata flowers were extracted with ethanol and the solid residue was further extracted with i) a sequence of organic solvents and ii) a sequence of greener stages (aqueous, acid and microwave hydrogravity extraction). The extraction yield and the phenolic content and antiradical activity of the extracts were analysed. The ethanolic extracts presented more than 25% phenolic content and showed 85% of the ABTS radical scavenging potency of Trolox. For selected extracts different properties, including antibrowning, antilipogenic, anti-inflammatory and cytotoxicity against tumoral cells, were also evaluated. Different extracts were protective against neutrophils oxidative burst and moderately cytotoxic against colon carcinoma HCT-116 and lung adenocarcinoma A549 cells.

4.3.2. INTRODUCTION The leguminosa Acacia dealbata Link is originary from Australia and in Europe became an invasive species (Hernández et al., 2014). Its flowers exert a key role in the colonizing capacity leading to scarcity of undergrowth species, due to the allelopathic interference caused by live flower washings and by the decomposition in the soil. The toxicity of soil percolates during the flowering stage is maintained for a long time due to decomposition (Carballeira and Reigosa, 1999). The inhibition of germination and seedling growth in the presence of A. dealbata extracts was confirmed (Souza-Alonso et al., 2014). The volatile fraction from flowers contains compounds exclusive to this fraction, such as heptadecadiene, n-nonadecane, ntricosane, octadecene, hexadecene, n-hexadecane, heptadecene, hexadecenal, n-octadecane, octadecene, octadecenal, n- eicosane, kaurene, manool, ndocosane, and n-tetracosane. The most abundant compounds in flowers were anisal, methyl p-hydroxycinnamate and p-anysil alcohol (Aguilera et al., 2015). A. dealbata flowers can be used in perfumes (Souza-Alonso et al., 2014), but the possibility of utilizing other fractions as colouring and antioxidant agents should be

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explored. Solvent extraction is the conventional method to obtain plant components, whuich can be further concentrated. The use of organic solvents may lead to thermal degradation, oxidative transformations of the components and production of undesirable residues or solvent traces in the final products. The utilization of a sequence using conventional organic solvents has been compared with a sequence of stages using greener solvents and a final step of microwave assisted extraction assisted by gravity. Microwave hydrogravity is a novel extraction technique based on the irradiation of material and drainage by gravity of the extract (Perino-Issartier et al., 2013; López-Hortas et al., 2016). The present study aims at evaluating the potential applications of the solvent extracts from A. dealbata flowers, based on their antiradical potency and other properties of interest in cosmetics and pharmaceuticals.

4.3.3. MATERIALS AND METHODS 4.3.3.1. Materials Acacia dealbata flowers, collected in Pereiro de Aguiar (7°50´11.7´´W, 45°19´59.7´´N, Ourense, Spain) in February 2014 and 2016, were manually separated from stems and leaves. After air-drying to a final moisture of 7.81 wt% were kept in a dry and dark place until use. Flowers were processed without previous milling stage to avoid disgregation and loss of compounds.

4.3.3.2. Solvent extraction The dry flowers were macerated with 96 % ethanol at a liquid of solid ratio of 20:1 (v:w) at 40 °C during 24 h on shaking extractors protected from light. Solids were separated by filtration through Whatman No.1 filter paper, extracted in two additional stages with fresh solvent and further processed. One part of the residual solids was air dried and then extracted using organic solvents, hexane (Sigma Aldrich, Israel) dichloromethane (Sigma Aldrich, France), ethyl acetate (Fisher Chemical, UK) and n- butanol (Sigma Aldrich, USA) was carried out in soxhlet. Another part of the residual solid was extracted with water at a liquid to solid ratio 1:10, at 25 °C during 12 h. After filtration, the solids were reextracted in the same conditions and the extracts were

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individually characterized. The residual solid was then extracted with acidified water (1.2 M HCl), at a liquid solid 1:10, at 25 °C during 12 h. The solid residue from this stage was treated in a microwave hydrogravity equipment (an open vessel NEOS-GR (Milestone Srl, Italy) microwave extractor with a 1.5 L Pyrex extraction vessel.The drained extract was collected and the solid residue was discarded. The solvent extracts were rotary evaporated to remove the extracts, and the aqueous extracts were freeze- dried and further characterized.

4.3.3.3. Analytical methods The total extractables were gravimetrically determined and the extraction yield was calculated as percentage of the dry raw material. The ABTS [2,2-azinobis(3-ethyl- benzothiazoline-6-sulfonate)] radical cation (ABTS·+) scavenging capacity was expressed as Trolox equivalent antioxidant capacity. The diluted ABTS·+ solution (1.0 mL) and extract or Trolox were mixed and the absorbance readings at 734 nm were taken for 6 min and the absorbance inhibition was expressed as TEAC value (Re et al., 1999).

Antibrowning The assay provides a measurement of the ability of samples and extracts to inhibit the melanin production in B16-F10 cells (American Type Culture Collection ATCC code CRL- 6475, Manassas, VA). Cells were cultured in Dulbecco´s Modified Eagle´s Medium (DMEM) free from phenol red, and supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomicin and 1% L-glutamin, which was stored at 4-8 °C°. The standard compound was arbutin (Sigma A4256), a natural compound derived from hydroquinone, inhibiting the action of tyrosinase and melanin production, used at 25-

500 M, the IC50 values were 50-100 M, being non cytotoxic under 500 M. Murine melanoma cells were were grown until confluence in T-75 culture flasks, then disgregation was performed by washing cells with 4-6 mL PBS, after addition of 2 mL trypsin were placed in the CO2 incubator during 2 min, collected in 8-10 mL culture medium and centrifuged (1200 rpm, 5 min). Cells were resuspended in 10 mL culture medium and cells were counted in a Neubauer chamber, using Tripan blue to allow the

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observation of viable cells. The negative control was DMEM medium without cells and the positive control was the cell suspension at 2 104 cells/mL. Extracts, 5 mg, dissolved in methanol:DMSO (9:1) were evaluated at 50-1000 g/mL. Cells were incubated at 37

°C in 5% CO2 for about 24 h. Melanin production was spectrophotometrically measured at 405 nm. Cytotoxicity of extracts was also determined, the assay consisted on dispensing 50 L Thiazolyl Blue Tetrazolium Bromide, MTT (Sigma) (1 mg/mL) and incubating during 2 h at 37 °C°. Then 100 L DMSO was added to solubilize the color. Optical density was measured at 490 nm, this value being proportional to the viable cells. The negative control was DMEM medium and cell survival was zero, the positive control were B6-F10 cells without treatment and survival was maximal.

Fibroblast growth The potential of an extract to stimulate the proliferation of a primary culture of human dermal fibroblast (HFF-1) was determined using as standard bFGF (fibroblast basic growth factor), a 18 kD protein stimulating the growth of fibroblast, endothelial cells, queratinocytes, osteoblasts, myoblasts, chondrocytes, neuronal cells, ..., which at doses of 1-5 ng/mL bFGF increases proliferation by 20-30%. Cells were grown in DMEM medium, supplemented with FBS (1-10%), 1% penicillin/streptomycin and 1% L- glutamin. Extracts, 5 mg, were previously dissolved in methanol: DMSO (9:1) and were tested at 25-500 g/mL. An extract is considered active if the optical density values are 20% or more superior to control. If an extract is active in the first assay, a second confirmation assays is required, and consists on a dose-response at eight concentrations in triplicate up to 1000 g/mL. When the extract presents color, this could interfere in the readings, indicating a false increase in cell survival. Cells were grown to confluence in T-75 culture medium with 10% FBS. The medium is renewed 24 h before the assay and replaced with 1% FBS supplement. After 24 h cells were disgregated and resuspended in 10 mL culture medium and counted. The negative control is DMEM medium without cells. A positive control was prepared with 3 ·104 cells/mL, without any extract. Cells were incubated during 48 h at 37 °C° and 5%

CO2. Then 50 L MTT (1 mg/mL) was added and the mixture was incubated during 2 h at 37 °C°. When the medium is separated, the color was removed after solubilization in

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100 L DMSO. Optical density was measured at 490 nm, this value being proportional to viable cells. FGFb (R and D Systems, 233 FB) was used as positive standard.

Cytotoxicity against human tumoral cell lines The cytotoxic activity was determined against lung adenocarcinoma epithelial cell line (A549), colon carcinoma (HCT-116), pancreatic adenocarcinoma (PSN1), and Caucasian human glioblastoma cells (T98G), from the ECACC (European Collection of Cell Cultures). All cells were grown in RPMI-1640 medium, for A549 and HCT-116 it was supplemented with 5% FBS, 1% L-glutamine and 1% penicillin/streptomycin; for PSN1 cells it was supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin and for T98G cells it was supplemented with 10% FBS, 1% L- glutamine and 1% penicillin/streptomycin, 1% sodium pyruvate and 1% aminoacid solution. The freeze-dried extracts (5 mg) were dissolved in 500 L methanol:DMSO (9:1) to prepare concentrations in the range of 25 - 500 g/mL, which were incubated during 48 h at 37 °C and 5% CO2. Staurosporine was used as a positive control and two negative controls were used, one is the specific medium without cells and the other is the untreated cells suspension. Cell viability was determined by MTT assay. To a cell suspension of 3 · 104 cells/mL incubated during 48 h at 37 °C° and 5% CO2, 50 L of 1 mg/mL MTT solution were incubated in a humidified atmosphere of 5% of CO2 during 2 h at 37 °C. The medium was removed and the colored compounds generated were dissolved in 100 L of pure DMSO. Optical density (490 nm) was read as a measurement of cell survival. The IC50 was calculated as the extract concentration, which inhibits 50% of cellular growth, in relation to non-treated control cells.

Antiinflammatory activity, neutrophils' oxidative burst - Oxidation of luminol Venous blood was collected from healthy human volunteers, following informed consent, by antecubital venipuncture, into K3EDTA vacuum tubes. The density gradient centrifugation method was used to isolate human neutrophils using tris-glucose (25 mM Tris, 1.26 mM CaCl2·2H2O, 5.37 mM KCl, 0.81 mM MgSO4, 140 mM NaCl, and 5.55 mM D-(+)-glucose) as the incubation media used (Ribeiro et al., 2013).

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The neutrophils’ oxidative burst was measureed by monitoring ROS-induced oxidation of luminol (Fluka Chemie GmbH, Steinheim, Germany). The reaction mixtures contained neutrophils (1 · 106 cells/mL), the extracts ton be tested at concentrations in the range 1-500 µg/mL, luminol (500 µM) and PMA (160 nM) in a final volume of 250 µL. Cells were pre-incubated with luminol and the tested compounds for 5 min before the addition of PMA. Kinetic readings, immediately initiated after cell stimulation and carried out at 37 °C, under continuous soft shaking, were taken at the peak of the curve, observed at around 10 min. Effects are expressed as the percent inhibition of luminol oxidation, using quercetin (Sigma) as positive control.

4.3.4. RESULTS AND DISCUSSION 4.3.4.1. Extraction yields, phenolic content and antiradical properties The extraction yields and the phenolic content and antiradical activity of the extracts from A. dealbata flowers are shown in Table 4.3.1. The highest extraction yields were obtained in the first stage of ethanolic extraction, followed by the hexane extract of the residual solid after ethanol extraction. The highest phenolic content was observed in the extracts from the first and second stages with ethanol (27-28%), and the TEAC values correlated linearly with the phenolic content, exceot in the samples extracted with butanol, that are higher. Some extracts were selected for further evaluation of properties of interest for cosmetic and pharmaceutical applications.

4.3.4.2. Inhibition of melanogenesis Melanin is synthesized in the basal layer of the epidermis and has an important role in the protection against ultraviolet damage. However, overproduction and accumulation of melanin occur in several skin disorders and in order to control the skin darkening, several natural and synthetic components have been added in the cosmetics. The antibrowning activity, evaluated on murine melanoma B16F10 cells as the IC50 values of melanin production inhibition and cytotoxicity (Table 4.3.2) confirmed that no growth inhibition was observed under 1000 g/mL however, some of these extracts were cytotoxic. Viability at 500-1000 g/mL was reduced to 40-60%, and for ethanol extracts 26% and ERWA 10%. Arbutin showed IC50 values in the range 50-100 M,

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without showing toxicity. Some pure compounds have shown moderate cytotoxicity on

B16-F10 melanoma cells, thymol and carvacrol with IC50 < 100 g/mL and p-cymene and menthol < 150 g/mL (Satooka and Kubo, 2012).

Table 4.3.1.Extraction yield, phenolic content and ABTS radical scavenging capacity of solvent extracts from Acacia dealbata

Extraction yield Phenolic content TEAC

(g/100 g A.d.) (g GAE/ g extract) (g Trolox/g extract)

ADEE1 36.41 0.266 0.852

ADEE2 7.15 0.279 0.829

ADEE3 - - -

ADLEE-H1 9.45 0.070 0.037

ADLEE-H2 - - -

ADLEE-D 0.49 0.094 0.159

ADLEE-EA1 1.86 0.025 0.019

ADLEE-B1 5.46 0.159 0.044

ADLEE-EA2 2.06 0.099 0.317

ADLEE-B2 4.41 0.258 0.069

ADLEE1 1.92 0.066 0.125

ADLEE-HClE 4.95 0.043 0.234

ADLEE-HClE-MW 0.01 0.038 0.000

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Other crude extracts required high concentration and some of them also showed high cytotoxicity. Extracts from Rosa berberifolia, Punica granatum and Casiia angustifolia showed more than 80% inhibition at 500 mg/mL, comparable with that of the control, kojic acid, and showed less than 50% cytotoxicity at the concentrations tested (Vidyalakshmi et al., 2016). Tyrosinase inhibition (16-66%) was found in methanolic fractions of plants and mushrooms at 500 μg/mL (Nannapaneni et al., 2016). The whitening effect of Salvia miltorrhiza water extract in human epidermal melanocyte caused cell viability reduction of 50% at 20 µg/mL, although mushroom tyrosinase inhibitory effect of 42% required 1000 μg/mL (Park et al., 2015). Other extracts were more effective for melanogenesis inhibition, i.e. those from Gaillardia aristata flower extract, observed in B16F10 and normal human epidermal melanocyte cells with IC50 of 20 µg/mL (Kim et al., 2015), or a lignan and two flavonoid aglycones isolated from the

CHCl3 fraction of the methanol-water extract from Centaurea diluta flowers showed cytotoxic effects on B16-F10 at lower concentrations, with IC50 under 15 μg/L (Zater et al., 2016). Limonoids and flavonoids isolated from a methanolic extract of Azadirachta indica flowers presented melanogenesis-inhibitory activity in B16 melanoma cells with lower IC50 (3-50 μg/mL) with no toxicity. Tetrahydrocurcumin showed maximum decrease in melanin synthesis in B16-F10 cells at 0.1 µg/mL, and similar inhibition was observed with 10 µg/mL kojic acid (Trivedi et al., 2017). Oenothera laciniata methanol extract (66.3 mg polyphenols/g), treatment at 50 μg/mL was more effective than arbutin, but reduced cell viability to 53% at 200 μg/mL, whereas arbutin did not reduce cell viability at 25-200 μg/mL (Kim et al., 2017). Ethanolic extract from Asphodelus microcarpus flower (68 mg GAE/g) exhibiting strong anti-tyrosinase activity (an important approach towards controlling hyper pigmentation) and antimelanogenesis effect in B16F10 cells superior to kojic acid, without cytotoxic effect until 150 μg/mL FEE (Di Petrillo et al., 2016).

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4.3.4.3. Induction of HFF-1 cell proliferation Extracts 1, 6, 7, 9 were not active as HFF-1 cell proliferation inductors, since the percentage of positive induction was observed at concentrations where the extracts showed color, whereas at lower concentration the extracts showed a slight toxicity. The extract 2 was cytotoxic at 250 and 110 µg/mL, extract 10 showed activity at concentrations where some color was developed and also at some lower concentrations where color was less pronounced, although no dose-response effect was observed. In an additional test the effect of color was assessed. Extract 10 was not active since the optical density of the mixtures extract+medium+cells both before and after the addition of MTT are higher than the values of their respective controls. The color in the extracts difficulted the evaluation of the survival ratio. The inducing activity on human dermal fibroblasts was evaluated at four concentrations and the extracts 3 and 4 were discarded because they were not active. Extracts 2, 5 and 8 were cytotoxic. Extracts 1, 6, 7, 9 and 10 showed activity at some concentrations, although their color could be interfering showing false positives. Extracts 1, 6, 7, 9 and 10 were evaluated in a response dose triplicate assay at eight concentrations, and extracts 1, 6, 7 and 9 were discarded due to color interferences, and at lower concentrations the extracts showed cytotoxicity. Fig. 4.3.2.a and b shows that extracts 1, 2, 6 and 7, after dispensing and evaporating the extracts, these present some yellowish color at 500 and 250 g/ml, whereas extracts 9 and 10 show an orange color at the same concentration.

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a

b b

Figure 4.3.2.Color of a) some extracts and b) well 1 of confirmation after dispensing and evaporating the extracts

Extract 10 did not present a dose-response effect and in a test comparing optical densities under different conditions (cells+medium, cells+medium+extract without MTT and cells+medium+ extract with MTT), the observed optical density increase could be due to a coloured effect, indicating that these extracts were not active). The studied extracts did not induce cell proliferation, according to the IC50 values (Table 2).

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Table 4.3.2.Cytotoxicity of the selected Acacia dealbata extracts against different cell lines

IC50 IC50 melanin IC50 HFF-1 IC50 IC50 IC50 IC50 cytotoxicity inhibition (g/mL) A549 HCT-116 PSN1 T98G B16-F10 (g/mL) (g/mL) (g/mL) (g/mL) (g/mL) (g/mL)

1 ADEE (2014) >1000 500 >500 >500 250 >500 >500

2 ADEE (2016) >1000 500 50/>500 >500 250 >500 >500

3 ADEE 1+ADEE2 >1000 500 >500 >500 250 >500 >500

4 ADLEE1+ADLEE2 >1000 500/1000 >500 >500 250 >500 >500

5 ADLEE-HClE >1000 1000 25/250 250 250/500 >500 500

6 ADSR1 >1000 500 >500 >500 250 >500 >500

7 ADSR2 >1000 500/1000 >500 >500 250 >500 >500

8 ADLEEH1+ADLEEH2 >1000 500 500/>500 >500 250 >500 >500

9 ADLEE-EA2 >1000 500 >500 >500 250 >500 >500

10 ADLEE-B2 >1000 100/500 >500 >500 >500 >500 >500

1.ADEE (2014): A. dealbata ethanol extract (2014); 2.ADFF (2016): A. dealbata freeze-dried flower; 3. ADEE1+ADEE2: A. dealbata ethanol extracts from stages 1 and 2; 4. ADLEE1+ADLEE2: A. dealbata aqueous extract of the residue after ethanol extraction. 5. ADLEE-HClE: Extract in acidified water of the solid residue after ethanol and water extraction, 6. ADSR1: A. dealbata solid residue after Soxhlet extraction sequence; 7. ADSR2: A. dealbata solid residue after Soxhlet extraction sequence; 8. ADLEEH1+ADLEEH2: Extract in hexane of the solid residue after 3 stages of ethanol extration; 9: ADLEE-EA2: Extract in ethyl acetate after hexane extraction of the solid residue after 3 stages of ethanol extraction, 10: ADLEE-B2: Extract in butanol after ADLEE-EA2 extraction

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4.3.4.4. Antiinflammatory The extracts tested for inhibition of neutrophils oxidative burst. Data in Figure 4.3.3 shows a dose dependent inhibition of the ethanolic extracts, although at concentrations lower than 100 g/mL the fractions in butanol, ethyl acetate, or in hexane of the residue remaining after ethanol extraction were more effective.

Figure 4.3.3.Inhibition of luminol oxidation in the presence of Acacia dealbata flowers extracts

4.3.4.5. Cytotoxicity All extracts showed similar cytotoxicity against the tumoral cell lines tested. The extract in water-HCl (ADLEE-HClE) shows the highest against A549 cells. IC50 values of 250-500 ug/mL against the colon tumoral cell line HCT-116 were observed for all tested extracts, except for the extract in butanol. Concentration of 500 g/mL and higher were needed for PSN1 and T98G cells. According to literature information, anticancer activities of galenic preparations with root, leaf and flower extracts from Rosa rugosa against lung cancer (A549) cells, evaluated using the BrdU test, confirmed the considerable impact of polyphenols on the anticancer activity of extracts (Olech et al., 2012). Different phenolic compounds from flower extracts have been reported as cytotoxic against different tumoral cell lines. Compounds from the flowers of Chorisia chodatii, including sterols, furanoids, - 130 -

coumarins, phenolic acids and esters, flavonoids, in addition to mono-octyl phthalate and succinic acid showed antiradical properties and cytotoxic activities against the human lung cancer cell line A549 (Refaat et al., 2015). Limonoids and flavonoids from

Azadirachta indica flowers were cytotoxic against A549 human cancer cells, with IC50 values in the range of 4.5-9.9 μM (Kitdamrongtham et al., 2014). A moderately polar extract (CHCl3 soluble fraction of the methanol-water extract) from the flowers from Centaurea diluta and its isolated secondary metabolites after fractionation by separation and purification on silica gel 60 showed in vitro cytotoxic activity in the A549 lung carcinoma cells using a MTT colorimetric assay. The raw extract reduced cell viability (IC50 27 μg/mL) (Zater et al., 2016). Thymol and carvacrol exhibited moderate cytotoxicity on B16-F10 melanoma cells, with IC50 under 100 g/mL and p-cymene and menthol under 150 g/mL (Satooka and Kubo, 2012). Methanol extracts from magnolia flowers, showed IC50 values of 3340 and 10550 g/mL (Loizzo et al., 2012).

4.3.5. Conclusions Acacia dealbata flowers were sequentially extracted with different solvents with different solvent to obtain fractions with up to 27% phenolic content and equivalent to 0.8 g Trolox per gram of extract. The extracts obtained with ethanol and with the sequence of green solvents were more active than those obtained with organic solvents. The ethanol extracts were the most enriched in phenolic compounds and the most potent antioxidants. They also showed cytotoxic properties against lung and colon carcinoma cells.

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4.4 . Supercritical CO2 extracts from Acacia dealbata flowers

4.4.1. ABSTRACT

Supercritical (SC) CO2was used for the production of extracts from Acacia dealbata flowers in a semi continuous flow equipment at laboratory scale. Extraction conditions at pressures from 100 to 350 bar and at temperatures from 35 to 55 °C were studied to assess their influence on the yield, antiradical properties and composition of the volatiles. Using pure sc-CO2 at 30 MPa and 45 °C maximum extraction yields of 8.92% (% of the ethanol extractables) were obtained. The extract showed an antiradical capacity equivalent to 0.034 g Trolox/g extract. The addition of 10% ethanol as cosolvent increased up to 6% the extraction yields and radical scavenging activity by

1.3. The best conditions of extraction were 30 MPa, 45 °C° and 1.5 kg/h CO2.

4.4.2. INTRODUCTION Natural molecules remained the traditional products in the flavor and fragrance field despite the arrival of new synthetic molecules. Environmental restrictions, progressively constrained public health regulations and new market trends towards ecology have prompted the search for new raw materials and for the development of new eco-friendly processes, with renewable non toxic solvents (Lavoine-Hanneguelle et al., 2014). The conventional methods with organic solvents may lead to thermal degradation, oxidative transformations of the components and production of undesirable residues or solvent traces in the final products. Hydrodistillation and steam distillation require high temperature, affecting thermostable components and could also induce hydrolysis. Supercritical fluid extraction (SFE) is an important alternative for recovering fragrances and perfumery chemicals from natural products, avoiding the decomposition of labile compounds in operation at low temperatures while the absence of light and oxygen prevents oxidation reactions, and the natural odour and flavour of the plant are maintained. SFE offers the possibility of modulating the solvating power and selectivity. The most used supercritical fluid is carbon dioxide (Tc= 318 K and Pc= 74 bar), which is safe, non-toxic, noninflammable, odorless, colorless,

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nontoxic, readily available and no solvent residue is left in the final product (Grosso et al., 2009). Supercritical carbon dioxide (sc-CO2) extraction renders not only the higher recovery but also a better quality product (Lavoine-Hanneguelle et al., 2014). Sc-CO2 extraction of fragrances offered higher recovery and better quality product than hexane extraction (Rao et al., 1992), but the fragrance compounds having less molecular weight and polarity, are more soluble in sc-CO2 than the higher molecular weight and polar constituents, which would require the addition of a suitable co- solvent. The content of bioactive compounds in SCF extracts was generally much higher than in conventional extracts the SFE extracts can contain the active undegraded compounds, which could be predominant in the extract obtained by steam distillation (Kotnik et al., 2007). SFE is more effective than the conventional hydrodistillation in the extraction of fatty acids and the preservation of its quality, the yields of fatty acids and essential oils are higher, shorter extraction time are required and energy cost are lower (Ramandi et al., 2011). Based on the parameters determined in the lab scale the extraction process was successfully transferred to pilot scale (Kotnik et al., 2007). Despite the equipment initial cost is high, the supercritical process presents interesting solvent properties and the economically viable extraction from flowers requires that the cost of the raw material is minimized and that the operational conditions are optimized (Mezzomo et al., 2011).

Sc-CO2 was proposed for obtaining extracts from a number of flowers, such as Turkish lavender (Ada et al., 1994), marigold (Campos et al., 2005; Palumpitag et al., 2011),), chamomile (Kotnik et al., 2007), Tanacetum parthenium (Čretnik et al., 2005), Satureja fruticosa Béguinot (Coelho et al., 2007), Santolina chamaecyparissus L. (Grosso et al., 2009), Vitex agnus-castus L. (Marongiu et al., 2010), Borago officinalis (Ramandi et al., 2011), Helichrysum italicum (Ivanovic et al., 2011), Lavandula hybrida (Kamali et al., 2012), Achyrocline satureioides (Hatami et al., 2012), Coffea arabica (Stashenko et al., 2013), Eupatorium intermedium (Czaikoskiet al., 2015).

Supercritical CO2 extraction was also proposed to fractionate conventional flower extracts (Reverchon et al., 1999). Acacia dealbata Link is an Australian leguminosa introduced in Europe for ornamental purposes, which became a widespread invader in Mediterranean type ecosystems with a rapid expansion (Hernández et al., 2014). Its

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great colonizing capacity leads to a very low covering and scarcity of undergrowth species. A. dealbata flowers play a crucial role in the allelopathic interference by means of live flower washings and by the flower’s soil decomposition. Soil percolates during the blossoming period retained toxicity for a longer time, due perhaps to the decomposition of flowers fallen on the ground (Carballeira and Reigosa, 1999). Souza- Alonso et al. (2014) proposed that volatile organic compounds released from flowers, leaves and/or litter strongly inhibited germination and seedling growth of selected species and on its own seedlings, causing a reduction in root and stem length in different species. GC/MS analyses revealed 27 compounds in the volatile fraction from flowers, 12 of which were exclusive to this fraction. Within them, heptadecadiene, n- nonadecane, ntricosane, and octadecene represent 62 % of the fraction, four compounds accounting for 62 %: heptadecadiene, n-nonadecane, n-tricosane and octadecene. Some compounds were found exclusively in the flowers: hexadecene, n- hexadecane, heptadecene, hexadecenal, n-octadecane, octadecene, octadecenal, n- eicosane, kaurene, manool, ndocosane, and n-tetracosane, accounting for 20% of the isolate. Leaves had higher diversity of compounds but phytol accounted for almost half of the sample. The flowers are used in perfumes and the essential oil composition was reported (Perriot et al., 2010; Souza-Alonso et al., 2014). This work is aimed at evaluating the influence of the operational conditions during scCO2 extraction of Acacia dealbata flower, regarding the solubles extraction yields, radical scavenging properties and volatiles composition.

4.4.3. MATERIALS AND METHODS Acacia dealbata flowers were collected in Pereiro de Aguiar, located at 7°50´11.7´´W and 45°19´59.7´´N (Ourense, Spain) in February 2016, were manually separated from stems and leaves, were air-dried until reaching a moisture content of 7.81 wt%, and stored in a dry and dark place until use. Carbon dioxide (99.99% purity) was purchased from Praxair (Spain). Conventional solvent extraction. The air dried flowers were contacted with 96 % ethanol at a liquid of solid ratio of 20:1 (v:w) at 40 °C during 24 h on shaking extractors protected from light to limit the possibility of oxidation. Solids were separated by

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filtration through Whatman No.1 filter paper. Flowers were used without milling, due to their characteristics and because some loss of volatiles with the decrease of particle size due to the milling process could occur (Coelho et al., 2007). Supercritical fluid extraction. The dried flowers (10 g) werer packed with glass beads into an extractor with a 1 L cylinder extractor and two 500 mL separators (Thar Designs

SFE-1000F-2-C10, Pittsburgh, USA). The CO2was precooled with a circulating bath (PolyScience, USA, model 9506) before being pumped by a P-200A piston pump (Thar

Design Inc., Pittsburgh, PA, USA) at a flow rate of 25 g CO2/min. The cosolvent was pumped by a HPLC pump (Scientific Systems, Inc., USA, model Series III), the flow rate was adjusted to achieve modifier concentrations in the range 0.5-10% (wt). The extract was collected in the first separator, operating at ambient temperature. Pressure was studied in the range 10-35 MPa and temperature from 35 °C to 55 °C. The separator was further washed with 96 % ethanol to recover the extract. Ethanol was removed in rotavapor and the extracts were kept under N2, at -20 °C in the dark until analysis. Analytical methods The total amount of extractable material was gravimetrically determined and the extraction yield was calculated as mass of extract/mass of dry raw material. ABTS [2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonate)]. The ABTS radical cation (ABTS·+) scavenging capacity was expressed as Trolox equivalent antioxidant capacity (TEAC) (Re et al., 1999). Absorbance readings of the mixture of diluted ABTS·+ solution (1.0 mL) and extract or Trolox were taken at 734 nm for 6 min. Absorbance inhibition was calculated as a function of the concentration of extracts and Trolox.

4.4.4. RESULTS AND DISCUSSION

4.4.4.1. Kinetics of sc-CO2 extraction

The sc-CO2 extraction of dried A. dealbata flowers at 25 MPa, 45 °C and 1.5 kg CO2/h yielded up to 1.1% of the initial material (d.b.). The yield attained with 96% ethanol was 12.77% (d.b.). The kinetics in Figure 4.4.1 confirms that an initial linear period at constant rate up to 30 min provided a quantitative extraction and a subsequent diffusion controlling mass transfer rate period.

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t cer 1 = 32.08 min tcer 2 = 53.42 min

Figure 4.4.1. Extraction kinetics from 10 g Acacia dealbata flowers at 25 MPa and 45 °C °with

25 g pure CO2/min and 30 MPa and 45 °C °with 10% ethanol.

The typical behavior of extraction curves with three phases: a constant extraction rate, a falling extraction rate and the third stage where diffusion is the main extraction mechanism. The three stages are not properly reached in some studies, where only two of them are observed (Czaikoskiet al., 2015; Kotnik et al., 2007).

4.4.1 Silva et al, 2009 Logistic model (Martínez et al., 2003)

4.4.2

Difussion model (Crank, 1980, modified by Reverchon, 1997; Melo et al., 2016)

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4.4.3 Simple single plate model (Gaspar et al., 2003)

4.4.4

Sovová’s model described experimental data with satisfactory high accuracy of H. italicum extract (Ivanovic et al., 2011). The high-pressure extraction of Tanacetum parthenium flower heads was analysed with mathematical model for initial constant rate period and subsequent time-dependant diffusion controlling mass transfer rate period. The time of extraction required to reach constant cumulative weight of extract varied from 120 to 240 min in experiments at pilot scale at high pressures (600 and 800 bar) and temperatures (60 and 80 °C) (Čretnik et al., 2005). The model assuming constant extraction rate (film controlling mass transfer mode) followed by falling extraction rate (diffusion controlling mass transfer mode) was

useful for extraction of chamomile flower heads with CO2 (Kotnik et al., 2007). Table 4.5.1 summarizes the calculated parameters, where tm negative values were obtained in logistic model, and possesses no physical meaning. This behavior was also reported by other authors (Domingues et al., 2012; Jesus et al, 2013).

Table 4.4.1.Modeling parameters and fitting indicators obtained with the proposed models

Empirical model Logistic model Diffusion model Simple Single Plate Model 8, - 2 2 be bL (min tm Dm/r Dm/δ MSE R2 MSE R2 MSE R2 MSE R2 (min) 1) (min) (min-1) (min-1) 1 10.42 0.0236 0.833 0.072 -0.016 0.0147 0.835 0.00198 0.0346 0.790 0.00198 0.0285 0.791 2 45.18 0.0236 0.973 0.019 0 0.0813 0.982 0.000689 0.0412 0.939 0.000981 0.0262 0.960 MSE: Mean Square error Exp 1: 25 MPa, 45 °C, 0% EtOH Exp 2: 30 MPa, 45 °C, 10% EtOH

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The extraction yields were increased with an increase in the pressure (Figure 4.4.2), due to the higher solvent density. However, whereas the lowest temperature was preferable at low pressures, the highest temperature was preferable at the highest pressures. An increase in the pressure at constant temperature enhanced the extraction yield, due to the increased density of the solvent, but the increase in the temperature can increase the solubility due to the effect of the vapor pressure of the solute or reduce the solubility due to a decrease in the density of the solvent. Cross over points were observed between 10 and 15 MPa and between 25 and 30 MPa. The effect of the density of the solvent was also more important than the volatility of the oil from S. chamaecyparissus (Grosso et al., 2009). These authors compared the cross over values of volatile oils, usually isolated at 8-12 MPa and 40-50 °C°, which is usually below the crossover pressure. Studies performed with other plants from Asteraceae family showed that the crossover region was around 15 MPa (for temperatures of 30 and 40 °C°) for marigold (Calendula officinalis L.) (Campos et al., 2005) and for wormwood (Artemisia annua L.) the crossover pressure for the 30 and 50 °C° was found to be 25 MPa (Quispe-Condori et al., 2005). Other authors reported optimal values in this range for other flowers. During extraction of fatty acids and essential oils of the flower from Boragoofficinalis (350 atm, 65 °C, with methanol as modifier, static and dynamic extraction time of 10 min) ranged from 0.02% to 1.96% (w/w), and hydrodistillation yielded 0.05% (v/w) (Ramandi et al., 2011), the total xanthophylls from marigold (60 °C, 40 MPa) yielded 74.4% (Palumpitag et al., 2011), yield higher than 80% was obtained from Turkish lavender flowers (85.77 bar, 36.6 °C) (Ada et al., 1994), and for Lavandula hybrid flowers (90.6 °C, 63 bar) (Kamali et al., 2012).

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Figure 4.4.2.Effect of extraction pressure and temperature on the (a) solubles yield and (b) ABTS radical scavenging capacity of extracts from Acacia dealbata flowers (30 min)

Increasing solvent density leads to increased extraction yields and faster rate of extraction due to positive effect on solvent power. Combination of pressure and 3 temperature providing sc-CO2 densities around 700 kg/m can be optimal for achieving a high rate of extraction and 4% (w/w) yield of H. italicum extract, whereas the yield of the essential oil by hydrodistillation was 0.23% (w/w). The highest percentage of

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lighter compounds (mono- and sesquiterpenes) was found in the extracts isolated at lowest density of sc CO2 and 9 MPa and 50 °C (Ivanovic et al., 2011). The pressure had a positive effect on the extraction yield Eupatorium intermedium flowers with maximum extraction yield (5.85 wt%), obtained at 25 MPa and 353.15K; with petroleum ether was 9.41 wt% (Czaikoski et al., 2015). Usually organic solvents provide higher extraction yields, but extracts contained higher amounts of undesirable compounds, moisture and non-volatile compounds (Čretnik et al., 2005; Kotnik et al., 2007). However, the selectivity can be lowered (Čretnik et al., 2005), the adverse effect of using high solvent density on selectivity was also observed at the initial period of extraction (Ivanovic et al., 2011). Under optimal pressures could be selected to avoid co-extraction of other undesirable components (Coelho et al., 2007), since the yield of the SFE of S. fruticose increased slightly with the pressure (90–100 bar) at 40 °C due to a higher amount of heavier molecular weight components, mainly waxes, but the increase in extraction pressure led to a decrease of the relative amounts of isomenthone and pulegone. The highest extraction yield from chamomile 3.81% and the highest content of active components in extracts were obtained at 250 bar and 40 °C, and two step separation was used to optimize the separation of essential oil and waxes. The total extraction yield obtained with pilot scale extraction was not the highest, however, the extract in second separator contained the highest amounts of matricine and -bisabolol. The SFE extracts mainly contained matricine and the amount of chamazulene, the degradation product of matricine, was very low (Kotnik et al., 2007). The yield and composition of S. chamaecyparissus volatile oil was greatly influenced by the density of the sc-CO2. However, although with 9 MPa and 40 °C the method yielded more oil, the composition was different from that obtained by HD, and at 8 MPa the composition and yield were similar to that obtained for HD (Grosso et al., 2009). Significantly higher pressures were used for Tanacetum parthenium flower heads extracted with sc-CO2 at pilot scale (600 and 800 bar, at 60 and 80 °C). The best result was obtained at 600 bar and 80 °C, with 9.11% yield of extraction. These high values were required (600 bar, 60 °C) to achieve approximately the same amount of

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parthenolide isolated (328.8 mg/100 g raw material) as with acetonitrile extraction (Čretnik et al., 2005). An increase in the extraction pressure led to decreased ABTS radical scavenging properties of the extracts, but the effect of the operation temperature was less marked.

4.4.4.2. Extraction with etanol modified CO2

Supercritical CO2 is a poor solvent for polar components and the extraction efficiency can be greatly improved by the addition of polar modifier such as methanol or ethanol. Also vegetal oils, i.e. palm, soybean or olive oil have been proposed for lutein fatty acid esters from marigold flower (Palumpitag et al., 2011), and the increased moisture content of H. italicum flowers positively influences solubility of lighter and oxygenated compounds and changed selectivity towards aroma compounds (sesquiterpenes) (Ivanovic et al., 2011).

Addition of methanol, acetone or DMSO at 3.5 mol % to the CO2 enhanced by 200, 266 and 600 % the fragrance recovery from jasmine flowers, respectively (confirmer), but the choice of the cosolvent is important since the product quality is affected by selectively facilitating the extraction of different fragrant components (Rao et al., 1992). In the present work ethanol was selected for its non toxic and biorenewable character; the beneficial influence of the ethanol concentration in the extracting solvent is shown in Figure 4.4.3. The SFE yield of Achyrocline satureioides at 303.15 K and 30 MPa increased with greater quantities of co-solvent, with an optimal value at 16.4% (Hatami et al., 2012). The methanol modifier volume is another important factor that has a positive significant effect on extraction of the oil from B. officinalis (Ramandi et al., 2011). Achyrocline satureioides ethanol as co-solvent at 303.15-318.15 K and 20-30 MPa (Hatami et al., 2012).

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Figure 4.4.3.Influence of the ethanol concentration on (a) the solubles extraction yield and (b) the ABTS radical scavenging capacity of the extracts from Acacia dealbata flowers with sc-CO2 at 45 °C, 35 MPa, 30 min

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The comparison of the kinetics during extraction with and without cosolvent is presented in Figures 4.4.5 and in Figure 4.4.6, which also show the behaviour proposed by the studied models.

Figure 4.4.4.Experimental data obtained at 25 MPa and 45 °C and modelling results

Figure 4.4.5. Experimental data obtained at 30 MPa and 45 °C with 10 % ethanol and modelling results

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4.4.4.3. Sequential Natural products contain components of essential oil such as hydrocarbon terpenes, oxygenated terpenes, and sesquiterpenes and also waxes, coloring material, and resinoids. The extraction conditions affect these components due to the complexity of the mixtures involved. A fractionation step after extraction can provide pure oil fraction (Coelho et al., 2007), two step separation techniques on separation of essential oil from chamomile (Kotnik et al., 2007).

Supercritical CO2 extraction was also proposed to fractionate a semi-solid orange flower conventional extract. Operating at 90 bar and 40 °C no undesired compounds were co-extracted with the volatile oil except waxes, which were separated using two separators operated in series (Reverchon et al., 1999). We proposed operation in a sequence of different conditions to obtain different fractions: a static stage, followed by a dynamic stage with pure CO2 and with ethanol- modified CO2. The application of a static stage allowed the extraction of 0.4% and 0.6% at 10 and 30 min, respectively (Figure 4.4.6). However, the active compounds were maximum at 20 min. The joint application of a static 30 min period followed by a dynamic one of 30 min allowed 1.2%.

0.6 0.06

0.5 0.05

0.4 0.04

0.3 0.03

0.2 0.02 Extraction Extraction Yield

0.1 0.01 (g extract/100(g dry flowers) g

0 TEAC (g extract)Trlox/g 0 F1 F2 F3 F1 F2 F3

Figure 4.4.6.Comparison of combinations of static and dynamic operation

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4.4.4.4. CONCLUSIONS

Acacia dealbata flowers can be processed by supercritical scCO2 extraction providing yields equivalent to 9% of the total ethanol extractables when pure scCO2 was used, operation at 30 MPa and 45 °C was selected. The addition of 10% ethanol as cosolvent increased the extraction yields and ABTS radical scavenging activity.

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4.5. Sequential extraction of Hericium erinaceus using green

4.5.1. ABSTRACT More than 40% of Hericium erinaceus can be solubilized with the green processes proposed below, and provide extracts containing phenolic and polysaccharide fractions, through a sequence of steps consisting of microwave hydrogravity (400 W), supercritical CO2 extraction (20 MPa (3% of protease + cellulase + pectinase, 50 °C, 3 h) or non-isothermal autohydrolysis with water (200 °C) was added to the remaining refined samples. The phenolic components could be further concentrated by adsorption on polymeric resins and desorption with 96% ethanol to produce ABTS radical scavenging concentrates equivalent to 0.6 grams of Trolox/g extract.

4.5.2. INTRODUCTION Hericium erinaceus, known as the lion Mane Mushroom or Hedgehog Mushroom, is commonly used for medicinal or nutritional purposes and has attracted recent attention because of its health effects. Most of the therapeutic polysaccharides found in H. erinaceus are β-glucans (Han et al., 2013; Khan et al, 2013; Zhu et al., 2014), but fungi also contain other bioactive compounds, such as phenolics, Tocopherols, ascorbic acid and carotenoids. Preliminary separation of other components increased the purification of saccharide compounds (Vegas et al., 2005), providing an additional stream with valuable compounds that could benefit the economy of the process. The combination of microwave assisted water extraction and other technologies can be advantageous (Pérez et al., 2014; Flórez et al., 2015) and meet green chemistry and sustainability criteria (Barba et al., 2014; Deng et al., 2015). Because the use of organic toxic solvents should be limited for food purposes, a simple extraction technology is based on microwave irradiation and gravity. This mode of operation is suitable for samples with a high water content and for the extraction of volatile compounds (Bousbia et al., 2009; Flórez et al., 2014), in addition this process is suitable for the fractionation with different solvents and helped in The extraction of phenols from wild edible fungi (Kim et al., 2011; Özyürek et al., 2014). Therefore,

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sequential extraction of antioxidants from fungi and antimicrobial agents was proposed using a solvent of increasing polarity (Smolskaitė et al., 2015). Other green extraction technologies for the solubilization of phenolic fractions are supercritical CO2 extraction and subcritical water extraction or autohydrolysis.

The objective of this work is to evaluate the viability of a sequence of different methods of extraction with green solvents to fractionate Hericium erinaceus, these stages include aqueous extraction, microwave hydrogravity, supercritical carbon dioxide extraction and treatment with hydrolytic enzymes for the solubilization of The phenolic and saccharide components of the remaining solid.

4.5.3. METHODOLOGY 4.5.3.1. Material Hericium erinaceus samples were cultivated by Hifas da Terra (Pontevedra, Spain). Strains were cultured on potato dextrose agar medium (PDA: potato 200 mg/mL glucose 20 mg/mL and agar 20 mg/mL) and incubated at 25 °C for 7-10 days (Fen et al., 2014). These cultures were used to seed Populus alba autoclaved wood pellets previously humidified. Mycelium was allowed to grow on wood pellets for 7-10 days at 25 °C, and fruit bodies developed after 20 days. Fresh fruit bodies were cut into pieces of aprox. 2 cm x 2 cm before use. The average moisture content of the fresh mushroom was 70.22  1.36% of the wet weight. In dry basis the mushroom contained 7.06 % ash, 35 % carbohydrates, 26% proteins and 28% ethanol extractives. Sepabeads SP700 was purchased from Resindion S.R.L. (Mitsubishi Chemical Corp.).

4.5.3.2. Extraction and processing technologies 4.5.3.3. Microwave hydrogravity extraction (MHG) Drained liquid obtained after the microwave gravity extraction (MHG) process was collected at irradiation powers comprised in the interval 70-900W, and analyzed for total solubles, total phenolic compounds and ABTS radical cation removal activity. For this the mushrooms were contacted overnight with distilled water at 4 °C. Trials were performed in triplicate

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4.5.3.4. Supercritical fluid extraction (SFE) The freeze-dried mushroom and the MHG processed mushroom were ground in an IKA grinder (M20, Germany) and samples of 10 g were packed with glass beads into a Thar Designs extractor, model SFE-1000F-2-C10 (Pittsburgh, USA) with a 1 L cylinder extractor and two 500 mL separators. The CO2 was precooled with a water bath (PolyScience model 9506, USA) and pumped with a piston pump (P-200A, Thar Design

Inc.). The solvent flowed at 25 g CO2/min and the cosolvent was pumped by a HPLC pump (Series III, Scientific Systems Inc., USA). The modifier was added in the concentration range 0.5-15% (wt basis). The extraction vessel was covered with polypropylene wool to avoid solid particles from entering the extraction line. Pressure was fixed at 20 MPa and temperature at 40 C, based on literature (Kitzberger et al., 2007; Mazzutti et al., 2012). The extract was collected during 2 h in the first separator at ambient temperature and further recovered with 96% ethanol. Ethanol was removed in rotavapor and the extracts were kept under N2, at -20 °C in the dark until analysis.

4.5.3.5. Enzyme aided extraction (EAE) 10 grams of fresh mushroom or the solid residue of MHG in sodium acetate-acetic acid buffer at pH 4.5 in a liquid to solid ratio of 25 (v:w) were placed in 500 mL Erlenmeyer flasks placed in a Orbital agitator (INNOVA 4000, New Brunswick) at 50 °C for 3 h for enzymatic extraction with the commercial enzymes Viscozyme:Celluclast: Flavourzyme (2:1.5:1.5) (Novozymes) at an enzyme to substrate ratio of 5 % (weight). For enzymatic inactivation it was heated at 100 °C for 10 min to inactivate the enzymes and centrifuged (4500 rpm, 8 min) to remove the solid residue. The supernatant was filtered through 0.45 μm membranes and the filtrate was stored at -20 °C in the absence of light until analysis. Trials were performed in triplicate.

4.5.3.6. Autohydrolysis (NIAH) The wet ground fungus or the depleted solid after MHG with water (liquid ratio: solid 12:1, w: w) were contacted and heated in a Parr Insul. Co., USA under a Parr. Non- isothermal operation at 200 °C (based on results from previous unpublished data).

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After cooling, the reactor was opened and the liquid phase was filtered off for further characterization.

4.5.3.7. Adsorption-desorption (AD) The wet SP700 resin (1: 3 w / v) was contacted with the aqueous extract obtained after steeping Hericium erinaceus with distilled water overnight, in Erlenmeyer flasks at 175 rpm and at 25 °C for 60 min on an orbital shaker. To remove the non-adsorbed compounds the resin was washed with distilled water, at 175 rpm and 25 °C for 20 min. The desorption was carried out by contacting the saturated resins with 96% ethanol 1: 3 (w: v) at 175 rpm and 25 °C for 60 min. Trials were performed in triplicate.

4.5.3.8. Analytical methods The total phenolic content was determined by the Folin-Ciocalteu assay (Singleton and Rossi, 1965) and expressed as Gallic Acid Equivalents (GAE). The TEAC assay improved by Re et al. (1999), based on the scavenging of ABTS radical (2,2’-azinobis-(3-ethyl- benzothiazoline-6-sulfonate)) was used. ABTS•+ was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate, diluted with phosphate buffer saline (PBS) and equilibrated at 30 °C. After addition of 1.0 mL of diluted ABTS•+ solution to 10 μL of antioxidant compounds or Trolox standards in ethanol or PBS, the absorbance was read for 6 min. The percentage of absorbance inhibition at 734 nm was expressed as Trolox equivalents. The FRAP assay (Benzie and Strain, 1996) was used to measure the ability to reduce the ferric 2,4,6-tripyridyl-s-triazine (TPTZ) complex under acidic conditions. The reagent was made by mixing 25 mL of 300 mmol/L acetate buffer (pH 3.6) and 2.5 mL of a 10 mmol TPTZ/L solution in 40 mmol/L

HCl and 20 mmol/L FeCl3·6 H2O in distilled water. Samples (100 μL) were mixed with 3 mL of the reagent, and the absorbance was monitored at 593 nm, using ascorbic acid as standard. Glucuronic acid, glucose, arabinose, and the sum of xylose+galactose+mannose were determined by HPLC-RI (1100 series Hewlett-Packard) using a 300 × 7.8 mm Aminex

HPX-87H column (BioRad, Hercules, CA) at 60 °C with 0.003 M H2SO4 as mobile phase. The concentration of oligosaccharides was determined after acid hydrolysis (4%

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sulfuric acid, 121 °C, 20 min). Xylose, mannose and galactose were determined with a 300 × 7.8 mm CARBOsep CHO 682 column (Transgenomic, Glasgow, UK) operating at 80°C using distilled water as the mobile phase. Fatty acid methyl esters (FAME) were prepared by transmethylation (Carreau and Dubacq, 1978). FAMEs were extracted by hexane and, after evaporating the organic phase under reduced pressure, were analyzed in a GC–MS QP 2010 (Shimadzu, Japan) using a SP-2380 column. The temperature gradient increased from 50 °C (2 min) at 40 °C/min to 250 °C, which was maintained for 15 min. FAMEs were identified by comparing their mass spectra with those of authentic standards and NIST MS Search 2.0 library. Mineral content (Ca, Mg, Mn, Zn, Cu, Cr, Pb, Cd) of samples was determined by atomic absorption and by atomic emission (Na, K) spectrophotometry (220 Fast Sequential Spectrophotometer, Varian, Palo Alto, CA) after a previous digestion of 0.3 g of ashes with 10 mL of HNO3 and 1 mL of H2O2 in a microwave station (Marsxpress, CEM), operating at 1600 W during 15 min and maintained 10 min at 200 °C°. Mushroom samples were freeze-dried, fixed onto aluminium stubs, sputter coated with gold in an Emitech K550X equipment and examined in a FEI Quanta 200 scanning electron microscope at an accelerating voltage of 12.5 kV.

4.5.4. RESULTS To evaluate the carbohydrates of the fungi and to facilitate the purification of the fractions of saccharides a methodology of extraction in multiple stages was carried out with technologies and green solvents. These extracts have been studied for their radical scavenging and biological properties and the phenolic and flavonoid components may be the major contributors to the inhibitory effect on reactive species (Mau et al, 2002, Özyürek et al., 2014).

4.5.4.1. Microwave hydrogravity (MHG) To the biomass of fresh mushrooms wit 70.22% moisture was applied microwave technology (MHG). For a higher yield of the drained liquid phase the fungi were previously hydrated overnight at a temperature of 4 °C. Figure 4.5.1 shows the typical

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temperature profile during the MHG, with an initial rapid increase and a plateau until the complete drainage of the aqueous phase; A warming beyond this point could lead to burning (observed at the highest irradiated powers tested) and avoided. Separation of the liquid phase allowed the extraction of 5.15% of 0.69% solubles, containing 1.96 x 0.11 g of GAE extracts (gallic acid equivalents) / 100 grams with a scanning capacity of Radicals equivalent to 0.04 g Trolox extract /g. Up to 42.17  1.49% of these phenolic compounds could be adsorbed onto SP700. During elution with 96% ethanol, 96.72  2.35% of the adsorbed compounds could be efficiently desorbed. The dried desorbed fraction contained 13.30  1.05% phenolics and was equivalent to 0.59  0.05 g Trolox/g.

Figure 4.5.1.Effect of irradiation power during MHG on the temperature profile and liquid volume drained for fresh and water-extracted Hericium erinaceus samples

Figure 4.5.2 shows the extraction yield, phenolic content and antiradical properties of the different fractions extracted for irradiation power in the range 70-900 W, the phenolic content expressed as gallic acid equivalents and ABTS radical scavenging capacity of the fractions collected. The irradiation power has hardly any effect on the extraction yields and on the phenolic content of each collected fraction, except for the

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higher values found in the latest fractions regardless the power. Slightly higher activity was also noticed on the last fractions and in the extracts from 400-500 W. In the short periods needed in this process no decrease associated to thermal degradation was noticed. During microwave assisted extraction of edible mushrooms with methanol, the antioxidant properties of the extract increased with temperature up to 80 °C°, and then decreased, probably due to partial thermal degradation of heat sensitive constituents (Özyürek et al., 2014).

Figure 4.5.2.Effect of irradiation power during MHG on a) extraction yield, b) phenolic concentration and c) TEAC of the fractions from water extracted Hericium erinaceus

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The extraction yield was lower than that attained with conventional solvents, reaching 28% with ethanol from this mushroom or with methanol from other mushrooms (25.5- 33.1%) (Mau et al., 2002; Wang et al., 2005). The MHG process allowed total phenolics concentrations of 2-3%, comparable to those attained with conventional solvents, i.e. methanolic extracts (12.05-31.20 mg GAE/g extract) (Mau et al., 2002; Wong et al., 2009), chloroform, n-hexane, and n-butanol subfractions from methanolic extracts (11.2-35.2 mg ferulic acid equivalents/g) (Li et al., 2012) or hot water extracts (10.2 mg GAE/g) (Abdullah et al., 2012). The TEAC activity was comparable or higher to that reported for the chloroform subfraction of a methanolic extract from this mushroom, with a phenolic content of 1.1-3.5% being the identified phenolic acids chlorogenic acid, p-hydroxybezoic acid, vanillic acid, ferulic acid, sinapic acid, syringic acid and p- anisic acid and flavonoids (catechin, epicatechin, rutin, myricetin, hesperidin and quercetin) (Li et al., 2012). Benzoic acids, hydroxycinnamic acids and flavonoids have been identified in the microwave-assisted extraction methanolic extraction of polyphenols from edible mushrooms (Özyürek et al., 2014). The antioxidant capacity of Hericium erinaceus was relatively high among methanolic extracts of several specialty mushrooms (Mau et al, 2002) and among water extracts of several culinary mushrooms (Abdullah et al., 2012). However, the IC50 for DPPH was significantly higher for standard antioxidants (Mau et al., 2002; Abdullah et al., 2012), and the inhibition of the lipid peroxidation of phospholipids in egg yolk induced by Fe2+ was lower than for quercetin, BHA and ascorbic acid (Abdullah et al., 2012). The FAME composition of the MHG extracts showed that the most abundant compound was oleic acid (C18:1), accounting for 73.3  0.23% of the total FAME, followed by linoleic acid (C18:2), with 12.0  0.11%, palmitic acid (C16:0), 7.9  0.31% and stearic acid, (C18:0), 6.8  0.03%. After MHG processing the remaining solid sample was dry (average moisture in the range 10.28% and 8.30%, for samples processed at 100 W and 500 W, respectively) and presented suitable characteristics for stable storage. In addition, the microwave irradiation during these short periods caused structural damage of the cell walls and thus, it could be a suited pretreatment before extraction with apolar solvents. Figure 4.5.3 shows the demolition effect on the cell walls from both internal and external

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parts of the mushroom exposed to 100 and to 500 W in comparison to a freeze-dried sample.

a b c

d e f

Figure 4.5.3.Microscopic structure of Hericium erinaceus internal parts for: a) freeze-dried, b) after MHG at 100 W and c) at 500 W and external parts, d) freeze-dried, e) after MHG at 100 W and f) at 500 W

4.5.4.2. Supercritical CO2 extraction (SFE) The operating conditions for the freeze-dried samples (humidity 8.08%) of Hericium erinaceus for the extraction with supercritical CO2 were at 20 MPa and 45 °C°. These conditions were selected from preliminary trials and bibliographic information on other edible fungi (Kitzberger et al., 2007; Mazzutti et al., 2012). The literature reports on the beneficial effect of the phenolic fraction and for this reason the effect of a polar modifier during the extraction during 30 min under the same conditions of pressure and temperature was evaluated. The use of ethanol increased by almost 4 times the extraction yields, but the extracted compounds were less active and the value of TEAC was only favored in 5% ethanol, whereas in ethanol at 15% the activity was reduced by half (Figure 4.5.4.b). The use of ethanol as a modifier caused a similar increase in the extraction yield of Agaricus brasiliensis (Mazzutti et al., 2012) and Lentinula edodes (Kitzberger et al., 2007).

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Figure 4.5.4.Supercritical extraction of total solubles and TEAC of the extracts from freeze-dried

H. erinaceus: a) extraction kinetics (20 MPa, 40 °C) with pure CO2 and b) effect of ethanol concentration (20 MPa, 40 °C, 30 min)

The previous hydration-extraction of samples improves the SFE yields and the antiradical properties. The influence of the irradiation power during MHG of water hydrated-extracted samples was evaluated on the supercritical CO2 extraction at 20

MPa and 45 °C during 30 min with pure and with ethanol modified CO2. Enhanced - 154 -

yields were attained after irradiation at 500-700 W, with an increase in the antiradical capacity. The use of cosolvent improved the extraction of active components, the yields were increased by 20%, and the ABTS radical cation scavenging was doubled (Figure 4.5.5). The cosolvent also favoured the extraction yields and radical scavenging activity of extracts from non hydrated mushroom samples (data not shown).

Figure 4.5.5.Effect of irradiation power of the water-extracted H. erinaceus on the extraction yield, phenolic content of the extracts and TEAC values during supercritical extraction with pure

( grey symbol) or with ethanol modified CO2 (black symbol) of the solids remaining after MHG

4.6.4.3. Water-based fractionation The flowchart of Figure 4.5.6 shows sequential extraction with green technologies and mass balances at each stage. The application of additional extraction stages allowed the fractionation of the raw material and the selective separation of components. Preliminary extraction of organic solvent facilitates the separation of polysaccharides (Ruthes et al., 2015). Also a combination of enzymes and autohydrolysis can be proposed for the solubilization of the polysaccharide fraction. Table 4.5.1 summarizes the performance and characteristics of the extracts obtained in the different

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processing steps. There was only some difference in the radical scavenging properties of the supercritical extracts. No significant influence of irradiation potency during MHG on soluble and phenolic yields was observed.

Figure 4.5.6.Flow diagram of the sequence of stages using green solvents for the extraction of phenolic components from Hericium erinaceus and phenolics and saccharide yields during further enzyme aided or subcritical water extraction of the exhausted solids. AD: adsorption- desorption; MHG: microwave hydrogravity; SFE: supercritical fluid extraction; EAE: enzyme aided extraction: NIAH: non isothermal autohydrolysis

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Table 4.5.1.Extraction yield, phenolic content and TEAC of Hericium erinaceus extracts Extraction technology (Extract) WE (E1) Extraction yield (g E1/100 g H.e.) 5.15  0.69 Phenolic content (g GAE/100 g E1) 1.96  0.11 TEAC (g Trolox/g E1) 0.04  0.01 WE-AD (E2) Recovery yield (g E2/100 g H.e.) 1.57  0.07 Phenolic content (g GAE/100 g E2) 13.30  1.05 TEAC (g Trolox/g E2) 0.59  0.05 Irradiation power in MHG 100 W 500 W WE-MHG (E3) Extraction yield (g E3/100 g H.e.) 4.50  0.00 4.22  0.00 Phenolic content (g GAE/100 g E3) 5.16  0.04 0.08  0.01 TEAC (g Trolox/g E3) 0.13  0.00 0.24  0.02 WE-MHG-SCF (E4) Extraction yield (g E4/100 g H.e.) 0.76  0.01 1.62  0.00 Phenolic content (g GAE/100 g E4) 1.71  0.04 3.44  0.02 TEAC (g Trolox/g E4) 0.07  0.02 0.22  0.01 WE-MHG-SCF-EAE (E5) Extraction yield (g E5/100 g H.e.) 26.70  0.38 32.36  1.74 Phenolic content (g GAE/100 g E5) 1.44  0.12 1.30  0.09 Protein content (g BSA/100 g E5) 11.31  0.24 8.17  0.14 Monomers (g/100 g E5, 5.55;0.04;0.08; 5.29;0.05;0.03; GA;G;X+Gal+Man;FA;AA) 0.28;0.08 0.48;- Oligomers (g/100 g E5, GA;G;X;Gal;Man;Ar) 12.04;1.16;0.88;- 11.21;1.27;0.02; ;0.19;0.22 0.26;-;- TEAC (g Trolox/g E5) 0.02  0.00 0.02  0.00 FRAP (mg AA/g E5) 2.73  0.18 2.45  0.36 WE-MHG-SCF-NIAH (E6) Extraction yield (g E6/100 g H.e.) 34.25  4.85 33.19  2.19 Phenolic content (g GAE/100 g E6) 3.33  0.05 3.42  0.02

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Protein content (g BSA/100 g E6) 21.28  0.39 17.73  1.53 Monomers (g/100 g E6, 0.14;0.00;0.07; 0.11;0.01;0.03; GA;G;X+Gal+Man;FA;AA) 0.05;0.02 0.06;0.15 Oligomers (g/100 g E6, GA;G;X;Gal;Man;Ar) 1.73;9.36;0.53: 1.70;10.99;0.42;0.7 0.75;1.33;2.82 3;1.16;4.67 TEAC (g Trolox/g E6) 0.05  0.00 0.05  0.00 FRAP (mg AA/g E6) 9.57  0.37 9.68  0.28

WE: water extraction; AD: adsorption-desorption; MHG: microwave hydrogravity; SCF: ethanol

modified supercritical CO2; EAE: Enzyme assisted extraction, NIAH: non isothermal autohydrolysis GA: Glucuronic acid; G: Glucose; X: Xylose; Gal: Galactose; Mal: Mannose; Ar: Arabinose; FA: Formic acid; AA: Acetic acid

The enzyme assisted extraction of mushroom samples previously treated in MHG at 100 W and at 500 W, yielded 26.70  0.38 % and 32.36  1.74%, respectively. Non isothermal autohydrolysis was performed at 200 °C, leading to maximal solubilization yields and phenolic content of the extracts; optimal temperature was in the range of that reported for the extraction of I. obliquus under subcritical conditions using a different device and combination of time and temperature (Seo and Lee, 2010). The enzyme-assisted extraction of Hericium erinaceus in shorter incubation times was reported and the polysaccharides were mainly composed of mannose, glucose, xylose, and galactose (Zhu et al., 2014). The optimum temperature for autohydrolysis was found at 200 °C, resulting in the highest solubilization and phenolic contents of the extracts. Enzyme assisted extraction of pre-treated fungus samples at 100 W and 500 W MHG yielded 26.70 ± 0.38% and 32.36 ± 1.74%, respectively. Non-isothermal autohydrolysis of the MHG solids at 100 W and at 500 W, further extracted with supercritical carbon dioxide, yielded 34.25 ± 4.85% and 33.19 ± 2.19%. Non-isothermal autohydrolysis allows more effective fractionation of H. erincaus since glucose oligomers were significantly more abundant in the extracts accounting for 9-11% (wt).

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H. erinaceus polysaccharides, composed of xylose (7.8%), ribose (2.7%), glucose (68.4%), arabinose (11.3%), galactose (2.5%) and mannose 5.2%), can significantly decrease the level of lipid peroxidation and increase the activities of antioxidant enzymes in experimental mice at a dose of 300 mg /kg for 15 days (Han et al., 2103). Thus the ROS-generating activity and the antioxidant activity of hot-water extracts of culinary-medicinal mushrooms correlated strongly with their content of polysaccharides and polyphenols (Wei and Van Griensven, 2008). The mineral content of solid wastes and products is summarized in Table 4.5.2. Chromium levels were below 9 mg / kg; The limits of Cd for other fungi are regulated (EU, 2014), with limits of 1.0 mg / kg fw (Giannaccini et al., 2012) and the values obtained for the solids processed in this work are under these limits. The importance of processing and not the cultivation conditions of H. erinaceus mycelium on the antioxidant properties of the extracts has been highlighted for the production of standardized formulations for either human nutrition or therapy (Wong et al., 2009) and the development of green processes could be suited for the integral utilization of this raw material and the production of components for nutraceutical, food and cosmetic industries.

4.5.5. CONCLUSIONS The sequential fractionation of Hericium erinaceus components can be accomplished through a sequence of extraction stages with green solvents. Direct processing by

MHG allows drying and structural alterations that facilitates supercritical CO2 extraction. The remaining solid phase can be further hydrolyzed with either enzymes or with pressurized hot water (autohydrolysis) to solubilize the saccharidic fraction. Autohydrolysis is suited for the extraction of oligomeric glucose-rich fractions, which could be valuable for their biological properties. Further chemical characterization of these components is ongoing.

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Table 4.5.2.Mineral composition of Hericium erinaceus initial freeze dried samples (H. e.) and the solids remaining after the processing stages shown in the flow diagram of Figure 6

Mg (mg/kg) Ca (mg/kg) Zn (mg/kg) Cu (mg/kg) Mn (mg/kg) Cd (mg/kg) Na (mg/kg) K (g/kg) H. e. (freeze dried) 881.00  29.16 333.93  3.36 64.94  0.57 10.55  1.87 8.88  0.16 <1,5 93.85  4.81 31.57  2.25 WE-MHG 100 W 909.26  39.14 354.11  55.32 131.63  0.35 15.62  0.01 12.02  0.33 1.91  0.02 53.75  5.28 18.13  1.01 WE-MHG 500 W 889.27  16.87 277.85  45.07 152.38  5.20 16.71  0.26 13.94  0.40 1.96  0.08 76.31  17.23 15.48  0.11 WE-MHG 100 W-SCF-EAE 135.35  3.31 225.67  13.74 136.41  4.36 19.54  0.97 2.25  0.27 1.80  0.03 1897.75  297.33 17.89  0.22 WE-MHG 500 W-SCF-EAE 213.59  3.30 177.15  20.34 160.77  1.20 19.99  0.35 4.28  0.03 2.03  0.02 1507.36  17.27 13.35  2.40 WE-MHG 100 W-SCF-NIAH 575.04  22.15 281.25  62.62 188.36  20.98 26.46  13.37 14.77  1.71 3.32  0.28 104.41  3.06 4.11  4.85 WE-MHG 500 W-SCF-NIAH 785.95  27.55 207.27  34.19 207.06  4.19 28.82  5.74 17.52  0.91 3.33  0.04 43.26  32.58 2.30  2.72

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5. GENERAL CONCLUSIONS

GENERAL CONCLUSIONS

- The utilization of innovative extraction technologies can be applied for the extraction of bioactives from underutilized sources of different origin, terrestrial and marine.

- Hydrothermal processing (autohydrolysis) is more effective than enzyme assisted hydrolysis of brown seaweeds, but both are suited to solubilize the bioactive fractions.

- The adsorption-desorption technology is suited for the selective recovery of phenolic compounds from aqueous extracts of different sources and technologies.

- Microwave irradiation facilitates further solvent extraction of bioactives and causes severe disruption of cell walls.

- Autohydrolysis and enzyme assisted aqueous extraction can be combined with previous microwave pretreatment and supercritical fluid extraction to attain a fractionation of the bioactives from terrestrial and marine vegetal biomass.

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6. APPENDIX

NOMENCLATURE

A, adsorbent

AB, acetate buffer ABTS, 2,2´‐azino‐bis(3‐ethylbenzothiazoline ‐6 sulfonic acid AC, acetic acid Ac, acetone AD, Adsorption-desorption ADEE1, Acacia dealbata ethanolic extraction 1 ADEE2, Acacia dealbata ethanolic extraction 2 ADEE3, Acacia dealbata ethanolic extraction 3 adHyp, adhyperforin ADLEE-B1, Acacia dealbata ethanolic extraction-dicloromethane 1

ADLEE-B2, Acacia dealbata ethanolic extraction-dicloromethane 2

ADLEE-D, Acacia dealbata ethanolic extraction-dicloromethane

ADLEE-EA1, Acacia dealbata ethanolic extraction-ethyl acetate 1 ADLEE-EA2, Acacia dealbata ethanolic extraction-ethyl acetate 2 ADLEE-H1, Acacia dealbata ethanolic extraction-hexane 1

ADLEE-H2, Acacia dealbata ethanolic extraction-hexane 2 ADLEE-HClE,Ac acia dealbata

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ethanolic extraction-HCLextraction ADLEE-HClE-MW, Acacia dealbata ethanolic extraction-HCL extraction- microwave AESm, alginate exhausted Sargassum muticum Afl, amentoflavone Antc, anthocyanins Ap, apigenin BHA, butylhydroxyanisole

BHT, butylhydroxytoluene

BP, biphenol-1

BV, bed volume C, catechin

C, colum CA, caffeic acid Ca, carnosol CaA, carnosic acid Car, carvacrol Cell, cellulose ChA, chlorogenic acid ChiAc, chicoric acids Chr, chrysanthenol Con, conocarpan Cou, coumarin DAD, diode‐array detector DHF, dihydroxyflavone

DhK, dihydrokaempferol DhQ, dihydroquercetin

DMSO, dimethylsulfoxide

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DPPH, 2,2‐Diphenyl‐1 picrylhydrazyl

Dt, dynamic extraction time E, ethanol

EA, ellagic acid EA, ethyl acetate

EAE enzymatic-assisted extraction

EC, epicatechin

Ed, eriodictyol

Eu, eugenol Eup, eupomatenoid-5 F, flavonoids FA, formic acid Flv, flavone Flvl, flavonols FLvn, flavanone FLvn, flavanone

FRAP, ferric reducing antioxidant power GA, gallic acid MW, microwave

My, myricetin Mys, myristicin

N, naringenin NIAH, Autohydrolysis

NL, neolignans PAL, enzyme phenylalanine ammonolase

PB, phosphate buffer PCA, protocatechuic aciD p-Cy, p-Cymene

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PEP, phosphoenolpyruvate

PG, phloroglucinol PMA, phorbol-12-myristate-13- acetate Q, quercetin Q-3-g, quercetin 3-d-glucoside RAC, radical absorbance capacity ROS, oxygen and nitrogen reactive pecies

Ros, rosmanol RosA, rosmarinic acid

RS, reactive species ( Ru, rutin S, stirring rate

SC-CO2, supercritical carbon dioxide

Sco, scopoletin SEM, Scanning electron microscopy

SFE, supercritical fluid extraction

SLR, solid liquid ratio Sm, Sargassum muticum

SOD, superoxide dismutase

St, static extraction time SWE, subcritical water extraction SWE, subcritical-water extraction SyA, syringic acid

T, terpinene TEAC, trolox equivalent antioxidant capacity Thy, thymol TPC, total phenolic compounds TTC, total tannins content

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TXBSI, thromboxane synthase inhibitor USE, ultrasound and enzyme assisted extraction VA, vanillic acid W, water

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