Universidade Federal Do Rio De Janeiro Laís De Oliveira Silva

Universidade Federal do Rio de Janeiro

Laís de Oliveira Silva

VALORIZATION OF POMEGRANATE (PUNICA GRANATUM L.) AGROINDUSTRIAL RESIDUES: EXTRACTION OF SEED OIL AND FRACTIONATION OF BIOACTIVE COMPOUNDS FROM PEEL USING

SUPERCRITICAL CO2 AND CONVENTIONAL METHODS

RIO DE JANEIRO 2019 0

Laís de Oliveira Silva

VALORIZATION OF POMEGRANATE (PUNICA GRANATUM L.) AGROINDUSTRIAL RESIDUES: EXTRACTION OF SEED OIL AND FRACTIONATION OF BIOACTIVE COMPOUNDS FROM PEEL USING

SUPERCRITICAL CO2 AND CONVENTIONAL METHODS

Tese de Doutorado apresentada ao Programa de Pós- graduação em Ciência de Alimentos, Instituto de Química, Universidade Federal do Rio de Janeiro, como requisito parcial à obtenção do título de Doutor em Ciência de Alimentos.

Orientador: Prof. Dr. Alexandre Guedes Torres

Rio de Janeiro 2019 1

Laís de Oliveira Silva

VALORIZATION OF POMEGRANATE (PUNICA GRANATUM L.) AGROINDUSTRIAL RESIDUES: EXTRACTION OF SEED OIL AND FRACTIONATION OF BIOACTIVE COMPOUNDS FROM PEEL USING

SUPERCRITICAL CO2 AND CONVENTIONAL METHODS

Prof. Dr. Alexandre Guedes Torres

DEDICATION

Aos meus pais, por todo amor, cuidado, dedicação e infinito amor. Meus grandes exemplos de vida e amor.

ACKNOWLEDGEMENTS

Agradeço a Deus pelo seu imenso amor, por me sustentar em todos os momentos de fraqueza, desespero e angústia, e me conduzir até aqui. Agradeço pelo dom da vida, pelas bênçãos e vitórias recebidas. Aos meus pais, pelo amor incondicional, carinho, compreensão, por serem meus maiores exemplos de força, determinação e fé. Agradeço por estarem ao meu lado em todos os momentos, sobretudo, nos momentos difíceis, por me ajudar e incentivar sempre. Sem vocês na minha vida... nos meus dias, eu não conseguiria! Muito obrigada! A minha irmã, por ser simplesmente o meu tudo! Minha grande amiga e confidente! Obrigada por me apoiar e me incentivar a ir sempre mais longe. Ao meu marido, por todo o seu amor, apoio, companheirismo, compreensão e paciência ao longo desta longa caminhada. Por estar sempre ao meu lado me ajudando a vencer os obstáculos ao longo desse caminho. Meu anjo, sempre! Ao meu orientador, professor Alexandre Torres, pelo apoio ao longo da construção deste trabalho, pela confiança e por seus ensinamentos que foram essenciais para o meu crescimento profissional. Aos queridos amigos do LBNA: Ana Beatriz, Ana Rafaela, André, Andressa, Carol, Desirée, Ellen, Emília, Fabricio, Genilton, Isabele, Kim, Natália Sales, Nívea, Suellen e Talita agradeço por toda a colaboração e ajuda recebida, pela amizade e descontração na rotina do dia-a-dia. À Aline e Vanessa, “as meninas dos óleos”, pelo apoio, amizade, companhia e ajuda nas análises. À Layla, minha querida IC, obrigada pela dedicação e empenho no trabalho, e por fazer parte desta história. E a todos aqueles, que de alguma forma contribuíram para que este sonho fosse concretizado, os meus sinceros agradecimentos.

ABSTRACT

Silva, Laís de Oliveira. Valorization of pomegranate (Punica granatum L.) agroindustrial residues: extraction of seed oil and fractionation of bioactive compounds from peel using supercritical CO2 and conventional methods. Rio de Janeiro, 2019. Tese (Doutorado em Ciência de Alimentos). Instituto de Química, Universidade Federal do Rio de Janeiro.

The extraction technique may influence the bioactive compounds profile of the extracts obtained, may affect its bioactivity potential. The aim of this study was to evaluate the influence of different extraction techniques on the bioactive compounds profile of pomegranate (Punica granatum L.) byproducts, seed oil (PSO) and peel extract fractionated.

PSO was extracted by expeller pressing (P-PSO), alcohol (A-PSO) or supercritical CO2 (SC- PSO without co-solvent), and experimental design was performed to improve conditions of SC-PSO extraction with alcohol as co-solvent. Pomegranate peels were extracted by supercritical CO2 (non-fractionated extract; N-FE) and for the sequential fractionation two methods were developed: fractionation 1 (F1) composed by three steps and fractionation 2 (F2) composed by two steps. P-PSO and SC-PSO presented higher conjugated linolenic acid (cLnA) contents, while A-PSO showed higher contents of phenolic compounds, tocopherols and phytosterols, and higher antioxidant capacity and oxidative stability than P-PSO and SC- PSO. The fractional factorial design of the SC-PSO with co-solvent indicated two improved extraction conditions, one resulting in an oil enriched with tocopherols and the other in an oil enriched with phenolics. Sequential fractionation and extraction by SC-CO2 with ethanol were able to provide an extract and fractions rich in bioactive compounds, being N-FE and the last fractions (fractions 2 and 3 of F1; fraction 2 of F2) enriched in phenolic compounds, especially punicalagin, and first fractions (fractions 1 of F1 and F2) concentrated in tocopherols and β- carotene. Therefore, A-PSO and SC-PSO with alcohol as co-solvent can be considered specialty oils, rich in cLnA, phenolics and tocopherols. The fraction 2 of F2 from pomegranate peel can be considered a potential source of antioxidants enriched in punicalagin. A-PSO, SC-

PSO and the fraction 2 of F2 from pomegranate peel are promising as nutraceutical ingredients with applications in the pharmaceutical, cosmetic and food industries, and also fraction 2 of F2 can be used as analytical standards for laboratory assays.

Keywords: pomegranate seed oil, pomegranate peel, supercritical CO2, alcohol-extraction, cLnA, phenolic compounds, tocopherols. 6

RESUMO

Silva, Laís de Oliveira. Valorização de resíduos agroindustriais da romã (Punica granatum L.): extração de óleo da semente e fracionamento de compostos bioativos da casca usando

CO2 supercrítico e métodos convencionais. Rio de Janeiro, 2019. Tese (Doutorado em Ciência de Alimentos). Instituto de Química, Universidade Federal do Rio de Janeiro.

O método de extração pode influenciar o perfil de compostos bioativos dos extratos obtidos, podendo afetar sua potencial bioatividade. O objetivo deste estudo foi avaliar a influência de diferentes técnicas de extração no perfil de compostos bioativos de sub-produtos da romã (Punica granatum L.), óleo da semente (OSR) e extrato fracionado da casca. O OSR foi extraído por prensagem (OSR-P), com álcool (OSR-A) ou CO2-supercrítico (OSR-SC). Um planejamento experimental foi realizado a fim de melhorar as condições de extração do OSR-

SC com álcool. As cascas da romã foram extraídas por CO2-supercrítico sem fracionamento

(ESF), e para o fracionamento sequencial 2 métodos foram desenvolvidos: F1 composto por 3 etapas e F2 composto por 2 etapas. OSR-P e OSR-SC apresentaram elevado conteúdo de ácido linolênico conjugado (cLnA), enquanto que OSR-A apresentou elevado conteúdo de compostos fenólicos, tocoferóis e fitoesteróis, além de maior capacidade antioxidante e estabilidade oxidativa. O planejamento fatorial fracionário indicou duas condições de extração melhoradas, sendo uma enriquecida em tocoferóis e a outra em compostos fenólicos. A extração e o fracionamento sequencial por CO2-supercrítico foram capazes de proporcionar um extrato e frações ricos em compostos bioativos, sendo o ESF e as últimas frações do fracionamento (frações 2 e 3 do F1; fração 2 do F2) enriquecidas em compostos fenólicos, especialmente punicalagina, e as primeiras frações (frações 1 do F1 e F2) concentradas em tocoferóis e β-caroteno. Portanto, OSR-A e OSR-SC com álcool podem ser considerados

óleos especiais, ricos em cLnA, compostos fenólicos e tocoferóis. A fração 2 do F2 da casca da romã pode ser uma potencial fonte de antioxidantes, enriquecida em punicalagina. OSR-A,

OSR-SC e a fração 2 do F2 são promissores como ingredientes nutracêuticos, com potenciais aplicações nas indústrias farmacêuticas, de cosméticos e de alimentos, além da fração 2 do F2 pode ser usada como padrão analítico em ensaios laboratoriais.

Palavras-chave: óleo de semente de romã, casca da romã, CO2 supercrítico, extração com etanol, cLnA, compostos fenólicos, tocoferóis.

PUBLICATIONS ARISING FROM THIS THESIS

Scientific journal paper:

L.O. Silva, L.G. Ranquine, M.C. Monteiro, A.G. Torres, Pomegranate (Punica granatum L.) seed oil enriched with conjugated linolenic acid (cLnA), phenolic compounds and tocopherols: improved extraction of a specialty oil by supercritical CO2, The Journal of Supercritical Fluids, 147 (2019) 126–137. (Published)

Scientific conference abstracts:

A.M.M. Costa, L.O. Silva, V.N. Castelo-Branco, J.C. Nunes, A.G. Torres, Influência do método de extração no perfil de ácidos graxos, composição de fitoesteróis e capacidade antioxidante de óleos de semente de romã (Punica granatum). Seminário Internacional de Processamento de Óleos e Gorduras: Tendências e Desafios, Florianópolis, Brazil, 2015.

L.O. Silva, L.G. Ranquine, A.G. Torres, Influence of extraction method on bioactive compounds and antioxidant capacity of Brazilian pomegranate seed oils. 17th AOCS Latin American Congress and Exhibition on Fats, Oils, and Lipids, Cancun, Mexico, 2017.

LIST OF FIGURES

Literature Review

Figure 1. Pomegranate (Punica granatum L. cv. Ruby) structures produced in the São Francisco river valley, in the State of Pernambuco, Brazil………………………………..24 Figure 2. Variation in the size of the pomegranate and in the color of peel, arils and juice, depending on the pomegranate variety in USA...... 24 Figure 3. Chemical structure of the cLnA isomers present in the PSO…………………...30 Figure 4. Biosynthesis of conjugated fatty acids by (A) Calendula officinalis and (B) Aleurites fordii Hemsl (Tung tree)………………………………………………………...31 Figure 5. Chemical structure of phenolic compounds of the PSO………………………..33 Figure 6. Chemical structures of the tocopherols isoforms present in the PSO…………..35 Figure 7. Chemical structures of the phytosterols present in the PSO……………………36 Figure 8. Chemical structures of the sex sterols present in the PSO……………………...37 Figure 9. Chemical structures of the major phenolic compounds of the pomegranate peel ……………………………………………………………………………………………..38

Figure 10. Pressure-temperature phase diagram of CO2………………………………….42

Chapter 1

Figure 1. Bioactive compounds in pomegranate (Punica granatum L. cv. Ruby) seed oils,

obtained with either expeller pressing, alcohol or supercritical CO2 (without co-solvent). A. Tocopherol contents (mg/100 g) in PSOs; B. β-carotene contents (mg/100 g) in PSOs; C. Phytosterol contents (mg/100 g) in PSOs………………………………………………….65 Figure 2. Antioxidant capacity and oxidative stability of pomegranate (Punica granatum

L. cv. Ruby) seed oils, obtained with either expeller pressing, alcohol or supercritical CO2 (without co-solvent). A. Antioxidant capacity (mmol Trolox Equivalent/kg) of PSOs; B. Oxidative stability (hours) of PSOs………………………………………………………..67 Figure 3. Pareto charts of experimental design of the Brazilian pomegranate (Punica

granatum L. cv. Ruby) seed oils obtained by supercritical CO2 (SC-PSO) extraction with added co-solvent in function of response variables: A. Tocopherols; B. Phenolic compounds; C. cLnA; D. Total Antioxidant Capacity…………………………………….68

Figure 4. Surface response charts of the experimental design results, showing the tocopherol content (mg/100 g) in pomegranate (Punica granatum L. cv. Ruby) seed oils

obtained by supercritical CO2 extraction with added co-solvent, as a function of temperature (°C) and pressure (bar)……………………………………………………….63 Figure 5. Surface response charts of the experimental design results, showing the phenolic compound contents (mg/100 g) in pomegranate (Punica granatum L. cv. Ruby) seed oils

obtained by supercritical CO2 extraction with added co-solvent, as a function of the significant factors: A. Temperature (°C) and pressure (bar); B. Temperature (°C) and co-

solvent (% alcohol); C. Temperature (°C) and co-solvent concentration in SC-CO2 (%); D.

Co-solvent (% alcohol) and co-solvent concentration in SC-CO2 (%)……………………69 Figure 6. Distribution of phenolic compounds in the Brazilian pomegranate (Punica

granatum L. cv. Ruby) seed oils obtained by supercritical CO2 extraction (SC-PSOs) with added co-solvent…………………………………………………………………………...70 Figure 7. Principal component analysis (PCA) plot of the pomegranate (Punica granatum

L. cv. Ruby) seed oils obtained by supercritical CO2 extraction with added co-solvent, as a function of the response variables used in the statistical experimental design. A. Correlation matrix of the factors of PCA; B. Grouping of the experiments in factors’ plane……………………………………………………………………………………….72

Chapter 2

Figure 1. Bioactive compounds in extracts and fractions of the pomegranate (Punica granatum L. cv. Ruby) peel, obtained with extraction and sequential fractionation by

supercritical CO2 or methanol (conventional extraction). A. Total phenolic contents (mg GAE/g) in extracts and fractions of the pomegranate peel; B. Total flavonoids contents (mg QE/g) in extracts and fractions of the pomegranate peel; C. Hydrolysable tannins contents (mg TAE/g) in extracts and fractions of the pomegranate peel. Different letters indicate significant differences between the extracts and fractions of the pomegranate peel……...85 Figure 2. Representative fragmentograms achieved by HPLC-MS/ESI of the major ellagitannins of the pomegranate peel in N-FE (non-fractionated extract), fractions 2 and 3

of F1 (fractionation 1) and fraction 2 of F2 (fractionation 2) obtained with extraction and

sequential fractionation by supercritical CO2 or methanol (conventional extraction). A. Galloyl-HHDP-hexoside; B. Punicalin; C. Pedunculagin; D. Punicalagin………………..87

Figure 3. β-carotene contents (µg/g) in extracts and fractions of the pomegranate (Punica granatum L. cv. Ruby) peel, obtained with extraction and sequential fractionation by

supercritical CO2 or methanol (conventional extraction). Different letters indicate significant differences between the extracts and fractions of the pomegranate peel……...91 Figure 4. Antioxidant capacity of extracts and fractions of the pomegranate (Punica granatum L. cv. Ruby) peel, obtained with extraction and sequential fractionation by

supercritical CO2 or methanol (conventional extraction). A. Antioxidant capacity by FRAP assay (µmol Fe2+/g) of extracts and fractions of the pomegranate peel; B. Antioxidant capacity by TEAC assay (µmol TE/g) of extracts and fractions of the pomegranate peel..91

Supplementary Figures – Chapter 1

Supplementary Figure 1. Representative chromatogram, by GC-FID on an Omegawax- 320 column (30 m × 0.32 mm i.d., 0.25 μm), of fatty acid methyl esters from methylated Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil……………………...…112 Supplementary Figure 2. Representative chromatograms by reversed-phase HPLC-ELSD on a C18 column (3.5 µm, 150 mm  3 mm), of major lipid classes (TAG, triacylglycerols; DAG, diacylglycerols; MAG, monoacylglycerols; FFA, free fatty acid) in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil…………………………………...113 Supplementary Figure 3. Representative chromatogram, by reversed-phase HPLC-PDA- MS on a C18 column (5 μm, 250 mm × 4.6 mm), of non-anthocyanin phenolic compounds in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil…………………...…114 Supplementary Figure 4. Representative chromatogram, by reversed-phase HPLC-PDA on a C18 column (5 μm, 250 mm × 4.6 mm), of anthocyanins in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil…………………………………………………115 Supplementary Figure 5. Representative chromatogram, by normal-phase HPLC-PDA- Fluorescence (HPLC-PDA-Fluo) on an unmodified silica column (5 µm, 4.6 mm × 250 mm), of tocopherols in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil…………………………………………………………………………………………116 Supplementary Figure 6. Representative chromatogram, by normal-phase HPLC-PDA- Fluorescence (HPLC-PDA-Fluo) on an unmodified silica column (5 µm, 4.6 mm × 250 mm), of carotenoid in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil...117

Supplementary Figure 7. Representative chromatogram, by reversed-phase HPLC-PDA on a C18 column (5 μm, 50 mm × 2.1 mm), of phytosterols in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil………………………………………………....118 Supplementary Figure 8. Representative chromatogram, by GC-MS on a fused silica column 5% phenyl/95% methylpolysiloxane (30 m × 0.32 mm i.d., 3 µm film), of volatile compounds from alcohol-extracted Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil…………………………………………………………………………………....119

Supplementary Figures – Chapter 2

Supplementary Figure 1. Weight gain of the extract and fractions of the pomegranate

peel obtained by SC-CO2 in function of time…………………………………………….120 Supplementary Figure 2. Representative chromatogram, by reversed-phase HPLC-PDA on a C18 column (5 μm, 250 mm × 4.6 mm), of hydrolysable tannins (punicalagin and

ellagic acid) from fraction 2 of F2 of the pomegranate (Punica granatum L. cv. Ruby) peel…………………………………………………………………………………….…121

LIST OF TABLES

Literature Review

Table 1. Fatty acid profile in cold-pressed PSO………………………………………..…29

Chapter 1

Table 1. Operational conditions adopted for the experimental design (24-1) used for the

extraction of pomegranate (Punica granatum L. cv. Ruby) seed oil with supercritical CO2 with co-solvent…………………………………………………………………………….59 Table 2. Quality indices of pomegranate (Punica granatum L. cv Ruby) seed oil using

different extraction methods: expeller pressing, alcohol or supercritical CO2 extractions without co-solvents………………………………………………………………………...61 Table 3. Fatty acid (g/100 g total fatty acids) composition* in pomegranate (Punica

granatum L. cv. Ruby) seed oil extracted by expeller pressing, alcohol or supercritical CO2 without co-solvents………………………………………………………………………...62 Table 4. Phenolic compounds profile (mg/100 g)* in pomegranate (Punica granatum L.

cv. Ruby) seed oils extracted by expeller pressing, alcohol or supercritical CO2 without co- solvents…………………………………………………………………………………….63 Table 5. Volatile compounds profile of pomegranate (Punica granatum L. cv. Ruby) seed oil extracted by alcohol…………………………………………………………………..66

Chapter 2

Table 1. Phenolic compounds profile by HPLC-ESI-MS in the extracts and fractions of the

pomegranate peel obtained with supercritical CO2 and conventional extraction using methanol…………………………………………………………………………………...88 Table 2. Punicalagin and ellagic acid contents (mg/g of dry peel) in the extracts and

fractions of the pomegranate peel obtained with supercritical CO2 and conventional extraction using methanol………………………………………………………………....89

Table 3. Tocopherol contents (µg/g of dry peel) in the extracts and fractions of the

pomegranate peel obtained with supercritical CO2 and conventional extraction using methanol………………………………………………………………………………..….90

Supplementary Tables – Chapter 1

Supplementary Table 1. Yield, extraction efficiency and extraction time of pomegranate (Punica granatum L. cv Ruby) seed oil using different extraction methods: expeller

pressing, alcohol or supercritical CO2 (without or with co-solvent) extractions………...110 Supplementary Table 2. Total contents of tocopherols, phenolic compounds and cLnA, and total antioxidant capacity of pomegranate (Punica granatum L. cv. Ruby) seed oil

obtained by extraction with supercritical CO2 using alcohol as co-solvent, following the experimental conditions determined by statistical experimental design (24-1)…………...111

LIST OF ABREVIATIONS

ABTS 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt ANOVA Variance analysis A-PSO Alcohol-extracted pomegranate seed oil ASFE Automatic supercritical fluid extractor CE Catechin equivalent cLnA Conjugated linolenic acid DAG Diacylglycerol ddMS2 Data-dependent MS2 EAE Ellagic acid equivalent ELSD Evaporative light scattering detector ESI Electrospray ionization

F1 Fractionation 1

F2 Fractionation 2 FAME Fatty acid methyl esters FFA Free fatty acid FID Flame ionization detector FRAP Ferric Reducing Antioxidant Power GAE Gallic acid equivalent GC Gas chromatography HHDP Hexahydroxydiphenoyl HPLC High performance liquid chromatography HSCC High-speed countercurrent chromatography LC Liquid chromatography LRI Linear retention indexes MAG Monoacylglycerol MPLC Medium pressure liquid chromatography MS Mass spectrometer N-FE Non-fractionated extract PCA Principal component analysis PDA Photodiode array detector P-PSO Pressed pomegranate seed oil

PSO Pomegranate seed oil PTFE Polytetrafluorethylene RF Fluorescence detector

SC-CO2 Supercritical carbon dioxide SC-PSO Supercritical carbon dioxide extracted pomegranate seed oil SFE Supercritical fluid extraction SI Similarity index SIM Selected ion monitoring SPME Solid phase microextraction TAE Tannic acid equivalent TAG Triacylglycerol TE Trolox equivalent TEAC Trolox equivalent antioxidant capacity TPTZ 2,4,6-Tris(2-pyridyl)-S-triazine UHPLC Ultra-high performance liquid chromatography UV Ultraviolet

SUMMARY

THESIS INTRODUCTION………………………………………………………………...20 THESIS OBJECTIVES……………………………………………………………………..21

Literature review…………………………………………………………………………….22 1. Pomegranate (Punica granatum L.)……………………………………………………23 1.1 Pomegranate chemical composition…………………………………………………26 1.2 Economical application of pomegranate and its residues…………………………...27 2. Pomegranate seed oil (PSO)…………………………………………………………….28 2.1 Pomegranate seed oil chemical composition………………………………………...28 2.1.1 Major compounds: Fatty acids………………………………………………….28 2.1.2 Minor compounds: Phenolic compounds……………………………………….32 2.1.3 Minor compounds: Tocopherols………………………………………………..34 2.1.4 Minor compounds: Phytosterols………………………………………………...36 3. Pomegranate peel…………………………………………………………………………37 4. Extraction methods……………………………………………………………………….40 4.1 Pomegranate seed oil (PSO)…………………………………………………..……..40 4.1.1 Extraction by pressing…………………………………………………………..40 4.1.2 Solvent extraction……………………………………………………………….41

4.1.3 Supercritical CO2 (SC-CO2) extraction…………………………………………42 4.2 Pomegranate peel…………………………………………………………...……….45

Chapter 1 - Pomegranate (Punica granatum L.) seed oil enriched with conjugated linolenic acid (cLnA), phenolic compounds and tocopherols: improved extraction of a specialty oil by supercritical CO2

1. Introduction……………………………………………………………………………….49 2. Materials and Methods…………………………………………………………………...50 2.1. Solvents and reagents……………………………………………………………….50 2.2. Pomegranate acquisition and processing………………………………………...…51 2.3. Extraction of PSOs………………………………………………………………..…51 2.4. Pomegranate seed oil quality………………………………………………………..52

2.5. Chemical composition of PSOs……………………………………………………...52 2.5.1. Fatty acid composition by GC-FID…………………………………………….52 2.5.2. Lipid classes distribution by HPLC-ELSD…………………………………….53 2.5.3. Phenolic compound composition by HPLC-PDA/MS…………………………54 2.5.3.1 Extraction………………………………………………………………...54 2.5.3.2 Analysis by HPLC……………………………………………………..…54 2.5.4. Tocopherol, carotenoid and chlorophyll profiles by HPLC-RF/PDA……….…56 2.5.5 Phytosterol composition by HPLC-PDA……………………………………….56 2.5.6. Profile of volatile compounds by SPME-GC-MS……………………………...57 2.6. Total antioxidant capacity and oxidative stability of PSOs…………………………58

2.7. Experimental design for SC-CO2 extraction of PSO, with alcohol as co-solvent…..58 2.8. Statistical analysis………………………………………………………………..…60 3. Results……………………………………………………………………………………..61 3.1. Fatty acid composition and distribution of lipid classes…………………………....61 3.2. Phenolic compounds profile………………………………………………………...63 3.3. Tocopherols, β-carotene and phytosterols in PSO………………………………….64 3.4. Volatile compounds profile………………………………………………………….66 3.5. Total antioxidant capacity and oxidative stability…………………………………..66

3.6. Experimental design for SC-CO2 extraction of PSO with alcohol as co-solvent and oil grouping by PCA……………………………………………………………………..67 4. Discussion……………………………………………………………………………….…71 5. Conclusions………………………………………………………………………….…….75

Chapter 2 - Sequential fractionation of pomegranate (Punica granatum L.) peels by supercritical CO2 provides a fraction enriched with punicalagin

1. Introduction…………………………………………………………………...…………..77 2. Materials and Methods……………………………………………………………….…..79 2.1 Solvents and reagents………………………………………………………………..79

2.3 Extraction and sequential fractionation of the pomegranate peel by SC-CO2……....79 2.4 Characterization of the extracts and fractions of the pomegranate peel…………….80 2.4.1 Total phenolic compounds content (TPC)…………………………………...…81 2.4.2 Total flavonoid content…………………………………………………………81

2.4.3 Hydrolysable tannin content…………………………………………………….81 2.4.4 Phenolic compounds profile by HPLC-MS/ESI and MS2 analysis……………..81 2.4.5 Punicalagin and ellagic acid contents by HPLC-PDA………………………….82 2.4.6 Tocopherols and β-carotene contents by HPLC-PDA/Flu……………………...83 2.4.7 Antioxidant Capacity…………………………………………………………....83 2.5 Statistical analysis………………………………………………………………...…84 3. Results……………………………………………………………………………………..84 3.1 Phenolic compounds…………….……………………………………..…………….84 3.2 Tocopherols and β-carotene………………………………………………………....89 3.3 Antioxidant capacity…………………………………………………………………90 4. Discussion………………………………………………………………………………….92 5. Conclusion…………………………………………………………………………………96

THESIS GENERAL CONCLUSION……………………………………………………...97

Literature cited………………………………………………………………………………98

Supplementary Materials………………………………………………………………….110

THESIS INTRODUCTION

Pomegranate’s (Punica granatum L.) seed and peel are residues of pomegranate processing industry rich in bioactive compounds with potential health benefits, such as conjugated linolenic acid (cLnA), tocopherols and polyphenols, especially hydrolysable tannins. It is known that the oil and bioactive compounds extraction method might have a great influence on the chemical composition of the extract obtained and, therefore, in the bioactive potential. In addition, aiming at develop a more sustainable process, supercritical

CO2 (SC-CO2) extraction could provide extracts rich in bioactive compounds, improving the potential functionality of the extract obtained using non-polluting solvents. For this purpose, this thesis was divided into two chapters. Chapter one was dedicated to the evaluation of the chemical composition, antioxidant capacity and oxidative stability of pomegranate seed oils (PSOs) extracted by different techniques. PSOs were extracted by expeller pressing, alcohol and SC-CO2 (without co- solvent). An experimental design was performed to improve the conditions of SC-CO2 extraction with co-solvent, in order to obtain PSO concentrated in bioactive compounds from the seeds. Chapter two was dedicated to the evaluation of the fractions enriched in bioactive compounds of the pomegranate peel obtained with sequential fractionation by SC-CO2 using co-solvent. In order to fractionate the bioactive compounds from pomegranate peel and concentrate the punicalagin, different sequential fractionation methods were performed. In parallel, a conventional extraction using methanol was conducted as a standard to compare with pomegranate peel extract and fractions obtained by SC-CO2.

THESIS OBJECTIVES

1. General

The general objective of this study was to evaluate the influence of different extraction techniques on the bioactive compounds profile of pomegranate (Punica granatum L.) byproducts, seed oil and peel fractionated extract.

2. Specifics

Chapter 1: Evaluate the influence of expeller pressing, alcohol-extraction and SC-CO2 extraction on the initial quality, chemical composition (fatty acids, lipid classes, phenolic compounds, tocopherols, carotenoids, chlorophyll, phytosterols and volatile compounds), antioxidant capacity and oxidative stability of PSO; and investigate the improved conditions (tocopherols, phenolics, cLnA and

antioxidant capacity) of SC-CO2 extraction with added co-solvent, by applying experimental design.

Chapter 2: Investigate the influence of sequential fractionation by SC-CO2 with co-solvent in the production of fractions enriched with pomegranate peel’s bioactive compounds, especially punicalagin; and compare the bioactive compounds profile (phenolic compounds, tocopherols and carotenoids) and antioxidant activity of the

pomegranate peel extract and fractions obtained by SC-CO2 with a conventional extraction method using methanol.

Literature

Review

1. Pomegranate (Punica granatum L.)

Pomegranate (Punica granatum L.) belongs to the monogeneric family Punicaceae, subclass Rosidae, and its name comes from the Latin pomum (apple) and granatus (full of seeds) (Adiletta et al., 2018; Ambigaipalam et al., 2016). It was praised in ancient times in the Old Testament of the Bible, the Jewish Torah and the Babylonian Talmud as a sacred fruit conferring powers of fertility, longevity, health, abundance and good luck. Pomegranate also stood out in the ceremonies, art and mythology of the Egyptians and Greeks and was the personal emblem of the Holy Roman Emperor, Maximilian (Jurenka, 2008). Among fruit trees, it is one of the oldest cultivated species and is widely grown in tropical and subtropical regions of the world. The fruit is native to Afghanistan, Iran, China and the Indian sub- continent, but pomegranate cultivation has spread throughout the Mediterranean region, and several countries of Central and South America (Brighenti et al., 2017; Adiletta et al., 2018). The pomegranate tree is a large shrub (5–10 m) that has many spiny branches and can be extremely long lived, known to be over 200 years old. The leaves are glossy and lance shaped, and the peel of the tree turns gray as the tree ages. The flowers are large, red, white or multicolored and have a tubular calyx that eventually becomes the fruit. The ripe pomegranate fruit can be up to 13 cm wide, in grenade-shaped, crowned by the pointed calyx, with a deep red peel being often thought to be a large berry (Jurenka, 2008; Brighenti et al., 2017). The fruit can be divided into three main parts: the arils, translucent juice-containing sacs which surround the seeds, called sarcotesta; the mesocarp, or albedo, the white tissue where the arils are attached inside the fruit; and the exocarp, or pericarp, the external fibrous layer of the fruit (Ambigaipalam et al., 2016; Adiletta et al., 2018; Russo et al., 2018) (Figure 1). The aril is the edible portion of fruit, representing about 50 % of the total fruit weight and consists of about 80 % juice and 20 % seeds, while pomegranate peels constitute approximately 40 % of the whole fruit (Çam & Hisil, 2010; Fernandes et al., 2017; Russo et al., 2018). The size and color of the pomegranate, and the amount of arils and seeds may vary depending on the variety, geographical location, growing conditions, maturity stage, among others (Figure 2) (Fernandes et al., 2015).

Figure 1. Pomegranate (Punica granatum L. cv. Ruby) structures produced in the São Francisco river valley, in the State of Pernambuco, Brazil. A. Pomegranate fruits; B. Pomegranate fruit open with arils and white septal membranes; C. Pomegranate arils; D. Pomegranate juice; E. Pomegranate seeds; F. Pomegranate peels. Font: Author.

Figure 2. Variation in the size of the pomegranate and in the color of peel, arils and juice, depending on the pomegranate variety in USA. Font: Sarkhosh, 2018.

There are over 500 cultivars of pomegranate identified, which might be recognized by different names depending on the harvesting region, fruit size, color, pulp content, fruit cracking and drought resistance (Calani et al., 2013). Iranian, Turkish, Croatian, Moroccan, Spanish, Italian, Chinese and USA pomegranate cultivars have already been studied (Mousavinejad et al., 2009; Jing et al., 2012; Qu et al., 2012; Fernandes et al., 2017; Adiletta et al., 2018). Although, Iran is worldwide known by pomegranate’ quality and quantity produced with wide variety pomegranate cultivars grown in different regions, highlighting the 24 cultivars “Naderi”, “Ravandi”, “Malas”, “Shahvar”, “Zagh” and “Shirin” (Holland & Bar- Ya’akov, 2018). New production and consumers markets are emerging, in Europe, Spain is one of the main pomegranate producers and its production is mainly located in the provinces of Alicante and Murcia, being the cultivars “Mollar de Elche” and “Valenciana” the most cultivated, showing very attractive sensorial characteristics (Nuncio-Jauregui et al., 2015; Fernandes et al., 2017). Moreover, in USA, the cultivar “Wonderful” is the most cultivated and marketed, which own brightly-red coloration in the outside peel and arils (Qu et al., 2012). Pomegranate genotypes differ in sensory and agronomic characteristics, these properties conduct them to distinct application in food processing industry, based in fruits’ sweetness, sourness, juiciness, and seeds texture (Adiletta et al., 2018). The global production of pomegranate was approximately 1,500,000 tons in 2009, being Iran the largest producer and exporter, contributing with 47 % to the total (Fischer et al., 2011; Ambigaipalam et al., 2016). Brazil has favorable soil and climatic conditions to vegetative growth, flowering, fruiting and quality fruit production of pomegranate tree, with economic potential for the irrigated areas of the Brazilian semi-arid region. The Brazilian Northeast presents ideal conditions for fruit cultivation, which has aroused the interest of region producers. Pomegranate cultivation was introduced at 2010 and evaluated has begun ever since in the experimental field of Embrapa Semiarid in Petrolina (Pernambuco) with the cultivars “Wonderful” and “Bagawa” imported from the USA and Israel, respectively, with the objective of finding a new cultivation option for the region’s producers. In Paraíba state, Sousa city, is installed one of the largest Brazilian orchards of the pomegranate culture, with about 70 hectares. The cultivation started with the “Molar” and “Wonderful” varieties that were imported from California (USA) and propagated by seeds. The orchard is in full production phase, and one of the biggest producers’ concerns is in natura fruit quality improvement and the implementation of technologies for fruits’ conservation (Moreira et al., 2015). The pomegranate market has shown a increasing grow, presumably due to the rising demand of health-conscious consumers for products with potential beneficial effects on human health. Pomegranate is considered a functional food that provides physiological benefits and plays an important role in disease prevention or slows the progress of chronic diseases (Adiletta et al., 2018). Since ancient times, the pomegranate has been regarded as a “therapeutic food” with numerous beneficial properties in several diseases. Indeed, the pomegranate was commonly used in popular medicine for eliminating parasites, as an

25 anthelmintic and vermifuge, and to treat and cure aphtae, ulcers, diarrhea, dysentery, hemorrhage, microbial infections and respiratory pathologies (Viuda-Martos et al., 2010). The increasing interest in the health-promoting properties of pomegranate is justified by the most recent findings, where the fruit is a well-known source of many valuable substances that show high antioxidant activity and can be a useful agent for the prevention and treatment of a wide range of human disorders and diseases, such as infectious and cardiovascular diseases, diabetes and cancer. Pomegranate juice is used in cosmetics and food for its well-known antioxidant activity. Pomegranate peel extracts are documented as having anti-inflammatory, antimicrobial and antiproliferative properties, besides to exert a cardiovascular protective role (Nuncio-Jauregui et al., 2015; Brighenti et al., 2017; Fernandes et al., 2017; Russo et al., 2018). The beneficial health effects attributed to the consumption of pomegranate are related to presence of bioactive compounds (Russo et al., 2018). In the last years, several studies have been focused on demonstrated the pharmacological mechanisms of pomegranate and the individual constituents responsible for them. The therapeutic and medicinal properties of pomegranate and its parts (arils, seed, peel, besides leaves, roots and peel of the pomegranate tree) are associated with pomegranate components conferred especially by the polyphenols, such as ellagitannins (including punicalagin), flavonoids, anthocyanidins, anthocyanins, flavonols and flavones, and bioactive lipids, as conjugated linolenic acid (cLnA), highlighting punicic acid (Jurenka, 2008; Viuda-Martos et., al. 2010).

1.1 Pomegranate chemical composition

Pomegranate fruit has gained popularity due to its nutritional properties. The chemical composition of pomegranate is strongly dependent on the cultivar type, growing region, climate, maturity, cultivation practice and storage conditions (Viuda-Martos et al., 2010). Significant variations in minerals, water-soluble vitamins, organic acids, sugars and phenolic compounds have already been shown in pomegranate fruits (Lansky & Newman, 2007). The arils (or pomegranate pulp) present 85 % water, 1.5 % pectin, 10 % total sugars, mainly fructose and glucose, organic acids as ascorbic acid, citric acid and malic acid, minerals (Fe, Ca, Ce, Cl, Co, Cr, Cs, Cu, K, Mg, Mn, Mo, Na, Rb, Sc, Se, Sn, and Zn), tocopherols and coenzyme Q10. Furthermore, pomegranate arils are a source of bioactive compounds such as phenolic acids and flavonoids, mainly anthocyanins, which provide a red-

26 brilliant color that increases in intensity during ripening and decays after processing (Lansky & Newman, 2007; Viuda-Martos et al., 2010; Russo et al., 2018). Pomegranate seeds are a rich source of total lipids, which comprises 12 % to 20 % of total seed weight (Kalamara et al., 2015). The oil extracted from pomegranate seed is characterized by a high content of polyunsaturated fatty acids such as linoleic and linolenic, and other lipids as oleic acid, stearic acid and palmitic acid. The oil consists of approximately 80 % conjugated octadecatrienoic fatty acids, with a high content of punicic acid (cis9,trans11,cis13). Besides of lipids, the seeds present important phytochemicals such as polyphenols, tocopherols, phytosterols and sex steroids. Moreover this part, also contains protein, vitamins, minerals, pectin, lignin and sugars (Lansky & Newman, 2007; Jurenka, 2008; Viuda-Martos et al., 2010). Pomegranate peel is an important source of bioactive compounds, characterized by substantial amounts of phenolics compounds, including flavonoids (anthocyanins, catechins and other complex flavonoids) and hydrolyzable tannins (ellagic acid, punicalin, pedunculagin and punicalagin). Additionally, minerals, mainly K, N, Ca, P, Mg and Na, and complex polysaccharides have already been described in the peel (Lansky & Newman, 2007; Jurenka, 2008; Viuda-Martos et al., 2010; Ismail et al. 2012).

1.2 Economical application of pomegranate and its residues

Pomegranate is consumed as a fresh fruit or processed into juice, syrup, sauce, jam, jelly, vinegar, wine, and dietary supplements, where the peels and seeds of the fruits are discarded (Çam & Hisil, 2010; Fernandes et al., 2017; Russo et al., 2018). The food and agricultural products processing industries produce significant amounts of residues that are rich in components with high nutritional value and functional potential, wich could be important sources of antioxidant compounds of natural origin. Thereby, great attention has been paid not only to the edible parts of pomegranate, but also to the processing residues such as seeds and peels (Kalamara et al., 2015). These by-products of pomegranate are valuable potential sources of bioactive compounds arousing the interest of the food and nutraceutical industry (Russo et al., 2018). The oil extraction from pomegranate seeds seems to be an advantageous alternative for the use of this residue with high-value added, since pomegranate seed oil (PSO) shows an exceptional fatty acid profile with potential bioactivity already described, such as anti-obesity,

27 anticarcinogenic, anti-inflammatory and antioxidant activities (Lansky & Newman, 2007; Jurenka, 2008; Verardo et al., 2014; Kalamara et al., 2015). Likewise, the extraction of the bioactive compounds from the pomegranate peel seems to be promising, considering that this by-product is rich in phenolic compounds of health interest. The bioactivity of pomegranate peel extracts has already been described, as cardiovascular protective role, anti-inflammatory, anti-allergic and anticancer properties, and antioxidant, antimicrobial and wound healing potential (Ismail et al., 2012; Ambigaipalam et al., 2016).

2. Pomegranate seed oil (PSO)

The PSO extraction is a way of recovering the bioactive compounds from a by-product that is a viable renewable source. PSO present unique nutritional and functional properties which differ from other fruit seed oils; can be considered as a specialty oil rich in bioactive compounds, such as cLnA, phenolic compounds and tocopherols, being promising as a nutraceutical ingredient in the pharmaceutical industry (Costa et al., 2019).

2.1 Pomegranate seed oil chemical composition

2.1.1 Major compounds: Fatty acids

The main components of edible vegetable oils are triacylglycerols (TAGs), which are formed by a three fatty acids esterified in a glycerol skeleton, making up about 95 to 99 % of the vegetable oil composition, providing energy for the human body (Lansky & Newman, 2007). TAGs of the PSO contains conjugated unsaturated fatty acids and exhibit a distinctive biological activity (Topkafa et al., 2015). The bioactive fatty acids of the PSO are distributed majorly in position sn-2 of the TAG, providing a good bioavailability. The TAGs composition of the PSO is varied and the most importants identified are cLnA-cLnA-cLnA and cLnA- cLnA-Palmitic acid (Melo et al., 2014). The fatty acids of the PSO are predominantly unsaturated, including oleic acid, linoleic acid and, especially, high levels of cLnA, being highly recommended for human consumption, due to low contents of saturated fatty acids, such as C14:0 and C16:0 pro- atherogenics, and high contents of fatty acids beneficial to health (Table 1) (Costa et al., 2019).

Table 1. Fatty acid profile in cold-pressed PSO. Fatty acids (g/100 g) PSO Saturated Palmitic (16:0) 1.99 ± 0.05 Stearic (18:0) 1.64 ± 0.07 Arachidic (20:0) 0.53 ± 0.04 Total Saturated 4.16 ± 0.16 Unsaturated Oleic (18:1n-9) 4.00 ± 0.15 Linoleic (18:2n-6) 4.67 ± 0.17 Cis-11-eicosenoic (20:1n-9) 0.69 ± 0.08 Punicic (18:3 cis9,trans11,cis13) 60.6 ± 5.41 α-eleostearic (18:3 cis9,trans11,trans13) 6.04 ± 0.56 Catalpic (18:3 trans9,trans11,cis13) 4.79 ± 0.35 β-eleostearic (18:3 trans9,trans11,trans13) 1.41 ± 0.17 Unknown cLnAs 1.79 ± 0.24 Total Unsaturated 83.9 ± 7.13 cLnA: conjugated linolenic acid. Font: Costa et al., 2019.

cLnA is a collective term for the positional and geometric isomers of linolenic acid (octadecatrienoic acid, C18:3). The three double bonds present in cLnA can occur in positions Δ8,10,12, Δ9,11,13, Δ10,12,14 and Δ11,13,15, and can exist in both cis and trans geometrical isomer forms (ttt, ctt, ctc, ccc, tct, ttc, etc.) (Sassano et al., 2009; Topkafa et al., 2015). The PSO exhibits an exclusive fatty acids composition due to the high content of cLnA isomers. Besides PSO, others special edible oils presents cLnAs isomers in their composition, such as tung seed, bitter gourd, catalpa seed and pot marigold seed oils, in concentrations below to 0.2 % of total fatty acids (Sassano et al., 2009; Turtygin et al., 2013; Siano et al., 2016). However, PSO may contain up to 80 % of cLnA, with varied isomeric distribution, being punicic acid (cis9,trans11,cis13) the major cLnA, followed by α-eleostearic acid (cis9,trans11,trans13), catalpic acid (trans9,trans11,cis13) and β-eleostearic acid (trans9,trans11,trans13) (Figure 3) (Pande & Akho, 2009; Costa et al., 2019). The fatty acid profile of the PSO is a quality marker of this oil, where abusive conditions in the extraction process and storage can be noted based on modification of this profile (Sassano et al., 2009). cLnAs are chemically unstable due to the reactivity of the 29 conjugated double bonds and can easily suffer oxidative deterioration, especially when exposed to oxygen, moisture, light and heat. The oxidative degradation of fatty acids impairs the shelf stability, sensory properties and nutritional quality of the oil (Siano et al., 2016).

Figure 3. Chemical structure of the cLnA isomers present in the PSO. Font: PubChem database.

The occurrence of the conjugated fatty acids in seed oils is result of the action of divergent forms of the following enzymes, fatty acid desaturases and conjugase on oleic or linoleic acids, as depicted in Figure 4. Oleic acid (cis9-C18:1) is desaturated in the cells endoplasmic reticulum to linoleic acid (cis9,cis12-C18:2), then linolenic acid (C18:3) isomers by the sequential action of fatty acid desaturases. The successive formation of cLnAs is catalyzed by fatty acid conjugases. cLnA isomers are generated by mean of the action of the enzymes that convert the double bounds of linoleic acid in position 12 into 2 conjugated double bonds in positions 11 and 13. Moreover, the cis9,trans11,cis15 cLnA and trans9,trans11,cis15 cLnA isomers may be produced through the isomerization of α-linolenic acid by intestinal and ruminal bacteria via the action of the enzyme linoleic acid isomerase (Melo et al., 2014; Henessy et al., 2011).

Figure 4. Biosynthesis of conjugated fatty acids by (A) Calendula officinalis and (B) Aleurites fordii Hemsl (Tung tree). Font: Henessy et al., 2011.

The conjugation process can result in the production of bioactive fatty acids, the mechanisms of health promoting are associated with their ability to modulate the expression of genes associated with disease pathogenesis, compete with pro-inflammatory fatty acids for incorporation into the cell membrane, among others. There is evidence to suggest that cLnA isomers may suffer elongation and desaturation reactions like α-linolenic acid. This process results in the production of conjugated derivatives of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which also possess potent anti-carcinogenic and anti- adipogenic properties (Melo et al., 2014; Henessy et al., 2011). The bioactivity related to PSO is due the presence of punicic acid, which may contribute to resistance to lung, prostate, breast and colorectal cancers as well as human monocytic leukemia, and reducing both total and HDL cholesterol, which is associated with a lowered incidence of coronary heart disease and atherosclerosis (Henessy et al., 2011, Khoddami et al., 2014). Despite this, have already been reported that cLnA isomers present a low absorption in human organism, differently of the linolenic acid and conjugated linoleic acid (cLA). Studies in vivo associate the bioactive effects of the cLnA to its metabolic transformation in cLA, because part of the absorbed cLnA is quickly converted to cLA by an enzymatic Δ13-saturation in small intestine, liver and kidney (Yuan et al., 2009; Henessy et al., 2011). Thus, PSO can be considered as a much concentrated cLA source, since this oil presents cLnA contents ranging 600 to 800 mg/g of oil, whereas the principal natural sources of cLA, butter (2.5 mg/g of lipid) and whole milk (8.4 mg/g of lipid), show very lower contents (Yuan et al., 2009; Henessy et al., 2011; Melo et al., 2016).

2.1.2 Minor compounds: Phenolic compounds

Some studies reported that the potential beneficial effects on health attributed to PSO can also be due the presence of the phenolic compounds from seeds (Lansky & Newman, 2007; Viuda-Martos et al., 2010). Polyphenols are the main compounds responsible for most of the functional properties of pomegranate (Viuda-Martos et al., 2010). The profiles of phenolic compounds of the pomegranate and its parts (arils, juice, peels, seeds and mesocarp) are already well described (Pande & Akoh, 2009; Fischer et al., 2011; Ambigaipalam et al., 2016); however, data on phenolic compounds profile of the PSO and its bioactive potential are still poorly studied.

Recently, two works reported the exceptional phenolic compounds composition in this oil. PSO exhibits phenolic compounds chemically different, belonging to three groups, namely phenolic acids, flavonoids and ellagitannins (Costa et al., 2019). Among the class of the phenolic compounds, the phenolic acids are the main compounds identified in vegetable oils, owning an aromatic ring bound to one or more hydrogenated substituents, including their functional derivatives (Viuda-Martos et al., 2010). The phenolic acids described in PSO are 3,4-dihydroxyphenylacetic, 5-caffeoylquinic, ferulic, m-coumaric, p-coumaric, p-hydroxybenzoic, rosmarinic, syringic, trans-cinnamic and vanillic acids (Figure 5) (Costa et al., 2019). These compounds have antioxidant activity as chelators and free radical scavengers, interacting primarily with hydroxyl, peroxyl, superoxide and peroxynitrite radicals (Aslani & Ghobadi, 2016). The flavonoids are synthesized from the aromatic amino acids phenylalanine and tyrosine, and from malonate, presenting flavan nucleus with (C6-C3-C6) backbone, consisting of 15 carbon atoms, arranged in two aromatic rings and an oxygenated heterocyclic ring. Based on the pattern of substitutions and oxidation states of the heterocyclic rings, the flavonoids can be separated into six groups: flavonols, flavones, flavanones, flavanols, isoflavones and anthocyanins (Viuda-Martos et al., 2010). Within this class of phenolics, the anthocyanins are of great importance in the pomegranate, giving the red color to fruit and juice (Viuda-Martos et al., 2010; Aslani & Ghobadi, 2016). The flavonoids present in PSO are naringenin and quercetin, belonging to flavonol and flavanone groups, respectively, and the anthocyanins cyanidin-3-O-glucoside, cyanidin-3,5-O-diglucoside and delphinidin (Figure 5) (Costa et al., 2019). The presence of phenolic pigments in vegetable oils is not common; however anthocyanins have been identified in PSO and jussara-berry oil, both extracted with polar solvent (Silva et al., 2017).

Figure 5. Chemical structure of phenolic compounds of the PSO. Font: Pubchem database; Khan et al., 2017.

The flavonoids exhibit antioxidant and anti-inflammatory potential, and inhibit lipid peroxidation and inflammatory agents such as cyclooxygenase 1 and 2. The anthocyanidins (aglycons) show higher ability to scavenge free radicals than anthocyanins (glycosides), and this activity reduces with increase in the number of sugar molecules (Aslani & Ghobadi, 2016). Tannins are high-molecular-weight polyphenols, divided into three chemically and biologically distinct groups, as follows: condensed tannins or proanthocyanidins, hydrolyzable tannins or ellagitannins and gallotannins (Viuda-Martos et al., 2010; Ismail et al., 2012). The ellagitannins are esters of hexahydroxydiphenic acid and a polyol, usually glucose or quinic acid, being the only group found in PSO among tannins class. PSO displays one ellagitannin, the hexahydroxydiphenoyl (HHDP)-hexoside (Figure 5). Overall, ellagitannins exhibit anticarcinogenic, antimicrobial, antioxidant and anti-inflammatory activities. Nevertheless, the bioavailability of ellagitannins is low, and in the human gastrointestinal tract they release ellagic acid, which is further metabolized by the colonic microbiota to urolithins (hydroxy-6H-dibenzopyran-6-one derivatives) (García-Villalba et al., 2015). The biological effects of urolithins have been reported (Espín et al., 2013). Due to potential health properties of ellagitannins, their correct characterization and quantification are of great relevance, but this is hampered by their complex structure, their diversity and the lack of commercial standards of high purity (García-Villalba et al., 2015). PSO contains polyphenols of biological importance, and can be considered as a functional food with potential application in the formulation of nutraceuticals for human nutrition. However, the content and variety of phenolic compounds in the PSO depends on the pomegranate cultivar, geographical origin and, in particular, extraction method used (Costa et al., 2019).

2.1.3 Minor compounds: Tocopherols

Edible oils are sources of minor compounds such as tocopherols (α-, β-, γ- and δ- tocopherol), which are the main lipophilic antioxidants natural occurring in vegetable oils, with biological activity of vitamin E. The isoforms of tocopherols differ from each other by the number and location of methyl groups in their chemical structures (Figure 6). Tocopherols positive biological effects have already been described, through the following mechanism of action: regulating gene expression, signal transduction and cell functions

34 modulation by protein-membrane interactions. Moreover, tocopherols play a very important role in protecting polyunsaturated fatty acids oxidation, which may also explain the high concentration of these antioxidants in highly unsaturated edible oils (Górnas et al., 2014; Shahidi & Camargo, 2016). Antioxidants as the tocopherols present behavior like a chain- breaking electron donor antioxidant by competing with the substrate for peroxyl oxidation reaction, stopping lipid peroxidation chain reaction. This class of lipophilic antioxidants reacts more readily to peroxyl radicals than lipids, providing the formation of a resonance stabilized radical that does not propagate the chain reaction (Drotleff et al., 2015).

Figure 6. Chemical structures of the tocopherols isoforms present in the PSO. Font: Pubchem database.

In several studies, γ-tocopherol is described as the most concentrated tocopherol in PSOs, but the other isoforms also have already been identified (δ-tocopherol < α-tocopherol < β-tocopherol). Total tocopherol contents in PSO shows a wide range, from 194 to 4561 mg/100 g of oil, depending on the pomegranate variety and extraction method (Fernandes et al., 2015; Siano et al., 2016; Costa et al., 2019). PSO is highly concentrated in tocopherols, showing contents about 30-fold higher than highly consumed vegetable oils, such as soybean oil (49.7 mg/100 g), corn oil (104 mg/100 g), sunflower oil (80.8 mg/100 g), olive oil (17.8 mg/100 g) and flaxseed oil (53.4 mg/100 g) (Akil et al., 2015; Melo et al., 2016). The presence of antioxidants as α- and γ-tocopherol in PSO makes it even more special, because α-tocopherol is the main bioactive form of vitamin E in humans, and γ-tocopherol is able to inhibit lipid oxidation in vegetable oils (Shahidi & Camargo, 2016). The bioactivity of α- tocopherol, as vitamin E, is related to the decreased risk of developing cardiovascular disease, diabetes and cancer (Drotleff et al., 2015; Aslani & Ghobadi, 2016).

2.1.4 Minor compounds: Phytosterols

The plant sterols are known as phytosterols, and exhibit biologic functions similar to those of mammalian cholesterol, but due to small differences in their chemical structure, they are much less absorbed (2–5 %) than cholesterol (56 %). Phytosterols comprise great part of the unsaponifiable fraction in oils; can be found as free or esterified sterols (Ghazani et al., 2014; Fernandes et al., 2015). Phytosterols are present at high concentrations in PSO. Campesterol, β-sitosterol, stigmasterol, sitosterol, Δ-5-avenasterol and citrostadienol have already been described in PSO (Figure 7). Several studies reported the β-sitosterol as the major phytosterols in PSO. The content of total phytosterols in PSO can vary 363 to 3100 mg/100 g, depending on the pomegranate cultivar, cultivate location and, mainly, the oil extraction method (Melo et al., 2014; Verardo et al., 2014; Fernandes et al., 2015; Siano et al., 2016). The interesting composition of phytosterols in the PSO can provide potential beneficial health effects, this might encourage its use as a nutraceutical for inhibition of cholesterol absorption. Therefore, acting as lipid-lowering agent and preventing cardiovascular diseases, obesity, diabetes and cancer, moderating inflammatory responses and wound healing (Lansky & Newman, 2007; Ghazani et al., 2014; Verardo et al., 2014; Fernandes et al., 2015).

Figure 7. Chemical structures of the phytosterols present in the PSO. Font: Pubchem database.

Besides phytosterols, PSO exhibits sex sterols that are compounds from the same class of plant sterols. There are few studies evaluating sex sterols in PSO; however some compounds have already been described, such as estrone, 17-α-estradiol, estriol and testosterone (Figure 8) (Lansky & Newman, 2007).

Figure 8. Chemical structures of the sex sterols present in the PSO. Font: Pubchem database.

3. Pomegranate peel

Pomegranate is an interesting source of phenolic compounds due to their presence in different parts of the fruit, such as peels, arils, seeds and leaves. Recently, the interest and the popularity of the pomegranate have increased because of the potential health-promoting benefits related to consumption of the fruit and its derivative products (Viuda-Martos et al., 2010; Russo et al., 2018). Among seed, peel and juice of the pomegranate, the peel is the constituent that possesses higher content of phenolic compounds, which is the main bioactive compound found in this residue. The therapeutic potential of pomegranate peel has been extensively studied in different cultures. In Egyptian and Indian culture, pomegranate peel extract have already been used to treat inflammation, diarrhea, intestinal worms, cough and infertility (Ismail et al., 2012; García-Villalba et al., 2015; Russo et al., 2018). Pomegranate peels are characterized by substantial amounts of phenolic compounds, being considered a rich source of ellagitannins. The pomegranate peel extracts have a noticeably higher antioxidant capacity than the aril extracts (juice). Thus, the utilization of pomegranate peel, residues generated in juice production, might be used as potential nutraceuticals and natural food preservatives. Encouraging results have already been described improving stability in bread, probiotic ice creams, juices, wines and jams (Lansky & Newman, 2007; Ismail et al., 2012; Ambigaipalam et al., 2016; Varzakas et al., 2016). Furthermore, from the economic point of view, pure compounds of high value, high demand and limited sources, could be obtained from pomegranate residues (Varzakas et al., 2016). 37

The exceptional antioxidant potential and strong medicinal properties of pomegranate peel are attributed to presence of the polyphenols. The profile of phenolic compounds of the pomegranate peel is well established. Pomegranate peel is composed by phenolic acids (gallic acid, ellagic acid, caffeic acid, chlorogenic acid and p-coumaric acid), flavonoids (catechin, gallocatechin, epigallocatechin-3-gallate, kaempferol, kaempferol-3-O-glucoside, kaempferol- 3-O-rhamnoglucoside, luteolin, naringin, quercetin and rutin; anthocyanins: cyanidin, pelarginidin and delphinidin) and ellagitannins (HHDP-hexoside, punicalin, punicalagin, corilagin, casuarinin, pedunculagin, punigluconin, granatin A and granatin B). Moreover, some ellagic acid derivatives (ellagic acid-hexoside, -pentoside, etc.) are also present in pomegranate peel. Although there is a variety of polyphenols in the pomegranate peel, the gallic and ellagic acids, and the punicalagin are the main phenolics (Figure 9), the latter being the most abundant (Çam & Hisil, 2010; Viuda-Martos et al., 2010; Fischer et al., 2011; Qu et al., 2012; García-Villalba et al., 2015; Ambigaipalam et al., 2016).

Figure 9. Chemical structures of the major phenolic compounds of the pomegranate peel. Font: Pubchem database.

Punicalagin is a high-molecular-weight polyphenol with great biological importance, found in high amounts in the pomegranate peel (Viuda-Martos et al., 2010). The α and β forms of punicalagin are isomers of 2,3-(S)-hexahydroxydiphenoyl-4,6-(S,S)-gallagyl-ᴰ- glucose. Punicalagin from pomegranate peel exhibit high antioxidant, antifungal and antibacterial potential (Ismail et al., 2012). The health benefits of the punicalagin also are related with the prevention and treatment of obesity, inhibiting the fatty acid synthase and adipogenesis of 3T3-L1 adipocyte. The cytoprotective and inhibitory effects of the peel demonstrate reduction in cell proliferation and induction of apoptosis in breast cancer,

38 suppression of inflammatory cell signaling in colon cancer, down-regulation of expression of androgen synthesizing genes in human prostate cancer cells, and overexpressing the androgen receptor, besides potential to prevent stomach ulcers, cardiovascular diseases and digestive disorders. Furthermore, the presence of ellagic acid and gallic acid in the pomegranate peel may be responsible for the antimutagenicity (Lansky & Newman, 2007; Jurenka, 2008; Ambigaipalam et al., 2016). The content of punicalagin in the pomegranate peel can vary to 11–20 g/kg of the peel powder; it is water-soluble, thus hydrolyzes into smaller phenolic compounds in the small intestine under normal physiological conditions (Ismail et al., 2012). As well as others ellagitannins, the punicalagin is hydrolyzed to ellagic acid in the gut, resulting in a prolonged release of this acid into the blood. Studies in vivo has been found that ellagic acid is metabolized by the colon microflora to form urolithins A and B. Urolithins can be absorbed into the enterohepatic circulation, which implies that urolithins are in the systemic bloodstream for a short time and then can be excreted in the urine over 12–56 h. The metabolite profile is different among subjects, probably due to differences in colonic microflora, where the ellagitannins are metabolized. Onde in the bloodstream, ellagic acid and urolithins can arrive and accumulate in many target organs, including intestine and prostate, where the effects of pomegranate ellagitannins are observed (Viuda-Martos et al., 2010; García-Villalba et al., 2015; Turrini et al., 2015). The potential antioxidant, anti-inflammatory and anti-infective activity renders this inedible part of the pomegranate fruit nutraceutically more active as compared to pomegranate juice. Punicalagin from pomegranate peel is still few utilized in food systems. Despite its outstanding nutritional and functional potential, the astringency is the limiting factor that impairs pomegranate peel utilization in food. Some papers have focused on the applications of the punicalagin and of the pomegranate peel extract as food additives, functional food ingredients or biologically active components in nutraceutical preparations (Akhtar et al., 2015; Varzakas et al., 2016). The use of the pomegranate peel and its fractionated bioactive compounds for health promoting purposes, food preservatives, stabilizers, supplements and probiotics, and the efficacy of their nutraceutical role as supplements in food, the stability of their active ingredients under distinct food processing conditions and organoleptic alterations in finished food products, need to be explored thoroughly to ensure the effectiveness of its functional properties (Akhtar et al., 2015).

4. Extraction methods

4.1 Pomegranate seed oil (PSO)

The choice of oil extraction method will influence in the nutritional and functional properties of the oil obtained. In addition, some factors must be taken into consideration, such as water content of the raw material, hardness and location of the oil in the plant tissue. The traditional extraction methods are the pressing and the extraction with solvents; however, there are other efficient and innovative methods, such as extraction with supercritical fluid, ultrasound, pressurized liquid and hydrodistillation, but still poorly used in the food industry (Freitas et al., 2007; Tian et al., 2013; Akbari et al., 2015). PSO is considered a high-quality oil with potential health benefits. Several studies have already reported PSO extraction by different methods, such as Soxhlet, cold-pressing, normal stirring with solvent, microwave irradiation, ultrasonic irradiation and supercritical carbon dioxide (SC-CO2) (Abassi et al., 2008; Liu et al., 2009; Tian et al., 2013; Akbari et al., 2015).

4.1.1 Extraction by pressing

In the extraction method by pressing, high-pressure and generally flexible presses are used to operate different types of raw materials. Expeller-press is widely used and have several advantages, such as ease operation of the equipment, not requiring a skilled employee for its handling, has a system of wide use adaptable for different raw materials with few simple mechanical adjustments, and the process of expulsion of the oil is continuous and done in a short time (Pighinelli et al., 2008; Tian et al., 2013; Khoddami et al., 2014). This process extracts the oil without the use of chemical products, being considered safer, allowing the use of the extraction waste (cake), which is considered a byproduct of the mechanical extraction, normally used as fertilizer and animal feed. However, a peculiarity of this method is the oil residue in the extraction byproduct. In order to increase the extraction efficiency, it is necessary to adjust the moisture content around 15 %, since high moisture content reduces friction, causing low extraction yields and very low moisture content affects the press operation (Pighinelli et al., 2008; Akbari et al., 2015; Carvalho et al., 2019). Moreover, the pressing process presents the possibility of working in combination

40 with the solvent extraction, being called mixed process. This process can be used on a large scale and can also be adapted to any raw material. Although this combination of extraction methods exists to obtain a better result, solvent extraction is still the main industry choice because of its recognized efficient (Pighinelli et al., 2008; Baümler et al., 2017). The extraction of PSO by cold-press exhibits a good quality crude oil, being a valuable source of fatty acid with health‐promoting properties. This oil has substantial industrial uses, such as pharmaceutical applications in the production of anti‐aging skin creams (Khoddami et al., 2014).

4.1.2 Solvent extraction

The solvent extraction method is characterized by ensuring the complete defatted of the sample, which is caused by the removal of the components contained in the solid matrix, which are extracted and dissolved by the liquid solvent, a process also known as leaching or solid-liquid extraction. The solution composed of oil and solvent, also called micelle, is evaporated to remove the solvent. After complete removal of the solvent the oil is obtained (Tian et al., 2013; Toda et al., 2016; Baümler et al., 2017). The main organic solvent of choice in the industry is hexane, because it is the most selective and water immiscible, therefore it has high solubility in oil. Despite this, this solvent is highly flammable, as well as shows negative impacts on the environment and human health. Other solvents have been studied as an alternative to the use of hexane, such as petroleum ether and dichloromethane, both widely used in the extraction of volatile oils, however these are also considered pollutants (Ferreira-Dias et al., 2003; Toda et al., 2016; Baümler et al., 2017). Due to this, studies have been performed to improve the techniques of vegetable oils extraction in order to reduce damages caused to human health and environment. It is worth mentioning that the solvent will influence the composition of the oil, the sensory quality and the extraction yield (Silva et al., 2017; Carvalho et al., 2019). The use of solvents from renewable sources, such as ethanol, can represent a sustainable, economically viable and less polluting alternative, as it shows lower flammability, lower toxicity and is obtained from sugarcane. In addition, ethanol possesses affinity with the antioxidants present in the vegetal raw materials. The vegetable oil extraction with ethanol provides a more effective extraction of polar compounds, such as polar bioactive compounds, phospholipids, and pigments, being a add value to the oil obtained (Toda et al.,

2016; Baümler et al., 2017; Silva et al., 2017; Carvalho et al., 2019). Therefore, the use of ethanol in the extraction of PSO may provide a special oil enriched in bioactive compounds with a promising application in the pharmaceutical industry.

4.1.3 Supercritical CO2 (SC-CO2) extraction

SC-CO2 extraction is an innovative technique and seems to be advantageous, having high selectivity for solute extraction. Furthermore, is considered environmentally safe (when is done the recycling of CO2), present low cost and is readily available at high purity. This method allows supercritical operations at relatively low pressures (73.8 bar) and at near-room temperatures (31.1 °C), minimizing thermal damage to bioactive compounds (Czaikoski et al.,

2015; HadiNezhad et al., 2015). The SC-CO2 exhibits physicochemical properties intermediate between those of a gas and a liquid, which increases its function as solvent. The physical state of CO2 can be described by the pressure and temperature diagram (Figure 10).

The pressure-temperature phase diagram of CO2 shows three curves: sublimation, melting and boiling curves which in turn limit three distinct regions corresponding to solid, liquid and gaseous states. The boiling curve ends at the so-called critical point. After this point, there is the so-called supercritical region of CO2.

Figure 10. Pressure-temperature phase diagram of CO2. Font: Laboureur et al., 2015, with adaptations.

The most important properties of SC-CO2 are its density, viscosity, diffusivity, heat capacity and thermal conductivity. The high densities of SC-CO2 contribute to greater solubilisation of compounds, while low viscosities enable penetration into solids and allow 42 for flow with less friction. Manipulating the temperature and pressure above the critical points affects the properties of SC-CO2 and enhances the ability of the SC-CO2 to penetrate and extract targeted molecules from the source materials (Sahena et al., 2009; Temelli, 2009).

Moreover, CO2 is a good solvent for extracting lipid-soluble compounds and enables a high level of recovery (Sahena et al., 2009; Duba & Fiori, 2015).

The SC-CO2 present characteristics, such as: it dissolves non-polar or slightly polar compounds; the solvent power for low molecular weight compounds is high and decreases with increasing molecular weight; SC-CO2 has high affinity with oxygenated organic compounds of medium molecular weight, proteins, polysaccharides, sugars and mineral salts are insoluble; and SC-CO2 is capable of separating compounds that are less volatile, have a higher molecular weight and/or are more polar, as pressure increases (Sahena et al., 2009; Temelli, 2009; HadiNezhad et al., 2015).

SC-CO2 extraction provide extracts considered as completely natural and are given the GRAS (generally recognized as safe) status by the FDA (Food and Drug Administration,

USA), for products allowed in food applications. The extracts obtained by SC-CO2 extraction is especially useful in the food and pharmaceutical industry, where toxicity of the extraction medium, residual solvent and thermal stability of the materials are major concerns (Czaikoski et al., 2015; HadiNezhad et al., 2015).

The lipids extraction by SC-CO2 has received attention as an alternative to conventional extraction methods and has been shown to be an ideal method for extracting certain lipids (Sahena et al., 2009; Sánchez-Vicente et al., 2009; Temelli, 2009; de Melo et al.,

2014). The use of the SC-CO2 extraction is a promising alternative that can achieve comparable oil yield with respect to the conventional organic solvent extraction with better product quality, and similar to of mechanical pressing (Duba & Fiori, 2015). The oil extraction by SC-CO2 occurs with the exposition of the sample to the SC-CO2 under the controlled conditions of temperature and pressure, allowing the dissolution of the lipids from the sample in the SC-CO2. The dissolved lipids will be then separated from the SC-CO2 by a significant drop in solution pressure (Sahena et al., 2009). The inherent variability in density and chemical composition of the raw materials make it possible the extraction of many substances by SC-CO2, in which modification of the extraction conditions, especially temperatures, pressures and extraction time, may be necessary to obtain maximum extraction efficiency (Abassi et al., 2008).

SC-CO2 extraction has been successfully used to obtain oil from seeds: apricot, palm,

43 canola, rape, soybean, sunflower, jojoba, sesame, celery, parsley, neem, amaranth, borage, flax, grape and pomegranate; and nuts: acorn, walnut, almond and pistachio (Salgin, 2007;

Sánchez-Vicente et al., 2009; de Melo et al., 2014; Duba & Fiori, 2015). The SC-CO2 extraction of the PSO result in a more selective extraction of different categories of the compounds present in natural matrices, presenting an oil with chemical properties and quality very similar to pressed PSO, being enriched with bioactive fatty acids, tocopherols and phytosterols (Abassi et al., 2008; Liu et al., 2009).

The use of SC-CO2 as only extraction solvent tends to result in an oil with low content of bioactive compounds of high or intermediate polarity, such as phenolic compounds which have potential beneficial effects on human health and oils’ oxidative stability (Sánchez-

Vicente et al., 2009; HadiNezhad et al., 2015). In SC-CO2 extraction is common the use of modifiers (co-solvents), such as ethanol, methanol, isopropanol, acetone and water, which can alter the physiochemical properties of SC-CO2. The components that are added to the primary fluid to enhance extraction efficiency are known as co-solvents. The addition of 1–10 % of co-solvent to SC-CO2 can expand its extraction range to include polar compounds (Sánchez- Vicente et al., 2009; Sahena et al., 2009; Temelli, 2009). The application of co-solvents has been dominated by ethanol, which was selected in

53 % of the SC-CO2 extraction studies from vegetable matrices (de Melo et al., 2014; Solana et al., 2014). The presence of ethanol positively affects the extraction of polyphenols, promoting the obtaining of an oil with better functional and antioxidant properties, and possibly with greater oxidative stability. The use of water as SC-CO2 extraction co-solvent favor the extraction of specific polar compounds from the matrix, but the process working conditions needs higher pressure and temperature. SC-CO2, ethanol and water have different polarities, however, when they are mixture in different proportions a homogeneous mixture can be obtained, depending on the temperature and pressure conditions and the individual molar fraction of the solvents (Da Porto et al., 2014).

The main drawback in SC-CO2 extraction is the cost of the equipment; however the long-term cost can be reduced compared to the traditional solvent extraction method, as materials used in the SC-CO2 extraction are reduced and can be reused at the end of the process (Sahena et al., 2009; Temelli, 2009). Therefore, the use of the SC-CO2 in the vegetable oils extraction is viable and of great relevance to obtain products enriched with bioactive compounds naturally present in the solid matrix (Sánchez-Vicente et al., 2009; HadiNezhad et al., 2015).

4.2 Pomegranate peel

The recovery of bioactive compounds from solid residues of food industry has been reported using conventional and alternative technologies (Paes et al., 2014). Phenolic compounds of the pomegranate peels have been extracted mostly by methanol and/or combinations of methanol and other organic solvents by classical extraction techniques. However, the bioactive compounds from pomegranate peel have already been extracted by innovative methods such as SC-CO2 extraction and pressurised water extraction (Çam & Hisil, 2010; Mushtaq et al., 2015). Several studies already showed the pomegranate peel exhibit an exceptional phenolic compounds profile. Nonetheless, the quality of extracts obtained is strongly related to the employed extraction technique, and can be evaluated through characterization techniques of their phytochemical profile (Reátegui et al., 2014). The extraction of phenolic compounds from plant materials has been traditionally carried out by Soxhlet, maceration, infusion and vapor distillation, with the following solvents methanol, ethanol, acetone and ethyl acetate (Ashraf-Khorassani & Taylor, 2004; Çam & Hisil, 2010; Paes et al., 2014; Solana et al., 2014; Mushtaq et al., 2015). Among the solvents traditionally used in polyphenols extraction of raw materials or residues, the methanol is the most effective extractant solvent for a broad range of phenolic compounds, being frequently used for both in laboratory scale and industrial extraction process. Methanol is cheap and easily accessible, and in the manufacturing of herbal medicine usually uses methanol as a solvent to extract natural ingredients. The residual methanol concentration in these products is a concerning fact (Çam & Hisil, 2010; Da Porto et al., 2014; Solana et al., 2014; Mushtaq et al., 2015).

The use of SC-CO2 in the extraction of natural products has significant advantages over more conventional solvent extraction techniques, showing considerable potential in the extraction and isolation of high-value natural products and bioactive compounds. The superiority of this technique lies in the recovery of relatively pure and clean extracts especially useful for functional food and nutraceutical products development (Ashraf- Khorassani & Taylor, 2004; Mushtaq et al., 2015). Furthermore, this technique is relatively safer and applicable for the extraction of thermally labile and oxidation-susceptible plant materials, besides the absence of light and air during the extraction, reducing components degradation (de Melo et al., 2014; Solana et al., 2014). Considering the non-polar nature of

SC-CO2, the phenolic compounds extraction performance is limited due to polarity. The use of co-solvents has been proposed as an alternative to enhance the solubility of the target compounds and to increase the extraction selectivity (Ashraf-Khorassani & Taylor, 2004; Díaz-Reinoso et al., 2006; Reátegui et al., 2014; Solana et al., 2014; Mushtaq et al., 2015).

For the food industry, the great benefits of extracts obtained by SC-CO2 are their natural origin and extract composition controlled by process selectivity (Paes et al., 2014).

The possibility of extraction and fractionation of vegetable raw materials with SC-CO2 receives widespread interest due to the direct applications in the food and pharmaceutical industries for the generation of high-value products (Sahena et al., 2009; Temelli, 2009). In theory, under different conditions of temperature, pressure and time in SC-CO2 extraction, each compound possesses a unique extractability. Thus, the components in a sample are extracted in an ordered manner, under optimized conditions of SC-CO2 extraction, allowing for convenient fractionation of the extract. This can reduce the cost and time involved in the separation, compared to traditional solvent extraction (Díaz-Reinoso et al., 2006; Reverchon & De Marco, 2006; Sahena et al., 2009).

The fractionation of plant materials by SC-CO2 extraction has been used to concentrate minor compounds, such as those already reported, isolation of tocopherols from soybean and canola oils, sterols and tocopherols from olive oil, phenolics and tocopherols from olive leaves, carotene, vitamin E, sterols and squalene from palm-pressed fiber and palm leaves, bixin from annatto seed, oil and phenolics from grape seeds, and caffeine and catechins from green tea (Ashraf-Khorassani & Taylor, 2004; Lau et al., 2008; Flores & Meireles, 2016; Sökmen et al., 2018).

The fractionation by SC-CO2 is perform in successive steps changing pressure, temperature and co-solvent, to obtain the fractional extraction of the soluble compounds contained in the organic matrix, selected by decreasing solubilities in the supercritical solvent. The scope of this operation is to induce the selective precipitation of different compound families as a function of their different saturation conditions in the SC-CO2 (Díaz-Reinoso et al., 2006; Reverchon & De Marco, 2006). A SC-CO2 extraction in steps successfully divides the bioactive compounds of the matrix into dissimilar fractions with different antioxidant activities. In this process, it is possible to separate the lipophilic compounds and polar compounds with different molecular weight in distinct fractions, based on the concept that molecular weight might be the dominant factor in the SC-CO2 extractions (Ashraf-Khorassani & Taylor, 2004; Díaz-Reinoso et al., 2006; Lau et al., 2008).

The conditions employed to extract phenolic antioxidants by SC-CO2 are usually harsher than those used in the extraction of oils. Usually, oils are soluble at pressure lower than 100 bar and temperatures in the range of 40–55 °C, whereas increased pressures result in enhanced extraction of polar and high molecular mass compounds (including phenolic compounds) (Díaz-Reinoso et al., 2006). Similarly, the use of increasing amounts of modifier allows extraction of, first, the low polar and then the more polar compounds. A progressive increase in pressure and modifier proportion allows the extraction of phenols with increasing molecular weight. Theoretically, ethanol as co-solvent enhances the SC-CO2 extraction of phenols, where just low molecular weight phenols can be extracted with 2% ethanol, catechin and epicatechin can be separated with 5% ethanol, epicatechin gallate can be extracted with 10% ethanol and proanthocyanin dimers can be recoved with 15% ethanol. Higher ethanol proportions also improve the yields of low molecular weight phenols (Díaz-Reinoso et al., 2006; Reverchon & De Marco, 2006; de Melo et al., 2014). Punicalagin, the polyphenol most abundant in pomegranate peel has already extracted by SC-CO2. The optimal condition to extract phenolic compounds from pomegranate peel is 399 bar, 48 °C and 19.9 % of co-solvent (Bustamante et al., 2017). Studies about the sequential extraction of bioactive compounds from pomegranate peel by SC-CO2 are still scarce. Therefore, the sequential extraction by SC-CO2 from pomegranate peel can be attractive for the production of antioxidant extracts enriched with bioactives of interest, especially punicalagin that might be a useful active ingredient for application in food and pharmaceutical industries.

Chapter 1

Pomegranate (Punica granatum L.) seed oil enriched with conjugated linolenic acid (cLnA), phenolic compounds and tocopherols: improved extraction of a

specialty oil by supercritical CO2

Published on The Journal of Supercritical Fluids v. 147, p. 126–137, 2019

1. Introduction

Pomegranate (Punica granatum L.) seed oil (PSO) is rich in bioactive polyunsaturated fatty acids, among which conjugated linolenic acid (cLnA) stands out (Kýralan et al., 2009). cLnA is a set of positional and geometric isomers of linolenic acid (18:3), characterized by three conjugated double bonds, which occurs in products of plant origin, such as seed oils of some fruits, in concentrations below 0.2% of total fatty acids. Interestingly, PSO may contain up to 80 % of cLnA, with varied isomeric distribution (Sassano et al., 2009). Among these, punicic (cLnA cis9,trans11,cis13), catalpic (cLnA trans9,trans11,cis13), α-eleostearic (cLnA cis9,trans11,trans13) and β-eleostearic (cLnA trans9,trans11,trans13) acids have been previously identified in PSO (Sassano et al., 2009; Turtygin et al., 2013). In addition to high contents of cLnA, PSO also contains other bioactive compounds such as phenolic compounds (Elfalleh et al., 2011), tocopherols and phytosterols (Fernandes et al., 2015). These PSO components might confer bioactivity to the oil. cLnA bioactivity is related to the inhibition of certain types of cancer, modulation of the immune system, and reducing the risk of obesity and cardiovascular disease risk factors (Hennessy et al., 2011; Saha et al., 2012). Additionally, phenolic compounds and tocopherols might modulate oxidative stress and inflammation, leading to reduced risk of various chronic diseases (Aslani & Ghobadi, 2016; Zhang & Tsao, 2016). The profile of PSO bioactive compounds depends on the extraction techniques and conditions employed, which can provide oils that contain compounds with varied chemical characteristics, from non-polar triacylglycerol containing cLnA to the moderately polar phenolic compounds. Conventionally, vegetable oils are extracted by mechanical pressing or by using organic solvents, such as n-hexane (Tian et al., 2013). However, environmental and potential health hazards concerns raise the interest for the use of sustainable techniques that are recognized as economically viable and safer (Baümler et al., 2017). Ethanol has been used as an alternative solvent to n-hexane due to the good operational safety, low toxicity, renewable sourcing and for being produced on a large scale (Toda et al., 2016). Furthermore, vegetable oil extraction with ethanol promotes more effective extraction of polar compounds, such as phospholipids, pigments and polar bioactive compounds (e.g., phenolics), adding value to the bioactive specialty oil obtained (Silva et al., 2017).

In this sense, supercritical CO2 (SC-CO2) extraction emerges as an innovative technique that seems to be advantageous, due to SC-CO2 having high selectivity for solute

49 extraction, combining its ability to penetrate the matrix bed, due to its high diffusivity, with its solvation ability, besides being environmentally safe (when recirculated in an industrial plant), presenting low cost and being readily available (Abbasi et al., 2008). SC-CO2 plant oil extraction involving a polar co-solvent may extract bioactive compounds highly effectively

(HadiNezhad et al., 2015). SC-CO2 extraction might result in PSO highly concentrated in cLnA, phenolic compounds and tocopherols, leading to an improved specialty oil, but this hypothesis was not previously tested. The aim of this study was to investigate the influence of different extraction techniques (expeller pressing, alcohol-extraction and SC-CO2 extraction) on the chemical composition, antioxidant capacity and oxidative stability of PSO and to determine, by means of experimental design, improved conditions of SC-CO2 extraction with added polar co- solvent to obtain PSO concentrated in natural bioactive compounds.

2. Materials and Methods

2.1. Solvents and reagents

Standards of tocopherols (α-, β-, γ- and δ-), phytosterols (β-sitosterol, campesterol, cholestanol and stigmasterol), fatty acid methyl esters (37 FAME mix, Supelco), 6-hydroxy- 2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and 2,2’-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma- Aldrich Brazil (São Paulo, SP, Brazil). cLnA isomers (punicic acid, α-eleostearic acid, β- eleostearic acid and catalpic acid) were purchased from Larodan AB (Solna, Stockholm, Sweden). Anthocyanins and non-anthocyanin phenolic compound standards were purchased respectively from Indofine Chemical Co. (Hillsborough, NJ, USA) and Sigma-Aldrich Brazil. Carotenoids (α- and β-carotene) were isolated from carrot by open column chromatography (Rodriguez-Amaya & Kimura, 2004). Standards of chlorophylls a and b were obtained from spinach by paper chromatography (Yentsch & Menzel, 1963). All pigment standards showed purity grade higher than 95 %, determined by HPLC-PDA, and their concentrations in stock solutions were determined spectrophotometrically, based on published absorptivity data (Yentsch & Menzel, 1963; Rodriguez-Amaya & Kimura, 2004). Anhydrous reagent alcohol (90 % ethanol, 5 % methanol, 5 % 2-propanol; v/v) was used in PSO extraction, as extraction media of alcohol-extracted-PSO (A-PSO) as well as in SC-CO2 extractions, and was referred

50 to simply as alcohol throughout this work. All solvents used were HPLC grade from Tedia Brazil (Rio de Janeiro, RJ, Brazil). HPLC grade water (Milli-Q system, Millipore, Bedford, MA, USA) was used throughout the experiments.

2.2. Pomegranate acquisition and processing

Pomegranates (Punica granatum L. cv. Ruby) were produced in the São Francisco river valley, in the State of Pernambuco (Brazil), by Boa fruta® company and were purchased in a Central Distributor of fruits and vegetables in the State of Rio de Janeiro (CEASA). Pomegranates were processed in a pilot-plant in Embrapa Agroindústria de Alimentos, Rio de Janeiro, Brazil. After fruit selection, washing and sanitization (immersing in 100 ppm NaClO for 20 min), the pomegranates were longitudinally cut into four parts with a sharp knife, manually processed for the separation of peels and arils, and depulped in a pilot-scale mechanical pulper (Industrial pulper, Etal®, Araraquara, SP, Brazil). The seeds were freeze- dried (FreeZone 2.5 Liter; Labconco, Kansas City, MO, USA) for 48 h, ground (MF 10 Basic analytical mill, IKA®, Staufen, BW, Germany), sieved (< 30 mesh) and stored in polypropylene bags under partial vacuum at -20 °C until oil extraction.

2.3. Extraction of PSOs

PSOs were extracted from the freeze-dried ground seeds with alcohol, by expeller pressing or with SC-CO2, and all extractions were performed in triplicate and the oils obtained from each extraction method were combined in order to have enough sample to perform all the analyses. A-PSO was obtained as follows: anhydrous reagent alcohol as extraction solvent, 6:1 (v/w) solvent to sample ratio, under orbital shaking (100 rpm; KS 4000 i control, IKA®, Staufen, BW, Germany), at 75 °C for 2 h, based on preliminary tests. Afterwards, samples were filtered through paper filter (Whatman Nº 1) and the solvent was evaporated under vacuum at 21 °C (Rotavapor® R-215, BÜCHI, Flawil, AR, Switzerland). A-PSO was decanted at room temperature for a few minutes to separate the oil from a viscous higher density polar fraction, probably composed of alcohol-soluble carbohydrates and proteins from the seeds. The upper-phase, consisting of the clear A-PSO was collected.

Pressed-PSO (P-PSO) was obtained by pressing the freeze-dried ground seeds through an expeller press (CA 59 G, Oekotec IBG Monforts®, Monchengladbach, NRW, Germany), followed by vacuum filtration.

SC-CO2-PSO (SC-PSO) was obtained in an automatic supercritical fluid extractor (ASFE) system (MV-10 ASFE, Waters®, Milford, MA, USA) using supercritical carbon dioxide as solvent. This system was equipped with a column oven, a back-pressure regulator, and pumps including carbon dioxide delivery pump, co-solvent pumps, and makeup solvent ® pump. SC-CO2 system was controlled by Chrom Scope TM software (Waters ). Freeze-dried ground seeds (20 g, < 30 mesh) were placed in 25 mL stainless steel extraction cells. The supercritical fluid extraction (SFE) procedure was carried out at 40 °C and pressure of 200 bar, which were conditions optimized elsewhere (Abbasi et al., 2008), and CO2 flow of 2.0 mL/min, until constant weight of the extract to the nearest 0.01 g plus an additional 20 min, which in practical terms was considered as the solvent’s extraction capacity was exhausted (Supplementary Table 1). The extraction mode was set as dynamic mode, and SC-PSO was collected in 100 mL glass-flasks. All PSOs were stored in screw-capped amber vials under nitrogen at -20 °C until analysis.

2.4. Pomegranate seed oil quality

Peroxide, p-anisidine and iodine values were determined according to AOCS official methods (AOCS, 2009). Acid value was assessed by potentiometric titration in an automatic titrator (Mettler Toledo, Greifensee, ZH, Switzerland) and the results were expressed as % punicic acid (Costa et al., 2019). Refractive index was determined in a portable digital refractometer (N-3E, ATAGO, Tokyo, Japan).

2.5. Chemical composition of PSOs

All analyses were done in triplicate from sample preparation.

2.5.1. Fatty acid composition by GC-FID

The lipids of PSOs were transesterified by alkaline catalysis (Kramer et al., 1997), and fatty acid methyl esters (FAME) were analyzed in a capillary gas chromatograph (GC-2010,

Shimadzu®, Kyoto, Japan) equipped with a split/splitless injector, flame ionization detector and it was separated into a polar capillary column (30 m × 0.32 mm i.d., 0.25 mm film; Supelco Co., Belefonte, USA). The injector and detector temperatures were set up at 260 °C and 280 °C, respectively. The oven was temperature-programmed as follows: 160 °C for 2 min, increased to 190 °C at 2.5 °C/min, held at 190 °C for 5 min, increased to 220 °C at 3.5 °C/min, and held at 220 °C for 20 min. Helium was used as the carrier gas (Costa et al., 2019). Fatty acid peaks were identified by comparison of relative retention times with commercial standards of common fatty acids (37 FAME mix, Supelco, Bellefonte, PA, USA) and individual standards of the four major cLnA isomers (punicic acid, α-eleostearic acid, β- eleostearic acid and catalpic acid). Quantitative analysis was based on the use of C17:0 as an internal standard.

2.5.2. Lipid classes distribution by HPLC-ELSD

The distribution of the major lipid classes in PSOs was determined on a high performance liquid chromatograph (HPLC; Shimadzu®, Kyoto, Japan) equipped with a quaternary pump LC-20AT, system controller CBM-20A, degasser DGU-20A5 and evaporative light scattering detector ELSD-LT II. Nitrogen was used as nebulizing gas at 0.65 mL/min, and detector temperature was set at 40 °C. Chromatographic separation was achieved using a reversed-phase C18 column (3.5 µm, 150 mm × 3 mm; Kromasil, Bohus, Ale, Sweden) and a mobile phase gradient at 1.0 mL/min flow rate were used. PSOs (2.0 g) were dissolved in 1.5 mL acetonitrile:isopropanol:hexane (2:2:1 v/v/v), centrifuged (10,000 xg, 5 min) and 1.0 mL of the solvent phase was filtered through a PTFE syringe filter (0.45 µm), followed by injection of 20 µL in the system (Silva et al., 2017). Identification of the lipid classes: free fatty acids + monoacylglycerol (FFA+MAG), diacylglycerol (DAG) and triacylglycerol (TAG) were based on retention. Quantitative analysis was based on internal normalization.

2.5.3. Phenolic compound composition by HPLC-PDA/MS

2.5.3.1 Extraction

Non-anthocyanin phenolic compounds were extracted from PSOs in diol solid phase extraction cartridges (Bond Elut 2-OH, 3 mL, 500 mg; Agilent Technologies; CA, USA). Briefly, cartridges were conditioned with 5 mL of methanol and 5 mL of n-hexane, and then 2.5 g of sample in n-hexane (1:2 w/v) were added. The cartridge was washed with 5 mL of n- hexane:ethyl acetate (9:1, v/v) and non-anthocyanin phenolic compounds were eluted with methanol and collected in a 5 mL volumetric flask (Siger et al., 2008). Anthocyanins were extracted by liquid-liquid extraction, dissolving PSOs in n-hexane (1:2, w/v) and extracting with 80 % aqueous methanol. Solutions were vortex-mixed for 10 min and the lower phase was removed and transferred to a round bottom flask. Extraction was repeated three times and all extracts were combined. Solvent was completely evaporated (Rotavapor® R-215) and the residue was reconstituted with 4 mL of 80 % aqueous methanol (Tuberoso et al., 2007).

2.5.3.2 Analysis by HPLC

Analysis of non-anthocyanin phenolic compounds was performed in a HPLC system (Shimadzu®, Kyoto, Japan) consisting of two parallel pumps LC-20AD, automatic injector SIL-20AHT, photodiode array detector (PDA) SPD-M20A, system controller CBM-20A, degasser DGU-20A5 and a quadrupole mass spectrometer LCMS-2020 with electrospray ionization (ESI). A nitrogen generator (NM32LA, Peak Scientific, Inchinnan, SCT, UK) was coupled to the LC-MS. Chromatographic separation of non-anthocyanin phenolic compounds was achieved using a reversed-phase C18 column (5 μm, 250 mm × 4.6 mm, Kromasil®, Bohus, Ale, Sweden) (de la Torre-Carbot et al., 2005). The mobile phase consisted of a gradient of water:formic acid:acetonitrile (98.7:0.3:1.0, v/v; eluent A) and methanol:acetonitrile (99:1, v/v; eluent B), at 1.0 mL/min flow rate. Prior to injection, the column was equilibrated with 24.2 % B, followed by a gradient elution program: 24.2 % (B) at 8 min, 28.2 % (B) at 18 min, 33.3 % (B) at 30 min, 65.6 % (B) at 60 min, 24.2 % (B) at 60.1 min, followed by 15 min re-equilibration, for a total analysis cycle time of 75 min. Non- anthocyanin phenolic compounds were monitored by PDA, from 190 to 370 nm. The ESI interface was operated by selective ion monitoring (SIM), in negative and positive modes for

54 phenolic compound ionization. Mass spectrometer operation conditions were as follows: detector voltage, 3.0 kV; interface temperature, 350 °C; dessolvation line temperature, 250

°C; nebulizing gas (ultra-pure N2) flow, 1.5 L/min; heat block, 200 °C; drying gas (ultra-pure ® N2) flow, 15 L/min. Data were acquired with LabSolutions software (Shimadzu Corporation , Kyoto, Japan, version 5.82 SP 1, 2015). Analysis of anthocyanins was performed in an HPLC system (Shimadzu®, Kyoto, Japan) consisting of a quaternary pump LC-20AT, photodiode array detector SPD-M20A, system controller CBM-20A and degasser DGU-20A5. Chromatographic separation of anthocyanins was achieved using a reversed-phase C18 column (5 μm, 250 mm × 4.6 mm, Kromasil®, Bohus, Ale, Sweden). The mobile phase consisted of a gradient of 1 % aqueous formic acid (eluent A), 1 % formic acid in methanol (eluent B) and acetonitrile (eluent C), at 2.0 mL/min flow rate. Eluent C concentration was kept constant at 2 % during analysis. Prior to injection, the column was equilibrated with 18 % B. The gradient elution program was as follows: 18 % (B) at 2 min, 32 % (B) at 6 min, 52 % (B) at 8 min, 18 % (B) at 8.1 min, followed by 15 min re-equilibration, for a total analysis cycle time of 23 min. Anthocyanins were monitored at 530 nm (Brown & Shipley, 2011). Data were acquired with LC solution software (Shimadzu Corporation®, Kyoto, Japan, version 1.25, 2009). Phenolic compounds were identified based on retention times of commercial standards, and peak identity was confirmed by standard co-elution, and by comparison of the absorption spectra of peaks in the samples before and after spiking, and those of peaks of commercial standards (2,4-dihydroxybenzoic, 2,4-dihydroxyphenylacetic, 4- hydroxyphenylacetic, 5-caffeoylquinic, caffeic, ellagic, ferulic, gallic, m-coumaric, p- coumaric, p-hydroxybenzoic, rosmarinic, syringic, trans-cinnamic and vanillic acids, cyanidin, cyanidin-3-O-glucoside, cyanidin-3,5-O-diglucoside, cyanidin-3-O-rutinoside, delphinidin, kaempferol, naringenin, pelargonidin, quercetin and rutin). In the case of non- anthocyanin phenolic compounds, peak identity was confirmed by the pseudo molecular ions (LC-MS analysis) of the suspected compounds in the samples. Quantitative analysis was based on external calibration with commercial standards. Calibration curves were linear for all non-anthocyanin phenolic compounds and anthocyanins (1.0 to 20.0 μg/mL, R2 > 0.999, p < 0.0001).

2.5.4. Tocopherol, carotenoid and chlorophyll profiles by HPLC-RF/PDA

Tocopherol, β-carotene and chlorophyll contents in PSOs were determined simultaneously in an HPLC system (Shimadzu®, Kyoto, Japan) consisting of a quaternary pump LC-20AT, system controller CBM-20A, degasser DGU-20A5, photodiode array detector SPD-M20A and fluorescence detector RF-10AXL. PSOs were dissolved in n-hexane, centrifuged (10,000 xg, 5 min) and the oil solution was filtered through a PTFE syringe filter (0.45 µm) (Gimeno et al., 2000). Chromatographic separation was achieved using a normal phase silica column (Zorbax-SIL 5 µm, 4.6 mm × 250 mm; Agilent Technologies, Santa Clara, CA, USA) with isocratic elution (hexane:isopropanol, 99:1, v/v) at 1.0 mL/min. Fluorescence detector was operated at 290 nm (excitation) and 330 nm (emission) for tocopherols. PDA detector was used for quantification of carotenoids (λ: 450 nm) and chlorophylls (λ: 665 nm) (Silva et al., 2017). Commercial standards of tocopherols and standards of carotenoids and chlorophylls (chromatographically isolated) were used for identification and quantification by external calibration. Concentrations of each tocol, carotenoid and chlorophyll standard stock solutions were determined spectrophotometrically after appropriate dilutions using the following specific extinction coefficients (λ; dL/g.cm) in ethanol: α-tocopherol (292 nm; 75.8); β- tocopherol (296 nm; 89.4); γ-tocopherol (298 nm; 91.4); δ-tocopherol (298 nm; 87.3); α- carotene (450 nm; 2800); β-carotene (450 nm; 2620); lutein (450 nm; 2550); lycopene (450 nm; 3450) and zeaxanthin (450 nm; 2540); and in acetone: chlorophyll a (665 nm; 670) and chlorophyll b (665 nm; 518). Calibration curves were linear for all tocols (0.5 to 3.0 μg/mL, R2 > 0.999, p < 0.0001), β-carotene (0.5 to 5.0 μg/mL, R2 = 0.999, p < 0.0001) and chlorophylls (0.5 to 10.0 μg/mL, R2 = 0.9991, p < 0.0001). Data were acquired by LC solution software (Shimadzu Corporation®, version 1.25, 2009).

2.5.5 Phytosterol composition by HPLC-PDA

Phytosterol contents in PSOs were determined in an HPLC system (Shimadzu®, Japan) consisting of a quaternary pump LC-20AT, system controller CBM-20A, degasser DGU- 20A5 and photodiode array detector SPD-M20A. After saponification, the chromatographic separation was achieved using a reversed-phase C18 column (5 μm, 50 mm × 2.1 mm; Phenomenex®, Torrance, CA, USA) with isocratic elution (acetonitrile:isopropanol, 98:2, v/v)

56 at 0.4 mL/min. PDA was monitored to 210 nm for identification and quantification of phytosterols (Bauer et al., 2013). Commercial standards of β-sitosterol, campesterol, cholestanol and stigmasterol were used for phytosterols identification and quantification by external calibration. Phytosterols were identified based on retention times of commercial standards, and peak identity was confirmed by standard spiking and by comparison of absorption spectra between injections (samples, spiked samples and pure standards). Calibration curves were linear for all phytosterols (0.5 to 3.0 μg/mL, R2 > 0.9991, p < 0.0001). Data were acquired by LC solution software (Shimadzu Corporation®, version 1.25, 2009).

2.5.6. Profile of volatile compounds by SPME-GC-MS

Volatile compounds were extracted from PSOs by solid phase microextraction (SPME) using a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber (Supelco, Bellefonte, PA, USA) and were analyzed in a gas chromatograph GC-17A coupled to a mass spectrometer QP5050A (Shimadzu®, Kyoto, Japan) equipped with a split/splitless injector and a fused silica column 5 % phenyl/95 % methylpolysiloxane (007-5, 30 m × 0.32 mm i.d., 3 µm film; Quadrex, Bethany, CT, USA) (Akil et al., 2015). In SPME, PSOs (1 g) were weighed in a headspace vial, 20 µL of internal standard (0.1 mg/mL of bromobenzene in methanol; Supelco, Bellefonte, PA, USA) was added and they were sealed with a PTFE-lined septum. Headspace vials were placed in a glycerol bath (40 °C) for 30 min, after this equilibration time, septum was pierced and the fiber was exposed to the sample headspace for 10 min. Volatile compounds were desorbed from the SPME fiber in the injection port at 260 °C for 5 min, in the splitless mode, and after 5 min sampling the split purge valve was open at 3.0 ml/min. Helium was used as the carrier gas and the column pressure was set at 49 kPa. The column oven temperature was held at 30 °C for 10 min, then increased at 3 °C/min to 200 °C and held for 10 min. The mass spectrometer was operated in electron impact mode at 70 eV. The interface and ion source temperatures were 260 °C. Analyses were performed in full scan acquisition mode, in the mass range 40-500 m/z at 0.5 scan/s. A mixture of standard hydrocarbons (C7-C30 saturated alkanes; Supelco, Bellefonte, PA, USA) was run under the same conditions to allow calculation of linear retention index (LRI) values for the volatile compounds (Viegas & Bassoli, 2007). Compounds were tentatively identified utilizing the comparison of mass spectra with those of the National Institute of Standards (NIST) library,

57 calculation of similarity indexes (SI) provided by the instrument’s software (Lab Solutions GC-MS, Kyoto, Japan, version 1.21, 2008) and calculation of the experimental LRI of each compound.

2.6. Total antioxidant capacity and oxidative stability of PSOs

Total antioxidant capacity of the PSOs was determined by TEAC assay (Trolox Equivalent Antioxidant Capacity). The ABTS•+ solution was prepared by reacting aqueous ABTS (7 mmol/L) with potassium persulfate (2.45 mmol/L). The ABTS•+ solution was diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm. For the assay, 1 mL of ABTS•+ solution reacted with 10 µL of PSO or standard solution in n-hexane. The reaction was monitored at 734 nm for 4 min in a spectrophotometer (UV-1800; Shimadzu, Japan). Total antioxidant capacity of oils was determined by calibration curve with Trolox, in concentrations ranging from 0.02 to 0.75 mmol/L. The area under the curve (AUC) of absorbance at 734 nm vs. reaction time was calculated, and the results were expressed as mmol of Trolox equivalents/kg (mmol TE/kg) (Castelo-Branco & Torres, 2012). The oxidative stability of PSOs was determined in Rancimat® equipment (Metrohm 743; Metrohm Co., Herisau, Switzerland). PSOs (3 g) were heated at 80 °C with a 20.0 L/h air flow rate and the time required for a sharp increase in water conductivity was calculated with the instrument software, which corresponds to the induction period (h) and is directly related to the oils’ oxidative stability.

2.7. Experimental design for SC-CO2 extraction of PSO, with alcohol as co-solvent

Experimental design was used to determine the influence of the extraction factors on the composition of PSO extracted by SC-CO2. For these experiments, alcohol was used as co- solvent, in the same equipment (MV-10 ASFE, Waters, Milford, MA, USA) detailed in section 2.3. Fractional factorial design (24-1) was applied to evaluate the effects of pressure (bar), temperature (°C), co-solvent (anhydrous or aqueous reagent alcohol) and co-solvent concentration in SC-CO2 (%, v/v) over the response variables investigated (phenolic profile, tocopherol and cLnA contents, and total antioxidant capacity), with the purpose of finding improved conditions for the extraction of PSO enriched in bioactive compounds, through the combination of SC-CO2 with a polar co-solvent. The operational conditions adopted for the

58 experimental design (24-1) used for the extraction of SC-PSO with co-solvents and their respective levels are listed in Table 1. Pressure and temperature intervals used in the experimental design were established based on a previously published optimized extraction of PSO (Abassi et al., 2008) and preliminary tests (data not shown); moreover, the temperature interval was chosen so as to minimize the thermal degradation of compounds. Anhydrous reagent alcohol was used as co-solvent for SC-PSO, with 100 % alcohol or mixed with ultra- pure water for the concentrations of 10 and 50 % alcohol (v/v). Solvent composition (100 % or 10 % alcohol, v/v) and its concentration in SC-CO2 (10 % and 1 %, v/v) were, respectively, the maximum and minimum values used in the experimental design as polar modifiers to improve the extraction of polar compounds in PSO.

Table 1. Operational conditions adopted for the experimental design (24-1) used for the extraction of pomegranate (Punica granatum L. cv. Ruby) seed oil with supercritical CO2 with co-solvent. Co-solvent Experiment Pressure Temperature 1 Concentration in number (bar) (°C) Alcohol 2 SC-CO2 (%, v/v)

5 200 40 10% 10

9 3 250 50 50% 5

2 300 40 100% 1

3 200 60 100% 10

4 300 60 100% 1

1 200 40 100% 10

7 200 60 10% 10

10 3 250 50 50% 5

8 300 60 10% 1

6 300 40 10% 1

1 Listed randomly, in the order that they were performed; 2 Anhydrous reagent alcohol was used (mixture of 90 % ethanol, 5 % methanol and 5 % iso-propanol; v/v), either pure (100 % alcohol) or mixed with ultra-pure water (10 and 50 % alcohol; v/v); 3 Internal points in experimental design.

Response variables were analyzed as described in sections 2.5.1, 2.5.3, 2.5.4 and 2.6. Freeze-dried ground pomegranate seeds (20 g, < 30 mesh) were placed in 25 mL stainless steel extraction cells. SFE procedure was carried out in random order under the experimental conditions set-up in the experimental design until the lipids of the raw material were depleted, using the extraction mode set as dynamic. The extraction was carried out until no more visible oil droplets mixed in the co-solvent came out of the extractor, plus an additional 20 min, which was considered as the apparent time for sample exhaustion by the extraction solvent. The average extraction times for each experiment are shown in Supplementary Table 1. These extractions were repeated at least three times, and the extracted oil for each experiment was combined, to have enough sample for all the analyses. SC-PSO was collected in 100 mL glass flasks and sealed with silicone septa aluminum seals until they were used within 48 h. The co-solvent used for SC-PSO extraction was evaporated under a gentle nitrogen stream at 30 °C following day, until constant weight was reached, to the nearest 0.1 mg. When aqueous alcohol was used as co-solvent, drying was concluded in the following day. Oils were stored in sealed vials under nitrogen at -20 °C until analysis.

2.8. Statistical analysis

All results are presented as mean ± standard deviation of analytical triplicates of the composite oil samples (at least three extraction replicates in each experiment). Data normality distribution was assessed by the Kolmogorov-Smirnov normality test. One-way ANOVA with Tukey´s post-test was used to compare PSOs’ quality indices, chemical compositions, antioxidant capacities and oxidative stabilities. Fractional factorial design (24-1) was applied in order to find the improved conditions for the extraction of potentially bioactive SC-PSO with added co-solvent, and response surface methodology (RSM) was used to estimate each response variable as a function of the significant factors in each model. Principal Component Analysis (PCA) was performed to assess sample grouping according to the chemical composition of PSO from the experimental design. For all analyses, two-sided p-values < 0.05 were considered as statistically significant. All data were analyzed using GraphPad Prism software (version 6.01, 2012, GraphPad Software, San Diego, CA, USA). For the experimental design and the PCA, STATISTICA software (version 7.0, 2004, StatSoft. Inc., Tulsa, OK, USA) was used.

3. Results

The method of PSO extraction, either by expeller pressing, alcohol or SC-CO2, affected the chemical composition of the oil obtained. Despite this, the general fatty acid, vitamin E, phytosterol and polyphenolics profile was similar between extraction techniques, and the quality indices acid value, peroxide value, p-anisidine value, iodine value, and refractive index were adequate (Table 2) according to current technical regulations (Codex Alimentarius Commission, 1999).

Table 2. Quality indices of pomegranate (Punica granatum L. cv Ruby) seed oil using different extraction methods: expeller pressing, alcohol or supercritical CO2 extractions without co-solvents.

Pomegranate seed oil extraction method

Quality indices Expeller Alcohol- Supercritical CO2 pressing extraction (no added co-solvents) Acid value (% punicic acid) 1.48 ± 0.08b 2.41 ± 0.01a 1.34 ± 0.15c c a b Peroxide value (mEq O2/kg) 1.68 ± 0.20 4.02 ± 0.74 3.24 ± 0.30 p-anisidine value 6.81 ± 0.19b 2.04 ± 0.01c 8.59 ± 0.04a Iodine value 258.1 ± 6.38a 198.6 ± 9.08c 229.5 ± 4.27b Refractive index 1.4295 ± 0.0003c 1.5019 ± 0.0001b 1.5382 ± 0.0004a

All values are mean  SD of analytical triplicates; extraction experiments were performed in triplicate and the PSO for each extraction method were combined. Different superscript letters in the same line indicate significant differences (p<0.05; one-way ANOVA with Tukey´s post-test).

3.1. Fatty acid composition and distribution of lipid classes

Although the general fatty acid profiles were similar among the PSOs, for most individual fatty acids, the contents varied significantly (p<0.05) (Table 3). Three saturated fatty acids were identified in PSOs samples and, on average, represented only 3.3 % of total fatty acids. Eleven unsaturated fatty acids were identified in PSOs, from which four were the major cLnA isomers identified with the aid of commercial cLnA standards, and four isomers of unknown cLnAs (Supplementary Figure 1).

Table 3. Fatty acid (g/100 g total fatty acids) composition* in pomegranate (Punica granatum L. cv. Ruby) seed oil extracted by expeller pressing, alcohol or supercritical CO2 without co-solvents.

Method used for pomegranate seed oil extraction

Fatty acids Expeller Alcohol- Supercritical CO2 pressing extraction (no added co-solvents) Saturated Palmitic (16:0) 1.71 ± 0.03a 1.45 ± 0.16b 0.98 ± 0.10c Stearic (18:0) 1.85 ± 0.11a 1.24 ± 0.26b 0.76 ± 0.03c Arachidic (20:0) 0.43 ± 0.17a 0.36 ± 0.07b 0.21 ± 0.02c Total Saturated 3.99 ± 0.31a 3.05 ± 0.49b 1.95 ± 0.15c Unsaturated Oleic (18:1n-9) 4.15 ± 0.24a 2.99 ± 0.53b 1.58 ± 0.07c Linoleic (18:2n-6) 3.84 ± 0.23a 4.36 ± 0.55a 3.43 ± 0.16a Cis-11-eicosenoic (20:1n-9) 0.65 ± 0.07a 0.39 ± 0.10b 0.48 ± 0.02b Punicic (18:3 c9,t11,c13) 75.9 ± 3.09b 60.6 ± 0.96c 80.7 ± 1.45a α-eleostearic (18:3 c9,t11,t13) 6.05 ± 2.23a 2.97 ± 1.32b 4.89 ± 0.31a Catalpic (18:3 t9,t11,c13) 2.38 ± 1.08b 1.12 ± 0.23c 3.69 ± 0.22a β-eleostearic (18:3 t9,t11,t13) 0.99 ± 0.32a 0.58 ± 0.13b 0.56 ± 0.05b Unknown cLnAs 2.37 ± 0.33a 1.88 ± 0.40b 2.80 ± 0.10a Total Unsaturated 96.3 ± 7.59a 74.9 ± 4.22b 98.1 ± 2.38a

* Determined by GC-FID. All values are mean  SD. Different superscript letters in the same line indicate significant differences (p<0.05; one-way ANOVA with Tukey´s post-test). c: cis; t: trans.

The predominant fatty acids in the PSOs were cLnA isomers, of which punicic acid was the most prevalent (75.9 – 80.7 % of total fatty acids). Contents of total cLnA in P-PSO and SC-PSO were, respectively, 1.3- and 1.4-fold higher than in A-PSO (67.1 g/100 g). As expected, PSOs were composed mainly of triacylglycerols (Supplementary Figure 2), as observed for most vegetable oils (Tranchida et al., 2007). TAG contents were, as follows: A-PSO, 93.7 %; P-PSO, 99.4 %; SC-PSO, 99.8 %. Consequently, A-PSO showed high contents of the other two major lipid classes (FFA+MAG, 3.7 %, and DAG, 2.6 %), consistently with the higher acid value found in A-PSO (Table 2) that indicates higher contents of free fatty acids.

3.2. Phenolic compounds profile

As expected, A-PSO presented higher contents of phenolic compounds than P-PSO and SC-PSO (Table 4).

Table 4. Phenolic compounds profile (mg/100 g)* in pomegranate (Punica granatum L. cv. Ruby) seed oils extracted by expeller pressing, alcohol or supercritical CO2 without co-solvents. Contents of phenolic compounds (mg/100 g) for each pomegranate seed oil extraction method

Phenolic compound Expeller Alcohol- Supercritical CO2 pressing extraction (no added co-solvents)

Phenolic acids 2,4-Dihydroxyphenylacetic acid nd nd 0.14 ± 0.00 3,4-Dihydroxyphenylacetic acid 0.07 ± 0.00 nd nd 5-Caffeoylquinic acid nd 7.62 ± 0.01 nd Ferulic acid 0.22 ± 0.02b 4.21 ± 0.06a 0.29 ± 0.02b p-Coumaric acid 0.07 ± 0.00b 2.49 ± 0.05a nd p-Hydroxybenzoic acid nd 1.37 ± 0.00a 0.10 ± 0.00b Rosmarinic acid 0.05 ± 0.00b 6.17 ± 0.00a nd Trans-Cinnamic acid 0.11 ± 0.00b 1.63 ± 0.00a 0.14 ± 0.00b Vanillic acid 0.07 ± 0.00c 6.69 ± 0.22a 0.19 ± 0.00b Total phenolic acids 0.59 ± 0.02c 30.2 ± 0.34a 0.86 ± 0.02b Ellagitannin HHDP hexoside (eq. ellagic acid) 0.11 ± 0.00b 22.4 ± 0.30a 0.11 ± 0.00b Flavonoids Cyanidin-3-O-glucoside nd 0.90 ± 0.00 nd Cyanidin-3,5-O-diglucoside nd 20.0 ± 0.61 nd Delphinidin nd 0.02 ± 0.00 nd Naringenin 0.31 ± 0.03b 3.37 ± 0.33a 0.14 ± 0.00c Total flavonoids 0.31 ± 0.03b 24.3 ± 0.94a 0.14 ± 0.00c Total phenolic compounds 1.01 ± 0.05b 76.9 ± 1.58a 1.11 ± 0.02b

*Determined by HPLC-PDA-MS. All values are mean  SD, of analytical triplicates of the composite oil samples, coming from at least three extraction replicates in each experiment. Different superscript letters in the same line indicate significant differences (p<0.05; one-way ANOVA with Tukey´s post-test). nd: not detected. HHDP: hexahydroxydiphenoyl. eq: equivalent 63

In A-PSO twelve phenolic compounds were identified, seven phenolic acids being 5- caffeoylquinic, ferulic, p-coumaric, p-hydroxybenzoic, rosmarinic, trans-cinnamic and vanillic acids, one ellagitannin being hexahydroxydiphenoyl (HHDP) hexoside and four flavonoids being cyanidin-3-O-glucoside, cyanidin-3,5-O-diglucoside, delphinidin and naringenin (Supplementary Figures 3 and 4). However, only eight and seven phenolic compounds were found in P-PSO and SC-PSO, respectively. The content of total phenolic compounds in A-PSO (76.9 mg/100 g) was 76- and 69-fold higher than in P-PSO and SC- PSO, respectively. Regarding the flavonoids cyanidin-3-O-glucoside, cyanidin-3,5-O- diglucoside and delphinidin, these phenolic pigments were undetectable in both P-PSO and SC-PSO (Table 4).

3.3. Tocopherols, β-carotene and phytosterols in PSO

The contents of tocopherols, β-carotene and phytosterols in PSOs were significantly influenced by the extraction methods (Figure 1). Four tocopherol isoforms (α-, β-, γ- and δ-) were identified in A-PSO, while three isoforms (α-, γ- and δ-) were present in P-PSO and SC- PSO (Supplementary Figure 5). In all PSOs, γ-tocopherol was the major tocol (on average 98.9% of total tocols), followed by δ-tocopherol and α-tocopherol (Figure 1A). Furthermore, the content of γ-tocopherol in A-PSO (2888 mg/100 g) was approximately 2.6- and 1.2-fold higher than that of P-PSO and SC-PSO, respectively. Although found in low concentrations, -carotene was the only natural pigment found in all PSOs (Figure 1B; Supplementary Figure 6). P-PSO showed the highest content of β- carotene (0.17 mg/100 g) when compared to A-PSO (0.11 mg/100 g) and SC-PSO (0.02 mg/100 g). Chlorophylls a and b were also investigated as natural pigments in this study but were undetectable in all PSOs. All PSOs presented high contents of phytosterols (Figure 1C; Supplementary Figure 7). Total phytosterol content in A-PSO and SC-PSO was, on average, 1.6-fold higher than in P-PSO (1964 mg/100 g). In A-PSO and P-PSO, β-sitosterol was the major sterol, whereas in SC-PSO, campesterol was the major sterol.

Figure 1. Bioactive compounds in pomegranate (Punica granatum L. cv. Ruby) seed oils, obtained with either expeller pressing, alcohol or supercritical CO2 (without co-solvent). A. Tocopherol contents (mg/100 g) in PSOs; B. β-carotene contents (mg/100 g) in PSOs; C. Phytosterol contents (mg/100 g) in PSOs. Different letters indicate significant differences between the extraction techniques (one-way ANOVA with Tukey´s post-test, p<0.05).

3.4. Volatile compounds profile

Volatile compounds were undetectable in P-PSO and SC-PSO. But, A-PSO showed seven volatile compounds belonging to different chemical classes (Table 5 and Supplementary Figure 8). Of these compounds, the following five present flavor notes resembling fruit, sweet, roses, and toasted aroma: ethyl acetate; propanoic acid, 2-hydroxy-, ethyl ester; hexylene glycol; ethanedioic acid, diethyl ester; 2(3H)-furanone, 5-ethoxydihydro- (Table 5).

Table 5. Volatile compounds profile of pomegranate (Punica granatum L. cv. Ruby) seed oil extracted by alcohol. Alcohol-extracted Brazilian pomegranate seed oil Volatile compounds SI Content Aroma LRI (%) (µg/g of oil) notes Alcohols 2-Pentanone, 4-hydroxy-4-methyl- 841 87 11.82 ± 0.97 - Hexylene glycol 918 93 20.60 ± 0.29 Sweet Esters Ethyl acetate 505 89 36.81 ± 1.86 Pineapple Propanoic acid, 2-hydroxy-, ethyl ester 812 90 15.96 ± 0.75 Fruit Ethanedioic acid, diethyl ester 983 95 74.95 ± 4.88 Sweet, roses Others 2-Hydroxypropanoic acid 870 93 7.68 ± 0.67 - 2(3H)-Furanone, 5-ethoxydihydro- 1063 89 7.46 ± 0.29 Toasted

LRI: Linear Retention Index. SI: Similarity Index.

3.5. Total antioxidant capacity and oxidative stability

Total antioxidant capacity and oxidative stability were significantly different among PSOs (Figure 2). A-PSO presented total antioxidant capacity 1.1- and 1.2-fold higher than P- PSO and SC-PSO, respectively (Figure 2A). Similarly, oxidative stability was 2.5- and 2.3- fold higher in A-PSO than in P-PSO and SC-PSO, respectively (Figure 2B).

Figure 2. Antioxidant capacity and oxidative stability of pomegranate (Punica granatum L. cv. Ruby) seed oils, obtained with either expeller pressing, alcohol or supercritical CO2 (without co-solvent). A. Antioxidant capacity (mmol Trolox Equivalent/kg) of PSOs; B. Oxidative stability (hours) of PSOs. Different letters indicate significant differences between the extraction techniques (one-way ANOVA with Tukey´s post-test, p<0.05).

3.6. Experimental design for SC-CO2 extraction of PSO with alcohol as co-solvent and oil grouping by PCA

Contents of tocopherols, phenolic compounds and cLnA, and antioxidant capacity of

PSO extracted with SC-CO2 with polar co-solvent, using the experimental conditions of the fractional factorial design (24-1) are shown in Supplementary Table 2. In this study, the effects of processing conditions (pressure, temperature, co-solvent and co-solvent concentration in SC-CO2) were analyzed referring to the experimental outcomes of tocopherols, phenolic compounds, cLnA and total antioxidant capacity, represented in Pareto charts (Figure 3).

Figure 3. Pareto charts of experimental design of the Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oils obtained by supercritical CO2 (SC-PSO) extraction with added co-solvent in function of response variables: A. Tocopherols; B. Phenolic compounds; C. cLnA; D. Total Antioxidant Capacity.

As noted in sections 3.1-3.3, SC-PSO presented high contents of cLnA, although lower contents of phenolic compounds and tocopherols, therefore, a fractional factorial design (24-1) was used to determine improved conditions for the extraction of PSO containing high concentrations of bioactive compounds by SC-CO2 with a polar modifier. The coefficient of determination (R2) of the fractional factorial design (24-1) for tocopherols, phenolic compounds, cLnA and total antioxidant capacity were 0.92, 0.95, 0.30 and 0.77, respectively; thus, for tocopherols and phenolics the R2 values indicated a strong correlation between the experimental data and the predicted values. Tocopherol contents were positively affected by temperature and by the interaction between pressure versus temperature, according to Pareto charts from the experimental design (Figure 3A). Independently of the co-solvent composition (% alcohol), at higher pressures, increasing temperature led to higher contents of tocopherols (1.9-fold on average), whereas at lower pressures the opposite was observed (1.2-fold on average) (Supplementary Table 2). Response surface plots showed that the highest tocopherol content was achieved at higher temperature (60 °C) and pressure (300 bar) (Figure 4).

Figure 4. Surface response charts of the experimental design results, showing the tocopherol content (mg/100 g) in pomegranate (Punica granatum L. cv. Ruby) seed oils obtained by supercritical CO2 extraction with added co- solvent, as a function of temperature (°C) and pressure (bar).

Total phenolic contents were positively affected by temperature, co-solvent composition and co-solvent concentration in SC-CO2 and were negatively affected by the interaction of pressure versus temperature, shown in the Pareto charts (Figure 3B). At lower 69 pressures, increasing temperature led to higher contents of phenolics (5.5-fold on average), whereas at higher pressure the influence of temperature depended on the co-solvent (% alcohol) (Supplementary Table 2). In this way the highest content of phenolic compounds was observed at the lowest pressure (200 bar), the highest temperature (60 °C), the highest alcohol proportion (100 %) and the highest co-solvent concentration in SC-CO2 (10 %) (Figure 5).

Figure 5. Surface response charts of the experimental design results, showing the phenolic compound contents

(mg/100 g) in pomegranate (Punica granatum L. cv. Ruby) seed oils obtained by supercritical CO2 extraction with added co-solvent, as a function of the significant factors: A. Temperature (°C) and pressure (bar); B.

Temperature (°C) and co-solvent (% alcohol); C. Temperature (°C) and co-solvent concentration in SC-CO2

(%); D. Co-solvent (% alcohol) and co-solvent concentration in SC-CO2 (%).

Overall, the extraction conditions sensibly affected the profile of phenolic compounds in SC-PSOs. Using a polar co-solvent promoted the co-extraction of ten phenolic compounds in SC-PSO: eight phenolic acids being 2,4-dihydroxybenzoic, 3,4-dihydroxyphenylacetic, ferulic, p-coumaric, p-hydroxybenzoic, rosmarinic, trans-cinnamic and vanillic acids, one

70 ellagitannin being HHDP hexoside and one flavonoid being naringenin, but these compounds were not identified in all samples (Figure 6).

Figure 6. Distribution of phenolic compounds in the Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oils obtained by supercritical CO2 extraction (SC-PSOs) with added co-solvent.

Experimental design factors of SC-PSO extraction with alcohol were not significant for cLnA content and total antioxidant capacity, as dependent variables, as depicted in Pareto charts (Figures 3C and 3D). PCA was used to discriminate the SC-PSO based on bioactive profile, and it confirmed the results from the experimental design. Tocopherols, phenolic compounds, and cLnA were included in the PCA matrix, and the resulting factors 1 and 2 explained more than 80% of the total variance in the correlation matrix (Figure 7A), indicating a robust statistical model capable of identifying the trends and groupings of data. Figure 7B depicts graphically sample grouping based on chemical composition.

4. Discussion

Different extraction methods were used to obtain PSOs enriched in bioactive compounds. Alcohol was used as a low toxicity solvent that resulted in PSO with high contents of phenolic compounds, tocopherols and phytosterols, besides presenting high

71 antioxidant capacity and volatile compounds from the fruit, some of fruity character, with attractive aromatic notes. Differently, cLnA content was higher in PSO extracted by expeller pressing or with SC-CO2 without polar modifier. The experimental design showed that tailor- made PSO could be extracted by SC-CO2 depending on the particular interest on each of the natural antioxidant classes investigated. Additionally, the detailed chemical characterization of oils extracted from pomegranate seeds acclimated and cultivated in Brazil is described for the first time in this study.

Figure 7. Principal component analysis (PCA) plot of the pomegranate (Punica granatum L. cv. Ruby) seed oils obtained by supercritical CO2 extraction with added co-solvent, as a function of the response variables used in the statistical experimental design. A. Correlation matrix of the factors of PCA; B. Grouping of the experiments in factors’ plane.

PSOs fatty acid profiles were predominantly formed of unsaturated fatty acids (74.9 g/100 g to 98.1 g/100 g), especially cLnA isomers. This typical fatty acid profile was previously reported for Turkish PSO extracted by cold pressing or n-hexane, in which the major fatty acid was punicic acid, ranging from 70.4 % to 84.5 %, depending on the pomegranate cultivar (Kýralan et al., 2009; Topkafa et al., 2015). cLnA isomers are bioactive lipids with functionalities already shown in humans (Yuan et al., 2009). The biological properties of the cLnA isomers previously described were immunomodulatory activity, reduced risk of obesity, improved cardiovascular health, and strong anticarcinogenic activity in in vitro and animal studies (Hennessy et al., 2011). The analysis of volatile compounds revealed a unique character of A-PSO, which was the only oil that showed volatile compounds in the present study. Interestingly, these aroma compounds come from pomegranate’s seeds, residual pulp and recovering skin. The profile of volatile compounds identified in A-PSO was consistent with the aroma released by this oil, with a sweet and fruity character. Future investigations, using olfatometric analysis by GC- MS-sniffing port, are necessary to identify which of the aroma active compounds in this oil (ethyl acetate; propanoic acid, 2-hydroxy-, ethyl ester; hexylene glycol; ethanedioic acid, diethyl ester; and 2(3H)-furanone, 5-ethoxydihydro-) or compounds combinations could impart flavor to A-PSO. The extraction technique affected PSO phenolic profile, in which A-PSO presented higher contents of phenolic compounds than P-PSO and SC-PSO; and this was due to the extraction media polarity, meaning that alcohol was able to drag higher amounts of phenolic compounds to the oil (Silva et al., 2017). On the other hand, in P-PSO and SC-PSO the extraction media were, respectively, the seed oil and SC-CO2, which are non-polar and present lower capacity as carrier for these polar compounds with the oil (Temelli, 2009). Similar results were observed in ethanol extracted jussara-berry oil, which showed high contents of phenolic compounds (71.5 mg/100 g) (Silva et al., 2017). Data about the phenolic compound profile of PSO is scarce; however, its composition in other pomegranate parts, such as, peel, mesocarp, arils and seeds, and pomegranate juice has previously been published (Pande & Akoh, 2009; Fischer et al., 2011). Pomegranate seeds from Georgia showed phenolic compound classes similar to those found in PSO in the present study, that were phenolic acids, flavonoids and ellagitannins, of which p-coumaric and ferulic acids, and the hydrolyzable tannins stand out (Pande & Akoh, 2009). Furthermore, the pomegranate fruit presents several anthocyanins (Fischer et al., 2011), and among some of the major

73 anthocyanins, cyanidin-3-O-glucoside, cyanidin-3,5-O-diglucoside and delphinidin were found in A-PSO. Previous studies of pomegranate and its industrial byproducts have shown antioxidant, anti-inflammatory, antiatherogenic, antihypertensive and antiproliferative activities attributed to their phenolic compounds, as well as their effects on subcellular signaling pathways, induction of cell-cycle arrest and apoptosis (Seeram et al., 2005; Danesi et al., 2014; Turrini et al., 2015). Thus, alcohol extraction increased the potential bioactivity of PSO, by increasing the diversity and the contents of phenolic compounds compared to other extraction methods. Brazilian PSOs presented higher tocopherol contents than other PSOs, as follows: P- PSO, A-PSO and SC-PSO showed total tocopherol contents on average 3-, 9- and 7-fold higher than previously reported (Fernandes et al., 2015; Melo et al., 2016). Additionally, total tocopherol contents in Brazilian PSOs were higher than those reported for common vegetable oils, such as soybean oil (49.7 mg/100 g), sunflower (61 mg/100 g) and olive oil (17.8 mg/100 g) (Akil et al., 2015), and fruit seed oils, such as grape seed oil (24.9 mg/100 g) (Beveridge et al., 2005). PSOs showed -tocopherol contents within the range of soybean oil, providing similar vitamin E activity (Akil et al., 2015). In contrast, PSO was highly concentrated in - tocopherol, showing contents on average 30-fold higher than those in soybean oil, which is known as a major dietary source of this tocopherol. Consistently, γ-tocopherol was previously reported as the predominant tocol in PSOs (Fernandes et al., 2015; Melo et al., 2016). The importance of γ-tocopherol lies on its high antioxidant activity, and for inhibiting lipid oxidation in vegetable oils (Melo et al., 2016), although its bioactivity remains largely unknown. The total phytosterol contents in A-PSO, P-PSO and SC-PSO were on average 2-to-3- fold higher than those found in plant oils considered dietary sources of these compounds, such as corn, rapeseed, soybean and sunflower (Verleyen et al., 2002), adding potential bioactivity to PSO, because of the health benefits attributed to phytosterols (Uddin et al., 2015). Tocopherol contents were increased in SC-PSO (with polar co-solvent) with pressure and temperature at the maximum values tested (300 bar and 60 °C). Although the co-solvent polarity (100 % vs. 10 % alcohol) was not significant to tocopherol in PSO according to Pareto chart (Supplementary Figure 3A), there was a clear tendency otherwise, because 10 % alcohol seemed to extract tocopherols more effectively (Supplementary Table 2). Co- solvents, such as ethanol, methanol and water are often used to increase SC-CO2 polarity and favor tocopherol extraction, for instance from canola oil (Quancheng et al., 2004). The

74 improved extraction of phenolic compounds required a more polar solvent, consisting of 100

% alcohol at 10 % in SC-CO2; and this was probably because alcohol has higher solubility in

SC-CO2 than water (Da Porto et al., 2014), providing higher solubilization of the phenolic compounds from within the pomegranate seeds into the extracted oil. In addition, the percentage of co-solvent in SC-CO2 may influence the phenolic profiles of the extracts (Murga et al., 2000). SC-PSO extraction with added co-solvent can provide a PSO concentrated on each of these natural bioactive compounds depending on the specific interest.

5. Conclusions

A-PSO and SC-PSO with added co-solvent can be considered as specialty oils rich in bioactive compounds, such as cLnA, phenolic compounds and tocopherols that can be used by the pharmaceutical industry as promising nutraceutical ingredients. Future scaled-up studies using pure ethanol for PSO extraction, either directly or as co-solvent in SC-CO2, are desirable, because of its lower toxicity compared to reagent alcohol used in this study. The findings of this study provide information to add value to pomegranate seeds’ that are obtained as residue of juice production, and are, therefore, a viable renewable source of different bioactive compounds. These results might stimulate future investigations about the bioactivity of A-PSO and SC-PSO with polar co-solvent.

Chapter 2

Sequential fractionation of pomegranate (Punica granatum L.) peels by

supercritical CO2 provides a fraction enriched with punicalagin

To be published on The Journal of Supercritical Fluids

1. Introduction Pomegranate (Punica granatum L.) is an interesting source of natural antioxidants and bioactive compounds (Ismail et al., 2012; Ambigaipalan et al., 2016). Pomegranate consumption popularity increased in recent years, because of the potential health-promoting benefits associated to the consumption of the fresh fruit or of numerous processed products such as juices, jams, jellies, vinegar, wine, syrups, among other products containing the fruit pulp [1]. Pomegranate peels and seeds are by-products formed in the fruit processing, that have high contents of antioxidants (Ismail et al., 2012; Russo et al., 2018). Pomegranate peel is of high interest, corresponding to approximately 40 % in weight of the whole fruit (Çam & Hisil, 2010; Li et al., 2015) and presenting high contents of biologically active compounds, it is considered a nutraceutical product with high functional added-value (Hasnaoui et al., 2014; Ambigaipalan et al., 2016). Health benefits have been related to pomegranate peel use, such as anti-inflammatory, antioxidant, anticarcinogenic, antibacterial, antifungal and anti-atherosclerotic activities, besides aiding wound healing (Ismail et al., 2012; Khan et al., 2017). The potential bioactivity conferred to pomegranate peel is attributed to polyphenols, especially ellagitannins, of which stand out punicalagin (α and β), punicalin, hexahydroxydiphenoyl (HHDP)-hexoside, ellagic acid and its derivatives (ellagic acid-hexoside, ellagic acid-pentoside and others) (Fischer et al., 2011; Ambigaipalan et al., 2016; Russo et al., 2018). Among these, punicalagin is the most abundant phenolic compound in pomegranate peel and to it are attributed most of the beneficial health effects of the fruit (Bustamante et al., 2017). Being bulky and rich in bioactive phenolic compounds makes the pomegranate peel of great interest for use as raw material to extract natural food preservatives, functional ingredients, dietary supplements, and also analytical standards for laboratory assays (Hasnaoui et al., 2014; Aguilar-Zárate et al., 2017; Bustamante et al., 2017; Russo et al., 2018). Pomegranate peel bioactive compounds have been extracted by organic solvents, such as water, methanol, diethyl ether, acetone, ethyl acetate, ethanol and aqueous-ethanol, via classical extraction techniques (Ismail et al., 2012; Bustamante et al., 2017; Tyskiewicz et al., 2018). Methanol is frequently used in laboratory scale and industrial extraction process, where the residual level of this solvent in the extracts is a matter of concern (Çam & Hisil, 2010).

Supercritical CO2 (SC-CO2) extraction is an environment-friendly technology when

CO2 is recycled, that emerges as an alternative technique for the extraction of natural products

77 and can be very effective as an integrated fractionation technique of bioactive compounds

(Paviani et al., 2010; Da Porto et al., 2014; Mushtaq et al., 2015). SC-CO2 presents numerous interesting properties as extraction medium, such as high diffusivity and low viscosity, allowing extraction at low temperatures (near 30 °C), at relatively low pressures (near 73.8 bar), and also, has a low cost (Da Porto et al., 2014; Tyskiewicz et al., 2018). SC-CO2 is non- polar, thus being less efficient to extract polar components, such as phenolic compounds. Therefore, the addition of small amounts of a polar co-solvent, such as ethanol, can increase the polarity of SC-CO2 and improve the extractability of polyphenols (Murga et al., 2000;

Mushtaq et al., 2015; Tyskiewicz et al., 2018). Using SC-CO2 to extract bioactive compounds has the additional advantage to allow integrated fractionation of compounds of interest.

The fractionation by SC-CO2 is a selective separation of the food matrix components in sequential steps, based on their polarity and molecular weight, in order to obtain fractions enriched with different bioactive compounds (Murga et al., 2000; Reverchon & De Marco, 2006; Lau et al., 2008). In this process selective precipitation of different compounds takes place as a function of their different saturation conditions in the SC-CO2 (Reverchon & De

Marco, 2006). Although SC-CO2 extracts non-polar compounds more easily, increasing pressure and density increases SC-CO2 capacity to extract more polar components (Paviani et al., 2010), and this property is used in favor of integrating extraction with fractionation.

Fractionation of plant materials by SC-CO2 extraction has been previously used to isolate and concentrate minor bioactive components (Ibañez et al., 1999; Murga et al., 2000; Ashraf- Khorassani et al., 2004; Lau et al., 2008; Paviani et al., 2010; Sökmen et al., 2018). However, to the best of our knowledge, pomegranate peel bioactive compounds’ fractionation by SC-

CO2 has not been previously reported. Other methods have been proposed for the isolation and concentration of ellagitannins from pomegranate waste, such as preparative high-performance liquid chromatography (HPLC), high-speed countercurrent chromatography (HSCC) and medium pressure liquid chromatography (MPLC) (Zhou et al., 2010; Zhou et al., 2011; Aguilar-Zárate et al., 2017; Russo et al., 2018). However, these methods require expensive equipment and the operating process is time consuming (Aguilar-Zárate et al., 2017; Russo et al., 2018), especially considering scaled-up processes, when compared to the SC-CO2 extraction systems.

In the present study, the sequential fractionation by SC-CO2 with ethanol as polar co- solvent was applied to obtain fractions enriched in bioactive compounds of the pomegranate peel, especially punicalagin. A conventional extraction method using methanol was

78 performed in parallel to use as a reference to compare the bioactive compounds profile and antioxidant activity of the pomegranate peel extract and fractions obtained by SC-CO2.

2. Materials and Methods

2.1 Solvents and reagents

Standards of tocopherols (α-, β-, γ- and δ-), Folin–Ciocalteu reagent, 2,4,6-tris(2- pyridyl)-S-triazine (TPTZ), 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate, (±)-6-hydroxy-2,5,7,8-tetramethylchromane- 2-carboxylic acid (Trolox), fluorescein and potassium phosphate were purchased from Sigma- Aldrich Brazil (São Paulo, SP, Brazil). Sodium carbonate and aluminum chloride were purchased from Spectrum Chemical Manufacturing Corp. (Gardena, CA, USA). Iron (II) sulfate was purchased from Merck KGaA (Darmstadt, HE, Germany). Phenolic compounds standards were purchased respectively from Indofine Chemical Co. (Hillsborough, NJ, USA) and Sigma-Aldrich Brazil (São Paulo, SP, Brazil). Beta-carotene was isolated from carrot by open column chromatography (Rodriguez-Amaya & Kimura, 2004). All solvents used were HPLC grade from Tedia Brazil (Rio de Janeiro, RJ, Brazil). HPLC grade water (Milli-Q system, Millipore, Bedford, MA, USA) was used throughout the experiments.

2.2 Pomegranates acquisition and processing

Pomegranates (Punica granatum L. cv. Ruby) were cultivated in the São Francisco river valley, in the State of Pernambuco (Brazil), by Boa fruta® company and were acquired in a Central Distributor of fruits and vegetables in the State of Rio de Janeiro (CEASA). Pomegranates were processed in a pilot-plant in Embrapa Agroindústria de Alimentos, Rio de Janeiro, Brazil. After fruit selection, washing and sanitization (immersing in 100 ppm NaClO for 20 min), the pomegranates were longitudinally cut into four parts with a sharp knife, manually processed for the separation of peels and aryls. The peels were dried in an air convection dryer (330 drier, FANEM®; São Paulo, SP, Brazil) at 40 °C for 24 h (on average, 1.3 % moisture content), ground (MF 10 Basic analytical mill, IKA®, Staufen, BW, Germany), sieved (< 30 mesh) and stored in polypropylene bags under partial vacuum at -20 °C.

2.3 Extraction and sequential fractionation of the pomegranate peel by SC-CO2

The extract and fractions of the pomegranate peel were obtained in an automatic supercritical fluid extractor (ASFE) system (MV-10 ASFE, Waters®, Milford, MA, USA) using SC-CO2 as solvent. This system was equipped with a column oven, a back-pressure regulator, and pumps including carbon dioxide delivery pump, co-solvent pumps, and makeup ® solvent pump. SC-CO2 system was controlled by Chrom Scope TM software (Waters ). Dried ground peels (20 g, < 30 mesh) were placed in 25 mL stainless steel extraction cells. The continuous extraction technique was used in this study with isothermal condition at 40 °C and constant CO2 flow at 2.0 mL/min. SC-CO2 extraction procedure was carried out starting from an optimized extraction condition for punicalagin (Bustamante et al., 2017), with pressure at 350 bar, temperature at 40 °C and 20 % co-solvent (ethanol), for 8 h (non-fractionated extract, N-FE). Based on preliminary extractions (unpublished data), two methods for the sequential fractionation of the pomegranate peel by SC-CO2 were developed to obtain fractions enriched in bioactive compounds and, especially, concentrated in punicalagin. The first fractionation method (F1) was performed in a three-step process: step 1 – 350 bar and 40 °C, for 6 h (fraction 1); step 2 – 100 bar, 40 °C and 5 % ethanol, for 2 h (fraction 2); step 3 – 350 bar, 40 °C and 10 % ethanol, for 6 h (fraction 3); with a total extraction time of 14 h. The second fractionation method (F2) was performed in two-step process: step 1 – 350 bar, 40 °C and 2 % ethanol, for 2 h (fraction 1); step 2 – 350 bar, 40 °C and 10 % ethanol, for 6 h (fraction 2); with a total extraction time of 8 h. The extraction mode was set as dynamic mode, and the extract and fractions of the pomegranate peel were collected in 100 mL flasks with aluminum seals. Pomegranate peels were also extracted with methanol under orbital shaking (200 rpm and 40 °C, for 1 h) (Çam & Hisil, 2010), in order to compare with the results obtained of the extraction and sequential fractionation by SC-CO2. All extracts and fractions were stored in screw-capped amber vials at -20 °C until analysis.

2.4 Characterization of the extracts and fractions of the pomegranate peel

Prior to analysis, the extract and fractions of the pomegranate peel obtained by SC-

CO2 were transferred into a 50 mL of volumetric flasks and the total volume was adjusted with ethanol. Then, all the extracts and fractions, including methanol extract, were filtered through a PTFE syringe filter (0.45 µm) and used in the analyses.

2.4.1 Total phenolic compounds content (TPC)

Extracts and fractions of the pomegranate peel were analyzed spectrophotometrically by the Folin–Ciocalteu reagent assay at 725 nm (Singleton et al., 1999). Results were expressed as mg of gallic acid equivalents (GAE) per g dry peel.

2.4.2 Total flavonoid content

Total flavonoid content in extracts and fractions of the pomegranate peel was determined spectrophotometrically by the assay with Aluminum Chloride (AlCl3) (Taie et al., 2008). Samples absorbance was read at 425 nm and compared to blank solution. The results were expressed as mg catechin equivalent (CE) per g of dry peel.

2.4.3 Hydrolysable tannin content

Hydrolysable tannin content in extracts and fractions of the pomegranate peel was determined as described previously (Çam & Hisil, 2010). Briefly, a 1.0 mL aliquot of the extracts and fractions, properly diluted, was added to 5 mL of 2.5% KIO3 and vortexed by 10 s. Samples absorbance was read at 550 nm and compared to blank solution. The results were expressed as mg tannic acid equivalent (TAE) per g of dry peel.

2.4.4 Phenolic compounds profile by HPLC-MS/ESI and MS2 analysis

The profile of phenolic compounds in extracts and fractions of the pomegranate peel was determined in a Dionex UltiMate 3000 Ultra-High Performance Liquid Chromatography system (UHPLC; Thermo Fisher Scientific, Waltham, MA, USA) consisting of a quaternary solvent delivery pump (Dionex UltiMate 3000 UHPLC; Thermo Fisher Scientific, Waltham, MA, USA), a column oven compartment, an automatic injector (TriPlus RSH Autosampler, Thermo Fisher Scientific, Waltham, MA, USA) and a mass spectrometer (Q Exactive Plus, Hybrid Quadrupole-Orbitrap Mass Spectrometer, Thermo Fisher Scientific, Waltham, MA,

USA). A N2 generator (NM32LA, Peak Scientific, Inchinnan, SCT, UK) was coupled to the UHPLC-MS. Chromatographic separation of phenolic compounds was achieved using a reversed-phase C18 column (2.7 μm, 100 mm × 2.1 mm, Poroshell®; Agilent Technologies,

Santa Clara, CA, USA) maintained at 40 °C, as described previously (Bustamante et al., 2017) with modifications. The mobile phase consisted of a gradient of water:formic acid (99.9:0.1, v/v; eluent A) and acetonitrile (eluent B), at 0.4 mL/min flow rate. Prior to injection, the column was equilibrated with 5 % B, followed by a gradient elution program: 5 % (B) at 1 min, 25 % (B) at 6 min, 95 % (B) at 17–20 min, 5 % (B) at 20.5 min, followed by 4.5 min re-equilibration, for a total analysis cycle time of 25 min. The electrospray ionization (ESI) source was operated in selective ion monitoring (SIM), in negative and positive modes for phenolic compounds ionization. Mass spectrometer operation conditions were as follows: detector voltage, 3.5 kV; temperature, 280 °C; scan range of m/z 120 to 2,000 at resolution of 70,000, followed by data‐dependent MS2 (ddMS2) using a resolution of 17,500. Phenolic compounds were tentatively identified based on peak identity by the pseudo-molecular ions of the suspected compounds in samples, analyzing their exact mass (confirmation of composition with < 5 ppm mass error), interpretation of their fragmentograms obtained from mass spectra (MS), and the abundances of the isotope peaks of the molecular ions. Data acquisition and analysis were performed by Thermo Xcalibur software (Thermo Fisher Scientific, version 2.2, 2011, Waltham, MA, USA).

2.4.5 Punicalagin and ellagic acid contents by HPLC-PDA

Punicalagin and ellagic acid contents in extracts and fractions of the pomegranate peel were determined in a HPLC system (Shimadzu®, Kyoto, Japan) consisting of a quaternary pump (LC-20AT), photodiode array detector (PDA; SPD-M20A), system controller (CBM- 20A) and degasser (DGU-20A5). Chromatographic separation was achieved using a reversed- phase C18 column (2.7 μm, 100 mm × 2.1 mm, Poroshell®; Agilent Technologies, Santa Clara, CA, USA) as previously described in the section 2.4.4 (Bustamante et al., 2017). PDA detector scanned from 200 to 400 nm, and λ at 258 and 378 nm were used for quantification of punicalagin and ellagic acid, respectively. Punicalagin α and β were tentatively identified based on the results from the UHPLC-ESI-MS analysis (section 2.4.4) using retention time data, and comparison of ultraviolet (UV) absorption spectra with literature data. Commercial standard of ellagic acid was used for identification and quantification by external calibration. The calibration curve for ellagic acid was linear (1.0 to 20.0 μg/mL, R2 > 0.999, p < 0.01). Data were acquired by LC solution software (Shimadzu Corporation®, version 1.25, 2009,

Kyoto, Japan). Analyses were performed in duplicate, and results were expressed as mg of ellagic acid equivalent (EAE) per g dry peel.

2.4.6 Tocopherols and β-carotene contents by HPLC-PDA/Flu

The contents of tocopherols and β-carotene in extracts and fractions of the pomegranate peel were determined simultaneously in a HPLC system (Shimadzu®, Kyoto, Japan) consisting of a quaternary pump (LC-20AT), system controller (CBM-20A), degasser (DGU-20A5), photodiode array detector (SPD-M20A) and fluorescence detector (RF- 10AXL). Samples were evaporated under nitrogen, dissolved in n-hexane and the sample solution was filtered through a PTFE syringe filter (0.45 µm) (Gimeno et al., 2000). Chromatographic separation was achieved using a normal phase silica column (Zorbax-SIL 5 µm, 4.6 mm x 250 mm; Agilent Technologies; Santa Clara, CA, USA) with isocratic elution (hexane:isopropanol, 99:1, v/v) at 1.0 mL/min. Fluorescence detector was operated at 290 nm (excitation) and 330 nm (emission) for tocopherols (Tan & Brzuskiewicz, 1989). PDA detector scanned from 390 to 600 nm, and λ at 450 nm was used for quantification of carotenoids (Rahmani & Csallany, 1991). Data were acquired by LC solution software (Shimadzu Corporation®, version 1.25, 2009, Kyoto, Japan). Commercial standards of α-, β-, γ- and δ-tocopherols and standard of β-carotene chromatographically isolated were used for identification and quantification by external calibration. Concentrations of each tocol and carotenoid standard stock solutions were determined spectrophotometrically after appropriate dilutions using the following specific extinction coefficients (λ; dL/g.cm) in ethanol: α-tocopherol (292 nm; 75.8); β-tocopherol (296 nm; 89.4); γ-tocopherol (298 nm; 91.4); δ-tocopherol (298 nm; 87.3); β-carotene (450 nm; 2620) (Rodriguez-Amaya & Kimura, 2004; Franke et al., 2007). Calibration curves were linear for all tocols (0.5 to 3.0 µg/mL, R² > 0.999, p < 0.02) and β-carotene (0.5 to 3.0 µg/mL, R² > 0.999, p < 0.01). Analyses were performed in duplicate, and results were expressed in µg/g.

2.4.7 Antioxidant Capacity

Antioxidant capacity in extracts and fractions of the pomegranate peel was determined by FRAP (Ferric Reducing Antioxidant Power) and TEAC (Trolox Equivalent Antioxidant

Capacity) assays, exactly as previously described (Benzie & Strain, 1996; Re et al., 1999). The results of FRAP were expressed as µmol of Fe2+ equivalents per g dry peel, and the results of TEAC were expressed as µmol of Trolox equivalents per g dry peel.

2.5 Statistical analysis

All results are presented as mean ± standard deviation. One-way ANOVA with Tukey´s post-test was used to compare the bioactive compounds content and the antioxidant capacity of pomegranate peel extracts and fractions. Pearson correlation analysis was used to evaluate associations between variables. For all analyses, two-sided p-values < 0.05 were considered as statistically significant. All data were analyzed using GraphPad Prism software (GraphPad Software, version 6.01, 2012, San Diego, CA, USA).

3. Results

The extraction and sequential fractionation of the pomegranate peel by SC-CO2 added with co-solvent were able to provide an extract and fractions rich in bioactive compounds, being the extract and some fractions enriched in phenolic compounds and other fractions concentrated in tocopherols and β-carotene. The weight gain of extract and fractions of the pomegranate peel obtained by SC-CO2 varied according to the conditions used for extraction and fractionation (Supplementary Figure 1).

3.1 Phenolic compounds

Total phenolic compounds, total flavonoids and hydrolysable tannin contents, determined spectrophotometrically, were significantly (p<0.05) affected by the extraction conditions of the pomegranate peel (Figure 1). Similar behavior was observed among the extracts non-fractionated, fraction 3 of F1 and fraction 2 of F2, obtained with SC-CO2, and methanol extract (conventional extraction), which presented the highest contents of total phenolic compounds, total flavonoids and hydrolysable tannins from pomegranate peel. In contrast, fractions 1 of F1 and F2 showed the lowest contents of total phenolic compounds, total flavonoids and hydrolysable tannins. Total phenolic compounds content in fraction 2 of

F2 was similar to methanol extract and fraction 3 of F1, and was approximately 1.3-fold higher than the extract non-fractionated (211.6 mg GAE/g) (Figure 1A).

Figure 1. Bioactive compounds in extracts and fractions of the pomegranate (Punica granatum L. cv. Ruby) peel, obtained with extraction and sequential fractionation by supercritical CO2 or methanol (conventional extraction). A. Total phenolic contents (mg GAE/g) in extracts and fractions of the pomegranate peel; B. Total flavonoids contents (mg QE/g) in extracts and fractions of the pomegranate peel; C. Hydrolysable tannins contents (mg TAE/g) in extracts and fractions of the pomegranate peel. Different letters indicate significant differences between the extracts and fractions of the pomegranate peel (one-way ANOVA with Tukey´s post- test, p<0.05). N-FE, non-fractionated extract; F1, fractionation 1; F2, fractionation 2. 85

Otherwise, the total flavonoid content in methanol extract (83.3 mg QE/g) was approximately 3.6-, 4.1- and 5.5-fold higher than extract non-fractionated, fraction 2 of F2 and fraction 3 of F1, respectively (Figure 1B). Hydrolyzable tannins content contributed significantly for the total phenolics content in extracts and fractions of the pomegranate peels. The content of hydrolyzable tannins in methanol extract (283.4 mg TAE/g) was on average

1.3-, 1.5- and 1.5-fold higher than fraction 2 of F2, fraction 3 of F1 and extract non- fractionated, respectively (Figure 1C). Pomegranate peel presented an appealing phenolic compounds profile, showing to be an excellent source of antioxidant compounds. The UHPLC-MS/ESI-MS2 analysis allowed the attempt to elucidate the phenolic compounds profile of the pomegranate peel extracts and fractions based on analyzing their exact mass (< 5 ppm mass error), interpretation of their fragmentation patterns obtained from mass spectra (MS2; Figure 2) using published data as reference, and the contribution of the molecular ions isotope peaks. The profile of phenolic compounds was different in pomegranate peel fractions (Table

1), especially for the initial fractions of the fractionation process (fractions 1 of F1 and F2).

Pomegranate peel extract non-fractionated, fractions 2 and 3 of F1, and fraction 2 of F2 obtained with SC-CO2, and the methanol extract (conventional extraction) showed similar phenolic compounds profile, in which fifteen compounds were identified, one phenolic acid being gallic acid, two flavonoids being (+)-catechin and (+)-gallocatechin and twelve ellagitannins being ellagic acid, monogalloyl-hexoside, ellagic acid-pentoside, ellagic acid- deoxyhexoside, ellagic acid-hexoside, digalloyl-hexoside, galloyl-HHDP-hexoside, punicalin, peduncalagin, punigluconin, punicalagin α and punicalagin β (Table 1). In contrast, five and eight phenolic compounds were found in fractions 1 of F1 and F2 obtained by SC-CO2, respectively. The class of ellagitannins was the most abundant class of phenolic compounds in all extracts and fractions of the pomegranate peel, except for fraction 1 of F1. Anthocyanins were undetectable in extracts and fractions of pomegranate peel.

The extraction and sequential fractionation by SC-CO2 seems to have been efficient, since it provided for obtaining an extract and fractions rich in punicalagin. The contents of punicalagin α and β, and ellagic acid in the extracts and fractions of the pomegranate peel analyzed by HPLC-PDA were significantly (p<0.05) influenced by the extraction conditions used (Table 2).

Figure 2. Representative fragmentograms achieved by HPLC-MS/ESI of the major ellagitannins of the pomegranate peel in N-FE (non-fractionated extract), fractions 2 and 3 of F1 (fractionation 1) and fraction 2 of F2

(fractionation 2) obtained with extraction and sequential fractionation by supercritical CO2 or methanol (conventional extraction). A. Galloyl-HHDP-hexoside; B. Punicalin; C. Pedunculagin; D. Punicalagin.

Table 1. Phenolic compounds profile by HPLC-ESI-MS in the extracts and fractions of the pomegranate peel obtained with supercritical CO2 and conventional extraction using methanol. Presence (+) or absence (–) of phenolic compounds in [M – H]– (m/z) MS2 RT Error extracts and fractions of the pomegranate peel Phenolic compounds ion fragments (min) (ppm) F1, F1, F1, F2, F2, Experimental Theoretical (m/z) Methanol N-FE fr. 1 fr. 2 fr. 3 fr. 1 fr. 2 Phenolic acids Gallic acid 2.86 169.01369 169.01335 2.01 125 + + + + + + + Flavonoids (+)-Catechin 7.33 289.07176 289.0719 0.49 179, 205, 245 + + + + + + + (+)-Gallocatechin 4.93 305.06667 305.06683 0.52 179 + + – + + – + Ellagitannins Ellagic acid 9.38 300.99899 300.99915 0.53 229, 257, 284, 301, 302 + + – + + – + Monogalloyl-hexoside 1.59 331.06706 331.06729 0.69 125, 169, 211, 241, 271 + + + + + + + Ellagic acid-pentoside 9.03 433.04124 433.04144 0.46 300, 301 + + – + + + + Ellagic acid-deoxyhexoside 8.99 447.05689 447.05719 0.67 300, 301 + + – + + + + Ellagic acid-hexoside 8.07 463.05181 463.05246 1.40 300, 301, 302 + + + + + + + Digalloyl-hexoside 6.95 483.07802 483.07837 0.72 169, 193, 313, 331 + + – + + + + Galloyl-HHDP-hexoside 7.92 633.07333 633.07385 0.82 249, 331 + + – + + – + Punicalin 3.16 781.05299 781.05383 1.07 575, 601 + + – + + – + Pedunculagin 6.57 783.06809 783.06927 1.51 249, 275, 301, 633 + + – + + – + Punigluconin 8.25 801.55911 801.56121 2.62 301, 347 + + + + + + + Punicalagin α 6.29 1083.05926 1083.05994 0.63 275, 601, 781 + + – + + – + Punicalagin β 7.00 1083.05926 1083.05933 0.06 275, 601, 781 + + – + + – +

RT, retention time; N-FE, non-fractioned SC-CO2 extract; fr., fraction; HHDP, hexahydroxydiphenoyl.

Table 2. Punicalagin and ellagic acid contents (mg/g of dry peel) in the extracts and fractions of the pomegranate peel obtained with supercritical CO2 and conventional extraction using methanol.

Extracts and fractions of Punicalagin α Punicalagin β Ellagic acid the pomegranate peel (mg EAE/g) (mg EAE/g) (mg /g) Methanol 18.3 ± 0.04a 24.0 ± 0.27a 3.12 ± 0.09b Non-fractioned 13.4 ± 0.10c 20.6 ± 0.88b 2.17 ± 0.06c

F1, fraction 1 nd nd nd e d e F1, fraction 2 0.26 ± 0.00 0.68 ± 0.01 0.08 ± 0.00 d c d F1, fraction 3 4.44 ± 0.16 10.9 ± 0.01 1.00 ± 0.00

F2, fraction 1 nd nd nd b b a F2, fraction 2 15.7 ± 0.02 22.1 ± 0.83 3.92 ± 0.05 * Determined by HPLC-PDA. All values are mean ± SD. Different superscript letters in the same column indicate significant differences (p< 0.05; one-way ANOVA with Tukey´s post-test). EAE: ellagic acid equivalent. nd: not detected

As expected from the SC-CO2 polarity, punicalagin α and β, and ellagic acid were absent in the fraction 1 of F1 and F2. Methanol extract (conventional extraction) presented the highest content of punicalagin (42.3 mg EAE/g), followed by the non-fractionated and the fraction 2 of F2 extracted by SC-CO2 that also stood out with high contents of this compound

(34.0 and 37.8 mg EAE/g, respectively). Moreover, the fraction 2 of F2 showed the highest content of ellagic acid (3.92 mg/g) among all the extracts and fractions analyzed.

3.2 Tocopherols and β-carotene

The contents of tocopherols and β-carotene in the pomegranate peel extracts and fractions were significantly (p<0.05) affected by the extraction conditions (Table 3 and Figure 3). The α-, β- and γ- isoforms of tocopherols were found in the pomegranate peel extracts and fractions obtained in this work, but these compounds were not identified in all samples (Table 3). All three isoforms were identified only in the fraction 1 of F1, while α- and γ-tocopherol were present in the non-fractioned and methanol extracts. Likewise, β- and γ- tocopherol were found in the fraction 2 of F1 and fraction 1 of F2, and in the fraction 3 of F1 and fraction 2 of F2 only β-tocopherol was identified. The highest contents of total tocopherols were found in the fraction 1 of F1 and the non-fractionated extract (18.1 and 18.0 µg/g, respectively), which were, on average, 1.7-, 1.9- and 5.1-fold higher than methanol 89 extract, fraction 1 of F2 and fraction 2 of F1, respectively. The other fractions, fraction 3 of F1 and fraction 2 of F2, showed contents of total tocopherol lower than 1 µg/g. Exception for fraction 3 of F1 and fraction 2 of F2, in all the extracts and fractions, γ-tocopherol was the major tocol isoform.

Table 3. Tocopherol contents (µg/g of dry peel) in the extracts and fractions of the

pomegranate peel obtained with supercritical CO2 and conventional extraction using methanol. Tocopherol contents Extracts and fractions of α-tocopherol β-tocopherol γ-tocopherol the pomegranate peel (µg/g) (µg/g) (µg/g) Methanol 2.11 ± 0.10c nd 8.70 ± 0.45b Non-fractioned 7.34 ± 0.25a nd 10.68 ± 0.42a b b a F1, fraction 1 6.48 ± 0.10 1.39 ± 0.03 10.25 ± 0.22 a d F1, fraction 2 nd 1.58 ± 0.05 1.93 ± 0.16 c F1, fraction 3 nd 0.57 ± 0.02 nd a c F2, fraction 1 nd 1.55 ± 0.10 7.95 ± 0.02 d F2, fraction 2 nd 0.20 ± 0.01 nd * Determined by HPLC-FLU. All values are mean ± SD. Different superscript letters in the same column indicate significant differences (p<0.05; one-way ANOVA with Tukey´s post-test); nd: not detected.

-carotene was the single natural pigment found in all extracts and fractions of the pomegranate peel (Figure 3). The fraction 1 of F1 showed the highest content of β-carotene

(7.6 µg/g), being approximately 1.4-, 1.7-, 2.7- and 3.3-fold higher than fraction 1 of F2, non- fractionated extract, fraction 2 of F1 and methanol extract, respectively. The fraction 2 of F2 and fraction 3 of F1 presented the lowest contents of β-carotene (≤ 0.5 µg/g).

3.3 Antioxidant capacity

Antioxidant capacity measured by FRAP and TEAC assay varied significantly (p< 0.05) among the extracts and fractions of the pomegranate peel (Figure 4). Although FRAP and TEAC have different reaction mechanisms, the extracts and fractions of the pomegranate peel presented similar behavior in both the assays.

Figure 3. β-carotene contents (µg/g) in extracts and fractions of the pomegranate (Punica granatum L. cv. Ruby) peel, obtained with extraction and sequential fractionation by supercritical CO2 or methanol (conventional extraction). Different letters indicate significant differences between the extracts and fractions of the pomegranate peel (one-way ANOVA with Tukey´s post-test, p<0.05). N-FE, non-fractionated extract; F1, fractionation 1; F2, fractionation 2.

Figure 4. Antioxidant capacity of extracts and fractions of the pomegranate (Punica granatum L. cv. Ruby) peel, obtained with extraction and sequential fractionation by supercritical CO2 or methanol (conventional extraction). A. Antioxidant capacity by FRAP assay (µmol Fe2+/g) of extracts and fractions of the pomegranate peel; B. Antioxidant capacity by TEAC assay (µmol TE/g) of extracts and fractions of the pomegranate peel. Different letters indicate significant differences between the extraction techniques (one-way ANOVA with Tukey´s post- test, p<0.05). ENF, extract no fractionation; F1, fractionation 1; F2, fractionation 2.

Methanol extract showed the highest antioxidant capacity among all the extracts and fractions, independently of the assay used. Among the samples obtained by SC-CO2, the fraction 2 of F2 and the non-fractioned extract stood out by the high antioxidant capacity. On the other hand, the fractions 1 of F1 and F2 showed the lowest antioxidant capacity among the extracts and fractions of the pomegranate peel. The antioxidant capacity in fraction 2 of F2 and the non-fractionated extract analyzed by the FRAP and TEAC assays was, on average, 91

2+ 3.1- and 4.9-fold higher than fraction 2 of F1 (851.2 µmol Fe /g; 826.2 µmol TE/g), and 1.6- 2+ and 1.5-fold higher than fraction 3 of F1 (1610.8 µmol Fe /g; 2668.7 µmol TE/g), respectively. A positive correlation (r > 0.895, p < 0.04) was observed between total phenolic compounds and punicalagin contents, and antioxidant capacity in the non-fractionated extract, fraction 3 of F1 and fraction 2 of F2 obtained with SC-CO2, and in methanol extract (conventional extraction), independently of the assay used. The other fractions of the pomegranate peel showed no correlation between the bioactive compounds analyzed and the antioxidant capacity.

4. Discussion

SC-CO2 extraction is an innovative and environment-friendly technique that was used in this study for extraction and sequential fractionation of bioactive compounds of the pomegranate peel in respective fractions enriched with phenolic compounds, and tocopherols and carotenoid. Extraction and fractionation methods of the pomegranate peel using SC-CO2 added with co-solvent were determined based on optimized extraction condition for punicalagin (Bustamante et al., 2017) and previous tests (data not shown). As well as the methanol extract (reference extract obtained by conventional extraction), the non-fractionated extract, fraction 3 of F1 and fraction 2 of F2 were rich in phenolic compounds, mainly ellagitannins such as punicalagin, besides showing high antioxidant capacity. Differently, the initial fractions, fractions 1 of F1 and F2, presented higher content of tocopherols and β- carotene than the other fractions and extracts. As expected, the profile of antioxidant compounds found in the fractions obtained with sequential F1 and F2 by the SC-CO2 extraction system is due to the extraction conditions, in which the initial steps of the fractionations there was no co-solvent (ethanol) or the co-solvent was used in small amounts, aiming the cleaning of the extract with the removal of lipophilic compounds and polar compounds of low molecular weight, so as to concentrate the punicalagin in the last step of fractionation. In addition, the sequential fractionation of bioactive compounds of the pomegranate peel by SC-CO2 added with co-solvent is described for the first time in this study. The remarkable variation in the yield of the non-fractionated extract and of the fractions obtained by SC-CO2 was due to the use of different pressures, presence or absence of ethanol, percentage of ethanol and, mainly, different times in extraction and sequential

92 fractionation. Pressure is an important parameter for SC-CO2 extraction, since density and solvent capacity increases with increased pressure. Usually, the extraction conditions employed to extract antioxidants compounds with high molecular weight are more extreme, whereas low molecular weight compounds can be extracted with low pressure (Ibañez et al., 1999; Díaz-Reinoso et al., 2006; Lau et al., 2008; Paviani et al., 2010), thus different pressures were used in the sequential fractionation in order to perform a selective extraction of bioactive compounds present in the pomegranate peel. Being CO2 non-polar explains the high extraction of low polarity compounds and, even at high pressure, pure SC-CO2 is not very efficient in extraction of polar antioxidant compounds, being necessary the addition of polar co-solvents. SC-CO2 extraction yield increased directly with solvent polarity (Castro- Vargas et al., 2010; Paes et al., 2014). Ethanol is the co-solvent most frequently used in combination with SC-CO2 as it enhances the extraction of polar antioxidant compounds, besides being easily accessible, non-toxic, environmentally friendly and non-hazardous to health (Murga et al., 2000; Díaz-Reinoso et al., 2006; Castro-Vargas et al., 2010). The gradual increase of co-solvent and pressure allowed the extraction of, first, the low polarity compounds and, then, the more polar compounds with high molecular weight. Therefore, the use of ethanol as co-solvent for the extraction and sequential fractionation of the pomegranate peel by SC-CO2 provided enhanced extraction of phenolic compounds and also increased the yield, as shown in extraction of guava seed (Castro-Vargas et al., 2010) and blueberry fruit, leaves and residue (Paes et al., 2014; Pereira et al., 2016) by SC-CO2. At constant temperature, the increase of pressure, ethanol (co-solvent) percentage and time of extraction and fractionation by SC-CO2 led to higher yield and increased extraction of polar antioxidant compounds (mainly, high molecular weight compounds), as occurred in the non-fractionated extract, fraction 3 of F1 and fraction 2 of F2. Pomegranate peel is an industrial residue considered an appealing source of phenolic compounds (Çam & Hisil, 2010; Hasnaoui et al., 2014; Bustamante et al., 2017; Russo et al.,

2018). The extract and fractions of pomegranate peel obtained by SC-CO2 presented an exceptional quality, mainly in the non-fractionated extract, fraction 3 of F1 and fraction 2 of

F2 that showed the highest phenolic compound contents. This fact was due to the use of the co-solvent, 10 % ethanol in extraction and fractionation by SC-CO2, which interacted strongly with the compounds by means of hydrogen bonding and dipole-dipole interactions, being capable to extract more phenolic compounds (Murga et al., 2000; Díaz-Reinoso et al., 2006).

Total phenolic compound contents of the fraction 2 of F2 was similar to fraction 3 of F1 and

93 methanol extract, which were on average 1.2-, 1.3-, 2.9- and 1.6-fold higher than pomegranate peel extracts obtained by conventional extraction with methanol (Çam & Hisil, 2010), extraction with ethanol:acetone mixture (Hasnaoui et al., 2014), ethyl acetate extraction

(Fischer et al., 2011) and SC-CO2 extraction with ethanol as co-solvent (Mushtaq et al., 2015), respectively. Hydrolyzable tannins contributed with significant amount to total phenolic compounds of the pomegranate peel, on the other hand, flavonoids constitute only a small part of phenolic compounds of the pomegranate peel. Hydrolyzable tannins and total flavonoid contents in the methanol extract was higher than in the extract and fractions of the pomegranate peel obtained by SC-CO2. Similarly, conventional extraction of the pomegranate peel using methanol as solvent showed higher contents of hydrolyzable tannins and total flavonoid than extraction with ethanol, water or pressurized water; indicating the efficiency of methanol in the extraction of these bioactive compound (Çam & Hisil, 2010; Orak et al., 2012). Although the higher contents of total flavonoid and hydrolyzable tannins were found in the methanol extract, the non-fractionated extract, fraction 3 of F1 and fraction 2 of F2, obtained by SC-CO2, presented high content of these bioactive compounds, being about 1.6-, 1.1- and 1.4-, and 1.4-, 1.4- and 1.6-fold higher than the methanol extract previously reported (Orak et al., 2012), respectively. Phenolic compounds of the pomegranate peel extracts and fractions were tentatively identified by the combining the information obtained with MS detector (exact mass, < 5 ppm mass error and fragmentation patterns) with literature data. As expected, some fractions of the pomegranate peel obtained by SC-CO2 showed different phenolic compound profiles and this was due to the different conditions (pressure and co-solvent) employed during the sequential fractionation of the pomegranate peel. Phenolic compound profiles of the non-fractionated extract, fractions 2 and 3 of F1, and fraction 2 of F2 obtained with SC-CO2, and the methanol extract were similar, where three classes of compounds were identified; namely, phenolic acids, hydrolyzable tannins and flavonoids, which were consistent with the polyphenols found in pomegranates peel cultivated in Turkey, Canada and Italy (Çam & Hisil, 2010; Ambigaipalan et al., 2016; Russo et al., 2018). These extracts and fractions of the pomegranate peel showed richness in phenolic compounds, highlighting the presence of the ellagitannins that are the majority of the compounds identified in the pomegranate peel, representing about 80 % (Russo et al., 2018), being the punicalagin α and β the most abundant ellagitannins (Fischer et al., 2011; Li et al., 2015; Aguilar-Zárate et al., 2017; Bustamante et al., 2017; Russo et al., 2018), which were confirmed with the UV absorption spectrum

(absorbance maximum at 258 nm and a second less intense absorbance at 378 nm) and were in agreement with data already reported (Fischer et al., 2011; Russo et al., 2018). Among the pomegranate peel extracts and fractions studied, methanol extract, non-fractionated extract and fraction 2 of F2 showed the highest contents of punicalagin, being this content in methanol extract 1.2-fold higher than pomegranate peel aqueous-methanol extract (conventional extraction) (Russo et al., 2018), whereas the punicalagin content of the non- fractionated extract and of the fraction 2 of F2 were similar to aqueous-methanol extract. The fraction 2 of F2 obtained by SC-CO2 showed the highest content of ellagic acid, being 1.4 and 3.1-fold higher than pomegranate peel methanol extract described in the literature (Çam & Hisil, 2010; Li et al., 2015). Besides of the punicalagin (α and β) and ellagic acid, other phenolic compounds of the pomegranate peel also stood out, as galloyl-HHDP-hexoside and punicalin, which are derived from the partial or complete hydrolysis of punicalagin (Aguilar- Zárate et al., 2017). Several therapeutic effects have been attributed to the presence of ellagitannins of the pomegranate peels, such as anticarcinogenic, antiatherogenic, antiangiogenic, anti-hyperglycemic, anti-hyperlipedemic and anti-obesity activities (Li et al., 2015; Ambigaipalan et al., 2016; Aguilar-Zárate et al., 2017). Furthermore, there is a great interest in punicalagin due to their health benefits, as antioxidant, antifungal and antibacterial properties (Ismail et al., 2012; Bustamante et al., 2017). The pomegranate peel's unique red color is conferred by anthocyanins (Fischer et al., 2011; Ambigaipalan et al., 2016); however, in the present study these phenolic pigments were undetectable in the extracts and fractions of the pomegranate peel, as well as described previously (Russo et al., 2018). Pomegranate peel is not a source of tocopherols and carotenoids, despite this, the extraction and sequential fractionation by SC-CO2 provided an extract and a fraction enriched in these lipophilic compounds. Data on the content of tocopherols and carotenoids in pomegranate peel are scarce. The fraction 1 of F1 was the only among all the extracts and fractions that presented three isoforms of tocopherols (α-, β- and γ-), showing the highest content of total tocopherols and β-carotene, together with the nonfractionated extract. This finding in fraction 1 of F1 is due to the conditions applied in the fractionation, since the purpose of the initial steps of the process is to remove the lipophilic compounds so that in the later steps there may be concentration of the phenolic compounds. However, methanol extract, non-fractionated extract and the fraction 1 of F1 obtained by SC-CO2 stood out by the presence of α- and γ-tocopherol, which have vitamin E activity and important antioxidant action, respectively (Silva et al., 2017).

In antioxidant capacity assays, FRAP and TEAC, methanol extract, non-fractionated extract and the fraction 2 of F2 showed high antioxidant capacity, probably due to richness in polyphenols of the pomegranate peel. The non-fractionated extract, the fraction 3 of F1 and fraction 2 of F2 showed antioxidant capacity by TEAC assay similar to reported for pomegranate peel aqueous-methanol extract (conventional extraction), 1291–3998 µmol TE/g (Russo et al., 2018), while methanol extract was higher than this reported previously. In contrast, SC-CO2 extraction with ethanol as co-solvent was approximately 4-fold higher than the extracts and fractions of pomegranate peel obtained in the present study (Mushtaq et al., 2015). Regarding to antioxidant capacity by FRAP assay, methanol extract, non-fractionated extract, fractions 2 and 3 of F1, and fraction 2 of F2 were higher than in ultrasonic-assisted extraction using aqueous-ethanol, 243–634 µmol Fe2+/g (Tabaraki et al., 2012). The non- fractionated extract, fraction 3 of F1 and fraction 2 of F2, obtained with SC-CO2, and methanol extract (conventional extraction) showed a positive correlation between total phenolic compounds and punicalagin contents, and antioxidant capacity, for both assays, indicating that these bioactive compounds from pomegranate peel contribute extensively to antioxidant capacity of the extracts and fractions.

5. Conclusion

Pomegranate peel, which is a byproduct of juice production, was confirmed as a very rich source of bioactive compounds by extracting with SC-CO2 or methanol. The sequential fractionation of pomegranate peel by SC-CO2 was an effective mean of obtaining fractions with different compositions in bioactives that might have varied applications according to the intended use. F2 was more efficient for the fractionation of pomegranate seed, because it combined a lower fractionation time with an appealing composition of phenolic compounds, especially by the concentration of punicalagin with high antioxidant capacity, appealing for future use in pharmaceutical, cosmetic and food industries, besides as analytical standard for laboratory assays. These results might stimulate future investigations to improve sequential fractionation technologies to obtain semi-purified high value-added bioactives from the pomegranate peel, such as extracts concentrated in punicalagin, by means of scaling-up and optimizing the fractionation using statistical experimental designs.

THESIS GENERAL CONCLUSION

The extraction techniques used in this study, expeller pressing, alcohol-extraction and

SC-CO2, allowed to obtain the special oils with exceptional nutritional and functional properties. Additionally, the sequential fractionation of pomegranate peel by SC-CO2 was an effective mean of obtaining fractions with different compositions in bioactives that might have varied applications. The both pomegranate byproducts exhibit high antioxidant compounds contents with potential bioactive and possible future applications in pharmaceutical, cosmetic and food industries.

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Supplementary Table 1. Yield, extraction efficiency and extraction time of pomegranate (Punica granatum L. cv Ruby) seed oil using different extraction methods: expeller pressing, alcohol or supercritical CO2 (without or with co-solvent) extractions.

Pomegranate seed oils Yield Extraction Extraction time (n) (g/100 g) efficiency (%) (h) P-PSO (3) 8.72 ± 0.30c 51.5 ± 0.90d 1.50 ± 0.50g A-PSO (3) 13.2 ± 0.53b 77.9 ± 1.70c 3.00 ± 0.33f SC-PSO (without co-solvent; 11) 16.0 ± 0.79a 94.7 ± 4.70a 4.00 ± 0.17e SC-PSO with co-solvent Exp. 1 (3) 14.7 ± 0.79a 86.9 ± 4.69a,b 5.25 ± 0.97c,d,e Exp. 2 (3) 14.5 ± 1.22a,b 85.8 ± 7.27b,c 4.25 ± 0.87e Exp. 3 (3) 12.7 ± 1.37b 75.1 ± 8.13b,c 5.42 ± 0.25d Exp. 4 (3) 16.0 ± 0.95a 94.4 ± 5.62a 5.29 ± 0.13d Exp. 5 (6) 5.02 ± 0.21e 29.6 ± 1.24f 9.92 ± 0.18a Exp. 6 (7) 4.26 ± 0.53f 25.2 ± 3.16f 8.00 ± 0.67b Exp. 7 (7) 4.51 ± 0.35e,f 26.7 ± 2.11f 10.2 ± 0.32a Exp. 8 (6) 6.63 ± 0.59d 39.2 ± 3.53e 7.58 ± 0.21b Exp. 9 (4) 8.52 ± 0.76c 50.4 ± 4.50d 6.17 ± 0.87c Exp. 10 (4) 7.96 ± 0.81c 47.1 ± 4.78d 6.67 ± 0.50c

All values are mean  SD of at least three extraction experiments. Different superscript letters in the same column indicate significant differences (p<0.05; one-way ANOVA with Tukey´s post-test).

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SUPPLEMENTARY TABLE 2 – CHAPTER 1 Supplementary Table 2. Total contents of tocopherols, phenolic compounds and cLnA, and total antioxidant capacity of pomegranate (Punica granatum L. cv. Ruby) seed oil obtained by extraction with supercritical CO2 using alcohol as co-solvent, following the experimental conditions determined by statistical experimental design (24-1).

Co-solvent Response Factors Pressure Temperature 1 Exp. Alcohol Concentration Tocopherols Phenolic compounds cLnA Antioxidant capacity (bar) (°C) 2 concentration in SC-CO2 (%) (mg/100 g) (mg/100 g) (g/100 g) (mmol TE/kg)

1 200 40 100% 10 1783.2 4.79 89.9 27.2

2 300 40 100% 1 1182.3 18.7 90.8 27.7

3 200 60 100% 10 1535.8 22.4 91.0 26.5

4 300 60 100% 1 2200.5 6.46 91.2 27.5

5 200 40 10% 10 2132.1 2.03 91.8 29.5

6 300 40 10% 1 1383.2 6.88 90.7 27.5

7 200 60 10% 10 1613.8 12.8 88.9 19.8

8 300 60 10% 1 2613.0 8.11 88.7 24.8

93 250 50 50% 5 1813.5 12.2 90.1 30.3

103 250 50 50% 5 1425.4 7.14 90.0 26.8 cLnA: conjugated Linolenic Acid. TAC: Total Antioxidant Capacity. 1 Experiments were performed in random order, as presented in Table 1; 2 Anhydrous reagent alcohol was used (90 % ethanol, 5 % methanol and 5 % iso-propanol), either pure (100 % alcohol) or mixed with ultra-pure water (10 and 50 % alcohol); 3 Internal points in experimental design.

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SUPPLEMENTARY FIGURE 1 – CHAPTER 1

Supplementary Figure 1. Representative chromatogram, by GC-FID on an Omegawax-320 column (30 m × 0.32 mm i.d., 0.25 μm), of fatty acid methyl esters from methylated Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil. The column oven was temperature programmed as follows: 160 °C for 2 min, increased to 190 °C at 2.5 °C/min, kept at 190 °C for 5 min, increased to 220 °C at 3.5 °C/min, and kept at 220 °C for 20 min. Helium was used as the carrier gas (72 kPa), the injector were operated at 1:30 split ratio and 260 °C, and FID at 280 °C.

112

SUPPLEMENTARY FIGURE 2 – CHAPTER 1

Supplementary Figure 2. Representative chromatograms by reversed-phase HPLC-ELSD on a C18 column (3.5 µm, 150 mm  3 mm), of major lipid classes (TAG, triacylglycerols; DAG, diacylglycerols; MAG, monoacylglycerols; FFA, free fatty acid) in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil. The mobile phase consisted of a gradient of isopropanol (eluent A) and acetonitrile (eluent B), at a flow rate of 1.0 mL/min. The gradient elution program was as follows: 0 to 69% (B) from 0 to 60 min; 69% (B) from 60 to 75 min; 0% (B) at 75.1 min and solvent re-equilibration for 10 min. Nitrogen was used as nebulizing gas at 0.65 mL/min.

113

SUPPLEMENTARY FIGURE 3 – CHAPTER 1

Supplementary Figure 3. Representative chromatogram, by reversed-phase HPLC-PDA- MS on a C18 column (5 μm, 250 mm × 4.6 mm), of non-anthocyanin phenolic compounds in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil. The mobile phase consisted of a gradient of water:formic acid:acetonitrile (98.7:0.3:1, v/v; eluent A) and methanol:acetonitrile (99:1, v/v; eluent B), at 1.0 mL/min flow rate. Prior to injection, the column was equilibrated with 24.2% B, followed by a gradient elution program: 24.2% (B) at 8 min, 28.2% (B) at 18 min, 33.3% (B) at 30 min, 65.6% (B) at 60 min, 24.2% (B) at 60.1 min, followed by 15 min re-equilibration, for a total analysis cycle time of 75 min.

114

SUPPLEMENTARY FIGURE 4 – CHAPTER 1

Supplementary Figure 4. Representative chromatogram, by reversed-phase HPLC-PDA on a C18 column (5 μm, 250 mm × 4.6 mm), of anthocyanins in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil. The mobile phase consisted of a gradient of 1% aqueous formic acid (eluent A), 1% formic acid in methanol (eluent B) and acetonitrile (eluent C), at 2.0 mL/min flow rate. Eluent C concentration was kept constant at 2% during analysis. Prior to injection, the column was equilibrated with 18% B. The gradient elution program was as follows: 18% (B) at 2 min, 32% (B) at 6 min, 52% (B) at 8 min, 18% (B) at 8.1 min, followed by 15 min re-equilibration, for a total analysis cycle time of 23 min.

115

SUPPLEMENTARY FIGURE 5 – CHAPTER 1

Supplementary Figure 5. Representative chromatogram, by normal-phase HPLC-PDA- Fluorescence (HPLC-PDA-Fluo) on an unmodified silica column (5 µm, 4.6 mm  250 mm), of tocopherols in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil. Analytes were eluted with a binary isocratic solvent system (hexane:isopropanol, 99:1, v/v) at 1.0 mL/min.

116

SUPPLEMENTARY FIGURE 6 – CHAPTER 1

Supplementary Figure 6. Representative chromatogram, by normal-phase HPLC-PDA- Fluorescence (HPLC-PDA-Fluo) on an unmodified silica column (5 µm, 4.6 mm  250 mm), of carotenoid in Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil. Analytes were eluted with a binary isocratic solvent system (hexane:isopropanol, 99:1, v/v) at 1.0 mL/min.

117

SUPPLEMENTARY FIGURE 7 – CHAPTER 1

118

SUPPLEMENTARY FIGURE 8 – CHAPTER 1

Supplementary Figure 8. Representative chromatogram, by GC-MS on a fused silica column 5% phenyl/95% methylpolysiloxane (30 m × 0.32 mm i.d., 3 µm film), of volatile compounds from alcohol-extracted Brazilian pomegranate (Punica granatum L. cv. Ruby) seed oil. Volatile compounds were desorbed from the SPME fiber in the injection port at 260 °C for 5 min, in the splitless mode, and after 5 min sampling the split purge valve was open at 3.0 mL/min. Helium was used as the carrier gas and the column pressure was set at 49 kPa. The column oven temperature was held at 30 °C for 10 min, then increased at 3 °C/min to 200 °C and held for 10 min. The mass spectrometer was operated in electron impact mode at 70 eV. The interface and the ion source temperature was set at 260 °C. Analyses were performed in full scan acquisition mode, in the mass range 40-500 m/z at 0.5 scan/s.

119

SUPPLEMENTARY FIGURE 1 – CHAPTER 2

Supplementary Figure 1. Weight gain of the extract and fractions of the pomegranate peel obtained by SC-CO2 in function of time.

120

SUPPLEMENTARY FIGURE 2 – CHAPTER 2

Punicalagin β

Punicalagin α a

Ellagic acid

Supplementary Figure 2. Representative chromatogram, by reversed-phase HPLC-PDA on a C18 column (5 μm, 250 mm × 4.6 mm), of hydrolysable tannins (punicalagin and ellagic acid) from fraction 2 of F2 of the pomegranate (Punica granatum L. cv. Ruby) peel. The mobile phase consisted of a gradient of water:formic acid (99.9:0.1, v/v; eluent A) and acetonitrile (eluent B), at 0.4 mL/min flow rate. Prior to injection, the column was equilibrated with 5% B, followed by a gradient elution program: 5% (B) at 1 min, 25% (B) at 6 min, 95% (B) at 17–20 min, 5% (B) at 20.5 min, followed by 4.5 min re-equilibration, for a total analysis cycle time of 25 min.

121