Instituto Tecnológico y de Estudios Superiores de Monterrey Campus Monterrey School of Engineering and Sciences

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Instituto Tecnológico y de Estudios Superiores de Monterrey

Campus Monterrey

School of Engineering and Sciences

General Protocol to Design Highly Effective Food Products against Chronic Degenerative Diseases by using Nutraceutical

Combinations as Novel Element

A thesis presented by

Jesús Santana Gálvez

Submitted to the

School of Engineering and Sciences

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Biotechnology

Monterrey, Nuevo León, México June 5, 2020

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DEDICATION

To my parents, Irene María Gálvez Hinojosa and Jesús Santana Blanco; my sister, Anakaren Santana Gálvez; my brother, Andrés Santana Gálvez; my love, Brenda Aranda de la Garza; my relatives, and friends.

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ACKNOWLEDGEMENTS

I would like to express deeply my gratitude to my advisor, Dr. Daniel Alberto Jacobo Velázquez, and my co-advisor, Dr. Luis Alberto Cisneros Zevallos, for their support, recommendations, contributions to this project, and for letting me develop my ideas. Ever since I was studying my bachelor’s degree, I thought that the combination of compounds (nutraceuticals and/or drugs) could be the solution to chronic degenerative diseases. Hence, I reached Dr. Jacobo and Dr. Cisneros, who had also the same idea, and together we conceptualized this thesis. I also thank Dr. Jacobo for funding acquisition and providing many of the resources that were required.

I further thank greatly my other co-advisor, Dr. Sergio Román Othón Serna Saldívar, for his support, recommendations, contributions to this project, funding acquisition, and providing many of the resources that were needed.

I am grateful as well with my committee members, Dr. Yolanda Arlette Santacruz López and Dr. Jorge Alejandro Benavides Lozano, for their advice.

Special thanks to M.S. Javier Villela Castrejón, who taught me how to perform cell culture and viability studies, cultured and maintained all cell lines, seeded most microplates with cells, helped acquire materials, and contributed in reviewing some of the articles that were product of this thesis.

Special thanks as well to Dr. Beatriz Quiroz, Fernando Viacava, Ernesto Paredes, Dr. Ana Mariel Torres, Erika Ortega, Dr. Marilena Antunes, Dr. Beatriz Acosta, and Gabriela Montemayor for your advice and/or helping me with my experiments.

My deepest gratitude to my parents, Irene María Gálvez Hinojosa and Jesús Santana Blanco, for their unconditional love and support. You gave me the

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vi foundations that determined the professional and human being that I am today.

This great achievement would not have been possible without you, which means that this is also your achievement. You also gave me one of the greatest gifts through your work and effort: school and university education. There will never be enough words to thank you. I love you very much!

Thank you as well to my sister, Anakaren Santana Gálvez, and my brother, Andrés Santana Gálvez, for showing interest in my work and for their support. I love you very much too!

I thank profoundly my love, Brenda Aranda de la Garza, for your unconditional love and support. Thank you for being by my side. You made this hard path easier to follow. I love you with all my heart!

My special gratitude to my relatives, friends, and colleagues for their interest and support, as well as all the professors that I have had throughout my lifetime who contributed to my professional and personal formation.

I acknowledge Bioprocess & Synthetic Biology and NutriOmics research groups of Tecnológico de Monterrey for their support, as well as the Consejo Nacional de Ciencia y Tecnología (CONACYT) for the scholarship #291137.

Finally, I thank very much Tecnológico de Monterrey for the Tec scholarship and for providing me with all the necessary knowledge, skills, equipment, and installations to accomplish this thesis.

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General Protocol to Design Highly Effective Food Products against Chronic Degenerative Diseases by using Nutraceutical

Combinations as Novel Element by

Jesús Santana Gálvez

Abstract

Chronic degenerative diseases (CDDs) are the leading causes of death worldwide. Nutraceuticals are compounds naturally found in foods that have preventive and therapeutic activities against CDDs. However, no single nutraceutical has been successful in preventing or curing CDDs. This can be attributed to the complexity of CDDs, which are multifactorial and multisymptomatic. Nutraceutical combinations that attack different targets of a CDD have the potential to exert a synergistic effect, i.e., an effect greater than the sum of the effects of the individual compounds. Therefore, synergistic nutraceutical combinations can be a powerful solution, as they can serve as a basis to design novel and highly effective food products against CDDs. However, many factors need to be considered to successfully formulate these products.

Hence, to make the job easier and less overwhelming for academia and food industries, the main objective of this thesis was to develop a general protocol to design highly effective food products for the prevention/co-treatment of CDDs by using nutraceutical combinations as novel element, and partially validate it with colon cancer as model CDD. The method consists of a 10-step procedure that considers all factors to successfully design these products. Some steps are

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viii routinely applied by academia and industry, such as in vitro gastrointestinal digestion, animal, and clinical studies. However, novel steps were incorporated that cover the design and evaluation of synergistic nutraceutical and food ingredient combinations. To partially validate the protocol, the first five steps were undertaken, which cover from the selection of the target CDD (in this case, colon cancer) up to selecting and preparing the food ingredients based on the best nutraceutical combination. First, the effect of dihydrocaffeic acid (D, a chlorogenic acid metabolite) over the viability of several human cancer cell lines, including MCF-7 (breast), Hep-G2 (liver), PC-3 (prostate), and HCT-116 (colon), and healthy dermal fibroblasts (HDFa), was tested to determine its anticancer potential. Then curcumin (C), sulforaphane (S), and dihydrocaffeic acid were evaluated, individually and in combination (CD, SC, SD, and CSD at different doses and proportions), over the viability of HT-29 and Caco-2 colon cancer cells, and compared with healthy fetal human colon (FHC) cells. The cytotoxic concentrations to reduce cell viability by 50%, 75%, and 90% (CC50, CC75, and CC90, respectively) were obtained by using the MTS assay. Combination effects (i.e., synergy, additivity, or antagonism) were determined with the combination index (CI) method. Broccoli sprouts and carrot juice were chosen as the sources of sulforaphane and dihydrocaffeic acid, respectively. To elevate the levels of chlorogenic acid (and consequently, dihydrocaffeic acid) in carrot juice, wounding stress was applied to carrots. The effects of wounding intensity, storage, peeling, blanching, filtration, and pasteurization over physicochemical, nutritional, nutraceutical, and sensory properties of carrot juice were evaluated.

Dihydrocaffeic acid was significantly more cytotoxic for most cancer cell lines, including MCF-7, PC-3, and HCT-116, but less cytotoxic for Hep-G2 compared

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ix with HDFa. Thus, dihydrocaffeic acid is a relevant candidate for cancer prevention and treatment. Furthermore, the SD(1:1) combination exerted synergistic effects against HT-29 at 90% cytotoxicity level (doses 90:90 µM), whereas CD(1:4) was synergistic at all cytotoxicity levels (9:36–34:136 µM) and CD(9:2) at 90% (108:24 µM) against Caco-2. SD(1:1) was significantly more cytotoxic for cancer cells than FHC healthy cells, while CD(1:4) and CD(9:2) were similarly or more cytotoxic for FHC. Consequently, SD(1:1) was chosen as the best combination. A model explaining SD(1:1) synergy was proposed. Moreover, juices from unpeeled carrots had 7–40% more minerals, 0.46–1.6 less °Brix, and 1.16x more titratable acidity. The carrot juice with the highest phenolic content was obtained by cutting unpeeled carrots into slices and storing them for 48 h at 15 °C before blanching (80 °C for 6 min) (stressed unpeeled carrot juice, SUCJ).

SUCJ had 3600% more chlorogenic acid, 195% more total phenolics, and similar carotenoid content than conventional carrot juice. Sensory evaluation of SUCJ was acceptable and willingness-to-pay increased by providing information about health benefits. Mechanistic tests are needed to elucidate the anticancer mode of action of dihydrocaffeic acid, and to validate the proposed model of synergy between sulforaphane and dihydrocaffeic acid. Additionally, the remaining five steps must be carried out to fully validate the protocol, including in vitro gastrointestinal digestion and combination studies of broccoli sprouts and SUCJ, formulation of a synergistic beverage against colon cancer, and shelf-life, animal, and clinical evaluations. The study of nutraceutical and food ingredient combinations is an emerging field. Therefore, the solution to CDDs could be just a matter of finding the right combination, along with a healthy lifestyle.

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Protocolo General para el Diseño de Productos Alimenticios Altamente Efectivos contra las Enfermedades Crónico- Degenerativas mediante el uso de las Combinaciones de

Nutracéuticos como Elemento Novedoso por

Jesús Santana Gálvez

Resumen

Las enfermedades crónico-degenerativas (ECDs) son las principales causas de muerte a nivel mundial. Los nutracéuticos son compuestos que se encuentran naturalmente en los alimentos que tienen actividades preventivas y terapéuticas contra las ECDs. Sin embargo, ningún nutracéutico ha prevenido o curado exitosamente las ECDs. Esto se puede atribuir a la complejidad de las ECDs, las cuales son multifactoriales y multisintomáticas. Las combinaciones de nutracéuticos que atacan diferentes blancos de una ECD tienen el potencial de ejercer un efecto sinérgico, es decir, un efecto más grande que la suma de los efectos de los compuestos individuales. Consecuentemente, las combinaciones sinérgicas de nutracéuticos pueden ser una solución poderosa, ya que pueden servir de base para el diseño de nuevos productos alimenticios, altamente efectivos contra las ECDs. No obstante, muchos factores se necesitan considerar para formular exitosamente estos productos. Por lo tanto, para hacer el trabajo más fácil y menos abrumador para la academia y las industrias de alimentos, el objetivo principal de esta tesis fue desarrollar un protocolo general para el diseño de productos alimenticios altamente efectivos para la

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xi prevención/co-tratamiento de las ECDs mediante el uso de las combinaciones de nutracéuticos como elemento novedoso, y parcialmente validarlo con el cáncer de colon como ECD modelo. El método consiste en un procedimiento de 10 pasos que considera todos los factores para diseñar exitosamente estos productos. Algunos pasos son rutinariamente aplicados por la academia y las industrias, como los estudios de digestión gastrointestinal in vitro, animales y clínicos. Sin embargo, nuevos pasos fueron incorporados que cubren el diseño y la evaluación de combinaciones sinérgicas de nutracéuticos e ingredientes alimenticios. Para validar parcialmente el protocolo, los primeros cinco pasos se llevaron a cabo, los cuales abarcan desde la selección de la ECD objetivo (en este caso, cáncer de colon) hasta la selección y preparación de los ingredientes alimenticios basados en la mejor combinación nutracéutica. Primero, el efecto del ácido dihidrocafeico (D, un metabolito del ácido clorogénico) sobre la viabilidad de varias líneas celulares cancerígenas humanas, incluyendo MCF-7 (mama), Hep-G2 (hígado), PC-3 (próstata) y HCT-116 (colon), y fibroblastos dérmicos sanos (HDFa), fue estudiado para determinar su potencial anticancerígeno. Posteriormente, la curcumina (C), el sulforafano (S) y el ácido dihidrocafeico se evaluaron, individualmente y en combinación (CD, SC, SD y CSD a diferentes dosis y proporciones), sobre la viabilidad de las células cancerígenas de colon HT-29 y Caco-2, y se comparó con células sanas de colon de feto humano (FHC). Las concentraciones citotóxicas para reducir la viabilidad celular en 50%, 75% y 90% (CC50, CC75 y CC90, respectivamente) se obtuvieron, utilizando el ensayo MTS. Los efectos combinatorios (es decir, sinergia, aditividad o antagonismo) se determinaron con el método del índice de combinación (IC). Los germinados de brócoli y el jugo de zanahoria se eligieron

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xii como fuentes de sulforafano y ácido dihidrocafeico, respectivamente. Para elevar los niveles de ácido clorogénico (y en consecuencia, ácido dihidrocafeico) en jugo de zanahoria, se aplicó estrés de corte a las zanahorias. Los efectos de la intensidad de corte, almacenamiento, pelado, escaldado, filtración y pasteurización sobre las propiedades fisicoquímicas, nutricionales, nutracéuticas y sensoriales del jugo de zanahoria fueron examinados. El ácido dihidrocafeico fue significativamente más citotóxico para la mayoría de las líneas celulares cancerígenas, incluyendo la MCF-7, PC-3 y HCT-116, pero menos citotóxico para la Hep-G2 comparado con la HDFa. Consiguientemente, el ácido dihidrocafeico es un candidato relevante para la prevención y tratamiento del cáncer. Además, la combinación SD(1:1) ejerció efectos sinérgicos contra la HT- 29 al nivel de 90% de citotoxicidad (dosis 90:90 µM), mientras que la CD(1:4) fue sinérgica en todos los niveles de citotoxicidad (9:36–34:136 µM) y la CD(9:2) al 90% (108:24 µM) contra la Caco-2. La SD(1:1) fue significativamente más citotóxica para las células cancerígenas que la células sanas FHC, mientras que la CD(1:4) y CD(9:2) fueron similarmente o más citotóxicas para la FHC. En consecuencia, la SD(1:1) fue elegida como la mejor combinación. Un modelo que explica la sinergia de la SD(1:1) fue propuesto. Más aún, los jugos de zanahorias no peladas tuvieron 7–40% más minerales, 0.46–1.6 menos °Brix y 1.16x más acidez titulable. El jugo de zanahoria con el contenido más alto de fenólicos se obtuvo mediante el cortar zanahorias no peladas en rodajas y almacenándolas por 48 h a 15 °C antes de escaldarlas (80 °C por 6 min) (jugo de zanahoria estresada no pelada, JZEN). El JZEN tuvo 3600% más ácido clorogénico, 195% más fenólicos totales y un contenido similar de carotenoides que un jugo convencional de zanahoria. La evaluación sensorial del JZEN fue

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xiii aceptable y la disposición a comprar incrementó al proveer información sobre los beneficios a la salud. Se necesitan pruebas mecanísticas para elucidar el modo de acción anticancerígeno del ácido dihidrocafeico y validar el modelo de sinergia propuesto entre el sulforafano y el ácido dihidrocafeico. Adicionalmente, los cinco pasos restantes se deben de realizar para validar completamente el protocolo, incluyendo estudios de digestión gastrointestinal in vitro y combinatorios de los germinados de brócoli y el JZEN, la formulación de una bebida sinérgica contra el cáncer de colon, y pruebas de vida de anaquel, animales y clínicas. El estudio de combinaciones de nutracéuticos e ingredientes alimenticios es un área emergente. Por lo tanto, la solución a las ECDs podría ser sólo una cuestión de encontrar la combinación correcta, acompañado de un estilo de vida saludable.

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List of Figures

2.1 Hypothetical isobolograms of binary mixtures of nutraceuticals.

2.2 Chemical structure of curcumin.

2.3 Chemical structure of sulforaphane.

2.4 Chemical structure of chlorogenic acid.

2.5 Chemical structures of caffeic and dihydrocaffeic acids.

3.1 Proposed general protocol for designing highly effective food products against chronic degenerative diseases (CDDs).

4.1 Combination index (CI) values of equimolar combinations of curcumin (C), sulforaphane (S), and dihydrocaffeic acid (D) on the viability of HT-29 colon cancer cells at (A) 50%, (B) 75%, and (C) 90% cytotoxicity levels.

4.2 Combination index (CI) values of equimolar combinations of curcumin (C), sulforaphane (S), and dihydrocaffeic acid (D) on the viability of Caco-2 colon cancer cells at (A) 50%, (B) 75%, and (C) 90% cytotoxicity levels.

4.3 Combination index (CI) values of nonequimolar combinations of curcumin (C), sulforaphane (S), and dihydrocaffeic acid (D) on the viability of Caco- 2 colon cancer cells at (A) 50%, (B) 75%, and (C) 90% cytotoxicity levels.

4.4 Cytotoxicity of synergistic combinations of curcumin (C), sulforaphane (S), and dihydrocaffeic acid (D) on colon cancer cells (HT-29 and Caco-2) and healthy fetal human colon (FHC) cells.

4.5 Effect of wounding intensity, blanching, storage, and peeling on total phenolics of carrot.

4.6 Effect of blanching, storage, and peeling on (A) chlorogenic acid and (B) β-carotene of carrot slices.

4.7 Effect of wounding stress and peeling on (A) chlorogenic acid and (B) total phenolics in carrot juice.

4.8 Proposed process to increase phenolic content of carrot juice.

4.9 Hypothetical model of the synergistic mechanism of sulforaphane and dihydrocaffeic acid against the viability of HT-29 colon cancer cells.

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List of Tables

2.1 Advantages of nutraceutical combinations with study examples.

2.2 Dietary supplements available in the market with scientifically proven health benefits at in vitro, animal, or human level.

2.3 Nutraceutical combination studies against colon cancer

4.1 Cytotoxic doses of dihydrocaffeic acid in cancer and healthy cell lines.

4.2 R-values of linearized dose-effect curves of curcumin (C), sulforaphane (S), dihydrocaffeic acid (D), and their constant-ratio combinations.

4.3 Doses and dose-reduction index (DRI) values of curcumin (C), sulforaphane (S), dihydrocaffeic acid (D), and their equimolar combinations at different cytotoxicity levels of HT-29 colon cancer cells.

4.4 Preliminary combination index (CI) values of nonequimolar combinations of curcumin (C), sulforaphane (S), and dihydrocaffeic acid (D) on the viability of Caco-2 colon cancer cells.

4.5 Doses and dose-reduction index (DRI) values of curcumin (C), sulforaphane (S), dihydrocaffeic acid (D), and their nonequimolar combinations at different cytotoxicity levels of Caco-2 colon cancer cells.

4.6 Proximate, dietary fiber, and mineral analyses of carrot juices.

4.7 Physicochemical properties of carrot juices.

4.8 Effect of processing, wounding stress, and peeling on phenolics and carotenoids during carrot juice production.

4.9 Sensory evaluation of carrot juices.

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With the advent of organic chemistry in the 19^th century and the development of the pharmaceutical industry in the 20^th century, Western medicine enjoyed its golden age with the production of many drugs to combat disease by following a monotherapy model, i.e., attacking one target of a disease with a single drug (Efferth & Koch 2011; Lila & Raskin 2005). This method, also known as the reductionism approach, is still used today and involves the identification of biologically active extracts from bacteria, fungi, and plants, and subsequent isolation of the main active ingredient. Then the active ingredient is manufactured directly from the source organism, synthesized de novo, or chemically modified to improve safety and efficacy. This approach has been successful against infectious diseases and for the development of drugs with analgesic, anticholinergic, and muscle relaxant activities (Lila & Raskin 2005).

However, a series of complex diseases arose afterwards, characterized by being of long duration and deteriorating. These disorders are known as chronic degenerative diseases (CDDs).

Nowadays, CDDs are the leading causes of death worldwide, including cancer, diabetes, cardiovascular (ischaemic heart disease and stroke), respiratory (chronic obstructive pulmonary disease), and neurological disorders (Alzheimer’s disease and other dementias) [World Health Organization (WHO) 2018a]. Unfortunately, no cure for CDDs has been found so far. In other words, the reductionism approach of modern medicine does not seem to be the most appropriate method to defeat these diseases. The reason for this is that CDDs are multifactorial and multisymptomatic. For instance, cancer can be caused by

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2 internal factors (e.g., inherited mutations, hormones, immune conditions, and oxidative stress) and environmental factors (e.g., tobacco, diet, radiation, and infectious organisms) (Shukla & George 2011; Singh & Shukla 2015).

Furthermore, the metabolic syndrome consists of many symptoms that include elevated levels of plasma glucose, total cholesterol, low-density lipoproteins, and triglycerides; low levels of high-density lipoproteins, high blood pressure, insulin resistance, chronic proinflammatory and prothrombotic states, non-alcoholic fatty liver, and sleep apnea, which vary among individuals (Santana-Gálvez et al.

2017). It is therefore very complicated that a single compound can defend an individual against all possible causes or treat all symptoms of a CDD.

Consequently, a new research field has emerged in modern medicine:

combination of drugs that work together for treating CDDs more effectively. This strategy, which follows a more holistic approach (Efferth & Koch 2011), is based on two facts: 1) CDDs are multi-target diseases, meaning that different symptoms or different targets can be attacked at the same time; 2) compounds can interact with each other to produce more powerful effects, a phenomenon known as synergy, which will be discussed later on. A metaphor to describe this approach has been used: instead of trying to defeat CDDs with a “magic bullet,” use a

“shotgun” (Efferth & Koch 2011). Understanding how compounds interact with each other and what factors influence those interactions are key to develop effective products against CDDs.

Combining compounds to treat disease is not new in the history of nature and humankind. Plants learned this strategy millions of years ago. Unlike animals, plants are immobile, and consequently they are exposed to attack by different organisms, including pathogens, insects, and herbivores. They respond to these

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3 threats by synthesizing a great variety of phytochemicals. In fact, phytochemicals have been compared to the immune system of animals (Efferth & Koch 2011). It seems that synthesizing a cocktail of multi-targeted phytochemicals gives several evolutionary advantages to plants: 1) defense against several predators at once without the need of adapting to specific organisms (Efferth & Koch 2011), 2) avoidance of pathogen resistance, as it is easier for a pathogen to develop resistance to a single compound than a mix of compounds (Lila & Raskin 2005), and 3) saving of energy and resources, as smaller doses of each compound are needed. On the other hand, humans have been using a holistic approach for thousands of years. Ancient medicine systems, such as traditional Chinese medicine and Ayurveda of India, have treated diseases by using complex mixtures of medicinal plants (Efferth & Koch 2011; Lila & Raskin 2005). Still, the modern medical community did not adopt it until recently, since these systems lacked scientific validation and standardization (Lila & Raskin 2005).

The real novelty is that modern medicine in current years has been giving scientific validation to blends of compounds to treat disease more successfully.

Fixed-dose combinations (FDCs) are pharmaceutical products composed of two or more active ingredients in a single dose (Hao et al. 2015). FDCs have been increasing in use and authorization in recent years (Sawicki-Wrzask et al. 2015).

The WHO has recognized FDCs as products with real clinical benefits, including increased efficacy and/or reduced incidence of adverse effects (WHO 2005).

Other advantages include therapy simplification (e.g., reduction of pill burden) and extension of patents, drug exclusivity, and marketability (Hao et al. 2015;

Sawicki-Wrzask et al. 2015).

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4 Drug combinations are a promising alternative for finding a cure against CDDs; however, this implies people getting sick, and getting sick has its disadvantages, such as discomfort (e.g., taking pills), treatments with adverse effects (e.g., injections and chemotherapy), and high costs (e.g., medicines, doctor appointments, and hospitals). Hence, prevention is a better and more economical solution.

Nutraceuticals are compounds naturally found in foods that possess preventive and therapeutic activities against disease, including CDDs. Just as in the case of drugs, no single nutraceutical or food has been found to be effective against CDDs. Nutraceutical combinations can be used, therefore, to formulate novel and highly effective food products for the prevention (and even co- treatment) of CDDs.

1.2. Hypothesis

At least one combination of curcumin, sulforaphane, and/or dihydrocaffeic acid will have a synergistic effect and will be more selective towards colon cancer cells than healthy colon cells.

1.3. General objective

Develop a general protocol to design highly effective food products against CDDs by using nutraceutical combinations as novel element, and partially validate it with colon cancer as model CDD.

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5 1.4. Specific objectives

1) Develop a general methodology to design highly effective food products against CDDs.

2) Evaluate the anticancer potential of dihydrocaffeic acid.

3) Select nutraceutical candidates to perform combination studies in colon cancer cells.

4) Evaluate the effect of curcumin, sulforaphane, and dihydrocaffeic acid, individually and in combination, over the viability of HT-29 colon cancer cells.

5) Evaluate the effect of curcumin, sulforaphane, and dihydrocaffeic acid, individually and in combination, over the viability of Caco-2 colon cancer cells.

6) Compare the cytotoxicity of synergistic combinations of curcumin, sulforaphane, and dihydrocaffeic acid between colon cancer cells and healthy colon cells.

7) Determine the best combination against colon cancer cells.

8) Select ingredients that contain the nutraceuticals of the best combination.

9) Evaluate the effect of wounding stress and peeling over physicochemical, sensory, nutritional, and nutraceutical properties of carrot juice.

10) Determine best conditions of wounding stress and peeling to obtain maximum increase of chlorogenic acid (precursor of dihydrocaffeic acid) in carrot juice.

1.5. Thesis structure

The thesis is composed of 5 chapters. Chapter 1 presents a general introduction to the topic. Chapter 2 consists of a literature review about nutraceutical combinations, colon cancer, and postharvest stresses. Chapter 3

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6 contains the materials and methods. Chapter 4 covers the results and discussion.

And chapter 5 closes with conclusions and future studies.

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7 Chapter 2. Theoretical framework

2.1. Nutraceuticals 2.1.1. General aspects

The term nutraceutical was coined in 1989 by Stephen DeFelice from the words “nutrition” and “pharmaceutical”, and defined it as a food or part of a food that provides health benefits, including the prevention and treatment of disease beyond basic nutritional functions (DeFelice 1989; DeFelice 1995).

Nutraceuticals are chemically classified as isoprenoid derivatives [terpenoids, carotenoids, saponins, terpenes, and all forms of vitamin E (tocotrienols and tocopherols), phenolic compounds, carbohydrate derivatives (vitamin C, oligosaccharides, and non-starch polysaccharides), fatty acid and structural lipids (mono and polyunsaturated fatty acids, conjugated linoleic acid, sphingolipids, and lecithins), amino acid derivatives (amino acids, allyl-S compounds, capsaicinoids, isothiocyanates, indoles, folate, and choline), microbes and associated compounds (probiotics, prebiotics, and synbiotics), and minerals (Ca, Zn, Cu, K, and Se) (Singh & Shukla 2015).

2.1.2. Advantages and disadvantages

Compared with drugs, nutraceuticals are natural, less expensive, and generally have none or few of the adverse effects frequently associated with drugs after long-term administration (DiMarco-Crook & Xiao 2015; Shukla &

George 2011). As they are present in food, nutraceuticals can be delivered through the oral route, which is the preferred route among consumers, since it is non-invasive, does not involve special techniques or complex instructions, and follows the same natural process of food and nutrient consumption in the body

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8 (Braithwaite et al. 2014). Furthermore, nutraceuticals can have preventive and therapeutic activities against CDDs. Examples are antioxidant, anti-inflammatory, antiobesity, antihypertensive, antidiabetic, anticancer activities, among others, which have been observed in vitro, animals, and humans (Daliu et al. 2019;

Naveen & Baskaran 2018; Santana-Gálvez et al. 2017; Singh & Shukla 2015). In addition, specific nutraceuticals may have multiple beneficial activities. For instance, chlorogenic acid has been reported to have antioxidant, anti- inflammatory, antiobesity, antidiabetic, and antihypertensive activities (Santana- Gálvez et al. 2017). The preventive and therapeutic activities of nutraceuticals have led some researchers to recommend them as complementary or alternative treatments against CDDs (Braithwaite et al. 2014). Also, nutraceuticals do not require extensive toxicity tests and rigorous human trials (Lila & Raskin 2005), which can significantly diminish the time to reach the market.

Nevertheless, nutraceuticals have important disadvantages too. Based on their biological activities, secondary metabolites of plants can be classified into two main groups: 1) highly active compounds with great selectivity for cellular targets, and 2) broad-spectrum compounds with moderate or weak activity, which attack multiple cellular targets. It has been estimated that only 10% of all thoroughly described medicinal plants have compounds of the first kind, while 90% has the second kind (Efferth & Koch 2011). Compounds of the first class frequently belong to the class of alkaloids (e.g., atropine, colchicine, galanthamine, physostigmine, and digoxin), while most nutraceuticals belong to the second class (e.g., flavonoids, coumarins, tannins, monoterpenes, triterpenes, sesquiterpenes, saponins, iridoids, lignans, and anthracenes) (Efferth & Koch 2011). Moreover, nutraceuticals are normally found in low

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9 concentrations in natural foods (e.g., fruits and vegetables) (Liu 2003).

Consequently, high doses of nutraceuticals are normally required to achieve the desired effects in humans, which may imply the consumption of unrealistic amounts of food or physiological concentrations that are impossible to achieve in the body (Singh & Shukla 2015; DiMarco-Crook & Xiao 2015). Also, nutraceuticals may cause toxic effects by excessive consumption, interaction with drugs, or in patients with certain diseases (Ronis et al. 2018). In addition, most nutraceuticals, when administered through the oral route, are extensively metabolized by the body (low stomach pH, enzymes, intestinal alkaline pH, liver, among other factors), which can diminish or nullify their activity (Braithwaite et al.

2014). Furthermore, the efficacy of nutraceuticals can be compromised by interaction with other compounds present in the food matrix (Braithwaite et al.

2014).

2.2. Nutraceutical combinations for more effective management of CDDs 2.2.1. Types of combination effects

Different effects can occur as a result of the combination of nutraceuticals.

When the combined effect is equal to the sum of the effects of the individual nutraceuticals, is known as an additive effect; if greater than the sum, a synergistic effect; and if less than the sum, an antagonistic effect. Moreover, a special kind of effect, known as potentiation, happens when an inactive compound enhances the effect of an active one (Efferth & Koch 2011). For instance, (-)-epicatechin (EC) was inert against PC-9 lung cancer cells, but enhanced the apoptotic effects of (-)-epigallocatechin-3-gallate (EGCG) (Suganuma et al. 1999).

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10 Combination effects can also be classified according to the source from which the nutraceuticals come from. Endointeractions occur between nutraceuticals within a source (e.g., an ingredient, a whole food, or extract), while exointeractions occur between nutraceuticals of different sources (e.g., mixing different foods or ingredients) (Lila & Raskin 2005).

2.2.1.1. Methods for determining the combination effect

Two main methods have been applied to determine the combination effect between compounds: the isobologram and the combination index (CI). Both methods have been mostly used for studying drug combinations, but they can also be used for nutraceuticals. The isobologram is a graphical method used for binary mixtures, where the individual and combined doses of the nutraceuticals that achieve a desired effect x (e.g., kill a certain percentage of cancer cells) are plotted in a double-axis diagram (Lee et al. 2007; Phan et al. 2018). First, a straight line is traced by connecting the individual doses of nutraceuticals A and B that produce effect x [Dx(A) and Dx(B), respectively], which will serve as a reference line (Figure 2.1A). Then the nutraceutical doses in combination that produce the same effect x are graphed. Any point below the reference line means that the effect of the nutraceutical combination is synergistic at those specific doses; if it is above the line, then it is an antagonistic effect; and if it is in the line, then it is an additive effect. Furthermore, if many points are plotted, a more general conclusion can be obtained about the combination based on the shape of the curve. If the curve is concave up, the combination is synergistic (Figure 2.1B); if it is concave down, it is antagonistic (Figure 2.1C); and if it is a straight line, it is additive (Figure 2.1D). Furthermore, synergistic and antagonistic effects

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11 Figure 2.1. Hypothetical isobolograms of binary mixtures of nutraceuticals.

Dx(A) and Dx(B) are the individual doses of nutraceuticals A and B, respectively, to achieve a desired effect x. (A) A straight line is traced by connecting Dx(A) and Dx(B), which will serve as a reference line. Then the nutraceutical doses in combination that produce the same effect x are graphed. The shape of the curve determines the combination effect. (B) Synergistic effect. (C) Antagonistic effect.

(D) Additive effect. (E) Synergistic and antagonistic effects.

can be observed for an established dose range. In that case, the curve would have a sigmoidal shape (Figure 2.1E). Graphing many points is useful to find the most synergistic or antagonistic combination. A tendency curve could be plotted to obtain that information.

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12 On the other hand, the CI is a mathematical method (Chou 2006). Similar to the isobologram, the individual and combined doses of nutraceuticals are determined to achieve an effect x. Then the CI is determined with the following equation:

(CI ) = (D)

(D ) = (D)

(D ) + (D)

(D ) + ⋯ + (D) (D ) (Eq. 2.1)

where (CIx)n = combination index for n nutraceuticals that achieve effect x, (D)j = dose of each nutraceutical in the mixture that achieves effect x, and (Dx)j = individual dose of each nutraceutical that produces the same effect x. The CI- value determines the type of effect. If CI < 1, = 1, or > 1, then the combination is synergistic, additive, or antagonistic, respectively.

2.2.2. Advantages of synergistic nutraceutical combinations

Synergistic nutraceutical combinations have several advantages, which are summarized in Table 2.1. Mixtures can reduce substantially the dose needed of each compound to achieve an effect. Dagne et al. (2011) studied the effects of indole-3-carbinol (I3C) and silibinin, individually and in combination, on the viability of the lung cancer cell line A549. I3C was tested in the dose range of 25–

400 µM, and only at 400 µM a significant reduction was observed, which corresponded to a nearly 20% viability. Silibinin was tested in the 25–100 µM range, and at 100 µM the viability was reduced to about 40%. But by combining I3C and silibinin at 50 µM each, viability was decreased to about 40%, meaning a dose reduction of 87% and 50%, respectively. Treatment with berberine alone

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13 Table 2.1. Advantages of synergistic nutraceutical combinations with study examples.

Advantage Foods/Nutraceuticals Disease/condition Model Study details Experimental findings Reference

Dose reduction I3C, silibinin Lung cancer A549 cells I3C (25–400 µM) and silibinin (25–100 µM) were tested individually and in combination

(50 µM each).

The combination reduced 87% and 50%

the necessary dose of I3C and silibinin, respectively, to decrease cell viability to

30–40%.

Dagne et al., 2011

Berberine, curcumin Breast cancer MCF-7 and MDA-MB-

231 cells Berberine and curcumin were administered alone and in

combination at various concentrations.

Co-treatment with berberine and 10 μM curcumin decreased 68% and 92% the IC50 values of berberine for MCF-7 and

MDA-MB-231, respectively.

Wang et al., 2016

Quercetin, 6-gingerol Type 2 diabetes,

hyperlipidemia Wistar rats Quercetin (10 mg/kg) and 6- gingerol (3 mg/kg) were administered individually and in

mixture to rats.

The combination significantly decreased several parameters compared with either compound alone, including fasting

glucose, total cholesterol, triglycerides, among others.

Shao et al., 2016

Resveratrol, piperine Neurological associated

functions

Humans Healthy adults received resveratrol (250 mg), or resveratrol (250 mg) and piperine

(20 mg)

Participants showed a significantly greater cerebral blood flow with the

mixture compared with resveratrol alone.

Wightman et al., 2014

Bioavailability

enhancement EC, curcumin Lung cancer PC-9 and A549 cells EC and curcumin were tested at various concentrations both individually and in combination

EC increased the bioavailability of curcumin, causing a significant increase in inhibition of cell growth and apoptosis.

Saha et al., 2010

Resveratrol, piperine Bioavailability C57BL mice Mice were administered resveratrol (100 mg/kg) or resveratrol (100 mg/kg) and

piperine (10 mg/kg)

The AUC and Cmax of resveratrol were increased 229% and 1544%, respectively, with the addition of

piperine.

Johnson et al., 2011

Curcumin, piperine Bioavailability Sprague–Dawley rats Rats were given curcumin and piperine at various dose ratios

(1:1 to 100:1).

Piperine increased the bioavailability of curcumin through inhibition of UGTs and

SULTs

Zeng et al., 2017

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14 Table 2.1. Continued.

Advantage Foods/Nutraceuticals Disease/condition Model Study details Experimental findings Reference

Adverse effects

reduction Daidzein, genistein Prostate cancer Balb/c nu/nu nude

mice Mice were fed 21.5 mg/kg genistein, 10.5 mg/kg daidzein, or

50 mg/kg soy isoflavones.

Genistein promoted metastasis, but the effect was cancelled if combined with

daidzein.

Singh-Gupta et al., 2010

β-carotene, quercetin, naringenin, α-

tocopherol

Inflammation HL-60 cells β-carotene was tested alone or in combination with quercetin, naringenin, or α-tocopherol

Pro-inflammatory effects of 20 µM β- carotene were partially suppressed by quercetin, naringenin, or α-tocopherol.

Yeh et al., 2009

Better food combination recommendations

Fruits, vegetables,

legumes Oxidative stress FRAP, DPPH, ORAC Fruits (raspberry, blackberry, apple), vegetables (broccoli, tomato, mushroom, purple

cauliflower), and legumes (soybean, adzuki bean, red kidney

bean, black bean) were combined in pairs.

Combining foods from different categories increased the chances of

synergy (particularly, fruits with legumes). Raspberry and adzuki bean were the only combination that produced

synergy in all antioxidant assays.

Wang et al., 2011

Butyrate, corn oil, fish

oil Colon cancer Sprague-Dawley rats Rats were fed butyrate capsules

with corn oil or fish oil. Increase in apoptosis and decrease in ACF was observed with butyrate + fish oil, but not with butyrate + corn oil.

Crim et al., 2008

Mediterranean diet,

Western diet, curcumin Neurofibromatosis

type 1 Humans Patients were fed a

Mediterranean diet or a Western diet, both with or without curcumin

(1200 mg)

Only patients who consumed the Mediterranean diet enriched with curcumin presented a significant reduction in the number and volume of

neurofibromas.

Esposito et al., 2017

Abbreviations: ACF = aberrant crypt foci, AUC = area under the curve, Cmax = maximum serum concentration, DPPH = 2,2-diphenyl-1-picryl-hydrazyl-hydrate, EC = epicatechin, FRAP = ferric antioxidant power, I3C = indole-3-carbinol, IC50 = 50% growth inhibition dose,ORAC = oxygen radical absorbance capacity, SULTs = sulfotransferases, and UGTs = UDP-glucuronyltransferases.

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15 caused 50% growth inhibition (IC50) in MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines at a concentration of 65.27 and 178.87 μM, respectively (Wang et al. 2016). However, co-treatment with berberine and 10 μM curcumin decreased the IC50 values of berberine to 21.05 and 14.85 μM for MCF-7 and MDA-MB-231, respectively, which means 68% and 92% dose reductions.

Quercetin (10 mg/kg) and 6-gingerol (3 mg/kg) were evaluated individually and in mixture (same doses as individually) on several metabolic parameters in rats with type 2 diabetes (treatments administered for 6 weeks) and hyperlipidemia (treatments administered for 24 h), induced by streptozotocin and poloxamer P- 407, respectively (Shao et al. 2016). The combination significantly decreased several parameters compared with either compound alone, including fasting glucose, total cholesterol, triglycerides, among others. These results suggest that a greater dose of the individual compounds would be needed to achieve the same effect as the combination; therefore, a dose reduction is implied. Likewise, in a randomized, double-blind, placebo-controlled clinical trial, 23 healthy adults (4 males and 19 females, and 19–34 years old) received three single-doses (conducted 2–14 days apart) of either placebo, resveratrol (250 mg), or resveratrol (250 mg) and piperine (20 mg) (Wightman et al. 2014). After a 40 min rest, participants showed a significantly greater cerebral blood flow with the resveratrol/piperine blend compared with placebo and resveratrol alone, while cognitive function, mood, and blood pressure remained unaffected.

Mixtures can also enhance bioavailability, the measure of how much of the compound remains available for physiological functions after ingestion. EC enhanced the bioavailability of curcumin, causing a significant increase in inhibition of cell growth and apoptosis of lung cancer cell lines (Saha et al. 2010).

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16 The area under the curve (AUC) and maximum serum concentration (Cmax) of resveratrol were increased 229% and 1544%, respectively, with the addition of piperine in C57BL mice (Johnson et al. 2011). Piperine also increased the bioavailability of curcumin in rats through inhibition of UDP- glucuronyltransferases (UGTs) and sulfotransferases (SULTs) (Zeng et al. 2017).

Adverse effects of some nutraceuticals may be decreased by other nutraceuticals. It was observed that genistein promoted metastasis of prostate cancer in mice, but the effect was cancelled when combined with daidzein (Singh- Gupta et al. 2010). The pro-inflammatory effects of 20 µM β-carotene were partially suppressed on HL-60 cells by quercetin, naringenin or α-tocopherol (Yeh et al. 2009).

Knowledge of how nutraceuticals interact can allow dietitians to give better recommendations of what foods to combine. Wang et al. (2011) tested the antioxidant capacity of different food blends. Foods from three different categories were combined in pairs, including fruits (raspberry, blackberry, and apple), vegetables (broccoli, tomato, mushroom, and purple cauliflower), and legumes (soybean, adzuki bean, red kidney bean, and black bean). Three antioxidant assays were performed, including FRAP, DPPH, and ORAC.

Synergistic, additive, and antagonistic effects were observed by mixing foods within the same category and between categories. Combining foods from different categories increased the chances of synergy (particularly, fruits with legumes) compared with food mixtures of the same category. The combination of raspberry and adzuki bean was the only one that produced synergistic effects in all three antioxidant assays. The effect of mixtures of butyrate and different fat sources on colon cancer was tested in rats (Crim et al. 2008). Sprague-Dawley

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17 rats were fed butyrate capsules with either corn oil or fish oil, and were injected with azoxymethane (AOM). Increase in apoptosis and decrease in aberrant crypt foci (ACF) was observed with butyrate + fish oil, while butyrate + corn oil caused an increase in ACF and no effect on apoptosis. In a human trial, 11 patients (8 males and 3 females, and 18–59 years old) with neurofibromatosis type 1 were fed a Mediterranean diet or a Western diet, both with or without curcumin (1200 mg/day) for 6 months (Esposito et al. 2017). Only in patients who consumed the Mediterranean diet enriched with curcumin was observed a significant reduction in the number and volume of neurofibromas. Neither the unenriched Mediterranean diet nor the enriched Western diet presented any significant positive effect. The authors hypothesized that the combination of a polyphenol- rich Mediterranean diet and curcumin was responsible for the beneficial effect observed.

Finally, economic benefits can be obtained by developing new synergistic nutraceutical combinations. First, there is a growing demand for natural products and a rising awareness of personal responsibility in health (Efferth & Koch 2011).

Second, a more complex and synergistic formulation allows competitive differentiation and lower costs of raw material (Cicero et al. 2015; DiMarco-Crook

& Xiao 2015). Third, nutraceutical blends with scientifically proven benefits can be patented. For instance, Emulin^TM is a patented blend of chlorogenic acid, myricetin, and quercetin with proven antidiabetic effects in humans (Ahrens &

Thompson 2013). Some examples of dietary supplements available in the market that are based on mixtures of nutraceuticals, medicinal herbs, and medicinal mushrooms with scientifically proven health benefits in vitro, animals, or humans are shown in Table 2.2.

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18 Table 2.2. Dietary supplements, composed of a mixture of nutraceuticals and other ingredients, with scientifically proven health benefits at in vitro, animal, or human level.

Name Composition Disease (study level) Reference

AOB Wheat germ, soybean, rice bran, tear grass, sesame, wheat, citrus lemon, green tea, green leaf extract, and malted rice.

Colon cancer (animals)

Minamiyama, Takemura, Hirohashi, &

Okada, 2004 BreastDefend Curcuma longa, diindolylmethane, quercetin,

medicinal mushrooms (Coriolus versicolor, Ganoderma lucidum, and Phellinus linteus), and medicinal herbs (Scutellaria barbata and Astragalus membranaceus).

Breast cancer

(animals) Jiang et al., 2012b

Coltect Curcuma longa, green tea, and L-

selenomethionine. Colon cancer

(animals) Aroch et al., 2010 Emulin Chlorogenic acid, myricetin, and quercetin Diabetes (humans) Ahrens &

Thompson, 2013 Iberogast Iberis amara, peppermint, chamomile flower,

liquorice root, Angelica root, caraway fruit, milk thistle fruit, lemon balm leaves, and greater celandine.

Colon cancer (in vitro) Bonaterra, Kelber, Weiser, &

Kinscherf, 2013 Kepar Curcuma longa, silymarin, guggul, chlorogenic

acid, and inulin. Metabolic syndrome

(humans) Patti et al., 2015 ProstaCaid 33 ingredients including Curcuma longa,

quercetin, mushrooms, herbs, among others.

Prostate cancer (in vitro and animals)

Jiang et al., 2012a; Yan &

Katz, 2010

2.2.3. Factors for designing highly effective food products against CDDs 2.2.3.1. Activity

The first factor to consider to successfully design a highly effective food product against a CDD is to choose nutraceuticals that have activity against the disease (e.g., anticancer, antidiabetic, antiobesity, or antihypertensive activities).

Another alternative is the potentiation effect, where inactive compounds increase the impact of active compounds when mixed together, as discussed previously.

However, from the probabilistic point of view, it is more likely to succeed if all nutraceuticals used are active against the CDD of interest.

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19 2.2.3.2. Bioavailability

Before considering performing combination studies, it is highly recommended to make sure that the nutraceutical candidates can reach their destination and in sufficient quantities. In other words, the bioavailability of the nutraceuticals must be evaluated. Since food products are delivered through the oral route, nutraceuticals must encounter the gastrointestinal digestive tract;

therefore, digestion, absorption, and metabolism need to be considered. Gawlik- Dziki (2012) performed simulated gastrointestinal digestion of onion, lettuce, garlic and tomato, and compared the antioxidant activity of raw, digested, and dialyzed extracts, using the ABTS assay. Onion, lettuce, and tomato showed significant decreases in antioxidant activity in digested extracts compared to raw extracts, while garlic showed a significant increase. Dialyzed extracts of onion and tomato showed no difference in antioxidant activity compared with digested extracts, but it was significantly lower in lettuce. Although the digested extract of garlic showed significantly higher activity compared with the raw extract, the dialyzed extract had significantly lower activity than the raw extract.

Durak et al. (2014) studied the effect of in vitro digestion over the interaction between extracts of coffee and cinnamon on lipoxygenase (LOX) activity. Before digestion, coffee and cinnamon showed synergistic effects in LOX inhibition, while digested extracts showed antagonistic effects. Opposite results were observed for coffee and ginger (Durak et al. 2015). Sun et al. (2015) also used in vitro digestion to investigate the changes in antioxidant and cytoprotective effects of purple rice anthocyanins against H2O2 in BRL-3A immortalized rat liver cells.

Antioxidant capacity, cytoprotective effects, and the level of anthocyanins did not change significantly during gastric digestion. Nonetheless, 76% of anthocyanins

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20 were degraded during intestinal digestion, and antioxidant activity decreased more than half. Interestingly, combining gastric and intestinal samples led to synergistic antioxidant and cytoprotective effects.

An important part of metabolism, which has been increasingly investigated in recent years, is the gut microbiota (microbiota from here on), i.e., the community of microorganisms living in the human intestine. The microbiota can be found in different sections of the intestines, but it is mostly concentrated in the colon. It is composed of various types of microorganisms, including bacteria, viruses, fungi, archaea, bacteriophages, and protozoans. The most abundant are bacteria. About 100 trillion bacteria and 1000 different species have been identified (Yang & Yu 2018). Increasing evidence suggests that the microbiota is essential for human health, including immune system development, maintaining homeostasis, and influencing autoimmune diseases and allergies (Brennan &

Garrett 2016). In addition, the microbiota seems to play a role in the development of CDDs such as cancer (Brennan & Garrett 2016). It has been found that the microbiota can metabolize nutraceuticals as well. Polyphenols, the largest family of nutraceuticals known to date, are present in food mostly as glycosides (e.g., flavonoids) or in the form of high molecular weight oligomers and polymers (e.g., proanthocyanidins and ellagitannins), but they are extensively metabolized by the microbiota, first into aglycones and monomers, and then into simpler phenolics via dehydroxylation, decarboxylation, and ring breakage reactions (Rowland et al. 2018). For instance, flavonoids, the biggest subgroup of polyphenols, are converted into ring-fission products and phenolic acids (Mansoorian et al. 2019;

Murota et al. 2018). Chlorogenic acid, one of the most studied polyphenols, is degraded into hippuric acid, caffeic acid, ferulic acid, derivatives of caffeic and

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21 ferulic acids, among others (Ludwig et al. 2013; Olthof et al. 2003). Hence, in cases like this, more valuable information can be obtained by studying the nutraceutical metabolites rather than the nutraceutical itself.

2.2.3.3. Mechanisms

Although information is limited for most nutraceuticals, knowledge of their mechanisms is ideal for formulating synergistic blends. One way by which nutraceuticals produce synergistic effects is by acting on different mechanisms.

Combination of ellagic acid and urolithin A exhibited a synergistic antiproliferative effect against PC-3 prostate cancer cells (Vicinanza et al. 2013). It was found that each compound inhibited proliferation via two different mechanisms. Ellagic acid induced cell-cycle arrest in S phase, while urolithin A induced G2/M cycle arrest.

Pretreatment with diindolylmethane (DIM) enhanced apoptosis by butyrate in colon cancer cells expressing a mutant adenomatous polyposis coli (APC) gene (Bhatnagar et al. 2009). Butyrate can induce apoptosis in colon cancer cells by inhibiting histone deacetylase, but the mutations in the APC gene confer the cells resistance due to the failure of butyrate to downregulate survivin, an antiapoptotic protein. Nevertheless, DIM downregulated survivin, producing the synergistic effect observed.

Mixtures of nutraceuticals can also activate new mechanisms that none of the compounds can trigger alone, leading to synergy. Curcumin and docosahexaenoic acid (DHA) showed a synergistic antiproliferative effect against SK-BR-3 breast cancer cells (Altenburg et al. 2011). Microarray analysis revealed that some mechanistic pathways were triggered by the combination that neither compound alone managed to activate, partly explaining the results. Hsu et al.

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22 (2011) studied the effects of soy and green tea on inflammation in a rat model of prostate cancer. Their combination decreased expression of NF-κB levels, tumor necrosis factor alpha (TNF-α), and interleukins (IL)-6 and IL-1β; increased Bax/BcL2 ratio, and attenuated prostate malignancy by decreasing prostate hyperplasia. These effects were not apparent with soy or tea alone.

Another way in which combinations can produce synergistic effects is by increasing the bioavailability of active nutraceuticals. A nutraceutical can enhance the bioavailability of another nutraceutical through two mechanisms: 1) increasing its intracellular concentration, or 2) inhibiting its metabolization. EC increased the intracellular concentration of curcumin in lung cancer cell lines, leading to a superior inhibition of cell growth compared to the individual compounds (Saha et al. 2010). The flavonoids quercetin, fisetin, myricetin, kaempferol, and apigenin could inhibit resveratrol sulphation in human liver and duodenum, which might improve its bioavailability (De Santi et al. 2000).

Quercetin combined with EGCG demonstrated enhanced inhibition of proliferation of prostate cancer cells by increasing the intracellular concentration of EGCG and decreasing EGCG methylation (Wang et al. 2012b).

2.2.3.4. Doses and proportions

The dose of each nutraceutical in the combination can change the effect, even though the same proportion is maintained. For instance, Pappa et al. (2007) evaluated the impact of mixtures of sulforaphane and DIM (ratios 1:4, 1:2, 1:1, 2:1, and 4:1) on cell proliferation, cell-cycle progression, and apoptosis in colon cancer cells. At a total concentration of 2.5 µM, all blends of sulforaphane and

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23 DIM were antagonistic. However, with increasing concentrations, the antagonistic effect gradually turned into a synergistic effect.

In contrast, different proportions of each nutraceutical in a combination can cause different effects. Liu et al. (2016) studied the effect of several proportions (7:1, 5:1, 3:1, 1:1, 1:3, 1:5, and 1:7 ratios) of Potentilla fruticose L. extract (PFE) and green tea polyphenols (GTP) on antioxidant activity. The most synergistic effect was achieved at a 3:1 ratio (PFE:GTP).

Most in vitro studies use higher concentrations than the plasma or tissue levels reported in vivo (Phan et al. 2018). Thus, it is important to formulate mixtures where the concentrations of each compound can be achieved in the human body.

2.2.3.5. Simultaneous vs. sequential combinations

Usually, nutraceutical combination studies are done by applying all components simultaneously. Nonetheless, it is possible to administer each component at different times, and the order of the sequence can result in different effects. Montgomery et al. (2016) incubated colon cancer cells for 48 h with curcumin and silymarin, and found synergistic effects on proliferation inhibition and apoptosis when administered together. Pre-exposure of cells with curcumin for 24 h and subsequent treatment with silymarin for 48 h greatly decreased viability, while the opposite sequence had no effect.

2.2.3.6. Raw material quality control

Raw material quality control refers to all possible factors that can affect the nutraceutical composition of ingredients before processing. As most

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24 nutraceuticals come from plant sources, temperature, humidity, sunlight, soil, and postharvest conditions are all factors that can alter the nutraceutical composition of horticultural crops, which may cause great batch-to-batch variations (Efferth &

Koch 2011). Thus, these elements must be controlled as much as possible to minimize variation.

2.2.3.7. Vehicle

The appropriate vehicle to deliver nutraceuticals (food, beverage, or dietary supplement) must be chosen, as variables, such as processing, presence of other compounds, and the food matrix, can alter the efficacy and stability of nutraceuticals.

Processing conditions can have a huge impact on the nutraceuticals of interest. First, the choice of whether the product will be a food, beverage, or supplement will define the processing conditions, which can vary greatly, such as the thermal treatment (e.g., cooking/sterilization for foods, pasteurization for beverages, and dehydration for supplements), leading to differences in temperature and application time, and the ingredient parts that will be used (e.g., for juices, normally the peel and pulp are removed, while foods are generally more whole). These variations will affect physically (amount) and chemically (structure) the nutraceuticals. The use of nonthermal processing technologies (NTPTs) can be considered to minimize degradation and alteration of nutraceuticals, which will be discussed later on. Second, key enzymes might be degraded that are required to transform an inactive compound into a nutraceutical (e.g., glucoraphanin to sulforaphane by myrosinase activity, as previously discussed). Third, certain technologies, such as PAS, can increase nutraceutical

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