Development of nanoparticles loaded with bioactive compounds for application as nutraceuticals

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INTEGRATED MASTER IN BIOENGINEERING

Development of Nanoparticles Loaded with

Bioactive Compounds for Application as

Nutraceuticals

Natacha Alexandra Branco Rosa

Dissertation presented according to the requirements for the degree in INTEGRATED MASTER IN BIOENGINEERING – SPECIALIZATION IN BIOMEDICAL

ENGINEERING

___________________________________________________________ President of the Jury: Professor Ana Paula Gomes Moreira Pego

(Assistant Researcher of the Chemical Engineering Department in the Faculty of Engineering of Oporto University)

__________________________________________________________ Supervisor: Professor Marlene Susana Dionísio Lúcio

(Research Associate of the Faculty of Pharmacy of Oporto University) ___________________________________________________________

Co-Supervisor: Professor Dr. Maria de La Salette Freitas Fernandes Hipólito Reis Dias Rodrigues

(Assistanted Professor of the Faculty of Pharmacy of Oporto University) ___________________________________________________________

Examiner: Professor Sandra Cristina Pinto da Rocha

(Assistant Researcher of the Chemical Engineering Department in the Faculty of Engineering of Oporto University)

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Desde o início da Era Industrial os estilos de vida mudaram drasticamente, reflectindo-se nos hábitos alimentares da população. Conreflectindo-sequentemente, as pessoas foram empurradas para regimes de alimentação “fast-food” que são facilmente consumidos, mas apresentam menor valor nutricional. No entanto, estudos têm demonstrado que diferentes componentes da dieta constituem importantes factores de risco modificáveis para doenças crónicas, especialmente para doenças cardiovasculares que são as principais causas de morte nos Estados Unidos e outros países ocidentais. Como resultado, houve um crescente interesse por parte dos profissionais de saúde e cientistas em produtos nutracêuticos como o resveratrol, que é um polifenol natural presente em diversas plantas incluindo legumes, frutas, grãos, raízes, flores, sementes, chá e vinho.

Dados os benefícios apresentados pelo resveratrol, nomeadamente o seu efeito protector contra doenças cardiovasculares, neurodegenerativas e cancerígenas, o principal objectivo desta dissertação foi desenvolver nanopartículas lipídicas encapsuladas com resveratrol. Estas nanopartículas são biocompatíveis e capazes de transportar e proteger este importante composto bioactivo contra a degradação, aumentando a sua estabilidade física bem como a sua biodisponibilidade. As nanopartículas lipídicas (LN) testadas foram nanopartículas lipídicas sólidas (NLS) e nanotransportadores lipídicos (NLC) e foram produzidos usando uma técnica de homogeneização modificada em que alguns dos parâmetros deste método também foram optimizados (tempos de agitação; tempos de sonicação e intensidade de sonicação). Estas nanopartículas permitirão a ingestão oral deste composto o qual pode funcionar como um nutracêutico e pode ser adicionado aos alimentos ou bebidas para aumentar o seu valor nutricional. A fim de avaliar a qualidade das nanopartículas desenvolvidas, uma caracterização adequada e apropriada foi realizada. As nanopartículas lipídicas foram caracterizadas de acordo com: morfologia da superfície; parâmetros do tamanho das partículas, ou seja, o diâmetro médio do índice de polidispersão usando dynamic light scattering (DLS); o potencial zeta também foi determinado utilizando DLS; pH; o grau de cristalinidade e a modificação de lípidos

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in vitro de resveratrol foi avaliada por meio da técnica de diálise em condições de

imersão. A estabilidade das nanopartículas também foi verificada para um período de dois meses, em que durante esse tempo as formulações foram armazenadas à temperatura ambiente (25°C) e à luz do dia, tendo sido realizadas medições do tamanho das partículas, potencial zeta, pH e de encapsulamento de resveratrol.

Palavras-chave: Resveratrol, nutracêutico, nanopartícula lipidica sólida, nanotransportadores lipídicos, cerveja

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Abstract

Since the start of the Industrial Age, lifestyles have dramatically changed; reflecting in the eating habits of the population. Consequently people were pushed into fast-food regimes which are easily consumed, but present less nutritional value. However, studies have demonstrated that different dietary components constitute important modifiable risk factors for chronic diseases, especially for cardiovascular diseases, which are the leading causes of death in the United States and other westernized countries. As a result, there was a rising interest from health professionals and scientists in nutraceuticals like resveratrol which is a natural polyphenol present in a variety of plants including vegetables, fruits, grains, roots, flowers, seeds, tea, and wine.

Given the benefits presented by resveratrol, namely its protective effect against cardiovascular and neurodegenerative diseases, and cancer, the main goal of this dissertation was to develop resveratrol loaded lipidic nanoparticles which are biocompatible and capable of transporting and protecting this important bioactive compound against degradation, increasing its physical stability and bioavailability. The lipid nanoparticles (LN) tested were solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) and were produced using a modified hot homogenization technique were some of the parameters of this method were also optimized (times of stirring; times of sonication and sonication intensity). These nanoparticles will allow oral intake of this compound which can function as a nutraceutical that can be added to food or drinks to increase their nutritional value. In order to evaluate the quality of the developed nanoparticles, an adequate and proper characterization was accomplished. LN were characterized according to their: surface morphology; particle size parameters i.e., the average diameter size and polydispersity index using dynamic light scattering (DLS); zeta potential also determined using DLS; pH; degree of crystallinity and lipid modification (polymorphism) using a differential scanning calorimetry (DSC). Drug loading and release were measured by UV-Visible spectroscopy and in vitro resveratrol release was evaluated using dialysis bag diffusion technique under sink conditions. The stability of the nanoparticles was also verified for a period of two months, in which

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during that time the formulations were stored at room temperature (25°C) and under day light and measurements of particle size, zeta potential, pH and resveratrol encapsulation were made.

Key words: resveratrol, nutraceutical, SLN, NLC, beer

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Acknowledgements

I’m very grateful to my supervisor and co-supervisor, Marlene Lúcio and Sallete Reis, for their always constructive critique and encouragement, which helped me to conceptualize and accomplish my Dissertation.

My sincere gratitude to the constant support, encouragement and unconditional love of my family and friends.

I would also like to give special thanks to everyone in the Departamento de Química da Faculdade de Farmácia da Universidade do Porto laboratory for making me feel so welcome.

Last but not least, I would like to say thank you to my yoga teacher, mentor and friend Clara Alexandra for her tireless afford to keep my mind in the good direction.

The number of people giving me their support has been crucial along the way and I’m very grateful to each and every person support that contributed to the accomplishment of this dissertation.

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List of Figures

Figure ... Page 1 Isomeric forms of resveratrol ... 9 2 NOW®Natural Resveratrol from Genuine Whole ... 17 3 The impacts and needs of nanotechnology applications in foods and

food processing ... 18 4 Enzymes secreted and nutrients absorption along the

gastrointestinal tract ... 21 5 High shear homogenization and ultrasound method for the

preparation of lipid nanoparticles ... 25 6 Mechanism of absorption promotion effect of lipid being

formulated as lipid nanoparticles ... 27 7 Three drug incorporation models ... 30 8 Types of NLC ... 33 9 UV spectra of the standard solutions of resveratrol prepared in

ethanol containing concentrations of resveratrol from 13,89 to 159,72 μM ... 44 10 Calibration curve of resveratrol in ethanol ... 45 11 Average diameter and respective standard error of SLN in day 1 and

2 months after, in formulations containing different concentrations of resveratrol ... 47 12 Average diameter and respective standard error of NLC in day 1 and

2 months after, in formulations containing different concentrations of resveratrol ... 48 13 Zeta potential and respective standard error of SLN in day 1 and 2

months after, in formulations containing different concentrations of resveratrol ... 50 14 Zeta potential and respective standard error of NLC in day 1 and 2

months after, in formulations containing different concentrations of resveratrol ... 50

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ii 15 Encapsulation efficiency of SLN in day 1 and 2 months after, in

formulations containing different concentrations of resveratrol ... 52 16 Encapsulation efficiency of NLC in day 1 and 2 months after, in

formulations containing different concentrations of resveratrol ... 53 17 SEM image of solid lipid nanoparticles placebo and containing

resveratrol ... 55 18 Average diameter of the LN for different concentrations of

resveratrol in the formulations. Each bar corresponds to the average of three formulations measured independently ... 56 19 Zeta potential of LN for different concentrations of resveratrol in

the formulations. Each bar corresponds to the average of three formulations measured independently ... 58 20 Schematic representation of the polymorphic forms of the lipids in

lipid nanoparticles. Arrows indicate the influence of the polymorphic transitions in: the drug incorporation; thermodynamic stability and lipid packing ... 60 21 DSC thermogram showing the phase transitions to the different

polymorphic forms ... 61 22 DSC melting curves of cetyl palmitate bulk material (CP) first and

second heating ... 63 23 DSC melting curves of cetyl palmitate bulk material (CP) and

polysorbate 60 (P60) and this mixture B with resveratrol ... 64 24 DSC melting curves of cetyl palmitate bulk material (CP) and

polysorbate 60 (P60) and Miglyol and this mixture A with resveratrol ... 65 25 DSC melting curves of NLC placebo and NLC loaded with resveratrol .... 67 26 DSC melting curves of SLN placebo and sln loaded with resveratrol ... 68 27 Encapsulation efficiency of SLN and NLC, in formulations of

different concentrations of resveratrol ... 69 28 Dialysis process ... 71 29 In vitro Controlled release set-up ... 74

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iii 30 spectra of the standard solutions of resveratrol prepared in beer 0

to 131,94 μM ... 75 31 Calibration curve of resveratrol in beer ... 76 32 Resveratrol release (%) from the dialysis bag containing only

resveratrol molecules to receptor compartment containing different solvents (water, water and ethanol and beer) and at different temperatures (25ᵒC and 4ᵒ) ... 77 33 Resveratrol release (%) from the dialysis bag containing SLN with

resveratrol to receptor compartment containing different solvents (water, water and ethanol and beer) and at different temperatures (25ᵒC and 4ᵒ) ... 78 34 Resveratrol release (%) from the dialysis bag containing NLC with

resveratrol to receptor compartment containing different solvents (water, water and ethanol and beer) and at different temperatures (25ᵒC and 4ᵒ) ... 79 A.1 Arrangement of Ultra-Turrax T25 and sonication devices ... 90 A.2 SLN and NLC formulations ... 90

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List of Tables

Table ... Page 1 Wines produced in years between 1996 and 1999 and respective

concentration of pure trans-Resveratrol in mg/L... 16 2 Times of ultra-turrax and sonication and sonication intensity for

different formulations ... 34 3 Composition and respective percentage of each component in the

SLN and NLC formulations ... 35 4 Average diameter, polydispersity index and Zeta potential of the

SLN formulations ... 39 5 Average diameter, polydispersity index and Zeta potential of the

NLC formulations ... 39 6 Best results selected for each type of nanoparticle and

corresponding production method ... 40 7 Average diameter, polydispersity index and Zeta potential of the LN

formulations with and without filtering ... 41 8 Best results selected for each type of nanoparticle and

corresponding production method ... 42 9 Average diameter, polydispersity index of the LN formulations with

water solvent and with 1mM KCl solvent. ... 43 10 Maximum absorbance verified in the standard solutions of

resveratrol with concentrations from 13.89 to 159.72 μM and correspondent wavelength where the maximum absorbance was observed. ... 45 11 Polidispersity Index of SLN and NLC in day 1 and 2 months after, to

different concentrations of resveratrol in the formulations ... 49 12 pH of SLN and NLC in day 1 and 2 months after, to different

concentrations of resveratrol in the formulations ... 51 13 Polydispersity index of LN, in formulations containing different

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v 14 pH of LN, to different concentrations of resveratrol in the

formulations ... 59 15 Name of each formulation and respective composition and quantity ... 62 16 Melting point (peak maximum), onset and enthalpy of cetyl

palmitate obtained fromfirst and second heating in the DSC analysis ... 64 17 Melting point (peak maximum), onset and enthalpy of bulk mixtures

of cetyl palmitate with polysorbate 60 ... 65 18 Melting point (peak maximum), onset and enthalpy of bulk mixtures

of cetyl palmitate with polysorbate 60 and miglyol ... 66 19 Melting point (peak maximum), onset and enthalpy of NLC placebo

and NLC containing resveratrol ... 67 20 Melting point (peak maximum), onset and enthalpy of SLN placebo

and SLN containing resveratrol ... 68 21 In vitro Formulations for the studies of resveratrol release ... 72 22 In vitro Formulations for the studies of resveratrol release ... 75 A.1 Quantities used for SLN formulations and the accuracy of the

weights ... 91 A.2 Quantities used in the NLC formulations and the accuracy of the

weights ... 91 A.3 Quantities used in the SLN formulations and the accuracy of the

weights ... 91 A.4 Quantities used in the NLC formulations and the accuracy of the

weights ... 92 A.5 Quantities used in the SLN formulations and the accuracy of the

weight ... 92 A.6 Quantities used in the NLC formulations and the accuracy of the

weights ... 92 A.7 Quantities of each component in the two types of formulations

(SLN, NLC) ... 93 A.8 Quantities used of stock solution and ethanol and correspondent

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Abbreviations and symbols

ADP Adenosine diphosphate

Ca2+ Calcium ion

CEMUP Centro de materiais da Universidade do Porto

D Translational diffusion coefficient

DLS Dynamic light scattering

d(H) Hydrodynamic diameter

DSC Differential scanning calometry

ELS Electrophoretic light scattering

EU European Union

FDA Food and Drug Administration

GALT Gut-associated lymphoid tissue

GRAS Generally Recognized as Safe

K Boltzmann’s constant

K-1 Debye length

KCl Potassium chloride

kcps Kilo-counts per second

LCT Oil-derived long chain triglycerides

LDL Low-density lipoprotein

LN Lipid nanoparticles

MCT Oil-derived medium chain triglycerides

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NLC Nanostructures lipid carriers

NO Nitric oxide

NIOSH National Institute for Occupational Safety and Health

O2- Superoxide anion

PCS Photon correlation spectroscopy

r Distance between particles

RES Reticuloendothelial system

RNA Ribonucleic acid

rpm Revolutions per minute

SEM Scanning electron microscopy

SLN Solid lipid nanoparticles

T Absolute temperature

TXA2 Thromboxane A2

USA United State of America

η Viscosity of solution

μep - electrophoretic mobility εr - relative permittivity of the liquid

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1. Introduction

1.1. Motivation

Resveratrol is a natural polyphenol nonflavonoid of the subclass hydroxylated stilbene, being grapes and wines the most important food sources of this compound [1]. This compound provides health or medical benefits which includes prevention and/or treatment of diseases. The heath promoting effects reported for resveratrol are: antioxidant, neuroprotective, anti-inflammatory, cardioprotective and anti-platelet effects. Furthermore, resveratrol acts as a phytoestrogen and it also inhibits cancer [2-4]. Given the benefits of resveratrol, and trying to overcome the disadvantages of this lipophilic bioactive compound like its low bioavability and cytotoxic effect in a dose and time-dependent manner, this dissertation aimed primarily to develop lipid nanoparticles (LN), loaded with resveratrol to be applied as a nutraceutical this way adding the health benefits of resveratrol to beverages other than the wine.

1.2. General and specific objectives

The aim of the current work is to include resveratrol in lipid nanoparticles (SLN and NLC) for further dispersion in alcoholic or non alcoholic beverages (e.g. beer) improving their nutritional value and developing a healthy drink with cardiovascular protection similar to the one attributed exclusively to red wine consumption.

Lipid nanoparticles (LN) will be employed in this study since lipids are known to promote oral absorption of drugs or vitamins. Indeed, there are quite a number of lipophilic compounds for which increased oral bioavailability is reported. Given that resveratrol is also a lipophilic compound, the formulation purposed provides an opportunity to exploit the chemopreventive, antidiabetic and cardiovascular protective properties of resveratrol together with the advantages of lipid nanoparticles such as their good physical stability, their protection of incorporated resveratrol from degradation, controlled release and compatibility. Therefore, in order to, accomplish the principal objective of developing a valid nanocarrier system to encapsulated resveratrol, several other specific goals had to be established and achieved:

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 Parameters of the modified hot homogenization technique for the production

of the LN had to be optimized, e.g. the time of stirring, time of sonication and sonication intensity for each type of nanoparticle tested;

 Optimization of the drug incorporation through the experiment of different

concentrations of resveratrol in the formulations;

 Evaluation of the stability of the nanoparticles during 2 months allowing

inferring about the nanoparticles shelf life and the behavior of the nanoparticles during storage. The stability was determined through: (i) particle size measurements i.e., determination of the average hydrodynamic diameter and polydispersity index using dynamic light scattering (DLS); (ii) zeta potential measurements using electrophoretic light scattering (ELS) and entrapment efficiency measured with UV-spectrophotometer;

 Nanoparticles produced have to be characterized by the determination of the

particles’ size; zeta potential, pH, degree of crystallinity and changes in the lipid polymorphism by means of differential scanning calorimetry (DSC), entrapment efficiency measured with UV-spectrophotometer and resveratrol release

studies through a dialysis technique and UV-spectrophotometer

measurements.

1.3. Challenges and progress beyond state-of-the-art

The beneficial use of resveratrol as an additive to increase nutritional value is limited by intrinsic features that lead to low bioavailability and formulation challenges; the most serious being low water-solubility and instability. Frequently, these formulation challenges are difficult to overcome and are responsible for the products being abandoned early on the development process; or the product is lunched with suboptimal properties including poor bioavailability, lack of optimal dosage, presence of extra excipients that pose limitations with respect to dose and ultimately, poor consumer compliance [5]. So, the creation of drug delivery nanotechnology seems to be a promising alternative to overcome these barriers. But the delivery of lipophilic compounds is extremely demanding being one of the sources of frustration in the pharmaceutical sciences. In this context, the methodology adopted for this

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dissertation is very challenging since it will try to succeed in one of the most elusive objectives in the pharmaceutical area which is the nanodelivery of a lipophilic drug. Within the timeframe available for the development of this work, the main concern was to focus on a successful encapsulation of resveratrol into nanoparticles and the careful characterization of these nanoparticles regarding their morphology, size and chemical aspects, such as charge, pH and lipid polymorphism. The stability of the nanocarriers systems developed was also a major concern, and it was assured a periodic control of the formulations in a 2 months period. The developed system was thought to be orally administrated, and in this regard, optimization of the nanoparticles’ synthesis was set to produce small mean diameters of the LN and to use surfactants in the formulations favoring drug solubilization and this way it is expected to further enhance the oral absorption of resveratrol [6]. Besides the improvement of resveratrol absorption and protection against gastric degradation, it was intended that the LN developed had also the advantage of being composed by lipids that are biocompatible and biodegradable. In view to this, the monoester cetyl palmitate and the medium chain triglyceride Miglyol 812 were chosen to be part of the lipid matrix due to their excellent physical properties [7, 8]. Furthermore, both the lipids and surfactants, selected for this study, are generally recognized as safe (GRAS) excipients [7].

Although the research developed in the ambit of this work is aimed to reach the development of a nanoformulation for oral administration of resveratrol, the studies required to assure the viability of this route of administration are out of the scope of this work. Nevertheless the research described herein signifies a promising approach of a resveratrol nanocarrier system that can be further developed to conclude this aim of oral administration with improved bioavailability. Furthermore, this is to our knowledge one of the few attempts to encapsulate resveratrol in lipid particles [9-11], and the first attempt with the aim of developing a nutraceutical.

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1.4. Organization of the dissertation

The dissertation is presented according to the policies of the International Standard ISO 7144-1986 [12] and is constituted by:

1. Introduction, where basic necessary information provides a framework to the research topic, with an explanation why the subject was chosen for study, the description of the main and secondary goals of the dissertation, the innovative aspects of the studied research and the general organization of the dissertation;

2. State-of-the-art where a relevant literature review is made on the subject of this work and that includes: definition of resveratrol and its relation with nutraceuticals, some examples of patent food, drinks or nutraceuticals containing resveratrol,

3. Work Plan where it is justified the selection of the type of carrier system for resveratrol delivery; it is explained and justified the selected type of method of nanoparticle’s production and a brief description is given about the lipid nanoparticles used during the experimental work. It is also mentioned all the methodologies used in the course of the work, and the results and discussion of the results in the context of the literature review;

4. Conclusions and future research where it is summarized in some way the advantages and utility of the formulation developed. It will also be presented the main conclusions based on the results actually obtained and a comparison between the results obtained and the initial goals of the dissertation. Suggestions of possible future extension and modification of the developed work and personal appreciation of the work developed are also included in this part.

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2. State-of-the-art

2.1. Overview

The modern society has become more complex and since the beginning of the Industrial age, lifestyle has dramatically changed. Fueled by urbanization and the advent of global economy, the productivity has increased worldwide promoting longer work schedules and a demanding daily life with less time available to eat properly. Consequently, people were pushed into fast-food regimes which are easily consumed, but present less nutritional value. Furthermore, the changes in dietary habits are radical in many developed and developing nations and are replacing traditional eating patterns by a Western diet rich in animal products and refined carbohydrates and low in whole grains, fruits, and vegetables [13-15]. The decrease of the nutritional value of the diet together with other consequences of the industrialization, that include for example air and water pollution, have led to an increased incidence of numerous pathologies such as diabetes, obesity, cancer, vascular diseases, as well as, other degenerative diseases. Information from the Portuguese National Institute of Statistic shows that diseases affecting the circulatory system (32.4%) and tumors/neoplasms (23.0%) remain the major causes of death in 2008 and, among cardiovascular diseases, cerebrovascular disease remains the leading cause of death in Portugal. Hypertension problems, high levels of cholesterol, eating habits and sedentary lifestyles, are factors which are able to explain the percentages of death causes in Portugal, when compared with others EU countries [16].

Current knowledge and scientific advances have proved that nutritional value of the diet is directly correlated with better health and reduction of the risk of diseases. In this context, it has been suggested that only by changing nutritional habits and introducing some specific nutrients in the diet, it is possible to limit the risk factors for chronic diseases such as cardiovascular diseases, cancer, arteriosclerosis, hepatic diseases, and diabetes, among others [14, 17, 18].

Therefore, understanding the nutritional value of food and food additives has become an essential factor to control disease and increase life time expectation of the general population [14].

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6 2.2. Functional food and nutraceuticals

The term “Nutraceutical” was combined from “Nutrition” and “Pharmaceutical” in 1989 by Dr. Stephen DeFelice. Nutraceuticals are nutrients or part of a food (purified or concentrated food components) that provide health or medical benefits, including prevention and/or treatment. Such products can range from: isolated nutrients; dietary supplements in the form of capsules; vitamins and minerals (e.g. selenium); herbal products; processed foods such as cereals, soups and beverages; plants (e. g. garlic, ginger and Ginkgo biloba), and animal extracts (e.g. carnosine, carnitine and chitosan). No specific regulation exists in Europe to control nutraceuticals, although they are considered under the same laws that regulate medicine and drug. In the United States of America, the Food and Drug Administration (FDA) regulates dietary supplements under a different set of guidelines than those covering conventional foods and drug products [14, 15, 19-21]

The aim of nutraceuticals is significantly different from functional food, for several reasons. Prevention and treatment of diseases are relevant to the nutraceuticals’ action, while in the case of functional food only reducing the risk of disease (not prevention and treatment of disease) are involved. Moreover, nutraceuticals include dietary supplements and other forms of food whereas functional food must be a common type of food.

Nutraceuticals can be:

 Probiotics and prebiotics. Probiotics, are live microbial ingredients that can be

added as supplements in adequate amounts in the diet and that confer benefice to the health of the host, the bacterial genera, most often used as probiotics, are Lactobacillus (e. g. L. casei) and Bifidobacterium (e. g B. bifidum) [14, 22]. The only prebiotics for which sufficient data have been generated to allow an evaluation of their possible classification as functional food ingredients are the inulin-type fructans, which include native inulin,

enzymatically hydrolyzed inulin or oligofructose, and synthetic

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 Food containing sulfur and nitrogen compounds like broccolis and cauliflower

are used as protectors against carcinogenesis and mutagenesis, and activators of liver detoxification enzymes [14];

 Antioxidant vitamins like vitamin C, glutathione, uric acid, vitamin E and

carotenoids [14, 23], can prevent or reduced through their activity the lesions caused by free radicals in cells by directly involved in the neutralization of the action of free radicals or having an indirect influence over enzyme systems functioning [14, 19, 20];

 Phenolic compounds can be found in red wine, tea, fruit and vegetables like

soybeans [14, 24-26]. As part of the human diet, they have been associated with several health promoting activities such as: decreasing blood sugar levels, reducing body weight and acting as thrombotic, carcinogenic, anti-inflammatory and anti-aging (reduce the risk of age-related neurological disorders, such as dementia of the Alzheimer’s type AD, stroke and Parkinson disease). However, the major claimed activity of phenolic compounds has been their antioxidant effect. Their antioxidant activity is mainly due to their redox properties, which can play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides [14, 21, 25, 27];

 Polyunsaturated fatty acids which include the families of omega-3 fatty acids

that may help prevent or treat a variety of diseases, including heart disease, cancer, arthritis, depression and Alzheimer's disease among others and omega-6 fatty acids that play an important physiological role as part of the structure of cell membranes, influencing the blood viscosity, vessel permeability, anti-aggregation action, blood pressure, inflammatory response and platelet functions (this compounds are found in coldwater fish like salmon, tuna, sardines and cod, vegetable oils, flaxseed, walnuts and some types of vegetables) [14, 28];

 Fibers also known as oligosaccharides can be found in grains (rice, soybeans,

wheat, oats, beans, peas), vegetables (lettuce, broccoli, cabbage, cauliflower, cabbage, chayote, cucumber), and roots (carrot, radish) and lead to reduction in blood cholesterol levels and decreased risk of developing cancer, according

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to the ability to retain toxic substances ingested or produced in the gastrointestinal tract during the digestive process; reducing the transit time and promoting a rapid elimination of fecal material, which reduces the contact time of intestinal tissue with mutagenic and carcinogenic substances and formation of protective substances by bacterial fermentation of food compounds [14, 29].

2.3. Resveratrol as a nutraceutical

In 1940, resveratrol was first identified as the medicinal component of grapes, and it was extracted from the dried roots of Polygonum cuspidatum, popularly known as Ko-jo-kon in Japan, and used to treat hyperlipidemic diseases. However, it was only until 1997, that its applicability to human health was first proved, on cancer prevention. Since then, resveratrol received considerable attention by researchers. Nowadays, resveratrol demonstrates; a high popularity in the scientific literature (as on March 2008, a MEDLINE search revealed 2,009 hits for the term “resveratrol,” with 361 citations from 2007 alone); potential for use in Medicine field, and an emerging commercial availability [2, 3].

Chemically, resveratrol is a natural polyphenol nonflavonoid of the subclass hydroxylated stilbene and exists in two isomeric forms as trans- (3,4,5-trihydroxystilbene) and cis-resveratrol (cis-3,4,5-(3,4,5-trihydroxystilbene), with trans-resveratrol having the greater biological activity. The two isomeric forms of trans-resveratrol are shown in Figure 1 [1, 3, 30-32].

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Figure 1 – Isomeric forms of resveratrol. (a) – trans-Resveratrol; (b) – cis-Resveratrol. Source: [30]

Resveratrol is present in a variety of 72 plant species including: vegetables [25]; herbs; fruits [25] e.g. mulberries [2, 31], bilberry [2], lingonberry [2], sparkleberry [2], deerberry [2], partridgeberry [2], cranberry [2], blueberry [2], jackfruit [2], and peanuts

(present a maximum concentration of resveratrol of 1.92 μg/g) [1-3, 33]; grains; roots

like Polygonum Cuspidatum roots which have a resveratrol concentration of 0.524 mg/g [2, 3]; flowers and leaves including gnetum, white hellebore, corn lily, butterfly orchid tree, eucalyptus, spruce, poaceae, scots pine, and rheum; seeds and tea [25, 26, 31]. Although resveratrol is widespread in a great variety of plants; grapes [1, 3, 31, 33-36] and wine [1, 25, 26, 31, 33, 35-37] are the most important food sources of this nutraceutical [30].

Red wine is known for being the most common source of resveratrol, with concentrations of trans-resveratrol between 0.1 to 10 mg/L and cis-resveratrol between 0.1 to 3 mg/L. Whereas in white wines the concentration of trans- and cis-resveratrol is generally less than 0.1 mg/L, with a few exceptions having up to 2 mg/L. The concentration of resveratrol is dependent upon the type of grape used, the region of the world where it is produced, and others environmental factors which may vary from year to year [3, 30]. Even in grapes, resveratrol has a beneficial protective effect being increasingly produced in response to stress or to attack by nonpathogenic or avirulent bacteria, viruses or fungi [38]. This fact is also the reason why there is high variability of resveratrol found in grapes and ultimately in wine. Grape juice also contains resveratrol, with concentrations of 0.5 mg/L in red juices and 0.05 mg/L in

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white juices and the whole grapes themselves do not have high concentrations of

resveratrol, with a maximum of 3.54 μg/g (3.54x10-3 mg/g), being the grape skin

responsible for containing about 50 to 100 mg/resveratrol [2, 3].

Trans-resveratrol is found mainly in red wine and in red grape skin, demonstrating the

importance of grape and wine color to the concentration of this compound (the more intense wine or grape color, the bigger their polyphenol content), as well as the importance of the concentration of polyphenols within the grapes’ skin [3, 39].

Resveratrol has been described to exert different biological activities and possesses diverse biochemical and physiological reactions that lead to promotion of several benefits to human health, demonstrating a broad spectrum of pharmacological and therapeutic health effects [2, 3, 40].

The heath promoting effects reported by resveratrol are: antioxidant, neuroprotective, anti-inflammatory, cardioprotective and anti-platelet effects. Furthermore, resveratrol acts as a phytoestrogen and it also inhibits cancer.

It has been demonstrated that resveratrol acts as an antioxidant. This polyphenol is capable of scavenging some intracellular reactive oxygen species like hydroxyl radicals

and can also scavenge the superoxide anion (O2-). Resveratrol antioxidant properties,

probably arise from its ability to increase the synthesis of nitric oxide (NO) which behaves as a potent antioxidant when has an unpaired electron. Nitric oxide scavenges superoxide radicals and rapidly reacts near the diffusion-limited rate with O2. The

affinity of nitric oxide for O2, is far greater than the affinity of superoxide dismutase for

O2. It has also been demonstrated that trans-resveratrol suppresses lipid peroxidation

both by chelating copper and by scavenging of the free radicals and that in human lymphocytes, resveratrol increase the amounts of several antioxidant enzymes, including glutathione peroxidase, glutathione-S-transferase and glutathione reductase [2, 30]. Although, in vivo and at low concentration, resveratrol possesses antioxidant properties, yet at high concentration it can induce redox signaling and behave like a prooxidant. In vitro, this compound does not function as a strong antioxidant. [2, 26]. Resveratrol demonstrates a promising approach for the treatment of neurodegenerative disorders. Indeed, the antioxidant action of resveratrol can be somewhat related with its beneficial role in neurodegenerative diseases, since it has been documented that elevation in reactive oxygen and reactive nitrogen species can

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cause damage to the neural membrane by damaging proteins, nucleic acids, and membrane polyunsaturated fatty acids, causing lipid peroxidation and leading to loss of membrane integrity, reducing of mitochondrial membrane potential, and increasing

permeability to Ca2+in plasma membrane. These are the major factors associated with

the pathogenesis of a number of neurodegenerative diseases including Alzheimer’s disease (progressive age-dependent neurodegenerative disorder affecting the cortex and hippocampus and eventually leading to cognitive impairment), Parkinson’s disease (chronic and progressive neurodegenerative disease associated with impairment of motor function), alcohol-induced neurodegenerative disorder (alcohol can participate in the free radical reaction to form ethoxyl radical), stroke (associated with loss of brain functions due to the disturbance in blood supply to the brain and can be due to ischemia, lack of blood supply, or a hemorrhage), Huntington’s disease (neurodegenerative disorder that results from the neuronal damage in basal ganglia) and amyotrophic lateral sclerosis (progressive neurodegenerative disorder characterized by loss of upper and lower motor neurons). Resveratrol reveals an ability to exert a protective effect in the referred neurodegenerative diseases by the activation of sirtuin 1 and production of vitagenes [26]. Furthermore, resveratrol acts as neuroprotector by protecting the hippocampal neurons against toxicity induced by the free radical donor sodium nitroprusside and beta-amyloid (Aβ) (induces oxidative stress and apoptosis in cells) or islet amyloid polypeptide peptides which have severe implications in neurodegenerative diseases like Alzheimer’s, Parkinson’s disease or in affecting peripheral tissues as in the case of diabetes mellitus. These studies also suggested that mechanisms underlying the neuroprotective action of resveratrol are not solely attributable to its antioxidant properties, but involve its abilities to modulate intracellular effectors such as protein kinase C or to interact with Aβ peptides by inhibiting Aβ fibril formation and reducing the secreted and intracellular Aβ level and inhibit islet amyloid polypeptide aggregation also in the presence of aggregation-fostering negatively charged lipid interfaces (inhibition takes place during the very early stages of the fibrillogenesis and is efficient already at stoichiometric concentrations of resveratrol and inhibit islet amyloid) [25, 36, 37].

Besides the already mentioned therapeutic actions, resveratrol demonstrates anti-inflammatory role which can be compared to non-steroidal anti-anti-inflammatory drugs

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such as aspirin and indomethacin [30, 34]. Studies concluded that, in a dose-dependent manner, resveratrol has the ability to effectively suppress the aberrant expression of tissue factor by inhibiting the transcription of the gene tissue factor and cytokines in endothelium vascular cells. This nutraceutical also inhibited, in a dose-dependent manner, the arachidonate-dose-dependent synthesis of the inflammatory agents thromboxane B2, hydroxyheptadecatrienoate and 12-hydroxyeicosatetraenoate [2]. Resveratrol is also phytoestrogen, which is functionally and structurally similar to steroidal estrogens like diethylstilbestrol. At physiologic concentrations, resveratrol does not appear to induce any changes in uterine weight, uterine epithelial cell height, or serum cholesterol. Only at very high concentrations does resveratrol interfere with the serum cholesterol lowering activity of estradiol, because when combined with estradiol, resveratrol acts as superagonist to induce the expression of estrogen-regulated genes [40]. Elsewhere, resveratrol given orally or subcutaneously did not affect uterus weight at any concentration (0.03 to 120 mg/kg/day) in in vivo studies. This pholyphenol also enhanced endothelin-dependent vascular relaxation in response to acetylcholine and, in a manner similar to estradiol, prevented ovariectomy-induced decreases in femoral bone strength. Another recent study showed that both isomers of resveratrol possessed superestrogenic activity only at moderate concentration (>10 μM), whereas at lower concentrations (<1 μM), anti-estrogenic effects prevailed. Resveratrol also acts as an estrogen receptor agonist in breast cancer cells stably transfected with estrogen receptor, so the anti-estrogenic properties of this nutraceutical may be involved in the reduction of breast cancer risk. Although more data accumulate on the estrogenic behavior of resveratrol, the controversy about it continues to persist since several studies failed to confirm estrogenic potentiality of resveratrol [2, 31].

Resveratrol is thought to have diverse antiatherogenic activities (‘atherogenic’ implies the deposition of atheromas, lipids, and calcium in the arterial lumen), such as: the inhibition of LDL against copper-catalyzed oxidation; regulation of vascular smooth muscle proliferation; and modulation of nitric oxide production, as well as, suppression of platelet aggregation, which is induced by collagen, thrombin or ADP [2, 30, 34, 41-43].

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Besides all the abovementioned therapeutical effects of resveratrol the renewed interest in monitoring the presence and effectiveness of this compound was caused by the existence of the so-called “French paradox”. It has been found out and statistically confirmed that, in certain parts of France, the death rate caused by coronary artery diseases is lower despite relatively high fat consumption in the human diet. The consumption of wine is one of the dietary factors that might partially explain the low mortality caused by atherosclerosis. This fact led to an assumption that it is possible to enjoy wine, moderately consumed during the meals as a protector against the deleterious effects of high-fat-diets and therefore limit incidence and extent of coronary artery diseases. Previous research have shown that resveratrol exerts a marked inhibitory effect on collagen- and epinephrine-induced aggregation of human platelets and that it also inhibits ADP-induced platelet responses being this inhibition less than the nutraceutical effect on collagen-induced responses. Studies performed in hypercholesterolemic rabbits have shown that the ADP-induced platelet aggregation can be blocked by treatment with resveratrol, suggesting a role for resveratrol in interrupting the generation of eicosanoids involved in pathological processes [2, 38, 41].

Coronary heart disease, acute myocardial infarction and stroke are multifactorial diseases, whose etiology is related to: dysfunctional platelets-platelet adhesion;

aggregation triggered by ADP/TXA2; and platelet-derived growth factor release and

may be further attributed to the development of atherosclerosis, proliferation of smooth muscle cell, and acute thrombosis (formation or presence of a blood clot in a blood vessel). Thrombosis is known to play a very important role in the development, progression, and clinical squeal of atherosclerosis [2, 42, 44, 45]. Tissue factor is expressed by macrophages, smooth muscle cells, and endothelial cells in atherosclerotic arterial wall and is also the primary initiator of the coagulation cascade in both hemostasis and pathogenesis. In the atherosclerotic plaques there is accumulation of tissue factor, but little or no tissue factor is found in the intima or media of normal arteries. Hence, activation of the tissue factor-mediated coagulation pathway not only plays a major role in determining plaque thrombogenicity, but could also have other effects on the vessel wall (e.g. to promote vascular smooth cell proliferation and thus may play a role in the development of intima hyperplasia). In a

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dose-dependent manner, resveratrol suppresses the induction of tissue factor expression in both endothelial cells and monocytes. The resveratrol-mediated inhibition of tissue factor activity in endothelial cells is caused by inhibition of transcriptional activation of the tissue factor gene and suppression of tissue factor activity is associated with the lower accumulation of tissue factor mRNA) [2, 42]. It has been demonstrated that resveratrol significantly improved post-ischemic ventricular function and reduced myocardial infarct size compared to non-treated control group. The amount of pro-adhesive molecules, including soluble intracellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1 were each significantly decreased during reperfusion in the resveratrol-treated group. Nitro-L-arginine methyl ester, an nitric oxide blocker, completely support an anti-inflammatory action of resveratrol through a nitric oxide dependent mechanism [2].

Besides the applicability of resveratrol as a cardioprotective and anti-platelet agent, there are also studies that suggest that high-risk cardiovascular failure of aspirin resistant patients (in clinical practice inhibition of platelet activity with aspirin significantly reduces the odds of serious atherothrombotic vascular events and death in patients at high risk), will especially benefit from resveratrol consumption [41]. Resveratrol has also been described as having a cancer chemopreventive activity (prevention of cancer by the reduction of the risk of carcinogenesis), in assays representing three major stages of carcinogenesis (i.e. initiation, promotion, and progression). Indeed, resveratrol is an antiproliferative agent for cancer; induces apoptosis in tumor cells and sensitizes cancer cells by inhibiting cell survival signal transduction and anti-apoptotic pathways [2, 30, 31].

Most recent studies, have shown that resveratrol inhibit growth of several types of cancer such as: prostate and colon cancers, as well as, prevents pancreatic, gastric and thyroid cancer. Studies in animal models of human neuroblastoma show a very potent inhibitory effect of resveratrol on tumor growth in these animal models since after 5 weeks of treatment, the average tumor volume in the animal models were 70% to 80% less than that of the control groups. Resveratrol inhibited the outgrowth of tumors by as much as 80%. In oral squamous carcinoma cells, resveratrol inhibited growth, both alone and in combination with quercetin (antioxidant found in the skin of apples and

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red onions) and inhibited the growth of highly metastatic B16-BL6 melanoma cells. Moreover, the intra-peritoneal administration of resveratrol increased apoptosis and reduced tumor growth. Another investigation research demonstrated that resveratrol affected the three major stages of carcinogenesis and inhibited the formation of preneoplastic lesions in a mouse mammary organ culture model [1, 2, 46].

Though the molecular mechanism is not fully understood, several studies have demonstrated that the anti-tumor effect of resveratrol is via reactive oxygen species-dependent apoptosis pathway and that resveratrol efficiency makes it a novel and potential anti-cancer agent [1].

Although it is becoming increasingly clear that resveratrol has a dual behavior, having different reactions within the same health problem when the conditions or the nutraceutical concentrations are not the same; resveratrol likely fulfills the definition of a pharmaceutical preconditioning compound and gives hope to its applicability as a successful therapeutic drug in medicine [2]. Furthermore, besides all the potential benefits presented by resveratrol in therapeutics, the use of this bioactive compound can be extended to the nutraceutical notion, where the purpose is not healing a disease, but is about preventing a disease trough the association of bioactive effects and nutrients. However, nutraceuticals and functional foods present an economically robust opportunity on a global scale, manufactures and marketers of these products are confronted with the difficult task of developing finished goods that are simultaneously healthy and pleasant to eat, and that are able to create sustainable revenues and yield attractive investment returns [25].

2.4. Patented food, drinks or nutraceuticals containing resveratrol

Several eating and drinking patented food containing resveratrol are being commercialized. The red wine is a good example, since it is known for being a good source of this nutraceutical. As stated before, there are even several epidemiological studies that suggest that coronary heart disease mortality is lowered by moderate consumption of red wine due to the presence of this compound, as well as, other polyphenols [3, 42]. Table 1 presents some examples of well-known and

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commercialized wines and the respective concentration of trans-resveratrol by the year of production.

Table 1 – Wines produced in years between 1996 and 1999 and respective concentration of pure trans-Resveratrol in mg/L. Source: [32]

Wines

Concentration of pure trans

-3,4,5-trihydroxystilbene (mg/L) Year Merlot 5,10 1999 Cabernet Sauvignon 2,33 1998 Cabernet Franc 2,10 1997 Pinot Noir 4,21 1996 Gamay 1,64 1999 Pinotage 3,43 1997 Sangiovese 5,75 1993 Tannat 4,17 1997

Individuals who hope to benefit from resveratrol cannot simply eat grapes or most other resveratrol-containing products besides red wine. As a nutraceutical, resveratrol is commercially available in the United States and Europe at between 50 mg and 60 mg per dosage form. Therefore, a number of resveratrol supplements have been developed and commercialized. Resveratrol supplements are commercialized in the form of pills, liquids or powders. Some are nearly 100% pure resveratrol, while others may be as little as 10 % and can be sold has a combination with others components such as quercetin, pterostilbene, rice bran, grape seed, grape extract, and red wine extract. Therefore, resveratrol supplements are available in a wide range of styles and qualities. An example of a supplement containing resveratrol is NOW® Natural Resveratrol from Genuine Whole Food. This health supplement comes in a 473 mL volume bottle and contains a mixture of polyphenols, including natural resveratrol, proanthocyanins (grape seed) and catechins (green tea extract) (see Figure 2) [3, 47-49].

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Figure 2 - NOW®Natural Resveratrol from Genuine Whole Food. Source [47]

Besides health supplements, resveratrol is also commercialized within formulations and drugs. Some examples of these types of products which are already being commercialized and patented or that are being developed are the following:

- Resveratrol from Fluoxome® - Fluoxome is a company that develops products and commercializes naturally derived trans-resveratrol that is manufactured by fermentation using a yeast strain obtaining a pure product in the form of a white, crystalline powder with minimum 98% of trans-resveratrol that according to Independent Expert Panel can be safely used as a dietary ingredient in dietary supplements at intakes up to 450 mg/day [50];

- SRT501 - is a formulation of resveratrol with improved bioavailability developed by Sirtris Pharmaceuticals® that is at the present time, being tested in phase II trial in patients with multiple myeloma since the phase I human clinical trials was found to be safe and well tolerated (this formulation will not be available for 4 or 5 years pending FDA approval) [51];

- Solumer™ formulation of resveratrol - Solumer™ is a unique solubilization platform developed by SoluBest® based on the self-assembly of selected components, enabling the design and production of new polymer-drug constructs with well-defined physical-chemical properties. The Solumer™ formulation of resveratrol yields a composition including only the active form

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of the compound trans-resveratrol. Pharmacokinetic studies demonstrated a significantly higher bioavailability using Solumerised resveratrol, not only for the total resveratrol metabolites but also for intact resveratrol [52].

Nanotechnology may take an active part, to encapsulate and deliver the bioactive compounds, and to include them in healthier finished goods without compromising their taste. The field of food nanotechnology is experiencing significant growth due to the confluence of interests of industry, government and academia and is seen as a new and fast emerging field. The application of nanotechnology to the food sectors is relatively recent compared with their use in drug delivery and pharmaceuticals. Smart delivery of nutrients, food preservation using nanosensores and nanoencapsulation of nutraceuticals and delivery of bioactive components are some of the emerging topics of food nanotechnology (see Figure 3) [53-55].

Figure 3 - The impacts and needs of nanotechnology applications in foods and food processing. Source: [56]

The application of nanoparticles in electronics, medicine, textiles, defense, food, agriculture, cosmetics, and other areas is already a reality and the applications are beginning to impact food-associated industries. The field of nanoparticle delivery systems for nutraceuticals with poor water solubility has been expanding, almost exponentially, over the last years. In order to apply resveratrol as a nutraceutical, in

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drinks and food, it is of great interest and desire to use biodegradable nanocarriers. The nanocarriers will allow an oral administration of this nutraceutical, protecting it from metabolization and degradation. This approach will also permit the transport and release of the pholyphenol at a specific and desired location and allow superior ability to penetrate cell membranes, increasing this way the cellular uptake of this compound. This strategy also aims to overcome some of the undesired characteristic of resveratrol like its low bioavailability, hydrofobicity and instability. It has been proposed that other factors such as the increase in the rate of release (due to the large surface area), the increase in the retention time due to the small size of the nanoparticles (entrapment in the mucous layer), or the direct uptake of the particle (as suggested before) are important elements, that explain the improved absorption of the nanoparticle systems. Therefore the application of nanoparticles might result in a higher consumer exposure to resveratrol [1, 9, 54, 55].

The earliest reported nanoformulation of resveratrol comes from a study by Yao and his team, where they prepared resveratrol chitosan nanoparticles with free amine groups on the surface to conjugate ligands, which actively target special tissues or organs. Another liposomal nano based approach was done by Wang and coworkers, where they showed that resveratrol release from nanoliposomes in vitro fitted the log-normal distribution equation and had a property of sustained release. These studies allow concluding that resveratrol-nanoliposomes, could as well have an in vivo sustained release of resveratrol. Interestingly, the absorption was a first-order process with the passive diffusion mechanism and also supported the notion that resveratrol-nanoliposomes could promote the absorption of resveratrol in rat small intestine. Amphiphilic block copolymer-based polymeric micelles receive most attention because they can self-assemble into nanoparticles with hydrophilic outer shells and hydrophobic inner cores, which capture hydrophobic drug in the core and disperse easily in solution with the protection of hydrophilic shells. These drug loaded polymeric micelles are not bigger than 100 nm, which are easy to be internalized by cells. By incorporating in these nanoparticles, drugs and nutraceutical are prevented from being quickly degraded and enabled to sustained release at the expected site [36]. Shao and his co-workers developed an interesting clinical study, were resveratrol was incorporated into mPEG-PCL based nanoparticles. The polymeric nanoparticles

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obtained presented high encapsulation efficiency compared to equivalent dose of free resveratrol.

Recently, studies with resveratrol-loaded polymeric micelles also revealed that the polymeric micelles were able to protect from Aβ-induced reactive oxygen species generation [41].

2.5. Nanodelivery system for resveratrol

To design effective nanodelivery systems for resveratrol, it is necessary to understand the biological processes that regulate uptake and bioavailability [54].

The orally administered food or pharmaceutical formulations containing resveratrol primarily suffer digestion in the oral cavity mainly by mastigation. Then the food and/or resveratrol go through a dissolution process in the stomach at acidic conditions were the pH is between 1 to 2, during a period of time that ranges from 1 to 3 h. Various enzymes (pepsin and others) are released in the stomach to help breaking down some of the proteins and carbohydrates. As the digested food (now in the form of a suspension) and/or resveratrol leave the stomach and enter the duodenum, they mix with the bile salts (such as sodium glycocholate, sodium taurocholate, and lecithin) released by the gall bladder. These bile salts are able to emulsify the fats and hydrophobic compounds such as resveratrol present in the suspension. In addition to the release of bile salts, a bicarbonate solution containing a cocktail of enzymes (trypsin among others) is also released in the duodenum. The pH of the intestine content during digestion in the duodenum is around 6 and increases slowly in distal direction to values up to 8. The pH in the duodenum and jejunum is very constant during the digestion period. The suspension then enters the largest part of the small intestine, where it resides for about 3 to 5 h before entering the large intestine [54, 57].

The inner surface of the small intestine is covered with small “finger-like” protuberances called villi. Each epithelial cell is covered with even smaller protuberances called microvilli that help to increase the surface area for nutrient absorption. A mucous layer of an anionic glycoprotein, mucin, typically covers the surface of the microvili and represents a key factor in the absorption of compounds

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through the intestinal walls [54]. Figure 4 shows a schematic representation of the route taken by nutrients in the gastrointestinal track, the enzymes secreted during that path and the nutrients uptake. This will allow a better visualization of the process suffered by the resveratrol along the gastrointestinal track since its ingestion and until its uptake by inner surface cell of the small intestine.

Figure 4 – Enzymes secreted and nutrients absorption along the gastrointestinal tract. Source: [58]

In the intestine and in the liver, resveratrol is rapidly metabolized by glucuronidation and sulfonation and excreted by the kidneys. This extensive and rapid metabolization makes the antioxidant effect of resveratrol to be considerably diluted. The distribution of resveratrol to the tissues by plasma proteins is majorly done by albumin and studies demonstrated that intra-peritoneal administration of this compound leads to its

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transport by the circulatory system and that resveratrol has the ability to pass through the blood–brain barrier and reach the brain tissue rapidly [3, 26, 36].

The extent of uptake of a compound is a function of the difference in its activity across the epithelial tissue and the activity of a component is calculated as the product of its concentration times its activity coefficient. Due to its high hydrophobicity, resveratrol has a large activity coefficient, thus inducing a large driving force for the nutraceutical to permeate across the epithelial cells of the intestine. However, resveratrol concentration in plasma is very sparse (only several micromoles after addition) and presents a short half-life time because of its poor water-solubility (less than 0.03 g/L) and instability. Therefore, although presenting good membrane permeation, the low bioavailability of resveratrol is thus reflected by its clearance, apparent volume of distribution, and urinary excretion and also by its not so elevated maximum plasma concentrations reached within an hour following oral administration. Similar indications of a low bioavailability of this compound have been presented in studies evidencing that only 16% to 24.6% of the resveratrol dose consumed were recovered in the form of the intact compound and its conjugates in urine within 24 hours. High concentrations of resveratrol are often used in in vitro studies, but several studies demonstrated that this compound shows a cytotoxic effect in a dose and time-dependent manner. Although it is difficult to estimate the exact concentrations in tissues, beneficial effects have been verified at a variety of plasma concentrations ranging from 100 nM to 1 μM. There are also evidences that small amounts of resveratrol may accumulate over long periods of time until it reaches sufficient concentrations for its beneficial effects [3, 36, 39, 49].

Although many different delivery systems are now available to deliver bioactive components in nutraceuticals and functional foods, clear in vitro or in vivo evidences of their biological efficacies are still limited [53]. Drug carriers were investigated for many years, including microemulsions, nanoemulsions, oil-in-water emulsions, liposomes, micro and nanoparticles based on synthetic polymers or natural macromolecules and lipid nanoparticles composed of pharmacological acceptable excipients have all been examined and tested. Since resveratrol, besides its nutraceutical properties, can also be considered as a bioactive compound, the potentialities evaluated for the mentioned systems as drug carriers can also be applied in the transport of this pholyphenol [9, 59,

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60]. Hence, among the various nanodelivery systems available and described in the literature it is necessary to evaluate which nanosystem would be most adequate for the delivery of resveratrol.

Despite the fact that the emulsion is a very interesting delivery system there are reasons for not applying it as drug carrier. One of the reasons is the physical instability which can be caused by the incorporated drug. In addition, the registered oils such as soybean oil, MCT (oil-derived medium chain triglycerides) and LCT (oil-derived long chain triglycerides) and mixtures show an insufficient solubility for drugs of possible interest to be incorporated into emulsions. There is necessity to search for new oils with improved solubility properties which would of course also require an expensive toxicity study, therefore the number of products on the market containing this type of delivery system are relatively low, being an indication of their limited success [60]. The same reasoning can be applied to liposomes, which display many advantages as nanocarriers, but are not considered ideal as resveratrol carrier systems since besides some technological problems they are not an economic valuable alternative [59-62]. Furthermore, besides the advantages shown by the polymeric particles, their commercialization on the market is also limited. There are quite a few well-known reasons for this, of which three should be highlighted: the cytotoxicity of polymers (in nanometer and micrometers size range, the polymer can be internalized by cells like macrophages and the degradation inside the cell can lead to cytotoxic effects), the lack of a suitable large scale production method (being cost effective and leading at the same time to a product having a quality that should be acceptable by the regulatory authorities) and residues from the organic solvents used in the production method. Since, the commercialization of resveratrol loaded nanoparticles (for further addition to food and drinks) requires a production that can potentially be scaled up; thus polymeric micro and nanoparticles do not seem suitable for this application [1, 33, 36, 59, 60, 63]

LN being colloidal drug carriers, combine the advantages of polymeric nanoparticles and liposomes while simultaneously, avoiding some of their disadvantages such as acute and chronic toxicity (fact that the lipid matrix of the solid lipid nanoparticles is made from physiologically tolerated lipid components decreases the potential acute and chronic toxicity) or low stability [63, 64]. Therefore, these nanoparticles will be