1--TESE-COMPLETA-Davi-Vieira

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CHARACTERIZATION OF BETANIN FROM RED BEET (BETA VULGARIS L.), 
BIOACCESSIBILITY AND BIOACTIVITY EVALUATIONS, USE AS A MEAT 
PRESERVATIVE AND INTAKE EFFECTS ON OXIDATIVE STRESS IN A RODENT 
MODEL 
 
 
 
Davi Vieira Teixeira da Silva 
 
 
 
 
Rio de Janeiro 
2019 
 
UNIVERSIDADE FEDERAL DO RIO DE JANEIRO 
INSTITUTO DE QUÍMICA 
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIA DE ALIMENTOS 
 
DAVI VIEIRA TEIXEIRA DA SILVA 
 
CHARACTERIZATION OF BETANIN FROM RED BEET (BETA VULGARIS L.), 
BIOACCESSIBILITY AND BIOACTIVITY EVALUATIONS, USE AS A MEAT 
PRESERVATIVE AND INTAKE EFFECTS ON OXIDATIVE STRESS IN A RODENT 
MODEL 
 
 
 
 
 
 
 
 
 
Orientador: Profa. Dra. Vânia Margaret Flosi Paschoalin 
Coorientador: Prof. Dr. Eduardo Mere Del Aguila 
 
 
Rio de Janeiro 
 
Tese de Doutorado apresentada ao 
Programa de Pós-graduação em 
Ciência de Alimentos, Instituto de 
Química, Universidade Federal do 
Rio de Janeiro, como requisito parcial 
à obtenção do título de Doutor em 
Ciência de Alimentos. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AGRADECIMENTOS 
 
Aos órgãos de fomento, CAPES, CNPq e FAPERJ, pelo apoio financeiro concedido a 
mim e ao presente projeto. 
Ao Programa de Pós-Graduação em Ciência dos Alimentos pela oportunidade que me 
foi dada e pelo conteúdo transmitido para que eu alcançasse o grau de doutor. 
A Profa. Dra. Vânia Paschoalin e ao Prof. Dr. Eduardo Mere Del Aguila pelo apoio 
incansável nas correções da tese e pela experiência acadêmica que foi de grande importância 
para que realizasse um bom trabalho e concluísse esta etapa. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
DAVI VIEIRA TEIXEIRA DA SILVA 
 
CHARACTERIZATION OF BETANIN FROM RED BEET (BETA VULGARIS L.), 
BIOACCESSIBILITY AND BIOACTIVITY EVALUATIONS, USE AS A MEAT 
PRESERVATIVE AND INTAKE EFFECTS ON OXIDATIVE STRESS IN A RODENT 
MODEL 
 
Tese de Doutorado apresentada ao Programa de Pós-graduação em Ciência de Alimentos, 
Instituto de Química, Universidade Federal do Rio de Janeiro, como requisito parcial à 
obtenção do título de Doutor em Ciência de Alimentos. 
 
Aprovada por: 
 
________________________________________________ 
Profa. Dra. Vânia Margaret Flosi Paschoalin – IQ/UFRJ 
 
_______________________________________________________ 
Prof. Dr. Carlos Adam Conte Júnior – IQ/UFRJ 
 
_______________________________________________________ 
Profa. Dra. Eveline Lopes Almeida – EQ/UFRJ 
 
_______________________________________________________ 
Prof. Dr. Sérgio Borges Mano – UFF 
 
________________________________________________________ 
Prof. Dr. Vitor Francisco Ferreira – UFF 
 
 
ABSTRACT 
Silva, Davi Vieira Teixeira. Characterization of betanin from red beet (Beta Vulgaris L.), 
bioaccessibility and bioactivity evaluations, use as a meat preservative and intake effects on 
oxidative stress in a rodent model. Rio de Janeiro, 2019. Tese (Doutorado em Ciência de 
Alimentos). Instituto de Química, Universidade Federal do Rio de Janeiro. 
 
Betanin is a heterocyclic and water-soluble nitrogen natural pigment belonging to the class 
of betalains, approved for use in food and pharmaceutical products as a natural red colorant. 
Herein, betanin was purified by semi-preparative HPLC and identified by LC-ESI(+)-
MS/MS as the pseudomolecular ion m/z 551.16. Betanin displayed significant stability up to 
-30 ºC and mild stability at chilling temperature. The stability and antioxidant capacity of 
this compound were assessed during a human digestion simulation and ex vivo colon 
fermentation. Half of the betanin amount was recovered in the small intestine digestive fluid 
and no traces were found after colon fermentation. The high antioxidant ability of betanin 
was retained even after a simulated small intestine digestion. Betanin, besides displaying an 
inherent colorant capacity, was equally effective as a natural antioxidant presenting peroxy-
radical scavenger ability in pork meat. Betanin should be considered a multi-functional 
molecule able to confer an attractive color to frozen or refrigerated foods, but with the 
capacity to avoid lipid oxidation, thereby preserving food quality. The short-term intake of 
betanin was assessed in a rodent model to test its effects against oxidative stress, a common 
condition described in several risk factors for CVD, such as hyperlipidemia, diabetes, 
hypertension and metabolic syndrome. The consumption of this phytochemical may 
promote beneficial effects in the cellular redox status of living organisms. Oxidative stress 
was induced in Winstar rats by a hyperlipidic diet (60% fat) for 60 days, followed by intra-
gastric administration of betanin (20 mg·kg-1 b.w) for 20 days. The hyperlipidic diet caused 
hyperglycemia, hyperinsulinemia, insulin resistance, increased serum alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST) levels, reduction of 
antioxidant enzymes, increased lipid peroxidation and histopathological liver alterations. 
Betanin administration regulated glucose, insulin and HOMA-IR levels. Hepatic damage 
was reversed as evidenced by the reduction of ALT and AST levels and confirmed by 
histological analyses. Betanin also reduced hepatic malondialdehyde (MDA) and increased 
the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase 
(CAT) enzymes. Betanin intake for a short period reversed hepatic tissue damage, 
modulated biochemical parameters and attenuated oxidative stress in the rodent model. 
Keywords: betanin; semi-preparative RP-HPLC; in vitro human gastrointestinal digestion; 
ex vivo colon fermentation; bioaccessibility; antioxidant activity; malondialdehyde, 
oxidative stress, hepatic damage reversal. 
 
 
RESUMO 
Silva, Davi Vieira Teixeira. Caracterização da betanina da beterraba vermelha (Beta 
Vulgaris L.), avaliação da sua bioacessibilidade, bioatividade, uso como conservante de 
carne e efeito da ingestão sobre o estresse oxidativo em ratos. Rio de Janeiro, 2019. Tese 
(Doutorado em Ciência de Alimentos). Instituto de Química, Universidade Federal do Rio 
de Janeiro. 
 
Betanina é um pigmento heterocíclico e hidrossolúvel pertencente a classe das betalaínas, e 
o único composto desta classe aprovado para uso, como corante natural, em produtos 
alimenticios e farmacêuticos. Neste estudo, a betanina foi purificada por HPLC semi-
preparativo e identificada por LC-ESI(+)-MS/MS como o íon m/z 551,16. A betanina 
apresentou alta estabilidade a -30º C e estabilidade moderada a 4º C. A estabilidade química 
da molécula e capacidade antioxidante deste composto foram avaliadas durante uma 
simulação de digestão gastrointestinal humana seguida de fermentação colônica ex vivo. 
Metade da concentração de betanina foi recuperada no fluido digestivo do intestino delgado 
e nenhum traço foi encontrado após a fermentação no cólon. A alta capacidade antioxidante 
da betanina foi mantida mesmo após a digestão simulada no intestino delgado. A betanina, 
além de exibir uma capacidade inerente de corante, foi igualmente eficaz como um 
antioxidante natural, que apresenta capacidade de remoção de espécies oxidantes em carne 
de porco moída. Baseado nestes dados, a betanina deve ser considerada uma molécula 
multifuncional capaz de conferir coloração atrativa aos alimentos congelados ou 
refrigerados, e com capacidade de evitar a oxidação lipídica, preservando a qualidade dos 
alimentos. Em ratos, a suplementação de betanina por período curto, foi estudada com a 
finalidaade de testar a proteção desse composto contra o estresse oxidativo, uma condição 
comum descrita em várias patologias que representam fatores de risco para DCV, como 
hiperlipidemia, diabetes, hipertensão e síndrome metabólica. O consumo de betanina 
promoveu efeitos benéficos no status redox celular dos animais testados. O estresse 
oxidativo foi induzido em ratos Winstar pela administração de uma dieta hiperlipídica(60% 
de gordura) por 60 dias, seguida de ingestão intra-gástrica de betanina (20 mg·kg-1 b.w) por 
20 dias. A dieta hiperlipídica causou hiperglicemia, hiperinsulinemia, resistência à insulina, 
elevação dos níveis séricos das enzimas alanina aminotransferase (ALT) e aspartato 
aminotransferase (AST), redução da atividade de enzimas antioxidantes, como superóxido 
dismutase (SOD), glutationa peroxidase (GPx) e catalase (CAT), aumento da peroxidação 
lipídica e alterações histopatológicas do fígado. A administração de betanina regulou os 
níveis de glicose, insulina e HOMA-IR. O dano hepático foi revertido como evidenciado 
pela redução dos níveis de ALT e AST e confirmado pela análise histológica. A betanina 
também reduziu os níveis de malondialdeído (MDA) hepático e aumentou a atividade das 
enzimas SOD, GPx e CAT. A ingestão de betanina por curto período reverteu o dano 
tecidual hepático, modulou os parâmetros bioquímicos e atenuou o estresse oxidativo nos 
animais. 
 
Palavras chave: betanina; RP-HPLC semi-preparativo; digestão gastrointestinal humana in 
vitro; fermentação colonica ex vivo; bioacessibilidade; atividade antioxidante; 
malondialdeído, estresse oxidativo, reversão do dano hepático. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
LIST OF FIGURES 
 
CHAPTER 1 
Figure 1. Chemical structure of betacyanins, betaxanthines and betalamic acid 
(Reproduced from Stintzing et al., 2004) .............................................................................. 23 
Figure 2. Betanin structure (Reproduced from Stintzing et al., 2004) ................................... 23 
Figure 3. Degradation pathways of betanin (Reproduced from Stintzing et al., 2004) ......... 25 
 
CHAPTER 2 
Simulated digestion scheme (as Supplementary data figure S5). .......................................... 45 
Figure 1. Betanin separation by high-performance liquid chromatography diode array 
detector (HPLC-DAD) monitored at 536 nm. (A) Betanin standard chromatographed in 
the analytical HPLC column, (B) fresh beetroot juice sample chromatographed in semi-
preparative HPLC, (C) betanin purified by semi-preparative HPLC and separated using an 
analytical HPLC column and (D) betanin evaluated after 275 days of freezing and 
chromatographed using an analytical HPLC column. Betanin (peak 1) and isobetanin 
(peak 1’). The betanin chemical structure from red beet was reproduced from Cai et al. 
[19]. ........................................................................................................................................ 47 
Figure 2. Identification of purified betanin by HPLC-ESI(+)-MS/MS. (A) betanin (m/z 
551 [M+H]+), (B) fragmentation of purified betanin m/z from the MS/MS of 551[M +H]+. 
................................................................................................................................................ 48 
Figure 3. Lipid oxidation in ground pork loin evaluated by the production of 
malondialdehyde (MDA) during 9 days of storage at 4°C. Control H2O-DD, BHA 
(buthylated hydroxyanisole), BHT (butylated hydroxytoluene), Betanin 2% (w/w). Data 
are expressed as the means ± SD of three independent determinations. Different letters 
indicate differences between days at a significance level of p < 0.01. The symbol *(p< 
0.05) indicates differences compared to day 0. The symbol **(p < 0.05) indicates 
differences compared to day 3 ……..................................................................................... 50 
Supplementary data 
Figure S1: Chemical structure of betanin (A) and betanidin (B) .......................................... 62 
Figure S2: Betanin chromatograms before and after each in vitro digestion phase assessed 
by RP-HPLC equipped with DAD detector (536 nm) run after each gastrointestinal 
phase....................................................................................................................................... 63 
 
Figure S3: Betanin total antioxidant potential (TAP). Hydroxyterephthalic acid (HTPA) 
chromatograms of generated in the Fenton reaction without any sample (A), after betanin 
addition (B), after oral digestion (C), after gastric digestion (D), after small intestine 
digestion (E). ......................................................................................................................... 
64 
Figure S4: Influence of pH on betanin chemical structure charge changes in an aqueous 
solution according to Frank et al. (2005). ............................................................................. 65 
Figure S5: Simulated digestion scheme. ............................................................................... 65 
 
 
CHAPTER 3 
Figure 1. Experimental design of the study .......................................................................... 73 
Figure 2. Antioxidant activity of GPx (A), CAT (B) and SOD (C) after 60 days to GPx 
(D), CAT (E) and SOD (F) after 80 days of high-fat chow. The symbol* in figures A, B 
and C indicate differences between CONT 60 and HF 60 at p < 0.05 significance level. 
Different letters in figures D, E and F indicate difference at p < 0.05 significance 
level........................................................................................................................................ 75 
Figure 3. Liver malondialdehyde (MDA) concentration of high-fat group after 60 days 
(A) and after 80 days (B). The symbol* in figure A indicates difference between CONT 
60 and HF 60 at p < 0.05 significance level. Different letters in figure B indicate 
difference at p < 0.05 significance level................................................................................ 78 
Figure 4. Histopatologycal liver alterations of rats fed with high-fat chow during 60 days 
(A – C). Figure A shows the thickening of connective tissue capsule in portal triad (a), 
proliferation of bile ducts (b) and dilation of branch of portal vein (c). Figure B shows the 
presence of inflammatory infiltrate of mononuclear cells (a) H&E,40x. Figure C indicates 
the presence of areas with hepatocyte necrosis (a) and micro and macrovesicular steatosis 
(b). Figure D represents the liver of animals fed with standard chow. Photographs 
recorded at 20× and 40× magnifications (Figure B) ............................................................. 79 
Figure 5. Histopatologycal liver alterations of rats fed with high-fat chow during 80 days 
(A) and those fed with high-fat chow plus betanin (B, C). Figure A shows macrovesicular 
degeneration (a), necrosis (b) and centrolobular veins congestion (c). Figure B represents 
normal histologycal architecture of betanin supplemented animals. Figure C indicate 
hepatocytes suggestive of a regenerative cellular process (a). Photographs rcorded at 20× 
magnifications (H&E staining) ............................................................................................ 79 
 
 
 
 
 
LISTS OF TABLES 
 
CHAPTER 2 
Table 1. Betanin concentrations during in vitro simulated gastrointestinal digestion. 
............................................................................................................................................ 51 
Table 2. Total antioxidant potential and antioxidant activity pre and post in vitro 
simulated gastrointestinal digestion. ................................................................................. 53 
 
CHAPTER 3 
Table 1. Ingredients and chow nutritional composition ................................................... 72 
Table 2. Plasma biochemical parameters after 60 days of distinct chows ....................... 77 
Table 3. Biochemical parameter analysis after 80 days of chow supplementation .......... 77 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
LIST OF ABREVIATIONS 
 
ABTS - 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) 
ALT - Alanine transaminaseAST - Aspartate transaminase 
BHA - Butyrate hydroxyanisole 
BHT - Butylatedhydroxytoluene 
CAT – Catalase 
CT – Total cholesterol 
CHO - Carbohydrate 
CVD - Cardiovascular disease 
DNA - Deoxyribonucleic acid 
EFSA - European Food Safety Authority 
FDA - Food and Drug Administration 
FRAP - Ferric reducing ability of plasma 
GPx - Glutatione Peroxidase 
Glu - Glucose 
HOMA-IR - Homeostatic model assessment- Insulin Resistence 
HPLC - High performance liquid chromatography 
HTPA - Hydroxyterephthalicacid 
LC-ESI-MS/MS - Liquid chromatography/electrospray ionization tandem mass 
spectrometry 
LIP - Lipid 
m/z - Mass/charge ratio 
MDA - Malondialdehyde 
NO - Nitric oxide 
NO2
- - Nitrite 
NO3
- - Nitrate 
eNOS - Nitric oxide synthase 
ORAC - Oxygen radical antioxidant capacity 
ROS - Reactive oxygen species 
RNS - Reactive nitrogen species 
SOD - Superoxide dismutase 
 
TAP - Total antioxidant potential 
TEAC - Trolox equivalent antioxidant capacity 
TG - Triglycerides 
UFF - Universidade Federal Fluminense 
UFRJ - Universidade Federal do Rio de Janeiro 
UNESA - Universidade Estácio de Sá 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SUMARY 
INTRODUCTION .......................................................................................................... 17 
OBJECTIVES ................................................................................................................ 20 
• General ................................................................................................................. 20 
• Specifics ............................................................................................................... 20 
• Hypotesis .............................................................................................................. 20 
• Justification .......................................................................................................... 21 
 
CHAPTER 1 – LITERATURE REVIEW .................................................................... 22 
Betalains ........................................................................................................................... 22 
• Structural origin, occurrence and use as Additive ................................................ 22 
• Degradation pathways of betacyanins .................................................................. 24 
• Bioavailability of betanin ..................................................................................... 26 
• Biologycal effects of betanin ............................................................................... 27 
Reactive oxygen and nitrogen species ............................................................................. 27 
Antioxidant defense system ............................................................................................. 31 
Relationship between cardiovascular risk factors, atherosclerosis and oxidative stress 
.......................................................................................................................................... 33 
CHAPTER 2 .................................................................................................................... 36 
Title page .......................................................................................................................... 36 
Abstract ............................................................................................................................ 37 
Introduction ...................................................................................................................... 38 
Material and methods ....................................................................................................... 39 
• Standards and reagentes ........................................................................................ 39 
• Betanin purification and Identification ................................................................. 40 
• Storage Stability .................................................................................................... 41 
• Betanin ability to inhibit lipid peroxidation in meat ............................................. 41 
• TAP determination ............................................................................................... 42 
• FRAP determination ............................................................................................. 42 
• TEAC determination ............................................................................................. 42 
• ORAC determination ............................................................................................. 43 
 
• Simulated in vitro human gastrointestinal digestion and ex vivo colon 
fermentation ……………………………………………………………………. 44 
• Statistical analyses ................................................................................................. 45 
Results and Discussion ……………..…………………………………………………… 46 
• Betanin purification and identification …..………..……………………………. 47 
• Storage Stability ………………………………………………………………… 48 
• Lipid Peroxidation Inhibition in Meat …………………………………..……… 49 
• Betanin chemical stability during in vitro simulated gastrointestinal digestion 
…………………………………………………………………………………. 51 
• Betanin antioxidante activity throughout simulated human gastrointestinal 
digestion ……………………………………………………….……………….. 53 
Conclusions ……………………………………………………….........………………. 55 
References ........................................................................................................................ 57 
 
CHAPTER 3 ................................................................................................................... 65 
Title page .......................................................................................................................... 66 
Abstract ............................................................................................................................ 67 
Introduction ...................................................................................................................... 68 
Material and methods ....................................................................................................... 70 
• Reagents ................................................................................................................ 70 
• Processing of chow ............................................................................................... 70 
• Animals ................................................................................................................ 71 
• Plasma analysis ..................................................................................................... 73 
• Biochemical analysis ................................................................................ 73 
• Antioxidant enzyme activity ..................................................................... 73 
• Tissue analysis ...................................................................................................... 74 
• Thiobarbituric acid reactive substances (TBARS) ................................... 74 
• Histopathological analysis .................................................................... 74 
• Statistical analysis .............................................................................................. 74 
Results ............................................................................................................................. 75 
• Biochemical analysis .......................................................................................... 75 
• Antioxidant enzyme activity ............................................................................... 76 
 
• Thiobarbituric acid reactive substances (TBARS) ..............................................77 
• Histopathological analysis ................................................................................... 78 
Discussion .......................................................................................................... 80 
Conclusion ....................................................................................................................... 83 
References ........................................................................................................................ 84 
GENERAL DISCUSSION ............................................................................................. 90 
CONCLUSION ................................................................................................................ 97 
REFERENCES ................................................................................................................ 98 
ATTACHMENT 1 
Certificate Ethics Committee on the Use of Animals (CEUA-UFF) .................................. 110 
ATTACHMENT 2 
List of publications arising from this thesis ...................................................................... 111 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
17 
 
INTRODUCTION 
Cardiovascular diseases (CVD) are the leading cause of death globally. It comprises a 
class of disorders, which involves blocking blood supply to cardiac muscle and brain due to 
the formation of an aggregate formed by inflammatory and immune cells, platelets, oxidized 
lipids and smooth muscle cells proliferated in response to vascular endothelial injury 
(WHO, 2017; Fearon et al., 2009). Disorders like peripheral arterial disease, coronary heart, 
and cerebrovascular disease are the major clinical manifestations of CVD (Celermajer et al., 
2012). 
Pathological conditions such as systemic hypertension, hyperlipidemia, diabetes and 
obesity are considered risk factors for CVD since they usually precede the onset of vascular 
disease when aggravated (Drummond et al., 2011). In addition, the risk factors for 
developing CVD are characterized by excessive production of reactive oxygen and nitrogen 
species (ROS, RNS) and reduced detoxification capacity by the antioxidant defense system 
leading to a pathophysiological condition called oxidative stress (Drummond et al., 2011; 
Lassegue & Griendling, 2004; Newsholme et al., 2016; Yang et al., 2008). 
ROS and RNS produced above physiological levels lead to oxidative damage to DNA 
molecules, cell membranes, lipids and proteins, and reduce the availability of nitric oxide 
(NO), being responsible for the pathogenesis of many chronic diseases including CVD 
(Zhang et al., 2015). 
However, epidemiological evidence and clinical trials have demonstrated the protective 
effect of vegetable consumption on oxidative stress and CVD, and this protection is exerted 
by bioactive phytochemicals that make up the nutrients of the matrix of these foods (Pollock 
et al., 2016; Kikuchi et al., 2015; Zhang et al., 2015). Bioactive compounds have different 
physiological defences mechanisms, many of these show antioxidant action due to their 
potential for oxi-reduction, while others have the capacity to compete for active enzymatic 
and receptor sites in various subcellular structures or may modulate the expression of genes 
encoding proteins involved in intracellular mechanisms of defence against oxidative damage 
processes of molecules and cellular structures (Baião et al., 2017a). 
Beetroot (Beta Vulgaris L.) is a member within the Chenopodiaceae family, which is 
considered a source of important bioactive compounds, such as nitrate (NO3
-), phenolic 
18 
 
compounds and mainly betanin (300-600 mg/kg), being the richestfood source of this 
compound (Baião et al., 2017a,b). 
Betanin (betanidin 5-O-β-D-glucoside) is a heterocyclic and water-soluble nitrogen 
compound that is the responsible for the red-violet color of beetroot (Silva et al; 2016). 
Betanin is used as a natural dye in foods, cosmetics and pharmaceuticals, and its use is 
regulated by the European Union (EFSA) and Food and Drug Administration (FDA) (Silva 
et al., 2019). 
Betanin has emerged as a bioactive compound capable of modulating the generation of 
ROS, inflammatory cytokines, gene expression and antioxidant enzyme activities, being 
therefore a potential compound against the pathophysiological changes caused by the 
oxidative stress that lead to CVD diseases (Zielińska-Przyjemska et al., 2012; Tan et al., 
2015; Dhananjayan et al., 2017; Sutariya & Saraf, 2017). 
 ROS and RNS can oxidize the LDL molecule, which in turn damages the endothelium 
and inhibits NO production via nitric oxide synthase. In addition, the superoxide anion can 
react directly with NO and reduce its availability by producing the peroxynitrite radical 
(Radi, 2018). The low production of NO increases the expression of ICAM-1, that regulates 
leukocyte and platelet adhesion (Xu et al., 2013). Previous studies have shown that betanin 
is capable of acting on different oxidative and inflammatory processes that leading to CVD. 
 Betanin was able to inhibit linoleate peroxidation and LDL oxidation in a model of 
lipid peroxidation induced by citocrome c and metmyoglobin/H2O2 (Kanner et al., 2001). At 
micromolar concentration, betanin inhibited expression of the cell-adhesion molecule 
ICAM-1 induced by TNF-α in endothelial cells (Gentile et al., 2004). Betanin was found to 
inhibit overpoduction of ROS and transforming growth factor-beta (TGF-β) expression, 
induced by high glucose in tubular epithelial cells (Sutariya et al., 2017). 
In addition to the deleterious effects of ROS on atherogenesis, TGF- β1 is known to control 
cell proliferation, cell migration, matrix synthesis, calcification and the immune response, 
all being major components of the atherosclerotic process (Toma et al., 2016). 
Recently, different beet derived formulations, including juice, chips, gel and ceral-bar, 
have been developed with the purpose of offering an attractive food, easy to administer due 
to the reduced volume, and concentrated in bioactive compounds, as a tool to promote 
19 
 
health (Baião et al., 2016b, Vasconcellos et al., 2016; da Silva et al., 2016; Baião et al., 
2017b). 
In the present study, betanin, the main bioactive compound in beet, used in the industry 
as a dye, was purified and evaluated as an antioxidant additive in foods. Moreover, its 
stability, considering the chemical structure and antioxidanty capacity, during 
gastrointestinal digestion were evaluated, in order to be used in the treatment of oxidative 
stress in animals and maybe, in the future, as a non-drug treatment in humans with risk 
factors for the development of CVD. 
For this purpose, this thesis was divided into three chapters: 
Chapter 1 was dedicated to brief review of the structural and biological characteristics 
of betalaines emphasizing betanin, bioavailability and degradation pathways. Main reactive 
species of oxygen and nitrogen with potential implications to human health was present and 
the characterization and mechanism of action of the antioxidant defense system was 
discussed. Relation between oxidative stress, the main risk factors for cardiovascular disease 
and atherosclerosis was also addressed. 
Chapter 2 was dedicated to the purification of betanin, evaluation of its stability during 
storage, ability to reduce lipid peroxidation in food in order to obtain data concerning to 
evaluate the use of betanin, as a candidate to natural antioxidant additive. Evaluation of its 
chemical structure stability and antioxidant activity during simulated gastrointestinal 
digestion in vitro was carried out to observe the behavior of betanin after gastrointestinal 
ingestion and estimate the possibility of absorption of absorption 
Chapter 3 was dedicated to the in vivo evaluation of the effect of betanin intake in a 
rodent model induced to oxidant imbalanceredox by a hyperlipid diet. The physiopathology 
of animals induced to oxidant stress was evaluated through biochemical parameters, effects 
on the antioxidant enzymes and morphological alterations in the hepatic tissue. 
 
 
 
20 
 
 
 
OBJECTIVES 
General 
The objective of the study was to extract betanin from beetroot juice, to evaluate the 
stability, bioaccessibility, bioactivity and its effect as a food preservative. Furthermore, the 
effect of purified betanin intake was tested in animals induced to oxidative stress by high-fat 
diet. 
 
Specifics 
Chapter 2: To extract betanin from beetroot juice, determine its chemical stability 
during storage in refrigeration and freezing, evaluate its capacity as an antioxidant additive 
on lipid peroxidation in meat and compare with synthetic antioxidants. The bioaccessibility 
and bioactivity of betanin during simulated gastrointestinal digestion in vitro was addressed. 
 
Chapter 3: To evaluate the effect of betanin intake by Winstar rats induced to oxidative 
stress with hyperlipidic diet on changes in the following biochemical and metabolic 
parameters in the blood, insulin, glucose, triglycerides, total cholesterol, and the activity of 
the several enzymes aspartate transaminase, alanine transaminase, glutathione peroxidase, 
catalase and superoxide dismutase were studied. Additionaly, malondialdehyde was 
determined in the hepatic tissue as an indicator of lipid peroxidation. All these evaluations 
were evaluated after the histological analysis of hepatic tissue altefrations promoted by 
high-fat-diet and by betanin intake. 
 
Hypothesis 
Betanin can be used as an antioxidant additive in substitution of synthetic antioxidants 
and its ingestion can reduce oxidative damage to organs and tissues and the manifestation of 
cardiovascular events through its antioxidant activity on free radicals and its ability to 
modulate antioxidant enzymes in animals with oxidative stress and risk factors for CVD. 
 
 
21 
 
 
 
 
Justification 
This study proposes the use of betanin as a preservative ingredient in foods, taking 
advantage of its bioactive properties in promoting health to be used in the formulation of 
new products to aid in the treatment of CVD. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
22 
 
 
 
 
CHAPTER 1 – REVISÃO DA LITERATURA 
Betalains 
Structural origin, occurrence and use as additive 
Betalaínas are nitrogenous and watersoluble pigments that occur in a wide variety of 
plant tissues, including leaves, stems, fruits, flowers, roots and seeds belonging to the order 
of Caryophyllales. Betalaínas are synthesized from tyrosine, an aromatic amino acid that is 
produced primarily in plants via the chiquimato pathway (Herrmann, 1995, Tzin & Galili, 
2010). Tyrosine is initially hydroxylated to form 3,4-dihydroxy-L-phenylalanine (L-DOPA) 
(Steglich and Strack, 1990). L-DOPA is then converted to betalamic acid [4- (2-
oxoethylidene) -1,2,3,4-tetrahydropyridine-2,6-dicarboxylic acid] in a two-step reaction 
initiated by the enzyme DOPA 4,5 -dioxygenase (Girod and Zryd, 1991, Christinet et al., 
2004). Alternatively, L-DOPA is oxidized and cyclized to cyclo-DOPA [cyclo-L- (3,4-
dihydroxyphenylalanine)], which spontaneously condenses with betalamic acid, the basic 
structure of all betalaisins (Schliemann et al. 1999). The condensation of betalamic acid 
with cyclo-DOPA or its glycosidic derivatives gives rise to the subclass of the betacyanins 
(red pigments) or to condense with amino acids or aminoderivative compounds giving the 
subclass betaxanthins (yellow pigments). The structural differences between betacyanins 
and betaxanthines reflect the light-absorbing capacity of these compounds (Gandía-Herrero 
et al., 2013; Stintzing & Carle, 2004; Cai et al., 2003; 2005). Betacyanins and betaxanthines 
exhibit maximum absorption in the range of 535 to 540 nm and 460 to 480 nm respectively 
(Figure 1). Due to the structural variety of betacyanins and betaxanthines, these pigments 
can be classified into several types, where approximately 50 betacyanins and 20 
betaxanthines have been described. The subclass of betaxanthines is represented mainly by 
the pigment vulgaxanthin present in yellow beet (Beta Vulgaris L.) and indicaxantin present 
in the cactus pear (Opuntia fícus indica (L.) Mill.). In the structure of the betacyanins, 
glycosylation and acylation reactions can occur in the hydroxyls located at the C-5 or C-6 
positions giving rise to several betacyanins of which betanin (betanidin 5-O-β-D-glucoside) 
23 
 
is the most common ( Figure 2), besides being the most studied and will be the focus of the 
present review (Azeredo, 2009; Cliford et al.; Slimen et al., 2016). 
 
 
Figure 1: Basic structure of the betacyanins (left), betaxanthines (right) and the common precursor 
betalamic acid (medium) (Stintzing et al., 2004). 
 
 
Figure 1: Betanin (betanidin 5-O-β-D-glucoside) structure (Stintzing et al., 2004). 
 
Red beet (Beta vulgaris L.) is the largest source of betanin (300-600 mg/kg) and has 
low concentrations of isobetanin, betanidin and betaxanthines (Kanner et al., 2001; Nemzer 
et al., 2011; al., 2012; Vulic et al., 2013). The betanin content of red beet may be affected by 
several factors, including the growing conditions (temperature during the growing season, 
soil fertility, soil moisture, etc.) and storage temperature (Kujala et al., 2000; 2002). 
In the food industry, betanin is extracted from beet and used as a food additive having 
applications in foods such as sorbets, dairy products and meats (i.e. sausage) as a natural 
food coloring. The use of betanin as a dye is regulated by the Food and Drug Administration 
(FDA) and European Union under E-number E162 (EFSA, 2015, FDA, 2009, Nemzer et al., 
2018). The use of betanin as a natural red-violet dye becomes more advantageous in the 
industry than the use of anthocyanins also used as dye in the industry, because betanin is 
soluble in water and its color is largely pH independent. Betanin is stable in the pH range 
between 3 and 7 and therefore more suitable for coloring acidic to neutral foods, replacing 
24 
 
anthocyanins, due to instability at pH values above 3 (Polturak & Aharoni, 2018). Betanin is 
also used as a dye in cosmetics and pharmaceuticals (Esatbeyoglu et al., 2015). 
In addition to its utility as a food coloring additive, betanin has recently been shown to 
be a possible natural food preservative as an alternative to synthetic antioxidants (i.e BHA 
and BHT) because of its ability to inhibit the oxidation of foods susceptible to lipid 
peroxidation (Raikos et al., et al., 2016, Sucu et al., 2018, Silva et al., 2019). 
 
Degradation pathways of betacyanins 
Betalaines exhibit different chemical loads depending on the pH of the medium they are 
in (Wyler, 1969) but present higher resistance to hydrolytic cleavage compared to 
anthocyanins. In addition, betacyanins maintain their stability over a wide pH ranging from 
3 to 7, but degrade at pH below 2 and pH above 9 (Jackman & Smith, 1996; Polturak & 
Aharoni, 2018). Comparing the stability between different betacyanins, the glycosylated 
structures are more stable than the aglycones (i.e., betanin> betanidin), probably due to the 
higher oxidation-reduction potentials (von Elbe & Attoe, 1985). 
During processing, reactions of isomerization, decarboxylation or cleavage of 
betacyanins may occur by the action of heat or acid (Figure 3). During the process of 
isomerization and decarboxylation there are no visible changes in the staining of the 
betacyanins, but the cleavage reaction is accompanied by total loss of color (Stintzing et al., 
2000). Betacyanins have the ability to degrade and regenerate continuously during storage 
(Han et al., 1998; Silva et al., 2019). In the case of betanin, regeneration consists of a partialresynthesis of betanin from its hydrolysis products, which involves condensation of the 
amine group of the Dopa-5-Oglycoside cycle with the aldehyde group of betalamic acid; 
betanin forms rapidly when both compounds are mixed in solution (Huang & von Elbe, 
1985). Therefore, allowing food sources of newly processed betacyanins to remain for some 
time at pH values of about 5 and temperatures below 10° C can compensate for the loss of 
yield due to the possibility of regeneration of betacyanins from degradation products ( 
Figure 3) (Huang & Von Elbe, 1985, 1987; Schwartz & von Elbe, 1983). Different 
endogenous enzymes may contribute to degradation of betacyanins during processing, 
including beta-glycosidases, polyphenol oxidases and peroxidases (Escribano et al., 2002; 
Azeredo et al., 2009). The degradation products resulting from the enzymatic action are 
25 
 
similar to those promoted by thermal, alkaline and acidic degradation (Escribano et al., 
2002; Stintzing & Carle, 2004). 
In addition to the enzymatic action, the presence of O2 increases the degradation of 
the pigment while low levels of O2 favor its partial recovery (Von Elbe et al., 1975; Huang 
& von Elbe, 1987). Another factor affecting the stability of betacyanins is exposure to light, 
of which their intensity (in the range of 2200 to 4400 lux) was shown to have an inverse 
relationship with pigment stability. Absorption of UV or visible light excites electrons from 
the pigment chromophore to a more energetic state, increasing its reactivity or decreasing 
activation energy, favoring the onset of chemical reactions (Jackman & Smith, 1996). 
In short, color loss during food processing of betacyanin sources can be minimized 
by respecting the temperature and pH stability ranges as well as minimizing exposure to 
light and oxygen (Delgado-Vargas et al., 2000; Stintzing et al., 2000) 
Figure 3: Betanin degradation pathways (Stintzing et al., 2004). 
 
 
26 
 
Bioavailability of betanin 
The small intestine is designated as the largest site of absorption of betanin (including 
other betalaines), but the oral bioavailability is considered low (Moreno et al., 2008; Frank 
et al., 2005). In animal model studies it was shown that 2.7% of betanin administered orally 
was excreted in the urine and feces (Krantz et al., 1980). 
As demonstrated by previousstudies, it is suggested that most of the ingested betanin is 
degraded in the gastrointestinal tract where the highest rate of degradation occur after gastric 
digestion, followed by intestinal and then colonic digestion (Krantz et al., 1980, Tesoriere et 
al. al., 2008, Silva et al., 2019). The exact mechanism of absorption, metabolism and 
excretion of betanin is not fully elucidated (Tesoriere et al., 2004; Watts et al., 1993; Netzel 
et al., 2005) and the betalain profile as well as the detection of glucuronides , sulfates or 
conjugates of methylated betalain in plasma and urine are still not well established (Frank et 
al., 2005; Sawicki et al., 2017). It is known that the hydrolysis of the glycosidic bond in the 
betanin structure is not a requirement for its absorption, since it is detected intact in the 
plasma unlike its betanidin aglycone (Tesoriere et al., 2004; Khan, 2016). According to 
these findings, the trans-epithelial transport of betanin was studied in Caco-2 cells, where 
the absorption of betanin through intestinal epithelial cells occurred without metabolic 
transformation (Tesoriere et al., 2013). 
 In humans, betanin can be detected in the plasma after 60 min of oral administration, 
reaching maximum plasma concentration in ~ 3 h and no longer detected in the plasma after 
12 h of ingestion (Tesoriere et al., 2004). Absorbed bethanin is estimated to be excreted 
primarily by the urine route, including reddish urine (beeeturia) in some individuals after 
oral administration, however renal excretion is low, less than 3% of the dose applied (Watts 
et al., 1993; Mitchell, 2001; Frank et al., 2005; Netzel et al., 2005). According to Kanner et al. 
(2001), betacyanins absorbed through the intestine into the blood system were identified in 
the urine 2-4 h and for 12 h after the ingestion of 300 ml of beet juice (120 mg of betanin), 
but only about 1% of the dose ingested was excreted as isobethanine (present in low 
concentrations in beet), suggesting the occurrence of isomerization of betanin due to the 
relatively high temperature of the human organism. 
 The low excretion of betalains has also been reported by Netzel et al. (2005), after 
ingesting 363 mg of betalains (500 ml of beet juice), only 0.28% of the administered dose, 
27 
 
identified as bethanin and isobetanin, were excreted in the urine with a half-life of 7.5 h 
after oral intake. 
Biologycal effects of betanin 
The biological effect of plant sources has been well documented in the literature and 
this property is mainly attributed to bethanin (Khan, 2016). 
Betanin has attracted attention because of its anti-inflammatory and hepatoprotective 
functions in human cells. Betanin is capable of modulating redox mediated signal 
transduction pathways involved in inflammation responses in endothelial cell culture, by 
inhibiting the intercellular cell adhesion molecule-1 (ICAM-1) and performs 
antiproliferative effects on tumor cells (Gentile et al., 2004; Kapadia et al., 2011). In healthy 
and tumorous hepatic cells, betanin induces the translocation of nuclear factor 2 related to 
factor 2 (Nrf2) / antioxidant response element (ARE), from the cytosol to the nucleus, which 
controls messenger RNA (mRNA) levels and GSTM, GSTT, GSTA (glutathione S-
transferases), NQO1 (NAD (P) H quinone dehydrogenase 1) and HO- (heme oxygenase-1) 
in cells, exerting hepatoprotective effects and anticarcinogenic agents (Krajka-Kuźniak et 
al., 2013). 
In vivo assays betanin has antidiabetic effects and attenuates the complications of 
hyperglycemia by modulating the activity of hepatic enzymes in the metabolism of 
carbohydrates and hepatic glycogen with a consequent reduction in insulin resistance, 
hyperglycemia, glycated hemoglobin and other advanced glycation end products (AGEs), 
attenuating renal fibrosis in diabetes-induced animals (Han et al., 2015, Dhananjayan et al., 
2017). Betanin also inhibited the expression of TGF-β and collagen type IV in rats induced 
by streptozotocin (Sutariya & Saraf, 2017). 
Regarding the ability to remove reactive species, the antioxidant power of betanin lies 
in the presence of cyclic amine in its structure, similar to ethoxyquin, a strong antioxidant, 
besides the presence of hydroxyl groups (-OH), which are good hydrogen donors (Kanner et 
al., 2001). 
The antioxidant activity of betanin was demonstrated in vitro, where betanin reduced 
the production of ROS in human polymorphonuclear neutrophils, inhibited lipid 
peroxidation induced by cytochrome c, metmioglobin / H2O2 and lipoxygenase in lipid 
28 
 
peroxidation models (Zielińska-Przyjemska et al. 2012; Kanner et al., 2001; Vidal et al., 
2014). 
The relationship between the strong antioxidant activity of betanin and its structure in 
different pH ranges (2 to 9) was previously evaluated by the TEAC assay, homolytic O-H 
phenolic bond dissociation energy (OH BDE), ionization potential (IP) and deprotonation 
energy (Gliszczyńska-Swigło et al., 2006). Through the TEAC assay it was observed that 
the antioxidant activity of betanin is pH dependent, being elevated above pH 4. Moreover, 
with the gradual increase in the deprotonation of the betanin molecule (mono, bi- and tri-
deprotonated / anionic) as a function of the increase in pH, the BDE and PI values decreased 
significantly increasing their H + and electron donation capacity. These findings are 
associated with the model proposed by Nilson (1970) and Frank et al. (2005), where betanin 
is structurally modified according to the pH of the medium. It has thefollowing forms: 
cationic form at pH <2, neutral at pH 2, mono anionic at pH between 2 and 3.5 (with C2-
COOH and C15-COOH groups deprotonated), bi-anionic from pH 3.5 to 7 (with C2 -
COOH, C15-COOH and C17-COOH groups deprotonated) and trianionic from pH 7 to 9.5 
(with all carboxyls including C6-OH hydroxyl groups deprotonated). Later, corroborating 
these data, purified betanin from fresh beet juice had high antioxidant activity in artificial 
duodenal alkaline fluid and low activity in the acidic pH of gastric fluid in the TAP, FRAP, 
TEAC and ORAC assays during in vitro human digestion conducted by Silva et al. (2019). 
Betanin also appears to inhibit oxidative stress by modulating the activity and 
expression of endogenous enzymes. In a study performed by Han et al. (2015) and Tan et al. 
(2015), betanin restored glutathione level (GSH), activity of GPx, SOD, CAT enzymes and 
reduced NF-κB. In addition, betanin modulated the expression of inducible nitric oxide 
synthase (iNOS) and cyclooxygenase (COX). 
Reactive oxygen and nitrogen species 
It is currently believed that the development of various chronic-degenerative and 
cardiovascular diseases are carried out by the oxidizing action of reactive oxygen species 
(ROS) and nitrogen (RN), which have been the reason for several research and therapeutic 
strategies carried out with the aim of neutralizing the damages caused by these molecules 
(Halliwell, 2001; 2002; Halliwell & Whiteman 2004). 
29 
 
Reactive oxygen species (ROS) and nitrogen (RN), traditionally called free radicals, are 
oxidizing molecules with one or more electrons mismatched in the last electron layer, 
among which we can highlight the superoxide anion (O2
•-), peroxide (H2O2), hydroxyl 
radical (OH•) and peroxynitrite (ONOO-), these being the main reactive species with 
potential implication on human health (Halliwell, 1987). The ROS generation is due to the 
univalent reduction of oxygen (O2) during the respiration process that occurs in the 
mitochondrial electron transport chain (Green et al., 2004; Cadenas & Davies, 2000). 
Reactive oxygen species generated at the physiological level regulate multiple cellular 
functions, including angiogenesis, apoptosis, vascular tone, host defense and genomic 
stability (Freed et al., 2013; Nita & Grzybowski, 2016). 
The oxygen (O2) consumed in respiration is metabolized in the mitochondria at the 
electron transport chain, and the rest is used by several oxidases and oxigenases enzymes. 
Almost all cells and tissues continuously convert a small proportion of molecular oxygen 
into ROS. In the terminal phase of the electron carrier chain, the cytochrome oxidase 
enzyme oxidizes four molecules of cytochrome c by abstracting one electron from each 
molecule by adding them to O2 to form water (O2 + 4e
- + 4H+ = 2H2O). About 2 to 5% of 
the O2 metabolized in this reaction is diverted to another metabolic pathway where it is 
reduced to the univalent form giving rise to the O2
•- radical (Halliwell & Gutteridge, 1999). 
In addition to the mitochondrial generation of O2
•-, NADPH oxidase, xanthine oxidase and 
decoupled eNOS enzymes (not bound to cofactor for NO synthesis) act in the production of 
this radical, mainly under the stimulation of risk factors for cardiovascular disease, such as 
in conditions of hypertension, hypercholesterolemia, diabetes and obesity. Other sources of 
ROS in the vasculature include lipoxygenases, cyclooxygenases, and cytochrome P450 
(Münzel et al., 2017). The O2
•- radical is subsequently dissociated to H2O2 in the reaction 
catalyzed by the enzyme superoxide dismutase (SOD). H2O2 has low reactivity but can cross 
membranes and in the presence of iron (Fe2+) results in the formation of highly reactive OH• 
radical in the so-called Fenton reaction (Schneider & Oliveira, 2004). The OH• radicals can 
also be generated by the Haber-Weiss reaction, where iron and copper metals catalyze the 
reaction between the O2
•- radical and H2O2, again generating the OH
• radical (Schneider & 
Oliveira, 2004; Karamac et al., 2009). The O2
•- radical generated in the respiratory process 
can also react with nitric oxide (NO) synthesized in the endothelial cells by the enzyme 
30 
 
nitric oxide synthase (eNOS) to form the radical ONOO- (NO- + O2 = ONOO
-), a highly 
reactive nitrogen specie (Green et al., 2004; Kirsch & Groot, 2002; Squadrito & Pryor, 
1998). 
Although ROS play important roles in cell physiology, excessive production of these 
molecules promotes disease, including CVD, through oxidative damage to macromolecules 
such as DNA, lipids and proteins, as well as the interruption of signaling pathways in the 
redox-dependent vasculature (Münzel et al., 2017, Welch et al., 2002, Radi et al., 2001). 
Hydroxyl radical is considered the most reactive and most harmful radical to biological 
systems. Its action on DNA results in single or double strand breaks, 
depuration/depirimidation, or chemical modification of bases or deoxyribose, allowing the 
occurrence of mutations and apoptosis (Chevion, 1988). In proteins, the OH• radical is 
capable of altering its structure by oxidation of sulfhydryl groups (-SH) with altered 
biological function, and may also cause oxidation of the side chain of amino acids leading to 
the conversion of some of them to carbonyl derivatives, loss of catalytic activity, and 
increased protein susceptibility to proteolytic degradation (Ferreira & Matsubar, 1997; 
Welch et al., 2002). 
It has been suggested that the OH• radical is the main initiator of lipid peroxidation by 
abstracting hydrogen atoms from lipid membranes, altering their permeability, resulting in 
loss of selectivity for entry and exit of nutrients and toxic substances into the cell, leading to 
damage and even cell death (Vasconcelos et al., 2007; Hensley et al., 2000; Lima & 
Abdalla, 2001). 
Hydrogen peroxide is not a free radical, because it does not present an electron unpaired 
in the last electronic layer, even though it is potentially deleterious to biological systems, 
because unlike the other ROS, H2O2 is stable in pH and physiological temperatures, besides 
being able to cross membranes and generate the radical OH• (Barbosa et al., 2010; Bienert et 
al., 2006; Halliwell & Whiteman, 2004). 
The ONOO- radical is a potent oxidant generated by the reaction between the O2
•- 
radical and nitric oxide (NO), which diffuses easily into the mitochondria (Green et al., 
2004). ONOO- has a reactivity comparable to the OH• radical and causes damage to several 
biological molecules including hydroxylation reactions, nitration of aromatic compounds, 
damage to sulfhydryl groups (-SH) of proteins and oxidation of LDL, which in turn 
31 
 
damages the vascular endothelium (Radi et al., 2001; Vasconcelos et al., 2007). The 
production of ONOO- also results in the reduction of NO bioavailability, which plays an 
important role in vasodilation and normal endothelial function. The low NO bioavailability 
is a key factor in the atherogenesis process. ONOO- is involved in the pathogenesis of a 
number of diseases such as neurodegenerative disorders, sepsis, acute and chronic 
inflammatory processes (Radi et al., 2001; Halliwell & Whiteman, 2004; Henzler & Steudle, 
2000). 
In short, the vasculature is O2
•- generated through NADPH oxidase, xanthine oxidase, 
mitochondria or uncoupled eNOS. The enzyme SOD converts O2
•- to H2O2 (O2
•- can also 
react with NO and generate ONOO-). Through the Fenton reaction, H2O2 can convert to OH
• 
radical, which due to its high reactivity along with the ONOO- radical causes oxidative 
damage to macromolecules. 
Antioxidant defense system 
The respiratory chain generates the formation of reactive species that mediate oxidation 
reactions and can result in tissue damage and cell death when generated above physiological 
levels. Antioxidants have the function of inhibiting or reducing theformation of reactive 
species and cascade oxidation reactions through hydrogen (H+) donation, stabilizing the 
reactive species, or by chelating transition metals such as Fe2 + and Cu+ that are involved in 
the generation of reactive species and in the catalysis of oxidation reactions (Brewer, 2011). 
The antioxidant system comprises the enzymatic and non-enzymatic systems. The non-
enzymatic system is composed by a variety of antioxidants, which may have both 
endogenous and exogenous (dietetic) origin. The endogenous non-enzymatic defense system 
includes compounds such as glutathione (GSH), uric acid, bilirubin, ceruloplasmin and 
coenzyme Q (Wu et al., 2004, Ferashtehnejad et al., 2012, Verma et al. 2005; James et al., 
2004). Exogenous antioxidants include ascorbic acid (vitamin C), α-tocopherol (vitamin E), 
β-carotene (vitamin A), phenolic compounds and betanin for example. These compounds 
have been reported to have potential for inhibiting oxidative processes (Brewer, 2011; 
Hillstrom et al., 2003; Ozsoy et al., 2009; Brown & Kelly; Baião et al., 2017). 
The enzymatic system includes glutathione peroxidase (GPx), superoxide dismutase 
(SOD) and catalase (CAT), which are antioxidant enzymes able to convert reactive 
32 
 
molecules (superoxide radical, hydrogen peroxide and hydroxyl radical) to forms with lower 
reactivity in the organism (Nema et al. al., 2009; Flora et al., 2009). 
The enzyme SOD is responsible for the catalysis of the dismutation reaction which 
consists of the conversion of the superoxide radical (O2
•-) into hydrogen peroxide (H2O2). 
SOD is present in microorganisms, plants and animals and to show biological activity, they 
have to be associated with enzymatic cofactors in their active site such as copper, zinc and 
manganese metals. SOD can be found in two forms: in the cytoplasm and nucleus, it occurs 
bound to copper and zinc (SOD-Cu/Zn), while in the mitochondria, SOD requires 
manganese (SOD-Mn) as the enzymatic cofactor (Huang et al. al., 2012; Shin et al., 2009; 
Noor et al., 2002). 
The glutathione peroxidases (GPx) are selenoproteins belonging to a family of 
phylogenetically related enzymes, which have in their active site selenium and exert their 
antioxidant function by reducing H2O2 to alcohol or water. Selenium present in the active 
site of GPx is derived from the diet and is bound to methionine in foods of plant origin 
(selenomethionine), while in foods of animal origin it is bound to cysteine (selenocysteine). 
GPx catalyzes the reduction of H2O2 or organic hydroperoxides to water or alcools 
respectively, using selenocysteine as a catalytic moiety and glutathione (GSH) as a reducing 
agent, ensuring a rapid reaction with hydroperoxide and a rapid reducibility by GSH 
(Brigelius-Flohé et al., 2013, Vasconcelos et al., 2007). 
The enzyme catalase (CAT) is a heme-enzyme where the heme group is at its active site 
and is present mainly in mammalian cell peroxisomes. Catalase plays an important role in 
protecting cells against oxidative damage by catalyzing the degradation reaction of two 
H2O2 molecules by converting them to two molecules of water and one of oxygen (2 H2O2 = 
2 H2O + O2). The catalysis of this reaction occurs in two stages: in the first stage the ferric 
heme (Fe3+) reduces a molecule of hydrogen peroxide to water and generates a covalent 
oxyferril specie (Fe4+=O) with a π-cation porphyrin radical (referred to as compound I). In 
the second step, compound I oxidizes the second peroxide molecule to a molecular oxygen 
releasing the ferril oxygen species as water (Alfonso-Prieto et al., 2009; Ścibior & Czeczot, 
2006; Putnam et al., 2000). 
 
 
33 
 
 
Relationship between cardiovascular risk factors, atherosclerosis and oxidative 
stress 
Atherosclerosis is an inflammatory state of the arterial wall strongly associated with 
oxidative stress (Evrard et al., 2016). ROS generated at the physiological level regulate 
multiple cellular and vascular functions, including smooth muscle cell growth, endothelial 
cells, proliferation and migration, angiogenesis, apoptosis, vascular tone, host defenses, and 
genomic stability (Freed et al., 2013; Nita & Grzybowski, 2016). 
Excessive levels of ROS, above the ability of regulation by the antioxidant system, 
cause a redox shift in favor of oxidants, a pathological condition defined as oxidative stress. 
Oxidative stress mediates vascular disease through direct and irreversible oxidative damage 
to macromolecules, as well as the interruption of redox-dependent signaling pathways in the 
vascular wall (Stocker et al., 2004; Münzel et al., 2017). 
The increase of ROS favors the gene expression of pro-inflammatory molecules, 
including vascular cell adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-1 
(L-1), monocyte-1 chemotactic protein and E- selectin, which in turn are transcribed by 
redox-activated transcription factors, such as the factor NF-kB, activator protein-1, HIF-1β, 
and early-response protein to growth-1. These genes are crucially involved in the early 
stages of atherogenesis in addition to leukocyte-endothelial interactions (Kunsch et al., 
1999). The same transcription factors also play a role in proliferative signaling associated 
with smooth muscle cell growth and vascular remodeling (Stocker et al., 2005). 
Oxidative stress was previously reported to directly promote the transition from 
endothelial cells to mesenchymal cells (End-MT) (Evrard et al., 2016). Although such a 
process is indispensable for the development of the cardiovascular system (Kovacic et al., 
2005), End-MT in adults is generally considered pathological in the context of chronic 
inflammatory diseases such as atherosclerosis and this transition was found to occur in 
response to high concentration of H2O2 (Evrard et al., 2016). 
Oxidative stress is also a critical component of hypertension, where the enzyme 
NADPH oxidase, activated via angiotensin II, represents the primary source of ROS. In this 
sense, the role of oxidative stress in hypertension is counteracted by the effects of lowering 
34 
 
blood pressure or pharmacological inhibition of NADPH oxidase (Landmesser et al., 2002 
& 2007; Li et al., 2006). 
In hyperglycemia, oxidative stress is promoted via the mitochondrial respiratory chain 
as the primary source of O2
•- (Giacco et al., 2010). The O2
•- mitochondrial mediates the 
protein kinase C stimulus and the generation of advanced glycation end products (AGEs), a 
complication of diabetes (Giacco et al., 2010). Protein kinase C and AGEs in turn activate 
the enzyme NADPH oxidase and inhibit eNOS. In addition, eNOS uncoupled via NADPH 
oxidase promotes more oxidative stress due to higher production of O2
•- (Hink et al., 2001). 
Another proposed mechanism is the possible effect of hyperglycemia on mitochondrial 
fragmentation, reducing the activity of the electron transport chain and, consequently, higher 
ROS generation (Yu et al., 2011). 
Insulin resistance, a characteristic condition in type 2 diabetes, may inhibit the activity 
of GTP cyclohydrolase (GTPCH) -1 and, therefore, the synthesis of BH4 (eNOS cofactor for 
NO synthesis). In addition, insulin resistance inhibits insulin-mediated activation of eNOS 
via the phosphoinositide 3 kinase (PI3K)/Akt pathway. Another characteristic of 
hyperglycemia involved in oxidative stress is the presence of increased levels of Ang II 
found in patients with diabetes, which may contribute to the activation of NADPH oxidase 
(Chalupsky et al., 2005). 
In hypercholesterolemia, NADPH oxidase and xanthine oxidase are the main sources of 
O2
•- (Guzik et al., 2006); while the eNOS decoupling is possibly secondary to the oxidative 
stress induced by these enzymes (Pritshard et al., 1995; Stepp et al., 2002), corroborating 
previous studies in which endothelial cells were treated with LDL cholesterol and in studies 
with human hypercholesterolemies(Stroeet al., 1997; Xia et al., 2010). 
LDL cholesterol, more precisely the oxidized cholesterol of low-density lipoprotein 
(oxLDL), promotes the generation of O2
•- and ONOO-, and thus regulate monocyte 
adhesion, vascular inflammatory gene expression, and redox-sensitive transcription factors 
such as NF-kB and activator protein-1. Moreover, cellular lipoxygenases have been 
proposed as a possible enzymatic source of LDL-ox cholesterol. As previously reported, 
elevated levels of lipoxygenases have been observed in atherosclerotic lesions of the human 
and animal aorta (Yla-Hertualla et al., 1991). In addition to the direct effects of LDL 
cholesterol on the production of vascular ROS, dyslipidemia has also been found to 
35 
 
potentiate the effects of Ang II (NADPH oxidase activator) through the positive regulation 
of the AT-1 receptor, a cellular membrane component protein that binds to angiotensin II 
and promotes various cellular reactions including vasoconstriction and ROS production 
(Nickenig et al., 1999). 
In aging, endothelial dysfunction and central arterial stiffness are considered the main 
factors related to oxidative stress (Camici et al., 2015). In addition, the endothelial 
dysfunction is mediated by reduced NO bioavailability, either by decreased synthesis or by 
increased degradation (Donato et al., 1989; van der Loo et al., 2000). However, data in the 
literature suggest greater decoupling of eNOS as a function of age (Yang et al., 2009), 
favoring oxidative stress by decreasing the availability of NO and increasing production of 
O2
•- and ONOO-. The imbalance in redox state promotes inflammation, which leads to 
increased expression of NADPH oxidase and the production of more O2
•- through mediators 
such as tumor necrosis factor alpha (TNF-α). In parallel, the advancement of age seems to 
be directly related to the activation of the renin-angiotensin-aldosterone system, and may 
further stimulate NADPH oxidases through angiotensin II (Dikalov et al., 2013). 
However, central arterial stiffness involves degradation of elastic fibers by proteases 
with elastinolytic activity, including matrix metalloproteinases. These proteases have been 
associated with the activation of ROS-induced cyclophilin A, which mediates matrix 
metalloproteinase-9, TNF-α, IL-6 and NF-kB and ERK factor expression (Dikalov et al., 
2013). 
 
 
 
 
 
 
 
 
 
 
 
36 
 
 
 
 
CHAPTER 2 
Manuscript published: Davi Vieira Teixeira da Silva, Diego dos Santos Baião, 
Fabrício de Oliveira Silva, Genilton Alves, Daniel Perrone, Eduardo Mere Del 
Aguila and Vania M. Flosi Paschoalin. Betanin, a Natural Food Additive: Stability, 
Bioavailability, Antioxidant and Preservative Ability Assessments Molecules 2019, 
24(3),458; https://doi.org/10.3390/molecules 24030458 
 
 
GRAPHICAL ABSTRACT 
 
 
 
 
 
 
 
https://doi.org/10.3390/molecules%2024030458
37 
 
 
 
Article 
Betanin, a Natural Food Additive: Stability, 
Bioavailability, Antioxidant and Preservative Ability 
Assessments 
Davi Vieira Teixeira da Silva, Diego dos Santos Baião, Fabrício de Oliveira Silva, Genilton Alves, Daniel 
Perrone, Eduardo Mere Del Aguila and Vânia M. Flosi Paschoalin* 
 
 Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos 149, 21941-909 Rio de 
Janeiro, Brazil. daviufrj@outlook.com (D.V.T.d.S.); diegobaiao20@hotmail.com (D.d.S.B.); silvafo@live.com (F.d.O.S.); 
geniltonalves@gmail.com (G.A.); perrone@iq.ufrj.br (D.P.); emda@iq.ufrj.br (E.M.D.A.) 
* Correspondence: paschv@iq.ufrj.br; Tel: +55-21-3938-7362; Fax: +55-21-3938-7266 Received: 11 January 2019; 
Accepted: date; Published: date 
Abstract: Betanin is the only betalain approved for use in food and pharmaceutical products as a natural 
red colorant. However, the antioxidant power and health-promoting properties of this pigment have been 
disregarded, perhaps due to the difficulty in obtaining a stable chemical compound, which impairs its 
absorption and metabolism evaluation. Herein, betanin was purified by semi-preparative HPLC-LC/MS and 
identified by LC-ESI(+)-MS/MS as the pseudomolecular ion m/z 551.16. Betanin showed significant stability 
up to -30°C and mild stability at chilling temperature. The stability and antioxidant ability of this compound 
were assessed during a human digestion simulation and ex vivo colon fermentation. Half of the betanin amount 
was recovered in the small intestine digestive fluid and no traces were found after colon fermentation. Betanin 
high antioxidant ability was retained even after simulated small intestine digestion. Betanin, besides displaying 
an inherent colorant capacity, was equally effective as a natural antioxidant displaying peroxy-radical 
scavenger ability in pork meat. Betanin should be considered a multi-functional molecule able to confer an 
attractive color to frozen or refrigerated foods but with the capacity to avoid lipid oxidation, preserving food 
quality. Long-term supplementation by beetroot, a rich source of betanin, should be stimulated to protect 
organisms against oxidative stress. 
Keywords: Beetroot, betalains, semi-preparative RP-HPLC, in vitro human gastrointestinal digestion, ex 
vivo colon fermentation, antioxidant ability, malonildialdehyde 
________________________________________________________________________________ 
1. Introduction 
Beetroot (Beta vulgaris L.) is a vegetable presenting significant scientific interest, 
mainly because it is a rich source of nitrate (NO3
-), a compound with beneficial 
cardiovascular health effects, through the endogen production of nitric oxide (NO) [1]. 
Moreover, beetroots are the main source of betalains, a heterocyclic compound and water-
soluble nitrogen pigment, which can be subdivided into two classes according to their 
chemical structure: betacyanins, such as betanin, prebetanin, isobetanin and neobetanin, 
responsible for red-violet coloring, and betaxanthins, responsible for orange-yellow 
38 
 
coloring, comprising vulgaxanthin I and II and indicaxanthin [2,3]. Betalains are present in 
the tuberous part of beetroots, conferring its red-purple coloration. 
Betanin (betanidin 5-O-β-D-glucoside) is the most abundant betacyanin and the only 
one approved for use as a natural colorant in food products, cosmetics and pharmaceuticals, 
under code EEC No. E 162 by the European Union and under Section 73.40 in Title 21 of 
the Code of Federal Regulations (CFR) stipulated by the Food and Drug Administration 
(FDA) in the United States [4-6] (Supplementary file - Figure S1A). 
In the food industry, synthetic antioxidants are added to foods containing fat, especially 
meats, with the purpose of delaying oxidative processes that result in undesirable sensorial 
changes, decreased shelf life and nutritional value and the formation of secondary 
compounds potentially harmful to health [7,8]. However, data in the literature have 
associated the synthetic antioxidants BHT (butylated hydroxytoluene) and BHA (butylated 
hydroxyanisole) to possible deleterious health effects, as they have been reported as 
potential tumor promoters, following the chronic administration of these compounds to 
animals [9,10]. This has motivated the replacement of synthetic antioxidants by natural 
antioxidants extracted from foodstuffs [11,12]. 
Betanin can be used as a powerful antioxidant in the food industry in extract or powder 
form, and is also applied as a natural pigment. Its antioxidant activity in biological lipid 
environments has been demonstrated in human macromolecules, such as low density 
lipoproteins, membranes and whole cells [13]. Furthermore, betanin has attracted attention 
due to its anti-inflammatory and hepatic protective functions in human cells. This compound 
is able to modulate redox-mediated signal transduction pathways involved in inflammation 
responses in cultured endothelia cells, and has alsodisplayed antiproliferative effects on 
human tumor cell lines [14,15]. In both healthy and tumoral human hepatic cell lines, 
betanin can induce the translocation of the erythroid 2-related factor 2 (Nrf2) antioxidant 
response element (ARE) from the cytosol to the nuclear compartment, which controls the 
mRNA and protein levels of detoxifying/antioxidant enzymes, including GSTP, GSTM, 
GSTT, GSTA (glutathione S-transferases), NQO1 (NAD(P)H quinone dehydrogenase 1) 
and HO- (heme oxygenase-1), in these cells, exerting hepatoprotective and anticarcinogenic 
effects [16]. 
39 
 
Several betanin purification techniques have been reported, involving distinct steps and 
methodologies in order to purify this compound from vegetal sources, including complex 
food matrices such as beetroot. Among the methods employed for betanin purification, 
HPLC and other chromatographic methods using reverse phase columns seem to provide the 
best balance between speed and efficiency [17]. However, no other studies have evaluated 
the stability of this molecule during storage conditions and its antioxidant capability after 
purification and during storage with the aim of use as a food additive. 
In this context, the aim of the present study was to optimize a methodology applied for 
betanin purification in large amounts, using fresh juice obtained from red beetroot (Beta 
vulgaris L. species). In addition, the chemical stability and bioactivity of the purified 
molecule were also assessed, through two different view points: i) as a food preservative 
and ii) as a in natura or processed food matrix component after consumption and simulated 
gastrointestinal and ex vivo colon fermentation processes. 
2. Material and methods 
2.1. Standards and reagents 
The Betanin standard (C24H26N2O13), sulfuric acid (H2SO4), boric acid (H3BO3), formic 
acid (CH2O2), hydrochloric acid (HCL), terephthalic acid (TPA, C8H6O4), ethylenediamine 
tetra acetic acid (EDTA), 6-hydroxy-2-5-7-8-tetramethylchromo-2-carboxylic acid (Trolox), 
ascorbic acid (C6H8O6), sodium hydroxide (NaOH), potassium permanganate (KMnO4), 
hydrogen peroxide (H2O2), potassium sulphate (K2SO4), ferrous sulphate (FeSO4), methyl 
red, bromocresol green, petroleum ether, anhydrous sodium acetate (CH3COONa), 
tetrabutylammonium perchlorate (C16H36N.H2PO4), vanillin, tripyridyltriazine (TPTZ, 
C18H12N6), iron chloride (FeCl3), dibasic sodium phosphate (NaH2PO4.H2O), monobasic 
sodium phosphate (NaH2PO4.H2O), sodium chloride (NaCl), anhydrous monobasic sodium 
phosphate (Na2HPO4), sodium bicarbonate (NaHCO3) C-211,2,2′-Azobis (2-
methylpropionamidine), dihydrochloride (AAPH), 2,2’-azinobis [3-ethylbenzothiazoline-6-
sulfonic acid]-diammonium salt (ABTS, C18H24N6O6S4), sodium fluorescein (C20H12O5), 
potassium hydroxide (KOH), ammonium thiocyanate (NH4SCN), trichloroacetic acid 
(Cl3CCOOH) were purchased from Sigma-Aldrich Chemical Co. (MO, USA). Methanol 
(MeOH), ethanol, acetone, and acetonitrile were purchased from Tedia Company Inc. (OH, 
40 
 
USA). Butylated hydroxytoluene (BHT), 1,1,3,3-tetramethoxypropane 
((CH3O)2CHCH2CH(OCH3)2) and 2-thiobarbituric acid (C4H4N2O2S) were purchased from 
Sigma-Aldrich Co. HPLC grade Milli-Q water (Merck Millipore, MA, USA) was used 
throughout the experiments. 
2.2. Betanin purification 
2.2.1. Sample preparation 
Red beetroot was peeled, sliced and homogenized using a centrifuge food processor EC 
700 (Black & Decker, SP, BRA). The homogenates were centrifuged at 15,000 x g for 30 
min at 25°C and filtered through a PTFE filter membrane 25 mm, pore size 0.45 µm 
(Merck-Millipore). The supernatants (4 mL) were concentrated under reduced pressure (18 
mbar, 25°C) and resuspended in 2 mL deionized water. 
2.2.2. HPLC betanin purification 
Concentrated beetroot juice was purified by reverse phase high-performance liquid 
chromatography (RP-HPLC). The HPLC apparatus consisted in an LC-20A Prominence, 
(Shimadzu®, Kyoto, JPN) equipped with a quaternary pump and a diode-array detector 
model SPD-M20A (Shimadzu®). A 15 µm Phenomenex C18 column (250 x 21.2 mm I.D., 
Torrance, CA, USA) connected to an FRC-10A fraction collector (Shimadzu®) was used in 
the semi-preparative HPLC. The elution conditions were performed according to Cai et al. 
[39] with modifications. Solvent A was 1% formic acid, and solvent B was 80% methanol at 
a linear gradient (0 – 25 min, 11 – 55%). The injection volume was 100 μL and a flow rate 
of 5.5 mL/min was used. Separations were monitored at 536 nm and, after purification, 
magenta fractions, containing betanin, were concentrated by a rotary evaporator 
(Rotavapor® R-215, Buchi, SP, BRA) at 24°C, 150 rpm and a water bath at 40°C. The 
extracts were then suspended in 1 mL deionized water and stored at -30°C under an N2 
atmosphere for further analysis. The purified betanin was analyzed using a Nucleosil 100-
C18 column (250 × 4.6 mm I.D., 5 μm) with 30 µl injection volume and a flow rate of 1.0 
ml/min. The mobile phase and gradient conditions were similar to the purification step and 
betanin concentrations were quantified in comparison to a betanin standard solution (Sigma-
Aldrich Co.). 
41 
 
2.3 Betanin identification by liquid chromatography positive ion electrospray ionization 
tandem mass spectrometry (LC-ESI(+)-MS/MS) 
Mass spectrometry was performed as described by Gonçalves et al. [17]. The RP-HPLC 
purified fraction was ionized in the positive mode and ions were monitored in the full scan 
mode (range of m/z 50–1500). The ESI(+)-MS/MS analysis was carried out on a Bruker 
Esquire 3000 Plus Ion Trap Mass Spectrometer (Bruker Co., WI, USA) equipped with an 
electrospray source in the positive ion mode. Nitrogen was used as the nebulizing (45 psi) 
and drying gas (6 L∙min-1, 300°C) and helium as the buffer gas (4 × 10-6 mbar). The high 
capillary voltage was set to 3500 V. To avoid space–charge effects, smart ion charge control 
(ICC) was set to an arbitrary value of 50.000. Betanin identification was based on its mass 
(550 g.mol-1) and by similarity with the commercial standard and literature-available spectra 
[39]. 
2.4. Storage stability 
The stability of purified betanin during refrigeration (4°C) and freezing (-30°C) was 
evaluated by RP-HPLC-DAD, monitoring changes in the area under the chromatogram peak 
obtained at 536 nm, in similar conditions as those described for the betanin analysis. 
2.5. Betanin ability to inhibit lipid peroxidation in meat 
Betanin ability to inhibit lipid peroxidation was evaluated by MDA determination in 
meat TBARS assay, as described previously [40] with modifications. A sample of ground 
pork loin (500 g) from a local butcher shop in Rio de Janeiro, Brazil and divided into 4 
portions and treated as follow: i) ground pork loin non-treated by antioxidants; ii) ground 
pork loin treated with betanin (2%; w/w); iii) ground pork loin treated with BHT (0.01%); 
iv) ground pork loin treated with BHA (0.01%). MDA extraction was performed in 3.0 g of 
each meat sample homogenized with 9 mL of 7.5% TCA. The homogenate was centrifuged 
at 3,000 x g for 15 min at 25°C and filtered through Whatman n° 4 paper (Merck Millipore 
Co). TMP (the MDA standard) at 3.2 mM in 0.1 M HCl (stock solution) was kept for 2 h at 
room temperature in the dark. After hydrolysis, the TMP solution was diluted with 7.5% 
TCA to the concentrations of 1, 2, 4, 8, 16 and 32 µM. After, 1 mL of MDA at different 
concentrations or 7.5% TCA solution (blank) was transferred into a screw-cap tube and 1 
mL of 20 mM TBA solution was added. The tubes were heated in a boiling water bath at 
42 
 
90°C for 30 min and cooled in tap water for 10 min. Absorbance of the MDA-TBA adduct 
was measured at 532 nm on a spectrophotometer DU®530 (Beckman Coulter Inc., CA, 
USA). Because betanin absorbs light in the range of 530-540 nm, additional blanks 
containing betanin (1 or 2%), TCA or TBA (no meat)