Lídia Cristina Rocha

FAC

ULD

AD

E DE FA

RM

ÁC

IA

Lídia C

ristina Ro

cha. Phenolic compounds of w

hite wine from

the Douro region: T

he relationship betw

een chemistry and bioactivity in the context of A

lzheimer

s disease and Type 2 diabetes

Phenolic com

pounds of white w

ine from the

Douro region: T

he relationship between

chemistry and bioactivity in the context of

Alzheim

ers disease and Type 2 diabetes

Lidia Cristina Rocha

Phenolic compounds of white wine from the Douro region: The relationship between chemistry and bioactivity in the context of Alzheimer s disease and Type 2 diabetes

Lídia Cristina Rocha

M 2018

M.FFU

P 2018

MESTRADO EM CONTROLO DE QUALIDADE

FÁRMACOS E PLANTAS MEDICINAIS

Phenolic compounds of white wine

from the Douro region: The relationship

between chemistry and bioactivity in

the context of Alzheimer's disease and

Type 2 diabetes

Lídia Cristina Alves da Rocha

Dissertação do 2º Ciclo de Estudos Conducente ao Grau de Mestre em

Controlo de Qualidade

Área de especialização: Fármacos e Plantas Medicinais

Trabalho realizado sob a orientação da Professora Doutora

Paula B. Andrade e do Doutor Romeu Videira

Novembro de 2018

ii

A presente Dissertação enquadra-se no projeto “IBERPHENOL- Red cooperativa de

investigación en el ámbito de polifenoles y sus aplicaciones industriales”, financiado por

POCTEP – Programa de Cooperação Transfronteiriça Portugal – Espanha.

Phenolic compounds of white wine from the Douro region: The relationship between chemistry and bioactivity

in the context of Alzheimer's disease and Type 2 diabetes

_______________________________________________________________________________________

iii

De acordo com a legislação em vigor, não é permitida a reprodução de qualquer parte

desta dissertação.



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iv

Agradecimentos

Quero expressar o meu profundo agradecimento a todas as pessoas que me

acompanharam nesta jornada.

À Professora Doutora Paula Branquinho de Andrade, minha orientadora, agradeço

por toda a disponibilidade, orientação e pela oportunidade de desenvolver este estudo no

Laboratório de Farmacognosia.

Ao Doutor Romeu, meu co-orientador, quero agradecer a forma incansável com que

sempre me acompanhou neste percurso, por todos os ensinamentos e pela sua dedicação

e paciência.

Aos restantes elementos do laboratório, agradeço a prontidão com que sempre me

ajudaram.

Aos meus amigos agradeço todo o apoio e compreensão. Sublinho, de um modo

especial, o apoio incondicional da Rita, da Rute e da Daniela, que sempre me

acompanharam e motivaram. Sinto-me muito grata pela partilha. Que os nossos trilhos se

continuem a cruzar e que esta amizade perdure pela vida.

Ao Leonardo, sempre presente, agradeço todo o carinho, encorajamento e

paciência.

Expresso ainda a minha profunda gratidão a um dos grandes pilares da minha vida

– a minha família, em especial, aos meus pais.



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v

Resumo

Evidências experimentais recentes interligam a diabetes mellitus tipo 2 e a doença

de Alzheimer, ampliando a dimensão social e económica destas patologias. Assim, o

desenvolvimento de novas estratégias terapêuticas capazes de abordar o quadro

fisiopatológico comum a estas patologias é um desafio científico e uma prioridade

biomédica. Assim, o presente estudo tem como objetivo avaliar o potencial terapêutico

e/ou preventivo de uma mistura de compostos fenólicos, considerando os efeitos sobre

alvos bioquímicos com relevância na diabetes mellitus tipo 2 e/ou na doença de Alzheimer.

Os compostos fenólicos foram obtidos a partir do polímero polivinilpolipirrolidona

(PVPP), utilizado no tratamento preventivo de um vinho branco da região do Douro, e

analisados por cromatografia líquida de alta eficiência com um detetor de fotodíodos

(HPLC-DAD). O extrato de vinho branco obtido por adsorção seletiva ao PVPP é uma

mistura rica em compostos fenólicos (cerca de 80% da massa seca), dominada por ácidos

fenólicos (e.g. ácido caftárico) e por proantocianidinas (e.g. oligómeros de catequina). A

avaliação da atividade antioxidante revelou que o extrato tem capacidade para sequestrar

o radical superóxido e de proteger as membranas celulares da oxidação lipídica induzida

pelo sistema Fe2+/ascorbato. Estudos enzimáticos mostraram que o extrato de vinho

branco apresenta capacidade para inibir a α-glicosidase e a acetilcolinesterase através de

um mecanismo de inibição não competitiva, e não afeta a atividade da α-amilase. O perfil

toxicológico do extrato de vinho branco foi avaliado em células de neuroblastoma SH-

SY5Y, considerando os efeitos sobre a viabilidade celular e a integridade da membrana.

Na gama de concentrações testadas, não foram detetados quaisquer efeitos tóxicos.

Adicionalmente, a pré-incubação das células SH-SY5Y com o extrato de vinho branco

protege as células da toxicidade induzida pelo glutamato, como detetado pela redução da

produção de espécies reativas de oxigénio e pelo aumento da razão GSH/GSSG.

Em conclusão, este estudo revelou que uma mistura de compostos fenólicos tem

capacidade para modular vários alvos bioquímicos relevantes no contexto da doença de

Alzheimer e da diabetes mellitus tipo 2. Consequentemente, o extrato de vinho branco,

obtido por adsorção seletiva ao PVPP, apresenta potencial para suportar estratégias para

tratar e/ou prevenir o quadro fisiopatológico comum à doença de Alzheimer e à diabetes

mellitus tipo 2.

Palavras-chave: Doença de Alzheimer; Diabetes mellitus tipo 2; Polifenóis; Inibição

enzimática; Atividade antioxidante; Neuroprotecção.



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vi

Abstract

Recent experimental evidences link type 2 diabetes mellitus to Alzheimer´s disease,

increasing the social and economic dimension of these health problems. Thus, the

development of new therapeutic strategies to address these overlapping health issues is a

scientific challenge and a biomedical priority. Thus, the present study aims to evaluate the

therapeutic and/or preventive potential of a mixture of phenolic compounds considering the

effects on biochemical targets with relevance in type 2 diabetes mellitus and/or Alzheimer’s

disease.

The phenolic compounds were obtained from polyvinylpolypyrrolidone polymer

(PVPP), used in the preventive treatment of a Douro white wine and analysed by high

performance liquid chromatography with a photodiode detector (HPLC-DAD). The wine

extract obtained by selective adsorption to PVPP (PVPP-white wine extract) is a mixture

rich in phenolic compounds (about 80% of dry mass), dominated by phenolic acids (e.g.

caftaric acid) and by proanthocyanidins (e.g. catechin oligomers). The evaluation of the

antioxidant activity revealed that the extract has the capacity to scavenge superoxide radical

and to protect the cell membranes from lipid oxidation induced by the Fe2+/ascorbate

system. Enzymatic studies shown that PVPP-white wine extract has the ability to inhibit α-

glucosidase and acetylcholinesterase through a non-competitive inhibition mechanism and

does not affect α-amylase activity. The toxicological profile of the PVPP-white wine extract

in neuroblastoma SH-SY5Y cells was evaluated, considering the effects on cell viability and

membrane integrity. In the range of concentrations tested, no toxic effects were observed.

In addition, pre-incubation of SH-SY5Y cells with the PVPP-white wine extract protects the

cells from glutamate-induced toxicity, as detected by a decrease in production of reactive

oxygen species and an increase in intracellular GSH/GSSG ratio.

In conclusion, this study revealed that a mixture of phenolic compounds has the

ability to modulate several relevant biochemical targets in the context of Alzheimer's disease

and type 2 diabetes mellitus. Consequently, the white wine extract obtained by selective

adsorption to PVPP presents potential for support strategies to treat and/or prevent the

pathophysiological framework common to Alzheimer's and type 2 diabetes mellitus.

Keywords: Alzheimer’s disease; Type 2 Diabetes mellitus; Phenolic compounds;

Enzymatic inhibition; Antioxidant activity; Neuroprotection.



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vii

Index

Index ................................................................................................................................ vii

Index of Figures ............................................................................................................... ix

Index of Tables .................................................................................................................. x

List of Abbreviations ......................................................................................................... xi

1. Introduction ................................................................................................................... 1

1.1. Phenolic compounds from wine and winery by-products: medicinal applications

and standardization needs ............................................................................................. 2

1.2. Type 2 diabetes mellitus and Alzheimer’s disease ............................................. 7

1.2.1. Type 2 diabetes mellitus .......................................................................... 7

1.2.2. Alzheimer’s disease ................................................................................. 9

1.2.3. Link between Type 2 diabetes mellitus and Alzheimer’s disease ........... 10

1.3. Phenolic compounds to treat and/or prevent Type 2 diabetes mellitus and

Alzheimer’s disease ..................................................................................................... 13

2. Objectives ................................................................................................................... 15

3. Material and Methods .................................................................................................. 17

3.1. Standards and reagents .................................................................................. 18

3.2. Preparation of white wine polyphenolic extract ................................................ 18

3.3. Determination of chemical composition of white wine extract by HPLC-DAD ... 19

3.4. Antioxidant activity – In vitro assays ................................................................ 20

3.4.1. Evaluation of superoxide radical scavenging activity ............................. 20

3.4.2. Cis-parinaric acid assay ......................................................................... 22

3.5. Effects on the activity of enzymes with therapeutic relevance ......................... 22

3.5.1. Determination of α-amylase activity ....................................................... 22

3.5.2. Determination of α-glucosidase activity .................................................. 23

3.5.3. Determination of acetylcholinesterase in rat brain homogenates ........... 24

3.6. Cell-based assays ........................................................................................... 25

3.6.1. Culture conditions .................................................................................. 25

3.6.2. Determination of cell viability and membrane integrity ........................... 25



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viii

3.6.3. Measurement of intracellular reactive species ....................................... 26

3.6.4. Determination of GSH and GSSG levels ............................................... 26

3.7. Statistical analysis ........................................................................................... 27

4. Results and Discussion ............................................................................................... 28

4.1. Chemical composition of PVPP-white wine extract - HPLC-DAD analysis ....... 29

4.2. Antioxidant activity – In vitro assays ................................................................ 32

4.2.1. Superoxide radical scavenging activity .................................................. 32

4.2.2. Protective effects against lipid peroxidation ........................................... 33

4.3. Effects on the activity of enzymes with relevance as therapeutic targets ......... 34

4.3.1. α-amylase activity .................................................................................. 34

4.3.2. α-glucosidase activity ............................................................................ 37

4.3.3. Acetylcholinesterase activity .................................................................. 40

4.4. Cell-based assays ........................................................................................... 44

4.4.1. Effects of the PVPP-white wine extract on SH-SY5Y cell viability and

membrane integrity .................................................................................................. 44

4.4.2. Protective effects against glutamate-induced neurotoxicity in SH-SY5Y

cells .......................................................................................................................... 45

5. Conclusions and Future Perspectives ......................................................................... 50

6. References .................................................................................................................. 53



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ix

Index of Figures

Figure 1 – Chemical structure of hydroxybenzoic, hydroxycinnamic and

hydroxycinnamoyltartaric acids present in grapes and wine. ............................................. 4

Figure 2 – Stilbenes present in grapes and wine. ............................................................. 6

Figure 3 – Free radical–mediated cellular injury.. ........................................................... 12

Figure 4 – Chemical structure of PVPP - (C6H9NO)n, used to stabilize the white wine of

Douro region. .................................................................................................................. 18

Figure 5 – Conversion of xanthine to uric acid with formation of formazan in the presence

of NBT. ............................................................................................................................ 21

Figure 6 – HPLC polyphenolic profile of PVPP-white wine extract.. ................................ 30

Figure 7 – Free-radical scavenger activity of PVPP-white wine extract as function of

increasing concentrations of phenolics.. .......................................................................... 33

Figure 8 – Evaluation of the capacity of PVPP-white wine extract to protect the lipids of the

cellular membranes from the oxidation induced by Fe2+ (50 µM) and ascorbic acid (250 µM)

system ............................................................................................................................. 34

Figure 9 – Kinetic of α-amylase activity in the absence and presence of increasing

concentrations of PVPP-white wine extract. .................................................................... 36

Figure 10 – α-amylase inhibitory activity of acarbose, an inhibitor of this enzyme with

approval clinical use. ....................................................................................................... 36

Figure 11 – α-glucosidase inhibitory activity of PVPP-white wine extract (A) and acarbose,

an inhibitor of this enzyme with approval clinical use (B).. ............................................... 38

Figure 12 – Kinetics of α-glucosidase activity in the absence and presence of increasing


Figure 13 – Kinetics of rat brain AChE activity in the absence and presence of increasing


Figure 14 – Evaluation of the effects of the PVPP-white wine extract on SH-SY5Y cell

viability and membrane integrity. ..................................................................................... 44

Figure 15 – Evaluation of protective effect of PVPP-white wine extract on the viability of

SH-SY5Y cells treated with glutamate ............................................................................. 46

Figure 16 – Evaluation of intracellular generation of reactive oxygen species triggered by

50 mM glutamate exposure and the protective effect of increasing concentrations of PVPP-

white wine extract to the toxic stimulus.. .......................................................................... 47

Figure 17 – Evaluation of the effects triggered by glutamate 50mM exposure on

GSH/GSSG ratio of the SH-SY5Y cells and the potential protective effect of increasing

concentrations of PVPP-white wine extract to the toxic stimulus.. ................................... 49



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x

Index of Tables

Table 1 – Main classes of phenolic compounds (Harborne, 1989). ................................... 3

Table 2 – Some classes of flavonoids presents in grapes and wine. ................................. 5

Table 3 – Reverse phase gradient program for determination of phenolic composition of

PVPP-white wine extract. ................................................................................................ 20

Table 4 – Phenolic composition of PVPP-white wine extract determined by HPLC-DAD. 31

Table 5 – Regression equations, LOD and LOQ data for the quantification of the identified

phenolic compounds in PVPP-white wine extract. ........................................................... 32

Table 6 – Kinetic parameters (Km and Vmax), chi-squared (2), correlation factor (R) of α-

amylase in the absence and presence of PVPP-white wine extract. ................................ 37

Table 7 – Characterization of the type of inhibition induced by PVPP-white wine extract on

α-glucosidase. Kinetic parameters (Km and Vmax), chi-squared (2), correlation factor (R),

and inhibition constants used to determine the type of inhibition. .................................... 40

Table 8 – Characterization of the type of inhibition induced by PVPP-white wine extract on

rat brain AChE. Kinetic parameters (Km and Vmax), chi-squared (2), correlation factor (R),

and inhibition constants used to determine the type of inhibition. .................................... 43

Table 9 – Levels of reduced, oxidized and total glutathione in presence and absence of

glutamate and/or PVPP-white wine extract. ..................................................................... 49



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xi

List of Abbreviations

AChE Acetylcholinesterase

AD Alzheimer’s disease

ATP Adenosine triphosphate

BSA Bovine serum albumin

DAD Diode array detector

DCFH-DA 2′,7′-dichlorofluorescein diacetate

DMEM Dulbecco’s Modified Eagle Medium

DNA Deoxyribonucleic acid

DTNB 3,5 – dinitrosalicylic acid, 5,5′-dithiobis(2-nitrobenzoic acid)

EC50 Half maximal effective concentration

FBS Fetal bovine serum

GSH Reduced glutathione

GSSG Oxidized glutathione

HBSS Hank’s balanced salt solution

HO• Hydroxyl radical

HPLC High-performance liquid chromatography

IC50 Half maximal inhibitory concentration

LDH Lactate dehydrogenase

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NADH Nicotinamide adenine dinucleotide (reduced form)

NBT Nitro blue tetrazolium chloride

O2•- Superoxide anion

OPT o-Phthalaldehyde

p-NGP 4-nitrophenyl α-D-glucopyranoside

PVPP Polyvinylpolypyrrolidone

ROS Reactive oxygen species

T2DM Type 2 Diabetes mellitus

X Xanthine

XO Xanthine oxidase

WHO World Health Organization



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1

1. Introduction



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2

1.1. Phenolic compounds from wine and winery by-products:

medicinal applications and standardization needs

Phenolic compounds comprise a diverse group of secondary metabolites (more than

8000 molecules) involved in plants’ responses against biotic and abiotic stresses (1,2). As

a common distinctive feature, these compounds contain at least one aromatic ring

substituted by at least one hydroxyl group, free or engaged in another function (ether ester,

or glycoside) (3,4). The monosaccharides associated with phenolics include, for instance,

glucose, galactose and rhamnose (5). However, this purely chemical definition is not

sufficient to characterize the phenolic compounds, since other secondary metabolites also

have these structural elements, thus, the biosynthetic origin is another criterion to define

this group. Plant phenolics arise from two main pathways: shikimate and acetate (3–5). The

simplest phenols are compounds that have a single aromatic ring with one or more hydroxyl

groups, whereas polyphenols are compounds with multiple phenol rings within a single

structure (6). According to Harborne (5), phenolic compounds can be divided into different

classes depending on their basic chemical structure (Table 1).

Grapes (Vitis spp.) are one of the richest sources of polyphenols among fruits.

Phenolic compounds are the third most abundant constituent in grapes, after carbohydrates

and fruit acids. The total extractable phenolics in grapes are present at approximately 10%

in the pulp, 60% to 70% in the seeds, and 28% to 35% in the skin (7). Phenolic compounds

play an important role on the sensorial characteristics of both grapes and wine, because

they are responsible for some organoleptic properties, such as aroma, color, flavor,

bitterness and astringency (8).

Polyphenols contained in grapes and wines can be classified into different classes

and subclasses: (1) phenolic acids, subdivided in hydroxybenzoic and hydroxycinnamic

acids; (2) flavonoids (flavanols, flavones, flavanones, flavonols, flavanonols and

anthocyanins); (3) hydrolysable tannins and proanthocyanidins; (4) stilbenes.

Phenolic acids constitute a class of phenolic compounds characterized by at least

one carboxyl group and one phenolic hydroxyl group. These substances can be

subclassified into hydroxycinnamic and hydroxybenzoic acids (3).

Hydroxycinnamic acids are aromatic compounds displaying a C6-C3 structure that

belong to the phenylpropanoid group and are very often found esterified (Figure 1) (3,5).

The most referenced compounds of this phenolic group in wine includes, for example,

caffeic, caftaric (caffeoyltartaric), chlorogenic, coutaric (coumaroyltartaric), fertaric

(feruloyltartaric), ferulic and p-coumaric acids. These acids are associated with wine

browning process (8).



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3

Table 1 – Main classes of phenolic compounds (Harborne, 1989) (5).

Number of carbon atoms Basic skeleton Class Basic structure

6 C6 Simple phenols

7 C6- C1 Phenolic acids

8 C6- C2

Acetophenones

Phenylacetic acids

9 C6- C3

Hydroxycinnamic acids

Phenylpropenes

Coumarins

Chromones

10 C6- C4 Naphthoquinones

13 C6- C1- C6 Xanthones

14 C6- C2- C6 Stilbenes

15 C6- C3- C6 Flavonoids



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4

In other hand, hydroxybenzoic acids are hydroxylated derivatives of benzoic acid,

that present a C6-C1 structure (Figure 1). These compounds are quite common in the free

state, as well as combined into esters or glycosides (3,5). The most abundant in wine are

gallic, gentisic, p-hydroxybenzoic, protocatechuic, syringic, and vanillic acids (8).

Figure 1 – Chemical structure of hydroxybenzoic, hydroxycinnamic and hydroxycinnamoyltartaric

acids present in grapes and wine.

Flavonoids are low molecular weight compounds composed by fifteen carbon atoms,

consisting in a C6-C3-C6 configuration and are mainly water-soluble compounds. Essentially

the structure consists of two aromatic rings, A and B, linked through three carbons that

usually form an oxygenated heterocyclic (C) (5). Biogenetically, the A ring usually comes

from a condensation of three molecules of malonyl-CoA with an ester of coenzyme A

synthetized in the acetate pathway, whereas B ring derived from phenylalanine through the

shikimate pathway (3,5). This basic structure is share by different subclasses, as flavanols

(also known as flavan-3-ols), flavonols, flavones, flavanonols (also known as

dihydroflavonols) and anthocyanidins, that differ in in the oxidation degree of the central

pyran-ring (3). In Table 2 are presented some subclasses of flavonoids as well as examples

of compounds present in grapes and wine (8).

Flavonoids are very effective scavengers of free radicals in in vitro tests and they

are very important antioxidants because of their high redox potential and their ability to

chelate metals (9).

R=R1=H: p-hydroxybenzoic acid

R=OH; R1=H: protocatechuic acid

R=OCH3; R1=H: vanillic acid

R=R1=OH: gallic acid

R=R1=OCH3=syringic acid

R= R=R1=H: p-coumaric acid

R=OH; R1=H: caffeic acid

R=OCH3; R1=H: ferulic acid

R=R1=OCH3=sinapic acid

R=H: p-coumaroyltartaric acid (coutaric acid)

R=OH: caffeoyltartaric acid (caftaric acid)

R=OCH3: feruloyltartaric acid (fertaric acid)



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5

Table 2 – Some classes of flavonoids presents in grapes and wine.

Flavonoids Basic structure Wine/grape compound

Flavanols

Catechin

Flavones

Luteolin

Flavanones

Eriodictyol

Flavonols

Quercetin

Flavanonol / Dihydroflavonol

Taxifolin

Anthocyanidins

Malvidin

Unlike the previous described groups of wine phenolics, tannins are compounds of

intermediate to high molecular weight. These polyphenolic compounds are highly

hydroxylated molecules and can form insoluble complexes with proteins, displaying

astringent properties. Plant tannins are usually divided in two major groups: condensed and



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6

hydrolysable tannins (2). The natural tannins present in wine are predominantly of the

condensed type (8). Condensed tannins, also known as proanthocyanidins, are polymers

of flavanols units (e.g. catechin, epicatechin) linked through C4-C8 bonds. The four most

common dimmers are B-type procyanidins (e.g. B1, B2, B3 e B4) (3). Proanthocyanidins

are responsible for the sensory characteristics of wine (color, taste, astringency and

bitterness), playing an important role in the wine aging process due to their oxidation,

condensation and polymerization capabilities (8).

In other hand, hydrolysable tannins are esters of glucose and gallic acid or ellagic

acid (gallotannins and ellagitannins, respectively) (3,10). These oligomers are not found in

Vitis vinifera L., but are in oak wood and Vitis rotundifolia Michx (muscadine grapes) (6).

Stilbenes are phenolic compounds chemically constituted by two aromatic rings

linked by an ethene bridge. These compounds occur free (aglycone) or as glycosides.

Within this group, trans-resveratrol (trans-3,5,4’-trihydroxystilbene) is the most referenced

as present in wine (3,8). Other stilbenes detected in grape are trans-piceid (trans-

resveratrol-3-O-glucoside) and ε-viniferin (resveratrol dimer) (8).

ε-viniferin

Figure 2 – Stilbenes present in grapes and wine.

In last years, polyphenols have been associated with the bioactive potential of

grapes, wine and winery by-products due to their antioxidant, anti-inflammatory,

anticarcinogenic and antibacterial activities (11–15). The discovery that winery by-products

contain these compounds that are beneficial to human health has led to rapidly expanding

markets for a wide range of products: functional foods, nutraceuticals, food supplements

and cosmetics. Indeed, some of these polyphenols have been tested in clinical trials (16).

The number of herbal medicinal products obtained from winery by-products should

increase substantially soon, making the implementation of an efficient standardization of

phenolic extracts obtained from winery by-products imperative. Actually, the phenolic

extracts are usually standardized in a physical and chemical perspective (17). Winery by-

products exhibit a great chemical complexity and variability, and the chemical composition

R1=R2=H: trans-resveratrol

R1=H; R2=Gluc: trans-piceid



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7

of their extracts is highly dependent of extraction procedures, which hinder to obtain the full

chemical profile by using only one analytic procedure (18). Thus, the standardization of the

phenolic extracts obtained from winery by-products considering only the extraction

procedures and the chemical quantification of one (or small number) bioactive/marker

compounds may not be able to ensure that the same product obtained in different batches

(as well as similar products produced by different companies) exhibited uniform bioactivity.

Thus, additional assays are required to promote the effective standardization of herbal

medicinal products obtained from winery by-products.

Considering that the intrinsic antioxidant activity of phenolic compounds has

biological relevance, it can be considered to implement biological assays in order to

promote the effective standardization of herbal medicinal products obtained from grape

processing by-products as well as from other resources rich in phenolic compounds. This

goal can be reach through cell assays designed to assess the ability of these medicinal

products (e.g. food supplements) to modulate the redox state of cells under normal health

condition or under oxidative stress condition promoted by well-defined oxidant agents.

Therefore, we propose the chosen of a small panel of standard cell lines,

representative of different human tissues and currently used in many labs, to perform some

biological standardization assays. Glutathione is a major non-protein thiol in human cells

and the ratio of its reduced and oxidized forms (GSH/GSSG) is the best indicator of cellular

redox status (19,20). Cell assays performed with different concentration of herbal medicinal

products in absence and in presence of a standard oxidant agent can be used to determine

the GSH/GSSG ratio. With this type of results, a graduated scale to indicate the effective

bioactivity of herbal medicinal products can be constructed. Thus, this parameter may be

included in cells assays that aim the standardization of herbal medicinal products rich in

phenolic compounds. To increase the robustness of this standardization methodology,

another cell parameter with universal biological relevance - energetic charge (levels of

adenine nucleotides), can be included in the graduated scale of bioactivity of these

products. Note that cellular levels of adenosine nucleotides can be measured (by high

performance liquid chromatography (HPLC) (21)) in the same samples used to determine

the GSH/GSSG ratio.

1.2. Type 2 diabetes mellitus and Alzheimer’s disease

1.2.1. Type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a chronically progressive disease that is

characterized by inherited and acquired insulin resistance and by high blood sugar levels

over a prolonged period. On the other hand, Type 1 diabetes is caused by an autoimmune



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8

destruction of pancreatic islet beta cells, leading to insulin deficiency (22). According data

of World Health Organization (WHO), in 2016, diabetes mellitus killed 1.6 million people,

occupying the seventh place on top ten causes of death worldwide (23).

In T2DM, the peripheral tissues (liver, muscle and adipose tissue) develop a

progressive insulin resistance, which affects the glucose uptake into cells and its metabolic

storage as glycogen and triacylglycerols. Consequently, abnormal glucose levels remains

in blood with potential to promote cell damage (24). In the first stages of disease, pancreatic

β-cells increase insulin secretion (hyperinsulinemia) to overcome insulin resistance,

maintaining blood glucose levels within the physiological range, i.e. below the T2DM range.

However, this condition promotes the progressive functional decline of β-cells, and insulin

production becomes inadequate to overcome insulin resistance, consequently blood

glucose levels rise triggering first a prediabetes condition and then T2DM (25). The

deregulation of blood glucose levels could lead to several disorders, including retinopathy,

nephropathy, neuropathy and angiopathy, usually designated as secondary complications

of diabetes (26).

T2DM is the most common type of diabetes, accounting for about 90-95% of

diagnosed cases of diabetes, and is often associated with a variety of risk factors, such as

hypertension, cardiovascular disease, dyslipidemia (high triglyceride levels and low levels

of high-density lipoproteins), and high cholesterol levels (22,27,28).

1.2.1.1. Current therapy

The main goal of the therapeutic approaches for diabetes mellitus is to control the

blood glucose levels within the physiological range. Thus, adequate diet and exercise are

the first steps in the treatment of T2DM. In case of failure of life-style changes, various oral

hypoglycemic agents may be used (29). To date, six classes of oral antihyperglycemic

drugs are available to complement the therapy: biguanides (metformin), sulphonylurea (e.g.

tolbutamide), glinidines (e.g. repaglinide), thiazolidinediones (e.g. pioglitazone), dipeptidyl

peptidase IV inhibitors (e.g. sitagliptin) and carbohydrate hydrolysis enzymes inhibitors (e.g.

acarbose, miglitol, and voglibose) (30,31).

An important therapeutic approach for treating T2DM is to decrease the postprandial

hyperglycemia by retarding the absorption of glucose through the inhibition of the

carbohydrate-hydrolyzing enzymes, α-amylase and α-glucosidase, in the digestive tract.

Enzyme inhibitors determine a reduction in the rate of glucose absorption, blunting the post-

prandial plasma glucose rise (32). The most important digestive enzyme is the pancreatic

alpha-amylase, a calcium metalloenzyme that catalyzes the hydrolysis of the α-1,4

glycosidic linkages of starch, amylose, amylopectin, glycogen and various maltodextrins



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9

and is responsible of most of starch digestion in humans. The enzyme α-glucosidase or

maltase catalyzes the final step of the digestive process of carbohydrates acting upon 1,4-

alpha bonds and giving as a result glucose (31).

1.2.2. Alzheimer’s disease

Alzheimer’s disease (AD) is the most common cause of dementia, currently

accounts for 60-80% of cases. This pathology was first described in 1906 by Alois Alzheimer

(33) as a rare disease brain. However, Alzheimer Europe association estimated that in 2013

more than 9 million of European citizens were affected for dementia (34). Deaths due to

Alzheimer’s disease and other dementias more than doubled between 2000 and 2016,

making it, according to WHO, the fifth leading cause of global deaths in 2016 (23).

Additionally, Prince et al (35) estimated that people lived with dementia worldwide would

increase from 35.6 million in 2010 to 65.7 million in 2030 and to 115.4 million in 2050. Thus,

the development of effective medicine to break or delay the progressive brain degeneration

underlying AD and other dementia diseases is one greatest challenge of our time.

AD is a severe, chronic and progressive neurodegenerative disorder, clinically

characterized by progressive memory loss and a gradual decline in cognitive function,

eventually leading to premature death of the individual (33). At the macroscopic level, AD

brains are characterized by significant atrophy due to cell loss. The neuropathological

hallmarks of AD are the extracellular accumulation of senile plaques, mostly formed by the

accumulation of amyloid-beta peptide (Aβ) and the intracellular formation of twisted strands

of hyperphosphorylated tau protein – the neurofibrillary tangles (36,37). Plaques and

tangles are present mainly in cortex and hippocampus, regions involved in learning and

memory. These regions typically exhibit a dramatic synaptic and neuronal loss, with

cholinergic and glutamatergic neurons being the most affected (37).

Based on its age of onset, AD is classified into early-onset AD (onset <65 years),

accounting for only 1-5% of all cases, and late-onset AD (onset ≥65 years). Early onset AD

is an inherited familial form of the disease, caused by mutations in three genes: amyloid

precursor protein, presenilin-1 and presenilin-2 (38,39). In other hand, the sporadic AD

form, like other chronic diseases, develops as a result of multiple factors rather than a single

cause. Older age, having a family history of Alzheimer’s and presence of the APOE-e4 gene

are the greatest risks for the late-onset AD (33).

In the last decades, several alterations in multiple biochemical pathways have been

identified as key players in multifactorial AD pathology. Those include aberrant amyloid-

beta precursor protein metabolism (40); changes in tau phosphorylation (41),

neuroinflammation (37), dysregulation of intracellular calcium signalling (42), oxidative



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10

stress (43), mitochondrial dysfunction (44) and aberrant lipid metabolism (45,46). However,

the disease aetiology remains to be elucidated.

1.2.2.1. Current therapy

Despite the extensive research in this field, there are no therapies with ability to able

to break the progressivity of brain degeneration and cognitive decline underlying AD

progression. In fact, the current available treatment, designed to modulate the cholinergic

(e.g. acetylcholinesterase inhibitors), and glutamatergic (e.g. N-methyl D-aspartate (NMDA)

receptor antagonist) neuronal networks, demonstrate only modest symptomatic benefits

that are not sustained (47). However, they have a scientific valid rationalization and they

are also important to improve the patient's life. For instance, a marked loss of cholinergic

activity is detected in Alzheimer’s brains. Thus, by blocking the enzyme responsible for

acetylcholine breakdown, acetylcholinesterase inhibitors increase the levels of this

neurotransmitter in the brain, increasing also the probability of post-synaptic acetylcholine

receptors activation, which may improves the cholinergic network connection (48). There

are four drugs approved for this purpose: tacrine, donepezil, rivastigmine, and galantamine

(49).

On the other hand, glutamatergic neurons regulate synaptic plasticity, neuronal

growth and differentiation, cognition, learning and memory (50–52) and the brains regions

more affected by AD are also dominated by glutamatergic neurons. Memantine is an

uncompetitive NMDA receptor antagonist that blocks the receptor by trapping it in open

conformation, which protects against neurotoxic increased glutamate concentration (49,53).

Currently, memantine is the only drug approved for clinical use in moderate to severe AD

in USA and Europe (53).

1.2.3. Link between Type 2 diabetes mellitus and Alzheimer’s

disease

Recent work supports the idea that the overall pathophysiological changes

associated with AD phenotype emerge as a consequence of unbalanced metabolism

associated with abnormal lipid metabolism and mitochondrial dysfunctions, which are relate

to a decline of mitochondrial complex I activity and with a specific abnormal cardiolipin

profile (45). Similarly to the brain, deregulation of lipid and bioenergetic metabolisms

accompanied by an abnormal production/accumulation of amyloid-β peptides, signs of

chronic inflammation and oxidative stress are also detected in peripheral tissues (e.g.

skeletal muscle), suggesting AD as a systemic metabolic disorder (54).



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11

Moreover, other scientific advances reveal that metabolic dysfunctions connected

with insulin resistance, such as type 2 diabetes, increase significantly the relative risk of

AD. Indeed, it has been suggested that AD represents a form of diabetes that selectively

involves the brain, being often described as “type 3 diabetes” (55–57).

In fact, numerous epidemiological studies have explored the relationship between

diabetes and AD, and most have identified diabetes as a risk factor for AD (25). The

magnitude of the risk is a matter of debate, but a recent meta-analysis of longitudinal studies

suggests that the relative risk for AD is approximately 1.5-fold higher among persons with

T2DM (58,59).

Besides the epidemiological studies, the link between T2DM and AD is suggested

by: progressive brain insulin resistance and insulin deficiency in AD; cognitive impairment

in experimental models of T2DM; AD-type neurodegeneration and cognitive impairment in

experimentally induced brain insulin resistance and insulin deficiency; improved cognitive

performance in experimental models and humans with AD after treatment with insulin

sensitizer agents or intranasal insulin; shared molecular, biochemical, and mechanistic

abnormalities in T2DM and AD (56,57,60). Experimental evidences indicate that AD and

T2DM share several molecular mechanisms of degeneration, such as protein misfolding

(aggregation of amyloid peptides and formation of hyperphosphorylated proteins), insulin

signalling impairments, and cytotoxic processes (e.g. induction of apoptosis connected with

high generation of reactive oxygen species and mitochondrial dysfunction) (28). Although

both disorders possess several overlapping features, mitochondrial dysfunction is one of

the most relevant (61).

Oxidative stress can be defined as a redox state resulting from an imbalance

between the generation and detoxification of reactive oxygen species (ROS), which results

in oxidative damage. Oxidative stress can be due to either an increase in ROS production

or a decrease in the activity of the antioxidant enzymes (e.g. superoxide dismutase,

catalase and glutathione peroxidase) or nonenzymatic antioxidants (e.g. glutathione,

vitamin E) (51). ROS can produce cellular dysfunction by reacting with lipids, proteins,

carbohydrates, and DNA to extract electrons (summarized in Figure 3). Briefly, superoxide

and the hydroxyl radical initiate lipid peroxidation in the cellular, mitochondrial, nuclear, and

endoplasmic reticulum membranes. The increase in cellular permeability results in an influx

of Ca2+, which causes further mitochondrial damage. The cysteine sulfhydryl groups and

other amino acid residues on proteins are oxidized and degraded. Nuclear and

mitochondrial DNA can be oxidized, resulting in strand breaks and other types of damage

(24). Thus, oxidative stress can cause necrosis, adenosine triphosphate (ATP) depletion,

and disruptions in normal mechanisms of cellular signalling (51).



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12

The brain is especially prone to oxidative stress-induced damage due to its high

oxygen consumption, high levels of polyunsaturated fatty acids susceptible to free radical

attack, high content in transition metals, poor antioxidant defences (poor catalase activity

and moderate superoxide dismutase and glutathione peroxidase activities). Additionally,

high content in iron and ascorbate can be found in some regions of the central nervous

system, enabling generation of more reactive species through the Fenton/Haber Weiss

reactions (44).

Figure 3 – Free radical–mediated cellular injury. Adapted from (24).

Mitochondria play a critical role in the regulation of cell survival and death. These

organelles are essential for the production of ATP through oxidative phosphorylation and

regulation of intracellular calcium (Ca2+) homeostasis. Thus, dysfunction of mitochondrial

energy metabolism culminates in a decrease of ATP production, Ca2+ buffering impairment

and high rates of ROS generation (62).

Although the human brain represents only a few percent of body weight, it consumes

nearly 20% of total body oxygen. Neurons need high quantity of oxygen to create ATP,

which fuels the intracellular machinery, such as the maintenance of ion gradients across

the plasma membrane, that is critical for the generation and propagation of action potentials

and neurosecretion (61). Neurons depend almost exclusively on mitochondria to obtain

energy due to their limited glycolytic capacity. However, the aerobic oxidative

phosphorylation is a major source of endogenous toxic free radicals, including hydrogen

peroxide (H2O2), hydroxyl (HO●-) and superoxide (O2●-) radicals that are products of normal

cellular respiration (44).

With inhibition of electron transport chain, electrons accumulate in complex I and

coenzyme Q, where they can be donated directly to molecular oxygen to give O2●- that can

be detoxified by the mitochondrial manganese superoxide dismutase to give H2O2 that, in

turn, can be converted to H2O by glutathione peroxidase. However, O2●- in the presence of

O2•-



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13

nitric oxide (●NO), formed during the conversion of arginine to citrulline by nitric oxide

synthase, can lead to peroxynitrite (ONOO-). Furthermore, H2O2 in the presence of reduced

transition metals can be converted to toxic HO●- via Fenton and/or Haber Weiss reactions.

Several efficient enzymatic processes are continuously operational to quench the reactive

species including superoxide dismutase, glutathione peroxidase, superoxide reductase,

catalase, peroxiredoxin and thioredoxin/thioredoxin reductase. Inevitably, if the amount of

free radical species produced overwhelms the neuronal capacity to neutralize them,

oxidative stress occurs, followed by mitochondrial dysfunction and neuronal damage (44).

ROS generated by mitochondria have several cellular targets, including

mitochondrial components themselves (lipids, proteins, and DNA). The lack of histones in

mitochondrial DNA and the diminished capacity for DNA repair render the mitochondria an

easy target to oxidative stress events. The accumulation of mitochondrial DNA mutations

enhances oxidative damage, causes energy depletion, and increases ROS production in a

vicious cycle (44,62).

The literature shows that mitochondrial dysfunction and oxidative stress are

important in the early pathology of AD. Indeed, there are strong indications that oxidative

stress occurs before the onset of symptoms in AD and that oxidative damage is found not

only in the vulnerable regions of the brain affected in disease but also peripherally (55).

Moreover, oxidative damage has been shown to occur before Aβ plaque formation,

supporting a causative role of mitochondrial dysfunction and oxidative stress in AD (63).

1.3. Phenolic compounds to treat and/or prevent Type 2 diabetes

mellitus and Alzheimer’s disease

Considering that dietary conditions that promote metabolic dysfunctions

connected with insulin resistance, such as type 2 diabetes, increase significantly the

relative risk of AD, the modulation of the brain metabolic act ivity by specific nutritional

conditions and bioactive compounds emerges as an attractive multitarget strategy to

prevent and/or slow down the progression of T2DM and AD-related brain degeneration

(64).

Epidemiological studies support the idea that long-term consumption of diets

rich in polyphenols (e.g. grape products) is associated with a decreased risk for both

T2DM (65,66) and AD (67–70). Based on several in vitro, animal models and some

human studies, polyphenol-rich products modulate carbohydrate and lipid metabolism,

attenuate hyperglycemia, dyslipidemia and insulin resistance, improve adipose tissue

metabolism, and alleviate oxidative stress, stress-sensitive signaling pathways and

inflammatory processes (71). For example, a work reported that phenolic-rich red and



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14

white grape pomace extract suppresses postprandial hyperglycemia in diabetic mice

following a potato starch challenge, most likely due to α-glucosidase activity inhibition

(72). Additionally, several in vitro and in vivo studies show that dietary polyphenols

exhibit neuroprotective effects and attenuate the progression of AD-like pathology and

memory deterioration in transgenic AD mice models. Polyphenols attenuate AD

phenotypes targeting multiple disease mechanisms, including reducing the amyloid-β

peptides generation and aggregation, decreasing the hyperphosphorylation of Tau

protein and neurofibrillary tangles formation, reducing the oxidation of cellular

biomolecules and attenuating neuroinflammation (73–79).



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15

2. Objectives



_______________________________________________________________________________________

16

Objectives

The general goal of the present work is to evaluate the potential of a mixture of white

wine polyphenols, recovered from the polyvinylpolypyrrolidone polymer (PVPP) used to

stabilize the white wine of Douro Region, to support preventive and/or therapeutic

approaches for type 2 diabetes mellitus and Alzheimer’s disease.

Thus, the specific objectives are:

• Characterization of the phenolic composition of PVPP-white wine extract by

high performance liquid chromatography with a photodiode detector (HPLC-

DAD);

• Assessment of the antioxidant activity of PVPP-white wine extract,

considering its ability to scavenge the superoxide radical and to prevent the

membrane lipids from the oxidation induced by Fe2+/ascorbate system;

• Evaluation of the capacity of the PVPP-white wine extract to modulate the

activity of α-amylase, α-glucosidase as well as the activity of brain

acetylcholinesterase, enzymes with relevance as therapeutic targets in the

context of T2DM and AD, respectively;

• Characterization of the toxicological profile of the PVPP-white wine extract

in SH-SY5Y cells, considering effects on cell viability and membrane

integrity;

• Evaluation of the ability of the PVPP-white wine extract to protect SH-SY5Y

cells against the excitotoxicity induced by glutamate, considering the effects

on cell viability, ROS generation and cell redox state.



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17

3. Material and Methods



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18

3.1. Standards and reagents

Catechin were purchased at Extrasynthese (Genay, France). Acarbose, α-amylase

(from porcine pancreas - Enzyme Commission number 3.2.1.1), α-glucosidase (from

Saccharomyces cerevisiae - Enzyme Commission number 3.2.1.20), ascorbic acid, bovine

serum albumin (BSA), caftaric acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), 3,5 – dinitrosalicylic acid,

5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), gallic acid, L- glutathione reduced (GSH), L-

glutathione oxidized (GSSG), L-glutamic acid monosodium salt monohydrate, iron(II)

sulfate heptahydrate, nitro blue tetrazolium chloride (NBT), 4-nitrophenyl α-D-

glucopyranoside (p-NGP), o-phthalaldehyde, resveratrol-3-O-glucoside, sodium hydroxide,

sodium phosphate, soluble starch, trypan blue, Triton™ X-100, xanthine (X), xanthine

oxidase (XO) (from bovine milk) (EC number 1.17.3.2) were purchased from Sigma-Aldrich

(St. Louis, MO, USA). ortho-phosphoric acid 85% was obtained from Panreac (Barcelona,

Spain). Ammonia solution 25%, potassium dihydrogen phosphate, potassium sodium

tartrate tetrahydrate, ethanol, methanol and formic acid of HPLC purity were obtained from

Merck (Darmstadt, Germany). Cis-parinaric acid probe, Dulbecco’s Modified Eagle Medium

(DMEM) + GlutaMAXTM‑I, heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin

solution (penicillin 5000 units/mL and streptomycin 5000 µg/mL), Hank’s balanced salt

solution (HBSS), 0.25% trypsin-EDTA were acquired from GIBCO, InvitrogenTM (Grand

Island, NY, USA). Water was deionized using a Milli-Q water purification system (Millipore,

Bedford, MA, USA). The SH-SY5Y cell line (ATCC® CRL-2266™) was acquired to American

Type Culture Collection (Manassas, Virginia, USA).

3.2. Preparation of white wine polyphenolic extract

PVPP is a water-insoluble synthetic polymer, which is used to adsorb phenolic

compounds from beverages (80). In wine Douro region, PVPP is used to treat white wine

(legal maximum limit: 80 g/hL (81)) in order to prevent adverse effects due to phenolic

oxidation and polymerization. PVPP adsorbs phenolic compounds, including those

responsible for browning reactions and bitterness, improving the stability of wine (82).

Figure 4 – Chemical structure of PVPP - (C6H9NO)n, used to stabilize the white wine of Douro region.



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19

In local winery, PVPP with adsorbed phenolic compounds was removed from the

wine by filtration and transported to the laboratory, keeping the temperature at 4ºC.

Polyphenolic compounds were extracted from the PVPP (100 g wet weight) with 250 mL of

alkaline ethanol solution (ethanol:100 mM of aqueous ammonia solution, 90:10, v/v) by

shaking for 30 minutes. The extract was filtrated in a Büchner filter (Whatman no. 1 filter

paper), neutralized with an aqueous solution of formic acid and evaporated by rotatory

evaporation to remove the ethanol. After evaporation step, the pH of the water extract was

adjusted to 5.5 and the extract was freeze-dried under vacuum at -50ºC to eliminate both

formic acid and ammonia, as volatile ammonium formate. The dried extract, referred as

PVPP-white wine extract in the present work, was stored at -20ºC for further analyses.

3.3. Determination of chemical composition of white wine extract

by HPLC-DAD

For HPLC-DAD, dried PVPP-white wine extract was redissolved in methanol, filtered

through 0.45 μm size pore membrane (Millipore, Bedford, MA, US) and analysed by reverse

phase chromatography to determine its phenolic composition. Analysis were performed

using a Gilson HPLC unit (Gilson Medical Electronics, Villiers le Bel, France) and a reverse

phase Spherisorb ODS 2 column (25.0 x 0.46 cm; 5 µm particle diameter; Waters, Milford,

MA, USA). Phenolics compounds were analyzed following a previously described method,

with some modifications (83). The mobile phase consisted of 5% aqueous formic acid

(eluent A) and 100% HPLC grade methanol (eluent B), using a gradient program (described

in the Table 3), with a flow rate of 0.9 mL/min and injection volume of 20 µL.

Detection was achieved by an Agilent 1100 series diode array detector (Agilent

Technologies, Waldbronn, Germany). Spectral data from all peaks were collected in the

range of 200–700 nm and chromatograms were recorded at 280, 320 and 350 nm. The data

were processed on a Clarity Software, version 5.04.158 (DataApex Ltd, Prague, Czech

Republic). The phenolic compounds were identified by comparing their retention times and

UV spectra with those of authentic standards.

Phenolic compounds quantification was achieved by interpolation with external

standard curves, as follows: hydroxybenzoic acids were quantified as gallic acid (280 nm),

hydroxycinnamic acids as caftaric acid (320 nm), flavan-3-ols and proanthocyanidins as (+)-

catechin (280 nm) and stilbenes as resveratrol-3-O-glucoside (320 nm). Standards and

samples were analysed in triplicate. Limit of quantification (LOQ) and limit of detection

(LOD) were calculated from the residual standard deviation (σ) of the regression equation

curve and the slope (S), according with the following equations: LOD = 3σ/S; LOQ = 10σ/S.



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20

Table 3 – Reverse phase gradient program for determination of phenolic composition of PVPP-white

wine extract.

Time (min) Eluent A Eluent B

0 95% 5%

3 85% 15%

13 75% 25%

25 70% 30%

35 65% 35%

39 55% 45%

47 45% 55%

56 25% 75%

60 0% 100%

66 95% 5%

3.4. Antioxidant activity – In vitro assays

3.4.1. Evaluation of superoxide radical scavenging activity

Superoxide radicals were generated by xanthine-xanthine oxidase (X/XO) system in

the presence of NBT, to allow its spectrophotometric quantification, according with coupled

reaction display in Figure 5. Superoxide radical scavenging activity was determined in the

presence and absence of PVPP-white wine extract (44, 88, 175 and 350 µg/mL).

Superoxide radicals were generated by the X/XO system, in which XO catalyses the

oxidation of xanthine to uric acid, generating O2•-, following a described procedure (84).

To each plate-well was added 206 µL of phosphate buffer (KH2PO4 50 mM, EDTA

1 mM, pH=7) (control), or 156 µL of phosphate buffer plus 50 µL of PVPP-white wine extract

solution with the required concentrations to obtain the above-mentioned final

concentrations, 20 µL of NBT (final concentration of 0.16 mM), and 4 µL of XO (final

concentration of 0.08 U/ml). The mixtures were incubated at 37 ºC for 5 min. The reaction

was initiated with the addition of 20 µL of xanthine (final concentration of 0.16 mM) and the

reaction was followed at 560 nm, during 5 minutes in Multiskan Ascent (Thermo Electron

Corporation) plate reader. For each concentration, a blank containing the PVPP-white wine

extract, but not the enzyme, was prepared, to evaluate the influence of samples absorption



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21

on the assay. Three independent experiments, each one in triplicate, were performed.

Results were expressed in percentage (%) from the following equation: Scavenging

activity= ((Abscontrol - Absextract)/Abscontrol) × 100), where Abscontrol and Absextract are the mean

of observed absorbance, respectively, in absence and presence of PVPP-white wine

extract. The half maximal effective concentration (EC50) was assessed considering the plots

of scavenging activity as a function of the extract concentration.

Figure 5 – Conversion of xanthine to uric acid with formation of formazan in the presence of NBT.

3.4.1.1. Effect on XO activity

The activity of XO was evaluated in the absence and presence of PVPP-white wine

extract (44, 88, 175, 350 µg/mL), by measuring the formation of uric acid from xanthine.

The reaction mixtures contained the same proportion of components as in the enzymatic

assay for superoxide radical scavenging activity, except NBT, which was substituted by

phosphate buffer. The mixtures were incubated at 37 ºC for 5 min. The reaction was initiated

with the addition of 20 µL of xanthine (final concentration of 0.16 mM) and the reaction was

followed at 293 nm, during 5 minutes in Multiskan Ascent (Thermo Electron Corporation)

plate reader. Three independent experiments, each one in triplicate, were performed. The

effect of the PVPP-white wine extract on XO are expressed as % of control, considering the

activity of XO in the absence of PVPP-white wine extract as 100%.

XO

2 O2 2 O2• -

Xanthine Uric acid

Formazan Tetrazolium



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22

3.4.2. Cis-parinaric acid assay

Cis-parinaric acid (9,11,13,15-octadecatetraenoic acid) is a fluorescent

polyunsaturated fatty acid usually used as a sensitive probe for the initial stages of lipid

oxidation (85). The ability of PVPP-white wine extract to protect the membrane lipids from

the oxidation promoted by oxidant agents was evaluated following the fluorescence decay

of cis-parinaric acid incorporated in membranes, as previously described by Mendes and

collaborators (64), with the required modifications for evaluation in microplate.

A mixture of Fe2+/ ascorbate (1:1) was used as oxidant agent and the matrix chosen

to induce lipid peroxidation was whole cell membranes of neuroblastoma SH-SY5Y cells.

Cells, cultured as monolayer as will be described below, were washed with HBSS,

trypsinized, centrifuged, resuspended in HBSS and stored at -20ºC. The protein

concentration of the cell suspensions was determined by the Biuret method using bovine

serum albumin (BSA) as standard (86).

The assays were performed at 30°C in the microplate-well, each one with 250 µL

potassium phosphate buffer (110 mM NaCl, 20 mM phosphate, pH 7.4), supplemented with

0.5 mg/mL of SH-SY5Y cell protein and 12.0 μM of cis-parinaric acid. PVPP-white wine

extract was incubated with the cell membranes for 5 min before probe addition. The reaction

was initiated by addition of a mixture of 250 µM ascorbic acid and 50 µM Fe2+ and monitored

by fluorescence intensity decay of cis-parinaric acid at 413 nm, setting excitation at 324 nm,

using a Synergy™ HT spectrofluorimeter (Biotek Instruments, Winooski, USA) operated by

Gen5 Software. The ability of PVPP-white wine extract to protect the lipids of cell

membranes against the oxidative degradation induced by Fe2+/ascorbate system was

determined using the value of the normalized cis-parinaric acid fluorescence 14 min after

the beginning of the reaction.

3.5. Effects on the activity of enzymes with therapeutic relevance

3.5.1. Determination of α-amylase activity

α-amylase activity was assessed as previously reported (87), with some

modifications. Equal volumes (200 μL) of extract and porcine pancreatic α-amylase (0.25

mg/ml) was added to each tube and samples were incubated at room temperature for 5

min. A volume of 1% starch solution in 20 mM sodium phosphate buffer (pH 6.9 with 6 mM

sodium chloride) were incubated in microtubes at 25 °C for 10 min. The reaction was

stopped with 400 μL of dinitrosalicyclic acid colour reagent and tubes were incubated at 100

°C for 5 min. Once samples had cooled to room temperature, 50 μL was removed from each

tube and transferred to the wells of 96-well microplate. The reaction mixture was diluted by



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23

adding 150 μL of water to each well and absorbance was measured at 500 nm. For each

concentration, a blank containing the PVPP-white wine extract, but not the enzyme, was

prepared to evaluate the influence of PVPP-white wine extract absorption on the assay. The

pharmacological inhibitor, acarbose, was used as a positive control. The activity of α-

amylase was calculated as follows: % activity= absorbance of sample/absorbance of control

* 100.

α-amylase activity was also evaluated in the absence and presence of three

concentrations of PVPP-white wine extract (50, 100 and 200 µg/mL), as a function of

substrate concentration, using seven concentrations of starch from 110 to 8800 µM, to

determine the Michaelis–Menten kinetic parameters Km (Michaelis constant) and Vmax

(maximal velocity).

3.5.2. Determination of α-glucosidase activity

The activity of α-glucosidase was assessed as previously reported (88), with some

modifications. Briefly, α-glucosidase activity was determined in the presence and absence

of PVPP-white wine extract (36.5 – 700.0 µg/mL), as well as in the presence of increasing

concentration of acarbose (10-600 µg/mL), an enzyme inhibitor with approval for clinical

use. To each plate-well was added 180 µL of phosphate buffer (KH2PO4 10 mM, pH=7), or

130 µL of phosphate buffer plus 50 µL of phenolic extract or acarbose at the desire

concentrations, and 20 µL of enzyme 0.05 U/ml. The mixtures were incubated at 37 ºC for

5 min and an absorbance initial was read at 405 nm. The reaction was initiated with a quick

addition of 100 µL of substrate - 4-nitrophenyl α-D-glucopyranoside (p-NGP) 2500 µM and

the kinetic reaction was followed at 405 nm for 4 minutes in Multiskan Ascent (Thermo

Electron Corporation) plate reader. The rates of reactions were calculated by Skanlt

Software version 4.1 (Thermo Scientific) and used to calculate the percentage of α-

glucosidase inhibition, as follows: % inhibition = 100 –((msample/mcontrol) * 100); m = slope.

α-glucosidase activity was also evaluated in the absence and presence of three

concentrations of PVPP-white wine extract (36.5, 146 and 389 µg/mL), as a function of

substrate concentration, using seven concentrations of p-NGP, 20 to 1700 µM, to determine

the Michaelis–Menten kinetic parameters Km (Michaelis constant) and Vmax (maximal

velocity). The mode of enzyme inhibition promoted by PVPP-white wine extract was studied

by using Lineweaver-Burk plots (LB). The enzyme inhibitor (Ki) and the enzyme substrate

inhibitor (Ki’) constants were calculated from secondary plots of LB: Plotting of slopes

obtained from the LB vs inhibitor concentrations gave the Ki values and intercepts vs

inhibitor concentrations gave the Ki’ values from the x-axis intercepts.



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24

3.5.3. Determination of acetylcholinesterase in rat brain

homogenates

Acetylcholinesterase (AChE) activity was evaluated in brain supernatants

(mitochondria-free cytosolic fraction) obtained from Wistar rat brain homogenates, as

previously described (89). Brains were diced and homogenized (1: 10 w/v) in cold buffer

containing 160 mM sucrose, 10 mM Tris–HCl and pH 7.2. The homogenates were

centrifuged at 16 000 g for 10 min, at 4ºC. The pellet was discarded, and the supernatant

used for the enzymatic assays. The protein concentration of supernatants was determined

by the biuret method using BSA as a standard (86). In supernatant, rat brain AChE activity

was determined colorimetrically by using the Ellman’s method (90) with the changes

previously described (89).

AChE activity was determined in the absence and presence of phenolic extracts of

PVPP (175, 350 and 700 µg/mL), as a function of substrate concentration by using seven

concentrations of acetylthiocholine iodide (15 to 2000 µM). To each plate-well was added

10 µl of brain homogenate, 125 µL of Ellman’s reagent (3 mM DTNB in 100 mM phosphate

buffer pH 7.4), 65 µL phosphate buffer (KH2PO4 100 mM, pH 7.4), or 15 µL of phosphate

buffer plus 50 µL of phenolic extract. The mixtures were incubated at 37 ºC for 2 min and

the absorbance was read at 405 nm. The reaction was initiated with the addition of 50 µL

of acetylthiocholine iodide solution at the required concentrations. Catalytic activity

(variation of the absorbance per unit of time and amount of protein in supernatant) was

measured by following the increase of the absorbance at 405 nm (i.e. the yellow anion 5-

thio-2-nitrobenzoate produced from thiocholine when it reacts with DTNB) for 3 min (kinetics

intervals of 10s), using Multiskan Ascent (Thermo Electron Corporation) plate reader. The

rates of reactions were calculated by Skanlt Software version 4.1 (Thermo Scientific).

Results were used to plot the AChE activity as function of substrate concentration in

the absence and presence of PVPP-white wine extract to determine the Michaelis–Menten

kinetic parameters Km (Michaelis constant) and Vmax (maximal velocity). The model of

enzyme inhibition promoted by PVPP-white wine extract was studied by using Lineweaver-

Burk plots (LB). The enzyme inhibitor (Ki) and the enzyme substrate inhibitor (Ki’) constants

were calculated from secondary plots of LB: Plotting of slopes obtained from the LB vs

inhibitor concentrations gave the Ki values and intercepts vs inhibitor concentrations gave

the Ki’ values from the x-axis intercepts.



_______________________________________________________________________________________

25

3.6. Cell-based assays

3.6.1. Culture conditions

The human neuroblastoma cell line (SH-SY5Y) from the American Type Culture

Collection (LGC Standards S.L.U., Barcelona, Spain) was cultured as monolayer T75 flasks

with DMEM + GlutaMAXTM medium, supplemented with 10% FBS and 1%

penicillin/streptomycin, at 37ºC, in a humidified atmosphere of 5% CO2. When cells reached

70-80% confluence, cells were washed with HBSS, treated with 2 mL of 0.25% trypsin-

EDTA solution for 3 min at 37 ºC. Afterwards, cell suspension was diluted with 5 mL of

culture medium, collected and centrifuged at 390 g for 4 min. The supernatant was removed,

and the cell pellet was resuspended in 3 mL of culture medium. A small aliquot of cell

suspension was diluted in trypan blue solution and used to estimate the number of viable

cells by using a Neubauer chamber.

3.6.2. Determination of cell viability and membrane integrity

3.6.2.1. MTT reduction assay

SH-SY5Y cell viability was evaluated using the MTT [(3-(4,5-dimethylthiazol 2-yl)-

2,5-diphenyltetrazolium] reduction. This method, first described by Mosmann (91) to

determine viable cell number, is based on reduction of MTT to formazan mainly promoted

by mitochondrial enzymes, which require cells with active metabolism. The effect of PVPP-

white extract on cell viability was assessed 24 h after exposure. The toxicity induced by

glutamate (25-400 mM) was evaluated after 4 hours of incubation. The protective effect of

PVPP-white extract against glutamate toxicity was assessed considering the pre-exposure

of cells to PVPP-white wine extract for 20h, before the exposure to glutamate (50 mM) for

4 hours.

To determine the cell viability, SH-SY5Y cells were seeded in 96-well plates at a

density of 30 000 cells/well. After 24 h, PVPP-white wine extract (1.8-58.4 µg/mL) were

added and plates were incubated for 24 h. Then, the medium was removed by vacuum

suction and cells incubated for 2 h, at 37 °C, with culture medium containing 0.5 mg/mL

MTT. Afterwards, the solution was removed, and formazan crystals were solubilized by the

addition of a DMSO:isopropanol mixture (3:1, v/v) and quantified spectrophotometrically at

560 nm using a microplate reader Multiskan Ascent (Thermo Electron Corporation). The

results of cell viability were expressed as the percentage of control. For each experimental

condition, at least, three independent experiments, each one in triplicate, were performed.



_______________________________________________________________________________________

26

3.6.2.2. Membrane integrity assay

Lactate dehydrogenase (LDH) activity was evaluated spectrophotometrically

recording the time-dependent nicotinamide adenine dinucleotide (NADH) oxidation at 340

nm, associated to the conversion of pyruvate to lactate, using a microplate reader Multiskan

Ascent (Thermo Electron Corporation) (92,93). The reaction was conducted using 50 μL of

cell-free supernatant of extracellular medium or 25 μL of supernatant of cell lysates

(obtained with 1% Triton X-100 in 10 mM phosphate buffer, pH = 7.2) in phosphate buffer

(50 mM KH2PO4, pH 7.4), supplemented with 10 μM pyruvate and 300 μM of NADH. Results

were expressed as the % of LDH activity in extracellular medium, considering as 100% the

total (intracellular plus extracellular) LDH activity. For each experimental condition, at least,

three independent experiments, each one in triplicate, were performed.

3.6.3. Measurement of intracellular reactive species

Intracellular reactive species were measured with DCFH-DA probe, as previously

described (94), with some modifications. Cells were incubated in black 96 well-plates with

a density of 30 000 cells/well in growth medium as described for MTT assay. SH-SY5Y cells

were treated with PVPP-white wine extract (0- 58.4 µg/mL) for 24 hours, before to be

submitted to excitatory toxic stimulus promoted by addition of 50 mM glutamate. After 1

hour incubated with glutamate, the medium was removed, cells were washed with HBSS

and 100 µL of a solution DCFH-DA (10 µM) in HBSS was added to each well. Kinetic of

intracellular ROS production was recorded for 45 minutes, at 37ºC, following the

fluorescence emission of dichlorofluorescein at 520 nm under excitation at 490 nm in a

microplate spectrofluorimeter (SynergyTM HT, Biotek Instruments Winooski, USA) operated

by Gen5 Software.

3.6.4. Determination of GSH and GSSG levels

To evaluate the intracellular levels of GSH and GSSG, SH-SY5Y cells were

incubated in 12-well plates with a density of 250 000 cells/well in growth medium and

submitted to the experimental condition described above: in the absence and presence of

PVPP-white wine extract (7.3 and 14.6 µg/mL, 20 hours) without and with glutamate (50

mM, 4 hours) addition. After cell treatments/incubation, the medium was removed, cells

were washed with HBSS and frozen at -80 ºC, for 24 h, to stop the metabolism. Then, cells

were collected with 300 µL of lyse solution (2 % Triton X-100 in 5 mM phosphate buffer, 5

mM EDTA, pH = 7.2). An aliquot of cell lysates was used to assess the protein concentration

by Bradford method (95,96), and the remaining (about 0.3 mg of protein) were reconstituted

in 500 mL of buffer (100 mM NaH2PO4, 5 mM EDTA, pH=8), 100 µL of H3PO4 2.5% was



_______________________________________________________________________________________

27

added, and the cell lysates were centrifuged at 16 000 g for 30 min at 4°C to promote

deproteinization. Supernatants were collected, neutralized with NaOH and used to measure

GSH and GSSG levels after reaction with fluorochrome o-phthalaldehyde (OPT) by a

fluorimetric assay, using a microplate spectrofluorimeter (SynergyTM HT, Biotek Instruments

Winooski, USA), setting excitation at 339 nm and emission at 426 nm, as previously

described (64).

To determine GSH content, supernatants were incubated at room temperature in

2.5 mL of phosphate buffer (100 mM potassium phosphate, 5 mM EDTA, pH 8.0)

supplemented with 200 µg OPT for 15 min. To determine GSSG content, supernatants were

first incubated at room temperature with N-ethylmaleimide for 30 min. After that, 1.3 mL of

0.1 M NaOH were added, followed by the addition of 200 μg OPT to the reaction followed

by 15-min incubation at room temperature. Concentrations were calculated using standard

curves prepared with different concentrations of GSH and GSSG which underwent the

same sample treatment. The results were expressed as GSH/GSSG ratio. The levels of

GSH and GSSG were normalized by the amount of protein and GSH total were calculated

as: [GSH total] = [GSH] + 2[GSSG]. Protein content was measured using Bradford method

with BSA as standard (95,96).

3.7. Statistical analysis

All results were expressed as mean ± standard error of the mean (SEM) from at

least three independent assays, performed in triplicate. All statistical calculations were

made with GraphPad Prism version 6.00 (GraphPad Software, San Diego, California, USA).

Statistical comparison between groups was estimated using the parametric method of one-

way ANOVA on ranks followed by the Bonferroni’s post hoc test. In all cases, p values lower

than 0.05 were considered statistically significant.



_______________________________________________________________________________________

28

4. Results and Discussion



_______________________________________________________________________________________

29

4.1. Chemical composition of PVPP-white wine extract - HPLC-DAD

analysis

One of most serious problem in white wine stability is oxidative browning, which is

intrinsically related to the oxidation of phenolic compounds by polyphenoloxidases to form

quinones, which are highly reactive species involved in different reaction pathways, leading

to the formation of yellow or brown products (97,98). Caftaric acid and flavan-3-ols are

determinant compounds in browning (99,100). To avoid the browning phenomenon, it is

common to subject the white wine to a fining process.

Water insoluble-PVPP is a synthetic polymer, produced by cross-linking

polyvinylpyrrolidone (PVP), which is used to adsorb phenols from beverages in fining

process, including white wines. It is thought that PVPP interact with polyphenols via H bonds

between their CO-N linkages and the hydroxyl functions and by hydrophobic interactions

between the phenolic and pyrrolidinone rings (80). Thus, at low PVPP concentration, the

phenolic compounds affinity by the polymer increases with increasing of the number of

phenolic hydroxyl groups, since they exhibit higher ability to form hydrogen bonding

(80,101). Therefore, treatment of white wine with PVPP (80 g/100 L) allows not only to

improve the color and redox stability of wine but also the recovery of high-purified phenolic

extracts, enriched in catechins and their derivatives (e.g. proanthocyanidins), which have

potential to be used in food and pharmacological applications (64).

HPLC-DAD analysis revealed that PVPP-white wine extract obtained from a white

wine of Douro region is a complex mixture, composed by different classes of phenolic

compounds, including, at least, two hydroxybenzoic acids, four hydroxycinnamic acids,

several catechin derivatives and two stilbenes, as resveratrol derivatives (Figure 6 and

Table 4). Compounds identification was established according to the retention time and UV

spectra features, while the quantification of each phenolic compound was determined by

the external standard method (Table 5).

Results display in Figure 6 and Table 4 show PVPP-white wine extract is particularly

rich in phenolic acids (gallic, caftaric and chlorogenic acids) and proanthocyanidins. The

amount of total phenolic compounds obtained from PVPP was 803.70 µg/mg of freeze-dried

extract, indicating high purity in terms of phenolic compounds. Proanthocyanidins (465.65

µg/mg) and caftaric acid (105.56 µg/mg) are the dominant compounds, representing more

than 70% of the total phenolic content. In other hand, stilbenes are the minor compounds,

account only for 2% of total content. These data are in accordance with those reported by

Mendes et al (64), where PVPP was used to treat a white wine of Douro region from harvest

2015. As in the present work, in the PVPP extract obtained by Mendes et al (64) the



_______________________________________________________________________________________

30

proanthocyanidins and caftaric acid are the compounds with higher relative abundance and

the total phenolic compounds represent about 80% of freeze-dried weight extract.

Figure 6 – HPLC polyphenolic profile of PVPP-white wine extract. Phenolic compounds were

analyzed by HPLC-DAD, using a reverse phase column (Spherisorb ODS 2; 25.0 x 0.46 cm; 5 µm,

particle diameter) with spectra acquisition in continuous scan mode from 200 to 700 nm.

Chromatograms were recorded at 280, 320 and 350 nm. Identification and quantification of the

phenolic compounds corresponding to each one of the numbered peaks in chromatograms are

display in Table 4.

According to literature, hydroxycinnamic acids and their tartaric esters are the main

class of phenolics in white wines (8,102). Therefore, these data support the idea that PVPP

allows to obtain extracts with a consistent phenolic composition, but different from that of

the wine to which it was added.

0

50

100

150

200

250

2 4 6 8 12 14 16 18 22 24 26 28 32 34 36

1020

30

l350 nm

l320 nm

l280 nm

1

2

3

45

6

7

8

9

10

11



_______________________________________________________________________________________

31

Table 4 – Phenolic composition of PVPP-white wine extract determined by HPLC-DAD.

Peak

number

Retention time

(min) Compound

Quantification

(µg/mg PVPP-white wine

extract)

1 6.45 Gallic acid 34.26 ± 5.26

2 8.75 Protocatechuic acid 10.36 ± 3.54

3 10.15 Caftaric acid 105.56 ± 23.81

4 11.56 Hydroxycinnamic acid 7.17 ± 0.80

5 12.67 Hydroxycinnamic acid 21.59 ± 1.31

6 15.72 Chlorogenic acid 57.24 ± 10.10

7 17.51 Catechin derivative 53.44 ± 9.27

8 21.21 Resveratrol-3-O-glucoside 10.47 ± 1.86

9 22.96 Proanthocyanidin 465.65 ± 88.64

10 25.56 Resveratrol derivative 7.28 ± 0.23

11 30.50 Catechin derivative 67.06 ± 16.43

Total phenolic compounds 803.70 ± 88.64

Hydroxybenzoic acids 44.62 ± 7.38

Hydroxycinnamic acids 191.58 ± 19.05

Catechins plus proanthocyanidins 556.02 ± 101.77

Stilbenes 15.32 ± 4.34

Peak number, retention time, identification and quantification of each phenolic compound, considering three

extractions by interpolation with external standard curves, as follows: gallic and protocatechuic acid as gallic

acid, hydroxycinnamic acids as caftaric acid, catechin derivatives and proanthocyanidins as (+)-catechin, and

resveratrol derivatives as resveratrol-3-O-glucoside. The linear equations are presented in Table 5. The

concentrations of identified phenolic compounds are expressed as mean ± standard deviation (SD).



_______________________________________________________________________________________

32

Table 5 – Regression equations, LOD and LOQ data for the quantification of the identified phenolic

compounds in PVPP-white wine extract.

Standard Regression equation R2 Range of

concentrations (µg/mL) LODa LOQb

Gallic acid y = 62.81 – 19.31 0.999 2.9-57.5 0.66 2.19

Caftaric acid y = 48.86 𝑥 + 111.21 0.996 5.5-44.0 1.01 3.37

Catechin y = 19.90 𝑥 + 2.75 0.999 1.55-31.0 0.41 1.37

Resveratrol-3-

glucoside y = 106.17 𝑥 + 30.15 0.999 6.55-131.0 0.26 0.88

a LOD – limit of detection; b LOQ – limit of quantification.

4.2. Antioxidant activity – In vitro assays

4.2.1. Superoxide radical scavenging activity

Phenolic compounds are recognized as valuable exogenous antioxidants since they

counteract the oxidative stress through direct radical scavenging and enzymatic inhibition

or indirectly by modulation of signal pathways (103).

Superoxide anion radical (O2•-) is an endogenous reactive oxygen species (ROS)

essential for life of the aerobic organisms, since it is produced by several biochemical

pathways and works as a signalling molecule with impact on regulation of numerous

physiological processes, including apoptosis, aging, and senescence. Nevertheless, when

overproduction of O2•- occurs and/or antioxidant defences are deficient, it can promote the

oxidative damage of biomolecules altering their physiological function. Additionally, it is also

precursor of other important reactive oxygen species, like hydroxyl radical and peroxynitrite,

which are also involved in the pathological process of many human diseases, including type

II diabetes and Alzheimer’s disease (24,104). Thus, the scavenging effect of PVPP-white

wine extract against superoxide anion radical produced by the X/XO system was assessed

in cell-free assays by the decreasing of the NBT reduction, and the results are display in

Figure 7A. PVPP-white wine extract showed a strong ability to scavenge superoxide anion

radical in a concentration dependent manner, with an EC50 equal to 153.5 µg/mL.



_______________________________________________________________________________________

33

Figure 7 – Free-radical scavenger activity of PVPP-white wine extract as function of increasing

concentrations of phenolics. (A) Superoxide anion radical (O2●-) scavenging activity of PVPP-white

wine extract. The radicals were generated by xanthine/xanthine oxidase system. (B) Xanthine

oxidase activity in absence and presence of increasing concentrations of PVPP-white wine extract.

Data are expressed as mean ± SEM of three independent assays, each one performed in triplicate.

Taking in account that a putative inhibition of xanthine oxidase by the phenolic

compounds may lead to a decrease in NBT reduction, the effect of the PVPP-white wine

extract on the XO activity were evaluated following the conversion of xanthine to uric acid.

As shown in Figure 7B, the extract did not present significant effects on the activity of the

enzyme. Thus, PVPP-white wine extract is able to scavenge the superoxide anion radicals,

but not to prevent its generation by xanthine oxidase.

4.2.2. Protective effects against lipid peroxidation

Ascorbic acid (vitamin C) can act as an antioxidant as well as a prooxidant

depending on the conditions. Its antioxidant activity is derived from its ability to scavenge

radicals in the water phase, while it may also enhance radical formation by reductive

recycling of metal ions (105,106). In fact, the combination of iron with ascorbic acid is

frequently used as model systems to induce oxidative stress (106–108), including to

evaluate the lipid peroxidation based on the oxidation of cis-parinaric acid, a membrane

fluorescent probe (109).

In the present work, the capacity of PVPP-white wine extract to protect membrane

lipids against oxidative degradation induced by Fe2+ (50 µM)/ascorbic acid (250 µM) system

was also evaluated. Cis-parinaric fluorescent probe was incorporated into the membranes

of SH-SY5Y cells, in the absence and presence of different concentrations of the PVPP-

white wine extract, and the oxidation of the cis-parinaric acid was measured following the

fluorescence decay at 413 nm (excitation 324 nm) as a function of the time (Figure 8). The

addiction of the Fe2+/ascorbic acid system promotes a time-dependent oxidation of the

O2

•-

sc

av

en

gin

g a

cti

vit

y (

%)

1 .0 1 .5 2 .0 2 .5 3 .0

0

2 0

4 0

6 0

8 0

E C 5 0 = 1 5 3 .5 g /m L

L o g [P V P P -w h ite w in e e x tra c t ] ( g /m L )

Xa

nth

ine

Ox

ida

se

Ac

tiv

ity

(% o

f c

on

tro

l)

044

88

175

350

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

[P V P P -w h ite w in e e x tra c t ] ( g /m L )

A B



_______________________________________________________________________________________

34

membrane probe, detected by a decrease in the fluorescence intensity. The kinetics of

fluorescence decay can be described by a single-exponential first-order equation. The pre-

incubation with increasing concentrations of PVPP-white wine extract decreases in a

concentration-dependent manner the apparent kinetic constant of fluorescence decay. In

fact, plotting the percentage of protection of cis-parinaric oxidation 14 min after the addition

of the oxidative agent as function of PVPP-white wine extract concentration, an EC50 of 119

µg/mL was estimated (Figure 8B).

Results of the Figure 8 indicate that the phenolic compounds of PVPP-white wine

extract may be able to protect the membrane probe, and supposedly also the

polyunsaturated acyl chains of the phospholipids of the SH-SY5Y cell membranes, by the

oxidative degradation promoted by Fe2+/ascorbic acid system, suggesting that they have a

activity not only in aqueous system but also in biological membranes, as previously reported

(64).

Figure 8 – Evaluation of the capacity of PVPP-white wine extract to protect the lipids of the cellular

membranes from the oxidation induced by Fe2+ (50 µM) and ascorbic acid (250 µM) system. (A) A

typical experiment representing the kinetic of the oxidation of the cis-parinaric acid incorporated into

the cellular membrane in the absence and presence of increasing concentrations of PVPP-white

wine extract; (B) The percentage of protection of cis-parinaric oxidation 14 min after the addition of

the oxidative agent, as function of different concentrations of PVPP-white wine extract. Results are

expressed as mean ± SEM for three independent experiments, performed in triplicated.

4.3. Effects on the activity of enzymes with relevance as

therapeutic targets

4.3.1. α-amylase activity

Post-prandial hyperglycemia was recognized as an important therapeutic target in

T2DM, since a stronger relationship between the postprandial glycemia and microvascular

and macrovascular complications in diabetes patients was established. Thus, the inhibition

T im e (m in )

No

rm

ali

ze

d f

luo

re

sc

en

ce

(F

/F0

)

0 2 4 6 8 1 0 1 2 1 4 1 6

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

400

200

100

50

0

[PV

PP

-wh

ite w

ine

ex

tra

ct]

(

g/m

L)

C o n t r o l (w ith o u t p r o -o x id a n t )


An

tio

xid

an

t a

cti

vit

y o

r

Pro

tec

tio

n o

f p

ro

be

ox

ida

tio

n(%

)

1 .5 2 .0 2 .5 3 .0

0

2 0

4 0

6 0

8 0

1 0 0

E C 5 0 = 1 1 9 .0 g /m L

A B



_______________________________________________________________________________________

35

of the carbohydrate hydrolysing enzymes, α-amylase and α-glucosidase, to regulate the

availability of glucose for intestinal absorption by modification of carbohydrate digestion is

considered one of the most effective approaches for diabetes care (30,31). Currently, there

are some antidiabetic drugs, namely, acarbose, miglitol and voglibose, which act by

inhibiting α-amylase and α-glucosidase activity. While efficient in attenuating the rise in

blood glucose levels in many patients, the continuous use of these drugs is often associated

with undesirable side effects, such as liver toxicity and adverse gastrointestinal symptoms

(30,31). Thus, there is a need for natural inhibitors of these enzymes which have low

secondary effects.

The effect of PVPP-white wine extract on the activity of α-amylase was investigated

using starch as substrate, and the results are display in Figure 9. The α-amylase activity as

a function of substrate concentration follows a Michaelis–Menten kinetics equation, with the

values of apparent kinetic parameters (Km and Vmax) indicated in the Table 6. The Michaelis–

Menten kinetic equation fits reasonably well with the experimental data as reflected by low

2 values, approaching zero, and high correlation factors (R). Results of the Figure 9 show

that the extract did not inhibit α-amylase up to concentration of 200 µg/mL. Acarbose, which

is an inhibitor of this enzyme with approval for clinical use, was used as positive control and

an IC50 of 4.24 µg/mL was obtained (Figure 10), in agreement with the values previously

reported by others researchers that have use the similar procedures (110). However, some

previous studies present grape phenolic compounds as strong inhibitors of α-amylase.

Yilmazer-Musa et al (111) showed that a grape seed extract, rich in flavan-3-ols monomers

and procyanidins (dimeric, trimeric and oligomeric flavan-3-ols), is a potent inhibitor of

human salivary α-amylase (2.5 mU/mL), with similar results to those obtain with acarbose,

suggesting that procyanidins of grape seed strongly inhibit α-amylase activity. Other study

also report that an extract obtained from white grape skins recovered from wineries, rich in

monomeric flavanols, monomeric flavonols and either galloylated and non-galloylated

oligomeric proanthocyanidins, inhibits mammalian α-amylase (and α-glucosidase) more

efficiently than acarbose (112).



_______________________________________________________________________________________

36

Figure 9 – Kinetic of α-amylase activity in the absence and presence of increasing concentrations of

PVPP-white wine extract. Data were fitted into a Michaelis–Menten kinetic equation using GraphPad

Prism software error-minimization procedure with 2 between successive iterations of < 0.001%.

Each point represents the mean ± SEM of three independent assays, each one performed in

triplicate.

Figure 10 – α-amylase inhibitory activity of acarbose, an inhibitor of this enzyme with approval clinical

use. Results are express as mean ± SEM of four independent experiments performed in triplicated.

IC50 was calculated by GraphPad Prism software and presented in graph.

Additionally, other plant phenolic compounds, including flavonoids, such as

naringenin, kaempferol, luteolin, apigenin, (+)-catechin, (-)-epicatechin, daidzein and

epigallocatechin gallate, have been described as potential inhibitors of α-amylase, being

the flavonols more potent than flavan-3-ols. Therefore, the lack of inhibitory capacity by

PVPP-white wine extract may emerge from its chemical composition, characterized by the

absence of flavonols and dominated by proanthocyanidins and phenolic acids (113).

[S ta rc h ] ( M )

0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

0 .1 2

C o n tro l

5 0 g /m L

1 0 0 g /m l

2 0 0 g /m LV

ap

p

A

bs

t

(UA

/min

)

L o g [A c a rb o s e ] ( g /m L )

-a

my

las

e I

nh

ibit

ion

(%)

0 .0 0 .5 1 .0 1 .5 2 .0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

IC 5 0 = 4 .2 4 g /m L



_______________________________________________________________________________________

37

Table 6 – Kinetic parameters (Km and Vmax), chi-squared (2), correlation factor (R) of α-amylase in

the absence and presence of PVPP-white wine extract.

Kinetic

model

Kinetics

parameters

PVPP-White wine extract (µg/mL)

0 50 100 200

Michaelis-

Menten

Vmax 0.121 ± 0.006 0.122 ± 0.008 0.134 ± 0.007 0.133 ± 0.007

Km 3323.0 ± 380.4 2909.0 ± 440.2 2812.0 ± 338.4 2846.0 ± 351.0

2 0.0059 0.0060 0.0054 0.0054

R 0.966 0.968 0.979 0.978

Data relative to Vmax and Km are expressed as mean ± SEM.

4.3.2. α-glucosidase activity

The effect of PVPP-white wine extract on the activity of α-glucosidase was evaluated

using p-NPG as substrate, and the obtained results are display in the Figures 11 and 12.

Acarbose was used as positive control. Considering the array of assays carry out in the

presence of excess of enzyme substrate, PVPP-white wine extract as well as acarbose

decrease the activity of the enzyme in a dose-dependent manner. Plotting the % of α-

glucosidase inhibition as function of PVPP-white wine extract (Figure 11A) or as function of

acarbose concentration (Figure 11B) allowed to determine the IC50 values, i.e.

concentration of extract (or acarbose) required to decrease in 50% the original enzyme

activity. The IC50 values were used to evaluate the effectiveness of the extract to inhibit the

enzyme. For acarbose, an IC50 value of 252.2 µg/mL was obtained, which was 60 times

higher than IC50 value obtained for α-amylase, indicating that the acarbose are most

effective to inhibit α -amylase than α-glucosidase. Interestingly, PVPP-white wine extract,

while not inhibiting α-amylase, is able to inhibit α-glucosidase with an IC50 of 337.4 µg/mL,

which is very similar to the value obtained with acarbose.

There have been many reports of natural compounds with α-glucosidase inhibitory

activity (29). Tadera et al (113) demonstrated that yeast α-glucosidase is potently inhibited

by anthocyanidin, isoflavones and flavonol groups and epigallocatechin gallate, while other

flavan-3-ols, such as catechin, epicatechin and epigallocatechin, also inhibit the enzyme,

but with lower effectiveness. In addition, proanthocyanidins have also ability to inhibit this

enzyme (114). Thus, the inhibitory activity exhibited by PVPP-white wine extract may be

connected with its high content in catechins and proanthocyanidins.



_______________________________________________________________________________________

38

Figure 11 – α-glucosidase inhibitory activity of PVPP-white wine extract (A) and acarbose, an

inhibitor of this enzyme with approval clinical use (B). Results are express as mean ± SEM of at least

three independent experiments performed in triplicated. IC50 values were calculated by GraphPad

Prism software and presented in graphs.

Results from a study of Yilmazer-Musa et al showed that grape seed extract strongly

inhibit yeast α-glucosidase (20 mU/mL) (IC50 = 1.2 µg/mL), with much higher potency than

acarbose, which in their experimental procedure exhibited an IC50 of 91.0 µg/mL (111).

Lavelli et al also demonstrated that extracts of white grape skins could inhibit mammalian

α-glucosidase more efficiently than acarbose (112). The fact of PVPP-white wine extract

not present better efficacy than acarbose may be justified by the absence of flavonols.

Indeed, studies on purified phenolic compounds have revealed that flavonol compounds,

such as quercetin and its derivatives have a high affinity towards the α-glucosidase enzyme

in vitro (115). However, it is worth noting that although PVPP-white wine extract is not more

efficient than acarbose, it has an additional advantage, since it is available in large quantity

as a low-cost byproduct of winemaking. Thus, PVPP-white wine extract could provide

sustainable inhibitors of α-glucosidase.

An inhibitor can interact with an enzyme by different ways: the inhibition can be

competitive, uncompetitive, noncompetitive, or mixed. Studies of kinetics allows to

distinguish between inhibitory mechanisms (116–118). The type of inhibition by PVPP-white

wine extract on α-glucosidase activity was determined by both nonlinear and linear method

using Michaelis-Menten and Lineweaver-Burk plots. As shown in Figure 12, the α-

glucosidase activity as a function of substrate concentration follows a Michaelis–Menten

kinetics equation, with the values of apparent kinetic parameters (Km and Vmax) indicated in

the Table 7. The Michaelis–Menten kinetic equation fits reasonably well with the

experimental data as reflected by low 2 values, approaching zero, and high correlation

factors (R).


-g

luc

os

ida

se

In

hib

itio

n

)

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5

-2 0

0

2 0

4 0

6 0

8 0

1 0 0

IC 5 0 = 3 3 7 .4 g /m L

L o g [A c a rb o s e ] ( g /m L )

-g

luc

os

ida

se

In

hib

itio

n(%

)

1 .0 1 .5 2 .0 2 .5 3 .0

0

2 0

4 0

6 0

8 0

1 0 0

IC 5 0 = 2 5 2 .2 g /m L

A B



_______________________________________________________________________________________

39

According to the Michaelis-Menten plots, the PVPP-white wine extract decreased

the activity of the enzyme (Figure 12A). The Vmax was significantly reduced by the addiction

of the extract, compared to the control (Table 7). However, no significative changes were

notice on values of Km, suggesting that compounds of PVPP-white wine extract are non-

competitive inhibitors. To further confirm the inhibition type, double reciprocal plots

(Lineweaver-Burk plots) were obtained, presenting a family of straight lines with different

slopes with a common intersect in the X-axis (Figure 12B). Ki, and Ki’, which are the

dissociation constants of the enzyme-inhibitor complex and the enzyme-substrate-inhibitor

complex, respectively, were determined from the slope and intercept on the vertical axis

against inhibitor concentrations as 161.6 and 162.4 (Figure 12C,D). Therefore, these results

are consistent with the idea that the inhibition is reversible and that the compounds of the

PVPP-white wine extract reduces the enzyme activity but does not affect the binding of

substrate to enzyme.

Figure 12 – Kinetics of α-glucosidase activity in the absence and presence of increasing

concentrations of PVPP-white wine extract. (A) Data were fitted into a Michaelis–Menten kinetic

equation using GraphPad Prism software error-minimization procedure with 2 between successive

iterations of < 0.001%. (B) Lineweaver-Burk plot analysis of the inhibition kinetics of α-glucosidase.

(C) Linear secondary plot of slope of the straight lines of 1/v vs. 1/[S] against the inhibitor

concentration to determine Ki, the disassociation constant for competitive inhibition. (D) Linear

secondary plot of 1/Vmax against the inhibitor concentration to determine the value of Ki’, the inhibitor

constant for non-competitive inhibition. Each point represents the mean ± SEM of three independent

assays, each one performed in triplicate.

[ p -N G P ] ( M )

-g

luc

os

ida

se

ac

tiv

ity

(Ab

s/m

in)

0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

C o n tro l

3 6 .5 g /m L

1 4 6 g /m L

3 8 9 g /m L

1/V

1/(

M

/min

)

-0 .0 2 5 0 .0 2 5 0 .0 5 0

-2 0 0

2 0 0

4 0 0

6 0 0

C o n tro l

3 6 .5 g /m L

1 4 6 g /m L

3 8 9 g /m L

1 /[S u b s tra te ] ( M-1

)

In h ib ito r ( g /m L )

Slo

pe

-3 0 0 0 3 0 0 6 0 0

4 0 0 0

8 0 0 0

1 2 0 0 0

K iIn h ib ito r ( g /m L )

1/V

ma

x'

-3 0 0 0 3 0 0 6 0 0

1 0

2 0

3 0

4 0

5 0

K ii

A

C D

B



_______________________________________________________________________________________

40

Table 7 – Characterization of the type of inhibition induced by PVPP-white wine extract on α-

glucosidase. Kinetic parameters (Km and Vmax), chi-squared (2), correlation factor (R), and inhibition

constants used to determine the type of inhibition.

Kinetic

model

Kinetics

parameters


0 36.5 146 389

Michaelis-

Menten

Vmax 0.076 ± 0.004 0.055 ± 0.003*** 0.046 ± 0.001*** 0.022 ± 0.001***

Km 193.3 ± 26.58 172.2 ± 24.63 225.5 ± 18.68 238.3 ± 26.35

2 0.003103 0.002395 0.001067 0.0006750

R 0.985 0.983 0.995 0.990

Lineweaver

-Burk

Slope

(=Km/Vmax) 3228 ± 125.8 3699 ±57.57* 4821 ± 184.0*** 10157 ± 252.9***

1/Vmax 10.19 ± 2.496 16.51 ± 1.142 22.37 ± 3.650 51.25 ± 5.015

1/Km -0.003158 -0.004465 -0.004640 -0.005046

2 5.385 2.464 7.874 10.82

R 0.991 0.999 0.991 0.996

Secondary

plots

Ki 161.6

Ki’ 162.4

Type of

inhibition Non-competitive

Data relative to Vmax, Km, slope and 1/Vmax are expressed as mean ± SEM. Comparisons relative to

the control were performed using one-way ANOVA with Bonferroni as post-test (*p < 0.05; ***p <

0.001).

4.3.3. Acetylcholinesterase activity

The pharmacological modulation of cholinergic brain activity supported by AChE

inhibitors is an approved therapeutic strategy for the treatment of AD and other forms of

dementia (49). AChE inhibitors lead to the accumulation of acetylcholine synapses region,

increasing the probability of the nicotinic and muscarinic receptors in the post-synaptic

neurons to be stimulated, which have positive outcomes on Alzheimer brains characterized

by depressed acetylcholine-dependent neuronal networks. However, the current available



_______________________________________________________________________________________

41

drugs with AChE inhibitory activity (e.g. tacrine, rivastigmine and donezepil) demonstrate

only modest symptomatic benefits and also exhibit negative side effects (48). Consequently,

there is a need to finding new drugs to treat the brain diseases, including AD.

Figure 13A shows the AChE activity in mitochondria-free rat brain homogenates in

the absence and presence of increasing concentrations of PVPP-white wine, as function of

substrate concentration. For all experimental conditions, data follow a Michaelis-Menten

kinetics equation, with the values of apparent kinetic parameters (Km and Vmax) display in

Table 8. In the absence of PVPP-white wine extract, the values of Vmax of 0.0563 ± 0.0013

µmol.min.mg protein and a Km of 58.79 ± 5.938 µM was estimated for rat brain AChE, which

is in agreement with the values previously reported (89). Data of Figure 13 also reveals that

PVPP-white wine extract promotes a concentration-dependent AChE inhibition, decreasing

Vmax without significant effects on the Km of the enzyme, suggesting a possible inhibition of

non-competitive type. However, the effects on Vmax not did not allow to estimate the IC50

value, since the inhibition only reach 30% for the maximal concentration of extract tested.

To characterize the type of inhibition promoted by PVPP-white wine extract on brain

AChE, data were also analyzed by linear method using Lineweaver-Burk plots, which reveal

a family of straight lines with different slopes with a common intersect in the 1/[S] axis

(Figure 13B). Plotting the slope and intercept on the vertical axis as function of inhibitor

concentration (PVPP-white wine extract) it was possible to estimate a value of 844.5 for Ki,

and a value of 769.5 for Ki’, as shown in Figure 13C,D, suggesting that PVPP-white wine

extract works as reversible noncompetitive inhibitor.

Some works, carry out with commercially available AChE, have reported that

several phenolic compounds, including catechin, epicatechin, anthocyanins and

chlorogenic acid, show a strong ability to inhibit AChE, and sometimes that inhibition reach

100% within the concentration range that was used in the present work (119–122). Since in

the present study the AChE inhibition promoted by PVPP-white wine extract only reach a

maximal of 30%, it can be considered a week inhibitor, in particular when compared with

the above-mentioned studies. However, it is important to highlight that the commercially

available AChE(s) are water-soluble monomeric structure, while in mammalian brains the

AChE occurs attached to plasma membrane mainly as high functional tetramers (123).

Thus, in present study mitochondria-free rat brain homogenates were used to evaluate the

enzyme activity, which mimic the conditions that occurs in biological system. In fact, the

mitochondria-free brain homogenates have not only the AChE but also many other proteins

as well several types of membranes, including the plasma membrane of neurons that

anchors the AChE as tetramers, all of them with potential to compete with AChE for the

phenolic compounds. Thus, the present assays show high biological relevance and PVPP-

white wine extract exhibits an interesting AChE inhibition.



_______________________________________________________________________________________

42

Figure 13 – Kinetics of rat brain AChE activity in the absence and presence of increasing

concentrations of PVPP-white wine extract. (A) Data were fitted into a Michaelis–Menten kinetic

equation using GraphPad Prism software error-minimization procedure with 2 between successive

iterations of < 0.001%. (B) Lineweaver-Burk plot analysis of the inhibition kinetics of AChE. (C)

Linear secondary plot of slope of the straight lines of 1/v vs. 1/[S] against the inhibitor concentration

to determine Ki, the disassociation constant for competitive inhibition. (D) Linear secondary plot of

1/Vmax against the inhibitor concentration to determine the value of Ki’, the inhibitor constant for non-

competitive inhibition. Each point represents the mean ± SEM of three independent assays, each

one performed in triplicate.

[A c e ty lth io c h o lin e ] ( M )

AC

hE

ac

tiv

ity

(

mo

l.m

in.m

g p

ro

tein

)

0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0

0 .0 0

0 .0 2

0 .0 4

0 .0 6

C o n tro l

1 7 5 g /m L

3 5 0 g /m L

7 0 0 g /m L

1/V

1/(

M

/min

)

-0 .0 2 5 0 .0 2 5 0 .0 5 0

-5 0

5 0

1 0 0

1 5 0

C o n tro l

1 7 5 g /m L

3 5 0 g /m L

7 0 0 g /m L

1 / [S u b s tra te ] ( M-1

)


Slo

pe

-1 0 0 0 -5 0 0 0 5 0 0 1 0 0 0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

K i


1/V

ma

x'

-1 0 0 0 -5 0 0 0 5 0 0 1 0 0 0

1 0

2 0

3 0

4 0

K ii

A B

C D



_______________________________________________________________________________________

43

Table 8 – Characterization of the type of inhibition induced by PVPP-white wine extract on rat brain

AChE. Kinetic parameters (Km and Vmax), chi-squared (2), correlation factor (R), and inhibition

constants used to determine the type of inhibition.

Kinetic

model

Kinetics

parameters


0 175 350 700

Michaelis-

Menten

Vmax 0.056 ± 0.001 0.053 ± 0.002 0.037 ± 0.002*** 0.031 ± 0.002***

Km 58.79 ± 5.938 56.19 ± 7.197 62.78 ± 10.48 67.72 ± 16.62

2 0.003412 0.004063 0.003694 0.004485

R 0.961 0.935 0.904 0.801

Lineweaver

-Burk

Slope

(=Km/Vmax) 1010 ± 42.82 1220 ± 71.44 1648 ± 101.6*** 1857 ± 148.1***

1/Vmax 17.82 ± 0.7453 18.57 ± 1.244 27.29 ± 1.769 34.93 ± 2.578

1/Km -0.01764 -0.01522 -0.01656 -0.01881

2 1.522 2.539 3.611 5.264

R 0.991 0.983 0.981 0.969

Secondary

plots

Ki 844.5

Ki’ 769.5

Type of

inhibition Non-competitive

Data relative to Vmax, Km, slope and 1/Vmax are expressed as mean ± SEM. Comparisons relative to the control

were performed using one-way ANOVA with Bonferroni as post-test (***p < 0.001).



_______________________________________________________________________________________

44

4.4. Cell-based assays

4.4.1. Effects of the PVPP-white wine extract on SH-SY5Y cell

viability and membrane integrity

The human neuroblastoma SH-SY5Y cell line has been extensively used as cell

model for neurodegenerative diseases, such as Parkinson’s Disease and AD, and for

mitochondrial dysfunction and neuronal cell death induced by oxidative stress (94,124–

129). These cells, when undifferentiated, express functional nicotinic and muscarinic

acetylcholine receptors (130), as well as both ionotropic and metabotropic glutamate

receptors (52). To screen the neurotoxic or neuroprotective effects of individual compounds

and/or extracts, undifferentiated SH-SY5Y cells are considered more suitable than its

differentiated counterparts, since, it is difficult to control all aspects underlying the cell

differentiation process with consequent loss of results reproducibility (131).

The effect of PVPP-white wine extract on SH-SY5Y cell viability was evaluated by

MTT reduction assay, while membrane integrity was assessed by the LDH release assay.

After 24 h of exposure to PVPP-white wine extract, no significant toxicity was observed in

the concentrations range from 1.825 to 58.4 µg/mL in MTT reduction assay (Figure 14A).

In the same range of concentrations, no increase in the leakage of the LDH to the

extracellular media was observed when compared to the control group, and, therefore the

hypothesis of cell death by necrosis can be excluded (Figure 14B).

Figure 14 – Evaluation of the effects of the PVPP-white wine extract on SH-SY5Y cell viability and

membrane integrity. (A) Effect of PVPP-white wine extract on the viability of SH-SY5Y cells,

assessed by the MTT reduction assay after a 24 h incubation period; (B) Effect of PVPP-white wine

extract on the membrane integrity of SH-SY5Y cells, assessed by the LDH release assay after a 24

h incubation period. Results are expressed as mean ± SEM of, at least, three independent

experiments performed in triplicated.

Ce

ll V

iab

ilit

y

(% o

f c

on

tro

l)

Co

ntr

ol

1.8

25

3.6

57.3

14.6

29.2

58.4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

P V P P -w h ite w in e e x tra c t (g /m L )

LD

H r

ele

as

e

(% o

f c

on

tro

l)

Co

ntr

ol

1.8

25

3.6

57.3

14.6

29.2

58.4

0

2 0

4 0

6 0

8 0

1 0 0


A B



_______________________________________________________________________________________

45

4.4.2. Protective effects against glutamate-induced

neurotoxicity in SH-SY5Y cells

Glutamate and mitochondria are two important players in the oxidative stress

process that underlie the pathogenesis of several neurodegenerative diseases, including

AD (51). Glutamate is one of the main excitatory neurotransmitters of the central nervous

system and it acts as a mediator of both interneuronal and neuronal-glial signaling (132).

However, excessive extracellular glutamate concentration can lead to uncontrolled

continuous depolarization of brain cells (neurons and glial cells), a toxic process called

excitotoxicity, leading eventually to neuronal death (51,52). Glutamate excitotoxicity triggers

neuronal and glial toxicity events connected with changes in intracellular calcium

homeostasis (mainly mediated by the activation of the NMDA subtype of glutamate

receptor), mitochondrial dysfunction and the generation of ROS (52).

In recent years, the potential of natural products to prevent glutamate-induced

neuronal damage has been explored as therapeutic strategies for neurodegenerative

diseases, including AD (94,133–135). Thus, the neuroprotective capacity of PVPP-white

wine extract against glutamate induced toxicity in SH-SY5Y cells was also evaluated in the

present work. In the first step, the effects of increasing concentration of glutamate (0-400

mM) on SH-SY5Y cells were evaluated, after 4 h of exposure, by MTT reduction assay

(Figure 15A). The results of Figure 15 show that the glutamate promotes toxicity for SH-

SY5Y cells. In fact, exposure for 4 h of SH-SY5Y cells to 25, 50 and 100 mM glutamate

decrease cell viability to about 70% of control, and the subsequent increase of glutamate

concentration further decrease cell viability. Microdialysis studies have been reported an in

vivo brain glutamate concentration in µM range (136). However, the glutamate is in a

dynamic equilibrium with glutamine, and the brain levels of glutamine are in mM

concentration range. Thus, a glutamate concentration of 50 mM was chosen to evaluate the

ability of PVPP-white wine extract to protect SHSY5Y neuroblastoma cells from the toxicity

promoted glutamate (Figure 15B).

Cells were incubated with increasing concentration PVPP-white wine extract for 20

h and then the medium was removed, and cell were incubated with 50 mM glutamate for 4

h. Data of Figure 15B show that the pre-incubation of cells with PVPP-white wine extract

did not show ability to protect glutamate-damaged SH-SY5Y cells.



_______________________________________________________________________________________

46

Figure 15 – Evaluation of protective effect of PVPP-white wine extract on the viability of SH-SY5Y

cells treated with glutamate. (A) Viability of SH-SY5Y cells treated for 4 hours with glutamate; (B)

Viability of SH-SY5Y cells subjected to a pretreatment for 20 h with the PVPP-white wine extract,

followed by 4 h of exposure to 50 mM glutamate. Results are expressed as mean ± standard error

of the mean of at least three independent experiments performed in triplicate. Comparisons relative

to the control were performed using one-way ANOVA with Bonferroni as post-test (***p < 0.001). No

significant differences were observed with respect to cells exposed to glutamate in the absence of

extracts (“Glut 50 mM” bar).

4.4.2.1. Effects on cell generation of reactive oxygen species

Oxidative stress has been shown to contribute to AD neuropathology, in both animal

models for AD and AD patients (137,138). Antioxidant therapy has been suggested for the

prevention and treatment of neurodegenerative diseases (139). Additionally and as referred

above, an interplay among glutamate excitotoxicity, mitochondria and oxidative stress is a

well-documented process (51,140). Thus, intracellular reactive oxygen species generated

by SH-SY5Y cells exposed to 50 mM glutamate for 1 h without and with pre-treatment with

increasing concentrations of PVPP-white wine extract was evaluated by using DCFH-DA

assay. DCFH-DA diffuses through the cell membrane of viable cells, being deacetylated to

2’,7’-dichlorofluorescein (DCFH), which is not fluorescent. This compound reacts

quantitatively with reactive oxidant species inside the cell to produce the fluorescent

dichlorofluorescein (DCF), which can be measured to evaluate the intracellular generation

of reactive oxygen species (141).

In the present work, the generation of intracellular reactive oxygen species by SH-

SY5Y cells under the above-mentioned experimental conditions were evaluated during 45

min (Figure 16A,B). Data of Figure 16A show that in the absence of excitatory insult, PVPP-

white wine extract decreases significantly the kinetic of reactive oxygen species generation

by SH-SY5Y cells, an effect that is dependent of concentration. The ability of PVPP-white

Ce

ll V

iab

ilit

y

(% o

f c

on

tro

l)

Co

ntr

ol

25

50

100

200

300

400

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

G lu ta m a te (m M )

* * * * * * * * *

* * * * * *

* * *

Ce

ll V

iab

ilit

y

(% o

f c

on

tro

l)

Co

ntr

ol

Glu

t 50 m

M

1.8

25

3.6

57.3

14.6

29.2

58.4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0


+

5 0 m M G lu ta m a te

* * ** * * * * *

* * ** * * * * *

* * *

A B



_______________________________________________________________________________________

47

wine extract to decrease the kinetic of cell generation of reactive oxygen species are more

evidenced in the assay performed with cells exposed to glutamate, as display in Figure 16B.

In fact, the exposure lead to a significant (p < 0.001) increase of the rate of reactive oxygen

species generation, and it is under these conditions that the beneficial effects of the phenolic

compounds of white wine extract are more well-evidenced, as shown in Figure 16C

constructed with of overall intracellular reactive oxygen species generated by cells for 45

minutes. These results indicate that PVPP-white wine extract shows potential to counteract

glutamate early phase excitotoxicity via oxidative damage.

Figure 16 – Evaluation of intracellular generation of reactive oxygen species triggered by 50 mM

glutamate exposure and the protective effect of increasing concentrations of PVPP-white wine

extract to the toxic stimulus. Toxicity was induced over one hour in SH-SY5Y cells, with or without

pre-treatment with the extract (24 hours), followed by addiction of DCFH-DA (10 µM). (A) ROS

generation, over 45 minutes, by SH-SY5Y cells in absence of glutamate; (B) ROS generation, over

45 min, by SH-SY5Y cells in presence of glutamate; (C) Levels of intracellular reactive oxygen

species generated at the end of 45 minutes after probe addiction, expressed as percent of control

(without glutamate), in absence and presence of glutamate. Results are expressed as mean ± SEM

of three independent experiments, performed in triplicate. *p < 0.05, ***p < 0.001 compared to the

control without glutamate; # # # p < 0.001 compared to the control with glutamate.

T im e (m in )

RO

S g

en

era

tio

n k

ine

tic

s

(FL

DC

F)

0 1 0 2 0 3 0 4 0 5 0

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

C o n tro l

3 .65 g /m L

1 4 .6 g /m L

5 8 .4 g /m L

T im e (m in )

RO

S g

en

era

tio

n k

ine

tic

s

(FL

DC

F)

0 1 0 2 0 3 0 4 0 5 0

0

7 0 0 0

1 4 0 0 0

2 1 0 0 0

2 8 0 0 0

3 5 0 0 0

P V P P -w h ite w in e

e x tra c t

+

g lu ta m a te 5 0 m M

3 .65 g /m L

1 4 .6 g /m L

5 8 .4 g /m L

G lu t 5 0 m M


Intr

ac

ell

ula

r R

OS

ge

ne

ra

tio

n

(% o

f c

on

tro

l)

0

3.6

5

14.6

58.4

0

5 0

1 0 0

1 5 0

2 0 0

W ith o u t g lu ta m a te

W ith g lu ta m a te

# # #

# # #

# # #

# # #

# # #

# # #

# # #

* * *

* * *

*

* * *

* * *

* * *

* * *

A B

C



_______________________________________________________________________________________

48

4.4.2.2. Effects on the intracellular redox state of cells,

evaluated by glutathione pool

The redox balance of cells is preserved by an interconnected network of biochemical

pathways involving both non-enzymatic and enzymatic antioxidant defenses. However,

glutathione is the major non-protein antioxidant in human cells and the ratio of its reduced

and oxidized forms (GSH/GSSG) is the best indicator of cellular redox status (19,20).

In the present work, the intracellular levels of reduce and oxidized glutathione as

well as the GSH/GSSG ratios and total (GSH+2GSSG) pool of glutathione were evaluated

in SH-SY5Y cells without any treatment, and in cells exposed to 50 mM glutamate in the

absence and presence of pre-incubation with increasing concentration of PVPP-white wine

extract (Figure 17 and Table 9). In the absence of glutamate addition, data of Figure 17 and

Table 9 suggest that PVPP-white wine extract promotes changes in the redox state of cells,

since the intracellular GSH/GSSG ratio decrease with increasing concentration of PVPP-

white wine extract, and the differences reach statistical significance for the highest

concentration tested (14.6 µg extract/mL). However, the changes in the levels of GSH and

GSSG not reach the statistical significance (Table 9).

Regarding to glutamate effects, data also show that the exposure of cells to

glutamate promotes a significant decrease (p < 0.001) of GSH/GSSG ratio, which emerge

from an increase of the GSSG level. Therefore, glutamate decreased the antioxidant

defenses of cell in agreement with its ability to increase the generation of reactive oxygen

species, which is consistent with the idea that glutamate toxicity is associated with oxidative

stress. Pretreatment of cells with 7.3 and 14.6 µg/mL of PVPP-white wine extract, for 20 h,

prior to exposure to the 50 mM of glutamate increases the resistance of cells to the oxidant

effects of glutamate, since the decreased intracellular GSH/GSSG ratio promoted by

glutamate was attenuated with the increasing concentration of extract (Figure 17).

These data suggest that PVPP-white wine extract has ability to protect SH-SY5Y

cells against the toxicity induced by glutamate, modulating the redox state of cells and

decreasing the generation of reactive oxygen species. Therefore, it can influence key

cellular mechanisms in neuronal cells with relevance in the context of degenerative brain

disorders, such as Alzheimer’s disease.



_______________________________________________________________________________________

49

Figure 17 – Evaluation of the effects triggered by glutamate 50mM exposure on GSH/GSSG ratio of

the SH-SY5Y cells and the potential protective effect of increasing concentrations of PVPP-white

wine extract to the toxic stimulus. Results are expressed as mean ± SEM of four independent

experiments, performed in duplicated. Comparisons relative to the control (*p < 0.05; **p < 0.01; ***p

< 0.001) and glutamate 50 mM (# # p < 0.01; # # # p < 0.001) were performed using one-way ANOVA

with Bonferroni as post-test.

Table 9 – Levels of reduced, oxidized and total glutathione in presence and absence of glutamate

and/or PVPP-white wine extract.

Culture conditions GSH GSSG GSH total

(GSH + 2 GSSG)

Control 5.968 ± 0.062 1.698 ± 0.148 # # 9.363 ± 0.249

7.3 µg/mL 6.562 ± 0.655 1.706 ± 0.095 # # 9.973 ± 0.818

14.6 µg/mL 6.232 ± 0.357 1.879 ± 0.130 # 9.989 ± 0.611

Glut 50 mM 5.871 ± 0.571 3.055 ± 0.480 ** 11.98 ± 1.503

7.3 µg/mL + Glut 50 mM 5.844 ± 0.257 1.948 ± 0.114 # 9.739 ± 0.478

14.6 µg/mL + Glut 50 mM 5.383 ± 0.107 2.120 ± 0.182 9.623 ± 0.331

Results are expressed as mean ± SEM of four independent experiments in nmol/mg protein, performed in

triplicated. Comparisons relative to the control (**p < 0.01) and glutamate 50 mM (# p < 0.05; # # p < 0.01) were

performed using one-way ANOVA with Bonferroni as post-test.

P V P P -w h ite w in e e x tra c t ( g /m L )

GS

H/G

SS

G r

ati

o

07.3

14.6

0

1

2

3

4

5

W ith o u t g lu ta m a te

W ith g lu ta m a te

* * *

* **

* * *

# # #

# # #

# #

# # #



_______________________________________________________________________________________

50

5. Conclusions and Future

Perspectives



_______________________________________________________________________________________

51

Conclusions and Future Perspectives

T2DM and AD are common chronic disorders, and for both diseases the incidence

increases with aging. Experimental evidences have shown that the same

pathophysiological factors (e.g. deregulation of glucose metabolism, mitochondrial

dysfunction, oxidative stress, chronic low-grade inflammation, membrane lipid alterations)

operate in both conditions, affecting several tissues and organs. Additionally, recent

epidemiological data reveal a strong interlink between T2DM and AD, and each one

increases the risk of development of the other. Thereby common preventive and therapeutic

approaches may be used in their prevention and treatment.

Considering the scientific framework of the T2DM and AD, the modulation of

biochemical networks involving cells in different tissues and organs should be required to

reach well-succeed therapeutic and/or preventive approaches. Therefore, the traditional

Western vision for diseases therapeutics, one drug for one biochemical target, is not

adequate to deal with the systemic metabolic deregulation underlying AD and T2DM. On

the other hand, epidemiological studies support the idea that dietary polyphenols, including

the mixture present in wine, have a strong impact on human health, and experimental data

have revealed that wine polyphenols are able to modulate several biochemical pathways in

many types of cells. Thus, a mixture of white wine polyphenols, recovered from the PVPP

used to stabilize the white wine of Douro Region, was studied in order to evaluate its

potential to support preventive and/or therapeutic approaches for T2DM and AD. Several

concluding remarks can be drawn from the data obtained in this research project.

HPLC-DAD analysis revealed that PVPP-white wine extract is rich in phenolic acids

and proanthocyanidins. This phenolic mixture showed a notable antioxidant activity:

scavenging of superoxide anion radical and protection of lipid peroxidation in biological

membrane matrices, suggesting its potential to avoid/reduce the oxidative stress connected

with degeneration mechanisms in both T2DM and AD.

Cell-free assays showed that PVPP-white wine extract displays an effective

inhibitory activity towards α-glucosidase (similar to acarbose), suggesting its potential to

modulate glucose absorption and, consequently, to prevent and/or attenuate hyperglycemia

in T2DM. However, the extract did not inhibit α-amylase up to concentration of 200 µg/mL.

Regarding the potential of brain cholinergic system modulation, mitochondria-free rat brain

homogenates were used to evaluate the AChE activity, which mimic the conditions that

occurs in biological systems, and PVPP-white wine extract exhibited an interesting enzyme

inhibition, suggesting its potential for alleviate some symptoms of AD. The phenolic extract



_______________________________________________________________________________________

52

behaved in both situations (α-glucosidase and AChE) as a reversible non-competitive

inhibitor.

Considering the cell-based assays, no significant toxicity was observed in the SH-

SY5Y cells after the exposure to the PVPP-white wine extract. Additionally, the extract

revealed neuroprotective effects on glutamate-induced oxidative stress in SH-SY5Y cells,

decreasing the ROS generation and increasing the intracellular GSH/GSSG ratio. Thus, the

obtained results point to the valuable multimodal-effect of PVPP-white wine extract over

distinct central nervous system targets.

Moreover, the typology of assays used in the present work allowed not only the

evaluation of the potential of an extract rich in phenolic compounds in the prevention and/or

treat of diseases that share oxidative stress as a pathological mark, but also the validation

of a methodology (e.g. GSH/GSSG ratio assessment) to improve the standardization of the

bioactivity of polyphenol rich-products. In the future, it would be also important to evaluate

the energetic charge of cells by measuring adenine nucleotide levels in order to create a

robust bioactivity scale of polyphenols rich-products.

It should be note that PVPP-white wine extract is available in large amount as it is a

low-cost byproduct of winemaking, thus providing sustainable therapeutic options. For all

the potentialities of this winery by-product mentioned above, further studies should focus

on the outcome of investigating the effects in in vivo studies, namely, with models that

represent each pathology and even with animal models that mimic both the pathologies

(T2DM and AD). It would also be interesting to evaluate in the future another enzyme with

relevance in the context of T2DM – the aldose reductase, since it is a key enzyme in the

polyol pathway, which catalyzes the reduction of glucose to sorbitol and provides a common

link in the onset of diabetic complications in various parts of the human body.



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53

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