Extraction, identification and antioxidant activity of the phenolic secondary metabolites isolated

259 Pages·2017·6.59 MB·English

Extraction, identification and antioxidant activity of the
phenolic secondary metabolites isolated from the leaves,
stems and fruits of two shrubs of the Ericaceae family
Oana-Crina Bujor
To cite this version:
Oana-Crina Bujor. Extraction, identification and antioxidant activity of the phenolic secondary
metabolites isolated from the leaves, stems and fruits of two shrubs of the Ericaceae family. Other.
Université d’Avignon, 2016. English. ￿NNT: 2016AVIG0261￿. ￿tel-01722698￿
HAL Id: tel-01722698
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―Gheorghe Asachi‖ Technical University of Iasi
Faculty of Chemical Engineering and Environmental Protection, Romania

University of Avignon – National Institute for Agricultural Research (INRA)
UMR 408 Safety and Quality of Plant Products, Avignon, France

Extraction, identification and antioxidant
activity of the phenolic secondary metabolites
isolated from the leaves, stems and fruits
of two shrubs of the Ericaceae family

- PhD THESIS -

Chemist Oana-Crina Bujor

Supervisors:
Professor emeritus Valentin I. POPA
Dr. Claire DUFOUR

IASI, April 19th 2016
The composition of the thesis jury:

President:
Professor Nicolae Hurduc
Faculty of Chemical Engineering and Environmental Protection
―Gheorghe Asachi‖ Technical University of Iasi, Romania

Supervisors:
Professor emeritus Valentin I. Popa
Faculty of Chemical Engineering and Environmental Protection
―Gheorghe Asachi‖ Technical University of Iasi, Romania

Dr. Claire Dufour
UMR408 SQPOV ―Sécurité et Qualité des Produits d‘Origine Végétale‖
National Institute for Agricultural Research, Avignon, France

Reviewers:
Dr. Hélène Fulcrand
UMR1083 ―Sciences Pour l‘Oenologie‖
National Institute for Agricultural Research, Montpellier, France

Professor Florin Dan Irimie
Faculty of Chemistry and Chemical Enginerring
Babes-Bolyai University of Cluj-Napoca, Romania

Professor Teodor Măluţan
Faculty of Chemical Engineering and Environmental Protection
―Gheorghe Asachi‖ Technical University of Iasi, Romania
To Rares, for his love and patience
To my niece and my nephew, Alesia and Denis: the chemistry is all around us!
ACKNOWLEDGEMENTS

This PhD thesis was elaborated in the joint supervision between the ―Gheorghe
Asachi‖ Technical University of Iasi, Faculty of Chemical Engineering and
Environmental Protection (FCEEP), Romania and the University of Avignon and
Vaucluse, France. The present work was carried out at the Department of Natural and
Synthetic Polymers at the FCEEP, and in ―Chemistry of antioxidants‖ team of UMR 408
―Safety and Quality of Plant Products (SQPOV)‖ at the National Institute for Agricultural
Research (INRA) from Avignon. The travel costs to INRA-SQPOV Unit were financed
by the Erasmus Program, which I gratefully acknowledge for the student mobility grants
and particularly to Mrs. Liliana Lazăr and Mrs. Claudia Haasis for their support. I am
also grateful to Dr. Catherine Renard, Head of the INRA-SQPOV Unit, for providing the
facilities for my work in her unit.
I am most grateful to my supervisors, Professor emeritus Valentin I. Popa and
Dr. Claire Dufour, for their guidance throughout the thesis and for always had the door
open for all questions and discussions concerning all aspects of the work … and not
only. I am very grateful to Professor Valentin I. Popa for his encouragement to start this
work and for all support, kindly guidance, advices, and insightful comments. I express
my special thanks to Dr. Claire Dufour for introducing me to the world of antioxidant
polyphenols and lipid oxidation and more important for knowledge on mass
spectrometry. I appreciated their never failing support and suggestions, and the
constructive advice and criticism without the scientific results of this work would not
attain this high level. Her kindness and friendship were and are also very precious to
me. ―Merci, Merci!‖.
I would like to extend my gratitude to the members of my examination committee,
Professor Nicolae Hurduc, Dr. Hélène Fulcrand, Professor Florin Dan Irimie and
Professor Teodor Măluţan, for their time and interest in evaluating the scientific quality
of this thesis.
I wish to express my sincere acknowledgment to Professor Olivier Dangles,
Head of Doctoral School 536 "Agricultural Sciences & Sciences" of University of
Avignon, who promptly responded to my interest in collaboration with its university
and give me the chance to do my research at the SQPOV Unit. I would like to thank him
for his time, friendly attitude, advice and help in my grant and thesis applications.
Two persons at the Faculty of Chemical Engineering and Environmental
Protection of Iasi are specially acknowledged for their contributions to this study. I am
grateful to Associate Professor Irina Volf for the opportunity to work at her laboratory
and use the facilities available there for my researches and her advice and support
during my thesis. I also thank Associate Professor Camelia Mihăilescu for her
proficiency and guidance in experimental extraction analysis.
Many sincere thanks go to the members of the ―Chemistry of antioxidants‖ team
of INRA-SQPOV Unit for kindness and warm welcome and pleasant working
atmosphere in the laboratory. Special thanks go to: Mrs. Marie- José Vallier for her
sympathy and providing me guidance and help on my lab work whenever needed; Mrs.
Michèle Loonis for her help and guidance in UPLC/MS analyses and especially for her
gentleness and good ideas; Mr. Christian Ginies for helping me through the hard
laboratory work in the digestion: «Je saisμ c‘est pour la recherche!»; Dr. Michel Carail
for his advice on lab «trucs» and for trying to learn romanian.
I would like to thank all the staff of INRA-SQPOV Unit for welcoming me as
another member of their unit. Especially, I thanks to Dr. Carine Le Bourvellec for
teaching me about the procyanidin analyses and their fruitful and pleasant contribution
to this study. An extra thank is also addressed to the unit informaticians,
Dominique Loonis and Eric Pietri, for helping me to solve the ―everyday‖ IT problems.
I thank the Forestry Direction of Neamt for the access to Borca Mountains
(Neamt, Romania) for collecting bilberry and lingonberry samples for my research. I
also want to thank to Dr. Gina Tiron from National Meteorological Administration -
Moldova Meteorological Center of Iasi, for providing the meteorological data.
I am grateful to all my colleagues and friends at the Faculty of Chemical
Engineering and Environmental Protection of Iasi and the INRA-SQPOV Unit of
Avignon for making my work easier during these years: Andrei and Cornel are thanked
for their ―useful advice‖ at the beginning of the thesis and helpfulness; Roxana is
thanked for her all support and beautiful friendship; Adina for her collaboration for
review, confident spirit and help while I was in France; Dan is thanked for his help with
the lyophilisation of samples and friendship; my colleagues at the INRA-SQPOV Unit,
Katérina, Anna-Lena, Rachel, Béa, Joseana, Dimitra, Albert, Romain, Sara, Julien,
Aurélie, Djidji deserve great thanks for all their help, friendly welcome, kindness,
lunches, coffee breaks and all the good moments that we have shared together during
my work in Avignon.
Finally, I am heartily thankful to my family, and especial to my sister for his
encouragement and unconditional support. In particular, my dearest thanks are
addressed to Rares for understanding me and my work, and for his love, tireless support
and patience.

Contents

Contents

INTRODUCTION...........................................................................................................7

LITERATURE REVIEW ........................................................................................................ 9

I. Phenolic compounds as secondary metabolites found in plants and foods .............. 11
I.1. Structure and classification ................................................................................ 12
I.1.1. Non-flavonoid polyphenols ............................................................................... 12
I.1.2. Flavonoids ........................................................................................................15
I.2. Methods for extraction and characterisation of phenolic compounds ............. 18
I.3. Applications of phenolic compounds in different biological systems ............... 20
I.3.1. Applications of phenolic compounds in the plants development ...................... 20
I.3.1.1. As plants growth bioregulators ................................................................ 20
I.3.1.2. As amendments in bioremediation .......................................................... 22
I.3.2. Applications of phenolic compounds in microorganisms development ............ 23

II. The antioxidant activity of phenolic compounds .......................................................... 25
II.1. The antioxidant action of phenolic compounds and their mechanisms ......... 25
II.1.1. Through reducing effects: H-atom and/or electron transfer ........................... 26
II.1.2. Through non-reducing effects ......................................................................... 28
II.1.2.1. Metal-ion chelating activity ................................................................... 28
II.1.2.2. Inhibition of enzymes implied in the production of ROS ...................... 28
II.1.3. Inhibition of lipid oxidation ............................................................................. 29
II.2. Methods to evaluate the antioxidant activity of phenolic compounds ............ 30
II.2.1. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method ............... 30
II.2.2. Folin–Ciocalteu redox method ........................................................................ 31
II.2.3. Other methods ................................................................................................. 32

III. Bilberry and lingonberry, two shrubs of the Ericaceae family as sources of
phenolic secondary metabolites ............................................................................................. 33
III.1. Bilberry (Vaccinium myrtillus L.) ................................................................... 33
III.1.1. General description ........................................................................................ 33
III.1.2. Chemical composition: phenolic compounds and other constituents ............ 36
III.2. Lingonberry (Vaccinium vitis-idaea L.) ......................................................... 42
III.2.1. General description ........................................................................................ 42
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III.2.2. Chemical composition: phenolic compounds and other constituents ............ 44
III.3. Extraction and analysis of bilberry and lingonberry phenolic compounds .. 48
III.4. Health benefits of bilberry and lingonberry ................................................... 48
III.4.1. Cardioprotective activity ................................................................................ 49
III.4.2. Anti-cancer activity ........................................................................................ 50
III.4.3. Antidiabetic activity ....................................................................................... 51
III.4.4. Vision improvement activity ........................................................................... 52
III.4.5. Bacterial anti-adhesion activity ..................................................................... 52
III.5. Other applications of bilberry and lingonberry extracts ................................ 53

PERSONAL CONTRIBUTION ........................................................................................... 54

Chapter I. PHENOLIC CONSTITUENTS IN BILBERRY (VACCINIUM
MYRTILLUS L.): ACCUMULATION IN LEAVES, STEMS AND FRUITS AT
DIFFERENT HARVEST PERIODS AND ANTIOXIDANT ACTIVITY ................... 55
1. Background ........................................................................................................... 57
2. Methodology .......................................................................................................... 59
2.1 Materials ..............................................................................................................59
2.1.1. Bilberry samples ........................................................................................ 59
2.1.2. Chemicals and solvents .............................................................................. 59
2.2. Extraction of phenolic compounds ...................................................................... 60
2.3. Qualitative and quantitative analyses of phenolic compounds ........................... 61
2.3.1. Identification of phenolic compounds by UPLC/MS ................................ 61
2.3.2. Quantification of phenolic compounds ...................................................... 62
2.4. Analysis of procyanidins using thioacidolysis .................................................... 63
2.4.1. Freeze-dried extracts .................................................................................. 63
2.4.1.1. HPLC analysis without thiolysis ......................................................... 63
2.4.1.2. HPLC analysis after thiolysis .............................................................. 63
2.4.2. Freeze-dried fruits ...................................................................................... 64
2.4.2.1. HPLC analysis without thiolysis ......................................................... 64
2.4.2.2. HPLC analysis with thiolysis .............................................................. 64
2.5. Antioxidant activity by applying spectrophotometric methods ........................... 65
2.5.1. Total Phenolic Contents by the Folin Ciocalteu method ........................... 65
2.5.2. DPPH (2,2-diphenyl- 1-picrylhydrazyl) radical scavenging test ............... 65
2.6. Statistical analyses .............................................................................................. 66
3. Results and discussion .......................................................................................... 67
3.1. Optimal extraction conditions: the preliminary test ........................................... 67
3.2. Phenolic profile and content of bilberry extracts ............................................... 69
3.2.1. Caffeic acid derivatives ............................................................................. 69
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Contents

3.2.2. Coumaric acid derivatives.......................................................................... 73
3.2.3. Flavonol glycosides ................................................................................... 75
3.2.4. Flavanols .................................................................................................... 76
3.2.5. Anthocyanins ............................................................................................. 85
3.3. Influence of the harvest period on the phenolic composition in bilberry leaves,
stems and fruits……..... .............................................................................................. 86
3.4. Characterization of flavan-3-ol oligomers ......................................................... 91
3.4.1. In freeze-dried extracts .............................................................................. 91
3.4.2. In freeze-dried fruits .................................................................................. 92
3.5. Antioxidant activity of bilberry extracts .............................................................. 94
4. Conclusions ........................................................................................................... 99
ANNEXES ............................................................................................................... 101

Chapter II. PHENOLIC PROFILE AND ANTIOXIDANT ACTIVITY OF LEAF,
STEM AND FRUIT EXTRACTS OF LINGONBERRY (VACCINIUM VITIS-IDAEA
L.) AT THREE VEGETATIVE STAGES ....................................................................... 105
1. Background ......................................................................................................... 107
2. Methodology ........................................................................................................ 109
2.1. Materials ...........................................................................................................109
2.1.1. Lingonberry samples ................................................................................ 109
2.1.2. Chemicals and solvents ............................................................................ 109
2.2. Extraction of phenolic compounds .................................................................... 110
2.3. Qualitative and quantitative analyses of phenolic compounds ......................... 110
2.4. Analysis of procyanidins using thioacidolysis .................................................. 111
2.4.1. Freeze-dried extracts ................................................................................ 111
2.4.2. Freeze-dried fruits .................................................................................... 111
2.5. Antioxidant activity by applying spectrophotometric methods ......................... 111
2.5.1. Total Phenolic Contents by the Folin Ciocalteu method ......................... 111
2.5.2. DPPH (2,2-diphenyl- 1-picrylhydrazyl) radical scavenging test ............. 112
2.6. Statistical analyses ............................................................................................ 112
3. Results and discussion ........................................................................................ 113
3.1. Phenolic profile and content of lingonberry extracts ....................................... 113
3.1.1. Hydroxycinnamic acid derivatives .......................................................... 116
3.1.1.1. Caffeic acid and ferulic acid derivatives ........................................... 116
3.1.1.2. Coumaric acid derivatives ................................................................. 117
3.1.2. Flavonol glycosides ................................................................................. 118
3.1.3. Flavanols .................................................................................................. 119
3.1.4. Other compounds in lingonberry extracts ................................................ 121
3.1.5. Anthocyanins ........................................................................................... 131
3.2. Influence of the harvest period on the phenolic composition in lingonberry
leaves, stems and fruits ……………………………………………………………………132
3