evaluation of antioxidant and biological properties

138 Pages·2016·2.62 MB·English

by  France 2010

KAUNAS UNIVERSITY OF TECHNOLOGY
UNIVERSITY OF TOULOUSE
LINA SMOLSKAITĖ
RARE FOREST AND COASTAL-DUNE
MUSHROOM: EVALUATION OF
ANTIOXIDANT AND BIOLOGICAL
PROPERTIES

Doctoral dissertation
Physical Sciences, Chemistry (03P)

2016, Kaunas
UDK 582.28 (043.3)

Doctoral research was carried out at Kaunas University of Technology, Faculty
of Chemical Technology, Department of Food Science and Technology and in
France at the Institut National Polytechnique de Toulouse, Ecole Nationale
Supérieure des Ingénieurs en Arts Chimiques Et Technologiques de Toulouse.
Part of the research was performed in Latvia University of Agriculture, Faculty
of Food Technology, Department of Food Technology. This study was
supported, by Midi-Pyrénées Regional Council and the SMI (International
mobility support) program of INP Toulouse.

Scientific supervisors:

Prof. Dr. Petras Rimantas VENSKUTONIS (Kaunas University of Technology,
Physical Sciences, Chemistry - 03P).

Dr. Thierry TALOU (University of Toulouse, Physical Sciences, Chemistry - 03P).

Doctoral dissertation has been published in: http://ktu.edu

Editor:

UAB “Synergium”

©L. Smolskaitė, 2016

ISBN 978-609-02-1262-2
KAUNO TECHNOLOGIJOS UNIVERSITETAS
TULŪZOS UNIVERSITETAS
LINA SMOLSKAITĖ
RETIEJI MIŠKO IR PAJŪRIO GRYBAI:
ANTIOKSIDACINIŲ IR BIOLOGINIŲ
SAVYBIŲ ĮVERTINIMAS

Daktaro disertacija
Fiziniai mokslai, chemija (03P)

2016, Kaunas
UDK 582.28 (043.3)

Dvigubo laipsnio disertacija parengta 2010–2016 metais Kauno technologijos
universitete, Cheminės technologijos fakultete, Maisto mokslo ir technologijos
katedroje, ir Prancūzijoje, - Nacionaliniame Tulūzos politechnikos institute,
Chemijos inžinerijos ir technologijų padalinyje. Papildomi tyrimai atlikti Latvijos
Žemės ūkio universitete, Maisto technologijos fakulteto, Maisto technologijos
katedroje. Mokslinius tyrimus rėmė Midi-Pyrénées (Pietų Pirėnų) regiono mokslo
taryba ir Tulūzos politechnikos instituto tarptautinė mainų programa.

Moksliniai vadovai:

Prof. dr. Petras Rimantas VENSKUTONIS (Kauno technologijos universitetas,
fiziniai mokslai, chemija – 03P).

Dr. Thierry TALOU (Nacionalinis Tulūzos politechnikos institutas, fiziniai mokslai,
chemija – 03P).

Interneto svetainės, kurioje skelbiama disertacija, adresas: http://ktu.edu

Redagavo:

UAB “Synergium”

©L. Smolskaitė, 2016

ISBN 978-609-02-1262-2
ACKNOWLEDGEMENTS
I would like to gratefully acknowledge all the people who have walked
alongside with me in recent years as I have worked on this thesis. They have guided
me, placed opportunities in front of me and showed me the doors that might be
useful to open. I would like to thank each and every one of them. First, I owe an
enormous debt of gratitude to my supervisors Prof. Ph.D. Rimantas Petras
VENSKUTONIS and Dr Thierry TALOU, for their expertise, understanding,
patience and generous guidance. They encouraged and challenged me to be faithful
throughout the years.
I am grateful to Prof. Eric DEHARO and Prof. Louis CHAVANT for their
pleasant words, suggestions, new experiences and the skills obtained regarding the
topic of my research.
A huge thanks to Dr Anne-Cécille LE LAMER and Dr Cynthia GIRARDI for
being an excellent guide throughout the time together and for encouraging me with
your kind words when I was struggling.
I would like to express my warmest thanks to Dr sc. ing. Zanda KRUMA for
the helpful discussions and recommendations.
I would also like to thank the Research Council of Lithuania, Midi-Pyrénées
Regional Council and SMI (International mobility support) program of INP
Toulouse for their financial support.
I would like to thank all my colleagues at the Department of Food Science and
Technology, either the laboratory of Agro-Industrial Chemistry in Toulouse for an
inspiring scientific and social environment.
A special thanks to my family and my parents who have always been ready to
help me, for giving the right advice at the right time and for being a source of
motivation.
The heartfelt thanks to my husband Vytautas, for his comprehension and belief
in me. I really appreciate his tolerance and all his encouragement during this time.
CONTENT
ABBREVIATIONS ................................................................................................. 9
INTRODUCTION ................................................................................................. 11
1. LITERATURE REVIEW ................................................................................ 15
1.1. HISTORY, DIVERSITY AND IMPORTANCE OF MUSHROOMS ............. 15
1.1.1. General characterisation and definition of mushrooms ............................... 15
1.1.2. History, traditions and importance of mushrooms ...................................... 15
1.1.3. Diversity, composition and characteristics of mushrooms .......................... 17
1.2. THE MUSHROOM AS A SOURCE OF NATURAL BIOACTIVE
MOLECULES ..................................................................................................... 19
1.2.1. Brief review on the investigated wild mushrooms bioactive molecules ...... 21
1.2.1.1. Heterocyclic compounds in mushrooms ................................................ 23
1.2.1.2. Polyketides in mushrooms ..................................................................... 24
1.2.1.3. Sterols in wild mushrooms .................................................................... 26
1.2.1.4. Terpene compounds in mushrooms ....................................................... 27
1.2.1.5. Miscellaneous compounds in mushrooms ............................................. 28
1.3. BRIEF REVIEW OF COLLECTED MUSHROOM SPECIES AND THEIR
SECONDARY METABOLITES ........................................................................ 29
1.4. THE POTENTIAL ACTIVITY OF MUSHROOM SUBSTANCES AND
THEIR RECOVERY ........................................................................................... 32
1.4.1. Antioxidants in mushrooms and their mode of action ................................. 33
1.4.2. Antimicrobials in mushrooms and their mode of action ............................. 37
2. MATERIALS AND METHODS ..................................................................... 39
2.1. MATERIALS .................................................................................................. 39
2.1.1. Research objects ......................................................................................... 39
2.1.2. Reagents ..................................................................................................... 39
2.2. PREPARATION OF EXTRACTS .................................................................. 46
2.2.1. Isolation of active compounds from P. schweinitzii extract ........................ 47
2.3. RESEARCH METHODS ................................................................................ 47
2.3.1. ANTIOXIDANT ANALYSIS ...................................................................... 48
2.3.1.1. DPPH•-scavenging capacity ................................................................... 48
2.3.1.2 ABTS•+ decolourisation assay ................................................................ 49
2.3.1.3. Ferric-reducing antioxidant power assay ............................................... 49
2.3.1.4. Oxygen radical absorbance capacity ...................................................... 49
2.3.1.5. Determination of total phenolic content ................................................. 50
2.3.2. MICROBIAL ANALYSIS ............................................................................ 50
2.3.2.1. Disc diffusion assay ............................................................................... 50
2.3.2.2. Agar-overlay assay (TLC bioautography) ............................................. 51
2.3.3. BIOLOGICAL ANALYSIS .......................................................................... 52
2.3.3.1. Antileishmanial activity on amastigotes of wild mushrooms species .... 52
2.3.3.2. Antileishmanial activity on promastigotes of isolated compounds ........ 53
2.3.3.3. Cytotoxicity assay ................................................................................. 53
2.3.3.4. Larval exsheathment assay .................................................................... 54
2.3.3.5. Detailed biological activity assays of hispidin ....................................... 54
2.3.3.5.1. Cell culture and cytogenetic procedures .............................................. 54
2.3.3.5.2. Cytokinesis-block micronucleus assay ................................................ 55
2.3.3.5.3. Chromosome aberration and sister chromatid exchange assay ............ 55
2.3.3.5.4. Comet assay ........................................................................................ 55
2.3.3.5.5. Salmonella/microsome test ................................................................. 56
2.3.4. CHROMATOGRAPHIC ANALYSIS .......................................................... 57
2.3.4.1. Thin layer chromatography on selected mushroom extracts .................. 57
2.3.4.2. Bioautography using DPPH• as the detection reagent ............................ 57
2.3.4.3. HPLC-preparative chromatography ....................................................... 58
2.3.4.4. UHPLC- analytical chromatography ..................................................... 58
2.3.5. STRUCTURE ELUCIDATION OF ISOLATED COMPOUNDS ................ 58
2.4. STATISTICAL ANALYSIS ........................................................................... 58
3. RESULTS AND DISCUSSION ....................................................................... 60
3.1. YIELDS AND ANTIOXIDANT PROPERTIES OF WILD MUSHROOM
EXTRACTS ........................................................................................................ 60
3.1.1. Yield of mushroom extracts fractionated with different solvents .................. 60
3.1.2. Antioxidant capacity differences between mushroom species ....................... 68
3.1.3. Antioxidant capacity differences between extractants ................................... 69
3.1.4. Antioxidant capacity differences between extraction methods ...................... 72
3.2. ANTIBACTERIAL AND BIOLOGICAL PROPERTIES OF COLLECTED
WILD MUSHROOM SPECIES .......................................................................... 75
3.2.1. Antibacterial properties of mushroom extracts ........................................... 75
3.2.2. Agar-overlay (TLC bioautography) technique on mushroom species ......... 81
3.2.3. Biological properties of mushroom extracts ............................................... 83
3.3. ISOLATION AND IDENTIFICATION OF SECONDARY METABOLITES
IN METHANOLIC EXTRACT FROM PHAEOLUS SCHWEINITZII (FR.)...... 85
3.3.1. Detection of secondary metabolites by thin layer chromatography ............. 86
3.3.2. Detection of secondary metabolites by TLC-DPPH• bioautography ........... 87
3.3.3. Isolation of active substances from P. schweinitzii ..................................... 87
3.3.4. Purification and structure elucidation of active compounds ........................ 87
3.3.5. Structure elucidation of hispidin (compound 3) .......................................... 90
3.3.6. Structure elucidation of hispolon (compound 1) ......................................... 92
3.3.7. Structure elucidation of inonotic acid methyl ester (compound 2) .............. 94
3.4. ASSESSMENT OF ANTIOXIDANT, BIOLOGICAL AND ANTIPHRASTIC
ACTIVITY OF ISOLATED COMPOUNDS FROM P. SCHWEINITZII ........... 97
3.4.1. Compound hispidin antioxidant activity ..................................................... 98
3.4.2. Isolated compounds and P. schweinitzii and I. hispidus extracts biological
activity ........................................................................................................ 104
3.4.2.1. Determination of antileischmanial and cytotoxicity activity of isolated
compounds ......................................................................................................... 104
3.4.2.2. Detailed biological activity of mushroom metabolite hispidin............. 105
3.4.2.2.1. Induction of micronuclei in human lymphocytes in vitro .................. 105
3.4.2.2.2. Induction of chromosome aberrations and SCEs in human lymphocytes
in vitro ......................................................................................................... 106
3.4.2.2.3. Induction of primary DNA damage detected by comet assay ............ 107
3.4.2.2.4. Salmonella/microsome test ............................................................... 108
3.4.2.3. Determination of anthelmitic effect on gastrointestinal nematodes ..... 109
4. CONCLUSIONS ............................................................................................ 118
REFERENCES .................................................................................................... 120
ABBREVIATIONS

ABTS•+  2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
FRAP  ferric reducing antioxidant power
ASE  ‘antioxidant score’ of extract
ASM  antioxidant scores of mushrooms
C  cyclohexane
CC50  cytotoxic concentration of the substance to cause death to 50% of viable
cells in the host
CFU  colony forming unit per ml
CIU  comparative integrated units
COSY  correlation spectroscopy
D  dichloromethane
DMSO  deuterated dimethyl sulfoxide
DPPH  2,2-Diphenyl-1-picrylhydrazyl hydrate radical
EDW  extract dry weight
EC  effective concentration 50%
50
ESI  electrospray ionisation
EX  exsheathment
EY  extract yields
FCS  fetal calf serum
FID  flame ionisation detector
FL  fluorescein
GAE  gallic acid equivalent
GLM  general linear model
HAT  hydrogen atom transfer
HMBC  heteronuclear multiple-bond correlation spectroscopy
HPLC  high performance liquid chromatography
HSQC  heteronuclear single quantum correlation spectroscopy
IC50  inhibition concentration 50%
M  methanol
MIC  minimal inhibitory concentration
MS  mass spectrometry
MTT  3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
N.D.  not detected
N.T.  not tested
NMR  nuclear magnetic resonance spectroscopy
ORAC  oxygen radical absorbance capacity
PBS  phosphate buffered saline
PDA  photo diode array detector
Q-TOF  quadrupole time-of-flight
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R  correlation coefficient
R  retention factor
f
RPM  rotation per minute
RPMI  Roswell Park Memorial Institute
RSC  radical scavenging capacity
Rt  retention time
SD  standard deviation
SET  single electron transfer
SI  selectivity index
SQD  simple quadrupole detector
TE  Trolox equivalents
TEAC  Trolox antioxidant activity coefficient
TLC  thin layer chromatography
TPC  total phenolic content
TPTZ  2,4,6-tripyridyl-s-triazine
U/ml  units per volume
UPLC  ultra performance liquid chromatography
UV  ultraviolet
V/V  volume to volume

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