Nematicidal, antimicrobial and acaricidal activity of plant secondary metabolites

134 Pages·2014·2.55 MB·English

Università degli Studi di Cagliari
XXVI ciclo di corso di dottorato in Tossicologia

Nematicidal, antimicrobial and acaricidal activity of plant
secondary metabolites

Nadhem Aissani
Department of Live and Environmental Sciences,
Cagliari, Italy

Supervised by
Prof. Pierluigi Caboni

Degree of
European Doctor of Philosophy

Final exam academic year 2012-2013
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ACKNOWLEDGEMENTS

Firstly, thanks to God for finalizing this PhD thesis
, Laboratorio di Igiene degli
Alimenti, Medicina Clinica e Molecolare at the University of Cagliari, Italy and Instituto di
Ciencias Agrarias (ICA-CSIC)-Madrid, Spain, during the years 2011-2014.
I would like to express my sincere gratitude to my Professor Pierluigi Caboni, for giving me
the opportunity to peruse a PhD with him. Pierluigi, thank you for the guidance you have
giving me over the past of years, providing excellent facilities for my work, enthusiasm,
support and mainly for his friendship and sympathy. I have learned so much from you.
I thank Professor Valentina Coroneo for giving me the possibility to work in her laboratory,
introducing me to test methods for determining antimicrobial activity of plant extracts as well
as giving me her confidence.
My special thanks to Professor Azucena Gonzalez Coloma at Instituto di Ciencias Agrarias
(ICA-CSIC)-Madrid, for giving me the possibility to spend a stage period in her laboratory,
sympathy and help.
I owe thanks to Professors Alberto Angioni, Carlo Tuberoso and Giorgia Sarais for their help,
suggestions for using the analytical instruments, and friendship.
I am grateful to the official reviewers of my thesis, Professors Cedric Bertrand from the
University of Perpignan, France and Urania Menkissoglu–Spiroudi from Aristotle University
of Thessaloniki, Greece for their valuable comments, suggestions and expert criticism on the
manuscript.
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I wish to thank all my colleagues for providing a good atmosphere to work, inspiring
discussions and for mental support during the creation of this thesis.
My special thanks go to my dear friend Marco Saba for his encouragement and friendship,
both at work and during free time.
I am grateful to my dear parents Lamine and Hayet, my sisters Rania and Roeya, my brother
Nadim and my uncle Slah for their love, support and confidence during all my academic steps
and difficult moments. I would never have got to the end without you. Thank you very much.
My deep and warm thanks are due to my dear Rosy, for her endless love, support and
encouragement during all the years of this thesis work. Thank you for everything.
Thanks to my friends Yassin, Dhia and Carlo for their friendship and encouragement.

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TABLE OF CONTENTS

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INTRODUCTION 9

Chapter 1. Nematicidal activities of aldehydes, ketones, isothiocyanates and
polyphenols against Meloidogyne Spp 19

I. Nematicidal Activity of (E,E)-2,4-decadienal and (E)-2-decenal from
Ailanthus altissima against Meloidogyne javanica 20
I. 1. Ailanthus altissima 20

I. 2. Materials and Methods 20
I. 2. a. Chemicals 20
I. 2. b. Plant Materials 21
I. 2. c. A. altissima Methanol Extracts 21
I. 2. d. GC-MS Analysis 21
I. 2. e. Effect of A. altissima Extracts on J2 Motility 22
I. 2. f. Statistical Analysis 23
I. 2. g. Scanning Electronic Microscopy (SEM) Analysis 24
I. 3. Results 24
II. Potent Nematicidal Activity of Phthalaldehyde, Salicylaldehyde,
and Cinnamic Aldehyde against Meloidogyne incognita 33
II. 1. Materials and Methods 33
II. 1. a. Chemicals 33
II. 1. b. General Procedure for the Formylation of Phenols I and III 33
II. 1. c. General Procedure for Aldehyde Immobilization on PEG 34
II. 1. d. Effect of Different Aromatic Aldehydes on J2 Motility 35
II. 1. e. SEM Characterization 35
II. 1. f. LC-MS Detection of Reaction Products 37
II. 1. g. Cysteine Reduction and Alkylation Experiments 38

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II. 2. Results 39
III. Nematicidal Activity of 2-thiophenecarboxaldehyde and
Methylisothiocyanate from Caper (Capparis spinosa) against
Meloidogyne incognita 56
III. 1. Capparis spinosa 56
III. 2. Materials and Methods 57
III. 2. a. Chemicals 57
III. 2. b. Experimental Chemistry 57
III. 2. c. Synthesis of 2,5-Thiophenedicarbonitrile 58
III. 2. d. Synthesis of 5-Formylthiophene-2-carbonitrile 58
III. 2. e. Synthesis of 2-Thiophenecarboxaldehyde oxime 58
III. 2. f. Synthesis of Thiophene-2-carbonitrile (F) 59
III. 2. g. Synthesis of 2-Pyridinecarboxaldehyde Oxime 59
III. 2. h. Synthesis of Pyridine-2-carbonitrile 60
III. 2. i. Plant Materials 60
III. 2. j. GC-MS Analysis 60
III. 3. Results 61
IV. Nematicidal Activity of Allylisothiocyanate from Horseradish
(Armoracia rusticana) Roots against Meloidogyne incognita 70
IV. 1. Armoracia rusticana 70
IV. 2. Materials and Methods 71
IV. 2. a. Chemicals 71
IV. 2. b. Plant material and extraction 71
IV. 2. c. GC-MS Analysis 71
Roots Extract 72
Isothiocyanates Degradation Studies in Soils 72
IV. 2. d. LC-MS/MS Analysis 73
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IV. 3. Results 74
V. Nematotoxic Phenolic Compounds from Melia azedarach
Against Meloidogyne incognita 80
V. 1. Melia azedarach 80
V. 2. Materials and Methods 81
V. 2. a. Chemicals 81
V. 2. b. Extraction of Plant Materials 82
V. 2. c. Apparatus and Chromatography 82
HPLC-MS/MS Analysis 82
HPLC-DAD Analysis 82
V. 3. Results 83
Chapter 2. Antimicrobial activity of polyphenols 89
Inhibitory Effect of Carob (Ceratonia siliqua) Leaves Methanolic
Extract on Listeria monocytogenes 90
I. Ceratonia siliqua 90
II. Materials and Methods 91
II. a. Chemicals 91
II. b. Plant material, extraction and analysis 91
II. c. Microbiological Assay 92
MIC determination 92
Stability and Heat Inactivation of Carob Leaves Extract 93
Effect of Carob Extract on Some Enzyme Activity 93
II. d. Statistical Analysis 94
III. Results 94
Chapter 3. Acaricidal activity 102
Acaricidal activity of Lavandula luisieri hydrolates 103
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I. Lavandula luisieri 103
II. Materials and Methods 104
II. a. Chemicals 104
II. b. Plant material, extraction and analysis 104

Plant material and cultivation 104
Extract preparation 105

Water residue extraction and fractionation 105
Essential oil and organic fractions analysis 105
HPLC-MS analysis 106
II. c. Acaricidal assay 106
III. Results 107
FINAL DISCUSSION 111
REFERENCES 114

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INTRODUCTION

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It has been estimated that less than 1-10 % of the large diversity of 250.000-500.000 plant
species on the Earth have been studied chemically and pharmacologically for their medicinal
properties.1,2 This is especially true for the tropical flora, as at date only 1 % of the species in
these habitats have been studied for their pharmaceutical potential.2 Traditions of collecting,
processing and applying plants and plant-based medications, have been handed down from
generation to generation.3 In African countries, traditional medicines, with medicinal plants as
their most important components, are sold in marketplaces or prescribed by traditional healers
at their homes.4 Because of this strong dependence on plants as medicines, it would be
important to study their safety and efficiency.5 The value of ethno-medicine and traditional
pharmacology is nowadays gaining increasing recognition in modern medicine because the
search for new, potential medicinal plants is more successful if the plants are chosen on an
ethno-medical basis. It has been estimated that 74% of the pharmacologically active plant-
derived components were discovered after the ethno-medical uses of the plants started to be
investigated.6
Since plants are long-lived stationary organisms, they must resist attackers over their lifetime
by producing and exuding secondary metabolites.
In biology, the concept of secondary metabolite can be attributed to Kossel7. He was the first
to define these metabolites as opposed to primary ones. Thirty years later an important step
forward was made by Czapek8 who dedi ‘ y’
w ‘endproduckt’. A g w
g y w ‘ y fi ’ .
Compared to the main molecules found in plants, these secondary metabolites were soon
defined by their low abundance, often less than 1% of the total carbon, or a storage usually
occurring in dedicated cells or organs. Plant secondary compounds are usually classified
according to their biosynthetic pathways.9 Three large molecule families are generally
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