Chemical Study and Biological Activities (Insecticide and Antiplasmodial) of Plant Extracts from Benin

MEMBERS OF THE JURY

READING COMMITTEE Prof. Dr. ir. Bruno De MEULENAER Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University

Prof. Taofiki AMINOU Laboratory of Expertise and Research in Environment and Water Chemistry, Faculty of Sciences and Techniques, University of Abomey-Calavi, Benin

Prof. Gnon BABA Laboratory of Sanitation, Water Sciences and Environment, Faculty of Sciences and Technology, University of Kara, Togo

EXAMINATION COMMITTEE Prof. Dr. ir. Peter BOSSIER Department of Animal production, Faculty of Bioscience Engineering, Ghent University

Prof. Dr. ir. Guy SMAGGHE Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University

- PROMOTERS Prof. Félicien AVLESSI Laboratory of Study and Research in Applied Chemistry, Polytechnic School of Abomey-Calavi, University of Abomey-Calavi, Benin

Prof. Dr. ir. Norbert DE KIMPE; Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Belgium

Prof. Dr. ir. Sven MANGELINCKX Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Belgium

- CHAIRMAN OF THE EXAMINATION COMMITTEE Prof. Dominique C. K. SOHOUNHLOUE Laboratory of Study and Research in Applied Chemistry, Polytechnic School of Abomey-Calavi, University of Abomey-Calavi, Benin

DEAN OF THE FACULTY DEAN OF THE FACULTY Prof. Dr. ir. Guido VAN HUYLENBROECK Prof. Felix HONTINFINDE

RECTOR OF THE UNIVERSITY RECTOR OF THE UNIVERSITY Prof. Dr. Anne DE PAEPE Prof. Brice SINSIN

UNIVERSITE D’ABOMEY-CALAVI GHENT UNIVERSITY ------FACULTE DES SCIENCES ET TECHNIQUES FACULTY OF BIOSCIENCE ENGINEERING ------FORMATION DOCTORALE CHIMIE ET APPLICATIONS DEPARTMENT OF SUSTAINABLE ORGANIC CHEMISTRY AND ------TECHNOLOGY LABORATOIRE D’ETUDE ET DE RECHERCHE EN CHIMIE ------APPLIQUEE (LERCA) ------N° d'ordre : 29-2014/FDCA/FAST/UAC

Thesis submitted in fulfillment of the requirements for the degree of: Doctorat de l'Université d'Abomey-Calavi – Spécialité : Chimie Organique et Chimie des Substances naturelles Doctor (PhD) in Applied Biological Sciences (UGENT)

CHEMICAL STUDY AND BIOLOGICAL ACTIVITIES (INSECTICIDE AND ANTIPLASMODIAL) OF PLANT EXTRACTS FROM BENIN

Defended By

Annick Flore Arlette Dohoué BOSSOU

Chairman Prof. Dominique C. K. SOHOUNHLOUE, Laboratory of Study and Research in Applied Chemistry, UAC: Polytechnic School of Abomey-Calavi, University of Abomey-Calavi, Benin Chairman Prof. Dr. ir. Peter BOSSIER, Department of Animal production, Faculty of Bioscience Engineering, UGENT: Ghent University Members: Prof. Dr. ir. Bruno De MEULENAER, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University Prof. Taofiki AMINOU, Laboratory of Expertise and Research in Environment and Water Chemistry, Faculty of Sciences and Techniques, University of Abomey-Calavi, Benin Prof. Gnon BABA, Laboratory of Sanitation, Water Sciences and Environment, Faculty of Sciences and Technology, University of Kara, Togo Prof. Dr. ir. Guy SMAGGHE, Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University Prof. Félicien AVLESSI, Laboratory of Study and Research in Applied Chemistry, Polytechnic School of Abomey-Calavi, University of Abomey-Calavi, Benin Prof. Dr. ir. Norbert DE KIMPE, Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Belgium Prof. Dr. ir. Sven MANGELINCKX, Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Belgium

Academic year 2014-2015

Dutch translation of the title: Chemische studie en biologische activiteiten (insecticide en anti-malaria) van plantenextracten uit Benin

To refer to this thesis: Bossou, A. F. A. D. (2014). Chemical study and biological activities (insecticide and antiplasmodial) of plant extracts from Benin. Doctoral dissertation, Joint PhD, Faculty of Sciences and Techniques, University of Abomey-Calavi, Benin - Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.

ISBN-number: : 978-90-5989-743-4

The author and the promoters give the authorisation to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. Ghent , 4 November 2014

Abomey-calavi, 4 November 2014

The author The promoter The promoter The promoter

Annick F. A. Prof. Félicien Prof. Dr. ir. Prof. Dr. ir. Sven D. BOSSOU AVLESSI Norbert DE KIMPE MANGELINCKX

This research was made possible by a fellowship allowed to the PhD student by the Belgian Technical Cooperation (BTC).

This research was also performed with the financial support of the Research Foundation - Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen).

TABLE OF CONTENT Dedication…………………………………………………………………………………………………… v Acknowledgements………………………………………………………………………………………… vi List of figures……………………………………………………………………………………………….. viii List of tables………………………………………………………………………………………………… ix Chapter 1: Introduction and aim…………………………………………………………………………. 1 Chapter 2: Background……………………………………………………………………………………. 5 2.1. Essential oils: general information…………………………………………………………………. 6 2.1.1. Definition……………………………………………………………………………………………………………………………….. 6 2.1.2. Extraction of essential oils ……………………………………………………………………………………………………… 6 2.1.2.1. Hydrodistillation………………………………………………………………………………………………………………….…….. 6 2.1.2.2. Simultaneous distillation extraction (SDE) ………………………………………………………………………………………. 7 2.2. Background on the chromatographic methods used………………………………………………………………. 8 2.2.1. Gas chromatography coupled with mass spectrometry (GC-MS) ……………………………………………… 8 2.2.2. Headspace-solid phase microextraction (SPME) ……………………………………………………………………… 9 2.3. Background on malaria……………………………………………………………………………………………………………. 10 2.3.1. Geographical distribution of malaria transmission and resistance…………………………………………….. 10 2.3.2. Symptoms and diagnosis ……………………………………………………………………………………………………….. 12 2.3.2.1. The symptoms of malaria……………………………………………………………………………………………………………. 12 2.3.2.2. Diagnosis of malaria…………………………………………………………………………………………………………………... 12 2.3.3. Malaria treatment ………………………………………………………………………………………………………………….. 13 2.3.3.1. Pharmacology of antimalarials……………………………………………………………………………………………………… 15 2.3.3.2. Effect of medicines on malaria transmission…………………………………………………………………………………….. 19 2.3.4. Malaria prevention, vector control ………………………………………………………………………………………...... 21 2.3.4.1. Larval control…………………………………………………………………………………………………………………………… 21 2.3.4.2. Reduction of human-vector contact……………………………………………………………………………………………….. 22 2.3.4.3. Increase adult vector mortality……………………………………………………………………………………………………… 22 2.3.5. Treatment of malaria by traditional medicine in Benin…………………………………………………………….. 22 2.4. Overview on Plasmodium falciparum…………………………………………………………………………………...….. 25 2.5. Overview on Anopheles gambiae…………………………………………………………………………………………….. 27 2.6. Overview on selected plants …………………………………………………………………………………………………… 29 2.6.1. Chenopodium ambrosioides L. (Amaranthaceae) ………………………………………………………………………. 29 2.6.2. Cochlospermum planchonii Hook. f. ex Planch. (Bixaceae) …………………………………………………………. 32 2.6.3. Cochlospermum tinctorium A.Rich (Bixaceae) ………………………………………………………………………..... 35 2.6.4. Cymbopogon citratus (DC.) Stapf (Poaceae) ……………………………………………………………………………... 37 2.6.5. Cymbopogon giganteus Chiov. (Poaceae) …………………………………………………………………………………. 39 2.6.6. Cymbopogon schoenanthus (L.) Spreng. (Poaceae) …………………………………………………………………… 41 2.6.7. Erythrophleum suaveolens (Guil. & Perr.) Brenan (Fabaceae) …………………………………………………… 43

2.6.8. Eucalyptus citriodora Hook (Myrtaceae) ………………………………………………………………………………….. 45 2.6.9. Eucalyptus tereticornis Sm. (Myrtaceae) ………………………………………………………………………………….. 47 2.6.10. Guiera senegalensis J.F. Gmel. (Combretaceae) ……………………………………………………………………….. 49 2.6.11. Securidaca longepedunculata Fresen. (Polygalaceae) …………………………………………………………….. 51 2.6.12. Spondias mombin L. (Anarcardiaceae) …………………………………………………………………………………… 53 2.7. Overview on Tribolium castaneum (Herbst, 1797) …………………………………………………………………. 55 Chapter 3: Experimental………………………………………………………………………………………………………………. 60 3.1. Plant material…………………………………………………………………………………………………………………………... 61 3.2. Plant extracts ………………………………………………………………………………………………………………………….. 61 3.2.1. Essential oils extracted by hydrodistillation……………………………………………………………………………. 61 3.2.2. Essential oils extracted by stimultaneous distillation extraction (SDE) …………………………………… 62 3.2.3. Ethanolic extracts…………………………………………………………………………………………………………………... 62 3.3. Gas chromatography coupled with mass spectrometry (GC-MS)..…………………………………………... 63 3.4. Headspace solid-phase microextraction (HS-SPME/GC-MS)..……………………………………………….. 63 3.5. Biological tests ………………………………………………………………………………………………………………………... 64 3.5.1. Insecticidal tests on Anopheles gambiae…………………………………………………………………………………… 64 3.5.1.1. Mosquito collection and determination of the status …….……………………………………………………………………. 64 3.5.1.2. Bioassay on adult mosquitoes………………………………………………………………………………………………………. 65 3.5.1.3. Susceptibility status of A. gambiae………………………………………………………………………………………………… 65 3.5.1.4. Data analysis for the bioassays…………………………………………………………………………………………………….. 66 3.5.2. Antiplasmodial tests on Plasmodium falciparum………………………………………………………………………. 66 3.5.2.1. In vitro cytotoxicity……………………………………………………………………………………………………………………... 66 3.5.2.2. In vitro antiplasmodial assays……………………………………………………………………………………………………… 67 3.5.3. Insecticidal tests on Tribolium castaneum……………………………………………………………………………….. 67 3.5.3.1. Stock of T. castaneum………………………………………………………………………………………………………………... 67 3.5.3.2. Contact "no choice" test……………………………………………………………………………………………………………… 67 3.5.3.3. Fumigant test…………………………………………………………………………………………………………………………… 68 3.5.3.4. Tests with isolated compounds……………………………………………………………………………………………………... 69 3.5.3.5. Procurement of the isolated compounds…………………………………………………………………………………………. 69 Chapter 4: Results and discussion……………………………………………………………………………………………... 71 4.1. Chemical composition obtained by hydrodistillation, simultaneous distillation extraction and solid phase microextraction…………………………………………………………………………………………………………….. 72

4.1.1. Chenopodium ambrosioides L. (Amaranthaceae) ………………………………………………………………………. 72 4.1.2. Cochlospermum planchonii Hook. f. ex Planch. (Bixaceae) ………………………………………………………. 73 4.1.3. Cochlospermum tinctorium A.Rich (Bixaceae) ………………………………………………………………………… 75 4.1.4. Cymbopogon citratus (DC.) Stapf (Poaceae) ……………………………………………………………………………. 77 4.1.5. Cymbopogon giganteus Chiov. (Poaceae) ……………………………………………………………………………….. 79 4.1.6. Cymbopogon schoenanthus (L.) Spreng. (Poaceae) ………………………………………………………………….. 80 4.1.7. Erythrophleum suaveolens (Guil. & Perr.) Brenan (Fabaceae) ………………………………………………….. 82

4.1.8. Eucalyptus citriodora Hook (Myrtaceae) ………………………………………………………………………………… 84 4.1.9. Eucalyptus tereticornis Sm. (Myrtaceae) ………………………………………………………………………………… 86 4.1.10. Guiera senegalensis J.F. Gmel. (Combretaceae) ……………………………………………………………………... 87 4.1.11. Securidaca longepedunculata Fresen. (Polygalaceae) …………………………………………………………….. 89 4.1.12. Spondias mombin L. (Anarcardiaceae) …………………………………………………………………………………… 90 4.1.13. Analysis of the chemical compositions…………………………………………………………………………………….. 93 4.1.13.1. Influence of extraction methods on chemical composition…………………………………………………………….. 93 4.1.13.2. Influence of extraction conditions on the chemical composition………………………………………………………. 94 4.1.13.3. Influence of climatic conditions on the chemical composition…………………………………………………………. 96 4.1.13.4. Variability of the chemical composition of the duplicates……………………………………………………………… 97 4.2. Ethanolic extracts…………………………………………………………………………………………………………………….. 98 4.3. Insecticidal tests on Anopheles gambiae…………………………………………………………………………………. 98 4.3.1. Adult bioassay on susceptible strain "Kisumu" of Anopheles gambiae………………………………………. 98

4.3.2. KDT50 and KDT95…………………………………………………………………………………………………………………….. 99 4.3.3. Mortality rates……………………………………………………………………………………………………………………… 99 4.3.4. Diagnostic concentrations …………………………………………………………………………………………………….. 101 4.3.5. Discussion on the insecticidal test against A. gambiae………………………………………………………………. 103 4.4. Cytotoxicity and antiplasmodial tests on Plasmodium falciparum…………………………………………….. 105 4.4.1. In vitro cytotoxicity………………………………………………………………………………………………………………… 106 4.4.2. In vitro antiplasmodial activity………………………………………………………………………………………………... 108 4.4.2.1. Chenopodium ambrosioides………………………………………………………………………………………………………… 108 4.4.2.2. Cochlospermum planchonii………………………………………………………………………………………………………… 109 4.4.2.3. Cochlospermum tinctorium………………………………………………………………………………………………………….. 109 4.4.2.4. Eucalyptus citriodora………………………………………………………………………………………………………………….. 110 4.4.2.5. Eucalyptus tereticornis……………………………………………………………………………………………………………….. 110 4.4.2.6. Cymbopogon citratus…………………………………………………………………………………………………………………. 110 4.4.2.7. Cymbopogon giganteus……………………………………………………………………………………………………………… 111 4.4.2.8. Cymbopogon schoenanthus………………………………………………………………………………………………………… 111 4.4.2.9. Securidaca longepedunculata………………………………………………………………………………………………………. 112 4.4.2.10. Guiera senegalensis………………………………………………………………………………………………………………… 112 4.4.2.11. Spondias mombin……………………………………………………………………………………………………………………. 113 4.4.2.12. Erythrophleum suaveolens………………………………………………………………………………………………………… 113 4.4.2.13. Discussion on the results obtained with the antiplasmodial tests………………………………………………………….. 113 4.5. Insecticidal tests against Tribolium castaneum………………………………………………………………………... 115 4.5.1. In vitro Contact "no choice" against Tribolium castaneum…………………………………………………………. 115 4.5.2. Discussion on the results obtained with the contact "no choice" tests……………………………………….. 120 4.5.2.1. C. citratus………………………………………………………………………………………………………………………………... 120 4.5.2.2. E. citriodora……………………………………………………………………………………………………………………………... 120 4.5.3. In vitro fumigant test against Tribolium castaneum…………………………………………………………………... 121

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4.5.4. Discussion on the results obtained with the fumigant tests………………………………………………………. 121 Chapter 5: Conclusion and perspectives……………………………………………………………………………………. 125 Chapter 6: Summary…………………………………………………………………………………………………………………….. 128 Chapter 7: Samenvatting…..……………………………………………………………………………... 133 References …………………………………………………………………………………………………………………………………… 139 Curriculum vitae ………………………………………………………………………………………………………………………….. 153

DDEEDDIICCAATTIIOONN

To God

To my dead parents

To my two kids and my husband

To all my family

ACKNOWLEDGMENTS

The current research is the result of four years work between two laboratories, under the supervision of eminent supervisors and colleagues that I would like to thank for their availability and hard working.

- To Prof. Félicien AVLESSI Thank for his simplicity, availability, and his aim to help us to become a very good researcher.

- To Prof. Norbert De KIMPE He gave to me the opportunity to find very good conditions and to perform the phytochemical part of this thesis. I would like to present to him my heartfelt gratefulness.

- To Prof. Sven MANGELINCKX His hard work and close supervision allowed to give the best of myself, and increase the scientific value of this doctoral thesis.

- To Prof. Dominique C. K. SOHOUNHLOUE His strictness at work and love of work well done were crucial in the success of this thesis.

- To Prof. Mansourou SOUMANOU By finding my Belgian promoter, he allowed me to achieve conditions to obtain a scholarship from the Belgian Technical Cooperation. Big thanks.

- To Dr. Edwige AHOUSSI-DAHOUENON (Maître de conférence- CAMES)

She was the support of my family during these four years, during which this PhD thesis was performed. I would like to say to her all my gratitude.

- To Dr. Ir. An ADAMS She initiated me to all phytochemical analysis, and allowed me to identify volatiles of the plants species by several techniques. Despite her departure from Ghent University, I would like to thank her for her invaluable assistance.

- To all my professors and colleagues from UAC and from UGENT Thank you for their unwavering support and wise advices.

- To all the members of the Jury All my gratitude for your suggestions and critics, which have contributed to the scientific quality of this research.

- To all my friends for which I was not available during this four years especially Akibou OSSENI, Sophie Bogninou Agbidinoukoun, Fifa T. Diane BOTHON, Arnaud SAGBO, Anne Marthe LANIAN, Lahana MANZA, Amina MANZA, Lucie FASSINOU and Lucie MOUSSOUGAN, big thanks for their support.

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LIST OF FIGURES

Figure 1: Clevenger apparatus for hydrodistillation……………………………………………………………….. 7 Figure 2: Likens-Nickerson apparatus for simultaneous distillation extraction………………………. 8 Figure 3: Head of the Anopheles female (top) and male (bottom)…………………………………………. 10 Figure 4: Diagram of the adult Anopheles………………………………………………………………………………. 10 Figure 5: Malaria-free countries and malaria-endemic countries in phases of control, pre- elimination, elimination, prevention and reintroduction by end 2008…………………….. 11 Figure 6: Malaria-endemic countries in Africa reporting resistance to pyrethroids to at least one malaria vector………………………………………………………………………………………………..... 12 Figure 7: Mode of action of antigen-detecting malaria RDTs…………………………………………………. 14 Figure 8: Transmission of Plasmodium falciparum and the effects of antimalarials……………….. 21 Figure 9: Map of Benin showing the collection sites of the plant species………………………………. 25 Figure 10: Life cycle of Plasmodium spp…………………………………………………………………………………… 26 Figure 11: Life cycle of the mosquito……………………………………………………………………………………….. 29 Figure 12: Freshly blood-fed mosquito Anopheles with a dilated abdomen……………………………… 29 Figure 13: Chenopodium ambrosioides……………………………………………………………………………………. 30 Figure 14: Cochlospermum planchonii……………………………………………………………………………………… 33 Figure 15: Cochlospermum tinctorium……………………………………………………………………………………… 36 Figure 16: Cymbopogon citratus………………………………………………………………………………………………. 38 Figure 17: Cymbopogon giganteus…………………………………………………………………………………………… 40 Figure 18: Cymbopogon schoenanthus…………………………………………………………………………………….. 42 Figure 19: Erythrophleum suaveolens………………………………………………………………………………………. 44 Figure 20: Eucalyptus citriodora………………………………………………………………………………………………. 46 Figure 21: Eucalyptus tereticornis……………………………………………………………………………………………. 48 Figure 22: Guiera senegalensis………………………………………………………………………………………………… 50 Figure 23: Securidaca longepedunculata…………………………………………………………………………………. 52 Figure 24: Spondias mombin……………………………………………………………………………………………………. 54 Figure 25: Tribolium castaneum………………………………………………………………………………………………. 56 Figure 26: Life cycle of Tribolium castaneum……………………………………………………………………………. 58 Figure 27: Chemical transformation of -terpinene during hydrodistillation…………………………… 96

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LIST OF TABLES Table 1: Pharmacology of antimalarials ...... 15 ...... Table 2: Effects of some used antimalarials on the infectivity of P. falciparum ...... 20...... Table 3: Ethnobotanical survey of the selected medicinal plant species used to prevent or treat malaria in Benin ...... 24 Table 4: Taxonomic position of Plasmodium ...... 25 ...... Table 5: Taxonomic position of Anopheles gambiae ...... 27 Table 6: Taxonomic position of Tribolium castaneum ...... 56 Table 7: Locations, soils and organs extracted from selected plant species ...... 61 Table 8: Volumes and concentrations in fumigant test ...... 71 Table 9: Quantities used in contact "no choice" test ...... 71 Table 10: Chemical composition and essential oil yields of Chenopodium ambrosioides ...... 74 ...... Table 11: Chemical composition and essential oil yields of Cochlospermum planchonii ...... 75 ...... Table 12: Chemical composition and essential oil yields of Cochlospermum tinctorium ...... 77 ...... Table 13: Chemical composition and essential oil yields of Cymbopogon citratus ...... 79 ...... Table 14: Chemical composition and essential oil yields of Cymbopogon giganteus ...... 81 ...... Table 15: Chemical composition and essential oil yields of Cymbopogon schoenanthus ...... 82 ...... Table 16 Chemical composition and essential oil yields of Erythrophleum suaveolens ...... 84 ...... Table 17: Chemical composition and essential oil yields of Eucalyptus citriodora ...... 86 ...... Table 18: Chemical composition and essential oil yield of Eucalyptus tereticornis ...... 87 ...... Table 19: Chemical composition and essential oil yields of Guiera senegalensis ...... 89...... Table 20: Chemical composition and essential oil yields of Securidaca longepedunculata ...... 90 ..... Table 21: Chemical composition and essential oil yields of Spondias mombin ...... 92...... Table 22: Main compounds identified by hydrodistillation, SDE and SPME coupled to GC-MS ...... 93 Table 23: Variation of chemical composition of Chenopodium ambrosioides essential oil by climatic conditions ...... 97 ..... Table 24: Location soil and relative variation of major compounds amounts of Chenopodium ambrosioides ...... 98 ...... Table 25: Variability of the chemical composition of Cochlospermum tinctorium during SPME analysis ...... 98...... Table 26: Yield of ethanolic extracts ...... 99 Table 27: Knock down times (KDT), mortality and susceptibility of essential oils on sensitive Anopheles gambiae “Kisumu” ...... 101......

Table 28: LC50, LC99 and diagnostic concentration for all essential oils tested ...... 103

Table 29: KDT50, KDT95 and mortality of essential oils tested on the resistant strain of Anopheles gambiae ...... 103 ...

Table 30: Cytotoxicity and efficacity of extracts against Pf-K1 ...... 108 ...... Table 31: Efficiency of essential oils by "contact no choice" test against Tribolium castaneum ...... 118 Table 32: Mortalities obtained by contact no choice test against Tribolium castaneum ...... 119 ...... Table 33: Lethal concentrations of essential oils and isolated compounds against Tribolium castaneum, by fumigation ...... 120 Table 34: Mortalities obtained by fumigation against Tribolium castaneum ...... 120...... Table 35: Recapitulative of insecticide activities of essential oils against Anopheles gambiae and Tribolium castaneum ...... 123 Table 36 Antiplasmodial activites of studied plant extracts ...... 123......

CHAPTER 1: INTRODUCTION AND AIM

Malaria is a life-threatening disease affecting 3.3 billion people worldwide with 80% of cases and 90% of deaths occurring in sub-Saharan Africa (SSA). Of those affected, children under the age of five and pregnant women are the most vulnerable [1]. The key factor for the reduction of human mortality is their protection against the malaria parasite vectors, the Anopheles mosquito, by the use of long-lasting insecticidal nets (LLIN) and application of indoor residual spraying (IRS) as recommended by the WHO [1]. Pyrethroids are the only insecticides recommended by WHO for the treatment of bednets [1] while in West Africa, Anopheles gambiae Giles is the major vector of malaria [2]. This mosquito species is now subjected to selection pressure, and has become resistant to dichlorodiphenyltrichloroethane (DDT) and pyrethroids because of the use of these insecticides both for agricultural and public health purposes [2, 3]. During the last decade, the emergence of resistance to synthetic insecticides in Anopheles populations has been widely described in many African countries such as Benin [4-10], Ivory Coast [2, 11], Niger [12], Burkina-Faso [3, 13, 14], Mali [15], Nigeria [16], Kenya [17], Cameroun [18-20], Zanzibar [21], Uganda [22], Equatorial Guinea [23], and Ghana [24]. Because of the widespread occurrence of this resistance, there is a reduction of the efficacy of synthetic insecticides used in the field. Therefore, it is pertinent to explore the pesticidal activity of natural products [25], such as essential oils. Indeed, the use of essential oils has been recognized as a potential alternative in the control of vectors of mosquito-borne diseases [26-30] such as A. gambiae. A lot of research on the pesticidal activities of essential oils has been conducted and has proven that essential oils could be considered as potent bioactive compounds against various pests and mosquitoes [28, 31-34].

Following the above, the first objective of the current study was to investigate the chemical composition, the insecticidal and the antiplasmodial activities of twelve plant extracts traditionally used in Benin as a natural method of protection against A. gambiae, and Plasmodium falciparum, in order to confirm the traditional knowledge of Benin's population and to find new valuable sources of bioactive molecules to conquer the resistance of the vector and the parasite of malaria. Indeed, these plant species have been reported to possess repellent and insecticidal activities against various mosquitoes, stored product beetles and other pests such as Anopheles

species [30, 35], Culex quinquefasciatus [36], Callosobruchus species [37, 38], Sitophilus species [39, 40] and Tribolium castaneum [41].

The second objective of the present PhD study was to evaluate the efficacy of some essential oils against Tribolium castaneum, the "red flour beetle". The choice of Tribolium castaneum was based on its economic importance expressed by its worldwide distribution and its preference for several stored products and grocery stores [42]. Since a very long time, the control of T. castaneum has required the use of chemicals and has led to the appearance and development of resistance in the genus Tribolium [43] as well as adverse effects in animals and humans [44]. It has been reported that botanical extracts and essential oils have been successfully evaluated against this pest as natural repellent or natural insecticide [45-49]. In order to control T. castaneum in a benign manner with respect to human health and environment, the current work has also screened the efficacy of some essential oils as potential natural pesticides against this pest.

To achieve our goals, the following research steps were undertaken: 1. The essential oils have been extracted from twelve plant species namely by hydrodistillation and simultaneous distillation extraction. The plant species were selected for their good scientific interest according to our area of study (no publication in Benin on their insecticidal and antiplasmodial properties).

2. The volatile profiles of each plant species have been established by hydrodistillation, simultaneous distillation extraction, and solid phase microextraction coupled with gas chromatography and mass spectrometry.

3. The standard WHO susceptibility test has been used with various concentrations of essential oils under laboratory conditions [50], to evaluate the insecticidal properties of essential oils used, against A. gambiae.

4. The ethanolic extracts and essential oils have been tested to determine the

susceptibility of the K1 strain of P. falciparum to these plant extracts.

5. The essential oils of some plant species have been tested by contact test and fumigation test, to identify the susceptibility of T. castaneum to these natural extracts.

The selected plants species included Chenopodium ambrosioides L. (Amaranthaceae), Cymbopogon citratus (DC.) Stapf (Poaceae), Cymbopogon schoenanthus (L.) Spreng. (Poaceae) and Cymbopogon giganteus Chiov. (Poaceae), Eucalyptus citriodora Hook. (Myrtaceae), Eucalyptus tereticornis Sm. (Myrtaceae), Cochlospermum planchonii Hook. f. Ex Planch. (Bixaceae), Cochlospermum tinctorium A. Rich. (Bixaceae), Erythrophleum suaveolens (Guil. & Perr) Brenan (Fabaceae), Guiera senegalensis J. F. Gmel. (Polygalaceae), Securidaca longepedunculata Fresen. (Polygalaceae) and Spondias mombin L. (Anarcadiaceae).

CHAPTER 2: BACKGROUND

CHAPTER 2: BACKGROUND 2.1. Essential oils: general information

2.1.1. Definition According to the European Pharmacopoeia Commission, an essential oil is a: "Fragrant product, generally with a complex composition, obtained from a raw vegetable botanically defined, either by steam distillation, or dry distillation, or a proper mechanical process without heating. The essential oil is most often separated from the aqueous phase by a physical process involving no significant change in its composition" [51].

2.1.2. Extraction of essential oils For the purpose of this research, the following two methods were used to obtain essential oils: hydrodistillation and simultaneous distillation extraction (SDE).

2.1.2.1. Hydrodistillation According to the European Pharmacopoeia, hydrodistillation is a process during which a quantity of material is submitted to steam distillation [51] in an alembic such as a Clevenger-type apparatus (Fig 1) and extracted until no more essential oil is obtained. The essential oil is condensed in a liquid phase, in the extractor's refrigerant, inside which it is separated from the water by decantation. The essential oil obtained is often dried over anhydrous sodium sulfate, and stored in dry, dark glass bottles in a fridge at 4°C until analysis and/or uses [52, 53]. This technique of extraction is useful to obtain essential oils from aromatic samples such as aromatic plant species [29, 39, 54-56].

Figure 1: Clevenger apparatus for hydrodistillation

2.1.2.2. Simultaneous distillation extraction (SDE) Simultaneous distillation extraction is a liquid-liquid extraction technique, using an apparatus such as Likens-Nickerson apparatus (Fig 2). The Likens-Nickerson method is based on the relative solubility of an analyte in two immiscible phases. Analytes in solution are extracted by direct partitioning with an immiscible solvent. Distilled water is used to boil the sample in one flask while the extracting solvent is boiled in another flask [57]. During this process, a solvent lighter than water or heavier than water can be used. Water and solvent vapors are condensed on the surface of the cold tube. The volatiles are removed from the matrix by the water vapor and transferred to the organic phase when the liquids condense together in the cold tube. Then both water and solvent are collected in the extractor body after their condensation and returned into the corresponding flasks, allowing continuous reflux [58]. After these steps, the mixture (solvent and essential oil) obtained is concentrated at ambient temperature, with a rotavapor to obtain the essential oil.

Figure 2: Likens-Nickerson apparatus for simultaneous distillation extraction

2.2. Background on the chromatographic methods used 2.2.1. Gas chromatography coupled with mass spectrometry (GC-MS) Gas chromatography is a technique of separation of a mixture into its different compounds. When coupled to mass spectrometric detection, this technique provides detailed information about the identity, the molecular composition and the amount of a compound through a chromatogram which is the graphical image of a detector output [59]. A gas chromatograph uses a carrier gas and is composed of an injector, an oven containing a column, a detector with its amplifier, a computer for the handling of data and a printer [60, 61]. The separation is based both, on the mobile phase, which is an inert gas, and the stationary phase, which is a solid, or a fixed liquid packed into a column. With a syringe (injector port), the sample is quickly injected into the column, where the chromatographic behaviour of each compound depends on the affinity between the compound, the stationary phase and the

temperature. It follows that compounds with different physical and chemical properties will reach the detector at different times called retention times [60, 61]. In the mass spectrometer, analytes are bombarded with electrons and are broken into several fragments [62]. For each analyte, a single ion fragment is tracked using selected ion monitoring (SIM), and the quantification is done by comparison of the ion current with a standard curve [63].

2.2.2. Headspace-solid phase microextraction (HS-SPME) Solid phase microextraction (SPME) is a solvent free sample extraction method invented by Janusz Pawliszyn in 1989 [64] developed by Supelco [65], as a result of the new regulatory restrictions on solvent uses [66], and to permit rapid sample preparation both in situ and in laboratory [67]. For this purpose, a small amount of the sample is introduced in SPME vials, and an extraction fiber is placed in the sample matrix for a predetermined amount of time. This phase permits the establishment of concentration equilibrium between the sample matrix and the extraction phase. The extraction phase and the transport of analytes begin as soon as the sample is in contact with the fiber [67]. The fiber to be used depends on the molecular weight of compounds inside the mixture, and determines the volatile profile obtained. For instance, 75 μm/85 μm carboxen/polydimethylsiloxane fibers are suitable for gases and low molecular weight compounds (MW 30-225), whereas 100 μm polydimethylsiloxane fibers are useful for the identification of volatiles (MW 60-275). 65 μm polydimethylsiloxane/divinylbenzene fibers and 85 μm polyacrylate fibers are the most efficient for volatiles, amines, nitro-aromatic compounds (MW 50-300) and polar semi-volatiles (MW 80-300), respectively. For the identification of flavor compounds C3-C20 (MW 40-275) and trace compounds (MW 40-275) it is recommended to use a 50/30 μm divinylbenzene/carboxen on polydimethylsiloxane on a StableFlex fiber. In practice, the HS-SPME method allows a retractile needle covered with a suitable phase inside an isolated environment (Headspace) to trap analytes and to put them into an injector where they are desorbed [65]. This method can be used for several types of material, solid or liquid [68, 69], in vitro and in vivo [70-72], for the determination of volatiles in environmental samples [70, 73, 74], contaminants in food [57, 75-77], phenolic compounds in foods as well as volatiles in essential oils [69], to name a few applications.

2.3. Background on malaria 2.3.1. Geographical distribution of malaria transmission and resistance Malaria is a parasitic disease transmitted by mosquito bites, and can be fatal. The parasite called Plasmodium enters in its human host when he is bitten by a mosquito of the genus Anopheles, infected by a blood meal. Only the female of Anopheles can take blood meals and transmit malaria because of her morphology: on the female antennae, hairs are few and short, whereas the male has very long hairs, which have a bushy texture (Fig 3 and 4), thus preventing the latter from taking blood meals.

Figure 3: Head of the Anopheles female (top) Figure 4: Diagram of the adult Anopheles [78] and male (bottom) [78]

There are four forms of malaria in humans due to four different species of Plasmodium: Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium falciparum. The most commonly encountered are malaria due to P. vivax and P. falciparum, and the most lethal is P. falciparum. Malaria due to P. falciparum is widespread in sub-Saharan Africa particularly in Benin and is largely responsible for the high mortality recorded in this region [79]. Recently, resistance to antimalarial compounds is widespread and the approach chosen by the international community for malaria control is an integrated approach including prevention and treatment. The first antimalarial used to fight malaria was chloroquine but now this drug is ineffective in most countries where P. falciparum malaria occurs. P. falciparum resistance to other antimalarials such as amodiaquine, mefloquine, quinine,

and sulfadoxine-pyrimethamine was also noticed and, more recently, to artemisinin derivatives [80]. The geographical distribution of malaria, of its resistance and its speed of propagation showed considerable variation (Fig 5). This P. falciparum resistance is linked to mutations of five genes namely dihydrofolate reductase (dhfr) gene, dihydropteroate synthase (dhps) gene, P. falciparum chloroquine-resistance transporter (pfcrt) gene, P. falciparum multidrug-resistance gene 1 (pfmdr1) and the cytochrome b gene [81]. Besides the resistance of Plasmodium species to antimalarials, the vector of malaria Anopheles gambiae became resistant to dichlorodiphenyltrichlorethane (DDT) and pyrethroids because of the long use of these insecticides both for agricultural and health purposes [3, 82]. During the last decade, the emergence of this resistance to DDT and pyrethroids has been shown in African countries such as Benin [6, 83-85], Ivory coast [82, 86], Niger [12], Burkina-Faso [3], Mali [15], Nigeria [16], Kenya [17], Cameroon [19] etc. The resistance to pyrethroids in Africa is summarized in Fig 6.

Figure 5: Malaria-free countries and malaria-endemic countries in phases of control, pre-elimination, elimination, prevention and reintroduction by end 2008 [87].

Figure 6: Malaria-endemic countries in Africa reporting resistance to pyrethroids to at least one malaria vector [88]

2.3.2. Symptoms and diagnosis 2.3.2.1. The symptoms of malaria The first symptoms of malaria are headache, lassitude, fatigue, abdominal discomfort, muscle and joint aches, fever, chills, perspiration, anorexia, vomiting and worsening malaise [80]. If the patient is treated with an effective antimalarial specific to the region, he improves quickly. Other severe symptoms, due to the continuous burden of the parasite, appear within a few hours. These severe symptoms are coma (cerebral malaria), metabolic acidosis, severe anemia, hypoglycemia, acute renal failure or acute pulmonary œdema. At this stage of the disease, 10-20% of the treated patients will die [80].

2.3.2.2. Diagnosis of malaria Two principal methods are used in the diagnosis of malaria: clinical diagnosis and parasitological or confirmatory diagnosis.

2.3.2.2.1. Clinical diagnosis Clinical diagnosis of malaria is performed on the basis of the history of fever, taking into account the epidemiological settings as indicated below [80]: - In the case that the risk of malaria is low and if any symptoms of other illness appear, the clinical diagnosis will be done on the history of fever in the previous three days; - If the risk of malaria is high, the clinical diagnosis will be performed on the basis of the fever history during the previous 24 hours and/or the presence of anemia (pallor of palm) - In the two previous cases the clinical diagnosis needs to be confirmed by a parasitological diagnosis

2.3.2.2.2. Parasitological diagnosis To perform parasitological diagnosis of malaria, two tests can be conducted: light microscopy and rapid diagnostic tests (RDTs). The goal of light microscopy is to identify the species, to quantify the parasites responsible for the symptoms observed (also for other causes of fever), to evaluate the response to the antimalarial treatment chosen and it also has a lower cost than RDTs. However, this diagnosis technique requires qualified and well-trained staff as well as the presence of an energy source to power the microscope.

RDTs objectives (Fig 7) are to detect parasite-specific antigens or enzymes and/or to differentiate species by the detection of histidine-rich protein 2 (HRP2), specific for P. falciparum, pan-specific or species-specific Plasmodium lactate dehydrogenase (pLDH) or pan-specific aldolase [80].

2.3.3. Malaria treatment The emergence of resistance of P. falciparum to monotherapies and its rapid propagation through variable continents have forced WHO to recommend antimalarial combination therapies (ACTs) for the treatment of uncomplicated malaria due to P. falciparum and other Plasmodium. In ACTs, two or more antimalarials with independent modes of action are combined. This simultaneous administration of two

or more schizontocides1 has the aim to improve the efficacy of the treatment and to delay the emergence of resistance against each of these schizontocides, because even if the parasite is resistant to one drug it cannot be resistant to all schizontocides associated and will be killed by one of them. The potential disadvantages of ACTs are the potential risk to increase side effects [80].

Figure 7: Mode of action of antigen-detecting malaria RDTs [89] (a) Dye-labeled antibody (Ab), specific for target antigen (Ag), is present on the lower end of the nitrocellulose strip or in a well provided with the strip. Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line, and either antibody specific for the labeled antibody, or antigen, is bound at the control line. (b) Blood and buffer, which have been placed on the strip or in the well, are mixed with the labeled antibody and are drawn up the strip across the lines of bound antibody. (c) If antigen is present, some labeled antibody will be trapped on the test line. Other labeled antibody is trapped on the control line.

1 A schizonticide is a drug, which is selectively destructive of the schizont. The latter being the multinucleated cells which appear during the asexual phase of Plasmodium, when the parasite becomes active, after the incubation phase (see more detail in page 26).

2.3.3.1. Pharmacology of antimalarials According to the areas of transmission and resistance, several antimalarials are used. Names, structures and mechanisms of action of the commonly used molecules are summarized in Table 1. Table 1: Pharmacology of antimalarials

Antimalarials Chemical structures Mechanisms of action

Chloroquine Chloroquine is a 4-aminoquinoline, which cannot be used against P. falciparum malaria because of the N resistance of this strain; nevertheless, it can still be HN efficient in the case of vivax malaria, ovale malaria and malariae malaria. Chloroquine interferes with parasite haem detoxification on its site of action which is the food vacuole of the parasite [80].

Cl N

Sources: [90-92] Amodiaquine OH Amodiaquine is also a 4-aminoquinoline which has a similar mode of action of that of chloroquine

N (interference with parasite haem detoxification) and HN which is effective against some chloroquine-resistant strains of P. falciparum, despite the existence of cross- resistance [80].

Cl N

Sources: [90-92] Sulfadoxine Sulfadoxine is a sulfonamide. Sulfonamides are N N O O antimetabolites that are competitive inhibitors of

S dihydropteroate synthase (the bacterial enzyme that is Me N O responsible for the incorporation of p-aminobenzoic H acid), important in the synthesis of folic acid [80]. All O cells need folic acid to grow. In humans, this substance H N 2 Me can diffuse or be actively transported into cells but cannot cross the protozoan wall the same way and

needs to be synthetized from p-aminobenzoic acid by Source: [90-92] Plasmodium for its growth [93]. Pyrimethamine Pyrimethamine is a diaminopyrimidine, which is Cl effective against all four human malaria types by

NH2 inhibiting plasmodial dihydrofolate reductase and which is used in combination with a sulfonamide (sulfadoxine or dapsone) to slow down resistance. By N its mechanism of action, pyrimethamine prevents the synthesis of purines and pyrimidines, the nucleic acids H2N N in Plasmodium that are essential for its DNA synthesis and its cell multiplication. Because of this mode of action pyrimethamine is active against schizont Source: [90-92] formation in erythrocytes and liver and sporozoites development in Anopheles species [80, 94].

Table 1: Pharmacology of antimalarials (continued) Antimalarials Chemical structures Mechanisms of action

Mefloquine Mefloquine is a 4-hydroxymethylquinoline, related NH H to quinine, and is effective against all forms of malaria. Mefloquine is a schizonticide active against OH erythrocytes. With free heme, mefloquine develops toxic complexes that break P. falciparum membrane and interact with other plasmodial components, to provoke the enlargement of P. falciparum food vacuole [80, 95]. F N F F F F F Sources: [90-92] Artemisinin Artemisinin (qinghaosu in Chinese), is a sesquiterpene trioxane lactone extracted from the leaves of Artemisia annua with an endoperoxide bridge which confers to this molecule its O O antiplasmodial activity by killing schizonts and O gametocytes. Its mechanism of action is still on debate. One of the probable mechanisms of action O H of artemisinin and its derivatives (artemether, H artesunate, dihydroartemisinin, artemotil), is the O H inhibition of calcium adenosine triphosphatase, PfATPase 6. Another probable mechanism is the bioactivation of artemisinin in the parasites. Indeed Sources : [90-92] to create space within its digestive vacuole, and to have enough material (peptides and amino acids), for its development, the red cell degrades its hemoglobin and releases hematin which is toxic to the parasite. To survive into its host, the parasite has developed a mechanism to transform hematin into insoluble non-toxic hemozoin, which is malaria pigment. Artemisin provokes the bioactivation of 1,2,4-trioxanes triggered by iron(II) to generate toxic activated oxygen [96]. Artemether Artemether is used to treat malaria due to P. falciparum by inhibiting nucleic acid and protein

O O synthesis on the erythrocytes of the parasite [97]. After administration, this drug is metabolized into O dihydroartemisinin, the active metabolite, and oral O route gives a better performance than the H H intramuscular administration [80]. Me O H

Sources: [90-92]

Table 1: Pharmacology of antimalarials (continued) Antimalarials Chemical structures Mechanisms of action

Dihydroartemisinin Dihydroartemisinin is the main active metabolite of the artemisinin derivatives and its mechanism of O O action is similar to artemisinin [80]. O

O H H

HO H

Sources: [90-92] Artesunate Artesunate is the sodium salt of the hemisuccinate of artemisinin [80]. For its mechanism of action, see O O artemisinin. O

O O H H HO O H O Sources :[90-92] Artemotil Artemotil is the ethyl ether of artemisinin, given only by intramuscular route [80]. The mechanism of O O action of artemotil is the same of that of artemisinin. O

O H H

O H

Source: [91] Lumefantrine Lumefantrine is an aryl-alcohol existing only in (Benflumetol) combination with arthemether. This combination is very effective against all human malarial parasites, N including multi-drug resistant P. falciparum [80, 92]. Lumefantrine is also a schizonticide active against erythrocytic stages of P. falciparum. Its mechanism HO of action is unknown but available data suggest the formation of a complex with hemin and the Cl inhibition of the synthesis of nucleic acids and proteins by P. falciparum [98]. (The mechanism of action of arthemether is the same of that of Cl artemisinin).

Sources : [90-92]

Table 1: Pharmacology of antimalarials (continued) Antimalarials Chemical structures Mechanisms of action

O Me

N Primaquine Primaquine is an 8-aminoquinoline effective against HN P. vivax, P. ovale and P. falciparum in their stage in NH2 the liver. It exerts its antiplasmodial action by

interference with the mitochondrial function of the parasite [80, 92, 99]. Source: [90-92] Atovaquone O Atovaquone is a hydroxynaphthoquinone active HO against all Plasmodium species, in which it exerts its

H antiplasmodial activity by interference with the cytochrome bc1 complex called Complex III with at least the inhibition of nucleic acid and ATP synthesis. O Atovaquone is also effective against Anopheles

species on pre-erythrocytic stage in the liver, and

H oocyst [80, 100].

Source: [91] Proguanil Proguanil is a biguanide compound, which is H H H transformed in the body into cycloguanil as its active N N N metabolite. Only 3% of Caucasian and African populations are able to make this transformation. In NH NH the plasmodia, this drug is active against primary Cl exoerytrocytic forms of P. falciparum and asexual blood forms of all Plasmodium species but possess a weak action on gametocytes. Proguanil exerts on plasmodia a weak antimalarial activity by inhibiting

the dihydrofolate reductase. The consequence is the Sources: [91, 92] lack of synthesis of purines and pyrimidines required for the synthesis of DNA and cell multiplication [80, 101, 102]. Quinine Quinine is an alkaloid extracted from Cinchona tree N bark. This drug is still very efficient against severe P. falciparum malaria in areas such as Benin where OH resistance to chloroquine has been demonstrated. H H Quinine blocks the plasmodium capacity to detoxify O haem in the food vacuole by a mechanism not well Me known. Quinine is active against immature gametocytes and schizonts and is therefore very effective for clinical uses [80, 102, 103]. N Sources: [90-92]

Table 1: Pharmacology of antimalarials (continued)

Antimalarials Chemical structures Mechanisms of action

Tetracycline Me Me Tetracycline, doxycycline and clindamycine, are N three antibiotics with antimalarial activity, which are HO H H OH an interesting alternative for the treatment of multidrug-resistant falciparum malaria. The tetracyclines are active against primary NH2 exoerythrocytic form of P. falciparum, and should OH only be used to treat malaria due to resistant strain OH O OH O O of Plasmodium [102]. They are inhibitors of the Source: [91] binding aminoacyl-tRNA during protein synthesis [80]. Doxycycline Me Me Doxycycline is a tetracycline derivative, which may OH N be preferred to tetracycline because of its longer H H OH half-life, its reliable absorption and its safety profile when it is used by patients with renal insufficiency [80]. NH2

OH OH O OH O O

Source: [91]

Clindamycine Me Clindamycin is a chlorinated derivative of lincomycin. O Cl It inhibits the early stages of protein synthesis by a N H mechanism similar to that of the macrolides [80].

N H H OH

OH S

Me OH Source: [91]

2.3.3.2. Effect of medicines on malaria transmission Two mechanisms can explain the reduction of malaria transmission: early and effective treatment of the malaria blood infection and the lowering of the parasite infectivity (Table 2). Early and effective treatment of the malaria blood infection with any antimalarial will eliminate the asexual blood stages of the parasite, of which gametocytes derive. In

malaria due to P. vivax, P. malariae and P. ovale, immature gametocytes cannot survive more than 2-3 days, whereas mature gametocytes are short-lived. On the contrary, in case of P. falciparum, gametocytes take about 12 days for their developmental period and mature from a young parasite (merozoite) and mature gametocytes may remain infective for up to several weeks. Regarding this previous information, it is evident that an effective treatment of asexual blood parasites is sufficient to cure malaria covered by P. vivax, P. malariae, and P. ovale. It is not the case with malaria due to P. falciparum, in which infection can remain for several weeks after the patient has been treated by an efficient specific anti-gametocyte medication [80]. Medicines can also lower the parasite infectivity through a direct gametocytocidal effect or a sporonticidal effect (Table 2; Figure 8). Indeed: - Chloroquine is effective against young gametocytes, but enhances the infectivity of mature infective gametocytes; - In contrast, sulfadoxine-pyrimethamine increases gametocyte carriage but reduces their infectivity; - Artemisinins are the most gametocytocidal drugs currently used. They destroy young gametocytes, prevent new infective gametocytes, but have a small effect on mature gametocytes; - Primaquine acts on mature gametocytes. In addition to ACTs, in the treatment of malaria due to P. falciparum, primaquine is useful because primaquine acts on mature gametocytes, on which artemisinins have no or very little effect [80].

Table 2: Effects of some used antimalarials on the infectivity of P. falciparum

Effect of treatment Gametocytocidal Sporonticidal Sporonticidal Overall effect on Drug Viability of young Infectivity of suppressing Viability of mature sequestered gametocytes to infectivity circulating gametocytes gametocytes mosquitoes Chloroquine Reduces No effect Enhances Moderate effect Sulfadoxine- No effect Increases Decreases No overall effect pyrimethamine Artemisinin Greatly reduces Little effect Unknown Very high effect derivatives Primaquine Unknown Greatly reduces Unknown Very high effect Quinine No effect No effect No effect None Source: [80]

*When parasites are sensitive to the drug unless otherwise stated. Positive and negative arrows indicate the effect of the drug, enhancement (+) and suppression (–), respectively, on the parasite stage or its development. Figure 8: Transmission of Plasmodium falciparum and the effects of antimalarials, Source: [80]

2.3.4. Malaria prevention, vector control Prevention of malaria involves the prevention of contact with the vector of the disease, the female of Anopheles gambiae. The control of this vector incorporates three major components namely larval control, control of human-vector contact and control of adult mosquitoes [78].

2.3.4.1. Larval control Larval control is indicated to control vectors if a large proportion of the breeding sites are accessible and can be easily identified and treated. It is effective in areas with dense human population with few gites, in arid climates during periods of drought, in endemic areas where the breeding sites are well located, accessed, and treatable and in refugee camps located in low rainfall risk areas and in development areas.

This control involves the larval source reduction to eliminate mosquito-breeding sites or to prevent the breeding of mosquitoes, the use of larvivorous fish, the larviciding by chemical and biological larvicides, the use of petroleum oils, common chemical insecticides, and insect growth regulators.

2.3.4.2. Reduction of human-vector contact The World Health Organization recommends several strategies to prevent mosquito bites as malaria prevention and control intervention. These strategies include the use of long lasting insecticidal mosquito nets and other insecticide-treated materials like curtains on doors and windows, the improvement of housings and locations near the breeding sites to prevent the mosquito’s entry, the use of repellents, mosquito coils and protective clothing.

2.3.4.3. Increase adult vector mortality Control of adult mosquito population is achieved mainly by two methods: indoor residual spraying (IRS) and space spraying. - Indoor residual spraying (IRS) IRS is still a viable option for malaria control when applied in appropriate circumstances. However, three problems are associated with this method, in particular high prices, resistance acquired by the vector and environmental risks. - Space spraying Space spraying is defined as the destruction of flying mosquitoes by contact with insecticides in the area, especially when the vector activity is at its peak, and should be used as a complementary measure to reduce the density of the vector by increasing its mortality.

2.3.5. Treatment of malaria by traditional medicine in Benin In many parts of the world and in Africa and particularly in Benin, traditional medicine is widespread and treats several diseases especially those most faced in each country. Many diseases are transmitted through the bite of infected insects. These diseases include malaria, yellow fever, dengue fever, trypanosomiasis and onchocerciasis. Some diseases such as cholera and typhoid fever are transmitted through contaminated food and water. Other diseases which can be transmitted through intimate contact include AIDS/HIV, hepatitis B and meningococcal meningitis

[104]. Regarding malaria, its treatment in Benin is achieved using several plants. To choose the plant species, studied in the current work, an ethnobotanical survey has been conducted. This ethnobotanical survey has been realized to identify plants used in traditional medicine against malaria and its symptoms such as fever, headache, and pain. During this survey, fourthy-two traditional practitioners and herborists (persons who sell dried plant material and advise people) have been interviewed from May 2009 to June 2010. Following this first step, a literature review has been conducted to identify twelve plant species which have, according to our area of study, a good scientific interest (no publication in Benin on their insecticidal and antiplasmodial properties). Subsequently, with Prof. Hounnankpon Yedomonhan, the curator of the National Herbarium of Benin, the sites of harvesting of these twelve plants have been identified, and three periods of campaign of harvesting have been identified and performed during the great rainy season of Benin which occurs from mid-March to mid-July. These periods of harvest were from May 2009 to June 2009, from March 2011 to May 2011 and from March 2012 to June 2012. During our survey, the following information was collected for each plant species retained: vernacular names, the parts used, preparation of the herb tea and its administration. The different villages of harvesting have been identified in the Fig 9 and the uses of plant species were summarized in Table 3. The selected plant species for this study include three species of Cymbopogon namely Cymbopogon citratus (DC.) Stapf (Poaceae), Cymbopogon schoenanthus (L.) Spreng. (Poaceae) and Cymbopogon giganteus Chiov. (Poaceae), two species of Eucalyptus which are Eucalyptus tereticornis Sm. (Myrtaceae), Eucalyptus citriodora Hook. (Myrtaceae), two species of Cochlospermum namely Cochlospermum planchonii Hook. f. Ex Planch. (Bixaceae), Cochlospermum tinctorium A. Rich. (Bixaceae), as well as Chenopodium ambrosioides L. (Amaranthaceae), Securidaca longepedunculata Fresen. (Polygalaceae), Erythrophleum suaveolens (Guill. & Perr.) Brenan (Fabaceae), Spondias mombin L. (Anacardiaceae), Guiera senegalensis J.F. Gmel. (Combretaceae). Their use in the treatment of malaria in Benin is secular and needs to be proven scientifically, hence the interest of our study. For prevention purposes, these plants are burnt and the smoke is detrimental to mosquitoes, whereas malaria treatment is achieved by oral absorption of infusion, aqueous extract, or ethanolic decoction.

Table 3: Ethnobotanical survey of the selected medicinal plant species used to prevent or treat malaria in Benin

Plant species Vernacular Cities Plant parts Uses names used Cymbopogon citratus Cha, tigbe Cotonou Leaves Decoction or infusion of leaves is (DC.) Stapf (Poaceae) used against malaria, fever and as repulsive agent against mosquitoes Cymbopogon giganteus Gbèzin Koudo Leafy stems The whole plant is burnt to repel Chiov. (Poaceae) mosquitoes Cymbopogon Susumè Nalohou 2 Leafy stems Leaves are burnt as repellent and schoenanthus L. Spreng. insecticide (Poaceae) Eucalyptus citriodora Eucalyptus Abomey-calavi Leaves Leaves are burnt as repellent and Hook. (Myrtaceae) essential oil is used as deodorant Eucalyptus tereticornis Eucalyptus Abomey-calavi Leaves Leaves are burnt as repellent and Sm. (Myrtaceae) insecticide Cochlospermum Adjinakuvokafun Mount Kassa Roots Decoction of the roots is used in planchonii Hook. f. Ex the treatment of malaria, and Planch. (Bixaceae) against fever Cochlospermum Atinyi vokanfun Mount Kassa Roots Decoction of roots is used against tinctorium A. Rich. jaundice, malaria and diabetes (Bixaceae) Chenopodium Amahun kokwe, Savalou Leafy stems Leaves are burnt as repulsive and ambrosioides L. amatluzu insecticide against mosquitoes (Amaranthaceae) Securidaca Kpata Oucagourou Leafy stems Decoction of roots is used against longepedunculata malaria and snakes bites Fresen. (Polygalaceae) Erythrophleum Obo Manigri Leaves Decoction of leaves and roots is suaveolens (Guill. & used in the treatment of malaria Perr.) Brenan (Fabaceae) Spondias mombin L. Akikontin Abomey-calavi Leaves Decoction of leaves is used as (Anacardiaceae) antimalarial, anti-inflammatory, diuretic and febrifuge Guiera senegalensis J.F. Hlikon Alpha kouara Leaves Leaves decoction is used in the Gmel. (Combretaceae) treatment of malaria

Figure 9: Map of Benin showing the collection sites of the plant species

2.4. Overview on Plasmodium falciparum Malaria is caused by single-celled protozoan parasites of the genus Plasmodium (Table 4).

Table 4: Taxonomic position of Plasmodium [105]

Phylum : Protozoa Subphylum : Apicomplexa Class : Sporozoa Subclass : Coccidia Order : Coccidiida Suborder : Haemosporina Family : Plasmodiidae Genus : Plasmodium Species : Plasmodium falciparum, malariae, ovale, vivax

The parasites are transmitted from an infected person to a healthy person by females of Anopheline mosquitoes whereas the males of Anopheles mosquitoes only feed on plant juices and nectar and are not a malaria vector. The life cycle of Plasmodium falciparum is realized into two hosts: in the female Anopheles and in human (Fig 10).

Figure 10: Life cycle of Plasmodium spp. [78]

Life cycle in the female Anopheles During a blood meal of the female Anopheles, male and female gametocytes are ingested and transformed in the insect stomach, into male and female gametes. Male and female gametes unite to form a zygote called mobile ookinete. The latter penetrates the wall of the stomach and becomes a spherical oocyst inside which the nucleus divides several times into sporozoites. When the sporozoites development is complete (10 to 15 days depending on the Plasmodium species and environmental conditions), the oocyst breaks down releasing them into the general cavity of female

Anopheles from where they migrate to its salivary glands [78]. This phase is the sporogonic cycle of Plasmodium falciparum.

Life cycle in humans The parasite development is realized in two phases: the exo-erytrocytic cycle and the erytrocytic cycle. During the exo-erytrocytic cycle, sporozoites are injected into healthy human blood with the mosquito's saliva during the female Anopheles' bite. Then they migrate into liver cells and during 7 to 12 days, they multiply several times and transform inside the liver schizont into merozoites. During the erytrocytic cycle, merozoites are released into the bloodstream and invade red blood cells inside of which they multiply again into trophozoite. After maturation, the latter is transformed into blood schizont whose rupture releases male and female gametocytes that migrate to peripheral bloodstream and the cycle begins again [106]. Malaria could also be transmitted accidentally by blood transfusions containing malaria parasites, or through contaminated needles or syringes. During pregnancy, fetuses could be infected with parasites from the blood of the mother (transplacental transmission). Each female mosquito needs a blood meal to ensure the maturation of eggs, and as the mosquito lays eggs several times during its existence, it will take several blood meals and will have several opportunities to take malaria parasites and transmit them.

2.5. Overview on Anopheles gambiae The life cycle of A. gambiae (taxonomic position in Table 5) occurs during four distinct stages: egg, larva, pupa and adult (Fig 11).

Table 5: Taxonomic position of Anopheles gambiae [107] Phylum : Arthropoda Class : Insecta Sub-class : Pterygota Order : Diptera Sub-order : Nematocera Family : Culicideae Sub-family : Anophelinea Genus : Anopheles Species : Anopheles gambiae

Eggs: The females usually mate only once throughout their life but produce eggs at intervals. To be able to do so, females need blood meals. The digestion of the blood meal is simultaneous with the development of eggs that can take 2-3 days below the tropics but longer in temperate zones. Males feed only on the nectar of flowers [108]. Eggs are laid on water surface in various places (small amounts of water in footprints, puddles of rain water or larger sites such as rivers, canals, swamps, lakes, rice fields) [78, 106].

Larvae: After 2-3 days, eggs hatch to release the larvae. The latter grow within 4 to 7 days throughout 4 stages called instars. At the surface of the water, with their siphon, Anopheles larvae can feed on yeasts, bacteria, small aquatic organisms and breathe. The size of the larvae varies from 1.5 mm (first instar) to 8-10 mm (fourth instar) [78, 106, 108].

Pupa: At the end of the larval stage, the fully-grown larva transforms into a comma- shaped pupa of Anopheles. The pupal stage is the transition from aquatic to aerial life of the mosquito. This pupa does not feed and its life cycle takes 2 or 3 days after which its cuticule splits freeing the adult. This adult rests on the surface of the water until it is able to fly [78, 106, 108].

Adult: The copulation takes place shortly after the emergence of the adult mosquito. This only one copulation allows the female to receive enough sperm, to fertilize all the successive batches of eggs. After the first or the second blood meal depending on the species (Fig 12), the female lays its first batch of eggs. The later batches of eggs will follow each blood meal. The females of most species of mosquitoes bite at night but some bite after sunset, around midnight or even early morning hours, in houses (endophagic), or outdoors (exophagic). Some mosquitoes prefer human's blood (anthropophilic mosquitoes) and others, animals' blood (zoophilic mosquitoes). Anthropophilic mosquitoes are the most dangerous because of their transmission of malaria throughout the human population [78, 108].

Figure 11: Life cycle of the mosquito [108]

Figure 12: Freshly blood-fed mosquito Anopheles spp. with a dilated abdomen [108]

2.6. Overview on selected plants 2.6.1. Chenopodium ambrosioides L. (Amaranthaceae) 2.6.1.1. Common names Benin vernacular names: amahun kokwe, mawanwan, amatluzu, godo, azogbidiwa (Fon) English: Epazote, wormseed, Jesuit's tea, Mexican tea, Paico or Herba Sancti Mariæ French : Chenopode, épazote, thé du Mexique, anserine, chénopode vermifuge, herbe aux vers

2.6.1.2. Botanical classification Kingdom: Plantae Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák ex Takht. Superorder: Caryophyllanae Takht. Order: Caryophyllales Juss. ex Bercht. & J. Presl Family: Amaranthaceae Juss.

Genus: Chenopodium L. Species: Chenopodium ambrosioides L. [109]

2.6.1.3. Botanical description C. ambrosioides is an annual herb, sometimes perennial, planted, and ruderal with a strong odor (Figure 13). Originally, from Mexico, it is found worldwide except in cold countries. This plant has lanceolate leaves, large panicles of flowers, grouped, sessile on the distal branches. Flowers and fruit appear throughout the year [110].

Figure 13: Chenopodium ambrosioides

2.6.1.4. Traditional uses In Benin, the leaves of C. ambrosioides are burnt as repulsive and insecticide against A. gambiae. They are used in infusion as an anthelmintic and for the treatment of skin diseases and fractures [110]. The decoction of the leaves of C. ambrosioides and Citrus aurantifolia is used as anthelmintic and the sap is used in the treatment of broken bones and dermatosis [111]. Pounded leaves are applied to sores and its veterinary use against Ascaris and Ankylostoma is widespread [112].

2.6.1.5. Previous studies The chemical composition of essential oil extracted from C. ambrosioides in China has showed as main compounds p-cymene (12.7%), Z-ascaridole (29.7%) and isoascaridole (13.0%) [39], like the sample analysed in Madagascar that contained ascaridole (41.8%), isoascaridole (18.1%) and p-cymene (16.2%) [113]. On the contrary, the sample originating from India has revealed another chemical profile with the following major components: m-cymene (43.9%) and myrtenol (13.3%) [114], while the one from Argentina contained ascaridole at 99.4% [115]. The main compounds identified in a sample from Togo were ascaridole (51.1%), p-cymene (19.8%), neral (8.7%) and geraniol (7.5%) [116], whereas chromatographed in Rwanda, the main compounds identified were -terpinene (72.7%), p-cymene (15.3%) and ascaridole (7.2%) [117]. A sample from Nigeria, unlike all the others does not contain ascaridole, but -terpinene (56.0%), p-cymene (15.5%) and terpinyl acetate (15.7%) [118].

C. ambrosioides has exhibited various biological activities. Indeed, the alcoholic leaf extracts of C. ambrosioides (Chenopodiaceae) has exhibited a strong insecticidal activity against a pest in commercial tomato plantation such as Tuta absoluta. After 24 hours post spraying, the population of adults of Tuta absoluta was reduced to a few individuals [119]. The strong fumigant activity of the crude essential oil of C. ambrosioides and Z-ascaridole as one of the main compounds against Sitophilus −1 zeamais adults with a LC50 value of 3.08 and 0.84 mg L air, respectively, has been reported whereas by contact toxicity the lethal dose 50 (LD50) obtained against S. zeamais adults were 2.12 and 0.86 μg g−1 body weight, respectively [39]. Using a concentration of 90%, C. ambrosioides essential oil has demonstrated a mosquito repellent activity against Aedes aegypti after 60 min and this repellency has been attributed to ascaridole and cis-carveol [115]. The wide spectrum antifungal activity of this C. ambrosioides essential oil and ascaridoles [120], its antimycotic activity against the dermatophytes Trychophyton rnentagrophytes and Microsporum audouinii, at a concentration of 50 ppm [121] were reported. In vitro, the fungitoxicity of C. ambrosioides essential oil has been demonstrated as it exhibited at 100 μg/ml an extensive fungitoxic spectrum against Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, Botryodiplodia theobromae, Fusarium oxysporum, Sclerotium

rolfsii, Macrophomina phaseolina, Cladosporium cladosporioides, Helminthosporium oryzae and Pythium debaryanum. In vivo, investigation has demonstrated protection of stored wheat from different storage fungi for one year [122]. The evaluation in vitro of the antifungal efficacy of Chenopodium ambrosioides essential oil against dermatophytes Microsporum gypseum and Trichophyton rubrum has been demonstrated at 700 ppm and 350 ppm, respectively, whereas values of 5500 ppm, 5000 ppm and 4500 ppm, 4500 ppm were obtained using griseofulvin and ketoconazole, respectively [114]. The ethanolic extract of C. ambrosioides showed a good repellence index (66%) when applied at 2.20 mg/cm2 and 1.10 mg/cm2 against Amblyomma cajennense, a result similar to the one obtained with DEET [123]. When the powder obtained from dry ground leaves was tested against Callosobruchus chinensis, C. maculatus, Acanthoscelides obtectus, Sitophilus granarius, S. zeamais and Prostephanus truncates, at 0.4% a mortality higher than 60% of all the bruchids and 100% mortality at 6.4% against S. granarius and S. zeamais, was noticed two days after the treatment. Also all the tested concentrations

(0.05-6.4% w/w) of the dry ground leaves inhibited F1 progeny production and adult emergence of all the pests tested. Furthermore 80-100% mortality of the beetles were recorded at the concentration of 0.2 µl/cm2 of essential oil within 24 h except for C. maculatus and S. zeamais, for which only 20% and 5% mortality, respectively, were observed [40].

2.6.2. Cochlospermum planchonii Hook. f. ex Planch. (Bixaceae) 2.6.2.1. Common names Benin vernacular names: Adjinakuvokanfun (Fon) English: False cotton French: Faux cotonnier

2.6.2.2. Botanical classification Kingdom: Plantae Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák ex Takht. Superorder: Rosanae Takht. Order: Malvales Juss.

Family: Bixaceae Kunth Genus: Cochlospermum Kunth Species: Cochlospermum planchonii Hook. f. ex Planch. [109]

2.6.2.3. Botanical description Cochlospermum planchonii is a semi woody plant having the appearance of a shrub, 1- 1.5 m high (Figure 14); the leaves are alternate palmately lobed; the inflorescences are terminal, the flowers are yellow gold and fruits are capsular ovoid [110].

Figure 14 : Cochlospermum planchonii

2.6.2.4. Traditional uses Our investigations have revealed the use of decoction of Cochlospermum planchonii in the treatment of malaria in Benin. The decoction of the leaves C. planchonii together with the bark of Afzelia africana is used against anasarca and the aqueous extract of the macerated young leaves is used as antiemetic and antidiarrhoeal [111]. A mixture of the roots of Cochlospermum planchonii and leaves of Cassia alata and Phyllanthus amarus called “saye” is used in Burkina-Faso to treat malaria and jaundice; the tuberous roots decoction of Cochlospermum planchonii Hook “N'Dribala” is used against malaria [124, 125]. The fibre obtained from the stem bark, is used for making string and rope in northern Sierra Leone and in northern Nigeria

and for binding mats in Burkina-Faso. In Benin, Sudan and Nigeria a yellow dye extracted from the rhizome is used by Hausa people by addition of indigo to obtain green shades. In Benin, after crushing and drying the rhizome produced a reddish powder, which is used in the preparation of sauces. The rhizome decoction is drunk against gonorrhea (Sierra Leone), against jaundice in addition to Lophira lanceolata and shea butter, against fever and malaria and as diuretic, drunk as a tonic when diluted in water and mixed with lemon juice; the tea of the leaves with a few peppercorns is drunk against gastrointestinal problems (Mali). In Ivory Coast decoction of the leaves is used for babies' bath and against their diarrhea. In Nigeria, this plant is used against AIDS, in the control of menstruations and in the treatment of broken bones [126].

2.6.2.5. Previous studies The chemical profile of C. planchonii from Burkina-Faso revealed the presence of 3- tetradecanone (69.8%), dodecyl acetate (4.7%) and tetradecyl acetate (14.4%) [127]. Some other additional compounds such as 3-tetradecenone (15.3%), 2-tridecanone (7.8%) and -elemene (6.0%), in addition to 3-tetradecanone (30.6%), dodecyl acetate (12.4%) and tetradecyl acetate (15.0%) were identified in another sample from Burkina-Faso [128].

The tuberous roots decoction of Cochlospermum planchonii Hook "N’Dribala" has been tested versus chloroquine, in the treatment of uncomplicated P. falciparum malaria and was revealed to be as efficient as chloroquine with 52% of cured patients and 90% of treated patients with no symptom [124]. The antiplasmodial activity of the essential oil of this plant species has been evaluated in vitro on the chloroquine- resistant strains FcB1-Columbia and the chloroquine sensitive Nigerian strain. It follows that after 72 hours, the oil has induced 50% inhibition of the parasite growth

(IC50) at 15 µg/mL on the resistant strains FcB1-Columbia and 21 µg/mL on the sensitive Nigerian strain [127]. The antibacterial activity of C. planchonii with 0.25% MIC (minimum inhibitory concentration) has been demonstrated when the essential oil was tested on Bacillus cereus, Escherichia coli, Listeria innocua and Streptococcus pyogenes [128].

2.6.3. Cochlospermum tinctorium A. Rich (Bixaceae) 2.6.3.1. Common names Benin vernacular names: atinyi vokanfun English: - French: Cochlospermum à teinture

2.6.3.2. Botanical classification Kingdom: Plantae Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák ex Takht. Super order: Rosanae Takht. Order: Malvales Juss. Family: Bixaceae Kunth Genus: Cochlospermum Kunth Species: Cochlospermum tinctorium A. Rich.[109]

2.6.3.3. Botanical description Cochlospermum tinctorium is a vivacious plant with semi woody and tuberous stump, emitting leafy stems during the raining season (Figure 15). The leaves are alternate palmatilobed. Its yellow flowers appear after the vegetation fires at ground level or at the top of the racemes 5-10 cm high [110].

2.6.3.4. Traditional uses According to our ethnopharmacological survey in Benin, the roots of this plant are mainly used against jaundice, malaria and diabetes. It is also used against headaches, and hepatic diseases [111], as natural colorant in food and tincture for clothes, and also as an antibiotic [129]. Uses against abdominal pain, healing wounds, hemorrhoids, intestinal worms, schistosomiasis and hepatitis have been reported [130]. In Mali, this plant is used to treat jaundice, gastro-intestinal diseases or ailments, malaria, schistosomiasis and dysurea [131].

Figure 15: Cochlospermum tinctorium

2.6.3.5. Previous studies The essential oil extracted in Burkina-Faso from the whole tubercle of C. tinctorium has revealed the presence of four main compounds, namely 3-tetradecanone (64.6%), tetradecyl acetate (11.7%), 3-hexadecanone (5.6%) and dodecyl acetate (4.6%) [127].

The good analgesic activities of the aqueous methanol leaf extract of C. tinctorium at the dose of 80 mg/kg was reported. The aqueous methanol leaf extract significantly and dose dependently inhibited at 96.65% the acetic acid-induced writhing test in mice, greater than that of the standard agent, ketoprofen (82.30%) [132].

The investigation of the antiplasmodial activity of fractions obtained from the ethanolic extract of the root of C. tinctorium has revealed the good antiplasmodial activity of 3-O-E-p-coumaroylalphitolic acid with IC50 values of 2.3 µM against the P. falciparum chloroquine sensitive strain 3d7, and 3.8 µM against the P. falciparum chloroquine resistant strain Dd2 [133]. The extracts obtained by infusion and decoction from the tubercle have demonstrated in vitro an antiplasmodial activity against the P. falciparum chloroquine resistant strain FcBl-Colombia and the

chloroquine sensitive strain F32-Tanzania with IC50 values ranged between 1 µg/mL and 2 µg/mL [134]. In addition, the essential oil extracted from the whole tubercle has been tested against the Nigerian sensitive strain of P. falciparum and the P. falciparum FcBl resistant strain, showing after 72 hours an IC50 of 8 µg/mL and 5 µ/mL, respectively [127].

2.6.4. Cymbopogon citratus (DC.) Stapf (Poaceae) 2.6.4.1. Common names Benin vernacular names: cha (Fon), tigbé (Mina), tiikoosu (Dendi) English: lemon grass, fever grass, ginger grass, citronella grass French: citronnelle, herbe citron, verveine des Indes

2.6.4.2. Botanical classification Kingdom: Plantae Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák ex Takht. Super order: Lilianae Takht. Order: Poales Small Family: Poaceae Barnhart Genus: Cymbopogon Spreng. Species: Cymbopogon citratus (DC.) Stapf [109]

2.6.4.3. Botanical description Cymbopogon citratus is a perennial grass with short rhizome up to 2 m high (Figure 16). Fertile culms are rare and the leaves are highly aromatic [110]. The flowers are bisexual or unisexual and the fruit is a dry indehiscent caryopsis with a thin pericarp [126].

2.6.4.4. Traditional uses In Benin, C. citratus is used to treat malaria and fever and to repel mosquitoes. The decoction of the leaves associated with the roots of Securidaca longepedunculata is administered orally in the treatment of snakebites and in the treatment of edema, jaundice and anemia [110]. In Nigeria, C. citratus leaves are used to treat malaria

[135] and in Congo, a decoction of the leaves of Cymbopogon citratus is used for its hypotensive properties [136] and against stomachaches [137].

Figure 16: Cymbopogon citratus

2.6.4.5. Previous studies The essential oils extracted from C. citratus, collected in Brazil, showed that the fresh leaves contain myrcene (11.1%), neral (31.5%) and geranial (47.5%) [138], and the essential oils obtained from blades, sheaths and rhizomes of C. citratus contain myrcene (10.7%-14.2%), neral (31.5-32.1%) and geranial (39.9-41.3%) [54]. The same main compounds were also identified in the sample originated from Burkina- Faso with myrcene (10.7%), neral (33.0%) and geranial (44.6%) [56], and the one originating from Portugal with myrcene (11.5%), neral (32.5%) and geranial (45.7%) [139].

Various biological activities of C. citratus have been demonstrated. Indeed the essential oil extracted from C. citratus in Brazil revealed to be active against larvae of

Aedes aegypti with LC50 (0.28 µg/ml) and LC90 (0.56 µg/ml) [140]. When tested against larvae of Culex tritaeniorhynchus and Anopheles subpictus, a good repellency and larvicidal activity was observed and the lethal concentrations LC50 and

LC90 were 136.58 ppm and 243.18 ppm for Cx. tritaeniorhynchus and 77.24 ppm and

128.39 ppm for A. subpictus [141]. Larvicidal, insecticidal and repellent activities have been detected against A. arabiensis with LC50 = 74.02 ppm and LC90 = 158.20 ppm [142, 143], and Tribolium castaneum with a mean repellent dose after 4 hours exposure of 0.021 ml/L [41]. Anti-Leishmania activity of C. citratus essential oil and a mixture of its main compounds obtained from 40% neral (Z-citral) and 60% geranial

(E-citral), has been noticed against L. infantum, L. tropica and L. major, with IC50 concentrations ranged from 25 to 52 µg/ml for C. citratus essential oil, and from 34 to 42 µg/ml for the mixture of citral [139]. A potent antimicrobial activity has been demonstrated against various microorganisms with minimal inhibitory concentrations determined as follows: Enterococcus faecalis (1.0 mg/ml), Salmonella enterica (2.1 mg/ml), S. typhimurium (2.5 mg/ml) [144]. The essential oil of C. citratus, originating from Congo, has shown antibacterial activity against 17 different bacteria species [145]. C. citratus lipid- and essential oil-free leaves infusion, and its polyphenolic compounds, were shown to be natural and safe sources of new anti-inflammatory drugs [146]. Other antioxidant, antiradical, and anti-inflammatory properties were also noticed [56, 146-148].

2.6.5. Cymbopogon giganteus Chiov. (Poaceae) 2.6.5.1. Common names Benin vernacular names: gbèzin; yakimooribu; monuuso English: kachi grass French: citronnelle de Madagascar

2.6.5.2. Botanical classification Kingdom: Plantae Division: Magnoliophyta Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák Ex Takht. Superorder: Lilianae Takht. Order: Poales Small Family: Poaceae Barnhart Genus: Cymbopogon Spreng. Species: Cymbopogon giganteus Chiov. [109]

2.6.5.3. Botanical description C. giganteus is a perennial grass, 2.5 meters high, with slightly aromatic glaucous leaves (Figure 17). Its dense panicles are narrow and its flowers appear throughout the year [110].

Figure 17: Cymbopogon giganteus 2.6.5.4. Traditional uses In Benin, the whole plant is burnt to repel mosquitoes (ethnobotanical survey). Associated to Ocimum basilicum, the decoction of the leaves is used in the treatment of drepanocytosis and the decoction of leafy stems quiet epilepsy crises [110, 111]. In Burkina-Faso, the fresh roots decoction is used against toothache, gingivitis and sores in the mouth, the tongue and on the lips [149]. In Congo, the infusion of roots and leaves is drunk against stomachaches [137].

2.6.5.5. Previous studies The chemical composition of the essential oil of C. giganteus, originating from Burkina-Faso, has revealed the presence of limonene (42%) and a set of monoterpene alcohols, namely E-p-mentha-1(7),8-dien-2-ol (14.2%), Z-p-mentha- 1(7),8-dien-2-ol (12%), E-p-mentha-2,8-dien-1-ol (5.6%) and Z-p-mentha-2,8-dien-1- ol (5.2%) [144]. These five main compounds were also detected when the essential

oil was characterized in Ivory-Coast [150, 151], Cameroon [152], Benin [153], Togo [28] and Mali [154].

Concerning the biological activities, C. giganteus essential oil has shown insecticidal, larvicidal and ovicidal activity against Callosobruchus maculatus through the destruction of growing eggs or larvae [153], against C. maculatus and C. subinnotatus with an oviposition reduction at 5 µL/L by 91% in C. subinnotatus population versus 81% in C. maculatus population [28].

2.6.6. Cymbopogon schoenanthus (L.) Spreng. (Poaceae) 2.6.6.1. Common names Benin vernacular names: susumè; tsable English: camelgrass, geranium grass French: gazon de chameau, Herbe à Chameau

2.6.6.2. Botanical classification Kingdom: Plantae Division: Magnoliophyta Subclass: Magnoliidae Novák Ex Takht. Superorder: Lilianae Takht. Order: Poales Small Family: Poaceae Barnhart Genus: Cymbopogon Spreng. Species: Cymbopogon schoenanthus (L.) Spreng. [109]

2.6.6.3. Botanical description C. schoenanthus is an aromatic vivacious herb of drier parts of tropical Africa that can reach 60 to 80 cm high (Figure 18). This plant species possesses linear and fragrant leaves and its inflorescences are contracted in panicles [110].

Figure 18: Cymbopogon shoenanthus

2.6.6.4. Traditional uses In Benin, C. schoenanthus is used as insect repellent and natural insecticide. The whole plant, crushed and mixed with leaves of Vitex simplicifolia is indicated in traditional medicine against schizophrenia [110]. In Tunisia, Cymbopogon is eaten in salads and used to prepare traditional meat recipes [155] and in Congo-Brazaville the infusion of the leaves is used to treat stomachache [137].

2.6.6.5. Previous studies The volatile profile of C. schoenanthus leaves, determined in Brazil, was dominated by geraniol (62.5%), geranial (12.5%) and neral (8.2%) [156]. In contrast, the essential oil of C. schoenanthus from Togo was dominated by piperitone (61.0- 69.0%) and -2-carene (16.9-23.4%) [38, 157, 158]. A similar profile was also identified in Burkina-Faso where the main compounds were piperitone (42.0%) and - 2-carene (8.2%) [159]. A different volatile profile in the leaves essential oil originated from Tunisia was noticed as its main compounds were limonene (24.2-27.3%), - phellandrene (13.4-16.0%), -terpinene (8.4-21.2%) and -terpineol (9.1-11.7%) [155].

The insecticidal activity of C. schoenanthus essential oil and its main constituent, piperitone has been demonstrated on Callosobruchus maculatus with LC50 values of 1.6 μl/l and 2.7 μl/l, respectively [38]. Its adulticidal activity against Dinarmus basalis has been also proven [157]. Its activity against ovine trichostrongylids and gastrointestinal nematodes (Haemonchus contortus and Trichostrogylus spp.) has been demonstrated [156]. Furthermore, the antioxidant, antiacetylcholinesterase and antimicrobial properties of C. schoenanthus have been shown [155, 158, 160].

2.6.7. Erythrophleum suaveolens (Guil. & Perr.) Brenan (Fabaceae) 2.6.7.1. Common names Benin vernacular names: obo English: red water tree, sasswood tree French: Bois rouge, poison d’épreuve

2.6.7.2. Botanical classification Kingdom: Plantae Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák Ex Takht. Superorder: Rosanae Takht. Order: Fabales Bromhead Family: Fabaceae Lindl. Genus: Erythrophleum Afzel. Ex R. Br. Species: Erythrophleum suaveolens (Guill. & Perr.) Brenan

2.6.7.3. Botanical description Erythrophleum suaveolens is a tree of 25 to 33 meters height, with branches alternate composed and bipinnate of 2-4 pairs of pinnae (Figure 19). The inflorescences are in the form of axillary panicles and the flowers are bisexual, regular yellowish-white to greenish yellow [110, 126].

Figure 19: Erythrophleum suaveolens

2.6.7.4. Traditional uses In Benin and in Uganda, leaves and roots of Erythrophleum suaveolens are boiled and drunk against malaria [161]. The bark is used as emetic and purgative. The crushed bark is applied to swellings caused by Filaria. In DR Congo, the dried powdered bark is taken as a snuff to cure headache. In Kenya, a diluted decoction of the roots is used as an anthelminthic, especially against tapeworm. In Malawi, a decoction of the roots and the bark is applied to soothe general body pain. In West Africa, the powdered bark is mixed with the residue of palm oil, and after boiling is mixed with seeds of maize, cowpea or cotton, to reduce pest damage in the seeds. Dried leaves are mixed with stored grains and beans to repel or kill storage insects [126].

2.6.7.5. Previous studies The aqueous and the chloroform extracts from E. suaveolens have revealed the presence of saponins, tannins, steroids and alkaloids [162]. Diterpenic alkaloids (erythrosuavine) have been isolated from its chloroform extract and characterized as N-acetyl-9,11-dehydro-6-ketocassaidinyl-3-acetate [163], norcassaide and

norerythrosuaveolide characterized as 7-hydroxy-7-deoxo-6-oxonorcassaide [164]. It also contains procyanidin monomers ((+)-catechin and (−)-epicatechin), monomeric and oligomeric procyanidins [165]. The aqueous and the chloroform extracts from E. suaveolens at a concentration of 10 mg/mL, exhibited bactericidal activity with MIC ranged from 0.625 mg/mL to 2.5 mg/mL for the aqueous extract, and from 0.313 mg/mL to 5.0 mg/mL for the chloroform extract [162]. The aqueous extract at 1.5% has revealed its preservative property on wood [166]. The hexane extract of the stem bark has demonstrated anti- inflammatory and analgesic properties [165].

2.6.8. Eucalyptus citriodora Hook (Myrtaceae) 2.6.8.1. Common names Benin vernacular names: eucalyptus English: lemon-scented gum, broad-leaved peppermint-tree French: eucalyptus citronné

2.6.8.2. Botanical classification Kingdom: Plantae Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák ex Takht. Superorder: Rosanae Takht. Order: Myrtales Juss. ex Bercht. & J. Presl Family: Myrtaceae Juss. Genus: Eucalyptus L'Hér. Species: Eucalyptus citriodora Hook.

2.6.8.3. Botanical description E. citriodora is a handsome evergreen tree, 24-40 (max. 50) m height. It has alternate leaves, which have the smell of lemon. Its inflorescences are terminal and compositae or axillary and simple (Figure 20). The flower bud is white, clavate with hemispherical operculum, the fruit is ovoid or urceolate and the seeds are few, irregularly elliptical, large, shiny and black [167].

Figure 20: Eucalyptus citriodora

2.6.8.4. Traditional uses E. citriodora in Benin is used for its repellent properties against mosquitoes and its deodorizing properties (ethnobotanical survey). In Brazil, hot aqueous extracts of its dried leaves are traditionally used as analgesic, anti-inflammatory, and antipyretic remedies for the symptoms of respiratory infections, such as cold, flue, and sinus congestion [168].

2.6.8.5. Previous studies The glycoside rutin has been isolated from the aqueous methanol extract of E. citriodora [169]. The volatile profile of its essential oil of the leaves has revealed the presence of citronellal (72.3%) and citronellol (6.3%) [145]. These main compounds was also detected in Ethiopia (73.3% and 11.9%) [170]. In contrast, volatile profiles, obtained in Brazil and India, were quite different since their main compounds were (-) isopulegol (7.3%), -citronellal (71.7%) [27], and -thujene (11.9%), -pinene (18.3%), sabinene (19.6%), -pinene (25.7%) [171], respectively.

The larvicidal activity of E. citriodora was demonstrated since the essential oil of the leaves has exhibited insecticidal activity against C. quinquefasciatus larvae with LC50 of 245.5 mg/mL [36]. Its insecticidal effects on Lutzomyia longipalpis has been

demonstrated with 100% mortality at 6.5 mg/mL on the larvae and 88.1% and 100% at 10 mg/mL on the adults after 24 hours and 48 hours [27]. The acaricidal property of its essential oil against Anocentor nitens larvae has been observed, with mortalities of 20.1%, 84.5%, 89.2%, and 100.0% when it was tested at concentrations of 6.25%, 12.5%, 25.0% and 50.0%, respectively [172]. The repellent property of its essential oil against S. zeamais was revealed at 0.50 µL/cm2 (82%), 2 with a RD50 (dose that repels 50% of exposed insects) of 0.094 µL/cm [173]. The antibacterial activity of its essential oil at 5 µl/disc has been demonstrated against bacteria such as Bacillus subtilis, Citrobacter diversus, Citrobacter sp., Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Salmonella typhimurium, Staphylococcus aureus and Shigella flexneri [145].

2.6.9. Eucalyptus tereticornis Sm. (Myrtaceae) 2.6.9.1. Common names Benin vernacular names: eucalyptus English: Queensland blue gum, blue gum French: eucalyptus bleu

2.6.9.2. Botanical classification Kingdom: Plantae Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák ex Takht. Superorder: Rosanae Takht. Order: Myrtales Juss. ex Bercht. & J. Presl Family: Myrtaceae Juss. Genus: Eucalyptus L'Hér. Species: Eucalyptus tereticornis Sm.

2.6.9.3. Botanical description Eucalyptus tereticornis is an evergreen tree that has a height ranged from 20 to 50 meters and a circumference of up to 2 meters (Figure 21). The bark surface is white, grey, or grey-blue, peeling in large plates or thin sheets and becoming a mottled surface with rough patches white, grey or bluish in color. The leaves are narrowly lance-shaped and green. The inflorescences are composed by 7 to 11 flowers, the

fruit is thin-walled globular to ovoid capsule and seed are rough and brown-black [126, 167].

Figure 21: Eucalyptus tereticornis

2.6.9.4. Traditional uses In Benin, E. tereticornis is used in traditional medicine for its repellent properties. In the Philippines, E. tereticornis is used as anti-diabetic [174].

2.6.9.5. Previous studies The volatile profile of E. tereticornis from Benin has revealed the presence of p- cymene (31.1%), -phellandrene (9.8%), spathulenol (8.1%), -terpinene (7.0%), - phellandrene (6.8%) and 1,8-cineole (5.4%) [175]. In other African countries, such as Nigeria, -pinene (21.4%), -pinene (39.4%) and limonene (8.0%) were identified [176], whereas -pinene (8.3%), p-cymene (28.6%), cryptone (17.8%), -terpineol (5.6%) and cuminaldehyde (6.5%) were the main compounds in the sample from Congo [145]. Elsewhere, -pinene (6.8%), 1,8-cineole (81.0%), limonene (4.1%) were identified in a sample from Algeria [177]; -pinene (24.4%), limonene (7.3%), 1,8-cineole (34.5%), -terpineol (6.3%) were the main compounds of a sample from Ethiopia [170], whereas in Tunisia, limonene (10.1%), p-cymene (17.4%),

caryophyllene oxide (9.1%), spathulenol (12.7%) and cryptone (9.1%) were revealed. In Argentina, -phellandrene (9.44%), p-cymene (14.5%), -phellandrene (22.6%), 1,8-cineole (18.6%), 4-terpineol (5.8%) and spathulenol (6.8%) were detected [178], whereas -pinene (9.0%) in addition to p-cymene (13.8%), 1,8-cineole (23.2%), E- pinocarveol (6.0%) and cryptone (6.9%) were detected in a sample from Cuba [179].

The bactericidal property of E. tereticornis essential oil against Saccharomyces spp., Sporobolomyces and Hansenula, its fungicidal activity against Corynebacteriaceae spp, its fungistatic activity against Torulopsis candida and its insecticidal activity against ticks have been demonstrated [175]. The adulticide activity of E. tereticornis against Aedes aegypti was demonstrated with a knock down time (KDT50) of 10.5 min, and this bioactivity was correlated with the concentration of 1,8-cineole in the essential oil [26]. The larvicidal activity of the pure essential oil has been demonstrated with a LC50 value of 22.1 mg/L 24 hours post-exposure [178]. This larvicidal and adulticide activities were confirmed by the results obtained by Nathan et al, 2007 since the leaf extracts suppressed the pupal and adult of Anopheles stephensi at a dose of 160 ppm which induced 100% mortality in larvae population [180].

2.6.10. Guiera senegalensis J.F. Gmel. (Combretaceae) 2.6.10.1. Common names Benin vernacular names: hlikon, tiepienu, yeloko, geloki, ndieloki English: - French: Nguère

2.6.10.2. Botanical classification Kingdom: Plantae Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák ex Takht. Superorder: Rosanae Takht. Order: Myrtales Juss. ex Bercht. & J. Presl Family: Combretaceae R. Br. Genus: Guiera Adans. ex Juss. Species: Guiera senegalensis J.F. Gmel.

2.6.10.3. Botanical description Guiera senegalensis is a shrub of 1 to 2 meters long, with black peltate scales present over the entire plant (Figure 22). The leaf blade is grayish, elliptical rounded sub-cordate at the base, rounded and mucronate at the top. The flower is 5 mm long, with a triangular-acute calyx. The fruit is covered with a gray rose silk. The flowers and the fruits come out throughout the year but especially in October [110].

Figure 22 : Guiera senegalensis

2.6.10.4. Traditional uses According to our ethnobotanical survey in Benin and referring to Adjanohoun et al. (1989), the decoction of the leaves of G. senegalensis is used to treat malaria [111]. The leaves are used as an antiseptic, as astringent, as antibiotic and in the treatment of diabetes; leaves and roots are used against arthritis, diarrhoea, gout, edema of articulations and rheumatism [129]. These leaves are also used in the treatment of uncomplicated malaria in Burkina-Faso [181]. In traditional medicine in Nigeria, the aqueous root extract of G. senegalensis is used as anti-diarrhoeal and ulcer- protective [182].

2.6.10.5. Previous studies

The chloroform extract of the roots of G. senegalensis was demonstrated to have antimalarial activity since it showed effectiveness against P. falciparum W2 strain with an IC50 lower than 25 µg/mL. Two alkaloids isolated from the active fraction, namely harman and tetrahydroharman, showed antiplasmodial activity with a IC50 value of 2.2 µg/mL and 3.9 µg/mL, respectively [183]. Furthermore, alkaloids isolated from G. senegalensis (harman, harmanlan, tetrahydroharman and guieranone A) have confirmed the antiplasmodial activity with IC50 values of 3.29 µg/mL, 22.43 µg/mL, 8.56 µg/mL and 1.29 µg/mL, respectively [184]. The infusion and the decoction of G. senegalensis have displayed both antiplasmodial activities against P. falciparum strains FcB1a and F32b with IC50 values of 0.79 µg/mL and 7.03 µg/mL, respectively [185]. G. senegalensis exhibited a good activity against diarrhoea both by the reduction of the frequency of defecation as well as the wetness of the faeces, and an ulcer-protective effect as it provoked a significant delay in gastrointestinal transit [182].

2.6.11. Securidaca longepedunculata Fresen. (Polygalaceae) 2.6.11.1. Common names Benin vernacular names: kpata, kpeta, ikpata, yokore English: violet tree, krinkhout, rhodesian violet tree French: arbre à serpent, arbre aux hachettes, arbre videtté, arbre violette, médicament du serpent

2.6.11.2. Botanical classification Kingdom: Plantae Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák ex Takht. Superorder: Rosanae Takht. Order: Fabales Bromhead Family: Polygalaceae Hoffmanns. & Link Genus: Securidaca L. Species: Securidaca longepedunculata Fresen.

2.6.11.3. Botanical description S. longepedunculata is a shrub up to 6 meters high, with oblong lanceolate leaves that are rounded at the end (Figure 23). It possesses purplish flowers that are gathered in racemes, and its winged fruits have sometimes a second small and imperfect wing [110].

Figure 23: Securidaca longepedunculata

2.6.11.4. Traditional uses S. longepedunculata is recognized in the treatment of malaria (ethnobotanical survey), in the treatment of madness [186] and in the treatment of snake bite [110]. In Burkina-Faso this plant species is used in the treatment of uncomplicated malaria [181], and its dried roots powder or the decoction of its root combined with root of Waltheria indica is applied against toothache, gingivitis and sores [149]. In Tchad, the decoction of the root is used to treat rheumatism, back pain, stomachache, venereal diseases, snakebites, dysentery, malaria, migraine, bronchitis, leprosy, hepatitis, ear infections, sore eyes and diarrhea [186].

2.6.11.5. Previous studies The identification of the volatile profile of S. longepedunculata in Nigeria, Benin, Ghana, and Burkina-Faso has revealed the presence of one main compound, which is methyl salicylate [187-190]. In the ethanolic, hexane and acetone extracts, several xanthones were identified [191-193] as well as the presence of glycosides, tannins, saponins, alkaloids and proteins was confirmed [194].

The aqueous root extract of Securidaca longepedunculata has demonstrated antinociceptive and antidepressant like effects at the safe dose of 2 g/kg body weight [195]. When used at the dose of 150 mg/kg b.w. intraperitoneally, two times daily for 3 days, the root dichloromethane extract of Securidaca longepedunculata has induced 48% reduction of parasitaemia in mice, experimentally infected with Trypanosoma brucei brucei [196]. Some flavonoids, isolated from S. longepedunculata, have shown antimicrobial activity [194] and isolated xanthones revealed efficacy in the treatment of erectile dysfunction [192, 193]. The aqueous root extract has demonstrated central nervous system depressant and anxiolytic activities since it produced at 100-400 mg/kg, a significant dose increase in onset of convulsion, a significant dose prolongation of the cumulative time spent in the open arms of the elevated plus maze and Y maze, and a significant reduction of the sleeping time induced by hexobarbitone. The lethal doses LD50 for acute toxicity were 1.74 g/kg by oral administration and 19.95 mg/kg by intraperitonal route [197].

2.6.12. Spondias mombin L. (Anacardiaceae) 2.6.12.1. Common names Benin vernacular names: akikontin, sèma, akukon, ahlinhon, aklokon, jogbema, jogbi, iyeye, okika, ekika, ekan English: java plum, yellow mombin, hog plum French: Prunier mombin, prune myribalan, prune d’or

2.6.12.2. Botanical classification Kingdom: Plantae Class: Equisetopsida C. Agardh Subclass: Magnoliidae Novák ex Takht. Superorder: Rosanae Takht.

Order: Sapindales Juss. ex Bercht. & J. Presl Family: Anacardiaceae R. Br. Genus: Spondias L. Species: Spondias mombin L.

2.6.12.3. Botanical description Spondias mombin is a deciduous tree up to 25 m with a trunk exceeding 50 cm in diameter and a spiny bark when young (Figure 24). It has pinnate leaves and its inflorescence is pending with more than 12 ovoid, orange-yellow fruits [110].

Figure 24: Spondias mombin

2.6.12.4. Traditional uses Our investigations have revealed in Benin that S. mombin is used as antimalarial, anti-inflammatory, diuretic and febrifuge. S. mombin is also used in Venezuela in malaria treatment [198]. In Igbo land (Nigeria), the fresh leaves extract is used to cure eye ailment, dizziness especially after childbirth, while the leaves decoction is used against gonorrhea, and the leaves infusion is used as laxative and to treat sore throats, colds and diarrhea [199, 200]. The fruits are used in the treatment of infertility [135].

2.6.12.5. Previous studies Different profiles were detected by Solid Phase Micro Extraction (SPME) and Simultaneous Distillation Extraction (SDE), in the fruits of S. mombin harvested from two locations in Argentina (Tapereba and Caja). Indeed E-caryophyllene (18.7%), ethyl butyrate (10.0%) and ethyl hexanoate were identified by SPME, whereas by SDE, Z-caryophyllene (13.2%) and limonene (9.5%) were the main compounds of the fruit from Tapereba. In addition, the fruit from Caja has revealed the presence of myrcene (42.1%) and -phellandrene (8.5%) by SPME and myrcene (38.0%) and p- cymene (6.2%) by SDE [69]. The phytochemical analysis of the leaves demonstrated the presence of tannins, saponins, flavonoids, alkaloids, phenols and vitamins [201, 202].

In Nigeria, the presence of tannins, saponins, and anthraquinone glycosides in the methanol extract of the leaves of Spondias mombin was reported, which has exhibited wide spectrum antibacterial effects comparable to those of ampicillin and gentamycin [203]. The tannic acid, isolated from S. mombin, has demonstrated in vitro a good anti-leishmania activity with an IC50 of 11.26 µg/mL and 0.27 µg/mL against promastigotes and amastigotes, respectively [204]. S. mombin displayed good cardioprotective property comparable to ramipril [205]. The leaf ethanolic and aqueous extract of S. mombin have shown anthelmintic activity in sheep, with LC50 values of 0.456 mg/mL and 0.907 mg/mL, respectively [206]. This anthelminthic activity against earthworm has also been demonstrated [207]. The methanolic and ethanolic extracts of the leaves of S. mombin demonstrated sedative and antidopaminergic effects [208].

2.7. Overview on Tribolium castaneum (Herbst, 1797) T. castaneum (Table 6), or "red flour beetle" is a worldwide pest of variable stored products including stored grains, flours, cereals, beans, nuts, pastas, chocolate, which can survive over three years [209, 210] and can provoke allergic reaction [211]. T. castaneum and T. confusum are known to be the two most important pests of stored products in home and grocery stores. The life cycle of T. castaneum is the result of four development steps (Figure 25, Figure 26): eggs, larvae, pupae, and adult. The eggs are white and microscopic and the larvae are creamy yellow to light brown with two dark pointed projections, at the extremity of their last segment

whereas the pupae are white to yellowish. At 30-35°C and 60-80% relative humidity, the duration of the different steps of development is 2-3 days for eggs, 15-20 days for larvae, 3-4 days for pupae and 4-5 days for reproductive maturation which results to a total time egg to egg of 32 days at 30°C and 24 days at 34°C [42, 212, 213].

Figure 25: Tribolium castaneum

Table 6: Taxonomic position of Tribolium castaneum

Phylum Arthropoda Class Insecta Order Coleoptera Family Tenebrionidae Genus Tribolium castaneum

The chemical control of T. castaneum was performed with organophosphates, pyrethroids and fumigants, with side effects such as persistence in environment and impact on the human health. This large use of chemical insecticides has also promoted the development of resistance in the genus Tribolium [43]. The resistance of Tribolium species to insecticides has been worldwide noticed following the repeated chemical treatments [214-217]. This resistance to insecticides has been observed in America [218-220], Malawi [221], Australia [222-224], India [217], Philippines [225], Nigeria [226], Brazil [227] and Italy [216].

The genus Tribolium has developed resistance against almost all insecticides commonly used such as malathion [222, 225, 228], lindane [222], phosphine [224, 229], deltamethrin [217] and cyclodiene [218]. The spread of multiple insecticide resistance in Tribolium castaneum worldwide noticed has been attributed to several factors, namely the innate ability of T. castaneum to develop resistance more quickly than some other pests [222], international grain trade [215, 227], migration [230] and local selection [227]. The resistances in Tribolium castaneum have been linked to several genetic mutations [218]. Indeed, malathion resistance is controlled by a single factor located on chromosome IV [222, 231]. Two incompletely dominant alleles Rmal-l and Rmal-2 responsible of the high and low resistance to malathion, respectively, have been located at the same locus, in the sixth linkage group or near [232]. Another study has located the single dominant gene responsible of the resistance to lindane and cyclodiene on chromosome III [219]. A single point mutation in the Rdl locus

(Resistance to dieldrin) has been identified to be responsible of cyclodiene resistance [219]. The high level in phosphine resistance has been also identified as provoked by two resistant genes, which work together, whereas the weak resistance may be governed by a single major gene [224]. Pyrethroids resistance was controlled by two autosomal incompletely dominant factors [223].

To conquer the resistances in the genus Tribolium, it is important to find new bioactive compounds to replace the synthetic ones, safer for non-target organisms and humans, but effective and biodegradable [233]. Indeed, plant extracts and essential oils have been recognized since a long time as good fumigants and very efficient insecticides and insect growth regulators [226, 233- 240]. Indeed, when tested against T. castaneum, essential oils from Mentha microphylla, Artemisia judaica and Eucalyptus camaldulensis collected in Egypt were

2 2 very effective by contact test with LC50 values of 0.01 mg/cm , 0.15 mg/cm and 0.15 mg/cm2 respectively, compare to a value from malathion of 0.16x10-3 mg/cm2, whereas the fumigant toxicity of Mentha microphylla was very interesting with an

LC50 value of 4.51 µL/L [241]. Essential oils from some plant species encountered in Choco (Colombia), such as Piper pseudolanceifolium and Ocimum campechianum were more repellent than the commercial repellent IR3535 [236]. Moreover, the essential oil isolated from Piper auritum specie showed a high insecticidal activity

since 100% mortality was reached, at 0.9 µl/cm2, after 24 hours of exposure compared to IR3535 (0% mortality) [235]. By fumigation, essential oil extracted from Pistacia terebinthus growing in Turkey, has led to 100% mortality of adult Tribolium after 48 hours exposure, at 160 µL/L air [242]. Moreover, in several countries, plant species traditionally used by populations for their insecticidal/repellency properties against stored products pests, were tested efficiently against T. castaneum. That was the case for Mentha spicata from India [243], wild Salvia aucheri subsp blancoana from Morocco [244], Origanum vulgare from Korea [245], Piper guineense from Nigeria [226], Rosmarinus officinalis and Citrus limonum from Australia [246], Cinnamomum aromaticum, Syzygium aromaticum and Elletaria cardamomum growing in Bengladesh [247], Achillea species from Egypt [239], Laurelia sempervirens and Drimys winteri collected in Chile [240].

Figure 26: Life cycle of Tribolium castaneum [42]

Following the previous results, essential oils extracted from Cymbopogon nardus, Origanum vulgare, Rosmarinus officinalis collected in Italy and Eucalyptus citriodora from Colombia have been efficiently tested as repellent in food packaging [237, 248]. All these results are good indications for the use of essential oils as new bioactive insecticides, to fight resistance in the genus Tribolium, and are today valuable alternatives for food industry to limit loss due to attack of food packages by Tribolium species.

CHAPTER 3: EXPERIMENTAL

CHAPTER 3: EXPERIMENTAL 3.1. Plant material Twelve plant species belonging to eight botanical families were collected in several regions of Benin (Table 7). Collected plant material was dried, free from light, at room temperature. All these plants have been the subject of classification and botanic description at the National Herbarium of the University of Abomey-Calavi, Benin [110]. These plants include Chenopodium ambrosioides L. (Amaranthaceae), Securidaca longepedunculata Fresen. (Polygalaceae), Cochlospermum planchonii Hook. f. Ex Planch. (Bixaceae), Cochlospermum tinctorium A. Rich. (Bixaceae), Eucalyptus tereticornis Sm. (Myrtaceae), Eucalyptus citriodora Hook. (Myrtaceae), Cymbopogon citratus (DC.) Stapf (Poaceae), Cymbopogon schoenanthus (L.) Spreng. (Poaceae) and Cymbopogon giganteus Chiov. (Poaceae), Erythrophleum suaveolens (Guill. & Perr.) Brenan (Fabaceae), Spondias mombin L. (Anacardiaceae), Guiera senegalensis J.F. Gmel. (Combretaceae). Locations of collection, organs extracted and nature of soil are summarized in Table 7. Table 7: Locations, soils and organs extracted from selected plant species Plant Cymbopogon Cymbopogon Cymbopogon Cochlospermum Cochlospermum Eucalyptus species citratus giganteus schoenanthus planchonii tinctorium citriodora Locations Cotonou Koudo Nalohou 2 Mount Kassa Mount Kassa Abomey Calavi Soils Sandy Ferralitic Gravelly ferruginous Ferruginous Ferruginous Ferralitic Organs Leaves Leafy stems Leafy stems Roots Roots Leaves Certification AA6463/HNB AA6464/HNB AA6465/HNB AA6466/HNB AA6467/HNB AA6468/HNB numbers Plant Eucalyptus Chenopodium Securidaca Spondias Erythrophleum Guiera species tereticornis ambrosioides longepedunculata mombin suaveolens senegalensis Locations Abomey Savalou Oucagourou Abomey Manigri Alpha kouara Calavi Calavi (Goungoun forest) Soils Ferralitic Ferruginous Ferruginous Ferralitic Ferruginous Gravelly ferruginous Organs Leaves Leafy stems Root bark/Root Leaves Leaves Leaves Certification AA6469/HNB AA6470/HNB AA6471/HNB AA6472/HNB AA6473/HNB AA6474/HNB numbers

3.2. Plant extracts 3.2.1. Essential oils extracted by hydrodistillation Essential oils from all plants were extracted by hydrodistillation using a Clevenger- type apparatus for two to four hours until essential oils were extracted completely from the plant part (organs). The extracted oils were dried over anhydrous

magnesium sulfate and stored at 4°C, away from light, before use. Each essential oil was diluted tenfold with diethyl ether. One milliliter of each diluted solution was charged into a sampler flask for GC-MS analysis.

3.2.2. Essential oils extracted by simultaneous distillation extraction (SDE) Essential oils were extracted by simultaneous distillation extraction (SDE) using a Likens-Nickerson-type apparatus. Practically, one-hundred grams of each dried plant material was added to 1 liter of distilled water into a round-bottom flask, whereas 10 mL of diethyl ether was added to a conical flask. Both flasks were connected to the extremities of the apparatus, then water and diethyl ether were added to the central arm of the Likens-Nickerson apparatus. The round-bottom flask was heated using an oil bath at 120°C, whereas the conical flask was heated with a water bath at 35°C. Simultaneous distillation was carried out for one hour after reaching the boiling point. Mixtures obtained (essential oil and diethyl ether) were dried over anhydrous magnesium sulfate, concentrated using a rotavapor and the extracted oils obtained were stored at 4°C, away from light, before use. Essential oils were diluted at 10% with diethyl ether and one milliliter of each diluted solution was charged into a sampler flask for GC-MS analysis. To avoid or limit oxidation reactions due to the possible presence of peroxides in diethyl ether, the extraction time was limited to just one hour, and the dilution for GC- MS analysis was prepared just before the analysis. No essential oil was stored diluted in this solvent.

3.2.3. Ethanolic extracts A sample of each dried organ was transformed into powder using a traditional mortar. The extraction was carried out at room temperature, by mixing 100 grams of each powder with 1 liter of 70% ethanol for 24 hours, repeating this extraction two times. Each mixture was filtered with a vacuum flask two times, firstly through a Büchner funnel and then through a borosilicated funnel filter. The filtrate was concentrated in a rotavapor under vacuum, in a water bath at 40-50°C, frozen with liquid nitrogen and lyophilized for 48 hours. The powders obtained were stored at 4°C. The waste was dried at room temperature, and kept in a plastic bag in the fridge, for further utilization.

3.3. Gas Chromatography coupled with Mass Spectrometry The GC-MS analysis of the essential oils was performed on an Agilent 6890 GC Plus automatic sampler system, coupled to a quadrupole mass spectrometer 5973 MSD (Agilent Technologies, Diegem, Belgium) and equipped with a capillary HP-5MS column fused with silica (length: 30 m; diameter: 0.25 mm, film thickness: 0.25 microns) in the split mode 1:100. The oven temperature was programmed at 60°C for 3 min and to 350°C at a rate of 5°C/min. The injector was kept at 250°C programmed with a rate of 10°C/s. Helium was used as carrier gas at a flow rate of 1 ml/min. All analyses were performed at constant flow. The mass detector conditions were: transfer line temperature 260°C; ionization mode electron impact: 70 eV. The identification of sample compounds was carried out in single runs. The Kovats retention indices were calculated for all volatile constituents using a series of n- alkanes C7-C17 [249]. Quantification of each compound was performed using percentage peak area calculations. The identification of volatile compounds of the oil was done by comparing their retention indices with those of reference compounds in the literature and confirmed by GC-MS by comparison of their mass spectra with those of reference compounds [249-252]. The relative concentration of each compound in the essential oil was quantified according to the peak area integrated by the analysis program (Chemstation data analysis).

3.4. Headspace solid-phase microextraction (HS-SPME/GC-MS) SPME extraction and desorption were performed automatically by means of an MPS- 2 autosampler (Gerstel). One gram of powder of each sample was incubated at 30°C for 2 min with an agitation speed of 250 rpm. Extraction of volatiles by solid-phase microextraction were carried out using a 50/30 µm divinylbenzene/carboxen/ polydimethylsiloxane (DVB/Car/PDMS) fiber (Supelco, Bornem, Belgium) appropriate for the analysis of mixtures with polar and non-polar components [253], but also for the analysis of volatiles and semi-volatiles flavor compounds, with C3-C20 and which molecular weights are ranged between 40 and 275 g/mol [254]. The determination of each component was done by exposure of the fiber to the headspace for 60 min at 35°C, followed by desorption for 120 s. The GC-MS analyses of the SPME extracts were performed with an Agilent 6890 GC Plus, coupled to a mass selective- quadrupole spectrometer 5973 MSD (Agilent Technologies, Diegem, Belgium),

equipped with a Varian CP-PoraBOND Q capillary column (25 m length; 0.32 mm id; coating 5 µm film thickness) and a CIS-4 Programmed Temperature Vaporization (PTV) injector. Working conditions were as follows: transfer line to MSD 250°C, injector 250°C, carrier gas (He) 1.2 ml/min, SPME desorption in a CIS-4 PTV injector (Gerstel) in split mode; ionization: EI 70 eV. The oven temperature was programmed from 60 to 350°C and analyses were performed in SCAN mode. Each analysis was done with three repetitions.

3.5. Biological tests 3.5.1. Insecticidal tests on A. gambiae 3.5.1.1. Mosquito collection and determination of the status Larvae of the resistant strain of A. gambiae were collected three times a week, from March 2012 to June 2012, from the natural breeding site in “Ladji” in Cotonou. Ladji is a neighborhood of the outskirts of Cotonou, the capital of Benin. Larvae were brought and reared in the insectary of the Cotonou Research Center of Entomology (CREC). Larvae were fed with honey juice and the adults were placed in cages and fed in the same way. Emerging adult female mosquitoes of the resistant strain were used to carry out the susceptibility tests, whereas a susceptible strain of A. gambiae “Kisumu”, originating from Kenya and maintained at the insectary, was used as a reference. Before the realization of the bioassays with essential oils, the status of the collected mosquitoes was determined. The resistance in the genus Anopheles was due to mutations. Indeed two mutations have been identified in Benin: the Leu-Phe kdr (knock-down resistance) and the ace-1R (insensitive acetylcholinesterase) mutation [4, 5, 7, 8, 10]. To identify mutation in the genus Anopheles in Benin, Polymerase Chain Reactions (PCRs), which involved only An. gambiae sensu stricto (s.s.), were performed. PCR tests for species identification were performed to identify the members of An. gambiae complex collected from each site as described by Scott et al. [255]. From PCR positive specimens of An. gambiae s.s., aliquots of DNA were extracted and subjected to PCR assays to identify the two molecular forms, An. gambiae s.s. M and An. gambiae s.s. S, as described by Favia et al. [256]. “M” and “S” forms in An. gambiae sensu stricto (s.s.) are due to two independent mutational events or to an introgression between one molecular form and the other [257]. The next series of

PCR diagnostic tests, which determined the presence of kdr “Leu-Phe” mutation, was carried out on dead and alive An. gambiae mosquitoes from a bioassay, as described by Martinez-Torres et al. [258]. The PCR-RFLP diagnostic test, a test that detects the presence of the G119S mutation in single mosquitoes, was performed for the detection of the presence of the G119S mutation (ace-1R gene) as described by Weill et al. [259].

3.5.1.2. Bioassay on adult mosquitoes Susceptibility tests were carried out using WHO insecticide susceptibility test-kits and standard procedures [50]. Four replicates of batches of 20-25 unfed females, aged 2- 5 days old were exposed to filter papers (Whatman N°1) 12 cm x 15 cm, impregnated with 2 ml of various doses of the essential oil obtained by hydrodistillation, diluted in acetone. The susceptible strain A. gambiae “Kisumu” was used as reference to determine the diagnostic doses. The filter papers of the control holding tubes were impregnated with acetone only. Tests were carried out at 25°C ( 2°C) and 70-80% relative humidity. Six doses were tested for each essential oil: 0.25% (w/v), 0.50% (w/v), 1% (w/v), 2% (w/v), 4% (w/v) and 8% (w/v). The number of mosquitoes knocked down was recorded every 5 min. Permethrin 0.75% was used as reference insecticide. Permethrin impregnated papers were obtained from the WHO reference center of “the Vector Control Research Unit of The University Sains Malaysia". The quality of filter paper used is Whatman  1 for chromatography, density 90 g/m², thickness 0.16 mm. After the exposure time, mosquitoes were transferred to holding tubes and were fed with 10% honey juice for 24 hours. Subsequently the mortality was recorded. Data were plotted to determine the diagnostic doses to which the resistant strain from Ladji was exposed.

3.5.1.3. Susceptibility status of A. gambiae The status of mosquito samples was determined according to the WHO criteria summarized as follows [50]: - 98-100% mortality indicates susceptibility of the mosquito strain to the tested essential oil - Mortality less than 98% is suggestive of the existence of a resistance to the essential oil that needs to be confirmed by two additional tests - Mortality less than 90% suggests resistance in the mosquito population

3.5.1.4. Data analysis for the bioassays Times at which 50% or 95% of mosquitoes fell down on their back or on their side, i.e. knockdown time (KDT50 and KDT95) was calculated by means of the log time- probit using SPSS statistics 17.0 software. The relation between the KDT, the mortality, and the doses were assessed by probit regression. The diagnostic concentration which is the double of the minimum concentration at which 100% of the susceptible strain died [260] was also taken into account. All the results obtained for

KDT (KDT50 and KDT95) and lethal doses (LC50 and LC99), were expressed with 95% confidence limits.

3.5.2. Antiplasmodial tests on Plasmodium falciparum All the in vitro cytotoxicity and antiplasmodial bioassays were performed in collaboration with Prof. Louis Maes and Prof. Paul Cos from the Laboratory of Microbiology, Parasitology and Hygiene of Antwerp University – Belgium.

3.5.2.1. In vitro cytotoxicity

Diploid human embryonic lung fibroblasts cells (MRC-5SV2) were used. The cells were cultured in MEM + Earl’s salts-medium, supplemented with L-glutamine,

NaHCO3 and 5% inactivated fetal calf serum. Stock solutions of the test extracts (ethanolic extracts and essential oils obtained by SDE) were prepared at 20 mg/ml in 100% DMSO and serially diluted. An intermediate dilution was done in demineralized water to keep the final in-test concentration inferior to 1%. Extracts were tested at 5 concentrations: 64, 16, 4, 1 and 0.25 µg/ml. Assays were performed in sterile 96-well microtiter plates, each well containing 10 μl of the watery extract dilutions together 4 with 190 μl of MRC-5SV2 inoculum (3x10 cells/ml). Firstly, the cell growth was determined by comparison of the untreated-control wells (100% cell growth) and medium-control wells (0% cell growth). Secondly, after 3 days incubation, cell viability is determined fluorimetrically after addition of 50 μl of resazurin2 per well. Thirdly, after 4 hours at 37°C, fluorescence was measured (λex 550 nm, λem 590 nm). The results are expressed as % reduction in cell growth/viability compared to control wells

2 Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) is a blue fluorescent dye, until it is irreversibly reduced into a red fluorescent resorufin by living cells. Resazurin is used as an oxidation-reduction indicator in cell viability assays and thereby as an indicator for these mammalian cells viability.

and a IC50 (50% inhibitory concentration) is determined. The extract is classified non- toxic when the IC50 is higher than 30 µg/ml. When the IC50 is lower than 10 µg/ml, the extract is classified as highly toxic. Tamoxifen3 was used as cytotoxic reference compound [261].

3.5.2.2. In vitro antiplasmodial assays

The K1 strain of P. falciparum was used and chloroquine was used as reference drug. Assays were performed in 96-well microtiter plates, each well containing 10 μl of the watery extract (ethanolic extracts and essential oils obtained by SDE) and 190 μl of parasite inoculum (1% parasitaemia, 2% haematocrit). After 72 h incubation at 37°C, plates were frozen at -20°C. After thawing, 20 μl of each well was transferred into another plate and mixed with 100 μl MalstatTM reagent and 20 μl of a 1/1 mixture of PES (phenazine ethosulfate, 0.1 mg/ml) and NBT (Nitro Blue Tetrazolium Grade III, 2 mg/ml). The plates were kept protected from light for 2 hours and measured spectrophotometrically at 655 nm. The results are expressed as % reduction in parasitaemia compared to control wells and IC50 values were calculated. The extract is classified as inactive when the IC50 is higher than 10 μg/ml. When the IC50 is lower than 2 μg/ml, the extract is classified as highly active on the condition of absence of cytotoxicity [261].

3.5.3. Insecticidal tests on Tribolium castaneum 3.5.3.1. Stock of T. castaneum The rearing of T. castaneum was done in the Laboratory of Agrozoology, Faculty of Bioscience Engineering, Ghent University, Belgium. Beetles were reared in a box endowed with aeration, which contained whole-wheat flour nutritionally supplemented by 5% (w/w) brewer's yeast, and which was closed very well to avoid the escape of beetles. The boxes were maintained in a growth incubator, in total obscurity, at 30°C and 60% relative humidity.

3.5.3.2. Contact "no choice" test Contact "no choice" tests were performed in petri dishes 8.5 cm in diameter. Six replicates of batches of ten or twenty adult beetles were exposed to impregnated

3 Tamoxifen (Z-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine citrate) is an anti-estrogen drug, which blocks estrogen receptors in cells and prevents their multiplication.

filter paper (Whatman  1 for chromatography, density 90 g/m², thickness 0.16 mm), 8.5 cm diameter. Impregnation was performed with 0.7 ml of various doses of solutions of the essential oils obtained by hydrodistillation in acetone, and dried during 5 min at room temperature under flow. After the drying, the impregnated filter papers were inserted into the petri dishes, ten or twenty unsexed adult beetles were added into each petri dish and the lid was sealed in place with parafilm to avoid the escape of the beetles. Five doses were tested: 0.5% (w/v), 1% (w/v), 2% (w/v), 4% (w/v), and 8% (w/v). Acetone was used as negative control and permethrin 0.75% as positive control. Permethrin impregnated papers were obtained from the WHO reference center of “the Vector Control Research Unit of The University Sains Malaysia". Acetone was purchased from "Sigma Aldrich" (Bornem, Belgium). Beetles were fed in the same way as during rearing and the experiment was performed in the same environmental conditions. Mortality rates were recorded at 24 hours, 48 hours, and 72 hours of exposure. Lethal concentrations (LC50, LC90 and LC99), at which 50%, 90% and 99% of beetles died, were calculated by means of the log time-probit using SPSS statistics 17.0 software and expressed with 95% confidence limits. The beetle was considered dead when no antenna or leg movement was detected when gently touched with a brush. Differences among lethal concentrations (LC) were considered significant when the 95% CL of the LC50, LC90, or LC99 values failed to overlap.

3.5.3.3. Fumigant test Glass vials of 30 ml were used to assess the fumigant toxicity of essential oils used. Practically, filter paper (Whatman  1 for chromatography, density 90 g/m², thickness 0.16 mm) disc of 2 cm in diameter, were impregnated with the crude oil obtained by hydrodistillation using 30 µl, 60 µl, 120 µl. Impregnated filter paper was then attached to the under surface of the vial screw cap. The cap was screwed on the vial to generate the following concentrations: 1 ml/L air, 2 ml/L air, and 4 ml/L air, respectively. Six replicates of batches of ten adult beetles were performed and acetone was used as negative control. Permethrin 0.75% was used as positive control, and all the vials were kept under the same environmental conditions as during the rearing of T. castaneum. Mortality was recorded after 24 hours of exposure and LC50, LC90 and LC99 values were calculated by means of the log probit regression using SPSS statistics 17.0 software for Windows and expressed with 95%

confidence limits. The beetles were considered dead when no leg or antennal movement were observed. Differences among lethal concentrations (LC) were considered significant when the 95% CL of the LC50, LC90, or LC99 values failed to overlap.

3.5.3.4. Tests with isolated compounds Following the good mortality obtained with C. schoenanthus and E. citriodora, their main isolated compounds, piperitone for C. schoenanthus, citronellal, citronellol and citronellyl acetate for E. citriodora, respectively, were also tested against T. castaneum. The volume/quantity used for these isolated compounds were calculated taking into account their percentages in the crude oils. The two previous tests described above were performed. Quantities, volumes, and concentrations of each isolated compounds were presented in Table 8 and Table 9.

3.5.3.5. Procurement of the isolated compounds The isolation of piperitone has been performed in collaboration with MSc. Ewout Ruysbergh, PhD researcher at the department. Piperitone was obtained from the purification of the essential oil of C. schoenanthus via flash chromatography on SiO2 with hexane:diethyl ether 4:1. 7.26 g of the crude oil was used during the process of purification at the end of which four fractions were obtained. Each fraction was analyzed by GC-MS following the conditions described above. Fraction 1 (0.89 g), contained -2-carene (72.8%) as major compound and limonene (13.8%) as minor compound. Fraction 2 (3.21 g) contained pure piperitone (99.9%), while Fraction 3 (1.37 g) contained piperitone (93.7%) as the only major compound with some other very small minor peaks and Fraction 4 (0.68 g) contained elemol (50.8%) and -eudesmol (23.6%) as major compounds, E-p-menth-2-en-1-ol (5.8%), -terpineol (10.5%), -eudesmol (0.4%) and eremoligenol (6.26%) as minor compounds. The combined yield of piperitone obtained was 63%. Following the purity of fraction 2 in piperitone (99.9%), this fraction was used to perform the tests on T. castaneum. ()-Citronellal 80-90%, citronellol 97% and citronellyl acetate 97% were purchased from Fluka Chemie AG CH-9471 Buchs, ALDRICH Europe-Belgium and ACROS Organics-Belgium, respectively.

Table 8: Volumes and concentrations in fumigant test Essential oils Volume vial Volume crude oil Concentration Volume Concentration crude oil compound compound C. citratus 30 ml 30 µl C1=1 ml/l ND ND 60 µl C2=2 ml/l ND ND 120 µl C3=4 ml/l ND ND C. giganteus 30 ml 30 µl C1=1 ml/l ND ND 60 µl C2=2 ml/l ND ND 120 µl C3=4 ml/l ND ND C. schoenanthus piperitone piperitone 30 ml 30 µl C1=1 ml/l 17.67 µl C1=0.59 ml/l 60 µl C2=2 ml/l 35.34 µl C2=1.18 ml/l 120 µl C3=4 ml/l 70.68 µl C3=2.36 ml/l E. citriodora citronellal citronellal 30 ml 30 µl C1=1 ml/l ND ND 60 µl C2=2 ml/l 31.68 µl C2=1.06 ml/l 120 µl C3=4 ml/l 63.38 µl C3=2.11 ml/l citronellol citronellol ND ND 12 µl C2= 0.4 ml/l 24 µl C3=0.8 ml/l citronellyl acetate citronellyl acetate ND ND 5.4 µl C2=0.18 ml/l 10.8 µl C3=0.36 ml/l ND: not determined

Table 9: Quantities used in contact "no choice" test Identified Essential oils Quantity Concentrations Compounds Crude oil at 0.5% (CC, CG, CS, EC)1 - 3.5 mg 0.5% Crude oil at 1% (CC, CG, CS, EC)1 - 7 mg 1% Crude oil at 2% (CC, CG, CS, EC)1 - 14 mg 2% Crude oil at 4% (CC, CG, CS, EC)1 - 28 mg 4% Crude oil at 8% (CC, CG, CS, EC)1 - 56 mg 8.00% C. schoenanthus Piperitone 32.94 mg 4.70% E. citriodora Citronellal 29.56 mg 4.22% Citronellol 11.2 mg 1.60% Citronellyl 5.04 mg 0.72% acetate 1 = CC = C. citratus, CG = C. giganteus, CS = C. schoenanthus, EC = E. citriodora

CHAPTER 4: RESULTS AND DISCUSSION

4.1. Chemical composition obtained by hydrodistillation, simultaneous distillation extraction and solid phase microextraction The chemical composition of all selected plant species have been investigated using essential oils extracted both by hydrodistillation and simultaneous distillation extraction (SDE) as well as by solid phase microextraction (SPME). Chemical compositions obtained by these three methods of extraction, and essential oil yields expressed as oil wt./wt. of dried organ extracted, are presented in the following tables (Tables 10 to 21). By hydrodistillation the essential oils yields varied from 0.2% for Cochlospermum species to 4.6% for E. citriodora, whereas by SDE, the values obtained ranged from 0.03% for G. senegalensis to 1.5% for E. citriodora. The main compounds identified by these three methods are summarized in Table 22.

4.1.1. Chenopodium ambrosioides L. (Amaranthaceae) By hydrodistillation, C. ambrosioides essential oil was extracted with a yield of 1.3% (w/w). Its essential oil contained mainly ascaridole (41.9%). Some other components, such as α-terpinene (16.5%), p-cymene (14.4%) and isoascaridole (7.5%), were identified as well (Table 10). This chemical composition is similar to the one of a sample from China [39] whose main compounds were p-cymene (12.7%), Z- ascaridole (29.7%) and isoascaridole (13.0%) [39], and from Madagascar that contained ascaridole (41.8%), isoascaridole (18.1%) and p-cymene (16.2%) [113]. On the contrary, a sample from India has revealed other main compounds namely m- cymene (43.9%) and myrtenol (13.3%) [114], and a sample from Argentina contained ascaridole at 99.4% [115]. Other chemical profiles were identified in Togo with ascaridole (51.1%), p-cymene (19.8%), neral (8.7%) and geraniol (7.5%) [116] whereas in a sample from Rwanda, the main compounds identified were -terpinene (72.7%), p-cymene (15.3%) and ascaridole (7.2%) [117]. Unlike all the other samples, a sample from Nigeria does not contain ascaridole and isoascaridole, but - terpinene (56.0%), p-cymene (15.5%) and -terpinyl acetate (15.7%) [118].

By SPME/GC-MS, ascaridole (47.3%) and isoascaridole (33.3%) were identified as the main compounds, whereas by SDE/GC-MS, the current work has demonstrated

the presence of -terpinene (53.5%), p-cymene (19.7%) and E-p-mentha-1(7),8-dien- 2-ol (5.5%) (Table 10, Table 22).

Table 10: Chemical composition and essential oil yields of Chenopodium ambrosioides

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % % 960 961 MS, RI Benzaldehyde 0.1 987 988 MS, RI Myrcene 0.5 1010 1014 MS, RI -Terpinene 16.5 53.5 2.5 1018 1020 MS, RI p-Cymene 14.4 19.7 4.3 1021 1024 MS, RI Limonene 0.4 0.3 1050 1054 MS, RI -Terpinene 0.3 1086 1089 MS, RI p-Cymenene 0.3 1117 1119 MS, RI E-p-Mentha-2,8-dienol 0.2 4.1 2.5 1125 MS Cyclooctanone 0.2 1131 1133 MS, RI Z-p-Mentha-2,8-dienol 2.3 0.6 1145 1148 MS, RI Citronellal 0.1 1175 1178 MS, RI Naphthalene 0.1 1184 1187 MS, RI E-p-Mentha-1(7),8-dien-2-ol 5.5 2.3 1211 1215 MS, RI E-Carveol 1.0 1227 1227 MS, RI Z-p-Mentha-1(7),8-dien-2-ol 4.1 1.1 1235 1234 MS, RI Ascaridole 41.9 4.4 47.3 1240 1239 MS, RI Carvone 0.7 1247 1252 MS, RI E-Piperitone epoxide 1.1 1266 MS 5-Undecen-4-one 0.5 1288 1289 MS, RI Thymol 0.4 1299 1299 MS, RI Isoascaridole 7.47 5.4 33.3 1347 1349 MS, RI Thymol acetate 0.2 1410 1417 MS, RI E-Caryophyllene 0.2 1477 MS E-β-Ionone 0.1 1581 MS 3-Tetradecanone 0.4 Yield 1.3 0.1 Total identified 83.6 100 96.7 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.2. Cochlospermum planchonii Hook. f. ex Planch. (Bixaceae) The essential oil of C. planchonii was prepared by hydrodistillation with a yield of 0.20% (w/w), which is lower than from the tubercle from Burkina-Faso (0.30%) [127]. In the current study the composition of this essential oil was dominated by 3- tetradecanone (24.7%), ethyl tetradecanoate (11.4%) and isoamyl dodecanoate (14.1%), accompanied by 2-tridecanone (6.8%), dodecyl acetate (4.9%), 2- pentadecanone (5.7%), n-tetradecanol (3.0%), 3-hexadecanone (2.0%) and 4- octadecanone (2.2%) (Table 11). In the sample from Burkina-Faso, 3-tetradecanone (69.8%) was the main compound, followed by tetradecyl acetate (14.4%), dodecyl

acetate (4.7%), 3-hexadecanone (2.5%) and n-tetradecanol (1.0%) [127]. Another sample, originating from Burkina-Faso and characterized by Ouattara et al. [128], was similar to the previous one with 3-tetradecanone (30.6%), dodecyl acetate (12.4%) and tetradecyl acetate (15.0%), as well as 3-tetradecenone (15.3%), 2- tridecanone (7.8%) and -elemene (6.0) as the main compounds [128].

In the current work, when characterized by SDE/GC-MS or SPME/GC-MS, C. planchonii has revealed the following main compounds: piperitone (38.1%), 3- tetradecanone (12.0%), neo-isopulegol (6.5%) and 2-tridecanone (4.5%) by SDE/GC- MS, and piperitone (20.9%), citronellal (12.9%), 3-tetradecanone (10.4%) and neo- isopulegol (6.4%) by SPME/GC-MS (Table 11, Table 22).

Table 11: Chemical composition and essential oil yields of Cochlospermum planchonii

a b c d Hydro. SDE SPME KI exp KI lit ID Compounds % % % 801 801 MS, RI Hexanal 0.1 973 974 MS, RI β-Pinene 0.1 987 988 MS, RI Myrcene 0.8 1000 1001 MS, RI δ-2-Carene 2.9 1003 1002 MS, RI α-Phellandrene 0.2 1013 1014 MS, RI α-Terpinene 3.5 1021 1020 MS, RI p-Cymene 0.3 0.2 2.9 1024 1024 MS, RI Limonene 2.7 1026 1025 MS, RI β-Phellandrene 0.1 1028 1026 MS, RI 1,8-Cineole (Eucalyptol) 0.1 1033 1032 MS, RI Z-β-ocimene 0.1 1043 1044 MS, RI E-β-ocimene 0.1 1050 1055 MS, RI 2,6-Dimethyl-Hept-5-en-1-al 0.1 1054 1054 MS, RI -Terpinene 0.3 1085 1086 MS, RI Terpinolene 0.2 1086 1089 MS, RI p-Cymenene 0.1 1098 1098 MS, RI Linalool 0.1 1100 1099 MS, RI Undecane 0.1 1141 1144 MS, RI Neo-Isopulegol 6.5 6.4 1149 1148 MS, RI Citronellal 0.2 0.2 12.9 1152 1155 MS, RI Iso-Isopulegol 4.0 4.3 1174 1178 MS, RI Naphthalene 0.1 0.5 1189 1186 MS, RI α-Terpineol 0.6 0.2 1192 1190 MS, RI Methyl salicylate 4.4 1.1 1224 1223 MS, RI Citronellol 0.2

1258 1249 MS, RI Piperitone 0.1 38.1 20.9 1303 1299 MS, RI Isoascaridole 0.5 1309 1305 MS, RI Undecanal 0.2 0.2 1348 1350 MS, RI Citronellyl acetate 0.3 0.4 1368 1374 MS, RI α-Copaene 0.1 1377 1387 MS, RI β-Bourbonene 0.1 1385 1389 MS, RI β-Elemene 0.8 2.0 1391 1398 MS, RI Cyperene 0.1 1393 1389 MS, RI β-Elemene 1.0

1400 1398 MS, RI Cyperene 0.1 1410 1408 MS, RI Dodecanal 0.1 0.2 1411 1417 MS, RI E-Caryophyllene 0.9 1.9 1419 1424 MS, RI 2,5-Dimethoxy-p-cymene 0.8 0.6 1423 1431 MS, RI β-Gurjunene 0.7 1428 1432 MS, RI α-E- Bergamotene 0.1 1446 1452 MS, RI α-Humulene 0.2 1469 1478 MS, RI - Muurolene 0.6 1478 1475 MS, RI -Gurjunene 1.9 1487 1494 MS, RI α-Selinene 1.0 1490 1495 MS, RI 2-Tridecanone 6.8 4.5 2.2 1492 1490 MS, RI 1-Dodecanol 0.5 1493 1500 MS, RI α-Muurolene 0.3 0.7 1498 1504 MS, RI Cuparene 0.3 0.5 1502 1506 MS, RI β-Bisabolene 0.2 0.3 1507 1513 MS, RI -Cadinene 1.0 0.8 1512 1509 MS, RI Tridecanal 0.3 1517 1522 MS, RI -Cadinene 0.6 1.0 1525 1529 MS, RI E--Bisabolene 0.1 1533 MS Cyclododecane 0.1 1544 1548 MS, RI Elemol 0.9 0.2 1580 MS 1-Hydroxyundecan-3-one 2.8

1578 1576 MS, RI Dodecanoic acid 1.2 1585 1582 MS, RI Caryophyllene oxide 0.3 1598 MS 3-Tetradecanone 24.7 12.0 10.4 1599 1607 MS, RI Dodecyl acetate 4.9 0.3 1600 1609 MS, RI Tetradecanal 0.3

1623 1630 MS, RI -Eudesmol 0.2

1642 1649 MS, RI β-Eudesmol 0.4

1646 1652 MS, RI -Eudesmol 0.4

1663 1658 MS, RI neo-Intermedeol 0.1 1672 1671 MS, RI n-Tetradecanol 3.0 1.8 1702 1697 MS, RI 2-Pentadecanone 5.7 0.5 1775 1780 MS, RI Tetradecanoic acid 0.5 1795 1795 MS, RI Ethyl tetradecanoate 11.4 1798 1797 MS, RI 3-Hexadecanone 2.0 1.0 1846 1844 MS, RI Isoamyl dodecanoate 14.1 1874 MS, RI Pentadecanol 2.0

2003 MS 4-Octadecanone 2.2

Yield 0.2 0.1 Total identified 82.0 82.6 89.4 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.3. Cochlospermum tinctorium A. Rich (Bixaceae) By hydrodistillation, the essential oil yield of C. tinctorium was 0.2% (w/w) which is higher than the one obtained in Burkina-Faso (0.10%) by Benoit-Vical et al. [127]. The essential oil extracted from C. tinctorium in the current study is dominated by 3- tetradecanone (48.3%), cyclododecanone (7.8%), 3-hexadecanone (7.4%),

1-tetradecanyl acetate (4.3%), 2-tridecanone (3.4%), dodecyl acetate (2.0%) and methyl tetradecanoate (2.3%) (Table 12). This chemical composition is similar to the result obtained by Benoit-Vical et al. [127], from the whole tubercle essential oil which contained four main compounds, namely 3-tetradecanone (64.6%), tetradecyl acetate (11.7%), 3-hexadecanone (5.6%), 3-hexadecenone (5.6%) and dodecyl acetate (4.6%).

The 3-tetradecanone was also detected as the main compound both by SDE/GC-MS (54.5%) and SPME/GC-MS (42.4%) associated to neo-isopulegol (10.1%), iso- isopulegol (6.2%) and 3-hexadecanone (5.2%) by SDE/GC-MS, and -terpinene (13.1%) and p-cymene (11.3%) by SPME/GC-MS (Table 12, Table 22).

Table 12: Chemical composition and essential oil yields of Cochlospermum tinctorium

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % % 973 974 MS, RI β-Pinene 0.1 987 988 MS, RI β-Myrcene 1.1 978 981 MS, RI 6-Methyl-5-Hepten-2-one 0.1 994 1001 MS, RI δ-2-Carene 0.1 4.3 1003 1002 MS, RI α-Phellandrene 0.1 1014 1014 MS, RI α-Terpinene 0.1 13.1 1021 1020 MS, RI p-Cymene 0.3 11.3 1025 1024 MS, RI Limonene 0.1 4.2 1028 1026 MS, RI 1,8-Cineole 0.1 0.2 1033 1032 MS, RI Z-β Ocimene 0.2 1054 1054 MS, RI -Terpinene 0.3 1085 1086 MS, RI Terpinolene 0.1 1087 1089 MS, RI p-Cymenene 0.2 0.2 1093 1095 MS, RI Linalool 0.1 1143 1144 MS, RI neo-Isopulegol 10.1 1.2 1149 1148 MS, RI Citronellal 0.2 1153 1155 MS, RI iso-Isopulegol 6.2 1 1175 1178 MS, RI Naphthalene 0.1 1185 1186 MS, RI -Terpineol 0.1 1190 1190 MS, RI Methyl salicylate 2.6 2.2 1222 1223 MS, RI Citronellol 0.3 1233 1235 MS, RI Neral 0.1 1248 1249 MS, RI Piperitone 2.7 0.7 1263 1264 MS, RI Geranial 0.1 1298 1305 MS, RI Undecanal 0.2 1301 1299 MS, RI Isoascaridole 0.3 1345 1350 MS, RI Citronellyl acetate 0.1 1368 1374 MS, RI α-Copaene 0.0 1380 1389 MS, RI 3-Dodecanone 0.1 1382 1389 MS, RI β-Elemene 0.6 0.1 1388 1398 MS, RI Cyperene 0.1 0.1 0.2 1400 1408 MS, RI Dodecanal 0.1 0.5 1403 1410 MS, RI α-Cedrene 0.2

1405 1411 MS, RI Z--Bergamotene 0.1 0.1 0.3 1408 1417 MS, RI E-Caryophyllene 0.1 0.6 0.6 1419 1424 MS, RI 2,5-dimethoxy-p-Cymene 0.2 0.1 1428 1432 MS, RI E--Bergamotene 0.6 0.9 2.1 1440 1445 MS, RI epi-β-Santalene 0.1 0.2 0.3 1442 1443 MS, RI Z-Prenyl limonene 0.1 1453 1454 MS, RI β-Santalene 0.1 1467 1469 MS, RI n-Dodecanol 0.5 1487 1495 MS, RI 2-Tridecanone 3.4 3.2 1.9 1493 1506 MS, RI Z--Bisabolene 0.3 0.5 1499 1505 MS, RI β-Bisabolene 2.2 2.6 3.8 1505 1506 MS, RI α-Bisabolene 0.1 1504 1514 MS, RI Butylated Hydroxytoluene 0.6 1508 1514 MS, RI Z--Bisabolene 0.1 0.4 1513 1511 MS, RI -Amorphene 0.1 0.1 1517 1522 MS, RI -Cadinene 0.1 1515 1524 MS, RI Methyl dodecanoate 0.5 1523 1529 MS, RI E--Bisabolene 0.3 0.6 0.9 1572 1582 MS, RI Caryophyllene oxide 0.1 1576 MS Cyclododecanone 7.8 1580 MS 1-Hydroxyundecan-3-one 0.2 1587 MS 3-Tetradecanone 48.3 54.5 42.4 1599 1607 MS, RI Dodecyl acetate 2.0 1602 1609 MS, RI Tetradecanal 0.3 0.8 0.5 1652 1652 MS, RI -Eudesmol 0.1 1674 1671 MS, RI n-Tetradecanol 0.6 1682 1685 MS, RI -Bisabolol 0.1 0.1 1691 1697 MS, RI 2-Pentadecanone 0.7 0.3 1704 MS Tridecyloxirane 0.1 1717 1722 MS, RI Methyl tetradecanoate 2.3 1786 MS 3-Hexadecanone 7.4 5.2 1.7 1798 MS 1-Tetradecanyl acetate 4.3 0.8 1818 1822 MS, RI Hexadecanal 0.6 0.2 2003 MS 4-Octadecanone 0.8 0.3 Yield 0.2 0.1

Total identified 83.3 97.7 96.7 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.4. Cymbopogon citratus (DC.) Stapf (Poaceae) The oil yield of C. citratus was 1.7% (w/w) by hydrodistillation. This value is higher than the one obtained with C. citratus (1.3%) collected in northern Brazil [54]. The essential oil of C. citratus was characterized by myrcene (12.4%), neral (33.1%) and geranial (44.3%) (Table 13). This result corroborates with previous results [54], which showed that the aerial part of C. citratus contains myrcene (10.7%), neral (30.8%) and geranial (53.9%). Furthermore, myrcene, neral and geranial were also the main

compounds identified in C. citratus characterized in Burkina-Faso, Brazil and Portugal [56, 138, 139]. By SDE/GC-MS and SPME/GC-MS, the chemical profiles obtained were dominated by geraniol (41.2%), neral (23.6%), selina-6-en-4-ol (7.2%) by SDE/GC-MS, and geranial (23.9%), neral (19.7%) and E-caryophyllene (5.0%) were detected by SPME/GC-MS (Table 13, Table 22).

Table 13: Chemical composition and essential oil yields of Cymbopogon citratus

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % % 978 988 MS, RI 6-Methyl-5-hepten-2-one 0.5 0.4 1.6 987 988 MS, RI Myrcene 12.4 1.2 1.2 999 1002 MS, RI δ-2-Carene 1.2 1013 1014 MS, RI α-Terpinene 1.6 1021 1020 MS, RI p-Cymene 2.1 1024 1024 MS, RI Limonene 0.8 1028 1026 MS, RI Eucalyptol 0.1 1033 1032 MS, RI Z-β-Ocimene 0.3 0.1 1039 1044 MS, RI E-β-Ocimene 0.2 1050 1051 MS, RI 2,6-Dimethyl-5-heptenal 0.1 1054 1054 MS, RI -Terpinene 0.1 1069 1067 MS, RI Z-linalool oxide 0.1 1084 1084 MS, RI E-linalool oxide 0.1 1085 1090 MS, RI 6,7-Epoxymyrcene 0.2 0.3 1093 1095 MS, RI Linalool 1.1 1.1 3.0 1141 1144 MS, RI Neo-Isopulegol 0.4 0.4 1149 1148 MS, RI Citronellal 0.1 0.7 3.8 1150 1155 MS, RI iso-Isopulegol 0.1 0.6 1171 1173 MS, RI Rose furan epoxide 0.2 0.3 2.0 1175 1177 MS, RI E-Isocitral 0.6 1188 1190 MS, RI Methyl salicylate 0.9 1.7 1216 1224 MS, RI 2,3-epoxyneral 0.1 0.3 1222 1223 MS, RI Citronellol 1.1 0.6 1234 1234 MS, RI Ascaridole 0.2 1237 1235 MS, RI Neral 33.1 23.6 19.7 1250 1249 MS, RI Piperitone 1.6 1252 1249 MS, RI Geraniol 1.0 41.2 2.0 1268 1264 MS, RI Geranial 44.3 23.9 1289 1293 MS, RI 2-Undecanone 0.1 0.2 0.6 1298 1298 MS, RI Geranyl formate 0.2 0.2 1301 1299 MS, RI Isoascaridole 2.0 1351 1359 MS, RI Geranic acid 0.1 1364 1369 MS, RI Cyclosativene 0.2 1368 1374 MS, RI α-Copaene 0.1 1376 1379 MS, RI Geranyl acetate 0.8 0.5 1384 1389 MS, RI β-Elemene 0.2 0.9 1411 1417 MS, RI E-Caryophyllene 0.1 1.0 5.0 1419 1424 MS, RI 2,5-dimethoxy-p-Cymene 0.1 1428 1432 MS, RI -E-Bergamotene 0.9 2.3 1431 1437 MS, RI α-Guaiene 0.6 1433 1439 MS, RI Aromadendrene 0.4

1436 1438 MS, RI Iso-caryophyllene 0.6 1445 1452 MS, RI α-Humulene 0.7 1448 1451 MS, RI E-Muurola-3,5-diene 0.4 1450 1454 MS, RI E-β-Farnesene 0.2 0.6 1455 1451 MS, RI E-Muurola-3,5-diene 0.1 1466 1461 MS, RI Z-Cadina-1(6),4-diene 0.2 1466 1465 MS, RI Z-Muurola-4(14),5-diene 0.1 1469 1471 MS, RI Dauca-5,8-diene 0.3 1473 1478 MS, RI -Muurolene 0.5 1.4 1476 1477 MS, RI γ-Selinene 1.4 1484 1492 MS, RI -Selinene 0.9 1487 1492 MS, RI Z-β-Guaiene 0.2 1489 1495 MS, RI 2-Tridecanone 0.1 0.6 0.3 1493 1500 MS, RI -Muurolene 0.8 0.6 1498 1496 MS, RI Viridiflorene 0.5 1502 1505 MS, RI β-Bisabolene 0.2 0.3 1506 1513 MS, RI -Cadinene 1.4 1.2 1516 1522 MS, RI -Cadinene 1.3 1.1 1525 1527 MS, RI Cadina-1,4-diene 0.2 1527 1537 MS, RI -Cadinene 0.2 1576 1582 MS, RI Caryophyllene oxide 0.1 1.4 0.3 1581 MS, RI 3-Tetradecanone 0.3 1594 1607 MS, RI 5Epi, 7Epi--Eudesmol 0.3 1609 1615 MS, RI Selina-6-en-4-ol 0.4 7.2 1637 1638 MS, RI epi-α-Cadinol 1.2 0.1 1639 1644 MS, RI -Muurolol 0.1 1648 1652 MS, RI -Cadinol 1.6 Yield 1.7 1.2 Total identified 95.2 92.5 92.5 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.5. Cymbopogon giganteus Chiov. (Poaceae) The oil yield of C. giganteus was 1.4% (w/w) by hydrodistillation and the main constituents of its essential oil were limonene (9.6%) and a set of monoterpene alcohols: E-p-mentha-1(7),8-dien-2-ol (19.6%), E-p-mentha-2,8-dienol (19.3%), Z-p- mentha-2,8-dienol (10.2%), Z-p-mentha-1(7),8-dien-2-ol (2.1%), Z-carveol (17.0%) and E-carveol (6.0%) together with p-menth-6-en-2,3-diol (3.2%) and carvone (3.2%) (Table 14). This composition is similar to that of C. giganteus from Burkina-Faso which was characterized by E-p-mentha-1(7),8-dien-2-ol, E-p-mentha-2,8-dienol, Z-p- mentha-2,8-dienol and Z-p-mentha-1(7),8-dien-2-ol [56] and from many West and Central African countries [28, 152, 154, 262]. The chemical profile identified by SDE/GC-MS contained mainly the same compounds, namely E-p-mentha-1(7),8-dien-2-ol (28.3%), Z-p-mentha-1(7),8-dien-2- ol (17.3%), E-p-mentha-2,8-dienol (15.3%), Z-p-mentha-2,8-dienol (10.4%) and by

SPME/GC-MS E-p-mentha-1(7),8-dien-2-ol (22.8%), E-p-mentha-2,8-dienol (17.7%), Z-p-mentha-1(7),8-dien-2-ol (15.9%), Z-p-mentha-2,8-dienol (6.8%) and isoascaridole (6.8%) (Table 14, Table 22).

Table 14: Chemical composition and essential oil yields of Cymbopogon giganteus

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % % 800 801 MS, RI Hexanal 0.2 984 988 MS, RI Dehydro-1,8-cineole 0.1 999 1001 MS, RI δ-2-Carene 0.1 1021 1020 MS, RI p-Cymene 0.5 1.2 2.2 1024 1024 MS, RI Limonene 9.6 3.7 0.2 1085 1089 MS, RI p-Cymenene 0.2 0.3 1.7 1104 1108 MS, RI 1,3,8-p-Menthatriene 0.3 1119 1119 MS, RI E-p-Mentha-2,8-dienol 19.3 15.3 17.7 1131 1133 MS, RI Z-p-Mentha-2,8-dienol 10.2 10.4 6.8 1133 1137 MS, RI E-Limonene Oxide 0.2 1138 1144 MS, RI Neo-Isopulegol 0.1 1145 1148 MS, RI Citronellal 0.7 1148 MS 4-Isopropenylcyclohexanone 0.4 0.4 1161 MS 6-methyl-Bicyclo[3.3.0]oct-2-en-7-one 0.5 1178 1179 MS, RI p-Methyl-acetophenone 0.3 1186 1187 MS, RI E-p-Mentha-1(7),8-dien-2-ol 19.6 28.3 22.8 1193 1200 MS, RI E-Dihydrocarvone 0.2 1194 MS p-Menth-6-en-2,3-diol 3.2 1195 1196 MS, RI E-4-Caranone 6.9 1214 1215 MS, RI E-Carveol 6.0 8.4 6.0 1224 1226 MS, RI Z-Carveol 17.0 0.5 1226 1227 MS, RI Z-p-Mentha-1(7),8-dien-2-ol 2.1 17.3 15.9 1239 1239 MS, RI Carvone 3.2 3.2 3.9 1247 1249 MS, RI Piperitone 0.1 1269 1269 MS Perilla aldehyde 0.8 0.5 0.6 1273 1273 MS, RI E-Carvone oxide 0.2 1284 MS 2,6-Dimethyl-2,4-heptadiene 0.3 1297 1299 MS, RI Isoascaridole 0.5 6.8 1571 1582 MS, RI Caryophyllene oxide 0.1 1636 1640 MS, RI 2-Phenylethyl hexanoate 0.1 Yield 1.4 0.3 Total identified 93.4 97.5 86.2 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.6. Cymbopogon schoenanthus (L.) Spreng. (Poaceae) The yield of C. schoenanthus essential oil obtained by hydrodistillation was 2.6% (w/w). This result is similar to Ketoh et al. [157]. In the current work, the major constituents were δ-2-carene (15.5%) and piperitone (58.9%) (Table 15). These

results are consistent with the results obtained by Koba et al. and Ketoh et al. (2005 and 2006) with the essential oils of C. schoenanthus from Burkina-Faso and Togo, as the main compounds identified in Togo samples were piperitone (68.0, 61.0%, 69.0%) and -2-carene (16.5, 23.4%, 16.9%), [38, 157, 158], and in Burkina-Faso the main compounds were piperitone (42.0%) and -2-carene (8.2%) [159]. In contrast, the volatile profile of C. schoenanthus leaves achieved in Brazil was dominated by geraniol (62.5%), geranial (12.5%) and neral (8.2%) [156], whereas the one isolated from the leaves, originating from Tunisia, contained as its main compounds limonene (24.2-27.3%), -phellandrene (13.4-16.0%), -terpinene (8.4-21.2%) and -terpineol (9.1-11.7%) [155]. By SDE/GC-MS and SPME/GC-MS, two main compounds have been detected. By SDE/GC-MS piperitone (53.1%) and elemol (9.3%) were detected, whereas by SPME/GC-MS piperitone (56.6%) and E-caryophyllene (6.5%) were identified (Table 15, Table 22).

Table 15: Chemical composition and essential oil yields of Cymbopogon schoenanthus

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % % 984 988 MS, RI 2,3-Dehydro-1,8-cineole 0.1 0.1 987 988 MS, RI Myrcene 0.1 999 1001 MS, RI δ-2-Carene 15.5 4.4 1.3 1003 1002 MS, RI α-Phellandrene 0.2 0.1 1013 1014 MS, RI α-Terpinene 0.2 0.4 1021 1020 MS, RI p-Cymene 0.1 0.4 1024 1024 MS, RI Limonene 3.6 0.8 0.5 1033 1032 MS, RI Z-β-Ocimene 0.1 1039 1044 MS, RI E-β-Ocimene 0.1 1081 1083 MS, RI L-Fenchone 0.2 0.1 1118 1118 MS, RI Z-p-Menth-2-en-1-ol 1.4 1.1 1.6 1136 1136 MS, RI E-p-Menth-2-en-1-ol 0.7 0.7 0.6 1141 1144 MS, RI Neo-Isopulegol 0.6 1148 1148 MS, RI Citronellal 1.6 1.0 1152 1155 MS, RI iso-Isopulegol 0.4 0.1 1160 1166 MS, RI α-Phellandren-8-ol 0.1 1173 1169 MS, RI Lavandulol 0.1 1179 1178 MS, RI Naphthalene 0.1 1180 1179 MS, RI p-Cymen-8-ol 0.1 1182 1184 MS, RI Dill ether 0.1 1183 1186 MS, RI α-Terpineol 1.5 1.4 1.5 1188 1195 MS, RI Z-Piperitol 0.3 0.6 1192 1190 MS, RI Methyl salicylate 2.8 1205 1207 MS, RI E-Piperitol 0.2 0.1 1253 1249 MS, RI Piperitone 58.9 53.1 56.6 1268 1264 MS, RI E-Citral (Geranial) 0.1 1302 1299 MS, RI Isoascaridole 1.0

1343 1345 MS, RI α-Cubebene 0.1 1368 1374 MS, RI α-Copaene 0.1 1378 1378 MS, RI α-Bourbonene 0.6 1382 1389 MS, RI β-Elemene 0.4 1.3 5.1 1400 1407 MS, RI α-Barbatene 0.1 1408 1417 MS, RI E-Caryophyllene 1.1 2.3 6.5 1419 1424 MS, RI 2,5-Dimethoxy-p-cymene 0.1 1424 1431 MS, RI β-Gurjunene 0.1 0.3 0.9 1428 1436 MS, RI Isobazzanene 0.1 1433 1440 MS, RI β-Barbatene 0.1 1443 1452 MS, RI α-Humulene 0.1 0.2 0.6 1455 1451 MS, RI E-Muurola-3,5-diene 0.1 1460 1458 MS, RI allo-Aromadendrene 0.1 1469 1474 MS, RI Z-Prenyl limonene 0.3 0.6 1470 1471 MS, RI Dauca-5,8-diene 0.9 1475 1475 MS, RI -Gurjunene 0.3 1484 1489 MS, RI β-Selinene 3.0 1487 1498 MS, RI α-Selinene 0.7 1490 1500 MS, RI α-Muurolene 0.1 0.5 0.6 1494 1496 MS, RI Valencene 1.5 1497 1504 MS, RI Cuparene 1.0 1507 1513 MS, RI -Cadinene 0.1 0.8 1.1 1513 1519 MS, RI β-Bazzanene 0.1 1517 1522 MS, RI -Cadinene 0.4 2.0 1.2 1525 1529 MS, RI E--Bisabolene 0.3 0.7 1530 1537 MS, RI α-Cadinene 0.2 1545 1546 MS, RI Elemol 5.3 9.3 2.2 1576 1582 MS, RI Caryophyllene oxide 0.4 1600 1607 MS, RI 5-Epi-7-epi--eudesmol 0.3 0.6 0.1 1614 1622 MS, RI 10-Epi--eudesmol 0.2 0.3 0.1 1623 1630 MS, RI -Eudesmol 1.1 1.9 0.1 1624 1630 MS, RI β-Muurola-4,10(14)-dien-1-ol 0.1 1626 1629 MS, RI Eremoligenol 1.9 0.3 1634 1640 MS, RI Hinesol 0.7 2.7 0.2 1643 1649 MS, RI β-Eudesmol 1.2 2.6 0.5 1646 1652 MS, RI α-Eudesmol 2.1 3.5 0.5 Yield 2.6 1.4 Total identified 98.4 96.7 96.5 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.7. Erythrophleum suaveolens (Guil. & Perr.) Brenan (Fabaceae) The analysis of the volatile profile of E. suaveolens was done by SDE/GC-MS and SPME/GC-MS since by hydrodistillation no essential oil was obtained. By SDE/GC-MS the following compounds have been detected: citronellal (23.0%), piperitone (22.5%), neo-isopulegol (11.0%), methyl salicylate (8.4%), iso-isopulegol (8.3%), 3-tetradecanone (6.8%). On the contrary, by SPME/GC-MS, -terpinene

(25.8%), p-cymene (18.1%), -2-carene (13.4%), citronellal (9.6%), limonene (8.9%), myrcene (6.3%) were identified (Table 16, Table 22).

Table 16: Chemical composition and essential oil yields of Erythrophleum suaveolens

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % % 793 801 MS, RI Hexanal 0.4 0.2 846 844 MS, RI 3E-Hexenol 0.6 0.1 863 863 MS, RI n-Hexanol 0.2 911 906 MS, RI 2-Methylpropyl isobutyrate 0.1 923 924 MS, RI α-Thujene 0.1 930 932 MS, RI α-Pinene 0.3 969 969 MS, RI Sabinene 0.2 973 974 MS, RI β-Pinene 0.6 984 981 MS, RI 6-Methyl-5-hepten-2-one 0.5 987 988 MS, RI Myrcene 6.3 999 1001 MS, RI -2-Carene 0.9 13.4 1003 1002 MS, RI α-Phellandrene 0.3 1009 1007 MS, RI Isoamyl butyrate 0.1 1014 1014 MS, RI -Terpinene 0.9 25.8 1021 1020 MS, RI p-Cymene 2.3 18.1 1022 1024 MS, RI Limonene 0.7 8.9 1028 1026 MS, RI 1,8-Cineole 0.6 1033 1032 MS, RI Z-β-Ocimene 0.6 1043 1044 MS, RI E-β-Ocimene 0.1 1050 1056 MS, RI 2,6-Dimethyl-5-heptenal 0.2 1054 1054 MS, RI -Terpinene 0.7 1084 1086 MS, RI Terpinolene 0.5 1093 1095 MS, RI Linalool 0.7 1099 1102 MS, RI Perillene 0.1 1142 1144 MS, RI Neo-Isopulegol 11.0 0.6 1148 1148 MS, RI Citronellal 23.0 9.6 1152 1155 MS, RI iso-Isopulegol 8.3 0.8 1191 1190 MS, RI Methyl salicylate 8.4 4.0 1221 1223 MS, RI Citronellol 2.1 1230 1232 MS, RI Thymol Methyl Ether 0.1 1249 1249 MS, RI Piperitone 22.5 2.5 1301 1299 MS, RI Isoascaridole 1.4 1346 1350 MS, RI Citronellyl acetate 0.8 1385 1389 MS, RI β-Elemene 0.5 1411 1417 MS, RI E-Caryophyllene 3.6 0.4 1419 1424 MS, RI 2,5-dimethoxy-p-Cymene 1.0 0.1 1428 1432 MS, RI E-α-Bergamotene 0.1 1498 1505 MS, RI β-Bisabolene 0.6 1503 1514 MS, RI Butylated hydroxytoluene 1.5 1581 MS 3-Tetradecanone 6.8 Yield 0.05 Total identified 96.6 97.6 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.8. Eucalyptus citriodora Hook (Myrtaceae) The essential oil from E. citriodora was extracted with a yield of 4.6%. The main compounds detected by GC-MS were citronellal (52.8%), citronellol (20.0%), citronellyl acetate (9.0%) and neo-isopulegol (7.8%) (Table 17). Components like citronellal, citronellol and isopulegol were also detected in E. citriodora essential oil analyzed in Colombia [27, 41], the Democratic Republic of Congo [145] and Ethiopia [170]. On the contrary, the volatile profiles obtained in Brazil and India were quite different since their main compounds were (-)- isopulegol (7.3%), -citronellal (71.7%) [27] and -thujene (11.9%), -pinene (18.3%), sabinene (19.6%), -pinene (25.7%) [171], respectively. The chemical profile identified by SDE/GC-MS is composed of neo-isopulegol (31.5%), citronellal (28.4%), iso-isopulegol (15.7%), citronellol (11.8%) (Table 17, Table 22), whereas by SPME/GC-MS the following compounds have been detected: citronellal (48.8%), neo-isopulegol (15.2%), E-caryophyllene (9.2%), citronellyl acetate (5.8%) (Table 17, Table 22).

Table 17: Chemical composition and essential oil yields of Eucalyptus citriodora

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % % 832 MS 3-Methylcyclopentanol 0.5 911 908 MS, RI Isobutyl isobutyrate 0.1 930 932 MS, RI α-Pinene 0.1 0.2 969 969 MS, RI Sabinene 0.1 973 974 MS, RI β-Pinene 0.2 0.1 0.7 982 788 MS, RI Myrcene 0.1 0.3 999 1001 MS, RI -2-Carene 0.6 1009 1007 MS, RI Isoamyl isobutyrate 0.1 1013 1014 MS, RI α-Terpinene 0.1 0.2 1017 1020 MS, RI p-Cymene 0.1 0.4 1020 1024 MS, RI Limonene 0.2 0.5 1027 1026 MS, RI 1,8-Cineole 1.5 0.8 0.9 1033 1032 MS, RI Z-β-Ocimene 0.1 1049 1051 MS, RI 2,6-dimethyl-5-Heptenal 0.2 0.2 0.3 1050 1054 MS, RI -Terpinene 0.1 0.1 1085 1086 MS, RI Terpinolene 0.1 1093 1095 MS, RI Linalool 0.4 0.6 0.7 1102 1103 MS, RI Isoamyl isovalerate 0.1 1104 1106 MS, RI Z-Rose oxide 0.1 1.0 0.1 1120 1122 MS, RI E-Rose oxide 0.4 1143 1144 MS, RI Neo-Isopulegol 7.8 31.5 15.2 1150 1148 MS, RI Citronellal 52.8 28.4 48.8 1152 1155 MS, RI iso- Isopulegol 3.0 15.7 3.9 1188 1186 MS, RI α-Terpineol 0.3 0.1 0.1 1192 1190 MS, RI Methyl salicylate 0.5 1224 1223 MS, RI Citronellol 20.0 11.8 4.5 1246 1249 MS, RI Piperitone 0.6 1256 1257 MS, RI Methyl citronellate 0.2 1268 1274 MS, RI Neo-Isopulegyl acetate 0.3 0.2 0.3 1301 1299 MS, RI Isoascaridole 0.1 1330 1335 MS, RI -Elemene 0.1 1349 1350 MS, RI Citronellyl acetate 9.0 3.5 5.8 1354 1356 MS, RI Eugenol 0.6 1384 1389 MS, RI β-Elemene 0.4 1390 1393 MS, RI 2-Phenylethyl isobutanoate 0.1 1391 1392 MS, RI Z-Jasmone 0.2 0.1 1412 1417 MS, RI E-Caryophyllene 0.4 1.6 9.2 1435 1442 MS, RI 6,9-Guaiadiene 0.1 1445 1452 MS, RI α-Humulene 0.5 1452 1458 MS, RI allo-Aromandendrene 0.1 1478 1482 MS, RI Citronellol isobutanoate 0.1 1485 1500 MS, RI Bicyclogermacrene 0.1 1568 1577 MS, RI Spathulenol 0.1 1576 1582 MS, RI Caryophyllene oxide 0.2 Yield 4.6 1.5 Total identified 97.1 97.0 96.5 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.9. Eucalyptus tereticornis Sm. (Myrtaceae) E. tereticornis essential oil was extracted with a yield of 1.0% (Table 18, Table 22). This yield is lower than the one obtained from leaves of E. tereticornis (3.4%) isolated in Nigeria [176]. In our study, this oil was characterized by the presence of p-cymene (16.7%), cryptone (11.4%), spathulenol (13.5%), caryophyllene oxide (14.2%). Furthermore, compounds such as 4-terpineol (4.4%), phellandral (4.2%), cumin aldehyde (3.1%), β-phellandrene (2.9%), 1,8-cineole (2.2%) and humulene epoxide II (2.2%) were detected in significant amounts (Table 18). p-Cymene, β-phellandrene, 1,8-cineole, 4-terpineol, cryptone and spathulenol were also identified in the oil of E. tereticornis analyzed in Benin, by Alitonou et al. [175] but in different amounts. Other differences were that it did not contain cumin aldehyde, humulene epoxide II and phellandral, whereas α-phellandrene, bicyclogermacrene and α-, β- or γ- isomers of eudesmol were detected. The presence of these main compounds was also noticed in the oil extracted in Argentina [178, 263]. In contrast, the major constituents of the fresh leaf oil analyzed in India and Ethiopia were α-pinene and 1,8-cineole [170, 264], whereas the main compounds in the essential oil from Nigeria were α- and β-pinene [176], the one from Cuba were characterized by 1,8-cineole and p-cymene [179], and the one from Algeria contained α-pinene, 1,8-cineole, β-ocimene, allo- aromadendrene and 4-terpineol [177]. In addition, the sample from Congo contained -pinene (8.3%), p-cymene (28.6%), cryptone (17.8%), -terpineol (5.6%) and cuminaldehyde [145].

Table 18: Chemical composition and essential oil yield of Eucalyptus tereticornis

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % %

918 924 MS, RI -Thujene 1.7

925 932 MS, RI -Pinene 0.7

964 969 MS, RI Sabinene 0.5

968 974 MS, RI β-Pinene 0.1

982 988 MS, RI Myrcene 0.3

998 1002 MS, RI α-Phellandrene 0.5

1009 1014 MS, RI α-Terpinene 0.5

1018 1020 MS, RI p-Cymene 16.7

1021 1025 MS, RI β-Phellandrene 2.9

1023 1026 MS, RI 1,8-Cineole 2.2

1049 1054 MS, RI -Terpinene 0.7

1080 1086 MS, RI Terpinolene 0.1

1081 1089 MS, RI p-Cymenene 0.3

1093 1095 MS, RI Linalool 0.1

1109 1112 MS, RI E-Thujone 0.2

1114 1118 MS, RI Z-p-Menth-2-en-1-ol 0.5

1132 1136 MS, RI E-p-Menth-2-en-1-ol 0.4

1145 1148 MS, RI Citronellal 0.1

1150 1154 MS, RI Sabina ketone 0.1

1166 1167 MS, RI Umbellulone 0.1

1170 1174 MS, RI Terpinen-4-ol 4.4

1181 1183 MS, RI Cryptone 11.4

1183 1186 MS, RI -Terpineol 0.5

1200 1192 MS, RI -Phellandrene Epoxide 0.2

1223 1224 MS, RI m-Cumenol 0.5

1232 1238 MS, RI Cumin aldehyde 3.1

1236 1239 MS, RI Carvone 0.1

1239 1244 MS, RI Carvotanacetone 0.1

1246 1249 MS, RI Piperitone 0.2

1267 1273 MS, RI p-Menth-1-en-7-al 4.2

1276 1283 MS, RI -Terpinen-7-al 0.3

1296 1298 MS, RI Carvacrol 1.4

1301 1289 MS, RI Cymen-7-ol 0.3

1326 1330 MS, RI 3-Oxo-p-menth-1-en-7-al 0.2

1382 1389 MS, RI β-Elemene 0.1

1408 1417 MS, RI E-Caryophyllene 0.5

1443 1452 MS, RI -Humulene 0.3

1450 1458 MS, RI allo-Aromadendrene 0.9

1513 1521 MS, RI E-Calamenene 0.2

1570 1577 MS, RI Spathulenol 13.5

1574 1582 MS, RI Caryophyllene oxide 14.2

1592 1590 MS, RI Globulol 0.6

1598 1608 MS, RI Humulene epoxide II 2.2

1631 1631 MS, RI Isospathulenol 1.6

1643 1639 MS, RI Caryophylla-4(12),8(13)-dien-5-β-ol 0.9

1648 1652 MS, RI -Cadinol 0.3

1729 1733 MS, RI Isobicyclogermacrenal 0.8

1753 1759 MS, RI Cyclocolorenone 0.6

Yield 1.04

Total identified 92.3 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.10. Guiera senegalensis J.F. Gmel. (Combretaceae) The analysis of the volatile profile of G. senegalensis was done by SDE/GC-MS and SPME/GC-MS since by hydrodistillation no essential oil was obtained. neo-Isopulegol (24.9%), iso-isopulegol (13.4%), methyl salicylate (17.3%), and piperitone (29.7%) have been detected as main compounds by SDE/GC-MS and α- terpinene (17.4%), p-cymene (16.5%), citronellal (15.5%), limonene (9.1%), δ-2- carene (8.8%), neo-isopulegol (7.5%) were identified by SPME/GC-MS (Table 19, Table 22).

Table 19: Chemical composition and essential oil yields of Guiera senegalensis

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % % 923 924 MS, RI α-Thujene 0.1 930 932 MS, RI α-Pinene 0.1 969 969 MS, RI Sabinene 0.1 973 974 MS, RI β-Pinene 0.3 984 981 MS, RI 6-Methyl-5-hepten-2-one 0.2 987 988 MS, RI Myrcene 2.4 999 1002 MS, RI δ-2-carene 0.3 8.8 1003 1002 MS, RI α-phellandrene 0.3 1010 1007 MS, RI Isoamyl isobutyrate 0.1 1014 1014 MS, RI α-Terpinene 17.4 1022 1020 MS, RI p-Cymene 1.7 16.5 1025 1024 MS, RI Limonene 0.4 9.1 1028 1026 MS, RI Eucalyptol (1,8-Cineole) 0.8 1033 1032 MS, RI Z-β-Ocimene 0.2 1043 1044 MS, RI E-β-Ocimene 0.1 1050 1051 MS, RI 2,6-Dimethyl-5-heptenal 0.2 1054 1054 MS, RI -Terpinene 0.5 1085 1086 MS, RI Terpinolene 0.2 1087 1089 MS, RI p-Cymenene 0.3 0.1 1098 1095 MS, RI Linalool 0.3 0.2 1109 1106 MS, RI Z-Rose Oxide 0.1 1119 1118 MS, RI Z-p-menth-2-en-1-ol 0.3 1133 1133 MS, RI Z-p-Mentha-2,8-dien-1-ol 0.1 1141 1144 MS, RI neo-Isopulegol 24.9 7.5 1145 1145 MS, RI Isopulegol 0.1 1148 1148 MS, RI Citronellal 1.2 15.5 1152 1155 MS, RI iso-Isopulegol 13.4 4.3 1165 1167 MS, RI Neoiso-Isopulegol 0.4 0.2 1179 1178 MS, RI Naphthalene 0.1 1180 1187 MS, RI E-p-Mentha-1(7),8-dien-2-ol 1.0 1187 1186 MS, RI α-Terpineol 0.6 0.1 1191 1190 MS, RI Methyl salicylate 17.3 2.6 1225 1223 MS, RI Citronellol 4.4 0.4 1234 1235 MS, RI Neral 0.4 0.4 1250 1249 MS, RI Piperitone 29.7 3.5 1266 1264 MS, RI Geranial 0.2 1271 1275 MS, RI Isopulegyl acetate 0.1 1301 1299 MS, RI Isoascaridole 3.9 1348 1350 MS, RI Citronellyl acetate 2.1 0.6 1385 1389 MS, RI β-Elemene 0.1 1411 1417 MS, RI E-caryophyllene 1.1 0.3 Yield 0.03

Total identified 99.5 98.1 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.11. Securidaca longepedunculata Fresen. (Polygalaceae) The essential oil of S. longepedunculata was extracted by hydrodistillation with a yield of 0.4% (w/w). This yield is similar to the result obtained (0.30-0.52%) by Alitonou et al. [188] in Benin and Adebayo et al. in Nigeria [187]. The S. longepedunculata essential oil was characterized by only one major constituent, namely methyl salicylate (98.9%) (Table 20). Indeed, Nebie et al. [190] has shown that the essential oil of S. longepedunculata from Burkina-Faso contains only one compound which is methyl salicylate. The same compound was also found in the methanol extract of S. longepedunculata from Ghana [189] and in essential oil extracted in Nigeria and Benin [187, 188]. When characterized by SDE/GC-MS, the chemical profile of S. longepedunculata, identified in the current work, is composed mainly of methyl salicylate (92.9%), whereas by SPME/GC-MS the profile identified is composed of methyl salicylate (35.3%), citronellal (29.2%), piperitone (11.2%) and neo-isopulegol (6.9%) (Table 20, Table 22).

Table 20: Chemical composition and essential oil yields of Securidaca longepedunculata

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % % 973 974 MS, RI β-Pinene 0.1 985 981 MS, RI 6-Methyl-5-hepten-2-one 0.1 987 988 MS, RI Myrcene 0.4 999 1001 MS, RI -2-Carene 0.9 1013 1014 MS, RI α-Terpinene 1.1 1021 1020 MS, RI p-Cymene 1.3 1024 1024 MS, RI Limonene 0.8 1027 1026 MS, RI 1,8-Cineole 0.4 1050 1051 MS, RI 2,6-Dimethyl-5-heptenal 0.1 1054 1054 MS, RI -Terpinene 0.1 1085 1086 MS, RI Terpinolene 0.1 1098 1095 MS, RI Linalool 0.2 1102 1100 MS, RI Nonanal 0.1 1119 1118 MS, RI Z-p-Menth-2-en-1-ol 0.3 1141 1144 MS, RI Neo-Isopulegol 1.9 6.9 1145 1145 MS, RI Isopulegol 0.9 0.1 1151 1148 MS, RI Citronellal 1.0 29.2 1153 1155 MS, RI Iso-Isopulegol 3.1 1165 1167 MS, RI Neo-Iso-Isopulegol 0.3 1173 1173 MS, RI Rosefuran epoxide 0.1 1178 1178 MS, RI Naphthalene 0.1 1187 1186 MS, RI α-Terpineol 0.1 1195 1190 MS, RI Methyl salicylate 98.9 92.9 35.3 1237 1235 MS, RI Neral 0.2 1250 1249 MS, RI Piperitone 1.1 11.2 1268 1264 MS, RI Geranial 0.2

1302 1301 MS, RI Isoascaridole 1.3 1348 1350 MS, RI Citronellyl acetate 0.5 1384 1389 MS, RI β-Elemene 0.3 1411 1417 MS, RI E-Caryophyllene 0.2 2.2 1419 1424 MS, RI 2,5-Dimethoxy- p-cymene 0.2 1423 1431 MS, RI β-Gurjunene 0.1 1428 1432 MS, RI α-E-Bergamotene 0.1 1439 MS Methyl 4-methoxysalicylate 1.0 0.2 1445 1452 MS, RI α-Humulene 0.2 1473 1471 MS, RI Dauca-5,8-diene 0.1 1487 1492 MS, RI Z-β-Guaiene 0.1 1503 1514 MS, RI Butylated hydroxytoluene 0.3 1507 1513 MS, RI - Cadinene 0.1 1517 1522 MS, RI - Cadinene 0.1 1587 MS 3-Tetradecanone 1.0 0.2 Yield 0.4 0.1

Total identified 99.9 99.5 98.3 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

4.1.12. Spondias mombin L. (Anarcardiaceae) The analysis of the volatile profile of S. mombin was done by SDE/GC-MS and SPME/GC-MS since by hydrodistillation no essential oil was obtained. Piperitone (53.2%), neo-isopulegol (12.8%), methyl salicylate (9.0%), iso-isopulegol (7.7%), and p-cymene (17.4%), -2-carene (12.4%), α-terpinene (12.1%), limonene (8.4%), neo- isopulegol (7.9%), iso-isopulegol (7.1%) and myrcene (7.0%) were the main compounds identified, respectively, by SDE/GC-MS and SPME/GC-MS (Table 21, Table 22).

Table 21: Chemical composition and essential oil yields of Spondias mombin

a b c d KI exp KI lit ID Compounds Hydro. SDE SPME % % % 923 924 MS, RI α-Thujene 0.1 930 932 MS, RI α-Pinene 0.2 973 974 MS, RI β-Pinene 0.8 983 988 MS, RI 6-Methyl-5-hepten-2-one 1.8 987 988 MS, RI Myrcene 7.0 999 1001 MS, RI -2-Carene 12.4 1003 1002 MS, RI α-Phellandrene 0.3 1013 1014 MS, RI α-Terpinene 12.1 1021 1020 MS, RI p-Cymene 0.4 17.4 1025 1024 MS, RI Limonene 8.4 1027 1026 MS, RI 1,8-Cineole 0.8 1033 1032 MS, RI Z-β-Ocimene 0.6 1043 1044 MS, RI E-β-Ocimene 0.2 1054 1054 MS, RI -Terpinene 0.7 1084 1086 MS, RI Terpinolene 0.4 1086 1089 MS, RI p-Cymenene 0.2 1108 1106 MS, RI Z-Rose oxide 0.1 1118 1118 MS, RI Z-p-2-Menthenol 0.1 1141 1144 MS, RI neo-Isopulegol 12.8 7.9 1148 1148 MS, RI Citronellal 4.2 1151 1155 MS, RI iso-Isopulegol 7.7 7.1 1173 1167 MS, RI Neoiso-Isopulegol 0.1 1183 1186 MS, RI -Terpineol 0.8

1191 1190 MS, RI Methyl salicylate 9.0 2.0 1222 1223 MS, RI Citronellol 0.7

1231 1232 MS, RI Thymol, methyl ether 0.1 1250 1249 MS, RI Piperitone 53.2 5.4 1301 1299 MS, RI Isoascaridole 2.0 1368 1374 MS, RI α-Copaene 0.1 1384 1389 MS, RI β-Elemene 0.6 0.1 1411 1417 MS, RI E-Caryophyllene 2.7 3.6 1420 1424 MS, RI 2,5-dimethoxy-p-Cymene 0.8 0.1 1428 1432 MS, RI α-E-Bergamotene 0.1 1445 1452 MS, RI α-Caryophyllene 0.2 1473 1478 MS, RI -Muurolene 0.1 1478 1475 MS, RI -Gurjunene 1.0 0.1 1487 1489 MS, RI β-Selinene 0.2 1507 1513 MS, RI - Cadinene 2.5 1517 1522 MS, RI -Cadinene 1.0 0.1 1540 1548 MS, RI Elemol 0.7 1581 MS 3-Tetradecanone 3.7 Yield 0.07

Total identified 97.6 97.1 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold. Extraction methods: Hydro. = hydrodistillation; SDE = simultaneous distillation extraction; SPME = solid phase microextraction.

Table 22: Main compounds identified by hydrodistillation, SDE and SPME coupled to GC-MS Main compounds by Main compounds by Main compounds by Plant species % % % Hydrodistillation/GC-MS SDE/GC-MS SPME/GC-MS Chenopodium Ascaridole 41.9 -Terpinene 53.5 Ascaridole 47.3 ambrosioides -Terpinene 16.5 p-Cymene 19.7 Isoascaridole 33.3 p-Cymene 14.4 E-p-Mentha-1(7),8-dien-2-ol 5.5 Isoascaridole 7.47 Cochlospermum 3-Tetradecanone 24.7 Piperitone 38.1 Piperitone 20.9 planchonii Isoamyl dodecanoate 14.1 3-Tetradecanone 12.0 Citronellal 12.9 Ethyl tetradecanoate 11.4 neo-Isopulegol 6.5 3-Tetradecanone 10.4 2-Tridecanone 6.8 2-Tridecanone 4.5 neo-Isopulegol 6.4 2-Pentadecanone 5.7 Dodecyl acetate 4.9 Cochlospermum 3-Tetradecanone 48.3 3-Tetradecanone 54.5 3-Tetradecanone 42.4 tinctorium Cyclododecanone 7.8 neo-Isopulegol 10.1 α-Terpinene 13.1 3-Hexadecanone 7.4 Iso-Isopulegol 6.2 p-Cymene 11.3 3-Hexadecanone 5.2 Cymbopogon Geranial 44.3 Geraniol 41.2 Geranial 23.9 citratus Neral 33.1 Neral 23.6 Neral 19.7 Myrcene 12.4 Selina-6-en-4-ol 7.2 E-Caryophyllene 5.0 Cymbopogon E-p-Mentha-1(7),8-dien-2-ol 19.6 E-p-Mentha-1(7),8-dien-2-ol 28.3 E-p-Mentha-1(7),8-dien-2-ol 22.8 giganteus E-p-Mentha-2,8-dienol 19.3 Z-p-Mentha-1(7),8-dien-2-ol 17.3 E-p-Mentha-2,8-dienol 17.7 Z-Carveol 17.0 E-p-Mentha-2,8-dienol 15.3 Z-p-Mentha-1(7),8-dien-2-ol 15.9 Z-p-Mentha-2,8-dienol 10.2 Z-p-Mentha-2,8-dienol 10.4 Z-p-Mentha-2,8-dienol 6.8 Limonene 9.6 Isoascaridole 6.8 E-Carveol 6.0 Cymbopogon Piperitone 58.9 Piperitone 53.1 Piperitone 56.6 schoenanthus δ-2-Carene 15.5 Elemol 9.3 E-Caryophyllene 6.5 Elemol 5.3 β-Elemene 5.1 Erythrophleum Citronellal 23.0 -Terpinene 25.8 suaveolens Piperitone 22.5 p-Cymene 18.1 Neo-Isopulegol 11.0 -2-Carene 13.4 Methyl salicylate 8.4 Citronellal 9.6 iso-Isopulegol 8.3 Limonene 8.9 3-Tetradecanone 6.8 Myrcene 6.3 Eucalyptus Citronellal 52.8 neo-Isopulegol 31.5 Citronellal 48.8 citriodora Citronellol 20.0 Citronellal 28.4 Neo-Isopulegol 15.2 Neo-Isopulegol 7.8 iso- Isopulegol 15.7 E-Caryophyllene 9.2 Citronellyl acetate 9.0 Citronellol 11.8 Citronellyl acetate 5.8 Eucalyptus p-Cymene 16.7 tereticornis Caryophyllene oxide 14.2 Spathulenol 13.5 Cryptone 11.4 Guiera senegalensis neo-Isopulegol 24.9 α-Terpinene 17.4 iso-Isopulegol 13.4 p-Cymene 16.5 Methyl salicylate 17.3 Citronellal 15.5 Piperitone 29.7 Limonene 9.1 δ-2-Carene 8.8 neo-Isopulegol 7.5 Securidaca Methyl salicylate 98.9 Methy lsalicylate 92.9 Methyl salicylate 35.3 longepedunculata Citronellal 29.2 Piperitone 11.2 neo-Isopulegol 6.9 Spondias mombin Piperitone 53.2 Myrcene 7.0 neo-Isopulegol 12.8 -2-Carene 12.4 Methyl salicylate 9.0 α-Terpinene 12.1 iso-Isopulegol 7.7 p-Cymene 17.4 Limonene 8.4 neo-Isopulegol 7.9 iso-Isopulegol 7.1

4.1.13. Analysis of the chemical compositions 4.1.13.1. Influence of extraction methods on chemical composition A representative characterization of the volatile chemical composition of plant species or other material depends on the suitable choice of the sample preparation method, because of the specificity of the material, for instance the presence of thermally or chemically labile analytes [58]. Hydrodistillation is still commonly used to extract essential oils with the aim to determine their volatile profiles and in fact provides chemical profiles including terpenoids and aliphatic compounds [58]. This method is solvent free, which is a good benefit but is time-consuming and requires a lot of plant material. In contrast, by SDE or the Licken-Nickerson method, extraction is realized using a solvent (diethyl ether in the current work), and essential oil cannot be obtained without the concentration step utilizing a rotavapor. This last step is a disadvantage because of the possibility of losing some volatiles during the concentration process [265]. However, SDE permits to extract the main compounds and to isolate them if needed. Besides the above, two variables must be taken into account to minimize the alteration of the volatile composition, which are the duration of the extraction to avoid thermal isomerization and the choice of the solvent by selecting one which could provide the extraction of a wide range of volatiles [69]. Indeed non-polar compounds should be extracted by a non-polar solvent such as pentane or hexane and more polar compounds by a more polar solvent such as diethyl ether. The choice for diethyl ether was made because it is a very common solvent widely used for extraction of volatiles; because of its low boiling point and good solvent properties for most flavor compounds. The low boiling point of the solvent is an advantage during the concentration step to avoid the loss of volatiles with lower boiling points [266]. Compared to distillation methods, SPME has several advantages including the avoidance of loss of volatile compounds that occurs during distillation or concentration processes, the absence of solvent, the simplicity, the rapidity, the automatization of the analysis and the minimizing of the sample preparation method. Moreover, it can be used to analyze several matrices such as solid or liquid [68, 69], in vitro and in vivo [70-72], for the determination of volatiles in environmental samples [70, 73, 74], to identify contaminants in food [57, 75-77], phenolic compounds in foods [267] as well as volatiles in essential oils [69]. SPME enables us to obtain an extensive chemical profile including several low-boiling and minor compounds

whereas in SDE profiles more compounds are present with lower volatility and higher molecular weight like noticed in some previous works [69, 265, 268]. Despite these advantages, the choice for SPME to analyze a matrix should be done by taking into account some parameters including the choice of the SPME fiber coating, the amounts and the polarity of compounds inside the mixture which is to be analyzed [253]. To have an idea on the composition of the mixture by SPME, a preliminary analysis should be done using a suitable fiber. Different SPME fiber coatings which are commonly used are PDMS (polydimethyl siloxane), DVB/PDMS (divinylbenzene/polydimethyl siloxane), CARB-PDMS (Carboxen/polydimethyl siloxane) and PA (polyacrylate) [253]. 100 µm PDMS coated fiber is useful for low molecular volatile compounds (MW 60-275) and 7 µm or 30 µm PDMS fiber is suitable for extraction of non polar higher molecular weight compounds (MW 125- 600) or non polar semi-volatile compounds (MW 80-500) while the very polar semi- volatile compounds (MW 80-300) are more effectively extracted with 85 µm PA coated fiber [254]. A 65 µm PDMS/DVB fiber is efficient for the extraction of more volatile polar analytes, such as alcohols or amines (MW 50-300) [254, 269, 270] whereas 75 µm /85 µm PDMS/CARB fiber is suitable for gases, low molecular weight compounds (MW 30-225), for compounds with lower Kovats index [254, 271, 272], but also non-polar compounds with high molecular weight [273]. The analysis of mixtures with a large range of polar and non-polar analytes (C3-C20), including compounds with higher Kovats indexes should be analyzed using a 50/30 µm PDMS/DVB/CARB fiber [254, 272]. In the current work a 50/30 µm PDMS/DVB/CARB fiber has been used and has confirmed the efficiency of this fiber to identify volatile compounds in essential oils. A PDMS/DVB/CARB fiber has also been used by Vichi et al. to identify aroma compounds in olive oil [274] and by Kraujalyte et al. for the characterization of volatiles in Aronia melanocarpa [268] and has revealed profiles with the largest range of volatiles in the matrix.

4.1.13.2. Influence of extraction conditions on the chemical composition The conditions of extraction could provoke some changes in the chemical composition. Indeed, in the essential oil of Chenopodium ambrosioides, there is a correlation between the amount of -terpinene+p-cymene, and the amount of ascaridole+isoascaridole (Table 10, fig 27), because -terpinene+p-cymene are

primary metabolites which could be oxidised into ascaridole or isoascaridole [275]. Moreover, in a pentane extract of Chenopodium ambrosioides obtained by cold extraction, the amount of -terpinene+p-cymene decreased, whereas the amount of ascaridole+isoascaridole increased. The decrease of the amount of isoascaridole is accompanied by the presence of oxygenated compounds such as piperitone, piperitone oxide, carvenone oxide and ascaridole glycol [275]. The decrease of the amount of -terpinene+p-cymene is possibly due to oxidation of these compounds into ascaridole+isoascaridole, which was promoted by the presence of the solvent (pentane).

Figure 27: Chemical transformation of -terpinene during hydrodistillation [275] Because of the presence of protons coming from the hot water and the high temperature of extraction during hydrodistillation or SDE, isoascaridole is isomerized and hydrolyzed into other compounds such as piperitone oxide and carvenone oxide, which explained the low amount of this compound in the essential oil extracted from C. ambrosioides [275].

4.1.13.3. Influence of climatic conditions on the chemical composition The chemical composition can be also modified by climatic conditions (rainfall, temperature, soil) of the area where the plant material was collected. Indeed, the chemical composition of the essential oil of C. ambrosioides collected in different areas of Benin is summarized in Table 23.

Table 23: Variation of chemical composition of Chenopodium ambrosioides essential oil obtained by hydrodistillation by climatic conditions

Area 1: Area 2: Area 3: a b c d KI exp KI lit ID Compounds Savalou Dogbo Gankpintin % % % 987 988 MS, RI Myrcene 0.2 0.1

999 1001 MS, RI -2-Carene 0.1 0.1 1004 1008 MS, RI -3-Carene 0.1 0.1 1010 1014 MS, RI -Terpinene 16.5 42.4 26.8 1018 1020 MS, RI p-Cymene 14.4 17.5 19.4 1021 1024 MS, RI Limonene 0.4 0.9 0.6 1050 1054 MS, RI -Terpinene 0.3 1.1 0.6 1117 1119 MS, RI E-p-Mentha-2,8-dienol 0.2

1145 1148 MS, RI Citronellal 0.1

1175 1178 MS, RI Naphthalene 0.1

1080 MS, RI 0.2 1086 Terpinolene 1179 MS, RI 0.2 1179 p-Cymen-8-ol 1183 1186 MS, RI -Terpineol 0.1 0.1 1235 1234 MS, RI Ascaridole 41.9 25.7 33.7 1247 1252 MS, RI E-Piperitone epoxide 1.1 0.4 0.7 1288 1289 MS, RI Thymol 0.4 0.1

1299 1299 MS, RI Isoascaridole 7.47 7.0

1347 1349 MS, RI Thymol acetate 0.2

1477 MS E-β-Ionone 0.1

1581 MS 3-Tetradecanone 0.4

Total identified 83.6 88.8 82.4 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; the names and the percentages of main compounds are indicated in bold.

The same compounds have been identified (Table 24). However, their relative amounts were variable. Indeed, concerning the major compounds a decreasing amount of -terpinene+p-cymene was noticed whereas the amount of ascaridole+isoascaridole increased, as indicated by Alitonou [275]. These data confirm the hypothesis that -terpinene+p-cymene are primary metabolites, which can be oxidized into ascaridole or isoascaridole.

The low amount of -terpinene+p-cymene, can be linked to the different climatic conditions that occur in these three areas. Indeed Savalou and Gankpintin are two close locations were climatic conditions especially rainfall and soil are the same compared to Dogbo where different climatic conditions occur.

Table 24: Location, soil and relative variation of amount of major compounds of Chenopodium ambrosioides Localities Dogbo Gankpintin Savalou Soil Sandy Ferruginious Ferruginious Major compounds

-terpinene+p-cymene 59.9 46.2 30.9 Ascaridole+isoascaridole 32.7 33.7 49.4

Sub-total 92.6 79.9 80.3

4.1.13.4. Variability of the chemical composition of the duplicates Few modifications can be observed in the amount of chemical compounds during analysis, but these differences are not important. Indeed, the amounts of chemical compounds of C. tinctorium obtained by SPME revealed a very low standard error ranged from 0.0 to 2.5 (Table 25).

Table 25: Variability of the chemical composition of Cochlospermum tinctorium by SPME analysis Duplie Duplie Duplie Duplie Mean a b c d Standard KIexp KIlit ID Compounds 1 2 3 4 error % % % % % 973 974 MS, RI β-Pinene 0.1 0.1 0.1 0.1 0.1 0.0 987 988 MS, RI β-Myrcene 1.2 1.0 1.1 1.1 1.1 0.0 999 1001 MS, RI δ-2-Carene 3.7 3.7 5.2 4.6 4.3 0.4 1003 1002 MS, RI α-Phellandrene 0.1 0.1 0.2 0.2 0.1 0.0 1014 1014 MS, RI α-Terpinene 19.0 14.6 8.8 9.9 13.1 2.3 1021 1020 MS, RI p-Cymene 14.9 13.1 7.7 9.7 11.3 1.6 1025 1024 MS, RI Limonene 5.2 4.3 3.6 3.9 4.2 0.4 1028 1026 MS, RI 1,8-Cineole 0.2 0.2 0.1 0.3 0.2 0.0 1033 1032 MS, RI Z-β Ocimene 0.2 0.2 0.2 0.3 0.2 0.0 1054 1054 MS. RI -Terpinene 0.4 0.3 0.2 0.3 0.3 0.0 1085 1086 MS, RI Terpinolene 0.1 0.1 0.1 0.2 0.1 0.0 1087 1089 MS, RI p-Cymenene 0.1 0.2 0.2 0.2 0.2 0.0 1141 1144 MS, RI Neo-Isopulegol 0.5 0.4 1.6 2.3 1.2 0.4 1149 1148 MS, RI Citronellal 0.4 0.0 0.1 0.3 0.2 0.1 1152 1155 MS, RI Iso-Isopulegol 0.50 0.31 1.1 1.8 0.9 0.3 1191 1190 MS, RI Methyl salicylate 3.0 2.8 1.4 1.7 2.2 0.4 1251 1249 MS, RI Piperitone 0.1 0.1 1.6 0.9 0.7 0.4 1301 1299 MS, RI Isoascaridole 0.4 0.0 0.3 0.4 0.3 0.1

1384 1389 MS. RI β-Elemene 0.1 0.1 0.2 0.1 0.1 0.0 1390 1398 MS. RI Cyperene 0.2 0.2 0.2 0.2 0.2 0.0 1403 1410 MS. RI α-Cedrene 0.2 0.2 0.3 0.2 0.2 0.0 1408 1411 MS. RI α-Z-Bergamotene 0.3 0.3 0.3 0.3 0.3 0.0 1411 1417 MS. RI E-Caryophyllene 0.5 0.5 0.7 0.6 0.6 0.0 1419 1424 MS. RI 2.5-dimethoxy-p-Cymene 0.1 0.1 0.1 0.1 0.1 0.0 1428 1432 MS. RI α-E-Bergamotene 1.9 2.1 2.1 2.2 2.1 0.1 1440 1445 MS. RI Epi-β-Santalene 0.3 0.3 0.3 0.3 0.3 0.0 1489 1495 MS. RI 2-Tridecanone 1.6 1.9 2.2 1.9 1.9 0.1 1502 1505 MS. RI β-Bisabolene 3.5 3.9 4.1 3.9 3.8 0.1 1505 1506 MS. RI α-Bisabolene 0.1 0.1 0.1 0.1 0.1 0.0 1508 1514 MS. RI Z--Bisabolene 0.3 0.3 0.4 0.4 0.4 0.0 1517 1522 MS. RI - cadinene 0.1 0.1 0.2 0.1 0.1 0.0 1525 1529 MS. RI E--Bisabolene 0.8 0.9 1.0 0.9 0.9 0.0 1580 MS 1-Hydroxyundecan-3-one 0.2 0.2 0.3 0.2 0.2 0.0 1587 MS 3-Tetradecanone 35.6 42.0 47.3 44.6 42.4 2.5 1607 1609 MS. RI Tetradecanal 0.3 0.5 0.6 0.5 0.5 0.1 1786 MS 3-Hexadecanone 1.2 1.8 2.2 1.8 1.8 0.2 Total identified 97.4 97.0 95.9 96.6 96.7 0.3 a KIexp = retention indices are determined using n-alkanes (C7-C17). bKIlit = retention indices of reference compounds from literature. cID = Identification methods; MS = comparison of the mass spectrum with those of the computer mass libraries, and Adams (2007); RI = comparison of calculated RI with those reported in the literature. dCompounds are listed in order of their retention time; e = Duplicate

4.2. Ethanolic extracts The yields of ethanolic extracts of the different plant species ranged between 9.1- 28.4% are summarized in Table 26.

Table 26: Yield of ethanolic extracts

Plant CC CG Cs Cp CT CA EC ET ES Gs SLa SLb Sm species Yield 22.6 11.9 19.6 11.9 10.6 10.0 16.2 ND 20.0 20.8 19.4 9.1 28.4 (%) CC = C. citratus; CG = C. giganteus; Cs = Cymbopogon schoenanthus; Cp = C. planchonii ; CT = C. tinctorium; CA= C. ambrosioides; EC = E. citriodora; ET = E. tereticornis; ES = E. suaveolens; SL = S. longepedunculata; Sm = Spondias mombin; Gs = Guiera senegalensis; a= root bark ; b = root

4.3. Insecticidal tests on Anopheles gambiae 4.3.1. Adult bioassay on susceptible strain "Kisumu" of Anopheles gambiae The resistant status of mosquito samples was determined according to the WHO criteria summarized as follows [50]: - 98-100% mortality indicates susceptibility of the mosquito strain to the tested essential oil;

- Mortality less than 98% is suggestive of the existence of a resistance to the essential oil that needs to be confirmed by two additional tests; - Mortality less than 90% suggests resistance in the mosquito population. The results are presented in Table 27.

4.3.2. KDT50 and KDT95

KDT50 and KDT95 were calculated with 95% confidence limits and are summarized in

Table 28. The lowest KDT50 and KDT95 values were obtained with C. citratus and are 2.1 min and 13.9 min at 0.5%, respectively, and 1.2 min, 6.6 min at 1%, respectively, whereas for permethrin (0.75%) these values were 11.3 min and 21.6 min, respectively. The highest KDT50 and KDT95 values were recorded with C. planchonii at 8% and were 11.3 min and 20.6 min, respectively.

4.3.3. Mortality rates Mortality rates to different essential oils are shown in Table 27. At 0.25%, the mortality rate of A. gambiae “Kisumu” varies from 0.0% to 72.5%. However, the mortality rates increased with the dosage. At 0.50%, mortality has reached 100% for C. citratus, whereas it was at 5.6% for S. longepedunculata. At 1%, mortality was still 100% for C. citratus whereas it was 6.7% for C. schoenanthus. At 2%, the mortality rate was 29.6% for S. longepedunculata and 100% for C. citratus, E. citriodora, E. tereticornis and C. ambrosioides. At 4% and 8%, the mortality rates varied from 79.2% to 100% for C. tinctorium and C. planchonii. To summarize these results, the susceptibility tests on the sensitive strain A. gambiae “kisumu” have demonstrated its susceptibility status on essential oils tested. The most efficient essential oil was C. citratus at 0.50%, followed by E. tereticornis at 1%, E. citriodora and C. ambrosioides at 2%, C. schoenanthus, C. giganteus, C. planchonii and S. longepedunculata at 4%.

Table 27: Knock down times (KDT), mortality and susceptibility of essential oils on sensitive Anopheles gambiae “Kisumu”

Doses 0.25% 0.50% 1% a a b a a b a a b Essential oils and controls KDT50 (min) KDT95 (min) Mortality (%) Susceptibility KDT50 (min) KDT95 (min) Mortality (%) Susceptibility KDT50 (min) KDT95 (min) Mortality (%) Susceptibility C. citratus 62.0 119.8 55.6 R 2.1 13.9 100 S 1.2 6.6 100 S C. giganteus - - 10.7 R 204.4 366.5 10.0 R 33.5 100.2 29.6 R C. schoenanthus - - 0.0 R 263.5 440.7 6.4 R 49.0 117.8 6.7 R E. citriodora - - 4.3 R 125.2 207.3 10.4 R 32.2 61.7 75.5 R E. tereticornis 12.4 49.8 72.5 R 5.4 18.2 86.7 R 2.5 16.1 98 S C. tinctorium 44.3 115.6 23.3 R 10.6 19.4 44.0 R 8.1 16.7 72.7 R C. planchonii 34.5 74.1 13.0 R 25.1 52.8 23.6 R 20.9 39.9 24.5 R S. longepedunculata - - 5.9 R 314.1 539.5 5.6 R 76.2 91.3 8.6 R C. ambrosioides 736.3 1290.0 9.8 R 127.8 201.3 18.8 R 110.9 178.9 41.9 R Permethrin 0.75% 11.3 21.6 100 S 11.3 21.6 100 S 11.3 21.6 100 S Negative control 0 0 0 - 0 0 0 - 0 0 0 - Doses 2% 4% 8% a a b a a b a a b Essential oils and controls KDT50 (min) KDT95 (min) Mortality (%) Susceptibility KDT50 (min) KDT95 (min) Mortality (%) Susceptibility KDT50 (min) KDT95 (min) Mortality (%) Susceptibility C. citratus - 3.8 100 S - 3.8 100 S - - - - C. giganteus 6.2 9.5 62.7 R 2.6 3.8 100 S - - - - C. schoenanthus 6.9 14.5 83.0 R 2.6 3.8 100 S - - - - E. citriodora 4.5 9.0 100 S 2.6 3.8 100 S - - - - E. tereticornis - 3.8 100 S - 3.3 100 S - - - - C. tinctorium 6.9 12.3 78.1 R 5.2 9.1 79.2 R - - - - C. planchonii 16.3 31.3 58.5 R 12.1 21.1 98.3 S 11.3 20.6 100 S S. longepedunculata 10.9 81.0 29.6 R 2.6 3.8 98.2 S 2.6 3.8 100 S C. ambrosioides 12.8 25.2 100 S 2.6 3.8 100 S - - - - Permethrin 0.75% 11.3 21.6 100 S 11.3 21.6 100 S 11.3 21.6 100 S Negative control 0 0 0 - 0 0 0 - 0 0 0 - a The KDT (KDT50 and KDT95) values were expressed with 95% confidence limits. bS = Susceptible defined as 98-100% of mortality; RS = Resistance suspected defined as 90-97% of mortality; R = Resistance defined as <90% of mortality.

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4.3.4. Diagnostic concentrations The diagnostic concentration is defined as twice the lethal concentration (LC) for 99% mortality (LC99) on sensitive strains [50]. The diagnostic concentration is used to detect the resistant status of Anopheles since after standard bioassay resistance test, any survivors at this concentration is considered as resistant [50].

Lethal concentration for 50% mortality (LC50), lethal concentration for 99% mortality

(LC99) expressed with 95% confidence limits and diagnostic concentrations for all essential oils tested are summarized in Table 28. The lowest diagnostic concentration of 0.77% for C. citratus was not significantly different from the diagnostic dose of permethrin (0.75%). Other interesting values were also obtained for E. tereticornis (2.80%), E. citriodora (3.37%), and C. ambrosioides (4.26%). These plant species were followed by C. schoenanthus and C. giganteus whose diagnostic concentrations were 5.48% and 7.36%, respectively. The highest diagnostic doses were obtained with S. longepedunculata (9.84%), C. tinctorium (11.56%) and C. planchonii (15.22%). All diagnostic doses obtained above were tested on the resistant strain of A. gambiae and results obtained were summarized in Table 29. Concerning the diagnostic doses tested, the lowest KDT50 and KDT95 were observed with C. schoenanthus (2.58 min, 3.84 min), followed by S. longepedunculata, C. giganteus, E. tereticornis, C. ambrosioides and E. citriodora for which the KDT50 and KDT95 were lower than 5 min and 6 min, respectively. A moderate knock down effect (KDT50  12 min and KDT95  20 min) was observed with C. planchonii, C. tinctorium and C. citratus.

The KDT50 observed was 6 to 35 fold lower than for permethrin, the positive control, and the KDT95 was 5 to 38 fold lower than permethrin. This observation demonstrates the promising insecticidal properties of these plants species on the A. gambiae resistant strain used.

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Table 28: LC50, LC99 and diagnostic concentration for all essential oils tested

a a Essential oils LC50 LC99 Diagnostic Diagnostic Diagnostic concentration concentration concentration % % % mg/ml mg/cm2 C. citratus 0.237 0.386 0.77 7.7 0.085 C. giganteus 1.600 3.682 7.36 73.6 0.82 C. schoenanthus 1.570 2.739 5.48 54.8 0.60 E. citriodora 0.900 1.685 3.37 33.7 0.37 E. tereticornis 0.148 1.401 2.80 28.0 0.31 C. tinctorium 1.16 5.781 11.56 115.6 1.28 C. planchonii 2.314 7.608 15.22 152.2 1.69 S. longepedunculata 2.489 4.919 9.84 98.4 1.09 C. ambrosioides 0.997 2.131 4.26 42.6 0.47

LC50: lethal concentration for 50% mortality; LC99: lethal concentration for 99% mortality. a The lethal doses (LC50 and LC95) values were expressed with 95% confidence limits.

Table 29: KDT50, KDT95 and mortality of essential oils tested on the resistant strain of Anopheles gambiae

a a b Essential oils Diagnostic doses KDT50 KDT95 Mortality Susceptibility (%) (min) (min) (%) C. citratus 0.77 15.77 26.00 100 S C. giganteus 7.36 2.92 5.53 100 S C. schoenanthus 5.48 2.58 3.84 100 S E. citriodora 3.37 4.02 5.93 100 S E. tereticornis 2.80 3.56 5.27 100 S C. tinctorium 11.56 12.01 20.67 90.4 RS C. planchonii 15.22 11.35 30.72 100 S S. longepedunculata 9.84 2.87 4.47 94.8 RS C. ambrosioides 4.26 3.96 5.84 98.0 S Permethrin 0.75% 0.75 90.87 145.37 62.3 R

KDT50: 50% knock down in mosquito’s population; KDT95: 95% knock down in mosquito’s population; a The KDT (KDT50 and KDT95) values were expressed with 95% confidence limits. bS = Susceptible defined as 98-100% of mortality; RS = Resistance suspected defined as 90-97% of mortality; R = Resistance defined as <90% of mortality.

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4.3.5. Discussion on the insecticidal test against A. gambiae Apart from the essential oil from C. tinctorium and S. longepedunculata for which resistance was suspected because the mortality was less than 97%, the resistant strain of A. gambiae was susceptible to all essential oils at diagnostic doses tested (Table 28). The resistance to permethrin in southern Benin has been demonstrated and was explained by the massive use of DDT in house spraying and agriculture, during the WHO malaria eradication program, which has permitted the apparition and the increase of kdr mutation in A. gambiae populations in Benin [7, 10]. We have noticed that the knock-down times, the mortalities, and the resistance status of A. gambiae did not only depend on the values of doses used but mainly on the chemical composition of essential oils used.

Larvicidal activity of C. citratus essential oil on Aedes aegypti at lower concentrations

(LC50 = 0.28 µl/ml and LC90 = 0.56 µl/ml) and its major component citral (neral and geranial) has been demonstrated by Freitas et al. [140]. C. citratus has also demonstrated a very good repellency against A. aegypti [30]. In the current study, C. citratus has shown the best insecticidal activity against A. gambiae, but its KDT50 and

KDT90 were higher than some of the other essential oils. The same conclusion has been found by Phasomkusolsil et al. [33] when the essential oil was tested on A. aegypti, C. quinquefasciatus and Anopheles dirus. The topical application of C. citratus showed high toxicity against Sitophilus oryzae [138]. C. citratus essential oil and citral have been shown to be potential anti-Leishmania agents [139]. Insecticidal and larvicidal activities of C. citratus have been attributed to citral that has demonstrated 100% mortality against A. aegypti, at 2.5 µl/ml with LC50 = 0.02 µl/ml and LC90 = 0.28 µl/ml, respectively [140], and its repellent effect at 15% (v/v) is comparable to 5% C. citratus essential oil [276]. In conclusion, the presence of geranial and neral is potentially responsible for the insecticidal activity of the essential oil of C. citratus, as demonstrated in the current work.

C. giganteus essential oil, rich in limonene and (Z and E)-p-mentha-1(7),8-dien-2-ol such as the current sample from Benin, has proven to be toxic by fumigation to Callosobruchus species [28]. The insecticidal properties noticed in this study against A. gambiae might also be attributed to these main compounds.

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Several studies on essential oils, rich in piperitone such as the essential oil from C. schoenanthus have demonstrated insecticidal activity against some pests. This is the case for Cymbopogon olivieri, which demonstrated good larvicidal activity against A. stephensi with LD50 = 321.9 mg/l [277]. Exposure of Callosobruchus maculatus to C. schoenanthus essential oil for 24 hours resulted in 90% of adult mortality at 6.7 µl/l [157]. Piperitone has been reported to be powerful against ants of Crematogaster spp [278]. Piperitone was very toxic to adults of C. maculatus with an LC50 recorded at 1.6  0.14 µl/l versus 2.7  0.2 μl/l for the crude oil. Furthermore, newly laid eggs and neonate larvae, and all eggs were aborted at 6.7 µl/l with a total inhibition of the neonate larvae penetration in the seed [38]. Piperitone isolated from Artemisia judaica L. was studied against the third larvae of Spodoptera littoralis (Boisd) and has revealed a high insecticidal and antifeedant activity against this pathogen, with an

LD50 = 0.68 µg/larvae [279]. Following this previous research, the insecticidal activity of C. schoenanthus could be attributed to its main compound, i.e. piperitone.

The essential oil from E. citriodora, rich in citronellal, citronellol and isopulegol, has been found to be repellent against Tribolium castaneum at 0.084 ml/l and was more active than the commercial product IR3535 at 0.686 ml/l [41]. The insecticidal activity of E. citriodora has been demonstrated at 5 mg/ml against Lutzomyia longipalpis [27]. E. citriodora has also demonstrated larvicidal activity against C. quinquefasciatus [36] and acaricidal activity against larvae of Amblyomma cajennense and Anocentor nitens [172]. The presence of citronellal, citronellol and isopulegol could well explain the insecticidal activity of E. citriodora against A. gambiae.

The essential oil from E. tereticornis leaf extract has shown a larvicidal activity against A. stephensi at 160 ppm which has provoked 100% of oviposition deterrence [35]. The sensitivity of adults of A. aegypti has been shown, resulting from the presence of 1,8-cineole, α-pinene and p-cymene and is correlated to the amount of 1,8-cineole in the extract [26, 178]. The insecticidal activity of the essential oil of E. tereticornis observed in the current work, might be explained by the presence of one of its major components (p-cymene) but also by a minor compound (1,8-cineole), which both have demonstrated insecticidal activity.

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In essential oils from Cochlospermum species one minor compound (2-tridecanone) has been found to have insect repellent properties. Indeed, 2-tridecanone has demonstrated repellent activity against the granary weevil Sitophilus granarius and S. zeamais at 100 ppm and 500 ppm on wheat [280]. Its repellent activity was confirmed against ticks since 0.63 mg/cm2 was repellent to 87% of Amblyomma americanum after 12 hours and to 72% of Dermacentor variabilis after 15 hours [281]. The weak insecticidal activity of the essential oils of these two Cochlospermum species could be due to the low abundancy of 2-tridecanone.

Root powder, the methanol extract, and the main volatile component of S. longepedunculata (methyl salicylate) have proven to exhibit repellent and toxic effects against S. zeamais. In the same study, methyl salicylate has demonstrated a dose dependent fumigant effect with an LD100 of 60 µl in a 1-l container after 24 hours exposure on S. zeamais, Rhyzopertha dominica and Prostephanus truncates and after 6 days exposure, 100% mortality could be recorded with 30 µl in a 1-l container [31].

The C. ambrosioides essential oil has demonstrated a larvicidal activity against A. arabiensis and A. aegypti after 24 hours exposure with LC50 and LC90 equal to 17.5 ppm and 33.2 ppm for A. arabiensis and 9.1 ppm and 14.3 ppm for A. aegypti under laboratory conditions [282]. Contact and fumigant toxicity of isolated compounds from this plant species have shown that ascaridole (LC50 = 0.84 mg/l) followed by isoascaridole (LC50 = 2.45 mg/l) were the most efficient insecticidal compounds by fumigation and contact with LC50 = 0.86 mg/l (ascaridole) and 2.16 mg/l

(isoascaridole). The crude oil was less active with LC50 = 3.08 mg/l by fumigation and 2.12 mg/l by contact [39]. The insecticidal activity of C. ambrosioides, noticed in the study, might be explained by the presence of ascaridole and isoascaridole, which were among its major constituents.

4.4. Cytotoxicity and antiplasmodial tests on Plasmodium falciparum The cytotoxicity test and the antiplasmodial test has been performed using the ethanolic extract as well as the essential oils of plant species against the MRC-5SV2 cell lines and the K1 strain of P. falciparum, respectively. These tests were performed in the Laboratory of Microbiology, Parasitology and Hygiene of Antwerp University.

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4.4.1. In vitro cytotoxicity

The % reduction in cell growth/viability on MRC-5SV2 cell lines was examined and IC50 values obtained are summarized in Table 30. It was noticed that the growth/viability effects of all extracts were generally low with IC50 > 30 µg/ml, a value which classifies these extracts as non-toxic. The ethanolic extracts of C. citratus (17.55 µg/ml), S. longepedunculata root bark (16.00 µg/ml) and S. longepedunculata root (12.70 µg/ml) are classified moderately toxic, whereas E. suaveolens (1.93 µg/ml) is classified highly toxic (IC50<10 µg/ml).

This lack of toxicity of most of these extracts, is an indication of the good selectivity of these extracts, and supports the secular use of these plants in traditional medicine in Benin, without acute toxicity effect.

Godano et al. (2002, 2006, 2007) has revealed the decreasing of the mitotic index value in lymphocyte cultures exposed in vitro to the aqueous extracts of the nine specimens of C. ambrosioides which is an indication of a cytotoxic effect of C. ambrosioides aqueous extracts related with the essential oil of the plant [283-285]. Furthermore, the in vitro cytotoxicity bioassays on human cell line HaCaT have indicated moderate cytoxicity of C. ambrosioides essential oil in the model used [116].

Concerning the cytotoxicity of C. planchonii essential oil, it was revealed that C. planchonii oils has a weak cytotoxicity on the human erythroblastic cell line K562, with

IC50 of 320 µg/ml for the whole tubercle oil versus  50000 µg/ml for chloroquine [127]. The cytotoxicity of C. planchonii root ethanolic extract on L-6 rat skeletal myoblast cells, investigated by Kamanzi Atindehou et al. (2004), was higher than that obtained in the current study (246.3 µg/ml) versus 942 µg/ml for chloroquine [286]. The root dichloromethane extract studied by Vonthronc-sénécheau et al. (2003) on the rat skeletal muscle myoblasts has resulted in weak cytotoxicity with an LC50 of 67.3 µg/ml) [287]. The oils extracted from whole tubercle and epiderma of C. tinctorium have demonstrated high cytotoxicity on the K562 cell line with IC50 of 80 and 90 µg/ml respectively, versus  50000 µg/ml for chloroquine [127].

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Table 30: Cytotoxicity and efficacity of extracts against Pf-K1

Essential oils MRC-5SV2 Pf-K1 IC50 IC50 (µg/ml) (µg/ml) Chenopodium ambrosioïdes >64.00 >64.00 Cochlospermum planchonii 30.09 32.00 Cochlospermum tinctorium >64.00 >64.00 Eucalyptus citriodora >64.00 >64.00 Eucalyptus tereticornis >64.00 36.76 Cymbopogon citratus >64.00 34.90 Cymbopogon giganteus >64.00 8.00 cymbopogon schoenanthus >64.00 >64.00 Securidaca longepedunculata(roots bark) >64.00 >64.00 Securidaca longepedunculata (roots) ND ND Spondias mombin >64.00 >64.00 Guiera senegalensis >64.00 >64.00

Ethanolic extacts MRC-5SV2 Pf-K1 IC50 (µg/ml) IC50 (µg/ml) Chenopodium ambrosioïdes >64.00 35.17 Cochlospermum planchonii >64.00 29.31 Cochlospermum tinctorium >64.00 37.95 Eucalyptus citriodora 31.53 14.98 Cymbopogon citratus 17.55 11.47 Cymbopogon giganteus >64.00 30.99 Cymbopogon schoenanthus >64.00 23.69 Securidaca longepedunculata (roots bark) 16.00 32.66 Securidaca longepedunculata (roots) 12.70 >64.00 Spondias mombin >64.00 23.35 Guiera senegalensis >64.00 42.92 Erythrophleum suaveolens 1.93 43.07 Chloroquine 0.310.10 Tamoxifen 7.230.07

Concerning G. senegalensis, the cytotoxicity of the total alkaloids extracted from roots and leaves, studied on human monocyte THP1 cells was >100 µg/ml and 50

µg/ml, respectively [184]. In addition, the cytotoxicity on THP1 cells, studied by Ancolio et al. (2002), was >25 µg/ml for root methanol extract and 32.2 µg/ml for root chloroform extract [183].

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The minimum toxic concentration value of the root crude dichloromethane extract of S. longepedunculata was higher than 500 µg/ml on the fibroblast-like cells [196]. The lethal concentrations of its methanol and chloroform extracts on THP1 cells were 5.3 µg/ml and 13 µg/ml, respectively [183].

Different fractions obtained from S. mombin, exhibited at 100 µg/ml variable cytotoxicity (% mortality) on RAW 264.7 cells, summarized as followed: 0% for chloroform : ethyl acetate (80:20), 31.3% for ethyl acetate : chloroform (90:10), 22.9% for ethyl acetate : methanol (80:20), and 40.2% for methanol (100%).

4.4.2. In vitro antiplasmodial activity Twelve plants were selected and twenty-four extracts were tested (ethanolic extracts and essential oils extracted by simultaneous distillation extraction) for antiplasmodial activity against the PfK1 chloroquine-resistant strain of Plasmodium falciparum, and the results are summarized in Table 30.

4.4.2.1. Chenopodium ambrosioides

Against the K1 chloroquine-resistant strain of Plasmodium falciparum, the essential oil of C. ambrosioides has no effect (IC50 > 64 µg/ml) whereas the reduction of parasitemia induced by its ethanolic extract was weak (IC50 = 35.17 µg/ml). To our knowledge, no report was published on the antiplasmodial properties of C. ambrosioides. Nevertheless, some other reported properties of this plant species, related to fever and anxiety, could support its use in malaria treatment. Indeed Bum et al., (2011) has demonstrated antipyretic activity of C. ambrosioides with reduction of the body temperature as well as anxiolytic-like properties. In stress-induced hyperthermia test, C. ambrosioides, significantly antagonised the increase of temperature with a reduction of ΔT° which decreased from 1°C in the control group to -1.1°C at a dose of 120 mg/kg [288]. Furthermore, the anti-inflammatory and the antinociceptive properties of C. ambrosioides have been proven, since this plant species has demonstrated its ability to inhibit the mediators and the enzymes involved in inflammatory and pain processes [289].

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4.4.2.2. Cochlospermum planchonii

Extracts of C. planchonii demonstrated a weak reduction of parasitemia with IC50 = 29.31 µg/ml and 32.00 µg/ml for the ethanolic extract and the essential oil, respectively. These results are in contrast with some previous works where it was demonstrated that the decoction of C. planchonii from Burkina-Faso showed in vivo a significant antiplasmodial activity. Indeed N’dribala (tuberous roots decoction of Cochlospermum planchonii) was evaluated for the treatment of uncomplicated Plasmodium falciparum malaria in Burkina-Faso, and was as efficient as chloroquine since 57% of chloroquine treated volunteers and 52% of N’dribala treated volunteers were cured with no detectable parasitemia and more than 90% of all patients were asymptomatic [124]. Moreover, the essential oil of the whole tubercle of C. planchonii obtained by hydrodistillation in Burkina-Faso, has been tested in vitro on Plasmodium falciparum sensitive Nigerian strain and Plasmodium falciparum resistant strain FcB1, and a reduction of parasitemia after 72 hours with

IC50 of 21 µg/ml and 15 µg/ml, respectively, has been shown [127]. Our result is in accordance with that obtained for the ethanolic extract of C. planchonii obtained in

Ivory Coast [286] which has no significant antiplasmodial activity with IC50 > 5 µg/ml in the model used.

4.4.2.3. Cochlospermum tinctorium

When tested against the K1 resistant strain of P. falciparum, the essential oil of C. tinctorium was inactive (IC50 > 64 µg/ml), whereas its ethanolic extract gave a weak reduction of parasitemia with IC50 = 37.95 µg/ml. On the contrary, the infusion and the decoction of C. tinctorium have exhibited in vivo a great antiplasmodial activity against the FcB1-colombia resistant strain and the F32-Tanzania chloroquine sensitive strain of P. falciparum with an IC50 ranged between 1 µg/ml and 2 µg/ml [134]. Following this experiment, Ballin et al., (2002), has isolated five compounds from the root ethanolic extract of C. tinctorium, among which 3-O-E-p- coumaroylalphitolic acid which had a good antiplasmodial activity against the chloroquine sensitive strain of P. falciparum (3d7) and the Dd2 chloroquine resistant strain of P. falciparum. The IC50 values were 2.3  1.1 µM and 3.8  1.9 µM respectively, [133].

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4.4.2.4. Eucalyptus citriodora The ethanolic extract of the leaves of E. citriodora has revealed in the current work a moderate reduction of parasitemia against the K1 P. falciparum strain, with IC50 = 14.98 µg/ml. To our knowledge, no result has been published regarding the antiplasmodial properties of E. citriodora. However, Sahouo et al. (2003) has disclosed the ability of E. citriodora essential oil to inhibit the synthesis of two enzymes [Soybean lipoxygenase (L-1) and Cyclooxygenase function of prostaglandine H synthase-1 (PGHS)] involved in the production of mediators of inflammation [290]. This anti-inflammatory property of its main compound, i.e. citronellal, has been also confirmed by Sepúlveda-Arias et al., (2013) [291]. With inflammation being one of the worst symptoms of malaria, this property could explain (in addition to the reduction of the parasitemia), the use and efficacy of E. citriodora, as one of the primary steps in the fight against malaria by the population of Benin.

4.4.2.5. Eucalyptus tereticornis In the current work, the essential oil extracted from E. tereticornis has a very weak reduction of parasitemia with IC50 = 36.76 µg/ml against the K1 strain of P. falciparum. Despite this weak reduction of parasitemia, the use of this plant species to treat malaria could also be attributed to its analgesic and anti-inflammatory properties which have been demonstrated by Silva et al. (2003) since the essential oil of E. tereticornis has induced a dose-related analgesic effect in mice [168].

4.4.2.6. Cymbopogon citratus

Among the ethanolic extracts tested, C. citratus was the most efficient against the K1 strain of P. falciparum (IC50 = 11.47 µg/ml) whereas the antiplasmodial activity of its essential oil against Pf-K1 was weak with IC50 = 34.90 µg/ml. Dapper et al. (2008) have studied the blood schizonticidal activity of C. citratus against of P. berghei using Swiss albino mice. Using the crude aqueous extract of C. citratus a significant blood schizonticidal activity against P. berghei was noticed, higher to that of pyrimethamine and sulphadoxine/pyrimethamine, respectively. Indeed, an average suppressive repository activity of 65.8% for the extract and a blood schizonticidal activity which increased from 68.33% in the 24-hour Rane test to 92% in the 72-hour Rane test at a dose of 310 mg/kg were found [292]. The petroleum ether, dichloromethane, ethyl acetate, methanol and aqueous extracts of

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C. citratus have demonstrated antiplasmodial activities against the asexual erythrocytic stages of the chloroquine sensitive strain D10 of P. falciparum, with IC50 values summarized respectively as follows: 9.1 µg/ml, 7.6 µg/ml, 12.1 µg/ml, 15.9 µg/ml and >50 µg/ml [293]. Other biological activities of C. citratus extracts against malaria symptoms have been also demonstrated. As the production of nitric oxide by inflammatory cells has been demonstrated to be involved in the pathogenesis of acute and chronic inflammation, Figueirinha et al. (2010) and Francisco et al. (2011) have evaluated the effects of the infusion of dried leaves from C. citratus on this production and have demonstrated the anti-inflammatory properties of C. citratus which have been attributed to its phenolic fractions [146, 147].

4.4.2.7. Cymbopogon giganteus Among the essential oils tested, the essential oil of the leafy stems of C. giganteus has demonstrated the best reduction of parasitemia (IC50 = 8 µg/ml) whereas the antiplasmodial of the ethanolic extract effect was weak (IC50 = 30.99 µg/ml). The aqueous extract of C. giganteus has demonstrated in vivo a good reduction of parasitemia in albinos mice infected with the chloroquine resistant strain Plasmodium yoelii nigeriensis [294]. C. giganteus extracts could also reduce some malaria symptoms such as inflammation and pain. Indeed, the moderate inhibition of the proteins prostaglandin H synthase (PGHS) and lipoxygenase-5LO (model Lipoxygenase L-1), implicated in the production of mediators of inflammation, was demonstrated by the essential oil of C. giganteus [290].

4.4.2.8. Cymbopogon schoenanthus The ethanolic extract of the leafy stems of C. schoenanthus also exhibited a weak reduction of parasitemia (IC50 = 23.69 µg/ml) whereas its essential oil was inactive against the K1 strain of Plasmodium falciparum (IC50 > 64 µg/ml). Its traditional uses against tiredness [112], associated with the reduction of parasitemia observed, could explain its use in the treatment of malaria. No study of the antiplasmodial activity of C. schoenanthus is reported, according to our knowledge. However, some other biological activities of C. schoenanthus have been demonstrated. Indeed, the antimicrobial activity of its proanthocyanidin extract against Streptococcus sobrinus at

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low concentration (4 mg/ml) and its great acetylcholinesterase inhibitory activity (IC50 = 0.23  0.04 mg/ml) have been shown [160].

4.4.2.9. Securidaca longepedunculata The essential oil extracted from the root bark of S. longepedunculata, as well as the ethanolic extract obtained from the root, have no effect (IC50 > 64 µg/ml) on the K1 strain of P. falciparum whereas the ethanolic extract of the root bark has demonstrated a weak reduction of parasitemia (IC50 = 32.66 µg/ml). Previous study has also revealed a weak antiplasmodial activity of the methanol and the chloroform root extract of S. longepedunculata with IC50 > 25 µg/ml [183]. Nevertheless, S. longepedunculata possesses some biological properties, which can explain its use in malaria treatment in Benin. Indeed, its aqueous root extract demonstrated sedative, anticonvulsant and anxiolytic properties [197, 295]. The relaxation properties of the xanthones [193], as well as the antinociceptive and antidepressant-like effects of S. longepedunculata have been also demonstrated [195].

4.4.2.10. Guiera senegalensis G. senegalensis was the one of the selected plants whose extracts showed the poorest antiplasmodial activity with IC50 = 42.92 µg/ml for its ethanol extract and >64 µg/ml for its essential oil. Nevertheless, the antiplasmodial activities of other extracts of G. senegalensis from Mali has been already proven. Indeed, the good antiplasmodial activity of the root chloroform extract of G. senegalensis has been demonstrated on P. falciparum strain W2 with an IC50 < 25 µg/ml, and this activity has been attributed to harman and tetrahydroharman, two alkaloids isolated from G. senegalensis root with IC50 lower than 4 µg/ml [183]. Fiot et al. (2006) have proven the antiplasmodial potential of isolated alkaloids from Guiera senegalensis with IC50 = 121.93 µM, 18.10 µM and 4.08 µM for harmalan, harman and guieranone A, respectively [184]. This difference in antiplasmodial activity could be explained by the difference between the extracts used and the different Plasmodium strains used. The persistence of the use of this plant species to treat malaria could be due to its traditionally known properties against fever, pains and headache, which are some malaria symptoms [111, 296].

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4.4.2.11. Spondias mombin S. mombin is one of the plants most used to treat malaria by traditional healers and population from Benin. In our study, the ethanolic extract of the leaves of this plant species has demonstrated a poor reduction of parasitemia with IC50 = 23.35 µg/ml and its essential oil was completely inactive IC50 > 64 µg/ml. According to our knowledge, no study of the antiplasmodial activity of S. mombin has been reported. The low reduction of parasitemia of the parasite tested is associated with a complete absence of cytotoxicity. Further antiplasmodial assays on other stages of Plasmodium with other extracts are needed to confirm the use of S. mombin as effective and safe antiplasmodial product. Nevertheless, other activities of S. mombin have been indicated by Abo et al. (1999) [203], who have proven that S. mombin leaves extract exhibited wide antibacterial effect comparable to ampicillin and gentamycin. The leaves sedative effect has also been demonstrated by Ayoka et al., (2006) [208]. The sedative activity, associated to the weak reduction of parasitemia demonstrated by our study, supports the efficacy of S. mombin in the treatment of malaria in Benin.

4.4.2.12. Erythrophleum suaveolens The ethanolic extract of the leaves of E. suaveolens has demonstrated in the current work the worst antiplasmodial activity with IC50 = 43.07 µg/ml, besides a strong cytotoxic activity. However, Dongmo et al. (2001) has demonstrated the anti-inflammatory and analgesic properties of E. suaveolens by the significant 5-lipoxygenase inhibitory activity since the hexane extract at the concentration of 19.2 µg/ml inhibits 39% of the synthesis of this enzyme [165].

4.4.2.13. Discussion on the results obtained with the antiplasmodial tests All the plants investigated in this study are used in Benin by traditional healers for antimalarial applications, but their extracts (ethanolic extracts and essential oils)

have presented a variable reduction of parasitemia against the K1 strain of P. falciparum. These mitigated results do not mean that these plants are not active against Plasmodium falciparum. Indeed, in our study the tests were done using one strain of the parasite and with one type of extract. These extracts or other extracts

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could be active on other strains of P. falciparum. This is the case for G. senegalensis whose chloroform extract has already been reported to be active against the strain W2 of P. falciparum [286]. Moreover, the extracts obtained from these plant species possess some other therapeutic activities against malaria symptoms since the antipyretic, anti-anxiolitic, anti-inflammatory, analgesic and sedative properties of these extracts have been demonstrated as reported above, and could explain the amelioration of the user's health. These results could as well be explained by the fact that the plants used in this study could be more active in vivo, potentially containing prodrugs, non-active themselves in vitro but which can be, in vivo, metabolized into active drugs [297]. The differences observed could also be explained by the variable composition of different extracts used, which can exhibit differential results. Indeed, Ménan et al. (2006) have shown that methanol, pentane and chloroform extracts of the same plant could exhibit different biological activity against the same parasite [298]. In conclusion, it seems that the nature of the solvent and its affinity with the organ extracted could be important to demonstrate the efficiency of an extract and bring out its antiplasmodial properties. Our further investigations will prospect the antiplasmodial activity of other solvent extracts of these plants, on other sensitive and resistant strains of P. falciparum.

The current research is a preliminary study, which has permitted to find some scientific justifications for the use of the plant species investigated and traditionally used against malaria in Benin. Antiplasmodial assays of the ethanolic plant extracts have shown a reduction of parasitemia with IC50 values ranged from 8.00 µg/ml to 43.07 µg/ml. The existence of alternative biological activity against some malaria symptoms such as pain, headache, vomiting and fever associated with these reductions of parasitemia could well explained the amelioration of the user health. The absence of cytotoxicity of most of these extracts supported their use as traditional medicine in Benin. To our knowledge, this is the first report in which the antiplasmodial activity of extracts obtained from C. ambrosioides, E. citriodora, E. tereticornis, C. schoenanthus, S. mombin, E. suaveolens and leaves of G. senegalenis have been investigated.

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4.5. Insecticidal tests against Tribolium castaneum Tables 31 to 34 summarize for each plant species, the lethal doses, and mortalities obtained during the in vitro contact no choice test and the fumigant test against T. castaneum.

4.5.1. In vitro contact "no choice" test against Tribolium castaneum

The lethal concentrations (LC50, LC90, LC99) obtained by contact no choice test are summarized in Table 31. These lethal concentrations varied by mean times and concentrations used. It was noticed that the most efficient crude oil was E. citriodora followed by C. schoenanthus. The LC50, LC90 and LC99 values of E. citriodora were ranged from 6.86% to 15.04% after 24 hours of exposure, 5.05% to 13.22% after 48 hours of exposure and 4.54% to 12.52% after 72 hours of exposure. Concerning C. schoenanthus these values were 7.52% to 16.33%, 6.37% to 13.70% and 5.61% to

12.30% after 24 hours, 48 hours and 72 hours of exposure, respectively. The LC values obtained after 24 hours, 48 hours, and 72 hours of exposure for E. citriodora essential oil and C. schoenanthus essential oil were not significantly different based on the non-overlapping 95% CL for doses.

Regarding the mortalities (Table 32), the same tendencies were observed since after 24 hours the most efficient essential oil was the one extracted from E. citriodora (58.33% at 8% concentration), followed by C. schoenanthus essential oil (50.39% at 8% concentration).

The major compound isolated from C. schoenanthus essential oil (piperitone) was more efficient than the crude essential oil, since the mortality values obtained after 24 hours, 48 hours and 72 hours were 63.33%, 76.67% and 86.67%, respectively, for piperitone versus 50.39%, 65.12% and 72.87%, respectively, for the crude oil extracted from C. schoenanthus (Table 33). The results obtained with piperitone were better than those of permethrin as positive control (60%, 75% and 83.33%).

The tested compounds of E. citriodora were not as effective as the crude oil since no or mortality less than 4% was obtained for citronellyl acetate and citronellol, whereas for citronellal a mortality of 40% could be obtained after 72 hours.

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From these results, it can be confirmed that the major compound of C. schoenanthus (piperitone) is responsible for the insecticidal properties against T. castaneum.

Concerning E. citriodora, since the isolated compounds are not as efficient as the crude oil, the insecticidal properties of this essential oil could be rationalized by the synergistic action of its major compounds (citronellal and citronellol) and some other minor compounds such as 1,8-cineole, α-pinene and p-cymene, whose insecticidal properties have been already reported. Indeed the insecticidal properties of 1,8- cineole, α-pinene and p-cymene have been demonstrated against A. aegypti and correlated to the amount of 1,8-cineole in the extract [26, 178]. The insecticidal property of citronellol has not been demonstrated according to our knowledge, but the repellent properties of (+)-citronellol against T. castaneum has been already reported [299]. The nature of the biological properties of this compound (repellent) might explain its inefficiency in the contact "no choice" test.

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Table 31: Efficiency of essential oils by "contact no choice" test against Tribolium castaneum LC (%) LC (%) LC (%) Chi- Exposure Plant species 50 90 99 Intercept Slope df Sig. (95%CL) (95%CL) (95%CL) Square 24 hours C. citratus 21.76* 34.07* 44.10* -2.2660.143 0.1040.030 47.60 40 0.191 (15.09-44.96)a (23.05-72.85)a (29.51-95.61) a C. giganteus 10.26 15.43 19.64 -2.5460.177 0.2480.029 30.762 34 0.627 (9.14-11.96)b (13.42-18.60)b (16.87-24.06) b C. schoenanthus 7.52 12.38 16.33 -1.9870.111 0.2640.022 54.486 34 0.014 (6.71-8.65)c (10.85-14.66)b (14.15-19.64) b E. citriodora 6.86 11.37 15.04 -1.9540.157 0.2850.031 38.029 34 0.291 (6.09-7.91)c (9.95-13.49)b (13.01-18.13) b Piperitone ND** ND** ND** ND** ND** ND** ND** ND** Citronellal ND** ND** ND** ND** ND** ND** ND** ND** Citronellol ND** ND** ND** ND** ND** ND** ND** ND** Citronellyl acetate ND** ND** ND** ND** ND** ND** ND** ND** Permethrin 0.75% ND** ND** ND** ND** ND** ND** ND** ND** 48 hours C. citratus 19.96 32.14 42.28 -2.0290.117 0.1030.026 54.678 40 0.061 (13.49-42.91)a (21.42-72.83) a (27.84-97.26) a C. giganteus 8.50 14.11 18.68 -1.9400.113 0.2280.022 47.156 34 0.066 (7.46-10.03)b (12.13-17.21) b (15.88-23.13) b C. schoenanthus 6.37 10.41 13.70 -2.0180.110 0.3170.022 57.788 34 0.007 (5.75-7.14)c (9.31-11.95)) c (12.13-15.96) b E. citriodora 5.05 9.55 13.22 -1.4380.121 0.2850.029 49.998 34 0.038 (4.29-6.07)c (8.11-11.87) c (11.09-16.73) b Piperitone ND** ND** ND** ND** ND** ND** ND** ND** Citronellal ND** ND** ND** ND** ND** ND** ND** ND** Citronellol ND** ND** ND** ND** ND** ND** ND** ND** Citronellyl acetate ND** ND** ND** ND** ND** ND** ND** ND** Permethrin 0.75% ND** ND** ND** ND** ND** ND** ND** ND** 72 hours C. citratus 14.99 26.04 35.04 -1.7410.094 0.1160.021 37.267 40 0.594 (11.73-21.79)a (19.91-38.99) a (26.55-53.04) a C. giganteus 6.06 10.92 14.88 -15970.092 0.2640.020 56.715 34 0.009 (5.36-6.98)b (9.55-12.93) b (12.88-17.87) b C. schoenanthus 5.61 9.29 12.30 -1.9520.106 0.3480.022 48.036 34 0.056 (5.13-6.17)c (8.45-10.40) b (11.09-13.92) b E. citriodora 4.54 8.94 12.52 -1.3260.116 0.2920.030 53.456 34 0.018 (3.82-5.51)c (7.55-11.20) b (10.44-15.98) b Piperitone ND** ND** ND** ND** ND** ND** ND** ND** Citronellal ND** ND** ND** ND** ND** ND** ND** ND** Citronellol ND** ND** ND** ND** ND** ND** ND** ND** Citronellyl acetate ND** ND** ND** ND** ND** ND** ND** ND** Permethrin 0.75% ND** ND** ND** ND** ND** ND** ND** ND**

LCx = concentration of essential oil required to kill X% of T. castaneum. LCx values within a column followed by the same letter are not significantly different , based on non-overlapping 95% CL for the LCx (X = 50, 90, 99) * = Values obtained by extrapolation since a mortality higher than 50% has not been obtained ND** - not determined since only one concentration was tested for each identified compound

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Table 32: Mortalities obtained by contact no choice test against Tribolium castaneum Plant Mortalitya Mortalitya Mortalitya Mortalitya; Mortalitya Exposure species/isolated at 0.5% at 1% at 2% at 4% at 8% compounds 24 hours C. citratus 1.53% 0.9 1.64% 1.0 1.67% 1.0 4.92% 2.8 6.67% 2.8 C. giganteus 0.83% 0.7 0.83% 0.7 0.83% 0.7 10.00% 1.7 26.67% 5.6 C. schoenanthus 1.61% 1.5 3.13% 0.9 9.92% 2.8 24.63% 4.6 50.39% 7.0 E. citriodora 1.67% 1.5 3.33% 1.9 13.33% 5.1 26.67% 7.7 58.33% 2.8 Piperitone ND ND ND ND 63.33 8.4 Citronellal ND ND ND ND 40,00 3.3 Citronellol ND ND ND ND 0.00 0.0 Citronellyl acetate ND ND ND ND 0.00 0.0 Permethrin 0.75% 60% 7.8 48 hours C. citratus 2.29% 0.9 2.46% 1.6 5.83% 2.2 6.56% 3.2 10.00% 3.9 C. giganteus 2.46% 1.0 4.17% 1.8 9.17% 1.8 20.83% 1.4 41.66% 9.5 C. schoenanthus 1.61% 1.5 3.13% 1.4 10.69% 4.3 30.60% 5.1 65.12% 4.1 E. citriodora 11.67% 3.7 13.33% 3.8 25.00% 6.1 41.67% 7.2 76.67% 8.4 Piperitone ND ND ND ND 76.67 6.5 Citronellal ND ND ND ND 40,00 3.3 Citronellol ND ND ND ND 1.67  1.5 Citronellyl acetate ND ND ND ND 0.00 0.0 Permethrin 0.75% 75%  7.0 72 hours C. citratus 3.79% 1.2 8.94% 1.9 10.83% 2.2 11.48% 2.2 18.33% 3.0 C. giganteus 4.92% 1.6 12.50% 4.7 20.83% 2.2 30.83% 4.9 66.67% 5.2 C. schoenanthus 2.42% 1.5 3.91% 1.3 9.16% 1.6 44.03% 5.0 72.87% 3.1 E. citriodora 15.00% 3.9 16.67% 3.8 30.00% 8.2 45.00% 8.4 81.67% 6.4 Piperitone ND ND ND ND 86.67 4.5 Citronellal ND ND ND ND 40,00 3.3 Citronellol ND ND ND ND 3.33 1.9 Citronellyl acetate ND ND ND ND 0.00 0.0 Permethrin 0.75% 83.33% 7.3 a: Mortality values = mean SE of the six replicates (SE= standard error)

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Table 33: Lethal concentrations of essential oils and isolated compounds against Tribolium castaneum, by fumigation LC50 LC90 LC99 Plant species / Chi- ml/L air ml/L air ml/L air Intercept Slope df Sig. compounds Square (95%CL) (95%CL) (95%CL) C. citratus 4.19* 7.59* 10.36* -1.5830.182 0.3770.068 28.221 22 0.168 (3.52-5.41)a (6.14-10.55)a (8.21-14.81)a C. giganteus 2.25 5.02 7.28 -1.0430.149 0.4630.063 40.061 22 0.011 (1.71-2.92)b (4.04-7.08)b (5.72-10.69)a C. schoenanthus 2.10 4.72 6.85 -1.0260.149 0.4890.064 55.426 22 0.000 (1.48-2.83)b (3.71-7.05)b (5.26-10.76)a E. citriodora 2.01 4.63 6.77 -0.9820.147 0.4890.065 32.14 22 0.075 (1.55-2.52)b (3.82-6.14)b (5.47-9.30) a Piperitone 0.543 0.94 1.29 -1.4710.229 2.9390.342 85.869 22 0.000 (0.34-0.74)c (0.74-1.36)c (1.01-2.01)b Citronellal 1.18 2.17 2.98 -1.5300.223 1.2940.158 25.022 16 0.069 (0.93-1.43)d (1.86-2.71))d (2.50-3.87)c Citronellol ND ND ND ND ND ND ND ND Citronellyl acetate ND ND ND ND ND ND ND ND Permethrin 0.75% ND ND ND ND ND ND ND ND

LCx = concentration of essential oil or identified compound required to kill X% of T. castaneum. LCx values within a column followed by the same letter are not significantly different, based on non-overlapping 95% CL for the LCx (X = 50, 90, 99) * = Values obtained by extrapolation since a mortality higher than 50% has not been obtained

Table 34: Mortalities obtained by fumigation against Tribolium castaneum Plant species / Mortality a Mortality a Mortality a

isolated compounds at C1 at C2 at C3 C. citratus 15.00% 5.7 28.33% 5.5 41.67% 4.4

C. giganteus 38.33% 5.5 63.33% 6.5 68.33% 3.7 C. schoenanthus 40.00% 7.8 66.67% 3.8 71.67% 8.0

E. citriodora 48.33% 4.4 60.00% 2.4 75.00% 3.1 Piperitone 78.33% 6.8 91.67% 6.0 100% 0.0

Citronellal - 60.00% 2.4 81.67% 5.5

Citronellol - 0.00% 0.0 0.00% 0.0 Citronellyl acetate - 0.00% 0.0 1.67% 1.5

Permethrin 0.75% 8.33% 3.7 a = C1, C2 and C3 values for crude oils and isolated compounds are indicate in Table 8

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4.5.2. Discussion on the results obtained with the contact "no choice" tests Biological properties of essential oils extracted from plant species are related to their chemical profiles which depend from the extracted organ, its age, the geographical area of collection, the method of extraction [41, 300] and the method used to test the extract.

4.5.2.1. C. citratus The essential oil extracted from the whole plant of C. citratus, collected in Colombia [41], was rich in neral (28.4%), geraniol (11.5%) and geranial (34.4%). This composition is a bit different from the one obtained in the current study in view of the high amount of geraniol (11.5% in Colombia versus 1% in Benin) and the absence of myrcene which was at 12.4% in the current work. The mortality obtained at 40 ml/L against T. castaneum by Olivero-verbel et al. with the essential oil, originating from Colombia (65% after 24 hours, 70% after 48 hours and 75% after 72 hours of exposure), might be explained, by the different chemical profiles of this plant when originated from different areas, also by the different method of dilution used, w/v in the current work and v/v by Olivero-verbel et al., which can be correlated with the amount of the active compound used.

4.5.2.2. E. citriodora The chemical profile of the essential oil extracted from the leaves of E. citriodora in Colombia revealed as main compounds the presence of citronellal (40%), isopulegol (14.6%), and citronellol (13.0%). Its chemical profile revealed the presence of two minor compounds, namely 1,8-cineole (3.4%) and citriodiol [5-methyl-2-(2-hydroxy-2- propyl)cyclohexanol] (4.7%). This crude oil has generated in adults population of T. castaneum, mortalities of 55%, 57% and 60%, at 40 ml/L (4%) after 24 hours, 48 hours and 72 hours, respectively, [41].

The insecticidal property of 1,8-cineole against stored grain insects [26, 27, 29], fumigant property against Lutzomyia longipalis [301], acaricidal and repellent properties against Tetranychus urticae and Culex pipiens [302, 303] have been already reported. Citriodiol has also demonstrated repellent properties against Ixodes ricinus tick [304] and land leeches of the genus Haemadipsa [305]. The presence of 120

citriodiol and 1,8-cineole in the extract from Colombia might explain the differences in response of T. castaneum to these essential oils extracted from E. citriodora. To our knowledge, no research on the insecticidal activity of C. schoenanthus and C. giganteus against T. castaneum, by contact "no choice test" has been already published.

4.5.3. In vitro fumigant test against Tribolium castaneum The fumigant test against T. castaneum has demonstrated the susceptibility of this beetle to the essential oil tested by fumigation, especially to C. schoenanthus and E. citriodora with LC50, LC90, LC99 values ranged respectively as followed: 2.10 ml/L air, 4.72 ml/L air, 6.85 ml/L air for C. schoenanthus and 2.01 ml/L air, 4.63 ml/L air, 6.77 ml/L air for E. citriodora (Table 33). These results are not significantly different according to the non-overlapping 95% CL for doses.

Concerning piperitone, the LC50, LC90, LC99 values were 0.54 ml/L air, 0.94 ml/L air, 1.29 ml/L air and 1.18 ml/L air , 2.17 ml/L air , 2.98 ml/L air ) for citronellal (Table 33). Piperitone, the major compound of C. schoenanthus was more than three times as efficient as compared to the crude oil; citronellal was about two times as efficient as compared to the crude essential oil.

Regarding the mortalities (Table 34), the more efficient essential oils were the same.

Indeed at C3 (Table 8), the mortalities obtained were 75% for E. citriodora, 71.67% for C. schoenanthus, 100% for piperitone and 81.67% for citronellal. Citronellol has no insecticidal effect by fumigation (0% mortality) whereas citronellyl acetate (1.67%) and permethrin, the positive control (8.33%) have demonstrated very weak mortalities.

4.5.4. Discussion on the results obtained with the fumigant tests To our knowledge, no result on the fumigant properties of plant species tested against T. castaneum, has been already reported. Nevertheless, the good insecticidal properties of piperitone have been already reported. Indeed, piperitone isolated from the essential oil of Artemisia judaica, has demonstrated a complete antifeedant activity at a concentration of 1000 µg/ml, against the third instar larvae of Spodoptera littoralis using non-choice leaf disc assay [279]. Furthermore when tested against Callosobruchus maculatus, piperitone, 121

isolated from C. schoenanthus, was more toxic by fumigation to adults with a LC50 value of 1.6 μl/l vs. 2.7 μl/l obtained with the crude extract [38].

The crude oil of E. citriodora, rich in beta-citronellal (71.8%) has demonstrated its good insecticidal activity against the adult of Lutzomya longipalpis, by fumigation. Indeed at the concentration of 2 mg/ml, 4 mg/ml, 6 mg/ml, 8 mg/ml and 10 mg/ml, the following mortalities have been respectively recorded after 24 hours: 10.60%, 31.01%, 48.41%, 68.14%, and 88.13% [27]. The other tested compound, i.e citronellol, has developed no repellent activity against A. aegypti, since at the concentration of 0.25% and 1%, 3 mosquito bites have been noticed within 8 seconds and 4 seconds, respectively [306].

All results obtained from the biological activity testing of plant extracts (Table 35 and Table 36), as mixtures of chemical compounds, i.e. insect tests against A. gambiae and T. castaneum, cytotoxicity against the MRC-5sv2 cells and antiplasmodial activity against the resistant strain K1 of P. falciparum, have been compared to reference compounds which are single compounds, namely permethrin 0.75%, tamoxifen and chloroquine, respectively. All these tests, which are preliminary tests, were performed to establish the basis of the biological properties (insecticidal and antiplasmodial) of these mixtures. The reference compounds are commonly used references in these kind of tests. The next steps of our research could be to test the major isolated compounds, and minor isolated compounds with known biological properties, of the active plant extracts, against the same cell lines. This would allow a direct comparison of isolated compounds at equal concentrations. In this case, synergistic action of compounds in the mixtures would be eliminated and the compounds possibly responsible for biological activities observed would be identified more easily.

Concerning insecticidal tests (Table 35), by contact test, it was noticed that the susceptibility of T. castaneum to essential oils is lower than the susceptibility of A. gambiae. It is important to notify that by contact test, A. gambiae was exposed to essential oils just for one hour whereas T. castaneum exposure time was 24 hours repeated three times. Nevertheless, by fumigation, the essential oils were more toxic to T. castaneum, especially the essential oils extracted from C. schoenanthus and E. citriodora. 122

Table 35: Summary of insecticide activities of essential oils against Anopheles gambiae and Tribolium castaneum

Against Tribolium castaneum Against Tribolium castaneum Against Anopheles gambiae by contact “no choice” test by fumigation (24 hours (24 hours post exposure) Essential oils (24 hours exposure) exposure)

LC50 (%) LC99 (%) LC50 (%) LC99 (%) LC50 (%) LC99 (%) C. citratus 0.24 0.39 21.76 44.10 4.19 10.36 C. giganteus 1.60 3.68 10.26 19.64 2.25 7.28 C. schoenanthus 1.57 2.74 7.52 16.33 2.10 6.85 E. citriodora 0.90 1.68 6.86 15.04 2.01 6.77 E. tereticornis 0.15 1.40 ND ND ND ND C. tinctorium 1.16 5.78 ND ND ND ND C. planchonii 2.31 7.61 ND ND ND ND S. longepedunculata 2.49 4.92 ND ND ND ND C. ambrosioides 0.99 2.13 ND ND ND ND

Table 36: Antiplasmodial activites of studied plant extracts

Extracts and cell lines Essential oils Ethanolic extacts

Plant species MRC-5SV2 Pf-K1 IC50 MRC-5SV2 Pf-K1 IC50 (µg/ml) (µg/ml) IC50 (µg/ml) IC50 (µg/ml) Chenopodium ambrosioïdes >64.00 >64.00 >64.00 35.17 Cochlospermum planchonii 30.09 32.00 >64.00 29.31 Cochlospermum tinctorium >64.00 >64.00 >64.00 37.95 Eucalyptus citriodora >64.00 >64.00 31.53 14.98 Eucalyptus tereticornis >64.00 36.76 ND ND Cymbopogon citratus >64.00 34.90 17.55 11.47 Cymbopogon giganteus >64.00 8.00 >64.00 30.99 cymbopogon schoenanthus >64.00 >64.00 >64.00 23.69 Securidaca longepedunculata(roots >64.00 >64.00 16.00 32.66 bark) Securidaca longepedunculata (roots) ND ND 12.70 >64.00 Spondias mombin >64.00 >64.00 >64.00 23.35 Guiera senegalensis >64.00 >64.00 >64.00 42.92

Erythrophleum suaveolens 1.93 43.07

Chloroquine 0.310.10 0.310.10 Tamoxifen 7.230.07 7.230.07

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The variability of the susceptibility of A. gambiae and T. castaneum towards insecticides could be rationalized by the involvement of different detoxification mechanisms or different targets. Several glutathione S-transferases (GSTs), which are important enzymes involved in the process of resistance to insecticides, have been identified in the genome of T. castaneum and A. gambiae. Indeed, fourthy-one GSTs (36 putative cytosolic GSTs and 5 microsomal GSTs) and thirty-five GSTs (32 cytosolic GSTs and 3 microsomal GSTs) were found in the genome of T. castaneum and A. gambiae, respectively. Among these GSTs, T. castaneum possesses nineteen Epsilon GSTs genes whereas A. gambiae has only eight. These Epsilon GSTs are involved in the process of detoxification [307]. Furthermore, G protein coupled receptors (GPCRs), which are key drug targets for more than 50% pharmaceutical drugs [308, 309], have been identified in the genome of T. castaneum and A. gambiae. Indeed, twenty GPCR-Bs (family B of GPCR) have been identified in T. castaneum versus fourteen in A. gambiae [310]. Bioactive compounds such as insecticides especially the neonicotinoids target nicotinic acetylcholine receptors (nAChR) genes families [311]. In the genome of T. castaneum and A. gambiae twelve nAChR subunits and ten nAChR subunits were identified, respectively [311]. These neonicotinoid receptor based compounds were the fasted-growing class of insecticides in modern crop protection, considered as safe and efficient, because of their selectivity for insect nicotinic receptors over mammalian nAChRs [312]. This safety consideration is important to be taken into account in the control of pests such as T. castaneum living in human food sources. More data on nAChR subunits are required to improve the chemical control of vectors such as A. gambiae [311].

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CHAPTER 5: CONCLUSION AND PERSPECTIVES

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Chemical profiles of plant species During the current study, firstly, the chemical profiles of twelve plant species have been established by hydrodistillation/GC-MS, SDE/GC-MS and SPME/GC-MS. Advantages and disadvantages of each method have been discussed above. It follows that the method of choice to establish the chemical profile is SPME because of its numerous advantages, which allows detecting very low concentrations, large range of volatiles and prevents hydrolysis/pyrolysis of volatiles.

Insecticidal test against Anopheles gambiae In the next step of this study, nine essential oils extracted from these plant species have been tested against A. gambiae using the WHO susceptibility test protocol. The susceptibility of this vector of malaria to the essential oils has been established especially to C. citratus, E. tereticornis, E. citriodora and C. ambrosioides whereas this malaria vector is resistant to Permethrin 0.75%. The insecticidal properties of these essential oils have been correlated to their major constituents namely citral, piperitone, 1,8-cineole, citronellal, 2-tridecanone and methyl salicylate, whose insecticidal properties have been already reported. To our knowledge, it was the first time that diagnostic doses of essential oils on A. gambiae were determined, using the WHO susceptibility test protocol. These plant species, as well as the compounds identified above, may be included in malaria vector control programs, since these plant species occur in the natural environment of local populations, could be obtained at lower cost and represent today a valuable source of bioactive compounds for the protection of populations against malaria.

Antiplasmodial test against Plasmodium falciparum The third part of the current research has dealt with the antiplasmodial properties of essential oils extracted by SDE and ethanolic extracts obtained from these twelve plant species. The antiplasmodial assays have revealed a reduction of parasitemia with IC50 ranged from 8.00 µg/ml to 43.07 µg/ml. In the model used, these plant extracts have shown weak to no antiplasmodial properties. Nevertheless, these plant species possess other biological activities against malaria symptoms such as pain, headache, vomiting, and fever, which support the amelioration of the user health.

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The absence of cytotoxicity of most of these extracts supported their use as traditional medicine in Benin. To our knowledge, this is the first report in which the antiplasmodial activity of extracts obtained from C. ambrosioides, E. citriodora, E. tereticornis, C. schoenanthus, S. mombin, E. suaveolens and leaves of G. senegalensis, have been investigated.

Insecticidal tests on T. castaneum The last step of the current study consisted of testing the essential oils extracted from four plant species against T. castaneum, by fumigation and by contact no choice test. The most promising ones were from C. schoenanthus and E. citriodora, as well as their isolated compounds piperitone and citronellal, respectively. It was noticed that the insecticidal activity of piperitone was better than the crude oil from which it was isolated.

Perspectives The chemical analysis of the volatiles of the plant species has permitted the identification of several chemical profiles, which allowed correlation with the biological properties of the different plant species studied.

Concerning the insecticidal activities, it is planned to continue the in vitro tests on A. gambiae, by testing the main compounds of each essential oil, as well as some minor compounds, the insecticidal properties of which have been already reported. Another perspective is to test these essential oils and isolated compounds against other pests such as Sitophilus zeamais and S. oryzae as well as Callosobruchus species to establish their susceptibilities to these natural extracts.

Regarding the antiplasmodial assays, a future plan consists of testing our ethanolic extracts, as well as other extracts obtained with dichloromethane and chloroform, against other strains of P. falciparum such as the sensitive 3D7 and/or FcB1- colombia, and the resistant W2 and/or F32-Tanzania.

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CHAPTER 6: SUMMARY

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Malaria is a worldwide disease especially occurring in sub-Saharan Africa, which presents 80% of cases and 90% of death. The vector of malaria belongs to the genus Anopheles, of which the most imported in West Africa is A. gambiae Giles. Pregnant women and children under five are the most vulnerable [1]. In many African countries, especially in Benin, A. gambiae is developing resistance to all classes of insecticides used for its control [3-5, 11, 13, 20, 23]. Furthermore, pyrethroids are the only option of insecticide for the treatment of bed nets because of the safety for humans at low dosage, the excito-repellent properties, the good knock-down and the killing effects on mosquitoes [313]. The malaria parasite also became resistant to common drugs used in the treatment of malaria such as amodiaquine or chloroquine [314-316]. Following the above, the current study has been conducted to justify or counter indicate the use of plant species originating from Benin as first medication against malaria and/or to avoid mosquito's bites. Twelve plant species have been selected in Benin for which no research has proven scientifically their use in the prevention or the treatment of malaria. These plant species include Chenopodium ambrosioides L. (Amaranthaceae), Cymbopogon citratus (DC.) Stapf (Poaceae), Cymbopogon schoenanthus (L.) Spreng. (Poaceae) and Cymbopogon giganteus Chiov. (Poaceae), Eucalyptus citriodora Hook. (Myrtaceae), Eucalyptus tereticornis Sm. (Myrtaceae), Cochlospermum planchonii Hook. f. Ex Planch. (Bixaceae), Cochlospermum tinctorium A. Rich. (Bixaceae), Erythrophleum suaveolens (Guil. & Perr) Brenan (Fabaceae), Guiera senegalensis J. F. Gmel. (Polygalaceae), Securidaca longepedunculata Fresen. (Polygalaceae) and Spondias mombin L. (Anarcadiaceae). To reach the objectives, the following research steps were undertaken:

- The essential oils have been extracted from these plant species by hydrodistillation and simultaneous distillation extraction (SDE);

- The chemical profiles of all these aromatic plant species have been elucidated using three different methods: SDE/GC-MS, Hydrodistillation/GC-MS and SPME/GC-MS;

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- The essential oils obtained by hydrodistillation have been tested against A. gambiae sensitive strain "Kisumu" and the resistant strain "Ladji", using the WHO standard protocol [50];

- The antiplasmodial assays have been performed against the K1 strain of P. falciparum, using essential oils extracted by SDE and ethanolic extracts;

- A final research part has been conducted to determine the susceptibility of Tribolium castaneum to essential oils extracted from C. citratus, C. giganteus, C. schoenanthus and E. citriodora. The objective was to find new bioactive compounds to conquer the resistance of the red flour beetle to synthetic insecticides used for its control. In this case, fumigation tests, as well as a contact "no choice" tests have been performed. Furthermore, the major compounds of the most promising plant species, namely piperitone from C. schoenanthus as well as citronellal, citronellol and citronellyl acetate from E. citriodora, have been tested against this pest, to identify the source of the bioactivity of these crude oils against T. castaneum.

The different results obtained in the current study were summarized as followed and presented in Table 35 and Table 36.

- Chemical profiles Different chemical profiles have been identified using the three methods described above and are summarized in Table 10 to Table 22. It was noticed that the same plant species could result in different chemical profiles, using different methods of extraction. This variability in the chemical profiles is responsible for the different biological activities of the extracts, due to the variable presence of bioactive compounds in the extract used.

- Insecticidal tests against A. gambiae Different chemical compositions were obtained from the essential oils of the plant species extracted by hydrodistillation and used to perform these tests against A. gambiae. The major constituents identified were neral and geranial for C. citratus, Z- carveol, E-p-mentha-1(7),8-dien-2-ol and E-p-mentha-2,8-dienol for C. giganteus, 130

piperitone for C. schoenanthus, citronellal, citronellol, citronellyl acetate and neo- isopulegol for E. citriodora, p-cymene, caryophyllene oxide and spathulenol for E. tereticornis, 3-tetradecanone for C. tinctorium and C. planchonii, methyl salicylate for S. longepedunculata and ascaridole for C. ambrosioides.

The diagnostic dose of the essential oils was 0.77% for C. citratus, 2.80% for E. tereticornis, 3.37% for E. citriodora, 4.26% for C. ambrosioides, 5.48% for C. schoenanthus and 7.36% for C. giganteus. The highest diagnostic doses were obtained with S. longepedunculata (9.84%), C. tinctorium (11.56%) and C. planchonii (15.22%), compared to permethrin 0.75%. A. gambiae cotonou, which is resistant to pyrethroids, showed significant tolerance to essential oils from C. tinctorium and S. longepedunculata as expected but was highly susceptible to all the other essential oils at the diagnostic dose. These doses were presented in %, mg/ml and mg/cm2 to facilitate further research on these plant species. KDT50 and KDT95 as well as LC50 and LC99 and results obtained have proven that all essential oils from these plant species are more effective against the resistant strain of A. gambiae than permethrin 0.75% at the diagnostic doses tested.

With the aim to find new bioactive compounds to conquer Anopheles resistance, at a low cost and friendly for the environment, the essential oils extracted from C. citratus, E. tereticornis, E. citriodora and C. ambrosioides as well as their insecticide constituents namely citral, piperitone, 1,8-cineole, citronellal, 2-tridecanone, methyl salicylate, may be included in malaria vector control programs.

- Antiplasmodial tests against the K1 strain of P. falciparum The current research is a preliminary study, which has permitted to find some scientific justifications for the use of the plant species investigated and traditionally used to prevent or treat malaria in Benin. Antiplasmodial assays of the ethanolic plant extracts and the essential oils have shown a reduction of parasitemia with an

IC50 of 8.00 µg/ml to 43.07 µg/ml, on the K1 strain of P. falciparum. These results

have shown the lack of activity of many of these extracts on the K1 strain of P. falciparum. The existence of alternative biological activities against some malaria symptoms such as pain, headache, vomiting and fever, in combination with to these 131

weak reductions of parasitemia could be part of a scientific basis which allows to rationalize the amelioration of the user's health. The absence of cytotoxicity of most of these extracts supports their safe use as traditional medicine in Benin.

- Insecticidal tests against T. castaneum By fumigation and contact "no choice" test, C. schonanthus and E. citriodora have demonstrated a good insecticidal activity against T. castaneum. Indeed, by fumigation, the LC99 values for C. schoenanthus and E. citriodora were 6.85 ml/L air and 6.77 ml/L air, respectively. For piperitone and citronellal, these LC99 values were 1.29 ml/L air and 2.98 ml/L air. The mortalities obtained were 100%, 81.67%, 75% and 71.67% for piperitone, citronellal, E. citriodora essential oil and C. schoenanthus essential oil when tested at the doses of 4 ml/L air for the crude oils, 2.36 ml/L air for piperitone and 2.11 ml/L air for citronellal.

Concerning the contact "no choice" tests, the LC99 values obtained for the crude oils were after 72 hours 12.30% (w/v), 12.52% (w/v), 14.88% (w/v) and 35% (w/v) for C. schoenanthus, E. citriodora, C. giganteus and C. citratus, respectively. Concerning the mortalities, at 8% (w/v) for the crude oils, after 72 hours, 72.87%, 81.67%, 66.67% and 18.33% were noticed for the same four plants, respectively. For the same time of exposure, piperitone at 4.70% w/v (Table 9), and citronellal at 4.22% w/v (Table 9) have provoked mortalities of 86.67% and 40.00% in the population of T. castaneum.

Following the above, the essential oil from C. schoenanthus and E. citriodora as well as piperitone and citronellal represent potential valuable alternative pesticides for use against the resistance of T. castaneum to synthetic insecticides.

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CHAPTER 7: SAMENVATTING

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Malaria is een wereldwijd voorkomende ziekte, die voornamelijk in afrika ten zuiden van de sahara vele slachtoffers eist. Zo komen 80% van de nieuwe besmettingen en 90% van de malariadoden uit dit gebied. De vector van malaria behoort tot het genus anopheles, waarvan a. Gambiae giles de voornaamste vertegenwoordiger is in west- afrika. De meest kwetsbare groepen zijn zwangere vrouwen en kinderen jonger dan vijf jaar [1]. In vele afrikaanse landen, en in het bijzonder benin, ontwikkelt a. Gambiae resistentie ten opzichte van de verschillende klassen insecticiden die gebruikt worden tegen deze mug [3-5, 11, 13, 20, 23]. Pyrethroïden zijn bovendien de enige optie voor de behandeling van muggennetten voor boven bedden gezien de onschadelijkheid voor mensen bij lage dosissen, de “excito-repellent” eigenschappen, het goede “knock-down” effect en het afdoden van muggen [307]. De malariaparasiet verwierf ook resistentie tegenover algemeen gebruikte medicijnen in de behandeling van malaria zoals amodiaquine en chloroquine [308-310]. Bovenvermelde elementen leidden ertoe dat dit onderzoekswerk werd uitgevoerd om het gebruik van plantensoorten uit Benin als medicatie tegen malaria en/of voor het vermijden van muggenbeten te rechtvaardigen of net te verwerpen. Twaalf plantensoorten, waarvan het gebruik bij de preventie of behandeling van malaria nog niet wetenschappelijk werd onderzocht, werden geselecteerd in Benin. Deze plantensoorten omvatten Chenopodium ambrosioides L. (Amaranthaceae), Cymbopogon citratus (DC.) Stapf (Poaceae), Cymbopogon schoenanthus (L.) Spreng. (Poaceae), Cymbopogon giganteus Chiov. (Poaceae), Eucalyptus citriodora Hook. (Myrtaceae), Eucalyptus tereticornis Sm. (Myrtaceae), Cochlospermum planchonii Hook. f. Ex Planch. (Bixaceae), Cochlospermum tinctorium A. Rich. (Bixaceae), Erythrophleum suaveolens (Guil. & Perr) Brenan (Fabaceae), Guiera senegalensis J. F. Gmel. (Polygalaceae), Securidaca longepedunculata Fresen. (Polygalaceae) en Spondias mombin L. (Anarcadiaceae). Om de vooropgestelde objectieven te behalen, werden volgende onderzoeksstappen uitgevoerd:

- De essentiële oliën werden geëxtraheerd uit deze plantensoorten via stoomdestillatie en simultane destillatie extractie (SDE);

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- Het chemisch profiel van al deze aromatische plantensoorten werd opgehelderd via drie verschillende methoden: SDE/GC-MS, stoomdestillatie/GC-MS en SPME/GC-MS;

- De essentiële oliën, bekomen via stoomdestillatie, werden getest op de nog gevoelige A. gambia Kisumu en de resistente A. gambia Ladji, volgens het standaard WHO protocol [50];

- De anti-plasmodium assays werden uitgevoerd op de K1 stam van P. falciparum, gebruik makend van essentiële oliën geëxtraheerd met SDE en de ethanolextracten.

- Een laatste onderzoeksluik werd uitgevoerd om de vatbaarheid van Tribolium castaneum voor essentiële oliën, geëxtraheerd uit C. citratus, C. giganteus, C. schoenanthus en E. citriodora te bepalen. Het doel hiervan was nieuwe bioactieve verbindingen te vinden om zo de resistentie van de kastanjebruine rijstmeelkever tegenover synthetische insecticiden, die gebruikt worden tegen deze plaag, te omzeilen. Hierbij werden zowel fumigatie testen als “no choice” contact testen uitgevoerd. De hoofdverbinding van de meest belovende plantensoorten, met name piperiton voor C. schoenanthus en citronellal, citronellol en citronellyl acetaat voor E. citriodora werden getest tegen deze plaag, om alzo de oorsprong van de bioactiviteit van deze ruwe oliën tegenover T. castaneum te achterhalen.

De verschillende resultaten behaald in de huidige studie werden samengevat en worden weergegeven in Tabel 35 en Tabel 36.

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- Chemische profielen De verschillende chemische profielen werden geïdentificeerd, gebruikmakend van de drie hierboven beschreven methoden en zijn samengevat in Tabel 10 tot en met Tabel 22. Verschillende extractiemethoden konden voor eenzelfde plantensoort tot een verschillend chemisch profiel leiden. Deze variabiliteit is verantwoordelijk voor de verschillende biologische activiteit van de extracten, te wijten aan de variabele aanwezigheid van bioactieve verbindingen in het gebruikte extract.

- Insecticidale testen tegen A. gambiae De verschillende chemische samenstellingen werden bepaald voor de essentiële oliën, geëxtraheerd door middel van stoomdestillatie en gebruikt voor deze testen tegen A. gambiae uit te voeren. De hoofdcomponenten die geïdentificeerd werden waren neral en geranial bij C. citratus, Z-carveol, E-p-mentha-1(7),8-dien-2-ol en E-p- mentha-2,8-dienol bij C. giganteus, piperiton bij C. schoenanthus, citronellal, citronellol, citronellyl acetaat en neo-isopulegol bij E. citriodora, p-cymeen, caryofylleen oxide en spathulenol bij E. tereticornis, 3-tetradecanon bij C. tinctorium en C. planchonii, methyl salicylaat bij S. longepedunculata en ascaridol bij C. ambrosioides.

De diagnostische dosis van de essentiële oliën was 0,77% in C. citratus, 2,80% in E. tereticornis, 3,37% in E. citriodora, 4,26% in C. ambrosioides, 5,48% in C. schoenanthus en 7,36% in C. giganteus. De hoogste diagnostische dosis werd geobserveerd in S. longepedunculata (9,84%), C. tinctorium (11,56%) en C. planchonii (15,22%), vergeleken met permethrin 0,75%. Zoals verwacht vertoonde A. gambiae cotonou, resistentie tegen pyrethroïden, een significante tolerantie tegenover de essentiële oliën van C. tinctorium en S. longepedunculata, maar A. gambiae cotonou was zeer gevoelig aan alle andere essentiële oliën bij hun diagnostische dosis. Deze dosissen werden weergegeven in %, mg/ml en mg/cm2 om verder onderzoek op deze plantensoorten te vergemakkelijken. Zowel de KDT50 en KDT95 als de LC50 en LC99 en de behaalde resultaten bewijzen dat al de essentiële oliën van deze plantensoorten (getest in de diagnostische dosis) effectiever zijn tegen de resistente stam van A. gambiae dan permethrin 0,75%

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In de zoektocht naar nieuwe bioactieve verbindingen om, op een goedkope en milieubewuste manier, de resistentie van Anopheles te overwinnen, is het dus een goed idee om zowel de essentiële oliën, geëxtraheerd uit C. citratus, E. tereticornis, E. citriodora en C. ambrosioides, als hun insecticidale verbindingen, zijnde citral, piperiton, 1,8-cineol, citronellal, 2-tridecanon, en methylsalicylaat, op te nemen in controleprogramma’s tegen de malaria vector.

- Antiplasmodium testen tegen de K1 stam van P. falciparum Het huidig onderzoek is een verkennende studie, die enkele wetenschappelijk verantwoorde drijfredenen vond om het gebruik van de onderzochte en in traditionele medicijnkunde gebruikte plantensoorten in de preventie en behandeling van malaria in Benin aan te moedigen. Antiplasmodium assays met de ethanolextracten van de planten en met de essentiële oliën vertoonden een reductie

van parasitemie met een IC50 van 8,00 µg/ml tot 43,07 µg/ml tegen de K1 stam van P. falciparum. Deze resultaten toonden een gebrek aan activiteit van vele van deze

extracten tegenover de K1 stam van P. falciparum. De aanwezigheid van alternatieve biologische activiteiten tegenover sommige malariasymptomen zoals pijn, hoofdpijn, overgeven en koorts, in combinatie met deze matige reductie van parasitemie kan een wetenschappelijke verklaring bieden om het verbeteren van de gezondheidstoestand van de patiënt te verklaren. Het gebrek aan cytotoxiciteit van de meeste van deze extracten ondersteunt het veilige gebruik van deze planten in de traditionele medicijnkunde in Benin.

- Insecticidale testen tegen T. castaneum Bij fumigatie en contact "no choice" testen, vertoonden C. schoenanthus en E. citriodora een goede insecticidale activiteit tegenover T. castaneum. Bij de fumigatie testen bedroegen de LC99 waarden van C. schoenanthus en E. citriodora 6,85 ml/L lucht en 6,77 ml/L lucht, respectievelijk. Voor piperiton en citronellal waren de LC99 waarden 1,29 ml/L lucht en 2,98 ml/L lucht. De behaalde mortaliteiten waren 100%, 81,67%, 75% en 71,67% voor piperiton, citronellal, de essentiële olie van E. citriodora en C. schoenanthus, getest in een dosis van 4 ml/L lucht voor de ruwe essentiële oliën, 2,36 ml/L lucht voor piperiton en 2,11 ml/L lucht voor citronellal.

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In het geval van de contact "no choice" testen, werden na 72 h LC99 waarden van 12,30% (w/v), 12,52% (w/v), 14,88% (w/v) en 35% (w/v) behaald voor de ruwe essentiële oliën van C. schoenanthus, E. citriodora, C. giganteus en C. citratus, respectievelijk. Bij 8% (w/v) werd na 72 h voor de ruwe essentiële oliën een mortaliteit van 72,87%, 81,67%, 66,67% en 18,33% geobserveerd voor dezelfde, bovenvermelde planten. Bij eenzelfde blootstellingstijd brachten piperiton bij 4,70% w/v (Tabel 9) en citronellal bij 4,22% w/v (Tabel 9) mortaliteiten van 86,67% en 40,00% teweeg in de T. castaneum populatie.

Hieruit kan besloten worden dat zowel de essentiële olie van C. schoenanthus en E. citriodora als piperiton en citronellal waardevolle alternatieve pesticiden zijn tegenover T. castaneum, die resistent zijn tegenover synthetische insecticiden.

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CURRICULUM VITAE

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CURRICULUM VITAE Personal data Name: BOSSOU First Name: Annick Flore Arlette Dohoué Age: 15 July 1971 Emails: [email protected] [email protected] Status: Married, 2 children

Education Year Institute Diploma Currently University of Abomey-calavi- Doctor in Applied Chemistry (UAC) Benin/Ghent University-Belgium Doctor in Applied Biological Sciences (UGENT) Topic Chemical study and biological activities (insecticide and antiplasmodial) of plant extracts from Benin Promoters Prof. Félicien Avlessi Prof. Dr. ir. Norbert De Kimpe Prof. Dr. ir. Sven Mangelinckx Locations University of Abomey-calavi Polytechnical School of Abomey-Calavi Laboratory of Study and Research in Applied Chemistry Ghent University Faculty of Bioscience Engineering Department of Sustainable Organic Chemistry and Technology 2006 University of Abomey-calavi-Benin Master - Environmental Chemistry 2001 University of Abdou Moumouni- Master-Environmental Protection Niamey -Niger 1996 University of Abomey-calavi-Benin Master Degree - Natural sciences 1995 University of Abomey-calavi-Benin Bachelor - Natural sciences

Scientific publications in international journals with peer review 1- Bossou A, Mangelinckx S, Yedomonhan H, Boko P, Akogbeto M, De Kimpe N, Avlessi F, Sohounhloue DC: Chemical composition and insecticidal activity of plant essential oils from Benin against Anopheles gambiae (Giles). Parasit Vectors 2013, 6:337-353.

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2- Noudogbessi JPA, Natta AK, Tchobo FP, Bogninou GS, Bothon FTD, Bossou AD, Figueredo G, Chalard P, Chalchat JC, Sohounhloué DCK: Phytochemical Screening of Pentadesma butyracea Sabine (Clusiaceae) Acclimated in Benin by GC/MS. ISRN Analytical Chemistry 2013, 2013:1-8.

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