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How to cite this thesis

Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujcontent.uj.ac.za/vital/access/manager/Index?site_name=Research%20Output (Accessed: Date).

DETERMINATION OF BIOLOGICAL ACTIVITY OF CELTIS

AFRICANA EXTRACTS AND ITS ENDOPHYTIC MICROFLORA AND

MYCOFLORA.

A Dissertation submitted to the Faculty of Science,

University of Johannesburg

In partial fulfilment of the requirement for the award of a

Master’s Degree in Technology: Biotechnology

EVONIA KANYANE NCHABELENG

STUDENT NUMBER: 201182098

Supervisor: Dr. V. Mavumengwana

Co-supervisor: Dr. D.T. Ndinteh

Co-supervisor: Dr. N. Niemann

JANUARY, 2017 ABSTRACT With the rapid rise in untreatable diseases, researchers are compelled to search for new drugs that can combat these diseases. Plants are recognized as unlimited sources of bioactive compounds that can be used to treat different ailments and also be applied in various sectors including agriculture, pharmaceutical and food. In addition, plants are also known to be associated with endophytes such as bacteria and fungi, which are also regarded as fertile sources of bioactive constituents. Celtis africana is an ornamental and medicinal plant that is used to treat different ailments. In the study reported herein, the aims were to identify and characterize secondary metabolites produced by both the plant and its endophytes and to further investigate the antibacterial activity of C. africana extracts against fourteen pathogenic bacteria.

To achieve these aims, endophytes were isolated from fresh and apparently healthy aerial parts (leaves, stems and fruit) of C. africana. The isolated endophytes were then cultivated and secondary metabolites extracted sequentially with hexane, dichloromethane and ethyl acetate. Dried plant aerial parts were screened for the presence of phytochemicals, extracted successively with hexane, ethyl acetate and dichloromethane: methanol (1:1 v/v) and the crude extracts tested for antibacterial activity. The crude extracts of both the plant parts and endophytes were analyzed using two dimensional gas chromatography coupled with time of flight mass spectrometry(GC×GC-TOF/MS ) to determine their volatile secondary metabolites constituents.

From the results obtained, it is evident that C. africana has endophytic bacteria and fungi in the stem and fruit, but not in the leaves. Seven bacteria (Kocuria sp., Micrococcus luteus, Staphylococcus hominis, Bacillus sp., Staphylococcus saprophyticus, Brachybacterium conglomeratum and Arthrobacter sp) were isolated and identified. Four fungal endophytes, all belonging to the same genus (Aspergillus) were also isolated and identified, of which two were identified to genus level and two were found to be A. niger and A. flavus. The plant crude extracts showed antibacterial activity against seven of the test microorganisms, viz., Escherichia coli, Proteus mirabilis, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter aerogenes. These microorganisms are causal agents of various ailments ranging from food-borne illnesses to skin infections, urinary tract infections and pneumonia.

The profiles of volatile secondary metabolites of both the endophytes and the plants revealed the presence of numerous compounds with reported biological importance ranging from antimicrobial to antioxidant, anticancer and flavoring. Furthermore, the endophytes had similarities in the type of secondary metabolites they produce with specificity to the alkaloidal compounds (diketopiperazines). The current study has laid a groundwork on the types of secondary metabolites that C. africana and its endophytes produce, and thus both the plant and endophytes are suggested to be a potential source of compounds with therapeutic uses.

Keywords: Antibacterial, bioactive compounds, Celtis africana, endophytes, secondary metabolites

DECLARATION I, Evonia Kanyane Nchabeleng hereby declare that this study titled “Determination of biological activity of Celtis africana extracts and its endophytic microflora and mycoflora” is my own work, completed under the supervision of Dr. V. Mavumengwana, Dr. N. Niemann, and Dr. D.T. Ndinteh. Furthermore I declare that this work has never been submitted elsewhere as a fulfillment for any qualification. In case where other sources have been used, acknowledgement has been fully given with citations and full references.

______

EVONIA KANYANE NCHABELENG

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DEDICATION This work is dedicated to my parents Mr. T.J. and Mrs. M.M. Nchabeleng, I am where I am today because of the immense support and sacrifices they have made.

THANKS AND ACKNOWLEDGEMENTS My deepest gratitude goes out to the below individuals and organizations for their support throughout this study:  To my supervisors Dr. V. Mavumengwana, Dr. N. Niemann, and Dr. D.T. Ndinteh, this study has been a success solely because of your guidance, constructive criticism, patience, and for providing all the needed materials throughout the course of this study. Most importantly thank you, for being helping out even when you were in your personal space. Not only have you groomed me academically but you have also grown me personally as an individual. A big thank you.

 To my parents, Mr. and Mrs. Nchabeleng, thank you for the support you have given me throughout my life. To my siblings, Johanna, Mpho, Thabo, and my cousin Phummy, your support, encouragement and inspiration to do better are much appreciated. You all have been my pillars of strength and source of motivation. “Kea leboga Batau ba Seokodibeng”

 My laboratory colleagues, Bongeka Mbambo and Letlhabile Moyaha, thank you for your guidance through the fruitful discussions we would have over the late night and weekend lab experiments. I also sincerely thank Tendani Sebola, Natasha Samongoe, Nkem Ucheokafor and Mbali Webb with whom I have also worked with.

 Dr. Judith Phoku thank you for the assistance with the fungal isolates identification and fermentation. Dr. S. Sekar, for your help with the bacterial fermentation and construction of the phylogenetic trees. Mr. O. Adebo, your assistance with reviewing part of this work is much valued.

 I would also like to thank the National Research Foundation for funding this study, the University of Johannesburg Merit bursary and also Alumni Bursary for partially funding this study.

 Lastly I would to thank I AM, the Omnipresent Almighty God, whom through His grace and love I am still breathing. Greater is He that is within me than he that is in the world (1 John 4:4).

ARTICLES PREPARED FOR PUBLICATION Nchabeleng, E.K., Ndinteh, D.T., Niemann, N., Mavumengwana, V. In vitro antibacterial activity testing and profiling of volatile constituents of the leaves, stem, and fruit of Celtis africana from South Africa. Prepared for submission to South African Journal of Botany. Nchabeleng, E.K., Ndinteh, D.T., Niemann, N., Mavumengwana, V. Identification and characterization of volatile secondary metabolites produced by endophytic fungi associated with Celtis africana from South Africa. Prepared for submission to Journal of Microbiology and Biotechnology. Nchabeleng, E.K., Ndinteh, D.T., Niemann, N., Mavumengwana, V. Identification and characterization of volatile secondary metabolites produced by endophytic bacteria associated with Celtis africana from South Africa. Prepared for submission to Journal of Microbiology and Biotechnology.

TABLE OF CONTENTS ABSTRACT ...... i DECLARATION...... iii DEDICATION...... iv THANKS AND ACKNOWLEDGEMENTS ...... v ARTICLES PREPARED FOR PUBLICATION …...... vi TABLE OF CONTENTS ...... vii LIST OF FIGURES ...... xii LIST OF TABLES ...... xiii LIST OF ABBREVIATIONS AND ACRONYMS ...... xiv LIST OF UNITS ...... xv DISSERTATION OUTLINE ...... xvi CHAPTER ONE ...... 1

1.0 GENERAL INTRODUCTION ...... 1 1.1 Background ...... 1 1.2 Problem Statement ...... 2 1.3 Aims ...... 2 1.4 Objectives ...... 3

CHAPTER TWO ...... 4 2.0 LITERATURE REVIEW ...... 4 2.1 MEDICALLY IMPORTANT AND COMMON PATHOGENS ...... 4 2.1.1 Gram positive pathogens………………………………………………………………4

2.1.1.1 Staphylococcus aureus and Staphylococcus epidermidis (Family: Staphylococcae) ...... 4

2.1.1.2 Enterococcus faecalis (Family: Enterococcaceae) ...... 5

2.1.1.3 Bacillus cereus and B. subtilis (Family: Bacillaceae) ...... 5

2.1.2 Gram negative pathogens ...... 5 2.1.2.1 Klebsiella pneumonia and Klebsiella oxytoca (Family: Enterobacteriaceae) . 5

2.1.2.2 Enterobacter cloacae and E. aerogenes (Family: Enterobacteriaceae) ...... 6

2.1.2.3 Proteus mirabilis and P. vulgaris (Family: Enterobacteriaceae) ...... 6

2.1.2.4 Escherichia coli (Family: Enterobacteriaceae) ...... 7

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2.1.2.5 Pseudomonas aeruginosa (Family: Pseudomonadaceae) ...... 7

2.2 MEDICINAL PLANTS AS TRADITIONAL MEDICINE…………………………...8

2.3 NATURAL PRODUCTS FROM PLANTS…………………………………………..8

2.3.1 Classes of phytochemicals…...... 9

2.3.1.1 Terpenes…………………………………………………………………………...... 9

2.3.1.1.1 Saponins……………………………………………………………………10

2.3.1.1.2 Steroids/ Plant sterols……………………………...... 11

2.3.1.2 Phenolic compounds…………………………………………………………………11

2.3.1.2.1 Tannins.……………………………………………………………...... 11

2.3.1.2.2 Flavonoids……………………………...... 12

2.3.1.3 Nitrogen Containing Phytochemicals………………………………………………...13

2.3.1.3.1 Alkaloids.…………………………………………………...... 13

2.4 FAMILY CANNABACEAE: OVERVIEW………………………………………...... 14

2.5 THE GENUS CELTIS……………………………………………………………...... 15

2.6 THE SPECIES CELTIS AFRICANA…………………………………………………16

2.6.1 Botanical description of C. africana…………………………………...... 16

2.6.2 Distribution and habitat of C. africana………………………………………17

2.6.3 Ethnobotany of C. africana…………………………………………...... 18

2.6.4 Phytochemistry and pharmacology of C. africana…………………...... 18

2.7 PLANT AND MICROORGANISM RELATIONS……………………………...... 19

2.7.1 Symbiosis of plants and microorganisms…………………………………….19

2.8 BENEFICIAL PLANT BACTERIA/FUNGI……………………………………...... 19

2.8.1 Root colonizing bacteria……………………………………………………...19

2.8.2 Endophytes…………………………………………………………………...20

2.8.2.1 Location of endophytes………………………………………………………21

2.8.2.2 Advantages of endophytes…………………………………………………...21

2.8.2.3 Endophytes associated with members of Cannabaceae plant family………..22

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2.8.2.4 Natural products from endophytes…………………………………………...22

2.9 Concluding remarks ...... 24

References ...... 25

CHAPTER THREE ...... 45

ISOLATION AND IDENTIFICATION OF ENDOPHYTIC MYCO- AND MICROFLORA FROM CELTIS AFRICANA (STINKWOOD) TREE ...... 45 Abstract ...... 45

3.1 Introduction ...... 46 3.2 Materials and methods ...... 47

3.2.1 Sample collection ...... 47 3.2.2 Endophytes isolation ...... 47 3.2.3 Morphological identification of fungal endophytes ...... 48 3.2.4 Molecular identification ...... 48 3.3 Results and Discussion ...... 49

3.3.1 Morphological identification of fungal endophytes ...... 49 3.3.2 Molecular identification of fungal endophytes ...... 52 3.3.3 Identification of bacterial endophytes ...... 55 3.4 Conclusion ...... 57

References ...... 57 CHAPTER FOUR ...... 61

IN VITRO ANTIBACTERIAL ACTIVITY TESTING AND PROFILING OF VOLATILE CONSTITUENTS OF THE LEAVES, STEM AND FRUIT OF CELTIS AFRICANA FROM SOUTH AFRICA ...... 61

Abstract ...... 61 4.1 Introduction ...... 62 4.2 Materials and methods ...... 63

4.2.1 Sample collection ...... 63 4.2.2 Plant crude extract preparation ...... 63 4.2.3 Preliminary phytochemical screening ...... 64 4.2.3.1 Test for tannins ...... 64 4.2.3.2 Test for alkaloids ...... 64

4.2.3.3 Test for flavonoids ...... 64 4.2.3.4 Test for saponins ...... 64 4.2.3.5 Test for steroids ...... 65 4.2.3.6 Test for reducing sugars ...... 65 4.2.4 Antibacterial screening and minimum inhibitory concentration (MIC) determination ...... 65

4.2.4.1 Sample preparation ...... 65 4.2.4.2 Experimental procedure ...... 66 4.2.5. Profiling of volatile constituents ...... 66

4.2.5.1 GCxGC-TOF/MS conditions ...... 66 4.2.5.2 GCxGC-TOF/MS data processing ...... 67 4.3 Results and Discussion ...... 67

4.3.1 Preliminary phytochemical screening ...... 67 4.3.2 Antibacterial activity screening and minimum inhibitory concentration (MIC) determination ...... 69

4.3.3 Profiling of volatile constituents ...... 71

4.4 Conclusion ...... 89

References ...... 89

CHAPTER FIVE ...... 98

IDENTIFICATION AND CHARACTERIZATION OF VOLATILE SECONDARY METABOLITES PRODUCED BY ENDOPHYTIC FUNGI ASSOCIATED WITH C. AFRICANA FROM SOUTH AFRICA ...... 98 Abstract ...... 98 5.1 Introduction ...... 99 5.2 Materials and methods ...... 100

5.2.1 Fungal isolates resuscitation ...... 100

5.2.2 Fermentation and secondary metabolite extraction ...... 100 5.2.3 Profiling of volatile secondary metabolites ...... 100 5.2.3.1 Sample preparation ...... 100 5.2.3.2 GC×GC-TOF/MS conditions ...... 101 5.2.3.3 GCxGC-TOF/MS data processing ...... 101

5.3 Results and Discussion ...... 101

5.3.1 Extracellular secondary metabolites from the fungal endophytes ...... 102

5.3.2 Intracellular metabolites investigations of the fungal endophytes ...... 112

5.4 Conclusion ...... 117

References ...... 117

CHAPTER SIX ...... 123

IDENTIFICATION AND CHARACTERIZATION OF SECONDARY METABOLITES PRODUCED BY ENDOPHYTIC BACTERIA ASSOCIATED WITH CELTIS AFRICANA FROM SOUTH AFRICA ...... 123 Abstract ...... 123 6.1 Introduction ...... 124 6.2 Materials and methods ...... 125

6.2.1 Bacterial isolates resuscitation ...... 125

6.2.2 Fermentation and secondary metabolite extraction ...... 125 6.2.3 Profiling of volatile secondary metabolites ...... 126 6.2.3.1 Sample preparation ...... 126 6.2.3.2 GC×GC-TOF/MS conditions ...... 126 6.2.3.3 GCxGC-TOF/MS data processing ...... 126 6.3 Results and Discussion ...... 126

6.4 Conclusion ...... 138 References ...... 138

CHAPTER SEVEN ...... 142

7.0 GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATIONS ... 142

7.1 GENERAL DISCUSSION AND CONCLUSION ...... 142

7.2 RECOMMENDATIONS ...... 144

References ...... 145

Appendices……………………………………………………………………………..147-215

LIST OF FIGURES Figure 2.1: Terpenes chemical structures...... 10 Figure 2.2: Chemical structures representing the two classes of saponins...... 10 Figure 2.3: Common and widely distributed plant sterols ...... 11 Figure 2.4: Representative chemical structures of monomers for hydrolysable tannins (Ellagic and gallic acids) and condensed tannins (catechin and gallocatechin) ...... 12 Figure 2.5: Examples of compounds from four subclasses of flavonoids. …...... 13 Figure 2.6: Some examples of pharmaceutically significant alkaloids ...... 14 Figure 2.7: Images of C. africana...... 17 Figure 2.8: Geographical distribution of C. africana in South Africa ...... 17 Figure 2.9: The structure of the two novel compounds isolated from the plant as studied by Perveen et al. (2011)...... 19 Figure 2.10: General mechanisms of entry of endophytes in host plants ...... 21 Figure 3.1: Macroscopic illustrations depicting the morphology of the fungal endophytes ... 50 Figure 3.2: Microscopic images of the fungal isolates...... 51

Figure 3.3: Phylogenetic tree based on the ITS sequences showing the evolutionary relationships of the fungal endophytes from C. africana with the closest fungal relatives. .... 54 Figure 3.4: Phylogenetic tree based on the 16S rRNA sequences with sequences of close in close relations in the GenBank database...... 56 Figure 4.1: Some of the significant ketone based compounds detected from the plant extracts by 2D-GC-TOF/MS………………………………………………………………………….73

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LIST OF TABLES Table 2.1: Representatives of species in the Cannabaceae family ...... 15 Table 2.2: Examples of various bioactive natural products from endophytes ...... 23 Table 3.1: General Table showing the endophytes isolated from C. africana...... 49 Table 3.2: Identification of the endophytic fungal strains from C. africana ...... 53 Table 3.3: Identification of bacterial endophytic strains from C. africana ...... 55 Table 4.1: Phytochemical components present in the crude extracts of C. africana ...... 68 Table 4.2: Antibacterial screening of the crude extracts...... 70 Table 4.3. Minimum Inhibitory Concentrations of C. africana crude extracts...... 71 Table 4.4: Ketones detected in the crude extracts of C. africana ...... 74 Table 4.5: Fatty acids and aldehydes detected from the crude extracts of C. africana ...... 76 Table 4.6: Alcohol compounds detected from the crude extracts of C. africana ...... 79

Table 4.7: Ester compounds detected from the crude extracts of C. africana ...... 83 Table 4.8: Hydroxypyrones, Nitrogenous bases and other compounds detected in the crude extracts of C. africana...... 86 Table 5.1: Extracellular secondary metabolites detected from the hexane broth crude extracts ...... 104 Table 5.2: Extracellular secondary from the ethyl acetate crude extracts ...... 108 Table 5.3: Extracellular secondary metabolites detected in the DCM crude extracts ...... 111 Table 5.4: Intracellular secondary metabolites detected in the hexane crude extracts ...... 114 Table 5.5: Intracellular secondary metabolites detected in the ethyl acetate crude extracts. 115 Table 5.6: Intracellular secondary metabolites detected in the DCM crude extracts...... 116 Table 6.1: Secondary metabolites from the hexane crude extracts of the bacterial endophytes ...... 128 Table 6.2: Secondary metabolites from ethyl acetate crude extracts of the bacterial endophytes ...... 132 Table 6.3: Secondary metabolites from DCM crude extracts of the bacterial endophytes. .. 136

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LIST OF ABBREVATIONS AND ACRONYMS 2D-GC-TOF/MS 2 Dimensional Gas Chromatography Time of Flight Mass Spectrometry ACC Amino Cyclopropane Carboxylate BLAST Basis Local Alignment Search Tool DCM Dichloromethane DCM: MeOH Dichloromethane: Methanol DMSO Dimethyl Sulfur Oxide ESBL Extended Spectrum Beta-Lactamases GCxGC-TOF/MS 2-Dimensional Gas Chromatography Time of Flight Mass Spectrometry ITS Internal Transcribed Spacer MeOH Methanol MHB Muller-Hinton Broth NA Nutrient Agar NAD National Agricultural Directory NB Nutrient Broth NCBI National Center for Biotechnology Information ND Not Detected PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PDA Potato Dextrose Agar PDB Potato Dextrose Broth PGPR Plant Growth Promoting Rhizobacteria rRNA ribosomal Ribo-nucleic Acid THP Traditional Health Practitioner TSS Toxic Shock Syndrome UTI Urinary tract infection

xiv

LIST OF UNITS % Percentage °C Degree Celsius µL Microliter cfu/ mL Colony forming units/ Milliliter g Gram h Hour L Liter mg/ mL Milligrams/Milliliter min Minutes mL Milliliter rpm Revolutions per minute s Second v/v Volume/ Volume w/v Weight/ Volume

DISSERTATION OUTLINE The dissertation is structured into seven chapters, the description of each chapter* is given below:

*Chapters three to six are written in an article format.

Chapter 1: General Introduction This chapter addressed the problem statement in relation to the study, in addition the background of the study subject is given, with the aims and objectives of the study ending the chapter.

Chapter 2: Literature Review This Chapter is an in depth review on the subject of study. Where the medicinal properties and phytochemistry of the plant (Celtis africana) under study are fully reviewed. The chapter further discusses endophytes associated with higher plants and different classes of phytochemicals from plants in general are discussed.

Chapter 3: Isolation and identification of endophytic myco- and microflora from Celtis africana (stinkwood) tree This chapter is written up in a paper format and it presents the isolation and identification of endophytes from the aerial parts of the plant under study (C. africana).

Chapter 4: In vitro antibacterial activity testing and profiling of volatile constituents of the leaves, stem, and fruit of Celtis africana from South Africa. The fourth chapter of the dissertation focuses on the types of constituents present in the plant and as such, identify compounds produced by the plant. The chapter further describes the antimicrobial activity of the different extracts from the plant. Chapter 5: Identification and characterization of volatile secondary metabolites produced by endophytic fungi associated with Celtis africana from South Africa. The metabolites produced by the fungal endophytes from C. africana are described in this chapter. Chapter 6: Identification and characterization of volatile secondary metabolites produced by endophytic bacteria associated with Celtis africana from South Africa. This chapter presents the types of secondary metabolites extracted with different solvents from the bacterial endophytes of C. africana.

xvi

Chapter 7: General discussion, conclusion and recommendations This constitutes the last chapter of the dissertation, in this chapter the outcomes from the studies done (Chapter three to Chapter six) are generally discussed and conclusions drawn, the chapter further discusses the challenges observed in the study and recommendations for future work.

Appendices The complete 2D-GC-TOF/MS data, fungal and bacterial sequence data are outlined in the appendices from page 147-215

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CHAPTER ONE

1.0 GENERAL INTRODUCTION 1.1 BACKGROUND For centuries, humans have always depended on plants for food, medicine and shelter. Plants have also had other uses such as clothing and religious ceremonies. It was through ancestral observations that plants were noticed to have healing powers, it was then later discovered through scientific studies that the healing properties are from active chemical compounds present in those plants (Afzal et al., 2015). Traditionally, when these plants are used for medicinal purpose, they are usually used as they are, prepared as decoction or pastes (de Brum et al., 2016).

According to Camou-Guerrero et al. (2016), ethnobotany is a scientific discipline that resulted from the co-evolution of anthropology and botany. Gonsalves (2010), defines ethnobotany as the study of how indigenous plants are used by people of a certain culture and region. From the definition by Gonsalves, it shows that ethnobotany is multidisciplinary. Ethno medicine is one discipline of ethnobotany which focuses on the study of traditional medicines. This discipline can be further divided into medicinal ethnobotany and ethno-pharmacology.

In medicinal ethnobotany, the traditional uses of plants with respect to human healthcare are studied, it deals with the nature of the traditional medicine, preparation and how it is applied in the traditional medical system (Rehman et al., 2015). Furthermore, it also investigates how certain ethnic groups use the plants to prevent or manage diseases (Rehman et al., 2015). The ethno-pharmacology discipline is mainly the scientific evaluation of the medicinal plants, this provides experimental evidence about the uses of the medicinal plants (Leonti, 2011). In these kinds of studies, the plants are extracted, different compounds from that particular plant are identified and also the plant is tested for bioactive properties (de Brum et al., 2016).

Every plant on earth has an association with microorganisms. The microorganisms can be ecto or endosymbionts. Endophytes are microorganism that are known to be non-pathogenic and are found inside the plants. The plant and the endophytes have a mutual relationship. As such, the inhabitation of an endophyte inside a plant benefit both the microorganism and the plant. Almost all the plants studied have been found to have endophytes (Kusari et al., 2014).

Plants and microorganisms both can display primary and secondary metabolism, the former involves basic biochemical pathways that allow for the production of compounds that are

1 essential for the survival and growth of the cells, whilst the latter includes pathways that allow for the production of compounds which allow for the cells to be interactive and competitive within its ecosystem and not necessarily needed for basic survival of the cell (Ahmed et al., 2015; Seigler, 1998). These secondary metabolites are generally useful in various sectors e.g., food, medicine and cosmetics. Consequently, both plants and endophytes (fungi and bacteria) are considered as outstanding sources of bioactive natural compounds which are used in agriculture, pharmaceutical industries, and as such they could be alternative sources of therapeutic compounds to help eradicate infections affecting the human population (Ma et al., 2015; Venugopalan and Srivastava, 2015).

1.2 PROBLEM STATEMENT Ever since antibiotics were discovered about eighty years ago, they were believed to be a perpetual solutions for the eradication of infectious diseases, however, these antibiotics and other antimicrobials are now less effective in treating these diseases (Paphitou, 2013). This is due to the re-emergence of old infectious diseases caused by pathogens which are resistant to the present antibiotics, furthermore, new infections caused by various agents such as viruses, fungi, bacteria, and parasites are also increasing (Muzzamal et al., 2012). These new infections and the antimicrobial resistance of already existing pathogens is a global health concern (While, 2016; Sibanda and Okoh, 2007). The World Health Organization (WHO, 2014) global surveillance report on antimicrobial resistance has revealed that common pathogens which are associated with common and community acquired infections have shown high rates of drug resistance. Moreover, traditional synthetic drugs being used currently have hostile side effects, they have also become less effective due to over prescription and misuse, in contrast to conventional drugs, natural products have less side effects and higher pharmacological activity (Bonifacio et al. 2014). Therefore, these public health issues necessitate the need for the development of new drugs that can control these diseases. On this basis, this project focused on the deciduous plant species Celtis africana, which is used as a traditional medicinal and ornamental plant. The plant parts (leaves, bark and roots) are used for treating symptoms associated with ailments ranging from eye infections, rheumatism, cancer and sexually transmitted diseases. Previous studies of the plant were focused more on its phytochemistry, where new compounds were isolated and identified.

1.3 AIMS The study aimed to extract, characterize, identify and assess the bioactivity (antimicrobial activity) of the secondary compounds from C. africana. The study was further aimed at

2 isolating, characterizing and the identification of potential bioactive compounds from isolated endophytic bacteria and/or fungi of C. africana.

1.4 OBJECTIVES For the aims stated in Section 1.3 to be achieved, specific objectives for the proposed study were:  To isolate and identify endophytic bacteria and/ fungi from the plant  To extract and identify secondary metabolites from the endophytic isolates using GCxGC- TOF/MS  To screen the plant for the presence of phytochemicals  To extract phytochemicals from C. africana and test their bioactivity against pathogenic microbes  To characterize and identify the secondary metabolites of C. africana using GCxGC- TOF/MS.

CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 MEDICALLY IMPORTANT AND COMMON PATHOGENS The on-going ability of pathogens to develop resistance against multiple drugs is a serious challenge, and it is claimed that unless something is done to eradicate this problem, the world might find itself facing the same challenges as those before the antibiotic era (Edward-Jones, 2013). Discovery of new therapeutic agents has always had challenges, for example the targeted microbial pathogens always develop resistance to that particular antimicrobial. Presently, while the discovery of new antimicrobials has detrimentally decreased, the emergence of these drug resistant pathogens and new infections is rapidly growing (Rajan and Kannabira, 2014).

Gene mutations and development of resistance genes that were transferred from other bacteria through plasmids are the general mechanisms of drug resistance in bacteria (Burgess and Traub-Dargatz, 2014). Both Gram positive and Gram negative bacteria are able to be resistant to current drugs. Discussed in sections 2.1.1 and 2.1.2, are bacteria associated with hospital acquired (nosocomial) infections (Trubiano and Padiglione, 2015), which are also noted for resistance to antimicrobials currently in use.

2.1.1. Gram positive pathogens 2.1.1.1 Staphylococcus aureus and Staphylococcus epidermidis (Family: Staphylococcae) Staphylococcus aureus is one of the most common Gram positive bacteria that is normally a skin flora. However, some strains are pathogenic and responsible for various ailments including foodborne related diseases, endocarditis, toxic shock syndrome (TSS), pneumonia, bacteremia and sepsis (Raffaella et al., 2017; Barbosa et al., 2016). This microbe is also is stated to be one of those pathogens that develop resistance to drugs over a short period of time, and in some instances can be resistant to widely used antibiotics such as streptomycin, erythromycin, vancomycin and methinicillin (Ma et al., 2017; Rueda, 2013).

S. epidermidis is another Staphylococci of medical importance, this microbe was previously considered as a non- pathogenic inhabitant of the human skin, however it this currently known as an opportunistic pathogen that is responsible for hospital acquired infections (Li et al., 2016). The infections include bacteremia and infections that result due to the pathogen’s ability to form biofilms on nervous central shunts and prosthetic joints (Hijazi et al., 2016; Gomes et al., 2014). Although S. epidermidis does not have many virulent genes, its ability to form biofilms

4 enhances its pathogenicity and thus it has become resistant to antimicrobials such as vancomycin, fluoroquinolones and erythromycin (Sharma et al., 2016; Gomes et al., 2014).

2.1.1.2 Enterococcus faecalis (Family: Enterococcaceae) According to Fernandes et al. (2015), over 80% of human infections are caused by E. faecalis. This microbe belong into the genus Enterococcus and is known to cause infections in individuals with compromised immune systems. The microbe is in addition resistant to common antibiotics such as aminoglycosides, penicillins and glycopeptides (Hollenbeck and Rice, 2012; McBride et al., 2010). Infections caused by E. faecalis include bacteremia, wound infections, urinary tract infections, and endocarditis (Hollenbeck and Rice, 2012; Kau et al., 2005).

2.1.1.3 Bacillus cereus and B. subtilis (Family: Bacillaceae) Bacillus genus consists of species that are abundant in the environment (Fernadenz-No et al., 2011). Of the species in this genus, B. cereus is the one most commonly associated with foodborne diseases, and can survive harsh conditions like high temperatures and dry conditions. Therefore that is the reason it is responsible for food poisoning (Vaughan et al., 2003). The diseases are caused as results of the species to produce cytotoxins, hemolysins and enterotoxins which will induce emesis (vomiting) and diarrhea when the B. cereus contaminated food are consumed (Thorsen et al., 2015). Nosocomial infections where B. cereus is the causative agent are those affecting the eyes, soft tissues, central nervous system and diseases such as pneumonia and bacteremia (Ikeda et al., 2015). Like many Gram positive pathogenic bacteria, B. cereus is resistant to antibiotics including gentamicin, methicillin, tetracycline and kanamycin (Fenselau et al, 2008; Schlegelova et al., 2003). In comparison with the other members in the Bacillus genus, the B. subtilis is the one less associated with pathogenicity (Fernadenz-No et al., 2011). Although B. subtilis has beneficial uses, like using them as probiotics in food (Lefevre et al., 2017), there has been a report where strains of this species were found to produce toxins that caused food poisoning (Pavic et al., 2005).

2.1.2. Gram negative pathogens 2.1.2.1 Klebsiella pneumonia and Klebsiella oxytoca (Family: Enterobacteriaceae) One of the most common Gram negative pathogens is K. pneumoniae, this pathogen is a causative agent of different ailments such as wound infections, pneumonia and urinary tract infections (UTI). In addition the nosocomial infections caused by this pathogen normally affect immunocompromised individuals, infants and the elderly (Wyres and Holt, 2016). Most strains

5 of K. pneumoniae have acquired genes for enzymes (carbapenemases and extended spectrum beta-lactamases (ESBLs)) that have the ability to hydrolyze antibiotics and thus render them ineffective (Doorduijn et al., 2016; Weterings et al., 2015; Arnold et al., 2011). In a study by Elemam et al. (2009), antibiotics such as tigecyline and some beta lactams (ampicillin, piperacillin-tazobactam and cefazolin) proved ineffective against a K. pneumoniae strain, an observation supporting the notion that the pathogen has enzymatic abilities that overcome antibacterial agents.

K. oxytoca is another Klebsiella species that is an opportunistic pathogen, responsible for causing diseases such as enterocolitis, urinary tract infections, pneumonia, and bacteremia and in rare cases this opportunistic pathogen is the causal agent of inflammation of testicles in men (Alikhani et al., 2016; Lee et al., 2016; Fujita et al., 2015). There are reports that certain strains in this species have carbapenemases, extended-spectrum β-lactamases and β-lactamases genes encoded in their plasmids (Singh et al., 2016; Fujita et al., 2015; Decre et al., 2004) and thus this enables them to be resistant to multiple antimicrobials.

2.1.2.2 Enterobacter cloacae and E. aerogenes (Family: Enterobacteriaceae) E. cloacae and E. aerogenes are important pathogenic members of the Enterobacter genus (Talon et al., 2000). According to Jha et al (2016), species in the Enterobacter genus rank in the top 8 of common nosocomial infections in the USA. Furthermore E. cloacae and E. aerogenes together constitute 90 % of isolates from health-care setting/hospitals (Talon et al., 2000). In humans, E. cloacae is responsible for hospital acquired infections including urinary tract infections, pneumonia, sepsis and wound infections (Akbari et al., 2016; Yap et al., 2016). E. aerogenes like its relative (E. cloacae) is responsible for various human ailments including urinary and respiratory tracts infections, post-chirurgical infections, sepsis and bacteremia (Jha et al., 2016; Philippe et al., 2015).

Like other pathogenic members in the Enterobacter genus, both E. cloacae and E. aerogenes strains have been reported to have acquired plasmids that have carbapenem and β-Lactam resistant genes (Flury et al., 2016; Jha et al., 2016; Girlich et al., 2015; Majewski et al., 2014). Thus it shows that these pathogens are a huge burden and are clinically important microbes.

2.1.2.3 Proteus mirabilis and P. vulgaris (Family: Enterobacteriaceae) All members of the Proteus genus are considered human pathogens, and have enhanced virulence as a result of the cilia structures on their cells and in addition these structures also increase their resistance to antibiotics (Kwiecinska-Pirog et al., 2016). Furthermore members

6 in this genus are causal agents of health-care acquired diseases such as prostatitis, pyelonephritis, urinary tract infections and cystis. P. mirabilis is furthermore claimed to be a causative agent of rheumatoid arthritis (Jacobsen and Shirtliff, 2011; Zych et al., 2007). Persistent Proteus infection of the urinary tract, in most cases leads to the formation of kidney and bladder stones (Toukach et al., 2003). P. vulgaris is also associated with food poisoning where it can result in gastroenteritis (Myszka and Czaczyk, 2011).

2.1.2.4 Escherichia coli (Family: Enterobacteriaceae) E. coli (extra-intestinal and intra-intestinal) forms part of the microbiome that inhabits the intestinal tract of animals and humans (Carlet, 2012). While not all strains of E. coli are non- pathogenic, some have fatal effects on humans and animals. Extra-intestinal E. coli are known causal agents of different human diseases including meningitis, urinary tract infections and septicemia (Jafari et al., 2012) whereas, the intra-intestinal type are mainly responsible for diarrhea as a result of consuming contaminated food, water or through person to person contact (Schwarts et al., 2009). There are different types E. coli that are responsible for diarrhea in humans namely: shiga toxin-producing E. coli which include the enterohaemorrhagic E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, enteroinvasive E. coli and diffuse-adherent E. coli (Akinduti et al., 2016; Clarke et al., 2002). These E. coli types are said to be prevalent in developing countries, especially the enterotoxigenic type (Akinduti et al., 2016; Qadri et al., 2005). There is a rapid rise of E. coli resistance of antibiotics, specifically to the cephalosporins and fluoroquinolones types (Collignon, 2009).

2.1.2.5 Pseudomonas aeruginosa (Family: Pseudomonadaceae) P. aeruginosa is a ubiquitous opportunistic pathogen that is recognized as the most medically important pathogen from the Pseudomonas genus (Selim et al., 2015). It is predominantly associated with inflammation of the external ear (otitis externa) (Bateman et al., 2012). Other health-care associated infections caused by P. aeruginosa include those affecting the skin, wounds and septicemia infections (Song et al., 2016). According to Hammer et al. (2017), the outer membrane’s low permeability in addition to its chromosomal resistant genes makes it resistant to various antibiotics.

Despite diseases caused by bacteria, there are other incurable chronic conditions such as cancer, diabetes, and fungal infections that need to be controlled.

2.2 MEDICINAL PLANTS AS TRADITIONAL MEDICINE Before the era of modern medicine, medicinal plants have been traditionally used to treat numerous ailments (Huang et al., 2016). It is estimated that there are over 21000 medicinal plants out of the 350 000 known plants in the whole world (Sheik et al., 2016). Although drugs are being developed to help improve and contribute to human healthcare, a great proportion of the world population cannot afford to buy these drugs and therefore are not able to access modern medical treatments (Ansari, 2016). Relative to modern medicine, Chan (2015), stated that herbal medicines and other traditional treatments are more affordable and easily accessible, especially to people living in rural areas of developing countries, in addition to that Patil et al. (2013), also reported that approximately 80% of the population globally relies on medicinal plants for primary healthcare needs. Countries such as China, utilize a combination of western and traditional medicine to treat ailments (Mokgolodi et al., 2011).

In South Africa, approximately 70 % of the population is said to be using traditional medicine or relying on traditional health practitioners (THPs) (Nethathe et al., 2016). In addition to that, South Africa has a rich plant diversity with over 24 000 higher plant species inhabiting it. From the 24 000 plants, about 3000 species have been reported to be used as medicines by different ethnic groups (de Wet et al., 2013). Because of the popularity of traditional medicines, medicinal plant demand is high in both rural and urban areas of the country. These traditional medicines can be bought from street traders (informal trading) or in African medicine shops (muthi shops) where the packaging is modernized (Mander et al., 2007). According to the National Agricultural Directory (NAD) of South Africa (2011), an estimated amount of R2.9 billion per annum is generated from traditional medicine trades, which represents 5.6 % of the National Health budget. This sector has over 133 000 people earning an income through the trade of medicinal plants.

2.3 NATURAL PRODUCTS FROM PLANTS It is through ethnopharmacology and ethnobotany methodologies that allow for the isolation of compounds responsible for healing properties. The isolation therefore enables the development of new drugs or lead compounds (active compounds within the plant) (Graca et al., 2016). Montanher et al. (2002) stated that it is important/preferred to investigate plants that have been used as traditional medicine in order to isolate bioactive compounds.

Like any other living organisms, plants undergo primary and secondary metabolism. The primary metabolites (sugars, amino acids and fatty acids) are mainly for the basic survival of

8 the plant, these molecules take part in pathways such as glycolysis, photosynthesis and respiration to name but a few (Seigler, 1998). On the other hand during secondary metabolism, natural products (which may be referred to as phytochemicals) are synthesized and constitute a wide range of non-protein molecules that are not essential for basic development and plant growth (do Nascimento and Fett-Neto, 2010). For a long time, secondary metabolites were regarded as waste products that had no apparent use to the plant. As such they were sometimes referred to as “accessory plant metabolites”. However, they are now generally accepted as metabolites that assist the plants to have an association with their respective environments, thus they are responsible for different functions such as growth and metabolism regulation, pigmentation of plant parts, plant protection against pathogenic microbes and pests, they are also responsible for lignification in plants and serve as signal compounds to attract pollinators (Sinha, 2004; Kintzios and Barberaki, 2003; Verpoorte, 2000). The type and quantity of phytochemicals in plants is dependent on exogenous (climatic conditions, soil type and plant growth state) and endogenous (phytohormones) stimulus on the plant (Pourcel and Grotewold, 2009).

2.3.1. Classes of phytochemicals According to Sinha (2004), phytochemicals can be generally categorized into three groups (phenolic compounds, terpenoids/ terpenes and nitrogen containing compounds) with the main classes divided into distinct subgroups. Since there are various types of subgroups from the three main classes only a few that are common to most plants will be discussed.

2.3.1.1 Terpenes Terpenes are a large class of phytochemicals with around 36 000 structures elucidated (Campos-Vega and Oomah, 2013). Although hydrocarbons are the main compounds in this class, there are also oxygen containing terpenes (Dembitsky, 2006). Terpenes are major biosynthetic building blocks within nearly every living creature (Raaman, 2006). These compounds are biosynthetically derived from the linkage of isoprene (C5H8) units (figure 2.1). As the chains of isoprene units are built, resulting terpenes are classified sequentially by size as in hemiterpene (one), monoterpenes (two), sesquiterpenes (three), diterpenes (four), sesterterpenes (five), triterpenes (six), and tetraterpenes (eight). Most of the compounds in this class of phytochemicals are volatile and aromatic with biological properties that include antibacterial, immunostimulant and anticancer (Badal et al., 2017; Manivasagan et al., 2014; Dembitsky, 2006).

A B

Figure 2.1: Chemical structures of terpenes. A: Isoprene unit/repeating unit of terpenes and B: Geraniol (monoterpene) (Chen et al., 2016).

2.3.1.1.1 Saponins Saponins are a subclass of triterpene phytochemicals that are mainly valued for their detergent properties, these compounds form a stable soap-like foam when dissolved in water. They are also toxic to fish and have been observed to have hemolytic properties and numerous other beneficial bioactivities that include but not limited to antimicrobial, anticancer, antiinflammatory, antiviral and immunomodulatory (Makkar et al., 2007; Hostettmann and Marston, 2005; Mroczek, 2015; Sahu et al., 2008). In general, the structure of these compounds consists of a sugar moiety that is linked to an aglycone that can either be of a steroid or a triterpene nature (Makkar et al., 2007). The sugar moiety can consist of galactose, glucose, glucuronic acid, rhamnose, ormethylpentose or xylose, and they are linked to the aglycone moiety by a glycosisdic bond (Das et al., 2012). The surface-active properties of these compounds arise from their structure (the polar groups in the sugars and the nonpolar groups in the aglycones) making saponin compounds ampiphillic (Wina, 2012). The number of sugars beared on the aglycone ring and modifications on the aglycone ring can result in the formation of structurally diverse saponin compounds that posess a number of different bioactive properties (Das et al., 2012).

O OH OH H O HO O H O HO O H OH HO O O OH HO O O HO HO OH H O OH HO HO OH

Soyasaponin III Asparanin A

Figure 2.2: Chemical structures representing the two classes of saponins. Triterpene saponin (Soyasaponin III) (Makkar et al., 2007), and a steroidal saponin (Asparanin) (Liu et al., 2009).

2.3.1.1.2 Steroids/ Plant sterols Steroids are another group phytochemicals derived from triterpnes, of which many of them are used as drugs, prodrugs and hormones, in addition they have the ability to control many physiological reactions. Brassinosteroids are a good example of plant steroids responsible for the growth regulation of the plant (Dean et al., 2017; Talapatra and Talapatra, 2015). Bioactive properties of steroids include anticancer and anti-inflammatory (Genrshenzon and Kreis, 1999). Plant sterols are type of steroid compounds, similar to the cholesterol found in humans and animals. They are synthesized in the plant through cyclization of S-squalene-2, 3-epoxide to cycloartenol which is then further transformed to various sterols such as stigmasterol, campestorol, etc. (Kamal et al., 2014).

HO HO HO

Campesterol  -Sitosterol Stigmasterol

Figure 2.3: Common and widely distributed plant sterols (Fahy et al., 2004).

2.3.1.2 Phenolic compounds Phenolic compounds are widely distributed in the plant kingdom and they are generally characterized by a benzene ring that has at least one hydroxyl group as a substituent (Vermerris and Nicholson, 2008). Phenolic compounds can be further categorized into different subclasses based on the differences in their chemical structures. The subclasses include phenolic acids, lignans, tannins, coumarins, aldehydes, polyphenols, simple phenolics and flavonoids (Patra, 2012).

2.3.1.2.1 Tannins Plant tannins constitute the second largest category of phenolics following lignin. These compounds are normally distributed in the leaves, bark and the cell walls of the plant. They help to protect the plant against attacks from predators, microbial infections and also desiccation (Sinha, 2004). According to Serrano et al. (2009), tannins (figure 2.4) can be grouped into four classes based on their chemical structures, the four classes are hydrolysable tannins, proanthocyanidins, phlorotannins (derived from 1, 3, 5-trihydroxybenzene and found in brown algae) and complex tannins which are featured by structural elements of the other tannin groups. However, the hydrolysable tannins and condensed tannins (proanthocyanidins)

11 are considered as the two major classes of tannins. The hydrolysable tannins (ellagi- and gallotannins) are synthesized from partial or whole esterification of a sugar alcohol molecule such as glucose or glusitol with gallic acid (for gallotannins synthesis) or hexahydroxydiphenic acid (for ellagitannins synthesis) (Patra et al., 2012), whilst condensed tannins are derivatives resulting from oligomerization of monomeric flavan-3-ol units. The flavan-3-ol units can be fisetinidol, epigallocatechin and (epi) catechin (Quideau et al., 2011). Tannins have been reported to posess antiviral, antibacterial, antioxidant and anticarcinogenic properties (Serrano et al, 2009; Chung et al., 1998).

OH O O OH OH OH O HO O HO O OH OH

HO OH OH OH O OH OH OH O

Gallic acid Ellagic acid Catechin Gallocatechin Figure 2.4: Representative chemical structures of monomers for hydrolysable tannins (Ellagic and gallic acids) and condensed tannins (catechin and gallocatechin) (Patra et al., 2012).

2.3.1.2.2 Flavonoids Flavonoids are another group of phenolic phytochemicals that are generally known to have bioactive properties such as antiviral, anti-inflammatory, anti-tumor and antioxidant activities (Li et al., 2016; Menezes et al., 2016; Patra, 2012). These compounds are derived from a chalcone synthase mediated condensation reaction between 3-malonyl COA groups and cinnamic acid. Their chemical structures consist of a phenlychromanone unit that has two benzene rings linked by three carbons and an oxygen atom to form a pyrone ring (Menezes et al., 2016). Because of the differences in the types of structures formed from the flavonoid production, this class of phytochemicals is divided into six subclasses, namely: flavones, flavonols, isoflavones, anthocyanidins, flavonones and flavanols (figure 2.5) (Fredes and Montenegro, 2013).

OH OH OH OH OH OH

HO O HO O HO O

OH OH O OH O OH O

Apigenin Hesperetin Quercetin OH OH

HO O OH

OH OH

Delphinidine

Figure 2.5: Examples of compounds from four subclasses of flavonoids. Flavonone (Apigenin) (Kanwal et al., 2016), Flavanone (Hesperetin) (Urpi-Sarda et al., 2012), Flavonol (Quercetin) (Piskula et al., 2012), and Anthocyanidin (Delphinidine) (Takasawa et al., 2010).

2.3.1.3 Nitrogen Containing Phytochemicals 2.3.1.3.1 Alkaloids Alkaloids are recognized as the major class of N-containing phytochemicals with over 10 000 compounds reported to occur in approximately 300 families of plant species (Mates, 2013). These biomolecules are derived through a transamination process or from amino acids. They are characterized by heterocyclic structures containing a nitrogen atom. Because of the nitrogen, most of the alkaloids are basic. The compounds are mostly toxic and their physiological action is also strong (Aniszewski, 2015). Bioactive properties of this class of plant secondary metabolites include antimalarial, anticancer, anti- inflammatory, antimicrobial and analgesia (Klein-Junior et al., 2016). The classification of alkaloids can be based on chemical structures, taxonomic nature of the source and also on the pharmacological activities (Roberts and Wink, 1998). Based on chemical classifications, the main groups of alkaloids include indoles, isoquinolines, pyrrolizidines, pyrrolidines, pyridines, and steroidal alkaloids (Nordegren, 2002). Figure 2.6 shows some examples of vinca alkaloids (pharmaceutically significant and potent antitumour agents).

O H OH O O N HO NH N H O O O H O HO N O H HO O HO O O N H O O O O O O

Taxol Vinblastine

N HO N O O H H H N N N H H HO OH O O N O O O O O O

Vincristine Vincamine

Figure 2.6: Some examples of pharmaceutically significant alkaloids (Aniszewski, 2015).

2.4 FAMILY CANNABACEAE: OVERVIEW The plant under study previously belonged to the Ulmaceae but recent phylogenetic studies place it under the Cannabaceae family (Gil, 2011) which form part of the dicotyledonous flowering plants (angiosperm) (Epple and Epple, 2012). This family is quite small, with only 200 species partitioned in 11 genera distributed in the Northern hemisphere. The members in this family can be wind pollinated herbs, trees, vines and shrubs with simple sexual apetolous flowers that are unisexual or bisexual. The fruits are usually either drupes or nuts (Friis et al., 2011; Zarafshar, 2010).

Members in this family are characterized by the production of flavone C-glycosides (Spitaler et al., 2009). Some of the species in Cannabaceae family representing twining and erect herbs (Humulus and Cannabis, respectively) and trees (Celtis), which are medicinal, common and economically important (Cannabis and Humulus) are tabulated in Table 2.1 together with some of their phytochemicals and traditional uses.

Table 2.1: Representatives of species in the Cannabaceae family

Plant Ethnomedicinal uses Compounds reported Potential Reference/s name Bioactivity of compounds Cannabis Treatment of symptoms Humulene, Antifungal Wanas et al., sativa associated with Caryophyllene. 2016; asthma, cancer. Ayenigbara, Cannabinoids(Cannabidi Anticaner, 2014; ol tetrahydrocannabinol, Brown et al., Cannabicyclol, 2013 Cannabinol) Humulus Treatment of Xanthohumol Antiviral Chen et al., lupus respiratory infections Antiprolifirative 2012; Wang inflammation, anorexia, against cancer et al., 2008 Insomnia cells Wang et al., 2004 Celtis Treatment of digestive 2''-galactosylvitexin Antioxidant Abbouyi et australis ailments associated al., 2015; El- with peptic ulcers, Alfy et al., Pains, dysentery 2011

C. africana*

* Discussed in Section 2.6

2.5 THE GENUS CELTIS The widely spread Celtis is a large genus with around 70 species of shrubs and trees. Plants belonging to this genus are commonly known as nettle trees or hackberries (Bonner, 2008). This genus is wide spread in areas that are tropical and temperate and most of the trees are valued for their ornamental qualities or wood (Gianguzzi et al., 2014; Demir et al., 2002). The tree boasts of leaves that are simple and alternate, with flowers that occur in small axillary clusters. General traditional medicinal uses of Celtis include the treatment of pains, inflammation, disinfection of wounds, sore throat and prevention of miscarriages in humans (Umberto, 2012; Bradford, 2008; Miller and Nyberg, 1996).

2.6. THE SPECIES CELTIS AFRICANA Celtis africana is a multipurpose tree that is in addition to being used both as an ornamental and medicinal plant, it is also valued for its forage and timber which is usually used in flooring, furniture and construction and for purposes of energy (charcoal production and firewood) (Nyunai, 2012). The plant has various English common names such as white stinkwood, camdeboo, camdeboo stinkwood, African white stinkwood, African elm and common celtis. Whereas, in South Africa it is multingually referred to as mothibadifate/mohlatlakgomo (Sepedi), mbholovhisi/muyilakaya (Tsonga), modutu (Tswana), mpopano (Venda), ndwandwazwane (Zulu), umvumvu (Xhosa) and witstinkhout/kamdeboostinkhout (Afrikaans) (Umberto, 2012; Jooste, 2005). The plant English name (whitestinkwood) is derived from the unfriendly smell the wood releases when cut fresh (Van Wyk et al., 2000).

2.6.1 Botanical description of C. africana Celtis africana (figure 2.7) is a perennial deciduous tree that can grow up to 30 m long. The growth and form of this plant is dependent on the condition of the environment from which it grows. If grown on rocky soil, it can only be a few feet high shrub whereas in wild it grows up to 24 m high with a trunk that is straight, tall and clean. However, in deep soil this plant has a spread crown and a short trunk (Palmer and Pitman, 1972).

Morphologically this tree is characterized by green-greyish, and smooth stem that has raised dots, the branchlets often have lenticels. The leaves are egg shaped, simple and alternate. They have three veins from an asymmetrical base. The leaf margin is whole but is sometimes toothed on the upper half or two thirds especially on young leaves. The leaves further have hairs that range from scattered to dense. Young leaves are pale green while mature leaves are dark green (Boon, 2010; Schmidt et al., 2002). The bark of the tree is smooth and pale grey to whitish in color. The plant is further characterized by its flowers which are greenish, small and unobtrusive and occur simultaneously with the new leaves in spring (Thomas and Grant, 2002). The male and female sexes are on the same plant but separate. Male flowers are usually densely clustered on new branchlets and female flowers occur in isolation or in groups of two or three. The fruits are egg-shaped and thinly-fleshed drupes that are yellow to red-brownish when ripe with short hairs (Palgrave, 2002; Thomas and Grant, 2002; van Wyk et al., 2000).

Figure 2.7: Images of C. africana. A: The whitish grey bark surface. B: Unripe fruits, with mature green short haired leaves. C: Young leaves with pollinated flowers developing into fruit.

2.6.2 Distribution and habitat of C. africana Although Celtis africana is common in tropical Africa and as far north as Ethiopia. It is also widely spread in South Africa (figure 2.8) with all nine provinces (Limpopo, Gauteng, Free State, Kwa-Zulu Natal, Northen Cape, Eastern Cape, Western Cape, North West) being natural habitats (Mbambezeli and Notten,2008). The plant can grow in a wide range of habitats, from bushveld, grasslands, woodlands and rocky areas often dolomite areas. It can grow in any type of soil (Jooste, n.d). Although the plant prefers temperate conditions, it can sometimes withstand extreme environments as they are fairly drought and frost resistant (Palgrave, 1983).

Figure 2.8: Geographical distribution of C. africana in South Africa (Foden and Potter, 2005).

2.6.3 Ethnobotany of C. africana People in Kenya use the leaves of the tree to treat cattle with indigestion or trypanosomiasis edema. The leaves are consumed for indigestion or they are pounded and applied on the edema affected tissue (Krief et al., 2005). Nyunia (2012), reported that some Nigerian cultures use ground bark for the treatment of general pain (discomfort), headache and fever. Whereas in Lesotho the leaves are claimed to treat pleurisy (Moffett, 2010). A number of South African ethnic groups use the infusion of pounded, sundried bark and root in water or milk to treat symptoms associated with cancer, the mixture is normally taken orally until signs of relief are observed (Koduru et al., 2007). Unspecified plant parts have also been reported for the treatment of rheumatism, pains and sexually transmitted diseases (syphilis) (Seukep et al., 2014). The plant has also been reported to have magical powers as it is believed to chase lightning by mixing the wood with crocodile fat, and other ethnic groups believe that if the wood is put in the ground, it will chase witches or evil spirits away, whilst other cultures in Botswana believe that if the tree is used to stir cooked meat, livestock will multiply (Tshehlana, 2005; Palmer and Pitman, 1972). Other ethnobotanical uses of the plants is for making clothing and ropes in Congo and Lesotho, respectively (Nyunai, 2012).

2.6.4. Phytochemistry and pharmacology of C. africana Al-Taweel et al. (2012) reported for the first time the isolation of phenolic amides (trans-N- feruloyltyramine, trans-N-coumaroyltyramine, trans-N-caffeoyltyramine), sterol (β-sitosterol), triterpenoids (lupeol, oleanic acid) and fatty acids (palmitic acid, oleic acid, lauric acid) from C. africana. Whilst these isolates were later shown to possess unappreciable to satisfactory inhibition on acetyl cholinesterase enzyme, they however, proved to have strong anti- inflammatory and antioxidant activities.

In another study by Perveen et al. (2011), an n-butanol soluble fraction from an ethanol extract led to the isolation of seven C-glycosylflavonoids. Two of these compounds (celtisides) shown in figure 2.9 were novel to the Celtis species while the other five (vitexin, orientin, isoswertiajaponin, and isoswertisin and 2–O-rhamnosyl vitexin) were reported for the first time from the plant itself. The compounds showed high to moderate antioxidant activity although the crude n-butanol soluble fraction had more activity than the pure compounds. Very strong urease inhibition was detected with vitexin, orientin, isoswertiajaponin and isoswertisin.

OR2 HO R HO O OH R1O

O O

OH O

1: R = R1= H, R2 = Rhamnose 2: R = OH, R1 = Rhamnose, R2 = H

Figure 2.9: The structures of the two novel compounds isolated from the plant as studied by Perveen et al. (2011). Celtiside 1 was assigned as 8-C-[α -L-rhamnopyranosyl-(1 →6) β -D-glucopyranoside]. The 2nd Celtiside was assigned as 7-methoxy luteolin 8-C-[α-L-rhamnopyranosyl-(1→2) β-D-glucopyranoside].

Recently a glucosphingolipid with strong anti-cancer activity against mouse lymphoma cells was also reported from an ethyl acetate fraction of an ethanol extract (Perveen et al., 2015). Other biological activity studies on the plant revealed antidiarrheal activity at higher doses and laxative properties at low doses of aqueous-ethanol extracts of the plant. The aqueous-ethanol extracts also showed prokinetic properties when tested in mice (Khan et al., 2012). Mokoka et al. (2010), reported that the acetone extracts of the plant showed good bioactivity against a pathogenic fungal species of Cryptococcus neoformans.

2.7. PLANT AND MICROORGANISM RELATIONS 2.7.1 Symbiosis of plants and microorganisms There has always been a symbiotic microbe-host association between plants and microorganisms, where the relationship can be mutualistic (where both the host plant and the microorganism benefit), parasitic (where the microorganism benefits at the expense of the plant), or commensalistic (the microorganism benefits and the plant is unaffected) (Govindasamy et al., 2014; Singh et al., 2011; Hirsch, 2004).

2.8. BENEFICIAL PLANT BACTERIA/FUNGI 2.8.1 Root colonizing bacteria Due to the presence of nutrients in the root exudates, this allows for the formation of microbial communities on the roots of plants (Nadeem et al., 2015). When plants are engaged with root- associated fungi/bacteria, the association may help the plant expand on its access to nutrients (Rey and Schornack, 2013). One example is the association with plant growth promoting rhizobacteria (PGPR’s), which promote the growth of the plant either by stimulation (direct) or by reducing stress on the plant (indirect). The PGPR’s can be found in the root interiors,

19 rhizosphere and rhizoplane (Ramprasad et al., 2014) where they act as biocontrol agents, assist with plant nutrient uptake and they also help the plant thrive against biotic stresses such as drought, temperature, salinity and heavy metals (Nadeem et al., 2015). In unfavorable conditions PGPR’s are able to help the plant thrive through different mechanisms such as nitrogen fixation, production of sideophores and phytohormones, hydrolytic and ACC deaminase (1-aminocyclopropane-1-carboxylate) activities (Szymanska et al., 2016; Ramprasad et al., 2014; Abou‐Shanab et al., 2003).

2.8.2 Endophytes Endophytes are microorganisms that are regarded as non-pathogenic symbionts found inside plants. Therefore, they cause no apparent symptoms of disease on the host plant, (Muzzamal et al., 2012). The first case of endophytes discovery was in 1904, and ever since then, various definitions for the term endophyte have emerged depending on the prospective focus of that researcher (Zhao et al., 2012). Carrol (1986), claimed that endophytes are fungi which cause symptomless infections in the internal tissues of the host plant. Stone et al. (2000), proposed that endophytes are organisms which cause inconspicuous infections and the infected host tissues does not have apparent symptoms, furthermore, for these organisms to be true endophytes, there must be conclusive evidence that the microorganisms are derived from the internal structures of the host plant. The demonstration can be by isolation from tissues that have been strongly disinfected to remove epiphytic organisms or through histological means. The most accepted description was by Schulzs and Boyle (2005), who defined endophytes as fungi that can be detected at a particular time of their lifecycle within the tissues of apparently healthy plant hosts. Even though a lot of these descriptions are applied to fungi, endophytic bacteria are treated exactly the same way.

Kusari and Spiteller (2012), describes the term Endophytism, as a distinctive cost-benefit plant- microbe (fungi and/bacteria) association defined by ‘‘location’’, furthermore this association is transiently symptomless, unobtrusive, and entirely established inside the living host plant tissues. Endophytes have been found in almost all the plants investigated to date (Kusari et al., 2014).

There are two classes of endophytes depending on their living strategies, namely facultative and obligate endophytes. The obligate endophytes are those that depend entirely on the host plant for growth and survival, they can be transmitted from one host to the next through plant- plant transmission, transmission by a vector or through seeds. The facultative endophytes are

20 able to live outside of their host at a particular stage in their life cycle (Christina et al., 2013; Hardoim et al., 2008).

2.8.2.1 Location of endophytes Although the colonization of endophytes into the plant is mainly through the roots, the upper/aerial parts of the plant such as the leaves, flowers or stem can also be a point of entry. Since there are openings that already exist on the plant, the endophytes can also enter through the stomata or epidermis of the plant. Once inside the plant, these microorganism can disseminate to other parts of the plant or they can actually be localized at that point of entry. The microorganisms can colonize inter or intracellular spaces within the plant. (Bernardi- Wenzel et al., 2010; Zinniel et al., 2002). According to Haque et al. (2015) endophytes utilize hydrolytic extracellular enzymes to penetrate the plant cells. In other cases the endophytes are disseminated through seed transmission (Santoyo et al., 2016).

Figure 2.10: General mechanisms of entry of endophytes in host plants (Santoyo et al., 2016).

2.8.2.2 Advantages of endophytes Like the rhizosphere microorganisms, endophytes have beneficial effects on the host plant and are reported to be more efficient in their interaction with the host than the rhizosphere microbes (Santoyo et al., 2016).

The presence of the endophytes in the plant can contribute to the host plant being tolerant to either biotic or abiotic stress, these stresses include diseases caused by phytopathogens, pests

21 salt, drought and heat (Venugopalan and Srivastava, 2015; Joseph and Priya, 2011). In microbial plant infections, endophytes control phytopathogens by synthesizing antimicrobial compounds and by competing for nutrient resources with the pathogens thus helping the plant to develop phytopathogens resistance (Mohanty et al., 2017; Aly et al., 2010).

Endophytes have also been found to be able to directly promote plant growth, this can be done by assisting the plant acquire essential nutrients (i.e iron, phosphorus, nitrogen or sulfur) or by controlling the levels of hormones in the plant, i.e. through ACC deaminase activity, which reduces the level of the plant stress hormone ethylene (Santoyo et al., 2016). In addition to plant growth promotion, they are also very essential in promoting heavy metal concentrations tolerance by the host plant (Jasim et al., 2014; Mirzahosseini et al., 2014; Spaepen et al., 2007).

2.8.2.3 Endophytes associated with members of Cannabaceae plant family Although literature does provide some information on endophytic studies in this family, there have been a few reports of endophytes isolated from species in the Cannabaceae family. Members known to harbor beneficial endophytes (fungi/bacteria) include Celtis occidentalis (Errasti et al., 2010) and Cannabis sativa (Gautam, 2014; Kusari et al., 2014; Kusari et al., 2013).

2.8.2.4 Natural products from endophytes Other research studies posited that through co-evolution, endophytes were able to take up the plant’s DNA, thus acquiring the ability to produce phytochemicals originating from their host plants. The diverse chemicals that endophytes produce include peptides, steroids, phenolic compounds, aliphatic compounds, terpenoids, alkaloids, lignans, and isocoumarins just to name a few. Some of these compounds have been shown to have bioactive properties. As such, a number of endophytes have been proven to biosynthesize similar compounds as those synthesized by the host plant (Rai et al., 2014; Zhao et al., 2010). Because of the role that the compounds produced by endophytes have on the physiology and nature of their host plant, they therefore have the potential for applications in the agriculture, biotechnology and pharmaceutical industry (Miller et al., 2012).

Table 2.2: Examples of various bioactive natural products from endophytes

Endophytic source Compound name Plant host Application/Pot References ential Bioactivity Fungi

Pestalotiopsis mangiferae 4-(2,4,7-trioxa- Mangifera indica Antimicrobial Subban et al., bicyclo[4.1.0]heptan-3-yl) 2013 phenol Taxomyces andreanae Taxol Taxus brevifolia Anticancer Fatima et al., 2016 Mucor fragilis Podophyllotoxin Sinopodophyllum antiviral,anticanc Huang et al., hexandrum er, antioxidant, 2014; Zhao et anti-rheumatic, al., 2010. antibacterial,imm unostimulation Aspergillus fumigatus Asperfumin, Asperfumoid Cynodon Antifungal Liu et al., dactylon 2004 Periconia sp. F-31 Periconianone A and B Annonsa Antiinflammator Zhang et al., Muricata y 2014 Eupenicillium parvum Azadirachtin A and B Azadirachta Insecticidal Kusari et al., indica 2012 Epicoccum nigrum quinizarin beauvericin, Entada Antibacterial, Dzoyem et al., indole-3-carboxylic acid abyssinica antioxidant 2016 parahydroxybenzaldehyde Aspergillus flavipes Flavipesin A Acanthus Antibacterial Bai et al., Ilicifolius 2014 Penicillium sp Helvolic acid, Pinellia ternata Antibacterial Yang et al., 2017 Bacteria Streptomyces sp. Coronamycin Monstera sp. Antifungal, Ezra et al., antibacterial, 2004 antimalarial

Table 2.2 (Continued) Endophytic source Compound name Plant host Application/Pot References ential Bioactivity Streptomyces sp. Kakadumycin A Grevillea Antibacterial, Castillo et al., pteridofolia antiparasitic 2003 Bacillus Indole acetic acid Moringa Plant growth Khan et al., Subtilis peregrina hormone 2016 Pseudomonas Asparaginase Hibiscus Reduction of Bhagat et al., oryzihabitans rosasinensis acrylamide 2016 formation in food industry, used to treat leukemia Pantoea ananatis Indole derivative Musa sp. Antifungal Aman and V, 2016 Paenibacillus polymyxa Camptothecin Camptotheca Precursor for Pu et al., 2015 acuminata synthesis of anticancer drugs

2.9 Concluding remarks From the literature explored in previous sections, it is apparent that the world needs a more diverse arsenal of biologically active compounds for various applications. It further shows that both plants and their associated microorganisms have the ability to produce various bioactive compounds that can be applied in those sectors. Furthermore, the available literature reveals that the family Cannabaceae consists of members which are medicinally valuable. Celtis africana like other members in its family has the potential to produce bioactive compounds, however the available information on its phytochemistry is deficient and its endophytic microbe relations are still lacking. As such, more studies on these two aspects are needed to provide more information and establish a foundation of the host-endophyte interaction of Celtis africana and the consequent secondary metabolites they both produce including their potential downstream applications.

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CHAPTER THREE ISOLATION AND IDENTIFICATION OF ENDOPHYTIC MYCO- AND MICROFLORA FROM CELTIS AFRICANA (STINKWOOD) TREE. Nchabeleng1, E.K., Ndinteh2, D.T., Niemann1, N and Mavumengwana*1, V. 1Department of Biotechnology and Food Technology, Faculty of Science, University of Johannesburg, P. O. Box 17011, Doornfontein Campus, Johannesburg, South Africa. 2Department of Applied Chemistry, Faculty of Science, University of Johannesburg, P. O. Box 17011, Doornfontein Campus, Johannesburg, South Africa *Corresponding author: Email: [email protected]; Tel: +27115596915

Abstract White stinkwood (Celtis africana) is a medicinal plant, with a dearth of information about its endophytic myco or micro-flora (fungi or bacteria that reside in its endosphere). The aim of this study was therefore to isolate and identify the endophytic mycoflora and/or microflora present in the aerial parts (stem, fruit and leaves) of C. africana. The aerial parts of the plant were surface sterilized and culturable endophytes were isolated and identified using morphological and molecular (internal transcribed spacer (ITS) region for fungi and 16S rRNA for bacteria) identification techniques. Results obtained showed that endophytes were only present in the stem and fruit and were represented by two bacterial classes (Actinobacteria and Bacilli) involving seven species (Kocuria sp., Micrococcus luteus, Staphylococcus hominis, Bacillus sp., Staphylococcus saprophyticus, Brachybacterium conglomeratum and Arthrobacter sp.). In a similar vein, four fungal strains belonging to the Aspergillus genus were also isolated and characterized from the stem and fruit. The genus was represented by A. flavi (Aspergillus sp. and A. flavus) and A. nigri (A. niger and Aspergillus sp.). To the best of our knowledge, this is the first report on the endophytic microflora/mycoflora in C. africana indicating that both bacteria and fungi co-exist inside the stem and fruit parts of C. africana and has further provided the type of endophytes inhabiting this plant. Future studies to determine the beneficial effects of these endophytes to the plant, or the isolation of valuable secondary metabolites from the endophytes are necessary.

Keywords: Celtis africana, endophytes, medicinal plant, microflora, mycoflora

3.1 Introduction The above-ground (phyllosphere) and the below-ground portions (rhizosphere) of plants are associated with various microorganisms, especially endophytes (Bringel and Couee, 2015). These endophytes refer to fungi and/or bacteria that reside symbiotically in the internal healthy tissues of a host plant, without damaging the host (Arora et al., 2016). Endophytes and epiphytic microbes (fungi and/or bacteria on the surface of the plant) in the rhizosphere have been extensively studied and are known to have a symbiotic relationship with their respective host plants (Szymanska et al., 2016; Wani et al., 2016; Bringel and Couee, 2015; Abou‐Shanab et al., 2003). As compared to the rhizosphere, there is a dearth of information on the micro and mycoflora communities in the phyllosphere of plants (Ortega et al., 2016; Costa et al., 2012). Further to this, is the fact that these endophytes have been found to have useful effects on the plants (Costa et al., 2012). The ability of plants to fight biotic and abiotic stresses such as drought, high temperatures (heat) and diseases caused by plant pathogens and attacks by pests is said to be attributed to the presence of endophytes in those plants (Venugopalan and Srivastava, 2015; Joseph and Priya, 2011).

In order to explore the potential of endophytes, understand their diversity and investigate possible bioactive secondary metabolites, it is necessary to isolate and characterize them (Sun and Guo, 2012). As indicated by Guo et al. (2000), and Hallmann et al. (2006), caution must however be taken during isolation as the method used must guarantee efficient recovery of these endophytes in addition to being strong enough to kill fungi/bacteria on the plant surface. There are also other factors that will determine the type of endophytic communities within a host plant, these include: geographic locations (soil conditions), season, climate, the identity of the host plant and physiology of the host tissues colonized by the endophytes (Huang et al., 2015; Sun et al., 2013).

Various endophytes have been isolated from different plant types, ranging from terrestrial plants (shrubs, grass and trees) (Larran et al., 2016; Khorsandy et al., 2016; Gherbawy and Gashgari, 2014; Zinniel et al., 2002) to marine plants such as macro algae (Flewelling et al., 2015). With approximately 300 000 plant species on earth, it has been stated that, each plant is believed to be a host to a minimum of one endophyte, which translates to over 300 000 endophytes on the planet (Stepniewska and Kuzniar, 2013). According to the literature conducted, C. africana is unexplored with regard to endophytes. Hence, the main aim of this study was to determine the endophytic diversity of C. africana.

3.2. Materials and methods 3.2.1 Sample collection Fresh, healthy (showing no apparent symptom of disease) aerial parts (leaves, fruit and stem) of the C. africana plant were collected from the University of Johannesburg, Doornfontein Campus, located in Johannesburg, South Africa. The plant specimen was identified and deposited in the University Herbarium with the specimen code BTN NE 01. The samples were transferred to the laboratory, thoroughly washed with sterile distilled water and used within four hours.

3.2.2 Endophytes isolation The leaves, fruit and stem were surface sterilized separately using the method described by Jasim et al. (2014), with slight modifications. Briefly, each sample (approximately 10 g) was treated with 5 % Tween 20 (enough to cover the plant material) and vigorously shaken for five minutes. The Tween 20 was removed by rinsing several times with sterile distilled water, followed by disinfection with 70 % ethanol for one minute. Traces of the ethanol were removed by rinsing with sterile distilled water five times. The sample was then treated with 1 % Sodium Hypochlorite (NaHClO) for ten minutes and rinsed five times with sterile distilled water. The last rinse was used as a control and 100 µL of it was plated on Potato Dextrose agar (PDA) (HiMedia) and Nutrient Agar (NA) (Oxoid). The sample was then macerated in sterilized phosphate buffered saline (PBS) with the outer surface trimmed out. The macerated sample was serially diluted up to 10-3 dilution and each dilution inoculated (using spread plate method) in triplicates on PDA (for fungi enumeration) and NA (for bacteria enumeration). The PDA plates were incubated at 25 ºC, (IncoTherm, Labotec, South Africa), whilst the NA plates were incubated at 30 ºC, (IncoTherm, Labotec, South Africa). The growth was monitored periodically (5 days for NA, 7 days for PDA) during the incubation period. Effectiveness of the sterilization was monitored on the wash control plate, with growth indicating poor sterilization. Under such circumstances, the plates for that particular plant part were discarded and the sterilization repeated.

Distinct colonies were selected and subcultured on the appropriate media (PDA for fungi, NA for bacteria) to obtain pure isolates. Pure fungal isolates were preserved by cultivating them on PDA slants and storing at 4 ºC, this was repeated every two months to maintain the purity and viability of the isolates. Pure bacterial isolates were preserved in 50% glycerol on a ratio of 1 mL glycerol: 1 mL overnight broth culture and kept at -80 ºC.

3.2.3 Morphological identification of fungal endophytes The primary identification of the fungal isolates was determined by macroscopic and microscopic studies described by Pitt and Hocking (2009; 1997) and Klich (2002). Briefly each isolate was grown on PDA agar for five days at 25 ºC. After, incubation, phenotypic characteristics like the colony color analysis on the reverse and front, colony diameter, mycelia and color of the conidia were analyzed. For the microscopic characteristics, a wet mount for each isolate was prepared using lactophenol cotton blue stain. The microscopic-morphological character analyzed where the conidia, vesicles, stipes and philiades and metula. A drop of the stain was placed onto a microscope slide to form a suspension with needle picked growth of the isolate. The suspension was covered with a cover slip and viewed with a light microscope (BX51, Ultra 20 soft imaging system (Olympus, Japan). For the bacterial isolates, Gram staining was done to determine cell morphology.

3.2.4 Molecular identification To identify fungal isolates, the DNA of each isolate was extracted using the Zymo ZR fungal/Bacterial DNA kitTM (California, USA) following the manufacturer’s method, subsequently polymerase chain reaction (PCR) was performed to amplify the Internal Transcribed Spacers (ITS) regions. The primers used were ITS1 5’TCCGTAGGTGAACCTGCGG 3’ and ITS4 5’ TCCTCCGCTTATTGATATGC 3’. The PCR products were extracted, purified and sequenced using ABI Prism 3500xl genetic analyzer (Applied Biosystems(ThermoFisher Scientific), California, USA), after which the sequences were analyzed using a Geneious software, followed by comparison of the sequences with Basic Local Alignment Search (BLAST) on the National Center for Biotechnology Information using the GenBank database to identify the isolates. The DNA isolation and 16S rRNA regions amplification of the bacterial endophytes isolates was done as described above except that universal primers: 27F 5’AGAGTTTGATCMTGGCTCAG3’ and 1492R 5’ CGGTTACCTTGTTACGACTT 3’ were used instead to amplify the 16S rRNA. Furthermore, amplified products also underwent the same process as described above. MEGA 6 software was used to construct a phylogenetic tree for both the fungal and bacterial isolates, this was to determine the relation among the isolated endophytes and other fungal/bacterial species.

3.3 Results and Discussion In this study, a total of 11 endophytes (Table 3.1) were isolated and identified. Since the control plates did not reveal any fungal/bacterial growth, it was concluded that the isolates reported are endophytic to the plant under study. No endophytes were isolated from the leaves. They were only observed from the fruit and stems, with the former having a higher number of endophytes. There could be factors that would suggest why there were no endophytes isolated from the leaves, it could be that the sterilization method was too severe and thus penetrated through the leaf cells and killed any endophytes present, or there could be certain compounds in the leaf which do not allow for the survival of endophytes. One more reason could be that the leaves of Celtis africana generally are not inhabited by endophytes. This is supported by Zinniel et al. (2002), who asserted that populations of endophytes are generally less in the leaves and stems and more abundant in the roots.

Table 3.1: General Table showing endophytes isolated from C. africana. Plant Sample code Sample name Sample nature part Fruit EKFA1 Aspergillus sp. Fungi EKFA2 Aspergillus sp. Fungi EKFF Aspergillus sp. Fungi EKF1 Kocuria sp. Bacteria EKF2 Micrococcus luteus Bacteria EKF3 Staphylococcus hominis Bacteria EKF4 Bacillus sp. Bacteria Stem EKB1 Staphylococcus saprophyticus Bacteria EKB2 Brachybacterium conglomeratum Bacteria EKB3 Arthrobacter sp. Bacteria EKBE Aspergillus sp. Fungi

3.3.1 Morphological identification of fungal endophytes From the results obtained, four fungal endophytes were isolated and identified from the stem (EKBE) and fruit (EKFA1, EKFA2 and EKFF). Macroscopic identification of the isolates (Figure 3.1) showed similar characteristics. Isolate EKFA1 and EKFA2 had colonies which dispersed all-round the plate, with a size of approximately 75 mm. While the front view of EKFA1 had a green colony color with a pale green-yellowish rear and wrinkled mycelia,

EKFA2, exhibited a yellow colony front color, dense-white cottony mycelia in the center with green-yellowish margins. The reverse side of the latter was white-yellowish also with a wrinkled mycelium. The other two isolates (EKFF and EKBE), were also observed to have similar colony morphology. Their colony color was both blackish-grey with a dull white mycelia, though EKBE had a lower color intensity as compared to EKFF. EKFF and EKBE had colony diameters of 27 mm and 36 mm respectively.

Figure 3.1: Macroscopic illustrations depicting the morphology of the fungal endophytes. Two isolates (EKFF and EKBE) showed similar colony morphology.

Observations from further microscopic investigations of isolates is presented in figure 3.2. EKFA1 and EKFA2 showed the best images at 10× magnification, while EKBE and EKFF isolates showed best images at 40× magnification. The microscopic examination showed similarities among the stipes of the four isolates. Both EKFA1 and EKFA2 had similar conidiophore vesicles, which were semi-globose in shape, the metulae of both isolates were covering about ¾ of the vesicle. Both isolates had conidia which were also spherical. At 40× magnification, the EKBE showed a globule with slightly brown or dark-yellow vesicle and singlet conidia that have a dark-yellowish color and others black, while the EKFF isolate had a pale yellow and slightly brown conidia. When compared to the features of Aspergillus in

50 literature (Pitt and Hocking, 1997; 2009; Klich, 2002), the characteristics of EKFA1 were similar to those of Aspergillus flavus. On the other hand, EKFA2 could not be confidently identified, but had similarities to EKFA2 and hence could be posited to be an Aspergillus sp. The isolates EKFF and EKBE can be suggested to be Aspergillus niger due to their properties (Pitt and Hocking, 2009; Klich, 2002; Pitt and Hocking, 1997). Although macro and micro identification have suggested the possible genus and species of the isolates, these forms of identification could only be primary. As further stated by Reddy et al. (2010), due to similarities in phenotypic characteristics, it is difficult to effectively distinguish and identify fungal species based on morphological properties. This therefore necessitated DNA sequencing of the isolates to ascertain their identity and confirm the observations of the primary identification.

Figure 3.2: Microscopic images of the fungal isolates. A1: (EKFA1), A2: (EKFA2), E :( EKBE) and F :( EKFF)

3.3.2 Molecular identification of fungal endophytes Based on ITS region sequencing and phylogenetic evolution (figure 3.3), the fungal isolates in this study were found to belong to the Aspergillus genus. The sequences were susbequently compared with other fungal sequences in the GenBank database, it was observed that all the isolates had 100% similarity with another strain, with the exception of EKFF (Table 3.2). Although the ITS region sequencing is a powerful molecular technique for identifying fungi at species level, in the predicted organism column in Table 3.2, there are more than one type of predicted organism, as such the intergration of the earlier morphological identification presented in section 3.3.1 guided the appropriate selection of the closest organism.

As hinted from the primary identification, EKFA1 and EKFA2 have the same predicted fungal strains from the BLAST search. All the possible organisms for EKFA1 and EKFA2 belong to three closely related Aspergillus species which fall under the Flavi section (A. flavus, A. oryzae and A. parvisclerotigenus). There are earlier studies in the literature which have also reported the closeness of these species (Klich et al., 2015; Varga et al., 2011; Godet and Munaut, 2010; Lee et al., 2004). In addition, these fungal species have been reported to be producers of toxic secondary metabolites called “mycotoxins” (Njobeh et al., 2010). The strains now forms part of a microorganism collection bank by the Department of Biotechnology and Food Technology, University of Johannesburg, South Africa. EKFF and EKBE were also found to have have the same identity through the molecular identification. The possible names for EKFF and EKBE were A. tubingensis and A. niger, with the former having an additional predicted fungal species (A. awamori). According to Perrone et al. (2011) these fungal species are also closely related and belong to the Aspergillus “Nigri” section, A. awamori is suggested to be another name for A. niger. From the present investigations it can be said that two fungal isolates belong to the Aspergillus “Flavi” section and two belong to the “Nigri” section. The Aspergillus species reported herein have been reported to be endosymbionts of other plants, A. niger has been reported as endophyte in various plants (Gautam et al., 2013; Talontsi et al., 2013; Wani et al., 2010), A. flavus has also been reported as an endophtyte in earlier studies (Uddandarao and B, 2016; Russo et al., 2016; Gautam et al., 2013).

Table 3.2: Identification of the endophytic fungal strains from C. africana Sample Predicted microorganism Similarity code percentage (%) EKFA1 A. flavus/A. oryzae/A. parvisclerotigenus 100 EKFA2 A. flavus/ A. oryzae/ A. parvisclerotigenus 100 EKFF A. tubingensis/ A. niger/ A. awamori 99 EKBE A. niger/A. tubingensis 100

With specificity to EKFA1, the primary identification guided that the isolate is an A. flavus, because the morphological characteristics strongly agree more with the key fungal identification of A. flavus strain, thus ruling out the possibility of the EKFA1 being A. oryzae or A. parvisclerotigenus. Therefore, the combination of the secondary and primary identification of EKFA1 strongly suggest that this isolate is A. flavus. Although EKFA2 exhibited phenotypes fairly similar to EKFA1, the other two possible predicted organisms cannot be ruled out, thus it is concluded that EKFA2 is Aspergillus sp., the phylogenetic analysis (figure 3.3) also supports the stated observations. EKFA1 is in the same clade/branch as A. flavus whereas EKFA2 is observed to be equally related to all the three organisms. The isolates EKFF and EKBE intensely suggested through primary identification that they are both A. niger strains, however when compared to other fungal species as depicted in the phylogenetic tree (figure 3.3), EKBE is the one isolate that is in the same clade as A. niger, and the former is equally related to the predicted organisms. Therefore the fungal endophytes resulted in two Aspergillus sp., namely A. flavus and A. niger.

Figure 3.3: Phylogenetic tree based on the ITS sequences showing the evolutionary relationships of the fungal endophytes from C. africana with the closest fungal relatives. A: EKFA1, B: EKFA2, C: EKFF and D: EKBE.

3.3.3 Identification of bacterial endophytes The isolation of bacterial endophytes led to the identification of seven isolates. Table 3.3, shows the sample code with its colony morphology, Gram stain results, predicted microorganism biological names as found on BLAST (Appendix 1 for complete Blast results) and also the similarity percentage between the sample isolate and the predicted organism. The colony morphology column shows the color, shape, elevation and margin of the colony as observed on a petridish. The macroscopic features of the isolates varied from each other. All the seven bacterial isolates exhibited Gram positive cocci characteristic with the exception of one (EKF4) which was a Gram negative short rod.

From the observed results (summarized in Table 3.3, with the phylogenetic tree in figure 3.4), the seven isolated bacterial endophytes were affiliated with only two classes, Actinobacteria and Bacilli class. While the former was represented by four genera (Brachybacterium, Arthrobacter, Kocuria and Micrococcus), the latter had two genera (Staphylococcus and Bacillus). Most of the species belonging in the Actinobacteria class are said to be producers of biologically important compounds such as antibiotics (Kugler et al., 2015).

Table 3.3: Identification of bacterial endophytic strains from C. africana Sample code Colony morphology Gram Predicted microorganism Similarity stain percentage (%) Isolates from the fruit EKF1 Orange, round, convex, + cocci Kocuria sp. 100 entire EKF2 Yellow ,round, convex, + cocci Micrococcus luteus 99 entire EKF3 White, round, flat, entire + cocci Staphylococcus hominis 100

EKF4 Creamy dry, Irregular, - short rod Bacillus sp. 100 flat, lobate Isolates from the stem EKB1 Glossy cream, round, + cocci Staphylococcus saprophyticus 99 entire

Table 3.3 (Continued) Sample code Colony morphology Gram Predicted microorganism Similarity stain percentage (%) EKB2 Yellow, round, flat, + cocci Brachybacterium 100 entire. conglomeratum

EKB3 Creamy with white + cocci Arthrobacter sp. 98 center, round, raised entire.

Although most of the bacterial endophytes isolated and identified in the current study, are known to be opportunistic pathogens such as Kocuria (Savini et al., 2010), they have also been reported to be endophytic to other plants. Species belonging in the Kocuria, Micrococcus, Bacillus and Arthrobacter, Brachybacterium, Staphylococcus genera have also been found coexisting as endophytes in vine stems (Andreolli et al., 2016; Szymanska et al., 2016; Lins et al., 2014; Cho et al., 2007 ).

Figure 3.4: Phylogenetic tree based on the 16S rRNA sequences with sequences of close relations in the GenBank database.

3.4 Conclusion The present study revealed the co-existence of bacteria and fungi in the medicinal plant C. africana, although there is little diversity of endophytic microorganisms in the plant. The endophytes have been reported to occur in other plants as beneficial endophytes. According to the literature conducted this is to the best of our knowledge the first report on the presence of endophytes in this plant, this has thus provided information on the microbial community in the endosphere of C. africana. Further studies are therefore needed to determine how the isolated endophytes are of benefit to the C. africana.

Acknowledgements The authors would like to thank the National Research Foundation (NRF) for the Masters Innovation Scholarship and the University of Johannesburg for financial assistance granted to Ms. Nchabeleng E.K.

Author Contribution E.K. Nchabeleng planned and performed the experiments, analyzed the data and wrote the paper.

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CHAPTER FOUR IN VITRO ANTIBACTERIAL ACTIVITY TESTING AND PROFILING OF VOLATILE CONSTITUENTS OF THE LEAVES, STEM, AND FRUIT OF CELTIS AFRICANA FROM SOUTH AFRICA. Nchabeleng1, E.K., Ndinteh2, D.T., Niemann1, N and Mavumengwana1*, V. 1 Department of Biotechnology and Food Technology, Faculty of Science, University of Johannesburg, P. O. Box 17011, Doornfontein Campus, Johannesburg, South Africa. 2 Department of Applied Chemistry, Faculty of Science, University of Johannesburg, P. O. Box 17011, Doornfontein Campus, Johannesburg, South Africa *Corresponding author: Email: [email protected]; Tel: +27115596915

Abstract Phytochemicals are key components responsible for the medicinal properties and defense systems of plants. As such, knowledge about these phytochemicals is necessary for the development of new drugs and to understand the healing mechanism of these plants as medicinal preparations. The study was therefore carried out to identify various phytochemicals in Celtis africana and their subsequent antimicrobial activity. The aerial parts (stem, leaves and fruit) of C. africana were extracted with solvents of varying polarities viz., hexane, ethyl acetate and dichloromethane:methanol and then screened for the presence of phytochemicals and antibacterial activity against fourteen bacterial strains. The crude extracts were further analyzed to determine the volatile chemical constituents of the plant using two dimensional gas chromatography coupled with time of flight mass spectrometry (GCxGC-TOF/MS). Results obtained revealed the presence of alkaloids, tannins, saponins, steroids, and reducing sugars with the chemical profiles revealing a total of 45 volatile phytocompounds of pharmaceutical importance. Only the hexane crude extracts of the fruit and leaves and the ethyl acetate crude extract of the stem showed inhibitory activity against Escherichia coli, Proteus mirabilis, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter aerogenes. The compounds identified together with the biological activity further indicate the potential of C. africana as a medicinal plant. This study has in addition provided information on compounds which can inspire the synthesis of bioactive drugs.

Key words: Antimicrobial activity, Celtis africana, medicinal properties, secondary metabolites, volatile compounds

4.1 Introduction The emergence and spread of antimicrobial resistant pathogens is progressively increasing, thus suggesting that the current available antimicrobials are less effective and in need of reviewing (While, 2016). For example, approximately 440,000 new cases of multidrug- resistant tuberculosis (MDR-TB) are reported yearly, leading to deaths of about 150,000 people worldwide (Srivastava et al., 2014). This global health concerns aggravates the need for development of new drugs to combat the current human diseases. Kintzios (2006), suggested that plant-derived compounds with biological activity can have a significant part in the commercial development of new drugs.

It has been reported by Patil et al. (2013), that almost 80% of the people living in rural areas rely on plants as sources of medicine for primary healthcare. As such, plants are considered as one of the main sources of biologically active compounds since some forms of plant extracts have been used for thousands of years for medicinal purposes (Ibrahim et al., 2014; Raina et al., 2014). Medicinal plants that have been traditionally used have gained the attention in biological scientific research. This is because the active components conferring these medicinal properties have been attributed to their secondary metabolites (phytochemicals). Various studies have thus isolated and identified the secondary metabolites produced by these plants (Chang et al., 2016; Vikram et al., 2014; Ferreres et al., 2011). The structural properties and molecular arrangements of these plant’s secondary metabolites subsequently enable them to be used as lead compounds for new drugs (Tariq and Rey, 2013). It is therefore important to know the phytoconstituents of these plants in order to discover new therapeutic agents, determine the active principles of the medicinal preparation and also to discover new phytocompounds that can be used for the synthesis of new drugs (Keerthiga and Anand, 2015).

In addition to other chromatographic techniques, gas chromatography is one of the chromatographic techniques that can also be used to separate and identify compounds (as either a screening step or a confirmatory one) within plant crude extracts. Two dimensional gas chromatography (GCxGC) is a multidimensional gas chromatography that involves two columns that are coupled directly and provides a simultaneous separation of volatile and semi- volatile components (Marriot et al., 2001). Gas Chromatography coupled with Mass Spectrometry (MS) serves as a powerful technique for the identification of bioactive compounds. While the former is valued for the separation, the latter serve as an identification tool. The constituents from plants identified by this method may range from long chain

62 hydrocarbons, esters, steroids, alcohols, amino compounds, acids, nitro compounds and alkaloids (Jadhav et al., 2014).

Celtis africana belongs in the Celtis genus under the family Cannabaceae and in South Africa referred to as White stinkwood (English); Ndwandwazane/umvumvu (Zulu) (Gil, 2011). Human ailments known to be treated by this plant include pains, rheumatism, syphilis, conjunctivitis sore eyes, cancer, indigestion and edema (Seukep et al., 2014; Koduru et al., 2007; Krief et al., 2005; Jooste, 2003). The major plant parts used for traditional medicine from this plant are the stem bark, leaves and roots (Nyunai, 2012).

Different phenolic compounds including trans-N-coumaroyltyramine, trans-N- feruloyltyramine, vitexin, and isoswertisin have earlier been reported to occur in the plant (Al- Taweel et al., 2012; Perveen et al., 2011). Likewise, few studies have been presented in the literature on the antifungal activity and other bioactivities of the plant extracts (Mokoka et al., 2010; Adedapo et al., 2009). From the literature search conducted, it can be said that there is still a gap on the phytochemistry and bioactivity studies on the plant. In this article a full profile of volatile phytochemicals and the antibacterial activity against various bacterial pathogens is reported.

4.2. Materials and methods 4.2.1 Sample collection Celtis africana (stems, fruits and leaves) samples were collected from the University of Johannesburg, Doornfontein campus, South Africa. The samples were washed under running water, cut into small pieces, after which they were air dried. Following drying, they were blended into fine powder separately, stored in airtight bottles, and kept at room temperature until analysis. The plant has been deposited into the University of Johannesburg Herbarium with specimen code BTN NE 01.

4.2.2 Plant crude extract preparation The plant extracts were prepared according to Ogueke et al. (2014) with slight modifications. Three solvents of different polarities were used for the extraction such that each plant part produced three crude extracts. The organic solvents used were hexane, ethyl acetate, and equal volumes of dichloromethane:methanol (1:1 v/v) (DCM:MeOH). Briefly, 100g of plant material was added to 1L of organic solvent and shaken on an orbital shaker (Stuart SSL1, Keison products, UK) for 48 h at 25 °C. This was followed by filtering the extract using a Buchner funnel and Whatman No.1 filter paper and concentrated under reduced pressure using a rotary

63 evaporator (IKA RV 10, Staufen, Germany). The extraction process was repeated until an adequate crude extract was obtained. An overall of nine crude extracts (for the stem, fruit and leaves) were obtained.

4.2.3 Preliminary phytochemical screening The crude organic solvent and water extracts of the plant parts were tested for the presence or absence of phytochemicals. Standard procedures by Trease and Evans (1983) and Harbourne (1983), were used to qualitatively analyze the presence of alkaloids, tannins, reducing sugars, flavonoids, saponins, steroids. The water extracts of the plant (leaves, fruit and stem) were prepared by adding 10 g of plant material into 100 mL of water, followed by shaking on an orbital shaker (IKA RV 10, Staufen, Germany) at 25 °C for 24 h. After which the extracts were steam boiled and filtered using a Whatman No. 1 filter paper. To get a concentrated water extract, the filtrates were further brought to boil.

4.2.3.1 Test for tannins

Two to three drops of 10% (v/v) Iron Chloride (FeCl3) solution were added to 2 mL of the crude extract. A blackish green or blackish blue coloration indicated the presence of tannins.

4.2.3.2 Test for alkaloids 0.5 g of crude extract/crushed plant material was added to 5 mL of 1% (v/v) HCl solution, and was further placed in a boiling water bath, after which 1 mL was aliqouted into a clean container and three drops of (potassium bismuth iodide solution) Dragendorff’s reagent was added. Turbidity or formation of a precipitate indicated the presence of alkaloids.

4.2.3.3 Test for flavonoids 0.5 g of crude extract/crushed plant material was heated with 10 mL of ethyl acetate for three minutes over a steam bath. Thereafter 4 mL of the heated sample was shaken with 1 mL of dilute ammonia solution. Presence of flavonoids was indicated by a yellow coloration that disappeared after a while.

4.2.3.4 Test for saponins Ten mL of crude extract was vigorously shaken to form a stable resistant foam that lasted at least ten minutes. The foam was then collected into a clean container to which, it was mixed with three drops of olive oil (Rio Largo) and vigorously shaken again to form an emulsion which indicated a positive result.

4.2.3.5 Test for steroids

0.5 g of crude extract/ crushed plant material was dissolved in 5 mL of chloroform (CHCl3), the sample was then filtered. The filtrate was mixed with a concentrated H2SO4 solution. The layers were then allowed to separate. Presence of steroids was indicated by a reddish brown steroidal ring.

4.2.3.6 Test for reducing sugars The presence of reducing sugars was determined by adding 2 mL of Benedict’s solution to 10 mL of the crude extract. The mixture was then boiled in a water bath until color change was observed. The presence of reducing sugars in relative of the abundance is indicated by different colors. Blue indicate that there are no reducing sugars, whilst the presence of reducing sugars in terms of increasing concentration are indicated by green, yellow, red, and brown.

4.2.4 Antibacterial activity screening and Minimum Inhibitory Concentration (MIC) determination There were 14 test microorganisms used for the screening of antibacterial activity, namely: Mycobacterium smegmatis (MC 2155), Bacillus subtilis (ATCC 19659), Klebsiella pneumoniae (ATCC 13882), Enterococcus faecalis (ATCC 13047), Bacillus cereus (ATCC 10876), Staphylococcus aureus (ATCC 25923), Enterobacter cloacae (ATCC 13047), Staphylococcus epidermidis (ATCC 14990), Proteus vulgaris (ATCC 6380), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 7002), Enterobacter aerogenes (ATTC 13048) and Klebsiella oxytoca (ATCC 8724). These strains are common and important pathogens, they were purchased from Davies diagnostics (Johannesburg, South Africa). These test organisms were preserved in 50% glycerol and stored at -80 °C until use.

4.2.4.1 Sample preparation Each crude extract was prepared such that the concentration was 32 mg/mL. The dissolving solvent used was Dimethylsulfoxide (DMSO) and sterile Muller Hinton Broth (MHB). I.e. to prepare 5.5 mL of 32 mg/mL crude extract, 0.176 g of crude extract was weighed into a sterile bottle, to which 1 mL of DMSO was added to dissolve the crude extract. The volume was then topped up to 5.5 mL with sterile MHB.

4.2.4.2 Experimental procedure The antibacterial activity of the plant crude extracts were tested following the method of Andrews (2001), with minor modifications. The test organisms were resuscitated in 10 mL of sterile MHB and incubated at 37 °C overnight. Concentration of the overnight culture of each test organism was visually adjusted using the 0.5 McFarland standard such that the concentration is 107 to 108 cfu/mL.

On a microtiter plate, the wells on the edge of the plate were filled with sterile distilled water

(dH2O) to minimize the evaporation of the liquid media when incubated. For each test organism 100 µL of 107 to 108 cfu/mL was placed in the well of the microtiter plate, followed by 100 µL of 32 mg/mL crude extracts, the components were mixed. This was done in replicates of five for each crude extract. In addition clean broth alone served as a sterility control, test microorganism mixed with DMSO (1:1) served as negative control, test microorganism alone served as a growth control and the test microorganism with streptomycin (1:1) at a concentration of 10 mg/mL served as a positive control. The plate was then covered with tin foil to seal the plate, and incubated at 37 °C for 18 hours. After incubation, 10 µL of sterile 0.02 % (w/v) Resazurin sodium salt solution was added to each well containing crude extract with bacteria and all the controls. The plates were covered with tin foil and incubated for further 2 hours after which the results were read, where blue color indicated positive results(growth inhibition) and pink color indicated negative results(no growth inhibition).

The crude extracts that showed antibacterial activity were further tested to determine their minimum inhibitory concentrations (MIC). Each crude extract had prepared solutions of concentrations ranging from 32 mg/mL to 1 mg/mL. The experiment was then performed as outlined above.

4.2.5. Profiling of volatile constituents 4.2.5.1 GCxGC-TOF/MS conditions The samples were first prepared by dissolving a small amount (picked with a pipette tip) of each of the crude extracts (hexane, ethyl acetate and DCM:MeOH plant extracts) in 1 mL of HPLC grade methanol (Sigma-Aldrich, Aston manor, South Africa). The extracts were then analyzed using the method described by Ralston-Hooper et al. (2008), with minor changes.

The volatile compounds were analyzed using Leco’s Pegasus 4D GCXGC-TOFMS (USA) fitted with an Rxi-5Sil MS (30 m, 250 µm i.d, 0.25 µm d.f) (Restek, USA) as a primary column. The secondary column was Rxi- 17Sil MS (2 m, 250 µm i.d, 0.25 µm d.f) (Restek, USA). The

66 oven temperature (T°) was maintained at 50 °C for 0.5 minutes and then ramped to 300 °C at 10 °C/min, after which it was held at 300 °C for ten minutes. The transfer line T° was 250 °C. Helium gas was used as a carrier gas with a flow rate of 1 mL/min and a split mode injection of 1:10 ratio. The inlet T° was at 250 °C. The mass spectrometer was operated at an electron energy of -70 eV with an ion source at 250 °C. The mass range used was 40-660 with an acquisition rate of 10 spectra/second.

4.2.5.2 GCxGC-TOF/MS data processing ChromaTOF software was used to collect the mass spectra, the compounds were identified by comparing the mass spectra with those on various libraries (Replib, Mainlib, and NIST MSMS). Only compounds with similarity values of 70 % were considered.

4.3 Results and Discussion 4.3.1 Preliminary phytochemical screening The screening of the presence of phytochemicals is essential to determine the different classes of phytochemicals present in the C. africana. Table 4.1 summarizes the observations from the qualitative phytochemicals test of different crude extracts of the plant. The presence/absence of alkaloids, flavonoids and steroids under the water extract columns in Table 4.1 were tested from the dry plant material and not the water extract itself.

Numerous phytochemical constituents are contained in plants, and a lot of these constituents are known to be medicinally active and thus responsible for various pharmacological activities (Al-Owaisi et al., 2014). From the qualitative analysis of phytochemicals in the plant (Table 4.1), it was observed that all the phytochemicals tested for (sections 4.2.3.1 to 4.2.3.6) were present in the C. africana with the exception of flavonoids, this could be as a result of seasons and climate changes influencing the type of phytochemicals produced by plants at any given point in time (Pourcel and Grotewold, 2009). The water extracts of the stem and fruit were the only extracts in which saponins were observed to be present. Saponins are reported to have anti-inflammatory properties and are known to exhibit anticancer, antioxidant and antimicrobial activities (Sidana et al., 2016; Xia et al., 2009). The presence of saponins in the plant could explain the traditional use of the plant as an anti-inflammatory to treat pains such as sore eyes or use as veterinary medicine to treat edema (Krief et al., 2005; Jooste, 2003).

Tannins were observed in all the crude extracts with the exception of all the hexane extracts and the fruit ethyl acetate extract. In addition, the tannins were more abundant in the stem and leaf water extracts and the leaf ethyl acetate extract. Although tannins are commonly known to

be antinutritional, they also possess antihemorrhoidal, antidiarrheal and hemostatic activities (Gomathy et al., 2012). The stem and fruit showed moderate presence of steroids, weak concentration of this class of compounds were detected in all the other organic solvents extracts of the fruit and stem. Steroids are reported to exhibit a wide range of biological activities (Sun et al., 2016). Aiyelaagbe and Osamudiamen (2009) stated that steroids have antimicrobial, cardiotonic activities and insecticidal properties. The study revealed the presence of alkaloids only in the water and DCM:MeOH crude fruit extracts, with moderate and weak presence of the alkaloids, respectively. Although cytotoxicity is one common biological property of alkaloids (Yadav and Agarwala, 2011), other alkaloids have been found to have therapeutic effects against rheumatoid arthritis as a results of their anti-inflammatory and analgesic activities (Xu et al., 2016). Other reports have associated them with antibacterial and antioxidant activities (Qin et al., 2015; Milugo et al., 2013).

Flavonoids are phenolic compounds that are mostly known for their ability to scavenge free radicals and as such, they are anticarcinogenic and antioxidative (Sharma and Janmeda, 2014). Interestingly, although there have been flavonoids reported from C. africana (Adedapo et al., 2009), no flavonoids were detected in this study. The fruit water extract was found to be rich in reducing sugars, followed by the fruit ethyl acetate crude extract, fruit DCM:MeOH extract and stem water extract, all with partially strong presence, while the leaves water extract had lower concentration of the reducing sugars. Due to the important role that phytochemicals play in defending plants against, pathogens and prey, this therefore makes phytochemical screening a necessary step in order to predict the kind of active compounds present in a plant (Chew et al., 2011). The presence of the various phytochemicals (alkaloids, steroids, tannins, reducing sugars and saponins in the plant correlate with the reported ethnomedicinal uses of the plant for treatment of cancer, rheumatism and sexually transmitted infections, in particular, syphilis (Seukep et al., 2014; Koduru et al., 2007).

Table 4.1: Phytochemical components present in the crude extracts of C. africana Phytochemical Water extracts Hexane extracts Ethyl acetate 1DCM:1MeOH Test extracts extracts

Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit Alkaloids - - ++ ------+ Flavonoids ------

Table 4.1 (Continued) Phytochemical Water extracts Hexane extracts Ethyl acetate 1DCM:1MeOH Test extracts extracts

Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit Steroids - ++ ++ - + + - + + - + + Tannins +++ +++ + - - - +++ + - ++ + + Reducing sugars + ++ +++ - - + - - ++ - - ++ Saponins - +++ ++ ------

Where – shows absence of phytochemical, + shows weak presence of phytochemical, ++ shows partially strong presence of phytochemicals and +++ shows strong presence of phytochemicals.

4.3.2 Antibacterial activity screening and minimum inhibitory concentration (MIC) determination. The antimicrobial activity screening and MIC value determination of the nine crude extracts tested are shown in Tables 4.2 and 4.3 respectively.

Only three extracts showed antibacterial activity, the leaf hexane extract inhibited the growth of E. aerogenes and P. aeruginosa whilst the stem ethyl acetate crude extracts was active against K. pneumonia, P. aeruginosa, S. aureus, P. mirabilis, and E. coli. The fruit hexane extract on the other hand exhibited activity against B. cereus, S. aureus, P. mirabilis and E. coli (Table 4.2).

When the crude extracts were screened for antibacterial activity using conventional methods such as the disc diffusion assay (data not shown), no activity was observed on all the crude extracts. However when the extracts were tested for antimicrobial activity using the 96-well plate broth assay (Table 4.2), some activity was observed. The extracts were tested using an initial concentration of 32 mg/mL for antibacterial screening. It was observed that the leaf hexane extract (L1), stem ethyl acetate extract (S2) and fruit hexane extract (F1) exhibited antibacterial activity against two (E. aerogenes & P. aeruginosa), five (K. pneumonia, P. aeruginosa, S. aureus, P. mirabilis & E. coli) and four(B. cereus, S. aureus, P. mirabilis, E. coli) test organisms, respectively. Of all the seven microorganisms inhibited, five of them were Gram negative bacteria.

Overall, the results showed that Gram negative bacteria were not tolerant to the plant extracts than Gram positive bacteria. However this contradicts what other authors have reported, it has been found that Gram positive bacteria are likely to be inhibited by plant extracts than Gram negative, because Gram negative bacteria have a selective outer membrane that limits or exclude numerous compounds getting inside the cell (Rakholiya et al., 2013 ; Parakh et al., 2005).

Table 4.2: Antibacterial screening of the crude extracts. Test organism Crude extract + Control at antibacterial activity at 32 mg/ml 10 mg/mL L1 L2 L3 S1 S2 S3 F1 F2 F3 Streptomycin Sulphate M. tuberculosis ------+ B. cereus ------+ - - + B. subtilis ------+ K. pneumoniae - - - - + - - - - + K. oxytoca ------+ E. aerogenes + ------+ E. cloacae ------+ P. aeruginosa + - - - + - - - - + P. vulgaris ------+ S. epidermidis ------+ S. aureus - - - - + - + - - + P. mirabilis - - - - + - + - - + E. coli - - - - + - + - - +

+ Antibacterial activity observed and – indicates no growth inhibition observed. L1: leaf hexane extract, L2: leaf ethyl acetate extract, L3: leaf DCM: MeOH extract, S1: stem hexane extract, S2: stem ethyl acetate extract, S3: stem DCM: MeOH extract, F1: fruit hexane extract, F2: fruit ethyl acetate extract, F3: fruit DCM: MeOH extract.

In this study the MIC value is the lowest concentration of the crude extract at which the test microorganism is susceptible/inhibited (Table 4.3).

For the minimum inhibitory concentration determination of the crude extracts that showed growth inhibition on the test microorganisms, doses of the test extract were used with the highest concentration being 32 mg/mL and the lowest concentration being 1 mg/mL. The fruit

70 hexane crude extract showed MIC value of 4 mg/mL against S. aureus, and all the other extracts that showed antibacterial activity exhibited inhibitory effects at MIC values of 32 mg/mL.

Considering that the samples tested for antibacterial activity were crude extracts and not pure compounds, the antibacterial activity exhibited thereof is notable, especially for the hexane fruit extract. Although Wintola and Afolayan (2015), asserted that an MIC value of 0.1 mg/mL is considered active for crude extracts, this is arguable, since at times the active compound in the crude extract could be in low concentration, and hence activity of the crude extract could be observed at only slightly higher concentrations. The test organisms that were susceptible to the plant extracts are mostly associated with nosocomial infections and are known to be multidrug resistant. S. aureus is known to cause septic arthritis and other skin and soft tissues diseases like abscess (Changchien et al., 2016). The presence of phytochemicals such as tannins, steroids and reducing sugars may have possibly contributed to the antibacterial activity of the plant crude extracts.

Table 4.3. Minimum Inhibitory Concentrations of C. africana crude extracts. Test organism MIC value(mg/mL) L1 S2 F1 E. coli NT 32 32 S. aureus NT 32 4 P. mirabilis NT 32 32 B. cereus NT NT 32 P. aeruginosa 32 32 NT K. pneumoniae NT 32 NT E. aerogenes 32 NT NT

NT: Not Tested, L1: Leaf hexane crude extract, S2: Stem ethyl acetate crude extracts, F1: Fruit ethyl acetate extract.

4.3.3 Profiling of volatile constituents Chemical compounds in the crude extracts are known to be biologically active ingredients. These compounds are directly responsible for many bioactivities like conferring anticancer, antioxidant, antifungal and antimicrobial effects (Hossain et al., 2013). The plant parts were extracted with organic solvents of increasing polarity, nonpolar (Hexane), dipolar (ethyl acetate) and most polar (DCM:MeOH). Using 2D-GC-TOF/MS, the active compounds in the

71 different extracts were determined. A total number of 45 major phytoconstituents were found in the overall plant which are presented in Tables 4.4-4.8 together with their retention time (R.T), molecular formula, molecular weight and the peak percentage area.

The identified compounds were organized into 7 groups according to their functional groups, namely: ketones, hydroxypyrones, nitrogenous bases, aldehydes, esters, alcohols, fatty acids. Each of the seven chemical groups are discussed individually and the relative importance of the identified compounds is also noted.

Ketones In the present investigation, a total number of six compounds (figure 4.1) bearing a ketone functionality were identified (Table 4.4), the complete list of compounds (including those deemed less significant) detected by 2D-GC-TOF/MS is given in Appendix 2. Three (3.4- dimethyldihydrofuran-2,5-dione, 2,3-pentanedione and 2,3-heptanedione) of the six compounds identified were detected in the hexane extracts whilst 2,3-pentanedione was detected in all the hexane extracts. Since the peak percentage area is directly proportional to the concentration, it shows that the leaf and stem had more of 2,3-pentanedione than the fruit. It represented a peak percentage area of 4.804, 4.439 and 1.747 in the leaf, stem and fruit, respectively. This diketone is used as a flavoring agent (Zaccone et al., 2015) while another diketone, 2,3-heptanedione also used as a flavoring agent (Winter, 2009) was detected in the leaf and stem hexane extracts, 3,4-dimethyldihydrofuran-2,5-dione on the other hand was detected only in the stem hexane extract. The remaining three other compounds only detected in the stem extract were 1-(4-hydroxy-3-methoxyphenyl)ethanone, 3,5,6,7,8,8a-hexahydro- 4,8a-dimethyl-6-(1-methylethenyl)-2(1H)naphthalenone and Friedelan-3-one (Table 4.4). Friedelan-3-one is a triterpene that has been reported to have anti-inflammatory and antioxidant properties (Bindu et al., 2015).

In a study by Kuete et al. (2007), Friedelan-3-one showed antibacterial activity against E. cloacae and P. vulgaris amongst others. Recently Odeh et al. (2016) also reported antibacterial activity of this compound against S. aureus and E. coli. The compound 3,5,6,7,8,8a-hexahydro- 4,8a-dimethyl-6-(1-methylethenyl)-2(1H)naphthalenone has been noted to possess anti- inflammatory activities (Vaithiyanathan and Mirunalini, 2015), while 1-(4-hydroxy-3- methoxyphenyl)ethanone is known as an inhibitor of the enzyme, NADPH oxidase which produces reactive oxygen species (ROS) (Sharma et al., 2016).

O O

O O O O 3.4-dimethyldihydrofuran-2.5-dione 2.3-pentanedione Friedelan-3-one

O O

OH O 1-(4-hydroxy-3-methoxyphenyl)ethanone 2.3-heptanedione

3,5,6,7,8,8a-hexahydro-4,8a-dimethyl-6-(1-methylethenyl)-2(1H)naphthalenone Figure 4.1: Some of the significant ketone based compounds detected from the plant extracts by 2D-GC-TOF/MS

Table 4.4: Ketones detected in the crude extracts of C. africana Compound name (R.T)(s) Molecular Molecu Peak area % formula lar mass Hexane extracts Ethyl acetate extracts DCM:MeOH extracts Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit Ketones

2,3-Pentanedione 121.48 C5H8O2 100.05 4.804 4.439 1.747 ND ND ND ND ND ND

2,3-Heptanedione 123.66 C7H12O2 128.08 2.136 2.079 ND ND ND ND ND ND ND

3,4-Dimethyldihydrofuran-2,5-dione 109.74 C6H8O3 128.05 ND 0.452 ND ND ND ND ND ND ND

Friedelan-3-one 2023 C30H50O 426.39 ND ND ND ND 0.576 ND ND 2.756 ND

1-(4-hydroxy-3- 851.88 C9H10O3 166.06 ND ND ND ND ND ND ND 0.015 ND methoxyphenyl)ethanone

3,5,6,7,8,8a-hexahydro-4,8a-dimethyl- 1883.7 C15H22O 218.17 ND ND ND ND ND ND ND 0.298 ND 6-(1-methylethenyl)- 2(1H)naphthalenone

Where ND indicates no detection of the particular compound in the crude extract

Fatty acids As shown is Table 4.5, fatty acids were detected in all the crude extracts of the plant in varying concentrations. The fatty acids in the C. africana crude extracts were represented by two unsaturated fatty acids; 9,12,15-octadecatrienoic acid, (Z, Z, Z) -; 9,12-octadecadienoic acid (Z, Z) - and one saturated fatty acid; n-hexadecanoic acid. They were present in all the crude extracts, with the exception of 9,12,15-octadecatrienoic acid, (Z, Z, Z) - which was absent in the fruit hexane extract. The DCM:MeOH extracts had higher concentrations of the fatty acids than the ethyl acetate and hexane extracts. Compounds in this class are well known for their antibacterial activity (Abdel-Aal et al., 2015). Rajendra et al. (2014), reported n-hexadecanoic acid to be a free radical scavenger (antioxidant) whilst Al-Taweel et al. (2012), also reported the presence of this compound in the same plant. Although the compound 9,12,15- octadecatrienoic acid, (Z, Z, Z) – has been reported to possess promising biological properties such as neuroprotective, cardioprotective, and anti-inflammatory, it is further used in pharmaceutical drugs for genital disorders and contraceptives (Rawal and Sonawani, 2016; Stark et al., 2008).

Aldehydes Five aldehydes were found in the C. africana plant. In the hexane extracts (Table 4.5), 2- propylhexanal was the only aldehyde found in the fruit hexane extract with a peak percentage area of 3.434. In the DCM:MeOH crude extracts only the fruit had two aldehydes (2-heptenal and deca-2, 4-dienal) and it was the only extract with aldehyde compounds. Meanwhile, in the ethyl acetate crude extracts, the stem was the only one found to have aldehydes (Table 4.5), with two being the same compounds found in the fruit DCM:MeOH crude extract and the other two being hexanal and 2,4-heptanedienal. From the results observed, no aldehydes were detected in all the leaf extracts. In plants, aldehydes are produced in response to insect attack and several reports have been made on their pesticidal and antimicrobial activities (Hubert et al., 2008). In addition to other pharmacological activities including antifungal effects, hexanal has also found widespread usage as a food additive (Baggio et al., 2014).

Table 4.5: Fatty acids and aldehydes detected from the crude extracts of C. africana Compound name Retention Molecular Molecu Peak area % time(R.T) Formula lar (s) mass Hexane extracts Ethyl acetate extracts DCM:MeOH extracts Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit Fatty acids

n-Hexadecanoic acid 1084.78 C16H32O2 256.24 0.084 0.038 0.019 0.084 0.118 0.056 0.243 0.152 0.390

9,12,15-Octadecatrienoic acid, (Z,Z,Z)- 1188.8 C18H30O2 278.22 0.189 0.049 ND 0.234 0.083 0.207 0.377 0.194 0.207

9,12-Octadecadienoic acid (Z,Z)- 1184.04 C18H32O2 280.24 0.056 0.060 0.028 0.080 0.134 0.075 0.199 0.229 0.423

Aldehydes

2-Propylhexanal 115.94 C9H18O 142.13 ND ND 3.434 ND ND ND ND ND ND

2,4-Heptadienal, (e,e)- 360.78 C7H10O 110.07 ND ND ND ND 0.017 ND ND ND ND

2-Heptenal, (z)- 324.62 C7H12O 112.09 ND ND ND ND 0.062 ND ND ND 0.296

Hexanal 200.78 C6H12O 100.09 ND ND ND ND 0.040 ND ND ND ND

Table 4.5 (Continued) Compound name Retention Molecular Molecu Peak area % time(R.T) Formula lar (s) mass Hexane extracts Ethyl acetate extracts DCM:MeOH extracts Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit

Deca-2,4-dienal 642.34 C10H16O 152.12 ND ND ND ND 0.022 ND ND ND 0.204

Where ND indicates no detection of the particular compound in the crude extract

Alcohols The volatile constituent analysis of C. africana revealed the presence of 15 types of alcoholic compounds (eicosanol and n-tridecan-1-ol, , phytol, A-friedooleanan-3-ol, (3α)-, D-L-α- tocopherol, 2H-1-benzopyran-6-ol, 3,4-dihydro-2,7,8-trimethyl-2-(4,8,12-trimethyltridecyl)-, ç-tocopherol); á-sitosterol, 1,2,3 benzenetriol, 1, 2, 3-propanetriol, diacetate, 3, 4, 4-trimethyl- 3-pentanol, 3-hexen-1-ol, (Z) - and 3-hexanol, 4, 4-dimethyl-). The hexane extracts had more alcohol compounds with a total of 9 compounds, followed by ethyl acetate extracts with a total of 8 compounds while the DCM:MeOH had the lowest number of alcohols. Like its ketone form, D: A-friedooleanan-3-ol, (3α) - was only exclusive to the stem extracts exhibiting the same peak percentage area of 0.916 in the ethyl acetate and DCM:MeOH extracts. This triterpene has been reported for its antifungal and anti-inflammatory properties (Zhou et al., 2011), whereas, 1,2,3-benzenetriol is phenolic compound that has been noted for its antimicrobial activity (Lima et al., 2016).

A steroidal compound, α-sitosterol was found in the leaf and stem hexane crude extracts and also the fruit portion of the DCM:MeOH extract. The beta form of sitosterol was also reported by Al-Taweel et al. (2012) in the same plant species. Compounds like sitosterol and other phytosterols have been shown to have anti-atherogenic, anticancer and anti-atherosclerotic properties (Rubis et al., 2008). The fatty alcohols 1-eicosanol and n-tridecan-1-ol are stated to have antifouling and antibacterial effects, respectively (Togashi et al., 2007; Ganti et al., 2006). Furthermore, 1-eicosanol is used in cosmetics as an emollient and thickening agent (Begoun, n.d). 1-eicosanol was detected in the leaf ethyl acetate crude extract only. Muthukrishnan et al. (2016), reported that the diterpene, phytol has antimicrobial, anticancer, antidiuretic and diabetic properties. The identification of tocopherols from Celtis africana was of particular interest given the fact that these compounds are well-known for their free radical scavenging activities and are associated with the relief of ailments such as rheumatoid arthritis (Sears, 2004; Sigounas et al., 1997). The detection of 3-Hexen-1-ol, (Z) from C. africana may have implications in the biotechnological due to the compound’s potential application in the perfumery industry (Furuhata et al., 1982).

As such, in addition to the therapeutic aspects of the plant, numerous other applied biological sciences can benefit from the plant’s secondary metabolites as enumerated by the 2D-GC- TOF/MS results.

Table 4.6: Alcohol compounds detected from the crude extracts of C. africana Compound name (R.T)(s) Molecular Molecula Peak % area Formula r mass Hexane extracts Ethyl acetate extracts DCM:MeOH extracts Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit

3-Hexen-1-ol, (Z)- 139.22 C6H12O 100.09 1.357 ND ND ND ND ND ND ND ND

1-Propanol, 2- 111.5 C6H15NO 117.12 ND ND 0.734 ND ND ND ND ND ND (dimethylamino)-2-methyl

1-Eicosanol 1540.58 C20H42O 298.32 0.070 ND 0.014 ND ND ND ND ND ND

3-Hexanol, 4,4-dimethyl- 274.9 C8H18O 130.14 0.022 ND ND ND ND ND ND ND ND

D:A-Friedooleanan-3-ol, 2020.26 C30H50O 426.37 ND 0.054 ND ND 0.912 ND ND 0.916 ND (3à)-

ç-Tocopherol 1621.4 C28H48O2 416.37 0.002 ND ND ND ND ND ND ND ND

dl-α-Tocopherol 1666.46 C29H50O2 430.38 0.007 ND ND ND ND 0.011 1.166 ND 0.049

α-Sitosterol 1806.5 C29H50O 414.39 0.110 0.074 ND ND ND ND ND ND 0.330

Table 4.6 (Continued) Compound name (R.T)(s) Molecular Molecula Peak % area Formula r mass Hexane extracts Ethyl acetate extracts DCM:MeOH extracts Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit

n-Tridecan-1-ol 1540.08 C13H28O 200.21 ND ND ND ND 0.147 ND ND ND ND

3,4,4-Trimethyl-3-pentanol 168.04 C8H18O 130.14 ND ND ND ND ND 0.079 ND ND ND

1-Eicosanol 1454.46 C20H42O 298.32 ND ND ND 0.087 ND ND ND ND ND

ND2H-1-Benzopyran-6-ol, 1620.6 C28H48O2 416.37 ND ND ND ND ND 0.012 ND ND ND 3,4-dihydro-2,7,8-trimethyl- 2-(4,8,12-trimethyltridecyl)

Phytol 1172.5 C20H40O 296.31 ND ND ND 0.042 ND ND 0.052 ND ND

1,2,3-Propanetriol, diacetate 576.72 C7H12O5 176.07 0.037 0.021 ND 0.037 0.021 ND ND 0.066 0.377

Table 4.6 (Continued)

Compound name (R.T)(s) Molecular Molecula Peak % area Formula r mass

Hexane extracts Ethyl acetate extracts DCM:MeOH extracts

Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit

1,2,3-Benzenetriol 690.12 C6H6O3 126.03 ND ND ND ND ND ND ND ND 0.034

Where ND indicates no detection of the particular compound in the crude extract

Esters Seven different types of esters were found in the different crude extracts. In the hexane crude extracts (Table 4.7), all the esters detected were only from the leaf. Esters such as sulfurous acid, dibutyl ester, and an ethyl ester derived from hexadecanoic acid was the second in the leaf hexane extract, whereas, the DCM:MeOH and ethyl acetate fruit extracts respectively gave three fatty acid esters derived from 9,12-octadecadienoic acid (Z,Z)- and a methyl ester of 9,12,15-octadecatrienoic acid, (Z,Z,Z)- derivative. While some of the esters obtained from the analysis have interesting biological applications, others like benzyl benzoate are used as insectides (Mounsey and McCarthy, 2013) and ethyl (9Z, 12Z)-9, 12-octadecadienoate was reported by Park et al. (2014), to possess anti-inflammatory property.

Table 4.7: Ester compounds detected from the crude extracts of C. africana Compound name R.T.(s) Chemical Molecula Peak Area % formula r mass Hexane extracts Ethyl acetate extracts DCM:MeOH extracts Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit

Sulfurous acid, dibutyl ester 115.92 C8H18O3S 194.10 3.067 ND ND ND ND ND ND ND ND

Hexadecanoic acid, ethyl ester 1104.3 C18H36O2 284.27 0.025 ND ND ND ND ND ND ND ND 8

9,12,15-Octadecatrienoic 1186.2 C19H32O2 292.24 ND ND ND ND ND 0.024 ND ND ND acid,methylester, (Z,Z,Z)- 2

9,12-Octadecadienoic acid (Z,Z)-, 2- 1455.2 C21H38O4 354.27 ND ND ND ND ND ND ND ND 0.098 hydroxy-1-(hydroxymethyl)ethyl ester 6

Benzyl benzoate 974.6 C14H12O2 212.08 ND ND ND ND ND ND ND 0.046 ND

Methyl octadeca-9,12-dienoate 1162.4 C19H34O2 294.26 ND ND ND ND ND ND ND ND 0.305 6

Table 4.7 (Continued) Compound name R.T.(s) Chemical Molecula Peak Area % formula r mass Hexane extracts Ethyl acetate extracts DCM:MeOH extracts Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit

Ethyl(9Z,12Z)-9,12-octadecadienoate 1198.7 C20H36O2 308.27 ND ND ND ND ND ND ND ND 0.311 8

Where ND indicates no detection of the particular compound in the crude extract

Hydroxypyrones; Nitrogenous bases and others Hydroxypyrones and nitrogenous bases (Table 4.8) were only found in the fruit and stem DCM:MeOH crude extracts, respectively. The two hydroxypyrone compounds are both products of the Maillard reaction and thus it makes sense that they were only found in the fruit crude extract because they are responsible for the browning/color and also flavor of the fruit. Moreover, the bioactivity of these compounds (e.g. 2,3-dihydro-3,5-dihydroxy-6-methyl-4H- pyran-4-one) has also been reported by Yu et al. (2013) where it was noted for the antioxidant activity of Maillard reaction products. Another study by Ban et al. (2007), reported that the compound has antiproliferative activity against cancer cells. On the other hand, the second hydroxypyrone detected (3-hydroxy-2-methyl-4H-pyran-4-one) has been implicated in pharmaceutical applications where it functions as a metal chelator with the resultant metal complexes being bioactive enough towards the alleviation of diseases like anaemia and diabetes (Zborowski et al., 2005).

In general, the DCM:MeOH crude extracts of C. africana revealed eight other compounds that included mome inositol (Table 4.8). This polysaccharide is a component of vitamin B and is reported to be responsible for a number of bioactivities like, anti-cirrhotic, anti-neuropathic, and lipotropic (Das et al., 2014). Phenolic compounds, such as benzyl α-D-glucoside was detected in the leaf DCM:MeOH crude extract. A triterpene, squalene was also detected in the leaf DCM:MeOH crude extracts and is stated to have numerous biological activities including hepatoprotective, antioxidant, gastropreventive, antimicrobial and anticancer (Muthukrishnan et al., 2016). A compound which was observed to be dominant in both the leaf and stem was 1,2-bis(dicyanmethylen)-3-(2,3 dimorpholinocyclopropenyliothio)-cyclopropanid. It had a peak percentage area of 9.7 % in the leaf and 6.7 % in the stem extracts analyzed. A toxic compound (3-nitropropionic acid) which was also found in the fruit ethyl acetate extract that has been reported in other plants such as Lotus pedunculata and Hippocrepis comosa, and is reported to be responsible for neurodegenerative disorders (Gregory et al., 2000). Although the fruits are reported to be used in ethno-medicine, they have to be used with caution because of the presence of 3-nitropropionic acid, nonetheless there are more bioactive compounds of pharmaceutical value than this poisonous compound.

Table 4.8: Hydroxypyrones, Nitrogenous bases and other compounds detected in the crude extracts of C. africana. Compound name R.T.(s) Chemical Molecula Peak % area formula r mass Hexane extracts Ethyl acetate extracts DCM:MeOH extracts Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit Nitrogeneous bases

2-Amino-9-(3,4- 734.92 C10H13N5O5 283.09 ND ND ND ND ND ND 0.152 0.703 0.364 dihydroxy-5- hydroxymethyl- tetrahydro-furan-2-yl)- 3,9-dihydro-purin-6- one

2,4(1H,3H)- 429.68 C5H6N2O2 126.04 ND ND ND ND ND ND ND 0.053 ND pyrimidinedione, 5- methyl-

Hydroxy pyrones

3-hydroxy-2-methyl- 429.78 C6H6O3 126.03 ND ND ND ND ND ND ND ND 0.083 4H-pyran-4-one

Table 4.8 (Continued) Compound name R.T.(s) Chemical Molecula Peak % area formula r mass Hexane extracts Ethyl acetate extracts DCM:MeOH extracts

Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit

2,3-dihydro-3,5- 494.86 C6H8O4 144.04 ND ND ND ND ND ND ND ND 0.769 dihydroxy-6-methyl- 4H-pyran-4-one others

3-Nitropropinoic acid 167.8 C3H5NO4 119.02 ND ND ND ND ND 0.079 ND ND ND

Mome inositol 879.18 C7H14O6 194.08 ND ND ND ND ND ND 0.478 0.398 0.201

1,2- 103.88 C20H16N6O2S 404.11 ND ND ND ND ND ND 9.683 6.668 ND bis(dicyanmethylen)-3- (2,3- dimorpholinocycloprop enyliothio)cyclopropan id

Table 4.8(Continued) Compound name R.T.(s) Chemical Molecula Peak % area formula r mass Hexane extracts Ethyl acetate extracts DCM:MeOH extracts Leaf Stem Fruit Leaf Stem Fruit Leaf Stem Fruit

Piperidine, 1,1'- 611.4 C11H22N2 182.18 ND ND ND ND ND ND ND 0.012 ND methylenebis

Benzyl α-D-glucoside 1245.7 C13H18O6 270.11 ND ND ND ND ND ND 0.029 ND ND 6

Squalene 1513.9 C30H50 410.39 ND ND ND ND ND ND 0.063 ND ND

Where ND indicates no detection of the particular compound in the crude extract

4.4.1 Conclusion It is evident from the results, that C. africana has many bioactive chemical constituents, which correlate with its ethnomedicinal uses. The presence of various compounds like Friedelan-3-one, phytol, the fatty acids and other compounds with reported antimicrobial activity may suggest why the plant is used to treat infectious diseases like syphilis and may also be the active ingredients that allowed for the plant extracts to inhibit the growth of the test bacterial species as discussed (in section 4.3.2). Inflammation is associated with a number of conditions like cancer and rheumatism, hence some of the esters (Ethyl (9Z,12Z)-9,12-octadecadienoate), tocopherols and sterols found in the plant may also attribute to the traditional uses of the plant to treat inflammation related conditions (cancer, rheumatism, sore eyes) and because of their antioxidant activity this could also explain the use of the plant to treat symptoms associated with cancer. Although most of the compounds have been identified or reported in other plants, the present study according to the best of our knowledge, is the first report on the full chemical profile of this plant and antibacterial activity against a spectrum of bacterial pathogens. Though the presence of bioactive compounds was established in this study, further research in isolating and purifying these compounds is needed for additional proof of their bioactivity, and to allow large scale production of these valuable compounds.

Author Contribution E.K. Nchabeleng planned and performed the experiments, helped analyze the gas chromatography data and wrote the paper.

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CHAPTER FIVE IDENTIFICATION AND CHARACTERIZATION OF VOLATILE SECONDARY METABOLITES PRODUCED BY ENDOPHYTIC FUNGI ASSOCIATED WITH CELTIS AFRICANA FROM SOUTH AFRICA. Nchabeleng*1, E.K., Ndinteh2, D.T., Niemann1, N and Mavumengwana1, V. 1 Department of Biotechnology and Food Technology, Faculty of Science, University of Johannesburg, P. O. Box 17011, Doornfontein Campus, Johannesburg, South Africa. 2 Department of Applied Chemistry, Faculty of Science, University of Johannesburg, P. O. Box 17011, Doornfontein Campus, Johannesburg, South Africa *Corresponding author: [email protected] , Tel: +27115996915

Abstract Fungal endophytes produce secondary metabolites that have many biological activities and thus have various application in the pharmaceutical, agricultural and food industries. The current study was aimed at determining the volatile metabolites produced in the secondary metabolism of fungal endophytes that were previously isolated from Celtis africana. The four fungal endophytes (Aspergillus flavus, A. niger and two Aspergillus sp.) were grown for three weeks on potato dextrose broth (PDB) after which secondary metabolites were extracted from the broth media and mycelia using different organic solvents (hexane, ethyl acetate and dichloromethane). The extracts were then analyzed on a two-dimensional gas chromatography coupled with time of flight mass spectrometry to determine the nature of these secondary metabolites. The results obtained showed the presence of various chemical compounds including aldehydes, alkaloids, steroids, ketones and esters which have reported bioactive properties. It was further observed that the crude extracts of the four fungal endophytes contained similar major compounds (alkaloidal pyrazines and triazole compounds (propiconazole and cyproconazole)). The obtained data suggest that compounds from the endophytes could be responsible for the protection of the plant against various factors. This study has therefore provided a basis for the determination of the bioactive constituents of these endophytes.

Keywords: Aspergillus flavus, Aspergillus niger, fungal endophytes, secondary metabolites, 2D- GC-TOF/MS.

5.1 Introduction Fungi are eukaryotic organisms that have the same features as plants, but without chlorophyll (Nisa et al., 2014). Natural products derived from endophytic fungi have interesting biological properties such as being antiviral and conferring anticancer, antioxidant, antimicrobial and pesticidal properties (Hussain et al., 2014). Accordingly, fungal endophytes continue to form a major part of the fungi that have not been adequately explored (Nisa et al., 2014). Moreover these endophytes are a reservoir of structurally diverse bioactive compounds, with which they can be used to synthetically produce new compounds of biological importance (Malysheva et al., 2014; Aly et al., 2010).

According to Cardoso-Martinez et al. (2015), most of the secondary metabolites of fungal origin are mainly produced by the species in the Aspergillus genus. The species in this genus are filamentous, most common and ubiquitous in the environment (Soltani and Moghaddam, 2014). This genus is the oldest and large with over 180 anamorphic species, it is further divided into seven subgenera that are also divided into sections (Rodrigues et al., 2007). The Flavi and Nigri sections are among the 18 sections reported (Afzal et al., 2013). Secondary metabolites reportedly produced by the Aspergillus species include mycotoxins, and other useful ones such as antimicrobials, cholesterol lowering agents and immune suppressants (Afzal et al., 2013; Njobeh et al., 2010).

A. flavus belongs to the Flavi section and though is notorious for aflatoxin production, it is also known to be an opportunistic phytopathogen of many plants (Chen et al., 2011). Despite this deleterious factor, the species has been found in certain plants as an endophyte where it has produced compounds that have exhibited interesting biological activities (Yadav et al., 2014; Gautam et al., 2013). Likewise, numerous studies in literature have been reported on endophytic A. niger, this fungus is known to produce secondary metabolites of biotechnological importance (Govindappa and Govindappa, 2014; Yadav et al., 2014; Talontsi et al., 2013; Zhang et al., 2007).

Although there have been studies on the aforementioned species as endophytes from other plants, plant hosts are stated to have the ability to influence the biology of the endophytes and the type of secondary metabolites that they produce (Ludwig-Muller, 2015; Strobel and Daisy, 2003). This consequently means that the same endophytic species inhabiting different plants could produce different metabolites that may be beneficial in other systems. In this regard, the study focused on the profiles of secondary metabolites produced by four fungal endophytes that were isolated from

C. africana. To the best of our knowledge, this is the first report of the screening of secondary metabolites of the C. africana’s fungal endophytes.

5.2 Materials and methods 5.2.1. Fungal isolates resuscitation Each of the four isolates was resuscitated by picking a small growth from the slant culture (preserved stock culture) and culturing on potato dextrose agar (PDA) (HiMedia) plate for five days at 25 ºC.

5.2.2. Fermentation and secondary metabolite extraction A modified method of Pragathi et al. (2013), was used to grow the isolates and extract secondary metabolites. Each isolate was grown in 1 L potato dextrose broth (PDB) (Himedia) that was halved between two 1 L conical flasks, each containing 500 mL of (PDB). The flasks were incubated at 25 ºC (Amerex Gyromax, ISS, Carlifonia, USA), for 21 days with intermittent shaking at 100 rpm. After fermentation the broth was separated from the mycelia by first filtering with a three layered muslin cloth followed by using a three layered Whatman No.1 filter paper. Secondary metabolites were sequentially extracted from the broth with equal volumes of hexane, dichloromethane (DCM) and lastly ethyl acetate (extracted three times with each solvent). The organic phase layer was concentrated using a rotary evaporator, and the resulting mass constituted the broth crude extract. The residue after filtration (mycelial mat) was blotted on a sterile filter paper and dried for 24 hours at 34 ºC, after which it was crushed using a sterile pestle and mortar. This was then soaked in an organic solvent on a ratio of 1 g (crushed mycelia):10 mL (organic solvent (hexane, DCM, ethyl acetate)) on an orbital shaker for 24 h and filtered, the filtrate was concentrated by means of a rotating evaporator under reduced pressure. The organic solvents were also used sequentially as above.

5.2.3. Profiling of volatile secondary metabolites 5.2.3.1 Sample preparation Prior to analysis on GC×GC-TOF/MS, each crude extract was dissolved in 1 mL of HPLC grade methanol (Sigma-Aldrich, Aston Manor, South Africa).

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5.2.3.2 GC×GC-TOF/MS conditions A slightly modified method by Ralston-Hooper et al. (2008) was adopted to analyze the crude extracts using Leco’s Pegasus 4D GC×GC-TOFMS (USA) fitted with an Rxi-5Sil MS (30 m, 250 µm i.d, 0.25 µm d.f) (Restek, USA) as a primary column and Rxi- 17Sil MS (2 m, 250 µm i.d, 0.25 µm d.f) (Restek, USA) as a secondary column. The oven temperature (T°) was set at 50 °C for half a minute and then ramped at a rate of 10 °C/min to 300 °C, where it was held for ten minutes. The inlet T° was at 250 °C and the transfer line T° was 250 °C. Helium gas was used as a carrier gas at a flow rate of 1 mL/min using a split mode injection ratio of 1:10. The mass spectrometer was operated at an electron energy of -70 eV with an ion source at 250 °C. The mass range used was 40-660 with an acquisition rate of 10 spectra/second.

5.2.3.3 GCxGC-TOF/MS data processing ChromaTOF software was used to analyze the mass spectra of the analyzed extracts. The compounds’ intergrity were then identified by comparing the mass spectra with those on different chemical databases (NIST MSMS, Replib and Mainlib). Only compounds with similarity values of 70 % and above were considered.

5.3. Results and Discussion Volatile secondary metabolites with beneficial biological properties are commonly produced by fungi (Schalchli et al., 2015), hence the present study focused on investigating the chemical constituents of the fungal endophytes isolated from C. africana.

The four fungal endophytes were respectively grown for 21 days after which metabolites were extracted from both the broth media and the mycelia with hexane, ethyl acetate and dichloromethane. Subsequent analysis on GC×GC-TOF/MS showed different volatile secondary metabolites (Tables 5.1-5.6), discussed herein are compounds considered significant (refer to Appendix 2 for complete lists of compounds from all the endophytes including those considered less significant). While Tables 5.1-5.3 show extracellular metabolites (released into the media), Tables 5.4 – 5.6 show those of the mycelia crude extracts representing the intracellular metabolites (those that were retained inside the fungal cells). The subsequent compounds tabulated (Tables 5.1-5.6) showed similarity values of 70 % and above with the spectral data from the libraries used. Overall results obtained, showed the presence of compounds of various biochemical structures including hydrocarbons, alcohols, esters, nitrogen containing compounds and sulfur containing

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compounds. Most of the compounds that were detected from the endophytes under the current investigation have been observed in other studies and observed to have biological properties (Diblasi et al., 2015; Krishnaveni, 2015; Chen and Viljoen, 2010).

5.3.1 Extracellular secondary metabolites from the fungal endophytes. Tables 5.1-5.3 show the compounds extracellular volatile secondary metabolites together with their retention time (R.T), chemical formula, molecular weight and peak percentage (%) area which also relate to the amount of that particular compound present. In each Table, the detected compounds are grouped in terms of their functional groups.

The extracellular secondary metabolites, revealed the presence of a total of 21, 13, 16 and 15 compounds for isolates EKFA1 (A. flavus), EKFA2 (Aspergillus sp), EKFF (Aspergillus. sp.) and EKBE (A. niger) respectively. Compared to the other organic solvents that were used to extract the secondary metabolites, it is evident that ethyl acetate extracts had more compounds detected, followed by hexane then lastly DCM. This was observed in the case of the two isolates in the Aspergillus flavi section. This suggests that most of the compounds produced by the Flavi species are polar. On the other hand, it was not the same case in the Aspergillus Nigri section isolates, in EKFF the hexane had more compounds detected and identified than the other organic solvents suggesting that, more of the compounds produced by EKFF isolate were non-polar. Whilst the EKBE isolates had more compounds detected in the ethyl acetate, followed by DCM and lastly hexane with the least number of compounds detected.

From the hexane broth crude extracts (Table 5.1), 8 compounds were identified from the A. flavus (EKFA1), which was the endophyte with the highest number of compounds detected, followed by A. niger (EKBE), and the two Aspergillus sp. (EKFF; EKFA2) with 6, 7 and 5 compounds respectively. Overall, a total of 6 esters were detected across all the hexane broth crude extracts of the endophytes, of which two were sulfur containing compounds. A particular observation of interest was that EKFA2 afforded no ester compounds extractable by the non-polar solvent (hexane). Most sulfur-containing compounds have been shown to have protecting effects against numerous types of cancer (Gianni and Fimognari, 2015). Gahan and Schmalenberger (2014), reported that sulfur in the soil is available in an unutilized form, and microorganisms that have symbiotic relationship with a plant can help to demineralize the sulfur so that the plant can be able

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to absorb it. The observation of the sulfur compounds could suggest that they produce them in order for C. africana to be able to utilize the sulfur.

The isolate EKFA1 was found to produce fatty acids, and it was the only isolate with these compounds in the hexane extracts. This includes three fatty acid methyl esters (hexadecanoic acid methyl ester; 9,12-octadecadienoic acid methyl ester (E,E) and octadecanoic acid methyl ester) and one fatty acid (9-octadecenoic acid (Z)). The latter is an unsaturated fatty acid, and has been previously extracted from a plant-root associated bacterium, and was suggested to have antimicrobial activity by inhibiting the formation of biofilms (Singh et al., 2013). In addition, fatty acids are important as they are products of a genetically conserved pathway (acetyl-CoA pathway) and therefore can be used to chemically characterize microorganisms (Tomaz et al., 2012). Another ester worth mentioning is the ethyl ester of benzenepropanoic acid (benzenepropanoic acid α-oxo-, ethyl ester) that was detected in the broth hexane extract of EKFF, this ester is reported to be used as a flavoring agent (Burdock, 2001) and also used as an intermediate in the production of various compounds. In a study by Shehab and Ghoneim (2011), the compound was used to synthesize 4H-pyran derivatives which exhibited antimicrobial activity.

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Table 5.1: Extracellular secondary metabolites detected from the hexane broth crude extracts. Compound name R.T(s) Chemical Molecula Peak % area formula r weight EKFA1 EKFA2 EKFF EKBE Esters

Benzenepropanoic acid, α-oxo-, ethyl ester 338 C11H12O3 192.08 ND ND 0.0001 ND

Hexadecanoic acid, methyl ester 1080.2 C17H34O2 270.26 0.1146 ND ND ND

9,12-Octadecadienoic acid, methyl ester, (E,E)- 1266.5 C19H34O2 294.26 0.0362 ND ND ND

Octadecanoic acid, methyl ester 952.8 C19H38O2 298.29 0.0151 ND ND ND

Sulfurous acid, decyl 2-ethylhexyl ester 1203.7 C18H38O3S 334.25 0.0737 ND 0.0356 0.0182

Sulfurous acid, dodecyl 2-propyl ester 1225.2 C15H32O3S 292.21 0.0154 ND ND 0.0116 Alcohols

1-dodecanol 1135.8 C12H26O 186.20 0.0173 ND ND ND

1-Octadecanol 1277.7 C18H38O 270.29 0.0012 ND 0.0013 ND

1-Hexadecanol 759.5 C16H16O2 240.12 ND ND ND 0.0013

Isooctanol 1166.1 C8H18O 130.14 ND ND ND 0.0098

Phenol, 2,4-bis(1,1-dimethylethyl)- 803.5 C14H22O 206.17 ND ND 0.0054 ND

Phenol, 5-methyl-2-(1-methylethyl)- 625.6 C10H14O 150.10 ND 0.1668 ND ND Acids

9-Octadecenoic acid (Z)- 1223.8 C18H34O2 282.26 0.0084 ND ND ND

Tetronic acid 1329.2 C4H4O3 100.01 ND ND 0.0167 ND

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Table 5.1 ( Continued) Compound name R.T(s) Chemical Molecula Peak % area formula r weight EKFA1 EKFA2 EKFF EKBE Ketones

2(1H)-Pyrazinone, 1-hydroxy-6-(1-methylpropyl)-3-(2- 922.6 C12H20N2O2 224.15 ND 25.456 ND ND methylpropyl)-, (+)-

Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- 1086.3 C11H18N2O2 210.13 ND 0.3766 ND ND methylpropyl)-

(5as-cis)-octahydro-5H,10H-dipyrrolo[1,2-a:1’,2’-d]pyrazine- 1105.7 C10H14N2O2 194.10 ND 0.0737 ND ND 5,10-dione

hexahydropyrrolo[1,2-a]pyrazine-1,4-dione 998 C7H10N2O2 154.07 ND 10.385 ND ND Others(N- containing, aldehydes and hydrocarbon)

2H-tetrazole, 2-acetyl- 205.6 C3H4N4O 112.03 ND 1.5542 ND ND

Dimethomorph 853 C21H22ClNO4 387.12 ND ND 0.0232 ND

Simetryn 878.9 C8H15N5S 213.10 ND ND 0.0587 ND

Stigmastan-3,5-diene 1678.4 C29H48 396.37 ND ND ND 0.0644 Total # compounds 8 6 7 5

Where ND indicates no detection of the particular compound in the crude extract, EKFA1: A. flavus, EKFA2: Aspergillus sp. (Flavi group), EKFF: Aspergillus sp. (Nigri group) and EKBE (A. niger).

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There were compounds detected in the hexane extracts that were only unique to specific endophytes. For example, the steroidal hydrocarbon stigmastan-3,5-diene, reported to have antimicrobial effects (Krishnaveni, 2015), was only detected in the broth crude extract of EKBE (Table 5.1) at a trace amount of 0.0644 %. One other compound worth mentioning, is tetronic acid which was found in the hexane crude extract of EKFA2, this compound is a precursor for the synthesis of a class of tetronic acids with butenolides cores, these compounds have multiple medicinal utilities, such as antiviral, antibiotic, anticoagulant and also antineoplastic (Abdou et al., 2015). It was also reported as a metabolite from an endophytic Penicillium griseoroseum (da Silva et al., 2013).

In the hexane extracts (Table 5.1), four diketopiperizine/pyrazine compounds were detected only in the EKFA2 isolate. Observation of the presence of pyrazines is notable, since nitrogen containing compounds are generally important compounds with biological activities and some of them are precursors for alkaloidal compounds. The pyrazines have been isolated from bacteria (Munjal et al., 2016), fungi (Lau et al., 2014) and plants (Beck et al., 2003), and they were reported to exhibit chemo-preventive and antibiotic properties (Beck et al., 2003). For example Pyrrolo [1, 2-a]pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl) was isolated from a Penicillium species and is reported to have antibacterial effects (Diblasi et al., 2015). Furthermore, Southon and Buckingham (1989), reported the compound to be antifungal and antitumureous. Another pyrazine compound, hexahydropyrrolo[1,2-a]pyrazine-1,4-dione was found to be the compound responsible for antioxidant activity in a study by Ser et al. (2015), whereas, (5as-cis)-octahydro- 5H,10H-dipyrrolo[1,2-a:1’,2’-d]pyrazine-5,10-dione was isolated from an insect and found to possess antibacterial effects (Huberman et al., 2007).

Compounds detected in the ethyl acetate crude extracts (Table 5.2) are to some extent similar to the compounds detected in the hexane broth extracts (Table 5.1). EKFA1 was the isolate with more secondary metabolites (14 compounds), followed by EKBE and EKFA2, with 10 and 8 compounds, respectively. EKFF had the least number of metabolites with only 6. Two of the diketopiperizine compounds that were observed in the hexane broth extracts of EKFA2 isolate were also detected, but in this case these compounds were dispersed among the four isolates with Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- occurring in all the endophytes and hexahydropyrrolo[1,2-a]pyrazine-1,4-dione occurring in the three endophytes EKFA1,

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EKFA2 and EKBE only. Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)- and 2,5- Piperazinedione, 3,6-bis(phenylmethyl)- were detected in the ethyl acetate extracts but not the hexane extracts. They were both found in the EKFA1 isolate, with the former additionally occurring in the EKBE isolate. The peptide, 2,5-Piperazinedione, 3, 6-bis (phenylmethyl) - is known as an active ingredient for supplements used to improve people’s motivation to learn (Matsubayashi et al., 2012). Pyrrolo[1, 2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl) - is another peptide pyrazine, known to possess antifungal activity (Sanjenbam et al., 2014).

The nitrogen containing metabolites (propiconazole and cyproconazole), bis(2-ethylhexyl) adipate were generally detected across all the four fungal endophytes. Interestingly, the latter compound has utilities as a hydraulic fluid component and in the make-up of plastics and medical devices (Masse et al., 2017). It is thus intriguing to detect this compound in the fungal endophytes of C. africana and as such, further studies are required to determine its biosynthesis or how to enhance its production as it has widespread industrial usage. Propiconazole and cyproconazole are used widely in agricultural settings as they are valued for their fungicidal properties (Hemalatha et al., 2016; Huang et al., 2016). These triazoles were also detected from all four endophytic fungi. One other compound of agricultural importance is simetryn, the compound has herbicidal properties (Kasai and Hanazato, 1995). Its presence in the endophytes of C. africana could suggest a competitive advantage of the plant over plant weeds. This compound was detected in the two Flavi section Aspergillus species EKFA2 (Table 5.1) and EKFA1 (Table 5.2).

According to Geetha et al. (2015), alcohols are generally antioxidative and antibacterial, while fatty alcohols are particularly used in the cosmetic industry. In the present study, the volatile component profile of the ethyl acetate crude extracts showed the presence of six alcohols, with each EKFA1, EKFF and EKBE having two alcoholic compounds detected. Fatty alcohols such as 1-tetradecanol were among the compounds detected in EKBE isolate with a peak percentage area of 1.1042%, this compound is used in the cosmetic industry as an emollient to soften skin (Avci et al., 2014). There were also phenolic alcohols (phenol,2,6-bis(1,1-dimethylethyl)-4-methyl-; phenol,3,5-bis(1,1-dimethylethyl)-; and phenol,2,4-bis(1,1-dimethylethyl)-) detected in the broth crude extracts of ethyl acetate of EKFA1, EKBE and EKFF isolates. Phenol, 3,5-bis(1,1- dimethylethyl)- and Phenol, 2,4-bis(1,1-dimethylethyl)- are well-known for their antimicrobial properties (Gao et al., 2016; Dhanya et al., 2016).

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Table 5.2: Extracellular secondary from the ethyl acetate crude extracts Compound name R.T(s) Chemical formula Molecula Peak % area r weight EKFA1 EKFA2 EKFF EKBE Ketones

Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- 1095.4 C11H18N2O2 210.14 1.0269 0.0879 1.3687 1.3854 methylpropyl)-

Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 1346 C14H16N2O2 244.12 0.3298 ND ND 0.11283 (phenylmethyl)-

hexahydropyrrolo[1,2-a]pyrazine-1,4-dione 991.3 C7H10N2O2 154.07 0.3917 0.2175 ND 0.1211

2,5-Piperazinedione, 3,6-bis(phenylmethyl)- 1543.4 C18H18N2O2 294.14 0.0169 ND ND ND Alcohols

1-Undecanol 870.8 C11H24O 172.18 ND ND 0.2024 ND

1-Tetradecanol 869.6 C14H30O 214.23 ND ND ND 1.1042

Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl- 1139.1 C15H24O 220.18 0.0422 ND ND ND

1-Octadecanol 909.2 C18H38O 270.29 0.0115 ND ND ND

Phenol, 3,5-bis(1,1-dimethylethyl)- 802.9 C14H22O 206.17 ND ND ND 0.0275

Phenol, 2,4-bis(1,1-dimethylethyl)- 803.4 C14H22O 206.17 ND ND 0.0199 ND Esters

bis(2-ethylhexyl) adipate 1332.4 C22H42O4 370.31 0.1898 0.2921 0.2847 0.2372

Sulfurous acid, dodecyl 2-propyl ester 1399.8 C15H32O3S 292.21 0.0122 ND ND 0.0391

Sulfurous acid, decyl 2-ethylhexyl ester 1306.9 C18H38O3S 334.25 0.0077 0.0813 ND 0.0640

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Table 5.2 ( Continued) Compound name R.T(s) Chemical formula Molecula Peak % area r weight EKFA1 EKFA2 EKFF EKBE Aldehydes

3-[2',4'-Dimethylphenyl]-2,2-dimethylpropanal 426.6 C13H18O 190.14 ND 0.0294 ND ND

2-Phenylpropanal 435.3 C9H10O 134.07 ND 0.0098 ND ND

Benzaldehyde 342.5 C7H6O 106.04 0.0186 ND ND ND

Acetaldehyde, (acetyloxy)-, 1-oxime, (E)- 866.4 C4H7NO3 117.04 0.3254 ND ND ND Others

Propiconazole 1325.5 C15H17Cl2N3O2 341.07 0.6588 0.7752 0.7245 0.8309

Cyproconazole 1262.4 C15H18ClN3O 291.11 0.1032 0.2390 0.2721 0.2071

Simetryn 565.5 C8H15N5S 213.10 0.0131 ND ND ND Total # compounds 14 8 6 10

Where ND indicates No Detection of that particular compound in the crude extract, EKA1: A. flavus. EKA2: Aspergillus sp. (Flavi group) EKFF: Aspergillus sp. (Nigri group) and EKBE: A. niger

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Other phenolic compounds detected in the EKFF and EKFA1 ethyl acetate broth crude extracts include 3-(2',4’-dimethylphenyl)-2;2-dimethylpropanal; 2-phenylpropanal and benzaldehyde respectively.

When the endophytes broth media were extracted with a solvent of medium polarity (DCM), the number of compounds was not as high as expected, instead, these extracts yielded the smallest number of metabolites identified, as compared to the other two solvents discussed before with the exception of EKFF which yielded the same number of compounds when compared with its hexane broth crude extract (Table 5.1) and EKBE which had higher number of compounds detected when compared to its hexane broth extract. EKFA1 in this case, afforded only 7 compounds identified, followed by the two “Nigri” section Aspergillus species (EKFF and EKBE) each of which had 6 compounds identified. The least number of compounds was observed in isolate EKFA2 with only 4 compounds. One observation of interest was that all the alkaloidal compounds (pyrazines) that were detected in the hexane and ethyl acetate broth crude extracts (Table 5.1 and Table 5.2) were also detected in the DCM extracts. Moreover unlike in the other two groups (hexane and ethyl acetate) of extracts where these compounds were found to be in other isolates and not in others, in this case, the pyrazine compounds were occurring in all the endophytes at varying amounts. They were also detected in higher peak percentage area, thus constituting them the major compounds in the DCM crude extracts of the isolates, the peak % area of these compounds ranged from 0.8008 to 16.797. As discussed before (Table 5.1 and 5.2) these compounds are significant for their various biological activities. The ester, bis(2-ethylhexyl) adipate was absent in the EKFA2 isolate but present in all the other three.

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Table 5.3: Extracellular secondary metabolites detected in the DCM crude extracts Compound name R.T(s) Chemical Molecular Peak % area formula weight EKFA1 EKFA2 EKFF EKBE Ketones

Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 994.6 C7H10N2O2 154.07 14.7530 4.5574 16.7460 13.9390

Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- 1095.9 C11H18N2O2 210.14 8.0830 16.797 14.065 11.2560 methylpropyl)-

Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 1323.8 C14H16N2O2 244.12 0.8008 7.4436 2.9948 0.88774 (phenylmethyl)-

5H,10H-dipyrrolo[1,2-a:1',2'-d]pyrazine-5,10-dione, 1103.8 C10H14N2O2 194.11 4.1865 2.0052 7.3942 6.0669 octahydro-, (5as-cis)- Esters

bis(2-ethylhexyl) adipate 1332.2 C22H42O4 370.30 1.0177 ND 1.9383 1.5065 Alcohols

1-Heptadecanol 861.7 C17H36O 256.23 0.0609 ND ND ND

1-Heptanol 1004.1 C7H16O 116.12 ND ND 0.0322 ND Acids

Phenylacetic acid 1394 C8H8O2 136.05 ND ND ND 0.0006

3.75 Butanoic acid-2-methyl- 238.9 C5H10O2 102.07 0.0373 ND ND ND Total # compounds 7 4 6 6

Where ND indicates No Detection of that particular compound in the crude extract, EKA1: A. flavus. EKA2: Aspergillus sp. (Flavi group) EKFF: Aspergillus sp. (Nigri group) and EKBE: A. niger

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Phenylacetic acid was detected in the EKBE isolate, although it was in very little amounts with a peak percentage area of 0.0006 %, this compound is an auxin hormone and is known to be a plant growth regulator (Leuba and Le-Tourneau, 1990). Corresponding to the current study, this compound was previously found in A. niger and observed to have antimicrobial activity (Nair and Burke, 1988). One of the benefits of endophytes to the plant host is to enhance their growth and therefore the presence of this compound in one of the C. africana endophytic isolates further suggests that they are true endophytes. There has been a study whereby a plant hormone of this type was found in endophytes and was observed to enhance the growth of those particular plants (Khan et al., 2016).

5.3.2 Intracellular metabolites investigations of the fungal endophytes To determine the types of metabolites that are retained inside the cells after being produced as products of secondary metabolism, each fungal isolate was grown for three weeks after which the mycelia mat was extracted with solvents of different polarities. The volatile compounds of all the endophytes forming part of intracellular secondary metabolites are tabulated in Table 5.4 - 5.6.

Compared to the broth crude extracts (Section 5.3.1), the mycelial extracts showed a fewer number of compounds identified. The endophytes of EKFA1, EKFA2, EKFF and EKBE isolates had 4, 2, 1 and 6 compounds identified respectively. Evidently it shows that secondary metabolites produced by these endophytes are released into the media (extracellular). However, there were a few of the compounds that were detected in higher peak % area in the mycelial extract than in the broth extract, for example, the triazine, simetryn was detected in the mycelial ethyl acetate crude extract of EKFA1 at a peak % area of 4.5 (Table 5.5), on the other hand this compound was in ethyl acetate broth crude extract of the same isolate at a trace amount of 0.01 % (Table 5.2). The EKFF isolate had the compound at a peak % area of 0.0587 in the hexane broth crude extract (Table 5.1).

Of the 6 total metabolites identified in the hexane extracts, EKBE isolate was the one which produced more metabolites, where five were identified, followed by EKFA1 and EKFF both with 1 metabolite identified. No compounds were observed in the EKFA2. Two esters were identified and they were all detected in the EKBE isolate, namely: Sulfurous acid, 2-ethylhexyl isohexyl ester and 9,12-octadecadienoic acid, methyl ester, (E, E). The fatty acid methyl ester, ((9,12- octadecadienoic acid methyl ester, (E, E)-) was found at a peak percentage area of 2.1177 %. 2,3-

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Heptanedione was one hexane extractable ketone that was found in at least one endophyte. It was found in EKFA1 and EKFF, at a peak percentage area of 0.0291 % and 0.1802 %. This diketone is valued for its flavor and thus is used as a flavoring agent in the food industry (Winter, 2009).

The alcohols content of isolate EKBE was represented by two compounds. All the other three isolates had no alcohol compounds detected. One alcohol of interest from the aforementioned isolate, is geraniol which constituted 0.01 % peak percentage area. This monoterpene has previously been shown to have inhibiting effect against pathogenic fungi (Sharma et al., 2016) and antibacterial properties (Jirovetz et al., 2007). Other properties of geraniol include antiproliferative against cancer cells, insecticidal and insect repelling (Chen and Viljoen, 2010). The compound is in addition reported to be used as an ingredient in cosmetics (Chen and Viljoen, 2010). Other compounds of interest that were detected in the hexane extracts include the steroid, 3à, 5-Cyclo- 5à-ergosta-6, and 8(14).22t-triene which was detected only in the A. niger isolate (EKBE).

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Table 5.4: Intracellular secondary metabolites detected in the hexane crude extracts Compound name R.T(s) Chemical Molecular Peak % area formula weight EKFA1 EKFA2 EKFF EKBE Ketones

2,3-Heptanedione 970.30 C7H12O2 128.08 0.0291 ND 0.1802 ND Esters

Sulfurous acid, 2-ethylhexyl isohexyl ester 1517.50 C14H30O3S 278.19 ND ND ND 0.0984

9,12-Octadecadienoic acid, methyl ester, (E,E)- 1213.30 C19H34O2 294.26 ND ND ND 2.1177 Alcohols

1-Hexadecanol 997.20 C16H16O2 240.12 ND ND ND 0.3027

Geraniol 368.00 C10H18O 154.14 ND ND ND 0.0096 Others

3à, 5-Cyclo-5à-ergosta-6,8(14).22t-triene 1556.00 C28H42 378.33 ND ND ND 0.0318 Total # compounds 1 0 1 5

Where ND indicates No Detection of that particular compound in the crude extract, EKFA1: A. flavus. EKFA2: Aspergillus sp. (Flavi group) EKFF: Aspergillus sp. (Nigri group) and EKBE: A. niger

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Ethyl acetate extracted very few identifiable intracellular compounds, which were detected in the EKFA1 isolate, while all the other isolates had no compounds identified. The two compounds were the aldehyde (hexanal) which was observed to be an intracellular compound as it was not observed in the broth extracts of the endophyte. The second compound was the triazine (simetryn) which was observed in the broth extract of the same isolate (Table 5.2) and EKFFF (Table 5.1) isolate.

Table 5.5: Intracellular secondary metabolites detected in the ethyl acetate crude extracts. Compound R.T(s) Chemical Molecular Peak % area name formula weight

EKFA1 EKFA2 EKFF EKBE

Aldehyde

Hexanal 209.2 C6H12O 100.09 0.1358 ND ND ND Triazine

Simetryn 374 C8H15N5S 213.10 4.5295 ND ND ND Total 2 0 0 0 compounds

Where ND indicates No Detection of that particular compound in the crude extract, EKFA1: A. flavus, EKFA2: Aspergillus sp (Flavi group), EKFF: Aspergillus sp. (Nigri group) and EKBE: A. niger.

Showing similar observations, EKFA1 produced more metabolites that were extracted by DCM with three being identified followed by the isolates EKFA2 and EKBE with two and one metabolite/s respectively. In a similar vein like the ethyl acetate extract, the EKFF isolate had no identifiable secondary metabolites. From the alkaloidal pyrazine compounds that have been consistently detected in the broth extracts, only two were observed to be intracellular to the endophytes, pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, (3s-trans)- (0.0383 % ) was observed in the EKFA1 and pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- was observed in the EKBE (3.5796 %). These have been earlier discussed (Section 5.3. 1). The dicrotophos-(E) isomer was only detected in the DCM mycelial extract of EKFA1 and not in any of the other three isolates. This compound is valued for its pesticidal properties (Barbosa et al., 2016).

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Table 5.6: Intracellular secondary metabolites detected in the DCM crude extracts. Compound name R.T(s) Chemical Molecular Peak % area formula weight EKFA1 EKFA2 EKFF EKBE Ketones

Pyrrolo[1,2-a]pyrazine-1,4-dione, 1102.7 C11H18N2O2 210.14 0.0383 ND ND ND hexahydro-3-(2-methylpropyl)-, (3s- trans)-

Pyrrolo[1,2-a]pyrazine-1,4-dione, 990.1 C7H10N2O2 154.07 ND ND ND 3.5796 hexahydro- Others

Sulfurous acid, 2-ethylhexyl isohexyl 1335.7 C14H30O3S 278.19 ND 0.5659 ND ND ester

Hexanal 207.6 C6H12O 100.09 12.6750 3.3138 ND ND

Dicrotophos-(E)-Isomer 1282 C8H16NO5P 237.08 0.5067 ND ND ND Total # compounds 3 2 0 1

Where ND indicates No Detection of that particular compound in the crude extract, EKFA1: A. flavus, EKFA2: Aspergillus sp (Flavi group), EKFF: Aspergillus sp. (Nigri group) and EKBE: A. niger.

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5.4 Conclusion The secondary metabolites investigations of C. africana fungal endophytes carried out in this study revealed the presence of various compounds, such as the alkaloids, pyrazines and various alcohols compounds. However, there were also bioactive compounds that were mainly found in particular endophytes, such as Stigmastan-3, 5-diene, geraniol and Phenylacetic acid which were produced by A. niger (EKBE) only. Hexanal was only produced by the Flavi section endophytes (EKFA1 and EKFA2). Most of the various compounds that were detected from the fungal endophytic of C. africana have been observed by other researchers to have biological properties. The compounds detected have a range of applications, from perfumery to flavoring, cosmetics, pharmaceuticals and agriculture. Although, the production of these compounds by the endophytes could attribute to the beneficial nature of the endophytes to the C. africana, nonetheless more research is needed to determine the roles that these endophytes play in their host plant. Studies on biological control and structural elucidation of these compounds is still very much needed.

Author contribution E.K. Nchabeleng planned and performed the experiments, assisted with analyzing the gas chromatography data and wrote the paper.

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CHAPTER SIX IDENTIFICATION AND CHARACTERIZATION OF SECONDARY METABOLITES PRODUCED BY ENDOPHYTIC BACTERIA ASSOCIATED WITH CELTIS AFRICANA FROM SOUTH AFRICA. Nchabeleng*1, E.K., Ndinteh2, D.T., Niemann1, N. and Mavumengwana1, V. 1 Department of Biotechnology and Food Technology, Faculty of Science, University of Johannesburg, P. O. Box 17011, Doornfontein Campus, Johannesburg, South Africa. 2 Department of Applied Chemistry, Faculty of Science, University of Johannesburg, P. O. Box 17011, Doornfontein Campus, Johannesburg, South Africa *Corresponding author: [email protected], Tel: +27115596915 Abstract The principal aim of this study was to determine the secondary metabolites from seven bacterial endophytes (Kocuria sp., Micrococcus luteus, Staphylococcus hominis, S. saprophyticus, Brachybacterium conglomeratum, Arthobacter sp. and Bacillus sp.) obtained from Celtis africana (white stinkwood). The endophytes were cultivated in broth growth media for 48 hours. Following that, the growth media was separated from the cells by centrifugation, the resulting supernatant was then extracted with three solvents (hexane, dichloromethane and ethyl acetate) successively. Gas chromatography coupled with mass spectrometry was used to analyze the extracts and thus determine the metabolites identity. The results obtained showed that the bacterial endophytes produce metabolites of different structural nature from alkaloids to phenolics and terpenes. Some of the alkaloidal compounds were the piperazine compounds which were the most commonly produced among the seven endophytes. A bioactive terpene (squalene) was found to be produced by the endophyte Arthrobacter sp. One of the phenolic compounds was the antimicrobial, phenol, 2,4-bis(1, 1-dimethylethyl)– which was observed to be a metabolite produced by B. conglomeratum and Arthobacter sp. One other interesting observation was that all the endophytes produced a fungicidal triazole (propiconazole). This study has therefore revealed the presence of various volatile bioactive metabolites from these endophytes and thus has suggested that these endophytes are potential sources of interesting compounds which can be useful in various sectors.

Keywords: Endophytic bacteria, gas chromatography; secondary metabolites, white stinkwood.

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6.1 Introduction According to Brader et al. (2014), over 20 000 microbial bioactive compounds have been identified from bacteria. The association of endophytes with their host plants have led to the suggestion that these endophyte have the ability to produce metabolites that improve the plant’s ability to fight diseases. Therefore these endophytes are a potential source of bioactive natural products (Krishnan et al., 2012). In contrast to fungal endophytes, less studies have been done on the metabolic potential of bacterial endophytes (Brader et al., 2014).

The Actinobacteria belong in the oldest and largest phylum (Actinobacteria) within the bacteria domain (Verma et al., 2013). The bacteria in this class are considered as one of the richest sources of biologically active metabolites (Hamedi et al., 2015). According to Manivasagan et al. (2014), the Streptomyces genus in this Actinobacteria class, is regarded as a dominant producer of bioactive metabolites. However, four of the seven bacterial endophytes (Micrococcus luteus, Arthobacter sp., Brachybacterium conglomeratum and Kocuria sp.) that were isolated from C. africana belong to this class and there have been previous studies that reported on their ability to synthesize bioactive constituents. For example a Micrococcus sp. associated with a marine organism was reported to produce bioactive secondary metabolites (diketopiperazines) (Stierle et al., 1988). Other studies showed antimicrobial properties of species in the Micrococcus genus and specifically the M. luteus strain (Eltamany et al., 2014; Umadevi and Krishnaveni, 2013). Members in The Arthrobacter genus are known for the production of pigments which are sometimes used in pharmaceuticals and food (Sutthiwong et al., 2014). Amaresan et al. (2012), made note of an Arthrobacter sp. endophyte strain that has antifungal activity and the ability to produce a phytohormone that enhances growth. Very little information is available with regards to members in the Kocuria and Brachybacterium genera as sources of biologically active compounds.

The three bacterial endophytes that represented the Bacilli class isolated from C. africana, were Staphylococcus hominis, S. saprophyticus, and Bacillus sp., from the literature search conducted it shows that these strains are also capable of producing bioactive compounds or have shown biological activity of some sort. In a study by Kim et al. (2010), it was observed that S. hominis produces a peptide compound that has antibacterial activity. Furthermore, Lins et al. (2014) found that the aforementioned Staphylococcus species have the ability of produce indole acetic acid, which is a plant growth promoting hormone. Species in the Bacillus genus are known to produce

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various metabolites of biological importance such as antibacterial, pesticidal and bio-surfactant compounds (Osouli and Afsharmanesh, 2016; Bacon et al., 2015; Cho et al., 2002).

This thus shows that bacteria are potential fertile sources of bioactive compounds with diverse biotechnological applications, therefore in this regard, as part of our aimed study on determining the bioactive compounds from endophytes associated with C.africana, investigation of secondary metabolites produced by the seven endophytic bacteria associated with the aerial parts of C. africana was carried out.

6.2. Materials and methods 6.2.1 Bacterial isolates resuscitation The stock cultures of the bacterial isolates were resuscitated by inoculating a loopful of the glycerol stock culture into 100 mL of Nutrient broth (NB)(Oxoid), each broth culture was incubated at 37 ºC (Labotec, South Africa) for 24 h. This was used as inoculum for the fermentation of the bacterial endophytes.

6.2.2 Fermentation and secondary metabolite extraction Following a modified method by Melo et al. (2009), the endophytes were grown in large quantities with the aim of extracting secondary metabolites. Each isolate was grown in a total of 3 L NB, with 1 L flask each containing 500 mL of NB using an inoculum of 5 % v/v. The flasks were then incubated in shaking incubator (Labotec, South Africa) at 120 rpm and 37 ºC for 48 h. After the set incubation period, the broth was centrifuged in batches at a maximum speed of 10 000 x g at 4 ºC for 20 minutes to separate the bacterial cells from the broth. The resulting supernatant was then filtered using Whatman No. 1 filter paper and a separating funnel. Afterwards the filtrate was extracted three times with equal volume of organic solvent (hexane, ethyl acetate and dichloromethane (DCM)). After the extraction, each organic phase was concentrated under reduced pressure using a rotary evaporator. As such, the seven bacterial endophytes afforded 21 crude extracts. Sterile NB was also extracted with organic solvents as described and served as a control.

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6.2.3 Profiling of volatile secondary metabolites 6.2.3.1 Sample preparation Each crude extract was scrapped with a tip of a pipette and dissolved in 1 mL of HPLC grade methanol (Sigma-Aldrich, Aston manor, South Africa).

6.2.3.2 GC×GC-TOF/MS conditions The bacterial endophytes crude extracts were analyzed using a GC×GC-TOF/MS following the outline by Ralston-Hooper et al. (2008), with minor modifications. Helium gas was used as a carrier gas with a flow rate of 1 mL/min and a split mode injection of 1:10 ratio on a Pegasus 4D GCXGC-TOF/MS (Leco, USA) that was fitted with an Rxi-5Sil MS (30 m, 250 µm i.d, 0.25 µm d.f) (Restek, USA) as a primary column and Rxi- 17Sil MS (2 m, 250 µm i.d, 0.25 µm d.f) (Restek, USA) as secondary column. The oven temperature (T°) was set at 50 °C for 0.5 minutes and then ramped to 300 °C at a rate of 10 °C/min, after which it was held at 300 °C for ten minutes. The transfer line T° was 250 °C. The inlet T° was at 250 °C. The mass spectrometer was operated at an electron energy of -70 eV with an ion source at 250 °C. The mass range used was 40-660 with an acquisition rate of 10 spectra/second.

6.2.3.3 GCxGC-TOF/MS data processing ChromaTOF software was used to collect the mass spectra, and the resultant compounds were identified by comparing the mass spectra with those on various libraries (Mainlib, NIST MSMS, and Replib). Only compound with similarity value of 70 % were considered.

6.3 Results and Discussion This study was done as a result of bacterial endophytes largely known for their ability to produce compounds of significant properties. As such, it was a necessity to determine the chemical constituents of endophytic bacteria from C. africana. After the fermentation of the endophytes, each bacterial growth media was sequentially extracted with three solvents. Thus the focus was only on the metabolites that were released into the growth media. Furthermore, only compounds deemed significant and which their mass spectra matched the mass spectra in the libraries with at least 70 % are reported in this study (refer to Appendix 2 for a complete list of compounds as detected from 2D-GC-TOF/MS including those deemed less important).

Compounds in the hexane broth crude extracts of the seven endophytes are tabulated in Table 6.1. Isolates F1, and F2 had the least number of compounds identified, with only one compound each.

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The B2 isolate had the highest number of compounds (six in total) extracted with hexane that were identified, followed by B1 with 4 compounds and B3, F3 and F4 with 3, 2 and 2 compounds respectively. Although the numbers are few some of these compounds have been reported to have useful properties. For example one of the ketones (2,5-di-tert-Butyl-1, 4-benzoquinone) that was detected in the F2, F3, B2 and F4 isolates has also been reported to be a metabolite from a Streptomyces in an earlier study and found to possess antimicrobial properties (Jannu et al., 2015). The phenolic alcohol (phenol, 2,4-bis(1,1-dimethylethyl)-) was detected in the F1 and B2 isolates, with the former having a peak percentage area of 10.345 % and the latter having 0.0203 %. This phenolic compound was recently reported in an endophytic Bacillus species and was observed to exhibit antifungal properties (Gao et al., 2016). In another study by Teresa et al. (2014), this compound was further suggested to interfere with the reactive oxygen species of pathogenic plant fungi.

The six ester compounds that are tabulated in Table 6.1 are partitioned between isolates F3, B2 and F4. F4 isolate had the two sulfur esters: Sulfurous acid, decyl 2-ethylhexyl ester and sulfurous acid, hexyl pentadecyl ester. Isolate F3 showed three esters (2-ethylhexyl nonyl ester, bis(2- ethylhexyl) adipate and isopropyl tetradecanoate). Whilst isolate B2 had only, 6- tetradecanesulfonic acid, butyl ester with a peak % area of 3.4403 %. The synthetic form of the fatty acid ester, isopropyl tetradecanoate (that was detected in F3) has various uses including being used as a skin soother in the preparation of topical medicinal creams and it also used in the food industry as a flavoring agent (Ash and Ash, 2004). Therefore, this means that bacterial metabolites can inspire the chemical synthesis of other compounds. Isolate B3, also afforded an important compound (squalene) as its secondary metabolite which has a fatty hydrocarbon tritepene chemical nature. This compound is said be a precursor for the synthesis of sterols, furthermore it is reported to occur in various life forms, from animals to plant and microbes. A major bioactive property of squalene is mainly the ability to scavenge reactive oxygen species, thus denoting the compound to have antioxidative properties and anticancer (Rudzinska et al., 2017; Xu et al., 2016).

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Table 6.1: Secondary metabolites from the hexane crude extracts of the bacterial endophytes Compound name R.T(s) Chemical Molecula Peak % area formula r weight F1 F2 F3 B2 B3 F4 B1

Ketones

7,9-Di-tert-butyl-1- 1075.5 C17H24O3 276.17 ND ND 0.5693 ND 0.2923 0.4626 0.7775 oxaspiro(4,5)deca-6,9-diene-2,8- dione

2,5-di-tert-Butyl-1,4-benzoquinone 1116.9 C14H20O2 220.15 ND 0.2605 0.0133 0.4470 ND 0.0475 ND Alcohols

1-Dodecanol, 2-methyl-, (S)- 1124.2 C13H28O 200.21 ND ND ND ND ND ND 3.2794

1-Decanol, 2-hexyl- 1012.9 C16H34O 242.26 ND ND ND 0.0565 ND ND ND

Phenol, 2,4-bis(1,1-dimethylethyl)- 802.5 C14H22O 206.17 10.345 ND ND 0.0203 ND ND ND Esters

Sulfurous acid, decyl 2-ethylhexyl 1202.3 C18H38O3S 334.25 ND ND ND ND ND ND 0.1283 ester

Sulfurous acid, hexyl pentadecyl 1310.7 C21H44O3S 376.30 ND ND ND ND ND ND 0.0490 ester

Sulfurous acid, 2-ethylhexyl nonyl 1402.8 C17H36O3S 320.24 ND ND ND 0.1003 ND ND ND ester

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Table 6.1 (Continued) Compound name R.T(s) Chemical Molecula Peak % area formula r weight F1 F2 F3 B2 B3 F4 B1

bis(2-ethylhexyl) adipate 1331.7 C22H42O4 370.31 ND ND ND 0.0068 ND ND ND

Isopropyl tetradecanoate 1015.9 C17H34O2 270.26 ND ND ND 0.0056 ND ND ND

6-Tetradecanesulfonic acid, butyl 1124.4 C18H38O3S 334.25 ND ND ND ND 3.4403 ND ND ester Others

Squalene (Fatty hydrocarbon) 1526.7 C30H50 410.39 ND ND ND ND 0.0518 ND ND Total # of compounds 1 1 2 6 3 2 4

Where ND: Not Detected, F1: Kocuria sp., F2: M. luteus, F3: S. homonis, B2: B. conglomeratum, B3: Arthobacter sp., F4: Bacillus sp. and B1: S. saprophyticus

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Table 6.2 shows the compounds that were detected in the ethyl acetate crude extracts of the endophytes. In general the ethyl acetate had more compounds detected and identified from the endophytes crude extracts as compared to the hexane extracts. Crude extracts from isolates B1 and F3 were the ones with lower compounds identified with 4 and 7 compounds, respectively. F1 and B2 crude extracts both had 10 compounds identified each. And the crude extracts with the highest number of compounds were from B3 and F4 isolates. From the data, a lot of compounds detected from all the endophytes were nitrogen containing, from diketo peptides to triazoles and amines. The ketone profile showed the presence of diketopiperazine compounds which are simple alkaloids. Furthermore, most of the cyclo peptides/diketopiperazines have important biological properties like immunosuppressant, antimicrobial and antitumor, they are further reported to be used for the biosynthesis of bioactive compounds in the host of that particular microbe (Guimarres et al., 2010; Zheng et al., 2005). One such compound detected in this study was 2,5- piperazinedione, 3,6-bis (2-methylpropyl)- which was found in all the endophytes ethyl acetate crude extracts with the exception of B3 and B1. According to Bhaderka and Parhi (2016), this compound was found to be produced by a food pro-biotic microbe and has antifungal activity. 3,6- diisopropylpiperazine-2, 5-dione was only observed in two of the endophyte isolates, namely F2 and F4. This compound has been isolated from a marine-derived bacterium which possessed antimicrobial activity (Zheng et al., 2005).

Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- and pyrrolo[1,2-a]pyrazine- 1,4-dione, hexahydro- were common across all the crude extracts. The latter compound was in earlier studies, isolated from marine Bacillus species by Gopi et al. (2014) and found to possess antioxidant properties. The cyclo peptide, pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- methylpropyl)- also has reported antimicrobial activity (Diblasi et al., 2015). 3- Benzylhexahydropyrrolo [1, 2-a] pyrazine-1, 4-dione is another cyclo peptide that was common in all the ethyl acetate crude extracts of the endophytes, this compound was found to be a secondary metabolite from Bacillus cereus with antifungal and antibacterial properties (Kumar et al., 2014). Hexahydropyrrolizin-3-one was the ketone with the lowest molecular weight and retention time and it was observed in the B2 isolate.

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Some of the ketones detected in the ethyl acetate extract of the endophytes have similar structures with the ones already discussed (Table 6.1). Furthermore, some of the pyrazine ketone compounds reported in this study have also been reported in our previous studies of the metabolites produced by the endophytic fungi from the same plant (C. africana) (Chapter 5). This includes pyrrolo[1,2- a]pyrazine-1,4-dione,hexahydro-; pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-(2- methylpropyl)-; pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-(phenylmethyl)-; 2,5-and piperazinedione, 3,6-bis(phenylmethyl)-. Other compounds detected in the extracts were aldehydes. 1H-indole-3-carboxaldehyde, is an indole derived alkaloidal compound that has been found in the B3 isolate only and compounds that have an indole center are said to be biologically active (Rosu et al., 2012). This compound was isolated from an endophyte and observed to exhibit antiviral activity (Wang et al., 2014). Two phenolic aldehydes, 2-phenylpropanal and Benzaldehyde, 4-(4-methylbenzyloxy) - were observed in the B2 isolate at a peak percentage area of 7.9274 % and 2.8773 %, respectively. Some compounds detected in the ethyl acetate extract were also detected in the hexane extracts (Table 6.1). For example the phenolic compound, phenol, 2, 4-bis(1,1-dimethylethyl)- was detected in the hexane extract of B2, and also in the B3 ethyl acetate extract. Squalene was found in the same isolate (B3) but different solvent extracts (the hexane extract afforded this compound at peak percentage area of 0.0518 %, whereas the ethyl acetate had a slightly higher % than the hexane with 0.2023 %). The fungicide and thus a popularly used biocontrol agent, propiconazole was observed in all the bacterial endophytes. This compound was also observed to be produced by the fungal endophytes of C. africana (Chapter 5).

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Table 6.2: Secondary metabolites from ethyl acetate crude extracts of the bacterial endophytes. Compound name R.T(s) Chemical Molecular Peak % area formula weight

F1 F2 F3 B2 B3 F4 B1 Ketones

2,5-Piperazinedione, 3,6- 1096.7 C12H22N2O2 226.17 3.4892 2.5095 6.6055 2.3816 ND 2.5095 ND bis(2-methylpropyl)-

3- 1347.9 C14H16N2O2 244.12 1.1596 0.76912 1.8181 0.74132 2.2093 0.5360 1.4791 Benzylhexahydropyrrolo[1,2- a]pyrazine-1,4-dione

Pyrrolo[1,2-a]pyrazine-1,4- 1102.9 C11H18N2O2 210.14 4.5292 0.7461 1.4044 2.5778 1.3710 0.7461 0.7711 dione, hexahydro-3-(2- methylpropyl)-

2,5-Piperazinedione, 3- 1227.3 C12H14N2O2 218.11 ND ND 0.20517 ND 0.0790 ND ND methyl-6-(phenylmethyl)-

3-Benzyl-6-isopropyl-2,5- 1287.9 C14H18N2O2 246.14 0.2008 ND ND ND ND ND ND piperazinedione

Pyrrolo[1,2-a]pyrazine-1,4- 992.8 C7H10N2O2 154.07 1.4387 0.2929 0.78429 0.26851 0.618 0.2929 1.2637 dione, hexahydro-

2,5-Piperazinedione, 3- 1248 C11H12N2O2 204.0899 ND 0.0639 ND ND 0.0975 0.0639 ND (phenylmethyl)-

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Table 6.2 ( Continued) Compound name R.T(s) Chemical Molecular Peak % area formula weight F1 F2 F3 B2 B3 F4 B1

3,6-Diisopropylpiperazin- 1044.5 C10H18N2O2 198.14 ND 0.3426 ND ND ND 0.3426 ND 2,5-dione

Hexahydropyrrolizin-3-one 957.7 C7H11NO 125.08 ND ND ND 0.1155 ND ND ND

5,8-Tridecadione 1046.2 C13H24O2 212.18 0.6358 ND ND ND ND ND ND Aldehydes

2-Propenal, 3-(1-aziridinyl)- 960.9 C7H12N2O 140.10 0.1992 0.1079 ND ND 0.2515 0.1079 ND 3-(dimethylamino)-

1H-Indole-3-carboxaldehyde 1027.2 C9H7NO 145.05 ND ND ND ND 0.3612 ND ND

2-Phenylpropanal 370.1 C9H10O 134.07 ND ND ND 7.9274 ND ND ND

Benzaldehyde, 4-(4- 338 C15H14O2 226.10 ND ND ND 2.8773 ND ND ND methylbenzyloxy)- Esters

bis(2-ethylhexyl) adipate 1333.5 C22H42O4 370.31 0.1851 0.0794 ND 0.08919 0.1865 0.0794 ND

Squalene 1526.5 C30H50 410.39 ND ND ND ND 0.2023 ND ND Alcohols

Phenol, 2,4-bis(1,1- 802.5 C14H22O 206.17 ND ND ND ND 0.0395 ND ND dimethylethyl)-

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Table 6.2 ( Continued) Compound name R.T(s) Chemical Molecular Peak % area formula weight F1 F2 F3 B2 B3 F4 B1

1-Pentanol, 2,2-dimethyl- 273.4 C7H16O 116.12 ND ND ND 0.1708 ND ND ND Others

Propiconazole 1325.4 C15H17Cl2N3O2 341.07 1.1248 0.4344 1.2237 0.45185 1.292 0.43435 1.2682

Pyrazine, trimethyl- 377.2 C7H10N2 122.0844 ND 0.0570 ND ND ND 0.0569 ND

dl-Alanyl-l-leucine 972.8 C9H18N2O3 202.13 0.3095 0.1692 0.3195 ND ND 0.2031 ND Number of compounds 10 11 7 10 11 11 4

Where ND: Not Detected, F1: Kocuria sp., F2: M. luteus, F3: S. homonis, B2: B. conglomeratum, B3: Arthobacter sp., F4: Bacillus sp. and B1: S. saprophyticus

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Apart from the other organic solvents utilized in the extraction, dichloromethane extracts afforded very few or no metabolites identified. For example, isolate F1 had no compounds identified, whilst isolate B3 and B2, had 1 and 3 compounds respectively. In these three aforementioned isolates, their ethyl acetate crude extracts had more compounds identified, followed by the hexane crude extracts. Of the 14 compounds that were collectively detected in the DCM extracts , more than half (9) of them were also detected in the ethyl extracts: namely, pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-; pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-; pyrrolo[1,2- a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-; 2,5-Piperazinedione, 3,6-bis(2- methylpropyl)-; 3-benzyl-6-isopropyl-2,5-piperazinedione ; 3,6-diisopropylpiperazin-2,5-dione; 2-propenal, 3-(1-aziridinyl)-3-(dimethylamino)-; bis(2-ethylhexyl) adipate; and D-L-alanyl-l- leucine.

However, the peak percentage area of the compounds varied between the two solvent extracts, the ethyl acetate generally afforded the extract at lower peak % area when compared to DCM. For example, the alkaloid, pyrrolo [1,2-a]pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl)- was found to be the dominant compound in the DCM crude extract of isolate B3 with a peak percentage area of 84.018 % (Table 6.3), whilst the ethyl acetate crude extract of the same endophyte (Table 6.2) had the compound at 25.066 %. This observation constitute the pyrazines as dominant compounds from their respective bacteria, in addition, it evidently suggests that the compounds are better extracted with DCM than with ethyl acetate. Corresponding to this observation, a similar result was observed in the pyrazine content of the fungal crude extracts of DCM and ethyl acetate extract (Chapter 5).

5H,10H-dipyrrolo[1,2-a:1',2'-d]pyrazine-5,10-dione,octahydro-,(5as-cis)- and 3-benzyl-6-methyl- 2,5-piperazinedione are two diketopiperazine that were not observed in any of the ethyl acetate extracts but were present in the DCM extracts. The latter compound was observed in the extracts of F2 and F3, and the former was found to be produced by F2, F3, B2 and B1. Although the literature review conducted afforded no useful potential/uses of these structurally important molecules. They can only be assumed to be significant molecules as diketopiperazine compounds are generally regarded as biologically active compounds.

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Table 6.3: Secondary metabolites from DCM crude extracts of the bacterial endophytes. Compound name R.T(s) Chemical Molecular Peak % area formula weight F1 F2 F3 B2 B3 F4 B1 Ketones

Pyrrolo[1,2-a]pyrazine-1,4-dione, 999.2 C7H10N2O2 154.07 ND ND 3.591 ND ND 11.268 8.5071 hexahydro-

Pyrrolo[1,2-a]pyrazine-1,4-dione, 1085.1 C11H18N2O2 210.14 ND ND 12.408 8.3952 84.018 3.0453 25.066 hexahydro-3-(2-methylpropyl)-

Pyrrolo[1,2-a]pyrazine-1,4-dione, 1325.4 C14H16N2O2 244.12 ND 4.5764 2.0383 ND ND 0.80534 4.8572 hexahydro-3-(phenylmethyl)-

2,5-Piperazinedione, 3,6-bis(2- 1098.8 C12H22N2O2 226.17 ND 15.586 19.769 13.291 ND ND ND methylpropyl)-

5H,10H-dipyrrolo[1,2-a:1',2'- 1104 C10H14N2O2 194.11 ND 5.2266 6.8547 8.3763 ND ND 5.5095 d]pyrazine-5,10-dione, octahydro-, (5as-cis)-

3-Benzyl-6-methyl-2,5- 1227.4 C12H14N2O2 218.12 ND 0.25246 0.17177 ND ND ND ND piperazinedione

3-Benzyl-6-isopropyl-2,5- 1284.7 C14H18N2O2 246.14 ND 0.2659 0.2079 ND ND ND ND piperazinedione

3,6-Diisopropylpiperazin-2,5-dione 1046.7 C10H18N2O2 198.14 ND 3.5121 ND ND ND ND ND

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Table 6.3 ( Continued) Compound name R.T(s) Chemical Molecular Peak % area formula weight F1 F2 F3 B2 B3 F4 B1

2-Propenal, 3-(1-aziridinyl)-3- 959.3 C7H12N2O 140.10 ND ND 1.3421 ND ND ND 2.1767 (dimethylamino)- Amines

Pyrrolidine, 2,5-dimethyl-1-nitroso- 975.9 C6H12N2O 128.10 ND ND ND ND ND 0.3659 ND

Pyrimido[1,2-a]azepine, 1128.9 C9H16N2 152.13 ND ND ND ND ND 0.1857 ND 2,3,4,6,7,8,9,10-octahydro-

1,3,5-Trimethylhexahydro-1,3,5- 203.1 C6H17N3 131.14 ND 0.7200 ND ND ND ND ND triazine Others

bis(2-ethylhexyl) adipate ( ester) 1331.9 C22H42O4 370.31 ND 0.1081 0.1626 ND ND 0.3898 0.6894

dl-Alanyl-l-leucine(Peptide) 981.5 C9H18N2O3 202.13 ND 0.7544 0.6515 ND ND 0.2628 ND

1-Dodecanol(Alcohol) 959.5 C12H26O 186.20 ND 1.4863 ND ND ND 1.8085 ND Total # compounds 0 10 10 3 1 8 6

Where ND: Not Detected, F1: Kocuria sp., F2: M. luteus, F3: S. homonis, B2: B. conglomeratum, B3: Arthobacter sp., F4: Bacillus sp. and B1: S. saprophyticus

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The compound, bis(2-ethylhexyl) adipate is one of the compounds which was detected in all the bacterial endophytes under investigation. There were also amines present in the F2 DCM broth crude extracts, in particular 1,3,5-trimethylhexahydro-1,3,5-triazine. Pyrrolidine, 2,5-dimethyl-1- nitroso- and Pyrimido[1,2-a]azepine, 2,3,4,6,7,8,9,10-octahydro- were observed in isolate F4 only.

6.4 Conclusion In summary, the aim of the study which was to determine metabolites produced by bacteria endophytic to C. africana has been achieved. The results obtained have shown that the endophytes have the ability to produce bioactive metabolites, with the piperazine compounds being most common amongst the bacterial species. Although this investigation is a first report on the metabolites from C. africana bacterial endophytes, it is necessary to further fractionate these compounds and purely isolate to test them for their reported bioactivity and also test for other bioactive properties except for the ones already in literature. This study has therefore provided a foundation on what type of bioactive molecules are produced in the secondary metabolism of these endophytes.

Author contribution E.K. Nchabeleng planned and performed the experiments, assisted with analyzing the gas chromatography data and wrote the paper.

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CHAPTER SEVEN GENERAL DISCUSISION, CONCLUSION AND RECOMMENTDATIONS 7.1 General discussion and conlusion The lack of novel compounds to eradicate various diseases affecting either plants, animals and humans, compelled the need to therefore search for natural products that can have potential activity against these disease causing agents. Hence the current study focused on the ornamental and medicinal plant (Celtis africana) and its endophytes.

The C. africana plant was observed to have activity against seven pathogenic bacteria viz., Escherichia coli, Proteus mirabilis, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter aerogenes, These pathogenic bacteria are associated with diseases ranging from those affecting the skin, to food, and the respiratory tract. It was further observed that of the three organic solvent extracts, the hexane fruit and leaf extracts together with the ethyl acetate stem extracts were the ones that showed activity against these pathogens. Since crude extracts, and not pure isolated compounds were tested it can be suggested that the compounds within those extracts were inhibiting the growth of these microorganism in synergy. The compounds activity against the pathogens could be part of the various classes of phytochemicals that were observed to be present in the plant. The data generated from the 2D-GC- TOF/MS of the crude extracts of the three plant parts (leaves, stem and fruit) showed the presence of various compounds with different chemical nature. The compounds ranged from fatty acids to esters, alcohols and aldehydes just to name a few. In addition, most of the compounds that were detected in the plant have bioactivities such as anti-inflammatory, antimicrobial, antipesticide and other biotechnological significance, and as such they can be used in the food, agriculture, cosmetic and pharmaceutical industries. Despite that, there were also compounds that suggested that the plant should be used cautiously, i.e. the presence of the toxic compound 3-nitropropinoic acid in the fruit. Since the solvent had different polarities the hexane was expected to extract non-polar compounds, the ethyl acetate (medium polar compounds) and the DCM:MeOH was expected to extract polar compounds. It was observed that the fatty acid compounds detected were extracted by all these three solvents and occurred in all the plant plants. There were however some active compounds that were only specific to a certain plant part, for example the friedelan-3-one and its counter alcohol were specific to the stem and extracted only by ethyl acetate and the DCM:MeOH. The ketone was detected in higher peak % area in the DCM:MeOH than in the ethyl acetate,

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whereas its alcohol counterpart was observed in a similar peak % area in both (DCM:MeOH and ethyl acetate). The chemical profile of the plant parts supported the use of the plant by different ethnic groups to treat various ailments.

Studies done on endophytes, do not only intend on understating the relationship between the host and the endophytes, but they can also be studied for screening bioactive metabolites. In this study, a total of 11 endophytes (7 bacteria and 4 fungi) were isolated from the stem and fruit of the plant. Four of the bacterial endophytes belonged to the Actinobacteria class which is well-known for its ability to produce valuable secondary metabolites. The other three bacterial endophytes belonged to the bacterial class Bacilli. The four endophytic fungi isolated all belonged to the Aspergillus genus. Although Aspergillus species are mostly known to be mycotoxin producers, they have also been reported as endophytes. For example, the two Aspergillus species (A. flavus and A. niger) that were observed from the plant in this study, were earlier reported to be endophytic to a plant in the same family as C. africana (Gautam et al., 2013). When the 11 endophytes were evaluated for their volatile secondary metabolites, it was observed that the endophytes produced similar secondary metabolites. Secondary metabolites from the fungal endophytes were extracted from the growth media and the mycelia (chapter 5) since some bioactive compounds can also be retained inside the cells. Observations evidently suggested that all the four fungal endophytes released their secondary metabolites in to the growth media as very few metabolites were detected in the mycelial crude extracts in comparison to the growth media crude extracts. The similar compounds that were detected throughout the fungal endophytes include the diketopiperazines with the alkaloidal chemical nature, however amongst the three solvents used to extract, dichloromethane crude extracts had these compounds detected in higher peak % area which is also directly proportional to the abundance /amount of the compounds. Other bioactive compounds observed to be commonly produced, among the fungal endophytes, were propiconazole and cyproconazole. The similarity in the kinds of secondary metabolites produced by the endophytes is expected since it is most likely that members in the same genus would produce similar types of compounds (Talapatra and Talapatra, 2015).

When the bacteria were analyzed for their secondary metabolites (Chapter 6), the observation were similar to those in the fungal endophytes (Chapter 5). The intracellular metabolites from the bacteria could not be analyzed as there were negligible yield from the cell mass. In a similar vein,

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the dichloromethane crude extracts of the extracellular metabolites had most of the alkaloidal piperazine compounds detected in higher peak percentage area as compared to other solvents (hexane and ethyl acetate). There were also other bioactive compounds that were observed in the crude extracts of the bacterial endophytes, ranging from antibacterial to antifungal, antifungal, pesticidal, antiviral and etc. in terms of the polarity of solvents and the number of compounds detected, the endophytes had varying relationships in the number of compounds and the extracting solvent. Other endophytes had more compounds detected in the dichloromethane, while others in the ethyl acetate and hexane. For example the Aspergillus Flavi species in chapter 5 had most of their secondary metabolites in the dichloromethane.

In comparison to their host plant, there was to some extent similarities in the compounds produced by the plant and the endophytes. Although the endophytes did not produce exactly the same compounds as the host plant, the classes of compounds detected were similar and they also have similar reported bioactivity. There were similarities observed in the types of secondary metabolites in the endophytes, it has also been reported that the endophytes in the plant are capable of producing similar compounds and as such they maybe exhibit similar biological activities (Yadav et al., 2014).

Another observation was the abundance of N-containing compounds detected in the endophytes of C. africana especially the diketopiperazine. Dragendoff test is able to detect any nitrogen containing compounds, from tertiary amine to alkaloids. The test for alkaloids in the plant was indicative of the presence of this class, and however no alkaloidal compounds were detected in the crude extracts of the plant. Therefore it could be suggested that some compounds from the plant could be ascribed to the presence of the endophytes.

This study according to the best of the author’s knowledge was the first to report on the full chemical profile of C. africana and its endophytic community and their chemical profiles. Thus both the endophytes and their host are suggested to be promising sources of pharmaceutically relevant metabolites and biotechnologically useful compounds.

7.2 Recommendations Based on the observations in this study, the following can be recommended for future studies on the plant:

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Information about the diversity of endophytes from C. africana has been provided. However the diversity of the endophytes was narrow compared to other studies where tens of endophytes were isolated. For future work, it can be recommended that the method of isolation of endophyte be modified, in this study the endophytes were isolated by macerating sterile plant material in PBS and plating the dilutions of that plant material suspension. In other studies, the sterilized plant material were cut into small pieces and placed on to growth media, this was found to enumerate more isolates (Huang et al., 2015). Moreover since no endophytes were detected in the leaves of the plant in the current study, it can be suggested that future experiments adjust the concentration of the solutions used to disinfect and also the time of exposure (Martinez-Klimova et al., 2016), this might give room for endophytes to be isolated from the leaves. Two fungal endophytes (EKFA 1 and EKFA2) were observed to be morphologically similar, future studies can focus on detailed investigation on the extract profiles to fully examine the two endophytes.

All the plant parts of the plant showed antibacterial activity, therefore further investigations are necessary to determine the active compounds that are responsible for the antibacterial activity and also to determine if the compounds work in synergy or not . Furthermore, the chemical constituents under investigation of both the endophytes and the host plant showed the presence of important bioactive compounds, as such, more research is needed on the isolation of these compounds and tested for various bioactivities like anticancer, antioxidant, antifungal, antibacterial and etc., this could validate the use of this plant in traditional medicine and can thus allow for the isolation of these compounds for drug development. Although Gas Chromatography is a powerful technique to identify and separate compounds, it has its shortfall as it can only detect volatile compounds, as such future work can also investigate on the bioactive non-volatile compounds from the endophytes and host plant using techniques such as liquid chromatography.

References Gautam, A.J., Kant, M. and Thakur, Y. (2013). Isolation of endophytic fungi from Cannabis sativa and study their antifungal potential. Archives of Phytopathology and Plant Protection, 46(6):627-635. Huang, Q., An, H., Song, H., Mao, H., Shen, W. and Dong, J. (2015). Diversity and biotransformative potential of endophytic fungi associated with the medicinal plant Kadsura angustifolia. Research in Microbiology, 166:45-55.

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Martinez-Klimova, E., Rodriguez-Pena, K. and Sanchez, S. (2016). Endophytes as sources of antibiotics. Biochemical Pharmacology (2016). In press, DOI: http://dx.doi.org/10.1016/j.bcp.2016.10.010. Talapatra, S.K. and Talapatra, B. (2015). Chemistry of plant natural products; Stereochemistry, conformation, synthesis, biology, and medicine. Vol 1, Springer-Verlag, Heidelberg, pp. 246 Yadav, M., Yadav, A. and Yadav, J.P. (2014). In vitro antioxidant activity and total phenolic content of endophytic fungi isolated from Eugenia jambolana Lam. Asian Pacific Journal of Tropical Medicine, 7(1):256-261.

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APPENDICES Appendix 1: Sequences of the endophytes

A1.1. Fungal endophytes sequences

>EKFA1 TAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCA TTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGAT CTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGC ATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCT TTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAA GCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCAC CGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAAC GCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAA TAAGCGGAGGGGATCATTACCGAGTGTAGGGTTCCTAGCGAGCCAACCTCCACCGTGTTTACTGTACC TTAGTTGCTTCGGCGGGCCCGCATTTAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTAC CTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCC GGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTT CAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTG CAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCT GTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGA CGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTG TAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGA

>EKFA2 CTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGC CGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAA GTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGA AGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGC ACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGC TTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCC GATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATC AATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAGCGGA GGACTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCAT GGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGT GAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGA TGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAA CGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCAC GGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCG TCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAA ATCAATCTTTTTCCAGGTTGACCTCGGATCAGA

>EKFF CAACCTCCCATCCGTGTCTATTATACCCTGTTGCTTCGGCGGGCCCGCCGCTTGTCGGCCGCCGGGGGG GCGCCTTTGCCCCCCGGGCCCGTGCCCGCCGGAGACCCCAACACGAACACTGTCTGAAAGCGTGCAGT CTGAGTTGATTGAATGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAG AACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCAC ATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGT GTGTTGGGTCGCCGTCCCCCTCTCCGGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGATC CTCGAGCGTATGGGGCTTTGTCACATGCTCTGTAGGATTGGCCGGCGCCTGCCGACGTTTTCCAACCAT TTTTTCCAGGTTGACCTCGGATCCAACCTCCCATCCGTGTCTATTATACCCTGTTGCTTCGGCGGGCCCG CCGCTTGTCGGCCGCCGGGGGGGCGCCTTTGCCCCCCGGGCCCGTGCCCGCCGGAGACCCCAACACGA

147

ACACTGTCTGAAAGCGTGCAGTCTGAGTTGATTGAATGCAATCAGTTAAAACTTTCAACAATGGATCT CTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGA ATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATT GCTGCCCTCAAGCCCGGCTTGTGTGTTGGGTCGCCGTCCCCCTCTCCGGGGGGACGGGCCCGAAAGGC AGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACATGCTCTGTAGGATTGGCCGGC GCCTGCCGACGTTTTCCAACCATTTTTTCCAGGTTGACCTCGGATCTGA

>EKBE GAGTGCGGGTCTTTGGGCCAACCTCCCATCCGTGTCTATTATACCCTGTTGCTTCGGCGGGCCCGCCGC YTGTCGGCCGCCGGGGGGGCGCCTTTGCCCCCCGGGCCCGTGCCCGCCGGAGACCCCAACACGAACAC TGTCTGAAAGCGTGCAGTCTGAGTTGATTGAATGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGG TTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCAT CGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGC CCTCAAGCCCGGCTTGTGTGTTGGGTCGCCGTCCCCCTCTCCGGGGGGACGGGCCCGAAAGGCAGCGG CGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACATGCTCTGTAGGATTGGCCGGCGCCTG CCGACGTTTTCCAACCATTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCA TATCAATAAGGTGAACCTGCGGAAGGATCATTACCGAGTGCGGGTCCTTTGGGCCCAACCTCCCATCC GTGTCTATTATACCCTGTTGCTTCGGCGGGCCCGCCGCTTGTCGGCCGCCGGGGGGGCGCCTTTGCCCC CCGGGCCCGTGCCCGCCGGAGACCCCAACACGAACACTGTCTGAAAGCGTGCAGTCTGAGTTGATTGA ATGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAAT GCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTG GTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGTCGCC GTCCCCCTCTCCGGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATG GGGCTTTGTCACATGCTCTGTAGGATTGGCCGGCGCCTGCCGACGTTTTCCAACCATTTTTTCCAGGTT GACCTCGGATCAGGTAGGGATAC

A1.2 Bacterial endophytes sequences

>F1 GAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCAC GTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCA AAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACC TTACCAAGGCTTGACATACACCAGACCGGCCCAGAGATGGGTTTTCCTCTTTGAGGTTGGTGTACAGG TGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTC GTTCTATGTTGCCAGCACGTGATGGTGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGT GGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAA TGGGTTGCGATACCGTGAGGTGGAGCTAATCCCAAAAAGCTGGTCTCAGTTCGGATCGTGGTCTGCAA CTCGACCACGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGG GCCTTGTACACACCGCCCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGGTGGCCTAA

>F2 GCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGACCATTCC ACGGTTTCCGCGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACT CAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAA CCTTACCAAGGCTTGACATGTTCCCGATCGCCGTAGAGATACGGTTTCCCCTTTGGGGCGGGTTCACAG GTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT CGTTCCATGTTGCCAGCACGTAATGGTGGGGACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAG GTGAGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTAC

148

AATGGGTTGCGATACTGTGAGGTGGAGCTAATCCCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGC AACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCC GGGCCTTGTACACACCGCCTCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGG

>F3 AGCTTGCTCCTTTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTACCTATAAGACTGGG ATAACTTCGGGAAACCGGAGCTAATACCGGATAATATTTCGAACCGCATGGTTCGATAGTGAAAGATG GCTTTGCTATCACTTATAGATGGACCTGCGCCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGC AACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGACTCCTA CGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGA TGAAGGTCTTCGGATCGTAAAACTCTGTTATTAGGGAAGAACAAACGTGTAAGTAACTGTGCACGTCT TGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCA AGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCA CGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCA TGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTA ACTGACGCTGATGTGC

>F4 AGCTTGCTCCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGA TAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGAAAGACG GTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGGGGTAATGGCTCACCAAG GCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGT GATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCGAGAGTAACTGCTCGCAC CTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGC AAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCC CCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCC ACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGT AACTGACGC

>B1 AGCTTGCTCCTTTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTACCTATAAGACTGGG ATAACTTCGGGAAACCGGAGCTAATACCGGATAACATTTGGAACCGCATGGTTCTAAAGTGAAAGATG GTTTTGCTATCACTTATAGATGGACCCGCGCCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGC AACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGACTCCTA CGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGA TGAAGGGTTTCGGCTCGTAAAACTCTGTTATTAGGGAAGAACAAAAGTGTAAGTAACTGTGCACGTCT TGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCA AGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCA CGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCA TGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTA ACTGACGCTGA

149

>B2 ACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAG TACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCTGATT AATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGCACTGGACGGCTGCAGAGATGTGGCTT TCTTTGGACTGGTGCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC CGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTAATGGTGGGGACTCATAGGAGACTGCCGG GGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCAAGCAT GCTACAATGGTCGGTACAATGGGTTGCGAAACTGTGAGGTGGAGCGAATCCCAAAAAGCCGGCCTCA GTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGC TGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTCGGTAACACCCGAA GCCAGTGGC

>B3 AGCTTGCTCTTGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTCTGGGATA AGCCTGGGAAACTGGGTCTAATACCGGATATTCACTTTTCACCGCATGGTGGGGGGTGGAAAGCTTTT GTGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGG GTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC AGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCC TTCGGGTTGTAAACCTCTTTCAGTAGGGAACAAGGCCATACGTTGTGTGGTTGAGGGTACTTGCAGAA GAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTAT TGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCCGTGAAAGTCCGGGGCTCAACTCCGGATCT GCGGTGGGTACGGGCAGACTAGAGTGCAGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGC GCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTGTAACTGACGCTGAGGAGCGA AA

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Appendix 2: 2D-GC-TOF/MS profiles of Celtis africana and the endophytes Appendix 2.1: Volatile compounds from the nine crude extracts of Celtis africana

Table A1: Volatile compounds from the leaf hexane of C. africana

Peak Compound name Area % Formula Similarity Library Height Quant S/N R.T. (s) Exact # Mass 32 2,3-Pentanedione 4.8039 C5H8O2 881 DATABASE 15644072 14777 121.48 100.0524 45 Hexane, 2-methyl- 4.0601 C7H16 832 replib 15302565 14455 131.72 100.1252 49 Heptane 4.0601 C7H16 919 DATABASE 15302565 14455 133.98 100.1252 21 Disulfide, diheptyl 3.5182 C14H30S2 970 DATABASE 13432459 12688 115.74 262.1789 50 1-Propanamine, N-ethyl-N-methyl- 2.3887 C6H15N 782 replib 14294513 13502 135.8 101.1204 53 2-Butenoic acid, 3-methyl-, 2,6,6,9- 2.3887 C25H34O5 842 DATABASE 14294513 13502 136.74 414.2406 tetramethyl-11- oxotricyclo[5.4.0.0(2,8)]undec-9-ene- 3,5-diyl ester, stereoisomer 35 2,3-Heptanedione 2.1357 C7H12O2 871 DATABASE 15367898 14516 123.66 128.0837 42 Pentane, 3,3-dimethyl- 1.818 C7H16 923 replib 10777204 10180 129.28 100.1252 24 1-Butene, 4-bromo- 1.476 C4H7Br 709 replib 13094620 12369 117.42 133.9731 54 3-Hexen-1-ol, (Z)- 1.3574 C6H12O 713 mainlib 5681771 5366.9 139.22 100.0888 5 5H-Benzocyclohepten-5-one, 0.76529 C15H22O2 870 DATABASE 6979821 6593 103.74 234.162 1,4,4a,6,7,9a-hexahydro-4-methoxy- 4a,7,7-trimethyl-, (4à,4aà,9aà)-(.+-.)- 16 Diazene, bis(1,1-dimethylethyl)- 0.58188 C8H18N2 848 mainlib 3994767 3773.4 112.1 142.147 31 2,4,6-Octanetrione 0.45915 C8H12O3 841 DATABASE 12903525 12188 120.46 156.0786 68 Cyclohexane, 1,1-dimethyl- 0.24691 C8H16 748 DATABASE 1470544 1389.1 157.3 112.1252 102 9,12,15-Octadecatrienoic acid, 0.18935 C18H30O2 893 mainlib 1072219 1012.8 1188.8 278.2246 (Z,Z,Z)- 88 Ethanone, 1-(1,2,2,3- 0.14387 C11H20O 743 mainlib 1021874 965.24 265.5 168.1514 tetramethylcyclopentyl)-, (1R-cis)- 94 Hexadecane 0.12356 C16H34 954 replib 944686 892.33 853.84 226.2661 113 á-Sitosterol 0.10953 C29H50O 892 replib 373749 353.04 1806.5 414.3862 100 Eicosane 0.10611 C20H42 921 replib 728976 688.58 1107.24 282.3287

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36 (Z,E)-4-Oxo-2,6-decadien-9-olide 0.1056 C10H12O3 757 DATABASE 2139697 2021.1 124.82 180.0786 96 n-Hexadecanoic acid 0.083579 C16H32O2 912 replib 527350 498.13 1084.78 256.2402 108 Heneicosane 0.069737 C21H44 921 replib 335230 316.65 1539.72 296.3443 109 1-Eicosanol 0.069737 C20H42O 833 DATABASE 335230 316.65 1540.58 298.3236 86 Octane, 3-methyl- 0.062244 C9H20 872 DATABASE 405146 382.69 251.56 128.1565

87 p-Xylene 0.062244 C8H10 replib 405146 382.69 252.68 106.0783 101 9,12-Octadecadienoic acid (Z,Z)- 0.057738 C18H32O2 881 replib 271397 256.36 1184.04 280.2402 38 3-(Azidomethyl)cyclohexene 0.05639 C7H11N3 742 DATABASE 980408 926.08 126.58 137.0953 93 Tetradecane 0.031951 C14H30 947 replib 246143 232.5 706.12 198.2348 66 Tetrahydrofuran, 2,2-dimethyl- 0.03142 C6H12O 786 mainlib 294991 278.64 154.94 100.0888 74 Cyclopentane, 1,2,4-trimethyl-, 0.02686 C8H16 862 DATABASE 219766 207.59 165.18 112.1252 (1à,2à,4á)- 98 1-Docosene 0.025248 C22H44 856 replib 162610 153.6 1103.86 308.3443 99 Hexadecanoic acid, ethyl ester 0.025248 C18H36O2 829 DATABASE 162610 153.6 1104.38 284.2715 111 Pentatriacontane 0.021968 C35H72 893 DATABASE 111114 104.96 1633 492.5634 91 3-Hexanol, 4,4-dimethyl- 0.021615 C8H18O 702 mainlib 128759 121.62 274.9 130.1358 82 Cyclohexane, 1,2-dimethyl-, cis- 0.016929 C8H16 885 DATABASE 117873 111.34 187.8 112.1252 112 dl-à-Tocopherol 0.006751 C29H50O2 847 mainlib 37481 35.404 1666.46 430.3811 97 Dibutyl phthalate 0.005727 C16H22O4 868 mainlib 32384 30.589 1091.36 278.1518 110 ç-Tocopherol 0.002072 C28H48O2 823 replib 15074 14.238 1621.4 416.3654

Table A2: Volatile compounds from the fruit hexane crude extract

Peak Compound name Area % Formula Similarity Library Height Quant R.T. (s) Exact # S/N Mass 47 Pentane, 2,3-dimethyl- 4,5506 C7H16 842 DATABASE 16357314 15564 134,1 100,1252 45 2H-tetrazole, 2-acetyl- 4,5177 C3H4N4O 794 DATABASE 16275492 15486 132,92 112,0385 46 2-Oxabicyclo[4.1.0]heptan-3-one, 5-methyl-, 4,5177 C7H10O2 727 DATABASE 16275492 15486 133,56 126,0681 (1à,5à,6à)-(.+-.)- 19 2-Propylhexanal 3,434 C9H18O 914 DATABASE 12500572 11895 115,94 142,1358 26 2,4,6-octanetrione 2,3701 C8H12O3 807 DATABASE 15188042 14452 120,36 156,0786

152

27 Cyclopentanol, 2-ethenyl-2-methoxy-, trans- 2,3701 C8H14O2 835 DATABASE 15188042 14452 120,68 142,0994 29 2,3-Pentanedione 1,7474 C5H8O2 837 DATABASE 14963835 14238 121,72 100,0524 22 Cyclohexane, [3-(2,2,2- 1,7394 C11H19F3O 737 DATABASE 13080053 12446 117,62 224,1388 trifluoroethoxy)propyl]- 52 butanoic ACID, 2-HEXENYL ESTER, (E)- 1,5102 C10H18O2 741 DATABASE 5493629 5227,3 139,62 170,1307 2 1,1-Cyclopentanedicarboxylic acid, 3,4- 1,2781 C13H18O4 773 DATABASE 6953218 6616,1 101,26 238,1205 bis(methylene)-, diethyl ester 28 Cyclopentane, (1,1-dimethylethyl)- 0,75252 C9H18 721 mainlib 14781016 14064 120,98 126,1409 14 Oxalic acid, diallyl ester 0,73436 C8H10O4 833 mainlib 4314809 4105,6 112,22 170,0579 15 Acetonyl decyl ether 0,73436 C13H26O2 802 DATABASE 4314809 4105,6 112,36 214,1933 16 1,1-Ethanediol, diacetate 0,73436 C6H10O4 824 DATABASE 4314809 4105,6 112,52 146,0579 32 Propanoic acid, 2,2-dimethyl-, 1- 0,67261 C12H20O5 838 DATABASE 8963502 8529 123,94 244,1311 [(acetyloxy)methyl]-4-oxobutyl ester 35 Cyclohexene, 3-(3-nitro-2-propenyl)-, (e)- 0,076611 C9H13NO2 776 DATABASE 1201063 1142,8 126,84 167,0946 96 Eicosane C20H42 replib 402421 382,91 1106,58 282,3287 0,067234 914 87 Octane, 3-methyl- 0,064679 C9H20 873 replib 393067 374,01 252,02 128,1565 88 p-Xylene C8H10 replib 393067 374,01 253,1 106,0783 0,064679 955 89 Ethanone, 1-(1,2,2,3-tetramethylcyclopentyl)-, 0,047898 C11H20O 735 mainlib 310084 295,05 265,9 168,1514 (1R-cis)- 75 Hexane, 2,3-dimethyl- 0,042165 C8H18 939 replib 305561 290,75 175,46 114,1409 98 Triacontane 0,029073 C30H62 911 DATABASE 181947 173,13 1216,52 422,4852 65 Tetrahydrofuran, 2,2-dimethyl- 0,0284 C6H12O 791 replib 256089 243,67 155,32 100,0888 97 9,12-Octadecadienoic acid (Z,Z)- 0,028337 C18H32O2 896 replib 157865 150,21 1182,22 280,2402 72 Hexane, 3,3-dimethyl- 0,024385 C8H18 870 mainlib 188638 179,49 165,26 114,1409 64 Butane, 2,2,3,3-tetramethyl- 0,024165 C8H18 924 replib 239263 227,66 154,76 114,1409 95 n-Hexadecanoic acid 0,018881 C16H32O2 899 replib 121404 115,52 1082,96 256,2402 91 Nonane 0,011579 C9H20 910 DATABASE 79780 75,913 274,74 128,1565

Table A3: Volatile compounds from the stem hexane crude extract

153

Peak # Compound name Area % Formula Similarity Library Height Quant R.T. (s) Exact S/N Mass 29 2,3-Pentanedione 4,4393 C5H8O2 760 mainlib 15918202 14738 121,68 100,0524 44 Hexane, 2-methyl- 4,0481 C7H16 840 DATABASE 16105665 14912 132,44 100,1252 20 Sulfurous acid, dibutyl ester 3,0674 C8H18O3S 959 DATABASE 12822362 11872 115,92 194,0977 51 2-Ethylthiolane, S,S-dioxide 2,3127 C6H12O2S 755 mainlib 14222635 13168 137,06 148,0558 27 2,3-Heptanedione 2,0787 C7H12O2 864 DATABASE 15736940 14570 120,32 128,0837 33 Propanoic acid, 2,2-dimethyl-, 1- 2,0521 C12H20O5 841 DATABASE 15671467 14510 123,9 244,1311 [(acetyloxy)methyl]-4-oxobutyl ester 40 Hydroperoxide, 1-methylpentyl 1,8629 C6H14O2 761 DATABASE 10550940 9768,7 129,38 118,0994 14 Diazene, bis(1,1-dimethylethyl)- 0,65396 C8H18N2 850 mainlib 4377976 4053,4 112,34 142,147 15 2,5-Dioxotetrahydro-3-furanyl acetate # 0,65396 C6H6O5 783 DATABASE 4377976 4053,4 112,52 158,0215 103 Octacosane 0,6282 C28H58 917 replib 3745869 3468,2 1540,3 394,4539 8 4,4-Dideuteriomethoxycyclohexane 0,45161 C7H12D2O 853 DATABASE 3134401 2902 108,82 116,117 11 3,4-Dimethyldihydrofuran-2,5-dione 0,45161 C6H8O3 808 mainlib 3134401 2902 109,74 128,0473 67 Pentane, 2-isocyano-2,4,4-trimethyl- 0,23553 C9H17N 739 replib 1393753 1290,4 157,68 139,1361 77 Heptane, 4-methyl- 0,19611 C8H18 894 mainlib 1142303 1057,6 179,02 114,1409 83 Octane 0,1055 C8H18 932 mainlib 840280 777,98 199,26 114,1409 104 Triacontane 0,10461 C30H62 905 DATABASE 476127 440,83 1632,5 422,4852 89 Ethanone, 1-(1,2,2,3-tetramethylcyclopentyl)-, 0,08573 C11H20O 739 mainlib 650517 602,29 265,96 168,1514 (1R-cis)- 105 á-Sitosterol 0,074704 C29H50O 886 replib 251011 232,4 1805,98 414,3862 88 p-Xylene 0,064665 C8H10 956 replib 439809 407,2 253,16 106,0783 98 9,12-Octadecadienoic acid (Z,Z)- 0,059937 C18H32O2 911 Replib 342364 316,98 1183 280,2402 99 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- 0,049034 C18H30O2 887 Replib 284954 263,83 1186,88 278,2246 97 Heneicosane 0,048801 C21H44 922 replib 360246 333,54 1106,64 296,3443 52 2-Propenoic acid, anhydride 0,040439 C6H6O3 745 mainlib 1640390 1518,8 138,1 126,0317 96 n-Hexadecanoic acid 0,037859 C16H32O2 901 replib 262263 242,82 1083,62 256,2402 65 3-Buten-2-ol, 2,3-dimethyl- 0,032777 C6H12O 793 DATABASE 304042 281,5 155,32 100,0888 93 Tetradecane 0,03275 C14H30 947 replib 272893 252,66 705,9 198,2348 64 Butane, 2,2,3,3-tetramethyl- 0,031589 C8H18 914 replib 298448 276,32 154,76 114,1409

154

73 Cyclopentane, 1,2,4-trimethyl- 0,027166 C8H16 872 mainlib 219114 202,87 165,6 112,1252 100 Pentacosane 0,025239 C25H52 837 DATABASE 170530 157,89 1216,46 352,4069 91 Nonane 0,016539 C9H20 860 DATABASE 112004 103,7 274,84 128,1565 82 Cyclopentane, 1-ethyl-2-methyl- 0,01232 C8H16 875 DATABASE 70318 65,105 196,14 112,1252 101 Heptacosane 0,011681 C27H56 916 replib 76251 70,598 1317,38 380,4382 84 Heptane, 2,5-dimethyl- 0,003874 C9H20 860 replib 38617 35,754 224,48 128,1565

Table A4: Volatile compounds from the leaf ethyl acetate crude extract

20 Butane, 1-ethoxy- 3,1338 C6H14O 830 DATABASE 8172249 7623,1 146,26 102,1045 15 Acetic acid, 1-methylethyl ester 2,0377 C5H10O2 933 DATABASE 8767594 8178,5 135 102,0681 19 Di-n-propyl ether 0,42751 C6H14O 917 mainlib 1595308 1488,1 143,34 102,1045 36 9,12,15-Octadecatrienoic acid, 0,23411 C18H30O2 896 mainlib 575300 536,65 1187,16 278,2246 (Z,Z,Z)- 39 Heptacosane 0,14725 C27H56 880 replib 241096 224,9 1538,82 380,4382

22 Butane, 1-propoxy- 0,11754 C7H16O 812 mainlib 251127 234,25 166,9 116,1201 23 3-Hexanol, 4-methyl- 0,11754 C7H16O 758 replib 251127 234,25 167,24 116,1201 21 Erythro-2-ethyl-3-ethoxybutan-1- 0,11137 C8H18O2 815 DATABASE 438659 409,19 156,84 146,1307 ol 37 1-Eicosanol 0,087282 C20H42O 901 replib 213754 199,39 1454,46 298,3236 33 n-Hexadecanoic acid 0,083802 C16H32O2 909 replib 229871 214,43 1083,3 256,2402 28 p-Xylene 0,082038 C8H10 955 replib 278424 259,72 254,1 106,0783 35 9,12-Octadecadienoic acid (Z,Z)- 0,080293 C18H32O2 898 replib 183963 171,6 1182,82 280,2402 38 Tricosyl acetate 0,051613 C25H50O2 867 mainlib 114081 106,42 1503 382,3811 34 2-Hexadecen-1-ol, 3,7,11,15- 0,04245 C20H40O 919 DATABASE 106321 99,178 1172,5 296,3079 tetramethyl-, [r-[r*,r*-(e)]]- 14 Butane, 2-ethoxy- 0,039893 C6H14O 867 mainlib 273536 255,16 132,84 102,1045 30 1,2,3-Propanetriol, diacetate 0,037298 C7H12O5 941 DATABASE 109213 101,87 576,72 176,0685 27 Ethylbenzene 0,031046 C8H10 951 replib 118044 110,11 248,02 106,0783 31 2,3-Dihydroxypropyl acetate 0,024184 C5H10O4 908 DATABASE 52456 48,932 579,82 134,0579

155

26 1,3-Dimethylcyclohexane,c&t 0,01354 C8H16 917 mainlib 42542 39,684 189,82 112,1252 32 1-Dodecanamine, n,n-dimethyl 0,006411 C14H31N 908 DATABASE 15632 14,582 784,74 213,2457 18 3-Methoxy-1-pentene 0,001477 C6H12O 739 mainlib 11616 10,835 138,86 100,0888

TABLE A5: Volatile compounds from the fruit ethyl acetate crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. (s) Exact S/N Mass 8 1-Propylethanoate 23,122 C5H10O2 707 DATABASE 19356686 18606 116,92 102,0681 21 Butane, 1-ethoxy- 2,1887 C6H14O 854 DATABASE 7964193 7655,3 146,12 102,1045 16 Acetic acid, 1-methylethyl ester 1,3763 C5H10O2 924 mainlib 8316127 7993,6 134,84 102,0681 4 Sulphuric acid dibutyl ester 0,5779 C8H18O4S 871 mainlib 1898908 1825,3 109,56 210,0926 20 Di-n-propyl ether 0,30427 C6H14O 917 mainlib 1610018 1547,6 143,2 102,1045 23 Oxalic acid, butyl propyl ester 0,079114 C9H16O4 787 mainlib 243359 233,92 166,82 188,1049 25 3-Nitropropanoic acid 0,079114 C3H5NO4 811 DATABASE 243359 233,92 167,8 119,0219 22 Erythro-2-ethyl-3-ethoxybutan-1-ol 0,078744 C8H18O2 814 DATABASE 432982 416,19 156,72 146,1307 33 9,12-Octadecadienoic acid (Z,Z)- 0,075275 C18H32O2 907 replib 275629 264,94 1182,34 280,2402 32 n-Hexadecanoic acid 0,057704 C16H32O2 910 replib 244655 235,17 1083,32 256,2402 30 p-Xylene 0,055816 C8H10 958 replib 258376 248,36 254,02 106,0783 29 Ethylbenzene 0,025063 C8H10 950 replib 111923 107,58 247,96 106,0783 34 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- 0,023735 C19H32O2 831 replib 74233 71,354 1186,22 292,2402 28 Cyclohexane, 1,3-dimethyl-, cis- 0,013481 C8H16 784 replib 51455 49,46 189,7 112,1252 35 2H-1-benzopyran-6-ol, 3,4-dihydro-2,7,8-trimethyl- 0,012007 C28H48O2 809 DATABASE 46703 44,892 1620,6 416,3654 2-(4,8,12-trimethyltridecyl)- 36 dl-à-Tocopherol 0,01123 C29H50O2 826 mainlib 34443 33,107 1666,24 430,3811

Table A6: Volatile compounds from stem ethyl acetate crude extract

Peak Name Area % Formula Similarity Library Height Quant S/N R.T. (s) Exact # Mass 6 Acetic acid, anhydride 5,42 C4H6O3 854 DATABASE 17966591 17015 114,48 102,0317 20 Butane, 1-ethoxy- 2,7646 C6H14O 895 DATABASE 7919762 7500,3 146,22 102,1045

156

15 Acetic acid, 1-methylethyl ester 1,6952 C5H10O2 944 DATABASE 8248944 7812,1 134,98 102,0681 16 n-Propyl acetate 1,6952 C5H10O2 872 replib 8248944 7812,1 135,18 102,0681 44 Triacontane 0,68724 C30H62 911 DATABASE 1803605 1708,1 1539,36 422,4852 47 Friedelone 0,57584 C30H50O 868 DATABASE 567337 537,29 2023 426,3862 18 Di-n-propyl ether 0,38483 C6H14O 914 mainlib 1608692 1523,5 143,32 102,1045 46 D:A-Friedooleanan-3-ol, (3à)- 0,18166 C30H52O 787 replib 181895 172,26 1992,48 428,4018 40 n-Tridecan-1-ol 0,14725 C13H28O 832 mainlib 241096 224,9 1540,08 200,214 40 9,12-Octadecadienoic acid (Z,Z)- 0,13355 C18H32O2 906 replib 384129 363,79 1182,82 280,2402 39 n-Hexadecanoic acid 0,11806 C16H32O2 901 replib 347210 328,82 1083,64 256,2402 23 Oxalic acid, allyl hexyl ester 0,10748 C11H18O4 802 mainlib 256890 243,28 166,88 214,1205 25 Erythro-2-ethyl-3-ethoxybutan-1-ol 0,10748 C8H18O2 811 DATABASE 256890 243,28 167,86 146,1307 22 2-Ethoxypentane 0,1062 C7H16O 811 mainlib 459824 435,47 156,82 116,1201 5 Ethanedioic acid, dibutyl ester 0,088091 C10H18O4 791 DATABASE 445880 422,27 112,82 202,1205 41 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- 0,083118 C18H30O2 894 mainlib 199594 189,02 1186,56 278,2246 31 p-Xylene 0,077434 C8H10 954 replib 283968 268,93 254,08 106,0783 33 2-Heptenal, (Z)- 0,062049 C7H12O 930 mainlib 215837 204,41 324,62 112,0888 43 1-Decanol, 2-hexyl- 0,051465 C16H34O 813 replib 138577 131,24 1454,82 242,261 29 Hexanal 0,040436 C6H12O 886 DATABASE 122251 115,78 200,78 100,0888 38 Deca-2,4-dienal 0,021502 C10H16O 808 DATABASE 63232 59,883 642,34 152,1201 35 1,2,3-Propanetriol, monoacetate 0,020929 C5H10O4 925 Mainlib 73019 69,152 445,08 134,0579 36 1,2,3-Propanetriol, diacetate 0,020669 C7H12O5 927 DATABASE 73237 69,359 576,76 176,0685 34 2,4-Heptadienal, (e,e)- 0,016718 C7H10O 904 DATABASE 56354 53,37 360,78 110,0732 28 1,3-Dimethylcyclohexane,c&t 0,009447 C8H16 924 Mainlib 35039 33,183 189,78 112,1252 42 4,8,12,16-Tetramethylheptadecan-4-olide 0,008906 C21H40O2 869 Mainlib 31218 29,565 1301,94 324,3028

Table A7: Volatile compounds from the leaf DCM: MeOH crude extract

Pea Name Area % Formula Similarit Library Height Quant S/N R.T. (s) Exact k # y Mass 6 1,1-Cyclopentanedicarboxylic acid, 3,4- 20,133 C13H18O4 774 DATABASE 11225037 10736 107,5 238,1205 bis(methylene)-, diethyl ester

157

3 Cyclopropanecarboxylic acid, 2-(1,1- 11,642 C11H20O2 658 DATABASE 10470874 10014 104,24 184,1463 dimethylethyl)-1,2-dimethyl-, methyl ester, cis- 2 1,2-bis(dicyanmethylen)-3-(2,3- 9.6825 C20H16N6 849 DATABASE 9908964 9476,9 103,88 404,1055 dimorpholinocyclopropenyliothio)cyclopropanid O2S 19 Mome inositol 0,4775 C7H14O6 826 DATABASE 122961 117,6 879,18 194,079 23 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- 0,37731 C18H30O2 898 mainlib 377414 360,96 1186,46 278,2246 22 9,12-Octadecadienoic acid (Z,Z)- 0,19853 C18H32O2 900 replib 183593 175,59 1182,36 280,2402 20 n-Hexadecanoic acid 0,15941 C16H32O2 902 replib 167158 159,87 1083,02 256,2402 24 1-Docosene 0,12555 C22H44 922 replib 122397 117,06 1454,52 308,3443 15 Butanoic acid, methyl ester 0,073237 C5H10O2 738 DATABASE 47852 45,766 156,84 102,0681 30 Squalene 0,063327 C30H50 891 replib 97555 90.643 1513.9 410.9313 21 Phytol 0,052203 C20H40O 890 replib 47476 45,406 1172,46 296,3079 25 Heptacosane 0,031825 C27H56 815 replib 74210 70,974 1538,9 380,4382 29 Benzyl á-d-glucoside 0,029258 C13H18O6 850 mainlib 43243 40.179 1245.76 270.1103

Table A8: Volatile compounds from the fruit DCM: MeOH crude extract

Peak Name Area % Formula Similar Library Height Quant R.T. (s) Exact # ity S/N Mass 8 Bicyclo[1.1.1]pentane-1,3-dithiol 3,0181 C5H8S2 844 DATABASE 6198217 5746,8 110 132,0067 30 2(3H)-Furanone, dihydro-4-hydroxy- 0,90583 C4H6O3 879 mainlib 336784 312,26 501,14 102,0317 28 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- 0,76958 C6H8O4 885 replib 351439 325,84 494,86 144,0423 40 9,12-Octadecadienoic acid (Z,Z)- 0,42285 C18H32O2 915 replib 520772 482,85 1182,7 280,2402 4 38 n-Hexadecanoic acid 0,39054 C16H32O2 897 replib 471053 436,75 1083,8 256,2402 2 32 1,2,3-Propanetriol, diacetate 0,3767 C7H12O5 821 DATABASE 381631 353,84 577,12 176,0685 35 2-Amino-9-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro- 0,3644 C10H13N5O5 761 DATABASE 116799 108,29 735,84 283,0917 furan-2-yl)-3,9-dihydro-purin-6-one 39 Ethyl (9z,12z)-9,12-octadecadienoate # 0,31127 C20H36O2 913 DATABASE 525098 472,19 1198,7 308,2715 8 36 Methyl octadeca-9,12-dienoate 0,30501 C19H34O2 911 DATABASE 498815 448,56 1162,4 294,2559 6

158

45 á-Sitosterol 0,33028 C29H50O 871 replib 210215 194,91 1805,4 414,3862 6 19 Acetic anhydride 0,25759 C4H6O3 823 mainlib 134276 124,5 181,14 102,0317 17 2-Heptenal, (Z)- 0,29631 C7H12O 924 mainlib 451482 405,99 325,04 112,0888

22 Propanoic acid, 2-oxo-, methyl ester 0,23088 C4H6O3 727 mainlib 138054 128 257,86 102,0317 41 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- 0,20749 C18H30O2 861 mainlib 177214 164,31 1186,3 278,2246 6 36 Mome inositol 0,20168 C7H14O6 702 DATABASE 67571 62,65 888,28 194,079 29 Deca-2,4-dienal 0,20383 C10H16O 929 DATABASE 238979 214,9 642,46 152,1201 39 9,12-Octadecadienoic acid, methyl ester, (e,e)- 0,17376 C19H34O2 906 DATABASE 242415 224,76 1162,4 294,2559 8 42 9,12-Octadecadienoic acid (Z,Z)-, 2-hydroxy-1- 0,097612 C21H38O4 841 mainlib 125989 116,81 1455,2 354,277 (hydroxymethyl)ethyl ester 6 33 Butanedioic acid, 2-hydroxy-2-methyl-, (S)- 0,089717 C5H8O5 756 mainlib 78639 72,912 660,24 148,0372 31 Valeric anhydride 0,084685 C10H18O3 779 replib 76931 71,328 552,42 186,1256 26 4H-pyran-4-one, 3-hydroxy-2-methyl- 0,083375 C6H6O3 70 DATABASE 94841 87,934 429,78 126,0317 16 Hydroperoxide, hexyl 0,070622 C6H14O2 728 DATABASE 63521 58,895 132,74 118,0994 43 2H-1-benzopyran-6-ol, 3,4-dihydro-2,7,8-trimethyl-2- 0,060783 C28H48O2 822 DATABASE 59223 54,91 1620,9 416,3654 (4,8,12-trimethyltridecyl)- 6 37 Hexadecanoic acid, methyl ester 0,048906 C17H34O2 901 replib 60945 56,506 1063,8 270,2559 2 44 dl-à-Tocopherol 0,048906 C29H50O2 806 Mainlib 28396 26,328 1665,9 430,3811 2 34 1,2,3-Benzenetriol 0,033536 C6H6O3 745 DATABASE 36181 33,546 690,12 126,0317

Table A9: Volatile compounds from stem DCM:MeOH crude extract

Peak Name Area % Formula Similarity Library Height Quant R.T. (s) Exact # S/N Mass 2 1,2-Bis(dicyanmethylen)-3-(2,3- 6,6681 C20H16N6O2S 869 DATABASE 9693318 8783,2 103,8 404,1055 dimorpholinocyclopropenyliothio)cyclopropanid 17 Mome inositol 0,398 C7H14O6 837 DATABASE 134150 126,91 887,7 194,079 19 9,12-Octadecadienoic acid (Z,Z)- 0,22874 C18H32O2 912 replib 176382 166,87 1182,26 280,2402 21 Heptacosane 0,20482 C27H56 913 replib 137334 129,93 1538,72 380,4382

159

20 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- 0,19441 C18H30O2 884 mainlib 121094 114,56 1186,06 278,2246 18 n-Hexadecanoic acid 0,15267 C16H32O2 896 replib 128433 121,51 1082,92 256,2402 12 Butanoic acid, methyl ester 0,11726 C5H10O2 916 DATABASE 47228 44,681 156,72 102,0681 16 1,2,3-Propanetriol, diacetate 0,06616 C7H12O5 792 DATABASE 40955 38,746 575,8 176,0685 37 Friedelan-3-one 2,7568 C30H50O 851 replib 1429204 1295 2024,14 426,3862 33 Pentatriacontane 0,95682 C35H72 907 DATABASE 1132535 1026,2 1539,02 492,5634 36 D:A-Friedooleanan-3-ol, (3à)- 0,91553 C30H52O 792 replib 492275 446,06 1993,16 428,4018 24 2-Amino-9-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro- 0,70324 C10H13N5O5 775 DATABASE 186324 168,83 740,1 283,0917 furan-2-yl)-3,9-dihydro-purin-6-one 18 2,4(1H,3H)-pyrimidinedione, 5-methyl- 0,053363 C5H6N2O2 70 DATABASE 61548 55,769 429,68 126,0429

20 2,3-Dihydro-3,5-dihydroxy-6-methyl-4h-pyran-4-one 0,11318 C6H8O4 874 DATABASE 148080 134,18 494,58 144,0423 35 2(1H)Naphthalenone, 3,5,6,7,8,8a-hexahydro-4,8a- 0,29854 C15H22O 788 mainlib 140541 127,35 1883,7 218,1671 dimethyl-6-(1-methylethenyl)- 27 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol 0,10577 C10H12O3 892 mainlib 150934 136,76 953,56 180,0786 28 Benzyl Benzoate 0,045635 C14H12O2 924 replib 70904 64,247 974,6 212,0837 25 Ethanone, 1-(4-hydroxy-3-methoxyphenyl)- 0,015236 C9H10O3 801 DATABASE 34381 31,153 851,88 166,063 23 Piperidine, 1,1'-methylenebis- 0,011762 C11H22N2 mainlib 20258 18,356 611,4 182,1783

Appendix 2.2 Volatile compounds from the fungal endophytes*

* The intracellular metabolites profiles are not included as the ones identified were already discussed in Chapter 5.

Table A10: Volatile compounds from EKFA1 broth hexane crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. Exact S/N (s) Mass 95 Octadecane 21,396 C18H38 938 DATABASE 56705272 5742,4 1004,8 254,2974 64 Hexadecane 17,352 C16H34 935 replib 54175546 5486,2 869,9 226,2661 167 Pentacosane 8,0833 C25H52 902 DATABASE 33354552 3377,7 1237 352,4069 131 Oxalic acid, allyl nonyl ester 4,709 C14H24O4 856 Mainlib 44986849 4555,7 1126,7 256,1675 63 Tetradecane, 2-methyl- 3,9618 C15H32 887 DATABASE 38390069 3887,7 868,7 212,2504 42 Tetradecane 3,9222 C14H30 941 DATABASE 22057870 2233,7 719,1 198,2348

160

212 Heptacosane 1,1147 C27H56 912 Replib 4160675 421,34 1432,1 380,4382 93 1-Nonadecene 0,59186 C19H38 941 Replib 2657886 269,16 998,5 266,2974 65 7-Hexadecene, (Z)- 0,55487 C16H32 808 Mainlib 3624674 367,06 871,6 224,2504 229 Eicosane, 2-methyl- 0,4041 C21H44 909 Mainlib 1594843 161,51 1519,3 296,3443 114 7,9-Ditert-butyl-1-oxaspiro[4.5]deca-6,9- 0,33291 C17H24O3 923 DATABASE 1414758 143,27 1076,5 276,1725 diene-2,8-dione 166 1-Docosene 0,30789 C22H44 929 Replib 1754563 177,68 1232,9 308,3443 102 Octadecane, 4-methyl- 0,15368 C19H40 864 Mainlib 693034 70,182 1038 268,313 171 Heptadecane, 4-methyl- 0,13966 C18H38 862 Replib 540290 54,714 1265,4 254,2974 137 Eicosane, 2-methyl- 0,1181 C21H44 890 mainlib 580931 58,829 1156,8 296,3443 72 Hexadecane, 4-methyl- 0,11633 C17H36 888 mainlib 560654 56,776 907,5 240,2817 117 Hexadecanoic acid, methyl ester 0,11461 C17H34O2 898 DATABASE 398607 40,366 1080,2 270,2559 14 Benzene, 1,3-dimethyl- 0,084548 C8H10 948 DATABASE 303482 30,733 260,3 106,0783 156 Sulfurous acid, hexyl pentadecyl ester 0,07956 C21H44O3S 818 mainlib 333465 33,769 1208,2 376,3011 86 Tridecane, 5-propyl- 0,077548 C16H34 863 mainlib 383870 38,874 965,7 226,2661 154 Sulfurous acid, decyl 2-ethylhexyl ester 0,073665 C18H38O3S 819 mainlib 313929 31,791 1203,7 334,2542 155 (2RS,3SR)-2-butyl-3-methylbutane-1,4-diol 0,073665 C9H20O2 765 DATABASE 313929 31,791 1203,9 160,1463 22 Benzene, 1,2,4-trimethyl- 0,072637 C9H12 932 DATABASE 293972 29,77 368,8 120,0939 124 N,N-Bis(2-hydroxyethyl)dodecanamide 0,072026 C16H33NO3 780 DATABASE 452861 45,86 1106,5 287,246 201 Octadecane, 2-methyl- 0,064547 C19H40 844 replib 299624 30,342 1401 268,313 88 Tetradecane, 4-ethyl- 0,062423 C16H34 868 mainlib 301177 30,5 970 226,2661 233 8-Heptylpentadecane 0,047163 C22H46 872 DATABASE 225107 22,796 1575,5 310,36 97 1-Decanol, 2-hexyl- 0,046617 C16H34O 869 mainlib 228239 23,113 1014,1 242,261 245 Sulfurous acid, butyl dodecyl ester 0,046537 C16H34O3S 857 mainlib 199110 20,163 1723,8 306,2229 177 Tetradecane, 1-iodo- 0,045685 C14H29I 848 mainlib 213773 21,648 1287,6 324,1314 243 1-Iodo-2-methylundecane 0,04387 C12H25I 796 mainlib 179036 18,131 1679,1 296,1001 136 Undecane, 2-methyl- 0,04351 C12H26 847 replib 177908 18,016 1146,9 170,2035 218 Tetracosane, 3-ethyl- 0,042165 C26H54 836 DATABASE 225602 22,846 1457,2 366,4226 205 1,2-Benzenedicarboxylic acid, diisooctyl 0,040609 C24H38O4 875 replib 205092 20,769 1405,5 390,277 ester 204 Sulfurous acid, decyl 2-ethylhexyl ester 0,039513 C18H38O3S 871 mainlib 222385 22,52 1404,2 334,2542

161

134 4,6-di-tert-Butyl-m-cresol 0,038227 C15H24O 710 mainlib 174427 17,664 1140,6 220,1827 122 2,6-di-butyl-2,5-cyclohexadiene-1,4-dione 0,036492 C14H20O2 710 DATABASE 170504 17,267 1100,6 220,1463 172 9,12-Octadecadienoic acid, methyl ester, 0,036269 C19H34O2 733 DATABASE 231567 23,45 1266,5 294,2559 (e,e)- 226 Sulfurous acid, 2-propyl tridecyl ester 0,034578 C16H34O3S 809 mainlib 144629 14,646 1508,2 306,2229 66 7-Hexadecene, (Z)- 0,032205 C16H32 885 mainlib 158539 16,055 879,6 224,2504 123 1,2-Benzenedicarboxylic acid, dibutyl ester 0,03187 C16H22O4 906 DATABASE 194455 19,692 1103,8 278,1518 91 Benzene, (1-ethyldecyl)- 0,026083 C18H30 759 replib 309767 31,369 984,2 246,2348 41 3-Tetradecene, (z)- 0,024936 C14H28 882 DATABASE 133426 13,512 712 196,2191 80 Benzene, (1-methyldecyl)- 0,020834 C17H28 835 DATABASE 105363 10,67 942,5 232,2191 232 Sulfurous acid, 2-propyl tetradecyl ester 0,019078 C17H36O3S 763 mainlib 108293 10,967 1561,7 320,2385 111 9-Oxabicyclo[6.1.0]non-2-ene, cis- 0,018325 C8H12O 824 DATABASE 157153 15,915 1066,7 124,0888 132 1-Dodecanol 0,01726 C12H26O 758 DATABASE 118470 11,997 1135,8 186,1984 163 Sulfurous acid, dodecyl 2-propyl ester 0,015428 C15H32O3S 750 mainlib 124601 12,618 1225,2 292,2072 81 Octadecanoic acid, methyl ester 0,015097 C19H38O2 737 DATABASE 93949 9,514 952,8 298,2872 25 Benzene, 1-ethyl-3-methyl- 0,01354 C9H12 906 DATABASE 84949 8,6025 394,6 120,0939 231 Sulfurous acid, 2-pentyl undecyl ester 0,013054 C16H34O3S 836 mainlib 108330 10,97 1543,2 306,2229 228 2-Hexyl-1-octanol 0,012813 C14H30O 833 mainlib 179426 18,17 1517,5 214,2297 236 Docosane 0,01142 C22H46 762 DATABASE 77803 7,8789 1627,8 310,36 45 Nonane, 5-propyl- 0,009864 C12H26 875 DATABASE 68625 6,9495 754,8 170,2035 127 Dodecanoic acid, 3-methylbutyl ester 0,008615 C17H34O2 768 DATABASE 96668 9,7893 1116,8 270,2559 161 9-Octadecenoic acid (z)- 0,008365 C18H34O2 735 DATABASE 110595 11,2 1223,8 282,2559 105 1,2-Benzenedicarboxylic acid, bis(2- 0,008147 C16H22O4 803 DATABASE 74022 7,496 1045,4 278,1518 methylpropyl) ester 164 Acetic acid, trifluoro-, 3,7-dimethyloctyl 0,008041 C12H21F3O2 837 mainlib 48793 4,9412 1229,6 254,1494 ester 217 Benzenepropanol, à,à-dimethyl-ç- 0,007327 C14H24OSi 884 DATABASE 66614 6,7458 1451,5 236,1596 (trimethylsilyl)- 2 Cyclopentane, 1-ethyl-2-methyl-, cis- 0,006964 C8H16 825 Replib 60813 6,1584 203,8 112,1252 73 Cyclobutanecarboxylic acid, 3-methylbut-2- 0,005804 C10H16O2 737 mainlib 67799 6,8659 910,1 168,115 enyl ester 158 Z-10-Pentadecen-1-ol 0,004778 C15H30O 727 mainlib 65377 6,6206 1218,1 226,2297

162

239 Sulfurous acid, 2-propyl undecyl ester 0,004663 C14H30O3S 795 mainlib 51939 5,2597 1639,1 278,1916 96 Benzene, (1-methylundecyl)- 0,004505 C18H30 837 mainlib 52353 5,3016 1010,6 246,2348 225 Sulfurous acid, hexyl pentadecyl ester 0,004373 C21H44O3S 826 mainlib 69389 7,0268 1504,9 376,3011 170 Decane, 2,4,6-trimethyl- 0,003637 C13H28 773 DATABASE 49435 5,0061 1258,1 184,2191 241 Methanone, (2-indanyl)(4-morpholyl)- 0,00362 C14H17NO2 755 mainlib 51501 5,2154 1676,6 231,1259 85 Benzene, (1-butyloctyl)- 0,003246 C18H30 739 DATABASE 48393 4,9006 960 246,2348 55 2-[(Benzoyloxy)methyl]-1-chloro-1- 0,003122 C13H12Cl4O 709 DATABASE 50085 5,072 829,7 323,9642 (trichlorovinyl)cyclopropane 71 Benzene, (1-propyloctyl)- 0,002808 C17H28 742 mainlib 36190 3,6648 900,5 232,2191 12 Propanoic acid, 2-hydroxy-2-methyl-, 0,00161 C5H10O3 831 DATABASE 34531 3,4968 257,8 118,063 methyl ester 152 Octane, 2,4,6-trimethyl- 0,001602 C11H24 790 DATABASE 27203 2,7548 1198,2 156,1878 175 Decane, 4-methylene- 0,001281 C11H22 762 mainlib 19054 1,9295 1274,8 154,1722 89 Cyclohexane, propyl- 0,001226 C9H18 741 DATABASE 24666 2,4979 982,1 126,1409 176 1-Octadecanol 0,001156 C18H38O 755 DATABASE 23016 2,3308 1277,7 270,2923 133 Glycylglycine 0,000934 C4H8N2O3 784 nist_msms 36969 3,7438 1139,5 132,0535 150 1,2-Butanediol, 2-[2,2-bis(1-methylethyl)- 0,000666 C15H30O4 791 DATABASE 19198 1,9441 1193,6 274,2144 1,3-dioxolan-4-yl]-3,3-dimethyl- 37 Naphthalene, 2-methyl- 0,000253 C11H10 786 DATABASE 6198,4 0,62769 659,6 142,0783 220 Methylmalonic acid 0,000178 C4H6O4 732 nist_msms 12631 1,2791 1470,4 118,0266 44 Naphthalene, 2,3-dimethyl- 0,000138 C12H12 862 DATABASE 4418,1 0,44741 746,7 156,0939

Table A11: Votalile compounds from EKFA1 ethyl acetate crude extract

Peak Name Area % Formula Similarity Library Height Quant R.T. (s) Exact Exact Mass # S/N mass 93 Pentadecane 21,79 C15H32 928 DATABASE 48908318 4962,5 868,4 212,2504 61 Tetradecane 15,38 C14H30 937 DATABASE 42485512 4310,8 718,8 198,2348 167 Octadecane 3,5567 C18H38 916 DATABASE 8909156 903,98 1123,7 254,2974 160 Pyrrolo[1,2-a]pyrazine-1,4-dione, 1,0269 C11H18N2O2 887 mainlib 1788229 181,44 1095,4 210,1368 hexahydro-3-(2-methylpropyl)- 124 1-Nonadecene 0,69458 C19H38 923 replib 1686646 171,14 997,2 266,2974

163

206 Propiconazole 0,6588 C15H17Cl2N3O2 886 Restek Pesticides 2010 1298211 131,72 1325,5 341,0698 Library 43 Dodecane 0,59462 C12H26 926 mainlib 1536519 155,9 551,2 170,2035 19 Benzene, 1,2,3-trimethyl- 0,47722 C9H12 878 DATABASE 876231 88,908 369,5 120,0939 209 1-Iodo-2-methylundecane 0,46709 C12H25I 894 mainlib 1031172 104,63 1336,4 296,1001 121 Pyrrolo[1,2-a]pyrazine-1,4-dione, 0,39169 C7H10N2O2 887 mainlib 569877 57,823 991,3 154,0742 hexahydro- 166 1-Nonadecene 0,35377 C19H38 938 replib 891527 90,46 1119,7 266,2974 210 Pyrrolo[1,2-a]pyrazine-1,4-dione, 0,32984 C14H16N2O2 841 mainlib 524646 53,234 1346 244,1212 hexahydro-3-(phenylmethyl)- 91 Acetaldehyde, (acetyloxy)-, 1-oxime, 0,32535 C4H7NO3 797 DATABASE 3216035 326,32 866,4 117,0426 (e)- 71 Oxalic acid, heptyl isohexyl ester 0,23833 C15H28O4 745 mainlib 501979 50,934 753,1 272,1988 236 1,2-Cyclohexanedicarboxylic acid, 0,20094 C24H42O4 729 mainlib 525057 53,275 1510,1 394,3083 cyclohexylmethyl nonyl ester 74 Tetradecane, 4-methyl- 0,19269 C15H32 893 mainlib 505717 51,313 762,3 212,2504 207 Hexanedioic acid, bis(2-ethylhexyl) ester 0,18983 C22H42O4 900 replib 473418 48,036 1332,4 370,3083 128 Acetonyl decyl ether 0,16618 C13H26O2 798 DATABASE 412109 41,815 1006,6 214,1933 221 Eicosane, 2-methyl- 0,1399 C21H44 900 mainlib 335354 34,027 1430,4 296,3443 107 Hexadecane 0,1289 C16H34 927 replib 386590 39,226 935,9 226,2661 191 Cyproconazole 0,10319 C15H18ClN3O 787 Korean Pesticide 265664 26,956 1262,4 291,1138 158 2,6-Dimethyloctadecane 0,099093 C20H42 873 DATABASE 229253 23,261 1089 282,3287 116 Tetradecane, 4-ethyl- 0,096535 C16H34 855 mainlib 228408 23,176 969,1 226,2661 96 3-Hexadecene, (z)- 0,093011 C16H32 851 DATABASE 229643 23,301 878,7 224,2504 21 Benzene, 1-ethyl-3-methyl- 0,087721 C9H12 918 DATABASE 186659 18,94 395,2 120,0939 41 Dehydromevalonic lactone 0,084813 C6H8O2 906 mainlib 236620 24,009 522,2 112,0524 5 Butanoic acid, 2-methyl- 0,075136 C5H10O2 895 DATABASE 175256 17,782 242 102,0681 14 3-Pentanone, 2,2,4,4-tetramethyl- 0,074155 C9H18O 903 DATABASE 181957 18,462 343,6 142,1358 122 1-Undecene 0,072494 C11H22 843 DATABASE 312394 31,697 992,7 154,1722 83 Pentadecane, 7-methyl- 0,070431 C16H34 875 mainlib 201220 20,417 827,8 226,2661 145 Benzene, butoxy- 0,070189 C10H14O 701 replib 203530 20,651 1047,6 150,1045 129 1-Octanol, 2-butyl- 0,069221 C12H26O 736 mainlib 184353 18,706 1012,8 186,1984

164

114 Heptadecane, 2,6-dimethyl- 0,068725 C19H40 898 mainlib 263585 26,745 964,8 268,313 26 4-Methyl-2-oxovaleric acid 0,060826 C6H10O3 751 mainlib 172228 17,475 422,6 130,063 208 10-Heneicosene (c,t) 0,060096 C21H42 795 mainlib 304277 30,874 1334,1 294,3287 157 Dodecane, 2-methyl- 0,059211 C13H28 851 mainlib 175724 17,83 1087,2 184,2191 68 Pentane, 2,2,3-trimethyl- 0,051978 C8H18 750 DATABASE 127317 12,918 747,8 114,1409 69 Oxirane, (1,1-dimethylbutyl)- 0,051978 C8H16O 713 mainlib 127317 12,918 747,9 128,1201 88 1-dodecene, 2-ethyl- 0,048648 C14H28 848 DATABASE 153080 15,532 856,8 196,2191 153 7,9-Ditert-butyl-1-oxaspiro[4.5]deca-6,9- 0,048347 C17H24O3 816 DATABASE 167721 17,018 1074,3 276,1725 diene-2,8-dione 16 Benzene, 1-ethyl-2-methyl- 0,046426 C9H12 917 replib 149245 15,143 355,2 120,0939 172 Phenol, 2,6-bis(1,1-dimethylethyl)-4- 0,042184 C15H24O 711 DATABASE 123999 12,582 1139,1 220,1827 methyl- 62 Cyclooctane, methyl- 0,039741 C9H18 836 mainlib 163673 16,607 721,3 126,1409 56 Tridecane, 3-methyl- 0,039401 C14H30 903 replib 115368 11,706 694,1 198,2348 201 1-Iodo-2-methylundecane 0,039214 C12H25I 763 mainlib 134660 13,663 1304,4 296,1001 15 Benzene, 1,2,3-trimethyl- 0,039062 C9H12 921 mainlib 121204 12,298 346,4 120,0939 162 Dibutyl phthalate 0,038546 C16H22O4 880 replib 196946 19,983 1102 278,1518 64 2,2-Dimethyl-3-heptanone 0,037745 C9H18O 812 mainlib 135760 13,775 732,2 142,1358 183 1-Iodo-2-methylundecane 0,035975 C12H25I 835 mainlib 105445 10,699 1219,2 296,1001 123 trans-7-pentadecene 0,035527 C15H30 864 DATABASE 155583 15,786 994,2 210,2348 228 2H-Pyran-2-carboxylic acid, 6-butoxy- 0,034927 C12H20O4 707 mainlib 97356 9,8783 1470 228,1362 3,6-dihydro-, ethyl ester 220 Cyclotetradecane 0,031861 C14H28 861 replib 96365 9,7778 1428,8 196,2191 46 Dodecane, 4-methyl- 0,026528 C13H28 823 replib 86790 8,8062 601,4 184,2191 182 Sulfurous acid, hexyl pentadecyl ester 0,02438 C21H44O3S 832 mainlib 108091 10,968 1207 376,3011 219 1,2-Benzenedicarboxylic acid, diisooctyl 0,021863 C24H38O4 895 replib 97389 9,8817 1403,9 390,277 ester 203 Octane, 3,4,5,6-tetramethyl- 0,019123 C12H26 806 mainlib 65495 6,6455 1311,1 170,2035 13 Benzaldehyde 0,018587 C7H6O 790 DATABASE 120583 12,235 342,5 106,0419 173 1-(2-Hydroxyethoxy)tridecane 0,017798 C15H32O2 780 DATABASE 67998 6,8994 1145,5 244,2402 42 Azulene 0,017175 C10H8 708 DATABASE 83974 8,5205 549,5 128,0626 195 Thiocyanic acid, phenylmethyl ester 0,016999 C8H7NS 752 mainlib 82627 8,3838 1281,5 149,0299

165

247 2,5-Piperazinedione, 3,6- 0,016934 C18H18N2O2 776 mainlib 68524 6,9529 1543,4 294,1368 bis(phenylmethyl)- 141 1-(2-Thienyl)-1-propanone 0,016247 C7H8OS 729 mainlib 73496 7,4573 1040,2 140,0296 12 Benzene, 1-ethyl-2-methyl- 0,01574 C9H12 838 DATABASE 120583 12,235 342,2 120,0939 66 Methyl 2-oxohexanoate 0,014482 C7H12O3 845 DATABASE 63150 6,4076 736,1 144,0786 73 Sulfurous acid, hexyl heptyl ester 0,013831 C13H28O3S 780 mainlib 76060 7,7175 757,8 264,1759 59 7-Tetradecene, (Z)- 0,013595 C14H28 884 mainlib 65272 6,6229 707,6 196,2191 118 3-Isopropyl-6-methyl-piperazine-2,5- 0,013524 C8H14N2O2 701 DATABASE 64762 6,5712 973,9 170,1055 dione 45 Simetryn 0,013135 C8H15N5S 708 KeTa_PestHe V_1_00 81787 8,2986 565,5 213.10 213,1048 217 Sulfurous acid, dodecyl 2-propyl ester 0,012187 C15H32O3S 825 mainlib 60986 6,188 1399,8 292,2072 102 Decane, 2,5,9-trimethyl- 0,011756 C13H28 770 mainlib 66472 6,7446 916,2 184,2191 100 1-Octadecanol 0,011461 C18H38O 814 DATABASE 90991 9,2325 909,2 270,2923 108 Sulfurous acid, hexyl pentadecyl ester 0,010468 C21H44O3S 807 mainlib 44043 4,4688 949,9 376,3011 20 Benzene, 1-methyl-2-(1-methylethyl)- 0,010231 C10H14 765 mainlib 52976 5,3752 392 134,1096 185 Cyclopentane, 1,2,4-trimethyl-, 0,008763 C8H16 720 DATABASE 47498 4,8194 1228,2 112,1252 (1à,2à,4á)- 155 Decane, 2-methyl- 0,008758 C11H24 782 mainlib 44325 4,4975 1078,6 156,1878 202 Sulfurous acid, decyl 2-ethylhexyl ester 0,007669 C18H38O3S 858 mainlib 54926 5,5731 1306,9 334,2542 174 Sulfurous acid, hexyl pentadecyl ester 0,007626 C21H44O3S 743 mainlib 45776 4,6447 1147,9 376,3011 249 1-Iodoundecane 0,007534 C11H23I 828 mainlib 67725 6,8717 1608 282,0844 196 Dodecyl acrylate 0,003135 C15H28O2 841 replib 27771 2,8178 1285 240,2089 198 Oxalic acid, allyl octyl ester 0,002517 C13H22O4 784 mainlib 40625 4,122 1286,7 242,1518 197 Cyclohexanone, 2-acetyl-2-(3-ethoxy-2- 0,002104 C13H20O3 952 DATABASE 37202 3,7747 1286,3 224,1412 propenyl)-, (e)- 81 Decane, 2,5,9-trimethyl- 0,001165 C13H28 799 mainlib 14643 1,4858 819,7 184,2191 178 Cyclopentane, nitro- 0,000213 C5H9NO2 705 DATABASE 8129,5 0,82487 1165,5 115,0633 6 Benzene, ethyl- 0,000205 C8H10 841 DATABASE 5024,9 0,50986 255,4 106,0783

Table A12: Volatile compounds from EKFA1 DCM crude extract

166

Peak # Name Area % Formula Similarity Library Height Quant R.T. Exact Exact Mass S/N (s) mass 155 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 14,753 C7H10N2O2 901 mainlib 2000478 208,35 994,6 154,0742 190 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 8,083 C11H18N2O2 887 mainlib 1630186 169,79 1095,9 210,1368 3-(2-methylpropyl)- 194 5H,10H-dipyrrolo[1,2-a:1',2'-d]pyrazine-5,10- 4,1865 C10H14N2O2 857 DATABASE 813154 84,692 1103,8 194,1055 dione, octahydro-, (5as-cis)- 226 Hexanedioic acid, bis(2-ethylhexyl) ester 1,0177 C22H42O4 878 Replib 324739 33,822 1332,2 370,3083 157 Octadecane 0,94119 C18H38 896 DATABASE 237384 24,724 1001,3 254,2974 223 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 0,80084 C14H16N2O2 769 Mainlib 183303 19,091 1323,8 244,1212 3-(phenylmethyl)- 198 Heptadecane, 2-methyl- 0,52037 C18H38 769 Replib 158525 16,511 1123 254,2974 213 Docosane 0,24605 C22H46 738 DATABASE 121478 12,652 1234 310,36 227 1-Iodo-2-methylundecane 0,13942 C12H25I 859 Mainlib 77312 8,0522 1336,1 296,1001 128 Decane, 6-ethyl-2-methyl- 0,13667 C13H28 862 Mainlib 60171 6,2669 785,9 184,2191 145 Pentadecane 0,10155 C15H32 857 DATABASE 74073 7,7148 964,8 212,2504 91 Pentanoic acid, 4-methyl-2-oxo-, methyl ester 0,098182 C7H12O3 728 Mainlib 56001 5,8326 426,7 144,0786 174 Benzene, butoxy- 0,095813 C10H14O 725 Replib 46954 4,8903 1048,2 150,1045 241 Dodecane, 2-methyl- 0,075954 C13H28 802 Mainlib 53640 5,5867 1475,2 184,2191 232 Heptadecane, 2,6,10,14-tetramethyl- 0,068641 C21H44 813 Mainlib 69725 7,262 1384,3 296,3443 220 2-Pentanol, 3-methylene- 0,06752 C6H12O 715 DATABASE 39363 4,0998 1286 100,0888 244 Nonadecane 0,065686 C19H40 757 DATABASE 52053 5,4214 1518 268,313 137 1-Heptadecanol 0,060943 C17H36O 778 DATABASE 36879 3,841 861,7 256,2766 108 Dehydromevalonic lactone 0,058779 C6H8O2 710 Mainlib 68893 7,1753 528,4 112,0524 209 Undecane, 3,8-dimethyl- 0,052162 C13H28 837 DATABASE 38506 4,0105 1191,5 184,2191 29 Benzene, 1,2-dimethyl- 0,048888 C8H10 809 DATABASE 39225 4,0854 261,1 106,0783 181 2-Bromo dodecane 0,039512 C12H25Br 845 Mainlib 40589 4,2275 1070,4 248,114 197 Oxalic acid, allyl octadecyl ester 0,036445 C23H42O4 719 Mainlib 23497 2,4472 1119,3 382,3083 53 Bicyclo[1.1.1]pentane-1,3-dithiol 0,03497 C5H8S2 779 DATABASE 27547 2,8691 321 132,0067 126 Pentane, 2,2-dimethyl- 0,024708 C7H16 721 DATABASE 22700 2,3642 753,6 100,1252 130 Hexadecane 0,024668 C16H34 861 Replib 35082 3,6539 819,5 226,2661

167

215 CIS-5,5-Dimethyl-6-heptyl-2- 0,024268 C14H23Cl3O3 773 DATABASE 29506 3,0731 1271,1 344,0713 trichloromethyl-1,3-dioxan-4-one 236 1,2-Benzenedicarboxylic acid, bis(1- 0,023675 C24H38O4 716 DATABASE 22428 2,3359 1403,8 390,277 methylheptyl) ester 208 Dodecane, 1,1-difluoro- 0,019048 C12H24F2 731 DATABASE 34500 3,5933 1179,8 206,1846 132 1,2-Butanediol, 2-[2,2-bis(1-methylethyl)- 0,011305 C15H30O4 760 DATABASE 22074 2,2991 834,1 274,2144 1,3-dioxolan-4-yl]-3,3-dimethyl- 86 Benzene, 1-propynyl- 0,008736 C9H8 710 Replib 15097 1,5723 417,4 116,0626 28 1,2,5-Thiadiazolidine-2,5-dicarboxylic acid, 0,008206 C16H20N2O4S2 713 DATABASE 15324 1,5961 259,9 368,0864 3-ethenyl-4-[[(phenylmethyl)thio]methyl]-, dimethyl ester, cis-(.+-.)- 245 Sulfurous acid, 2-propyl undecyl ester 0,004069 C14H30O3S 807 Mainlib 8443,5 0,8794 1560,8 278,1916 221 Sulfurous acid, 2-ethylhexyl isohexyl ester 0,002841 C14H30O3S 820 Mainlib 6262,7 0,65227 1301,9 278,1916 217 Benzene, [[1-(1- 3,7E-05 C13H18O2 790 DATABASE 163,19 0,016996 1281,6 206,1307 propenyloxy)propoxy]methyl]-, (e)-

Table A13: Volatile compounds from EKFA2 broth hexane crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. Exact Exact S/N (s) mass Mass 67 2(1H)-pyrazinone, 1-hydroxy-6-(1- 25,456 C12H20N2O2 746 DATABASE 3303388 350,74 922,6 224,1525 methylpropyl)-3-(2-methylpropyl)-, (+)- 75 Pyrrolo[1,2-a]pyrazine-1,4-dione, 10,385 C7H10N2O2 900 mainlib 1359751 144,37 998 154,0742 hexahydro- 4 2H-Tetrazole, 2-acetyl- 1,5542 C3H4N4O 878 DATABASE 475093 50,444 205,6 112,0385 88 7,9-Ditert-butyl-1-oxaspiro[4.5]deca- 0,42252 C17H24O3 789 DATABASE 171585 18,218 1074,5 276,1725 6,9-diene-2,8-dione 91 Pyrrolo[1,2-a]pyrazine-1,4-dione, 0,37655 C11H18N2O2 793 mainlib 107534 11,418 1086,3 210,1368 hexahydro-3-(2-methylpropyl)- 52 Phenol, 5-methyl-2-(1-methylethyl)- 0,16683 C10H14O 815 DATABASE 104293 11,073 625,6 150,1045 95 5H,10H-dipyrrolo[1,2-a:1',2'- 0,073666 C10H14N2O2 744 DATABASE 48084 5,1054 1105,7 194,1055 d]pyrazine-5,10-dione, octahydro-, (5as- cis)- 89 Cyclohexan-2,2,3,3,4-d5-ol, 4-methyl-, 0,039146 C7H9D5O 742 DATABASE 33670 3,5749 1079 119,1358 (1s-trans)- 44 Cyclobutane, 1,3-difluoro-1,3-dimethyl- 0,035269 C6H10F2 745 DATABASE 24082 2,5569 291,3 120,0751 , trans-

168

87 Disulfide, ethyl 1-methylpropyl 6,37E-05 C6H14S2 841 DATABASE 223,56 0,023737 1050,6 150,0537

Table A14: Volatile compounds from EKFA2 broth ethyl acetate crude extract.

Peak Name Area % Formula Similarity Library Height Quant R.T. Exact # S/N (s) Mass 83 Hexadecane 28,19 C16H34 930 replib 8422460 909,6 867,3 226,2661 55 Tetradecane 20,54 C14H30 944 replib 6168440 666,17 717,8 198,2348 109 Octadecane 15,457 C18H38 928 replib 4198817 453,46 1001,4 254,2974 139 Eicosane 5,8461 C20H42 916 replib 1548847 167,27 1122,8 282.3287 282,3287 152 Heptacosane 2,0971 C27H56 902 replib 565602 61,083 1233,7 380,4382 22 Benzene, 1,2,3-trimethyl- 1,6884 C9H12 932 replib 342894 37,031 370,1 120,0939 108 1-Nonadecene 1,4062 C19H38 927 replib 397076 42,883 996,9 266,2974 43 Tridecane 0,991 C13H28 892 DATABASE 323753 34,964 551,3 184,2191 41 Acetic acid, 2-ethylhexyl ester 0,78642 C10H20O2 917 replib 230509 24,894 503,1 172,1463 165 Propiconazole 0,77518 C15H17Cl2N3O2 851 Restek Pesticides 246578 26,63 1324,8 341,0698 2010 Library 138 1-Docosene 0,67966 C22H44 821 replib 225252 24,326 1119,1 308,3443 84 Cyclooctane, methyl- 0,60958 C9H18 817 mainlib 243564 26,304 869,8 126,1409 66 Tridecane, 4-methyl- 0,5937 C14H30 844 replib 161559 17,448 762 198,2348 164 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)- 0,51559 C15H17Cl2N3O2 838 mainlib 163461 17,653 1317,9 341,0698 4-propyl-1,3-dioxolan-2-yl]methyl]- 54 Cyclopropane, 1-ethyl-2-heptyl- 0,37002 C12H24 911 mainlib 126192 13,628 711,2 168,1878 26 Benzene, 1-ethyl-3-methyl- 0,36369 C9H12 902 DATABASE 103593 11,188 395,6 120,0939 96 Pentadecane 0,36018 C15H32 784 DATABASE 134594 14,536 935,5 212,2504 189 1,2-Cyclohexanedicarboxylic acid, 0,31249 C24H42O4 707 mainlib 132884 14,351 1509,1 394,3083 cyclohexylmethyl nonyl ester 166 Hexanedioic acid, bis(2-ethylhexyl) ester 0,2921 C22H42O4 889 DATABASE 101602 10,973 1332 370,3083 90 2,3,5,8-Tetramethyldecane 0,29077 C14H30 836 DATABASE 101800 10,994 906,4 198,2348 103 Hexane, 2,2,3,3-tetramethyl- 0,26136 C10H22 837 mainlib 96397 10,411 968,8 142,1722 154 Cyproconazol, R*,S* / R*,S* same as 0,23895 C15H18ClN3O 766 KeTa_PestHe 96075 10,376 1261,5 291,1138 cyproconazole V_1_00

169

106 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 0,21753 C7H10N2O2 819 mainlib 100080 10,808 990,5 154,0742 2 Cyclopentane, 1-ethyl-3-methyl-, cis- 0,20399 C8H16 879 mainlib 99472 10,743 203,9 112,1252 56 Cyclooctane, methyl- 0,17935 C9H18 810 mainlib 127536 13,773 720,9 126,1409 102 Heptadecane, 2,6-dimethyl- 0,17614 C19H40 885 mainlib 76392 8,25 964,4 268,313 77 Tridecane, 3-methyl- 0,17574 C14H30 853 replib 96682 10,441 845,7 198,2348 117 Pentane, 2,3,3-trimethyl- 0,17179 C8H18 854 DATABASE 86171 9,3061 1036,8 114,1409 17 Propanethioic acid, 2,2-dimethyl-, 0,14498 C10H18O2S 858 DATABASE 61452 6,6366 344,8 202,1028 anhydrosulfide 20 Benzene, 1-ethyl-2-methyl- 0,13797 C9H12 882 replib 78071 8,4314 355,5 120,0939 105 Undecane, 3,9-dimethyl- 0,13464 C13H28 814 mainlib 69259 7,4797 982,3 184,2191 23 Nonane 0,12332 C9H20 795 DATABASE 107753 11,637 371,9 128,1565 19 Benzene, 1,3,5-trimethyl- 0,12308 C9H12 894 DATABASE 56102 6,0588 346,7 120,0939 129 Heptane, 2,3-dimethyl- 0,11987 C9H20 836 DATABASE 69904 7,5493 1093 128,1565 196 1,2-Cyclohexanedicarboxylic acid, 0,10279 C24H42O4 710 mainlib 63339 6,8404 1532,6 394,3083 cyclohexylmethyl nonyl ester 182 Undecane, 2-methyl- 0,10159 C12H26 704 DATABASE 46034 4,9716 1477,4 170,2035 125 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 0,087954 C11H18N2O2 742 DATABASE 59503 6,4261 1082,3 210,1368 (2-methylpropyl)-, (3s-trans)- 127 Sulfurous acid, decyl 2-ethylhexyl ester 0,081291 C18H38O3S 808 mainlib 57041 6,1602 1088,6 334,2542 79 2-Undecene, 3-methyl-, (z)- 0,080686 C12H24 769 DATABASE 47723 5,1539 856,4 168,1878 74 7-Methylpentadecane 0,074954 C16H34 771 DATABASE 59721 6,4496 827,6 226,2661 11 Benzene, 1,2-dimethyl- 0,067887 C8H10 891 DATABASE 50540 5,4582 262,5 106,0783 85 7-Tetradecene, (Z)- 0,060196 C14H28 811 mainlib 39937 4,3131 878,4 196,2191 27 1H-Indene, 2,3-dihydro- 0,057554 C9H10 861 DATABASE 34684 3,7457 409,5 118,0783 146 Tetradecane, 1-iodo- 0,052581 C14H29I 802 mainlib 44251 4,779 1199,8 324,1314 147 Sulfurous acid, 2-ethylhexyl nonyl ester 0,045547 C17H36O3S 754 mainlib 34111 3,6838 1202,1 320,2385 148 Hexane, 2,2,3,3-tetramethyl- 0,03977 C10H22 769 mainlib 27024 2,9184 1206,4 142,1722 110 Oxalic acid, allyl dodecyl ester 0,039184 C17H30O4 831 mainlib 35286 3,8108 1012,6 298,2144 33 3-[2',4'-Dimethylphenyl]-2,2-dimethylpropanal 0,02948 C13H18O 713 DATABASE 33927 3,664 426,6 190,1358 39 Benzene, 1-methyl-2-(1-methylethyl)- 0,02421 C10H14 865 DATABASE 35281 3,8103 482,1 134,1096 149 2-Propyldecan-1-ol 0,021245 C13H28O 804 DATABASE 20515 2,2156 1218,5 200,214

170

51 Tridecane, 3-methyl- 0,020408 C14H30 860 replib 21761 2,3501 693,9 198,2348 113 Syn-dimethyl [(e)-1-ethyl-2,6,6-trimethyl-3- 0,017204 C17H28O5 731 DATABASE 25630 2,768 1025,4 312,1937 oxo-4-heptenyl]malonate 163 Pentane, 3-ethyl-2,4-dimethyl- 0,015063 C9H20 828 DATABASE 14374 1,5523 1310,5 128,1565 34 2-Phenylpropanal 0,009779 C9H10O 805 DATABASE 14370 1,5519 435,3 134,0732 59 Propanethioic acid, 2,2-dimethyl-, 0,008841 C10H18O2S 817 DATABASE 18139 1,959 736,8 202,1028 anhydrosulfide 64 Vinyl 2,2-dimethylpropanoate 0,007596 C8H14O2 721 DATABASE 17413 1,8806 753,4 142,0994 31 Benzene, 1-methyl-4-propyl- 0,00357 C10H14 874 DATABASE 11193 1,2088 421,4 134,1096 65 Disulfide, dipentyl 0,002432 C10H22S2 844 DATABASE 8324,2 0,89898 753,9 206,1163 12 Methyllaurate 0,001256 C8H10 810 DATABASE 5408,4 0,58409 281,3 106,0783 131 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 0,001109 C11H18N2O2 794 DATABASE 3560,9 0,38456 1098,9 210,1368 (2-methylpropyl)-, (3s-trans)- 38 Dodecane, 1,1-difluoro- 0,000339 C12H24F2 864 DATABASE 1459,9 0,15766 462,4 206,1846 136 1-Octene, 3-ethyl- 0,000138 C10H20 802 DATABASE 594,71 0,064226 1115,4 140,1565

Table A15: Volatile compounds from EKFA2 broth DCM crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. (s) Exact S/N Mass 164 Pyrrolo[1,2-a]pyrazine-1,4-dione, 16.797 C11H18N2O2 883 mainlib 3405617 369.23 1096.9 210.1368 hexahydro-3-(2-methylpropyl)- 189 Pyrrolo[1,2-a]pyrazine-1,4-dione, 7.4436 C14H16N2O2 849 mainlib 894431 96.971 1345.6 244.1212 hexahydro-3-(phenylmethyl)- 133 Pyrrolo[1,2-a]pyrazine-1,4-dione, 4.5574 C7H10N2O2 908 mainlib 912617 98.943 993.3 154.0742 hexahydro- 167 5H,10H-dipyrrolo[1,2-a:1',2'- 2.0052 C10H14N2O2 835 DATABASE 731526 79.31 1103.6 194.1055 d]pyrazine-5,10-dione, octahydro-, (5as- cis)- 186 Hexanedioic acid, bis(2-ethylhexyl) 0.86758 C22H42O4 865 replib 189742 20.571 1331.5 370.3083 ester 120 Pentadecane 0.33565 C15H32 835 DATABASE 122647 13.297 935.5 212.2504 145 6-Amino-2-(methylamino)-4(3h)- 0.29775 C5H8N4O 730 DATABASE 152204 16.502 1027.3 140.0698 pyrimidinone 30 Benzene, 1,2-dimethyl- 0.23541 C8H10 785 DATABASE 75400 8.1747 260.8 106.0783

171

124 Hexadecane 0.17149 C16H34 781 DATABASE 59451 6.4455 964.5 226.2661 181 Benzene, [(1-propenyloxy)methyl]- 0.13948 C10H12O 731 DATABASE 62655 6.7928 1280.4 148.0888 198 Eicosane 0.11342 C20H42 835 DATABASE 70401 7.6327 1430 282.3287 193 Decane, 1-iodo- 0.062005 C10H21I 745 mainlib 24750 2.6834 1383.5 268.0688 103 Propanethioic acid, 2,2-dimethyl-, 0.049587 C10H18O2S 906 DATABASE 41506 4.5 736.9 202.1028 anhydrosulfide 108 2,3,5,8-Tetramethyldecane 0.048429 C14H30 743 DATABASE 39041 4.2327 785.5 198.2348 183 1-Iodoundecane 0.029173 C11H23I 845 mainlib 22598 2.45 1301.5 282.0844 14 Cyclopropanecarboxylic acid, 2-(1,1- 0.027338 C11H20O2 886 DATABASE 20923 2.2684 227.9 184.1463 dimethylethyl)-1,2-dimethyl-, methyl ester, cis- 2 Phosphonic acid, (2-oxo-1,3- 0.025367 C15H20O 809 DATABASE 23738 2.5736 202.7 216.1514 propanediyl)bis-, tetramethyl ester 200 1-Iodo-2-methylnonane 0.023068 C10H21I 800 mainlib 22385 2.4269 1474.4 268.0688 111 2-Propyldecan-1-ol 0.022436 C13H28O 814 DATABASE 14620 1.585 819.1 200.214 141 Thiophene, 2-methyl-5-propyl- 0.017635 C8H12S 701 DATABASE 18159 1.9688 1017 140.066 3 1,2,5-Thiadiazolidine-2,5-dicarboxylic 0.005771 C16H20N2O4S2 771 DATABASE 9612.7 1.0422 209.2 368.0864 acid, 3-ethenyl-4- [[(phenylmethyl)thio]methyl]-, dimethyl ester, trans-(.+-.)- 195 1,2-Benzenedicarboxylic acid, 0.005528 C24H38O4 721 DATABASE 11727 1.2714 1402.8 390.277 diisooctyl ester 37 o-Xylene 0.003732 C8H10 815 replib 5727.8 0.62099 279.8 76 Phosphonic acid, (2-oxo-1,3- 0.001168 C15H20O 721 DATABASE 3320.2 0.35996 462.5 216.1514 propanediyl)bis-, tetramethyl ester 44 1,1-Cyclopentanedicarboxylic acid, 3,4- 0.000904 C13H18O4 727 DATABASE 2571.8 0.27883 307.2 238.1205 bis(methylene)-, diethyl ester 208 bis-(octylphenyl)-amine 0.000323 C28H43N 731 DATABASE 919 0.099635 1597.9 393.3396 202 Dodecanamide 0.000315 C12H25NO 722 DATABASE 895.44 0.097081 1515 199.1936

Table A16: Volatile compounds from EKFF broth hexane crude extract

Peak # Name Area % Formula Similarity Library Height Quant S/N R.T. (s) Exact Mass 110 Octadecane 27.727 C18H38 924 DATABASE 50008804 5143.1 1003.3 254.2974

172

73 Pentadecane 24.638 C15H32 932 DATABASE 49431712 5083.7 868.8 212.2504 180 Pentacosane 6.3156 C25H52 910 DATABASE 15276212 1571.1 1235.2 352.4069 45 Tetradecane 4.8531 C14H30 943 replib 12747863 1311 718.4 198.2348 203 Heptacosane 2.3865 C27H56 914 replib 4872364 501.09 1337 380.4382 223 Nonadecane, 2-methyl- 1.0568 C20H42 912 mainlib 2070909 212.98 1431.2 282.3287 109 1-Nonadecene 0.81806 C19H38 940 replib 1710613 175.93 997.6 266.2974 179 1-Docosene 0.37188 C22H44 947 replib 1005154 103.37 1231.8 308.3443 91 Heptadecane 0.25175 C17H36 924 DATABASE 630228 64.815 936.1 240.2817 117 Octadecane, 4-methyl- 0.17053 C19H40 874 mainlib 367447 37.79 1037.3 268.313 24 Benzene, 1,2-dimethyl- 0.1678 C8H10 943 DATABASE 295738 30.415 260.5 106.0783 141 1,2-Benzenedicarboxylic acid, dibutyl 0.16709 C16H22O4 905 DATABASE 296051 30.447 1102.3 278.1518 ester 106 Tridecane, 3-methyl- 0.13924 C14H30 862 replib 266828 27.442 982.8 198.2348 156 Eicosane, 2-methyl- 0.12959 C21H44 873 mainlib 328542 33.789 1155.8 296.3443 132 7,9-Ditert-butyl-1-oxaspiro[4.5]deca-6,9- 0.11299 C17H24O3 771 DATABASE 268933 27.658 1074.5 276.1725 diene-2,8-dione 208 Sulfurous acid, 2-pentyl undecyl ester 0.096576 C16H34O3S 842 mainlib 196137 20.171 1364 306.2229 102 Tetradecane, 4-ethyl- 0.090003 C16H34 857 mainlib 230184 23.673 969.2 226.2661 169 Eicosane 0.086499 C20H42 840 DATABASE 204980 21.081 1200.5 282.3287 170 Undecane, 2,3-dimethyl- 0.086499 C13H28 811 mainlib 204980 21.081 1200.7 184.2191 46 Cyclooctane, 1,4-dimethyl-, cis- 0.085423 C10H20 837 mainlib 203700 20.949 721.3 140.1565 107 Benzene, (1-ethyldecyl)- 0.084887 C18H30 705 DATABASE 266828 27.442 983.3 246.2348 198 Octadecane, 2-methyl- 0.083983 C19H40 857 replib 176950 18.198 1322.9 268.313 137 Octadecane, 2,6-dimethyl- 0.079495 C20H42 857 mainlib 194436 19.996 1089.2 282.3287 171 Undecane, 6-ethyl- 0.068658 C13H28 836 replib 148013 15.222 1202.7 184.2191 138 1-Iodo-2-methylundecane 0.067365 C12H25I 854 mainlib 170689 17.554 1093.7 296.1001 61 Pentadecane, 7-methyl- 0.059124 C16H34 854 mainlib 140873 14.488 828.1 226.2661 75 Simetryn 0.058741 C8H15N5S 714 KeTa_PestHe 172769 17.768 878.9 213.1048 V_1_00 216 1,2-Benzenedicarboxylic acid, mono(2- 0.053604 C16H22O4 755 mainlib 167483 17.225 1404 278.1518 ethylhexyl) ester 21 Ethylbenzene 0.049979 C8H10 908 mainlib 135413 13.926 253.5 106.0783

173

140 2,6-di-butyl-2,5-cyclohexadiene-1,4-dione 0.048954 C14H20O2 721 DATABASE 192699 19.818 1098.8 220.1463 25 Benzene, 1,3-dimethyl- 0.04813 C8H10 934 DATABASE 119205 12.259 279.5 106.0783 112 2-Ethyl-1-dodecanol 0.044765 C14H30O 889 mainlib 135016 13.886 1013.1 214.2297 172 Sulfurous acid, hexyl pentadecyl ester 0.040231 C21H44O3S 787 mainlib 135453 13.931 1203 376.3011 226 Tetracosane, 3-ethyl- 0.039334 C26H54 853 DATABASE 135299 13.915 1456.5 366.4226 218 Nonadecane, 2-methyl- 0.038705 C20H42 832 mainlib 124271 12.781 1418.5 282.3287 195 Sulfurous acid, decyl 2-ethylhexyl ester 0.035572 C18H38O3S 871 mainlib 116047 11.935 1307 334.2542 240 2-Bromo dodecane 0.035557 C12H25Br 853 mainlib 120225 12.364 1574.7 248.114 211 1-Iodo-2-methylundecane 0.032218 C12H25I 847 mainlib 89065 9.1598 1384.6 296.1001 23 Octane, 3-methyl- 0.023265 C9H20 876 DATABASE 84159 8.6552 258.6 128.1565 69 Dimethomorph (E) 0.023178 C21H22ClNO4 701 KeTa_PestHe 80040 8.2316 853 387.1237 V_1_00 94 Oxalic acid, allyl nonyl ester 0.02264 C14H24O4 809 mainlib 75765 7.7919 950.2 256.1675 217 Sulfurous acid, hexyl tetradecyl ester 0.019942 C20H42O3S 869 mainlib 86958 8.9431 1407.1 362.2855 205 Nonane, 5-(2-methylpropyl)- 0.019698 C13H28 823 mainlib 102229 10.514 1355 184.2191 199 Tetronic acid 0.016763 C4H4O3 715 nist_msms 64274 6.6101 1329.2 100.016 111 Benzene, (1-methylundecyl)- 0.015249 C18H30 815 mainlib 53847 5.5378 1009.7 246.2348 93 Benzene, (1-methyldecyl)- 0.013216 C17H28 766 DATABASE 56902 5.852 941.7 232.2191 44 4-Dodecene, (e)- 0.013171 C12H24 890 DATABASE 50694 5.2135 713.7 168.1878 230 Pentadecane, 7-methyl- 0.012845 C16H34 853 mainlib 64108 6.5931 1492.9 226.2661 167 Heptane, 3-ethyl-5-methyl- 0.012707 C10H22 816 mainlib 68073 7.0009 1192.7 142.1722 122 5-Methyloctene-1 0.012528 C9H18 744 DATABASE 95786 9.851 1047.2 126.1409 108 2-Undecene, 9-methyl-, (E)- 0.011879 C12H24 833 mainlib 50116 5.1541 992.9 168.1878 105 Cyclohexane, octyl- 0.011369 C14H28 747 DATABASE 65038 6.6887 981.3 196.2191 38 1-Methylbutyl nitrite 0.011004 C5H11NO2 841 DATABASE 67968 6.99 676.9 117.079 42 Decane, 2,5,9-trimethyl- 0.010498 C13H28 746 DATABASE 54214 5.5756 698 184.2191 158 Acetyl valeryl 0.010466 C7H12O2 786 replib 59571 6.1265 1165.1 128.0837 181 Tridecane, 6-methyl- 0.010323 C14H30 720 DATABASE 58436 6.0098 1243.2 198.2348 72 1,2-Benzenedicarboxylic acid, diethyl 0.007204 C12H14O4 818 DATABASE 60238 6.1951 864.3 222.0892 ester 103 Hexane, 3,3-dimethyl- 0.007132 C8H18 831 DATABASE 56097 5.7693 973.6 114.1409

174

224 Sulfurous acid, 2-propyl undecyl ester 0.006907 C14H30O3S 839 mainlib 35794 3.6812 1447.9 278.1916 204 Octane, 2,4,6-trimethyl- 0.006898 C11H24 813 DATABASE 36144 3.7172 1343.9 156.1878 95 Sulfurous acid, decyl 2-ethylhexyl ester 0.006264 C18H38O3S 849 mainlib 47081 4.842 953.6 334.2542 14 Heptane, 3,5-dimethyl- 0.005909 C9H20 874 DATABASE 44176 4.5433 230.7 128.1565 90 3-Tetradecene, (z)- 0.005829 C14H28 777 DATABASE 31321 3.2212 931.4 196.2191 36 Octane, 2,3,7-trimethyl- 0.005824 C11H24 733 mainlib 44895 4.6171 612.5 156.1878 57 Phenol, 2,4-bis(1,1-dimethylethyl)- 0.00539 C14H22O 773 replib 29074 2.9901 803.5 206.1671 80 Benzene, (1-propyloctyl)- 0.005035 C17H28 753 mainlib 29540 3.038 899.8 232.2191 207 Decane, 2,4-dimethyl- 0.004831 C12H26 846 DATABASE 30052 3.0907 1357.6 170.2035 88 2-Oxepanone, 7-methyl- 0.004704 C7H12O2 701 mainlib 34417 3.5395 922.1 128.0837 200 Oxalic acid, 2-ethylhexyl hexyl ester 0.004633 C16H30O4 784 mainlib 43116 4.4342 1332 286.2144 92 Nonane, 5-propyl- 0.004083 C12H26 755 DATABASE 26543 2.7298 940.7 170.2035 84 Benzene, (1-ethylnonyl)- 0.003576 C17H28 759 DATABASE 37002 3.8054 915.4 232.2191 101 Benzene, (1-propylhexyl)- 0.003431 C15H24 748 DATABASE 33936 3.4901 968.1 204.1878 166 2-Hydroxy-16-cyano-hexadecane 0.001636 C17H33NO 787 DATABASE 15193 1.5625 1190.6 267.2562 82 Cyclopentane, butyl- 0.001286 C9H18 800 replib 15091 1.552 909.5 126.1409 189 1-Octadecanol 0.001256 C18H38O 807 DATABASE 13248 1.3625 1276.7 270.2923 77 2,2-Dimethyl-1-[3-(phenylmethoxy)-3- 0.000712 C16H24O2 752 DATABASE 17871 1.838 889.5 248.1776 methylbutyl]oxirane 27 Methyl 2-methylidene-4-phenyl-4- 0.000105 C12H12O3 910 DATABASE 3625.4 0.37285 305.1 204.0786 oxobutanoate 29 Benzenepropanoic acid, á-oxo-, ethyl ester 9.32E-05 C11H12O3 849 DATABASE 3212.7 0.33041 338 192.0786

Table A17: Volatile compounds from EKFF broth ethyl acetate crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. Exact S/N (s) Mass 91 Pentadecane 25,585 C15H32 936 DATABASE 55088211 5503,5 869,1 212,2504 126 Octadecane 18,227 C18H38 922 DATABASE 48983436 4893,6 1003 254,2974 57 Tetradecane 18,019 C14H30 942 DATABASE 51207539 5115,8 719,2 198,2348 163 Eicosane 6,427 C20H42 896 DATABASE 22891991 2287 1124,2 282,3287

175

90 Tetradecane, 2-methyl- 5,8306 C15H32 890 DATABASE 41060343 4102 867,9 212,2504 153 Pyrrolo[1,2-a]pyrazine-1,4-dione, 1,3687 C11H18N2O2 887 mainlib 3070124 306,71 1095,3 210,1368 hexahydro-3-(2-methylpropyl)- 124 1-Nonadecene 0,82509 C19H38 940 replib 2561831 255,93 997,5 266,2974 39 Dodecane 0,80558 C12H26 929 DATABASE 2762381 275,97 551,4 170,2035 206 Propiconazole 0,72447 C15H17Cl2N3O2 871 Restek 1856983 185,52 1326 341,0698 Pesticides 2010 Library 209 Heptacosane 0,60456 C27H56 922 replib 1807262 180,55 1336,6 380,4382 99 Hexadecane, 4-methyl- 0,30022 C17H36 903 mainlib 943077 94,216 906,8 240,2817 152 Undecane, 2,4-dimethyl- 0,28664 C13H28 838 replib 1397302 139,59 1093,6 184,2191 207 Hexanedioic acid, bis(2-ethylhexyl) 0,28477 C22H42O4 897 DATABASE 563257 56,271 1332,6 370,3083 ester 190 Cyproconazole 0,27215 C15H18ClN3O 861 Korean 657462 65,682 1264,6 291,1138 Pesticide 233 1,2-Cyclohexanedicarboxylic acid, 0,25282 C24H42O4 739 mainlib 614755 61,416 1510,1 394,3083 cyclohexylmethyl nonyl ester 15 Benzene, 1,2,3-trimethyl- 0,21768 C9H12 940 DATABASE 635212 63,46 369,6 120,0939 218 Octadecane, 2-methyl- 0,21705 C19H40 915 mainlib 646471 64,584 1430,9 268,313 92 1-Undecanol 0,2024 C11H24O 792 DATABASE 1803656 180,19 870,8 172,1827 121 Heptadecane, 3-methyl- 0,14129 C18H38 895 replib 434642 43,422 982,8 254,2974 168 Eicosane, 2-methyl- 0,13891 C21H44 865 mainlib 431147 43,073 1155,7 296,3443 230 Undecane, 2-methyl- 0,13609 C12H26 721 replib 474697 47,424 1489,1 170,2035 11 4-Heptanone, 2,6-dimethyl- 0,069688 C9H18O 904 DATABASE 207110 20,691 343,7 142,1358 151 Heptadecane, 2,6-dimethyl- 0,068951 C19H40 864 mainlib 262810 26,256 1089,2 268,313 199 1-Iodo-2-methylundecane 0,066746 C12H25I 826 mainlib 211797 21,159 1304,3 296,1001 93 3-Hexadecene, (z)- 0,065777 C16H32 894 DATABASE 257865 25,761 878,9 224,2504 12 Benzene, 1,2,3-trimethyl- 0,06391 C9H12 913 DATABASE 179841 17,967 346,5 120,0939 237 Nonadecane, 2-methyl- 0,062361 C20H42 891 mainlib 256470 25,622 1518,4 282,3287 150 Undecane, 2,3-dimethyl- 0,049513 C13H28 863 mainlib 186092 18,591 1087,5 184,2191 133 1-Undecene, 4-methyl- 0,048331 C12H24 839 mainlib 178353 17,818 1027,7 168,1878 215 1,2-Benzenedicarboxylic acid, 0,044116 C24H38O4 880 replib 170374 17,021 1404,2 390,277 diisooctyl ester

176

40 Benzene, 1-ethyl-4-(1-methylethyl)- 0,039898 C11H16 748 DATABASE 409495 40,91 552,5 148,1252 224 Sulfurous acid, 2-pentyl undecyl ester 0,039318 C16H34O3S 749 mainlib 189808 18,962 1456,3 306,2229 88 4-Decene, 8-methyl-, (E)- 0,035339 C11H22 855 mainlib 158529 15,838 857 154,1722 178 Hexadecane, 3-methyl- 0,034113 C17H36 848 mainlib 144277 14,414 1219,3 240,2817 61 3-Tetradecene, (z)- 0,031133 C14H28 854 DATABASE 122475 12,236 729,7 196,2191 137 2,3,5,8-Tetramethyldecane 0,031055 C14H30 789 DATABASE 124804 12,468 1040,9 198,2348 213 Sulfurous acid, 2-pentyl tridecyl ester 0,029164 C18H38O3S 824 mainlib 129571 12,945 1364,1 334,2542 164 5-Octadecene, (e)- 0,026057 C18H36 865 DATABASE 99220 9,9123 1134,4 252,2817 203 Decane, 3,8-dimethyl- 0,025945 C12H26 706 mainlib 204105 20,391 1323 170,2035 201 Tetradecane, 4-ethyl- 0,020554 C16H34 775 mainlib 117869 11,775 1311,2 226,2661 78 Phenol, 2,4-bis(1,1-dimethylethyl)- 0,019911 C14H22O 838 replib 111049 11,094 803,4 206,1671 85 1-Iodo-2-methylundecane 0,019786 C12H25I 727 mainlib 81365 8,1286 841,5 296,1001 179 2-Propyldecan-1-ol 0,01608 C13H28O 836 DATABASE 83702 8,3621 1224,2 200,214 84 Hexane, 3,3-dimethyl- 0,012023 C8H18 809 DATABASE 104722 10,462 836,4 114,1409 195 2-Propenoic acid, oxybis(methyl-2,1- 0,011772 C15H28O2 717 mainlib 73843 7,3771 1285 240,2089 ethanediyl) ester 181 5-Undecene, 3-methyl-, (E)- 0,011568 C12H24 748 mainlib 73708 7,3637 1228,4 168,1878 29 Benzene, 1-methyl-2-(1-methylethyl)- 0,010097 C10H14 731 mainlib 60429 6,0371 452,1 134,1096 38 Naphthalene 0,009479 C10H8 795 DATABASE 122042 12,192 549,8 128,0626 14 2-Heptanone, 4,6-dimethyl- 0,00717 C9H18O 791 mainlib 39337 3,9299 359,5 142,1358 112 Heptadecane, 2,6-dimethyl- 0,006965 C19H40 837 mainlib 61724 6,1664 953,5 268,313 18 Benzeneacetaldehyde, à-methyl- 0,002871 C9H10O 796 DATABASE 26829 2,6803 383,5 134,0732 217 2-Propyldecan-1-ol 0,002414 C13H28O 851 DATABASE 40240 4,0201 1418,1 200,214 198 6-Oxabicyclo[3.1.0]hexan-2-ol, 2-ethyl- 0,001789 C7H12O2 840 DATABASE 27933 2,7906 1291 128,0837 , (1à,2à,5à)- 249 Oxirane, 2,2'-[oxybis(methylene)]bis- 0,00095 C6H10O3 833 DATABASE 16776 1,676 2068,8 130,063

Table A18: Volatile compounds from EKFF DCM broth crude extract

Peak # Name Area % Formula Similarity Library Height Quant S/N R.T. (s) Exact Mass

177

122 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 16.746 C7H10N2O2 920 mainlib 1262087 131.97 992.4 154.0742 133 3-Isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4- 14.065 C11H18N2O2 884 DATABASE 1212931 126.83 1094.2 210.1368 dione 137 5H,10H-dipyrrolo[1,2-a:1',2'-d]pyrazine-5,10-dione, 7.3942 C10H14N2O2 817 DATABASE 657818 68.787 1103.7 194.1055 octahydro-, (5as-cis)- 166 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 2.9948 C14H16N2O2 840 mainlib 289887 30.313 1345.6 244.1212 (phenylmethyl)- 164 Hexanedioic acid, bis(2-ethylhexyl) ester 1.9383 C22H42O4 888 replib 270733 28.31 1332.5 370.3083 134 Undecane, 3,8-dimethyl- 0.5557 C13H28 823 DATABASE 149770 15.661 1096.1 184.2191 165 1-Iodo-2-methylundecane 0.38724 C12H25I 778 mainlib 70617 7.3843 1336.1 296.1001 130 Hexane, 3,3-dimethyl- 0.36297 C8H18 758 replib 69391 7.256 1075.5 114.1409 129 Tetradecane, 1-iodo- 0.34372 C14H29I 851 mainlib 76758 8.0264 1070.5 324.1314 119 Hexadecane 0.26887 C16H34 862 replib 67561 7.0647 965.4 226.2661 111 Decane, 2,4-dimethyl- 0.16878 C12H26 818 DATABASE 51679 5.404 786.7 170.2035 177 Eicosane 0.11563 C20H42 843 DATABASE 34728 3.6314 1518.3 282.3287 145 Hexadecane 0.07149 C16H34 857 replib 41898 4.3812 1214.5 226.2661 174 Octane, 2,7-dimethyl- 0.068581 C10H22 842 DATABASE 40193 4.2029 1475.3 142.1722 139 Pentadecane 0.045834 C15H32 808 DATABASE 21932 2.2933 1123.4 212.2504 143 Heptane, 2,5,5-trimethyl- 0.036194 C10H22 845 mainlib 20549 2.1488 1191.8 142.1722 125 1-Heptanol 0.032201 C7H16O 703 DATABASE 19359 2.0243 1004.1 116.1201 178 Docosane 0.026152 C22H46 766 DATABASE 18754 1.9611 1560.9 310.36 147 1-Iodoundecane 0.007 C11H23I 824 mainlib 8081.9 0.8451 1234.1 282.0844 114 Undecane, 5-methyl- 0.005799 C12H26 867 DATABASE 5805.1 0.60703 834.9 170.2035 26 1,1-Cyclopentanedicarboxylic acid, 3,4- 0.003832 C13H18O4 831 DATABASE 4389.6 0.45902 273.7 238.1205 bis(methylene)-, diethyl ester 74 Thiirene, methylphenyl-, 1,1-dioxide 0.00371 C9H8O2S 811 DATABASE 7110 0.74348 417.4 180.0245 58 1,2,5-Thiadiazolidine-2,5-dicarboxylic acid, 3- 0.00143 C16H20N2O4S2 781 DATABASE 2741.1 0.28663 358.6 368.0864 ethenyl-4-[[(phenylmethyl)thio]methyl]-, dimethyl ester, trans-(.+-.)-

Table A19: Volatile compounds from EKBE broth hexane crude extract

178

Peak # Name Area % Formula Similarity Library Height Quant S/N R.T. Exact (s) Mass 97 Octadecane 18.879 C18H38 938 DATABASE 84089188 8216.2 1004.7 254.2974 65 Hexadecane 17.598 C16H34 937 replib 84640819 8270.1 870 226.2661 129 Eicosane 13.616 C20H42 764 mainlib 69335848 6774.7 1126 282.3287 163 Heneicosane 6.2392 C21H44 905 replib 47246562 4616.4 1236.8 296.3443 32 Tetradecane 5.851 C14H30 940 DATABASE 54149880 5290.9 719.2 198.2348 64 Tetradecane, 2-methyl- 3.2771 C15H32 893 DATABASE 55220760 5395.5 868.5 212.2504 188 Heptacosane 1.8906 C27H56 909 replib 15852776 1549 1338.3 380.4382 95 1-Nonadecene 0.5251 C19H38 928 replib 3682021 359.77 998.4 266.2974 127 1-Docosene 0.4567 C22H44 943 replib 3755478 366.94 1120.9 308.3443 225 Octadecane, 2-methyl- 0.32709 C19H40 912 mainlib 1907552 186.38 1519.3 268.313 131 2,6-di-butyl-2,5- 0.27834 C14H20O2 735 DATABASE 2175547 212.57 1141 220.1463 cyclohexadiene-1,4-dione 162 1-Docosene 0.21539 C22H44 945 replib 2215187 216.44 1232.7 308.3443 123 Eicosane, 2-methyl- 0.16763 C21H44 876 mainlib 925696 90.448 1107.2 296.3443 112 Nonadecane 0.15731 C19H40 910 replib 1108018 108.26 1064.4 268.313 233 Eicosane, 2-methyl- 0.15235 C21H44 901 mainlib 842146 82.285 1609.1 296.3443 2 Octane 0.15176 C8H18 938 DATABASE 1419685 138.72 206 114.1409 248 Sulfurous acid, dodecyl 0.14863 C17H36O3S 753 mainlib 1535194 150 1881.3 320.2385 pentyl ester 72 Hexadecane, 4-methyl- 0.13006 C17H36 901 mainlib 1029758 100.62 907.4 240.2817 103 Octadecane, 4-methyl- 0.11938 C19H40 904 mainlib 945875 92.42 1037.7 268.313 89 Heptadecane, 2,6-dimethyl- 0.10235 C19H40 889 mainlib 621047 60.682 965.3 268.313 114 7,9-ditert-butyl-1- 0.090845 C17H24O3 893 DATABASE 669391 65.405 1075.8 276.1725 oxaspiro[4.5]deca-6,9- diene-2,8-dione 118 Heptadecane, 2,6-dimethyl- 0.082456 C19H40 893 mainlib 534190 52.195 1089.8 268.313 93 Heptadecane, 3-methyl- 0.07844 C18H38 885 replib 648654 63.379 983.4 254.2974 122 1,2-Benzenedicarboxylic 0.069805 C16H22O4 871 DATABASE 439344 42.928 1103.5 278.1518 acid, dibutyl ester 90 Tetradecane, 4-ethyl- 0.064659 C16H34 882 mainlib 509282 49.761 969.8 226.2661

179

241 Stigmastan-3,5-diene 0.064444 C29H48 709 mainlib 272966 26.671 1678.4 396.3756 16 BENZENE, 1,3- 0.060345 C8H10 952 DATABASE 394521 38.548 260.1 106.0783 DIMETHYL- 59 Tetradecane, 1-iodo- 0.059691 C14H29I 852 mainlib 526794 51.472 846.6 324.1314 149 Undecane, 2,3-dimethyl- 0.057794 C13H28 870 mainlib 443603 43.344 1201.4 184.2191 151 Decane, 2-methyl- 0.054202 C11H24 786 mainlib 378942 37.026 1203.3 156.1878 117 Undecane, 2,3-dimethyl- 0.048879 C13H28 859 mainlib 433256 42.333 1088.2 184.2191 153 Sulfurous acid, hexyl 0.048203 C21H44O3S 841 mainlib 405984 39.668 1207.8 376.3011 pentadecyl ester 120 Octadecane, 2-methyl- 0.046164 C19H40 857 replib 424776 41.504 1094.3 268.313 70 Tetradecane 0.043187 C14H30 883 DATABASE 333078 32.545 898 198.2348 179 Tridecane, 5-propyl- 0.042646 C16H34 844 mainlib 396541 38.745 1307.9 226.2661 23 Tridecane 0.042332 C13H28 932 DATABASE 368100 35.967 636.7 184.2191 193 Sulfurous acid, 2-pentyl 0.039588 C18H38O3S 856 mainlib 432563 42.265 1365 334.2542 tridecyl ester 54 Pentadecane, 5-methyl- 0.039145 C16H34 870 DATABASE 334059 32.64 832.3 226.2661 152 Pentadecane 0.038885 C15H32 812 DATABASE 406227 39.692 1203.7 212.2504 217 Undecane, 2,3-dimethyl- 0.036951 C13H28 833 mainlib 343894 33.601 1491.5 184.2191 126 1-Butanone, 1-(5-acetyl-2- 0.036833 C13H16O3 742 DATABASE 1332582 130.2 1119.9 220.1099 hydroxyphenyl)-3-methyl- 199 Dodecane, 2-methyl- 0.035874 C13H28 849 mainlib 322343 31.496 1401.7 184.2191 180 Sulfurous acid, hexyl 0.035846 C19H40O3S 823 mainlib 273974 26.77 1312 348.2698 tridecyl ester 99 2-Hexyl-1-octanol 0.034478 C14H30O 859 mainlib 353226 34.513 1013.8 214.2297 101 Tridecane, 3-methyl- 0.03264 C14H30 804 replib 208324 20.355 1028 198.2348 30 1-Dodecene 0.03222 C12H24 902 DATABASE 269859 26.368 711.7 168.1878 204 Nonadecane, 2-methyl- 0.029188 C20H42 854 mainlib 228937 22.369 1419.1 282.3287 150 Octane, 3-ethyl- 0.026466 C10H22 765 DATABASE 409560 40.018 1203.1 142.1722 67 3-Tetradecene, (Z)- 0.025945 C14H28 848 mainlib 269565 26.339 879.5 196.2191 212 Tetracosane, 3-ethyl- 0.023967 C26H54 873 DATABASE 230238 22.496 1457 366.4226 21 Dodecane 0.023833 C12H26 954 DATABASE 225673 22.05 550.9 170.2035 177 Hexadecane, 4-methyl- 0.023611 C17H36 843 mainlib 322264 31.488 1304.5 240.2817

180

12 Octane, 2-methyl- 0.022438 C9H20 805 mainlib 190044 18.569 252.8 128.1565 13 Ethylbenzene 0.022438 C8H10 932 mainlib 190044 18.569 253.2 106.0783 94 2,4,6-Cyclooctatrien-1-one, 0.020911 C9H10O 722 DATABASE 467287 45.658 984 134.0732 8-methyl- 200 Dodecane, 2,6,10-trimethyl- 0.020145 C15H32 858 mainlib 219157 21.414 1404.1 212.2504 197 Nonadecane, 2-methyl- 0.020143 C20H42 824 mainlib 206559 20.183 1385.2 282.3287 178 Nonadecane 0.019743 C19H40 872 replib 429060 41.923 1305.3 268.313 17 p-Xylene 0.019362 C8H10 913 replib 164683 16.091 279.1 106.0783 202 Sulfurous acid, hexyl 0.01924 C20H42O3S 872 mainlib 141457 13.822 1407.7 362.2855 tetradecyl ester 218 Sulfurous acid, decyl 2- 0.018178 C18H38O3S 861 mainlib 245089 23.947 1493.2 334.2542 ethylhexyl ester 229 Sulfurous acid, pentyl 0.017768 C19H40O3S 827 mainlib 204625 19.994 1543.1 348.2698 tetradecyl ester 221 1-Iodo-2-methylundecane 0.017365 C12H25I 816 mainlib 218142 21.314 1507.6 296.1001 242 Sulfurous acid, butyl 0.016466 C16H34O3S 823 mainlib 207051 20.231 1679.3 306.2229 dodecyl ester 165 2,3-Dimethyldecane 0.01613 C12H26 787 mainlib 152249 14.876 1255.3 170.2035 166 Dihexylsulfide 0.01613 C12H26S 763 DATABASE 152249 14.876 1255.5 202.1755 243 Nonadecane, 2-methyl- 0.016126 C20H42 873 mainlib 286336 27.978 1723.9 282.3287 219 Sulfurous acid, hexyl 0.01604 C21H44O3S 828 mainlib 162548 15.882 1497.1 376.3011 pentadecyl ester 27 Tridecane, 3-methyl- 0.014206 C14H30 897 replib 144459 14.115 694.3 198.2348 214 Sulfurous acid, 2-propyl 0.011952 C16H34O3S 851 mainlib 144655 14.134 1475.8 306.2229 tridecyl ester 61 1-Undecene 0.011838 C11H22 853 DATABASE 167458 16.362 857.8 154.1722 113 Tetracosane 0.011665 C24H50 803 DATABASE 125578 12.27 1070.8 338.3913 174 Sulfurous acid, dodecyl 2- 0.011622 C15H32O3S 812 mainlib 104372 10.198 1291.3 292.2072 propyl ester 145 Oxalic acid, allyl hexadecyl 0.011472 C21H38O4 826 mainlib 91971 8.9864 1185 354.277 ester 198 Tridecane, 3-methyl- 0.0103 C14H30 847 replib 125095 12.223 1387.8 198.2348 201 1,2-Benzenedicarboxylic 0.010104 C24H38O4 871 replib 180227 17.61 1405.8 390.277 acid, diisooctyl ester 75 Tetradecane, 1-iodo- 0.009793 C14H29I 801 mainlib 126082 12.319 916.7 324.1314

181

140 Isooctanol 0.009772 C8H18O 769 DATABASE 136822 13.369 1166.1 130.1358 125 1-Octene, 3-ethyl- 0.009461 C10H20 740 DATABASE 126795 12.389 1116.9 140.1565 28 Dodecane, 2,6,10-trimethyl- 0.009427 C15H32 846 DATABASE 93089 9.0956 698.1 212.2504 73 Cyclopentane, butyl- 0.009287 C9H18 775 mainlib 183685 17.948 909.9 126.1409 220 Sulfurous acid, pentadecyl 0.009006 C18H38O3S 839 mainlib 121188 11.841 1504.9 334.2542 2-propyl ester 18 Nonane 0.007529 C9H20 934 DATABASE 95624 9.3433 281.2 128.1565 228 Undecane, 2,4-dimethyl- 0.007468 C13H28 767 DATABASE 130026 12.705 1534.4 184.2191 115 Sulfurous acid, 2-ethylhexyl 0.007437 C17H36O3S 867 mainlib 117328 11.464 1079 320.2385 nonyl ester 82 2,6-Dimethylheptadecane 0.006942 C19H40 810 DATABASE 90704 8.8626 953.9 268.313 209 Undecane, 2-methyl- 0.006728 C12H26 789 DATABASE 90198 8.8131 1448.1 170.2035 31 1-Dodecene 0.006691 C12H24 811 replib 101080 9.8764 713.8 168.1878 47 Tetradecane 0.00666 C14H30 829 DATABASE 102793 10.044 802.7 198.2348 189 Cyclooctane, 1,4-dimethyl-, 0.005661 C10H20 744 DATABASE 68696 6.7122 1348.4 140.1565 trans- 159 1-Heptene, 5-methyl- 0.005558 C8H16 829 mainlib 81761 7.9888 1229.4 112.1252 78 3-Tetradecene, (z)- 0.00536 C14H28 891 DATABASE 66796 6.5265 931.8 196.2191 110 Heptane, 2,2,3,3,5,6,6- 0.005178 C14H30 754 replib 80100 7.8265 1059.5 198.2348 heptamethyl- 42 Tridecane, 3-methyl- 0.005027 C14H30 857 replib 69449 6.7858 767 198.2348 50 Tridecane 0.004694 C13H28 821 mainlib 95211 9.303 820.1 184.2191 15 Octane, 3-methyl- 0.004278 C9H20 861 DATABASE 81606 7.9736 258.2 128.1565 108 Oxalic acid, allyl hexadecyl 0.004227 C21H38O4 808 mainlib 78809 7.7004 1049.7 354.277 ester 7 Heptane, 2,5-dimethyl- 0.00413 C9H20 899 DATABASE 64941 6.3453 230.4 128.1565 38 Nonane, 5-propyl- 0.004125 C12H26 877 DATABASE 58407 5.7069 754.5 170.2035 80 Benzene, (1-methyldecyl)- 0.004041 C17H28 736 DATABASE 76541 7.4788 942.3 232.2191 43 Tetradecane, 1-iodo- 0.00389 C14H29I 758 mainlib 86852 8.4862 772.5 324.1314 69 Benzene, (1-butylheptyl)- 0.003717 C17H28 793 mainlib 54417 5.317 892.6 232.2191 56 1-Iodoundecane 0.002898 C11H23I 729 mainlib 53311 5.2089 841.8 282.0844 92 Heptane, 3,4-dimethyl- 0.002729 C9H20 801 replib 65988 6.4476 979.4 128.1565

182

84 Heptane, 2,3,5-trimethyl- 0.002575 C10H22 758 DATABASE 54532 5.3282 958.5 142.1722 24 Octane, 2,3,7-trimethyl- 0.002218 C11H24 755 DATABASE 50759 4.9596 677 156.1878 9 Cyclohexane, 1,1,3- 0.002206 C9H18 785 DATABASE 41181 4.0237 236.9 126.1409 trimethyl- 147 Diisoamylene 0.002195 C10H20 756 mainlib 52932 5.1719 1191.6 140.1565 10 Heptane, 2,3-dimethyl- 0.002161 C9H20 850 DATABASE 39296 3.8395 246.4 128.1565 196 5-Methyloctene-1 0.002153 C9H18 799 DATABASE 40230 3.9309 1374 126.1409 3 Cyclohexane, 1,2-dimethyl-, 0.001994 C8H16 774 DATABASE 61086 5.9686 210.1 112.1252 cis- 14 Propanoic acid, 2-hydroxy- 0.001793 C5H10O3 794 DATABASE 46805 4.5732 257.6 118.063 2-methyl-, methyl ester 109 Dodecane, 2-methyl- 0.001745 C13H28 705 DATABASE 25050 2.4476 1053.7 184.2191 63 1,2-Benzenedicarboxylic 0.001661 C12H14O4 746 DATABASE 95072 9.2894 865.1 222.0892 acid, diethyl ester 49 Heptane, 3-ethyl-5-methyl- 0.001612 C10H22 851 mainlib 38501 3.7619 816.4 142.1722 74 Benzene, (1-nitropropyl)- 0.001579 C9H11NO2 783 mainlib 54222 5.298 916 165.079 236 Undecane, 2,8-dimethyl- 0.001414 C13H28 811 mainlib 38167 3.7293 1638.3 184.2191 39 1-Hexadecanol 0.001256 C16H16O2 801 DATABASE 41805 4.0848 759.5 240.115 6 Heptane, 2,6-dimethyl- 0.001207 C9H20 894 DATABASE 29767 2.9084 225.6 128.1565 35 1,5-Dimethylnaphthalene 0.000959 C12H12 801 DATABASE 31188 3.0473 746.1 156.0939 45 Dodecane 0.000948 C12H26 832 DATABASE 21664 2.1168 786 170.2035 26 1-Iodoundecane 0.000876 C11H23I 868 mainlib 30396 2.9699 688.8 282.0844 4 Cyclohexane, 1,4-dimethyl- 0.000851 C8H16 853 replib 25418 2.4836 214.2 112.1252 237 Benzeneacetaldehyde, à- 0.000454 C9H10O 703 DATABASE 17215 1.6821 1649.3 134.0732 methyl- 22 Octane, 1-iodo- 0.000258 C8H17I 751 DATABASE 14525 1.4192 612.5 240.0375 184 L-Prolyl-glycine 0.000103 C7H12N2O3 770 nist_msms 6403.4 0.62566 1332.8 172.0848

Table A20: Volatile compounds from EKBE broth ethyl acetate crude extract

Peak # Name Area % Formula Similarity Library Height Quant S/N R.T. (s) Exact Mass 81 Pentadecane 32.21 C15H32 929 DATABASE 20042751 2135.8 867.5 212.2504

183

50 Tetradecane 17.248 C14H30 942 replib 11227761 1196.5 717.9 198.2348 115 Octadecane 16.822 C18H38 927 replib 9552934 1018 1001.6 254.2974 150 Eicosane 6.1067 C20H42 920 replib 367.79 1122.9 282.3287 140 Pyrrolo[1,2-a]pyrazine-1,4-dione, 1.3854 C11H18N2O2 867 mainlib 643656 68.589 1092.8 210.1368 hexahydro-3-(2-methylpropyl)- 82 1-Tetradecanol 1.1042 C14H30O 709 DATABASE 786576 83.819 869.6 214.2297 114 1-Nonadecene 1.0976 C19H38 932 replib 658920 70.216 996.7 266.2974 190 Propiconazole 0.83094 C15H17Cl2N3O2 868 Restek 377932 40.273 1324.8 341.0698 Pesticides 2010 Library 148 3-Octadecene, (E)- 0.55784 C18H36 881 mainlib 349965 37.293 1119.1 252.2817 193 Octadecane, 2-methyl- 0.48408 C19H40 894 mainlib 447896 47.729 1335.5 268.313 89 Hexadecane, 4-methyl- 0.48057 C17H36 884 mainlib 258157 27.51 906.3 240.2817 51 Cyclooctane, methyl- 0.35621 C9H18 817 replib 215433 22.957 720.7 126.1409 38 Tridecane 0.32992 C13H28 913 DATABASE 228968 24.399 551.1 184.2191 229 1,2-Cyclohexanedicarboxylic acid, 0.2572 C24H42O4 721 mainlib 162657 17.333 1521.1 394.3083 cyclohexylmethyl nonyl ester 122 Octadecane, 4-methyl- 0.2563 C19H40 868 mainlib 169231 18.034 1036.6 268.313 169 1-Nonadecene 0.25026 C19H38 845 replib 188292 20.065 1230.7 266.2974 191 Hexanedioic acid, bis(2-ethylhexyl) ester 0.23719 C22H42O4 886 DATABASE 172912 18.426 1331.7 370.3083 48 3-Tetradecene, (z)- 0.23673 C14H28 916 DATABASE 160040 17.054 711.1 196.2191 76 Tridecane, 3-methyl- 0.23264 C14H30 846 replib 126599 13.491 845.6 198.2348 225 1,2-Cyclohexanedicarboxylic acid, 0.22094 C24H42O4 721 mainlib 172153 18.345 1509.1 394.3083 cyclohexylmethyl nonyl ester 37 Acetic acid, octyl ester 0.21915 C10H20O2 904 DATABASE 131585 14.022 502.9 172.1463 175 Cyproconazol, R*,S* / R*,S* 0.20706 C15H18ClN3O 814 KeTa_PestHe 192321 20.494 1263.2 291.1138 V_1_00 105 2-(Dibromomethyl)tetrahydropyran 0.19092 C6H10Br2O 846 DATABASE 122263 13.029 963.8 255.9098 107 Tetradecane, 4-ethyl- 0.17935 C16H34 741 DATABASE 127685 13.606 968.5 226.2661 58 Tridecane, 3-methyl- 0.17135 C14H30 870 replib 161117 17.169 761.8 198.2348 112 Pyrrolo[1,2-a]pyrazine-1,4-dione, 0.12114 C7H10N2O2 875 mainlib 79049 8.4236 989.7 154.0742 hexahydro- 40 Dodecane 0.11434 C12H26 915 DATABASE 101023 10.765 636.3 170.2035

184

189 3-Benzylhexahydropyrrolo[1,2-a]pyrazine- 0.11283 C14H16N2O2 728 DATABASE 101020 10.765 1322.7 244.1212 1,4-dione # 72 Pentadecane, 5-methyl- 0.11018 C16H34 747 DATABASE 98871 10.536 831.4 226.2661 231 1,2-Cyclohexanedicarboxylic acid, 0.1087 C24H42O4 723 mainlib 107743 11.481 1532.6 394.3083 cyclohexylmethyl nonyl ester 83 1-Pentadecene 0.10636 C15H30 860 DATABASE 66788 7.117 878.2 210.2348 23 Benzene, 1-ethyl-3-methyl- 0.097558 C9H12 921 DATABASE 85903 9.1539 395.8 120.0939 145 Tridecane, 3-methyl- 0.095856 C14H30 823 replib 71803 7.6515 1105.7 198.2348 192 1-Docosene 0.091808 C22H44 858 replib 125231 13.345 1333 308.3443 228 Nonadecane, 2-methyl- 0.084046 C20H42 779 mainlib 77523 8.261 1517.4 282.3287 15 4-Heptanone, 2,6-dimethyl- 0.067731 C9H18O 930 DATABASE 90662 9.6611 345 142.1358 139 Sulfurous acid, decyl 2-ethylhexyl ester 0.064008 C18H38O3S 861 mainlib 71082 7.5746 1088.4 334.2542 71 Pentadecane, 7-methyl- 0.06012 C16H34 865 mainlib 73496 7.8319 827.5 226.2661 198 Undecane, 3,8-dimethyl- 0.058597 C13H28 787 mainlib 55539 5.9183 1363 184.2191 138 Sulfurous acid, 2-ethylhexyl hexyl ester 0.040117 C14H30O3S 816 mainlib 69103 7.3638 1086.9 278.1916 86 Dodecane, 2-methyl- 0.040001 C13H28 869 mainlib 43455 4.6306 897 184.2191 157 PENTADECANE 0.03991 C15H32 814 DATABASE 41365 4.4079 1179.2 212.2504 201 Sulfurous acid, dodecyl 2-propyl ester 0.039067 C15H32O3S 740 mainlib 50627 5.395 1398.9 292.2072 113 2-Ethyl-1-decene # 0.032599 C12H24 772 DATABASE 64319 6.8539 992.2 168.1878 4 Cyclopentane, 1-ethyl-2-methyl- 0.029332 C8H16 770 DATABASE 31489 3.3555 205.6 112.1252 65 Phenol, 3,5-bis(1,1-dimethylethyl)- 0.027576 C14H22O 705 replib 41462 4.4183 802.9 206.1671 164 Dodecane, 5-methyl- 0.026667 C13H28 847 DATABASE 41231 4.3937 1206.2 184.2191 147 Cyclobutane, 1,1,2,3,3-pentamethyl- 0.026551 C9H18 771 mainlib 28077 2.992 1115.3 126.1409 55 Nonane, 5-propyl- 0.025784 C12H26 806 DATABASE 47377 5.0486 753.8 170.2035 100 Oxalic acid, allyl decyl ester 0.020291 C15H26O4 835 mainlib 40746 4.342 949.5 270.1831 78 Octane, 3-methyl-6-methylene- 0.018113 C10H20 811 mainlib 44243 4.7146 856.4 140.1565 17 Benzene, 1-ethyl-4-methyl- 0.01501 C9H12 886 replib 28748 3.0634 355.5 120.0939 117 Oxalic acid, allyl dodecyl ester 0.014697 C17H30O4 859 mainlib 45712 4.8712 1012.3 298.2144 28 Benzene, 2-ethyl-1,4-dimethyl- 0.012268 C10H14 875 replib 19003 2.025 426.6 134.1096 173 1-Iodotetradecane 0.011853 C14H29I 751 DATABASE 28451 3.0318 1253.7 324.1314 101 Docosane 0.011005 C22H46 760 DATABASE 23088 2.4603 952.8 310.36

185

180 Benzene, [(1-propenyloxy)methyl]- 0.010948 C10H12O 880 DATABASE 23607 2.5156 1280.6 148.0888 153 Dodecane, 2,6,10-trimethyl- 0.00942 C15H32 790 DATABASE 18407 1.9615 1145.2 212.2504 184 Sulfurous acid, 2-propyl undecyl ester 0.009201 C14H30O3S 829 mainlib 22719 2.421 1303.7 278.1916 26 Benzene, 1,4-diethyl- 0.008575 C10H14 789 DATABASE 24679 2.6299 418.1 134.1096 41 1-Methylbutyl nitrite 0.004409 C5H11NO2 705 DATABASE 17436 1.858 654 117.079 27 Benzene, (1-methylpropyl)- 0.003464 C10H14 705 DATABASE 19229 2.0491 421.3 134.1096 133 2-Octyldodecan-1-ol 0.002211 C20H42O 800 DATABASE 8060.7 0.85896 1075.3 298.3236 96 2-Propyldecan-1-ol 0.002049 C13H28O 773 DATABASE 11183 1.1917 921.7 200.214 29 1-Propanone, 2-(2-nitrocyclopentyl)-1- 0.001752 C14H17NO3 743 DATABASE 10287 1.0962 435.1 247.1208 phenyl- 33 Decane 0.001126 C10H22 865 DATABASE 1.065 462.1 142.1722 74 3,4-Hexanedione, 2,2,5-trimethyl- 0.000419 C9H16O2 815 mainlib 3720 0.39641 840.6 156.115 30 1,3,8-p-Menthatriene 0.000402 C10H14 729 mainlib 3569.4 0.38037 444.1 134.1096 165 2-Propyldecan-1-ol 2.61E-05 C13H28O 830 DATABASE 231.44 0.024662 1218.6 200.214

Table A21: Volatile compunds from EKBE DCM broth crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. (s) Exact S/N Mass 126 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 13.939 C7H10N2O2 910 mainlib 1844899 201.37 994.8 154.0742 142 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- 11.256 C11H18N2O2 892 mainlib 1944344 212.23 1094.6 210.1368 methylpropyl)- 147 5H,10H-dipyrrolo[1,2-a:1',2'-d]pyrazine-5,10- 6.0669 C10H14N2O2 792 DATABASE 1153850 125.94 1103.4 194.1055 dione, octahydro-, (5as-cis)- 179 Hexanedioic acid, bis(2-ethylhexyl) ester 1.5065 C22H42O4 898 DATABASE 374105 40.834 1331.6 370.3083 181 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 0.88774 C14H16N2O2 828 mainlib 253424 27.662 1344.7 244.1212 (phenylmethyl)- 144 (R,R)-3,8-dimethyldecane 0.8272 C12H26 774 DATABASE 606905 66.245 1095.6 170.2035 176 Undecane, 3,8-dimethyl- 0.27797 C13H28 831 mainlib 172468 18.825 1322.2 184.2191 191 Hexadecane 0.25033 C16H34 872 replib 100559 10.976 1429.8 226.2661 157 Nonadecane, 2-methyl- 0.19386 C20H42 799 mainlib 80506 8.7874 1214.1 282.3287 135 Eicosane 0.18835 C20H42 857 DATABASE 148354 16.193 1070.1 282.3287

186

118 Undecane, 3,8-dimethyl- 0.13113 C13H28 865 mainlib 99290 10.838 965 184.2191 128 Undecane, 3-methyl- 0.11727 C12H26 731 replib 78412 8.5588 1001.4 170.2035 24 Methyllaurate 0.11709 C8H10 743 DATABASE 51092 5.5768 253.9 106.0783 28 Benzene, 1,2-dimethyl- 0.089916 C8H10 827 DATABASE 48188 5.2598 260.8 106.0783 162 1-Iodo-2-methylnonane 0.085195 C10H21I 763 mainlib 57339 6.2586 1233.4 268.0688 119 Undecane, 5-methyl- 0.061232 C12H26 724 DATABASE 40412 4.411 968 170.2035 180 Pentadecane 0.057815 C15H32 843 DATABASE 62696 6.8434 1335.4 212.2504 199 1-Iodo-2-methylundecane 0.038214 C12H25I 866 mainlib 33029 3.6052 1517.3 296.1001 30 Phosphonic acid, (2-oxo-1,3-propanediyl)bis-, 0.035183 C15H20O 849 DATABASE 40183 4.386 266.8 216.1514 tetramethyl ester 186 Docosane 0.030818 C22H46 780 DATABASE 43118 4.7064 1402.6 310.36 187 Phenacetin 0.027435 C10H13NO2 788 nist_msms 20481 2.2355 1403.2 179.0946 154 Dodecane, 1,1-difluoro- 0.025649 C12H24F2 821 DATABASE 26891 2.9352 1179.2 206.1846 205 Docosane 0.021366 C22H46 757 DATABASE 31447 3.4325 1659.3 310.36 22 1,2,5-Thiadiazolidine-2,5-dicarboxylic acid, 3- 0.018064 C16H20N2O4S2 772 DATABASE 15583 1.7009 251.7 368.0864 ethenyl-4-[[(phenylmethyl)thio]methyl]-, dimethyl ester, trans-(.+-.)- 133 Disulfide, ethyl 1-methylpropyl 0.013452 C6H14S2 753 DATABASE 19016 2.0756 1044.8 150.0537 156 2-Butenal 0.009312 C4H6O 792 DATABASE 23488 2.5638 1197.8 70.0419 188 Hexane, 3,3,4,4-tetraethyl- 0.001456 C14H30 730 DATABASE 5966.3 0.65123 1417.1 198.2348 185 Phenylacetic acid 0.000636 C8H8O2 970 nist_msms 2605.8 0.28443 1394 136.0524

Appendix 2.3: Volatile compounds from endophytic bacteria

Table A22: Volatile compounds from F1 hexane crude extract

Peak Name Area % Formula Similarity Library Height Quant R.T. (s) Exact # S/N Mass 45 Octadecane 10.674 C18H38 942 DATABASE 57252326 8636.6 1007.1 254.2974 46 1-Undecanol 10.345 C11H24O 802 DATABASE 56503557 8523.6 1008 172.1827 68 Eicosane 10.146 C20H42 931 replib 52387443 7902.7 1128.2 282.3287

187

21 Hexadecane 9.761 C16H34 935 DATABASE 56901700 8583.7 872.1 226.2661 22 Nonadecane 9.6832 C19H40 858 replib 56725120 8557.1 872.8 268.313 84 Tetracosane 8.1123 C24H50 858 DATABASE 44784363 6755.8 1239.7 338.3913 82 E-2-Octadecadecen-1-ol 8.08 C18H36O 681 mainlib 44750955 6750.7 1237.9 268.2766 83 Heneicosane 7.9615 C21H44 925 replib 44628297 6732.2 1238.4 296.3443 13 Tetradecane 3.6837 C14H30 951 replib 47568020 7175.7 719.8 198.2348 66 Hexadecane, 2,6,10,14-tetramethyl- 1.7195 C20H42 734 DATABASE 40646180 6131.5 1125.7 282.3287 69 Tetradecane, 2-methyl- 1.3933 C15H32 786 DATABASE 21926451 3307.6 1130.9 212.2504 47 Tridecane, 2-methyl- 1.2381 C14H30 885 DATABASE 26868514 4053.2 1008.6 198.2348 23 7-Hexadecene, (z)- 1.1768 C16H32 863 DATABASE 29371803 4430.8 873.2 224.2504 65 Oxalic acid, heptyl propyl ester 0.70624 C12H22O4 818 mainlib 27660609 4172.6 1124.9 230.1518 64 1-Docosene 0.49603 C22H44 938 replib 11425123 1723.5 1121.8 308.3443 95 Oxalic acid, allyl nonyl ester 0.48513 C14H24O4 833 mainlib 22488441 3392.4 1338.5 256.1675 43 1-Nonadecene 0.44272 C19H38 944 replib 6732625 1015.6 999.1 266.2974 56 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8- 0.25786 C17H24O3 933 replib 3353786 505.92 1078.1 276.1725 dione 94 1-Docosene 0.16483 C22H44 939 replib 2650614 399.85 1335.9 308.3443 39 Heptadecane, 3-methyl- 0.1224 C18H38 904 replib 1591462 240.07 983.8 254.2974 40 Benzene, (1-ethyldecyl)- 0.1224 C18H30 866 replib 1591462 240.07 984.4 246.2348 10 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.12 C10H18O 936 DATABASE 2340398 353.05 404.8 154.1358 72 Eicosane, 2-methyl- 0.11716 C21H44 871 mainlib 1848285 278.82 1157 296.3443 51 Octadecane, 4-methyl- 0.11412 C19H40 923 mainlib 1827694 275.71 1038.4 268.313 9 Benzene, 1-methyl-2-(1-methylethyl)- 0.087585 C10H14 952 mainlib 1755079 264.76 396.3 134.1096 73 Triacontane 0.080377 C30H62 906 DATABASE 1211657 182.78 1181 422.4852 33 Benzene, (1-methyldecyl)- 0.056671 C17H28 902 DATABASE 715487 107.93 942.7 232.2191 5 p-Xylene 0.055399 C8H10 956 replib 848370 127.98 260.2 106.0783 19 Pentadecane, 3-methyl- 0.053282 C16H34 892 mainlib 932191 140.62 846.9 226.2661 74 Pentacosane 0.052529 C25H52 866 DATABASE 925290 139.58 1201.8 352.4069 59 Heptadecane, 2,3-dimethyl- 0.05021 C19H40 866 mainlib 862979 130.18 1094.7 268.313 61 Dibutyl phthalate 0.033351 C16H22O4 916 mainlib 678080 102.29 1104 278.1518

188

60 2,5-Cyclohexadiene-1,4-dione, 2,6-bis(1,1- 0.031375 C14H20O2 773 DATABASE 607969 91.713 1101.1 220.1463 dimethylethyl)- 91 Heneicosane, 5-methyl- 0.029702 C22H46 816 mainlib 593914 89.593 1312.4 310.36 85 Triacontane 0.029477 C30H62 875 DATABASE 513713 77.494 1255.9 422.4852 29 Benzene, (1-propyloctyl)- 0.028013 C17H28 879 mainlib 336898 50.821 900.8 232.2191 7 Bicyclo[3.1.0]hexane, 4-methyl-1-(1-methylethyl)-, 0.027748 C10H16 915 mainlib 464743 70.107 306.6 136.1252 didehydro deriv. 31 Benzene, (1-ethylnonyl)- 0.025077 C17H28 892 DATABASE 409578 61.785 916.5 232.2191 97 1-Iodo-2-methylundecane 0.023295 C12H25I 864 mainlib 281349 42.442 1355.9 296.1001 35 Benzene, (1-butyloctyl)- 0.021391 C18H30 896 DATABASE 305724 46.119 960.2 246.2348 70 4,6-di-tert-Butyl-m-cresol 0.019602 C15H24O 738 mainlib 338956 51.132 1141 220.1827 24 2-Tridecene, (E)- 0.017703 C13H26 898 mainlib 412398 62.211 880.1 182.2035 37 Benzene, (1-propylnonyl)- 0.016906 C18H30 865 DATABASE 587761 88.664 968.9 246.2348 6 Benzene, 1,3-dimethyl- 0.016681 C8H10 941 replib 322009 48.575 279.3 106.0783 34 Benzene, (1-pentylheptyl)- 0.016 C18H30 830 mainlib 271535 40.961 957 246.2348 27 Benzene, (1-butylheptyl)- 0.014314 C17H28 897 mainlib 269319 40.627 893.3 232.2191 52 Phthalic acid, 2-cyclohexylethyl butyl ester 0.010523 C20H28O4 653 mainlib 209958 31.672 1045.7 332.1988 42 Octane, 3-methyl-6-methylene- 0.008785 C10H20 820 mainlib 144363 21.777 994 140.1565 8 Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl- 0.008708 C10H16 925 DATABASE 172162 25.971 314.7 136.1252 79 Undecane, 3-methylene- 0.008431 C12H24 837 mainlib 163635 24.684 1229.7 168.1878 4 Octane, 3-methyl- 0.008265 C9H20 890 DATABASE 181622 27.398 258.1 128.1565 26 Benzene, (1-pentylhexyl)- 0.005502 C17H28 859 DATABASE 108087 16.305 890.8 232.2191 48 Benzene, (1-methylundecyl)- 0.000313 C18H30 840 DATABASE 26724 4.0313 1011.6 246.2348

Table A23: Volatile compounds from F1 ethyl acetate crude extract

Peak Name Area % Formula Similarity Library Height Quant R.T. Exact # S/N (s) Mass 22 Octadecane 14.039 C18H38 937 replib 34605010 5391.1 1002.9 254.2974 15 Hexadecane 7.3184 C16H34 935 replib 24229886 3774.7 867.9 226.2661 36 Heneicosane 5.9901 C21H44 917 replib 19604886 3054.2 1124.4 296.3443

189

30 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 4.5292 C11H18N2O2 891 mainlib 5810455 905.2 1100.5 210.1368 (2-methylpropyl)- 38 Triacontane 4.3802 C30H62 922 DATABASE 5227465 814.38 1202.4 422.4852 29 2,5-Piperazinedione, 3,6-bis(2-methylpropyl)- 3.4892 C12H22N2O2 796 mainlib 5144667 801.48 1100.1 226.1681 50 Heptacosane 2.2043 C27H56 922 replib 3741180 582.83 1407.7 380.4382 20 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 1.4387 C7H10N2O2 830 mainlib 2063247 321.43 996.5 154.0742 46 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 1.1596 C14H16N2O2 844 mainlib 1806954 281.5 1328.7 244.1212 (phenylmethyl)- 45 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)-4- 1.1248 C15H17Cl2N3O2 872 mainlib 1789593 278.8 1327.4 341.0698 propyl-1,3-dioxolan-2-yl]methyl]- 28 Docosane 1.0666 C22H46 672 DATABASE 1873005 291.79 1087.8 310.36 21 1-Nonadecene 1.0518 C19H38 860 replib 1798747 280.22 997.6 266.2974 2 Octane 0.70091 C8H18 826 DATABASE 780452 121.59 207.6 26 5,8-Tridecadione 0.6358 C13H24O2 684 mainlib 911529 142.01 1046.2 212.1776 11 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.6 C10H18O 928 DATABASE 1864580 290.48 404.8 154.1358 1 2(3H)-furanone, dihydro-3,5-dimethyl- 0.51069 C6H10O2 730 DATABASE 785574 122.38 205.5 114.0681 8 Benzene, 1,2,4-trimethyl- 0.50269 C9H12 939 mainlib 1362539 212.27 369.2 120.0939 10 Benzene, 1-methyl-2-(1-methylethyl)- 0.50164 C10H14 945 mainlib 1093234 170.31 396.6 134.1096 60 1,2-Cyclohexanedicarboxylic acid, 0.42263 C24H42O4 767 mainlib 643227 100.21 1523 394.3083 cyclohexylmethyl nonyl ester 18 dl-Alanyl-l-leucine 0.4098 C9H18N2O3 876 mainlib 507343 79.038 977 202.1317 65 Heptacosane 0.39983 C27H56 913 replib 539263 84.011 1669.4 380.4382 39 3-Benzyl-6-methyl-2,5-piperazinedione # 0.39844 C12H14N2O2 838 DATABASE 764649 119.12 1230.9 218.1055 37 Eicosane, 2-methyl- 0.36627 C21H44 861 mainlib 582773 90.789 1156.1 296.3443 44 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)-4- 0.36157 C15H17Cl2N3O2 882 mainlib 806281 125.61 1320.5 341.0698 propyl-1,3-dioxolan-2-yl]methyl]- 51 Octadecane, 2-methyl- 0.35647 C19H40 905 mainlib 613026 95.502 1431.5 268.313 55 Undecane, 2-methyl- 0.34564 C12H26 715 replib 672594 104.78 1499.9 170.2035 53 1,2-Cyclohexanedicarboxylic acid, 0.24708 C24H42O4 714 mainlib 336598 52.438 1489.8 394.3083 cyclohexylmethyl nonyl ester 54 Eicosane, 2-methyl- 0.23948 C21H44 865 mainlib 492545 76.733 1497.2 296.3443 43 3-Benzyl-6-isopropyl-2,5-piperazinedione # 0.2008 C14H18N2O2 739 DATABASE 342634 53.378 1287.9 246.1368 17 2-Propenal, 3-(1-aziridinyl)-3-(dimethylamino)- 0.19917 C7H12N2O 663 mainlib 368597 57.423 960.9 140.095

190

47 Hexanedioic acid, bis(2-ethylhexyl) ester 0.18512 C22H42O4 912 DATABASE 393622 61.322 1333.5 370.3083 24 Octadecane, 4-methyl- 0.16602 C19H40 911 mainlib 496355 77.326 1037.5 268.313 31 1,2-Benzenedicarboxylic acid, dibutyl ester 0.15242 C16H22O4 729 DATABASE 879591 137.03 1103.4 278.1518 14 Tetradecane 0.15148 C14H30 933 DATABASE 450375 70.163 717.9 198.2348 16 Nonadecane 0.11603 C19H40 896 replib 367009 57.176 936.1 268.313 3 Bicyclo[3.1.0]hexane, 4-methyl-1-(1- 0.10239 C10H16 920 mainlib 323205 50.352 307.5 136.1252 methylethyl)-, didehydro deriv. 7 Benzene, 1-ethyl-2-methyl- 0.081436 C9H12 932 replib 232981 36.296 354.8 120.0939 62 1,2-Cyclohexanedicarboxylic acid, dinonyl ester 0.075494 C26H48O4 790 mainlib 216551 33.736 1534.3 424.3553 5 Benzene, 1-ethyl-4-methyl- 0.067934 C9H12 914 replib 191216 29.789 341.5 120.0939

Table A24: Volatile compounds from F1 DCM crude extract

Peak Name Area % Formula Similarity Library Height Quant R.T. (s) Exact # S/N Mass 1 2(3H)-furanone, dihydro-3,5-dimethyl- 22.621 C6H10O2 724 DATABASE 903380 146.79 204.7 114.0681 12 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 16.146 C10H18O 934 DATABASE 1668458 271.1 404.5 154.1358 9 Benzene, 1,2,3-trimethyl- 15.386 C9H12 941 replib 1171651 190.38 369.1 120.0939 11 Benzene, 1-methyl-2-(1-methylethyl)- 13.641 C10H14 942 mainlib 830774 134.99 396.7 134.1096 15 Hexadecane 4.917 C16H34 877 replib 321642 52.262 868.4 226.2661 7 Benzene, 1,2,3-trimethyl- 3.4812 C9H12 922 replib 311183 50.563 345.9 120.0939 3 Bicyclo[3.1.0]hexane, 4-methyl-1-(1- 2.8749 C10H16 927 mainlib 315379 51.245 307.5 136.1252 methylethyl)-, didehydro deriv. 4 1R-à-Pinene 2.3559 C10H16 734 replib 198770 32.297 315.5 136.1252 2 Benzene, 1,3-dimethyl- 1.3981 C8H10 954 mainlib 128081 20.811 261.8 106.0783

Table A25: Volatile compounds from F2 hexane crude extract

Peak # Name Area % Formula Similarity Library Height Quant S/N R.T. Exact (s) Mass 21 Nonadecane 17.487 C19H40 911 replib 49346570 7600.5 1002.6 268.313 22 Octadecane 17.428 C18H38 732 mainlib 49308657 7594.7 1002.9 254.2974

191

15 Hexadecane 15.559 C16H34 951 DATABASE 50259061 7741.1 868.3 226.2661 28 1-Iodo-2-methylundecane 13.389 C12H25I 855 mainlib 46039397 7091.1 1124.7 296.1001 27 Eicosane 13.153 C20H42 936 mainlib 45938673 7075.6 1124.4 282.3287 33 Heneicosane 6.2881 C21H44 931 replib 31756049 4891.2 1235.1 296.3443 12 Tetradecane 6.1541 C14H30 955 replib 37463996 5770.3 717.8 198.2348 35 Triacontane 1.9378 C30H62 911 DATABASE 11740100 1808.2 1336.7 422.4852 1 Octane 0.45541 C8H18 935 DATABASE 3592525 553.33 207 114.1409 9 2-Oxabicyclo[2.2.2]octane, 0.27981 C10H18O 939 DATABASE 1919129 295.59 404.1 154.1358 1,3,3-trimethyl- 29 2,5-di-tert-Butyl-1,4- 0.26052 C14H20O2 788 replib 1537596 236.83 1138.7 220.1463 benzoquinone 32 1-Docosene 0.23796 C22H44 955 replib 1417385 218.31 1231 308.3443 8 Benzene, 1-methyl-2-(1- 0.1885 C10H14 954 mainlib 1233920 190.05 395.9 134.1096 methylethyl)- 24 Nonadecane 0.14732 C19H40 914 replib 787336 121.27 1063 268.313 19 Heptadecane, 2,6-dimethyl- 0.1183 C19H40 904 mainlib 556413 85.7 964.2 268.313 17 Hexadecane, 4-methyl- 0.11408 C17H36 899 mainlib 586683 90.363 906.1 240.2817

4 p-Xylene 0.10065 C8H10 957 replib 488843 75.293 260.9 106.0783 31 Eicosane, 2-methyl- 0.084254 C21H44 769 mainlib 505564 77.868 1155.1 296.3443 6 Bicyclo[3.1.0]hexane, 4- 0.043873 C10H16 915 mainlib 312434 48.122 307 136.1252 methyl-1-(1-methylethyl)-, didehydro deriv. 3 Ethylbenzene 0.043378 C8H10 847 replib 227026 34.967 254 106.0783 25 1,2-Benzenedicarboxylic acid, 0.02674 C16H22O4 912 DATABASE 123962 19.093 1101.5 278.1518 dibutyl ester

Table A26: Volatile compounds from F2 ethyl acetate extract

Peak Name Area % Formula Similarity Library Height Quant R.T. Exact # S/N (s) mass 69 Hexadecane 7.4861 C16H34 939 DATABASE 45006942 6563.4 868.6 226.2661 79 Nonadecane 6.1593 C19H40 852 replib 41277306 6019.5 1002.2 268.313 80 Octadecane 6.1208 C18H38 933 replib 41219265 6011 1002.5 254.2974

192

64 Tetradecane 5.6892 C14H30 951 replib 43751968 6380.4 718.8 198.2348 23 (2R*,3R*)-2,4-dimethyl-1,3-diphenyl-4-nitro- 5.1917 C18H19NO3 713 DATABASE 37348906 5446.6 371.2 297.1365 1-pentanone 22 Benzene, 1,2,3-trimethyl- 5.1914 C9H12 917 DATABASE 37348029 5446.5 370.6 120.0939 21 Decane 5.1649 C10H22 873 DATABASE 37264139 5434.2 370.1 142.1722 56 2,4-Dimethylstyrene 3.8314 C10H12 773 mainlib 44483477 6487 503.7 132.0939 94 Heneicosane 2.7335 C21H44 931 replib 29209918 4259.7 1123.7 296.3443 88 2,5-Piperazinedione, 3,6-bis(2-methylpropyl)- 2.5095 C12H22N2O2 792 mainlib 17761380 2590.1 1098.5 226.1681 14 1,3,5-Cycloheptatriene, 7,7-dimethyl- 2.3741 C9H12 804 mainlib 31294230 4563.6 338.3 120.0939 15 Benzene, 1-ethyl-2-methyl- 2.3722 C9H12 929 DATABASE 31284875 4562.3 338.8 120.0939 60 Dodecane 1.7465 C12H26 936 mainlib 26534152 3869.5 550.9 170.2035 40 Benzene, (2-methylpropyl)- 1.624 C10H14 855 replib 18868602 2751.6 425.3 134.1096 20 Benzene, (1-methyldodecyl)- 1.1291 C19H32 747 mainlib 31314800 4566.6 369 260.2504 35 Benzene, 2-propenyl- 0.94465 C9H10 925 replib 13569239 1978.8 408.5 118.0783 104 3-Benzylhexahydropyrrolo[1,2-a]pyrazine-1,4- 0.76912 C14H16N2O2 844 DATABASE 5494084 801.2 1349 244.1212 dione # 81 Cyclopentanol, 1-[1-(4-morpholinyl)ethyl]- 0.76854 C11H21NO2 662 DATABASE 8706743 1269.7 1004.7 199.1572 100 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)- 0.43435 C15H17Cl2N3O2 878 mainlib 3571506 520.83 1326 341.0698 4-propyl-1,3-dioxolan-2-yl]methyl]- 7 p-Xylene 0.37421 C8H10 958 replib 4055709 591.44 261.6 106.0783 85 3,6-Diisopropylpiperazin-2,5-dione 0.34264 C10H18N2O2 652 mainlib 1839986 268.33 1044.5 198.1368 84 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 0.34019 C11H18N2O2 753 mainlib 1836600 267.83 1043.2 210.1368 (2-methylpropyl)- 8 1-Pentanol, 2,2-dimethyl- 0.32891 C7H16O 772 DATABASE 4858724 708.55 273.7 116.1201 76 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 0.29288 C7H10N2O2 868 mainlib 1683438 245.5 994.6 154.0742 37 Benzene, 1-propynyl- 0.29231 C9H8 927 DATABASE 4121625 601.06 417.4 116.0626 65 7-Tetradecene, (Z)- 0.28719 C14H28 843 replib 3852534 561.82 721 196.2191 34 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.2704 C10H18O 922 DATABASE 4069857 593.51 404.7 154.1358 73 dl-Alanyl-l-leucine 0.20306 C9H18N2O3 829 mainlib 1132316 165.13 975.9 202.1317 1 1,3,5-Trimethylhexahydro-1,3,5-triazine 0.11641 C6H17N3 752 DATABASE 694854 101.33 202.7 131.1422 2 Cyclopentane, 1-ethyl-3-methyl-, cis- 0.11462 C8H16 780 mainlib 681891 99.44 203.7 112.1252 51 Benzene, 1,2,3,4-tetramethyl- 0.10833 C10H14 927 mainlib 1445677 210.82 481.1 134.1096

193

72 2-Propenal, 3-(1-aziridinyl)-3- 0.10789 C7H12N2O 673 mainlib 919170 134.04 958.4 140.095 (dimethylamino)- 6 Ethylbenzene 0.10736 C8H10 957 replib 1582900 230.83 254.8 106.0783 99 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)- 0.10533 C15H17Cl2N3O2 886 mainlib 1146604 167.21 1319.1 341.0698 4-propyl-1,3-dioxolan-2-yl]methyl]- 3 Butanoic acid, 3-methyl- 0.1043 C5H10O2 797 DATABASE 781567 113.98 235.5 102.0681 4 2-Propanol, 1-propoxy- 0.10316 C6H14O2 879 DATABASE 778123 113.47 236.5 118.0994 9 Benzene, 1,3-dimethyl- 0.10231 C8H10 950 replib 1438478 209.77 280.5 106.0783 114 1,2-Cyclohexanedicarboxylic acid, dinonyl 0.099725 C26H48O4 794 mainlib 600799 87.615 1521.8 424.3553 ester 71 Hexadecane 0.097113 C16H34 926 replib 1101368 160.61 935.4 226.2661 70 Hexadecane, 4-methyl- 0.090432 C17H36 923 mainlib 1190454 173.6 906.3 240.2817 24 Benzene, 2-propenyl- 0.08388 C9H10 906 replib 763372 111.32 374.5 118.0783 17 3-Pentanone, 2,2,4,4-tetramethyl- 0.082886 C9H18O 927 DATABASE 1708202 249.11 343.2 142.1358 102 Hexanedioic acid, bis(2-ethylhexyl) ester 0.079401 C22H42O4 906 replib 650272 94.829 1332.2 370.3083 106 Octadecane, 2-methyl- 0.079369 C19H40 911 mainlib 709098 103.41 1430.2 268.313 109 1,2-Cyclohexanedicarboxylic acid, 0.078554 C24H42O4 770 mainlib 579701 84.538 1489 394.3083 cyclohexylmethyl nonyl ester 62 Tridecane 0.074604 C13H28 935 DATABASE 1088812 158.78 636 184.2191 66 Tetradecane, 4-methyl- 0.072743 C15H32 901 mainlib 971774 141.71 761.8 212.2504 96 2,5-Piperazinedione, 3-(phenylmethyl)- 0.063937 C11H12N2O2 812 mainlib 331041 48.276 1248.4 204.0899 111 1,2-Cyclohexanedicarboxylic acid, 0.063097 C24H42O4 756 mainlib 453076 66.072 1502.4 394.3083 cyclohexylmethyl nonyl ester 83 Octadecane, 4-methyl- 0.063033 C19H40 919 mainlib 844484 123.15 1036.7 268.313 112 1,2-Cyclohexanedicarboxylic acid, dinonyl 0.060025 C26H48O4 763 mainlib 632223 92.197 1509.5 424.3553 ester 25 Pyrazine, trimethyl- 0.05695 C7H10N2 828 replib 472286 68.874 377.2 122.0844 10 Nonane 0.052187 C9H20 910 DATABASE 870563 126.95 282.2 128.1565 32 Benzene, 2-propenyl- 0.050831 C9H10 876 replib 602968 87.931 399.1 118.0783 110 1,2-Cyclohexanedicarboxylic acid, 0.049842 C24H42O4 741 mainlib 433434 63.208 1498.5 394.3083 cyclohexylmethyl nonyl ester 75 Heptadecane, 3-methyl- 0.04975 C18H38 856 replib 704190 102.69 982.2 254.2974 33 Cyclohexene, 1-methyl-4-(1-methylethenyl)-, 0.035903 C10H16 904 replib 454652 66.302 401 136.1252 (S)-

194

12 1,3-Cyclohexadiene, 2-methyl-5-(1- 0.029581 C10H16 910 DATABASE 368903 53.797 307.5 136.1252 methylethyl)- 57 Benzene, 1-methyl-4-(2-propenyl)- 0.028276 C10H12 920 mainlib 422231 61.574 512.5 132.0939 59 Azulene 0.026581 C10H8 929 DATABASE 881759 128.59 549.2 128.0626 105 1,2-Benzenedicarboxylic acid, diisooctyl ester 0.024748 C24H38O4 830 replib 257772 37.591 1403.6 390.277 90 Dibutyl phthalate 0.021931 C16H22O4 898 mainlib 735072 107.2 1102.2 278.1518 47 Benzene, 1-butenyl-, (E)- 0.014557 C10H12 893 mainlib 200017 29.169 454.5 132.0939 78 (Z)-2-Fluoro-3-methyl-2,6-heptadienal 3.14E-05 C8H11FO 831 DATABASE 3281.4 0.47853 999.2 142.0794

Table A27: Volatile compounds from F2 DCM crude extract

Peak Name Area % Formula Similarity Library Height Quant R.T. (s) Exact # S/N Mass 28 2,5-Piperazinedione, 3,6-bis(2-methylpropyl)- 15.586 C12H22N2O2 70 mainlib 23775133 3633.1 1101.2 226.1681 30 5H,10H-Dipyrrolo[1,2-a:1',2'-d]pyrazine-5,10- 5.2266 C10H14N2O2 737 DATABASE 10638547 1625.7 1104.7 194.1055 dione, octahydro-, (5as-cis)- 38 3-Benzylhexahydropyrrolo[1,2-a]pyrazine-1,4- 4.5764 C14H16N2O2 845 DATABASE 7729671 1181.2 1349.5 244.1212 dione 25 3,6-Diisopropylpiperazin-2,5-dione 3.5121 C10H18N2O2 688 mainlib 5922408 905.02 1046.7 198.1368 21 Methyl (3s*,4s*)-2,3,4,5-tetrahydro-4-methyl-1,5- 3.35 C13H13NO4 689 DATABASE 3483848 532.38 1001.1 247.0845 dioxo-1h-benz[c]azepine-3-carboxylate 15 1-Dodecanol 1.4863 C12H26O 683 DATABASE 2352906 359.55 961.1 186.1984 17 dl-Alanyl-l-leucine 0.75441 C9H18N2O3 879 mainlib 933815 142.7 980 202.1317 1 1,3,5-Trimethylhexahydro-1,3,5-triazine 0.72004 C6H17N3 838 DATABASE 768688 117.47 203.1 131.1422 11 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.42982 C10H18O 932 DATABASE 1529083 233.66 404.5 154.1358 22 Cycloglycylserine 0.40152 C5H8N2O3 717 DATABASE 989768 151.25 1001.9 144.0535 10 Benzene, 1-methyl-2-(1-methylethyl)- 0.33214 C10H14 942 mainlib 790212 120.75 396.7 134.1096 35 3-Benzyl-6-isopropyl-2,5-piperazinedione # 0.26587 C14H18N2O2 777 DATABASE 574198 87.745 1286.1 246.1368 33 3-Benzyl-6-methyl-2,5-piperazinedione # 0.25246 C12H14N2O2 864 DATABASE 439204 67.116 1228.9 218.1055 16 3-Dodecene, (E)- 0.24947 C12H24 677 mainlib 736845 112.6 977.6 168.1878 14 Hexadecane 0.11349 C16H34 932 replib 410158 62.677 867.1 226.2661 37 Hexanedioic acid, bis(2-ethylhexyl) ester 0.10805 C22H42O4 918 DATABASE 345337 52.772 1332.5 370.3083

195

3 Bicyclo[3.1.0]hexane, 4-methyl-1-(1-methylethyl)-, 0.067995 C10H16 913 DATABASE 267617 40.895 307.6 136.1252 didehydro deriv. 7 Benzene, 1-ethyl-2-methyl- 0.062937 C9H12 915 DATABASE 182980 27.962 354.8 120.0939

Table A28: Volatile compounds from F3 hexane crude extract

Peak Name Area % Formula Similarity Library Height Quant R.T. (s) Exact # S/N Mass 33 Octadecane 9.7716 C18H38 950 replib 53892541 13330 1005.4 254.2974 52 Eicosane 9.1786 C20H42 931 replib 50241095 12427 1126.7 282.3287 20 Hexadecane 9.1451 C16H34 947 DATABASE 54770890 13548 870.6 226.2661 21 Pentadecane 9.0465 C15H32 763 DATABASE 54528738 13488 871.3 212.2504 62 Docosane 7.4244 C22H46 860 replib 43095610 10660 1236.5 310.36 10 Tetradecane 5.0797 C14H30 931 DATABASE 49186984 12166 719.2 198.2348 70 Eicosane, 2-methyl- 3.659 C21H44 835 DATABASE 37393453 9249.3 1338 296.3443 69 Heneicosane 3.5811 C21H44 921 replib 37307863 9228.1 1337.8 296.3443 50 Nonadecane 1.4873 C19H40 910 replib 38690720 9570.1 1123.8 268.313 74 Triacontane 1.3773 C30H62 922 DATABASE 21812883 5395.4 1431.5 422.4852 34 Cyclotetradecane 1.263 C14H28 829 mainlib 29187877 7219.6 1006.4 196.2191 49 1-Docosene 0.96524 C22H44 954 replib 17865692 4419.1 1120.1 308.3443 22 Cyclooctane, methyl- 0.90832 C9H18 825 mainlib 22974391 5682.7 871.8 126.1409 19 1-Hexadecene 0.69605 C16H32 931 replib 11822445 2924.3 861.9 224.2504 77 Sulfurous acid, octadecyl pentyl ester 0.67246 C23H48O3S 760 mainlib 10715800 2650.5 1882.9 404.3324 53 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene- 0.56928 C17H24O3 784 replib 13869747 3430.7 1140.1 276.1725 2,8-dione 11 7-Tetradecene, (Z)- 0.19993 C14H28 817 replib 5228874 1293.4 721.2 196.2191 27 Heptadecane, 2,6-dimethyl- 0.17033 C19H40 900 mainlib 2533639 626.69 964.5 268.313 24 Hexadecane, 4-methyl- 0.14404 C17H36 924 mainlib 2751084 680.48 906.3 240.2817 8 Tridecane 0.12391 C13H28 932 DATABASE 2541336 628.6 636 184.2191 29 Heptadecane, 3-methyl- 0.12149 C18H38 889 replib 1936288 478.94 982.4 254.2974 72 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) 0.081616 C16H22O4 856 mainlib 886275 219.22 1403.6 278.1518 ester

196

18 Pentadecane, 3-methyl- 0.077283 C16H34 889 DATABASE 1559422 385.72 845.6 226.2661 12 Tetradecane, 4-methyl- 0.076496 C15H32 883 mainlib 1461987 361.62 761.7 212.2504 58 Eicosane, 2,4-dimethyl- 0.067454 C22H46 880 mainlib 967416 239.29 1206.6 310.36 3 Benzene, 1-methyl-2-(1-methylethyl)- 0.062551 C10H14 949 mainlib 1211733 299.72 395.7 134.1096 44 Decane, 2,3,5,8-tetramethyl- 0.061565 C14H30 864 mainlib 815370 201.68 1095.6 198.2348 4 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.052862 C10H18O 923 DATABASE 1089654 269.53 403.9 154.1358 41 Undecane, 2,3-dimethyl- 0.051222 C13H28 860 mainlib 1045533 258.61 1087.1 184.2191 15 1-Iodo-2-methylundecane 0.048833 C12H25I 892 mainlib 845625 209.17 818.9 296.1001 36 1-Docosene 0.04064 C22H44 889 replib 769970 190.45 1013.1 308.3443 45 Dibutyl phthalate 0.040534 C16H22O4 921 mainlib 572604 141.63 1101.5 278.1518 2 p-Xylene 0.031911 C8H10 915 replib 492310 121.77 260 26 Benzene, (1-methyldecyl)- 0.031144 C17H28 889 mainlib 349560 86.464 941.1 232.2191 38 Phthalic acid, butyl undecyl ester 0.026692 C23H36O4 765 mainlib 383178 94.779 1043.2 376.2614 63 Hexadecane, 5-butyl- 0.025009 C20H42 870 mainlib 455286 112.61 1254.1 282.3287 47 1-Octene, 3-ethyl- 0.013346 C10H20 820 DATABASE 233309 57.709 1115.7 140.1565 48 2,5-di-tert-Butyl-1,4-benzoquinone 0.013346 C14H20O2 696 replib 233309 57.709 1116.9 220.1463

Table A29: Volatile compounds from F3 ethyl acetate crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. Exact S/N (s) Mass 19 Hexadecane 23.044 C16H34 938 replib 44037908 6481.5 868 226.2661 28 Octadecane 18.919 C18H38 929 replib 40894588 6018.9 1002.2 254.2974 14 Tetradecane 11.344 C14H30 953 replib 35855671 5277.3 717.9 198.2348 39 Heneicosane 8.9275 C21H44 921 replib 27006751 3974.9 1123.4 296.3443 33 2,5-Piperazinedione, 3,6-bis(2-methylpropyl)- 6.6055 C12H22N2O2 701 mainlib 12862187 1893.1 1096.7 226.1681 41 Triacontane 2.1208 C30H62 922 DATABASE 7604456 1119.2 1234.1 422.4852 50 3-Benzylhexahydropyrrolo[1,2-a]pyrazine-1,4- 1.8181 C14H16N2O2 849 DATABASE 4016124 591.1 1347.9 244.1212 dione # 36 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 1.4044 C11H18N2O2 885 mainlib 2890070 425.36 1102.9 210.1368 (2-methylpropyl)-

197

46 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)-4- 1.2237 C15H17Cl2N3O2 876 mainlib 2918145 429.5 1325.4 341.0698 propyl-1,3-dioxolan-2-yl]methyl]- 27 1-Nonadecene 1.1958 C19H38 912 replib 4168577 613.53 996.8 266.2974 35 Dibutyl phthalate 0.96218 C16H22O4 800 mainlib 2413295 355.19 1101.9 278.1518 18 1-Hexadecene 0.90061 C16H32 915 replib 2706569 398.36 861.5 224.2504 26 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 0.78429 C7H10N2O2 917 mainlib 1384339 203.75 992.8 154.0742 11 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.55977 C10H18O 932 DATABASE 1754518 258.23 404.5 154.1358 10 Benzene, 1-methyl-2-(1-methylethyl)- 0.44639 C10H14 940 mainlib 949263 139.71 397 134.1096 8 Benzene, 1,2,4-trimethyl- 0.44334 C9H12 945 mainlib 1183373 174.17 369.3 120.0939 23 dl-Alanyl-l-leucine 0.3195 C9H18N2O3 832 mainlib 612201 90.104 972.8 202.1317 22 Cyclododecane 0.27135 C12H24 689 DATABASE 603583 88.836 957.8 168.1878 20 Hexadecane, 4-methyl- 0.2523 C17H36 905 mainlib 911037 134.09 906.2 240.2817 48 Hexanedioic acid, bis(2-ethylhexyl) ester 0.24988 C22H42O4 911 DATABASE 569619 83.837 1331.9 370.3083 55 1,2-Cyclohexanedicarboxylic acid, 0.2331 C24H42O4 771 mainlib 490959 72.26 1502.2 394.3083 cyclohexylmethyl nonyl ester 40 2,5-Piperazinedione, 3-methyl-6-(phenylmethyl)- 0.20517 C12H14N2O2 843 replib 397770 58.544 1227.3 218.1055 56 1,2-Cyclohexanedicarboxylic acid, dinonyl ester 0.19338 C26H48O4 772 mainlib 533918 78.583 1509.5 424.3553 30 Octadecane, 4-methyl- 0.18354 C19H40 905 mainlib 705552 103.84 1036.6 268.313 15 Tetradecane, 4-methyl- 0.147 C15H32 886 mainlib 559569 82.358 761.7 212.2504 6 Benzene, 1,2,3-trimethyl- 0.12243 C9H12 910 replib 300830 44.276 346 120.0939 25 Heptadecane, 3-methyl- 0.095027 C18H38 856 replib 359496 52.911 982.1 254.2974 7 Benzene, 1-ethyl-4-methyl- 0.091813 C9H12 914 replib 251107 36.958 354.8 120.0939 3 Bicyclo[3.1.0]hexane, 4-methyl-1-(1- 0.086641 C10H16 919 mainlib 318289 46.846 307.6 136.1252 methylethyl)-, didehydro deriv. 4 Benzene, 1-ethyl-2-methyl- 0.069754 C9H12 941 DATABASE 247225 36.387 338.7 120.0939 51 1,2-Benzenedicarboxylic acid, diisooctyl ester 0.049627 C24H38O4 900 replib 140269 20.645 1403.6 390.277 1 1,3,5-Trimethylhexahydro-1,3,5-triazine 0.001633 C6H17N3 790 DATABASE 43207 6.3593 203.1 131.1422

Table A30: Volatile compounds from F3 DCM crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. (s) Exact S/N Mass

198

12 2,5-Piperazinedione, 3,6-bis(2-methylpropyl)- 19.769 C12H22N2O2 738 mainlib 21715897 3389.6 1098.8 226.1681 8 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- 12.408 C11H18N2O2 734 mainlib 13985442 2183 1028.1 210.1368 methylpropyl)- 15 Hydroperoxide, heptyl 7.0421 C7H16O2 709 mainlib 10826025 1689.8 1104.2 132.115 14 5H,10H-dipyrrolo[1,2-a:1',2'-d]pyrazine-5,10-dione, 6.8547 C10H14N2O2 808 DATABASE 10652193 1662.7 1104 194.1055 octahydro-, (5as-cis)- 7 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 3.591 C7H10N2O2 889 mainlib 4425469 690.77 996.6 154.0742 19 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 2.0383 C14H16N2O2 832 mainlib 3051117 476.25 1326.7 244.1212 (phenylmethyl)- 3 2-Propenal, 3-(1-aziridinyl)-3-(dimethylamino)- 1.3421 C7H12N2O 678 mainlib 1638548 255.76 959.3 140.095 4 dl-Alanyl-l-leucine 0.65149 C9H18N2O3 833 mainlib 702218 109.61 975.4 202.1317 18 3-Benzyl-6-isopropyl-2,5-piperazinedione # 0.20786 C14H18N2O2 727 DATABASE 359995 56.192 1284.7 246.1368 17 3-Benzyl-6-methyl-2,5-piperazinedione 0.17177 C12H14N2O2 852 DATABASE 279317 43.599 1227.4 218.1055 2 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.16392 C10H18O 927 DATABASE 371852 58.042 404.2 154.1358 20 Hexanedioic acid, bis(2-ethylhexyl) ester 0.16261 C22H42O4 755 DATABASE 296010 46.204 1331.7 370.3083 1 4-Penten-2-ol 0.12537 C5H10O 807 DATABASE 291098 45.438 216.7 86.0732

Table A31: Volatile compounds from F4 hexane crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. (s) Exact S/N Mass 32 Octadecane 15.782 C18H38 949 replib 52475741 7479 1004.5 254.2974 50 Eicosane 14.706 C20H42 925 replib 48640086 6932.3 1126.2 282.3287 14 Hexadecane 12.748 C16H34 943 replib 51137787 7288.3 869.2 226.2661 61 Oxalic acid, allyl dodecyl ester 10.052 C17H30O4 853 mainlib 42592184 6070.4 1237.1 298.2144 60 Pentacosane 9.8639 C25H52 921 DATABASE 42477411 6054 1236.3 352.4069 69 Heptacosane 4.2789 C27H56 933 replib 30740661 4381.3 1337.6 380.4382 49 Heneicosane 3.7305 C21H44 907 replib 42316498 6031.1 1124.4 296.3443 15 Butyl 2,4-dimethyl-2-nitro-4-pentenoate 2.1803 C11H19NO4 675 DATABASE 23158802 3300.7 870.8 229.1314 16 7-Hexadecene, (z)- 1.9013 C16H32 706 DATABASE 21146042 3013.8 871 224.2504 76 Triacontane 1.6261 C30H62 922 DATABASE 18856132 2687.4 1431.1 422.4852 31 1-Nonadecene 1.2725 C19H38 948 replib 13953600 1988.7 997.4 266.2974

199

48 1-Docosene 0.8575 C22H44 956 replib 10509913 1497.9 1120.1 308.3443 10 Tetradecane 0.57963 C14H30 945 replib 8082061 1151.9 717.4 198.2348 40 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene- 0.46256 C17H24O3 934 replib 5787416 824.84 1075.3 276.1725 2,8-dione 39 Nonadecane 0.2327 C19H40 928 replib 2319764 330.62 1063.4 268.313 1 Octane 0.17274 C8H18 949 DATABASE 2384411 339.83 206 114.1409 52 Eicosane, 2-methyl- 0.16525 C21H44 877 mainlib 1568194 223.5 1155.4 296.3443 36 Octadecane, 4-methyl- 0.16154 C19H40 926 mainlib 1778294 253.45 1036.8 268.313 20 Hexadecane, 4-methyl- 0.14337 C17H36 915 mainlib 1639161 233.62 906.3 240.2817 29 Heptadecane, 3-methyl- 0.14226 C18H38 910 replib 1447923 206.36 982.3 254.2974 30 Benzene, (1-ethyldecyl)- 0.14226 C18H30 890 replib 1447923 206.36 982.8 246.2348 53 Triacontane 0.13492 C30H62 908 DATABASE 1261043 179.73 1179.4 422.4852 42 Octadecane, 2,6-dimethyl- 0.12576 C20H42 890 mainlib 1090594 155.43 1088.8 282.3287 28 Tetradecane, 4-ethyl- 0.12161 C16H34 869 mainlib 1054741 150.32 968.7 226.2661 55 Sulfurous acid, decyl 2-ethylhexyl ester 0.11455 C18H38O3S 867 mainlib 888860 126.68 1202.3 334.2542 7 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.10854 C10H18O 932 DATABASE 1235028 176.02 403.9 154.1358 74 Phthalic acid, cyclohexyl 2-pentyl ester 0.10288 C19H26O4 716 mainlib 704734 100.44 1403.5 318.1831 6 Benzene, 1-methyl-2-(1-methylethyl)- 0.10055 C10H14 950 mainlib 1084295 154.54 395.7 134.1096 35 2,3-Dimethyldecane 0.067466 C12H26 770 mainlib 449942 64.127 1027 170.2035 38 2,5-Cyclohexadiene-1,4-dione, 2,6-bis(1,1- 0.066631 C14H20O2 748 mainlib 565300 80.568 1045.8 220.1463 dimethylethyl)- 41 Undecane, 2,3-dimethyl- 0.064457 C13H28 865 mainlib 770491 109.81 1087 184.2191 23 Benzene, (1-methyldecyl)- 0.062112 C17H28 908 DATABASE 513882 73.24 941.1 232.2191 3 p-Xylene 0.06195 C8H10 956 replib 580097 82.677 260 106.0783 45 1,2-Benzenedicarboxylic acid, bis(2- 0.057261 C14H18O6 933 DATABASE 574494 81.879 1101.6 282.1103 methoxyethyl) ester 34 1-Decanol, 2-hexyl- 0.047454 C16H34O 896 replib 580057 82.671 1012.9 242.261 21 Benzene, (1-ethylnonyl)- 0.041411 C17H28 858 DATABASE 317961 45.317 914.9 232.2191 70 Docosane, 11-decyl- 0.03965 C32H66 833 DATABASE 345999 49.313 1354.3 450.5165 17 7-Hexadecene, (z)- 0.031712 C16H32 880 DATABASE 436615 62.228 878.5 224.2504 25 Benzene, (1-butyloctyl)- 0.030839 C18H30 843 replib 270345 38.53 958.5 246.2348

200

37 Phthalic acid, butyl undecyl ester 0.029379 C23H36O4 806 mainlib 275654 39.287 1043.2 376.2614 2 Ethylbenzene 0.023994 C8H10 897 replib 262812 37.457 253.1 106.0783 5 1,3-Cyclohexadiene, 2-methyl-5-(1- 0.021634 C10H16 780 DATABASE 283608 40.421 306.5 136.1252 methylethyl)- 19 Benzene, (1-propyloctyl)- 0.018533 C17H28 897 DATABASE 220863 31.478 899.1 232.2191 24 Benzene, (1-pentylheptyl)- 0.018463 C18H30 774 DATABASE 228513 32.568 955.4 246.2348 47 Tridecane, 3-methylene- 0.016741 C14H28 848 mainlib 172441 24.577 1115.7 196.2191 27 Benzene, (1-propylnonyl)- 0.013243 C18H30 901 replib 314032 44.757 967.3 246.2348

Table A32: Volatile compounds from F4 ethyl acetate broth crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. (s) Exact S/N Mass 69 Hexadecane 7.4861 C16H34 939 DATABASE 45006942 6563.4 868.6 226.2661 79 Nonadecane 6.1593 C19H40 852 replib 41277306 6019.5 1002.2 268.313 80 Octadecane 6.1208 C18H38 933 replib 41219265 6011 1002.5 254.2974 64 Tetradecane 5.6892 C14H30 951 replib 43751968 6380.4 718.8 198.2348 23 (2R*,3R*)-2,4-dimethyl-1,3-diphenyl-4-nitro-1- 5.1917 C18H19NO3 713 DATABASE 37348906 5446.6 371.2 297.1365 pentanone 22 Benzene, 1,2,3-trimethyl- 5.1914 C9H12 917 DATABASE 37348029 5446.5 370.6 120.0939 21 Decane 5.1649 C10H22 873 DATABASE 37264139 5434.2 370.1 142.1722 56 2,4-Dimethylstyrene 3.8314 C10H12 773 mainlib 44483477 6487 503.7 132.0939 94 Heneicosane 2.7335 C21H44 931 replib 29209918 4259.7 1123.7 296.3443 88 2,5-Piperazinedione, 3,6-bis(2-methylpropyl)- 2.5095 C12H22N2O2 792 mainlib 17761380 2590.1 1098.5 226.1681 60 Dodecane 1.7465 C12H26 936 mainlib 26534152 3869.5 550.9 170.2035 40 Benzene, (2-methylpropyl)- 1.624 C10H14 855 replib 18868602 2751.6 425.3 134.1096 20 Benzene, (1-methyldodecyl)- 1.1291 C19H32 747 mainlib 31314800 4566.6 369 260.2504 35 Benzene, 2-propenyl- 0.94465 C9H10 925 replib 13569239 1978.8 408.5 118.0783 81 Cyclopentanol, 1-[1-(4-morpholinyl)ethyl]- 0.76854 C11H21NO2 662 DATABASE 8706743 1269.7 1004.7 199.1572 82 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 0.74607 C11H18N2O2 746 mainlib 5715589 833.5 1027 210.1368 (2-methylpropyl)- 49 Undecane 0.59609 C11H24 929 DATABASE 11006870 1605.1 461.2 156.1878

201

46 Benzene, 1-methyl-2-(1-methylethyl)- 0.56144 C10H14 922 mainlib 9226384 1345.5 451.5 134.1096 101 3-Benzylhexahydropyrrolo[1,2-a]pyrazine-1,4- 0.536 C14H16N2O2 792 DATABASE 3813517 556.13 1326.3 244.1212 dione 100 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)- 0.43435 C15H17Cl2N3O2 878 mainlib 3571506 520.83 1326 341.0698 4-propyl-1,3-dioxolan-2-yl]methyl]- 7 p-Xylene 0.37421 C8H10 958 replib 4055709 591.44 261.6 106.0783 85 3,6-Diisopropylpiperazin-2,5-dione 0.34264 C10H18N2O2 652 mainlib 1839986 268.33 1044.5 198.1368 8 1-Pentanol, 2,2-dimethyl- 0.32891 C7H16O 772 DATABASE 4858724 708.55 273.7 116.1201 76 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 0.29288 C7H10N2O2 868 mainlib 1683438 245.5 994.6 154.0742 37 Benzene, 1-propynyl- 0.29231 C9H8 927 DATABASE 4121625 601.06 417.4 116.0626 65 7-Tetradecene, (Z)- 0.28719 C14H28 843 replib 3852534 561.82 721 196.2191 34 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.2704 C10H18O 922 DATABASE 4069857 593.51 404.7 154.1358 13 Benzene, propyl- 0.23562 C9H12 946 replib 3429841 500.17 332.1 120.0939 73 dl-Alanyl-l-leucine 0.20306 C9H18N2O3 829 mainlib 1132316 165.13 975.9 202.1317 1 1,3,5-Trimethylhexahydro-1,3,5-triazine 0.11641 C6H17N3 752 DATABASE 694854 101.33 202.7 131.1422 2 Cyclopentane, 1-ethyl-3-methyl-, cis- 0.11462 C8H16 780 mainlib 681891 99.44 203.7 112.1252 72 2-Propenal, 3-(1-aziridinyl)-3-(dimethylamino)- 0.10789 C7H12N2O 673 mainlib 919170 134.04 958.4 140.095 6 Ethylbenzene 0.10736 C8H10 957 replib 1582900 230.83 254.8 106.0783 3 Butanoic acid, 3-methyl- 0.1043 C5H10O2 797 DATABASE 781567 113.98 235.5 102.0681 4 2-Propanol, 1-propoxy- 0.10316 C6H14O2 879 DATABASE 778123 113.47 236.5 118.0994 9 Benzene, 1,3-dimethyl- 0.10231 C8H10 950 replib 1438478 209.77 280.5 106.0783 114 1,2-Cyclohexanedicarboxylic acid, dinonyl ester 0.099725 C26H48O4 794 mainlib 600799 87.615 1521.8 424.3553 71 Hexadecane 0.097113 C16H34 926 replib 1101368 160.61 935.4 226.2661 70 Hexadecane, 4-methyl- 0.090432 C17H36 923 mainlib 1190454 173.6 906.3 240.2817 24 Benzene, 2-propenyl- 0.08388 C9H10 906 replib 763372 111.32 374.5 118.0783 17 3-Pentanone, 2,2,4,4-tetramethyl- 0.082886 C9H18O 927 DATABASE 1708202 249.11 343.2 142.1358 102 Hexanedioic acid, bis(2-ethylhexyl) ester 0.079401 C22H42O4 906 replib 650272 94.829 1332.2 370.3083 106 Octadecane, 2-methyl- 0.079369 C19H40 911 mainlib 709098 103.41 1430.2 268.313 109 1,2-Cyclohexanedicarboxylic acid, 0.078554 C24H42O4 770 mainlib 579701 84.538 1489 394.3083 cyclohexylmethyl nonyl ester 42 1,2-Diethylbenzene 0.078292 C10H14 831 DATABASE 946696 138.06 427.8 134.1096

202

66 Tetradecane, 4-methyl- 0.072743 C15H32 901 mainlib 971774 141.71 761.8 212.2504 96 2,5-Piperazinedione, 3-(phenylmethyl)- 0.063937 C11H12N2O2 812 mainlib 331041 48.276 1248.4 204.0899 83 Octadecane, 4-methyl- 0.063033 C19H40 919 mainlib 844484 123.15 1036.7 268.313 25 Pyrazine, trimethyl- 0.05695 C7H10N2 828 replib 472286 68.874 377.2 122.0844 50 Benzene, 2-ethyl-1,4-dimethyl- 0.055249 C10H14 928 replib 778965 113.6 470 134.1096 108 1,2-Cyclohexanedicarboxylic acid, 0.053382 C24H42O4 737 mainlib 487073 71.03 1477.4 394.3083 cyclohexylmethyl nonyl ester 32 Benzene, 2-propenyl- 0.050831 C9H10 876 replib 602968 87.931 399.1 118.0783 33 Cyclohexene, 1-methyl-4-(1-methylethenyl)-, 0.035903 C10H16 904 replib 454652 66.302 401 136.1252 (S)- 12 1,3-Cyclohexadiene, 2-methyl-5-(1- 0.029581 C10H16 910 DATABASE 368903 53.797 307.5 136.1252 methylethyl)- 57 Benzene, 1-methyl-4-(2-propenyl)- 0.028276 C10H12 920 mainlib 422231 61.574 512.5 132.0939 59 Azulene 0.026581 C10H8 929 DATABASE 881759 128.59 549.2 128.0626 105 1,2-Benzenedicarboxylic acid, diisooctyl ester 0.024748 C24H38O4 830 replib 257772 37.591 1403.6 390.277 90 Dibutyl phthalate 0.021931 C16H22O4 898 mainlib 735072 107.2 1102.2 278.1518

Table A33: Volatile compounds from F4 DCM crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. (s) Exact S/N Mass 9 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 11.268 C7H10N2O2 914 mainlib 9370126 1432.7 999.2 154.0742 10 Benzene, m-diethoxy- 10.857 C10H14O2 714 DATABASE 9252291 1414.7 999.3 166.0994 14 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- 3.0453 C11H18N2O2 827 mainlib 5321427 813.68 1085.1 210.1368 methylpropyl)- 5 1-Dodecanol 1.8085 C12H26O 682 DATABASE 2052038 313.77 959.5 186.1984 22 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 0.80534 C14H16N2O2 766 mainlib 860454 131.57 1325.4 244.1212 (phenylmethyl)- 23 Hexanedioic acid, bis(2-ethylhexyl) ester 0.38983 C22H42O4 921 DATABASE 782191 119.6 1331.9 370.3083 6 Pyrrolidine, 2,5-dimethyl-1-nitroso- 0.36591 C6H12N2O 655 mainlib 351271 53.711 975.9 128.095 7 dl-Alanyl-l-leucine 0.26284 C9H18N2O3 778 mainlib 231886 35.457 981.5 202.1317 3 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.22402 C10H18O 925 DATABASE 508631 77.773 404.2 154.1358

203

20 Pyrimido[1,2-a]azepine, 2,3,4,6,7,8,9,10- 0.1857 C9H16N2 696 replib 255884 39.126 1128.9 152.1313 octahydro- 17 1,2-Benzenedicarboxylic acid, dibutyl ester 0.15415 C16H22O4 845 DATABASE 1494234 228.48 1102 278.1518 2 Benzene, 1-methyl-2-(1-methylethyl)- 0.12442 C10H14 807 mainlib 294638 45.052 395.9 134.1096

Table A34: Volatile compounds from B1 hexane crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. (s) Exact S/N Mass 38 Octadecane 14.773 C18H38 950 DATABASE 55124121 7963.6 1005.3 254.2974 57 Eicosane 14.037 C20H42 930 replib 51076772 7378.9 1126.6 282.3287 17 Hexadecane 12.442 C16H34 950 DATABASE 53239682 7691.4 870 226.2661 69 Heneicosane 11.463 C21H44 940 replib 44282905 6397.4 1236.8 296.3443 80 1-Iodo-2-methylundecane 5.497 C12H25I 861 mainlib 37461406 5411.9 1338.1 296.1001 56 1-Dodecanol, 2-methyl-, (S)- 3.2794 C13H28O 782 mainlib 41483380 5993 1124.2 200.214 68 Nonadecane 2.4405 C19H40 880 replib 37596364 5431.4 1235.2 268.313 89 Triacontane 2.1901 C30H62 924 DATABASE 23426146 3384.3 1431.6 422.4852 39 7-Tetradecene, (Z)- 1.9807 C14H28 820 replib 30452035 4399.3 1006.3 196.2191 36 1-Nonadecene 1.6636 C19H38 946 replib 19461271 2811.5 997.4 266.2974 19 Cyclohexane, 1,1'-(1,2-dimethyl-1,2- 1.5232 C16H30 691 mainlib 21371242 3087.4 871.3 222.2348 ethanediyl)bis-, (R*,R*)-(ñ)- 55 1-Docosene 1.4623 C22H44 957 replib 17635318 2547.7 1120.1 308.3443 10 Tetradecane 0.99628 C14H30 945 replib 14859926 2146.8 717.4 198.2348 47 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene- 0.77745 C17H24O3 928 replib 10581028 1528.6 1075.5 276.1725 2,8-dione 92 Heptacosane 0.62603 C27H56 923 replib 5343809 772 1518.4 380.4382 43 Octadecane, 4-methyl- 0.20591 C19H40 926 mainlib 2542322 367.28 1036.8 268.313 60 Eicosane, 2-methyl- 0.19791 C21H44 896 mainlib 2061464 297.81 1155.4 296.3443 33 Heptadecane, 3-methyl- 0.19503 C18H38 907 replib 2153234 311.07 982.2 254.2974 34 Benzene, (1-ethyldecyl)- 0.19503 C18H30 874 replib 2153234 311.07 982.7 246.2348 24 Hexadecane, 4-methyl- 0.19284 C17H36 918 mainlib 2639103 381.26 906.2 240.2817 1 Octane 0.19006 C8H18 950 DATABASE 3129478 452.11 205.9 114.1409

204

30 Heptadecane, 2,6-dimethyl- 0.18551 C19H40 905 mainlib 1688244 243.9 964.2 268.313 12 Pentadecane 0.14935 C15H32 926 DATABASE 2135621 308.53 793.8 212.2504 49 Octadecane, 2,6-dimethyl- 0.14296 C20H42 899 mainlib 1578323 228.02 1088.6 282.3287 63 Sulfurous acid, decyl 2-ethylhexyl ester 0.12827 C18H38O3S 864 mainlib 1216018 175.67 1202.3 334.2542 85 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) 0.11586 C16H22O4 826 mainlib 884400 127.77 1403.7 278.1518 ester 77 Hexadecane, 3-methyl- 0.10365 C17H36 873 mainlib 824015 119.04 1322.4 240.2817 7 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.10175 C10H18O 933 DATABASE 1400190 202.28 403.8 154.1358 27 Benzene, (1-methyldecyl)- 0.094402 C17H28 904 DATABASE 852638 123.18 941.1 232.2191 6 Benzene, 1-methyl-2-(1-methylethyl)- 0.093506 C10H14 949 mainlib 1222465 176.61 395.6 134.1096 48 Tridecane, 3-methyl- 0.088445 C14H30 875 replib 1203965 173.93 1087 198.2348 3 p-Xylene 0.083164 C8H10 958 replib 801825 115.84 260 106.0783 45 2,5-Cyclohexadiene-1,4-dione, 2,6-bis(1,1- 0.079142 C14H20O2 761 mainlib 795614 114.94 1045.6 220.1463 dimethylethyl)- 52 Dibutyl phthalate 0.076083 C16H22O4 930 mainlib 780286 112.73 1101.5 278.1518 42 2,3-Dimethyldecane 0.075555 C12H26 868 mainlib 562931 81.325 1027 170.2035 87 Docosane, 11-decyl- 0.072436 C32H66 854 DATABASE 612150 88.435 1417.9 450.5165 76 Sulfurous acid, hexyl pentadecyl ester 0.049024 C21H44O3S 864 mainlib 635566 91.818 1310.7 376.3011 59 2,3-Dimethyldecane 0.047593 C12H26 847 mainlib 569137 82.221 1145.6 170.2035 20 7-Hexadecene, (z)- 0.038065 C16H32 922 DATABASE 567725 82.017 878.5 224.2504 25 Benzene, (1-ethylnonyl)- 0.038 C17H28 889 DATABASE 421453 60.886 914.7 232.2191 44 Phthalic acid, butyl undecyl ester 0.035599 C23H36O4 779 mainlib 389846 56.32 1043.1 376.2614 2 Ethylbenzene 0.034848 C8H10 897 replib 382736 55.293 253.1 106.0783 58 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene- 0.034397 C17H24O3 763 replib 490381 70.844 1138.5 276.1725 2,8-dione 23 Benzene, (1-propyloctyl)- 0.028705 C17H28 869 DATABASE 374233 54.064 899.1 232.2191 5 Bicyclo[3.1.0]hexane, 4-methyl-1-(1-methylethyl)-, 0.026666 C10H16 892 DATABASE 321425 46.435 306.4 136.1252 didehydro deriv. 31 Benzene, (1-propylnonyl)- 0.025229 C18H30 896 replib 565619 81.713 967.2 246.2348 54 Tridecane, 3-methylene- 0.019207 C14H28 836 mainlib 230749 33.336 1115.7 196.2191 66 Undecane, 3-methylene- 0.018202 C12H24 838 mainlib 253346 36.6 1228 168.1878 35 Octane, 3-methyl-6-methylene- 0.014971 C10H20 687 DATABASE 250959 36.255 992.4 140.1565

205

40 Benzene, (1-methylundecyl)- 0.000621 C18H30 878 DATABASE 33555 4.8476 1009.5 246.2348

Table A35: Volatile compounds from B1 ethyl acetate crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. Exact S/N (s) Mass 22 Octadecane 31.611 C18H38 928 replib 39432848 5882 1002.1 254.2974 15 Hexadecane 21.484 C16H34 943 replib 34914729 5208.1 867.4 226.2661 31 Heneicosane 15.934 C21H44 923 Replib 27525307 4105.8 1123.4 296.3443 34 Triacontane 3.7567 C30H62 917 DATABASE 8519424 1270.8 1234.2 422.4852 37 3-Benzylhexahydropyrrolo[1,2-a]pyrazine-1,4- 1.4791 C14H16N2O2 819 DATABASE 1864378 278.1 1324.3 244.1212 dione 21 1-Nonadecene 1.4675 C19H38 939 Replib 2754360 410.86 996.6 266.2974 38 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)-4- 1.2682 C15H17Cl2N3O2 875 mainlib 1792291 267.35 1325.3 341.0698 propyl-1,3-dioxolan-2-yl]methyl]- 20 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 1.2637 C7H10N2O2 914 mainlib 1383460 206.36 991.8 154.0742 10 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.83215 C10H18O 933 DATABASE 1765803 263.4 404.4 154.1358 23 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- 0.77105 C11H18N2O2 738 mainlib 1175663 175.37 1024.1 210.1368 methylpropyl)- 7 Benzene, 1,2,4-trimethyl- 0.73142 C9H12 947 mainlib 1188728 177.32 369.1 120.0939 30 1-Docosene 0.70393 C22H44 947 replib 1495381 223.06 1119.1 308.3443 9 Benzene, 1-methyl-2-(1-methylethyl)- 0.6817 C10H14 937 mainlib 753929 112.46 396.7 134.1096 36 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)-4- 0.57208 C15H17Cl2N3O2 886 mainlib 1085926 161.98 1318.4 341.0698 propyl-1,3-dioxolan-2-yl]methyl]- 39 Hexanedioic acid, bis(2-ethylhexyl) ester 0.51563 C22H42O4 891 DATABASE 605949 90.387 1331.8 370.3083 46 1,2-Cyclohexanedicarboxylic acid, 0.50716 C24H42O4 756 mainlib 518246 77.305 1488.7 394.3083 cyclohexylmethyl nonyl ester 14 7-Hexadecene, (Z)- 0.47374 C16H32 917 mainlib 856879 127.82 861.2 224.2504 43 Octadecane, 2-methyl- 0.42198 C19H40 908 mainlib 778867 116.18 1430.2 268.313 52 1,2-Cyclohexanedicarboxylic acid, dinonyl ester 0.3715 C26H48O4 726 mainlib 463644 69.16 1521.8 424.3553 32 Eicosane, 2-methyl- 0.3593 C21H44 883 mainlib 518599 77.357 1154.9 296.3443 24 Octadecane, 4-methyl- 0.34944 C19H40 894 mainlib 651386 97.165 1036.5 268.313 13 Tetradecane 0.31011 C14H30 932 replib 645001 96.212 717.3 198.2348

206

1 1,3,5-Trimethylhexahydro-1,3,5-triazine 0.30335 C6H17N3 851 DATABASE 507305 75.673 202.2 131.1422 18 1-Dodecanol 0.22473 C12H26O 693 DATABASE 310395 46.3 957.3 186.1984 5 Benzene, 1,2,3-trimethyl- 0.20804 C9H12 939 replib 319949 47.725 345.9 120.0939 2 Bicyclo[3.1.0]hexane, 4-methyl-1-(1-methylethyl)-, 0.12641 C10H16 915 mainlib 287647 42.907 307.5 136.1252 didehydro deriv. 28 Dibutyl phthalate 0.097059 C16H22O4 901 mainlib 430298 64.186 1101.5 278.1518 42 1,2-Benzenedicarboxylic acid, diisooctyl ester 0.083442 C24H38O4 922 replib 182349 27.2 1403.4 390.277

Table A36: Volatile compounds from B1 DCM crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. Exact S/N (s) Mass 21 Docosane 25.066 C22H46 705 DATABASE 10678403 1619.1 1095.8 310.36 22 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- 25.066 C11H18N2O2 887 mainlib 10678403 1619.1 1096.9 210.1368 methylpropyl)- 16 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 8.5071 C7H10N2O2 914 mainlib 2877585 436.3 994.8 154.0742 24 5H,10H-dipyrrolo[1,2-a:1',2'-d]pyrazine-5,10- 5.5095 C10H14N2O2 815 DATABASE 2625860 398.14 1103.8 194.1055 dione, octahydro-, (5as-cis)- 28 3-Benzylhexahydropyrrolo[1,2-a]pyrazine-1,4- 4.8572 C14H16N2O2 852 DATABASE 1985415 301.03 1347 244.1212 dione 15 2-Propenal, 3-(1-aziridinyl)-3-(dimethylamino)- 2.1767 C7H12N2O 681 mainlib 954483 144.72 958.5 140.095 11 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 2.1705 C10H18O 930 DATABASE 1581017 239.72 404.5 154.1358 8 Benzene, 1,2,3-trimethyl- 2.0655 C9H12 937 DATABASE 1110612 168.39 369.2 120.0939 10 Benzene, 1-methyl-2-(1-methylethyl)- 1.799 C10H14 936 mainlib 671494 101.81 396.8 134.1096 17 Nonadecane 0.8387 C19H40 879 replib 487241 73.876 1001 268.313 27 Hexanedioic acid, bis(2-ethylhexyl) ester 0.68944 C22H42O4 909 DATABASE 457220 69.324 1331.9 370.3083 25 Eicosane 0.53094 C20H42 903 replib 313959 47.603 1122.5 282.3287 3 1,3-Cyclohexadiene, 2-methyl-5-(1-methylethyl)- 0.31796 C10H16 915 DATABASE 283209 42.941 307.5 136.1252 2 p-Xylene 0.2348 C8H10 937 replib 118617 17.985 261.8 106.0783

Table A37: Volatile compounds from B2 hexane crude extract

207

Peak Name Area % Formula Similarity Library Height Quant R.T. Exact # S/N (s) Mass 51 Octadecane 9.5005 C18H38 947 replib 62625987 34420 1007.5 254.2974 29 Hexadecane 8.5922 C16H34 950 DATABASE 63744608 35035 873.1 226.2661 72 Eicosane 7.399 C20H42 943 replib 58500191 32152 1128.4 282.3287 52 E-7-Octadecene 6.3166 C18H36 796 mainlib 53031554 29147 1008.2 252.2817 88 Heneicosane 5.2801 C21H44 929 replib 53207867 29244 1238.2 296.3443 27 Benzene, (1-methylnonyl)- 4.7712 C16H26 835 mainlib 57929746 31839 870.4 218.2035 14 Tetradecane 4.5182 C14H30 937 replib 56203769 30890 719.7 198.2348 49 7-Tetradecene, (Z)- 1.95 C14H28 831 replib 28356049 15585 998.5 196.2191 25 Diethyl Phthalate 0.91883 C12H14O4 668 replib 16841238 9256.1 863.1 222.0892 68 1-Docosene 0.60446 C22H44 957 replib 10690024 5875.3 1121 308.3443 74 2,5-di-tert-Butyl-1,4-benzoquinone 0.44703 C14H20O2 793 replib 10769405 5919 1140.8 220.1463 76 Eicosane, 2-methyl- 0.22313 C21H44 901 mainlib 4870189 2676.7 1155.6 296.3443 56 Octadecane, 4-methyl- 0.19595 C19H40 921 mainlib 5298162 2911.9 1037 268.313 123 Docosane 0.14191 C22H46 794 DATABASE 1873490 1029.7 1722.6 310.36 45 Heptadecane, 3-methyl- 0.12793 C18H38 918 replib 3239007 1780.2 982.5 254.2974 46 Benzene, (1-ethyldecyl)- 0.12793 C18H30 766 replib 3239007 1780.2 982.7 246.2348 44 Tetradecane, 4-ethyl- 0.11993 C16H34 885 mainlib 2661509 1462.8 968.7 226.2661 1 Octane 0.11626 C8H18 944 DATABASE 4685441 2575.2 206.9 114.1409 104 Nonadecane, 2-methyl- 0.11524 C20H42 849 mainlib 2513317 1381.3 1363.8 282.3287 106 Sulfurous acid, 2-ethylhexyl nonyl ester 0.10033 C17H36O3S 864 mainlib 1836317 1009.3 1402.8 320.2385 65 Eicosane, 2-methyl- 0.099746 C21H44 909 mainlib 2453704 1348.6 1106.3 296.3443 55 2,3-Dimethyldecane 0.077954 C12H26 869 mainlib 1271491 698.82 1027.2 170.2035 20 Pentadecane, 5-methyl- 0.077831 C16H34 893 DATABASE 1935449 1063.7 831.3 226.2661 79 Hexadecane, 4-methyl- 0.077473 C17H36 863 mainlib 2060446 1132.4 1200.3 240.2817 61 Undecane, 2,3-dimethyl- 0.064101 C13H28 864 mainlib 1968756 1082 1087.1 184.2191 53 1-Decanol, 2-hexyl- 0.056487 C16H34O 893 mainlib 1592582 875.3 1013.9 242.261 113 Tetracosane, 3-ethyl- 0.052051 C26H54 884 DATABASE 1193775 656.11 1456 366.4226 64 Dibutyl phthalate 0.04649 C16H22O4 924 mainlib 1173894 645.18 1101.5 278.1518

208

116 Undecane, 2,3-dimethyl- 0.041854 C13H28 844 mainlib 1406493 773.02 1490 184.2191 8 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.038844 C10H18O 939 DATABASE 1137171 625 404.1 154.1358 4 p-Xylene 0.037636 C8H10 955 replib 867895 477 260.8 106.0783 39 Benzene, (1-methyldecyl)- 0.035547 C17H28 892 DATABASE 691183 379.88 941 232.2191 58 Cyclopentane, decyl- 0.035458 C15H30 829 DATABASE 818912 450.08 1043.7 210.2348 7 Benzene, 1-methyl-2-(1-methylethyl)- 0.034936 C10H14 955 mainlib 1057213 581.05 395.8 134.1096 23 4,9-Dioxacyclodeca-1,6-diene 0.034822 C8H12O2 734 DATABASE 756506 415.78 856.6 140.0837 89 Heptadecane, 2,3-dimethyl- 0.032944 C19H40 878 mainlib 863664 474.68 1254.3 268.313 112 Eicosane, 7-hexyl- 0.023528 C26H54 862 mainlib 687618 377.92 1447 366.4226 37 Benzene, (1-ethylnonyl)- 0.02143 C17H28 872 DATABASE 488355 268.4 914.9 232.2191 18 Phenol, 2,4-bis(1,1-dimethylethyl)- 0.020307 C14H22O 824 mainlib 448477 246.49 802.5 206.1671 35 Benzene, (1-propyloctyl)- 0.01859 C17H28 852 mainlib 551419 303.07 899.4 232.2191 3 Ethylbenzene 0.016986 C8H10 938 mainlib 391032 214.92 253.9 106.0783 73 10-Heneicosene (c,t) 0.016361 C21H42 851 mainlib 657695 361.48 1135.2 294.3287 5 Benzene, 1,3-dimethyl- 0.012528 C8H10 947 replib 332101 182.53 279.7 106.0783 48 Heptane, 3-ethyl-5-methylene- 0.009981 C10H20 788 mainlib 202976 111.56 993.1 140.1565 83 Tridecane, 3-methylene- 0.009755 C14H28 819 mainlib 311772 171.35 1228.2 196.2191 43 Benzene, (1-propylnonyl)- 0.00903 C18H30 788 DATABASE 647911 356.1 967.3 246.2348 6 à-Phellandrene 0.007678 C10H16 806 replib 238983 131.35 306.9 136.1252 97 2-Undecene, 9-methyl-, (Z)- 0.006818 C12H24 817 mainlib 286750 157.6 1331.3 168.1878 98 Hexanedioic acid, bis(2-ethylhexyl) ester 0.006818 C22H42O4 827 replib 286750 157.6 1331.7 370.3083 33 Benzene, (1-butylheptyl)- 0.00657 C17H28 901 mainlib 214654 117.98 892 232.2191 54 Isopropyl tetradecanoate 0.005584 C17H34O2 814 DATABASE 413957 227.51 1015.9 270.2559 21 Benzene, (1-ethyloctyl)- 0.003541 C16H26 843 DATABASE 210636 115.77 842.8 218.2035

Table A38: Volatile compounds from B2 ethyl acetate crude extract

Peak Name Area % Formula Similarity Library Height Quant R.T. Exact # S/N (s) Mass 58 Hexadecane 9.5163 C16H34 943 replib 43892257 6667.1 867.6 226.2661

209

18 2-Phenylpropanal 7.9274 C9H10O 804 DATABASE 36965163 5614.9 370.1 134.0732 17 Benzene, 1,3,5-trimethyl- 7.8709 C9H12 899 DATABASE 36858917 5598.8 369.7 120.0939 54 Tetradecane 7.5094 C14H30 956 replib 42580027 6467.8 717.9 198.2348 66 Octadecane 7.2605 C18H38 937 replib 39918442 6063.5 1001.7 254.2974 46 2,4-Dimethylstyrene 4.9014 C10H12 761 mainlib 37081735 5632.6 503.2 132.0939 45 3-Methylheptyl acetate 4.8908 C10H20O2 853 mainlib 37070114 5630.9 502.4 172.1463 44 2-Ethylhexyl acetate 4.8862 C10H20O2 739 DATABASE 37065078 5630.1 502.2 172.1463 76 Heneicosane 3.5525 C21H44 920 replib 25437916 3864 1123 296.3443 11 Benzaldehyde, 4-(4-methylbenzyloxy)- 2.8773 C15H14O2 698 mainlib 25338533 3848.9 338 226.0994 71 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- 2.5778 C11H18N2O2 900 mainlib 11734529 1782.4 1096.7 210.1368 methylpropyl)- 70 2,5-Piperazinedione, 3,6-bis(2-methylpropyl)- 2.3816 C12H22N2O2 777 mainlib 11407813 1732.8 1096.3 226.1681 50 Dodecane 1.8621 C12H26 934 DATABASE 17194614 2611.8 550.6 170.2035 30 Benzene, 1-methyl-4-propyl- 1.4266 C10H14 885 replib 10509353 1596.3 424.6 134.1096 83 3-Benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione # 0.74132 C14H16N2O2 857 DATABASE 2973055 451.6 1347.4 244.1212 78 Triacontane 0.72027 C30H62 919 DATABASE 5446615 827.33 1233.8 422.4852 26 Benzene, 2-propenyl- 0.67948 C9H10 932 replib 6059679 920.45 408.1 118.0783 39 Undecane 0.50953 C11H24 922 replib 4874941 740.49 460.8 156.1878 5 p-Xylene 0.45782 C8H10 958 replib 2821709 428.61 261.4 106.0783 80 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)-4- 0.45185 C15H17Cl2N3O2 695 mainlib 2143778 325.63 1325.1 341.0698 propyl-1,3-dioxolan-2-yl]methyl]- 65 1-Nonadecene 0.41333 C19H38 923 replib 2917023 443.09 996.4 266.2974 28 Benzene,1-ethynyl-2-methyl- 0.29932 C9H8 929 mainlib 2009377 305.22 417.1 116.0626 10 Benzene, propyl- 0.27541 C9H12 947 replib 2120729 322.13 331.8 120.0939 64 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 0.26851 C7H10N2O2 909 mainlib 969078 147.2 992.4 154.0742 25 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.24792 C10H18O 931 DATABASE 1931611 293.41 404.4 154.1358 1 1-Propanamine, N,2-dimethyl-N-nitroso- 0.18959 C5H12N2O 698 mainlib 549123 83.41 203.3 116.095 6 1-Pentanol, 2,2-dimethyl- 0.17081 C7H16O 774 DATABASE 1203149 182.76 273.4 116.1201 75 1-Docosene 0.14813 C22H44 944 replib 1097493 166.71 1118.9 308.3443 21 Benzene, 1,4-dichloro- 0.14507 C6H4Cl2 913 DATABASE 1058058 160.72 389.4 145.969 4 Ethylbenzene 0.14283 C8H10 955 mainlib 1111249 168.8 254.5 106.0783

210

7 p-Xylene 0.12248 C8H10 946 replib 896275 136.14 280.2 106.0783 56 Pentadecane 0.11554 C15H32 932 DATABASE 975947 148.24 793.5 212.2504 61 Hexahydropyrrolizin-3-one 0.11548 C7H11NO 671 mainlib 520682 79.09 957.7 125.0841 59 Hexadecane, 4-methyl- 0.099036 C17H36 924 mainlib 773677 117.52 905.9 240.2817 14 3-Pentanone, 2,2,4,4-tetramethyl- 0.094774 C9H18O 938 DATABASE 1085428 164.87 342.8 142.1358 81 Hexanedioic acid, bis(2-ethylhexyl) ester 0.08919 C22H42O4 904 DATABASE 408813 62.098 1331.5 370.3083 2 Butanoic acid, 3-methyl- 0.078798 C5H10O2 933 DATABASE 303443 46.092 237 102.0681 85 Decane, 5-ethyl-5-methyl- 0.057391 C13H28 716 mainlib 297021 45.117 1488.1 184.2191 89 1,2-Cyclohexanedicarboxylic acid, cyclohexylmethyl 0.05637 C24H42O4 782 mainlib 306762 46.596 1520.9 394.3083 nonyl ester 47 Benzene, 1-methyl-4-(2-propenyl)- 0.051537 C10H12 915 mainlib 245901 37.352 512.2 132.0939 9 Bicyclo[3.1.0]hexane, 4-methyl-1-(1-methylethyl)-, 0.026811 C10H16 908 mainlib 245165 37.24 307.3 136.1252 didehydro deriv.

49 Azulene 0.021104 C10H8 862 replib 485167 73.696 548.9 128.0626

Table A39: Volatile compounds from B2 DCM crude extract

Peak Name Area % Formula Similarity Library Height Quant R.T. Exact # S/N (s) Mass 31 2,5-Piperazinedione, 3,6-bis(2-methylpropyl)- 13.291 C12H22N2O2 733 mainlib 7521822 1234.5 1098.3 226.1681 34 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- 8.3952 C11H18N2O2 875 mainlib 3948137 647.96 1104.7 210.1368 methylpropyl)- 33 5H,10H-dipyrrolo[1,2-a:1',2'-d]pyrazine-5,10-dione, 8.3763 C10H14N2O2 865 DATABASE 3941907 646.93 1103.7 194.1055 octahydro-, (5as-cis)- 13 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 2.261 C10H18O 937 DATABASE 2660587 436.65 404.3 154.1358 10 Benzene, 1,2,4-trimethyl- 1.9974 C9H12 943 mainlib 1831095 300.51 368.8 120.0939 12 Benzene, 1-methyl-2-(1-methylethyl)- 1.8063 C10H14 945 mainlib 1335945 219.25 396.4 134.1096 21 1-DODECANOL 1.7484 C12H26O 671 DATABASE 1028140 168.74 959.1 186.1984 1 1,3,5-Trimethylhexahydro-1,3,5-triazine 1.6497 C6H17N3 828 DATABASE 600872 98.613 202.9 131.1422 19 Hexadecane 0.69591 C16H34 931 replib 664516 109.06 867 226.2661 17 Tetradecane 0.5641 C14H30 933 replib 435298 71.44 718.3 198.2348 26 Pentadecane 0.42855 C15H32 916 DATABASE 466392 76.543 1000.8 212.2504

211

38 3-Benzyl-6-isopropyl-2,5-piperazinedione # 0.39466 C14H18N2O2 834 DATABASE 248616 40.802 1284.5 246.1368 2 Benzene, 1,3-dimethyl- 0.2454 C8H10 809 mainlib 209953 34.457 261.6 106.0783 40 Hexanedioic acid, bis(2-ethylhexyl) ester 0.24158 C22H42O4 722 replib 221676 36.381 1332 370.3083 36 Nonadecane 0.24121 C19H40 909 replib 253731 41.642 1122.5 268.313 23 Hexahydropyrrolizin-3-one 0.22941 C7H11NO 662 mainlib 229152 37.608 976.2 125.0841 4 Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, (ñ)- 0.17695 C10H16 918 mainlib 218229 35.815 315.3 136.1252 14 Benzene, 2-propenyl- 0.1386 C9H10 913 replib 122870 20.165 408.3 118.0783

Table A40: Volatile compounds from B3 hexane extract

Peak Name Area % Formula Similarity Library Height Quant S/N R.T. (s) Exact # Mass 37 Octadecane 12.703 C18H38 892 DATABASE 54524768 8361.5 1004.4 254.2974 19 Pentadecane 12.351 C15H32 896 DATABASE 56193190 8617.4 869 212.2504 20 Hexadecane 12.351 C16H34 850 replib 56193190 8617.4 870 226.2661 21 Nonadecane 12.128 C19H40 852 replib 55832584 8562.1 870.8 268.313 47 Eicosane 11.027 C20H42 865 replib 51054997 7829.4 1125.4 282.3287 10 Tetradecane 7.6735 C14H30 944 replib 55446435 8502.9 718.9 198.2348 53 1-Iodo-2-methylundecane 6.8045 C12H25I 856 mainlib 46106759 7070.6 1236.4 296.1001 52 Heneicosane 6.7609 C21H44 938 replib 46073938 7065.6 1236.2 296.3443 46 6-Tetradecanesulfonic acid, butyl ester 3.4403 C18H38O3S 703 mainlib 47954368 7353.9 1124.4 334.2542 56 Heptacosane 2.5788 C27H56 929 replib 30145086 4622.8 1337.5 380.4382 35 1-Nonadecene 0.53415 C19H38 948 replib 5870963 900.33 997.2 266.2974 17 7-Hexadecene, (Z)- 0.41954 C16H32 931 mainlib 4145473 635.72 861.6 224.2504 45 1-Docosene 0.38937 C22H44 958 replib 4634134 710.66 1119.7 308.3443 7 Dodecane 0.38524 C12H26 928 DATABASE 5202311 797.79 550.3 170.2035 48 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9- 0.29233 C17H24O3 783 replib 3633913 557.27 1139.3 276.1725 diene-2,8-dione 13 Pentadecane 0.26397 C15H32 922 DATABASE 3437073 527.09 793.7 212.2504 62 Eicosane, 2-methyl- 0.15579 C21H44 910 mainlib 1208419 185.31 1607.7 296.3443 30 Heptadecane, 2,6-dimethyl- 0.14561 C19H40 907 mainlib 1380080 211.64 964.1 268.313

212

9 Tridecane 0.14072 C13H28 937 DATABASE 1881488 288.53 635.8 184.2191 24 Hexadecane, 4-methyl- 0.12821 C17H36 925 mainlib 1613229 247.39 906 240.2817 39 Octadecane, 4-methyl- 0.11656 C19H40 928 mainlib 1393468 213.69 1036.5 268.313 33 Heptadecane, 3-methyl- 0.11323 C18H38 914 replib 1339428 205.41 982.1 254.2974 34 Benzene, (1-ethyldecyl)- 0.11323 C18H30 873 replib 1339428 205.41 982.5 246.2348 32 Tetradecane, 4-ethyl- 0.1067 C16H34 894 mainlib 914478 140.24 968.4 226.2661 41 Octadecane, 2,6-dimethyl- 0.089092 C20H42 884 mainlib 867737 133.07 1088.5 282.3287 50 Pentacosane 0.087796 C25H52 916 DATABASE 972134 149.08 1179.2 352.4069 3 Undecane 0.080371 C11H24 924 replib 1096264 168.12 460.6 156.1878 16 Pentadecane, 3-methyl- 0.079788 C16H34 889 mainlib 974655 149.47 845.4 226.2661 58 1,2-Benzenedicarboxylic acid, diisooctyl 0.067135 C24H38O4 923 replib 570593 87.502 1403.2 390.277 ester 27 Benzene, (1-methyldecyl)- 0.056584 C17H28 914 DATABASE 572590 87.808 940.8 232.2191 61 Squalene 0.051799 C30H50 919 replib 583237 89.441 1526.7 410.39 18 1,2-Benzenedicarboxylic acid, diethyl 0.040691 C12H14O4 910 DATABASE 1277352 195.89 863 222.0892 ester 29 Benzene, (1-butyloctyl)- 0.028724 C18H30 815 replib 290052 44.48 958.2 246.2348 12 Cycloheptasiloxane, tetradecamethyl- 0.028533 C14H42O7Si7 743 replib 249678 38.289 764.7 518.1315 25 Benzene, (1-ethylnonyl)- 0.027459 C17H28 890 DATABASE 278825 42.759 914.5 232.2191 4 Benzene, 1-methyl-2-(1-methylethyl)- 0.024993 C10H14 888 mainlib 243822 37.391 484 134.1096 28 Benzene, (1-pentylheptyl)- 0.018324 C18H30 788 DATABASE 264962 40.633 955.1 246.2348 22 Benzene, (1-butylheptyl)- 0.015993 C17H28 896 mainlib 188758 28.947 891.3 232.2191 6 Azulene 0.011777 C10H8 929 DATABASE 296324 45.442 548.8

Table A41: Volatile compounds from B3 ethy acetate crude extract

Peak Name Area % Formula Similarity Library Height Quant S/N R.T. Exact Exact Mass # (s) mass 27 Hexadecane 21.555 C16H34 947 replib 49153492 8190.3 868.2 226.2661 37 Oxalic acid, isobutyl nonyl ester 16.195 C15H28O4 865 mainlib 45843749 7638.8 1002.5 272.1988 36 Octadecane 16.163 C18H38 925 replib 45833976 7637.1 1002.2 254.2974

213

20 Tetradecane 11.396 C14H30 957 replib 43537341 7254.5 718 198.2348 48 Eicosane 6.3202 C20H42 922 replib 27885860 4646.5 1123.4 282.3287 282.3287 60 3-Benzylhexahydropyrrolo[1,2-a]pyrazine-1,4- 2.2093 C14H16N2O2 863 DATABASE 4314471 718.9 1348.3 244.1212 dione # 51 Pentacosane 1.4052 C25H52 915 DATABASE 5233745 872.08 1234 352.4069 45 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3- 1.371 C11H18N2O2 872 mainlib 2951677 491.83 1103.2 210.1368 (2-methylpropyl)- 56 1H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)- 1.292 C15H17Cl2N3O2 884 mainlib 2758723 459.68 1325.4 341.0698 4-propyl-1,3-dioxolan-2-yl]methyl]- 34 1-Nonadecene 0.84268 C19H38 934 replib 3010627 501.65 996.5 266.2974 26 1-Hexadecene 0.66672 C16H32 927 replib 2371082 395.08 861.2 224.2504 13 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl- 0.66326 C10H18O 936 DATABASE 2612209 435.26 404.2 154.1358 33 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- 0.61812 C7H10N2O2 916 mainlib 1128082 187.97 993.6 154.0742 10 Benzene, 1,2,4-trimethyl- 0.52732 C9H12 945 mainlib 1783519 297.18 368.8 120.0939 12 Benzene, 1-methyl-2-(1-methylethyl)- 0.51129 C10H14 948 mainlib 1397528 232.86 396.4 134.1096 59 Triacontane 0.50732 C30H62 916 DATABASE 1878194 312.96 1335.7 422.4852 1 1,3,5-TRIMETHYLHEXAHYDRO-1,3,5- 0.46394 C6H17N3 747 DATABASE 537756 89.604 202.7 131.1422 TRIAZINE 39 1H-Indole-3-carboxaldehyde 0.36119 C9H7NO 899 replib 1073921 178.94 1027.2 145.0528 53 Heneicosane, 11-cyclopentyl- 0.35396 C26H52 666 replib 681015 113.47 1263.6 364.4069 64 1,2-Cyclohexanedicarboxylic acid, 0.33123 C24H42O4 746 mainlib 486880 81.127 1488.4 394.3083 cyclohexylmethyl nonyl ester 30 2-Propenal, 3-(1-aziridinyl)-3- 0.25153 C7H12N2O 676 mainlib 713284 118.85 957.6 140.095 (dimethylamino)- 23 Pentadecane 0.22981 C15H32 938 DATABASE 1013511 168.88 793.5 212.2504 28 Hexadecane, 4-methyl- 0.22753 C17H36 919 mainlib 899678 149.91 906 240.2817 69 1,2-Cyclohexanedicarboxylic acid, dinonyl 0.22276 C26H48O4 769 mainlib 475086 79.162 1521.4 424.3553 ester 62 Heptacosane 0.21071 C27H56 910 replib 617112 102.83 1430.1 380.4382 70 Squalene 0.20233 C30H50 901 replib 777806 129.6 1526.5 410.39 410.3913 58 Hexanedioic acid, bis(2-ethylhexyl) ester 0.18654 C22H42O4 910 DATABASE 539508 89.896 1331.7 370.3083 40 Octadecane, 4-methyl- 0.18049 C19H40 898 mainlib 744085 123.98 1036.4 268.313 50 1-Docosene 0.16808 C22H44 911 replib 702244 117.01 1230.5 308.3443

214

31 Heptadecane, 2,6-dimethyl- 0.15096 C19H40 904 mainlib 408380 68.047 963.8 268.313 49 Eicosane, 2-methyl- 0.13447 C21H44 886 mainlib 446637 74.421 1154.8 296.3443 32 Heptadecane, 3-methyl- 0.10971 C18H38 869 replib 441342 73.539 981.9 254.2974 3 Benzene, 1,3-dimethyl- 0.10796 C8H10 950 replib 191228 31.864 261.5 106.0783 4 Bicyclo[3.1.0]hexane, 4-methyl-1-(1- 0.10678 C10H16 927 DATABASE 412375 68.712 307.4 136.1252 methylethyl)-, didehydro deriv. 52 2,5-Piperazinedione, 3-(phenylmethyl)- 0.097523 C11H12N2O2 835 mainlib 300324 50.042 1248 204.0899 5 Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, (ñ)- 0.073074 C10H16 931 mainlib 239383 39.887 315.3 136.1252 61 1,2-Benzenedicarboxylic acid, diisooctyl ester 0.064676 C24H38O4 752 replib 179982 29.99 1403 390.277 54 1H-indole-3-ethanol 0.058497 C10H11NO 655 DATABASE 152738 25.45 1276.5 161.0841 24 Phenol, 2,4-bis(1,1-dimethylethyl)- 0.039531 C14H22O 760 mainlib 169811 28.295 802.5 206.1671

Table A42: Volatile compounds from B3 DCM crude extract

Peak # Name Area % Formula Similarity Library Height Quant R.T. Exact S/N (s) Mass 2 Pyrrolo[1,2-a]pyrazine-1,4-dione, 84.018 C11H18N2O2 869 mainlib 206059 35.477 1092.1 210.1368 hexahydro-3-(2-methylpropyl)-

215

216