Chemical Composition of Lantana (Lantana Camara L.) Leaves Essential Oil and Its Antimicrobial Activity

Chemical Composition of Lantana (Lantana camara L.) Leaves Essential Oil and its Antimicrobial Activity

Nosiba Hassan Elsheikh Idris

B.Sc. (Honors) in Agricultural Sciences (Horticultural Sciences) Faculty of Agricultural Sciences, University of Gezira (1998) M.Sc. in Horticultural Sciences, University of Gezira, (2008)

A Thesis Submitted to the University of Gezira in Fulfillment of the Requirements for the Award of the Degree of Doctor of Philosophy

in Oil Chemistry National Oil Seed Processing Research Institute (NOPRI) University of Gezira

August, 2013

Chemical Composition of Lantana (Lantana camara L.) Leaves Essential Oil and its Antimicrobial Activity

By Nosiba Hassan Elsheikh Idris

Supervision Committee: Name Position Signature Dr. Nour Ahmed Osman Main Supervisor ……… Prof. Nafisa Elmahi Ahmed Co- Supervisor ………. Dr. Atif Abdel monium Ahmed Co- Supervisor ………

i Chemical Composition of Lantana (Lantana camara L.) Leaves Essential Oil and its Antimicrobial Activity

By Nosiba Hassan Elsheikh Idris

Examination Committee: Name Position Signature Dr. Nour Ahmed Osman Chairman ………………. Dr. Salah Mohamed Nour External Examiner …….………… Dr. Salih Mohamed A. Abbaker Internal Examiner ………………..

Date of Examination: 15/8/2013

ii Declaration

I here declare that the results and findings of this research are original graphs, tables , figures and photos are genuine and they are not reproduced or extracted from any other research.

Nosiba

iii

Dedication

For My Brothers Elsir and Zahir

ACKNOWLEDGEMENTS

I would like to thank all those who participated in this work. Special thanks to my supervisor Dr. Nour Ahmed Osman who generously provided her time, effort and advice to make this work possible. Her support has been valuable during all the stages of the work. She spent much of her time organizing and discussing the different aspects especially in the identification and elucidation of the active ingredients my heart felt gratitude to my co-supervisors Prof. Nafisa Elmahi Ahmed, ARC and Dr. Atif Abdelmonium Ahmed, NOPRI, for all the kind efforts exerted in guiding me and the valuable comments and suggestions offenet during this study. My deeps thanks are due to Prof. Salah Ahmed Ali Elhussien for this topic, his genuine guidance and invaluable support. I am also greatly indebted to the staff of the food Technology Laboratory, Ustaz Hassan Ansary and his steam, University of Gezira, Ustaz Khalid Dafalla Abu Idris (ARC) Plant Pathology Lab and his team, Ustaz Betijowk (ARC) Plant Pathology Lab Research Program for their great help and advice. Finally my family my father, my mother, brothers, sister, husband and daughters for their support. Sincere thanks are due to Miss Ayda Yousif for formatting this thesis.

Chemical Composition of Lantana (Lantana camara L.) Leaves Essential Oil and its Antimicrobial Activity Nosiba Hassan Elsheikh Idris A Ph.D. in Oil Chemistry (August , 2013) National Oil Seed Processing Research Institute (NOPRI) University of Gezira ABSTRACT Lantana camara (L), of the family Verbenaceae, is listed as one of the important medicinal plants of the world. The plant is an evergreen aromatic plant, found in tropical and subtropical areas around the world. In Sudan it is commonly grown as an ornamental plant and displays several flower colors combing different shades of red- yellow- purple- violet- white- orange and yellow and orange. The essential oil of Lantana camara showed a wide spectrum of antibacterial, antimicrobial, and antifungal activities. The aims of this study were to evaluate chemical composition of locally grown Lantana plants, to evaluate their content of leaf essential oil and to assay biological activities of crude and purified constituents of the essential oil against a selected bacterium and a fungus. The plants, collected from the Gezira area around Wad-Medani, were extracted by using hydro- distillation method and cold extraction technique. The crude essential oil extracts of lantana Camara Var. aculeata were tested for growth inhibition of Agrobactrium tumefaciens and Aspergillus niger using the disc diffusion method and well fungal- growth disc diffusion method. Chemical fractionations and component identification were performed using TLC, PTLC, GC/MS, MS, UV, and FTIR. The results indicated that the crude essential oils of lantana Camara Var. aculeate possessed considerable antibacterial activities, with lethal concentration (LC50) between 160- 200μl/ml and minimum inhibitory concentration (MIC) between 4 and 8 μl/ml. On the other hand, the essential oil showed significant antifungal activity resulting in 100% kill at doses of 280 and 300 μl/ml, and LC50 of 160μL/ml, and 8-12μL/ml for MIC. Of nine separated components, eluted from PTLC-plate, only two (C1 and C6) were found active against A. niger (causing antifungal inhibition of 14% and 55% respectively). The major constituents of the essential oil of Lantana camara dependent on GC/MS were limonene, C6-Ketone, the sesquiterpene -cayrophyllene, representing more than 30% of the oil. Compared with Iran(14%), China(12%), and Nigeria(13%).

vi المكونات الكيميائية للزيت الطيار من أوراق الالنتانا وتضاده الميكروبي نسيبة حسن الشيخ إدريس دكتوراه الفلسفة في كيمياء الزيوت )أغسطس 2013م( المعهد القومي لبحوث تصنيع الحبوب الزيتية )نوبري( جامعة الجزيرة

الخالصة نبات الالنتانا .Lantana camara L أحد النباتات الطبية والعطريةة الااةةة ةل العةالي ونةل تنت ةل للعائلة الفيربينية وتوجد ل ال ناطق ذات ال ناخ ال عتدل وغير ال عتدل حول العالي. ل السةواا تةع ك بنبةات للعينةة حةول ال نةةا ل وتت يةع بالواناةةا ال تلفةة التةةل ت تةون سلةة البنفسة – األصةةفر – الةو ان – األح ةةر – األبةةي – البرتقةةالل – األصةةفر والبرتقةةالل. أااةةر العيةة الطيةةا لالنتانةةا ةقةةد ساليةةة للت ةةاا الب تيةةرن وال ي روبل والفطرن. الادف ةن نذه الد اسةة نةو تقيةيي عاليةة العية العطةرن ال ةا وة وناتة دةد النطةاط اإلحيائل و تي تصنيف النبات بنا ًء سل لةو األ نةا ولة ل السةاب إل باإلدةا ة ل عر ةة النسةبة ال زويةة للعية الطيا ل أنواك األ نا ة تلفة األلوا . تي ج ع أو اب الالنتانةا ةةن حةول ةدينةة واةةدنل ةل وجيةة ال عيةر واسةةت لا العيةة ةناةةا بواسةةطة طريقةةة التقطيةةر ال ةةائل وباجسةةت الب البةةا ا إل تةةي ااتبةةا عاليةةة العيةة ال سةةت لا ةةةن Lantana camara var. aculeata دةةد ب تريةةا العقةةد ال ذ يةةة Agrobacterium tumefaciens و طر العفن األسوا Aspergillus niger باسةت دا طريقةة اجنتطةا سةن طريةق نةرب ةةن الةةو ب وبطريقةةة اجنتطةةا سةةن طريةةق نةةرب نةةاةل ةةةن الفطةةر ةةل األجةةا . أجريةة ت اليةةل بي يائيةةة ل عر ةةة ال ونات الرئيسية للعي الطيا لعدا تسع سينات ةن الالنتانا باست دا )بروةاتوجرا يا الغا /ةطيا ية ال تلةة وةطيا ية ال تلة ب ا ت ةعر ة نوية ال ربب الفعال باست دا ةطياف األلةعة ت ة ال ةراء ال ةعوا ب ةول و ييةإل ةطيا ية األلعة وب البنفس يةإل بروةاتوجرا يا الطبقة الرنيقةإل )بروةاتوجرا يا الغا /ةطيا ية ال تلة وةطيا ية ال تلة. ألا ت النتائ إل أ ي الالنتانةا الطيةا أبةدع عاليةة دةد الب تريةا و صةدت ني ةة جرسةة

التربيةةع القاتلةةة LC50 وبانةة بةةين 160-200 ةةةاي روليتر /ةةةل ب ةةا صةةدت ني ةةة ال ةةد األانةة للقتةةل MIC وبانةة بةةين 4-8 ةةةاي روليتر /ةةةةل. أةةةا الفطةةر قةةةد وصةةل نسةةبة القتةةةل إلةة 100% سنةةد ال رسةةةات 280

ةاي روليتر /ةةل و 300 ةةاي روليتر /ةةلإل أي ةاً صةدت ني ةة LC50 وبانة سنةد ال رسةة 160 ةةاي روليتر /ةل و MIC وبان بين 8-12 ةاي روليتر /ةل. تي صل تسعة ةرببات للعي الطيا Lantana camara var. aculeata ب روةات را يةا الطبقةة الرنيقةة ووجةد أ ا نةين قة (C6, C1) لا ةا عاليةة دةد طةر العفةن األسوا بنسبة 14% و55% سلل التوالل وند وجد أ ال ونات الرئيسية للعي الطيا لالنتانا نةل الالي ةونين و ك6-بيتو و سس وتربين بيتا با يو يللين (β- caryophyllene) والذن تفوب نسبت 30% ةقا نة بالنسةبة ال وجوا ل إيرا 14% والصين 12% وني يريا %13.

vi LIST OF CONTENT Page Declaration ……………………………………………………………….. iii Dedication …………...…………………………….……..……..………... iv Acknowledgements …..…………………………..…………..…………... v English Abstract …...…..………………………………….……..……...... vi Arabic Abstract ………...………………………………………….……... vi List of Contents...... vii List of Tables …...... viii List of Figures…...... ix List of Abbreviations……………………………………………………... x CHAPTER ONE:…………………………………………..……………. 1 1. INTRODUCTION …..……………………………………………… 1 1.1. Importance of natural products………………………………… 1 1.2. The plant Lantana camara..…………………………………… 3 CHAPTER TWO: ………………………………………………………. 6 2. LITERATURE REVIEW…………..……………………………… 6 2.1. Natural products……………………………………………….. 6 2.2. Plant-derived natural product …………………………………. 6 2.2.1. Primary plant products ………………………………... 6 2.2.1.1. Carbohydrates ……………………………… 7 2.2.1.2. Amino acids and proteins…………………... 7 2.2.1.3. Lipids ………………………………………. 8 2.2.2. Secondary plant products ……………………………... 10 2.2.2.1. Alkaloids……………………………………. 10 2.2.2.2. Biosynthesis………………………………… 12 2.2.2.3. Phenolic compounds………………………... 12 2.2.2.4. Flavonoid pigments……………………….. 14 2.2.2.5. Classification of flavonoids………………… 14 2.2.2.6. Isoflavonoids……………………………….. 15 2.2.2.7. Terpenoids………………………………….. 15 2.2.2.8. General properties of Terpenoids…………... 16 2.2.2.9. Biosynthetic pathways of terpenoids in plants……………………………………….. 16 2.2.2.10 Tri terpenoids…………………………...... 19 2.2.2.11 Essential (volatile) oils……………………... 22 2.2.2.12 Essential oil classes…………………………. 22 2.2.2.13 Sources of Natural Essential Oils…………. 23 2.2.2.14 Essential Oil Constituents…………………. 29 2.2.2.15 Chemical structures of essential oils……….. 29

vii 2.2.2.16 Mono terpenes……………………….…………. 31 2.2.2.17 Sesqui terpenes…………………………………. 31 2.2.2.18 Phenolic essential oils………………………..…. 32 2.2.2.19 Alcohols…………………………………………. 32 2.2.2.20 Ethers / Esters……………………..……………. 32 2.2.2.21 Ketones…………………………………...……... 32 2.2.2.22 Aldehydes………………………………………... 33 2.2.2.23 Coumarins ……………………………..……….. 33 2.2.2.24 Biosynthesis of essential oils……………..…….. 33 2.2.2.24.1 Essential oils are biosynthesized in plants two pathway………………… 36 2.2.2.25 Extraction of essential oils…………………..….. 37 2.2.2.25.1 Distillation………………………….. 37 2.2.2.25.2 Steam distillation…………………… 37 2.2.2.25.3 Cold pressing……………………….. 38 2.3. Microbes of the study…………………………….. 38 2.3.1. Cell structure and metabolism………………………… 39 2.3.2. Ecology………………………………………………... 39 2.3.3. Aspergillus niger……………………………………… 40 2.3.4. Toxin production by A. niger…………………………. 40 2.3.5. Industrial use………………………………………….. 41 2.3.6. Symptoms of Aspergillus niger:…………………….. 41 2.3.7. Antifungal medicines…………………………………. 41 2.3.8. Antifungal azoles……………………………………… 42 2.4. Lantana camara L……………………………………………... 42 2.4.1. Vernacular names…………………………………….. 42 2.4.2. Botany…………………………………………………. 42 2.4.3. Taxonomy……………………………………………... 43 2.4.4. Poisonous principles…………………………………... 44 2.4.5. Distribution……………………………………………. 44 2.4.6. Folk-medicinal uses…………………………………… 44 2.4.7. Chemical composition………………………………… 45 2.4.8. Biological activities of the Essential oil of L .camara 46 2.4.9. Commercial production……………………………….. 47 2.4.10 Sudanese Lantana camara……………………………. 47 CHAPTER THREE……………………………………………………... 48 3. MATERIALS AND METHODS…………………………………… 48 3.1. Plant collection and preparation……………………………….. 48 3.2. Chemicals……………………………………………………… 48 3.3. Equipments…………………………………………………….. 48 3.4. Essential oil extraction…………………………………………. 50

vii 3.5. Thin layer chromatography (TLC)…………………………….. 52 3.5.1. Preparation of plates…………………………………... 52 3.5.2. Preparative TLC………………………………………. 52 3.5.3. TLC solvent system and detection reagent…………… 53 3.6. Bioassay techniques……………………………………………. 53 3.6.1. Culture and subculture of testing microorganism…….. 53 3.6.2. Testing microorganism……………………………….. 53 3.6.3. Preparation of media………………………………….. 54 3.6.3.1. Potato Dextrose Agar (PDA)………………. 54 3.6.3.2. Nutrient agar media (NA)………………….. 56 CHAPTER FOUR………………………………………………………. 57 4. RESULTS AND DISCUSSIONS…………………………………… 57 4.1. Morphological variability among Lantana camara species growing in Sudan………………………………………………. 57 4.2. Essential oils content (%) of lantana species in Sudan………. 57 4.3. TLC-separated components of Lantana-essential oil……….. 59 4.4. Biological activity of Lantana essential oil………………….. 63 4.4.1.Antibacterial activity…………………………………… 63 4.4.2.Antifungal activity……………………………………… 68 4.4.3.Disc diffusion method…………………………………. 72 4.4.4.Agar diffusion method…………………………………. 73 4.4.5.Biological activity of dry leaf-essential oil…………….. 73 4.4.6.Biological stability of Lantana camara essential oil…… 76 4.5. Determination of the active antifungal ingredient of( LC1)…… 78 4.6. GC/MS analysis of lantana camara (LC1)essential oil……… 84

CONCLUSION AND RECOMMENDATIONS………………..……. 100 5.1. Conclusion & Recommendation………………………………... 100 References ………………………………………………………….. 101 Appendices……………………………………………………………….. Appendix1 ……………………………………………………………...... 108 Appendix 2 ………………………………………………………………. 110 Appendix 3 ……………………………………………………………… 118

vii LIST OF TABLES

Table No. Page 2.1. Number of isoprene units incorporated in the basic molecular skeleton……………………………………………………….. 17 2.2. Family – Specific plant tissues responsible for production or 26 storing essential oil…………………………………………….. 2.3. Plant organ containing natural essential oils………………... 28 2.4. Heterogeneous groups present in essential oils…………….. 30 2.5. Classification of Essential oil compounds…………………... 34 2.6. Essential oil classification based on molecular class or family.. 35 3.1. Indigenous varieties of lantana camara L…………………… 51 4.1. Botanical variability of indigenous lantana camara L……….. 58 4.2. Essential oil content(%) indigenous lantana varieties……..… 64 4.3. Some phyosico chemical properties of lantana essential oil… 65 4.4. TLC separated components of essential oil of indigenous lantana nine varieties…………………………………………. 65 4.5. Antibacterial activity of Lantana camara essential oil (20l/ml/disc) fresh leaves on different bacteria ……………. 69 4.6. Inhibition (%) of essential oil of Lantana camara on the growth of bacteria……………………………………………. 69 4.7. Inhibition (%) of essential oil of Lantana camara on the growth of fungi………………………………………………………. 72 4.8. Stability of Antifungal activity of L. camara essential oil ….. 77 4.9. Stability of Antibacterial activity of( LC1)…………………. 77 4.10 Antifungal activity of TLC separated components of E.O of 79 (LC1) leaves……………………………………………………

viii 4.11 Antifungal activity of C1 and C6 ……………………………. 81 4.12 Observed FTIR spectrum for compound( LC1)……………… 89 4.13. Interpretation from FTIR ,U.V. spectroscopy &GC/MS analysis. .99

viii LIST OF FIGURE

Figure Page No. 2.2 Structure of some phenolic compounds 13 2.4 Structure of two "true" tri terpenes 20 2.5 Example of structural types of tri terpenes steroids 21 2.6 Representative chemical structure for mono terpene 24 2.7 Example for structure of sesqui terpenes 25 4.1.a Photos of Lantana flower color 59 4.1.b Photos of Lantana leaf type 60 4.1.c Photos of Lantana stem shape 61 4.2 TLC separated components of fresh leaves of five varieties of 66 Lantana E.O.(S5, S9, S4, S6, &S7) 4.3 TLC separated components of fresh leaves of four varieties of 67 Lantana E.O.(S2, S3, S8, S1, &S11) 4.4 Photographs of Lantana (S1)* essential oil against 70 Agrobacterium Tumefaciens 4.5 Inhibition of Aspergillus niger by (LC1) E.O. 74 4.6 A photograph of TLC separated components of E.O 80 4.7 Activity of C6(TLC0fraction ,fig 4.6) against A.niger 81

4.8 TLC separation of Lantana camara E.O.(LC1) &commercial 84 standard Essential oil 4.9 GC/MS chromatogram of LC1 85 4.10 FTIR spectrum of C6 active against A. niger 90 4.11 UV spectrum of C6 active against A. niger 91 4.12 GC/MS chromatogram of compound 6 active against A. niger 92

ix List of Abbreviations

 Micro liter E.O Essential Oil DCM Dichloromethane DMAPP Di methyl aellyl Pyrophosphate FID Flame Ionization Detector FTIR Fourier Transform Infrared MVA Mevalonic Acid MEP Mevalonic Pyruvate GPP Gernyl Pyrophosphate IPP Iso pentyl pyrophosphate I.R Infrared

LC50 Lethal Concentration which Kill 50% MIC Minimum Inhibitory Concentration ML Mill liter NA Nutrient Agar PDA Potato Dextrose Agar r.t Room Temperature T.L.C. Thin lager Chromatography UV Ultra Violet Light GCLMS Gas Chromatography / Mass spectra MS Mass Spectra W/V Weight Per Volume

CHAPTER ONE INTRODUCTION 1.1. Importance of natural products Plants produce a diverse range of bioactive molecules, making them a rich source of different types of medicines. Higher plants, as sources of medicinal compounds, have continued to play a dominant role in the maintenance of human health since ancient times. Over 50% of all modern clinical drugs are of natural product origin, however, natural products also play an important role in drug development programs in the pharmaceutical industry. It is estimated that 60% of the world population and 80% of the population of developing countries rely on traditional medicine, mostly plant drugs, for their primary health care needs, (Shrestha and Dhillions, 2003). The potential of new drug discovery of natural products attract scientists of various disciplines (organic chemistry, bioorganic chemistry, pharmacology and biology etc), however, recent attention has been paid to extracts of biologically active components that isolated from plant species. The medicinal value of plants lies in some chemical substances that produce a definite physiological action in human body. Plants are rich in a variety of secondary metabolites; the most important of these are the bioactive constituents: including alkaloids, terpenoids, volatile oils, flavonoids and other phenolics (Edeoga, et al., 2005 and Abubakar, et al., 2008). The increasingly growing rate of antibiotic resistance of microorganisms necessitates the development and research of new antimicrobial agents or resistance modifiers. The plant kingdom provides a wide source for new drugs, therefore substances of herbal origin with

1 antimicrobial properties may be potential candidates for the development of new anti-infective agents. Reversal of multidrug resistance may be another attempt to mitigate the spread of resistance. Interestingly essential oils from some herbs and spices were reported to possess anti-bacterial as well as cancer chemo preventive activities (Lai and Roy, 2004). The spread of drug resistant pathogens is one of the most serious threats to successful treatment of microbial diseases (Hawkey, 2008). University of Birmingham U.K. On the other hand, antibiotics are sometimes associated with adverse effects. Therefore, there is a need to develop alternative antimicrobial medicines for the treatment of infectious diseases from other sources such as plants (Cordell, 2000). Essential oils and other extracts of higher plants may be a new source of antimicrobial agents possibly with novel mechanisms of action (Barbour et al., 2004). Antimicrobial agents represent a main therapeutic tool to control and treat a variety of bacterial infectious diseases. At the highest level, antimicrobial agents can be classified as either bactericidal or bacteriostatic (Hancock. 2005). Bactericidal kill bacteria directly while bacteriostatic prevent them from dividing. However, in practice, both of these are capable of ending a bacterial infection. Lantana cammara is the important medicinal and aromatic plant. Belong to verbenceae family which comprises 100 genera and 2600 species that grow as her 65, shrubs or trees it used as an ornamental plant.

Objectives: The objective of this study is to determine the antimicrobial (antibacterial and antifungal) activities of the essential oil extracted from

2 indigenous Lantana var aculrata against two pathogenic organisms, using the following: (1) Collection and preparation of plant material. (2) Botanical characterization (3) Preparation of the essential oils by steam distillation methods. (4) Micro extraction method. (5) In vitro study of crude and purified extracts using different bioassays technique, including disc diffusion-method and agar- hole disc diffusion method.

(6) Determination of maximum lethal concentration (LC50) and minimum inhibitory concentration (MIC) for biological activity. (7) Perform studies on the biological activities of essential oil constituents separated by thin layer chromatography technique. (8) Identification of active ingredient using preparative thin layer chromatography (PTLC), gas chromatography and mass spectrometry (GC/MS), mass spectrometry (MS), FTIR, and UV spectra.

1.2 . The plant Lantana Camara Lantana Camara, also known as Spanish flager yellow sageor red, belongs to the family Verbenaceae. This family, which comprises 100genera and about 2600 species, is widely distributed in tropical and subtropical region of the world. Members of the genus Lantana are grown in many parts of the world as ornamental plants. This genus consists of about 150 species occurring in tropical and subtropical countries (Innocent et al., 2008). Redsage is the most wide-spread species of this genus (Gaujewala et al., 2009).

All species of Lantana do well in a sunny weather and can stand rigorous conditions of poor soil; but some prefer light fertile soils with good drainage (Lantanea culeata and Lantana mista). Lantana is mostly used as an ornamental plant and often used for hedges and erosion control. In west India lantana used as house hold for nature and used in butter flygandens in United State. Several species of the genus Lantana are used in folk medicine in gastrointestinal, dermatological and respiratory affections (Hernandez et al., 2008). The fruits are used as a famine food by the Zulu in South Africa while people of northern Tanzania regard the plant as poisonous if eaten in large amounts but non-poisonous to sheep and goats (Innocent et al., 2008). Extracts of Lantana camara were reported to possess pharmacological activities as carminative, antispasmodic, antiemetic and to treat respiratory infections as cough, cold, asthma, malaria and bronchitis previous studies related antitumor, antifungal, antimalaria, analgesic and hepatotoxic activities (Barretofs et al., 2010). Extract of the fresh leaves are antibacterial and are traditionally used in Brazil as antipyretic lantana triterpen ester so useful in biological activities (ElGhisalberli, 2000). Lantana is an important aromatic plant as the leaves and flowers contain an essential oil with potential uses in perfumery and flavor industries. Egyptian grown Lantana Camara contained 29 chemical compounds, the essential oil of leaves and flowers the major ones being Caryphyllene, Cineole and pinene (Abdel-hady et al., 2005). This composition is different from that reported for other species, Lantana achyiantifolia (Hernandez et al., 2005) and lantana xenica (Juliani et al., 2002).

Essential oils are odorous products obtained from natural raw materials such as leaves, fruits, roots, flowers and wood of many seasonal or perennial plants. They are generally of complex composition and contain alcohol, aldehydes, ketones, phenols, esters, ethers and terpens varying proportions. Estimated 3.000 essential oils are known of approximately 300 are of industrial importance the majority of them are obtained from agricultural plants but some 28 essential oils are collected in commercial quantities from wild sources. World total production of essential oils is estimated at about 100.000 metric tons. The major exporters are the United States, the European Union and a number of developing Countries. Lantana Camara was introduced to Sudan as an ornamental plant. Different varieties exist as judged by flower colour. In the last few years more introductions were made especially of dwarvranieties or species. So far the essential oil of Sudanese grown lantana has not been chemically nor biologically evaluated, through the plant grows quite successfully in the country. The objectives of this study is collection, identification and au thentification of the different "lantana" growing in Sudan and to determinate of the chemical composition of the essential oil of leaves identified of preserved Lantana's. Study of some biological activities of Lantana essential oils e.g. antibacterial and antifungal.

CHAPTER TWO LITERATURE REVIEW 2.1. Natural products A natural product is a chemical compound or substance produced by a living organism-found in nature that usually has pharmacological or biological activity for use in pharmaceutical drug discovery and drug design. A natural product can be considered as such even if it can be prepared by total synthesis. Natural products may be extracted from tissues of terrestrial plants, marine organism or microorganism fermentation broths. The natural product compound has some form of biological activity and that compound is known as the active principle. Such a structure can act as a lead compound. Not all natural products can be fully synthesized and many natural products have very complex structures that are too difficult and expensive to be synthesized on an industrial scale. Natural product synthesis is heavily integrated with medicinal and combinatorial chemistry as well as the traditional organic disciplines.

2.2. Plant-derived natural product Plants produce a huge number of natural products (mostly so- called secondary metabolites). These compounds, also called phytochemicals, have important ecological functions (Anne et al., 2009). Chemical compounds produced by plants fall into two groups, primary and secondary products.

2.2.1. Primary plant products: Primary natural products are those that are involved in primary metabolic pathways such as carbohydrates, nucleic acids, lipids and amino acid synthetic and degradative pathways (Brian, 2011).

2.2.1.1. Carbohydrates: A carbohydrate is an organic compound that is composed of the atoms of carbon, hydrogen and oxygen. Some carbohydrates are relatively small molecules, called monosaccharide's, the most important of which are the sugars glucose and fructose. Other more complex carbohydrates, called polysaccharides, are built from monosaccharide units. Polysaccharides more complex carbohydrates polymers consisting of ten to hundreds to several thousand monosaccharide's units. Polysaccharides are synthesized by plants, animals and humans to be stored as food, structural support or metabolized for energy. Plants store glucose as the polysaccharide starch (Chares and Ophardt, 2003). Polysaccharides are mostly insoluble in water. Simple carbohydrates are digested quickly while complex carbohydrates are digested more slowly (Jon Mohrman, 2011). The primary function of carbohydrate polysaccharides such as starch is for short term storage of energy, yielding metablizable monosaccharide's when needed for cellular metabolism. Other polysaccharides including cellulose play important structural roles, resulting in rigidity of plant stems. Carbohydrates are found in fruits, vegetables and grains which are important energy sources for humans.

2.2.1.2. Amino acids and proteins: Amino acid serve as the building blocks of proteins. An amino acid contains both a carboxyl group (COOH) and an amino group (NH2). There are 20 amino acids derivable from proteins. Plants, in addition, contain a large number of amino acids not used in protein synthesis, the so called non-protein amino acids.

One of the most common ways to classify amino acids is to group them according to the nature of their side chains. The nine so-called essential amino acids occupy a key position in that they are not synthesized in animals and humans but must be ingested with feed or food (Wolfgon et al., 2005). There are amino acids with polar, uncharged side chains e.g. serine and thereonine which have hydroxyl groups. Asparagine and glutamic acid have amide groups. Histidine and tryptophan have heterocyclic aromatic amine side chains while tyrosine has a phenolic side chain. Non polar side chain amino acids include glycine, alanine, proline, valine and phenyl alanine. There are four amino acids with charged side chains. These include aspartic acid and glutamic acid which have carboxyl groups on their side chains. Arginine and lysine have side chains with amino groups. Proteins made by linking amino acids together end to end with a peptide bond. The peptide bond made between two amino acids is created by a condensation reaction. Dipeptides have two amino acids linked together, tripeptides have three and polypeptides have many amino acids linked together. Amino acids are important in nutrition and are commonly used in nutrition supplements, fertilizers, food technology and industry.

2.2.1.3. Lipids: Lipids are organic compounds that contain hydrocarbon structures and are related by their solubility in non polar organic solvent such as ether, chloroform, acetone and benzene and also by their general insolubility in water. Lipids are essential constituent of plant cells. The vegetative cells of plants contain 5 to 10% lipid by dry weight. Lipids are the major form

8 of carbon storage in the seeds of many plant species, their major site of synthesis is within the plastid (John Browse et al., 1995). Lipids function as energy storage molecules (e.g. fatty acids) as well as components of cellular structures. Fatty acids, building blocks of many complex lipids, usually have long chains which are either saturated or unsaturated (contain at least one carbon-carbon double bond). Lipids are variable in structure, many are insoluble in water because they lack polar groups. Lipids provide insulation in animals, are energy storage forms, are components of cell membranes (phospholipids) and lipids such as steroids are important cell messengers. Waxes, which are esters of fatty acids and long-chain alcohols, are found in animal, plant and microbial tissues and they have a variety of functions, such as acting as energy stores and water proofing. The nutritionally important lipids are fats (solid) and oils (liquids) that consist of fatty acids of up to 20 carbons in their chains. Most of these lipids are found in food in the form of triglycerides which are fatty acid esters of glycerol. Other types of dietary lipids are cholesterol and phospholipids. Glycerophospholilipds are major structural lipids in cellular membrane systems and play key roles as suppliers of the first and second messengers in the signal transduction and molecular recognition processes. Triacylglycerols, the major energy storage forms of lipids in plants and animals, are composed of glycerol (1, 2, 3-trihydroxy propane) and 3 fatty acids. The old name was triglycerides. Fatty acids may be classified as essential or non essential. Essential fatty acids are those that our bodies can not synthesize and must be obtained from our diets. Non essential fatty acids are those that we can make in our bodies because we have the proper enzymes present.

There are two classes of phospholipids, the first are the glycerophospholipids, in which case the molecule is composed of glycerol substituted with two fatty acid esters and at the third position a phosphate unit connects to the alcohol. The other subclass of glycerophospholipids are the plasma an logins. Steroids are non hydrolysable lipids with large molecular weights. The only steroids discussed are animal steroids and more specifically ones found in humans, steroids also occur in plants and the cardiac steroids from an important class.

2.2.2. Secondary plant products: Secondary plant products include most of the important natural products used by man. These chemicals are not normally considered as primary metabolites because they are not present as part of the primary metabolic pathways universal to all living cells. Most of them are unique to plants in their occurrence. Their exact functions in plants that produce them have been debatable. Their presence is often restricted to certain plants, e.g., the alkaloids caffeine and nicotine (Brain et al., 2011). The most important sub-classes of secondary plant products are terpenoids (including essential oils), alkaloids and phenolic compounds, discussed below.

2.2.2.1. Alkaloids: Alkaloids are naturally occurring chemical compounds containing basic nitrogen atoms. The name derives from the word alkaline and was used to describe any nitrogen-containing base. Alkaloids are produced by a large a variety of organisms, including bacteria, fungi, plants and animals and are part of the group of natural products (secondary metabolites).

N N O

N N H O Nicotine Coniine HO Atropine

N glc O rha gal O O Solanine (a steroidal glycoalkaloid)

Fig 2.1 Structures of some alkaloids.

There are three main types of alkaloids: True, proto, and pseudo alkaloids. Colchicine is an example of a proto alkaloids, pseudo-alkaloids can be derived from, terpenoids and purines. The basic unit in the biogenesis of the true alkaloids are amino acids. The non-nitrogen containing rings or side chains are derived from terpene units and acetate, while methionine us responsible for the addition of methyl groups to nitrogen atoms. Alkaloids trivial names usually end in-ine (morphine, a tropine, colchicines). Alkaloids are synthesized by plants and are found in the leaf, bark, seed or other parts.

2.2.2.2. Biosynthesis: Biosynthesis pathways to alkaloids depending on the nitrogen source. Alkaloids are biosynthesized from various kinds of amino acids. Complex alkaloids are also biosynthesized by attaching another alkaloid to the original skeleton.

2.2.2.3. Phenolic compounds: Compound of one or more aromatic benzene rings with one or more hydroxyl groups (C-OH). The essential oils are often classified as terpene, many of these volatile chemicals are actually phenolic compounds such as eucalyptol from (Eucalytusglopulus) many phenolic compounds are attached to sugar molecules and are called glycosides or glycosides depending on the type of sugar the double-ring phenolic compound called coumarin imparts the distinctive sweet small to newly mownhay. Some phenolic compounds occur as polymers (often combined with glucose). Tannins are phenolic polymens that combine with the protein of animals skins (collagen) forming leather. Lignin is a valuable

O OH OH O

OH HO OH Phenol OH Cinnamic acid (parent structure) Gallic acid (C6-C3 compound) (a simple phenolic)

O O

HO Eugenol Flavone O (a phenyl propene; (parent of flavonoids) an essential oil component)

OH OH OH OH

+ HO O HO O

OH OH OH O OH Quercetin Cyanidin (a typical flavonoid compound) (of the anthocyanidin subgroup of flavonoids)

Fig 2.2 Structures of some phenolic compounds.

13 phenolic polymers that give wood its characteristic brown colour, density and mass. Flavonoids the largest group of phenolic which are 3-rings phenolic compounds consist of a double ring attached by a single bond to third ring. In leaves they block far ultra violet (UV) light (which is highly destructive tonucleicacids and proteins) while selectively admitting light of blue and red wave lengths which is crucial for photosynthesis. Mostly anthocyanins are responsible for the colour of many flowers and can range from red to blue, depending on the pH of the water gsapin the vacuoles.

2.2.2.4. Flavonoid pigments: Flavonoids are colorless or yellow flavonoids found in leaves and many flowers. Quercetin is the yellow flavonol pigment of oakpollen. The fall coloring decido-ustrees may involve carotenoid pigments (terpens) as well as flavonoids. In some trees, such as red scarlet oak (Quercus coccinea), colorless flavonoils are converted into red anthocyanin as the chlorophyll breaks down. Flavonoids with glucose side chains are called glucoflavonoids or glucosides (glycoside if the sugar is not specified) while the flavonoid component without sugar is called anaglycone. Some nutritionists recommend flavonoids (bio flavonoids and is flavonoes) in order to maintain healthy tissues and promote the proper balance of hormones and antioxidants in the body. They may be obtained supplements and from good diet of fruits, vegetables and soy protein.

2.2.2.5. Classification of flavonoids: The flavonoids are poly phenolic compound possessing 15 carbon atoms, two benzene rings joined by linear three carbon chain. The skeleton above, can be represented as the C6-C3-C6 system. The chemical structure of flavonoids are based on a C15 skeleton with a chromane ring bearing a second aromatic ring B in position 2, 3 or 4. Most of these (flavonones, flavones, flavonols and anthocyanins) bear in ring B in position 2 of the hetero cyclic ring. In isoflavonoids, ring B occupies position 3.

2.2.2.6. Isoflavonoids: Most flavonoids, isoflavonoids have another limited taxonomic distribution, mainly within the leguminosae. The isoflavonoids are all colorless. It has been established that acetate gives rise to ring A and that phenylalanine, cinnamate derivatives are incorporated into ring B and C- 2,-3, and 4 of the heterocyclic ring.

2.2.2.7. Terpenoids: Terpenoids form a group of naturally occurring compounds majority of which occur in plants, a few of them have also been obtained from other sources. Some terpenoids are volatile substances which give plants and flowers their fragrance. They occur widely in the leaves and fruits of higher plants, conifers, citrus and eucalyptus. The term ‘terpene’ was given to the compounds isolated from terpentine, a volatile liquid isolated from pine trees. The simpler mono and sesqui terpenes are chief constituent of the essential oils obtained from sap and tissues of certain plants and trees. The di and tri terpenoids are not steam volatile. They are obtained from plant and tree gums and

15 resins. Tetra terpenoids form a separate group of compounds called ‘Carotenoids’.

2.2.2.8. General properties of Terpenoids: 1. Most of the terpenoids are colorless, fragrant liquids which are lighter than water and volatile with steam. A few of them are solids e.g. camphor. All are soluble in organic solvent and usually insoluble in water. Most of them are optically active. 2. They are open chain or cyclic unsaturated compounds having one or more double bonds. Consequently they undergo addition reaction with hydrogen, halogen, acids, etc. A number of addition products have antiseptic properties. 3. They undergo polymerization and dehydrogenation 4. They are easily oxidized nearly by all the oxidizing agents. On thermal decomposition, most of the terpenoids yields isoprene as one of the product. The terpenoids, sometimes called isopernoids, are a large and diverse class of naturally occurring organic plant chemicals. Plant terpenoids include the essential oils, used extensively for their aromatic qualities. Terpenoids may be defined as a group of molecules whose structure is based on a various but definite number of isoprene units (methyl buta-1,3-diene, named hemiterpene, with 5 carbon atoms). Isoprenoid units are also found within the frame work of other natural molecules. Thus, indole alkaloids, several quinones, alcohols, formed from b-carotene, phenols, isoprenoid alcohols also contain terpenoid fragments (Bouvier, 2005).

2.2.2.9. Biosynthetic pathways of terpenoids in plants: Most natural terpenoid hydrocarbon have the general formula (C5H8). The basic biological isopreneoid units are isopentenyl pyrophosphate (IPP). In plants, IPP may be synthesized in either the cytosol or in the stroma of plastids, using different precursors at each site. The cytosolic pathway utilizes acetate and is named the mevalonate (MVA) pathway after the important intermediate mevalonic acid. This is the only pathway that operates for terponoid biosynthesis in fungi and animals Fig. The plastidic pathway in the green parts of plants and in algae involves glyceraldehydes 3-phosphate and pyruvate, and again the pathway is named after an important intermediate, methyl erythirtol phosphate (MEP) (Eisenreich, 2001). It is generally accepted that the cytosolic pool of IPP serves as a precursor of sesquiterpenes, triterpenes, sterols and poly terpenes whereas the plastid pool of IPP provides the precursors of mono-,di-and tetraterpens (Bohlmann, 1998). The most accepted classification of terpenes is based on the Table 2.1number of isoprene units incorporated in the basic molecular skeleton. Name of Terpenoid No. of isoprene units Carbon atoms per sub-class No. of C5 molecule Mono terpenes 2 10 Sesqui terpens 3 15 Di terpenes 4 20 Sesterterpenes 5 25 Tri terpenes 6 30 Carotenoids 8 40 Rubber >100 >500

Mono-, sesqui-, di-, and sester-terpenes contain the isoprene units linked in a head to tail fashion. The tri terpenes and carotenoids (tetra terpenes) contain two C15 and C20 units, respectively, linked head to head.

Acetyl-Co A G3P + pyruvate MVA MVP pathway pathway HMG – Cp A D px Fasmidomycin

Mevalonic NEP acid

DMAPP IPP HMBPP

GPP IPP DMAPP

EPP(C15) GPP

x x2 Mono terpens

Sqyakebe (C30) GGPP

Brassinosteroids sterols Phytoene(C40)

Phytol(C20) ent-kaurene (C20)

Cnt-Kaurcnoicacid

GA12 Carotenoids Other Gibberellins

Fig.2.3: Two biosynthetic pathways to terpenoids in plant. The MVA pathway is in the cytosolm the MEP pathway in the stroma of plastiods Many terpens are purely hydrocarbons, but oxygen-containing compounds such as alcohols, esters, aldehydes or ketones re also found. These derivatives are frequently named terpenoids. Mono- and sesqui-terpenes are the chief constituents of the essential oils while the other terpenes are constituents of resins, waxes and rubber.

Oleoresin is a mixtures of turpentine (85% C10-monoterpens and 15%

C15-sesquiterpen) and rosin (C20-diterpene) that acts in many conifer species to seal wound and is toxic to both invading insects and their pathogenic fungi (Steel, 1998). A number of inducible terpenoid defensive compounds (phytoalexins) from angiosperm species are well known (Stoessl, 1976). These include both sesquiterpenoid and diterpenoid types.

2.2.2.10. Tri terpenoids: Members of this class of compounds have molecular skeletons containing 30 carbon atoms. Tri terpenes are assembled from C5 isoprene unit through the cytosolic merlonate pathway to make C30 compounds. Some are steroidal in nature. Cholesterol is one example of a tri terpene. The tri terpenes are subdivided into some 20 groups depending on their particular structures. Mono- and sesqui-terpenens, the two subclasses of terpenoids that constitute the essential oils are separately discussed below.

Abietic acid COOH Hardwickiic acid COOH

Two structures of diterpenes.

COOH

COOH HO Oleanolic acid HO Ursolic acid

Fig 2.4 : Structures of two ‘true’ triterpenes

Stigmasterol HO Squalene ( a plant sterol) (precursor for steroids)

Cholesterol Ergosterol HO HO ( an animal sterol; (a fungal sterol) steroid hormone precursor)

Estrone (a female steroid hormone)

Fig 2.5: Examples of structural types of tri terpene steroids

2.2.2.11. Essential (volatile) oils: An oils is any substance that is liquid at ambient temperatures an does not mix with water but may mix with other oils and organic solvents. This general definition includes vegetables oils, volatile essential oils, petrochemical oils, and synthetic oils. The oils of inters there are the essential oils of plant origin. An essential oil is a concentrated hydrophobic liquid containing volatile aroma compounds from botanical plant parts such as flower, seed, leaves bark and wood. Essential oils are also known as volatile oils, ethereal oils or aetherolea. They are generally extracted by distillation and used mainly by the perfume and cosmetic industries. Pure essential oils or plant organs that contain them have been used in cooking, medicine and in religious rituals in every culture. Aroma therapy is probably the most popular of essential oil. It is used here to enhance mood and balance and for relaxation and stress relief. Perhaps the most important essential oils are lavender, lemon, rosemary, eucalyptus and tea tree oils. Essential oils have been extracted from over 300 plants, of which 200 to 300 are commonly traded on world markets. 2.2.2.12. Essential oil classes: Mono- and sesqui-terpenes could be subdivided into subclasses according to the number of rings present in the structure. i) Acyclic Terpenoids: They contain open structure. ii) Monocyclic Terpenoids: They contain one ring in the structure. iii) Bicyclic Terpenoids: They contain two rings in the structure. iv) Tricyclic Terpenoids: They contain three rings in the structure.

v) Tetra cyclic Terpenoids: They contain four rings in the structure.

2.2.2.13. Sources of Natural Essential Oils: Plant organs containing natural essential oils are illustrated in Table (2.2): Essential oils are generally derived from one or more plant parts, such as flowers (e.g. rose, jasmine, carnation, clove, mimosa, rosemary, lavender), leaves (e.g. mint, Ocimum spp., lemongrass, jamrosa), leaves and stems (e.g. geranium, patchouli, petitgrain, verbena, cinnamon), bark (e.g. cinnamon, cassia, canella), wood (e.g. cedar, sandal, pine), roots (e.g. angelica, sassafras, vetiver, saussurea, valerian), seeds (e.g. fennel, coriander, caraway, dill, nutmeg), fruits (bergamot, orange, lemon, juniper), rhizomes (e.g. ginger, calamus, curcuma, orris) and gums or oleoresin exudations (e.g. balsam of Peru, balsam of Tolu, storax, myrrh, benzoic).

a) Acyclic:

OH OH

geraniol myrecene linalool b) Monocyclic:

alpha-terpineol limonene

c) Bicyclic:

alpha-pinene thujone d) Irregular:

.. H glc .. O .. O O O HO

Loganin Thujaplicin O O

Fig (2.6 ): Representative chemical structures for mono terpenes

24 a) Acyclic:

HOH2C OH Farnesol Neroliool b) Monocyclic:

COOH O

Bisabolene Abscisic acid

c) Bicyclic:

Cadinene Sterene d) Sesquiterpene lactones:

O O O O O Santonin O Xanthinin

Fig (2.7 ): Examples for structures of sesquiterpenes.

Table (2.2): Family – Specific plant tissues responsible for production or storing essential oil.

Resin Modified Glandular Gum canals Umbelliferae Schizogenous Lysigenous parenchyma Cavities cavities canals hairs cells

Conifers Piperaceae Labiatae Cistaceae Vitae Rutaceae Conifers Verbenaceae burseraceae (oils tubes ) Geraniaceae

Specialized plant structures that produce and store essential oils are shown in Table (2.1). Depending upon the plant family, essential oils may occur in specialized secretary structures such as glandular hairs (Labiatae, Verbenaceace, Geraniaceae), modified parenchyma cells (Piperaceae), resin canals (conifers), oil tubes called vittae (Umbelliferae), lysigenous cavities (Rutaceae), schizogenous passages (Myrtaceae, Graminae, Compositae) or gum canals (Cistacae, Burseraceae). It is well known that when a geranium leaf is lightly touched, an odor is emitted because the long stalked oil glands are fragile. Similarly, the application of slight pressure on a peppermint leaf will rupture the oil gland and release oil. In contrast, pine needles and eucalyptus leaves do not release their oils until the epidermis of the leaf is broken. Hence, the types of structures in which oil is contained differ depending on the plant type and are plant- family specific. Unfortunately, not enough is known even today about these oil secretary structures to carefully categorize them. From the practical standpoint, they can be categorized into superfi cial and subcutaneous oils. Based on the currently available information, it may be inferred that oils of the Labiatae, Verbenaceae and Geraniaceae families are the only superfi cial oils known; consequently, the others are considered subcutaneous oils. During handling, some flowers continue to produce aroma while other quickly loose their odor. Flowers collected at different times may also give different perfumery values. Regarding the rose, half-open flowers with plump anthers give higher oil yield than fully opened flowers with shriveled anthers. Humidity, wind, rain and surface temperature also affect the oil yield considerably. Harvesting schedule affects both quantity and quality of the oil.

Table (2.3): Plant organ containing natural essential oils. Leaves Leaves Flowers Fruits Seeds Roots Woods Barks Rhizome stems Mentha Geranium Rose Bergamot Fennel coriander Angelica Cedar Cinnamon Ginger Ocimum Patchouli Jasmine Orange Caraway Sassafras Santal Cassia Calamus Lemongrass Petitgrain Carnation Lemon Dill Vetiver Pine Canella Orris Jamrosa Verbena Orange Juniper Nutmeg Saussurea Curcuma Cinnamon Clove Valerian Mimosa Rosemary Lavender

2.2.2.14. Essential Oil Constituents Major constituents of essential oils are shown in Table (2.4), from which it is clear that most essential oils consist of hydrocarbons, esters, terpenes, lactones, phenols, aldehydes, acids, alcohols, ketones, and esters. Among these, the oxygenated compounds (alcohols, esters, aldehydes, ketones, lactones, phenols) are the principal odor source. They are more stable against oxidizing and resinifying influences than other constituents. On the other hand, unsaturated constituents like mono terpenes and sesquiterpenes have the tendency to oxidize or resinify in the presence of air and light. The knowledge of individual constituents and their physical characteristics, such as boiling point, thermal stability and vapor-pressure-temperature relationship, is of paramount importance in technology development of oxygenated compounds, (ICS, 2008).

2.2.2.15. Chemical structures of essential oils: The total essential oil content of plants is generally very low (<1%). Essential oils chemistry is very complex in nature as essential oils themselves have many chemical ingredients. Some play a major part and others a minor part. The ingredients found in essential oils are organic due to their molecular structure which is based on carbon atoms held together by hydrogen atoms. Oxygen atoms and sometimes nitrogen and sulphur atoms are also present. Below is a list of the main chemical components found in essential oils and information on each one:

 Mono terpenes

 Sesqui terpenes

 Phenols

 Alcohols

 Ethers / Esters

Table (2.4): Hetero geneous groups present in essential oil. Alcohols Aldehydes Acids Terpene Hydrocarbon Phenols Phenol Ester Oxides Ketones ethers Ceraniol Citral Benzoic Limonene Cymene Eugenol Anethol Benzoates Cineol Camphor Citronellol Citronellal Cinnamic Phellandrene Myrcene Thymol Safrol Acetates Carvone Menthol Benzaldehyde Myristic Pinene Sabinene Carvacrol Salicylates Menthone Linalool Cinnamaldehyde Isovaleric Camphene Storene Cinnamates Pulegone Terpineol Vanillin Cedrene Thujone Borneol

 Ketones

 Coumarins

2.2.2.16. Mono terpenes: These chemicals are found in most essential oils, with citrus oils having a very large percentage of them. They have high antiseptic and tonic qualities and are very good air purifiers. Mono terpenes contain 10 carbon atoms. They are colorless and highly volatile. They can deteriorate very quickly therefore need to be kept at cool temperatures. Examples of mono terpenes are limonene (found in most citrus oils such as lemon), pinene (found in pine), and camphene (found in camphor) (Essential oil recipes, 2011). Limonene for example, is the precursor of the main constituents of the mono terpenes in mints Menthe spp (Lamiaceae), including carvone and menthol . Limonene and other citrus oils have anti tumor effect in mice, though the action may be lost or even reversed as the hydrocarbons gradually become oxidized .

2.2.2.17. Sesqui terpenes: These terpenes are not as volatile as mono terpenes. They contain 15 carbon atoms (hence the name 'sesqui' which means one and a half). Sesqui terpenes have a calming effect as well as being anti inflammatory and anti- infectious. Examples of these include zingiberene (found in ginger), cedrene (found in cedar wood), and caryophellene (found in clove).

2.2.2.18. Phenolic essential oils Phenols are the most antiseptic chemicals found in plants. They stimulate the body and can be beneficial in small doses however large doses can be a poison to the nervous system. Large doses can also cause skin irritations as well as digestive discomfort to sensitive people. Examples of phenols are thymol (found in thyme), and eugenol (found in clove).

2.2.2.19 Alcohols: Alcohols are also highly antiseptic, antibacterial, anti-fungal and antibiotic. They are a good tonic to the nervous system and can stimulate the immune response. They are far less aggressive than phenols and examples include lavendulol (found in lavender), nerol (found in neroli), and geraniol (found in geranium).

2.2.2.20. Ethers / Esters: Ethers are much stronger than esters but both have similar properties. They are a powerful antispasmodic, antibacterial, and anti-inflammatory. They are very gentle on the skin and particularly efficient in relaxing and rebalancing the nervous system. Examples of ethers and esters include cinnamyl acetate (found in cinnamon), and myrtinyl acetate (found in myrtle).

2.2.2.21. Ketones In small doses, ketones can relax and sedate. They can promote the healing of scar tissues and are known to be anticoagulants. They can be useful in stimulating the immune system as well as treating respiratory complaints. In large doses, however, they can have the opposite effect and can be a poison to the nervous system. This can cause miscarriage, convulsions, and even epileptic fits. Examples of ketones include thyone (found in sage), pinocamphone (found in hyssop), and carvone (found in peppermint).

2.2.2.22. Aldehydes The properties of aldehydes are similar to the properties of both alcohols and ketones. They can calm the nervous system and they are anti- inflammatory. Aldehydes can be quite harsh however, and can cause major irritation to both the skin and mucous membranes. Examples include furfural (found in lavender, sandalwood, cinnamon and cypress), and aldehyde benzoic (found in benzoin)

2.2.2.23. Coumarins These chemicals have a relaxing and sedative effect. They are known to have properties that are anticonvulsant and anti-coagulant. Coumarins, in particular, furocourmains can be photosensitive therefore it is best to avoid exposure to the sun when using essential oils with these constituents. Examples of coumarins include bergaptene (found in bergamot), angelicine (found in angelica), and citroptene (found in most citrus oils). (Essential oil recipes,2011). The major subclasses or families of terpene essential oil constituents are listed in

Table (2.5): Classification of essential oil compounds : Compound Description Hydrocarbon Contain only carbon and hydrogen atoms. Alcohol Contains a hydroxyl group (OH) attached to the terpene structure. Aldehyde Terpenoids with a carbonyl group(C=O) and hydrogen bonded to a carbon. Cyclic Aldehyde group attached to benzene ring aldehyde Ketone Contains a carbonyl group bonded to two carbon atoms. Phenol Hydroxyl group attached to benzene ring. Phenolic ether Contains an O between C and benzene ring. Oxide Has and O bridging 2 or more carbons. Ester Condensation product of acid and alcohol.

Table (2.6): Essential oil classification based on molecular class or family:

Molecular class Ending Compound Essential oil examples Hydrocarbons Ene Pinene, limonene Citrus, pine . Alcohols ol Linalool, menthol, Coriander, tea tree, terpinen-4-ol. peppermint. Sesquiterpene ol Alpha –bisabolol, German chamomile, alcohols santolol, farnesol. sandalwood Phenols ol Thymol, borneol. Thyme, oregano. Aldehydes al Citral Citronella, lemon balm, lemon myrtle. Cyclic aldehydes Hyde Cinnamic Cinnamon , bitter aldehyde, almond, cumin. benzaldehyde. Ketones One Pulegone, thujone, Pennyroyal, thuja, piperitone, sage, eucalyptus camphor. radiate Esters Ate Methyl salicylate, Lavender, linalyl acetate wintergreen, clary sage. Oxides Ole 1-8 cineole, Eucalyptus , ascaridole, linalool wormseed, cajeput. oxide. Phenyl propanes Ol Eugenol, safrol, Aniseed, clove, elimicin, anethol. tarragon, myrtle leaf. Sesquiterpenes Ene Bisabolene, German chamomile, chamazulene, yarrow. caryophyllene. Diterpenes Ol Scalreol, carnosol Clary sage, rosemary. . Lactones in, one Coumarins- Angelica, celery, umbelliferone, bergamot. bergapten Sesquiterpene In Helenalin, Elecampane, arnica lactones

2.2.2.24. Biosynthesis of essential oils 2.2.2.24.1. Essential oils are biosynthesized in plants two pathway 1) The terpenoid pathway There are two biosynthetic pathway, the mevalonate path way and the non-mevlonate pathway. For the terpenoid, building blocks: isopentenyl di phosphate (ipp) and di methyl allyl pyrophosphate (DMAPP). The action of prenyltrans order buiding blokes; geranyl di phosphate (GPP), farsenyldi phosphate (FPP) and gernayl gernayldi phosphate (GGPP), which are the precnrsors of mono terpenoids (C10), sesquiterpenoids (C15) and diterpenoids

(C20), respectively. Condensations there building blocks gives rise to the precnrosors of sterols (C30) and carwotenoids (C40). The non-mevalonate pathway plants is coexist in separate cellule compartemts. The non-mevalonate pathway, operating in the platids, is responsible for the formation of essential oil mono terpenes and linalyl acetate some sequi terpenes, diterpenes and cartenoids and phytol. The mevalonate pathway, operating in the cytosol gives rise to tri terpens, sterols and mostsesquterpenes.

2) The Shikimic acid pathway In plants the biosynthesis of prenylipods and isoprenoids proceeds via two independent pathways: The cytosolic classical acetate/mevalonate pathway for the biosynthesis of sterols, sesqui terpenses, tri terpenoids. The alternative, non-mevlonate 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for the biosynthesis of plastid isopernoids, such as carotenoids, phytol (aside-chain of chlorophylls), plastoquinone-9, isoprene, mono-and diterpenes. Both pathways from the active C5-unit isopentyl di phosphate (IPP) as the precursor from which all other isoprenids are formed via heat-to-tail addition. This review memorizes current knowledge of the

novel 1-deoxy-D-xylulose-5-phosphate biosynthesis apparently located in plastids. The (DOXP) pathway of (IPP) formation starts from D- glyceraldehyde-3-phosphate (GA-3-P) and pyruvate, with DOXP-synthesis as the starting enzyme. This pathway provides new in sight into the regulation o chloroplast metabolism (Hatmutk, 1999).

2.2.2.25. Extraction of essential oils: There are many methods of essential oil extraction, the most popular being steam distillation.

2.2.2.25.1. Distillation: The vast majority of true essential oils are produced by distillation. There are different processes used, however, distillation of them, water is heated to produce steam. Which carried the most volatile chemicals of the aromatic material with it. The steam chilled (in condenser) and the resulting distillate is collected the essential oil will normally float on top of the hydrosol (the distilled water component) and may be separated off.

2.2.2.25.2. Steam distillation True steam distillation uses an outside source of steam which pipes the steam into the distillation unit, sometimes at high pressure. The steam passes through the aromatic material, and exits into the condenser. The composition of essential oil components are also determined by analytical methods such as GC-MS and GLC.

2.2.2.25.3. Cold pressing: Cold pressing is used to extract the essential oils from citrus rinds such a orange, lemon, grapefruit and bergamot. The rinds are separated from the

fruit, are ground or chopped and are then, pressed. The result is a watery mixture of essential oil and liquid which will separate given time. The EOV 2000 is a true vertical steam distillation unit made from scientific-grad borosilicate glass. EOV 2000 offers true "dry steam" distillation in all glass (Pyrex) system. Coupled together with ground glass joints, there are no hoses or rubber stoppers in contact with your product. Simple design of EOV 2000 addresses a number of problem associated with competitive stills, because of its "vertical" design, the biomass flask stays dry, none of the " boiling water" is waste fully condensed and trapped in the biomass flask, and because it uses separate flask for boiling and biomass, there's further insurance that over heating or possibly burning your plant material will never happen.

2.3. Microbes of the study: Agrobactrium tumefaciens of the family Rhizobiales Genus: Agrobacterium, species: A. tumefaciens. Scientific name: Rizobium radiobacter. The micro Agrobacterium tumefaciens is harmful to plants and useful to scientists for the same reason. It transfers DNA into plant genomes. Found in soil worldwide, and causes diseases in plants by transferring its own DNA into plant cells (Edward, 2001). The genetic material that is introduced is called TDNA A. bacterium is the only cellular organisms on earth that is naturally capable of transferring genetic material between the kingdoms of life (Agrobacterium: from biological technology (2008) (transferred DNA) which is located on a Tiplasmid. A tiplasmid is a circular piece of DNA found in almost bacteria. Agrobcterium tumefaciens is the causal agent of crown gall disease. Crown affects hundreds of species, particularly fruits, nuts and ornamental plants such as roses. A. tumerfaciens infects a plant, the bacterium travels

throughout the root system, and can wipe out an entire crop. The only option for farmer is to destroy the plants. Agrobcterium can be used to inset a piece of DNA in the middle of a plant gene, thus in activating the gene. The genome has a very unusual structure, some 5.400 genes reside on four DNA elements-a circular chromosome, a linear chromosome, and two small circular structures called plasmids. A. tumefaciens causes disease in humans-by dirty hands or shoes, more than 50 cases in hospital have been reported in the literature of patients with severely compromised immune system being infected by the microbe. Good new has three strains isolate from patients and will be comparing them with the newly sequenced C58 strain.

2.3.1. Cell structure and metabolism: A. tumefaciens contain flagella, which are important in their life cycle as it helps them swim through the soil to find their plant hosts. The bacterium swims towards concentration of phenolic compounds and also small metabolites such as glucose and amino acids which are usually exuded from plant wounds.

2.3.2. Ecology: A. tumefaciens can either freely in the soil or inside plants as a parasite when it is a parasite it used its plant host to produce energy for it (Moore et al., 1997). 2.3.3. Aspergillus niger: Aspergillus niger is a member of the genus Aspergillus which includes a set fungi that are generally considered a sexual. Aspergillus are ubiquitous in nature they are geographically widely distributed.

Aspergillus niger commonly found as apophyte growing and dead leaves, stored grain, compost piles, and other decaying vegetation. The primary uses of A. niger are for the production of enzymes and organic acids by fermentation, food industry and the organisms itself. The taxonomy of Aspergillus primary based on morphological features, rather than the physiological bio-chemical features and genetic characteristics often used to classify bacteria. The major distinction currently separation A. niger from the other species of Aspergillus is the production of carbon black or very dark brown spores from viseriate philides (Raper and Fenmell, 1965).

2.3.4. Toxin production by A. niger Aspergillus niger produce a variety of fungal metabolites termed mycotoxin, depending upon growth condition and the strain of the organisms. A. niger produces oxalic acid and Kojc acid abundantly these to products have only slight acute toxicity (Uenoandueno, 1978). Malformins produced by A. niger are more potent toxin (Kobbe et al., 1977). The majority of human illness is caused by Aspergillus fumigatus and Aspergillus niger and less frequently by Aspergillus flavus and Aspergillus clavatus. The transmission of fungal spores to human host is inhalation. Aspergillus may cause a board spectrum of disease in the human host ranging from hypersensitivity reaction to direct angioin vision. Aspergillus niger is isolated from house dust, soil, plant litter, dried fruit, nuts and seeds, untreated textiles such as jute, hemp and cotton bracts and in horns of rose bushes. A. niger damages foods such as stoned fruits and vegetables, nuts and corn, oil seeds, grains and dairy products.

2.3.5. Industrial use: A. niger is industrially important since it decomposes plastic and cellulose it is used commercially in the degradation of organic waste such as squeeze remains from apple, potatograbage, sugar beet waste water, in been production, and in the production of organic acid and enzymes.

2.3.6. Symptoms of Aspergillus niger: In plant, young plants will wilt suddenly. Areas affected by the disease will become covered in dark fungal growth. Most plant will die with in 30 days of being planted. In animal A. niger production of oxalic acid in animal feed has been shown to adversely affect animal. Symptoms include calcium depletion, physiological abnormalities and lordeath. In human A. niger it has been linked to the formation of lung and idquo, fungal ball and idquo. This ball is created by the growth of A. niger in the lung, however it do senand isquo; tinuade the lung tissue. Symptoms may exhibit as impaired lung function and wheezing. Aolditionally, while some people will be asymptomatic; others may experience weight loss, shortness of breath, fever, tiredness, chest discomfort or may be coughing up blood (Vrabe et al., 1997).

2.3.7. Antifungal medicines: Antifungal medicines are used to treat fungal in factions, fungiane plant like organisms but, unlike plants, they can not turn sunlight into food (photosynthesis) to feed fungi have to break down living tissue instead, which include human tissue. Fungi that cause infections in human are known as dematophytes. Dermatophytes are particularly attracted to a type of tissue called keratin,

which is a tough, water proof tissue that can be found in many parts of the body such as in the nails, hair, skins outer surface.

2.3.8. Antifungal azoles: A zole antifungal drugs inhibit the enzyme tanosterol-14-α-demethy lase, the enzyme necessary to convert lano sterol to ergostrol. Depletion of ergosterol in fungal membrane disrupts the structure and many functions of fungal member leading to inhibition of fungal growth.

2.4. Lantana camara L. 2.4.1. Vernacular names: In English: lantana, German: wandelroschen, Malaysia: Bungapagar and in Brazil and Uruguay are popularly known as cambara chumbinho (Barrelofs et al., 2003, 2010).

2.4.2. Botany: Lantana camara belong to family Verbenaceae this family is wide spread in the tropical and subtropical region. There are many varieties in lantana camara different in flower colour. Lantana camara var. aculata, lantana camara var mistand lantana camara var. triliifolia. The plant can be easily propagated by using matured stem cuttings. The cutting having at least three modes should be taken from nigorous mother plant. The plant start to germinate after 2-3 weeks and ready for planting when the new branches attain the length of about 5-8 cm. the use of the rooting hormones such as IBA (indole butyric acid) and NAA (Naphthalene acetic acid) can help in the development of roots.

2.4.3. Taxonomy: Synonym: lantana Camara aculeata L. Aculeata: with thorns. Prickly and pointed camara: South American vernacular name for a species of lantana. Flower colour: purple, violet, pink, red, orange, yellow, beige. Lantana camara use as an ornamental garden plant and as a notorious weed. The used of flower colour as a primary identification tool need to be evaluated and has found various uses in folk medicine in many parts of the world. The volatile compound of the fresh leaves and flower of lantana camara L. grown in Guangxi-provice resulted in 44 peaks were separated from the leaves and 39 compounds were identified, accounting for 98.83%of the total constituents and from the flowers 50 peaks were separated and 43 compounds were identified, accounting for 98.54%of the total constituents. The major compounds from the leaves were Nerolidol (Journal of Science Edition, 2009). Lantana becomes bushy and produces abundant flowers, stems square, leaves: opposite, orate crenate-dentate, to 25 cm long, rough abore, aromatic when crushed; inflorescence: flat: topped heads to 5 cm across orange-yellow or orange changing to red, or white; peduncles: axilary, longer that the leaves; flowers, pink, orange, white, yellow, lilac and other shades according to the variety, tubular, 4-parted, small; fruit; black 9greenish when immature) fleshy, one seeded drupes, 6 mm in diameter. Soil: well drained, tolerates, season: summer to fall propagation cutting. Poisonous parts: the green, un ripened fruit is very drought dangerous, leaves of lantana also have yielded toxic principle upon extraction, feeding studies indicate that lantana is quite poisonous, about 1% (green-weghtbasis) of body weight is sufficient for bovine toxic reactions. Fresh lantana fed to sheep produce acute symptoms and death with in 50 days at about 2% of the animal weight.

The degree of poisoning depends on the amount of plant consumed and the degreed exposure to sunlight. Lantana contains toxins that cause organism toreact when exposed to the sun.

2.4.4. Poisonous principles: The alkaloid lantana in and tuiter penedrivative, lantadene A, are implicated in poisonings, species of animals affected: children have died after consumption of unripened berries. Beef and dairy cousanere ported to succumb to browsing of lantana.

2.4.5. Distribution: Lantana camara is found in home, shopping and green houses, it is most frequently sold in hanging-baskets. The native range of lantana camara includes Mexico, Central America, Colombia, Venezuela, Kenya, India, Australia and much of Africa it colonizes new area when its seeds are dispersed by birds.

2.4.6. Folk-medicinal uses: Several species of the genus lantana are used in folk medicine in gastrointestinal, dermatological and respiratory affections (Hernandez et al., 2008). In South Africa while the Lou of Northern Tanzania regard the plant as poisonous if eaten in large amount but non-poisonous to sheep and goats (Innocent Ester et al., 2008). The use of flower colour as a primary identification tool needs to be reevaluated. Extract of lantana camara find many pharmacological uses as carminative, antispasmodic, antiemetic and to treat respiratory infections as cough, cold, asthma, malaria and born chit is previous studies related anti

tumor, antifungal, anti malaria, analgesic and hepato toxic activities (Barretofs et al., 2010). Some tri terpene ester so use falin biological activity (El Ghisalberli, 2000). Extracts of the fresh leaves are antibacterial and are traditionally used in Brazil as an antipyretic.

2.4.7. Chemical composition: Chemical composition of leaves essential oils of lantana camara in the India were analyzed and resulted in the identification of 71 constituents, the major constituents were germacrene-D, Y-elemenc, β-caryophyllene, β- elemenc, α-copaene and α-cadirene (M-Khan et al., 2002). The chemical composition of fruit and stem essential oils of lantana camara from northern plains of India resulted in the identification of 52 and 66 constituents, representing 98.1% and 96.6% of the oils respectively. The major constituents in the fruits oil were palmitic acid, stearic acid and frmacrenw-D while the major constituents in the stem oil were palmitic acid and stearic acid (M-Khan et al., 2003). The components characterizing the pink-violet flower chemo type are sabinene, 1,8-cineole, linalool, β-caryophyllene, α-humulene, β-bi sabolene, Y-cadinene-ar-curcumene, canyophyllene oxide and davanone. The components characterizing the yellow-orange flower chemo type are sabinene, 1,8-cineaole, linalool, β-caryophyllene, α-humulene, caryophyllene oxide, β-bisabolene, Y-cadinene-ar-curcumene and davanone. Yellow-orange and pink-violet colour of flower plants of lantana camara essential oils were studied (Jean et al., 2005).

2.4.8. Biological activities of the essential oil of Lantana camara: The essential oil of L. camara, obtained by distillation, has been shown to possess several biological activities including herbicidal, antimicrobial, insecticidal and antioxidant properties. The lantana camara oil was assayed against several microorganisms, showing moderate antibacterial activity against the human pathogen and antifungal activity, fungicidal, insectidal and nematicidal activity (J-Chill- Chen, 2009). It leaves are used as, anti tumor, antibacterial and tihypertensive agent (Taoubi et al., 1997). Several tri-terpenoids, naphaquinones, flavonoides, alkaloids and glycosides isolated from this plant are known to exert diverse biological activities including cytotoxic and anticancer properties (Ghisalberti, 2000). The presence of toxic lanthanide e.g.: lantadene A (65) and lantadene B (66) was reported. In Lantana camara var. anovel tri terpenoid has been isolated, from the leaves, lantadene-D. It's structure has been established as 22-β-isobutyroy- toxy-3-oxoolean-12-en-28-oic acid (Ompsharma et al., 1990) (Fig. lantadene- D). Lantanoside (1), lantanone (2) and the known compounds linaro side (3) and camarinic acid (4) are flavonoids isolated from aerial parts of lantana camara. Compound 1,3 and 4 were tested for nematicidal activity against root- knot nematode meloidogyne in cognate and showed 90, 85 and 100% mortality, respectively, at 1, 0% concentration. Linaro side (1) and lantano side (2) and their common acetyl ederivative (3) were examined for anti mycobacterium activity against mycobacterium tube culosis, strain (H[37]) Rv these compounds exhibited 30, 37 and 98% inhibition, respectively 6.25 micro gm (-1) concentration. Among these flavonoids acetylated compound was found to be the most activity (Begum et al., 2008; Begum S et al., 2000).

2.4.9. Commercial production: Lantana camara is highly variable species. It has been cultivated for over 300 years and now has hundred cultivar and hybrids. Lantana has several uses, mainly as a her biomedicine and in some areas as firewood and mulch (Sharma et al., 1988; Sharma and Sharma, 1989; In Day et al., 2003). In some counties, it is planted as a hedge lo contain or keep out live stock (Ghisalberti, 2000, In Day et al., 2003). Verbascoside, which possess antimicrobial and antitumor activities, has been isolated (Mahato et al., 1994; In Day et al., 2003). Lantanoside, linaro side and camaninic acid have been isolated and are being investigated as potential nemsto Sides (Begum et al., 2000; In Day et al., 2003). Lantana oils is sometimes used for the treatment of skin itches as an antiseptic for wounds (Anon, 1962) plant extracts are used in folk medicine for a treatment of cancers chicken pox, asthma, tumor, high blood pressure, malaria (Anon, 1962; Gisalberti, 2000, In Day et al., 2003).

2.4.10. Sudanese Lantana camara: Lantana camara was introduced to Sudan as an ornamental plant. Different varieties exist as judge by flower colour. In Sudan there is no use as medicinal plant. Lantana in Sudan consist chemical compounds so far the essential oil of Sudanese grown lantana has not been chemically nor biologically evaluated, through the plant grows quits successfully in the country. Now work was carried out to evaluate and chemically characterize the essential oils of Sudanese Lantana camara. Chemistry not studied/ against agrobacterium. Or asp. not reported

CHAPTER THREE MATERIALS AND METHODS

3.1 Plant collection and preparation: Leaves of nine varieties of Sudanese plant lantana camara used in this study were collected from different parts of Wad Medani, Central of Sudan shown in Table (1). The collected plant materials (leaves) were air dried at room temperature (35 ± 2°C), for two weeks , then after, they were ground in a laboratory grinding mill (Model ED-5) just before extraction.

3.2 Chemicals: All chemicals, solvents and reagents used were of analytical grade. Most of the chemicals used were supplied by British Drug Houses "BDH", England. Solvent purification, when required, was carried out according to standard chemical procedures. Silica gel (G60 F254 "BDH") containing 13%

Ca SO4 was used as thin layer chromatographic materials for plates.

3.3 Equipments: (i) Fourier Transform Infra Red (FTIR) spectrophotometer Model FTIR- 8400S, Shimadzu Corporation was used for identification of isolated compounds. FTIR spectra were calibrated against spectra sol chloroform. (ii) Mass spectra (MS) were determined on Regional Centre for Mycology (RCMB), Fairmont Heliopolis and Tower, Cairo, Egypt. The MS spectrum of isolated compounds was recorded on FAB – MS techniques (V G 70 S E) Mass –electronic U.K., London. MS conditions of operation including, MS ionization (EI) at 70 eV; M/Z range 30-300°C, scan/rate/sec [scan (188), mass peak (140), Ret time

(1.567), base peak (250.40)]. Ionization chamber at 180°C, transfer line at 280 °C Tetrad Tetrad cane was used as internal standard. The identification of separated compounds was evaluated by comparing the MS Kovat’s retention indices (KI) with the MS library. (iii) Gas liquid chromatography–Mass spectra (GC-MS), were determined on GC–MS, model QP2010 at Faculty of Industrial Sciences and Technology, University of Malaysia Pahang (UMP). (iv) A Schimadzu GC-MS GP 2010 was used by kind permission of the Central Laboratory, Ministry of Science Technology, and Khartoum, Sudan. Crude essential oil and its isolated active components were identified by GC–MS spectrum, under the following conditions: Capillary column (50m × 0.20 mm with a film thickness of 0.33µm), split (1: 20), injection temperature (250°C), carrier gas(He) flow (1ml/min), velocity (26 -4 /cm) ,oven temperature (60 – 40 °C at 30 °C / min ), detector temperature (280°C), time run (60 min) and MSD (at 70 eV with scan range 40 -300 amu). (v) An ultra violet lamp having a short wave length (254nm and a long one 365nm) was used for identification of different compounds separated on TLC-plate. (vi) An autoclave model LSB50L was used for sterilization under the following conditions: temp-(121°C) and pressure (15 lb/inch). (vii) Laboratory incubator, model 6307 OGAWA SEIKLO. Ltd. was used for preparation of media. (viii) Standard laboratory equipments, were used, for extraction (steam distillation apparatus), weighing (analytical balance), micro-volumetric (Pipette), TLC-developed plate, spraying (manual-sprayer, SPU 1), grinding (laboratory mill, Model ED-5), drying (oven OSK type 6284),

disc preparation (cork poorer, T. Searle Co.), and fungal culture (Petri – dish). (ix) Glass used in all experiments were cleaned with chromic acid or /and nitric acid followed by distilled water before drying. (x) Glass ware used in all experiments was cleaned with chromic or nitric acid followed by distilled water before drying on an oven. (xi) Standard essential oils: eugenol, linalool, geraniol, limonene, Thymol, methyl trans-cinnamate, citral, Eucalyptol and Lanisole were purchased from educational laboratory services and supplies, England.

3.4 Essential oil extraction: Essential oil extraction was carried out according to the official method (Wagner et al., 1984). Lantana leaves (200 grams) were chopped to reduce their size and to ease the extraction. The prepared plant material was added to 500 ml water in a 1000 ml round bottom flask. A glass U- tube (10-15 cm long; ca. 5mm) is placed between the distillation flask and the receiver (250 ml round bottom flask). The contents of the flask are heated to boiling (boiling stones) and distillation via the U–tube performed slowly until about 50 ml of distillate has been collected in the receiver .Then the distillate was extracted by shaking with 50 ml dichloromethane (DCM). The DCM solution was evaporated at room temperature and then used for either TLC (20 -100µl) or subjected for antimicrobial bioassay.

Table (3.1): Indigenous varieties of lantana camara L.

Sample No. Stem-shape Flower color

(1) Erect Pink

(2) Erect Yellow

(3) Erect Red

(4) Erect Purple

(5) Erect White

(6) Dwarf Orange

(7) Dwarf White

(8) Dwarf Yellow

(9) Dwarf Violet

3.5 Thin layer chromatography (TLC) 3.5.1 Preparation of plates: The TLC thickness layers of. 0.25 mm and 0.5 mm were prepared using equipment from Shandon Scientific Instruments Ltd. The kissl gel DF and

G60F254 "BDH" of silica gel containing 13% CaSO4 was shaken vigorously for about one minute with a volume of distilled water equivalent to twice the weight of gel "w/v" and applied to 20 x 20 cm glass plate set to the required thickness. The plates were heated in an oven for half an hour at 100 0C before cooling in desiccators. The sample was applied to the plate and equilibrated with developing solvent before chromatographic development. After chromatographic development residual solvent was separated spots or bands were visualized using a UV lamp or by spray with a specific reagent.

3.5.2 Preparative TLC: A few micro liters (about 10-20) of lantana leaves extract (Essential oil) dissolved in a small volume of dichloromethane (DCM) were used for separation of essential oil constituents. The solution was applied as band using a micro siring on a TLC-plate coated with silica gel (0.5mm thickness). The plates were developed in a tank containing the solvent mixture toluene: ethyl acetate (93:7) for 45 minutes. After solvent drying at room temperature the developed plate covered with another clean glass plate exposing silica gel about 2cm, at one edge. The two plates were clamped together and provision was made to ensure that only this narrow zone has been reached by the detection reagent in the exposed edge using anisaldehyde reagent. The corresponding bands which were underneath the covered plate were scraped individually with care, transferred to conical flask (100 ml) and eluted with chloroform. After filtration through Whatman filter paper (№ 5 or 4), the filtration was dried at r.t. and the residue (EO) was kept and used for further analysis (i.e. FTIR identification, GC-MS and other biological activity).

3.5.3 TLC solvent system and detection reagent: A) The solvent mixture toluene/ethyl acetate (93:7) and chloroform were used for essential oil separation and elution respectively. B) Anisaldehyde-sulfuric acid reagent: This reagent was prepared freshly by adding 0.5 ml anisaldehyde to 10 ml glacial acetic acid, followed by 85 ml methanol and 4 ml concentrated sulfuric acid. The developed TLC plate was sprayed with about 10 ml of the prepared reagent, and then heated at 100C for 5-10 minutes, until the developing colors were observed (Note that this reagent has only limited stability and is no longer useful when the color has turned to red-violet).

3.6 Bioassay techniques: Essential oils of Lantana camara var aculeate L. obtained by steam distillation was evaluated for antibacterial and antifungal activity as described below.

3.6.1 Culture and subculture of testing microorganism: The pathogenic of test microorganism including bacteria and fungi were cultured at r.t., either on nutrient agar medium or PDA media, for 24 and 72 hours incubation time respectively.

3.6.2 Testing microorganism: (a) The Agrobacterium tumefaciens pathogen was obtained from Medical Laboratory University of Gezira, Wad-Medani, Sudan. The bacteria were maintained on nutrient agar (NA) medium for sensitivity test of plant extract. (b) The Aspergillus niger pathogen isolates were obtained from Department of Plant Pathology at Agriculture Research Corporation

(ARC), Wad – Medani, Sudan. The fungus is identified by Prof. Dr. Nafisa Elmahi Ahmed Khalifa.

3.6.3 Preparation of media: 3.6.3.1 Potato Dextrose Agar (PDA): The Potato Dextrose Agar (PDA) media was prepared by mixing 200 gm of potato, 20gm dextrose and 20gm of agar in one liter of distilled water, the contents were mixed and boiled until the agar melted. The mixture was distributed into conical flasks and covered with cotton plugs and aluminum foil; thereafter they were sterilized in an autoclave at 121°C and pressure (15 P.S.I) for 15 minutes. Rose pingal (10ml) were added to the mixture to prevent contamination. (i) Preparation of Petri –plates: The solid medium was boiled and cooled to 45°C and poured into Petri – plates (8cm in diameter), to be used for further tests. The test organisms (fungi) were aseptically inoculated on Petri-plates containing autoclaved, cooled and settled medium. Petri-plates were incubated at 31°C for 72 hours.

(ii) Antifungal activity assay: Biological activity of the crude and purified essential oil of Lantana camara was tested by using disc diffusion method and agar hole disc diffusion method.

(A) Disc diffusion method: This method was carried out according to the following steps: (1) Isolated fungi (Aspergillus niger) was grown on PDA media. (2) Micro-fiber glass discs (0.5cm diameter) saturated with crude EO were placed in the middle of the plate before culturing.

(3) Two discs of 0.5cm diameter cylindrical type of fungal growth were corked out and placed by the two sides of each Petri–plate containing saturated disc. (4) Disc saturated with dichloromethane or chloroform solvent only was used as control. (5) Growth inhibition was periodically measured for up to three days. (6) Three replicas were done for each test. Inhibition diameter to disc Inhibition (%) was calculated as = Inner diameter of Petri-dish x 100

(B) Well-fungal growth disc diffusion method: A preparation of different concentrations of LEO was used in this method in which PDA media was prepared with or without LEO. Cylindrical discs of fungal growth (0.5cm diameter) were corked out and placed in the middle of the control and treated PDA media. Fungal growth was allowed to ensure by incubation at room temperature for 72 hours. Thereafter fungal growth was measured from the center of inoculated discs. The decrease in growth diameter was calculated according to the following equation: control diameter  treatment diameter %decrease  100 Control diameter

3.6.3.2 Nutrient agar media (NA): The nutrient agar medium was prepared by mixing 2 grams yeast extract, 5 grams of peptone, 15 grams agar, one gram lablemco powder, 5 grams sodium chloride and one liter of distilled water. The mixture was boiled in a water bath until the agar melted, the pH was adjusted to 7.1 up to 7.5, then the mixture were distributed into four conical flask (250 ml) covered

with cotton plugs and aluminum foil. Then they were adjusted in an autoclave at 1210C and pressure 15 P.S.I. for 15 minutes.

(i) Preparation of Petri-plates: The solid prepared media of (NA) was boiled, cooled at 45ºC and poured in Petri-plates (8cm in diameter) to be used for subculture and for further tests. The test organism (agrobacteria) was incubated at room temperature (350C).

(ii) Anti-bacterial activity assay: The biological activity of LEO extract was assayed by disc-diffusion method on NA media.

A) Disc-Diffusion Method: In this method micro-glass fiber discs of 0.5 in diameter were saturated with LEO extract were placed on the middle of Petri-plates separately. The test organism (agro bacterium) was streaked on each test Petri-plate before saturated disc was added in the middle of this plate. Treated plates (three replicas for each test) were incubated at room temperature. Zones of inhibition were determined after 24 hours.

CHAPTER FOUR

RESULTS AND DISCUSSIONS

4.1 Morphological variability among lantana camara species growing in Sudan

Indigenous Lantana camara species collected from or around Wad- Medani area can possibly be distinguished morphologically according to their variation in flowers color (Fig 4.1a); leaves type (Fig 4.1b) and stem shape (Fig 4.1c) in this study as shown in Table (4.1) and Figure (4.1). Lantana camara growing in Sudan is a low erector dwarf (see Fig 4.1c), however, other species will grow to 6 H high and way spread to 8H. Dwarf species will grow to 1-2H, root system is very strong, stem and leaves are covered with rough hairs and give aroma when crushed (Fig 4.1b). The small flowers are held in clusters (called umbels) that are typically 1-2 in a cross. Leaf wide ovate in erect species and narrow ovate in dwarf ascorbic on both side (Fig 4.1c). The leaves are 2-5 in long by 1-2 in wide (in erect spp.) and 1-2in long by 0.5-1 in wide (in dwarf spp) with rounded tooth edges and textured surfaces; orange to red, pink to red in unlimited combinations, in florescence's are compact, dome-shaped, contain 20-40 sessile flowers. Fruit small, greenish when it immature-turn to black immature poisoning dispersed binds.

4.2 Essential oils content (%) of Lantana species in Sudan

Essential oil (EO) percentages of lantana leaves varieties growing in Central Sudan (Wad-Medani area) were extracted according to Wagner et al, method (1984), and calculated on fresh and dry bases. Table (4.2) showed the essential oil content per each sample extracted from 200g of lantana leaves. The essential oil content (%) of nine analyzed sample were varied from 0.12% up to

0.01% when calculated on dry base (leaf) and on the other hand, fresh

leaf showed high oil content that varies from 1.8 – 0.3%.

Table (4.1): Botanical variability of Indigenous lantana camara L

Sample No. Possible scientific name Flower-color Stem-shape

(1) Lantana aculeate Pink (erect)

(2) Lantana flava Yellow (erect)

3) Lantana mista Red (erect)

(4) Lantana montevidensis Purple (erect)

(5) Lantana montevidnsis White (erect)

6) Lantana mista Orange (dwarf)

(7) Lantana montevidensi White (dwarf)

(8) Lantana montevidnsis Yellow (dwarf)

(9) Lantana montevidnsis Violet (dwarf)

pink-flower-erect violet-flower –dwarf orange - flower- dwarf

red-flower-erect yellow-flower-erect white-flower-dwarf

purple-flower-erect yellow-flower –dwarf white leaves- erect

Fig ( 4.1.a ) : photos of lantana flower color

Violet – dwarf purple – erect pink- erect

Orange-dwarf white-dwarf yellow-dwarf

yellow – erect red – erect

Fig ( 4.1.b ) : photos of lantana leaf type

Purple-erect-s stem length -white dwarf stem length -violet

Stem length-pink red-erect stem stem lehgth –purpel

stem length- yellow dwarf stem length- yello

Fig ( 4.1.c ) : photos of lantana stem shape

Love et al, (2009) found that the yields of essential oils varied from 0.1 to 0.79% in foliar hydro-distillates of eleven morpho types of Lantana species. However, for all analyzed Sudanese samples, the highest oil content (1.8%) was represented by pink flower-color sample with an erect stem for fresh leaf and 0.12% for dry leaf. These obtained results showed that lantana Fresh leave contain high content of the essential oil, however, subsequent work was carried out only on fresh leaf material for biological activity mainly: antibacterial and antifungal bioassay (i.e. bioassay against Salmonella typhimurium (S. typhi), para typhimurim (S. paratyphi), Agrobacterium tumefaciens, and against the fungus , Aspergillus niger) .

Table (4.3) showed some physicochemical properties of fresh and dry leaves of lantana essential oil (sample 1). The percentages of crude essential oil (EO) of fresh and dry leaves of lantana were 1.8 % and 0.12%, respectively and the color of extracted EO are light green and dark green(see Table 4.3). Fresh leaf EO was selected for further biological activity.

4.3 TLC-separated components of lantana-essential oil

This part of study was carried out to compare the chemical separated components according to: a) Essential oils extracted from nine collected samples of indigenous lantana were subjected to TLC separation for their EO constituents as shown in Fig (4.2 & 4.3) and Table (4.4)

Essential oil components prepared by TLC technique for all lantana analyzed samples in addition to dry leaves (11DL) of indigenous lantana (sample 1), were prepared from lantana leaves by hydro-distillation method, separated by TLC solvent system Toluene/ethyl acetate (93:7 v/v), and identified by Anisaldehyde–Sulfuric acid reagent color.

In Table (4.4), the higher number of TLC- separated compounds was obtained by sample (1), followed by sample (3), (6), (7), (8), and (9) which represented the same number of eight separated compounds, then after, sample (4) & (11) showed only seven, however, the lowest number of separated compounds (six) was shown by sample (2) & (5) as shown in Fig (4.2) & (4.3) respectively.

Fig (4.3) and Table (4.4) showed the extracted essential oil of lantana fresh leaf (FL) sample1), containing more numbers of separated compounds than the dry one (11(DL) sample 1), i.e. containing 9 and 7 TLC-separated compounds respectively.

4.4 Biological activity of lantana essential oil

4.4.1 Antibacterial activity

Table (4.5) showed the essential oil extracted from lantana fresh leaves (sample № 1, Fig 4.3 (1FL)) tested against two bacteria in a dependent dose manner of 20 µl/ml using disc diffusion method. No inhibition zone was detected against S. typhi or S. paratyphi. However, Agrobacterium tumefaciens (A. tumefaciens) seems to be more sensitive towards lantana essential oil resulting in 10% inhibition by only 20 µl/ml/ a dose. Table (4.6) and Fig (4.4) showed the percentage of growth inhibition of lantana camara sample (1) on A. tumefaciens according to different doses.

Table (4.2): Essential oil content (%) of indigenous Lantana varieties

(The EO % was calculated on dry and fresh weight bases)

Oil content (%)

Sample No. Flower-color Fresh-leaf Dry-leaf

(1) Pink (erect) 1.8 0.12

(2) Yellow(erect) 1.0 0.01

3) red(erect) 0.6 0.02

(4) purple(erect) 0.4 0.03

(5) white(erect) 0.6 0.02

6) Orange(dwarf) 0.5 0.03

(7) white(dwarf) 0.3 0.02

(8) yellow(dwarf) 0.8 0.02

(9) violet(dwarf) 1.4 0.01

Table (4.3): Some physicochemical properties of Lantana essential oil

Type of Lantana leaf Oil% Oil color

Fresh 1.8 Dark green Dry 0.12 Light green

Table (4.4): TLC separated components of essential oils of indigenous lantana nine varieties

Sample NO. Lantana/flower color NO. of TLC separated components

(1) Fresh Pink (ES)* 9

(2) Yellow(ES) 6

)3) red(ES) 8

(4) purple(ES) 7

(5) white(ES) 6

)6) Orange(DS)** 8

(7) white(DS) 8

(8) yellow(DS) 8

(9) violet(DS) 8

(11) dry pink(ES) 7

(ES)* = erect stem

(DS)** = dwarf stem

5 9 4 6 7 2

Fig (4.2): TLC separated Components of fresh leaves of five varieties

of lantana essential oil (S5, S9, S4, S6, & S7).

- The developing TLC solvent system : Toluene / ethyl acetate (93 : 7) - TLC reagent= Anisaldehyde

2 1(FL) 8 3 11 (DL) 11 (DL)

Fig (4.3): TLC separated Components of fresh leaves of four varieties

of lantana essential oil (S2, S3, S8, S1, & S11).

- 1 (FL) = lantana fresh leaves of sample (1) - 11(DL)= lantana dry leaves of sample (1) - The developing TLC solvent system : Toluene / ethyl acetate (93 : 7) - TLC reagent= Anisaldehyde

(Volumes) extracted essential oil from lantana plant leaves, using disc

diffusion method.

The applied volumes (doses) of the essential oil on the glass discs are varied from 200 µl to 4µl equivalent to concentrations of 100% up to 0.02 % µl/ml. More than 20 % kill was obtained by 200µl, however, doses of 160,120 ,100,80,60 and 50, 40 µl showed more than 10 % of bacteria inhibition as shown in Table (4.6) and Fig (4.4). Lantana essential oil against A. tumefaciens showed that the MIC can be obtained in less than 4 µl/ml. However, no reports were available in the literature on antibacterial activity of essential oils of lantana camara L varieties against A. tumefaciens. On the other hand, the activity of lantana– essential against other bacteria (Enterococcus faecalis and Staphylococcus aureus) was reported by Tesch et al, (2011), and also Kurade et al,(2010) was studied the activity against Arthrobacterprotophormiae, Micrococcus luteus, Rhodococcusrhodochrous, and Staphylococcus aurous. No literature reports available of lantana camara against A. tumefaciens.

4.4.2 Antifungal activity

In vitro treatments for fungal inhibition were carried out according to disc diffusion method and/or agar diffusion method. Micro-fiber glass disc was saturated by DCM solvent only for control in disc-diffusion method and for dilution for other prepared concentrations.

Table (4.5): Antibacterial activity of the Lantana essential oil (20 l/ml/disc) fresh leaves on different bacteria

Name of organism Inhibition %

A. tumefaciens 10.0 S. typhi 0.0 S. paratyphi 0.0

Table (4.6): Inhibition % of essential oil of lantana camara on the growth of bacteria.

Doses (volumes)l/ml Inhibition (%) 200 21.1 160 18.2 120 15.3 100 14.7 80 14.5 40 14.1 20 10.0 16 7.5 12 3.7 8 3.2 4 1.6 Control 0.0

160 200

120 100

Control 80

Fig (4.4): Photographs of lantana (S1)* essential oil against Agrobacterium tumefaciens .

(S1)* = sample No. (1)

4.4.3. Disc diffusion method:

Table (4.7) and Fig (4.5) showed the percentage of growth inhibition on Aspergillus niger according to different doses of L. camara essential oil extracted from plant leaves (sample (1FL), Fig 4.3) using disc diffusion method. Table (4.7) and Fig (4.5) showed the percentage of growth inhibition on Aspergillus niger according to different doses of L. camara essential oil applied on the glass disc are varied from 300l to 4l equivalent to concentration of 100% and 0.02% l/ml. The 100% kill was obtained by doses of 300 and 280, 250, 200l, however, doses of 160 and 120 l showed more than 75% inhibition on (see Table 4.7).On the other hand, volumes (doses) of 100, and 80l/ml, showed inhibition zones more than 50% while volume of 40l gave 24% kill and 20l showed 14% (Table 4.7). However, low inhibition % (4 & 2) was represented by 16 and 12l, but no inhibition zone on fungal growth was detected below 12l/ml (Table 4.7). The LC50 of lantana camara (LC1) essential oil against A. niger is obtained by 50l/ml, while MIC lie between 12l and 8l.

No literature available about essential oil of lantana camara varieties against A. niger. However, Tzasna et al. (2008) reported about activity of lantana achyranthifolia an lippie-essential oil against fusarium monitiforme and Trichophytonmentagrophytes. Also Passos et al, (2012), found that the plant Lantana radula essential oil had fungi static activity more than the oils of L. camara on the phyto pathogenic fungi Corynesporacassiicola.

Table (4.7): Inhibition% of essential oil of lantana camara on the growth of fungi .

Doses (volume) in l /ml Inhibition (%) 300 100.0 280 100.0 250 (100)*93.8 200 (100)*87.5 160 85.0 120 75.5 100 62.0 80 50.0 40 24.0 20 13.8 16 3.8 12 2.1 8 0.00 4 0.00 Control 0.00

4.4.4. Agar diffusion method

More quantities method was carried out on lantana essential oil for sample

(1), using different prepared concentrations, varied from 4% to 0.03%

(oil/media, see Material & Method). A cylindrical disc of 1cm in diameter of

No observed inhibition zone was detected in fungal growth for all applied concentrations. For this reason A. niger inhibition was taken for further

studies only by using disc-diffusion method.

4.4.5 Biological activity of dry leaf-essential oil

Dried leaves (30 grams) of lantana camara (11DL, sample 1), were extracted by shaking for 8 hours with 300 ml DCM solvent at room temperature (cold method). The suspension was filtered and the clear filtrate evaporated to dryness to tenth of original volume (i.e. 30 ml). The extracted essential oil obtained by either cold method or by hydro-distillation method were tested against A. niger inhibition using disc diffusion method as describe above. Different volume (doses) varied from 500 l/ml to 0.5l/ml were

applied in this trial, while control was saturated with DCM solvent only.

The results showed no inhibition zone was detected in fungal growth in all

applied doses.

280 300

200 250

Control

Fig (4.5): Inhibition of Aspergillus niger by LC1 essential oil.

120 160

100 80

Control

Fig (4.5): Continued

4.4.6 Biological stability of L. camara essential oil

An in vitro test was carried out for a fixed volume (20µl/ml) of extracted essential oil of lantana camara leaves (LC1) were applied to microfiber glass discs, placed onto the growth media (PDA or/and NA) at room temperature and allowed to stand for, 24, 48, 72, 96 and 120 hrs for fungus " A. niger "

(Table 4.8), and 12, 24, 48 and 72 hrs for bacteria " A. tumefaciens" (Table

4.9) before organisms inoculation. Table (4.8) showed that the anti fungal activity was maximal after 120 hours incubation time (35% inhibition) and

Table (4.9) represented antibacterial activity after 24 hours incubation time

(13% inhibition).

These results for both test organisms pointed to a chemical relationship between a fungus-PDA media, a bacterium NA-media, and LC1-essential oil constituents according to incubation time for oil diffusion. Both results indicated that inhibition% increases with incubation time increase reaching

14% after 24 hrs incubation and 1% at zero time (Table 4.9) for test bacteria.

On the other hand, reaching 35% after 120 hrs incubation and 10% at zero time (Table 4.8) for test fungus. More study of chemical and physicochemical characteristics is needed for this interesting relationship.

Table 4.8: Stability of antifungal activity of L. camara (LC1) essential oil

Time /hours Inhibition (%) 0.00 10 24 .0 28 48.0 31 72.0 33 96.0 34 120.0 35 control 0.0

Table 4.9: Stability of antibacterial activity of (LC1) essential oil

Time /hours Inhibition (%) 0.0 1 12 4 24 14 48 13 72 10 control 0.0

4.5 Determination of the active antifungal ingredient of (LC1):

Thin layer chromatographic fractionation of lantana camara (LC1) leaves essential oil result in at least nine components (Fig 4.6). The Rf values for these compounds together with a description of their color according to detection reagent (Anisaldehyde) were shown in Table (4.10).

These nine compounds were eluted with chloroform/methanol

(2:1), individually and then after followed by bioassay of each of the separated component against A . niger using disc-diffusion method. Only two out of the nine TLC separated compounds showed biological activity against A. niger. These two compounds are numbered one and six calculated from origin of TLC-plate as shown in Fig (4.6).

Compound six (C6) which having dark violet color with the detection reagent possess the highest activity (55% kill) and compound number one

(C1) represented the dark brown color with the same reagent showed a lower

activity (14 %) compared with C6 as shown in Table (4.11) and Fig (4.7).

Standard essential oils including Eugenol, Linalool, Geraniol, Limonene,

Thymol, Methyl trans-cinnamate, Citral, Eucalyptol, Methyl Chavicol, and

Cineol, were applied to TLC plate with LC1 sample for comparison study to identified TLC separated compounds according to positive color of

Anisaldehyde reagent. Possible identification showed that lantana camara .

Table (4.10): Antifungal activity of TLC separated components of essential oil of LC1 leaves( The applied dose is 20 µl/ml )

TLC bands* Rf% Band color ** Inhibition (%)

1 10 Dark brown 13.7

2 28 Light brown 0.0

3 34 Light green 0.0

4 51 Violet 0.0

5 68 Dark violet 0.0

6 79 Dark blue 55.0

7 86 Blue 0.0

8 97 Dark orange 0.0

* TLC band* = Band separated from the TLC origin.

** The inhibition% calculated according disc diffusion method

TLC solvent system = Tolerant/ethyl acetate (93.7)

Detection reagent: Anisaldehyde.

6 9

3 2 1

Fig (4.6): A Photograph of TLC separated components of the essential oil of lantana camara leaves ( sample LC1)

Table (4. 11): Antifungal activity of C1and C6

Compound Inhibition (%) Color Rf (%)

C1 13.7 Dark brown 10

C6 55.0 Dark violet 79

C6 Control

Fig (4.7): Activity of C6 (TLC-fraction, Fig 4.6) against A. niger

LC1 1 2 3 4 5 6 7 8 9

Fig (4.8): TLC separation of lantana camara essential oil (LC1) and commercial standard essential oils TLC solvent system = Toluene / ethyl acetate (93:7) TLC reagent = Anisaldehyde-sulphuric acid Standard commercial essential oils numbered/name: 1- Linalool 2- Lanisole 3- Geraniol 4- Eugenol 5- Limonene 6- Eucalyptol 7- Citral 8- Thymol 9- Methyl trans–Cinnamete (LC1) contained Eugenol compound in contrast to Rf value and TLC reagent color (see Fig 4.8).

No literature was found for TLC separated components of lantana camara essential oil.

4.6 GC/MS analysis of Lantana camara (LC1) essential oil:

The analysis of GC/MS chromatogram was carried out in University Malaysia

Pahang (UMP), Faculty of Industrial Sciences and Technology (FIST),

Chemical Engineering Laboratory.

The essential oil of indigenous Sudanese Lantana camara, sample № (1) a signed by LC1 was prepared by hydro-distillation method and analyzed by

GC/MS chromatogram. However, GC/MS chromatogram and data for crude extracted essential oil were shown in Fig (4.9).

Fig (4.9) shows GC/MS separation / identification of the essential of LC1, the most active against A. niger. Ten peaks were separated and partially identified. The major components of essential oil of LC1 leaves, determined by GC/MS-separated essential oil constituents were limonene, C6-ketone(2- cyclohexen-1-one,2-methyl-5-(1-methylethenyl) and caryophllene. Passos et al. (2012), report showed that one of the main components of essential oil of

L. camara was E-caryophyllene (19.7%), and this agree with our indigenous

analyzed active sample of L. camara against A. niger and A. tumefaciens.

Fig. (4.9): GC/MS chromatogram of LC1

Fig. (4.9): Continued

Also we agree with Tesch et al (2011) in two major compounds of L. camara essential oil beta-caryophyllene (14.8%), and limonene (5.7%).

Table (4.12) and Fig (4.10) represented the i.r. spectrum of compound six

(C6) of essential of Lantana camara sample (1) as shown in Fig (4.6), the most active compound against A. niger.

UV spectrum showed absorption of 337nm that indicated appearance of benzene ring ( i.e. aromatic ring) as shown in Fig (4.11).

Table(4.12): Observed FTIR spectrum for compound LC1and possible interpretation of the absorbing groups according to Williams and Fleming, (1980).

FTIR Interpretation of possible chemical groups absorption per according to literature cm 3560 H – bonded OH (3600 – 3200) 1725 Aromatic 2100 – 1700 1720 (1) Aromatic (2100 – 1700) 1715 (2) Ketone (1750 – 1705) 1705 (3) Aromatic alkene (1715 – 1695) (4) Methyl, methylene (1730 – 1470) 1640 C = C ( - unsaturated carbonyl compound 1610 (1640 – 1590) 1605 1500 1520 Aromatic rings 1530 1430 = C – O – C – {ether (1275 – 1200)} 1440 C – O (str) for ether (1260 – 1050) 1265 C – O, str (1300-1000)

737 Aromatic alkene (870 – 680) 669

Fig (4.10): FTIR spectrum of C6 active against A. niger

Fig (4.11): UV spectrum of C6 active against A. niger

Fig (4.12) :GC/ MS chromatogram of compound 6 active against A .niger

Continued

Interpertation from FTIR, U.V spectrum and GC/MS analysis:

The FTIR analysis results of compound 6 showed that, this compound has alcoholic moiety (i.e. containing- OH group), (Table 4.12) this comfirmed by

GC/MS and MS suggestion (Fig 4.9) represented alcoholic compounds. More over, the FTIR spectrum (table 4.12), for compound 6, on the other hand, MS suggestion represented atypical compound with M. wt = 220, and structure formula C15H24O1 However, the other alcoholic compounds containing steroidal nucleus has not been shown in FTIR absorption (980, 960, 900 cm-1) as shown in (table 4.12). Depending on FTIR results, the antifungal active ingredient of L. camara essential oil suggested to be alcoholic compound.

From GC/MS result atypical compound with M.Wt of 202 and structure

-1 formula C15H22 is confirmed by absortion on 1500/1530 cm and 337nm the benzene ring (i.e. aromatic ring) with side chail of oxgen additive compound .

Table (4.13) Interpertation from FTIR, U.V. spectroscopy and GC/MS analysis.

Method Compound Absorb M. Formula

FTIR (-OH) group. 3560/ cm-1 - Ar-OH

Benzene ring (aromatic ring) 1520/cm-1

Unsaturated 7 th member 1530/ cm-1

Carbonyl group (side chain) 1590 cm-1

U.V. Benzene ring (aromatic ring) 337nm - -

GC /MS Caryophyllene oxide - 220 C15H12O1

Benzene, 1-(1, 5, Dimethyl- 202 C15H22

4-hexenyl) -4-methyl-

Conclusion And Recommendation

Conclusion : In Sudanese Lantana Camara

 Flower color was varied (pink, yellow, white, violet, orange and purple

. While stem varied between erect and dwarf.

 Antimicrobial activity evaluate and could be found fungi more

sensitive than bacteria .

 The major constituents of the essential oil of Lantana Camara was

determined by GC /MS found sesquiterpene – caryophllene contain

more than 30% of the oil .

Recommendations:

Further studies are needed to covered others Sudanese Lantana Camara varieties for evaluation fruit , flower , root and stem essential oil .

REFERENCES

Abdel Hady, N.M. Abdel Halim A.S. Al – Ghadban, A.M. (2005). Chemical composition and insecticidal activity of the volatile oils of leaves and flowers of Lantana camara L. cultivated in Egypt, 35(2): 687 – 98.

Altren, B. MPH. M.S. P. (2011). Secondary Natural Products. Public Health, Medicine and History.

Anne E., Lanzotti and Virginia , (2009). Plant sciences [springer].

Anon, (1962). The wealth of India: Raw Materials, P. 31 – 34, CSIR, New Delhi.

Barbour, E.K., AL-Sharif, M., Sagherian ,V.K., Habre ,A.N ., Talhouk, R.S. and Talhouk ,S.N. (2004). Screening of selected in digeneous plants of Lebanon for antimicrobial activity. J. Ennthnopharmacol. American University of Beirut, Beirut, Lebanon. 94: 1 – 7.

Barreto F.S., Sousa E.O., Campos A.R., Costa J.G.M. and Rodriguez ,F .Jan . (2010). Antibacterial activity of Lantana camara Linn and Lantana montevidensis Brig Extracts from Cariri – Ceara, Brazil, 2120, 136, 162. 2(1): 42 – 4. National Library of Medicine National Institutes of Health.

Begum, S., Wahab, Siddqui, A. and Gamar, S.F. (2000 – 2006). Nematicidal constituents of the aerial parts of Lantana camera. Journal of Natural Products.

Begum, S.A., Wahab and Siddiq, B. (2008). Antimycobacterial activity of flavonoids from Lantana camara Linn. 22(6): 467 – 470.

Bohlman, J. (1995 – 1998). Proc Natl Acadsci USA. 4126.

Bouvier, F. (2005). Prog lipid Res 44,357

Burt, S.A. (2004). Essential oils: their antibacterial properties and potential application in foods: are views. International journal of food microbiology, 94: 223 – 253. Elsevier B.V. University of Utrecht. Netherland.

Chares, E. Ophar D.T., C. (2003). Carbohydrates, Starch. Virtual Chem. Book, Elmhurst College.

Cordell, G.A. (2000). Biodiversity and drug discovery a symbiotic relationship, photochemistry. University of IUinois, Chicago, USA. 55: 4(463 – 480).

Edward, R. W. (2001). The genome of agrobacterium tumefactions, a plant pathogenwitha taken for trans foreignness. GNN Genome News Net Work.

Eisenreich, (2001). Plant physiology online. Gibberellins Biosynthesis. Chapter 20. Topic 203.

Elghisalberti, (2000). Lantana commara L. (Verbenaceae). Science Direct, ELSEVIER (Fitoterapia) 71(5): 467 – 486.

Innocent, E., Cosam, C. Joseph , N. G., Mainen , J. Moshi, M. H. N. and Ahmed Hassana L.F. (2008). Mosquito larvicidal constituents from Lantana vibur noids sp. Viburnoidesvar Kisi (Arich) verde (Verbenaceae). 240 – 244.

Ganjewala, D., Sam ,S. and Khan, K.H. (2009). Biochemical composition and antibacterial activities of Lantana camara Plants with yellow, lavender, Red and whiter flower. 3, 10, 69 – 77.

Hancock, R.E.W. (2005). Mechanisms of action of newer antibiotics for gram – positive pathogens. The lancet infections diseases. University of British Columbia, Canada 5: 209 – 218.

Hatmutk, L. (1999). Plant physiology and biochemistry. University of Karlsruhe, Karlsruhe, D – 76128 Germany.

Hawkey, P.M. (2008). The growing burden of antimicrobial resistance. Journal of antimicrobial chemotherapy. 62(1): 1 – 9, British society for antimicrobial chemotherapy. University of Birmingham. U.K.

Hernandez, T., Canales, M., Avila, J.G., Garcia, A.M., Martinez, A., Caballero, J. , devivar, A.R. and Lina, R. (2005). Composiiton and antibacterial activity of essential oil of Lantana achyranthifolia Desf. (Verbenaceae) 96(3): 551 – 4.

Hernandez, T., Canales, M., Grasia, A.M., Duran, A., Meraz, S., Davila, P. and Avila, J.G. (2008). Antifungal activity of the essential oils of two verbenaceae: Lantana achyranthifolia and Lippia graveolens of zapotitlandelas Salinas, Puebla (Mexico). 7(4): 202 – 206.

Jean-Aime, R. , Panja, AR. R., Jean, RE, Rsoarahona and Emile M. G. (2005). Seasonal and chemotype influences on the chemotype influences on the chemical composition of Lantana camara L. Essential oil from Madagascar. Analytical Chimica Acta ELSEVIER. 545(1): 46 – 52.

Jchil – Chen, Soc. (2009). Composition and biological activity of the essential oil of Peruvian Lantana camara, Escielo. Journal of the Chilean Chemical Society, 54(4): 379 – 384.

John, O. and John, B. (1995). The plant cell, American society of plant physiologists lipid biosynthesis, 7: 957 – 970.

Jon Mohrman, (2011). Healthy food with carbohydrates. Food and Health.

Juliani, H.R., Biurrun,Jr., Koroch, F., Olivamm,A.R., Demo, M.S. Trippivs, and Zygadlo J.A. (2002). Chemical constituents and antimicrobial activity of the essential oil of Lantana xenica. 68(8): 762 – 4.

Khan, M., S.K., Srivastava, K.V., Syam A.M., Singh, A.A. and Nagvi, (2002). Chemical Composition of Leaf and Flower Essential Oil of Lantana camara for India. 17(1): 75 – 77.

Khan, M., Srivastara, S.K., Neetujain, S., K.V. and Anink, Y. (2003). Chemical composition of fruit and stem essential oils of Lantana camara from northern India. Flavour and Fragrance Journal, 18(5): 376 – 379.

Kobbe, B., Cushman, M., Wogan, G.N. and Demain, A.L. (1977). Production and bacterial activity of malforminc, a toxic metabolite of Aspergillus niger. Appl.and environ. Microbial, 33(1): 996- 997.

Kurade, NP. , Jaitak, V. Kaul ,V.K.. , Sharma O.P. (2010). "Chemical composition and antibacterial activity of essential oils of Lantana camara, Ageratum houstonianum and Eupatorium adenophorum".Pharm Biol. May;48(5):539-44.

Lai, P.K. and Roy, J. (2004). Antimicrobial and chemo preventive properties of herbs and spices. Curr. Medchem. Salem west Virginia, USA II, 1451 – 1460.

Love, A., Naik ,D., Basak, S.K., Babu, S. , Pathak ,N., Babu, C.R. (2009). "Variability in foliar essential oils among different morph types of Lantana species complexes, and its taxonomic and ecological significance". ChemBiodivers.; 6(12):2263-74.

Mahato, S.B., Sahu. N.P., Toy, S.K., and Sharma, O.P. (1994). Potential antitumoragents from Lantana camara structures of flavonoid and phengl propanoict glycosides; 50: 94, 39 – 46.

Mohrman, J. (2011). Healthy foods with carbohydrate, Food and Health.

Moore, L.W., Chitton, W.S. and Canfield, M.L., (1997). Diversity of opines and opine catabolizing bacteria isolated from naturally occurring grown calltumows. APPI Environ. Microbial. 63(1): 201 – 207.

Passos, J.L., Barbosa, L.C, Demuner, AJ., Alvarenga, E.S.,daSilva, CM. and Barreto R.W. (2012). "Chemical characterization of Volatile. Compounds of Lantana Camara L. radula Sw . and their antifungal Activity " .Molecules. Sep 27;17 (10): 11447-55

Raper, K.B. and Fennell, D.I. (1965). The genus Aspergillus. Williams and Wikins company, Baltimore, M.D.

Sharma, O.P., Makkar, H.P. and Dawaa, R.K. (1988). A review of the noxious plant Lantana camara. 26(1): 975 – 87.

Sharma, O.P., Sharma, P.D. (1989). Natural products of the Lantana plant. The present and prospects. Journal of Scientific and Industrial Research. 48, 471 – 474.

Sharma, R., Dawra,K. and Dandala R. (1990). Atriterpenoid acid, Lantadene. D. from Lantana camara var. Aculeata. Photochemistry, Elsevier. 29(12): 3961 – 3962.

Singh, M., Nagvi, A.A., Khan, M., Snivastava, S. K. and Syamasnndar, K.V., (2002). chemical composition of leaf and flower essential oil of Lantana from India. Flavour and Fragrance Journal, 17(1):75 – 77.

Shresth, P.M. and Dhillion, S.S. (2003). Medicinal plant diversity and use in the high lands of Dolakhadistrict, Nepal. J. Wthnopharmacol., 86: 81 – 96.

Steelecl , (1998). Plant physiology: 116, 1497.

Stoessl (1976). Phytochemistry, these include both sesquiterpenoids and diter penoids type: 15, 855.

Taoubi, K., Fauvel, M.T., Gleye, J., Moulis, C. and Fouraste, (1997). Phenyl propanoids glycosides from Lantana camara and lippie multifora. Plant a med., 63: 192 – 193.

Tesch, N.R, Mora, F., Rojas, L., Díaz ,T., Velasco, J., Yánez, C., Rios, N., Carmona, J. and Pasquale, S. (2011). "Chemical Composition and antibacterial activity of the essential oil of Lantana camara var. moritziana"Nat Prod Common. Jul;6(7):1031-4.] .

Ueno, Y. and Ueno,I. (1978). Toxicology and biochemistry of mycotoxins. In ,Toxicology, biochemistry and pathology of mycotoxins. (Uraguchi and M. Yamazaki), eds, Johuwiley and sons, Halstead Press, N.Y.

Underbrin, M., Vrabec. J., and Quinn ,J.r. (1997). Symptoms of Fungal Exposure (Mycotoxicosis) Mayoclinic: Aspergillosis Symptoms.

Environmental Protection. Agency. Aspergillus niger Final Risk Assessment: 1997.

Wagner, H., Bladt, S. and Zgainski ,E. (1984). "Plant drug analysis springer verlag Berlin".

Wolfgon, L. B., Huthmacher,K. and Drauz, K. (2005). Biotechnological Production of amino acids and derivatives: Current status and prospects, 69: 1-8.

Appendices

Appendix № (1):

Possible identification of C1- TLC separated compound:

FTIR spectrum of C1 active against A. niger

UVspectrum of C1 active against A. niger

Appendix ( II)

Identification of second Active Antifungal compound (c1) isolated from

Lantana Camarac.v.a Essential oils .

Introduction :

Compound c1 re chromatogram with the active antifungal compound c6 was

separated by PTLC (see fig (4.7))and Table (4.1) .

Methodology :

Compound c1 was separated by TLC and identified by GC/MS , MS , and

FTIR spectrum . This compound was bioassay against A. niger using disc

diffusion method .

Results and Discussions :

Compound c1 Table shows 13.7 %growth inhibition against A. niger in vitro test –when subjected to TLC separation with to toluene /ethyl acetate (93: 7)

(v:v) solvent system and detection reagent Anisaldehyde its shows light or

with Rf value (10%) Table (4.11) .

The FTIR spectrum showed on alcoholic (OH) functional group (Table 4.12) .

More ever , the MS analysis showed 10 suggested compound. This compound

is need more identification .

GC/MC chromatogram of C1 active against A. niger

Continued:

MS possible identification of C1 active against A. niger

Continued:

MS possible identification of C1 active against A. niger

Continued:

MS possible identification of C1 active against A. niger

Appendix (2) :

Photographs of LC1 biological Stability for fungus & bacterium

Control

Stability of essential oil of (LC1)leaves against A. niger

Control

Stability of essential oil of LC1leaves against agrobacterium tumefactions

Appendix (iii) :

GC/MS chromatogram of indigenous Sudaneseof nine species of Lantana camarasubjected for essential oil content (%) according to their chemical composition.

Appendix (III)

GC/MS analysis of nine species of Lantana Camara essential oils .

Introduction :

The analysis of GC/MS chromatograph was carried out in university Malaysia (UMP) ,Faculty Fairmont and Tower , Cairo ,

Egypt.

Methodology :

The essential oil of Lantana Camara species were prepared by hydro –distillation method and analyzed by GC/MS chromatogram .

Results and Discussions :

Three to ten peaks were separated and partially identified in nine species . The major component of essential oils of Lantana Camara leaves determined by GC/MS –separated essential oil constituents were

Limonene , c6-ketone , caryophuene ,1,8 cineol , camphor and

Eucalyptol.

Major components of E.O. of Lantana camara leaves determined

byGC/MS

LC1

No. Component Retention Component time (RT) (%) 1 Limonet 4.797 1 2 C6-Ketone (2-cyclohexan-1 –onem 7.818 5 2-methyl -5- (1-methylethenyl) 3 caryophllene 10.107 1

LC2

No. Component Retention Component time (RT) (%) 1 Cyclobutane, ethyl 1.656 26 2 Pentane, 3-mehyl 1.318 5 3 1,2-Benzenedicarboxylicacid- mono 23.382 1 (2-ethyl hexyl) ester

LC3

No. Component Retention Component time (RT) (%) 1 2, 2,6,2,6 – tetramethyl heptane 1.318 5 2 Cyclobutane, ethyl 1.656 27 3 Phthalicacid, 6- ethyloct- 3yl 2-e 23.382 0.3 thylhexlester

LC4

No. Component Retention Component time (RT) (%) 1 2,2,6,- Tetramethyl heptane 1.318 5 2 Cyclobutane, ethyl 1.661 26 3 Phthalic acid, 6-ethyloct- 3- yl 2- 23.376 1 ethlhexyester

LC1

LC1 :Continued

Continued

LC2

Continued

LC3

Continued

LC4

Continued

LC5

No. Component Retention Component time (RT) (%) 1 Eucalyptol 7.83 2 2 1.8-cineole 7.83 2 3 copaene 15.90 2

LC6

No. Component Retention Component time (RT) (%) 1 Sabinene 5.71 5 2 Eucalyptol 7.86 4 3 Camphor 17.31 1 4 Caryophyllene oxide 32.89 6

LC7

No. Component Retention Component time (RT) (%) 1 Cyanoacetylene 3.02 77 2 1,8- cineole 7.82 5 3 Eucalyptol 7.82 5

LC8

No. Component Retention Component time (RT) (%) 1 Trans-canyophyllene 21.59 2 2 Aromandendrene 19.16 8 3 Eucalyptol 7.82 8 LC9

No. Component Retention Component time (RT) (%) 1 Eucalyptol 7.82 8 2 Camphor (AS) 17.28 4

3 Trans-Caryophyllene 20.50 9 4 Caryophyllene oxide 30.71 2

LC5

LC6

LC7

LC8 LC8