Bioactive secondary metabolites from the endophytic microorganisms of the medicinal plant Bidens pilosa [Elektronische Ressource] / Randa Abdou. Gutachter: Christian Hertweck ; Dirk Hoffmeister ; Jörg Durner

Randa Abdou

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• Gesundheit

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• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2011
• Nombre de lectures: 60
• Poids de l'ouvrage: 3 Mo

Extrait

Bioactive secondary metabolites from the endophytic
microorganisms of the medicinal plant Bidens pilosa


Dissertation
zur Erlangung des akademischen Grades doctor rerum naturalium
(Dr. rer. nat.)




vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena





von Apothekerin Randa Abdou
geboren am 21.02.1976 in Kairo, Ägypten

















Gutachter:
1. Prof. Dr. Christian Hertweck, Friedrich-Schiller-Universität Jena und Hans-Knöll-
Institut (HKI) Jena
2.Prof. Dr. Dirk Hoffmeister, Friedrich-Schiller Universität Jena
3. Prof. Dr. Jörg Durner, Technische Universität München und Helmholtz Zentrum
München

Tag der öffentlichen Verteidigung : 14.01.2011



List of contents

List of contents

1. Introduction .................................................................................................................... ..1
1.1. Endophytes ................ ….1
1.2. . Plant-endophyte interaction ................................................................... ………………..2
1.3. Biologically active natural products from endophytes……..……………..…………………..3
1.3.1. Antimycotic agents from endophytes .......................................................................... ..3
1.3.2. Antibiotic agents from endophytes ............... .4
1.3.3. Antiviral agents from endophytes…………………………………………………………….4
1.3.4. Anticancer agents from endophytes………………………………………………………....5
1.4. Plant selection for investigation of endophytes..................................................................6
1.5. The plant Bidens pilosa……………………………………………………………………….....6
2. Aim of the study..................................................................................................................8
3. Results and discussion………………………………………………………………………….9
3.1. Isolation of endophytes and cultivation..............................................................................9
3.2. Strain 1: Botryosphaeria rhodina……....………………………………………….......…..10
3.2.1. Structure elucidation of compounds 1-4 ………………………………………………......10
3.2.1.1. Bioactivity of compounds 1-4 ………………...…………………………………………..14
3.2.2. Structure elucidation of compound 5 ………………………………….............................18
3.2.3. Structure elucidation of compound 6 ………………………………….……..……………21
3.2.4. Identification of compound 7 ………..…………………………………………..................25
3.2.5. Identification of compound 8 ………………..…………………………………..................26
3.2.6 Discussion of the results of B. rhodina………………………………………….................26
3.3. Strain 2: Aspergillus neoniger ……… ………. …… ………… ……… … ………… ……. ….28
3.3.1. Structure elucidation of compounds 9-12……………………………….….….................28
3.3.2. Structure elucidation of compounds 13 and 14…..……….………………………………34
3.3.3. Bioactivity of compounds 9-14………………………………………………………………38
3.4. Strain 3: Epicoccum nigrum ……… ……… ………… ……… ……… … ………… …….......40
Page II
List of contents

3.4.1. Structure elucidation of compound 15………………………………………..……………41
3.4.2. Structure elucidation of compound 16…………………………………………..………....43
3.4.3. Structure elucidation of compound 17………………………………………………..……47
3.5. Strain 4: Khuskia oryzae …… ………… …… ………… ……… ……… … ………… ……. ….47
3.5.1. Structure elucidation of compound 18…………………..………………………………....48
3.5.2. Structure elucidation of compound 19…………………………..………………….……...49
3.5.3. Structure elucidation of compound 20……………………………………………………..53
3.6. Strain 5 (20076005)……………………………………...…………………………………….54
3.6.1. Structure elucidation of compound 21………………………………………………..........55
3.6.2. Identification of compound 22……………………………………………………………….57
3.6.3. Identification of compound 23…………………………………………………………........57
3.6.4. Identification of compound 24…………………………………………………………........58
3.6.5. Detection of indole carboxylic acid………………………………………………………….59
3.7. Strain 6 (20076002)……...…………………………………………………………………….59
3.7.1. Identification of compound 25……………………………………………………………….60
3.8. Testing endophyte-endophyte interaction…….………………………………………….62
3.8.1. Effect of antifungal endophytic extracts on neighboring endophytes…………………...62
3.8.2. Effect of mixed fermentations on the metabolic profile of endophytes……….…...……62
3.9. Plant immune assay……………………………………………………………………..……64
3.10. Discussion……………………………………………….…………………………………...67
4. Materials and Methods………………………………………………………………………….72
4.1. General experimental procedures…………………………………………………………….72
4.2. Spray reagents………………………………………………………………………………….72
4.3. Microbiological and analytical methods………………………………………………………73
4.4. Ingredients of different media………………………………………………………………….73
4.5. General laboratory chemicals…………………………………………………………………74
4.6. Plant collection and endophyte isolation……………………………….…………………….75
4.7. cultivation of pure fungal strains……………………………………………..…………..……75
Page III
List of contents

4.8. Cultivation of dual cultures………………………………………………………….…….…...75
4.9. Fungal identification…………………………………………………….…………..75
4.10. Cultivation for screening and isolation of secondary metabolites………………………..75
4.11. Biological screening methods………………………………………………………..………76
4.11.1. Antimicrobial screening………………………………………………………….…………76
4.11.2. Antiproliferative and cytotoxic assays……………………………………….……………77
4.11.3. Spectrofluorometric assay………………………………………………………………….78
4.12. Isolation and identification of metabolites…………………………………………………..79
4.12.1. Botryosphaeria rhodina…………………………………………………………………….79
4.12.2. Aspergillus neoniger………………………………………………………………………..79
4.12.3. Epicoccum nigrum…………………………………………………………………………..80
4.12.4. Khuskia oryzae………………………………………………………………………………80
4.12.5. Strain 20076005………………………………………………………….……..................80
4.12.6. Strain 20076002…………………………………………………………………………….81
5. Summary………………………………………………………………………………...………..82
6. Zusammenfassung……………………………….……………………………………………..85
7. References………………………………………………………………………………...……...88
8. Appendix……………………………………………………………………………………...…..96
8.1. Physicochemical data of the isolated compounds…………………………….…………….96
8.2. NMR spectra of some of the isolated compounds…………….…………………….……..99


Page IV
List of abbreviations

List of abbreviations

1D = One Dimensional
2D = Two Dimensional
CC = Concentration at which 50% cytotoxicity (cell death) is observed 50
CDCl = Deuterated Chloroform 3
13C NMR = Carbon Nuclear Magnetic Resonance
COSY = Correlated Spectroscopy
DEPT = Distortionless Enhancement by Polarization Transfer
DMSO = Dimethylsulfoxide
D OD = Deuterated Methanol 3
Fig = Figure
GI = Concentration at which 50% growth inhibition is achieved 50
HeLa = Human immortal cell line from the cervical cancerous cells of Henrietta Lacks
HMBC = Heteronuclear Multiple-Bond Correlation
HMQC = Heteronuclear Multiple Quantum Coherence
1H NMR = Proton Nuclear Magnetic Resonance
HPLC = High Performance Liquid Chromatography
HPLC-MS = High Performance Liquid Chromatography Mass Spectrometry (coupling)
HRESIMS = High Resolution Electron Spray Ionization Mass Spectrometry
HSQC = Heteronuclear Single-Quantum Coherence
HUVEC = Human Umbellical Vein Endothelial Cells
K-562 = human immortal erythroleukaemia cells
MeOH = Methanol
MS = Mass Spectrometry
NMR = Nuclear Magnetic Resonance
ppm = Parts Per Million
RP = Reversed Phase
rpm = Rotation Per Minute
TFA = Trifluoroacetic Acid
TLC = Thin Layer Chromatography
UV = Ultraviolet

Introduction
1. Introduction
1.1. Endophytes
The term endophyte includes all organisms that during a variable period of their life
symptomlessly colonise the living internal tissues of their hosts. This definition includes any
organisms residing inside a plant host [1]. However the most frequently isolated endophytes
are the fungi. Endophytic fungi are found in almost all plants including trees, grasses, algae,
mosses and herbaceous plants [1]. Under normal conditions they live within the host plant
without causing any symptoms of disease.
Presently no one knows exactly the role of endophytes in nature and their relationship to
various host plant species. While some endophytic fungi appear to be ubiquitous (e.g.
Fusarium spp., Pestalotiopsis spp., and Xylaria spp.), one cannot state that endophytes are
host specific [2]. It is more propable that their associations are developed by chance. Many
endophytes of the same species are often isolated from the same plant and only one or a few
strains produce a highly biologically active compound in culture. There is a lack of knowledge
about what an endophyte produces in culture and what it may produce in symbiosis. The
production of certain bioactive compounds by the endophyte in situ may facilitate the
domination of its biological niche within the plant or even provide protection to the plant from
harmful invading pathogens [2].
Many endophytes are closely rela