Phytochemical Characterization and Antimicrobial Activity of Sudanese Medicinal Plants

522 Pages·2012·11.53 MB·English

Phytochemical Characterization and Antimicrobial Activity of
Sudanese Medicinal Plants

by
Mohamed Gamaleldin Elsadig Karar
A thesis submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
in Chemistry

Approved Dissertation Committee
Prof. Dr. Nikolai Kuhnert
Professor of Chemistry, Jacobs University Bremen
Prof. Dr. Gerd Roeschenthaler
Professor of Chemistry, Jacobs University Bremen
Dr. Robert B. Smith
Senior Lecturer in Organic/Medicinal Chemistry, University
of Central Lancashire, UK
Date of Defense: 29.06.2015
To my Mother, Souls of my Father and my Son

ii
Statutory Declaration
(on Authorship of a Dissertation)

I, Mohamed Gamaleldin Elsadig Karar hereby declare that I have written this PhD thesis
independently, unless where clearly stated otherwise. I have used only the sources, the data and
the support that I have clearly mentioned. This PhD thesis has not been submitted for conferral
of degree elsewhere.

I confirm that no rights of third parties will be infringed by the publication of this thesis.

Bremen, July 11, 2015

Signature ___________________________________________________________
iii
Acknowledgements
All praises to AllAH, the Almighty for giving me the strength to complete this work.
I am very grateful to my advisor, Prof. Dr. Nikolai Kuhnert for his comprehensive support in all
stages of my study. I had the excellent opportunity of being a PhD student in his department
during my doctoral studies. Prof. Dr. Nikolai Kuhnert introduced me to the world of LC-MS
among countless of other areas, and for this I am forever thankful. His knowledge,
understanding, motivation and consideration contributed to my personal and professional
improvement. “Prof. Kuhnert, vielen herzlichen Dank!”
I express my sincere thanks to the members of my defense committee Prof. Dr. Gerd
Roeschenthaler and Dr. Robert B. Smith for their time to go through my dissertation and being
part of my final exam.
I am indebted to DAAD (Deutscher Akademischer Austausch Dienst) for giving me the
opportunity to carry out my PhD study in Germany.
I am grateful to Dr. Rakesh Jaiswal for his constant help, fruitful suggestions, scientific
discussions and considerable contribution to my studies. I would like to thank Anja Müller for
excellent technical support. I wish to express my warmest thanks to my current and former Prof.
Dr. Nikolai Kuhnert’s group members for providing me with supportive company during my
research work. I would also like to give a special thanks to Abhinandan Shrestha for his alliance
and help with certain computer issues. I would like to express my greatest gratitude to Marius-
Febi Matei for revision and language suggestions for parts of this thesis.
I would also like to thank Dr. Ahmed Rezk, Dr. Daniel Pletzer, Laura Quiet, Maren Rehders, Dr.
Susanne Illenberger, Dr. Hiba Ali and Prof. Dr. Sami A. Khalid for their help with my biological
assays.
I have reverence and gratitude for my wife Tasneem Husein, who gave me the determination and
strength I needed on many occasions. My sincere love goes to my sisters Hanadi, Hala and Nona,
and my brothers Bakri, Magdi and Elsadig for always being there for me.
iv
The following is my appreciation to those people who made me feel at home in Germany, in
particular:
 I wish to express my warmest thanks to Karin Janssen for standing behind me and my
family in the dark and the bright days. Thanks for always being there for us.
 I owe a huge debt to my host family Mrs. Karin Bleyens and Mr. Siegfried Bleyens for
all happy moments we had together.
 I am also thankful to John Marcus for being like my father and for the nice time we spent
together. I appreciated that I had the chance to meet you here in Germany.
 I have special gratitude for Beatrix Tetzlaff, you have helped me in so many ways in
particular in translating and understanding several German’s medical reports.
 Finally, I offer my sincerest thanks to Monika Weissenhorn, Eichen-Apotheke for her
kindness and for preparing prescriptions for me and my family over the last four years.
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Abstract
In Sudan, a tropical country rich in biodiversity and biological resources, medicinal plants have
been used for the treatment of several diseases and microbial infections in traditional medicine.
However, the actual compounds responsible for the biological effects are still poorly
characterized. In this project, extracts prepared from traditional medicinal plants native to Sudan
and Europe were investigated for their phytoconstituents and biological effects. 457 phenolic
compounds were unambiguously assigned to their regioisomeric level using LC–MSn. 332 of
them were extracted for the first time from their sources, and 46 of them were not reported
previously in nature. Chlorogenic acids, chlorogenic acid glycosides, phenolic acids,
hydroxycinnamates, proanthocyanidins, flavonoid glycosides, flavonoid aglycones and
derivatives thereof were identified as major phytoconstituents. Next, using preparative HPLC, 18
bioactive compounds including chlorogenic acids, flavonoid glycosides, coumarins and
derivatives were isolated and identified in the plant extracts. Concerning the biological activities,
various extracts showed antibacterial, antiparasitic and antioxidant activities. For example
Acacia nilotica, Ocimum basilicam, Ziziphus spina-christi, Balanites aegyptiaca, Sonchus
oleraceus, Punica granatum, Mimosa pigra and Ixora coccinea were the most interesting ones
with antioxidative, antiplasmodial, antitrypanosomal and antibacterial activities. Moreover, the
antibacterial activities of some naturally occurring phenolic compounds were investigated.
Among the tested individual phytochemicals, phloretin, resveratrol, luteolin 7-O-glucoside and
epigallocatechin gallate showed the highest antimicrobial activities. Most importantly, we
investigated the influenza virus neuraminidase (NA) inhibitory activities of naturally occurring
chlorogenic acids and synthesized derivatives against several viral NAs. The enzyme inhibition
results indicated that chlorogenic acids and caffeic acid derivatives exhibited high antiviral
effects.
This study provides a detailed molecular level understanding of the phytoconstituents in a wide
selection of Sudanese medicinal plants. Consequently, the phytochemical and pharmacological
knowledge is better understood in support of the use of such plants in traditional medicine.
As a highlight, the project presents a comprehensive LC-MS analysis for model mono and
oligosaccharides based on their binding behavior to the sodium ion.
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This contribution shows that the modern MS methods such as UPLC-ESI-Q-TOF-MS/MS and
HPLC-ESI-MSn can be used as unambiguously assignment of regioisomers of flavonoids
glycosides and phenolic natural products. They can also be applied as predictive reliable
techniques in the assignment of mono and oligosaccharide relative stereochemistry.
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Contents
Statutory Declaration ……………………………………………………………………. iii
Acknowledgements ……………………………………………………………………….. iv
Abstract …………………………………………………………………………………… vi
Publications ……………………………………………………………….......................... ix
Glossary of Abbreviations ……………………………………………………………….. xii
Thesis Overview ………………………………………………………………………….. xiv
1. Introduction …………………………………………………………………………….. 1
1.1. Polyphenols ………………………………………………………………………. 1
1.2. Aim of the Thesis ………………………………………………………………… 17
2. Herbal Drugs from Sudan: A review (submitted) ………………………………………. 18
3. Carbohydrates Analysis (submitted) ……………………………………………………. 77
4. Inhibition of Neuraminidase Activity by Chlorogenic Acids (submitted) ……………... 98
5. Mimosa pigra Phenolics (submitted) …………………………………………………… 123
6. Phenolics, Cytotoxicity and Antibacterial Activity of Ziziphus spina-christi (submitted) 159
7. Crataegus species & Crataegutt Tropfen Phenolics (submitted) ……………………….. 198
8. Biological Activities of Medicinal Plants from Sudan (submitted) …………………….. 251
9. Quince (Cydonia oblonga) Phenolics ………………………………………………… 277
10. Ixora coccinea Phenolics ……………………………………………………………… 286
11. Ilex glabra Phenolics ………………………………………………….......................... 296
12. Lonicera henryi Phenolics …………………………………………….......................... 311
13. Proanthocyanidins of Rhododendron Genus ………………………………………….. 323
14. Conclusions ……………………………………………………………………………. 341
Appendix ………………………………………………………………………………….. 343
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Publications
1. Karar M.G.E., Pletzer D., Jaiswal R., Weingart H., Kuhnert N.* Identification,
Characterization, Isolation and Activity Against Escherichia coli of Quince (Cydonia
oblonga) Fruit Polyphenols. Food Res Int 2014; 65: 121-129.
2. Jaiswal R, Karar M.G.E., Gadir HA, Kuhnert N*. Identification and Characterisation of
Phenolics from Ixora coccinea L. (Rubiaceae) by Liquid Chromatography Multi-stage
Mass Spectrometry. Phytochem Anal 2014; 25: 567-576.
3. Jaiswal R, Halabi EA, Karar M.G.E., Kuhnert N*. Identification and Characterisation of
the Phenolics of Ilex glabra L. Gray (Aquifoliaceae) Leaves by Liquid Chromatography
Tandem Mass Spectrometry. Phytochemistry 2014; 106: 141-155.
4. Jaiswal R, Mueller H, Mueller A, Karar M.G.E., Kuhnert N*. Identification and
Characterization of Chlorogenic Acids, Chlorogenic Acid Glycosides and Flavonoids
from Lonicera henryi L. (Caprifoliaceae) Leaves by LC-MSn. Phytochemistry 2014; 108:
252-263.
5. Jaiswal R, Karar M.G.E., Kuhnert N. Identification and Characterization of Trimeric
Proanthocyanidins of Two Members of the Rhododendron Genus (Ericaceae) by Liquid
Chromatography Multi‐Stage Mass Spectrometry. In: Robert A. Meyers, editor.
Encyclopedia of Analytical Chemistry. Marietta: Wiley Online Library; 2014.
6. M.G.E. Karar, N. Kuhnert*. Herbal Drugs from Sudan: Traditional Uses and
Phytoconstituents. A review. Pharmacognosy review, submitted.
7. M.G.E. Karar, A. Müller and N. Kuhnert*. A hierarchical Scheme for the Determination
of Hexose and Pentose relative Stereochemistry in Oligosaccharides and Glycosides
using Tandem Mass Spectrometry, Journal of Agricultural and Food Chemistry,
submitted.
8. M.G.E. Karar, MF. Matei, R. Jaiswal, S. Illenberger, N. Kuhnert*. Neuraminidase
Inhibition of Chlorogenic Acids and Derivatives from Ambrosia maritima and Sonchus
oleraceus. Phytomedicine, submitted.
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9. M.G.E. Karar, R. Jaiswal and N. Kuhnert*. Profiling the Antioxidant (Poly)phenols of
Mimosa pigra L. (Fabaceae) by LC-DAD-ESI-MSn and LC-DAD-ESI-HRMS.
Fitoterapia, submitted.
10. M.G.E. Karar, L. Quiet, A. Rezk, R. Jaiswal, M. Rehders, M. S. Ullrich, K. Brix, N.
Kuhnert*. Phenolic Profile and In Vitro Assessment of Cytotoxicity and Antibacterial
Activity of Ziziphus spina-christi Leaf Extracts. Plant Medica, submitted.
11. M.G.E. Karar, and N. Kuhnert*. UHPLC-ESI-Q-TOF-MS Characterization of Phenolics
from Crataegus monogyna and Crataegus laevigata (Hawthorn) Leaves, Fruits and the
Herbal Derived Syrup (Crataegutt Tropfen). Fitoterapia, submitted.
12. M.G.E. Karar, A. Rezk, O. T. A. Abdalla, A. A. Y. Ebrahim, M. S. Ullrich, N. Kuhnert*.
Antimicrobial, Antiparasitic and Antioxidant Activities of Medicinal Plants from Sudan.
Journal of Ethnopharmacology, submitted.
Poster Presentations
1. M.G.E. Karar, N. Kuhnert*. Identification and Characterization of Phenolics from
Mimosa pigra L. (Fabaceae) by LC-MSn. Bremen mass spectrometry seminar. July 2014,
Bremen, Germany.
2. M.G.E. Karar, N. Kuhnert*. Phenolics in the Sudanese Medicinal Plants Ziziphus spina-
christi. Retreat-MOLIFE Center. Jacobs University Bremen. March 2013, Bad Bevensen-
Germany.
3. M.G.E. Karar, N. Kuhnert*. Polyphenols in Sudanese Plant Ixora coccinea L.
(Rubiaceae). Retreat-NanoFun Center and Nanomol Graduate Programe, Jacobs
University Bremen, June 2012, Herz, Clausthal-Zellerfeld, Germany.
Oral Presentations
1. M.G.E. Karar, N. Kuhnert*. Characterization and Identification of Quince (Cydonia
oblonga) Fruit and Ziziphus spina-christi (L.) Desf. Leaves Polyphenols by LC-MSn.
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