Extraction, isolation and characterization of oleanolic acid and its analogues from syzygium aromaticum (cloves) and evaluation of their biological activities. A ―Dissertation submitted in Partial Fulfilment of the Requirements for the Degree of Masters of Science (MSc.) in Chemistry in the Faculty of Science and Agriculture, at the University of Fort Hare‖ By Khwaza Vuyolwethu (201209227) Supervisor: Prof. O. O. Oyedeji Co-supervisor: Prof. B. A. Aderibigbe 2019 i DECLARATION I, ―Khwaza Vuyolwethu, student number 201209227 declare that this Dissertation entitled, Extraction, isolation and characterization of oleanolic acid and its analogues from Syzygium aromaticum (cloves) and evaluation of their biological activities, which I hereby submit to the university of Fort hare in partial fulfilment of the requirements for the Masters of Science (Chemistry) is my own original work, it has never been submitted for any academic award to any other institution of higher learning.‖ ……………………………. …………………………………… Khwaza Vuyolwethu Date ……………………………. …………………………………… Prof O.O Oyedeji Date Supervisor ……………………………. …………………………………… Prof. B. A. Aderibigbe Date Co-Supervisor ii PLAGIARISM DECLARATION I, Khwaza Vuyolwethu, student number 201209227 hereby declare that I am fully aware of the University of Fort Hare‘s policy on plagiarism and I have taken every precaution to comply with the regulations. 1. I declare that this dissertation is the results of my own original work. Where someone else‘s work was used (others sources i.e internet, printed sources etc.) due acknowledgment was given and reference was made according to the department requirements. 2. I did not use another student‘s work and submit it as my own work. 3. I did not and will not allow anyone to copy my work with the intention of presenting it as his/her own work.‖ iii CONFERENCE AND PUBLICATIONS Conferences Vuyolwethu Khwaza, Opeoluwa O. Oyedeji and Blessing A. Aderibigbe (2018). Isolation and Characterization of oleanolic acid and its analogues from syzygium aromaticum. Poster presentation at the BIOAFRICA CONVENTION: International conference Durban ICC South Africa. Publications Vuyolwethu Khwaza, Opeoluwa O. Oyedeji and Blessing A. Aderibigbe (2018). Antiviral Activities of Oleanolic Acid and Its Analogues. Molecules 2300 (23) 1-14. Khwaza V, Opeoluwa O. Oyedeji, Mike O.Ojemaye, Adebola O.Oyedeji and Francis B.Lewu (2018) ―Assessment of the heavy metal content of wild and cultivated Pelargonium inquinans: an herbal plant used for the treatment of divers ailments in South Africa‖. Fresenius Environmental Bulletin 27(6) 3914-165. iv ACKNOWLEDGEMENTS In Africa they say ―It takes a village to raise a child‖. Nothing in this world is ever successful without a corporate of many talented people who are willing to work and submit their talents, experiences, and passion for a common goal. Firstly I would like thank God Almighty for making it possible for me to complete this work in spite of many difficulties experienced along the way. I wish to thank my supervisors Prof. O. O. Oyedeji and Prof. B.A. Aderibigbe for their assistance, guidance and encouragement throughout the study, you both played a major role in my life. Thank you and may you continue to make tremendous difference in South Africa and the world at large. It is an honour to have been under your supervision. My Mentor ―from Sasol Dr Du Plessis Esna, thank you for your words of encouragement you have carried me to where I am today. For that, God bless you and your family. I would like to thank Mr T. Mcako for helping with the FTIR spectroscopy. I wish to thank Prof. R. Krause and Dr S. N. Xavier from Rhodes University for their excellent assistance with NMR, and MS. I would like to thank National Research Foundation (NRF), Sasol inzalo foundation and Medical Research Council for funding.‖ The Department of chemistry, University of fort hare. I would like to send my sincere gratitude to my brother Mtobeli Khwaza ―Madala‖ and my dear sister Tobeka Khwaza for your support, encouragement and for being there for me throughout. A special thanks to Khatywa Ongeziwe. You‘ve been a big brother to me since my first day in UFH. v I want to thank all my lab mates for lending their expertise when required. My family at large thank you for your prayers, it is not my by power that I managed to finish, but through your prayers. From the bottom of my heart I love you so much. vi DEDICATION I dedicate this work to my Mom (Nozuzile khwaza) for being the positive motivating force in my life. You‘ve struggled against all odds to get me educated. May God Almighty shower you with all the blessings that you deserve, I love you Mazaka. vii ABSTRACT Pathogenic microorganisms have serious impact on people's lives. Every year, millions of people around the world die of bacterial infections. Resistance to common antibacterial drugs has proven to be a challenging problem in control of bacterial infections. In an attempt to develop an effective and affordable treatment for bacterial infections, oleanolic acid isolated from syzygium aromaticum conjugates incorporating other pharmaceutical scaffolds such as chloroquine derivatives, curcumin, and ergocalciferol etc have been developed. Based on the previous successes of testing combination of antimicrobial drugs and pharmaceutical drugs which appeared to be the promising strategy to overcome treatment failure; a series of hybrid compounds containing oleanolic acid and other pharmaceutical scaffolds were synthesized. 4- Aminoquinoline derivatives were first hybridized with selected organic compounds to form a class of hybrid compounds containing either amide bond or ester bond as a linker between the precursor molecules. Analogues/hybrid compounds can overcome the disadvantages of combination therapy such as drug-drug interaction. The structural effects of this type of conjugation of oleanolic acid and other pharmaceutical scaffolds were characterised by FT- IR, Mass Spec and NMR spectroscopy. These compounds were studied along with the mono- substituted oleanolic acid analogues and the organic components in order to compare the effects of the substitution on their biological response.‖ All the synthesized analogues were tested against 11 bacterial strains on both Gram-positive and Gram-negative bacteria. The synthesized compounds showed selectivity and higher activity against Enterococcus faecalis (EF), Klebsiella oxytoca (KO), Escherischia coli (EC), Staphylococcus aureous (SA), Proteus vulgaris (PV) and Bacillus subtilis (BS) with MIC values; ranging between of 1.25 mg/mL to 0.072 mg/mL. Key words: Oleanolic acid, Antibacterial, Syzygium aromaticum, Analogues and Pharmaceutical scaffolds. viii LIST OF FIGURES Figure 2. 1: phenol (parent structure of all phenolic compounds) ........................................... 13 Figure 2. 2: examples of phenolic compounds ........................................................................ 14 Figure 2. 3: biosynthesis of different classes terpenes. ............................................................ 16 Figure 2. 4: examples of monoterpenes ................................................................................... 17 Figure 2. 5: Sesqueterpene compounds ................................................................................... 18 Figure 2. 6: examples of diterpene compounds ....................................................................... 19 Figure 2. 7: different classes of triterpenes structures. ............................................................ 20 Figure 2. 8: Examples of terpene compounds. ......................................................................... 21 Figure 2. 9: Common modern drugs derived from plant sources ............................................ 22 Figure 2. 10: antimicrobial drugs ............................................................................................. 24 Figure 2. 11: 3-acetoxy, 28-methyloleanolic acid (2.42), 3-acetoxyoleanolic acid (2.43). ..... 27 Figure 2. 12: oleanolic acid derivatives studied for PTP-1B inhibition. ................................. 29 Figure 2. 13: Oleanolic derivatives with anti-HIVactivity ...................................................... 31 Figure 2. 14: derivatives of oleanolic acid with anti-HIV activities. ...................................... 32 Figure 2. 15: Previously modified anti-HIV triterpene derivatives. ....................................... 32 Figure 3. 1: World‘s distribution of syzygium aromaticum (clove) ......................................... 52 Figure 3. 2: Fresh (A) and dried (B) syzygium aromaticum .................................................... 53 Figure 3. 3: Clove‘s oil constituents. ....................................................................................... 54 Figure 3. 4: TLC plate showing the isolated three compounds from ethylacetate crude extract. .................................................................................................................................................. 56 Figure 3. 5: formula for calculating Rf value of a compound. ................................................. 57 Figure 3. 6: Structure of Eugenol (Eu)..................................................................................... 58 ix Figure 3. 7: Structure of oleanolic acid (OA). ......................................................................... 60 Figure 3. 8: Structure of Maslinic acid. ..................................................................................