Isolation and characterisation of bioactive compounds from Antidesma venosum E.Mey. ex Tul. and Euphorbia cooperi N.E.Br. ex A.Berger Theses submitted in accordance with the requirements for the degree Master of Science By Sbonelo Sanele Hlengwa School of Chemistry and Physics University of KwaZulu-Natal Pietermaritzburg Supervisor: Professor Fanie R. van Heerden November 2018 i | P a g e Abstract The need for the discovery of new pharmacologically active compounds is a global priority as new diseases and drug resistance mechanisms are emerging and spreading throughout the world. In the past natural products have played an important role in drug discovery. Plant- derived natural products are important in the discovery of new drugs for treatment of infectious diseases and cancer. Many biologically active compounds with diverse structures have been isolated from the Euphorbiaceae family. In South Africa, Euphorbiaceae is the largest flowering plant family and some species within the family have not been investigated for unique secondary metabolites. However, many species have not been researched. This project aimed to identify bioactive compounds in South Africa Euphorbiaceae species. Two species within the Euphorbiaceae family, Euphorbia cooperi N.E.Br. ex A.Berger and Antidesma venosum E.Mey. ex Tul. were selected for investigation. The crude extracts were fractionated using column chromatography and centrifugal thin-layer chromatography to yield single compounds. A mixture of hexane, dichloromethane, chloroform, ethyl acetate and methanol were used as eluting solvents. The structures of the compounds isolated were elucidated by 1D and 2D NMR experiments (1H, 13C NMR, DEPT- 135, COSY, HSQC, HMBC and NOESY). The compounds isolated from A. venosum were identified as loliolide, β-sitosterol 3-O-β-D- glucoside, lutein, 4-hydroxyphenylethyl trans-ferulate as well as pheophytin A and pheophytin B. Although these compounds are known compounds, only pheophytin A has previously been identified in A. venosum. A phytochemical study of the aerial part of E. cooperi has led to the isolation of a novel norsesquiterpenoid named euphorbilactone and its glucoside, arachiside A, along with a triterpenoid, glutinol, a known phorbol ester, 16-angeloyloxy-13α-isobutanoyloxy-4β,9α,20- trihydroxytiglia-1,5-diene-3,7-dione, and a novel phorbol ester, 20-acetoxy-16-angeloyloxy- 13α-isobutanoyloxy-4β,9α,20-tetrahydroxytiglia-1,5-diene-3-one. This is the first report on the isolation of these compounds from E. cooperi. The crude extracts and loliolide, p-hydroxyphenylethyl trans-ferulate and β-sitosterol 3-O-β- D-glucoside, were assayed for antimicrobial activities against one Gram-positive (Staphylococcus aureus ATCC 12600), two Gram-negative (Pseudomonas aeruginosa ATCC i | P a g e 10145, Klebsiella pneumoniae ATCC 13883), and one drug-resistant Gram-negative (Escherichia coli ATCC 25218) bacteria. The crude extracts of the plants showed moderate but promising antimicrobial activity. The pure compounds did not show any antimicrobial activity. This investigation has shown that South African Euphorbiaceae contains novel bioactive compounds. The two usual phorbol esters isolated from E cooperi warrant further investigation of the bioactivity of these compounds. ii | P a g e Declaration The experimental work described in this dissertation was carried out in School of Chemistry and Physics, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Pietermaritzburg, under the supervision of Professor Fanie R. van Heerden. I hereby declare that these studies represent original work by the author and have not otherwise been submitted in any form for any degree or diploma to any tertiary institution. Where the use of published information from other authors has been made and it dully acknowledged in the text Signed …………………………. Sbonelo Sanele Hlengwa I hereby certify that this statement is correct Signed …………………………. Professor Fanie R. van Heerden (Supervisor) iii | P a g e Plagiarism I, Sbonelo Sanele declare that: 1. The research reported in this thesis, except where otherwise indicated, is my original research. 2. This thesis has not been submitted for any degree or examination at any other university. 3. This thesis does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This thesis does not contain other persons' writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written but the general information attributed to them has been referenced b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This thesis does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References sections. Signed………………………… Date……………………… (Sbonelo Sanele Hlengwa) iv | P a g e Acknowledgements I would like to express my sincere graduate to: My supervisor Professor Fanie van Heerden for her encouragements, advice guidance and support throughout this project, without you this theses would never have been a reality. My lovely Family for always being there for me through tough times and supporting me The Natural Products Research Group for creating a good atmosphere The academic and the technical staff in the School of Chemistry and Physics for their assistance National Research Funding for financial assistance All friends for creating a good environment for me “Ngiyabonga” v | P a g e Table of Contents Isolation and characterisation of bioactive compounds from Antidesma venosum E.Mey. ex Tul. and Euphorbia cooperi N.E.Br. ex A.Berger Abstract ....................................................................................................................................... i Declaration ................................................................................................................................ iii Plagiarism.................................................................................................................................. iv Acknowledgements .................................................................................................................... v Table of Contents ...................................................................................................................... vi List of Figures ........................................................................................................................... ix List of Tables .......................................................................................................................... xiii List of schemes......................................................................................................................... xv Abbreviations .......................................................................................................................... xvi CHAPTER 1 .............................................................................................................................. 1 Introduction and Aims of the Project ......................................................................................... 1 1.1 African traditional medicine .................................................................................... 1 1.2 Natural products and drug discovery ........................................................................... 2 1.3 Problem statement ................................................................................................... 4 1.4 Aims and objectives ..................................................................................................... 5 CHAPTER 2 .............................................................................................................................. 6 A Literature Review on the Euphorbiaceae and Diterpenoids ............................................... 6 2.1 Euphorbiaceae (Spurge family) ................................................................................... 6 2.2 Tigliane diterpenes isolated from Euphorbiaceae plant species .................................. 9 CHAPTER 3 ............................................................................................................................ 16 Phytochemistry of Antidesma venosum E.Mey. ex Tul. .......................................................... 16 3.1 Introduction .................................................................................................................... 16 3.1.1 Antidesma Genus .................................................................................................... 16 3.1.2 Bioactive compounds from Antidesma ................................................................... 16 3.1.3 Antidesma venosum E.Mey. ex Tul. ....................................................................... 18 3.2 Results and discussion ................................................................................................... 19 vi | P a g e 3.2.1 Structural elucidation of loliolide (37) .................................................................... 20 3.2.2 Structural elucidation of lutein (38) ........................................................................ 25 3.2.3 Structural elucidation of pheophytin A (39) and pheophytin B (40) .....................