Southern Cross University ePublications@SCU Theses 2009 Phytochemical studies and bioactivity of Centipeda and Eremophila species Karren D. Beattie Southern Cross University, [email protected] Suggested Citation Beattie, KD 2009, 'Phytochemical studies and bioactivity of Centipeda and Eremophila species', PhD thesis, Southern Cross University, Lismore, NSW. Copyright KD Beattie 2009 ePublications@SCU is an electronic repository administered by Southern Cross University Library. Its goal is to capture and preserve the intellectual output of Southern Cross University authors and researchers, and to increase visibility and impact through open access to researchers around the world. For further information please contact [email protected]. Phytochemical Studies and Bioactivity of Centipeda and Eremophila Species Thesis submitted by Karren Deanne Beattie B.Sc. (Hons.). A thesis submitted in fulfillment of the requirements for the award of the degree of Doctor of Philosophy School of Natural and Complementary Medicine Southern Cross University 2009 Thesis Declaration I certify that the work presented in this thesis is, to the best of my knowledge and belief, original, except as acknowledged in the text, and that the material has not been submitted, either in whole or in part, for a degree at this or any other university. I acknowledge that I have read and understood the University's rules, requirements, procedures and policy relating to my higher degree research award and to my thesis. I certify that I have complied with the rules, requirements, procedures and policy of the University (as they may be from time to time). Name: ……………………………………………………………… Signature: ………………………………………………………………… Date: …………………………………………………..…………………. ii Preface Some of the bioassays described in Chapter 6 of this thesis were performed by others; • The termiticidal, repellency and fumigant properties as well as the barrier studies of the E. mitchellii wood oil were performed by Associate Professor Robert Spooner-Hart and Dr Albert Basta of the University of Western Sydney, (Centre for Plant and Food Sciences) Hawkesbury. • Efficacy of the E. mitchellii wood oil fractions against termites as well as investigation of the termiticidal properties of the E. mitchellii leaf oil and its constituents were also performed by Associate Professor Robert Spooner-Hart and Dr Albert Basta. • Determination of the acute dermal toxicity IC50 and IC95 for the eremophilanes was performed collaboratively with Associate Professor Robert Spooner-Hart and Dr Albert Basta. Some of the bioassays described in Chapter 4 of this thesis were performed by others; • Antioxidant and anti-inflammatory assays on C. cunninghamii extracts and fractions were performed by Dr Denise Hunter and Ms Kelly Shepherd at the Centre for Phytochemistry and Pharmacology at Southern Cross University. Some of the spectroscopic analyses described in this thesis were performed by others; iii • Optical rotations were performed by Dr Kim Dastlick at the Institute for Molecular Biology at the University of Queensland. • High Resolution Mass Spectroscopy was performed by Chemical Analysis Laboratories, Bulleen, Victoria. • X-ray crystallography studies were performed by Dr Donald Craig at the Department of Chemistry, University of New South Wales. • NMR spectroscopy of compounds from C. cunninghamii was performed by Dr Myrna Deseo at the Centre for Phytochemistry, Southern Cross University. Some photographs in this thesis were provided by others, as credited in the text. iv Abstract The aim of this study was to isolate and characterise biologically active compounds from endemic Australian plants. A total of 6 novel, and 26 known compounds have been isolated throughout the course of this work. A comprehensive investigation of the GC-MS chemical profile of C. cunninghamii leaf essential oil found that thymol (1) cis-chrysanthenyl acetate (4), myrtenyl acetate (2), myrtenol (3) and cis-chrsanthenol (5) were the major constituents. The essential oil and crude solvent extracts of C. cunninghamii possessed significant antioxidant and anti- inflammatory activity. A 50% aqueous ethanol extract was demonstrated to possess multiple modes of anti-inflammatory action. The crude extract was found to significantly inhibit both COX-1 and COX-2 cyclooxygenases and was comparable to the positive controls; Ibuprofen and Celebrex respectively. The crude extract also exhibited anti-inflammatory activity in the nitric oxide (NO) and tumor necrosis factor- alpha (TNF-α) assays, but did not show inhibition in the lipoxygenase (LO) assay. A total of seventeen compounds, of which 10, 6, 7, 8 and 9 are novel, have been identified from the aqueous-ethanolic extract of C. cunninghamii. Five flavonoids; axillarin (16), isokaempferide (17), 4’,5,7-trihydroxy-3,6-dimethoxyflavone (18), jaceidin (19), and 2’,4’,5,7-tetrahydroxy-6-methoxyflavone-3-O-β-glucopyranoside (10) were isolated from the flowers of C. cunninghamii. A series of caffeic acids were isolated as the major component of the stems, these included; chlorogenic acid (12) and its methyl ester (13), caffeic acid ethyl ester (11), isochlorogenic acid A (14) v macroantoin G (15) and the novel 4ξ,5ξ-di-O-caffeoyl-2,6ξ-dihydroxyhept-2-ene-1,7- dioic acid (6) and its 1-methyl ester (7), 7-methyl ester (8) and 1,7-dimethylester (9) derivatives. Lastly, arnicolide C (20) a sesquiterpene lactone, 3-hydroxykaura-9(11),16- diene-18-oic acid (21) and 8-hydroxy-9,10-diisobutyryloxythymol (23) were characterised by spectroscopic methods. All of the compounds were evaluated for anti-inflammatory activity, as determined by the inhibition of prostaglandin E2 in 3T3 fibroblast cells. All compounds, inhibited PGE2 production to some extent, at a concentration of 31.25 µg/mL. The flavonoids 10 and 16-19 were the most active compounds. The caffeic acids 6-9, 12-14 and the thymol derivative 23 also significantly inhibited PGE2 production. The IC50 values were determined for the novel compounds; 10, 6, 7, 8 and 9, as 1.47, 2.48, 4.73, 5.54 and 1.26 µM, respectively. These novel compounds were more potent than the positive control, aspirin, which was found to inhibit PGE2 production by 42% at a concentration of 18 µM. Antioxidant activity, as determined by oxygen radical absorbance capacity (ORAC) has also been attributed to both the flavonoids; 10, 16-19 and caffeic acid compounds; 6-9 and 12-14. The antioxidant capacity of these compounds was found to be comparable to epicatechin, a major antioxidant constituent of green tea. A detailed analysis of the wood, leaf, branch and root oil of E. mitchellii was carried out by a combination of GC-FID, GC-MS, LC/MS and NMR spectroscopy. The wood, root, leaf and branch oils were found to be predominantly composed of sesquiterpenes. The three major compounds identified in the leaf oil, which accounted for 44% of the vi oil, were α-pinene (40), (+) spathulenol (15) and an unidentified sesquiterpene alcohol. The composition of the leaf oil was complex and chemically distinct from the wood and root oils, whereas the branch oil was found to exhibit a chemical composition that was intermediate between the leaf and the wood oil. After fractionation by preparative HPLC six components of the wood oil were characterized and accounted for 80% of the oil. The major constituents of the wood oil were; eremophilone (30), 9-hydroxy-7(11),9-eremophiladien-8-one (36), santalcamphor (35) and the novel 9-hydroxy-1,7(11),9-eremophilatrien-8-one (42). Two minor constituents, 8-hydroxy-10,11-eremophiladien-9-one (32) and 8-hydroxy-1,11- eremophiladien-9-one (33) were also isolated in this study. The two major constituents of the root oil of E. mitchellii were found to be eremophilone (30) and the zizaene, sesquithuriferone (43). These, together with the minor constituents 32, 33, 42, 35 and 36 accounted for 92% of the root oil. The insecticidal properties of E. mitchellii were evaluated against several species of termites Nasutitermes walkeri (Hill), Nasutitermes exitiosus (Hill) and Coptotermes acinaciformis (Froggatt). Bioassay-guided fractionation of E. mitchellii wood oil was undertaken to investigate the termiticidal metabolites. Of the major components, it has been determined that eremophilone (30) was the most active constituent of the wood oil followed by 8-hydroxy-1,11-eremophiladien-9-one (33), 9-hydroxy-7(11),9- eremophiladien-8-one (36) and santalcamphor (35). vii The methanolic extracts from a total of 36 species have been evaluated for cytotoxicity against P388D1 mouse lymphoblast cells. A collection of fifteen species of Eremophila from Western Australia and a further twenty from the Northern Territory were surveyed. Cytotoxicity was found to be largely non-selective across a range of human cancer cell lines, including MCF7 (mammary adenocarcinoma), Hep G2 (hepatocellular carcinoma), A2780 (ovarian carcinoma), A-375 (malignant melanoma) and PC-3 (prostate cancer). Fractionation of the leaf material of E. racemosa afforded the six major metabolites. Isolation and structural elucidation of these polar compounds revealed the cyanogenetic glycoside prunasin (65), the flavonoid luteolin (74), the furofuran lignans, phillygenin (75), its 4-O-β-D-glucoside phillyrin (76), pinoresinol-4-O-β-D-glucoside (77) and epipinoresinol-4-O-β-D-glucoside (78). Fractionation of the leaf material of E. maculata var. brevifolia afforded piceine (81) and epipinoresinol-4-O-β-D-glucoside (78). Quercetin (79) and nepetin (80) were isolated from the methanolic extracts of E. bignoniflora.