The aim of this study was to isolate and characterise novel anti-inflammatory compounds from native Australian plants which were important to the D'harawal Aboriginal people for antiinflammatory and related activities. A total of thirty two plants were screened for their antiinflammatory and neuroprotective activity. In chapter 2, ethanolic extracts of seventeen Eucalyptus spp. (Myrtaceae) were screened for their nitric oxide (NO) and tumour necrosis factor-I± (TNF-I±) downregulation activity and cytotoxicity in lipopolysaccharide (LPS) and interferon-I³ (IFN-I³) activated RAW 264.7 macrophages. Extracts from seven Eucalyptus spp. demonstrated strong activity with IC50 values between 7.58 - 19.77 µg/mL for NO inhibition and IC50 values for suppression of TNF-I³ production were between 2.06 "" 19.02 µg/mL. These extracts also showed a wide range of cytotoxicity with LC50 values between 22.34 "" 236.5 µg/mL. In chapter 3, two of the highly active Eucalyptus spp. (Myrtaceae), E. viminalis and E. bosistoana were sequentially extracted and screened to find out the most active extracts which were then fractionated to identify bioactive compounds. From E. viminalis a new chromone (compound 1) has been identified together with two known compounds 8-I² C glucopyranosyl-5,7 dihydroxy 2isobutylchromone and globuluside. The anti-inflammatory and cytotoxic activities of all three compounds were evaluated against RAW 264.7 macrophage and N11 microglial cell line. In RAW 264.7 macrophage, the IC50 values for NO down regulation were 44.0, 47.0 and 37.6 µg/mL whereas the IC50 of TNF I± suppression were 41.0, 38.3 and 43.2 µg/mL for compound 1, 8"I²"Cglucopyranosyl"5,7"dihydroxy"2"isobutylchromone and globuluside respectively. In N11 microglia, the IC50 values for NO down regulation were 43.4, 34.1 and 21.8 µg/mL whereas the IC50 of TNF"I± suppression were 20.4, 34.3 and 19.0 µg/mL for compound 1, 8"I²"C"glucopyranosyl"5,7"dihydroxy2"isobutylchromone and globuluside respectively. In both cell lines all of the compounds were nontoxic up to the highest concentration (36 µg/mL) tested. Oleuropeic acid which was obtained as a hydrolyzed product of compound 1 was also tested for its anti-inflammatory activity. The NO inhibitory IC50 values were 18.7 and 10.2 µg/mL and TNF"I± inhibitory IC50 values were 21.5 and 17.0 µg/mL for oleuropeic acid against RAW 264.7 macrophage and N11 microglial cell line respectively. From E. bosistoana (chapter 3a) one known metabolite, 4"coumaroylquinic acid was identified whose anti-inflammatory and cytotoxic activities were evaluated in RAW 264.7 macrophage and N11 microglial cell line as well. In RAW 264.7 macrophages the compound exhibited IC50 values of 95.74 and 52.56 µg/mL for NO and TNF-I± inhibition respectively. Whereas in N11 microglia the NO and TNF-I± inhibitory IC50 values were 44.31 and 35.50 µg/mL. In both cell line the compound was nontoxic up to the highest concentration (36 µg/mL) tested. In chapter 4, ethanolic extracts of fifteen plant species from 8 different families and 11 different genera were screened for their NO and TNF-I± downregulation activity and cytotoxicity in LPS and IFN-I³ activated RAW 264.7 macrophages. Extracts from four of the plants, Syncarpia glomulifera subsp. glomulifera, Melaleuca linariifolia, Baeckea ramosissima subsp. ramosissima and B. imbricata exhibited very strong activity with IC50 values between 8.25 "" 16.78 µg/mL for NO inhibition and IC50 values for suppression of TNF-I± production were between 8.31 "" 23.30 µg/mL. These extracts were found less toxic compared to plants from Eucalyptus spp. with LC50 values between 52.09 "" 130.0 µg/mL. xxi In chapter 5, Melaleuca linariifolia was extracted sequentially with low to high polar solvents and the sequential extracts were screened for their anti-inflammatory activity to identify the most potent one, which was then purified using HPLC. From this plant, two known flavonoids 3, 3', 4', 5, 7pentahydroxyflavan and 3, 3', 5, 5', 7- pentahydroxyflavan were identified along with one known triterpenoid betulinic acid. The anti-inflammatory activities as well as cytotoxicity of all three compounds were evaluated in RAW 264.7 macrophage and N11 microglial cell line. In RAW 264.7 macrophage, the IC50 values for NO down regulation were 72.81, 39.69 and 2.73 µg/mL whereas the IC50 of TNF-I± suppression were 58.88, 80.70 and 4.11 µg/mL for 3, 3', 4', 5, 7pentahydroxyflavan, 3, 3', 5, 5', 7- pentahydroxyflavan and betulinic acid respectively. In N11 microglia, the IC50 values for NO down regulation were 66.27, 58.05 and 2.23 µg/mL whereas the IC50 of TNF-I± suppression were 17.34, 21.84 and 6.76 µg/mL for 3, 3', 4', 5, 7-pentahydroxyflavan, 3, 3', 5, 5', 7- pentahydroxyflavan and betulinic acid respectively. In chapter 6, Syncarpia glomulifera subsp. glomulifera was extracted sequentially with low to high polar solvents and the sequential extracts were screened for their anti-inflammatory activity to identify the most potent one, which was then purified using HPLC. From this plant, two new compounds compound 6.1 and 6.4 were identified along with three known compounds tetragocarbone B (compound 6.2), sideroxylin (compound 6.3) and lumaflavanone A (compound 6.5). The anti-inflammatory and cytotoxic activities of all five compounds were evaluated in RAW 264.7 macrophage and N11 microglial cell line. In RAW 264.7 macrophage, the IC50 values for NO down regulation were 3.91, 35.15, 2.76, 29.42 and 7.84 µg/mL whereas the IC50 of TNF-I± suppression were 16.90, 32.12, 20.80, 37.57 and 33.35 µg/mL for compound 6.1, 6.2, 6.3, 6.4 and 6.5 respectively. In N11 microglia, the IC50 values for NO down regulation were 4.52, 21.17, 3.87, 39.64 and 4.51 µg/mL whereas the IC50 of TNF-± suppression were 6.50, 27.01, 13.66, 33.06 and 5.46 µg/mL for compound 6.1, 6.2, 6.3, 6.4 and 6.5 respectively. In chapter 7, Baeckea ramosissima subsp. ramosissima and B. imbricata leaves were extracted sequentially with low to high polar solvents and the sequential extracts were screened for their antiinflammatory activity to identify the most potent one. Sequential EtOAc extract from both plants showed highest anti-inflammatory activity for NO inhibition (IC50 = 9.33 and 8.98 I¼g/mL for Baeckea ramosissima subsp. ramosissima and B. imbricata respectively) in LPS + IFN-I³ activated 264.7 RAW macrophages and subjected to HPLC for fractionation. From Baeckea ramosissima subsp. ramosissima, two known flavonoids quercetin and kaemferol were identified and evaluated for antiinflammatory and cytotoxic activities in RAW 264.7 macrophage and N11 microglial cell line. In RAW 264.7 macrophage, the IC50 values for NO down regulation were 10.95 and 9.18 µg/mL whereas the IC50 of TNF-I± suppression were 17.35 and 11.26 µg/mL for quercetin and kaemferol respectively. In N11 microglia, the IC50 values for NO down regulation were 19.71 and 16.06 µg/mL whereas the IC50 of TNF"I± suppression were 8.84 and 8.12 µg/mL for quercetin and kaemferol respectively. Isolation and characterization of other active constituents are in progress.
| Date of Award | 2018 |
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| Original language | English |
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