Year

2015

Degree Name

Doctor of Philosophy

Department

School of Chemistry

Abstract

Chapter 1 of this thesis is a review of the literature on the structure, biological activities and isolation of Stemona alkaloids from the Stemonaceae family of plants. This chapter also outlines the aims of this project, which were to isolated and determine the structures of the Stemona alkaloids and other chemical constituents in four species of Stemonaceae; three of them collected in peninsular Malaysia and another one in Indonesia. Another aim of this project was to determine the biological activities of these alkaloids.

Chapter 2 reviews and describes the genus of Stemona and Stichoneuron including their distribution and morphologies, and also the field work undertaken to collect the plants for this study. Stemona curtisii Hook F., Stichoneuron halabalensis Hook F. and Stichoneuron caudatum Ridley were collected from different sites in Malaysia by Ramli, R. A., while Stemona javanica (Kunth) Engl. was collected in Indonesia by our collaborator, Pudjiastuti, P.

Chapter 3 reviews the earlier studies on S. curtisii from Thailand. This chapter also describes the results of the isolation of the chemical constituents from the roots extract of S. curtisii from Malaysia using chromatographic techniques, including column chromatography and preparative thin layer chromatography. Six known alkaloids were successfully isolated and their structures were confirmed by NMR and ESI-MS analysis and from camparisons made with the spectroscopic data from the literature. The isolated compounds were (2'S)-hydroxystemofoline, stemocochinine, 1- hydroxyprotostemonine, oxystemokerrin-N-oxide, isostemofoline and stemofoline. Eleven Stemona alkaloids were reported from S. curtisii in Thailand; five of them, were pyrrolo[1,2-a]azepine alkaloids while the others were pyrido[1,2-a]azepine alkaloids. In contrast, in this study only one pyrido[1,2-a]azepine alkaloid, oxystemokerrin-N-oxide, was isolated from S. curtisii from Malaysia. Stemofoline was the major alkaloid found in the roots of S. curtisii from Thailand while (2'S)-hydroxystemofoline was the major alkaloid from the roots of the plant material collected in Malaysia.

Chapter 4 describes the isolation of Stemona alkaloids from the root extracts of Stemona javanica using chromatograpic techniques. Two new protostemonine-type alkaloids, javastemonine A and B were isolated together with four known Stemona alkaloids, 13-demethoxy-11(S*),12(R*)-dihydroprotostemonine, isoprotostemonine, protostemonine and isomaistemonine. The structures and relative configurations of the new alkaloids were determined by spectroscopic analysis and molecular modeling studies.

Chapter 5 describes the isolation of the chemical components from the roots and leaf extracts of the hitherto unreported Stichoneuron halabalensis. This study led to the characterization of the known compounds (+)-α-tocopherol, (R)-(+)-goniothalamin, four known Stemona alkaloids, bisdehydrostemoninine A, stemoninine, sessilistemoamine C, sessilistemoamine A; and three new Stemona alkaloids, stichoneurines C, D and E. The structures and relative configurations of the new alkaloids were determined by spectroscopic analysis, and from comparisons made with published spectroscopic data and molecular modeling studies.

Chapter 6 provides a discussion of the isolation of four novel stichoneurine-type alkaloids, stichoneurines F and G and sessilistemonamines E and F, Stemona alkaloids, from the root extracts of Stichoneuron caudatum. The isolation and purification of these chemical constituents were achieved using various chromatographic techniques. The structures and relative configurations of the new alkaloids have been determined by spectroscopic methods and molecular modeling experiments. A possible biosynthesis of these new alkaloids from stichoneurine B involving an intramolecular Mannich reaction to form the cyclopentane ring of these alkaloids was proposed.

Chapter 7 provides an introduction and discussion of the biological assays and the biological activities of the isolated chemical components. The results of the AChE inhibition studies showed that stemoninine and bisdehydoxystemoninine A had the highest inhibitory activities against human acetylcholinestrase (hAChE), with IC50 values of 3.74±0.09 μM and 5.52±0.13 μM, respectively. Both were far less active againt electric eel AChE (eeAChE). In contrast, stichoneurine E showed the highest activity against eeAChE (IC50 = 5.90 + 0.08 μM) when compared to hAChE (IC50 = 34.63±0.81 μM). The crude extracts of S. curtisii, St. halabalensis and St. caudatum showed significant inhibitory activities against hAChE (IC50 values of 41.8±0.05, 32.94±0.88 and 41.8±0.05 μg/mL, respectively).

The MDR-reversing properties of eight selected Stemona alkaloids on the cytotoxicities of two cancer drugs, doxorubicin and paclitaxel, were performed by our collaborator, Assoc. Prof. Pornngarm Limtrakul from the Departement of Biochemistry, Faculty of Medicine, Chiang Mai University, Thailand. Among the tested compounds, isostemofoline showed the highest modulating effect on drug resistant K562-Adr cells by decreasing the IC50 of doxorubicin from 22.33 ± 2.08 μM to 2.52 ± 0.03 μM and decreasing the IC50 of paclitaxel from 1.48 ± 0.01 μM to 0.23 ± 0.01μM. The other compounds that showed MDR-reversing properties were (11Z)-1',2'-didehydrostemofoline, and (11E)-didehydrostemofoline. In the presence of these compounds, doxorubicin had IC50 values of 6.00 ± 0.00 and 4.83 ± 0.29 μM, respectively, while for paclitaxel, the IC50 values decreased to 0.47 ± 0.01 and 0.38 ± 0.01 μM, respectively.

Thirteen isolated Stemona alkaloids were examined for their antiplasmodial activities. Compounds 13-demethoxy-11(S*),12(R*)-dihydroprotostemonine, isoprotostemonine, protostemonine, sessilistemoamine F, stichoneurine F, and 1- hydroxyprotostemonine demonstrated moderate in vitro antiplasmodial activity against the P. falciparum strains, TM4 (a wild type chloroquine and antifolate sensitive strain) with IC50 values of 17.7μg/mL, 16.8 μg/mL, 16.0 μg/mL, 18.5 μg/mL, 20.2 μg/mL, and 27.1 μg/mL, respectively, and K1 (multidrug resistant strain) with IC50 values of 16.8 μg/mL, 14.1 μg/mL, 11.9 μg/mL, 17.7 μg/mL, 16.5 μg/mL, 20.6 μg/mL, respectively. Stichoneurine G showed only moderate antiplasmodial activity against TM4 with an IC50 value of 26.8 μg/mL. The other compounds did not show antiplasmodial activity, even at the highest tested concentration of 38.9-42.3 μg/mL.

Thirteen isolated Stemona alkaloids were also examined for their cytotoxicities. None of the tested alkaloids showed toxicity against mammalian cell lines, KB (human mouth epidermal carcinoma cells) and Vero (kidney epithelial cells from an African green monkey) cells, even at the highest tested concentration of 38.9-42.3 μg/mL.

The conclusions arising from this study are described in Chapter 8. In addition, all the results and methodology of this study are presented in Chapter 9.

FoR codes (2008)

0304 MEDICINAL AND BIOMOLECULAR CHEMISTRY

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.