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Title:	Pemencilan dan pencirian aktinomiset endofit bioaktif daripada tumbuhan ubatan di Malaysia
Authors:	Nurul 'Izzah Mohd Sarmin (P43589)
Supervisor:	Noraziah Mohamad Zin, Prof. Madya Dr.
Keywords:	Endophytic actinomycetes Medicinal plants Malaysia
Issue Date:	2-Jul-2013
Description:	Hutan di Malaysia yang mempunyai diversiti tinggi menyediakan persekitaran yang sesuai untuk pemencilan aktinomiset endofit. Strain aktinomiset yang novel merupakan sumber yang berpotensi untuk menghasilkan sebatian farmaseutikal bioaktif baru yang boleh dibangunkan sebagai calon dadah baru. Kajian ini memfokus kepada pemencilan aktinomiset endofit daripada tumbuhan ubatan, mencirikan strain novel dan menilai sebatian bioaktif daripada metabolit strain tersebut. Sebanyak sembilan aktinomiset endofit telah berjaya dipencilkan daripada tumbuhan di pelbagai kawasan di Malaysia. Pencilan aktinomiset endofit ini dikenalpasti melalui pemerhatian morfologi dan analisis jujukan gen 16S rRNA. Spesis novel Streptomyces sp. iaitu SUK 12 telah dipencilkan daripada pokok Portulaca oleracea Linn. dan dicirikan selanjutnya dengan menggunakan pendekatan taksonomi polifasik. Kesemua pencilan ini diuji terhadap aktiviti antimikrobnya. Sebatian bioaktif daripada strain SUK 12 dipencilkan dengan menggunakan pendekatan pemisahan berpandukan bioasai dan struktur kimianya ditentukan dengan menggunakan analisis spektrometri. Kesan sitotoksisiti sebatian bioaktif ini diuji terhadap sel selanjar bukan malignan (hepar Chang) dan sel hepatoma (HepG2) dengan menggunakan asai metil tiazol tetrazolium (MTT). Pencilan aktinomiset endofit yang dipencilkan terdiri daripada lapan Streptomyces spp. dan satu Microbispora sp. Pencilan tersebut dikelaskan ke dalam kumpulan berdasarkan warna miselia arialnya. Kumpulan siri warna kelabu membentuk satu clade filogenetik yang sama berdasarkan data jujukan 16S rRNA. Sebanyak tiga pencilan iaitu SUK 8, SUK 10 dan SUK 15 yang dipencilkan daripada kawasan pensampelan yang sama tergolong di dalam satu clade filogenetik dan menunjukkan corak aktiviti biologi yang hampir serupa. Pencilan aktif iaitu SUK 8, SUK 10, SUK 12 dan SUK 15 berupaya merencat atau membunuh satu atau lebih organisma patogenik sehingga 100%. Jujukan penuh data 16S rRNA menunjukkan bahawa strain SUK 12 paling berkaitan dengan S. corchorusii DSM 40340T (98.2% similariti). Peratus similariti hibridisasi DNA-DNA di antara strain SUK 12 dan S. corchorusii DSM 40340T adalah sebanyak 18.85±4.55% mengesahkan bahawa strain SUK 12 adalah spesis baru di dalam genus Streptomyces dan dicadangkan untuk dinamakan sebagai Streptomyces kebangsaanensis sp. nov. DSM 42048T (= NRRL B-24860T). Sebanyak dua sebatian bioaktif berjaya dipencilkan daripada strain SUK 12 iaitu fenazin-1-asid karboksilik (tubermycin B) dan sebatian baru 6-((2-hidroksi-4-metoksifenoksi)karbonil)fenazin-1-asid karboksilik yang aktif terhadap Bacillus subtilis ATCC 6633 masing-masing dengan nilai MIC 0.3125 mg/mL dan 0.0781 mg/mL. Ujian sitotoksisiti menunjukkan sebatian tubermycin B tidak merangsang aktiviti sitotoksisiti terhadap sel selanjar ujian di mana nilai IC50 terhadap sel hepar Chang ialah 80 μg/mL manakala tiada nilai IC50 terhadap sel HepG2. Kesimpulannya, kaedah taksonomi polifasik berjaya dibangunkan untuk pencirian spesis Streptomyces novel yang menghasilkan sebatian antibakteria yang menarik untuk dibangunkan sebagai calon dadah baru.,High biodiversity of forest in Malaysia provides suitable environment for the isolation of endophytic actinomycetes. Novel strains of actinomycetes are potential sources in producing new bioactive pharmaceutical compounds that can be developed as new drug candidates. This study focused on the isolation of endophytic actinomycetes from medicinal plants, characterization of novel strain and evaluation of bioactive compounds from its metabolites of the strain. A total of nine endophytic actinomycetes were successfully isolated from plants of various places in Malaysia. These endophytic actinomycetes were identified using morphological observation and 16S rRNA gene sequence analysis. The novel species of Streptomyces sp., namely SUK 12 was isolated from plant Portulaca oleracea Linn. and further characterized using polyphasic taxonomy approach. All isolates were tested for their antimicrobial activities. Bioactive compounds from SUK 12 were isolated using bioassay guided fraction approach and its chemical structures were determined by using spectrometry analyses. Cytotoxicity effect of the bioactive compound was tested on non-malignant cell line (Chang liver) and hepatoma cell (HepG2) by using the methyl thiazole tetrazolium (MTT) assay. The isolated endophytic actinomycetes consist of eight Streptomyces spp. and a Microbispora sp. These isolates were classified into groups based on the colour of aerial mycelium. The grey series formed the same phylogenetic clade based on the 16S rRNA sequence data. Three isolates of SUK 8, SUK 10 and SUK 15 which were isolated from the same sampling area fall within a phylogenetic clade and showed similar patterns of biological activities. Active isolates which were SUK 8, SUK 10, SUK 12 and SUK 15 can inhibit or kill up to 100% of one or more pathogenic organisms. Full sequence of 16S rRNA data indicated that strain SUK 12 was most closely related to S. corchorusii DSM 40340T (98.2% similarity). The percentage of DNA-DNA hybridization similarity between strain SUK 12 and S. corchorusii DSM 40340T is 18.85±4.55% confirming that strain SUK 12 is a new species of the genus Streptomyces and is proposed to be named as Streptomyces kebangsaanensis sp. nov. DSM 42048T (= NRRL B-24860T). Two bioactive compounds were successfully isolated from strain SUK 12 namely phenazine-1-carboxylic acid (tubermycin B) and a new compound 6-((2-hydroxy-4-methoxyphenoxy)carbonyl)phenazine-1-carboxylic acid which were active against Bacillus subtilis ATCC 6633 with MIC values of 0.3125 mg/mL and 0.0781 mg/mL, respectively. Cytotoxicity test showed that tubermycin B did not induce cytotoxic activity against tested cell lines which IC50 value of Chang liver cell was 80 μg/mL but no IC50 value for HepG2 cell. In conclusion, a polyphasic taxonomy approach has been successfully developed for the characterization of a novel species of Streptomyces that produced exciting antibacterial compounds to be developed as new drug candidates.,PhD
Pages:	275
Call Number:	QV766.N974p 2013 9 tesis
Publisher:	UKM, Kuala Lumpur
Appears in Collections:	Faculty of Health Sciences / Fakulti Sains Kesihatan

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