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Title:	Fraksinasi berpandukan bio asai dan pemencilan anti Staphylococcus aureus rintang metisilin daripada kulit batang Phyllanthus columnaris Mull.Arg. dan kajian mod tindakan
Authors:	Siti Noor Adnalizawati Adnan (P57665)
Supervisor:	Nazlina Ibrahim, Profesor Madya Dr
Keywords:	Staphylococcus aureus Phyllanthus Staphylococcus aureus infections
Issue Date:	27-May-2014
Description:	Kajian ini telah dijalankan untuk mengenal pasti sebatian bioaktif daripada kulit batang tumbuhan Phyllanthus columnaris Müll.Arg. dan menilai kesan anti bakteria terhadap Staphylococcus aureus rintang methicillin(MRSA). Penyaringan anti bakteria melalui ujian asai sebaran cakera, asai pencairan kaldu dan asai pembunuhan minima telah dijalankan menggunakan ekstrak metanol, aseton dan akuas. Kedua-dua ekstrak metanol dan aseton mempamerkan aktiviti anti-MRSA yang baik dengan nilai MIC kedua-duanya 0.78 mg/ml dan nilai MBC 1.56 mg/ml. Penyaringan fitokimia juga telah dilakukan dan mendedahkan kehadiran saponin dan tannin. Kromatografi berpandu-bioasai telah dijalankan melalui tiga kaedah kromatografi lapisan nipis-bioautografi (KLN-B) (ujian langsung, sentuh dan rendaman) dan berjaya menunjukkan dua sebatian bioaktifanti-MRSA daripada kedua-dua ekstrak metanol dan aseton bebas tannin. Pengasingan kedua-dua sebatian bioaktif telah dilakukan melalui KLN persediaan dan dicirikan sebagai stigmasteroldan lupeol melalui data spektrum resonans magnetik nuclear (RMN). Kedua-dua sebatian bioaktif dan fraksi yang mengandungi kedua-dua sebatian telah dipencilkan melalui gabungan kaedah kromatografi cecair vakum dan kromatografi turus sebagai persediaan untuk kajian selanjutnya. Analisis melalui asai masa-membunuh menunjukkan bahawa kadar pembasmian bakteria semakin meningkat dengan kepekatan stigmasterol, lupeol, fraksi dan ekstrak kasar dan dianggap mempunyai aktiviti bakterisid bergantung-kepekatan. Tannin didapati lebih aktif terhadap MRSA diikuti dengan ekstrak kasar, lupeoldan stigmasterol. Kesan sinergi di antara lupeol dan stigmasterol hanya ditunjukkan terhadap MRSA BM1. Pengamatan melalui mikroskop elektron imbasan dan transmisi terhadap MRSA yang dirawat dengan stigmasterol dan lupeol menunjukkan kerosakan dinding sel, pelepasan kandungan sitoplasma dan pengurangan isipadu sel. Analisa profil transkripsi genom keseluruhan strain rujukan MRSA ATCC 43300 yang dirawat dengan lupeol, stigmasterol, tanninatau ekstrak kasar telah dibandingkan dengan menggunakan teknologi penjujukan high-throughput next-generation. Pendedahan MRSA kepada stigmasterol, lupeolatau ekstrak kasar menunjukkan modulasi pengekspresan gen yang signifikan yang mengawalatur pelbagai sasaran di dalam tapak jalan pembahagian sel, aminoacyl-tRNA synthetasedan ribosom. Tannin juga menunjukkan pengawalaturan yang signifikan di dalam dua tapak jalan penting iaitu aminoacyl-tRNA synthetase dan ribosom tetapi bukannya pembahagian sel.Stigmasteroldan tannin mempunyai nilai CC50 17.47mg/mL dan 2.11mg/mL manakala lupeol, fraksi dan ekstrak kasar mempunyai nilai CC50 melebihi 25 mg/mL menunjukkan semua bahan uji tidak sitotoksik terhadap sel Vero dan berpotensi digunakan sebagai agen anti-MRSA yang selamat.,This study was conducted to identify bioactive compounds from Phyllanthus columnaris Müll.Arg. stems bark and evaluates the antibacterial activity against methicillin resistant Staphylococcus aureus(MRSA). Antibacterial screening by disk diffusion, broth microdilution and minimum bactericidal assays was done using different methanol, acetone and aqueous extracts. Both methanol and acetone extracts exhibited good anti-MRSA activity with the lowest MIC value for both were0.78 mg/ml and the lowest MBC were1.56 mg/ml. Phytochemical screening was also performed and revealed the presence of saponin and tannin. Bioassay-guided chromatography was conducted by three methods of thin layer chromatography (TLC) bioautography (direct, contact and immersion/agar overlay tests) and successfully revealed two active anti-MRSA compounds from both tannin-free methanol and acetone extracts. Isolation of the two active compounds were performed by means of preparative TLC and characterized as stigmasterol and lupeol from nuclear magnetic resonance (NMR) spectral data. Both bioactive compounds and fraction that contains both compounds only isolated by using combination of vacuum liquid chromatography and column chromatography techniques in preparation for further investigations. Time kill analysis showed that the rate of killing increases with concentration of stigmasterol, lupeol, fraction or crude and considered as concentration-dependent bactericidal activity. Tannin was more active against MRSA followed by crude extract, lupeol and stigmasterol. Synergistic effect between lupeol and stigmasterol can only be seen on MRSA BM1. Scanning and transmission electron microscopy of MRSA treated with stigmasterol and lupeol showed cell wall disruption, release of cytoplasmic compounds and decrease in cellular volume. Analysis of a genome-wide transcriptional profile of reference strain of ATCC 43300MRSAin response tolupeol, stigmasterol, tannin or crude treatment were compared by using high-throughput next-generation sequencing technology. Exposure of MRSA to stigmasterol, lupeolor crude revealed significant modulation of gene expression that regulates multiple desirable targets in cell division, aminoacyl-tRNA synthetase and ribosome pathways. Tannin also showed significant regulation in two of the essential pathways which were aminoacyl-tRNA synthetase and ribosome pathways but not in cell division. Stigmasterol and tannin have CC50 values of 17.47 mg/mL and 2.11 mg/mL respectively while lupeol, fraction and crude have CC50 value of more than 25 mg/mL indicating the non-cytotoxicity of all the tested materials towards Vero cells and potentially safe use of these as anti-MRSA agents.,PhD
Pages:	329
Call Number:	QR201.S68S588 2014 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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