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Title:	The potential of Marrubium vulgare as anti-herpes simplex type 1 agent
Authors:	Amal G.S. Fayyad (P57429)
Supervisor:	Nazlina Ibrahim, Associate Professor Dr.
Keywords:	Marrubium vulgare Anti-herpes simplex Herpes simplex virus
Issue Date:	5-May-2014
Description:	Kajian ini bertujuan mengkaji potensi aktiviti antivirus Marrubium vulgare dikenali juga sebagai "Horehound" dan kesannya terhadap kitar replikasi virus herpes simpleks jenis-1 (HSV-1). Tumbuhan diekstrak dengan campuran air-metanol untuk menghasilkan ekstrak mentah dan difraksi menggunakan heksana, kloroform, etil asetat dan n-butanol. Analisis kualitatif fitokimia menunjukkan kehadiran flavon aglikon, steroid, karbohidrat, saponin, antrakuinon, tanin dan/atau terpena. Nilai kepekatan ekstrak/fraksi yang mengakibatkan 50 % kematian sel (CC50) adalah antara 140 ke 400 μg/mL menunjukkan tidak toksik terhadap sel Vero. Dalam asai penurunan plak, hanya ekstrak mentah, fraksi heksana (HF) dan kloroform menunjukkan aktiviti antivirus dengan indeks pemilihan (SI) masing-masing 3.11, 2.8 dan 1.28. Hasil fraksi lanjutan seluruh tumbuhan dan daun menunjukkan tiada kesitotoksikan namun nilai SI lebih rendah kecuali untuk fraksi heksana daun tumbuhan. Nilai CC50 fraksi heksana 180 μg/mL dan nilai SI 2.8 pula menyamai fraksi heksana seluruh tumbuhan. Penentuan lanjutan kesan fraksi heksana terhadap kitar replikasi HSV-1 dijalankan. Asai prarawatan sel dan penembusan virus menunjukkan tiada perencatan dalam pembentukan plak berbanding kawalan. Ini bermakna fraksi heksana tidak menganggu reseptor sel lalu membenarkan virus menembusi membran sel. Pra-rawatan virus dengan fraksi HF merencatkan 100 % pembentukan plak menunjukkan penyahaktifan virus menjangkiti sel. Pengesahan melalui mikroskopi elektron transmisi mendapati sampul virus separa rosak setelah dirawat dengan fraksi. Rawatan virus dengan HF pada 100 μg/mL, 90 μg/mL atau 80 μg/mL diikuti jangkitan kepada sel Vero menghasilkan perencatan 44 %, 33 % serta 18 %, menunjukkan keupayaan mengurangkan pelekatan virus kepada sel. Dalam asai hasil virus, sel terjangkit virus dirawat selama 30 j diikuti pentitratan virus tereplikasi. Perencatan pembentukan plak berlaku pada kurang dari 1×102 pfu/mL, 2.4×102 pfu/mL dan 4.7×103pfu/mL berbanding kawalan tidak rawat (9.4×103pfu/mL), menunjukkan perencatan adalah bersandar dos. Dalam asai penambahan fraksi kepada sel terjangkit pada sela masa berbeza, perencatan tertinggi melebihi 88% berlaku 2 j pasca jangkitan (pi), menurun dari 4 ke 8 j dan tiada perencatan pada 10 dan 12 j. didapati merencat pada fasa paling awal kitaran hidup virus. Dalam asai ambilan sampel pada sela masa 2 hingga 48 j, perencatan plak dicerap selepas 12 jpi serta mencecah lebih 88% perencatan pada 48 jpi. Gen virus paling awal (UL54), awal (UL23) dan lewat (US6) dipilih untuk penentuan tahap pengekspresan. RNA jumlah dipencilkan dari sel terjangkit yang dirawat dan kawalan digunakan di dalam tindak balas rantaian polimerase-masa nyata kuantitatif (qRT-PCR). Pengekspresan UL54 tidak terganggu oleh fraksi heksana pada 4j pi tetapi pengekspresan berkurangan selepas itu. Perencatan UL23 pada 8 jpi hingga 24 jpi dan perencatan US6 pada 16 jpi hingga 24 jpi. Ini mengesahkan fraksi heksana memberi kesan kepada replikasi virus apabila diberi selepas pos-jangkitan. Teknik imunopedapan menggunakan antibodi terhadap ICP27, TK dan gD digunakan bagi melihat kesan fraksi heksana ke atas protein virus. Sintesis protein tiga gen terencat sama sekali pada semua masa rawatan. Dicadangkan fraksi heksana memberi kesan kepada kedua-dua transkripsi dan translasi gen-gen UL23 dan US6 dan menghasilkan TK dan gD tetapi memberi kesan juga terhadap translasi UL54 atau aktiviti pos-transkripsi,This study aims to screen for the potential antiviral activity of Marrubium vulgare also known as "Horehound" and determine the effects on herpes simplex virus type-1 (HSV-1) replication phases. The whole plant was extracted in water–methanol mixture to produce the crude extract and further fractioned using hexane, chloroform, ethyl acetate and butanol. Qualitative phytochemical analysis revealed the presence of flavones aglycones, steroid, carbohydrates, saponins, anthraquinones, tannin and/or terpenes in the crude extract and all fractions. The cytotoxicity towards Vero cells was evaluated with the extract/fraction concentration that causes 50 % of cell death (CC50) value ranges between 140 to 400 μg/mL indicating no toxicity towards Vero cells. Plaque reduction assay showed only crude extract, hexane and chloroform fractions showed antiviral activity with the selected indices (SI) of 3.11, 2.8 and 1.28, respectively. Further fractionation on whole plant and leaf showed no cytotoxicity but lower SI except for the hexane fraction (HF) of plant leaf with CC50 value of 180 μg/mL. In the antiviral activity, the SI value was 2.8 which is almost similar to the hexane fraction of the whole plant. Subsequent determination of the hexane fraction effect on HSV-1 replication cycle was done. The cell pretreatment and penetration assays showed no inhibition in plaque formation compared to control indicating that the fraction does not affect cell receptors thus allowing the virus to penetrate the cell membrane. When the virus is pretreated with the fraction, 100 % inhibition of virus plaque formation was observed, indicating the possibility of virus deactivation in infecting the cells. This is confirmed by transmission electron microscopy with virus envelope was partially dissolved if treated with the fraction. Virus treated with fraction at 100 μg/mL, 90 μg/mL or 80 μg/mL and infected to Vero cells showed inhibition of 44 %, 33 % and 18 %, respectively indicating the ability to reduce attachment of virus to cells. In the virus yield assay, virus infected cells were treated with the hexane fraction as above for 30 hours, after which titration of releasing virus was done. Plaque formation was inhibited with less than 1×102 pfu/mL, 2.4×102 pfu/mL and 4.7 ×103 pfu/mL compared to the non-treated control (9.4×103 pfu/mL) indicating that inhibition is dose dependent. In time of addition assay, infected cells were treated with the fraction at different time points. The highest inhibition was more than 88 % at 2 h post infection (pi) and declined gradually from 4 to 8 hours and no inhibition at 10 and 12 hours. This indicated that the fraction inhibited the intermediate early phase of the virus life cycle. In time removal assay, virus infected cells were treated and aliquots of medium were taken at 2 hour intervals until 48 hours pi and subject to plaque reduction assay. Plaque inhibition was observed after 12 hours pi and reached more than 88% inhibition at 48 h pi. The virus intermediate early (UL54), early (UL23) and late (US6) genes were selected to determine for the expression level. Total RNA extracted from treated infected cells and control infected cells were subjected in the quantitative Real-Time Polymerase Chain Reaction (QRT-PCR). The expression of UL54 was not affected by the hexane fraction at 4 h pi, but expression reduces at later times. UL23 was inhibited at 8 hpi to 24 hpi and US6 was inhibited at 16 hpi to 24 hpi. This confirms that the HF affects the viral replication when given after post-infection. The effect of the hexane fraction on viral proteins, dot blot and western blot techniques using antibodies against ICP27, TK and gD were used. Protein synthesis of the three genes was completely inhibited at all time-points. It is suggested that the hexane fraction affects both the transcription and translation of the UL23 and US6 genes producing TK and gD but only affects UL54 translation or post-transcription activity.,PhD
Pages:	148
Call Number:	QR400.2.H47F349 2014 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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