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Effects of Anti-Oxidative and Cholesterol Lowering Capacities of Selected Edible- Medicinal Mushrooms Towards Amelioration of Alzheimer’S Disease

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Effects of Anti-Oxidative and Cholesterol Lowering Capacities of Selected Edible- Medicinal Mushrooms Towards Amelioration of Alzheimer’S Disease EFFECTS OF ANTI-OXIDATIVE AND CHOLESTEROL LOWERING CAPACITIES OF SELECTED EDIBLE- MEDICINAL MUSHROOMS TOWARDS AMELIORATION OF ALZHEIMER’S DISEASE MOHAMMAD AZIZUR RAHMAN FACULTY OF SCIENCE UNIVERSITY OF MALAYA UniversityKUALA LUMPUR of Malaya 2017 EFFECTS OF ANTI-OXIDATIVE AND CHOLESTEROL LOWERING CAPACITIES OF SELECTED EDIBLE-MEDICINAL MUSHROOMS TOWARDS AMELIORATION OF ALZHEIMER’S DISEASE MOHAMMAD AZIZUR RAHMAN THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY FACULTY OF SCIENCE UNIVERSITY OF MALAYA UniversityKUALA LUMPUR of Malaya 2017 UNIVERSITY OF MALAYA ORIGINAL LITERARY WORK DECLARATION Name of Candidate:Mohammad Azizur Rahman Registration/Matric No:SHC120054 Name of Degree: Doctor of Philosophy Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”):Effects of Anti-oxidative and Cholesterol Lowering Capacities of Selected Edible-Medicinal Mushrooms Towards Amelioration of Alzheimer’s Disease Field of Study:Biochemistry I do solemnly and sincerely declare that: (1) I am the sole author/writer of this Work; (2) This Work is original; (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this Work; (4) I do not have any actual knowledge nor do I ought reasonably to know that the making of this work constitutes an infringement of any copyright work; (5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained; (6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM. UniversityCandidate’s Signature of Malaya Date: Subscribed and solemnly declared before, Witness’s Signature Date: Name: Designation: ii ABSTRACT Alzheimer’s disease (AD) is a neuro-degenerative disorder affecting mainly the elderly people. Though more than 40 million people are suffering from AD complications up to present date, there is hardly any treatment effective in withstanding the progression of AD. Thus, demand for natural, safe and cost-effective AD therapeutics has got momentum. Oxidative stress (OS) and increased plasma cholesterol levels have been implicated among the numerous pathomechanistic factors of AD. Despite some discrepencies, strategies aimed at lowering OS and hypercholesterolemia seems promising as AD therapeutics. Mushrooms have been highly hailed for providing numerous health benefits including anti-oxidative and hypocholesterolemic potentialities. However, combined anti-oxidative and hypocholesterolemic effect of edible-medicinal mushrooms have not been reported though these two aspects have been studied separately. Also, as an AD therapeutic agent, mushroom has not been interpreted through their anti-oxidative and hypocholesterolemic properties. Thus, the present study has been designed to elucidate the anti-oxidative, hypocholesterolemic and AD ameliorating properties of the selected edible-medicinal mushrooms: H. erinaceus, L. edodes, F. velutipes and G. lucidum. Five solvent-solvent partitioned fractions (methanol:dichloromethane, dichloromethane, hexane, ethylacetate, aqueous and hot water extract (HWE) of each of the four mushroom species were screened through in vitro anti-oxidant tests including 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicalUniversity scavenging, Folin-Ciocalteu assay, of lipid peroxidationMalaya (LPO) and low density lipo-protein (LDL) oxidation inhibition tests. Among all, the HWE of G. lucidum had been found possessing the best in vitro anti-oxidative capacity and highest content of polyphenols, tri-terpenoids and sterols. Thus, this mushroom extract had been chosen for the rest of the studies. In vivo anti-oxidative and hypocholesterolemic studies upon rats showed increasing effect towards plasma and liver anti-oxidant enzymes (catalase, iii glutathione peroxidase and superoxide dismutase) while total cholesterol (TC), triacylglycerol (TG), LDL-cholesterol were decreased and HDL-cholesterol was increasd. This mushroom extract demonstrated absence of adverse effect upon the organ function tests. As derangement of memory and learning abilities is the most notable complication associated with AD, the effect of the HWE of G. lucidum had been tested upon the AD model rats. Feeding of HWE of G. lucidum was found to improve memory and learning abilities of the AD rats. This cognitive improvement has been supported by elevated levels of memory related neurotransmitters in the respective rats. Transmission electron microscopic (TEM) studies demonstrated pronged neuronal dendrites in the G. lucidum HWE treated rats than those of the non-treated. Finally, brain comparative proteomics have identified differentially expressed proteins involved in neurotransmission, metabolism, cellular stress response and misfolding repairment. Improved functional network had been observed among the proteome of the G. lucidum HWE treated rats through STRING analysis. Ingenuity pathway analysis (IPA) identified nervous system development, cell-cell signaling and interaction, molecular transport and cell death and survival among the top-most functional networks among the experimental subjects. Thus, AD ameliorating effect of G. lucidum through anti- oxidative and hypocholesterolemic performances might be implicated in AD therapeutics. University of Malaya iv ABSTRAK Penyakit Alzheimer (AD) adalah gangguan degeneratif neuro yang mempunyai kesan ke atas orang tua lazimnya. Walaupun lebih daripada 40 juta orang yang menderita komplikasi AD sehingga kini, tiada rawatan yang berkesan yang wujud untuk menahan perkembangan AD. Oleh itu, keperluan untuk sebatian terapeutik bercirikan semulajadi, selamat dan kos efektif telah mendapat perhatian. Tekanan oksidatif (OS) dan peningkatan paras kolesterol plasma adalah faktor penting di antara pelbagai faktor-faktor yang menyebabkan AD. Walaupun terdapat beberapa perbezaan, strategi untuk menurunkan OS dan hiperkolesterolemia berpotensi sebagai terapeutik AD. Cendawan dikenali dengan pelbagai manfaat kesihatan termasuk anti-oksida dan potensi hipokolesterolemik. Walau bagaimanapun gabungan ciri anti-pengoksidaan dan kesan hipokolesterolemik cendawan yang boleh dimakan atau ubatan tidak dilaporkan sedangkan kedua-dua aspek telah dikaji secara berasingan. Juga, cendawan masih tidak dianggap sebagai agen terapeutik AD, berdasarkan ciri-ciri antioksida dan hipokolesterolemik. Oleh itu, kajian ini telah dilakukan untuk menjelaskan sifat-sifat anti-pengoksidaan dan hypocholesterolemia cendawan terpilih (H. erinaceus , L. edodes, F. velutipes dan G. lucidum) dalam mencegah AD. Lima pecahan pelarut- pelarut (methanol : diklorometana, diklorometana, heksana, etil asetat, akueous dan ekstrak air panas (HWE ) bagi setiap empat spesies cendawan telah disaring melalui ujian in vitro antioksida termasuk memerangkap radikal bebas 2,2-diphenyl-1- picrylhydrazylUniversity (DPPH), esei Folin-Ciocalteu, of pengoksidaan Malaya lipid (LPO) dan ujian perencatan pengoksidaan lipoprotein ketumpatan rendah. Antara semua, HWE G. lucidum telah didapati memiliki potensi terbaik kapasiti anti-oksida in vitro dan kandungan tertinggi polifenol, tri-terpenoid dan sterol. Oleh itu, ekstrak cendawan ini telah dipilih untuk kajian selanjutnya. Kajian in vivo anti-oksida dan hipokolesterolemik ke atas tikus menunjukkan kesan peningkatan paras enzim anti-oksida plasma dan hati v (katalase, glutation peroksidase, superoxide dismutase) manakala jumlah kolesterol (TC), triasigliserol (TG), LDL-kolesterol berkurangan dan HDL-kolesterol meningkatkan kesan dari ekstrak cendawan ini tanpa memberi kesan buruk kepada ujian fungsi organ. Oleh kerana gangguan daya ingatan dan kebolehanan belajar adalah komplikasi yang paling ketara yang dikaitkan dengan AD, kesan HWE G. lucidum telah diuji ke atas tikus model AD. Pemakanan HWE G. lucidum telah didapati meningkatkan daya ingatan dan kebolehan pembelajaran tikus AD. Peningkatan kognitif telah disokong oleh peningkatan tahap neurotransmitter memori yang berkaitan dalam tikus masing-masing. Bukti lanjut dari kajian histopatologi (DAPI) yang menunjukkan peningkatan sel-sel neuron dan pemanjangan ketara dendrit dalam tikus yang dirawat dengan G. lucidum HWE (kajian TEM). Akhirnya, perbandingan proteomik otak telah mengenal pasti protein terzahir yang terlibat dalam penghantaran maklumat, metabolisme, tindak balas tekanan selular dan pembaikan pelipatan. Pemuliharaan rangkaian berfungsi telah meningkat di kalangan proteome tikus dirawat oleh G. lucidum HWE berdasarkan analisis STRING. Analisis laluan ‘ingenuity’ (IPA) mengenal pasti pembangunan sistem saraf, isyarat sel-sel dan interaksi, pengangkutan molekul dan sel mati dan hidup di kalangan rangkaian berfungsi paling atas di kalangan subjek eksperimen. Oleh itu, G. lucidum membendung kesan AD melalui ciri-ciri anti- oksida dan hipokolesterolemik mungkin berpotensi sebagai calon untuk terapeutik AD. University of Malaya vi ACKNOWLEDGEMENTS All praise to Almighty ALLAH Whose constant blessings have enabled me to be engaged in
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