Final thesis pdf (a4)

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Final thesis pdf (a4)

  1. 1. IDENTIFICATION OF COMPONENTS IN EXTRACTSOF HERICIUM ERINACEUS (BULL.: FR.) PERS THAT STIMULATE IN VITRO NEURITE OUTGROWTH OF NG108-15 WONG YUIN TENG FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2012
  2. 2. IDENTIFICATION OF COMPONENTS IN EXTRACTS OF HERICIUM ERINACEUS (BULL.: FR.) PERS THAT STIMULATE IN VITRO NEURITE OUTGROWTH OF NG108-15 WONG YUIN TENGDISSERTATION SUBMITTED IN FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE INSTITUTE OF BIOLOGICAL SCIENCES FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2012
  3. 3. ABSTRACT Hericium erinaceus, locally known as cauliflower mushroom, and elsewhere aslion’s mane mushroom, Houtou (monkey head mushroom), Yamabushitake andHarisenbon (balloon fish), is an edible mushroom. It is well known for its medicinal andnutritional values. Hericium erinaceus is reported to have good anti-tumor propertiesand nerve tonic effects. Although H. erinaceus is a temperate mushroom, it has beensuccessfully cultivated in Malaysia. However, there are very few reported studies on thechemical constituents that stimulate the neurite outgrowth for the locally cultivatedspecies. The crude aqueous ethanol extract of H. erinaceus and its fractionated extracts(hexane, ethyl acetate and water) were evaluated for their effect in stimulating theneurite outgrowth using neural cell line NG108-15 whilst the Nerve Growth Factor(NGF) was used as the positive standard. The crude aqueous ethanol extract of H.erinaceus showed 15.0 % increase in neurite outgrowth at the concentration of 10.0µg/ml. However, the crude aqueous ethanol extract showed decreased neurite growth asthe dose was increased. The hexane, ethyl acetate and water fractions showed anincrease in neurite outgrowth when the dose was increased exponentially (10.0, 25.0,50.0 and 100.0 µg/ml). Maximum stimulation of neurite outgrowth was recorded withethyl acetate fraction with 68.5 % increase compared to negative control followed byhexane fraction with 65.2 % increase. The combined fraction of hexane and ethyl acetate was further subjected to flashcolumn chromatography. Among the 7 isolated fractions (fraction E1-E7), fraction E1and fraction E2 show relatively higher neurite stimulation activity compared to otherfractions. Maximum stimulation was recorded as 160.6 % increase and 149.1 % ii
  4. 4. increase compared to negative control at the concentration of 100 µg/ml for fraction E1and fraction E2 respectively. The chemical compositions of the fraction E1 of H. erinaceus were analayzed byGCMS. Four components were identified from fraction E1 comprising about 80.5 % ofthe total. Fraction E1 was made up of ethyl palmitate (29.8 %), ethyl stearate (2.3 %),ethyl oleate (18.6 %) and ethyl linoleate (29.9 %). Further isolation of fraction E2 usingpreparative TLC and HPLC gave subfraction sub4b_4 and subfraction sub4b_6.Subfraction sub4b_4 showed better neurite stimulation activity compared to subfractionsub4b_6 with 187.1 % increase in comparison. The chemical compositions of subfraction sub4b_4 and sub4b_6 were analyzedby NMR and LC/MS/MS. The components identified from subfraction sub4b_4 werehericenone C (and its isomer) and 4-(3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-3-hydroxy-5-methoxylbenzyl oleate (and its isomer). On the other hand, subfractionsub4b_6 comprised of hericenone C, 4-(3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-3-hydroxy-5-methoxylbenzyl oleate and a phenolic component attached to thefatty ester side chain contained 26 carbons with 3 double bonds. iii
  5. 5. ABSTRAK Hericium erinaceus, dikenali sebagai cendawan bunga kobis di Malaysia dancendawan “lion’s mane”, Houtou (cendawan kepala monyet), Yamabushitake danHarisenbon di tempat lain. Cendawan ini boleh dimakan dan ia terkenal dari segi nilaiperubatan dan nutrisi. Kebelakangan ini, laporan saintifik menunjukkan H. erinaceusmempunyai nilai anti-tumor yang baik dan sebagai tonik terhadap saraf. Walaupun H.erinaceus merupakan cendawan yang ditanam di kawasan sederhana tetapi kini berjayaditanam di Malaysia yang beriklim tropika. Walaubagaimanapun, tidak banyak terbitanlaporan termpatan yang melaporkan tentang komposisi kimia dalam H.erinaceus yangditanam secara tempatan dalam rangsangan pertumbuhan saraf. Ekstrak mentah akueus etanol dan fraksi-fraksi (heksana, etil asetat dan air) dariH.erinaceus telah diselidik dalam rangsangan pertumbuhan saraf pada sel saraf NG108-15 dan NGF digunakan sebagai kawalan positif. Ekstrak mentah akueus ethanolmenunjukkan peningkatan sebanyak 15.0 % dalam pertumbuhan saraf pada kepekatan10.0 µg/ml. Walaubagaimanapun, peningkatan kepekatan ekstrak mentah akueusethanol akan menyebabkan penurunan dalam pertumbuhan saraf. Fraksi hexana, etilasetat dan air akan menyebabkan peningkatan dalam pertumbuhan saraf apabilakepekatan fraksi-fraksi ditingkatkan secara eksponen (10.0, 25.0, 50.0 and 100.0 µg/ml).Pertumbuhan maksimum saraf direkodkan oleh fraksi etil asetat dengan 68.5 %peningkatan berbanding dengan kawalan negatif dan diikuti oleh fraksi hexana dengan65.2 % peningkatan berbanding dengan kawalan negatif. Pengasingan komponen daripada gabungan fraksi heksana dal etil asetatmenggunakan kaedah kromatografi kolum kilat menghasilkan 7 fraksi (fraksi E1-E7).Fraksi E1 dan E2 menunjukkan aktiviti pertumbuhan saraf yang lebih tinggi jikaberbanding dengan fraksi-fraksi lain. Peningkatan pertumbuhan maksimum sebanyak iv
  6. 6. 160.6 % dan 149.1 % direkodkan oleh fraksi E1 dan E2 pada kepekatan 100µg/mlberbanding kawalan negatif. Komposisi kimia fraksi E1 bagi H. erinaceus dianalisis dengan menggunakanGCMS. Empat komponen telah dikenalpasti daripada fraksi E1 dan komponen-komponen tersebut adalah terdiri daripada 80.5 % daripada keseluruhan fraksi E1.Komponen yang terkandung dalam fraksi E1 adalah etil palmitat, etil stearat, etil oleatdan etil linoleat. Subfraksi sub4b_4 dan subfraksi sub4b_6 adalah hasil daripada isolasifraksi E2 dengan menggunakan preparatif TLC dan HPLC. Subfraksi sub4b_4menunjukkan aktiviti pertumbuhan saraf yang lebih baik daripada subfraksi sub4b_6iaitu 187.1 % peningkatan pada kepekatan 100 µg/ml jika dibandingkan dengankawalan negatif. Komposisi kimia subfraksi sub4b_4 dan sub4b_6 dianalisis denganmenggunakan NMR dan LC/MS/MS. Komponen-komponen yang dikenalpasti daripadasubfraksi sub4b_4 termasuk hericenone C (dan isomernya) dan 4-(3’,7’-dimetil-5’-oxo-2’,6’-octadienil)-2-formil-3-hidroksi-5-metoksibenzil oleat (dan isomernya). Identifikasisubfraksi sub4b_6 menunjukkan kehadiran hericenone C, 4-(3’,7’-dimetil-5’-oxo-2’,6’-octadienil)-2-formil-3-hidroksi-5-metoksibenzil oleat dan satu komponen fenolik yangmengandungi rantai ester asid lemak yang mempunyai 26 karbon dan 3 ikatan dubel. v
  7. 7. ACKNOWLEDGEMENT Writing a significant scientific thesis is hard work and it would be impossiblewithout support from various people. First of all, I wish to express my greatestappreciation towards my supervisor Professor Datin Dr. Sri Nurestri Abdul Malek, myco-supervisor, Professor Dr. Noorlidah binti Abdullah, the project leader of this research,Professor Dr. Vikineswary Sabaratnam from Mushroom Research Centre and Dr.Murali Naidu from the Faculty of Medicine for the intellectual guidance, valuableadvices and help that was given to me during my research. The thesis would not havebeen written successfully without their continuous supervision and guidance. I wouldlike to thank to University Malaya for the grant (PS191/2009A) and fellowship support. My special appreciation to my labmates and friends’ enthusiasm and support inproviding relevant assistance and help to complete this study. Thanks to Wong Kah Hui,Lai Puei-Lene, Priscilla Ann, Joanna Eik Lee Fang, Hong Sok Lai, Lee Guan Serm,Phang Chung Weng, Sujatha Ramasamy, Sharifah Nur Syed Abdul Rahman, GowriKanagasabapathy, Jaime Stella Richardson, Ong Kia Ju and Mamalay. A special thankto Madam Chang May Hing for her kind gesture in helping me especially in thelaboratory system operation procedures. Last but not least, I would like to express my appreciation to my parents, MrWong Wai Yew and Madam Chia Saw Meng, and other members of my family for theiremotional, financial support and providing a lovely environment for me. vi
  8. 8. CONTENTS PAGEABSTRACT iiABSTRAK ivACKNOWLEDGEMENTS viLIST OF FIGURES xLIST OF TABLES xiiLIST OF APPENDICES xiiiLIST OF SYMBOLS AND ABBREVIATIONS xvCHAPTER IINTRODUCTION 1CHAPTER IILITERATURE REVIEW2.1 Medicinal mushrooms and its usages 52.2 Hericium erinaceus 2.2.1 Origin 7 2.2.2 Classification 8 2.2.3 Medicinal properties, nutritional and bioactive components derived 8 from Hericium erinaceus2.3 Nervous system and neurodegenerative diseases 2.3.1 Neurite 12 2.3.2 Neurodegenerative diseases (factors, therapies to cure and prevent) 122.4 Neuroprotective, neurotrophic, neuronal differentiation and neurite 16 stimulation effects of Hericium erinaceus2.5 Neurite outgrowth bioassay system of neural hybrid cell line NG108-15 2.5.1 Formation of NG108-15 hybrid cell 19 2.5.2 Characteristics of NG108-15 hybrid cell 19CHAPTER IIIMATERIALS AND METHODS3.1 Extraction 3.1.1 Preparations of fruitbodies 21 vii
  9. 9. 3.1.2 Preparation of aqueous ethanol crude extract 21 3.1.3 Solvent-solvent extraction (fractionation) 213.2 Neurite outgrowth activity assay 3.2.1 Preparation of stock solutions 23 3.2.2 Cell culture 23 3.2.3 Preparation of medium and buffer for cell culture 3.2.3.1 Dulbecco’s Modified Eagle’s Medium (DMEM) 23 3.2.3.2 Phosphate buffer saline 24 3.2.4 Cell culture techniques 3.2.4.1 Revival of frozen cells 24 3.2.4.2 Subculture of cells 25 3.2.4.3 Medium renewal 25 3.2.4.4 Cryopreservation of cells 26 3.2.5 Effect of Hericium erinaceus on stimulation of neurite outgrowth 26 activity of NG108-15 3.2.6 Scoring of neurites 26 3.2.7 Statistical analysis 273.3 Isolation of bioactive constituents 3.3.1 Column chromatography 28 3.3.2 Analytical thin layer chromatography 28 3.3.3 Preparative thin layer chromatography 29 3.3.4 High performance liquid chromatography (HPLC) 3.3.4.1 HPLC samples and mobile phase preparation 29 3.3.4.2 Analytical HPLC 30 3.3.4.3 Semipreparative HPLC 303.4 Identification 3.4.1 Gas chromatography-mass spectrometry (GCMS) 32 3.4.2 Nuclear magnetic resonance spectroscopy (NMR) 32 3.4.3 Liquid chromatography- mass spectrometry (LC/MS/MS) 32CHAPTER IVRESULTS & DISCUSSION4.1 Extraction, fractionation and isolation 4.1.1 Extraction, fractionation and isolation of aqueous ethanol extract of 33 Hericium erinaceus viii
  10. 10. 4.2 Neurite outgrowth activity 4.2.1 Effect of aqueous ethanol extract and fractions of Hericium 37 erinaceus on the neural cell line NG108-15 4.2.2 Effect of the fraction E1-E7 of Hericium erinaceus on the neural 45 cell line NG108-15 4.2.3 Effect of the subfraction sub4b_4 and sub4b_6 of Hericium 57 Erinaceus on the neural cell line NG108-154.3 Overall comparison of aqueous ethanol extract, hexane fraction, ethyl 62 acetate fraction, water fraction, fraction E1-E7, subfraction sub4b_4 and subfraction sub4b_64.4 Identification of chemical constituents 4.4.1 Identification of chemical constituents of the fraction E1 64 4.4.2 Identification of chemical constituents of the subfraction sub4b_4 66 4.4.3 Identification of chemical constituents of the subfraction sub4b_6 704.5 Overall comparison of the identified compounds and the neurite 75 stimulation activity in fraction E1, fraction E2 (subfraction sub4b_4 and subfraction sub4b_6) of Hericium erinaceusCHAPTER VGENERAL DISCUSSION & CONCLUSION 78REFERENCES 83APPENDICES 91 ix
  11. 11. LIST OF FIGURESFIGURE TITLE PAGE 2.1 Hericium erinaceus (Bull.: Fr.) Pers. 7 2.2 HeLa cell growth inhibitory substances isolated from Hericium 10 erinaceus 2.3 Hericenones isolated from fruiting body of Hericium erinaceus 16 which showed NGF synthesis promoting activity 2.4 Erinacines isolated from fruiting body of Hericium erinaceus 18 which showed NGF synthesis promoting activity 3.1 A schematic diagram showing the extraction and fractionation 22 procedures, process of biological investigations and isolation of active fractions of Hericium erinaceus 3.2 A schematic diagram showing the isolation of active fractions, 31 process of biological investigations and identification of the active fractions 4.1 Aqueous ethanol extraction of Hericium erinaceus 33 4.2 Fractionation of aqueous ethanol extract of Hericium erinaceus 34 4.3 Isolation of combined hexane and ethyl acetate extract of 35 Hericium erinaceus obtained through flash column chromatography 4.4 Isolation of fraction E2 of Hericium erinaceus by using 36 preparative thin layer chromatography and high performance liquid chromatography 4.5 Percentage of neurite bearing cells incubated with varying 37 concentrations of aqueous ethanol crude extract, hexane fraction, ethyl acetate fraction and water fraction of Hericium erinaceus 4.6 The morphology of the NG108-15 cells treated with various 39 concentrations of crude aqueous ethanol extract of Hericium erinaceus 4.7 The morphology of the NG108-15 cells treated with various 40 concentrations of hexane fraction of Hericium erinaceus 4.8 The morphology of the NG108-15 cells treated with various 41 concentrations of ethyl acetate fraction of Hericium erinaceus 4.9 The morphology of the NG108-15 cells treated with various 42 concentrations of water fraction of Hericium erinaceus x
  12. 12. 4.10 The morphology of the NG108-15 cells treated with various 48 concentrations of fraction E1 of Hericium erinaceus4.11 The morphology of the NG108-15 cells treated with various 49 concentrations of fraction E2 of Hericium erinaceus4.12 The morphology of the NG108-15 cells treated with various 51 concentrations of fraction E3 of Hericium erinaceus4.13 The morphology of the NG108-15 cells treated with various 52 concentrations of fraction E4 of Hericium erinaceus4.14 The morphology of the NG108-15 cells treated with various 53 concentrations of fraction E5 of Hericium erinaceus4.15 The morphology of the NG108-15 cells treated with various 55 concentrations of fraction E6 of Hericium erinaceus4.16 The morphology of the NG108-15 cells treated with various 56 concentrations of fraction E7 of Hericium erinaceus4.17 Percentage of neurite bearing cells incubated with varying 57 concentrations of subfractions sub4b_4 and sub4b_6 of Hericium erinaceus4.18 The morphology of the NG108-15 cells treated with various 59 concentrations of subfraction sub4b_4 of Hericium erinaceus4.19 The morphology of the NG108-15 cells treated with various 60 concentrations of subfraction sub4b_6 of Hericium erinaceus4.20 Compounds (I and II) identified in subfraction sub4b_4 684.21 Compounds (I, II and III) identified in subfraction sub4b_6. 73 xi
  13. 13. LIST OF TABLESTABLE TITLE PAGE 2.1 Neuronal properties of neuroblastoma x glioma hybrid cells 20 NG108-15 4.1 Stimulation of neurite outgrowth activity in the NG108-15 cells 38 with varying concentrations of aqueous ethanol extract and fractions of Hericium erinaceus 4.2 Stimulation of neurite outgrowth activity in the NG108-15 cells 46 with varying concentrations of fractions (E1-E4) of Hericium erinaceus 4.3 Stimulation of neurite outgrowth activity in the NG108-15 cells 47 with varying concentrations of fractions (E5-E7) of Hericium erinaceus 4.4 Stimulation of neurite outgrowth activity of the NG108-15 cells 58 with varying concentrations of sub4b_4 and sub4b_6 of Hericium erinaceus 4.5 Identified constituents of fraction E1 of Hericium erinaceus 64 1 4.6 H- and 13C-NMR for subfraction sub4b_4 in CDCl3 69 1 4.7 H- and 13C-NMR for subfraction sub4b_6 in CDCl3 74 xii
  14. 14. LIST OF APPENDICESAPPENDIX TITLE PAGE 1 Calculation for sample yield 91 2 Statistical analysis for percentage of neurite bearing cells of 92 aqueous ethanol extract by using one way ANOVA 3 Statistical analysis for percentage of neurite bearing cells of 93 hexane fraction by using one way ANOVA 4 Statistical analysis for percentage of neurite bearing cells of 94 ethyl acetate fraction by using one way ANOVA 5 Statistical analysis for percentage of neurite bearing cells of 95 water fraction by using one way ANOVA 6 Statistical analysis for percentage of neurite bearing cells of 96 fraction E1 by using one way ANOVA 7 Statistical analysis for percentage of neurite bearing cells of 97 fraction E2 by using one way ANOVA 8 Statistical analysis for percentage of neurite bearing cells of 98 fraction E3 by using one way ANOVA 9 Statistical analysis for percentage of neurite bearing cells of 99 fraction E4 by using one way ANOVA 10 Statistical analysis for percentage of neurite bearing cells of 100 fraction E5 by using one way ANOVA 11 Statistical analysis for percentage of neurite bearing cells of 101 fraction E6 by using one way ANOVA 12 Statistical analysis for percentage of neurite bearing cells of 102 fraction E7 by using one way ANOVA 13 Statistical analysis for percentage of neurite bearing cells of 103 subfraction sub4b_4 by using one way ANOVA 14 Statistical analysis for percentage of neurite bearing cells of 104 subfraction sub4b_6 by using one way ANOVA 15 The total ion chromatogram (TIC) of fraction E1 of 105 Hericium erinaceus 16 Mass spectrum of fraction E1 of Hericium erinaceus 106-107 1 17 H-NMR spectrum of subfraction sub4b_4 108- 110 13 18 C NMR spectrum of subfraction sub4b_4 111-113 xiii
  15. 15. 19 DEPT NMR spectrum of subfraction sub4b_4 114-116 120 H-NMR spectrum of subfraction sub4b_6 117-119 1321 C NMR spectrum of subfraction sub4b_6 120-12222 DEPT NMR spectrum of subfraction sub4b_6 123-12623 Chromatogram and mass spectrum data of peak in 127 LC/MS/MS at retention time 5.76 min in subfraction sub4b_4 with [M+H]+ of 148.824 Chromatogram and mass spectrum data of peak in 128 LC/MS/MS at retention time 7.66 min in subfraction sub4b_4 with [M+H]+ of 571.325 Chromatogram and mass spectrum data of peak in 129 LC/MS/MS at retention time 7.92 min in subfraction sub4b_4 with [M+H]+ of 597.326 Chromatogram and mass spectrum data of peak in 130 LC/MS/MS at retention time 8.72 min in subfraction sub4b_4 with [M+H]+ of 571.327 Chromatogram and mass spectrum data of peak in 131 LC/MS/MS at retention time 9.01 min in subfraction sub4b_4 with [M+H]+ of 597.328 Chromatogram and mass spectrum data of peak in 132 LC/MS/MS at retention time 6.24 min in subfraction sub4b_6 with [M+H]+ of 162.929 Chromatogram and mass spectrum data of peak in 133 LC/MS/MS at retention time 8.02 min in subfraction sub4b_6 with [M+H]+ of 569.330 Chromatogram and mass spectrum data of peak in 134 LC/MS/MS at retention time 9.11 min in subfraction sub4b_6 with [M+H]+ of 571.331 Chromatogram and mass spectrum data of peak in 135 LC/MS/MS at retention time 9.31 min in subfraction sub4b_6 with [M+H]+ of 597.3 xiv
  16. 16. LIST OF SYMBOLS AND ABBREVIATIONSAc AcetoneAD Alzheimer’s diseaseADFM Alzheimer’s Disease Foundation MalaysiaADI Alzheimers Disease Internationalα AlphaANOVA Analysis of varianceApoE4 Apolipoprotein E4ATCC American Tissue Culture Collectionβ betaCa2+ Calcium ionCO2 Carbon dioxideCHCl3 Chloroformcm Centimeter°C Degree celciusCDCl3 Deuterated chloroformDa DaltonDLPE Dilinoleoyl-phosphatidylethanolamineDMSO Dimethyl sulfoxideDMEM Dulbecco’s Modified Eagle’s MediumEDTA Ethylenediaminetetraacetic acidER Endoplasmic reticulumGC-MS Gas Chromatography-Mass Spectroscopyg Gramg/l Gram per litreHMG-CoA 3-hydroxy-3-methyl-glutaryl-CoA xv
  17. 17. HPLC High-performance liquid chromatographyhr HourHIV Human immunodeficiency virusHCl Hydrochloric acidHAT Hypoxanthine- aminopterine- thymidinekg Kilogramλ Lambda< Less thanLC/MS/MS Liquid chromatography-mass spectrometryL LitreLDL Low-density lipoproteinm/z Mass-to-charge ratioMHz MegahertzmRNA Messenger RNAMeOH Methanolµg/ml Microgram per mililitreµM Micromolarmg/ml Miligram per mililitreml Mililitremm Milimetremin Minuteng/ml Nanogram per mililitrenm NanometerNGF Nerve Growth FactorNO Nitric oxideN Normality xvi
  18. 18. NMR Nuclear magnetic resonance spectroscopy% PercentagePTFE PolytetrafluoroethyleneKH2PO4 Potassium hydrogen phosphatepsi Pounds per square inch± Plus-minusRP Reverse phaserpm Rotation per minuteNa2HPO4 Disodium hydrogen orthophosphateNaHCO3 Sodium bicarbonateNaCl Sodium chlorideNaOH Sodium hydroxideNa+ Sodium ionTMS TetramethylsilaneTLC Thin layer chromatographyUSP-NF The United States Pharmacopeia–National FormularyUPLC Ultra pure liquid chromatographyUV Ultravioletv/v Volume per volumew/v weight per volume xvii
  19. 19. CHAPTER I INTRODUCTION Neurodegenerative diseases can be defined as hereditary and sporadic conditionswhich are characterized by progressive nervous system dysfunction. Alzheimer’sdisease (AD) is one of the major neurodegenerative diseases. According to a report fromAlzheimers Disease International (ADI), it is estimated that there are currently about 18million people with AD worldwide. According to Alzheimer’s Disease FoundationMalaysia (ADFM), approximately 50,000 Malaysians are currently diagnosed with theillness. The production of reactive oxygen species during oxidative stress is speculatedto be pathologically important in neurodegenerative diseases. Degeneration ofcholinergic neurons and concomitant impairment of cortical and hippocampalneurotransmission lead to cognitive and memory deficits (Schorderet, 1995). Therefore,the characterization of neurite formation, maturation and collapse/ resorption is an areaof interest because these cellular processes are essential for the interconnection ofneuronal cell bodies. Choline supplementation (lecithins) and/ or acetylcholinesterase inhibitors(Tacrine) have been used to attenuate the cognitive and memory deficits. However,these agents have showed several side effects such as gastrointestinal troubles, hepatitisand reversible hepatotoxicity. The use of neurotrophin NGF (nerve growth factor) hasbeen initiated to treat neurodegenerative diseases. However, NGF cannot pass throughthe blood-brain barrier. Therefore, it needs to be injected directly into the brain to beeffective (Kawagishi et al., 2002). If a substance can permeate the membrane andstimulates the NGF synthesis in brain, this may result in the repair of the damagednervous functions. 1
  20. 20. Mushrooms, belonging to the kingdom fungi, are well-known for their medicinaland therapeutic values for centuries, since every culture has a written or oral tradition ofusing mushroom for their healing powers (Hobbs, 1995). There are over 1.5 millionspecies of fungi on earth, but mushrooms only constitute 14,000 species (Hawksworth,2001). However, the well investigated known species of mushrooms are still very low.Only 700 species are eaten as food and 50 species are poisonous (Halpern, 2007). In recent years, studies on the medicinal values of the edible mushrooms havegained a great deal of interests from researchers, as there is demand for more naturalremedies for lifes ailments. Mushrooms are valuable health food - low in calories, highin vegetable proteins, chitin, iron, zinc, fiber, essential amino acids, vitamins andminerals. Besides that, mushrooms have been used as bioengineering resources in thedevelopment of food materials (functional foods) as well as starting materials in theproduction of drugs. For example, the hot water extracts from dried fruitbodies of H.erinaceus are used as health drink (Yang and Jong, 1989). It has been pickled in brewedwine to give a health drink (Mizuno, 1999). Mushrooms possess many medicinal properties, pharmacological effects andphysiological properties such as bioregulation, maintenance of homeostasis, regulationof biorhythm, prevention and improvement in cancer, cerebral stroke and heart diseases,decreasing blood cholesterol, antifungal, anti-inflammatory, antiviral, antibacterial andantiseptic, antidiabetic, serve as kidney and nerve tonic, hepatoprotective and sexualpotentiator (Wasser and Weis,1999). Hericium erinaceus, belonging to the Basidiomycetes class, is an ediblemushroom occurring widely in Japan and China. These mushrooms grow on dead ordying wood. Hericium erinaceus, known as Yamabushitake (mountain hiddenmushroom), Jokotake (drinker fungus), Usagitake (rabbit fungus) and Harisenbon 2
  21. 21. (balloon fish) in Japan; Houtou (monkey head mushroom) and Hedgehog mushroom inChina and cauliflower mushroom (cendawan ‘bunga kobis’) in Malaysia. As a culinarydelicacy, H. erinaceus is one of the few mushrooms imparting the flavor of lobster andshrimp when cooked. Hericium erinaceus has served as traditional medicines in many regions. InChina, it is prescribed for stomach disorders, ulcers and gastrointestinal ailments. InNorth American, native Americans used H. erinaceus as a styptic, applied as a driedpowder to cuts and scratches to stop them from bleeding. Some compounds have been successfully isolated from the fruiting bodies andmycelia of H. erinaceus which showed NGF stimulation. Hericenones isolated from thefruiting bodies of H. erinaceus have been shown to promote NGF synthesis (Kawagishiet al., 1991). Erinacines isolated from mycelium of H. erinaceus have been identified asstimulators of nerve growth factor (NGF) synthesis (Kawagishi et al., 1996; Shimbo etal., 2005). Dilinoleoyl-phosphatidylethanolamine (DLPE) isolated from the fruitingbodies of H. erinaceus may reduce the risk of neurodegenerative diseases by reducingthe endoplasmic reticulum (ER) stress (Nagai et al., 2006). The screening for neurite outgrowth activity by H. erinaceus in an in vitro modelprovides important preliminary data to select mushroom extracts for isolation purposes.The neural hybrid clone, NG108-15 cell line is most widely used as an in vitro model ofneuronal differentiation because of its high proliferative activity and rapid elaborationof neurites (Smalheiser, 1991). The advantages of this bioassay is that it uses acontinuous cell line, thus avoiding the need for dissection. Hericium erinaceus is a temperate mushroom reported to fruit in cooltemperature. Currently, it is successfully cultivated in Malaysia. The mushroom nowgrown in tropical climate, may have bioactive profiles different from temperate grown 3
  22. 22. H. erinacius. However, Wong et al. (2007, 2009) have shown that the cultivationtemperature did not affect this. Both the ethanol and water extract of H. erinaceusgrown locally displayed stimulation of the neurite outgrowth using an in vitro model.Further, antioxidant and antimicrobial activities have been reported (Wong et al., 2009).It was reported that extracts of H. erinaceus enhanced nerve regeneration (Wong et al.,2009; 2011). It was therefore of interest to identify the chemical constituents in themushroom extract which may be responsible for stimulating neurite outgrowth.Objectives of studyThe objectives of the study were to: (a) evaluate the crude and fractionated extracts of H. erinaceus for their effects in stimulating the neurite outgrowth using the neural cell line NG 108-15. (b) identify the most active fraction. (c) identify the components present in the most active fraction . 4
  23. 23. CHAPTER II LITERATURE REVIEW2.1 MEDICINAL MUSHROOMS AND ITS USAGES Fleshy mushrooms (members of the class basidiomycetes) have long been usedfor their medicinal and therapeutic values. The term ‘medicinal mushroom’ is nowincreasingly gaining worldwide recognition due to its value in the prevention andtreatment of diseases. Furthermore, it can be easily obtained from the naturalenvironment. Medicinal properties of mushrooms have been widely studied. It was recordedthat mushrooms can exert a number of beneficial physiological effects. Auriculariaauricula-judae has been identified as a mushroom with reducing effect on the riskfactors of cardiovascular diseases. It has been reported to lower down the totalcholesterol and low density lipoprotein (LDL) level in hypercholesterolemic rats(Cheung, 1996; Chen et al., 2008) and reduced blood platelet binding which will causearterial thromboses (Fan et al., 1989). Cordyceps sinensis, Grifola frondosa andLentinus edodes were effective in reduce the triglyceride level (Francia et al., 1999). Besides that, Ganoderma lucidum and G. frondosa reduced blood pressure inspontaneously hypertensive rats (Kabir et al., 1988; 1989). There were few species ofmushrooms which possessed hypoglycemic action such as Agaricus bisporus(Swanston-Flatt et al., 1989), Agrocybe aegerita (Kiho et al., 1994), C. sinensis (Kihoet al., 1996) and G. frondosa (Kubo et al., 1994). Ergosterol, an antitumor compoundwhich has been isolated out from the mushroom Agaricus blazei, reduced the tumorgrowth with no side effects (Takaku et al., 2001). Mushrooms are also good candidates for promoting neuronal differentiation andsurvival. For example, polysaccharides in aqueous extract of G. lucidum induce 5
  24. 24. neuronal differentiation of rat pheochromocytoma PC12 cells and prevent NGF-dependent PC12 neurons from undergoing apoptosis (Cheung et al., 2000; Silva, 2004).Cyathane diterpenoid, termed scabronines, have been isolated from Sarcodonscabrosus, a bitter mushroom (Ohta et al., 1998), and have been reported to stimulateneurite outgrowth in rat pheochromocytoma cells (PC12) cultivated with theconditioned medium of human astrocytoma cells (1221N1) (Obara et al., 1999). Waterextract of Tremella fuciformis induced neurite outgrowth in PC12 cells and improvedthe memory deficit in rats by increasing the central cholinergic activity (Kim et al.,2007). Wu Ri, a famous Chinese physician from the Ming Dynasty (A.D. 1368- 1644),claimed that L. edodes contain the ability to increase energy, cure colds, eliminateworms and improve blood circulation. In “Shen Nong Ben Cao Jing”, G. lucidum, isranked under the superior medicine reported to be effective for multiple diseases andmostly responsible for maintaining and restoring the body balance with no unfavorableside effects. In the Taoist tradition, G. lucidum is said to enhance spiritual receptivityand it was used by monks to calm the spirit and mind. It is also considered a symbol offeminine sexuality as it refines the beauty and complexion. 6
  25. 25. 2.2 HERICIUM ERINACEUS2.2.1 Origin Hericium erinaceus (Bull.: Fr.) Pers. (Figure 2.1), a member of thebasidiomycetous fungus, is well known as a traditional medicine or food in Japan andChina. In Japan, H. erinacues is called Yamabushitake because it resembles theornamental cloth worn by Yamabushi. It is also called Jokotake (drinker fungus),Usagitake (rabbit fungus), Harisenbon (balloon fish) due to its shape. Figure 2.1: Hericium erinaceus (Bull.: Fr.) Pers. This mushroom is called Houtou (monkey head mushroom) in China due to theclose resemblance of fruiting body to the head of a baby monkey. It also known asHedgehog mushroom according to its shape. A Chinese traditional drug prepared bydrying this mushroom is also called Houtou. The hot water extracts from driedfruitbodies are used as health drink (Yang and Jong, 1989). It can be pickled in brewedwine to give a health drink (Mizuno, 1999). Hericium erinaceus is a wood destroying fungus and grows in standing anddecayed broadleaf trees such as oak, beech, and walnut. The cultivation of H. erinaceus 7
  26. 26. has been established using artificial logs made with agricultural residues in either bottlesor polypropylene bags (Mizuno, 1999; Chang and Miles, 2004).2.2.2 Classification Kingdom : Fungi Phylum : Basidiomycota Class : Basidiomycetes Order : Russulales Family : Hericiaceae Genus : Hericium Species : erinaceus2.2.3 Medicinal properties, nutritional and bioactive components derived fromHericium erinaceus Medicinal properties of H. erinaceus have been widely studied. Both the fruitingbodies and mycelia of H. erinaceus contain bioactive polysaccharides which exhibitvarious pharmacological activities including immunomodulatory effect, as well as anti-tumor, hypoglycemic and anti-aging properties (Zhang et al., 2007). Fifteenpolysaccharides have been successively extracted out with hot water. Five types ofpolysaccharides which showed relatively strong antitumor activity and a good lifeprolongation effect were glucoxylan, xylan, heteroxyloglucan, glucoxylan proteincomplex and galactoxyloglucan protein complex (Zhang et al., 2007). Besides the polysaccharides, an ergosterol derivative was also isolated from H.erinaceus. This compound showed cytotoxic effects on the cervical carcinoma HeLacells (Mizuno, 1999), antitiumor activity against Walker carcinosarcoma and humanmammary adenocarcinoma cell lines in vitro (Jong and Donovick, 1989), human gastric 8
  27. 27. tumor cell line, human hepatoma cell line, human colorectal tumor cell line and murinesarcoma-180. It also showed antivenom, anti-inflammatory (Keyzers and Davies-Coleman, 2005) and antimicrobial activity (Lu et al., 2000). Hericenones, erinacines, hericerin and hericenes, the aromatic compounds thatidentified in H. erinaceus, showed a wide range of in vitro and in vivo bioactivities(Shang et al., 2012). The novel oxyketo acid, Y-A-2, cytotoxic phenols, hericenone Aand hericenone B (Kawagishi et al., 1990), two novel γ- pyrones, erinapyrone A anderinapyrone B (Figure 2.2) (Kawagishi et al., 1992) extracted from the fruiting body ofH. erinaceus using ethanol or acetone, showed inhibition against the proliferativeactivity of HeLa cells. Besides that, various acidic phenol-like and neutral fatty acid-like compoundssuch as hericenones and hericerins found in H. erinaceus (Kim et al., 2000) wereeffective against pathogenic microorganisms and showed antibacterial activity at lowconcentrations against S. aureus, B. subtilis and E. coli respectively. Two novel and aknown chlorinated orcinol derivaties were also isolated from the mycelium of H.erinaceus. These three compounds exhibited antimicrobial activities against Bacillussubtilis, Saccharomyces cerevisiae, Vetticillium dahlia and Aspergillus niger (Okamatoet al., 1993). Ethanol extract of mycelia or fruitbodies promoted better antimutagenic effectsthan water extract examined with the Ames test (Wang et al., 2001). On the other hand,methanol extract of fruitbodies was found to have hypoglycemic effect and reduceelevation rates of serum triglyceride and total cholesterol levels when administered tostreptozotocin-included diabetic rats (Wang et al., 2005). Yang et al., (2003)investigated the hypolipidemic effect of an exo-biopolymer produced from a submergedculture of H. erinaceus in dietary-included hyperlipidemic rats. The exo-biopolymerreduced the level of plasma total cholesterol, low density lipoprotein cholesterol, 9
  28. 28. triglyceride, phospholipids, atherogenic index and hepatic HMG-CoA reductaseactivity; and preserving the high density lipoprotein at relatively high level. Theseeffects would help to reduce the risk of atherosclerosis. OH O COOH OH Y-A-2 (Novel oxyketo acid) O OH O O H3CO Hericenone A O OH O N H3CO Hericenone B O O HO OH O O Erinapyrone A Erinapyrone BFigure 2.2: HeLa cell growth inhibitory substances isolated from Hericium erinaceus 10
  29. 29. This mushroom has been reported to exhibit significant antioxidant activitywhich might help to reduce the oxidative damage caused by the uncontrolled productionof oxygen-derived free radicals (Mau et al., 2001). The reduction of free radicals mightlower the risk in the onset of many diseases such as cancer, rheumatoid arthritis,artherosclerosis, degenerative processes and deterioration of physiological functionsassociated with aging. Besides that, total polyphenols were the major natural antioxidantcomponents found in the methanol extract from dried H. erinaceus fruit bodies (Mau etal., 2002). In Chinese traditional medicine, it is used for the treatment for neurasthenicgastritis and gastroduodenal ulcer. In recent year, cultures or their extracts processed intablets have been produced in large scale for curing gastric ulcer and chronic gastricism. Nitric oxide (NO) is a pleiotropic biological molecule involved in a myriad ofphysiological and pathological processes such as regulation of blood pressure,neurotransmission, signal transduction, anti-microbial defense, immunomodulation,cellular redox regulation and apoptosis. The water extract of H. erinaceus activated themacrophages and induce NO production in peritoneal macrophages and RAW 264.7 cellline through the activation of transcription factor NF-KB (Son et al., 2006). A 63kDa laccase, with a novel N-terminal sequence isolated from the waterextract of H. erinaceus dried fruiting bodies showed inhibitory effect towards HIV-1reverse transcriptase (Wang and Ng, 2004). HIV-1 reverse transcriptase was involved inHIV replication; inhibitors of this enzyme are potential therapeutic agents in the battleagainst HIV (Sarafianos et al., 2009). 11
  30. 30. 2.3 NERVOUS SYSTEM AND NEURODEGENERATIVE DISEASES2.3.1 Neurite The characterization of neurite formation, maturation and collapse/ resorption isan area of interest because these cellular processes are essential for the interconnectionof neuronal cell bodies. Neurites are particularly interesting in relation toneuropathological disorders, neuronal injury/ regeneration and neuropharmacologicalresearch and screening (Smit et al., 2003). Neurites emerging from cloned neural celllines have been studied extensively over the past 15 years (Smalheiser & Schwartz,1987). It was appreciated very early that some clones can express neuritesspontaneously, even without inducing them to differentiate, but most neurobiologistshave ignored this class of neurites in favor of studying clones such as the PC12 cell line,whose neurites are under inducible control and contain characteristics of axons indifferentiated neurons. Bioassay which uses the PC12 cell line of ratpheochromocytoma was described by Greene (1977) and Greene & Tischler (1982).The matured neurite, called neuron, is responsible for receiving stimuli, producing andtransmiting electrical signal called nerve impulses, or action potentials. It alsosynthesizes and releases neurotransmitters.2.3.2 Neurodegenerative diseases (factors, therapies to cure and prevent) Neurodegenerative diseases can be defined as hereditary and sporadic conditionswhich are characterized by progressive nervous system dysfunction. These disorders areoften associated with atrophy of the affected central or peripheral nervous systemstructures. Neurological disorders are quite diverse, chronic, challenging to treat, andoften disabling. They can be caused by many different factors, including (but notlimited to): inherited genetic abnormalities, problems in the immune system, injury to 12
  31. 31. the brain or nervous system, or diabetes. Many mental illnesses are believed to beneurological disorders of the central nervous system, but they are classified separately. The production of reactive oxygen species during oxidative stress is speculatedto be pathologically important in neurodegenerative diseases which include Alzheimer’sdisease, Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease(Halliwell and Gutteridge, 1999). Alzheimer’s disease is the most common form ofsenile dementia. Alzheimer’s disease could be caused by both environmental andgenetic factors. This has been proved by the genetic linkage studies on thechromosomes 14, 21 (early-onset) and 19 (late-onset). Trisomy and mutations of the β-amyloid precursor protein gene on chromosome 21 are the causes that lead to the early-onset of the Alzheimer’s disease (Goate et al., 1991). Early-onset familial forms ofAlzheimer’s disease could be caused by genetic mutations which may affectchromosome 14 (Mullan et al., 1993). Mutations on chromosome 19 and theconcomitant expression of variant apolipoprotein E4 (ApoE4) from ApoE gene (ε4allele) are associated with sporadic and late-onset familial forms of Alzheimer’s disease(Uterman, 1994). However, less than 1% of patients who suffer from the disease are dueto these genetic causes. It is probable that the majority of the cases is caused by avariety of environmental factors which may be either sufficient to trigger disease bythemselves, or sufficient when acting synergistically with the patient genotype. A director indirect role has been attributed to normal or structurally altered amyloid β-protein(concentrated in senile plaques) and/or excessively phosphorylated tau protein (locatedin neurofibrillary tangles) (Schorderet, 1995). Degeneration of cholinergic neurons andconcomitant impairment of cortical and hippocampal neurotransmission lead tocognitive and memory deficits (Schorderet, 1995). Neuronal cell death is an essential feature of neurodegenerative disease. Manytypes of neuronal cell death, for example, these which are associated with amyloid-β, 13
  32. 32. glutamate and nitric oxide are thought to be caused by endoplasmic reticulum stress.Glutamate toxicity is a major contributor to pathological cell death within the nervoussystem and appears to be mediated by reactive oxygen species (Lee et al., 2003). Thus,it is reasonable to suspect that molecules which are able to attenuate endoplasmicreticulum stress might reduce both the risk for and the extent of the damage inneurodegenerative disease (Nagai et al., 2006). Treatments for reducing neuronal cell death are important for preventing as welltreating neurodegenerative disease, including dementia and motor dysfunction.However, because neurodegenerative diseases have, typically, a long incubation periodprior to diagnosis and are symptom-free; hence, there is a late diagnosis of the disease.This is a severe problem because once neurons are dead or neuronal circuits destroyed,lost of brain function associated with the neurons or neuronal circuits is almostimpossible to restore. Attenuation of the cognitive deficits by using choline supplementation(lecithins) and/ or acetylcholinesterase inhibitors might enhance the cholinergic activityif the cognitive deficits are due to the loss of cholinergic activity. Tacrine(tetrahydroaminoacridine, Cognex®), a potent, centrally active and reversibleacetylcholinesterase inhibitor, was used together with morphine to lessen respiratorydepression without affecting analgesia in the mid 1940s. Tacrine has been used alone, orin combination with lecithin, to treat symptoms of the Alzheimer’s disease (Chatellier etal., 1990; Farlow et al., 1992). It showed a slight but statistically significantimprovement in the physicians score on the visual analogue scale (Chatellier et al.,1990). Only a small percentage of patients, moderately affected or treated at an earlystage of Alzheimer’s disease, seem to benefit from the drug (Farlow et al., 1992).However, it showed several side effects such as gastrointestinal troubles, hepatitis andreversible hepatotoxicity. 14
  33. 33. Investigation on the projection of neurotrophin NGF (Nerve Growth Factor)which could counter the degeneration of cholinergic neurons to the hippocampus, arecognized memory center, were recently initiated. NGF is a protein that is essential forsupporting the growth and maintenance of peripheral sympathetic neurons as well asfacilitating the development of some sensory neurons for a brief period during earlydevelopment (Shimbo et al., 2005). Infusions of NGF into the brain of a patient canimprove performance in memory test and prevent or stabilize the processes ofcholinergic pathway degeneration (Schorderet, 1995). Alternatively, antioxidants, free redical scavengers and/ or non-steroidal anti-inflammatory agents such as α-tocopherol (vitamin E), ubiquinols (coenzyme Q),retinoic acid (vitamin A), and ascorbic acid (vitamin C), may be screened as potentialtherapies for neurodegenerative disease induced by multiple endogenous and/ orexogenous factors (Schorderet, 1995). 15
  34. 34. 2.4 NEUROPROTECTIVE, NEUROTROPHIC, NEURONALDIFFERENTIATION AND NEURITE STIMULATION EFFECTS OFHERICIUM ERINACEUS Hericenone C, D, E, F, G, H have been successively isolated from H. erinaceus(Kawagishi et al., 1991, 1993). Among them, hericenone C, D, E (Figure 2.3) have beenproven to show NGF synthesis promoting activity (Kawagishi et al., 1991). O OH CHO 16" O H3CO O Hericenone C O OH CHO 18" O H3CO O Hericenone D O OH CHO 18" O H3CO 9" 12" O Hericenone EFigure 2.3: Hericenones isolated from fruiting body of Hericium erinaceus whichshowed NGF synthesis promoting activity. 16
  35. 35. This mushroom also produces erinacines A (Shimbo et al., 2005), B, C(Kawagishi et al., 1994), D (Kawagishi et al., 1996a), E, F, G (Kawagishi et al., 1996b)which have been identified as stimulators of nerve growth factor (NGF) synthesis(Figure 2.4). Stimulators of NGF synthesis have been used as medicines fordegenerative neuronal disorders such as Alzheimer’s disease and peripheral nerveregeneration. NGF is a protein that is essential for supporting the growth andmaintenance of peripheral sympathetic neurons as well as facilitating the developmentof some sensory neurons for a brief period during early development (Shimbo et al.,2005). NGF cannot pass through the blood-brain barrier, the semi-permeable membranebetween the blood and brain. Only small and lipid soluble molecules can pass throughthe membrane. NGF is too large to permeate it. Therefore, it needs to be injecteddirectly into the brain to be effective (Kawagishi et al., 2002). If a substance canpermeate the membrane and stimulates the NGF synthesis in brain, this may result inrepairing the damaged nervous functions. Dilinoleoyl-phosphatidylethanolamine (DLPE), an endoplasmic reticulum (ER)stress- attenuating molecule which might reduce the ER-stress, has been isolated fromthe fruit bodies of H. erinaceus and these may reduce the risk of gettingneurodegenerative diseases (Nagai et al., 2006). ER stress is the major cause of theneuronal cell death which leads to the neurodegenerative diseases. 17
  36. 36. O O O O H OH OH O HO OH OH CHO CHO Erinacine A Erinacine B O O O O H H OH OH O HO OH OH CH2OH C2H5O CHO Erinacine C Erinacine D H O O O O H H OH H H OH H H OH OH HO OH HO OH Eriancine E Erinacine F O O O O H H OH H OH HO OH Erinacine GFigure 2.4: Erinacines isolated from mycelium of Hericium erinaceus which showedNGF synthesis promoting activity. 18
  37. 37. 2.5 NEURITE OUTGROWTH BIOASSAY SYSTEM OF NEURAL HYBRIDCELL LINE NG108-152.5.1 Formation of NG108-15 hybrid cell 6-thioguanine-resistant clonal mouse neuroblastoma cells N18TG2 and thebromodeoxyuridine-resistant rat glioma cells C6-BU-1 were fused with the aid ofinactivated Sendai virus to generate the neuroblastoma x glioma hybrid cell clone,NG108-15 (Hamprecht et al., 1985). Cells were grown in selective hypoxanthine-aminopterin-thymidine (HAT) medium, which was known to select for the wild-typehybrid cells and against the parental cell lines and their corresponding homokaryocytes(Littlefield, 1964).2.5.2 Characteristics of NG108-15 hybrid cell The hybrid cell is used as model neurons because every characteristic generallyascribed to neurons has been observed with the hybrid cell. The properties of NG108-15are summarized in Table 2.1 (Hamprecht et al., 1985). Due to the complexity of themammalian nervous system, it is hard to assign a certain effect observed to a certain celltype. Problems that are difficult to solve with animal or tissue experiments can betackled with the aid of cultured cells. Then, it is like having at one’s disposal thenumerous cell types as homogenous cell populations for studying their individualdifferentiated functions and their mechanisms of intercellular communication. 19
  38. 38. Table 2.1: Neuronal properties of neuroblastoma x glioma hybrid cells (NG108-15)Neuronal PropertiesExtension of long processesClear and dense core vesiclesExcitable membranes (inward current of action potentials carried by Na+ or Ca2+Formation of functional synapsesNeurotransmitter enzymes - Choline acetyltransferase - Dopamine-β-hydroxylaseSynthesis of neurohormones - Acetylcholine - Leu- and Met- enkephalin - Dynorphine-(1-8), α-neoendorphine - β-Endorphine - Vasoactive intestinal peptide - Angiotensin - Hydra head activator- like activityUptake system for - Catecholamines - TaurineDepolarization- induced Ca2+- dependent release of acetylcholineReceptors for neurohormones - Acetylcholine - Noradrenaline - Opioids (Morphine, enkephaline) - Prostaglandin E1 - Adenosine 20
  39. 39. CHAPTER III MATERIALS AND METHODS3.1 EXTRACTION3.1.1 Preparation of fruitbodies Hericium erinaceus fresh fruitbodies were obtained from Ganofarm Sdn. Bhd. inTanjung Sepat, Selangor. The fruitbodies were freeze-dried at 50± 2 C for 48 hours.The dried fruit bodies were blended in the commercial Waring blender and stored inairtight containers prior to assay.3.1.2 Preparation of aqueous ethanol crude extract The powdered freeze-dried fruit bodies of H. erinaceus was soaked in 80 %(v/v) aqueous ethanol for 3 days. The residue was then resoaked in 80 % (v/v) aqueousethanol and the extraction and filtration process was repeated three times. The solventcontaining extract was then concentrated under vacuum using a rotary evaporator.3.1.3 Solvent- solvent extraction (fractionation) The crude extract obtained was successively fractionated with hexane, ethylacetate and water using a separating funnel. All the fractions (hexane, ethyl acetate andwater) were filtered and concentrated under vacuum using a rotary evaporator to givehexane, ethyl acetate and water fractions Figure 3.1 shows the flow chart of the extraction and fractionation procedures,process of biological investigations and isolation of active fractions of H. erinaceus. 21
  40. 40. Fresh Hericium erinaceus Freeze-dried and ground to fine powder Dried and ground H. erinaceus i. Extraction with 80% ethanol (3 times) ii. Concentration under reduced pressure Aqueous ethanol crude extract i. Extraction with hexane ii. Concentration under reduced pressure Hexane soluble fraction Hexane insoluble fraction i. Partition (v/v) between ethyl acetate and water (Ratio 1:2) ii. Concentration under reduced pressure Ethyl acetate fraction Water fraction In vitro neurite outgrowth assay by using NG108-15 hybrid clone Isolation of active fractionsFigure 3.1: A schematic diagram showing the extraction and fractionation procedures,process of biological investigations and isolation of active fractions of Hericiumerinaceus 22
  41. 41. 3.2 NEURITE OUTGROWTH ACTIVITY ASSAY3.2.1 Preparation of stock solutions Each extract and fractions were dissolved in dimethylsulfoxide (DMSO) to formstock solutions 20 mg/ml for neurite outgrowth assay and kept at -20 ºC for future use.The concentration of samples was prepared according to the requirements for the assayby serial dilutions using the media or media with Tween 80.3.2.2 Cell culture The neural hybrid clone NG108-15 was chosen for this purpose. NG108-15 cellswere purchased from American Type Culture Collection (ATCC) and cultured inDulbecco’s Modified Eagle’s Medium (DMEM).3.2.3 Preparation of medium and buffer for cell culture3.2.3.1 Dulbecco’s Modified Eagle’s Medium (DMEM)Basic medium Final volume cell culture grade water (80 - 90 %) was measured. Watertemperature should be 15-30 C. Dry powder medium (13.38 g/l) was added slowly andallowing mixing time between additions (original package was rinsed with smallamount of water to remove all traces of powder and added to solution). The solutionwas mixed for 30 minutes. 3.7 g/l of sodium bicarbonate (NaHCO3) and hypoxanthine-aminopterine- thymidine (HAT) were added and stirred until dissolved. While mixing,the pH of the medium was adjusted to 6.9-7.1 using 1N NaOH or 1N HCl. Additionalwater was added to bring the solution to final volume and continue mixing for at least30 minutes. The medium was sterilized by filtration using a membrane with a pore sizeof 0.22 microns and aseptically dispensed into sterile container. 23
  42. 42. Complete growth medium Basic medium described above supplemented with 10 % (v/v) of fetal bovineserum, 100units/ml penicillin and 100 µg/ml streptomycin.Revival medium Complete growth medium described above supplemented with 20 % (v/v)instead of 10 % (v/v) fetal bovine serum.Cryoprotectant medium Basic growth medium described above supplemented with 10 % (v/v) dimethylsulfoxide (DMSO) and 50 % (v/v) fetal bovine serum.3.2.3.2 Phosphate Buffer Saline 1.52 g of sodium hydrogen phosphate (Na2HPO4), 0.58 g of potassium hydrogenphosphate (KH2PO4) and 8.5 g of sodium chloride (NaCl) were dissolved in 1L distilledwater and pH was adjusted to 7.2. The solution was filtered with filter paper andautoclaved for 15 minutes at 121 C, 15 psi. The solution was stored at roomtemperature.3.2.4 Cell culture techniques3.2.4.1 Revival of frozen cells The vial containing frozen cells was thawed by gentle agitation in a 37 C waterbath. To reduce the loss of viability, the vial was thawed rapidly. Once the contentswere thawed, the contents were transferred aseptically into a centrifuge tube bypipetting. 1 ml of revival medium was added and centrifuged at 1000 rpm for 5 minutes. 24
  43. 43. Supernatant was discarded and pellet was resuspended in 1 ml of revival medium. Thesuspended cells were pipetted into a cell culture flask containing 5- 10 ml revivalmedium. The culture was incubated at 37C in a 5% CO2 incubator at atmosphericpressure.3.2.4.2 Subculture of cells The culture was examined with an inverted microscope to check for anyevidence of microbial contamination and to determine whether the majority of the cellswere attached to the bottom of the flask. When the color of the medium changed fromreddish to yellowish (the medium become acidic) or every 2- 3 days, the culturemedium was removed and discarded. Trypsin-EDTA (1 ml) and phosphate buffer salinesolution (3 ml) were added to the flask and cells were observed under invertedmicroscope until cell layer was detached from the bottom of the flask. The contents ofthe flask were transferred aseptically into a centrifuge tube containing 2 ml of completegrowth medium by pipetting. The cells were centrifuged at 1000 rpm for 5 minutes.Supernatant was discarded and pellet was resuspended in 3- 4 ml of complete growthmedium. Appropriate amounts of suspended cell were added to a new culture flaskcontaining complete growth medium. Cultures were incubated at 37 C in a 5 % CO2incubator at air atmosphere.3.2.4.3 Medium renewalThe medium was changed 3 to 4 times weekly. 25
  44. 44. 3.2.4.4 Cryopreservation of cells Cryopreservation of cells was carried out in accordance with subculturingprocedure except that the pellet obtained after centrifugation was suspended incryoprotectant medium instead of complete growth medium. The suspended cells werethen transferred to a cryogenic vial and stored in liquid nitrogen vapor phase.3.2.5 Effect of Hericium erinaceus on stimulation of neurite outgrowth activity ofNG108-15 The neural hybrid cell NG 108-15 was cultured until 60-70 % confluent prior toassay. Cells were detached from the flask with 0.25 % (w/v) solution of trypsin inphosphate buffer saline solution and washed in culture medium. The cell pellet wasobtained by centrifugation at 1000 rpm for 5 minutes. The density of the cells wascounted by 0.4 % (w/v) of tryphan blue exclusion method in a haemocytometer. Thecells were plated into 6 well plates coated with 2 x 10-5 % (w/v) of poly-D-lysine at acell density of 10000 cells per well in medium containing 4 various concentrations (10,25, 50, 100 µg/ml) of extract and fractions and subfractions of fruit bodies. Negativecontrol is the well with the untreated cells whilst positive control is the well with thecells treated with Nerve Growth Factor (NGF). Plates were incubated at 37 C in a 5 %CO2 humidified incubator. Cells were observed for neurite outgrowth, branching ofneurites after 24 hours.3.2.6 Scoring of neurites A cell was considered as positive for bearing neurites if it had at least one thinextension longer than one full diameter of its cell body. Specifically excluded wereextensions associated with clearly different patterns of cell responses, such as broad, 26
  45. 45. sheet-like spreading of cells or the rare radially oriented processes apparently arising by“shrinkage” (Smalheiser and Schwartz, 1987). Cell clumps containing more than fivecells were also not included in the results. If more relaxed criteria had been used, manyshort extensions would have been counted as neurites, and the assay would not havebeen useful to detect stimulatory effects upon neurite formation (Smalheiser andSchwartz, 1987). Duplicates were set up for each concentrations tested. Approximately300 cells in each well were evaluated. Neurite formation was quantified by scoring thenumber of cells processing neurites and expressed as a percentage of the total number ofcells counted. Neurite bearing cells (%) = number of cells processing neurites x 100 % total number of cells countedThe result was also expressed in percentage increase in neurite bearing cells incomparison to negative control. Percentage increase compared to negative control = neurite bearing cells of extract – neurite bearing cells of negative control x 100 % neurite bearing cells of negative control3.2.7 Statistical analysis The means of data were subjected to a one way analysis of variance (ANOVA)and the significance of the difference between means was determined by the Duncan’smultiple range test at 95 % least significance difference (P<0.05). 27
  46. 46. 3.3 ISOLATION OF BIOACTIVE CONSTITUENTS3.3.1 Column chromatography The combined fraction of hexane and ethyl acetate was subjected to silica gelflash column chromatography. Column chromatography was performed by using Mercksilica gel. The gel was packed onto the column. After the sample was introduced to thecolumn, solvent with increasing polarity gradient was used to elute the column[developing solvent: chloroform (100 % chloroform) → chloroform-acetone mixtures(20 % Ac/CHCl3, 40 % Ac/CHCl3) → chloroform-methanol mixtures (10 %MeOH/CHCl3, 30 % MeOH/CHCl3) → methanol (100 % MeOH)]. Fractions weremonitored by thin layer chromatography (TLC) and fractions that possess the samespots/bands on the TLC were combined and where necessary subjected to furtherseparation. Nine fractions were obtained (E1-E9) from the flash columnchromatography which monitored by TLC. Figure 3.2 shows the flow chart of the isolation of active fractions of H.erinaceus.by using different types of chromatographic technique, process of biologicalinvestigations and identification of the active fractions.3.3.2 Analytical thin layer chromatography TLC was routinely used to detect and separate the various compounds. Thefractions from column chromatography were examined by TLC using precoated glassplates, 0.25 mm thickness, silica gel F254 (Merck, Darmstadt, G.F.R). The TLC plateswere spotted with a piece of fine glass capillary tube and then developed in saturatedchromatography tanks with various solvent systems at room temperature. The spotswere visualized by examination of the TLC plates under UV light, followed by applyingiodine vapor. 28
  47. 47. 3.3.3 Preparative thin layer chromatography Subfraction E2 was successively subjected to preparative thin layerchromatography. The sample was dissolved by using chloroform. The TLC precoated(silica gel F254) glass plates (Merck, Darmstadt, G.F.R) with 0.25 mm thickness, 10 cm(width) x 10 cm (height) were used. A line was drawed (about 1 cm) from the bottom ofthe plate. ). The line on the TLC plates was spotted with a piece of fine glass capillarytube and then developed in saturated chromatography tanks (100 % CHCl3) at roomtemperature. After the solvent reach the solvent front, the plates were taken out from thechromatography tanks. When the plates were dried enough, the bands were visualizedby using UV light. The bands were marked lightly by using pencil. The bands werescrapped off onto a clean, white paper by using the edge of a spatula. The compoundswere washed off from the silica gel by using chloroform. The solvent containingsamples were filtered and concentrated under vacuum using a rotary evaporator.3.3.4 High performance liquid chromatography (HPLC)3.3.4.1 HPLC samples and mobile phase preparation The samples were dissolved in methanol/acetonitrile mixture. The samples werefiltered through 0.45 µm Sartorius minisart PTFE-membrane syringe filter to removeany particular matter that might clog the column. The mobile phase that used wasacetonitrile. The mobile phase was filtered by using 0.45 µm Sartorius PTFE-membraneand degassed before introduced to the system. All the solvents that used were in HPLCgrade. 29
  48. 48. 3.3.4.2 Analytical HPLC Analytical HPLC analysis were carried out by using the instrument Waters DeltaPrep consists of water Prep LC controller, quaternary pump, vacuum degasser, UVdetector (water 2487, Dual λ Absorbance Detector). The separation profiles of thesamples can be improved by changing the solvent system of the mobile phase, flow rateand column. The column that used for analytical HPLC was performance RP-18encapped column 100-4.6 mm purchased from Merck. The analysis was carried out inisocratic mode at a flow rate of 1 ml/min, with column effluent being monitored at thewavelength of 214 nm and 254 nm.3.3.4.3 Semipreparative HPLC The samples were then further isolated out by using Chromolith Semiprep RP-18 column encapped 100-10 mm purchased from Merck. The separation was carried outin isocratic mode by using 100 % acetonitrile as mobile phase at a flow rate of 3ml/min, with column effluent being monitored at the wavelength of 214 nm and 254nm. The separated subfraction was collected manually. 30
  49. 49. Combined fraction of hexane and ethyl acetate Flash column chromatograhy E1 E2 E3 E4 E5 E6 E7 In vitro neurite outgrowth assay by using NG108-15 hybrid clone Identification of the Preparative thin layer chemical constituents chromatography by GC-MS sub4b HPLC sub4b_4 sub4b_6 Identification of the chemical constituents by NMR Identification of the chemical constituents by LC/MS/MS In vitro neurite outgrowth assay by using NG108-15 hybrid cloneFigure 3.2: A schematic diagram showing the isolation of active fractions, process ofbiological investigations and identification of the active fractions. 31
  50. 50. 3.4 IDENTIFICATION3.4.1 Gas chromatography-mass spectrometry (GCMS) GCMS analysis was performed on fraction E1 using Network GasChromatography System (Agilent Technologies 6890) and Inert Mass SelectiveDetector (Agilent Technologies 5975) (70eV direct inlet) on a HP-5MS (5 % phenylmethyl siloxane) capillary column (30 m x 250 µm x 0.25 µm) initially set at 150 ºC,then programmed to 300 ºC at 5 ºC min-1 and held for 10 minutes at 300 ºC usinghelium as the carrier gas. The total ion chromatogram obtained was autointegrated bychemstation and the constituents were identified by comparison with the accompanyingmass-spectra database (NIST 05 Mass Prectral Library, USA) wherever possible.3.4.2 Nuclear magnetic resonance spectroscopy (NMR) All the NMR experiments were performed on a JEOL 400MHz NMRspectrometer that install with the JEOL Delta software. Subfractions sub4b_4 andsub4b_6 were dissolved in deuterated chloroform and 1H, 13C and 1H-1H-COSY spectrawere collected. The internal standard for 1H NMR was TMS (δ: 0.00) and 13 C wasCDCl3 (δ: 77.00).3.4.3 Liquid chromatography-mass spectrometry (LC/MS/MS) LC/MS/MS analysis were carried out on subfractions sub4b_4 and sub4b_6 byusing the instrument Applied Biosystems 3200Q Trap LCMS/MS with Shimadzu ultrapure liquid chromatography (UPLC) system. Full scan with MS/MS data collection wasused. Positive ionization mode was set. The column that was used is Phenomenex AquaC-18 with dimension 50.0 mm x 2.0 mm x 5.0 µM. Rapid screening was performedwith 10 min run time. 32
  51. 51. CHAPTER IV RESULTS & DISCUSSION4.1 EXTRACTION, FRACTIONATION AND ISOLATION4.1.1 Extraction, fractionation and isolation of aqueous ethanol extract of HericiumerinaceusThe flow chart shows the extraction and fractionation procedures for H. erinaceus(Figure 4.1 and 4.2) Fresh H. erinaceus (1.33 kg) Dried and ground to fine powder Dried and ground H. erinaceus (200.00 g) i. Extraction with 80 % ethanol (3 times) ii. Concentration under reduced pressure Aqueous ethanol extract (52.29 g)Figure 4.1: Aqueous ethanol extraction of Hericium erinaceus. Fresh H. erinaceus (1.33 kg) yielded 200.00 g of dried and ground H.erinaceus.The dried H. erinaceus sample (200.00 g) yielded 52.29 g of crude aqueous ethanolextract. 33
  52. 52. Aqueous ethanol extract i. Extraction with hexane ii. Concentration under reduced pressure Hexane soluble fraction Hexane insoluble fraction (3.85 g, 7.36 %) i. Partition (v/v) between ethyl acetate and water (Ratio 1:2) ii. Concentration under reduced pressure Ethyl acetate fraction Water fraction (0.77 g, 1.47 %) (44. 34 g, 84.80 %)Figure 4.2: Fractionation of aqueous ethanol extract of Hericium erinaceus. The aqueous ethanol extract was further extracted with hexane to give hexane-soluble fraction (3.85 g, 7.36 %) and hexane-insoluble residues. The hexane-insolubleresidues were further partitioned between ethyl acetate-water (ratio 1:2) to give the ethylacetate-soluble fraction (0.77 g, 1.47 %) and water fraction (44. 34 g, 84.80 %). 34
  53. 53. The combined hexane and ethyl acetate fraction were subjected to flash columnchromatography to yield 7 fractions, which were E1 (384.0 mg, 0.73 %), E2 (780.8 mg,1.49 %), E3 (438.2 mg, 0.84 %), E4 (62.4 mg, 0.12 %), E5 (39.7 mg, 0.08 %), E6(183.1 mg, 0.35 %), E7 (1068.2 mg, 2.04 %) (Figure 4.3). The percentage yields werecalculated based on the crude aqueous ethanol extract. Combined fraction of hexane and ethyl acetate Flash column chromatography (developing solvent: CHCl3 → CHCl3/Ac → CHCl3/MeOH →MeOH) E1 (384.0mg, E3 (438.2mg, E5 (39.7mg, E7 (1068.2mg, 0.73 %) 0.84 %) 0.08 %) 2.04 %) E2 (780.8mg, E4 (62.4mg E6 (183.1mg, 1.49 %) 0.12 %) 0.35 %)Figure 4.3: Isolation of combined hexane and ethyl acetate extract of Hericiumerinaceus obtained through flash column chromatography. 35
  54. 54. Fraction E2 obtained from flash column chromatography were further subjectedto preparative thin layer chromatography to yield subfraction sub4b (187.7 mg, 0.36 %)(Figure 4.4). Sub4b was then subjected to high performance liquid chromatography(HPLC) to give sub4b_4 (68.5 mg, 0.13 %) and sub4b_6 (38.5 mg, 0.07 %) (Figure 4.4). E2 Preparative thin layer chromatography Sub4b (187.7 mg, 0.36 %) High perfomance liquid chromatography (HPLC) Sub4b_4 (68.5 mg, Sub4b_6 (38.5 mg, 0.13 %) 0.07 %)Figure 4.4: Isolation of fraction E2 of Hericium erinaceus using preparative thin layerchromatography and high performance liquid chromatography. 36
  55. 55. 4.2 NEURITE OUTGROWTH ACTIVITY4.2.1 Effect of aqueous ethanol extract and fractions of Hericium erinaceus on theneural cell line NG108-15 Aqueous ethanol extract and fractions of H. erinaceus were screened for the invitro neurite outgrowth activity on the neural hybrid cell line NG108-15 at variousconcentrations (µg/ml) (Figure 4.5; Table 4.1). Cells were observed under a phasecontrast microscope for the neurite outgrowth and branching of neurites. The effect ofthe various extracts on the morphology and neurite extension of the NG108-15 cells aregiven in Figure 4.6 (crude aqueous ethanol extract), Figure 4.7 (hexane fraction), Figure4.8 (ethyl acetate fraction) and Figure 4.9 (water fraction). 50 45 Neurite- bearing cells (%) 40 35 ethanol crude extract 30 Hexane fraction 25 Ethyl acetate fraction 40.6 20 Water fraction 34.5 34.1 32.3 27.8 27.4 15 26.5 25.6 25.4 24.6 24.2 24.1 24.0 23.7 23.5 23.0 22.9 22.5 21.9 20.9 20.5 19.9 19.0 18.6 10 5 0 Negative NGF 10 25 50 100 control (20ng/ml) Concentrations (µg/ml)Figure 4.5: Percentage of neurite bearing cells incubated with varying concentrations ofaqueous ethanol crude extract, hexane fraction, ethyl acetate fraction and water fractionof Hericium erinaceus (nerve growth factor, 20 ng/ml, used as positive control). 37
  56. 56. Table 4.1: Stimulation of neurite outgrowth activity in the NG108-15 cells with varying concentrations of aqueous ethanol extract andfractions of Hericium erinaceus. NG108-15 cells without extract was negative control. 20 ng/ml of nerve growth factor (NGF) was used aspositive control. Ethanol crude extract Hexane fraction Ethyl acetate fraction Water fraction Treatment Neurite Increase Neurite Increase Neurite Increase Neurite Increase concentration bearing cells compared to bearing cells compared to bearing cells compared to bearing cells compared to (µg/ml) (%) control (%) (%) control (%) (%) control (%) (%) control (%) Negative control 19.9±1.5ab - 24.6±1.5a - 20.5±1.5a - 19.0±1.7a - Positive control (NGF) 23.0±1.8c 15.5 28.9±0.9bc 11.3 24.1±0.2b 17.8 24.2±0.2b 27.1 10 22.9±0.5c 15.0 25.4±1.3ab 3.3 23.7±0.8b 15.9 21.9±1.8ab 14.9 25 22.5±0.7bc 13.2 32.3±2.6bc 31.4 26.5±2.0bc 29.4 24.0±1.6b 26.3 50 20.9±0.9abc 4.8 34.1±0.1c 38.7 27.8±1.6c 35.4 23.5±1.9b 23.3 100 18.6±1.3a -6.4 40.6±2.5d 65.2 34.5±0.9d 68.5 25.6±1.3b 34.4Note: Data are expressed as means ± standard deviation (n = 2). Means with different letters in the same column are significantly different(P < 0.05), one-way analysis of variance/ANOVA) 38
  57. 57. A B neurite C D E F neuriteFigure 4.6: The morphology of the NG108-15 cells treated with various concentrationsof crude aqueous ethanol extract of Hericium erinaceus [24hr of incubation at 37 ºC in a5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20ng/ml) was negative and positive control, respectively.]A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);C: 10 µg/ml of crude aqueous ethanol extract;D: 25 µg/ml of crude aqueous ethanol extract;E: 50 µg/ml of crude aqueous ethanol extract;F: 100µg/ml of crude aqueous ethanol extract 39
  58. 58. A B neurite C D neurite neurite E F neurite neuriteFigure 4.7: The morphology of the NG108-15 cells treated with various concentrationsof hexane fraction of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml)was negative and positive control, respectively.]A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);C: 10 µg/ml of hexane fraction; D: 25 µg/ml of hexane fraction;E: 50 µg/ml of hexane fraction; F: 100 µg/ml of hexane fraction 40
  59. 59. A B neurite neurite C D neurite neurite E F neurite neurite neuriteFigure 4.8: The morphology of the NG108-15 cells treated with various concentrationsof ethyl acetate fraction of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 %CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20ng/ml) was negative and positive control, respectively.]A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);C: 10 µg/ml of ethyl acetate fraction; D: 25 µg/ml of ethyl acetate fraction;E: 50 µg/ml of ethyl acetate fraction; F: 100 µg/ml of ethyl acetate fraction 41
  60. 60. A B neurite C D neurite E F neurite neuriteFigure 4.9: The morphology of the NG108-15 cells treated with various concentrationsof water fraction of Hericium erinaceus [24hr of incubation at 37 ºC in a 5 % CO2humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml)was negative and positive control, respectively.]A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);C: 10 µg/ml of water fraction; D: 25 µg/ml of water fraction;E: 50 µg/ml of water fraction; F: 100 µg/ml of water fraction 42

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