พฤกษเคมีและฤทธิ์ทางชีวภาพของ Knema glauca และ Knema furfuracea นายนพดล รังแกว วิทยานพนธิ นี้เปนสวนหนึ่งของการศึกษาตามหลักสูตรปริญญาวิทยาศาสตรดุษฎีบัณฑิต สาขาวิชาเภสชเคมั ีและผลิตภัณฑธรรมชาต ิ คณะเภสัชศาสตร จุฬาลงกรณมหาวิทยาลยั ปการศึกษา 2551 ลิขสิทธิ์ของจุฬาลงกรณมหาวิทยาลัย PHYTOCHEMICAL AND BIOACTIVITIES OF KNEMA GLAUCA AND KNEMA FURFURACEA Mr.Noppadon Rangkaew A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Program in Pharmaceutical Chemistry and Natural Products Faculty of Pharmaceutical Sciences Chulalongkorn University Academic year 2008 Copyright of Chulalongkorn University vi ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my thesis advisor, Associate Professor Dr. Rutt Suttisri of the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University. I greatly appreciate his valuable advice, kindness, patience, and constant encouragement throughout my research study. My appreciations are extended to Dr. Kazuko Kawanishi and Dr. Masataka Moriyasu of the Department of Natural Medicinal Chemistry, Kobe Pharmaceutical University, for their kindness, hospitality and guidance in the testing of AGEs inhibitor during my research at Kobe Pharmaceutical University, Kobe, Japan. I would like to thank all members of my thesis committee for their constructive suggestions and critical review of this thesis. I am particularly indebted to the Thailand Research Fund (TRF) for financial support (grant no. PHD/0033/2547) through the 2004 Royal Golden Jubilee Ph.D. program. I would also like to acknowledge BIOTEC laboratories, NSTDA, Thailand for evaluation of antituberculosis, antimalarial, antiviral and anticancer activities. I would like to thank all staff members and graduate students of the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University for their friendship, kind support and encouragement throughout the period of my study. Finally, I would like to express my deepest appreciation to my parents for their love, understanding and encouragement. CONTENTS Page ABSTRACT (Thai)…………………………………………………………... iv ABSTRACT (English)...……………………………………………………... v ACKNOWLEDGEMENTS………………………………………………….. vi CONTENTS………………………………………………………………….. vii LIST OF TABLES………………...…………………………………….…… xiv LIST OF FIGURES…………………………………….……..………….….. xvii LIST OF SCHEMES……………………………………...……………….… xxviii LIST OF ABBREVIATIONS………….……………………………….….... xxix CHAPTER I INTRODUCTION…………………………………..…………………. 1 II HISTORICAL………………………………………..………………… 4 1. The family Myristicaceae…………….………….….……………….. 4 2. The genus Knema……………………………………………………. 4 3. Knema glauca var. glauca and K. furfuracea…………………………... 5 4. Linear Diterpenes in Higher Plants………………………………….. 8 5. Arylnaphthalene Lignans in Higher Plants………………………….. 45 6. Chemical Constituents of Plants in the Genus Knema………………. 61 III EXPERIMENTAL……………………………………......……………. 73 1. Sources of Plant Materials……………….….….…………………. 73 viii CHAPTER Page 2. General Techniques……………………….……..………………... 73 2.1 Solvents……………………………………………………. 73 2.2 Analytical Thin-Layer Chromatography (TLC)….………... 73 2.3 Column Chromatography…………………………………. 73 2.3.1 Conventional Column Chromatography…….…… 73 2.3.2 Gel Filtration Chromatography………………….. 74 2.3.3 High Pressure Liquid Chromatography (HPLC)….. 74 2.4 Spectroscopy……………………………………………….. 74 2.4.1 Ultraviolet (UV) Spectra………………………….. 74 2.4.2 Infrared (IR) Spectra……………………………… 74 2.4.3 Mass Spectra……………………………………… 74 2.4.4 Proton and Carbon-13 Nuclear Magnetic Resonance (1H and 13C NMR) Spectra…………… 75 2.4.5 Fluorescence Spectrophotometer…………………. 75 2.5 Physical properties…………………………………………. 75 2.5.1 Melting points…………………………………….. 75 2.5.2 Optical Rotations…………………………………. 75 3. Extraction and Isolation 3.1 Extraction and Isolation of Compounds from Knema glauca var. glauca…………………………………………. 75 3.1.1 Extraction of the fruits of K. glauca var. glauca… 75 ix CHAPTER Page 3.1.2 Isolation of Compounds from the First Batch of EtOAc Extract of K. glauca var. glauca Fruits…… 75 3.1.2.1 Isolation of Compound KG-F1 (2,6-Dihydroxyphenyl-tetradecan-1-one) 76 3.1.2.2 Isolation of Compounds KG-F2 (Asarinin) and KG-F3 (Sesamin)……… 77 3.1.2.3 Isolation of Compound KG-F4 (Glaucaic acid)…………………………. 77 3.1.2.4 Isolation of Compound KG-F5 (Myristinin F)…………………….……. 77 3.1.3 Isolation of Compounds from the Second Batch of EtOAc Extract of K. glauca var. glauca Fruits…… 78 3.1.3.1 Isolaton of Compound KG-F6 (Malabaricone A)………………………. 78 3.1.3.2 Isolation of Compounds KG-F7 [Dodecanoylphloroglucinol] and KG-F8 [1-(2,4,6-Trihydroxyphenyl)-9- phenylnonan-1-one]………………….. 79 3.1.4 Extraction of the Leaves of K. glauca var. glauca... 79 3.1.5 Isolation of Compound from the EtOAc extract of K. glauca var. glauca Leaves…………………….. 79 3.1.5.1 Isolation of Compound KG-L1 (Myristinin C)………………………….. 80 3.1.6 Extraction of the Stems of K. glauca var. glauca… 80 3.1.7 Isolation of Compound from the EtOAc extract of K. glauca var. glauca Stems……………………… 81 3.1.7.1 Isolation of Compound KG-S1 ([(±)-7,4΄-Dihydroxy-3΄-methoxy- flavan]………………………………….. 81 x CHAPTER Page 3.2 Extraction and Isolation of Compounds from Knema furfuracea………………………………………….. 88 3.2.1 Extraction of the Stems of K. furfuracea…………. 88 3.2.2 Isolation of Compounds from the Hexane Extract of K. furfuracea Stems……………………………. 88 3.2.2.1 Isolation of Compounds KF-S1 [(+)- trans-1,2-Dihydrodehydro-guaiaretic acid] and KF-S2 (Fragransin A2)………. 88 3.2.3 Isolation of Compounds from the EtOAc Extract of K. furfuracea Stems……………………………. 89 3.2.3.1 Isolation of Compounds KF-S3 (Biochanin A) and KF-S4 (Mixture of Anarcardic Acid and Ginkgoic Acid)…. 89 3.2.3.2 Isolation of compound KF-S5 (Mixture of 2-hydroxy-6-(12- phenyldodecyl) benzoic acid and 2- hydroxy-6-(12-phenyldodecen-8΄Z-yl) benzoic acid)…………………………... 90 3.2.4 Extraction of the Leaves of K. furfuracea………… 90 3.2.5 Isolation of Compounds from Hexane Extract of K. furfuracea Leaves…………………………………. 90 3.2.5.1 Isolation of Compound KG-L1 (Myristinin A)…………………………. 91 4. Physical and Spectral Data of Isolated Compound………………... 96 4.1 Compound KG-F1 (2,6-Dihydroxyphenyl-tetradecan-1- one)………………………………………………………… 96 4.2 Compound KG-F2 (Asarinin)……………………………… 96 4.3 Compound KG-F3 (Sesamin)……………………………... 96 xi CHAPTER Page 4.4 Compound KG-F4 (Glaucaic acid)………………………... 97 4.5 Compound KG-F5 (Myristinin D)…………………………. 97 4.6 Compound KG-F6 (Malabaricone A)……………...……... 97 4.7 Compound KG-F7 (1-(2,4,6-Trihydroxy-phenyl)-undecan-1-one)…………… 98 4.8 Compound KG-F8 (1-(2,4,6-Trihydroxy-phenyl)-9-phenylnonan-1-one)……... 98 4.9 Compound KG-L1 (Myristinin A)………………………… 98 4.10 Compound KG-S1 ((±)-7,4΄-Dihydroxy-3΄-methoxyflavan)………………….. 99 4.11 Compound KF-S1 ((+)-trans-1,2-Dihydrodehydroguaiaretic acid)…………… 99 4.12 Compound KF-S2 (Fragransin A2)………………………... 100 4.13 Compound KF-S3 (Biochanin A)………………………….. 100 4.14 Component KF-S4 (Mixture of Anarcardic Acid and Ginkgoic Acid)…………………………………………….. 100 4.15 Component KF-S5 (Mixture of 2-hydroxy-6-(12- phenyldodecyl) benzoic acid and 2-hydroxy-6-(12- phenyldodecen-8΄Z-yl) benzoic acid)……………………… 101 4.16 Compound KF-L1 (Furfuracin)……………………………. 101 5. Evaluation of Biological Activities………………………………... 101 5.1 Determination of Antimycobacterial Activity……………... 101 5.2 Determination of Antimalarial Activity…………………… 102 5.3 Determination of Cytotoxic Activity……………………… 103 xii CHAPTER Page 5.3.1 Human Small Cell Lung Carcinoma (NCI-H187)... 102 5.3.2 Human Epidermoid Carcinoma (KB) and Breast Cancer (BC and MCF-7)…………………………. 103 5.3.3 Vero Cells………………………………………… 104 5.4 Determination of Anti-Herpes Simplex Activity………….. 104 5.5 Determination of Advance Glycation Endproducts (AGEs) Formation Inhibition Activity……………………………… 104 IV RESULT AND DISCUSSION…………………………………………. 106 1. Structure Determination of Compounds Isolated from Knema glauca var. glauca…………………………………………………. 106 1.1 Identification of Compound KG-F1 [1-(2,6- Dihydroxyphenyl)-tetradecan-1-one]……………………… 106 1.2 Identification of Compound KG-F2 [Asarinin]……………. 109 1.3 Identification of Compound KG-F3 [Sesamin]……………. 112 1.4 Structure Elucidation of Compound KG-F4 [Glaucaic acid] 114 1.5 Identification of Compound KG-F5 [Myristinin D]……….. 116 1.6 Identification of Compound KG-F6 [Malabaricone A]……. 119 1.7 Identification of Compound KG-F7 [Dodecanoylphloroglucinol]……………………………….. 122 1.8 Identification of Compound KG-F8 [1-(2,4,6- Trihydroxyphenyl)-9-phenyl-nonan-1-one]……………….. 124 1.9 Identification of Compound KG-L1 [Myristinin A]………. 126 1.10 Identification of Compound KG-S1 [(±)-7,4΄-Dihydroxy- 3΄-methoxy- flavan]………………………………………... 130 xiii CHAPTER Page 2. Structure Determination of Compounds Isolated from Knema furfuracea………………………………………………………….. 133 2.1 Identification of Compound KF-S1 [(+)-trans-1,2-Dihydrodehydroguaiaretic acid]…………… 133 2.2 Identification of Compound KF-S2 [Fragransin A2]………. 135 2.3 Identification of Compound KF-S3 [Biochanin A]………... 138 2.4 Identification of Compound KF-S4 [Mixture of Anarcardic Acid and Ginkgolic Acid]………... 140 2.5 Identification of Compound KF-S5 [Mixture of 2-hydroxy-6-(12-phenyldodecyl) benzoic acid and 2-hydroxy-6-(12-phenyldodecen-8΄Z-yl) benzoic acid]. 145 2.6 Structure Elucidation of Compound KF-L1 [Furfuracin]…. 150 3. Bioactivity Evaluation of Compounds Isolated from Knema glauca and Knema furfuracea…………………………………….. 153 3.1 Bioactive Compounds from Knema glauca……………….. 153 3.1.1 Cytotoxic activity…………………………………. 153 3.1.2