Studies on As arum hongkongense LEE, Kit Lin A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Philosophy in Chinese Medicine ©The Chinese University of Hong Kong September 2007 The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part of whole of the materials on the thesis in a proposed publication must seek copyright release the Dean of the Graduate School. lifmjAN 2iji| NS^^UB'iARY SYSTEM Thesis/Assessment Committee Professor Lin Zhi-xiu (Chair) Professor Che Chun Tao (Thesis Supervisor) Professor Wong Yum Shing (Committee Member) Professor Ko Kam Ming Robert (External Examiner) Abstract of thesis entitled: Studies on Asarum honskonsense Submitted by LEE Kit Lin for the degree of Master of Philosophy in Chinese Medicine At The Chinese University of Hong Kong in September, 2007 Abstract The favorable climate and geographical conditions support the wide diversity of flora in Hong Kong. According to the information provided by the Hong Kong Herbarium, there are 3，164 species of vascular plants with 16 species named after Hong Kong. Asarum hongkongense is a potential medicinal herb which was only discovered in 1988 and is endemic to Hong Kong. Herba Asari is a Chinese medicinal material known as Xixin (糸田辛)，which is derived from the entire plant of several species of Asarum. The plant parts used as medicine have recently been restricted to the roots and rhizomes, and thus re-named as Radix et Rhizoma Asari, due to the presence of the toxic ingredient aristolochic acid in the aerial parts. Herba Asari is a "warming and surface-relieving herb" with a diversity of medicinal functions including dispelling excessive cold, relieving pain and eliminating inflammation. In this study, Asarum hongkongense was investigated by employing four distinct approaches, including macroscopic features, microscopic features, aristolochic acid content and molecular DNA sequencing. The form and structure of the leaf, rhizome, root and flower of Asarum hongkongense are described in the first chapter. The leaf is ovate-cordate with densely ciliated margin. The plant has elongated prostrate rhizome, spicy root and i bell-shaped flowers with three sepals. In chapter two, the internal structures of the root, rhizome, stem and powders are characterized. Stomata are only exists on the lower epidermis of the leaf blade and the subsidiary cells are mostly tetracytic. The central xylem of the root appears from triarch to hexarch. The vascular bundles of the rhizome form an incomplete ring and the cambium between vascular bundles is indistinct. The starch grains are from simple to compound form of 6 units. The vessel is reticulate or spiral. The DNA sequence of Asarum hongkongense was studied. The length of DNA nucleotide of ITS 1 region and ITS2 region are 256 bp and 219 bp，respectively. The aristolochic acid content was determined by means of High Performance Liquid Chromatography. The detection limit of the method was 50 ng/mL. The aristolochic acid-I content in the leaf part was found to be 4.8 mg/kg while its concentration in the root part was 1.5 mg/kg, which is only one third of that of the leaves. Being an endemic potential herb that possesses medicinal potentials, Asarum hongkongense deserves further studies in detail. The present studies provide foundation information to support further research and development of this herb. ii Abstract of thesis entitled: Studies on Asarum honskonsense Submitted by LEE Kit Lin for the degree of Master of Philosophy in Chinese Medicine At The Chinese University of Hong Kong in September, 2007 撮要 根據香港標本室所提供的資料，現時本港維管束植物有3164個品種，其中16 個品種是以香港命名的。香港細辛於1988年被發現，是本港特有品種，也是具 有藥用潛質的草本植物。 藥用細辛（英文名稱爲HerbaAsari)是採用全株植物入藥。最近，由於地上部 分發現含有有毒成份馬鬼鈐酸，所以近年規限只可利用該植物的根或根莖作藥 用，並重新命名爲Radix et Rhizoma Asari�細辛是一種辛溫解表藥，具有驅寒、 止痛、消炎等多方面的用途。 本論文探討了香港細辛的形態特徵、顯微特徵、馬贺鈐酸含量和DNA序四個 方面。 首章闡述了香港細辛形態特徵’包括根、根楚、葉及花的形態和結構：葉爲心 形及邊緣有毛；根華成長匍狀；根有辛味；花是圓鐘形和三瓣。 第二章顯微特徵中，描述了根、根華、楚、花及粉末的結構特徵。氣孔只存在 於葉的下表皮，副衛細胞呈四輪裂形。木質部是三元型至六元型；根莖的導管 形成一個不完全的環，導管間的形成層不甚明顯。澱粉粒由單一至六個分粒組 成。導管是網紋狀或螺旋狀。 從DNA序分析發現，ITS1區及ITS2區的DNA長度分別爲256bp和219bp� iii 本文採用HPLC法測定了馬鹿鈐酸的含量，檢測限度爲50ng/ml�發現地上部位 的馬宽鈐酸的含量爲4.8 mg/kg，而根部則爲1.5 mg/kg�根部的馬宽鈐酸含量僅 爲地上部位的三分之一。 總括來說，香港細辛是一個香港特有品種而且具有藥用的潛質。本論文能爲曰 後相關開發硏究奠定基礎。 iv Acknowledgements I would like to express my sincerest gratitude to my supervisor, Prof. CHE Chun-tao, for his profound and continuous guidance, advice，supervision, help and encouragement throughout my study. My heartfelt gratitude is given to Mr. TSAI Sam-hip of the School of Chinese Medicine. I deeply thank him for his advice, comments, patience and technical support. I would also like to express my appreciation to Ms. Zong Yuying of the School of Chinese Medicine. She has given me a great deal of excellent advice, support, and assistance in the area of morphology and microscopic features. My deepest gratitude must give to Prof. Kwan Hoi-shan in the Biology Department for his support in providing the facilities for the study of DNA sequencing. I would also express great appreciation to Ms. Anna YIP for her assistance, technical support and advice. In addition, I deeply thank Dr. LEE Lin-hon and Mr. CHOW Ping who have provided me support in sample collection. I am also like to express my sincere thanks all the staff and students of the School of Chinese Medicine and the staff in the Herbarium of the Agriculture, Fisheries and Conservation Department, who have offered me supports, technical assistance, valuable advice, comments and suggestions. viii Last but not least, I greatly thank my family, especially my husband and daughters, who provide me with constant support, love, care and encouragement throughout my study. vi Table of Contents Abstract i 撮要 iii Acknowledgements v Table of contents vii List of Tables x List of Figures xi List of Abbreviations xiii Chapter 1: Literature Review 1 1.1 Introduction 1 1.1.1 History of Botanical Studies in Hong Kong 1 1.1.2 Plant Species Named after Hong Kong 2 1.2 Botanical Background of Asarum Plants 5 1.2.1 Plant Species under the Family of Aristolochiaceae 5 1.2.2 HerbaAsari 6 1.2.3 Classification of Asarum hongkongense 8 1.2.4 Growing Habitat of Asarum hongkongense 8 1.3 Medicinal Properties of Asari 10 1.4 Chemical Constituents of Asari 10 1.5 Aristolochic acid and Health Issues 12 1.5.1 Aristolochic Acid Intoxication 12 1.5.2 Description of Aristolochic Acid 13 1.5.3 Toxicities of Aristolochic Acid 13 1.5.4 Aristolochic Acid-Containing Plants 15 1.5.5 Control of Aristolochic Acid-Containing Products 17 1.5.6 Control of Aristolochic Acid-Containing Products in Hong Kong 18 1.6 Objectives of Study 19 Chapter 2: Macroscopic Features of Asarum hongkongense 20 2.1 Introduction 20 2.2 Plant Material 20 2.2.1 Asarum hongkongense 20 2.2.2 HerbaAsari 23 2.3 Macroscopic Characteristics of Aarum hongkongense 23 2.3.1 Leaf 23 2.3.2 Root and Rhizome 25 vii 2.3.3 Flower 27 2.4 Macroscopic Characteristics of Herba Asari heterotropoidis 31 (Liaoxixin) 2.4.1 Leaf 31 2.4.2 Root and Rhizome 31 2.4.3 Flower 34 2.5 Discussion 36 Chapter 3: Microscopic Features oiAsarum hongkongense 38 3.1 Introduction 38 3.2 Plant Materials 39 3.3 Chemical, Reagents and Instrumentation 39 3.4 Methods 39 3.5 Microscopic Characteristics of Asarum hongkongense 40 3.5.1 Transverse Section of Leaf 40 3.5.2 Surface View of Leaf 40 3.5.3 Transverse Section of Root 43 3.5.4 Transverse Section of Rhizome 43 3.5.5 Powder 47 3.5.5.1 Pollens 47 3.5.5.2 Vessels 47 3.5.5.3 Starch Grains 47 3.6 Microscopic Characteristics of Herba Asari heterotropoidis 49 (Liaoxixin) 3.6.1 Transverse Section of Leaf 49 3.6.2 Surface View of Leaf 49 3.6.3 Transverse Section of Root 53 3.6.4 Transverse Section of Rhizome 53 3.6.5 Powder 56 3.6.5.1 Starch Grains 56 3.6.5.2 Vessels 56 3.7 Discussion 58 Chapter 4: Molecular DNA Sequencing of Asarum hongkongense 61 4.1 Introduction 61 4.2 Sample Preparation 64 4.3 Method 64 4.3.1 Extraction of Total DNA 64 viii 4.3.2 PGR Amplification of ITS 1 and ITS2 Regions of rRNA Gene 65 4.3.3 Purification of PGR Products 65 4.3.4 Sequencing of ITS Regions . 66 4.3.4.1 Cycle Sequencing Reaction 66 4.3.4.2 Purification of Sequencing Extension Products 67 4.3.4.3 Electrophoresis by Genetic Analyzer 67 4.3.4.4 Sequence Analysis and Alignment 67 4.4 Results and Discussion 68 4.4.1 Extraction of Total DNA 68 4.4.2 PGR Amplification of ITS 1 and ITS2 Regions of rRNA Gene 68 4.4.3 Sequence Analyses 68 Chapter 5: Determination of Aristolochic Acid oiAsarum hongkongense 80 5.1 Introduction 80 5.2 Sample Preparation 81 5.3 Standard Preparation 81 5.4 Experimental 83 5.4.1 Chemical and Reagents 83 5.4.2 Methods 83 5.4.2.1 High-Performance Liquid Chromatography 83 5.4.2. 2 Mass Spectrometry 85 5.4.3 Other Instrumentation 85 5.5 Method Validation 85 5.5.1 Calibration 85 5.5.2 Precision 87 5.5.3 Recovery Test 88 5.5.4 Limit of Detection 89 5.6 Results and Discussion 90 Chapter 6: Conclusion 92 References 94 ix List of Tables Table 1.1 List of plants named after Hong Kong 3 Table 1.2 Chemical constituents in some Asarum species 11 Table 1.3 Botanicals known or suspected to contain Aristolochic 16 Acid Table 4.1 PGR primers used for the amplification of ITSl and ITS2 70 regions of rRNA gene Table 5.1 Gradient program of the HPLC mobile phase 84 Table 5.2 Precision data for AA-I 87 Table 5.3 The result of recovery rate 88 xiii List of Figures Figure 1.1 Living plant oi Asarum hongkongense 4 Figure 1.2 Asarum hongkongense in dried product 4 Figure 1.3 Herba Asari obtained from local market 7 Figure 1.4 Growing