Clemson University TigerPrints All Dissertations Dissertations 12-2008 CHARACTERIZATION OF BIOACTIVE PHYTOCHEMICALS FROM THE STEM BARK OF AFRICAN MAHOGANY Khaya senegalensis (MELIACEAE) Huaping Zhang Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Food Science Commons Recommended Citation Zhang, Huaping, "CHARACTERIZATION OF BIOACTIVE PHYTOCHEMICALS FROM THE STEM BARK OF AFRICAN MAHOGANY Khaya senegalensis (MELIACEAE)" (2008). All Dissertations. 305. https://tigerprints.clemson.edu/all_dissertations/305 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. CHARACTERIZATION OF BIOACTIVE PHYTOCHEMICALS FROM THE STEM BARK OF AFRICAN MAHOGANY Khaya senegalensis (MELIACEAE) A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Food Technology by Huaping Zhang December 2008 Accetped by: Dr. Feng Chen, Committee Chair Dr. Alex Kitaygorodskiy Dr. R. Kenneth Marcus Dr. Xi Wang i ABSTRACT African mahogany Khaya senegalensis (Desr.) A. Juss (Meliaceae) is a large tree growing mainly in the sub-Saharan savannah forests from Senegal to Uganda. This plant is one of the most popular medicinal meliaceous plants in traditional African remedies, used as a bitter tonic, folk and popular medicine against malaria, fever, mucous diarrhea, and venereal diseases as well as an anthelmintic and a taeniacide remedy. Its extracts and chemical constituents have been the subject of extensive phytochemical and pharmacological investigations since 1960s. Anti-inflammatory, anti-bacterial, anthelmintic, and antiplasmodial activities of plant extracts have been reported. Limonoids with anti-feeding, feeding deterrent and growth inhibitory properties, anti- fungal, and anti-sickling activity, as well as dimeric flavonoids with immunostimulating activity have been isolated from different parts of this plant. However, none of extracts or pure chemicals has been screened for biological activity such as antioxidant activity and anti-proliferative capacity against human cancer cell lines. Therefore, the main object of this study was to screen bioactive ingredients with anticancer and antioxidant activities from the plant through purification, isolation, structural elucidation, and bioassays. Eleven natural products were isolated from the methanolic extract of stem bark of Khaya senegalensis after extraction and purification, especially through crystallization and modern column chromatographic techniques using normal phase and reverse phase silica gel columns, Sephadex LH-20, MCI CHP20P, prep-HPLC, etc . Their structures were determined to be two ring D-seco limonoids 3α, 7 α-dideacetylkhivorin (1) and 1α, 3α, 7 α-trideacetylkhivorin ( 2), mexicanoloid limonoid khayanone ( 3), five khayanolide ii limonoids 1-O-deacetylkhayanolide B (4), khayanolide B (5), khayanolide E ( 6), 1-O- deacetylkhayanolide E ( 7), and novel 6-dehydroxykhayanolide E ( 8), three lignans (-)- lyoniresinol ( 9), (-)-lyoniresin-9-yl-β-D-xylopyranoside ( 10 ), and (-)-lyoniresin-4'-yl-β- D-glucopyranoside (11 ), respectively, through various spectroscopic methods including IR, EI-MS (HREI-MS), LC-ESI-MS (accurate ESI-MS), extensive 1D and 2D NMR (1H , 13 C, DEPT, 1H-1H COSY, HMQC or HSQC, HMBC, NOESY), and X-ray diffraction experiments. The structures and stereochemistry of 1, 2, 3, 4, 6, and 8 were confirmed through X- ray crystallography. Based on the X-ray diffraction analyses and NMR data, two reported khayanolides 1α-acetoxy-2β,3 α,6,8 α,14 β-pentahydroxy-[4.2.1 10,30 .1 1,4 ]-tricyclomeliac-7- oate 12 and 1α,2 β,3 α,6,8 α,14 β-hexahydroxy-[4.2.1 10,30 .1 1,4 ]-tricyclomeliac-7-oate 13 are, in fact, 1-O-acetylkhayanolide B 4 and khayanolide B 5, respectively, and two reported phragmalins methyl 1α-acetoxy-6,8 α,14 β,30 β-tetrahydroxy-3-oxo-[3.3.1 10,2 .1 1,4 ]- tricyclomeliac-7-oate 14 and methyl 1α,6,8 α,14 β,30 β-pentahydroxy-3-oxo-[3.3.1 10,2 .1 1,4 ]- tricyclomeliac-7-oate 15 are, in fact, khayanolide E 6 and 1-O-deacetylkhayanolide E 7, respectively. In the anti-proliferative bioassay, 1 and 2 showed significant growth inhibitory activities against MCF-7, SiHa, and Caco-2 tumor cells with IC 50 values in the range of 35-69 µg/ml, while other compounds did not show anticancer activity even at high concentration 200 µg/ml; 9, 10 , 11 exhibited strong antioxidant activities comparable to that of BHT, regarding the DPPH free scavenging activity. iii 23 23 22 22 F O 20 MeO O O 20 18 21 7 18 12 12 21 17 11 17 11 H OR 19 C O HO O 30 13 19 H 13 D 9 OH 14 16 6 14 16 2 1 9 8 A 10 O O 8 15 O 3 B O 15 H 4 5 10 1 1 R=Ac 5 4 30 7 OH 3 HO 6 2 R=H 2 28 3 28 23 H 29 29 22 O O 2' 7' 9' MeO 20 MeO 1' O 18 12 21 3' OH 7 17 8' 11 H 7 OR R4 13 O 1 19 4' 6' 8 R2O 6 OH 14 16 5' 9 1 9 OMe 5 8 O 2 10 15 6 4 1 30 O New compound 8 OR 28 1 MeO 5 3 OMe 29 4 2 4 R 1=Ac R 2=OH R 3=H R 4=OH OH R3 3 5 R 1=H R 2=OH R 3=H R 4=OH R2 6 R 1=Ac R 2=R 3=O R 4=OH 9 R 1=H R 2=H 7 R 1=H R 2=R 3=O R 4=OH 10 R 2=H R 1=xyl 23 8 R 1=H R 2=R 3=O R 4=H 11 R 1=H R 2=glc 22 O 20 O 18 21 12 MeO MeO 17 O O 11 7 O HO OH O H OH 13 HO 19 14 16 9 6 10 8 15 O O OR1 OH OH 5 1 OR 4 29 30 1 OH OH 2 28 3 H 12 R =Ac 14 R =Ac 1 O 1 R =H OH 13 R 1=H 15 1 iv DEDICATION This dissertation is dedicated to my parents, Baocheng Zhang and Juxiang Xie and my parents in law, Zhenglun Chen and Fengying He, my beautiful wife Qing Chen, my lovely son Bowen Zhang. They not only shared every step of my study and research progress, but also are always the source of strength, support and encouragement. v ACKNOWLEDGEMENTS At first, I would like to thank my advisor, Dr. Feng Chen, for his guidance and patience of my graduate study in Clemson University. His high expectation, encouragement, knowledge, support and mentorship are greatly appreciated. I am grateful to other committee members, Dr. Xi Wang (Department of Genetics and Biochemistry), Dr. R. Kenneth Marcus (Department of Chemistry), and Dr. Alex Kitaygorodskiy (Department of Chemistry). Their professional knowledge initiates my passion in the theory of separation science, instrumental analysis and bioassays. My special thanks go to Dr. Don VanDerveer (Department of Chemistry) for his much great help to do the X-ray diffraction analysis of the isolates, to Dr. Michael J. Wargovich (Department of Cell and Molecular Pharmacology, Hollings Cancer Center, Charleston) for the NIH grant, to Ms. Barbara Ramirez (Director of the Writing Center at Clemson University) for her help in the preparation of this manuscript. I am grateful to my colleagues Mr. Xiaohu Fan, Miss. Yen-Hui Chen, Ms. Juanjuan Yin, the visiting scholar Dr. Xiaowen Wang, Dr. Huarong Tong, for their constructive suggestions on the experiments and help on data analyses. I also appreciate Mrs. Kimberly Collins and Mr. James Findley for their generous administrative help. The help from inter-loan library is especially appreciated. I thank all other faculty and staff of the Department of Food Science and Human Nutrition for their friendship. In addition, I would express my gratitude to Dr. Liangwei Qu, Dr. Ping He, Dr. Caoxi Luo, Dr. Xiaofei Jiang, Dr. Changfeng Wu, Dr. Limei Jin, Dr. Cao Li and Dr. Feng Xu for their help in my daily life. vi TABLE OF CONTENTS Page TITLE PAGE....................................................................................................................i ABSTRACT.....................................................................................................................ii DEDICATION.................................................................................................................v ACKNOWLEDGEMENTS............................................................................................vi LIST OF TABLES..........................................................................................................ix LIST OF FIGURES .........................................................................................................x CHAPTER 1. INTRODUCTION .........................................................................................1 1.1 Botany of Khaya genus..........................................................................1 1.2 Botany and distribution of Khaya senegalensis .....................................1 1.3 Medicinal usage of Khaya senegaleneis ................................................2 1.4 Khayanolides from Khaya senegalensis ................................................3 1.5 Purpose of our study ..............................................................................4 1.6 References cited.....................................................................................6 2. PURIFICATION AND ISOLATION..........................................................11 2.1 General experimental procedures .......................................................11 2.2 Plant material ......................................................................................11 2.3 Extraction and partition.......................................................................11 2.4 Purification and isolation ....................................................................12 3. STRUCTURAL DETERMINATION OF THE ISOLATES ......................15 3.1 Introduction.........................................................................................15