"V‘wc' 1v.- tn!" va-vw- ‘ .- ... ,... .. N...“ fitus V .. a .. . 2 s. QOOI LIBRARY Michigan SW University 'W F This is to certify that the dissertation entitled BIOACTIVE CONSTITUENTS FROM PIPER METHYSTICUM FORST (KAVA KAVA) ROOTS presented by Di Wu has been accepted towards fulfillment of the requirements for Ph .D . degree in Horticulture Major professor $724 /0l Date August 21, 2001 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 chlRC/DateDuepGErp. 15 .c lltfi Eli BIOACTIVE CONSTITUENTS FROM PIPER ME TH YS TIC UM FORST (KAVA KAVA) ROOTS By Di Wu A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 2001 ABSTRACT BIOACTIVE CONSTITUENTS FROM PIPER METHYSTICUM FORST (KAVA KAVA) ROOTS By Di Wu Earlier research on Piper methysticum (kava kava) roots was focused on phytochemistry and its application as anesthetic, muscle relaxation, anti-anxiety and analgesic agents. Little work has been done on antiinflammatory, antioxidant and anticancer activities of crude extracts and purified compounds of P. methysticum roots. My research on solvent extracts of P. methysticum roots and bioassay-directed isolation and characterization resulted in several antioxidant, cyclooxygenase (COX) and topoisomerase enzyme inhibitory compounds. The isolation and characterization of these compounds were accomplished by chromatographic (MPLC, TLC and HPLC) and spectral methods (1D- and 2D-NMR, FTIR and MS). P. methysticum roots were sequentially extracted with hexane, ethyl acetate and MeOH. In another extraction, P. methysticum roots were extracted sequentially with hot water and MeOH. Bioassy- directed isolation and purification of the ethyl acetate extract yielded dihydrokawain (l), desmethoxyyangonin (2), flavokawain A (3), kawain (4), dihydromethysticin (5), yangonin (6) and methysticin (7). The COX enzyme inhibitory assay directed purification of the MeOH extract yielded a novel bomyl ester of 3,4-methylene dioxy cinnamic acid (8), bomyl ester of cinnamic acid (9), pinostrobin (10), flavokawain B (11) and 5,7- dimethoxyflavonone (12). Compound 8 is novel and 9, 10 and 12 were isolated for the first time from P. methysticum roots. At pH 7 and 100 pg mL", compounds 1-12 demonstrated cyclooxygenase-1 (COX-1) and cyclooxygenase-II (COX-II) enzyme inhibitory activities. This is the first report of COX enzyme inhibitory activities of compounds 1-12. Compounds 6 and 7 showed moderate antioxidant activities in free radical scavenging assay at 2.5 mg mL'l. Topoisomerase (top-I and -II) enzyme inhibitory and gap junctional intercellular communication (GJIC) assays were used to test anticancer properties of compounds 1, 3- 7 and 11. Compound 1 is a moderate top-II inhibitor when tested at 250 pg concentration in plate agar assays. In ras-transduced rat liver epithelial cell line, compounds 1, 3 and 4 showed growth inhibition at 287, 46.8 and 217 uM, respectively. In ras- and myc/ras- transduced rat liver epithelial cell lines, compounds 5 and 7 showed growth inhibition at 181 and 182 uM, respectively, while 5 distinctly changed cell morphology and acted as a reversible cytostatic compound. It also slightly upregulated GJIC at 181 uM in ras- transduced rat liver epithelial cells. This is the first report of potential anticancer activities of compounds 1 and 5 using topoisomerase enzyme inhibitory and GJIC assays. COX-I and —II enzyme inhibitory activities of compounds 1-12 provided scientific support for the anecdotal claims on the traditional use of kava kava roots for controlling inflammatory pain. Antiinflammatory, antioxidant and GJIC activities of isolated compounds might also help to explain the low cancer incidence in Fiji where the kava drink is consumed regularly. The isolation and characterization of antioxidant, cyclooxygenase and topoisomerase enzyme inhibitory compounds from P. methysticum roots might support the consumption of P. methysticum roots use as a healthy beverage and alternative medicine and also help to develop clinically useful products for preventing inflammation and cancers in human. To HY PARENTS M SISTER ACKNOWLEDGMENTS I express my deep gratitude to my major advisor Professor Muraleedharan G. Nair for his invaluable guidance, constant encouragement, profound instruction and rigorous training. I am grateful to his support and patience in my academic growth. Without his sincere help I would not have made so much progress in these three years. I owe special thanks to my advisory committee, Drs. James E. Trosko, Gale M. Strasburg and Robert E. Schutzki for their instruction, invaluable suggestions and encouragement. I am very grateful to be able to perform the gap junctional intercellular communication (GJIC) assays in Dr. Trosko’s Analytical Toxicology Lab and also very thankful to Drs. Brad L. Upham and Yasushi Nakamura for their instruction and assistance in carrying out GJIC assay. I owe thanks to Dr. Long Lee and Mr. Kermit Johnson for their assistance in conducting various NMR experiments. I am thankful to Dr. Kathryn G. Severin for her assistance in conducting FTIR experiments. I express genuine thanks to my fellow members, Dr. Seeram, Dr. Momin, Dr. Zhang, Dr. Kelm, Dr. Ramsewak, Dr. Balasubramanian, Dr. Henry, Dr. Wang, Laura Clifford, Robert Cichewicz, Marvin Weil and Andrew Erickson for their support and assistance. I owe heartfelt thanks to my parents, my younger sister and my close friends for their tremendous love, sacrifice and moral support. TABLE OF CONTENTS LIST OF FIGURES ............................................................................................... VIII LIST OF ABBREVIATIONS .................................................................................. X INTRODUCTION .................................................................................................... l CHAPTER-1 ............................................................................................................. 4 LITERATURE REVIEW Introduction ............................................................................................................... 5 Botanical Aspects of P. methysticum Roots ............................................................. 7 Chemical Composition of P. methysticum Roots ..................................................... 7 Pharmacology and Bioactivity of the Constituents in P. methysticum Roots .......... 15 CHAPTER-2 ............................................................................................................ 21 Cyclooxygenase Enzyme Inhibitory Compounds with Antioxidant Activities from Piper methysticum Forst (kava kava) Roots Abstract .................................................................................................................... 21 Introduction .............................................................................................................. 22 Material and Methods .............................................................................................. 23 General Experimental Procedures .......................................................................... 23 Plant Material ......................................................................................................... 24 Cyclooxygenase Enzyme Inhibitory Assay ........................................................... 24 Lipid Oxidant Assay .............................................................................................. 25 Total Free Radical Scavenging Assay Using DDPHO ........................................... 26 Extraction of P. methysticum Roots ....................................................................... 27 CD Spectral analyses of compounds 1, 4, 5 and 7 ................................................. 30 Results ...................................................................................................................... 31 Discussion ................................................................................................................ 34 CHAPTER-3 ............................................................................................................ 36 Novel Compounds from Piper methysticum Forst (kava kava) Roots and Their Effects on Cyclooxygenase Enzyme Abstract .................................................................................................................... 36 Introduction .............................................................................................................. 37 Material and Methods ............................................................................................ 38 General Experimental Procedures .......................................................................... 38 Plant Materials ....................................................................................................... 39 Extraction of Piper methysticum Roots ................................................................. 39 Cyclooxygenase Enzyme Inhibitory Assay ........................................................... 43 Results and Discussion ............................................................................................ 44 VI CHAPTER-4 ............................................................................................................ 51 Up Regulation of Gap Junctional Intercellular Communication in Oncogene Transformed Rat Liver Cells and Topoisomerase Inhibition by Compounds from Piper methysticum Forst (kava kava) roots. Abstract .................................................................................................................... 51 Introduction .............................................................................................................. 52 Material and Methods .............................................................................................. 54 Topoisomerase Enzyme Inhibitory Assay ............................................................. 54 Cell Culture ............................................................................................................ 54 Test Compounds .................................................................................................... 55 Cytotoxicity ............................................................................................................ 56 Treatment with test compounds ............................................................................. 56 GJIC Assay ............................................................................................................ 57 Results ...................................................................................................................... 58 Topoisomerase Enzyme Inhibitory Assay ............................................................. 58 Cytotoxicity ............................................................................................................ 58 Treatment with test compounds ............................................................................. 59 Discussion ................................................................................................................ 63 CHAPTER-5 ............................................................................................................ 65 Summary and Conclusion LITERATURE CITED ............................................................................................ 70 V11 LIST OF FIGURES Figure 2.1. Percentage COX-I inhibition of compounds from kava kava roots at 100 pg mL". Ibuprofen, aspirin and naproxen were tested at 2.1, 180 and 2.5 pg mL", respectively. Vertical bars represent the standard deviation of each data point (n = 2) ............................ 32 Figure 2.2. Percentage COX-II inhibition of compounds from kava kava roots at 100 pg mL". Ibuprofen, aspirin and naproxen were tested at 2.1, 180 and 2.5 pg mL", respectively. Vertical bars represent the standard deviation of each data point (n = 2) ...... 33 Figure 2.3. Kinetics of Yangonin(6)-DPPH- and Methysticin(7)-DPPH0 reactions. Control = solvent with no antioxidant. The concentration in final reaction mixtures of compound 6 and 7 was 2.5 mg mL", respectively .............................................................. 33 Figure 2.4. Comparison of DPPH- radical scavenging activities of compounds 6 and 7 with vitamin E, vitamin C, and BHT at 40 min. Vit E = vitamin E, Vit C = vitamin C, BHT = butylated hydroxytoluene, Control = solvent control containing no antioxidant. The concentrations of antioxidants in final reaction mixtures were 2.5 and 2.5 mg mL’l, 10 pM, 25 pM, and 50 pM for yangonin and methysticin, vitamin E, vitamin C, and BHT, respectively. Vertical bars represent the standard deviation of each data point (n = 3) ................................................................................................................................... 34 Figure 3.1. Significant HMBC correlations observed in compound 8 ................................ 45 VIII Figure 3.2. Fragmentation pattern of compound 8 under EIMS conditions ........................ 47 Figure 3.3. Percentage COX-I inhibition of compounds from kava kava roots at 100 pg mL'l. Ibuprofen, naproxen and aspirin were tested at 2.1, 2.5 and 180 pg mL", respectively. Celebrex and Vioxx were tested at 1.67 pg mL'l. Vertical bars represent the standard deviation of each data point (n = 3) ....................................................................... 49 Figure 3.4. Percentage COX-II inhibition of compounds from kava kava roots at 100 pg mL'l. Ibuprofen, naproxen and aspirin were tested at 2.1, 2.5 and 180 pg mL", respectively. Celebrex and Vioxx were tested at 1.67 pg mL". Vertical bars represent the standard deviation of each data point (n = 3) ....................................................................... 49 Figure 4.1. Morphological images in the ras-transformed rat liver epithelial cells treated with compound 5. (A) control (36 h exposure); (B) treated with compound 5 (36 h exposure); (C) control (64 h exposure); (D) treated with compound 5 (64 h exposure) ..... 61 Figure 4.2. Scrape loading dye transfer images in the ras-transformed rat liver epithelial cells treated with compound 5. (A) control (120 h); (B) treated with compound 5 (181 pM, 120 h); (C) treated with compound 5 (181 pM, 144 h) ............................................... 62 Figure 4.3. The effect of compound 5 on gap junctional intercellular communication (GJIC) in res-transduced rat liver epithelial cells ............................................................... 62 BHA BHT CD CHC13 cox COX-I COX-II l3CNMR DMSO d dd DDPH. DEPT DPA-PA DQFCOSY EIMS EtOAc FOC FTIR GJIC 'HNMR HMBC HMQC HPLC IR J m MeOH MOPS mp MPLC MS m/z NMR PPm PTLC LIST OF ABBREVIATIONS Butylated hydroxyanisole Butylated hydroxytoluene Circular Dichroism Chloroform Cyclooxygenase Cyclooxygenase-I Cyclooxygenase-II Carbon nuclear magnetic resonance Dimethyl sulfoxide Doublet Doublet of doublet 2,2-diphenyl-1-picryhydrazyl radical Distortionless enhancement polarization transfer 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid double quantum filter correlation spectroscopy Electron impact ionization mass spectroscopy Ethyl acetate Fraction of control Fourier transfer infrared spectroscopy Gap junctional intercellular communication Proton nuclear magnetic resonance Heteronuclear multiple bond coherence Heteronuclear correlation through multiple quantum coherence High pressure liquid chromatography Infrared Coupling constant Multiplet Methanol 3-[N-morpholino]propanesulfonic acid Melting point Medium pressure liquid chromatography Mass spectroscopy Mass-to-charge ratio Nuclear magnetic resonance Parts per million Preparative thin layer chromatography Reference value Retention time Singlet 2-thiobarbituric acid tert-butylhydroquinone Thin layer chromatography Topoisomerase-I top-II TPA YPDA [9] Topoisomerase-II 12-O-tetradecanoylphorbol- 1 3 -acetate Ultraviolet Yeast potato dextrose agar Chemical shifts Molar ellipticity (in circular dichroism) Wavelength XI INTRODUCTION Piper methysticum F orst (kava kava) is a perennial herb of Piperaceae family grown widely in the Pacific Islands. The rootstock of kava kava is commonly used to prepare a beverage for ceremonial activities by the native Pacific Islanders. Also, it is used in the traditional herbal medicine for treating gonorrhea, menstrual pain, tuberculosis, respiratory tract infections and chronic pain related to gout and arthritic conditions. Pacific islanders routinely apply the extract of kava kava roots as an analgesic and as a mouthwash for toothache and canker sores. In Europe, kava kava has been approved at the beginning of the twentieth century for the treatment of chronic inflammations of the urinary tract. Since P. methysticum roots have been used in folkloric medicine in Pacific Islands for centuries and approved for clinical use in Europe, kava kava is considered as a valuable herb and requires further studies. From early 1990’s, kava kava roots have become an important merchandise in the USA. However, it is not approved by Food and Drug Administration (FDA) for pharmaceutical use in the United States. Most of the health claims attributed to kava kava are anecdotal. Earlier research on P. methysticum root extract was primarily focused on its phytochemical constituents. The application of P. methysticum root extracts as an alternative medicine for muscle relaxation, anti-anxiety, analgesics, insomnia and antibacterial agents were also reported. However, little work has been done on antiinflammatory, antioxidant and anticancer activities of crude extracts and purified compounds of P. methysticum roots. Based on the traditional use of kava kava roots to treat arthritic and gout related pain and the inverse relationship between the consumption of kava kava beverage and cancer incidence in Pacific Island nations, my working hypothesis is that the roots of P. methysticum contain anti-inflammatory, anticancer and antioxidant agents. In general, anti-inflammatory, antioxidant and anticancer activities of compounds in kava kava roots have not been investigated in detail. A bioassay-directed identification of components of kava kava roots and detailed evaluation of these purified compounds are required to understand various health claims associated with kava kava roots. Therefore, I have conducted a bioassay-guided isolation and characterization of compounds in P. methysticum root extracts using chromatographic and spectral methods, and I have determined the efficacies of purified compounds, using antioxidant, cyclooxygenase (COX) and topoisomerase enzyme inhibitory and gap junctional intercellular communication (GJIC) assays. My dissertation depicts the biossay-directed fractionation and purification of compounds with antioxidant, topoisomerase and COX enzyme inhibitory activities. The dissertation comprises of five chapters. A literature review, discussing the past work on the chemistry and biological activitites of crude extracts and some purified compounds from P. methysticum roots, is recorded in Chapter 1. The isolation of kava lactones and flavokawin A and their COX enzyme inhibitory activities are discussed in Chapter 2. The isolation of novel compounds and their effects on COX enzymes are also detailed in Chapter 3. Chapter 2 is accepted for publication in Phytomedicine and Chapter 3 is being reviewed by Journal of Agricultural and Food Chemistry. The evaluation of anticancer properties of kava lactones and chalcones from P. methysticum roots are discussed in Chapter 4. Finally, the conclusions derived from my research on P. methysticum roots are summarized in Chapter 5. Chapter 1 Literature Review The literature on the botanical, chemistry and pharmacological aspects of crude extract and pure compounds from Piper methysticum roots is reviewed in this chapter. Previous research on biological activities of crude extract and pure compounds from P. methysticum roots has been focused on anesthetic, muscle relaxation, analgesic, anti- anxiety, antibacterial, anti-fungal, anti-thrombotic, anti-stress and sedative activities. Little work on anti-inflammatory, antioxidant and anticancer studies of P. methysticum roots has been reported. Introduction Kava kava is a native name given to the plant Piper methysticum Forst and to a beverage prepared from the rhizome of this plant. It is also called ava, awa, kawa, yaqona, kava, kawa-kawa or wati in Pacific Islands (Keller et al., 1963). Piper, the generic name, came from the Latin for pepper and methysticum, the species name, originated from the Greek “methustikos” for intoxicating drink (Singh, 1992). Kava kava is consumed as an intoxicating and ethnopharmacological herbal drink and as a dietary supplement around the world. There are several versions of myths and legends on the origin of the plant and the beverage prepared from its roots (Singh et al., 1997). Since Captain James Cook first described the native use of kava kava drink in his travelogue to the South Seas in 1768, kava kava has received intensive attention and investigation around the world for more than two centuries (Keller et al., 1963). The ceremonial preparation and everyday use of kava kava beverage play an important role in social, economic and religious life of Pacific Islanders. Its cultural status in the Pacific Islands can be compared with that of wine in Southern Europe. Most of the Pacific Islands had built up the tradition of drinking kava kava since the beginning of recorded history of the region. The rituals employed for its preparation and drinking of kava kava beverage among participants reveal the historical and ethnic features of the island communities of the Pacific Islands (Singh et al., 1997). The part of P. methysticum plant used for the preparation of kava kava drink is its rootstock. The kava drink was historically prepared by two different methods, either by chewing or by pounding. Chewing, also called “Tonga method”, required young men or women to chew the root until it was fine and fibrous. Then, the masticated kava roots were immersed in water, decanted, and imbibed as an infusion. Pounding or grating, also called “Fiji method”, consisted of stone (now mechanical) pounding and extraction of the powder with water. The fibrous residue was strained and the resulting extract was served (Shulgin, 1973). It was reported by Lewin (1886) and Van (1938) that chewing broke up the root and emulsified the resin containing active compounds and facilitated its extraction into water. Schubel (1924) attributed that the chewing induced an increase in potency of kava kava extract due to enzymatic breakdown of starch in the root which then resulted into a more efficient extraction of active compounds. Colonial and missionary influences led to the abandonment of the chewing of kava kava root to prepare the kava kava drink in favor of pounding (Singh, 1992). There were various reports on the taste of kava kava infusion. In one such report, it was stated as “pleasant, cooling, aromatic, numbing effect in the mucous membrane of the tongue, while others told to the world the bitterness and burning taste in the mout .” (Emerson, 1903). Kava kava also produced different physiological effects after drinking. It was mentioned that someone could experience a decreased fatigue and anxiety, and a pleasant cheerful feeling immediately after the kava drink. Hocart described it as follow, “It gives a pleasant, warm and cheerful, but lazy feeling, sociable, though no hilarious or loquacious, the reason is not obscured” (Hocart, 1929). In Pacific Islands, kava kava is considered as a healthy beverage if consumed properly. Also, it is considered as a traditional medicine. For example, a decoction of rootstock was applied to treat gonorrhea and was consumed as a remedy for menstrual problems, tuberculosis, sleeping problems and respiratory tract infections (Leung et al., 1996). Kava kava was reported as a good remedy for chronic pain, reducing sensitivity and relaxing tensed muscles (Chevallier, 1996). Other beneficial effects attributed to kava kava were an analgesic and diuretic effect and for treating rheumatic and arthritic problems and as a mouthwash for toothache and canker sores. Also, the relaxing pr0perty of kava kava beverage can alleviate anxiety (Chevallier, 1996; Leung et al., 1996). Botanical Aspects of P. methysticum The kava kava plant, P. methysticum Forst, is a perennial and deciduous shrub grown up to 2.5 m in height with fairly succulent leaves and well branched. The leaves are thin, smooth, cordate, alternate, petiolate (petioles to 6 cm long), 8-25 cm wide and green on both sides. The flowers are in irregular spadices. Rootstock is knotty, 5-8 cm thick at maturity (3-5 years afier planting), sometimes tuberous, with lateral roots up to 3 m in length (Lebot et al., 1992). Rootstocks yielded voluminous root mass under cultivation. Average fresh weight of kava kava root system at 10 months from planting is about 1 kg. The kava kava plant grows throughout the South Pacific from Hawaii to New Guinea, all the islands groups of Oceania, with the exception of New Zealand, New Caledonia, and most of the Solomon Islands. However, the botanic origin of kava is unknown. It is interesting to note that almost 72 different varieties of P. methysticum were reported from Vanvatu archipelago, one of the Pacific Islands (Lebot et al., 1992). Chemical Composition of P. methysticum roots Kava rootstock consists of 43% starch, 20% fiber, 12% water, 3.2% sugars, 3.6% proteins, 3.2% minerals and 3-20% of kava lactones on a dry weight basis. However, these numbers may vary depending on the age and cultivar of the plant. Fresh rootstock contains 80% of water on an average (Lebot et al., 1992). The chemical study of P. methysticum has been conducted since 1860 (Gobley et a1, 1860; Cuzent, 1861). a-pyrones, chalcones and several other compounds, such as alkaloids, ketones and flavones, were purified and elucidated from kava rhizome (Lebot etaL,1992) The kava lactones present in the resin are a-pyrones bearing a C4-methoxyl and C6-styryl moieties. An unsaturated lactone ring connected by a double or single bond with a benzene ring constitutes the basic l3-carbon back bone of kava lactones. There are numerous kava lactones identified from kava kava roots. Methysticin is the first compound isolated as a white, crystalline and neutral compound from P. methysticum (Gobley et al., 1860; Cuzent, 1860). Previously it was called kawahin, kawakin, kavatin or kanakin. This compound was obtained in pure form and was assigned the molecular formula as C15H1405 (Pomeranz, 1889). Seventy years later, dl-methysticin was synthesized and the complete structure of the methysticin was elucidated by Klohs et a1. (1959). OMe \ \ o 0 0 O \_—o methysticin A crystalline compound, yangonin, was first isolated in 1874 (Nolting et al., 1874) and named as yangonin by Lewin (1886). Yangonin was considered as an y-pyrone initially (Borsche et al., 1930) and assigned the correct structure as a-pyrone later (Macierewicz, 1950). A decade later, the same conclusion was reached by the polish workers by synthetic methods (Herbst et al., 1960). OMe yangonin A white neutral compound from kava kava extract was identified as a mixture of methysticin and dihydromethysticin by Borsche and Peitzsch (1929). The presumption was confirmed with the evidence that dihydromethysticin was produced by the hydrogenation of methysticin (Borsche, 1929a). OMe \ O 0 Lo dihydromethysticin The compounds, obtained from the saponification of kava kava resin after having isolated methysticin, dihydromethysticin and yangonin by crystallization, were studied by Borsche et al. (1929b). The presence of kawain in kava kava resin was confirmed by comparing it with a synthetic sample. Also, the reinvestigation of kava kava resin yielded kawain and dihydrokawain (Borsche et al., 1929b). dl-Kawain was synthesized by different methods in 1950 (Kostermans, 1950; Fowler et al., 1950). Reformatsky condensation of cinnamaldehyde with ethyl-4-bromo- 3-methoxy-crotonate was employed to synthesize dl-kawain (Kostennans, 1950). dl- Dihydrokawain was synthesized by replacing cinnamaldehyde by dihydrocinnamaldehyde following Kostermans’ procedure for the synthesis of dl-kawain (Viswanathan et al., 1960). Dihydrokawain was also obtained from kava kava extract by column chromatography using an acid washed alumina as the adsorbant (Van, 193 8). OMe \ \ O O Kawain Desmethoxyyangonin, also called 5,6-dehydrokavain, was purified from the chloroform extract of kava roots (Klohs et al., 1959). Also, ll-methoxyyangonin and 6- dehydromethysticin were isolated in 1962 (Mors et al., 1962). 10 \ \ | O desmethoxyyangonin 1 l-methoxy-yangonin A total of 15 lactones from P. methysticum rhizome were published by Hansel (1968). The major compounds found in the kava rootstock were yangonin, methysticin, dihydromethysticin, kawain, dihydrokawain and desmethoxyyangonin. The minor constituents reported were: 5,6-dehydro-methysticin, ll-methyoxyyangonin, cis-S- hydroxykawain, ll-hydroxyyangonin, IO-methoxyyangonin, 7,8-dihydroyangonin, 5,6- dihydroyangonin, 1l-methoxy-l2-hydroxydehydrokawain and 5,6,7,8-tetrahydro- yangonin (Duve, 1981). Me dehydromethysticin cis-S-hydroxykawain ll Me OMe \ \ I I MeO MeO OMe OH 1 l-hydroxyyangonin 10-methoxyyangonin OMe OMe I \ \ O 0 0 O \ 0 0 MeO MeO 7,8-dihydroyangonin 5,6-dihydroyangonin OMe OMe I \ \ HO M O OMe e 1 l-methoxy-l2-hydroxydehydrokawain 5,6,7 ,8-tetrahydroyangonin Two yellow compounds, flavokawain A and flavokawain B, were isolated from kava kava resin using thin layer chromatography (Hansel et al., 1963). Flavokawain C was isolated from P. methysticum roots and synthesized by Dutta et al. (1976). 12 The presence of alkaloidal compounds in P. methysticum was known from as early as 1889 (Lavialle, 1889). However, attempts to separate these compounds were not successful. An alkaloid present in kava kava leaves was named as pipermethystin (Smith, 1979). However, it was very unstable and decomposed during chromatographic purification. OMe O OMe . O / O O / O MeO OH Me MeO OH flavokawain A flavokawain B OMe 0 0 l? 0 / O UOCMe O N flavokawain C pipermethystin Cepharadione A, a highly fluorescent and orange-colored compound, was isolated by Gerken (1974) as a trace component from P. methysticum. The structural elucidation of this compound was achieved by Jaggy et al. (1992). 13 cepharadione A Two other alkaloids, l-cinnamoylpyrrolidine and 1-(m-methoxycinnamoyl)- pyrrolidine, were isolated from the MeOH extract of kava kava roots by Achenbach (1970). In addition, dihydrokavain-S-ol, an alcohol, was identified by NMR, MS, UV, IR and CD spectral methods (Achenbach, 1970). Two cinnamyl derivatives, cinnamylideneacetone and 3,4-(methylenedioxy)cinnamylideneacetone were also identified from kava kava rhizome (Jossang et al., 1967). dihydrokavain-S—ol cinnamylideneacetone Me () \.—() 3,4-(methy1enedioxy) cinnamylideneacetone In recent years researchers have established and validated rapid quantitative analytical methods for the analysis of kava lactones. Gas chromatography and methane chemical ionization mass spectrometry were employed to analyze the constituents of P. methysticum (Duffield et al., 1986). Electrospray HPLC-MS and supercritical fluid extraction techniques were successfully applied to separate and quantitatively determine kava pyrones from the root of the plant (He et al., 1997; Avila et al., 1997). The enantiomers of kava pyrones were quantified by HPLC using a chiral column (Boonen et aL,1997) Pharmacology and Bioactivity of the constituents in P. methysticum roots In 1886, Lewin published the first pharmacological evaluation of kava pyrones. In this study, the kava kava resin remaining after the crystallization of methysticin and yangonin showed local anesthetic action on frogs (Lewin, 1886). Kava kava resin paralyzed sensory nerves by stimulation followed by paralysis of smooth muscles. Also, the hydrolysis of the crude extract led to similar actions as in the case of crude extract (Schubel, 1924). The local anesthetic action of kava kava extract was attributed to compounds containing benzoic and cinnamic acids moieties. Both dihydrokawain (DHK) and dihydromethysticin (DHM) were found to be active and caused sleep in white mice and white rats when orally administered as an emulsion using a stomach tube (Hansel et al., 1959). DHM and DHK were implicated to have sedative effect when administered intraperitoneally or orally in mice, rats, rabbits and cats (Meyer et al., 1960). The mechanisms by which kava kava extract caused muscle paralysis were suggested to be similar in action to local anesthetics (Singh, 1983). Glietz et al. (1995) supported this point when they found that kawain blocked the voltage-dependent sodium ion channel similar to local anesthetics. The sedative action of kava lactones was considered to occur through their effects on the GABA (gamma aminobutyric acid) receptor binding site (Jussogie et al., 1994). However, interaction between kava lactones and GABA was not observed (Davies et al., 1992). Meyer’s team (1964) tested the analgesic effect of DHK and DHM. The results showed that these compounds were comparable to dimethylaminophenazone, an analgesic drug. The analgesic effects of DHK and DHM (120 mg kg") were comparable to 200 mg kg”1 body weight of aspirin. The lactones from kava kava tested showed significant action on central nervous system in producing muscular relaxation in all species of laboratory animals studied (Meyer, 1962). They were also proved to be more effective than mephenesin against convulsions and strychnine-induced death in mice (Meyer, 1966). The crude extract of kava kava roots, methysticin and DHM were effective in protecting the mice from the lethal effects of strychnine, while yangonin and dihydroyangonin (DHY) showed no 16 effect (Klohs et al., 1959). The muscle-relaxing activity in mice was not due to the inhibition of neuromuscular transmission but was a direct effect on muscle contraction (Singh, 1983). Antibacterial assays revealed that kawain, dihydrokawain, tetrahydroyangonin, methysticin, yangonin, dihydromethysticin, desmethoxyyangonin, flavokawain A and flavokawain B, did not inhibit the growth of a large number of Gram-positive, Gram- negative, pathogenic and non-pathogenic bacteria (Hansel et al., 1966). However, kawain and DHK effectively inhibited the growth of the fungus Aspergillus niger. The pyrones from kava kava had a selective effect on certain species of fungi and they did not inhibit the growth of yeasts at all (Hansel et al., 1966). Flavokawain C showed very little antibacterial activity against Salmonella typhi, perhaps due to the presence of a free phenolic group in ring B (Som etal., 1985). Kava kava has been classified as a narcotic and hypnotic. However, studies revealed that it is neither hallucinogenic nor stupefying, is nonaddictive, and does not cause dependency (Shulgin, 1973). DHM showed the greatest effect on barbituric narcosis, while all of the a-pyrone compounds increased barbiturate-induced sleeping time (Klohs et al., 1959; Meyer, 1962). Synergistic activity of kava pyrones was observed by Meyer (1967). In combination with other constituents in kava roots, yangonin and desmethoxyyangonin showed considerably higher activity in mice against maximal electroshock seizure (Meyer, 1967). The highest concentrations of kawain and DHK administered were very effective in protecting mice from maximal electroshock seizure (Keledjian et al., 1988). In order to test the effects of kava drink on alertness and speed of access of information in long-term memory, a study was conducted among the native people who consumed kava drink at normal or higher dose than the social consumption. The results revealed that kava kava had no effect on reaction times among the native drinkers tested (Russell et al., 1987). No changes in visual, stereo acuity or in ocular refractive error were found in a study on the visual effect by drinking kava kava (Garner et al., 1985). The pharmacological effects of kava kava were considered to be mainly due to the lipid-soluble compounds in kava resin (Duffield et al., 1989). The water-soluble fraction of kava kava roots, relatively free from pyrones, also showed strong physiological activity such as muscular relaxation and anti-serotonin activity (Buckley et al., 1967). However, most researchers attribute the pharmaceutical effects of kava kava to the compounds present in the lipid soluble fraction of the resin. A long-term clinical trial involving 101 patients suffering from anxiety and tension was conducted with kava kava extract, WS 1490, which contained kawain, dihydrokawain, methysticin, dihydromethysticin, yangonin and desmethoxyyangonin (Volz et al., 1997). The study demonstrated that the short-term (first eight weeks) and long-term (twenty five weeks) effectiveness of kava kava were both superior to that of a placebo against depression and anxiety. Adverse reactions from the consumption of kava kava extracts were not observed among the patients. The results from similar studies on kava kava extract were consistent with this study (Kinzler E., 1991; Lehmann et al., 1996) Heavy usage of kava kava has been reported to have adverse effects based on comparing health status among thirty-nine kava kava users and thirty-four non-users in a 18 coastal aboriginal community in Arnhem Land (Mathews et al., 1988). The adverse health conditions included a typical scaly rash, slightly increased patellar reflexes, haematuria, increased levels of y-glutamyl transferase and high-density lipoprotein cholesterol, decreased levels of albumin, plasma protein, urea and bilirubin. One of the side effects reported caused by heavy kava kava consumption was skin darkness. Shulgin (1973) proposed that the skin discoloration might be due to the accumulation of chalcone pigments, flavokawain A, B and C. However, no scientific findings have been published to support the proposal. In Europe, kava kava was used for the treatment of chronic inflammation of the urinary tract at the beginning of the twentieth century (Mors et al., 1962). The results of a clinical trial conducted in Germany revealed that kawain is as effective as benzodiazepene in relieving anxiety, stress and unrest (Chevallier, 1996). In 1990, the Federal Board of Health in Germany approved kava kava for treating anxiety disorders (Sahelian, 1998). Kawain was identified to have antithrombotic action on human platelets (Gleitz et al., 1997). It was also shown to prevent the formation of prostaglandin E2 and thromboxane A2 by inhibiting the action of cyclooxygenase and thromboxane synthase enzymes (Gleitz et al., 1997). Combined with pumpkin seed oil, kava kava extracts have been used in the treatment of irritable bladder syndrome. Contraindication of the treatment is applied to pregnancy, lactation and depression. The German monograph on the plant also notes that the chronic use may cause temporary yellow discoloration of the skin, hair and nails or rare allergic skin reactions. Medicinal preparation of kava kava might interact with alcohol, barbiturates and other psychopharmaceuticals or interfere l9 with the operation of machinery or vehicles (Leung, 1996; Chevallier, 1996). In USA. the pharmacological effect of kava kava is not known or studied (Leung, 1996). Several epidemiological studies of cancer incidence in the Pacific Islands showed low rate of many cancers. Although a high percentage of the population have smoking habit, the cancer incidence in Fiji, especially the lung cancer, was lower in the 1970’s. Also, some cancers which are common in the developed countries were rarely observed in Fiji (Reed, 1977; Henderson et al., 1985; Steiner, 2000). These included lung, stomach and colon-rectal cancers (Reed, 1977; Henderson et al., 1985; Steiner, 2000). The preliminary research conducted by a Japanese group showed that desmethoxyyangonin significantly inhibited the release of TNFa in BALB/3T3 cells which is an endogenous tumor promoter and a central mediator of cancer development (Fujiki, 1999). Kava kava, consumed as a daily beverage in Fiji, might play a role in cancer prevention as an alternative medicine (Fujiki, 1999). A study was conducted to compare the cancer incidence among a number of Pacific Island nations which consume kava beverage (Steiner, 2000). The data revealed that there was a close inverse relationship between cancer incidence and kava consumption (Steiner, 2000). 20 Chapter 2 Cyclooxygenase Enzyme Inhibitory Compounds with Antioxidant Activities from Piper methysticum Forst (kava kava) Roots* ABSTRACT- Cyclooxygenase enzyme inhibitory assay-guided purification of the ethyl acetate extract of Piper methysticum Forst (kava kava) roots yielded six biologically active compounds (1-7), which were purified using MPLC, preparative TLC and HPLC methods. These compounds were also evaluated for antioxidant activities. Dihydrokawain (1) and yangonin (6) showed the highest COX-1 and COX-II inhibitory activities at 100 pg mL", respectively. The lipid oxidation assay did not reveal antioxidant activities for dihydrokawain (1), desmethoxyyangonin (2), kawain (4), dihydromethysticin (5) and methysticin (7) at 50 pg mL". The antioxidant activities of flavokawain A (3) and yangonin (6) could not be tested in lipid oxidation assay due to solubility problems. However, yangonin and methysticin showed moderate antioxidant activities in free radical scavenging assay at 2.5 mg mL". * Phytomedicine, in press. 21 INTRODUCTION Piper methysticum Forst (kava kava) is a herb consumed as a drink and used in traditional medicine by many people in the Pacific Islands (Keller et al., 1963). Kava kava is considered as a healthy beverage (Singh, 1992) and is also reported as a good remedy for treating gonorrhea, menstrual pain, tuberculosis, sleeping problems, respiratory tract infections, chronic pain related to gout and arthritic conditions (Singh, 1992; Singh and Blumenthal, 1997). Other beneficial effects of kava kava reported include analgesic and diuretic effects, relaxation of muscle tension and alleviation of anxiety (Singh, 1992; Singh and Blumenthal, 1997). Pacific islanders use kava extract as an analgesic and as a mouthwash for toothache and canker sores (Singh and Blumenthal, 1997). The rootstock of kava kava is traditionally used for the preparation of kava drink by brewing the roots in water at room temperature and as a medicine (Singh, 1992; Singh and Blumenthal, 1997). Prostaglandin endoperoxide H synthase (PGHS) or cyclooxygenase (COX) enzymes have been used widely to evaluate the anti-inflammatory activities of natural products (Goda et al., 1992). It is hypothesized that one of the mechanisms in which non- steroidal anti-inflammatory drugs (N SAIDs) control inflammation is that NSAIDs inhibit the synthesis of prostaglandin, an inflammatory mediator produced at the site of tissue injury, by inhibiting COX-1 and -II enzymes responsible for converting arachidonic acid to prostaglandins. Prostaglandins mediate inflammation through which nerves are sensitized to painful stimuli (Winzeler and Rosenstein, 1998). COX enzymes exist in two isoforrns, COX-1 and COX-II. The COX-II enzyme is considered to be the principal isoforrn that participates in inflammatory process in the body (Cryer and DuBois, 1998). 22 The analgesic effectiveness of dihydrokawain and dihydromethysticin at 120 mg kg'l was reported to be equivalent to aspirin at 200 mg kg'1 body weight (Lebot et al., 1992). However, no study on mechanisms by which kava extract or kava lactones work against inflammation in the body was reported. In order to verify the anecdotal claims that kava extract has numerous phytoceutical benefits, we have investigated kava kava roots for cyclooxygenase inhibitory and antioxidant compounds. MATERIALS AND METHODS General Experimental Procedures 1H-NMR spectra were recorded at 300 and 500 MHz, respectively, l3C-NMR spectra were recorded at 126 MHz in CDCl3, and values were presented in 8 (ppm) based on residual 8 value of CHC13 at 7.24 ppm. Coupling constants J are in Hz. CD spectra were recorded on JASCO J710 Spectropolarimeter. Medium pressure liquid chromatography (MPLC) was carried out on silica gel 60 and C13 (25-40 pm). Preparative TLC was conducted over silica gel GF glass plate (20 x 20 cm, 250 or 500 pm thickness, Analtech, Newark, Delaware). Bands were viewed under UV light (254 and 366 nm). Positive controls naproxen, ibuprofen and aspirin for the COX enzyme inhibitory assays, tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) for lipid oxidation assays and 2,2-diphenyl-l- picryhydrazyl radical (DPPHO), vitamin E and vitamin C for free radical scavenging assay were purchased from Sigma Chemical Company. All solvents were ACS reagent grade and purchased from Aldrich Chemical Company. 23 Plant Material Dried kava kava roots were purchased from Meetex Fiji Ltd., Fiji. The roots were stored at -20°C in plastic shipping bags prior to extraction. The roots were milled using Thomas-Wiley Laboratory Mill Model 4 (Thomas Scientific, USA, 2mm filter) prior to extraction. Cyclooxygenase Enzyme Inhibitory Assay This assay is based on measuring cyclooxygenase enzyme activities by monitoring the rate of 02 uptake using an oxygen electrode YSI Model 5357 (INSTECH Laboratory, Plymouth Meeting, PA) (Meade et al., 1993; Wang et al., 2000). The assay mixture consisted of 600 pL of 0.1 M Tris-lmM phenol buffer (pH 7.0), 17 pg hemoglobin and 10 pM arachidonic acid. Cyclooxygenase I (COX-I) enzyme was prepared from ram seminal vesicles purchased from Oxford Biomedical Research, Inc., Oxford, MI. COX-I enzyme solution was prepared by dissolving 0.46 mg of protein/mL in 30 mM Tris buffer (pH 7.0) (Wang et al., 2000). Cyclooxygenase II (COX-II) enzyme was isolated from PGHS-II cloned insect cell lysate and diluted with Tris buffer (pH 7.0) till the concentration was 1.5 mg of protein/mL (Wang et al., 2000). Reactions were initiated by adding 5-30 pg of microsomal protein in a volume of 10-20 pL of Tris-buffer in the assay mixture contained in a 600 pL microchamber. Instantaneous inhibition of COX enzymes was determined by measuring the cyclooxygenase activity at 37 °C controlled by a circulation bath (Model- 1166, VWR Scientific Products, Chicago, IL). The enzyme activity was monitored by a Biological Oxygen Monitor and the data were collected using Quicklog data acquisition 24 and computer software (Strawberry Tree Inc., Sunnyvale, CA, USA). Finally, the data were transformed into Microsofi Excel. The assay was conducted in duplicate. Lipid Oxidation Assay The lipid oxidation assay was conducted by detecting liposome oxidation using fluorescence spectroscopy. A mixture containing 5 pmol of 1-stearoyl-2-linoleoyl-sn- glycerol-3-phosphocholine (Avanti Polar Lipids, Inc., Alabaster, AL) and 5 pmol of the fluorescence probe 3-(p.(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (Molecular Probes, Inc., Eugene, OR) in CHC13, was evaporated under vacuum pressure. The resulting lipid film was suspended in 500 pL of a solution containing NaCl (0.15 M), EDTA (0.1mM), MOPS (0.01M), and exposed to ten freeze-thaw cycles using a dry ice/ethanol bath. The liposomes in buffer was then treated with chelating resin (Chelex 100) to remove trace metal ions. The lipid-buffer suspension was extruded 29 times through a LiposoFast extruder (Avestin, Inc., Ottawa, Canada) containing a polycarbonate membrane with a pore size of 100 nm to produce unilamellar liposomes. A 20 pL aliquot of this liposomal suspension was diluted to 2 mL with chelex 100-treated HEPES buffer (100 pL, pH 7.0), 1M NaCl (200 pL), deionized water sparged with N2 (1.64 mL) and DMSO solution with or without test compounds (20 pL). The liposomal suspension was incubated for 5 min at room temperature, and then incubated at 23°C in the cuvette holder of a Turner Model 450 Digital Fluorometer (Barnstead Thennolyne, Dubuque, IA). Peroxidation was initiated by the addition of 2 mM FeClz solution (20 pL) to achieve a final concentration of 20 pM of Fe2+ in the absence or presence of test compounds or crude extracts. Controls contained no test compounds. Fluorescence 25 intensity was measured at an excitation wavelength of 384 nm for every 3 min over a period of 21 min. The positive controls BHA, BHT and TBHQ were tested at 10 pM in 2 mL of final solution. The decrease of fluorescence intensity with time indicated the rate of peroxidation (Arora and Strasburg, 1997). Total Free Radical Scavenging Assay Using DPPH- Total free radical scavenging capacities of kava kava compounds were determined and compared to vitamin E, vitamin C, and BHT according to the previously reported procedure using stable 2,2-diphenyl-1-picryhydrazyl radical (DPPHO) (Blois, 1958; Chen and Ho, 1997). An ethanolic solution of DPPH- (800 pL) was added into a DMSO solution (800 pL) of test compound to start the reaction. Absorbance of each reaction mixture at 517 nm was measured against an ethanol control at O, 5, 10, 20, 40, and 80 min, respectively. The percent DPPH- radical remaining at each time point was calculated using a DPPH- standard curve. A preliminary evaluation of the free radical scavenging assay revealed that only compounds 6 and 7 were active. Therefore, the kinetics of methysticin(6)-DPPHO and yangonin(7)-DPPHO reactions were determined by plotting the percent of unreacted DPPH- against time for each compound tested. The concentration of compounds 6 and 7 in the reaction mixtures was 2.5 mg mL'l, respectively, while the concentration of DPPH- was 100 pM. The test concentrations for vitamin C, vitamin E, and BHT were 25, 10 and 50 pM, respectively. Reactions were carried out in triplicate for each antioxidant tested. 26 Extraction of P. methysticum Roots The ground roots (749g) were extracted sequentially with hexane (3 x 1.96 L, 72h), ethyl acetate (3 x 1.2 L, 72h) and methanol (3 x 0.25 L, 72h). The ethyl acetate extract (1.46 g) showed the highest cyclooxygenase inhibitory activity and was further fractionated by MPLC on silica gel using hexane-acetone (10:1, 110 mL; 6:1, 350 mL; 3:1, 660 mL; 1:1, 400 mL) followed by 100% acetone (140 mL) and MeOH (100 mL). The fractions collected were: hexane-acetone, 3:1; A (75mL, 5.7mg), B (210 mL, 18.6 mg), C (180 mL, 6.4 mg), D (120 mL, 22.1 mg), E (75 mL, 0.9 mg); hexane-acetone 1:1; F (195 mL, 317.8 mg), G (60 mL, 458.7 mg), H (30 mL, 109 mg), I (90 mL, 362 mg); 100% acetone, J (30 mL, 15.5 mg), K (75 mL, 20.2 mg), L (30 mL, 8.8 mg) and 100% MeOH, M (100 mL, 84.6 mg). The cyclooxygenase inhibitory assay on fractions A-M revealed that fractions D-I were the most active. Compounds 1-3. Fraction F (150.9 mg) was purified by preparative TLC (hexane-ethyl acetate 4:1) to yield two major bands, I (36.5 mg, Rf 0.8) and II (84.4 mg, Rf 0.6), respectively. Both bands were active when tested for COX-I and COX-II inhibitory activities. Band H was further purified by repeated preparative TLC (hexane-ethyl acetate 3:1) and C13 TLC (methanol-water 7:3) followed by crystallization to yield compound 1 (67 mg, mp 55-56 °C). Spectral analysis confirmed that compound 1 was identical to (+)- dihydrokawain (Spino et al., 1996). Compound 2 (22.1 mg, mp 137-139 °C) was obtained after crystallization of Band I. 1H- and l3C-NMR analyses confirmed that compound 2 was identical to desmethoxyyangonin (Itokawa et al., 1981). Fraction D yielded pure compound 27 3 (22.1 mg) and its NMR analysis confirmed that compound 3 was flavokawain A (Shukla et al., 1973; Parmar et al., 1990). Compounds 4-6. Fraction G (458.7 mg) was dissolved in MeOH to yield MeOH-soluble (415.5 mg) and MeOH-insoluble (43.1 mg) fractions. The MeOH solution was further precipitated with ACN-Hzo (60:40 v/v), centrifuged and the supernatant was evaporated under vacuum. The residue from the supernatant (333 mg) was fractionated by C13 MPLC uSing ACN-HZO gradient and followed with 100 % ACN. The fractions collected were: ACN-HZO, 60:40; 1 (300 mL, 262.2 mg), 2 (60 mL, 7.9 mg), 3 (50 mL, 6.1 mg), 4 (50 mL, 10.6 mg), 5 (30 mL, 14.8 mg); ACN-H20, 70:30; 6 (90 mL, 17.7 mg), 10 (50 mL, 2.8 mg), 28 11 (300 mL, 2.4 mg). The fraction 1, active in cyclooxygenase inhibitory assay, was further purified by preparative HPLC, on two Jaigel S-343-15 ODS columns in tandem, under gradient conditions using MeOH-ACN-HZO (20:20:60) to 100% ACN at a flow rate of 3.5 mL/min and detected at 246 nm. This yielded compound 4 (46.3 mg, R, 98.27 min, mp 108-110 °C) and compound 5 (3 mg, R 94.32 min, mp 116-118 °C). NMR and CD spectral analyses revealed that compounds 4 and 5 were identical to (+)-kawain (Klohs et al., 1959; Abramson et al., 1981) and (+)—dihydromethysticin (Shao et al.,1998; Franca et al., 1973), respectively. The fractions 4 and 5, also active in cyclooxygenase inhibitory assay, were combined and purified by preparative TLC using CHCl3 as the mobile phase to yield compound 6 (13.2 mg, mp 152-153 °C). NMR data indicated that compound 6 was identical to yangonin (Shao et al., 1998). The ACN-H20 (60:40)-insoluble portion of fraction G (69.9 mg) and the MeOH-insoluble fraction of the fraction G (43.1 mg) were purified by preparative TLC with hexanezethyl acetatezether (3:0.524) and CHCI3, respectively, followed by crystallization to yield the additional supply of compound 6 (68.1 mg). The fraction H (109 mg) was purified by preparative TLC with CHC13, CHCl3zethyl acetate (16:0.5), hexanezethyl acetate (2:1) and hexanezacetone (4:1), respectively, and the resulting product was crystallized with hexane-acetone to yield an additional supply of compound 5 (44.8 mg). 29 CH3 6 Compound 7. The fraction I (100.6 mg) was purified by preparative TLC with hexanezacetone (4:1) and hexanezetherzacetone (4:1221). The resulting band (Rf 0.6) was eluted with CHCl3 and the residue was crystallized to yield compound 7 (33.5 mg, mp 138- 139 °C). NMR and CD spectral data of compound 7 showed that it was identical to (+)- methysticin (Shao et al., 1998; Dutta et al., 1972). CD Spectral Analyses of Compounds 1, 4, 5 and 7. Compounds 1, 4, 5 and 7 for CD spectral analysis were dissolved in MeOH at 0.25, 3.4, 0.3 and 0.6 mg mL", respectively and measured at 20°C. The CD spectra of compounds 1, 4, 5 and 7 gave a positive Cotton 30 effect at 262.9, 299.6, 263.6 and 332 nm with CD values of 42.5, 0.64, 12.14 and 1.14 mdeg, respectively. RESULTS The kava kava roots purchased from Meetex Fiji, Ltd. was a mixture of fibrous and tap roots. The purification of the ethyl acetate extract using various chromatographic techniques yielded 9.7, 3.2, 1.5, 3.2, 3.5, 4.9 and 2.3% of dihydrokawain, demethoxyyangonin, flavokawain A, kawain, dihydromethysticin, yangonin and methysticin, respectively. Both COX-I and COX-II enzymes were used in the bioassay-directed isolation of potential anti-inflammatory compounds present in kava kava roots. Positive controls used for cyclooxygenase inhibitory assays were ibuprofen, aspirin and naproxen and were dissolved in DMSO at their ICso concentration of 2.1, 180 and 2.5 pg mL", respectively. In COX-I inhibitory assay, ibuprofen, naproxen and aspirin showed 25, 32 and 27 % inhibition, respectively. Ibuprofen, naproxen and aspirin gave 21, 28 and 16 % inhibition, respectively, in COX-II inhibitory assay. The concentrations of each test compound assayed were 100, 50, 25 and 12.5 pg mL'1 at pH 7.0 for both COX-1 and -II inhibitory assays. The percentage of inhibition of cyclooxygenase activities for each compound at 100 pg m1.“ are shown in Figures 2.1 and 2.2. Compounds 1-7 showed 58, 39, 33, 34, 25, 36 and 42 % inhibition, respectively, in COX-I inhibitory assay. Similarly, they gave 28, 23, 15, 25, 32, 34 and 26 % inhibition, respectively, in COX-II inhibitory assay. The lipid oxidation assay did not show antioxidant activities for compounds 1, 2, 4, 5 and 7. The addition of compound 3 to the lipid oxidation assay mixture made the solution 31 opaque and hence the fluorescent data of the solution could not be determined by fluorescence spectroscopy. Compound 6 also precipitated when added into the buffer containing the liposomal suspension. However, compounds 6 and 7 showed free radical scavenging capacity against stable DPPH radicals (Figures 2.3 and 2.4). Compounds 6 and 7 showed 64.7 and 64.3 % of DPPH- radical scavenging activities at 2.5 mg mL", respectively, compared to 14.3, 8.3 and 31.7% of DPPH. radical scavenging activities for vitamin E, vitamin C and BHT at 10, 25 and 50 pM, respectively. The kinetics of yangonin(6)-DPPH and methysticin(7)-DPPH reactions gave the similar activity profile for compounds 6 and 7 at 2.5 mg mL'l. Figure 2.1. Percentage COX-I inhibition of compounds from kava kava roots at 100 pg mL’l. Ibuprofen, aspirin and naproxen were tested at 2.1, 180 and 2.5 pg mL", respectively. Vertical bars represent the standard deviation of each data point (n = 2). 70 C O E a .c m .5 1...: z j O :' 0 :7: g '. 32 Figure 2.2. Percentage COX-II inhibition of compounds from kava kava roots at 100 pg mL". Ibuprofen, aspirin and naproxen were tested at 2.1, 180 and 2.5 pg mL'l, respectively. Vertical bars represent the standard deviation of each data point (n = 2). % COX-ll lnhlbltlon 20* 151 , 1o? . 5. or - s a t\ t‘ t‘ w “9‘0“ “3 9,010 “9m ’3 A 5 6 1 Figure 2.3. Kinetics of Yangonin(6)-DPPHO and Methysticin(7)-DPPH0 reactions. Control = solvent with no antioxidant. The concentration in final reaction mixtures of compound 6 and 7 was 2.5 mg mL", respectively. % DPPH Remaining 80- . ‘i + Control + 6 l - A ~ 7 C o 20 40 so so Tlme (min) 33 Figure 2.4. Comparison of DPPH. radical scavenging activities of compounds 6 and 7 with vitamin E, vitamin C, and BHT at 40 min. Vit E = vitamin E, Vit C = vitamin C, BHT = butylated hydroxytoluene, Control = solvent control containing no antioxidant. The concentrations of antioxidants in final reaction mixtures were 2.5 and 2.5 mg mL", 10 pM, 25 pM, and 50 pM for yangonin and methysticin, vitamin E, vitamin C, and BHT, respectively. Vertical bars represent the standard deviation of each data point (11 = 3). 100 ‘ so .... _._ 7 so . ' 7 ‘ 7o . so . so 1 4o 1 30 20 o a, .4; , , 1_... D 1...... VitE VitC °/o DDPH Remaining BHT Control DISCUSSION In the COX-1 assay, compound 1 at 100 pg mL'l gave 58% of inhibition and was the most active among the kava kava compounds tested. Compound 2 at all four concentrations tested showed better cyclooxygenase enzyme inhibition than ibuprofen, aspirin and naproxen at 2.1, 180 and 2.5 pg mL", respectively. All kava kava compounds tested at 100 or 50 pg mL'l demonstrated better or similar COX-I inhibition activities Compared to ibuprofen, aspirin and naproxen. Also, compounds 1, 2 and 7 displayed 48, 43 and 37 % COX-I inhibition, respectively, at 50 pg mL". Compounds 1, 2, 4 and 7 at 25 34 pg mL'l concentration gave excellent COX-I inhibitory activities with 34, 41, 21and 24 %, respectively. COX-II inhibitory assay exhibited good enzyme inhibition for kava kava compounds 1-7 when tested at 100 and 50 pg mL", respectively. However, compound 6 showed the highest percentage inhibition of enzyme as shown in Figure 2.2. Compounds 1, 4 and 5 at 25 pg mL'l also gave COX-II enzyme inhibition comparable to ibuprofen, aspirin and naproxen at 16, 13 and 20 %, respectively. The antioxidant activities of compounds 3 and 6 in lipid oxidation assay could not be evaluated due to their poor solubility in DMSO and in the buffer containing the liposome reacted with the fluorescence probe. Compounds 1, 2, 4, 5 and 7 did not have solubility problems but were not active. However, compounds 6 and 7 gave moderate antioxidant activity when compared to vitamin E, vitamin C, and BHT in free radical scavenging assay. The kinetics of yangonin(6)-DPPH and methysticin(7)-DPPH reactions showed an increase in scavenging activities for compounds 6 and 7 with longer reaction times. The findings of our study provide some scientific basis for the traditional use of kava kava roots for managing inflammatory pain. The compounds isolated from the ethyl acetate extract of kava kava roots exhibited both COX-1 and COX-II enzyme inhibitory activities and therefore might be responsible for the folkloric use of kava kava roots preparation to alleviate arthritic and gout related pain. This is the first report of the cyclooxygenase inhibitory activities of compound 1-7 isolated from kava kava roots. 35 Chapter 3 Novel Compounds from Piper methysticum Forst (kava kava) Roots and Their Effects on Cyclooxygenase Enzyme* ABSTRACT - Milled Piper methysticum roots were sequentially extracted with hot water and MeOH. Cyclooxygenase (COX)-assay-directed purification of the MeOH extract yielded bomyl esters of 3,4-methylene dioxy cinnamic acid (8) and cinnamic acid (9), pinostrobin (10), flavokawain B (11) and 5,7—dimethoxyflavonone (12). The structures of compounds 1-5 were accomplished by spectral experiments. The aqueous extract contained previously published kava lactones, as confirmed by TLC analysis. Compound 8 is a novel natural product and compounds 9, 10 and 12 were isolated for the first time from kava kava roots. Compound 11 showed the highest COX-I inhibitory activity at 100 pg mL". All the compounds tested gave good COX-1 and moderate COX-II enzyme inhibitory activities at 100 pg mL". This is the first report of COX-1 and —11 inhibitory activities for compounds 8-12. * Journal of Agricultural and Food Chemistry, submitted, July, 2001. 36 INTRODUCTION Piper methysticum Forst (kava kava) is a perennial herb of Piperaceae family grown widely in the Pacific Islands (Keller and Klohs, 1963). The rootstock of kava kava is commonly used to prepare a beverage for ceremonial activities by the native Pacific Islanders. Also, it is used in traditional herbal medicine for treating gonorrhea, menstrual pain, tuberculosis, respiratory tract infections and chronic pain related to gout and arthritic conditions (Singh, 1992; Singh and Blumenthal, 1997). Pacific Islanders routinely apply kava kava root extract as an analgesic and as mouthwash for toothache and canker sores (Chevallier, 1996; Leung and Foster, 1996). In Europe, kava kava has been used since the beginning of the twentieth century for the treatment of chronic inflammations of the urinary tract (Mors et al., 1962; Sahelian, 1998). The anti-inflammatory activities of many natural products are determined by evaluating their ability to inhibit cyclooxygenase (COX) enzymes (Cryer and DuBois, 1998). One of the mechanisms by which anti-inflammatory agents control inflammation is by inhibiting the synthesis of prostaglandins. Prostaglandins, inflammation causing hormones, are formed by the conversion of membrane lipid catalyzed by COX and other downsream enzymes. Two isoforms of the COX enzymes responsible for the prostaglandins formation are cyclooxygenase-1 (COX-1) and cyclooxygenase-II (COX-II) enzymes (Cryer and DuBois, 1998; Winzeler and Rosenstein, 1998). However, they differ in their distribution in the body by location and physiological activities. COX-I enzyme is a constitutive form and expressed in most cells. It keeps ‘housekeeping’ functions such as maintaining gastrointestinal mucosa, protecting renal blood flow function and influencing platelet aggregation. COX-II enzyme, on the other hand, is an inducible form of the enzyme which is expressed in response to inflammatory and other physiological stimuli. The 37 prostaglandins produced by COX-II enzyme mediate pain and the inflammatory process. The over-the-counter (OTC) pain relieve agents such as aspirin, ibuprofen and naproxen inhibit both COX-1 and COX-II enzymes at therapeutic doses (Winzeler and Rosenstein, 1998). Kawain, one of the six major kavalactones present in kava kava roots, was identified to have antithrombotic action on human platelets (Gleitz et al., 1997). It was also shown to prevent the formation of prostaglandin E2 and thromboxane A2 by inhibiting the action of COX and thromboxane synthase enzymes (Gleitz et al., 1997). In order to evaluate anti-inflammatory effect of the constituents in kava kava roots, we have investigated compounds other than kava lactones present in the MeOH extract of kava kava roots. In this paper, we report a novel natural product and three other compounds with COX-1 and COX-II inhibitory activities isolated for the first time from kava kave roots. MATERIALS AND METHODS Genaral Experimental Procedures. lH-NMR spectra were recorded at 300 or 500 MHz, and l3C-NMR spectra at 75 or 126 MHz. All compounds were dissolved in CDC13 and lH- and l3C-NMR shift values were presented in 5 (ppm) based on residual 8 value of CHCI3 at 7.24 and 77.0 ppm, respectively. Electron ionization mass spectra (EIMS) were obtained on JEOL AX-SOSH double focusing mass spectrometer at 70 ev. IR spectra analysis were conducted on Galaxy Series FTIR 5020. Medium pressure liquid chromatography (MPLC) was carried out on silica gel 60 and preparative TLC was conducted over silica gel GF glass plate (20 x 20 cm, 250 or 500 pm thickness, Analtech, Newark, Delaware). Bands were viewed under UV light (366 and 254 nm). 38 Preparative HPLC was conducted on LC-20 (Japan Analytical Industry Co., Tokyo) with a JAIGEL—ODS column (A-343-10, 20 mm x 250 mm, 10 pm, Dychrom, Santa Clara, CA). A UV detector was used and peaks were recorded by a model D-2500 Chromato-integrator (Hitachi Co., Tokyo). Positive controls, naproxen, ibuprofen and aspirin for COX enzyme inhibitory assays were purchased from Sigma Chemical Company (St. Louis, MO). Celebrex capsules and Vioxx tablets, as physician’s professional samples, were provided by Dr. Subash Gupta, Sparrow Pain Center, East Lansing, MI. All solvents were ACS reagent grade and purchased from Aldrich Chemical Company. Plant Materials. Dried kava kava roots were purchased from Meetex Fiji Ltd., Fiji. The roots were stored at -20°C in plastic shipping bags till extraction. The roots were milled using Thomas-Wiley Laboratory Mill Model 4 (Thomas Scientific, USA, 2mm filter) prior to extraction. Extraction of Piper methysticum Roots. The ground roots (200g) were extracted twice with H2O (3L, 3h and IL, 1h) at 60°C. The water extracts were combined, cooled to room temperature and centrifiiged (10000 rpm, 4 °C, 15 min) and the supernatant was lyophilized. The precipitant from the aqueous extract mainly contained very fine particles of plant material and hence combined with the residue prior to the MeOH extraction. The residue, plant material after aqueous extraction, was extracted with MeOH (3L, 24h). This MeOH extract (8 g) showed the highest COX inhibitory activity when compared to the water extracts. Therefore, the MeOH extract was used for this study and fractionated by MPLC on silica gel using hexane-acetone (10:1, 750 mL; 7:3, 400 mL; 1:1, 200 mL; 3:7, 200 mL) followed by 100% acetone (1.05 L) and MeOH (2.3 L). The fractions yielded were: A (0.505g, hexane-acetone 10:1 and 7:3, 1.15 L), B 39 (3.038 g, hexane-acetone 1:1, 250 mL), C (1.798g, hexane-acetone 3:7, 250 mL), D (1.279g, 100% acetone, 250 mL), E (0.161 mg, 100% acetone, 910 mL) and F (0.674g, 100% MeOH, 2 L). The COX inhibitory assay revealed that fractions A-D were the most active. Fraction A (0.505g) was further purified by preparative TLC with hexane- acetone (4:1) to yield eight bands. Band 1 (101.5 mg, Rf 0.7), Band II (33.7 mg, Rf 0.5), Band IV (77.1 mg, Rf 0.4), Band V (212mg, Rf 0.3) and Band VII (63.5 mg, Rf 0.2) were active in COX-1 and COX-II enzyme inhibitory assays. Isolation of Compound 8 and 9. Compound 8 (5.1 mg) was obtained from the crystallization of band 11 from MeOH. The band 11 from fraction A was further purified by TLC with hexane-acetone (8:1) to yield compound 9 (40.3 mg, Rf 0.8). Compound 8. White needle-like crystals; mp. 138-140°C; IR (KBr), 2954, 1700, 1635, 1520, 1444, 1255, 1180, 931, 807, 755 cm"; lH-NMR (CDC13, 500MHz), 6 7.03 (1H, s, aromatic H), 6.9 (2H, dd, J = 8.1, 2.1 Hz, aromatic ortho-H), 6.88 (2H, dd, J = 15.9 Hz, H-9/H-10), 5.98 (2H, s, CH2-7’), 5.00 (1H, m, H-Ze), 2.40 ( 1H, m, H- 3e), 2.02 (1H, m, H-6a), 1.75 ( 1H, m, H-Se), 1.68 (1H, t, H-4e), 1.36 (1H, m, H-6e), 1.26 (1H, m, H-Sa), 1.03 (1H, dd, J = 13.8, 1.8 Hz, H-3a), 0.92/ 0.89 (2 x 311, 23, 2CH3-C7), 0.85 (3H, s, CH3-C1); l3C-NMR (CDC13, 126 MHz): 5 167.5 (C-8), 149.5, 148.3, 143.9, 128.9, 124.4, 124.3 (C-1’-C-6’), 124.3 (C-10), 116.7 (C-9), 101.6 (C- 7’), 79.8 (02), 44.9 (04), 48.9/ 47.8 (C-l/C-7), 36.8 (C-3), 28.0 (C-5), 27.2 (C-6), 19.7/18.8 (2CH3 -C7), 13.5 (CH3-C1); EIMS (% rel intensity), m/z 328.1673 [Mi] (100), 192 (50), 175 (100), 145 (80). Compound 8 was deduced to be bomyl ester of 3,4-methylene dioxy cinnamic acid. 40 Compound 9. Pale yellow oil; IH-NMR (CDC13, 500 MHz): 6 7.03 (5H, m, aromatic H), 6.91 (2H, dd, J = 15.9 Hz, H-9/H-10), 5.01 (1H, m, H-2e), 2.41 (1H, m, H-3e), 2.04 (1H, m, H-6a), 1.77 (1H, m, H-Se), 1.70 (1H, t, H-4e), 1.36 (1H, m, H-6e), 1.29 ( 1H, m, H-Sa), 1.05 (1H, dd, J = 13.8, 1.8 Hz, H-3a), 0.93/ 0.89 (2 x 3H, 23, 2CH3- C7), 8 0.87 (3H, s, CH3-C1); l3C-NMR (CDC13, 126 MHz): 5 167.3 (C-8), 144.2, 134.5, 130.1, 128.8, 128.8, 128.0 (C-1’-C-6’), 128.0(C-10), 118.0 (C-9), 79.9 (C-2), 44.9 (C-4), 48.9/ 47.8 (C-1/C-7), 36.8 (C-3), 28.0 (OS), 27.2 (C-6), 19.7/ 18.8 (2CH3 -C7), 13.5 (CH3-C2); EIMS (% rel intensity), m/z 284 [Mi] (25), 153 (72), 131 (100), 109 (30), 103 (30). Compound 9 was identified as cinnamic acid bomyl ester. The spectral data of 2 were in agreement with the published values (Monteiro et al., 1997). 41 Isolation of Compounds 10-12. Band IV from fraction A was further purified by repeated preparative TLC with hexane-acetone (4:1, x 2) to yield one major band (66 mg, Rf 0.7). This band, both COX-1 and COX-II inhibitory, was purified by preparative TLC with toluene-ethyl acetate (13:1) and hexane-acetone (8:1,x 4) followed by crystallization from hexane-acetone to afford compound 10 (14.5 mg). Compound 10 was identified as pinostrobin based on the published chemical shift values for pinostrobin (Burke and Nair, 1986). Band V from fraction A was crystallized from hexane-ether to afford compound 11 (73.1 mg) and confirmed as flavokawain B. The NMR data of 11 was identical to the published values of flavokawain B (Sirat et al., 1996; Itokawa et al., 1981). The mother liquor (76.3 mg) after the crystallization of compound 11 was further purified by preparative TLC using toluene-ethyl acetate (8:1) as the mobile phase to afford five bands. Band 1 (49.1mg, Rf 0.8) was further purified by preparative TLC using hexane-ethyl acetate (8:3) to yield additional supplies of compound 10 (10.7 mg) and compound 11 (39.0 mg). H3C O H3C C I O O OH O OH 10 11 Fraction B fi'om the MeOH extract was further fractionated by silica MPLC using hexane-acetone (10:1, 550 mL; 6:1, 560 mL; 4:1, 150 mL; 3:1, 160 mL; 2:1, 420 mL; 1:1, 200 mL). Six fractions were collected: hexane-actone 6:1; 1 (48.2 mg, 300 mL), 2 (24.5 mg, 100 mL); hexane-acetone 6:1 and 4:1, 3 (111.6 mg, 300 mL); 42 hexane-acetone 3:1 and 2:1; 4 (489.2mg, 300 mL), 5 (636.7 mg, 200 mL); hexane- acetone 1:1; 6 (1.35 g, 300 mL). Fraction 6 was active in COX enzyme inhibitory assays and 500 mg of it was further purified by preparative HPLC on a Jaigel-ODS column under isocratic condition using MeOH-H2O (60:40) at a flow rate of 3 mL/min and detected at 210 nm. This yielded compound 12 (7mg, R, 174.7 min) and identified as 5,7-dimethoxyflavonone. The NMR data of 12 were in agreement with the published data (Mayer, 1990; Bick, 1972). H3C O OCH3 0 12 Cyclooxygenase Enzyme Inhibitory Assay. This assay is based on measuring COX enzyme activities by monitoring the rate of 02 uptake using an oxygen electrode YSI Model 5357 (INSTECH Laboratory, Plymouth Meeting, PA) (Meade et al., 1993; Smith et al., 2000; Wang et al., 2000). The assay mixture consisted of 600 pL of 0.1 M Tris-lmM phenol buffer and 17 pg hemoglobin. COX-I enzyme was prepared from ram seminal vesicles purchased from Oxford Biomedical Research, Inc., Oxford, MI. COX-I enzyme solution was prepared by dissolving 0.46 mg of protein/mL in 30 mM Tris buffer (pH 7.0). COX-II enzyme was isolated from prostaglandin endoperoxide H synthases-Z cloned insect cell lysate and diluted with Tris buffer (pH 7.0) till the concentration was 1.5 mg of protein/mL. Reactions were initiated by adding 10 pL of arachidonic acid (0.25 mg/0.5 mL Tris buffer) in the assay mixture. The test samples 43 or controls as DMSO solutions and 5-25 pg of COX enzymes in 10-20 pL were pre- incubated for 10 min prior to injection of 10 pL of arachidonic acid in a 600 pL microchamber. Instantaneous inhibition of COX enzymes was determined by measuring the COX enzyme activity at 37 °C (Model-1166, VWR Scientific Products, Chicago, IL). The enzyme activity was monitored by a Biological Oxygen Monitor and the data were collected using Quicklog data acquisition and computer software (Strawberry Tree Inc., Sunnyvale, CA, USA). Finally, the data were transformed into Microsoft Excel. The assay was conducted in triplicate. RESULTS AND DISSCUSSION Compounds 8, 9, 10 and 12 were isolated for the first time from kava kava roots. The structure of compound 8 was deduced by using 1H and l3C-NMR, DEPT, Decoupling, DQF-COSY, HMQC, HMBC, IR and MS spectral techniques. 1H-NMR spectrum of 8 gave a singlet at 8 7.03 and a doublet of doublet at 8 6.91 suggested a trisubstituted aromatic moiety in 8. The singlet at 8 5.98, integrated for two protons, and a corresponding carbon chemical shift at 8 101.6 suggested a methylene dioxy group in the molecule. 'H-NMR spectrum of 8 gave a doublet of doublet signal, integrated for two protons at 8 6.88 with a coupling constant of 15.9 Hz, was corresponded to a trans olefinic group. A singlet at 8 5.00 was determined to be a proton connected to an oxygenated carbon. Three singlets at 8 0.92, 0.89 and 0.85, each integrated for three protons, were indicative of three CH3 groups. I3C-NMR spectrum of compound 8 revealed nineteen peaks. They were assigned to one carbonyl carbon (8 167.3), six aromatic carbons (8 149.5, 148.3, 143.9,128.9, 124.4, 124.3), two olefinic carbons (8 124.3, 116.7), two quaternary carbons (8 48.9, 47.8), 44 two CH (8 79.8, 44.9), four CH2 (8 101.6, 36.8, 28.0, 27.2) and three CH3 (8 19.7, 18.8 and 13.5) as confirmed by the DEPT spectrum of 8. DQF-COSY, HMQC and HMBC spectral data provided additional evidence to confirm the structure of compound 8. The important connectivities in DQF-COSY spectrum of 8 were those observed for H-3 & H-4, H-2 & H-3 and H-5 & H-6. The decoupling spectrum of compound 8 revealed long range couplings between H-3e & H-5e as well as between H-2e & H-6e. Also, decoupling spectrum of 8 indicated that dihedral angles 8 between H-4e and H-5a as well as between H-4e and H-3a were almost at 90° (Lambert et al., 1998). HMBC spectrum of compound 8 (Figure 3.1) showed important correlation observed for C-1 and H-4. The signals for O4 correlated to H-3, H-5 and H-6a while C-l displayed cross signals to H-3e, H-4e, H-Sa and H-6. Also, the correlation of C-5 to H-3 and H-6 as well as the correlation of O2 to H-3, H-4e and H-6 supported the proposed structure. One of the methyl groups at 8 0.85 correlated to C-2. Figure 3.1. Significant HMBC correlations observed in compound 8. 45 The presence of a carbonyl group in 8 was suggested by a strong peak at 1700 cm'l in its IR spectrum. Another strong peak at 1635 cm'l was assigned to C=C and indicated a conjugated system in the molecule, a carbonyl group connected to a C=C group. Peaks at 1255 and 1180 cm'1 were corresponded to C-0 functionality. The MS analysis of 8 gave a strong M+ ion peak at m/z 328.1673 (100%) which suggested the molecular formula of 8 as C20H24O4. The fragmentation pattern of 8 under EIMS condition is shown in Figure 3.2. The EIMS fragments at m/z 192, 175 and 145 supported the proposed structure for compound 8. Based on all the spectral analysis, compound 8 was deduced to be bomyl ester of 3,4-methylene dioxy cinnamic acid, a novel natural product. The structure of compound 9 was deduced from 1H and l3C-NMR, DEPT, HMQC, HMBC and MS spectral data. 111- and l3C-NMR spectra of compound 9 were similar to compound 8 except minor changes in the aromatic moiety. The multiplet at 8 7.03, integrated for five protons, were assigned to a mono-substituted aromatic moiety. Based on 'H- and I3C-NMR spectra data of 9, it was proposed to be cinnamic acid bomyl ester. HMQC and HMBC spectral data supported the proposed structure for 9. The MS analysis of 9 gave a 25% intensity of W ion peak at m/z 284. The EIMS fragments at m/z 153, 131, 109 and 103 provided further evidence to confirm the structure of 9. Based on all the spectra data, compound 9 was assigned as cinnamic acid bomyl ester. 46 Figure 3.2. Fragmentation pattern of compound 8 under EIMS conditions. o '7‘“ l \ \ {(03% // V0 0 IT m/z 328 (100%) \H\ \ | \ l \ \ 1: / o/ / o \___O m/z 175 (100%) m/z 192 (50%) \ %_l+ / r°\_. 0 m/z 145 (80%) 47 COX-1 and COX-II enzyme inhibitory assays were used in the bioassay- directed isolation of potential anti-inflammatory compounds present in kava kava roots. Ibuprofen, naproxen, aspirin, Celebrex and Vioxx were used as positive controls in these assays and were dissolved in DMSO at their ICso concentrations of 2.1, 2.5, 180, 1.67 and 1.67 pg mL", respectively. In the COX-1 enzyme inhibitory assay, ibuprofen, naproxen, aspirin, Celebrex and Vioxx demonstrated 30, 63, 78, 47 and 23 % inhibition, respectively. Similarly, in the COX-II enzyme inhibitory assay ibuprofen, naproxen, aspirin, Celebrex and Vioxx showed 44, 61, 24, 88 and 67 % inhibition, respectively. All test compounds were assayed at 100 pg mL‘1 and pH 7 .0 in both COX-1 and COX-II enzyme inhibitory assays. The percentage of inhibitions of COX enzymes activities for test compounds at 100 pg mL'l are shown in Figures 3.3 and 3.4. Compounds 8-12 showed 16, 66, 36, 77 and 42 % inhibition, respectively, in COX-I enzyme inhibitory assay while they gave 18, 19, 7, 16 and 19 % inhibition, respectively, in the COX-II enzyme inhibitory assay. Since compound 11 was the most COX-I active, it was fithher tested at 50, 25, 12.5 and 6.25 pg mL’lin the COX-1 enzyme inhibitory assay and revealed 79, 64, 55 and 20 % inhibition at 50, 25, 12.5 and 6.25 pg mL", respectively. In COX-1 inhibitory assay, compound 11 exhibited 77% inhibition and was the most active among the compounds tested at 100 pg mL". It also showed good COX-I enzyme inhibitory activities at 6.25 pg mL". However, it demonstrated only 16% COX-H inhibition at 100 pg mL". This indicated that compound 11 is not a preferred anti-inflammatory agent. Compounds 9, 10 and 12 also displayed higher COX-I inhibition at 100 pg mL". However, compounds 8-12 showed relatively low percentage of COX-II inhibition at 100 pg mL". 48 Figure 3.3. Percentage COX-I inhibition of compounds from kava kava roots at 100 pg mL". Ibuprofen, naproxen and aspirin were tested at 2.1, 2.5 and 180 pg mL", respectively. Celebrex and Vioxx were tested at 1.67 pg mL". Vertical bars represent the standard deviation of each data point (n = 3). 100 ~ 90 l 80 a O 4; - 1 % inhibition dethN oooooco l . 1 ML... Figure 3.4. Percentage COX-II inhibition of compounds from kava kava roots at 100 pg mL". Ibuprofen, naproxen and aspirin were tested at 2.1, 2.5 and 180 pg mL", respectively. Celebrex and Vioxx were tested at 1.67 pg mL". Vertical bars represent the standard deviation of each data point (11 = 3). 100 7 90 - 80 - 7o « 60 - 50 - 40 l % inhibition 49 The results of our study provided some scientific support for the anecdotal claims on the traditional use of kava kava roots for controlling inflammatory pain by Pacific Islanders. The compounds yielded from the MeOH extract of kava kava roots after extraction with hot water demonstrated substantial COX-1 and moderate COX-II inhibitory activities. Therefore, a combination of kava lactones and compounds 8-12 might account for the traditional use of kava kava roots to alleviate arthritic and gout related pain. This is the first report of cyclooxygenase enzyme inhibitory activities for compounds 8-12. 50 Chapter 4 Up Regulation of Gap Junctional Intercellular Communication (GJIC)) in Oncogene Transformed Rat Liver Cells and Topoisomerase Inhibition by Compounds from Piper methysticum Forst (kava kava) Roots. ABSTRACT- Gap junctional intercellular communication (GJIC) and topoisomerase enzyme inhibitory assay have been conducted on compounds 1, 3-7 and 11 isolated from kava kava roots. Compound 1 is a moderate topoisomerase-II inhibitor when tested at 250 pg concentration. In ras— and myc/ras-transduced rat liver epithelial cell lines, compounds 5 and 7 showed growth inhibition at 181 and 182 pM, respectively. In ras-transduced rat liver epithelial cells, compounds 1, 3 and 4 showed growth inhibition at 287, 46.8 and 217 pM, respectively. Compound 5 distinctly changed cell morphology and showed a reversible cytostatic effect. It also slightly upregulated GJIC at 181 pM in ras-transduced rat liver epithelial cells. This is the first report of potential anticancer activities of compounds 1 and 5. 51 INTRODUCTION Several epidemiological study of cancer incidence in the Pacific Islands showed unusually low rate of many cancers, including cancers of lung, stomach and colon-rectum (Reed, 1977; Henderson et al., 1985; Steiner, 2000). Desmethoxyyangonin, one of the six major kava lactones in P. methysticum roots, was found to significantly inhibit the release of TNFa in BALB/3T3 cells which is an endogenous tumor promoter and a central mediator of cancer development (Fujiki, 1999). A close inverse relationship between cancer incidence and kava consumption was also found when a study was conducted on comparing the cancer incidence in a number of Pacific Island nations with local kava consumption (Steiner, 2000). Therefore, kava kava, when consumed as a daily beverage, might play a role in cancer prevention as an alternative medicine. It might also contain some chemopreventive and chemotherapeutic compounds. One of the mechanisms of action for anticancer drugs is to inhibit topoisomerase I and/or 11 enzymes. Some antitumor compounds act as topoisomerase enzyme inhibitors by stabilizing an intermediate of cleavage complex of the topoisomerase reaction in which DNA strands are broken and the enzyme is covalently attached to the target DNA molecules. Camptothecin and its derivatives have been proven to have effect on eukaryotic type I topoisomerase by stabilizing DNA cleavage products (Hsiang et al., 1985). Compounds in kava kava roots might act as topoisomerase 1 and/or 11 enzyme inhibitors. Another possible mechanism of action for anti-cancer agents is induction of gap junctional intercellular communication (GJIC). GJIC plays a homeostasis role in growth 52 control and terminal differentiation (Trosko et al., 1996). GJIC can be reversibly down- regulated or inhibit either at the posttranslational levels or transcriptional level in in vitro and in vivo experiments with tumor-promoting chemicals such as 12-O- tetradecanoylphorbol-l3-acetate (TPA), l-methylanthracene, dieldrin and dicumyl peroxide (Trosko et al., 1993; Trosko et al., 1996). As evidenced by a GJIC-deficient mutant, GJIC is a necessary, but not sufficient to initiate the mitogenic process in the promotion process of carcinogenesis (Trosko et al., 1993). Retinoid and c-AMP, antitumor promoters, can up regulate GJIC. The enhancement of GJIC by retinoids has been reported to correlate with their ability to inhibit neoplastic transformation (Hossain et al., 1989). Caffeic acid phenethyl ester has been reported to restore GJIC in a ras- transforrned rat liver epithelial cell line (Na et al., 2000). Up regulation of diminished GJIC in oncogene transformed cells might lead to differentiation of tumor cells. Tumor cell differentiation has been shown to be associated with restoration of a diminished GJIC that is often observed in transformed cells (Carystinos et al., 2001). Based on these scientific reports, compounds in kava kava roots might act as antitumor promoters to restore diminished GJIC caused by tumor promoters. Also, they might upregulate GJIC in oncogene transformed cells (Trosko and Chang, 2001). Kava lactones and chalcones are major components isolated from kava kava roots with cyclooxygenase (COX) inhibitory activities (Chapters 1 and 2). In this chapter, we report tumor growth inhibition and up regulation of GJIC by kava lactones and chalcones, as well as a topoisomerase inhibitor. 53 MATERIAL AND METHODS Topoisomerase Enzyme Inhibitory Assay Compounds 1-7 and 11, isolated from kava kava roots, were evaluated for topoisomerase-I (Top-I) and —II (Top-II) inhibitory activities. Saccharomyces cerevisiae mutant cell cultures of JN394, JN394t.1 and JN394L2-5 were used in the assay. The cultures were grown in petri dishes containing YPDA medium (20g/L of bacto peptone, IOg/L of yeast extract, 20g/L of dextrose, 17g/L of agar and 2le of 0.5% adenine sulfate). A 5x106 CFU/mL suspension of each mutant cell culture in physiological saline was obtained by diluting concentrated cell culture suspension with physiological saline. The cell suspension (50 pL) was spread evenly on petri dishes containing YPDA medium. To these dishes containing test cultures, DMSO (20 pL) or DMSO solution of test compound (250mg/20 pL) were spotted. The plates were allowed to stand in a laminar flow hood for 10 min and then incubated at 27°C for 72 h. The zone of inhibition was measured in mm (Nair et al., 1989; Chang et al., 1995; Roth et al., 1998). Cell Culture The normal rat liver epithelial cell lines, WB-F344, were obtained from Grisharn J. W. and Tsao M. S. at the University of North Carolina (Chapel Hill, NC) (Tsao et al., 1984). WB-ras cells are WB-344 cell line infected with a virus carrying c-Ha-ras oncogene and neomycin-resistance markers (deFeijter et al., 1990). WB myc-ras cells are co-transfected with v-myc and c-Ha-ras oncogenes (Hayashi et al., 1998). All the cells used in the experiment were of low passage. 54 Cells were cultured in 2 mL D-media (formula no. 78-5470EG; Gibco Laboratories, Grand Island, NY) with 5 % fetal bovine serum (Gibco) and 50 pg mL'l gentarnicin (Gibco), and grown in 35 mm diameter plastic petri dishes (Corning Glass Works, Corning, NY) and incubated at 37 °C in a humidified atmosphere containing 5 % CO2 and 95 % air. Bioassays were conducted on confluent cells. Test Compounds TPA, l-methylanthracene, dicumyl peroxide, dieldrin, formaldehyde (37%) and acetonitrile were obtained from Aldrich Chemical Co., Inc (Milwaukee, WI). The lithium salt of Lucifer yellow was obtained from Sigma Chemical Co. (St. Louis, MO). Compounds 1, 3-7 and 11 were isolated and purified from kava kava roots (Chapters 2 and 3). Their structures and purity were identified using spectral, TLC and HPLC methods. Compound 2 was not evaluated due to its short supply (Chapters 2 and 3). Stock Solution All test compounds and tumor promoters were dissolved separately in acetonitrile. Concentrations for stock solutions of TPA, l-methylanthracene, dicumyl peroxide and dieldrin were 3 pM, 4, 10 and 5 mM, respectively. The volume of the stock solutions that were added directly to 2 mL of culture media in 35 mm diameter plastic petri dish was 10 pL. The volume of acetonitrile for control added to cell culture was equivalent to that of test compounds added. 55 Cytotoxicity Different concentrations of solutions of each compound were prepared. Cells were exposed to different doses of the compounds for 24 h. The morphological shape of cells and GJIC were checked after 24 h exposure. The highest non-cytotoxic doses for the compounds were determined based on normal morphological form and GJIC as observed in control. Treatment with test compounds Normal WB-F344 cells were exposed to the highest non-cytotoxic dose of each compound for 15 min or 24h. After 15 min or 24 h exposure, the culture medium was changed with culture medium containing 10 pL of stock solution of a tumor promoter. Cells were exposed to TPA, l-methylanthracene and dicumyl peroxide for 10 min and to dieldrin for 5 min. The control cells were either not treated or treated with equal amount of acetonitrile. GJIC assay was conducted on the cells following chemical treatment. Normal WB-F344 cells were incubated for 24 h to get 100 % confluence and treated with culture medium containing 10 pL of one tumor promoter and one compound, respectively. Culture medium was changed before the chemical treatment. The exposure time for TPA, 1-methylanthracene and dicumyl peroxide with corresponding compounds was 10 min and for dieldrin was 5 min. The control cells were either not treated (untreated) or treated with equal amount of acetonitrile (control). The GJIC assay was conducted on the cells following chemical treatment. WB ras- or myc/ras-transduced cells were grown to approximately 50-60 % confluence and treated with the highest non-cytotoxic dose of each compound for 2 days. 56 Culture medium was changed every two days. The control cells were either not treated or treated with equal amount of acetonitrile. The GJIC assay was conducted on the cells following chemical treatment. WB ras- and myc/ras- transformed cells with 216.8 x 104 cells /2 mL were seeded at 25 % confluence and a significant number of cells were allowed to attach to the plate bottom in 4 h. Then cells were exposed to 10 pL of each compound at the highest non- cytotoxic dose till the cells were grown to 100 % confluence. Culture medium was changed every two days. Controls were prepared using same and less number of cells since preliminary test showing these compounds inhibited cell growth. The GJIC assay was conducted on the confluent cells following chemical treatment. GJIC Assay GJIC was determined by using the scrape loading/ dye transfer (SL/DT) method (El-Fouly et al., 1987). Immediately following the chemical exposure, cells were rinsed with phosphate-buffered saline (PBS) three times and then approximately 1 mL of 0.01 % Lucifer yellow (PBS as solvent) was added into the cell dish. A surgical steel blade was used to make 5-6 scrapes through the monolayer of cells under low light intensities. After 3 min incubation time at room temperature, the Lucifer yellow was removed and cells were rinsed with PBS for 3 times and then fixed with approximately 0.5 mL of 4 % formalin. The distance of the dye migration was measured on photographs of 200x magnified microscopic images made of fixed cells by a Nikkon Diaphot-TMD epifluorescence phase-contrast microscope illuminated with an Osram HBO 200W lamp 57 and equipped with a 35-mm FA Nikkon camera. The perpendicular distance of migration of the dye was measured from the scrape line to the edge of the dye front. Ten measurements were made, one cm apart, along the scrape line and ten values were averaged and divided by control. A fraction of control (FOC) was reported with standard variance at the 95% confidence interval as results. An F OC value of approximately 1.0 indicates normal GJIC as control; FOC values more than 1.0 indicates up regulation of GJIC; FOC values less than 1.0 indicates down-regulation of GJIC. RESULTS Topoisomerase Inhibitory Assay Only compound 1 showed a weak top-H inhibitory activity when tested at 250 pg in 20pL of DMSO. The zone of inhibition was 15 mm for JN394t-l and 12 mm for JN394. There was no zone of inhibition for JN394t-2-5. Cytotoxicity. The highest non-cytotoxic doses for compounds 1, 3-7 and 11 were determined at 143.5, 93.5, 108.5, 90.5, 19.35, 91.0 and 3.05 pM, respectively, in normal WB-F344 rat liver epithelial cells with FOC values at 1.0 approximately. Similarly, the highest non- cytotoxic doses for compounds 1, 3-7 and 11 were determined at 287, 46.8, 217, 181, 38.7, 182 and 6.1 pM, respectively, in ras- and myc/ras- transduced rat liver epithelial cells with FOC values at 1.0 approximately. Lower doses of 11 were determined than other chemicals based on its cytotoxicity, which indicates that compound 11 is very 58 cytotoxic at high doses. Low doses of 6 were determined due to its low solubility in acetonitrile and cell culture medium. The highest non-cytotoxic concentrations for all test compounds were used in the chemical treatment. Treatment with test compounds GJIC was almost completely inhibited when the normal WB-F344 rat liver epithelial cells were treated with tumor promoters following incubation with test compounds for 15 min or 24 h. Also, GJIC was almost completely inhibited when the normal WB-F344 rat liver epithelial cells were treated simutanously with tumor promoters and test compounds. Ras- or myc/ras-transduced rat liver epithelial cells, were treated with test compounds at 50-60 % confluent stage till cells were fully grown to 100 % confluence, GJIC were checked and was not restored on 100 % confluent cells treated with any compound. However, cells, treated with compounds 5 and 7, became confluent 36 and 24h later, respectively, than controls treated with equivalent volume of acetonitrile as the testing chemicals. The myc/ras-transduced rat liver epithelial cells were seeded at 25 % confluence followed by being incubated for four hours to allow the significant number of cells attached to the bottom. Then the cells were treated with test compounds till the cells fully covered the plate bottom. GJIC was checked and was not restored on 100 % confluent cells exposed to any compound. However, cells, treated with compounds 5 and 7, became confluent 48h later than controls treated with equivalent volume of acetonitrile as the chemicals. 59 The ras-transduced rat liver epithelial cells were seeded at 25 % confluence and incubated for four hours to let the significant number of cells attached the plate bottom. Then cells were exposed to test compounds till 100 % confluence. GJIC was checked at the various time points. GJIC was not restored on cells treated with 1, 3, 4, 6 and 7. However, cells, treated with compounds 1, 3, 4 and 7, became confluent 48h later than controls treated with equivalent volume of acetonitrile as the chemicals. The morphological shape of ras-transformed rat liver epithelial cells, treated with compound 5, was distinctly different from the normal spindle-shaped morphology of ras-transduced rat liver epithelial cells (Figure 4.1). Some cells became larger and flatter and appeared to become senescent. GJIC was slightly restored on ras-transduced rat liver epithelial cells, treated with compound 5, at 52, 68, 96, 120 and 144 h (Figures 4.2 and 4.3), compared with controls treated with equivalent volume of acetonitrile as the compounds. The first time point selected to check GJIC was at 52 h based on approximately 80 % confluence of cells obtained. 60 Figure 4.1. Morphological images in the ras-transformed rat liver epithelial cells treated with compound 5. (A) control (36 h exposure); spindle-shaped and confluent; (B) treated with compound 5 (36 h exposure); some cells became larger and flatter and appeared to be senescent; (C) control (64 h exposure); spindle-shaped and overconfluent; (D) treated with compound 5 (64 h exposure); not confluent, different cell morphology including larger and flatter cells, some cells appeared to be senescent. 61 Figure 4.2. Scrape loading dye transfer images in the ras-transformed rat liver epithelial cells treated with compound 5. Brighter spots indicate dye migration. (A) control (120 h); diminished GJIC; (B) treated with compound 5 (181 pM, 120 h); slightly increased GJIC; (C) treated with compound 5 (181 pM, 144 h); slightly increased GJIC. (A) (C) , a . ' ' *‘kil‘wfiflfi‘r' _ Figure 4.3. The effect of compound 5 on GJIC in ras-transduced rat liver epithelial cells. GJIC activity (FOC) 144h 62 DISCUSSION Except compound 1, the rest of the compounds tested gave negative results in top-I and -II inhibitory assays. The mutant strains of S. cerevisiae, strain JN394, is hypersensitive to top-I and -II enzymes. JN394L1, lacking top-I gene, responds to top-II inhibitors. JN394L2-5 responds only to top-I inhibitors since it carries the mutant top-II gene which is resistant to top-II poisons. In top-I and -II inhibitory assays, compound 1 showed moderate top-H inhibitory activity when tested at 250 pg concentration. This result indicated that compound 1 is a potential top-II inhibitor. Compounds 1, 3, 4, 5 and 7 demonstrated growth inhibitory activities against ras- and myc/ras-transduced rat liver epithelial cells under the experimental conditions. Especially, compounds 5 and 7 inhibited the transduced cells growth distinctly. The mechanism of action by which these compounds, as COX inhibitors, inhibited the growth of ras- and myc/ras-transduced rat liver epithelial cells might be by inhibiting the synthesis of prostaglandins that stimulated the tumor cell growth (Langenbach et al., 1999; Williams et al., 1999; Masferrer et al., 2000; Williams et al., 2000; Liu et al., 2001). Since the highest non-cytotoxic doses used for compounds 6 and 11 were relatively low, compound 6 and 11 did not show inhibitory activities on cell growth at 38.7 and 6.1 pM, respectively. However, compounds 1, 3, 4 and 7 failed to up regulate or restore the inhibited GJIC in these two transformed cell lines at the highest noncytotoxic dose. Compound 5 slightly up regulated GJIC in ras-transduced rat liver epithelial cells where chemical treatment was conducted following incubation for 4 h after inoculation and changed the cell morphology. However, these results could not be confirmed when cells were treated at 50-60 % of confluence with compound 5 until full confluence was 63 attained by ras-transduced rat liver epithelial cells. After 5 days of treatment with compound 5 in this cell line, culture medium containing 5 was changed with culture medium without the compound. The cells were grown to 100 % confluence at the end of 9 days. It indicated that compound 5 did not cause cells to apoptosis but acted as a reversible cytostatic agent. Up regulation of GJIC by compound 5 in this cell line might lead to suppression of tumor cell growth and restore normal contact inhibition. Further studies such as soft agar and western blot analysis will determine whether compound 5 could suppress tumor cell growth and provide the mechanism of cytostatic effect independent growth, a marker for tumorigenicity. The research conducted in this chapter showed that compound 5 has potential as an anticancer agent in ras-transduced rat liver epithelial cells. This is the first report of potential anticancer activities of compounds 1 and 5 using topoisomerase enzyme inhibitory and GJIC assay. 64 Chapter 5 Summary and Conclusion Piper methysticum F orst is a perennial herb of Piperaceae family grown widely in the Pacific Islands. The rootstock of P. methysticum is commonly known as kava kava and used to prepare a beverage for ceremonial and social activities and also used in the traditional herbal medicine by the native Pacific Islanders. Prior research covering phytochemistry and pharmacology and bioactivities of crude extracts and purified compounds were reviewed in Chapter 1. The literature review in Chapter I focused on the bioactivities of crude extracts and purified compounds with anti-anxiety, muscle relaxation, analgesic, anesthetics, insomnia, antibacterial and antifungal activities. In my research, I hypothesized that P. methysticum roots contain antioxidant, anti-inflammatory and anticancer compounds. Therefore, attempts were made only to isolate and evaluate bioactive compounds with antioxidant, cyclooxygenase and topoisomerase enzyme inhibitory activities. This research yielded twelve compounds with antioxidant and cyclooxygenase and topoisomerase inhibitory activities. These compounds were also evaluated for their potential anticancer activities using gap junctional intercellular communication (GJIC) assay. Bioassay-guided isolation and purification of compounds from P. methysticum roots were discussed in Chapters 2 and 3. The structures of purified compounds were elucidated by using spectroscopic techniques such as 1H- and l3C-NMR, DQF-COSY, HMQC, HMBC, F TIR and MS. Kava kava roots were sequentially extracted with hexane, ethyl acetate and MeOH. Bioassy-directed isolation and purification of the active 65 ethyl acetate extract afforded seven compounds, dihydrokawain (1), desmethoxyyangonin (2), flavokawain A (3), kawain (4), dihydromethysticin (5), yangonin (6) and methysticin (7) (Chapter 2). The structures of compounds 1-12 were determined by IH- and 13C- NMR spectral experiments and confirmed their optical activities by CD measurements. Milled P. methysticum roots were also extracted sequentially with hot water and MeOH. Cyclooxygenase enzyme inhibitory assay directed purification of the active MeOH extract yielded bomyl ester of 3,4-methy1ene dioxy cinnamic acid (8), bomyl ester of cinnamic acid (9), pinostrobin (10), flavokawain B (11) and 5,7-dimethoxyflavonone (12) (Chapter 3). Compound 8 is a novel natural product and compounds 9, 10 and 12 were isolated for the first time from P. methysticum roots. The structures of compounds 8 and 9 were determined by using ‘11- and l3c-NMR, DQF-COSY, HMQC, HMBC, FTIR and MS spectral techniques. Isolation and purification of these compounds were performed by MPLC, repeated TLC and HPLC on a C13 column. Biological activities of compounds 1-12 were discussed in Chapters 2-4. Antioxidant, cyclooxygenase and topoisomerase enzyme inhibitory and gap junctional intercellular communication (GJIC) assay were carried out for compounds 1-12. Cyclooxygenase enzyme inhibitory and antioxidant activities of compounds 1-7 were detailed in Chapter 2. Compounds 1-7 showed 58, 39, 33, 34, 25, 36 and 42 % inhibition at 100 pg mL'1 and pH 7, respectively, in COX-I inhibitory assay. Similarly, they gave 28, 23, 15, 25, 32, 34 and 26 % inhibition at 100 pg mL'1 at pH 7, respectively, in COX-II inhibitory assay. A dose response study of 1-7 were conducted for the COX-1 and COX-II inhibitory activities. Compounds 1-7 showed a dose response in both COX-1 and COX-II inhibitory assays. Compounds 6 and 7 showed 64.7 and 64.3 % of DPPH- 66 radical scavenging activities at 2.5 mg mL", respectively, compared to 14.3, 8.3 and 31.7 % of DPPH. radical scavenging activities for vitamin E, vitamin C and BHT at 10, 25 and 50 pM, respectively. Cyclooxygenase enzyme inhibitory activities of compounds 8-12 were discussed in Chapter 3. Compounds 8-12 showed 16, 66, 36, 77 and 42 % inhibition at 100 pg mL'1 and pH 7, respectively, in COX-I enzyme inhibitory assay while they gave 18, 19, 7, 16 and 19 % of inhibition at 100 pg mL'1 and pH 7, respectively, in COX-II enzyme inhibitory assay. Compound 11 showed a dose response in COX-I inhibitory assay and revealed 77, 79, 64, 55 and 20 % of COX-1 inhibition at 100, 50, 25, 12.5 and 6.25 pg mL'l, respectively. Compounds 1, 3-7 and 11 were evaluated for topoisomerase enzyme inhibitory and gap junctional intercellular communication activities (Chapter 4). Compound 1 is a moderate topoisomerase-II inhibitor when tested at 250 pg concentration. In ras- and myc/ras-transduced rat liver epithelial cell lines, compounds 5 and 7 showed growth inhibition at 181 and 182 pM, respectively. In ras-transduced rat liver epithelial cells, compounds 1, 3 and 4 showed growth inhibition at 287, 46.8 and 217 pM, respectively. Compound 5 distinctly changed cell morphology and acted as a reversible cytostatic compound. It also slightly upregulated GJIC at 181 pM in ras-transduced rat liver epithelial cells. This is the first report of potential anticancer activities of compound 1 and 5 using topoisomerase enzyme inhibitory and GJIC assay. The results of my research on cyclooxygenase enzyme inhibitory and antioxidant activities of crude extract and purified compounds of kava kava roots provided additional scientific support for the anecdotal claims on the traditional use of kava kava roots for 67 controlling inflammatory pain by Pacific Islanders. Antioxidant, cyclooxygenase and topoisomerase enzyme inhibitory and cytostatic activities of purified kava compounds may account for the low cancer incidence in Fiji and other Pacific Island nations where the drink prepared from P. methysticum roots is consumed on a regular basis. Although P. methysticum roots have been approved for treatment of anxiety disorders in Germany since 1990, it only became popular in the US. recently as an anti- anxiety agent in alternative health field. Antioxidant and anticancer properties of crude extract and purified compounds from P. methysticum roots have rarely been reported and unknown for a long time. My research on P. methysticum roots yielded antioxidant, anti- inflammatory and potential anticancer natural products including a novel compound and determination of their efficacy in human might be worthwhile and should be studied further. Additional research on compound 5 is needed to identify the anticancer activities of this compound which has shown cytostatic effect and growth inhibition of oncogene transformed cells. Since kava kava compounds identified in my research demonstrated antioxidant, cyclooxygenase and topoisomerase enzyme inhibitory and cytostatic activities in in vitro assay, they have potential for pharmaceutical or clinical applications in human against inflammation and cancers. Determination of mode of actions of these compounds would be helpful to decide whether these natural products can be used as pharmaceuticals or phytoceuticals since anti-inflammatory and antioxidant processes are involved in the anticarcinogenesis as well. The results of my research support the consumption of kava kava beverage and its 68 use in alternative medicine. It also might provide new chemical templates for the development of novel anticancer and anti-inflammatory pharmaceuticals. 69 LITERATURE CITED Abramson, H. 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