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CHAPTER 1 INVESTIGATION AND PROJECT OVERVIEW Introduction Over 20,000 plant species are used as a primary form of medicine

throughout the world. Over 3 million people depend on medicinal plants as a source of healing (Anonymous, 2002). Most of these species are angiosperms, of which there are over 257,000 species (Judd, et al., 2002). However, only 0.5% of angiosperm species have been researched for pharmacological properties. Traditional use of plants as medicine has been found to be a major indicator of bioactivity (Verpoorte, 1998). Conventional medicine in the United States today is wary of the use of medicinal plants and botanical dietary supplements, regardless of Congressional law, which solidly supports public access to these products (US Congress, 1994). Similarly, many scientific disciplines sustain an active interest in the potential application of botanical products to health and disease, though a cautious tenor concerning safety and efficacy is apparent. As evidenced by the robust academic literature, science and medicine are nonetheless poised to embrace a direction in plant-based medicinal products. One particularly important application of medicinal plants involves the amelioration of pathological disorders related to heightened stress sensitivity and the dysregulation of stress-coping mechanisms, which contribute to nearly two-thirds of acute and chronic disease in the United States (Sabban and Kvet!ansk", 2001). Stress has been shown to have a crucial role in insomnia, chronic anxiety disorder and depression, fatigue, sleep disorders, addictive behavior, neurodegeneration, allergic and autoimmune disorders, metabolic syndrome, gastrointestinal diseases, and pre-term labor (Vgontzas et al., 1998; Grammatopoulos and Chrousos, 2002). Stress is linked to many chronic diseases such as syndrome X, diabetes, and other autoimmune conditions. Hyper- and hypofunctional symptoms of stress include infertility, impotence, anxiety, depression, illness, fatigue, weakness, panic disorder, chronic fatigue, free radical neural damage, memory deficits, and increased aging of the brain (Chrousos and Gold, 1992; McEwen, 1999; McEwen and Wingfield, 2003). Stress also plays a role in vulnerability to infectious diseases such as in stomach ulcers (Helicobacter pylori), progression of HIV, influenza, and the common cold (Elenkov and Chrousos, 1999). Stress hormones such as catecholamines themselves, have been shown to enhance bacterial growth in vitro and may play a role in the progression of infection in the host (Belay et al., 2003). Many classes of conventional drugs are used to treat stress-related conditions, although their safety and efficacy is still uncertain (Carrasco and Van de Kar, 2003). The ethnobotanical, medical and phytochemical literature is replete with plant remedies used by various world cultures to ameliorate stressrelated pathologies. Among these, the adaptogen remedies are the most recently developed and most broadly used; they even have been suggested for use in central nervous system ailments (Carlini, 2003). As expanded upon in Chapter 4, an adaptogen is a substance that has the ability to increase resistance to stress and to ameliorate tissue damage caused by stress, as well as ameliorate some chronic diseases linked to stress. The adaptogenic activity is an accepted pharmacological property of specific drugs in Russia, Sweden and Armenia. The original definition of an adaptogen requires that the substance be innocuous, nonspecific (any type of stress), and be able to adjust to normal excess or deficient pathological symptoms (Brekhman and Dardymov 1969; Davydov and Krikorian, 2000). The adaptogen class of plant remedies will be the primary focus of this thesis because all of the recent developments in this field need to be synthesized. The scientific research of these remedies includes hypotheses concerning the exact chemical compounds believed to be responsible, along with potential mechanisms of action. A cursory review of adaptogenic species indicates they are widespread throughout angiosperm plant families. The chemical characteristics of related species may help explain why adaptogens are so widespread in angiosperms, a trend common to phylogenies of secondary metabolites (Wink, 2003). Recent restructuring of angiosperm plant families due to new DNA techniques affords an excellent opportunity to study the evolution of adaptogens and determine if phylogeny can be used as a predictor of adaptogenic properties in species never before chemically analyzed. The development of new tools, such as the Angiosperm Phylogeny Database and Phylomatic, enable the non-systematist and nonphylogeneticist to access up-to-date information on species relationships and thus the ability to study the evolution of plant traits, and questions pertaining to the origins of species with adaptogenic properties.

Goals of Investigation My hypothesis is that by determining the phylogenetic relationships of plant species with adaptogenic activity, we can begin to search for other potentially adaptogenic plant species that are presently unknown. The phylogenetic presentation of the origins of adaptogenic properties in the angiosperms will be the topic of the final chapter. A phylogenetic tree that represented all of the major subgroups of angiosperms was required in this study because species with adaptogenic properties are found in diverse angiosperm families. An extensive angiosperm phylogeny is needed in order to ascertain whether species with adaptogenic properties tend to be related (i.e., adaptogenic properties tend to be inherited from common ancestors) and/or isolated among groups otherwise lacking adaptogenic properties (i.e., adaptogenic properties tend to be independently evolved). If adaptogenic properties tend to be inherited from common ancestors, the relatives of known adaptogenic species should be targeted for phytochemical and pharmacological study. Methods and Materials The following steps involving a thorough search of the scientific literature were needed to meet this goal. Step 1: A literature search was made for reports of angiosperm species having adaptogenic activity, including medicinal and other uses, exact chemical makeup, and potential mechanism of action of adaptogenic species. This represented an effort to establish the potential homology of the chemical properties among a set of adaptogenic species chosen for this analysis. That is, a preliminary assessment was made that chemical similarity among adaptogenic species could be due to inheritance from a common ancestor. This step is described in Chapters 2, 3, 5, and 6. Step 2: Species were selected that met a precise definition of adaptogen, which is to be described in Chapter 4 along with the adaptogenic species selected for this study. Step 3: A representative angiosperm phylogeny was derived from exemplar species that represented all of the major subgroups or clades of angiosperms without regard to phytochemical constituents or medicinal use. The source used to supply angiosperm species to contrast with the adaptogenic species was the Angiosperm Phylogeny Database (http://www.mobot.org/MOBOT/research/APweb/). This source also contributed up-to-date information related to the higher-level classification for each sampled species. Both the adaptogenic species and background species was then submitted to Phylomatic (http://www.phylodiversity.net/phylomatic/) in order generate a tree description, which could be submitted to MacClade (Maddison and Maddison, 2001), PAUP (Swofford, 2001), or TreeView (Page, 2000) for graphical output. These steps are described in Chapters 7. Step 4: The selected adaptogenic species were then included in the phylogeny of exemplar angiosperm species, and the degree of relatedness among the adaptogens was then determined using standard methods, as described in Chapter 7. Summary The field of natural products and the use of these products in medicine have been extremely valuable to our society. The fields of phytochemistry and ethnopharmacology maintain a keen interest in medicinal plants and willcontinue to provide a source of novel drugs in the future (Breinbauer et al., 2002; Gottlieb et al., 2002; Raskin et al., 2002; Newman et al., 2003; Wu et al., 2003a; Wu et al., 2003b; Rouhi, 2003). Needless to say, there are still important challenges to overcome in finding treatments for serious diseases, induced by exposure to stress. Therefore, an exploration and identification of adaptogenic species is relevant today. This thesis will contribute not only a shopping list of adaptogenic species, but also an introduction and overview of the subject with which to guide future research. The phylogeny of

adaptogenic species could help identify many more unrecognized adaptogens, which undoubtedly will contribute novel mechanisms of action to pharmacology. This thesis is unique in that no attempt has yet been made to investigate the evolutionary relationships of adaptogenic plant species, especially in light of the recent rearrangements in the major angiosperm groups, especially at family and subfamily levels. This thesis provides a valuable and timely investigation into the identity and phylogenetic relationships of adaptogenic plants. This will facilitate a search for additional species that have adaptogenic properties, but that are unknown in this regard.

Robyn Klein 2006 www.rrreading.com Phylogenetic and phytochemical characteristics of plant species with adaptogenic properties MS Thesis, 2004, Montana State University Chapter 1 of 8

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