44 CHAPTER 8 PHYLOGENETIC ANALYSIS Adaptogenic Trait of Plant Species My hypothesis is that the most current phylogenetic angiosperm hypothesis

can predict the occurrence of adaptogenic plant species. To describe known adaptogenic plant species and to investigate the evolutionary relationships of these plant species I used the current angiosperm phylogeny database, which is based on recent DNA sequence data banks, such as Gen Bank (http://www.ncbi.nlm.nih.gov/Genbank/) and TreeBase (http://www.treebase.org/treebase/). Phylogeny is the genealogy of species and higher level taxa. Modern systematic classification of living taxa is derived from genealogy. Species formally classified into a subgenus, genus, tribe, subfamily, family, etc., are expected to meet the criterion of monophyly. A monophyletic lineage comprises species whose most recent common ancestor is not shared with any other species. Understanding the phylogenetic history of a group of plants is the most important element in classification (Bohm and Stuessy, 2001). In cladistic analysis, branching patterns are constructed from shared derived character states (synapomorphies). Maximum numbers of these states are used to form common branches between taxa and this is achieved via a parsimonious mapping of synapomorphies onto the phylogeny. The guiding principle is that the simplest explanation is most likely the correct one, which is actually a complex philosophical concept. Cladistics provides a method of quantifying information by mapping traits onto a tree, thus showing hypothetical evolutionary origins of those traits. In a phylogenetic analysis, random origins of a trait suggest independent evolution, whereas, clustering of traits on a phylogeny suggests inheritance from common ancestry in explaining shared similarity (chemical or otherwise) among species. The degree of clustering can be quantified to show the amount of prediction. Targeting species that belong to clades rich in adaptogenic species, for example, can lead to the discovery of new species with novel properties. There is little known about the functional or adaptive significance of chemical compounds that plants synthesize, although this remains an active area of research in the plant sciences (Wink, 1999; Harrewjin et al., 2001; Grassman et al., 2002; Havsteen, 2002; Nurnberger and Brunner, 2002; Winkel-Shirley, 2002). While it has been common to use phylogeny to indicate chemical compound relatedness (Grayer et al., 1999; Bohm and Stuessy, 2001; Gottlieb, 2002), to my knowledge, this is the first time a pharmacological class of medicinal plants, i.e., adaptogens, has been analyzed phylogenetically. In the case of this investigation, the trait mapped onto an evolutionary tree was the adaptogenic activity of angiosperm plant species. Similar analyses have used molecular phylogenies as a framework to map and interpret the distribution of plant secondary metabolites (Wink, 2003). Instead of secondary metabolites, I have used the molecular phylogeny of the Angiosperm Phylogeny Group and Phylomatic program to indicate evolutionary relationships between plant species having adaptogenic activity, a functional, pharmacological trait that is likely linked to secondary compounds. I focused on those species tested and found to have adaptogenic activity and mapped them with selected species from well-known plant groups which have never been shown as potential adaptogen candidates. These latter species provided a contrasting background. Methods This thesis involved the use of many scientific databases and software programs available on the Internet. The essential methods employed are provided in a brief format in Table 8 below.

45 Table 8. The essential methods employed to build a phylogenetic tree of angiosperm species with and without the adaptogenic trait. Developed a list of angiosperm species that had some evidence of potential adaptogenic traits. Identified species with best evidence of the adaptogenic trait. Built list of species from same families with poor evidence of adaptogenic trait to provide framework. Checked correct plant nomenclature of all species Submitted list of combined species with family name to Phylomatic database. Downloaded output Nexus file and added in the adaptogenic trait. Submitted edited Nexus file to MacClade and PAUP. Manually resolved polytomies using recent phylogenetic research. Entered data from the output file into tree-building software programs. Used tree statistical approaches to evaluate the degree of homology.

The Missouri Botanical Gardens VAST (VAScular Tropicos) nomenclatural database was accessed from the Missouri Botanical Garden website (http://mobot.mobot.org/W3T/Search/vast.html) in order to secure a consistent and agreed-upon nomenclature for all the species discussed in this thesis. The Missouri Botanical Garden also hosts the Angiosperm Phylogeny Group (APG) (http://www.mobot.org/MOBOT/research/APweb/) that provides a comprehensive phylogeny-based treatment of angiosperms that is regularly updated to reflect newly published research contributions. Another important source consulted was the recently published text, Plant Systematics: A Phylogenetic Approach (Judd et al., 2002) that reflects and incorporates new systematic data that has resulted from the prior two decades of scientific contributions involving angiosperm taxonomy, systematics and phylogeny. This source, along with newly published research papers was used for current evolutionary placement of plant families, subfamilies, and species. Four main software programs, Phylomatic, PAUP (Phylogenetic Analysis Using Parsimony), McClade and TreeView were employed to build a phylogenetic tree and show evolutionary relationship. Phylomatic (http://www.phylodiversity.net/phylomatic/) takes any list of angiosperm taxa and matches them by genus to the APG megatree, or failing that, by family name. Species are occasionally found to be absent from the APG website. If a genus is missing from the megatree, the returned phylogeny will show a polytomy or unresolved relationship for that genus. The APG megatree may also not be able to resolve the order in which species descended from a single common ancestor. Thus, relationships among species with a genus will be unresolved. I consulted Judd, et al. (2002) and the recent phylogenetic literature to manually resolve relationships in areas of the phylogeny not resolved in the Phylomatic output. These unresolved relationships are produced on a tree as a multifurcating node (polytomy) because the order in which these species descended from a single common ancestor is not resolved. The Angiosperm megatree contains some unresolved polytomies. Hypothesized ancestral relationships can sometimes be found in recently published phylogenetic studies. Because sampling in this study occurred primarily at the genus level, there were not many polytomies to manually resolve. To name a few of these amendments, I consulted phylogenetic studies such as, Dong et al. (2003) for Astragalus membranaceus and Astragalus membranaceus var. mongholicus; Saunders (1998) for Schisandra chinensis and Schisandra glabra; and Bohm and Stuessy (2001) for the Cardueae species, Rhaponticum uniflorum, Saussurea costus, Centaurea

46 maculosa, Leuzea carthamoides, and Serratula coronata. Thus, polytomies were manually resolved after the returned output file from the Phylomatic database (see next). The list of taxa was formatted appropriately and input to the Phylomatic database on the Internet as shown in Table 9. Table 9. List of taxa applied to Phylomatic in formatted style.
Dioscoreaceae Dioscoreaceae Dioscoreaceae Dioscoreaceae Dioscoreaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Magnoliaceae Menispermaceae Menispermaceae Menispermaceae Menispermaceae Berberidaceae Berberidaceae Berberidaceae Berberidaceae Berberidaceae Berberidaceae Amaranthaceae Amaranthaceae Amaranthaceae Amaranthaceae Amaranthaceae Amaranthaceae Crassulaceae Crassulaceae Crassulaceae Crassulaceae Crassulaceae Crassulaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Cucurbitaceae Cucurbitaceae Cucurbitaceae Cucurbitaceae Cucurbitaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Gentianaceae Stenomeris Tacca Dioscorea Trichopus Trichopus Schizandra Schisandra Magnolia Liriodendron Michelia Tinospora Menispermum Abuta Cissampelos Epimedium Epimedium Epimedium Berberis Caulophyllum Podophyllum Achyranthes Achyranthes Chenopodium Atriplex Amaranthus Celosia Rhodiola Rhodiola Rhodiola Sedum Crassula Echeveria Senna Mimosa Acacia Indigofera Dalea Glycyrrhiza Glycyrrhiza Astragalus Astragalus Bryonia Sicyos Cucurbita Gynostemma Momordica Hesperis Capparis Lepidium Rorippa Arabis Swertia dioscoreifolia pinnatifida sativa sempervirens zeylandicus chinensis glabra virginiana tulipifera champaca cordifolia canadense rufescens pareira brevicornu koreanum saggitatum vulgaris thalictroides peltatum bidentata aspera album hortensis caudatus argentea rosea sacra sachalinensis acre perfoliata coccinea Alexandria sensitive scorpioides tinctoria alopecuroides glabra uralensis membranaceus membranaceus var mongholicus alba angulatus pepo pentaphyllum balsamina matronalis spinosa meyenii sylvestris alpina perennis

47
Gentianaceae Gentianaceae Gentianaceae Gentianaceae Lamiaceae Lamiaceae Lamiaceae Lamiaceae Lamiaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Scrophulariaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Araliaceae Araliaceae Araliaceae Araliaceae Araliaceae Araliaceae Araliaceae Araliaceae Araliaceae Araliaceae Araliaceae Campanulaceae Campanulaceae Campanulaceae Campanulaceae Campanulaceae Campanulaceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Halenia Gentiana Hoppea Hoppea Scutellaria Stachys Ocimum Salvia Hyptis Scrophularia Selago Manulea Bacopa Verbascum Cestrum Nicotiana Lycianthes Lycium Solanum Withania Eryngium Sanicula Eleutherococcus Oreopanax Oplopanax Schefflera Hedera Dendropanax Aralia Panax Panax Lobelia Centropogon Siphocampylus Campanula Codonopsis Codonopsis Hieracium Rhaponticum Leuzea Serratula Saussurea Centaurea Aster Eupatorium Artemisia sibirica lutea dichotoma fastigiata galericulata sylvatica sanctum officinalis capitata nodosa corymbosa cheiranthus monniera thapsus nocturnum tabacum lycioides afrum nigrum somnifera maritimum europaea senticosus capitatus elatus digitata helix arboreus mandshurica ginseng quinquefolium cardinalis surinamensis westinianus latifolia pilosula lanceolata murorum uniflorum carthamoides coronata costus maculosa amellus cannabinum vulgaris

Phylomatic produces an output document in a format called Nexus. This Nexus document has two main sections: the list of the species that were entered with an added tab space followed by a question mark; and at the end, a list of the species entered in paragraph form and grouped within sets of parentheses. See Figure 7 below. Figure 7. The Nexus file resulting from Phylomatic. Presence or absence of adaptogenic trait using 0/1 codes was added. ?0 represents absence of trait; ?1 represents presence of trait.
#NEXUS [! Online output from Phylomatic; www.phylodiversity.net/phylomatic] [! Run at: Mon Nov 17 18:52:15 EST 2003 ]

48
BEGIN DATA; DIMENSIONS NTAX=98 NCHAR=1; FORMAT MISSING=? GAP=- DATATYPE=STANDARD; MATRIX [ [ ] ]

Swertia_perennis ?0 Halenia_sibirica ?0 Gentiana_lutea ?0 Hoppea_dichotoma ?1 Hoppea_fastigiata ?1 Menispermum_canadense ?0 Abuta_rufescens ?0 Tinospora_cordifolia ?1 Cissampelos_pareira ?0 Sedum_acre ?0 Rhodiola_rosea ?1 Rhodiola_sacra ?1 Rhodiola_sachalinensis ?1 Echeveria_coccinea ?0 Crassula_perfoliata ?0 Podophyllum_peltatum ?0 Caulophyllum_thalictroides ?0 Berberis_vulgaris ?0 Epimedium_brevicornu ?1 Epimedium_koreanum ?1 Epimedium_saggitatum ?1 Rorippa_sylvestris ?0 Lepidium_meyenii ?1 Capparis_spinosa ?0 Arabis_alpina ?0 Hesperis_matronalis ?0 Scrophularia_nodosa ?0 Selago_corymbosa ?0 Manulea_cheiranthus ?0 Bacopa_monniera ?1 Verbascum_thapsus ?0 Bryonia_alba ?1 Sicyos_angulatus ?0 Gynostemma_pentaphyllum ?1 Cucurbita_pepo ?0 Momordica_balsamina ?0 Serratula_coronata ?1 Rhaponticum_uniflorum ?1 Aster_amellus ?0 Eupatorium_cannabinum ?0 Centaurea_maculosa ?0 Leuzea_carthamoides ?1 Artemisia_vulgaris ?0 Saussurea_costus ?0 Hieracium_murorum ?0 Eleutherococcus_senticosus ?1 Sanicula_europaea ?0 Oreopanax_capitatus ?0 Oplopanax_elatus ?1 Eryngium_maritimum ?0 Dendropanax_arboreus ?0 Hedera_helix ?0

49
Schefflera_digitata ?0 Panax_ginseng ?1 Panax_quinquefolium ?1 Aralia_mandshurica ?1 Ocimum_sanctum ?1 Hyptis_capitata ?0 Stachys_sylvatica ?0 Salvia_officinalis ?0 Scutellaria_galericulata ?0 Atriplex_hortensis ?0 Celosia_argentea ?0 Chenopodium_album ?0 Amaranthus_caudatus ?0 Achyranthes_bidentata ?1 Achyranthes_aspera ?0 Mimosa_sensitive ?0 Indigofera_tinctoria ?0 Acacia_scorpioides ?0 Senna_Alexandria ?0 Astragalus_membranaceus ?1 Astragalus_membranaceus var mongholicus Glycyrrhiza_glabra ?1 Glycyrrhiza_uralensis ?1 Dalea_alopecuroides ?0 Michelia_champaca ?0 Liriodendron_tulipifera ?0 Magnolia_virginiana ?0 Schizandra_chinensis ?1 Schisandra_glabra ?0 Cestrum_nocturnum ?0 Solanum_nigrum ?0 Lycianthes_lycioides ?0 Nicotiana_tabacum ?0 Lycium_afrum ?0 Withania_somnifera ?1 Tacca_pinnatifida ?0 Trichopus_sempervirens ?1 Trichopus_zeylandicus ?1 Dioscorea_sativa ?0 Stenomeris_dioscoreifolia ?0 Lobelia_cardinalis ?0 Codonopsis_pilosula ?1 Codonopsis_lanceolata ?1 Siphocampylus_westinianus ?0 Centropogon_surinamensis ?0 Campanula_latifolia ?0 ; END;

?1

BEGIN TREES; TREE 'main' = [&R] ((Tacca_pinnatifida,(Trichopus_sempervirens,Trichopus_zeylandicus),Dioscorea_sa tiva,Stenomeris_dioscoreifolia),((((Liriodendron_tulipifera,Magnolia_virginiana ),Michelia_champaca),(Schisandra_glabra,Schisandra_chinensis)),(((Menispermum_c anadense,Abuta_rufescens,Tinospora_cordifolia,Cissampelos_pareira),(Caulophyllu m_thalictroides,Berberis_vulgaris,((Epimedium_brevicornu,Epimedium_koreanum,Epi medium_saggitatum),Podophyllum_peltatum))),((Celosia_argentea,Chenopodium_album ,(Amaranthus_caudatus,Atriplex_hortensis),(Achyranthes_bidentata,Achyranthes_as pera)),((Sedum_acre,(Rhodiola_rosea,Rhodiola_sacra,Rhodiola_sachalinensis),Eche veria_coccinea,Crassula_perfoliata),((((Senna_alexandria,(Mimosa_sensitive,Acac ia_scorpioides)),(Dalea_alopecuroides,(Indigofera_tinctoria,((Glycyrrhiza_glabr

50
a,Glycyrrhiza_uralensis),(Astragalus_membranaceus_var_mongolicus,Astragalus_mem branaceus))))),(Bryonia_alba,Sicyos_angulatus,Cucurbita_pepo,Gynostemma_pentaph yllum,Momordica_balsamina)),(Hesperis_matronalis,Capparis_spinosa,(Lepidium_mey enii,Rorippa_sylvestris,Arabis_alpina)))),(((Swertia_perennis,Halenia_sibirica, Gentiana_lutea,(Hoppea_dichotoma,Hoppea_fastigiata)),(((Scutellaria_galericulat a,(Ocimum_sanctum,Hyptis_capitata,Salvia_officinalis),Stachys_sylvatica),(Scrop hularia_nodosa,Selago_corymbosa,Manulea_cheiranthus,Bacopa_monniera,Verbascum_t hapsus)),(Cestrum_nocturnum,Nicotiana_tabacum,(Lycianthes_lycioides,Lycium_afru m,Solanum_nigrum,Withania_somnifera)))),(((Eryngium_maritimum,Sanicula_europaea ),(Eleutherococcus_senticosus,Oreopanax_capitatus,Oplopanax_elatus,Schefflera_d igitata,Hedera_helix,Dendropanax_arboreus,((Panax_ginseng,Panax_quinquefolium), Aralia_mandshurica))),(((Lobelia_cardinalis,Centropogon_surinamensis,Siphocampy lus_westinianus),((Codonopsis_pilosula,Codonopsis_lanceolata),Campanula_latifol ia)),(((Rhaponticum_uniflorum,Serratula_coronata,Leuzea_carthamoides,Centaurea_ maculosa,Saussurea_costus),(Aster_amellus,Eupatorium_cannabinum,Artemisia_vulga ris)),Hieracium_murorum)))))))); END;

The paragraph of taxa at the end of the Nexus file is arranged using parentheses to denote phylogenetic groupings according to the APG megatree. To reiterate, there were some unresolved relationships or polytomies which were amended using recent literature before input to the tree drawing programs, PAUP, MacClade and TreeView. Once the Nexus file was produced it was copied to Microsoft! Word 2000 software program. In order to signify absence or presence of the adaptogen trait, the numbers, 0 or 1, were added after the question mark (?) on the list of species in the first section. The Nexus format can be read directly by many phylogenetic tree programs such as PAUP, MacClade and TreeView, allowing easy interchange of data between programs. These high-speed computer programs analyze molecular and morphological data in order to infer phylogenetic relationships and evolutionary trees. After manually coding for absence or presence of adaptogenic trait, the Nexus file was then input into PAUP (Swafford, 2000) (http://paup.csit.fsu.edu/about.html), MacClade (Maddison and Maddick, 2001) (http://macclade.org/) and Treeview (Page, 2003) (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) to graphically illustrate the phylogenetic trees. The tree that resulted from TreeView is shown in Figure 8 with family names added and adaptogenic species bolded. The non-bolded species have not been tested for the adaptogenic trait. However, there is neither any strong evidence from ethnobotanical or traditional medicine that would signify adaptogenic activity. The resulting phylogeny shows that out of 33 species having adaptogenic traits, there are at least 14 cases of independent evolution. This suggests that the adaptogenic trait is prone to independent evolution. It is not unusual for phylogenetic analyses using secondary compound traits to show an erratic distribution (Wink, 2003). Since the adaptogenic trait is likely linked to

51

Dioscoraceae

Magnoliaceae

Menispermaceae

Berberidaceae

Amaranthaceae

Crassulaceae

Brassicaceae

Cucurbitaceae

Fabaceae

to Figure 8.

Tacca pinnatifida Stenomeris dioscoreifolia Dioscorea sativa Trichopus sempervirens Trichopus zeylanicus Schisandra glabra Schisandra chinensis Michelia champaca Liriodendron tulipifera Magnolia virginiana Menispermum canadense Cissampelos pareira Tinospora cordifolia Abuta rufescens Caulophyllum thalictroides Berberis vulgaris Podophyllum peltatum Epimedium brevicornu Epimedium saggitatum Epimedium koreanum Celosia argentea Chenopodium album Achyranthes bidentata Achyranthes aspera Amaranthus caudatus Atriplex hortensis Sedum acre Crassula perfoliata Echeveria coccinea Rhodiola rosea Rhodiola sachalinensis Rhodiola sacra Hesperis matronalis Capparis spinosa Lepidium meyenii Arabis alpina Rorippa sylvestris Bryonia alba Momordica balsamina Gynostemma pentaphyllum Cucurbita pepo Sicyos angulatus Senna alexandria Mimosa sensitive Acacia scorpioides Dalea alopecuroides Indigofera tinctoria Glycyrrhiza glabra Glycyrrhiza uralensis Astragalus membranaceus var mongholicus Astragalus membranaceus

Figure 8. Phylogenetic tree resulting from Phylomatic of selected angiosperm taxa including adaptogenic species in bold.

52

Gentianaceae
from Figure 8.

Solanaceae

Lamiaceae

Scrophulariaceae Apiaceae Araliaceae

Campanulaceae

Asteraceae

Swertia perennis Gentiana lutea Halenia sibirica Hoppea dichotoma Hoppea fastigiata Cestrum nocturnum Nicotiana tabacum Lycianthes lycioides Withania somnifera Solanum nigrum Lycium afrum Scutellaria galericulata Stachys sylvatica Ocimum sanctum Salvia officinalis Hyptis capitata Scrophularia nodosa Verbascum thapsus Bacopa monniera Manulea cheiranthus Selago corymbosa Eryngium maritimum Sanicula europaea Eleutherococcus senticosus Dendropanax arboreus Hedera helix Schefflera digitata Oplopanax elatus Oreopanax capitatus Aralia mandshurica Panax ginseng Panax quinquefolium Lobelia cardinalis Siphocampylus westinianus Centropogon surinamensis Campanula latifolia Codonopsis pilosula Codonopsis lanceolata Hieracium murorum Aster amellus Artemisia vulgaris Eupatorium cannabinum Rhaponticum uniflorum Saussurea costus Centaurea maculosa Leuzea carthamoides Serratula coronata

53 secondary compounds, a similar trend should be expected. Wink (2003) attributes such highly distributed and erratic trends to either convergence, in which the responsible biosynthetic genes evolved independently, or to genes that have been turned off. In the present phylogeny, there is evidence of shared, adaptogenic similarity due to inheritance from common ancestry in the eight clades as illustrated in Table 10 below. Table 10. Monophyletic groups resulting from the phylogenetic tree (Figure 8) in which shared similarity of adaptogenic traits is due to inheritance from the most recent common ancestor. Dioscoreaceae Berberidaceae Trichopus sempervirens (H. Perrier) Caddick & Wilkin Trichopus zeylandicus Gaertn. Epimedium brevicornu Maxim. Epimedium koreanum Nakai Epimedium saggitatum (Siebold & Zucc.) Maxim. Rhodiola rosea L. Rhodiola sacra (Prain ex Raym.-Hamet) S.H. Fu Rhodiola sachalinensis Boriss. Glycyrrhiza glabra L. Glycyrrhiza uralensis Fisch ex. D.C. Astragalus membranaceus Moench Astragalus membranaceus var mongholicus (Bunge) P.K. Hsiao Hoppea dichotoma Willd. Hoppea fastigiata (Grisebach) Clarke Panax ginseng C.A. Mey Panax quinquefolium L.

Crassulaceae

Fabaceae

Gentianaceae Araliaceae

Campanulaceae Codonopsis pilosula (Franch.) Nannf. Codonopsis lanceolata (Siebold & Zucc.) Trautv. Asteraceae Leuzea carthamoides (Willd.) DC. Rhaponticum uniflorum (L.) DC. Serratula coronata L.

The present phylogenetic analysis involving adaptogenic activity as a trait should be seen as a rudimentary effort that needs further refinement. In particular, all plant species have not been tested thoroughly for the adaptogenic trait. Therefore, many species closely related to the adaptogenic species are lacking sufficient evidence of adaptogenic activity and thus were not included. Nonetheless, some of these species may have similar bioactivity evidenced by the tonic properties in Aralia schmidtii (Baranov, 1982), cardiotonic properties in Panax notoginseng (Dong et al., 2003), the aphrodisiac uses of Panax pseudoginseng (Rai and Sharma, 1994), the similar chemistry of Schisandra glabra (Saunders, 1998), the similar chemistry of Achyranthes fauriei (Ida et al., 1998), and the radioprotective and memory enhancement properties of Amaranthus paniculatus (Bhatia and Jain, 2003). Thus, further testing of angiosperm species for adaptogenic activity could add more species to these same monophyletic clades, as well as add monophyletic clades to lone adaptogens as identified in this phylogenetic analysis.

54 Independent evolution can also be viewed in the placement of the 33 adaptogenic species within the major angiosperm orders. It can be seen in Table 11 that adaptogenic species are widely dispersed among angiosperm orders though missing in many orders. Monophyletic Clades with Adaptogen Trait The previously listed eight clades of adaptogenic species are enumerated morphologically, ecologically, and biogeographically in this section. After a brief

55 Table 11. Major angiosperm orders with adaptogenic species (Judd et al., 2002). MAGNOLIID COMPLEX Magnoliales Schisandra chinensis Laurales Canellales Piperales MONOCOTS Acorales Alismatales Liliales Nartheciales Asparagales Dioscoreales Malpighiales Fabales

Glycyrrhiza glabra G. uralensis Astragalus membranaceus A. membranaceus var mongholicus Bryonia alba Gynostemma pentaphyllum

Rosales Cucurbitales Fagales Myrtales Trichopus sempervirens T. zeylanicus EUROSIDS II Brassicales Malvales Sapindales

Lepidium meyenii

COMMELINOID CLADE Arecales Commelinales Poales Zingiberales EUDICOTS Ranunculales Tinospora cordifolia Epimedium brevicornu E. saggitatum E. koreanum

ASTERID CLADE Cornales Ericales EUASTERIDS I Garryales Solanales Withania somifera Gentianales Hoppea dichotoma H. fastigiata Lamiales Ocimum sanctum Bacopa monniera EUASTERIDS II Aquifoliales Apiales Eleutherococcus senticosus Oplopanax elatus Aralia mandshurica Panax ginseng P. quinquefolium Dipsacales Asterales Codonopsis pilosula C. lanceolata Leuzea carthamoides Rhaponticum uniflorum Serratula coronata

CORE EUDICOTS Caryophyllales Achyrathes bidentata Polygonales Santalales ROSID CLADE Saxifragales Rhodiola rosea R. sachalinensis R. sacra Vitales Geraniales EUROSIDS I Zygophyllales Oxalidales Celastrales

67 presentation of each constituent adaptogenic species, a summary of future prospects and research direction is provided for each of the 8 subclades. Trichopus in the Dioscoraceae The Dioscoraceae (yam family) is made up of species that are twining vines with thick rhizomes or a large tuberlike swelling. Flowers are small, inconspicuous and often greenish and are pollinated mainly by flies. Plants are usually dioecious. These species are mainly found in tropical and warm temperate areas. There are six genera with over 600 species. Three genera are native to Europe, two to North America. The two major genera found in North America are Dioscorea and Stenomeris (Cullen, 1997; Judd et al., 2002). A recent phylogenetic analysis has shown that Dioscorea , Stenomeris, Tacca and Trichopus form a strongly supported monophyletic group (Caddick et al., 2002). Tacca and Trichopus have very similar morphology and are most closely related. Avetra and Trichopus have also been shown to have a close relationship, so much so that Avetra sempervirens has now been reclassified as a second species in Trichopus (T. sempervirens). Thus, there are only two known species of Trichopus (Caddick et al., 2002). Both species grow as perennial climbers or as understory herbs in low lying, wet tropical regions. Trichopus zeylanicus is native to Sri Lanka, Southern India, and peninsular Malaysia. Trichopus sempervirens is found only in Madagascar. The fruit of Trichopus zeylanicus is edible. Insects are suspected pollinators of Trichopus, but this has not been verified (Caddick et al., 2002). There has been very little study of the chemistry of Trichopus species in the literature. Steroidal saponins are common in the Dioscoraceae, along with various alkaloids. For example, Dioscorea has been widely exploited as raw source material for the production of corticosteroid drugs and in the manufacture of estrogen for oral contraceptives. Trichopus sempervirens. The fruit of this species is used by tribal peoples to enhance physical performance, old age, health and vitality (Evans et al. 2002). According to one study using mice, the leaf extract increased swimming performance and decreased glucose. There was an increased efficiency of fuel substrate utilization via a shifting of skeletal muscle energy production towards oxidation of fatty acids (Evans et al., 2002). This bioactivity is descriptive of the adaptogenic effect. Trichopus zeylanicus. The glyco-peptido-lipid fraction in Trichopus zeylanicus has been shown to be a source of adaptogenic activity in rats (Singh et al., 2001a). Subramoniam et al. (1999) found that extracts of this species beneficially affected stamina, old age disorders, increased swimming performance, increased resistance to stress, and showed an immunomodulatory effect. Singh et al., (2000; 2001a) reported that Trichopus zeylanicus decreased adrenal enlargement, enhanced endurance, inhibited stress-induced hypothermia, and increased resistance to nonspecific stress in mice. As an adaptogenic remedy, Trichopus zeylanicus is the focus of intellectual property rights and product development by indigenous tribes in India (Wilder, 2001). Epimedium in the Berberidaceae The Berberidaceae (barberry family) are herbs or shrubs mainly found in Northern temperate zones and the Andes of South America (Judd et al., 2002). There are 15 genera and 650 species. Ten of these genera are found in North America. Berberis, Epimedium and Mahonia are widely cultivated as ornamentals. The major genus, Berberis (600 spp.), along with Achlys, Caulophyllum, Diphyllea, Jeffersonia, Nandina, Podophyllum, and Vancouveria occur in North America. Epimedium, Vancouveria, and Jeffersonia form a monophyletic group (Kim and Jansen, 1998). There are 20 species of Epimedium. In Epimedium , the flowers are bisexual and tend to be showy. Pollination is mainly by bees that gather nectar and pollen. Epimedium species are woodland perennials native to China, Japan, Italy, and Europe and are popularly cultivated. Epimedium species have been heavily researched especially in China. A recent paper extensively reviews the pharmacology and chemistry of the species (Wu et al., 2003b). Though the isoquinoline alkaloid, berberine, is common to many genera in Berberidaceae, it is not a feature in Epimedium species.

68 Instead, the main classes of compounds in Epimedium species are the flavonoid glycosides, flavones, and icarisides, which are all phenolic-based structures. In all, 130 different compounds have been identified in 16 different species of Epimedium (Wu et al., 2003a). Sixty different flavonoid glycosides are found with icariin being present in each species, and therefore used as a marker compound for positive identification in testing for adulterants in medicinal products. Epimedium species vary in amount of icariin from 0.0031% to 3.69%, with an average content of 0.34% on a dry weight basis. Experimental data (both in vitro and in vivo) show that the icariin series of flavonoids and glycosides have androgenic/antiestrogenic and antioxidant properties similar to the flavonoid luteolin (Wu et al., 2003a). Other compounds include 30 flavones, 50 icarisides and many other compounds such as cetyl alcohol, essential oil, fatty acids, tannins, oleic acid, linoleic acid, palmatic acid, phytosterols, phytosteryl glucosides and polysaccharides (Wu et al, 2003a). The phytosterols and polysaccharides in Epimedium species have been shown to stimulate the human immune system (Wu et al., 2003a). Icariin can increase estradiol production of granulosa cells, has a stimulant effect on corticosterone production, has been shown to modulate the hypothalamus-pituitary-testis axis, and has many other effects, including antioxidant, antiinflammatory, immunomodulatory, antitumor and cardiovascular properties (Wu et al., 2003a). These researchers also report that icariin has been shown to increase nitric oxide release which has a role in penile erection, and therefore explains the use of Epimedium as an aphrodisiac. The pharmacological properties of Epimedium are believed to have multiple mechanisms of action and multiple targets (Wu et al., 2003a). Eight of sixteen Epimedium species have been used in China for many centuries to treat a wide range of conditions. The earliest record of medicinal use of Epimedium species in China is dated from 200-300 B.C. (Wu et al., 2003a). Then as now, it was touted as a rejuvenator, able to reinforce muscle and bone and used as a bodybuilding agent. It was especially known to enhance sexual functions in impotence and be useful in asthenic (weakened) conditions. Though few clinical studies have been conducted on humans, Epimedium has been used for millennia in the indigenous medical practice of China (Wu et al., 2003a). A short mention here, but continued later in this chapter, concerns the monophyletic group containing Epimedium and Vancouveria. Vancouveria hexandra is endemic to the Pacific Coast of North America and has virtually no historical or current use in herbal practices. Some chemical analyses of Vancouveria hexandra suggest that it contains many of the same phenylpropanoid types of compounds as species of Epimedium. Vancouveria hexandra is an example of the potential targeting of species known to be closely related to known adaptogens. However, it should be emphasized that Vancouveria has a limited distribution, thus its population would suffer greatly if exploited as a wild harvested medicinal plant. Efforts should be made to enhance its cultivation and thus produce a sustainable source, if in fact, it is found to have adaptogenic properties. Epimedium brevicornu. Both E. brevicornu and E. saggitatum have been shown to have a protective effect in humans on the HPA axis by decreasing levels of ACTH and corticosterone, reducing lymphocyte proliferative reaction, activating monoamine transmitters in the hypothalamus, decreasing the weight of the pituitary, adrenal and thymus, as well as the number of CRH positive neurons in the hypothalamic paraventricular nucleus (Cai et al, 1998). Other potential adaptogenic effects of both these Epimedium species include decreasing CRH positive neurofibrilin median eminence and anterior pituitary ACTH positive secretory cells, reducing adrenal fasciculate zone and thymus cortex atrophies, reducing NK cell cytotoxicity and the level of IL-2 and gamma-IFN in rats, and have benefical effects on neuroendocrineimmune factors inhibited by exogenous glucocorticoid (Cai et al., 1998). Epimedium koreanum. This species also contains glucopyranosides(Sun et al., 1998) and is considered a tonic, aphrodisiac, antirheumatic, and immunostimulant (Kim et al., 2001). Chiba et al. (2002) showed that Epimedium koreanum extracts enhanced immune cells in vitro , improved aging-related amnesia and impotence, and possess nerve growth properties. Epimedium saggitatum . This species has been used to treat side effects from long-term glucocorticoid exposure (Wu et al., 1996), and has been shown to cause nerve cell growth in vitro in cultured cells (Kuroda et al., 2000).

69 Rhodiola in the Crassulaceae Species in the Crassulaceae (stonecrop family) are herbs and shrubs with crassulacean acid metabolism (CAM). The species are worldwide except for Australia. There are 30 genera and about 1,400 species. All are succulent and almost all genera are in cultivation. Rhodiola species are widespread from tropical to boreal regions and often occur in arid habitats, and are mainly pollinated by insects. Thirteen genera are native to Europe and twelve to North America (Cullen, 1997; Judd et al., 2002). Rhodiola rosea. The nomenclature of Rhodiola species is confusing because there are many synonyms, Sedum is the most common. Rhodiola rosea (syn. Sedum integrifolium and Sedum roseum) grows at high altitudes in Europe, Asia, and North America. The chemical investigation of Rhodiola has focused on R. rosea and has identified six main classes of compounds: phenylpropanoids (rosavin, rosin, rosarin), phenylethanol derivatives (salidroside, tyrosol), flavonoids (rodiolin, rodionin, rodiosin, acetylrodalgin, tricin), monoterpenes (rosiridol, rosaridin), triterpenes (phytosterols: daucosterol, beta-sitosterol), and phenolic acids (cholorogenic and hydroxycinnamic, gallic acids) (Brown et al., 2002). This particular species has been heavily exploited in Russia and Scandinavia as a traditional medicine for centuries. More than 180 pharmacological, phytochemical and clinical studies on Rhodiola rosea have been published since 1960 (Brown et al., 2002). Rhodiola rosea is the primary species used in traditional medicine and botanical supplements. Rhodiola rosea is listed in the 1755 Pharmacopoeia of Sweden. Historical reports suggest that its use goes back to the Vikings who used the herb to enhance their physical strength and endurance. Rhodiola rosea has been included in official Russian medicine since 1969. Rhodiola rosea has been the focus of 17 human and 32 animal studies (i.e., Spasov et al., 2000; Darbinyan et al., 2000; Shevtsov et al., 2003). The main use of Rhodiola in Russia are for asthenia (fatigue), somatic and infectious illnesses, psychiatric and neurological conditions, and to relieve fatigue and increase attention span, memory and work productivity (Brown et al., 2002). Some have suggested that other species are not adaptogenic (Brown et al., 2002), however I have found enough evidence to include Rhodiola sacra and Rhodiola sacchalinensis in this study (Ohsugi et al. 1999; Li and Chen 2001). Many other species have also been studied for possible adaptogenic properties, suggesting that other Rhodiola species may also be a source of valuable remedies. Rhodiola sacra. Uses for Rhodiola sacra have been reported to demonstrate anti-anoxia, anti-fatigue, anti-radiation effects, and improvement of memory and learning (Ohsugi et al., 1999). Rhodiola sachalinensis. This species is also considered to have adaptogenic and hepatoprotective properties (Song et al., 2003; Wu et al., 2003). It is used in China to enhance survival in adverse environments, resist anoxia, radiation and fatigue (Li and Chen, 2001). Astragalus and Glycyrrhiza in the Fabaceae The legume family (Fabaceae) is a very large family of about 630 genera and 18,000 species and is sometimes divided into three separate families (Mimosoideae, Caesalpinioideae, and Faboideae). Many genera are native to Europe and/or North America, and many are cultivated. The flowers of Fabaceae are variable in color, shape. Pollinators are varied as well, though bees pollinate most species. In particular, the genera Glycyrrhiza and Astragalus, are in the Faboideae subfamily. Both Glycyrrhiza and Astragalus have the typical bilateral flower with banner, wing and keel petals. Species in both genera have a long history of medicinal use. Astragalus and Glycyrrhiza are known to be somewhat closely related. They both belong to a monophyletic subclade of legumes referred to as the Iverted-Repeat-Loss Clade (Wojciechowski, et al., 2000). One of the most well known Astragalus species is Astragalus gummifera, tragacanth gum. It is used for its polysaccharides that help stabilize drug suspensions and emulsions in pharmacy and is a thickening agent in the food industry (Bruneton, 1995). Glycyrrhiza species are commonly known as licorice and also

70 have industrial uses as flavoring agents and as a sweetener. The root of Glycyrrhiza species are known to contain about 25-30% starch, 3-10% sugars, coumarins, triterpene saponins, steroids as well as numerous flavonoids and isoflavonoids (Hayashi et al., 2003). Glycyrrhizin is the major saponin and makes up 3-5% of the weight of the root. Glycyrrhiza root is one of the oldest and most commonly traded herbs in ethnomedicine. Both species G. glabra and G. uralensis, have been interchangeably used as a sweetener and for medicinal use (Hayashi et al., 2003). From an adaptogenic perspective, the most important medicinal uses of Glycyrrhiza are the antiinflammatory and adrenal tonic properties. The latter involves the triterpene, glycyrrhetic acid, which has been shown to potentiate endogenous corticoids such as cortisol and aldosterone, possibly by inhibiting the enzyme 11"-hydroxysteroid dehydrogenase (Bruneton, 1995). Hyperaldosteronism has been a problem for some who are sensitive to this effect and for those who abusively consume the root tea. Astragalus membranaceus and A. membranaceus var mongholicus. The monophyletic relationship of these two species has recently been phylogenetically supported (Dong et al., 2003). Astragalus is one of the major genera of the Fabaceae with about 2000 species, mostly found in northern temperate regions, but also in the cool, arid regions of South America. Many of the Astragalus species are poisonous, unlike the two species in this treatment which have a long and active history of use in herbal medicine. The original traditional Chinese medicine species used as radix Astragali (pharmaceutical Astragalus) are Astragalus membranaceus, Astragalus membranaceus var mongholicus, and Hedysarum polybotrys (Dong et al., 2003). The inclusion of a Hedysarum species in Chinese herbal medicine suggests yet another closely related genera to explore phytochemically. Other closely related Astragalus species have presented quality control problems in the medicinal herb industry. Thus, there has been great interest in analyzing all Astragalus species. This same phytochemistry research has not been extended to North America, except for taxonomic and systematic purposes (Lavin and Marriott, 1997). The popular use of certain species of Astragalus as adaptogens has been encouraged by its frequent use by traditional Chinese medicine. Astragalus products are considered to be among the most important adaptogens, but also as a mild tonic and immunostimulant. Astragalus membranaceus and Astragalus membranaceus var mongholicus have a very similar morphology and chemical composition and are the two main species representing huang qi, or radix Astragli, the pharmaceutical name for Astragalus. The morphology between these species is so close that it is fairly easy to either purposely or mistakenly collect the wrong species in the wild. Twelve different species have been involved in such problems, therefore chemical and phylogenetic analysis are common research foci of quality control efforts. In particular, Astragalus membranaceus and Astragalus membranaceus var mongholicus are distinctive in that they contain higher amounts of astragaloside IV, though these compounds have been shown to vary according to habitat, season, and age of the plant (Ma et al., 2002; Dong et al., 2003). The constituents most often associated with the activity of Astragalus are isoflavonoids, triterpenoid saponins (astragalosides), and polysaccharides (Ma et al., 2002). Glycyrrhiza glabra. This heavily researched species has been found to contain more than 50 triterpenoids (25%), more than 200 phenolic compounds (3-6%), dozens of polysaccharides (20%), many amino acids (10%), and many lipids (3-4%) (Pavlova et al., 2003). The roots and stolons contain the flavonoid, glabridin, specific to this species (Hayashi et al, 2003). Pharmacological properties mainly center on antiinflammatory, antihyperlipidemic, hepatoprotective, antiulcerogenic effect. It has been shown to decrease cortisol and ACTH, potentiate corticosteroids, and reduce serum testosterone. Glycyrrhiza can induce sodium retention and hypertension in some sensitive individuals especially in high doses (Al-Qaraw et al., 2002). Glycyrrhiza uralensis. The roots and stolons contain the flavonoid, glycycoumarin, specific to this species (Hayashi et al, 2003). The uses of this species are also well-known in Asian herbal medicine and it has antiulcer, hepatoprotective, antitumor, antimutagenic, and antimicrobial properties (Bai et al., 2003)

71 Hoppea in the Gentianaceae The Gentianales is placed within the Asteridae, which comprises the tube-flowered eudicot groups. Gentianales is a large monophyletic group comprising three major and three minor subclades; these correspond to Gentianaceae, Rubiaceae, Apocynaceae, Gelseminaceae, and Loganiaceae (Struwe and Albert, 2002). Of these, the Gentianaceae, with over 1600 species and 87 genera, is the third largest family of the Gentianales (Struwe and Albert, 2002). At least nine genera occur in Europe and 17 in North America. Species of Gentiana are popularly cultivated. Like the Asteridae, nearly all species in the Gentiaceae are sympetalous. Hoppea is described based on H. dichotoma Willd. Only two species are known to occur. They are found in the tropical countries India, Sri Lanka and Pakistan. Hoppea fastigiata is also found in Senegal, Africa. Hoppea is placed in the subtribe Canscorinae, within the Chironieae tribe of 23 genera and 159 species. Schinziella tetragona is the only sister species to Hoppea, forming a separate clade. The description of the species within the Chironieae include mostly cream-colored corollas (Struwe and Albert, 2002). The presence of iridoid compounds or complex indole alkaloids are common among the genera of Gentianaceae. Gentiana lutea is one of the most commonly known medicinal plants in Europe and North America due to its use as a bitter stomach tonic. In the case of Hoppea, these compounds are not in evidence. These species are found in open, moist places such as cultivated soils. Hoppea dichotoma Willd. and Hoppea fastigiata (Grisebach) Clarke. The compounds thought to have adaptogenic properties in Hoppea are the flavan glycosides dichotosin, dichotosinin and diffutin (Ghosal et al., 1985; Wagner et al., 1994) and tetraoxygenated xanthones in both species (Peres et al., 2000). The genus Hoppea is not as well known in modern herbal medicine and is identified as adaptogenic by one source, Wagner et al. (1994). Hoppea is also not commonly found in modern Ayurvedic medicine texts, possibly because access to Hoppea is limited. Panax in the Araliaceae The Araliaceae (ginseng) family has been a topic of controversy in systematics because of its uncertain relationship with the Apiaceae (carrot family). This is because of taxa characterized by a mixture of features typical of both families (Plunkett and Lowry, 2001). Apiaceae is often viewed as a large, specialized group derived from within Araliaceae, and grouped under the Apiales as a part of the core asterid clade. The Apiales thus comprises two large core clades, corresponding broadly to Apiaceae and Araliaceae and at least two other additional segregate clades (Plunkett and Lowry, 2001; Judd et al, 2002). The flowers in Apiaceae and Araliaceae tend to be small and sometimes inconspicuous, mostly greenish-white, and are pollinated by a wide range of insects. The Araliaceae are mostly woody herbs, trees and shrubs. But Panax and Aralia have lost the woody habit; yet have retained other Araliaceae characteristics. Both Araliaceae and Apiaceae species commonly include distinctive resin/ethereal oil canals and a presence of polyacetylenes, which are also characteristic of some Asteraceae species. The monoterpenes and resins of Apiaceae and Araliaceae species are antimicrobial, vasodilating and stimulating to various organ systems in the human body and thus have had a long history of medicinal use. A perplexing synonomy of medicinal Araliaceae species afflicts the phytotherapy literature. Eleutherococcus senticosus and Acanthopanax senticosus refer to the same species, yet Acanthopanax sessiliflorus is a different species altogether (Zhuravlev et al., 2003). Panax quinquefolium is often spelled P. quinquefolius. Aralia elata and A. mandshurica refer to the same species (http://mobot.mobot.org/W3T/Search/vast.html). Thus, correct identification of species with adaptogenic properties can be problematic. There are likely many other genera in Araliaceae that have adaptogenic properties, such as Aralia schmidtii (Baranov, 1982) and Acanthopanax sessiliflorum (Brekhman and Dardymov, 1969), but lack of research limits their inclusion in this thesis. Panax notoginseng has a long history of use for coronary heart disease and cerebral vascular disease, yet not enough evidence of adaptogenic activity (Dong et al., 2003).

72 However, close relatedness of species suggests that other Panax, Aralia, and Acanthopanax (Eleutherococcus) species would be worth targeting for future studies on adaptogenic properties. Panax ginseng. The use of Panax ginseng is legion and has been traced to the Han dynasty, 2000 years ago (Liu et al., 2003). This species contains at least 31 ginsenosides (triterpene glycosides) (Li et al., 2000). Panax ginseng has many adaptogenic activities including increase in protein synthesis, increased physical performance, and anti-fatigue effects (Wagner et al., 1994; Attele et al., 1999); enhancement of memory and nerve growth (Lee et al., 2000; Attele et al., 1999); and has been shown to improve cell gap junction communication in cell culture (Zhang et al., 2001). There are at least 34 controlled and uncontrolled clinical studies involving Panax ginseng as reviewed in Bucci (2000). Panax quinquefolium. This species is often spelled P. quinquefolius. Although polysaccharides and polyacetylenes are implicated in some of the effects, much of the bioactivity is associated with ginsenosides (Rg1, Re, Rb1, Rc, Rb2, and Rd) (Li et al., 2000). Panax quinquefolium is used for treatment of fatigue and as an immunostimulant in times of stress. It has been shown to have estrogenic, antimutagenic and hypoglycemic effects, and to enhance memory and learning (Assinewe et al., 2003). Codonopsis in the Campanulaceae The Campanulaceae are grouped under the Asterales. The species are herbs, often with milky sap and are rarely woody. The family of 87 genera and 2,000 species is widespread. Fifteen genera are native to Europe and 23 to North America. Flowers are usually showy and pollinated by both bees and birds. Many species are cultivated ornamentals (Cullen, 1997; Judd et al., 2002). Lobeliaceae is considered a sister family. Mention here is to emphasize the long history of use of Lobelia inflata by herbalists for severe infectious diseases such as yellow fever, with reportedly spectacular cures (Haller, 2000). Though chemical analysis of this species centers around an alkaloid, lobeline, L. inflata has a curious, unexplained activity found only in small doses, of increased resistance to infectious pathogens. Lobelia inflata would be a potential species to test for presence of anabolic ecdysteroids. A chemotaxonomic study of Codonopsis found that the species show a high degree of similarity of chemical compounds, particularly triterpenes and sterols (Wang et al., 1995). Among the closest species to those with adaptogenic properties is C. tangshen Oliv. which may also have the same activities. Again, this suggests that closely related species should be tested for adaptogenic activity. Codonopsis lanceolata. This species contains sapogenins and prosapogenins (codonoposide, echinocystic acid) (Lee et al., 2002), and is reported to promote intelligence, reduce lipid peroxide and increase superoxide dismutase in mice (Han et al., 1999). Codonopsis pilosula. The adaptogenic activity of C. pilosula is considered similar to Panax ginseng but not as strong (Wagner et al., 1994; Bensky and Gamble, 1986). It is used in traditional Chinese medicine to increase resistance, boost energy, improve digestion, and increase lung vitality (Hsu et al., 1986). Codonopsis pilosa also contains glucopyranosides along with some phytosterols (Chen et al., 1995), neolignan glycosides, tanshenosides, syringin, glucose, and sucrose (Yuda et al., 1990). Cardueae in the Asteraceae The species in this monophyletic group in Asteraceae (Rhaponticum uniflorum, Serratula coronata , Leuzea carthamoides) are part of a larger group of genera encompassing the Cardueae tribe that have not been tested for adaptogenic activity. The adaptogenic phytochemistry of these three Asteraceae species (Leuzea carthamoides, Rhaponticum uniflorum, Serrulata coronata) are attributed to phytoecdysteroids (Khodolova, 2001). Though Asterales is sister to Apiales (including Araliaceae), there does not seem to be

73 a close chemotaxonomic relationship. However, to my knowledge the Araliaceae has not been tested or found to contain phytoecdysteroids (EcdyBase search 11/25/03). The adaptogenic properties of the three Cardueae species were not explained until they were discovered to produce ecdysteroids (Kholodova, 2001). This suggests that all adaptogenic plant species should be tested for presence of ecdysteroids and all species containing edysteroids should be tested for adaptogenic properties. Many of the Cardueae genera such as Atractylis, Carduus, Carthamus, Centaurea, Cnicus, Cynara, Saussurea, and Silybum have been used in both indigenous medical practices and in modern herbal medicine. Saussurea is one of the larger genera in Cardueae having at least 300 species widely distributed in Eurasia (Bohm and Stuessy, 2001). Saussurea species are currently harvested from the wild for both medicinal and religious purposes in India. Due to habitat specificity and its narrow range of distribution almost all the Saussurea species growing in the high altitude of the Himalayas need conservation (C. Prakash Kala, personal communication, October 27, 2003). Some Saussurea species, in particular, Saussurea costus and Saussurea medusa, may be adaptogenic, as suggested by use in Tibetan and Chinese medicine for wasting of muscles and memory deficit (Tsarong, 1994; Fan and Yue, 2003). However, neither species is yet supported enough by scientific research to include them in this study. Brekhman and Dardymov (1969) mention Carlina biebersteinii Bernh. ex Hornem as a species that was studied for adaptogenic properties. I did not find any other reference to the medicinal use of this species. It is noteworthy that Carlina biebersteinii is placed in Carlininae, a subtribe of the Cardueae. The history of medicinal use of species in the Cardueae thus suggest that this tribe and closely related species may have a tendency toward adaptogenic activity. Leuzea carthamoides. Synonyms for this species are Cnicus carthamoides, Centaurea carthamoides, Cirsium carthamoides, Leuzea carthamoides, Serrulata carthamoides, Stemmacantha carthamoides and Rhaponticum carthamoides. Thus, Leuzea is commonly confused with Rhaponticum uniflorum. Leuzea carthamoides is reported as having antistress and anabolic properties (Brekman and Davydov, 1969), and considered a rasayana tonic and adaptogen (Rege et al., 1999). Rhaponticum uniflorum. Synonyms for this species are Cnicus uniflorus and Leuzea uniflora. It is reported to have anabolic properties (Syrov and Kurmukov, 1976), to stimulate production of red blood cells (Syrov et al., 1997), and to inhibit peroxidation of membrane lipids (Zhang et al., 2002). Serratula coronata . Synonyms for this species are S. coronaria and S. wolffii. This species is reported to have anabolic properties (Syrov and Kurmukov, 1976), to stimulate production of red blood cells (Syrov et al., 1997), and to inhibit peroxidation of membrane lipids (Zhang et al., 2002). It has been used as a tonic and adaptogenic remedy, for treatment of overstrain, for weakening of the restoration process, and to improve mental and physical ability to work (Kholodova, 2001). Discussion The presentations of the foregoing monophyletic clades represent preliminary findings primarily because all these species (and all plant species) have not been tested for the adaptogenic trait. Many related species are lacking sufficient evidence to include them as part of these subclades having adaptogenic properties. However, the evidence accumulated in this study suggests that adaptogenic properties are homologous among closely related species. That is, an adaptogen species tends to have a close relative that is also adaptogenic. Thus, future testing for adaptogenic activity should target close relatives of known adaptogens, especially those that produce triterpenoid saponins, phytoecdysteroids, phenylpropanes, and oxylipins. It could be argued that the inconsistent adaptogenic profile among angiosperm species is due to either loss of an adaptive trait or gene expression that has simply been switched off. For example, it is believed that most plants retain the genetic capacity to produce ecdysteroids, but expression is turned off in most species (Dinan, 2001). Aggregate (i.e., multiple origins) distribution is also seen in phylogenetic analyses involving secondary metabolites as traits plants employ in order to adapt to environmental and defense challenges. The focus here is on the bioactivity in mammals and humans, not the role of these compounds

74 in the plant itself, though this is an additional consideration for future research. This adaptogenic trait is most likely linked to not one, but many classes of secondary metabolites. It is not clear which precise structure-activity of secondary metabolites affords the adaptogenic activity, though the answer is likely a complex one. Thus, a focus on pharmacological activity, instead of single secondary metabolites, is more effective. The use of the trait, adaptogenic activity, as a taxonomical marker does have value for showing distribution. However, just like any adaptive trait, the resulting analysis must include careful critical evaluation. Phytogeography The traditional systems of medical practice in China (traditional Chinese medicine) and India (Ayurveda) are among the oldest in the world. These indigenous systems have had 2,000 to 7,000 years to explore and perfect the use of their medicinal natural products (materia medica). This lengthy history has significantly influenced the focus of natural products research. During this investigation it was discovered that, with three exceptions, Lepidium meyenii (South America), Panax quinquefolium (North America) and Trichopus sempervirens (Madagascar), the majority of the 33 well-characterized adaptogenic species are native to China, India, and Russia. This could be an artifact of the vigorous natural products research focusing on well-known medicinal species. This phenomena could also be due to the ability of the indigenous traditional medical practices in these countries to advance and develop therapeutic products. The phytogeography of North America is quite dissimilar, having undergone severe geologic and climatic changes (Flannery, 2001), despite its flora being related to Asian species. North American plants are generally genetically closer to each other than to their Asian relatives (Wen, 1999; Liu et al, 2002). That is, it is generally agreed that North American plant lineages show much evidence of endemic speciation and synthesize similar but different chemical compounds compared to Asian relatives. For example, the North American species, Panax trifoliata, does not contain the triterpenoid saponin compounds found in Panax ginseng, though another North American adaptogenic species, Panax quinquefolium, retained them. Many factors are to blame for the loss of indigenous healing knowledge systems in North America. Massive epidemics, poverty, displacement throughout history (Marles, 2000), as well as acculturation (Johns, 1990), have accelerated a distancing of medicinal plant knowledge among indigenous tribes of North America. Kindscher (1992) and Etkin (1998) have reported disinterest of both white and Indian cultures in an exchange of information concerning medicinal plants of North America. Medicinal plant knowledge is purposely kept secret from non-tribal members of the Confederated Salish & Kootenai Tribes of the Flathead Reservation in Montana by recording in Salish all information gathered from elders (Bigcrane, 1999). These factors may explain the low number of North American species collected in the ethnobotanical literature survey described in Chapter 5. Conversely, traditional herbal medicine practices in China and India were maintained despite intermittent political and social upheaval. The history and sociology of North America suggests an explanation for the striking differences between Old World versus New World herbal medicine practices, such as a more extensive materia medica. Nonetheless, herbal medicine has a very active history in North America, revealed by the rich narration of the Eclectic physicians (1850s 1930s) (Haller, 1994). The Eclectics widely utilized native medicinal plants from North America, and in fact, were responsible for popularizing the Native American herbs, Echinacea purpurea and Echinacea angustifolia, in the late 1800s and early 1900s. Modern herbalists and naturopathic physicians today still actively employ Eclectic publications and their materia medica. Despite such an extensive use of native plants to North America, modern herbal medicine can claim few, if any, adaptogen remedies from this continent. Only one North American species, Panax quinquefolium, was found to meet both the definition and have enough scientific substantiating adaptogenic activity. Yet it seems highly unlikely the North American flora is bereft of other candidates. The North American flora includes plant families known to contain adaptogenic species, in particular, Schisandraceae and Berberidaceae. There is potential for adaptogenic species in these North American plant families. In a recent monograph on Schisandraceae, the North American species, Schisandra glabra, is placed in the same clade as the adaptogenic Asian species, Schisandra chinensis (Saunders, 1998). A chemical

75 analysis also indicated similar compounds. Yet, there was found no mention of the use of this North American Schisandra species in the literature either by North American indigenous peoples, in Eclectic medicine, or by current cultures today. Another intriguing example is the close phylogenetic relationship of Epimedium species and Vancouveria hexandra, both from the Berberidaceae family. Vancouveria hexandra, is endemic to the Pacific Coast of North America, and is sister to Epimedium from the Asian continent. At least eight Epimedium species have been used in traditional Chinese medicine for conditions related to stress dysfunction, whereas, there are no reports of medicinal use of Vancouveria hexandra either by North American cultures or by modern herbalists today. However, V. hexandra contains the glycosides, icariin, epimedin B, and epimedosides A and E (Mizuno et al., 1991), which are also found in Epimedium brevicornu, E. koreanum, and E. saggitattum (Kim et al., 2001), along with similar prenylated flavonoids and glucopyranosyls (Ngadjui et al., 2002). These compounds are thought to be responsible for the adaptogenic properties in Epimedium species (Wu, et al., 2003). Other suspected North American adaptogens are saw palmetto, Serenoa repens, a native palm of Florida (Winston, 1999) and nettles, Urtica dioica (Grieve, 1931). Though their chemistry indicates a different mechanism of action, being high in essential fatty acids. Recent research suggests that Urtica dioica has a glucose-lowering property, similar to adaptogens (Bnouham et al., 2003). More of this type of research needs to be encouraged to identify new adaptogens. Schisandra glabra and Vancouveria hexandra are but two examples of North American plant species not known for medicinal use, yet related to Asian adaptogenic species and having similar chemistry. It was only through the investigation of the phylogeny and chemistry in this investigation that these two North American species could now be suggested as potential adaptogenic candidates. Thus, for North American plant species, ethnobotany may be a poor predictor of adaptogenic activity. It may, therefore, fall to phylogeny and chemotaxonomy to find more adaptogens among North American plant species. The strong indication of adaptogenic activity due to close relationship and similar chemistry of Schisandra glabra and Vancouveria hexandra suggest that there exist more heretofore-unknown adaptogenic plant species throughout the world. Summary The contribution of plant compounds to pharmacology has been estimated in the billions of dollars, though the interest in plants as sources of novel medicines has diminished recently because of political and technological reasons (Newman et al., 2003). Yet many scientists feel that billions of dollars worth of natural products still remain undiscovered and unexplored (Raskin et al., 2002; Carlini, 2003; Newman et al., 2003; Wu et al., 2003). An investigation of the scientific literature demonstrated that there is an increasing need for novel medicines to treat stress-related conditions and that many scientific disciplines are actively involved in researching the medicinal properties of plants and their compounds. Further, according to the National Institutes of Health, complex diseases require a holistic understanding of genetics, diet, infectious agents, environment, behavior, and social structures (Zerhouni, 2003). If so, then surely complex remedies such as adaptogens require interdisciplinary contributions. Plant-derived adaptogen remedies have had little investigation despite their common use by the public. Adaptogens present a novel approach to treating pathological disorders related to heightened stress sensitivity and dysregulation of stress-coping mechanisms. Future research from a wide variety of disciplines will help unravel the pharmacological properties of plant adaptogens and their compounds. There is, in particular, a lack of research on the presence of ecdysteroid compounds in adaptogens. For the same reason, plant species known to contain ecdysteroids should be tested for adaptogenic activity. Bridging the disciplines of plant science, phytochemistry and natural products, human physiology and pathophysiology, and plant-animal co-evolution has provided an integrative perspective on the distribution of the adaptogenic trait in angiosperm species. This phylogenetic study is the first to use a pharmacological class of medicinal plants as an adaptive trait, and the first phylogeny of plants with adaptogenic properties. Phylogenetic analysis may offer a successful tool to investigate other pharmacological classes of medicinal

76 plants and to help predict bioactivity. My hypothesis suggests 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. Despite the seemingly high degree of independent evolution of adaptogenic properties among higher taxa of angiosperms, targeting species known to be closely related to known adaptogens should prove very worthwhile for finding more plant species with adaptogenic activity. Robyn Klein 2006 www.rrreading.com Phylogenetic and phytochemical characteristics of plant species with adaptogenic properties MS Thesis, 2004, Montana State University Chapter 8 of 8