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Occurrence and activity of ecdysterone (insect moulting hormone) in plants Dreier, Susan I. 1987

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O C C U R R E N C E AND  ACTIVITY  (INSECT MOULTING  OF  ECDYSTERONE  HORMONE) IN P L A N T S by  SUSAN  I. D R E I E R  B.Sc, The University of Guelph, 1977  A  T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F THE  REQUIREMENTS MASTER  FOR T H E D E G R E E  OF  OF SCIENCE  in THE  FACULTY OF GRADUATE  STUDIES  Department of Botany  We  accept this thesis as conforming to the required  THE  standard  U N I V E R S I T Y O F BRITISH  COLUMBIA  June 1987  c  SUSAN  I. DREIER, 1987  In  presenting  degree  at  this  the  thesis in  University of  partial  fulfilment  of  of  department  this or  publication of  thesis for by  his  or  her  y  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D  t  DE-6(3/81)  representatives.  June 25, 1987  for  an advanced  Library shall make  it  agree that permission for extensive  It  this thesis for financial gain shall not  B o t a n  that the  scholarly purposes may be  permission.  Department of  requirements  British Columbia, I agree  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  head of copying  my or  be allowed without my written  ABSTRACT The  occurrence  hormone) was A  aqueous  method  was  elution  reverse-phase extract was  activity  developed  in plant extracts. The  methanolic  gradient  biological  of  ecdysterone  (insect  extract  of  the  cartridge),  of  plant  freeze-dried and  for  procedure  the  L  H  2  the  semiquantitative  consists of repeated with  aqueous  high-performance  purified on Sephadex  petroleum extract liquid  ether,  on  found  were  examined  in a number of plant  been  examined  ferns  (Aspidotis  Blechnum  with  a  SepPak  gymnosperms  (L.)  {Taxus  slight  increase  Roth,  brevifolia  of angiosperms (Trillium  Applied  Lellinger.,  Polypodium  of ecdysterone.  L,  (CIS Crude  Trillium  had  no  effect  elongation  of  mung  D.C.  canadensis ovatum  in a bean  species of  Ecdysterone  was  not previously  crispa Eat);  Marsh.);  (L.)  two  and  R.Br.,  species  two  of  species  Pursh.).  cytokinin epicotyls  bioassay, but (GA  3  elongation of excised dwarf pea hypocotyl hooks (auxin bioassay).  ii  five  include four species of  Cryptogramma  glycyrrhiza  Nutt., Taxus  cernuum  ecdysterone in  presence  species, the chemistry of which has  (Brackenr.)  by  for spectral analysis.  0  the  of  followed  chromatography.  respect to phytoecdysteroids. These  densa  spicant  for  analysis  washing of an  Twelve species of ferns, three species of gymnosperms, and angiosperms  moulting  examined in a number of plant species.  simple  ecdysterone  and  elicited  bioassay)  a  and  T A B L E OF  CONTENTS  ABSTRACT  ii  ACKNOWLEGEMENTS  vii  1. I N T R O D U C T I O N 1.1. Discovery of Ecdysteroids 1.2. Chemistry of Ecdysteroids 1.3. Distribution and Concentration of Ecdysteroids in Plants 1.3.1. Distribution 1.3.2. Concentration 1.4. Biological Activity of Ecdysteroids 1.4.1. Insects and Other Invertebrates . 1.4.2. Plants ; 1.5. Occurrence and Biological Activity of Steroids in Plants and Fungi 1.6. Outline of Research  1 1 3 8 8 10 12 12 15 17 26  2. M A T E R I A L S A N D M E T H O D S 27 2.1. Plant Material 27 2.2. Solvent Extraction 28 2.3. Chromatography 30 2.3.1. Thin Layer Chromatography (TLC) 30 2.3.2. Column Chromatography 30 2.3.3. High Performance Liquid Chromatography (HPLC) 31 2.3.4. Droplet Countercurrent Chromatography (DCC) 32 2.3.5. Gas Chromatography (GC) 32 2.4. Spectroscopy 33 2.5. Plant Growth Bioassays 33 2.5.1. Mung Bean Epicotyl Elongation 33 2.5.2. Elongation of Cucumber Hypocotyls 35 2.5.3. Promotion of Expansion of Cucumber Cotyledons 37 2.5.4. Expansion of Dwarf Pea Hooks 37 2.5.5. Thiarubrine Production in Tumour Cultures of Chaenactis  41  douglasii  3. R E S U L T S A N D D I S C U S S I O N 42 3.1. Methods of Extraction and Isolation of Ecdysterone from Plant Material 42 3.1.1. Solvent Extraction 44 3.1.2. Chromatography 45 3.2. Occurrence of Ecdysterone in Plants Surveyed 50 3.3. Biological Activity of Ecdysterone in Plant Tissues 58 3.3.1. Production of Thiarubrine in Callus Cultures 58 3.3.2. Plant Growth Bioassays 60  Gibberellin Bioassays Auxin Bioassay Cytokinin Bioassay Summary iii  60 66 70 72  4. C O N C L U S I O N  74  5. B I B L I O G R A P H Y  75  6. A P P E N D I X  85  1  iv  LIST Table Table Table Table Table  OF  TABLES  I. Chemical and Physical Data for Ecdysterone II. Plant Species Containing High Concentrations of Ecdysterone III. Dates and Locations of Plant Collections IV. Occurrence of Ecdysterone in Plants Surveyed V. Production of Thiarubrine in Callus Cultures from Transformed Chaenactis douglasii  v  7 11 27 53 59  LIST Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.  OF  FIGURES  1. Common ecdysteroids 2. Cholesterol 3. Amasterol 4. Brassinolide 5. Conformational formulae of ecdysterone and brassinolide 6. Sterols 7. Antheridiol; oogoniol 8. Extraction and isolation of ecdysterone from plants 9. Material for Elongation of Mung Bean Epicotyls bioassay 10. Material for Elongation of Cucumber Hypocotyls bioassay 11. Material for Expansion of Cucumber Cotyledons bioassay 12. Material for Expansion of Dwarf Pea Hypocotyl Hooks bioassay 13. U V absorption spectrum of ecd3'sterone 14. H P L C trace of a mixture of ecdysteroids 15. H P L C trace of crude extract of A. densa following partial purification by SepPak 16. D C C elution profile of 0.5 mg ecdysterone 17. Major steps in the mevalonic acid pathway, showing branch point to G A and ecdysterone 18. 24-epibrassinolide 19. Elongation of cucumber hypocotyls 20. Elongation of mung bean epicotyls (response to ecdysterone) 21. Elongation of mung bean epicotyls (response to 24-epibrassinolide) 22. Elongation of dwarf pea hypocotyl hooks 23. Fresh weight increase of dwarf pea hypocotyl hooks 24. Expansion of cucumber cotyledons , 3  Fig. Fig. Fig. Fig. Fig. Fig. Fig.  vi  2 4 5 18 20 22 24 29 34 36 38 39 43 47 47 50 61 62 64 65 65 68 69 71  ACKNOWLEGEMENTS  I and  thank  Dr. Towers  for his support  thank advice,  all those and  and enthusiasm  people  Risa  analyses, support,  for introducing me throughout  to the topic this  study.  in the lab and the department  Smith  who  and an island  gave  me  valuable  of  who  assistance  phytochemistr} , 7  I appreciate and offered with  help and statistical  retreat. Special thanks to Nesrin Tanrisever for  her advice and encouragement during the later stages of this work. Finally,  I would  like  to thank  my  encouragement.  vii  family for their  endless  support and  1. INTRODUCTION  1.1. DISCOVERY OF ECDYSTEROIDS  Ecdysis, the periodic shedding and regeneration of the cuticle during insect development,  was suspected to be under  demonstrated  that  caterpillars Fraenkel, brain  from  1935; Wigglesworth,  Becker  production  they  were  (1941)  (1954)  managed  1934) indicated  of a "moulting  obtained  erythrocephala but the active Karlson  the brain  control  active  named ecdysone, from  for pupation  of  studies (Hachlow, 1931;  the blowfly  was not isolated  500 kg of silkworm  (1922)  (MH) by the prothoracic  from  25 mg of pure  Kopec  substances secreted by the  hormone"  fractions  compound  to obtain  that  after  necessary  of the gypsy moth, Lymantria dispar. Later  stimulate  glands.  secretions  hormonal  until  crystalline  Calliphora  Butenandt and hormone,  which  (Bombyx mori) pupae. Karlson  (1956) isolated a second active compound from B. mori and named it p'-ecdysone to distinguish it from the a-ecdysone isolated earlier. The structure of a-ecdysone was finally determined by Huber and Hoppe (1965) and it was first synthesized in 1966 simultaneously by two groups (Siddall et al, 1966; Kerb et al, 1966).  Structures and nomenclature of a- and j3-ecdysone are shown in F i g . 1, and  further  information  on the chemistry  and structure  of ecdysteroids (the  generic name for this group of compounds) is given in Section 1.2.  Owing to the difficult}  7  of synthesis of a- and /3-ecdysone and the trace  1  INTRODUCTION / 2  Fig. 1.  Common phytoecdysteroids. A: ecdysterone; 20-OHecdysone; /3-ecdysone; crustecdysone B: ecdysone; a-ecdysone C: ponasterone B D: ponasterone B E: polypodine B F: inokosterone  INTRODUCTION / 3 amounts  present  extreme  scarcity  ecdysteroids  in  insects, research  of the  were  on  compounds. It  unexpected^  found  moulting  was  in  with  plant  hormones great  interest  sources at  same  time  Podocarpus  that  Galbraith  elata.  Takemoto  plants  have  Horn  et al  /3-ecdysone from Achyranthes  Many  therefore,  (1966)  (1967a)  reported  isolated  by that  several  (1966) reported  (Fig. 1-C) from the conifer Podocarpus and  impeded  concentrations  orders of magnitude greater than in insects. Nakanishi et al isolation of ponasterone A  was  nakaii  at  in  leaves  j5 -ecdysone  inokosterone  the  (Fig.  1-F)  the of  and  radix.  since  been  screened  for  MH  ecdysteroids have been isolated from plants (phytoecdysteroids)  activity.  Over  70  and 20 are known,  in animals (zooecdysteroids) (Horn and Bergamasco, 1985).  1.2.  CHEMISTRY  Ecdysteroids  OF  are  ECDYSTEROIDS  polyhydroxylated  steroids  containing  27  to  29  carbon  atoms, with a structure similar to that of cholesterol. The structure of cholesterol and  conventional  system  of  molecules is shown in Fig. 2.  numbering  carbon  atoms  and  rings  of  steroid  INTRODUCTION / 4  Fig. 2.  All structure.  ecdysteroids (2)  have  6-keto-7-ene  the  moiety,  Cholesterol  following (3)  features  in  common:  cis-fused A / B ring junction,  (1)  steroidal  and  (4)  full  sterol side chain. These four features are necessary for M H activity (Bergamasco and  Horn,  1983).  The  effects on M H activity.  presence and  position  of  hydroxyl  groups  have  Presence of a 14a-hydroxyl group is important for  varied high  activity, O H groups at C3 and C22 are less important, and O H groups at C 2 , C20  and C25 have a marginal  effect  on M H  activity'  (Bergamasco and Horn,  1980).  The most widespread and abundant phytoecdysteroid is ecdysterone (20-OH ecdysone). The nomenclature of this compound has been rather  confusing in the  past  20-OH ecdysone.  but  it  is  now  usually  referred  to  as  ecdysterone  or  Structures of ecdysterone and other common phytoecdysteroids are shown in Fig. 1.  INTRODUCTION / 5 Ecdysteroids  are  derived  from  cholesterol,  but  the  steps  of  their  biosynthesis in plants and animals are not well understood. The probable order of events  in  arrangement  the  biosynthesis  in  mevalonic  acid  (Heftmann  et al,  and  plants. is  Isaac,  Labelled  converted  1968; Rees,  1974; Morgan and Poole, Amaranthus  viridus  (Roy  1985).  insects  is:  formation  by hydroxylations  Very  cholesterol to  is  ecdysterone  little  is  of  the  cis  of the sidechain and  1977). Amasterol et al,  1982).  This  known  incorporated in  seedlings  1971; Lloyd-Jones et al,  has been proposed as an intermediate Amaranthus  in  at C 2 , based on combined results of studies with a number  species (Rees  biosynthesis  ecdysone  of A and B rings, followed  a hydroxylation insect  of  about  into  ecdysteroid  ecdysterone  of  of  various  and plants  1973; Rees and Goodwin,  (Fig. 3) was isolated from roots compound, containing  of ecdysone biosynthesis  of  a A5,7-diene,  in insects. Since  spp. also contain ecdysterone, amasterol may well be a precursor of  ecdj'sterone in these plants. Few studies of ecdysteroid synthesis in plants been carried out.  HO  Fig. 3.  Amasterol  have  INTRODUCTION Physical  and chemical  data  for ecdysterone  Ecdysteroids are water-soluble and are among steroids  known  (Bergamasco  and  Horn,  are summarized  / 6  in Table I.  the most polar naturally occurring  1983).  Their  polarity  presents  problems in the extraction and isolation of ecdysteroids from plants.  special  INTRODUCTION  Table 1.  MW  Physical and Chemical Data  / 7  for Ecdysterone  = 480.6  m.p. = 2 3 7 - 2 3 9 ° C  UV:  Xmax 240nm, e = 12,000  IR:  O-H  MS:  m/z  3440 cm" ,  C=0  1  1650 cm" ,  462 (M-H 0), 444 (M-4H 0), 99, 81 2  2  Morgan and Wilson (1980) Banerji et al (1971)  1  C=C  (M-2H 0), 2  1612  426  cm"  1  (M-3H 0), 2  408  INTRODUCTION / 8 1.3.  DISTRIBUTION  AND  CONCENTRATION  OF  ECDYSTEROIDS  IN  PLANTS  1.3.1. Distribution  Soon after the discovery of phytoecdysteroids, a large number of Japanese plants  were screened for  M H activity  with the  aim of finding new sources of  moulting hormones and looking for chemotaxonomic relationships (Takemoto et 1967b; Imai et al, means  of  suppressalis  1969a; Hikino et al,  a  bioassay  in  or  Calliphora  which  1973). M H activity  ligated  segments  erythrocephala) were  of  scored for  al,  was determined by  insect  larvae  pupation  24  (Chilo h  after  injection of, or immersion in, a crude extract of the plant (Karlson and Hanser, 1953; Sato el al, and  plant  1968). Pupation was taken as an indication of M H  extracts  which  resulted  in  pupation  were  assumed  to  activity, contain  phytoecdysteroids.  Takemoto  et  al  (1967b)  obtained  ferns and 27/74 species of higher MH  activity  in  fungi  (mushrooms)  active  plants or  extracts  from  22/39  surveyed. They found  species of  no evidence of  algae. Phytoecdysteroids have,  however,  recently been isolated from the red alga, Laurencia pinnata Yamada (Fukuzawa et al, ' 1986).  Imai et al  (1969a)  screened  1056  species of Japanese plants  from  186  families of ferns, Gymnosperms and Angiosperms. Ferns and Gymnosperms had the  highest  incidence of  MH  activity,  in  particular  the  families Polypodiaceae,  INTRODUCTION / 9 Taxaceae and Podocarpaceae. They also found M H activity plants  such  as  horsetails  (Equisetaceae)  and  clubmosses  in primitive  vascular  (Selaginellaceae  and  Lycopodiaceae).  In  a later  study,  283 species (76 genera from  20 families) of Japanese  ferns were surveyed, 170 of which were MH-active (Hikino et al, noted  that M H  Pteridophytes. Zealand  activity  Russell  ferns,  with  1973). It  was most common in the more evolutionarily  and most  Fenemore  (1974)  found  species of Blechnum,  MH  Todea,  activity  in  Rumohra  was  advanced  24/64  and  New  Adiantum  being active. MH-active species are found in most families of Gymnosperms, and the  probability  of  activity  in  species of Angiosperms  is  much  lower  than  for  Pteridophytes and Gymnosperms. Of 410 families of Angiosperms tested, 74 have species with M H the probability  activity.  of activity  higher than the probability  Phytoecdysteroids • are widespread in Angiosperms  but  in more than one species of the same genus is much of activity  in more than one genus per active  family  (Bergamasco and Horn, 1983).  To date, over 110 plant families are known to be MH-active or to contain phytoecdysteroids.  The  compounds  have  been  found  in  all  plant  parts  and  concentrations within a single plant may vary from part to part.  The  five  ecdysteroids  frequency)  are  ecdysterone,  pterosterone  (Horn and Bergamasco, 1985). Comprehensive lists of plant families,  genera and species from  most  commonly  ponasterone  A,  isolated  (in  polypodine  order B,  of  decreasing  ecdysone  which ecdysteroids have been isolated can be found  and  in  I N T R O D U C T I O N / 10 reviews by Bergamasco and Horn (1983) and Horn and Bergamasco (1985).  1.3.2. Concentration  Phytoecdysteroids  commonly  0.1% of the dry weight reported  in amounts  hand,  occur  of the plant  as high  arachnoidae C B . Clarke  occur  (Chou  at concentrations  at  concentrations  (Jones and Firn,  between  1978), but have been  as 2.9% of the dry weight and Lou, 1980). of 10"  9  to  0.001 and  of roots of Cyanotis  Zooecdy steroids,  1 0 " % in insects 5  on the other  (Bergamasco and  Horn, 1983). The highest concentrations of ecdysteroids have been found in plants from a wide range of families. These are listed in Table II.  Some  authors  have  noted  that  M H activity  tended  to  be greater  in  autumn than in the spring, in extracts of the same species (Hikino et al, 1973; Yen et al, 1974). In bracken fern (Pteridium aquilinum (L.) Kuhn), concentrations of ecdysone and ecdysterone were ver3' low throughout the growing season (<0.3 Mg/kg fresh weight) until October, when the concentration Mg/kg fresh concentrations  weight) in  (Jones and Firn,  autumn  1978).  may be due to  Apparent a  was at its peak (53  increases in ecdysteroid  decrease  in  protein  compounds concurrent with no change in actual ecdysteroid levels.  or  other  Table  II  Plant  Species  Containing  High Concentrations  of  Ecdysterone  DIVISION SUBDIVISION FAMILY  SPECIES  %  EC  REFERENCE  Pterldophyta Polypodlaceae  PoIypodIum  1.0  vulgare  L.  FW  Jlzba  e f al  (1967)  (rhlzomes)  Spermatophyta Gymnospermae Podocarpaceae  Dacrydlum 1nt  1.0  DW  (bark)  Russell  et  al  (1972)  ermed!um  Kirk . 0.45  Podocarpus elatus  DW  (bark)  R.Br.  Galbralth  8  Horn  (1966)  Anglospermae Ranuncu1aceae  He I mult  Ieborus  0.36  IfIdus  ssp.  DW  parts  (aerial  Hardman  S flowers)  (1976)  S Benjamin  serv(cus (Adamovlc) Merzm. & CommelInaceae  Podl.  CyanotIs  2.9  DW  (roots)  Chou  8 Lu  (1980)  arachnoidae CB. Alzoaceae  Clarke  Sesuv(um  0.35  portuIacastrum  plant)  DW  (whole  Banerjl  0.20  DW  (leaves)  Takemoto  0.20  DW  (leaves)  Imai  et  al  (1971)  L . Amaranthaceae  Achyrant Japontca  Lablateae  Ajuga  hes  e t al  Japontca  et  al  (1969)  MI q u e 1 Asteraceae  Source:  Serratula  0.33  xeranthemotdes  (Inflorescence)  Horn and Bergamasco  (196B)  Nakal  (19B5)  DW  Kholodova (1979)  et  aI  I N T R O D U C T I O N / 12 1.4. B I O L O G I C A L A C T I V I T Y  OF  ECDYSTEROIDS  1.4.1. Insects and Other Invertebrates  Ecdysone and ecdysterone are the most commonly occurring ecdysteroids in invertebrates.  Ecdysteroids  have  been  arthropods including the spider Pisaura tick  Amblyomma  polyphemus  hebraeum  (Winget  found  mirabilis  and Herman,  1976).  as well  as in  They  have  edulis  also  been  (Takemoto et al,  (Romer, 1979), the zoanthid Gerardia  and the parasitic fluke Schistosoma  mansonii  other  Limulus  1978) and the crustacean isolated  phyla. The parasitic nematode Ascaris  (Horn et al, 1974), the mussel Mytilus  pomatia  insects  (Bonaric and DeReggi, 1977), the  (Delbecque et al,  members of other invertebrate  Helix  in  savaglia  (Torpier  et al,  from  lumbricoides  1967c), the snail  (Sturaro et al, 1'982) 1982) have all been  reported to contain one or more of these compounds.  Ecdysteroids  are  present  at  all  development. In insects, they are primarily but  stages  of  insect  and  crustacean  synthesized by the prothoracic glands,  are also synthesized and accumulated in the ovaries of adult females, then  transferred of events (PTTH) Changing  to eggs (Sail et al, and interaction  from  the brain,  titres  of  1983). Insect moulting involves a complex series  of a number stimulates  ecdysone  of hormones. Prothoracicotropic  the prothoracic  and  juvenile  glands  hormone  hormone  to release ecdysone. interact  to  regulate  cuticulogenesis and metamorphosis. There is a large body of literature concerning the hormonal  regulation  of insect moulting.  see Kerkut and Gilbert (1985), Vol. 8.  For more information  on this  topic,  I N T R O D U C T I O N / 13 Studies  on the  activity  of  ecdysteroids  at  the  gene  level have  had  important influence on ideas regarding the mode of action of vertebrate  steroid  hormones such as testosterone and estrogen. Clever and Karlson (1960) that  puffing a  Chironomus indication (1974)  occurred few  that  in  isolated  minutes  steroid  after  hormones  salivary  gland  application may  act  of  polytene  on  proposed a general model of steroid hormone  passively  diffuses  receptor-steroid  into  the  complex  cell,  then  where  moves  it  binds  to  the  reported  chromosomes  ecdysone. This  directly  was  the  a  receptor  nucleus  and  of first  genes. Ashburner  action in which the to  an  et  al  steroid  protein.  binds  to  The  specific  chromosomal sites, inducing transcription of m R N A at those loci. The presence of ecdysteroid Drosophila  receptor  was  first  demonstrated  in  1978,  in  imaginal discs ("Yund and Fristrom, 1978) and Drosophila  (Maroy  et al,  affinity  and  ecdysone  proteins  1978).  These proteins,  specificity.  Gronemeyer  bound  to  salivary  by and  gland  definition, Pongs  bind  (1980)  polytene  cells  cell cultures  ecdysterone with were  able  chromosomes  to  of  to  the  locations  of  high  visualize Drosophila.  Ecdysteroids were photocrosslinked to their binding site and visualized by labelling techniques. Brightly  of  affinity  fluorescing regions on the chromosomes corresponded  ecdysteroid-induced  puffs.  The  originally  proposed  model  of  steroid hormone action has been altered somewhat since O'Connor's group (Sage et al,  1982; O'Connor, 1983) demonstrated the presence of ecdysteroid  receptors  resident in the nucleus. See O'Connor (1983) for a discussion of ways in which ecdysteroids might influence transcription of specific proteins in insects.  The biosynthetic they  cannot  synthesize  precursor the  sterol  of ecdysteroids nucleus,  most  in  insects is cholesterol. Since  insects  require  cholesterol  or  I N T R O D U C T I O N / 14 phytosterols  (primarily  sitosterol  and stigmasterol)  in their  diet.  There  is some  debate regarding the fate of ingested ecdysteroids. If the compounds evolved in plants as a means of defense against insects (first suggested by Galbraith and Horn,  1966),  However,  a number  conjugation, Hetru,  one would  expect  them  of insects have  oxidation,  epimerization  to have  adverse effects  been shown  to inactivate  and hydroxylation  1983). Robbins et al (1968)  when ingested. ecdysterone by  (at C26) (Hoffmann and  reared housefly (Musca domestica) larvae on  an artificial diet containing either ecdysterone or ponasterone A . Ecdysterone was inactive  at the highest  dose tested  (150 ppm) but ponasterone  A  (150 ppm)  resulted in growth and metamorphosis abnormalities. The effects of ingestion of a mixture been  of ajugasterone  used  A and ecdysterone by silkworm  by the silk  industry.  Administration  larvae  of M H at  (B. mori) has  appropriate  times  shortens or prolongs the feeding period of last instar larvae, synchronizes coccoon spinning, and increases silk production (increase of approximately coccoon weight compared to controls) fed  mori larvae  B.  Effects of dietary duration  (Chou and L u , 1980). Kubo et al (1983a)  diet  containing  25 to  100 ppm ecdysterone.  ecdysterone varied with concentration, developmental stage and  of exposure and included death without moulting, death after  moulting, larvae  an artificial  19% in average  and growth  of the moth  abnormalities  inhibition.  In  another  Heliothis sp. exhibited  following ingestion of greater  study  (Kubo  and Klocke,  no developmental  than  prolonged 1983),  or morphological  3000 ppm phytoecdysteroids in  an artificial diet. In the same study, phytoecdysteroids added to an aqueous diet were (1984)  shown  to deter  reported  that  migratoria was rapidly  feeding  in the aphid Schizaphis graminim.  ecdysterone converted  ingested into  its  by  fifth  3-acetate  instar  larvae  Modde et al of Locusta  and 3-acetate-2-phosphate  I N T R O D U C T I O N / 15 derivatives. These metabolites were rapidly excreted and ecdysterone never entered the  hemolymph.  Locusts  (Schistocerca  fed  gregaria)  bracken  fern  (high  in  ecdysterone) showed no growth or developmental abnormalities (Carlisle and Ellis, 1968).  In  aquilinum  a  study  of  the  role  of  ecdysteroids  in  bracken  fern  (Pteridium  (L.) Kuhn.), Jones and Firn (1978) tested the effect of ecdysteroids on  the feeding behaviour of seven species of insects. They concluded that the levels of ecdysterone in P. aquilinum lowest  levels  which  would  were "2 x 1 0  be  required  to  to 9 x 1 0  6  affect  times lower than the  3  development,  reproduction,  or  survivorship of insects feeding on the plant". They also point out that levels of ecdysterone  are  highest  in  the  autumn,  a  time  of  year  during  which  insect  herbivory is declining.  The theory  of phytoecdysteroids as defensive compounds has been neither  confirmed nor refuted.  1.4.2. Plants  It  is  possible that  ecdysteroids have  This  has been investigated  that  MH-active fractions  bioassay.  The  significantly  locust  over  that  in  of  whole  extract of  very  few locust  elongation of GA -treated plants. It 3  and  and  Jones,  1972).  extracts  These  were  elongation elongation  of was  active dwarf  in  (1963) in  plants. reported  a  gibberellin  pea  internodes  approximately  10%  of  has since been pointed out that the extract  was of unknown composition and may (Hendrix  physiological function  studies. Carlisle et al  stimulated  controls  a  have contained gibberellin-like compounds  latter  investigators  tested  the  activity  of  I N T R O D U C T I O N / 16 ecdysterone  in four  gibberellin  bioassays.  They  found  no change in: a-amylase  production by barley half-seeds, dwarf pea internode length, leaf sheath length of dwarf  corn,  and dark  Gibberellin-type inokosterone  activity  germination has been  in one study,  of spores  reported  however.  of the fern  for ponasterone  Anemia phyllitidis. A , ecdysterone and  These compounds stimulated  the growth of  the second leaf sheath and root of rice seedlings (Matsuoka et al, 1969).  The (1971)  effect  observed  of ecdysterone no significant  on effect  flowering  is unclear.  of ecdysterone  Jacobs  and Suthers  on flowering  of  cultured  shoot-tips of Xanthium pennsylvanicum. The same compound inhibited formation of female flowers on Cucurbita pepo (Felippe, 1979).  Following the isolation of a number of Pteridium aquilinum gametophytes, were  tested  for  activity  in  ponasterones  antheridium  compounds displayed any activity  of ecdysteroids from culture A  induction  with gametophytes  medium  and C, and ecdysterone bioassays.  None  of the  of Pteridium, Onoclea, and  Anemia (McMorris and Voeller, 1971).  The question plants manner.  of a physiological function  has not been  resolved  (or lack  and has not been  of) for ecdysterone in  investigated  in a  thorough  I N T R O D U C T I O N / 17 1.5.  OCCURRENCE  PLANTS  AND  AND  BIOLOGICAL  ACTIVITY  OF  STEROIDS  IN  compounds,  the  FUNGI  Ecdysteroids  are  members  of  a  group  of  triterpenoid  steroids. These are C30 compounds with a characteristic four-ring nucleus, formed via the mevalonic acid pathway by condensation of six isoprene units. The major classes  of  phytosteroids  are:  1)  sterols,  2)  progestagens,  androgens, 4) cardiac glycosides, and 5) saponins. Activity development bioassays has been demonstrated interesting  since the  compounds are  similar  for in  3)  estrogens  in plant  and  growth  and  a number of steroids. This is structure  to  ecdysteroids. It  is  logical to consider that steroidal compounds may have a physiological role within the plants that produce them, in view of their strong biological activity in animal systems. Relevant studies of biological activity  of plant steroids are reviewed in  this section.  There  are  sidechain at as:  a  large  number  C17. They are often  of  known  phytosterols,  all  with  a  classified on the basis of substitution  long  at C 3 ,  1) free sterols (OH at C3), 2) steryl esters (fatty acid esterified at C3), 3)  steryl glycosides (carbohydrate  at C3), and 4) acylsteryl glycosides (carbohydrate  at C 3 , with a fatty acid esterified to C6 of the carbohydrate). The sterol most often  isolated  phytosterols  from  higher  is largety  plants  unknown,  is  0-sitosterol  although  (Fig.  6-A).  some have exhibited  The  function  activity  in  of  plant  growth bioassays.  The most notable of these is brassinolide (Fig. 4), a steroidal plant  growth  I N T R O D U C T I O N / 18 hormone isolated in extremely low concentrations from pollen of Brassica (Grove et al,  1979).  Fig. 4.  Brassinolide which involves Mitchell, with  IAA  napus L.  (BR)  has  a  Brassinolide  very  strong  growth-promoting  effect  on  plants,  an increase in both cell elongation and cell division (Worley  1971). BR exhibits (Yopp et al,  auxin  1979,  activity,  1981). It  in  some cases  has effects  similar  acting  and  synergistically  to kinetin  in some  cytokinin bioassays and is active in gibberellin assays (no synergism with G A ) 3  (Mandava  et  investigated. It  al,  on  The  mode  of  action  of  brassinosteroids  appears to be distinct from the activity  growth regulators. effect  1981).  Ancymidol, an inhibitor  responses to  brassinolide  (Yopp  is  being  of other groups of plant  of gibberellin-mediated  growth has no  1979;  and Mandava.  et  al,  Gregory  1982), and brassinosteroids are inactive in some assays which are very sensitive to gibberellins  (Takeno and Pharis,  1982). Likewise, brassinolide is not active in  all cytokinin bioassays (Mandava et al,  1981). It  acts synergistically with  auxin  INTRODUCTION in  some  tests, but has  segments,  the growth  1986). It is possible et  (Arteca  resemblance  al,  effect  of which that  1985).  to each  an  opposite  is inhibited by  endogenous  Brassinolide  auxin  and  other, but there  Stereochemical]}',  differences. Fusion ecdysterone,  giving  ecdysterone  of the A brassinolide  ecdysterone, which is kinked C2  and  C3  in the A  ring  a  may  (Yokota  bear  important  (vs six-membered brassinolide  rings  relatively  (see structures are oriented  a  in maize and  be necessary  ecdysterone  and  and B  of auxin  auxin  are certain  ring of brassinolide is a seven-membered ring.  to that  activity  structural  differences. The  B  in ecdysterone) lactone  displa}'  some  is trans in brassinolide planar  root  Takahashi,  for BR strong  / 19  configuration  important and cis in  as opposed to  in Fig. 5). The hydroxyl groups at differently (j3 in ecdysterone,  brassinolide), and' the hydroxyl at C22 in the sidechain  is oppositely  a in  oriented in  the two compounds. These differences almost certainly have serious effects on the biological activity of these molecules.  INTRODUCTION / 20  Fig. 5.  Conformational structure of ecdysterone  (A), and brassinolide (B).  I N T R O D U C T I O N / 21 Activity sterols.  in  Steryl  exhibited  plant  growth  bioassaj's  glucosides, particularly  auxin  activity,  and  has  been  reported  for  stigmasteryl-/3-D-glucoside  acted  synergistically  with  elongation of Avena coleoptile segments (Kimura et al,  other  (Fig.  auxin  in  plant  6-B),  have  assays  1975; Tietz et al,  of  1977;  Smith and VanStaden, 1978).  Various functions act  to  stabilize  membranes  comparing  the  effects  membrane  permeability,  ethanol-induced  have been proposed for  of  by free  found  leakage  of  controlling  phytosterols.  their  Some sterols  permeability.  sterols,  steryl  that only  free  sterols  were  effective  in  from  barley  roots.  There  are  electrolytes  esters  and  Grunwald  steryl  may  (1971),  glycosides  on  reducing certain  structural requirements for activity of sterols as membrane stabilizers. These are: 1)  presence  of  phospholipids),  a  free  C3  hydroxyl  2) a relatively  group  (for  flat configuration  interaction  with  membrane  (ie trans-fused rings), and 3) a  ring system with at least one double bond (Grunwald, 1980). It  is important to  consider  of  these  characteristics  Ecdysterone is unlikely  in  comparison  with  the  structure  to be membrane-stabilizing because:  1) it  is  ecdysterone. hydrophilic,  and 2) its A and B rings are cis-fused. Steryl esters and steryl glycosides may be  involved  in  the  transport  of  sterols  and  glucose  within  plants.  Steryl  glycosides may also be a storage form of plant sterols (Grunwald, 1980).  Estrogens and androgens  (Fig. 6-C and 6-D)  are C18 and C19 steroids  with strong hormonal activity  in mammals. There are a few reports of estrogens  in  in  plants  Androgens,  at  concentrations  including  testosterone,  the were  ng  kg" found  range  1  in  pollen  (see of  Grunwald,  1980).  Scotch pine Pinus  INTRODUCTION / 22  Fig. 6.  Sterols. A: /3-sitosterol B: stigmastery]-j3-D-glucoside C: estradiol D: testosterone E:  Cortisol  F: corticosterone  I N T R O D U C T I O N / 23 sylvestris L.  (Saden-Krehula et al,  1971). Applied estrogens and androgens have  been reported to influence sex expression of Ecballium elaterium L. (Cucurbitaceae) grown  in  increased  natural the  ratio  opposite effect. but  field  conditions  of  female  (Kopcewicz,  to  male  1970).  flowers,  whereas  Cortisone, a corticosteroid, increased the  had no effect on sex expression. Geuns (1974;  various corticosteroids Cortisol  and  on etiolated  Estrogens  significantly  androgens  total number  of  the  flowers  1977) studied the effect  mung bean seedlings and found  that  (Fig. 6-E and 6-F) doubled the number of  corticosterone  had  of  applied  lateral  roots  formed. Cortisol stimulated elongation of root cells and hypocotyls. There has only been  one  report  of  isolation  of  a  corticosteroid  from  a  plant.  11-deoxycorticosterone was isolated from rice {Oryza sativa) husk oil (Bahadur and Srivastava,  1971).  sodium-potassium implications  of  Corticosteroids  ion these  regulate  concentrations studies  with  in  respect  carbohydrate  mammals to  a  metabolism  (Grunwald,  possible  1980).  hormonal  function  and The of  estrogens, androgens or corticosteroids in plants, are unclear, since occurrence of the compounds in plants has been reported so rarely.  Another  interesting  compounds, antheridiol aquatic  fungus  initiate  and  Achlya  coordinate  instance of biological activity  of sterols  concerns  two  and oogoniol (Fig. 7), isolated from various species of the (Barksdale, sexual  1969;  reproduction  McMorris, in  1978).  Achlya  spp.  These in  the  compounds following  manner. Antheridial hyphae secrete oogoniol, which stimulates female mycelium to form  oogonial  chemotropic  initials.  Antheridiol  growth of antheridial  secreted  by  the  oogonial  initials  stimulates  hyphae to the oogonia. The oogonia are  enveloped by the antheridial hyphae prior to fertilization.  then  INTRODUCTION /  Fig. 7.  A: antheridiol; B: oogoniol.  I N T R O D U C T I O N / 25 It significant more  has  been  biological  demonstrated that activity  comprehensive for the  expressed  by  Parthier  hormone  action  activities  and  to  the  functions  in some animal  (1985), plains  as  sterols  of  cases. Our kingdom  "looking  of phytohormone  of phytosterols  knowledge  than  down  plants  and  This  was  the summits  effects". Further  is needed. It is quite  sterols have a hormonal function in plants.  do  of hormones  for plants. from  fungi  likely  have is far aptly  of animal  research that  on some  I N T R O D U C T I O N / 26  1.6. O U T L I N E  The  OF  aims  RESEARCH  of  this  study  extraction  and  isolation  Columbia,  and  relevant plant  for  the presence  were:  of ecdysterone,  examining  of plant growth bioassays.  to  (b) to  species from  of ecdysterone, and  phytoecdysteroids by  (a)  develop screen  reliable  selected  groups other than  (c) to address  the biological  a  ferns  method  for  of British  the Pteridophytes,  the question of the role of  activity of ecdysterone in a number  2. MATERIALS AND  2.1.  METHODS  PLANT MATERIAL  Plant  material  collected, weighed  used  for  extraction  (fresh weight) and stored  within 24 h of collection. Table III each species tested. Plant material indicated. Plant material  for  and at  isolation -80°C.  All  of  ecdysterone  material  was  III.  Species Aspidotis densa Cryptogramma crispa Pityrogramma triangularis  was collected by the  author  unless  otherwise  Dates and Locations of Plant Collections Date 9/7/84 18/7/84 9/7/84  Location Nanoose Hill, V.I. Yale, B.C. Nanoose Hill, V.I. Squamish, B.C.  2/9/84  Mission, B . C . *  Athyrium filix-femina  18/6/84  Bowen Is.,B.C.  Cystopteris fragilis  18/7/84  Yale, B.C.  Dryopteris arguta -  18/6/84  Bowen Is., B.C.  Polystichum munitum  18/6/84  Bowen Is., B.C.  Blechnum spicant  18/6/84  Bowen Is., B.C.  Polypodium glycyrrhiza  18/6/84  Bowen Is., B.C.  Taxus baccata  20/7/85  UBCEL  Thelypteris phegopteris  for  plant growth bioassays is described in Section 2-5,  10/6/84  Adiantum pedatum  frozen  shows the date and location of collection  this Chapter. Table  was  27  M A T E R I A L S A N D M E T H O D S / 28  Taxus  brevifolia  29/7/85  Bot. Gdns., U B C  Taxus  canadensis  30/6/85  Cambridge, N . S . *  28/5/86  Kemptown, N . S . * *  Trillium  cernuum  Trillium  ouatum  Zebrina  pendula  Tradescantia Murdannia  virginiana scapiflora  *  collected by Dr. W.B. Schofield  **  collected by Dr. W. Maas  ***  collected by Dr. G . H . N . Towers  1/6/86  Victoria, B.C.  2/1/85  UBC  Bot. Greenhouse  6/1/85  UBC  Bot. Greenhouse  9/1/86  Thailand***  Voucher specimens were placed in the U.B.C. Herbarium.  2.2. S O L V E N T  A  EXTRACTION  number of solvent extraction procedures were tested (Sauer et al, 1968;  Morgan and Poole, 1976a; Hikino, 1981; Kubo and Klocke, 1983). Fig. 8 outlines the procedure that was ultimately from -10°C.  solvent  partitioning  used for  procedures  were  all extracts. Crude extracts freeze-dried,  weighed  and  obtained stored  at  MATERIALS  HOMOGENIZE  I  6 0 % AQUEOUS MEOH  AND  M E T H O D S / 29  I N MEOH  PET,  ETHER  EVAPORATE  FREEZE-DRY  SEP-PAK SEPHADEX  Fig. 8.  •HPLC - ( N M R ) , MS  Extraction and isolation of ecdysterone from plants.  M A T E R I A L S A N D M E T H O D S / 30 2.3.  CHROMATOGRAPHY  2.3.1. T h i n L a y e r Chromatography ( T L C )  Silica with  gel 60 G254,  chloroform:95%ethanol  0.2 mm precoated (4:1)  as  the  T L C plates  solvent  system.  (Merck)  were  used,  Ecdysterone was  visualized as a dark spot under U V or by spraying with vanillin-H SO « reagent 2  (Stahl,  1962). Plates were sprayed with a mixture  concentrated H S O 2  of 1.5 g vanillin  and 1 g  in 50 m l 95% ethanol, then heated to 110°C for 5 min.  t t  Preparative T L C was carried out using a Chromatotron (Harrison Research Associates).  This  is a spinning  T L C plate  coated  with  silica  gel 60 GF254  (thickness 2 mm). Fractions were eluted in chloroform:95%ethanol (4:1) at a flow rate of 4 ml m i n " . The sample (1 mg ecdysterone) was applied in 2 ml of 1  the solvent system.  In order to test recovery from T L C , a band containing a known amount of ecdysterone (Sigma) was scraped off and eluted by centrifugation in MeOH (3 times,  15  min  each).  The  concentration  of  ecdysterone  in  the  combined  supernatants was determined by H P L C .  2.3.2. C o l u m n  Silica  Chromatography  gel columns  (80:20) in 20 ml fractions.  (Si gel G 6 0 ; Merck)  were  eluted  with  CHCl :MeOH 3  MATERIALS XAD-4 mg  Taxus  brevifolia  volume =15 MeOH in  Amberlite  15  redissolved  ml  Sephadex  Aspidotis dia.  spotted  in a gradient  of increasing  30 m l 70%;  which  LH20  (Pharmacia) extract  A l l fractions  were  w a s performed  variable  Ecdysterone  wavelength  Fifty  the  column  concentrations  3 0 m l 100%).  w a s presoaked w a s applied  evaporated,  in  Eluent  (column  of aqueous w a s collected  evaporated  MeOH.  to t h e c o l u m n  rate  of 5 ml/hr  redissolved  2  partially  purified  cartridges  (isocratic; prior  (SepPak,  100/il  1 0 % aqueous  eluted  with  on a Varian  flow  redissolved  rate  to injection  Waters  to  dryness,  in  T w o hundred (length  mg  45 c m ;  and 5 m l 2 0 0 n\  int.  fractions  M e O H and  1  (HPLC)  5000  a Varian  ml  on H P L C  (Rees  (maximum methanol, a n d Isaac,  i n 2 0 0 p\ M e O H .  HPLC  MCH10  with  Series column.  a t 2 4 3 n m , a n d w a s eluted  in2 0 %  min" ).  Reverse  Varian Phase  Crude  1  plant  extracts  b y the u s e of C 1 8 reverse  Associates). T h e sample  methanol  methanol  using  Model  b y U V absorbance  2 m l 1 0 % aqueous  aqueous  Chromatography  detector,  w a s detected  acetonitrile/H O  and  and hot M e O H .  on T L C .  HPLC  60%  / 31  to  subsequently  w a s r u n at a flow  2.3.3. H i g h Performance L i q u i d  634  were  METHODS  a n d screened on T L C .  densa f r e e z e - d r i e d  collected.  w a s applied  30 ml 50%;  2 cm). T h e column  were  in hot water  extract  fractions  in M e O H  w a s prewashed  freeze-dried  m l ) a n d eluted  (45 m l 30%;  ten  resin  AND  w a s applied  1 m g sample  p e r cartridge)  4 m l 2 0 % aqueous 1979).  were  phase  SepPak  in  a n d serially  methanol,  T h e 6 0 % eluate  2 0 p\ s a m p l e s  to a  were  and 6 ml  w a s evaporated  run on H P L C  with and  MATERIALS without  ecdysterone (Sigma)  MeOH  fraction  collected  added.  from  For each  the SepPak  plant  AND  species  was also  tested  METHODS  examined, on H P L C  / 32  the 2 0 % to ensure  that all ecdysterone had eluted in the 6 0 % M e O H fraction.  2.3.4. Droplet Countercurrent Chromatography (DCC)  DCC between (Tokyo  is a  form  of chromatography  two immiscible  liquid  Rikakai Co.) using  phases.  a solvent  v/v) in the ascending mode  in which  Samples  system  (Kubo et al,  were  the sample  is partitioned  chromatographed  by  DCC  of chloroform:methanol:water (13:7:4 (maximum  2 mg)  was  injected in 3 ml of a. 1:1 (v/v) mixture of the mobile and stationary  phases  and  eluent  monitor  was  proved  the detection  collected  unreliable  cell, so each  in ' 5  ml  1983b). The sample  fractions  (35 min/fraction).  Use of a  due to bubble formation of the immiscible fraction  was evaporated, redissolved  UV  solvents in  in methanol and  optical density (O.D.) measured at 243 nm.  2.3.5. Gas Chromatography (GC)  Ecdysterone ethers. These (1986).  was derivatized  for GC  by formation of trimethylsilyl (TMS)  were prepared and purified on T L C by the method of Bielby et al  M A T E R I A L S A N D M E T H O D S / 33 2.4.  SPECTROSCOPY  UV  spectroscopy  was performed  on  a  Pye Unicam  SP8-100  UV/VIS  spectrophotometer.  Mass  spectroscopy  was carried  out by F. Balza  on a Finnigan 1020  GC/MS.  2.5. P L A N T  GROWTH  Compounds  BIOASSAYS  used in plant  Chemical Co. unless otherwise  The  Bean Epicotyl  bioassays were  obtained  from  Sigma  indicated. 24-epibrassinolide was prepared by Dr.  B. Abeysakara in the laboratorj  2.5.1. M u n g  growth  7  of Dr. G . H . N . Towers.  Elongation  procedure described by Gregory and Mandava (1982) was used. Mung  beans (Phaseolus aureus) were grown in moist vermiculite in a growth chamber at  27°C  on  ten-day-old  a  16  h  seedlings with  photoperiod epicotyls  (120 ^Einsteins  m~  2  sec" ).  35 to 60 mm long were  1  Nine-  harvested,  to their  cotyledons removed and hypocotyls cut to 3 cm. Cuttings were placed in vials (5 cuttings length were  per vial)  containing  2 ml of test  solution or water  was measured for each seedling, and from placed on the epicotyl  to identify  individual  one to five  (Fig. 9) Epicotyl India  seedlings within  ink dots each vial.  Epicotyl length was taken as the distance from the most apical cotyledonary scar  MATERIALS AND  Fig. 9.  METHODS / 34  Material for Elongation of Mung Bean Epicotyls bioassay.  M A T E R I A L S A N D M E T H O D S / 35 to the point where the primary leaves join the epicotyl. Test vials contained 1.98 ml distilled water  and 20jul  ecdysterone (EC) or brassinolide (BR) in methanol.  Controls contained distilled water only. E C and B R were tested at concentrations of  l O ' , 1 0 " , 1 0 " , 10" , 6  7  8  9  10 "  1  0  , 10'  11  and 1 0 '  1 2  M . There were two  vials per concentration, each containing five seedlings. Vials were maintained in a growth chamber with a 16 h photoperiod, at 27°C. Distilled water was added to vials if they dried out. A second measurement of epicotyl length was taken after 48 h.  2.5.2. Elongation of C u c u m b e r  Hypocotyls  This bioassay was based on the methods Mandava  et al (1981).  distilled water  Seeds  of Cucumis  of Katsumi et al (1965) and  sativus (Marketer)  for 2 h, surface sterilized in 5% hypochlorite  were  aerated in  for 5 min, rinsed  and sewn on wet paper towels. The seedlings were grown in the dark at 21 °C and aerated once a day under was approximately  green light. After  5 days, when the hypocotyl  4 cm long, it was cut 5 mm below the concave surface of  the hypcotyl hook. The apical segments of cut seedlings were incubated in Petri dishes containing 3 ml of test  solution or water,  dish (Fig. 10). Petri dishes were kept light for 24 h (110 /jEinsteins m " were  used:  10" M 4  gibberellic  2  acid  in groups of 5 seedlings per  in a growth chamber  s e c " ) at 22°C 1  (GA ), 3  under  continuous  *-2°C. Four test solutions  10" " M I A A (indole-3-acetic  acid),  10* M ecdysterone and 10" M 24-epibrassinolide. There were three Petri dishes 5  per test solution. After  5  24 h in continuous light, hypocotyl length was measured  and mean elongation calculated for each Petri dish.  M A T E R I A L S A N D M E T H O D S / 36  Fig. 10.  Material for Elongation of Cucumber Hypocotyls bioassay.  M A T E R I A L S A N D M E T H O D S / 37 2.5.3. Promotion of Expansion of Cucumber  The procedure of Green and Muir  Cotyledons  (1978)  and Mandava et al (1981) was  followed. Seedlings of Cucumis satiuus (Marketer)  were dark-grown  as described in  Section 2.5.2. Under a green safety light, cotyledons of 10-day-old seedlings were excised so that  all  of  the  hypocotjd  hook  was  removed. Cotyledon pairs  were  weighed to the nearest mg in groups of 5 pairs, and floated adaxial side down, on 5 ml of test solution in a Petri dish (5 cotyledon pairs per dish) Test compounds were dissolved in a solution of pH  6.0.  10" " M  24-epibrassinolide, contained buffer a  dark  solutions  kinetin,  of  the  lOmM C a C l  following  6-benzylaminopurine  and 40mM KC1,  2  compounds  (BAP)  and  were  ecdysterone.  only. There were 5 Petri dishes per treatment. After  growth chamber  at  25 °C,  cotyledons  were  (Fig. 11).  drained  used: Controls  4 days in  and weighed  as for  initial weighing.  2.5.4. E x p a n s i o n of Dwarf Pea  Dwarf  pea seeds  hypochlorite for depth  of  1  Hooks  (Pisum sativum Laxton's  15 min, rinsed thoroughly, cm  and  grown  in  Progress) were  soaked in  5%  sown in moist, sterile vermiculite at a  complete  darkness  at  room  temperature.  Seven-day-old seedlings were harvested by the methods of Adamson et al (1968), Sasse et al (1972), and Yopp er al (1981). Hypocotyl hooks were cut as shown in Fig. 12, and measured (at their longest point) to the nearest 0.2 mm using a stage micrometer. just  passed,  over  Only the  seedlings in hook  were  which used.  the Hook  scale leaf was  passing, or  segments  weighed  were  to  had the  MATERIALS AND  Fig. 11.  METHODS /  Materia] for Expansion of Cucumber Cotyledons bioassay.  38  M A T E R I A L S A N D M E T H O D S / 39  ig. 12.  Material for Expansion of Dwarf Pea Hypocotyl Hooks bioassay.  M A T E R I A L S A N D M E T H O D S / 40 nearest mg in groups of 10, then transferred to a 35 5  ml  green  of the  test  safe-light.  Test  solutions of I A A (pH  5.9).  In  solution  (Fig  12). All  solutions  were  procedures were carried out  made  by  and ecdysterone in unbuffered  order  to  dissolve  test  mm Petri dish containing  serial dilution  a  10" * M  stock  2% sucrose (Sasse et al,  1972)  compounds  of  under  completely,  they  were  first  dissolved in a small volume (200 ul) of ethanol. A l l possible combinations of I A A (0,  10" , 7  10" , 6  tested. Ten hook continuous  light  which time they groups of 10.  and  10" M ) 5  and  segments were (110  E C (0,  floated  ^Einsteins m "  2  10" , 1  10" ,  on each test sec ) - 1  at  and  6  20°C  10' M ) 5  solution and kept ( l°C) i  were again measured individually, blotted dry  for  24  were under h,  at  and weighed in  M A T E R I A L S A N D M E T H O D S / 41 2.5.5. Thiarubrine Production in Tumour Cultures of Chaenactis douglasii  Callus  cultures  from  a  crown  (Hook.) H . & A . were transferred  gall  tumor  line  of  Chaenactis douglasii  to agar plates of S H medium  (Schenck  and  Hildebrandt, 1972) (Appendix 1) without hormones. Experimental plates had either ecdysterone (10.5 |zM) or I A A (50 JIM) added to the medium. Controls contained agar  and  SH  medium  only. There  Cultures were grown in the dark was  removed,  dried  24 h at  callus was ether.  ground  were  at  replicate  27°C for 4 weeks.  and thoroughly  75 ° C ,  three  extracted  then weighed.  The  methanolic  (e o o=3000; M W = 228) (Cosio 9  Dr. E. Cosio/Dr. G . H . N .  Towers.  was  determined  et al,  The  treatment. plate,  twice into  cultures  O.D. at were  callus  residue  fraction obtained  by measuring  1986). Callus  per  For each  into methanol.  diluted to 60% aqueous methanol and extracted  Thiarubrine concentration  plates  from  was the  petroleum 490 nm  provided  by  3. RESULTS AND  METHODS  3.1.  OF  EXTRACTION  DISCUSSION  AND  ISOLATION  OF  ECDYSTERONE  FROM P L A N T MATERIAL  Numerous methods have been published ecdysteroids and  from both plants and  chromatography  pose light.  It is not  spectrum. Absorption  by  single  nm  peak  at  is not  distinguishable  243  the  in  chromatographic  water-soluble  coloured,  from  (Fig. 13).  systems.  compounds  the  with  Many  nature of ecdysteroids and  other  cyclohexenone  strongly at this wavelength. For  extraction and  isolation of  insects. Many combinations of solvent extraction  have been used, but  problems. Ecdysterone  for the  does  compounds B  ring  other  not on  fluoresce the  produces organic  basis  a  continues  to  under  UV  of its  UV  spectrum  compounds  absorb  In  addition,  solubilities  purification of ecdysteroid-containing  this  section  study, leading  is a to  UV  ecdysteroids  similar  to  are  other  polar  strongly  polar,  phytochemicals partial  plant extracts.  discussion  the  a  these reasons, it is difficult to detect ecdysterone  such as glycosides. This limits the efficiency of solvent partitioning in the  This  with  adoption  of the of  a  results of various simple  technique  isolation of ecdysterone from fresh or frozen plant material.  42  procedures used in for  semi-quantitative  RESULTS AND  DISCUSSION  ,._|..  ir-  -Jks  _! — i.  r  1  /  —!—/•!  1  -.-  • '; -  -:-  .-;  :-(•-  !—  :  j — \ — • • !  — ••'  :-  :  r--;  :  ;  [  j  i  i  1  i  1  i l  :  r - H -  .  i  1  1 : 1  i i  ,  :5t T  Fig. 13.  UV  i=~ i ;  ~r~—c  UZZC  !— ' '  absorption spectrum of ecdysterone.  ;  / 43  R E S U L T S AND  DISCUSSION / 44  3.1.1. Solvent Extraction  A little  number of solvent extraction procedures were tested (see Methods),  success,  opposing their  until  it became  apparent  that  methods in order to extract the  extraction  procedure,  Morgan  between water and  ethyl acetate and  other  et  hand,  Kubo  al  two  groups  used  diametrically  same compound. In the  and  Poole  (1976a)  with  final  step of  the  extract  partitioned  discarded the ethyl acetate fraction. On  (1983a) partition  samples  between  ethyl  the  acetate  and  water and  keep the ethyl acetate fraction. Following this discovery, the solubility  properties  of  partitioning ended up and  pure  ecdysterone  followed by  TLC  were  of the  for  contents  a  number  of both  of  phases.  solvents,  by  All ecdysterone  in the aqueous phase when partitioned between equal volumes of water  petroleum ether. When ecdysterone dissolved in water is extracted with ethyl  acetate once,  most of the ecdysterone  washes  with  ethyl  present  in both  each  tested  time.  ecdysterone into  the  that  ethyl  and  ethyl  procedure. mixtures (1984).  acetate, approximately  phases.  Water remains  remains  and  This  was  n-butanol  in the  equal  repeated  acetate Further  were,  useful  but  after  two  in the  information on be  extraction  partitioning  found  After  one  in an  compound identical  the  it was  behaviour article by  solvent  some  all moved concluded  of ecdysterone. Both from  are  results  extraction  extractions, it has  to evaporate, and  eliminated  the  of the  times, with  tested.  therefore,  of various solvents can  three  also  water,  aqueous phase. After six  amounts  were  butanol phase. Butanol is difficult acetate is not  in the  butanol  extraction  of ecdysterone in Mamatkhanov  et  al  R E S U L T S AND It  is desirable  compound simple with  may  and  be  to  lost  use in  as  each  consists of repeated  petroleum  ether  only  few  extraction  step.  The  extraction  (until  the  ether  steps  procedure of a  60%  DISCUSSION / 45  as  possible,  finally  since  some  was  very  adopted  aqueous methanolic  is colourless). This  crude  extract  extract is  then freeze-dried.  3.1.2. Chromatography  TLC  is  useful  for  extracts. Ecdysterone has compound gel  can  be  containing  which  to  (Morgan and  Wilson, 1980).  Preparative  two 68%  TLC  replicates. 58% from  the  which  (F254),  crude  plant  (80:20).  2) as  on  colour a  the  detection  as  a  from  compound  means  of  silica was  further  gel plates recovered  deemed too low  second method of preparative from  it eluted  a  spinning over such  circular a  large  with  from  TLC  TLC  colour  by  these values  purification  was  and  spot  their  ecdysterone  of  silica  yellowish-brown basis  of  The  reaction  jug, which is in agreement with published  applied  eluted  applied but  and  appears  of  Percent recovery was  fractions are  ecdysterone was  limit  considered  of use. A  in  3  ecdysteroids  of ecdysterone  of the  other.  this method to be in  was  extracts. Recovery  other  0.5  ecdysterone  in CHC1 :95% E t O H  Ecdysterone  The  2  of  ways: 1) absorbance of visible light on  indicator  vanillin-H SO <j. determined as  crude  in two  from  methods was  detection  value of 0.2  reagent.  is distinguishable  reaction  Rf  fluorescent  spray  2  an  visualized  a  vanillin-H SOy  preliminary  tested one  of  using  replicate,  too variable for was  investigated,  plate. One  mg  volume of solvent  of that  R E S U L T S A N D D I S C U S S I O N / 46 it was not detectable in any of the fractions collected.  Many HPLC  have been published for  (Gilgan, 1976; Lafont  involved injection was  methods  techniques on H P L C  adapted  from  in  et al,  which  determination  1980; Baltaev et al,  ecdysteroids  are  of ecdysteroids by  1984), including more  fluorescently  labelled  prior  (Kubo and Komatsu, 1986). The method used in this Lafont  et  al  (1976).  Good  separations  of  to  study  mixtures  of  ecdysteroids are obtained using this elution program, as shown in Fig. 14. The retention time for ecdysterone is 9 to The  10 min and the detection limit is 60 ng.  best results are obtained when the crude extract is partially  to H P L C ,  by means of a SepPak C18 cartridge  (modified  purified  prior  method of Rees and  Isaac, 1979). SepPaks are a very convenient means of purification of mixtures of polar compounds. The sample is loaded in a small volume of solvent and eluted in  a  HPLC  step-gradient trace  of  obtained  increasing concentrations for  Aspidotis  densa  of  MeOH.  crude  Fig.  extract,  15  shows  following  the  partial  purification using a SepPak.  Isolation NMR)  can  of larger amounts of ecdysterone for LH20  column,  molecules on the basis of size and, to a lesser extent,  polarity.  Other  be  carried  out  chromatographic  on  a  Sephadex  spectroscopic analysis (MS,  techniques  tested  included  which  GC,  separates  reverse-phase  adsorption chromatography on X A D - 4 resin, silica gel column chromatography and droplet countercurrent chromatography.  R E S U L T S A N D D I S C U S S I O N / 47  Fig.  14.  H P L C trace of a mixture of ecdysteroids. A : ecdysterone; B: ecdysone; C: ponasterone A .  INJECT  TIME  b<e:Zi:Zi  14.  Fig.  15.  /h^:'dj.hs, dl~^«.  CtO^io^-P  firT-lfj,  .  8*E?.«  H P L C trace of crude extract of A. densa following partial by SepPak. Arrow indicates ecdysterone peak.  purification  R E S U L T S A N D D I S C U S S I O N / 48 Ecdysterone  is non-volatile  and must  (Morgan and Poole, 1976b; Bielby et al, at  each hydroxyl  hydroxyl in the  group,  but  due to  be derivatized  for  analysis by G C  1986). Trimethylsilyl ethers are formed  the  large  number  of variously  positioned  groups (six) in ecdysterone, incomplete silylation often occurs, resulting formation  of a number  of different derivatives. Although this method is  used by a number of workers in the field, it was attempted, but discarded for use in this study, for the abovementioned reason.  Adsorption chromatography on X A D - 2 resin is described by Schooley et al (1972)  as "an extremely  from plants". A study,  but  slightly  efficient more  ecdysterone was  present in all fractions  not  and simple extraction  adsorptive retained  resin  procedure for ecdysones  (XAD-4) was employed in  on this  material.  this  The compound was  collected. This technique may bear further  investigation,  using X A D - 7 or X A D - 9 resins.  Although silica gel columns are often used in the isolation of ecdysterone, their value is questionable for this type of compound since: 1) its retention time is very long, requiring large volumes of solvent, 2) as demonstrated by the T L C recoverj'  tests  mentioned  earlier,  significant  amounts  of  ecdysterone  will  irreversibly bind to the silica gel, and 3) ecdysterone cannot be visualized on the column nor can it be distinguished from other compounds on the basis of its U V spectrum.  Droplet countercurrent  chromatography  (DCC) has a large sample capacity  (up to 2.5 g crude extract per injection) and has been successfully used by Kubo  R E S U L T S AND and  coworkers  et  (Kubo injected and  al, onto  in the isolation of mg  DCC  was  the compound  after  sample  addition,  ecdysterone  spectrophotometrically absorbs U V to  the  up  tested  eluted  injection.  over  The  nm.  of bubbles  1985).  Recovery  was  the  very 243  broad  solvent  mobile  could  ecdysterone  (0.5  is a  procedure  slow  not very not  contains  to 26  phase  really  be  the  30  40  FRACTION NUMBER  Fig. 16.  DCC  SO  (5  60  70  ml/f raction )  elution profile of 0.5 mg  due  eventually abandoned as  of phytoecdysterone.  20  which  stationary  7 -  10  h  detected  chloroform,  passing through was  mg)  reproducible. In  absorption traces were erratic  phase in the detection cell. For these reasons, DCC a method for purification  and  system  nm,  plant sources  fractions (Fig. 16), 17.5  nm)  Even at 254 of the  of pure  present study. DCC  fifteen five ml  max.  because  quantities of ecdysteroids from  in the  band  (UV  to 245  presence  al,  et  1983b; Kubo  DISCUSSION / 49  ecdysterone  tO  R E S U L T S A N D D I S C U S S I O N / 50 In summary, although a wide variety  of purification  methods exist in the  literature, many of these are used in the extraction and isolation of ecdysteroids from  insects, not plants.  behave  in  systems.  a  manner  The  Plant  similar  purification  straightforward,  extracts to  ecdysterone  scheme  reproducible  and  ecdysterone in different plants  contain  many'  in  ultimately  polar  solvent  and  selected  semiquantitative  can be determined).  (ie.  compounds  in  which  chromatographic this  relative  study  is  amounts  of  Fig. 8 outlines this scheme,  and relevant physical and chemical data for ecdysterone are shown in Table I.  For  more accurate analysis of trace quantities  probably  turn  (1972;  1974)  to radioimmunoassay  chemiluminescent  and  O'Connor  derivative  (RIA) techniques (1985).  Another  of ecdysterone, one should (see Borst  and O'Connor  immunoassay  of ecdysone has also been developed  using  a  (Reum et al,  1984).  3.2. O C C U R R E N C E  OF E C D Y S T E R O N E IN PLANTS S U R V E Y E D  Twelve species of ferns, three species of gymnosperms, and five species of angiosperms were examined for the presence of ecdysterone. A detailed  summary  of the results of this part of the study is given in Table IV. For each species, the presence of ecdysterone was determined by T L C and H P L C .  The behaviour  of the compound was the same for all species in which ecdysterone is recorded as present, and co-chromatography with commercially obtained ecdysterone resulted in peak  enhancement  ecdysterone  peak  on H P L C .  was  isolated  For one species of fern (Aspidotis densa), the on  Sephadex  LH20  and  mass  spectrometry  Table IV:  Occurrence of Ecdysterone  DIVISION SUBDIVISION ORDER FAMILY  PterIdophyta PterIda 1es S1nopter1daceae  SPECIES  Aspidotis  PLANT  densa  fronds  In Plants  PART  Surveyed  EC  PREVIOUS  (s+f )  NO  REPORTS  REPORTS  (Brackenr.) Lei 1 Inger. Cryptogrammaceae  Crypt  ogramma  fronds  (s)  no MH  activity  HlkIno et  a'(1973)  fronds  (s)  no MH  act 1v1ty  Hlk Ino  aH 1973 )  crispa(L.)R.Br.  Gymnogrammaceae  f ronds  Pityrogramma triangnIari  Maxon.  The  3 fronds  Adiantum pedatum  Asp1d1ales Thelypter(daceae  L.  Iypteris  fronds  Athyrlaceae  Hlklno et  no MH  H1k1no et  activity  a'(1973)  fronds  I  Ix-femina  (L . )  Roth.  high MH  act1v1ty  r co  a!(1973)  species)  (other species)  CO  > co  Sloss.  Athyrium fi  s l i g h t MH a c t i v i t y (some c o l l e c t i o n da tes)  (another  phegopteris  (L.)  REPORTS  s  (Kaulf.) Adlantaceae  NO  et  Hlklno ef  a'(1973)  o d  CO  co  o z  Cyst  opteris  EC i s o l a t e d  Htklno &  fronds  no MH  Hlklno et  *M 1973)  fronds  s l i g h t MH  Hlklno et  a)( 1973)  fronds  no MH a c t i v i t y (another species)  Hlklno ef  3/(1973)  EC i s o l a t e d spec 1es)  Takemoto et  activity  Hlklno (1970)  frag(!is(L.)  Bernh. Aspidlaceae  Dryopteris  activity  (Kaulf.)  arguta  Watt . Polysti mm  chum  i turn  (Kaulf.) Blechnales B1echnaceae  Presl  fronds  0Iechnum  (s+f)  +  spicant(L.)  (other  «M1973)  Roth. Polypodla 1es Polypodlaceae  rhlzomes  PoIypodium gIycyrrhiza  EC Isolated from other species  Bergamasco 8 (1983)  Horn  EC Isolated  Hoffmelster cf  EC Isolated from T. cuspi dat a Sieb 8 Zucc.  Ima1  D.C.Eat . Spermatophyta Gymnospermae Taxa1es Taxaceae  Taxus  baccata  1 eaves  aM 1967)  L. leaves  Taxus brevIfolI  Ncitt.  a  +++  ef  al(1967  )  T  1 eaves  axus  canadensI  s  Marsh. Anglospermae LiHales LI 11aceae  Trt  cernuum  TriI  rhizomes  nIum L.  I Ium  E C Isolated from other species  ovatum  rh1 zones  pendula  1 eaves  NO REPORTS  1 eaves  NO REPORTS  roots  NO REPORTS  Imai et  al(19G9)  Pursh. CommelInales Commelinaceae  Zebrina  Schlzl. TradescantI v i r g i n I  a ana  L.  Murdannia  tubers  s c a p i f l o r a  EC Isolated from another species  Hou et  al(1980)  H CO  f EC ED (s) (f ) +  ecdysterone ecdysterold(s) s t e r i l e fronds f e r t 1 I G fronds Indicates r e l a t i v e concentration number of "+" no E C detected  Pterldophyta c l a s s i f i e d  according to the  scheme of  CO  >  a of EC CO  O Plcchl-Serm o111 (1958)  a  CO CO  o  •z  Oi CO  R E S U L T S AND  DISCUSSION / 54  confirmed the identity of the peak as ecdysterone.  Ecdysterone Cryptogramma  was  crispa  found  in fronds of Aspidotis  densa (Brackenr.) Lellinger.,  (L.) R.Br., Dryopteris arguta  spicant (L.) Roth., and  in the rhizomes  (Kaulf.) Watt,  of Polypodium  glycyrrhiza  and  D.C.  Blechnum Eat. These  five species belong to five separate families of Pteridophytes (Pichi-Sermolli, 1958).  The FW.  concentration of ecdysterone in Aspidotis  There  are no  This  sterile and  species was  of  showed  slight  Japanese  Sterile  and  (Takemoto  In  the  hormone  same  study,  activity, which  moulting  fronds of Blechnum  extracts  concurs  small amount of ecdysterone was  and  spicant contain  B. et  moulting hormone activity was niponicum al,  Makino  1973)  2-deoxy-20-hydroxyecdysterone Strong  no  hormone  activity in  of Dryopteris  with  the  and  (0.01%  moulting hormone activity  contain  >0.001%  was  observed  FW)  from  observed  results of the  FW  et  in B.  A al  Blechnum  ecdysterone,  as  three other species  in a fourth. B.  ponasterone  Chong  arguta  detected in Dryopteris arguta.  in this study. Ecdysteroids have been isolated from  of Blechnum Makino  have  crispa contain ecdysterone.  Hikino et al (1973) using extracts of a large number of  ferns.  moulting  present study. A  determined  by  been studied previously.  fertile fronds of Cryptogramma  previously reported to  bioassays performed species  0.01%  previous reports of ecdysteroids or moulting hormone activity  in this species of fern, nor has the genus Aspidotis  Both  densa is approximately  and (1970)  amabile  ecdysterone isolated  minus (R.Br.) Ettingsh.  castaneum L. but extracts  R E S U L T S A N D D I S C U S S I O N / 55 of B. orientale L. showed no activity  whatsoever (Hikino et al, 1973). The genus  Blechnum has a high incidence of ecdysteroid-containing species, with moderate to low concentrations of ecdysteroids.  Various  members  of  the  Polypodium  complex,  most  notabfy  vulgare L. contain ecdysteroids, with reports of concentrations  Polypodium  as high as 1% of  the fresh weight of rhizomes (Jizba et al, 1967; Horn and Bergamasco, 1985).  It  was therefore expected that P. glycyrrhiza D.C. Eat. from British Columbia might contain  high  concentrations  of  the  compound,  but  rhizomes  of  this  species  contained relatively low amounts of ecdysterone.  For  most  of  the  species of  ferns  examined  in  which  ecdysterone  was  undetectable, the results correspond with published information on those species or members  of  the  same genus  (see Table  IV).  No ecdysterone  was  detected  in  fronds of Pityrogramma triangularis (Kaulf.) Maxon. This genus has not previously been examined.  There  does not  appear,  from  this  study,  presence of ecdysterone and degree of evolutionary  to  be  a relationship  between  advancement in the ferns, but  the number of species examined is too low for any meaningful conclusions to be drawn. When Hikino et al (1973) MH  activity,  evolutionarily  they  noticed  advanced  that  families.  screened 283 families  MH  activity  Ecdysterone  was may  more be  of  of Japanese ferns common use  in  in  the  for most  distinguishing  between species of confusing groups such as the Polypodium complex, but further screening of all Polypodium species is required, using more quantitative,  sensitive  R E S U L T S AND techniques  To  such as  DISCUSSION / 56  RIA.  shed more light on the question of ecdysterone  as an  agent, it would be interesting to compare insect herbivory on the  same  genus  ecdysteroids. considers  The  the  present, 7  Blechnum)  problem  number  each  secondarj  (eg.  with  of  which  becomes  contain  increasingly  ecdysteroids  and  different effects on  a  range  herbivore defense  sympatric of  complex  concentrations  however,  ecdysteroid-glycosides  insects, as  well  as  species of  the  when  which other  of one  may  be  classes of  metabolites involved in plant-insect interactions.  Several  species of plants  also examined  in the  which  species  certain  course are  from  of the known  groups study.  to  other  These  contain  than  the  Pteridophyta  were chosen  relatively  high  from  were  families in  concentrations  of  ecdysteroids.  In the gymnosperms, members of the genus Taxus, are reported to contain up  to  0.002%  isolated from  (fresh  T.  from  1967). The  UBC  Endowment  leaves. The T.  1967), and  Lands  and  Manchuria. T. analyzed  for  species contained  American one  yew  Eastern  significant  (T.  canadensis  Marsh.).  been  ecdysterone  Two  and  collected  mainly  expected,  and  Africa  baccata L. was  quantities of ecdysterone.  were tested as well: one  has  cuspidata Sieb.  means of confirming the validity of the isolation technique. As this  steroid  former species is native to Europe, N.  Asia; the latter to Japan, Korea and the  in their  baccata L. (Hoffmeister et al,  Zucc. (Imai et al, W.  weight) ecdysterone  as  a  leaves of  species of North  Western species (7\ brevifolia Nutt.) and These  species  have  not  previously  been  R E S U L T S AND examined  with  approximately  respect to ecdysteroid content. The  baccata contains an  The  production  Another  from  the  genera  family. No leaves  or  species  ecdysterone  contain  an  triquetra (Wall.) collected present  China,  of  the  known  in T.  Clarke  Lu,  1980)  where  this  (Commelinaceae). The  dry roots  and  in the leaves  (Santos  et al,  was  amounts  detected  of  based  Bruckn.  Polypodine  (Wang  et  B  al,  is grown  is used  L.  These  has  1984).  contain detectable  for silk 0.7%  1972). Three other  on  the  may  be  plants  in the  contains  possibility found  of Zebrina pendula  species of the genus Murdannia  ecdysteroid.  plant  1970;  ecdysterone  in leaves  virginiana  the  vaga Schult.,  C.  large  in any  compound  examined,  species.  species to  genus,  were  canadensis. T.  plant  same  in Thailand, did not  have  in  that this  Schizl., or in not  previouslj'  (Commelinaceae) is reported  been  isolated  Tubers  Murdannia  of  amounts  from  Murdannia scapiflora,  of ecdysterone  in the  study.  The  number  of  species  conclusions to be  drawn  ecdysterone.  compound  range  in  of Tradescantia  been examined. One to  and  Commelinaceae  producing  roots  brevifolia than  of ecdysterone  (Chou  weight) ecdysterone  genera other  ecdysterone  of ecdysterone  industry. (fresh  concentration  in Cyanotis arachnoidae C B .  2.9%  concentration of ecdysterone is  amount intermediate between the other two  highest  date, occurs contain  greater in T.  four to five times  DISCUSSION / 57  The  of concentrations,  future, when  a  greater  examined  in  this  survey  is  too  regarding chemotaxonomical relationships with is so  that  widespread  it may  number  be  in plants, and  of chemotaxonomic  present  small  respect to in such  significance  of species have been examined  for  a  in  the  chemically.  The  RESULTS results that  of this  study  ecdysterone  will  ecdysteroid-containing  confirm  earlier  observations  not necessarily family,  occur  but is often  AND  DISCUSSION / 58  (Bergamasco  in more  present  than  and Horn, one  in varying  genus  1983) of an  concentrations  in  more than one species of the same genus. The findings of this survey are useful as  an addition  to the growing body of information  concerning the occurrence of  ecdysterone in plant species.  3.3. B I O L O G I C A L A C T I V I T Y  OF  3.3.1. Production of Thiarubrine  ECDYSTERONE  i n Callus  A.  produce  activity  (Cosio  thiarubrines et  al,  —  sulfur-containing  1986). Production  TISSUES  Cultures  Callus cultures of crown gall tumours from &  IN P L A N T  Chaenactis douglasii (Hook.) H.  polyacetylenes  of secondary  with  metabolites  antimicrobial  has, in some  instances, been enhanced by administration  of "elicitor" compounds to plant tissue  and  effect  cell  production  cultures  (Bohm,  1980).  The  in callus cultures of transformed  of  ecdysterone  on  Chaenactis douglasii was tested. The  results are shown in Table V. Ecdysterone had no discernible effect on of thiarubrine in this tissue culture system.  thiarubrine  production  RESULTS  Table  V.  Production Chaenactis  of Thiarubrine  in Callus  Cultures  DISCUSSION  from  / 59  Transformed  douglasii  Treatment  *Values  AND  Total Thiarubrine* (mg/g dry weight)  Control  4.4 (1.6)  EC  3.7 (0.94)  IAA  4.7 (2.3)  are means of three replicates. Values  in brackets are standard errors.  R E S U L T S A N D DISCUSSION / 60 3.3.2. Plant Growth Bioassays  Ecdysterone defensive  compound  has a widespread  distribution in plants,  remains in the realm  group, tend  to exhibit high  by  (1983): "Why are so many  a  Karlson  biological  and its role as a  of speculation. Steroid molecules, as a  activity in many organisms  (see an article  hormones steroids?"), and plants  contain  large number of sterols, the functions of which are still a mystery. Very few  studies  have  plants  (see Introduction).  ecdysteroids testing  investigated  These  and the potent  of ecdysterone  brassinolide  the physiological  exhibits  facts,  plant  for activity  high  activity.  activity  and the structural  growth  similarity  between  substance, brassinolide, prompted the  in several Two  (if any) of ecdysteroids in  plant  growth  gibberellin assays,  bioassays  in which  one auxin,  and one  cytokinin assay were carried out. The results of these tests are discussed  below. Gibberellin Bioassays  Gibberellins are a group of diterpenoid phytohormones which have a range of biological activities in excised  tissue from  intact plants. Gibberellins and ecdysteroids the mevalonic acid pathway compound isoprene  species of the Cucurbitaceae, and in  share a common biosynthetic origin in  (see Fig. 17), with gibberellins arising from  farnesylpyrophosphate,  and ecdysteroids  arising  from  the C15  addition  of an  unit to farnesylpyrophosphate, followed by cyclization and oxidations.  Due biosynthetic  to  some  structural  origin, it has often  similarities been  to  suggested  gibberellins,  that  and  the ecdysteroids  a  common  may  play  RESULTS  AND  DISCUSSION  / 61  ACETYL COA  MEVALONIC ACID ((WA)  ISOPRENE (C5)  2 1SOPRENES  GERANYLPP  MONOTERPENES  (CIO)  FARNESYLPP  SESQUITERPENES  IPP  (C15)  C20  2 FARNESYLPP  TRITERPE'lES ( C 3 0 )  DITERPE'IES ( C 2 0 )  I GIQBERELLINS  SQUALENE CHOLESTEROL  ECDYSTERONE AND OTHER STEROIDS  OH OH  Fig.  17.  Major steps in the mevalonic GA and ecdysterone. 3  acid  I  pathway, showing branch  point to  RESULTS AND some  gibberellin-like  opposing four  role  results. Hendrix  gibberellin  within and  bioassays,  plants.  This  has  Jones  (1972) found  whereas  Matsuoka  been no et  tested  activity al  DISCUSSION / 62 only  twice, with  of ecdysterone in  (1969)  had  observed  stimulation of growth in rice.  The was  effect of ecdysterone on  re-examined  semi-intact  tissue  hypocotyls, compared  in  and  in  this  study.  two  elongation in gibberellin-sensitive plant tissues Ecdysterone  gibberellin  was  bioassays:  2) elongation of mung  bean  applied 1)  to  elongation  epicotyls. Ecdysterone  excised of  and  cucumber  effects  were  with those of 24-epibrassinoIide (Fig. 18). Brassinolide is active in both  bioassays (Mandava et al,  1981;  Gregory  Fig. 18.  and  Mandava,  24-epibrassinolide  1982).  R E S U L T S AND Ecdysterone with  cotjdedons  no  attached  (Katsumi et al,  system of  had  GA -treated  effect  (Fig. 19). 1965)  was  brassinolide-treated hypocotyls no  significant  The  These  tissue being length  of  indicated  were  were  cm  that  does  not  and  3  between  not  shown was  as  are  in  a  Fig.  shown  in  the  because  analysis of variance epicotyl  difference between trials. for to  and  bean  of  the  controls  for  the  elongation  seedlings  9).  The  initial  effect  of  elongation  repeated  leaves  and  hypocot3d was trial  of  ecdysterone  tested for  difficult  showed  the  this  a  (p = 0.05),  as  experiment  concentration  well. The  ecdysterone to draw  was  20)  these  significant effect of there  results  (Fig.  from  also  a  the  cut to a  a total of four times. In two  graphs are  (ie.  of  on of of and  results,  ecdysterone significant  Comparisons between means of initial length of epicotyls  each trial showed significant differences as well. It might, therefore, be useful repeat the experiment, germinating  enough seedlings with that the may  greater,  mung  significant  (Fig. 21). Conclusions  on  times  the  clear-cut  24-epibrassinolide  concentration  this  and  of 24-epibrassinolide was  activity  although  in  elongation  the  experiment  elongation  was  experiment was  the  seven  (the epicotyl) were intact, but the  as  there  the  promote  hypocotyls  four times more, than controls. There  epicotyl elongation. The trials,  cucumber  24-epibrassinolide do. Elongation  approximately  epicotyls of semi-intact  measured 3  of excised  hypocotyls.  results  epicotyls.  IAA  elongated  difference  ecdysterone-treated  elongation  but both G A  hypocotyls  3  on  DISCUSSION / 63  equal  epicotyl lengths  effects of ecdysterone,  have  no  biological  a large number of seeds in order to obtain  while  (+  statistically  significance. Jones  and  1 mm).  It is important  significant, are very Matloff (1986) have  to note  small  and  pointed  out  RESULTS AND  DISCUSSION / 64  10-  U3  5-  EC  10-4  BR  GA3 10-5  CONCENTRATION  Fig. 19.  IAA 10-5  (M)  Elongation of cucumber hypocotyls. Values are means of three replicates. Bars indicate standard deviations. Controls (C), ecdysterone (EC), 24-epibrassinolide (BR), gibberellic acid ( G A ) , indole-3-acetic acid (IAA). 3  RESULTS  AND  DISCUSSION  / 65  7.0 6.0E  5.0-  5  «.o-  3 ?  rh  rh  rh rf, rh th  3.02.0I.O 0  10"12 10* : 1  IO - 1 0  IO'3  IO"8  Ecnrs-so* :onciiTi«r:oii  Fig.  20.  io-7  10"5  rn >  Elongation of mung bean epicotyls. Values Bars represent standard deviations.  are means  of four  trials.  of two  trials.  s 9.0E  S  rh  rh  0  10"12  rh rh  2.01.0-  10' 1 J  IO - 1 3  ID"3  IC" 8  IO"7  ID-'-  2»-i»ir.asi»:'.K tw.trvr.w, < r i Fig.  21.  Elongation of mung bean epicotyls. Bars represent standard deviations.  Values  are means  R E S U L T S AND the  dangers  of  assigning  significant, effects. The concentration Comparison treatment  on of  ANOVA  epicotyl means  had  biological  showed  elongation, for  significantly  each  and  epicotyls from  the  three  the lowest concentration  Maximum times  greater  concentration (Gregory  elongation  than  procedure, a  (10" M)  of  1982),  a  regression  showed than  less  strict  with  a  in this more  system, and  strict  size  only  controls test  (10~ ,  statistically ecdysterone of  the  0.07.  10"  (Scheffe  6  M  test,  (p = 0.05), indicated 10 ,  8  and  - 7  application of 6.2  (10' M) 6  at  times that of controls. In elicited  3  it may  limitation  seedlings  24-epibrassinolide  a  3.7-fold  controls in this bioassaj'. It appears that ecdysterone elongation  but  coefficient  that  the  concentrations  ecdy steroid-treated  GA  slight,  10"  6  M)  were all significantly longer than controls.  1 2  controls, and  Mandava,  with  elongation  highest  very  significant, linear effect of  concentration  resulted in elongation  and  a  but  greater  p = 0.01). Student-Newman-Keuls that  significance to  DISCUSSION / 66  be  may  worthwhile  placed  on  to repeat  initial  length  1.8  the  same  another  study  elongation have  was  relative  to  some effect  on  the of  experiment  epicotyls, as  previously mentioned. Auxin Bioassay  Auxins plant  promote  tissues. Applied  ones, due  growth auxin  to creation of a  which  does not  auxin  in certain auxin  by  cell  stimulates  extension growth  in particular, types  in excised  deficiency of endogenous  auxin  stems, but in excised  exist in intact plants. Brassinolide shows strong bioassays, such as  of excised not  intact  segments,  synergism  with  inhibition of opening of etiolated bean  RESULTS AND hypocotyl  hooks  et al,  (Yopp  1979); elongation  of azuki  DISCUSSION / 67  bean  epicotyl segments;  and  expansion of dwarf pea hooks (Yopp et al, 1981). In the expansion of dwarf  pea  hooks, brassinolide alone  IAA  alone  elicited  did not elicit  elongation  or fresh weight increase.  little or no elongation, but did promote  lateral  expansion, as  indicated by an increase in fresh weight of the hook segments (39% greater than controls).  When  synergistic (10" M)  applied  manner.  resulted  6  A  simultaneous^, combination  in elongation  brassinolide  of IAA  67%  greater,  and  (3 x and  IAA  10" M )  with  6  fresh  acted  weight  in a  brassinolide  increase  78%  greater, than those of control segments (Yopp et al, 1981).  The not  effect  observed  significant exhibited  of brassinolide on the elongation  unless  elongation a  slight  IAA  is added  and fresh  effect  response in this  to the system,  weight  on elongation  increases  under  occur.  in the mung  which  Since  bean  bioassay is conditions,  ecdysterone  epicotyl  had  bioassay, it  seemed possible that it might elicit significant elongation in the presence of IAA in  a  manner  bioassays  similar  to brassinolide. Brassinolide enhances  specific to either IAA or G A  In  the  combinations  present  study,  of ecdysterone  10" M.  Figs.  increase  respectively. Percent  5  calculated  as  22  and 23  (increase/initial  3  excised  dwarf  the results  elongation x  pea  hooks  were  of .0,  for elongation  and percent  100). The data  fresh  were  treated  increased  with  the magnitude  of the response,  7  and fresh  weight  6  weight  increase  log-transformed  thus  with  1 0 " , 10" , and  statistical analysis by two-way analysis of variance, because the variance means  in stem  (Mandava et al, 1981).  and IAA at concentrations show  elongation  obscuring  were  prior to around possible  RESULTS  Fig. 22.  Elongation trials.  of dwarf pea hypocotyl  AND  hooks. Values  DISCUSSION / 68  are means  of three  RESULTS AND  Fig. 23.  Fresh weight increase means of three trials.  of dwarf  pea  hypocotyl  DISCUSSION / 69 .  hooks.  Values  are  R E S U L T S A N D D I S C U S S I O N / 70 effects. As expected from the results of Yopp et al (1981), I A A had a significant effect on fresh weight increase, but not elongation (p = 0.01). Ecdysterone had a significant,  but  slight,  effect  on  elongation,  but  (p = 0.05). There was no interaction between I A A  not  fresh  weight  and ecdysterone for  increase elongation  or fresh weight increase. It is difficult to numerically compare these results with those reported by Yopp et al (1981). They present elongation as the mean final length of hooks (mm), and fresh weight increase as mean final weight (mg). This is  not  a  valid  presentation  of  results,  since  initial  lengths  and  weights  of  segments are not at all uniform when prepared according to the methods for this bioassay.  It  is  interesting  that  GA  3  elicits  a  slight,  but  significant  increase  elongation in this system, but no increase in fresh weight (Adamson et al, This response to G A the  present  study.  3  in  1968).  is comparable to the response to ecdysterone observed in  Possible  implications  of this  are discussed in  the  summary  section of this chapter.  Cytokinin Bioassay  Applied cytokinin results  weight  are  fresh  increases significantly enhanced  a concentration  expansion of  experiment  increase in  (10 *M) -  bioassay of  of the  percent  ecdysterone at  cucumber  shown in  weight.  of  10" " M  had  no activity  in  a  (Cucumis sativus) cotyledons. The  F i g . 24. Expansion was measured as  Ecdysterone-treated  cotyledons  did  not  have  different from controls, whereas 6-benzylaminopurine  expansion  3  times  relative  to  controls  and  RESULTS  AND  DISCUSSION / 71  140120i LU CO  100-  CO LU  601020-  EC  BR  BAP  TEST SOLUTION  Fig. 24.  KIN  ( lO"*1 M )  Expansion of cucumber cotyledons. Values are means of five replicates. Bars represent standard deviations. Controls (C), ecdysterone (EC), 24-epibrassinolide (BR), 6-benzylaminopurine (BAP), kinetin (KIN).  R E S U L T S A N D D I S C U S S I O N / 72 kinetin(10~ M)-treated cotyledons had fresh weight increases over 4 times greater 4  than controls. 24-epibrassinolide (10" M ) 4  cotyledon expansion. In concentration  the  was also tested and had no effect  same bioassay, Yopp et al  of brassinolide resulted in  a  significant  (1981)  fresh  found  weight  that  increase  on this 1.5  times greater than that of controls. 6-Benzylaminopurine and kinetin are synthetic c.ytokinins which probably do not occur in plants, but their structures are similar to  naturally  occurring  (in  plants)  cytokinins  such  as  zeatin.  They  are  all  adenine-derived compounds with a purine ring and their structure and biosynthetic origins are different from those of ecdysterone. This is probably the reason that ecdysteroids  have  explain their  not  previously  been  tested  activity  in  in  cytokinin  bioassays,  and  may  inactivity. Summary  Ecdysterone  showed  no  a  cytokinin  bioassay  and  in  one  gibberellin bioassay. In both tests, the response of excised tissue from Cucumus sativus to applied ecdysterone was measured. Elongation and lateral expansion of dwarf pea hypocotyl hooks are elicited by G A  3  (Adamson et al,  1968) and I A A  (Yopp et al, 1981) respectively. Ecdysterone had no effect on lateral expansion of hook segments, and no interaction with I A A , but it did elicit a slight increase in elongation  (the  gibberellin-responsive parameter  of this  system). Ecdysterone also  elicited an increase in elongation in a gibberellin bioassay using semi-intact mung bean epicotyls.  Although the effects of ecdysterone were not great, they were statistically  RESULTS AND significant and it is interesting  to note that for the tests in which  was seen: 1) both are responsive  to G A ,  observed  are elongation  for ecdysterone;  DISCUSSION / 73  to approximately  3  2) both  a response  the same degree as  responses;  and 3) both involve  non-Cucurbitaceous species.  The role  for ecdysteroids  biological using in  results of this study  activity  and there  in plant  do not exclude is some  plants have  classes outlined  of plant  demonstrated growth  the presence  regulators  subject  one  could  of much  research.  demonstrate  the  Combining or  follow  that  ecdysterone  does  should be studied in greater detail.  have  absence  binding  this  area  of receptors,  physiological role  for both 1986). As  cells  of these  receptors  sites  et al,  in plant tissues. If these a  have  and auxin  in Drosophila  the technologies  could  to pursue  1986; Libbenga  receptors  high-affinity binding sites, for ecdysterone would  of high-affinity  of a physiological  ecdysterone  for gibberellin  (Stoddart,  presence  that  be interesting  searches  in the Introduction, ecdysteroid  the  indication  tissues. It would  a different approach. Ongoing  the possibility  have three  or  at  been areas, least  sites exist, it  in plants  which  4. CONCLUSION  A  simple, reliable  ecdysterone number to  in plant  method  material. By  of plant species which  phytoecdysteroids. These  and  two  species  ecdysterone  plant  of  be  this  have  Trillium.  species  should  be  of  provides  studies  for  was  found with  in  a  respect  species of Taxus,  indicated  that  applied  directed along the following lines. ecdysteroid  content,  using  more  may  prove  such as immunoassay. Phytoecdysteroids concentrations. The  in plants would probably  receptors in plant tissues. Much  best be  question of a physiological answered by  more could be  searching for  learned regarding the  the localization of these compounds in plant  cells. So little is known about this group of compounds, that for  are still largely regarded  research  species of ferns, two  screened  biosynthesis of phytoecdysteroids, and  they  ecdysterone  previous!}' been examined  work in this area would best be  role for ecdysterone  tissues and  not  Physiological  ubiquitous in plants at low  ecdysteroid  for semi-quantitative analysis of  procedure,  include four  sensitive analytical techniques to  developed  could have biological activity in plant tissues.  Future More  was  is full  some  of  as a curiosity of plant biochemistry, and  interesting  suggestions  regarding  questions the  which should be most fruitful.  74  with  types  elusive  of research  answers. on  now  this area  This  study  phytoecdysteroids  5. 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APPENDIX 1  S H Growth Medium  Stock Solutions (per 100 ml): #1 M g S o , . 7 H 0 20.0 g #2 C a C l . 2 H 0 20.0 g #3 F e S O „ . 7 H 0 1.5 g + Na EDTA.2H 0 2.0 2  2  2  2  2  2  Vitamins: Nicotinic acid Thiamine.HCl Pyridoxine.HCl  500 mg 500 mg 50 mg  Micronutr ients: MnSO„.4H 0  1320 mg  2  H3BO3  600  mg  KI ZnSO„.7H 0 CuSO .5H 0 CoCl .6H 0 Na MoO .2H 0  100 100 20 10 10  mg mg mg mg mg  2  f l  2  2  2  2  a  2  B. Medium: Stock #1 2 ml/1 Stock #2 1 ml/1 Stock #3 1 ml/1 Vitamins 1 ml/1 Micronutrients 1 ml/1 inositol 1.0 g/1 KNO3  2.5  g/1  NH,H P0, 2  sucrose agar  0.3 g/1 30 g/1 7.0 g/1  * Adjust p H to 5.7 From: Schenck and Hildebrandt (1972). 85  


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