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Determination of some phytoestrogens in alfalfa sprouts Yada, Sylvia Ann (Duffek) 1984-12-31

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DETERMINATION OF SOME PHYTOESTROGENS I N ALFALFA SPROUTS  by SYLVIA ANN (DUFFEK) YADA B.Sc.(Agr.),  U n i v e r s i t y of B r i t i s h Columbia,  A t h e s i s submitted  in partial  the requirements f o r Master  of  fulfillment  the degree of  Science  in THE FACULTY OF GRADUATE STUDIES Department  of Food S c i e n c e  We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA February 1984  0  S y l v i a Ann (Duffek)  Yada,  1984  1978  of  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may  be  department o r by h i s o r her  granted by  the head o f  representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department of  Food Science  The U n i v e r s i t y of B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3  March 14,  1984  Columbia  written  (ii)  ABSTRACT  Alfalfa determine rinse gens  sprouts  the  effect  frequency were  isolated  was  graphy  of  A method f o r  water  system  solvent  to  Baseline  time  of  the  dark  cantly trol  (0 h l i g h t )  greater than  Although alfalfa  those the  would  common v e g e t a b l e s , not yet been  1.0%  acetic  sprout  less  growth  be  octadecylsilane  and  0.1  methanol/ M ammonium  daidzein,  content  Alfalfa  period  daidzein, chosen  phytoestrogens to  an  chromato-  formonone-  e x t r a c t s was achieved with an  employed.  longer  liquid  than 2 ppm f r e s h  other  appear  acid  phytoestrogen  from of  phytoestro-  254- nm and a g r a d i e n t  of the phytoestrogens  the p h y s i o l o g i c a l  determined.  at  of  sprouts  The  of  high-performance  contents  contribution  sprouts  for  conditions  (P<_ 0.05)  volume and  evapora-  (or  the  to  rinse  phytoestrogens. extract  order  remaining f o l l o w i n g e t h y l ether  Total  alfalfa  for  duration,  in  sprouts  containing  growth  conditions  alfalfa  UV d e t e c t i o n  30 m i n u t e s .  on the  light  methanol  from the a l f a l f a  22 ppm dry weight  depending  of  selected  HPLC was developed using  resolution  and coumestrol  elution  crude  i n methanol  column,  tin  a  The r e s i d u e  reversed-phase  acetate.  accumulation  from  redissolved  (HPLC).  grown under  growth p e r i o d ,  on the  using e t h y l e t h e r . tion  were  markedly  ranged  from 1  weight  alfalfa)  sprouts  grown i n  (148  h)  had  signifi-  formononetin and treatment to  the  higher  significance  of  coumes-  combinations.  human than  diet from  from other  such an i n t a k e  has  (iii)  TABLE OF CONTENTS P  ABSTRACT  a  9  e  ii  TABLE OF CONTENTS  iii  LIST OF FIGURES  v  LIST OF TABLES  vii  LIST OF APPENDICES  viii  ACKNOWLEDGEMENTS  ix  INTRODUCTION  1  LITERATURE REVIEW  3  A.  Nature and d i s t r i b u t i o n of phytoestrogens  B.  B i o s y n t h e s i s and a c c u m u l a t i o n 1. Biosynthesis 2. E f f e c t s of m i c r o b i a l i n f e c t i o n 3. Fungitoxic a c t i v i t y 4. E f f e c t s of l i g h t  14 14 16 21 22  C.  Metabolism 1. Isoflavones 2. Coumestans 3. Absorption  23 24 27 28  D.  E x t r a c t i o n of phytoestrogens  29  E.  Detection techniques 1. Paper chromatography 2. T h i n - l a y e r chromatography 3. Column chromatography/UV spectrophotometry 4. High-performance l i q u i d chromatography  35 35 38 39 41  MATERIALS AND METHODS  3  45  A.  Materials  45  B.  P hy to e s tr o g e n e x t r a c t i o n  45  C.  Moisture determination  48  D.  Measurement of phytoestrogens  E.  G e r m i n a t i o n of a l f a l f a seeds 1. Sprouting preparation 2. Growth s t u d i e s (a) Standard c o n d i t i o n s (b) T r i a l conditions (c) Percent g e r m i n a t i o n 3. Sprout h a r v e s t i n g  53 53 55 57 57 60 60  F.  S t a t i s t i c a l analysis 1. S i n g l e factor a n a l y s i s of variance 2. F a c t o r i a l a n a l y s i s of v a r i a n c e  62 62 62  by HPLC  49  (iv)  RESULTS AND DISCUSSION  63  A.  Extraction 1. E x t r a c t i o n methodology 2. Recovery s t u d i e s  63 63 64  B.  High-performance l i q u i d chromatography 1. HPLC methodology 2. Chromatographic parameters 3. Linear regression analysis  67 67 70 77  C.  Growth s t u d i e s 1. Moisture determination 2. Growth c o n d i t i o n s (a) Percent g e r m i n a t i o n (b) Light (c) Temperature  78 78 80 80 80 82  3.  D.  A l f a l f a sprout development (a) Standard c o n d i t i o n s (b) T r i a l conditions  Phytoestrogen a n a l y s i s 1. Daidzein 2. Formononetin 3. Coumestrol 4.  General d i s c u s s i o n  82 83 83 86 87 89 93 98  CONCLUSIONS  101  REFERENCES CITED  103  APPENDIX  113  (v)  L I S T OF FIGURES  Page Figure 1 .  Structural  formulas  for  Figure 2.  Structural  formulas  for estrogenic  isoflavones  9  Figure 3.  Structural  formulas  for estrogenic  coumestans  10  F i g u r e 4-.  Proposed b i o s y n t h e t i c pathway of via  Figure 5.  s t e r o i d estrogens  4  isoflavones  a chalcone  15  P o s s i b l e b i o g e n e t i c r e l a t i o n s h i p s among isoflavonoids  Figure 6.  Metabolism of some e s t r o g e n i c  Figure 7.  G e r m i n a t i o n and development of a l f a l f a  20  isoflavones  i n sheep  seed  ....  to  sprout  31  Figure 8.  Phytoestrogen e x t r a c t i o n  Figure 9.  R e t e n t i o n time and peak width at  Figure 10.  Rack used  Figure 1 1 .  A l f a l f a s p r o u t s growing under f l u o r e s c e n t ( l i g h t treatment) and under aluminum f o i l (dark treatment)  Figure 12.  Figure 13.  F i g u r e 1A-.  Figure 15.  Figure 16.  25  scheme f o r a l f a l f a  sprouts  base measurements  for d r a i n i n g sprouting j a r s  D r a i n i n g of a l f a l f a freezing  sprouts  prior  ...  46  ....  51 54  lamps covers  56  to sampling and 61  HPLC chromatogram of phytoestrogen s t a n d a r d s . Peaks: D = d a i d z e i n , F=formononetin, C=coumestrol  73  HPLC chromatogram of phytoestrogen standards extract. Peaks: D=daidzein, F=formononetin, C=coumestrol  7k  HPLC chromatogram of a l f a l f a e x t r a c t s p i k e d w i t h phytoestrogen standards p r i o r to e x t r a c t i o n . Peaks: D=daidzein, F=formononetin, C=coumestrol  75  HPLC chromatogram of a l f a l f a e x t r a c t (Treatment X) Peaks: D = d a i d z e i n , F=formononetin, C=coumestrol  76  (vi)  Page Figure 17.  Figure 18.  Figure 19.  Figure 20.  Figure 2 1 .  E f f e c t curve of growth and l i g h t (G x L ) i n t e r a c t i o n f o r d a i d z e i n accumulation i n a l f a l f a s p r o u t s  91  E f f e c t curve of l i g h t and volume (L x V) i n t e r a c t i o n f o r d a i d z e i n accumulation i n a l f a l f a s p r o u t s  92  E f f e c t curve of growth and l i g h t (G x L) i n t e r a c t i o n f o r formononetin accumulation i n a l f a l f a s p r o u t s  94  E f f e c t curve of growth and frequency (G x F) i n t e r a c t i o n f o r formononetin accumulation i n sprouts  95  alfalfa  E f f e c t curve of growth and l i g h t (G x L) i n t e r a c t i o n f o r coumestrol accumulation i n a l f a l f a s p r o u t s  97  (vii)  LIST OF TABLES  Page  Table 1 .  R e l a t i v e b i n d i n g a f f i n i t y of some n a t u r a l l y o c c u r r i n g e s t r o g e n s f o r mammalian estrogen receptors  11  Table 2.  Standard  58  Table 3.  Trial  Table  Recovery o f phytoestrogens e x t r a c t i o n method  Table 5.  Table 6. Table 7 . Table 8.  growth c o n d i t i o n s f o r a l f a l f a  growth c o n d i t i o n s f o r a l f a l f a using  sprouts  sprouts  59  developed 66  R e t e n t i o n times ( t R ) , c a p a c i t y f a c t o r s ( k ) , s e p a r a t i o n f a c t o r s ( « ) and r e s o l u t i o n ( R s ) of s e l e c t e d phytoestrogens on MicroPak MCH-10 using l i n e a r g r a d i e n t e l u t i o n as d e s c r i b e d  71  M o i s t u r e content of a l f a l f a s p r o u t s grown under standard and t r i a l c o n d i t i o n s  79  P h y t o e s t r o g e n c o n t e n t s of a l f a l f a s p r o u t s grown under standard and t r i a l c o n d i t i o n s  88  S i g n i f i c a n c e of c a l c u l a t e d F - v a l u e s from g e r m i n a t i o n f a c t o r s and f a c t o r i n t e r a c t i o n s f o r phytoestrogen accumulation as determined by f a c t o r i a l a n a l y s i s of v a r i a n c e  90  (viii)  LIST OF APPENDICES Page Appendix A - 1 .  Appendix A - 2 .  Appendix B.  Appendix C.  Appendix D.  A n a l y s i s of v a r i a n c e f o r d a i d z e i n content of a l f a l f a s p r o u t s from Treatments AA to Z (1 to 14)  113  A n a l y s i s of v a r i a n c e f o r d a i d z e i n content of a l f a l f a s p r o u t s from Treatments AA to Z (1 to 14 without C-3 or Y-1 v a l u e s )  114  A n a l y s i s of v a r i a n c e f o r formononetin content o f a l f a l f a s p r o u t s from Treatments AA to Z (1 to 14)  115  A n a l y s i s of v a r i a n c e f o r coumestrol content o f a l f a l f a s p r o u t s from Treatments AA to Z (1 to 14)  116  F a c t o r i a l a n a l y s i s of v a r i a n c e for d a i d z e i n , formononetin and coumestrol c o n t e n t s of a l f a l f a s p r o u t s from Treatments A to Z  117  (ix)  ACKNOWLEDGEMENTS  I wish to express my deep a p p r e c i a t i o n h i s patience course  and s u p p o r t ,  of t h i s  the committee, their  financial,  throughout  the  I a l s o wish to thank the members o f  D r . Shuryo N a k a i , D r . Brent Skura and D r . Bob Bose,  I would l i k e to thank my f a m i l i e s ,  t h e i r constant  years,  project.  w e l l as  for  for  s u g g e s t i o n s and a s s i s t a n c e . Finally  for  research  moral as  to D r . John Vanderstoep  support  and a s p e c i a l  criticism,  both Duffek and Yada,  and understanding d u r i n g the past  thanks to my husband Rickey f o r  i n s p i r a t i o n and  encouragement.  his  several  invaluable  -  1 -  INTRODUCTION  Several  plants  of  contain  isoflavonoids  species  (Wong,  stans,  the Leguminosae  which are  1975).  known as  estrogenically  Two main  groups,  comprise the m a j o r i t y of these Alfalfa,  (also  c l o v e r and soybeans  the  active  as  well  phytoestrogens dietary  for  exposure  The  alfalfa  important sources  are  However,  response  Lookhart  coumestrol sprouts,  to m i c r o b i a l  et  al.  accumulated  but  Sherwood,  are 1973).  from  plant  they  primarily  quantitate  (1979a) during  detect  as  dietary  on  man  source  of  yet unknown.  can accumulate i n  1974;  that  germination  of  longterm  plants  and have been shown to  demonstrated  the  phytoestro-  the  of  be  Lyon and Wood, phytoestrogen  soybeans  to  soy  of fungal i n f e c t i o n .  and coumestrol occur n a t u r a l l y as w a t e r - s o l u b l e g l y c o -  hydrolyzed  to  aglycones  in  infected  plants  Many methods p r e v i o u s l y used to e x t r a c t  tissues  are  only  isolate  the  the e s t r o g e n i c  variety  is  (Nairn et a l . ,  useful  for  qualitative  alcohol-soluble  of  chromatographic  and measure these e s t r o g e n i c  (Olah  aglycones.  and  phytoestrogens  purposes,  p o t e n t i a l of a food or f e e d ,  g l y c o s i d e forms o f the i s o f l a v o n o i d s must be A  effect  infestation  p o s s i b l y a l s o as a r e s u l t  Isoflavones sides  the  of  Soybeans and soybean  potential  and coumestans  to i n v a d i n g f u n g i and b a c t e r i a  1975).  a  to these p l a n t c o n s t i t u e n t s isoflavones  and the coume-  "phytoestrogens".  are  sprouts  humans.  estrogenic  i n apparent toxic  as  family  i n d i v e r s e animal  isoflavones  gens i n the feeds o f g r a z i n g and domestic a n i m a l s . products  Fabaceae)  In  because order  to  both aglycone and  extracted.  techniques  have  been  developed  to  compounds. T h i n - l a y e r chromatography  - 2 -  has  p r e d o m i n a t e d , „ however,  graphy means and  (HPLC) for  rapid,  Dolphin,  1982a).  systems  isoflavones  have  Murphy,  and  of  coumestans  been  analysis  1981;  application  high-performance  recently  quantitative  1980;  The  several  HPLC  in  to  actual  demonstrated  of  Daigle  liquid  flavonoid  to  plant  a  of  the  (Carlson  1982;  mixture  extracts  provide  mixtures  and C o n k e r t o n ,  resolve  chromato-  Eldridge, estrogenic  requires  further  study. The extraction  object  of  procedure  the  present  research  for  alfalfa  with  the most predominant p h y t o e s t r o g e n s , tin;  secondly,  isolation study  and  the e f f e c t  duration,  rinse  phytoestrogens  high  of  selected  volume  in alfalfa  and  and  thirdly,  sprouting rinse  sprouts.  to  first,  extraction  coumestrol,  to develop an HPLC system quantitation;  was:  apply  conditions  frequency)  develop  efficiencies  an for  d a i d z e i n and formonone-  resolve  to  to  on  these compounds these  techniques  (growth p e r i o d , the  for to  light  accumulation  of  -  3 -  LITERATURE REVIEW  A.  NATURE AND DISTRIBUTION OF PHYTOESTROGENS Estrogens  can  be d e f i n e d  to the normal  sexual  primarily  the  by  hormones  in  the  steroid  development of  ovary  the  term e s t r o g e n  can a l s o  the  (Prosser,  circulation  incorporating  which w i l l  as  are  used  of e s t r u s ,  female r e p r o d u c t i v e system a s s o c i a t e d  workers  induce e s t r u s or  have  of e s t r o g e n i c  examined  i n animals,  phytoestrogens.  subterranean  clover  reproductive  abnormalities  Bradbury and White isoflavones,  various  et  al.  (Trifolium  (1951)  genistein  grazing  subsequently  and  Curnow  (1954)  that  x  if  consumed  isoflavones  could  plants to  identify  reported L.)  naturally  As c i t e d by Bradbury and White  Cheng  in  in  (1954).  i n plants  these a  western  strain  al.  subterranean  of for  of  two  extracts.  estrogenically normal  (1955) and  -  formononetin  clover was  to  Australia.  the presence and  time  plant  responsible  affecting et  i n the  ability  that  was  genistein  be a f a c t o r  The  substance  Since that their  subterranean  that  all  (Prosser,.1973).  for  sheep  by other . a n i m a l s .  occurring  any  a s e r i e s of changes  demonstrated  in  showed  a c t i v e i n m i c e , and t h e r e f o r e duction  describe  (5,7,4'-trihydroxyisoflavone)  (7-hydroxy-4'-methoxyisoflavone), Biggers  to  subterraneum  of  estriol,  shown i n F i g u r e 1.  and F e l l n e r 1 .  (1946)  secreted estrogenic  substances being present  and have attempted  Bennetts  and  with o v u l a t i o n  were made i n 1926 by Loewe, Dohrn et a l . , numerous  principal  estrone  nucleus as  be more b r o a d l y  essential  mammal and are  The  estradiol,  18 carbon s t e r o i d  reports  female  1973).  produce c h a r a c t e r i s t i c s  The f i r s t  hormones which are  repro-  determined  red  clovers,  -  F i g u r e 1.  Structural  formulas  4  -  for steroid  estrogens.  -  including  daidzein  (7,4'-dihydroxyisoflavone),  droxy-4'-methoxyisoflavone), activity  40  species  were r e p o r t e d  sativa  L.)  that  listed  as  time  established  that  estrogenic  of  and  both  but  (Bickoff  et  partial  (Lyman  or  of  rats  et  Cheng et  1957;  Bickoff  method,  known  cornified estrogen  of  al.,  as  the  (5,7-dihy-  have  estrogenic  clover  repens  L.)  alfalfa were  experiments  displayed  significant  Engle  of  the  et  al.,  ladino  1957). clover  component was not an  which  1958).  detected  in  reported  as  at  later  1956;  estrogenic  al.,  potency  was  named  coumestrol  Coumestrol other the  (7,12-dihy-  leguminous  dominant  iso-  forage  estrogen  in  1961). activity  enlargement with  Pieterse  1958;  (Bickoff  due to  of  test, in  al.,  primarily  o v a r i e c t o m i z e d or extracts  al.,  (Verdeal the  based  and Andrews,  cells et  were  plant  Lyman et  Allen-Doisy  criticisms  possible  et  estrogenic  However,  derivative  and  epithelial  stimulation  are  1959)  1955;  et  to  Bickoff  treatment  al.,  (Trifolium  ladino  the major  al.,  some  characterization  estrogenic  uterine  vaginal  subject  results  A  The common forages  plants.  subsequently  al.,  after  1954;  been  was  ( L i v i n g s t o n et  Reports  mice  1957;  (1954).  and Andrews,  a benzofurocoumarin al.,  showed  clover  and  (Pieterse  and  droxycoumestan)  measurement  which  ladino  alfalfa  estrogen(s) revealed that flavone,  plants  non-estrogenic  activity  isolation  alfalfa  formononetin and g e n i s t e i n ,  by Bradbury and White  (Medicago  plants  biochanin  i n mice.  Over  The  5 -  1959).  was the  1969). and  method of  Bickoff  on  animal  Both of  Ryan,  and  An e a r l i e r  based test  immature  (Biggers 1956;  the  female Curnow, et  al.,  detection  the  evidence  of  in  response  to  these assays  1979),  treatment  on  have  and  misleading  and the  metabolism  - 6 -  of  the  changes  compounds  being  studied.  Nevertheless,  induced by phytoestrogens  are observed as  normal e s t r u s induced by endogenous estrogens An endogenous on the uterus of  the  still  cells.  to  the  coworkers (1968) triggers  for  It  of  et  1975).  uterine  estrogen  receptor  induced  protein  (Ruh  by Ruh et  synthesis,  followed  Only  et  al. by  a  estradiol-17g,  which  specific  proteins al.,  (1973)  of  exerts  its  effect  was  can  into  the  al.,  1968).  later  estrogen  be most  induced  the  effective  other  endogenous  immediate  effect  of  G o r s k i and  to  protein  will  bind  synthesis  estrogen  the  while  established  which  regulate The  nucleus  e s t r o g e n - p r o t e i n complex  compounds  1973). to  tissue  p r o t e i n r e c e p t o r i n the cytoplasm  ( G o r s k i et  those  and  the c h a r a c t e r i s t i c s  transported  RNA s y n t h e s i s ,  (Gorski  observed  then  organ  i n r a t s and m i c e .  further hypothesized that t h i s  synthesis  al.,  is  protein receptor  messenger  necessary  such as  by b i n d i n g to a s p e c i f i c  uterine bound  estrogen,  the  (IP)  to of  estrogens  the this  estradiol-170 in inducing  be  was  protein  estriol  and  estrone. Another imbibition the u t e r u s  more  of water by the u t e r i n e t i s s u e s , (Notebloom and G o r s k i ,  u t e r i n e weight and s i z e on  a  combination o f  uterine  estrogen  1963).  resulting  protein  synthesis  the  of  activity is  and water  increased thus  uptake  based by the  tissues.  Studies  on the phytoestrogens  have shown t h a t  a l s o complex with the e s t r o g e n - p r o t e i n r e c e p t o r s and i n t u r n i n d u c e p r o t e i n s y n t h e s i s Gorski,  is  i n enlargement of  The measurement  i n determining estrogenic  induced  administration  1963;  Shemesh et a l . ,  1972;  these compounds can  i n the u t e r i n e c y t o s o l ,  and water i m b i b i t i o n Kitts,  1974;  (Notebloom and  M a r t i n et a l . ,  1978).  - 7 -  In it  is  order  to  believed  be r e c o g n i z e d  that  a  by estrogen  compound must  Plant  activity  possess a v a r y i n g degree of s t r u c t u r a l The  high  presence  of at  least  phenolic  hydroxyl  and  specific  ments.  steroids.  isoflavones  meet  receptors  binding  coumestans  affinity  one aromatic  group  on t h i s  that  of  ring, ring,  structural  requireestrogenic  s i m i l a r i t y to  estrogenic  is  presence  and  cells,  demonstrate  steroids  the  in target  the  related  to  and p o s i t i o n  nature  and  the of  position  a of  oxygen f u n c t i o n s on r i n g D. More s p e c i f i c a l l y , ring  A is  considered  al.,  1973).  Busetta  of  an estrogen  in  binding a f f i n i t y  strated  that  alcoholic lic  (Figure  1).  be  et  al.  on  to  or  C-3  of  on r i n g  of  an  reported  that  binding  aromatic  (Hahnel  the pentagonal  proteins.  Hahnel for  unsaturated of  the  a  et  C-3  ring  A,  estrogenic  D or a l k y l nucleus  groups  by  the  ring D  decrease  (1973) demonoccurs  as  in  with  an  steroid  is  estradiol-17g to  the  decreased  addition  binding  being about  10 A  upon removal  of  other  on r i n g A , and by f u r t h e r  (Korenman, 1969;  et  g c o n f i g u r a t i o n and a pheno-  Binding a f f i n i t y hydroxyl,  al.  steroid  two hydrogen bonding c e n t r e s  1973).  the  t i o n of the s t e r o i d  receptor  ( r i n g D) i n the  The attachment  methylation  et a l . ,  (1977)  for  binding a f f i n i t y  on C-17  (Hahnel et a l . ,  functions  essential  receptor  s i t e may depend on these apart  p h e n o l i c hydroxyl at C-3 on the  cannot be m o d i f i e d without an important  highest  hydroxyl  hydroxyl  to  steroid  the  a free  oxygen  unsatura-  Shutt and Cox, 1972;  Hahnel  1973).  The s t r u c t u r a l and e s t r o g e n i c  s i m i l a r i t y among e s t r o g e n i c  steroids  is  isoflavones,  e v i d e n t by comparing F i g u r e s  important aromatic r i n g A with a p h e n o l i c hydroxyl  1,  coumestans  2 and 3.  group at  C-3  The  (C-7 i n  - 8 -  isoflavone the  and  exception  vones  and  coumestan of  the  numbering)  isoflavone prunetin).  coumestans  have  an  methoxyl group) at o p p o s i t e  largely  site  ends  to  the  of  the  molecules  reviewed  the  related  compounds and  ring  affinity  more unsaturated The  that  has  compound  Martin  hydroxyl et  that  the  estrogenic  of  furan  to  (Korenman, 1969; Results  parallel  the  (C) was  an  et a l . closely  important  O v e r a l l , the  from  competitive Cox,  1972;  (Martin  relationship  terms  the  of  of  order  However, r e l a t i v e b i n d i n g a f f i n i t y non-steroid  estrogens  ( B i c k o f f et a l . , 1962). metabolic  uterotrophic  Shemesh et a l . , 1972;  receptors  Ryan, 1979).  and  oppo-  of  binding than a  of an e s t r o g e n i c compound to u t e r i n e  estradiol  determining  ring  sites  nucleus.  et a l . , 1978).  v a r i a t i o n and  genis-  at  Bickoff  coumestrol  activity.  human tumor c e l l  the  groups p o s i t i o n e d  or  because of a d d i t i o n a l s u b s t i t u e n t groups and  out with u t e r i n e c y t o s o l (Shutt and  for  hydroxy1  coumestans would be expected to be l e s s  been r e p o r t e d  in  a  isofla-  Coumestrol and  a l . , 1972).  activity  r e l a t i v e binding a f f i n i t y  receptors  (either  (with  e s t r o g e n i c i t y of some coumestans; opening  of i s o f l a v o n e s and estrogens  a d d i t i o n , these  function  (Shemesh  reported  g r e a t l y decreased  t h a t of s t e r o i d  two  estrogenic  f a c t o r c o n t r i b u t i n g to the this  In  a l l structures  compete f o r u t e r i n e c y t o s o l b i n d i n g  presence of  (1969)  oxygen  to  ends of the molecule.  t e i n are a b l e to s i g n i f i c a n t l y due  i s common  values  than  do  properties  Ruh  binding  Verdeal  et a l . , assays  estrogenic  support  potency  Lin  vivo  uterine  weight  Presumably, the d i f f e r e n c e s are due e f f e c t s , but  (Table  i n d i c a t e a much higher  to  1973;  carried  et a l . , 1980)  et a l . , 1978)  of  and this 1).  potency assays species  i t i s not yet c l e a r which method of  e s t r o g e n i c potency i s most a p p l i c a b l e to humans ( V e r d e a l  and  -  F i g u r e 2.  Structural  F O R M O N O N E T I N  9 -  formulas f o r e s t r o g e n i c  isoflavones.  PRUNETIN  - 10 -  Figure 3 .  Structural  formulas f o r e s t r o g e n i c  coumestans.  COUMESTROL  4'- M E T H O X Y C O U M E S T R O L  REPENSOL  - 11 -  Table 1.  R e l a t i v e b i n d i n g a f f i n i t y o f some n a t u r a l l y gens f o r mammalian e s t r o g e n r e c e p t o r s .  occurring estro-  Receptors Estrogens estradiol-17B  Rat u t e r i n e cytosol 1  100  Sheep u t e r i n e cytosol 2  Human cancer c e l l l i n e MCF-7 3  100  100 10  coumestrol  4.9  5  genistein  1.3  0.9  2  daidzein  0.09  0.1  -  <0.01  formononetin biochanin A  0.07  Vrom V e r d e a l e t a l . (1980). 2  From S h u t t and Cox (1972).  3  From M a r t i n e t a l . (1978).  ^Not  determined.  -  <0.01 -  -  Very flavonoids genic  few  naturally  ubiquitous types  are  occurring  have been r e p o r t e d  activity,  aside  from  constituents often  of  those green  encountered  are f l a v o n o i d s  of  Leguminosae  (Harborne  (1975)  listed  crops  over  have been i s o l a t e d In  addition  plants, in  and  al.,  1975);  and  as  40  the  reported  estrogenic  of g e n i s t e i n ,  vone)  isolated  estrogenic isoflavone (Nairn et  isoflavone  to one s u b f a m i l y  this as  subfamily  in  mice  pratensein  than  Wong  glycosides  which  et  al.  for  (1955)  as  genistein, found  estrogenic  was e s t r o g e n i c  1973),  namely  A  as  (Flux  i n mice prune-  when i n j e c t e d i n  Of the  from  glycitein  naturally  reported  significant  activity  for  strated  that  alfalfa  estrogenic  4'-methoxycoumestrol. repensol  and  occurring  et  al.,  1964).  less  Another  an important component of  soybeans  (7,4'-dihydroxy-6-methoxyisofla-  are b i o g e n e t i c a l l y r e l a t e d  primarily  that  (5,7,3'-trihydroxy-41-methoxyisofla-  biochanin  which has been i s o l a t e d al.,  are  soybeans.  from c l o v e r s by Wong (1963) was shown to be s l i g h t l y  Coumestans  1969).  (Markham,  Bradbury and White (1954) demonstrated t h a t  vone) , has not been examined f o r e s t r o g e n i c  isolated  flavonoid  groups  activities  was e q u a l l y  (5,41-dihydroxy-7-methoxyisoflavone) The  are  plants.  the g l u c o s i d e  mice.  well  estro-  Flavonoids  plant  isoflavone  genistin,  tin  related  demonstrating  within  and c l o v e r ,  or  characteristic  particular  formononetin and b i o c h a n i n A , Cheng  itself.  as  mentioned.  daidzein,  as g e n i s t e i n  coumestans  generally restricted  isoflavones  from to  et  alfalfa  70  already  only  Isoflavones  the common forage  isoflavones,  i n the l i t e r a t u r e  1982). the  12 -  activity. to isoflavones  ladino  clover  coumestans,  activity  for  Livingston  and have been  (Bickoff  Bickoff  et  al.  (7,10,12-trihydroxycoumestan)  al.,  al.  (1960)  and  lesser  (1964)  demon-  coumestrol et  et  had  comparable  -  estrogenic  activity  trifoliol  coumestrol  in  are  other  coumestans  sativol,  no r e p o r t s  in  the  identified  medicagol,  coumestan.  literature in  roots  and l e a v e s  of  crops  into  to  of  livestock, and  phytoestrogens  has  the  very  and  the  coumestrol focussed  Research i n legumes and  soybeans  (Lookhart  Bengalgram  pulses  Samples  alfalfa  higher  of  quantities  (Knuckles et a l . , of  genistein,  namely  has  been  et al.,  et  al.,  1971),  however,  its  from estro-  date. real  importance  observed  isolated  of  estrogenic  leguminous effects  from these c r o p s , specific  of  research  compounds.  to be r e l a t i v e l y i n a c t i v e i n compari-  1976);  1976);  al.,  (Dziedzic  and  1976).  daidzein  protein food  Eldridge,  Dick,  1982),  products  were  coumestrol  than  phytoestrogens  common v e g e t a b l e s ,  leaf  and other  1979a;  and soybean of  of  lucernol,  isolated  i d e n t i f i c a t i o n and q u a n t i f i c a t i o n o f  et  activity  1979).  (Knuckles  (Knuckles  estrogenic  inactive.  7-hydroxy-11,12-dimethoxy-  has r e c e n t l y expanded to i n c l u d e :  grains  alfalfa  into  coumestan,  relatively  p r i m a r i l y on those  Other c l a s s e s o f f l a v o n o i d s appear son ( V e r d e a l and Ryan,  or  coumestrol  to  another  clover,  (Olah and Sherwood,  because  isoflavones  was  and  3'-methoxycoumestrol  A 7-monoglycoside  Presumably  several  while  citing  alfalfa  g e n i c a c t i v i t y has not been e v a l u a t e d  forage  mice,  (7,10-dihydroxy-12-methoxycoumestan)  There  alfalfa  to  13 -  most  concentrates  products  1982b; all  Murphy,  for  found to  their  use  i n t o processed  soy  protein products.  phytoestrogens  in  these  foods  is  not  glucosides  The s i g n i f i c a n c e yet  understood,  from  1982)  by  or  humans.  c o n t a i n markedly  non-leguminous  respective  from  prepared  food  Murphy (1982) r e p o r t e d a s i g n i f i c a n t and  legumes  from  plants  carryover soybeans  of the l e v e l s and  must  of  await  -  further  studies  on  14 -  how phytoestrogens  are  metabolized  i n man  (Verdeal  and Ryan, 1979).  B.  BIOSYNTHESIS AND ACCUMULATION  1.  Biosynthesis The common b i o s y n t h e t i c  all to  flavonoids form  rings  a  involves  chalcone  (A  and  B)  (Grisebach,  of  the  three  linkage  labelled can  11+  C have  include  (1972)  have  three from  of  separate r i n g in Figure  different  origins.  acetate u n i t s ,  a  conversion  and  other  less  p-coumaric  reviewed  the  of  closure  where r i n g B i s has  been  of  the  chalcone  1975).  derived  positioned  shown to  occur  at  carbon-1  (Grisebach  to  the  from  to  of  these  is the an  using ring B  shikimic  of  of  two A  Studies  acid.  aromatic  plants.  formation  (Figure  and D o e r r ,  Ring  precursors  common s y n t h e s i s  leads  the  phenylalanine  compounds from glucose v i a the s h i k i m i c a c i d pathway i n Ring  4,  w h i l e r i n g B and  effective  acid  skeleton  intermediates  As shown  (Hahlbrock and G r i s e b a c h ,  shown that  tyrosine  Ribereau-Gayon  of  arise  cinnamic a c i d  1959).  chalcone  condensation  activated  the b a s i c C 6 - C 3 - C 6  the condensation  formed by the carbon  pathway f o r  of  4).  An a r y l  1960)  at,  chalcone stage where r i n g B migrates from C-1 to C-2 f o r  or  a  flavone  migration after,  the  s y n t h e s i s of an  isoflavone. In the formation of an i s o f l a v o n e aglycone, (Hahlbrock the  the  sugar  moiety  and  Grisebach,  glycosidic  isoflavones  is  glycoside  transferred  1975).  Nairn et  via al.  from the a  glycosyl  (1974)  form,  for  transferase  established  i n soybeans were 7 - 0 - g l u c o s i d e s .  t i o n serves to convert the f l a v o n o i d i n t o a l e s s r e a c t i v e soluble  corresponding  s t o r a g e i n the c e l l  vacuole  that  Glycosyla-  and more water  (Markham, 1982).  Figure  Proposed b i o s y n t h e t i c pathway of i s o f l a v o n e s v i a a c h a l c o n e .  Adapted from Hahlbrock and G r i s e b a c h (1975).  -  Grisebach biosynthesis rather the  of  than  to  highest  (Wong,  and  Barz  of  1975).  methyl  intact  for  was  possible  1979a).  migration  converted to a  4'-methoxycoumestan.  influenced  by  of  stage  reviewed by B i c k o f f levels in  of  coumestrol  coumestrol  alfalfa  (1965)  alfalfa of  et  content  isoflavones, represent  isoflavonoid  skeleton  to  be  a  formononetin  precursor  of  then  well-  with  its  V-methoxycoumes-  proposed  would  the  a group  shown  whereas  extensively  and r e p o r t e d  growth, al.  as  was  that  that  be  methylation isoflavonoid  more  readily  infection  Hanson and coworkers content  the  the  step a s s o c i a t e d with  4'-methoxyisoflavone  of m i c r o b i a l  coumestrol  as  The authors  a  Effects  Coumestans  daidzein  biosynthesis;  2.  of  for  coumestrol,  aryl  to  demonstrated  that  a more e f f i c i e n t  (Dewick and M a r t i n , occur d u r i n g the  further  analogous  isoflavone  precursor  could  1963b)  coumarins.  level  The  incorporated  trol  was  the  oxidation  group  (1963a;  coumestrol that  16 -  variety  (1969)  alfalfa  that  and  on  the  variation  coumestrol  location.  and L i v i n g s t o n  and c l o v e r  depending  reviewed the  (1978)  hays matured, geographical  content  Other noted and  in was  studies increased  variations  location  where  was grown.  These v a r i a b l e s  affecting  coumestrol  accumulation were observed  be u l t i m a t e l y r e l a t e d  to d i s e a s e i n c i d e n c e i n the p l a n t s  1965).  99  Furthermore,  percent  of  was determined to be n o n - g e n e t i c . commonly coumestrol  associated content  with  the  i n coumestrol  Advancing m a t u r i t y i n p l a n t  increased  among v a r i e t i e s  variation  (Hanson et  disease  incidence;  may be due to v a r i e t a l  to  al.,  content  growth  is  differences  in  differences  in  -  r e s i s t a n c e to f o l i a r  pathogens,  a s s o c i a t e d with l e s s f o l i a r Bickoff organism  increase  extent  of  et  tissue  al.  averaged  contain  2,600  reported  in  the  an  absence  levels  pathogens.  of  genetic  common  flavone  there  Several and  also  leafspot  content  was a  shown  to  of  significant  other  with  coumestans  4'-methoxycoumestrol)  leafspot  as  accumulate  in  levels  healthy  was  parts  fungal  pathogens.  up to  that of  organism  plant,  maturing Stuthman  ppm but (1965),  the  leaflet and  Millington  medic et  species  al.  (1966) which  had no obvious  fungal  these workers  of a l f a l f a  cultivars  et  areas  L.  coumestrol  Loper  in  Medicago s a t i v a  i n the  by  al.  suggested  concentrations for  (1967)  resistance resulted  in  contents.  accumulate  pathogenic  coumestrol the  26  Francis  in  including  implicated  shown to  to i n f e c t i o n by s e v e r a l found  coumestrol  Selection  diseased  However,  in  lowered coumestrol  Coumestrol  coumestrol  than 3 ppm whereas  of a l f a l f a  was  cultivars.  common  that  to Hanson et a l .  variation  significantly  other  medicagol  coumestrol  In c o n t r a s t  among a l f a l f a  to  infection.  were  increase  of  workers  the  and  found that  ppm c o u m e s t r o l .  contained  the  1969).  of  on coumestan  found  less  species  to  effect  often  day growth p e r i o d which c o r r e l a t e d  plant  (1967)  examined s e v e r a l  that  et a l . ,  a r e a s are  plants.  Loper  even  the  The workers  7 , 4 ' - d i h y d r o x y f lavone  infected  (1965a)  medicaginis  3'-methoxycoumestrol,  as  could  studied  i n coumestrol over a H  (sativol,  alfalfa  and c e r t a i n g e o g r a p h i c a l  disease (Bickoff  (1967)  varieties.  visible  well  al.  Pseudopeziza  two a l f a l f a  the  et  17 -  was which  fungi not  in alfalfa  in direct  (Sherwood et a l . ,  translocated  indicated  that  response  1970).  from i n f e c t e d coumestrol  These areas  synthesis  -  could  be c a r r i e d  alfalfa  leaves  f o r the h y p o t h e s i s  coumestrol 1976;  (1979a)  of  compared Lookhart  sprouts  infecting  to et  the  most  distilled  water  unrinsed c o n t r o l s . the beans, thus  It  1971 ed  and 1973; wide  large  Saba et  varietal  variations  separate 1965b;  been  Sachse,  flavonoid isoflavones  1974)  provided  or  Lookhart  could a were  not  marked rinsed  during germination  suggested that  the  increase  (Knuckles  concentration  noted  further  accumulation.  seed  1982).  but  coumestrol  cause a marked  bean  sprouts  daily)  observed al.,  1974;  Biggs,  in  regions  had f o r  of  the  rinsing  et  with  et al.  those  identify  the  reduction  in  periodically  as  compared  served  estrogenic  by numerous  isoflavone  1974).  it  also  in  of  found i n d i s e a s e d  flavonoid  Murphy,  infected,  result  Injury  to  to  cleanse  infection.  accumulation, as  al.,  coumestrol  accumulation  geographical  infection.  levels  shown to  1979b;  differences in  been  authors  times  limiting  also  Saba et  ungerminated  al.,  was  of  and f l a v o n e  when soybean  (4  Pathogen-induced legumes has  has  noticeably  concentration  site  pathogen-induced  the  The  the  means d i d not  1971;  highest  organisms.  coumestrol  of  legumes  associated  soybean  with  by mechanical  (Wong and L a t c h ,  Germination  al.,  at  I n c r e a s e d coumestan  white c l o v e r  in  independently  simply  accumulation.  support  out  18 -  Again, although  workers  1975).  isoflavone patterns  (Beck, the it  contents  not  yet  of  and  between been  in  Sherwood, report-  forages,  cultivars  Francis  relationship had  (Olah and  E a r l i e r studies  between  1964;  isoflavones  grown  and in  Millington, disease  and  recognized  for  coumestans, may have played a r o l e .  -  Plants  have  been  shown  19 -  to  accumulate  antimicrobial  known as  phytoalexins,  i n response t o i n f e c t i o n by pathogenic  bacteria  (Hare,  Kuc,  phytoalexins Leguminosae flavonoid carpans cated  1966;  are  1972;  flavonoids,  family  1975).  produced  5,  the  isoflavonoids,  probably  opening  1975).  (Wong,  The o b s e r v a t i o n tissue  i n response  microbially studying  that  however,  is  in  coumestans  separating  the  et  al.  disease  and other  workers  demonstrated  oid  compounds,  infected to be  (1970)  stress  but  that  of  likely  is  in  most  on coumestrol do not  studied  the iso-  ptero-  As  indi-  group  accumulate  The  (Dewick and M a r t i n ,  of  the  reduced  presumably  processes  Many  by r e d u c t i v e  microbially  metabolic  are  and  of  1979b).  phytoalexins.  extensively  fungi  the  1974).  classes  plants  and i s o f l a v o n e s  infection  biosynthesis  those of the i n f e c t i n g organism Sherwood  represent  accumulation  isoflavonoid  leguminous  fungi  by p l a n t s  abundant  from the pterocarpans  to m i c r o b i a l  induced  produced  (Dewick and M a r t i n ,  isoflavans derived  Van E t t e n ,  The most  by  and 2 ' - h y d r o x y i s o f l a v a n s  in Figure  and  i n c l u d i n g those  (McClure,  phytoalexins  Pueppke  compounds,  leaf  to  the  difficulty  infected of  ring  in  related  the  of  in  plants,  plant  from  1979b). the  accumulation  effect  of  fungal  in a l f a l f a .  These  independently synthesize  flavon-  produce enzymes  or m e t a b o l i t e s  that  p l a n t t i s s u e and cause coumestrol and other f l a v o n o i d  enter  the  aglycones  released. Olah  and  Sherwood  (1971)  glycosides  from h e a l t h y a l f a l f a  of  infection,  fungal  concentrations.  It  flavonoid was  were plant  only tissue,  aglycones  suggested  able  that  to  isolate  flavonoid  but d u r i n g the development  were  detected  infection  could  in have  increasing impaired  F i g u r e 5.  Adapted  Possible  biogenetic  from Wong (1975).  r e l a t i o n s h i p s among  isoflavonoids.  1  - 21 -  glycosylation likely,  that  release  of  Sherwood  at  the  the  the  Additional  infection 1971).  rather  Moreover,  precede these  but  of  the two  events  glycosides  and  synthesis  Sherwood,  1973).  Fungitoxic Lyon  and  a  are  plant's  in  activity  had  The i n c r e a s e d  in  the  shown  implying levels  pathways  fungal  when  during  been  1970)3,  normal  during  may  (Olah and Sherwood,  related.  stimulated  resistance  provided  decrease  (Olah,  that  and  fungal  activity  was  aglycones  suggests  Olah of  disease  theory  d i d not  enzyme  caused  1975).  the same or i n c r e a s e d  Wood  phytoalexin  (1975)  demonstrated  with Pseudomonas by  inhibiting  Unlike  fungitoxic  activity.  coumestrol,  4'-methoxycoumestrol,  As c i t e d  probably  glycosidase  glycosides  pathogens.  3  the  study  more  of  infection  to  that  of  both  flavonoid (Olah  and  activity  bean l e a v e s i n f e c t e d like  the  closely  aglycones  In  activity,  or  enzyme  subsequent  glycosidase  increase of  a  by fungal  the  remained  were  glycoside  3.  for  appearance  In  biosynthesis  glycosidase  growth (McClure,  flavonoid  the  a  3-glucosidase  released  support  flavonoid  via  infection.  fungal  concentrations  organism  that  aglycones  of  glycosides.  during  turn i n h i b i t further  the  from  found  increased  mechanism  stages  infecting  aglycones  (1973)  origin,  final  phytoalexins, Screening  by Olah and Sherwood  that  coumestrol  spp.  and that  the  growth  coumestans tests medicagol  (1971).  do  have and  accumulated  the coumestrol of  these  not  appear  been  for  acted  bacterial to  performed  trifoliol  in  have with  mycelial  - 22 -  growth  inhibition  (Bickoff  et  1969;  precursor via  a  ity.  1973;  contrast,  daidzein,  McClure,  and  al.  higher f u n g i s t a t i c  activity,  activity.  findings  fungally  infected  resistance  plants  may  that  of  than do coumestans.  pathways  involved  diseased  plants,  in  a  significantly  role  easily  through  to  isoflavones play  understanding  the  compounds  both  mechanism  of  in  the  in of  in  pathogen metabolic  healthy  and  phytoalexin  light studies  found between l i g h t  flavonoid  activ-  1974).  fungitoxic  biosynthesis,  1975).  tests.  (Nairn et a l . ,  greater  (Wong,  the  a  more  i n d u c t i o n can be e x p l a i n e d  In  fungistatic  pass  before  of  isoflavones  would  needed  Effects  the  showed  1971;  1979a).  lipophilic  be  4.  reflect  anti-fungal  more  A better  isoflavonoid  will  of  the  these  have  The  the 7 - 0 - g l u c o s i d e s had only l i m i t e d  suggest  accumulation  1969). simply  that  pronounced  isoflavones  the c y t o p l a s m i c membranes of the fungus the  results  (Wong and L a t c h ,  demonstrated  (methylated>hydroxylated>glycosylated)  Thus  then  production  pathway  exert  whereas  negative  Dewick and M a r t i n ,  (1974)  methylated  may  the  1975;  glycitein  corresponding  These  for  with  Cruickshank,  coumestans  formation  Nairn et  genistein  The  of  fungi,  and  common b i o s y n t h e t i c  Olah and Sherwood, In  pathogenic  Perrin  accumulation  or  phytoalexins  numerous  al.,  fungal-induced by-product  of  by Hanson  et  al.  (1965)  no  correlation  could  i n t e n s i t y and coumestrol content of a l f a l f a  The m a j o r i t y accumulation  of  reported  in plants  research has  on  pertained  the to  effect  of  be  in field light  anthocyanins,  and  on is  - 23 -  reviewed  by McClure  according tions  to  the  plant  estrogenic  and  Beck  isoflavone  formononetin,  (1967)  sity  light,  of  However, or  light  and can  strated in  the  have  quite  visible  that  for  the  different  was  was  not  generaliza-  essential  for  concentrations  attributed light  vary  formation.  detected  that  to  in  leaves  to  of  darkness.  high  inten-  environment  did  in clover. either  requirements  of  The d i f f e r e n t effects  below 290  nm,  no  Substantial  germination,  light  and  those grown i n complete  1975).  range 400-700  light  concluded  influence  on  study  in flavonoid  clover.  levels  by i n h i b i t i o n with l i g h t  Poljakoff-Mayber,  C.  can  were r e p o r t e d  b i o c h a n i n A were  authors  Poljakoff-Mayber, also  under  synthesis  isoflavone  light  in  and a l s o  the  i n h i b i t i o n dependng  (Mayer  and  isoflavone  however,  not markedly a f f e c t  found  formation  p l a n t s grown i n d a y l i g h t  effects  the r o l e of l i g h t  genistein  Some s t i m u l a t i o n  The observed  and anthocyanins  were made r e g a r d i n g Rossiter  of  (1975).  via  stimulation  particular  spectral  on g e r m i n a t i o n , nm and  especially  with  seeds  ranges as  demon-  promotion with red  light  of  light  (Mayer  and  1975).  METABOLISM Although a b i o s y n t h e t i c  isoflavones, appear  to  the  metabolic  be d i f f e r e n t  relationship  pathways  (Bickoff  et  of al.,  e x i s t s between coumestans and  these  1969).  ences among animal s p e c i e s i n t h e i r metabolism which can p r o f o u n d l y a f f e c t vones  (Lindner,  1967;  Shutt,  two  of  There are  of e s t r o g e n i c  the e s t r o g e n i c potency 1976).  groups  flavonoids  also  differ-  isoflavones  of p a r t i c u l a r  isofla-  - 24 -  As  mentioned  of p l a n t test  species.  1954;  (Verdeal potent  et  Bickoff al.,  Both JLn v i v o et  al.,  1980)  phytoestrogen,  studies  to  evaluate  out using  uterine  weight  and i n v i t r o  indicated  that  in  by g e n i s t e i n ,  of d e c r e a s i n g  r a t s and mice as  the  assays  and  cytosol  rats  estrogenicity  (Bradbury  binding  coumestrol  daidzein,  was  studies the  most  b i o c h a n i n A and  potency.  Isoflavones Both g e n i s t e i n  ean  clover  1946).  of  Since  activity factor  and formononetin were  (Bradbury  infertility  and  "clover  in rats  of  et  Braden,  did it  al.  not  was  not w i t h i t s  metabolic  isolated  which  grazing  (Bennetts  significant  et  in al.,  estrogenic  was  the  major  ( B i g g e r s and Curnow, 1954).  strains  for  subterran-  implicated  genistein  correlated  the  with the amount  content.  of  estrogenic formononetin  The f i n d i n g t h a t  estrogen than g e n i s t e i n , pathway  the  been  sheep  that  however,  genistein  from  had  demonstrate  thought  (1964), clover  o n e t i n was a more potent different  in  clover disease  subterranean  i n the c l o v e r ,  1954)  disease"  or m i c e ,  responsible for  activity  White,  formononetin  Millington  a  1962)  followed  formononetin i n order  1.  early  compounds were p r i m a r i l y c a r r i e d  animal  White,  previously,  formononetin  formon-  was shown to be due in  sheep  (Shutt  and  1968).  A comparison isoflavone  of  circulating  composition  of  clover  isoflavones ingested,  in  sheep  indicated  that  plasma  to  the  genistein  and  b i o c h a n i n A were degraded more r a p i d l y than formononetin and d a i d z e i n sheep  to  (Shutt  suggested  et  that  al.  1967;  Shutt  formononetin  and  and  Braden,  biochanin  1968). A  were  The r e s u l t s demethylated  in  also to  Figure 6.  Metabolism of some estrogenic isoflavones i n sheep.  1  +  ^rom  Shutt (1976).  PHENOLIC  ACID  - 26 -  daidzein  and  genistein  respectively,  (1967) and Braden et a l .  6,  Lindner,  occurs  primarily  1967).  position  to  yield  in  to  ring  1967;  Shutt,  1976).  Equol,  in contrast, of  response  i n sheep  al.,  1965;  et  al.,  1971), guinea p i g s  and Common, 1968)  i n the rumen (Shutt has  tive" period  appears and  degradation evidenced  of by  reduced  to  al.,  (from  ring  B)  and a l s o  and Braden,  Braden et  al.,  (Shutt and Braden,  and  a  the  isoflavan, which  (Braden et  al.,  1968)  i n sheep  five  days  plasma  activ-  significant  Equol  has  been  (Batterham  cattle  (Braden  and domestic fowl  et et  (Tang  c o n v e r t e d to equol i n  to undergo f u r t h e r  that  as  (Shutt,  the metabolism  A  1976).  In  these i s o f l a v o n e s  (Braden  biochanin  a  sheep  1971),  is  acid  elicits  of  C-4'  metabolism  1970).  to be s h o r t e r  high  at  1965;  phenolic  1968).  formononetin i s  does not appear  et a l . ,  excreted  al.,  genistein  i n a c t i v e metabolites  (Shutt  1967;  Equol  about  Lindner  indicated in  the  to i n a c t i v e phenols becomes more e f f i c i e n t  p e r i o d of  conjugated  et  i n the u r i n e and plasma  been observed  and g e n i s t e i n  turn  i n mice,  i n d i c a t i n g that  these s p e c i e s .  by  has been r e p o r t e d to have an e s t r o g e n i c  genistein  as a m e t a b o l i t e Shutt  (Batterham  d e m e t h y l a t i o n to y i e l d  A ) , both e s t r o g e n i c a l l y  estrogenic  It  in  p-ethylphenol  (from  to t h a t  rumen  as  is  which  degraded  all  the  i n sheep,  daidzein  further  detected  isoflavones  O-demethylation  B i o c h a n i n A undergoes  equal  supported  Formononetin undergoes  equol.  ity  finding  (1967).  The metabolism of e s t r o g e n i c Figure  a  and  et  al.,  genistein  concentrations  is of  In not  both  biochanin A  over an  cows  are  1971).  of  this  rapidly the as  "adap-  adaptive degraded,  guinea efficient  isoflavones  pig, as after  - 27 -  consumption. genic  As a r e s u l t  activity  i n the  guinea  biochanin A to g e n i s t e i n 1968). as  These  workers  a metabolite  been r e p o r t e d  Lindner  further  also  for  reported  flora  that  i n ovine t i s s u e s  Griffiths  isoflavones and  Further  experiments  ingested  flavonoids  of  from  with  (Tang and Common,  of  however, t h i s  equol  were  found  c o n v e r s i o n has  (Cox and Braden,  isoflavones  not  1974).  et  al.,  1970),  i n j e c t i o n of  i n v i t r o incubation of  microflora  genistein  germ-free  (Griffiths,  (Shutt  following parenteral  of  and  rats  do not undergo r i n g  intestinal microflora  Demethylation o f  fowl  traces  estro-  some b i o c h a n i n A and formononetin c o u l d  intestinal  equol  ruminants.  that  (1975) showed t h a t  with  have g r e a t e r  i n the rumen are b e l i e v e d to be p r i m a r i l y  O-demethylation  (1967) demonstrated  p-ethylphenol  in  i n domestic  i n other animal s p e c i e s  isoflavones.  2.  than  of biochanin A i n fowl,  be demethylated  genic  pig  occurs  Although b a c t e r i a l responsible  b i o c h a n i n A and g e n i s t e i n  the  rat  daidzein  gave  estrorise  to  respectively.  demonstrated  fission  these  that  orally  i n the absence o f normal  1975).  Coumestans Metabolism  studied  i n sheep  and domestic genically  of  coumestrol  (Braden  fowl  coumestans,  appear to be demethylated  repensol greater  to  al.,  give  respectively estrogenic  some  1967;  (Cayen and Common,  inactive  isoflavones  et  and  related  Shutt  et  1965).  coumestans  al.,  1969;  has  Kelly,  Two methylated and  4'-methoxycoumestrol  and  been 1972) estro-  trifoliol,  i n sheep i n a manner s i m i l a r to the methylated  rise (Shutt  activity  to et of  estrogenically al.,  1969;  coumestrol  active  Saba  et  compared  coumestrol al.,  to  the  1974).  and The  isoflavones  - 28 -  (Bickoff  et a l . ,  1962)  coumestrol  (Shutt  Breakdown  products  equol  (Cayen  metabolites sheep  to  or  Kelly  reported  do  and L i n d s a y  and L i n d s a y ,  not  previously  which i s  for  Kelly  metabolism  1965)  1976).  1972;  develop an i n s e n s i t i v i t y to  appear  to  identified  (1978)  1978). include  isoflavone  demonstrated  coumestrol  after  that  prolonged  not s i m i l a r to the mechanism or  biochanin  and  A  genistein  metabolism  1976).  Absorption The r e l a t i v e  and  possibly  mice  also  estrogencity cows  ( V e r d e a l and Ryan,  coumestrol,  is  the rumen (Shutt gut  et  al.,  are  (Lindner,  1967;  serves  inactivate  their 1970;  1967).  conjugated, Shutt  formononetin and g e n i s t e i n  opposite  to  The most  potent  remain active  in  the a  forms  very "free"  situation  metabolites  phytoestrogen  The major  al.,  appear  Cox and Braden,  probably  et  in  1967;  to  sheep  rats is  and still  biochanin  1974)  p o r t i o n of  the  Shutt  liver, et  or the  small  portion  of  unconjugated  ( L i n d n e r , 1967,  Shutt  et  part  estrogenic  glucuronides  1970).  Conjugation  Harper,  then  excreted,  1975).  circulating  form  represent 1969).  from  form  the  al.,  upper  absorbed  to  al.,  be absorbed  the c i r c u l a t i n g compounds which are  or  in  f o r m o n o n e t i n , g e n i s t e i n and  u s u a l l y v i a the u r i n e (Cox and Braden, 1974; Only  the  in  potency.  and  (Lindner,  compounds  to  then  1979).  i n order of d e c r e a s i n g  the  of  f o l l o w e d by d a i d z e i n ,  Phytoestrogens  of  Kelly,  coumestrol  Common,  period  (Shutt,  A,  of  1969;  to t h i s p h y t o e s t r o g e n ,  adaptive  3.  and  al.,  (Shutt,  appear  exposure  et  may be due to slower metabolism and e x c r e t i o n of  estrogens the  Factors  which  biologically which  affect  - 29 -  the  liver's  ability  malnutrition levels  or  of free  cant  liver  toxins,  the  (Linder,  effects  i s maintained at  Shutt,  1976).  Very  show  lating  levels  Without  this  of  high l e v e l s  it  exposure  estrogens, when  such  increases  in  as  plasma  their  for  estrogen  they can e x e r t  signifi-  concentration  ( L i n d n e r , 1967;  known of  phytoestrogens  knowledge  term d i e t a r y  is  i n marked  low b i n d i n g a f f i n i t i e s  on animals  little  isoflavonoids,  1967).  i n comparison to endogenous  estrogenic  absorbed  may r e s u l t  phytoestrogens  plasma  long  detoxify  estrogens  Although receptors  to  in  blood  Shutt and Cox,  1972;  the metabolism  in  humans  is  difficult  to  to  phytoestrogens  and normal  (Martin  assess  et  the  al.,  circu1978).  significance  i n man ( V e r d e a l  of  and Ryan,  1979).  D-  EXTRACTION OF PHYTOESTROGENS Bickoff  et a l .  Andrews,  1956;  initially  established  (1962) and o t h e r s  Lyman  et  al.,  establish  Wada,  the e s t r o g e n i c i t y  e n t i r e p l a n t or crude p l a n t to  1959;  (Cheng et a l . , 1963;  1955;  Saba  of c e r t a i n p l a n t s  e x t r a c t s to t e s t a n i m a l s .  they o c c u r r e d i n p l a n t s ,  et  al.,  and 1974)  by feeding  the  However, i n order  which compounds c o n t r i b u t e d to the t o t a l  i t y and at what l e v e l s  Pieterse  estrogenic  more s p e c i f i c  activ-  extraction  and d e t e c t i o n methods had to be d e v e l o p e d . Early extractions coumestrol clover.  isolation such as  methods  described  in alfalfa,  95% ethanol and f i l t e r e d  to  involved  by L i v i n g s t o n et  and Wong  F r e s h or r e h y d r a t e d  commonly employed to  usually  (1962)  for  plant material give  a  crude  al.  series (1961)  detecting  of  for  solvent detecting  phytoestrogens  was macerated  extract.  remove waxes and l i p i d  a  and soaked  Petroleum ether  material  and  the  in in was  remaining  - 30 -  mixture was evaporated and then further extracted with ethyl ether to isolate the compounds of interest.  Modifications of this system were  made primarily through the choice of solvents for extraction and purification (Bickoff et a l . , 1969; Wong and Latch, 1971; Saba et a l . , 1974; Sharma, 1979). Beck (1964) modified  the previous  procedure after finding that  bound isoflavones in the form of glycosides were not released by alcohol extraction  of intact  plant  material.  glycosides  in plants  in general  Flavonoids  commonly occur as  (Markham, 1982), and as noted, in  legumes (Beck, 1964; Olah and Sherwood, 1971; Nairn et a l . , 1974), so only the limited proportion of aglycone flavonoids would be extracted. Hydrolysis leaves  prior  of the glycosides  to ethanolic  was effected  by crushing  extraction, indicating that  enzyme was present in the leaves (Beck, 1964).  clover  a hydrolytic  The enzyme was inacti-  vated by placing intact leaves in boiling water or ethanol, and was not found in clover petioles (Figure 7). strated  B-glucosidase  activity  Olah and Sherwood (1973) demon-  in healthy  alfalfa  leaves  and roots,  which they noted could be involved along with other glucosidases in the hydrolysis of flavonoids. Francis leaves  and Millington  minutes before  crushed  treatments  prior to ethanolic extraction and obtained  yields i f leaves  two-fold  (1965b) compared  were crushed and allowed  adding  increase  ethanol.  Glencross  in formononetin  in water before  content  of clover  maximum isoflavone  to stand  a maximum of 10  et a l . (1972) reported a when clover  extraction with ethanol,  leaves  were  and concluded that  endogenous enzymic hydrolysis of the isoflavone glycoside had occurred.  -  31 -  Figure 7. Germination and development o f a l f a l f a seed t o s p r o u t .  COTYLEDON EPICOTYL PETIOLE  COTYLEDONS  0  YPOCOTYL ESTA  HYPOCOTYL  SEED  RADICLE  -  Sherwood  et  al.  (1970)  did  coumestrol when a l f a l f a Studies not  of  the  isolated  genistin,  by  not,  leaves  daidzein,  ethanolic  also  did  author  not  liberate genistein  extraction  proposed  protein  that  isolates  (Eldridge,  aglycones.  from  their  soy  in  water  (1982)  Ohta  glucosides,  flour  but  did  et  al.  daidzin  and  not  product a f f e c t e d  although  in a  attempted  the  of  isofla-  soybean meal paper  glycosides  losses to  determine  subsequent  isoflavone  soluble  of  have g e n e r a l l y  by E l d r i d g e (1982a)  decrease  Murphy  profiles  flavonoid and  levels  extraction. products  soy f l o u r  matter,  the  arose  1982b).  isoflavonoid  this  increased  i n soybean  of  A similar analysis  address  detect  were ground before  whether p r o d u c t i o n o f the i n i t i a l vonoid p r o f i l e .  however,  phytoestrogens  employed h y d r o l y s i s to  (1979)  32 -  during  explain  the  in  soy  production  the  different  of soybean p r o d u c t s i n terms of p r o c e s s i n g  effects  such as water s o l v a t i o n and o x i d a t i o n . In c o n t r a s t , a  clover  express  leaf  would  protein concentrate  juice,  Subsequent  at  (1982)  extraction  which  analysis  therefore  Thompson  G l e n c r o s s et a l .  of  not  treatments, depends  of  pulping  h y d r o l y s i s would  that  additional  from  intact  the  hydrolysis.  clover  to  leaves  to  have o c c u r r e d .  isoflavone  some h y d r o l y s i s may have  flavonoid  glycosides  employing  reviewed  primarily  first  aglycones  Nicollier occurred  account  for  and  during  the  high  detected.  conditions as  enzymic  require  isoflavones  Hydrolysis  involved  the p r o t e i n c o n c e n t r a t e for  reported  l e v e l s of a g l y c o n e s  controlled  of  point  (1972) noted that the p r e p a r a t i o n of  by  either  Markham  on the nature  of  can  be  acidic,  (1982).  carried  enzymic  The c h o i c e  the g l y c o s i d e  to  or of  out  under  alkaline treatment  be h y d r o l y z e d .  As  - 33 -  mentioned of  previously,  coumestrol  sides.  isolated  According  completely tions,  in  to  15  namely  (Harborne,  the to  40  of  heating  the  Nicollier  and  glycosides hours at  in  at  estrogenic Thompson intact  clover  recent  research  literature  with  2N  found that  isoflavones  as  and  7-0-monoglyco-  would be h y d r o l y z e d  acid  hydrolysis  HC1:95%  EtOH  condi-  (1:1,  h y d r o l y s i s of c l o v e r  v/v) samples  1 hour was adequate to a c h i e v e and  obtained  coumestrol  full  leaves  using  reports  were  1979;  the e x t r a c t i o n  isoflavones  has  i n feeds  Lookhart,  as  hydrolysis  conditions  of  total  aglycones.  of  isoflavone  4N HC1 f o r  and  foods  Murphy,  Lookhart,  1979;  2  1981;  Lookhart reported  a  (1979)  maximum  system.  1981;  of  The  64  when soybeans  water  ethyl  extraction  ether  efficiencies  phytoestrogens  other  procedure  used  was  have thus  were  with  than instead  the  by  prior  Lookhart  Murphy  al.,  methods  to  obtain  (Knuckles et a l . ,  a  content  1976;  to  a  series  of  procedure  when  (1981)  to  mechanical extract  of  methanol/  reported  hour  and  spiked  efficiency  (1981)  A 2  only  coumestrol  extraction  Murphy  coumestrol.  Several  been developed  from  enhanced  extractions.  Knuckles et  coumestrol  recovery  defatted  1974;  of  1982b).  soybeans f o r  found  much of  determinations  1982a).  p l a n t product  percent  author  coumestrol and  Eldridge,  Eldridge,  examined  al.,  identification  in plants,  quantitative  (Nairn et  of phytoestrogens  Murphy,  concerned with  and coumestans  emphasized  maximum r e c o v e r y from a p a r t i c u l a r  soybean  identified  standard  isoflavones  (1982)  estrogenic  70°C.  phytoestrogens  for  100°C  the  such a l i n k a g e  under  bath f o r  and a c t i v i t y of e s t r o g e n i c  1976;  been  have  Sachse (1974)  While e a r l i e r  the  date  minutes  w i t h 1N HC1 i n a hot water levels  of  Markham (1982)  to  1965).  glycosides  very  applied  low to  agitation  defatted  soy  -  flakes,  and  products (25:5,  later  (Murphy,  v/v)  tives.  for 1982).  genistein  flakes.  defatted  A solvent  soybeans  system  of  and  other  soybean  a c e t o n i t r i l e r O . 1 N HC1  was r e p o r t e d to y i e l d maximum r e c o v e r y and minimum  Recoveries  ery),  dried,  34 -  were measured  (86%), d a i d z e i n  Eldridge  genistein,  (1982b)  glycitein  and  for  standards  (57%)  genistin  and coumestrol  reported their  of  (91%  (55%)  maximum  extraction  corresponding  glucosides  coextracrecov-  added to of  soy  daidzein,  from  soybean  products by r e f l u x i n g with 80 percent methanol for 4 h o u r s . Knuckles determination the  et of  coumestrol  al.,  1976).  (1975)  Briefly,  alcohol  percent,  phyll  soybeans  dried  samples  chlorophyll  extract  to  however,  removal s t a g e ,  t i o n procedure. alfalfa  in alfalfa  of  i s o l a t e the c o u m e s t r o l . 98  developed a procedure  coumestrol content  p er cen t e t h a n o l ; the  al.  and  also  soybean  the  quantitative  used  to  products  were r e h y d r a t e d ,  pH  10,  and  ethyl  ether  evaluate  (Knuckles  then soaked  was removed with c h l o r o f o r m a f t e r was  used  et  i n 95  adjusting to  finally  Recovery of added coumestrol was r e p o r t e d to be  the  standard  the  chloro-  and thus was not c a r r i e d through the e n t i r e  extrac-  Quantitative  was  added  recoveries  just  prior  for estrogenic  to  isoflavones  from  have not been r e p o r t e d .  Sachse (1974) demonstrated vones  which was  for  on the  estrogens. hydrolysis, Markham although efficient  development Isoflavones  of in  even under m i l d  (1982). this than  several  suitable general  solvent  was  petroleum  extraction were  conditions,  Formononetin was  as  found  reported ether  restrictions  for  found  (Bickoff the  be et  isofla-  techniques  for  phyto-  susceptible  to  alkali  supported to  imposed by  by Wong (1975)  soluble al.,  removal  of  and  in chloroform,  1969) waxes  to  be more  and  lipid  -  material renders  from p l a n t  The low p o l a r i t y  these compounds more s o l u b l e  chloroform, (Markham,  E.  extracts.  35 -  in solvents  and only moderately s o l u b l e  of  isoflavone  such as  aglycones  ethyl  ether and  i n e t h a n o l , methanol and  acetone  1982).  DETECTION TECHNIQUES The  based  on  simplest  observations  consumption Andrews, nature  of  1956;  or  of  specific Wada,  Since isoflavones  the  methods  feeds  of  those  (Bickoff  have  been  flavonoid  in  al.,  However,  little  compounds  involved  initial  i d e n t i f i c a t i o n of  et  al.,  1957),  for  the  a  1951;  literature  animals 1946;  were  following  Pieterse  information  and  about  the  in estrogenicity  was  up u n t i l  the l a s t  (HPLC)  analysis  emerged  analysis  (eg.  Wulf  and  of of  et  al.,  components  1955)  and  chromatographic the  estrogenic  years  Nagel,  coumes-  flavonoids. research  when high-performance  a suitable  quantitative  1976;  as  techniques  predominated phytoestrogen  several as  some e s t r o g e n i c  Cheng  variety  and t h i n - l a y e r chromatography  chromatography  et  the  studies.  developed  literature  in  stimulation  (Bennetts  (Bradbury and White,  tans  reported  estrogenic  1963).  quantities  r e v e a l e d i n such  Paper  detection  liquid  technique  Vande C a s t e e l e  et  for al.,  1982).  1.  used  Paper chromatography The  technique  of  for  flavonoid  analysis,  separations, (Markham,  even  1975).  of  paper  large  chromatography largely amounts  (PC)  because of  it  is  still  extensively  produces  flavonoids,  at  acceptable a  low  cost  -  Paper legume  plants  compound found  chromatography of  the  when exposed  to  UV l i g h t  as  little  p h i c system  for  that  developed  acetic  adapted tion  reported  of  al.,  1959).  could  be  coumestrol  et  the  al.  (1961)  studies  1967; method  detection  reported  the  relative  to  al., spots  spots 1961). after  benzidine.  detected  in this  (1962)  under UV  chromatogra-  (in fresh  and d r i e d  The chromatogram v/v).  and p l a n t  Comparisons  extract  for  resolution of  measuring  coumestrol  (Stuthman  et  al.,  was of  spots on deve-  concentrations.  other  characteristic  paper  coumestans  and  fluorescence  Livingston  fluorometric  1966;  1970).  two-dimensional  of  the  the  made use  Sherwood et a l . , to  with  content Bickoff  of  intensities  in of  alfalfa al.,  (1967)  chromatography flavones  system  PC method of  et  Loper et a l .  1967; further  for  separa-  alfalfa,  these  in  and  compounds  coumestrol.  D a i d z e i n and formononetin were a l s o d e t e c t e d escent  of  Wong  improved a paper  (50:35:15,  standard  coumestrol  fluorescence  2 ppm c o u m e s t r o l .  good  Numerous workers  al.,  and  analysis to  blue  were used to determine coumestrol  and  disease  Loper et  further  detect  (1967) compared t e n d e v e l o p i n g systems f o r the PC s e p a r a t i o n  mentioned. Livingston  coumestrol  (1961)  between  to  (Lyman et  acid/water/HCl  readings  coumestrol  foliar  al.  used  characteristic  ug  sensitive  loped chromatograms Chury  0.1  quantitative  was  in  fluorescence  of  as  L i v i n g s t o n et  alfalfa)  easily  because  that  light.  was  36 -  under  UV l i g h t  (long  Genistein  and  spraying  developed  wave,  biochanin  365  nm)  A could  chromatograms  be  as  blue v i o l e t  using  PC (Guggolz  visualized with  fluor-  as  et  brown  bis-diazotized  - 37 -  Wong method  (1962)  for  the  developed  separation  The b e n z e n e / a c e t i c dimension front.  was  reported  noting  to  partial of  pounds  of  were  for tein  interest  of  isoflavonoids well  the  other spray reagents spots  Color  Subsequent 30% or 60% a c e t i c  are  were  dark of  from  the  flavonoids  et  the  acid  was  al.  extracts  separation  These workers solvent  chose  because  the  for  the  separated  (1975)  aside  did  be most  isoflavones  of a  to  with  genisseveral study,  concentrations  maxima u s i n g a UV s p e c t r o p h o t o m e t e r .  second  for  dimension s o l v e n t  hydrochloric  system  (Wong, 1962).  identification  reported  that  the  from  50% a c e t i c acid  was  using rather  A mixture of  based on UV s p e c t r a  removal  chromatography was  coumestrol  simple  the  suitable  In the same and  not  The com-  chromatogram;  upon comparison with a u t h e n t i c  p r i o r to  first  solvent  author  corresponding  chromatogram  the pigment from enhancing the f l u o r e s c e n c e with  the  system.  final  the  clover.  s t u d i e s by Wong and Latch (1971) were c a r r i e d out  and chromatographic p r o p e r t i e s Knuckles  for  near  the  summarized by Wong (1962).  eluted  in  ammonia, but  alkaline  spots  than the a l k a l i n e system p r e v i o u s l y r e p o r t e d  alfalfa  A,  on the  reactions  were o b t a i n e d from the absorbance  from  used  2N aqueous  biochanin  separated  non-fluorescent  and b i o c h a n i n A .  twelve  system  and l i p i d s  in this  chromatographic  phytoestrogens  v/v)  was  paper  d i a z o t i z e d s u l p h a n i l i c a c i d was found t o  visualizing  isoflavone  of  move c h l o r o p h y l l  degradation  instability  reagent  (125:72:3,  dimension system  detect  spray  two-dimensional  and measurement  acid/water  The second  from  a  of  necessary  samples. chlorophyll to  prevent  of coumestrol or i n t e r f e r i n g other  acid  fluorescing  system  found  to  as  the  result  compounds. developing in  fragile  - 38 -  chromatograms measurements  2.  with  high  background  were c a r r i e d out as  fluorescence.  per L i v i n g s t o n et  Fluorescence  al.  (1961).  T h i n - l a y e r chromatography The  use  flavonoids 1960's  of  has  the  thin-layer  chromatography  been reviewed  development  of  by Markham (1975).  thin-layer  methods  s m a l l s c a l e chromatography began to r e p l a c e separation  of e s t r o g e n i c  (89:11, tory  isoflavones  v/v)  as  Genistein  biochanin  and with  fumes.  However,  Beck  al.,  1964;  i n soybeans  1969),  and i n plasma Dissatisfied plates,  solvent  the mid  paper chromatography f o r  the  made  mixtures  clover.  al.,  as  acid  1974),  exposing  not  workers  by  ammonia separate  used in  1965b; Shehata generally  nm).  spots  to  isoflavones  i n legumes  could  (257  adequately  numerous of  the  satisfac-  orange-brown  and  could  analysis  this clover  et  al.,  (Harborne,  1967).  isoflavonoid (1971)  to g i v e a  under UV l i g h t  visible  system  al.,  to r e s o l v e  Chloroform/methanol  system  spots  Nevertheless,  (Braden et  Sachse  of  and  F r a n c i s and M i l l i n g t o n ,  (Nairn et  with  rapid  in  Formononetin and d a i d z e i n  fluorescent  semi-quantitative  1982),  cellulose  from  sulphanilic  genistein.  for  ( M i l l i n g t o n et  this  analysis  analytical  and s o l v e n t  plates.  A were  diazotized  from  system  gel  blue-white  spraying  coumestrol  isolated  on s i l i c a  be v i s u a l i z e d  the  Beginning  for  was found to be the only s o l v e n t  separation  for  isoflavonoids.  Beck (1964) t e s t e d 60 s o l v e n t s estrogenic  (TLC)  separations  developed  a  system  ether/benzene/methylethylketone/methanol/acetic  on  silica  employing  acid  gel  or  petroleum  (30:30:20:20:2,  -  v/v)  with  polyamide  separation biochanin  of  as  quantitative  was  reported  to  Lookhart analysis  plates  he r e p o r t e d  genistein,  Increasing  the  d i d not improve the s e p a r a t i o n .  spectrophotometry require  et  al.  of  which  (88:12,  3  days  extracts  better  formononetin,  polarity  of  the  The same system  was  (Sachse,  v/v).  (1978)  in  developed  Fluorescence  1974),  and was  the  compounds  spot was not Many flavone  workers  by  1975;  to  range  of  rapid,  spotting  procedure  the  quantitative  The system  1  minute  of  in  extract  ppm.  with  the  used  extract  method  for  silica  gel  chloroform/acetone spots  standards to  50-100  interfering  of  the  (under  estimate  coumestrol  The p o s s i b i l i t y barely  UV 365  of  developed  other  coumestrol  discussed. reported  purification  separated  time  a  only  those of  However,  measurement.  intensities  concentration fluorescent  the  soybeans.  for  compared to  in  from  reported  coumestrol  were  analysis.  quantitative  nm) were v i s u a l l y  and  using  TLC p r i m a r i l y  identification  column chromatography Sharma,  identify  1979).  isoflavones  are w e l l d e s c r i b e d  3.  system  daidzein,  standards.  onto the p l a t e to the f i n a l  Biggs,  this  to i n c l u d e development i n a second dimension and f i n a l l y e l u t i o n  for  used  as  a l s o employed to s e p a r a t e c l o v e r  adapted  TLC  Using  well  pratensein  developing solvent later  TLC p l a t e s .  coumestrol  A and  39 -  by Sachse  after  (Nairn et  Several  a  method  extracts al.,  chemical  and coumestans  as  and  1974;  were  for  iso-  initially  Sachse,  1974;  physical  treatments  on the developed  TLC p l a t e s  (1971).  Column chromatography/UV spectrophotometry Column chromatography has a l s o been used as a s e p a r a t i o n  followed  by UV s p e c t r o p h o t o m e t r y  studies  for  identification  technique and  often  - 40 -  quantitation soybean  (Glencross  extracts  fractions magnetic  were  gel  identified  soybean  r e p o r t e d l y most as  al.,  by s i l i c a  resonance.  separate  et  isoflavone  Ohta et  et  al.  isoflavone for  aglycones  al.  (1979)  fractionated  column chromatography and the  by UV s p e c t r a ,  Nairn  useful  1972).  infra  (1974)  used  glycosides.  the s e p a r a t i o n (Markham,  red a  spectra  1982)  less  but  and  polyamide  Silica of  isoflavone  gel  polar  many  proton  column  to  columns  are  flavonoids  such  applications  using  other systems are reviewed by Markham (1975). In  UV spectrum  mined  for  many  isoflavones  band  at  (Markham,  310-330 1982).  absorption  (band  (band II) Certain  be added  istic and  shifts the  of  been  the  for a  as  ranges IV),  of  by  high  generally nm (band  band  exhibit I),  reagents  such  as  sodium  and  low  II  at  high  and  these  interpretations  of  the  resulting  spectra  "shift  are  Bickoff 1975;  and/or  et a l . ,  coumestrol  1969;  Ohta et a l . ,  Glencross  1979).  in et  a al.,  number 1972;  of  studies  Nairn et  1969).  al.,  characterreagents"  summarized  Markham (1982) and have been a p p l i e d to the i d e n t i f i c a t i o n of isoflavones  III)  aluminum c h l o r i d e  In a b s o r p t i o n  of  nm  300-320 nm  et a l . ,  which cause  The use  intensity  i n t e n s i t y UV  to the f l a v o n o i d - m e t h a n o l s o l u t i o n s spectra.  of  (1961).  245-275  i n the  (Bickoff  methoxide  spectra  Jurd  a  deter-  230-250 nm (band  absorption  evidenced by a s h o u l d e r peak  usually  Reference  include  intensity  low i n t e n s i t y  is  Horowitz  isoflavones  340-355  and  spectrum  flavonoid.  compiled  maxima  nm and  flavonoid  The coumestans  i n the  and 200-215  can  solution  have  UV a b s o r p t i o n  range  the  a methanol  Typical I  studies,  by  estrogenic  (Wong,  1962;  1974;  Biggs,  - 41 -  4.  High-performance l i q u i d High-performance  increased rapid  attention  quantitative  tion  and  Yost  well  et  the  liquid  i n the  al.,  field  (Markham,  described 1980).  literature  chromatography of  technique which  sensitivity  HPLC are  chromatography  in  dealing  with  can p r o v i d e a  high  (Snyder  Kingston  level  of  and  it  of  theories  extensively  HPLC  to  is  a  resolu-  and K i r k l a n d ,  (1979)  applications  receiving  because  The technique  literature  In a d d i t i o n ,  is  flavonoid analysis  1982).  the  (HPLC)  of  1979;  reviewed  natural  plant  products. The v a s t m a j o r i t y out  using  Markham, used  reversed-phase  1982;  are  (RP)  as  'reversed-phase  hydrocarbon groups  c o n j u n c t i o n with  water,  to e f f e c t  (Yost et a l . ,  of  a  more  workers  large  et a l . , variety  C-18  was c o n s i d e r e d  to be due to  the  well  stationary  acceptor form  as  a  is  as  systems.  its  phase.  and 1982) of  types,  which  silica  the s t a t i o n a r y phase,  internal  Nagel,  1976;  for  consist  of  packing.  The  phase and are example  its  Daigle  have s t u d i e d phenolic  used  methanol-  and C o n k e r t o n ,  the mechanisms of  acids  The e l u t i o n  and  sequence  of  separ-  flavonoids  using  a g i v e n compound  hydrogen bond donating and/or  accepting  c o n t r i b u t i o n to the hydrophobic i n t e r a c t i o n with In  isoflavones  the c a r b o n y l group at  strong  1979;  The columns most commonly  C-18'  mobile  (Kingston,  of n o n - p o l a r to moderately p o l a r compounds  (Wulf  reversed-phase  ability  polar  a separation  Vande C a s t e e l e  ation  1983).  carried  1980).  Numerous 1982;  a  as  have been  systems  c h e m i c a l l y bound to  n o n - p o l a r o c t a d e c y l s i l y l groups a c t in  flavonoids  chromatography  Andersen and P e d e r s e n ,  known  octadecyl  of HPLC s t u d i e s of  hydrogen  C-3;  the  strongest  however,  bond with  this  hydrogen  an OH group at C-3  carbonyl  C-5  bond will  group  so  - M  that  the c a r b o n y l can no l o n g e r  (Vande of  Casteele  aglycone  were  found  et  al.,  interact  1982).  be  longer  without  the 5-OH group  further  generalized  whereas  m e t h y l a t i o n of  Glycosylation  of  than  Additional  (such  those  as  of  the  genistein  the  that  additional  OH groups  hydroxyl  prevents  introduces  i n decreased  tp  groups  the  and et  (Lookhart et Dick,  al.,  1982;  hexane  al.  to  system  achieved,  et  The authors  of  tp  values  these  hydrophilic  groups.  moiety  and  values.  isoflavones, Ohta  1982a;  Dolphin resolve  consisted  mixture.  systems  1978;  Eldridge,  and  column  solvent  isoflavones  workers u s i n g RP C-18 columns  et  al.,  Nicollier  the p r e v i o u s  their glycosides 1979;  Murphy,  findings  and coumes-  1981;  Dziedzic  and Thompson, 1982;  Patroni  1982).  Carlson silica  al.,  (tp)  b i o c h a n i n A)  reduce  effect  a more  HPLC s t u d i e s by d i f f e r e n t  r e l a t i o n to the e s t r o g e n i c  trol  and  corresponding  and methanol-water mobile phases g e n e r a l l y support in  r e t e n t i o n times  (namely d a i d z e i n and f o r m o n o n e t i n ) .  an OH group  would a l s o r e s u l t  s t r o n g l y with the mobile phase  As a r e s u l t ,  5-hydroxyisoflavones to  -  A  however,  compare  (1980)  of  good  (1982) a d v i s e d  phase  extract.  The  a methylene d i c h l o r i d e / e t h a n o l / a c e t i c  acid/  isoflavones  separation  many of  favorably  normal  some  reported  to  the  of  isoflavone  this  the in  use a  of  soybean  daidzein  and  separations  normal phase system.  a  genistein reported Vande  RP  Casteele  c o u l d become  adsorbed.  Although only methanol-water mixtures mentioned  for  a g a i n s t the use of normal phase columns when d e a l -  ing with p l a n t e x t r a c t s because other more p o l a r f l a v o n o i d s irreversibly  was  RP work with  isoflavonoids,  were employed i n the  various modifiers  have been  aforeadded  - 43 -  to  the  mobile  phase  concerning  other  were added  to  the  from  place  of the e l u t i n g  et  reported  phenolic  hydroxy groups  levels  1978;  of 0.019  to  nm f i x e d  (UV)  been u s e f u l  (Murphy,  ion  sup-  mechanisms tailing  hydrogen  polar  movement  Proksch et was  bonding  al.,  of  (Murphy  1981).  essential  for  At clear  p h e n o l i c compounds.  in  HPLC also  nm f i x e d  known as  The a d d i t i o n of ammonium  ammonium a c e t a t e  and  (Wulf and N a g e l ,  severe peak  intramolecular  1980;  acids  ionization  retention  prevents  1980).  analysis  to  P a t r o n i et  flavonoids (FL).  phytoestrogens  wavelength  detectors  of  fluorescence  and coumestan  1981;  ionic  studies  formic  suppress  technique,  cause abnormal  related  employed  wavelength  al.,  can  M the  t i o n maxima i n the 254 254  et  in  A c e t i c or  and f l a v o n o i d s  avoid  and S t u t t e ,  the i s o f l a v o n e  characteristics  1 to 5% to  This  to  prevent  that  to 0.09  ultraviolet  previously,  used  (Yost  Hardin  detectors  primarily  of  column, and thus  s e p a r a t i o n s among c l o s e l y The  1982).  is  compounds  was  Stutte,  levels  al.,  on the  acetate  and  at  of the p h e n o l i c a c i d s  chromatography,  taking  separation  and p h e n o l i c a c i d s .  solvents  Vande C a s t e e l e  pression  improve  flavonoids  of the a c i d groups 1976;  to  regions.  isoflavonoid al.,  1982;  include  As mentioned have  an  absorp-  The a p p l i c a t i o n of detection  has  Vande C a s t e e l e et  thus al.,  1982). Other wavelengths et  al.,  1979;  Eldridge, maxima at at al.  280  1982a),  and  which  could  developed  a  in  and 280  Dolphin,  presumably  these wavelengths.  nm,  (1978)  Carlson  of 262  because  nm have a l s o 1980;  Dziedzic  specific  sensitive  affect  detection  UV d e t e c t i o n  and  isoflavones  However, the coumestans turn  been employed (Ohta Dick, have  1982; peak  absorb only weakly  limits.  method  Lookhart  specifically  et for  - 44 -  coumestrol  at  absorption  at  The  343  fluorescent using  compounds  in  maxima  et in  The  t h i s wavelength  exploited  Lookhart  nm.  (Markham,  properties  fluorescence  plant al.  isoflavones  extracts  (1978)  ethanol  at  Murphy (1982) used a f l u o r e s c e n c e 360 nm and a secondary  filter  daidzein  (Lookhart  and  for  exhibit  significant  et  nm  detector  and  coumestrol  identification al.,  fluorescence 411  not  1982).  detectors  reported 348  of  do  1978;  Murphy,  excitation  respectively,  of  for  and  were these 1982).  emission  coumestrol.  with an e x c i t a t i o n maximum at  (range 460-700 nm).  - 45 -  NATERIALS AND METHODS  A.  MATERIALS Daidzein  ceuticals,  (7,4'-dihydroxyisoflavone)  Inc.,  Plainview,  Kodak C o . , R o c h e s t e r , was  kindly  B. C .  to  R.  solutions  contain  obtained  Coumestrol was  from ICN Pharma-  obtained  from  Eastman  Formononetin ( 7 - h y d r o x y - 4 ' - m e t h o x y i s o f l a v o n e )  p r o v i d e d by D r .  Separate stock  prepared  NY.  NY.  was  30.0  3.  Bose,  U n i v e r s i t y of  B . C . , Vancouver,  f o r each of the above phytoestrogens  yg/mL i n  HPLC grade  glass-distilled  were  methanol  ( F i s h e r S c i e n t i f i c C o . , F a i r l a w n , NO). Both  cultivars  germination  of  studies  alfalfa  were  seeds,  purchased  Moapa and  from  Vernal,  Richardson  used  Seed  Co.  in  the  Ltd.,  Burnaby, B . C .  B.  PHYTOESTROGEN EXTRACTION A method  from  alfalfa  followed sprouts.  to  suitable  was  unless  before  use.  extract  washed,  used  dried  hydrolysis  glycosidases.  sand for of  in  the  fresh  using  The sample  selected  illustrates  extraction  phytoestrogens  the  basic  scheme  from  alfalfa  or f r o z e n a l f a l f a and  analytical  reagent  sprouts  pestle.  were ground  The ground  per F r a n c i s and M i l l i n g t o n  phytoestrogen was  were  and water was d i s t i l l e d and d e i o n i z e d  a mortar  10 min as the  8  of  formononetin and coumestrol  otherwise s p e c i f i e d ,  was allowed t o stand permit  extraction  Figure  daidzein,  Approximately 20 g of with  the  developed.  A l l solvents  grade,  for  glycosides  then r e f l u x e d  with  by  sample  (1965b)  the  to  alfalfa  20 mL 2N HC1 and  80  - 46 -  Figure  8.  Phytoestrogen e x t r a c t i o n  scheme f o r a l f a l f a  sprouts.  ALFALFA add sand g r i n d , stand  alfalfa  1  add 2N HC1 + MeOH reflux, cool  filter,  wash with MeOH  filtrate evaporate extract  with PE  discard PE crude a l f a l f a  extract extract  with EE  discard aqueous l a y e r ether  extract evaporate to @ 1 mL a d j u s t volume, f i l t e r  PHYTOESTROGEN EXTRACT  MeOH PE EE  = = =  methanol petroleum ether e t h y l ether  - 47 -  mL methanol f o r  30 min to  ing  period,  the a l f a l f a  50  (hardened)  filter  further  s l u r r y was  paper  Etobicoke,  mL  discarded.  and  portion  of  methanol  Ont.)  in  flash  conjunction  filter  The f i l t e r  was  evaporator  with  a  was  water  through Whatman No.  aid  "Hyflo Super-Cel"  cake was  washed  transferred  to  (2 X 5 mL w a t e r ) .  filtrate  A f t e r a 10 min c o o l -  filtered  The f i l t r a t e  i n the  25 min u s i n g a r o t a r y  suction  Ont.).  round bottom f l a s k with r i n s i n g a  hydrolysis.  with 4 g C e l i t e  (Oohns-Manville, methanol  the  w i t h 50  a 1000 mL  E v a p o r a t i o n to  carried  out  under vacuum  (Brinkmann Instruments, bath  at  30°C.  remove  This  for  Rexdale,  temperature  allowed r a p i d methanol removal yet l i m i t e d the amount of heat a p p l i e d the sample. 250  The remaining aqueous methanol e x t r a c t was t r a n s f e r r e d  mL s e p a r a t o r y  funnel,  again  adjusted  rinsing  mL).  Volume was  mark.  L i p i d s and c h l o r o p h y l l pigments  X 100 mL p o r t i o n s tion  of  of  petroleum  petroleum ether  allowed  to  pigments  to t r a n s f e r  The  stand  final  with water  for  the 10  min  to  to  flask  a previously  (b.p.  was  37-58°C).  shaken  permit  the  phases  30 s  and  and  the  settle  layer.  from  min  before  removal  e x t r a c t s were combined and evaporated approximately  1 mL.  volumetric  flask  The  extract  final  of  the  alfalfa  with HPLC grade methanol filtered  anhydrous  layer.  under vacuum at 3 2 ° C  The remaining r e s i d u e  was  ether  of  crude  Each ether/aqueous l a y e r mixture was shaken f o r 30 s and allowed 10  mL p o r t i o n s  the  ether.  for  X 50  to  addi-  was  stand  with 4  phytoestrogens  with 3  extract  to  accomplished  of  75 mL  A f t e r each for  to a ( 2 x 5  calibrated  vigorously  to the upper petroleum ether  extraction  with water  were removed by e x t r a c t i o n  ether  mixture  the  to  through  was t r a n s f e r r e d  The for  Swinny  filter  ether  15 min to  i n t o a 10 mL  r i n s e s and d i l u t e d a  ethyl  to  unit  volume. fitted  - 48 -  with  a 0.2  Samples  urn M i l l i p o r e  were  Millville, required  stored  NO) at  Type EG f i l t e r in  borosilicate  5°C until  approximately  4  (Millipore glass  analyzed.  h  per  Corp.,  vials  The e n t i r e  sample,  B e d f o r d , MA).  (Wheaton  extraction  however,  several  Sci.,  procedure extractions  c o u l d be run c o n c u r r e n t l y . All  sprouting  duplicate  treatments  extractions  treatment.  were  were  carried  performed  The number of  from each  extractions  was  s p r o u t s t h a t c o u l d be grown i n the j a r s Recovery tions  of  the  studies three  were  also  pg/mL stock  tion  procedure c a r r i e d out as  each  30.0  ug/mL  prior  to  grinding  stock and  sprouting by  jar  the  In  using  one  prepared  experiment  previously described.  this  study.  solution  was  completing  In another  added  the  c o n d i t i o n s as  and  within  quantity  a of  used.  2.0  stock  solu-  mL of  to  g  extraction  described  extrac-  experiment 2.0  20  of  alfalfa  procedure.  each  extrac-  Triplicate  were performed f o r each of the two a l f a l f a  ated under " s t a n d a r d "  C.  triplicate,  s o l u t i o n was added to the mortar and the e n t i r e  t i o n s were c a r r i e d out f o r  extractions  in  limited  performed  phytoestrogens.  30.0  out  mL of sprouts  Duplicate  c u l t i v a r s germin-  in Section E.  MOISTURE DETERMINATION Moisture  method  for  samples  of  content  plants alfalfa  (A0AC,  Sprout  1980)  sprouts  from every s p r o u t i n g j a r analyzed.  of a l f a l f a  of  s p r o u t s was determined u s i n g with  some  modifications.  approximately  f o r moisture a n a l y s i s  samples  were  chopped  10  g  each  and s t o r e d  lightly  while  the A0AC  Triplicate  had  been  taken  at - 3 0 ° C  until  still  frozen,  - 49 -  and  weighed  into  aluminum d i s h e s  pre-dried  (1  h),  desiccator  (57 mm diam X 17 mm depth)  Samples were d r i e d to c o n s t a n t vacuum oven  (National  inches Hg).  Sprouts  desiccator  were d r i e d f o r  containing  Samples were r e t u r n e d reweighed  to  check  silica  gel  5.5  constant  to  cool  weight.  MEASUREMENT OF PHYTOESTROGENS BY HPLC  401  equipped  data  Rheodyne Model out (4  system 7125  30°C.  with  a  (Varian  loop  at  moisture  h)  95-100°C 91.5  before  desiccator weights  valve  UV-50  variable  Instrument injector  kPa  mm I . D .  X 30 cm),  fitted  Palo  weighing. cooled,  D e t e c t i o n of  the  was  and  CA) and  a  was  (octadecylsilane)  0DS-10  made  Liquid  Separation  Column temperature  phytoestrogens  and  after  dectector  Alto,  yL l o o p ) .  with a B i o - S i l  Richmond, C A ) .  (27  into a  recorded  wavelength  Group, (10  in a  contents.  on a r e v e r s e - p h a s e V a r i a n MicroPak MCH-10  (Bio-Rad L a b o r a t o r i e s , at  weighed  HPLC a n a l y s e s were performed u s i n g a V a r i a n Model 5060  Chromatograph  column  (0.5  Final  O.  carried  g)  OR) under  1 h, a g a i n  h d r y i n g were used to c a l c u l a t e  Vista  h),  h and immediately p l a c e d  6.5  All  (0.5  Richmond, B . C . ) .  (±0.002  Portland,  to the oven f o r  for  (Canlab,  weight  Appliance C o . ,  cooled  at  guard  column  was maintained  254  nm  (optical  d e i o n i z e d and  filtered  bandwidth 8 nm). All through  water a  0.45  for  HPLC a n a l y s i s  ym M i l l i p o r e  was d i s t i l l e d ,  filter  (Millipore  Corp.,  Bedford,  Methanol was g l a s s d i s t i l l e d HPLC-grade ( F i s h e r S c i e n t i f i c C o . ) . a t i o n was a c h i e v e d by u s i n g a l i n e a r methanol-water flow  r a t e of  1 .0  mL/min.  1.0%  glacial  acetic  acid  Methanol and water (v/v)  and 0.01  gradient  reservoirs  Separ-  system at  each  M ammonium a c e t a t e  MA).  a  contained  (HPLC grade,  -  Fisher  Scientific  Co.).  53% to 58% r e s e r v o i r sample,  The g r a d i e n t  B (methanol)  programmed to  over 30 m i n .  Total  increase  analysis  from  time  per  integrator.  The  and peak areas were computed a u t o m a t i c a l l y by  retention  chromatographic parameters: the  was  i n c l u d i n g column e q u i l i b r a t i o n , was 60 m i n .  Peak r e t e n t i o n times the  50 -  separation  factor  times  the c a p a c i t y  (relative  The f o l l o w i n g e q u a t i o n s  were  (Yost et  to  factor,  retention),  were used  used  «;  al.,  calculate  k (also  and the  several  known as  k');  resolution,  Rs.  1980):  k =  Eqn. 1  Eqn. 2  R  =  S  where  t  t^  b1'  +  Eqn. 3 W  b2  = mobile phase holdup time k2  At W  b1  = r e t e n t i o n time of compound  K  ki,  — W  W  = capacity  factors  = distance  between two peak maxima  b2 ~ P e a k width at  and a l l measurements  are  of compounds 1 and 2,  base of compounds 1 and 2 r e s p e c t i v e l y ,  expressed  i n the same time u n i t s  F i g u r e 9 shows how these measurements The s e p a r a t i o n for  factors  daidzein/formononetin  (min or sec) .  were o b t a i n e d from a chromatogram.  were c a l c u l a t e d and  respectively  as  ratios  formononetin/coumestrol.  of  the k Ratios  values were  -  Figure 9.  Adapted  51  -  R e t e n t i o n time and peak width at base measurements . 1  from Yost et a l . (1980).  -  obtained  for  (Vernal) for  the  extract  tentative  and  alfalfa  mix,  standards  and a l f a l f a  only  (Treatment  of  coumestrol,  sentative  standards  peak  The r e s o l u t i o n  the  was  two c l o s e l y  calculated. of  (Vernal) extract  prepared  from  ug/mL. curve f o r  stock  of 2 0 . 0 ,  10.0,  methanol  each phytoestrogen  was  each standard  Monroe 1880 calculate  100.  of  content  of  were  used  2.0, for  the  and  values.  from  repre-  standards e x t r a c t ,  spiked  1.0, all  extract. coumestrol  were  ug/mL to  give  0.8,  0.6,  dilutions.  from the peak area Triplicate  counts  analysis  Regression  corresponding  compared  formononetin  obtained  the s t a n d a r d s .  regression  alfalfa  these «  c o n t a i n i n g 30.0  s o l u t i o n and area  Linear  of  formononetin and  4.0,  programmable c a l c u l a t o r .  the  extract,  (Treatment X)  was prepared  were made f o r nearest  only  spiked  phytoestrogens,  values  solutions 6.0,  10-uL i n j e c t i o n s  the  Rs  daidzein,  responses f o r  to  X)  the standards mix,  of  respective  HPLC-grade  eluting  and a l f a l f a  solutions  concentrations  extract,  i d e n t i f i c a t i o n based on agreement  chromatograms  Standard  52 -  was  (in  0.4  and 0.2  A  standard  integrator injections  uV-s)  recorded  performed  equations  phytoestrogen  using  were used in  the  a to  sample  extracts. Duplicate of was in  four used  injections  determinations in calculating  each j a r .  were made f o r  (two the  sample  (three  sample  calculate  phytoestrogen  preparations,  phytoestrogen  For the e x t r a c t s  minations  each sample  of  the s t a n d a r d s ,  preparations, recovery.  content  each  extract.  each of  The mean  injected  the  s p rou t s  the mean of s i x  injected  twice)  twice)  was  grown deter-  used  to  -  Preliminary retention sample  times  peak of  extracts.  grams  of  the  standards alfalfa  identification  phytoestrogen  To f u r t h e r  alfalfa  before  samples  were  1.  Sprouting  to  limit  before  bands  permitted  use.  were r e p l a c e d  (22°C)  wide mouth j a r s  microbial contamination,  was  rapid  in  stand air jars  rinsed  order  peaks  in  the  the chromato-  spiked  phytoestrogen  to  with  those  of  the  unspiked  used  as  sprouting  and t h o r o u g h l y r i n s e d  yet  (10  (v/v) with  solution distilled  cm X 10 cm) secured by rubber  retained  seeds  and  sprouts;  covers  seeds  into  the  each  were soaked  volume  of  for  jar,  300  4 h at  room  p e r i o d , water  water  temperature  was decanted  designated  The growth p e r i o d was measured  mL d i s t i l l e d  i n the  and  rinsing  from the time  the  soaking. thoroughly  1/2  at  circulation. in place:  were  a 1% c h l o r i n e b l e a c h  At the end of t h i s  with  to  from  inverted  apart.  unknown  of  identities,  mL v o l )  were weighed  and the  seeds began  constructed  drainage  15 g l o t s  schedule and d r a i n e d .  In  comparison  daily.  added,  were  alfalfa  (800  C h e e s e c l o t h covers  i n the l i g h t .  seeds  a  alone.  Oars were washed with  water  water  had been  on  preparation  glass  Seeds  and  compared  and the standards  GERMINATION OF ALFALFA SEEDS  containers.  based  standards  which  E.  Clear  was  confirm the peak  extraction  extracts  53 -  inch  (1.3  a 4 5 ° angle  drain cm)  the  sprouting  wire mesh t h a t  and p e r m i t t e d  Figure  10  illustrates  two rows  of  three  jars  the each  jars,  racks  allowed the  jars  adequate  illumination  draining  rack  placed  were to and  system  with  approximately  3 cm  -  F i g u r e 10.  54  -  Rack used f o r d r a i n i n g s p r o u t i n g  jars.  -  2.  Growth s t u d i e s Growth s t u d i e s  duration, alfalfa  tions  rinse  studies  were undertaken  frequency,  cultivar  In a l l  on the  rinse  content  the ambient  beneath  the  distilled.  The r i n s e  volume  time,  and  volume.  Daily  observations  Light  was  provided  by  15-watt  that  the  sprouts  (Lambda  Instrument  density  (PPFD) sensor was used  t i o n r e c e i v e d at the  available  to  Corp.)  the sprout  quantity plants  dark foil  darkness period, (Figure  used  of  light  well  fluctua-  on each were  for  water  rack  taken  rinsing  used  "washes" sprout  as  sprouts.  for  readings  stage of  cool-white  sprouting  for  the  were  equipped  jar  of  to  jars  at  3  was each  of  equal  development  a  to.  those  A Li-cor  level  racks  Light  level  11). 2  covers were 0 yEm" s"  1  .  of  photon  light  active  treatments  were  or  the E i n s t e i n  requiring  completely  measurements  covered  taken  a  flux  radia-  yEm~2s-1,  radiation  ( T i b b i t t s and K o z l o w s k i , 1979).  sprouting  10  photometer  The u n i t of measure,  photosynthetically  sprouting  posi-  shown i n F i g u r e s  photosynthetic  measure the  surface.  as  tubes  continuous hours of l i g h t per  exposed with  fluorescent  i n c o n t r o l l e d environments,  d e f i n e d as a mole of photons total  as  alfalfa  placed  successive  size,  L i g h t d u r a t i o n was the number of  indicates  and  of  s i g n s of c o n t a m i n a t i o n were r e c o r d e d .  was  h period  period,  was  quantity  two  color,  effects  in  A l l water  the  in  t i o n e d d i r e c t l y over each row of and 11.  openings,  was  applied of  growth  A thermometor  jar  the  was monitored d a i l y  8 h intervals.  rinsing  and any v i s i b l e  determine  phytoestrogens  temperature  sprout  times per 24 h p e r i o d at  to  volume and of  d u r i n g the growth p e r i o d .  directly  24  55 -  (E)  (PAR) being  To p r o v i d e 12  to  24 h  with aluminum  under  these  foil  -  (a)  Standard  The purpose that  could  alfalfa  conditions  of  serve  sprouts.  this  as  a  experiment standard  High q u a l i t y  tability  and  radicles;  3 to 5 cm l e n g t h .  used  to  content  of  chosen  incorporate  p r o d u c t i o n as  the  two  was  as  production  cotyledons;  alfalfa  known  to  conditions  jar  of  sprouts  s t u d i e s as Vernal Each  of  described  alfalfa  jar  each  was  in Section  sprouts  labelled  cultivar  were  used  in  Trial  The  purpose  environment  on  as  sprouts.  e.g.  of  the  phytoestrogen  Conditions commercial  were  alfalfa  of s p r o u t s .  Sprouts  al. of  was  extraction  B.  designated  for  In a d d i t i o n , for  treatment  this  experiment  phytoestrogen  i n Table 3.  "A".  The 24  conditions  recovery  four  preliminary code  and  replicate extraction  sample  jars  One  of  trials.  number and  was  racks.  conditions  summarized  t i c code,  and  jars.  c o n d i t i o n s were chosen to r e p r e s e n t ties  accep-  recommended by Hesterman et  randomly a s s i g n e d a p o s i t i o n on the s p r o u t i n g  (b)  quality  were grown i n four r e p l i c a t e  grown  with a  high  hypocotyls  The  measured.  be  conditions  of v i s u a l  white  cutivars.  subsequently  Moapa and V e r n a l ,  of  the standard  (1981) to improve q u a l i t y and f r e s h weight y i e l d each c u l t i v a r ,  growth  d e f i n e d here i n terms  green  those  well  establish  T a b l e 2 summarizes  from  cultivars  was to  for  is  bright  sprouts  both  to  sprout  yield:  grow  57 -  was  to  evaluate  content  of  sprouts.  Each treatment  and 24  h dark  effect  The  a wide range of treatment  W i t h i n each treatment  h light  the  treatments  "trial"  possibili-  was a s s i g n e d an  were t h r e e  of  replicate  were c a r r i e d  alphabejars  of  out on  -  Table 2 .  Standard  58 -  growth c o n d i t i o n s f o r a l f a l f a  sprouts.  Treatment Condition  AA  Cultivar Growth p e r i o d , h Light duration, h R i n s e volume, L Rinse frequency/24  h  BB  Moapa  Vernal  100  100  12  12  1  1  2  2  - 59 -  T a b l e 3.  Trial  growth c o n d i t i o n s  for a l f a l f a  sprouts.  Conditions  Treatment  Growth p e r i o d (h)  Light duration (h)  Rinse volume (L)  Rinse  Frequency /24 h  A B C D E  76 76 76 76 76 76  24 24 24 0 0 0  1 .0 0.4 1.0 1.0 0.4 1.0  2 5 5 2 5 5  U  148 148 148 148 148 148  24 24 24 0 0 0  1.0 0.4 1.0 1.0 0.4 1 .0  2 5 5 2 5 5  F  V  w  X Y Z  -  separate were  sprouting  randomly  assigned  Vernal a l f a l f a  (c) A  racks;  sprouting  a  position  simple  germination  trial  the  each  treatment  corresponding  class  racks.  Only  studies.  and  Seeds  allowed  (15 to  at  room  was  g)  were  up  soaked  germinate  temperature  set  to  measure  the  percent  seeds under c o n d i t i o n s used i n the germin-  Ungermfnated seeds were separated dried  on  within  Percent germination  studies.  drained  jars  was used throughout these  germination of Vernal a l f a l f a ation  60 -  for  for  4  h  with  24  h  in  300  mL water,  periodic  rinsing.  manually from v i a b l e , germinated  overnight  and  weighed.  seed,  The d i f f e r e n c e  in  weight between the o r i g i n a l q u a n t i t y of seeds and the ungerminated seeds was used to c a l c u l a t e  3.  Sprout  harvesting  Alfalfa schedule vigorously paper  sprouts  immediately shaken  to  were  given  before remove  the  being excess  two samples  (@ 20 g each)  phytoestrogen a n a l y s i s ,  analysis.  A l l samples  final  water  and  the  1 h (Figure  of  Each  the  treatment  jar  was  sprouts 12).  emptied  From each  then onto jar's  were weighed i n t o p o l y e t h y l e n e bags  and t h r e e samples  were f r o z e n at  rinse  harvested.  towels and allowed to d r a i n f o r  contents, for  percent germination.  (<§> 10 g each)  -30°C until  analyzed.  for  moisture  - 61 -  F i g u r e 12.  D r a i n i n g of a l f a l f a  sprouts  prior  to sampling and  freezi  -  F.  STATISTICAL ANALYSIS  1.  Single factor The  analysis  standard grow  (2)  alfalfa  of  62 -  variance  and t r i a l  to  (14)  and analyzed i n a one-way a n a l y s i s  computer. used  to  1978)  available  perform m u l t i p l e comparisons  The  data  of  collected  growth c o n d i t i o n s  factors  (growth  quency)  each  available  as  of  for  single  variance, use  conditions  factor using  treatments  the UBC MFAV  on the UBC Amdahl 470 V/8  among means  for  (Zar,  each  1974)  of the  for were  period,  with  2  treatment  each coded  light  levels  of  as  the  for  phytoestrogen  variables  21*  design  a  duration, listed  in  factorial  rinse  volume  Table  3.  combinations we're performed,  and  Not  since  rinse  all  of  only  combinations  package (Greig mine the  for  sprout were  the  significance  accumulation  comparisons  analyzed  an unbalanced  and B j e r r i n g ,  among  of  growth.  design  1978). of each  the  As a r e s u l t , using  (GENLIN)  These  phytoestrogen means  using the Student-Newman-Keuls t e s t .  of  adapted  for  (of  factor  alfalfa  fre-  the  16  insuffi12  variance the  were c a r r i e d  and/or in  for  analysis  analyses  factor(s)  phytoestrogen  an  data  4  the r i n s i n g com-  cient  alfalfa  under  having  to be  ment  three  variance  b i n a t i o n of a 0.4 L volume a p p l i e d t w i c e / 2 4 h was assumed for  was  variables.  Factorial analysis  trial  treated  combination  The Student-Newman-Keuls m u l t i p l e range t e s t  phytoestrogen  2.  (Le,  were  treatment  used  program package  s pr o u ts  (12)  treatprogram  UBC computer out  to  deter-  interaction(s) sprouts.  3 replicates)  were  on  Multiple performed  - 63 -  RESULTS AND DISCUSSION  A.  EXTRACTION  1.  E x t r a c t i o n methodology The  method  developed  s p r o u t s was adapted and  Francis  Several the  and M i l l i n g t o n  larger  (1965b)  were made to  sample s i z e  for  phytoestrogen  from the e x t r a c t i o n  modifications  extract  for  i n the  subsequent  from  alfalfa  reported  by Beck  (1964)  methods  for  the  analysis  these methods  present  analysis  extraction  by  study high  and  of  clover  in part to  to account  prepare  performance  forage.  the  liquid  for  sample  chromato-  graphy. The and  ground  Millington  alfalfa  (1965b)  present  as  glycosides.  further  hydrolysis  sample to  permit  from  recovery  of  alfalfa  and  improved  coumestans A  during  enzymatic  to  stand  based  extractability v/v)  obtained The a c i d  the  period on  as  hydrolysis  extraction  could  of  30  evidence  of  indicated be  acid  may a l s o process  aid  chosen  showed  poorer of  the  isoflavones  and  1975).  for  7-0-glycosidic  by  Chromato-  i n recovery  since  that  achieved  without  this  Francis  isoflavones  mixture.  min was that  per  to the r e f l u x  are very s t a b l e i n an a c i d medium (Wong,  refluxing  hydrolysis  extracts  phytoestrogens.  phytoestrogens  allowed  However, p r e l i m i n a r y experiments  adding 2N HC1 (20:80 2N HCl/MeOH, grams  was  the  methanol/acid  linkages  would  be  c o m p l e t e l y h y d r o l y z e d i n 15 to 40 min under s i m i l a r c o n d i t i o n s (Markham, 1982).  F u r t h e r improvements i n phytoestrogen  when r e f l u x i n g attempted.  conditions  of  45  min with  r e c o v e r y were not a c h i e v e d  50:50  2N HCl/MeOH  (v/v)  were  - 64 -  The a l f a l f a  filtrate  a large portion  of  the  chlorophyll  lipids  methanol  and  volume,  the  was evaporated  a  methanol  layers  miscible  methanol.  with  chlorophyll was  present.  to  the  to a  total  i n an improved p a r t i t i o n  removal  most  pigmented  sludge  the f i n a l  petroleum ether  Ethyl  layer  that  alfalfa  mixture  soluble  in  solvent.  rapid  and  this the  for  HPLC  2.  Recovery  remaining  standards  conditions). contributed obtained tracted  the  petroleum  was  little  phase  if  ether  all  the  approximately  the  the  crude  interface  was  and  ether  transfer  the two phases and an from  total  is of  methanol 70  to  75  efficient  extract.  A  discarded  with  extract. the e x t r a c t i o n  because  both  Evaporation residue  could  of p h y t o e s t r o g e n s  isoflavones of  ethyl  be  easily  and  ether  from the  coumestrol  are  under vacuum was  dissolved  i n methanol  studies  r e c o v e r y by t h i s a  of  of  analysis.  As p r e v i o u s l y  of  formed at  extraction  because petroleum  volume of of  ether  presence  the  ether  pigments  ether was used f o r  aqueous  of  there  petroleum  chlorophyll  the  be a c h i e v e d  mL r e s u l t e d of  In  Furthermore,  Evaporation  petroleum  separation  c o u l d not  pigments  to  pigments.  complete  water/methanol  prior  i n a subsequent step to remove  (by from  extraction mix  In by  added  order the  HPLC the  described,  out  method of a phytoestrogen to  to  analysis) count  alfalfa  account  alfalfa  area  s t u d i e s were c a r r i e d  for  sprouts, from  sprouts  quantity  the  mean  unspiked  responses  of  the  under of  area  alfalfa spiked  the  s t a n d a r d s mix and  (grown  the  to e v a l u a t e  "standard"  phytoestrogens count  extracts alfalfa  response was  sub-  extracts.  - 65 -  These adjusted  area  count  phytoestrogen c o n t e n t s . to  be present  The mean contents  (Table  minimal  added case  to  to  insoluble  for  as  a  the  value of 6.0  standards  result  of  recovered calculated  ug/ml_ to o b t a i n  mix i n d i c a t e  extraction  samples  were  from  lower;  alfalfa,  these  Moapa  and  Sachse  sludge  layer  (1974) that  formed  pigments with petroleum e t h e r .  of  the  1979;  were  involving standards  except  recoveries  losses  during  there  procedures  however,  (Lookhart,  reported  that  R e c o v e r i e s of  markedly  p r e v i o u s l y reported values  (1962)  calculate  phytoestrogens  e v a p o r a t i o n and p h a s i c d i s t r i b u t i o n .  coumestrol  favorably Wong  losses  alfalfa  of  of  to  4).  The r e c o v e r y data  refluxing,  were then used  were compared to an expected  % r e c o v e r y data  only  values  in  did  the  compare  Murphy,  1981).  formononetin i n extraction  the  of  clover  These l o s s e s were a t t r i b u t e d t o  formon-  o n e t i n ' s low s o l u b i l i t y i n the a q u e o u s / a l c o h o l l a y e r .  Low r e c o v e r i e s  coumestrol have been r e p o r t e d i n soybean e x t r a c t i o n as  a result  ing  to  lipid  coumestrol  fractions  in  common  (Lookhart, solvents  and  1979).  The  aqueous  c o n t r i b u t e d to e x t r a c t i o n l o s s e s i n the present Cultivar documented 1966;  (Beck,  Francis  reports  differences  in  efficiencies.  et the  1964; al.,  in  phytoestrogen  F r a n c i s and M i l l i n g t o n , 1967;  literature  Loper et of  al.,  cultivar  low  systems  of  of  bind-  solubilities  of  may  also  have  have  been  well  Stuthman et  al.,  study. contents 1965b;  1967);  however, t h e r e  differences  in  are no  extraction  Table 4.  Recovery of phytoestrogens  ug/mL  1.  2.  3.  Standards mix  using developed e x t r a c t i o n method.  Daidzein % Recovery  6.110.1 1  Formononetin ug/mL % Recovery  Coumestrol ug/mL % Recovery  102  5.6±0.2  94  6.2±0.2  103  Standards + Moapa a l f a l f a  4.7 ± 0 . 2  78  3.8+0.3  63  2.5+0.5  42  Standards + Vernal a l f a l f a  5.0±0.2  83  4.7+0.2  78  3.4±0.1  57  Mean values  and standard  d e v i a t i o n of 6 d e t e r m i n a t i o n s .  - 67 -  B.  HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY  1.  HPLC methodology Numerous HPLC systems  d e t e c t i o n of selected al.,  Murphy,  1981;  adequately  resolve  the  phase  Varian  sprout  peaks o f  systems  were  60:40,  (1982)  studies  MCH-10  standards  interest  for  in  separation  and  o f these systems (Ohta et d i d not appear  quantitative  in this  column,  study,  attempts  1.0  out with i s o c r a t i c  mL/min) showed that  f o l l o w e d the expected  based p r i m a r i l y on r i n g  in  the  order  Daidzein  eluted  presence  of  of  purposes.  using to  a  to A  reversed-  resolve  alfalfa  methanol/water  systems  followed  a methoxy group  Resolution however,  o f Vande C a s t e e l e et  of  the f l a v o n o i d s .  which  is  C-V  vs.  a  phytoestrogen  of  reversed-  polar  hydroxy group  e l u t i n g immediately a f t e r  the  (less  standards  extracts,  extracts  resolution  contained  a  number  volume and i n t e r f e r i n g  with  peaks c o u l d correspond  to p o l a r ,  acids.  Ohta  et  al.  of  the  the r e s o l u t i o n  (1979)  peaks  due to  for  the  daidzein)  alone  was  excellent,  was  eluting  to  separ-  poor.  with  The  the  void  the d a i d z e i n peak.  These  n o n - f l a v o n o i d compounds such as  pheno-  reported  of  (1978).  formononetin.  phytoestrogens  of  al.  Elution  by Murphy and S t u t t e  by formononetin  at  typical  when the same high (>55%) methanol systems were used  alfalfa  alfalfa  of  polarity  and a l s o demonstrated  first,  the e l u t i o n of the phyto-  order  substitution  decreasing  and f i n a l l y c o u m e s t r o l ,  lic  the  and Thompson, 1982)  examined  carried  MeOH/H 2 0;  phase chromatography  ate  Several  for  extracts.  estrogen  was  Nicollier  MicroPak  Initial (e.g.  been developed  phytoestrogens.  1979;  number of other  have  a  similar  observation  with  - 68 -  soybean e x t r a c t s .  E a r l y e l u t i n g peaks were c o n s i d e r e d to be h y d r o p h i l i c  lower m o l e c u l a r weight compounds but not Using improved  solvent  the  peaks  but  times  of  systems  resolution  resulted the  trol  described  of  MeOH/H 2 0  alfalfa  extract  continued  at  a  reasonable  flow  to  to  rate  Further  improve the  early  increased  coumestrol  eluting  retention  peaks.  Trial  from 20 to 50% methanol over 20 resolve  isoflavones  and coumes-  column.  Formon-  by 60 min because the e l u t i n g An i s o c r a t i c  of  separation  chromatogram.  i n order  and  formononetin and  system was too low.  (v/v)  a  more p o l a r  spreading  had not even e l u t e d  of the s o l v e n t  give  the  but without success on the present  strength  to  band  from  methanol g r a d i e n t  o n e t i n and coumestrol  found  severe  by E l d r i d g e (1982a)  were attempted,  52:48  a lower methanol c o n c e n t r a t i o n (<50%)  daidzein  eluting  runs u s i n g an aqueous min as  of  in  later  with  flavonoids.  1.0  of  mL/min  the  resolution  was  early  fine-tuning of  of  solvent  system  subsequently  portion the  the l a t t e r  of  the  program  was  half  of  the  the  alfalfa  out.  Steep  chromatogram. To  reduce  extract,  various  gradients greatly  the  as  fluctuations  gradient  used  reduced  total  by Murphy  drift.  not  interfere  with  of  (1981) the  were later  to  greater  resolution  between  to  peaks  quantitation.  found adequate  Increases  found  to  of  carried  unsuitable as  a  of  starting  because b a s e l i n e  A gradual reduce  increase  coumestrol.  Faster  they  baseline at  53%  effects to 58%  program e l u t i o n  methanol c o n c e n t r a t i o n s  formononetin and  because  result  program  to be more d e s i r a b l e  peak  elution  were  gradient  methanol over 30 min was 30 min.  for  programs  A linear  methanol was demonstrated did  required  solvent  resolution  and  time  time  decreased  the  gradients  had  -  a minimal e f f e c t  on r e t e n t i o n  a l s o caused more b a s e l i n e In acetic found  an  effort  acid that  was  to  times  of  formononetin  and coumestrol  to  peak  the  tailing  water  a 1% c o n c e n t r a t i o n  and  of  the  methanol  eluting  compounds,  reservoirs.  was  i n the  sol-  of  glacial  acetic  vent mixture  improved peak shape of  the  eluting  phytoestrogen  effectively  reduced  coumestrol.  Acid  Vande C a s t e e l e on f l a v o n o i d s Methods). phenolic  et  been used  al.,  The most  to  Proksch et  al.,  to  abnormal  column.  Murphy  compounds,  such  polar  and as  bonding so  Neither and  effective  as  the  described  which  workers  is is  acid  and 1976;  groups  (Materials  considered present  movement  of  (1978)  phenolic  that  their  polar  formononetin  However,  in  compounds reported  acids,  would no longer  order  (Murphy  and  ammonium a c e t a t e as  and  to be the  on a l l  three  was this  e l u t i n g peaks.  could  Stutte,  a buffering  on  the  that  1978; reagent  reversed-phase  ortho-hydroxylated  undergo  intramolecular  hydroxy groups would be bound and be based on t r u e or  coumestrol  not  affected  study  i n improving r e s o l u t i o n ,  d a i d z e i n from e a r l y  and N a g e l ,  i o n i z a t i o n of  previously  1982)  formononetin  (Wulf  isoflavonoids  some  elution  ammonium a c e t a t e . found  suppress  other  Stutte  daidzein,  their  of  peaks and  here.  have used  e l u t i o n and s e p a r a t i o n  lated,  to  (Markham,  acid,  1981)  especially  by other workers  group of  C-7  studied  addition  prevent  1982)  acid  h y d r o x y l at  In  times,  and p h e n o l i c a c i d s  phytoestrogens  hydrogen  retention  acid  It  (v/v)  has  and  fluctuations.  reduce  added  69 -  0.01  polarity.  are by  ortho-hydroxy-  the  presence  M ammonium a c e t a t e  particularly  R e t e n t i o n times  the  separation  of was of  were a l s o reduced i n  - 70 -  comparison to  to  the methanol/water  only s o l v e n t  system,  and  the r e t e n t i o n times o b t a i n e d with the 1% a c e t i c a c i d In  ium  combination,  a c e t a t e to both  provided three  f o r an  (reservoir  the  methanol  improved  phytoestrogen  under 30 min  the a d d i t i o n of 1.0%  peaks  are  over a 30  min  sharp,  water the  period.  The  pH  the  guidelines  Assoc.,  2.  Chromatographic  k  to  7.5  for Varian  retention  (capacity  times  (tp)  factor),  «  and  the  (separation  t o 58%  elute  methanol system  which i s w e l l  at  within  columns  (Varian  chromatographic  parame-  factor)  for  HPLC runs of standards and a l f a l f a  and  measurements  (Figure  the p r e v i o u s s e c t i o n obtained  tion  and  solvent  MicroPak  other  the p h y t o e s t r o g e n peaks were c a l c u l a t e d  from  9)  HPLC a n a l y s i s  (Treatment  X).  of the a l f a l f a  used  of  only extract  The  were g i v e n i n  Table 5 l i s t s  extract  and  an  the v a l u e s  in a  unspiked  X s p r o u t s were chosen because  (resolution)  s  e q u a t i o n s (1 to 3)  three phytoestrogens  spiked a l f a l f a Treatment  R  calculations  Methods).  the  and  from chromatograms o b t a i n e d  extracts.  f o r these  ( M a t e r i a l s and  standards e x t r a c t ,  extract  4.5  where a l l  parameters  for  mix,  baseline,  of t h i s  was  reservoirs  1978).  The ters  2.0  to  M ammon-  extract  g r a d i e n t program from 53%  c o m p o s i t i o n c o n d i t i o n s (53:47, B/A) of  solvent  alfalfa  resolved  initial pH  (A)  similar  system.  a c e t i c a c i d and 0.01  and  s e p a r a t i o n of  u s i n g the l i n e a r  B)  (B)  were  standard alfalfa  for representa-  a l l three phytoestrogens  were  detectable. The minations  retention made  for  times r e p r e s e n t means of the t o t a l each  sample,  as  noted  on  Table  number of d e t e r 5.  Coefficients  Table 5.  Retention times ( t p ) , capacity factors ( k ) , separation factors ( « ) and r e s o l u t i o n (R s ) of selected phytoestrogens on MicroPak MCH-10 using l i n e a r gradient e l u t i o n as described.  k  tR (min) Sample  D  F  C  D  F  C  D/F  C  Rs  F/C  F/C  Standards 1 mix  10.78 ±0.02k  24.10 ±0.06  27.15 ±0.08  3.31  8.64  9.86  2.61  1.14  1.99  Standards extract  11.33 ±0.18  25.62 ±0.44  28.53 ±0.46  3.78  9.82  11.06  2.60  1.13  1.89  Alfalfa extract3 spiked  11 .678 ±0.04  26.38 ±0.08  29.38 ±0.09  3.86  9.99  11.24  2.59  1.13  1.91  Alfalfa extract3 (Treatment X)  11.10 ±0.14  24.62 ±0.38  27.62 ±0.47  4.05  10.19  11.55  2.52  1.13  1.54  tp i s the mean of 5 determinations, tp i s the mean of 6 determinations, tp i s the mean of 12 determinations. Standard d e v i a t i o n .  -  of  variation  of  r e t e n t i o n times  which  is  within  the  2.0%)  (Oohnson and Stevenson, The k v a l u e s  istics  for  unretained  required  analysis  cult.  the  time k  for  too  values  two peaks  tentatively  ing  the  of  could further  closely  of  the  was  eluting  tive  chromatogram  used  for  to  from  k>1  from  is the  peaks should have k<10-15  widths  the  character-  separation  tend  and  to  increase  detection  corresponding are  with  more  mean  within  pairs  to  determine the  diffi-  retention  this  recom-  only  can serve  and K i r k l a n d ,  1979;  by r e t e n t i o n  on temperature,  as  a reliable  Yost  et  times  and s p i k e d  calculated  to  manual measurements  13-16) of  as  the  alfalfa  determine  from  peak  index  al.,  three  for  1980).  peak The «  the  each  widths.  of  phytoestrogens.  with the extract  the  correspond-  (Table  5)  one  peaks.  actual  formononetin and c o u m e s t r o l .  (Figures  column and  e x t r a c t s were d e r i v e d from peaks  these <* D/F and « F / C v a l u e s  peaks,  retention  d a i d z e i n / f o r m o n o n e t i n and formononetin/  are dependent  standards  relative  confirm the i d e n t i t y of the phytoestrogen  Resolution the  Also,  the unspiked a l f a l f a  identified  « values  1.756  (0.2  (1980) s t a t e d t h a t  ensure  resolution  were c a l c u l a t e d  (Snyder  Based on agreement  to  Peak  makes  al.  formononetin and coumestrol  in  for  interest front.  mobile phase c o m p o s i t i o n and so  listed  identification  i n d i c a t e the r e t e n t i o n  Yost et  of  calculated  These v a l u e s  identification  peak  to  5).  values  coumestrol.  values  to  long.  which  (Table  The « the  is  daidzein,  mended range  peak  the s o l v e n t  values  The k  times  first  for  of 0.2  1978).  were c a l c u l a t e d  peaks at  increasing  of  were found i n the range  o f the chosen HPLC system.  desirable  or  precision  72 -  separation A  sample  An R s value of  of  representagroups 1.0  was  means  - 73 -  F i g u r e 13.  HPLC chromatogram o f p h y t o e s t r o g e n s t a n d a r d s . Peaks: D=daidzein, F=formononetin, C=coumestrol.  D  F  ,  0  ,  5  1  10  1  15  T I M E (min)  1  20  1  25  1  30  - 74 -  F i g u r e 14-.  HPLC chromatogram of phytoestrogen standards Peaks: D=daidzein, F=formononetin, C=coumestrol.  extract.  - 75 -  Figure 15.  HPLC chromatogram of a l f a l f a e x t r a c t s p i k e d with p h y t o e s t r o gen s t a n d a r d s p r i o r to e x t r a c t i o n . Peaks: D=daidzein, F=formononetin, C=coumestrol.  I  1  1  0  5  10  1— 15 TIME  (min)  1  1  1  20  25  30  - 76 -  F i g u r e 16.  HPLC chromatogram o f a l f a l f a e x t r a c t (Treatment D = d a i d z e i n , F=formononetin, C=coumestrol.  10  1 15 TIME (min)  —r20  -T" 25  X).  Peaks:  ~1  30  -  that  resolution  ficient sents  for  area  calculations,  separation  (Table 5 ) ,  .achieved,  approximately 90% complete,  peak  baseline  sented  is  77 -  (Yost  baseline  although  the  et  al.,  Rs  value  i n comparison  tions  formononetin and coumestrol  as  ments,  thus a f f e c t i n g  3.  very broad  0.2  to  30.0  peak  in  the  of  methanol  were  also  the  other  In a l l  alfalfa  1.5  repre-  samples  only  samples.  repre-  extract  was  The low c o n c e n t r a -  found i n the a l f a l f a i n large  occur  peak  retention  phytoestrogen  were  integrator  made  The standard  each  methanol  and the peak area  gens.  of  only  peak width  extract measure-  analysis  analysis  at  value  suf-  resolution.  solutions pg/mL  Rs  1980).  peaks which r e s u l t e d  Linear regression Standard  to  for  lower  eluted  an  considered  r e s o l u t i o n of formononetin and coumestrol was  markedly of  while  which i s  to  measure  times  curves  even  derived  injected  from in  concentrations  triplicate  responses r e c o r d e d . any in  baseline the  from c o l l e c t e d  of  found to be w e l l d e s c r i b e d by the f o l l o w i n g r e g r e s s i o n  that  the  peak  HPLC  Injections  response  absence  for  of  of  might  phytoestro-  area  data  were  equations:  Daidzein:  Y = -0.029 + 3.809X  r 2 = 0.9998  s  Formononetin:  Y =  0.168  + 3.813X  r 2 = 0.9982  s  Coumestrol:  Y =  0.592 + 2.707X  r 2 = 0.9992  s  y.x y.x  = 0.524 = 1 .516 = 0.771  yx Y = area  counts  (pV-sec)/10  X - c o n c e n t r a t i o n of phytoestrogen  Some d i f f i c u l t i e s grator  responses  for  (pg/mL i n methanol)  were encountered i n o b t a i n i n g r e p r o d u c i b l e i n t e the  lowest  concentrations  of  formononetin  and  -  coumestrol standard for  0.2  yg/ml_  included  solutions.  For t h i s reason the i n t e g r a t o r  formononetin and 0.2  in regression  equation  C.  GROWTH STUDIES  1.  Moisture determination Moisture  trial  78 -  contents  conditions  of  were  Moapa and V e r n a l  alfalfa  to  sprouts  range  difference sprouts  and dark  moisture period  (A to  contents  of  presumably  were a l l  the  148  (1981) a t t r i b u t e d  alfalfa  with  water a b s o r p t i o n . difference  sprouts,  but  no  moisture  contents  corresponding treatment  of  sprouts  (e.g.  periods  difference  sprouts  D vs.  grown  not  in  the  standard  94.8%  (Table  A ) , however, not a l l  The  (P<0.05)  than the  The l o n g e r by the  growth sprouts.  i n f r e s h weight y i e l d (4  to  8 days)  (1979) a l s o  between  light  6).  c o n d i t i o n s , both  to  sprouts  dark for  grown  a  greater  given  differences  for  the  study,  than  growth  were  sig-  h alfalfa  In the present were  of  greater  reported a  between 72 h and 120  the  and  conditions.  water  dark c o n d i t i o n s .  grown in  to  Z).  of  the i n c r e a s e  growing  same l e n g t h of time i n l i g h t v s .  were  i n the moisture content of  uptake  i n moisture c o n t e n t s  significant  88.5  (U to  Hamilton and Vanderstoep  nificant  under  s i g n i f i c a n t l y less  in greater  longer  grown  grown under t r i a l  h sprouts  Hesterman et a l . sprouts  coumestrol  grown under standard  F),  results  from  (P>0.05)  moisture c o n t e n t s of the 76 h s p r o u t s light  yg/mL  calculations.  alfalfa  found  There was no s i g n i f i c a n t  and 0.4  responses  the  period  significant  (P<0.05). Nevertheless, in  moisture  contents  it  was that  because  of  calculations  significant of  differences  each a l f a l f a  sprout  (P<0.05) sample's  -  T a b l e 6.  79 -  M o i s t u r e content of a l f a l f a and t r i a l c o n d i t i o n s .  s p r o u t s grown under  standard  Moisture1 (%)2  Treatment STANDARD Moapa  91.5  ± 0.433  Vernal  91.4  ±  A  88.5  + 0.5b  B  91.3  + 0.3a  C  91.6  + 0.1a  D  90.5  + 0.4C  E  91.4  + 0.5a  F  92.5  + 0.2d  U  93.3  + 0.26  V  93.5  + 0.3ef  W  94.1  +  X  94.2  f 0.21 + 0.1f  Y  93.7  + 0.6ef  Z  94.8  + 0.19  0.2a  TRIAL  Expressed  on wet weight  Mean of 3 r e p l i c a t e s  basis.  ± standard  deviation.  Means s h a r i n g the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t (P>0.05) as determined by Duncan's m u l t i p l e range  test.  -  dry  weight  content,  2.  were made  rather  the  corresponding  treatment's  than u s i n g an o v e r a l l mean moisture  mean  moisture  content.  Growth c o n d i t i o n s (a)  Percent germination  The  two  Vernal,  alfalfa  B.C.).  purposes  for  fungicide  in  the  "sprouting" a l f a l f a sold  for  human consumption  must  be  treatment,  present  study,  for  sprouting  and  (Richardson Seed Co. L t d . ,  home and  commercial  "untreated",  i.e.  (Kulvinskas,  1978;  sprouting  have  and should have germinaton percentages  desirable  Moapa  of  had  at  no  least  Hesterman and  1980).  Moapa greater  Alfalfa  used  seeds  85% to be most Teuber,  cutivars  were purchased as  Burnaby,  alfalfa  was  f r e s h weight y i e l d  (Hesterman et a l . , ation  (of  which  could s t i l l  for  using  80 -  3  1981).  replicates)  demonstrated  to  have  i n comparison to f i v e In the present of  Vernal  be c o n s i d e r e d  study  alfalfa  as  high  other a l f a l f a  was  level  found of  and  cultivars  the mean percent  seeds  a desirable  germination  to  germinbe  82%  seed v i a b i l i t y  sprouting.  (b)  Light  Light during  been  germination  indirectly thesis  has  affect  determines  demonstrated  1967).  influence  (Mayer and P o l j a k o f f - M a y b e r , the the  synthesis size  p r o d u c t i o n of the aromatic and Beck,  to  of  rings  of the  the  rate  1975).  f l a v o n o i d substances sugar  pool  which  of  respiration  Light since in  and Beck (1967)  turn  reported  also  photosyn-  v i a the s h i k i m i c a c i d pathway  However, R o s s i t e r  may  affects (Rossiter  appreciable  -  quantities plete  of  estrogenic  darkness,  81 -  isoflavones  i n d i c a t i n g that  the  synthesis  is  low.  Nevertheless,  determine  if  light  influences  in clover light  that  was  requirement  growth  studies  phytoestrogen  grown  for  were  i n com-  phytoestrogen  carried  accumulation  out  in  to  alfalfa  sprouts. The measured of  light i n terms  uEm~2s"1  photons. ation  lux  or  1979).  Einstein  plant  different  the its  alfalfa  nm)  the  is  that  to  r e l a t i o n to ski  the  light  In  was chosen as cular  to  the  the  present  rack.  human  one  spectral  was units  mole  of  radienvi-  Kozlowski,  pigments  in  in  that  Therefore, bands  of  plants each  differ-  light  with  1979). in taking l i g h t l e v e l  In f i e l d  the  studies,  centre  the top of the  of  the  positioned  obtained  plant  sprouting  with the sensor p l a c e d  lamps  in  T i b b i t t s and Kozlow-  be made at  raised  measurements  and p o s i t i o n of the sensor  the overhead room lamps was  and  vision  particular  The f l u o r e s c e n t  The same r e a d i n g  of  Tibbitts  spectrum.  the p o i n t f o r measurement  i n a d d i t i o n to  50 u E m ~ 2 s - 1 .  study  (PPFD) i n  in controlled  absorbing  for  (1979) recommended that measurements  canopy.  jars  1978;  depended on the angle lamps.  receive  and a c t i o n  (McFarlane,  overhead  to  f o r the d e t e r m i n a t i o n of the  responsible  respond  exposed  density  equivalent  plants  Some d i f f i c u l t y was e x p e r i e n c e d because the r e a d i n g s  the  were  the i n t e n s i t y of l i g h t produced by lamps  various  own a b s o r p t i o n  efficiencies  (E)  (McFarlane,  pigments  functions  sprouts  photon f l u x  favored  than measuring  foot-candles)  from has  is  and 700  Furthermore,  pigment ent  an  400  rather  the  of p h o t o s y n t h e t i c  where  (between  differ  that  T h i s measurement  ronments, (in  level  rack  perpendi-  directly  over  the  p r o v i d e d an average PPFD of when the  sensor  was  placed  - 82 -  in  the  centre  sprouts. by  of  the  T h i s l i g h t l e v e l was markedly lower than the 240 u E m ~ 2 s - 1  used  Hesterman  contrast studies  et  sprouting  al.  jars  (1981),  directly  however,  it  onto  was  the  (as  requires  measured (1978)  a light  (c)  with  a sensitivity  reported  that  the  l e v e l of only 0.1  surface  considered  with the dark c o n d i t i o n of 0 y E m ~ 2 s - 1 used  McFarlane  of 0.1  i n the other  viEnr2s-1).  germination  readings  indicated  light  found to taken al.  or  recorded  that  dark  the  from  thermometers  placed  temperature  inside  the  conditions.  (range 21.2 sprouting  found t h a t  Hamilton  germination  on  remained  Observations identifying  sprout  a  plant  of  1979;  at  Several Hsu et  con-  and r e g a r d l e s s temperature  Random temperature  constant  and other  sprouting  23°C.  readings  Hesterman  of 2 1 ° C was o p t i m a l workers  al.,  legumes  (Fordham et  1980) at  was  have  et for  al.,  conducted  comparable  tempera-  development r e c o r d e d d u r i n g the growth s t u d i e s p r o v i d e a means of in  growth c o n d i t i o n s .  i n a t i o n and/or  were  sprouts.  alfalfa  differences  length,  24.0°C).  the  25°C.  A l f a l f a sprout  various  of  relatively  The mean ambient  a g e r m i n a t i o n temperature  and Vanderstoep,  studies  t u r e s of 22 to  to  jars  f r e s h weight y i e l d of a l f a l f a  3.  growth  yEm"2s_1.  ambient  growth  be 2 2 . 2 ° C  (1981)  1975;  for  Furthermore,  response  s t a n t over a 24 h d u r a t i o n , throughout the growth p e r i o d s of  adequate  Temperature  The racks  of  color,  sprout  O b s e r v a t i o n s made at  maturity,  spoilage.  development  as  well  as  that  may  occur  24 h i n t e r v a l s  any v i s i b l e  signs  of  under  included contam-  -  (a)  Standard  After split. 7)  conditions  4 h soaking,  alfalfa  F o l l o w i n g the f i r s t  had emerged  83 -  seeds  were swollen and seed  24 h growth p e r i o d , white h y p o c o t y l s  from the seeds and averaged  1.0  cm i n l e n g t h .  c o a t s were only p a r t i a l l y removed from the c o t y l e d o n s . 24 h growth p e r i o d ,  the  h y p o c o t y l s averaged  dons had begun to develop green c o l o r as the  cotyledons.  elongated  to  At 76  an  remained l a r g e l y to  h  (3  average  days  of  2.0  and  and l a r g e r ,  subsequent  seedling Walter green pink  of  Jensen  cotyledons color  sp r ou t s  After study growth length  were  one  (1970). and  at  be  the  upper  at  the  were  had  green  but  appeared  sprouts  fleshier  to  i n terms  More to  rapid  the  another,  portion  3.0  of  germination  higher as  after  least  of  suggested  100  h had  cm long  the  inherent by  bright  with  some  hypocotyls.  The  spoilage.  conditions  a 4 h soaking swollen  period,  and  the  resulted  in  i n both 0 h l i g h t  "light"  Cotyle-  hypocotyls  seed white  ("dark")  the V e r n a l coats  were  hypocotyls  alfalfa  seeds used i n t h i s  split.  A subsequent  approximately  and 24 h l i g h t  ("light")  Seed c o a t s were p a r t i a l l y removed and c o t y l e d o n s appeared in  the second  s t a g e , Moapa s p r o u t s  harvested  hypocotyls  the  cotyledons  related  compared  The seed  separated away from  time)  open c o t y l e d o n s . may  After  had  (Figure  cm i n l e n g t h .  h e a l t h y with no v i s i b l e s i g n s of  Trial  period  The  At t h i s  Sprouts  white  soaking  cm.  cultivar  development  appeared  (b)  spread  1.5  seed c o a t s  than V e r n a l  photosynthesis  vigor and  +  folded together.  be d e v e l o p i n g more r a p i d l y  hypocotyls  coats  sprouts  and l i g h t  yellow i n  the  "dark"  1.0  24 h cm  in  conditions.  slightly  sprouts.  green After  -  the  second  24  averaged 2.0  h growth  cm i n l e n g t h  t o spread open. length  period,  and  84 -  the  and most  hypocotyls  cotyledons  The h y p o c o t y l s of the  were  much  straighter  "dark"  than  ences i n sprout  development  become more apparent at At  76  cotyledons, base,  h  the  this  A and U s p r o u t s  many seed  coats  well  as  rinsing.  at  the  (with  hypocotyls  base.  some were  remained  s p r o u t s averaged 4.0 of  the  vested at coats  sprout  this  was  of  white  After  or  100  in  the  sprout  light  Differhad  had  white at 3.0  mass.  sprouts  which  except  tip  Treatment  only  green  some  the  (radicle).  due to  pink  and  B and V  and most  seed  more  as  coats  frequent  small,  slightly  yellow  spread  pigmentation  open. at  the  cm h y p o c o t y l s and the  seed  Treatment  E and Y as w e l l as F and Z  and most  seed c o a t s  from Treatments  the 76. h growth p e r i o d . D-2.  the  had p r i m a r i l y  had  for  dark  cm long h y p o c o t y l s  presumably  "dark"  large,  There  were  no  had been A to  F were  A clump of other  washed har-  moldy seed  visible  signs  of  spoilage. h the  and a more pronounced were  sprouts.  growth c o n d i t i o n s  jars,  The s p r o u t s  jar  light  spread open.  cm i n l e n g t h  sprouts.  mass.  the  3.5  cm h y p o c o t y l s  stage f o r  found  contamination  tyls  remained  the  greening)  among the  cm i n  of  and  t h a t had become pink at  Treatment D and X s p r o u t s averaged 3.0  coats  out  but  averaged  The c o r r e s p o n d i n g  cotyledons  s p r o u t s averaged 2.5  generally  and h y p o c o t y l s  among  bottom  begun  begun t o  had approximately  remained  C and W s p r o u t s  accumulated  The  sprouts  centre  Treatment  green  sprouts  stage.  "light"  i n the  "light"  had  i n response to v a r i o u s  many spread open,  p a l e green  the  were  those  C o t y l e d o n s were yellow and some had a l s o  of  "light" long,  green,  sprouts  thin  with  exhibited  radicle,  pink  at  the  a  thickened  or primary base;  the  root.  hypocotyl  The hypoco-  radicles  remained  -  white.  85 -  S p r o u t s grown with Treatment U averaged 3.5  V and W s p r o u t s  averaged  appears to encourage spread  open;  cotyledons "dark"  sprout  epicotyls  (Figure  sprouts.  cm i n l e n g t h .  growth.  were  7).  There was  to  frequent  at  were a l s o present  the  i n the  base  but  open.  Sprouting  jar  Y-1  "light"  cm i n a l l  contained  a  sprouts,  much lower  treatments (X  and some were yield  the  otherwise  C o t y l e d o n s were predominantly y e l l o w with some g r e e n i n g ,  than those from the c o r r e s p o n d i n g  of  corresponding  the h y p o c o t y l s  l e n g t h averaged 5.5  rinsing  were dark green and  develop  some p i n k i n g of  Sprout  The more  The c o t y l e d o n s  beginning  Radicles  they remained w h i t e . to Z ) .  5.0  cm whereas Treatment  of  smaller spread  sprouts  than  j a r s Y-2 or Y - 3 . There was l i t t l e cm  increase  sprouts.  in  After in  sprout  Epicotyl  not yet apparent  length.  the r e s u l t  proposed the  and  radicles  of enzymatic browning.  of  crushed  discoloration  greater  2  to  1.0  in  all  s p r o u t s but  was  cm long  s p r o u t s averaged 5.0 cm  were  bruised  brown.  or  broken  and  This discoloration  was  F r a n c i s and M i l l i n g t o n  (1965b)  that a p o l y p h e n o l oxidase type of enzyme system was i n v o l v e d i n  browning  brown  to  "light"  the " l i g h t "  numerous Treatment U and V s p r o u t s were likely  1  than a 0.5  sprouts.  of 148 h growth, hypocotyls  were  c o n t i n u e d i n the  i n the " d a r k "  Many  124 h growth other  Radicles  development  a total  length.  change a f t e r  and  clover a  much f r e s h e r  volume and frequency  The " d a r k "  of water  s p r o u t s averaged 6.0  Y-1  showed s i g n s  all  samples  were  of  browning of  yellow  samples.  because  Treatment appearance,  applied  cm i n l e n g t h .  W sprouts likely  the  lack  to  no the  d u r i n g the growth p e r i o d . Only  the  h y p o c o t y l s and r a d i c l e s . of  due  had  of  light,  sprouts  in  jar  C o t y l e d o n s of however  those  -  sprouts  closest  sprouts  would have been u n a v o i d a b l y exposed  during  the  "light" Figure  to the g l a s s j a r  86 -  rinsing  procedure.  (Treatment 12.  s i d e s d i d e x h i b i t some g r e e n i n g .  U)  and  The s p r o u t s  to minimal  The d i f f e r e n c e  "dark"  from  (Treatment  in  X)  Treatment U to  amounts of  sprout  sprouts  Z were  These  color is  light  between  evident  harvested  at  in this  stage f o r the 148 h growth p e r i o d . Seed included that  that  i n the  had  had  sprout  accumulated  sampling. was  coats  not  samples at  the  rinsed  taken bottom  for of  made  in  this  study  to  identify  out  of  analysis; the  These d i s c a r d e d seed c o a t s f e l t  seed c o a t s or on the a l f a l f a  D.  been  jars  the  sprout  however, were  not  mass were seed  coats  included  in  s l i g h t l y s l i m y but no attempt  the m i c r o b i a l  flora  present  on the  sprouts.  PHYTOESTROGEN ANALYSIS Daidzein,  alfalfa lists  sprout  formononetin and coumestrol were d e t e c t e d extracts  as  i n many of  determined from HPLC chromatograms.  the  Table 7  the mean content o f each phytoestrogen c a l c u l a t e d to be present  alfalfa  sprouts  Analysis  of  corresponding  the  data  to  revealed  the that  various the  to the t r e a t m e n t s ,  phytoestrogens  discussed  first  be  treatments.  accumulation  phytoestrogen v a r i e d i n response will  growth  therefore,  individually.  in  of  each  the  three  Analysis  of  v a r i a n c e t a b l e s are p r o v i d e d i n the A p p e n d i c e s . The treatment two  levels  growth  combinations were coded f o r f a c t o r i a l a n a l y s i s  f o r each f a c t o r .  period,  light  l e v e l s were a s s i g n e d  The four f a c t o r s  duration, as  follows:  rinse  using  were coded i n the o r d e r of  volume and  rinse  frequency;  the  -  Growth p e r i o d  (G)  Light duration  1 = 76 h 2 = 148 h  (L)  1 = 2 =  R i n s e volume (V)  R i n s e frequency  1.  87 -  0 h 24 h  1 = 0.4 L 2 = 1.0 L (F)  1 = 2X 2 = 5X  Daidzein Single  factor  analysis  to Z) and the 2 standard  of  variance  treatments  of  of the observed  means of d a i d z e i n c o n t e n t s  are  three the  Treatments  replicates  C and  C and  which  Y means.  content  of  markedly  greater  than  higher  daidzein  in  Y-1.  jar  had  which  a  of  ± 4.5  the  content  other  Y.  Although  contaminated  seed  affected  total  the  ment means c a r r i e d  in  coats  very  to  the  yg/g  of  ±  from  the  there or  other  were  sprouts  excluding  the  were  (dry (6.9  visible  may  found  have  signs  An a n a l y s i s two extreme  the  of  in  values  a  was This  sprouts  from  jar  weight),  the  case  spoilage, and  variance  of  increased  (dry  present of  which  Sprouts  as  the  have  yg/g).  of  alfalfa  been  to  found an  plants.  of  deviation  and 3.7  also  yg/g  one  weight),  C replicates  no  Treatment  for  contamination  alfalfa 1.2  effects.  7.  Y-1  and 1973)  16.3  to  standard  alfalfa  (1971  was performed  large jar  to  (A  value  Y replicates  daidzein content. out  in  treatments  large  infected  content  greatly  i n Table  may be r e l a t e d  daidzein  daidzein  differed  Treatment  25.9  trial  treatment  a  sprouts  Olah and Sherwood  accumulation C-3  Y had  Alfalfa  daidzein  presented  contributed  12  (Moapa and V e r n a l )  determine the s i g n i f i c a n c e  Both  the  of  some  thereby  of  (assumed  treatto  be  - 88 -  Table 7.  P h y t o e s t r o g e n c o n t e n t s of a l f a l f a dard and t r i a l c o n d i t i o n s .  sprouts  Phytoestrogens Daidzein  Treatment  Formononetin  grown  (yg/g  under  stan-  alfalfa)1'2 Coumestrol  STANDARD Moapa (AA)  2.1 + 0 . 1 2  Vernal  1.0 + 0 . 2 0  (BB)  a b 3  2.9 ± 0 . 6 2  1.3 ± 0 . 6 4  a  N.Df4  a  b c  N.D3  TRIAL A  1.9 + 0A9 ah  B  2.2 + 0 . 2 9  C  9.3 + 7 . 0 0  D  0.2 + 0 . 0 6  E  0.9 + 0 . 0 6  F  0.7 + 0 . 1 2  U  5.0 + 0 . 7 4  V  2.0 + 0 . 8 9  w  2.5 + 0A9 ab  X  2A  ± 0Ak°  N.D3  a b  0.7 ± 0.67°  N.D!  a b  0.3 ± 0A6°  N.D3  0.5  a  1.6 ± 0 . 6 7  a b c  a  1.2 ± 0 . 2 1  b c  + 0.23  0.1  3  ± 0.12°  0.9  ± 0.90  a  1.0 ± 1 . 0 0  a  N.D3  a b  2.7 ± 0 . 5 5  a b  0.6 ± 0 . 2 1  C  2.1  ± 1.53  a  0.9 ± 1 . 1 7  C  0.9 ± 0 . 7 6  a  9.0 ± 1 . 3 6  d  5.5 ± 1 . 7 2  b  6.3 ± 0 . 7 6  6  3.6 ± 0 . 9 7  C  ± 0.87  6  6.0 ± 1 . 0 6  b  a b  Y  12.2 + 1 2 . 0 0  Z  6.2 + 0 . 3 2  b  a b  6A  a b  0.6 ± 0 . 5 1  Expressed on dry weight b a s i s . Mean of 3 r e p l i c a t e s ± standard d e v i a t i o n . t r e a t m e n t means i n a column s h a r i n g the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t (P>0.05) as determined by the Student-Newman-Keuls test. ^N.D. = Not d e t e c t a b l e . 2  3  -  missing  values)  Treatments (P<0.05)  more  C and  grow  analysis  alfalfa  and l i g h t  sprouts  (G x L) and of  (P<0.05) (Figure  (as  17)  possible  the  mentioned,  of v a r i a n c e revealed  daidzein levels (Figure  18)  decreased in  the at  the treatment  that  infection  that  daidzein  significant  of  that  only  the  sprouts  significantly  (Table 8 ) .  of  combinations  interactions  The e f f e c t  of  an  been  content  increase in  "light"  sprouts.  P = 0.048  and the  the  used  growth  significant  curve of G x L  76 h vs 148 h . sprouts  to  associated  1971). in  "dark" However,  this  different  in-  increased  curve of L x  resulted, in  but  an  interaction  Student-Newman-Keuls  means were not s i g n i f i c a n t l y  with  volume  sprouts  may be  As p r e v i o u s l y  The e f f e c t  rinse  It  infection  grown i n the dark.  has  greater  i n d a i d z e i n content over growth  alfalfa  are  from  (Appendix A - 2 ) .  the treatment  Olah and Sherwood,  shows  had  s p r o u t s grown f o r  susceptibility  (e.g.  Z)  alfalfa  l i g h t and r i n s e volume (L x V) were  "dark"  increased  that  from a l l other treatments  c r e a s e s with time i n s p r o u t s  2.  U and  shows that a marked i n c r e a s e  that  content  as  f o r d a i d z e i n accumulation  time o c c u r r e d i n the  V  indicated  well  daidzein contents  Factorial to  Y  clearly  89 -  test  a  increased is  only  showed  that  (P>0.05).  Formononetin The  determined T a b l e 7. Z were  significantly by  single  factor  treatment  analysis  The formononetin c o n t e n t s  shown to  treatments.  different  be  significantly  of  of  means  variance,  for  formononetin,  are  identified  s p r o u t s from Treatments  greater  (P<0.05)  than  from  as in  X, Y and the  other  - 90 -  Table 8.  S i g n i f i c a n c e of c a l c u l a t e d F - v a l u e s from g e r m i n a t i o n f a c t o r s and f a c t o r interactions f o r phytoestrogen a c c u m u l a t i o n as determined by f a c t o r i a l a n a l y s i s of v a r i a n c e .  Phytoestrogens Factors  Daidzein  Formononetin  Coumestrol  * *  (L)  NS  * *  R i n s e volume (V)  NS  NS  NS  NS  NS  Growth Light  (G)  NS  G x L  *  * *  G xV  NS  NS  NS  G x F  NS  *  NS  L xV  *  NS  NS  L x F  NS  NS  NS  G x L xV  NS  NS  *  G x L x F  NS  NS  NS  R i n s e frequency  (F)  * S i g n i f i c a n t (P<0.05). NS Not s i g n i f i c a n t (P>0.05).  *  - 91 -  lure 17.  E f f e c t curve of growth and l i g h t (G x L) d a i d z e i n accumulation i n a l f a l f a s p r o u t s .  _!  I  interaction  I 148  76  GROWTH  PERIOD  (h)  -  Figure  18.  92  -  E f f e c t curve of l i g h t and volume (L x V) d a i d z e i n accumulation i n a l f a l f a s p r o u t s .  _l  I  :  0.4  RINSE  V O L U M E (L)  I  interaction for  I 1.0  - 93 -  The Table  8.  sprouts  sprouts  factorial  daidzein,  for  "dark"  the  is a different  analysis  differences  grown  grown i n the  of  there  of  Significant  between  case  results  76  in  h  and  of  formononetin those  interaction  of  of  alfalfa  grown  148  G x L (Figure  may  proposed  be  associated  Again,  with  19)  to  were  h,  and  in  found between  As i n  indicates  the that  s p r o u t s than of  increased  microbially  by Olah and Sherwood (1971),  s p r o u t s may be more s u s c e p t i b l e  presented  "light".  formononetin response of the " d a r k "  formononetin s p r o u t s as  are  content  and those grown i n the  the " l i g h t " s p r o u t s over the growth p e r i o d . tions  variance  concentra-  contaminated  and the  i n f e c t i o n and subsequent  "dark"  phytoestro-  gen a c c u m u l a t i o n . Results  also  (P<0.05)  factor  found to  be  increase  in rinse  tin  content  lation  showed  that  rinse  frequency  i n formononetin a c c u m u l a t i o n .  significant  (P<0.05)  frequency  as  in Figure  It  appears that  rinse  frequency  suggested by Lookhart  accumulation.  Coumestrol The  significantly  determined by s i n g l e Table  7.  samples. than  where an  (1979a), and thereby reduce the l e v e l of c o n t a m i n a t i o n and phyto-  estrogen  3.  20,  formononetin accumu-  increasing  may serve to c l e a n s e the s p r o u t s d u r i n g growth, as et a l .  significant  i n a marked decrease i n formonone-  i n the 148 h s p r o u t s but d i d not a f f e c t  i n the 76 h s p r o u t s .  a  The G x F i n t e r a c t i o n was  illustrated  resulted  was  the  factor  Coumestrol Overall,  different  the  concentrations  analysis  could  not  coumestrol of  treatment  the  means  of v a r i a n c e ,  be  detected  other  coumestrol,  as  are a l s o i d e n t i f i e d i n  in  concentrations  for  many  alfalfa  appeared  phytoestrogens,  sprout  much  however,  one  lower must  - 94 -  - 95 -  F i g u r e 20.  E f f e c t curve of growth and frequency formononetin accumulation i n a l f a l f a  _l  I  (G x F) i n t e r a c t i o n f o r sprouts.  I  2X  I 5X  RINSE F R E Q U E N C Y  /24h  - 96 -  a l s o note that that  the e x t r a c t i o n  potentially  The coumestrol once  again  the  coumestrol  contents  found  efficiency  to  of  be  the  f o r coumestrol was only 57% and  content sprouts  could  be  considerably  from Treatments  significantly  greater  higher.  X, Y and Z were  (P<0.05)  from  the  other  treatments. Factorial a  significant  grown f o r  analysis effect  of v a r i a n c e determined that  on  coumestrol  concentration  148 h had a s i g n i f i c a n t l y  greater  trates  that  period et  differed  al.  soy  the  (1978)  sprouts  present  markedly reported  greatly  study,  significantly The been  trol  to  time  reported  1979b).  rinse  a  "dark"  removal the  sprouts  seed  between  The  increase  found  in this  by Knuckles et  disease  in  infection  Sherwood,  1971;  (Loper et a l . ,  that  Wong and L a t c h , 1971)  growth time by B i c k o f f et a l .  Lookhart  coats)  were of  from  In  the  observed  more  to  frequent  were not  shown  to  accumulation. coumestrol  supports  between  accumulation disease  content the  increases  been r e p o r t e d  Sherwood et a l . ,  al.  increasing in  coumes-  (1979a and  1970; to  has  other  i n response  has a l s o been r e l a t e d  (1969) and Lookhart et a l .  and  with  and Lookhart et  has  1967;  growth  content.  result  illus-  the  seed  frequency  than  (1976)  over  (or  treatments a  further  sprouts.  coumestrol  coumestrol  study  Coumestrol accumulation  hulls  and  extent  al.  the  rinse  coumestrol  sprouts  ( F i g u r e 21)  as  Sprouts  for 0 h l i g h t  "light"  coats  8).  had  content  and  some  factors  coumestrol  accumulation  total  from  much g r e a t e r  microbial  of  volume and  (P<0.05) a f f e c t  phytoestrogens. germination  that  dispersed  relationship  studied  coumestrol  reduced  of  However,  of  between  alfalfa  have lower l e v e l s rinsing.  The G x L i n t e r a c t i o n  response  (Table  (P<0.05)  than s p r o u t s grown f o r 76 h, and the same was t r u e vs 24 h l i g h t s p r o u t s .  several  to  Olah and increased  (1979a).  - 97 -  Figure 21.  E f f e c t curve of growth and l i g h t coumestrol accumulation i n a l f a l f a  (G x L) sprouts.  interaction  for  - 98 -  The  two  daidzein  (Y-1  and  formononetin) expected  alfalfa C-3)  than  if  the  these  samples  which  did  have  other  samples  degree of accumulation of fic  pathogens  as  Sherwood et a l . ,  4.  General  in  some g e n e r a l light of  19,  crease light  21) in  that  the  these  was  was  found to  by  many  to  of  found  high  of as  levels  might  infected.  workers  of  coumestrol  (or  have  been  However,  may be a response to (Hanson  et  the  speci-  al.,  Lookhart et a l . ,  could  be  as  to  be  of  content  the  affected  The f a c t o r  to of  show the  1965;  1979a).  phytoestrogens  by d i f f e r e n t  factors,  i n t e r a c t i o n of growth and i n the  The e f f e c t that  there  alfalfa  growth p e r i o d was  three  (P<0.05)  coumestrol.  interpreted  the  each  be s i g n i f i c a n t  formononetin and  curves was  sprouts  extended  accumulation (Figures  a marked i n grown  in  from 76 h to  0  increase  phytoestrogens et  conclude periods  those i n the l i g h t . of phytoestrogens  al., that  are  i n phytoestrogen accumulate 1970; those  more  Olah  in  alfalfa  and  alfalfa  susceptible  content. that  Sherwood,  sprouts to  grown  microbial  Based is  on  it  the  reports  may dark  infection  These s p r o u t s may have accumulated a h i g h e r  i n response to an i n f e c t i o n .  did  microbially  1971) in  h  148 h .  s p r o u t s grown i n 24 h l i g h t over the same growth p e r i o d s  (Sherwood  longer  isoflavonoid  accumulation  study  show a s i m i l a r  possible  each  microbially  t r e n d s were o b s e r v e d .  conditions  infected  were  levels  replicates  Wong and L a t c h , 1971;  phytoestrogen  The a l f a l f a not  the  this  (G x L)  daidzein,  17,  treatment  demonstrated 1970;  higher  very  discussion  Although examined  not  contained  be for  than  content  - 99 -  Rinse reported trol  frequency  relationship  content  and  alfalfa  (Lookhart  sprouts.  frequency  were  coumestrol 7).  standard  discussed  not  low,  c o n d i t i o n sprouts  not the purpose of t h i s it  would appear  that  able for a l f a l f a  sprouts in  total  (1  the  infected  coumestrol less,  forage  alone  in  have r e p o r t e d coumestrol  the  coumes-  factor  was  significant  of growth and  for  formononetin  Moapa and V e r n a l ,  were found to  formononetin content  content  i.e.  not  different  of  these  d a i d z e i n and  (P>0.05)  sprouts  significantly  of Treatments  but  A to F .  have a  (Table  grown  different  under  (P>0.05)  And a l t h o u g h i t  was  of  low phytoestrogen  content  c o n d i t i o n s most  and  desir-  germination.  is  of  phytoestrogens  minimal  et  to  al.,  various  al.,  detected  i n comparison to  which  different  healthy,  (Hanson et  neither  interaction  would make the standard  (Bickoff  that  be  significantly  alfalfa,  response  and decreased  However,  and the  to  combination  content  significantly  observed  1970).  sprout  to 22 ppm)  of  study to develop optimum g e r m i n a t i o n c o n d i t i o n s ,  good v i s u a l a c c e p t a b i l i t y  The  frequency  (P<0.05)  was  cleansing  because  f o r coumestrol or d a i d z e i n a c c u m u l a t i o n  phytoestrogen  conditions  examined  previously.  were  the  were  1979a).  cultivars,  contents  from t r i a l  (P<0.05)  different  Overall,  al.,  demonstrated  The two a l f a l f a significantly  et  Rinse  only  a c c u m u l a t i o n as  volume  between soy sprout  found to be s i g n i f i c a n t in  rinse  may  1967;  (P_<0.05) growth  contain Loper  Bickoff  several al.,  conditions.  et  contains  al.,  these  the c o n t e n t s  phytoestrogen  uninfected a l f a l f a 1965;  et  in  1967;  alfalfa reported  hundred  1967);  neverthe-  contents Previous less  were  workers  than 5 ppm  Sherwood et  A l f a l f a s p r o u t s from Treatments X, Y and Z c o n t a i n e d  ppm  al.,  potentially  -  9.6,  6.3  and 10.5  ppm coumestrol  which would appear to  indicate  100 -  respectively  that  (assuming  some coumestrol  57%  extraction)  accumulation beyond  the " h e a l t h y " l e v e l d i d o c c u r . Knuckles sprouts  et  al.  contained  5  (1976) and  reported  71  ppm  that  coumestrol  markedly higher than other v e g e t a b l e s t e s t e d study,  many  alfalfa  sprout  coumestrol,  and t h e r e f o r e  at  higher  a  many  level of  these  formononetin this  food.  consists by a  than  alfalfa which  samples  could also  sprout  samples  increase  the  However, u n l i k e the d i e t  of a great  minor d i e t a r y  physiological  variety  of  component  significance.  and  soy  which  was  ppm).  In the  greater  than  coumestrol  to  present 1.0  ppm  the  diet  common v e g e t a b l e s .  In  contained  daidzein  total  levels  of  phytoestrogen  addition,  content  and of  of a g r a z i n g a n i m a l , the human d i e t  foods such  (<1.0  contribute  i n other  sprouts  respectively  contained  that  would  alfalfa  as  But that  and the phytoestrogens alfalfa  sprouts  c o n c l u s i o n cannot  more knowledge of the metabolism of phytoestrogens  may  contributed have  little  be made without  i n man.  -  101 -  CONCLUSIONS  The  overall  performance estrogens  objective  liquid present  conditions.  of  this  chromatography in  alfalfa  A method f o r  research  to  project  examine  sprouts  the  grown  was  to  use  isoflavonoid  under  selected  the e x t r a c t i o n of phytoestrogens  from  high  phytogrowth alfalfa  s p r o u t s was developed which c o n s i s t e d of e x t r a c t i n g aglycone p h y t o e s t r o gens  from  ground  chlorophyll  lipids  and  pigments with petroleum ether and subsequently i s o l a t i n g  the  phytoestrogens ated  to  ranged  94%  spiked a l f a l f a tin)  and the  Recovery  from  samples  using ethyl  dryness  analysis.  alfalfa  final  of  residue  (formononetin)  sprouts  the  extracts  separation,  dissolved  103%  and  from  a  Murphy,  evaporfor  standards  recoveries  the  d e t e r m i n a t i o n of  and UV d e t e c t i o n at  HPLC  the s e l e c t e d  separation  at  a  flow  254  rate  phytoestrogens  appears  mix from  literature  1981).  employed a reversed-phase  0.01M ammonium a c e t a t e  HPLC  78% (formonone-  phytoestrogens  o c t a d e c y l s i l a n e column  nm.  Peak  area  integrator  phytoestrogen q u a n t i t a -  A l i n e a r g r a d i e n t methanol/water system c o n t a i n i n g 1% a c e t i c  r e s o l u t i o n of This  was  i n methanol  (coumestrol);  responses were compared t o a standard curve f o r tion.  extract  and compared f a v o r a b l y to r e p o r t e d  An HPLC method developed f o r  for  ether  phytoestrogens to  removing  were found to be 83% ( d a i d z e i n ) ,  ( L o o k h a r t , 1979;  i n the a l f a l f a  methanol,  The e t h y l  selected  and 57% ( c o u m e s t r o l ) ,  values  ether.  with  to  be very  d e t e r m i n a t i o n of the major phytoestrogens  of  1.0  mL/min gave  i n an e l u t i o n suitable in a l f a l f a  for  acid  baseline  time of 30 m i n . the  quantitative  sprouts.  -  The  phytoestrogens  detected  using  the  Concentrations cantly (148  of  greater  h vs  for  the dark f o r daidzein,  and  only a  all  the  limited  other  of  alfalfa  were  sprouts  factors  in  the  for  a  h light  was  and coumestrol  examined  conditions. in  on the  to  the  of  of  microbial  sprouts  and  be  sprouts  grown  greater  (P<0.05)  than the a l f a l f a  sprouts  study  of  were  rinse  of i n c r e a s e d  coats  not  to  in  have  alfalfa  d u r a t i o n on  susceptibility  however,  were  in  volume  found  phytoestrogens  infection;  seed  period  significant  growth p e r i o d and l i g h t  may be the r e s u l t  signifi-  vs 24 h l i g h t ) .  The l e v e l s  present  accumulation  influence  sprouts  contents  study.  longer  shown to  those a l f a l f a  were  present  found to be  grown (0  coumestrol  growth p e r i o d had s i g n i f i c a n t l y  accumulation  alfalfa  and  developed  phytoestrogens;  treatment  effect  alfalfa  flora  three  The observed  phytoestrogen the  in  growth and l i g h t  frequency  sprouts.  of  of  formononetin  rinse  methods  formononetin and coumestrol  the l o n g e r  grown under  formononetin  and those grown i n the dark  The i n t e r a c t i o n (P_< 0.05)  daidzein,  analytical  (P<0.05)  76 h)  102 -  the  microbial  enumerated  nor  i  identified. 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Prentice-Hall, Inc.,  Engle-  Appendix A-1  Analysis of variance  for daidzein  14).  content o f a l f a l f a  sprouts  from Treatments AA t o Z ( 1 t o  VARIABLE NAMES - DAIDZEIN DATA FORMAT  (I2.F4. 1 >  DAIDZEIN ANALYSIS OF  SOURCE TREAT ERROR TOTAL  13 28 41  GRAND MEAN  OF VARIANCE - DAIDZEIN  SUM SO  MEAN SO  480 42 390 .30 870 72  36.95S 13.939  F-VALUE . 2.6511  PROS 0.1491SE-01  3.4762  STANDARD DEVIATION OF VARIABLE  1 IS  4.6084  FREQUENCIES, MEANS, STANDARD DEVIATIONS ********************»******************* 1 10 MN DAIDZEIN  ERROR  2.067 2.000  2 11 1 .000 2.533  3 12 1 .867 2.367  STUDENTIZED RANGES FOR NEWMAN-KEUL'S TEST, 2.897 3.499 3.861 4.120 4.322 5.029 5.106 5.177  ***********  4 13 2.233 12.17 ALPHA=0.05 4.486 4.625  ******************  5 14 9.267 6.233 4.745  0.2333  4.850  0.9333  0.7333  4.944  THERE ARE 2 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS ( 6, 8. 7, 2. 3. 10. 1. 4. 12. 11, 9. 14, 5) ( 3. 10. 1. 4, 12. 11. 9. 14. 5. 13) TIME FOR MULTIPLE RANGE TESTS IS ANALYSIS  COMPLETE  0.1272E-01 SECONDS.  SIGNIFICANT RANGE  Appendix A - 2 .  A n a l y s i s of v a r i a n c e for d a i d z e i n 14 without C-3 or Y-1 v a l u e s ) .  content of a l f a l f a  sprouts  from Treatments AA to Z (1  to  VARIABLE NAMES - DAIDZEIN DATA FORMAT  (I2.1X.F3.1)  ANALYSIS  TREAT ERROR TOTAL  GRAND MEAN  OF VARIANCE - DAIDZEIN  DF  SUM SO  MEAN SO  13 26 39  139.19 33.192 172.38  10.707 1.2766  ERROR  F-VALUE  PROB  8.3869  0.26258E-05  2.5950  _^  STANDARD DEVIATION OF VARIABLE  1 IS  -P-  2.1024  I  FREQUENCIES. MEANS. STANDARD DEVIATIONS *•*****•**•*******•***********•****•****"•*******•*******•************••***•***** TREAT MN DAIDZEIN  1 10 2.067 2.000  2 1 1 1 .000 2.533  3 12 1 .867 2.367  4 13 2.233 5.300  STUDENTIZED RANGES FOR NEWMAN-KEUL'S TEST, ALPHA=0.05 2.907 3.514 3.879 4.141 4.346 4.511 4.652 5.061 5.140 5.211  5 14 S.750 6.233 4.774  0.7333  4.880  4.975  THERE ARE 2 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS ( 6, 8, 7, 2. 3, 10, 1, 4, 12, 11) ( 9, 13, 5 , 14) TIME FOR MULTIPLE RANGE TESTS IS ANALYSIS COMPLETE.  0.1616E-01 SECONDS.  SIGNIFICANT RANGE  Analysis to 14).  Appendix B.  of v a r i a n c e  for  formononetin content  of a l f a l f a  sprouts  from Treatments AA to Z (1  VARIABLE NAMES - FORMETIN DATA FORMAT  (I2.F4.1)  FORMETIN ANALYSIS SOURCE TREAT ERROR TOTAL  GRAND MEAN  13 28 41  OF VARIANCE - FORMETIN  SUM SO  MEAN SO  301.83 14.120 315.94  23.217 0.50429  MN FORMETIN  F-VALUE 4G.040  PROB 0.220G6E-14  2.450O  STANDARD DEVIATION OF VARIABLE FREQUENCIES.  ERROR  1 IS  2.7760  (  MEANS. STANDARD DEVIATIONS  .**•*#***«  1 10  2 11  3 12  4 13  5 14  2.900 0.5667  1 .267 0.8667  0.5000 9.033  0.7333 6.267  0.2667 6.367  STUDENTI ZED RANGES FOR NEWMAN-KEUL'S TEST. 2.897 3.499 3.861 4.120 4.322 5.029 5.106 5.177  ALPHA=0.05 4 486 4.625  THERE ARE 5 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS. FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED ( 8 . 5 . 3. 10. 4. 11. 7. 2. 6) ( 7 . 2 . 6. 9) ( 6 . 9 , 1) ( 13, 14) ( 12) TIME FOR MULTIPLE RANGE TESTS IS ANALYSIS COMPLETE.  0.1316E-01 SECONDS.  4.745  0.6667E-01  4.850  2.733  4.944  NO PAIR OF WHICH OIFFER BY MORE THAN THE SHORTEST  SIGNIFICANT RANGE  Appendix C .  A n a l y s i s of v a r i a n c e f o r coumestrol content of a l f a l f a 14).  sprouts  from Treatments AA to Z (T to  VARIABLE NAMES - COUMSTRL DATA FORMAT  (I2.F4.1)  COUMSTRL ANALYSIS  TREAT ERROR TOTAL  GRAND MEAN  OF VARIANCE - COUMSTRL  DF  SUM SO  MEAN SO  13 28 41  168.63 20.087 188.72  12.972 0.71738  MN COUMSTRL  F-VALUE 18.082  PROB 0.29041E-09  1.4643  STANDARD DEVIATION OF VARIABLE FREQUENCIES.  ERROR  MEANS,  1 IS  2.1454  STANDARD'DEVIATIONS  4 . * * * * * * * * * * * * * * . j * * * * * * . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * . * * * * *  1 10  2 11  3 12  4 13  5 14  0.0 2.067  0.0 0.8667  0.0 5.467  0.0 3.567  0.0 6.033  STUDENTIZED RANGES FOR NEWMAN-KEUL'S TEST, 2.897 3.499 3.861 4.120 4.322 5.029 5.106 5.177  ALPHA=0.05 4.486 4.625  4.745  ****•••  0.9000  4.850  4.944  THERE ARE 3 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS. NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS 8. 5, 4, 7, 10) 13) 12. 14) TIME FOR MULTIPLE RANGE TESTS IS ANALYSIS COMPLETE.  O.1232E-01  SECONDS.  0.5667  SIGNIFICANT RANGE  -  Appendix D .  117 -  F a c t o r i a l a n a l y s i s of v a r i a n c e f o r d a i d z e i n , formononetin and coumestrol contents of a l f a l f a s p r o u t s from T r e a t ments A to Z .  Coding of growth c o n d i t i o n f a c t o r s was e x p l a i n e d i n the R e s u l t s and D i s c u s s i o n S e c t i o n , and the treatment combinations are coded as f o l l o w s :  A  =  1  2  2  1  B  =  1  2  1  2  C  =  1  2  2  2  D  =  1  1  2  1  E  =  1  1  1  2  F  =  1 1 2  U  =  2  2  2  1  V  =  2  2  1  2  W  =  2  2  2  2  X  =  2  1  2  1  Y  =  2  1  1  2  Z  =  2  1  2  2  2  TITLE.ESTROGEN ANALYSIS: VARIABLES.G.L.V,F.OAIDZEIN.FORMETIN.COUMSTRL MODEL , DA IDZE IN. FORMET IN. COUMSTRL =G+L+G* L +V+G* V+L *V+G* I *V+F+G*F*L*F+G*L*F LEVELS G * 2 . L - 2 . V J , F = 2 INPUT FILE»PHYTO F0RMAT«(F1.0.3F2.0.3F5.1) OUTPUT OBSE.FREO.PRED TERMS'G,L,G*L,V,G*V,L*V,G*L*V,F,G*F,L*F,G*L*F MULRAN TYPE * D , N . T TERMS3G.L.G'L.V.G,V.L*V.G*L>V.F.G*F.L*F,G*L*F COMPUTE Time f o r c o n t r o l  c a r d p r o c e s s i n g was  0.10426  seconds.  Cumulative time Is  0.10871  seconds.  /*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/'•/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/'•/'•/*/*/*/*/*/••/*/*/'•/*/  Time f o r d a t a Input and c o r r e l a t i o n m a t r i x was Time f o r ANOVA t a b l e was 0.71316E-01 seconds.  0.53294E-01 seconds. Cumulative time i s Cumulative time i s 0.24115 seconds.  ESTROGEN ANALYSIS: A n a l y s i s f o r OAIOZEIN A n a l y s i s of v a r i a n c e Source  Sum of squares  G L G*L V G*V L»V G*L*V F G*F L*F G*L*F Residual Total  56.751 • 0.17778E-01 128.44 0.77042 56.120 70.384 0.22042 32.202 16.0OT 0.10667 66.002 390.19 842.48  DAIDZEIN  Overal1 mean 3.8OO0  table DF 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. 24. 35.  Mean square 56.751 0.17778E-01 128.44 0.77042 56.120 70.384 0.22042 32.202 16.0O7 0.10667 66 .002 16.258  18 MEAN 2.5444 MEAN 2 5444 STDV 3 9822 ERR M 0.95038  P r o b a b ! 1 i t y Test  3.4906 O.10935E -02 7.9004 0.47387E -01 3.4519 4.3292 0. 13558E -01 1.9807 0.98454 0.65609E -02 4.0596  0.07397 0.97389 0.00968 0.82951 0.07549 0.04831 0.90828 0.17214 0.33098 0.9361 1 0.05525  Overa11 standard d e v i a t i o n 4.9062  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s 1 . .. 2.. . 0 P O S  F-ratlo  for G  18 5.0556 5.0556 5.5101 0.95038  M u l t i p l e range t e s t s F-ratlo  i s not s i g n i f i c a n t  at p r o b a b i l i t y 0.07397  tern  RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIOUAL RESIDUAL RESIOUAL RESIOUAL  0.16872  seconds.  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r L . 1. . .2. . 0 P 0 S  18 MEAN 3.7778 MEAN 3.7778 STOV 6.0065 ERR M 0.95038  M u l t i p l e range F-ratio  18 3.8222 3.8222 3.67 13 0.95038 tests  Is not s i g n i f i c a n t  at p r o b a b i l i t y 0.97389  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r G*L 11.. 12.. 2 1.. 2 2.. 9 MEAN 0.63333 MEAN 0.63333 STDV 0.32016 ERR M 1.3440  0 P 0 S  M u l t i p l e range  9 4.4556 4.4556 5.0376 1.3440  9 6.9222 6.9222 7.3700 1.3440  9 3.1889 3.1889 1.5366 1.3440  tests  Duncan t e s t at 5% p r o b a b i l i t y l e v e l There are 2 homogeneous subsets which a r e l i s t e d as (  11...  22...  (  22...  12...  1 2 . ) 2  1..)  Newman-Keuls t e s t at 5% p r o b a b i l i t y l e v e l There are 2 homogeneous subsets which are l i s t e d as (  11...  (  22...  22...  11...  1 2 . )  22...  was  0.16081E-01 seconds.  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r V ..1. ..2. MEAN MEAN STDV ERR M  12 4.3333 3.5611 6.9942 1.2868  M u l t i p l e range  follows:  1 2 . )  ( 2 2 . . . 1 2 . . . 2 1.. ) Time f o r m u l t i p l e range t e s t  0 P 0 S  follows:  1 2 . . . 2 1 . )  Tukey t e s t at 5% p r o b a b i l i t y l e v e l There are 2 homogeneous subsets which a r e l i s t e d as (  follows:  24 3.5333 3.9194 3.6071 0.86758 tests  Cumulative time  0.30527  seconds.  f-ratio Frequencies, 1.1. 0 P 0 S  ( 3 not s i g n i f i c a n t  at p r o b a b i l i t y  0.82951  means, s t a n d a r d d e v i a t i o n s f o r G*V 1.2. 2.1. 2.2.  6 MEAN 1.5833 MEAN 0.81111 STDV 0.73598 ERR M 1.7352  12 3.0250 3.4111 4.8483 1.1959  6 7.0833 6.3111 9.4302 1.7352  12 4.0417 4.4278 1.7707 1.1959  M u l t i p l e range t e s t s F-ratlo  i s not s i g n i f i c a n t  at p r o b a b i l i t y 0.07549  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s .12. .1 1 . .2 1 . 0 P 0 S  MEAN MEAN STDV ERR M  6 6.5500 5.7778 9.7705 1.7352  12 2.3917 2.7778 2.4652 1.1959  for L*v .2.2.  6 2.1167 1.3444 0.60470 1.7352  12 4.6750 5.0611 4.2760 1.1959  M u l t i p l e range t e s t s Duncan t e s t at 5% p r o b a b i l i t y l e v e l There 1s 1 homogeneous subset which Is l i s t e d as (  .2  There (  .2  There  1.  follows:  .11.)  Newraan-Keuls t e s t at 5% p r o b a b i l i t y l e v e l Is 1 homogeneous subset which Is l i s t e d as f o l l o w s : 1.  .22.,  .1  1 . )  Tukey t e s t at 5% p r o b a b i l i t y l e v e l i s 1 homogeneous subset which Is l i s t e d as  follows:  ( .2 1., .1 2 . . .2 2 . , .1 1. ) Time f o r m u l t i p l e range t e s t was O.66795E-02 seconds.  Cumulative time i s  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r G * L * V 1 2 2. 111. 1 1 2. 1 2 1. 0 P 0 S  ro O  MEAN 0.93333 MEAN 0. 161 t 1 STDV 0.57735E-01 ERR M 2.3917 M u l t i p l e range F-ratio  6 0.48333 0.86945 0.28577 1.6686  3 2.2333 14611 0.28868 2.3917  6 5.5667 5.9528 6.0105 1 .6688  tests  Is not s i g n i f i c a n t  at p r o b a b i l i t y 0.90828  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s for F  2  0.34464  2 2 1.  11. 3 12.167 1 1.394 12.001 2 3917  6 3000 6861 1326  3 2.0000 1.2278 0.88882 2.3917  2 2 2. 6 3.7833 4 1694 1.4798 1.6688  O P 0 S  MEAN MEAN STDV ERR M  12 2.3750 2.2556 1 8445 1.2868  24 4.5125 4.5722 5.7804 0.86758  M u l t i p l e range t e s t s F-ratlo Frequencies, 1.1 0 P 0 S  i s not s i g n i f i c a n t  at p r o b a b i l i t y 0 17214  means, s t a n d a r d d e v i a t i o n s f o r G*F 1.2 2.1 2..2  6 MEAN 1 .0500 MEAN 0.93056 STOV 0.94816 ERR M 1.7352  12 3.2917 3.3514 4.7 193 1.1959  6 3.70OO 3.5806 1 .5401 1 .7352  12 5.7333 5.7931 6.6589 1.1959  M u l t i p l e range t e s t s F-ratto  Is not s i g n i f i c a n t  at p r o b a b i l i t y 0.33098  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r L * F .1.1 .1.2 .2.1 .2.2 0 P 0 S  MEAN MEAN STDV ERR M  6 1.3000 1.1806 1 .1781 1.7352  12 5.0167 5.0764 7.0784 1.1959  6 3.4500 3 .3306 1 .8229 1 .7352  12 4.0083 4.0681 4.3825 1.1959  M u l t i p l e range t e s t s F-ratlo  Is not s i g n i f i c a n t  at p r o b a b i l i t y  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s 1 1.2 12.1 1 1.1 0 P 0 S  3 MEAN 0.23333 MEAN 0.11389 STDV 0.57735E-O1 ERR M 2.3917  6 0.83333 0.89306 0.13663 1.6688  for  0.93611  G*L*F 1 2.2  3 1.8667 1.7472 0.49329 2.3917  6 5.7500 5.8097 5.8715 1.6688  2  1.1  3 2.3667 2.2472 0.23094 2.3917  2  1.2 6 9.2000 9.2597 8.2588 1.6688  2 2.1  2 2 2  3 5.0333 4.9139 0.7371 1 2.3917  6 2.2667 2.3264 0.70616 1 .6688  M u l t i p l e range t e s t s F-ratlo  Is not s i g n i f i c a n t  at p r o b a b i l i t y  0.05525  /*/•/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*//*/*/*/*/*/*/*/*/*/*/*•/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/^/*/*/*/*/*/*/*/*/*/*/*/*/*/*•/*/*•/  A n a l y s i s f o r FORMETIN A n a l y s i s of v a r i a n c e t a b l e Sum of  Mean  Source  squares  G L G*L V G*V L*V G*L*V F G*F L*F G*L*F Residual Total  1 15.92 88.3G0 65.610 0.51042 1.4504 0.2604 2 0 704I7E-01 14.727 2.9400 t.6017 O.B1667E-01 12.533 309.42  square  Overa11 mean 2.5111  115.92 88.360 65.610 0 . 5 1042 1.4504 0.26042 0.704I7E-01 14.727 2.9400 I.6017 0.81667E-01 0.52222  F-ratlo  P r o b a b i l i t y Test  221 98 169.20 125.64 0.97739 2.7774 0.49B67 O.13484 28 . 2O0 5.6298 3.0670 0.15638  0.00000 0.00000 0.00000 0.33271 0.10860 0.48688 0.71668 0.00002 0.02601 0.09267 0.69600  term  RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL  Overa11 standard d e v i a t i o n 2.9733  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r G 1. . . 2. . . O P 0 S  MEAN MEAN STDV ERR M  18 0.71667 0.71667 0.66355 0.17033  18 4.3056 4.3056 3.3078 0.17033  M u l t i p l e range t e s t s Duncan t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous subsets which a r e l i s t e d as (  1. . .  )  (  2...  )  Newman-Keuls t e s t at 5* p r o b a b i l i t y level There are 2 homogeneous subsets which a r e l i s t e d as (  1. . .  )  (  2...  )  Tukey t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous subsets which are l i s t e d as (  1. . .  follows:  follows:  )  ( 2... ) Time f o r m u l t i p l e range t e s t  was  0.66929E-02 seconds.  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r L . 1 .. .2. . 0 MEAN  follows:  18 4.0778  18 0.94444  Cumulative time Is  0.44469  seconds.  P MEAN 4.0778 0 STDV 3.4622 S ERR M 0.17033  0.94444 1.0083 0.17033  M u l t i p l e range t e s t s Duncan t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous subsets which are l i s t e d as (  .2..  (  . 1. . )  )  Newman-Keuls t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous subsets which a r e l i s t e d as (  .2..  (  . 1. . )  follows:  )  Tukey t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous subsets which are l i s t e d (  follows:  as  follows:  2. . )  ( .1. . ) Time f o r m u l t i p l e range t e s t  was  0.61455E-02 seconds.  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s 11.. 12.. 21.. 0 P 0 S  9 MEAN 0.93333 MEAN 0.93333 STDV 0.75993 ERR M 0.24088  9 0.50000 0.50000 0.50249 0.24088  Cumulative time  for G*L  9 7.2222 7 2222 1.6269 0.24088  22. 9 1.3B89 1.3889 1.2098 0.24088  M u l t i p l e range t e s t s Duncan t e s t at 5% p r o b a b i l i t y l e v e l There a r e 3 homogeneous subsets which a r e l i s t e d  as  follows:  Newman-Keuls t e s t at 5% p r o b a b i l i t y l e v e l There a r e 3 homogeneous subsets which a r e l i s t e d as  follows:  (  1 2 . 1 1 . )  (  1 1...  (  2  (  2 2..  )  1..)  1 2. . .  1 1. . )  (  1 1 . ., 2 2.. )  (  2  1 . )  Tukey t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous subsets which are l i s t e d as  follows:  Is  0.46163  seconds.  ( 2 1 . ) me f o r m u l t i p l e range t e s t  was  0.86842E-02 seconds.  Cumulat ve time Is  0.48031  seconds.  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r V . .1 . .2. 0 P 0 S  12 MEAN 2.1833 MEAN 2.7056 STDV 2.5135 ERR M 0.23063  24 2.6750 2.4139 3.2167 0.15549  M u l t i p l e range t e s t s F-ratlo Frequencies, 1.1. 0 P 0 S  i s not s i g n i f i c a n t  at p r o b a b i l i t y  0.33271  means, s t a n d a r d d e v i a t i o n s f o r G*V 1.2. 2.1. 2.2.  6 MEAN 0.95000 MEAN 1.4722 STDV 0.50100 ERR M 0.31098  12 0.60000 0.33889 0.72237 0.21433  6 3.4167 3.9389 3.1619 0.31098  12 4.7500 4.4889 3.4233 0.21433  M u l t i p l e range t e s t s F-ratio  i s not s i g n i f i c a n t  at p r o b a b i l i t y 0.10860  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r L*V .11. .12. .2 1. .2 2. 0 P 0 S  6 MEAN 3.7167 MEAN 4.2389 STDV 2.8379 ERR M 0.31098  12 4.2583 3 9972 3.8415 0.21433  6 0.6S0O0 1.1722 0.45055 0.31098  12 1.0917 0.83056 1.1866 0.21433  M u l t i p l e range t e s t s F-ratlo  Is not s i g n i f i c a n t  at p r o b a b i l i t y 0.48688  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r G * L * V 111. 112. 121. 122. MEAN MEAN STDV ERR M  3 1 . 1667 1 .6889 0.208 17 0.42865  6 81667 55556 92610 29909  0.73333 1.2556 0.66583 O.4 2865  6 0.38333 0.12222 0 42 151 O.29909  M u l t i p l e range t e s t s F-ratlo  i s not s i g n i f i c a n t  at p r o b a b i l i t y  0.71668  2  11.  3 6.2667 6.7889 0.76376 0.42865  2 1 2.  2 2 1.  2 2 2.  6 7.7000 7.4389 1 .7833 0 29909  3 0.56667 1 .0889 0 20817 O. 42865  6 1 8000 1 .5389 1 3100 0 29909  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r F ... 1 . . .2 12 MEAN 3.4583 MEAN 3.5556 3.534 1 STDV ERR M 0.23063  0 P 0 S  24 2.0375 I.9889 2.6033 O.15549  M u l t i p l e range t e s t s Ouncan t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous subsets which a r e l i s t e d as (  .  (  ...1 )  .2 )  Newman-Keuls t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous subsets which a r e l i s t e d as (  ...2  )  (  ...1  )  .2  follows:  ,  Tukey t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous subsets which a r e l i s t e d as (  follows:  follows:  ,  ,  ( ...1 ) Time f o r m u l t i p l e range t e s t  was  0.61197E-02 seconds.  Cumulative time i s  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r G*F 1.1 1.2 2.1 2.2 0 P 0 S  6 MEAN 1.0333 MEAN 1.1306 STDV 0.77115 ERR M 0.31098  12 0.55833 0.50972 0.57280 0.21433  6 5.8833 5 9806 3 5735 0.31098  12 3.5167 3.4681 3.0114 0.21433  M u l t i p l e range t e s t s Ouncan t e s t at 5% p r o b a b i l i t y l e v e l There a r e 3 homogeneous subsets which are l i s t e d as (  1.2.  (  2 . 2 )  (  2 . 1 )  1.1  1  2.  1.1  follows:  >  Newman-Keuls t e s t at 5% p r o b a b i l i t y l e v e l There are 3 homogeneous subsets which are l i s t e d as (  —* fNJ  )  follows:  0.54491  seconds.  (  2 . 2 )  (  2 . 1 )  Tukey t e s t at 5% p r o b a b i l i t y l e v e l There a r e 3 homogeneous subsets which are l i s t e d as (  1..2.  (  1..1  follows:  )  2 . 2 )  ( 2 . 1 ) Time f o r m u l t i p l e range t e s t Frequencies, .1.1  O.9O237E-02 seconds.  Cumulative time i s  0.56561  seconds.  means, s t a n d a r d d e v i a t i o n s f o r L*F .12 .2.1 .2.2 12 3.4667 3.4181 3.0467 0.21433  6  5.3000 MEAN 5.3972 MEAN 4.2005 STDV ERR M 0.31098  0 P 0 S  was  6 1.6167 1.7139 1.3014 0.31098  12 0.60833 0.55972 0.65707 0.21433  M u l t i p l e range t e s t s F-ratlo  i s not s i g n i f i c a n t  at p r o b a b i l i t y 0.09267  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r G * L * F 11.1 11.2 12.1 1 2.2 0 P 0 5  3 MEAN 1 .5667 MEAN 1.6639 STDV 0.665B3 ERR M 0.42865  6 0.61667 0.56806 0.62102 0 29909  3 0.50000 0.59722 0.43589 0.42865  6 0.50000 0.45139 0 57271 0.29909  o 2  1.1  3 9.0333 9.1305 1.3614 0.42865  2  1.2  2  6 6.3167 6.2680 0 73598 0. 29909  2.1  3 2.7333 2.8306 0.55076 0.42865  2 2 2 6 0.71667  0.66806 0.77050 0.29909  M u l t i p l e range t e s t s F - r a t l o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.69600 /+/*/*/*/+/*/+/*/+/*/+/*/*/'/*/*/*/*/*/*/*/*/+/+/+/*/*/+/+/*/*/*/'if*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/ A n a l y s i s f o r COUMSTRL A n a l y s i s of v a r i a n c e  table  Source  Sum of squares  DF  Mean square  G L G*L V G*V L'V G*L'V F G*F L*F  69.167 45.563 23.200 0.20417E -01 1.9837 2.6004 8.2838 0.41668E -03 1 . 1704 0. 15042  1. 69.167 1. 45.563 1. 23.200 1. 0.204 17E -01 1. 1.9837 1. 2.6004 1. 8.2837 1. 0.41668E -03 1 . 1. 1704 1. 0.15042  F-ratlo 82 642 54.439 27.720 0.24394E -01 2.3702 3.1070 9.8976 0.49786E -03 1.3984 0 17972  P r o b a b i l I t y Test 0 OOOOO 0.00000 0.O0O02 0.87719 0.13675 0.09069 0 004 38 0.98238 0.24857 0.67539  term  RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL  G*L * F Res iduat Total  O v e r a l l mean 1.7083  COUMSTRL Frequencies, 1... 0 P 0 S  I. 24. 35.  0.51042 20.087 173.71  MEAN MEAN STDV ERR M  O 51042 0.83G94  0.60986  O. 44248  RESIDUAL  Overa11 standard d e v i a t i o n 2 2278  means, s t a n d a r d d e v i a t i o n s f o r G 2...  18 0.32222 0.32222 0.65937 0.21563  18 3.0944 3.0944 2.3905 0 21563  M u l t i p l e range t e s t s Duncan t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous s u b s e t s which are l i s t e d as (  follows:  1 . )  (  2.. .  )  Newman-Keuls t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous s u b s e t s which are l i s t e d as (  1...  (  2.  ) . )  Tukey t e s t at S% p r o b a b i l i t y l e v e l There a r e 2 homogeneous s u b s e t s which are l i s t e d as  ( 2. . . ) Time f o r m u l t i p l e range t e s t  was  0.66795E-02 seconds.  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s . 1 . . .2. . 0 P 0 S  follows:  18 MEAN 2.B333 MEAN 2.8333 STDV 2 5652 ERR M 0.21563  follows:  Cumulativ  for L  18 O 58333 0 58333 0.97874 0.21563  M u l t i p l e range t e s t s Duncan t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous s u b s e t s which are l i s t e d as (  .2..  )  (  .1 . . )  follows:  time i s  0.63022  seconds.  Newman-Keuls test at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous s u b s e t s which are l i s t e d as (  .2..  (  .1 . . )  )  Tukey t e s t at 5% p r o b a b i l i t y l e v e l There are 2 homogeneous s u b s e t s which are l i s t e d (  .2..  as  follows:  )  ( . 1. . ) Time f o r m u l t i p l e range t e s t F r e q u e n c i e s , means, It.. 0 P 0 S  follows  9 MEAN 0.64444 MEAN 0.64444 STDV O 83083 ERR M 0.30495  M u l t i p l e range  was  0.63B10E-02 seconds.  standard d e v i a t i o n s 12.. 21.. 9  0 0 0 22335E-05 0.0 0.30495  Cumulative time  tests  Newman-Keuls t e s t at S% p r o b a b i l i t y l e v e l There a r e 3 homogeneous s u b s e t s which a r e l i s t e d as  follows:  (  2  (  1 1.  )  2 2..  )  1..)  1 2...  1 1..  )  (  1 1...  2 2..  )  (  2  1 . )  Tukey t e s t at 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous s u b s e t s which a r e l i s t e d (  0 66526  9 1.1667 1.1667 1.1269 0.30495  follows:  1 1 ...  (s  22..  9 5.0222 5.0222 1.5849 0.30495  as  (  0.64552  f o r G*L  Duncan t e s t at 5% p r o b a b i l i t y l e v e l There a r e 3 homogeneous s u b s e t s which are l i s t e d ( 1 2 . . .  is  1 2...  1 1..,  ( 2 1 . ) Time f o r m u l t i p l e Frequencies, . . 1. 0 MEAN P MEAN  2 2..  12 1.6667 1.6694  follows:  )  range t e s t means,  as  was  0.98696E-02 seconds.  standard deviations .2. 24 1.7292 1.7278  for V  Cumulative time  seconds.  2.5043 0. 19684  0 STDV 1.6356 S ERR M 0.29197 M u l t i p l e range t e s t s F-ratio  1s not s i g n i f i c a n t  at p r o b a b i l i t y  0.87719  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r G*V 1.1. 1.2. 2.1. 2.2. MEAN MEAN STOV ERR M  0 P 0 S  6 0.51667 0.51944 0.84951 0.39369  12 0.22500 0 22361 0.55942 0.27133  6 2.8167 2.8194 1.4106 0.39369  12 3.2333 3.2319 2.8043 0.27133  M u l t i p l e range t e s t s F-ratlo  i s not s i g n i f i c a n t  at p r o b a b i l i t y 0.13675  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r L * V .11. .12. .2 1. .2 2. 0 P 0 S  6 MEAN 2.3000 MEAN 2.3028 STDV 1.6444 ERR M 0.39369  12 3.1000 3.0986 2.9508 0.27133  6 1.0333 1.0361 1.4895 0.39369  12 0.35833 0.35695 0.55343 0.27133  ro NO  I  M u l t i p l e range t e s t s F-ratio  Is not s i g n i f i c a n t  at p r o b a b i l i t y 0.09069  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r G * L * V 111. 112. 1 2 1. 1 2 2. 0 P 0 S  3 MEAN 1.0333 MEAN 1.0361 STDV 1.0017 ERR M 0.54266  6 0.45000 0 44861 0.75299 0.37864  3 6 0.0 0.0 0.27793E-02 -0.13873E-02 0.0 0.0 0.54266 0.37864  M u l t i p l e range t e s t s Duncan t e s t at 5% p r o b a b i l i t y l e v e l T h e r e a r e 4 homogeneous subsets which a r e l i s t e d as (  12  (  2 2 2.,  2 . 1 2 1 .  (  2 2 1 . . 2 1 1 .)  (  2  follows:  1 1 2 , 2 2 2 .  1 1 1 ., 2 2 1 .  )  1 2 . )  Newman-Keuls t e s t at 5% p r o b a b i l i t y l e v e l There a r e 3 homogeneous subsets which are l i s t e d as  follows:  2  11.  3 3.5667 3.5694 0.97125 0.54266  2 1 2.  2 2 1.  2 2 2.  6 5.7500 5.7486 1.3172 0.37864  3 2.0667 2.0694 1 .5308 0.54266  6 0.71667 0.71528 0.60470 0.37864  (  1 2 2 . . 1 2 1 . . 1 1 2 . , 2 2 2 . . 1 1 1 . 2 2 1 . )  (  2 2 1 .. 2 1 1 . )  (  2  1 2 . )  Tukey t e s t at 5% p r o b a b i l i t y l e v e l There a r e 3 homogeneous s u b s e t s which a r e l i s t e d as (  1 2 2 .  1 2 1 .  (  2 2 1 ., 2 1 1 .  1 1 2 . 2 2 2 . . )  ( 2 1 1 ., 2 1 2 ) Time f o r m u l t i p l e range t e s t Frequencies, ... I 0 P 0 S  follows:  1 1 1 . 2 2 1 . )  was  0.14140E-01 seconds.  Cumulative time  means, s t a n d a r d d e v i a t i o n s for F ... 2  12 MEAN 1.7333 MEAN 1.7139 STDV 2.4325 ERR M 0.29197  24 1.6958 1.7056 2.1731 0.19684  M u l t i p l e range t e s t s F-rat1o  Is not s i g n i f i c a n t  at p r o b a b i l i t y  F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r G*F 1 . .1 1.2 2.1 0 P 0 S  6 MEAN 0.45000 MEAN 0.43056 STDV 0.75299 ERR M 0.39369  12 25833 26806 6331 1 27133  6 3.0167 2.9972 2.9151 0.39369  0.98238  2.2 12 3.1333 3.1431 2.2281 0.27133  M u l t i p l e range t e s t s F-ratio Frequencies, .1.1 0 P 0 S  1s not s i g n i f i c a n t  at p r o b a b i l i t y 0.24857  means, s t a n d a r d d e v i a t i o n s f o r L*F .1.2 .2.1 .2.2  6 MEAN 3.1833 MEAN 3.1639 STOV 2 7874 ERR M 0.39369  12 2.6583 2.6681 2.5568 0.27133  6 0.28333 0.26389 O 44907 0.39369  12 0 73333 0.74306 I 1468 0.27133  M u l t i p l e range t e s t s F-ratlo Frequencies,  Is not s i g n i f i c a n t  at p r o b a b i l i t y  0.67539  means, s t a n d a r d d e v i a t i o n s f o r G * L * F  0.72939  seconds.  0 P 0 S  MEAN MEAN STDV ERR M  0. 90000 0.88056 0.90000 0.54266  6 0.51667 0 52639 0.84951 0.37864  3 0.0 -O.19442E-01 00 0.54266  6 0.0 0 97240E-02 0.0 0.37864  M u l t i p l e range t e s t s F-ratlo  Is not s i g n i f i c a n t  at p r o b a b i l i t y 0.44248  3 5.4667 5.4472 1.7243 0.54266  6 4.8000 4.8097 1.6285 O 37864  3 0.56667 0.54722 0.51316 O 54266  6 1.4667 1.4764 1.2660 0.37864  

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