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Determination of some phytoestrogens in alfalfa sprouts 1984

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DETERMINATION OF SOME PHYTOESTROGENS I N ALFALFA SPROUTS by SYLVIA ANN (DUFFEK) YADA B . S c . ( A g r . ) , U n i v e r s i t y of B r i t i s h Columbia, 1978 A thes i s submitted i n p a r t i a l f u l f i l l m e n t of the requirements for the degree of THE FACULTY OF GRADUATE STUDIES Department of Food Science We accept t h i s thes i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1984 0 S y l v i a Ann (Duffek) Yada, 1984 Master of Science i n In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of 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 granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Food Science The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 March 14, 1984 ( i i ) ABSTRACT A l f a l f a sprouts were grown under se lected condi t ions in order to determine the ef fect of growth per iod , l i g h t dura t ion , r in se volume and r inse frequency on the accumulation of phytoestrogens. The phytoestro- gens were i s o l a t e d from a crude methanol extract of a l f a l f a sprouts using e thy l e ther . The residue remaining fo l lowing e thy l ether evapora- t i o n was red i s so lved i n methanol for high-performance l i q u i d chromato- graphy (HPLC). A method for HPLC was developed using an oc tadecy l s i l ane reversed-phase column, UV detect ion at 254- nm and a gradient methanol/ water solvent system containing 1.0% ace t i c ac id and 0.1 M ammonium acetate . Basel ine r e s o l u t i o n of the phytoestrogens da idze in , formonone- t i n and coumestrol from the a l f a l f a sprout extracts was achieved with an e l u t i o n time of 30 minutes. To ta l phytoestrogen content ranged from 1 to 22 ppm dry weight a l f a l f a (or less than 2 ppm fresh weight a l f a l f a ) depending on the growth condi t ions employed. A l f a l f a sprouts grown i n the dark (0 h l i g h t ) for the longer growth period (148 h) had s i g n i f i - cant ly greater (P<_ 0.05) contents of da idze in , formononetin and coumes- t r o l than those sprouts from other chosen treatment combinations. Although the cont r ibu t ion of phytoestrogens to the human die t from a l f a l f a sprouts would appear to be markedly higher than from other common vegetables , the p h y s i o l o g i c a l s i g n i f i c a n c e of such an intake has not yet been determined. ( i i i ) TABLE OF CONTENTS P a 9 e ABSTRACT i i TABLE OF CONTENTS i i i LIST OF FIGURES v LIST OF TABLES v i i LIST OF APPENDICES v i i i ACKNOWLEDGEMENTS ix INTRODUCTION 1 LITERATURE REVIEW 3 A. Nature and d i s t r i b u t i o n of phytoestrogens 3 B. B iosynthes i s and accumulation 14 1. Biosynthes i s 14 2. E f fec t s of microb ia l i n f e c t i o n 16 3. Fungi tox ic a c t i v i t y 21 4. E f fec t s of l i g h t 22 C. Metabolism 23 1. Isoflavones 24 2. Coumestans 27 3. Absorption 28 D. Ex t r ac t ion of phytoestrogens 29 E . Detect ion techniques 35 1. Paper chromatography 35 2. T h i n - l a y e r chromatography 38 3. Column chromatography/UV spectrophotometry 39 4. High-performance l i q u i d chromatography 41 MATERIALS AND METHODS 45 A. Mater i a l s 45 B. Phytoestrogen ex t rac t ion 45 C. Moisture determination 48 D. Measurement of phytoestrogens by HPLC 49 E . Germination of a l f a l f a seeds 53 1. Sprouting preparat ion 53 2. Growth studies 55 (a) Standard condi t ions 57 (b) T r i a l condi t ions 57 (c) Percent germination 60 3. Sprout harvesting 60 F. S t a t i s t i c a l ana lys i s 62 1. S ing le factor ana lys i s of variance 62 2. F a c t o r i a l ana lys i s of variance 62 ( iv ) RESULTS AND DISCUSSION 63 A. Ex t rac t ion 63 1. Ex t rac t ion methodology 63 2. Recovery studies 64 B. High-performance l i q u i d chromatography 67 1. HPLC methodology 67 2. Chromatographic parameters 70 3. Linear regress ion ana lys i s 77 C . Growth studies 78 1. Moisture determination 78 2. Growth condi t ions 80 (a) Percent germination 80 (b) Light 80 (c) Temperature 82 3. A l f a l f a sprout development 82 (a) Standard condi t ions 83 (b) T r i a l condi t ions 83 D. Phytoestrogen ana lys i s 86 1. Daidzein 87 2. Formononetin 89 3. Coumestrol 93 4. General d i scuss ion 98 CONCLUSIONS 101 REFERENCES CITED 103 APPENDIX 113 (v) L IST OF FIGURES Page F i g u r e 1 . S t r u c t u r a l formulas for s t e ro id estrogens 4 F i g u r e 2 . S t r u c t u r a l formulas for estrogenic i sof lavones 9 F i g u r e 3 . S t r u c t u r a l formulas for estrogenic coumestans 10 F i g u r e 4-. Proposed b iosynthet i c pathway of i sof lavones v i a a chalcone 15 F i g u r e 5 . Pos s ib le biogenet ic r e l a t i o n s h i p s among i so f l avono ids 20 F i g u r e 6 . Metabolism of some estrogenic i sof lavones in sheep . . . . 25 F i g u r e 7 . Germination and development of a l f a l f a seed to sprout 31 F i g u r e 8 . Phytoestrogen ext rac t ion scheme for a l f a l f a sprouts . . . 46 F i g u r e 9 . Retention time and peak width at base measurements . . . . 51 F i g u r e 1 0 . Rack used for dra ining sprouting j a r s 54 F i g u r e 1 1 . A l f a l f a sprouts growing under f luorescent lamps ( l i g h t treatment) and under aluminum f o i l covers (dark treatment) 56 F i g u r e 1 2 . Dra ining of a l f a l f a sprouts p r i o r to sampling and f reez ing 61 F i g u r e 1 3 . HPLC chromatogram of phytoestrogen standards. Peaks: D=daidzein, F=formononetin, C=coumestrol 73 F i g u r e 1A-. HPLC chromatogram of phytoestrogen standards e x t r a c t . Peaks: D=daidzein, F=formononetin, C=coumestrol 7 k F i g u r e 1 5 . HPLC chromatogram of a l f a l f a extract spiked with 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 F i g u r e 1 6 . HPLC chromatogram of a l f a l f a extract (Treatment X) Peaks: D=daidzein, F=formononetin, C=coumestrol 76 (v i ) Page F i g u r e 1 7 . Ef fect curve of growth and l i g h t (G x L) i n t e r a c t i o n for da idzein accumulation i n a l f a l f a sprouts 91 F i g u r e 1 8 . E f fec t curve of l i g h t and volume (L x V) i n t e r a c t i o n for da idzein accumulation i n a l f a l f a sprouts 92 F i g u r e 1 9 . E f fect curve of growth and l i g h t (G x L) i n t e r a c t i o n for formononetin accumulation i n a l f a l f a sprouts 94 F i g u r e 2 0 . Ef fect curve of growth and frequency (G x F) i n t e r a c t i o n for formononetin accumulation i n a l f a l f a sprouts 95 F i g u r e 2 1 . Ef fect curve of growth and l i g h t (G x L) i n t e r a c t i o n for coumestrol accumulation i n a l f a l f a sprouts 97 ( v i i ) LIST OF TABLES Page Table 1 . Re l a t ive binding a f f i n i t y of some na tura l ly occurr ing estrogens for mammalian estrogen receptors 11 Table 2. Standard growth condi t ions for a l f a l f a sprouts 58 Table 3. T r i a l growth condi t ions for a l f a l f a sprouts 59 Table Recovery of phytoestrogens using developed ex t rac t ion method 66 Table 5. Retention times ( tR) , capaci ty factors (k) , separat ion factors ( « ) and re so lu t ion (R s ) of se lec ted phytoestrogens on MicroPak MCH-10 using l i n e a r gradient e l u t i o n as described 71 Table 6. Moisture content of a l f a l f a sprouts grown under standard and t r i a l condi t ions 79 Table 7. Phytoestrogen contents of a l f a l f a sprouts grown under standard and t r i a l condi t ions 88 Table 8. S i g n i f i c a n c e of ca lcu la ted F-values from germination fac tors and factor i n t e r a c t i o n s for phytoestrogen accumulation as determined by f a c t o r i a l ana ly s i s of variance 90 ( v i i i ) LIST OF APPENDICES Page Appendix A - 1 . Ana lys i s of variance for daidzein content of a l f a l f a sprouts from Treatments AA to Z (1 to 14) 113 Appendix A -2 . Ana lys i s of variance for da idzein content of a l f a l f a sprouts from Treatments AA to Z (1 to 14 without C-3 or Y-1 values) 114 Appendix B. Ana ly s i s of variance for formononetin content of a l f a l f a sprouts from Treatments AA to Z (1 to 14) 115 Appendix C. Ana ly s i s of variance for coumestrol content of a l f a l f a sprouts from Treatments AA to Z (1 to 14) 116 Appendix D. F a c t o r i a l ana lys i s of variance for d a i d z e i n , formononetin and coumestrol contents of a l f a l f a sprouts from Treatments A to Z 117 ( ix) ACKNOWLEDGEMENTS I wish to express my deep apprec ia t ion to Dr. John Vanderstoep for h i s patience and support, moral as wel l as f i n a n c i a l , throughout the course of t h i s research p r o j e c t . I a l so wish to thank the members of the committee, Dr . Shuryo Nakai , Dr . Brent Skura and Dr . Bob Bose, for t h e i r suggestions and a s s i s t ance . F i n a l l y I would l i k e to thank my f a m i l i e s , both Duffek and Yada, for t h e i r constant support and understanding during the past several years , and a s p e c i a l thanks to my husband Rickey for his inva luable c r i t i c i s m , i n s p i r a t i o n and encouragement. - 1 - INTRODUCTION Several plants of the Leguminosae (also known as Fabaceae) family conta in i so f lavonoids which are e s t rogen ica l ly ac t ive i n d iverse animal species (Wong, 1975). Two main groups, the i sof lavones and the coume- stans , comprise the majority of these "phytoestrogens" . A l f a l f a , c lover and soybeans are important sources of phytoestro- gens i n the feeds of grazing and domestic animals . Soybeans and soybean products as wel l as a l f a l f a sprouts are a p o t e n t i a l d ie tary source of phytoestrogens for humans. However, the ef fect on man of longterm dietary exposure to these plant const i tuents i s as yet unknown. The estrogenic i sof lavones and coumestans can accumulate i n plants i n apparent response to microb ia l i n f e s t a t i o n and have been shown to be t o x i c to invading fungi and bacter ia (Nairn et a l . , 1974; Lyon and Wood, 1975). Lookhart et a l . (1979a) demonstrated that the phytoestrogen coumestrol accumulated during the germination of soybeans to soy sprouts , poss ib ly a l so as a re su l t of fungal i n f e c t i o n . Isoflavones and coumestrol occur na tura l ly as water-soluble glyco- sides but are hydrolyzed to aglycones i n infected plants (Olah and Sherwood, 1973). Many methods previous ly used to extract phytoestrogens from plant t i s sues are only useful for q u a l i t a t i v e purposes, because they p r i m a r i l y i s o l a t e the a l c o h o l - s o l u b l e aglycones. In order to quanti tate the estrogenic po ten t i a l of a food or feed, both aglycone and g lycos ide forms of the i so f l avonoids must be extracted . A v a r i e t y of chromatographic techniques have been developed to detect and measure these estrogenic compounds. Th in- l ayer chromatography - 2 - has p r e d o m i n a t e d , „ however, several high-performance l i q u i d chromato- graphy (HPLC) systems have recent ly been demonstrated to provide the means for r a p i d , quant i t a t ive ana lys i s of f lavonoid mixtures (Carlson and Dolphin , 1980; Murphy, 1981; Daigle and Conkerton, 1982; E l d r i d g e , 1982a). The a p p l i c a t i o n of HPLC to resolve a mixture of estrogenic i sof lavones and coumestans i n ac tua l plant extracts requires further study. The object of the present research was: f i r s t , to develop an ex t rac t ion procedure for a l f a l f a with high ex t rac t ion e f f i c i e n c i e s for the most predominant phytoestrogens, coumestrol , da idzein and formonone- t i n ; secondly, to develop an HPLC system to resolve these compounds for i s o l a t i o n and quant i t a t ion ; and t h i r d l y , to apply these techniques to study the ef fect of se lected sprouting condi t ions (growth per iod , l i g h t dura t ion , r in se volume and r inse frequency) on the accumulation of phytoestrogens i n a l f a l f a sprouts . - 3 - LITERATURE REVIEW A. NATURE AND DISTRIBUTION OF PHYTOESTROGENS Estrogens can be defined as s t e ro id hormones which are e s sen t i a l to the normal sexual development of the female mammal and are secreted p r i m a r i l y by the ovary (Prosser , 1973). The p r i n c i p a l estrogenic hormones i n the c i r c u l a t i o n are e s t r a d i o l , estrone and e s t r i o l , a l l incorpora t ing the 18 carbon s t e ro id nucleus as shown i n Figure 1. The term estrogen can a l so be more broadly used to descr ibe any substance which w i l l produce c h a r a c t e r i s t i c s of es trus , a se r ie s of changes i n the female reproduct ive system associated with ovulat ion (Prosser , .1973) . The f i r s t reports of estrogenic substances being present i n plants were made i n 1926 by Loewe, Dohrn et a l . , and F e l l n e r 1 . Since that time numerous workers have examined various plants for t h e i r a b i l i t y to induce estrus i n animals, and have attempted to i d e n t i f y these plant - or phytoestrogens. Bennetts et a l . (1946) reported that a s t r a i n of subterranean c lover ( T r i f o l i u m subterraneum L.) was respons ible for reproduct ive abnormal i t ies of grazing sheep i n western A u s t r a l i a . Bradbury and White (1951) subsequently demonstrated the presence of two i so f l avones , gen i s te in ( 5 , 7 , 4 ' - t r i h y d r o x y i s o f l a v o n e ) and formononetin (7-hydroxy-4'-methoxyisoflavone) , i n subterranean c lover ex t rac t s . Biggers and Curnow (1954) showed that gen i s te in was e s t r o g e n i c a l l y ac t ive i n mice, and therefore could be a factor a f f ec t ing normal repro- duct ion i f consumed by other .animals. Cheng et a l . (1955) determined that i sof lavones occurr ing na tura l ly i n subterranean and red c l o v e r s , xAs c i t e d by Bradbury and White (1954). - 4 - Figure 1. S t r u c t u r a l formulas for st e r o i d estrogens. - 5 - i n c l u d i n g da idzein (7 ,4 ' -d ihydroxy i so f l avone ) , biochanin A (5 ,7-dihy- droxy-4'-methoxyisof lavone) , formononetin and g e n i s t e i n , have es trogenic a c t i v i t y in mice. Over 40 species of plants which showed some estrogenic potency were reported by Bradbury and White (1954). The common forages a l f a l f a (Medicago s a t i v a L . ) and ladino c lover (Tr i fo l ium repens L . ) were at that time l i s t e d as non-estrogenic p l ant s . However, l a t e r experiments es tabl i shed that both a l f a l f a and ladino c lover displayed s i g n i f i c a n t estrogenic a c t i v i t y (Pieterse and Andrews, 1956; Engle et a l . , 1957). The i s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of the ladino c lover estrogen(s) revealed that the major estrogenic component was not an i so - f lavone, but a benzofurocoumarin d e r i v a t i v e which was named coumestrol (Bickof f et a l . , 1957; B ickof f et a l . , 1958). Coumestrol (7 ,12-dihy- droxycoumestan) was subsequently detected in other leguminous forage plants (Lyman et a l . , 1959) and reported as the dominant estrogen i n a l f a l f a (L iv ings ton et a l . , 1961). Reports of es trogenic a c t i v i t y were based p r i m a r i l y on the measurement of uter ine enlargement of ovariectomized or immature female mice or rats af ter treatment with plant extracts (Biggers and Curnow, 1954; Cheng et a l . , 1955; P ie ter se and Andrews, 1956; B ickof f et a l . , 1957; B ickof f et a l . , 1958; Lyman et a l . , 1959). An e a r l i e r detect ion method, known as the A l l en-Doi sy t e s t , was based on the evidence of c o r n i f i e d vag ina l e p i t h e l i a l c e l l s in the tes t animal i n response to estrogen s t imula t ion (Bickoff et a l . , 1969). Both of these assays have been subject to c r i t i c i s m s (Verdeal and Ryan, 1979), and misleading re su l t s are poss ib le due to the method of treatment and the metabolism - 6 - of the compounds being s tudied . Nevertheless , the organ and t i s sue changes induced by phytoestrogens are observed as the c h a r a c t e r i s t i c s of normal estrus induced by endogenous estrogens i n ra t s and mice. An endogenous estrogen, such as e s t rad io l -17g , exerts i t s e f fect on the uterus by binding to a s p e c i f i c prote in receptor i n the cytoplasm of the uter ine c e l l s . I t i s then transported into the nucleus while s t i l l bound to the prote in receptor (Gorski et a l . , 1968). Gorski and coworkers (1968) further hypothesized that t h i s es trogen-protein complex t r i g g e r s messenger RNA synthes i s , which was l a t e r es tabl i shed to be necessary for synthesis of a s p e c i f i c estrogen induced prote in (IP) (Gorski et a l . , 1975). Only those compounds which w i l l bind to the uter ine estrogen receptor prote ins can regulate the synthesis of t h i s induced prote in (Ruh et a l . , 1973). The estrogen e s t r a d i o l - 1 7 0 was observed by Ruh et a l . (1973) to be most e f f ec t ive in inducing prote in synthes i s , followed by the other endogenous estrogens e s t r i o l and estrone. Another more immediate ef fect of estrogen admini s t ra t ion i s the i m b i b i t i o n of water by the uter ine t i s sue s , r e s u l t i n g i n enlargement of the uterus (Notebloom and G o r s k i , 1963). The measurement of increased uter ine weight and s i ze i n determining estrogenic a c t i v i t y i s thus based on a combination of induced prote in synthesis and water uptake by the uter ine t i s s u e s . Studies on the phytoestrogens have shown that these compounds can also complex with the estrogen-protein receptors in the uter ine c y t o s o l , and i n turn induce prote in synthesis and water i m b i b i t i o n (Notebloom and G o r s k i , 1963; Shemesh et a l . , 1972; K i t t s , 1974; Martin et a l . , 1978). - 7 - In order to be recognized by estrogen receptors i n target c e l l s , i t i s bel ieved that a compound must meet s p e c i f i c s t r u c t u r a l require- ments. Plant i sof lavones and coumestans that demonstrate estrogenic a c t i v i t y possess a varying degree of s t r u c t u r a l s i m i l a r i t y to estrogenic s t e r o i d s . The high binding a f f i n i t y of s tero ids i s re l a ted to the presence of at l eas t one aromatic r i n g , the presence and p o s i t i o n of a phenol ic hydroxyl group on t h i s r i n g , and the nature and p o s i t i o n of oxygen functions on r ing D. More s p e c i f i c a l l y , a free phenolic hydroxyl at C-3 on the aromatic r ing A i s considered to be e s s e n t i a l for receptor binding (Hahnel et a l . , 1973). Busetta et a l . (1977) reported that the pentagonal r ing D of an estrogen s t e ro id cannot be modified without an important decrease i n binding a f f i n i t y to receptor p ro te ins . Hahnel et a l . (1973) demon- strated that the highest binding a f f i n i t y for a s t e ro id occurs with an a l c o h o l i c hydroxyl on C-17 ( r ing D) i n the g conf igura t ion and a pheno- l i c hydroxyl on C-3 of an unsaturated r ing A, as i n es t rad io l -17g (Figure 1) . The attachment of the estrogenic s t e ro id to the binding s i t e may depend on these two hydrogen bonding centres being about 10 A apart (Hahnel et a l . , 1973). Binding a f f i n i t y i s decreased upon removal or methylation of the C-3 hydroxyl , by the add i t ion of other oxygen funct ions on r ing D or a l k y l groups on r ing A , and by further unsatura- t i o n of the s t e r o i d nucleus (Korenman, 1969; Shutt and Cox, 1972; Hahnel et a l . , 1973). The s t r u c t u r a l s i m i l a r i t y among estrogenic i so f l avones , coumestans and estrogenic s tero ids i s evident by comparing Figures 1, 2 and 3. The important aromatic r ing A with a phenol ic hydroxyl group at C-3 (C-7 i n - 8 - isoflavone and coumestan numbering) i s common to a l l structures (with the exception of the isoflavone prunetin). In addition, these i s o f l a - vones and coumestans have an oxygen function (either a hydroxy1 or methoxyl group) at opposite ends of the molecule. Coumestrol and genis- t e i n are able to s i g n i f i c a n t l y compete for uterine cytosol binding s i t e s due l a r g e l y to the presence of two hydroxyl groups positioned at oppo- s i t e ends of the molecules (Shemesh et a l . , 1972). Bickoff et a l . (1969) reviewed the estrogenic a c t i v i t y of coumestrol and c l o s e l y related compounds and reported that the furan ring (C) was an important factor contributing to the e s t r o g e n i c i t y of some coumestans; opening of t h i s ring greatly decreased estrogenic a c t i v i t y . O v e r a l l , the binding a f f i n i t y of isoflavones and coumestans would be expected to be l e s s than that of st e r o i d estrogens because of a d d i t i o n a l substituent groups and a more unsaturated nucleus. The r e l a t i v e binding a f f i n i t y of an estrogenic compound to uterine receptors has been reported to p a r a l l e l the uterotrophic properties of that compound (Korenman, 1969; Shemesh et a l . , 1972; Ruh et a l . , 1973; Martin et a l . , 1978). Results from competitive binding assays c a r r i e d out with uterine cytosol (Shutt and Cox, 1972; Verdeal et a l . , 1980) and human tumor c e l l e s t r a d i o l receptors (Martin et a l . , 1978) support t h i s r e l a t i o n s h i p in terms of the order of estrogenic potency (Table 1). However, r e l a t i v e binding a f f i n i t y values indicate a much higher potency for the non-steroid estrogens than do Lin vivo uterine weight assays (Bickoff et a l . , 1962). Presumably, the differences are due to species v a r i a t i o n and metabolic e f f e c t s , but i t i s not yet c l e a r which method of determining estrogenic potency i s most applicable to humans (Verdeal and Ryan, 1979). - 9 - Figure 2. S t r u c t u r a l formulas for estrogenic i so f l avones . F O R M O N O N E T I N P R U N E T I N - 10 - Figure 3 . S t r u c t u r a l formulas for estrogenic coumestans. COUMESTROL 4'- METHOXYCOUMESTROL REPENSOL - 11 - Table 1. R e l a t i v e binding 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 - gens f o r mammalian estrogen receptors. Receptors Estrogens Rat u t e r i n e c y t o s o l 1 Sheep u t e r i n e c y t o s o l 2 Human cancer c e l l l i n e MCF-7 3 estradiol - 1 7 B 100 100 100 coumestrol 4.9 5 10 g e n i s t e i n 1.3 0.9 2 d a i d z e i n 0.09 0.1 - formononetin <0.01 <0.01 biochanin A 0.07 - - Vrom Verdeal et a l . (1980). 2From Shutt and Cox (1972). 3From Martin et a l . (1978). ^Not determined. - 12 - Very few n a t u r a l l y occurr ing i so f lavones , coumestans or re l a ted f lavonoids have been reported in the l i t e r a t u r e as demonstrating es t ro- genic a c t i v i t y , aside from those already mentioned. Flavonoids are ubiquitous const i tuents of green p lant s , and c h a r a c t e r i s t i c f l avonoid types are often encountered only i n p a r t i c u l a r plant groups (Markham, 1982). Isoflavones are f lavonoids genera l ly r e s t r i c t e d to one subfamily of the Leguminosae (Harborne et a l . , 1975); within t h i s subfamily are the common forage crops a l f a l f a and c l o v e r , as wel l as soybeans. Wong (1975) l i s t e d over 70 i sof lavones and 40 i sof lavone g lycos ides which have been i s o l a t e d from p l an t s . In addi t ion to the reported estrogenic a c t i v i t i e s for g e n i s t e i n , da idze in , formononetin and biochanin A, Cheng et a l . (1955) found that g e n i s t i n , the glucoside of g e n i s t e i n , was equal ly as estrogenic i n mice as gen i s te in i t s e l f . Bradbury and White (1954) demonstrated that prune- t i n (5,4 1 -dihydroxy-7-methoxyisoflavone) was estrogenic when in jec ted i n mice. The i sof lavone pratensein ( 5 , 7 , 3 ' - t r i h y d r o x y - 4 1 - m e t h o x y i s o f l a - vone) i s o l a t e d from c lovers by Wong (1963) was shown to be s l i g h t l y less estrogenic i n mice than biochanin A (Flux et a l . , 1964). Another i sof lavone which has been i s o l a t e d as an important component of soybeans (Nairn et a l . , 1973), namely g l y c i t e i n (7 ,4 ' -d ihydroxy-6-methoxyisof la- vone) , has not been examined for estrogenic a c t i v i t y . Coumestans are b i o g e n e t i c a l l y re l a ted to i sof lavones and have been i s o l a t e d p r i m a r i l y from a l f a l f a and ladino c lover (B ickof f et a l . , 1969). Of the na tura l ly occurr ing coumestans, B ickof f et a l . (1960) reported s i g n i f i c a n t estrogenic a c t i v i t y for coumestrol and le s ser a c t i v i t y for 4'-methoxycoumestrol. L iv ings ton et a l . (1964) demon- s t ra ted that repensol (7,10,12-trihydroxycoumestan) had comparable - 13 - estrogenic a c t i v i t y to coumestrol in mice, while another coumestan, t r i f o l i o l (7,10-dihydroxy-12-methoxycoumestan) was r e l a t i v e l y i n a c t i v e . There are no reports i n the l i t e r a t u r e c i t i n g estrogenic a c t i v i t y of other coumestans i d e n t i f i e d i n a l f a l f a and c l o v e r , namely l u c e r n o l , s a t i v o l , medicagol, 3'-methoxycoumestrol or 7-hydroxy-11,12-dimethoxy- coumestan. A 7-monoglycoside of coumestrol has been i s o l a t e d from a l f a l f a roots and leaves (Olah and Sherwood, 1971), however, i t s estro- genic a c t i v i t y has not been evaluated to date. Presumably because of the very r e a l importance of leguminous forage crops to l i v e s t o c k , and the observed estrogenic e f fec t s of severa l i sof lavones and coumestrol i s o l a t e d from these crops , research into phytoestrogens has focussed p r i m a r i l y on those s p e c i f i c compounds. Other c lasses of f lavonoids appear to be r e l a t i v e l y i n a c t i v e i n compari- son (Verdeal and Ryan, 1979). Research into 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 of phytoestrogens i n legumes has recent ly expanded to i n c l u d e : common vegetables , legumes and grains (Knuckles et a l . , 1976); l e a f prote in concentrates from a l f a l f a (Knuckles et a l . , 1976); and other food products prepared from soybeans (Lookhart et a l . , 1979a; E l d r i d g e , 1982b; Murphy, 1982) or Bengalgram pulses (Dziedz ic and Dick , 1982), a l l for use by humans. Samples of a l f a l f a and soybean products were found to contain markedly higher quant i t i e s of coumestrol than most non-leguminous food plants (Knuckles et a l . , 1976). Murphy (1982) reported a s i g n i f i c a n t carryover of g e n i s t e i n , da idzein and t h e i r respect ive glucosides from soybeans in to processed soy prote in products . The s i g n i f i c a n c e of the l e v e l s of phytoestrogens i n these foods i s not yet understood, and must await - 14 - fur ther studies on how phytoestrogens are metabolized i n man (Verdeal and Ryan, 1979). B. BIOSYNTHESIS AND ACCUMULATION 1. Biosynthes i s The common b iosynthet i c pathway for the basic C 6 - C 3 - C 6 skeleton of a l l f lavonoids involves the condensation of separate r ing intermediates to form a chalcone (Grisebach, 1959). As shown i n Figure 4, the two r ings (A and B) of the chalcone have d i f f e rent o r i g i n s . Ring A i s formed by the condensation of three acetate u n i t s , while r ing B and the three carbon l inkage a r i s e from a conversion of phenylalanine to an ac t ivated cinnamic acid (Hahlbrock and Grisebach, 1975). Studies using l a b e l l e d 1 1 + C have shown that other less e f f e c t i v e precursors of r ing B can include tyros ine and p-coumaric ac id derived from shik imic a c i d . Ribereau-Gayon (1972) reviewed the common synthesis of these aromatic compounds from glucose v i a the sh ik imic acid pathway i n p l an t s . Ring c losure of the chalcone leads to the formation of a flavone where r ing B i s pos i t ioned at carbon-1 (Figure 4 ) . An a r y l migrat ion has been shown to occur (Grisebach and Doerr, 1960) at , or a f t e r , the chalcone stage where r ing B migrates from C-1 to C-2 for synthesis of an i so f l avone . In the formation of an i sof lavone g lycos ide from the corresponding aglycone, the sugar moiety i s t rans ferred v i a a g l y c o s y l t ransferase (Hahlbrock and Grisebach, 1975). Nairn et a l . (1974) es tab l i shed that the g l y c o s i d i c i sof lavones i n soybeans were 7-0-glucos ides . G lycosy l a - t i o n serves to convert the f l avonoid into a less reac t ive and more water so luble form, for storage i n the c e l l vacuole (Markham, 1982). Figure Proposed biosynthetic pathway of isoflavones via a chalcone . Adapted from Hahlbrock and Grisebach (1975). - 16 - Grisebach and Barz (1963a; 1963b) further demonstrated that the biosynthes i s of coumestrol was analogous to that of the i so f l avones , ra ther than to that of the coumarins. Coumestans as a group represent the highest ox idat ion l e v e l poss ib le for the i so f l avono id skeleton (Wong, 1975). The i sof lavone da idze in was shown to be a w e l l - incorporated precursor for coumestrol , whereas formononetin with i t s methyl group in tac t was a more e f f i c i e n t precursor of V-methoxycoumes- t r o l (Dewick and Mar t in , 1979a). The authors proposed that methylation could occur during the a r y l migration step associated with i so f l avono id b iosynthes i s ; a 4 '-methoxyisoflavone would then be more r e a d i l y converted to a 4'-methoxycoumestan. 2. Ef fect s of m i c r o b i a l i n f e c t i o n Hanson and coworkers (1965) extens ive ly reviewed the v a r i a t i o n i n coumestrol content of a l f a l f a and reported that coumestrol content was inf luenced by stage of growth, va r i e ty and l o c a t i o n . Other s tudies reviewed by Bickof f et a l . (1969) and L iv ings ton (1978) noted increased l e v e l s of coumestrol as a l f a l f a and c lover hays matured, and v a r i a t i o n s i n coumestrol content depending on the geographical l o c a t i o n where a l f a l f a was grown. These var i ab le s a f f ec t ing coumestrol accumulation were observed to be u l t imate ly re la ted to disease incidence i n the plants (Hanson et a l . , 1965). Furthermore, 99 percent of the v a r i a t i o n i n coumestrol content was determined to be non-genet ic . Advancing maturity i n plant growth i s commonly associated with increased disease inc idence ; d i f ferences i n coumestrol content among v a r i e t i e s may be due to v a r i e t a l d i f ferences i n - 17 - re s i s tance to f o l i a r pathogens, and c e r t a i n geographical areas are often associated with le s s f o l i a r disease (Bickof f et a l . , 1969). B ickof f et a l . (1967) studied the ef fect of the common leafspot organism Pseudopeziza medicaginis on coumestan and flavone content of two a l f a l f a v a r i e t i e s . The workers found that there was a s i g n i f i c a n t increase i n coumestrol over a H day growth period which cor re l a ted with the v i s i b l e extent of plant i n f e c t i o n . Several other coumestans ( s a t i v o l , 3'-methoxycoumestrol, medicagol and 4'-methoxycoumestrol) as wel l as 7 ,4 ' -d ihydroxyf lavone were a l so shown to accumulate i n the infected p l an t s . Loper et a l . (1967) found that coumestrol l e v e l s i n healthy a l f a l f a t i s sue averaged less than 3 ppm whereas diseased l e a f l e t areas could contain 2,600 ppm coumestrol . However, Franc i s and M i l l i n g t o n (1965a) reported an increase i n coumestrol i n maturing medic species even i n the absence of fungal pathogens. Stuthman et a l . (1966) examined several species of a l f a l f a inc lud ing Medicago sa t iva L . which contained l e v e l s of coumestrol up to 26 ppm but had no obvious fungal pathogens. In contrast to Hanson et a l . (1965), these workers suggested that genetic v a r i a t i o n was impl icated i n the coumestrol concentrat ions among a l f a l f a c u l t i v a r s . Se l ec t ion of a l f a l f a c u l t i v a r s for re s i s tance to the common leafspot organism by Loper et a l . (1967) resul ted i n s i g n i f i c a n t l y lowered coumestrol contents . Coumestrol was shown to accumulate i n a l f a l f a i n d i r e c t response to i n f e c t i o n by several pathogenic fungi (Sherwood et a l . , 1970). These workers found that coumestrol was not t rans located from infected areas to other parts of the p l ant , which indica ted that coumestrol synthesis - 18 - could be c a r r i e d out independently at the s i t e of i n f e c t i o n . Injury of a l f a l f a leaves simply by mechanical means did not re su l t i n coumestrol accumulation. Increased coumestan and flavone l e v e l s found i n diseased white c lover (Wong and Latch , 1971; Saba et a l . , 1974) provided further support for the hypothesis of pathogen-induced f lavonoid accumulation. Germination of legumes has been shown to cause a marked increase i n coumestrol compared to the ungerminated bean or seed (Knuckles et a l . , 1976; Lookhart et a l . , 1979b; Murphy, 1982). Lookhart et a l . (1979a) associated the highest coumestrol concentrat ion with those soybean sprouts most not iceably i n f e c t e d , but could not i d e n t i f y the i n f e c t i n g organisms. The authors also noted a marked reduct ion i n coumestrol concentrat ion when soybean sprouts were r insed p e r i o d i c a l l y with d i s t i l l e d water (4 times da i ly ) during germination as compared to unrinsed c o n t r o l s . It was suggested that the r i n s i n g served to cleanse the beans, thus l i m i t i n g i n f e c t i o n . Pathogen-induced accumulation of the estrogenic i sof lavones i n legumes has also been observed by numerous workers (Olah and Sherwood, 1971 and 1973; Saba et a l . , 1974; Biggs, 1975). E a r l i e r s tudies report- ed wide v a r i e t a l d i f ferences in i sof lavone contents of forages, and large v a r i a t i o n s i n i sof lavone patterns between c u l t i v a r s grown i n separate geographical regions (Beck, 1964; Franc i s and M i l l i n g t o n , 1965b; Sachse, 1974). Again, the r e l a t i o n s h i p between disease and f lavonoid accumulation, although i t had not yet been recognized for i sof lavones as i t had for coumestans, may have played a r o l e . - 19 - Plants have been shown to accumulate a n t i m i c r o b i a l compounds, known as phytoalexins , i n response to i n f e c t i o n by pathogenic fungi and bacter ia (Hare, 1966; Kuc, 1972; Pueppke and Van E t t e n , 1974). Many phytoalexins are f l avonoids , inc lud ing those produced by plants of the Leguminosae family (McClure, 1975). The most abundant c lasses of i so- f lavonoid phytoalexins produced by leguminous plants are the ptero- carpans and 2 ' -hydroxyisof lavans (Dewick and Mar t in , 1979b). As i n d i - cated i n F igure 5, the i so f lavans represent the most reduced group of i s o f l a v o n o i d s , probably derived from the pterocarpans by reduct ive r ing opening (Wong, 1975). The observation that coumestans and i sof lavones accumulate i n l e a f t i s sue in response to mic rob ia l i n f e c t i o n i s presumably re la ted to the m i c r o b i a l l y induced accumulation of phytoalexins . The d i f f i c u l t y i n studying i so f l avono id b iosynthes i s i n m i c r o b i a l l y in fec ted p l an t s , however, i s i n separating the metabolic processes of the plant from those of the i n f e c t i n g organism (Dewick and Mar t in , 1979b). Sherwood et a l . (1970) extens ively studied the e f fect of fungal disease and other s tress on coumestrol accumulation i n a l f a l f a . These workers demonstrated that fungi do not independently synthesize f lavon- oid compounds, but l i k e l y produce enzymes or metabolites that enter the infected plant t i s sue and cause coumestrol and other f lavonoid aglycones to be re leased . Olah and Sherwood (1971) were only able to i s o l a t e f lavonoid glycosides from healthy a l f a l f a plant t i s sue , but during the development of fungal i n f e c t i o n , f lavonoid aglycones were detected i n increas ing concentrat ions . I t was suggested that i n f e c t i o n could have impaired F i g u r e 5. Possible biogenetic relationships among i s o f l a v o n o i d s . 1 A d a p t e d from Wong (1975). - 21 - g l y c o s y l a t i o n at the f i n a l stages of f lavonoid b iosynthes i s or more l i k e l y , that the i n f e c t i n g organism v ia a g lycos idase enzyme caused re lease of aglycones from g lycos ides . In a subsequent study Olah and Sherwood (1973) found that 3-glucosidase a c t i v i t y , probably of fungal o r i g i n , increased during i n f e c t i o n . In the p l a n t ' s disease res i s tance mechanism the aglycones released by fungal g lycos idase a c t i v i t y may i n turn i n h i b i t further fungal growth (McClure, 1975). A d d i t i o n a l support for the g lycosidase theory was provided when the concentrat ions of f lavonoid g lycos ides did not decrease during the i n f e c t i o n but rather remained the same or increased (Olah and Sherwood, 1971). Moreover, the increase i n enzyme a c t i v i t y had been shown to precede the appearance of the aglycones (Olah, 1970) 3 , implying that these two events were c l o s e l y r e l a t e d . The increased l e v e l s of both g lycos ides and aglycones suggests that normal pathways of f lavonoid g lycos ide synthesis are st imulated during fungal i n f e c t i o n (Olah and Sherwood, 1973). 3. Fungi tox ic a c t i v i t y Lyon and Wood (1975) demonstrated that coumestrol accumulated i n bean leaves infected with Pseudomonas spp. and that the coumestrol acted l i k e a phytoalexin by i n h i b i t i n g the growth of these b a c t e r i a l pathogens. Unl ike phytoa lex ins , coumestans do not appear to have fung i tox ic a c t i v i t y . Screening te s t s have been performed with coumestrol , 4'-methoxycoumestrol, medicagol and t r i f o l i o l for myce l i a l 3 As c i t e d by Olah and Sherwood (1971). - 22 - growth i n h i b i t i o n of numerous pathogenic fungi , with negative r e s u l t s (Bickof f et a l . , 1969; P e r r i n and Cruickshank, 1969). The fungal-induced accumulation of coumestans may then simply r e f l e c t by-product or precursor formation for the production of an t i - funga l phytoalexins v i a a common b iosynthet ic pathway (Wong and Latch , 1971; Olah and Sherwood, 1973; McClure, 1975; Dewick and Mar t in , 1979a). In contras t , Nairn et a l . (1974) demonstrated that the i sof lavones da idze in , gen i s te in and g l y c i t e i n exert pronounced f u n g i s t a t i c a c t i v - i t y . The corresponding methylated i sof lavones showed a s i g n i f i c a n t l y higher f u n g i s t a t i c a c t i v i t y , whereas the 7-0-glucosides had only l i m i t e d a c t i v i t y . These f indings suggest that the more l i p o p h i l i c compounds (methylated>hydroxylated>glycosylated) would pass more e a s i l y through the cytoplasmic membranes of the fungus (Nairn et a l . , 1974). Thus the accumulation of these fung i tox ic i sof lavones i n fungal ly infected plants may have a greater ro le to play in pathogen res i s tance than do coumestans. A better understanding of the metabolic pathways involved i n i so f l avono id b iosynthes i s , both i n healthy and diseased p lant s , w i l l be needed before the mechanism of phytoalexin induct ion can be explained (Wong, 1975). 4 . E f f ec t s of l i g h t In the studies by Hanson et a l . (1965) no c o r r e l a t i o n could be found between l i g h t i n t e n s i t y and coumestrol content of a l f a l f a i n f i e l d t e s t s . The majority of reported research on the ef fect of l i g h t on f l avonoid accumulation i n plants has pertained to anthocyanins, and i s - 23 - reviewed by McClure (1975). The observed e f fects were reported to vary according to the plant and anthocyanins under study and no genera l i za - t ions were made regarding the ro le of l i g h t i n f lavonoid formation. Ross i ter and Beck (1967) found that l i g h t was not e s sen t i a l for es trogenic i sof lavone formation in c l o v e r . Subs tant i a l concentrat ions of formononetin, gen i s te in and biochanin A were detected i n leaves of p lants grown i n dayl ight and a lso for those grown i n complete darkness. Some s t imula t ion of i sof lavone synthesis was a t t r i b u t e d to high in ten- s i t y l i g h t , however, the authors concluded that l i g h t environment d id not markedly a f fec t i sof lavone l e v e l s i n c l o v e r . However, l i g h t can inf luence germination, e i ther v ia s t imula t ion or i n h i b i t i o n dependng on the l i g h t requirements of p a r t i c u l a r seeds (Mayer and Pol jakoff-Mayber, 1975). The d i f f e r e n t spec t ra l ranges of l i g h t can a l so have qui te d i f f e r e n t e f fect s on germination, as demon- s trated by i n h i b i t i o n with l i g h t below 290 nm and promotion with l i g h t i n the v i s i b l e range 400-700 nm, e s p e c i a l l y with red l i g h t (Mayer and Pol jakoff-Mayber , 1975). C. METABOLISM Although a b io synthet i c r e l a t i o n s h i p ex i s t s between coumestans and i so f lavones , the metabolic pathways of these two groups of f lavonoids appear to be d i f f e r e n t (Bickof f et a l . , 1969). There are a l so d i f f e r - ences among animal species i n t h e i r metabolism of estrogenic i sof lavones which can profoundly a f fec t the estrogenic potency of p a r t i c u l a r i s o f l a - vones (Lindner , 1967; Shutt , 1976). - 24 - As mentioned prev ious ly , ear ly studies to evaluate e s t rogen ic i ty of plant compounds were p r imar i ly c a r r i e d out using rats and mice as the t e s t animal species . Both JLn vivo uter ine weight assays (Bradbury and White, 1954; B ickof f et a l . , 1962) and i n v i t r o cytoso l binding s tudies (Verdeal et a l . , 1980) indica ted that i n rats coumestrol was the most potent phytoestrogen, followed by g e n i s t e i n , d a i d z e i n , biochanin A and formononetin i n order of decreasing potency. 1. Isoflavones Both gen i s te in and formononetin were i so l a t ed from the subterran- ean c lover (Bradbury and White, 1954) which had been impl icated i n i n f e r t i l i t y of " c l o v e r disease" i n grazing sheep (Bennetts et a l . , 1946). Since formononetin d id not demonstrate s i g n i f i c a n t estrogenic a c t i v i t y i n rats or mice, i t was thought that gen i s te in was the major factor responsible for c lover disease (Biggers and Curnow, 1954). M i l l i n g t o n et a l . (1964), however, cor re l a ted the estrogenic a c t i v i t y of subterranean c lover s t r a i n s with the amount of formononetin in the c l o v e r , not with i t s gen i s te in content . The f inding that formon- onet in was a more potent estrogen than g e n i s t e i n , was shown to be due to a d i f f e r e n t metabolic pathway for formononetin i n sheep (Shutt and Braden, 1968). A comparison of c i r c u l a t i n g i sof lavones i n sheep plasma to the i so f lavone composition of c lover ingested, indica ted that gen i s te in and biochanin A were degraded more r a p i d l y than formononetin and da idze in i n sheep (Shutt et a l . 1967; Shutt and Braden, 1968). The r e s u l t s a lso suggested that formononetin and biochanin A were demethylated to Figure 6. Metabolism of some estrogenic isoflavones in sheep. 1 + P H E N O L I C A C I D ^rom Shutt (1976). - 26 - da idze in and gen i s te in r e s p e c t i v e l y , a f ind ing supported by Lindner (1967) and Braden et a l . (1967). The metabolism of estrogenic i sof lavones i n sheep, as ind ica ted i n F igure 6, occurs p r i m a r i l y i n the rumen (Batterham et a l . , 1965; L indner , 1967). Formononetin undergoes O-demethylation at the C-4' p o s i t i o n to y i e l d da idzein which i n turn i s reduced to the i s o f l a v a n , equol . Biochanin A undergoes demethylation to y i e l d gen i s te in which i s further degraded to p-ethylphenol (from r ing B) and a phenol ic ac id (from r ing A ) , both e s t rogen ica l ly i n a c t i v e metabolites (Braden et a l . , 1967; Shutt , 1976). Equol , i n contra s t , has been reported to have an estrogenic a c t i v - i t y equal to that of gen i s te in i n mice, and also e l i c i t s a s i g n i f i c a n t estrogenic response i n sheep (Shutt and Braden, 1968). Equol has been detected as a metabolite i n the urine and plasma of sheep (Batterham et a l . , 1965; Shutt et a l . , 1967; Braden et a l . , 1971), c a t t l e (Braden et a l . , 1971), guinea pigs (Shutt and Braden, 1968) and domestic fowl (Tang and Common, 1968) i n d i c a t i n g that formononetin i s converted to equol i n a l l these spec ies . Equol does not appear to undergo further metabolism i n the rumen (Shutt et a l . , 1970). I t has been observed in sheep that the metabolism of biochanin A and gen i s te in to i n a c t i v e phenols becomes more e f f i c i e n t over an "adap- t i v e " period of about f ive days (Shutt , 1976). In cows t h i s adaptive period appears to be shorter as these i sof lavones are r a p i d l y degraded, conjugated and excreted (Braden et a l . , 1971). In the guinea p i g , degradation of biochanin A and gen i s te in i s not as e f f i c i e n t as evidenced by high plasma concentrat ions of both i sof lavones a f ter - 27 - consumption. As a r e s u l t biochanin A and gen i s te in have greater estro- genic a c t i v i t y i n the guinea pig than i n ruminants. Demethylation of biochanin A to gen i s te in occurs a lso i n domestic fowl (Tang and Common, 1968). These workers further reported that traces of equol were found as a metabolite of biochanin A i n fowl, however, t h i s conversion has not been reported i n other animal species (Cox and Braden, 1974). Although b a c t e r i a l f l o r a i n the rumen are bel ieved to be p r i m a r i l y responsible for O-demethylation of i sof lavones (Shutt et a l . , 1970), Lindner (1967) demonstrated that some biochanin A and formononetin could be demethylated i n ovine t i s sues fo l lowing parentera l i n j e c t i o n of these i so f l avones . G r i f f i t h s (1975) showed that i n v i t r o incubat ion of estro- genic i sof lavones with i n t e s t i n a l micro f lora of the rat gave r i s e to p-ethylphenol and equol from gen i s t e in and da idze in r e s p e c t i v e l y . Further experiments with germ-free ra t s demonstrated that o r a l l y ingested f lavonoids do not undergo r ing f i s s i o n i n the absence of normal i n t e s t i n a l micro f lora ( G r i f f i t h s , 1975). 2. Coumestans Metabolism of coumestrol and some re la ted coumestans has been studied i n sheep (Braden et a l . , 1967; Shutt et a l . , 1969; K e l l y , 1972) and domestic fowl (Cayen and Common, 1965). Two methylated and estro- g e n i c a l l y i n a c t i v e coumestans, 4'-methoxycoumestrol and t r i f o l i o l , appear to be demethylated i n sheep i n a manner s i m i l a r to the methylated i sof lavones to give r i s e to e s t r o g e n i c a l l y a c t i v e coumestrol and repensol r e spec t ive ly (Shutt et a l . , 1969; Saba et a l . , 1974). The greater estrogenic a c t i v i t y of coumestrol compared to the i so f lavones - 28 - (B ickof f et a l . , 1962) may be due to slower metabolism and excret ion of coumestrol (Shutt et a l . , 1969; K e l l y , 1972; K e l l y and Lindsay, 1978). Breakdown products of coumestrol metabolism do not appear to include equol (Cayen and Common, 1965) or previous ly i d e n t i f i e d i sof lavone metabolites (Shutt, 1976). K e l l y and Lindsay (1978) demonstrated that sheep appear to develop an i n s e n s i t i v i t y to coumestrol a f ter prolonged exposure to t h i s phytoestrogen, which i s not s i m i l a r to the mechanism or adaptive period reported for biochanin A and gen i s te in metabolism (Shutt , 1976). 3. Absorption The r e l a t i v e es trogenci ty of formononetin and gen i s te in i n sheep and poss ib ly a lso cows i s then opposite to the s i t u a t i o n i n rats and mice (Verdeal and Ryan, 1979). The most potent phytoestrogen i s s t i l l coumestrol , followed by da idze in , formononetin, gen i s te in and biochanin A, i n order of decreasing potency. Phytoestrogens and t h e i r metabolites appear to be absorbed from the rumen (Shutt et a l . , 1970; Cox and Braden, 1974) or the upper part of the gut (Lindner, 1967). The major por t ion of absorbed estrogenic compounds are conjugated, probably i n the l i v e r , to form glucuronides (Lindner , 1967; Shutt et a l . , 1967; Shutt et a l . , 1970). Conjugation serves to i n a c t i v a t e the c i r c u l a t i n g compounds which are then excreted, usua l ly v i a the ur ine (Cox and Braden, 1974; Harper, 1975). Only the very small port ion of the c i r c u l a t i n g estrogens which remain i n a " f r ee " or unconjugated form represent the b i o l o g i c a l l y ac t ive forms (Lindner , 1967, Shutt et a l . , 1969). Factors which a f fec t - 29 - the l i v e r ' s a b i l i t y to detoxify the absorbed i so f l avono id s , such as ma lnut r i t ion or l i v e r tox ins , may re su l t in marked increases i n plasma l e v e l s of free estrogens (L inder , 1967). Although phytoestrogens show low binding a f f i n i t i e s for estrogen receptors i n comparison to endogenous estrogens, they can exert s i g n i f i - cant estrogenic e f fects on animals when t h e i r concentrat ion i n blood plasma i s maintained at high l e v e l s (Lindner , 1967; Shutt and Cox, 1972; Shutt , 1976). Very l i t t l e i s known of the metabolism and normal c i r c u - l a t i n g l e v e l s of phytoestrogens i n humans (Martin et a l . , 1978). Without t h i s knowledge i t i s d i f f i c u l t to assess the s i g n i f i c a n c e of long term d ie tary exposure to phytoestrogens i n man (Verdeal and Ryan, 1979). D- EXTRACTION OF PHYTOESTROGENS Bicko f f et a l . (1962) and others (Cheng et a l . , 1955; P ie ter se and Andrews, 1956; Lyman et a l . , 1959; Wada, 1963; Saba et a l . , 1974) i n i t i a l l y es tabl i shed the e s t rogen ic i ty of c e r t a i n plants by feeding the ent i re plant or crude plant extracts to test animals . However, i n order to e s t ab l i sh which compounds contr ibuted to the t o t a l estrogenic a c t i v - i t y and at what l e v e l s they occurred i n p lant s , more s p e c i f i c ex t rac t ion and detect ion methods had to be developed. Ear ly i s o l a t i o n methods usual ly involved a se r ie s of solvent ext rac t ions such as described by L iv ings ton et a l . (1961) for detect ing coumestrol i n a l f a l f a , and Wong (1962) for detect ing phytoestrogens i n c l o v e r . Fresh or rehydrated plant mater ia l was macerated and soaked i n 95% ethanol and f i l t e r e d to give a crude ex t rac t . Petroleum ether was commonly employed to remove waxes and l i p i d mater ia l and the 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 purifi- cation (Bickoff et al., 1969; Wong and Latch, 1971; Saba et al., 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. Flavonoids commonly occur as glycosides in plants in general (Markham, 1982), and as noted, in legumes (Beck, 1964; Olah and Sherwood, 1971; Nairn et al., 1974), so only the limited proportion of aglycone flavonoids would be extracted. Hydrolysis of the glycosides was effected by crushing clover leaves prior to ethanolic extraction, indicating that a hydrolytic enzyme was present in the leaves (Beck, 1964). The enzyme was inacti- vated by placing intact leaves in boiling water or ethanol, and was not found in clover petioles (Figure 7). Olah and Sherwood (1973) demon- strated B-glucosidase activity in healthy alfalfa leaves and roots, which they noted could be involved along with other glucosidases in the hydrolysis of flavonoids. Francis and Millington (1965b) compared treatments of clover leaves prior to ethanolic extraction and obtained maximum isoflavone yields i f leaves were crushed and allowed to stand a maximum of 10 minutes before adding ethanol. Glencross et al. (1972) reported a two-fold increase in formononetin content when clover leaves were crushed in water before extraction with ethanol, and concluded that endogenous enzymic hydrolysis of the isoflavone glycoside had occurred. - 31 - Figure 7. Germination and development of a l f a l f a seed to sprout. COTYLEDON EPICOTYL PETIOLE 0 SEED COTYLEDONS YPOCOTYL ESTA HYPOCOTYL RADICLE - 32 - Sherwood et a l . (1970) did not, however, detect increased l e v e l s of coumestrol when a l f a l f a leaves were ground before e x t r a c t i o n . Studies of the phytoestrogens i n soybean products have genera l ly not employed hydro ly s i s to l i b e r a t e f lavonoid aglycones. Ohta et a l . (1979) i so l a ted da idze in , gen i s te in and t h e i r g lucos ides , da idz in and g e n i s t i n , by e thanol ic ex t rac t ion of soy f lour but did not determine whether production of the i n i t i a l soy f lour product affected the i s o f l a - vonoid p r o f i l e . A s i m i l a r ana lys i s by E ldr idge (1982a) of soybean meal a l so did not address t h i s matter, although i n a subsequent paper the author proposed that the decrease i n i sof lavone g lycos ides i n soy pro te in i s o l a t e s arose from water so luble losses during production (E ldr idge , 1982b). Murphy (1982) attempted to expla in the d i f f e r e n t i so f l avono id p r o f i l e s of soybean products i n terms of processing e f fects such as water so lva t ion and o x i d a t i o n . In contra s t , Glencross et a l . (1972) noted that the preparat ion of a c lover l ea f prote in concentrate involved f i r s t pulping the leaves to express j u i c e , at which point enzymic hydro lys i s would have occurred . Subsequent ana lys i s of the prote in concentrate for i sof lavone aglycones would therefore not require a d d i t i o n a l h y d r o l y s i s . N i c o l l i e r and Thompson (1982) reported that some hydro lys i s may have occurred during ex t rac t ion of i sof lavones from i n t a c t c lover to account for the high l e v e l s of aglycones detected. Hydro lys i s of f lavonoid g lycos ides can be c a r r i e d out under c o n t r o l l e d condi t ions employing e i ther a c i d i c , enzymic or a l k a l i n e treatments, as reviewed by Markham (1982). The choice of treatment depends p r i m a r i l y on the nature of the g lycos ide to be hydrolyzed. As - 33 - mentioned prev ious ly , the g lycos ides of the estrogenic i sof lavones and of coumestrol i s o l a t e d to date have been i d e n t i f i e d as 7-0-monoglyco- s ides . According to Markham (1982) such a l inkage would be hydrolyzed completely in 15 to 40 minutes under standard ac id hydro ly s i s condi- t i o n s , namely heating at 1 0 0 ° C with 2N HC1:95% EtOH (1 :1 , v/v) (Harborne, 1965). Sachse (1974) found that hydro lys i s of c lover samples with 1N HC1 i n a hot water bath for 1 hour was adequate to achieve t o t a l l e v e l s of the estrogenic i sof lavones and coumestrol as aglycones. N i c o l l i e r and Thompson (1982) obtained f u l l hydro lys i s of i sof lavone g lycos ides i n in tac t c lover leaves using condi t ions of 4N HC1 for 2 hours at 7 0 ° C . While e a r l i e r research reports were concerned with i d e n t i f i c a t i o n and a c t i v i t y of estrogenic i sof lavones and coumestans i n p lant s , much of the recent l i t e r a t u r e has emphasized quant i t a t ive determinations of phytoestrogens i n feeds and foods (Nairn et a l . , 1974; Knuckles et a l . , 1976; Lookhart, 1979; Murphy, 1981; E l d r i d g e , 1982a). Several methods for the ex t rac t ion of phytoestrogens have thus been developed to obtain maximum recovery from a p a r t i c u l a r plant product (Knuckles et a l . , 1976; Lookhart , 1979; Murphy, 1981; E l d r i d g e , 1982b). Lookhart (1979) examined soybeans for coumestrol content only and reported a maximum of 64 percent recovery from a coumestrol spiked soybean system. The author found enhanced ext rac t ion e f f i c i e n c y of coumestrol when soybeans were defatted p r i o r to a ser ie s of methanol/ water and e t h y l ether ex t r ac t ions . Murphy (1981) reported very low ext rac t ion e f f i c i e n c i e s with the Lookhart procedure when appl ied to phytoestrogens other than coumestrol . A 2 hour mechanical a g i t a t i o n procedure was used instead by Murphy (1981) to extract defatted soy - 34 - f l ake s , and l a t e r for d r i e d , defatted soybeans and other soybean products (Murphy, 1982). A solvent system of a c e t o n i t r i l e r O . 1 N HC1 (25:5, v/v) was reported to y i e l d maximum recovery and minimum coextrac- t i v e s . Recoveries were measured for standards of g e n i s t i n (91% recov- e r y ) , gen i s te in (86%), da idzein (57%) and coumestrol (55%) added to soy f l akes . E ld r idge (1982b) reported maximum ex t rac t ion of d a i d z e i n , g e n i s t e i n , g l y c i t e i n and t h e i r corresponding glucosides from soybean products by re f lux ing with 80 percent methanol for 4 hours. Knuckles et a l . (1975) developed a procedure for the quant i t a t ive determination of coumestrol i n a l f a l f a which was a lso used to evaluate the coumestrol content of soybeans and soybean products (Knuckles et a l . , 1976). B r i e f l y , dr ied samples were rehydrated, then soaked i n 95 percent ethanol ; c h l o r o p h y l l was removed with chloroform af ter adjust ing the a l coho l extract to pH 10, and e thyl ether was used to f i n a l l y i s o l a t e the coumestrol . Recovery of added coumestrol was reported to be 98 percent, however, the standard was added jus t p r i o r to the chloro- p h y l l removal stage, and thus was not c a r r i e d through the e n t i r e extrac- t ion procedure. Quanti tat ive recover ies for estrogenic i sof lavones from a l f a l f a have not been reported . Sachse (1974) demonstrated several r e s t r i c t i o n s imposed by i s o f l a - vones on the development of su i t ab le ex t rac t ion techniques for phyto- estrogens. Isoflavones i n general were found suscept ib le to a l k a l i h y d r o l y s i s , even under mild cond i t ions , as supported by Wong (1975) and Markham (1982). Formononetin was found to be so luble i n chloroform, although t h i s solvent was reported (Bickof f et a l . , 1969) to be more e f f i c i e n t than petroleum ether for the removal of waxes and l i p i d - 35 - mater ia l from plant ex t rac t s . The low p o l a r i t y of i sof lavone aglycones renders these compounds more soluble i n solvents such as e thyl ether and chloroform, and only moderately soluble i n ethanol , methanol and acetone (Markham, 1982). E. DETECTION TECHNIQUES The simplest detect ion methods reported in the l i t e r a t u r e were based on observations of estrogenic s t imula t ion i n animals fo l lowing consumption of s p e c i f i c feeds (Bennetts et a l . , 1946; P ie ter se and Andrews, 1956; Wada, 1963). However, l i t t l e information about the nature or quant i t i e s of those compounds involved i n e s t r o g e n i c i t y was revealed i n such s tud ies . Since the i n i t i a l i d e n t i f i c a t i o n of some estrogenic components as i sof lavones (Bradbury and White, 1951; Cheng et a l . , 1955) and coumes- tans (Bickof f et a l . , 1957), a va r i e ty of chromatographic techniques have been developed for the ana lys i s of the estrogenic f l avonoids . Paper and t h i n - l a y e r chromatography predominated phytoestrogen research l i t e r a t u r e up u n t i l the l a s t several years when high-performance l i q u i d chromatography (HPLC) emerged as a su i t ab le quant i t a t ive technique for f lavonoid ana lys i s (eg. Wulf and Nagel , 1976; Vande Casteele et a l . , 1982). 1. Paper chromatography The technique of paper chromatography (PC) i s s t i l l ex tens ive ly used for f lavonoid a n a l y s i s , l a r g e l y because i t produces acceptable separat ions , even of large amounts of f l avonoids , at a low cost (Markham, 1975). - 36 - Paper chromatography was e a s i l y used to detect coumestrol i n legume plants because of the c h a r a c t e r i s t i c blue f luorescence of t h i s compound when exposed to UV l i g h t (Lyman et a l . , 1959). Wong (1962) found that as l i t t l e as 0.1 ug coumestrol could be detected under UV l i g h t . L iv ings ton et a l . (1961) further improved a paper chromatogra- phic system for quant i t a t ive ana lys i s of coumestrol ( in fresh and dr ied a l f a l f a ) that was s e n s i t i v e to 2 ppm coumestrol . The chromatogram was developed i n ace t i c ac id/water /HCl (50:35:15, v / v ) . Comparisons of f luorescence readings between standard and plant extract spots on deve- loped chromatograms were used to determine coumestrol concentra t ions . Chury (1967) compared ten developing systems for the PC separation of coumestrol and reported good r e s o l u t i o n with the L iv ings ton system mentioned. Numerous workers made use of the f luorometr ic PC method of L iv ings ton et a l . (1961) for measuring coumestrol content of a l f a l f a i n f o l i a r disease s tudies (Stuthman et a l . , 1966; B ickof f et a l . , 1967; Loper et a l . , 1967; Sherwood et a l . , 1970). Loper et a l . (1967) further adapted the method to two-dimensional paper chromatography for separa- t i o n and detect ion of other coumestans and flavones i n a l f a l f a , and reported the c h a r a c t e r i s t i c f luorescence i n t e n s i t i e s of these compounds r e l a t i v e to coumestrol . Daidzein and formononetin were also detected as blue v i o l e t f l u o r - escent spots under UV l i g h t (long wave, 365 nm) using PC (Guggolz et a l . , 1961). Geni s te in and biochanin A could be v i s u a l i z e d as brown spots a f ter spraying developed chromatograms with b i s - d i a z o t i z e d benzid ine . - 37 - Wong (1962) developed a two-dimensional paper chromatographic method for the separat ion and measurement of phytoestrogens in c l o v e r . The benzene/acetic acid/water (125:72:3, v/v) system used for the f i r s t dimension was reported to move c h l o r o p h y l l and l i p i d s near the solvent f r o n t . The second dimension system was 2N aqueous ammonia, but aside from noting p a r t i a l degradation of biochanin A, the author d id not detect i n s t a b i l i t y of i so f lavonoids i n t h i s a l k a l i n e system. The com- pounds of i n t e r e s t were wel l separated on the f i n a l chromatogram; the spray reagent d i azo t i zed s u l p h a n i l i c ac id was found to be most su i t ab le for v i s u a l i z i n g the non-fluorescent dark spots corresponding to genis- t e i n and biochanin A. Color react ions of the i sof lavones with severa l other spray reagents are summarized by Wong (1962). In the same study, i sof lavone spots were e luted from the chromatogram and concentrat ions were obtained from the absorbance maxima using a UV spectrophotometer. Subsequent s tudies by Wong and Latch (1971) were c a r r i e d out using 30% or 60% ace t i c ac id for the second dimension solvent system rather than the a l k a l i n e system previous ly reported (Wong, 1962). A mixture of twelve f lavonoids was separated for i d e n t i f i c a t i o n based on UV spectra and chromatographic propert ies upon comparison with authentic samples. Knuckles et a l . (1975) reported that the removal of c h l o r o p h y l l from a l f a l f a extracts p r i o r to chromatography was necessary to prevent the pigment from enhancing the f luorescence of coumestrol or i n t e r f e r i n g with the separat ion of coumestrol from other f luore sc ing compounds. These workers chose a simple 50% acet ic acid system as the developing solvent because hydrochlor ic ac id was found to r e s u l t i n f r a g i l e - 38 - chromatograms with high background f luorescence . Fluorescence measurements were c a r r i e d out as per L iv ings ton et a l . (1961). 2. Th in- l ayer chromatography The use of t h i n - l a y e r chromatography (TLC) for the ana lys i s of f lavonoids has been reviewed by Markham (1975). Beginning in the mid 1960's the development of t h i n - l a y e r methods for rapid a n a l y t i c a l and small scale chromatography began to replace paper chromatography for the separat ion of estrogenic i s o f l a v o n o i d s . Beck (1964) tested 60 solvents and solvent mixtures to resolve the es trogenic i sof lavones i s o l a t e d from c l o v e r . Chloroform/methanol (89:11, v/v) was found to be the only solvent system to give a s a t i s f a c - tory separation on s i l i c a gel p l a te s . Formononetin and da idzein could be v i s u a l i z e d as blue-white f luorescent spots under UV l i g h t (257 nm). Genis te in and biochanin A were made v i s i b l e as orange-brown spots by spraying with d i azo t i zed s u l p h a n i l i c ac id and exposing to ammonia fumes. However, t h i s solvent system could not adequately separate coumestrol from g e n i s t e i n . Nevertheless , numerous workers used t h i s Beck system for semi-quant i ta t ive ana lys i s of i sof lavones i n c lover ( M i l l i n g t o n et a l . , 1964; Franc i s and M i l l i n g t o n , 1965b; Shehata et a l . , 1982), in soybeans (Nairn et a l . , 1974), i n legumes genera l ly (Harborne, 1969), and i n plasma (Braden et a l . , 1967). D i s s a t i s f i e d with i so f l avono id separations on s i l i c a gel or c e l l u l o s e p l a te s , Sachse (1971) developed a system employing petroleum ether/benzene/methylethylketone/methanol/acet ic ac id (30:30:20:20:2, - 39 - v/v) with polyamide TLC p la te s . Using t h i s system he reported better separation of coumestrol as wel l as da idze in , g e n i s t e i n , formononetin, biochanin A and pratensein standards. Increasing the p o l a r i t y of the developing solvent did not improve the separat ion . The same system was l a t e r a l so employed to separate c lover extracts (Sachse, 1974), and was adapted to inc lude development i n a second dimension and f i n a l l y e l u t i o n for quant i t a t ive spectrophotometry a n a l y s i s . However, the procedure was reported to require 3 days from the time of spott ing the extract onto the p late to the f i n a l quant i t a t ive measurement. Lookhart et a l . (1978) reported a r a p i d , quant i t a t ive method for TLC ana lys i s of coumestrol i n soybeans. The system used s i l i c a gel p lates which were developed for only 1 minute i n chloroform/acetone (88:12, v / v ) . Fluorescence i n t e n s i t i e s of extract spots (under UV 365 nm) were v i s u a l l y compared to those of standards to estimate coumestrol concentrat ion i n the range of 50-100 ppm. The p o s s i b i l i t y of other f luorescent compounds i n t e r f e r i n g with the barely developed coumestrol spot was not d i scussed . Many workers reported using TLC p r i m a r i l y as a method for i so - flavone p u r i f i c a t i o n and i d e n t i f i c a t i o n a f ter extracts were i n i t i a l l y separated by column chromatography (Nairn et a l . , 1974; Sachse, 1974; Biggs , 1975; Sharma, 1979). Several chemical and phys ica l treatments used to i d e n t i f y i sof lavones and coumestans on the developed TLC plates are well described by Sachse (1971). 3. Column chromatography/UV spectrophotometry Column chromatography has a lso been used as a separat ion technique followed by UV spectrophotometry studies for i d e n t i f i c a t i o n and often - 40 - quant i t a t ion (Glencross et a l . , 1972). Ohta et a l . (1979) f rac t ionated soybean extracts by s i l i c a gel column chromatography and the i sof lavone f r a c t i o n s were i d e n t i f i e d by UV spectra , i n f r a red spectra and proton magnetic resonance. Nairn et a l . (1974) used a polyamide column to separate soybean i sof lavone g lycos ide s . S i l i c a gel columns are reportedly most useful for the separation of less polar f lavonoids such as i sof lavone aglycones (Markham, 1982) but many a p p l i c a t i o n s using other systems are reviewed by Markham (1975). In UV spectrum s tud ies , the f lavonoid spectrum i s usua l ly deter- mined for a methanol so lu t ion of the f l avono id . Reference spectra of many i sof lavones have been compiled by Horowitz and Jurd (1961). T y p i c a l UV absorpt ion maxima for i sof lavones inc lude a low i n t e n s i t y band I at 310-330 nm and a high i n t e n s i t y band II at 245-275 nm (Markham, 1982). The coumestans genera l ly exh ib i t high i n t e n s i t y UV absorption i n the ranges of 340-355 nm (band I ) , 230-250 nm (band III) and 200-215 (band I V ) , and low i n t e n s i t y absorption in the 300-320 nm range (band II) as evidenced by a shoulder peak (Bickof f et a l . , 1969). Cer ta in reagents such as sodium methoxide and aluminum c h l o r i d e can be added to the flavonoid-methanol so lu t ions which cause character- i s t i c s h i f t s In absorption spec t ra . The use of these " s h i f t reagents" and the i n t e r p r e t a t i o n s of the r e s u l t i n g spectra are summarized by Markham (1982) and have been appl ied to the i d e n t i f i c a t i o n of es trogenic i sof lavones and/or coumestrol i n a number of s tudies (Wong, 1962; B ickof f et a l . , 1969; Glencross et a l . , 1972; Nairn et a l . , 1974; Biggs, 1975; Ohta et a l . , 1979). - 41 - 4 . High-performance l i q u i d chromatography High-performance l i q u i d chromatography (HPLC) i s r e c e i v i n g increased a t t ent ion i n the f i e l d of f lavonoid ana lys i s because i t i s a rapid quant i t a t ive technique which can provide a high l e v e l of re so lu- t i o n and s e n s i t i v i t y (Markham, 1982). The technique and theor ies of HPLC are wel l described in the l i t e r a t u r e (Snyder and K i r k l a n d , 1979; Yost et a l . , 1980). In a d d i t i o n , Kingston (1979) extens ive ly reviewed the l i t e r a t u r e deal ing with a p p l i c a t i o n s of HPLC to natura l plant products . The vast majority of HPLC studies of f lavonoids have been c a r r i e d out using reversed-phase (RP) chromatography systems (Kingston, 1979; Markham, 1982; Andersen and Pedersen, 1983). The columns most commonly used are known as 'reversed-phase C-18' types, which cons i s t of octadecyl hydrocarbon groups chemical ly bound to s i l i c a packing. The non-polar o c t a d e c y l s i l y l groups act as the s ta t ionary phase and are used in conjunction with a more polar mobile phase, for example methanol- water, to e f fect a separation of non-polar to moderately polar compounds (Yost et a l . , 1980). Numerous workers (Wulf and Nagel , 1976; Daigle and Conkerton, 1982; Vande Casteele et a l . , 1982) have studied the mechanisms of separ- a t ion of a large va r i e ty of phenol ic ac ids and f lavonoids using reversed-phase C-18 systems. The e l u t i o n sequence of a given compound was considered to be due to i t s hydrogen bond donating and/or accepting a b i l i t y as wel l as i t s con t r ibu t ion to the hydrophobic i n t e r a c t i o n with the s tat ionary phase. In i sof lavones the strongest hydrogen bond acceptor i s the carbonyl group at C-3; however, an OH group at C-5 w i l l form a strong i n t e r n a l hydrogen bond with t h i s C-3 carbonyl group so - M - that the carbonyl can no longer in te rac t s t rongly with the mobile phase (Vande Casteele et a l . , 1982). As a r e s u l t , the re tent ion times (tp) of aglycone 5-hydroxyisoflavones (such as geni s te in and biochanin A) were found to be longer than those of the corresponding i sof lavones without the 5-OH group (namely daidzein and formononetin). The authors fur ther general ized that a d d i t i o n a l hydroxyl groups reduce tp values whereas methylation of OH groups prevents the ef fect of these groups. G lycosy l a t ion of an OH group introduces a more h y d r o p h i l i c moiety and would also r e su l t i n decreased tp va lues . A d d i t i o n a l HPLC studies by d i f f e rent workers using RP C-18 columns and methanol-water mobile phases genera l ly support the previous f indings i n r e l a t i o n to the estrogenic i so f lavones , t h e i r g lycos ides and coumes- t r o l (Lookhart et a l . , 1978; Ohta et a l . , 1979; Murphy, 1981; Dz iedz ic and Dick, 1982; E l d r i d g e , 1982a; N i c o l l i e r and Thompson, 1982; Patroni et a l . , 1982). Carlson and Dolphin (1980) reported the use of a normal phase s i l i c a column to resolve some i sof lavones i n a soybean e x t r a c t . The solvent system consis ted of 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 a c i d / hexane mixture. A good separat ion of daidzein and gen i s te in was achieved, however, many of the i sof lavone separations reported for RP systems compare favorably to t h i s normal phase system. Vande Casteele et a l . (1982) advised against the use of normal phase columns when deal- ing with plant extracts because other more polar f lavonoids could become i r r e v e r s i b l y adsorbed. Although only methanol-water mixtures were employed i n the afore- mentioned RP work with i so f l avono ids , various modif iers have been added - 43 - to the mobile phase to improve separation c h a r a c t e r i s t i c s i n s tudies concerning other f lavonoids and phenolic a c id s . A c e t i c or formic acids were added to the solvents at l e v e l s of 1 to 5% to suppress i o n i z a t i o n of the ac id groups of the phenolic acids and f lavonoids (Wulf and Nagel , 1976; Vande Casteele et a l . , 1982). This technique, known as ion sup- press ion chromatography, i s used to avoid i o n i c re tent ion mechanisms from taking place on the column, and thus prevents severe peak t a i l i n g of the e lu t ing compounds (Yost et a l . , 1980). The add i t ion of ammonium acetate was reported to prevent intramolecular hydrogen bonding of phenolic hydroxy groups that can cause abnormal polar movement (Murphy and S tu t te , 1978; Hardin and S tu t te , 1980; Proksch et a l . , 1981). At l e v e l s of 0.019 to 0.09 M the ammonium acetate was e s sen t i a l for c l ea r separations among c l o s e l y re la ted phenol ic compounds. The detectors employed i n HPLC ana lys i s of f lavonoids inc lude p r i m a r i l y u l t r a v i o l e t (UV) and a l so f luorescence ( F L ) . As mentioned prev ious ly , the i sof lavone and coumestan phytoestrogens have an absorp- t i o n maxima i n the 254 nm f ixed wavelength regions . The a p p l i c a t i o n of 254 nm f ixed wavelength detectors to i so f l avono id detect ion has thus been useful (Murphy, 1981; Patroni et a l . , 1982; Vande Casteele et a l . , 1982). Other wavelengths of 262 and 280 nm have a lso been employed (Ohta et a l . , 1979; Car lson and Dolphin , 1980; Dz iedz ic and Dick , 1982; E l d r i d g e , 1982a), presumably because s p e c i f i c i sof lavones have peak maxima at these wavelengths. However, the coumestans absorb only weakly at 280 nm, which could i n turn a f fect detect ion l i m i t s . Lookhart et a l . (1978) developed a s e n s i t i v e UV detect ion method s p e c i f i c a l l y for - 44 - coumestrol at 343 nm. The i sof lavones do not exh ib i t s i g n i f i c a n t absorption at t h i s wavelength (Markham, 1982). The f luorescent propert ies of da idze in and coumestrol were explo i ted using f luorescence detectors for i d e n t i f i c a t i o n of these compounds i n plant extracts (Lookhart et a l . , 1978; Murphy, 1982). Lookhart et a l . (1978) reported f luorescence e x c i t a t i o n and emission maxima i n ethanol at 348 and 411 nm r e s p e c t i v e l y , for coumestrol . Murphy (1982) used a f luorescence detector with an e x c i t a t i o n maximum at 360 nm and a secondary f i l t e r (range 460-700 nm). - 45 - NATERIALS AND METHODS A. MATERIALS Daidzein (7 ,4 ' -d ihydroxyi so f lavone) was obtained from ICN Pharma- c e u t i c a l s , I n c . , P la inview, NY. Coumestrol was obtained from Eastman Kodak C o . , Rochester, NY. Formononetin (7-hydroxy-4'-methoxyisoflavone) was k indly provided by Dr. R. 3. Bose, U n i v e r s i t y of B . C . , Vancouver, B. C . Separate stock so lu t ions for each of the above phytoestrogens were prepared to contain 30.0 yg/mL in HPLC grade g l a s s - d i s t i l l e d methanol (F i sher S c i e n t i f i c C o . , Fa i r lawn, NO). Both c u l t i v a r s of a l f a l f a seeds, Moapa and V e r n a l , used i n the germination s tudies were purchased from Richardson Seed Co. L t d . , Burnaby, B . C . B. PHYTOESTROGEN EXTRACTION A method su i t ab le for the ex t rac t ion of se lected phytoestrogens from a l f a l f a was developed. Figure 8 i l l u s t r a t e s the basic scheme followed to extract da idze in , formononetin and coumestrol from a l f a l f a sprouts . A l l solvents used i n the ex t rac t ion were a n a l y t i c a l reagent grade, unless otherwise s p e c i f i e d , and water was d i s t i l l e d and deionized before use. Approximately 20 g of fresh or frozen a l f a l f a sprouts were ground with washed, dr ied sand using a mortar and pe s t l e . The ground sample was allowed to stand for 10 min as per Franc i s and M i l l i n g t o n (1965b) to permit hydro ly s i s of the phytoestrogen g lycos ides by the a l f a l f a g lycos idases . The sample was then ref luxed with 20 mL 2N HC1 and 80 - 46 - Figure 8. Phytoestrogen ex t rac t ion scheme for a l f a l f a sprouts . ALFALFA add sand g r i n d , stand add 2N HC1 + MeOH r e f l u x , cool 1 f i l t e r , wash with MeOH a l f a l f a f i l t r a t e evaporate extract with PE d i scard PE crude a l f a l f a extract extract with EE d i scard aqueous layer ether extract evaporate to @ 1 mL adjust volume, f i l t e r PHYTOESTROGEN EXTRACT MeOH = methanol PE = petroleum ether EE = e thy l ether - 47 - mL methanol for 30 min to further the h y d r o l y s i s . Af ter a 10 min c o o l - ing per iod , the a l f a l f a s l u r r y was suct ion f i l t e r e d through Whatman No. 50 (hardened) f i l t e r paper with 4 g C e l i t e f i l t e r a id "Hyf lo Super-Ce l " (Oohns-Manvil le , Etobicoke, O n t . ) . The f i l t e r cake was washed with 50 mL methanol and discarded. The f i l t r a t e was t ransferred to a 1000 mL round bottom f lask with r i n s i n g (2 X 5 mL water) . Evaporation to remove a port ion of methanol in the f i l t r a t e was ca r r i ed out under vacuum for 25 min using a rotary f l a sh evaporator (Brinkmann Instruments, Rexdale, Ont.) in conjunct ion with a water bath at 3 0 ° C . Thi s temperature allowed rapid methanol removal yet l i m i t e d the amount of heat appl ied to the sample. The remaining aqueous methanol extract was t ransferred to a 250 mL separatory funnel , again r i n s i n g the f lask with water ( 2 x 5 mL). Volume was adjusted with water to a previous ly c a l i b r a t e d 75 mL mark. L i p i d s and c h l o r o p h y l l pigments were removed by ex t rac t ion with 3 X 100 mL port ions of petroleum ether (b .p . 3 7 - 5 8 ° C ) . A f te r each addi- t ion of petroleum ether the mixture was shaken v igorous ly for 30 s and allowed to stand for 10 min to permit the phases to s e t t l e and the pigments to t rans fer to the upper petroleum ether l a y e r . The f i n a l ex t rac t ion of phytoestrogens from the crude a l f a l f a extract was accomplished with 4 X 50 mL port ions of anhydrous e t h y l ether . Each ether/aqueous layer mixture was shaken for 30 s and allowed to stand for 10 min before removal of the ether l a y e r . The ether extracts were combined and evaporated under vacuum at 3 2 ° C for 15 min to approximately 1 mL. The remaining residue was t rans ferred into a 10 mL volumetr ic f la sk with HPLC grade methanol r inses and d i l u t e d to volume. The f i n a l extract was f i l t e r e d through a Swinny f i l t e r uni t f i t t e d - 48 - with a 0.2 urn M i l l i p o r e Type EG f i l t e r ( M i l l i p o r e C o r p . , Bedford, MA). Samples were stored i n b o r o s i l i c a t e glass v i a l s (Wheaton S c i . , M i l l v i l l e , NO) at 5 ° C u n t i l analyzed. The ent i re ex t rac t ion procedure required approximately 4 h per sample, however, several ex t rac t ions could be run concurrent ly . A l l sprouting treatments were c a r r i e d out i n t r i p l i c a t e , and dup l i ca te extract ions were performed from each sprouting j a r wi th in a treatment. The number of extract ions was l i m i t e d by the quantity of sprouts that could be grown i n the j a r s used. Recovery s tudies were a l so performed using prepared stock so lu- t ions of the three phytoestrogens. In one experiment 2.0 mL of each 30.0 pg/mL stock so lu t ion was added to the mortar and the e n t i r e extrac- t i o n procedure c a r r i e d out as previous ly descr ibed. T r i p l i c a t e extrac- t ions were c a r r i e d out for t h i s study. In another experiment 2.0 mL of each 30.0 ug/mL stock so lu t ion was added to 20 g of a l f a l f a sprouts p r i o r to gr inding and completing the ex t rac t ion procedure. Dupl ica te ext rac t ions were performed for each of the two a l f a l f a c u l t i v a r s germin- ated under "standard" condi t ions as described in Sect ion E . C. MOISTURE DETERMINATION Moisture content of a l f a l f a sprouts was determined using the A0AC method for p lants (A0AC, 1980) with some modi f i ca t ions . T r i p l i c a t e samples of a l f a l f a sprouts of approximately 10 g each had been taken from every sprouting j a r for moisture ana lys i s and stored at - 3 0 ° C u n t i l analyzed. Sprout samples were chopped l i g h t l y while s t i l l f rozen, - 49 - and weighed into pre-dr ied (1 h) , des iccator cooled (0.5 h) , weighed aluminum dishes (57 mm diam X 17 mm depth) (Canlab, Richmond, B . C . ) . Samples were dr ied to constant weight ( ± 0 . 0 0 2 g) at 9 5 - 1 0 0 ° C i n a vacuum oven (National Appliance C o . , Por t l and , OR) under 91.5 kPa (27 inches Hg). Sprouts were dr ied for 5.5 h and immediately placed into a des iccator conta ining s i l i c a gel to cool (0.5 h) before weighing. Samples were returned to the oven for 1 h, again des iccator cooled , and reweighed to check for constant weight. F i n a l weights recorded a f ter 6.5 h drying were used to c a l c u l a t e moisture contents . O. MEASUREMENT OF PHYTOESTROGENS BY HPLC A l l HPLC analyses were performed using a Var ian Model 5060 L i q u i d Chromatograph equipped with a UV-50 va r i ab le wavelength dectector and V i s t a 401 data system (Varian Instrument Group, Palo A l t o , CA) and a Rheodyne Model 7125 loop valve i n j e c t o r (10 yL l o o p ) . Separation was c a r r i e d out on a reverse-phase Varian MicroPak MCH-10 (octadecyls i lane) column (4 mm I . D . X 30 cm), f i t t e d with a B i o - S i l 0DS-10 guard column (Bio-Rad Laborator ie s , Richmond, CA). Column temperature was maintained at 3 0 ° C . Detect ion of the phytoestrogens was made at 254 nm ( o p t i c a l bandwidth 8 nm). A l l water for HPLC ana ly s i s was d i s t i l l e d , deionized and f i l t e r e d through a 0.45 ym M i l l i p o r e f i l t e r ( M i l l i p o r e C o r p . , Bedford, MA). Methanol was g lass d i s t i l l e d HPLC-grade (Fisher S c i e n t i f i c C o . ) . Separ- a t ion was achieved by using a l i n e a r methanol-water gradient system at a flow rate of 1 .0 mL/min. Methanol and water r e se rvo i r s each contained 1.0% g l a c i a l a c e t i c ac id (v/v) and 0.01 M ammonium acetate (HPLC grade, - 50 - F i sher S c i e n t i f i c C o . ) . The gradient was programmed to increase from 53% to 58% re se rvo i r B (methanol) over 30 min. Tota l ana lys i s time per sample, inc lud ing column e q u i l i b r a t i o n , was 60 min. Peak re tent ion times and peak areas were computed automatica l ly by the i n t e g r a t o r . The re tent ion times were used to ca l cu l a t e severa l chromatographic parameters: the capaci ty f a c t o r , k (also known as k ' ) ; the separation factor ( r e l a t i v e r e t e n t i o n ) , « ; and the r e s o l u t i o n , R s . The fo l lowing equations were used (Yost et a l . , 1980): k = Eqn. 1 Eqn. 2 R = — Eqn. 3 S Wb1 + Wb2 where t = re tent ion time of compound K t^ = mobile phase holdup time k i , k 2 = capaci ty factors of compounds 1 and 2, r e s p e c t i v e l y At = distance between two peak maxima W b1' Wb2 ~ P e a k width at base of compounds 1 and 2 r e s p e c t i v e l y , and a l l measurements are expressed in the same time uni t s (min or sec) . Figure 9 shows how these measurements were obtained from a chromatogram. The separat ion factors were ca l cu la ted as r a t i o s of the k values for daidzein/formononetin and formononetin/coumestrol . Ratios were - 5 1 - Figure 9 . Retention time and peak width at base measurements 1. Adapted from Yost et a l . (1980). - 52 - obtained for the standards mix, standards ext rac t , spiked a l f a l f a (Vernal) extract and a l f a l f a only (Treatment X) ex t rac t , and compared for tenta t ive peak i d e n t i f i c a t i o n based on agreement of these « va lues . The r e so lu t ion of the two c l o s e l y e l u t i n g phytoestrogens, formononetin and coumestrol, was c a l c u l a t e d . R s values were obtained from repre- sentat ive chromatograms of the standards mix, standards extract , spiked a l f a l f a (Vernal) extract and a l f a l f a only (Treatment X) ex t rac t . Standard so lu t ions of da idze in , formononetin and coumestrol were prepared from respect ive stock so lu t ions containing 30.0 ug/mL to give concentrat ions of 20.0 , 10.0, 6 .0 , 4 .0 , 2 .0 , 1.0, 0 .8 , 0 .6 , 0.4 and 0.2 ug/mL. HPLC-grade methanol was used for a l l d i l u t i o n s . A standard curve for each phytoestrogen was prepared from the peak area integra tor responses for 10-uL i n j e c t i o n s of the standards. T r i p l i c a t e i n j e c t i o n s were made for each standard so lu t ion and area counts ( in uV-s) recorded to the nearest 100. L inear regress ion ana lys i s was performed using a Monroe 1880 programmable c a l c u l a t o r . Regression equations were used to ca l cu la te the content of the corresponding phytoestrogen in the sample ex t rac t s . Dupl icate i n j e c t i o n s were made for each sample ex t rac t . The mean of four determinations (two sample preparat ions , each in jec ted twice) was used in c a l c u l a t i n g the phytoestrogen content of the sprouts grown i n each j a r . For the extracts of the standards, the mean of s ix deter- minations (three sample preparat ions , each in jec ted twice) was used to c a l c u l a t e phytoestrogen recovery. - 53 - Pre l iminary peak i d e n t i f i c a t i o n was based on a comparison of re tent ion times of phytoestrogen standards and unknown peaks i n the sample ex t rac t s . To further confirm the peak i d e n t i t i e s , the chromato- grams of the a l f a l f a samples which had been spiked with phytoestrogen standards before ex t rac t ion were compared to those of the unspiked a l f a l f a extracts and the standards alone. E. GERMINATION OF ALFALFA SEEDS 1 . Sprouting preparat ion Clear glass wide mouth jars (800 mL vol) were used as sprouting conta iner s . Oars were washed with a 1% c h l o r i n e bleach (v/v) so lu t ion to l i m i t mic rob ia l contamination, and thoroughly r insed with d i s t i l l e d water before use. Cheesecloth covers (10 cm X 10 cm) secured by rubber bands permitted rapid drainage yet reta ined seeds and sprouts ; covers were replaced d a i l y . Seeds i n 15 g l o t s were weighed into each j a r , 300 mL d i s t i l l e d water was added, and the seeds were soaked for 4 h at room temperature ( 2 2 ° C ) in the l i g h t . At the end of t h i s per iod , water was decanted and seeds were r insed with the volume of water designated i n the r i n s i n g schedule and dra ined . The growth period was measured from the time the a l f a l f a seeds began soaking. In order to thoroughly dra in the sprouting j a r s , racks were constructed from 1/2 inch (1.3 cm) wire mesh that allowed the j a r s to stand inver ted at a 4 5 ° angle and permitted adequate i l l u m i n a t i o n and a i r c i r c u l a t i o n . Figure 10 i l l u s t r a t e s the dra in ing rack system with jars i n p lace : two rows of three j ar s each placed approximately 3 cm apart . - 54 - Figure 10. Rack used for draining sprouting j a r s . - 55 - 2. Growth s tudies Growth s tudies were undertaken to determine the e f fects of l i g h t dura t ion , r in se frequency, r inse volume and growth per iod , as wel l as a l f a l f a c u l t i v a r on the content of phytoestrogens in a l f a l f a sprouts . In a l l s tudies the ambient temperature was monitored d a i l y for f luc tua- t ions during the growth p e r i o d . A thermometor was placed on each rack d i r e c t l y beneath the sprout j a r openings, and readings were taken 3 times per 24 h period at 8 h i n t e r v a l s . A l l water used for r i n s i n g was d i s t i l l e d . The r inse volume was the quanti ty of water used at each r i n s i n g time, and was appl ied i n two successive "washes" of equal volume. D a i l y observations of c o l o r , s i z e , stage of sprout development and any v i s i b l e signs of contamination were recorded. L ight was provided by 15-watt cool-white f luorescent tubes pos i - t ioned d i r e c t l y over each row of sprouting j a r s as shown in Figures 10 and 11. L ight durat ion was the number of continuous hours of l i g h t per 24 h period that the sprouts were exposed to . A L i - c o r photometer (Lambda Instrument Corp.) equipped with a photosynthetic photon f lux density (PPFD) sensor was used to measure the l e v e l of l i g h t or rad ia- t i o n received at the sprout j a r surface . The uni t of measure, y E m ~ 2 s - 1 , ind ica te s the quanti ty of photosynthet i ca l ly ac t ive r a d i a t i o n (PAR) a v a i l a b l e to plants i n c o n t r o l l e d environments, the E i n s t e i n (E) being defined as a mole of photons ( T i b b i t t s and Kozlowski , 1979). To provide t o t a l darkness for those sprouting treatments r equ i r ing a 12 to 24 h dark per iod , the sprouting racks were completely covered with aluminum f o i l (Figure 11). L ight l e v e l measurements taken under these f o i l 2 1 covers were 0 yEm" s" .  - 57 - (a) Standard condi t ions The purpose of t h i s experiment was to e s t ab l i sh growth condi t ions that could serve as a standard for the production of high q u a l i t y a l f a l f a sprouts . High q u a l i t y i s defined here in terms of v i s u a l accep- t a b i l i t y and y i e l d : br ight green cotyledons; white hypocotyls and r a d i c l e s ; 3 to 5 cm leng th . Table 2 summarizes the standard condi t ions used to grow sprouts from two a l f a l f a c u t i v a r s . The phytoestrogen content of both c u l t i v a r s was subsequently measured. Condit ions were chosen to incorporate those known to be used i n commercial a l f a l f a sprout production as well as condi t ions recommended by Hesterman et a l . (1981) to improve q u a l i t y and fresh weight y i e l d of sprouts . Sprouts of each c u l t i v a r , Moapa and V e r n a l , were grown i n four r e p l i c a t e j a r s . One j a r of sprouts of each c u l t i v a r was designated for recovery ex t rac t ion studies as described i n Sect ion B. In a d d i t i o n , four r e p l i c a t e jars of Vernal a l f a l f a sprouts were grown for pre l iminary ex t rac t ion t r i a l s . Each jar was l a b e l l e d with a treatment code and sample number and was randomly assigned a p o s i t i o n on the sprouting racks . (b) T r i a l condi t ions The purpose of t h i s experiment was to evaluate the ef fect of environment on the phytoestrogen content of sprouts . The " t r i a l " condi t ions were chosen to represent a wide range of treatment p o s s i b i l i - t i e s as summarized i n Table 3. Each treatment was assigned an alphabe- t i c code, e .g . " A " . Within each treatment were three r e p l i c a t e j a r s of sprouts . The 24 h l i g h t and 24 h dark treatments were c a r r i e d out on - 58 - Table 2 . Standard growth condi t ions for a l f a l f a sprouts . Treatment Condi t ion AA BB C u l t i v a r Moapa Vernal Growth per iod , h 100 100 L i g h t durat ion , h 12 12 Rinse volume, L 1 1 Rinse frequency/24 h 2 2 - 59 - Table 3. T r i a l growth condi t ions for a l f a l f a sprouts . Condit ions Growth period L ight durat ion Rinse volume Rinse Frequency Treatment (h) (h) (L) /24 h A 76 24 1 .0 2 B 76 24 0.4 5 C 76 24 1.0 5 D 76 0 1.0 2 E 76 0 0.4 5 F 76 0 1.0 5 U 148 24 1.0 2 V 148 24 0.4 5 w 148 24 1.0 5 X 148 0 1.0 2 Y 148 0 0.4 5 Z 148 0 1 .0 5 - 60 - separate sprouting racks; sprouting j a r s within each treatment c l a s s were randomly assigned a p o s i t i o n on the corresponding racks . Only Vernal a l f a l f a was used throughout these s tud ies . (c) Percent germination A simple germination t r i a l was set up to measure the percent germination of Vernal a l f a l f a seeds under condi t ions used i n the germin- a t ion s tud ie s . Seeds (15 g) were soaked for 4 h i n 300 mL water, drained and allowed to germinate for 24 h with p e r i o d i c r i n s i n g . Ungermfnated seeds were separated manually from v i a b l e , germinated seed, d r i ed at room temperature overnight and weighed. The d i f ference i n weight between the o r i g i n a l quanti ty of seeds and the ungerminated seeds was used to c a l c u l a t e percent germination. 3. Sprout harvest ing A l f a l f a sprouts were given the f i n a l r i n s e of the treatment schedule immediately before being harvested. Each j a r was then vigorous ly shaken to remove excess water and the sprouts emptied onto paper towels and allowed to dra in for 1 h (Figure 12). From each j a r ' s contents , two samples (@ 20 g each) were weighed into polyethylene bags for phytoestrogen a n a l y s i s , and three samples (<§> 10 g each) for moisture a n a l y s i s . A l l samples were frozen at - 3 0 ° C u n t i l analyzed. - 61 - Figure 12. Draining of a l f a l f a sprouts p r i o r to sampling and f r e e z i - 62 - F. STATISTICAL ANALYSIS 1. S ingle fac tor ana lys i s of variance The standard (2) and t r i a l (12) treatment combination condi t ions used to grow a l f a l f a sprouts were treated as s ing le factor treatments (14) and analyzed in a one-way analys i s of var iance , using the UBC MFAV program package (Le, 1978) ava i l ab le for use on the UBC Amdahl 470 V/8 computer. The Student-Newman-Keuls mul t ip le range tes t (Zar, 1974) was used to perform mul t ip le comparisons among means for each of the three phytoestrogen v a r i a b l e s . 2. F a c t o r i a l ana lys i s of variance The data c o l l e c t e d for each of the phytoestrogen var i ab le s under t r i a l growth condi t ions were coded for a 21* f a c t o r i a l design having 4 fac tors (growth per iod , l i g h t dura t ion , r inse volume and r inse f r e - quency) each with 2 l e v e l s as l i s t e d i n Table 3. Not a l l of the 16 a v a i l a b l e treatment combinations we're performed, s ince the r i n s i n g com- binat ion of a 0.4 L volume applied twice/24 h was assumed to be i n s u f f i - c ient for a l f a l f a sprout growth. As a r e s u l t , data for only 12 t r e a t - ment combinations were analyzed using an ana lys i s of variance program package for an unbalanced design (GENLIN) adapted for the UBC computer (Greig and B j e r r i n g , 1978). These analyses were c a r r i e d out to deter- mine the s i g n i f i c a n c e of the fac tor ( s ) and/or factor i n t e r a c t i o n ( s ) on the accumulation of each phytoestrogen i n a l f a l f a sprouts . M u l t i p l e comparisons among phytoestrogen means (of 3 r e p l i c a t e s ) were performed using the Student-Newman-Keuls t e s t . - 63 - RESULTS AND DISCUSSION A. EXTRACTION 1 . Ex t r ac t ion methodology The method developed for phytoestrogen ex t rac t ion from a l f a l f a sprouts was adapted from the ex t rac t ion methods reported by Beck (1964) and Franc i s and M i l l i n g t o n (1965b) for the ana lys i s of c lover forage. Several modi f icat ions were made to these methods in part to account for the l a rger sample s i ze in the present study and to prepare the sample extract for subsequent ana lys i s by high performance l i q u i d chromato- graphy. The ground a l f a l f a sample was allowed to stand as per F ranc i s and M i l l i n g t o n (1965b) to permit enzymatic hydro lys i s of i sof lavones present as g lycos ide s . However, pre l iminary experiments ind ica ted that further hydro ly s i s and improved e x t r a c t a b i l i t y could be achieved by adding 2N HC1 (20:80 2N HCl/MeOH, v/v) to the ref lux mixture. Chromato- grams from a l f a l f a extract s obtained without t h i s ac id showed poorer recovery of phytoestrogens. The ac id may a l so a id in recovery of the phytoestrogens during the ex t rac t ion process s ince i sof lavones and coumestans are very stable in an ac id medium (Wong, 1975). A re f lux ing per iod of 30 min was chosen for the methanol/acid h y d r o l y s i s based on evidence that 7 -0-g lycos id i c l inkages would be completely hydrolyzed i n 15 to 40 min under s i m i l a r condi t ions (Markham, 1982). Further improvements in phytoestrogen recovery were not achieved when re f lux ing condi t ions of 45 min with 50:50 2N HCl/MeOH (v/v) were attempted. - 64 - The a l f a l f a f i l t r a t e was evaporated in a subsequent step to remove a large por t ion of the methanol p r i o r to petroleum ether ex t rac t ion of the l i p i d s and c h l o r o p h y l l pigments. In the presence of the t o t a l methanol volume, a complete separation of the petroleum ether and water/methanol l ayers could not be achieved because petroleum ether i s m i s c i b l e with methanol. Furthermore, there was l i t t l e t rans fer of c h l o r o p h y l l pigments to the petroleum ether phase i f a l l the methanol was present. Evaporation to a t o t a l volume of approximately 70 to 75 mL resu l ted in an improved p a r t i t i o n of the two phases and an e f f i c i e n t removal of most c h l o r o p h y l l pigments from the crude ex t rac t . A pigmented sludge layer that formed at the inter face was discarded with the f i n a l petroleum ether ex t rac t . E t h y l ether was used for the ex t rac t ion of phytoestrogens from the aqueous a l f a l f a mixture because both i sof lavones and coumestrol are so luble in t h i s so lvent . Evaporation of e thyl ether under vacuum was rapid and the remaining residue could be e a s i l y d i s so lved in methanol for HPLC a n a l y s i s . 2 . Recovery s tudies As prev ious ly descr ibed, s tudies were c a r r i e d out to evaluate the recovery by t h i s ex t rac t ion method of a phytoestrogen standards mix and of a standards mix added to a l f a l f a sprouts (grown under "s tandard" c o n d i t i o n s ) . In order to account for the quanti ty of phytoestrogens contr ibuted by the a l f a l f a sprouts , the mean area count response obtained (by HPLC analys i s ) from unspiked a l f a l f a extracts was sub- tracted from the area count responses of the spiked a l f a l f a ex t rac t s . - 65 - These adjusted area count values were then used to c a l c u l a t e recovered phytoestrogen contents . The mean contents of phytoestrogens c a l c u l a t e d to be present were compared to an expected value of 6.0 ug/ml_ to obtain % recovery data (Table 4 ) . The recovery data for the standards mix ind ica te that there were only minimal losses as a r e s u l t of ex t rac t ion procedures i n v o l v i n g r e f l u x i n g , evaporation and phasic d i s t r i b u t i o n . Recoveries of standards added to a l f a l f a samples were markedly lower; however, except in the case of coumestrol from Moapa a l f a l f a , these recover ies d id compare favorably to previous ly reported values (Lookhart, 1979; Murphy, 1981). Wong (1962) and Sachse (1974) reported losses of formononetin i n the in so lub le sludge layer that formed during the ex t rac t ion of c lover pigments with petroleum ether . These losses were a t t r i b u t e d to formon- one t in ' s low s o l u b i l i t y i n the aqueous/alcohol l a y e r . Low recover ies of coumestrol have been reported i n soybean ex t rac t ion as a r e su l t of b ind- ing to l i p i d f r ac t ions (Lookhart, 1979). The low s o l u b i l i t i e s of coumestrol i n common solvents and aqueous systems may also have contr ibuted to ex t rac t ion losses in the present study. C u l t i v a r d i f ferences in phytoestrogen contents have been wel l documented (Beck, 1964; Franc i s and M i l l i n g t o n , 1965b; Stuthman et a l . , 1966; Franc i s et a l . , 1967; Loper et a l . , 1967); however, there are no reports i n the l i t e r a t u r e of c u l t i v a r d i f ferences i n ex t rac t ion e f f i c i e n c i e s . Table 4. Recovery of phytoestrogens using developed extrac t ion method. Daidzein ug/mL % Recovery Formononetin ug/mL % Recovery Coumestrol ug/mL % Recovery 1. Standards mix 6.110.1 1 102 5 . 6 ± 0 . 2 94 6 . 2 ± 0 . 2 103 2. Standards + Moapa a l f a l f a 4.7 ± 0 . 2 78 3.8+0.3 63 2.5+0.5 42 3. 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 deviat ion of 6 determinations. - 67 - B. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY 1. HPLC methodology Numerous HPLC systems have been developed for the separat ion and de tec t ion of se lected phytoestrogens. Several of these systems (Ohta et a l . , 1979; Murphy, 1981; N i c o l l i e r and Thompson, 1982) did not appear to adequately resolve the peaks of i n t e r e s t for quant i t a t ive purposes. A number of other systems were examined i n t h i s study, using a reversed- phase Varian MicroPak MCH-10 column, i n attempts to resolve a l f a l f a sprout ex t rac t s . I n i t i a l s tudies c a r r i e d out with i s o c r a t i c methanol/water systems (e .g . 60:40, MeOH/H20; 1.0 mL/min) showed that the e l u t i o n of the phyto- estrogen standards followed the expected order of Vande Casteele et a l . (1982) based p r i m a r i l y on r ing s u b s t i t u t i o n of the f l avono ids . E l u t i o n was i n the order of decreasing p o l a r i t y which i s t y p i c a l of reversed- phase chromatography and a l so demonstrated by Murphy and Stutte (1978). Daidzein eluted f i r s t , followed by formononetin ( less polar due to the presence of a methoxy group at C - V vs . a hydroxy group for daidzein) and f i n a l l y coumestrol , e lu t ing immediately a f ter formononetin. Resolut ion of the phytoestrogen standards alone was e x c e l l e n t , however, when the same high (>55%) methanol systems were used to separ- ate a l f a l f a ex t rac t s , r e so lu t ion of the phytoestrogens was poor. The a l f a l f a extracts contained a number of peaks e l u t i n g with the void volume and i n t e r f e r i n g with the r e so lu t ion of the da idzein peak. These peaks could correspond to po la r , non-flavonoid compounds such as pheno- l i c a c i d s . Ohta et a l . (1979) reported a s i m i l a r observat ion with - 68 - soybean ex t rac t s . E a r l y e l u t i n g peaks were considered to be h y d r o p h i l i c lower molecular weight compounds but not f l avonoids . Using solvent systems with a lower methanol concentrat ion (<50%) improved the r e s o l u t i o n of da idze in from the more polar ear ly e l u t i n g peaks but resu l ted in severe band spreading and increased re tent ion times of the l a t e r e l u t i n g formononetin and coumestrol peaks. T r i a l runs using an aqueous methanol gradient from 20 to 50% methanol over 20 min as described by E ldr idge (1982a) to resolve i sof lavones and coumes- t r o l were attempted, but without success on the present column. Formon- onetin and coumestrol had not even eluted by 60 min because the e l u t i n g strength of the solvent system was too low. An i s o c r a t i c solvent system of 52:48 MeOH/H20 (v/v) at a flow rate of 1.0 mL/min was subsequently found to give a reasonable separation of the ear ly por t ion of the a l f a l f a extract chromatogram. Further f ine- tuning of the program was continued in order to improve the r e so lu t ion of the l a t t e r ha l f of the chromatogram. To reduce the t o t a l time required for e l u t i o n of the a l f a l f a ex t rac t , var ious gradient solvent programs were c a r r i e d out. Steep gradients as used by Murphy (1981) were found unsuitable because they great ly reduced re so lu t ion of the l a t e r peaks as a r e su l t of base l ine f l u c t u a t i o n s and d r i f t . A l i n e a r gradient program s t a r t i n g at 53% methanol was demonstrated to be more des i rab le because base l ine e f fec t s d id not i n t e r f e r e with peak q u a n t i t a t i o n . A gradual increase to 58% methanol over 30 min was found adequate to reduce program e l u t i o n time to 30 min. Increases to greater methanol concentrat ions decreased the re so lu t ion between formononetin and coumestrol . Faster gradients had - 69 - a minimal e f fect on re tent ion times of formononetin and coumestrol and also caused more basel ine f l u c t u a t i o n s . In an e f for t to reduce peak t a i l i n g of the e l u t i n g compounds, a ce t i c acid was added to the water and methanol r e s e r v o i r s . It was found that a 1% concentrat ion (v/v) of g l a c i a l ace t i c ac id i n the s o l - vent mixture improved peak shape of the e lu t ing phytoestrogen peaks and e f f e c t i v e l y reduced re tent ion t imes, e s p e c i a l l y of formononetin and coumestrol . Acid has been used by other workers (Wulf and Nagel, 1976; Vande Casteele et a l . , 1982) to suppress i o n i z a t i o n of the ac id groups on f lavonoids and phenolic acids as previous ly described (Mater ia l s and Methods). The most acid group of i so f lavonoids i s considered to be the phenolic hydroxyl at C-7 (Markham, 1982) which i s present on a l l three phytoestrogens s tudied here. In add i t ion to a c i d , other workers (Murphy and S tu t te , 1978; Proksch et a l . , 1981) have used ammonium acetate as a buffer ing reagent to prevent abnormal polar movement of compounds on the reversed-phase column. Murphy and Stutte (1978) reported that ortho-hydroxylated compounds, such as some phenol ic ac id s , could undergo intramolecular hydrogen bonding so that t h e i r polar hydroxy groups would be bound and e l u t i o n and separat ion would no longer be based on true p o l a r i t y . Neither da idze in , formononetin or coumestrol are ortho-hydroxy- l a t e d , and t h e i r e l u t i o n order was not affected by the presence of ammonium acetate . However, i n t h i s study 0.01 M ammonium acetate was found e f f e c t i v e in improving r e s o l u t i o n , p a r t i c u l a r l y the separation of da idzein from ear ly e l u t i n g peaks. Retention times were also reduced i n - 70 - comparison to the methanol/water only solvent system, and were s i m i l a r to the retention times obtained with the 1% a c e t i c acid system. In combination, the addition of 1.0% a c e t i c acid and 0.01 M ammon- ium acetate to both the methanol (B) and water (A) solvent r e s e r v o i r s provided for an improved separation of the a l f a l f a extract where a l l three phytoestrogen peaks are sharp, resolved to baseline, and elute under 30 min using the l i n e a r gradient program from 53% to 58% methanol (reservoir B) over a 30 min period. The pH of t h i s solvent system at i n i t i a l composition conditions (53:47, B/A) was 4.5 which i s well within the pH guidelines of 2.0 to 7.5 for Varian MicroPak columns (Varian Assoc., 1978). 2. Chromatographic parameters The retention times (tp) and the other chromatographic parame- ter s k (capacity f a c t o r ) , « (separation factor) and R s (resolution) f o r the phytoestrogen peaks were calculated from chromatograms obtained for HPLC runs of standards and a l f a l f a extracts. The equations (1 to 3) and measurements (Figure 9) used for these c a l c u l a t i o n s were given in the previous section (Materials and Methods). Table 5 l i s t s the values obtained from HPLC analysis of the three phytoestrogens in a standard mix, standards extract, spiked a l f a l f a extract and an unspiked a l f a l f a extract (Treatment X). Treatment X sprouts were chosen for representa- tion of the a l f a l f a only extract because a l l three phytoestrogens were detectable. The retention times represent means of the t o t a l number of deter- minations made for each sample, as noted on Table 5. C o e f f i c i e n t s Table 5. Retention times (tp) , capacity factors ( k ) , separation factors ( « ) and resolut ion (R s) of selected phytoestrogens on MicroPak MCH-10 using l inear gradient e lut ion as described. tR (min) k C Rs Sample D F C D F C D/F F/C F/C Standards 1 mix 10.78 ± 0 . 0 2 k 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 A l f a l f a extract 3 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 A l f a l f a extract 3 (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 deviat ion. - 72 - of v a r i a t i o n of re tent ion times were found i n the range of 0.2 to 1.756 which i s within the p r e c i s i o n required for peak i d e n t i f i c a t i o n (0.2 to 2.0%) (Oohnson and Stevenson, 1978). The k values were ca l cu la ted to ind ica te the re tent ion character- i s t i c s of the chosen HPLC system. Yost et a l . (1980) stated that k>1 i s des i rab le for the f i r s t peak of in te re s t to ensure separation from the unretained peaks at the solvent f r o n t . A l s o , peaks should have k<10-15 or ana lys i s time i s too long . Peak widths tend to increase with increas ing k values which makes r e so lu t ion and detect ion more d i f f i - c u l t . The k values ca l cu la ted from the corresponding mean re tent ion times for d a i d z e i n , formononetin and coumestrol are wi th in t h i s recom- mended range (Table 5 ) . The « values were c a l c u l a t e d to determine the r e l a t i v e re tent ion of the two peaks i n the pa i r s daidzein/formononetin and formononetin/ coumestrol . These values are dependent only on temperature, column and mobile phase composition and so can serve as a r e l i a b l e index for peak i d e n t i f i c a t i o n (Snyder and K i r k l a n d , 1979; Yost et a l . , 1980). The « values l i s t e d for the unspiked a l f a l f a extracts were derived from peaks t e n t a t i v e l y i d e n t i f i e d by re tent ion times as the three phytoestrogens. Based on agreement of these <* D/F and « F/C values with the correspond- ing « values of the standards and spiked a l f a l f a extract (Table 5) one could further confirm the i d e n t i t y of the phytoestrogen peaks. Resolut ion was ca l cu la ted to determine the actua l separation of the c l o s e l y e l u t i n g peaks, formononetin and coumestrol . A representa- t i v e chromatogram (Figures 13-16) from each of the sample groups was used for manual measurements of peak widths. An R s value of 1.0 means - 73 - F i g u r e 1 3 . HPLC chromatogram of phytoestrogen standards. Peaks: D=daidzein, F=formononetin, C=coumestrol. D F , , 1 1 1 1 1 0 5 10 15 20 25 30 TIME (min) - 74 - Figure 14-. HPLC chromatogram of phytoestrogen standards extract. Peaks: D=daidzein, F=formononetin, C=coumestrol. - 75 - Figure 15. HPLC chromatogram of a l f a l f a extract spiked with phytoestro- gen standards 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 1 — 1 1 1 0 5 10 15 20 25 30 T I M E ( m i n ) - 76 - Figure 16. HPLC chromatogram of a l f a l f a extract (Treatment X ) . Peaks: D=daidzein, F=formononetin, C=coumestrol. 10 1 15 TIME (min) —r- 20 - T " 25 ~1 30 - 77 - that r e so lu t ion i s approximately 90% complete, which i s considered suf- f i c i e n t for peak area c a l c u l a t i o n s , while an R s value of 1.5 repre- sents basel ine separation (Yost et a l . , 1980). In a l l samples repre- sented (Table 5) , basel ine r e so lu t ion of formononetin and coumestrol was . achieved, although the R s value for the a l f a l f a only extract was markedly lower i n comparison to the other samples. The low concentra- t ions of formononetin and coumestrol found i n the a l f a l f a only extract e luted as very broad peaks which re su l ted i n large peak width measure- ments, thus a f f ec t ing peak r e s o l u t i o n . 3. L inear regress ion a n a l y s i s Standard so lu t ions of each phytoestrogen from concentrat ions of 0.2 to 30.0 pg/mL in methanol were in jec ted in t r i p l i c a t e for HPLC ana lys i s and the peak area integrator responses recorded. In jec t ions of methanol were a l so made to measure any basel ine response that might occur at peak re tent ion times even i n the absence of the phytoestro- gens. The standard curves derived from c o l l e c t e d peak area data were found to be wel l described by the fo l lowing regress ion equations: Da idze in : Y = -0.029 + 3.809X r 2 = 0.9998 s = y .x 0.524 Formononetin: Y = 0.168 + 3.813X r 2 = 0.9982 s = y .x 1 .516 Coumestrol: Y = 0.592 + 2.707X r 2 = 0.9992 s = y x 0.771 Y = area counts (pV-sec)/10 X - concentrat ion of phytoestrogen (pg/mL in methanol) Some d i f f i c u l t i e s were encountered in obtaining reproducible in te - grator responses for the lowest concentrat ions of formononetin and - 78 - coumestrol standard s o l u t i o n s . For t h i s reason the integra tor responses for 0.2 yg/ml_ formononetin and 0.2 and 0.4 yg/mL coumestrol were not included i n regress ion equation c a l c u l a t i o n s . C . GROWTH STUDIES 1. Moisture determination Moisture contents of a l f a l f a sprouts grown under standard and t r i a l condi t ions were found to range from 88.5 to 94.8% (Table 6 ) . There was no s i g n i f i c a n t d i f ference (P>0.05) in the moisture content of Moapa and Vernal a l f a l f a sprouts grown under standard c o n d i t i o n s . The moisture contents of the 76 h sprouts grown under t r i a l cond i t ions , both l i g h t and dark (A to F ) , were a l l s i g n i f i c a n t l y less (P<0.05) than the moisture contents of the 148 h sprouts (U to Z ) . The longer growth period presumably re su l t s in greater uptake of water by the sprouts . Hesterman et a l . (1981) a t t r i b u t e d the increase i n fresh weight y i e l d of a l f a l f a sprouts with longer growing periods (4 to 8 days) to greater water absorpt ion . Hamilton and Vanderstoep (1979) a lso reported a s i g - n i f i c a n t d i f ference i n moisture contents between 72 h and 120 h a l f a l f a sprouts , but no s i g n i f i c a n t d i f ference between sprouts grown for the same length of time i n l i g h t vs. dark cond i t ions . In the present study, moisture contents of sprouts grown in the dark were greater than the corresponding sprouts grown in the l i g h t for a given growth period treatment (e .g . D vs . A ) , however, not a l l d i f ferences were s i g n i f i c a n t (P<0.05). Nevertheless , i t was because of s i g n i f i c a n t d i f ferences (P<0.05) i n moisture contents that c a l c u l a t i o n s of each a l f a l f a sprout sample's - 79 - Table 6. Moisture content of a l f a l f a sprouts grown under standard and t r i a l c o n d i t i o n s . Moi s ture 1 Treatment (%)2 STANDARD Moapa 91.5 ± 0 . 4 3 3 Vernal 91.4 ± 0 . 2 a TRIAL A 88.5 + 0 . 5 b B 91.3 + 0 . 3 a C 91.6 + 0 . 1 a D 90.5 + 0 . 4 C E 91.4 + 0 . 5 a F 92.5 + 0 . 2 d U 93.3 + 0 . 2 6 V 93.5 + 0 . 3 e f W 94.1 + f 0 .2 1 X 94.2 + 0 . 1 f Y 93.7 + 0 . 6 e f Z 94.8 + 0 . 1 9 Expressed on wet weight bas 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 . Means sharing the same superscr ip 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 mul t ip le range t e s t . - 80 - dry weight were made using the corresponding treatment's mean moisture content, rather than using an o v e r a l l mean moisture content. 2. Growth condi t ions (a) Percent germination The two a l f a l f a cu t iva r s used i n the present study, Moapa and V e r n a l , were purchased as " sprout ing" a l f a l f a (Richardson Seed Co. L t d . , Burnaby, B . C . ) . A l f a l f a seeds sold for home and commercial sprouting purposes for human consumption must be "untreated" , i . e . have had no fungicide treatment, and should have germinaton percentages of at l ea s t 85% to be most des i rab le for sprouting (Kulvinskas , 1978; Hesterman and Teuber, 1980). Moapa a l f a l f a was demonstrated to have high germination and greater fresh weight y i e l d in comparison to f ive other a l f a l f a c u l t i v a r s (Hesterman et a l . , 1981). In the present study the mean percent germin- at ion (of 3 r ep l i ca te s ) of Vernal a l f a l f a seeds was found to be 82% which could s t i l l be considered as a des i rab le l e v e l of seed v i a b i l i t y for sprout ing . (b) L i g h t L ight has been demonstrated to inf luence the rate of r e s p i r a t i o n during germination (Mayer and Pol jakoff-Mayber, 1975). L ight may a l so i n d i r e c t l y a f fect the synthesis of f lavonoid substances s ince photosyn- thes i s determines the s i ze of the sugar pool which in turn a f fec t s production of the aromatic r ings v ia the sh ik imic ac id pathway (Ross i ter and Beck, 1967). However, Ross i ter and Beck (1967) reported appreciable - 81 - quant i t i e s of estrogenic i sof lavones i n c lover that was grown in com- plete darkness, i n d i c a t i n g that the l i g h t requirement for phytoestrogen synthesis i s low. Nevertheless , growth studies were c a r r i e d out to determine i f l i g h t inf luences phytoestrogen accumulation i n a l f a l f a sprouts . The l i g h t l e v e l that the a l f a l f a sprouts were exposed to was measured i n terms of photosynthetic photon f lux densi ty (PPFD) i n uni t s of uEm~ 2 s" 1 where an E i n s t e i n (E) i s the equivalent of one mole of photons. This measurement i s favored for the determination of the r a d i - a t ion (between 400 and 700 nm) that plants receive in c o n t r o l l e d env i - ronments, rather than measuring the i n t e n s i t y of l i g h t produced by lamps ( in lux or foot-candles) (McFarlane, 1978; T i b b i t t s and Kozlowski , 1979). Furthermore, the various l i g h t absorbing pigments i n p lants d i f f e r from the pigments responsible for human v i s i o n in that each pigment has i t s own absorption and ac t ion spectrum. Therefore , d i f f e r - ent plant functions respond to p a r t i c u l a r spec t ra l bands of l i g h t with d i f f e r e n t e f f i c i e n c i e s (McFarlane, 1979). Some d i f f i c u l t y was experienced in taking l i g h t l e v e l measurements because the readings depended on the angle and p o s i t i o n of the sensor i n r e l a t i o n to the overhead lamps. In f i e l d s tudies , T i b b i t t s and Kozlow- s k i (1979) recommended that measurements be made at the top of the plant canopy. In the present study the ra i sed centre of the sprouting rack was chosen as the point for measurement with the sensor placed perpendi- cular to the rack. The f luorescent lamps pos i t ioned d i r e c t l y over the j a r s in add i t ion to the overhead room lamps provided an average PPFD of 50 u E m ~ 2 s - 1 . The same reading was obtained when the sensor was placed - 82 - i n the centre of sprouting j a r s d i r e c t l y onto the surface of the sprouts . This l i g h t l e v e l was markedly lower than the 240 u E m ~ 2 s - 1 used by Hesterman et a l . (1981), however, i t was considered adequate for contrast with the dark condi t ion of 0 y E m ~ 2 s - 1 used in the other growth studies (as measured with a s e n s i t i v i t y of 0.1 v i E n r 2 s - 1 ) . Furthermore, McFarlane (1978) reported that the germination response of a plant requires a l i g h t l e v e l of only 0.1 y E m " 2 s _ 1 . (c) Temperature The readings recorded from thermometers placed on the sprouting racks ind ica ted that the ambient temperature remained r e l a t i v e l y con- stant over a 24 h dura t ion , throughout the growth periods and regardless of l i g h t or dark growth c o n d i t i o n s . The mean ambient temperature was found to be 2 2 . 2 ° C (range 21.2 to 2 4 . 0 ° C ) . Random temperature readings taken ins ide the sprouting jar s were constant at 2 3 ° C . Hesterman et a l . (1981) found that a germination temperature of 2 1 ° C was optimal for f resh weight y i e l d of a l f a l f a sprouts . Several workers (Fordham et a l . , 1975; Hamilton and Vanderstoep, 1979; Hsu et a l . , 1980) have conducted germination studies of a l f a l f a and other legumes at comparable tempera- tures of 22 to 2 5 ° C . 3. A l f a l f a sprout development Observations recorded during the growth studies provide a means of i d e n t i f y i n g d i f ferences i n sprout development that may occur under var ious growth c o n d i t i o n s . Observations made at 24 h i n t e r v a l s included sprout length , c o l o r , matur i ty , as wel l as any v i s i b l e signs of contam- i n a t i o n and/or spo i l age . - 83 - (a) Standard condi t ions After 4 h soaking, a l f a l f a seeds were swollen and seed coats had s p l i t . Fol lowing the f i r s t 24 h growth per iod , white hypocotyls (Figure 7) had emerged from the seeds and averaged 1.0 cm in l ength . The seed coats were only p a r t i a l l y removed from the cotyledons . Af ter the second 24 h growth per iod , the hypocotyls averaged 1.5 cm i n l eng th . Coty le - dons had begun to develop green co lor as seed coats separated away from the cotyledons . At 76 h (3 days + soaking time) the hypocotyls had elongated to an average of 2.0 cm. The cotyledons were green but remained l a r g e l y folded together . At t h i s stage, Moapa sprouts appeared to be developing more r a p i d l y than Vernal sprouts i n terms of f l e s h i e r hypocotyls and l a r g e r , spread open cotyledons . More rapid germination and subsequent photosynthesis may be re la ted to the higher inherent seedl ing v igor of one c u l t i v a r compared to another, as suggested by Walter and Jensen (1970). Sprouts harvested a f ter 100 h had br ight green cotyledons and white hypocotyls at l eas t 3.0 cm long with some pink co lor development at the upper por t ion of the hypocotyls . The sprouts appeared healthy with no v i s i b l e signs of spo i lage . (b) T r i a l condi t ions Af ter a 4 h soaking per iod , the Vernal a l f a l f a seeds used i n t h i s study were swollen and the seed coats were s p l i t . A subsequent 24 h growth period resu l ted i n white hypocotyls approximately 1.0 cm i n length i n both 0 h l i g h t ("dark") and 24 h l i g h t ( " l i g h t " ) c o n d i t i o n s . Seed coats were p a r t i a l l y removed and cotyledons appeared s l i g h t l y green i n the " l i g h t " sprouts and l i g h t yellow i n the "dark" sprouts . Af ter - 84 - the second 24 h growth per iod , the hypocotyls of the " l i g h t " sprouts averaged 2.0 cm in length and most cotyledons were green and had begun to spread open. The hypocotyls of the "dark" sprouts averaged 2.5 cm i n length and were much s t r a i gh te r than those of the l i g h t sprouts . Cotyledons were yellow and some had a lso begun to spread open. D i f f e r - ences i n sprout development in response to various growth condi t ions had become more apparent at t h i s stage. At 76 h the " l i g h t " sprouts general ly had l a rge , dark green cotyledons , many spread open, and hypocotyls that had become pink at the base, pale green in the centre but remained white at the t i p ( r a d i c l e ) . Treatment A and U sprouts had approximately 3.0 cm long hypocotyls and many seed coats remained among the sprout mass. Treatment B and V as w e l l as C and W sprouts averaged 3.5 cm i n length and most seed coats accumulated at the bottom of the j a r s , presumably due to more frequent r i n s i n g . The corresponding "dark" sprouts had p r i m a r i l y smal l , yellow cotyledons (with some greening) which had only s l i g h t l y spread open. The hypocotyls were white except for some pink pigmentation at the base. Treatment D and X sprouts averaged 3.0 cm hypocotyls and the seed coats remained among the sprouts . Treatment E and Y as wel l as F and Z sprouts averaged 4.0 cm hypocotyls and most seed coats had been washed out of the sprout mass. The sprouts from Treatments A to F were har- vested at t h i s stage for the 76. h growth per iod . A clump of moldy seed coats was found in jar D-2. There were no other v i s i b l e signs of contamination or spoi lage . Af te r 100 h the " l i g h t " sprouts exhib i ted a thickened hypocotyl and a more pronounced long, th in r a d i c l e , or primary root . The hypoco- t y l s were l i g h t green, with pink at the base; the r a d i c l e s remained - 85 - white . Sprouts grown with Treatment U averaged 3.5 cm whereas Treatment V and W sprouts averaged 5.0 cm in l ength . The more frequent r i n s i n g appears to encourage sprout growth. The cotyledons were dark green and spread open; e p i c o t y l s were beginning to develop at the base of the cotyledons (Figure 7 ) . Radic les were a lso present in the corresponding "dark" sprouts . There was some pinking of the hypocotyls but otherwise they remained white. Sprout length averaged 5.5 cm i n a l l treatments (X to Z ) . Cotyledons were predominantly yellow with some greening, smaller than those from the corresponding " l i g h t " sprouts , and some were spread open. Sprouting j a r Y-1 contained a much lower y i e l d of sprouts than j a r s Y-2 or Y-3 . There was l i t t l e change a f ter 124 h growth other than a 0.5 to 1.0 cm increase i n sprout length . Radic les were 1 to 2 cm long i n a l l sprouts . E p i c o t y l development continued in the " l i g h t " sprouts but was not yet apparent i n the "dark" sprouts . Af ter a t o t a l of 148 h growth, the " l i g h t " sprouts averaged 5.0 cm i n l ength . Many hypocotyls and r a d i c l e s were bruised or broken and numerous Treatment U and V sprouts were brown. This d i s c o l o r a t i o n was l i k e l y the r e s u l t of enzymatic browning. Franc i s and M i l l i n g t o n (1965b) proposed that a polyphenol oxidase type of enzyme system was involved i n the browning of crushed c lover samples. Treatment W sprouts had no brown d i s c o l o r a t i o n and a much fresher appearance, l i k e l y due to the greater volume and frequency of water appl ied during the growth per iod . The "dark" sprouts averaged 6.0 cm in length . Only the sprouts in j a r Y-1 showed signs of browning of hypocotyls and r a d i c l e s . Cotyledons of a l l samples were yellow because of the lack of l i g h t , however those - 86 - sprouts c loses t to the glass jar s ides did exh ib i t some greening. These sprouts would have been unavoidably exposed to minimal amounts of l i g h t during the r i n s i n g procedure. The d i f ference i n sprout c o l o r between " l i g h t " (Treatment U) and "dark" (Treatment X) sprouts i s evident i n F igure 12. The sprouts from Treatment U to Z were harvested at t h i s stage for the 148 h growth per iod . Seed coats that had not been r insed out of the sprout mass were included i n the sprout samples taken for ana ly s i s ; however, seed coats that had accumulated at the bottom of the j a r s were not included i n sampling. These discarded seed coats f e l t s l i g h t l y slimy but no attempt was made i n t h i s study to i d e n t i f y the microb ia l f l o r a present on the seed coats or on the a l f a l f a sprouts . D. PHYTOESTROGEN ANALYSIS Daidze in , formononetin and coumestrol were detected i n many of the a l f a l f a sprout extracts as determined from HPLC chromatograms. Table 7 l i s t s the mean content of each phytoestrogen ca lcu la ted to be present i n a l f a l f a sprouts corresponding to the various growth treatments. Ana lys i s of the data revealed that the accumulation of each phytoestrogen var ied i n response to the treatments, therefore , the three phytoestrogens w i l l f i r s t be discussed i n d i v i d u a l l y . Ana ly s i s of variance tables are provided i n the Appendices. The treatment combinations were coded for f a c t o r i a l ana ly s i s using two l e v e l s for each f a c t o r . The four factors were coded i n the order of growth per iod , l i g h t dura t ion , r in se volume and r inse frequency; the l e v e l s were assigned as fo l lows : - 87 - Growth period (G) 1 = 76 h 2 = 148 h L ight durat ion (L) 1 = 2 = 0 h 24 h Rinse volume (V) 1 = 0.4 L 2 = 1.0 L Rinse frequency (F) 1 = 2X 2 = 5X 1. Daidzein S ing le factor ana lys i s of variance of the 12 t r i a l treatments (A to Z) and the 2 standard treatments (Moapa and Vernal) was performed to determine the s i g n i f i c a n c e of the observed treatment e f f ec t s . Treatment means of da idzein contents are presented i n Table 7. Both Treatments C and Y had a very large value for one of the three r e p l i c a t e s which contr ibuted to the large standard dev ia t ion of the C and Y means. A l f a l f a sprouts i n jar Y-1 were found to have a da idzein content of 25.9 ± 4.5 yg/g a l f a l f a (dry weight) , which was markedly greater than the other Y r e p l i c a t e s (6.9 and 3.7 yg/g) . This higher daidzein content may be re la ted to contamination of the sprouts i n jar Y-1 . Olah and Sherwood (1971 and 1973) a lso found an increased accumulation of da idze in i n infected a l f a l f a p l ant s . Sprouts from jar C-3 had a da idze in content of 16.3 ± 1.2 yg/g a l f a l f a (dry weight) , which d i f f e r e d great ly from the other C r e p l i c a t e s as i n the case of Treatment Y. Although there were no v i s i b l e signs of spo i lage , some contaminated seed coats or sprouts may have been present and thereby af fected the t o t a l da idzein content. An ana lys i s of variance of t reat- ment means ca r r i ed out excluding the two extreme values (assumed to be - 88 - Table 7. Phytoestrogen dard and t r i a l contents of cond i t ions . a l f a l f a sprouts grown under stan- Phytoestrogens (yg/g a l f a l f a ) 1 ' 2 Treatment Daidzein Formononetin Coumestrol STANDARD Moapa (AA) 2.1 + 0 . 1 2 a b 3 2.9 ± 0.62 a N . D f 4 Vernal (BB) 1.0 + 0.20 a 1.3 ± 0 . 6 4 b c N . D 3 TRIAL A 1.9 + 0A9 ah 0.5 ± 0Ak° N . D 3 B 2.2 + 0 . 2 9 a b 0.7 ± 0.67° N . D ! C 9.3 + 7 . 0 0 a b 0.3 ± 0A6° N . D 3 D 0.2 + 0.06 a 1.6 ± 0 . 6 7 a b c 0.9 ± 0.90 a E 0.9 + 0.06 a 1 .2 ± 0 . 2 1 b c 1.0 ± 1.00 a F 0.7 + 0.12 3 0.1 ± 0.12° N . D 3 U 5.0 + 0 . 7 4 a b 2.7 ± 0 . 5 5 a b 0.6 ± 0 . 5 1 3 V 2.0 + 0 . 8 9 a b 0.6 ± 0 . 2 1 C 2.1 ± 1.53 a w 2.5 + 0A9 ab 0.9 ± 1.17 C 0.9 ± 0.76 a X 2A + 0 . 2 3 a b 9.0 ± 1.36 d 5.5 ± 1.72 b Y 12.2 + 1 2 . 0 0 b 6.3 ± 0.76 6 3.6 ± 0.97 C Z 6.2 + 0 . 3 2 a b 6A ± 0 . 8 7 6 6.0 ± 1.06 b Expressed on dry weight bas i s . 2Mean of 3 r e p l i c a t e s ± standard d e v i a t i o n . t rea tment means i n a column sharing the same superscr ipt 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 t e s t . ^N.D. = Not detectable . - 89 - missing values) more c l e a r l y ind ica ted that a l f a l f a sprouts from Treatments C and Y (as wel l as U and Z) had s i g n i f i c a n t l y greater (P<0.05) da idzein contents from a l l other treatments (Appendix A - 2 ) . F a c t o r i a l ana lys i s of variance of the treatment combinations used to grow a l f a l f a sprouts revealed that only the i n t e r a c t i o n s of growth and l i g h t (G x L) and of l i g h t and r inse volume (L x V) were s i g n i f i c a n t (P<0.05) for da idzein accumulation (Table 8) . The e f fect curve of G x L (Figure 17) shows that a marked increase in daidzein content over growth time occurred i n the "dark" sprouts grown for 76 h vs 148 h. I t may be poss ib le that the s u s c e p t i b i l i t y of a l f a l f a sprouts to i n f e c t i o n i n - creases with time i n sprouts that are grown i n the dark. As previous ly mentioned, increased i n f e c t i o n has been associated with increased da idze in l e v e l s (e .g . Olah and Sherwood, 1971). The e f fect curve of L x V (Figure 18) shows that an increase in r in se volume re su l t ed , in a decreased da idze in content in the "dark" sprouts but an increased content in the " l i g h t " sprouts . However, t h i s i n t e r a c t i o n i s only s i g n i f i c a n t at P = 0.048 and the Student-Newman-Keuls test showed that the treatment means were not s i g n i f i c a n t l y d i f f e ren t (P>0.05). 2. Formononetin The s i g n i f i c a n t l y d i f f e r e n t treatment means for formononetin, as determined by s ing le factor a n a l y s i s of var iance , are i d e n t i f i e d i n Table 7. The formononetin contents of sprouts from Treatments X, Y and Z were shown to be s i g n i f i c a n t l y greater (P<0.05) than from the other treatments. - 90 - Table 8. S i g n i f i c a n c e of ca lcu la ted F-values from germination factors and factor i n t e r a c t i o n s for phytoestrogen accumulation as determined by f a c t o r i a l ana lys i s of var iance . Phytoestrogens Factors Daidzein Formononetin Coumestrol Growth (G) NS * * Light (L) NS * * Rinse volume (V) NS NS NS Rinse frequency (F) NS * NS G x L * * * G x V NS NS NS G x F NS * NS L x V * NS NS L x F NS NS NS G x L x V NS NS * G x L x F NS NS NS * 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) i n t e r a c t i o n daidzein accumulation in a l f a l f a sprouts. _ ! I I 7 6 G R O W T H PERIOD ( h ) 148 - 92 - F i g u r e 18. 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 for daidzein accumulation i n a l f a l f a sprouts. _ l I : I I 0.4 1.0 RINSE VOLUME (L) - 93 - The r e s u l t s of f a c t o r i a l ana lys i s of variance are presented i n Table 8. S i g n i f i c a n t d i f ferences i n formononetin content were found between sprouts grown for 76 h and those grown 148 h, and between sprouts grown i n the "dark" and those grown in the " l i g h t " . As i n the case of da idze in , the i n t e r a c t i o n of G x L (Figure 19) ind ica te s that there i s a d i f f e r e n t formononetin response of the "dark" sprouts than of the " l i g h t " sprouts over the growth per iod . Again, increased concentra- t ions of formononetin may be associated with m i c r o b i a l l y contaminated a l f a l f a sprouts as proposed by Olah and Sherwood (1971), and the "dark" sprouts may be more suscept ib le to i n f e c t i o n and subsequent phytoestro- gen accumulation. Results a l so showed that r i n s e frequency was a s i g n i f i c a n t (P<0.05) factor i n formononetin accumulation. The G x F i n t e r a c t i o n was found to be s i g n i f i c a n t (P<0.05) as i l l u s t r a t e d in Figure 20, where an increase i n r in se frequency re su l ted i n a marked decrease i n formonone- t i n content in the 148 h sprouts but did not a f fec t formononetin accumu- l a t i o n i n the 76 h sprouts . I t appears that increas ing r in se frequency may serve to cleanse the sprouts during growth, as suggested by Lookhart et a l . (1979a), and thereby reduce the l e v e l of contamination and phyto- estrogen accumulation. 3. Coumestrol The s i g n i f i c a n t l y d i f f e r e n t treatment means for coumestrol , as determined by s ing le factor ana lys i s of var iance , are a l so i d e n t i f i e d i n Table 7. Coumestrol could not be detected i n many a l f a l f a sprout samples. O v e r a l l , the coumestrol concentrat ions appeared much lower than the concentrat ions of the other phytoestrogens, however, one must - 94 - - 95 - Figure 20. 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 for formononetin accumulation in a l f a l f a sprouts. _l I I I 2X 5X RINSE FREQUENCY / 2 4 h - 96 - a l so note that the ex t rac t ion e f f i c i e n c y for coumestrol was only 57% and that p o t e n t i a l l y the coumestrol content could be considerably higher . The coumestrol contents of the sprouts from Treatments X, Y and Z were once again found to be s i g n i f i c a n t l y greater (P<0.05) from the other treatments. F a c t o r i a l ana lys i s of variance determined that several factors had a s i g n i f i c a n t ef fect on coumestrol concentrat ion (Table 8 ) . Sprouts grown for 148 h had a s i g n i f i c a n t l y greater (P<0.05) coumestrol content than sprouts grown for 76 h, and the same was true for 0 h l i g h t sprouts vs 24 h l i g h t sprouts . The G x L i n t e r a c t i o n (Figure 21) further i l l u s - t ra te s that the response of coumestrol accumulation over the growth period d i f f e red markedly between "dark" and " l i g h t " sprouts . Lookhart et a l . (1978) reported that removal of the h u l l s (or seed coats) from soy sprouts grea t ly reduced the t o t a l coumestrol content . In the present study, a l f a l f a sprouts from some treatments were observed to have lower l e v e l s of dispersed seed coats as a r e su l t of more frequent r i n s i n g . However, r in se volume and r inse frequency were not shown to s i g n i f i c a n t l y (P<0.05) a f fect coumestrol accumulation. The r e l a t i o n s h i p between disease and coumestrol accumulation has been studied to a much greater extent than between disease and other phytoestrogens. The increase i n coumestrol content with increas ing germination time found i n t h i s study supports the increases i n coumes- t r o l reported by Knuckles et a l . (1976) and Lookhart et a l . (1979a and 1979b). Coumestrol accumulation that has been reported i n response to microb ia l i n f e c t i o n (Loper et a l . , 1967; Sherwood et a l . , 1970; Olah and Sherwood, 1971; Wong and Latch , 1971) has a lso been re la ted to increased growth time by B ickof f et a l . (1969) and Lookhart et a l . (1979a). - 97 - 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 for coumestrol accumulation in a l f a l f a sprouts. - 98 - The two a l f a l f a samples which contained very high l e v e l s of da idzein (Y-1 and C-3) did not have higher l e v e l s of coumestrol (or formononetin) than the other treatment r e p l i c a t e s as might have been expected i f these samples were m i c r o b i a l l y i n f e c t e d . However, the degree of accumulation of each i so f l avono id may be a response to speci- f i c pathogens as demonstrated by many workers (Hanson et a l . , 1965; Sherwood et a l . , 1970; Wong and Latch , 1971; Lookhart et a l . , 1979a). 4. General d i scus s ion Although the accumulation of each of the three phytoestrogens examined i n t h i s study was found to be af fected by d i f f e r e n t f ac tor s , some general trends were observed. The factor i n t e r a c t i o n of growth and l i g h t (G x L) was found to be s i g n i f i c a n t (P<0.05) in the accumulation of da idze in , formononetin and coumestrol . The e f fec t curves (Figures 17, 19, 21) could be interpre ted to show that there was a marked i n - crease i n phytoestrogen content of the a l f a l f a sprouts grown in 0 h l i g h t condi t ions as the growth period was extended from 76 h to 148 h. The a l f a l f a sprouts grown i n 24 h l i g h t over the same growth periods d id not show a s i m i l a r increase i n phytoestrogen content. Based on reports that these phytoestrogens accumulate in a l f a l f a that i s m i c r o b i a l l y in fec ted (Sherwood et a l . , 1970; Olah and Sherwood, 1971) i t may be poss ib le to conclude that those a l f a l f a sprouts grown in the dark for the longer periods are more suscept ib le to mic rob ia l i n f e c t i o n than those in the l i g h t . These sprouts may have accumulated a higher content of phytoestrogens i n response to an i n f e c t i o n . - 99 - Rinse frequency and r inse volume were examined because of the reported r e l a t i o n s h i p between soy sprout c leansing and decreased coumes- t r o l content (Lookhart et a l . , 1979a). However, neither factor was found to be s i g n i f i c a n t (P<0.05) for coumestrol or da idze in accumulation i n a l f a l f a sprouts . Rinse frequency and the i n t e r a c t i o n of growth and frequency were only demonstrated to be s i g n i f i c a n t for formononetin accumulation as discussed p r e v i o u s l y . The two a l f a l f a c u l t i v a r s , Moapa and V e r n a l , were found to have a 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) formononetin content but da idzein and coumestrol contents were 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) (Table 7 ) . O v e r a l l , the phytoestrogen content of these sprouts grown under standard condi t ions was low, i . e . 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) from t r i a l condi t ion sprouts of Treatments A to F . And although i t was not the purpose of t h i s study to develop optimum germination c o n d i t i o n s , i t would appear that the combination of low phytoestrogen content and good v i s u a l a c c e p t a b i l i t y would make the standard condi t ions most des i r - able for a l f a l f a sprout germination. The t o t a l content of phytoestrogens detected i n these a l f a l f a sprouts (1 to 22 ppm) i s minimal i n comparison to the contents reported in infected forage a l f a l f a , which may contain severa l hundred ppm coumestrol alone (Bickof f et a l . , 1967; Loper et a l . , 1967); neverthe- l e s s , 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) phytoestrogen contents were observed i n response to various growth cond i t ions . Previous workers have reported that heal thy, uninfected a l f a l f a contains less than 5 ppm coumestrol (Hanson et a l . , 1965; B ickof f et a l . , 1967; Sherwood et a l . , 1970). A l f a l f a sprouts from Treatments X, Y and Z contained p o t e n t i a l l y - 100 - 9 . 6 , 6.3 and 10.5 ppm coumestrol re spec t ive ly (assuming 57% extract ion) which would appear to ind ica te that some coumestrol accumulation beyond the "hea l thy" l e v e l did occur. Knuckles et a l . (1976) reported that a l f a l f a sprouts and soy sprouts contained 5 and 71 ppm coumestrol r e spec t ive ly which was markedly higher than other vegetables tested (<1.0 ppm). In the present study, many a l f a l f a sprout samples contained greater than 1.0 ppm coumestrol, and therefore could a lso contr ibute coumestrol to the d i e t at a l e v e l higher than that i n other common vegetables . In a d d i t i o n , many of these a l f a l f a sprout samples contained l e v e l s of da idze in and formononetin which would increase the t o t a l phytoestrogen content of t h i s food. However, unl ike the d ie t of a grazing animal , the human d ie t cons i s t s of a great va r i e ty of foods and the phytoestrogens contr ibuted by a minor d ie tary component such as a l f a l f a sprouts may have l i t t l e p h y s i o l o g i c a l s i g n i f i c a n c e . But that conclus ion cannot be made without more knowledge of the metabolism of phytoestrogens i n man. - 101 - CONCLUSIONS The o v e r a l l ob jec t ive of t h i s research pro ject was to use high performance l i q u i d chromatography to examine the i so f l avono id phyto- estrogens present i n a l f a l f a sprouts grown under se lected growth c o n d i t i o n s . A method for the ex t rac t ion of phytoestrogens from a l f a l f a sprouts was developed which consis ted of ex t rac t ing aglycone phytoestro- gens from ground a l f a l f a samples with methanol, removing l i p i d s and c h l o r o p h y l l pigments with petroleum ether and subsequently i s o l a t i n g the phytoestrogens using e thy l ether . The e thyl ether extract was evapor- ated to dryness and the f i n a l residue d i s so lved in methanol for HPLC a n a l y s i s . Recovery of se lected phytoestrogens from a standards mix ranged from 94% (formononetin) to 103% (coumestrol) ; recover ies from spiked a l f a l f a sprouts were found to be 83% (da idze in ) , 78% (formonone- t in ) and 57% (coumestrol) , and compared favorably to reported l i t e r a t u r e values (Lookhart, 1979; Murphy, 1981). An HPLC method developed for the determination of phytoestrogens i n the a l f a l f a extracts employed a reversed-phase oc tadecy l s i l ane column for the separat ion, and UV detect ion at 254 nm. Peak area in tegra tor responses were compared to a standard curve for phytoestrogen quant i ta- t i o n . A l i n e a r gradient methanol/water system containing 1% a c e t i c ac id and 0.01M ammonium acetate at a flow rate of 1.0 mL/min gave basel ine r e so lu t ion of the se lected phytoestrogens i n an e l u t i o n time of 30 min. Thi s HPLC separation appears to be very su i tab le for the quant i t a t ive determination of the major phytoestrogens in a l f a l f a sprouts . - 102 - The phytoestrogens da idze in , formononetin and coumestrol were detected using the a n a l y t i c a l methods developed in the present study. Concentrat ions of formononetin and coumestrol were found to be s i g n i f i - cant ly greater (P<0.05) i n a l f a l f a sprouts grown for a longer per iod (148 h vs 76 h) and those grown i n the dark (0 h l i g h t vs 24 h l i g h t ) . The i n t e r a c t i o n of growth and l i g h t factors was shown to be s i g n i f i c a n t (P_< 0.05) for a l l three phytoestrogens; those a l f a l f a sprouts grown in the dark for the longer growth period had s i g n i f i c a n t l y greater (P<0.05) d a i d z e i n , formononetin and coumestrol contents than the a l f a l f a sprouts grown under the other treatment c o n d i t i o n s . The l e v e l s of r inse volume and r inse frequency examined i n the present study were found to have only a l i m i t e d e f fect on the accumulation of phytoestrogens i n a l f a l f a sprouts . The observed inf luence of growth period and l i g h t durat ion on phytoestrogen accumulation may be the r e su l t of increased s u s c e p t i b i l i t y of the a l f a l f a sprouts to microb ia l i n f e c t i o n ; however, the mic rob ia l f l o r a of a l f a l f a sprouts and seed coats were not enumerated nor i i d e n t i f i e d . The combined phytoestrogen content of the a l f a l f a sprouts was found to range from 1 to 22 ppm on a dry weight basis (or less than 2 ppm fresh weight b a s i s ) . The p h y s i o l o g i c a l s i g n i f i c a n c e of t h i s l e v e l of phytoestrogens i n a human food item has not yet been determined. However, the p o t e n t i a l con t r ibu t ion of phytoestrogens to the human d ie t by a l f a l f a sprouts would appear to be minimal even from those a l f a l f a sprouts grown under unfavorable condi t ions of darkness and extended growth per iod . - 103 - REFERENCES CITED Andersen, 0. M. and Pedersen, W. B. 1983. 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Ana ly s i s of phenol ic ac ids and f lavonoids by high-pressure l i q u i d chromatography. 3. Chromatog. 116: 271. Yos t , R. W., E t t r e , L . S. and Conlon, R. D. 1980. P r a c t i c a l L i q u i d Chromatography - An I n t r o d u c t i o n . Perkin-Elmer C o r p . , Norwalk, CT. Zar, 3. H. 1974. B i o s t a t i s t i c a l A n a l y s i s . P r e n t i c e - H a l l , I n c . , Engle- wood C l i f f s , N3. Appendix A-1 A n a l y s i s of variance for daidzein content of a l f a l f a sprouts from Treatments AA to Z ( 1 to 1 4 ) . VARIABLE NAMES - DAIDZEIN DATA FORMAT ( I2 .F4 . 1 > DAIDZEIN ANALYSIS OF VARIANCE - DAIDZEIN SOURCE OF SUM SO MEAN SO ERROR F-VALUE . PROS TREAT 13 480 42 36.95S 2.6511 0.1491SE-01 ERROR 28 390 .30 13.939 TOTAL 41 870 72 GRAND MEAN 3.4762 STANDARD DEVIATION OF VARIABLE 1 IS 4.6084 FREQUENCIES, MEANS, STANDARD DEVIATIONS * * * * * * * * * * * * * * * * * * * * » * * * * * * * * * * * * * * * * * * * *********** ****************** 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 12.17 5 14 9.267 6.233 0.2333 0.9333 0.7333 STUDENTIZED RANGES FOR NEWMAN-KEUL'S TEST, ALPHA=0.05 2.897 3.499 3.861 4.120 4.322 4.486 4.625 4.745 4.850 4.944 5.029 5.106 5.177 THERE ARE 2 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE 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 0.1272E-01 SECONDS. ANALYSIS COMPLETE Appendix A-2 . Analys i s of variance for daidzein content of a l f a l f a sprouts from Treatments AA to Z (1 to 14 without C-3 or Y-1 va lues ) . VARIABLE NAMES - DAIDZEIN DATA FORMAT ( I2 .1X.F3 .1) TREAT ERROR TOTAL ANALYSIS OF VARIANCE - DAIDZEIN DF SUM SO MEAN SO ERROR F-VALUE 13 26 39 139.19 33.192 172.38 10.707 1.2766 8.3869 PROB 0.26258E-05 GRAND MEAN 2.5950 _ ^ -P- STANDARD DEVIATION OF VARIABLE 1 IS 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 5 14 S.750 6.233 0.7333 STUDENTIZED RANGES FOR NEWMAN-KEUL'S TEST, 2.907 3.514 3.879 4.141 4.346 5.061 5.140 5.211 ALPHA=0.05 4.511 4.652 4.774 4.880 4.975 THERE ARE 2 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE 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 0.1616E-01 SECONDS. ANALYSIS COMPLETE. Appendix B. Analys i s of variance for formononetin content of a l f a l f a sprouts from Treatments AA to Z (1 to 14). VARIABLE NAMES - FORMETIN DATA FORMAT ( I2 .F4 .1) FORMETIN SOURCE TREAT ERROR TOTAL 13 28 41 ANALYSIS OF VARIANCE - FORMETIN SUM SO MEAN SO ERROR F-VALUE 4G.040 301.83 14.120 315.94 23.217 0.50429 PROB 0.220G6E-14 GRAND MEAN 2.450O STANDARD DEVIATION OF VARIABLE 1 IS 2.7760 ( FREQUENCIES. MEANS. STANDARD DEVIATIONS .**•*#***« 1 10 MN FORMETIN 2.900 0.5667 2 11 1 .267 0.8667 3 12 0.5000 9.033 4 13 0.7333 6.267 5 14 0.2667 6.367 0.6667E-01 2.733 STUDENTI ZED RANGES FOR NEWMAN-KEUL'S TEST. ALPHA=0.05 2.897 3.499 3.861 4.120 4.322 4 486 4.625 4.745 4.850 4.944 5.029 5.106 5.177 THERE ARE 5 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS. NO PAIR OF WHICH OIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE 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 0.1316E-01 SECONDS. ANALYSIS COMPLETE. Appendix C . Analys i s of variance for coumestrol content of a l f a l f a sprouts from Treatments AA to Z (T to 14). VARIABLE NAMES - COUMSTRL DATA FORMAT ( I2 .F4 .1 ) COUMSTRL TREAT ERROR TOTAL ANALYSIS OF VARIANCE - COUMSTRL DF SUM SO MEAN SO ERROR F-VALUE 13 28 41 168.63 20.087 188.72 12.972 0.71738 18.082 PROB 0.29041E-09 GRAND MEAN 1.4643 STANDARD DEVIATION OF VARIABLE 1 IS 2.1454 FREQUENCIES. MEANS, STANDARD'DEVIATIONS 4 . * * * * * * * * * * * * * * . j * * * * * * . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * . * * * * * * * * * • • • 1 10 MN COUMSTRL 0.0 2.067 2 1 1 0 .0 0.8667 3 12 0.0 5.467 4 13 0 .0 3.567 5 14 0.0 6.033 0.9000 STUDENTIZED RANGES FOR NEWMAN-KEUL'S TEST, ALPHA=0.05 2.897 3.499 3.861 4.120 4.322 4.486 4.625 4.745 4.850 4.944 5.029 5.106 5.177 0.5667 THERE ARE 3 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS. NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS 8. 5, 4, 13) 12. 14) 7, 10) TIME FOR MULTIPLE RANGE TESTS IS O.1232E-01 SECONDS. ANALYSIS COMPLETE. - 117 - Appendix D . F a c t o r i a l ana lys i s of variance for da idze in , formononetin and coumestrol contents of a l f a l f a sprouts from Treat- ments A to Z . Coding of growth condi t ion factors was explained i n the Results and Discuss ion Sec t ion , and the treatment combinations are coded as fo l lows : 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 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 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 F 0 R M A T « ( F 1 . 0 . 3 F 2 . 0 . 3 F 5 . 1 ) OUTPUT OBSE.FREO.PRED T E R M S ' 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 T E R M S 3 G . L . G ' L . V . G , V . L * V . G * L > V . F . G * F . L * F , G * L * F COMPUTE Time for contro l card process ing was 0.10426 seconds. Cumulative time Is 0.10871 seconds. /*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/'•/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/'•/'•/*/*/*/*/*/••/*/*/'•/*/ Time for data Input and c o r r e l a t i o n matrix was 0.53294E-01 seconds. Cumulative time is 0.16872 seconds. Time for ANOVA table was 0.71316E-01 seconds. Cumulative time is 0.24115 seconds. ESTROGEN ANALYSIS: A n a l y s i s for OAIOZEIN Analys i s of var iance table Sum of Mean Source squares DF square F - r a t l o Probab!1i ty Test tern G 56.751 • 1. 56.751 3.4906 0.07397 RESIDUAL L 0.17778E-01 1. 0.17778E-01 O.10935E -02 0.97389 RESIDUAL G*L 128.44 1. 128.44 7.9004 0.00968 RESIDUAL V 0.77042 1. 0.77042 0.47387E -01 0.82951 RESIDUAL G*V 56.120 1. 56.120 3.4519 0.07549 RESIDUAL L»V 70.384 1. 70.384 4.3292 0.04831 RESIDUAL G*L *V 0.22042 1. 0.22042 0. 13558E -01 0.90828 RESIDUAL F 32.202 1. 32.202 1.9807 0.17214 RESIOUAL G*F 16.0OT 1. 16.0O7 0.98454 0.33098 RESIDUAL L*F 0.10667 1. 0.10667 0.65609E -02 0.9361 1 RESIOUAL G * L * F 66.002 1 . 66 .002 4.0596 0.05525 RESIOUAL Residual 390.19 24. 16.258 Total 842.48 35. Overa11 Overal1 mean standard dev ia t ion DAIDZEIN 3.8OO0 4.9062 Frequencies , means, standard devia t ions for G 1 . . . 2 . . . 18 18 0 MEAN 2.5444 5.0556 P MEAN 2 5444 5.0556 O STDV 3 9822 5.5101 S ERR M 0.95038 0.95038 M u l t i p l e range tes t s F - r a t l o 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 0.07397 Frequencies , means, . 1. . standard deviat ions for L .2 . . 0 MEAN P MEAN 0 STOV S ERR M 18 3.7778 3.7778 6.0065 0.95038 18 3.8222 3.8222 3.67 13 0.95038 M u l t i p l e range tes t s F - r a t i o 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 Frequencies , means, standard devia t ions for G*L 1 1 . . 1 2 . . 2 1.. 2 2 . . 9 9 9 9 0 MEAN 0.63333 4.4556 6.9222 3.1889 P MEAN 0.63333 4.4556 6.9222 3.1889 0 STDV 0.32016 5.0376 7.3700 1.5366 S ERR M 1.3440 1.3440 1.3440 1.3440 M u l t i p l e range tes t s Duncan test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( 1 1 . . . 2 2 . . . 1 2 . ) ( 2 2 . . . 1 2 . . . 2 1 . . ) Newman-Keuls test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( 1 1 . . . 2 2 . . . 1 2 . ) ( 2 2 . . . 1 2 . . . 2 1 . ) Tukey test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( 1 1 . . . 2 2 . . . 1 2 . ) ( 2 2 . . . 1 2 . . . 2 1.. ) Time for m u l t i p l e range test was 0.16081E-01 seconds. Cumulative time Frequencies , means, standard deviat ions for V . . 1 . . . 2 . 12 24 0 MEAN 4.3333 3.5333 P MEAN 3.5611 3.9194 0 STDV 6.9942 3.6071 S ERR M 1.2868 0.86758 M u l t i p l e range tests 0.30527 seconds. f - r a t i o (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 Frequencies , means, standard dev ia t ions for G*V 1.1. 1.2. 2 .1 . 2.2. 6 12 6 12 0 MEAN 1.5833 3.0250 7.0833 4.0417 P MEAN 0.81111 3.4111 6.3111 4.4278 0 STDV 0.73598 4.8483 9.4302 1.7707 S ERR M 1.7352 1.1959 1.7352 1.1959 M u l t i p l e range tes t s F - r a t l o 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 0.07549 Frequencies , means, standard devia t ions for L*v 0 MEAN P MEAN 0 STDV S ERR M .1 1 . 6 6.5500 5.7778 9.7705 1.7352 . 1 2 . 12 2.3917 2.7778 2.4652 1.1959 .2 1 . 6 2.1167 1.3444 0.60470 1.7352 .2 .2 . 12 4.6750 5.0611 4.2760 1.1959 M u l t i p l e range tests Duncan test at 5% p r o b a b i l i t y level There 1s 1 homogeneous subset which Is l i s t e d as fo l lows : ( .2 1. . 1 1 . ) Newraan-Keuls test at 5% p r o b a b i l i t y level There Is 1 homogeneous subset which Is l i s t e d as fo l lows : ( .2 1. . 2 2 . , .1 1 . ) Tukey test at 5% p r o b a b i l i t y leve l There is 1 homogeneous subset which Is l i s t e d as fo l lows : ( .2 1., .1 2 . . .2 2 . , .1 1. ) Time for mul t ip l e range test was O.66795E-02 seconds. Cumulative time is 0.34464 ro O Frequencies , means, standard devia t ions for G*L*V 0 MEAN P MEAN 0 STDV S ERR M 1 1 1 . 0.93333 0. 161 t 1 0.57735E-01 2.3917 1 1 2. 6 0.48333 0.86945 0.28577 1.6686 1 2 1. 3 2.2333 14611 0.28868 2.3917 1 2 2. 6 5.5667 5.9528 6.0105 1 .6688 2 1 1 . 3 12.167 1 1.394 12.001 2 3917 6 3000 6861 1326 2 2 1. 3 2.0000 1.2278 0.88882 2.3917 2 2 2. 6 3.7833 4 1694 1.4798 1.6688 M u l t i p l e range tests F - r a t i o 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 Frequencies , means, standard devia t ions for F 12 24 O MEAN 2.3750 4.5125 P MEAN 2.2556 4.5722 0 STDV 1 8445 5.7804 S ERR M 1.2868 0.86758 M u l t i p l e range tes t s F - r a t l o 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 17214 Frequencies , means, standard dev ia t ions for G*F 1 .1 1 . 2 2 . 1 2 . .2 0 MEAN P MEAN 0 STOV S ERR M 6 1 .0500 0.93056 0.94816 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 tests F-ra t to 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 Frequencies , means, standard dev ia t ions for L*F .1.1 .1.2 .2.1 .2.2 0 MEAN P MEAN 0 STDV S 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 tests F - r a t l o 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 0.93611 Frequencies , means, standard devia t ions for G*L*F 1 1.1 1 1.2 3 6 0 MEAN 0.23333 0.83333 P MEAN 0.11389 0.89306 0 STDV 0.57735E-O1 0.13663 S ERR M 2.3917 1.6688 M u l t i p l e range tes t s 1 2 . 1 3 1.8667 1.7472 0.49329 2.3917 1 2.2 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 3 5.0333 4.9139 0.7371 1 2.3917 2 2 2 6 2.2667 2.3264 0.70616 1 .6688 F - r a t l o 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 / * / • / * / * / * / * / * / * / * / * / * / * / * / * / * / * / * / * / / * / * / * / * / * / * / * / * / * / * / * • / * / * / * / * / * / * / * / * / * / * / * / * / * / * / * / * / * / ^ / * / * / * / * / * / * / * / * / * / * / * / * / * / * • / * / * • / Analy s i s for FORMETIN Analys i s of variance table Sum of Mean Source squares square F - r a t l o P r o b a b i l i t y Test term 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 12.533 309.42 115.92 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 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 RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL RESIDUAL Overa11 mean 2.5111 Overa11 standard dev ia t ion 2.9733 Frequencies , means, standard dev ia t ions for G 1. . . 2. . . 18 18 O MEAN 0.71667 4.3056 P MEAN 0.71667 4.3056 0 STDV 0.66355 3.3078 S ERR M 0.17033 0.17033 M u l t i p l e range tes t s Duncan test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( 1 . . . ) ( 2 . . . ) Newman-Keuls test at 5 * p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows: ( 1 . . . ) ( 2 . . . ) Tukey test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( 1 . . . ) ( 2 . . . ) Time for mul t ip l e range test was 0.66929E-02 seconds. Cumulative time Is 0.44469 seconds. Frequencies , means, standard devia t ions for L . 1 . . . 2 . . 18 18 0 MEAN 4.0778 0.94444 P MEAN 4.0778 0.94444 0 STDV 3.4622 1.0083 S ERR M 0.17033 0.17033 M u l t i p l e range tes t s Duncan test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( . 2 . . ) ( . 1. . ) Newman-Keuls test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( . 2 . . ) ( . 1. . ) Tukey test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( 2. . ) ( . 1 . . ) Time for m u l t i p l e range test was 0.61455E-02 seconds. Cumulative time Is 0.46163 seconds. Frequencies , means, standard devia t ions for G*L 1 1 . . 1 2 . . 2 1 . . 2 2 . 9 0 MEAN 0.93333 P MEAN 0.93333 0 STDV 0.75993 S ERR M 0.24088 9 0.50000 0.50000 0.50249 0.24088 9 7.2222 7 2222 1.6269 0.24088 9 1.3B89 1.3889 1.2098 0.24088 M u l t i p l e range tes t s Duncan test at 5% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as fo l lows : ( 1 2 . 1 1 . ) ( 1 1. . . 2 2 . . ) ( 2 1 . . ) Newman-Keuls test at 5% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as fo l lows : ( 1 2. . . 1 1. . ) ( 1 1 . . , 2 2 . . ) ( 2 1 . ) Tukey test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( 2 1 . ) me for m u l t i p l e range test was 0.86842E-02 seconds. Cumulat Frequencies , means, standard dev ia t ions for V . . 1 . .2. 12 24 0 MEAN 2.1833 2.6750 P MEAN 2.7056 2.4139 0 STDV 2.5135 3.2167 S ERR M 0.23063 0.15549 M u l t i p l e range tes t s F - r a t l o 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 0.33271 Frequencies , means, standard devia t ions for G*V 1.1. 1.2. 2 .1 . 2.2. 6 12 6 12 0 MEAN 0.95000 0.60000 3.4167 4.7500 P MEAN 1.4722 0.33889 3.9389 4.4889 0 STDV 0.50100 0.72237 3.1619 3.4233 S ERR M 0.31098 0.21433 0.31098 0.21433 M u l t i p l e range tes t s F - r a t i o 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 0.10860 Frequencies , means, standard deviat ions for L*V . 1 1 . . 1 2 . .2 1. .2 2. 6 12 6 12 0 MEAN 3.7167 4.2583 0.6S0O0 1.0917 P MEAN 4.2389 3 9972 1.1722 0.83056 0 STDV 2.8379 3.8415 0.45055 1.1866 S ERR M 0.31098 0.21433 0.31098 0.21433 M u l t i p l e range tes t s F - r a t l o 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 Frequencies , means, standard devia t ions for G*L*V 1 1 1 . 1 1 2 . 1 2 1 . 1 2 2 . 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 tests F - r a t l o 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 0.71668 ve time Is 0.48031 seconds. 2 1 1 . 3 6.2667 6.7889 0.76376 0.42865 2 1 2. 6 7.7000 7.4389 1 .7833 0 29909 2 2 1. 3 0.56667 1 .0889 0 20817 O. 42865 2 2 2. 6 1 8000 1 .5389 1 3100 0 29909 Frequencies , means, standard dev ia t ions for F . . . 1 . . .2 0 MEAN P MEAN 0 STDV S ERR M 12 3.4583 3.5556 3.534 1 0.23063 24 2.0375 I.9889 2.6033 O.15549 M u l t i p l e range tes t s Ouncan test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( . .2 ) ( . . . 1 ) Newman-Keuls test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( . . . 2 ) ( . . . 1 ) , Tukey test at 5% p r o b a b i l i t y level —* There are 2 homogeneous subsets which are l i s t e d as fo l lows: fNJ ( . 2 , , ( . . . 1 ) Time for mul t ip le range test was 0.61197E-02 seconds. Cumulative time is 0.54491 seconds. Frequencies , means, standard devia t ions for G*F 1 .1 1 . 2 2 . 1 2 . 2 6 12 6 12 0 MEAN 1.0333 0.55833 5.8833 3.5167 P MEAN 1.1306 0.50972 5 9806 3.4681 0 STDV 0.77115 0.57280 3 5735 3.0114 S ERR M 0.31098 0.21433 0.31098 0.21433 M u l t i p l e range tests Ouncan test at 5% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as fo l lows : ( 1 . 2 . 1 .1 > ( 2 . 2 ) ( 2 . 1 ) Newman-Keuls test at 5% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as fo l lows: ( 1 2 . 1 . 1 ) ( 2 . 2 ) ( 2 . 1 ) Tukey test at 5% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as fo l lows : ( 1. .2. 1..1 ) ( 2 . 2 ) ( 2 . 1 ) Time for m u l t i p l e range test was O.9O237E-02 seconds. Cumulative time is 0.56561 seconds. Frequencies , means, standard dev ia t ions for L*F .1.1 . 1 2 .2.1 .2.2 0 MEAN P MEAN 0 STDV S ERR M 6 5.3000 5.3972 4.2005 0.31098 12 3.4667 3.4181 3.0467 0.21433 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 tes 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 0.09267 Frequencies , means, standard devia t ions for 1 1 . 1 1 1 . 2 1 2 . 1 G*L*F o 0 MEAN P MEAN 0 STDV 5 ERR M 3 1 .5667 1.6639 0.665B3 0.42865 6 0.61667 0.56806 0.62102 0 29909 3 0.50000 0.59722 0.43589 0.42865 1 2.2 6 0.50000 0.45139 0 57271 0.29909 2 1.1 3 9.0333 9.1305 1.3614 0.42865 2 1.2 6 6.3167 6.2680 0 73598 0. 29909 2 2.1 3 2.7333 2.8306 0.55076 0.42865 2 2 2 6 0 . 7 1 6 6 7 0.66806 0.77050 0.29909 M u l t i p l e range tes 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*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/*/ Analy s i s for COUMSTRL Ana ly s i s of variance table Sum of Mean Source squares DF square F - r a t l o Probab i l I ty Test term G 69.167 1. 69.167 82 642 0 OOOOO RESIDUAL L 45.563 1. 45.563 54.439 0.00000 RESIDUAL G*L 23.200 1. 23.200 27.720 0.O0O02 RESIDUAL V 0.20417E -01 1. 0.204 17E -01 0.24394E -01 0.87719 RESIDUAL G*V 1.9837 1. 1.9837 2.3702 0.13675 RESIDUAL L'V 2.6004 1 . 2.6004 3.1070 0.09069 RESIDUAL G * L ' V 8.2838 1. 8.2837 9.8976 0 004 38 RESIDUAL F 0.41668E -03 1. 0.41668E -03 0.49786E -03 0.98238 RESIDUAL G*F 1 . 1704 1 . 1. 1704 1.3984 0.24857 RESIDUAL L*F 0. 15042 1. 0.15042 0 17972 0.67539 RESIDUAL G*L *F Res iduat Tota l 0.51042 20.087 173.71 I. O 51042 0.60986 24. 0.83G94 35. Overa11 Overa l l mean standard dev ia t ion COUMSTRL 1.7083 2 2278 Frequencies , means, standard devia t ions for G 1... 2 . . . 18 18 0 MEAN 0.32222 3.0944 P MEAN 0.32222 3.0944 0 STDV 0.65937 2.3905 S ERR M 0.21563 0 21563 M u l t i p l e range tes t s Duncan test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( 1 . ) ( 2 . . . ) Newman-Keuls test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( 1. . . ) ( 2. . ) Tukey test at S% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( 2. . . ) Time for mul t ip l e range test was 0.66795E-02 seconds. Cumulativ Frequencies , means, standard deviat ions for L . 1 . . .2. . 18 18 0 MEAN 2.B333 O 58333 P MEAN 2.8333 0 58333 0 STDV 2 5652 0.97874 S ERR M 0.21563 0.21563 M u l t i p l e range tes t s Duncan test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( . 2 . . ) ( . 1 . . ) O. 44248 RESIDUAL time is 0.63022 seconds. Newman-Keuls test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as follows ( . 2 . . ) ( . 1 . . ) Tukey test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows : ( . 2 . . ) ( . 1. . ) Time for m u l t i p l e range test was 0.63B10E-02 seconds. Cumulative time is 0.64552 seconds. Frequencies , means, standard dev ia t ions for G*L I t . . 1 2 . . 2 1 . . 2 2 . . 9 9 9 9 0 MEAN 0.64444 0 0 5.0222 1.1667 P MEAN 0.64444 0 22335E-05 5.0222 1.1667 0 STDV O 83083 0.0 1.5849 1.1269 S ERR M 0.30495 0.30495 0.30495 0.30495 M u l t i p l e range tests Duncan test at 5% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as fo l lows: ( 1 2 . . . 1 1. ) ( 1 1 . . . 2 2 . . ) ( 2 1 . . ) Newman-Keuls test at S% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as fo l lows: ( 1 2 . . . 1 1.. ) ( 1 1. . . 2 2 . . ) ( 2 1 . ) Tukey test at 5% p r o b a b i l i t y level There are 2 homogeneous subsets which are l i s t e d as fo l lows: ( 1 2 . . . 1 1. . , 2 2 . . ) ( 2 1 . ) Time for m u l t i p l e range test was 0.98696E-02 seconds. Cumulative time (s 0 66526 Frequencies , means, standard devia t ions for V . . 1. . 2 . 12 24 0 MEAN 1.6667 1.7292 P MEAN 1.6694 1.7278 0 STDV 1.6356 S ERR M 0.29197 2.5043 0. 19684 M u l t i p l e range tests F - r a t i o 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 Frequencies , means, standard devia t ions for G*V 1.1. 1.2. 2 .1 . 2.2. 6 12 6 12 0 MEAN 0.51667 0.22500 2.8167 3.2333 P MEAN 0.51944 0 22361 2.8194 3.2319 0 STOV 0.84951 0.55942 1.4106 2.8043 S ERR M 0.39369 0.27133 0.39369 0.27133 M u l t i p l e range tes 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 0.13675 Frequencies , means, standard devia t ions for L*V . 1 1 . . 1 2 . .2 1. .2 2. 6 12 6 12 0 MEAN 2.3000 3.1000 1.0333 0.35833 P MEAN 2.3028 3.0986 1.0361 0.35695 0 STDV 1.6444 2.9508 1.4895 0.55343 S ERR M 0.39369 0.27133 0.39369 0.27133 M u l t i p l e range tests F - r a t i o 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 Frequencies , means, standard devia t ions for G*L*V 1 1 1 . 1 1 2 . 3 6 0 MEAN 1.0333 0.45000 P MEAN 1.0361 0 44861 0 STDV 1.0017 0.75299 S ERR M 0.54266 0.37864 M u l t i p l e range tes t s 1 2 1. 1 2 2. 3 6 0 .0 0 .0 0.27793E-02 -0.13873E-02 0.0 0.0 0.54266 0.37864 Duncan test at 5% p r o b a b i l i t y level There are 4 homogeneous subsets which are l i s t e d as fo l lows : ( 1 2 2 . 1 2 1 . ( 2 2 2 . , 1 1 1 . ( 2 2 1 . . 2 1 1 . ( 2 1 2 . ) 1 1 2 , 2 2 2 . , 2 2 1 . ) ) 2 1 1 . 3 3.5667 3.5694 0.97125 0.54266 Newman-Keuls test at 5% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as fo l lows : ro NO I 2 1 2. 6 5.7500 5.7486 1.3172 0.37864 2 2 1. 3 2.0667 2.0694 1 .5308 0.54266 2 2 2. 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 test at 5% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as fo l lows: ( 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 1 . , 2 1 2 ) Time for m u l t i p l e range test was 0.14140E-01 seconds. Cumulative time Frequencies , means, standard deviat ions for F . . . I . . . 2 12 24 0 MEAN 1.7333 1.6958 P MEAN 1.7139 1.7056 0 STDV 2.4325 2.1731 S ERR M 0.29197 0.19684 M u l t i p l e range tes 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 0.98238 Frequencies , means, standard devia t ions for G*F 1 . . 1 1 . 2 2 . 1 2 . 2 6 0 MEAN 0.45000 P MEAN 0.43056 0 STDV 0.75299 S ERR M 0.39369 12 25833 26806 6331 1 27133 6 3.0167 2.9972 2.9151 0.39369 12 3.1333 3.1431 2.2281 0.27133 M u l t i p l e range tes t s F - r a t i o 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 Frequencies , means, standard deviat ions for L*F .1.1 .1.2 .2.1 .2.2 6 12 6 12 0 MEAN 3.1833 2.6583 0.28333 0 73333 P MEAN 3.1639 2.6681 0.26389 0.74306 0 STOV 2 7874 2.5568 O 44907 I 1468 S ERR M 0.39369 0.27133 0.39369 0.27133 M u l t i p l e range tests F - r a t l o 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 Frequencies , means, standard deviat ions for G*L*F 0.72939 seconds. 0 MEAN 0. 90000 P MEAN 0.88056 0 STDV 0.90000 S ERR M 0.54266 6 3 6 3 6 3 6 0.51667 0.0 0.0 5.4667 4.8000 0.56667 1.4667 0 52639 -O.19442E-01 0 97240E-02 5.4472 4.8097 0.54722 1.4764 0.84951 0 0 0 .0 1.7243 1.6285 0.51316 1.2660 0.37864 0.54266 0.37864 0.54266 O 37864 O 54266 0.37864 M u l t i p l e range tes t s F - r a t l o 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

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