Environmental and Experimental Botany, Vol. 26, No. 1, pp. 65-73, 1986. 0098~472/86 $3.00 + 0.00 Printed in Great Britain. © 1986. Pergamon Press Ltd.

PHENOLIC ACIDS AND FLAVONOIDS IN SOYBEAN R O O T AND LEAF EXTRACTS

P. M. PORTER, W. L. BANMIAIRT ~ d J. J. HASSETT

Department of Agronomy, University of Illinois, Urbana, IL 61801, U.S.A.

(Received 22 March 1985; accepted in revised form 6 June 1985)

PORTER P. M., BANWART W. L. and HASSETTJ. J. Phenolic acids andflavonoids in soybean root and leaf extracts. ENVIRONMENTAL AND EXPERIMENTAL BOTANY 26~ 65 73, 1986.--An HPLC technique and a GC-MS method were used to analyze extracts from soybean leaves and roots for phenolic acids and flavonoids. The reverse-phase HPLC technique involved comparing relative retention times and wavelength area ratios of standard compounds to unknown compounds in the soybean extracts. In addition, the standard compounds and extracts were derivatized with a silylation reagent, yielding trimethylsilyl (TMS) derivatives, and analyzed by GC-MS. The HPLC and GC MS analyses of the hydrolyzed soybean root extract revealed the presence of 4-hydroxy- benzoic, vanillic, ferulic, gentisic, syringic and protocatechuic acids, genistein, daidzein and coumestrol. Results from the HPLC analysis of the hydrolyzed leaf extract indicate the presence of salicylic, 4-hydroxybenzoic, vanillic, 4-hydroxycinnamic, ferulic, caffeic, gentisic, and syringic acids, naringenin, quercetin, genistein, and daidzein. GC-MS analysis of the leaf extract confirmed the presence of all of these compounds except salicylic acid. In addition, the GC-MS analysis of the leaf extract also indicated the presence of kaempferol.

INTRODUCTION

ONE OF the largest and most diverse groups of compounds implicated as agents in allelopathic interactions are the phenolic acids. ~ls) Several of these compounds have been identified in water leachates of various plants and some have been shown to have deleterious effects on the germin- ation and growth of other plants. ~8~ Numerous investigators have analyzed hydrolyzed soybean leaf extracts for phenolic and flavonoid com- pounds. Based on high pressure liquid chromato- graphy (HPLC) retention times of standard phenolic acids, MURPHY and STUTTE (9) tentatively identified the following eight compounds from the hydrolyzed soybean (var. Davis) leaf extracts: gallic, protocatechuic, 4-hydroxybenzoic, salicy- lic, vanillic, caffeic, 4-hydroxycinnamic, and fer- ulic acids. The same procedure was used by HARDIN and STUTTE (5) in their studies of the

Forrest cultivar. They reported the presence of sinapic acid, naringenin and quercetin in ad- dition to the eight compounds listed above.

There is little reason to believe that the pheno- lic acids and flavonoids thus far identified in the soybean exist only in the seed and above-ground portions of the plant. Below-ground plant parts may be equally important in assessing the poten- tial allelopathic effects, particularly as it may relate to root exudation or decomposition.

The purpose of this paper is to report our findings from the analyses of hydrolyzed soybean leaf extracts prepared according to the methods of HARDIN and STUTTE (5) and to compare those analyses with hydrolyzed extracts from the roots of the same plants. In addition to analysis by three separate HPLC gradients, the extracts were also subjected to analysis using gas chromatography- mass spectrometry (GC-MS).

65

66 P .M. PORTER et al.

MATERIALS AND METHODS

Chemicals All the chemicals and compounds were ob-

tained from commercial sources except daidzein, which was provided by Dr. John Ingham (University of Reading, England). Standards were prepared using 10/2g/ml of the compounds listed in Table 1. None of the standard com- pounds exhibited major impurity peaks when chromatographed independently.

Plant materials and extracts Soybeans (Glycine max) of the Forrest cultivar

were grown in 15 cm greenhouse pots (5 plants/pot) filled with sand. Leaf and root samples were taken when plants were at the 8th vegetative leaf stage of growth. ~3) The leaf extracts were prepared following the method of MURPHY and STUTTE (9) by extracting 1.0 g of fresh soybean leaf tissue from fifth trifoliolates in 6.0 ml of 2% acetic acid for 10 min in a boiling water bath. The

extract was filtered through a 5.0 #m Millipore filter, hydrolyzed in 1.0 N HC1 for 1 hr in a boiling water bath, and then extracted with approxi- mately 4 ml of anhydrous diethyl ether. The ether extract was reduced to dryness under a stream of purified nitrogen, and the resulting residue was then redissolved in 1.0 ml of spectral grade methanol.

To obtain root samples the plants were cut at the surface of the sand. Roots and sand were removed from the pots and the sand was separ- ated from the roots by shaking. Roots were cut into 2-3 cm lengths and mixed. One gram samples of fresh roots were then extracted using the same procedure described above for leaves.

Prior to HPLC analysis all samples were spiked with two internal references, 2,4-DHBA and indole. For G C - M S analysis, aliquots of the extracts to be analyzed were first evaporated to dryness under a stream of nitrogen. Trimethyl- silyl (TMS) derivatives were then made by dissolving a portion of the crystals formed in

Table 1. Selected standards analyzed by HPLC and GC-MS

Abbreviation Common name Chemical name (where applicable)

Gallic acid Gentisic acid Protocatechuic

acid fl-Resorcylic

acid*

Vanillic acid Caffeic acid Salicylic acid Syringic acid

p-Coumaric acid Ferulic acid Scopoletin Indole* Daidzein Naringenin O uercetin Genistein Coumestrol Kaempferol

3,4,5-Trihydroxybenzoic acid 2,5-Dihydroxybenzoic acid

3,4-Dihydroxybenzoic acid

2,4-Dihydroxybenzoic acid 4-Hydroxybenzoic acid 4-Hydroxy-3-methoxybenzoic acid 3,4-Dihydroxycinnamic acid 2-Hydroxybenzoic acid 4-Hydroxy-3,5-dimethoxybenzoic

acid 4-Hydroxycinnamic acid 4-Hydroxy-3-methoxycinnamic acid 7-Hydroxy-6-methoxycoumarin 1-Benzo(fl)pyrrole 4',7-Dihydroxyisoflavone 4',5, 7-Trihydroxyflavanone 3,3',4',5,7-Pentahydroxyflavone 4',5,7-Trihydroxyisoflavone 3,9-Dihydroxycoumestan 3,4',5,7-Tetrahydroxyflavone

3,4,5-THBA 2,5-DHBA

3,4-DHBA

2,4-DHBA 4-HBA 4-H,3-MBA 3,4-DHCA 2-HBA

4-H,3,5-DMBA 4-HCA 4-H,3-MCA

*The two internal references for the HPLC analyses of the soybean extracts.

PHENOLIC ACIDS AND FLAVONOIDS IN SOYBEAN ROOT AND LEAF EXTRACTS 67

approximately 0.2 ml of the silylation reagent bis (trimethylsilyl) trifluoroacetamide (BSTFA), and then gently heating the solution at 60°C for about 15 min.

length area ratio for each compound was ob- tained from one of the three gradient analyses by dividing the integrated area of the 254 nm peak by the integrated area of the 280 nm peak. (1)

HPLC analysis The extracts and standard compounds were

analyzed by a modification of the method of MURPHY and STUTTE tg) using a Beckman HPLC gradient elution system with mixtures of solvent A (98% water and 2% glacial acetic acid in 0.018 M ammonium acetate) and solvent B (68% water, 25% methanol, 5% butanol and 2% glacial acetic acid in 0.018 M ammonium acetate). Three separate gradients, as described by BANWART et al., ~1) were used to elute and help identify the compounds present. The column used was a 25 cm Ultrasphere Cls column and the sample size was 20 /21. Compounds were detected by u.v. absorption at 254 and 280 nm. Standard com- pounds were chromatographed alone and as mixtures. Relative retention times for the stan- dard compounds, as well as for the major peaks in the extracts, were determined for each gradient by dividing the compound's retention time by the retention time of an internal reference. A wave-

GC-MS analysis The GC-MS analyses of the TMS derivatized

extracts and standards were performed on a 5985 GC-MS Hewlett Packard system. The samples were analyzed by electron impact. The GC column was a narrow bore, 183 cm glass column, packed with 3% SP-2100. The carrier gas was helium and the flow rate was 30 ml/min. The temperature gradient was from 150 to 280°C at 5°C per min.

R E S U L T S A N D D I S C U S S I O N

The hydrolyzed leaf and root extracts were analyzed at 280 nm by gradients 1, 2 and 3 and at 254 nm by gradient 1. The relative retention times with respect to the two internal references for each of the three gradients, as well as the 254 : 280 nm wavelength area ratio for gradient 1, were determined and compared to data obtained

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T I M E IN M I N U T E S FIe:. 1. HPLC chromatogram of the hydrolyzed leaf extract (280 nm, gradient 2; indole

and 2,4-DHBA were added as internal references).

68 P .M. PORTER et al.

LU

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T I M E IN M I N U T E S

FIo. 2. HPLC chromatogram of the hydrolyzed root extract (280 nm, gradient 2; indole and 2,4-DHBA were added as internal references).

from selected standard compounds. The use of internal reference compounds, three separate gra- dients, and wavelength area ratios allow for the detection of more than 50 potential allelochemi- cals. (1) Typical HPLC chromatograms of the hydrolyzed leaf and the hydrolyzed root extracts are shown in Figs 1 and 2. The relative retention times and the wavelength area ratios for selected standards and the major peaks in GC analyses of the leaf and root extracts are shown in Table 2. Failure to observe any particular peak could indicate either the absence of that compound or a concentration in the hydrolyzed extract that was too low to be detected.

Based on relative retention times from the three gradients and the wavelength area ratio, the hydrolyzed leaf extract was found to contain the following compounds: gentisic (2,5-DHBA), 4- hydroxybenzoic (4-HBA), vanillic (4-H,3-MBA), caffeic (3,4-DHCA), salicylic (2-HBA), 4-hydro- xycinnamic (4-HCA) and ferulic (4-H,3-MCA) acids as well as naringenin, quercetin and genis- tein (see Table 1 for details of abbreviations). By these same methods the following compounds

were identified in the hydrolyzed root extract: 4- hydroxybenzoic (4-HBA), vanillic (4-H,3-MBA), ferulic (4-H,3-MCA) and gentisic (2,5-DHBA) acids, as well as genistein, daidzein and coumestrol.

The hydrolyzed leaf extract and the hydro- lyzed root extract were subjected to analysis by GC MS to substantiate results from the HPLC analysis. In Table 3 the GC retention times and the MS fragmentation pattern of selected stan- dard compounds are listed. The data in Tables 4 and 5 show the G C - M S results obtained for major compounds present in the hydrolyzed leaf and root extracts, respectively. Based on similar GC retention times and MS fragmentation patterns, many of the compounds present in the extracts were identified. The GC separations and com- pound names are given in Figs 3 and 4.

The G C - M S analysis of the hydrolyzed root extract confirmed the occurrence of the seven compounds initially identified by the HPLC analysis, and also revealed two additional compounds--protocatechuic and syringic acids. Although not shown in Fig. 2, later HPLC studies

P H E N O L I C A C I D S A N D F L A V O N O I D S IN S O Y B E A N R O O T A N D L E A F E X T R A C T S

Table 2. Relative retention times ( RR T* ) and 254 : 280 n m area ratios for the HPLC analysis of selected standard compounds ( Std) and the hydrolyzed extracts of soybean leaves and roots

69

G r a d i e n t 1 R R T G r a d i e n t 2 R R T G r a d i e n t 3 R R T A r e a rat ios C o m p o u n d s Std L e a f R o o t Std L e a f Roo t Std L e a f R o o t Std L e a f Roo t

U n k n o w n L1 . . . . 0.43 - - - - 0.44 - - - - 1.7 - - 3 , 4 , 5 - T H B A 0.40 - - - - 0.45 - - - - 0.46 - - - - 0.6 - - - - U n k n o w n L2 - - 0.44 - - - - 0.48 - - - - 0.49 - - - - 1.2 - - 2 , 5 - D H B A 0.59 0.59 0.60 0.65 0.67 0.70 0.65 0.68 0.66 1.0 0.8 1.0 3 , 4 - D H B A 0.72 - - - - 0.75 - - - - 0.77 - - - - 2.0 - - - - 2 , 4 - D H B A ( R e f ) t 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 3.5 2.7 2.6 4 - H B A 1.18 1.18 1.17 1.21 1.27 1.27 1.20 1.23 1.16 3.7 0.4 4.0 4 - H , 3 - M B A 1.55 1.52 1.55 1.52 1.47 1.48 1.46 1.44 1.43 2.0 0.2 2.7 3 , 4 - D H C A 1.76 1.66 - - 1.67 1.68 1.63 1.62 - - 0.6 0.5 - - 2 - H B A 1.80 1.74 - - 1.95 1.81 - - 1.89 1.86 - - 0.2 0.3 - - 4 - H , 3 , 5 - D M B A 1.87 - - - - 1.77 - - - - 1.65 - - - - 0.5 - - - - U n k n o w n L3 - - 2.22 - - - - 2.28 - - - - 2.14 - - - - 0.6 - - 4 - H C A 2.53 2.49 - - 2.66 2.64 - - 2.44 2.44 - - 0.2 0.2 - - 4 - H , 3 - M C A 2.83 2.83 2.83 3.13 3.06 2.96 2.74 2.73 2.72 1.2 0.6 1.0 U n k n o w n R1 - - - - 3.38 - - - - 4.32 - - - - 3.33 - - - - 1.0 Indole (Ref ) '~ 3.66 3.59 3.59 4.73 4.60 4.61 4.27 4.22 4.12 0.6 0.6 0.7 U n k n o w n R 2 - - 3.85 - - 5.18 - - 4 .14 - - - - 1.4 D a i d z e i n 4.41 - - 4.34 5.99 - - 5.95 5.89 - - 5.73 2.4 - - 2.7 N a r i n g e n i n 4.79 4.77 - - 6.57 6.81 - - 6.47 6.89 - - 0.1 3.0 - - U n k n o w n R3 . . . . . 6.04 - - - - 5.85 - - - - 2.7 Q u e r c e d n 4.95 5.01 - - 7.10 7.33 - - 7.21 7.42 - - 2.0 1.7 - - Gen i s t e in 4.96 4.86 4.93 7.00 6.98 7.09 6.99 7.01 2.5 3.0 2.0 C o u m e s t r o l 5.80 - - 5.90 8.21 - - 8 .34 8.55 - - 8.40 4.1 - - 4.9 Kaempferol :~ . . . . . . . . . . . .

* R e t e n t i o n t ime re la t ive to 2 , 4 - D H B A . t 2 , 4 - D H B A a n d indole were a d d e d to all s amples as i n t e rna l references pr ior to H P L C analysis . ~ K a e m p f e r o l was no t de tec ted by the H P L C m e t h o d s descr ibed b u t was f o u n d in the h y d r o l y z e d

lea f ex t r ac t by G C - M S analysis .

i n o u r l a b o r a t o r i e s u s i n g m o r e c o n c e n t r a t e d

h y d r o l y z e d r o o t e x t r a c t s s i m i l a r l y r e v e a l e d t h e

p r e s e n c e o f p r o t o c a t e c h u i c a n d s y r i n g i c a c i d s .

T h e G C M S a n a l y s i s o f t h e h y d r o l y z e d l e a f

e x t r a c t v e r i f i e d t h e p r e s e n c e o f 9 o u t o f t h e 10

c o m p o u n d s i d e n t i f i e d u s i n g t h e H P L C t e c h n i q u e .

O n l y s a l i c y l i c a c i d w a s n o t f o u n d b y t h e G C M S

a n a l y s i s . I t is p o s s i b l e t h a t t h i s c o m p o u n d o c c u r s

i n t h e e x t r a c t i n s m a l l q u a n t i t i e s b u t t h a t i t s

p r e s e n c e is m a s k e d b e c a u s e i t e l u t e s f r o m t h e G C

c o l u m n n e a r t h e s o l v e n t f r o n t w i t h t h e o t h e r m o r e v o l a t i l e c o m p o u n d s . S y r i n g i c a c i d , d a i d z e i n a n d

k a e m p f e r o l w e r e i n d i c a t e d b y G C - M S a n a l y s i s

b u t n o t t h e i n i t i a l H P L C a n a l y s i s . F u r t h e r a n a -

lyses in o u r l a b o r a t o r i e s w i t h m o r e c o n c e n t r a t e d

l e a f e x t r a c t s h a v e c o n f i r m e d t h e p r e s e n c e o f

s y r i n g i c a c i d a n d d a i d z e i n . T h e H P L C m e t h o d s

u s e d d o n o t a l l o w d e t e c t i o n o f l o w leve l s o f

k a e m p f e r o l d u e to u n d e f i n e d b r o a d p e a k s .

E i g h t o f t h e 13 c o m p o u n d s i d e n t i f i e d i n t h e l e a f

e x t r a c t b y t h e H P L C a n d G C M S a n a l y s e s

c o r r e s p o n d to t h e c o m p o u n d s r e p o r t e d b y

HARDIN a n d STUTTE. (5) G e n t i s t i c a n d s y r i n g i c

ac ids , g e n i s t e i n , d a i d z e i n a n d k a e m p f e r o l r e p r e -

s e n t f ive a d d i t i o n a l c o m p o u n d s i d e n t i f i e d in t h e s e

s t u d i e s . HARDIN a n d STUTTE [5) f o u n d t h r e e c o m -

p o u n d s ( s i n a p i c , p r o t o c a t e c h u i c a n d ga l l i c ac ids )

n o t i d e n t i f i e d i n o u r s t u d i e s . N o s i n a p i c a c i d

s t a n d a r d w a s a n a l y z e d b y o u r H P L C m e t h o d s

n o r w a s i ts e x p e c t e d f r a g m e n t a t i o n p a t t e r n

f o u n d b y G C - M S a n a l y s i s . N o d e t e c t a b l e a m o u n t

o f p r o t o c a t e c h u i c a c i d w a s i b u n d b y e i t h e r H P L C

70 P. M. PORTER et al.

Table 3. GC and MS data for the TMS derivatives of selected standard compounds

GC retention MS

Standard time fragmentation pattern compound (min) (M/Z)

2-HBA 3.3 281,267, 209, 149, 135 4-HBA 4.3 282, 267, 223, 193 4-H,3-MBA 6.3 312, 297, 282, 267, 253, 223, 188 2,5-DHBA 6.8 370, 355 3,4-DHBA 7.4 370, 355, 31 I, 193 4-H,3,5-DMBA 8.5 342, 327, 312, 297, 283, 253 4-HCA 9.2 308, 293, 249, 219, 179 3,4,5-THBA 10.I 458, 443, 281 Scopoletin 10.7 264, 249, 234, 206 4-H,3-MCA 11.9 338, 323, 308, 293, 279, 249, 219 3,4-DHCA 13.1 396, 381,219, 191 Naringenin 23.8 488, 473, 416, 355, 316, 297, 251,179 Daidzein 24.0 398, 383, 184 Genistein 24.7 486, 471,414, 399, 340, 192 Coumestrol 26.1 412, 397 Kaempferol 27.9 559, 502, 487, 430, 415, 216 Q uercetin 30.5 647, 590, 575, 487, 457, 415, 385, 221

Table 4. GC and MS data for the major compounds present in the TMS derivatized leaf extract

GC retention MS

Compounds time fragmentation pattern detected (rain) (M]Z)

4-HBA 4.3 282, 267, 257, 242, 223, 188 4-H,3-MBA 6.3 312, 297, 282, 267, 253, 223 2,5-DHBA 6.7 370, 355, 337, 321,297 Unknown 7.8 375, 363, 347, 285, 273, 192 4-H,3,5-DMBA 8.5 342, 327, 312, 297,283, 253 4-HCA 9.2 308, 293, 249, 219, 179 Unknown 10.2 396, 369, 312, 293, 281,257, 192, 188 Unknown 11.3 313, 297, 132, 117 4-H,3-MCA 11.9 338, 323, 308, 293, 279, 249, 219 3,4-DHCA 13.0 396, 381,249, 219, 191 Unknown 14.1 305, 219, 207, 171, 135 Unknown 16.9 259, 241,129, 117, 70 Naringenin 23.6 416, 386, 355, 308, 293, 280, 265, 249, 173 Daidzein 24.5 398, 383, 355, 341,184 Genistein 25.4 487, 414, 399, 370, 355, 340, 192 Kaempferol 27.6 559, 529, 502, 487 Ouereetin 30.1 590, 575, 559, 517, 487, 457, 415, 385, 221

PHENOLIC ACIDS AND FLAVONOIDS IN SOYBEAN ROOT AND LEAF EXTRACTS

Table 5. GC and MS data for the major compounds present in the TMS derivatized root extract

GC retention MS

Compounds time fragmentation pattern detected (min) (M/Z)

4-HBA 4.2 296, 282, 267, 223, 193, 170, 120 Unknown 5.6 303, 281,170, 120 4-H,3-MBA 6.2 312, 297, 282, 267,253, 223, 126 2,5-DHBA 6.6 370, 355, 170, 120 Unknown 6.8 355, 317, 297, 282, 267, 217, 201,188 3,4-DHBA 7.3 370, 355, 312, 193, 170, 147, 120 Unknown 7.7 465, 375, 363, 347, 312, 297, 285, 273 Unknown 8.1 312, 297, 267, 259, 99 4-H,3,5-DMBA 8.4 342, 327,312, 297, 267, 259 4-H,3-MCA 11.8 338, 323, 308, 297, 281,267, 249 Unknown 22.2 472, 457,429, 399, 355, 341,281,207 Daidzein 24.5 398, 383, 355, 184 Genistein 25 .3 471,429, 414, 399, 383, 355, 192 Coumestrol 26.6 412, 397, 191 Unknown 27.4 480, 465, 429, 412, 397, 383, 369, 355

71

or G C - M S analysis in hydrolyzed leaves in our studies but we did positively identify this com- pound in hydrolyzed root extracts. Gallic acid is a relatively polar compound which eluted very early from the HPLC column. The hydrolyzed leaf extract contained several relatively polar

compounds which were rapidly eluted off the HPLC column and which were not well resolved by the three gradients employed. Therefore posi- tive identification of gallic acid by this HPLC technique was difficult to obtain. Its GC MS identification was also complicated by difficulty

z 0

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T I M E IN M I N U T E S

FIO. 3. The GC of the hydrolyzed leaf extract (see Table 4 for retention times and fragmentation patterns of major peaks).

72 P . M . PORTER et al.

0q

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T I M E I N M I N U T E S

FIG. 4. The GC of the hydrolyzed root extract (see Table 5 for retention times and fragmentation patterns of major peaks).

in the der ivat iza t ion of this compound. I f gallic acid does occur in the hydrolyzed leaf extract , it is clearly present as only a very minor constituent.

I t should be emphasized that the compounds identif ied in the hydrolyzed leaf and root extracts are not necessarily those compounds that are present in the living tissue, or that are exuded by the plants as allelochemicals. However , the hydro lyzed leaf and root extracts conta ined com- pounds that are known allelochemicals. (t2) Work done by HARDIN and STUTTE (5) on leaf extracts, and work carried out in our laborator ies (11) on root extracts, indica ted that only a few if any of the phenolic acids present in hydrolyzed extracts were found in unhydro lyzed extracts. The i r ab- sence in unhydro lyzed extracts was not surprising since compounds that are al lelochemic are often stored in the p lan t as ethers with sugars or as esters with other phenolic compounds. ('m4)

One might speculate that kaempferol , quer- cetin and nar ingenin observed in the hydrolyzed leaf extract may act as feeding deterrents to insects. (13) Similarly, the daidzein, coumestrol and genistein observed in hydro lyzed root ex- tracts m a y act as phytoalexins, or possibly as precursors of phytoalexins. ¢2'6) The three la t ter compounds have also been found to possess some an t imicrobia l act iv i tyJ 7'1°)

R E F E R E N C E S

1. BANWART W. L., PORTER P. M., GRANATO T. C. and HASSETT J. J. (1985) HPLC separation and

wavelength area ratios of more than 50 phenolic acids and flavonoids. J. chem. Ecol. 11, 383-395.

2. DEWICK P. M. (1982) Isoflavonoids. Pages 620- 623 inJ. B. HAR~ORNE and T.J . MABRY, eds. The

flavonoids: advances in research. Chapman and Hall, New York.

3. FEHR W. R. and CAVINESS C. E. (1980) Stages of soybean development. Special Report 80, Agriculture and Home Economics Experiment Station, Iowa State University.

4. GRoss G. G. (1981) Phenolic acids. Pages 301-316 in P. K. STUMPF and E. E. C O N N , eds. The biochemistry of plants, Vol. 7, Secondary plant products. Academic Press, New York.

5. HARDINJ. M. and STUTTE C. A. (1980) Analyses of phenolic and flavonoid compounds by high- pressure liquid chromatography. Analyt. Biochem. 1 0 2 , 1 7 1 1 7 5 .

6. INGHAM J. L. (1982) Phytoalexins from the Leguminosae. Pages 21-80 in J. A. BAILEY and J. W. MANSFIELD, eds. Phytoalexins. Wiley, New York.

7. INGHAM J. L., KEEN N. T., MULHEIRN L. J. and LYNE R. L. (1981) Inducibly-formed isoflavonoids from leaves of soybean. Phytochemistry 20, 795--798.

8. KAmNSKY R. and MOLLER W. H. (1978) A recommendation against the use of alkaline soil extractions in the study ofallelopathy. Plant Soil 49, 641--645.

9. MURPHYJ. B. and STUTTE C. A. (1978) Analysis for substituted benzoic and cinnamic acids using high-pressure liquid chromatography. Analyt. Biochem. 86, 220-228.

10. NAIM M., GESTETNER B., ZIEKAH S . , BIRK Y. and BONDI A, (1974) Soybean isoflavones: characteriz- ation, determination and antifungal activity. J . agric. Fd Chem. 22, 806-810.

PHENOLIC ACIDS AND FLAVONOIDS IN SOYBEAN ROOT AND LEAF EXTRACTS 73

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