Environmental and Experimental Botany, Vol. 26, No. 1, pp. 65-73, 1986.

Printed in Great Britain.

0098~472/86 $3.00 + 0.00 1986. Pergamon Press Ltd.

P H E N O L I C ACIDS A N D F L A V O N O I D S IN SOYBEAN R O O T A N D LEAF E X T R A C T S

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

Department of Agronomy, University of Illinois, Urbana, IL 61801, U.S.A. (Received22 March 1985; acceptedin revisedform 6 June 1985) PORTERP. M., BANWART L. and HASSETTJ.J. Phenolicacids andflavonoids in soybean root and leaf W. extracts. ENVIRONMENTAL EXPERIMENTAL AND BOTANY 65 73, 1986.--An HPLC technique and 26~ 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-hydroxybenzoic, 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 germination and growth of other plants. ~8~ Numerous investigators have analyzed hydrolyzed soybean leaf extracts for phenolic and flavonoid compounds. Based on high pressure liquid chromatography (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, salicylic, vanillic, caffeic, 4-hydroxycinnamic, and ferulic 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 addition to the eight compounds listed above. There is little reason to believe that the phenolic 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 potential 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 chromatographymass spectrometry (GC-MS). 65

66

P . M . PORTER et al.
MATERIALS AND METHODS

Chemicals All the chemicals and compounds were obtained from commercial sources except daidzein, which was provided by Dr. J o h n Ingham (University of Reading, England). Standards were prepared using 10/2g/ml of the compounds listed in Table 1. None of the standard compounds 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 approximately 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 separated 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 H P L C 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. Trimethylsilyl (TMS) derivatives were then made by dissolving a portion of the crystals formed in

Table 1. Selectedstandards analyzed by HPLC and GC-MS

Common name 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

Chemical name 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

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

67

length area ratio for each compound was obtained 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 using a Beckman H P L C tg) 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 BANWARTet 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 compounds were chromatographed alone and as mixtures. Relative retention times for the standard 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-

G C - M S analysis
The G C - M S analyses of the TMS derivatized extracts and standards were performed on a 5985 G C - M S 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 280C at 5C per min.

RESULTS AND DISCUSSION

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

.5
t-

O
m I . o

0~

tJ)

,-li
"O ...I O
!

.c

o
t~ I

I
JI i

~'O
I~ j ri~ "1

"E .c

x"

lb

2'0

3'o

4b

s~

do

7'0

8'o

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
c

P . M . PORTER

et al.

~
m o

Ill I

LU Z 0 0.. OO LU O: nO I-" 0 U.I I'U.I C~

tN

_c
o =l

=E

< co

o
t "2

2'o

3'o
TIME

go
IN

5'0
MINUTES

;o

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 gradients, and wavelength area ratios allow for the detection of more than 50 potential allelochemicals. (1) Typical H P L C 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), 4hydroxybenzoic (4-HBA), vanillic (4-H,3-MBA), caffeic (3,4-DHCA), salicylic (2-HBA), 4-hydroxycinnamic (4-HCA) and ferulic (4-H,3-MCA) acids as well as naringenin, quercetin and genistein (see Table 1 for details of abbreviations). By these same methods the following compounds

were identified in the hydrolyzed root extract: 4hydroxybenzoic (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 hydrolyzed root extract were subjected to analysis by GC MS to substantiate results from the H P L C analysis. In Table 3 the GC retention times and the MS fragmentation pattern of selected standard 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 compound 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 H P L C analysis, and also revealed two additional compounds--protocatechuic and syringic acids. Although not shown in Fig. 2, later H P L C studies

PHENOLIC

ACIDS

AND FLAVONOIDS

IN SOYBEAN

ROOT

AND

LEAF EXTRACTS

69

Table 2. Relative retention times (RR T* ) and 254 : 280 n m area ratiosfor the HPLC analysis of selected standard compounds (Std) and the hydrolyzed extracts of soybean leaves and roots
Gradient 1 RRT Std L e a f R o o t . . . 0.40 --0.44 0.59 0.59 0.72 -1.00 1.00 1.18 1.18 1.55 1.52 1.76 1.66 1.80 1.74 1.87 --2.22 2.53 2.49 2.83 2.83 --3.66 3.59 -4.41 -4.79 4.77 . . . 4.95 5.01 4.96 4.86 5.80 -. . . . -0.45 --0.60 0.65 -0.75 1.00 1.00 1.17 1.21 1.55 1.52 -1.67 -1.95 -1.77 ---2.66 2.83 3.13 3.38 -3.59 4.73 3.85 -4.34 5.99 -6.57 . . -7.10 4.93 7.00 5.90 8.21 . . Gradient 2 RRT Std L e a f R o o t 0.43 -0.48 0.67 -1.00 1.27 1.47 1.68 1.81 -2.28 2.64 3.06 -4.60 -6.81 7.33 -. ---0.70 -1.00 1.27 1.48 ----2.96 4.32 4.61 5.18 5.95 -6.04 -6.98 8.34 . Gradient 3 RRT Std L e a f R o o t -0.46 -0.65 0.77 1.00 1.20 1.46 1.63 1.89 1.65 -2.44 2.74 -4.27 -5.89 6.47 -7.21 7.09 8.55 . . 0.44 -0.49 0.68 -1.00 1.23 1.44 1.62 1.86 -2.14 2.44 2.73 -4.22 -6.89 -7.42 6.99 -. ---0.66 -1.00 1.16 1.43 -----2.72 3.33 4.12 4.14 5.73 -5.85 -7.01 8.40 . A r e a ratios Std L e a f R o o t -0.6 -1.0 2.0 3.5 3.7 2.0 0.6 0.2 0.5 -0.2 1.2 -0.6 -2.4 0.1 -2.0 2.5 4.1 . 1.7 -1.2 0.8 -2.7 0.4 0.2 0.5 0.3 -0.6 0.2 0.6 -0.6 --3.0 -1.7 3.0 ----1.0 -2.6 4.0 2.7 -----1.0 1.0 0.7 1.4 2.7 -2.7 -2.0 4.9

Compounds U n k n o w n L1 3,4,5-THBA U n k n o w n L2 2,5-DHBA 3,4-DHBA 2,4-DHBA(Ref)t 4-HBA 4-H,3-MBA 3,4-DHCA 2-HBA 4-H,3,5-DMBA U n k n o w n L3 4-HCA 4-H,3-MCA U n k n o w n R1 Indole(Ref)'~ Unknown R2 Daidzein Naringenin Unknown R3 Quercedn Genistein Coumestrol Kaempferol:~

* R e t e n t i o n t i m e relative 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 a m p l e s as i n t e r n a l references p r i o r to H P L C analysis. ~ K a e m p f e r o l w a s n o t d e t e c t e d b y t h e H P L C m e t h o d s d e s c r i b e d b u t w a s f o u n d in t h e h y d r o l y z e d l e a f e x t r a c t b y G C - M S analysis.

in our laboratories using more concentrated hydrolyzed root extracts similarly revealed the presence of protocatechuic and syringic acids. The GC MS analysis of the hydrolyzed leaf 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 compounds identified using the HPLC technique. Only salicylic acid was not found by the GC MS 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 its p r e s e n c e is m a s k e d b e c a u s e it e l u t e s f r o m t h e G C column near the solvent front with the other more volatile compounds. Syringic acid, daidzein and kaempferol were indicated by GC-MS analysis but not the initial HPLC analysis. Further analyses in our laboratories with more concentrated leaf extracts have confirmed the presence of

syringic acid and daidzein. The HPLC methods used do not allow detection of low levels of 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 extract by the HPLC and GC MS analyses correspond to t h e c o m p o u n d s reported by 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 acids, genistein, daidzein and kaempferol repres e n t five 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 i n 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 pounds (sinapic, protocatechuic and gallic acids) not identified in our studies. No sinapic acid standard was analyzed by our HPLC methods n o r w a s its e x p e c t e d fragmentation pattern found by GC-MS analysis. No detectable amount ofprotocatechuic acid was ibund by either HPLC

70

P. M. PORTER et al.
Table 3. GC and MS data for the T M S derivatives of selected standard compounds

Standard compound 2-HBA 4-HBA 4-H,3-MBA 2,5-DHBA 3,4-DHBA 4-H,3,5-DMBA 4-HCA 3,4,5-THBA Scopoletin 4-H,3-MCA 3,4-DHCA Naringenin Daidzein Genistein Coumestrol Kaempferol Q uercetin

GC retention time (min) 3.3 4.3 6.3 6.8 7.4 8.5 9.2 10.I 10.7 11.9 13.1 23.8 24.0 24.7 26.1 27.9 30.5

MS fragmentation pattern
(M/Z)

281,267, 209, 149, 135 282, 267, 223, 193 312, 297, 282, 267, 253, 223, 188 370, 355 370, 355, 31 I, 193 342, 327, 312, 297, 283, 253 308, 293, 249, 219, 179 458, 443, 281 264, 249, 234, 206 338, 323, 308, 293, 279, 249, 219 396, 381,219, 191 488, 473, 416, 355, 316, 297, 251,179 398, 383, 184 486, 471,414, 399, 340, 192 412, 397 559, 502, 487, 430, 415, 216 647, 590, 575, 487, 457, 415, 385, 221

Table 4. GC and MS datafor the major compoundspresent in the T M S derivatized leaf extract

Compounds detected 4-HBA 4-H,3-MBA 2,5-DHBA Unknown 4-H,3,5-DMBA 4-HCA Unknown Unknown 4-H,3-MCA 3,4-DHCA Unknown Unknown Naringenin Daidzein Genistein Kaempferol Ouereetin

GC retention time (rain) 4.3 6.3 6.7 7.8 8.5 9.2 10.2 11.3 11.9 13.0 14.1 16.9 23.6 24.5 25.4 27.6 30.1

MS fragmentation pattern
(M]Z)

282, 267, 257, 242, 223, 188 312, 297, 282, 267, 253, 223 370, 355, 337, 321,297 375, 363, 347, 285, 273, 192 342, 327, 312, 297,283, 253 308, 293, 249, 219, 179 396, 369, 312, 293, 281,257, 192, 188 313, 297, 132, 117 338, 323, 308, 293, 279, 249, 219 396, 381,249, 219, 191 305, 219, 207, 171, 135 259, 241,129, 117, 70 416, 386, 355, 308, 293, 280, 265, 249, 173 398, 383, 355, 341,184 487, 414, 399, 370, 355, 340, 192 559, 529, 502, 487 590, 575, 559, 517, 487, 457, 415, 385, 221

PHENOLIC ACIDS AND FLAVONOIDS IN SOYBEAN ROOT AND LEAF EXTRACTS

Table 5. GC and MS datafor the major compoundspresent in the T M S derivatized root extract

71

Compounds detected 4-HBA Unknown 4-H,3-MBA 2,5-DHBA Unknown 3,4-DHBA Unknown Unknown 4-H,3,5-DMBA 4-H,3-MCA Unknown Daidzein Genistein Coumestrol Unknown

GC retention time (min) 4.2 5.6 6.2 6.6 6.8 7.3 7.7 8.1 8.4 11.8 22.2 24.5 25.3 26.6 27.4

MS fragmentation pattern
(M/Z)

296, 282, 267, 223, 193, 170, 120 303, 281,170, 120 312, 297, 282, 267,253, 223, 126 370, 355, 170, 120 355, 317, 297, 282, 267, 217, 201,188 370, 355, 312, 193, 170, 147, 120 465, 375, 363, 347, 312, 297, 285, 273 312, 297, 267, 259, 99 342, 327,312, 297, 267, 259 338, 323, 308, 297, 281,267, 249 472, 457,429, 399, 355, 341,281,207 398, 383, 355, 184 471,429, 414, 399, 383, 355, 192 412, 397, 191 480, 465, 429, 412, 397, 383, 369, 355

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

compounds which were rapidly eluted off the H P L C column and which were not well resolved by the three gradients employed. Therefore positive identification of gallic acid by this H P L C technique was difficult to obtain. Its GC MS identification was also complicated by difficulty

z 0
_1

-.r a

< E
cQ,~.
I '1" c c '-

I--

0
I. .c I

.i

1'2
TIME

1'6

2'0

2'4

&

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 Z

O L.

O
--I
0

<
< 113
"17

<
_ m ,
J

I O< r~ eo f"l r m T I ,W~,'

"~I [-" I

. . F ot. . v ~ -t . _ r ~ o
! -

12 TIME IN

16 MINUTES

20

2'4

2'8

FIG. 4. The GC of the hydrolyzed root extract (see Table 5 for retention times and fragmentation patterns of major peaks). wavelength area ratios of more than 50 phenolic acids and flavonoids. J. chem. Ecol. 11, 383-395. DEWICK P. M. (1982) Isoflavonoids. Pages 620623 inJ. B. HAR~ORNE and T.J. MABRY, eds. The flavonoids: advances in research. Chapman and Hall, New York. FEHR W. R. and CAVINESSC. E. (1980) Stages of soybean development. Special Report 80, Agriculture and Home Economics Experiment Station, Iowa State University. 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, Secondaryplant products. Academic Press, New York. HARDINJ. M. and STUTTE C. A. (1980) Analyses of phenolic and flavonoid compounds by highpressure liquid chromatography. Analyt. Biochem.
102,171 175.

in the derivatization of this c o m p o u n d . I f gallic acid does occur in the h y d r o l y z e d leaf extract, it is clearly present as only a very m i n o r constituent. It should be emphasized that the c o m p o u n d s identified in the h y d r o l y z e d leaf and root extracts are not necessarily those c o m p o u n d s that are present in the living tissue, or that are exuded by the plants as allelochemicals. However, the h y d r o l y z e d leaf a n d root extracts contained comp o u n d s that are k n o w n allelochemicals. (t2) W o r k done by HARDIN a n d STUTTE(5) on leaf extracts, a n d work carried out in our laboratories (11) on root extracts, i n d i c a t e d that only a few if any of the phenolic acids present in h y d r o l y z e d extracts were found in u n h y d r o l y z e d extracts. T h e i r absence in u n h y d r o l y z e d extracts was not surprising since c o m p o u n d s that are allelochemic are often stored in the p l a n t as ethers with sugars or as esters with other phenolic compounds. ('m4) O n e m i g h t speculate that kaempferol, quercetin and naringenin observed in the h y d r o l y z e d leaf extract m a y act as feeding deterrents to insects. (13) Similarly, the daidzein, coumestrol and genistein observed in h y d r o l y z e d root extracts m a y act as phytoalexins, or possibly as precursors of phytoalexins. 2'6) T h e three latter c o m p o u n d s have also been found to possess some a n t i m i c r o b i a l activityJ 7'1)
REFERENCES

2.

3.

4.

5.

1. BANWARTW. L., PORTER P. M., GRANATOT. C. and HASSETTJ. J. (1985) HPLC separation and

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. INGHAMJ. 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: characterization, determination and antifungal activity. J. agric. Fd Chem. 22, 806-810.

PHENOLIC ACIDS AND FLAVONOIDS IN SOYBEAN R O O T AND LEAF EXTRACTS 11. PORTER P. M. (1983) Identification of phenolic acids and flavonoids in the roots of the soybean (Glyeine max). MSc. Thesis, University of Illinois, Urbana, Illinois. 12. RICE E. L. (1979) Allelopathy--an update. Bot. Rev. 45, 15 54. l 3. SUTHERLAND . R. W., RUSSELLG. B., BIGGSD. R. 0 and LANE G. A. (1980) Insect feeding deterrent

73

activity of phytoalexin isoflavonoids. Biochem. Syst.

Ecol. 8, 73-75.
14. VmKERY M. L. and VmKERV B. (1981) Secondary plant metabolism. The Macmillan Press Ltd, Hong Kong, p. 166. 15. WmTTAKER R. H. and FEENY P. P. (1971) Allelochemics: chemical interactions between species. Science 171, 757 770.