Agricultural and Biological Chemistry

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Isolation of Zeanic Acid, a Natural Plant Growth-

regulator from Corn Steep Liquor and Its Chemical
Structure

Hirochika Matsushima, Hiroshi Fukumi & Kei Arima

To cite this article: Hirochika Matsushima, Hiroshi Fukumi & Kei Arima (1973) Isolation of
Zeanic Acid, a Natural Plant Growth-regulator from Corn Steep Liquor and Its Chemical Structure,
Agricultural and Biological Chemistry, 37:8, 1865-1871, DOI: 10.1080/00021369.1973.10860910

To link to this article: https://doi.org/10.1080/00021369.1973.10860910

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 Agr. BioI. Chem., 37 (8), 1865~ 1871,1973

Isolation of Zeanic Acid , a Natural Plant Growth-regulator from

Corn Steep Liquor and Its Chemical Structure t

Hirochika MATsusHIMA, Hiroshi FUKUMI* and Kei ARIMA

Department of Agricultural Chemistry, The University of Tokyo, Japan
* Sankyo Co., Ltd. Tokyo, Japan
Received December 22, 1972

Corn steep liquor markedly inhibited the growth of the grass, alfalfa, as previously
reported. The active principles from corn steep liquor were isolated. From the acidic
fraction of corn steep liquor, white needles and yellow needles were isolated. The compound
of white needles, which inhibited the germination of alfalfa seeds at the concentrations above
500 ppm, was found to be identical with ferulic acid known as germination inhibitor of plant
seeds. The compound of yellow needles did not inhibit the germination of alfalfa seeds, but
stimulated the growth of the plant. It has a structure related to p-acid which has been isolated
from rice-bran and has been identified as 2,6-dihydroxycinchoninic acid. This active substance
is a new quinoline, 2,8-dihydroxycinchoninic acid and was named zeanic acid.

In the course of screening research to look
for new plant growth-regulators among the
metabolites of microorganisms, corn steep
liquor which is often added to the growth
medium of microorganisms was found to in-
hibit markedly the germination of the grass,
alfalfa. 1I Corn steep liquor is obtained as a
by-product of the manufacture of starch from
corn. Corn is steeped for 2 to 3 days in
0.2 ~ 0.5 per cent sodium sulfite at the tempera-
ture of 46 ~ 50°C, and then the steep liquor is
condensed. 21
Many plant growth-inhibitors produced by
higher plants have been known. Germina-
tion inhibitors such as hydrogen cyanide, am-
monia, mustard oils, organic acids, phenolic
compounds, unsaturated lactones and aldehy-
active principles from corn steep liquor and
also their identification as zeanic acid (2,8-dihy-
droxycinchoninic acid), a new plant growth-
regulator and ferulic acid, a germination in-
hibitor. The physiological activities of zeanic
acid will be described in a following paper.
Corn steep liquor inhibited the germination
of alfalfa at the concentrations above 0.32 %.
On extraction with ethyl acetate, the activity
was detected in the acidic, neutral and resi-
dual water-soluble fractions (Table I). The
water-soluble fraction inhibited the germina-
tion completely at high concentrations.
Firstly, the isolation of active principles from
the acidic fraction was tried.
Corn steep liquor was diluted with water
and extracted with ethyl acetate at pH 2.
•

des were reported. 3 ) Growth inhibitors con-
1
tained in dormant organs are naringenin,4
catechin-tannin,51 pholoridzin 61 and abscisic
acid.7,8) Heliangine,9-12 1 portual,13I xanthi-
nin 141 and many other substances were also
isolated as growth inhibitors. In corn, the
. 15)
existence of plant hormones suc h as aUXlll,
161 171
cytokinin and gibberellin has been report-
ed. This paper reports the isolation of the
t Studies on Plant Growth-regulators. Part II.
See reference 1). A preliminary report has been
published in this Journal: 34, 1430 (1970).
The ethyl acetate phase was extracted with 2 %
aqueous sodium bicarbonate. The aqueous
phase of sodium biocarbonate was acidified
to pH 2 and extracted with ethyl acetate. The
solvent layer was concentrated in vacuo to
give an oily residue. The residue was purified
by chromatography on silica gel with increas-
ing concentrations of methanol in chloroform.
White needles and yellow needles were isolated
(Fig. I).
The white needles, which inhibited the ger-
mination of alfalfa at the concentrations above
1866 H. MATSUSHIMA, H. FUKUMI and K. ARIMA

TABLE I. GROWTH INHIBITION OF ALFALFA BY CORN STEEP LIQUOR

Inhibitory activity
Test solutio n Dilution

2 4 8 16 32 64

10 /~ corn steep liquor (I) ++ ++ + +

(I) was extd. with ethyl acetate at pH 7
Ethylacetate phase (II) + -L , +
Aq. phase (III)
(III) was extd. with ethyl acetate at pH 2
Ethyl acetate phase (IV) + + + + +
Aq. phase (V) ++ + +
Extraction was carried out with an equal volume of ethyl acetate.
+ +: almost no germination, +: growth inhibition,
, .
::I: • slight growth inhibition, -: normal growth.

Corn steep liquor (100 kg) spectra. The IR spectra of authentic ferulic
diluted with water acid and the white needles are shown in Fig. 2.
I
. Adjusted to pH 2 Ferulic acid has been known as a germination
iExtd. with ethyl acetate
inhibitor widely distributed in plants. 31
I
Ethyl acetate phase Aq. phase The yellow needles had no inhibitory ac-
I Extd. with 2 /'~ aq. NaHC0 3 tivity on the germination of alfalfa, but stimula-
i I ted the growth of plants. This compound is
Aq. phase Ethyl acetate phase
assumed to have a structure related to /:I-acid
! Adjust'=d to pH 2
, and xanthurenic acid. /:I-acid 1B1
has been
. Extd. with ethyl acetate
,
. _ ~ - _ .. _--
I
isolated from rice-bran and identified as 2,6-
Ethyl acetate phase Aq. phase dihydroxycinchoninic acid (1). and its pro-
Evapd. motive effects on the growth of mice/ 91 silk-
Oily residue (800 g) 201 211
1 grass and yeast have been reported.
Chromatographed on silica gel
Xanthurenic acid which has the structure of
with chloroform-methanol
I
4,8-dihydroxyquinaldic acid (II) is known to
White needles 1.7 g Yellow needles 76 mg be excreted by pyridoxine-deficient animals
(Ferulic acid) (Zeanic acid)
after the injection of tryptophan. 221
FIG. 1. Isolation Procedure of Ferulic Acid and Since the yellow needles obtained from corn
Zeanic Acid from Corn Steep Liquor.
steep liquor showed a little difference from these
acids in their physico-chemical properties,
(A)
COOH
HO~::?-v/J..~
v
to..
N'" 'OH T
N~ COOH
OH
2000 1600 1200 800 (I) ( II )
4000 3200
Wave number (cm- J )
COOH
FIG. 2. IR Spectra of Ferulic Acid (A) and White
Needles (B) in Nujol.
N OH N OH
500 ppm, were identified as ferulic acid OH OH
(3-methoxy-4-hydroxycinnamic acid) by ele-
( III ) (N)
mentary analysis and IR, UV, NMR and MS
 Isolation and Chemical Structure of Zeanic Acid 1867

this compound was considered to be a new

3
plant growth-regulator and was named ze .
acid (III). alllC
The molecular formula C H NO was
-
. '10 7 4,
asslgn~d to zeanic acid through elementary II
I I
I
lin
analysIs and molecular weight determination 2 I
I
I I , '
I I \,
from. its ~S spectrum. The UV spectra of I r.
"I 1:.
zealllC aCid at various pHs (Fig. 3) differed o
......
x J:1 'I'~ r,
w 1
1,'
II' I'} \
I 0'
I" I \
3 II 'j I ,'
I

1 1••• I
. ,
I,, I
I
I
\ /
\1 ~

\1,'
2
,, /I -
I ,
,,
I I 1
I .. I /1
300 400
M ,0
I I" /
I" , '\ Wavelength (m,u)
C>
...... 1' ,
X
w
If '
I,
I: ,
I I"'~",
.\
\
FIG. 5. UV Spectra of 8-Hydroxycarbostyril in
11 1 .. ' H 20.
I \ '\
1 Ii, 1 \
\ \ --- pH 4.47,5.43,6.09; ------ pH 7.88; - ' - pH 8.28;
\
\ - , 0.1 N NaOH.
\
\ /'" '\
v .

V v
..... -.
.....~.--::---
~---­

.-"
o --,
i(l)
300 400 (j)
(fj
,
I
'I v r_./ (~--' _"' ~"I.,
... -- ...
\ "'~/\ ,,)/
.-~
" ',I.!
, , ' ,v'... ...........
\ ,"'~'"

Wavelength (m,u) \', ,,r (II) ,

'" ,
L
',\11
•
,\
1 1 , ' .... 11
I
I ',"
'l"'''
I'
I
\'r-
V', ... - ,

FIG. 3. UV Spectra of Zeanic Acid in H 20.

. ,,.,,
\ I \
\ J I
\
I
I
I A"
Y', 11
, I , ~
\,. \,""
t'l'1I
~

", "",
--- pH 4.20,5.12,6.03; ------ pH 7.84; - ' - pH 8.40
- 0.1 N NaOH. , , " , , . ,
4000 3200 2000 1600 800
Wave number
3
FIG. 6. IR Spectra of Zeanic Acid (I) and P-Acid
~~) r\ . H 20 (II) in Nujol.
I.. I 1
' 1 I
\ I~I
\ I' ,

i. Y,. ,'\,, from those of ,B-acid (Fig. 4), but resembled to

2 , ,,
M
I
~
. I
I
,
,,
those of 8-hydroxycarbostyril (IV) (Fig. 5),
suggesting that zeanic acid has a 2,8-dihydro-
C> I :
...... I' ,
X
w I\ : xyquinoline nucleus. This is also supported
I' ,
I \ \ by the comparison of the pKa values of
I ' \
II \ ,\" phenolic hydroxyl groups in these acids.
1 I \ \
\ \ .\ ' Using the shift of the absorption bands of
'- ---\'-,',
\ '\,~
zeanic acid (257 mil to 274 mil), ,B-acid (232 mil
\\\. to 247 mil) and 8-hydroxycarbostyril (257 mil
\~
\ ,
\
'- to 273 mil) in an alkaline solution, the pKa
'--/

300 400 values of phenolic hydroxyls in these sub-

Wavelength (m,u) stances could be determined spectrophoto-
FIG. 4. UV Spectra of P-Acid in H 20. metrically as 8.02, 9.33, 8.04, respectively.
___ pH 4.70,5.10,6.03; - ' - pH 8.78; ------ pH 9.98; The IR spectrum presented in Fig. 6 showed
I
- 0.1 N NaOH. the presence of -C=N- (1655 cm- ), -COOH
 1868 H. MATSUSHIMA, H. FUKuMI and K. ARIMA

(a)
205 (W)

(1695, 2480~ 3000 em-I) and -OR (3160 em-I) HS COOH

groups. A~H3

The MS spectrum of zeanic acid showed

H' OH
almost the same fragmentation pattern as that
H'
of fJ-acid (Fig. 7). Prominent ions were found
at 111/e 205 (M +), 177, 160, 133, 105 and 104,
and metastable ions at m/e 153, 143 and 83
in the MS spectra of both compounds. There-
fore, the fragmentation of zeanic acid and
fJ-acid could be explained as follows.
-co -CH -HCN
205(M+) ) M+-28 'M+-45 ,
I -COOH i
-co
M+-72 'M+-lOO 9 8 7 ppm (8)
Generally, phenols have prominent peaks at FIG. 8. NMR Spectrum of /J-Acid in D s-Pyridine
M+-28 (CO) and M+-29 (CHO), and aromatic (l00 m Hz).
carboxylic acids have at M+ -17 (OR) and
M+-45 (COOH). The above scheme of the
fragmentation of zeanic acid and fJ-acid were
compatible with these general rules. On the N OH
other hand, the MS spectrum of xanthurenic OH
acid, which has a close resemblance in its
chemical structure, gave different peaks from
the above showing prominent peaks at M+-
•
18, M+ -46, M+-74. The fragmentation of H'
xanthurenic acid could be interpretated as H'
follows. HS
-H·,O
" -co -co
205(M+) , M+-18 ) M'-46 'M+-74

Therefore, the possibility that zeanic acid

has the chemical structure similar to that of
xanthurenic acid could be denied.
8 7 ppm (8)
The NMR spectrum of fJ-acid in pyridine
FIG. 9. NMR Spectrum of Zeanic Acid in D s"
is shown in Fig. 8 (a, fJ, y: the signals of the Pyridine (l00 MHz).
 Isolation and Chemical Structure of Zeanic Acid 1869

solvent). Each signal can be assigned as

shown in Fig. 8 from their spin coupling con-
stants (J5,7=2.5Hz and J7,s=8.5Hz). In the
NMR spectrum of zeanic acid (Fig. 9), singlet l-.
• •

was observed at 7.67 ppm, which indicated the Q \.

presence of 3-position proton in the quino-
line nucleus. But other signals of three pro- ~
!!
tons (7.11, 7.22, 8.34 ppm) differed from
~ o
those of jJ-acid. By a decoupling experiment, II
,/ "
I'"
these three protons were coupled with each II
"
.':
other with the coupling constants of J 5 ,6= ~ Q
II ,I ",, I
7.5 Hz, J 5 ,7=2.2Hz and J 6 ,7=8.0 Hz. From " ," ,'
II ,,', ,",I
these values these protons were interpreted II , I , ,
, I , ,
•
to lie next to another in a benzene ring, and ~ 1--

were assigned as shown in Fig. 9. This

ABC
denies the possibility of the presence of the Zeanic
hydroxyl group linked to 6-position or 7- acid
position in the quinoline nucleus, and leaves two FIG. 10. Thin-layer Chromatography of Corn Ex-
possible structures of 2,5-dihydroxycinchoninic tent (A), Corn Steep Liquor Hydrolized with HCI
(B), Non-hydrolized Corn Steep Liquor (C) and
acid or 2,8-dihydroxycinchoninic acid for
Zeanic Acid. The acidic fraction of each extract was
zeanic acid. spated on a silica gel plate and developed.
Inagami et al. 2S1 reported that rats fed a Solvent system; Benzene: MeOH: Acetic acid (9: 1: 1).
diet containing ground corn excreted 8- Detection of spots; Falin reagent.
hydroxycarbostyril. Tryptophan, kynurenine
and 3-hydroxykynurenine were not metabolic hydrochloric acid and non-hydrolyzed corn
precursors of 8-hydroxycarbostyril in rats. steep liquor were developed on a silica gel
Since 8-hydroxycarbostyril could not be isolat- plate (Fig. 10). The chromatograms of these
ed from corn, it appeared that it must be formed extracts showed a similar pattern. Many
from a precursor present in the corn. spots of phenolic acids including a spot which
Assuming that zeanic acid is 2,8-dihydroxy- had the same Rf value as that of zeanic acid
cinchoninic acid and contained in corn in some were observed in each extract.
form, zeanic acid could be one of the precur- Zeanic acid formed hardly soluble pre-
sors of 8-hydroxycarbostyril excreted by rats. cipitates with metallic salts in water or metha-
It is also possible that zeanic acid is an artifact nol. The metallic salts used and the color of
and formed in the process of steeping corn. the the precipitate formed (in parentheses)
The UV spectrum of zeanic acid (see above) were presented as follows. FeCl s ·6H20
is compatible with the structure of 2,8-dihy- (black), Pb(CHsCOO)z· 3H 20 (yellow), Zn
droxycinchoninic acid (III). The final proof (CHaCOO)2·2H20 (yellow), Co (CH SCOO)2'
of the structure was obtained by the synthesis 4H 2 0 (black), Cu (CH SCOO)2H zO (brown),
ofIII z4 ,251 and the chemical structure of zeanic Cd (CH SCOO)2' 2H 2 0(yellow), Ni(CH 3 COO)z'
acid was identified as 2,8-dihydroxycinchoninic 4H zO (yellowish green).
acid (III). The composition of the precipitates cor-
Thin-layer chromatography was performed responded to Cu(C 1o H 5 N0 4)· H 20 and Ni
to examine whether zeanic acid was contained (C OH 5 N0 4)· H 20. In case of f3-acid,181 Cu
in corn or not, and whether it existed as a (C OH 6 N0 4)·3H 20, Ba (C 1o H 6 N0 4)2' 2H zO and
conjugated form or as a free form in corn Na (C 1o H 6N0 4)· H 20 were already reported.
steep liquor. Each acidic fraction of a corn The ratio of zeanic acid to Cu was I: 1 in pre-
extract, corn steep liquor hydrolyzed with cipitate, but the ratio of f3-acid to Cu was 2: 1.
1870 H. MATSUSHIMA, H. FUKUMI and K. ARIMA
EXPERIMENTAL
IR spectra were measured with a JASCO IR-S
KCI spectrometer. NMR spectra (100 MHz) were
determined with a Varian HA-100 spectrometer.
MS spectra were determined with a JAM-01SG
spectrometer, equipped with direct inlet systems. The
electron accelerating energy was 75 eV and the
chamber temp. was 160'C for zeanic acid and 200'C
for p-acid. The UV spectra were determined with
a Hitachi 124 spectrometer. Melting points were
uncorrected.
Germination inhibition of alfalfa
For the test of germination, seeds of alfalfa were
placed on a piece of cotton moistened with test solu-
tions (pH 5 ~ 6), and placed in darkness at 27" C.
Materials to be tested were dissolved in water. Their
effects on the growth of alfalfa were examined after
two days. The result is shown in Table I.
TABLE II. THIN LAYER CHROMATOGRAPHY OF
ZEANIC ACID ON SILICA GEL PLATE
Solvent system Rfvalue
MeOH:BuOH:Benzene:H 20:NH,OH(28 ~;,;) 0.74
(40 :20 :20 :20: 1)
MeOH :BuOH-Benzene :H 20 :Acetic acid 0.57
(40 :20 :20 :20: 1)
Toluene :Ethylformate :Formic acid 0.95
(5:4:1)
Isolation of zeanic acid and ferulic acid from corn steep
liquor
A hundred kg of corn steep liquor was diluted with
water to a volume of 400 liters. The solution was
adjusted to pH 2 with sulfuric acid and extracted with
200 liters of ethyl acetate. The ethyl acetate phase
was extracted three times with 50 liters of 2 ~.~ aqueous
sodium bicarbonate. The aqueous phase of sodium
bicarbonate was acidified to pH 2 and extracted three
times with 50 liters of ethyl acetate. The solvent
layer was dehydrated with sodium sulfate and was
concentrated in vacuo. An oily residue (800 g) was
obtained. The residue was dissolved in chloroform
and chromatographed on a column of silica gel (silicic
acid 5 Ib + hyflo supercell 450 g) which was eluted
with increasing concentrations of methanol in chloro-
form. Biological activity was detected in the effluent
of 2~ 10% methanol, and from this fraction 76 mg of
yellow needles (zeanic acid) and 1.7 g of white needles
(ferulic acid) were obtained. White needles were
recrystallized from ether, and yellow needles from
methanol.
Properties of zeanic acid
Mp 340"C (decomp.). Anal. Found: C, 58.53;
H, 3.50; N, 6.93. Calcd. for ClOH 7 NO,: C, 58.54;
H, 3.44; N, 6.83 %. IR ).i;-;'~j;l em-I: 3170, 2480~
3000, 1695, 1655. Mass m/e: 205 (M+), 177, 160,
133, 104, 77. NMR og~-lzridine ppm: 7.11 (lH,
multiplet), 7.22 (1H, multiplet), 7.67 (1H, singlet),
8.34 (lH, quartet, J=7.5 Hz, J=2.2 Hz), 11.40 (lH,
broad). UV A:'~~H-H20 m!' (£): 257 (12900), 295
(3900), 340 (1800, shoulder). It was soluble in pyri-
dine and aqueous alkali hydroxide, slightly soluble
in methanol and acetone, but hardly soluble in water,
chloroform, benzene, hexane and petroleum ether.
It gave intence green color with the Folin reagent,
dark red color with the Millon reagent and brown
color with FeCb. The result of thin layer chromato-
graphy on a silica gel plate is shown in Table II.
Zeanic acid showed a Rf value similar to that of p-
acid.
Zeanic acid formed hardly soluble precipitates with
many metallic salts. Precipitation procedure (I):
5 per cent of metallic salt in methanol was added to
5 per cent of zeanic acid in methanol until new pre-
cipitates were not formed any more (the color of the
precipitate is presented in parenses); FeCb· 6H20
(black), Pb (CH 3COOh· 3H20 (yellow), Zn(CH3COOjz·
2H2 0 (yellow) and Co(CHaCOOh·4H2 0 (black).
Precipitation procedure (II): 5 per cent aqueous metal-
lic salt (pH 7, neutralized with NH3) was added to
5 per cent aqueous zeanic acid until new precipitates
were not formed any more: CU(CH3COOh-H 20
(brown), Cd(CH aCOOk2H20 (yellow) and Ni(CH3-
COOh-4H2 0 (yellowish green; no precipitate being
formed by mixing, the mixture was concentrated and
ethanol was added). These precipitates were insoluble
in benzene, chloroform, ethyl ether, acetone and metha-
nol, but the precipitate of Cu-zeanic acid was soluble
in pyridine, and the precipitate of Ni-zeanic acid was
soluble in water.
Precipitate (Cu-zeanic acid), Anal. Found: C,
44.0; H, 2.58; N, 4.77; Cu, 20.3. Calcd. for
C 1o H sNO,·Cu·H 2 0: C, 42.2; H, 2.48; N, 4.92; Cu,
22.3 /~. Precipitate (Ni-zeanic acid), Anal. Found:
C, 39.2; H, 2.60; N, 4.63; Ni, 20.1. Calcd. for
C1oHsNO,· Ni· H 2 0: C, 42.9; H, 2.53; N, 5.01; Ni,
21.0%.
Thin layer chromatography of corn extract and com
steep liquor
After corn (250 g) was allowed to stand with 500 ml
of 1 N hydrochloric acid at 50'C for 2 days, the material
was ground and filtered. The acidic fraction was
extracted with ethyl acetate from the filtrate and con-
centrated (oily residue A). Corn steep liquor (50 g)
was refluxed with 200 ml of 1.5 N hydrochloric acid
for 210 min. The acidic fraction was extracted with
ethyl acetate and concentrated (oily residue B). Com
steep liquor was diluted with a six-fold volume of
 Isolation and Chemical Structure of Zeanic Acid
water. The acidic fraction was extracted with ethyl
acetate and concentrated (oily residue C). Oily
residues A, Band C were developed on a silica gel
plate with a solvent system of benzene: methanol:
acetic acid (9: 1 : I). Spots were detected with the
Folin reagent.
Properties 0/ fJ-acid
IR l!;;'~~j cm- : 3240, 2480~ 3000, 1667, 1622.
j
Mass m/e: 205 (M+), 177, 160, 133, 105, 104, 77.
-Do-Pyridine
NMR dMe.,Si ppm:.743 'IH
( ,quartet, J=2.5Hz,
J=8.5 Hz), 7.57 (lH, doublet, J=8.5 Hz), 7.70 (lH,
singlet), 8.64 (I H, doublet, J = 2.5 Hz). UV A~;,~Hm,,,
(0): 274 (6200),357 (4200).
Properties 0/ white needles(ferulic acid)
IR lJ;;'~j;jcm-l: 3470, 2480~ 3000, 1690. Mass
m/e: 194 (M+), 179, 177, 161, 151, 134, 133, 105.
NMR aD6-Acetone ppm'" 387 .(3H , singlet) 631
Me4,Sl . , .(lH
,
doublet, J=10.5 Hz), 6.81 (lH, doublet, J=5 Hz),
7.08 (lH, quartet, J=2 Hz, J=5 Hz), 7.27 (lH,
doublet, J =2 Hz), 7.52 (lH, doublet, J = 10.5 Hz),
7.99 (lH, broad). UV A~':t~Hmfl (0): 295 (23,000,
shoulder), 310 (28,000). Mp 171 ~ 172'C. Anal.
Found: C, 61.66; H, 5.21. Calcd. for ClOHlOO,:
C, 61.85, H, 5.19 /~.
Acknowledgement. We wish to express our sincere
thanks to Professor Y. Sahashi for his kind supply
of a sample of fJ-acid.
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