Zoosporicidal activities of anacardic acids against Aphanomyces cochlioides.

Abstract

The EtOAc soluble constituents of the unripe fruits of Ginkgo biloba showed motility inhibition followed by lysis of zoospores of the phytopathogenic Aphanomyces cochlioides. We purified 22:1-omega7-anacardic acid (1), 24:1-omega9-anacardic acid (2) and 22:0-anacardic acid (3), together with other related compounds, 21:1-omega7-cardol (4) and 21:1-omega7-cardanol (5) from the crude extracts of Ginkgo fruits. Amongst them, compound 1 was a major active agent in quality and quantity, and showed potent motility inhibition (98% in 30 min) followed by lysis (55% in 3 h) of the zoospores at 1 x 10(-7) M. The 2-O-methyl derivative (1-c) of 1 displayed antibacterial activity against Bacillus subtilis, but practically inactive to Escherichia coli. A brief study on structure-activity relationships revealed that a carboxyl group on the aromatic ring and an unsaturated side chain in the anacardic acid derivative are important for strong motility inhibitory and lytic activities against the zoospore.

Full text

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Zoosporicidal Activities of Anacardic Acids against Aphanomyces cochlioides
Parvin Begum, Yasuyuki Hashidoko*, Md. Tofazzal Islam, Yuko Ogawa and
Satoshi Tahara
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Kita-Ku, Kita-9, Nishi-9, Sapporo 060-8589, Japan. Fax: +81-11-706-4182
E-mail: yasu-h@abs.agr.hokudai.ac.jp
* Author for correspondence and reprint requests
Z. Naturforsch. 57c, 874Ð882 (2002); received May 21/June 22, 2002
Anacardic Acids, Ginkgo biloba, Zoospore Lysis
The EtOAc soluble constituents of the unripe fruits of Ginkgo biloba showed motility
inhibition followed by lysis of zoospores of the phytopathogenic Aphanomyces cochlioides.
We purified 22:1-ω7-anacardic acid (1), 24:1-ω9-anacardic acid (2) and 22:0-anacardic acid
(3), together with other related compounds, 21:1-ω7-cardol (4) and 21:1-ω7-cardanol (5) from
the crude extracts of Ginkgo fruits. Amongst them, compound 1 was a major active agent in
quality and quantity, and showed potent motility inhibition (98% in 30 min) followed by lysis
(55% in 3 h) of the zoospores at 1 ¥ 10Ð7m. The 2-O-methyl derivative (1Ðc) of 1 displayed
antibacterial activity against Bacillus subtilis, but practically inactive to Escherichia coli. A
brief study on structure-activity relationships revealed that a carboxyl group on the aromatic
ring and an unsaturated side chain in the anacardic acid derivative are important for strong
motility inhibitory and lytic activities against the zoospore.
Introduction
Ginkgo biloba L. is one of the most important
medicinal plants that received a considerable in-
terest (Jaggy and Koch, 1997; Wang et al., 1998;
Briskin, 2000). It is a valuable source of several
groups of natural products such as phenolic lipids
(Zarnowska et al., 2000), terpenoids (ginkgolides
and bilobalide), and flavonoids (Ginkgo-flavone
glycosides) (Briskin, 2000). Previous works on
phenolic lipids of G. biloba revealed that it con-
tains anacardic acids, cardanols, and cardols with
alk(en)yl side chains differing in the chain length
and the unsaturation patterns (Lepoittevin et al.,
1989; Himejima and Kubo, 1991; Irie et al., 1996).
In particular, anacardic acids are known to inhibit
enzymes such as prostaglandin synthase (Grazzini
et al., 1991), tyrosinase (Kubo et al., 1994) and li-
poxygenase (Shobha et al., 1994), and also known
to exhibit antitumor (Itokawa et al., 1987) and an-
timicrobial (Himejima and Kubo, 1991) activities.
During the survey of physiologically active sec-
ondary metabolites toward zoospores of Aphano-
myces cochlioides, we observed that the EtOAc
soluble extracts of unripe Ginkgo fruits induced
potent motility inhibition followed by lysis of the
zoospores. This unusual phenomenon prompted us
to investigate the active constituents in the extract.
0939Ð5075/2002/0900Ð0874 $ 06.00
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D
This paper describes the isolation of 22:1-ω7-ana-
cardic acid (1) as a major active compound and
biological activities of 1 and related compounds
against the fungal zoospores and two bacteria.
Materials and Methods
General
Merck silica gel 60 F254 pre-coated on glass
plates was used for analytical and preparative
TLC. Column chromatography was conducted
using silica gel 60 (spherical, Merck 100Ð200
mesh). High performance liquid chromatography
(HPLC) was carried out by a Hitachi D-7500 equ-
ipped with a reversed phase column Prep-ODS
(RP-C18, 20 ¥ 250 mm, GL Sciences Inc., Tokyo)
using aq. MeOH as eluting solvent with a flow rate
at 10 ml/min, otherwise, specially mentioned in the
text. Chemical substances were detected and mon-
itored by a UV detector at wavelength of 254 nm.
1H NMR spectra were recorded on a JEOL JNM-
EX270 FT-NMR spectrometer at 270 MHz using
TMS as an internal standard in deuterated chloro-
form. FD-MS and EI-MS spectra were acquired
on a JEOL JMS-SX102A and a JEOL DX 500
spectrometers, respectively. Electron microscopic

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P. Begum et al. · Anacardic Acids Affect Aphanomyces Zoospores875
observation was done by a scanning electron
microscope (JEOL JSM-6301F).
Plant material and chemicals
The unripe Ginkgo (G. biloba) fruits were col-
lected in August 2000 at the campus of Hokkaido
University, Sapporo, Japan. The whole unripe
Ginkgo fruits (15 kg) were extracted with MeOH
(10 l) and the MeOH extract was concentrated in
vacuo to remove the solvent. The resulting aque-
ous solution was diluted with deionized water to
4 l and extracted successively with n-hexane (4 l)
and then EtOAc (4 l). The EtOAc solubles (ca.
70 g) were chromatographed on a silica gel (900 g)
column and the constituents were eluted with a
mixture of n-hexane and EtOAc (15:1 v/v) to yield
40 g of an anacardic acid-mixture (1 m). Fluazi-
nam (6) was purchased from Wako Pure Chemical
Ind. Ltd., Osaka.
Derivatization of a mixture of anacardic acids (1 m)
A part of the anacardic acid mixture (1 m, 1.5 g)
was refluxed in conc. H2SO4-MeOH (1:20 v/v) fol-
lowed by silica gel column chromatography to
yield a mixture of methyl esters (1 mÐa, 975 mg)
and a slowly eluting non-derivatizable constituent
(99 mg). Both carboxyl and phenolic OH groups
of 1 m were methylated by treating 1 m with
Me2SO4and K2CO3in acetone to give 1 m-b. The
resulting derivative 1 m-b was refluxed in a mix-
ture of MeOH-aq. 2 m NaOH (1:1) for 3 h to give
2-O-methylanacardic acids (1 m-c). Reduction of
1 m-a and 1 m-b with LiAlH4in EtOH/THF re-
sulted in yielding compounds 1 m-d and 1 m-e. Py-
rolytic decarboxylation of 1 m was done at 170 ∞C
for 3 h, using 2% acetone/glycerol mixture as the
matrix in sealed glass tubes. After the reaction
process, the reaction mixture was diluted with
water and then extracted with EtOAc to give de-
carboxylated product 1 m-f (a mixture of cardanol
homologues). Methylation of 1 m-f with Me2SO4
and K2CO3in acetone yielded compound 1 m-g
( = 2-O-methyl-1 m-f).
Isolation of anacardic acid homologues (1Ð3),
21:1-ω7-cardol (4) and 21:1-ω7-cardanol (5)
A mixture of methyl anacardates (1 m-a) was
applied to medium-pressure column chromatogra-
phy using an ODS column eluted with 3% H2O/
MeOH to give two major peaks (fr. 1 and fr. 2, 478
and 360 mg, respectively). The major component
in fr. 1 was further purified by HPLC (Prep-ODS
column, 1% H2O/MeOH, flow rate 5 ml/min,tR-
66.6 min) to yield 281 mg of 1a. From the latter
fraction, two major peaks were separable by
HPLC (Prep-ODS column, 3% H2O/MeOH) to
give 280 mg of crude 2a and 60 mg of crude 3a as
an oil, respectively. The compounds 2a and 3a
were purified from each HPLC fraction by re-
peated HPLC (100% MeOH, flow rate 5 ml/min)
as a colorless oil (180 and 24 mg, respectively).
Methylated products (1a, 2a and 3a) thus pu-
rified were separately hydrolyzed in MeOH-aq.
2 m KOH (1:1) and the resulting products purified
by preparative TLC (n-hexane-EtOAc-HCOOH,
14:2:1 v/v/v) to yield 1, 2 and 3, respectively in
good yields. The derivatization and transformation
reactions were similarly applied to highly purified
1 to prepare 1-b and 1-c. Pure 1 was hydrogenated
with H2/palladium carbon to yield a quantitative
amount of 3.
21:1-ω7-Cardol (4) containing fraction (66 mg)
eluted from the first silica gel column followed
after the anacardic acid mixture was purified
by preparative TLC in CHCl3-EtOAc-HCOOH
(60:10:3, v/v/v, Rf0.27, 35 mg). 21:1-ω7-Cardanol
(5) found the unchanged constituent (99 mg) in
the methylation reaction mixture of 1.5 g of 1 m
was separated by preparative TLC (Rf 0.65 in
CHCl3ÐEtOAcÐHCOOH = 60:10:3), and finally
purified by HPLC (Prep-ODS column, 3% H2O/
MeOH,tR-53.3 min) to give 40 mg of a colorless
syrup.
Culture of Aphanomyces cochlioides, preparation
of zoospore suspensions, and bioassay
Culture of A. cochlioides AC-5 and production
of its zoospores were done as described previously
(Horio et al., 1992). For zoospore assay, we used
both particle method (Tahara et al., 1999) and
homogeneous solution method (Shimai et al.,
2002). In the former method, particles of Chro-
mosorb W AW (60/80 mesh) were coated with a
test compound solved in EtOAc at a set concen-
tration, and some of the particles were then care-
fully dropped into 2 ml of a zoosopore suspension
(original zoospore suspension, ca. 2 ¥ 105/ml, was

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876P. Begum et al. · Anacardic Acids Affect Aphanomyces Zoospores
diluted 2Ð3 times before each experiment) in a
small petri dish (3 cm i.d.). The behavior of zoo-
spores around the particle was observed for sev-
eral hours after addition of the particles under a
microscope (x 53). As a control, particles treated
with solvent alone were used. The latter method
mainly employed in the present study was 100Ð
1000 fold sensitive in comparison with the particle
method. The test compounds were initially dis-
solved in DMSO and then diluted with water. To
each well of a 24-hole petri dish (16 mm i.d.),
450 µl of the zoospore suspension and 50 µl of the
test compound solution were added and gently
stirred. The final concentration of DMSO was ad-
justed to 0.1%. The original zoospore suspension
(ca. 2 ¥ 105/ml) was initially diluted (usually 2Ð3
times) with appropriate amount of water to adjust
the zoospore density which gives 30Ð60 zoospores
in each microscopic field when all zoospores are
halted swimming and settling down on the bottom
of the petri dish (16 mm i.d.). The numbers of zoo-
spores in the test solution were counted as follows:
once mechanically stimulated by a vortex for
30 sec, all zoospores in the suspension halted
swimming instantly and sunk to the bottom of the
petri dish within 5 min, which makes possible to
count the numbers of zoospores under a micro-
scope (x 53).
Evaluation of motility inhibitory and lytic activities
of the test compounds
Only preliminary evaluation of lytic activity
against the zoospores of the anacardic acid mix-
ture (1 m) and its derivatives (1 m-aÐ1 m-b) was
carried out by particle method (see Table I) and
the injured/burst spores were counted. Other ex-
periments using purified samples were conducted
by homogeneous solution method (Tables II and
III). When zoospores were exposed to 1 ¥ 10Ð5m
of fluazinam (6), nearly 100% zoospores were in-
stantly halted and shown remarkable lytic activity.
Therefore, compound 6 was used as the positive
control in our quantitative bioassay to measure
fungal zoospores halted-and-sunk and/or burst.
Compound 1 also showed almost the equivalent
halting activity to that of the reference fungicide
(6). The numbers of halted-and-sunk spores
effected by test compounds at 1 ¥ 10Ð6Ð 10Ð8m
up to 5Ð30 min were recorded by a digital camera
(OLYMPUS Camedia C-3040 200M) and numbers
of zoospores settling down on the bottom of each
petri dish were counted to compare with that of
vortex-stimulated zoospore suspension in each mi-
croscopic field. At 30, 60, 120, and 180 min after
the treatment, uninjured spores on the bottom of
each petri dish were counted in the same manner
described above to evaluate the lytic activity of
tested compounds. As a control solution for this
bioassay was used 0.1% DMSO alone.
Scanning electron microscopy
22:1-ω7-Anacardic acid (1) was first dissolved in
small quantities of DMSO and then diluted with
distilled water. Appropriate amounts of sample
suspension were directly added into the zoospore
suspension taken on a SEMpore membrane to
take final volume as 200 µl. The concentrations of
1 and DMSO in the zoospore suspension were
5 ¥ 10Ð5m and 0.1%, respectively. After a set in-
terval (10, 20 and 60 min) of treatments, the speci-
men was fixed with 2% buffered glutaraldehyde,
and the rest of the procedures for scanning elec-
tron microscopy were carried out as described pre-
viously (Islam et al., 2001).
Antimicrobial assay
As test microbes, two bacteria (Bacillus subtilis
AHU1036 (Laboratory of Applied Microbiology,
Graduate School of Agriculture, Hokkaido Uni-
versity, Japan) and Escherichia coli IFO 3301 (In-
stitute for Fermentation, Osaka)) and a fungus
(Pythium vexans) isolated from soil by Dr. Kasuya
(Yamaji et al., 2000) were used. The antimicrobial
tests of major compounds by means of paper disc
method were done on nutrient-broth agar and po-
tato-dextrose agar media for the bacteria and for
P. vexans, respectively. All of the tests were tripli-
cated.
Results and Discussion
Isolation and derivatization of anacardic acids
The procedures for isolation of anacardic acids
(1Ð3), cardol (4) and cardanol (5) from Ginkgo
fruits and derivatization of the anacardic acids
were described in the section of Materials and
Methods. Chemical structures of all isolated com-

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P. Begum et al. · Anacardic Acids Affect Aphanomyces Zoospores877
Fig. 1. Structures of anacardic acids and re-
lated compounds tested for their activities
toward zoospores of A. cochlioides and
some other microorganisms.
pounds (1Ð5) were confirmed on the basis of their
spectral data compared with the reported ones
(Itokawa et al., 1987; Yamagiwa et al., 1987; Irie
et al., 1996). Derivatization of an anacardic acid
mixture (1 m) was also described in Materials and
Methods.The structures
(1 m-aÐ1 m-f) from an anacardic acid mixture
(1 m) were spectroscopically confirmed.
of thederivatives
Biological activities of anacardic acids and related
compounds toward the Aphanomyces cochlioides
zoospores
When Chromosorb W AW particles coated with
1000-ppm solution of EtOAc solubles of unripe
Ginkgo fruits were subjected to a zoospore bioas-
say (particle method), the motility of zoospores
around the particles was suddenly inhibited and
followed by cell lysis. In homogeneous solution

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878P. Begum et al. · Anacardic Acids Affect Aphanomyces Zoospores
Table I. Lytic activity of a mixture of anacardic acids (1 m), 21:1ω7-cardol (4), 21:1ω7-cardanol (5) and derivatives
of 1 m (structures, in Fig. 1) against Aphanomyces cochlioides zoospores tested by particle method*.
Compound
1 m 1 m-a
Ð
1 m-b
Ð
1 m-c
40 ð 0.6
1 m-d
Ð
1 m-e
Ð
1 m-f
Ð
1 m-g
Ð
45
Ð
27 ð 0.66 ð 0.9
* In this experiment, Chromosorb W AW particles were coated with each 1000-ppm solution and 3 particles were
dropped into the petri dish (3 cm i.d.) containing 2 ml of a zoospore suspension. The lytic activity was observed
around the particles under a microscope (x 53) after 3 h. In this bioassay, the zoospore density was adjusted ca. 60
per microscopic field and injured/burst zoospores around the particles were counted. Results are shown as an
average of four microscopic fields ð SE.
method (Shimai et al., 2002), the active EtOAc
fraction exhibited lytic activity at a range of 0.1Ð
1 ppm (data not shown) toward A. cochlioides
zoospores. After adding the sample solution into
the zoospore suspension, cells quickly became im-
mobile or moved in an unusual circular fashion for
a few minutes and then halted. Initially the halted
zoospores became round-shaped spores by losing
their flagella and part of them were burst grad-
ually.
Table II. Motility inhibition of Aphanomyces cochlioides zoospores in the presence of anacar-
dic acids (1, 2 and 3), cardol (4), 2-O-methyl-22:1ω7-anacardic acid (1-c) and a reference
compound fluazinam (6).
Observed motility inhibition activity*
Tested
compound
Concentration
[m]
5 min 10 min20 min 30 min
1
1 ¥ 10Ð6
1 ¥ 10Ð7
1 ¥ 10Ð8
1 ¥ 10Ð6
1 ¥ 10Ð7
1 ¥ 10Ð8
1 ¥ 10Ð6
1 ¥ 10Ð7
1 ¥ 10Ð8
1 ¥ 10Ð6
1 ¥ 10Ð7
1 ¥ 10Ð8
1 ¥ 10Ð6
1 ¥ 10Ð7
1 ¥ 10Ð8
1 ¥ 10Ð6
1 ¥ 10Ð7
1 ¥ 10Ð8
Control
51ð2.5
37ð2.6
22ð2.5
39ð1.2
24ð1.2
9ð1.2
27ð3.2
15ð1.2
4ð1.4
47ð3.2
28ð1.9
16ð2.6
59ð2.1
44ð3.5
33ð3.7
65ð3.6
53ð2.7
43ð2.8
0
75ð1.9
60ð6.3
47ð2.1
55ð2.6
36ð3.1
21ð1.9
36ð5.0
23ð2.3
12ð1.9
56ð3.1
44ð4.4
32ð3.6
80ð4.3
65ð5.1
53ð3.3
84ð3.1
71ð3.9
59ð3.3
2ð0.9
98ð3.3
82ð4.8
66ð3.4
72ð2.0
58ð3.3
40ð5.4
54ð4.8
40ð4.2
26ð3.4
82ð2.8
68ð5.1
53ð3.0
100ð2.6
88ð2.3
72ð3.4
100ð2.6
90ð6.3
79ð2.5
4ð1.4
101ð2.5
98ð2.2
81ð2.0
99ð1.6
83ð3.9
69ð2.0
99ð2.0
61ð2.6
47ð1.5
99ð2.0
90ð4.5
77ð3.7
101ð2.5
100ð1.5
90ð4.8
101ð3.5
101ð3.5
95ð2.5
4ð0.9
2
3
4
1-c
6
* Time after treatment (min); % of motility inhibition activity ð SE; % of motility inhibi-
tion = (a-b)/(c-b) ¥ 100, a = average number of halted zoospores per microscopic field in
treated solution, b = avearge number of halted zoospores per microscopic field in control
solution, c = average number of halted zoospores per microscopic field from the vortexed
(30 sec) zoospore suspension, calculated after 15 min (33.75). Each test was replicated twice
and spores in 4 microscopic fields (randomly) were averaged.
Our preliminary bioassay by particle method re-
vealed that in the crude anacardic acid mixture
(1 m), chemically derivatized anacardates (1 m-a
to 1 m-g), cardol (4) and cardanol (5), 1 m and
2-O-methyl derivative (1 m-c) had significant lysis-
inducing activity against zoospores (Table I). 21:
1-ω7-Cardol (4), one of non-carboxylated Ginkgo
metabolites, also showed similar, but weak activity.
However, carboxyesters (1 m-a and 1 m-b), benzyl
alcohol-type derivatives (1 m-d and 1 m-e), and