U.S. patent application number 11/058368 was filed with the patent office on 2005-07-07 for plant activator.
This patent application is currently assigned to SHISEIDO CO., LTD.. Invention is credited to Iida, Toshii, Kobayashi, Koji, Kojima, Kiyotaka, Tanaka, Osamu, Yamaguchi, Shoko, Yokoyama, Mineyuki.
Application Number | 20050148474 11/058368 |
Document ID | / |
Family ID | 16997417 |
Filed Date | 2005-07-07 |
United States Patent
Application |
20050148474 |
Kind Code |
A1 |
Yokoyama, Mineyuki ; et
al. |
July 7, 2005 |
Plant activator
Abstract
An object of the present invention is to determine novel means
of activating plants; more particularly, means or controlling plant
growth, such as means of promoting growth, means of controlling
dormancy, means of imparting tolerance against stress for plants
(dryness, high or low temperatures, osmotic pressure, etc.), and
means of preventing aging. The present inventors have found that
the above object can be achieved by providing plant activators
containing, as an active ingredient, C.sub.4-C.sub.24 ketol fatty
acids, in particular, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic
acid.
Inventors: |
Yokoyama, Mineyuki;
(Kanagawa, JP) ; Yamaguchi, Shoko; (Kanagawa,
JP) ; Iida, Toshii; (Kanagawa, JP) ; Kojima,
Kiyotaka; (Shizuoka, JP) ; Kobayashi, Koji;
(Kanagawa, JP) ; Tanaka, Osamu; (Kyoto,
JP) |
Correspondence
Address: |
TOWNSEND & BANTA
c/o PORTFOLIO IP
PO BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
SHISEIDO CO., LTD.
|
Family ID: |
16997417 |
Appl. No.: |
11/058368 |
Filed: |
February 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11058368 |
Feb 16, 2005 |
|
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10069173 |
May 31, 2002 |
|
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10069173 |
May 31, 2002 |
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PCT/JP00/05614 |
Aug 22, 2000 |
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Current U.S.
Class: |
504/320 |
Current CPC
Class: |
A01N 37/42 20130101 |
Class at
Publication: |
504/320 |
International
Class: |
A01N 037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 23, 1999 |
JP |
11-236210 |
Claims
1-9. (canceled)
10. A method of promoting growth of a plant comprising applying to
the plant a C.sub.4-C.sub.24 ketol fatty acid.
11. The method according to claim 10, wherein the C.sub.4-C.sub.24
ketol fatty acid is applied to the plant during seedling or after
germination of its seeds.
12. The method according to claim 10, wherein the C.sub.4-C.sub.24
ketol fatty acid contains a carbon atom constituting a carbonyl
group and a carbon atom connected to a hydroxyl group, one of the
above carbon atoms being located at the .alpha. or .gamma. position
with respect to the other carbon atom.
13. The method according to claim 10, wherein the C.sub.4-C.sub.24
ketol fatty acid contains one to six carbon-carbon double bonds,
such that the number of the double bonds does not exceed the number
of carbon-carbon bonds in the ketol fatty acid.
14. The method according to claim 10, wherein the ketol fatty acid
contains 18 carbon atoms, and two carbon-carbon double bonds.
15. The method according to claim 10, wherein the C.sub.4-C.sub.24
ketol fatty acid is 9-hydroxy-10-oxo-12(Z), 15(Z)-octadecadienoic
acid.
16. A method for preventing dormancy of a plant comprising applying
to the plant a C.sub.4-C.sub.24 ketol fatty acid.
17. The method according to claim 16, wherein the C.sub.4-C.sub.24
ketol fatty acid is applied to the plant immediately after
germination to prevent dormancy or to a dormant plant to terminate
the dormancy.
18. The method according to claim 16, wherein the C.sub.4-C.sub.24
ketol fatty acid contains a carbon atom constituting a carbonyl
group and a carbon atom connected to a hydroxyl group, one of the
above carbon atoms being located at the .alpha. or .gamma. position
with respect to the other carbon atom.
19. The method according to claim 16, wherein the C.sub.4-C.sub.24
ketol fatty acid contains one to six carbon-carbon double bonds,
such that the number of the double bonds does not exceed the number
of carbon-carbon bonds in the ketol fatty acid.
20. The method according to claim 16, wherein the ketol fatty acid
contains 18 carbon atoms, and two carbon-carbon double bonds.
21. The method according to claim 16, wherein the C.sub.4-C.sub.24
ketol fatty acid is 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic
acid.
22. A method for enhancing tolerance of a plant against stress
comprising applying to the plant a C.sub.4-C.sub.24 ketol fatty
acid.
23. The method according to claim 22, wherein the C.sub.4-C.sub.24
ketol fatty acid is applied to the plant during or after
germination of its seeds.
24. The method according to claim 22, wherein the stress is dry
stress.
25. The method according to claim 22, wherein the C.sub.4-C.sub.24
ketol fatty acid contains a carbon atom constituting a carbonyl
group and a carbon atom connected to a hydroxyl group, one of the
above carbon atoms being located at the .alpha. or .gamma. position
with respect to the other carbon atom.
26. The method according to claim 22, wherein the C.sub.4-C.sub.24
ketol fatty acid contains one to six carbon-carbon double bonds,
such that the number of the double bonds does not exceed the number
of carbon-carbon bonds in the ketol fatty acid.
27. The method according to claim 22, wherein the ketol fatty acid
contains 18 carbon atoms, and two carbon-carbon double bonds.
28. The method according to claim 22, wherein the C.sub.4-C.sub.24
ketol fatty acid is 9-hydroxy-10-oxo-12(Z), 15(Z)-octadecadienoic
acid.
Description
TECHNICAL FIELD
[0001] The present invention relates to a plant activator.
BACKGROUND ART
[0002] Development of techniques for activating plants is a very
important issue for improving supply efficiency of grain plants and
garden plants.
[0003] Factors for determining growth rate of plants include
temperature, light, and nutrients. Conventionally, in order to
promote growth of plants, temperature conditions and sunlight
irradiation conditions have been controlled in accordance with
characteristics of plants to be grown. Along with techniques
utilizing temperature or light, manuring is a typical technique for
promoting growth of plants. Manuring has exerted reliable
effects.
[0004] However, the effect of manuring is limited, and even when
the amount of fertilizers employed is increased, the effect of
promoting growth of plants to a higher level cannot be expected.
Employment of excessive amounts of fertilizers may not only inhibit
growth of plants, but also induce contamination of soil.
[0005] Particularly, at an early stage of growth of a plant, growth
disorder attributed to manuring tends to occur, and therefore
manuring is generally not performed at this stage.
[0006] An object to be achieved by the present invention is to
determine means for activating plants different from conventional
activating means; specifically, means for controlling growth of
plants, such as means for promoting growth of plants, means for
preventing dormancy of plants, means for imparting to plants
tolerance against stresses (e.g., dryness, high or low
temperatures, and osmotic pressure), and means for preventing aging
of plants, to thereby potentiate plants, with the amounts of
fertilizers employed being reduced and contamination of soil being
prevented.
DISCLOSURE OF THE INVENTION
[0007] In order to achieve the object, the present inventors have
performed extensive studies. As a result, the present inventors
have found that, surprisingly, a specific ketol fatty acid exerting
"the effect of promoting flower bud formation" (see Japanese Patent
Application Laid-Open (kokai) No. 11-29410) also exerts "the effect
of activating plants," which is, in a sense, contrastive to the
effect of promoting flower bud formation. The present invention has
been accomplished on the basis of this finding.
[0008] Accordingly, the present invention provides a plant
activator comprising a C.sub.4-C.sub.24 ketol fatty acid as an
active ingredient (hereinafter the activator may be referred to as
"the present plant activator").
[0009] As used herein, the expression "activation of plants" refers
to controlling plant growth in a certain manner so as to activate
or maintain growth of plants, and encompasses actions for
controlling plant growth, such as promotion of growth (including
increase of size of stems and leaves and promotion of growth of
tubers and tuberous roots), control of dormancy, impartment of
tolerance against stresses, and prevention of aging. "Activation of
plants" is, in a sense, contrastive to "promotion of flower bud
formation" described in Japanese Patent Application Laid-Open
(kokai) No. 11-29410. Formation of flower buds is a phenomenon
associated with suppressing life activity of plants. In general,
flower buds are formed when growth of plants is inhibited. As is
well known in the horticultural field, when blooming of flowers is
desired, the following operations--which are considered to arrest
growth of plants--are performed: (1) the amount of a nitrogenous
fertilizer employed is reduced, (2) the frequency of water
sprinkling is reduced, (3) roots are cut, and (4) damage is
inflicted to stems. Formation of flowers is a generative phenomenon
at a mature stage of plants for transmitting their genes to the
next generation, and requires a large amount of energy.
[0010] As described above, the aforementioned ketol fatty acid
exerting the effect of promoting flower bud formation quite
unexpectedly exerts the effect of activating plants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on morning glory.
[0012] FIG. 2 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on lettuce.
[0013] FIG. 3 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on broad bean.
[0014] FIG. 4 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on Eustoma
russellianum.
[0015] FIG. 5 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on cyclamen.
[0016] FIG. 6 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on digitalis.
[0017] FIG. 7 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on chrysanthemum.
[0018] FIG. 8 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on geranium.
[0019] FIG. 9 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on Primula melacoides.
[0020] FIG. 10 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on Begonia
sempaflorens.
[0021] FIG. 11 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on Dianthus
caryophyllus.
[0022] FIG. 12 shows the results of evaluation of growth promotion
effect of specific ketol fatty acid (I) on Oryza sativa L.
[0023] FIG. 13 shows the results of evaluation of growth
controlling effect of specific ketol fatty acid (I) on Oryza sativa
L. in consideration of practical culture.
[0024] FIG. 14 shows the results of evaluation of dormancy
preventive effect of specific ketol fatty acid (I) on
strawberry.
[0025] FIG. 15 shows the results of evaluation of proliferation
enhancing effect of specific ketol fatty acid (I) on hyphae of
Pleurotus citrinopileatus Sing.
[0026] FIG. 16 is a photograph showing the results of evaluation of
growth promotion effect of specific ketol fatty acid (I) on
carpophore of Lentinu edodes (Berk.) Singer.
BEST MODE FOR CARRYING OUT THE INVENTION
[0027] Embodiments of the present invention will next be
described.
[0028] The present plant activator contains a specific ketol fatty
acid as an active ingredient.
[0029] As described above, the ketol fatty acid is a
C.sub.4-C.sub.24 ketol fatty acid (hereinafter the ketol fatty acid
may be referred to as "the specific ketol fatty acid").
[0030] Briefly, the specific ketol fatty acid is a C.sub.4-C.sub.24
fatty acid having a hydroxyl group of alcohol and a carbonyl group
of ketone in the molecule.
[0031] In the present invention, preferably, the specific ketol
fatty acid contains the carbon atom constituting the carbonyl group
and the carbon atom connected to the hydroxyl group at an .alpha.
or .gamma. position, in order to exert desired effects of
poteniating plants. From this viewpoint, particularly preferably,
the carbon atoms are present at an .alpha. position.
[0032] The specific ketol fatty acid preferably contains one to six
carbon-carbon double bonds (note: the number of the double bonds
does not exceed the number of carbon-carbon bonds in the ketol
fatty acid), in order to exert desired effects of poteniating
plants.
[0033] Preferably, the specific ketol fatty acid contains 18 carbon
atoms, and two carbon-carbon double bonds.
[0034] Specific examples of the specific ketol fatty acid include
9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid [hereinafter may
be referred to as "specific ketol fatty acid (I)"],
13-hydroxy-12-oxo-9(Z),1- 5(Z)-octadecadienoic acid [hereinafter
may be referred to as "specific ketol fatty acid (II)"],
13-hydroxy-10-oxo-11(E),15(Z)-octadecadienoic acid [hereinafter may
be referred to as "specific ketol fatty acid (III)], and
9-hydroxy-12-oxo-10(E),15(Z)-octadecadienoic acid [hereinafter may
be referred to as "specific ketol fatty acid (IV)].
[0035] The chemical formulas of specific ketol fatty acids (I) and
(IV) are as follows. 1
[0036] The chemical formulas of specific ketol fatty acids (II) and
(III) are described below in connection with the chemical synthesis
method of these fatty acids.
[0037] Among specific ketol fatty acids, at least some ketol fatty
acids are known as metabolic intermediates of fatty acids in
animals and plants, but the role that the ketol fatty acids play
directly in plants is not known.
[0038] For example, specific ketol fatty acid (I) is known as an
intermediate in a fatty acid metabolic pathway in which
.alpha.-linolenic acid, which is abundantly present in living
organisms, serves as a starting material. However, the specific
role that ketol fatty acid (I) plays directly in plants has
remained unknown.
[0039] The present inventors have found that the aforementioned
ketol unsaturated fatty acids related to the present invention
exert the effect of activating plants.
[0040] A. Production Method of Specific Ketol Fatty Acid
[0041] A desired specific ketol fatty acid can be produced by means
of a method in accordance with the specific structure of the fatty
acia.
[0042] Specifically, (1) a specific ketol fatty acid which is known
to be present in a naturally occurring product can be prepared from
the product by means of a method in which the naturally occurring
product is subjected to extraction and purification (hereinafter
the method will be referred to as an "extraction method"); (2) a
specific ketol fatty acid can be produced by means of a method in
which an unsaturated fatty acid is reacted with an enzyme such as
lipoxygenase in a manner similar to that of a fatty acid metabolic
pathway in plants (hereinafter the method will be referred to as an
"enzyme method"); and (3) a desired specific ketol fatty acid can
be produced by means of a known chemical synthesis method in
accordance with the specific structure of the fatty acid
(hereinafter the method will be referred to as a "chemical
synthesis method").
[0043] (1) Extraction Method
[0044] Specific ketol fatty acid (I) can be obtained from Lemna
paucicostata, which belongs to Lemnaceae, through extraction and
purification.
[0045] Lemna paucicostata used as a source material in this
extraction method is a small water plant floating on the surface of
a pond or a paddy field, and each thallus floating on the water
forms one root in the water. Lemna paucicostata is known to have a
relatively fast growth rate. The flower thereof is formed on the
side of the thallus in which two male flowers containing only one
stamen and a female flower containing one pistil are enveloped in a
small common bract.
[0046] The homogenate of Lemna paucicostata is subjected to
centrifugation (8,000.times.g, about 10 minutes), and the fraction
obtained by removing the supernatant from the resultant supernatant
and precipitate can be used as a fraction containing specific ketol
fatty acid (I).
[0047] As described above, specific ketol fatty acid (I) can be
isolated and purified from the aforementioned homogenate serving as
a starting material.
[0048] An aqueous solution obtained by floating or immersing Lemna
paucicostata in water may be used as a preferred starting material
in terms of preparation efficiency. No particular limitation is
imposed on the aqueous solution, so long as Lemna paucicostata is
viable in the solution.
[0049] Specific methods for preparing the aqueous solution are
described below in Examples.
[0050] The immersing time is not particularly limited, and may be
about two to three hours at room temperature.
[0051] When the starting material of specific ketol fatty acid (I)
is prepared by means of the aforementioned method, in consideration
of production efficiency of specific ketol fatty acid (I), Lemna
paucicostata is preferably subjected, in advance, to specific
stress which enables induction of specific ketol fatty acid
(I).
[0052] Specific examples of the aforementioned stress include dry
stress, heat stress, and osmotic pressure stress.
[0053] The dry stress may be imposed on Lemna paucicostata, for
example, by allowing Lemna paucicostata to spread on a dry paper
filter at low humidity (preferably at a relative humidity of 50% or
less) and at room temperature, preferably at 24-25.degree. C. In
this case, the drying time, which varies with the spreading density
of Lemna paucicostata to be dried, is about 20 seconds or more,
preferably five minutes to five hours.
[0054] The heat stress may be imposed on Lemna paucicostata, for
example, by immersing Lemna paucicostata in hot water. In this
case, the temperature of hot water is determined in accordance with
the immersing time. For example, when the immersing time is about
five minutes, the temperature of hot water is 40-65.degree. C.,
preferably 45-60.degree. C., more preferably 50-55.degree. C.
Preferably, immediately after the aforementioned heat stress
treatment, Lemna paucicostata is returned to water of ambient
temperature.
[0055] The osmotic pressure stress may be imposed on Lemna
paucicostata, for example, by bringing Lemna paucicostata into
contact with a solution of high osmotic pressure, such as a sugar
solution of high concentration. In the case, when a mannitol
solution is used, the sugar concentration is 0.3 M or more,
preferably 0.5-0.7 M. When a 0.5 M mannitol solution is used, the
treatment time is one minute or more, preferably two to five
minutes.
[0056] Thus, a desired starting material containing specific ketol
fatty acid (I) can be prepared.
[0057] No particular limitation is imposed on the strain of Lemna
paucicostata serving as a source material of the aforementioned
various starting materials, but a strain P441 is particularly
preferred when specific ketol fatty acid (I) is to be produced.
[0058] A starting material prepared as described above may be
subjected to the below-described separation and purification, to
thereby produce desired specific ketol fatty acid (I).
[0059] The separation method employed for producing specific ketol
fatty acid (I) from the aforementioned starting material is not
limited to the below-described example separation methods.
[0060] Firstly, the aforementioned starting material is preferably
subjected to extraction by use of a solvent, to thereby obtain an
extract containing specific ketol fatty acid (I). Examples of the
solvent include, but are not limited to, chloroform, ethyl acetate,
and ethers. Of these solvents, chloroform is preferred, since it
enables removal of impurities in a relatively easy manner.
[0061] The oil layer fractions obtained through the solvent
extraction are washed and concentrated by means of a conventionally
known method, and then subjected to high performance liquid
chromatography (HPLC) by use of a reversed-phase partition
chromatography column such as an ODS (octadecylsilane) column, to
thereby identify and isolate a fraction having ability to induce
flower bud formation, thereby potentially isolating specific ketol
fatty acid (I) [note: the specific ketol fatty acid is known to
have ability to induce flower bud formation (see Japanese Patent
Application Laid-Open (kokai) No. 10-324602)].
[0062] In accordance with properties of the starting material,
other conventionally known separation methods, such as
ultrafiltration and gel filtration chromatography, may be employed
in combination.
[0063] The production process of specific ketol fatty acid (I) by
means of the extraction method has been described above. When a
desired specific ketol fatty acid is present in a plant other than
Lemna paucicostata, the fatty acid can be produced by means of a
method similar to that described above or a modification of the
aforementioned method.
[0064] (2) Enzyme Method
[0065] Typical examples of the starting material employed in the
extraction method include C.sub.4-C.sub.24 unsaturated fatty acids
having carbon-carbon double bonds at positions corresponding to
those of carbon-carbon double bonds contained in a desired specific
ketol fatty acid.
[0066] Examples of the unsaturated fatty acids include, but are not
limited to, oleic acid, vaccenic acid, linoleic acid,
.alpha.-linolenic acid, .gamma.-linolenic acid, arachidonic acid,
9,11-octadecadienoic acid, 10,12-octadecadienoic acid,
9,12,15-octadecatrienoic acid, 6,9,12,15-octadecatetraenoic acid,
11,14-eicosadienoic acid, 5,8,11-eicosatrienoic acid,
11,14,17-eicosatrienoic acid, 5,8,11,14,17-eicosapentaenoic acid,
13,16-docosadienoic acid, 13,16,19-docosatrienoic acid,
7,10,13,16-docosatetraenoic acid, 7,10,13,16,19-docosapentaenoic
acid, and 4,7,10,13,16,19-docosahexaenoic acid.
[0067] These unsaturated fatty acids are generally present in
animals and plants. The fatty acids may be obtained from animals
and plants through extraction and purification by means of
conventionally known methods, or may be obtained through chemical
synthesis by means of conventionally known methods. Alternatively,
the fatty acids may be commercially available products.
[0068] In the enzyme method, the aforementioned unsaturated fatty
acid serving as a substrate is reacted with lipoxygenase (LOX), to
thereby introduce a hydroperoxy group (--OOH) into the carbon chain
of the unsaturated fatty acid.
[0069] Lipoxygenase is an oxidoreductase which introduces molecular
oxygen, as a hydroperoxy group, into the carbon chain of an
unsaturated fatty acid. As has been confirmed, lipoxygenase is
present in animals and plants, as well as in yeast such as
saccharomyces.
[0070] For example, the presence of lipoxygenase is recognized in
plants such as angiosperms [specifically, the below-described
dicotyledons and monocotyledons to which the present plant
activator can be applied].
[0071] Among the aforementioned plants, examples of the
particularly preferred origin of lipoxygenase include soybean,
flax, alfalfa, barley, broad bean, lupine, lentil, field pea,
potato, wheat, apple, bread yeast, cotton, cucumber, gooseberry,
grape, pear, kidney bean, rice, strawberry, sunflower, and tea.
Since chlorophyll tends to inhibit the aforementioned activity of
lipoxygenase, if possible, lipoxygenase is preferably obtained from
seeds, roots, fruits, etc. of the plants in which chlorophyll is
not present.
[0072] In the present invention, lipoxygenase of any origin may be
used, so long as it can introduce a hydroperoxy group into a
desired position of the carbon chain of an unsaturated fatty acid.
However, when specific ketol fatty acid (I) is produced, if
possible, lipoxygenase which enables selective oxidation of the
carbon-carbon double bond at position 9 of linoleic acid or
linolenic acid is preferably used.
[0073] Typical examples of the selective lipoxygenase include
lipoxygenase derived from rice germ [e.g., Yamamoto, A., Fuji, Y.,
Yasumoto, K., Mitsuda, H., Agric. Biol. Chem., 44, 443 (1980)].
[0074] Preferred examples of the unsaturated fatty acid serving as
a substrate with respect to the selective lipoxygenase include
linoleic acid and .alpha.-linolenic acid.
[0075] When an unsaturated fatty acid serving as a substrate is
treated with lipoxygenase, enzymatic reaction is preferably allowed
to proceed at an optimum temperature and an optimum pH of the
lipoxygenase to be employed.
[0076] Unwanted impurities generated through the aforementioned
lipoxygenase reaction process may be easily separated by means of
conventionally known methods, such as HPLC described above in
(1).
[0077] Lipoxygenase used herein may be obtained from, for example,
the aforementioned plants through extraction and purification by
means of conventionally known methods, or may be a commercially
available product.
[0078] Thus, a hydroperoxy unsaturated fatty acid can be produced
from the aforementioned unsaturated fatty acid.
[0079] The hydroperoxy unsaturated fatty acid may be considered an
intermediate in the production process of a specific ketol fatty
acid by means of the enzyme method.
[0080] Examples of the hydroperoxy unsaturated fatty acid include
9-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid, which serves
as an intermediate of the aforementioned specific ketol fatty acid
(I) and can be obtained by reacting .alpha.-linolenic acid with
lipoxygenase.
[0081] Of these hydroperoxy fatty acids, the former
9-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid will be
called "hydroperoxy fatty acid (a)" in relation to the present
invention, and the latter
13-hydroperoxy-9(Z),11(E),15(z)-octadecatrienoic acid will be
called "hydroperoxy fatty acid (b)" in relation to the present
invention. The chemical formulas of these hydroperoxy fatty acids
are described below. 2
[0082] Subsequently, the hydroperoxy unsaturated fatty acid serving
as a substrate is reacted with allene oxide synthase, to thereby
produce a desired specific ketol fatty acid.
[0083] Allene oxide synthase is an enzyme having the activity of
converting a hydroperoxy group, via epoxidation, into a ketol
structure. Similar to the aforementioned lipoxygenase, allene oxide
synthase is present in plants, animals, and yeast. For example,
allene oxide synthase is present in plants such as angiosperms
[specifically, the below-described dicotyledons and monocotyledons
to which the present plant activator can be applied].
[0084] The presence of allene oxide synthase is recognized in
plants such as barley, wheat, corn, cotton, eggplant, flax (e.g.,
flax seed), lettuce, oat, spinach, and sunflower.
[0085] In the present invention, no particular limitation is
imposed on the allene oxide synthase employed, so long as it
enables formation of an epoxy group through dehydration of the
hydroperoxy group at position 9 of, for example, the aforementioned
9-hydroperoxy-10(E),12(Z),15(Z)-octad- ecatrienoic acid, to thereby
produce a desired specific ketol fatty acid through nucleophilic
reaction of OH.sup.-.
[0086] When the aforementioned allene oxide synthase treatment is
performed, enzymatic reaction is preferably allowed to proceed at
an optimum temperature and an optimum pH of the allene oxide
synthase to be employed.
[0087] Allene oxide synthase used herein may be obtained from, for
example, the aforementioned plants through extraction and
purification by means of conventionally known methods, or may be a
commercially available product.
[0088] The aforementioned two enzymatic reaction processes may be
performed separately or successively. The aforementioned enzymatic
reaction may be allowed to proceed by use of the crude or purified
product of the aforementioned enzyme, to thereby produce a desired
specific ketol fatty acid. Also, the aforementioned enzyme may be
immobilized on a carrier, after which the enzyme substrate may be
subjected to column treatment or batch treatment by use of the
thus-immobilized enzyme, to thereby produce a desired specific
ketol fatty acid.
[0089] The enzymes employed in the aforementioned two processes may
be prepared by means of a genetic engineering technique.
Specifically, the enzymes can be produced as follows: genes
encoding the enzymes are obtained from, for example, plants through
extraction by means of a customary method, or the genes are
obtained through chemical synthesis on the basis of the genetic
sequence of the enzymes; microorganisms such as Escherichia coli
and yeast, animal cultured cells, or plant cultured cells are
transformed by use of the above-obtained genes; and the recombinant
enzyme protein is expressed in the resultant transformed cells.
[0090] When a specific ketol fatty acid is produced through
nucleophilic reaction of OH.sup.- (described above) after formation
of an epoxy group, depending on the reaction of the nucleophile in
the vicinity of the epoxy group, a .gamma.-ketol compound is formed
in addition to an .alpha.-ketol unsaturated fatty acid.
[0091] The thus-formed .gamma.-ketol compound can be easily
separated from the .alpha.-ketol compound by means of a
conventionally known separation method, such as HPLC described
above in (1).
[0092] (3) Chemical Synthesis Method
[0093] A specific ketol fatty acid may be produced by means of
conventionally known chemical synthesis methods.
[0094] For example, a saturated carbon chain having, at its one
end, a reactive group such as an aldehyde group and having, at the
other end, a carboxyl end group connected to a protective group is
synthesized by means of a conventionally known method, and
separately, an unsaturated carbon chain having, at a desired
position, an unsaturated group and having a reactive end group is
synthesized from a starting material such as an unsaturated alcohol
(e.g., cis-3-hexen-1-ol). Subsequently, the resultant saturated
hydrocarbon chain and unsaturated carbon chain are reacted with
each other, to thereby produce a specific ketol fatty acid. In the
aforementioned reactions, the protective group connected to any end
group which does not participate in reaction, and the catalyst for
promoting reaction may be appropriately chosen in accordance with
the specific reaction mode.
[0095] More specifically, specific ketol fatty acids may be
synthesized through, for example, the below-described
processes.
[0096] i) Synthesis of Specific Ketol Fatty Acid (I)
[0097] Nonanedioic acid monoethyl ester serving as a starting
material is reacted with N,N'-carbonyldiimidazole, to thereby yield
an acid imidazolide, and subsequently, the acid imidazolide is
reduced by use of LiAlH.sub.4 at low temperature, to thereby
synthesize the corresponding aldehyde. The aforementioned starting
material may be changed to, for example, a diol such as
1,9-nonanediol, to thereby synthesize a similar aldehyde.
[0098] Separately, cis-3-hexen-1-ol is reacted with
triphenylphosphine and carbon tetrabromide. The resultant bromide
is reacted with triphenylphosphine, and further reacted with
chloroacetaldehyde in the presence of n-BuLi, to thereby form a cis
olefin. The cis olefin is reacted with methylthiomethyl p-tolyl
sulfone, and then reacted with the above-synthesized aldehyde in
the presence of n-BuLi, to thereby yield a secondary alcohol. The
resultant secondary alcohol is protected by tert-butyldiphenylsilyl
chloride (TBDPSCl), and subjected to hydrolysis by use of an acid,
and then to deprotection, to thereby synthesize desired specific
ketol fatty acid (I).
[0099] A brief scheme of an embodiment of the synthesis process of
specific ketol fatty acid (I) is described below. 3
[0100] ii) Synthesis of Specific Ketol Fatty Acid (II)
[0101] Nonanedioic acid monoethyl ester serving as a starting
material is reacted with thionyl chloride, and the resultant acid
chloride is reduced by use of NaBH.sub.4, to thereby yield an acid
alcohol. Subsequently, the free carboxyl group of the resultant
acid alcohol is protected, and the resultant product is reacted
with triphenylphosphine and carbon tetrabromide. The resultant
bromide is reacted with triphenylphosphine, and further reacted
with chloroacetaldehyde in the presence of n-BuLi, to thereby form
a cis olefin. The cis olefin is reacted with methylthiomethyl
p-tolyl sulfone, and then reacted with, in the presence of n-BuLi,
an aldehyde which has been obtained through PCC oxidation of
cis-3-hexen-1-ol. The resultant product is subjected to
deprotection, to thereby accomplish synthesis of desired specific
ketol fatty acid (II).
[0102] A brief scheme of an embodiment of the synthesis process of
specific ketol fatty acid (II) is described below. 4
[0103] iii) Synthesis of Specific Ketol Fatty Acid (III)
[0104] Methyl vinyl ketone serving as a starting material is
reacted with trimethylsilyl chloride in the presence of LDA and
DME. MCPBA and trimethylaminehydrofluoric acid are added to the
resultant silyl ether at a low temperature (-70.degree. C.), to
thereby prepare a keto-alcohol. Subsequently, the carbonyl group of
the keto-alcohol is protected, and then, in the presence of
triphenylphosphine and trichloroacetone serving as reaction
reagents, reaction is allowed to proceed so as to prevent addition
of chlorine to the carbon-carbon double bond. The reaction product
is reacted with formic acid in the presence of tributylarsine and
K.sub.2CO.sub.3, to thereby form a trans olefin and then form a
chloride. Subsequently, the resultant chloride is reacted with an
aldehyde which has been obtained through PCC oxidation of
cis-3-hexen-1-ol. The resultant reaction product is bonded to
6-heptenoic acid, and then subjected to deprotection, to thereby
synthesize desired specific ketol fatty acid (III).
[0105] A brief scheme of an embodiment of the synthesis process of
specific ketol fatty acid (III) is described below. 5
[0106] B. The Present Plant Activator
[0107] When the present plant activator is applied to a plant, the
plant can be activated. Particularly, the present plant activator
exerts plant growth controlling effect in various manners so as to
activate growth of plants.
[0108] "Plant activation effect" and "plant growth controlling
effect" will next be described in detail.
[0109] (1) Growth Promoting Effect
[0110] Application of the present plant activator to a plant can
increase the growth rate of the plant and improve harvest
efficiency of the plant (as described above, increase of size of
stems and leaves and promotion of growth of tubers and tuberous
roots can be expected). Accordingly, the present invention also
provides a plant growth promoting agent which exerts more specific
effect; i.e., effect of promoting growth of plants.
[0111] When the present plant activator is used for promoting
growth of plants, particularly, promotion of growth of plants at an
early stage after germination--which has been difficult to attain
by use of a fertilizer--can be attained.
[0112] Therefore, when the present plant activator is used as a
plant growth promoting agent, application of the activator is
preferably carried out during seedling or at an early growth stage
after germination.
[0113] When the present plant activator is merely applied, for
example, through spraying, at an early growth stage after
germination, growth of plants is promoted, and the effect of
promoting plant growth is maintained. As described above, even when
the present plant activator is used in excessive amounts, growth
disorder of plants, which occurs when a fertilizer is used in
excessive amount, is not observed. Therefore, careful consideration
of the amount of the activator to be employed is not necessary.
[0114] In the horticultural or agricultural field, instead of
distribution of seeds, which require troublesome handling after
delivery, distribution of seedlings is becoming the mainstream.
Particularly, in the flower business, in most cases, gardening
amateurs purchase seedlings. When the present plant activator is
employed before distribution of seedlings, the grown seedlings can
be sold.
[0115] In the case of rice plants, in general, after seedlings are
grown in a seedbed at an early stage, the grown seedlings are
planted in a paddy field. When the present plant activator is
applied to seedlings in a seedbed, the growth of the seedlings is
promoted, and the number of stems per strain after planting is
increased. In the case of rice plants, the number of spikes per
strain can be increased, to thereby enhance harvest efficiency.
Similarly, when the present plant activator is employed, the
harvest efficiency of other cereal plants such as barley and corn
or fabaceous plants such as soybean can be enhanced.
[0116] The aforementioned properties of the present plant activator
are suitable for increasing the harvest of spinach, lettuce,
cabbage, broccoli, or cauliflower.
[0117] When the present plant activator is applied to ascomycete or
basidiomycete, the growth of hyphae thereof can be promoted, to
thereby increase the harvest yield of carpophores (mushrooms: for
example, Lentinus edodes, oyster mushroom, Lyophyllum decastes,
mushroom, Pholiota nameko, Grifola frondosa, and Celtis sinensis).
Furthermore, the present plant activator may contribute to
establishment of an artificial culture method of mushrooms which at
present are difficult to culture artificially (e.g., Tricholoma
matsutake).
[0118] (2) Dormancy Preventive Effect
[0119] When the present plant activator is applied to a plant, the
dormancy of the plant can be prevented. Specifically, when the
present plant activator is applied to a plant, the "dormancy
period" of the plant during which the growth of the plant is
stopped can be reduced or terminated.
[0120] Accordingly, the present invention also provides a plant
dormancy preventive agent which exerts more specific effect; i.e.,
effect of preventing dormancy of plants.
[0121] In the case in which the present plant activator is used as
a plant dormancy preventive agent, when the activator is applied to
a plant immediately after germination, the dormancy of the plant
can be prevented. Alternatively, the activator may be applied to a
dormant plant, to thereby terminate the dormancy of the plant.
[0122] (3) Anti-Stress Effect
[0123] When the present plant activator is applied to a plant, the
plant can be endowed with tolerance against various stresses, such
as dry stress, high-temperature stress, low-temperature stress, and
osmotic-pressure stress. Briefly, when the present plant activator
is employed, there can be reduced the effect of stresses--which are
attributed to climate variation, induction of germination of seeds,
etc.--on cultivated plants, the stresses potentially causing
reduction in yield of the plants.
[0124] In this sense, the present invention also provides a plant
stress suppressive agent which exerts more specific effect; i.e.,
effect of suppressing stresses imposed on plants.
[0125] In the case in which the present plant activator is used as
a plant stress suppressor, when the activator is applied to a plant
during germination of its seed or after germination, the plant can
be endowed with tolerance against stresses.
[0126] Application of the present plant activator to a plant may
prevent aging of the plant. For example, if the present plant
activator is applied to a therophyte plant which is in the period
in which the plant becomes weak and is dying, weakening (aging) of
the therophyte can be retarded.
[0127] No particular limitation is imposed on the upper limit of
the amount of a specific ketol fatty aci--which is an active
ingredient of the present plant activator--applied to plants. Even
when a specific ketol fatty acid is applied to plants in a large
amount through use of the present plant activator, negative effects
on plants, such as growth inhibition, are barely observed. In
contrast, when a conventionally used plant hormone agent is
excessively applied to plants, considerable negative effects on
plants are observed. Therefore, the plant hormone agent must be
used carefully so as not to be excessively applied to plants. From
this viewpoint, the present plant activator is more advantageous as
compared with the conventional plant hormone agent.
[0128] The lower limit of the amount of the aforementioned specific
ketol fatty acid applied to a single plant, which varies with the
type and size of the plant, is about 1 .mu.M per application.
[0129] The amount of a specific ketol fatty acid incorporated into
the present plant activator may be determined in accordance with
use of the activator, the type of a plant to which the activator is
to be applied, and the specific product form of the activator. A
specific ketol fatty acid may be used as the present plant
activator. However, in consideration of the aforementioned lower
limit of the application amount of a specific ketol fatty acid,
etc., the specific ketol fatty acid is preferably incorporated in
an amount of about 0.1-100 ppm, more preferably about 1-50 ppm, on
the basis of the entirety of the plant activator.
[0130] Examples of the product form of the present plant activator
include solutions, solid agents, powders, and emulsions. In
accordance with the product form, the present plant activator may
appropriately contain known pharmaceutically acceptable carrier
components and auxiliary agents for drug production, so long as
they do not impede the intended effect of the present invention;
i.e., plant growth promoting effect. When the present plant
activator assumes the form of powders or solid agents, for example,
the following carrier components may be incorporated: inorganic
substances such as talc, clay, vermiculite, diatomaceous earth,
kaolin, calcium carbonate, calcium hydroxide, terra alba, and
silica gel; and solid carriers such as flour and starch. When the
present plant activator assumes the form of solutions, for example,
the following carrier components may be incorporated: liquid
carriers including water; aromatic hydrocarbons such as xylene;
alcohols such as ethanol and ethylene glycols; ketones such as
acetone; ethers such as dioxane and tetrahydrofuran;
dimethylformamide; dimethyl sulfoxide; and acetonitrile. Examples
of the auxiliary agents for drug production which may be
incorporated include anionic surfactants such as alkyl sulfates,
alkyl sulfonates, alkyl aryl sulfonates, dialkyl sulfosuccinates;
cationic surfactants such as salts of higher aliphatic amines;
nonionic surfactants such as polyoxyethylene glycol alkyl ethers,
polyoxyethylene glycol acyl esters, polyoxyethylene polyalcohol
acyl esters, and cellulose derivatives; thickeners such as gelatin,
casein, gum arabi; extenders; and binders.
[0131] If desired, the present plant activator may further contain
typical plant growth controlling agents, benzoic acid, nicotinic
acid, nicotinamide, and pipecolic acid, so long as they do not
impede the intended effects of the present invention.
[0132] The present plant activator may be applied to various plants
in a manner in accordance with the product form of the activator.
For example, the present plant activator may be sprayed, dropped,
or applied, in the form of solution or emulsion, to the point of
growth of a plant, to a portion of the plant, such as stem or leaf,
or to the entirety of the plant; or may be absorbed, in the form of
solid agent or powder, in the root of the plant via earth.
Alternatively, when the present plant activator is used for
promoting growth of a water plant such as duckweed, the activator
may be absorbed in the root of the water plant, or the activator in
the form of solid agent may be gradually dissolve in the water.
[0133] The frequency of application of the present plant activator
to a plant varies with the type of the plant or the purpose of
application. Basically, desired effects can be obtained through
merely a single application. When the activator is applied several
times, application is preferably performed at an interval of one
week or more.
[0134] No particular limitation is imposed on the type of plants to
which the present plant activator can be applied, and the activator
is effective for angiosperms (dicotyledons and monocotyledons),
fungi, lichens, bryophytes, ferns, and gymnosperms.
[0135] Examples of dicotylendos of angiosperms include
Convolvulaceae such as Convolvulus (C. nil), Calystegia (C.
japonica, C. hederacea, and C. soldanella), Ipomoea (I. pescaprae,
and I. batatas), and Cuscuta (C. japonica, and C. australis),
Caryophyllaceae such as Dianthus, Stellaria, Minuartia, Cerastium,
Sagina, Arenaria, Moehringia, Pseudostellaria, Honkenya, Spergula,
Spergularia, Silene, Lychnis, Melandryum, and Cucubalus, and
furthermore, Casuarinaceae, Saururaceae, Piperaceae,
Chloranthaceae, Salicaceae, Myricaceae, Juglandaceae, Betulaceae,
Fagaceae, Ulmaceae, Moraceae, Urticaceae, Podostemaceae,
Proteaceae, Olacaceae, Santalaceae, Loranthaceae, Aristolochiaceae,
Mitrastemonaceae, Balanophoraceae, Polygonaceae, Chenopodiaceae,
Amaranthaceae, Nyctaginaceae, Theligonaceae, Phytolaccaceae,
Aizoaceae, Portulacaceae, Magnoliaceae, Trochodendraceae,
Cercidiphyllaceae, Nymphaeaceae, Ceratophyllaceae, Ranunculaceae,
Lardizabalaceae, Berberidaceae, Menispermaceae, Calycanthaceae,
Lauraceae, Papaveraceae, Capparaceae, Brassicaceae (Crusiferae),
Droseraceae, Nepenthaceae, Crassulaceae, Saxifragaceae,
Pittosporaceae, Hamamelidaceae, Platanaceae, Rosaceae, Fabaceae
(Leguminosae), Oxalidaceae, Geraniaceae, Linaceae, Zygophyllaceae,
Rutaceae, Simaroubaceae, Meliaceae, Polygolaceae, Euphorbiaceae,
Callitrichaceae, Empetraceae, Coriariaceae, Anacardiaceae,
Aquifoliaceae, Celastraceae, Staphyleaceae, Icacinaceae, Aceraceae,
Hippocastanaceae, Sapindaceae, Sabiaceae, Balsaminaceae,
Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvales,
Sterculiaceae, Actinidiaceae, Theaceae, Clusiaceae (Guttiferae),
Elatinaceae, Tamaricaceae, Violaceae, Flacourtiaceae,
Stachyuraceae, Possifloraceae, Begoniaceae, Cactaceae,
Thymelaeaceae, Elaeagnaceae, Lythraceae, Punicaceae,
Rhizophoraceae, Alangiaceae, Melastomataceae, Trapaceae,
Onagraceae, Haloragaceae, Hippuridaceae, Araliaceae, Apiaceae
(Umbelliterae), Cornaceae, Diapensiaceae, Clethraceae, Pyrolaceae,
Ericaceae, Myrsinaceae, Primulaceae, Plumbaginaceae, Ebenaceae,
Symplocaceae, Styracaceae, Oleaceae, Buddlejaceae, Gentianaceae,
Apocynaceae, Asclepiadaceae, Polemoniaceae, Boraginaceae,
Verbenaceae, Lamiaceae (Labiatae), Solanaceae (Solanum,
Lycoperisicon, etc.), Scrophulariaceae, Bignoniaceae, Pedaliaceae,
Orobanchaceae, Gesneriaceae, Lentibulariaceae, Acanthaceae,
Myoporaceae, Phrymaceae, Plantaginaceae, Rubiaceae, Caprifoliaceae,
Adoxaceae, Valerianaceae, Dipsacaceae, Cucurbitaceae,
Campanulaceae, and Asteraceae (Compositae).
[0136] Examples of monocotyledons include Lemnaceae such as
Spirodela (S. polyrhiza), and Lemna (L. paucicostata, and L.
trisulcata), Orchidaceae such as Cattleya, Cymbidium, Dendrobium,
Phalaenopsis, Vanda, Paphiopedilum, and Oncidium, Typhaceae,
Sparganiaceae, Potamogetonaceae, Najadaceae, Scheuchzeriaceae,
Alismataceae, Hydrocharitaceae, Triuridaceae, Poaceae (Gramineae)
(Oryza, Hordeum, Triticum, Secale, Zea, etc.), Cyperaceae,
Arecaceae (Palmae), Araceae, Eriocaulaceae, Commelinaceae,
Pontederiaceae, Juncaceae, Stemonaceae, Liliaceae (Asparagus,
etc.), Amaryllidaceae, Dioscoreaceae, Iridaceae, Musaceae,
Zingiberaceae, Cannaceae, and Burmanniaceae.
EXAMPLES
[0137] The present invention will next be described in detail by
way of Examples, which should not be construed as limiting the
invention thereto.
PRODUCTION EXAMPLE
Production of Specific Ketol Fatty Acid (I)
[0138] Specific ketol fatty acid (I)
[9-hydroxy-10-oxo-12(Z),15(Z)-octadec- adienoic acid] was produced
by means of an enzyme method as follows.
[0139] 1. Preparation of Rice-Germ-Derived Lipoxygenase
[0140] Rice germ (350 g) was washed with petroleum ether, defatted,
and then dried. The resultant rice germ (250 g) was suspended in a
0.1 M acetate buffer solution (pH 4.5) (1.25 L), and the resultant
suspension was homogenized.
[0141] Subsequently, the resultant homogenized extract was
subjected to centrifugation at 16,000 rpm for 15 minutes, to
thereby yield a supernatant (0.8 L). Ammonium sulfate (140.8 g)
(30% saturation) was added to the supernatant, and the resultant
mixture was allowed to stand at 4.degree. C. overnight. Thereafter,
the mixture was subjected to centrifugation at 9,500 rpm for 30
minutes, to thereby yield a supernatant (0.85 L). Ammonium sulfate
(232 g) (70% saturation) was added to the supernatant, after which
the resultant mixture was allowed to stand at 4.degree. C. for five
hours.
[0142] Subsequently, the mixture was subjected to centrifugation at
9,500 rpm for 30 minutes, to thereby yield a precipitate. The
above-yielded precipitates (fractions obtained from the rice germ
extract through addition of ammonium sulfate (30-70% saturation)
were dissolved in an acetate buffer solution (pH 4.5) (300 mL), and
then heated at 63.degree. C. for five minutes. Thereafter, the
precipitate was removed, and the supernatant was subjected to
desalting through dialysis (3 L.times.3) by use of an RC dialysis
tube (Pore 4, product of Spectrum: MWCO 12,000-14,000), to thereby
yield a crude solution containing desired rice-germ-derived
lipoxygenase.
[0143] 2. Preparation of Flaxseed-Derived Allene Oxide Synthase
[0144] Acetone (250 mL) was added to flaxseeds (200 g) purchased
from Ichimaru Pharcos Co., Ltd. The resultant mixture was
homogenized (20 s.times.3), and the resultant precipitate was
subjected to filtration by use of a perforated plate funnel, to
thereby remove the solvent.
[0145] Subsequently, the precipitate was again suspended in acetone
(250 mL), and the suspension was homogenized (10 s.times.3), to
thereby yield a precipitate. The precipitate was washed with
acetone and ethyl ether, and then dried, to thereby yield flaxseed
powder (150 g).
[0146] The thus-yielded flaxseed powder (20 g) was suspended in a
50 mM phosphate buffer solution (pH 7.0) (400 mL) under cooling
with ice. The resultant suspension was stirred by use of a stirrer
at 4.degree. C. for one hour to extraction.
[0147] The resultant extract was subjected to centrifugation at
11,000 rpm for 30 minutes. To the supernatant was added ammonium
sulfate (105.3 g) (0-45% saturation), and the mixture was allowed
to stand for one hour under cooling with ice. The mixture was
further subjected to centrifugation at 11,000 rpm for 30 minutes,
to thereby yield a precipitate. The precipitate was dissolved in a
50 mM phosphate buffer solution (pH 7.0) (150 mL), and the
resultant solution was subjected to desalting through dialysis (3
L.times.3), to thereby yield a crude solution containing desired
flaxseed-derived allene oxide synthase.
[0148] 3. Preparation of .alpha.-Linolenic Acid Sodium Salt
[0149] .alpha.-Linolenic acid serving as a starting material has
considerably low water solubility. Therefore, in order to cause
.alpha.-linolenic acid to function effectively as an enzyme
substrate, an .alpha.-linolenic acid sodium salt was prepared.
[0150] Specifically, sodium carbonate (530 mg) was dissolved in
purified water (10 mL), and then heated to 55.degree. C.
.alpha.-Linolenic acid (product of Nacalai Tesque, Inc.) (278 mg)
was added dropwise to the resultant solution, and the mixture was
stirred for three hours.
[0151] After completion of reaction, the reaction mixture was
neutralized with Dowex50W-X8 (H.sup.+ form) (product of Dow
Chemical Co.), to thereby yield a precipitate. The precipitate was
subjected to filtration to thereby remove a resin. Subsequently,
the precipitate was dissolved in MeOH, and then the solvent was
removed under vacuum.
[0152] The thus-obtained product was recrystallized with
isopropanol, to thereby yield a desired .alpha.-linolenic acid
sodium salt (250 mg, 83%).
[0153] 4. Production of Specific Ketol Fatty Acid (I)
[0154] The .alpha.-linolenic acid sodium salt yielded above in 3
(15 mg: 50 .mu.mol) was dissolved in a 0.1 M phosphate buffer
solution (pH 7.0) (30 mL). To the resultant solution was added the
rice-germ-derived lipoxygenase crude solution prepared above in 1
(3.18 mL) at 25.degree. C. under oxygen flow, and the mixture was
stirred for 30 minutes. The rice-germ-derived lipoxygenase crude
solution (3.18 mL) was further added to the mixture, and the
resultant mixture was stirred for 30 minutes.
[0155] After completion of stirring, the allene oxide synthase
crude solution prepared above in 2 (34.5 mL) was added to the
lipoxygenase reaction mixture under nitrogen flow, and the
resultant mixture was stirred for 30 minutes. Thereafter, dilute
hydrochloric acid was added to the reaction mixture under cooling
by use of ice, to thereby adjust the pH of the mixture to 3.0.
[0156] Subsequently, the reaction mixture was subjected to
extraction with a solvent mixture of CHCl.sub.3 and MeOH (10:1).
The thus-obtained organic layer was subjected to dehydration
through addition of magnesium sulfate, and the solvent was removed
under vacuum and then dried.
[0157] The thus-obtained crude product was subjected to HPLC, and a
fraction corresponding to the peak of specific ketol fatty acid (I)
(retention time: about 16 min.) was obtained. Chloroform was added
to the thus-obtained fraction, the separated chloroform layer was
washed with water, and the chloroform was removed by use of an
evaporator, to thereby yield a purified product.
[0158] In order to confirm the structure of the purified product,
the product was subjected to measurement of .sup.1H- and
.sup.13C-NMR spectra by use of a heavy methanol solution.
[0159] As a result, in the .sup.1H-NMR measurement, signals
corresponding to an end methyl group [.delta. 0.98 (t)], two
carbon-carbon double bonds [(.delta. 5.25, 5.40), (.delta. 5.55,
5.62)], a secondary hydroxyl group [.delta. 4.09 (dd)], and
numerous methylene groups were observed, and the product was
presumed to be specific ketol fatty acid (I).
[0160] Furthermore, the .sup.13C-NMR chemical shifts of the product
were identical to the .sup.13C-NMR chemical shifts of specific
ketol fatty acid (I) described in Japanese Patent Application
Laid-Open (kokai) No. 10-324602 (in [0054] and [0055], from line 2
of column 13, page 8), the fatty acid being produced in "Production
Example (extraction method)" described in the above publication
(from last line of column 11, page 7) (see Table 1).
[0161] Thus, the synthesized product obtained by means of the above
enzyme method was identified as
9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid.
1TABLE 1 6 Product synthesized by Standard product means of enzyme
method C-1 178.5 178.4 C-2 35.7 35.4 C-3 26.8* 26.9* C-4 31.1**
31.1** C-5 31.0** 31.0** C-6 31.1** 31.1** C-7 26.9* 26.9* C-8 35.4
35.4 C-9 78.6 78.6 C-10 213.8 213.8 C-11 38.4 38.4 C-12 123.0 123.0
C-13 133.5 133.4 C-14 27.5 27.5 C-15 128.4 128.4 C-16 134.6 134.0
C-17 22.3 22.3 C-18 15.4 15.4 *, **exchangeable
Test Example A
Evaluation of Plant Growth Promotion Effect of Specific Ketol Fatty
Acid (I) (Growth Promotion Test)
[0162] 1. Evaluation of Growth Promotion Effect on Morning
Glory
[0163] Seeds of morning glory (variety: murasaki) (9 g) were
subjected to concentrated sulfuric acid treatment for 20 minutes,
and then allowed to stand under water flow overnight. Subsequently,
the seeds were placed on wet sea sand such that the hila of the
seeds were directed upward for 24 hours, to thereby produce roots.
The seeds having roots were planted in sea sand at a position
1.5-2.0 cm below the surface of the sand, and then cultured under
continuous light irradiation (for about five days).
[0164] The entire plant bodies of the morning glory having leaves
produced through this culture were transferred into a culture
solution [KNO.sub.3 (250 mg), NH.sub.4NO.sub.3 (250 mg),
KH.sub.2PO.sub.4 (250 mg), MgSO.sub.4.7H.sub.2O (250 mg),
MnSO.sub.4.4H.sub.2O (1 mg), Fe-citrate n-hydrate (6 mg),
H.sub.3BO.sub.3 (2 mg), CuSO.sub.4.5H.sub.2O (0.1 mg),
ZeSO.sub.4.7H.sub.2O (0.2 mg), Na.sub.2MoO.sub.4.2H.sub.2O (0.2
mg), and Ca(H.sub.2PO.sub.4).sub.2.2H.sub.2O (250 mg) in distilled
water (1,000 mL)].
[0165] Water or a 100 .mu.M specific ketol fatty acid (I) aqueous
solution was sprayed to the cultured morning glory, which was then
placed in the dark overnight (for 14 hours). Thereafter, the
morning glory was grown under continuous light irradiation at
25.degree. C. for 16 days, and on the 16th day the height of the
plant was measured (N=8). The average of the heights of the plants
is shown in FIG. 1 [note: "I" shown in FIG. 1 refers to "specific
ketol fatty acid (I)"; the same shall apply to the below-described
Figs.]. As is clear from FIG. 1, the plant of the morning glory
becomes larger through application of specific ketol fatty acid
(I).
[0166] 2. Evaluation of Growth Promotion Effect on Lettuce
[0167] One month after seeding of lettuce, spraying of a 50 .mu.M
specific ketol fatty acid (I) aqueous solution was carried out for
five consecutive days, and growth of the lettuce (plant width) was
observed. The results are shown in FIG. 2. As shown in FIG. 2,
specific ketol fatty acid (I) exerts the effect of promoting growth
of the lettuce. The growth promotion effect was maintained 48 days
after the start of the test.
[0168] 3. Evaluation of Growth Promotion Effect on Broad Bean
[0169] One month after seeding of broad bean, spraying of a 50
.mu.M specific ketol fatty acid (I) aqueous solution was carried
out for five consecutive days, and growth of the broad bean (plant
width) was observed. The results are shown in FIG. 3. As shown in
FIG. 3, specific ketol fatty acid (I) exerts the effect of
promoting growth of the broad bean. The growth promotion effect was
maintained 48 days after the start of the test.
[0170] 4. Evaluation of Growth Promotion Effect on Eustoma
russellianum
[0171] Three months after seeding of Eustoma russellianum, a 50
.mu.M specific ketol fatty acid (I) aqueous solution was sprayed to
rosette leaves for five consecutive days, and bolting was observed
immediately after the spraying. Thereafter, growth of the plants of
the Eustoma russellianum was observed for 48 days. Although plant
width did not increase as had been expected, the plant height
continued to increase 48 days after observation of growth. The
results (plant height) are shown in FIG. 4.
[0172] 5. Evaluation of Growth Promotion Effect on Cyclamen
[0173] Four months after seeding of cyclamen, spraying of a 50
.mu.M specific ketol fatty acid (I) aqueous solution was carried
out for five consecutive days. Thereafter, the plant width and the
number of leaves were observed for 48 days. The plant width and the
number of leaves were both increased. The results are shown in FIG.
5.
[0174] 6. Evaluation of Growth Promotion Effect on Digitalis
[0175] Two weeks after seeding of digitalis, spraying of an 80
.mu.M specific ketol fatty acid (I) aqueous solution was carried
out for five consecutive days. Furthermore, three months after the
start of the test, spraying of an 80 .mu.M specific ketol fatty
acid (I) aqueous solution was carried out for six weeks (once a
week). Five-and-a-half months after the six-week spraying, the size
of leaves and the plant height were measured. The results show that
the size of leaves and the plant height were both increased (see
FIG. 6).
[0176] 7. Evaluation of Growth Promotion Effect on
Chrysanthemum
[0177] Two weeks after seeding of chrysanthemum, spraying of an 80
.mu.M specific ketol fatty acid (I) aqueous solution was carried
out for five consecutive days. Furthermore, three months after the
start of the test, spraying of an 80 .mu.M specific ketol fatty
acid (I) aqueous solution was carried out for six weeks (once a
week). Bolting was not observed at a nutrition growth stage of the
chrysanthemum. Four months after the last spraying, the plant width
was measured. The results show that the plant width of the
chrysanthemum is increased significantly (see FIG. 7).
[0178] 8. Evaluation of Growth Promotion Effect on Geranium
[0179] Two weeks after seeding of geranium, spraying of an 80 .mu.M
specific ketol fatty acid (I) aqueous solution was carried out for
five consecutive days. Furthermore, three months after the start of
the test, spraying of an 80 .mu.M specific ketol fatty acid (I)
aqueous solution was carried out for six weeks (once a week). Two
types of geranium; i.e., geranium having mottled leaves and
geranium having leaves of no mottles, were subjected to the test.
Five-and-a-half months after the last spraying, the size of the
leaves was measured. The results show that growth of the leaves of
these two types is promoted (see FIG. 8).
[0180] 9. Evaluation of Growth Promotion Effect on Primula
melacoides
[0181] One-and-a-half months after seeding of Primula melacoides,
spraying of an 80 .mu.M specific ketol fatty acid (I) aqueous
solution was carried out for five consecutive days. Furthermore,
four months after the start of the test, spraying of an 80 .mu.M
specific ketol fatty acid (I) aqueous solution was carried out for
six weeks (once a week). Bolting was not observed at a nutrition
growth stage of the Primula melacoides. Six-and-a-half months after
the last spraying, the plant width and the leave size were
measured. The results show that the plant width and leave size of
the chrysanthemum are increased (see FIG. 9).
[0182] 10. Evaluation of Growth Promotion Effect on Begonia
sempaflorens
[0183] Two weeks after seeding of Begonia sempaflorens, spraying of
an 80 .mu.M specific ketol fatty acid (I) aqueous solution was
carried out for five consecutive days. Furthermore, three months
after the start of the test, spraying of an 80 .mu.M specific ketol
fatty acid (I) aqueous solution was carried out for six weeks (once
a week). Four months after the last spraying, the leave size was
measured. The results show that growth of the leaves is promoted
(see FIG. 10).
[0184] 11. Evaluation of Growth Promotion Effect on Dianthus
caryophyllus
[0185] Seedlings of Dianthus caryophyllus (feeling scarlet) were
planted in early October, and then grown by means of a customary
method. In mid-April of the next year, spraying of a 100 .mu.M
specific ketol fatty acid (I) aqueous solution was carried out (5
mL per plant), and then the height of the plants was measured. The
results show that growth of the Dianthus caryophyllus plant was
promoted in the group to which the specific ketol fatty acid (I)
had been applied, although spraying of the specific ketol fatty
acid (I) had been carried out only once (see FIG. 11).
[0186] 12. Evaluation of Growth Controlling Effect on Oryza sativa
L.
[0187] (1) Good-quality seeds of Oryza sativa L. (variety:
koshihikari) (200 g) were immersed in water (800 mL) at 10.degree.
C. for 13 days. Thereafter, the seeds were equally divided into
four groups, and the respective seed groups were immersed in
specific ketol fatty acid (I) aqueous solutions (concentration: 0
.mu.M, 1 .mu.M, 10 .mu.M, and 100 .mu.M) (200 mL) at 30.degree. C.
for 1.5 days. The immersed seeds were planted in a four-divided
seedbed tray, and grown under no light irradiation at 27.degree. C.
for three days. Subsequently, the grown seedlings were exposed to
the typical outside environment.
[0188] Six days after the exposure, 18 seedlings were randomly
selected from each group, and the heights of the seedlings were
measured, and then averaged. The results are shown in FIG. 12. As
shown in FIG. 12, the degree of growth promotion of the seedlings
regarding the height is commensurate with the application amount of
the specific ketol fatty acid (I).
[0189] Thus, the plant growth promotion effect of the present plant
activator, which is confirmed in the aforementioned tests employing
various plants, is also observed in Oryza sativa L.
[0190] Subsequently, in consideration of practical handling of
seedlings of Oryza sativa L., the effect of application of specific
ketol fatty acid (I) was evaluated. Specifically, whether or not
specific ketol fatty acid (I) exerts the effect of controlling
growth of the third leaves of seedlings of Oryza sativa L. was
evaluated, since the time when the third leaves are grown in
seedlings of Oryza sativa L. is considered to be a suitable time
for transferring the seedlings from a seedbed to a paddy field. In
order to perform this evaluation, three weeks after the
aforementioned light irradiation treatment, seedlings of each group
were randomly selected, and the average of the proportions of the
second leaves and third leaves was obtained. The results are shown
in FIG. 13. As shown in FIG. 13, specific ketol fatty acid (I)
exerts the effect of controlling growth of the third leaves.
However, unlike the case of promotion of growth of the seedlings,
the optimum concentration of the specific ketol fatty acid (I)
aqueous solution is 1 .mu.M.
[0191] The results show that, when specific ketol fatty acid (I) is
used as an active ingredient of the present plant activator in
order to shorten the growth period of seedlings of Oryza sativa L.
in a seedbed, the application amount of the specific ketol fatty
acid (I) must be determined appropriately.
[0192] (2) Seedlings of Oryza sativa L. were grown in a seedbed in
a manner similar to that described in (1), except that the
seedlings were immersed in ion-exchange water at 10.degree. C. for
15 days, without application of specific ketol fatty acid (I)
described above in (1). Subsequently, the resultant seedlings which
had been divided into five groups, each group containing three
subgroups (only control group containing four subgroups) and each
subgroup containing 16 seedlings, were exposed to the outside
environment. Immediately after this exposure, spraying of specific
ketol fatty acid (I) was carried out (0 ppm for a first group
(control group), 25 ppm for second and third groups, 50 ppm for
fourth and fifth groups). Thirty days after the spraying, the
seedlings were planted in a paddy field, and additional spraying of
specific ketol fatty acid (I) (25 ppm) was carried out for two
groups; i.e., the third group (total amount of the acid (I): 25+25
ppm) and the fifth group (total amount of the acid (I): 50+25
ppm).
[0193] Subsequently, the seedlings of the Oryza sativa L. were
grown in the paddy field by means of a customary method. Forty-one
days after the above planting, in each group, the plant height and
the number of stems per plant (at a section at which four seedlings
were planted) were measured, and then averaged.
[0194] The plant height was 56 cm in the control group (first
group), 57 cm in the second group (amount of the specific ketol
fatty acid (I): 25 ppm), 58 cm in the fourth group (amount of the
specific ketol fatty acid (I): 50 ppm), 57 cm in the third group
(amount of the specific ketol fatty acid (I): 25+25 ppm), and 58 cm
in the fifth group (amount of the specific ketol fatty acid (I):
50+25 ppm). Therefore, significant difference was not observed
between these groups.
[0195] The number of stems per plant was 34 in the control group
(first group), 38 in the second group (amount of the specific ketol
fatty acid (I): 25 ppm), 38 in the fourth group (amount of the
specific ketol fatty acid (I): 50 ppm), 39 in the third group
(amount of the specific ketol fatty acid (I): 25+25 ppm), and 37 in
the fifth group (amount of the specific ketol fatty acid (I): 50+25
ppm). Briefly, the number of stems per plant in each of the second
through fifth groups was about 10% greater than that of stems per
plant in the control group. However, difference attributed to the
application manner of the specific ketol fatty acid (I) was not
observed.
[0196] The results show that specific ketol fatty acid (I) serving
as an active ingredient of the present plant activator exerts the
effect of controlling growth of Oryza sativa L.; i.e., the effect
of increasing the number of stems. Therefore, the specific ketol
fatty acid (I) exerts the effect of increasing the yield of rice on
the basis of unit area of the paddy field in which the seedlings
are planted; i.e., an considerably important effect in production
of rice.
[0197] The results of the aforementioned growth promotion tests
show that specific ketol fatty acid (I) exerts excellent effect of
promoting growth of various forms of many plants. Particularly, the
specific ketol fatty acid (I) exerts the effect of promoting growth
of a plant in an early stage of its growth, and the growth
promotion effect is continuous.
[0198] Thus, it is apparent that specific ketol fatty acid (I)
serving as an active ingredient of the present plant activator
exerts the effect of promoting growth of various plants, and the
present plant activator is useful.
[0199] As described above, it is clear that the present plant
activator can be used as a plant growth promoting agent or a plant
growth controlling agent.
Test Example B
Evaluation of Plant Dormancy Preventive Effect of Specific Ketol
Fatty Acid (I) (Plant Dormancy Preventive Test)
[0200] When strawberry seedlings are exposed directly to low
temperature conditions in winter, the seedlings enter dormancy, and
growth of the seedlings is stopped. Whether or not the present
plant activator exerts the effect of preventing dormancy was
evaluated.
[0201] Specific ketol fatty acid (I) aqueous solutions
[concentration: 10 .mu.M, 100 .mu.M, and 0 .mu.M (control)] were
applied through spraying to strawberry seedlings on August 27 (at
day 0), September 3, and September 8. Thereafter, the seedlings
were cultured outdoors without artificial treatment such as low
temperature treatment, and formation of flower buds was observed
with passage of time. In the control group, no flower bud formation
was observed. In contrast, in the groups in which the specific
ketol fatty acid (I) was applied through spraying, flower bud
formation proceeded, and the number of flowers increased (this
flower bud formation promotion effect agrees with the description
of Japanese Patent Application Laid-Open (kokai) No. 11-29410).
[0202] At the one hundred and eighth day, percent dormancy
(percentage of dormant plants--which are plants in which growth of
small leaf buds with markings is not observed 15 days after a
marking was applied to the leaf buds--with respect to the entirety
of the test plants) was measured. As a result, in the control
group, dormancy of the entire plants was observed. In contrast, in
the groups in which the specific ketol fatty acid (I) aqueous
solution was applied through spraying, dormancy of strawberry was
prevented. The results show that the dormancy preventive effect is
more significant in the group in which the specific ketol fatty
acid (I) of low concentration (10 .mu.M) was applied than in the
group in which the fatty acid (I) of high concentration (100 .mu.M)
was applied (see FIG. 14).
[0203] The results show that when the present plant activator of
low concentration is applied to a plant, the activator exerts the
effect of preventing dormancy of the plant; the activator can be
used as a plant dormancy preventive agent or a plant growth
controlling agent; and the activator is useful.
Test Example C
Evaluation of Plant Stress (Dry Stress) Suppressive Effect of
Specific Ketol Fatty Acid (I)
[0204] Seeds of lettuce (50 seeds per test group) were immersed in
specific ketol fatty acid (I) aqueous solutions [concentration: 2
.mu.M, 10 .mu.M, 20 .mu.M, and 0 .mu.M (control)] for 72 hours, and
dried in air for 48 hours. The resultant seeds were disposed on
water-containing filter paper, and allowed to germinate. In each
test group, germination rate--percentage (%) of germinated seeds
with respect to the entirety of seeds--as obtained.
[0205] The results are shown in Table 2.
2TABLE 2 Specific ketol Germination fatty acid (I) rate Number of
germinated concentration (.mu.M) (%: n = 50) seeds 0 10 5 2 86 43
10 98 49 20 90 45
[0206] As is apparent from the results, in the control group, most
seed failed to endure the dry stress in the drying step, resulting
in failure of germination. In contrast, most of the seeds which had
been immersed in the specific ketol fatty acid (I) aqueous solution
successfully germinated.
[0207] From the above results, it is clear that the present plant
activator exerts the effect of enhancing tolerance of plants
against dry stress; the activator can be used as a plant stress
suppressing agent or a plant growth controlling agent; and the
activator is useful.
Test Example D
Growth Controlling Effect of Specific Ketol Fatty Acid (I) on
Fungi
[0208] (1) Evaluation of effect of proliferating hyphae of P.
citrinopileatus Sing. (edible mushroom) belonging to Pleurotus of
basidiomycota.
[0209] A potato-dextrose-agar culture medium was sterilized by use
of an autoclave. After the medium was cooled to a temperature at
which the agar was not solidified, a 1 mM specific ketol fatty acid
(I) aqueous solution which had been sterilized by use of a membrane
filter was added to the medium, to thereby prepare four culture
media; i.e., a culture medium containing 0 .mu.M of the fatty acid
(I), a culture medium containing 10 .mu.M of the fatty acid (I), a
culture medium containing 30 .mu.M of the fatty acid (I), and a
culture medium containing 100 .mu.M of the fatty acid (I). Each of
the culture media was solidified in a 10-cm plate, and then hyphae
of P. citrinopileatus (one platinum loop) was inoculated on the
medium. Subsequently, the hyphae were cultured at 37.degree. C.,
and proliferation of the hyphae was observed (10 plates for each
group). Proliferation of the hyphae was evaluated on the basis of
the average of the diameters of the proliferated hyphae on the
plate. The results are shown in FIG. 15. As is clear from FIG. 15,
the degree of proliferation of the hyphae of P. citrinopileatus is
dependent on the concentration of the specific ketol fatty acid
(I).
[0210] (2) Evaluation of Growth Promotion Effect on Carpophore of
Lentinus edodes (Berk.) Singer
[0211] Wood (Quercus serrata) containing hyphae of Lentinus edodes
was cut into pieces having a length of about 15 cm, and the pieces
were immersed in 10.degree. C. water for 24 hours, after which the
pieces were allowed to stand in a container of high humidity.
Subsequently, specific ketol fatty acid (I) aqueous solutions
(concentration: 0 .mu.M, 3 .mu.M, 30 .mu.M, and 100 .mu.M) were
applied through spraying to the resultant pieces (six pieces for
each group). Each solution was applied to each group (5 mL for each
piece). Subsequently, the carpophores of Lentinus edodes were
cultured in the same container at 18.degree. C. under weak light
irradiation. This culture was continued for five days, and then
growth of the carpophores of Lentinus edodes was observed. FIG. 16
is a photograph showing the carpophores of Lentinus edodes cultured
in the groups (note: {circle over (1)} application of the specific
ketol fatty acid (I) (0 .mu.M), {circle over (2)} (3 .mu.M),
{circle over (3)} (30 .mu.M), and {circle over (4)} (100 .mu.M)).
The average of the carpophores per piece was 0 in the 0 .mu.M
application group, 0.17 in the 3 .mu.M application group, 1.0 in
the 30 .mu.M application group, and 1.0 in the 100 .mu.M
application group.
[0212] The results show that specific ketol fatty acid (I) exerts
the effect of promoting growth of carpophores of Lentinus edodes
during culture.
[0213] The results of the aforementioned tests (1) and (2) show
that when specific ketol fatty acid (I) is applied to ascomycete or
basidiomycete, proliferation of hyphae thereof can be promoted, and
harvest efficiency of carpophores can be enhanced. Furthermore, the
present plant activator may contribute to establishment of an
artificial culture method of mushrooms which at present are
difficult to culture artificially (e.g., Tricholoma matsutake).
Industrial Applicability
[0214] As described above, the present invention provides a plant
activator exerting excellent effect of controlling growth of
various plants.
* * * * *