Title of Invention

METHOD OF PRODUCING REDUCED COENZYME Q10 USING SOLVENT WITH HIGH OXIDATION-PROTECTIVE EFFECT

Abstract The present invention relates to a method of conveniently and efficiently producing reduced coenzyme Q10 having excellent qualities which is useful in foods, functional nutritive foods, specific health foods, nutritional supplements, nutrients, drinks, feeds, animal drugs, cosmetics, medicines, remedies, preventive drugs, etc. This method is suitable for industrial production thereof. In a method of synthesizing reduced coenzyme Q10 by reducing oxidized coenzyme Q10, followed by crystallization, at least one species selected from among hydrocarbons, fatty acid esters, ethers and nitriles is used as a solvent. Thus, the reduced coenzyme Q10 can be protected from oxidation, and as a result, the formation of the oxidized coenzyme Q10 as a by-product can be minimized, thereby giving reduced coenzyme Q10 having excellent qualities.
Full Text DESCRIPTION
METHOD OF PRODUCING REDUCED COENZYME Q10 USING SOLVENT WITH HIGH
OXIDATION-PROTECTIVE EFFECT
TECHNICAL FIELD
The present invention relates to a method of synthesizing
and a method of crystallizing reduced coenzyme Q10. Reduced
coenzyme Q10 shows a higher level of oral absorbability as
compared with oxidized coenzyme Q10 and is a compound useful
as an ingredient in good foods, functional nutritive foods,
specific health foods, nutritional supplements, nutrients,
drinks, feeds, animal drugs, cosmetics, medicines, remedies,
preventive drugs, etc.
BACKGROUND ART
Oxidized coenzyme Q10, which is a benzoquinone derivative
widely distributed in the biological world, is also called
vitamin Q because of its vitamin-like function and is an
ingredient acting as a nutrient in restoring the cell activity
that has been weakened to its healthy condition and rejuvenating
the body. On the other hand, reduced coenzyme Q10, which is
derived from oxidized coenzyme Q10 by two-electron reduction,
is as white crystals as compared with oxidized coenzyme Q10 being
as orange-colored crystals. Reduced coenzyme Q10 and oxidized
coenzyme Q10 are known to be localized in the mitochondrion,
lysosome, Golgi body, microsome, peroxisome, and cell membrane,
among others, and involved, as constituents of the electron
transport system, in ATP production and activation, in vivo
antioxidant activity, and membrane stabilization; they are thus
substances indispensable for body function maintenance.
It is known that reduced coenzyme Q10 can be prepared by
producing coenzyme Q10 in the conventional manner, for example
by synthesis, fermentation, or extraction from natural products,
and concentrating a reduced coenzyme Q10-containing eluate

fraction resulting from chromatography (JP-A-10-109933). On
that occasion, as described in the above-cited publication, the
chromatographic concentration may be carried cut after
reduction of oxidized coenzyme Q10 contained in the reduced
coenzyme Q10 with a conventional reducing agent such as sodium
borohydride or sodium dithionite (sodium hyposulfite), or
reduced coenzyme Q10 may be prepared by reacting the reducing
agent mentioned above with an existing highly pure grade of
coenzyme Q10 (oxidized form). However, the thus-obtained
reduced coenzyme Q10 cannot always be in a highly pure state
but tends to occur as a low-purity crystalline, semisolid, or
oily product containing such impurities as oxidized coenzyme
Q10.
JP-A-57-70834 discloses an example in which reduced
coenzyme Q10 was synthesized by dissolving coenzyme Q10 in hexane
and adding an aqueous solution of sodium hydrosulfite (sodium
hyposulfite) to the solution, followed by stirring. However,
sodium dithionite was used as a reducing agent in an amount
as large as twice the weight of coenzyme Q10. Thus, such method
of synthesis has problems from the economical viewpoint as well
as complexity viewpoint of the subsequent purification
procedure.
Reduced coenzyme Q10 is readily oxidized to oxidized
coenzyme Q10 by molecular oxygen. On a commercial
production scale, complete oxygen elimination is very
difficult to achieve and, furthermore, fairly long periods
of time are required for individual operations, unlike
laboratory scale production, so that residual oxygen exerts
a great adverse effect. The oxidation in question is
directly connected with such yield and quality problems as
the formation of hardly eliminable oxidized coenzyme Q10
and adulteration of the product therewith. For obtaining
highly pure reduced coenzyme Q10 in the form of crystals,
it is important to adequately protect, the reduced form from
the oxidation mentioned above.

SUMMARY OF THE INVENTION
In view of the foregoing, the present invention has an
object to provide a convenient and efficient method of
synthesizing and of crystallizing high-quality reduced
coenzyme Q10. Furthermore, the present invention has another
object to provide an outstanding method for obtaining
high-quality reduced coenzyme Q10 in the form of crystals which
is suited for the production thereof on a commercial scale.
The present inventors made intensive investigations, and
as a result, found that when placed in a specific solvent,
reduced coenzyme Q10 is favorably protected from oxidation by
molecular oxygen and, based on this finding, they have completed
the present invention.
Thus, the present invention provides a method of
producing reduced coenzyme Q10
which comprises using at least one species selected from
among hydrocarbons exclusive of hexane, fatty acid esters,
ethers and nitriles as a solvent in synthesizing reduced
coenzyme Q10 by reduction of oxidized coenzyme Q10
The invention also provides a method for producing
reduced coenzyme Q10
which comprises using a hydrocarbon as a solvent in a
deoxygenated atmosphere in synthesizing reduced coenzyme Q10
by reduction of oxidized coenzyme Q10.
The invention further provides a method for crystallizing
reduced coenzyme Q10,
which comprises crystallizing the reduced coenzyme Q10
using, as a solvent, at least one species selected from the group
consisting of hydrocarbons, fatty acid esters, ethers and
nitriles.
The invention further provides a method for stabilizing
reduced coenzyme Q10
which comprises handling reduced coenzyme Q10 in the form
of a solution in at least one solvent selected from among
hydrocarbons, fatty acid esters, ethers and nitriles to thereby

protect the reduced coenzyme Q10 from oxidation by molecular
oxygen.
The invention still further provides a reduced coenzyme
Q10 crystal with a reduced coenzyme Q10/oxidized coenzyme Q10
weight ratio of not lower than 96/4.
In accordance with the present invention, it is possible,
in synthesizing reduced coenzyme Q10 by reduction of oxidized
coenzyme Q10, to favorably protect the reduced coenzyme Q10 from
oxidation by molecular oxygen and, thus, it is possible to
synthesize high-quality reduced coenzyme Q10 in such a condition
that the formation of oxidized coenzyme Q10 as a byproduct is
minimized. Furthermore, the method of crystallizing reduced
coenzyme Q10 of the present invention makes it possible to
favorably protect reduced coenzyme Q10 from oxidation by
molecular oxygen and, thus it is possible to convert the same
into a crystalline state in such a condition that the formation
of oxidized coenzyme Q10 as a byproduct is minimized and thereby
obtain high-quality crystalline coenzyme Q10.
DETAILED DISCLOSURE OF THE INVENTION
In the following, the present invention is described in
detail.
In accordance with the invention, a solvent highly
effective in protecting reduced coenzyme Q10 from the
above-mentioned oxidation, namely at least one species selected
from among hydrocarbons, fatty acid esters, ethers, and
nitriles is used for synthesizing high-quality reduced coenzyme
Q10 and for crystallizing the same and, further, for handling
reduced coenzyme Q10 stably, while inhibiting the oxidation of
reduced coenzyme Q10 to oxidized coenzyme Q10 by molecular oxygen.
Surprisingly, reduced coenzyme Q10 is adequately protected from
oxidation by molecular oxygen in a state dissolved or suspended
in the solvents mentioned above rather in a crystalline state.
Such oxidation-protective effect of the solvents has been found
for the first time by the present inventors.

The hydrocarbons are not particularly restricted, but
there may be mentioned, for example, aliphatic hydrocarbons,
aromatic hydrocarbons, halogenated hydrocarbons, etc.
Preferred are aliphatic hydrocarbons and aromatic hydrocarbons,
and more preferred are aliphatic hydrocarbons.
The aliphatic hydrocarbons are not particularly
restricted, and may be cyclic or acyclic, or saturated or
unsaturated. However, generally they contain 3 to 20 carbon
atoms, and preferably 5 to 12 carbon atoms.
As specific examples, there may be mentioned, for example,
propane, butane, isobutane, pentane, 2-methylbutane,
cyclopentane, 2-pentene, hexane, 2-methylpentane,
2,2-dimethylbutane, 2,3-dimethylbutane, methylcyclopentane,
cyclohexane, 1-hexene, cyclohexene, heptane, 2-methylhexane,
3-methylhexane, 2,3-dimethylpentane, 2, 4-dimethylpentane,
methylcyclohexane, 1-heptene, octane, 2,2,3-trimethylpentane,
isooctane, ethylcyclohexane, 1-octene, nonane,
2,2,5-trimethylhexane, 1-nonene, decane, 1-decene, p-menthane,
undecane, dodecane, etc.
Among them, saturated aliphatic hydrocarbons having 5 to
8 carbon atoms are more preferred, and preferably used are
pentane, 2-methylbutane and cyclopentane, which have 5 carbon
atoms (referred to as "pentanes"); hexane, 2-methylpentane,
2,2-dimethylbutane, 2, 3-dimethylbutane, methylcyclopentane,
cyclohexane, which have 6 carbon atoms (referred to as
"hexanes"); heptane, 2-methylhexane, 3-methylhexane,
2,3-dimethylpentane, 2, 4-dimethylpentane, methylcyclohexane,
which have 7 carbon atoms (referred to as "heptanes") ; octane,
2,2,3-trimethylpentane, isooctane, ethylcyclohexane, which
have 8 carbon atoms (referred to as octanes) ; and a mixture of
these. In particular, the above heptanes are particularly
preferred since they have a tendency to show a very high
protection effect against oxidization, and heptane is most
preferred.
The aromatic hydrocarbons are not particularly

restricted, but generally they contain 6 to 20 carbon atoms,
preferably 6 to 12 carbon atoms, and more preferably 7 to 10
carbon atoms. As specific examples, there may be mentioned,
for example, benzene, toluene, xylene, o-xylene, m-xylene,
p-xylene, ethylbenzene, cumene, mesitylene, tetralin,
butylbenzene, p-cymene, cyclohexylbenzene, diethylbenzene,
pentylbenzene, dipentylbenzene, dodecylbenzene, styrene, etc.
Preferred are toluene, xylene, o-xylene, m-xylene, p-xylene,
ethylbenzene, cumene, mesitylene, tetralin, butylbenzene,
p-cymene, cyclohexylbenzene, diethylbenzene and pentylbenzene.
More preferred are toluene, xylene, o-xylene, m-xylene,
p-xylene, cumene and tetralin, and most preferred is cumene.
The halogenated hydrocarbons are not particularly
restricted, and may be cyclic or acyclic, or saturated or
unsaturated. However, acyclic halogenated hydrocarbons are
preferably used. More preferred are chlorinated hydrocarbons
and f luorinated hydrocarbons, and chlorinated hydrocarbons are
still more preferred. Additionally, ones containing 1 to 6
carbon atoms, preferably 1 to 4 carbon atoms, and more
preferably 1 to 2 carbon atoms are used.
As specific examples, for example,, there maybe mentioned
dichloromethane, chloroform, carbon tetrachloride,
1,1-dichloroethane, 1,2-dichloroethane,
1,1,1-trichloroethane, 1,1,2-trichloroethane,
1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane,
pentachloroethane, hexachloroethane, 1,1-dichloroethylene,
1,2-dichloroethylene, trichloroethylene, tetrachloroethylene,
1,2-dichloropropane, 1,2, 3-trichloropropane, chlorobenzene,
1,1,1, 2-tetrafluoroethane, etc. Preferred are
dichloromethane, chloroform, carbon tetrachloride,
1,1-dichloroethane, 1,2-dichloroethane,
1,1,1-trichloroethane, 1,1,2-trichloroethane,
1,1-dichloroethylene, 1,2-dichloroethylene,
trichloroethylene, chlorobenzene and
1,1,1, 2-tetrafluoroethane. More preferred are

dichloromethane, chloroform, 1,2-dichloroethylene,
trichloroethylene, chlorobenzene and
1,1,1,2-tetrafluoroethane. Still more preferred are
dichloromethane, chloroform, 1,2-dichloroethylene and
trichloroethylene.
The fatty acid esters are not particularly restricted,
but there may be mentioned, for example, propionates, acetates,
formates, etc. Preferred are acetates and formates, and more
preferred are acetates. Ester functional groups thereof are
not particularly restricted, but there may be mentioned alkyl
esters having 1 to 8 carbon atoms, aralkyl esters having 1 to
8 carbon atoms, etc. Preferred are alkyl esters having 1 to
6 carbon atoms, and more preferred are alkyl esters having 1
to 4 carbon atoms.
As the propionates, there may be mentioned, for example,
methyl propionate, ethyl propionate, butyl propionate,
isopentyl propionate, etc. Preferred is ethyl propionate.
As the acetates, there may be mentioned, for example,
methyl acetate, ethyl acetate, propyl acetate, isopropyl
acetate, butyl acetate, isobutyl acetate, sec-butyl acetate,
pentyl acetate, isopentyl acetate, sec-hexyl acetate,
cyclohexyl acetate, benzyl acetate, etc. Preferred are methyl
acetate, ethyl acetate, propyl acetate, isopropyl acetate,
butyl acetate, isobutyl acetate, sec-butyl acetate, pentyl
acetate, isopentyl acetate, sec-hexyl acetate and cyclohexyl
acetate. More preferred are methyl acetate, ethyl acetate,
propyl acetate, isopropyl acetate, butyl acetate and isobutyl
acetate. Most preferred is ethyl acetate.
As the formates, there may be mentioned, for example,
methyl formate, ethyl formate, propyl formate, isopropyl
formate, butyl formate, isobutyl formate, sec-butyl formate,
pentyl formate, etc. Preferred are methyl formate, ethyl
formate, propyl formate, butyl formate, isobutyl formate and
pentyl formate, and most preferred is ethyl formate.
The ethers are not particularly restricted, and may be

cyclic or acyclic, or saturated or unsaturated. But saturated
ones are preferably used. Generally, ones containing 3 to 20
carbon atoms, and preferably 4 to 12 carbon atoms and more
preferably 4 to 8 carbon atoms are used.
As specific examples, there may be mentioned, for example,
diethyl ether, methyl tert-butyl ether, dipropyl ether,
diisopropyl ether, dibutyl ether, dihexyl ether, ethyl vinyl
ether, butyl vinyl ether, anisol, phenetole, butyl phenyl ether,
methoxytoluene, dioxane, furan, 2-methylfuran,
tetrahydrofuran, tetrahydropyran, ethylene glycol dimethyl
ether, ethylene glycol diethyl ether, ethylene glycol dibutyl
ether, ethylene glycol monomethyl ether, ethylene glycol
monoethyl ether, ethylene glycol monobutyl ether, etc.
Preferred are diethyl ether, methyl tert-butyl ether,
dipropyl ether, diisopropyl ether, dibutyl ether, dihexyl ether,
anisol, phenetole, butyl phenyl ether, methoxytoluene, dioxane,
2-methylfuran, tetrahydrofuran, tetrahydropyran, ethylene
glycol dimethyl ether, ethylene glycol diethyl ether, ethylene
glycol dibutyl ether, ethylene glycol monomethyl ether and
ethylene glycol monoethyl ether. More preferred are diethyl
ether, methyl tert-butyl ether, anisol, dioxane,
tetrahydrofuran, ethylene glycol monomethyl ether and ethylene
glycol monoethyl ether. More preferred are diethyl ether,
methyl tert-butyl ether, anisol, etc., and most preferred is
methyl tert-butyl ether.
The nitriles are not particularly restricted, and may be
cyclic or acyclic, or saturated or unsaturated. However,
saturated ones are preferably used. Generally, ones
containing 2 to 20 carbon atoms, preferably 2 to 12 carbon atoms,
and more preferably 2 to 8 carbon atoms are used.
As specific examples, there may be mentioned, for example,
acetonitrile, propiononitrile, malononitrile, butyronitrile,
isobutyronitrile, succinonitrile, valeronitrile,
glutaronitrile, hexanenitrile, heptylcyanide, octylcyanide,
undecanenitrile, dodecanenitrile, tridecanenitrile,

pentadecanenitrile, stearonitrile, chloroacetonitrile,
bromoacetonitrile, chloropropiononitrile,
bromopropiononitrile, methoxyacetonitrile, methyl
cyanoacetate, ethyl cyanoacetate, tolunitrile,, benzonitrile,
chlorobenzonitrile, bromobenzonitrile, cyanobenzoic acid,
nitrobenzonitrile, anisonitrile, phthalonitrile,
bromotolunitrile, methyl cyanobenzoate, methoxybenzonitrile,
acetylbenzonitrile, naphthonitrile, biphenylcarbonitrile,
phenylpropiononitrile, phenylbutyronitrile,
methylphenylacetonitrile, diphenylacetonitrile,
naphthylacetonitrile, nitrophenylacetonitrile,
chlorobenzylcyanide, cyclopropanecarbonitrile,
cyclohexanecarbonitrile, cycloheptanecarbonitrile,
phenylcyclohexanecarbonitrile, tolylcyclohexanecarbonitrile,
etc.
Preferred are acetonitrile, propiononitrile,
butyronitrile, isobutyronitrile, succinonitrile,
valeronitrile, chloropropiononitrile, methyl cyanoacetate,
ethyl cyanoacetate, tolunitrile and benzonitrile. More
preferred are acetonitrile, propiononitrile, butyronitrile
and isobutyronitrile, and most preferred is acetonitrile.
In selecting the solvent to be used from among the solvents
mentioned above, such properties as boiling point and viscosity
are preferably taken into consideration; for example, the
solvent should have a boiling point which allows appropriate
warming for increasing the solubility and facilitates a solvent
removal from wet masses by drying and solvent recovery from
crystallization filtrates (about 30 to 150°C at 1 atm), a
melting point such that solidification hardly occurs in
handling at room temperature as well as upon cooling to room
temperature or below (not higher than about 20°C, preferably
not higher than about 10°C, still more preferably not higher
than about 0°C) , and a low viscosity (not higher than about 10
cp at 20°C) . From the industrial operation viewpoint, a solvent
which is scarcely volatile at ordinary temperature is

preferred; for example, one having a boiling point of not lower
than about 80°C is preferred, and one having a boiling point
of not lower than about 90°C is more preferred.
Reduced coenzyme Q10, when in a dissolved state, tends
to become more resistant to oxidation as the concentration
thereof increases. Reduced coenzyme Q10 is highly soluble in
the solvents mentioned above and, in this respect, too, the
above solvents are suitable for the protection from oxidation.
The concentration of reduced coenzyme Q10 which is preferred
from the viewpoint of protection thereof from oxidation may vary
depending on the solvent species, among others, hence cannot
be absolutely specified. Generally, however, the
concentration of reduced coenzyme Q10 in the above solvents is
generally not lower than 1 w/w%, preferably not lower than 2
w/w% . The upper limit is not particularly restricted but, from
the practical operability viewpoint, it is 400 w/w% or below,
preferably 200 w/w% or below, more preferably 100 w/w% or below,
still more preferably 50 w/w% or below.
Furthermore, the solubility of reduced coenzyme Q10 in
the solvents mentioned above shows a favorable temperature
dependency. Therefore, the use of the above solvents is
suitable also for favorably reducing the amount of reduced
coenzyme Q10 in solution and converting (crystallizing) the same
into a crystalline state.
Thus, when such a solvent as mentioned above is used, it
is possible to minimize the undesirable oxygen-involving side
reaction in the step of reduction reaction of oxidized coenzyme
Q10 and/or crystallization of reduced coenzyme Q10. In addition,
when reduced coenzyme Q10 is handled in the form of a solution
in any of the solvents mentioned above, the reduced coenzyme
Q10 is protected from oxidation by molecular oxygen and is thus
stabilized, so that such handling operations as extraction and
washing (extraction using any of the above solvents as added
from the outside and washing with water), concentration
(including solvent substitution by adding another solvent while

concentrating) and column chromatography may be carried out
successfully. In the practice of the invention, it is of course
preferred that the series of operations from the reduction
reaction of oxidized coenzyme Q10 to the crystallization
(purification by crystallization) of reduced coenzyme Q10
(inclusive of extraction of reduced coenzyme Q10, washing the
extract with water, and so forth, where necessary) be carried
out using the solvent or solvents mentioned above.
In the practice of the invention, the above solvents may
be used in combination with another solvent, if necessary,
within such an amount that the latter does not produce some or
other adverse effect. For example, an appropriate amount of
another solvent may be added to the above solvents in order to
improve the solubility of oxidized coenzyme Q10 and/or reduced
coenzyme Q10, the solubility of the reducing agent, the rate
of reaction and the like in the reduction reaction and, in the
crystallization of reduced coenzyme Q10, to improve the
solubility of reduced coenzyme Q10 as well as the
crystallization concentration, crystallization temperature,
yield, slurry properties, crystal properties, and the like.
Such other solvent is not particularly restricted but
includes, among others, water, alcohols, fatty acids, ketones,
nitrogen-containing compounds other than nitriles, and
sulfur-containing compounds, and the like.
The alcohols are not particularly restricted but may be
cyclic or acyclic, or saturated or unsaturated. Saturated ones
are preferred, however. Generally, they contain 1 to 20 carbon
atoms, preferably 1 to 12 carbon atoms, more preferably 1 to
6 carbon atoms, still more preferably 1 to 5 carbon atoms, and
most preferably 1 to 3 carbon atoms. Monohydric alcohols
containing 2 or 3 carbon atoms, dihydric alcohols containing
2 to 5 carbon atoms, and the trihydric alcohol containing 3
carbon atoms are preferred, among others.
As the monohydric alcohol, there may be mentioned, for
example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol,

2-butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol,
2-pentanol, 3-pentanol, 2-methyl-1-butanol, isopentyl alcohol,
tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol,
1-hexanol, 2-methyl-1-pentanol, 4-methyl-2-pentanol,
2-ethyl-1-butanol, 1-heptanol, 2-heptanol, 3-heptanol,
1-octanol, 2-octanol, 2-ethyl-l-hexanol, 1-nonanol, 1-decanol,
1-undecanol, 1-dodecanol, allyl alcohol, propargyl alcohol,
benzyl alcohol, cyclohexanol, 1-methylcyclohexanol,
2-methylcyclohexanol, 3-methylcyclohexanol,
4-methylcyclohexanol, etc.
Preferred are methanol, ethanol, 1-propanol, 2-propanol,
1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol,
1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol,
isopentyl alcohol, tert-pentyl alcohol, 3-methyl-2-butanol,
neopentyl alcohol, 1-hexanol, 2-methyl-1-pentanol,
4-methyl-2-pentanol, 2-ethyl-1-butanol and cyclohexanol.
More preferred are methanol, ethanol, 1-propanol, 2-propanol,
1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol,
1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol,
isopentyl alcohol, tert-pentyl alcohol, 3-methyl-2-butanol
and neopentyl alcohol. Still more preferred are methanol,
ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol,
isobutyl alcohol, 2-methyl-1-butanol and isopentyl alcohol.
Further preferred are methanol, ethanol, 1-propanol and
2-propanol, particularly preferred are ethanol, 1-propanol and
2-propanol, and most preferred is ethanol.
As the dihydric alcohol, there may be mentioned, for
example, 1,2-ethanediol, 1,2-propandiol, 1,3-propandiol,
1,2-butanediol, 1,3-butanediol, 1,4-butanediol,
2,3-butanediol, 1,5-pentanediol, etc. Preferred are
1,2-ethanediol, 1,2-propandiol and 1,3-propandiol, and most
preferred is 1,2-ethanediol.
As the trihydric alcohol, glycerol, etc. may be
preferably used, for example.
As fatty acids, there may be mentioned, for example,

formic acid, acetic acid, propionic acid, etc. Preferred are
formic acid and acetic acid, and most preferred is acetic acid.
The ketones are not particularly restricted, and ones
having 3 to 6 carbon atoms are preferably used. As specific
examples, there may be mentioned, for example, acetone, methyl
ethyl ketone, methyl butyl ketone, methyl isobutyl ketone, etc.
Preferred are acetone and methyl ethyl ketone, and most
preferred is acetone.
As the nitrogen compounds, there may be mentioned, for
example, nitromethane, triethylamine, pyridine, formamide,
N-methylformamide, N, N-dimethylformamide,
N,N-dimethylacetoamide, N-methylpyrrolidone, etc.
As the sulfur compounds, there may be mentioned, for
example, dimethyl sulfoxide, sulfolane, etc.
It is recommended that the above-mentioned other solvent
is used, together with at least one solvent selected from among
hydrocarbons, fatty acid esters, ethers, and nitriles, in an
adequate proportion depending on the solvent characteristics
thereof, including the known physical properties thereof, such
as melting point, boiling point, vapor pressure characteristics,
and phase transition, and the above-mentioned solvent amount
and, further, on the effect to the solubility of oxidized
coenzyme Q10, reduced coenzyme Q10 and/or the reducing agent.
Although the other solvent may be used within an amount
causing no adverse effect, without any other particular
restriction, it is preferred from the viewpoint of oxidation-
protective effect and/or crystallization yield (to be mentioned
later herein) , among others, that the above-mentioned solvent
with high oxidation-protective effect (at least one species
selected from among hydrocarbons, fatty acid esters, ethers,
and nitriles) is present in a larger proportion. It is more
preferred to use a solvent whose substantial main ingredient
is a solvent with high oxidation-protective effect.
In cases where the system comprises a homogeneous solvent
phase, at least one solvent selected from among hydrocarbons,

fatty acid esters, ethers, and nitriles is preferably used as
the reaction solvent or crystallization solvent or as the main
ingredient of the solvent for handling in the concentration,
extraction, column chromatography and/or other operations. In
cases where the system forms different solvent phases, it is
a preferred embodiment that the main ingredient of the solvent
phase having larger solubility of reduced coenzyme Q10 (in the
case of a mixed solvent system composed of water and an organic
solvent low in miscibility with water, for instance, reduced
coenzyme Q10 is scarcely soluble in the aqueous phase but soluble
in the organic solvent phase low in miscibility with water) is
at least one solvent selected from among hydrocarbons, fatty
acid esters, ethers, and nitriles.
From such viewpoint, the other solvent is used generally
in a volume ratio of lower than about 0.3, preferably lower than
about 0.2, more preferably lower than about 0.1, and still more
preferably lower than about 0.05, relative to the whole solvent
amount in the case of homogeneous systems or, in the case of
heterogeneous systems, to the volume of the solvent having
larger solubility of reduced coenzyme Q10. Needless to say, the
lower limit is 0 (zero).
Among the other solvents specifically given hereinabove,
alcohols and/or water are most preferably used either singly
or in combination from the reaction rate and yield improvement
viewpoint in the reduction reaction of oxidized coenzyme Q10.
In the step of crystallizing reduced coenzyme Q10, water can
be preferably used from the viewpoint of improvement in slurry
characteristics such as fluidity.
Now, a method of producing reduced coenzyme Q10 by reducing
oxidized coenzyme Q10 is described.
The oxidized coenzyme Q10 to be used in the practice of
the invention may be one prepared by synthesis, fermentation,
or extraction from a natural product, for instance, as mentioned
hereinabove, or may be an existing high-purity grade of coenzyme
Q10. It may consist of oxidized coenzyme Q10 alone or may be

a mixture of oxidized coenzyme Q10 and reduced coenzyme Q10.
In the practice of the invention, the reduction of
oxidized coenzyme Q10 is carried out using, as a solvent, the
above-mentioned solvent with high oxidation-protective effect,
namely at least one species selected from among hydrocarbons,
fatty acid esters, ethers, and nitriles, and, as the reducing
agent, a metal hydride compound, iron (metallic iron or iron
in a salt form), zinc (metallic zinc), dithionous acid or
a salt thereof, or ascorbic acid or a related compound thereof,
for instance.
The metal hydride compound is not particularly restricted
but includes, among others, sodium borohydride and lithium
aluminum hydride. The amount to be used of the metal hydride
compound may vary depending on the species thereof, hence cannot
be absolutely specified. generally, however, the reduction
can be favorably carried out by using it in an amount of 1 to
3 times the theoretical hydrogen equivalent.
The reduction using iron or zinc is generally carried out
using an acid. The acid is not particularly restricted but
includes, among others, fatty acids such as acetic acid,
sulfonic acids such as methanesulfonic acid, and inorganic
acids such as hydrochloric acid and sulfuric acid. Inorganic
acids are preferred, and sulfuric acid is more preferred.
The amount of iron to be used is not particularly
restricted but, for example, an amount o± about 1/5 by weight
or larger based on the charged weight of oxidized coenzyme Q10
is appropriate for carrying out the reaction. The upper limit is not
particularly restricted but,from the economical viewpoint, it is
about twice the weight of the above charged weight or lower. Iron
may be used not only in the form of metallic iron but also in the
form of a salt, for example iron (II)sulfate, etc.
The amount of zinc to be used is not particularly
restricted but, for example, an amount of about 1/10 by weight
or larger based on the charged weight of oxidized coenzyne Q10

is appropriate for carrying out the reaction. The upper limit is
not particularly restricted but, from the economic viewpoint, it
is about twice the weight of the above charged weight or lower.
The dithionous acid or a salt thereof is not
particularly restricted but a salt form of dithionous acid
is generally used. The salt of dithionous acid is not
particularly restricted but includes, as preferred species,
alkali metal salts, alkaline earth metal salts, ammonium salt
and the like. Alkali metal salts such as the lithium salt,
sodium salt, and potassium salt are more preferred, and the
sodium salt is most preferred. The amount to be used of the
dithionous acid or salt is not particularly restricted but
it is generally not smaller than about 1/5 by weight, preferably
not smaller than about 2/5 by weight, and more preferably not
smaller than about 3/5 by weight, based on the charged weight
of oxidized coenzyme Q10. Larger amounts may be used without
causing any particular trouble. From the economical viewpoint,
however, the amount to be employed is not larger than about twice
the weight of the above-mentioned charged weight, preferably
not larger than the charged weight. Thus, the reaction can be
more favorably carried out with employing an amount within the
range of about 2/5 by weight of the above-mentioned charge to
a weight roughly equal to that of the charged weight.
The ascorbic acid or related compounds thereof are not
particularly restricted, and include, for example, not only
ascorbic acid, but also rhamno-ascorbic acid, arabo-ascorbic
acid, gluco-ascorbic acid, fuco-ascorbic acid,
glucohepto-ascorbic acid, xylo-ascorbic acid,
galacto-ascorbic acid, gulo-ascorbic acid, allo-ascorbic acid,
erythro-ascorbic acid, 6-desoxyascorbic acid, and the like
ascorbic acid derivatives, and may be ester forms or salts of
these. Furthermore, these may be L-form, D-form or racemic form.
More specifically, there may be mentioned, for example,
L-ascorbic acid, L-ascorbyl palmitate, L-ascorbyl stearate,
D-arabo-ascorbic acid, etc. In producing the reduced coenzyme

Q10, any of the above-mentioned ascorbic acid and related
compounds thereof may be suitably used. However, the
water-soluble ones are suitably used in particular among the
above-mentioned ascorbic acid or related compounds thereof in
view of separatability with the generated reduced coenzyme Q10,
etc. And most preferred is a free form of L-ascorbic acid,
D-arabo-ascorbic acid, and the like in view of the ready
availability, price, etc.
The amount to be used of the ascorbic acid or a related
compound thereof mentioned above is not particularly restricted
but may be an amount effective in converting oxidized coenzyme
Q10 to reduced coenzyme Q10. Generally it is not smaller than
1 mole, preferably not smaller than 1.2 moles, per mole of
oxidized coenzyme Q10. The upper limit is not particularly-
restricted but, from the economical viewpoint, it is generally
not higher than 10 moles, preferably not higher than 5 moles,
and more preferably not higher than 3 moles, per mole of the
oxidized coenzyme Q10 •
Among the reducing agent species mentioned above, zinc,
dithionous acid and salts thereof, and ascorbic acid and
related compounds thereof are preferred from the viewpoint of
reducing ability, yield and/or quality, among others, and, in
particular, dithionous acid (specifically dithionous
acid salts) and ascorbic acid or related compounds thereof are
preferred.
In carrying out the reduction reaction, an alcohol and/or
water are/is suitably used singly or in combination, as
mentioned above. Water is preferred in particular when iron,
zinc, or dithionous acid or a salt thereof is used as the
reducing agent. When a metal hydride compound or ascorbic acid
or a related compound thereof is used as the reducing agent,
an alcohol can be used in combination. The combined use of water
and an alcohol exhibits the characteristics of both water and
the alcohol and contributes to improvements in reaction rate
and yield, among others.

In the following, a preferred method of reduction is
described in detail.
The reduction using dithionous acid or a salt thereof
is preferably carried out using water in combination, namely
in a mixed solvent system composed of at least one organic
solvent selected from among the above-mentioned hydrocarbons,
fatty acid esters, ethers, and nitriles, with water. On that
occasion, the reaction is preferably carried out generally at
a pH of not higher than 7, preferably at pH 3 to 7, mere preferably
at pH 3 to 6, from the viewpoint of yield, etc. The pH can be
adjusted using an acid (e.g. an inorganic acid sucn as
hydrochloric acid or sulfuric acid) or a base (e.g. an alkali
metal hydroxide such as sodium hydroxide).
In the reduction using dithionous acid or a salt
thereof, the amount of water is not particularly restricted but
may be an amount of water such that an appropriate amount of
the reducing agent, namely dithionous acid or a salt: thereof,
can be dissolved therein. Thus, for example, it is advisable
that the amount of the dithionous acid or a salt be adjusted
generally to not more than 30 w/w%, and preferably not more than
20 w/w%, relative to the weight of water. From the productivity
viewpoint, among others, it is advisable that the amount be
adjusted generally to not less than 1 w/w%, preferably not less
than 5 w/w%, and more preferably not less than 10 w/w%.
The reduction using the ascorbic acid or a related
compound thereof mentioned above is preferably carried out
using a solvent especially highly miscible with water as
selected from among the above-mentioned hydrocarbons, fatty
acid esters, ethers, and nitriles, in particular at least one
species selected from among ethers and nitriles, which are
highly miscible with water, and more specifically
tetrahydrofuran, dioxane, acetonitrile or the like. An
alcohol may also be used as another solvent. Furthermore, from
the viewpoint of reaction promotion (e.g. reaction temperature
lowering or reaction time shortening) in the production of

reduced coenzyme Q10, it is also possible to carry out the
reduction in the presence of an additive having a reaction
promoting effect, such as a basic substance or a
hydrogensulfite.
The basic compound is not particularly restricted but may
be either an inorganic compound or an organic compound. The
inorganic compound is not particularly restricted but includes,
among others, the hydroxides, carbonates, and
hydrogencarbonates of metals (preferably alkali metals,
alkaline earth metals, and the like) , and ammonia. As typical
examples thereof, there may be mentioned alkali metal
hydroxides such as sodium hydroxide, alkali metal carbonates
such as sodium carbonate, alkali metal hydrogencarbonates such
as sodium hydrogencarbonate, and alkaline earth metal
carbonates such as magnesium carbonate. The organic compound
is not particularly restricted but includes, among others,
amines such as triethylamine. Among the basic substances
specifically mentioned above, weakly basic substances (weak
bases or weak alkalis) such as the carbonates and
hydrogencarbonates of metals (preferably alkali metals,
alkaline earth metals, etc.), ammonia, and like inorganic
compounds; amines such as triethylamine, and like organic
compounds are preferably used. More preferred are the weakly
basic inorganic compounds mentioned above.
Preferred as the hydrogensulfite are, for example, alkali
metal hydrogensulfites such as sodium, hydrogensulfite.
The amount of the additive mentioned above is not
particularly restricted but may be such that the reaction
promoting effect of the additive can be produced to a desired
extent (effective amount). From the economical viewpoint,
however, the amount is generally not more than 2 0 moles,
preferably not more than 10 moles, more preferably not more than
5 moles, and still more preferably not more than 2 moles, per
mole of the ascorbic acid or a related compound thereof. The
lower limit is not particularly restricted but, generally, it

is not less than 0.01 mcies, preferably not less than 0.05 moles,
more preferably not less than 0.1 moles, and still more
preferably not less than 0.2 moles, per mole of the ascorbic acid
or a related compound thereof.
In the practice of the invention, the reduction reaction
is preferably carried out under forced flowing. The power
required for stirring to cause such flowing per unit volume is
generally not less than about 0.01 kW/m3, preferably not less
than about 0.1 kW/m3, and more preferably rot less than about
0.3 kW/m3. The above forced flowing is generally caused by the
turning of a stirring blade(s). The use of a stirring blade(s)
is not always necessary if the above flowing can be otherwise
obtained. For example a method based on Liquid circulation may
be utilized.
The reduction temperature may vary depending on the
reducing agent species and/or amount, hence cannot be
absolutely specified. In the reduction using dithionous
acid or a salt thereof, for instance, the reduction is generally
carried out at 100°C or below, preferably at 80°C or below, more
preferably at 60°C or below. The lower limit is the
solidification temperature of the system. Thus, the reduction
can be favorably carried out generally at about 0 to 100°C,
preferably at about 0 to 80°C, more preferably at about 0 to
60°C. In the reduction using ascorbic acid or a related
compound thereof, the reduction is carried out generally at 30°C
or higher, preferably at 40°C or higher, more preferably at 50°C
or higher. The upper limit is the boiling point of the system.
Thus, the reduction can be favorably carried out generally at
about 30 to 150°C, preferably about 40 tc 120°C, more preferably
at about 50 to 100oC.
The reaction concentration is not particularly
restricted but the weigh- of oxidized coenzyme Q10 relative to
the solvent weight is generally not less than about 1 w/w%,
preferably not less than 3 w/w %, more preferably not less than
10 w/w%, and still more preferably not less than 15 w/w%. The

upper limit is not particularly restricted but generally is net
higher than about 60 w/w%, preferably not higher than 50 w/w%,
more preferably not higher than 40 w/w%, and still more
preferably not higher than 30 w/w%. Thus, the reaction can be
favorably carried out at a reaction concentration of about 1
to 60 w/w%, preferably about 3 to 50 w/w%, and more preferably
about 10 to 40 w/w%.
The reduction reaction time may vary depending on the
reducing agent species and/or the amount thereof, hence cannot
be absolutely specified. Generally, however, the reaction can
be driven to completion wmhin 4 8 hours, preferably within 2 4
hours, more preferably within 10 hours, and still more
preferably within 5 hours.
It is exceedingly preferable to carry out the reduction
reaction in a deoxidized atmospnere. Surprisingly, It was
found that, in the reduction reaction using dithionous acid
or a salt thereof, in particular, such atmosphere greatly
contributes to an improvement in reduction reaction yield and
a reduction in reducing agent amount. The deoxidized
atmosphere can be attained by substitution with an inert gas,
pressure reduction, boiling, or a combination of these. It is
preferable to carry out at least the substitution with an inert
gas, namely to use an inert gas atmosphere. As the inert gas,
there may be mentioned, for example, nitrogen gas, helium gas,
argon gas, hydrogen gas, and carbon dioxide gas. Nitrogen gas
is preferred, however.
An organic phase containing the product reduced coenzyme
Q10 is recovered from the thus-obtained reduction reaction
mixture and, if necessary (preferably), the organic phase is
further washed repeatedly using water, an aqueous solution of
sodium chloride, or the like to achieve complete contaminant
elimination and, then, it can be subjected to crystallization.
When the dithionous acid or a salt thereof mentioned above, such
as sodium dithionite, in particular, is _sed as the
reducing agent, it is desirable to repeat washing with water

so that contaminants derived from the dithionous acid or salt
thereof may be removed completely and/or the pH of the aqueous
phase may be stabilized.
When oxidized coenzyme Q10 is reduced with ascorbic acid
or a related compound thereof using the above-mentioned highly
water-miscible solvent, in particular at least one species
selected from among those ethers and nitriles which are highly
miscible with water (e.g. tetrahydrofuran, dioxane,
acetonitrile, etc.), it is a very convenient and efficient
procedure to crystallize reduced coenzyme Q10 directly from the
reduction reaction mixture (direct isolation method (one-pot
method)).
The above treatment following the reduction reaction is
preferably carried out in a deoxidized atmosphere and, thereby,
che oxidation-protective effect can be furtner increased.
The crystallization of reduced coenzyme Q10 is now
described.
The reduced coenzyme Q10 to be subjected to
crystallization can be obtained in the conventional manner, for
example, by synthesis, fermentation, or extraction from a
natural source. Preferred is the product obtained by reduction
of oxidized coenzyme Q10 contained in reduced coenzyme Q10, or
reduction of oxidized coenzyme Q10 More preferred :..s the
product obtained by carrying out the reduction reaction in
accordance with the present invention, as described above.
While the method of crystallization according to the
invention can be applied also to products containing oxidized
coenzyme Q10 in relatively large amounts, the method is
particularly effective in crystallizing high-purity reduced
coenzyme Q10 prepared by the reduction method described above.
In the practice of the invention, it is very effective to purify
and crystallize reduced coenzyme Q10 with simultaneous removal
of impurities contained in the reaction mixture or extract
obtained in the conventional manner or produced by the
above-mentioned reduction method or the like. More

specifically, reduced coenzyme Q10 can be crystallized from the
reduction reaction mixture obtained by the reduction method
mentioned above, or a solution obtained by collecting the
reduced coenzyme Q10-containing organic phase from the above
reaction mixture, if necessary followed by washing of the
organic phase. On that occasion, the impurities should
preferably be eliminated into the mother liquor. This makes
it possible to remove coexisting impurities, in particular
analogous compounds having a similar structure and generally
not always easy to remove (specifically, reduced coenzyme Q9,
reduced coenzyme Q8, reduced coenzyme Q7, etc.). Needless to
say, it is possible to utilize the method as a method of
recrystallizing reduced coenzyme Q10 crystals obtained
beforehand by purification and crystallization.
The crystallization of reduced Q10 is carried out using,
as the solvent, at least one species selected from among the
above-mentioned hydrocarbons, fatty acid esters, ethers, and
nitriles. Among them, hydrocarbons are preferred, aliphatic
hydrocarbons and aromatic hydrocarbons are more preferred,
aliphatic hydrocarbons are still more preferred, and the
above-mentioned pentanes, hexanes, heptanes and octanes are
most preferred. From the viewpoint of crystallization yield
and/or unnecessariness of excessive cooling in crystallization,
among others, acyclic aliphatic hydrocarbons are particularly
preferred among the aliphatic hydrocarbons.
The method of crystallization is not particularly
restricted but the crystallization can be carried out by
utilizing a conventional crystallization method, namely at
least one of the cooling crystallization, concentration
crystallization, solvent substitution crystallization and
other methods. In particular, the cooling crystallization
method, or a combination of the cooling crystallization method
with some other method of crystallization is preferred.
The cooling temperature in the step of crystallization
is an important factor and, from the yield viewpoint, among

others, it is generally not higher than 20°C, preferably not
higher than 10°C, more preferably not higher than 5°C. The
lower limit is the solidification temperature of the system.
Thus, the crystallization can be advantageously carried out
generally at a cooling temperature of about -30°C to +10°C,
preferably about -20°C to +10°C, more preferably about -10°C
to +5°C.
In the process of crystallization, the amount of crystals
crystallizing out per unit time may be controlled to minimize
the immixture of various impurities into the obtained reduced
coenzyme Q10, or to obtain a slurry with good characteristics.
A preferred rate of crystallization per unit time is, for
example, not higher than the rate of crystallization which
causes crystallization of about 50%, per unit time, of the whole
amount of crystals to be obtained (i.e. at most 50%/hour),
preferably not higher than the rate of crystallization which
causes crystallization of about 25%, per unit time, of the whole
amount of crystals to be obtained (i.e. at most 25%/hour) . The
rate of cooling in the crystallization by cooling is generally
not higher than about 40°C/hour, and preferably not higher than
about 20°C/hour.
The crystallization concentration is also an important
factor and, when expressed in terms of the weight of reduced
coenzyme Q10 relative to the weight of the crystallization
solvent at the time of completion of crystallization, it is not
higher than about 15 w/w%, preferably not higher than about 13
w/w%, more preferably not higher than 10 w/w%. The preferred
crystallization concentration may vary depending on the solvent
species employed and, for attaining satisfactory
crystallization results using an aliphatic hydrocarbon or a
solvent whose main ingredient is an aliphatic hydrocarbon, the
crystallization concentration is not higher than about 13 w/w%,
preferably not higher than about 10 w/w%, more preferably not
higher than about 8 w/w% and, in the case of an acyclic aliphatic
hydrocarbon, which is most preferred, or a solvent whose main

ingredient is an acyclic aliphatic hydrocarbon, the
crystallization concentration is not higher than about 10 w/w%,
preferably not higher than about 8 w/w%, and more preferably
not higher than about 7 w/w%. By maintaining such a
concentration as mentioned above, it becomes possible to carry
out the crystallization favorably in a manner adapted to the
operability on an industrial scale. The lower limit to the
crystallization concentration is not particularly restricted
but, from the productivity viewpoint, it is generally not lower
than about 1 w/w%, preferably not lower than about 2 w/w%.
The crystallization is preferably carried out under
forced flowing. For preventing the state of supersaturation
from occurring and thereby allowing the nucleation and crystal
growth to proceed smoothly and, furthermore, from the viewpoint
of obtaining high-quality products, the flowing is generally
brought about by a stirring power per unit volume of not weaker
than about 0 . 01 kW/m3, preferably not weaker than about 0 .1 kW/m3,
and more preferably not weaker than about 0.3 kW/m3. The forced
flowing is generally provided by the turning of a stirring
blade (s) . However, the use of a stirring blade (s) is not always
necessary if the above flowing can be otherwise obtained. For
example, it is possible to utilize a method based on liquid
circulation.
In carrying out the crystallization, seed crystals are
preferably added so that the state of supersaturation may be
prevented from occurring and the nucleation and crystal growth
may be allowed to proceed smoothly.
In carrying out the crystallization according to the
invention, such another auxiliary solvent as mentioned above
may be used, according to need, in combination with the
above-mentioned solvent having a high protective effect against
oxidation if the other solvent will not produce any adverse
effect. While an appropriate amount of such other auxiliary
solvent may be added to the solvent mentioned above for
improving the solubility of reduced coenzyme Q10 as well as the

crystallization concentration, crystallization temperature,
yield, slurry characteristics, and crystal characteristics,
among others, the use of such a mixed solvent may result in a
drastic increase in solubility, whereby the crystallization
yield rather tends to decrease in some instances. Therefore,
from the viewpoint of oxidation-protective effect and/or
crystallization yield, among others, a solvent, whose
substantial main ingredient is the solvent mentioned above, is
preferably used. Thus, when the solvent is used in admixture
with another solvent, the proportion (ratio by volume) of the
other solvent is not particularly restricted but generally is
not higher than about 0.3, preferably not higher than about 0.2,
more preferably not higher than about 0.1, and still more
preferably not higher than about 0.05. Needless to say, the
lower limit is 0 (zero) . As mentioned hereinabove, the volume
ratio is the proportion of the volume of the other solvent to
the whole solvent volume in the case of homogeneous systems and,
in the case of heterogeneous systems, the proportion of the
volume of the other solvent (s) to the volume of the solvent phase
in which reduced coenzyme Q10 is well soluble.
When water is used combinedly as the other auxiliary
solvent, however, water can show its characteristics and can
contribute to improvements in slurry characteristics, for
instance. The amount of water in the crystallization of reduced
coenzyme Q10 influences on the slurry concentration of reduced
coenzyme Q10 crystallized out and/or on the slurry
characteristics. As the amount of water increases, the slurry
concentration decreases and the fluidity generally increases.
On the other hand, the decrease in slurry concentration as a
whole leads to a decrease in productivity. The amount of water
is not particularly restricted but, taking the above factors
into consideration, it may be varied so that it may be adjusted
to and maintained in an appropriate range.
The other auxiliary solvent mentioned above may be added
prior to or during the crystallization process or after

stabilization of the amount of the precipitated crystals.
The thus-obtained crystals of reduced coenzyme Q10 can
be recovered as a wet product, for example, by such a
solid-liquid separation technique as centrifugation, pressure
filtration, or vacuum filtration, if necessary followed by cake
washing. They can be recovered also as a dry product by further
charging the wet product in a reduced pressure drier (vacuum
drier) internally purged with an inert gas and drying the same
under reduced pressure. The recovery in a dry form is
preferred.
When the crystallization is carried out in a deoxidized
atmosphere, the protective effect against oxidation can be
further increased. The deoxidized atmosphere can be attained
by inert gas substitution, pressure reduction, boiling, or a
combination of these. It is preferable to carry out at least
the substitution with an inert gas, namely to use an inert gas
atmosphere. As the inert gas, there may be mentioned, for
example, nitrogen gas, helium gas, argon gas, hydrogen gas, and
carbon dioxide gas. Nitrogen gas is preferred, however.
In accordance with the present invention, high-quality
reduced coenzyme Q10 can be synthesized and crystallized in a
convenient and efficiency manner. The crystals of reduced
coenzyme Q10 as obtained in accordance with the present
invention are of very high quality and can be expected to have
a reduced coenzyme Q10/oxidized coenzyme Q10 weight ratio of not
lower than 96/4, preferably not lower than 98/2, more preferably
not lower than 99/1.
BEST MODE FOR CARRYING OUT THE INVENTION
The following examples illustrate the present invention
in further detail. These examples are, however, by no means
limitative of the scope of the present invention. In the
examples, the purity of reduced coenzyme Q10 and the reduced
coenzyme Q10/oxidized coenzyme Q10 weight ratio were determined
by the HPLC analysis specified below. The reduced coenzyme Q10

purity values as determined, however, are by no means indicative
of the limit purity value attainable in accordance with the
present invention. Likewise, the reduced ccenzyme
Q10/oxidized coenzyme Q10 weight ratio values obtained never
indicate the upper limit to that ratio.
(HPLC conditions)
Column: SYMMETRY C18 (product of Waters), 250 mm (in length),
4.6 mm (in inside diameter); mobile phase: C2H5OH :CH3OH = 4:3
(v/v); detection wavelength: 210 nm; flow rate: 1 ml/min;
retention time of reduced coenzyme Q10: 9.1 min; retention time
of oxidized coenzyme Q10: 13.3 min.
(Example 1)
Oxidized coenzyme Q10 (100 g; containing 0.40% of oxidized
coenzyme Q9, purity 99.4%) was dissolved in 1000 g of heptane
at 25°C. While stirring (power required for stirring: 0.3 kW/m3) ,
an aqueous solution prepared by dissolving 100 g of sodium
dithionite (purity: at least 75%), as the reducing agent, in 1000
ml of water was gradually added and the reduction reaction was
carried out at 25°C and at pH 4 to 6. After the lapse of 2
hours, the aqueous phase was removed frcm the reaction
mixture, and the heptane phase was washed 6 times with 1000 g
of a deaerated saturated aqueous sodium chloride solution to
give a heptane phase containing 100 g of reduced coenzyme Q10
(containing 0.40% of reduced coenzyme Q9) . This heptane phase
was cooled to 2°C while stirring (power required for stirring:
0.3 kW/m3) to give a white slurry. All the above operations
were carried out in a nitrogen atmosphere. The slurry obtained
was filtered under reduced pressure, and the wet crystals were
washed in sequence with cold heptane, cold ethancl, cold water,
cold ethanol and cold heptane (the cold solvents used for
washing having a temperature of 2°C). The wet crystals were
further dried under reduced pressure (20-40°C, 1-30 mmHg) to
give 93 g of white dry crystals (containing 0.2 9% of reduced

coenzyme Q9, percentage of elimination: 28%) (isolated product
yield: 93 mole percent). The reduced coenzyme Q10/oxidized
coenzyme Q10 weight ratio of the crystals obtained was 99. 6/0.4,
and the purity of the reduced coenzyme Q10 was 99.2%.
(Example 2 and Comparative Example 1)
One gram of the reduced coenzyme Q10 obtained in Example
1 was dissolved in 20 g of each of various solvents shown in
Table 1 at 25°C. After 24 hours of stirring in the air at 25°C,
the reduced coenzyme Q10/oxidized coenzyme Q10 weight ratio in
each solution was determined. The results thus obtained are
shown in Table 1 together with the results obtained by storing
the crystal under the same conditions as above (1 g of reduced
coenzyme Q10 stored in the air at 25°C for 24 hours) for
comparison.


One gram of the reduced coenzyme Q10 obtained in Example 1 was
dissolved in 100 g of each of various solvents shown in
Table 2 at 35°C. After 2 4 hours of stirring at 3 5°C in the air,
the reduced coenzyme Cho/oxidized coenzyme Q10 weight ratio in
each solution was determined. The results thus obtained are
shown in Table 2.

(Example 4)
Ten grams of oxidized coenzyme Q10 was dissolved in 100
g of each of various solvents shown in Table 3 at 25°C. While
stirring (power required for stirring: 0.3 kW/rrr), an aqueous
solution prepared by dissolving 10 g of a commercial grade of
sodium dithionite (purity: at least 75%), as the reducing agent,
in 100 ml of water was gradually added and the reduction reaction
was carried out at 25°C and at pH 4 to 6. After the lapse of
2 hours, the aqueous phase was removed from the reaction mixture,
and the organic phase was washed 6 times with 100 g of a deaerated
saturated aqueous sodium chloride solution. All the operations
were carried out in a nitrogen atmosphere. The reduced coenzyme
Cho/oxidized coenzyme Qlo weight ratio in the organic phase was
then determined. The results thus obtained are shown in Table 3.


(Example 5)
Ten grams of oxidized coenzyme Q10 was dissolved in 100
g of heptane at 25°C, 1.5 g of a zinc powder and 110 g of 2.9
N sulfuric acid were added and, after 6 hours of stirring (power
required for stirring: 0.3 kW/m3) at 25°C, 100 g of concentrated
hydrochloric acid was added. The reduced coenzyme Q10/oxidized
coenzyme Q10 weight ratio in the heptane phase was determined
and found to be 99.6/0.4. All the operations were carried out
in a nitrogen atmosphere.
(Example 6)
The reduction reaction and crystallization were carried
out in the same manner as in Example 1 except that hexane was
used as the solvent for dissolving oxidized coenzyme Q10- As
a result, 93 g of dry white crystals were obtained (isolated
product yield: 93 mole percent). The reduced coenzyme
Q10/oxidized coenzyme Q10 weight ratio of the crystals obtained
was 99.4/0.6, and the purity of the reduced coenzyme Q10 was
99.0%.

(Example 7)
Oxidized coenzyme Q10 (100 g; purity 99.4%) was dissolved
in 1000 g of hexane at 25°C. While stirring (power required
for stirring: 0.3 kW/m3) , an aqueous solution prepared by
dissolving 100 g of sodium dithionite (purity: at least 75%),
as the reducing agent, in 1000 ml of water was gradually added
and the reduction reaction was carried out at 25°C and at pH
4 to 6. After the lapse of 2 hours, the aqueous phase was removed
from the reaction mixture, and the hexane phase was washed 6
times with 1000 g of a deaerated saturated aqueous sodium
chloride solution. Methanol (50 g) was added to the hexane
phase, and the mixture was cooled to 2°C while; stirring (power
required for stirring: 0.3 kW/m3) to give a white slurry. All
the above operations were carried out in a nitrogen atmosphere.
The slurry obtained was filtered under reduced pressure, and
the wet crystals were washed in sequence with cold hexane, cold
ethanol, cold water, cold ethanol and cold hexane (the cold
solvents used for washing having a temperature of 2°C). The
wet crystals were further dried under reduced pressure (20-40°C,
1-30 mmHg) to give 92 g of dry white crystals (isolated product
yield: 92 mole percent). The reduced coenzyme Q10/oxidized
coenzyme Q10 weight ratio of the crystals obtained was 99.4/0.6,
and the purity of the reduced coenzyme Q10 was 99.0%.
(Example 8)
Oxidized coenzyme Q10 (LOO g; purity 99.4%) was dissolved
in 1000 g of ethyl acetate at 25°C. While stirring (power
required for stirring: 0.3 kW/m3) , an aqueous solution prepared
by dissolving 100 g of sodium dithionite (purity at least 75%),
as the reducing agent, in 1000 ml of water was gradually added
and the reduction reaction was carried out. at 25°C and at pH
4 to 6. After the lapse of 2 hours, the aqueous phase was
removed from the reaction mixture, and the ethyl acetate phase
was washed 6 times with 1000 g of a deaerated saturated aqueous

sodium chloride solution, Ethanol (300 g) and 50 g of water
were added to the ethyl acetate phase, and the mixture was cooled
to 2°C while stirring (power required for stirring: 0.3 kW/m3)
to give a white slurry improved in fluidity as compared with
Example 1. All the above operations were carried out in a
nitrogen atmosphere. The slurry obtained was filtered under
reduced pressure, and the wet crystals were washed in sequence
with cold ethanol, cold water, and cold ethanol (the cold
solvents used for washing having a temperature of 2°C). The
wet crystals were dried under reduced pressure (20--40°C, 1-30
mmHg) to give 89 g of dry white crystals (isolated product yield:
89 mole percent). The reduced coenzyme Q10/oxidized coenzyme
Q10 weight ratio of the crystals obtained was 99.5/0.5, and the
purity of the reduced coenzyme Q10 was 99.2%.
(Example 9)
Reduced coenzyme Q10 (100 g; reduced coenzyme Q10/oxidized
coenzyme Q10 weight ratio: 95/5; purity as coenzyme Q10: 99.4%)
was dissolved in 1000 g of heptane at 25°C. While stirring
(power required for stirring: 0.3 kW/m3) , an aqueous solution
prepared by dissolving 10 g of sodium dithionite (purity: at
least 75%), as the reducing agent, in 1000 ml of water was
gradually added and the reduction reaction was carried out at
25°C and at pH 4 to 6. After the lapse of 2 hours, the aqueous
phase was removed from the reaction mixture, and the heptane
phase was washed 6 times with 1000 g of a deaerated saturated
aqueous sodium chloride solution. This heptane phase was
cooled to 2°C while stirring (power required for stirring: 0.3
kW/m3) to give a white slurry. All the above operations were
carried out in a nitrogen atmosphere. The slurry obtained was
filtered under reduced pressure, and the wet crystals were
washed in sequence with cold heptane, cold ethanol, cold water,
cold ethanol and cold heptane (the cold solvents used for
washing having a temperature of 2°C). The wet crystals were
dried under reduced pressure (20-40°C, 1-30 mmHg) to give 93

g of dry white crystals (isolated product yield: 93 mole
percent) . The reduced coenzyme Q10/oxidized coenzyme Q10 weight
ratio of the crystals obtained was 99.6/0.4, and the purity of
the reduced coenzyme Q10 was 99.2%.
(Example 10)
The reduction reaction and crystallization were carried
out under exactly the same conditions as in Example 1 except
that the oxidized coenzyme Q10 used has a purity of 98.4%
(containing 1.0% of oxidized coenzyme Q9, 0.30% of oxidized
coenzyme Q8 and 0.04% of oxidized coenzyme Q7) • As a result,
93 g of dry white crystals (containing 0 . 72% of reduced coenzyme
Q9, percentage of elimination: 28%; and 0.11% of reduced
coenzyme Q8, percentage of elimination: 63%; reduced coenzyme
Q7: not detected) (isolated product yield: 93 mole percent) were
obtained. The reduced coenzyme Q10/oxidized coenzyme Q10 weight
ratio of the crystals obtained was 99.6/0.4, and the purity of
the reduced coenzyme Q10 was 99.0%.
(Example 11)
Ten grams of crystals of reduced coenzyme Q10 (containing
0.29% of reduced coenzyme Q9; purity 99.1%; reduced coenzyme
Q10/oxidized coenzyme Q10 weight ratio 99.4/0.6) as obtained in
the same manner as in Example 1 were dissolved in 140 g of
acetonitrile at 45°C, and the solution was cooled to 2°C while
stirring (power required for stirring: 0.3kW/m3) to give a white
slurry. All the above operations were carried out in a nitrogen
atmosphere. The slurry obtained was filtered under reduced
pressure, and the wet crystals were washed in sequence with cold
acetonitrile, cold water and cold acetonitrile (the cold
solvents used for washing having a temperature of 2°C). The
wet crystals were dried under reduced pressure (20-40°C, 1-30
mmHg) to give 9.5 g of dry white crystals (containing 0.25% of
reduced coenzyme Q9, percentage of elimination: 14%) (isolated
product yield: 95 mole percent). The reduced coenzyme

Q10/oxidized coenzyme Q10 weight ratio of the crystals obtained
was 99.4/0.6, and the purity of the reduced coenzyme Q10 was
99.1%.
(Example 12)
Ten grams of crystals of reduced coenzyme Q10 (containing
0.29% of reduced coenzyme Q9; purity 98.8%; reduced coenzyme
Q10/oxidized coenzyme Q10 weight ratio:: 99.1/0.9) as obtained
in the same manner as in Example 1 were dissolved in 100 g of
heptane at 25°C, and the solution was cooled to 2°C while
stirring (power required for stirring: 0.3kW/m3) to give a white
slurry. All the above operations were carried out in the air.
The slurry obtained was filtered under reduced pressure, and
the wet crystals were washed in sequence with cold heptane, cold
ethanol, cold water, cold ethanol and cold heptane (the cold
solvents used for washing having a temperature of 2°C). The
wet crystals were dried under reduced pressure (20-40°C, 1-30
mmHg) to give 9.3 g of dry white crystals (containing 0.20% of
reduced coenzyme Q9, percentage of elimination: 31%) (isolated
product yield: 93 mole percent). The reduced coenzyme
Q10/oxidized coenzyme Q10 weight ratio of the crystals obtained
was 99.0/1.0, and the purity of the reduced coenzyme Q10 was
98.8%.
(Comparative Example 3)
Ten grams of the crystals of reduced co>enzyme Q10 as
obtained in Example 1 were dissolved in 70 g of
N-methylpyrrolidone at 25°C. Furthermore, 10 g of water was
added, and the solution was cooled to 2°C while stirring (power
required for stirring: 0.3 kW/m3) to give a pale-yellow slurry.
All the above operations were carried out in the air. The slurry
obtained was filtered under reduced pressure, and the wet
crystals were washed in sequence with cold ethanol, cold water
and cold ethanol (the cold solvents used for washing having a
temperature of 2°C) . The wet crystals were dried under reduced

pressure (20-40°C, 1-30 mmHg) to give 9.6 g of pale-yellow dry
crystals (isolated product yield: 96 mole percent) . The
crystals obtained had a pale-yellow color, the reduced coenzyme
Q10/oxidized coenzyme Q10 weight ratio was 88.6/11.4, and the
purity of the reduced coenzyme Q10 was 88.3%.
(Example 13)
Oxidized coenzyme Q10 (100 g) was dissolved in 1000 g of
hexane at 25°C. While stirring (power required for stirring: 0.3
kW/m3) , an aqueous solution prepared by dissolving 40 g of
sodium dithionite (purity :at least 75%), as the reducing agent,
in 1000 ml of water was gradually added and the reduction
reaction was carried out at 25°C and at pH 4 to 6. After the
lapse of 2 hours, the aqueous phase was removed from the reaction
mixture, and the hexane phase was washed 6 times with 1000 g
of a deaerated saturated aqueous sodium chloride solution. All
the above operations were carried out in a nitrogen atmosphere.
The reduced coenzyme Q10/oxidized coenzyme Q10 weight ratio in
this hexane solution was 99.2/0.8.
(Example 14)
Oxidized coenzyme Q10 (100 g; purity 99.4%), 60 g of
L-ascorbic acid and 30 g of sodium hydrogencarbonate were added
to 1000 g of acetonitrile, and the reduction reaction was
carried out with stirring at 55°C. After the lapse of 40 hours,
1000 g of heptane and 1000 g of deaerated water were added to
the reaction mixture, and the resulting mixture was cooled to
25°C. The aqueous phase was removed from the reaction mixture,
and the heptane phase was washed 6 times with 1000 g of a
deaerated saturated aqueous sodium chloride solution. This
heptane phase was cooled to 2°C while stirring (power required
for stirring: 0.3 kW/m3) to give a white slurry. All the above
operations were carried out in a nitrogen atmosphere. The
slurry obtained was filtered under reduced pressure, and the
wet crystals were washed in sequence with cold heptane, cold

ethanol, cold water, cold ethanol and cold heptane (the cold
solvents used for washing having a temperature of 2°C). The
wet crystals were further dried under reduced pressure (20-40°C,
1-30 mmHg) to give 95 g of dry white crystals (isolated product
yield: 95 mole percent). The reduced coenzyme Q10/oxidized
coenzyme Q10 weight ratio of the crystals obtained was 99.5/0.5,
and the purity of the reduced coenzyme Q10 was 99.0%.
(Comparative Example 4)
The reduction reaction was carried out in the same manner
as in Example 13 except that all the operations were carried
out in the air. The reduced coenzyme Q10/oxidized coenzyme Q10
weight ratio in the hexane solution obtained was 45.3/54.7.
INDUSTRIAL APPLICABILITY
The invention, which has the constitution described above,
is a method suited for commercial scale production and can give
high-quality reduced coenzyme Q10 in a convenient and efficient
manner.

We Claim :
1. A method of crystallizing reduced coenzyme Q10 such as
herein described,which comprises crystallizing the reduced
coenzyme Q10 using, as a solvent such as herein described, at
least one species selected from the group consisting of
hydrocarbons such as herein described, fatty acid esters such
as herein described, ethers such as herein described and
nitriles such as herein described.
2. The method as claimed in claim 1,
wherein an aliphatic hydrocarbon is used as the solvent.
3. The method as claimed in claim 2,
wherein the aliphatic hydrocarbon is an acyclic aliphatic
hydrocarbon.
4. The method as claimed in claim 2,
wherein the aliphatic, hydrocarbon is at least one species
selected from among pentanes, hexanes, heptanes and octanes.
5. The method as claimed in any of claims 1 to 4,
wherein an impurity is eliminated into the mother liquor.
6. The method as claimed in claim 5,
wherein the impurity to be eliminated is at least one
species selected from among reduced coenzyme Q9, reduced
coenzyme Q8 and reduced coenzyme Q7.
7. The method as claimed in any of claims 1 to 6,
wherein the crystallization of reduced coenzyme Q10 is
carried out in the manner of crystallization by cooling or a
combination of crystallization by cooling with another method
of crystallization.

8. The method as claimed in claim 7,
wherein the cooling temperature in the step of
crystallization is not higher than 20°C.
9. The method as claimed in any of claims 1 to 8,
wherein the crystallization concentration as expressed
in terms of the weight of reduced coenzyme Q10 based on the weight
of the crystallization solvent at the time of completion of
crystallization is not higher than 15 w/w%.
10. The method as claimed in any of claims 1 to 9,
wherein the crystallization is carried out under forced
flowing caused by a power required for stirring per unit volume
of not weaker than 0.01 kW/m3.
11. The method as claimed in any of claims 1 to 10,
wherein a seed crystal is added in carrying cut the
crystallization.
12. The method as claimed in any of claims 1 to 11,
wherein the crystallization is carried out in a
deoxygenated atmosphere.
13. A reduced coenzyme Q10 crystal with a reduced coenzyme
Q10/oxidized coenzyme Q10 weight ratio of not lower than 96/4.
14. A method of producing reduced coenzyne Q10 such as
herein described
which comprises using at least one: species selected from
among aromatic hydrocarbons such as herein described,
halogenated hydrocarbons such as herein described, fatty acid
esters such as herein described and nitriles such as herein
described as a solvent in synthesizing reduced coenzyme Q10 by
reduction of oxidized coenzyme Q10.

15. The method as claimed in claim 14,
wherein at least one: species selected from among fatty
acid esters and nitriles is used as the solvent.
16. The method as claimed in claim 14 or 15,
wherein the reduction is carried out in a deoxygenated
atmosphere.
17. A method for preparing reduced coenzyme Q10
which comprises using a hydrocarbon and/or an ether as
a solvent in a deoxygenated atmosphere in synthesizing reduced
coenzyme Q10 by reduction of oxidized coenzyme Q10 •
18. The method as claimed in claim 17,
wherein a hydrocarbon is used as the solvent.
19. The method as claimed in claim 18,
wherein one of heptanes is used as the solvent.
20. The method as claimed in claim 17,
wherein an ether is used as the solvent.
21. The method as claimed in any of claims 14 to 20,
wherein a metal hydride compound, iron or zinc is used
as the reducing agent.
22. The method as claimed in any of claims 14 to 20,
wherein the reduction is carried out in the presence of
water using dithionous acid or a salt thereof as the reducing
agent.
23. The method as claimed in claim 22,
wherein the reduction is carried out at pH 3 to 7.

24. The method as claimed in claim 22 or 23,
wherein the amount of dithionous acid or a salt thereof
to be used equals to or is smaller than the amount of oxidized
coenzyme Q10 on the weight basis.
25. The method as claimed in any of claims 14 to 20,
wherein ascorbic acid or a related compound thereof is
used as the reducing agent.
26. The method as claimed in claim 25,
wherein at least one highly water-miscible species
selected from among ethers and nitriles is used as the solvent.
27. The method as claimed in claim 25 or 26,
wherein the amount of the ascorbic acid or a related
compound thereof as used is not smaller than 1 mole per mole
of oxidized coenzyme Q10.
28. The method as claimed in any of claims 2 5 to 27,
wherein the reduction is carried out in the presence of
a basic substance or a hydrogensulfite salt.
29. The method as claimed in any of claims 14 to 28,
wherein, after the reduction reaction, the organic phase
containing the product reduced coenzyme Q10 is recovered and
washed with water.
30. The method as claimed in claim 1,
wherein the reduced coenzyme Q10 to be subjected to the
crystallization is a product obtainable by the method as claimed
in any of claims 14 to 29.
31. The method as claimed in claim 1,
wherein the crystallization of reduced coenzyme Q10 is
carried out from a reduction reaction mixture obtainable by the

method as claimed in any of claims 14 to 29, or a solution
obtainable by separating an organic phase containing reduced
coenzyme Q10 and, if necessary, washing the organic phase.
32. The method as claimed in claim 1, a solvent other than the
hydrocarbons, fatty acid esters, ethers and nitriles is used
in a volume ratio of lower than 0 . 3 relat ive to the whole solvent
amount.
33. The method as claimed in claim 1, a solvent other than the
hydrocarbons, fatty acid esters, ethers and nitriles is used
in a volume ratio of lower than 0.3, relative to the whole solvent
amount in the case of homogeneous systems, or relative to the
volume of the solvent having larger solubility of reduced
coenzyme Q10 in the case of heterogeneous systems.

The present invention relates to a method of conveniently
and efficiently producing reduced coenzyme Q10 having excellent
qualities which is useful in foods, functional nutritive foods,
specific health foods, nutritional supplements, nutrients,
drinks, feeds, animal drugs, cosmetics, medicines, remedies,
preventive drugs, etc. This method is suitable for industrial
production thereof.
In a method of synthesizing reduced coenzyme Q10 by
reducing oxidized coenzyme Q10, followed by crystallization,
at least one species selected from among hydrocarbons, fatty
acid esters, ethers and nitriles is used as a solvent. Thus,
the reduced coenzyme Q10 can be protected from oxidation, and
as a result, the formation of the oxidized coenzyme Q10 as a
by-product can be minimized, thereby giving reduced coenzyme
Q10 having excellent qualities.

Documents:

68-KOLNP-2004-FORM-27.pdf

68-kolnp-2004-granted-abstract.pdf

68-kolnp-2004-granted-assignment.pdf

68-kolnp-2004-granted-claims.pdf

68-kolnp-2004-granted-correspondence.pdf

68-kolnp-2004-granted-description (complete).pdf

68-kolnp-2004-granted-examination report.pdf

68-kolnp-2004-granted-form 1.pdf

68-kolnp-2004-granted-form 13.pdf

68-kolnp-2004-granted-form 18.pdf

68-kolnp-2004-granted-form 2.pdf

68-kolnp-2004-granted-form 3.pdf

68-kolnp-2004-granted-form 5.pdf

68-kolnp-2004-granted-gpa.pdf

68-kolnp-2004-granted-reply to examination report.pdf

68-kolnp-2004-granted-specification.pdf


Patent Number 226784
Indian Patent Application Number 68/KOLNP/2004
PG Journal Number 52/2008
Publication Date 26-Dec-2008
Grant Date 24-Dec-2008
Date of Filing 20-Jan-2004
Name of Patentee KANEKA CORPORATION
Applicant Address 2-4 NAKANOSHIMA 3-CHOME, KITA-KU, OSAKA-SHI, OSAKA
Inventors:
# Inventor's Name Inventor's Address
1 UEDA TAKAHIRO 31-17-2018, SHIOYACHO 6-CHOME, TARUMI-KU, KOBE-SHI, HYOGO 655-0872
2 KITAMURA SHIRO 10-36-601, AIOICHO 1-CHOME, AKASHI-SHI, HYOGO 673-0882
3 UEDA YASUYOSHI 140-15, WAKU, ABOSHI-KU, HIMEJI-SHI, HYOGO 671-1227
PCT International Classification Number C07C 43/23, 41/26
PCT International Application Number PCT/JP02/07143
PCT International Filing date 2002-07-15
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2002-114854 2002-04-17 Japan
2 2001-214471 2001-07-13 Japan