Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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"ANTIHYPERCHOLESTERAEMIC AGENT, MONACOLIN K, AND ITS
PREPARATION"
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The present invention relates to a new compound
having antihypercholesteraemic activity and which we
5 have named Monacolin K. Monacolin K can be produced
by cultivating various microorganisms of the genus
! Monascus.
Thus the present invention consists in a compound,
Monacolin K, having the formula:
H W
,. ~fO
The invention further consists in a process for
. preparing an antihypercholesteraemic agent designated
Monacolin K, which comprises cultivating a Monacolin K-
producing microorganism of the genus Monascus in a
culture medium therefor.
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The invention still further consists in a
pharmaceutical composition comprising Monacolin K in
admixture with a pharmaceutically acceptable carrier
or diluent.
High blood cholesterol levels are recognized
as being one of the main causes of cardiopathy, such
as cardiac infarction or arteriosclerosis. As a
result, considerable research has been undertaken with
a view to discovering physiologically acceptable
substances which are capable of inhibitlng cholesterol
biosynthesis and thus reducing blood cholesterol
levels. One such compound is ML-236, which forms the
subject of our United Kingdom Patent Specification
No. 1,453,425. ML-236 is produced by cultivating
microorganisms of the genus Penicilllum.
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On investigating fungi of the genus Monascus,
it was found that these, particularly Monascus ruher
i strain 1~05 tFERM 4~22), produced an antihyper-
I cholesteraemic agent having substantially better
¦ 20 activity than that of ML-236. This agent was named
Monacolin K.
~' All microorganisms o~ the genus Monascus which
are capable of producing Monacolin K may be employed
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in the process of the present invention. Especially
useful are strains of Monascus ruber, particularly
Monascus ruber strain 1005 ~fERM 4822).
Monascus ruber strain 1005 (FERM 4822) is a
newly isolated microorganism having the following
microbiological properties. It was isolated -From
foodstuffs produced in Thailand and deposited on
16 February 1979 under the accession No. FERM 4822
with the Fermentation Research Institute, Agency of
Industrial Science and TechnologyJ Ministry of
International Trade and Industry, Japan and under
; the accession No, NRRL 12073 with the Agricultural
Research Service, Northern Regional Research
Laboratory, USA.
1. Growth
~ The growth on a potato-glucose-agar medium at
i 25C is fast and the diameter of the colony reaches
6 - 6,5 centimetres 10 days after inoculation. The
colony is flat and a relatively thin basal layer of
hyphae develops. Development of aerial hyphae is
poor; the aerial hyphae are white and most of them
are woolly. Many cleistothecia are formed on the
basal layer of hyphae and turn reddish-brown on
maturity. Both the surface and the reverse of the
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colony are brown to reddish-brown in colour.
The growth on Sabouraud's agar medium at
25C is very fast and the diameter of the colony
reaches 6 - 6.5 centimetres 10 days after inoculation.
The surface of the colony ls very flat, and basal
hyphae and aerial hyphae develop better than on
potato-glucose-agar medium. Cleistothecia counts
are very few. The surface of the colony is reddish-
yellow to reddish-brown in colour and the reverse
10 i9 reddish-brown to dark brown.
I The growth on oatmeal agar at 25C is slow
,~ and the diameter of the colony reaches 1.5 - 2 cænti-
metres 10 days after inoculation. The colony is
flat. Development of aerial hyphae and formation of
cleistothecia are both very poor. Both the surface
and the reverse of the colony are dark red to
reddish-brown in colour.
The growth on Czapek's agar medium at 25C
J is very slow and the diameter of the colony reaches
20 1.6 - 1.8 centimetres 10 days after inoculation.
The rates of growth on each of the above media
at 37C are substantially equal to tnoss at 25C.
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2. Mo~ l~ ical properties
The cleistothecia are spherical and 30 - 60
microns in diameter~ their walls are thin and mem-
branous, their stalks have septal walls and each
consists of a hypha o-F diameter 3.5 - 4.5 microns
and length 15 - ao microns. The ascus consists o~
8 spores and is nearly spherical and evanescent.
The ascospores are colourless and ovoid or ellipsoid~
they have a size of 4 - 5 x 4 - 7 micronsS and their
sur-Faces are smooth. The conidia are colourless
and spherical or pyriform; their size is 6 - 9 x
6 - 11 microns~ their bases are truncate and their
walls ars relatively thick and smooth. The
i conidia are linked basipetally as a type of meristem
arthrospore. The conidiophoreis like a vegetative
hypha and is branched or unbranched, the conidia
being formed at the top. The mycelia are colourless
; and branched and have septal walls~ most of them
have a diameter of 3 - 5 microns.
Z0 ~ased on the observations of its charactsristics
as reported above this microorganism was identified
as a strain of Monascus ruber van Tie~hem.
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Microbiological propsrties of Monascus
ruber have been reported in the following literature:
Takada, Transactions of the Micological Society of
Japan, 9, 125 - 13û (1969) [Materials for the
Fungus Flora of Japan (7)]~ and van Tieghem, Bull,
Soc. Botan. France, 31, 227 (1884). Ascospore
generation of the strain has been reported by
Cole et al in the Canadian Journal of Botany, 46, 987
(196B), "Conidium Ontogeny in hyphomycetes~ The
imperfect state of Monascus ruber and its meristem
arthrospores".
Although the use of Monascus ruber strain 1005
I is hereafter speci-Fically exemplified, it will be
appreciated that any strains o-F the genus Monascus,
including varieties and mutants, which are capable of
producing Monacolin K can be used in the process
- of the invention.
Monacolin K may be produced by cultivating
the chosen microorganism in a culture broth under
aerobic conditions, using the same techniques as
are well known in the art for the cultivation of
fungi and other microorganisms. For example, the
Monacolin K- producing microorganism may first be
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cultivated on a suitable medium and then the produced
microorganisms may be collected and inoculated into
and cultivated on another culture medium -to produce the -
desired Monacolin K; the culture media used for
multiplication of the microorganism and for production
of Monacolin K may be the same or differentO
~ ny culture medium well known in the art for
the cultivation of fungi may be employed, provided
that it contains, as is well known, the necessary
nutrlent materials, especially an assimilable carbon
source and an assimilable ni-trogen source. Examples
of suitable sources of assimilable carbon are
glucose, maltose, dextrin, starch, lactose, sucrose
and glycerine. Of -these sources, glucose, glycerine
and starch are particularly preferred for the
production of Monacolin K. ~xamples of suitable
sources of assimilable nitrogen are peptone, meat
- extract, yeast, yeast extract, soybean meal,
peanut meal, corn steep liquor, rice bran and
inorganic nitrogen sources. Of these nitrogen sources,
peptone is particularly preferred. When producing
Monacolin K, an inorganic salt and/or a metal salt
may, if necessary, be added to the culture mediu~
~urthermore, if necessary, a minor amount of a
heavy metal may also be added.
~ he microorganism is preferably cultivated
under aerobic conditions using cultivation methods
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well known in -the art, for example solid culture, shaken
culture or culture under aeration and agitation. ~he
microorganism will grow over a wide temperature range,
e.g. from 7 to 40 C, but, especially for -the pro-
duction of Monacolin K, the more preferred cultivationtemperature is within the range from 20 to 35 C.
During the cultivation of the microorganism,
the production of Monacolin ~ may be monitored by
sampling the culture medium and measuring the
physiological activity of the Monacolin K in the
culture medium by the test described hereafter.
~ultivation may then be continued until a substantial
accumulation of Monacolin K has been achieved in the
culture medium, at which time the Monacolin K may
be isola-ted and recovered from the culture broth
by any suita~le combination of isolation -techniques
; chosen having regard to its physical and chemical
properties. For example, any or all of the following
; isolation techniques may be employed extraction of
; 20 the liquor from the culture broth with a hydrophilic
solvent (for example, diethyl ether, ethyl aceta-te,
chloroform or benzene); extraction of the organism
with a hydrophilic solvent (such as ace-tone or
an alcohol); concentra-tion; dissolution into a more
polar solvent (e.g. acetone or an alcohol); removal
of impurities with a less polar solvent (such as
petroleum ether or hexane~; gel filtration through
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a column of a material such as Sephadex (a trade name
for a material available from Pharmacia, Co., Ltd.,
U.S.A.); absorptive chroma-tography with acti~e
- carbon or silica gel; and so onn By using a suitable
combination of these techniques, the desired Monacolin
K can be isolated from the culture broth as a pure ::
substance.
Monacolin K was ~ound to have the ~ollowing ~ :
properties~
1. Colour and form:
Colourless crystals.
2. Melting point:
157-159 C (with decomposition)-
3. ~lemental analysis:
C, 71.56%; H, 8~85%; 0, 19.59%.
4. Molecular weight:
. 404 (by mass spectrometry)
. 5. Molecular formula:
C2L~H3605 .
, 20 6. Ultraviolet absorp-tion spectrum (methanol):
As shown in Figure 1 of the accompanying
drawin~ss ha.ing maxima at 232, 238 and 246 m).
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7. Infrared absorption spectrum (KBr):
As shown in Figure 2 of the accompanying
drawings.
8. Nuclear magnetic resonance spectrum (60 MHz
proton):
As shown in ~igure 3 of the accompanying
drawings in deuterated chloroform, using
tetr~methylsilane as internal standard.
9. Nuclear magnetic resonance spectrum (13C):
As shown in Figure 4 of the accompanying
drawings, in deuterated methanol.
10. Solubility:
Soluble in lower alcohols (e.g. me-thanol,
ethanol and propanol), acetone, chloroform, ethyl
acetate and benzene.
Insoluble in petroleum ether and hexaneO
11. Specific rotation:
[C<~2D5 = +~07O6 (c=1, methanol).
12. Thin layer chromatography:
Rf = 0.47[No. 5715 Kieselgel 60~25L~ silica gel
~Merck & Co., ~td.) developed by a 4:1 by volume
mixture of methylene chloride and acetone,
detectable as an ultraviole-t radiation-absorbing
lump, 50% v/v sulphuric acid (a pale red to
reddish-brown colour develops on heating) or with
iodinel.
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The compound is neutral and is insoluble
in neutral or acidic aqueous media. It is cDnverted
to an acidic substance upon treatment with an alkali
and can then be dissolved in water. This acidic
substance can be extracted with ethyl acetate or
chloroform at an acid pH value and will revert to
Monacolin K on evaporation of the solvent.
The p~ysiological activity of Monacolin K
can be assayed and determined quantitatively by
the following in vivo test.
In vivo tsst with rabbits
In this test, the ability o-f Monacolin K to
reduce cholesterol levels in rabbit blood is measured.
The animals employed should weigh from 2.5 to 3.0 kg.
Immediately prior to starting the test, blood is
collected from the vein in an ear of each rabbit and
ths cholesterol level in the blood serum is measured
by a conventional method. A predetermined quantity of
Monacolin K is then administered orally continuously for
1 to 5 days and the cholesterol level in the blood serum
after administration is measured. The potency of the
Monacolin K or MDnacolin K-containing culture medium can be
determined quantitatively from the cholesterol values
obtained prior to and after administration of Monacolin K.
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We have demonstrated the ability of Monacolin K
to lower the blood and liver cholesterol levsls by
various in vivo tests
Reduction of blood cholesterol levels in rats
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The animals used were rats of the Wistar Imamichi
strain, each having a body weight of about 300 g. The
rests were conducted on groups of rats, sach group con-
sisting of 5 animals. Each animal was intravenusly
injected with 400 mg/kg of Triton WR-1339 ta trade ~ff-~e for
a material known to increase the blood cholesterol level)
whilst simultaneously administering intraperitoneally
10 mg/kg of Monacolin K. 1~ hours after intraperitoneal
administration, tlle rats were sacrificed by bleeding and
~ the blood was collected and its cholesterol level was
s 15 determined by conventional means. As a result, it was
established that blood cholesterol levels had been reduced,
as compared with a control group of animals to which
:f Triton WR-1339 alone had been administered, by 23.9%.
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Reduction o-f blood cholesterol levels in rabbits
ff 20 The test animals used were rabbits having a body
weight of from 2.7 kg to 2.9 kg. Each rabbit was given
j orally 1 mg/kg of Monacolin K twice each day ~morning and
evening) continuously for 5 days. Prior to administration
and at 3 and 5 days after administration, blood was
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collected from a vein in the ear and the cholesterol levels
in the blood serum were determined. As a result it was
found that the cholesteroi levels at 3 and 5 days
after administration of Monacolin K were 15~ and 29~j
respectivelyJ lower than the level prior to administrati~n
of Monacolin K,
~n addition to its valuable inhibitory effect
on the biosynthesis of cholesterol, Monacolin K has
a very low toxicity. Thus, the acute oral toxicity
(LD50) of Monacolin K in the mouss is 1 g/kg body weight
; or more.
The ~onacolin K may be administered orally or
parenterally in the form of a capsuleJ tabletJ
injectable preparation or any other known formulationJ
although we normally prefer to administer it orally.
The dose will vary, depending upon the ags and body
weight of the patient and the severity of the condition,
but,in general, the daily dose -for an adult would be
from 0.5 to 50 mg, either as a single dose or in
' 20 2 or 3 divided doses. However, in view of the low
; toxicity of the compound, higher doses may be employed
if required.
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The invention is further illustrated by the
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folowing non-limiting Example.
EXAMPLE
Monascus ruber 1005 strain was inoculated onto
a liquid culture medium containing 6% w/v glucose,
2.5% w/v peptone, 0.5% w/v corn steep li4uor and 0.5% w/v
ammonium chloride. Cultivation was continued under
aerobic conditions at a temperature of 28C for 10 days.
The resulting filtrate ~5 litrss) of the culture broth
; was adjusted to a pH value of 3 by the addition oF 6N
hydrochloric acid and then extracted with an equal volume
of ethyl acetats. The solvent was evaporated under
reduced pressure from the extract and the resulting
residue was dissolved in 100 ml of benzene. Insolubles
were filtered off.
The filtrate was washed twice, each time with 100 mI
~ of a 5% w/v aqueous solution of sodium bicarbonate.
;~ 100 ml of a 0.2N aqueous solution of sodium hydroxide
~ere then added to the washed -Filtrate and the mixture
was stirred at room temperature. After confirming
~' ~0 the disappearance of Monacolin K from the benzene layer
by thin layer chromatographyJ the aqueous layer was
separated off. The pH value of the aqueous layer was
then adjusted to 3 by addition of 6N hydrochloric acid
and the resulting solution was extracted twice, each time
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with 100 ml of ethyl acetate. The extract was
2vaporated -to dryness under reduced pressure, giving
260 mg of an oil. This oil was dissolved in benzene
and allowed to crystallize and then recrystallized
from an aqueous acetone solution to give 87 mg of
cnlourless needles of Monacolin K having the properties
: heretofor described.
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