U.S. patent application number 11/295601 was filed with the patent office on 2006-05-04 for food materials useful in preventing and ameliorating metabolic bone diseases and preventives/remedies for metabolic bone diseases comprising these materials.
This patent application is currently assigned to MEIJI DAIRIES CORPORATION. Invention is credited to Kouichiro Hashimoto, Kazuo Nagai, Yoshiro Sato, Naoki Taketomo, Hiroshi Tsuchida, Jetae Woo, Nobuo Yoda.
Application Number | 20060093651 11/295601 |
Document ID | / |
Family ID | 26560520 |
Filed Date | 2006-05-04 |
United States Patent
Application |
20060093651 |
Kind Code |
A1 |
Nagai; Kazuo ; et
al. |
May 4, 2006 |
Food materials useful in preventing and ameliorating metabolic bone
diseases and preventives/remedies for metabolic bone diseases
comprising these materials
Abstract
This invention relates to drinks and foods having osteogenetic
function, each of which comprises as an active ingredient an
effective amount of a culture of a propionic acid bacterium and/or
a lactic acid bacterium, said culture containing
2-amino-3-carboxy-1,4-naphthoquinone; and preventives and/or
remedies for metabolic bone diseases, each of which comprises as an
active ingredient a naphthoquinone compound selected from the group
consisting of 2-amino-3-carboxy-1,4-naphthoquinone,
1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone,
2,3-dichloro-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone,
8-hydroxy-1,4-naphthoquinone,
2-(.alpha.-hydroxy-.delta.-methylpentenyl)-5,8-dihydroxy-1,4-naphthoquino-
ne and salts thereof. These cultures and naphthoquinones promote
bone metabolism and increase bone mass and bone strength.
Inventors: |
Nagai; Kazuo; (Tokyo,
JP) ; Woo; Jetae; (Aichi, JP) ; Sato;
Yoshiro; (Kanagawa, JP) ; Hashimoto; Kouichiro;
(Kanagawa, JP) ; Taketomo; Naoki; (Kanagawa,
JP) ; Yoda; Nobuo; (Tokyo, JP) ; Tsuchida;
Hiroshi; (Tokyo, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
MEIJI DAIRIES CORPORATION
Tokyo
JP
|
Family ID: |
26560520 |
Appl. No.: |
11/295601 |
Filed: |
December 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10110206 |
Jul 9, 2002 |
|
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PCT/JP00/07217 |
Oct 18, 2000 |
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11295601 |
Dec 7, 2005 |
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Current U.S.
Class: |
424/439 ;
424/93.45; 514/682 |
Current CPC
Class: |
A23V 2002/00 20130101;
A61K 35/741 20130101; A61K 31/196 20130101; A61K 31/195 20130101;
A61P 19/00 20180101; A61K 35/747 20130101; A23L 29/065 20160801;
A61K 31/122 20130101; A23L 33/135 20160801; A23Y 2320/25 20130101;
A61P 3/14 20180101; A61P 19/10 20180101; A23L 33/10 20160801; A23V
2002/00 20130101; A23V 2250/206 20130101; A23V 2250/30 20130101;
A23V 2002/00 20130101; A23V 2250/206 20130101; A23V 2250/714
20130101 |
Class at
Publication: |
424/439 ;
424/093.45; 514/682 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61K 47/00 20060101 A61K047/00; A61K 31/12 20060101
A61K031/12 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 19, 1999 |
JP |
11/296052 |
Oct 19, 1999 |
JP |
11/296053 |
Claims
1-20. (canceled)
21. A method of treating a metabolic bone disease, comprising
administering, to a subject in need thereof, an effective amount of
at least one of a culture of a propionic acid bacterium and a
culture of lactic acid bacterium.
22. The method according to claim 21, wherein a culture of a
propionic acid bacterium is administered.
23. The method according to claim 21, wherein a culture of lactic
acid bacterium is administered.
24. The method according to claim 21, wherein a culture of a
propionic bacterium and a culture of lactic acid bacterium is
administered.
25. The method according to claim 21, wherein the culture comprises
a culture supernatant, cells or both.
26. The method according to claim 22, wherein the propionic acid
bacterium is Propionibacterium freudenreichii.
27. The method according to claim 23, wherein the lactic acid
bacterium is Lactococcus lactis subsp. lactis.
28. The method according to claim 21, wherein the administering
comprises providing a food or drink comprising the at least one
culture of a propionic acid bacterium and a culture of a lactic
acid bacterium; and at least one or more food additives.
Description
TECHNICAL FIELD
[0001] This invention relates to drinks and foods useful for the
prevention or amelioration of metabolic bone diseases led by
osteoporosis and also to preventives and/or remedies for such
metabolic bone diseases.
BACKGROUND ART
[0002] A bone is an important tissue, which serves as a structural
frame to support the body and also supports a mineral control
system for body fluid as a reservoir for various minerals such as
calcium and phosphorus. In osseous tissue, osteogenesis or bone
formation and resorption always remain active to replace old bone
by new bone. Especially in a bone the growth of which has finished,
osteoblasts in the bone formation system and osteoclasts in the
bone resorption system are closely related to each other to repeat
bone formation and bone resorption such that the osseous tissue is
maintained and minerals in the body fluid are kept constant. This
bone reconstitution is called "bone remodeling". This bone
remodeling is precisely controlled by physical factors, hormones,
cytokine and the like while maintaining a certain balance. Bone
mass, however, decreases if this balance is disturbed and the bone
resorption exceeds the bone formation. Of these pathologic
decreases in bone mass, one normal in bone components is called
"osteoporosis".
[0003] Osteoporosis is known to be one of health troubles from
which more people suffer as aging advances, and especially to occur
often on post menopausal females. Osteoporosis tends to result in a
fracture of the femoral neck, and like cerebrovascular accident, is
a cause of many "bedridden aged persons", thereby adversely
affecting on the society. There is, accordingly, a strong social
demand for performing appropriate treatment for osteoporosis to
avoid its aggravation and also for establishing an effective method
for its prevention. As drugs for osteoporosis, vitamin D
preparations, calcitonin preparations, bisphosphonate preparations
and the like have been developed and clinically applied to date. It
is, however, the current circumstance that these efforts have not
led yet to the development of satisfactorily effective drug.
[0004] Under the current circumstance that no satisfactory method
has been established yet for the treatment of osteoporosis as
mentioned above, its prevention is most important. Theoretically,
it is possible to keep the bone mass at a high level even when
advanced in age, provided that the bone mass can be increased as
much as possible in adolescence and maturity, the thus-increased
bone mass can be maintained for an extended time and a
postmenopausal reduction in bone mass can be suppressed even a
little. For this purpose, it is essential to continuously ingest
appropriate calcium throughout childhood, adolescence and maturity.
Ingestion of calcium alone, however, is not sufficient. If food
materials having function to prevent or ameliorate osteoporosis and
the like are available, their addition to foods which are taken
daily is expected to successfully prevent or ameliorate
osteoporosis through the everyday dietary. Further, identification
and separation of active ingredients in such food materials are
expected to make it possible to use them as preventives or remedies
for osteoporosis.
[0005] In the meantime, vitamin K which is widely known as a blood
coagulation factor was found to also take part in bone metabolism
and has been drawing interests, leading to the marketing of a
vitamin K.sub.2 preparation in Japan as a drug for ameliorating
bone mass and pains in osteoporosis. In addition, food materials
with vitamin K.sub.1 or vitamin K.sub.2 contained therein are being
increasingly put on the market.
[0006] With the foregoing in view, the present invention has as an
object thereof the provision of a novel food material useful for
the prevention and/or amelioration of a metabolic bone disease,
especially osteoporosis. The present invention also has as another
object thereof the provision of a pharmaceutical useful for the
prevention and treatment of a metabolic bone disease.
DISCLOSURE OF THE INVENTION
[0007] With a view to achieving the above-described objects, the
present inventors have proceeded with extensive screening and
research, concentrating on food materials. As a result, it has been
found that oral ingestion of a culture of a propionic acid
bacterium and/or a lactic acid bacterium makes it possible to
increase bone mass and bone strength. It has also been found that a
food containing an effective amount of the culture is effective for
the prevention or amelioration of metabolic bone diseases led by
osteoporosis. It has also been found that
2-amino-3-carboxy-1,4-naphthoquinone and its analogs contained in
the culture are effective in promoting differentiation and
functional expression of osteoblasts and also inhibiting formation
of osteoclasts, leading to finding that these compounds are useful
as preventives and/or remedies for metabolic bone diseases.
[0008] Described specifically, the present invention relates
to:
[0009] (1) A drink or food having osteogenesis promoting function,
comprising as an active ingredient a culture of a propionic acid
bacterium and/or a lactic acid bacterium (which may hereinafter be
called a "fermentation product"; and "a culture of a propionic acid
bacterium" may also be called "BGS") or a naphthoquinone compound
selected from the group consisting of
2-amino-3-carboxy-1,4-naphthoquinone, 1,4-naphthoquinone,
2-hydroxy-1,4-naphthoquinone, 2,3-dichloro-1,4-naphthoquinone,
5-hydroxy-1,4-naphthoquinone, 8-hydroxy-1,4-naphthoquinone,
2-(.alpha.-hydroxy-.delta.-methylpentenyl)-5,8-dihydroxy-1,4-naphthoquino-
ne and salts thereof.
[0010] (2) A drink or food as described above under (1), wherein
the active ingredient is a culture of a propionic acid bacterium
and/or a lactic acid bacterium.
[0011] (3) A drink or food as described above under (1), wherein
the culture is a culture supernatant and/or cells.
[0012] (4) A drink or food as described above under (1), wherein
the active ingredient is 2-amino-3-carboxy-1,4-naphthoquinone
(which may herein after be called "ACNQ").
[0013] (5) A drink or food as described above under (1), wherein
the propionic acid bacterium is Propionibacterium
freudenreichii.
[0014] (6) A drink or food as described above under (1), wherein
the lactic acid bacteriumis Lactococcus lactis subsp. lactis.
[0015] (7) Use of a culture of a propionic acid bacterium and/or a
lactic acid bacterium or a naphthoquinone compound selected from
the group consisting of 2-amino-3-carboxy-1,4-naphthoquinone,
1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone,
2,3-dichloro-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone,
8-hydroxy-1,4-naphthoquinone,
2-(.alpha.-hydroxy-.delta.-methylpentenyl)-5,8-dihydroxy-1,4-naphthoquino-
ne and salts thereof for the production of a drink or food having
osteogenesis promoting function.
[0016] (8) Use of a culture of a propionic acid bacterium and/or a
lactic acid bacterium as described above under (7).
[0017] (9) Use as described above under (7), wherein the culture is
a culture supernatant and/or cells.
[0018] (10) Use as described above under (7), wherein the propionic
acid bacterium is Propionibacterium freudenreichii.
[0019] (11) Use as described above under (7), wherein the lactic
acid bacterium is Lactococcus lactis subsp. lactis.
[0020] (12) A preventive or remedy for a metabolic bone disease,
comprising as an active ingredient a naphthoquinone compound, which
is selected from the group consisting of
2-amino-3-carboxy-1,4-naphthoquinone, 1,4-naphthoquinone,
2-hydroxy-1,4-naphthoquinone, 2,3-dichloro-1,4-naphthoquinone,
5-hydroxy-1,4-naphthoquinone, 8-hydroxy-1,4-naphthoquinone,
2-(.alpha.-hydroxy-.delta.-methylpentenyl)-5,8-dihydroxy-1,4-naphthoquino-
ne and salts thereof, or a culture of a propionic acid bacterium
and/or a lactic acid bacterium.
[0021] (13) A preventive or remedy as described above under (12),
wherein the naphthoquinone compound is
2-amino-3-carboxy-1,4-napthoquione.
[0022] (14) A preventive or remedy as described above under (12) or
(13), wherein the metabolic bone disease is osteoporosis.
[0023] 13. Use of a naphthoquinone compound, which is selected from
the group consisting of 2-amino-3-carboxy-1,4-naphthoquinone,
1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone,
2,3-dichloro-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone,
8-hydroxy-1,4-naphthoquinone,
2-(.alpha.-hydroxy-.delta.-methylpentenyl)-5,8-dihydroxy-1,4-naphthoquino-
ne and salts thereof, or a culture of a propionic acid bacterium
and/or a lactic acid bacterium for the production of a
pharmaceutical for prevention or treatment of a metabolic bone
disease.
[0024] (16) Use as described above under (15), wherein the
naphthoquinone is 2-amino-3-carboxy-1,4-naphthoquinone.
[0025] (17) Use as described above under (15), wherein the
metabolic bone disease is osteoporosis.
[0026] (18) A treatment method of a metabolic bone disease, which
comprises administering a naphthoquinone compound, which is
selected from the group consisting of
2-amino-3-carboxy-1,4-naphthoquinone, 1,4-naphthoquinone,
2-hydroxy-1,4-naphthoquinone, 2,3-dichloro-1,4-naphthoquinone,
5-hydroxy-1,4-naphthoquinone, 8-hydroxy-1,4-naphthoquinone,
2-(.alpha.-hydroxy-.delta.-methylpentenyl)-5,8-dihydroxy-1,4-naphthoquino-
ne and salts thereof, or a culture of a propionic acid bacterium
and/or a lactic acid bacterium.
[0027] (19) A treatment method as described above under (18),
wherein the naphthoquinone compound is
2-amino-3-carboxy-1,4-naphthoquinone.
[0028] (20) A treatment method as described above under (18),
wherein the metabolic bone disease is osteoporosis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a diagram showing tibial bone mineral densities
(mg/cm.sup.2) of SD rats after the SD rats were allowed to ingest
ad libitum refined feeds containing 2% or 10% of BGS powder,
[0030] FIG. 2 is a diagram illustrating femur fracture energies
(mJ) of the rats, and
[0031] FIG. 3 shows amounts of calcium (mg/g) per dry weight of the
femurs of the rats. In FIGS. 1 to 3, the bars each indicate an
average .+-.standard error (p<0.05).
[0032] FIG. 4 is a diagram showing alkaline phosphatase activities
of the stromal cell line ST-2 derived from mouse bone marrow after
the cells were cultured for 3 days at varied ACNQ concentrations,
and
[0033] FIG. 5 is a diagram illustrating viable cell counts of the
cells (absorbances at 595 nm) by MTT assay.
[0034] FIG. 6 is a diagram illustrating effects of Ca accumulation
by 1.alpha.,25(OH).sub.2D.sub.3, vitamin K.sub.2, ACNQ, and
combinations of 1.alpha.,25(OH).sub.2D.sub.3 and ACNQ on human
osteoblasts (SaM-1).
[0035] FIG. 7 is a diagram illustrating effects of P accumulation
by 1.alpha.,25(OH).sub.2D.sub.3, vitamin K.sub.2, ACNQ, and
combinations of active 1.alpha.,25(OH).sub.2D.sub.3 and ACNQ on
human osteoblasts (SaM-1).
[0036] FIG. 8 is a diagram illustrating cytotoxities of
1.alpha.,25(OH).sub.2D.sub.3, vitamin K.sub.2, ACNQ, and
combinations of active 1.alpha.,25(OH).sub.2D.sub.3 and ACNQ on
human osteoblasts (SaM-1).
[0037] FIG. 9 is a diagram illustrating effects of ACNQ and vitamin
K.sub.1 on TRAP-positive polykaryocytes in the culture of human
bone marrow cells.
[0038] FIG. 10 is a diagram showing effects on osteoclast formation
when osteoblast-like interstitial cells derived from the calvaria
of a ddY mouse newborn and bone marrow cells derived from the same
strain mouse were co-cultured in the presence of
1.alpha.,25(OH).sub.2D.sub.3 or ACNQ.
[0039] FIG. 11 is a diagram showing activity of osteoclasts when
osteoblast-like interstitial cells derived from the calvaria of a
ddY mouse newborn and bone marrow cells derived from the same
strain mouse were co-cultured in the presence of ACNQ.
BEST MODES FOR CARRYING OUT THE INVENTION
[0040] In the pharmaceutical and the drink or food according to the
present invention, the above-described naphthoquinone compounds,
the culture of the propionic acid bacterium and the culture of the
lactic acid bacterium are all usable. Among the naphthoquinone
derivatives, 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) is
particularly preferred. This ACNQ is known to be available, for
example, from a culture of a propionic acid bacterium (a
microorganism belonging to the genus Propionibacterium)
(JP-A-07-227207, JP-A-07-289273, JP-A-10-304871). On the other
hand, the naphthoquinone compounds other than ACNQ are also
available from the culture of the propionic acid bacterium
(JP-A-08-98677). These naphthoquinone derivatives and the culture
of the propionic acid bacterium are known to have activity to
proliferate bifid bacteria, but are not known at all whether or not
they are effective for metabolic bone diseases.
[0041] Illustrative of the propionic acid bacterium are propionic
acid bacteria for cheese, such as Propionibacterium freudenreichii,
P. thoenii, P. acidipropionici and P. jensenii; P. avidum; P.
acnes; P. lymphophilum; and P. granulosam. Examples of
Propionibacterium freudenreichii can include P. freudenreichii IFO
12424 and P. freudenreichii ATCC 6207.
[0042] Illustrative of the lactic acid bacterium are bacteria
belonging to the genus of Lactobacillus, the genus of
Streptococcus, the genus of Lactococcus, or the genus of
Leuconostoc. Lactobacillus bacteria can include, for example, Lb.
acidophilus and Lb. debrueckii subsp. bulgalicus; Streptococcus
bacteria can include, for example, Str. thermophilus; Lactococcus
bacteria can include, for example, Lc. lactis subsp. cremoris and
Lc. lactis subsp. lactis; and Leuconostoc bacteria can include, for
example, Leuc. mesentaroides subsp. cremoris and Leuc. lactis.
[0043] The culture of the propionic acid bacterium or the lactic
acid bacterium can be obtained by a known method. For example, as a
method for culturing a propionic acid bacterium at high
concentration, the propionic acid bacterium can be cultured on a
medium prepared by adding minerals and a monosaccharide to a whey
protein concentrate (WPC) or an enzymolysate thereof
(JP-A-10-304871). As an efficient method for culturing the
propionic acid bacterium, a bifid bacterium and the propionic acid
bacterium can be cultured while circulating a cultured liquor
through different culture tanks. These culturing methods can be
used for the practice of the present invention. It is also possible
for those skilled in the art to modify the compositions of the
media, the culturing conditions and the like (dissolved oxygen
concentration, etc.) in these known methods such that the methods
can be further optimized. Concerning a nitrogen source, for
example, optimization (an increase in cell count and an increase in
the osteometabolic activity of BGS) is feasible by conducting inter
alia an investigation on effects available from various amino acids
(and salts thereof) in addition to casein starch or WPC and an
investigation on culturing conditions (anaerobic culture or aerobic
culture). Accordingly, these modified methods are also encompassed
by the present invention.
[0044] The bifid bacteria proliferation activity of BGS is found in
a filtrate obtained by removing cells from a culture and also in a
methanol extract of the cells (Kaneko, T. et al.: J. Dairy Sci.,
77, 393-404, 1994). Osteometabolic activity is also considered to
be found in both of the culture supernatant and the cells. The term
"BGS" as used herein, therefore, includes all of a culture itself
of a propionic acid bacterium and/or a lactic acid bacterium, a
culture supernatant and cells, and their extracts, dry powders,
dilutions and the like, for example.
[0045] Processed forms of BGS can be divided roughly into a powder
form and a liquid form. To convert BGS into a powder form, skim
milk powder, whey powder, raw starch, dextrin or the like is
directly added as an excipient to a culture to adjust the solid
content to 30 to 40 wt. % in the culture, and the thus-prepared
culture is spray-dried; or a culture and a reconstituted solution
of an excipient are mixed together, the mixture is concentrated
until its solid content is increased to 30 to 40 wt. %, and the
thus-concentrated mixture is spray-dried. BGS so prepared can be
stored for an extended time by applying deoxidization treatment
upon filling (filling of nitrogen gas, addition of a deoxidizing
agent, or the like) As the excipient, it is possible to use, in
addition to the above-described excipients, WPC, a whey protein
isolate (WPI) and processed starches (soluble starch, British gum,
oxidized starch, starch esters, starch ethers or the like, in
addition to dextrin) as needed. Further, BGS may be formulated into
a triturated preparation (0.2% triturated powder) to facilitate its
use in foods.
[0046] On the other hand, the naphthoquinone compounds can be
collected by extraction and purification from a culture of a
propionic acid bacterium (JP-A-07-289273). Examples of
microorganisms, which produce fermentation products containing the
naphthoquinones, can include Bacteroides bacteria such as
Bacteroides vulgatus JCM5826T and Bacteroides fragilis ATCC 2375;
Bacillaceae bacteria; Enterococci bacteria; Bacteroidaceae
bacteria; Streptococcaceae bacteria; and Enterobacteriaceae
bacteria.
[0047] Further, the naphthoquinone compounds can also be obtained
by chemical syntheses. For example, ACNQ can be obtained through
four steps from commercially available 1,4-dihydroxynaphthoeic acid
as a starting material (JP-A-10-36238).
[0048] Illustrative salts of these naphthoquinone compounds are
salts acceptable from the standpoint of pharmaceutics or food
science. Representative salts can include the acetates,
benzenesulfonates, benzoates, hydrogencarbonates, lactates, and
citrates. It is, however, to be noted that these salts are merely
illustrative and the present invention is not limited to these
salts.
[0049] The naphthoquinone compounds for use in the present
invention may include stereoisomers and optical isomers. These
isomers can all be used in the present invention.
[0050] The above-described naphthoquinone compounds enhance the
alkaline phosphatase (ALP) activity of osteoblasts, which is an
index of osteogenesis, and promotes mineralization. While
inhibiting differentiation and maturation of osteoclasts, they
neither inhibit the activity of osteoclasts nor exhibit
cytotoxicity against osteoclasts. Namely, these naphthoquinone
compounds and the culture are useful as preventives and/or remedies
for metabolic bone diseases and also as drinks or foods for the
prevention or amelioration of metabolic bone diseases. These
preventives, remedies, drinks and foods are extremely unique in
that they have two effects, promotion of bone formation and
inhibition of bone resorption, in combination.
[0051] Concerning BGS and ACNQ, a description will below be made
about certain facets of bone metabolism, specifically the results
of investigations on bone forming action and bone sorbing action in
an in vitro assay system and an in vivo assay system.
[0052] In the in vivo assay system of BGS, oral ingestion of BGS
significantly enhanced femur bone mineral density and tibial bone
mineral density, the breaking energy of femur and the Ca content of
femur over a control group. Described specifically, it was
demonstrated in an in vivo experiment that oral ingestion of BGS
significantly enhances bone mass and bone strength (FIGS. 1, 2 and
3). To analyze this osteogenesis promoting effect of BGS, a further
investigation was conducted on effects of ACNQ, which is a
proliferation activating ingredient for bifidobacteria, on bone
metabolism.
[0053] A bone is composed of osteogenetic cells such as
osteoblasts, bone resorbing cells such as osteoclasts, various
interstitial cells (fibroblasts, adipocytes, reticular cells), and
hematopoietic cells. In a network where these cells cooperate with
each other, osseous tissue is formed.
[0054] Firstly, an investigation was made about effects of ACNQ on
osteogenesis. It is osteoblasts that play a principal role in
osteogenesis. In a bone after its growth, osteoblasts are mobilized
to bone resorption lacunae formed by osteoclasts, and osteogenesis
start to supplement the resorbed bone. Preosteoblasts actively
proliferate, and gradually acquire the differentiation character of
osteoblasts. The differentiation advances further, and by matured
osteoblasts, mineralization of a formed bone matrix takes place to
complete the osteogenesis. As indices of differentiation of
osteoblasts, expression of ALP and an increase in its activity are
most important in addition to synthesis of various matrical
proteins.
[0055] Therefore, ST-2 cells, established cells of bone marrow
stromal cells, were cultured in a medium with ACNQ added therein,
and as an index associated with differentiation of osteoblasts, ALP
activity was measured. ACNQ promoted formation of osteoblasts
concentration-dependently up to the concentration of 1 .mu.g/mL
(FIG. 4). Namely, ACNQ promoted differentiation from bone marrow
stromal cells to osteoblasts concentration-dependently. Further,
ACNQ did not show cytotoxicity up to the concentration of 1
.mu.g/mL (FIG. 5).
[0056] Although a relationship between ALP activity and
mineralization has not been elucidated yet, ALP is suggested to
play an important role in mineralization because a genetic defect
in this enzyme leads to occurrence of abnormality in the
development of a skeleton. Accordingly, an increase in ALP activity
is considered to promote the mineralization of osteoblasts. Bone
forming effect of ACNQ was, therefore, assayed depending upon
mineralization promoting effect of human osteoblasts (SaM-1). SaM-1
cells possess all the characteristics of osteoblasts, and are known
to calcify in the presence of 2 mM .alpha.-glycerophosphoric acid
depending upon the concentration of 1.alpha.,25(OH).sub.2D.sub.3
(active vitamin D.sub.3) (Koshihara, Y: Biolchem. Biophys. Res.
Commun., 145, 651-657, 1987). Accordingly, SaM-1 cells were
cultured in the presence of ACNQ at varied concentrations, and
amounts of Ca and P accumulated on matrices were measured. ACNQ
concentration-dependently promoted mineralization at 10.sup.-5 M to
10.sup.-7 M (FIGS. 6 and 7). According to the results of a
measurement of free LDH in culture supernatants, on the other hand,
ACNQ did not show cytotoxicity at 10.sup.-5 M to 10.sup.-7 M (FIG.
8).
[0057] From the above results, ACNQ has been found to promote
osteogenesis at extremely low concentrations of from 10.sup.-5 M to
10.sup.-7 M and to show no cytotoxicity at these
concentrations.
[0058] A still further investigation was next conducted about
effects of ACNQ on bone resorption. Osteoclasts are bone resorbing
cells, which originate from hematopoietic cells of the macrophage
cell lineage and are formed as a result of stimulation by a bone
resorbing factor under an environment specific to osseous tissue.
In vitro formation of osteoclasts from preosteoclasts which exist
in a bone marrow makes it possible to analyze ACNQ's function. As a
culture system for forming osteoclasts, culture of bone marrow
cells, co-culture of bone marrow cells and osteoblasts, co-culture
of splenocytes and osteoblasts, or the like can be used. Culture of
bone marrow cells together with 1.alpha.,25(OH).sub.2D.sub.3
results in formation of multinuclear osteoclasts which are positive
to tartrate-resistant acid phosphatase (TRAP), a marker enzyme for
osteoclasts.
[0059] Therefore, human monocytes were cultured in the presence of
1.alpha.,25(OH).sub.2D.sub.3 or ACNQ, and effects of ACNQ on the
formation of TRAP-positive polykaryocytes elicited by
1.alpha.,25(OH).sub.2D.sub.3 was investigated. ACNQ inhibited the
formation of osteoclasts concentration-dependently at 10.sup.-8 M
to 10.sup.-5 M (FIG. 9). Its inhibitory effect was significantly
stronger than that available from vitamin K.sub.1 at the same
concentration.
[0060] A still further investigation was also conducted on
TRAP-positive cells formed by a co-culture system of primary
osteoblast-like interstitial cells derived from the calvaria of a
mouse newborn and mouse bone marrow cells. ACNQ considerably
inhibited the formation of osteoclasts at 1.5 .mu.g/mL and higher
(FIG. 10). Effects of ACNQ on the activity of a bone re sorbing
system were then investigated. Measured were the formation of
resorption lacunae and cytotoxicity on a piece of dentinum in an
osteoclast forming system by co-culture of primary osteoblast-like
interstitial cells derived from the calvaria of the above mouse
newborn and mouse bone marrow cells. ACNQ did not inhibit the bone
resorbing activity of differentiated and matured osteoclasts (FIG.
11). Further, ACNQ did not exhibit cytotoxicity against osteoclasts
(FIG. 11). This indicates that ACNQ does not functionally impair
differentiated and matured osteoclasts.
[0061] From the above results, it has been found that, when BGS is
orally ingested, it shows effects in increasing bone mass and
enhancing bone strength. On the other hand, ACNQ which is
considered to be one of principal active ingredients of BGS
exhibited extremely unique osteometabolic effects in that it
enhances the formation of osteoblasts and promotes osteogenesis and
on the other hand, it inhibits the formation of osteoclasts but
formed osteoclasts function normally (possess bone resorbing
activity). The above-described osteogenesis promoting activity of
BGS is considered to be attributed to combined effects of various
ingredients in BGS, but its majority is undoubtedly believed to be
based on the effect of ACNQ which is contained in an extremely
small amount in BGS.
[0062] In the meantime, vitamin K which is widely known as a blood
coagulation factor has also been found to have a connection with
bone metabolism, leading to growing interests in this vitamin. In
the nature, vitamins K.sub.1 and K.sub.2 exist together. Vitamins
K.sub.1 and K.sub.2 commonly have the naphthoquinone skeleton, are
different only in the side chain bonded to the 3-position of the
skeleton, and commonly contain a methyl group bonded to the
1-position of the skeleton. Vitamin K.sub.1 is a single compound,
but vitamin K.sub.2 includes 14 homologs depending on the number of
isoprene bonded to the 3-position. Among these, only vitamin
K.sub.2-4 containing 4 isoprene units (geranylgeraniol) promotes
osteogenesis, inhibits bone resorption, and suppresses a reduction
in bone mass. Effects of the side chains of vitamin K.sub.1 or
vitamin K.sub.2 on bone resorption were investigated. As a result,
geranylgeraniol, the side chain of vitamin K.sub.2-4, was
recognized to have osteoclast-formation inhibitory effect of a
similar degree as vitamin K.sub.2-4. Incidentally, phytol which is
a side chain of vitamin K.sub.1 is weak in the inhibitory effect,
and the side chains of vitamin K.sub.2, said side chains containing
2 to 7 isoprene units (except for 4 isoprene units), respectively,
did not show the inhibitory effect but rather a positive effect
(Hara, K. et al.: Bone, 16, 179-184, 1995).
[0063] Similarly to vitamin K.sub.1 and K.sub.2, ACNQ in the
present invention also has the naphthoquinone skeleton. However, an
amino group and a carboxyl group are bonded to the 2-position and
the 3-position of the skeleton, respectively. Taking into
consideration that the osteometabolic activity of vitamin K.sub.1
or K.sub.2 relies upon its side chain as described above, they are
considered to be totally different from each other.
[0064] The above-described culture and naphthoquinone compounds
each promotes bone metabolism, and can be added in effective
amounts to daily ingested foods for the production of foods useful
in the prevention or amelioration of osteoporosis. No particular
limitation is imposed on the foods to which the culture and/or the
naphthoquinone compound may be added. In Japan, however, dietary
foods approved to claim health care applications or restricted
applications (foods for invalids, foods for elderly, specific
health foods) can be mentioned as preferred examples. In countries
outside Japan, foods approved to include health claims in labeling
(health-emphasized labeling) can be mentioned.
[0065] In drinks or foods for the prevention or amelioration of
metabolic bone diseases, the above-described naphthoquinone
compounds can be used. From the standpoint of safety, health and
hygiene, however, use of those derived from natural materials,
especially cultures of propionic acid bacteria is preferred. Each
culture includes not only itself but also its solvent extract and
processed or treated products thereof (for example, the culture
with cells contained therein, an extract of the cells and a
supernatant of the culture; and their concentrates, dried products,
dilutions and the like).
[0066] To make an active ingredient remain active in each food, it
is preferred to add, for example, sodium ascorbate as a stabilizer.
The amount of a culture to be added is calculated based on the
content of a naphthoquinone compound as an active ingredient (see,
for example, Referential Example 2). The culture may be added, for
example, in an amount of from 0.5 to 10 wt. %.
[0067] The composition according to the present invention, which is
suited for use in drinks or foods, may be used by itself. As an
alternative, the composition may also be added along with a carrier
acceptable from the standpoint of food science and one or more food
nutrients, for example, one or more of vitamins, minerals, amino
acids and the like. The term "a carrier acceptable from the
standpoint of food science" as used herein embraces binders,
excipients, preservatives, and color additives.
[0068] The above-described naphthoquinone compounds and culture can
be used generally in the form of ordinary pharmaceutical
compositions.
[0069] These pharmaceutical compositions can be prepared using a
pharmaceutically acceptable carrier, for example, a diluent, for
example, a filler, extender, binder, moistening agent,
disintegrator, surfactant or lubricant, or an excipient. For these
pharmaceutical compositions, a variety of forms can be chosen
depending on the therapeutic purpose. Representative examples of
these forms can include tablets, pills, powders, liquids,
suspensions, emulsions, granules, capsules, vaginal suppositories,
and injections (solutions, suspensions).
[0070] The dosage of the preventive or remedy for the metabolic
bone disease may desirably be set in view of the patient's
conditions such as his or her age and body weight, the
administration route, the nature and severity of the disease, and
the like. In general, however, the daily dosage may range from 0.5
to 100 mg/kg in terms of the amount of the naphthoquinone compound
in the case of oral administration. In some instances, however, a
dosage smaller than the above range may be sufficient or in
contrast, a dosage greater than the above range may be required.
The above dosage may be administered in 2 to 4 portions a day.
[0071] As calcium is needed for osteogenesis, a calcium preparation
may be used and administered in combination upon administration of
the drug according to the present invention. Further, a bone
sorption inhibitor [for example, active vitamin D.sub.2, active
vitamin D.sub.3, calcitonin, estrogen, iprifavone ("Osten", trade
name), or the like] may also be used and administered in
combination.
EXAMPLES
[0072] Certain specific embodiments of the present invention will
hereinafter be described based on Referential Examples and Tests.
It will, however, be understandable for those having ordinary skill
in the present field of art that various modifications are feasible
within the scope of the present invention.
Referential Example 1
Production of BGS Powder
[0073] After a 10 w/w % reconstituted solution of whey powder was
adjusted to pH 7.0 with 8 N NaOH, protease ("Amano A", product of
Amano Enzyme Inc.) was added at a rate of 6.79 mg per gram-whey
powder, followed by enzymolysis at 50.degree. C. for 2 hours.
During the reaction, the reaction mixture was maintained at pH 7.0
with 8 N NaOH. The reaction mixture was heated at 85.degree. C. for
10 minutes to deactivate the enzyme, and was then cooled to
35.degree. C. Subsequent to addition of 0.1 w/w % yeast extract,
the resulting mixture was adjusted to pH 6.7 with 4 N HCl and then
sterilized at 121.degree. C. for 7 minutes to provide a culture
medium.
[0074] A Propionibacterium freudenreichii strain, ATCC 6207 (this
strain number is an example), had been incubated stationary
(37.degree. C., 3 days) beforehand in a TPY medium (see Example 1
of JP-A-7-289273), and as an inoculum, was seeded at 2% to the
above culture medium. Setting the culture temperature at 35.degree.
C., the bacterium was cultured for 72 hours while maintaining the
culture at pH 6.0 with an 8 N aqueous solution of potassium
carbonate. Incidentally, the head space of a culture tank was
purged with nitrogen, and stirring was effected at 100 to 150 rpm.
Subsequent to addition of 0.5 w/w % sodium ascorbate, the culture
was sterilized at 121.degree. C. for 15 seconds. Skim milk powder
was added in an amount four times as much as the solid content of
the culture, followed by spray drying. The content of the
thus-dried powder was 0.56 .mu.g/g.
Referential Example 2
Analysis of ACNQ
(1) Culture
[0075] To the culture (0.5 m) with cells contained therein, 1 N
hydrochloric acid (0.05 m), purified water (2 mL) and acetone (2.5
mL) were added, and the resulting mixture was shaken at 200 rev/min
for 60 minutes. Ethyl acetate (2.5 mL) was added, and subsequent to
shaking at 200 rev/min for 30 minutes, centrifugation was conducted
(2,500 rpm, 4.degree. C., 5 minutes). Further, the water layer was
extracted twice with ethyl acetate (7.5 mL). The organic solvent
layers were concentrated, dissolved in methanol (200 .mu.L), and
analyzed by HPLC. The followings are HPLC conditions. [0076]
Column: "CAPCELL PAK C18 SG120" (.phi.4.6.times.250 mm, product of
Shiseido Co., Ltd.) [0077] Column temperature: 45.degree. C. [0078]
Mobile phase: Acetonitrile/methanol/7.94 mmol/L sodium perchlorate
solution/acetic acid (250/100/900/0.6, pH 5.6) [0079] Flow rate:
1.0 mL/min [0080] Detection: Multi-electrode electrochemical
detector ("Coulochem II", manufactured by ESA, Inc.) [0081] Guard
cell: -650 mV [0082] Analytical cells: DET. 1 -600 mV, DET. 2 +150
mV [0083] Sample temperature: Room temperature [0084] Injection
volume: 20 .mu.L (2) Lyophilized Powder
[0085] Methanol (50 mL) was added to a powder (1 g), followed by
shaking for 1 hour. Ethyl acetate (50 mL) was added further, and
the resulting mixture was stirred for 30 minutes. The suspension
was filtered. The filtrate was concentrated, dissolved in methanol
(5 mL), and analyzed by HPLC.
Test 1 [Effects of BGS on Bone Mass and Bone Strength (in Vitro
Tests)]
[0086] Male SD rats were allowed to ingest respective refined feeds
(prepared according to the AIN-93G composition)--which contained
the BGS powder of Referential Example 1 at 2% and 10%,
respectively--ad libitum for 4 weeks. Upon preparation of the test
feeds, equalization was effected between the test feeds in view of
proteins, lactose, calcium, phosphorus and magnesium originated
from the BGS powder. A control feed was prepared according to the
AIN-93G composition which contained casein at 20%.
[0087] As a result, no difference was recognized in post-raising
body weight gain or feed efficiency among the three groups. No
difference was recognized either in femoral or tibial dry weights
among the three groups. Concerning bone density, the group fed with
10% BGS powder showed a significantly higher value than the control
group, and the group fed with 2% BGS powder exhibited a tendency of
a higher value although the tendency was not significant compared
with the control group (FIG. 1). As for the breaking energy of
femur, the group fed with 10% BGS powder showed a significantly
higher value than the control and the group fed with 2% BGS powder,
and the group fed with 2% BGS powder exhibited a tendency of a
higher value although the tendency was not significant compared
with the control group (FIG. 2). With regard to femoral calcium
level, on the other hand, the group fed with 10% BGS powder showed
a significantly higher value than the control group, and the group
fed with 2% BGS powder exhibited a tendency of a higher value
although the tendency was not significant compared with the control
group (FIG. 3).
[0088] From the results of those in vivo tests, it has been found
that oral ingestion of BGS can increase bone mineral density, can
enhance breaking strength and can increase the accumulation of
calcium. In other words, BGS has been found to have effects that,
when it is orally ingested, osteogenesis is promoted to increase
bone mass and bone strength.
Test 2 [Osteogenesis Promoting Effect of ACNQ]
[0089] Cells of the stromal cell line ST-2 derived from mouse bone
marrow (Udagawa, N. et al.: Endocrinology 125, 1805-1813, 1989)
were used as osteoblasts. These cells have similar osteoclast
differentiation inducing ability for bone marrow cells as
osteoblasts.
[0090] The cells were seeded in the individual wells of a 96-well
plate at 4.times.10.sup.4 cells/well, and cultured for 1 day in
RPMI 1640 medium which contained 10% FBS, penicillin G and
streptomycin.
[0091] The medium was replaced by the same medium which contained
ACNQ, and cultured further for 3 days. After the culture, the ALP
activity of cells was assayed.
[0092] The cells were washed with PBS, and then immobilized with
ethanol for 1 minute. An enzyme reaction solution (a buffer
containing p-nitrophenyl phosphate, 0.1 M glycine, 1 mM MgCl.sub.2
and 0.1 mM ZnCl.sub.2 as substrates, pH 10.5) of 37.degree. C. was
added to have the cells reacted at 37.degree. C. for 30 minutes. An
equiamount of 0.05 N NaOH was added to quench the reaction. ALP
activity was expressed in terms of units, with 1 unit representing
the amount of p-nitrophenol formed in a minute per mg-protein as
measured in terms of absorbance at 415 nm) Protein contents were
determined using a protein assay kit (product of Bio-Rad Lab.).
Further, the cytotoxicity of ACNQ was measured by MTT assay (FIG.
5).
[0093] The ALP activity of the cells increased
concentration-dependently up to an ACNQ concentration of 1
.mu.g/mL, and the ALP activity reached the peak around the 1
.mu.g/mL concentration (FIG. 4). At that peak concentration of ALP
activity, cytotoxicity was not observed at all (FIG. 5).
Test 3 [Mineralizability Promoting Effect of ACNQ]
[0094] Employed were cultured human osteoblasts (SaM-1), which had
been established from the periosteum of an ulna of a 20-year-old
male obtained during a surgical operation of a fractured bone.
Those SaM-1 cells were equipped with all the characteristics of
osteoblasts (Koshihara, Y. et al.: In Vitro Cell, Dev. Biol., 25,
37-43, 1989). SaM-1 cells are known to promote mineralization in
the presence of 2 mM .alpha.-glycerophosphoric acid, depending upon
the concentration of 1.alpha.,25(OH).sub.2D.sub.3.
[0095] SaM-1 cells of 18 PDL (population doubling level) were
seeded to a 12-well plate, and cultured to a confluent state.
.alpha.-Glycerophosphoric acid, a mineralization promoter, was then
added to 2 mM. To the culture systems, 10.sup.-8 M to 10.sup.-9 M
1.alpha.,25(OH).sub.2D.sub.3, 10.sup.-5 M to 10.sup.-6 M vitamin
K.sub.2 (2-methyl-3-all-trans-teraphenyl-1,4-naphthoquinone;
menaquinone), 10.sup.-5 M to 10.sup.-7 M ACNQ, and 10.sup.-9 M
1.alpha.,25(OH).sub.2D.sub.3+10.sup.-5 M to 10.sup.-6 M ACNQ were
added, respectively, followed by culturing for 32 days. To the
control, the solvent, DMSO, was added such that its content reached
0.1% based on the culture. The cultures were replaced on every
second day by aliquots of the medium, said aliquots containing the
corresponding test substances, respectively. Degrees of
mineralization were each expressed in terms of the amounts of Ca
and P as constituents of hydroxyapatite.
(1) Quantitation of Calcium in Extracellular Matrices
[0096] Ca in each extracellular matrix was quantitated by using a
kit ("Calcium C Test Wako") based on the o-cresolphthalein
complexone method (OCPC method).
[0097] After completion of the culture, the cells were washed with
Hanks' solution. Chilled 5% perchloric acid was added at 0.5
mL/well, followed by extraction under shaking at 4.degree. C. for
15 minutes. Extracts (25 .mu.L) were each mixed with a buffer (2.5
mL). A staining solution (250 .mu.L) which contained OCPC (0.4
mg/mL) and 8-quinolinol was added, followed by stirring. Five
minutes later, the reaction mixtures were measured by an
absorptiometer (570 nm)(FIG. 6).
(2) Quantitation of Phosphorus in Extracellular Matrices
[0098] P in each extracellular matrix was quantitated by the method
proposed by Chen et al. (Chen, P. S. et al.: Anal. Chem., 28,
1756-1758, 1956).
[0099] 0.84% Ammonium molybdate, 2 N sulfuric acid and 10% ascorbic
acid were mixed at 3:3:1 (volume ratio) to prepare a reaction
reagent. The extracts (100 .mu.L) obtained in the above-described
Ca quantitation were each diluted with distilled water (800 .mu.L).
To each of the dilutions, the reaction reagent (2.1 mL) was added,
followed by incubation at 45.degree. C. for 20 minutes. Subsequent
to cooling with water, the reaction solutions were measured by the
absorptiometer (570 nm)(FIG. 7).
[0100] From FIG. 6 and FIG. 7, 1.alpha.,25(OH).sub.2D.sub.3 has
been ascertained to promote mineralization
concentration-dependently at 10.sup.-8 M to 10.sup.-9 m
concentrations. ACNQ, on the other hand, has been confirmed to
promote mineralization concentration-dependently at 10.sup.-5 M to
10.sup.-7 M concentrations.
(3) Cytotoxicity of ACNQ
[0101] Using "LDH-Cytotoxic Test Wako" (cytotoxicity assay kit),
free LDH in each culture supernatant was measured to investigate
the cytotoxicity of ACNQ. Culture supernatants on the 32.sup.nd day
of culture were sampled, and subsequent to a staining reaction,
their LDH concentrations were measured by using a microplate reader
(540 nm).
[0102] As a result, ACNQ was not recognized to have any significant
difference compared with the control at 10.sup.-5 M to 10.sup.-7 M
concentrations, and was not determined to show cytotoxicity at
these concentrations (FIG. 8).
Test 4 [Osteoclast Formation Inhibiting Effect of ACNQ]
[0103] (1) Human bone marrow cells obtained during an artificial
caput replacement operation were subjected to density-gradient
centrifugation to collect monocytes. Those monocytes were seeded
into .alpha.-MEM medium, which contained 20% equine serum and
10.sup.-8M 1.alpha.,25(OH).sub.2D.sub.3, to give 8.times.10.sup.5
cells/well (079 cm.sup.2), and were then cultured for 17 hours. The
cultures were each replaced twice a week by a half of its volume
each time. Addition of ACNQ was conducted at the beginning.
[0104] On the 16.sup.th day of culture, the cultures were treated
with pronase and EDTA to eliminate stromal cells. On the following
day, the cells were washed with PBS(-) and then immobilized with
formalin. The formalin-immobilized cells were subjected to TRAP
staining, and the TRAP-positive polykaryocyte counts of the
respective wells were determined. Vitamin K.sub.1 was used as a
control.
[0105] ACNQ inhibited the formation of osteoclasts
concentration-dependently at 10.sup.-8 M to 10.sup.-5 M
concentrations (FIG. 9).
[0106] (2) The calvaria of ddY mice newborn were enzymatically
treated (with a mixed solution of collagenase and disperse) to
obtain primary osteoblast-like interstitial cells. On the other
hand, bone marrow cells were isolated from the femurs and shinbones
of the same strain mice (males of 6 to 9 weeks of age). The primary
osteoblast-like interstitial cells and bone marrow cells were
co-cultured in the presence of ACNQ to investigate differentiation
induction effect of ACNQ on osteoclasts.
[0107] Using .alpha.MEM added with 10% FBS, a culture which
contained the primary osteoblast-like interstitial cells and the
bone marrow cells at a cell count ratio of about 1:20 was
co-cultured in the presence of 10.sup.-8 M to 10.sup.-10 M
1.alpha.,25(OH).sub.2D.sub.3 for 6 to 8 days. During the culture
period, ACNQ solutions of various concentrations and as a control
for comparison, vitamin K.sub.2 which is known to inhibit the
formation of osteoclasts, were added, respectively, upon culture
medium replacement (replaced by a half of the volume of the culture
medium; replaced on every third days).
[0108] Cells, which were positive to TRAP staining and internally
contained 3 or more nuclei per cell, were collected as
differentiated osteoclasts. The TRAP staining solution was prepared
shortly before its use by dissolving a substrate (naphthol AS-MX
phosphate; 5 mg) and a color additive (Fast red violet LB sat; 25
mg) in N,N-dimethylformamide (about 0.5 mL) and adding to the
resultant solution a 0.1 M sodium acetate buffer (pH 5.0) which
contained 50 mM sodium tartrate. After the culture medium was
removed from the cultured cells, the cells were washed with PBS,
immobilized for 5 minutes with PBS containing 10% formalin, and
then re-immobilized for 1 minute with ethanol-acetone (1:1). After
being dried in air, the cells were reacted with the TRAP staining
solution at room temperature for 10 to 15 minutes. The thus-reacted
cells were washed with water, and then dried. The cells were
observed under a microscope to determine the osteoclast count. ACNQ
inhibited differentiation and maturation of monocytes, which
existed in the bone marrow cells, into osteoclasts
concentration-dependently at concentrations of from 0.15 to 15
.mu.g/mL (FIG. 10).
Test 5 [Effect of ACNQ on Bone Resorption]
[0109] Investigated was the effect of ACNQ on the formation of bone
resorption lacunae on a piece of dentinum by osteoclasts.
[0110] As in the test (2) of Test 4, a culture (20 mL) containing
primary osteoblast-like interstitial cells, which had been derived
from the calvariae of ddY mice newborn, and bone marrow cells,
which had been isolated from the femurs and shinbones of the same
strain mice (males of 6 to 9 weeks of age), at a ratio of
1.times.10.sup.6 cells to 1.times.10.sup.7 cells in a 100 mm dish
was co-cultured for 6 to 8 days on a collagen gel in .alpha.MEM
medium in which 10% FEB and 10.sup.-8M 1.alpha.,25
(OH).sub.2D.sub.3 had been added. Medium replacement was conducted
on every third days. After the culture, a 0.2% collagenase solution
was added. The thus-obtained mixture was gently shaken at
37.degree. C. for 20 minutes to digest the collagen gel, and
differentiation-induced osteoclast-like polykaryocytes were
collected. The count of osteoclasts obtained by this method was at
most 4.times.10.sup.4 cells/100 mm-dish.
[0111] Those osteoclast-like polykaryocytes were prepared to
4.times.10.sup.3 cells/mL with .alpha.MEM medium containing 10%
FBS, seeded at 100 .mu.L/well into wells (96 wells) in which pieces
of dentinum were placed, respectively, and cultured at 37.degree.
C. for 2 hours. After the culture, the individual cultures were
transferred to 24 wells or 48 wells in which a culture medium was
placed at 0.5 to 1.5 mL/well. At the same time, ACNQ solutions of
various concentrations were added to the wells, respectively,
followed by culture at 37.degree. C. for 24 hours. After the
culture, the culture medium was removed, and the pieces of dentinum
were stained for a short time by Mayer's acidic hemalaun staining
solution. After the staining solution was removed, resorption
lacunae formed in the surface of each piece of dentinum as a result
of binging by osteoblasts were counted under a microscope. The
results were indicated as resorption lacunus counts (%) based on
the count of resorption lacunae in the control (ACNQ not added),
which was supposed to be 100%. ACNQ did not lower the bone
resorbing activity of osteoclasts even at the highest
concentration, i.e., 7.5 .mu.g/mL concentration (FIG. 11). This
indicates that ACNQ does not inhibit the bone resorbing activity of
differentiated and matured osteoclasts, and this indication is also
supported by the fact that ACNQ does not exhibit cytotoxicity
against osteoclasts.
INDUSTRIAL APPLICABILITY
[0112] The culture of the propionic acid bacterium and/or lactic
acid bacterium, which is useful in the present invention, can be
added to foods, and ingestion of the foods is expected to prevent
or ameliorate metabolic bone diseases, especially osteoporosis.
Further, 2-amino-3-carboxy-1,4-naphthoquinone and naphthoquinone
derivatives, which are contained in the culture, are useful as
preventives or remedies for metabolic bone diseases.
* * * * *