U.S. patent application number 11/381606 was filed with the patent office on 2006-10-05 for compositions having anti-dental caries function.
This patent application is currently assigned to Ezaki Glico Co., Ltd.. Invention is credited to Hiroshi KAMASAKA, Toshiyuki KIMURA, Takashi KURIKI, Takahisa NISHIMURA, Shigetaka OKADA, Reiichiro SAKAMOTO, Kenji TOO, Nobuo UOTSU.
Application Number | 20060222603 11/381606 |
Document ID | / |
Family ID | 26610385 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060222603 |
Kind Code |
A1 |
KAMASAKA; Hiroshi ; et
al. |
October 5, 2006 |
COMPOSITIONS HAVING ANTI-DENTAL CARIES FUNCTION
Abstract
The present invention relates to dietary compositions and oral
compositions having an anti-dental caries function. The present
invention provides dietary compositions and oral compositions
having an anti-dental caries functions which contain a buffering
agent having a pH buffering action in the oral cavity.
Inventors: |
KAMASAKA; Hiroshi; (Osaka,
JP) ; NISHIMURA; Takahisa; (Nara, JP) ; TOO;
Kenji; (Osaka, JP) ; KURIKI; Takashi; (Osaka,
JP) ; OKADA; Shigetaka; (Nara, JP) ; SAKAMOTO;
Reiichiro; (Ibaraki, JP) ; KIMURA; Toshiyuki;
(Chiba, JP) ; UOTSU; Nobuo; (Chiba, JP) |
Correspondence
Address: |
MARK D. SARALINO (GENERAL);RENNER, OTTO, BOISSELLE & SKLAR, LLP
1621 EUCLID AVENUE, NINETEENTH FLOOR
CLEVELAND
OH
44115-2191
US
|
Assignee: |
Ezaki Glico Co., Ltd.
|
Family ID: |
26610385 |
Appl. No.: |
11/381606 |
Filed: |
May 4, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10469478 |
Dec 10, 2003 |
|
|
|
PCT/JP02/01888 |
Feb 28, 2002 |
|
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11381606 |
May 4, 2006 |
|
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Current U.S.
Class: |
424/50 ; 424/52;
514/54 |
Current CPC
Class: |
A61K 8/365 20130101;
A61Q 11/00 20130101; A61P 43/00 20180101; A61K 8/73 20130101; A61K
8/60 20130101; A61K 31/715 20130101 |
Class at
Publication: |
424/050 ;
514/054; 424/052 |
International
Class: |
A61K 8/96 20060101
A61K008/96; A61K 8/21 20060101 A61K008/21; A61K 31/737 20060101
A61K031/737 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 2001 |
JP |
2001-056010 |
Feb 28, 2001 |
JP |
2001-056011 |
Claims
1.-2. (canceled)
3. A dietary composition having an anti-dental caries function,
wherein the composition comprises a buffering agent having a pH
buffering action in the oral cavity, wherein the buffering agent is
selected from the group consisting of: phosphorylated
oligosaccharides or sugar alcohol thereof, wherein the
phosphorylated oligosaccharides are glucan consisting of 3 to 5
glucoses with .alpha.-1,4 linkages, one phosphate group being
linked to the glucan, or glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages, two phosphate groups being linked to the
glucan; chondroitin sulfate; and chondroitin sulfate
oligosaccharides.
4.-9. (canceled)
10. A dietary composition having an anti-dental caries function,
wherein the composition comprises a buffering agent having a pH
buffering action in the oral cavity, a phosphorus-calcium
compensating agent, a phosphorus preparation, and/or a calcium
preparation, wherein the buffering agent is selected from the group
consisting of: phosphorylated oligosaccharides or sugar alcohol
thereof, wherein the phosphorylated oligosaccharides are glucan
consisting of 3 to 5 glucoses with a-1,4, linkages, one phosphate
group being linked to the glucan, or glucan consisting of 2 to 8
glucoses with .alpha.-1,4 linkages, two phosphate groups being
linked to the glucan; chondroitin sulfate; and chondroitin sulfate
oligosaccharides.
11.-16. (canceled)
17. An oral composition having an anti-dental caries function,
wherein the composition comprises a buffering agent having a pH
buffering action in the oral cavity, wherein the buffering agent is
selected from the group consisting of: phosphorylated
oligosaccharides or sugar alcohol thereof, wherein the
phosphorylated oligosaccharides are glucan consisting of 3 to 5
glucoses with .alpha.-1,4 linkages, one phosphate group being
linked to the glucan, or glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages, two phosphate groups being linked to the
glucan; chondroitin sulfate; and chondroitin sulfate
oligosaccharides.
18.-23. (canceled)
24. An oral composition having an anti-dental caries function,
wherein the composition comprises a buffering agent having a pH
buffering action in the oral cavity, a phosphorus-calcium
compensating agent, a phosphorus preparation, and/or a calcium
preparation, wherein the buffering agent is selected from the group
consisting of: phosphorylated oligosaccharides or sugar alcohol
thereof, wherein the phosphorylated oligosaccharides are glucan
consisting of 3 to 5 glucoses with .alpha.-1,4 linkages, one
phosphate group being linked to the glucan, or glucan consisting of
2 to 8 glucoses with .alpha.-1,4 linkages, two phosphate groups
being linked to the glucan; chondroitin sulfate; and chondroitin
sulfate oligosaccharides.
25.-30. (canceled)
31. A dietary composition according to claim 3, wherein the
buffering agent is in the form of an alkaline metal salt, an
alkaline earth metal salt, or an iron salt.
32. A dietary composition according to claim 31, wherein the
buffering agent is in the form of a sodium salt or a calcium
salt.
33. A dietary composition according to claim 3, further comprising
an effective amount of fluorine or a fluorine containing substance
for anti-dental caries.
34. A dietary composition according to claim 10, wherein the
buffering agent is in the form of an alkaline metal salt, an
alkaline earth metal salt, or an iron salt.
35. A dietary composition according to claim 34, wherein the
buffering agent is in the form of a sodium salt or a calcium
salt.
36. A dietary composition according to claim 10, further comprising
an effective amount of fluorine or a fluorine containing substance
for anti-dental caries.
37. An oral composition according to claim 17, wherein the
buffering agent is in the form of an alkaline metal salt, an
alkaline earth metal salt, a zinc salt or an iron salt.
38. An oral composition according to claim 37, wherein the
buffering agent is in the form of a sodium salt, a calcium salt, or
a zinc salt.
39. An oral composition according to claim 17, further comprising
an effective amount of fluorine or a fluorine containing substance
for anti-dental caries.
40. An oral composition according to claim 24, wherein the
buffering agent is in the form of an alkaline metal salt, an
alkaline earth metal salt, a zinc salt, or an iron salt.
41. An oral composition according to claim 40, wherein the
buffering agent is in the form of a sodium salt, a calcium salt, or
a zinc salt.
42. An oral composition according to claim 24, further comprising
an effective amount of fluorine or a fluorine containing substance
for anti-dental caries.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/469,478 filed on Aug. 27, 2003 which is hereby
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates to dietary compositions and
oral compositions having an anti-dental caries function. More
particularly, the present invention relates to dietary compositions
and oral compositions having an anti-dental caries function, such
as remineralization of teeth to reduce the development of dental
caries.
BACKGROUND ART
[0003] Dental caries is a demineralization of the tooth surface
caused by oral bacteria present thereon. Specifically, organic acid
produced by the oral bacteria is prevented from being diffused by
some obstructions and the teeth are exposed to a high concentration
of the organic acid, so that the tooth surface is demineralized. In
this definition, any oral bacteria having the ability to ferment
sugar to produce organic acid by metabolism can cause dental
caries. Substrates suitable for organic acid production are
saccharides, including monosaccharides and oligosaccharides (e.g.,
glucose and sucrose), and polysaccharides (e.g., starch) which are
polymers of monosaccharides.
[0004] The dispersion of organic acid is prevented roughly due to
(1) retention of starch taken from diet at the neck and root of
tooth, and (2) adhesion of insoluble glucan to tooth, which is
produced by bacteria using easily degradable sugars, such as
sucrose (i.e., fermentative sugars) as substrates.
[0005] As to factor (1), any oral bacteria having the ability to
ferment sugar, such as lactobacillus, is considered to be
responsible for dental caries. In this case, it is known that the
progression of dental caries is generally slow. The development of
an environment in which a high concentration of organic acid is
produced depends on passive factors.
[0006] Factor (2) is a major factor for dental caries today when
sucrose-containing foods are widely available. In this case,
Streptococcus mutans and Streptococcus sobrinus are believed to be
causative. Both bacteria are a type of Streptococcus which occurs
in chains, each cell having a diameter of about 0.6 .mu.m in a
round form. Both bacteria vigorously produce water-insoluble
.alpha.-glucan in the presence of sucrose. This glucan has a
property to adhere very well to the tooth surface. The bacteria
rapidly metabolize sucrose, exerting the ability to produce acid.
The bacteria per se have strong acid resistance and can survive in
an acid environment in which other bacteria cannot grow. The
adhesiveness of the water-insoluble glucan allows the bacteria to
be firmly adhered to the tooth surface and the like. The
water-insoluble glucan adsorbed to the tooth surface prevents the
dispersion of organic acid produced by the bacteria, resulting in
an environment in which the tooth surface is exposed to a high
concentration of organic acid. It is considered that as compared to
factor (1), the creation of the environment in which a high
concentration of organic acid is produced depends on an active
factor of the bacteria. In this case, the progression of dental
caries is faster than that caused by factor (1).
[0007] There is a new approach to prevent dental caries by
considering the health of teeth at microscopic levels, i.e.,
demineralization and remineralization of dentin (Yoichi Iijima,
Takashi Kumagaya; Kariesu Kontororu Dakkai-to
Saisekkaika-no-Mekanizumu [Caries Control--Mechanism of
Demineralization and Remineralization], Ishiyaku Shuppan K.K.;
21-51, 1999). The surface of a tooth is made of calcium and
hydroxyapatite [Ca.sub.10(PO.sub.4).sub.6(OH).sub.2] which is a
crystal of phosphate, and is called enamel. Enamel is the hardest
part of a tooth, and prevents important calcium or phosphate from
being dissolved from underneath the enamel (demineralization) due
to organic acid produced by bacteria in dental plaque, acid
contained in foods, etc.
[0008] The organic acid permeates enamel through gaps between
enamel rods which are filled with water, and dissolves
hydroxyapatite by a process called demineralization. A loss of
calcium and phosphate from enamel tissues leads to the development
of initial dental caries under the surface layer of enamel. As
described below, according to the present invention, dental caries
in the above-described stage can be repaired. Calcium and phosphate
ions permeate the dental caries portion under the enamel surface
and lost apatite can be restored by a process called
remineralization.
[0009] Each time that the diet containing fermentative
carbohydrates is taken in, the pH of plaque becomes acidic and
exceeds a critical pH at which demineralization begins. This
results from the action of acid-producing bacteria in the plaque.
When the plaque is buffered by saliva, the pH of the plaque is
returned to neutral, and calcium and phosphate ions in saliva are
reincorporated into dentin through the plaque (this process is
called remineralization).
[0010] Therefore, means for preventing and treating tooth decay
should not be a nutrient for oral bacteria which cause dental
caries to allow the bacteria to produce organic acid; should not be
a nutrient for mutans bacteria which cause dental caries to allow
the bacteria to produce water-insoluble glucan and organic acid;
should prevent pH reduction due to the organic acid from going
below the pH at which demineralization begins (e.g., should have a
buffering ability so as to prevent the pH reduction); should
promote remineralization; and the like.
[0011] To date various anti-dental caries agents have been
known.
[0012] Dental caries begin when mutans bacteria produce
water-insoluble glucan using sucrose as a nutrient and
glycosyltransferase as an enzyme. This glucan covers the tooth
surface, resulting in dental plaque. When the mutans bacteria
undergo acid fermentation within the dental plaque, the teeth is
dissolved away and tooth decay is formed.
[0013] As anti-dental caries saccharides, some oligosaccharides
which are not a nutrient for mutans bacteria have already been
proposed (S. Hamada et al., J. Jpn. Soc. Starch Sci., Vol. 31, pp.
83-91, 1984). One example of these anti-dental caries saccharides
is palatinit (Japanese Laid-Open Publication No. 2000-281550). When
palatinit is combined with fluorine or zinc, the remineralization
of teeth is promoted (Japanese Laid-Open Publication No.
2000-247852). However, palatinit has poor sweetness and is not
preferable for foods. Further, a concentration of as high as about
1 to 20 wt % is required for the remineralization effect of
palatinit.
[0014] Sugar alcohol (particularly, xylitol) is also known as an
anti-dental caries agent (e.g., Japanese Laid-Open Publication No.
2000-128752 and Japanese Laid-Open Publication No. 2000-53549).
Japanese Laid-Open Publication No. 11-12143 discloses an oral
composition comprising one or more sugar alcohols selected from
xylitol, mannitol, galactitol, and inositol. Japanese Laid-Open
Publication No. 11-12143 describes that these sugar alcohols can
promote the remineralization of teeth, but do not inhibit the
growth of the bacteria. Although sugar alcohol is effective only at
high concentrations, it is known that the intake of the sugar
alcohol in a large amount causes loose stool. As described in the
Examples below, the effect of xylitol was not substantially
confirmed.
[0015] Further, polyphenol which is a component of tea has been
reported and utilized as an anti-dental caries agent (S. Sakanaka
et al., Fragrance Journal, Vol. 11, pp. 42-49, 1990). However, use
of polyphenol also causes a problem with taste and is therefore
limited.
[0016] At present, fluorine is said to be most effective for the
remineralization effect. Fluorine can exert sufficient efficacy at
about 2 ppm. In regard to the efficacy of fluorine, the following
two points have been clarified: (1) promotion of remineralization;
and (2) fluorine is incorporated into a hydroxyapatite crystal
which is in turn converted to a hard crystal structure which resist
demineralization (fluorine is used in expectation of effect (2)
rather than (1)). Fluorine having such properties has been recently
added to various oral compositions. For example, Japanese Laid-Open
Publication No. 11-130643 discloses an oral composition containing
calcium carbonate and a soluble fluoride compound. It is known that
a combination of fluoride ions with sugar alcohol enhances the
ability of fluorine to remineralize teeth (For example, Japanese
Laid-Open Publication No. 11-21217, Japanese Laid-Open Publication
No. 2000-72638, and Japanese Laid-Open Publication No.
2000-154127). Japanese Laid-Open Publication No. 8-12541 discloses
a composition containing mutanase and a fluoride compound, which
enhances dentin and promotes remineralization to effectively
prevent dental caries.
[0017] It is known in the art that supply of calcium phosphate
promotes the remineralization of teeth (e.g., Japanese Laid-Open
Publication No. 11-228369 and Japanese Laid-Open Publication No.
10-310513).
[0018] Japanese Laid-Open Publication No. 11-29454 discloses an
oral composition containing calcium carbonate and alginate. This
composition enhances the ability of calcium carbonate to adhere and
remain on teeth so that satisfactory naturalization of pH and
promotion of remineralization are obtained, resulting in an
excellent dental caries-preventing effect.
[0019] Japanese Laid-Open Publication No. 8-104696 describes that
phosphorylated oligosaccharides disclosed therein suppress calcium
and phosporus from being deposited and crystallized (i.e.,
calcification), that the phosphorylated oligosaccharides are not a
nutrient for mutans bacteria which cause dental caries so that
water-insoluble glucan is not produced, and that the phosphorylated
oligosaccharides have a buffering ability and have the effect of
preventing pH reduction. The above-described properties prevent the
development of dental calculus and dental plaque, and the acid
fermentation by mutans bacteria. It is also disclosed that
phosphorylated oligosaccharides contained in a dietary composition
or an oral composition have the effect of preventing pH reduction
due to lactic acid, which is a product of fermentation within
dental plaque, without an influence on flavor. However, Japanese
Laid-Open Publication No. 8-104696 does not suggest that the
above-described phosphorylated oligosaccharides can have the
remineralization effect at a low concentration as described
herein.
DISCLOSURE OF THE INVENTION
[0020] Therefore, the present invention relates to materials having
an anti-dental caries function. Particularly, the objective of the
present invention is to provide dietary compositions and oral
compositions which reduce the development of dental caries by the
remineralization of teeth or the like.
[0021] The inventors have rigorously studied a technique for
preventing dental caries by using various substances. As a result,
the inventors found a buffering agent having a remineralization
effect on teeth, and completed the present invention.
[0022] According to one aspect of the present invention, a dietary
composition has an anti-dental caries function. The composition
comprises a buffering agent having a pH buffering action in the
oral cavity.
[0023] In one embodiment of this invention, the buffering agent is
a phosphorylated oligosaccharide or sugar alcohol thereof.
[0024] In one embodiment of this invention, the buffering agent is
selected from the group consisting of: phosphorylated
oligosaccharides or sugar alcohol thereof, in which the
phosphorylated oligosaccharides are glucan consisting of 3 to 5
glucoses with .alpha.-1,4 linkages, one phosphate group being
linked to the glucan, or glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages, two phosphate groups being linked to the
glucan; chondroitin sulfate; chondroitin sulfate oligosaccharides;
glucose-6-phosphate; oligogalacturonic acid; and tartaric acid.
[0025] In one embodiment of this invention, the buffering agent is
a phosphorylated oligosaccharide or sugar alcohol thereof. The
phosphorylated oligosaccharides are glucan consisting of 3 to 5
glucoses with .alpha.-1,4 linkages, one phosphate group being
linked to the glucan, or glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages, two phosphate groups being linked to the
glucan.
[0026] In one embodiment of this invention, the buffering agent is
in the form of an alkaline metal salt, an alkaline earth metal
salt, or an iron salt.
[0027] In one embodiment of this invention, the buffering agent is
in the form of a sodium salt or a calcium salt.
[0028] In one embodiment of this invention, the dietary composition
further comprises an effective amount of fluorine or a fluorine
containing substance for anti-dental caries.
[0029] According to another aspect of the present invention, a
dietary composition has an anti-dental caries function. The
composition comprises a buffering agent having a pH buffering
action in the oral cavity, a phosphorus-calcium compensating agent,
a phosphorus preparation, and/or a calcium preparation.
[0030] In one embodiment of this invention, the buffering agent is
a phosphorylated oligosaccharide or sugar alcohol thereof.
[0031] In one embodiment of this invention, the buffering agent is
selected from the group consisting of: phosphorylated
oligosaccharides or sugar alcohol thereof, in which the
phosphorylated oligosaccharides are glucan consisting of 3 to 5
glucoses with .alpha.-1,4 linkages, one phosphate group being
linked to the glucan, or glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages, two phosphate groups being linked to the
glucan; chondroitin sulfate; chondroitin sulfate oligosaccharides;
glucose-6-phosphate; oligogalacturonic acid; and tartaric acid.
[0032] In one embodiment of this invention, the buffering agent is
a phosphorylated oligosaccharide or sugar alcohol thereof. The
phosphorylated oligosaccharides are glucan consisting of 3 to 5
glucoses with .alpha.-1,4 linkages, one phosphate group being
linked to the glucan, or glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages, two phosphate groups being linked to the
glucan.
[0033] In one embodiment of this invention, the buffering agent is
in the form of an alkaline metal salt, an alkaline earth metal
salt, or an iron salt.
[0034] In one embodiment of this invention, the buffering agent is
in the form of a sodium salt or a calcium salt.
[0035] In one embodiment of this invention, the dietary composition
further comprises an effective amount of fluorine or a fluorine
containing substance for anti-dental caries.
[0036] According to another aspect of the present invention, an
oral composition has an anti-dental caries function. The
composition comprises a buffering agent having a pH buffering
action in the oral cavity.
[0037] In one embodiment of this invention, the buffering agent is
a phosphorylated oligosaccharide or sugar alcohol thereof.
[0038] In one embodiment of this invention, the buffering agent is
selected from the group consisting of: phosphorylated
oligosaccharides or sugar alcohol thereof, in which the
phosphorylated oligosaccharides are glucan consisting of 3 to 5
glucoses with .alpha.-1,4 linkages, one phosphate group being
linked to the glucan, or glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages, two phosphate groups being linked to the
glucan; chondroitin sulfate; chondroitin sulfate oligosaccharides;
glucose-6-phosphate; oligogalacturonic acid; and tartaric acid.
[0039] In one embodiment of this invention, the buffering agent is
a phosphorylated oligosaccharide or sugar alcohol thereof. The
phosphorylated oligosaccharides are glucan consisting of 3 to 5
glucoses with .alpha.-1,4 linkages, one phosphate group being
linked to the glucan, or glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages, two phosphate groups being linked to the
glucan.
[0040] In one embodiment of this invention, the buffering agent is
in the form of an alkaline metal salt, an alkaline earth metal
salt, a zinc salt, or an iron salt.
[0041] In one embodiment of this invention, the buffering agent is
in the form of a sodium salt, a calcium salt, or a zinc salt.
[0042] In one embodiment of this invention, the oral composition
further comprises an effective amount of fluorine or a fluorine
containing substance for anti-dental caries.
[0043] According to another aspect of the present invention, an
oral composition has an anti-dental caries function. The
composition comprises a buffering agent having a pH buffering
action in the oral cavity, a phosphorus-calcium compensating agent,
a phosphorus preparation, and/or a calcium preparation.
[0044] In one embodiment of this invention, the buffering agent is
a phosphorylated oligosaccharide or sugar alcohol thereof.
[0045] In one embodiment of this invention, the buffering agent is
selected from the group consisting of: phosphorylated
oligosaccharides or sugar alcohol thereof, in which the
phosphorylated oligosaccharides are glucan consisting of 3 to 5
glucoses with .alpha.-1,4 linkages, one phosphate group being
linked to the glucan, or glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages, two phosphate groups being linked to the
glucan; chondroitin sulfate; chondroitin sulfate oligosaccharides;
glucose-6-phosphate; oligogalacturonic acid; and tartaric acid.
[0046] In one embodiment of this invention, the buffering agent is
a phosphorylated oligosaccharide or sugar alcohol thereof. The
phosphorylated oligosaccharides are glucan consisting of 3 to 5
glucoses with .alpha.-1,4 linkages, one phosphate group being
linked to the glucan, or glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages, two phosphate groups being linked to the
glucan.
[0047] In one embodiment of this invention, the buffering agent is
in the form of an alkaline metal salt, an alkaline earth metal
salt, a zinc salt, or an iron salt.
[0048] In one embodiment of this invention, the buffering agent is
in the form of a sodium salt, a calcium salt, or a zinc salt.
[0049] In one embodiment of this invention, the oral composition
further comprises an effective amount of fluorine or a fluorine
containing substance for anti-dental caries.
[0050] According to another aspect of the present invention, a
method for investigating a remineralization effect of a sample
expected to have an anti-dental caries action on a tooth, comprises
the steps of: (A) subjecting a solution containing phosphorus,
calcium, and tooth components in the presence of the sample to a
calcium precipitation reaction; (B) measuring the concentration of
calcium in the solution or the amount of precipitated calcium after
the precipitation reaction; (C) subjecting the solution in the
absence of the sample to a calcium precipitation reaction; (D)
measuring the concentration of calcium in the solution or the
amount of precipitated calcium after the precipitation reaction;
and (E) comparing the concentration of calcium in the solution or
the amount of precipitated calcium in the steps (B) and (D).
[0051] In one embodiment of this invention, the solution comprises
hydroxyapatite, buffer solution, KH.sub.2PO.sub.4 and
CaCl.sub.2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1 is a graph showing mineral loss values due to dental
caries in a remineralization test system employing bovine teeth
sections.
[0053] FIG. 2 is a graph showing lesion depth in a remineralization
test system employing bovine teeth sections.
[0054] FIG. 3 is a graph showing the results of remineralization in
a simple test system of Example 4 employing a phosphorylated
oligosaccharide sodium salt.
[0055] FIG. 4 is a graph showing the results of remineralization in
a simple test system of Example 4 employing a phosphorylated
oligosaccharide calcium salt.
[0056] FIG. 5 is a graph showing the effect of phosphorylated
oligosaccharides on remineralization where P/Ca is 0.6.
[0057] FIG. 6A is a graph showing the effect of changes in P/Ca
concentration ratio on remineralization in the absence of
phosphorylated oligosaccharides. FIG. 6B is a graph showing the
influence of changes in P/Ca concentration ratio on
remineralization in the presence of a phosphorylated
oligosaccharide sodium salt. FIG. 6C is a graph showing the
influence of changes in P/Ca concentration ratio on
remineralization in the presence of a phosphorylated
oligosaccharide calcium salt.
[0058] FIG. 7A is a graph showing the remineralization effects of a
phosphorylated oligosaccharide calcium salt and a phosphorylated
oligosaccharide sodium salt in Example 5. FIG. 7B is a graph
showing the remineralization effects of xylitol and xylose. FIG. 7C
is a graph showing the remineralization effects of palatinit and
palatinose.
[0059] FIG. 8 is a photograph showing the results of TLC analysis
in Example 7.
[0060] FIG. 9 is a graph showing the synergistic action of
phosphorylated oligosaccharides and fluorine on remineralization in
Example 7.
[0061] FIG. 10 is a photograph showing the results of TLC analysis
of phosphorylated oligosaccharides having a standard solution
concentration in Example 8.
[0062] FIG. 11 is a photograph showing the results of TLC analysis
indicating the amount of elution over time when eating a
phosphorylated oligosaccharide containing gum in Example 8.
[0063] FIG. 12 is a graph showing the remineralization effect of
various substances in Example 12.
[0064] FIG. 13 is a graph showing the remineralization effect of
various substances in Example 13.
[0065] FIG. 14 is a graph showing pH changes in an artificial oral
device in Example 14.
[0066] FIG. 15 is a graph showing the amount of saliva when
masticating a POs Ca containing gum or a POs Ca-free gum in Example
16.
[0067] FIG. 16 is a graph showing the pH of saliva when masticating
a POs Ca containing gum or a POs Ca-free gum in Example 16.
[0068] FIG. 17 is a graph showing the P content of saliva when
masticating a POs Ca containing gum or a POs Ca-free gum in Example
16.
[0069] FIG. 18 is a graph showing the Ca content of saliva when
masticating a POs Ca containing gum or a POs Ca-free gum in Example
16.
[0070] FIG. 19 is a graph showing changes in Ca/P ratio when
masticating a POs Ca containing gum or a POs Ca-free gum in Example
16.
[0071] FIG. 20A is a graph showing lesion depth in each treated
tooth in Example 16. FIG. 20B is a graph showing a mineral loss
value in each treated tooth in Example 16.
[0072] FIG. 21 is a graph showing a remineralization rate in
Example 17.
[0073] FIG. 22 is a graph showing the pH of saliva secreted when
eating a POs Ca containing candy in Example 18.
[0074] FIG. 23 is a graph showing the amount of saliva secreted
when eating a POs Ca containing candy in Example 18.
[0075] FIG. 24 is a graph showing the Ca and P contents of saliva
secreted when eating a POs Ca containing candy in Example 18.
[0076] FIG. 25 is a graph showing the results of a remineralization
test employing POs Ca containing candies and POs Ca containing soft
candies.
[0077] FIG. 26 is a diagram showing the chemical structural
formulas of representative phosphorylated oligosaccharides.
BEST MODE FOR CARRYING OUT THE INVENTION
[0078] Hereinafter, the present invention will be described in more
detail.
[0079] The term "anti-caries function as used herein refers to both
functions of preventing dental caries and treating dental caries.
The function of treating dental caries means a function of
repairing a portion of a tooth which is once lost due to dental
caries. The term anti-dental caries function" as used herein refers
to one or more of the following properties: (1) a pH buffering
ability to prevent pH reduction due to acids produced by oral
bacteria; (2) an ability to prevent oral bacteria from producing
insoluble glucan; and (3) an ability to promote remineralization of
teeth in early dental caries. Preferably, the anti-caries function
has two of the above-described properties, and most preferably all
of the above-described properties.
[0080] The composition of the present invention can stably provide
phosphate and calcium to decayed teeth. The teeth supplied with
phosphate and calcium are remineralized, so that a portion of a
tooth lost due to dental caries can be repaired.
[0081] According to the present invention, a buffering agent is
added to the oral cavity, so that phosphate and calcium present in
saliva or the like in the oral cavity are stably used in the
remineralization of teeth. Therefore, the repair of teeth which are
conventionally considered to be difficult or impossible can be
realized.
[0082] A demineralized lesion can be repaired into a sound state,
if calcium or phosphate is supplied to a demineralized enamel
portion (remineralization) under appropriate conditions. To
maintain the sound state of teeth, minerals need to be supplied to
a demineralized lesion by saliva so that demineralization and
remineralization are balanced at microscopiclevels. Generally, the
pH in dental plaque tends to be lowered after eating or drinking,
and the balance between demineralization and remineralization is
altered. When demineralization>remineralization, the lesion
proceeds. Conversely, when remineralization>demineralization,
the demineralized lesion is restored due to the remineralization of
the tooth. The balance between demineralization and
remineralization depends largely on oral environments
(particularly, the pHs in saliva and dental plaque, and
concentrations of calcium and phosphate). The present invention can
provide an oral environment in which remineralization is likely to
occur, thereby preventing dental caries and treating demineralized
lesions (the early stage of dental caries) to obtain healthy and
robust teeth.
[0083] The term "buffering agent" as used herein refers to an agent
which exhibits a pH buffering action in the oral cavity.
Specifically, the buffering agent is a water-soluble salt obtained
from the anion or cation of the buffering agent, for example. The
presence of the buffering agent in the oral cavity can stabilize
the pH in the oral cavity. The buffering agent stabilizes phosphate
ions and calcium ions in saliva. Therefore, particularly, an agent
which has a good pH buffering action in the presence of phosphate
ions and calcium ions is preferable. More preferably, when the
buffering agent is added to an aqueous solution containing
phosphate ions and calcium ions, the stability of the phosphate
ions and calcium ions is not inhibited by the buffering agent. In
other words, a buffering agent which is likely to react with
phosphate ions and calcium ions and form precipitates is not
preferable.
[0084] Further, in the present invention, the pH buffering effect
is preferably obtained in dental plaque. If the pH buffering action
is exhibited in saliva, the pH buffering action is typically
exhibited in dental plaque. Therefore, a buffering agent which
exhibits the pH buffering action in saliva can be used to exhibit
the pH buffering action in dental plaque. A hydrogen ion sensitive
field effect transistor electrode (PH-6010: manufactured by Nihon
Kohden Corporation) may be placed on an enamel section and
incorporated into a tooth gap portion of a partial denture for the
lower jaw. Thereafter, the pH in dental plaque formed on the
sensitive portion of the electrode may be measured in accordance
with a method described in Yoshizumi Tamasawa et al. (Journal of
the Japan Prosthodontic Society, Vol. 40 special issue, P147,
1996), Kazuhiko Abe (DENTAL OUTLOOK, 90(3), 650-654, 1997),
Takahashi-Abbe, S et al (Oral Microbiol. Immunol., 16, P94-99,
2001). The pH in dental plaque is preferably 6 or more, more
preferably 7 or more. When the pH of plaque is caused by the
buffering action to return to neutral, phosphate ions and calcium
ions present in saliva in the oral cavity are supplied to the
surface of teeth, resulting in remineralization of dentin. The
upper limit of the pH of plaque is not particularly limited, but a
high alkaline condition is not intended for an actual organism. The
pH of plaque is preferably 10 or less, more preferably 8 or
less.
[0085] The buffering agent is typically used in the form of a salt,
and may be optionally used in the form of a free acid. Even if the
buffering agent is provided in the oral cavity in the form of a
free acid, since an alkaline metal and the like which can form a
salt together with a free acid are present in the oral cavity, it
can be substantially said that a salt of the free acid is provided
to the oral cavity.
[0086] A preferable buffering agent which can be used in the
present invention can be easily selected by a simple experiment.
Specifically, various known pH buffering agents are added to a
neutral aqueous solution (e.g., an aqueous solution of pH 6-8)
containing phosphate ions and calcium ions. The presence or absence
of precipitation is observed. A pH buffering agent which does not
form precipitate in such an experiment can be satisfactorily used
as the buffering agent that is added to the anti-caries composition
of the present invention.
[0087] When a buffering agent is not present, the oral cavity may
be acidified due to the effect of organic acids produced by oral
bacteria (i.e., saliva or dental plaque becomes acidic). When
saliva or dental plaque is acidified, calcium and phosphor of teeth
are eluted as Ca and P ions, resulting in the development of dental
caries. In this case, if a buffering agent is present, the pH of
saliva and dental plaque in the oral cavity becomes stable around
neutral pH, whereby formation of dental caries is unlikely to
proceed.
[0088] The pH of saliva is generally around neutral. Therefore, a
buffering agent which has a good buffering action at pH around
neutral is preferable.
[0089] Preferably, the buffering agent is an agent which does not
react with phosphate in saliva to form precipitate.
[0090] Preferably, the buffering agent is an agent which does not
react with calcium in saliva to form precipitate.
[0091] Preferably, the buffering agent has an acidic functional
group(s).
[0092] Preferably, the buffering agent has any of a phosphate
group, a carboxy group, and a sulfate group.
[0093] Preferably, the buffering agent has three or less acidic
functional groups in its molecule, more preferably two or less
acidic function groups. When a excessive number of acidic
functional groups are present in the molecule, its ability to
provide phosphor and calcium to hydroxyapatite is likely to be
reduced. For example, phosphorylated oligosaccharides having one or
two phosphate group in their molecules have an improved caries
treating function over phytic acid having 6 phosphate groups in its
molecule. Therefore, buffering agents other than a substance, such
as phytic acid, are preferably used.
[0094] A buffering agent having an excellent ability to provide
phosphor and calcium to hydroxyapatite is preferable. The ability
of the buffering agent to provide phosphor and calcium to
hydroxyapatite may be easily tested by a simple remineralization
test system method as described below.
[0095] Examples of the buffering agent include phosphorylated
oligosaccharides and sugar alcohols thereof. The term
"phosphorylated oligosaccharide" as used herein refers to an
oligosaccharide which has at least one phosphate group in its
molecule, preferably three or less phosphate groups, and more
preferably two or less phosphate groups. The term "neutral
oligosaccharide" as used herein refers to an oligosaccharide
without a phosphate group linked thereto. For example, the
phosphorylated oligosaccharide may be a glucan consisting of 3 to 5
glucoses coupled by .alpha.-1,4 linkages where one phosphate group
is linked to the glucan. Alternatively, the phosphorylated
oligosaccharide may be a glucan consisting of 2 to 8 glucoses with
.alpha.-1,4 linkages where two phosphate groups are linked to the
glucan. Examples of the buffering agent include, but are not
limited to, acidic saccharides and sugar alcohols thereof (e.g.,
oligogalacturonic acid, chondroitin sulfate, chondroitin sulfate
oligosaccharides, glucose-6-phosphate), organic acids (e.g.,
tartaric acid, citric acid, malic acid, lactic acid, fumaric acid,
and maleic acid), nucleic acids (e.g., phosphate esters of various
nucleosides or nucleotides), amino acids, and the sugar alcohols of
the above-described phosphorylated oligosaccharides.
[0096] The above-described buffering agents may be in the form of a
salt, such as a metal salt, in order to cause the buffering agents
to be effective. Examples of a metal which is used for the
formation of such a metal salt include alkaline metal, alkaline
earth metal, zinc, iron, chromium, and lead. For example,
potassium, sodium, calcium, and magnesium are included. As a metal
salt of a buffering agent contained in the dietary composition of
the present invention, a calcium salt and a sodium salt are
preferable. As a metal salt of a buffering salt contained in the
oral composition of the present invention, a calcium salt, a sodium
salt and a zinc salt are preferable. Although zinc salts are not
used for foods and drinks, it is known that zinc salts have the
effects of preventing halitosis and treating periodontal disease.
Therefore, zinc salts are preferable as metal salts contained for
oral compositions. Further, the buffering agent may be in the form
of an ammonium salt or a quaternary amine salt.
[0097] Chondroitin sulfate typically contains one sulfate group
every two sugars. A sulfate group is linked to the 4-position of
N-acetyl-D-galactosamine in chondroitin sulfate A, and the
6-position of N-acetyl-D-galactosamine in chondroitin sulfate C.
Chondroitin sulfate B (currently called dermatan sulfate) has a
repetition structure of disaccharide units of
N-acetyl-D-galactosamine-4-sulfate and L-iduronic acid. Chondroitin
sulfate can be degraded by chondroitinase up to disaccharides of
oligosaccharides having an unsaturated hexuronic acid at a
nonreducing terminal. For example, chondroitin sulfate can be
degraded up to unsaturated disaccharides having hexosamine at their
reducing terminals by chondroitinase ABC (derived from Proteus
vulgaris), chondroitinase ACI (derived from Flavobacterium
heparinum), or chondroitinase ACII (derived from Arthrobacter
aurescens) (the latter two enzymes do not act on dermatan sulfate).
Chondroitin sulfate, and unsaturated oligosaccharides (preferably,
disaccharide and tetrasaccharide) obtained by degrading chondroitin
sulfate with such enzymes have the remineralization effect.
[0098] Oligogalacturonic acid is an oligosaccharide of polymerized
galacturonic acids which is known as a constituent saccharide of
pectin. Oligogalacturonic acid preferably comprises 2 or more
saccharides, more preferably 3 or more, even more preferably 4 or
more, and preferably 10 or less, more preferably 8 or less, and
even more preferably 6 or less.
[0099] The term "sugar alcohol" as used herein refers to a sugar
whose reducing terminal is reduced. For example, the sugar alcohol
of phosphorylated oligosaccharide may be produced by adding
hydrogen to the reducing terminal of the phosphorylated
oligosaccharide. The addition of hydrogen can be conducted with any
method known to those skilled in the art. For example,
oligosaccharide can be reduced by preparing a weak alkaline
solution of 1 N aqueous sodium hydroxide solution, pH 8, adding 30
ml of 3% sodium boron hydroxide solution to 100 ml of the weak
alkaline solution, and allowing the mixture to stand at 40.degree.
C. for one hour. The sugar alcohol may be industrially produced by
a typical method using a nickel catalyst known to those skilled in
the art.
[0100] As the buffering agent contained in the dietary composition
and the oral composition of the present invention, phosphorylated
oligosaccharides which are glucans consisting of 3 to 5 glucoses
coupled by .alpha.-1,4 linkages where one phosphate group is linked
to the glucans, or phosphorylated oligosaccharides which are
glucans consisting of 2 to 8 glucoses with .alpha.-1,4 linkages
where two phosphate groups are linked to the glucans, are
preferable.
[0101] Such phosphorylated oligosaccharides can be prepared from
general crude plant starch, and preferably starch having a number
of phosphate groups. Examples of starting plants for starch which
is used to produce phosphorylated oligosaccharides include potato,
sweet potato, cassava, maize, wheat, rice, waxy rice, waxy maize,
waxy wheat, waxy potato, kudzu, yam, lily, and chestnut. Among
these things, the underground stems, rice, wheat, etc. contain much
linked phosphate groups and are suitable for materials for
phosphorylated oligosaccharides. For example, in potato starch, a
phosphate group is relatively often bound by an ester linkage to
the 3-position or 6-position of glucose as a constituent of the
starch. A phosphate group is mainly present in amylopectin. As
starch used to produce phosphorylated oligosaccharides, chemically
modified starch may also be preferably used. Chemically modified
starch is obtained by linking phosphorus to native starch as
described above. For example, starch from maize, waxy maize, or the
like is chemically coupled with phosphor to prepare phosphorylated
oligosaccharides.
[0102] The above-described phosphorylated oligosaccharides which
are contained in the dietary compositions and oral compositions of
the present invention may be produced as follows.
[0103] In order to enzymatically degrade starch or the like, at
least one selected from the group consisting of amylolytic enzymes
such as .alpha.-amylase (EC 3.2.1.1), .beta.-amylase (EC 3.2.1.2),
glucoamylase (EC 3.2.1.3), isoamylase (EC 3.2.1.68), pullulanase
(EC 3.2.1.41), and neopullulanase (Kuriki et al., Journal of
Bacteriology, vol. 170, pp. 1554-1559, 1988); and
glycosyltransferase such as cyclodextrin glucanotransferase (EC
2.4.1.19; hereinafter, referred to as CGTase) is allowed to act on
the starch. Alternatively, at least one of those enzymes is used in
combination with .alpha.-glucosidase (EC 3.2.1.20).
[0104] Phosphorylated saccharide having no branch structure can be
obtained by degrading starch with isoamylase or pullulanase to
cleave the .alpha.-1,6 branch structure in the starch. If
isoamylase or pullulanase is not used, phosphorylated saccharide
having an .alpha.-1,6 branch structure can be obtained. By
degrading phosphorylated saccharide with glucoamylase,
non-phosphorylated glucoses which are linked to nonreducing
terminals of the phosphorylated saccharide can be successively
liberated. With such an enzyme treatment, the number of phosphate
groups per unit molecular weight of purified phosphorylated
saccharide can be either increased or decreased.
[0105] Degradation by a plurality of kinds of enzymes can be
concurrently performed by allowing the enzymes to simultaneously
react with starch. Briefly, starch as raw material is dissolved in
water or a buffer with pH which is adjusted so that the enzymes can
act on starch. Liquefying .alpha.-amylase, pullulanase,
glucoamylase, etc. are simultaneously added to a reaction solution,
and the resulting solution is allowed to react while heating. With
this method, while starch is being gelatinized, neutral saccharide
can be liberated, non-phosphorylated glucose which is bound to a
nonreducing terminal of phosphorylated saccharide can be liberated,
or .alpha.-1,6 branch structure derived from a material in
phosphorylated saccharide structure can be cleaved. This method
makes it possible to obtain phosphorylated saccharide with an
increased phosphate content by a one-step reaction, rather than a
two-step reaction.
[0106] In the case where an enzyme reaction including two or more
steps is conducted by allowing a plurality of kinds of enzymes to
separately act on starch in respective steps, the sequence of
application of the enzymes is not limited to a particular order.
However, if the concentration of the starch is high, it is
preferable that the starch is first treated by enzymes including
liquefying amylase. If isoamylase or pullulanase is allowed to act
on the starch, the amylose content increases. Amylose is likely to
age and precipitate as compared to amylopectin and, therefore, the
starch ages and precipitates. As a result, the other enzymes no
longer act on the starch.
[0107] There is no particular limit to the origins of starch
degrading enzymes, glycosyltransferase, and .alpha.-glucosidase to
be used. For example, .alpha.-amylase is preferably a starch
degrading enzyme preparation derived from bacteria of the genus
Bacillus or Aspergillus. The reaction conditions for the enzymes
are any temperature and pH at which the enzymes can function. For
example, a temperature in the range of 25.degree. C. to 70.degree.
C., and pH in the range of 4 to 8 are preferably used.
[0108] First, starch as a raw material is dissolved in water or a
buffer with pH which is adjusted so that the enzymes can act on the
starch. Liquefying .alpha.-amylase is added to the resulting
solution and allowed to react while heating, whereby the starch is
liquefied while being gelatinized. Thereafter, the liquefied starch
is held at a temperature of 20 to 80.degree. C. for an appropriate
period of time. Any amount of the liquefying .alpha.-amylase can be
used as long as it can liquefy the starch. A preferable amount of
the liquefying .alpha.-amylase is 20 to 50,000 U. This holding time
is not limited as long as the starch is liquefied to a degree that
the starch will not age during the subsequent steps. Preferably,
the holding time is 30 minutes at a temperature of 20 to 80.degree.
C.
[0109] After completion of the liquefaction, inactivation of the
enzyme is not particularly required, but the enzyme may be
inactivated by a commonly used method, i.e., by being held at
100.degree. C. for 10 minutes. Further, insoluble substances may be
separated and removed using a commonly used method, such as
centrifugation or film filtration. Thereafter, phosphorylated
saccharide can be fractionated. When phosphorylated saccharide with
an increased phosphate content is desired, the additional steps
described below are conducted.
[0110] Briefly, after the material is liquefied, glucoamylase,
isoamylase, pullulanase, and .alpha.-glucosidase are added to the
liquefied material simultaneously or in an appropriate order so as
to saccharify the material. The saccharified material is allowed to
react at a temperature of 40 to 60.degree. C. for 30 minutes to 40
hours, for example, whereby neutral saccharide and
non-phosphorylated glucose which is linked to a non-reducing
terminal of phosphorylated saccharide can be liberated from the
material, and .alpha.-1,6 branch structure in the phosphorylated
saccharide structure derived from the material can be cleaved. When
glucoamylase, isoamylase, and pullulanase are used in combination,
the combination and the sequence of addition thereof are not
limited. The amount of additive enzymes and the holding time can be
determined depending on the required phosphate content of
phosphorylated saccharide. Preferably, 50 to 700 U of glucoamylase,
2 to 100 U of isoamylase, 2 to 100 U of pullulanase, and 50 to 700
U of .alpha.-glucosidase can be added. Immobilized enzymes can be
preferably used.
[0111] After completion of the reaction with each enzyme,
inactivation of the enzyme is not particularly required, but it may
be inactivated by a commonly used method, i.e., by being held at
100.degree. C. for 10 minutes. Further, insoluble substances may be
separated and removed using a commonly used method, such as
centrifugation or membrane filtration.
[0112] In order to purify phosphorylated oligosaccharides from a
saccharide mixture containing phosphorylated oligosaccharides, an
anion exchange resin can be used since the phosphorylated
saccharides are ionic substances unlike neutral saccharide. There
is no particular limit to the type of the resin. Preferable
examples of the resin include Chitopearl BCW 2500 type (produced by
Fuji Spinning Co., Ltd.), Anberlite IRA type (produced by Japan
Organo Co., Ltd.), DEAE-cellulose (produced by Whatman),
DEAE-Sephadex and QAE-Sephadex (produced by Pharmacia), and
QAE-CELLULOSE (produced by Bio Rad). The resin is equilibrated by
using a buffer whose pH has been appropriately adjusted. For
example, an about 10 to 50 mM acetate buffer (pH 4-5) is preferably
used. The equilibrated resin is packed into a column and a
saccharide mixture containing phosphorylated oligosaccharides is
loaded thereto. Neutral saccharides are removed by washing, and
then phosphorylated oligosaccharides adsorbed to the column is
eluted with an alkaline solution or a salt solution.
[0113] In the case where phosphorylated oligosaccharides are eluted
by increasing the ionic strength of an eluent, there is no
particular limit to the kind of a salt to be used. Preferable
examples of the salt include sodium chloride, ammonium bicarbonate,
potassium chloride, sodium sulfate, and ammonium sulfate.
[0114] In the case where phosphorylated oligosaccharides are eluted
by changing the pH of an eluent into alkaline, there is no
particular limit to the kind of an alkaline reagent to be used. For
example, ammonia, sodium carbonate, or sodium hydroxide may be
used. However, under a strong alkaline condition, phosphate groups
are liberated from saccharide or the reducing terminal of the
saccharide is oxidized. Therefore, phosphorylated oligosaccharides
are eluted preferably in the pH range of weakly acidic to weakly
alkaline, and more preferably in the pH range of 3 to 8.
[0115] In the above case, by eluting phosphorylated saccharide by
increasing the salt concentration or pH of the eluent gradually or
in a stepwise manner, the phosphorylated saccharides can be
fractionated depending upon the number of phosphate groups bound to
one phosphorylated saccharide molecule.
[0116] Activated charcoal can also be used instead of an anionic
exchange resin to purify phosphorylated oligosaccharides from a
saccharide mixture containing phosphorylated oligosaccharides.
There is no particular limit to the kind of activated charcoal to
be used, but granular activated charcoal capable of being packed
into a column is preferably used. Activated charcoal is prepared
using a buffer, an acid, an alkali, a salt solution, and distilled
water so that an ability to adsorb neutral saccharides excluding
glucose is obtained. For example, degassed activated charcoal
having a uniform grain size which has been packed into the column
and washed with distilled water may be preferably used.
Phosphorylated oligosaccharides can be obtained as a passed
fraction by applying a sample to the column and allowing neutral
saccharides to be adsorbed into the column.
[0117] Alternatively, phosphorylated oligosaccharides is
precipitated by addition of alcohol having 1 to 3 carbon atoms to
purify phosphorylated oligosaccharides from a saccharide mixture
containing phosphorylated oligosaccharides. Briefly, alcohol is
added to a sample solution to allow only phosphorylated
oligosaccharides to be precipitated. It is desired that if the
sample solution has a saccharide concentration of 10% or more, 3 or
more parts by volume of alcohol are added to one part by volume of
the sample solution.
[0118] Phosphorylated oligosaccharides form phosphorylated
saccharide metal salts and are likely to precipitate, in the
presence of a metal salt, preferably a calcium salt or an iron salt
in addition to alcohol. For this reason, in the presence of a metal
salt, phosphorylated oligosaccharides are recovered more easily
using even a small amount of alcohol, as compared with the case of
using alcohol alone. Preferably, the phosphorylated saccharide is
precipitated under an alkaline condition. There is no particular
limit to the kind of the salt to be used. For example, calcium
chloride, magnesium chloride, or ferrous chloride can be preferably
used because of their satisfactory solubility. The collection of a
precipitate generated by the addition of alcohol is conducted by a
commonly used method, such as decantation, filtration, and
centrifugation.
[0119] Phosphorylated oligosaccharide may be produced by removing
the metal salt from the phosphorylated oligosaccharide metal salt
which is precipitated by the addition of the metal salt. The
removal of the metal salt (desalting) can be conducted by a
commonly used method. The desalting can be easily conducted using,
for example, table-top desalting microacilyzer G3 (manufactured by
Asahi Chemical Industry Co., Ltd.).
[0120] The resultant phosphorylated saccharide solution,
phosphorylated saccharide, or phosphorylated saccharide derivative
can be condensed or powdered using a commonly used drying method,
such as hot-air drying, fluidized-bed drying, and vacuum drying. By
removing alcohol, if required, phosphorylated saccharide which can
be used in dietary or oral applications can be obtained.
[0121] In potato starch, a phosphate group is relatively often
linked by an ester linkage to the 3-position or 6-position of
glucose as a constituent of the starch. Therefore, phosphorylated
oligosaccharide prepared from potato starch using various amylases
may be an oligosaccharide in which a phosphate group is mainly
bound to the 3-position or 6-position of glucose. For example, if a
phosphate group is bound to the 6-position of glucose in the
phosphorylated oligosaccharide obtained by allowing glucoamylase to
act on potato starch, the starch can be cleaved immediately before
(at the non-reducing terminal side) the glucose having a phosphate
group at its 6-position. Thus, the phosphorylated oligosaccharide
is oligosaccharide having glucose with its 6-position bond with a
phosphate group at a nonreducing end or has a structure in which
the at least second glucose from the nonreducing terminal has its
6-position bond with a phosphate group. If a phosphate group is
bound to the 3-position of glucose in the phosphorylated
oligosaccharide, the second glucose from the nonreducing terminal
has its 3-position bond with a phosphate group. A representative
example of phosphorylated oligosaccharide obtained by hydrolyzing
potato starch using various amylase is shown in FIG. 26. Of course,
phosphorylated oligosaccharide having the above-described structure
is not limited to ones that are produced by hydrolyzing potato
starch by various amylase. Phosphorylated oligosaccharides having
like structure have like anti-dental caries functions.
[0122] The term "sugar alcohol of phosphorylated oligosaccharide"
as used herein refers to a compound obtained by reducing the
reducing terminal of the phosphorylated oligosaccharide. The sugar
alcohol of the above-described phosphorylated oligosaccharide may
be produced by adding hydrogen to the reducing terminal of the
phosphorylated oligosaccharide. The hydrogen addition may be
conducted by any method known to those skilled in the art. For
example, oligosaccharide can be reduced by preparing a weak
alkaline solution of 1 N aqueous sodium hydroxide solution, pH 8,
adding 30 ml of 3% sodium boron hydroxide solution, and allowing
the mixture to stand at 40.degree. C. for one hour. The sugar
alcohol may be industrially produced by a typical method using a
nickel catalyst known to those skilled in the art.
[0123] The above-described phosphorylated oligosaccharides or sugar
alcohols thereof may be in the form of a salt, such as a metal
salt. Examples of a metal which is used for the formation of such a
metal salt include alkaline metal, alkaline earth metal, zinc,
iron, chromium, and lead. For example, potassium, sodium, calcium,
and magnesium are included. As metal salts of phosphorylated
oligosaccharides contained in the dietary composition of the
present invention, a calcium salt and a sodium salt are preferable.
As metal salts of phosphorylated oligosaccharides contained in the
oral composition of the present invention, a calcium salt, a sodium
salt and a zinc salt are preferable. Although zinc salts are not
used for foods and drinks, it is known that zinc salts have the
effects of preventing halitosis and treating periodontal disease.
Therefore, zinc salts are preferable as metal salts contained for
oral compositions. Further, the phosphorylated oligosaccharides may
be in the form of an ammonium salt or a quaternary amine salt.
[0124] Such a metal salt can be produced as follows. A
phosphorylated oligosaccharide salt which is a compound of
phosphorylated oligosaccharide and a metal salt can be precipitated
by alcohol precipitation as described above. If necessary,
recovered precipitation may be redissolved in water or an
appropriate solution, followed by addition of alcohol. This
operation may be repeated. With this operation, impurities such as
neutral sugar and excessive salts can be removed. An
ultrafiltration film may be used to remove impurities such as a
salt.
[0125] It is known that the above-described phosphorylated
oligosaccharide has the following properties: (1) not to be
utilized by dental caries pathogenic bacteria (e.g., mutans
streptococci and sobrinus streptococci); (2) to suppress a
reduction in pH due to sucrose utilization by these bacteria in a
concentration-dependent manner; and (3) this suppression relies on
the buffering ability of the phosphorylated oligosaccharide (see
Japanese Laid-Open Publication No. 8-104696). According to the
present invention, it was further found that the salt-form
phosphorylated oligosaccharide and the sugar alcohol thereof have
the effect of promoting remineralization of teeth at a very low
concentration. By utilizing such properties of phosphorylated
oligosaccharide, dietary compositions and oral compositions having
an anti-dental caries function can be obtained. In particular, the
fact that the remineralization effect is sufficiently obtained at a
low concentration is very preferable for addition to foods.
[0126] The dietary compositions and oral compositions of the
present invention contain a buffering agent in an amount such that
the buffering agent effectively exhibits an anti-dental caries
function in the oral cavity. For example, in the case of a
phosphorylated oligosaccharide sodium salt, the amount may be such
that the concentration of the salt in the oral cavity is 0.01 to
20%, and preferably 0.03 to 1%. For example, in the case of a
phosphorylated oligosaccharide calcium salt, the amount may be such
that the concentration of the salt in the oral cavity is 0.01 to
20%, and preferably 0.03 to 1%. For example, in the case of a
phosphorylated oligosaccharide zinc salt, the amount may be such
that the concentration of the salt in the oral cavity is 0.01 to
20%, and preferably 0.03 to 1%. For all the phosphorylated
oligosaccharide sodium salt, the phosphorylated oligosaccharide
calcium salt, and the phosphorylated oligosaccharide zinc salt, and
most preferably, their concentrations in the oral cavity are about
0.2% where the inorganic calcium and phosphorus concentrations are
about 1.5 mM and 0.9 mM in the oral cavity.
[0127] The amounts of these additives may be determined by taking
into consideration the holding times in the oral cavity of the
dietary compositions and oral compositions of the present
invention. An will be given for the case of the dietary
compositions which require mastication behaviors. For example, in
the case of a chewing gum containing about 20% phosphorylated
oligosaccharide, phosphorylated oligosaccharide is eluted from the
dietary composition and a relatively high concentration (about 1%
to about 5%) of the phosphorylated oligosaccharide can be present
for about 10 minutes after mastication. After about 20 minutes to
30 minutes, only 0.25% or less phosphorylated oligosaccharide is
present in the oral cavity. Therefore, the concentration of
phosphorylated oligosaccharide in the oral cavity is diluted to one
fourth or less of the concentration in the food. Therefore, in the
case of such a food, a buffering agent may be added to a food at a
concentration which is four times or less the intended
concentration in the oral cavity (e.g., one to four times). On the
other hand, in the case of compositions which do not require
mastication behaviors (e.g., drinks), the holding time in the oral
cavity is within one minute. Such compositions are not
substantially diluted in the oral cavity. Therefore, phosphorylated
oligosaccharide is incorporated into a composition at a
concentration which is substantially equal to the intended
concentration in the oral cavity (e.g., 0.1% to 5.0%). The dietary
compositions and oral compositions of the present invention can
contain the above-described buffering agents alone or in
combination so that the above-described amount of the agents in the
oral cavity can be held.
[0128] In another aspect, the dietary compositions and oral
compositions of the present invention further contain any one of a
phosphorus-calcium compensating agent, a phosphorus preparation,
and a calcium preparation, or alternatively a combination of one or
more thereof in addition to the above-described buffering agent. In
particular, when the composition contains a calcium salt, an extra
amount of calcium is released from the calcium salt, so that the
ratio of calcium to phosphorus in the composition is changed.
Further, the buffering agent added may have an influence on elution
of calcium from teeth. In this case, if the ratio of phosphorus to
calcium concentrations in saliva of the oral cavity which is
changed by the buffering agent is compensated, remineralization of
teeth can be more effective. In the case of a normal human, the
mole ratio of phosphorus to calcium in saliva (hereinafter referred
to as "Ca/P") is generally 0.25 to 0.67 (P/Ca=1.45 to 3.9) and,
thus, phosphorus is present more than calcium (i.e., nearly 3 mole
phosphorus to 2 mole calcium to 3.9 mole phosphorus to 1 mole
calcium). Hydroxyapatite which is a component of teeth (represented
by Ca.sub.10(PO.sub.4).sub.6(OH).sub.2) has a Ca/P of 1.67
(P/Ca=0.6). A composition constituting the enamel of teeth has a
Ca/P of 1.0 to 1.67 (P/Ca=0.6 to 1.0). Therefore, by supplying
phosphorus and/or calcium along with the buffering agent to bring
the Ca/P close to 1.0 to 1.67 (P/Ca=0.6 to 1.0), and preferably
1.67 (P/Ca=0.6), it is possible to promote crystallization of these
substances into the hydroxyapatite.
[0129] An agent which can compensate for Ca/P is herein called a
"phosphorus-calcium compensating agent". Examples of such a
phosphorus-calcium compensating agent include calcium phosphate
monobasic {calcium bis (dihydrogenphosphate) monohydrate}, calcium
phosphate dibasic (calcium hydrogenphosphate dehydrate), calcium
phosphate tribasic, calcium pyrophosphate, hydroxyapatite powder,
amorphous calcium phosphate, bovine bone calcium, eggshell calcium,
coral calcium, pearl calcium, fish and shell fish calcium, and
.alpha.-tribasic calcium phosphate. To compensate for Ca/P herein
means to maintain Ca/P within a range which can be substantially
approximated to 1.0 to 1.67 (P/Ca=0.6 to 1.0). In this case, Ca/P
need not be strictly 1.0 to 1.67 (P/Ca=0.6 to 1.0). Ca/P may fall
outside the range of 1.0 to 1.67 (P/Ca=0.6 to 1.0) as long as Ca/P
can be substantially approximated to be approximately 1.0 to 1.67
(P/Ca=0.6 to 1.0). The amount of a compensating agent required for
compensation varies depending on the kinds of a buffering agent and
a compensating agent, but the range of such an amount can be
determined by those skilled in the art conducting a simple
experiment if necessary. In the case of a phosphorus-calcium
compensating agent, its appropriate amount is 1/20 parts to 20
parts by mole with respect to one parts of a buffering agent added,
and preferably 1/2 parts to 2 parts.
[0130] Since phosphorus is excessive in saliva, a calcium
preparation may be used to adjust Ca/P to 1.0 to 1.67 (P/Ca=0.6 to
1.0). In human saliva, the phosphorus concentration is 3 to 3.5 mM
and the calcium concentration is 0.9 to 2 mM. Therefore, calcium is
preferably added at about 4 to 5 mM to increase the calcium
concentration. Therefore, a calcium salt (buffering agent) can be
used as a phosphorus-calcium compensating agent. In the case of
phosphorylated oligosaccharide containing 3% calcium, addition of
about 0.7% phosphorylated oligosaccharide calcium is appropriate.
Preferable examples of the calcium preparation include, but are not
limited to, calcium carbonate, calcium chloride, calcium lactate,
calcium gluconate, whey calcium, organic acid calcium, colloidal
calcium carbonate, casein phosphopeptide calcium, and calcium
fluoride.
[0131] The dietary compositions and oral compositions of the
present invention may further contain a phosphorus preparation. The
term "phosphorus preparation" as used herein refers to a phosphate
compound. Examples of the phosphate compound include sodium
phosphate, sodium hydrogenphosphate, potassium phosphate, and
potassium hydrogenphosphate.
[0132] The above-described phosphorus-calcium compensating agent,
phosphorus preparation, or calcium preparation may be added alone
or in combination to the dietary compositions and oral compositions
of the present invention so as to bring Ca/P close to 1.0 to 1.67
(P/Ca=0.6 to 1.0), and preferably 1.67 (P/Ca=0.6).
[0133] The term "dietary composition" as used herein is a generic
name for human foods, feeds for animals or fish breeding, and pet
foods. Specifically, the dietary compositions of the present
invention include liquid and powdered drinks such as coffee, tea,
green tea, oolong tea, juice, processed milk, and sports drinks;
baked foods such as bread, pizza, and pie; baked confectionery such
as cookies, crackers, biscuits, and cake; pastas such as spaghetti
and macaroni; noodles such as wheat noodles, buckwheat noodles, and
Chinese noodles; sweets such as candy, soft candy, chewing gum, and
chocolate; snacks such as rice crackers, and potato chips; frozen
confectionery such as ice cream and sherbet; dairy products such as
cream, cheese, powdered milk, condensed milk, and milk beverage;
Western unbaked confectionery such as jelly, pudding, mousse, and
yogurt; Japanese confectionery such as a sweet bun, uirou
(square-cut rice cake obtained by adding saccharide to the powder,
followed by steaming), rice cake, and ohagi (rice dumpling covered
with bean jam or the like); seasonings such as soy sauce, sauce for
dipping, soup for noodles, Worcestershire sauce, broth stock, stew
stock, soup stock, mixed seasonings, curry powder, mayonnaise, and
ketchup; canned or retort foods such as curry, stew, soup, and rice
dishes; frozen and refrigerated foods such as ham, hamburg, meat
balls, croquette, Chinese-style dumpling, fried rice, and rice
ball; marine processed products such as tikuwa (tubular fish paste)
and kamaboko (fish paste cake); and rice products such as rice for
a picnic lunch and sushi. Furthermore, the dietary compositions of
the present invention include formulas, weaning foods, baby foods,
pet foods, feeds for animals, sports foods, nutrition auxiliary
foods, and health foods, because of its ability to allow calcium to
be readily absorbed.
[0134] In a preferred embodiment, the foods and drinks are ones
that are much masticated in eating, such as gum. In the case of the
foods and drinks which are much masticated, a buffering agent is
easily diffused in the oral cavity, resulting in a satisfactory
effect of anti-dental caries. In the case of the foods and drinks
which are much masticated, a buffering agent can be added to the
diet preferably at a proportion of 0.1 to 50% by weight, more
preferably 0.5 to 20% by weight, even more preferably 0.5 to 10% by
weight, and particularly preferably 0.5 to 5% by weight.
Specifically, for example, such a food is a gum containing 0.1 to
50% by weight of a buffering agent, or a tablet confectionary,
candy, gummy candy, etc. containing 0.1 to 50% by weight of a
buffering agent.
[0135] In another preferred embodiment, the foods and drinks are
ones that do not require mastication in eating, such as drinks
(e.g., juice or fresh water). In the case of the foods and drinks
that do not require mastication in eating, a buffering agent can be
mixed to the diet preferably at a proportion of 0.1 to 70% by
weight, more preferably 0.1 to 50% by weight, and even more
preferably 0.2 to 5% by weight. Specifically, for example, the diet
is juice containing 1 to 30% by weight of a buffering agent.
Preferably, the diet is vegetable juice, natural juice, milk
beverage, milk, soybean milk, sports drinks, near water drinks,
nutritional drinks, coffee beverage, or cocoa which contain 0.1 to
10% by weight of a buffering agent.
[0136] In still another preferred embodiment, the foods and drinks
are ones that are masticated as much as ordinary staple foods in
eating. The foods and drinks are preferably staple foods and
drinks. For example, such diet is rice. In the case of the staple
foods, since the diet is eaten in an abundant amount, even a small
concentration of a buffering agent added advantageously provides
the significant and long-term effect of preventing dental caries.
In the case of the diet that is masticated as much as ordinary
staple foods, a buffering agent can be added to the diet at a
proportion of preferably 0.01 to 20% by weight, more preferably
0.02 to 10% by weight, even more preferably 0.03 to 5% by weight,
and particularly preferably 0.05 to 3% by weight. Specifically, the
diet is, for example, rice containing 0.02 to 10% by weight of a
buffering agent, bread containing 0.01 to 20% by weight of
phosphorylated oligosaccharide, etc.
[0137] Of course, the present invention may be applied to foods and
drinks other than those of the above-described preferred
embodiments. Specifically, the present invention may be applied to,
for example, Chinese noodles containing 0.1 to 20% by weight of a
buffering agent, wheat noodles containing 0.1 to 20% by weight of a
buffering agent, rice cake containing 0.1 to 20% by weight of a
buffering agent, pretzel containing 0.1 to 20% by weight of a
buffering agent, agar containing 0.1 to 20% by weight of a
buffering agent, jelly containing 0.1 to 20% by weight of a
buffering agent, yogurt containing 0.1 to 20% by weight of a
buffering agent, cookies containing 0.1 to 20% by weight of a
buffering agent, tablet confectionary containing 0.1 to 20% by
weight of a buffering agent, tofu containing 0.1 to 20% by weight
of a buffering agent, chocolate containing 0.1 to 20% by weight of
a buffering agent, rice confectionary containing 0.1 to 20% by
weight of a buffering agent, Chinese dumpling containing 0.1 to 20%
by weight of a buffering agent, and ham containing 0.1 to 20% by
weight of a buffering agent.
[0138] The term "oral composition" as used herein refers to any
composition, which can be introduced into the oral cavity and can
be in contact with teeth, other than foods and drinks. The oral
compositions may be drugs or quasi-drugs or other compounds. For
example, the "oral compositions" further include cosmetics (more
particularly, dentifrices which have the effects of preventing
tooth decay, whitening teeth, removing dental plaque, cleansing the
oral cavity, preventing halitosis, removing tar, preventing
deposition of dental calculus, etc. (which may be acknowledged as
cosmetics under the Japanese Pharmaceutical Affairs Law (revised in
2001)). Specifically, the oral compositions of the present
invention include, for example, dentifrices, mouthwashes, troches,
gargles, gum massage creams, lozenges, artificial saliva, etc.
[0139] In one preferred embodiment, the oral compositions of the
present invention are dentifrices containing preferably 0.01 to 20%
by weight of a buffering agent, more preferably 0.02 to 10% by
weight, even more preferably 0.03 to 5% by weight, and particularly
preferably 0.05 to 3% by weight.
[0140] In one preferred embodiment, the oral compositions of the
present invention are mouthwashes containing preferably 0.01 to 20%
by weight of a buffering agent, more preferably 0.02 to 10% by
weight, even more preferably 0.03 to 5% by weight, and particularly
preferably 0.05 to 3% by weight.
[0141] In one preferred embodiment, the oral compositions of the
present invention are oral ointments containing preferably 0.01 to
20% by weight of a buffering agent, more preferably 0.02 to 10% by
weight, even more preferably 0.03 to 5% by weight, and particularly
preferably 0.05 to 3% by weight.
[0142] Preferably, the oral compositions of the present invention
are dentifrices, mouthwashes, troches, gargles, artificial saliva,
etc. containing 0.1 to 20% by weight of a buffering agent.
[0143] The artificial saliva has been used to improve xerostomia.
The artificial saliva contains substantially the same components,
such as minerals, as human saliva. The artificial saliva containing
the above-described buffering agent not only can wet the tongue and
the laryngeal mucosa to allow the tongue and the laryngeal mucosa
to move smoothly, but also can prevent and treat dental caries.
[0144] The dietary compositions and oral compositions of the
present invention further optionally contain fluorine. The dietary
compositions and oral compositions of the present invention contain
within the range of 1000 ppm or less, preferably 0.1 to 500 ppm and
more preferably 0.1 to 300 ppm. A buffering agent is suitable for
drugs, quasi-drugs, and cosmetics in order to increase the
effectiveness of fluorine of 100 ppm or more. The dietary
compositions and oral compositions of the present invention can
have a higher level of remineralization effect for teeth by further
containing fluorine. Here, "fluorine" includes fluorine ion. The
term "fluorine containing substance" refers to any material which
provides fluorine ion, preferably fluorine ion containing compounds
(e.g., sodium monofluorophosphate, sodium fluoride, potassium
fluoride, ammonium fluoride, amine salt fluoride, and stannous
fluoride). Use of sodium monofluorophosphate and sodium fluoride
are preferable.
[0145] Use of only fluorine or a fluorine containing substance
results in a low level of remineralization of teeth. Particularly,
fluorine and a fluorine containing substance are likely to be
insoluble at a high concentration of 100 ppm or more, resulting in
a significant reduction in the effectiveness. However, in the
present invention, it was found that use of a buffering agent along
with fluorine or a fluorine containing substance leads to an
increase of the effectiveness. As to foods, tea containing a high
amount of fluorine (200 to 300 ppm) and the like are preferable.
Fluorine or a fluorine containing substance is incorporated into
the crystal of teeth to produce the strong crystal resistant to
acids. Therefore, the dietary compositions and oral compositions of
the present invention are involved in the production of the strong
crystal of teeth as well as the remineralization of teeth, thereby
reducing the development of dental caries.
[0146] The dietary compositions and oral compositions of the
present invention may further contain other substances which it is
known to those skilled in the art that have an anti-dental caries
function. Examples of such a substance include various
oligosaccharides (panose (6.sup.2-glucosyl-maltose),
isomaltooligosaccharides, palatinose
(6-O-.alpha.-D-Glucopyranosyl-D-Fructofuranose), trehalose
(O-.alpha.-D-Glucopyranosyl(1-1)-.alpha.-D-Glucopyranoside),
maltooligosaccharides, lactosucrose.TM.
(4.sup.G-.beta.-D-Galactosylsucrose), fructooligosaccharides,
coupling sugars, xylosylfructoside, cyclodextrin, etc.); sugar
alcohols (xylitol, erythritol, palatinit, sorbitol, maltitol,
mannitol, etc.); tea extracts (fluorine, polyphenol, catechin,
etc.); herbs (e.g., mint, peppermint oil, camomile, sage, ginger,
rosemary, etc., see Shibuya et al., FRAGRANCE JOURNAL SPECIAL
ISSUE, 12, P150-155, 1992); enzymes (e.g., dextranase, mutase,
etc.); and vaccines (e.g., secreted immunogloblin A against mutans
streptococci). Sugar alcohols are preferable. Xylitol is more
preferable. The dietary compositions and oral compositions of the
present invention can have the increased effect of preventing
dental caries by containing the above-described substances.
[0147] The remineralization effect of a buffering agent may be
examined by a known method, such as a remineralization test system
using bovine tooth sections which is described in Inaba. D et al.,
Eur. J. Sci. 105:74-80, 1997; Inaba. D et al., J. Dent. Health.
47:67-74, 1997; and Iijima. Y et al., Dental Caries Research.
33:206-213, 1999.
[0148] To examine the remineralization effect of a buffering agent
contained in the dietary compositions and oral compositions of the
present invention, the inventors of the present invention have
developed a simpler test system compared to the above-described
remineralization test system. Conditions under which
remineralization easily occurs are, for example, the following:
quick supply of calcium and phosphorus to the contact surface of
the tooth surface (hydroxyapatite) and incorporation of them into a
component of teeth (hydroxyapatite); maintenance of higher calcium
or phosphorus concentration in a system including the tooth
surface; and no deposition or loss of calcium and phosphorus at a
place other than the tooth surface. These conditions for easy
remineralization are simplified as follows: in a system including
hydroxyapatite, calcium and phosphorus are supplied for
crystallization and soluble calcium is reduced; and in a system
including no hydroxyapatite, phosphorus and calcium are not
deposited and the high solubility thereof is maintained. Therefore,
the magnitudes of the solubility of calcium in the two systems were
compared to examine the remineralization effect. These simple test
systems will be described below. TMR (Transversal microradiography)
method has been used in a number of researches on demineralization
and remineralization as a standard method for measuring the
distribution of dentin mineral concentration in a quantitative
manner. For this method, there are the following constraints: a
long time required for evaluation; a high-level experimental
technique required; etc. Therefore, there is a demand for a simple
evaluation system capable of quickly capturing changes in dentin
mineral concentrations. Remineralization in an early dental caries
lesion in the enamel of teeth is considered to develop via the
following two processes:
[0149] (i) calcium (Ca) ions and phosphor (P) ions which are
constituents of enamel are supplied to a demineralized portion;
and
[0150] (ii) the supplied Ca ions and P ions are used in the crystal
growth of enamel in the demineralized portion.
[0151] Considering the above-described two processes, a substance
having the effect of promoting remineralization is considered to be
one that inhibits insolubilization and precipitation of Ca and P
but does not inhibit the crystal growth of hydroxyapatite under
neutral pH.
[0152] These test systems have a correlation with the
above-described conventional system using bovine teeth, and
constitute a simple and excellent method.
[0153] In one aspect of the present invention, the present
invention relates to a method for investigating the
remineralization effect on teeth of a sample which is expected to
have an anti-dental caries action. This method comprises the steps
of: (A) precipitating calcium in a solution containing phosphorus,
calcium, and tooth components in the presence of the sample; (B)
measuring a calcium concentration in the solution or the amount of
the precipitated calcium after the precipitation; (C) precipitating
calcium in the solution in the absence of the sample; (D) measuring
a calcium concentration in the solution or the amount of the
precipitated calcium after the precipitation; and (E) comparing
calcium concentrations or the amounts of the precipitated calcium
in steps (B) and (D). In a preferred embodiment, the
above-described solution may contain hydroxyapatite, a buffer,
KH.sub.2PO.sub.4 and CaCl.sub.2. The "tooth component" to be
contained in the above-described solution is any material that
precipitates phosphorus and calcium to produce hydroxyapatite due
to remineralization. Use of hydroxyapatite is preferable.
Alternatively, teeth of mammals such as a bovine, and sections or
fractions thereof may be used. When the solution for calcium
precipitation is prepared, the sequence of addition of the
above-described phosphorus, calcium, and the other tooth components
is not limited. Preferably, first the sample, then, phosphorus,
calcium chloride solution, and the tooth component suspension or
deionized water are added in this order to prepare the solution.
The pH of the solution is preferably adjusted after the addition of
KH.sub.2PO.sub.4. Calcium precipitation typically occurs by
incubation at room temperature for ten and several hours to several
days (preferably 10 hours to 7 days, more preferably 18 hours to 42
hours). The calcium solubility of the solution can be measured by
any procedure known to those skilled in the art. The calcium
solubility of the solution may be measured by OCPC method (using
calcium C test Wako manufactured by Wako Pure Chemicals).
Alternatively, the amount of precipitated calcium in the solution
can be measured. The amount of precipitated calcium in the solution
may be measured by any procedure known to those skilled in the art.
The calcium solubility of the solution may be measured by any
procedure known to those skilled in the art. Examples of such a
method include the ICP method (Inductive Coupled Plasma method),
atomic absorption analysis and an ion electrode method.
[0154] In order to examine an anti-dental caries function, an
artificial oral device is used to obtain demineralized enamel which
is as real as possible (see Jpn. J. Oral Biol. 20:288-291, 1984,
for example). For example, this device may comprise an electrode,
an enamel section attached around the electrode, and a mutans
streptococci cell suspension, a culture solution, and a means for
dropping a sugar solution. With this device, mutans streptococci
bacteria which synthesize water-insoluble glucan are attached to
the electrode surface to form an artificial plaque, thereby
creating low pH. Moreover, an artificial plaque is similarly formed
on the enamel piece, resulting in a significant reduction of the
hardness of the enamel.
EXAMPLES
[0155] Hereinafter, the present invention will be described in
detail byway of examples. These examples are not intended to limit
the present invention. Materials, reagents, etc. used in the
examples are commercially available unless otherwise mentioned.
Example 1
[0156] Example 1 shows a method for producing phosphorylated
oligosaccharide for use in the compositions of the present
invention.
[0157] First, a 1% solution of potato starch was rapidly heated to
100.degree. C. while being dissolved in 5 ml of a solution
containing 6 mM sodium chloride and 2 mM calcium chloride so as to
be gelatinized. Thereafter, 35 U of .alpha.-amylase (Fukutamirase)
produced by Hankyu Bioindustry Ltd.) was allowed to act on the
gelatinized mixture and held at 50.degree. C. for 30 minutes. A
small amount of the reaction solution was taken to prepare 0.2%
saccharide solution. 1/10 parts of 0.01 M iodine-potassium iodide
solution was added to one parts of the saccharide solution. The
resulting mixture was confirmed to be negative in iodometry.
Thereafter, 2 U of pullulanase (produced by Hayashibara Biochemical
Lab.) and 6 U of glucoamylase (produced by Toyobo Co., Ltd.) were
allowed to act on the mixture at 40.degree. C. for 20 hours
simultaneously. The reaction was terminated, followed by
centrifugation. The supernatant was applied to an anion exchange
resin column (Chitopearl BCW 2501; produced by Fuji Spinning Co.,
Ltd.) equilibrated with 20 mM acetate buffer (pH 4.5). The column
was thoroughly washed with the acetate buffer to remove neutral
saccharide, followed by elution with the acetate buffer containing
0.5 M sodium chloride. Each eluted fraction was condensed using an
evaporator, desalted, and lyophilized, thereby obtaining
phosphorylated oligosaccharide.
[0158] The thus-obtained phosphorylated oligosaccharide was applied
again to the anion exchange resin column (Chitopearl BCW 2501)
equilibrated with 20 mM acetate buffer (pH 4.5). The column was
thoroughly washed with the acetate buffer to remove neutral
saccharide. The column was subjected to elution with the acetate
buffer containing 0.15 M sodium chloride and then with the acetate
buffer containing 0.5 M sodium chloride. The collected fractions
were desalted and lyophilized. The analysis of these fractions in
accordance with the above-mentioned method for determining the
structure indicated that in phosphorylated saccharides obtained
from the 0.15 M sodium chloride-eluted fraction (PO-1 fraction),
one phosphate group was bound to glucan having 3 to 5 glucoses with
.alpha.-1,4 linkages; and in phosphorylated saccharide obtained
from the 0.5 M sodium chloride-eluted fraction (PO-2 fraction), two
or more phosphate groups were bound to glucan having 2 to 8
glucoses with .alpha.-1,4 linkages.
[0159] The above-described structural analysis of phosphorylated
oligosaccharide was conducted as follows.
[0160] First, phosphate groups were removed from phosphorylated
oligosaccharides. 100 .mu.l of 3% phosphorylated oligosaccharide
solution was mixed with 100 .mu.l of 60 mM sodium carbonate buffer
(pH 9.4) containing 10 mM magnesium chloride, 0.3 mM zinc chloride,
and 0.05% sodium azide. 100 .mu.l of 30 U/ml alkaline phosphatase
(EC. 3.1.3.1; derived from E. coli; manufactured by SIGMA) was
added to the mixture which was then allowed to react at 40.degree.
C. for 18 hours. The reaction was terminated by removing the
alkaline phosphatase using an ultrafiltration membrane, thereby
obtaining a reaction liquid (hereinafter referred to as reaction
liquid A) containing saccharides from which phosphate groups had
been removed (hereinafter referred to as dephosphorylated
saccharides).
[0161] To 10 .mu.l of the resultant reaction liquid A, 5000 U/ml of
.beta.-amylase (derived from sweet potato; manufactured by SIGMA)
dissolved in 10 .mu.l of 200 mM acetate buffer (pH 4.8) was added,
and the resultant mixture was held at 37.degree. C. for 2 hours
(the resultant liquid is referred to as reaction liquid B).
Similarly, 300 U/ml of glycoamylase (derived from Rhizopus;
manufactured by Toyobo Co., Ltd.) dissolved in 10 .mu.l of 60 mM
acetate buffer (pH 4.5) was added to 10 .mu.l of reaction liquid A,
and the resultant mixture was held at 35.degree. C. for 18 hours
(hereinafter the resultant liquid is referred to as reaction liquid
C).
[0162] Reaction liquids A to C were analyzed to confirm products
therein. The products of these reaction liquids were confirmed by
analyzing these liquids by high-performance liquid chromatography
using an anion exchange resin column, CarboPac PA-100
(.phi.4.times.250 mm, manufactured by Dionex Corp.) or thin layer
chromatography using silica gel, and comparing the analyzed results
with those of standard maltooligosaccharides having various degrees
of polymerization. The elution of dephosphorylated saccharides
using high-performance liquid chromatography was conducted by
increasing the concentration of 1 M sodium acetate, using 100 mM
sodium hydroxide as a basic solution. The detection of the
dephosphorylated saccharides was conducted by pulsed amperometric
detector (produced by Dionex Corp.). The analysis of the
dephosphorylated saccharides by thin layer chromatography can be
conducted by multi-developing the dephosphorylated saccharides with
acetonitrile/water (80/20), spraying a solution of sulfuric
acid/methanol (=1/1), and holding at 130.degree. C. for 3
minutes.
[0163] Reaction liquid A was analyzed so that the chain length of
the phosphorylated oligosaccharides was confirmed. When reaction
liquid B was analyzed, only maltose, or maltose and maltotriose
(and a slight amount of glucose) were detected. Therefore, the
dephosphorylated saccharide was confirmed to be glucan in which
glucoses are linked to each other by .alpha.-1,4 linkages. Further,
when reaction liquid C was analyzed, only glucose was detected.
Therefore, the dephosphorylated saccharides were confirmed to be
made of .alpha.-linked glucoses.
[0164] The average chain length of saccharides (hereinafter,
represented by DP, using glucose as one unit) was obtained from the
saccharide content of the dephosphorylated saccharides having
various degrees of polymerization. The total saccharide content of
the entire phosphorylated saccharide was determined by the
phenol-sulfuric acid method. The number of linked phosphate groups
was determined as inorganic phosphate obtained by subjecting the
dephosphorylated saccharide to wet incineration (Starch-related
saccharide experimental method, Biochemistry experimental method
19, M. Nakamura et al., p. 31, 1986, JSSP Tokyo). The number of
bound phosphate groups per molecule was calculated using the amount
of inorganic phosphate determined after the wet incineration of the
dephosphorylated saccharide and DP in accordance with the following
formula: ( The .times. .times. average .times. .times. number
.times. .times. of .times. .times. bound .times. .times. phosphate
groups .times. .times. per .times. .times. molecule ) = .times. [
Inorganic .times. .times. phosphate .times. .times. quantified
.times. .times. after wet .times. .times. incineration ] [ Total
.times. .times. sugar .times. .times. amount .times. .times. in
.times. .times. entire .times. .times. phosphorylated saccharides
.times. .times. ( g ) ] / [ Average .times. .times. molecular
.times. .times. weight .times. .times. of dephosphorylated .times.
.times. saccharide .times. .times. calculated .times. .times. from
.times. .times. DP ] ##EQU1##
Example 2
[0165] 10 g of each of a PO-1 fraction containing phosphorylated
oligosaccharides having one phosphate group per molecule and a PO-2
fraction containing phosphorylated oligosaccharides having two
phosphate groups was dissolved in 100 ml of distilled water. These
aqueous solutions were desalted using an electrodialyzer (Micro
acilyzer) G3, AC210-400 membrane: manufactured by Asahi Kasei Co.,
Ltd), and were then subjected to ion exchange using strong cation
exchange resin (Dowex 50w 20-50 MESH, H-Form: manufactured by
Nisshin Kasei), thereby obtaining a saccharide soluton of pH 2.7.
The resultant solution was neutralized with 1 N sodium hydroxide
solution or calcium hydroxide solution, followed by lyophilization,
thereby preparing a phosphorylated oligosaccharide sodium or
calcium salt.
[0166] Phosphorylated saccharides (in the form of a sodium salt or
a calcium salt) used in the following examples were phosphorylated
saccharide mixture containing 80% or more of the above-described
PO-1 fraction phosphorylated saccharides and the remainder of the
PO-2 fraction phosphorylated saccharides.
Example 3
[0167] In Example 3, a system using bovine tooth pieces was used to
clarify the effect of phosphorylated oligosaccharides on
remineralization of early dental caries.
[0168] This experiment was conducted basically in accordance with
Inaba. D et al., Eur. J. Sci. 105:74-80, 1997; Inaba. D et al., J.
Dent. Helth. 47:67-74, 1997; and Iijima. Y et al., Caries Research.
33:206-213, 1999.
[0169] Tooth pieces used in the experiment were prepared as
follows: cubic bovine tooth pieces 3 mm per side were placed so
that the enamel surfaces thereof are up. The pieces were covered
with composite resin except for the enamel surfaces. The enamel was
treated with wet abrasives and paper. Demineralization was
conducted as follows: the tooth pieces were immersed in 1% lactate
gel (pH 5.0) containing 6% carboxymethylcellulose gel at 37.degree.
C. for 3 weeks. Remineralization was conducted as follows: the
tooth pieces subjected to demineralization were immersed in 20 mM
2-[4-(2-hydroxyethyl)]-1-piperidinylethane sulfonate (HEPES) buffer
(pH 7.0) containing 1.5 mM CaCl.sub.2 and 0.9 mM KH.sub.2PO.sub.4
at 37.degree. C. for one week.
[0170] The following eight test groups were prepared: (1) only
demineralization (blank; "blank" in FIGS. 1 and 2); (2) only
remineralization (negative control; "control" in FIGS. 1 and 2);
(3) a remineralization solution+2 ppm fluorine (F) (positive
control; "2 ppm F" in FIGS. 1 and 2); (4) a remineralization
solution+4.0% phosphorylated oligosaccharide sodium salt ("POs Na
4%" in FIGS. 1 and 2); (5) a remineralization solution+1.0%
phosphorylated oligosaccharide sodium salt ("POs Na 1%" in FIGS. 1
and 2); (6) a remineralization solution+0.2% phosphorylated
oligosaccharide sodium salt ("POs Na 0.2%" in FIGS. 1 and 2); (7) a
remineralization solution+0.2% phosphorylated oligosaccharide
calcium salt ("POs Ca 0.2%" in FIGS. 1 and 2); and (8) a
remineralization solution+0.07% phosphorylated oligosaccharide
calcium salt ("POs Ca 0.07%" in FIGS. 1 and 2).
[0171] After each treatment, a 200 .mu.m-thick section was prepared
from each treated tooth piece, and the mineral concentration
distribution thereof was analyzed from the microradiographic images
(not shown). When the tooth pieces were subjected to
demineralization, minerals were eluted and lost from the tooth
pieces in which cavities were in turn produced (the onset of dental
caries). FIG. 1 shows the graph of the mineral loss value in
accordance with this mineral concentration analysis (the vertical
axis indicates the mineral loss value). FIG. 2 shows the depth of
demineralization (the vertical axis indicates the lesion depth
(.mu.m)). According to FIG. 1, in the case of both phosphorylated
oligosaccharide sodium and phosphorylated oligosaccharide calcium,
the mineral loss was minimum at the lowest concentration of the
tested concentrations. This mineral loss was less than that of (2)
positive control. In the case of phosphorylated oligosaccharide
sodium and phosphorylated oligosaccharide calcium, low lesion depth
was obtained (FIG. 2). This indicates that the cavities were filled
by remineralization. Interestingly, in the case of (2) positive
control with fluorine, the lesion depth was unchanged.
[0172] After each treatment, the calcium and phosphorus
concentrations of the post-remineralization solution were also
analyzed. The solution was centrifuged at 10,000 g for two minutes,
and the supernatant was analyzed. The phosphorus concentration was
determined by molybdic acid method ("Shin-ban Bunseki Kagaku Jikken
[New Edition Analyical Chemistry Experiment] (1st ed.), pp.
313-314, published by Kagaku Dojin K.K.), and the calcium
concentration was determined by OCPC method (manufactured by Wako
Pure Chemicals: measured by a "calcium C test Wako" kit). The
results are shown in Table 1. TABLE-US-00001 TABLE 1 Pi (mM) Ca
(mM) Control 0.34 0.68 2 ppm F 0.41 0.73 POs Na 4% 1.4 3.86 1% 1.2
1.86 0.2% 1.1 1.63 POs Ca 0.2% 1.2 2.66 0.07% 1.2 1.80
[0173] According to Table 1, it was found that by the addition of
phosphorylated oligosaccharides, the concentrations of calcium and
phosphorus dissolved in the solution remained high.
[0174] Therefore, this experiment suggests that by addition of
phosphorylated oligosaccharides, the concentrations of calcium and
phosphorus dissolved in the solution remains high, and as a result,
these solubilized phosphorus and calcium may be supplied to dental
caries portions and utilized for remineralization. Such a
phenomenon is considered to occur in the human oral cavity.
Example 4
[0175] In Example 4, a remineralization simple test system was used
to clarify an effect of phosphorylated oligosaccharides on
remineralization for early dental caries.
[0176] (Procedure of Remineralization Test System)
[0177] In order to examine the remineralization phenomenon in a
more simple manner, conditions under which remineralization occurs
more easily were simplified. In a system including hydroxyapatite,
calcium and phosphorus are supplied for crystallization and soluble
calcium is reduced. In contrast, in a system including no
hydroxyapatite, calcium and phosphorus are not precipitated so that
the solubility thereof is held at a high level. Based on these
facts, the following test system was designed.
[0178] 500 .mu.l of solution is prepared by mixing the following
materials in the following order: (1) 50 .mu.l of 200 mM HEPES
buffer (pH 7.0); (2) 200 .mu.l of deionized water or a sample; (3)
50 .mu.l of 18 mM KH.sub.2PO.sub.4 solution; (4) 50 .mu.l of 30 mM
calcium chloride solution; and (5) hydroxyapatite suspension (5
mg/ml) or deionized water. After the addition of (3), 0.1 N
potassium hydroxide solution is used to adjust the pH of the
solution. The resultant solution is stirred and incubated at
37.degree. C. for 1 to 7 days. Thereafter, the solution is
centrifuged at 12,000 rpm for 3 minutes. The calcium concentration
of the resultant supernatant was measured by OCPC method (as
above). To this end, absorbance is measured at 570-nm using calcium
C test Wako (Code; 272-21801). The percentage of soluble calcium is
obtained by dividing the calcium concentration of the supernatant
by the concentration of added calcium multiplied by 100. The
percentage of remineralization is obtained by calculating the
difference between the value obtained by the deionized water and
the value obtained at the time of the addition the hydroxyapatite
at (5).
[0179] (Effects of Phosphorylated Oligosaccharides having Various
Concentrations on Remineralization)
[0180] The above-described simple test system was used to incubate
a phosphorylated oligosaccharide sodium salt and a phosphorylated
oligosaccharide calcium salt having various concentrations at
37.degree. C. for 18 or 42 hours. The results of remineralization
in the case of the phosphorylated oligosaccharide sodium salt and
the phosphorylated oligosaccharide calcium salt are shown in FIGS.
3 and 4, respectively (in FIGS. 3 and 4, the vertical axis
indicates the remineralization rate (%), and the horizontal axis
indicates the sample (%), and control indicates no addition of the
samples; for each sample concentration, a bar to the left indicates
the 18 hour treatment and a bar to the right indicates the 42 hour
treatment). The phosphorylated oligosaccharide sodium salt even at
low concentration improved the ability to solubilize the added
calcium (FIG. 3). The phosphorylated oligosaccharide calcium salt
had a low ability to solubilize the exogenously added calcium salt,
and rather released extra calcium to change the ratio of calcium to
phosphorus in the solution so that calcium is more easily
precipitated and therefore the high calcium concentration cannot be
maintained (FIG. 4).
[0181] Therefore, the phosphorylated oligosaccharide sodium salt
could exhibit the solubilizing action without changing the ratio of
calcium to phosphorus concentrations in the system. In the case of
the phosphorylated oligosaccharide calcium salt, it was considered
that phosphorus (phosphate, a phosphorus compound, etc.) needs to
be concurrently supplied to maintain the Ca/P ratio at 1.67 (P/Ca
ratio=0.6). Alternatively, the concentration of the added
phosphorylated oligosaccharide calcium salt need have little
influence on the ratio.
[0182] (Effects of Phosphorylated Oligosaccharides at Ca/P
Concentration ratio=1.67 (P/Ca Concentration ratio=0.6) on the
Remineralization Effect)
[0183] The ratio of calcium to phosphorus concentrations was set to
be 1.67 (P/Ca concentration ratio=0.6) when phosphorylated
oligosaccharide calcium salt was used, the concentrations were set
so that the calcium was derived from the phosphorylated
oligosaccharides. The sodium salt was set to match the
phosphorylated oligosaccharide concentration. The concentration
settings are shown in Table 2 below. TABLE-US-00002 TABLE 2 Control
POs Na No. P (mM) Ca (mM) No. P (mM) Ca (mM) (%) -HAp -HAp 1 0.9
1.5 11 0.9 1.5 0.25 2 1.8 3.0 12 1.8 3.0 0.50 3 2.7 4.5 13 2.7 4.5
0.75 4 3.6 6.0 14 3.6 6.0 1.00 5 4.5 7.5 15 4.5 7.5 1.25 +HAp +HAp
6 0.9 1.5 16 0.9 1.5 0.25 7 1.8 3.0 17 1.8 3.0 0.50 8 2.7 4.5 18
2.7 4.5 0.75 9 3.6 6.0 19 3.6 6.0 1.00 10 4.5 7.5 20 4.5 7.5 1.25
POs Ca No. P (mM) Ca (mM) (%) -HAp 21 0.9 1.5 0.25 22 1.8 3.0 0.50
23 2.7 4.5 0.75 24 3.6 6.0 1.00 25 4.5 7.5 1.25 +HAp 26 0.9 1.5
0.25 27 1.8 3.0 0.50 28 2.7 4.5 0.75 29 3.6 6.0 1.00 30 4.5 7.5
1.25
[0184] Incubation at 37.degree. C. for 15 hours was conducted in
the above-described simple test system. The results are shown in
FIG. 5 (the vertical axis indicates the remineralization rate (%),
the horizontal axis indicates the Ca concentration (mM), filled
squares represent a control without a phosphorylated
oligosaccharide salt, diamonds represent a phosphorylated
oligosaccharide sodium salt (POs Na), circles represent a
phosphorylated oligosaccharide calcium salt (POs Ca)). As shown in
FIG. 5, when the Ca/P concentration ratio=1.67 (P/Ca concentration
ratio=0.6) was constant and the concentration of added calcium was
increased, similar results were obtained between the phosphorylated
oligosaccharide sodium salt and the phosphorylated oligosaccharide
calcium salt. When the added calcium salt was 6 mM or more, the
effect of the addition of phosphorylated oligosaccharides was
reduced.
[0185] (Effects of Phosphorylated Oligosaccharides at Various Ca/P
on the Remineralization Effect)
[0186] The above-described simple test system was incubated at
37.degree. C. for 17.5 hours or 1 week while The ratio of calcium
to phosphorus concentrations was changed as shown in Table 3 (Table
3 uses P/Ca). TABLE-US-00003 TABLE 3 No. X (P) Y (Ca) *1 *2 1 9 15
CaCl.sup.2 D.W. 2 18 15 CaCl.sup.2 D.W. 3 27 15 CaCl.sup.2 D.W. 4
36 15 CaCl.sup.2 D.W. 5 45 15 CaCl.sup.2 D.W. 6 9 15 CaCl.sup.2
2.4% POs-Na 7 18 15 CaCl.sup.2 2.4% POs-Na 8 27 15 CaCl.sup.2 2.4%
POs-Na 9 36 15 CaCl.sup.2 2.4% POs-Na 10 45 15 CaCl.sup.2 2.4%
POs-Na 11 9 15 2.4% POs-Ca D.W. 12 18 15 2.4% POs-Ca D.W. 13 27 15
2.4% POs-Ca D.W. 14 36 15 2.4% POs-Ca D.W. 15 45 15 2.4% POs-Ca
D.W.
[0187] The results are shown in FIGS. 6A to 6C (the vertical axis
indicates the remineralization rate (%) and the horizontal axis
indicates P/Ca). FIG. 6A indicates the results of a control without
phosphorylated oligosaccharides. Squares represent 17.5 hour
treatment and filled diamonds represent one week treatment. FIG. 6B
shows the results of a phosphorylated oligosaccharide sodium salt.
Triangles represent 17.5 hour treatment and filled triangles
represent one week treatment. FIG. 6C shows the results of a
phosphorylated oligosaccharide calcium salt. Circles represent 17.5
hour treatment and filled circles represent one week treatment. As
shown in FIGS. 6A to 6C, when Ca was fixed to 1.5 mM and the
phosphorus concentration was varied to change the P/Ca ratio, both
the phosphorylated oligosaccharide sodium salt and the
phosphorylated oligosaccharide calcium salt were considered to
cause relatively effectively remineralization. According to the
results, the calcium salt was considered to be more stable even at
a high concentration of phosphorus.
Example 5
[0188] Example 5 shows comparison of phosphorylated
oligosaccharides with other anti-dental caries agents in the
remineralization effect. As anti-dental caries agents, xylose,
xylitol, palatinose, and palatinit were used. The simple system of
Example 3 was used to examine the remineralization effect.
Incubatoin at 37.degree. C. for 8 days was conducted in the simple
system. The results are shown in FIGS. 7A to 7C (the vertical axis
indicates the remineralization rate (%) and the horizontal axis
indicate the sample concentration (%)). FIG. 7A shows the results
of a phosphorylated oligosaccharide salt where filled triangles
represent a calcium salt and open triangles represent a sodium
salt. FIG. 7B shows the results of xylitol where filled circles
represent xylitol and open circles represent xylose. FIG. 7C shows
the results of palatinit where filled squares represent palatinit
and open squares represent palatinose. According to FIGS. 7A to 7C,
the phosphorylated oligosaccharides of a concentration of as low as
about 0.1% exhibited a high remineralization effect, while the
other anti-dental caries agents (xylitol, palatinose, and
palatinit) exhibited a remineralization effect at a concentration
of 20% as previously reported (Japanese Laid-Open Publication No.
2000-128752, Japanese Laid-Open Publication No. 2000-247852, etc.).
In the case of xylose, the remineralization percentage was low at
any concentration.
Example 6
[0189] In Example 6, the effect of phosphorylated oligosaccharides
to inhibit demineralization was examined.
[0190] A demineralization solution having the following composition
was prepared: 6.0 mM calcium chloride solution; 3.6 mM potassium
dihydrogenphosphate; 2% lactate solution; and 5 mg/ml
hydroxyapatite solution, pH 5.0. 125 .mu.l of the demineralization
solution and 125 .mu.l of phosphorylated oligosaccharide sodium
salt solutions having final concentrations of 0.2% and 2% were
mixed and stirred, followed by incubation at 37.degree. C. for 2
days. Thereafter, the mixtures were centrifuged at 12,000 rpm for 3
minutes. The calcium concentration of the resultant supernatant was
measured by OCPC method. The added calcium concentration and the
calcium concentration after the treatment were compared with each
other. If the difference between the added calcium concentration
and the calcium concentration after the treatment in the presence
of the test sample was small as compared to a control (without a
test sample), the test sample was recognized to have the effect of
inhibiting demineralization. Comparing a control (5 mM) without
phosphorylated oligosaccharides, both the 0.2% and 2%
phosphorylated oligosaccharide sodium salt solutions had a small
difference (3 mM and 2 mM). Therefore, the phosphorylated
oligosaccharide sodium salt was considered to have the effect of
inhibiting demineralization.
Example 7
[0191] Example 7 shows a synergistic effect of phosphorylated
oligosaccharides with fluorine with respect to the remineralization
effect.
[0192] Compositions described in Table 4 below were used to examine
the remineralization effect in the presence or absence of
phosphorylated oligosaccharides. TABLE-US-00004 TABLE 4 No. POs Na
Ca (mM) P (mM) F (ppm) 1 0.20% 3.0 1.8 0 2 0.20% 3.0 1.8 3.91 3
0.20% 3.0 1.8 7.81 4 0.20% 3.0 1.8 15.63 5 0.20% 3.0 1.8 31.25 6
0.20% 3.0 1.8 62.50 7 0.20% 3.0 1.8 125.00 8 0.20% 3.0 1.8 250.00 9
0.20% 3.0 1.8 500.00 10 0.20% 3.0 1.8 1000.00
[0193] The simple system of Example 4 was used to examine the
remineralization effect. Incubation at 37.degree. C. for 5 days was
conducted in the simple system. Thereafter, the amount of soluble
calcium was measured by OCPC method. By thin layer chromatography
(TLC), phosphorylated oligosaccharides were qualitatively
confirmed. Conditions for TLC analysis are the following: silica
gel plate (manufactured by Merck); ethanol/deionized water/acetic
acid (=70/30/2); development one time at room temperature; 5 .mu.l
of a sample added; 1 .mu.l of 1% phosphorylated oligosaccharides
and 1 .mu.l of 1% maltotriose as markers.
[0194] The results of the TLC analysis are shown in Table 8. In
FIG. 8, each lane indicates fluorine having various concentrations
(ppm), upper spots represent maltotriose, and lower spots represent
phosphorylated oligosaccharides. FIG. 9 shows a synergistic action
of phosphorylated oligosaccharides with fluorine with respect to
remineralization (the vertical axis indicates the remineralization
rate (%) and the horizontal axis indicates the fluorine
concentration (ppm); in each value, a bar to the left indicates a
control without phosphorylated oligosaccharides and a bar to the
right belongs to a group of 0.2% phosphorylated oligosaccharides).
Fluorine is halogen elements which are highly reactive. The effect
of fluorine on phosphorylated oligosaccharides and the
quantification of calcium was examined. Under the conditions of the
experiment, it seemed that the influence of the addition of
fluorine was trivial (FIG. 8). The addition of fluorine alters the
balance of the concentration ratio of Ca to P so that the
insolubility is reduced. Therefore, the remineralization rate was
reduced due to an increase in the fluorine concentration. However,
when 0.2% phosphorylated oligosaccharide sodium salt was added, the
remineralization effect tended to be increased, so that a
significant synergistic effect could be confirmed (FIG. 9).
Example 8
[0195] Example 8 shows the mixture phosphorylated oligosaccharides
with a chewing gum and the elution of the phosphorylated
oligosaccharides to the human oral cavity.
[0196] Sheet gums (plate-like gums) containing phosphorylated
oligosaccharide calcium salts shown in Table 5 (the calcium content
was 3.2%) were prepared (a sheet gum had a weight of about 3.2 g).
TABLE-US-00005 TABLE 5 addition (%) Gum Base 25.2 POs Ca 22.7
Xylitol 50.4 Glycerol 0.7 Mint Oil 1.0 Total 100.0 3.2 g/slab
gum
[0197] The amount of the calcium salt eluted into the oral cavity
over time when the gum was chewed, was analyzed by thin layer
chromatography (TLC). Conditions for TLC were the following: the
development plate was a silica gel plate; the development eluent
was ethanol/deionized water/acetic acid=70/30/2; the development
temperature was room temperature, and development was done one
time; the amount of a spot sample was 3 .mu.l; detection was
conducted by spraying a detection solution (sulfate/ethanol=1:1) to
the plate, followed by processing at 130.degree. C. for 3 minutes,
whereby spots developed color.
[0198] FIG. 10 shows the results of the TLC analysis of
phosphorylated oligosaccharides having a standard solution
concentration. Each lane shows elution of phosphorylated
oligosaccharides having various concentrations (indicating 1%
xylitol as a control to the left and 1% maltotriose (G3) to the
right). Lower spots represent phosphorylated oligosaccharides,
while upper spots represent xylitol and maltotriose. FIG. 11 shows
the elution amount over time when a gum containing phosphorylated
oligosaccharides was chewed. Each lane indicates the elution over a
mastication time (indicating 1% phosphorylated oligosaccharides as
a control to the left and 1% xylitol and maltotriose (G3) to the
right). Lower spots represent phosphorylated oligosaccharides and
upper spots represent xylitol and maltotriose. The phosphorylated
oligosaccharides are not hydrolysed with saliva amylase. According
to these figures, it will be understood that about 10 minutes after
the beginning of mastication, a relatively high concentration of
phosphorylated oligosaccharides were present in the oral cavity,
and 20 minutes after, the phosphorylated oligosaccharides remained
at an about 0.25% concentration.
Example 9
[0199] Example 9 shows the effect of phosphorylated
oligosaccharides on fermentation of sucrose.
[0200] S. mutans strain 8148 was incubated in 1,000 ml of brain
heart infusion medium (manufactured by DIFCO Corporation) at
37.degree. C. for 14 hours. Thereafter, the bacteria were collected
by centrifugation at 6,000 rpm for 20 minutes. The bacteria was
washed with phosphate buffered saline (PBS, pH 7.2), and suspended
in the same PBS to 40% (v/v). To measure the pH, a reaction mixture
(250 .mu.l) was made of 125 .mu.l of 40% bacterial cell suspension,
62.5 .mu.l of 80 mM sucrose, and 62.5 .mu.l of an aqueous solution
containing various oligosaccharides (5% phosphorylated
oligosaccharide sodium salt and phosphorylated oligosaccharide
calcium salt). The pH of the reaction mixture was continuously
measured with a pH meter (manufactured by To a Denpa) while being
incubated at 37.degree. C.
[0201] When 0.684% sucrose or 0.684% glucose was added to the 20%
bacterial cell suspension containing S. mutans 8158 strain, the pH
of the reaction liquid was below 5.0 within 5 minutes, and was
reduced to 4.0 after 10 minutes. When 5% phosphorylated
oligosaccharides (PO-1 and PO-2) were concurrently present, the pH
reduction was clearly suppressed in either case (data not shown).
When 5% phosphorylated oligosaccharide sodium salt or
phosphorylated oligosaccharide calcium salt was added, the pH
reduction due to fermentation of 0.684% sucrose was efficiently
suppressed (data not shown).
Example 10
[0202] In Example 10, the sugar alcohol of phosphorylated
oligosaccharides was prepared.
[0203] 10 g of each of a PO-1 fraction containing phosphorylated
oligosaccharides having one phosphate group per molecule and a PO-2
fraction containing phosphorylated oligosaccharides having two
phosphate groups was dissolved in 100 ml of distilled water. The
solution was adjusted to weak alkaline solution (about pH 8) with 1
N sodium hydroxide solution. To 100 ml of the resultant solution,
30 ml of 3% sodium boron hydroxide solution was added. The mixture
was allowed to stand at 40.degree. C. for one hour so that
phosphorylated oligosaccharides were reduced. Thus, hydrogen was
added to the reducing terminals of phosphorylated oligosaccharides.
The hydrogen-added solution was adjusted to pH 7.5 with 1 N
hydrochloric acid solution. After the reaction was terminated, the
solution was subjected to dialysis using a 0.22 .mu.m membrane. The
resultant solution was desalted using an electrodialyzer (Micro
acilyser) G3, AC210-400 membrane: manufactured by Asahi Kasei
Corporation), and was then subjected to ion exchange using strong
cation exchange resin (Dowex 50w 20-50 MESH, H-Form: manufactured
by Nisshin Kasei), thereby obtaining a saccharide solution of pH
2.7. The resultant solution was neutralized with 1 N sodium
hydroxide solution or calcium hydroxide solution, followed by
lyophilization, thereby preparing a phosphorylated oligosaccharide
sodium or calcium salt.
Example 11
[0204] In Example 11, chondroitin sulfate oligosaccharides
(unsaturated disaccharide (dimer)) were prepared.
[0205] 4.8 g of sodium chondroitin sulfate (C type; manufactured by
Katayama Kagaku) was dissolved in 500 ml of distilled water (pH
6.0). 15 U chondroitinase ACII (derived from Arthrobacter
aurescens, manufactured by Seikagaku Kogyo) was added to the
resultant solution and allowed to react at 37.degree. C. for 23
hours. The reaction was terminated in a boiling bath, followed by
desalting as described in Example 10. Thus, a chondroitin sulfate
oligosaccharide sodium or calcium salt was prepared.
Example 12
[0206] In Example 12, the remineralization effect of various
substances were examined.
[0207] The simple remineralization test system of Example 4 was
used. As samples, substances shown in Table 6 below were used. All
of the substances were prepared to a final concentration of 0.1%.
TABLE-US-00006 TABLE 6 No. Sample 1 POs Na 2 PO--2Na 3 POsHNa 4 G3
5 PO--2HNa 6 Glc-6-P 7 Ser-P 8 Chondroitin Sulfate C 9
Oligogalacturonic acid 10 Dimer Na 11 D.W.
[0208] In the above-described Table 6, No. 1 POs Na indicates a
phosphorylated oligosaccharide (PO-1 fraction) sodium salt, No. 2
PO-2 Na indicates a phosphorylated oligosaccharide (PO-2 fraction)
sodium salt, No. 3 POsH Na indicates a phosphorylated
oligosaccharide (PO-1 fraction) sugar alcohol sodium salt, No. 4
PO-2H Na indicates a phosphorylated oligosaccharide (PO-2 fraction)
sugar alcohol sodium salt, No. 5 G3 indicates maltotriose (glucose
tertiary saccharide), No. 6 Glc-6-P indicates glucose-6-phosphate,
No. 7 Ser-P indicates phosphoserine, No. 8 indicates chondroitin
sulfate C, No. 9 indicates oligogalacturonic acid, No. 10 indicates
an unsaturated disaccharide of chondroitin sulfate (in Table 6 and
FIG. 12, Dimer Na), and No. 11 D. W. indicates deionized water.
[0209] The results are shown in FIG. 12 (the vertical axis
indicates the remineralization rate (%) and the horizontal axis
indicates the sample substances). In the figure, the substances
which had a remineralization proportion higher than that of the
deionized water were judged to have the remineralization effect.
The phosphorylated oligosaccharide alcohol sodium salt, the
glucose-6-phosphate, the chondroitin sulfate C sodium salt, and the
chondroitin sulfate unsaturated disaccharide sodium salt exhibited
the remineralization effect which is as good as or better than that
of the phosphorylated oligosaccharide sodium salt.
Example 13
[0210] In Example 13, the remineralization effect of various
substances was examined.
[0211] The simple remineralization test system of Example 4 was
used. As samples, substances shown in Table 7 were used.
TABLE-US-00007 TABLE 7 No. Sample Final (%) 1 POs Na 0.2 2 2.0 3
Palatinose 2.0 4 20 5 Xylitol 2.0 6 20 7 Treharose 2.0 8 20 9
Sorbitol 2.0 10 20 11 G3 2.0 12 D.W. 13 Oganic acid 0.2 14 1.4 15
Dextran sulfate 0.2
[0212] In Table 7, No. 1 POs Na indicates a phosphorylated
oligosaccharide (PO-1 fraction) sodium salt (final concentration of
0.2%), No. 2 POs Na indicates a phosphorylated oligosaccharide
(PO-1 fraction) sodium salt (final concentration of 2.0%), No. 3
indicates palatinose (final concentration of 2.0%), No. 4 indicates
palatinose (final concentration of 20%), No. 5 indicates xylitol
(Wako 244-0052) (final concentration of 2.0%), No. 6 indicates
xylitol (Wako 244-0052) (final concentration of 20%), No. 7
indicates trehalose (Wako 02252) (final concentration of 2%), No: 8
indicates trehalose (Wako 02252) (final concentration of 20%), No.
9 indicates sorbitol (Katayama 28-4770)(final concentration of 2%),
No. 10 indicates sorbitol (Katayama 28-4770) (final concentration
of 20%), No. 11 G3 indicates maltotriose (final concentration of
2%), No. 12 D.W. indicates deionized water (control), No. 13
indicates organic acid (tartaric acid) (final concentration of
0.2%), No. 14 indicates organic acid (tartaric acid) (final
concentration of 1.4%), and No. 15 indicates dextran sulfate (final
concentration of 0.2%).
[0213] The results are shown in FIG. 13 (the vertical axis
indicates the remineralization rate (%) and the horizontal axis
indicates sample substances). In the 20% addition group including
xylitol, palatinose, and sorbitol, the remineralization effect as
previously reported (as above) was confirmed. Further, similar to
chondroitin sulfate, dermatan sulfate was confirmed to have the
remineralization effect. The organic acid was also effective
similar to phosphorylated oligosaccharides.
Example 14
[0214] In Example 14, the effect of preventing dental caries was
examined in an artificial oral device.
[0215] S. sobrinus strain 6715 culture (preincubated in brain heart
infusion medium (manufactured by DIFCO Corporation)), heart
infusion liquid medium (manufactured by DIFCO Corporation), and a
sample solution (each solution was cooled during the testing), are
each supplied to a bovine tooth (about 5.times.5 mm) held in
constant temperature bath (37.degree. C.) at a rate of 6
ml/hour/tube. The pH of the tooth surface was measured over time.
The results are shown in FIG. 14 (the vertical axis indicates a
change in pH and the horizontal axis indicates the time lapse;
circles represent addition of only 1% sugar (GF) and filled
triangles represent addition of 1% GF+5% phosphorylated
oligosaccharides calcium salt (POs Ca)). After 16 hours, dental
plaque was scraped off the tooth, and the turbidity was measured at
500 nm. Further, the amount of water insoluble glucan (WIG) formed
was measured by a phenol-sulfuric acid method. The hardness of the
tooth was measured by a hardness meter. The difference between this
hardness and the hardness of an untreated tooth was obtained
(.DELTA.H). The results are shown in Table 8. TABLE-US-00008 TABLE
8 WIG Turbidity (.mu.g/mm.sup.2) (OD.sub.500/mm.sup.2) .DELTA.H 1%
GF Electrode 7.2 0.057 -- Enamel 10.8 .+-. 2.0 0.070 .+-. 0.012 240
.+-. 16.4 1% GF + Electrode 0.3 0.004 -- 5% POsCa Enamel 0.4 .+-.
0.3 0.016 .+-. 0.007 19 .+-. 10.4
[0216] It was clear that in the case of 1% GF (sugar), organic acid
was generated, and after about 10 hours, pH was 5.6 or less, and
the organic acid was held within plaque. Plaque was sufficiently
formed, and the tooth suffered from demineralization and became
brittle. In contrast, in the case of the solution containing 1% GF
and 5% phosphorylated oligosaccharides, no plaque was formed and
the pH was not reduced. That is, dental caries bacteria were
prevented from colonizing the tooth, so that plaque formation was
blocked and demineralization of the tooth was suppressed.
Therefore, the hardness of the tooth was not changed. According to
this result, it was clearly found that phosphorylated
oligosaccharides have the effect of preventing dental caries. This
phenomenon is considered to similarly occur in the human oral
cavity.
Example 15
[0217] In Example 15, phosphorylated oligosaccharides were prepared
from various starches.
[0218] Starches used in this example were from rice, starch (brand
name Better Friend.TM.: manufactured by Shimada Kagaku) and tapioca
starch (Sanwa Cornstarch Co., Ltd.).
[0219] 100 g of starch powder was added into 800 to 1000 ml of
water. To the resultant solution, 50 .mu.l of 5000 U/ml starch
liquefying .alpha.-amylase (BLA) derived from a bacterium, B.
lichenformis (available from Fukutamirase, from Hankyu Industries,
1%) was added. The solution was gelatinized at 5.degree. C. for 48
hours in water bath. Further, 50 .mu.l of 5000 U/ml BLA
(Fukutamirase, from Hankyu Industries, 1%), 50 .mu.l of 200 U/ml
pullulanase (Promozyme: manufactured Novo Nordisk), and 50 .mu.l of
glucoamylase (416 U/ml) (available from Toyobo) were added to the
gelatinized starch, followed by incubation at 50.degree. C. for 48
hours. The resultant mixture was centrifuged at 8,000 rpm for 20
minutes. The supernatant was applied to an anion exchange resin
(Chitopearl BCW 2501; produced by Fuji Spinning Co., Ltd.)
equilibrated with 10 mM acetate buffer (pH 4.5). The column was
thoroughly washed with the same buffer to remove neutral
saccharides, followed by elution with the same buffer containing
0.5 M sodium chloride. Each eluted fraction was condensed using an
evaporator, followed by desalting and lyophilization. Thus,
phosphorylated oligosaccharides were obtained.
[0220] The thus-obtained phosphorylated oligosaccharides were
applied again to an anion exchange resin column (Chitopearl
BCW2501) equilibrated with 20 mM acetate buffer (pH 4.5). The
column was thoroughly washed with the same buffer to remove neutral
saccharides. The column was subjected to elution first with the
same buffer containing 0.15 M sodium chloride and next with the
same buffer containing 0.5 M sodium chloride, thereby collecting
fractions. The collected fractions were desalted and lyophilized.
The analysis of these fractions in accordance with the
above-mentioned method for determining the structure indicated that
in phosphorylated saccharides obtained from the 0.15 M sodium
chloride-eluted fraction (PO-1 fraction), one phosphate group was
linked to glucan having 3 to 5 glucoses with .alpha.-1,4 linkages;
and in phosphorylated saccharide obtained from the 0.5 M sodium
chloride-eluted fraction (PO-2 fraction), two or more phosphate
groups were bound to glucan having 2 to 8 glucoses with .alpha.-1,4
linkages. The structural analysis of phosphorylated
oligosaccharides was conducted as described in Example 1.
Example 16
[0221] Example 16 shows that a chewing gum containing
phosphorylated oligosaccharides had the effect of promoting enamel
remineralization in early dental caries.
[0222] Two tablet gums (about 1.5 g/tablet): sugarless gums
containing 2.5% (mean content) of POs Ca derived potato starch
(containing 45% of xylitol) and sugarless gums containing no POs Ca
(containing 47.5% of xylitol), were produced by a commonly used
method. All experimental reagents were guaranteed reagents. The
content of each substance is a proportion with respect to the total
weight of a gum.
[0223] As a tooth material, the crown enamel of a bovine tooth was
used. A diamond saw (manufactured by LUXO) was used to cut the
enamel into blocks (7.times.7.times.3 mm) having a
standardized-size side. These enamel blocks (6 samples) were
embedded in an autopolymer resin (UNIFAST Trad, manufactured by
GC), which were shaped into plates having a size of 15.times.50 mm
and a thickness of 7 mm. Thereafter, the surface of the plates were
abraded with wet abrasive sandpaper (grit 800) to expose flat and
fresh enamel. On the other hand, the dentin side of the tooth was
previously embedded in an impression compound (manufactured by GC)
Each of the thus-prepared enamel block embedded plates was immersed
in 100 ml of 0.1 M lactic acid gel (6 wt % carboxymethylcellulose,
pH 5.0) at 37.degree. C. for 4 weeks, whereby dental caries
artificially occurred.
[0224] 17 healthy subjects participated in a test in which the
subjects masticate two grains of POs Ca containing gum or POs
Ca-free gum (3.0 g) for 20 minutes. In the test, the subjects were
not informed of the type of the gum. Saliva was collected into a 10
ml plastic test tube using a plastic funnel from the subjects
during a period of time after the beginning of gum mastication to
one minute later, 1 minute later to 3 minutes later, 3 minutes
later to 6 minutes later, 6 minutes later to 10 minutes later, and
10 minutes later to 20 minutes later. The amount and pH of the
saliva were measured immediately after the collection. Thereafter,
the saliva supernatant was diluted with distilled water by 10 fold,
followed by filtration with a 0.45 .mu.m filter (manufactured by
Millipore). The filtrate was subjected to quantification with
respect to the Ca and inorganic P contents using an OCPC method
(calcium C test Wako; manufactured by Wake Pure Chemicals) and a
molybdic acid method.
[0225] 12 healthy subjects participated in a test in which the
subjects masticate two tablets of POs Ca containing gum or POs
Ca-free gum (3.0 g) for 20 minutes. In the test, the subjects were
not informed of the type of the gum. Saliva was collected into a 50
ml plastic test tube using a plastic funnel from the subjects
during the first half 10 minutes of the 20-min mastication (saliva
A) and during the second half 10 minutes (saliva B). The amount and
pH of the saliva were measured immediately after the collection.
Immediately after the measurement, 7 ml of the saliva was poured
into a plastic vessel (10.times.30.times.60 mm) in which one enamel
block embedded plate with artificial dental caries was placed in
advance. This amount was such that the enamel block embedded plate
was sufficiently immersed in the saliva. After the plate was
immersed in saliva A for 10 minutes, the plate was immersed in
saliva B for 10 minutes. Thereafter, the plate was removed, and the
plate surface was thoroughly washed with distilled water. This
immersion operation was conducted at 37.degree. C., and repeated
consecutively four times a day. The enamel block embedded plate was
daily preserved in cool at a humidity of 100% after the operation.
The test was conducted in four consecutive days using human saliva
daily collected. As to the saliva used in the test, a portion of
the supernatant was used and diluted by 10 fold with distilled
water, followed by filtration of a 0.45 .mu.m filter (manufactured
by Millipore). The Ca and inorganic P of the filtrate was daily
measured by the above-described method.
[0226] After the immersion in the human saliva, each tooth enamel
was cut using a hard tissue cutter (Isomet, Buhler, USA) into
sections having a thickness of about 500 .mu.m. Each section was
abraded with wet abrasive sandpaper (grit 800) to about 200 .mu.m
thick. Each section was microradiographed (PW-1830, Philips, The
Netherlands). Conditions for the microradiography were that the
tube voltage was 25 kV; the tube current was 25 mA; and the
distance between the tube and the subject was 370 mm. Thereafter,
the lesion depth (Ld, .mu.m) and the mineral loss value .DELTA.Z
(vol %.mu.m) were measured by Inaba et al.'s image quantification
method (Eur. J. Oral. Sci. 105:74-84, 1997).
[0227] 17 healthy subjects masticated 2 grains (3.0 g) of POs Ca
containing gum or POs Ca-free gum for 20 minutes. In this case, the
amount of saliva (FIG. 15; the horizontal axis indicates a gum
mastication time and the vertical axis indicates the saliva amount
(ml)), the pH of saliva (FIG. 16; the horizontal axis indicates a
gum mastication time and the vertical axis indicates the pH), the
Ca content of saliva (FIG. 18; the horizontal axis indicates a gum
mastication time and the vertical axis indicates the calcium amount
(mg)), and the P content of saliva (FIG. 17; the horizontal axis
indicates a gum mastication time and the vertical axis indicates
the phosphorus amount (mg)), were measured over time where the
values are represented by integrated values from the start.
Moreover, a change in the Ca/P ratio of saliva (FIG. 19; the
horizontal axis indicates a gum mastication time and the vertical
axis indicates the Ca/P ratio) were calculated. In each of figures,
POs Ca containing gum (+POs Ca gum) and POs Ca-free gum (-POs Ca
gum) are indicated by squares and diamonds, respectively.
[0228] As a result the amount of secreted saliva (FIG. 15), pH
changes (FIG. 16), and changes in the P content (FIG. 17) did not
vary among gum types to a statistically significant level. In the
20-minute gum mastication, about 30 ml of saliva was secreted, and
the pH of saliva was increased from 7.0 at the beginning of the gum
mastication to about 7.5 after 5 minutes. The P content of saliva
secreted by the gum mastication was about 5 mg, which is sufficient
for remineralization as compared to the Ca content. In contrast, it
was clearly found that the amount of Ca dissolved in saliva at the
time point 20 minutes after the beginning of mastication in the
case of the POs Ca containing gum was about four times as much as
that in the case of the POs Ca-free gum (FIG. 18). Since a certain
amount of P is inherently present in saliva (FIG. 16), the Ca/P
ratio was also significantly high in the case of the POs Ca
containing gum (p<0.001) (FIG. 19). In the above-described
analysis results, there was no significant difference found between
the male and female subjects.
[0229] Table 9 shows the results of analysis of saliva A and saliva
B collected from the 12 healthy subjects who masticated two grains
(3.0 g) of POs Ca containing gum or POs Ca-free gum for 20 minutes.
TABLE-US-00009 TABLE 9 Comparison of salivary volumes, pH and
mineral content Gum spacies A saliva B saliva Saliva (ml) +POs-Ca
20.34 .+-. 4.13 9.35 .+-. 3.24 -POs-Ca 20.74 .+-. 4.43 9.65 .+-.
3.35 Ca (mM) +POs-Ca 6.29 .+-. 2.44** 1.72 .+-. 0.53* -POs-Ca 1.69
.+-. 0.41 1.51 .+-. 0.42 P (mM) +POs-Ca 5.62 .+-. 1.41 6.22 .+-.
1.31 -POs-Ca 6.15 .+-. 1.35 6.49 .+-. 1.15 Ca/P +POs-Ca 1.12 .+-.
0.31** 0.28 .+-. 0.08 -POs-Ca 0.28 .+-. 0.08 0.23 .+-. 0.06 Mean
.+-. SD, *p < 0.05, **p < 0.001, n = 12
[0230] In either gum, the amount of saliva A was about two times as
much as that of saliva B. In saliva A, there was a significant
difference in the Ca content between the POs Ca-free gum and the
POs Ca containing gum. However, in saliva B, such a difference was
small. As to the P amount, there was no difference recognized
between the gums and between saliva A and B. Therefore, in saliva
A, the Ca/P ratio when masticating the POs Ca containing gum was
about 4 times as high as that when masticating the POs Ca-free
gum.
[0231] Next, the results of evaluating the remineralization
promoting effect on the treated teeth of the 12 subjects are shown
as lesion depth and mineral loss value in FIG. 20A (the vertical
axis indicates the lesion depth (Ld, .mu.m)) and FIG. 20B (the
vertical axis indicates the mineral loss value .DELTA.Z (vol
%.mu.m)). In either figure, the horizontal axis indicates blank,
the POs Ca containing gum, and the POs Ca-free gum in this order.
The restoration of the demineralized tooth enamel was observed in
terms of both the lesion depth (Ld) and the mineral loss value
(.DELTA.Z) in the case of the POs Ca containing gum more
significantly than in the case of the POs Ca-free gum. That is, in
the POs Ca containing gum mastication group, promotion of
remineralization was obtained (p<0.001).
Example 17
[0232] Example 17 shows the remineralization promoting effect of a
chewing gum containing phosphorylated oligosaccharides on enamel in
the human oral cavity.
[0233] Similar to Example 13, two tablet gum (about 1.5 g/tablet),
i.e., a POs Ca containing gum and a non-POs Ca containing sugarless
gum, were prepared. All experimental reagents used were guaranteed
reagents.
[0234] Enamel disks (5 mm in diameter; 1.5 mm in thickness) were
prepared from the enamel portions of the crown parts of bovine
incisors. The head surfaces of the buccal surfaces were polished
with a wet abrasive sandpaper (grit 800) to expose a fresh and flat
plane of enamel. The thus-prepared enamel disks were immersed in
100 ml of 0.1 M lactic acid solution (pH 5.0) at 37.degree. C. for
3 days to generate artificial dental caries. After the
demineralization, three enamel disks were attached at the palatal
region of upper right molars in a removable palatal plate.
[0235] 12 healthy subjects (6 males and 6 female; mean age=21 years
old) masticated two pieces at a time (3.0 g) of a POs Ca containing
gum, a POs Ca-free gum, or a sucrose gum (containing 62% of
sucrose) (one grain; about 1.5 g) for 20 minutes. In this test,
each subject chewed one of the gums 4 times a day. A person in
charge of this test as well as the subjects were not informed of
the type of a gum which the subjects were chewing. For each gum,
the test was conducted for two straight weeks. There was a one-week
interval between each test. The palatal plate was attached for 20
minutes each during and after mastication of a gum. During the test
period, the subjects did not use a fluorine agent and the detached
palatal plate was stored in 100% humidity, avoiding drying.
[0236] The test tooth attached was removed from the palatal plate
of each subject after 1, 2 and 4 weeks. A section having a
thickness of about 200 .mu.m was cut from each enamel. Each section
was microradiographed (PW-1830, Philips, The Netherlands). The
microradiography conditions were the following: the tube voltage
was 25 kV; the tube current was 25 mA; and the distance between the
tube and the subject was 370 mm. Thereafter, the lesion depth (ld,
.mu.m) was measured by Inaba et al. 's image quantification method
(Eur. J. Oral. Sci. 105:74-84, 1997). The Id value was defined on
the mineral distribution profile as a distance from the head
surface of the tooth to the location of a lesion at which the
mineral content reaches 95% level of the mineral content of the
sound tissue. The remineralization rate was calculated as the
reduction rate of the Id value with respect to the initial ld value
after demineralization was calculated as the remineralization rate.
The results of remineralization are shown in FIG. 21. In FIG. 21,
the horizontal axis indicates the sucrose gum group (Suc), the POs
Ca-free gum group (Xyl), and the POs Ca containing gum group (POs)
in the order of week 1, week 2, and week 4. The vertical axis
indicates the remineralization rate (%).
[0237] The remineralization rates in the POs Ca containing gum
group (POs) were 67%, 54% and 76% at week 1, week 2 and week 4,
respectively. The remineralization rates in the POs Ca-free gum
group (Xyl) were 12 to 23%, which are lower than those of the POs
Ca containing gum group. The sucrose gum group (Suc) showed
positive remineralization rates by week 2, but finally reached to a
negative value by week 4, indicating demineralization.
[0238] The human intraoral evaluation showed the higher
remineralization promoting effect in the case of the POs Ca
containing gum than the POs Ca-free gum and the sucrose gum.
Specifically, all 12 subjects ate each kind of gum for two weeks
for each and a significant result was obtained in the case of the
POs Ca containing gum. Therefore, it was confirmed by the human
intraoral evaluation that the addition of POs Ca to a gum leads to
a high level of remineralization promoting effect. At the same
time, it was also confirmed in the oral cavities that by taking a
POs Ca containing gum product on a substantially daily basis,
remineralization of early dental caries was enhanced, thereby
preventing dental caries very effectively.
Example 18
[0239] In Example 18, the components of saliva when taking a candy
containing phosphorylated oligosaccharides were analyzed.
[0240] Candies containing the following ingredients in Table 10
were prepared. TABLE-US-00010 TABLE 10 Candy (% by weight)
Palatinit 95 POs-Ca 2.94 flavour 2.06
[0241] 4 healthy adult subjects ate a candy (4.7 g) and secreted
saliva was collected. The candies were present in the oral cavities
for about 10 minutes after being taken into the mouth. Saliva was
collected at the following four time periods: (i) 0 to 1 minutes;
(ii) 1 to 3 minutes; (iii) 3 to 6 minutes; and (iv) 6 to 10
minutes. The secreted saliva was collected through a funnel into a
15 ml test tube. Immediately after the collection, the secreted
saliva was stirred, and the pH and amount of the saliva were
measured. The results are shown in FIGS. 22 and 23. In FIG. 22, the
horizontal axis indicates the intake time (minute) and the vertical
axis indicates pH. The pH of saliva in the oral cavity was
constantly 7. In FIG. 23, the horizontal axis indicates the intake
time (min) and the vertical axis indicates the amount of saliva
(ml/min). The amount of secreted saliva is substantially constant
over the intake time.
[0242] Thereafter, 1800 .mu.l of saliva was put into four
centrifugal tubes. 200 .mu.l of 1N HCl solution was added to each
tube. The mixture was thoroughly mixed, followed by centrifugation
at 10,000.times.g for 3 minutes, and subjected to 0.5 .mu.m
membrane. 10 .mu.l of the resultant supernatant was measured by the
OCPC method to determine the calcium content. 50 .mu.l of the
supernatant was measured by the molybdenum method to determine the
phosphorus content. The calcium and phosphorus contents are shown
in FIG. 24. In FIG. 24, the horizontal axis indicates the intake
time (min), the left vertical axis indicates the calcium or
phosphorus content (mM), and the right vertical axis indicates the
Ca/P ratio. Both the calcium and phosphorus contents were
substantially constant over the intake time (remaining about
0.6).
Example 19
[0243] In Example 19, candies and soft candies containing
phosphorylated oligosaccharides were prepared to examine the
remineralization promoting effect.
[0244] Candies (4.7 g/each) containing ingredients and soft candies
(4.0 g/each) shown in Table 11 were prepared in accordance with a
commonly used method. TABLE-US-00011 TABLE 11 (% by weight) Candy
Palatinit 95 POs-Ca 2.94 flavour 2.06 Soft Candy Palatinit 47.5
Xylitol 47.5 POs-Ca 2.94 flavour 2.06
[0245] 10 ml of distilled water was added to each of the
above-described candies and soft candies, and dissolved in boiling
bath. The pH of the resultant extraction solution was measured by a
micro pH meter. Thereafter, the extraction solution was subjected
to centrifugation at 10,000.times.g for 3 minutes and subjected to
0.5 .mu.m membrane. 10 .mu.l of the resultant supernatant solution
was measured by the OCPC method to determine the calcium
concentration. 50 .mu.l of the resultant supernatant solution was
measured by vanadmolybdic acid method to determine the inorganic
phosphorus concentration. The results are shown in Table 12.
TABLE-US-00012 TABLE12 Mineral content of Ca and P in extract of
soft candy and candy Products Ca (mM) P (mM) Ca/P Soft candy 3.88
1.82 2.13 Candy 5.18 2.14 2.42
[0246] Further, based on the analysis result of Table 12, the 2-
and 10-fold diluted extraction solutions were adjusted to have the
calcium and phosphorus contents shown in Table 13. Thereafter, the
remineralization promoting effect on hydroxyapatite was evaluated.
TABLE-US-00013 TABLE13 Evaluation system for promoted
remineralization 3.0 mM CaCl2 soln. 1.8 mM KH2PO4 soln. 20 mM HEPES
buffer (pH7.0) (0.5 mg/ml Hydroxyapatite)
[0247] The results are shown in FIG. 24. In FIG. 24, the horizontal
axis indicates the intake time (min), the left vertical axis
indicates the calcium or phosphorus content (mM), and the right
vertical axis indicates the Ca/P ratio.
[0248] In either the candies or the soft candies, the 10-fold
diluted solution showed a high level of remineralization promoting
effect.
Example 20
[0249] A dentifrice having a composition shown in Table 14 was
prepared by a commonly used method. TABLE-US-00014 TABLE 14
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 sorbitol 10.00 xylitol 10.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine trace
sodium fluoride 0.15 POs Ca 4.00 disodium hydrogenphosphate 3.75
water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 21
[0250] A dentifrice having a composition shown in Table 15 was
prepared by a commonly used method. TABLE-US-00015 TABLE 15
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine 0.10
sodium fluoride 0.20 POs Ca 4.00 cetylpyridinium chloride 0.25
water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 22
[0251] A dentifrice having a composition shown in Table 16 was
prepared by a commonly used method. TABLE-US-00016 TABLE 16
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 sodium
pyrophosphate 0.50 sodium fluoride 0.20 POs Ca 4.00 dextranase 0.20
water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 23
[0252] A dentifrice having a composition shown in Table 17 was
prepared by a commonly used method. TABLE-US-00017 TABLE 17
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 POs Na 4.00 sucralose .TM. 0.20 water
remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 24
[0253] A dentifrice having a composition shown in Table 18 was
prepared by a commonly used method. TABLE-US-00018 TABLE 18
component % by weight silica 16.00 carboxymethylcellulose 1.50
sodium polyacrylate 2.00 Pluronic 1.00 xylitol 20.00 sodium
laurylsulfate 1.50 titanium dioxide 0.50 flavour 1.00 Triclosan
0.50 para-oxybenzoate ester 0.10 POs Ca 5.00 sodium
monofluorophosphate 0.80 stevia extract 1.50 water remaining
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 25
[0254] A dentifrice having a composition shown in Table 19 was
prepared by a commonly used method. TABLE-US-00019 TABLE 19
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 POs Na 4.00 stevia 0.20 water remaining
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 26
[0255] A dentifrice having a composition shown in Table 20 was
prepared by a commonly used method. TABLE-US-00020 TABLE 20
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 zinc chloride 0.20 POs Na 4.00 enzyme-treated stevia 0.20
water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 27
[0256] A dentifrice having a composition shown in Table 21 was
prepared by a commonly used method. POs Zn was prepared in the same
manner as that in Example 2, except that 1 N zinc hydroxide
solution was used for neutralization. TABLE-US-00021 TABLE 21
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 POs Zn 1.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 28
[0257] A dentifrice having a composition shown in Table 22 was
prepared by a commonly used method. TABLE-US-00022 TABLE 22
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 sodium tripolyphosphate 2.00 POs Ca
4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 29
[0258] A mouthwash having a composition shown in Table 23 was
prepared by a commonly used method. TABLE-US-00023 TABLE 23
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 POs Ca 5.00
cetylpyridinium chloride 0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 30
[0259] A mouthwash having a composition shown in Table 24 was
prepared by a commonly used method. TABLE-US-00024 TABLE 24
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 POs Ca 5.00 disodium
hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 31
[0260] A mouthwash having a composition shown in Table 25 was
prepared by a commonly used method. TABLE-US-00025 TABLE 25
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 POs Ca 5.00 sodium
fluoride 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 32
[0261] A mouthwash having a composition shown in Table 26 was
prepared by a commonly used method. TABLE-US-00026 TABLE 26
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 POs Na 5.00
.alpha.-calcium tertiary phosphate 4.00 disodium hydrogenphosphate
3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 33
[0262] A mouthwash having a composition shown in Table 27 was
prepared by a commonly used method. POs Zn was prepared in the same
manner as that in Example 2, except that 1 N zinc hydroxide
solution was used for neutralization. TABLE-US-00027 TABLE 27
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 POs Zn 5.00
cetylpyridinium chloride 0.25 .alpha.-calcium tertiary phosphate
4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 34
[0263] An oral ointment having a composition shown in Table 28 was
prepared by a commonly used method. TABLE-US-00028 TABLE 28
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 pectin 10.00 flavour 1.05 sodium fluoride 0.20 POs Ca 5.00
disodium hydrogenphosphate 3.75 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 35
[0264] An oral ointment having a composition shown in Table 29 was
prepared by a commonly used method. TABLE-US-00029 TABLE 29
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 xylitol 10.00 flavour 1.05 sodium fluoride 0.20 POs Ca 5.00
disodium hydrogenphosphate 3.75 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 36
[0265] A dentifrice having a composition shown in Table 30 was
prepared by a commonly used method. TABLE-US-00030 TABLE 30
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 sorbitol 10.00 xylitol 10.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine trace
sodium fluoride 0.15 chondroitin sulfate 4.00 disodium
hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 37
[0266] A dentifrice having a composition shown in Table 31 was
prepared by a commonly used method. TABLE-US-00031 TABLE 31
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine 0.10
sodium fluoride 0.20 chondroitin sulfate 4.00 cetylpyridinium
chloride 0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 38
[0267] A dentifrice having a composition shown in Table 32 was
prepared by a commonly used method. TABLE-US-00032 TABLE 32
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 sodium
pyrophosphate 0.50 sodium fluoride 0.20 chondroitin sulfate 4.00
dextranase 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 39
[0268] A dentifrice having a composition shown in Table 33 was
prepared by a commonly used method. TABLE-US-00033 TABLE 33
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 chondroitin sulfate 4.00 sucralose .TM.
0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 40
[0269] A dentifrice having a composition shown in Table 34 was
prepared by a commonly used method. TABLE-US-00034 TABLE 34
component % by weight silica 16.00 carboxymethylcellulose 1.50
sodium polyacrylate 2.00 Pluronic 1.00 xylitol 20.00 sodium
laurylsulfate 1.50 titanium dioxide 0.50 flavour 1.00 Triclosan
0.50 para-oxybenzoate ester 0.10 chondroitin sulfate 5.00 sodium
monofluorophosphate 0.80 stevia extract 1.50 water remaining
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 41
[0270] A dentifrice having a composition shown in Table 35 was
prepared by a commonly used method. TABLE-US-00035 TABLE 35
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 chondroitin sulfate 4.00 stevia 0.20
water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 42
[0271] A dentifrice having a composition, shown in Table 36 was
prepared by a commonly used method. TABLE-US-00036 TABLE 36
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 zinc chloride 0.20 chondroitin sulfate 4.00 enzyme-treated
stevia 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 43
[0272] A dentifrice having a composition shown in Table 37 was
prepared by a commonly used method. TABLE-US-00037 TABLE 37
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 chondroitin sulfate 1.00 water remaining
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 44
[0273] A dentifrice having a composition shown in Table 38 was
prepared by a commonly used method. TABLE-US-00038 TABLE 38
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 sodium tripolyphosphate 2.00
chondroitin sulfate 4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 45
[0274] A mouthwash having a composition shown in Table 39 was
prepared by a commonly used method. TABLE-US-00039 TABLE 39
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 chondroitin sulfate 5.00
cetylpyridinium chloride 0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 46
[0275] A mouthwash having a composition shown in Table 40 was
prepared by a commonly used method. TABLE-US-00040 TABLE 40
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 chondroitin sulfate 5.00
disodium hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 47
[0276] A mouthwash having a composition shown in Table 41 was
prepared by a commonly used method. TABLE-US-00041 TABLE 41
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 chondroitin sulfate 5.00
sodium fluoride 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 48
[0277] A mouthwash having a composition shown in Table 42 was
prepared by a commonly used method. TABLE-US-00042 TABLE 42
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 chondroitin sulfate 5.00
.alpha.-calcium tertiary phosphate 4.00 disodium hydrogenphosphate
3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 49
[0278] A mouthwash having a composition shown in Table 43 was
prepared by a commonly used method. TABLE-US-00043 TABLE 43
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 chondroitin sulfate 5.00
cetylpyridinium chloride 0.25 .alpha.-calcium tertiary phosphate
4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 50
[0279] An oral ointment having a composition shown in Table 44 was
prepared by a commonly used method. TABLE-US-00044 TABLE 44
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 pectin 10.00 flavour 1.05 sodium fluoride 0.20 chondroitin
sulfate 5.00 disodium hydrogenphosphate 3.75 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 51
[0280] An oral ointment having a composition shown in Table 45 was
prepared by a commonly used method. TABLE-US-00045 TABLE 45
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 xylitol 10.00 flavour 1.05 sodium fluoride 0.20 chondroitin
sulfate 5.00 disodium hydrogenphosphate 3.75 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 52
[0281] A dentifrice having a composition shown in Table 46 was
prepared by a commonly used method. TABLE-US-00046 TABLE 46
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 sorbitol 10.00 xylitol 10.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine trace
sodium fluoride 0.15 chondroitin sulfate dimer 4.00 disodium
hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 53
[0282] A dentifrice having a composition shown in Table 47 was
prepared by a commonly used method. TABLE-US-00047 TABLE 47
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine 0.10
sodium fluoride 0.20 chondroitin sulfate dimer 4.00 cetylpyridinium
chloride 0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 54
[0283] A dentifrice having a composition shown in Table 48 was
prepared by a commonly used method. TABLE-US-00048 TABLE 48
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 sodium
pyrophosphate 0.50 sodium fluoride 0.20 chondroitin sulfate dimer
4.00 dextranase 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 55
[0284] A dentifrice having a composition shown in Table 49 was
prepared by a commonly used method. TABLE-US-00049 TABLE 49
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 chondroitin sulfate dimer 4.00 sucralose
.TM. 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 56
[0285] A dentifrice having a composition shown in Table 50 was
prepared by a commonly used method. TABLE-US-00050 TABLE 50
component % by weight silica 16.00 carboxymethylcellulose 1.50
sodium polyacrylate 2.00 Pluronic 1.00 xylitol 20.00 sodium
laurylsulfate 1.50 titanium dioxide 0.50 flavour 1.00 Triclosan
0.50 para-oxybenzoate ester 0.10 chondroitin sulfate dimer 5.00
sodium monofluorophosphate 0.80 stevia extract 1.50 water remaining
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 57
[0286] A dentifrice having a composition shown in Table 51 was
prepared by a commonly used method. TABLE-US-00051 TABLE 51
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 chondroitin sulfate dimer 4.00 stevia
0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 58
[0287] A dentifrice having a composition shown in Table 52 was
prepared by a commonly used method. TABLE-US-00052 TABLE 52
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 zinc chloride 0.20 chondroitin sulfate dimer 4.00
enzyme-treated stevia 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 59
[0288] A dentifrice having a composition shown in Table 53 was
prepared by a commonly used method. TABLE-US-00053 TABLE 53
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 chondroitin sulfate dimer 1.00 water
remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 60
[0289] A dentifrice having a composition shown in Table 54 was
prepared by a commonly used method. TABLE-US-00054 TABLE 54
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 sodium tripolyphosphate 2.00
chondroitin sulfate dimer 4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 61
[0290] A mouthwash having a composition shown in Table 55 was
prepared by a commonly used method. TABLE-US-00055 TABLE 55
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 chondroitin sulfate dimer
5.00 cetylpyridinium chloride 0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 62
[0291] A mouthwash having a composition shown in Table 56 was
prepared by a commonly used method. TABLE-US-00056 TABLE 56
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 chondroitin sulfate dimer
5.00 disodium hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 63
[0292] A mouthwash having a composition shown in Table 57 was
prepared by a commonly used method. TABLE-US-00057 TABLE 57
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 chondroitin sulfate dimer
5.00 sodium fluoride 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 64
[0293] A mouthwash having a composition shown in Table 58 was
prepared by a commonly used method. TABLE-US-00058 TABLE 58
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 chondroitin sulfate dimer
5.00 .alpha.-calcium tertiary phosphate 4.00 disodium
hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 65
[0294] A mouthwash having a composition shown in Table 59 was
prepared by a commonly used method. TABLE-US-00059 TABLE 59
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 chondroitin sulfate dimer
5.00 cetylpyridinium chloride 0.25 .alpha.-calcium tertiary
phosphate 4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 66
[0295] An oral ointment having a composition shown in Table 60 was
prepared by a commonly used method. TABLE-US-00060 TABLE 60
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 pectin 10.00 flavour 1.05 sodium fluoride 0.20 chondroitin
sulfate dimer 5.00 disodium hydrogenphosphate 3.75 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 67
[0296] An oral ointment having a composition shown in Table 61 was
prepared by a commonly used method. TABLE-US-00061 TABLE 61
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 xylitol 10.00 flavour 1.05 sodium fluoride 0.20 chondroitin
sulfate dimer 5.00 disodium hydrogenphosphate 3.75 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 68
[0297] A dentifrice having a composition shown in Table 62 was
prepared by a commonly used method. TABLE-US-00062 TABLE 62
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 sorbitol 10.00 xylitol 10.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine trace
sodium fluoride 0.15 glucose-6-phosphate 4.00 disodium
hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 69
[0298] A dentifrice having a composition shown in Table 63 was
prepared by a commonly used method. TABLE-US-00063 TABLE 63
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine 0.10
sodium fluoride 0.20 glucose-6-phosphate 4.00 cetylpyridinium
chloride 0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 70
[0299] A dentifrice having a composition shown in Table 64 was
prepared by a commonly used method. TABLE-US-00064 TABLE 64
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 sodium
pyrophosphate 0.50 sodium fluoride 0.20 glucose-6-phosphate 4.00
dextranase 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 71
[0300] A dentifrice having a composition shown in Table 65 as
prepared by a commonly used method. TABLE-US-00065 TABLE 65
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 glucose-6-phosphate 4.00 sucralose .TM.
0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 72
[0301] A dentifrice having a composition shown in Table 66 was
prepared by a commonly used method. TABLE-US-00066 TABLE 66
component % by weight silica 16.00 carboxymethylcellulose 1.50
sodium polyacrylate 2.00 Pluronic 1.00 xylitol 20.00 sodium
laurylsulfate 1.50 titanium dioxide 0.50 flavour 1.00 Triclosan
0.50 para-oxybenzoate ester 0.10 glucose-6-phosphate 5.00 sodium
monofluorophosphate 0.80 stevia extract 1.50 water remaining
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 73
[0302] A dentifrice having a composition shown in Table 67 was
prepared by a commonly used method. TABLE-US-00067 TABLE 67
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 glucose-6-phosphate 4.00 stevia 0.20
water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 74
[0303] A dentifrice having a composition shown in Table 68 was
prepared by a commonly used method. TABLE-US-00068 TABLE 68
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 zinc chloride 0.20 glucose-6-phosphate 4.00 enzyme-treated
stevia 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 75
[0304] A dentifrice having a composition shown in Table 69 was
prepared by a commonly used method. TABLE-US-00069 TABLE 69
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 glucose-6-phosphate 1.00 water remaining
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 76
[0305] A dentifrice having a composition shown in Table 70 was
prepared by a commonly used method. TABLE-US-00070 TABLE 70
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 sodium tripolyphosphate 2.00
glucose-6-phosphate 4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 77
[0306] A mouthwash having a composition shown in Table 71 was
prepared by a commonly used method. TABLE-US-00071 TABLE 71
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 glucose-6-phosphate 5.00
cetylpyridinium chloride 0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 78
[0307] A mouthwash having a composition shown in Table 72 was
prepared by a commonly used method. TABLE-US-00072 TABLE 72
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 glucose-6-phosphate 5.00
disodium hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 79
[0308] A mouthwash having a composition shown in Table 73 was
prepared by a commonly used method. TABLE-US-00073 TABLE 73
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 glucose-6-phosphate 5.00
sodium fluoride 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 80
[0309] A mouthwash having a composition shown in Table 74 was
prepared by a commonly used method. TABLE-US-00074 TABLE 74
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 glucose-6-phosphate 5.00
.alpha.-calcium tertiary phosphate 4.00 disodium hydrogenphosphate
3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 81
[0310] A mouthwash having a composition shown in Table 75 was
prepared by a commonly used method. TABLE-US-00075 TABLE 75
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 glucose-6-phosphate 5.00
cetylpyridinium chloride 0.25 .alpha.-calcium tertiary phosphate
4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 82
[0311] An oral ointment having a composition shown in Table 76 was
prepared by a commonly used method. TABLE-US-00076 TABLE 76
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 pectin 10.00 flavour 1.05 sodium fluoride 0.20
glucose-6-phosphate 5.00 disodium hydrogenphosphate 3.75 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 83
[0312] An oral ointment having a composition shown in Table 77 was
prepared by a commonly used method. TABLE-US-00077 TABLE 77
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 xylitol 10.00 flavour 1.05 sodium fluoride 0.20
glucose-6-phosphate 5.00 disodium hydrogenphosphate 3.75 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 84
[0313] A dentifrice having a composition shown in Table 78 was
prepared by a commonly used method. TABLE-US-00078 TABLE 78
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 sorbitol 10.00 xylitol 10.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine trace
sodium fluoride 0.15 oligogalacturonic acid 4.00 disodium
hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 85
[0314] A dentifrice having a composition shown in Table 79 was
prepared by a commonly used method. TABLE-US-00079 TABLE 79
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine 0.10
sodium fluoride 0.20 oligogalacturonic acid 4.00 cetylpyridinium
chloride 0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 86
[0315] A dentifrice having a composition shown in Table 80 was
prepared by a commonly used method. TABLE-US-00080 TABLE 80
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 sodium
pyrophosphate 0.50 sodium fluoride 0.20 oligogalacturonic acid 4.00
dextranase 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 87
[0316] A dentifrice having a composition shown in Table 81 was
prepared by a commonly used method. TABLE-US-00081 TABLE 81
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 oligogalacturonic acid 4.00 sucralose
.TM. 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 88
[0317] A dentifrice having a composition shown in Table 82 was
prepared by a commonly used method. TABLE-US-00082 TABLE 82
component % by weight silica 16.00 carboxymethylcellulose 1.50
sodium polyacrylate 2.00 Pluronic 1.00 xylitol 20.00 sodium
laurylsulfate 1.50 titanium dioxide 0.50 flavour 1.00 Triclosan
0.50 para-oxybenzoate ester 0.10 oligogalacturonic acid 5.00 sodium
monofluorophosphate 0.80 stevia extract 1.50 water remaining
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 89
[0318] A dentifrice having a composition shown in Table 83 was
prepared by a commonly used method. TABLE-US-00083 TABLE 83
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 oligogalacturonic acid 4.00 stevia 0.20
water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 90
[0319] A dentifrice having a composition shown in Table 84 was
prepared by a commonly used method. TABLE-US-00084 TABLE 84
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 zinc chloride 0.20 oligogalacturonic acid 4.00 enzyme-treated
stevia 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 91
[0320] A dentifrice having a composition shown in Table 85 was
prepared by a commonly used method. TABLE-US-00085 TABLE 85
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 oligogalacturonic acid 1.00 water
remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 92
[0321] A dentifrice having a composition shown in Table 86 was
prepared by a commonly used method. TABLE-US-00086 TABLE 86
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 sodium tripolyphosphate 2.00
oligogalacturonic acid 4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 93
[0322] A mouthwash having a composition shown in Table 87 was
prepared by a commonly used method. TABLE-US-00087 TABLE 87
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 oligogalacturonic acid
5.00 cetylpyridinium chloride 0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 94
[0323] A mouthwash having a composition shown in Table 88 was
prepared by a commonly used method. TABLE-US-00088 TABLE 88
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 oligogalacturonic acid
5.00 disodium hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 95
[0324] A mouthwash having a composition shown in Table 89 was
prepared by a commonly used method. TABLE-US-00089 TABLE 89
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 oligogalacturonic acid
5.00 sodium fluoride 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 96
[0325] A mouthwash having a composition shown in Table 90 was
prepared by a commonly used method. TABLE-US-00090 TABLE 90
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 oligogalacturonic acid
5.00 .alpha.-calcium tertiary phosphate 4.00 disodium
hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 97
[0326] A mouthwash having a composition shown in Table 91 was
prepared by a commonly used method. TABLE-US-00091 TABLE 91
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 oligogalacturonic acid
5.00 cetylpyridinium chloride 0.25 .alpha.-calcium tertiary
phosphate 4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 98
[0327] An oral ointment having a composition shown in Table 92 was
prepared by a commonly used method. TABLE-US-00092 TABLE 92
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 pectin 10.00 flavour 1.05 sodium fluoride 0.20
oligogalacturonic acid 5.00 disodium hydrogenphosphate 3.75
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 99
[0328] An oral ointment having a composition shown in Table 93 was
prepared by a commonly used method. TABLE-US-00093 TABLE 93
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 xylitol 10.00 flavour 1.05 sodium fluoride 0.20
oligogalacturonic acid 5.00 disodium hydrogenphosphate 3.75
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 100
[0329] A dentifrice having a composition shown in Table 94 was
prepared by a commonly used method. TABLE-US-00094 TABLE 94
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 sorbitol 10.00 xylitol 10.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine trace
sodium fluoride 0.15 tartaric acid 4.00 disodium hydrogenphosphate
3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 101
[0330] A dentifrice having a composition shown in Table 95 was
prepared by a commonly used method. TABLE-US-00095 TABLE 95
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 saccharine 0.10
sodium fluoride 0.20 tartaric acid 4.00 cetylpyridinium chlorid
0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 102
[0331] A dentifrice having a composition shown in Table 96 was
prepared by a commonly used method. TABLE-US-00096 TABLE 96
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 glycerin 5.00 sodium
laurylsulfate 1.50 preservative 0.10 flavour 1.00 sodium
pyrophosphate 0.50 sodium fluoride 0.20 tartaric acid 4.00
dextranase 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 103
[0332] A dentifrice having a composition shown in Table 97 was
prepared by a commonly used method. TABLE-US-00097 TABLE 97
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 tartaric acid 4.00 sucralose .TM. 0.20
water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 104
[0333] A dentifrice having a composition shown in Table 98 was
prepared by a commonly used method. TABLE-US-00098 TABLE 98
component % by weight silica 16.00 carboxymethylcellulose 1.50
sodium polyacrylate 2.00 Pluronic 1.00 xylitol 20.00 sodium
laurylsulfate 1.50 titanium dioxide 0.50 flavour 1.00 Triclosan
0.50 para-oxybenzoate ester 0.10 tartaric acid 5.00 sodium
monofluorophosphate 0.80 stevia extract 1.50 water remaining
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 105
[0334] A dentifrice having a composition shown in Table 99 was
prepared by a commonly used method. TABLE-US-00099 TABLE 99
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 tartaric acid 4.00 stevia 0.20 water
remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 106
[0335] A dentifrice having a composition shown in Table 100 was
prepared by a commonly used method. TABLE-US-00100 TABLE 100
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 zinc chloride 0.20 tartaric acid 4.00 enzyme-treated stevia
0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 107
[0336] A dentifrice having a composition shown in Table 101 was
prepared by a commonly used method. TABLE-US-00101 TABLE 101
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 .alpha.-calcium tertiary phosphate
4.00 sodium fluoride 0.20 tartaric acid 1.00 water remaining
100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 108
[0337] A dentifrice having a composition shown in Table 102 was
prepared by a commonly used method. TABLE-US-00102 TABLE 102
component % by weight silica 15.00 carboxymethylcellulose 1.50
polyethyleneglycol 3.00 xylitol 20.00 sodium laurylsulfate 1.50
preservative 0.10 flavour 1.00 sodium tripolyphosphate 2.00
tartaric acid 4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 109
[0338] A mouthwash having a composition shown in Table 103 was
prepared by a commonly used method. TABLE-US-00103 TABLE 103
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 tartaric acid 5.00
cetylpyridinium chloride 0.25 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 110
[0339] A mouthwash having a composition shown in Table 104 was
prepared by a commonly used method. TABLE-US-00104 TABLE 104
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 tartaric acid 5.00
disodium hydrogenphosphate 3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 111
[0340] A mouthwash having a composition shown in Table 105 was
prepared by a commonly used method. TABLE-US-00105 TABLE 105
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 tartaric acid 5.00 sodium
fluoride 0.20 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 112
[0341] A mouthwash having a composition shown in Table 106 was
prepared by a commonly used method. TABLE-US-00106 TABLE 106
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 tartaric acid 5.00
.alpha.-calcium tertiary phosphate 4.00 disodium hydrogenphosphate
3.75 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 113
[0342] A mouthwash having a composition shown in Table 107 was
prepared by a commonly used method. TABLE-US-00107 TABLE 107
component % by weight ethylalcohol 10.00 sodium laurylsulfate 1.50
glycerin 10.00 menthol 1.00 xylitol 17.00 tartaric acid 5.00
cetylpyridinium chloride 0.25 .alpha.-calcium tertiary phosphate
4.00 water remaining 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 114
[0343] An oral ointment having a composition shown in Table 108 was
prepared by a commonly used method. TABLE-US-00108 TABLE 108
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 pectin 10.00 flavour 1.05 sodium fluoride 0.20 tartaric acid
5.00 disodium hydrogenphosphate 3.75 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 115
[0344] An oral ointment having a composition shown in Table 109 was
prepared by a commonly used method. TABLE-US-00109 TABLE 109
component % by weight liquid paraffin 25.00 sodium fragdecin 1.00
white petrolatum 25.00 silicone oil 4.00 carboxymethylcellulose
25.00 xylitol 10.00 flavour 1.05 sodium fluoride 0.20 tartaric acid
5.00 disodium hydrogenphosphate 3.75 100.00
With this composition, a satisfactory anti-dental caries function
could be achieved.
Example 116
[0345] An artificial saliva having a composition shown in Table 110
was prepared by a commonly used method. TABLE-US-00110 TABLE 110
(mg) Sodium chloride 42.2 Potassium chloride 60 Calcium chloride
7.3 Magnesium chroride 2.6 Potassium phosphate dibasic 17.1 POs Na
20 Total (ml) 50
[0346] The artificial saliva has an excellent remineralization
promoting effect and an ability to cause pH in the oral cavity to
return to neutral.
Example 117
[0347] An artificial saliva having a composition shown in Table 111
was prepared by a commonly used method. TABLE-US-00111 TABLE 111
(mg) Sodium chloride 42.2 Potassium chloride 60 POs Ca 10 Magnesium
chroride 2.6 Potassium phosphate dibasic 17.1 Total (ml) 50
The artificial saliva has an excellent remineralization promoting
effect and an ability to cause pH in the oral cavity to return to
neutral.
[0348] The artificial saliva can be similarly prepared by adding
buffering agents other than POs Ca and POs Na.
INDUSTRIAL APPLICABILITY
[0349] As described above, the present invention provides dietary
compositions and oral compositions which reduce the development of
dental caries by remineralization of teeth or the like.
* * * * *