Cariostatic materials and foods, and method for preventing dental caries

The decomposition of sugar and production of glucan by Streptococcus mutans which causes dental caries are prevented by a purified Gymnemic acid as a cariostatic material derived from Gymnema sylvestre. The Gymnema sylvestre itself is used as a cariostatic food.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to cariostatic materials and foods and 
further to a method for preventing dental caries. 
2. Description of the Prior Art 
Dental caries afflict humankind from ancient times, and particular 
attention has been given to a relationship between sugar and dental caries 
from the time when it was prevailingly said that "Degree of Consumption of 
Sugar is Barometer of Culture". Many results of researchers for prevention 
of dental caries have been accumulated. 
Basic points of view of methods for preventing dental caries have 
conventionally been in that Streptococcus mutans which are one kind of 
Streptcoccus are excluded from the mouth or oral cavity by administration 
of germicide or antibiotic; in that cariostatic artificial sweet materials 
are employed and sugar is not ingested; and in that dentine is 
strengthened using fluoridating to resist the attack by the Streptoccocus 
mutans. 
However, it has recently been proposed that dental caries start with 
decomposition of sugar present in the mouth by the Streptococcus mutans to 
produce a highly sticky glucan (polysaccharide) which is then adhered to 
surfaces of teeth to form bacterial plaque. Now, it is an established 
theory in the academic world that the formation of such plaque is the 
cause for growing up of dental caries. 
In view of this, if, in addition to the above caries preventing methods, 
the formation of plaque on tooth surfaces will be inhibited by preventing 
production of glucan, the growing up of dental caries can be avoided 
reliably. 
Thereupon, many researchers have made zealous efforts on studies in search 
of cariostatic materials, but at present, no effective cariostatic 
material has been found. 
SUMMARY OF THE INVENTION 
With the foregoing in view, it is an object of the present invention to 
provide cariostatic materials and foods and further a method for 
preventing dental caries, wherein a remarkable plaque formation inhibitory 
effect can be attained. 
To accomplish the above object, the present inventor has found as a result 
of repeated researches that a plant, Gymnema sylvestre, which belongs to 
the family of Asclepiadaceae and grows naturally in India, Africa and 
China, has a cariostatic property of inhibiting the production of the 
above-described glucan thereby to prevent the plaque formation on the 
tooth surfaces. 
A purified Gymnemic acid derived from the aforesaid Gymnema sylvestre 
particularly exhibits a significant cariostatic property. 
A tea produced by subjecting dried leaves of Gymnema sylvestre to a 
roasting treatment also has a cariostatic effect of inhibiting generation 
of an insoluble glucan which would cause dental caries by the 
Streptococcus mutans. 
Incidentally, Gymnemic acid has an effect of inhibiting the absorption of 
sucrose and the like through the intestinal tract when it is employed in 
low-caloric foods and beverages. This has already been proposed in U.S. 
patent application Ser. No. 745,161 now abandoned and its Continuation 
Application Ser. No. 8,081 now U.S. Pat. No. 4,761,286 by the present 
applicant. Gymnemic acid is non-toxic to a human body. 
Examples evidencing the effect exhibited by cariostatic materials and foods 
according to the invention will be described in conjunction with the 
accompanying drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
[Extraction and Purification of Gymnemic Acid] 
To achieve purified Gymnemic acid, 200 g of dried leaves of Gymnema 
sylvestre are first immersed in a warm water at 60.degree. C. for about 5 
hours, and this operation is repeated four or five times to provide a warm 
water-extraction solution containing crude Gymnemic acid. If this 
extraction is effected using an alcohol from the beginning, chlorophyll, 
lipid and the like are eluted. Hence, warm water is used in my extraction 
process to avoid this. 
The warm water-extraction solution is adjusted into pH of 3 with a 2N 
solution of sulfuric acid to give a precipitate containing crude Gymnemic 
acid. 
The precipitate is collected by a centrifugal sedimentation at 15,000 rpm 
for 15 minutes, and the collected material is washed with water and then 
extracted 4 or 5 times with ethanol to give an ethanol-extraction 
solution. This ethanol extraction is carried out for the purpose of 
removing protein, polysaccharide and the like from the precipitate. 
The ethanol-extraction solution is concentrated under a reduced pressure, 
and acetone is added to the resulting concentrate in an amount two times 
that of the latter in volume, followed by a centrifugal sedimentation. 
The supernatant fluid containing crude Gymnemic acid obtained after the 
sedimentation is concentrated and dried up under a reduced pressure, and 
extraction of the dried up material is repeated several times at a boiling 
point by adding diethyl carbonate as a solvent thereto. 
The precipitate from the solvent is collected and then evaporated for dry, 
and the resulting material is subjected to a fast liquid chromatography. A 
large-sized column is filled with a reversed phase carrier (C.sub.18), and 
a gradual elution is effected so that a mobile phase is converted from a 
proportion of water to methanol of 1:1 to a proportion of methanol of 
100%. As a result, purified Gymnemic acid is eluted in a phase having a 
methanol concentration of 60 to 70%. The eluted liquid is evaporated for 
dry and adjusted into pH of 7 to give the Gymnemic acid in the form of Na, 
K or NH.sub.4 salt. This purified Gymnemic acid is a saponin of one kind 
(glycoside). The yield of the purified Gymnemic acid is of 0.1 to 0.5% by 
weight per 100 g of the dried leaves of Gymnema sylvestre. 
ASSAY EXAMPLE I 
Selected specimens were a warm water-extracted material, a precipitate at 
pH of 3, a supernatant fluid at pH of 3 and a purified Gymnemic acid 
obtained in each step of the above described extracting process. For the 
warm water-extracted material, the precipitate and the supernatant fluid, 
solutions having concentrations of 0.1, 1 and 10 mg/ml were prepared. And 
in the case of the purified Gymnemic acid, solutions with concentrations 
of 0.1, 1 and 10 mM were prepared. 
Prepared culture media (TTY basic culture media) were a culture medium free 
of sucrose, a culture medium containing 30 mM of sucrose and culture media 
produced by individually incorporating 30 mM of sucrose into the 
afore-mentioned solutions. Streptococcus mutans, II D 973 strain were 
implanted at 100 .mu.l/4 ml into each of liquid media and subjected to a 
test tube culture at 37.degree. C. for 48 hours. 
In each of the liquid media, the degree of formation of plaque caused by 
the produced glucan was determined by pulverizing the plaque and then 
measuring the pulverized plaque for Klett unit by use of a turbidity meter 
with a wavelength of 660 nm. 
FIG. 1 shows the results of this determination. In the liquid medium (No 
Suc) free of sucrose, the formation of glucan and thus of plaque was not 
observed, and the measured value was not more than 2 Klett unit, whereas 
about 100 Klett unit was measured in the liquid medium (30 mM Suc) 
containing 30 mM of sucrose. 
In the liquid medium (GA) containing of 30 mM of sucrose and the purified 
Gymnemic acid, the measured value, when the purified Gymnemic acid was 
contained in an amount not less than 1 mM, was of 2 Klett unit similar to 
that in the aforesaid case of No Suc medium and hence, the formation of 
plaque was inhibited. However, with a content of the purified Gymnemic 
acid not more than 0.1 mM, such plaque formation inhibiting effect could 
not be provided. 
In the liquid medium (pH 3 ppt) containing 30 mM of sucrose and the 
precipitate at pH of 3, such plaque formation inhibiting effect could not 
be provided at the individual concentrations. 
On the other hand, a plaque formation promoting effect was revealed in the 
liquid medium (Sup) containing 30 mM of sucrose and the supernatant at pH 
of 3 as well as in the liquid medium (Deco) containing 30 mM of sucrose 
and the warm water solution. 
As apparent from the above results, it is desirable to contain 1 mM or more 
of a purified Gymnemic acid for the purpose of inhibiting the formation of 
plaque. In addition, it is an essential requirement to sufficiently purify 
a Gymnemic acid, because the plaque formation promoting effect was 
revealed with the aforesaid Sup and Deco media containing the crude 
Gymnemic acid. 
ASSAY EXAMPLE II 
The same Streptococcus mutans as in Assay Example I were implanted in a 
liquid medium (TTY basic medium) containing 56 mM of glucose, and a 
purified Gymnemic acid was added thereto at various concentrations. The 
resulting materials were subjected to a test tube culture at 37.degree. C. 
for 48 hours and examined for presence and absence of an effect of 
arresting the growth of the Streptococcus mutans. The number of 
Streptococcus mutans was determined by measuring Klett unit by use of a 
densitometer at a wave length of 660 nm. At the time of implanting, the 
number of Streptococcus mutans was of 10 Klett unit. 
After the test tube culture, the number of Streptococcus mutans was of 100 
Klett unit with the liquid medium free of the purified Gymnemic acid, but 
was extremely decreased to 40 Klett unit with the liquid medium containing 
10 mM of the purified Gymnemic acid added therein. However, with the added 
amount of purified Gymnemic acid of 1 mM, the number of Streptococcus 
mutans was less decreased and was of 80 Klett unit. 
In addition, it was confirmed that the Streptococcus mutans could not be 
propagated unless glucose was present, and the number of Streptococcus 
mutans as implanted in the liquid medium free of glucose did not vary from 
that at the time of implanting and was of 10 Klett unit. 
Further, it was also confirmed that even if the Streptococcus mutans were 
propagated in the presence of glucose, no glucan was produced and thus, no 
plaque was formed, unless sucrose was present. 
The above results showed that in order to significantly inhibit the growth 
of the Streptococcus mutans, it is necessary to contain 10 mM or more of 
the purified Gymnemic acid. 
Therefore, it is apparent from Assay Examples I and II that to prevent the 
formation of plaque and to inhibit the growth of the Streptococcus mutans, 
it is desirable to contain 10 mM or more of the purified Gymnemic acid. 
Specific examples of such applications include tooth powders, chewing 
gums, sugar and the like, which contain purified Gymnemic acid. Such 
purified Gymnemic acid can be used alone as a preventive. 
[Production of Tea] 
The tea according to the present invention can be produced through the 
following steps. 
These steps include an air drying step of drying fresh leaves of Gymnema 
sylvestre at ambient temperature for 7 days into a dried up condition, 
and a roasting step of placing the dried-up leaves into a roaster to roast 
them at 200.degree. to 300.degree. C. for 15 minutes while rotating the 
roaster. 
Therefore, the tea according to the present invention belongs to a coarse 
tea type in the classification of processes for producing Japanese tea and 
has a dark brown color. 
For comparison, the aforesaid leaves are used to produce teas of a green 
tea type, a half-fermented tea type and a full-fermented tea type. 
The green tea may be produced through the following steps and its color is 
green. 
These steps include a steaming step of steaming the fresh leaves until a 
grassy smell disappears and then cooling them, a dehumidification step of 
swishing water off from the steamed leaves while loosening the latter to 
facilitate rumpling of the leaves, a rotation-rumpling step of rumping the 
leaves with a sufficient force applied thereto to destroy the tissue and 
cells of the leaves for softening thereof, a step of loosening lumps of 
the leaves at a final stage of the rotation-rumpling step, a 
middle-rumpling of rumpling up the leaves while drying them so that the 
leaves may be twisted (in this step, the leaves get polish and diffuse a 
perfume or aroma), a finish-rumpling step of adjusting the shapes of 
leaves and improving the flavor of the leaves, and a step of drying the 
leaves at about 90.degree. C. for about 30 minutes. 
The half-fermented tea may be produced through the following steps and has 
a dark green color. 
These steps include a sunlight withering step of subjecting the 
above-described fresh leaves to sunlight for 2 to 3 hours with stirring 
them reversed after every lapse of 20 to 30 minutes and activating the 
evaporation of water and enhancing the activity of enzyme to accelerate 
the fermentation, a room withering step of stirring the stationary-left 
leaves after every lapse of one hour and continuing such stirring for 5 to 
7 hours to bring about a fermented condition which is inherent to 
half-fermented tea, a kiln treating step of subjecting the leaves to a 
high temperature to suppress the activity of enzyme and rapidly 
evaporating water to soften the tissue into a condition suitable for the 
subsequent rumpling, a rumpling step of leaving the leaves to stand for 20 
minutes and then rumpling such leaves for 10 minutes to loosen them, and a 
step of drying the leaves at about 90.degree. C. for about 30 minutes. 
The full-fermented type tea may be produced through the following steps and 
has a dark green color. 
These steps include a withering step of spreading over the above-described 
fresh leaves into a thin layer in a room to wither them, thereby 
facilitating the subsequent rumpling and twisting, a rumpling and twisting 
step of subjecting the leaves to a sufficient twisting to squeeze the sap 
from the leaves, thereby destroying the tissue and cells, so that the 
fermentation is well accelerated, a step of loosening the leaves in the 
form of lumps by dashing and then screening or sifting the leaves in order 
to provide a uniform fermentation, a fermentation step of bringing the 
fermentation already proceeding from the withering stage into a final 
fermentation stage (the flavor of the full-fermented type tea depends upon 
this step), and a drying step of evaporating water in the leaves into a 
level near to a content of 5% to stop the fermentation and then drying 
them. 
The yield of each of the above-described teas is of about 12% based on the 
weight of the fresh leaves of Gymnema sylvestre. 
Two grams of each of the aforesaid teas was immersed in 500 ml of hot water 
at about 80.degree. C. for 3 minutes to make a tea drink. The resulting 
tea drinks were measured for their optical densities by use of a 
spectrophotometer to determine the color. The results are given in Table 
1. 
TABLE I 
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Wave length (nm) 
440 450 520 570 620 660 
Type of tea 
(Purple) 
(Blue) 
(Green) 
(Yellow) 
(Orange) 
(Red) 
Color of tea drink 
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The present invention 
0.97 0.36 
0.15 0.09 0.06 0.04 
Light red brown 
(Coarse tea) 
Green tea 0.68 0.12 
0.04 0.04 0.03 0.03 
Yellowish green 
Half-fermented tea 
0.75 0.18 
0.07 0.05 0.03 0.03 
Yellowish brown 
Full-fermented tea 
1.15 0.26 
0.10 0.08 0.05 0.04 
Yellowish brown, 
Slightly clouded 
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It can be seen from Table 1 that the optical density is in a larger range 
of from 440 nm (purple) to near 520 nm (green) is the case of the coarse 
tea drink according to the present invention and hence, the color of such 
tea drink is brown near to red. In addition, with the green tea drink, the 
optical density up to 450 nm (blue) is relatively large and hence, the 
color of the green tea drink is yellowish green, while the colors of the 
half-fermented tea drink and the full-fermented tea drink are yellowish 
brown between the colors of the coarse tea drink and the green tea drink. 
In this case, it is believed that the reason why the optical density in 
the full-fermented tea drink is particularly larger in a region of shorter 
wavelength is because of clouding produced following the fermentation. 
A functional test was carried out at a temperature of 60.degree. C. for 
individual type tea drinks achieved in the above-described manner, of 
which results are given in Table II. Panels were four men of 27 to 45 
years old, and instructions were given to them to represent the strength 
of taste (bitterness, sweetness and astringency), the levels of smell and 
color, and the entire valuation in terms of 5-rank integers. The average 
values are shown in Table II. In the 5-rank representation, "very good" 
(or "very strong") is represented by 5; "good" (or "strong") is by 4; 
"mean" is by 3; "bad" (or "weak") is by 2; and "very bad" (or "very weak") 
is by 1. 
TABLE II 
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Strength of taste 
level Entire 
Type of tea 
bitterness 
sweetness 
astringency 
smell 
color 
valuation 
Note 
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Present Invention 
2.75 2.75 3.25 4.00 
4.00 
4.00 Nice-smelling, 
(Coarse tea) easy to drink 
Green tea 2.75 3.50 3.00 3.00 
4.67 
3.25 Grassy-smell, 
strange taste 
Half-fermented tea 
4.50 3.00 4.75 2.00 
3.00 
1.25 Bad-taste, 
strong 
bitterness 
Full-fermented tea 
3.33 3.33 4.00 1.33 
1.67 
1.33 Putrid odor, 
feel nausea 
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It can be seen from Table II that the coarse tea drink according to the 
present invention is at a mean level for the strength of taste, i.e., the 
bitterness, the sweetness and the astringency, but is favorable for the 
levels of smell and color and highest for the entire valuation. 
The green tea drink indicates a higher value in sweetness of the strength 
of taste, but is at mean level all over. Its color of clear yellowish 
green gives a good impression, but the entire valuation is lower than that 
of the coarse tea drink, because grassy-smell was partially commented. 
The half-fermented tea drink is lower in the entire valuation because of 
strong bitterness and astringency commented, and the full-fermented tea 
drink is likewise extremely low in the entire valuation because the smell 
is near a putrid odor and the tea drink is slightly cloudy. 
ASSAY EXAMPLE III 
Extracts were derived respectively from the above-described type individual 
teas in the following procedure. 
Five grams of each tea was immersed in 1,000 ml of hot water at 90.degree. 
C. for 10 minutes to produce a tea drink. This step was repeated twice by 
using the same tea leaves and tea drinks obtained by the first and second 
steps were mixed together, and the resulting mixture was concentrated in 
an evaporator and then subjected to a freeze-drying to provide an extract. 
The yield was of about 25%. 
Each of such extracts was added into a 1% solution of sucrose so that the 
concentration thereof was of 1 mg/ml, and Streptococcus mutans were 
implanted to observe the sequence of preparation of an insoluble glucan. 
The prepared insoluble glucan was subjected to a centrifugal sedimentation 
and then its amount was measured in an anthrone sulfuric acid process to 
provide the results given in FIGS. 2A and 2B. FIG. 2A illustrates the 
results provided after lapse of 12 hours, and FIG. 2B illustrates the 
results provided after lapse of 18 hours. In these Figures, Coa indicates 
the results when the extract from the coarse tea according to the present 
invention was added, and Gre, Ful and Hal indicate the results when 
extracts from the green and full-fermented and half-fermented teas were 
added, respectively. 1% Suc indicates the results with only sucrose. 
It is apparent from FIG. 2 that if the extract from the coarse tea 
according to the present invention is added, the preparation of an 
insoluble glucan is inhibited. 
The tea drink produced from the above method is substantially the same in 
taste as the commercially available common green teas and moreover, is 
nice-smelling and easy to drink and thus satisfies requirements for 
beverages. 
FIG. 3 illustrates an effect of inhibiting increase in the blood sugar 
content by an extract from the coarse tea according to the present 
invention in comparison with that provided by the other type teas. Namely, 
FIG. 3 illustrates the results obtained from an oral glucose tolerance 
test in which wister strain normal male rats (weighing 350 to 450 g) were 
used to examine increase in the blood sugar content (blood plasma level) 
from the time of an empty stomach. Dosage of glucose was 1 g per kg body 
weight of the rat, and the amount of each extract added was of 0.1 g per 
kg body weight, corresponding to one tenth of the amount of glucose. The 
number of cases in this test was of 10 to 27, and the blood sugar content 
at the time of an empty stomach was of 90.0.+-.2.6 mg/dl (mean.+-.S.E and 
so in the following, n=27). 
In FIG. 3, lines w, x and y indicate the results obtained from the addition 
of the individual extracts from the coarse tea according to the present 
invention and the green tea and half-fermented tea in the comparative 
examples, and a line z indicates the results obtained from the 
administration of only glucose. 
It is apparent from FIG. 3 that the rise in the blood sugar content is 
substantially inhibited are indicated by the line w when the extract from 
the coarse tea according to the present invention was added. For example, 
when only glucose was administered, an increase in blood sugar content to 
44.7.+-.4.0 mg/dl was exhibited in 30 minutes after administration, 
whereas when the extract from the coarse tea was added, the blood sugar 
content was decreased to 27.1.+-.4.4 mg/dl. 
When the extracts from the green tea and the half-fermented tea were added, 
the tendency to accelerate the increasing of the blood sugar content was 
observed. This was similar with the extract from the full-fermented tea. 
The similar test using 2 g/kg body weight of sucrose in place of glucose 
showed that rise of the blood sugar content could be inhibited by addition 
of the extract from the coarse tea according to the present invention. For 
example, when only sucrose was administered, an increase in blood sugar 
content to 44.1.+-.5.1 mg/dl was exhibited in 30 minutes after 
administration, whereas when the extract from the coarse tea was added, 
the blood sugar content was decreased to 32.9.+-.5.8 mg/dl.