Growth inhibitor for cariogenic bacteria

A growth inhibitor for cariogenic bacteria which comprises containing therein dihydroguaiaretic acid as an active ingredient. In particular, the effect of growth inhibition to Streptococcus mutans RIMD 3125001 strain can be obtained at a concentration of 1/50,000.

FIELD OF THE INVENTION 
The present invention relates to a growth inhibitor for cariogenic 
bacteria. More particularly, it relates to a growth inhibitor for 
cariogenic bacteria which comprises containing therein dihydroguaiaretic 
acid as an active ingredient. 
BACKGROUND OF THE INVENTION 
The caries is generally referred to as a decayed tooth. It is caused by 
Streptococcus mutans and other lactic acid bacteria indigenous to the oral 
cavity which form lactic acid in the bacterial plaque resulting from 
sucrose etc., in the food. The lactic acid dissolves calcium in the tooth. 
(This is called decalcification.) 
Heretofore, several attempts have been made to prevent caries. They include 
use of antibiotics, fungicides, an enzyme which dissolves cell walls and 
medicines having the antibacterial action to prevent the growth of 
cariogenic bacteria. They also include use of polysaccharide hydrolase to 
prevent the formation of bacterial plaque. However, those have a 
disadvantage of disturbing the bacterial flora in the oral cavity and 
intestine and destroying the natural balance among bacteria. In addition, 
the use of antibiotics tend to produce side effects. These disadvantages 
in practical use have not been overcome yet. 
In view of these circumstances, as a result of a series of investigations 
in search for a material having a high antibacterial activity specifically 
for cariogenic bacteria, it has been found that some of natural vegetable 
oleoresin have such antibacterial activity. 
As a result of further investigations on the vegetable oleoresin (extract 
by organic solvents) which is effective to inhibit the growth of 
cariogenic bacteria, it has been found that dihydroguaiaretic acid which 
is one component therein has the properties to well achieve the object of 
this invention. The present invention has been attained on the basis of 
this finding. 
SUMMARY OF THE INVENTION 
Accordingly, an object of this invention is to provide a growth inhibitor 
for cariogenic bacteria which comprises containing therein 
dihydroguaiaretic acid as an active ingredient. 
DETAILED DESCRIPTION OF THE INVENTION 
The dihydroguaiaretic acid used in this invention is the conventional 
compound represented by the structural formula: 
##STR1## 
This compound is a colorless crystal having a melting point of 87.degree. 
to 88.degree. C. 
The dihydroguaiaretic acid is contained in, for example, Machilus edulis 
K., Guaiacum officinale L. and Schizandra chinensis B. Banda Mace and 
Siaum Mace which are used in general spices for food and Penang Mace which 
is used in preparation of essential oils and/or oleoresin have the name: 
Myristica frangrans H. It is not yet reported that dihydroquaiaretic acid 
is present in the mace of such a species. 
On the other hand, according to the inventors' experiments, it has been 
found that Pupua Mace (name: Myristica argentea) having a bad odor and in 
which the yield of essential oil is poor contains dihydroguaiaretic acid. 
Therefore, the Pupua Mace can be used as the starting material for 
dihydroguaiaretic acid. 
Dihydroguaiaretic acid can be isolated from these oleoresins by the 
conventional method such as solvent extraction or silica gel column 
chromatography. 
The cariogenic bacteria growth inhibitor containing dihydroguaiaretic acid 
according to this invention can be used in the form of a solution by 
dissolving it in an organic solvent such as ethanol, propylene glycol or 
glycerin, which causes no problem in the oral cavity, because 
dihydroguaiaretic acid is hardly soluble in water but soluble in an 
organic solvent such as alcohols. The inhibitor can also be used in the 
form of an emulsion by emulsifying it in water using a surfactant such as 
Span 20 (a product of Atlas Powder Co.). Moreover, it is also possible to 
make such an emulsion into a water-dispersible powder by adding dextrin 
thereto followed by spray drying. Thus, the growth inhibitor of this 
invention can be used in various forms depending upon the purpose of use 
thereof. The inhibitor can, of course, be used in combination with other 
medicines, if desired. 
Dihydroguaiaretic acid performs bacteriostatic action on cariogenic 
bacteria, and its bactericidal action is mild. Therefore, 
dihydroguaiaretic acid can be suitably added to chewing gum, candy, 
troche, wheat gluten, and other foods which stay in the oral cavity for a 
long time, in the form of a propylene glycol or glycerin solution. 
Further, dihydroguaiaretic acid can be suitably added to toothpaste and 
mouth wash in the form of an emulsion and to tooth powder in the form of a 
powder. 
The growth inhibitor of this invention can completely inhibit the growth of 
Streptococcus mutans RIMD 3125001 strain which causes caries under the 
anaerobic condition in the medium, at a concentration of 1/50,000 of 
dihydroguaiaretic acid. On the other hand, the growth inhibitor of the 
invention was used at the same concentration to intestinal bacteria such 
as Bacteroides microfusus IPCR 1009 strain and Escherichia coli ATCC 10789 
strain which are aerobe, and Bifidobacterium adolescents ATCC 15705 strain 
which is obligate anaerobe; and general microorganisms such as Pseudomonas 
aeruginosa, Bacillus subtilis, Staphylococcus aureus, Aspergillus nigar, 
Candida albicans and Klebsiella pneumoniae. As a result, the growth 
inhibitor did not exhibit at all the growth inhibiting effect against the 
intestinal bacteria. Further, the growth inhibitor exhibited a slight 
antibacterial action against gram-positive bacteria such as Staphylococcus 
aureus or Bacillus subtilis under the aerobic conditions, but did not 
substantially exhibit any effect against the gram-negative bacteria and 
molds. 
The effectiveness of the growth inhibitor of this invention was not 
affected by a surfactant such as sodium laurylsulfonate or "Span 20" 
(ATLAS POWDER CO.). 
Where the cariogenic bacteria growth inhibitor of this invention is 
incorporated into foods or dentifrice, it is preferred that the 
concentration thereof be slightly higher than the effective concentration 
(1/50,000), because the contact time of the food or dentifrice to 
cariogenic bacteria is comparatively short. 
Oleoresin containing dihydroguaiaretic acid may be used for the object of 
this invention. It is, however, preferred that the oleoresin be purified 
completely or to a certain extent so as not to give off an unpleasant odor 
in the oral cavity due to other components. 
The cariogenic bacteria growth inhibitor of this invention is specifically 
effective to Streptococcus mutans and its effect of inhibiting the growth 
of bacteria is not affected by a surfactant. Dihydroguaiaretic acid is 
highly safe because it is a component contained in the oleoresin which is 
natural food. In addition, it tastes only a little and gives no unpleasant 
feeling when put in the mouth. 
This invention is now described in more detail by reference to the 
following Production Examples and Examples but is not limited thereto. 
Unless otherwise indicated, all percents, parts, ratios and the like are 
by weight.

PRODUCTION EXAMPLE 1 
100 g of Pupua Mace powder was dipped in 500 ml of 95% ethanol to extract 
for 2 days at room temperature. The extract was concentrated to obtain 9 g 
of a reddish brown oil. The oil was subjected to a column chromatography 
(silica gel 450 g) with a mixed solvent of n-hexane and ethyl ether (2:1 
by volume) and the resulting eluate was concentrated to obtain 0.9 g of 
colorless prism-like crystals. 
It was confirmed from the comparison between the following physical found 
values and the values mentioned in literature that the crystals were 
dihydroguaiaretic acid. 
m.p. 87.degree.-88.degree. C., [.alpha.].sub.D.sup.25 .+-.0.degree. 
IR (cm.sup.-1) 3,470, 1,820, 1,600 
NMR (CCl.sub.4) (.delta.): 0.84 (6H, .delta., J=6.8 Hz), 1.5-2.0 (2H, m), 
2.30 (2H, d.times.d, J=8.3, 14.4 Hz), 2.75 (2H, d.times.d, J=4.7, 14.4 
Hz), 3.85 (6H, s), 5.48 (2H, s), 6.6-6.95 (6H, m) 
PRODUCTION EXAMPLE 2 
4.6 kg of Schizandra chinensis B. was added to 23 l of petroleum ether to 
extract over day and night while sometimes stirring at room temperature. 
550 g (yield 12%) of a blackish brown oily product was obtained. The 
product was subjected to a silica gel column chromatography with a mixed 
solvent of benzene and acetone (1:1 by volume) and the resulting eluate 
was concentrated to obtain 11 g of a pale yellowish brown oil component. 
The oily component was further subjected to a column chromatography (silica 
gel 240 g) with a mixed solvent of ethyl acetate and n-hexane (4:1 by 
volume) and the solvent in the resulting eluate was evaporated to obtain 
520 mg of a substantially colorless oil component. 
This oil component was recrystallized with a mixed solvent iof n-hexane and 
acetone (7:3 by volume) to obtain 102 mg of prism-like crystals. 
EXAMPLE 1 
2.0 g of dihydroguaiaretic acid was dissolved in 98 g of official ethyl 
alcohol with stirring at room temperature to give 100 g of a solution. 
This solution was added to the heart infusion agar medium. Streptococcus 
mutans RIMD 3125001 (designated as A in Table) was transplanted by 
stabbing to this medium. Incubation was conducted at 37.degree. C. for 72 
hours. No growth was observed in the medium containing the solution at a 
concentration of 1/1,000 (i.e., 1/50,000 calculated as dihydroguaiaretic 
acid). Growth was barely observed in the medium containing the solution at 
a concentration of 1/1,600 (i.e., 1/80,000 calculated as dihydroguaiaretic 
acid). 
Then, Bacteroides microfusus IPCR 1009 (designated as B in Table) and 
Escherichia coli ATCC 10789 (designated as C in Table) were transplanted 
to the above-mentioned medium. Incubation was conducted under the aerobic 
condition at 37.degree. C. for 72 hours. Their growth was inhibited at a 
concentration of 1/2,000 as dihydroguaiaretic acid. 
Bifidobacterium adolescentis ATCC 15705 (designated as D in Table) was 
transplanted to the same medium as mentioned above, and incubation was 
conducted under the anaerobic condition at 37.degree. C. for 72 hours. The 
growth was inhibited at a concentration of 1/7,500 as dihydroguaiaretic 
acid. 
The same test as above was conducted under the aerobic condition for 
Pseudomonas aeruginosa (E), Bacillus subtilis (F), Staphylococcus aureus 
(G), Aspergillus nigar (H), Candida albicans (I), and Klebsiella 
pneumoniae (J). The results obtained are shown in Table below. 
TABLE 
______________________________________ 
Concentration for 
Growth Inhibition 
Designation (as dihydro- 
of Bacteria guaiaretic acid) 
______________________________________ 
A 1/50,000 
B 1/2,000 
C 1/2,000 
D 1/7,500 
E 1/500 
F 1/5,000 
G 1/5,000 
H 1/1,000 
I 1/2,000 
J 1/1,000 
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EXAMPLE 2 
2.0 g of dihydroguaiaretic acid was dissolved in 6 ml of official ethyl 
alcohol, and glycerin was then added thereto to give 100 g of a solution. 
This solution was added to two kinds of heart infusion agar media, one 
containing 1.0 g of sodium laurylsulfonate and the other, 1.0% of "Span 
20". Streptococcus mutans RIMD 3125001 was transplanted to the media. 
Incubation was conducted at 37.degree. C. for 72 hours. The concentration 
for growth inhibition was 1/1,000 (i.e., 1/50,000 calculated as 
dihydroguaiaretic acid) in both cases. This indicates that the 
effectiveness of the growth inhibitor is not affected by the surfactant 
used. 
EXAMPLE 3 
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parts by weight 
______________________________________ 
Calcium hydrogenphosphate, dibasic 
50 
Sodium carboxymethyl cellulose 
1 
Sodium lauryl sulfate 1.5 
Glycerin 25 
Saccharin 0.2 
Glycerin containing 1 wt % of dihydro- 
1 
guaiaretic acid 
Mint type flavor 1 
Water 20.3 
100 
______________________________________ 
The above components were sufficiently kneaded to prepare a tooth paste. 
EXAMPLE 4 
______________________________________ 
parts by weight 
______________________________________ 
90% Ethanol 20 
Saccharin 0.2 
Sodium N--acyl methyl taurate 
0.5 
Gelatin 0.5 
90% Ethanol containing 1 wt % of 
1.0 
dihydroguaiaretic acid 
Mint type flavor 0.5 
Water 77.3 
100 
______________________________________ 
The above components were mixed to prepare a mouth wash. 
EXAMPLE 5 
______________________________________ 
parts by weight 
______________________________________ 
Gum base 22 
Calcium carbonate 2 
Saccharin 0.1 
Sorbitol 14 
Lactose 60 
Glycerin containing 1 wt % of dihydro- 
1 
guaiaretic acid 
Mint type flavor 0.9 
100 
______________________________________ 
The above components were well kneaded at 0.degree. C., and after cooling, 
the blend was rolled and cut to prepare a chewing gum. 
EXAMPLE 6 
______________________________________ 
parts by weight 
______________________________________ 
Gum arabic 6 
Glucose 72 
Lactose 17 
Potassium phosphate, dibasic 
0.2 
Potassium phosphate, monobasic 
0.1 
Glycerin containing 1 wt % of dihydro- 
1 
guaiaretic acid 
Mint type flavor 0.7 
Magnesium stearate Proper amount 
______________________________________ 
The above components except magnesium stearate were sufficiently kneaded to 
prepare a plastic mass. The plastic mass was rolled and dried. After 
magnesium stearate was added, the mixture was cut to prepare a troche. 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it will be apparent to one skilled in the 
art that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.