JPH072687A - Cariostatic agent and antibacterial active substance - Google Patents

Abstract

PURPOSE:To provide the subject agent and substance suppressing the proliferation of Streptococcus in the oral cavity, free from risks of side effects, thus useful in troches or toothpaste for preventing or curing dental caries, containing, as active ingredient, Sophora exigua extract. CONSTITUTION:The objective agent/substance containing, as active ingredient, 5,7,2',6'-tetrahydroxy-8-lavandulyl flavanone of formula I or 5,7,2',4'- tetrahydroxy-8-lavandulyl flavanone of formula II which can be obtained by extracting Sophora exigua (pref. its roots), a kind of leguminous plant, with a solvent such as water, MeOH, EtOH or acetone, either singly or in the form of a combination thereof, or repeatedly.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anti-caries agent and an antibacterial active substance of a plant extract which can be used for caries prevention and treatment.

[0002]

2. Description of the Related Art Dental caries (dental caries) is two major diseases in dentistry as well as periodontal disease. The main cause of caries is due to various bacteria in dental plaque, and the development of periodontal disease is based on the development of dental plaque. Prevention of dental caries and suppression of plaque formation using antibacterial agents against these cariogenic bacteria in the oral cavity and plaque-forming bacteria are extremely important as well as dental treatment of the disease itself.

It is various bacteria belonging to the genus Streptococcus which are oral streptococci that play an important role as cariogenic bacteria and plaque forming bacteria. Conventionally, chlorhexidine and benzal chloride are used as the antibacterial agents. It is known that bactericides such as ruconium and antibiotics such as tetracycline and penicinline are effective. See also Shokoku 6
3-10133 is quercetin, kaempferol, etc.
JP-A-60-169413 discloses that shikonin contained in purple root and JP-A-62-210952 disclose that chrysin is effective in preventing and treating caries.

[0004]

However, bactericides such as chlorhexidine and benzalkonium chloride, and antibiotics such as tetracycline and penicin phosphorus have excellent antibacterial action, but since it is pointed out that side effects are possible, it is desirable. Preference is given to using plant extracts or substances extracted from plants.

However, quercetin, kaempferol and the like are mainly aimed at suppressing plaque formation of glucose polymer on the tooth surface and inhibiting synthesis of glucose polymer, and have an antibacterial activity that directly inhibits the growth of Streptococcus mutans. Since it is scarce, it is difficult to exert good antibacterial action. In addition, shikonin and chrysin contained in purple roots do not always exhibit a good antibacterial action against various bacteria of the genus Streptococcus.

An object of the present invention is to provide an anti-caries agent and an antibacterial active substance of a plant extract which can effectively suppress the growth of Streptococcus in the oral cavity.

[0007]

[Means for Solving the Problems] By the way, the oral cavity is a storage site of bacteria, and approximately 1100 g of bacteria of about 200 bacterial species inhabit 1 g of dental plaque. The genus Streptococcus occupies a large part of the bacterial flora, and plays an important role in the formation of plaque and subsequent caries and periodontal disease.

Oral Streptococcu
The genus s) usually includes the anaerobic Streptococcus mutans and Streptococcus rattu (Streptococcus rattu).
s), Streptococcus
us cricetus), Streptococcus oralis (Strep
tococcus oralis), Streptococcus sanguis, Streptococcus mitis, Streptococcus gordonii, Streptococcus pyogenus, and Streptococcus pyogenus.
livarius), most of which reside in plaque on the tooth surface and are closely associated with caries.

The pathogenicity of these mutans streptococcal groups is the ability to produce sticky glucose polymers from the sugars in the diet to firmly adhere to the tooth surface, and to metabolize dietary and endogenous sugars to produce lactic acid. It can be attributed to the ability to produce various organic acids such as the above and the ability to grow even under acidic conditions by the produced organic acids. Especially Streptococcus sanguis
), Streptococcus o
ralis) and Streptococcus gordonii (Strepto)
coccus gordonii) produces glucose polymers and is involved in early plaque formation. Furthermore, Streptococcus
Salivarius (Streptococcus salivarius) and Streptococcus mitis (Streptococcus mitis)
It is distributed predominantly on the tongue and buccal mucosa, and is involved in the bacterial flora formation of Streptococcus mutans, and thus in the formation of dental plaque and caries. That is, since all of these bacterial species actively bind to oral bacteria of other bacterial species, bacterial species that originally have no ability to directly attach to teeth and mucous membranes are also incorporated into plaque.

Thus, the characteristic of the genus Streptococcus in the oral cavity is cariogenicity or plaque forming ability. On the other hand, in edentulous patients, the genus Streptococcus decreases or disappears, but it increases again when the denture (denture) is attached. Streptococcus that attaches to dentures promotes the attachment of Candida albicans, the causative microorganism of denture stomatitis. It has also been pointed out that some strains of the genus Streptococcus are associated with the onset of endocarditis and Kawasaki disease.

Therefore, suppressing the growth of major bacteria of the genus Streptococcus in the oral cavity not only reduces the formation of plaque formation and caries, but also prevents the development of periodontal disease and stomatitis due to plaque development, It also helps prevent systemic infection.

Then, as a result of extensive studies to achieve the above object, it was found that an anti-caries agent containing Sophora exigua extract as a main component has an effect of effectively suppressing the growth of major bacteria of the genus Streptococcus in the oral cavity. I found it.

As a result of further investigation, among the Sophora exigua extracts, particularly 5,7,2 ′ of the following structural formula,
It has been found that a substance containing 6'-tetrahydroxy-8-labandryl flavanone (referred to as "Excigua flavanone A") as an active ingredient exhibits excellent antibacterial activity.

[0014]

[Chemical 3]

A substance containing 5,7,2 ', 4'-tetrahydroxy-8-labandrylflavanone (referred to as "sophoraflavanone G") having the following structural formula as an active ingredient also exhibits excellent antibacterial activity. I found that.

[0016]

[Chemical 4]

All of these have remarkably exerted a growth-inhibiting effect on various bacterial cells.

By the way, Sophra e
xigua) is a small shrub that belongs to the legume family and grows mainly in Southeast Asia. The plant of the same genus has been called broad root for a long time in China and has been used as a crude drug with anti-inflammatory and antitumor activity. Sophora exigua can be used as its tree, leaves, flowers and the like, but it is particularly preferable to use a root portion containing a large amount of an antibacterial substance that effectively inhibits the growth of cariogenic bacteria and the like.

The Sophora exigua extract is a solvent such as water, methyl alcohol, ethyl alcohol, acetone, ethyl acetate, benzene, ethyl ether, dichloromethane, chloroform and n-hexane, which may be used alone or in any combination, or may be repeated. Obtained.

[0020]

[Examples] 120 g of roots of Sophora exigua were crushed and then extracted with 0.5 liter of chloroform, and a part thereof was subjected to silica gel column chromatography (50 mmφ ×
500 mm), and mixed with chloroform-methanol mixture (1
It was developed at 0: 1) to obtain the extract extracts of fractions Fr1-4.

The extracted extracts of the obtained fractions Fr1 to 4 were measured for antibacterial activity at a constant concentration (125 μg / ml) against major Streptococcus involved in caries. The antibacterial activity was determined by the agar plate method according to the standard method of Chemotherapy Society. E. coli as a control strain
(ATCC 25922) was used. The medium used for both Streptococcus and E. coli was Muller Hinton.
ton) A medium to which 0.5% yeast extract and 0.5% glucose were added was used. The culture was anaerobic at 37 ° C. (gas phase, N ₂ 80%, H ₂ 10%, CO ₂ 10%, Ana-erob.
ic glove box, AZ type Hirasawa) for 3-5 days.

Table 1 shows the results. In the table,
(+) Indicates that antibacterial activity is recognized, and (-) indicates that antibacterial activity is not recognized.

[0023]

[Table 1]

From Table 1, the extract of fraction Fr4 was found to have antibacterial activity against major Streptococcus. To further evaluate the intensity of the activity, the extract of fraction Fr4 was treated with silica gel in the same manner as above. Column chromatography (50 mm
(φ × 500 mm), develop with a mixed solution of chloroform-methanol (10: 1), and collect fractions Fr4-1 to 4-
6 extracts were obtained. Obtained fraction Fr4-
Concerning the extracts 1 to 4-6, this time, the concentration is 50μ.
Antibacterial activity was measured in g / ml. The test method is the same as above. The results are shown in Table 2. (+) Indicates that antibacterial activity was observed, and (-) indicates that antibacterial activity was not observed.

[0025]

[Table 2]

From Table 2, it was observed that the fraction Fr4-1 had a strong activity in the intermediate fractions Fr4-2 and Fr4-3.

Therefore, even lower concentrations of these fractions (6.25 μg / ml and 25.0 μg / m) were obtained.
When various Streptococcus were examined in 1), as shown in the following table, the antibacterial activity was recognized especially in the fraction Fr4-2 even at 6.25 μg / ml.

[0028]

[Table 3]

Therefore, since it was found that the anti-caries substance contained in the root part of Sophora exigua was localized in the fraction Fr4-2, the compound contained in the fraction was isolated and its structure was determined. It was

40 g of the chloroform extract of the root of Sophora exigua was layered on silica gel column chromatography (50 mmφ × 500 mm) and developed with n-hexane-acetone (15: 1), and the content of acetone in the developing solvent was sequentially added. Continued to expand while increasing. Each fraction was collected by 500 ml and concentrated. About the fraction thus obtained, by thin layer chromatography,
Fractions containing similar components were collected using the above-mentioned fraction Fr4-2 as a reference substance, and again subjected to column chromatography (30 mmφ × 500 mm) to prepare a mixed solvent (10: 1) of n-hexane-acetone as a developing solvent. The compound 1 (Fr4-2-1) and the compound 2 (Fr
4-2-2) and compound 3 (Fr4-2-3) were each isolated in pure form.

For the isolated compounds 1, 2 and 3, the minimum inhibitory concentration (MI) was determined for various Streptococcus.
C) was determined. Table 4 shows the detailed results. The minimum inhibitory concentration (MIC) was performed under the same conditions as in the above-mentioned antibacterial activity test. In addition, for comparison, tetracycline, penicin phosphorus G, erythromycin, as natural antibiotics, quercetin, shikonin, chrysin, and chlorhexidine as fungicides were also evaluated. They are shown in Table 5 and Table 6, respectively.

[0032]

[Table 4]

[0033]

[Table 5]

[0034]

[Table 6]

From Table 4, Table 5 and Table 6, 50 or 100 μg / ml of conventional quercetin, shikonin and chrysin were obtained.
In contrast to the above, the compound of this example is the compound 1
Although the antibacterial activity was somewhat poor, the compounds 2 and 3 had an antibacterial activity of approximately 12.5 to 3.13 μg / ml, which was quite close to that of the bactericide chlorhexidine. Compared with antibiotics such as tetracycline, penicillin G, erythromycin, the antibacterial activity is weak, but considering the side effects of antibiotics and fungicides,
It can be seen that the compound of this example exhibits excellent antibacterial activity.

Further, even the above-mentioned extract exhibits antibacterial activity equivalent to or higher than that of conventional quercetin, shikonin, and chrysin, and it can be said that productivity is high.

The structures of Compounds 2 and 3 are as follows by analyzing various instrumental spectral data, and for Compound 1, 5,7,2 ′, 6′-tetrahydroxy-8-labandrylflavanone (“exciguaflavanone”) was obtained. A "),
Compound 2 was determined to be 5,7,2 ', 4'-tetrahydroxy-8-labandryl flavanone ("Sophora flavanone G").

5,7,2 ', 6'-tetrahydroxy-
8-Labandryl flavanone (Excigua flavanone A) Melting point 175-178 ° C (chloroform-hexane),
Colorless needles electron impact ionization mass spectrometry spectrum (EIMS) m
/ Z (relative intensity): 424 (70), 406 (4), 301 (78), 283 (84), 2
09 (12), 165 (100) High resolution electron impact ionization mass spectrometry spectrum (HRM
S): 424.1877 (calculated value from the C ₂₅ H ₂₈ 0 ₆ 424.1886
) Circular dichroism spectrum (CD) (Δε, methyl alcohol): 289 (-14.0), 333 (+1.5) UV absorption spectrum (UV) λ max nm (log ε):
293 (4.5), 340 (3.9), + NaOCH ₃ : 333; + AlCl ₃ : 315,392; + A
lCl ₃ -HCl: 315,392; + NaOCOCH ₃ : 293,340 proton-nuclear magnetic resonance spectrum ( ¹ H-NMR) (C
DCl ₃ ) δ: 1.49 (3H, br s.7 "-CH ₃ ), 1.56 (3h, br s,
6 "-CH ₃ ), 1.64 (3H, br s, 10" -CH ₃ ), 2.08 (2H, m, H-3 "), 2.
55 (1H, m, H-2 "), 2.67 (2H, m, H-1"), 2.77 (1H, dd, J = 17.2,2.
9 Hz, H-3 "), 3.07 (1H, dd, J = 17.2,13.2 Hz, H-3), 4.60 (2H,
br s, H-9 "), 5.02 (1H, m, H-4"), 5.68 (1H, dd, J = 13.2,2.9 H
z, H-2), 6.01 (1H, s, H-6), 6.42-6.48 (2H, m, H-3 ', 5'), 7.40
(1H, d, J = 8.1 Hz, H-6 '), 8.36,8.63,9.51 (1H, each s, OH),
_{12.19 (1H, s, C 5} -OH) Carbon 13 nuclear magnetic resonance spectrum ⁽¹³ C-NMR)
(Acetone-d ₆ ) δ: 718.3 (q, C-7 "), 19.6 (q, C-10"), 2
6.3 (q, C-6 "), 28.2 (t, C-1"), 32.4 (t, C-3 "), 43.3 (t, C-3),
48.3 (d, C-2 "), 75.8 (d, C-2), 96.7 (d, C-6), 103.7 (s, C-1
0), 103.9 (d, C-8,3 '), 111.6 (t, C-9 "), 118.3 (s, C-5'), 12
4.9 (d, C-4 "), 129.1 (d, C-6 '), 132.1 (s, C-5"), 149.6 (s, C-
8 "), 156.6 (s, C-2 '), 159.9 (s, C-4'), 162.6 (s, C-9), 163.5
(s, C-5), 165.8 (s, C-7), 198.7 (s, C-4)

5,7,2 ', 4'-tetrahydroxy-
8-Labandryl flavanone (Sophora flavanone G) Melting point 178.5-179 ° C. (hexane), colorless needle crystal Electron impact ionization mass spectrometry spectrum (EIMS) m
/ Z (relative intensity): 424 (11), 301 (100), 283 (93), 165 (7
3), 136 (4) High resolution electron impact ionization mass spectrometry spectrum (HRM
S) m / z: [M ] + 424.1917 (C 25 calcd 424.1886 from H ₂₈ 0 ₆₎ Circular dichroism spectrum (CD) ([Delta] [epsilon], methyl alcohol): 273 (-7.3), 303 (+3.1) Ultraviolet absorption spectrum (UV) λmax nm: 292,338; + A
lCl _3: 314,392 Proton - Nuclear magnetic resonance spectra ⁽¹ H-NMR) (acetone _{-d 6) δ: 1.48 (3H} , br s.7 "- CH 3), 1.54 (3h, b
rs, 6 "-CH ₃ ), 1.61 (3H, br s, 10"-CH ₃ ), 2.08 (2H, m, H-
3 "), 2.54 (1H, dd, J = 17,3Hz, H-3eq), 2.56-2.60 (3H, m, H-
1 ", 2"), 3.87 (1H, dd, J = 17,14 Hz, H-3ax), 4.55 (2H, br s, H
-9 "), 4.98 (1H, t like, H-4"), 6.01 (1H, s, H-6), 6.02 (1H, d
d, J = 14,3 Hz, H-2), 6.47 (2H, d, J = 8 Hz, H-3 ', 5'), 7.05 (1
H, t, J = 8 Hz, H-4 '), 8.50 (2H, br s, C _2.6 -OH), 9.45 (1H, br
s C ₇ -OH), 12.27 (1H, s, C ₅ -OH), 12.27 (1H, s, C ₅ -O
H) Carbon 13-nuclear magnetic resonance spectrum ( ¹³ C-NMR)
(Acetone-d ₆ ) δ: 18.3 (C-6 "), 19.6 (C-10"), 26.3 (C-
7 "), 28.3 (C-1"), 32.5 (C-3 "), 41.2 (C-3"), 48.4 (C-2 "), 7
4.3 (C-2), 96.8 (C-6), 103.8 (C-10), 108.4 (C-8), 108.9 (C
-C-3 ', 5'), 111.6 (C-9 "), 112.4 (C-1 '), 125.0 (C-4"), 131.
3 (C-4 '), 132.1 (C-5 "), 148.7 (C-8"), 158.2 (C-2', 6 '), 16
2.8 (C-9), 163.7 (C-5), 165.7 (C-7), 199.2 (C-4)

The present invention is not limited to the above embodiment. In particular, 5,7,2 ', 6'-tetrahydroxy-8-labandryl flavanone and 5,7,2', 4'-tetrahydroxy-8-labandryl flavanone are synthesizable substances.

[0041]

INDUSTRIAL APPLICABILITY As described above, the present invention provides Sophora exigua extract, 5,7,2 ', 6'-tetrahydroxy-8-labandryl flavanone, 5,7,2', 4 '.
-Because it is an anti-caries agent or an antibacterial active substance containing tetrahydroxy-8-labandryl flavanone as an active ingredient, there is no risk of side effects unlike conventional fungicides and antibiotics.
In addition, compared with conventional natural substances such as plants, the growth of Streptococcus in the oral cavity can be dramatically suppressed.

Therefore, not only the occurrence of dental caries and the formation of dental plaque are reduced, but it is also useful for the prevention of periodontal diseases and stomatitis due to the development of dental plaque, and for the prevention of systemic infection evidence.

Therefore, it can be applied to various medicines, quasi-drugs, foods and drinks, favorite foods and the like. Like troches, toothpaste,
Mouthwash, chewing gum, candy,
It can be mixed with candy, syrup, jelly, etc. The present invention is not limited to such use. In addition, since the present anti-caries agent and antibacterial active substance exhibit exceptional antibacterial activity against various cariogenic bacteria and various Streptococcus species which are plaque-forming bacteria, they exhibit excellent antibacterial activity against other periodontal bacteria. It is also possible to use it in combination with other oral plant extracts or natural substances that exert the above effects. For example, when used in combination with propolis or erythrina extract, it is extremely preferable as an oral composition. Further, in order to suppress side effects or further improve antibacterial activity, it is also possible to partially use a bactericidal agent or an antibiotic. Regarding the applied amount, it is acceptable to use the corresponding amount from the above examples according to various uses.

Claims (3)

[Claims] 1. An anti-caries agent containing Sophora exigua extract as an active ingredient. 2. An antibacterial active substance containing 5,7,2 ′, 6′-tetrahydroxy-8-labandrylflavanone of the following structural formula as an active ingredient. [Chemical 1] 3. An antibacterial active substance containing 5,7,2 ′, 4′-tetrahydroxy-8-labandrylflavanone of the following structural formula as an active ingredient. [Chemical 2]