KR20020018695A - Extracts showing excellent anti-microbial effect extracted from Pulsatilla koreana Nakai et Mori, a preparing method thereof and pharmaceutical compositions containing them - Google Patents

Abstract

PURPOSE: A process of preparing a Pulsatillae Radix extract and a pharmaceutically acceptable salt from Pulsatillae Radix is provided. Whereby, a composition containing the extract can be used as an oral rinse and a treating agent for periodontal disease having an antimicrobial effect. CONSTITUTION: The process comprises the following four stages. A ground Pulsatillae Radix is extracted and filtered after defatted to produce a methanol extract, which is fractionated several times into a chloroform layer and a water layer using chloroform and water to produce a chloroform extract, which is re-fractionated several times into an ethyl acetate layer and a water layer using ethyl acetate and water and then the ethyl acetate layer is concentrated to produce an ethyl acetate extract. The above water layer is fractionated several times into a butyl alcohol layer and a water layer using butyl alcohol and water to produce a butyl alcohol extract.

Description

Extracts showing excellent anti-microbial effect extracted from Pulsatilla koreana Nakai et Mori, a preparing method about and pharmaceutical compositions containing them}

The present invention relates to a baekduong extract, a preparation method thereof, and a pharmaceutical composition comprising the same, and more particularly, baekduong extract excellent in antibacterial effect obtained by extracting baekduong with an organic solvent, a pharmaceutically acceptable salt thereof, and a preparation method thereof. And it relates to a pharmaceutical composition for oral cleansing and periodontal disease treatment comprising the same as an active ingredient.

Periodontal sacs of normal teeth have Gram-positive bacteria and Streptococcus (Streptococcus)And actinomyces (Actinomyces) If the periodontal mixture such as gingivitis continues to occur, the percentage of streptococcus in the subgingival periodontal sac decreases and the proportion of anaerobic bacteria increases, leading to an increase in gingival fissure fluid, a decrease in bleeding and redox potential, and so on. As this worsens, periodontal disease is induced.

The periodontal disease is a disease in which periodontal tissue is destroyed by plaque and calculus mainly composed of bacteria, which is the main cause of tooth loss in modern people after middle age, and is divided into acute periodontal disease and chronic periodontal disease. In order to suppress such periodontal disease, proper plaque control ability is required, but the most important of the plaque control ability is the precise brushing method, so it is possible to suppress plaque formation primarily by using a toothbrush, floss and water jet. have. However, the use of chemical supplements is required to suppress plaque formation due to morphological weaknesses of the teeth.

The chemical adjuvant has generally been used in toothpaste, chewing gum and oral dentifrice, and is mainly used in oral dentifrice. Pharmaceuticals used for oral fern include bisguanides such as chlorhexidine, pyrimidine inducers such as hexidine, and vegetable alkaloids such as sanguinarine. alkaloid), quaternary ammonium compound preparations, and various antibiotics, and these have excellent antibacterial effects on oral bacteria.

However, quaternary ammonium compounds that exhibit excellent antimicrobial effects on oral bacteria are known to be difficult to maintain appropriate concentrations in the oral cavity. Also, a kind of benzophenan-threedine extracted from Sanguinaria Canadensis .

Sanguinerin, a plant alkaloid in quaternaryiminum ionic state that is fairly stable with (benzophenan-thridine), has excellent antibacterial effects on bacteria involved in gingivitis and periodontitis in vivo and ex vivo, The bond strength is too strong to reduce the drug efficacy. Sodium lauryl sulfate, which is added as an abrasive or ingredient in toothpaste, acts as an antagonist to the acid guinarin, resulting in lowering the antibacterial effect of the acid guinarin. (Johnson, JR et al., Scand. J. dent. Res. , 83, 288, 1975; Van der Ouderaa, FJ and Cummins, D., J. Dent. Res ., 68, 1722, 1989) . In addition, antibiotics are not widely used due to the emergence of resistant strains and induction of tissue hypersensitivity reactions. Chlorohexidine, the most widely used bisguanide agent, also has excellent plaque suppression effect, but the teeth are colored and tasted. damaged and can not be used for a long period of time can be the inflammation in the mucosa induced (Gusberti, FA et al, J. Clin periodontol 15, 60, 1988;...... Eriksen, HM et al, J. Clin Periodontol 12, 345 , 1985).

For the above reason, the necessity of developing a drug that exhibits strong antimicrobial activity against the pathogen of periodontal disease for a long time does not destroy the natural equilibrium of oral bacteria, does not form resistant strains, and has no body side effects. As part of this research, researches on natural extracts with high safety and low resistance to strains and tissue detriment have been conducted as drugs for treating and preventing periodontal disease that satisfies the above requirements.

Natural extracts that have long been used for periodontal disease in oriental medicine include green tea ( Theae folium ), myrrh ( Myrrha ), Mori radicis cortex , horse riding ( Cimicifugae rhizoma ) and ginseng saponine ( Ginseng Saponine ). It has the effect of inflammatory, anti-inflammatory and analgesic (handeokryong et al., modern pharmacognosy, 45, 76, 189, 248, 338, 1987 school use;. More handaeseok, pharmacognosy, 1983, 385, open branch).

Myrrh of the natural extracts are effective in gingivitis, thrush and oral preservation, and Sanggenon C, the main component of the extract of Sangbaekpi, inhibits the metabolism of Streptococcus mutans to form glucan. Decreases and interferes with bacterial attachment, the first stage of plaque formation. Also, polyphenols, which are components of green tea ( Theae folium ),

phenols) is an extensive antimicrobial agent that suppresses the induction of dental caries in animal experiments. Especially, triclosan is a nonionic substance that inhibits plaque formation and is mixed with zinc citrate. The application of plaque formation was reported to be better when applied (Saxton, CA et al., J. Periodontol. 9, 555-561, 1986).

In addition, research on the use of various natural extracts as herbal medicines for periodontal disease is underway, and Min et al.Zea Mays L.,Below "ZMLAbbreviated as ")When both ascorbic acid and ascorbic acid were administered, the epithelium was regenerated, connective tissue proliferated, and osteoblasts were observed.Korean Journal of Periodontal Science, 18 (2), 6-23. 1988), Choi et al after periodontal surgeryZMLof When administered, it was confirmed that the gingival index and tooth agitation decreased and the incidence of alveolar ringworm increased (Sang, Mook et al.,Korean Journal of Periodontal Science, 19 (1), 63-70, 1989), Kim et al.ZMLWas found to significantly reduce the depth of the periodontal pocket, plaque index, and loss of adhesion (Kim et al.,Korean Journal of Periodontal Science, 21 (2), 225-234, 1991).

In addition, Centella asiatica L Urban extract has been found to be effective in wounds, burns, bedsores and ulcers as a natural extract to help regenerate periodontal tissues. It has been reported to have been developed as a periodontal treatment (元 村 洋 一, 日 齒 周誌 . 36, 474-479, 1994), and Jung et al . Showed that Scutellaria baicalensis extract had anti-inflammatory activity It has been shown to enhance the cellular biological function of fibroblasts (Chung, CP et al., Planta. Medica. 61, 11-14, 1995).

In addition, it has been reported that Rhiszoma coptidis promotes the activity of periodontal ligament cells and inhibits the production of interleukin-6 (IL-6) (Song Ki-Bum et al . , 26 (3), 641-654). , 1996), Magnoliac cortex extract Magnolol and Honokiol are cytokines of inflammation.

ines) production and dental caries, have antimicrobial effect against plaque bacteria and periodontal disease pathogens, and inhibit collagen degrading enzyme production from gingival fibroblasts (Chang, BS et al. , Planta.Med . , 64, 367-369, 1998; Shen, D. et al., Chung Kuo Chung Hsi Chieh Ho Tsa Chih , 10, 589-591, 1994).

Lastly, sugar substitutes such as Mannitol and Xylitol, which are natural extracts from birch trees, were used to treat Streptococcus spp.

coccus) inhibits the reduction of plaque pH, but the effect of inhibiting plaque formation is very weak and the high water solubility of the sugar substitutes makes it difficult to maintain effective concentrations in the oral cavity. (Mhlemann, HR et al., Hev. Odontol. Acta. , 15, 107, 1971).

On the other hand, baekduong (白頭翁, Pulsatilla koreana Nakai et Mori ) is a perennial plant that country as well as belonging to Apiaceae (ranunculaceae) wild in northeastern China. Baekduong is commonly known to have effects such as toothache, antipyretic, edema, anti-inflammatory, astringent, hemostasis, dysentery, detoxification, scrotum subpopulation, antibacterial, soothing and anticancer (Chang, HM et al., Oriental Healing Arts Institute , 226, 1986). In addition, the stems and leaves of baekduong have an effect on anti-inflammatory, analgesic, antipyretic and detoxification, and the whole stomach except for the root has a strong heart effect, and it has been widely used as a medicinal herb since ancient times, especially on fresh stems and leaves of baekduong. It is known that the squeezed juice has antimicrobial activity against Staphylococcus aureus (Shin Min-kyo and Jung-sup Lee, Medicinal Herbal Medicine Metabolism , Yeonglamsa, 495, 1990). Baekduong is the main ingredient of saponin, herbicidal substance 11-hoxadecanoic acid, 14-methylpentadecanoic acid, 1,2-benzenedicarboxylic acid (1, 2-benzenedicarboxylic acid, 6-octadecenoic acid, 15-methyl-heptadecanoic acid, and the toxic substance anemonin. Has anti-inflammatory activity and is hydrolyzed and converted to triterpen-type genin, glucose, rhamnose and unknown sugars. In addition, SB-31β, an extract of baekduong, exhibits strong anti-cancer effects while being less toxic to various solid cancers, and methanol extract obtained from baekduong increased blood glucose levels by alloxan administration and total cholesterol content in serum. Have been reported to restore normal levels (Kim, SY and Kim, SB J. Kor. Cancer . 26 (6): 959-963, 1994).

Therefore, the inventors of the present invention tried to develop oral cleaning agent and periodontal disease treatment agent using the baekduong, four baekduong extract from the organic solvent from the baekduong and three of these baekduong extracts inhibit the growth of the periodontal disease pathogens The present invention was completed by confirming the excellent antimicrobial effect.

It is an object of the present invention to provide a baekduong extract and / or a pharmaceutically acceptable salt thereof, which exhibits excellent antimicrobial effect as a mouthwash and periodontal disease treatment.

It is also an object of the present invention to provide a method for extracting a baekduong extract showing an excellent antimicrobial effect from baekduong.

In addition, it is an object of the present invention to provide a pharmaceutical composition for oral cleansing and periodontal disease treatment excellent antimicrobial effect by containing the baekduong extract as an active ingredient.

Figure 1a shows the results of the growth inhibition effect on the various periodontal disease pathogens of the Baekduong extract of the present invention using a plate diffusion method (Kirby-Bauer), a bacterial growth inhibition effect test method,

A: Bacteroides forsythus

B: Streptococcus mutans (ATCC 25175)

C: Streptococcus sanguis

D: Prevotella nigrescens (ATCC 33563)

a: methanol extract of the baekduong extracts of the present invention

b: ethylacetate extract of the baekduong extracts of the present invention

c: Chloroform extract of the baekduong extracts of the present invention

d: Butyl alcohol (butylalcohol) extract of the extracts of the present invention

Figure 1b shows the results of confirming the growth inhibitory effect on various periodontal disease pathogens of the Baekduong extract of the present invention using the disc diffusion method,

A: Actinobacillus actinomycetemcomitans (ATCC 33384)

B: Eikenella corrodens (ATCC 23834)

C: Prevotella intermedia (ATCC 25611)

D: Actinomyces viscosus

a: methanol extract of the baekduong extracts of the present invention

b: ethyl acetate extract of the baekduong extracts of the present invention

c: Chloroform extract of Baekduong extracts of the present invention

d: butyl alcohol extract of the baekduong extracts of the present invention

Figure 1c shows the results of confirming the growth inhibitory effect on the various periodontal disease pathogens of the Baekduong extract of the present invention using the disc diffusion method,

A: Staphylococcus aureus (ATCC 65389)

B: Pseudomonas aeruginosa

C: E. coli

a: methanol extract of the baekduong extracts of the present invention

b: ethyl acetate extract of the baekduong extracts of the present invention

c: Chloroform extract of Baekduong extracts of the present invention

d: butyl alcohol extract of the baekduong extracts of the present invention

Figure 2 shows the bacterial arrangement in the medium of the disk diffusion method for investigating the minimum inhibitory concentration for the pathogen of periodontal disease pathogen extract of the present invention,

1: Bacteroid Four Cedars (ATCC 43037)

2: Streptococcus mutans (ATCC 25175)

3: Streptococcus sanguise (ATCC 10556)

4: Popillomonas Gingalis (ATCC 33277)

5: Actinobacillus Actinomycetem Comitans (ATCC 33384)

6: Akenella Corrodens (ATCC 23834)

7: Prebotella Intermediate (ATCC 25611)

8: Actinomyces Viscous (ATCC 15987)

9: Prevotela Nigressen (ATCC 33563)

10: Rosia Dentcariosa (ATCC 17931)

11: Fusobacterium Nuclitum (ATCC 25586)

12: Pseudomonas Aruginosa (ATCC 9027)

13: Staphylococcus aureus (ATCC 65389)

Figure 3 shows the results of the investigation of the minimum inhibitory concentration (Minimal inhibitory concentration, abbreviated as "MIC") for the periodontal disease pathogen of methanol extract of the extract of the present invention by the Agar dilution method,

A: control

B: Experimental group containing 20 μl / ml methanol extract

C: experimental group containing 10 μl / ml methanol extract

D: experimental group containing 5 μl / ml methanol extract

E: experimental group containing 2.5 μl / ml of methanol extract

Figure 4 shows the results of the investigation of the MIC for the periodontal disease pathogen of ethyl acetate extract of Baekduong extract of the present invention by agar dilution method,

A: control

B: Experimental group containing 20 μl / ml ethyl acetate extract

C: Experimental group containing 10 μl / ml ethyl acetate extract

D: Experimental group containing 5 μl / ml ethyl acetate extract

E: experimental group containing 2.5 μl / ml ethyl acetate extract

Figure 5 shows the results of the investigation of the MIC for the periodontal disease pathogen of the chloroform extract of the extract of the present invention by agar dilution method,

A: control

B: Experimental group containing 20 μl / ml chloroform extract

C: Experimental group containing 10 μl / ml chloroform extract

D: experimental group containing 5 μl / ml chloroform extract

E: experimental group containing 2.5 μl / ml chloroform extract

Figure 6 shows the results of the investigation of the MIC for the periodontal disease pathogen of the butyl alcohol extract of the baekduong extract of the present invention by agar dilution method.

A: control

B: Experimental group containing 20 μl / ml butyl alcohol extract

C: experimental group containing 10 μl / ml butyl alcohol extract

D: experimental group containing 5 μl / ml butyl alcohol extract

E: experimental group containing 2.5 μl / ml butyl alcohol extract

In order to achieve the above object, the present invention provides a baekduong extract and / or a pharmaceutically acceptable salt thereof showing excellent antibacterial effect.

Baekduong extract of the present invention is an acetylacetate extract, chloroform extract and butyl alcohol extract. Particularly preferred extracts of the extracts are butyl alcohol extracts.

Baekduong extract of the present invention is a natural extract showing an excellent antimicrobial effect than the existing periodontal disease treatment agent Honokiol and listerin or myrrh and ginseng saponin reported to have an antimicrobial effect against the periodontal disease pathogens. Specifically, ethyl acetate extract and chloroform extract of the Baekduong extract of the present invention show excellent antimicrobial effect against some periodontal disease pathogens, including prevotelella nigressen (ATCC 33563), and butyl alcohol extract is Staphylococcus aureus. It showed excellent antimicrobial activity against 13 periodontal disease pathogens consisting of Gram positive bacteria including U.S. (ATCC 65389) and Gram negative bacteria including E. coli. .

Baekduong extract of the present invention can be used in the form of a pharmaceutically acceptable salt, and the salt is an acid addition salt formed by a pharmaceutically acceptable free acid. The baekduong extract may form a pharmaceutically acceptable acid addition salt according to methods conventional in the art. Free acid and inorganic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuric acid, embonic acid, glutamic acid or aspartic acid Can be used.

In addition, the present invention provides a method for producing a baekduong extract showing an excellent antimicrobial effect.

The manufacturing method of the present invention

1) degreasing the ground baekduong, and extracted with methanol, filtered and dried in vacuo to obtain a methanol extract (step 1);

2) repeatedly fractionating the methanol extract of step 1 into a chloroform layer and a water layer using chloroform and water, and then concentrating the chloroform layer to obtain a chloroform extract (step 2);

3) repeatedly distilling the water layer of step 2 into an ethyl acetate layer and a water layer several times using ethyl acetate and water, and then concentrating the ethyl acetate layer to obtain an ethyl acetate extract (step 3); And

4) The water layer of step 3 is divided into butyl alcohol layer and water layer several times using butyl alcohol and water and then concentrated butyl alcohol layer to obtain a butyl alcohol extract (step 4).

In addition, the present invention provides a pharmaceutical composition for oral cleansing and periodontal disease treatment comprising a baekduong extract and / or a pharmaceutically acceptable salt thereof as an active ingredient.

Baekduong extract of the present invention exhibits excellent antimicrobial effect by inhibiting the growth of periodontal disease pathogens.

Baekduong extract of the present invention can be administered orally or parenterally during clinical administration, for example, can be applied intravenously, subcutaneously, intraperitoneally or topically, and can be used in the form of general pharmaceutical preparations.

The pharmaceutical composition of the present invention may be combined with a conventional carrier in the pharmaceutical field at the time of clinical use, and oral administration of a conventional agent in the pharmaceutical field, for example, tablets, capsules, crokies, solutions, suspensions, and the like. Preparations for use; It may be formulated into a variety of preparations, such as injectable solutions or suspensions, or injectable preparations in the form of ready-to-use injectable dry powders that can be prepared and used as injectable distilled water upon injection.

As a result of acute toxicity test, the extract of Baekduong of the present invention was very stable with MLD value of 120 g / kg or more.

Hereinafter, the present invention will be described in detail.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Baekduong Extract

In order to obtain four kinds of Baekduong extracts extracted from methanol, chloroform, ethyl acetate and butyl alcohol from the Baekduong, the inventors obtained the respective extracts from the well-dried Korean Baekduong commercially available using the above four organic solvents.

First, 150 mg of baekduong was crushed with a homogenizer, degreased with petroleum ether, and then extracted three times with 100 ml of 100% methanol to obtain an extract. The extract was filtered with filter paper and then vacuum dried (TYPE N-N, EYELA, Japan) to obtain a residue and methanol extract.

The methanol extract was repeatedly fractionated into chloroform layer and water layer several times using chloroform and water, and then the chloroform layer was concentrated to obtain a chloroform extract. The water layer, from which the chloroform layer was removed, was repeatedly partitioned into ethyl acetate layer and water layer several times using ethyl acetate and water, and then the ethyl acetate layer was concentrated to obtain an ethyl acetate extract. In addition, the water layer from which the ethyl acetate layer was removed was repeatedly fractionated several times into a water layer and a butyl alcohol layer using butyl alcohol and water, and the butyl alcohol layer was concentrated to obtain a butyl alcohol extract.

Experimental Example 1 Investigation of Antimicrobial Effect of Baekduong Extract against Periodontal Disease Pathogen

In order to examine whether the methanol extract, chloroform extract, ethyl acetate extract, and butyl alcohol extract extracted from Baekduong in Example 1 have antimicrobial effects against the periodontal disease pathogen, the present inventors prepared a standardized bacterium growth inhibition test. The antimicrobial effect was observed by the diffusion method (Kirby-Bauer method, AJCP., 45, 493-496, 1996), and the Agar dilution method-2-fold dilution method, NCCLS (the National Committee for Clinical Laboratory Standards) VOL. 5 NO. 2] was used to determine the minimum inhibition concentration (hereinafter, abbreviated as "MIC") of four Baekduong extracts of the present invention against 14 periodontal disease pathogens.

The 14 periodontal disease pathogens used to investigate the antimicrobial effect of Baekduong extract of the present invention are as shown in Table 1 below, and these are from Gangneung University, Yonsei University, Korean Collection for Type Culture (KCTC) and Korean Culture Center of Microorganism (KCCM). Strains that were distributed in BHIA (Brain Heart Infusion Agar, supplemented by Vit. K and Hemin Sol, Difco, USA) medium and BHIA medium containing fibrin-free 5% sheep blood (Korean medium, Korea) as needed Subculture was used for the experiment.

<Table 1> Periodontal disease pathogens used to investigate the antimicrobial effect of Baekduong extract of the present invention

Etiology Accession number Actinobacillus actinomycetem comitans ATCC 33384 Actinomyces viscosus ATCC 15987 Rothia dentocariosa ATCC 17931 Porphylomonas gingivalis ATCC 33277 Streptococcus sanguis ATCC 10556 Streptococcus mutans ATCC 25175 Fusobacterium nucleatum ATCC 25586 Eikenella corrodens ATCC 23834 Bacteroides forsythus ATCC 43037 Prevotella intermedia ATCC 25611 Prevotella nigrescens ATCC 33563 E. coli ATCC 8739 Staphylococcus aureus ATCC 65389 Pseudomonas aeruginosa ATCC 9027

The four baekduong extracts obtained in Example 1 were tested by the plate diffusion method, the most widely used standardized method for the antimicrobial susceptibility test of anaerobic bacteria, and a 6 mm paper disk was used.

MIC of Baekduong extract of the present invention for each bacterium was determined according to NCCLS, and the concentration of bacteria was diluted with physiological saline (0.9% NaCl) to unify at a concentration of 10 ⁴ to 10 ⁶ CFU (Colony forming Unit) per ml. Used.

First, the four kinds of Baekduong extract stock solution of the present invention obtained in Example 1 were adjusted to 20 µl / ml, respectively, and serially diluted 2 times to obtain the concentration of the Baekduong extract at 10 µl / ml, 5 µl / ml and 2.5 µl / ml. After adjustment, 20 μl each of these were used as solid plate medium (BHIA, Difco, USA, Brain heart infusion broth, Yeast extract, Distilled water, Resaqurin solution 0.025%, Cystein HCl 2H ₂ O, Hemin solution, Vitamin K1 solution, Agar) After smearing directly on and left for 30 minutes, it was used for the experiment. 14 microorganisms of Table 1 were inoculated into 1 μl of solid plate medium coated with the Baekduong extract of the present invention using a multipoint inoculator (Bootles, England), followed by 7-10% CO ² concentration and 37 Cultivation was carried out for 72 hours in an incubator maintained at ℃, and was used as a control was inoculated with 14 periodontal disease pathogens in a medium containing no baekduong extract of the present invention. The results of measuring the growth inhibition of the bacteria in the medium after the culture is shown in the following table, MIC of the Baekduong extract of the present invention for each bacteria is shown in Table 3 below.

TABLE 2 Growth Inhibitory Effects of Baekduong Extract of Example 1 on 14 Periodontal Disease Pathogens

Etiology A B C D E Actinobacillus Actinomycetem Comitans (ATCC 33384) - - - 7 - Actinomyces Viscous (ATCC 15987) - - - 8 - Rosia dentocariosa (ATCC 17931) - - - 8 - Popillomonas Gingalis (ATCC 33277) - - - - - Streptococcus sanguise (ATCC 10556) - 10 11 8 - Streptococcus mutans (ATCC 25175) - 8 8 8 - Fusobacterium nuclitum (ATCC 25586) - - - 8 - Akenella Corodens (ATCC 23834) - - - 7 - Bacteroid Four Cedars - - - 7 - Prebotella Intermediate (ATCC 25611) - - - 7 - Prebotella nigressen (ATCC 33563) - 9 - 8 - Escherichia coli (ATCC 8739) - - - 8 - Staphylococcus aureus (ATCC 65389) - - - 8 - Pseudomonas aruginosa (ATCC 9027) - - - 8 -

(Unit: mm, paper disc: 6 mm, concentration of each extract: 20 μl / ml)

A: Methanol Extract

B: ethyl acetate extract

C: Chloroform Extract

D: Butyl Alcohol Extract

E: control

Table 3 Minimum Inhibitory Concentration (MIC) of Baekduong Extract of the Present Invention Against Periodontal Disease Pathogens

Etiology A B C D E Actinobacillus Actinomycetem Comitans (ATCC 33384) > 20 > 20 > 20 > 20 - Actinomyces Viscous (ATCC 15987) > 20 > 20 > 20 20 - Rosia dentocariosa (ATCC 17931) > 20 > 20 > 20 20 - Popillomonas Gingalis (ATCC 33277) > 20 > 20 > 20 > 20 - Streptococcus sanguise (ATCC 10556) > 20 > 20 > 20 10 - Streptococcus mutans (ATCC 25175) > 20 > 20 > 20 20 - Fusobacterium nuclitum (ATCC 25586) > 20 > 20 > 20 20 - Akenella Corodens (ATCC 23834) > 20 > 20 > 20 > 20 - Bacteroid Four Cedars (ATCC 43037) > 20 > 20 > 20 > 20 - Prebotella Intermediate (ATCC 25611) > 20 > 20 > 20 > 20 - Prebotella nigressen (ATCC 33563) > 20 > 20 > 20 > 20 - Staphylococcus aureus (ATCC 65389) > 20 > 20 > 20 > 20 - Pseudomonas aruginosa (ATCC 9027) > 20 > 20 > 20 20 -

(Unit: μl / ml)

A: Methanol Extract

B: ethyl acetate extract

C: Chloroform Extract

D: Butyl Alcohol Extract

E: control

As shown in Table 2 and Table 3, in the medium containing the methanol extract obtained from the control group and Baekduong did not show a circular growth zone in all 14 periodontal disease pathogens, the methanol extract is the highest concentration used in the experiment 20 It was found that even in μl / ㎖ did not inhibit the growth of 14 periodontal disease pathogens.

In addition, in the medium containing the ethyl acetate extract obtained from Baekduong in Example 1, the developmental zone was shown in the prebotella nigressen (ATCC 33563), Streptococcus mutans (ATCC25175) and Streptococcus sangui (ATCC 10556). Especially in Streptococcus sanguis (ATCC 10556). Specifically, the ethyl acetate extract obtained from Baekduong in Example 1 showed a 100% growth inhibition rate against Prebotella nigressen (ATCC 33563) at a concentration of 20 μl / ml, Extinomyses biscusus (ATCC 15987), Rosia dentocariosa (ATCC 17931) and fusobacterium nucleatum (ATCC 25586) showed about 50% growth inhibition compared to the control group, and Streptococcus sanguise (ATCC 10556) compared to the control group. It showed growth inhibition of more than 80%. Therefore, it was confirmed that the MIC of the ethyl acetate extract for Prevotelella nigressen (ATCC 33563) was 20 µl / ml and the MIC of the ethyl acetate extract for the remaining 13 periodontal disease pathogens was 20 µl / ml or more.

In addition, in Example 1, the medium containing the chloroform extract obtained from Baekduong showed growth zones in Streptococcus mutans (ATCC 25175) and Streptococcus sanguise (ATCC 10556). The widest developmental zone was observed. Specifically, the chloroform extract showed a 100% growth inhibition rate against prebotella nigressen (ATCC 33563) at a concentration of 20 μl / ml, and more than 50% growth of Streptococcus sangui (ATCC 10556) compared to the control group. The inhibition rate was similar to that of the ethyl acetate extract. Therefore, the MIC of the chloroform extract obtained from Baekduong in Example 1 was 20 µl / ml and the MIC of the chloroform extract of the remaining 13 periodontal disease pathogens was 20 µl / ml. It confirmed that it was abnormal.

Finally, in the medium containing the butyl alcohol extract obtained from Baekduong in Example 1, all of the 13 periodontal disease pathogens except populomonas gingivalis (ATCC 33277) showed a developmental base. Specifically, the butyl alcohol extract is Staphylococcus aureus (ATCC 65389), Prebotella intermedia (ATCC 25611), Akenella corrodens (ATCC 23834), Actinobacillus ex at a concentration of 20 μl / ㎖ Although it did not completely inhibit the growth of Tinomycetcommitans (ATCC 33384), Bacteroid Forciders (ATCC 43037) and Popillomonas Gingivalis (ATCC 33277), Pseudomonas aeruginosa (ATCC 9027), Rosia den It showed 100% growth inhibition against Tocariosa (ATCC 17931), Actinomyces viscous (ATCC 15987), Prebotella nigressen (ATCC 33563) and Streptococcus mutans (ATCC 25175) For yeast (ATCC 10556), 100% growth inhibition was shown at a concentration of 10 μl / ml. Thus, the MIC for Streptococcus sangui (ATCC 10556) of the butyl alcohol extract obtained from Baekduong in Example 1 was 10 μl / ml, Pseudomonas aeruginosa (ATCC 9027), Rosia dentocariosa (ATCC 17931). , MIC for Actinomyces viscous (ATCC 15987), Prebotella nigressen (ATCC 33563) and Streptococcus mutans (ATCC 25175) is 20 μl / ml and Staphylococcus aureus (ATCC 65389) , Prebotella Intermediate (ATCC 25611), Akenella Corrodens (ATCC 23834), Actinovacillus ectinomycetem comitans (ATCC 33384), Bacterium Forciders (ATCC 43037) and Popillomonas Gingibal The MIC for leasing (ATCC 33277) was found to be at least 20 μl / ml.

From the above results, ethyl acetate extract and chloroform extract of the four baekduong extracts of the present invention showed excellent antibacterial effect against some periodontal disease pathogens, butyl alcohol extract containing Staphylococcus aureus (ATCC 65389) It was confirmed that the antimicrobial activity was very high because it showed excellent antibacterial effect against 13 periodontal disease pathogens consisting of Gram positive bacteria and Gram negative bacteria including E. coli. In addition, the first methanol extract extracted from baekduong of the four baekduong extract of the present invention contains ethyl acetate extract, chloroform extract and butyl alcohol extract with antimicrobial activity, but against all 14 periodontal disease pathogens used for antimicrobial investigation Although the methanol extract contained ethyl acetate extract, chloroform extract, and butyl alcohol extract having antimicrobial activity, the bacterium extract containing the antimicrobial effect was diluted in the methanol extract, indicating no antimicrobial effect. Judging.

Therefore, MIC against the periodontal disease pathogens of the Baekduong extract of the present invention is reported to exhibit an antimicrobial effect against the existing periodontal disease pathogens Honokiol and Listerin (MIC: 20 to 160 ul / ml) or myrrh and ginseng saponin (MIC) : 80 to 640 μl / ml) was significantly lower than the Baekduong extract of the present invention was confirmed to be a natural extract showing an excellent antimicrobial effect.

Experimental Example 2 Oral Acute Toxicity in Rats

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally administered at a dose of 3 g / kg / 15 ml by suspending the chloroform extract, the acetate extract, and the butyl alcohol extract, which were the extracts of the present invention, in 0.5% methylcellulose solution, respectively. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all three of the above-mentioned Baekduong extracts did not show toxicological changes in rats up to 50 g / kg, and the minimum oral dose (LD ₅₀ ) of 120 g / kg or more was determined to be a safe substance.

As described above, the baekduong extract of the present invention has excellent antimicrobial activity and can be formulated as a mouthwash and periodontal disease treatment.

Preparation Example 1 Manufacturing Method of Syrup

Syrup containing the baekduong extract (chloroform extract, ethyl acetate extract and butyl alcohol extract) of the present invention and a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) is prepared by the following method.

Acid addition salts, saccharin and sugars of the three baekduong extracts were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.

The components of the syrup are as follows.

Ethyl acetate extract, chloroform extract or butyl alcohol extract, hydrochloric acid salt, 2 g

Saccharin 0.8 g

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ·····················

Preparation Example 2 Preparation of Tablet

A tablet containing 15 mg of active ingredient is prepared by the following method.

250 g of baekduong extract (chloroform extract, ethyl acetate extract and butyl alcohol extract) and hydrochloride of the present invention were mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

Ethyl acetate extract, chloroform extract, or butyl alcohol extract, hydrochloride, ..., 250 g

Lactose ········ 175.9 g

Potato starch ········· 180 g

Colloidal silicic acid 32 g

10% gelatin solution

Potato starch · 160 g

Talc · 50 g

Magnesium stearate 5 g

Preparation Example 3 Manufacturing Method of Injection Solution

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

100 ml of the baekduong extract (chloroform extract, ethyl acetate extract, and butyl alcohol extract) of the present invention, 1 g of hydrochloride, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid were dissolved in distilled water. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

Acetylacetate extract, chloroform extract or butyl alcohol extract, hydrochloride, ..., 1 g

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ··················

As described above, the Baekduong extract of the present invention is a low-toxic natural extract having a high safety and low resistance to strains and tissue harm, including Gram-positive bacteria and Escherichia coli including Staphylococcus aureus (ATCC 65389). As well as showing excellent antimicrobial effect against various periodontal disease pathogens, such as Gram-negative bacteria, it can be usefully used as a mouthwash and periodontal disease treatment.

Claims (8)

Pulsatilla koreana Nakai et Mori extract and / or pharmaceutically acceptable salts thereof that exhibit an antimicrobial effect. 1) degreasing the ground baekduong, extracted with methanol, filtered and dried in vacuo to obtain a methanol extract (step 1); 2) repeatedly distilling the methanol extract of step 1 into chloroform layer and water layer using chloroform and water several times, and then concentrating the chloroform layer to obtain chloroform extract (step 2); 3) distilling the water layer of step 2 into ethyl acetate layer and water layer several times using ethylacetate and water to obtain an ethyl acetate extract by concentrating the ethyl acetate layer (step 3); And 4) The water layer of step 3 was repeatedly fractionated into butyl alcohol layer and water layer several times using butylalcohol and water, and then the butyl alcohol layer was concentrated to obtain a butyl alcohol extract (step 4). Method of Preparation of Extracts. The chloroform extract of step 2 and / or a pharmaceutically acceptable salt thereof according to claim 2, which exhibits an antimicrobial effect. The ethyl acetate extract of step 3 and / or a pharmaceutically acceptable salt thereof according to claim 2, which exhibits an antimicrobial effect. The butyl alcohol extract of step 4 and / or a pharmaceutically acceptable salt thereof according to claim 2, which exhibits an antimicrobial effect. A pharmaceutical composition for oral cleaning and periodontal disease, comprising the chloroform extract of claim 3 and / or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for treating mouthwashes and periodontal disease, comprising the ethyl acetate extract of claim 4 and / or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for oral cleansing and periodontal disease treatment, comprising the butyl alcohol extract of claim 5 and / or a pharmaceutically acceptable salt thereof as an active ingredient.