KR20030085169A - Active essential oil extract isolated from Compositae Chrysanthemum boreale having inhibitory effects on growing medical pathogens - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing an essential oil of Chrysanthemum boreale as an effective ingredient is provided. The extract of Chrysanthemum boreale shows excellent inhibiting activity against pathogenic bacteria and fungal microorganisms and is thus used as an antibacterial agent and/or antifungal agent. CONSTITUTION: An antibacterial agent and/or antifungal composition contain an extract of Chrysanthemum boreale and a pharmaceutically acceptable carrier or excipient. The extract of Chrysanthemum boreale is obtained by a steam distillation method. The desirable dose of the extract is in the range of 0.3 to 1,000mg/kg body weight. The composition has excellent growth inhibiting effect on causative agents of gingivitis, dental caries, gastrointestinal disease and major infections.

Description

Active essential oil extract isolated from Compositae Chrysanthemum boreale having inhibitory effects on growing medical pathogens}

The present invention provides a method for producing antimicrobial and / or antifungal activity of essential oil extracts from a country of origin and antimicrobial and / or antifungal compositions and pharmaceutical preparations having the same as an active ingredient and extracting and purifying the active ingredient. It is about.

In nature, tens of thousands of microorganisms inhabit a wide range of ecosystems and are closely related to our lives because of various diseases and pollution. Candida species, which form colonies on the surface of mucosal epithelial cells of the human body ( Candida species is a type of opportunistic infection-causing bacterium that exists as a normal microflora in the oral cavity and intestine, and causes severe diseases such as thrush or vaginitis in the oral cavity when the host resistance decreases. In many cases (Journal of Industrial Microbiology 21 (3): 263-268). The most common cause of stomatitis in denture wearers is not fully understood, but mucosal fungi have been reported (3rd Academic-Academic Symposium 27-35, Korean Journal of Biotechnology and Bioengineering 8 (1): 75-82). , Korean Journal of Oral Health 21 (3): 553-561). Candida albicans (C. albicans ) infections range from 50% to 100% in patients with denture stomatitis (J. Dent. Res. 91: 134-142, Acta.Odentol.Scand. 32: 329-333), Oral surg. Oral Meb. Oral Pathol. 47: 323-328), oral fungal infections include Candida albicans, Torulopsis glabrata, and Candida tropicalis (J. Dent. Tes. 82). : 151-190). The colonization process of microorganisms is the first step in attaching to host cells (Dent. Pract. Dent. Rec. 16: 138-144, Prot. Stom. 28: 223-232). It becomes possible to form colonies, and appears in a similar fashion to Candida species. There have been many reports on the ability to adhere to buccal mucosa cells against Candida species (Hiroshima J. Med. Sci. 35: 39-43), and the ability to adhere to epithelial cells depends on the species. Although there are differences, C. albicans has the best adhesion (J. Infect. Dis. 143: 325) and is known to exist in the oral mucosa and tongue (Top. Med. Mycol. 2:73). .

In addition, filamentous fungus Aspergillus nidulans (A. nidulans ), which is widely used in basic biology research, appears near the nasal sinus and shows secondary infections in patients undergoing chemotherapy. Opportunistic bacteria in the maxillofacial region that are scattered in the area (Infect. Immun. 32: 1234-1241). Oral candidosis is caused by secondary opportunistic infections in patients with a long-term administration of a wide range of antibiotics and in patients with compromised oral bacterial ecosystems or in patients with acquired immunodeficiency syndrome (AIDS) that has compromised the body's defense mechanisms. There is increasing interest in fungal infections (Infect. Immun. 27: 667).

In addition, dental caries, caused by gram-positive bacteria, is one of the most prevalent cultural diseases in the world, and the morbidity rate is gradually increasing in Korea (J. Med. Vet. Mycol. 24: 81-84). . In general, dental caries is caused by the fermentation of carbohydrates in food by bacteria and the destruction of the tooth surface (enamel or cementum) by the action of locally produced organic acids. In particular, oral bacteria are known to be caused mainly by Streptococcus mutans (S. mutans ) (Mycosis 39 (9-10): 361-366). Therefore, a very active research in terms of prevention of dental caries has been conducted along with basic research on the mechanism of dental caries. In particular, research on the prevention of dental caries is being conducted on new natural products such as green tea and oriental medicine as well as microorganisms and various compounds (J. Am. Dent. Assoc. 113: 586-589). . In 1984, Marshall and Warren of Australia observed Gram-negative curved bacillus from gastric mucosal biopsy tissue of patients with gastritis and gastric ulcers (Lancet 1: 1311-1315), which was the first in artificial media. The success of the net culture has been of great interest as to whether Helicobacter pylori ( H. pylori ) is a causative agent of human stomach disease. This gram-negative bacterium has proved to be the primary causative agent of human gastritis, plays a very important role in the development of peptic ulcers, and has recently been suggested to be the primary determinant of gastric cancer. There is an urgent need for a method that can be removed from mucous membranes (Korean Journal of Microbiology 35: 583-585, J. Kor. Soc. Microbiol. 23: 550-552). The only ecological habitat of H. pylori is localized to the junctions and mucous layers of the gastric mucosal epithelial cells of the human body (Journal of Korean Microbiology 26: 305-316, J. Infect Dis. 153: 658-663, J.). Pathol. 146: 355-362), the initial site of infection is presumed to be a gastric mucosa of the stomach. Because the gram-negative bacteria inhabit the gastric mucosa, it is likely to be a bacterium that is resistant to acidic environments, but the acidity (pH) of the medium required for optimal growth is 7.0-7.4 and does not grow under acidic or alkaline conditions outside this range ( Korean Journal of Microbiology 23: 17-26). The reason why H. pylori lives in the gastric mucosa where other bacteria are difficult to survive or settle in spite of the extremely difficult culture conditions is that H. pylori has very strong motility and urease production ability. . Hazell et al. Explained that the helical morphology and strong motility of these bacteria allow them to freely permeate or swim through the sticky gastric mucus layer, as evidence of gram-negative E. coli ( E) in motility experiments using methylcellulose artificial mucus. Coli) reported that the Helicobacter Philoly ( H. pylori) exhibits motility at more than 20 times higher viscosity than barely movable (Am. J. Gastroenterol. 81: 855). The urease produced by H. pylori is thought to neutralize the attack by hydrochloric acid in the gastric lumen by degrading urea in plasma exudates or tissue fluids of the gastric mucosa and alkalizing the cells around them. J. Clin. Pathol. 39: 353-365), it is hypothesized that ammonia accumulated in the gastric mucosa due to urease can cause gastritis or gastroduodenal ulcer by direct or indirect damage to the gastric mucosa ( Am. J. Clin. Pathol. 86: 575-582, Gastroenterol. 96: 615-625, Infect. Immun. 58: 1992-1994).

At this time, the development of antibacterial and antifungal agents in various fields has been actively conducted. This study also used the country of Asteraceae as a natural product to investigate the antibacterial and antifungal activity.

Chrysanthemum boreale is a perennial herb of the compositae, which blooms 1-1.5 cm of yellow flowers in wild mountains throughout the country from October to November. It is also known as C. indicum . In addition, it has been used mainly in headache, zephyr fever, and cheonghae detoxification in oriental medicine, and chrysanthemums and chrysanthemum teas have been consumed from the Silla era until today (Academic Book, Seoul p.329, Daewonsa, Seoul p.19). Ingredients in the country of origin include essential oils, linarin, glycosides of luteolin, chrysanthemin, chrysanthemaxanthin, polysaccharides, coumarins, and infants It contains lactone (yejuhua lactone), and the main components of essential oils are known as comphene, camphor and carvone (Planta Med. 64 (3): 289-290, Korean Journal of Agricultural Chemistry 40). (1): 85-87). The extracts of these flowers are known to have various physiological activities including central nervous sedation, blood pressure lowering effect, inhibitory effect on Mycobacterium tuberculosis bacteria and various viruses, and effects on lung cancer and liver cancer (Seoul National University Herbal Research Institute 14:69 -74, Oriental Resources Plant Society.

Accordingly, the present inventors continue to develop natural substances having antibacterial and / or antifungal activity, and in the case of using essential oils and / or selectively adding EDTA in a country of origin, pathogenic bacteria And a high inhibitory activity against fungi, finding the effect of treating various diseases caused by these pathogens has completed the present invention. Among the pathogenic bacteria and fungi, Staphylococcus aureus (Staphylococcus aureus, S. aureus),Streptococcus piogenes(Streptococcus pyogenes, S. pyogenes), Streptococcus mutans (Streptococcus mutans, S. mutans), Escherichia coli (Escherichia coli, E. coli), Helicobacter pylori (Helicobacter pylori, H. pylori), Fusobacterium nucleum (Fusobacterium nucleatum, F. nucleatum), Actino Bacillus Actinomycetem Comitans (Actinobacillus actinomycetemcomitans, A. actinomycetemcomitans), Prebotella Intermedia (Prevotella intermedia, P. intermedia), Prevotela Nigressen (Prevotella nigrescens, P. nigrescens) And Porphyromonas ginvalisPorphyromonas gingivalis, P. gingivalis)Aspergillus nidulans (such as bacteriaAspergillus nidulans, A. nidulans),Fusarium Oxis ForumFusarium oxysporum, F. oxysporum) And Alternaria Mali (Alternaria mali, Al. mali)Wow Such as filamentous fungi and Candida albicans (Candida albicans, C. albicansThe antimicrobial, antifungal activity and clinical effects of yeast fungi were excellent.

Accordingly, it is an object of the present invention to provide an antimicrobial and / or antifungal composition comprising an essential oil extract of an acid country as an active ingredient, and an agent comprising the same and a use thereof.

1 is a graph showing the growth curve (inhibitory activity) of Aspergillus nidulan on a complete medium containing Chrysanthemum boreale essential oil (essential oil).

FIG. 2 is a graph showing the growth curve (inhibitory activity) of the Furizium oxyforum on a complete medium containing Chrysanthemum boreale essential oil extract.

Figure 3 is a graph showing the growth curve (inhibitory activity) of alternalia Mali on potato dextrose medium containing Chrysanthemum boreale essential oil (essential oil).

Figure 4 is a graph showing the antimicrobial activity of Candida albicans of Chrysanthemum boreale essential oil extract (essential oil).

5 is a graph showing the antimicrobial activity of E. coli of essential oil extract ( Chrysanthemum boreale ) essential oil (essential oil).

Figure 6 is a graph showing the antimicrobial activity of Staphylococcus aureus of Chrysanthemum boreale essential oil (essential oil).

FIG. 7 is a graph showing the antimicrobial activity of Streptococcus pyogenes of Chrysanthemum boreale essential oil extract.

8 is a graph showing the antimicrobial activity against essential oil Streptococcus mutans of Chrysanthemum boreale .

9 is a graph showing the antimicrobial activity of Helicobacter pylori of Chrysanthemum boreale essential oil extract (essential oil).

In order to achieve the above another object, the present invention provides an antifungal and / or antimicrobial agent comprising the composition.

In order to achieve another object of the present invention, the present invention provides a novel use for the antifungal and / or antimicrobial activity of essential oil extract (essential oil) of the country.

Hereinafter, the present invention will be described in detail.

Through the antimicrobial and antifungal activity measurement experiment for the acid extract of the present invention, the extract showed a significant inhibitory activity against pathogenic bacteria and fungal microorganisms. Therefore, it can be usefully used for the treatment of diseases caused by the pathogenic bacteria and fungal microorganisms, in particular, oral diseases including stomatitis, as well as can be useful for the treatment of gastrointestinal diseases caused by pathogens. In addition, it can be usefully used as a mouthwash for therapeutic purposes.

Accordingly, in the present invention, it is possible to prepare an antimicrobial and antifungal composition comprising a volatile extract of the acid country as an active ingredient and a pharmaceutically acceptable carrier and a composition for treating various bacterial and fungal diseases. The composition can be formulated in a variety of oral or parenteral dosage forms. Formulations for oral administration include, for example, tablets, pills, hard soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These formulations may contain diluents (e.g. lactose, dextrose, sucrose) in addition to the active ingredients. , Mannitol, sorbitol, cellulose and / or glycine), glidants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid Or disintegrants or boiling mixtures such as sodium salts thereof and / or absorbents, colorants, flavors, and sweeteners. The formulations may be prepared by conventional mixing, granulating or coating methods. Representatives of non-oral dosage forms may also be water, Ringer's solution, isotonic physiological saline or suspension as a solvent for injection, and sterile fixed oils may be used as a solvent or suspending medium, mono-, di- Any non-irritating fixed oil can be used for this purpose, including glycerides, and fatty acids such as oleic acid can be used in the preparation of injectables. It may also be formulated into external preparations such as ointments, creams, gels, lotions, etc. using aqueous or oily ointments, antioxidants, preservatives, weights and the like.

The composition may contain sterile and / or adjuvant such as preservatives, stabilizers, hydrating or emulsifying accelerators, salts and / or buffers for the control of osmotic pressure and other therapeutically useful materials and may be formulated according to conventional methods. Can be.

When the extract of the present invention is administered for clinical purposes, it may be administered in a single dose or in separate doses and the total daily dose to be administered is preferably in the range of 0.3 to 1,000 mg / kg body weight, but specific dose levels for specific subjects may be used. It may vary depending on the specific compound, weight, sex, health condition, diet, time of administration, method of administration, rate of excretion, drug mixing and the severity of the disease.

When the volatile extract of the present invention is to be clinically administered to treat the desired pathogenic bacterial and fungal diseases, the EDTA component may be selectively administered simultaneously to the acid extract.

However, acid extract compositions and / or preparations according to the present invention for the purpose of treating pathogenic bacterial and fungal diseases using antifungal / antifungal activity are not limited to those described above, including pathogenic bacterial and fungal diseases Any agent useful for the treatment of can be included.

The acid extract of the present invention can be prepared by a known conventional plant extraction method such as steam distillation (steam distillation).

Hereinafter, the present invention will be described in more detail with reference to examples and examples. However, these reference examples and examples are merely to aid the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Reference Example 1. Preparation of Volatile Extracts from Country of Origin

In October-November 1998, after picking the country from Geumma-myeon, dried and dried, cut it into 3-4 cm, put about 180 g of dry weight in a 2,000 ml round flask, add 1,200 ml of distilled water, and then use Karlsruker's apparatus. Volatile components in the country of origin were collected, dissolved in ethyl ether (ethyl ether, Sigma Chemical Co., Mo. USA), stored frozen (-70 ℃ freezer), collected before the experiment, and collected in a rotary vacuum distillation ether. ) Component was used after removal (Allen, G. and Unwin (ed.) 208 p. Springer-Verlag).

Reference Example 2 Strains Used in the Experiment

As fungi include Aspergillus nidulans (A. nidulans) FGSC4, Alternaria Mali (Al. mali), Fusarium Oxy Forum (F. oxysporum) And the yeast fungus Candida albicans (C. albicans) Was used. Pathogenic microorganisms include Staphylococcus aureus (S. aureusATCC 25923, Streptococcus pyogenesS. pyogenesATCC 19615, Streptococcus mutans (S. mutans) ATCC 25175, Helicobacter Philly (H. pyloriATCC 26695 and Escherichia ColiE. coliATCC 25922, Fusobacterium Nucleum (F. nucleatumATCC 10953, 51190, 49256, Actino Bacillus Actinomycetem Comitans (A. actinomycetemcomitans) ATCC 43717. 43718, 33384, Prebotella Intermedia (P. intermedia) G8-9K-3, Prebotella nigresense (P. nigrescensATCC 9336 and Porphyromonas ginvalisP. gingivalis)ATCC 33277 was used.

2-1. Fungal Culture Conditions and Media

end. Filamentous fungi culture and conditions

Strain name Culture medium Incubation temperature Aspergillus nidulans Complete medium (CM) 37 ℃ Fusarium oxyporum Complete medium (CM) 30 ℃ Alternaria mali Potato Dextrose Badge (PD) 24 ℃

I. Composition of Minimal Media (Potato Dextrose, PD)

Potato Dextrose agar medium (Potato Dextrose agar, Difco, Lab. Co., Detroit, U. S. A.) was dissolved in 100 ml of distilled water and sterilized for 15 minutes at 120 ℃.

All. Composition of Complete Medium (CM)

1.0 g of dextrose (Merck Co., Darmstadt, Germany), 0.15 g of yeast extract (Deastco, Lab. Co., Detroit, USA), casein acid (Sigma Chemical Co., MO. USA) 0.15 g, 0.4 ml of adenine solution, 1 ml of vitamin solution (vitamin solution, 1 mg of riboflavin <100 mg, nicotinic acid> 100 mg, paraaminobenzoic acid <p-aminobenzonic acid> 500 mg , Pyridoxin hydrochloride <500 mg, thiamine <500 mg, biotin <biotin> 2 mg, ascorbic acid <ascorbic acid> 500 mg, folic acid <folic acid> 500 mg) 2 ml of minimal salt stock solution (76 g NaNO3, 26 g MgSO4 · 7H2O, 76 g KH2PO4 76 g, KCl 26 g, (NH4) 6Mo7O24) 0.4 g of ZnCl 2, 0.008 g of MnCl 2 · 4H 2 O, 0.032 g of CuSO 4 · 5H 2 O, and 0.1296 g of FeCl 2) were added thereto.

la. Yeast Fungus Culture and Conditions

After inoculating strains into tryptic soy broth (Difco, Lab. Co., Detroit, USA), the cells were incubated for 24 hours at 37 ° C and CO2 incubator, and then adjusted to turbidity 0.5 (McFarland 0.5). Used.

2-2. Bacteria Culture and Conditions

end. General bacterial culture and conditions

Each strain was inoculated in tryptic soy (Typtic soy, Difco, Lab. Co., Detroit, USA), followed by incubation for 24 hours in a carbon dioxide (CO2) incubator at 37 ° C and a turbidity of 0.5 (McFarland 0.5). Then used.

I. H. pylori culture and conditions

Strains used were Brucella agar medium (brucella agar, brucella broth 28 g (BBL, Becton Dickinson Co., MD. USA), agar 10 g (Junsei Chemical Co., DK. Japan), 50 ml of young bovine serum (fetal bovin serum) (GibcoBRL. Co., NY.USA), vancomycin 5 mg / ml (vancomycin, Sigma Chemical Co., MO.USA), 2 ml of nalididinic acid (12.5 mg / ml, Sigma) Chemical Co., MO. USA), amphotericin B (amphotericin B) (0.5 mg / ml, Sigma Chemical Co., MO. USA) inoculated in 2ml｝, 37 ℃, 4-5 in a carbon dioxide (CO2) incubator After culturing for one day, the inoculated in Brussela liquid medium to which antibiotics were added was used after adjusting the bacterial culture incubated for 4-5 days at 37 ℃, carbon dioxide (CO2) incubator to turbidity 0.5 (McFarland 0.5).

All. Porphyromonas P. gingivalis Culture and Conditions

After inoculating the used strain into brain heart infusion (BHI) agar, incubated in anaerobic incubator (80% N2, 10% H2, 10% carbon dioxide (CO2) for 5-7 days at 37 ° C) and then 0.4 μl / ml This was inoculated in BHI agar to which 0.125% EDTA was added, followed by incubation in an anaerobic incubator (80% N2, 10% H2, 10% carbon dioxide (CO2) for 5-7 days), and growth of the strain was observed. .

Example 1 Antibacterial Experiment

1-1 Antifungal activity measurement

After sterilizing the medium corresponding to each strain, the control group used the medium as it was, and added to the experimental group so that the final concentration of essential oil extract (essential oil) was 200 ppm, 400 ppm, 600 ppm, 800 ppm, 1,000 ppm. When the medium cools down to about 70 ° C, it is poured into a disposable Petri dish (Green Cross Medical Co., Seoul, Korea), waited until the medium is completely hardened, and then incubated for 3 days in Aspergillus nidulans (A. nidulans), Fusarium Oxy Forum (F. oxysporum) And Alternaria Mali (Al. maliThe growth changes were observed by measuring the diameter while incubating for 5 days after peaking in the middle of each medium prepared spores.

Antifungal Activity by Essential Oil Extracts from Domestic Countries

Three fungi Aspergillus nidulans (A. nidulans), Fusarium Oxy Forum (F. oxysporum) And Alternaria Mali (Al. maliThe essential oil extracts of the country were treated with concentrations of 0 ppm, 200 ppm, 400 ppm, 600 ppm, 800 ppm, and 1,000 ppm, followed by incubation for 5 days. Growth was inhibited as the concentration of essential oil extract (essential oil) increased. Alternaria Mali (Al. mali) Showed a slight tendency of growth at low concentrations of 200 ppm, but there was no significant difference (Fig. 1, 2, 3). In addition, the diameter of the fungi after 5 days of culture was measured and compared to the control group for each concentration, Aspergillus nidulans (A. nidulans) And the Fusarium Oxy Forum (F. oxysporum)silver It was found to be significantly suppressed in all experimental groups (P <0.001) and Alternaria Mali (Al. mali) Showed significance from a concentration of 400 ppm (P <0.01) (Table 1).

Table 1.Antifungal Effects of Domestic Essential Oil Extracts (Day 5)

Oil concentration (ppm) Colony diameter ± standard deviation (Colony diameter ± S.D.) Alternaria mali Aspergillus nidulans Fusarium oxyporum 0 4.175 ± 0.28 5.500 ± 0.00 5.200 ± 0.14 200 4.625 ± 0.22 4.200 ± 0.14 *** 3.700 ± 0.14 *** 400 3.225 ± 0.22 ** 2.725 ± 0.17 *** 2.600 ± 0.16 *** 600 2.850 ± 0.17 *** 2.100 ± 0.21 *** 2.075 ± 0.22 *** 800 1.925 ± 0.18 *** 1.750 ± 0.36 *** 1.700 ± 0.11 *** 1,000 1.325 ± 0.09 *** 0.625 ± 0.26 *** 1.300 ± 0.28 ***

***: P <0.001, **: P <0.01

1-2 Anti-Yeast Fungal Activity Determination

Tryptic Soy liquid medium is concentrated in a concentration of 0.0 μl / ml 0.2 μl / ml, 0.4 μl / ml, 0.6 μl / ml, 0.8 μl / ml, 1.0 μl / ml Dispense 1 ml into a plate (Falcon, Becton Dickinson Labware, NJ. USA), add 100 μl / ml / well of each microbial solution with a turbidity of 0.5, and incubate for 24 hours at 37 ° C in a CO2 incubator. Molecular Devices Co., CF. USA) and observed the change in growth by measuring the absorbance at 630 nm.

Wherein the yeast fungal activity and antibacterial activity of the essential oil extract of Country of origin

Yeast Fungus Candida albicans (C. albicans)And 5 kinds of bacteria Staphylococcus aureus (S. aureus), Streptococcus piogenes(S. pyogenes), Streptococcus mutans (S. mutans),Escherichia coli (E. coli)And Helicobacter Philly (H. pyloriEssential oil extract (essential oil) 0.0 μl / ml, 0.2 μl / ml, 0.4 μl / ml, 0.6 μl / ml, 0.8 μl / ml, 1.0 μl / ml The absorbance was measured by the reader and the yeast fungus Candida albicans (C. albicans)In the case of, it showed inhibition of essential oil extract from 0.2 μl / ml concentration and marked inhibition from 0.4 μl / ml concentration (Fig. 4).

1-3 Antimicrobial Activity Measurement

end. Antimicrobial Activity of Common Bacteria

Tryptic Soy liquid medium (TSB) is formulated with essential oil extracts of 0.0 μl / ml 0.2 μl / ml, 0.4 μl / ml, 0.6 μl / ml, 0.8 μl / ml and 1.0 μl / ml After dispensing 1 ml each into a 24 well plate (Falcon, Becton Dickinson Labware, NJ. USA), add 100 μl / ml / well of each microbial solution with a turbidity of 0.5, and incubate for 24 hours at 37 ° C and CO2 incubator. The change in growth was observed by measuring absorbance at 630 nm with a reader (Molecular Devices Co., CF. USA).

I. Determination of Antimicrobial Activity of H. pylori

Add essential oil extracts from country of origin to Brucella broth at concentrations of 0.0 μl / ml 0.2 μl / ml, 0.4 μl / ml, 0.6 μl / ml, 0.8 μl / ml and 1.0 μl / ml After dispensing 1 ml each into a 24 well plate, add 100 μl / ml / well of the microbial solution to a turbidity of 0.5, and incubate for 1-5 days at 37 ℃, CO2 incubator, and then use ELISA reader (Molecular Devices, Co., CF.USA). The change in growth was observed by measuring the absorbance at 630 nm by day.

Escherichia coli (E. coli)In the case of, there was no significant change in growth according to the concentration of essential oil.S. aureus), Streptococcus piogenes(S. pyogenes),Streptococcus mutans (S. mutans) And Helicobacter Philly (H. pylori), Growth was significantly inhibited as the concentration of essential oil extract increased (Figure 5-9).

1-4. Activity when mixing essential oils with EDTA 0.125%

Tryptic Soy liquid medium (TSB) was prepared by adjusting the essential oil extract of the country of origin to a concentration of 0.0 μl / ml 0.2 μl / ml, 0.4 μl / ml, 0.6 μl / ml, 0.8 μl / ml, 1.0 μl / ml, and then EDTA 0.125%. Dissolve 1 ml each in a 24 well plate (Falcon, Becton Dickinson Labware, NJ. USA), add 100 μl / ml / well of each microbial solution with a turbidity of 0.5, and incubate at 37 ° C for 24 hours in a CO2 incubator. Next, the change in growth was observed by measuring absorbance at 630 nm with an ELISA reader (Molecular Devices Co., CF. USA).

essential oilActivity in combination with 0.125% EDTA

The statistical significance of the antimicrobial effect of essential oil extract between yeast and bacteria and EDTA 0.125% was 0.2 μl / ml, 0.4 μl / ml, 0.6 μl / ml, 0.8 μl / ml, 1.0 μl / ml. Staphylococcus aureus at concentrationS. aureus), Streptococcus piogenes(S. pyogenes),Candida albicans(C. albicans),Streptococcus mutans (S. mutans) And Escherichia coli (E. coli)High growth inhibition was observed at. And Escherichia coli (E. coli)In the case of (P <0.05) it was significant with all the bacteria (Table 2).

Table 2. Essential Oil Extracts Antibacterial activity (%) of EDTA 0.125%

Essential Oil Extract Concentration Escherichia coli ATCC 25922 Staphylococcus aureus ATCC 25923 Streptococcus mutansATCC 25715 Streptococcus piogenes ATCC 19615 Candida albicans (0.125% removal of EDTA) 0.0 (μl / ml) 100.05 ± 6.66 * 100.12 ± 1.04 99.99 ± 1.38 99.75 ± 0.36 100.28 ± 1.08 (Added 0.125% EDTA) 0.0 (μl / ml) 37.79 ± 1.08a * 35.40 ± 0.58 0.43 ± 0.00 0.71 ± 0.20 1.24 ± 0.21 0.2 32.61 ± 1.46b * 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.4 30.10 ± 0.59c * 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.6 26.54 ± 0.47d * 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.8 23.66 ± 0.42e * 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 1.0 23.90 ± 0.55f * 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

mean ± standard deviation

* a: Significantly different among that of S. aureus , S. mutans, C. albicans and S. pyogenes (P <0.05).

* b: Significantly different among that of S. aureus , S. mutans, S. pyogenes and C. albicans (P <0.05).

* c: Significantly different among that ofS. aureus,S. mutans, S. pyogenesandC. albicans(P <0.05).

* d: Significantly different among that ofS. aureus,S. mutans, S. pyogenesandC. albicans(P <0.05).

* e: Significantly different among that ofS. aureus,S. mutans, S. pyogenesandC. albicans(P <0.05).

* f: Significantly different among that ofS. aureus,S. mutans, S. pyogenesandC. albicans(P <0.05).

Example 2 Liquid Dilution Method

Yeast fungi C. albicans , bacteria B. S. aureus , Streptococcus pyogenes , Streptococcus mutans and E. coli ( E) Liquid medium dilution was used to test the minimal inhibitory concentration (MIC ) of the country's volatile extracts. Each strain was inoculated in Mueller-Hinton liquid medium (Difco, Lab. Co., Detroit, USA) and incubated for 6-12 hours at 37 ℃, CO2 incubator (Vision Co., Seoul, Korea). The cultured fungi were adjusted to 0.5 tube turbidity on a McFarland nephelometer, diluted 1: 10, and inoculated in a Mueller-Hinton agar medium prepared by concentration using a volatile extract of the country of origin using an inoculator. Read after 24 hours incubation in CO2 incubator.

Minimum Inhibitory Concentration (MIC) of Bacteria on Essential Oil Extracts from Domestic Countries

5 countries of origin for bacteriaessential oilMIC for the extract was Staphylococcus aureus (S. aureus)1.4-1.8 μl / ml for Streptococcus piogenes(S. pyogenes0.2-0.6 μl / ml for Streptococcus mutans (S. mutans) 0.4-0.8 μl / ml, Escherichia coli (E. coli)For 4.0-6.0 μl / ml, Candida albicans (C. albicans)For 1.2-2.0 μl / ml (Table 3).

Table 3. Microorganism susceptibility to domestic essential oil extract

Microorganism (No. tested) MIC (μl / ml) Range Staphylococcus aureus ATCC 25923 ≤1.4 To ≥1.8 Streptococcus mutansATCC 25175 ≤1.4 To ≥1.8 Streptococcus mutansATCC 25175 ≤1.4 To ≥1.8 Escherichia coli ATCC 25922 ≤1.4 To ≥1.8 Candida albicans ≤1.4 To ≥1.8

Example 3. Cytotoxicity Measurement

The monolayer cells that reached densification in the culture dish were separated by 0.25% Trypsin / EDTA, and the cells were suspended in the culture medium and aliquoted to 10 × 104 cells in a 24-well plate. After 24 hours, the culture medium was removed, and 0.14% ethanol essential oil, 0.125% EDTA and 0.125% EDTA were added to 0.04% petroleum essential oil in the experimental group, and 1 × PBS was added to the control group.

After culturing for 2 days, the number of living cells was counted using a hemocytometer.

Effect of Domestic Essential Oils on Human Gingival Fibroblasts

In order to observe the effect of country essential oils on human gingival fibroblasts, we measured the number of cultured cells by adding 0.04% country essential oils, 0.125% EDTA and 0.04% country essential oils to 0.14% EDTA. As a result, treatment with 0.04% native essential oil did not affect the proliferation of human gingival fibroblasts.However, treatment with 0.125% EDTA and 0.14% EDTA with 0.04% native essential oil reduced cell proliferation. 4) .

Table 4. Effect of domestic essential oil extract and EDTA on the activity of human gingival fibroblasts (%)

density Cell activity Control group (phosphate buffer) 91.19 ± 0.84 0.125% EDTA 8.36 ± 2.11 * 0.04% Essential Oil Extract 92.65 ± 1.31 0.125% EDTA + 0.04% Essential Oil Extract 9.36 ± 3.31 *

* P <0.01, statistically significant

Example 4 Statistical Analysis

The measured OD value was calculated by the relative growth ratio (RGR) of the control group, and then one-way ANOVA was performed for significance test, and the DUNCAN multi-range test was performed.

Statistical significance test between yeast fungi and bacteria on essential oils

The statistical significance of the antimicrobial effect on essential oil extracts between yeast fungi and bacteria was Staphylococcus aureus at concentrations of 0.2 μl / ml, 0.4 μl / ml, 0.6 μl / ml, 0.8 μl / ml and 1.0 μl / ml.S. aureus), Streptococcus piogenes(S. pyogenes),Candida albicans (C. albicans),Streptococcus mutans (S. mutans) And Helicobacter Philly (H. pyloriCompared to E. coli (E. coli)Showed high growth (P <0.01). And Streptococcus piogenes at 0.2 μl / ml(S. pyogenes) Were significantly different (P <0.01), and 0.4, 0.6, and 1.0 μl / ml were significantly different from each other (P <0.01) and E. coli (0.8 μl / ml)E. coli)And Helicobacter pylori (H. pylori) Showed a significant difference (P <0.01) (Table 5). Porphyromonas ginvalis (P. gingivalis)ATCC 33277 did not grow regardless of the addition of EDTA at a concentration of 0.4 μl / ml.

Table 5. Antimicrobial Activities of Six Essential Bacteria Extracts from Domestic Oils

Escherichia coli ATCC 25922 Staphylococcus aureus ATCC 25923 Streptococcus mutansATCC 25175 Streptococcus piogenes ATCC 19615 density Helicobacter Philly ATCC 26695 0.0 (μl / ml) 100.00 ± 0.95 * 99.92 ± 7.04 100.00 ± 1.45 100.00 ± 2.17 99.95 ± 5.20 100.00 ± 1.24 0.2 98.59 ± 1.47 95.28 ± 9.80 94.84 ± 1.39 1.22 ± 0.72 * a 96.26 ± 11.44 88.38 ± 3.61 0.4 97.55 ± 1.47 * b 64.53 ± 12.20 * c 44.74 ± 25.05 * d 1.67 ± 0.82 * e 46.99 ± 10.61 * f 80.28 ± 4.37 * g 0.6 96.54 ± 1.23 * h 11.94 ± 2.47 * i 8.52 ± 2.96 * j 1.50 ± 0.68 * k 23.16 ± 5.81 * l 60.71 ± 1.27 * m 0.8 95.09 ± 1.47 * n 3.12 ± 0.99 4.85 ± 2.27 1.51 ± 1.57 5.05 ± 0.34 42.39 ± 1.93 * o 1.0 92.92 ± 2.05 * p 2.83 ± 1.07 * q 3.71 ± 1.11 * r 2.14 ± 2.41 * s 2.66 ± 0.36 * t 46.45 ± 4.91 * u

mean ± standard deviation

* a: Significantly different among that of E. coli , S. aureus , S. mutans, C. albicans and H. pylori (P <0.01).

b: Significantly different among that of S. aureus , S. mutans, S. pyogenes, C. albicans and H. pylori (P <0.01).

* c: Significantly different among that ofE. coli,S. mutans, S. pyogenes, C. albicansandH. pylori(P <0.01).

* d: Significantly different among that of E. coli, S. aurues, S. pyogenes and H. pylori (P <0.01).

* e: Significantly different among that of E. coli, S. aureus , S. mutans, C. albicans and H. pylori (P <0.01).

f: Significantly different among that of E. coli, S. aureus , S. pyogenes and H. pylori (P <0.01).

g: Significantly different among that of E. coli , S. aureus, S. mutans, S. pyogenes and C. albicans (P <0.01).

* h: Significantly different among that of S. aurues, S. mutans, S. pyogenes, C. albicans and H. pylori (P <0.01).

* i: Significantly different among that of E. coli, S. pyogenes, C. albicans and H. pylori (P <0.01).

j: Significantly different among that of E. coli , C. albicans and H. pylori (P <0.01).

* k: Significantly different among that of E. coli, S. aureus , C. albicans and H. pylori (P <0.01).

* l: Significantly different among that of E. coli , S. aurues, S. mutans, S. pyogenes and H. pylori (P <0.01).

m: Significantly different among that of E. coli, S. aureus , S. mutans, S. pyogenes and C. albicans (P <0.01).

* n: Significantly different among that of S. aureus, S, mutans, S. pyogenes, C. albicans and H. pylori (P <0.01).

o: Significantly different among that of E. coli, S. aureus, S, mutans, S. pyogenes and C. albicans (P <0.01).

* p: Significantly different among that of S. aureus, S. pyogenes, C. albicans and H. pylori (P <0.01).

* q: Significantly different among that of E. coli, S, mutans and H. pylori (P <0.01).

r: Significantly different among that of S. aureus, S, pyogenes, C. albicans and H. pylori (P <0.01).

* s: Significantly different among that of E. coli, S, mutans and H. pylori (P <0.01).

* t: Significantly different among that of E. coli, S, mutans and H. pylori (P <0.01).

u: Significantly different among that of E. coli, S. aureus, S, mutans, S. pyogenes and C. albicans (P <0.01).

Example 5. Clinical Study

One). Study subject

This study was conducted at Wonkwang University School of Dentistry, pregnant women, students within 3 months of delivery, those with systemic diseases, periodontal treatment within the last 6 months, and antibiotics within the last 3 months. The subjects of this study were 60 (male: 37, female: 23). For evaluation, 30 control groups and 30 experimental groups were used.

2). Research method

Sixty students were randomized by distillation without brushing for one week, and then divided into two groups, which were washed with the production country (C1) and the control group (E1) which was washed with distilled water containing no production country. At the start of the experiment (week 0), students were tested for microorganisms and no injections or antibiotics were given to avoid the effects of other drugs. One week later, microbial testing was performed in the same way as week 0.

The clinical index was calculated using the Sulcus Bleeding Index <SBI>: Muehlemann and Son 1971 to search for gingival areas and fissures in the teeth, and to check for bleeding, discoloration, edema, and ulcers. Evaluated.

0: The gingiva is healthy and there is no bleeding

1: gingival bleeding but no gingival discoloration and edema

2: gingival bleeding and discoloration but no edema

3: if it is accompanied by gingival bleeding, discoloration and edema

4: In case of gingival bleeding, discoloration, edema and ulcer

5: The gingival hemorrhage was spontaneous, discolored and marked with swelling and ulceration.

Microbiological examination was performed using a phase contrast microscope (Leitz Labolux S, D-6330 Wetzlar, Germany) to observe the type of bacteria. To determine the distribution of gingival bacteria, first remove the gingiva-associated gingival germ membrane with a scaler, and then put Cury (Cracey curette # 11) to the base of the periodontal pocket. Five sites randomly selected with a phase contrast microscope (× 1,000) by floating in saline were observed under grid intercepts. The observed bacteria were classified into cocci, non-motile rods, and spirochetes with focus on morphology and motility. The ratio of was measured.

3). Evaluation criteria and interpretation method

(1) Cocci distribution

The distribution of cocci after 1 week of the experiment was compared with the 0 strain of cocci. Incubation increment after 1 week of experiment in Experimental group E1 was included. When the increase was compared, it was not determined that the increase in the experimental group was the same or that the increase in the control group was not improved. However, when the distribution of non-motor bacilli was decreased in the experimental group E1, it was determined that the non-motile bacilli reduction in the experimental group was not improved even if it was less than the non-motor bacilli reduction in the control group.

(2) Distribution of nonmotile bacilli

The distribution of nonmotile bacilli at 1 week after the experiment was compared with the distribution of nonmotile bacilli at 0 weeks. When the distribution of nonmotile bacillus in the experimental group E1 was decreased, it was determined that the nonmotile bacillus reduction in the experimental group was not improved even if it was less than the nonmotile bacilli reduction in the control group.

(3) distribution of motility bacilli

The motility bacterium distribution after 1 week of the experiment was compared with the motility bacterium distribution of 0 weeks. The reduction in motility bacilli distribution after one week of experiment in Experimental Group E1 was included. When the reduction was compared, it was judged to be improved when the reduction in the experimental group was large. However, the increase in the distribution of motility bacillus in the experimental group E1 was not improved even if the decrease in motility bacilli in the experimental group was less than that in the control group.

Effects of Essential Oils from Country of Origin on Periodontal Disease-causing Bacteria

The minimum inhibitory concentration of F. nucleatum was 0.64-2.56 when the minimum inhibitory concentrations were determined by adding essential oils to observe the effect of volatiles on the periodontal disease-causing bacteria. Table 6).

Table 6. Minimum Inhibitory Concentrations for Periodontal Pathogens of Domestic Essential Oil Extracts (%)

division Country essential oil extract Amcillin Fusobacterium Nucleum 10953 1.28 0.01 Fusobacterium Nuclitum 51190 2.56 0.01 Fusobacterium Nuclitum 49256 0.64 0.01 Actino Bacillus Actinomycetem Comitans 43717 ^-a 0.01 Actino Bacillus Actinomycetem Comitans 43718 - 0.01 Actino Bacillus Actinomycetem Comitans 33384 - 0.01 Prebotella Intermediate G8-9K-3 - 0.01 Porphyromonas jinbali 33277 - 0.01 Prevotela Nigresense 9336 - 0.01

^a No inhibitory effect at 2.56% concentration.

Clinical examination

The group washed orally with EDTA had lower gingival bleeding index than the control group washed with distilled water (Table 7). As a result of the smear test on the bacterium, non-motor bacillus, motility bacillus, and spiral bacillus, it was found that the reduction of each bacterium in the experimental group was remarkable compared to the control group.

Table 7. Changes in the subgingival hemorrhage index after brushing with essential oil extract and 0.125% EDTA solution

(Mean ± S.D.)

Subgingival Hemorrhage Index Control Experimental group Week 0 0.00 ± 0.00 0.00 ± 0.00 1 week 1.00 ± 0.57 0.00 ± 0.00

Table 8. Reduced percentage of motility bacteria after brushing with essential oil extract and 0.125% EDTA solution

Number of people

division Control (%) Experimental group (%) coccus 13/30 (43.3) 21/30 (70.0) Non-motor bacilli 8/23 (34.8) 16/26 (61.5) Motility bacilli 13/25 (52.0) 20/27 (74.1) spirillum 10/21 (47.6) 21/22 (95.5)

In conclusion, the essential oil extract (essential oil) of the present invention is Aspergillus nidulans (A. nidulans), Fusarium Oxy Forum (F. oxysporum) And Alternaria Mali (Al. mali) As the concentration of essential oil extract increased, filamentous fungi, etc. showed a marked decrease in growth, and growth was significantly inhibited compared to the control group. Also, the Fusarium Oxy Forum (F. oxysporumExudates from three varieties that are resistant to) inhibit germination of spores, but unlike previous reports that exudates from plants susceptible to hospitals promote spore germination (Soc. 40: 145), Fusarium oxy ForumF. oxysporum) Growth was inhibited by the essential oil extract of the country, and thus the essential oil of the country was proved to have an inhibitory effect on the growth of the fungus. Meanwhile, as the concentration of essential oil extract (essential oil) increases, the yeast fungus Candida albicans (C. albicans)Inhibition of germination and the germination of spores of filamentous fungi showed that the volatile extracts of the country contained antifungal substances.

Tulipinolide and costunolide, germacranolide sesquiterpene lactones isolated from the flowers of the country of origin, were tested against hemolytic Vibrio parahaemolyticus, Bacillus subtilis, Bacillus cereus, and S. aureus. Strong antimicrobial activity at 100 μg / disk is reported (Kor. J. Pharmacogn. 29 (2): 67-70). Representative biological activities of sesquiterpene lactones are cytotoxic and anti-tumor activity, and sesquiterpene lactones having various skeletons such as helenalin, eupatoriopicrin, melampodinin A, parthenin, xanthatin, parthenolide, and artemisinin have cytotoxic or anti-tumor activity. B16 Melanoma cells are known to decrease melanin content with increasing concentrations of extracts from the country (Kor. J. Pharmacogn. 29 (3): 243-247, Cancer Res. 53: 701). -706, 1993).

In the present invention, five bacteria Staphylococcus aureus (S. aureus), Streptococcus piogenes(S. pyogenes), Streptococcus mutans (S. mutans),Escherichia coli (E. coli)And Helicobacter Philly (H. pyloriThe antimicrobial activity of E. coli essential oil extract was investigated.E. coli)In the case of, there was no particular change even when the concentration increased. Streptococcus piogenes(S. pyogenes) Showed a strong antimicrobial activity with no growth at 0.2 μl / ml concentration. Streptococcus mutans (S. mutans) Showed strong antimicrobial activity from 0.4 μl / ml concentration. Staphylococcus aureus (S. aureus)Also in the case of increased concentration showed a strong antibacterial activity, Helicobacter Philly (H. pylori) Also showed strong antimicrobial activity as the concentration increased.

The most characteristic change in the recent mechanism of dental caries is the deliming of hemorrhoids. The ability to form dental plaques, which are closely related to the deliming process, is a necessary feature of carniogenic bacteria and Streptococcus mutans, the causative agent of dental cariesS. mutans)to be

(Japanese Journal of Oral Microbiology 32 (1): 104-107, Agri. Biol. Chem. 52 (1): 3169-3171, Yiwu Cultural History 1993). Propolis (Caries Res. 25: 347-351, Ph.D. Dissertation, Graduate School of Chonbuk National University), Yellow White Water Extract (Korean Journal of Oral Health 19 (4): 439-446), Water Extract of Terminallia chebula ( The Journal of Korean Academy of Oral Health 19 (4): 495-503), 0.1% fluoride and 0.1% sucrose mixture, have the same antimicrobial activity against Streptococcus mutans . Strong antibacterial against Streptococcus mutans from concentrations as low as 0.4 μl / ml as treatment at concentrations of μl / ml, 0.4 μl / ml, 0.6 μl / ml, 0.8 μl / ml and 1.0 μl / ml The results of these studies confirmed that the essential oil extracts from the country of origin showed strong antibacterial activity against the causative agents of dental caries compared with previous studies.

In addition, staphylococcus aureus (which can cause oral infections)S. aureus),Streptococcus piogenes(S. pyogenes) And Candida albicans (C. albicans)Proliferation inhibitory effect of Staphylococcus aureusS. aureus)Showed antimicrobial activity from 0.4 μl / ml for(S. pyogenes) Showed strong antimicrobial activity at low concentration of 0.2 μl / ml, demonstrating that the essential oil extract of the country of origin has a strong antimicrobial activity, and Candida albicans (C. albicans)Also showed high antifungal activity from 0.4 μl / ml.

Helicobacter Philoly, a Gram-negative spiral bacillus that has been noted as an important causative agent of chronic active gastritis patients, gastritis and gastric ulcers, inhabiting junctions between gastric mucosal epithelial cells (H. pyloriVolatile extracts from country of origin from Helicobacter phylloli (0.2 μl / ml concentration with increasing concentrationH. pylori) It was shown that it showed high inhibition on proliferation (J. Med. Microbiol. 19: 157-167, Gut. 34: 1038-1041, Eur. J. Gastroenterol. Hepatol. 5 (Suppl 1): S35-S39, Am J. Gstroenterol. 82: 192, Am. J. Gastroenterol. 88: 1729-1733, Therapeutic Research 10: 1478-1485). Addition of 0.125% EDTA increased antimicrobial activity and increased cytotoxicity (Tables 2 and 4).

As described above, it can be seen that the natural chemical components contained in the country of origin not only have excellent antifungal activity and antibacterial activity, but also have no cytotoxicity to human fibroblasts. In addition, the combination of domestic oil refinery and EDTA showed much higher growth inhibition. Based on these antifungal and antimicrobial effects, it is possible to develop and utilize antifungal agents and antimicrobial agents (pharmaceuticals) using acid extract as active ingredients.

Claims (13)

Antimicrobial and / or Antifungal Compositions Comprising Essential Oil Extract of Chrysanthemum boreale The antimicrobial and / or antifungal composition of claim 1 further comprising a pharmaceutically acceptable carrier or excipient. The method according to claim 1 or 2, wherein the antimicrobial spectrum is (gram-positive Staphylococcus aureus <S. Aureus>, Streptococcus pyogenes <S. Pyogenes>, Streptococcus mutans <S. Mutans>, Gram-negative The bacterium Helicobacter Philoly <H. Pylori>, Fusobacterium nucleatum <F. Nucleatum>, Actinobacilli actinomycetem comitans <A. Actinomycetemcomitans>, Prebotella intermedia <P. Intermedia> , Prebotel nigrescens <P. Nigrescens>, Porphyromonas ginjivalis <P. Gingivalis> The antifungal spectrum according to claim 1 or 2, wherein the antifungal spectrum (A. nidulans>, Alternaria Mali, F. oxysporum, Candida albicans < C. albicans>) The composition according to claim 1, wherein the composition is for oral cleaning agents. The composition of claim 1, wherein the composition is for treating gastrointestinal diseases. The preparation for oral cleansing according to claim 1, comprising an essential oil extract of an acid country as an active ingredient. The preparation for treating dental caries according to claim 1, comprising an essential oil extract of an acid country as an active ingredient. The preparation for treating gastrointestinal disorders according to claim 1, comprising an essential oil extract of an acidic country as an active ingredient. 10. The formulation of claims 7 to 9, further comprising a pharmaceutically acceptable carrier or excipient. The preparation according to claim 7, wherein the preparation is a liquid, tablet, hard soft capsule, suspension, injectable preparation, ointment, cream, gel or lotion. Process for preparing essential oil extracts of country of origin, characterized by steam distillation Essential oil extract of the producing country prepared by the method of claim 12