KR20070057108A - Antifungal extract produced by supercritical fluid extraction - Google Patents

Abstract

An extract is provided to show excellent antifungal activity without cytotoxicity, thereby being used as medicines and foods for preventing and treating various fungal infections. The extract is prepared by extracting an antifungal plant such as oregano, Chamaecyparis obtusa, Cinnamomum loureirii and Scutellaria baicalensis using a supercritical fluid of 35-80 deg.C and 100-500 bar and the fungus is dermatophyte fungus such as Trichophyton rubrum, Microsporum audouinii, Trichophyton ferrugineum, Epidermophyton floccosum and Trichophyton mentagrophytes.

Description

Antifungal extract produced by supercritical fluid extraction

1 is a process diagram of supercritical extraction of the present invention using a supercritical fluid extraction method.

Figure 2 shows the antifungal activity by broiler supercritical extract.

Figure 3 shows the antifungal activity by the golden supercritical extract.

Figure 4 shows the antifungal activity by the oregano supercritical extract.

Figure 5 shows the antifungal activity by the protein extract supercritical shock.

The present invention relates to an antifungal extract by supercritical fluid extraction, and more particularly to antibiotics in antifungal plants and herbs such as oregano, cypress, broiler and gold using a supercritical fluid extraction process. It relates to the use as an antifungal agent to increase the extraction efficiency and activity.

Pathogenic microorganisms and fungi are distributed worldwide and cause disease in animals, including humans. Although many synthetic antibiotics have been developed and used for the purpose of treating diseases caused by pathogenic microorganisms and fungi, strains resistant to various antibiotics have recently emerged due to the misuse of antibiotics. Due to this, there are a lot of restrictions on the use of antibiotics, which shows a new powerful antibiotic effect on antibiotic resistant strains, and there is a need for the development of natural antifungal agents that can be used as a material of various food preservatives and medicines because there is no toxicity to the human body. Chemicals are increasing interest in replacing conventional synthetic antimicrobial and antifungal agents with natural and antifungal agents due to safety issues such as accumulation in the body and consumers' avoidance (Cherry, JP, Food Technol. 53: 54-57, 1999; Cho, SH, et al., J. Food Hyg.Saf., 10: 33-39. 1995; Kim.YS., et al., Korea J. Food Sci.Technol 35 (2) : 159-165, 2003; Cho.MH, at al., Food Sci. And Ind., 38 (2): 36-45, 2005).

The development of antifungal agents is very difficult due to the low correlation between in vitro antifungal activity and in vivo antifungal activity of antifungal agents and the fact that the fungal cells to which antifungal agents act are very similar to human cells as eukaryotic cells. However, research continues to develop antifungal agents that improve the disadvantages of human antitoxicity, drug resistance, and low selectivity against fungal infection strains, which are the biggest problems of existing antifungal agents. , 2000).

Representative antifungal agents include ketoconazole, itraconazole and terbinafine (Mattew JS, Int J Dermatology , 32, pp 16-21, 1993). However, these antifungal agents, except for terbinafin, have a long-term therapy because they are fungal, except for terbinafine. The disadvantage of terbinafine is that it can only be used as an external preparation because of side effects that cause digestive problems. Since the human body is composed of eukaryotic cells like fungi, the human body can also be seriously damaged by drugs during long-term treatment with these antifungal agents. As described above, synthetic antifungal agents have problems in toxicity to human cells, drug resistance to the site of action and chronic drug delivery (Yamaguchi H., Jap . J. Clinic. Med ., 49, pp 2176-2185, 1990 ).

As a result of this trend, the food industry is also reducing the use of artificial synthetic antifungal agents, and researches on the use of safe natural antifungal substances for the preservation of foods have been intensively conducted, including quasi-drugs, cosmetics, and other household goods. Products are being created to increase their use.

The market for natural antimicrobials is growing globally, and data published in the US market for natural antimicrobials show that the market is about $ 500 million in 2000 and $ 600 million in 2003. It is projected to continue to grow to about $ 1 billion a year (see US Market for Food Additives by Types and Materials, through 2006). Therefore, the market of natural antibacterial drugs is expected to form a very large market based on domestic well-being trend.

The common method of extracting active ingredients from copper and plants is to use organic solvents such as water, methanol, ethanol, hexane, etc. The extraction method using organic solvents is easy to apply, while selecting an appropriate solvent, remaining of organic solvent, Difficulties include removal of solvents, environmental pollution, low extraction yield, and usually require a long extraction time of about 24 to 48 hours. However, for commercial use, it is necessary to shorten the stability and extraction time of the solvent, to acquire the environmental pollution and the ingredients to be extracted and to have the activity, but there is no clear method for this.

Supercritical Fluid Extraction Technology is a technology that uses fluids above the critical temperature and pressure. The critical temperature and pressure are the temperature and pressure points at which the phase transition from the gas phase to the liquid phase changes the temperature, pressure and volume. It refers to the temperature and pressure of a state where a change does not occur at any point when a change in gas liquefaction, liquid vaporization, or the like occurs. For example, 132 ° C for ammonia, 31 ° C for carbon dioxide, and -119 ° C for oxygen are known as critical temperatures. At higher temperatures, no gas is liquefied, no matter how high the pressure is.

The supercritical extraction method is attracting attention as a new environmentally friendly clean technology that can replace existing processes in the fields of extraction and purification of pharmaceuticals, food processing and petrochemicals (CD Bevan and PS Marshall., Nat. Prod. Rep. 11, 451-576, 1994; WK Modey, DA Mullholl et al., Phytochem.Anal. 7, 1-15, 1996).

The areas where early supercritical fluid technology is mainly applied to extracts from natural products are limited to non-polar substances such as spices, cosmetics, fats, low-cost foods and flavoring ingredients, but recently, due to the development of several phenomenological characteristics and additional technologies related to this technology, It has been applied to the extraction, purification of polar, small amount and expensive natural medicines.

Accordingly, the present inventors are conducting a supercritical fluid extraction method while conducting research on the effective extraction and development of natural antimicrobial agents possessed by plants or herbs such as oregano, cypress, broiler and gold, which are known to have antibacterial and antifungal properties. The extract obtained using the present invention was found to have a higher antifungal effect on the skin fungus than the extract extracted by the conventional organic solvent extraction method, to complete the present invention.

It is an object of the present invention to provide an antifungal extract for skin fungi prepared by supercritical fluid extraction of antifungal plants.

Another object of the present invention is to provide an antifungal pharmaceutical composition comprising the antifungal extract as an active ingredient.

Another object of the present invention is to provide an antifungal food comprising the antifungal extract as an active ingredient.

Another object of the present invention to provide an external preparation for skin comprising the antifungal extract as an active ingredient.

Another object of the present invention is to provide a phytopathogenic fungi inhibiting pesticide, an antifungal paint, a phytonutrient or an additive thereof, an air freshener, an additive for household goods or clothing, a food additive or a cosmetic additive comprising the antifungal extract as an active ingredient. .

In order to achieve the object of the present invention, the present invention provides an antifungal extract prepared by supercritical fluid extraction of an antifungal plant, the fungus provides an extract, characterized in that the skin fungi.

The present invention is to extract a high concentration of useful components exhibiting antifungal activity from the plant having an antifungal through a supercritical fluid extraction method instead of a conventional organic solvent, the extract prepared by the supercritical fluid extraction of the present invention is a skin among fungi It can be particularly effective against fungi.

The present inventors have reported that the extract obtained from oregano, cypress, broiler and gold using various organic solvents and the antibacterial substance separated therefrom have an antimicrobial effect on various microorganisms (Lee. CK et al., J. Korea). Soc.Food Sci.Nutr., 34 (10): 1606-1610, 2005; F. Sahin., Et al., Food Control., 15: 549-557, 2004 ; Shang-Tzen C., et al., J. Pham., 77: 123-127, 2001). Based on this, hot water, organic solvents and supercritical extracts were prepared using oregano, cypress, broiler and gold as ingredients. . The antimicrobial and antifungal activity of each extract was determined by measuring the growth inhibitory ring size using the disk measurement method and the minimum inhibitory concentration and 'EC ₅₀ ' of the extract using the mycelial growth inhibitory activity on the solid medium by the method of Poison Food Technique. As a result, oregano, cypress, broiler and golden supercritical extracts were 15-20 times higher than hot water extracts and 5-10 times higher than organic solvent extracts. It was found that the extraction method by supercritical extraction was superior to general organic solvent or hot water extraction (see Tables 4 to 11).

The antifungal plant used in the preparation of the antifungal extract according to one embodiment of the present invention may be oregano, cypress, broiler, gold, etc., but is not limited thereto. The characteristics of the plant are as follows.

Origanum vulgare L is a perennial plant with mint originated from the Mediterranean coast, especially the sunny and sloped hills of Greece, Italy and Spain, with straight branches, red stems, buttocks, and purple flowers blooming at the top. It is effective against bacterial, viral and fungal infections and strengthens the immune system. It is used to treat acne, allergies, animal bites, arthritis, asthma diuresis, and wounds.

Cypress ( Chamaecyparis obtusa ) is an evergreen arboretum of the pine family, native to Japan, and is also called a cypress. It grows up to 40m in height and 2m in diameter, and its branches spread horizontally. The leaves face each other and have a white Y-shape on the back.

Essential oil extracted from baekbaek is known to emit phytoncide compared to other trees, and pharmacological action is to suppress the central nerve, soothing, analgesic, antipyretic, and expanding blood vessels By promoting sweating, subcutaneous swelling of the mucous membranes is alleviated.

Broiler ( Cinnamomum loureirii Nees) is the bark of the evergreen tall-key broiler tree of the camphoraceae. The bark of the stem and root is spicy and fragrant, so it can be used as a medicinal or sweet flavoring agent. Cinnamon is called broiler (cinnamon) and is collected from the kind growing in Sri Lanka and Indochina. Open the skin's pores in the early cold to sweat, relieve pain in the shoulders and back, pain in the extremities joints, promote circulation of blood, and cure shortages. Pharmacological action has been reported to inhibit the development of Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pneumococcus, and to inhibit influenza virus and diuretic effect.

Golden ( Scutellaria baicalensis ) belongs to Lamiaceae and is native to northern China in East Asia, and is found in mountainous regions of Korea, and is distributed in East Siberia, Mongolia, China, and Manchuria. It is a naturalized plant that is imported as a medicinal plant and is grown in herbalist farms everywhere. It is often grown in fields. In the herbal medicine, golden is dried roots. In spring or autumn, the roots are dug up, scrape off the husks and rotten genus, rinse in water, dry in the sun, and use it as a medicinal herb.

The extract extracted by the supercritical fluid extraction method according to the present invention includes an antibacterial and antifungal substance as an active ingredient and shows excellent antibacterial and antifungal activity against pathogenic bacteria, yeast and fungi. Pathogenic fungi are diseases that cause disease by parasitic predominantly in humans and animals. Skin fungi, which the extract of the present invention can exhibit antifungal activity, are described in Table 1 below, but are not limited thereto. Preferably, the skin fungus is Trichophyton rubrum KCCM 60443), Microsporum Order audouinii KCTC 6346), Trichophyton ferrugineum (KCTC 6351), Epidermophyton floccosum , KCTC 6587), Trichophyton mentagrophytes , KCTC 6077).

Table 1 Classification of skin fungi

Asexual state Sexual reproduction ecology Trichophyton      Human friendly skin fungus T. rubrum T. mentagrophytes   var. interdigitale T. violaceum T. tonsurans T. schoenleinii T. concentricum Microsporum M. ferrugineum M. audouinii Epidermophyton E. floccosum Trichophyton    Animal-Friendly Skin Fungi T. mentagrophytes  var. mentagrophytes A. vanbreuseghemii A. benhamiae T. verrucosum Micorsporum M. canis A. otae  var. canis M. nanum A. otae  var. distortum A. obtusa Trichophyton  Soil-friendly skin fungus T. simii A. simii Microsporum A. gypsea M. gypseum A. incurvata

In the antifungal extract according to an embodiment of the present invention, the supercritical fluid extraction may be performed by the following steps:

1) pretreatment step of crushing the antifungal plant;

2) filling the supercritical fluid extractor with the powder crushed in step 1;

3) extracting the extract by inputting a supercritical fluid through a heat exchanger to the extractor filled with the raw material powder in step 2;

4) separating the supercritical fluid and the extract mixture by depressurizing the pressure reducing valve through a pressure reducing valve in a pressure reducing separator; And

5) The supercritical fluid separated in step 4 is recycled by depressurizing by pump with the supercritical fluid supplemented from the outside after circulating the reservoir, and the separated extract is collected.

Extraction by various organic solvent extraction methods from various plants having antifungal properties, selection of solvents, residual organic solvents, difficulty in removing solvents, environmental pollution, extraction yield is low, and usually about 24 to 48 hours There are various problems such as extraction time. On the other hand, extraction using the supercritical fluid extraction method can eliminate the problems caused by the organic solvent extraction method, it is possible to obtain a high purity product.

1 is a process diagram of supercritical extraction of the present invention using a supercritical fluid extraction method. In the supercritical fluid extraction according to an embodiment of the present invention, the supercritical fluid is preferably carbon dioxide, carbon dioxide is not only infinitely present in nature, but also abundantly generated in the steel industry or petrochemical industry, colorless, odorless, human body This is because it is harmless and chemically very stable. First of all, it shows lower critical temperature (31.1 ° C) and critical pressure (7.4 MPa) than any other fluid, so it can be easily adjusted to supercritical conditions, resulting in high energy efficiency. In addition, it can solve the problems occurring in the existing process, that is, human toxicity by the organic solvent remaining in the final product.

In the supercritical fluid extraction according to the embodiment of the present invention, the temperature of the supercritical fluid in step 3 is preferably 35 to 80 ° C. and a pressure of 100 to 500 bar. This is because the supercritical fluid extraction efficiency is lowered when the temperature and pressure are out of the above ranges.

In supercritical fluid extraction according to an embodiment of the present invention, ethanol, methanol, acetone, hexane, ethyl acetate or methylene chloride may be further added to the extractor of step 2, and the concentration of the cosolvent It is preferably 0 to 50% by volume. If the concentration of the cosolvent is outside the above range, there is a problem of significant fragrance loss upon solvent removal and solvent residual in the final product.

In supercritical fluid extraction according to one embodiment of the present invention, the antifungal plant in step 1 is preferably broken into fine particles having a diameter of 2 mm or less.

As a result of using the supercritical fluid extraction method, the present inventors obtained an extract having no loss of useful components, high extraction efficiency, and antifungal activity about 0.5 to 2 times higher than that of the conventional extraction method. Since the extract exhibits antimicrobial and antifungal activity against pathogenic bacteria, yeasts and fungi, the composition containing the supercritical fluid extract may also be used for the prevention and treatment of diseases caused by the microorganisms.

In order to achieve another object of the present invention, the present invention provides an antifungal pharmaceutical composition comprising the antifungal extract of the present invention as an active ingredient.

The pharmaceutical composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

The present invention also provides an antifungal food comprising the antifungal extract of the present invention as an active ingredient.

Extracts of the present invention can be added to food for the purpose of preventing and treating fungal infections. When the extract according to the present invention and its fractions are used as food additives, the extract and its fractions can be added as such or used together with other food or food ingredients, and can be suitably used according to conventional methods. The blending amount of the active ingredient can be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. Is sure.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all foods in a conventional sense.

Extract of the present invention can be used in addition to food and health drinks, health drinks may contain a variety of flavors or natural carbohydrates and the like as an additional ingredient, like a conventional drink. Natural carbohydrates are sugars such as glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin, cyclodextrin, xylitol, sorbitol, and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used.

In addition to the above, the extract of the present invention contains various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. In addition, the composition of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination.

The present invention also provides an external preparation for skin comprising the antifungal extract of the present invention as an active ingredient. The external preparation for skin may be in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel, but is not limited thereto.

Skin external preparations in the general sense refer to any kind of preparation that can come into contact with the skin when in use, regardless of the gender and age of the user. Use forms of the topical skin preparation may be solid, semisolid, liquid and gas.

The present invention also provides phytopathogenic fungi inhibiting pesticides, antifungal paints, plant nutrients or additives thereof, fragrances, household goods or clothing additives, food additives or cosmetic additives comprising the antifungal extract of the present invention as an active ingredient. .

Hereinafter, the present invention will be described in detail by way of examples. However, the following Examples are only for illustrating the present invention and do not limit the present invention.

Example

< Example  1> Antifungal Plant Extract Manufacturing

Extracts obtained from oregano, cypress, broiler and gold using various organic solvents and antibiotics isolated therefrom have been reported to have antimicrobial effects (Lee. CK et al., J. Korea Soc. Food Sci Nutr., 34 (10): 1606-1610, 2005; F. Sahin., Et al., Food Control., 15: 549-557, 2004 ; Shang-Tzen C., et al., J. Pham., 77: 123-127, 2001). Based on this, extracts were prepared by selecting oregano, cypress, broiler, and gold as ingredients.

<1-1> Hydrothermal  Extraction method

Oregano (world spice), cypress (Napoli plantation farmer), broiler (herbal medicine market) and gold (herbal medicine market) were dried and pulverized finely by a grinder, and then heated in a boiling water to obtain a plant extract. At this time, about 2 g of distilled water was added to 100 g of each plant sample, and the mixture was heated and extracted for 3 hours in a boiling water of 100 ° C., maintained at a constant temperature of 90 ° C., and the filtrate obtained by filtering the extract under reduced pressure was subjected to rotary vacuum. Concentrated with a concentrator (EYELA, N-series, Japan).

<1-2> Organic Solvent Extraction Method

To prepare a solvent extract, oregano, cypress, broiler, and gold were dried and pulverized finely with a grinder, and then immersed in alcohol for 24 hours. After centrifugation, only the supernatant was separated and filtered under reduced pressure with a filter paper (Filter paper Whatman NO. 4, USA). The organic solvent extract was concentrated under reduced pressure with a rotary vacuum concentrator (EYELA, N-series, Japan) at 40 ℃ to prepare a sample.

<1-3> of antifungal plants Supercritical  Extract manufacturer

1 kg of oregano, cypress, broiler, and golden dried crushed samples and 500-700 ml of co-solvent ethanol (99.5%) were placed in a supercritical fluid extraction reactor (Natex, Autstria) and the temperature in the reactor was 65 ° C. and the pressure was 450. It was extracted under the condition of bar, S / F ratio (supercritical fluid kg / Feed kg) 35.

< Example  2> oregano  Confirmation of Antifungal Effect of Extracts

Of the extract extracted from Example 1, the oregano extract was carried out the following experiment to determine the antifungal effect on various microorganisms.

<2-1> Antifungal activity  Test strain culture

Disc-agar plate diffusion method was used to determine the antifungal activity of the extracted extract using a supercritical fluid. Trichophyton rubrum KCCM 60443, a major pathogenic fungus, was found at the Korea Microbiological Conservation Center, Microsporum. audouinii KCTC 6346), Trcihophyton ferrugineum , KCTC 6351), Epidermophyton flococcosum , KCTC 6587), Trichophyton mentagrophytes , KCTC 6077), were distributed by the Korea Gene Bank and used as strains for antifungal activity assay of supercritical extracts. Each strain and culture conditions are shown in Table 2 below, the medium used according to each strain, and the medium composition is shown in Table 3 below.

TABLE 2 Strains and Culture Conditions

Strain Strain number Incubation temperature category badge Trichophyton rubrum KCCM 60443 25 ℃ mold Sabourauds Agar Tricophyton ferrugineum KCTC 6351 28 ℃ mold Sabourauds Agar Tricophyton mentagrophytes KCTC 6077 28 ℃ mold Sabourauds Agar Microsporim audouinii KCTC 6346 28 ℃ mold Sabourauds Agar Epidermophyton floccosum KCTC 6587 30 ℃ mold Sabourauds Agar

TABLE 3 Composition of Badges

badge Ingredient Volume (g) / distilled water 1ℓ Sabourauds Agar peptone 10 g Glucose 40 g Agar 20 g

<2-2> Antifungal Activity Confirmation by Disc-diffusion Method

After sterilizing Sabourauds Agar (SA) medium, pour about 15 ml of Petri dishes into Petri dishes to prepare a basal medium. After mixing, an appropriate amount was dispensed into the substrate medium prepared in advance to harden. After attaching the sterilized paper disc (diameter 8 mm) to the medium inoculated with each bacterium, the extract obtained in Example 1 was dissolved in dimethyl sulfoxide to 1 mg / Samples were instilled at the concentration of the disc and placed on the medium. After incubating the medium inoculated with each bacterium for 5 days at an appropriate temperature, the size of the growth zone was measured. Figures 2 to 5 show antifungal activity by broiler, golden, oregano and cypress supercritical extract, respectively.

TABLE 4

Strain Unit: mm oregano SFE SE HWE  Epidermophyton floccosum - - -  Trichophyton rubrum 12 11 -  Trihcophyton mentagrophytes 14 13 -  Trichophyton ferrugineum 21 18 -  Microsporum audouinii 18 12 -  SFE: Supercritical Fluid Extraction SE: Solvent Extraction HWE: Hydrothermal Extraction

As a result, as shown in Table 4, the hydrothermal extract did not have antimicrobial activity, the supercritical extract was more excellent antifungal effect than the organic solvent extract.

<2-3> Minimum Inhibition Concentration

Mycelial inhibitory concentration was measured by the Poison Food Technique to investigate the mycelial inhibitory activity on solid medium. Savorauds agar medium (Sabourauds Agar, SA) was used as a solid medium and the compounds were treated at 500, 250, 125, 62.5, 31.25 μg / ml levels. Solid medium containing each compound was dispensed at 1 ml for each well of a 24-well plate. Agar disks with a diameter of 4 mm were removed from the well-growing cell tips and inoculated in the middle of the wells, and the control group was treated with SA medium containing 1% DMSO and inoculated with each bacterium. In the control group, the mycelial growth was measured after culturing until the tip of the cells reached the well edge. The diameter of the growth resistant ring was measured by culturing for 4 days at an appropriate temperature for each strain. Four replicates were performed for each treatment.

Four supercritical extracts, organic solvent extracts, and hot water extracts for five pathogenic fungi were shown as the minimum inhibitory concentration (MIC), that is, the minimum concentration necessary for complete inhibition of mycelial growth. Activity was measured as% inhibitory activity of the growing radius. In addition, 50% inhibition of growth of each pathogenic fungus (Effective Concentration, EC ₅₀ ) was calculated.

TABLE 5

Sample strain Unit: μg / ml oregano SFE SE HWE MIC EC ₅₀ MIC EC ₅₀ MIC EC ₅₀  Epidermophyton floccosum 500 286.20 > 500 854.79 > 500 15601  Trichophyton rubrum 250 142.05 > 500 506.33 > 500 1129.03  Trihcophyton mentagrophytes 250 120.05 > 500 504.53 > 500 1944.44  Trichophyton ferrugineum 250 122.70 > 500 434.78 > 500 4922.78  Microsporum audouinii 500 142.86 > 500 227.27 > 500 1577.39  SFE: Supercritical Fluid Extraction SE: Solvent Extraction HWE: Hydrothermal Extraction-: Non-inhibiting, ND: Non-Detection

As a result, as shown in Table 5, the oregano hydrothermal extract and the organic solvent extract have a minimum inhibitory concentration of about 500 ㎍ / ml or more against pathogenic fungi, whereas the supercritical extract is Epidermophyton. floccosum And Microsporum 500 ㎍ / mL for audouinii , Trichophyton rubrum , Trichophyton mentagrophytes And Trichophyton ferrugineum were completely inhibited at a concentration of 250 μg / ml.

< Example  3> Confirmation of antifungal effect of cypress extract

In order to confirm the antifungal effect on the protein extract extracted from Example 1, the experiment was carried out similarly to Example 2 except that the protein extract was used.

<3-1> Confirmation of antifungal activity by disk diffusion method

TABLE 6

Strain Unit: mm White SFE SE HWE  Epidermophyton floccosum 11 - -  Trichophyton rubrum 27 - -  Trihcophyton mentagrophytes 28 - -  Trichophyton ferrugineum 45 - -  Microsporum audouinii 35 - -  SFE: Supercritical Fluid Extraction SE: Solvent Extraction HWE: Hydrothermal Extraction

As a result, as shown in Table 6, the antifungal activity appeared only in the extract of the supercritical supercritical, Epidermophyton There was also an antifungal effect on floccosum , but Other strains showed better antifungal effects. In particular, it showed high antifungal effect against Trichophyton ferrugineum .

<3-2> Minimum Inhibition Concentration

TABLE 7

Sample strain Unit: μg / ml White SFE SE HWE MIC EC ₅₀ MIC EC ₅₀ MIC EC ₅₀  Epidermophyton floccosum > 500 833.33 > 500 930.23 > 500 858.56  Trichophyton rubrum 250 139.66 > 500 681.82 > 500 2143.65  Trihcophyton mentagrophytes > 500 135.14 > 500 444.44 > 500 2449.32  Trichophyton ferrugineum 125 86.21 > 500 344.83 > 500 1269.08  Microsporum audouinii 500 88.50 > 500 373.83 > 500 2574.78  SFE: Supercritical Fluid Extraction SE: Solvent Extraction HWE: Hydrothermal Extraction-: Non-inhibiting, ND: Non-Detection

As a result, as shown in Table 7, the minimum inhibitory concentrations of the supercritical extracts were 125 μg / ml and Trichophyton for Trichophyton ferrugineum . The concentration of 250 ㎍ / mL was shown for rubrum , but for other strains, the minimum inhibitory concentrations of supercritical extract, organic solvent extract, and hot water extract showed more than 500 ㎍ / mL.

< Example  4> Confirm antifungal effect of various broiler extracts

In order to confirm the antifungal effect on the broiler extract extracted from Example 1, the experiment was carried out similarly to Example 2 except that the broiler extract was used.

<4-1> Antifungal Activity Confirmation by Disk Diffusion Method

TABLE 8

Strain Unit: mm Broiler SFE SE HWE  Epidermophyton floccosum 55 16 -  Trichophyton rubrum > 90 36 -  Trihcophyton mentagrophytes > 90 56 -  Trichophyton ferrugineum > 90 43 -  Microsporum audouinii 80 14 -  SFE: Supercritical Fluid Extraction SE: Solvent Extraction HWE: Hydrothermal Extraction

As a result, as shown in Table 8, all of the supercritical extracts of broilers were found to be excellent antifungal effect against pathogenic microorganisms. Especially Trichophyton rubrum , Trichophyton mentagrohpytes , Trichophyton The antifungal effect of the supercritical extract on ferrugineum was found to be over 90 mm. In addition, all of the hot water extracts had no antifungal effect, and the supercritical extracts were superior to the organic solvent extracts.

<4-2> Minimum Inhibition Concentration

TABLE 9

Sample strain Unit: μg / ml Broiler SFE SE HWE MIC EC ₅₀ MIC EC ₅₀ MIC EC ₅₀  Epidermophyton floccosum 500 264.55 > 500 877.55 > 500 909.09  Trichophyton rubrum 31.25 - 500 321.97 > 500 747.13  Trihcophyton mentagrophytes 31.25 - 250 130.72 500 967.62  Trichophyton ferrugineum 62.5 59.88 500 409.84 > 500 5834.25  Microsporum audouinii 31.25 - 250 1148.81 > 500 971.70  SFE: Supercritical Fluid Extraction SE: Solvent Extraction HWE: Hydrothermal Extraction-: Non-inhibiting, ND: Non-Detection

As a result, as shown in Table 9, supercritical extract showed a strong antifungal effect at the concentration of 31.25 ~ 500 ㎍ / ㎖ at the minimum inhibitory concentration, especially Trichophyton rubrum , Trichophyton mentagrophytes and Microsporum It showed the strongest antifungal effect against audouinii .

< Example  5> Confirm antifungal effect of various golden extracts

In order to confirm the antifungal effect on the golden extract extracted from Example 1, the experiment was carried out similarly to Example 2 except that the golden extract was used.

<5-1> Antifungal Activity by Disk Diffusion Method

TABLE 10

Strain Unit: mm Gold SFE SE HWE  Epidermophyton floccosum 13 13 -  Trichophyton rubrum 42 40 21  Trihcophyton mentagrophytes 46 37 20  Trichophyton ferrugineum 45 40 20  Microsporum audouinii 42 35 -  SFE: Supercritical Fluid Extraction SE: Solvent Extraction HWE: Hydrothermal Extraction

As a result, as shown in Table 10, unlike the hydrothermal extract of other plants, Trichophyton rubrum , Trichophyton mentagrophytes And Trichophyton Anti- fungal effect was observed in golden hydrothermal extract against ferrugineum . When compared with the antifungal effect of the golden supercritical extract and the organic solvent extract on pathogenic microorganisms, it was confirmed that the supercritical extract had a slightly higher antifungal effect.

<5-2> Minimum Inhibition Concentration

TABLE 11

Sample strain Unit: μg / ml Gold SFE SE HWE MIC EC ₅₀ MIC EC ₅₀ MIC EC ₅₀  Epidermophyton floccosum > 500 1544.04 > 500 1819.32 > 500 3214.29  Trichophyton rubrum 31.25 - 62.5 32.47 250 210.11  Trihcophyton mentagrophytes 31.25 - 250 139.66 500 303.95  Trichophyton ferrugineum 250 121.95 > 500 434.00 > 500 1343.50  Microsporum audouinii 250 70.92 > 500 377.36 > 500 1636.04  SFE: Supercritical Fluid Extraction SE: Solvent Extraction HWE: Hydrothermal Extraction-: Non-inhibiting, ND: Non-Detection

As a result, as shown in Table 11, Epidermophyton Supercritical extracts showed excellent antifungal effects against 4 strains except floccosum , Trichophyton rubrum and Trihcophyton For mentagrophytes , a very strong antifungal effect was observed at a concentration of 31.25 ㎍ / mL.

< Example  6> Preparation of a food comprising the antifungal extract of the present invention

Food products comprising the antifungal extract or fractions thereof according to the present invention were prepared as follows.

<6-1> Preparation of Flour Food

0.5 to 5.0% by weight of the antifungal extract or fractions thereof according to the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

<6-2> Production of Dairy Products

0.5 to 10% by weight of the antifungal extract or fractions thereof according to the present invention was added to milk and various milk products such as butter and ice cream were prepared using the milk.

<6-3> Linear  Produce

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then roasted to prepare a powder having a particle size of 60 mesh.

The antifungal extract or fractions thereof according to the present invention were concentrated under reduced pressure in a vacuum concentrator and dried by spraying and drying with a hot air dryer to grind the dried product to a particle size of 60 mesh using a grinder to obtain a dry powder.

The grains, seeds, and anti-fungal extracts according to the present invention, or dry powders of the fractions thereof, were prepared by blending in the following ratios.

Cereals (30% by weight brown rice, 15% by weight radish, 20% by weight barley),

Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame),

Dry powder of the antifungal extract or fractions thereof according to the invention (0.5-3% by weight),

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

<6-4> Preparation of Carbonated Drink

Add 5 ~ 10% sugar, 0.05 ~ 0.3% citric acid, 0.005 ~ 0.02% caramel, 0.1 ~ 1% vitamin C, and add 75 ~ 93.57% purified water and antifungal extract or fractions thereof. Mixing to make a syrup, the syrup is sterilized for 20 to 180 seconds at 85 ~ 98 ℃ mixed with cooling water in a ratio of 1: 4 and then injected 0.5 to 0.82% carbon dioxide gas antifungal extract according to the present invention or its Carbonated beverages containing fractions were prepared.

<6-5> Health drink  Produce

Substances such as liquid fructose (0.5%), oligosaccharides (2%), sugars (2%), salts (0.5%), water (75%) and antifungal extracts according to the present invention or fractions thereof (0.5-5%) After homogeneous mixing and sterilization, and then packaged in a small packaging container such as glass bottles, plastic bottles to prepare a healthy drink.

<6-6> Vegetable juice  Produce

Antifungal extract or fractions thereof (0.5 to 5%) according to the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

<6-7> Fruit juice  Produce

Health promotion fruit juice was prepared by adding 0.5 to 5% of the antifungal extract or fractions thereof according to the present invention to 1,000 ml of apple or grape juice.

By applying the supercritical fluid extraction method as described above, there is no residual organic solvent, and it is possible to efficiently extract antifungal substances derived from plants or herbs such as oregano, cypress, broiler, gold, etc. within a short extraction time, and various bacteria, molds, yeasts It has a wide range of excellent antifungal effect, has the advantage that the resistant strain by the existing chemical synthesis agent is not produced, and because it is composed of natural substances harmless to the human body, there is no residual toxicity, so it can be used as an eco-friendly antifungal agent, food, medicine and It can be used in various industries such as cosmetics.

Claims (13)

An antifungal extract prepared by supercritical fluid extraction of an antifungal plant, wherein the fungus is a skin fungus. The antifungal extract according to claim 1, wherein the antifungal plant is at least one selected from the group consisting of oregano, cypress, broiler and gold. The method of claim 1, wherein the skin fungus is Trichophyton rubrum , Microsporum order ( Microsporum audouinii ), Trichophyton ferrugineum ), Epidermophyton floccosum or Trichophyton mentagrophytes mentagrophytes ) antifungal extract, characterized in that. The antifungal extract of claim 1, wherein the supercritical fluid extraction is performed by the following steps: 1) pretreatment step of crushing the antifungal plant; 2) filling the supercritical fluid extractor with the powder crushed in step 1; 3) extracting the extract by inputting a supercritical fluid through a heat exchanger to the extractor filled with the raw material powder in step 2; 4) separating the supercritical fluid and the extract mixture by depressurizing the pressure reducing valve through a pressure reducing valve in a pressure reducing separator; And 5) The supercritical fluid separated in step 4 is recycled by depressurizing by a pump with the supercritical fluid supplemented from the outside after circulating the reservoir, and the separated extract is collected. 5. The antifungal extract of claim 4, wherein the supercritical fluid is carbon dioxide. The antifungal extract of claim 4, wherein the supercritical fluid has a temperature of 35 to 80 ° C. and a pressure of 100 to 500 bar in step 3. 6. 5. The antifungal extract according to claim 4, wherein ethanol, methanol, acetone, hexane, ethyl acetate or methylene chloride is further added to the extractor of step 2. 8. The antifungal extract according to claim 7, wherein the concentration of the cosolvent is 0 to 50% by volume. An antifungal pharmaceutical composition comprising the antifungal extract of claim 1 as an active ingredient. An antifungal food comprising the antifungal extract of any one of claims 1 to 8 as an active ingredient. An external preparation for skin containing the antifungal extract of any one of claims 1 to 8 as an active ingredient. The skin external preparation according to claim 11, which is in the form of an ointment, a lotion, a spray, a patch, a cream, a powder, a suspension, a gel or a gel. Phytopathogenic fungal inhibitors, antifungal paints, phytonutrients or additives thereof, fragrances, additives for household goods or clothing, food additives or cosmetic additives comprising the antifungal extract of any one of claims 1 to 8 as an active ingredient .

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