KR20000059468A - Fungicidal composition comprising the extract from Cornu cervi - Google Patents

Abstract

PURPOSE: Provided is an antifungal composition containing the extract of cervi parvum cornu as an effective ingredient. The extract shows inhibiting activity of formation of pathogenic mycelium. CONSTITUTION: The extract of cervi parvum cornu having an antifungal effect is represented by the formula (1) and is obtained by the following steps of: extracting cervi parvum cornu with hexane; extracting the extract residues with chloroform; extracting the extract residues with ethanol; obtaining CN-E by distillation under reduced pressure, in which CN-E is further purified by solubilizing in chloroform/methanol/2.5M ammonia water(20:10:2, v/v/v) solution and isolated to obtain CN-Es, or CN-Es is further purified by solubilizing in chloroform/methanol/2.5M ammonia water(20:10:2, v/v/v) solution and isolated to obtain CN-Es and then passing through the silica gel column chromatography to obtain CN-Es-6, or CN-Es-6 is further purified by solubilizing in chloroform/methanol(2:1, v/v) solution and isolated to obtain CN-Es-6s, or CN-Es-6s is further purified by solubilizing in chloroform/methanol(2:1, 1:1, 1:1.5, v/v) solution, and chloroform/methanol/2.5M ammonia water(20:10:2, v/v/v) solution and passing through the silica gel column chromatography to obtain CN-Es-6s-F, and CN-Es-6s-F is further purified by solubilizing in chloroform/methanol(50:1, 2:1, 1:1, v/v) solution, and chloroform/methanol/2.5M ammonia water(20:10:2, v/v/v) solution and passing through the silica gel column chromatography to obtain CN-Es-6s-F-d.

Description

Antifungal agent extracted from deer antler {Fungicidal composition comprising the extract from Cornu cervi}

The present invention relates to an antifungal composition containing an ethanol extract of deer antler (plum) or a fraction isolated and purified therefrom, and a method for extracting, separating and purifying the ethanol extract of such deer antler and fractions thereof. In addition, the present invention confirms that the ethanol extract of Deer Antler contains the compounds represented by the following formula (1) as an active ingredient that inhibits the formation of mycelia, and provides an antifungal composition and a novel compound of the formula (1) containing them as active ingredients .

[Formula 1]

In the above formula

R represents octadecanoyl (stearyl), hexadecanoyl (palmitoyl), cis-9-octadecenoyl (oleoyl) or cis-9,12-octadecadenoyl (linoleoyl).

Deer antler is dried by collecting unhorned shells of deer belonging to the family Cornu cervi, and is one of the most widely used herbals in Northeast Asia as well as Korea. Among the deer antler origin animals used in Korea, there are Cervus nippon Temminick var. Mantchuricus Swinhoe and Cervus elaphus L. Cornu Ceriv Pavum.

Deer antler is reported to have pharmacological effects on the immune system, hematopoietic system, hepatic system, glucose metabolism, cardiovascular system, and the like. However, even though modern physiology, biochemistry, and pharmacological knowledge has been mobilized to reveal precisely the mechanisms of these effects of deer antler, little is known about the active ingredients or specific pharmacological action and mechanism of antler. . In this regard, the present inventors recently discovered the structure of a substance that promotes the proliferation of hematopoietic stem cells, platelet progenitor cells and macrophages in antler, but achieved the first structural analysis of the active ingredient, but the mechanism of action of the active ingredient It has not been revealed yet.

On the other hand, according to a report that antler is used as a tonic in oriental medicine and antler increases the body's overall immune function, antler extract can be used for the treatment of immune-related diseases based on the results of a study that measured the activity of natural killer cells with antler extract. It is estimated to be. However, to date, no studies have been carried out on whether the activity of increasing antler's immunity or disease resistance is due to activation of host cells or deterioration of physiological activity of bacteria.

Candida albicans, which is targeted in the present invention, is a type of fungus that causes opportunistic fungal infection. The fungal disease has rapidly increased in the last 20 years and has a high mortality rate. This high mortality rate is attributable to an increase in AIDS patients and an increase in immunocompromised patients due to the use of antibiotics for cancer chemotherapy and organ transplantation. In fact, in the mid-1980s, 40 percent of deaths from so-called hospital acquired infections were caused by fungi, not bacteria or viruses, and 80 percent were caused by the genus Candida. In addition, considering that 95% of Candida diseases found in the United States are caused by one species of Candida albicans, we can feel the power of Candida albicans species, and these data suggest that the early diagnosis and effective treatment of pathogenic fungal diseases in medicine To realize the need.

Ampoterisin B (amphotericin B) and the azole compounds ketoconazole, fluconazole and clotrimazole are currently known as the representative antifungal agents, all of which are chemically synthesized and not extracted from natural products. It is evaluated that it is not ideal for treating mycosis due to its various side effects. In particular, amphotericin B, known as the best antifungal agent, has a nickname 'amphoterrible' due to severe side effects such as high fever, lowering blood pressure, headache, vomiting, vasculitis, and kidney damage. Not only does it kill the fungus, it prevents it from spreading anymore, and there is also a lack of selective toxicity to the pathogen, causing many side effects in the host. In the case of ketoconazole, production has been stopped for three years.

A side effect of the fungal agent on a host is a problem due to the biological properties of the fungus that fungi also share many biochemical properties with host cells as eukaryotic organisms. Humans have been able to succeed in developing antibiotics because the target bacterial cells are prokaryotic organisms that are different from host cells, and because of their different biochemical properties, they have been able to apply active research to them. Therefore, in order to develop effective antifungal agents, it is necessary to search for targets that are specifically expressed in molds.The cell wall of the fungus, which is emerging as a target of antifungal agents, is a component lacking in eukaryotic organisms as host cells. Can be used as a good target at.

In this situation, the present inventors have made significant breakthroughs in the field of antifungal agents, which have so far relied on treatment with compounds prepared by chemical synthesis methods, if they can isolate the substance that affects the physiological activity of the fungus from antler and reveal its structure. I thought about it and started my research. As a result, it succeeded in clarifying the structure by separating from the antler a substance that completely inhibits the formation of pathogenic forms of mycelium involved in the development of Candida albicans, a type of fungus, thereby completing the present invention.

Figure 1 shows a thin layer chromatogram (TLC) of the fraction obtained in each step of further separating and purifying the 70% ethanol extract of Deer Antler (Plum) according to the method of Examples 2 to 4 [a, e: CN-Es-6s-Fd, b: CN-Es, c: CN-Es-6s, d: CN-Es-6s-F].

Figure 2 shows the fractionation method according to the process of Examples 2 to 4 of the antler 70% ethanol extract (CN-E).

Figure 3 shows the comparison of the effect of antler fraction and conventional antifungal agent on yeast growth of Candida albicans strain.

4 is a ¹ H- ¹ H COSY spectrum of the CN-Es-6s-Fd fraction according to the present invention.

5 is ¹³ C- ¹³ C COSY spectrum of the CN-Es-6s-Fd fraction according to the present invention.

6 is a FAB-MS spectrum of the CN-Es-6s-F-d fraction according to the present invention.

7 is a tandem mass spectrum (FAB-MS / MS) for m / z 546.07 corresponding to novel compound CHJ-1 according to the present invention.

8 is an infrared spectrum measured using Bio-Rad FTS 165 by coating the CN-Es-6s-F-d fraction according to the present invention on KBr cells in the form of a neat (neat).

It is a first object of the present invention to provide an antifungal composition containing ethanol extract of antler of the present invention or fractions separated and purified therefrom as an active ingredient.

Antifungal compositions isolated and purified from antler by the inventors can inhibit the conversion of Candida albicans to pathogenic forms, which account for 80% of pathogenic fungal diseases. In other words, Candida albicans is present in the non-pathogenic form of the yeast form and the pathogenic form of the hyphae form, and when it is converted from the yeast type to the mycelium form, the epithelial invasion enters the blood and the organ Hematogenously disseminated infection is known to be the pathogenesis. However, the antler component separated by the present inventors was shown to exhibit some degree of inhibition on the growth of the fungus to the yeast type and to completely inhibit the conversion to the pathogenic form of the mycelium type. Therefore, this component is very likely to be developed as a new antifungal agent without side effects compared to existing polyene or azole compounds.

The ethanol extract of antler used in the composition according to the present invention or the ethanol extract isolated from the purified fractions is a substance extracted by the following method. It is another object of the present invention to provide such extraction and separation methods.

According to the present invention, first, the antler is extracted with hexane, and the extraction residue is extracted again with chloroform, and then the extraction residue is extracted with ethanol again. The extract obtained is distilled under reduced pressure to obtain an ethanol extract of antler (hereinafter, extract CN-E). Is obtained).

The amount of the hexane, chloroform and ethanol extractant used in the extraction method as described above should be enough to immerse the used antler, and in general, each solvent in the ratio of about 4 to 5 l per 1 kg of antler. use.

Extract CN-E, an ethanol extract of antler obtained by this method, is further fractionated and purified by a series of silica gel column chromatography and TLC method. That is, the subsequent treatment step of extract CN-E, an ethanol extract of deer antler,

a) Extraction CN-E is treated with a mixture of chloroform / methanol / 2.5M ammonia water (20: 10: 2, v / v / v) and dissolved (extract CN-Es) and insoluble (extract CN-Ei). Fractionating;

b) Silica gel column chromatography using extract CN-Es as a eluent, a mixture of chloroform / methanol / 2.5M aqueous ammonia (20: 10: 2, v / v / v) solution, and the resulting fractions were run on TLC to give an Rf value. Obtaining nine extracts according to the extracts CN-Es-1 to CN-Es-9;

c) The part of the extract obtained in step b), which is treated by treating the extract CN-Es-6 with a chloroform / methanol (2: 1, v / v) mixed solution (extract CN-Es-6s) and the insoluble part ( Fractionation with extract CN-Es-6i);

d) extract CN-Es-6s with chloroform / methanol (2: 1, 1: 1, 1: 1.5, v / v), chloroform / methanol / 2.5M ammonia water (20: 10: 2, v / v / v) Performing silica gel column chromatography while changing to a mixed solution of the mixture, and expanding the obtained fraction on TLC to obtain seven extracts from CN-Es-6s-A to CN-Es-6s-G depending on the Rf value;

e) The extract CN-Es-6s-F in the extract obtained in step d) was added chloroform / methanol (50: 1, 2: 1, 1: 1, v / v), chloroform / methanol / 2.5M ammonia water (20 Silica gel column chromatography with a mixed solution of: 10: 2, v / v / v), and the resulting fractions were developed on TLC to extract CN-Es-6s-Fa to CN-Es-6s- according to Rf. It consists of obtaining four extracts up to Fd.

The fractionation and purification process of such antler ethanol extract CN-E is briefly shown in Figure 2, for example.

The extracts CN-Es, CN-Es-6, CN-Es-6s, CN-Es-6s-F and CN-Es-6s-Fd obtained in a series of purification processes as described above are also described in Examples below. It can be used as an active ingredient in the composition of the present invention in place of extract CN-E because it has mycelial formation inhibitory activity as demonstrated by.

Nuclear magnetic resonance analysis of the fraction CN-Es-6s-Fd finally obtained according to the method of the present invention to identify the active ingredient contained in the ethanol extract CN-E of deer antler used as an active ingredient in the composition of the present invention. In addition, the mass spectrometry and infrared spectrometry were performed, and the derivative formation reaction was performed. As a result, it was confirmed that the fraction contained the compound represented by Chemical Formula 1.

[Formula 1]

In the above formula

R represents octadecanoyl (stearyl), hexadecanoyl (palmitoyl), cis-9-octadecenoyl (oleoyl) or cis-9,12-octadecadenoyl (linoleoyl).

According to the present invention, as the compound of formula 1, which is an active ingredient found to be contained in the ethanol extract of deer antler and its fractions, specifically, the following four compounds may be mentioned.

1) Compound CHJ-1 (C ₂₆ H ₅₅ N ₂ O ₆ P; R = octadecanoyl)

2) Compound CHJ-2 (C ₂₄ H ₅₁ N ₂ O ₆ P; R = hexadecanoyl)

3) Compound CHJ-3 (C ₂₆ H ₅₃ N ₂ O ₆ P; R = cis-9-octadecenoyl)

4) Compound CHJ-4 (C ₂₆ H ₅₁ N ₂ O ₆ P; R = cis-9,12-octadecadienoyl)

As described above, there has been no report that the compound of formula 1, which has been identified as an active ingredient from the antler extract according to the present invention, is present in the antler. In particular, since the compound of Formula 1 is a novel compound that was not known before the present invention, it is another object of the present invention to provide novel compounds CHJ-1 to CHJ-4 represented by Formula 1.

According to the TLC results shown in FIG. 1, the four compounds of the above-mentioned compounds CHJ-1 to CHJ-4 ranged from CN-E, an ethanol extract of deer antler, to the fraction CN-Es-6s-Fd obtained in the final purification step. It can be seen that the material is maintained without denaturation by showing the same band, and the band becomes clearer as the purification step proceeds.

These compounds of formula 1 can be chemically synthesized by methods known or analogous to known methods. For example, the compound of Formula 1 may be prepared by using the compound of Formula 2 as a starting material, esterification, amide bond formation, reduction, and phosphorylcholine preparation. It can be prepared by the process of (see Scheme 1 below):

In Scheme 1, R is as defined above.

The preparation method of Scheme 1 uses a known reaction, and in the specific reaction conditions, conventional techniques commonly used in the art may be applied.

As described above, since the components showing mycelial formation inhibitory activity, i.e., antifungal activity, in the antler extract may be economically prepared by chemical synthesis method, these chemically synthesized chemical compounds are used instead of expensive antler, which is expensive and difficult to obtain. By using any one of the two substances or a mixture of two or more of them, a surprising effect equivalent to the antler extract can be obtained.

Accordingly, another object of the present invention is to provide an antifungal composition containing the compound of Formula 1 as one or more active ingredients.

The antifungal composition containing the ethanol extract of Deer Antler according to the present invention or a fraction thereof or a compound of Formula 1 as an active ingredient may be administered as an injectable preparation or an oral preparation as desired when administered for clinical purposes. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be prepared using suitable dispersing, wetting or suspending agents according to the known art. Pharmaceutically acceptable solvents that can be used include water, Ringer's solution and isotonic NaCl solution, and sterile fixed oils are conventionally employed as a solvent or suspending medium. Any non-irritating fixed oil may be used for this purpose, including mono- and diglycerides, and fatty acids such as oleic acid are also used in the preparation of injectables.

The solid dosage form for oral administration may be in the form of capsules, tablets, pills, powders and granules, and particularly, capsules and tablets are useful. Tablets and pills are preferably prepared with enteric agents. Solid dosage forms can be prepared by mixing the polymer polysaccharide fraction according to the present invention with one or more inert diluents such as sucrose, lactose, starch and the like and carriers such as lubricants such as magnesium stearate, disintegrants, binders and the like.

The dosage level for a particular patient of the composition according to the present invention is the type of extract, fraction or compound used as the active ingredient, the weight, sex, health condition, diet, time of administration, method of administration, excretion rate of the individual patient to which the composition is to be administered. It may be varied by the expert depending on the condition of the patient, and the mixture of the drug and the patient to use the composition of the present invention. For example, when the antler fraction CN-Es-6s-F-d is used, the daily dose per adult can range from about 600 mg to 1500 mg.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention in any sense is not limited to these examples.

Example 1

3 kg of deer antler (plum) was added to a finely crushed beaker, and hexane was added to the level of the deer antler (approximately 12 L), heated at 80 ° C. for 2 hours, extracted twice, and filtered (this filtrate was called extract 1). Extract 1 was distilled off under reduced pressure and dried to give 27.07 g of extract CN-H.

The extract was separated and added to the remaining residue soaked with chloroform (about 12 L), heated at 70 ° C. for 2 hours, extracted twice, and filtered (this filtrate was called Extract 2). Extract 2 was distilled off under reduced pressure to give 23.55 g of extract CN-C.

Extract 2 was separated again, and 70% ethanol (approximately 12 L) was added to the remaining residue, followed by cooling for 2-3 days, followed by extraction three times and filtering (this filtrate was referred to as extract 3). Extract 3 was distilled off under reduced pressure to obtain 66.99 g of extract CN-E, an ethanol extract of antler.

The extract CN-E, which is determined to be effective as an indicator of mycelial formation inhibitory activity as described in Example 5 below, for each of the extract CN-H, CN-C, and CN-E obtained above, was purified as follows. Used for the procedure.

Example 2

66.99 g of extract CN-E obtained in Example 1 was added to a mixed solution of chloroform / methanol / 2.5M ammonia (20: 10: 2, v / v / v) and centrifuged to melt 51.02 g (extract CN-Es). And 14.37 g (extract CN-Ei) of the insoluble portion were obtained. Of these, the melting part was divided into half (25.51g each) and the following method was performed about each.

4000 g of chloroform / methanol / 2.5M ammonia water (20: 10: 2, v / v / v) mixed solution was added to 1200 g of powdered silica gel, followed by swelling, followed by filling in an open column (bed volume = 2850 ml). . 25.51 g of the obtained extract CN-Es was dissolved in a minimum amount (the minimum amount capable of dissolving the extract) in a mixed solution of chloroform / methanol / 2.5M ammonia (20: 10: 2, v / v / v), followed by silica gel filling. To the applied column. After dissolving chloroform / methanol / 2.5M ammonia water (20: 10: 2, v / v / v) mixed solution as eluent in the column, do not dry out, and then take 100 ml of elution fraction per second. The volume (void volume) is 950 ml), which was developed by TLC (chloroform / methanol / 2.5M aqueous ammonia = 20: 10: 2, v / v / v), developed with iodine, and separated according to the Rf value. In this way, all nine major fractions were separated, distilled under reduced pressure, and dried to name the extracts CN-Es-1 to CN-Es-9, respectively. As a result of searching for mycelial formation inhibitory activity as in Example 5 described below, these nine extracts were separated and used for the following purification process by extracting CN-Es-6 [Rf = 0.30]. .

Example 3

3.82 g of extract CN-Es-6 obtained in Example 2 was added to a mixed solution of chloroform / methanol (2: 1, v / v) and centrifuged to melt 3.03 g (extract CN-Es-6s) and the insoluble part. 0.74 g (extract CN-Es-6i) were obtained.

To 276 g of powdered silica gel, 1000 ml of a chloroform / methanol (2: 1, v / v) mixed solution was added and swollen, followed by filling in an open column (bed volume = 600 ml). 3.03 g of the extract CN-Es-6s obtained above was dissolved in a minimum amount (the minimum amount capable of melting the extract) in a chloroform / methanol (2: 1, v / v) mixed solution, and then applied to a column filled with silica gel. The eluent was poured into the column in 1850 mL of chloroform / methanol (2: 1, v / v), 500 mL of (1: 1, v / v), 100 mL of (1: 1.5, v / v), and chloroform / methanol / 2.5 M ammonia water. (20: 10: 2, v / v / v) Changed to 250 ml of mixed solution, 50 ml of each drop was taken every three seconds (the volume is 200 ml), and then TLC (chloroform / Methanol = 2: 1, v / v) and developed with iodine and separated according to Rf. In this way, all seven major fractions were separated. The separated fractions were distilled under reduced pressure and dried to obtain extract CN-Es-6s-A to CN-Es-6s-G. These seven extracts were separated from the extract CN-Es-6s-F [Rf = 0.30], which was determined to be effective as a result of searching for mycelial formation inhibitory activity as in Example 5 to be described later. Used.

Example 4

200 g of a chloroform / methanol (50: 1, v / v) mixed solution was added to 50 g of powdered silica gel, followed by swelling, followed by filling in an open column (bed volume = 106 ml). After dissolving 334 mg of extract CN-Es-6s-F obtained in Example 3 in a minimum amount (the minimum amount capable of dissolving the extract) in a chloroform / methanol (50: 1, v / v) mixed solution, silica gel was charged. Applied to the column. Eluent was added to the column as in Example 3 with 200 ml of chloroform / methanol (50: 1, v / v), (2: 1, v / v) 200 ml, (1: 1, v / v) 40 ml, chloroform /Methanol/2.5M ammonia water (20: 10: 2, v / v / v) mixed with 200ml of solution, dropping 15ml of each drop every 5 seconds (the volume (void volume) 35 ml), which was developed by TLC (chloroform / methanol = 50: 1, v / v), developed with iodine, and separated according to the Rf value. In this way, all four major fractions were separated. The separated fractions were distilled under reduced pressure and dried to obtain extract CN-Es-6s-F-a to CN-Es-6s-F-d. 220 mg of the extract CN-Es-6s-F-d [Rf = 0.30], which was determined to be effective as a result of searching for mycelial formation inhibitory activity as in Example 5 described below, was isolated from these four extracts.

TLC results for each of the active fractions obtained in Examples 1 to 4 are shown in FIG. 1. As shown in Figure 1 it can be seen that the band (line b) appearing in the extract CN-Es appear the same in the process of purification. Therefore, it can be seen that the part showing activity is not denatured or lost in the process of purification. Initially, the band, which was obscured by other parts, developed deeper as it was refined.

Example 5

Screening of Efficacy of Deer Antler Fraction on Growth of Bacteria

The following experiment was performed to search for the effect of the antler extract obtained in Examples 1 to 4 and its purified extract on the growth of bacteria.

5-1. Effect of Candida Albicans on Yeast Growth

Candida albicans yeast growth medium Saburaud-dextrose medium (1% peptone, 2% dextrose) was inoculated with the strain, and then as a comparative material amphotericin B, ketoconazole, fluconazole and clotri Mazol, the extract CN-Es-6s-Fd purified as a test substance, were each added at a concentration of 120 μM. At this time, no antifungal agent was added to the control group. The growth of the bacteria was examined by measuring the absorbance at 660 nm at 2 hour intervals while growing the strain at 30 ° C. As a result, as shown in FIG. 3, the antler fraction inhibited the growth of the non-pathogenic form of the yeast by about 25%. Therefore, it was confirmed that the antler fraction according to the present invention exhibited a similar degree of inhibition to fluconazole, which is inferior to that of amphotericin B or ketoconazole, which strongly inhibits the growth of bacteria to yeast.

5-2. Effect of Candida Albicans on Mycelial Growth

After inoculating Candida albicans strain into RPMI-1640 (GIBCO BRL) medium, which is a mycelium forming medium, the antler fraction of each purification step according to Examples 1 to 4 was added thereto, followed by incubation for 7 hours at 37 ° C. of physiological conditions to form mycelia. Induced. The antler fraction was not added as a control. After incubation, the number of mycelial cells per total cell number was measured and the mycelial formation of the control group to which nothing was added was converted to 100, indicating the effect of each antler fraction in%. The results are as shown in Table 1 and FIG. 2.

When extract CN-Es was added to the medium at a concentration of 250 µg / ml, the mycelial formation rate was about 50% of the total cells. In addition, the activity of six fractions from CN-Es-4 to 9 obtained by purifying the CN-Es fraction according to Example 2 showed that the CN-Es-6 fraction showed the highest activity. In addition, the CN-Es-6s-F-d fraction was finally extracted from the CN-Es-6s-F fraction having the highest activity among the seven fractions (A-G) extracted from the CN-Es-6s fraction. As can be seen from the results of Table 1, the finally obtained CN-Es-6s-F-d fraction almost completely inhibits mycelial formation of Candida albicans strains.

Mycelial formation rate of each antler fraction Deer Antler fraction Mycelial formation rate (%) Control 100 CN-Es 51.28 CN-Es-6 17.65 CN-Es-6s-F 4.67 CN-Es-6s-F-d 0.43

5-3. Inhibitory Activity of Deer Antler Fraction and Antifungal Agents on Pathogenic Mycelia Formation

According to the same method as in 5-2, the mycelial formation inhibitory activity of the antler fraction according to the present invention was compared with the existing antifungal agents amphotericin B, ketoconazole, fluconazole and clotrimazole, and also the active ingredient in the antler fraction It was compared with sphingomyelin and phosphatidyl choline having a structure similar to that of the compound of formula (I). The results are shown in Table 2 below (However, the numerical values in Table 2 are 100% of the mycelial formation of the control group without the antifungal agent and are converted to the ratio thereof). As can be seen from the results of Table 2, the existing antifungal agent inhibited almost 100% of mycelial formation at a concentration of 60 μM or more, and phospholipid-based sphingomyelin and phosphatidyl choline, which are analogs of the antler fraction, were different from the antler fraction. Otherwise, it showed no inhibitory activity on conversion and formation to mycelium. On the other hand, the antler fraction CN-Es-6s-F-d purified according to the present invention showed almost 100% inhibition at a concentration of 400 μM. In other words, the antler fraction required a higher concentration than the existing antifungal agent in order to 100% inhibit the mycelial formation, but instead it is considered that it can be used more effectively as an antifungal agent because there are no various side effects that are problems of the existing antifungal agent.

Inhibition rate of mycelium formation of each drug according to the concentration change (%) Concentration (μM) 0 30 60 90 120 150 400 Amphotericin B 100 0 0 0 0 - - Clotrimazole 100 4.7 0 0 0 - - Fluconazole 100 2.3 0 0 0 - - Ketoconazole 100 0.65 2.75 3.75 0.42 - - CN-Es-6s-F-d 100 32.92 10.67 7.41 5.41 1.33 0.43 Sphingomyelin 100 100 100 100 100 100 100 Phosphatidyl choline 100 100 100 100 100 100 100

Example 6

Structural Analysis of Mycelial Formation Inhibitor in Deer Antler Fraction

Nuclear magnetic resonance spectra, mass spectrometry and infrared spectroscopy were used to analyze the structure of the substance showing the actual activity in the extract CN-Es-6s-F-d finally isolated from the antler. In addition, it was confirmed that the antler fraction was composed of the following four compounds by forming a derivative of CN-Es-6s-F-d through an acetylation reaction and a methylation reaction.

1) Compound CHJ-1 (C ₂₆ H ₅₅ N ₂ O ₆ P; R = octadecanoyl)

2) Compound CHJ-2 (C ₂₄ H ₅₁ N ₂ O ₆ P; R = hexadecanoyl)

3) Compound CHJ-3 (C ₂₆ H ₅₃ N ₂ O ₆ P; R = cis-9-octadecenoyl)

4) Compound CHJ-4 (C ₂₆ H ₅₁ N ₂ O ₆ P; R = cis-9,12-octadecadienoyl)

These four newly discovered compounds are phospholipid based neofunctional new substances that have not been reported in the literature to date.

6-1. Nuclear magnetic resonance spectrum

The CN-Es-6s-Fd fraction was investigated in two-dimensional ¹ H- ¹ H, ¹³ C- ¹³ C COZY spectra including ¹ H, ¹³ C spectra using a Bruker DPX 250 FT NMR. At this time, the solvent is CDCl ₃ was used, and made the CHCl ₃ in CDCl ₃ as internal standard included. Analyzes were made by comparing the respective spectra of sphingomyelin, ceramide, 1-oleyl-2-palmitoyl-3-phosphatidylcholine, presumed to have a structure similar to the active ingredient. ^{The 1} H, ¹³ C spectral data is shown in Tables 3 and 4, where the H-4, H-5, and H-6 peaks correspond to phosphocoline and these peaks are 1-oleyl-2- The same is seen with palmitoyl-3-phosphatidylcholine. ^The H-2 peak in the ¹ H spectrum is similar to sphingomyelin and ceramide, and thus it can be seen that the active ingredient according to the present invention has an amide structure. Meanwhile, -CH ₂ -peaks characteristic of fatty acids can be seen, and in addition to saturated fatty acids stearic acid and palmitic acid, oleic acid is unsaturated fatty acid due to H-9 and H-10 peaks. (oleic acid) and linoleic acid form part of the structure. The characteristic peak of linoleic acid appears at 2.74 ppm. ¹ H- ¹ H, ¹³ C- ¹³ C COZY spectra are shown in FIGS. 4 and 5.

¹ H NMR (250MHz) Spectrum Analysis H # δ One 3.81-3.87, 3.76-3.81 m, 2H 2 3.88-3.96 m, 1H 3 4.01-4.05 m, 2H 4 3.69-3.75 m, 2H 5 4.25-4.33 m, 2H 6 3.29 s, 9H -CH _2- 1.22-1.26 m, 20H 7 2.24-2.30 t, 2H 8 1.50-1.58 m, 2H 9 1.97-2.03 m, 2H 10 5.29-5.33 m, 2H 11 0.82-0.87 m, 3H NH, OH 5.02-5.27 m, 2H

¹³ C NMR (250MHz) Spectrum Analysis C # δ One 67.07 2 68.66 3 65.23 4 66.19 5 59.42 6 54.26 7 34.14 8 24.92 9 27.20 10 129.97, 129.66 11 14.09 CO 173.85 -CH _2- 31.90, 29.72, 29.31, 22.66

6-2. Mass spectrometry spectra

In mass spectrometry, the ionization method was FAB (fast atom bombardment) and the matrix was NBA (Na). 6 shows each peak corresponding to [M + H + Na] ⁺ as FAB-MS spectrum. In addition, the results of the mass spectrometry for the active compounds CHJ-1, 2, 3 and 4 present in the antler extract are summarized in Table 5 below.

compound Formula (M) [M + H + Na] ⁺ [M + H + Na-59 (NMe ₃ )] ⁺ CHJ-1 C ₂₆ H ₅₅ N ₂ O ₆ P (522.38) 546.07 487.03 CHJ-2 C ₂₄ H ₅₁ N ₂ O ₆ P (494.35) 518.04 458.90 CHJ-3 C ₂₆ H ₅₃ N ₂ O ₆ P (520.36) 544.06 484.99 CHJ-4 C ₂₆ H ₅₁ N ₂ O ₆ P (518.35) 542.05

FIG. 7 is a tandem mass spectrum (FAB-MS / MS) for m / z 546.07 corresponding to compound CHJ-1, m / z 487.6 is [M + H + Na-NMe ₃ ] ⁺ , m / z 334.3 is [M + Na-C ₁₅ H ₃₁ ] ⁺ , m / z 278.2 is [M + H + Na-C ₁₇ H ₃₅ CO] ⁺ , and m / z 275.2 is [M + Na-C ₁₅ H ₃₁ Peaks by -NMe ₃ ] ⁺ . In addition, stearic acid structure by m / z 334.3, 348.4, 362.4, 376.4, 390.4, 404.4, 418.4, 432.5, 446.5, 460.5, 474.5, 487.6, 502.6, 516.6, 530.6 You can see that it forms part of. Also shown is m / z 206.1 corresponding to the phosphocholine monomer.

6-3. Infrared spectrum

Infrared spectra were measured using Bio-Rad FTS 165 () compound coated in KBr cells in the form of neat (neat), the measurement results are shown in FIG. 8. In the spectrum, bands appearing over a wide range near the frequency of 3363 cm ^-1 are hydroxy groups in the molecular structure of the compound, bands at 1728 cm ^-1 are carbonyl groups at amide, bands at 3020-2853 cm ^-1 are saturated, and unsaturated carbon bonds are present. Confirm.

6-4. Structural analysis by derivative synthesis

After methylation of the extract CN-Es-6s-Fd using methyliodine, the formed compound was irradiated with nuclear magnetic resonance spectra. As a result, the characteristic peak ( ¹ H NMR: 3.39 ppm, ¹³ C NMR: 58.50 ppm) corresponding to the methyl group was confirmed. Similarly, in the case of acetylation reaction using acetic anhydride, characteristic peaks corresponding to the acetyl group ( ¹ H NMR: 2.03 ppm, ¹³ C NMR: 21.52 ppm) were observed. In addition, the extract CN-Es-6s-Fd also reacted with the permanganic acid coloring reagent used to identify the hydroxyl group, and thus the content of the hydroxyl group was confirmed.

As can be seen from the experimental results of the above examples, the ethanol extract of antler according to the present invention, the stepwise separated and purified fractions therefrom, and the compound of formula 1 are active to inhibit the formation of pathogenic fungi such as Candida albicans into mycelium. It is expected to be able to be used effectively as an antifungal agent because it is a substance extracted from herbal ingredients and thus the side effects are significantly reduced compared to conventional antifungal agents.

Claims (9)

As an active ingredient, antler was extracted with hexane, the extraction residue was again extracted with chloroform, and the extraction residue was further extracted by ethanol extract of antler (extract CN-E) obtained therefrom or further separated by silica gel column chromatography, An antifungal composition containing the purified fractions. The composition according to claim 1, wherein the extract contains soluble fraction CN-Es separated by dissolving extract CN-E in a mixed solution of chloroform / methanol / 2.5M ammonia (20: 10: 2, v / v / v) as an active ingredient. . The method of claim 2, wherein the extract is obtained by performing silica gel column chromatography using extract CN-Es as an eluent as a mixture of chloroform / methanol / 2.5M aqueous ammonia (20: 10: 2, v / v / v) solution. A composition containing fraction CN-Es-6. 4. The composition according to claim 3, wherein the active ingredient contains soluble fraction CN-Es-6s separated by dissolving extract CN-Es-6 in a chloroform / methanol (2: 1, v / v) mixed solution. The method according to claim 4, wherein the extract CN-Es-6s as an active ingredient is selected from chloroform / methanol (2: 1, 1: 1, 1: 1.5, v / v), chloroform / methanol / 2.5M ammonia water (20: 10: 2 , a composition containing the fraction CN-Es-6s-F obtained by performing silica gel column chromatography while switching to a mixed solution of v / v / v). The method according to claim 5, wherein the extract CN-Es-6s-F as an active ingredient is chloroform / methanol (50: 1, 2: 1, 1: 1, v / v), chloroform / methanol / 2.5M ammonia water (20:10 A composition containing the fraction CN-Es-6s-Fd obtained by performing silica gel column chromatography while switching to a mixed solution of: 2, v / v / v). The antler was extracted with hexane, the extraction residue was extracted with chloroform, and the extraction residue was extracted with ethanol to obtain an ethanol extract of antler, or the obtained ethanol extract of antler was further separated and purified by silica gel column chromatography. Characterized in that the ethanol extract of the deer antler according to any one of claims 1 to 6 and a method for producing a separated and purified fraction therefrom. A compound of formula [Formula 1] In the above formula R represents octadecanoyl (stearyl), hexadecanoyl (palmitoyl), cis-9-octadecenoyl (oleoyl) or cis-9,12-octadecadenoyl (linoleoyl). An antifungal composition containing one or more compounds of formula 1 according to claim 8 as an active ingredient.