KR101772680B1 - An antifungal composition containing Magnoloside A and its pharmaceutically acceptable salts thereof - Google Patents

Abstract

The present invention relates to an antifungal composition comprising magnoloside A or a pharmaceutically acceptable salt thereof as an active ingredient. More particularly, the present invention relates to a composition for antifungal agent comprising Magnolobide ( Magnolia obovata Thunb.), And that the extracts, fractions and fractions of the Magnoliaceae extract of Huang Magnolia have an antifungal effect against various strains of the present invention. Thus, it is possible to confirm that the magnoxides of the present invention, Pharmaceutically acceptable salts can be usefully used as compositions for antifungal agents.

Description

An antifungal composition containing Magnoloside A and its pharmaceutically acceptable salts as an active ingredient, which comprises Magnoloside A or a pharmaceutically acceptable salt thereof,

The present invention relates to a composition for an antifungal agent containing magnoloside A or a pharmaceutically acceptable salt thereof as an active ingredient.

The fungal diseases of the human body are mainly caused by superficial diseases such as dermatophytoses and onychomycoses, candidiasis, And aspergillosis. The most serious diseases are systemic fungal infections, which have rapidly increased in recent 20 years, with many opportunistic infections, despite many advances in medical technology (Georgopapadakou, NH et al., Science, 264: 371-373, 1994). The main reason for this increase was a significant increase in immunosuppression due to prolonged use of AIDS or anticancer drugs or in patients receiving immunosuppressive treatment after organ transplant. As other factors, fungal diseases are increasingly being caused by broad-spectrum antibacterial agents, glucocorticosteroid therapy, surgery, catheter, prosthetic and parenteral nutrition, and dialysis (Greene, SI Treatment of fungal infections in the human immunodeficiency virus-infected individual, p. 237-246. In Jacobs, PH, Nall, L. (eds.) Antifungal Drug Therapy, Marcel Dekker, Beck-Sague, CM et al., J. Infect. Dis., 167: 1247-1251, 1993). Representative pathogens of such opportunistic infection is Candida albicans (Candida albicans) and other Candida strains (C. glabrata, C. tropicalis, C. krusei) the Ruth Ars Peru quality incidence of Aspergillus (A. niger, A. flavus), or Histoplasma capsulatum ) and Cryptococcus Neoformans Increased Like (Cryptococcus neoformans) and have (Sternberg, S., Science, 266 : 1632-1634, 1994), recently Fusarium (Fusarium), isophorone tree Coles framework (Nguyen, MH et al., Am. J. Med., 100: 617-623, 1996). In the case of Trichosporon , Rhizopus and Mucor , . Fungal infections have become one of the major causes of death in hospitalized patients, and candidiasis has been detected in blood cultures to the fourth degree in American hospitals (Pfaller, MA et al., Diagn. Microbiol. Infect (Wenzel, RP et al., Clin. Infect. Dis., 20: 1531-1534, 1995), and that the mortality rate of patients with candidemia is 35% . Also aspergillus increase (aspergillosis) that occurs in the lungs has been a major cause of bone marrow transplant patients who died (Panutti, CS et al, Cancer , 69:. 2653-2662, 1992), lung carinii (Pneumocystis Carinii- induced pneumonia is the leading cause of death in AIDS patients in North America and Europe (Hughes, WT, Pediatr. Infect. Dis. J., 10: 391-399, 1991). Since most of the fungal diseases caused by opportunistic infections are difficult to diagnose by conventional blood cultures, they are treated only by experience in the treatment of severe immunodeficiency syndromes (Walsh, TJ, et al., Rev Infec, Dis., 13: 496-503,1991). Most fungal diseases have been reported to be predominant in patients with impaired immune function. However, in 1994, an earthquake near Los Angeles caused spores in the soil to spread into the air, causing hundreds of fungal diseases to last more than 10 times Mortality was increased (Sternberg, S., Science, 266: 1632-1634, 1994). In the late 1990s, Cryptococcus It has proven meningitis caused by neoformans) immune function, infection by more fungi became known to the cases of death by disease in the population from a normal healthy person does not develop only in people with immune dysfunction (Hoang, LMN et al. , J. Med. Microbiol., 53: 935-940, 2004). In addition, it is expected that fungal infections due to the deterioration of immunity function will increase more and more as the age of the aged population increases due to the increase in the elderly population. In addition, infectious diseases which appeared only in some strata tend to spread to the general people, Is expected to increase even more. As such, fungal diseases are becoming more and more widespread and become a major concern for many researchers, but there are many difficulties in controlling them. Amphotericin B (Amphotericin B), which was developed in 1960, has been used for systemic diseases in clinical studies even though there is a lot of toxicity due to the lack of antifungal drugs with low toxicity and excellent drug efficacy (Georgopapadakou, NH et al., Science, 264: 371-373,1994). Liposomes (Alder-Moore, JP et al., J. Liposomal Res., 3: 429-450, 1993; Ringden, O. et al., J. Antimicrob. F., Rev. Infect. Dis. (Suppl. 7): 1605-1609, 1989) or colloidal dispersion (see, for example, Development of new delivery systems such as Hanson, LH et al., Antimicrob. Agents Chemother., 36: 486-488, 1992; Patterson, TF et al., J. Infect. Dis., 159: 717-724, (McPherson, DT et al., Adventures in polymer macrolide chemistry: the derivatization of amphotericin B, pp. 205-222.) In PH Bentley and R. Ponsford (ed. , Recent advances in the chemistry of anti-infective agents, Royal Society of Chemistry, London, 1993). In addition, azole compounds, currently widely used in systemic fungal infections, act as fungistatic agents, but the frequency of resistance strains is increasing (Rex, JH et al. Antimicrob. Agents Chemother., 39: 8, 1995). The incidence of pathogenic non-pathogenic strains in patients with impaired immune function is increasing, and the need for new drugs with lower toxicity and better efficacy is increasing, but pathogenic fungi are difficult to cultivate, (Sternberg, S., Science, 266: 1632-1634, 1994). However, there is a lack of research on the microbiological and genetic studies on fungi that cause disease.

Cryptococcus Neoformans Cryptococcosis by neoformans is one of the most common fungal diseases in AIDS patients, and it is known that when the bacteria infect the central nervous system, life-threatening meningitis develops and leads to death. This strain develops when the inhaled basidiospore is inhaled mainly from the environment of soil or eucalyptus trees contaminated with pigeon excrement, and has four serotypes: A (var. Grubii ), B And C (var. Gatti ) and D (var. Neoformans ) forms. Of these, 99% of the world's hospitals, 99% of the AIDS patients, and more virulent than the anti-circular D, and the anti-circular B and C in the Vancouver region of Canada in the late 1990s (Hoang, L. et al., J. Med. Microbiol., 53: 935-940, 2004) have been reported to be causative agents of Cryptococcosis which occurs in normal persons. This strain is also a haploid emergence yeast with two mating types (a and a) (Hull, CM et al., Annu. Rev. Genet., 36: 557-615, 2002) It has been reported that most isolates isolated from the surrounding environment and hospitals are α-like, and that virulence is stronger than a-type (Kwon-Chung, KJ, et al., Infect. Immun 60: 602-605). The molecular level of this pathogenicity has been actively studied in advanced countries, mainly in signaling systems (Kraus, PR et al., Mol. Microbiol., 48: 1377-1387, 2003; Kojima, K. et al 17: 3122-3155, 2006; Roman, E., et al., Trends Microbiol., 15: 181 -190, 2007). Cryptococcus Neoformans The MAP kinase pathway is the main mechanism involved in regulating crucial processes in fungal pathogenesis such as capsule biosynthesis, morphogenesis and adaptation to oxidative stress (Idnurm , A. et al., Nat. Rev. Microbiol., 3: 753-764, 2005), mutants lacking MAP kinase (MAPK) have been reported to reduce pathogenicity in animal models. Adaptation to stress (osmotic, oxidative, acid and pH, etc.) occurs via the high osmolarity glycerol (HOG) pathway, which is known to be involved in the regulation of capsules associated with virulence of Cryptococcus neoformans (Roman, E. et al., Trends Microbiol., 15: 181-190, 2007). When HOG1 gene is deleted in the hog1 or indicate the proper sensitivity in osmotic medium (MAPK) mutant state or pbs2 (MAPKK) mutants despite that the capsule formation with melanin (melanine) generated increases and, as hog1 pbs2 Mutant strains have reduced pathogenicity in animal models, including cryptococcus (Bahn, YS et al., Mol. Biol. Cell, 16: 2285-2300, 2005), suggesting that the virulence of Neoformans is expressed by various factors.

On the other hand, a report on a compound acting on the signal transduction pathway has been reported in the late 2000's that the target of ambruticin, a polyketide antifungal compound, is the HOG pathway (Vetcher, L. et al., Antimicrob . Agents Chemother, 51:. the 3734-3736, 2007), calcineurin (calcineurin) or inhibitory mutations occurs aspergillus Puig tooth (Aspergillus Antimicrob. Agents Chemother., 51: 2979-2981, 2007) reported that the antifungal activity of caspofungin or nikkomycin Z, a cell wall inhibitor against fumigatus , is increased (Steinbach, WJ et al. ). In addition, tacrolimus (FK506) and cyclosporin A inhibit calcineurin and rapamycin inhibits interleukin-2 (T-cell) in the current clinical use of the immunosuppressants tacrolimus (FK506) and cyclosporin A T-cell, and geldanamycin has been reported to be an inhibitor of heat shock protein 90 (HSP90), all of which act on the signaling pathway (Cardenas, ME et al., Trends Biotechnol., 16: 427-433, 1998). In addition, the MAPK cascade plays an important role in the pathogenicity of plant pathogenic fungi, and the compound fludioxonil, which activates MAPK cascade, has been reported to be available as a fungicide (Kojima, K et al., Mol. Microbiol., 53: 1785-1796, 2004). In fungi, the signal transduction pathway is closely related to pathogenicity, and this signaling pathway is also found in mammalian cells and plant and prokaryotes, but the stress activity of fungi and mammalian cells, p38 / HOG1- MAPK system (stress-activated p38 / HOG1-MAPK system), there are many differences in the upstream signaling system. Because fungal, plant, and primitive nuclear cells use a two-component system that is not present in mammalian cells, this signaling system, a two-component regulatory system, is a major target for the development of new antifungal and antibiotic agents Bull. Cell, 17: 3122-3155, 2006). It is expected that research on virulence factors as well as signal transduction pathways will be activated in the future (Bahn, YS et al.

In this way, over the past 20 years, new targets have been discovered and new methods of exploration have been steadily developed, and many compounds have been reported but still have low toxicity, selective pharmacological effects, long lasting pharmacokinetic properties, The development of effective drugs for resistant bacteria is very insufficient. The development of the chemotherapeutic agent is very difficult because the host mammalian cells and fungi are all eukaryotic cells, so that the development of selective drugs is very difficult due to the similarity of many functions. However, due to the development of molecular biology, a new function using a mutant in which a specific function has been selectively deleted has been revealed, so that a mechanism-based screening for each target becomes possible, And the development of inhibitors that inhibit toxic factors is increasing, so there is a constant need for the development of new signaling regulators.

On the other hand, the plant Magnolia obovata Thunb. Is known as Hwanghak Magnolia or Liliaceae. It is widely distributed in Korea, China and Japan as the origin of Japan. It has been used as a grape herb in Japan, and leaves have been used for edible purposes in Korea and Japan. Various neolignan compounds such as obovatol, honokiol and magnolol have been reported from the plant, which have antioxidant activity (Lo, YC et al. Biochem. Pharmacol., 47: 549-553, 1994), 5-lipoxygenase inhibitory activity (Fukuyama, Y. et al. Tetrahedron Lett., 34: 1051-1054, 1993), ACAT inhibitory activity (Kwon, BM et al. -551, 1997), antifungal and antibacterial activity (Nitao, JK et al. Phytochemistry, 30: 2193-2195, 1991). In particular, Magnonol and Honokiol have been reported to exhibit excellent activity against pathogenic pathogens in periodontal tissues as well as low toxicity to gingival tissues and can be used as oral antiseptics (Chang, BS (Lin et al., Eur. J. Pharmacol., 537: 64-69, 2006). Recently, obovatol was reported to exhibit antiplatelet activity (Park et al., J. Atheroscler. Thromb., 18: 659-669, 2011), inhibit nitric oxide production (Seo, KH (Lee et al., J. Pharmacol. Sci., 111: 124-136, 2009), in an Alzheimer animal model (Choi, DY et al., J. Neurochem., 120: 1048-1059, 2012). In addition, many other Magnolia species have been reported to exhibit antispasmodic, antifungal and antibacterial activity and insecticidal activity against gastrointestinal seizures. Magnolytic diseases include alkaloids, coumarins, It has been reported to have at least 255 different components such as flavonoids, lignogens, neolignan, phenylpropanoids and terpenoids (Lee, YJ et al. Pharmacol. Ther., 130: 157-176, 2011). However, as described above, many compounds isolated from Magnolio have been reported to exhibit various biological and pharmacological activities, but the magnoloside A compound has been reported to be effective against Cryptococcus neoformans neoformans ) in the signal transduction process has not been revealed yet.

Accordingly, the present inventors have made efforts to develop a composition for an antifungal agent that acts on the signal transmission pathway of fungi, and found that the plant Magnolia obovata Thunb.) Acts as a calcineurin inhibitor of various fungi to exhibit an antifungal effect and can be effectively used as a composition for an antifungal agent. Thus, the present invention has been completed.

It is an object of the present invention to provide a composition for an antifungal agent containing magnoloside A or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to accomplish the above object, the present invention provides a composition for an antifungal agent comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient;

[Chemical Formula 1]

.

Also provided is a cosmetic composition for an antifungal agent comprising the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Also provided is an antifungal antiseptic containing a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides an antifungal pesticide containing the compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention separates the magnoloside A compound, which is an antifungal substance, from the ethyl acetate fraction of Magnolia obovata Thunb. The extract of Huang Magnolia, its fractions, and the magnoxidase A By confirming the antifungal effect of the compound, the magnoxoid compound of the present invention can be usefully used as an antifungal composition.

FIG. 1 is a crypto caucus's only neo-Fort (Cryptococus neoformans ) wild type strain and a calcineurin signal transduction mutant.
Fig. 2 shows the activity test of Magnolia obovata Thunb. Ethyl acetate fraction using plate B; Fig.
FIG. 3 shows the activity test of silica gel column chromatography fraction 13 after fractionation with ethyl acetate of sulfuric acid using fraction B plate.
FIG. 4 shows the activity test using the B plate of the Sulfur Magnolia fractions 13. FIG.
FIG. 5 is a schematic view showing a method of separating and purifying a signal regulatory substance from yellow magnolia. FIG.
Figure 6 is an HPLC chromatogram of a Magnoloside A compound isolated from sulfur magnolia.
7 is a view showing an activity test using a B plate of a magnosolide A using a B plate.
8 is an ESI-MS spectrum of a magnosolide compound separated from yellow magnolia.
9 is a graph showing a ¹ H-NMR spectrum of a magnoxoide compound separated from a yellow magnolia.
10 is a graph showing a ¹³ C-NMR spectrum of a magnoxoide compound separated from a yellow magnolia.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for an antifungal agent comprising a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient;

The compound is characterized in that it is separated from the Magnolia obovata Thunb. Extract, but includes all of those synthesized or commercially available.

The sulfur magnolia extract may be extracted with a solvent that is water, a C ₁ to C ₂ lower alcohol, or a mixture thereof, and the lower alcohol may be methanol or ethanol, but is not limited thereto.

The sulfur magnolia extract may be, but is not limited to, those produced by a process comprising the following steps:

1) adding an extracting solvent to the yellow magnolia;

2) cooling the extract of step 1) and filtering; And

3) Concentrating the filtered extract of step 2) under reduced pressure and drying.

In the above method, the yellow magnolia of step 1) may be used without limitation such as cultivated or commercially available. The Huang Magnolia may be, but not limited to, using the roots, leaves, stalks and fruit of Huang Magnolia.

The extract of Huang Magnolia can be extracted by a conventional method such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. The extract may be extracted one to five times by hot water extraction, More specifically, it may be repeated three times, but is not limited thereto. The extraction solvent may be added in an amount of 0.1 to 10 times, preferably 0.3 to 5 times, to the dried yellow magnolia. The extraction temperature may be 20 to 40, but is not limited thereto. In addition, the extraction time may be 24 to 96 hours, but is not limited thereto.

In this method, the vacuum concentration of step 3) may be by using a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. The drying may be, but not limited to, vacuum drying, vacuum drying, boiling drying, spray drying or lyophilization

The sulfur extract may be further fractionated by adding an organic solvent. The organic solvent may be one or more selected from the group consisting of ethyl acetate and butanol. According to a preferred embodiment of the present invention, Is most preferable.

The fractions obtained by fractionating the yellow magnolia extract are preferably subjected to column chromatography using silica gel or C ₁₈ using a mixed solvent of an organic solvent or water. However, the solvent used, the mixed solvent thereof, the volume of the solvent, etc., It can be changed easily.

The compound is characterized by inhibiting calcineurin.

The composition Cryptococcus neo Lactococcus's satiety (Cryptococcus neoformans ATCC 36556), Cryptococcus neoformans H99 ( Cryptococcus There is neoformans . grubii H99, serotype A), Cryptococcus Lactococcus Gatti R265 (Cryptococcus gatti R265, serotype B), Cryptococcus grit WM 276 ( Cryptococcus gatti WM276, serotype B), Cryptococcus neo Lactococcus satiety's JEC21 (Cryptococcus neoformans JEC21, serotype D). < / RTI >

The pharmaceutical compositions for antifungal use can be used for the prevention or treatment of all diseases caused by fungal infections. The disease may be selected from the group consisting of Cryptococcosis, Cryptococcus meningitis, skin cryptococcosis, European blastomycosis, superficial candidiasis, candidiasis nodal and nail inflammation, chronic skin, mucocutaneous candidiasis, But are not limited to, facial chronic granuloma, head chronic granuloma, oral chronic granuloma, candida endocarditis, Candida meningitis.

In a specific example of the present invention, the present inventors have isolated a magnocytosine-A compound from a sulfur magnolia extract (see FIGS. 6 to 9) and found that the magnocide-A compound inhibits the calcineurin signaling pathway of the Cryptococcus strain (See FIG. 7) and confirming that the compound exhibits an antimicrobial activity against human pathogenic fungus (see Table 8), the magnesium compound of the present invention can be effectively used as an antifungal composition.

The present invention includes not only the compounds represented by the above formula (1), but also pharmaceutically acceptable salts thereof, possible solvates, hydrates, racemates or stereoisomers thereof.

The compound represented by the formula (1) of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound represented by the formula (1) in an excess amount of an acid aqueous solution, and then dissolving the salt in a water-miscible organic solvent such as methanol, ethanol, Followed by precipitation using nitrile. It is also possible to prepare the mixture by evaporating a solvent or an excess acid in the mixture, or by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

The composition of the present invention contains, as an active ingredient, 0.1 to 99.9% by weight of the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, based on the total weight of the composition, and includes a pharmaceutically acceptable carrier, excipient or diluent can do.

The compositions of the present invention may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of suppositories include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may be administered orally or parenterally, and it is preferable to select the intraperitoneal, rectal, rectal, intravenous, intramuscular, subcutaneous, intrauterine or intracerebral injection methods for parenteral administration, It is used for external skin.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, Preferably from 30 to 500 mg / kg, more preferably from 50 to 300 mg / kg, based on the amount of the compound or the pharmaceutically acceptable salt thereof, and can be administered 1 to 6 times a day have.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The present invention also provides a cosmetic composition for an antifungal agent comprising, as an active ingredient, a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof;

[Chemical Formula 1]

.

In a specific example of the present invention, the present inventors have isolated a magnocytosine-A compound from a sulfur magnolia extract (see FIGS. 6 to 9) and found that the magnocide-A compound inhibits the calcineurin signaling pathway of the Cryptococcus strain (See FIG. 7) and confirming that they exhibit antimicrobial activity against human pathogenic fungus (see Table 8), the magnosolide compound of the present invention can be effectively used as a cosmetic composition for antifungal use.

When the compound represented by Formula 1 or the pharmaceutically acceptable salt thereof of the present invention is used as a cosmetic composition, for example, a solution, a gel, a solid or a paste anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, It may be provided in the form of microemulsions, microcapsules, microgranules or ionic forms (liposomes), non-ionic follicular dispersing agents, creams, skins, lotions, powders, ointments, sprays or conical sticks. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

The cosmetic composition may further contain, in addition to the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a surfactant, a water, an ionic or nonionic emulsifier, a filler, a sequestering and chelating agent, a preservative, a vitamin, a barrier, a wetting agent, an essential oil, a dye, a pigment, a hydrophilic or lipophilic active agent, Lipid vesicles or any other ingredient conventionally used in cosmetics, as well as adjuvants commonly used in the cosmetics field.

The present invention also provides antifungal preservatives containing the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In a specific example of the present invention, the present inventors have isolated a magnocytosine-A compound from a sulfur magnolia extract (see FIGS. 6 to 9) and found that the magnocide-A compound inhibits the calcineurin signaling pathway of the Cryptococcus strain (See FIG. 7) and confirming that the compound exhibits an antimicrobial activity against human pathogenic fungus (see Table 8), the magnesium compound of the present invention can be effectively used as an antifungal preservative.

The preservative may contain, in addition to the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, additional ingredients as long as it does not inhibit its preservative activity. The antifungal antiseptic can be widely used to achieve preservation purpose in medicines, cosmetics, foods, textiles, household goods, etc., and can be used in these products to enhance safety and stability of product quality. When the natural preservative of the present invention is contained in the above-mentioned products, the amount of the natural preservative is 0.001 to 30% by weight, preferably 0.001 to 20% by weight, more preferably 0.001 to 5% by weight Range. To prepare products comprising the antifungal preservatives, suitable formulations and additives may be prepared as is well known to those skilled in the art.

The present invention also provides an antifungal pesticide containing the compound represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In a specific example of the present invention, the present inventors have isolated a magnocytosine-A compound from a sulfur magnolia extract (see FIGS. 6 to 9) and found that the magnocide-A compound inhibits the calcineurin signaling pathway of the Cryptococcus strain (See FIG. 7), and confirming that the compound exhibits an antimicrobial activity against human pathogenic fungus (see Table 8), the magnesium compound of the present invention can be effectively used as an antifungal pesticide.

In addition to the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, the agricultural chemical preparation for antifungal use may further comprise a carrier according to the intended use, purpose and method.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples and Experimental Examples.

< Example  1> From the plant extract library Cryptococcus Neo-formal  Activity assays and derivations of signal transduction inhibitors that inhibit signal transduction pathways

The following experiments were conducted to detect signal transduction inhibitors that interfere with the signaling of the Hog1 MAP kinase pathway or calcineurin pathway of C. neoformans Respectively.

Specifically, C. neoformans wild state, and the serotypes (serotype) yi A is H99 (MAT α) strain and the calcineurin signaling mutant master KK1 (MATα cna1 △ :: NAT) strains each of YPD medium (1% yeast extract , 2% peptone, 2% glucose) at 30 ° C overnight. These strains were purchased from Yonsei University.

YPD agar medium at a constant concentration (A _{550 nm} = 1.75) was added to each of the above-mentioned culture broths in an amount of 0.5%, and the mixture was divided into 10 ml each on a YPD agar plate (diameter 15 cm) prepared in advance. Then, a plate (A) coated with a sample (25 μl, 5 mg / ml) to be tested on a wild-type (H99) plate, a sample to be tested (25 μl, 5 mg / fludioxonil) (10 μl, 0.1 mg / ml) were used for the activity assay. In addition, a flat plate (C) coated with a combination of fludioxonil (10 μl, 0.1 mg / ml) and a sample to be tested (25 μl, 5 mg / ml) was added to a plate medium coated with calcineurin mutant KK1 ) And a plate (D) coated with only the sample to be tested (25 μl, 5 mg / ml) were used for the activity assay. All four plates were cultured overnight in an incubator to determine whether the growth inhibition was increased or decreased by visual inspection. At this time, the plate (B) with the sample and the pyridoxonil added to the wild-type plate and the plate (D) coated with the sample only on the plate coated with the calcineurin mutant were selected as the compounds showing a large growth inhibition size , A sample with reduced inhibition of growth was more selective than pludyoxonil used as a control in the plate (C) to which the sample and fludioxonil were added to the calcineurin mutant strain plate, and showed inhibitory activity on both A to D plates The samples were excluded (Fig. 1).

As a result, as shown in Table 1 and Table 2, one of the about 540 plant extract library samples was detected as an activity inhibitor that inhibits the calcineurin pathway (Table 1), and furthermore, through a secondary screening process, (Table 2).

A first excavation list of signaling regulators showing inhibitory activity on the signaling pathway of Cryptococcus neoformans strains from plant libraries Number of samples processed Number of primary screened samples through activity assays Antifungal substances (A) The calcineurin inhibitor (B) Hog1 inhibitor (C) Hog1 activator (D) 540 0 7 3 0

 * A, B, C and D represent the inhibitory activity of the activity assay.

List of regulators of signal transduction that exhibit activity on calcineurin secondly screened from plant extracts Code name Academic name Kinds Growth restriction (mm) LBP0005-D3 Magnolia obovata Thunb. Calcineurin
Inhibitor (B) 12 LBP0005-D9 Arctium lappa linne Hog1
Inhibitor (C) 11 LBP0008-C8 Ephedra sinica Stapf Calcineurin
Inhibitor (B) 11 LBP0008-D2 Myristica fragans Houtt Calcineurin
Inhibitor (B) 10 LBP0008-D3 Aralia continentalis Kitagawa Calcineurin
Inhibitor (B) 11 LBP0008-D4 Myristica fragrans Houtt Calcineurin
Inhibitor (B) 11

 * B and C represent a reputation showing inhibitory activity on the activity assay.

< Example  2> From Hwang Magnolia Crude extract  Manufacturing and Calcineurin  Separation purification of inhibitory substances

In order to isolate and purify substances having a calcineurin inhibitory effect from the effective substances having the signal transduction regulating effect selected in Example 1 above, Magnolia obovata Thunb. Magnolia ovovata) was used to separate and purify the calcineurin inhibitor.

Specifically, 11.2 kg of dry yellow magnolia seeds purchased from the Korean Oriental Industry Promotion Agency were purchased. 35 ℓ of 80% methanol was added to the powder, and the mixture was stirred for 3 hours at room temperature for 24 hours. The mixture was filtered, And concentrated to obtain 740 g of methanol crude extract. 3,000 ml of distilled water and 3,000 ml of ethyl acetate were added to the concentrate, followed by fractionation with water and concentration under reduced pressure to obtain 238 g of an ethyl acetate fraction. To the remaining 3,000 ml of the remaining ethyl acetate layer was added n-butanol 2,800 ml was added, and the mixture was fractionated with water, and then concentrated under reduced pressure to obtain 134 g of an n-butanol fraction.

Each of the fractions was subjected to an activity assay using the plate B of Example 1, and as a result, the ethyl acetate fraction showed strong inhibitory activity as shown in FIG. 2, and the ethyl acetate fraction extract was further separated and purified 2 and Table 3).

reputation
After extraction with an organic solvent, the activity assay (growth inhibition: mm) The nucleic acid layer Butanol Ethyl acetate layer The control (fludioxonil) A - - - - B - - 13 12 C 14 14 18 15 D - - - -

200 g of the ethyl acetate fraction of the yellow magnolia extract was dissolved in hexane: ethyl acetate = 5: 1, followed by column chromatography on silica gel (Merck, 12 x 15 cm) 15 fractions were obtained in the order of hexane: ethyl acetate = 5: 1 → 2: 1 → 1: 2, CHCl ₃ : methanol = 3: 1 → 1: 1 → methanol. Among them, inhibitory activity was confirmed in fraction 13 (7.6 g) (FIG. 3 and Table 4).

reputation
Fractions (growth inhibition: mm) 1 to 11 12 13 14 15 A - - - - - B - - 13 - - C - - - - - D - - - - -

The fractions 13 were further purified by recrystallization from chloroform: methanol: distilled water (H ₂ O) = 36: 3: 1 → 33: 3: 1 → 30: 3: 1 → 27: 3: 1 → 24: 14 fractions were obtained by performing silica gel (Merck, 5 × 12 cm) column chromatography under the conditions of 1 → 10: 3: 1 → 6: 4: 1 (FIGS. 4, 5 and 5). The fraction 1 (3.02 g) was further purified by silica gel (Merck, 5 × 14 cm) column chromatography (chloroform: methanol: distilled water = 11: 3: 1 → 10: 3: 1 → 9: 4: 1) to obtain 12 fractions. Fraction 13 of this fraction (153.4 mg) was subjected to ODS (2.5 x 5 cm) column chromatography to obtain thirteen fractions. Among the fractions, a B plate inhibiting the calcineurin pathway (containing fluodioxonil Lt; RTI ID = 0.0 &gt; 8 &lt; / RTI &gt;

reputation
Fractions (growth inhibition: mm) 13-1 13-2 13-3 ~ 13-13 13-14 Control group
(fludioxonyl) A - - - - - B 12 - - - - C 15 16 14 16 15 D - - - - -

The final detached active inhibitor was subjected to high-speed liquid chromatography (HPLC) using a C ₁₈ column (4.6 ID x 250 mm, 5 탆, YMC-Pack Tri Art C ₁₈ ) at a flow rate of 1 mg / A single peak was observed at the time of separation (H ₂ O with 0.1% acetic acid / MeOH = 0 to 100, UV 254 nm) by a high performance liquid chromatography (HPLC) The compound had a retention time (RT) of 21.5 (FIG. 6).

HPLC conditions for qualitative analysis of magnosolide compounds from yellow magnolia plants item Operating Conditions HPLC system Agilent 1260 HPLC system with ELSD detector column YMC-Pack Tri art C ₁₈
(5 [mu] m, 4.6 ID x 250 mm, YMC Inc., USA) menstruum A; H ₂ O (0.1% trifluoroacetic acid),
B; Acetonitrile (0.1% trifluoroacetic acid)
60 minutes linear gradient from A to B Detector UV ₂₅₄ nm Flow rate 1 ml / min

As a result, as shown in FIG. 7 and Table 7, the compound isolated from Sulfur Magnolia showed strong inhibitory activity on the B plate, which was stronger than the control group, fludioxonil, and it was confirmed to act as a calcineurin inhibitor. In addition, these compounds showed a clear growth inhibition effect on the B plate exhibiting the calcineurin inhibitory activity, while the other plates showed no growth inhibition on the paper discs on the other plates such as A, C and D (Fig. 7 And Table 7).

reputation Active assay (growth inhibition: mm) Compound isolated from Hwang Magnolia The control (fludioxonil) A - - B 12 10 C 16 16 E - -

< Example  3> Identification of the structure of the separated compound

¹ H NMR (500 MHz), ¹³ C NMR (125 MHz) and ESI-NMR spectroscopy (Jeol JNM-ECA 500 FT-NMR spectrometer) were used to determine the molecular weight and molecular formula of the compound isolated and purified in Example 2, Structural analysis was performed using MS.

As a result, the compound had a molecular weight of 624 and a molecular weight of C ₂₉ H ₃₆ O _15. The molecular weight of the Magnoloside A compound of phenylpropanoid glycoside series, NMR ( ¹ H NMR Methanol- d 4 , 500 MHz); δ7.59 (1H, d, J = 15.8 Hz, H-7), 7.07 (1H, d, J = 1.8 Hz, H-2), 6.97 (1H, dd, J = 1.7, 8.0 Hz, H-6 ), 6.79 (1H, d, J = 8.0 Hz, H-5 '), 6.69 (1H, d, J = 2.0 Hz, H-2'), 6.66 (1H, d, J = 8.0 Hz, H-5 ), 6.57 (1H, dd, J = 2.0, 8.0 Hz, H-6 '), 6.36 (1H, d, J = 16.1 Hz, H-8), 4.90 (H-1'', ovelapped in H 2 O peak), 4.73 (1H, d , J = 8.0 Hz, H-1 '''), 4.03 (2H, m, H-8', 5 '''), 3.87 (1H, dd, J = 11.8 Hz, H-6 &quot;), 3.79 (1H, m, H-5 &apos;), 3.69 (5H, m, H-4 &quot;, 6 &quot;, 5 & H, 3 ''), 3.39 (1H, t, J = 9.5 Hz, H-4 '''), 2.77 (2H, t, J = 7.8 Hz, , d, J = 6.3 Hz, H-6 '''): ¹³ C-NMR (Methanol- d ₄ , 125 MHz); 147.9 (C-4), 146.2 (C-4 '), 144.8 (C-3'), 131.7 (C- 1 '), 127.9 (C-1), 123.2 (C-6), 121.5 (C-6'), 117.3 (C-8), 115.2 (C-2), 100.9 (C-1 '''), 98.6 73.8 (C-4 '''), 72.3 (C-5''), 72.2 5 '''), 67.2 (C-4''), 62.8 (C-6''), 36.9 (C-7'), 18.1 (Hasegawa, T. et al., Chem. Lett., 17: 163-166, 1988). The ESI-MS spectrum and NMR spectrum of the compound are shown in Figs. 8 to 10 (Figs. 8 to 10).

< Experimental Example  1> Magnoloside  Identification of antimicrobial activity against fungi of the a-compound

In order to confirm that the mangrocosidase compound isolated in Example 3 was an antimicrobial active substance against fungi, amphotericin B, which is a comparative compound and a magnesium compound, In vitro antibacterial activity against pathogenic fungi (Minimum Inhibitory Concentration, MIC) was measured.

Specifically, the crypto-neo Lactococcus satiety switch H99 to measure MIC (Cryptococcus neoformans var. Grubii H99, serotype A), Cryptococcus Lactococcus Gatti R265 (Cryptococcus gatti R265), Cryptococcus Lactococcus Gatti WM 276 (Cryptococcus gatti WM276) and Cryptosporidium Caucus Neo's satiety JEC21 (Cryptococcus neoformans JEC21) was the strain used under pre-sale in Yonsei University, Cryptococcus neo Lactococcus's satiety (Cryptococcus neoformans ATCC 36556, serotype D), Candida albicans (Candida albicans ATCC 10231), Candida Tropical faecalis (Candida Tropicalis ATCC 13803), Aspergillus fumigatus ATCC 16424 and Trichophyton mentagrophytes ATCC 9533 were purchased from the American Type Culture Collection (ATCC) Respectively.

According to the CLSI (Clinical and Laboratory Standards Institute, M27-A3, formerly NCCLS) method, the final concentration of the test organism in a 2-fold dilution series of the compound to be tested is 5 × 10 ² to 2.5 Lt; ³ &gt; cells / ml. After the inoculation, the cryptococcus strains were cultured for 72 hours in the RPMI 1640 medium and the rest for 48 hours, and the growth was visually observed. The minimum concentration of the culture which was not allowed to grow and which was kept in a transparent state was defined as the minimum growth inhibitory concentration (MIC). Amphotericin B was used in the positive control group.

As a result, as shown in Table 8, it was confirmed that the magnosolide compound, which is a calcineurin inhibitor in the signal transduction pathway, exhibits strong antibacterial activity at a concentration of 1 to 128 μg / ml or more for 9 yeast-type fungi Table 8). The above results showed that the magnoxidase inhibited not only the Cryptococcus neoformans signaling pathway but also the growth of other Cryptococcus strains strongly, and that some Candida strains, Aspergillus fumigatus and Tricobia But showed relatively weak antibacterial activity against ton mentagrophytes.

Antifungal Activity of Magnoxidase for Human Pathogenic Fungi Experimental strain
Minimum growth inhibition concentration (占 퐂 / ml) Magno to Side A Emptorisin B Candida albicans ATCC 10231 > 128 0.12 Candida tropicalis ATCC 13803 > 128 0.25 Cryptococcus gatti R265 4 <0.12 Cryptococcus gatti WM276 2 <0.12 Cryptococcus neoformans JEC21 2 <0.12 Cryptococcus neoformans ATCC 36556 One 0.12 Cryptococcus There is neoformans . grubii H99 2 0.12 Aspergillus fumigatus ATCC 16424 > 128 0.5 Trichophyton mentagrophytes ATCC 9533 > 128 0.25

Claims (8)

A pharmaceutical composition comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient and containing Cryptococcus neoformans ATCC 36556 , Cryptococcus neoformans var. Grubii H99 , serotype A ), which is selected from Cryptococcus Lactococcus Gatti the group consisting of R265 (Cryptococcus gatti R265, serotype B), Cryptococcus Lactococcus Gatti WM 276 (Cryptococcus gatti WM276, serotype B) and the crypto-Rhodococcus neo satiety's JEC21 (Cryptococcus neoformans JEC21, serotype D) Compositions for antifungal agents showing antifungal activity against one another:
[Chemical Formula 1]

.
The composition according to claim 1, wherein the compound is isolated from an extract of Magnolia obovata Thunb.
The method of claim 2, wherein the sulfur magnolia extract is water, C ₁ to antifungal composition, characterized in that the extraction with a lower alcohol or a solvent mixture thereof in the C _2.
The composition for an antifungal agent according to claim 1, wherein the compound inhibits calcineurin.
delete A pharmaceutical composition comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient and containing Cryptococcus neoformans ATCC 36556 , Cryptococcus neoformans var. Grubii H99 , serotype A ), which is selected from Cryptococcus Lactococcus Gatti the group consisting of R265 (Cryptococcus gatti R265, serotype B), Cryptococcus Lactococcus Gatti WM 276 (Cryptococcus gatti WM276, serotype B) and the crypto-Rhodococcus neo satiety's JEC21 (Cryptococcus neoformans JEC21, serotype D) A cosmetic composition for an antifungal agent exhibiting an antifungal activity against one:
[Chemical Formula 1]

.
A pharmaceutical composition comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient and containing Cryptococcus neoformans ATCC 36556 , Cryptococcus neoformans var. Grubii H99 , serotype A ), which is selected from Cryptococcus Lactococcus Gatti the group consisting of R265 (Cryptococcus gatti R265, serotype B), Cryptococcus Lactococcus Gatti WM 276 (Cryptococcus gatti WM276, serotype B) and the crypto-Rhodococcus neo satiety's JEC21 (Cryptococcus neoformans JEC21, serotype D) Antifungal preservatives that exhibit antifungal activity against one another:
[Chemical Formula 1]

.
A pharmaceutical composition comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient and containing Cryptococcus neoformans ATCC 36556 , Cryptococcus neoformans var. Grubii H99 , serotype A ), which is selected from Cryptococcus Lactococcus Gatti the group consisting of R265 (Cryptococcus gatti R265, serotype B), Cryptococcus Lactococcus Gatti WM 276 (Cryptococcus gatti WM276, serotype B) and the crypto-Rhodococcus neo satiety's JEC21 (Cryptococcus neoformans JEC21, serotype D) Antifungal pesticides that exhibit antifungal activity against one another:
[Chemical Formula 1]

.

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