KR101952715B1 - An antifungal composition for improving candidiasis comprising plant-derived natural compounds - Google Patents

Abstract

The present invention relates to an antifungal composition for bacteria causing demomycosis or candidiasis, and more specifically, to a pharmaceutical composition for preventing or treating candidiasis, comprising miconazole or magnoflorine having a structure of chemical formula 1, as an effective component. In particular, the antifungal composition contains a single natural compound as the effective component, and thus exhibits low toxicity to the human body and excellent antifungal effects against causative bacteria.

Description

TECHNICAL FIELD [0001] The present invention relates to an antifungal composition for improving candidiasis comprising a natural compound,

The present invention relates to a natural plant-derived antifungal candidate substance which inhibits growth of a causative organism causing dermatophytosis or candidiasis.

Fungus is a disease caused by a fungus parasitic on the body, Candida (Candida), actinomycetes (Actinomyces), Aspergillus (Aspergillus), Cryptococcal kusu (Cryptococcus), non-cor (Mucor), no carboxylic Dia (Nocardia) It is caused by about 40 kinds of fungi.

Most of the fungi invade the human body from the surrounding environment to cause illness. Kindida and actinomycetes are adhered to the mouth of a healthy person or other mucous membranes, and if any incentive occurs, they cause pathogenic fungi.

Mycosis varies from mild to severe, and can cause serious problems for older people and immunocompromised patients. Healthy adults have strong immunity to mycosis, but weak or weakened immune systems are susceptible to mycosis, such as children and the elderly, HIV / AIDS patients, transplant patients, chemotherapeutic patients and long-term patients with immunosuppressants.

Most fungal infections are not treated well if they are infected once, but they are recognized as chronic diseases but not as serious. However, infections and mortality from opportunistic infections such as Candida, Aspergillus and Cryptococcus have steadily increased worldwide in immunocompromised patients.

Candidiasis, a fungal infection caused by Candida, is classified as superficial and deep. The superficiality mainly occurs in the oral cavity of women with acquired immunodeficiency syndrome (AIDS) or vaginal vaginitis, and in the cases of diabetic and leukemia patients with immunocompromised organs and immunosuppressive agents, mortality is high. Candidiasis is mainly Candida albicans, but generated by the (Candida albicans), Recently, C. stellatoidea, C. parapsilosis, C. guilliermondii , C. tropcalis, C. pseudotopicalis, the infection by other Candida species such as C. krusei gradually .

The development of modern medicine has increased the number of recipients who undergo immunosuppressive transplant surgery and chemotherapy, thereby increasing diseases caused by various kinds of opportunistic infections such as mycosis. Accordingly, the demand for antifungal agents for inhibiting the growth of fungi has been increased. As a result, various kinds of antifungal agents such as azole, polyene and allyamines have been developed.

However, these antifungal agents may cause various kinds of side effects including hepatotoxicity. In the case of the most commonly used azole drugs, the resistance is rapidly increased. Therefore, new antifungal substitute materials with excellent antifungal effects, low side effects and low toxicity are required to be studied.

On the other hand, there are about 500,000 kinds of plants on the earth, and only about 10% are used for edible or medicinal purposes in humans or other animals. Many studies have been carried out in terms of industrialization since such plant or plant-derived antifungal substances have few cases of side effects.

Traditionally Berberine and Cinamaldehyde have been developed and are expected to be of value as novel antifungal agents. However, cervical toxicity has been reported in berberine, and cinnamaldehyde has been developed as a new inducer due to the weakness of the antifungal effect due to the nature of the substance.

Therefore, there is an urgent need to develop a leading substance which is more stable than conventional antifungal agents, less toxic, and directly or as a novel antifungal agent for synthesizing an inducer.

An object of the present invention is to provide an agent for inhibiting the growth of an environmentally friendly fungal-causing microorganism derived from plants which is excellent in human safety and can minimize resistance.

According to one aspect of the present invention, there is provided a composition for an antifungal agent against candidiasis causing bacteria comprising, as an active ingredient, a malopflone having a structure represented by the following formula (1).

[Chemical Formula 1]

In one embodiment, the causative bacterium may be Candida albicans .

In one embodiment, the composition may further comprise one or more antifungal agents.

In one embodiment, the antifungal agent may be at least one selected from the group consisting of amphotericin, fluconazole, flucytosine, natamycin, myconazole and ketoconazole.

According to another aspect of the present invention, there is provided a cosmetic composition for improving candidiasis comprising the composition for an antifungal agent.

According to another aspect of the present invention, there is provided a composition for external application for skin for improving candidiasis comprising the composition for an antifungal agent.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating candidiasis comprising the composition for an antifungal agent.

According to the present invention, the composition for antifungal use of the present invention contains a naturally occurring single compound as an active ingredient, and thus has low toxicity to humans and has excellent antifungal effect on causative microorganisms, so that it can be usefully used as a fungicide treatment or a leading substance thereof.

It should be understood that the effects of the present invention are not limited to the above effects and include all effects that can be deduced from the detailed description of the present invention or the configuration of the invention described in the claims.

FIG. 1 is a graph comparing susceptibility of Magnoflorine to Candida albicans, a Candida albicans strain, according to an embodiment of the present invention, and susceptibility of a conventional antifungal agent to a control group.
FIG. 2 shows the results of evaluating the inhibition of alpha-glucosidase by the concentration-dependent magnesium fluorophosphate according to an embodiment of the present invention.
FIG. 3 shows the results of evaluating cytotoxicity of magnesium fluorophosphate by concentration according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Further, in order to clearly illustrate the present invention in the drawings, portions not related to the description may be omitted.

As used herein, the terminology used herein is intended to encompass all commonly used generic terms that may be considered while considering the functionality of the present invention, but this may vary depending upon the intent or circumstance of the skilled artisan, the emergence of new technology, and the like. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term, not on the name of a simple term, but on the entire contents of the present invention.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The numerical range includes numerical values defined in the above range. All numerical limitations of all the maximum numerical values given throughout this specification include all lower numerical limitations as the lower numerical limitations are explicitly stated. All the minimum numerical limitations given throughout this specification include all higher numerical limitations as the higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include any better numerical range within a broader numerical range, as narrower numerical limitations are explicitly stated. The subject matter provided herein should not be construed as limiting the following embodiments in various aspects or as a reference throughout the specification.

According to one aspect of the present invention, there is provided a composition for an antifungal agent against candidiasis causing bacteria comprising, as an active ingredient, a malopflone having a structure represented by the following formula (1).

[Chemical Formula 1]

The Magnoflorine is referred to as Escholine or Thalictrine and includes (6aS) -2,10-dimethoxy-6,6-dimethyl-5,6,6a, 7-tetrahydro- Dibenzo [de, g] quinolin-6-ium-1,11-diol ((6aS) -2,10-Dimethoxy-6,6-dimethyl-5,6,6a, 7-tetrahydro-4H-dibenzo [de, g] quinoline-6-ium-1,11-diol.

The Magnoflorine can be isolated from rootstock extract of Sinomenium acutum .

The Bangui belongs to banggigwa (Menispermaceae), in Bangui (Sinomenium), it is distributed in Korea, China and Japan. The bug is mainly used in roots and stems. It contains Sinomenine, Magnoflorine, Syringaresinol, etc. as an index component and the efficacy of analgesic, anti-inflammatory and diuretic And is used as an antiallergic, anti-rheumatic agent and analgesic.

The bacterium can be extracted using a high-temperature water having a temperature of 60 ° C or higher as a solvent, or extracted with an alcohol as a solvent. The alcohol may be an aliphatic alcohol having 1 to 6 carbon atoms, and specifically, methanol, ethanol, isopropanol, butanol, hexane and the like may be used, but the present invention is not limited thereto.

The alcohol extract may be dispensed into an organic solvent and water. The organic solvent may be selected from aliphatic hydrocarbons having 1 to 10 carbon atoms, aliphatic halogenated hydrocarbons having 1 to 10 carbon atoms, and esters having 2 to 10 carbon atoms.

Further, it can be isolated by a chromatographic method well known in the art of magnesium fluoride contained in the abovementioned root root extract. For example, after the non-polar organic solvent is removed, it can be adsorbed on the non-ionic resin for column chromatography. The column chromatography can be carried out by a conventional method well known in the art.

Since the composition contains the compound of Formula 1 as an active ingredient, it may have an excellent antifungal activity against the causative bacteria of Candida albicans, and the causative bacteria may be Candida albicans .

That is, since the composition has a specific antimicrobial activity against a specific strain, it can not induce extensive use, can minimize the incidence of resistance, and is excellent in biosafety.

The composition may further comprise one or more antifungal agents. The antifungal agent may be at least one selected from the group consisting of amphotericin, fluconazole, flucytosine, natamycin, myconazole and ketoconazole.

Some antifungal agents that have been widely used have antifungal activity against the causative organisms, but their resistance is increased due to excessive use, and the use thereof is limited due to biotoxicity, so it is necessary to reduce the use amount.

Accordingly, the antifungal agent can be used in combination with the magnoflurane, whereby the amount of the antifungal agent to be used can be remarkably reduced, and the antifungal activity can be remarkably improved due to the synergistic effect.

According to another aspect of the present invention, there is provided a cosmetic composition for improving candidiasis or an external preparation for skin comprising the composition for an antifungal agent.

The cosmetic composition or external preparation for skin may be formulated by a conventional method. In the formulation of the external preparation for skin, Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can refer to, and in the formulation of the cosmetic composition, International cosmetic ingredient dictionary, 6th ed., The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995, which is incorporated herein by reference.

Specifically, the cosmetic composition may be prepared in the form of a general emulsified formulation and a solubilized formulation. For example, lotion such as soft lotion or nutrition lotion; Lotion such as facial lotion, body lotion; Nourishing cream, moisture cream, cream such as eye cream; essence; Makeup ointment; spray; Gel; pack; Sunscreen; Makeup base; A foundation such as a liquid type, a solid type or a spray type; powder; Make-up removers such as cleansing creams, cleansing lotions and cleansing oils; Or a cleansing agent such as a cleansing foam, a soap, a body wash, and the like, but is not limited thereto. The external preparation for skin may be formulated into ointments, patches, gels, creams or sprays, but is not limited thereto.

The external preparation for skin or the cosmetic composition according to the present invention may be appropriately blended with other ingredients within the range of not impairing the object of the present invention depending on the kind of the formulation or the purpose of use.

The external preparation for skin or the composition for cosmetics may contain a conventional acceptable carrier such as oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a pigment, a preservative and a flavoring agent. But is not limited thereto.

The acceptable carrier may vary according to the formulation. For example, when formulated into ointments, pastes, creams or gels, there may be used an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, Mixtures may be used.

Talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as the carrier component when the external preparation for skin or cosmetic composition is formulated into powder or spray, Propellants such as hydrocarbons, fluorohydrocarbons, propane, butane or dimethyl ether.

When the external preparation for skin or cosmetic composition is formulated into a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethylcarbonate, ethyl acetate, benzyl benzoate, Propylene glycol and 1,3-butyl glycol oil may be used, and in particular fatty acid esters of cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or sorbitan are used .

When the external preparation for skin or the cosmetic composition is formulated into a suspension, it is preferable to use, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester Suspending agent, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the cosmetic composition is formulated into soap, it is preferable to use, as a carrier component, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolizate, isethionate, a lanolin derivative, an aliphatic alcohol, a vegetable oil, Can be used.

The external preparation for skin or the cosmetic composition may be a lipid material, an organic solvent, a solubilizing agent, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent ), Perfumes, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants, chelating agents, preservatives, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, Such as any other ingredients used in cosmetics or dermatology.

However, the adjuvant and the mixing ratio thereof can be appropriately selected so as not to affect the preferable properties of the cosmetic composition according to the present invention.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating candidiasis comprising the composition for an antifungal agent.

The " prevention " means any action that reduces the frequency or extent of the pathological event. Prevention can be complete or partial. In this case, the symptoms caused by Candida in the subject may be reduced as compared with the case where the composition is not used.

The term " treatment " means any clinical intervention to alter the natural course of a subject or cell to be treated, and may be performed during or during the course of a clinical pathology. The desired therapeutic effect may be to prevent the occurrence or recurrence of the disease, to alleviate the symptoms, to reduce all direct or indirect pathological consequences of the disease, to prevent metastasis, to reduce the rate of disease progression, Relieving or temporarily alleviating the condition, or improving the prognosis.

The compositions can be administered in the form of oral delivery, parenteral delivery. The composition may be administered systemically or topically, and the administration may include oral administration and parenteral administration. The composition may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide a suitable dosage form.

The composition may further comprise a carrier, an excipient and a diluent used in the preparation of the pharmaceutical composition. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, But are not limited to, cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the composition may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, external preparation, suppository and sterilized injection solution.

The solid preparation for oral administration may be a tablet, a pill, a powder, a granule, a capsule, etc. The solid preparation may be prepared by adding to the compound and its fractions at least one or more excipients such as starch, calcium carbonate, sucrose, lactose , Or gelatin. In addition to the above excipients, lubricants such as magnesium stearate and talc may be used.

The liquid preparation for oral administration may be a suspension, a solution, an emulsion, a syrup, etc. In addition to water and liquid paraffin which are simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be used.

The preparation for parenteral administration may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. Examples of the non-aqueous solutions and suspensions may include injectable esters such as propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, and glycerogelatin.

The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The " pharmaceutically effective amount " means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dosage level will depend on the type of disease, severity, activity of the drug, Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is desirable to administer an amount that allows for all of the above factors to be maximally effected in a minimal amount without side effects, which can be readily determined by one skilled in the art.

Hereinafter, the present invention will be described in further detail with reference to the following examples. It is to be understood that the present invention is not limited by the following examples

Experimental Example  1: Minimum inhibitory concentration ( MIC Evaluation of Antifungal Activity by In vitro  antifungal activity)

Evaluate the minimum concentration of the antifungal for Candida albicans (Candida albicans) of the mageuno Florin (Magnoflorine).

Table 1 shows the anti- Canidia and the antifungal activity of the antifungal agent and the candidate substance (comparative example), which are widely used in the past, in addition to the albicans activity.

To determine the antifungal effect of C. albicans on the compounds, we used the broth microdilution assay presented in the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Each compound was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / mL.

Each compound was continuously diluted 2-fold and then added to a 96-well microtiter plate in an amount of 100 μL, and 100 μL of Candida The albicans suspension (5 × 10 ⁴ CFU / mL) was added to each well and cultured at 30 ° C. for 48 hours. The concentration of each compound was at least 1.5625 mg / L at a maximum of 100 mg / L. The MIC value for each material was the minimum concentration at which the strain did not grow in each plate.

As shown in Table 1, the MIC of the granule Growth inhibitory effect against albicans was confirmed. The activity of C. albicans was relatively inferior to that of Miconazole, which was widely used in the prior art, but was comparatively superior to those of Comparative Examples 2 and 3.

On the other hand, the comparative examples except for Comparative Example 1 had no or little inhibitory activity against the microorganisms.

That is, the above-mentioned magnoflurane has excellent antifungal activity against C. albicans causing candidiasis.

division MIC division MIC Example
(Magnoflorine) 50 Comparative Example 23
(Salvianolic acid B) > 100 Comparative Example 1
(Miconazole) 3.125 Comparative Example 24
(Curcumin) > 100 Comparative Example 2
(Berberine chloride) 100 Comparative Example 25
(cis-8,11,14-Eicosatrienoic acid) > 100 Comparative Example 3
(Cinnamaldehyde) 100 Comparative Example 26
(Linoleic acid) > 100 Comparative Example 4
(p-Anisaldehyde) > 100 Comparative Example 27
(y-Liolenic acid) > 100 Comparative Example 5
(Baicalin) > 100 Comparative Example 28
(Myrcene) > 100 Comparative Example 6
(Baicalin hydrate) > 100 Comparative Example 29
(Retinoic acid) > 100 Comparative Example 7
(Caffeic acid) > 100 Comparative Example 30
(D-Camphor) > 100 Comparative Example 8
(p-Coumaric acid) > 100 Comparative Example 31
(Citral) > 100 Comparative Example 9
(Coumarin) > 100 Comparative Example 32
(L-Carvone) > 100 Comparative Example 10
(Emodin) > 100 Comparative Example 33
((-) - caryophyllene oxide) > 100 Comparative Example 11
(Geranyl acetate) > 100 Comparative Example 34
(Ellagic acid) > 100 Comparative Example 12
((±) -Jasmonic acid) > 100 Comparative Example 35
(Furfural) > 100 Comparative Example 13
(Myricetin) > 100 Comparative Example 36
(Geraniol) > 100 Comparative Example 14
(Quercetin) > 100 Comparative Example 37
(Linalyl acetate) > 100 Comparative Example 15
(Quercetin hydrate) > 100 Comparative Example 38
(Methyl eugenol) > 100 Comparative Example 16
(Rhapontin) > 100 Comparative Example 39
(Nerol) > 100 Comparative Example 17
(Rhein) > 100 Comparative Example 40
((1R) - (+) - [alpha] -Pinene) > 100 Comparative Example 18
(Rotenone) > 100 Comparative Example 41
(Sennoside B) > 100 Comparative Example 19
(Sennoside A) > 100 Comparative Example 42
(Syringic acid) > 100 Comparative Example 20
(Shikimic acid) > 100 Comparative Example 43
(Aucubin) > 100 Comparative Example 21
(Chrysophanol) > 100 Comparative Example 44
(Macelignan) > 100 Comparative Example 22
(Salvianolic acid hydrate) > 100

Experimental Example  2: Various Candida on  Antifungal Active activity  evaluation( In vitro  antifungal activity)

Table 2 below shows the anti- Candida spp. And the antifungal activity of the candidate substance which is widely used in the prior art.

Candida Antifungal activity concentrations were measured in the same manner as in Experimental Example 1 by measuring the minimum inhibitory concentration (MIC) of the albicans , C. tropocalis , C. parapsilosis , and C. glabrata in Examples and Comparative Examples 1 to 3 ).

The results of Myconazole, which is a synthetic antifungal agent, were very good.

Berberine chroride (Comparative Example 2) and Cinnamaldehyde (Comparative Example 3) are excellent antifungal agents against C. albicans . Magnoflonin showed better antifungal effect against berberine and cinnamaldehyde, and antifungal effect against C. parapsilosis was also confirmed.

division C. albicans
( KCTC7965 ) C. albicans
( KCTC30071 ) C. tropocalis
( KCTC17762 ) C. parapsilosis
( KCTC45480 ) C. glabrata
( KCTC7219 ) Example
(Magnoflorine) 50 100 > 100 100 > 100 Comparative Example 1
(Miconazole) 3.125 6.25 6.25 0.7813 6.25 Comparative Example 2
(Berberine chloride) 100 100 > 100 100 > 100 Comparative Example 3
(Cinnamaldehyde) 100 100 > 100 > 100 > 100

Experimental Example 3: Evaluation of antifungal activity by disk diffusion test

Candida The disc diffusion test according to Examples and Comparative Examples was carried out on albicans .

1 is a Candida Compared to albicans , the antifungal activity of magnesium fluorophosphate (Example) and the widely used antifungal agents (Comparative Examples 1 to 3) were compared.

Table 3 shows numerical values of FIG. 1, wherein M is the average diameter (mm) of the inhibition size, and R is the range of the experimental values.

Candida for Compounds Antifungal susceptibility testing by disk diffusion assay as described in CLSI (formerly NCCLS) document M44-A was used to confirm the antifungal effect on albicans .

Each compound was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / mL. Each of the compounds was added to 6-mm diameter antibiotic test disks in an amount of 100 μg, and the suspension of Candida albicans (5 × 10 ⁴ CFU / mL) was applied to a YPD agar plate and cultured at 35 ° C. for 24 hours, 48 hours, Respectively. All experiments were repeated three or more times.

Referring to Table 3, the magnophore (example) is Candida showed antifungal effect against albicans .

In contrast to the effect of berberine (Comparative Example 2) and cinnamaldehyde (Comparative Example 3) disappearing after 72 hours, the size of the clear zone (the area in which fungal growth was inhibited) for 72 hours And the above results suggest that the antifungal activity can be maintained for a long time.

division 24 hours 48 hours 72 hours M R M R M R Example
(Magnoflorine) 14.5 14-15 12.3 12-13 11.5 11-12 Comparative Example 1
(Miconazole) 15.5 15-16 14 14 12 12 Comparative Example 2
(Berberine chloride) 12 11-13 7.3 7-8 - - Comparative Example 3
(Cinnamaldehyde) 20.3 20-21 13.3 13-14 - -

Table 4 is the measurement of the minimum inhibitory concentration (MIC) of Table 3 above.

Referring to FIG. 1 and FIG. 4, berberine and cinnamaldehyde disappeared after 72 hours, but the activity of magnesium was maintained. The above results suggest that the antifungal activity may be maintained for a long time because the magnophosphorin is more stable than berberine and cinnamaldehyde.

division 24 hours  After MIC
(μg / mL) 48 hours  After MIC
(μg / mL) 72 hours  After MIC
(μg / mL) Example
(Magnoflorine) 50 50 50 Comparative Example 1
(Miconazole) 3.125 3.125 3.125 Comparative Example 2
(Berberine chloride) 100 100 > 100 Comparative Example 3
(Cinnamaldehyde) 50 100 > 100

Experimental Example 4: Evaluation of alpha-glucosidase inhibitory activity

Candida of Magnoflurane In order to confirm the mechanism of growth inhibition in albicans , the inhibitory activity of alpha - glucosidase related to the viability and toxicity of Candida spp. was evaluated.

Of mageuno Florin alpha - to evaluate the inhibitory activity of the glucosidase, Candida from 0.6% maltose medium After incubation of albicans at 30 ° C for 24 hours, a crude enzyme was obtained using glass beads (0.4-0.6 mm diameter, DAIHAN-Scientific, Korea).

A mixture of 80 μL PCBS buffer (pH 6.8), 9 μL crude enzyme, and 1 μL of the compound at 10 mg / L was added to a 96-well plate and incubated for 30 minutes. P-nitrophenyl-alpha-D-glucopyranoside (PNPG, Sigma Aldrich) Respectively.

After 20 minutes, 100 μL of 0.1 M Na ₂ CO ₃ was added to stop the reaction. The activity was measured at 400 nm absorbance using a microplate reader (BioTek Instruments, Korea). All experiments were repeated at least 4 times.

2 is a Candida according to the concentration of mageuno Florin It is the result of confirming the inhibition of alpha-glucosidase activity of albicans .

Referring to FIG. 2, the activity of alpha-glucosidase rapidly decreased as the concentration of magnesium was increased.

Experimental Example 5: Cytotoxicity assay

In order to confirm the toxicity of the above-mentioned magnoflurane to cells, it was determined whether the growth of human-derived cells was inhibited.

In order to confirm the inhibitory effect of the compound, the magnophosphorin was treated with HepG2 cell line or HaCaT cell line according to the concentration and MTT assay was performed.

The cytotoxicity test was performed using a Hep-G2 cell line or a HaCaT cell line. Each _10-insert the 4cells / 0.1mL cells in each well of 96-well tissue culture plates (SPL ) was added to the culture plates at the concentration indicated for each material is dissolved in DMSO. Control DMSO was added to each plate so that the maximum concentration did not exceed 0.2%. After 48 hours, 100 μL of 5 mg / mL MTT (Sigma Cat. M2128) was added, followed by reaction at 37 ° C. for 3 hours.

After removing the medium, 0.1 ml of DMSO was added and reacted for 10 minutes. Activity was measured at 540 nm absorbance using a microplate reader (BioTek Instruments, Korea). All experiments were repeated three or more times.

FIG. 3 shows the results of evaluating cytotoxicity by treatment with the concentration of magnoflurane.

Referring to FIG. 3, when HaCaT cell line was treated with a high concentration of magnesium (100 mg / L), almost no cell growth was inhibited as compared to the case without treatment with magnesium fluoride, and in case of HepG2 cells, Growth was inhibited. The above results suggest that the toxicity to living organisms is very low if the magnesium fluorophosphate is used at a conventional concentration as an antifungal agent.

Experimental Example  6: Antifungal by combination application Synergistic effect  evaluation( Antifungal  synergy testing)

The antifungal activity of the above-mentioned magnoflurane was evaluated by the combined application with the existing antifungal agent.

Candida with Mixed Use of Myconazole and Magnoflurane The broth microdilution assay was used as a chequerboard fashion to confirm the antifungal effect against albicans . The concentration of each compound was continuously diluted 2 fold to a minimum of 1.564 mg / L at a maximum of 100 mg / L, and 100 μL was added to a 96-well microtiter plate. 100 μL of each suspension (1 × 10 ⁴ CFU / mL) And incubated at 35 ° C for 24 hours or 48 hours. The MIC values for each compound were confirmed on each plate at the minimum concentration at which the strain did not grow on each plate.

The synergistic effect according to the mixing use was expressed as Fractional inhibitory concentration indexes (FICIs) using FICI = (Ac / Aa) + (Bc / Ba). Ac and Bc are the MIC values of A and B substances, respectively, and Aa and Ba are the MIC values of A and B substances, respectively, when used alone.

A strong synergetic effect is obtained when the FICI value is 0.5 or less, a partial synergy effect is 0.5 to 0.75, an additive effect is 0.75 to 1.0, and an irrelevant effect is obtained when the FICI value is 4.0 or more. .

When myconezol and magnesium were combined, the FICI value was 0.75. This means that the MIC value of myconazole decreased from 6.25 mg / L to 3.125 mg / L when 25 mg / L of magnoflurane was administered.

The results showed that when 25 mg / L of magnoflurane was administered, Candida it means that the amount of myconezol that kills albicans can be reduced to half, and the amount required for realizing the antifungal effect can be remarkably reduced by increasing the activity of myconazole.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (7)

A pharmaceutical composition for the prevention and treatment of candidiasis comprising as an active ingredient Magnophosphorin and Myconazole having a structure represented by the following formula (1).
[Chemical Formula 1]

The method according to claim 1,
A pharmaceutical composition having antifungal activity against Candida albicans . delete delete 1. A cosmetic composition for improving candidiasis comprising as an active ingredient magnesiumphosphate and myconazole having the structure represented by the following formula (1).
[Chemical Formula 1]

A composition for external application for skin for the amelioration of candidiasis comprising, as an active ingredient, magnoflurane and myconazole having the structure represented by the following formula (1).
[Chemical Formula 1]

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