JP2008007464A - Antifungal composition of external preparation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition of an external preparation exhibiting excellent antimycotic actions and anti-inflammatory actions with slight irritation to skin or mucous membranes and excellent anti-inflammatory actions on atopic dermatitis. <P>SOLUTION: A high-purity (≥98%) composition of EGCG (epigallocatechin gallate) which is one kind of green tea polyphenol is directly used. Otherwise, the composition is mixed with a general antifungal antibiotic for use. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an external preparation composition having a therapeutic effect on fungal diseases such as candidiasis and dermatitis such as atopic dermatitis.

  Infections caused by fungi are referred to as mycoses, but common fungi are known as athlete's foot caused by ringworm, candidiasis caused by Candida, or cryptococcosis caused by cryptococcus. This mycosis is a superficial mycosis in which the affected area does not reach the skin dermis depending on which part of the body is violated, and a deep mycosis that affects the subcutaneous tissue after the dermis and organs such as the brain, lungs, and heart And classified. Further, deep mycosis is classified into a subcutaneous tissue infection type and a systemic infection type, and the systemic infection type is roughly divided into a primary infection and an opportunistic infection. These pathogens not only have a strong chitinous cell wall consisting of polysaccharides, but are also the same eukaryotic organism as the human body, so drugs that damage only the fungal cells and cause little harm to human tissues are extremely It will be limited. For this reason, the number of drugs with low side effects on fungi is low compared to infectious diseases against bacteria, which have many antibiotics that can cause serious damage to only bacteria.

  Since fungi do not secrete toxins, lesions are caused by tissue erosion and inflammatory reactions due to bacterial growth. It is known that when cellular immunity decreases due to HIV infection, resistance to fungi decreases and the frequency of mycosis, candidiasis and cryptococcosis, increases.

  Antimycotic agents include azole antifungal agents as polyene antibiotic species that target fungal-specific cell membranes. Polyene's amphotericin B and nystatin bind to sterols in cell membranes, and Azole's Clotrimazole, Miconazole, Ketoconazole, Fluconazole and Itraconazole are said to suppress the synthesis of ergosterol. In addition, 5-Fluorocytosine (5FU) that inhibits nucleic acid synthesis and Griseofulvin that suppresses association of microtubules are known.

  On the other hand, polyphenols contained in tea are known to have many physiological activities such as antioxidant action, antiviral action, antibacterial / bactericidal action, and bacterial toxin inhibitory action. The main bitter and astringent taste of tea is called tannin, but its main component is catechins. There are 8 types of catechins contained in green tea, but the main ones are (−)-epicatechin (EC; ratio in total tea leaf catechin = 10%), (−)-epigallocatechin (EGC; 22%) ), (−)-Epicatechin gallate (ECG; 11%), (−)-epigallocatechin gallate (EGCG; 54%), among which EGCG is the main component. Of these catechins, EGCG is said to have the highest antioxidant activity.

  It has been reported that catechin compounds such as EGCG have an antibacterial effect against bacteria (Patent Documents 1 to 3), and an antibacterial effect against methicillin-resistant Staphylococcus aureus (MRCA) has also been reported. In addition, Blanco (Non-patent Document 2) et al. Report that epigallocatechin gallate (EGCG), which is the main component of green tea polyphenols, can suppress bacterial enzyme activity and reduce bacterial entry. Furthermore, there are also reports on antifungal activity (Patent Document 3 and Non-Patent Document 1). In addition, it is reported also with respect to the antifungal activity by several other natural products (nonpatent literatures 5-7).

However, the catechin compound alone is considered to have insufficient antifungal action or the like (Patent Documents 1 and 2), and may contain an oligomeric proanthocyanidin obtained by extraction from pine bark (Patent Document 1). ) And the use of catechin derivatives with improved antifungal properties (Patent Document 2) have been proposed. It has also been proposed to use it in combination with existing antibiotics. It has been reported that EGCG is used in combination with penicillin in order to regulate the resistance of methicillin-resistant Staphylococcus aureus (MRSA) (Non-patent Document 3). It has also been proposed to use a mixture with amphotericin B (AMPH) or fluconazole (FLCZ) (Non-patent Document 4).
JP 2004-284975 A JP-A-9-110615 JP 2002-255810 A M. Hirasawa et al., J. Antimicrob. Chemother., 53, 225, 2004 AR Blanco, S. La Terra Mule, G. Babini, S. Garbisa, V. Enea, D. Rusciano, (-) Epigallocatechin-3-gallate inhibits gelatinase activity of some bacterial isolates from ocular infection, and limits their invasion through gelatine , Biochim. Biophys. Acta 1620 (2003) 273-281. PD Staplenton, PW Tayloy, Methiclillin resistance in Staphylococcus aureus: mechanisms and modulation, Sci. Prog. 85 (2002) 57-72. M. Hirasawa, K. Takada, Multiple effects of green tea catechin on the antifungal activity of antimycotics against Candida albicans, J. Antimicrob. Chemother. 53 (2004) 225-229. F. Mondello, F. De Bemardis, A. Girolamo, G. Salvatore, A. Cassone, In vitro and in vivo activity of tea tree oil against azole-susceptible and ≡resistant human pathogenic yeasts, J. Antimicrob. Chemother. 51 ( 2003) 1223-9. M. Donia, MT Hamann, Marine natural products and their potential applications as anti-infective agents, Lancet Infect.Dis. 3 (2003) 338-48. P. Lavermicocca, F Valerio, A. Viscnti, Antifungal activity of phenyllactic acid against molds isolated from bakery products, Appl.Environ. Microb. 69 (2003) 624-40. JV Higdon, B. Frei, Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions, Crit. Rev. Food Sci. Nutr. 43 (2003) 89-143. J. Jodoin, M. Demeule, R. Beliveau, Inhibition of the multidrug resistance P-glycoproten activity by green tea polyphenols, Biochim. Biophys. Acta1 542 (2002) 149-159 S. Okubo, M. Toda, Y. Hara, T. Shimamura, Antifungal and fungicidal activities of tea extract and catechin against Trichophyton, Nippon Saikingaku Zasshi 46 (1991) 509-14.

  Increasing cases of fungal infections have occurred over the past few years, and a variety of new antifungal agents are being used to treat patients, but the same drug-resistant species as Candida species are still on the rise. Amphotericin B (AMPH) is one of the Polyene antibiotic species and Fluconazole (FLCZ) is an Azole antifungal agent, but these are Candida albicans (Candida albicans). albicans) is known to exhibit potent antifungal activity. However, these antifungal agents have side effects, and antifungal resistant clinical isolates have been reported among Candida species.

  Therefore, the development of an effective and safe non-antibiotic formulation is a serious problem in eliminating all antibiotic sensitive and antibiotic resistant Candida species.

  In view of the above problems, the present inventors have intensively studied to replace antifungal external preparations with antibiotics using catechins and other safe natural compounds. As a result, it was found that when EGCG having a purity of 98% or more was used, very excellent antifungal properties were obtained. Such antifungal properties cannot be obtained at all with low-purity EGCG (for example, purity of 90% or less), and the mechanism is unknown.

  As described above, the present invention provides an antifungal topical preparation that provides an antifungal effect comparable to that of an existing general antibiotic-based antifungal topical agent, and that does not cause problems of side effects and resistant bacteria induction. It is intended to provide an agent.

  The external preparation composition of the present invention substantially contains only EGCG having a purity of 98% or more as a polyphenol compound. That is, 98% or more of the polyphenol component is EGCG. In another embodiment, the external preparation composition of the present invention comprises a polyphenol component having a purity of (−)-epigallocatechin gallate (EGCG) of 98% or more and an antifungal antibiotic, 99/1. It is characterized by including in the weight ratio of 0-1.0 / 99.

  An antifungal effect comparable to that of an existing general antibiotic antifungal topical agent can be obtained, and side effects and problems of inducing resistant bacteria do not occur.

  The external preparation composition of the present invention comprises only a polyphenol component as high-purity EGCG (purity of 98% or more) or a mixture of this with an antifungal antibiotic only as a substantially active ingredient. . However, it can contain various components for enhancing the effect of the active ingredient and performing other actions.

  First, water, ethanol, oil, etc. can be included as a diluent or a solvent. That is, the external preparation composition of the present invention can be used as a polyphenol component or a liquid in which antibiotics are mixed, and appropriately diluted or used as an aqueous solution, alcohol solution, cream, oil-containing emulsion or the like. be able to.

  The external preparation for skin of the present invention may contain ascorbic acid or a derivative thereof. Ascorbic acid is known not only to have antioxidative activity, but also to promote collagen synthesis and enhance immunity, thus suppressing inflammation and pain caused by fungal infections, especially ringworm And the effect of healing infections can be enhanced and skin conditioning can be expected. As ascorbic acid or a derivative thereof, ascorbic acid or a derivative thereof used as a food additive, for example, ascorbic acid glycoside, sodium ascorbate, magnesium ascorbate or the like is used. The external preparation for skin of the present invention contains oil, water, etc. according to its dosage form, application, etc., surfactant, moisturizer, lower alcohol, thickener, fragrance, antioxidant, chelating agent, Components such as pigments can be appropriately blended.

  Hereinafter, test examples will be described in detail.

<Comparison antifungal agent and evaluation strain>
The antifungal properties of high-purity EGCG (purity 98% or more) are compared with the existing six antifungal agents, ie, amphotericin B (AMPH), fluconagole (FLCZ), flucytosin (5FC), itraconolegole (ITCZ), micafungin (MCFG) ) And miconazole (MCZ). The antifungal evaluation was carried out by measuring the sensitivity to 6 clinical isolates belonging to Candida species by measuring the minimum growth arrest concentration (MIC) of each.

  As a clinical isolate, yeast-like fungus DP “Eiken” (produced by Eiken Chemical Co., Ltd.) was used. Specifically, Candida albicans (Candida albicans NBRC 0583 and 1388), Candida glabrada (Candida glabrada NBRC 0005 and 0622), and Candida parapsilosis (Candida parapsilosis NBRC 0708 and 0840), that is, a total of six types The bacterial species was used for the test. This is a susceptibility test kit based on a micro liquid dilution method, and can measure susceptibility to all drugs approved in Japan for the treatment of deep mycosis.

<Reagent>
As high-purity EGCG, TEAVIGO (RO-267624000, purity of 98% or more) from Roche Vitamin (Basel, Switzerland) was used as it was. For comparison, Sigma (Sigma-Aldrich) EGCG (purity 95% or more) was used. For media on well plates, filter sterilized RPMI-1640 medium (Sigma, USA) was used for all sensitivity tests. This medium contains L-glutamine and low concentration (2 mg / ml) glucose, but does not contain phenol red and sodium bicarbonate, and 0.165M 3- (N-morpholino) propanesulfonic acid (pH 7. 0) is used.

<Method for evaluating antifungal properties>
The minimum inhibitory concentration (MIC) was measured in accordance with the antifungal susceptibility measurement method reported by the Japanese Society for Medical Mycology Standardization Committee Report (Antifungal Susceptibility Test Method, Mycology Journal 36: 62-64.1995). Specifically, all antifungal susceptibility tests were performed according to Document M-27A published by the National Committee for Clinical Laboratory Standards (NCCLS).

  TEAVIGO (purity 98% or more), which is high purity EGCG, and EGCG made by Sigma having a purity of 95% or more were used at a concentration of 0.06 to 16 μg / mL. That is, dry plates were prepared as well plates containing high-purity EGCG at various concentrations within this range. Similarly, well plates each containing the above six types of antifungal agents at various concentrations within the following ranges were prepared. AMPH: 0.03-16 μg / mL, FLCZ: 0.125-64 μg / mL, 5FC: 0.125-64 μg / mL, ITCZ: 0.015-8 μg / mL, MCFG: 0.03-16 μg / mL and MCZ: 0.06-32 μg / mL.

On the other hand, all the above bacterial species were cultured in a PDA (potato dextrose agar) medium at 35 ° C. for 24 hours, and subcultured twice at intervals of 48 hours before use. Then, the pre-cultured strain was seeded in RPMI-1640 medium to prepare a suspension with a concentration of 2 × 10 ³ colony forming units (cfu) / mL. Thereafter, the strain was finally diluted to an inoculation concentration of 1 × 10 ³ cfu / mL to obtain a strain suspension.

Each well suspension containing EGCG and 6 types of antifungal agents was inoculated with 100 μL each of the strain suspension. At this time, a growth control group containing no drug was included in each test group. Thereafter, the growth inhibition degree was measured with an enzyme immunoassay (ELISA reader, wavelength 450 nm) after 24 and 48 hours while culturing the well plate at 35 ° C. The minimum drug concentration that produced 50% and 90% growth inhibition relative to the control group without drug was defined as the MIC value of EGCG and 6 antifungal agents (MIC ₅₀ and MIC _90, respectively).

<Evaluation results>
Table 1 shows the evaluation results of susceptibility to EGCG of clinical isolates belonging to Candida species and six antifungal agents, ie, AMPH, FLCZ, 5FC, ITCZ, MCFG, and MCZ.

The MIC range of EGCG was 0.5 mg / ml or more in all test strains, and some were evaluated to exceed 16 mg / ml (">16"), although it varies depending on the strain and the type of strain. When Candida glabrata is used as a test strain, the sensitivity to high purity EGCG (TEAVIGO) (MIC ₅₀ 0.5-1 μg / ml and MIC ₉₀ 1-2 μg / ml) is sensitive to FLCZ (MIC They were achieved significantly more sensitive than _{50 1~8μg} / ml and MIC ₉₀ 4~8μg / ml). The sensitivity to high purity EGCG (TEAVIGO) can be said to be at the same level as the sensitivity to AMPH (MIC ₉₀ 0.25 to 0.5 μg / ml), which is known to have significant side effects even at low concentrations. In contrast, Sigma EGCG (purity 95% or more) was less sensitive.

On the other hand, when Candida parapsilosis is used as a bacterial species, the sensitivity (MIC ₅₀ 1-4 μg / ml and MIC ₉₀ 2-16 μg / ml) to high-purity EGCG (TEAVIGO) is lower than that using Candida glabrata. , Quite sensitive. The sensitivity of these strains to EGCG is somewhat lower than the sensitivity to other antifungal agents.

In general terms, the sensitivity to comparative antifungal agents was comparable or slightly lower than the sensitivity to high purity EGCG (TEAVIGO). That is, for AMPH, MIC ₅₀ 0.125-0.5 μg / ml and MIC ₉₀ 0.25-2 μg / ml, and for 5FC, MIC ₅₀ <0.125 μg / ml and MIC ₉₀ 0.125 μg / ml. a MIC ₅₀ and MIC ₉₀ 0.03 [mu] g / ml for MCFG a MIC ₅₀ 0.06~0.5μg / ml and MIC ₉₀ 0.5~1μg / ml for ITCZ, MIC ₅₀ for MCZ < 0.06 to 0.25 μg / ml and MIC ₉₀ 0.125 to 2 μg / ml.

Of the six types of comparative antifungal agents used in this study, MCFG (MIC ₅₀ <0.03 to 0.5 μg / ml and MIC ₉₀ 0.5 μg / ml) is used regardless of the strain used. Showed the most sensitive sensitivity.

<Discussion>
EGCG is known to have a function of enhancing the antibacterial action and the effect of a specific antibiotic as a representative component of catechins present in green tea extracts. Many antibacterial actions of EGCG against several pathogenic bacteria have already been reported. (Non-patent documents 2-3, 8-9)
For fungi, Okubo et al. (Non-patent Document 10) 2.5% black tea extract suppresses the growth of Trichophyton mentagrophytes and T. rubrum, but the growth of Candida albican or Cryptococus neoformans cannot be suppressed even at 10% concentration. It is reported. However, Hirasawa et al. Reported the sensitivity of Candida albicans to catechins containing EGCG (Non-patent Document 4).

  As described above, it was confirmed that high-purity EGCG (TEAVIGO) exhibited antifungal activity against six clinical isolates classified by three Candida species (Table 1). Among the test strains, the Candida crablada strain showed the most sensitive sensitivity to EGCG (TEAVIGO). The susceptibility of these strains to EGCG is slightly less sensitive than other antifungal agents, but is more sensitive than FLCZ and is comparable to AMPH, which has significant side effects even at low concentrations. These results indicate that high purity EGCG is effective against candidiasis such as Candida pneumonia induced by Candida glabrata. In the case of EGCG, unlike antifungal agents by antibiotics, resistant bacteria and side effects are not induced.

Although specific data is not shown, when high-purity EGCG (TEAVIGO) was used, an excellent effect based on synergy was also obtained when used in combination with these six existing antifungal agents.

Claims (2)

  An antifungal external preparation composition comprising a polyphenol component having a purity of (-)-epigallocatechin gallate (EGCG) of 98% or more. It contains a polyphenol component having a purity of (−)-epigallocatechin gallate (EGCG) of 98% or more and an antifungal antibiotic in a weight ratio of 99 / 1.0 to 1.0 / 99. An antifungal composition for external use characterized by the above.