JP5239002B2 - Antibacterial active substances obtained from indigo grass and various compositions containing the same - Google Patents

Description

  The present invention relates to an antibacterial active substance obtained from cyanobacteria, an antifungal agent containing the antibacterial active substance as an active ingredient, in particular, an antifungal agent against fungi belonging to the genus Malassezia such as Malassezia / furfur, and the like It relates to various compositions.

  Bacteria belonging to the genus Malassezia represented by the basidiomycete-type incomplete yeast Malassezia furfur is a lipophilic skin resident fungus, and its presence has been confirmed in almost 90% of people, almost all of the body, especially It is frequently present in the head of hair, face, upper back, neck, etc., which are seborrheic sites.

  Recently, as shown in Non-Patent Documents 1 to 3 and the like listed below, this Malassezia genus fungus has various fungiforms such as folliculitis, intercalation rash, seborrheic dermatitis, psoriasis vulgaris, and atopic dermatitis. It has been strongly pointed out that it may be involved in various skin diseases, and it has been elucidated that its growth is the main cause of dandruff (J. Soc. Cosmet. Chem. Japan Vol. 22, No. 3). , 1988). Furthermore, mold that breeds in the outer ear of various pets (especially dogs with drooping ears) is also considered to be malassezia full.

  Patent Document 1 describes an antifungal agent against Malassezia sp. Containing tricomycin as an active ingredient.

  Patent Document 2 describes an anti-dandruff hair washing composition containing a saccharide alkenyl succinate ester salt, a cationic polymer, a higher fatty acid or a derivative thereof.

  Patent Document 3 describes a bioactive extract derived from indigo and various actions thereof. However, specific examples of antibacterial action against fungi are not disclosed.

JP-A-9-249571 JP 2000-273027 A JP 2001-31581 A Masaru Terui, et al., “Inflammation and Immune Responses Caused by Malassezia furufur”, Japan Journal Med. Mycol., 40, 1999, 63-67 Masaru Terui, et al., “Atopic dermatitis and fungal allergy”, Japan Journal Med. Mycol., 41, 2000, pp. 157-160 Masataro Hiruma, et al., “Fungus and Atopic Dermatitis”, Japan Journal Med. Mycol., 40, 1999, 79-83.

  Conventionally, many steroidal compounds have been used for the treatment of atopic dermatitis, but there are also many problems such as causing various side effects. Therefore, development of different types of drugs having an excellent antibacterial action against pathogenic bacteria such as atopic dermatitis is awaited.

  By the way, in Japan, indigo grass is used for dyeing clothing, and as a folklore, it is said that indigo-dyed clothing is effective for skin diseases. There were few skin diseases like.

  Therefore, the present inventor considers that there is a substance having an antibacterial action against fungi such as Malassezia fungi in the indigo-derived extract, and as a result of intensive studies for the purpose of finding such a substance The present invention has been completed.

That is, the first aspect of the present invention is an antibacterial active substance which is an organic solvent-soluble component derived from cyanobacteria and has the following physicochemical properties:
(1) Rf value = 0.65 by thin layer chromatography on silica gel plate using dichloromethane: ether: hexane (10: 1: 1) as developing solvent;
(2) NMR data:
NMR (CDCl3) δ (ppm):
11.16 (1H, s), 10.35 (1H, s), 9.65 (1H, s), 8.55 (2H, t),
8.40 (1H, d),
7.99 (2H, q), 7.90-7.57 (6H.m), 7.52 (1H, q), 7.38 (1H, t), 6.37 (1H, d), 6.22 (1H, t),
5.15 (1H, m), 4.47 (3H, m), 4.21 (3H, m), 4.06 (2H, q), 3.90 (3H, s), 3.67 (3H, s),
3.48 (1H, s), 3.37 (3H, s), 2.73-2.10 (3H, m), 1.94-1.75 (7H, m), 1.74-0.65 (67H, m),
Concerning.

  As a second aspect, the present invention relates to an antifungal agent against a fungus containing a Malassezia genus fungus such as Malassezia / fulfur, which contains the above antibacterial active substance as an active ingredient.

  As a third aspect, the present invention relates to an antifungal agent against fungi including malassezia fungi such as malassezia / furfur, which contains tryptanthrin, which is an organic solvent-soluble component derived from cyanobacteria, as an active ingredient.

  The present invention provides, as a fourth aspect, a pharmaceutical composition for skin diseases involving fungi belonging to the genus Malassezia, and various antibacterial active compositions or antibacterial active products, containing such an antibacterial active substance or antifungal agent. Concerning.

According to the present invention, it was confirmed that substances contained in tryptanthrin and the green fraction (Fr. T2B-1) have an excellent antibacterial action against fungi. Accordingly, an antifungal agent containing these substances as an active ingredient, in particular, an antifungal agent against fungi belonging to the genus Malassezia such as Malassezia / fluflur, and various compositions containing them, in particular, pharmaceutical compositions, are included in the present invention. Provided by.

  In this specification, “indigo” is an annual plant (Polygonum tinctorium) belonging to the family Tadeidae, and is also called “Kai-in”. For the extraction with an organic solvent, the entire above-ground part of indigo or an appropriate part thereof, for example, a leaf, a stem, or the like can be appropriately used.

  The antibacterial active substance of the present invention can be obtained by extraction methods well known to those skilled in the art. For example, the extraction operation can be performed using various organic compounds appropriately selected from dichloromethane, methyl acetate, ethyl acetate and the like in consideration of extraction efficiency and the like, or a mixture of two or more thereof.

  That is, after extracting with stirring at an appropriate temperature (usually room temperature) and time (overnight to several days) using an organic solvent, insoluble components are removed by operations such as filtration, liquid separation, fractional precipitation, and centrifugation. The organic solvent is distilled off using an evaporator or the like, and then dried with a vacuum pump or the like. Thereafter, the antibacterial active substance of the present invention can be separated and purified by using various means well known to those skilled in the art such as liquid chromatography, thin layer chromatography, and crystallization. For example, silica gel can be used as the chromatography filler, and n-hexane, ethyl acetate, dichloromethane, diethyl ether, and a mixed solvent thereof can be used as the developing solvent.

As a result, at least two kinds of substances were found as antibacterial activity extraction components that are active ingredients of the antifungal agent of the present invention. One of them is a novel antibacterial active substance according to the first aspect of the present invention, which is specified by Rf value and NMR spectrum data obtained by thin layer chromatography as shown in the following examples.
Furthermore, another antibacterial active substance has been found to be Tryptanthrin as described in the examples. Note that tryptanthrin is a commercially available compound that can be easily obtained, and can also be synthesized by an appropriate method.

In addition to malassezia furfur, fungi belonging to the genus Malassezia include malassezia furfur, malassezia pachydermatis, malassezia, due to differences in lipid requirement, growth temperature, cell morphology, and rDNA base sequence.・ Restorica (M.
restrica), Malassezia globosa (M. globosa), Malassezia obtusa (M. obtusa), Malassezia thrufie (M.
slooffiae) and M. sympodialis, a total of seven types. The antifungal agent of the present invention exhibits an excellent antibacterial action against fungi, in particular, against fungi belonging to the genus Malassezia, more preferably against Malassezia fluflur.

  In the antifungal agent of the present invention, various adjuvants known to those skilled in the art, for example, solubilizers, stabilizers, moisturizers, preservatives, and the like may be combined. The content of the active ingredient in the preparation of these adjuvants is usually about 0.01 to 90% by weight, preferably about 0.01 to 50% by weight.

  The pharmaceutical composition of the present invention contains the antibacterial active substance or the antifungal agent.

  The pharmaceutical composition of the present invention has effects such as treatment, prevention, and / or suppression for skin diseases involving fungi belonging to the genus Malassezia. In this specification, “a skin disease in which a fungus is involved” means that the fungus is directly or indirectly responsible for the onset of the skin disease, or that the fungus is involved in the onset, exacerbation or worsening of the skin disease, etc. It broadly means skin diseases that have some relationship with fungi, and there is no particular limitation on the onset mechanism.

  Examples of typical diseases that are currently known as examples of “dermatological diseases involving fungi” include, for example, atopic dermatitis, seborrheic dermatitis, psoriasis, fungal otitis externa, folding screen, Malassezia folliculitis (Summer acne, acne) and the like. Therefore, the pharmaceutical composition of the present invention can exert an effect particularly as a therapeutic, preventive and / or suppressive pharmaceutical agent for these skin diseases. Furthermore, it is clear that the pharmaceutical composition of the present invention has an effect on skin diseases in which it has become clear that fungi are involved in the future.

  Furthermore, the pharmaceutical composition of the present invention includes other substances having antifungal activity known to those skilled in the art, for example, clotrimazole, miconazole, econazole, isoconazole, imidazole substances such as ketoconazole, bifonazole, fluconazole, and the like, and Various agents known to those skilled in the art such as antipruritics, anti-inflammatory, analgesic or local anesthetics, bactericides, keratin softening penetrants, astringents or restoratives can be appropriately blended.

  The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and can be used as usual, for example, tablets, capsules, solutions, aerosols, gels, creams, powders, ointments, vaginal suppositories, and the like. . It is preferably used in an external dosage form. The content of the active ingredient in these preparations is about 0.01 to 50% by weight, preferably about 0.01 to 10% by weight.

  The pharmaceutical composition of the present invention can be formulated into various dosage forms as described above by blending with various bases and carriers well known to those skilled in the art of formulation by various conventional methods.

  That is, for example, a liquid carrier composed of water, lower alcohol, ethylene glycol, glycerol, and other various organic solvents; for example, an aerosol carrier in which the above liquid carrier and heptafluoropropane gas are blended; A gel base comprising a combination of a liquid carrier and a gel forming agent as described above; for example, a combination of a carboxyvinyl polymer and a base such as an organic amine or aqueous sodium hydroxide, or a cellulose ether such as hydroxypropylcellulose, sodium alginate, Gel formers such as propylene glycol alginate; emulsifiers such as nonionic surfactants; for example, peanut oil, olive oil, sesame oil, coconut oil, paraffin oil, lanolin, petrolatum, zinc oxide, beeswax, macrogol, stearyl alcohol , Propi Ointment bases such as Nglycol; inert diluents such as calcium carbonate, calcium phosphate, lactose, starch, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, magnesium stearate, aluminum stearate, talc, silicone, Various bases and carriers such as granules, dispersants, and binders can be used alone or in combination.

  These various preparations can be produced in a conventional manner, for example, in the same manner as described in the Japanese Pharmacopoeia.

The pharmaceutical composition of the present invention can be applied to the affected area by oral administration, application to the skin, spraying to the mucous membrane, etc. depending on the dosage form, and the dosage of the active ingredient at that time is the agent It is appropriately selected depending on the type, the type of causative bacteria to be treated, symptoms, etc., but generally 0.01 to 500 mg / kg / day, preferably 1 to 50 mg / kg / day per adult. It is applied in the range.
Incidentally, triptans Surin, as shown in toxicity studies in mice as described in JP-B-62-59685, LD ₅₀ is known to be extremely low toxic substance exceeding 1000 mg / kg.

  Furthermore, the present invention relates to various antibacterial active compositions containing the antibacterial active substance or antifungal agent of the present invention in addition to the above pharmaceutical composition. There are no particular restrictions on the type and use of such antibacterial active compositions, but examples include hair growth / hair restorer, pomade, hair stick, hair oil, hair cream, hair solid, hair liquid, set lotion, hair styling gel, water grease, Hair blow, aerosol hair conditioner, permanent wave liquid, hair cosmetics such as hair dye, shampoo, body shampoo, hair rinse, hair soap, cosmetic soap, cleansing foam and other cosmetics, lotion, cream, milky lotion, pack , Foundation, lipstick, cheek, eyeliner, mascara, eye shadow, eyebrows, nail polish, nail polish, skin cosmetics, powder toothpaste, toothpaste, toothpaste, toothpaste, medicated toothpaste, mouth freshener, mouthwash, etc. Mouth cosmetics, sunscreen cosmetics, shaving Use cosmetics, bath cosmetics, and even more, perfume, eau de cologne, body odor stop, baby powder, eye lotion, such as bleaching cream and the like.

  In particular, since the growth of Malassezia flufr is a major cause of dandruff, for example, a hair washing composition (such as shampoo or hair rinse) for preventing dandruff is effective. Furthermore, the antimicrobial active composition used for various pets, such as a dog, is also within the scope of the present invention.

  The antibacterial active composition described above includes, for example, an oily base, an aqueous base, a flavoring agent, a colorant, a dye, a refreshing agent, a wetting agent, an emollient depending on the purpose and application. Agents, emulsifiers, gelling agents, thickeners, softeners, solubilizers, surfactants, foam stabilizers, clearing agents, antioxidants, overfat agents, preservatives, film forming agents, propellants, etc. Various ingredients commonly used in cosmetics, as well as vitamins, amino acids, peptides, extracts, vasodilators, blood circulation promoters, cell activators, bactericides, anti-inflammatory agents, antipruritics, astringents, skin function enhancers, keratin softening It can mix | blend with 1 type or multiple of chemical | medical agents, such as an agent.

  Depending on the usage form of the antibacterial active composition, it is prepared in the form of a solution, emulsion, cream, paste, powder, granule, or any other desired shape. Depending on the purpose of use, for example, in the case of cosmetics, the antibacterial active substance is usually contained in an amount of 0.001% by weight or more, preferably 0.05% by weight or more.

  The present invention also relates to various antibacterial active products treated with the antibacterial active substance or the antifungal agent. Representative examples of such antibacterial active products include, for example, clothing such as jeans, trainers, jerseys, pajamas, underwear, stockings and socks, baby products such as diapers and underwear, and care products, and towels, futons, and blankets. And products that may come into direct contact with the skin, such as bedding such as skin covers and sheets.

  These products can be treated with the antibacterial active substance or the antifungal agent by any method known to those skilled in the art depending on the properties of the material to be treated. For example, it is produced using a fiber that has been previously adhered or adsorbed by impregnation or the like, or a fiber dyed with a dye containing the antibacterial active substance or the antifungal agent, or It can be produced by applying or spraying the antibacterial active substance or the antifungal agent to the finished product.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto. In the examples, “%” means% by weight.

Extraction and isolation method of tryptanthrin and antibacterial active substance from indigo leaves 100g dry leaves of indigo (Polygonum tinctorium) cultivated at Hirosaki University, Faculty of Education 800 ml of dichloromethane (or methyl acetate or ethyl acetate) was added and stirred overnight with a stirrer under a nitrogen stream. The obtained mixed liquid was subjected to suction filtration, divided into a filtrate and a precipitate, and the filtrate was used as the first extract. This deposit
Place in a 1 liter eggplant-shaped flask, add 600 ml of dichloromethane (or methyl acetate or ethyl acetate), stir overnight with a stirrer, suction filter, separate into filtrate and precipitate. Was used as the second extract. The solvent in the first and second extraction liquids was distilled off with an evaporator and dried with a vacuum pump to obtain 2.0736 g of an organic solvent extraction fraction.

This fraction was dissolved in a small amount of dichloromethane and adsorbed onto a flash column packed with 176 g of silica gel (silica gel 60N, Cat. No. 37561-79, Kanto Chemical), and then 4: 1 of n-hexane and ethyl acetate was used. Elute with mixed solvent. Fractions eluting from 1,350 ml to 2,600 ml of developing solvent were collected, and the solvent was distilled off to obtain 99.3 mg of Fr.1. Fr.1 for separation thin layer chromatography plate (20 × 20 cm, silica gel 60F ₂₅₄
, Cat.No. 1.05715, Kanto Chemical Co., Ltd.) (fraction solvent, dichloromethane: ether: hexane = 40: 1: 2), fraction Fr.T1 (yellow, 13.8 mg) with Rf value = 0.5 And Rf value =
A fraction Fr. T2 (green, 37.2 mg) of 0.27 was obtained. Fr. T2 is a thin layer chromatography plate for separation (20 × 20 cm, silica gel 60F ₂₅₄ , Cat.
1.05715, Kanto Chemical Co., Ltd.) (developing solvent, dichloromethane: ether: hexane = 10: 1: 1), fraction Fr.T2A (8.9 mg) with Rf value = 0.75 and Rf value = 0.70 Fraction of
Fr.T2B (11.8 mg) and a fraction Fr.T2C (3.5 mg) having an Rf value = 0.57 were obtained.
In addition, the fraction
Fr. T2B (11.8 mg) was separated using a separation thin-layer chromatography plate (20 × 20 cm, silica gel 60F ₂₅₄ , Cat. No. 1.05715, Kanto Chemical) (developing solvent, dichloromethane: air) Tellurium: hexane = 10: 1: 1) to obtain Fr.T2B-1 (4.8 mg) as a green fraction (Rf value = 0.62).
This green fraction (Fr.T2B-1) has an analytical thin layer chromatography plate (2 × 5
cm, silica gel 60F ₂₅₄ , Cat. No. 1.05715, Kanto Chemical; developing solvent, dichloromethane: ether: hexane = 10: 1: 1). As a result of analysis and confirmation, Rf value = 0.65 (average value) Obtained.

The NMR data of Fr.T1 (apparatus used: JEOL Alpha400) is as shown below, which includes commercially available tryptanthrin (Wako Pure Chemicals) and literature (K. Seifert).
and W. Unger, Verl. Zeitsch. Naturforsch., 1994, 49, 44). The substance was identified as tryptanthrin because it was in complete agreement with the NMR of tryptanthrin described in 1994, 49, 44).
NMR data:
NMR (CDCl ₃ ) δ (ppm): 8,58 (1H, ddd, J = 8.1, 0.9, 0.6
Hz), 8.39 (1H, ddd, J = 7.9, 1.5, 0.4 Hz), 7.99 (1H, ddd, J = 8.1, 1.2, 0.4 Hz),
7.88 (1H, ddd, J = 7.6, 1.4, 0.6Hz), 7.82 (1H, ddd, J = 8.1, 7.3, 1.5Hz), 7.75 (1H, ddd, J = 8.1,
7.6, 1.4 Hz,), 7.63 (1H, ddd, J = 7.9, 7.3, 1.2 Hz), 7.39 (1H, ddd, J = 7.6, 7.6, 0.9 Hz).

Similarly, NMR of the substance contained in the green fraction (Fr. T2B-1) was also measured using an NMR apparatus (apparatus used: JEOL JNM270). The results are shown below.
NMR data:
NMR (CDCl3) δ (ppm):
11.16 (1H, s), 10.35 (1H, s), 9.65 (1H, s), 8.55 (2H, t),
8.40 (1H, d),
7.99 (2H, q), 7.90-7.57 (6H.m), 7.52 (1H, q), 7.38 (1H, t), 6.37 (1H, d), 6.22 (1H, t),
5.15 (1H, m), 4.47 (3H, m), 4.21 (3H, m), 4.06 (2H, q), 3.90 (3H, s), 3.67 (3H, s),
3.48 (1H, s), 3.37 (3H, s), 2.73-2.10 (3H, m), 1.94-1.75 (7H, m), 1.74-0.65 (67H, m).

Antibacterial test The following antibacterial test was carried out using Malassezia full full 48428 (IFO 0656) from the Institute for Fungal Medicine, Chiba University as a test bacterium.
Potato dextrose agar medium for passage of test bacteria 3.9%
Polyoxyethylene (20) sorbitan monooleate (Tween 80) 1.0%.
The test bacteria were scraped from this medium, suspended and diluted with sterilized physiological saline, and used to obtain an inoculum amount of about 10 ⁵ CFU / mL. In this case, the test bacteria were used after 5 days of subculture at 35 ° C.
Preparation of test substance miconazole nitrate: dissolved in dimethyl sulfoxide
Tryptanthrin: Dissolved in dimethyl sulfoxide Green fraction (Fr.T2B-1): Dissolved in dimethyl sulfoxide
Test medium Potato dextrose agar medium 7.8%
Polyoxyethylene (20) sorbitan monooleate (Tween 80) 2.0%
Anti-malassezia fluflur activity test method Sterile purified water was added to each test substance solution described above to prepare a stock solution, which was further adjusted to a predetermined concentration by two-fold serial dilution using sterile purified water. A plate was prepared by adding 0.3 mL of these dilutions and 0.3 mL of a test medium at about 50 ° C. to each hole of a 24-well sterile plate for tissue culture and mixing. After the surface of this medium is dried for 30 minutes, 10 μL of the inoculum is inoculated into each well, cultured at 35 ° C. for 7 days, and then the minimum growth inhibitory concentration (MIC: μg / mL) that inhibits the growth of the test bacteria by the test substance. ) The test results are shown in Table 1 below. From the following results, it was found that substances contained in tryptanthrin and the green fraction (Fr. T2B-1) have excellent antibacterial activity.

Therapeutic effect of tryptanthrin ointment on atopic dermatitis The tryptanthrin external preparation irritation test was performed as follows.
(1) Date and time of patch test by healthy volunteers: March 24 to 26, 2003 Objective: Tolerance of tryptanthrin in normal skin was examined by a skin test (patch test) in healthy subjects.
Target: Total number of healthy adults without skin disease: 10 Drugs used: Tryptanthrin ointment (1% and 0.1% tryptanthrin / hydrophilic ointment)
Method: 1% tryptanthrin ointment, 0.1% tryptanthrin ointment, and control hydrophilic ointment were applied to the normal dorsal skin surface, and 48 hours later, judgment was made based on International Contact Dermatitis Society Standard (ICDRG Standard) (Table 2) .

  Results: The judgment results are as shown in Table 3, and one person showed irritation symptoms that seemed to be due to the base material, but nine were negative. Indigo, which is the source of tryptanthrin, is also used for the purpose of antipyretic and detoxification as one of the traditional Chinese medicines. Tryptanthrin, which is a component, can be said to have excellent safety even when applied externally.

(2) Clinical effect Ointment topical application Tryptanthrin ointment (0.5%) is applied to the affected area twice a day. Observe once every 1 to 2 weeks (daily in case of hospitalization) and stop immediately if the external disease may exacerbate the primary disease or if dermatitis occurs due to external use.
2. Target cases The diagnosis of atopic dermatitis in 2 cases was performed according to the criteria for diagnosis of atopic dermatitis of the Japanese Dermatological Association. That is, pruritic eczema lesions that persist for more than 6 months, and related laboratory results (eosinophils, IgE, LDH, etc.).

Case 1
Patient: 15-year-old boy.
First visit: September 18, 2003
Family history: No special mention.
Past history: There is no special mention matter.
Current medical history: From 4 years before the first visit, he began to see urticaria-like eruptions throughout the body, and eczema lesions on the face, auricles and lower limbs. Itching is seen throughout the body and insomnia continues.
Symptoms: Whole body skin tends to dry, scratches, erythema and vesicles are scattered or gathered throughout the body except the face. Both ears have cracks, scales, and erosion. Nodules are scattered in the lower limbs and buttocks, and follicular papules and rash formation are observed.
Progress: Hospitalized for treatment from September 26 to October 10. Meanwhile, tryptanthrin ointment (auricle, buttocks) is applied. No symptoms of irritation or contact dermatitis were observed on the 14th day after tryptanthrin application at the time of discharge. The pinna disappeared and the cracks were light. In the buttocks, alleviation of dry symptoms, erythema and papules disappeared. Itching is also decreasing. The laboratory results show improvement in LDH and CRP, which are indicators of inflammation, as well as reduction in eosinophils (normalization) and inflammation. The liver function was not affected (Table 4).

Case 2
Patient: 30 years old, male.
First visit: August 25, 2003
Family history: No special mention.
Past history: There is no special mention matter.
History of present illness: Pruritic rash appeared on the trunk from around the age of 15. It gradually expanded to the whole body. He had been treated with topical steroids at an acupuncture clinic, but was worried about the side effects of steroid therapy and visited a university hospital.
Onset: The whole body skin is highly dry, scales, and pigmented. Eczema lesions including the face, follicular papules, and scratch marks are conspicuous.
Progress: From September 4th to September 7th, hospitalized for the purpose of educating atopic dermatitis. Tryptanthrin ointment is externally applied to the trunk rash seen mainly in the shoulders. At the time of discharge, on the 4th day after application of tryptanthrin, there was no irritation symptom and the course was generally good. However, there were signs of hygiene (erythema, small blisters) in the restraint zone used for the purpose of suppressing scratching. Test results before hospitalization showed high levels of eosinophils and LDH, which was beneficial for the diagnosis of atopic dermatitis (Table 4). Subsequent follow-up will be followed in the outpatient department.

  In addition, Malassezia fungus is a skin resident fungus, and in the case of adult atopic dermatitis, 80% of patients have Malassezia fungus in the tape stripping test method, and scales and follicular papules are frequent. Therefore, the atopic dermatitis of the above case 1 and case 2 was determined to be a skin disease in which malassezia fungi are involved as a causative agent.

(3) Judgment of clinical effect of tryptanthrin In case 1, the clear infectious lesion was relieved, and the pruritus sensation was reduced accordingly. In addition, improvement in test results, which is an index of inflammation, was observed, and tryptanthrin ointment was determined to be effective. In case 2, although sufficient progress of treatment was not observed, dermatitis and dry tendency at the application site were relieved, and continuation was judged to be beneficial.

Effect of tryptanthrin on other skin diseases Malassezia folliculitis
Case 3
Patient: 22 years old, male.
First visit: February 13, 2003
Family history: No special mention.
Past history: Left sternoclavicular arthritis.
Current medical history: Since several days before the first visit, there have been frequent eruptions on the face and trunk that are free of autogenous diseases. Steroids (30 mg / day) have been administered systemically since January for the above diseases.
Onset: Many red papules occur on both cheeks on the face and on the front chest and upper back on the trunk, matching the pores.
Course: Malassezia folliculitis due to steroid administration was diagnosed. As a result of applying tryptanthrin ointment (0.5%) from the first visit, red papules almost disappeared as of March 3. There are no signs of brooding due to external use. After stopping the topical application, the eruption showed a tendency to relapse, but when it was used again, the red papule disappeared again. There is no blood test.

2. Screen (Pityriasis versicolor)
Case 4
Patient: 25 years old, female.
First visit: March 25, 2003
Family history: No special mention.
Past history: There is no special mention matter.
Current medical history: From 2 weeks ago, red spots appeared in the upper right limb, with mild pruritus. The acupuncturist treated with topical steroids but did not improve.
Present symptom: Money-sized red spots appear on the right upper limb, papules and scales appear on the margin. Similar skin eruptions are not observed at other sites.
Progress: Clinical image, microscopic examination (caustic specimen) showed malassezia, diagnosed as folding screen, and applied tryptanthrin ointment (0.5%) from the same day. As a result, erythema and papules disappeared rapidly, scales disappeared, and healed in about one week. There are no signs of brooding due to external use.

3. Determination of the clinical effect of tryptanthrin The skin lesions in cases 3 and 4 are caused directly by Malassezia. In case 3, it was difficult to see the positive findings of malassezia by microscopic examination in malassezia folliculitis, but we could diagnose malassezia folliculitis due to steroid administration from the common site and clinical features. After applying tryptanthrin ointment, tryptanthrin ointment was considered effective because of its rapid disappearance and disappearance after re-administration. Case 4 can be diagnosed as a folding screen in which topical use of steroids was an exacerbation factor. Because of the rapid disappearance, tryptanthrin ointment was considered effective against folding screens.

Claims (10)

The following steps:
(A) a step of obtaining an organic solvent soluble fraction from the dried green grass,
(B) The fraction obtained in step (a) was adsorbed on a flash column packed with silica gel and then eluted with a 4: 1 mixed solvent of n-hexane and ethyl acetate, and the solvent was distilled off. The obtained fraction was separated by thin layer chromatography for separation on a silica gel plate using dichloromethane: ether: hexane (40: 1: 2) as a developing solvent, and a fraction having an R f value = 0.27 was obtained. Obtaining step,
(C) The fraction obtained in step (b) is separated by thin layer chromatography for separation on a silica gel plate using dichloromethane: ether: hexane (10: 1: 1) as a developing solvent. The step of obtaining a fraction having an R f value of 0.62 to 0.70, and the fraction obtained in (d) step (c) were used dichloromethane: ether: hexane (10: 1: 1) as a developing solvent. Separating by analytical thin layer chromatography on a silica gel plate to obtain a fraction with R f value = 0.65 (average value);
An antifungal agent comprising , as an active ingredient , a substance having the following physicochemical properties prepared in :
(1) NMR data:
NMR (CDC l 3) δ (ppm):
1 1. 1 6 (1 H, s), 1 0. 3 5 (1 H, s), 9.6 5 (1 H, s), 8.5 5 (2 H, t), 8. 40 (1 H, d), 7. 9 9 (2 H, q), 7. 9 0-7. 5 7 (6 H. M), 7.5 2 (1 H, q), 7. 3 8 ( 1 H, t), 6. 3 7 (1 H, d), 6.2 2 (1 H, t), 5. 1 5 (1 H, m), 4.4 7 (3 H, m), 4.2 1 (3 H, m), 4.0 6 (2 H, q), 3.90 (3 H, s), 3.6 7 (3 H, s), 3.4 8 (1 H, s), 3.3 7 (3 H, s), 2. 7 3-2.10 0 (3 H, m), 1.9 4-1.75 5 (7 H, m), 1. 7 4-0.65 (6 7 H, m).
The antifungal agent according to claim 1, wherein the organic solvent is dichloromethane. The antifungal agent according to claim 2, against a fungus belonging to the genus Malassezia. The antifungal agent according to claim 3, wherein the fungus belonging to the genus Malassezia is malassezia furfur. A pharmaceutical composition for a skin disease involving a fungus belonging to the genus Malassezia, comprising the antifungal agent according to any one of claims 1 to 4. The pharmaceutical composition according to claim 5, wherein the skin disease is atopic dermatitis. The pharmaceutical composition according to claim 5, wherein the skin disease is malassezia folliculitis. The pharmaceutical composition according to claim 5, wherein the skin disease is folding screen. The antibacterial active composition containing the antifungal agent as described in any one of Claims 1-4. The antibacterial active product processed with the antifungal agent as described in any one of Claims 1-4.