Method of producing iturin A and antifungal agent for profound mycosis

The present invention is a method of producing iturin A, in which cells of Bacillus amyloliquefaciens are incubated and iturin A is collected from the culture. It also relates to an antifungal agent for profound mycosis, containing iturin A as the active ingredient. Heretofore, there have been known few medicines effective against profound mycosis, but iturin A may be an effective antifungal agent for profound mycosis.

DETAILED EXPLANATION OF THE INVENTION 
1. Industrial Field of the Invention 
The present invention relates to a method of producing iturin A and to an 
antifungal agent for profound mycosis comprising iturin A as the active 
ingredient. 
2. Prior Art 
Heretofore, infectious diseases to be caused by fungi such as mold, yeast 
and others have been essentially in local infection in the skin, 
respiratory organs, vagina and others, but recently, these tend to 
increase also in systemic infection. In particular, systemic infectious 
diseases such as profound infectious diseases to be caused by depression 
of immunological competence resulting from use of immunosuppressive 
agents, carcinostatics and others are increasing. Many cases of fatal 
serious infectious diseases have been reported in the clinical medicine 
(Nippon Byori Gakkai Kaishi, 74, 61 (1985)). However, as compared with 
noticeable progress of the chemotherapy for infectious diseases to be 
caused by bacteria, it must be said that the chemotherapy for infectious 
diseases to be caused by fungi is belated at present. One reason is that 
bacteria are prokaryocytes, being different from animal cells of 
eukaryocytes with respect to the cell background, and preparation of 
medicines having selective toxicity to them is easy; while fungi which are 
composed of eukaryocytes like animal cells are hardly differentiated from 
animal cells because of the similar cell background of them so that 
elevation of the selective toxicity specific only to fungi but not to 
animal cells is not easy. 
At present, only amphotericin B is known as a medicine effectively 
applicable to profound mycosis such as candidiasis, aspergillosis, 
cryptococcosis, mucormycosis, coccidioidomycosis, paracoccidioidomycosis, 
blastomycosis, histoplasmasis, sporotrichosis, etc. (Annu. Rev. 
Pharmacol., Toxicol., 23, 303 (1983)). 
PROBLEMS TO BE SOLVED BY THE INVENTION 
The object of the present invention is to provide a method of producing 
known iturin A and to provide an antifungal agent for profound mycosis 
which comprises iturin A as the active ingredient and which is useful as a 
medicine for preventing and curing profound, fungal infectious diseases.

MEANS FOR SOLVING THE PROBLEMS 
The present inventors broadly searched natural substances, especially 
microbial metabolites for antifungal substances and, as a result, have 
found that certain bacteria belonging to the genus Bacillus produce a 
substance which is effective against fungi in their cultures. They 
examined the microbiological properties of the strain, which was 
identified to be Bacillus amyloliquefaciens. This was deposited in the 
National Institute of Bioscience and Human-Technology, Agency of 
Industrial Science and Technology in Japan, as Bacillus amyloliquefaciens 
HSCC 124 (FERM BP-4758). 
The following Table 1 shows the microbiological properties of Bacillus 
amyloliquefaciens HSCC 124. 
TABLE 1 
______________________________________ 
Microbiological Properties of Bacillus 
amyloliquefaciens HSCC 124 
______________________________________ 
(A) Morphological Properties (in broth-agar medium): 
(1) Cell Size: 
3.mu. to 5.mu. .times. 0.5.mu. to 0.9.mu. 
(2) Cell Shape: 
bacillus form 
(3) Polymorphism 
Negative 
of Cell: 
(4) Motility: 
Positive (as peritrichous 
flagella) 
(5) Sporulation: 
Positive 
(6) Gram Stain: 
Positive after incubation for 
12 to 48 hours 
(B) Growth in Media: 
(1) Broth-agar 
The cells grew well 
Plate Culture: 
to form circular, flat yellowish-gray 
colonies, having endogenous gloss. 
(2) Broth-agar 
The cells grew well 
Slant Culture: 
to form spread cloth-like, yellowish-gray 
colonies, having endogenous gloss. 
(3) Broth Liquid 
The culture became slightly cloudy. 
Culture: The cells grew on the surface of the 
liquid culture to form film-like colonies. 
Precipitate formed in the culture was 
infinitesimal. 
(4) Decomposition 
+ 
of Gelatin: 
(5) Litmus Milk: 
The grown cells reduced litmus 
and decomposed milk. 
(C) Physiological Properties: 
(1) Reduction 
- 
of Nitrates: 
(2) MR Test: 
- 
(3) V-P Test 
+ 
(pH 5.5): 
(4) Formation of 
- 
Indole: 
(5) Hydrolysis of 
+ 
Starch: 
(6) Utilization of 
Citric Acid, 
Koser: + 
Christensen: 
+ 
(7) Utilization of 
Inorganic 
Nitrogen Sources, 
NO.sub.3 : - 
NH.sub.4 : + 
(8) Formation of 
- 
Dye: 
(9) Urease: 
- 
(10) Oxidase: 
+ 
(11) Catalase: 
+ 
(12) Range for 
Growth: 
pH for Growth: 
5 to 9 
Optimum 30.degree. C. 
Temperature: 
(13) Influence by 
aerobic 
Oxygen: 
(14) Formation of Acid and Gas from Saccharides: 
Acid Gas 
L-arabinose +/- - 
D-xylose + - 
D-glucose + - 
D-mannose + - 
D-fructose +/- - 
D-galactose - - 
Maltose +/- - 
Sucrose + - 
Lactose +/- - 
Trehalose + - 
D-sorbitol + - 
D-mannitol + - 
Inositol - - 
Glycerin +/- - 
Starch + - 
(15) NaCl Tolerance: 
NaCl 5%: + 
NaCl 7%: + 
(D) Chemotaxonomical Properties: 
(1) Base Composi- 
46.4 mol % 
tion of DNA 
(GC content): 
(2) DNA-DNA 
81% 
Homology with B. 
amyloliquefaciens 
DSM 7: 
______________________________________ 
Based on these findings, the present inventors further searched various 
strains of Bacillus amyloliquefaciens stored in the microorganisms-storing 
organ for those producing antifungal substances and found that the cells 
of Bacillus amyloliquefaciens IAM 1523 also produce the same antifungal 
substance in their culture. They isolated the antifungal substance from 
the cultures of Bacillus amyloliquefaciens HSCC 124 and Bacillus 
amyloliquefaciens IAM 1523 and identified its structure. As a result, they 
found that the substance was the same as iturin A that had been identified 
to be a mixture comprising 8 peptide compounds (Tetrahedron Lett., 23, 
3065-3068 (1982)). Iturin A is a substance that was found and identified 
by L. Delcambe et al. (C. R. Soc. Biol., 144, 1431-1434 (1950)), and there 
are known some examples demonstrating the production of iturin A by 
Bacillus subtilis. However, there is known no example demonstrating the 
production of the same by Bacillus amyloliquefaciens, and it is unknown 
that Bacillus amyloliquefaciens may produce iturin A. Specifically, the 
present invention relates to a method of producing iturin A by incubating 
Bacillus amyloliquefaciens and collecting iturin A from its culture. 
It is known that iturin A has an antifungal activity against various fungi 
(ARCH. BELGES. DERM. ET. SYPH., 14, 63-82 (1958)). The present inventors 
investigated the effectiveness of iturin A against fungi to cause profound 
mycosis which has been considered problematic in these days and have found 
that iturin A is effective against profound mycosis. Heretofore, there has 
been known no report suggesting the effectiveness of iturin A against 
profound mycosis. The present inventors are the first who have completed 
the antifungal agent for profound mycosis, comprising iturin A as the 
active ingredient, on the basis of the effectiveness of iturin A against 
profound mycosis. 
Accordingly, the present invention is a method of producing iturin A by 
incubating Bacillus amyloliquefaciens and collecting iturin A from its 
culture. In addition, it is also an antifungal agent for profound mycosis, 
comprising iturin A as the active ingredient. The active ingredient to be 
in the antifungal agent for profound mycosis of the present invention may 
be any of iturin A, preferably including iturin A2 to iturin A8. 
According to the method of the present invention for producing iturin A, 
any of strains of iturin A-producing Bacillus amyloliquefaciens, their 
variants or mutants is inoculated in a nutrient medium containing carbon 
sources and nitrogen sources and incubated therein under aerobic 
conditions (for example, by shaking culture, aerial stirring culture, 
etc.) whereby iturin A is produced in the culture. 
Any of carbon sources that may be assimilated by iturin A-producing 
microbes may be employed in the present invention. Preferred are glucose, 
sucrose, starch, fructose, glycerin and other carbohydrates. 
Like carbon sources, any of nitrogen sources that may be assimilated by 
iturin A-producing microbes may be employed in the present invention. 
Preferred are oat meal, yeast extract, peptone, gluten meal, cotton seed 
meal, soybean meal, corn steep liquor, dry yeast, wheat germ, peanut meal, 
chicken bone meal and others. Also advantageously usable are inorganic and 
organic nitrogen compounds, for example, ammonium salts (such as ammonium 
nitrate, ammonium sulfate, ammonium phosphate and others), urea, amino 
acids and others. Use of combinations of such carbon sources and nitrogen 
sources is advantageous, but it is not always necessary to use only pure 
ones. This is because impure ones often contain growth factors or micro 
nutrients. 
If desired, inorganic salts such as those mentioned below may be added to 
the medium. They are, for example, sodium carbonate, potassium carbonate, 
sodium phosphate, potassium phosphate, sodium chloride, potassium 
chloride, sodium iodide, potassium iodide, magnesium salts, copper salts, 
cobalt salts and others. 
In particular, where the medium is a strongly foaming one, liquid paraffin, 
animal oils, vegetable oils, mineral oils, silicones, higher alcohols and 
the like may be added thereto, if desired. 
For industrial mass-production of the intended product, the microorganisms 
are preferably incubated by aerial stirring culture like the case of 
producing other fermentation products. Where a small amount of the 
intended product is produced, they are preferably incubated by shaking 
culture in a flask. 
Where the incubation is effected in a large-size tank, it is preferred that 
the iturin A-producing microorganisms are first inoculated and cultivated 
in a relatively small amount of a medium and thereafter the resulting 
culture is transferred to a large-size production tank in which the 
previously grown microorganisms are further incubated to produce the 
intended product, for the purpose of preventing their delay in growing and 
producing iturin A. In this case, the composition of the medium to be used 
in the preculture and that of the medium to be used for the latter 
incubation to produce the intended product, iturin A may be the same one, 
or if desired, they may be different from each other. 
The incubation is preferably effected under the condition of aerial 
stirring. For example, any known method, such as stirring culture with 
propellers or other mechanical stirrers, rotary or shaking culture in a 
fermenter, pumping or air-blowing culture or the like, may suitably be 
employed. Air to be used for aeration of the culture system is desired to 
be previously sterilized. 
The incubation temperature may suitably be varied within the range where 
the iturin A-producing microorganisms may produce the present substance, 
iturin A. In general, the incubation may be effected at 10.degree. to 
40.degree. C., preferably 25.degree. to 35.degree. C. The incubation time 
varies, depending on the incubation condition and the amount for 
fermentation, and it is generally approximately from one day to one week. 
After completion of the fermentation, the culture obtained is recovered as 
iturin A, which is the active ingredient to be in the antifungal agent for 
profound mycosis of the present invention. The iturin A to be in the 
antifungal agent for profound mycosis of the present invention is purified 
by known methods to a pure one that is acceptable as a medicine. To obtain 
a pure product from the culture, for example, when iturin A is in the 
cells in the culture, the cells are directly extracted with water and/or 
organic solvent(s) or, alternatively, these are disrupted mechanically or 
ultrasonically or by any other known means and thereafter extracted with 
water and/or organic solvent(s). Then, the product is recovered and 
purified by ordinary methods. When iturin A is in the culture, the culture 
may directly be extracted with solvent(s) or, alternatively, it may be 
filtered through a filter membrane or the like. If desired, the culture 
may be brought into contact with an active charcoal, powdery cellulose, 
adsorbing resin or the like carrier so that the produced iturin A is 
adsorbed to the carrier, and thereafter the product may be desorbed 
therefrom by elution. 
For further purifying the product, any ordinary recovering and purifying 
method for harvesting antibiotics may be employed. For instance, usable 
are solvent extraction with water, organic solvents or mixed solvents of 
them; chromatography; recrystallization from a single solvent or a mixed 
solvent; and other ordinary means. These may be employed singly or in 
combination of them. 
Iturin A which is to be in the antifungal agent for profound mycosis of the 
present invention, as the active ingredient, may be produced by the 
above-mentioned microorganisms, and it may be a purified one. Apart from 
this, however, iturin A produced by any other iturin A-producing microbes 
as well as iturin A chemically synthesized may also be employed in the 
present invention. 
To use iturin A as an antifungal agent for profound mycosis according to 
the present invention, iturin A may be used as it is or, alternatively, it 
may be blended with a pharmaceutically-acceptable, non-toxic and inert 
carrier to form a drug composition containing, for example, from 0.1% to 
99.5%, preferably from 0.5% to 90% of iturin A. Iturin A or the drug 
composition containing iturin A is administered to animals including human 
beings. 
As the carrier, usable are one or more solid, semi-solid or liquid diluting 
agents, fillers and other ordinary drug additives. The drug composition is 
desired to be administered as its dose unit. The drug composition of the 
present invention may be administered by oral administration, tissue 
administration, local administration (such as endermic administration, 
etc.) or rectal administration. It may also be used for external 
application. Needless to say, the drug composition is administered as a 
form suitable to the administration route. 
It is desired that the amount of the antifungal agent of the present 
invention to be actually used is adjusted in consideration of the 
condition of the patient, such as its age or body weight, the 
administration route, the condition and degree of the disease and others. 
In general, the amount to be administered to one adult is from 10 to 2000 
mg a day, as the active ingredient of the present invention. As the case 
may be, the necessary amount may be smaller than the range or may be 
larger than the same. If a large amount of the medicine is administered, 
it is desired that the total amount thereof a day is divided into several 
parts for separate administration in one day. 
Oral administration of the medicine may be effected, using its solid or 
liquid dose unit of, for example, powder, powder mixture, tablets, 
dragees, capsules, drops, sublingual tablets and other preparation forms. 
Powder of the medicine may be produced by grinding the active substance 
into a desired fineness. Powder mixture of it may be produced by grinding 
the active substance into a desired fineness followed by blending the 
resulting powder with a fine powdery drug carrier, such as a fine powdery 
edible carbohydrate of, for example, starch, mannitol or the like. If 
desired, a flavoring, a preservative, a dispersing agent, a colorant, a 
perfume and other additives may be added to the preparation. 
Capsules of the medicine may be produced by encapsulating powder, powder 
mixture or granules of the medicine with a capsule coat such as a gelatin 
capsule coat. Prior to the encapsulation, a lubricant or fluidizing agent, 
such as colloidal silica, talc, magnesium stearate, calcium stearate, 
solid polyethylene glycol or the like powdery substance may be 
incorporated into the powdery or granular medicine. Addition of a 
disintegrator or a solubilizer, for example, carboxymethyl cellulose, 
calcium carboxymethyl cellulose, hydroxypropyl cellulose of low 
substitution degree, calcium carbonate or sodium carbonate, to the 
medicine preparation to be encapsulated is recommended so as to elevate 
the effectiveness of the medicine when the capsules have been ingested. 
If desired, the fine powdery medicine of the present invention may be 
suspended and dispersed in a vegetable oil along With polyethylene glycol, 
glycerin and a surfactant and the resulting dispersion may be wrapped with 
a gelatin sheet to give soft capsules. 
Tablets of the medicine may be produced by granulating or slagging the 
active substance-containing powder mixture followed by adding a 
disintegrator or lubricant thereto and forming the resulting blend into 
tablets. 
The powder mixture comprises a suitably powdered active substance and the 
above-mentioned diluting agent and base and/or may optionally contain a 
binder (for example, sodium carboxymethyl cellulose, alginates, gelatin, 
polyvinyl pyrrolidone, polyvinyl alcohol, etc), a slow-releasing agent 
(for example, paraffin, etc.), a reabsorbing agent (for example, 
quaternary salts) and/or an adsorbent (for example, bentonite, kaolin, 
dicalcium phosphate, etc.). The powder mixture may be wetted with a binder 
such as syrup, starch paste, gum arabic, cellulose solution or polymer 
substance solution and then forcedly sieved to give granules. In place of 
forming granules from the powder in this manner, the powder may directly 
be processed with a tabletting machine and thereafter the resulting slugs 
of an incomplete form may be pulverized to granules. 
A lubricant such as stearic acid, stearates, talc, mineral oils and others 
may be added to the granules to be obtained in this manner, so as to 
prevent their adhesion to each other. The lubricant-containing mixture is 
then formed into tablets. If desired, the active substance may directly be 
formed into tablets, after having been combined with a fluid inactive 
carrier, without taking the above-mentioned granulating or slagging step. 
As the case may be, a transparent or semi-transparent protective film 
composed of a shellac sealant film, or a sugar or polymer material film, 
or a waxy lubricant top coat film may be employed for overcoating the 
tablets. 
Other oral preparations, such as solution, syrup, elixir and the like may 
also be produced in the unit dose form of containing a determined amount 
of the active substance. Syrup may be produced by dissolving the active 
compound in a suitable aromatizing aqueous solution; and elixir may be 
produced by blending it with a non-toxic alcoholic carrier. Suspension may 
be formulated by dispersing the active compound in a non-toxic carrier. A 
solubilizer and an emulsifier (for example, ethoxylated isostearyl 
alcohols, polyoxyethylene sorbitol esters), a preservative, an aromatizing 
agent (such as peppermint oil, saccharin) and others may optionally be 
added to them. 
If desired, the unit dose formulation for peroral administration may be 
encapsulated into microcapsules. The formulation may be coated with a 
coating film or may be embedded into polymer substances, wax or the like 
so as to prolong the active life of the active substance or to attain slow 
release of the active substance. 
Parenteral administration may be effected, using a liquid dose unit form 
of, for example, a solution or suspension for subcutaneous, intramuscular 
or intravenous injection. This may be prepared by dissolving or suspending 
a determined amount of the active compound in a non-toxic liquid carrier 
suitable for injection of, for example, an aqueous or oily medium followed 
by sterilizing the resulting solution or suspension. Alternatively, a 
determined amount of the active compound may be put in a vial and 
thereafter the vial may be sterilized along with the content and then 
sealed. For dissolution or mixing of the powdery or freeze-dried active 
compound just before its administration, a preparatory vial or carrier may 
be prepared along with it. For making the injection containing the active 
compound isotonic, a non-toxic salt or salt solution may be added thereto. 
If desired, a stabilizer, a preservative, an emulsifier and the like may 
also be added thereto. 
Rectal administration may be effected, using a suppository composed of the 
active compound and a low melting point solid of, for example, 
polyethylene glycol, cacao butter, higher esters (e.g., myristyl 
palmitate) or a mixture of them. 
As other preparations for parenteral administration than the 
above-mentioned injection and suppository, which are employable in the 
present invention, mentioned are intravenous drip, liquid preparation for 
transfusion, ointment, lotion, tonic, spray, suspension, oil, emulsion, 
etc. These may also be formulated by ordinary methods. 
The effectiveness of the antifungal agent for profound mycosis of the 
present invention as a medicine may be identified by various tests. 
The microbicidal spectrum of iturin A, which is the active ingredient in 
the antifungal agent for profound mycosis of the present invention, may be 
obtained, for example, from the minimum growth inhibiting concentration 
(MIC) of iturin A to various fungi such as clinically-isolated strains, 
animal-infected fungi and others to be obtained on the basis of the method 
as designated by Japan Chemotherapy Association. 
Identification of the safety of iturin A may be effected by examining its 
toxicity, if any. For instance, employable for this purpose are a 
cytotoxicity test using animal cells and an acute toxicity test (oral 
single administration, intravenous single injection, intravenous repeated 
injection) using mice. 
As one example of the test of testing the effectiveness of the active 
substance, iturin A, it may be administered to systemically fungi-infected 
mice, to which various clinically-isolated strains (Candida albicans IFM 
40009, Aspergillus fumigatus Tsukuba No. 12, Cryptococcus neoformans 145A) 
were intravenously inoculated, whereby the in-vivo effectiveness of the 
administered substance may be examined. Of iturin A shown in Table 2 
below, iturin A2 and iturin A4 were tested in said manner, and the test 
results obtained are shown in Tables 3 to 5 below. 
TABLE 2 
______________________________________ 
##STR1## 
Name of Compound R 
______________________________________ 
A2 CH.sub.3 CH.sub.2 CH.sub.2 
A3 
##STR2## 
A4 (CH.sub.3).sub.2 CHCH.sub.2 
A5 CH.sub.3 CH.sub.2 CH.sub.2 CH.sub.2 
A6 (CH.sub.3).sub.2 CHCH.sub.2 CH.sub.2 
A7 CH.sub.3 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 
A8 
##STR3## 
______________________________________ 
TABLE 3 
__________________________________________________________________________ 
Number of Living Mice as Infected with C. albicans IFM40009 
Test Period (day) 
1 2 3 4 5 6 7 8 9 10 
11 
12 
13 
14 
15 
__________________________________________________________________________ 
Control 
10 
10 
4 2 2 2 2 2 1 1 1 0 0 0 0 
Iturin A2 
10 
10 
5 5 5 5 3 3 3 2 1 1 0 0 0 
60 mg/kg 
Iturin A4 
10 
10 
10 10 9 9 8 5 5 5 5 4 3 3 3 
60 mg/kg 
__________________________________________________________________________ 
TABLE 4 
__________________________________________________________________________ 
Number of Living Mice as Infected with A. fumigatus Tsukuba No. 12 
Test Period (day) 
1 2 3 4 5 6 7 8 9 10 
11 
12 
13 
14 
15 
__________________________________________________________________________ 
Control 
10 
10 
10 
10 
10 
9 
8 6 4 3 3 3 3 3 3 
Iturin A2 
10 
10 
10 
10 
10 
10 
9 7 5 4 4 3 3 3 3 
25 mg/kg 
Iturin A4 
10 
10 
10 
10 
10 
10 
10 10 10 9 9 9 9 9 9 
25 mg/kg 
__________________________________________________________________________ 
TABLE 5 
__________________________________________________________________________ 
Number of Living Mice as Infected with C. neoformans 145A 
Test Period (day) 
1 2 3 4 5 6 7 8 9 10 
11 12 
13 
14 
15 
__________________________________________________________________________ 
Control 
10 
10 
4 4 4 4 4 4 4 2 0 0 0 0 0 
Iturin A2 
10 
10 
8 8 8 7 7 7 5 3 2 1 1 1 1 
10 mg/kg 
Iturin A4 
10 
10 
6 5 5 5 5 5 5 5 3 1 0 0 0 
10 mg/kg 
__________________________________________________________________________ 
From the results, it is understood that iturin A to be in the antifungal 
agent for profound mycosis of the present invention has a selective 
toxicity to fungi and strongly affects various pathogenic fungi, that its 
toxicity is extremely weak and that it is effective against systemic 
mycosis in mice. Therefore, it has strongly been suggested that the 
antifungal agent for profound mycosis of the present invention is 
effective as a remedy for various fungi-infected diseases such as 
candidiasis, aspergillosis, cryptococcosis, etc. 
Next, the present invention will be explained in more detail by means of 
the following examples, which, however, are illustrative ones and are not 
intended to restrict the scope of the present invention. 
EXAMPLE 1 
As the seed medium, used was a medium comprising 2% of glucose, 0.5% of 
peptone, 0.1% of yeast extract, 0.01% of CaCO.sub.3, and 0.01% of NaCl. As 
the production medium, used was a medium comprising 2% of fructose, 1% of 
peptone, 0.1% of yeast extract, 0.01% of CaCO.sub.3, and 0.01% of NaCl. 
Both the two media were adjusted to have pH of 7.2 prior to their 
sterilization. 150 ml of the seed medium were put in a 500 ml-Erlenmeyer 
flask, which were sterilized at 120.degree. C. for 15 minutes and then 
cooled. To this, inoculated were from 2 to 3 platinum loops of a slant 
agar culture of Bacillus amyloliquefaciens HSCC 124 (FERM BP-4758) and 
cultivated at 30.degree. C. for 12 hours by shaking culture. Thus obtained 
was a seed culture of the strain. Next, 15 liters of the above-mentioned 
production medium were put in a 30 liter-jar fermenter, to which added 
were 10 ml of polypropylene glycol. This was sterilized at 120.degree. C. 
for 15 minutes and then cooled. To this, inoculated were 150 ml of the 
above-mentioned seed culture and incubated therein at 30.degree. C., at an 
aerating rate of 0.5 vvm and at a stirring rate of 150 rpm, for 12 hours. 
Then, 30 ml of polypropylene glycol were added thereto and the incubation 
was continued for further 24 hours. 
After the incubation, 15 liters of the culture were filtered through a 0.45 
.mu.m-filter membrane, by which a mixture comprising the cells and iturin 
A was collected on the membrane. The mixture was then fully washed with 
water to remove impurities therefrom. Next, to the fraction comprising the 
cells and iturin A, that had remained on the membrane, added was 
isopropanol of from 40% to 50%, by which a fraction containing iturin A 
was extracted. Thus, the extract was obtained as the filtrate fraction 
through the 0.45 .mu.m-filter membrane. 3 liters of the filtrate fraction 
were fractionated through an ultrafilter membrane having a fractionating 
molecular weight of 3000 to obtain a filtrate fraction passed 
therethrough, from which impurities were removed. Thus, 2.5 liters of an 
iturin A-containing liquid were obtained. The thus-fractionated, iturin 
A-containing fraction was passed through a column filled with 1.9 liters 
of ODS-C18 (made by Sohken Kagaku KK) and adsorbed to the adsorbent. The 
column was washed with 5 liters of 25%-acetonitrile and then eluted with 
40%-acetonitrile, by which seven peaks were separated. FIG. 1 shows the 
elution pattern. 
After having measured its biological activity, each active fraction was 
collected and concentrated under reduced pressure. Thus, seven fractions 
of iturin A were obtained, which were iturin A2 to iturin A8 shown in 
Table 2 above. Their yields were 300 mg, 75 mg, 120 mg, 12 mg, 40 mg, 40 
mg and 10 mg, respectively. 
EXAMPLE 2 
Bacillus amyloliquefaciens IAM 1523 which is an iturin A-producing strain 
was incubated in the same manner as in Example 1. Its culture was purified 
also in the same manner as in Example 1 and subjected to high performance 
liquid chromatography to separate into three peaks. FIG. 2 shows the 
elution pattern. After having measured its biological activity, each 
active fraction was collected and concentrated under reduced pressure. 
Thus, three fractions of iturin A were obtained, which were iturin A2 to 
iturin A4 shown in Table 2 above. Their yields were 20 mg, 80 mg and 50 
mg, respectively. 
EXAMPLE 3 
The antifungal activity of iturin A as produced in Example 1 was measured 
to identify its antifungal activity. Precisely, the minimum growth 
inhibiting concentration (MIC) of iturin A4 against fungi was measured on 
the basis of the method as designated by Japan Chemotherapy Association, 
and the results shown in Table 6 below were obtained. From these results, 
it has been verified that iturin A is strongly effective against various 
pathogenic fungi. 
In addition, MIC of each of iturin A2 to iturin A8 against Candida albicans 
7N was measured in the same manner as above, and the results shown in 
Table 7 below were obtained. 
TABLE 6 
______________________________________ 
Antifungal Activity of Iturin A 
MIC (.mu.g/ml) 
Tested Fungi of Iturin A4 
______________________________________ 
Candida albicans 
IFM 40001 &lt;0.05 
IFM 40002 1.56 
7N 25 
C. krusei IFM 40019 25 
C. tropicalis IFM 40018 6.25 
C. glabrata IFM 40065 6.25 
Cryptococcus neoformans 
IFM 40038 3.13 
IFM 40043 0.78 
Aspergillus fumigatus 
Tsukuba No. 12 
6.25 
A. flavas IAM 2044 2.4 
Mucor guilliermondii 
IFO 9403 1.2 
______________________________________ 
TABLE 7 
______________________________________ 
Antifungal Activity (MIC) of Iturin A2 to 
Iturin A8 against Candida albicans 7N 
Compound MIC (.mu.g/ml) 
______________________________________ 
Iturin A2 50 
Iturin A3 50 
Iturin A4 25 
Iturin A5 25 
Iturin A6 12.5 
Iturin A7 6.25 
Iturin A8 12.5 
______________________________________ 
Fungi used: Candida albicans 7N 
Medium used: 0.5% Glucose, Yeastnitrogen base (made by DIFCO Co.) 
EXAMPLE 4 
Iturin A as produced in Example 1 was tested with respect to its toxicity, 
by which its safety was verified. 
(1) Cytotoxicity Test: 
A floating suspension of CHL/IU cells, having a concentration of 
1.3.times.10.sup.5 cells/ml, was prepared, and this was separately put in 
a 96-well micro-plate in an amount of 100 .mu.l/well. The cells were 
incubated at 37.degree. C. for 48 hours, and the medium was exchanged for 
180 .mu.l/well of a fresh medium. Then, iturin A4 (obtained in Example 1) 
having a concentration of 10 mg/100 .mu.l was diluted to 1/10 with the 
medium to have a maximum concentration. The diluted one having a maximum 
concentration was further stepwise diluted to 1/2, 1/4 . . . at regular 
intervals in four stages. 20 .mu.l of the dilution having a varying 
concentration was added to each well and the cells therein were incubated 
for further 48 hours at 37.degree. C. After 48 hours, MTT/PBS solution 
having a concentration of 5 mg/ml was diluted to 1/2 and 20 .mu.l of the 
dilution was added to each well. After reacted for 4 hours, the plate was 
subjected to centrifugation at 1000 rpm for 5 minutes, 200 .mu.l of the 
supernatant was removed, and 100 .mu.l of 10% Triton X-100 containing 
0.04N HCl/isopropanol was added to the residue. The amount of MTT formazan 
as eluted was measured with a microplate reader (by Corona Co.; MTP-120) 
(main wavelength 570 nm; side wavelength 630 nm). 
Evaluation of the toxicity was effected in the manner mentioned below. 
Briefly, the average of the absorbance of the control well (Ac) and the 
average of the absorbance of the test well to which a varying 
concentration of iturin A4 had been added (At) were obtained, and 
drug-sensitivity of [At/Ac].times.100 was obtained therefrom for 
evaluation of the toxicity. The value is a criterion of the viability of 
the tested cells. The results obtained are shown in Table 8 below. As is 
understood from the results, iturin A4 had no cytotoxicity to the 
incubated animal cells in a concentration of from 1 to 0.5 mg/ml or less 
and therefore the extremely high safety of iturin A4 was verified. 
TABLE 8 
__________________________________________________________________________ 
Cytotoxicity Test 
__________________________________________________________________________ 
Amount of Active 
1.0 0.5 0.25 
0.125 
0.06125 
Control 
Substance Added 
to Each Well 
(mg/ml) 
Average 0.005 
0.036 
0.411 
0.429 
0.446 0.428 
Absorbance 
[At/Ac] .times. 100 (%) 
1.2 8.4 96.0 
100.2 
104.2 -- 
__________________________________________________________________________ 
(2) Acute Toxicity Test: 
Iturin A4 obtained in Example 1 was dissolved in an injectable distilled 
water to prepare an aqueous 10% (w/v) solution of iturin A4 to be tested. 
The test solution was forcedly oral-administered once to five male and 
five female ddy-N mice, after having been fasted for 13 hours, by the use 
of a stomach probe. The test ddy-N mice were about 4-week age ones and 
were obtained from Nippon Medico-Chemical Animal Sources Laboratories Co., 
Ltd. Prior to the test, all the test mice were preliminarily bred in the 
laboratory for one week so as to confirm the healthy condition of them. 
Then, the about 5-week age mice were subjected to the test. The body 
weight of the test mice just before the test was 28 g (male mice) and from 
18 to 22 g (female mice). 
To each of the male and female mice, the test solution was administered in 
an amount of 20 ml per kg of the body weight, whereby 2,000 mg/kg of the 
test substance was administered to them. Just after the administration, 
the vitality of all the test mice lowered slightly, but it came back to be 
normal in 2 hours after the administration. One hour after recovery of the 
vitality of them, they were fed, and the time-dependent mortality of the 
test mice was observed for 2 weeks. The results obtained are shown in 
Table 9 below. 
TABLE 9 
__________________________________________________________________________ 
Acute Toxicity Test 
Amount 
Administered 
Time-Dependent Mortality 
Mortality 
LD.sub.50 
Sex (mg/kg) 
1 2 3 4 5 6 . . . 14 days 
(%) (mg/kg) 
__________________________________________________________________________ 
Male Mice 
2,000 0/5 
. . . 0/50 
0 &gt;2,000 
Female Mice 
2,000 0/5 
. . . 0/50 
0 &gt;2,000 
__________________________________________________________________________ 
In the next test, mice of the same kind as mentioned above were used. 
Iturin A4 was dissolved in DMSO(dimethylsulfoxide)-castor oil-5% Glc 
solution, and the resulting solution was injected to the caudal vein of 
each of the five male mice once or plural times. For the single injection, 
the solution was administered to the mice each in an amount of 200 mg per 
kg of the body weight; and for the plural injections, it was administered 
to them 14 times each in an amount of 30 mg/kg/day. After the 
injection(s), the time-dependent mortality of the test mice was observed 
and the results shown in Tables 10 and 11 below were obtained. 
TABLE 10 
__________________________________________________________________________ 
Time-Dependent Mortality of Test Mice by Single Intravenous Injection 
Amount 
Administered 
Time-Dependent Mortality 
Mortality 
LD.sub.50 
Sex (mg/kg) 1 2 3 4 5 6 . . . 14 days 
(%) (mg/kg) 
__________________________________________________________________________ 
Male Mice 
200 0/5 
. . . 0/50 
0 &gt;200 
__________________________________________________________________________ 
TABLE 11 
______________________________________ 
Time-Dependent Mortality of 
Test Mice by Plural Intravenous Injections 
Amount 
Administered 
(mg/kg/ Time-Dependent Mortality 
Mortality 
Sex day .times. times) 
1 2 3 4 5 6 . . . 14 days 
(%) 
______________________________________ 
Male Mice 
30 .times. 14 
0/5 . . . 0/5 0 
______________________________________ 
As is obvious from the above-mentioned results, oral administration of 
iturin A resulted in no death of the test mice, and the anatomical view of 
the test mice gave no abnormality. In the animal tests, LD.sub.50 of the 
test substance, iturin A as calculated by a probit method was more than 
2000 mg/kg. The guide line by OECD (Apr. 11, 1986) and others have defined 
that the maximum peroral dose of a chemical substance to a mouse in the 
acute toxicity test is 2000 mg/kg, from which the low toxicity of iturin A 
has been verified. LD.sub.50 of iturin A by single intravenous injection 
and that by plural intravenous injections were both more than 200 mg/kg or 
more, from which the low toxicity and the safety of iturin A in injection 
have been verified like the case of its oral administration. 
EXAMPLE 5 
50 mg of iturin A7 were dissolved in 4 ml of DMSO. Apart from this, 42 mg 
of cholesterol were dissolved in 4 ml of methanol under heat, to which 16 
ml of hot 10% HCO-60 (made by Nippon Surfactant Co.) were added to prepare 
a cholesterol solution. After cooled, the cholesterol solution was added 
to the previously-prepared iturin A7 solution, to which 16 ml of water 
were added. The resulting solution was transferred into a vial and 
freeze-dried to prepare an iturin A7 preparation. 
This was dissolved in 40 ml of 5% glucose solution, which was mixed with 
500 ml of 5% glucose solution to prepare a preparation for dripping. 
EXAMPLE 6 
40 ml of 5% glucose solution were added to each of 10 vials containing the 
iturin A7 preparation produced in Example 1 to dissolve the preparation. 
These were mixed together and blended with 140 ml of 5% glucose solution 
to prepare a solution for oral administration. 
EXAMPLE 7 
Tablets were formed from (1) 50 g of iturin A4, (2) 90 g of lactose, (3) 29 
g of corn starch and (4) 1 g of magnesium stearate. Precisely, (1) and (2) 
were mixed along with (3) (17 g), and the mixture was granulated along 
with a paste prepared from (3) (7 g). (3) (5 g) and (4) were added to the 
resulting granules and well blended, and the resulting blend was tabletted 
with a compression tabletting machine to produce 1000 tablets containing 
50 mg/tablet of the active ingredient (1). 
ADVANTAGE OF THE INVENTION 
The present invention provides an antifungal agent for profound mycosis 
comprising, as the active ingredient, iturin A. Iturin A is extremely 
effective in preventing and curing human and animal profound mycosis 
caused by fungi. It can be administered to animals including human beings 
by oral administration, external application, endermic application, 
intravenous injection or other administration routes.