MXPA00006364A - Novel substances kf-1040 and process for producing the same - Google Patents

Abstract

A process for producing a substance KF-1040A represented by formula (I), and another substance KF-1040B represented by formula (II), which comprises culturing microorganisms capable of producing the substances KF-1040A and KF-1040B, thus accumulating these KF-1040A and/or KF-1040B in the liquid culture and harvesting the same from the culture. Because of the activities of inhibiting diacyloglycerol transferase and sphingomyelinase, the above substances are useful in preventing and treating arteriosclerosis, obesity, thrombus, inflammation and immune function-related diseases.

Description

SUBSTANCE KF-1040 NOVELTY AND PROCEDURE FOR ITS PRODUCTION FIELD OF THE INVENTION The present invention relates to a novel KF-1040 substance having an inhibiting activity for lipid metabolism as well as to a process for the production of said substance. PREVIOUS TECHNIQUES Numerous anti-obesity drugs and drugs against hyperlipidemia are known. For example, centrally acting anorectics suppress appetite, which can nevertheless be harmful to health. Therefore, the development of a new anti-obesity drug or a therapeutic drug for hyperlipidemia that has no side effects has been expected. On the other hand, it has recently become clear that the hydrolysis of sphingomyelin, which is one of the lipids that constitute a biomembrane, has been involved in the transduction of intracellular signals by cytokines, such as, interleukin-l or tumor necrosis factor alpha. [Y. A. Hannun, J. Biol. Chem. 269, 3125-3128 (1994) and R. Kolesnick & D. Golde, Cell, 77, 325-32 8 (1994)], and in the transduction of intracellular signals by activating T cells [L.M. Boucher et al., J. Exp. Med., 18, 2059-2068 (1995) and A. Ochi. Medicinal Im unol., 28, 397-401 (1994)] and has a role in diseases such as arteriosclerosis, inflammations, thrombosis, etc., and in the mechanisms of their immunoregulation. However, to date no prophylactic or therapeutic drug has been developed for these diseases from a specific and potential inhibition perspective or for sphingomyelinase, a sphingomyelin hydrolase. PROBLEM THAT THE INVENTION MUST RESOLVE In recent years, the increase in the population of patients with diseases related to lifestyle has posed great challenges to the medical sciences, both therapeutic and preventive. Especially, diseases of obesity and hyperlipidemia due to the accumulation of triacylglycerols due to the recent habit of excessive diet can frequently cause more severe diseases such as arteriosclerosis, fatty liver, hypertension, diabetes, coronary artery disease, stroke, gall bladder disease, osteoarthritis, respiratory problems and some types of cancer. Obesity refers to a physical state in which the accumulated fat, constituted mainly by triglycerides, accumulates excessively in the body, which is due to an increased synthesis of the triacylglycerols causing the abnormal accumulation of fat in adipose tissue. Likewise, it is believed that triacylglycerolemia is caused by the facilitation of triacylglycerol synthesis in intestines and in the liver, thus causing a lipoproteinemia with a high concentration of triacylglycerols in the blood. Accordingly, any substance that exhibits an inhibitory action on diacetylglycerolacyltransferase that includes the selective synthesis of triacylglycerols is considered to have the ability to suppress the accumulation of triacylglycerols in adipose tissue and blood and may be effective for the therapy of these diseases. Under these circumstances, it is believed that it is valuable for the therapy of obesity and hyperlipidemia and of degenerative diseases such as, for example, arteriosclerosis etc., which come from there, to offer a substance that has an activity of inhibiting diacetylglycerolacyltransferase. In addition, it is also expected that a substance that has a sphingomyelinase inhibiting activity that causes the hydrolysis of sphingomyelin, a liquid that constitutes the biomembranes, can be useful as a drug to fight arteriosclerosis, thrombosis and inflammation and as an immunosuppressant, based on a novel mechanism of action not found before. MEANS TO RESOLVE THE PROBLEM The inventors have carried out research into the metabolic products produced by microorganisms and have found that substances having activities that inhibit diacylglycerolacyltransferase and sphingomyelinase were produced in the culture medium by culturing a newly identified fungal KF-1040 strain isolated from seaweed. These active substances capable of inhibiting the metabolism of the lipids were then isolated from the aforementioned culture medium and purified, so that the chemical structures represented by the formulas (I) and (II) given below were determined. Since the substances represented by these formulas (I) and (II) were not known in the past, the inventors have named such substances "substance A of KF-1040" and "substance B of KF-1040", respectively, which are they know globally as the "substance KF-1040". The present invention has been prepared based on the aforementioned knowledge and refers to the substance KF-1040 comprising the substance A of KF-1040 represented by the following formula (I), and substance B of KF-1040, represented by the following formula (II), The present invention further relates to a process for the production of novel KF-1040 substance comprising the cultivation of a microorganism belonging to the genus Gliocladium and having the ability to produce substance A of KF-1040 and / or substance B of KF-1040 in a culture medium, causing the accumulation of substance A of KF-1040 and / or substance B of KF-1040 resulting in the culture medium and isolating substance A from KF-1040 and / or the substance B of KF-1040 of the culture medium. The present invention also relates to a process for the production of substance KF-1040, where the microorganism belonging to the genus Gliocladium and having the ability to produce substance A of KF-1040 and / or substance B of KF- 1040 is Gliocladium sp. KF-1040 (FERM BP-6251). The present invention further relates to a microorganism belonging to the genus Gliocladium and having the ability to produce substance A of KF-1040 and / or substance B of KF-1040.

The microorganism having the ability to produce the substance KF-1040 represented by formula (I) and (II) (hereinafter referred to as "fungus producing KF-1040 material") belongs to the genus Gliocladium and, for example, , the fungal strain Gliocladium sp. KF-1040 isolated by the inventors is an example to be used more effectively in accordance with the present invention. The taxonomic properties of this productive strain of KF-1040 appear below: 1. Morphological properties This strain grows relatively well in a medium containing 50% seawater (with a salt concentration of 3.4%), such as agar of potato glucose, corn flour agar, malt extract agar, Miura agar medium as well as marine starch agar, with an abundance of conidia. When observing through a microscope the growth of the colony on a medium of corn flour agar, the hypha is transparent and has a septum. The conidiophore assumes both the pen and verticilate forms. The pen-shaped conidiophore (which is 100 to 200 μm in length) rises or branches from the basal hypha and forms at the upper end or in the branch several pen-shaped cyclic fialides of sizes 2.5 at 3.0 μm x 10 - 23 μm, where a conidial mass is formed. On the other hand, the conidiophore in the form of verticilate (which has a length of 25-50 μm) rises from the basal hypha and forms at the upper end or branching fialides (sizes 3.0-5.0 μm X 17-25 μm) in the form of an elongated bottle or in shape of a cone that converges towards the upper part, from which a single conidial mass is formed. The conidium is colorless and has the shape of an ellipsoid or elongated ellipsoid with a size of 2.5-3.0 μm X 3.0-5.0 μm, rarely with a sharp tip at its end, for the conidiophore in the form of a feather. In the case of the verticilate form, the conidium is colorless and has an ellipsoidal or elongated elongated shape with a size of 2.5-3.0 μm X 6.0-8.5 μm. 2. Properties Cultivated in Various Media The results of visual observation of the culture status of this strain in various culture media at a temperature of 25 ° C for 14 days are given in the following table 1. Table 1 Color Condition Pigment Color growth on surface of re-soluble side colony half of the colony (diameter of the colony colony) Medium of glucose agar of good potato (20-30 mm) gray gray none flocose, bright glossy plane Good corn flour agar medium (24-30 mm) gray gray none flocose, with bright shiny bands Malt extract agar medium good (20-22 mm) gray ggrriiss none flocose, light pale plane Miura agar medium good (22-24 mm) gray gray none flocculent, with shiny shining backs Seaweed agar medium good (24-27 mm) gray bbllaannccoo none flocoso, milky bright plane 3. Physiological properties (1) Optimum growth conditions Optimal conditions of growth of the present strain are: pH 4-8, temperature: 17-27 ° C, seawater concentration *: 0-50% *: natural sea water is used with a salt concentration of 3.4% (2 ) Growth condition The growth range of the strain is: pH 3-10, temperature 9-32 ° C, seawater concentration *: 0-200% *: natural sea water is used with a salt concentration of 3.4% (3) Nature: aerobic As shown above, the morphological properties, culture condition and physiological properties of the present strain KF-1040 are given above, the inventors carried out a comparison of this strain with known fungal strains and arrived at the identification of the same as a strain belonging to the genus Gliocladium and which was named Gliocladium sp. KF-10 0. This strain was deposited on February 6, 1998, before the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Science and Technology (National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of Science and Technology International), under the deposit number FERM P-16629, and said strain was deposited again on February 12, 1998 before the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Science and Technology (National Institute of Bioscience and Human Technology, Agency for Industrial Science and Technology, Ministry of Science and International Technology), located at 1-3, Higashi 1-Chome, Tsukuva- shi, Ibaraki-ken, Japan, with the receipt number No. FERMP BP-6251 of the original deposit by transfer request to the deposit based on the Treaty of Bu dapest.

As a fungus producing the substance KF-1040 to be used according to the present invention, we can mention, in addition to the aforementioned strain Gliocladium sp. KF 1040, all the strains belonging to the genus Gliocladium and producing the substances KF-1040 represented by the formula (I) and by the formula (II) given above (hereinafter known comprehensively as the "substance KF-1040" in the measure unless otherwise specified), including natural mutants, artificial mutants resulting from the irradiation of X-rays and UV rays and by mutation treatment such as N-methyl-N '-nitro-N-nitrosoguanidine and 2-aminopurine, with fused strains and strains manipulated as to their genes. In the practical application of the present invention, the substance KF-1040 which produces fungi belonging to the genus Gliocladium is grown in a culture medium. As nutrient sources suitable for the production of the substance KF-1040, a nutrient medium containing carbon sources assimilable by the microorganism, source of nitrogen assimilable by the microorganism, and, if necessary, additives, including inorganic salts and vitamins As a carbon source, saccharides such as glucose, fructose, maltose, lactose, galactose, dextrin and starch can be incorporated, as well as vegetable oils such as soybean oil, etc., individually or good in combination. As a nitrogen source, peptone, yeast extract, meat extract, soybean powder, cottonseed powder, corn infusion liquor, malt extract, casein, amino acids, urea, ammonium salts and nitrates can already be used. either individually or in combination. further, salts such as phosphates, magnesium salts, calcium salts, sodium salts and potassium salts as well as heavy metal salts such as iron salts, manganese salts, copper salts, cobalt salts and zinc salts, and Vitamins and others adapted to the production of the substance KF-1040 can be added appropriately. In the culture can be added, if necessary, antifoam agent, based for example on liquid paraffin, animal oil, vegetable oil, silicone and surfactant, when there is a severe phenomenon of foam formation. The culture can usually be favorably carried out in a liquid culture medium, while liquid and solid media can be used insofar as they contain the sources of nutrients indicated above. In the case of small-scale production, the use of a bottle to carry out the cultivation may be favorable. For the production of material at the industrial level, a shaking aeration culture is preferred, as in the case of other fermentation products.

When the cultivation is carried out in a large tank, it is preferable to practice in such a way that the production fungus is first inoculated in a relatively small amount of culture medium, in order to avoid the retardation of growth of the fungus, and the The culture mixture is then transferred to a large tank in order to grow it there. It is possible that the composition of culture medium is the same or different in the case of the previous culture and the production culture. If necessary, the culture composition can be altered. In the case of which the cultivation is carried out under the condition of aeration with agitation, techniques which include mechanical agitation by blades or other devices, rotation or agitation of the fermenter, pumping agitation and bubbling can be used accordingly. of air. The aeration is carried out in air sterilization condition. The culture temperature can be suitably altered within the range in which the fungus producing the substance KF-1040 can produce the substance KF-1040, while usually the culture is carried out at a temperature within a range of 20 to 30 ° C, preferably at a temperature of about 27 ° C. The culture is usually carried out at a pH of 5 to 8, preferably at a pH of about 7. The duration of the Crop may vary according to specific growing conditions, usually 10 to 20 days. The substance KF-1040 produced in this way is present in the grown mycelia and in the cultivated filtrate. For the purification of the substance KF-1040 from the cultivated mass, the whole cultivated mass is extracted with an organic solvent miscible in water, such as acetone, and the extract is subjected to evaporation in vacuum to remove the organic solvent, so The resulting residue is extracted with an organic solvent which is immiscible with water, such as ethyl acetate. In addition to the aforementioned extraction technique, known practices used for the purification of lipid soluble substances, such as, for example, rption chromatography, gel filtration chromatography, thin layer chromatography, centrifugal countercurrent distribution chromatography, liquid chromatography High performance can be used in a suitable combination or in a repetitive manner to effect the separation of the substance KF-1040 in each component and to purify it. The physical-chemical properties of substance A of KF-1040 according to the present invention appear below: 1) Nature: white powder 2) Molecular weight: 776 (determined by fast atom bombardment mass spectrometry) 3) Molecular formula: CoH72O? 4 4) Specific rotation: [alpha] D25 = + 62 ° (c = 0.1 in methanol) 5) Maximum of UV absorption (in methanol): Figure 1: at 203 nm (e = 24900), 220 nm (e = 18000) and 275 nm (e = 1200) 6) Maximum IR absorption (KBr tablet): Figure 2 a 1637 c "1 and 3434 cm" 1 7) Proton NMR spectrum (in heavy hydrogen methanol): as illustrated in Figure 3 8) Spectrum 13C-NMR (in heavy hydrogen methanol): as illustrated in Figure 4 9) Solubility in solvents: soluble in methanol, benzene, chloroform and ethyl acetate; slightly soluble in water and hexane 10) Color reaction: positive to sulfuric acid and phosphoric acid 11) Acid or alkaline nature: neutral Through examination of physico-chemical properties, spectral analysis of KF substance A 1040 as indicated above, the chemical structure of substance A of KF-1040 represented by the following formula (I) was determined: The physical-chemical properties of substance B of KF-1040 according to the present invention appear below: 1) Nature: white powder 2) Molecular weight: 818 (by fast atom bombardment mass spectrometry) 3) Molecular formula : C2H7015 4) Specific rotation: [alpha] D25 = + 120 ° (c = 0.1 in methanol) '5) Maximum UV absorption (in methanol): Figure 5: at 204 nm (e = 43000), 218 nm ( e = 30300) and 272 nm (e = 2900) 6) Maximum IR absorption (KBr tablet): Figure 6 at 1633 cm "1 and 3417 cm" 1 7) Proton NMR spectrum (in heavy hydrogen methanol) : as illustrated in Figure 7 8) Spectrum 13C-NMR (in heavy hydrogen methanol): as illustrated in Figure 8 9) Solubility in solvents: soluble in methanol, benzene, chloroform and ethyl acetate; slightly soluble in water and hexane 10) Color reaction: positive for sulfuric acid and for phosphomolybdic acid 11) Acid or alkaline nature: neutral Through the examination of the physico-chemical properties, spectral analysis of data of substance B of KF-1040 as provided above, the chemical structure of substance B of KF-1040 was determined in accordance with that represented by the following formula: While the above description has been focused on the details of various physico-chemical properties of substance A of KF-1040 and of substance B of KF-1040, it is recognized that no compound having properties corresponding to the above-identified properties has been reported in the literature. Therefore, KF-1040 substances are considered to be novel substances. Now, the description focuses on the biological nature of substance A of KF-1040 and substance B of KF-1040. / \ (1) Action of inhibition of diacylglycerolacyltransferase originated in rat The activity of diacylglycerolacyltransferase was determined through the modified method of Mayorek and Bar-Tana [J. Biol. Chem. 260 6528-6532 (1985)]. Thus, a microsomal fraction prepared from rat liver was employed as the source of enzymes. To 175 mM Tris-HCl buffer (pH 8.0) containing 8 mM MgCl 2, 1 mg / ml bovine serum albumin and 2.5 mM diisopropyl fluorophosphate, 0.75 mM dioleoylglycerol and 30 μM [1-1 C] were added. -palmitoyl-CoA (0.02μCi) and the total volume was adjusted to 200 μl, after which the enzyme reaction mixture was incubated at 23 ° C for 15 minutes. The total lipids were extracted with a mixture of chloroform / methanol and each lipid was separated by TLC (with silica gel GF254 and a developer of petroleum ether / diethyl ether / acetic acid 80/20/1), followed by the determination of the radioactivity of the triacylglycerol fraction using RADIOSCANNER (from AMBIS System Inc.) to determine the diacylglycerolacyltransferase activity. The calculation of the drug concentration corresponding to a 50% inhibition of this enzyme gives the values of 16 μg / ml for substance A of KF-1040 and 9.0 μg / ml for substance B of KF-1040. (2) Inhibitory action on the formation of triacylglycerol in cells of human origin (raji cell originating from human Burkitt's lymphoma) The evaluation of the influence of the substances on the formation of triacylglycerol was carried out in accordance with the method of Tomoda et al [J. Biol. Chem. 266, 4214-4219 (1991)] using cells of human origin (Raji cells originating from human Burkitt's lymphoma). A dispersion of Raji cells of 2.7 x 10 6 cells per milliliter containing 0.36 nM [1-14 C] -oleic acid (0.02 μCi) in the presence or absence of the substance was filled to a total volume of 200 μl, so that caused the reaction at a temperature of 37 ° C for 30 minutes. The total lipids were extracted with a mixture of chloroform / methanol (2/1). Subsequent procedures were carried out in the same manner as in the aforementioned experiment (1) "action of inhibiting diacylglycerolacyltransferase of rat origin". The calculation of the drug concentration corresponding to a 50% inhibition of the formation of triacylglycerol gives the values of 10 μg / ml in the case of substance A of KF-1040 and 10 μg / ml in the case of the substance B of KF-1040. (3) Inhibition action of neutral sphingomyelinase originated in rat brain The evaluation of the influence of the substances on the neutral sphingomyelinase originating from rat brain was carried out in accordance with the modified method of Murakami & amp; amp;; Arima [J. Neurochem., 52, 611-618 (1989)]. Thus, a membrane fraction prepared from a rat brain was used as the source of enzymes and 20 mM of a buffer solution HEPES-NaOH (pH 7.4), 6.5 mM MgCl2, 0.1% Triton X-100 were added. and 25 μM [N-methyl-3H] -sphingomyelin (0.006 μCi) and the mixture was filled to a total volume of 50 μl. After the reaction at a temperature of 37 ° C for 30 minutes, 200 μl of the chloroform / methanol mixture (volume ratio 1/2) was added to the reaction mixture to separate the product from the reaction [3H] -phosphocholine from the initial material [3H] -sphingomyelin. The supernatant layer was collected in a 50 μl bottle. The amount of [3 H] -phosphocholine was quantitatively determined by a liquid scintillation counter to estimate neutral sphingomyelinase activity. The calculation of the concentration of substance KF-1040 corresponding to a 50% inhibition of the enzyme gives the values of 4.2 μg / ml for substance A of KF-1040 and 6.1 μg / ml for substance B of KF-1040. (4) Influence on the sphingomyelinase of acid that originates in the human placenta The evaluation of the influence of the substance on the sphingomyelinase acid originating in the human placenta was carried out according to the method of Jones et al [Biochem. Journal, 195, 373-382 (1981)] with partial modification. Thus, an acid sphingomyelinase that originates from the human placenta (a product of the company Sigma) was used as the enzyme source and to this 250 mM of a sodium acetate buffer solution (pH 5.0), 0.1 was added. % of NP-40 (from the same company Sigma), 25 μM of [N-methyl-3H] -sphingomyelin (0.006 μCi) and various substrate concentrations, and the mixture was filled to a total volume of 50 μl. After the reaction at a temperature of 37 ° C for 30 minutes, 200 μl of a mixture of chloroform / methanol (volumetric ratio 1/2) was added to the reaction mixture in order to separate the product from the reaction [ 3H] -phosphocholine from the initial material [3H] -sphingomyelin. The supernatant layer was collected in a 50 μl bottle. The amount of [3 H] -phosphocholine was quantitatively determined by a liquid scintillation counter to estimate the acid sphingomyelinase activity. The calculation of the concentration of the substance KF-1040 which corresponds to a 50% inhibition of this enzyme offers the values of 48 μg / ml for substance A of KF-1040 and 24 μg / ml for substance B of KF-1040. In accordance with that described above, the novel substance according to the present invention has an activity to inhibit diacylglycerolacyltransferase and sphingomyelinase and, therefore, is useful for the prophylaxis and therapy of patients with diseases that are related to atherosclerosis, obesity, thrombosis, inflammations and immunofunctional disorders. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the absorption spectrum of UV light (in methanol) of substance A of KF-1040 according to the present invention. Figure 2 shows an IR absorption spectrum (with KBr) of substance A of KF-1040 according to the present invention. Figure 3 shows the proton NMR spectrum (in heavy methanol) of substance A of KF-1040 according to the present invention. Figure 4 shows the 13 C-NMR spectrum (in heavy methanol) of substance A of KF-1040 according to the present invention. Figure 5 shows the absorption spectrum of UV light (in methanol) of substance B of KF-1040 according to the present invention. Figure 6 shows the absorption spectrum of IR (with KBr) of substance B of KF-1040 according to the present invention. Figure 7 shows the proton NMR spectrum (in heavy methanol) of substance B of KF-1040 according to the present invention. Figure 8 shows the 13 C-NMR spectrum (in heavy methanol) of substance B of KF-1040 according to the present invention. Example Two 500 ml Erlenmeyer flasks each loaded with 100 ml of a liquid culture medium (pH 6.0) prepared by dissolving 2.0% glucose, 0.5% polypeptone (from Nippon Seiyaku KK), 0.2% yeast extract (from Oriental Kobo Kogyo KK), 0.05% magnesium sulfate 7-hydrate, 0.1% potassium dihydrogen phosphate and 0.1% agar in 50% natural seawater were each inoculated with a loop of the Gliocladium sp. KF-1040 (FERM BP-6251), and each inoculated medium was cultured at a temperature of 27 ° C for 4 days with shaking. The resulting cultivated medium was used as the cultivated seed. Sixty Roux flasks each with a capacity of 1,000 ml each were charged with 300 ml of a liquid culture medium (pH 6.0) prepared by dissolving 100 g / 1 of portate and 1.0% of glucose in 50% of the water. natural sea After sterilization and cooling, Each vial was aseptically inoculated with 3 ml of the culture seed and the inoculated culture was cultured at a temperature of 27 ° C for 16 days at rest. To the total cultivated liquor 18 liters of acetone were added and stirred well, followed by a concentration under reduced pressure and the liquor concentrated in this way was extracted with ethyl acetate. The extract layer was subjected to concentration under reduced pressure, whereby 2.2 grams of a crude product were obtained. This crude product was dissolved in a small amount of acetonitrile and the resulting solution was passed to an ODS column (200 g, supplied by Senshu Kagaku K.K. ODS-SS-1020T) filled with 30% aqueous acetonitrile. After washing the column with 50% aqueous acetonitrile, the column was eluted with 60% aqueous acetonitrile, and then with 70% aqueous acetonitrile. From the eluates, 87 mg of a crude product of substance A and 104 mg of a crude product of substance B were obtained by concentration under reduced pressure. Each of the raw products was fractionated through high performance liquid chromatography (Shiseido Capsulepack, ODS-SG column, 20 mm X 250 mm, flow rate = 6.0 ml / min.; detection: 215 nm UV using an eluent of 70% aqueous acetonitrile). A fraction eluted in the retention time of 17 minutes for substance A and a fraction eluted in the retention time of 23 minutes for substance B were collected, respectively. Each fraction was treated by removing the organic solvent and extracting the aqueous layer with ethyl acetate, whereby 16 mg of substance A of KF-1040 and 40 mg of substance B of KF-1040 were obtained. EFFECT OF THE INVENTION As mentioned above, the novel substance KF-1040 according to the present invention exhibits an activity of inhibition of diacylglycerolacyltransferase and of sphingomyelinase and, therefore, is expected to be useful for the prophylaxis and therapy of patients. with diseases that are related to arteriosclerosis, obesity, thrombosis, inflammations as well as immunofunctional disorders.

Claims (3)

1. CLAIMS l.A novel KF-1040 substance comprising a substance A of KF-1040 represented by the following formula (I), and a substance B of KF-1040 represented by the following formula (II),
2. 2. A process for the production of a novel KF-1040 substance, characterized by the culture of a microorganism belonging to the genus Gliocladium and having the ability to produce a substance A of KF-1040 and / or a substance B of KF-1040 in a culture medium, causing substance A of KF-1040 and / or substance B of KF-1040 to accumulate in the culture medium and the crop of substance A of KF-1040 and / or substance B of KF-1040 from the culture medium.
3. 3. The method according to claim 2, wherein the microorganism belonging to the genus Gliocladium and having the ability to produce a substance A of KF-1040 and / or a substance B of KF-1040 is Gliocladium sp. KF-1040 (FERM BP-6251). A microorganism belonging to the genus Gliocladium and having the ability to produce a substance A of KF-1040 and / or a substance B of KF-1040.