WO2007061254A1

WO2007061254A1 - Genistein derivatives and anti-fungal composition containing the same

Info

Publication number: WO2007061254A1
Application number: PCT/KR2006/004979
Authority: WO
Inventors: Jong Woo Kim; Jae Jin Han; Eul Yong Park; Joo Won Suh; Tae Mi Yoon; Hyung Jin Kwon
Original assignee: B & C Biopharm. Co., Ltd.
Priority date: 2005-11-25
Filing date: 2006-11-24
Publication date: 2007-05-31

Abstract

Genistein derivative compound, method for producing the same from Kitasatospora kifunensis strain, anti-fungal composition containing the same and Kitasatospora kifunensis MJM-341 (accession number: KCCM- 10710P) are disclosed. The compound of the invention has excellent anti-fungal activity and low toxicity, which is useable as anti-fungal medicine.

Description

GENISTEIN DERIVATIVES AND ANTI-FUNGAL COMPOSITION CONTAINING THE SAME

Field of the Invention

The present invention relates to genistein derivatives represented by a following formula 1, preparing method thereof, anti-fungal composition containing the same and Kitasatospora kifunensis strain for isolating the genistein derivatives: [Formula 1]

wherein R represents hydrogen or

Description of the Related Art

It has been generally known that fungus affords benefits such as foods and antibiotics to human, while causing his life in danger by systemic mycosis including aspergillosis and dermatomycosis.

As for human being and animals, diseases based on fungal infection are typically classified into two types of systemic mycoses and dermatomycosis depending on site of infected tissues. In particular, the systemic mycoses are not developed for well and healthy men but normally causes opportunistic fungal infection for a man having weak immunity, which include cryptococcosis, candidasis, aspergillosis, etc. Cryptococcosis is represented by infection of Cryptococcus neoformans and developed over substantially all sites of whole body (including skins) of the human being who has reduced immunity, for instance, AIDS (Acquired Immune Deficiency Syndrome) patients. Especially, in case of infecting brain and meninges of a human, it may occur meningitis, brain abscess and/or brain tumor.

Candidasis is mycosis caused by Candida albicans and has been originally isolated from thrush patients, which develops vaginitis for women and diaper rash for infants.

Also, aspergillosis generally means common diseases caused by Aspergillus genus fungi, which includes ABPA (allergic bronchopulmonary aspergillosis), aspergilloma, IPA (invasive pulmonary aspergillosis) and the like. Such Aspergillus fungi exist all over natural environment, spread their spores in the air, and infect persons who breathe in and out the air containing the fungal spores. Such fungi can live in a wide range of sites in the human body including skin, eyes, lung and so on, and optionally, spinal cord and/or brain. Development of aspergillosis is absolutely related to condition of a host and generally occurred in a person having reduced immunity, which is caused by bone-marrow transplant, chemo-therapy, HIV (Human immunodeficiency virus) infection or AIDS, or administration of medicines for preventing rejection to transplant of internal organs. Representative drugs for remedying the above disease comprise, for example, itraconazole and amphotericin B.

Recently, attack rate of the systemic mycoses tends to rise along with increase in patients who has AIDS or reduced immunity caused by application of chemotherapy for treating cancer or administration of immune-suppressive drugs after transplant of internal organ, thereby acquiring greater importance more and more. In fact, it was demonstrated that patients suffering from cancer or AIDS die due to fungal infection of internal organs or blood rather than their disease as such. Furthermore, contaminants as causal microorganism of the fungal infection are increasingly varied and it is foreseeable that greater species of fungi induce invasive infection.

On the other hand, harmful influence or bad effect of the fungal infection is also extremely serious for plants. In particular, plant pathogenic fungus acts in harmful to most of plants and crops, therefore extremely decreases productivity thereof. However, it is not sufficient at present and still needs to develop novel and improved anti-fungal medicines with low toxicity for clinical uses and biopesticides for agricultural uses.

Representative anti-fungal medicines known and clinically available now are mostly azole-based synthetic materials in addition to amphotericin B. But, such medicines have disadvantage of seriously representing side effect during use.

Accordingly, it still requires development of a novel antifungal agent with low toxicity.

Detailed Description of the Invention Technical Subject

An embodiment of the present invention is desired to genistein derivative compound with excellent anti-fungal activity and pharmaceutically acceptable salts thereof, process for preparing the same, use thereof and Kitasatospora kifunensis strain for isolating the above compound.

Technical Solution

In accordance with an aspect of the present invention, provided at first is genistein derivative compound having a specific structure of the following formula 1 and pharmaceutically acceptable salts thereof: [Formula 1]

wherein R represents hydrogen or

Preferably, the compound represented by the above formula 1 comprises genistein 4',7-di-α-L-6-deoxy-talopyranoside of the following formula Ia and genistein 7-α-L-6-deoxy-talopyranoside of the following formula Ib. [formula Ia]

[formula Ib]

Among the above formulae, ¹C₄ conformation and ⁴Ci conformation in chair form of a sugar component can be converted each other depending on surrounding circumstance such as solvent, temperature, etc., therefore, are not represented by steric conformation.

The compound represented by the formula 1 is isolated from Kitasatospora kifunensis strain belonging to actinomycetes that are obtained from soil, has a defined structure and comprises original isoflavonoid including 6-deoxy-talose as one of physiologically active materials. The compound represented by the formula 1 may be also obtained by chemical synthesis other than isolation from actinomycetes.

Genistein derivative of the invention represented by the formula 1 may be used in the form of pharmaceutically acceptable salts. Such salts preferably comprise base adduct salt formed by pharmaceutically acceptable base. Such base may comprise inorganic base containing alkali metal or organic base such as amine with strong basicity. The salt formed by using the inorganic base may comprise adduct salt such as sodium salt, potassium salt, calcium salt, magnesium salt, etc. On the other hand, the salt formed by using the organic base may comprise adduct salt such as ethanolamine salt, propanolamine salt, ammonium salt or general tetraalkylamine salt, etc.

Another embodiment of the present invention is also directed to anti-fungal composition containing the compound represented by the formula 1 as active ingredient.

Still further, the present invention provides anti-fungal composition containing extract of Kitasatospora kifunensis MJM-341 (accession number: KCCM- 10710P). Herein, the extract contains the compound of the invention represented by the formula 1 as the active ingredient.

It was demonstrated that the compound represented by the formula 1 exhibited strong anti-fungal activity to Rhizoctonia solani as one of plant pathogenic fungi as well as Candida albicans, Cryptococcus neoformans and Aspergillus niger, all of which develop fungal infection to human being while having no inhibitory activity against general bacteria (see Table 4). Meanwhile, it was also found that the compound represented by the formula 1 has not cytotoxicity to human liver cells, i.e. HepG2 cell (see Table 5). This supports that the compound of the invention has a reliable possibility as a superior anti-fungal material since the compound shows considerably less toxicity to cells than amphotericin B which is common anti-fungal medicine available in clinical applications. In addition, as described above, the compound represented by the formula 1 has the strong anti-fungal activity to Rhizoctonia solani as one of plant pathogenic fungi, therefore, can be administered not to animals but also to plants. Accordingly, the antifungal composition of the invention is also useable as agricultural chemicals for preventing pathogenic fungi. The present invention provides pharmaceutical composition which contains at least one of compounds according to the present invention, or comprises at least one of active ingredients according to the present invention and, in addition to, non-toxic, inert, pharmaceutically suitable excipient. The present invention also provides a process for preparing the above pharmaceutical composition. The pharmaceutical composition according to the present invention is fabricated into formulations for oral administration including, for example, troches, lozenge, water-soluble or oil suspension, powder or granulate, emulsion, hard or soft capsule, syrup or elixirs, etc. In order to prepare the formulation in the form of tablet and/or capsule, the composition further includes: binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipient such as dicalcium phosphate; disingetrant such as corn starch or potato starch; and/or lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethyleneglycol wax.

Parenteral formulation can be prepared by adding active ingredient in solvent including, for example, sterilized solution for injection, non-toxic available diluent, or 1,3-butandiol to form a suspension, and administered as an injection formulation. As preferable excipient or solvent, used are water, Ringer's solution and isotonic saline solution. Also, co-solvent of ethanol, polyethyleneglycol and polypropyleneglycol is applicable in the formulation. Further, sterilized non-volatile oil is traditional solvent or suspension solvent to be used. Suppository formulation is in solid state at ambient temperature while being changed into liquid at desired temperature, for example, internal temperature of rectum and dissolved within the rectum to discharge drug. Such suppository formulation is prepared by admixing the drug with a proper non- sensitizing excipient, for example, cocoa butter or polyethyleneglycol, then, administered into the rectum.

If the composition of the invention is applied to cure disease, amount of the compound represented by the formula 1 as the active ingredient is varied according to age, weight, general health condition, gender, diet or meal, administration time, excretion rate, combination of drug use, severity of disease during remedy, but, usually ranges from 0.01 to 140mg/kg weight per day based on disease and 0.5mg to 7g per day for each person. Amount of the compound of the invention which is admixed with the carrier to define the formulation, is varied based on routes of drug administration and types of patient to be treated. For example, the formulation for oral administration to human contains 0.5mg to 5g of active ingredient as well as carrier materials in amount of 5 to 95% by weight to total composition of the formulation. While the formulation for parenteral administration to human contains O.lmg to 2.5g of active ingredient and 5 to 99% by weight of carrier material to total composition of the formulation.

As described above, it was found from a result of determining minimal inhibition concentration that genistein derivatives according to the present invention exhibited stronger anti-fungal activity to a wide range of pathogenic fungi compared with that of typical itaconazole. In addition, the present derivatives have non-toxicity to human liver cells. Alternatively, the present invention provides a method for producing the above compound by isolating the compound from Kitasatospora kifunensis strain, preferably Kitasatospora kifunensis MJM-341.

The isolation process is described in detail as follows.

At first, actinomycetes Kitasatospora kifunensis strain, preferably, Kitasatospora kifunensis MJM-341 is cultured in a medium, in particular, GSS medium which contains 1Og of soluble starch, 2Og of glucose, 25g of soybean meal, Ig of beef extract, 4g of yeast extract, 2g of NaCl, 0.25g of K₂HPO₄, 2g of CaCO₃ and 1 liter of distilled water and has pH 7.2, or a modified medium which contains: a mixture of soluble starch, glucose, soybean meal, saccharose and soybean powder in amount of Ig to 30g/L as carbon source; a mixture of yeast extract, beef extract and bacto-peptone in amount of Ig to 10g/L as nitrogen source; and inorganic salts including NaCl, K₂HPO₄, and CaCO₃ in amount of 0.1 g to 5g/L, at 25 to 34 ^°C with 100 to 350rpm for 6 to 8 days.

Next, after centrifugation of the cultured solution, only supernatant is taken for extracting of active ingredient by using butanol or is subjected to adsorption to Diaion HP-20 resin to isolate the active ingredient.

The active layer obtained by the isolation procedure is concentrated under reduced pressure and subjected to RP- 18 open column chromatography and finally, RP- 18 HPLC in due order.

Pure genistein 4',7-di-α-L-6-deoxy-talopyranoside represented by the formula Ia and pure genistein 7-α-L-6-deoxy-talopyranoside represented by the formula Ib are all isolated and purified by RP- 18 HPLC using acetonitrile in water as a mobile phase.

Genistein 4',7-di-α-L-6-deoxy-talopyranoside is a material isolated at peaks between 28 to 32 minutes in HPLC chromatogram as shown in Fig. 1. Genistein 7-α- L-6-deoxy-talopyranoside is a material isolated at peaks between 28 to 40 minutes in HPLC chromatogram as shown in Fig. 2.

The present invention still further provides actinomycetes Kitasatospora kifunensis strain that isolates and produces the compound represented by the formula 1.

The microorganism of the invention has been isolated from soil and, as a result of analysis for sequence of 16S rRNA, it was demonstrated that the microorganisms had the sequence defined as SEQ. ID. No. 1 and represented 99% homology to Kitasatospora kifunensis. Therefore, the microorganism of the invention was named to "Kitasatospora kifunensis MJM-341" and deposited in Korean Culture Center of Microorganisms, KCCM on November 21, 2005 (accession number: KCCM- 10710P).

Advantageous Effects

Genistein derivatives according to the present invention show remarkably superior anti-fungal effect against a variety of pathogenic fungi compared with known itraconazole. Also, as a result of toxicity test, it was determined that the derivatives are safe and effectively applicable in formulating anti-fungal medicines.

Description of Drawings Fig. 1 illustrates HPLC chromatogram for obtaining genistein 4',7-di-α-L-6- deoxy-talopyranoside.

Fig. 2 illustrates HPLC chromatogram for obtaining genistein 7-α-L-6-deoxy- talopyranoside.

Fig. 3 illustrates ¹H NMR spectrum of genistein 4',7-di-α-L-6-deoxy- talopyranoside.

Fig. 4 illustrates ¹³C NMR spectrum of genistein 4',7-di-α-L-6-deoxy- talopyranoside. Fig. 5 illustrates ¹H NMR spectrum of genistein 7-α-L-ό-deoxy-talopyranoside. Fig. 6 illustrates ¹³C NMR spectrum of genistein 7-α-L-6-deoxy-talopyranoside. Fig. 7 illustrates GC chromatogram of L-rhamnose-TMS derivative. Fig. 8 illustrates GC chromatogram of L-fucose-TMS derivative. Fig. 9 illustrates GC chromatogram of TMS derivative of sugar of genistein

4',7-di-α-L-6-deoxy-talopyranoside and genistein 7-α-L-6-deoxy-talopyranoside.

Fig. 10 illustrates ¹H-¹H COSY spectrum of TMS derivative of sugar of genistein 4',7-di-α-L-6-deoxy-talopyranoside and genistein 7-α-L-6-deoxy- talopyranoside.

Preferred Embodiments

Now, preferred embodiments and examples of the present invention will be described in detail. However, it will be obvious to those skilled in the art that the present invention is not restricted to the specific matters stated in the examples below.

EXAMPLE 1. Isolation and search of soil bacteria

Soil sample was taken from northern area of Gyeonggi province in Korea and suspended in (physiological) saline. After spreading the suspension on Bennet's medium which comprises 1Og of glucose, Ig of yeast extract, 2g of bacto-peptone, Ig of beef extract and 1 liter of distilled water, single colony was isolated while incubating the medium at 28 ^°C for two months. The isolated colony was suspended in 20% glycerol solution and stored at low temperature of about -70 ^°C .

Each of the strains isolated was cultured in GSS medium (which comprises 1Og of soluble starch, 2Og of glucose, 25g of soybean meal, Ig of beef extract, 4g of yeast extract, 2g of NaCl, 0.25g Of K₂HPO₄, 2g of CaCO₃ and 1 liter of distilled water and has pH 7.2) under agitating at 200rpm for 7 days. The cultured solution was subjected to both of anti-bacterial test and anti-fungal test to select actinomycetes Kitasatospora kifunensis MJM-341 with excellent anti-fungal activity and analyze 16S rRNA sequence thereof [Korean Agricultural Culture Collection (KACC), located in Rural Development Administration, Suwon, Gyeonggido, Korea]. As a result, it was found that the selected actinomycetes has 99% homology to Kitasatospora kifunensis and the sequence defined to SEQ. ID. No. 1. The present inventors named the isolated strain as "Kitasatospora kifunensis MJM-341" and deposited the strain in KCCM on November 21, 2005 (accession number: KCCM- 10710P).

EXAMPLE 2. Isolation and purification of anti-fungal materia!

40 liters of the above actinomycetes Kitasatospora kifunensis MJM-341 was cultured in GSS medium at 32 ^°C under agitating at 200rpm for 7 days.

After centrifugation of the cultured solution, supernatant was only taken and subjected to extraction using butanol. The butanol phase was concentrated under reduced pressure and subjected to RP- 18 open column chromatography and RP- 18 HPLC in due order for isolation and purification of pure genistein 4',7-di-α-L-6-deoxy- talopyranoside represented by the formula Ia and pure genistein 7-α-L-6-deoxy- talopyranoside represented by the formula Ib.

Genistein 4',7-di-α-L-6-deoxy-talopyranoside is the material isolated (3.3mg) at peaks between 28 to 32 minutes in HPLC chromatogram as shown in Fig. 1. Similarly, genistein 7-α-L-6-deoxy-talopyranoside is the material isolated (19.2mg) at peaks between 28 to 40 minutes in HPLC chromatogram as shown in Fig. 2.

EXAMPLE 3. Determination of the structure of the compound of the invention

In order to analyze the structure of the isolated pure materials, related data including ¹H-NMR, ¹³C-NMR, 2D-NMR and FAB-Mass information were combined together to define the structure as shown in the formulae Ia and Ib.

In order to identify the chemical structure of the pure genistein 4',7-di-α-L-6- deoxy-talopyranoside, determined were ¹H-NMR, ¹³C-NMR, ¹H-¹H COSY, HMQC, HMBC spectrum at 500MHz and 125MHz with using DMSO-d₆ as a solvent. As listed in Table 1, ¹H-NMR showed: two of olefin type methines and 1,4-disubstituted benzene ring at 6.50ppm and 6.77ppm; two anomeric protons at 5.70ppm and 5.54ppm; sugar derived hydroxylated methine at 3.53 to 3.83ppm; two methyl groups at 1.05ppm; and exchangeable proton at 8.46ppm. It was observed from C-NMR as follows: carbonyl carbon at 179.9ppm; seven of quarternary carbons at lόl.Oppm (x2), 156.9ppm, 155.7ppm, 123.8ppm, 121.9ppm and 105.8ppm; two of double-bonding carbons at l lό.Oppm and 129.9ppm; two of anomeric carbons at 98.5ppm and 98.4ppm; eight of hydroxylated methine carbons at 72 to 64ppm; two of methyl carbons at 16.3ppm and 16.4ppm. Consequently, the results indicated that the compound of the invention is glycoside of flavonoid consisting of isoflavonol moiety and two sugars. As a result of structural analysis based on ¹³C-NMR, HMQC and HMBC data, aglycone was genistein. From GC analysis and NMR explanation based on trimethylsilylation and existence of methyl at 1.05ppm, the sugar was 6-deoxy-talose. Further, HMBC demonstrated that two of 6-deoxy-taloses were bonded to the aglycone at 7 and 4' carbon members of the aglycone through O-glycosylation. Accordingly, the compound bonded with two sugars has the chemical structure determined for the genistein 4',7-di-α-L-6-deoxy-talopyranoside as shown in the formula Ia (see Fig. 3, Fig. 4, Table 1 and Table 2). Similarly, structure of the genistein 7~α-L-6-deoxy-talopyranoside which is bonded with one of sugar was also identified by the same procedure that was applied to the genistein 4',7-di-α-L-6-deoxy-talopyranoside (see Fig. 5, Fig. 6, Table 1 and Table 2).

Furthermore, the sugar has a chemical shift value of ¹³C-NMR near to that of 6- deoxy-talose rather than that of L-rhamnose, therefore, it was finally determined from sugar determination test performed in Example 4 below that the sugar contained in the compound of the invention wasα-L-6-deoxy-talose (see Table 3). [TABLE 1]

¹³C NMR and ¹H NMR data of genistein 7-α-L-6-deoxy-talopyranoside (1) and genistein 4',7-di-α-L-6-deoxy-talopyranoside (2).

1 2

Atom δ_H (J, Hz) δc δ H (J, Hz)

2 154.3 CH 8 .38 (IH. s) 154.7 CH 8.46 (IH, s)

3 122.6 C 121.9 C

4 180.2 C 179.9 C

5 161.7 C 161.0 C

6 99.9 CH 6 .46 (IH, s) 99.6 CH 6.50 (IH, s)

7 160.8 C 161.0 C

8 94.4 CH 6 .72 (lH.s) 94.5 CH 6.77 (IH, s)

9 157.1 C 156.9 C

10 106.4 C 105.8 C r 120.9 C 123.8 C

2',6' 131.1 CH 7 .38 (2H, d, 10.2) 129.9 CH 7.50 (2H,d, 10.2)

3\5' 115.0 CU 6 .83 (2H, d, 10.2) 116.0 CH 7.11 (2H, d, 10.2)

4¹ 157.6 C 155.7 C

1" 98.4 CH 5.54 (IH, brs)

2" 69.7 CH 3.75(111, m)

3" 64.8 CH 3.77 (IH, m)

4" 71.7 CH 3.53 (IH, m)

5" 67.6 CH 3.83 (IH, m)

6" 16.4 < 1.05 (3H, d.6.6)

1"' 98.8 ClI 5 .69 (IH, brs) 98.5 CH 5.70(111, brs)

2'" 69.8 CM 3 .77 (IH. m) 69.3 CH 3.75 (IRm)

3'" 65.0 ClI 3 .79 (IH. m) 64.6 CU 3.77 (III, m)

4'" 719 CII 3 .56 (IH. m) 71.5 CH 3.53 ( 1 H, m)

5'" 6X4 en 3 .82 (IH. m) 681 CH 3.83 (III, m)

6"' 16.4 ( ^"FI., I .11 (3H. d.66) 163 i C-H. 1.05 (311. d, 6.6)

[TABLE 2]

Physical and chemical properties of the compound of the invention

[TABLE 3]

Comparison of chemical shift values of ¹³C-NMR between sugar moieties of known 6- deoxy-talose and the compound of the invention

Example 4 was conducted to determine three-dimensional structure of the sugar moiety contained in the compound of the invention. EXAMPLE 4. Determination of sugar moiety of the compound of the invention

In order to determine sugar moiety bonded to aglycone of glycoside, the isolated material was subjected to acid hydrolysis to remove the sugar moiety from the aglycone. After derivation of the removed sugar moiety using TMS (trimethylsilane), the obtained material was analyzed by GC (gas chromatography). The same experimentation was repeatedly conducted three times. As a result, retention time of sugar moiety contained in the above material was 16.873 minutes, as shown in the following GC chromatogram. However, L-rhamnose and L-fucose had 16.109 minutes and 17.532 minutes of the retention time, respectively. Therefore, it was proved that the sugar moiety contained in the above material was neither L-rhamnose nor L-fucose (see Fig.7, Fig.8 and Fig.9). Lastly, TMS derivative of the sugar bonded to the compound of the invention was observed in ¹H NMR and ¹H-¹H COSY spectrum to determine coupling constant and bonding, thereby identifying 6-deoxy-talose as the sugar moiety (see Fig.10).

As described above, the sugar moiety has C₄ conformation and Ci conformation in chair form, which may be converted each other along with surrounding circumstance such as solvent, temperature, etc.

(1) Determination of L-form sugar

Absolute configuration of the sugar moiety in the above material was determined by optical rotation ([α]o, Jasco DIP-1000, Japan; determined by dissolving the material in methanol). Optical rotation of known L-6-deoxy-talopyranoside is - 13.7° (c. 0.15) as a negative value, and the present compound had -16.8° (c. 0.1, methanol) of the optical rotation which is also a negative value, thereby indicating the sugar in L-form (see, e.g., Shibuya N, Amanto K, Azuma JI, Nishihara T, Kitamura Y, Noguchi T, Koga T. 6-Deoxy-D-talan and 6-Deoxy-L-talan. The Journal of Biological Chemistry 266: 16318-16323, 1991, which is hereby incorporated by reference in its entirety).

(2) Determination of anomer in sugar (determination of ocβform) Structure of anomer in the sugar moiety was determined asα-form since the coupling constant of the trimethylsilylated sugar was small such as J_H-_I, _H-_{2 (}2.3HZ), as shown in a following formula 2:

[Formula 2]

EXAMPLE 5. Anti-fungal activity of the compound of the invention

Genistein 4',7-di-α-L-6-deoxy-talopyranoside and genistein 7-α-L-6-deoxy- talopyranoside isolated from the above actinomycetes Kitasatospora kifunensis MJM- 341 were subjected to determination of anti-fungal activity to a wide range of fungi including, for example, Cryptococcus neoformans, Candida albicans, Aspergillus niger, Trichophyton mentagrophytes and Rhizoctonia solani and the like in MIC (minimal inhibition concentration) values.

More particularly, the determination of anti-fungal activity was performed in the following procedure: strain sample to be tested was incubated in RPMI 1640 medium (which does not contain bicarbonate but L-glutamine; available from Sigma Chemical Co., St. Louis, Mo.) for 24 hours, then, dispensed into 96-well plate type of test tubes (which are round-bottom micro-titration plates) in desired concentration ranging from O.5^χlO³ to 2.5 x 10³ cells/ml. Thereafter, genistein 4',7-di-α-L-6-deoxy- talopyranoside and genistein 7-α-L-6-deoxy-talopyranoside for testing were added into the test tubes except controls, in a concentration ranging from 1 /ig/ml to 30 βg/πύ, and cultured at 35 ^°C for 48 hours. By using a spectrometer in visible ray range, it can be observed whether the strains were grown or not. MIC value was set up to a concentration at which 80% of the test tubes containing the compound of the invention had not grown, compared to the test tube containing no medicine.

As listed in the following Table 4, it was demonstrated that both of genistein 4',7-di-α-L-6-deoxy-talopyranoside and genistein 7-α-L-6-deoxy-talopyranoside had strong anti-fungal activity to various fungi causing systemic mycoses such as Cryptococcus neoformans, Candida albicans, Aspergillus niger, as well as Rhizoctonia solani which is the plant pathogenic fungus, although they exhibited no anti-fungal activity to Trichophyton mentagrophytes which develops Athlete's foot.

[TABLE 4]

Anti-fungal activity of the compound of the invention (MIC value)

EXAMPLE 6. Cytotoxicity of the compound of the invention to liver cells

Genistein 4',7-di-α-L-6-deoxy-talopyranoside and genistein 7-α-L-6-deoxy- talopyranoside isolated from the above actinomycetes Kitasatospora kifunensis MJM- 341 were subjected to cytotoxicity test to liver cells, particularly, HepG2 cells.

100 μi test solution containing l*10⁴ of HepG2 cells per well was added to 96-well plates, incubated in an incubator at 37^°C, under CO₂ atmosphere for 12 hours, then, 100 μi of the compound of the invention to be examined was added to the incubated solution. After further incubating the solution for 72 hours, the number of cells proliferated was determined by MTT detection method (Promega, USA) according to the instructions provided by the manufacturer.

As listed in the following Table 5, the compound of the invention did not represent cytotoxicity since the concentration CC₅₀ leading to 50% of fatality rate for liver cells was 100 μg/ml. Accordingly, it is expected that the compound of the invention has a possibility of applying to develop antifungal medicines with no toxicity in clinical use. [TABLE 5] Cytotoxicity of the compound of the invention to liver cells

Although the preferred embodiments and examples of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. Sequence Listings

Sequence listing is enclosed.

Claims

What is Claimed is:

1. Genistein derivative compound represented by the following formula 1 and pharmaceutically acceptable salts thereof: [Formula 1]

wherein R represents hydrogen or

2. The genistein derivative compound and pharmaceutically acceptable salts thereof according to claim 1, wherein the compound represented by the formula 1 comprises genistein 4',7-di-α-L-6-deoxy-talopyranoside of the following formula Ia or genistein 7-α-L-6-deoxy-talopyranoside of the following formula Ib: [formula Ia]

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3. A method for producing the compound of claim 1, wherein the compound is isolated from Kitasatospora kifunensis MJM-341 (accession number: KCCM- 10710P).

4. Anti-fungal composition comprising the compound of claim 1 and pharmaceutically acceptable salts thereof.

5. Anti-fungal composition comprising the extract of Kitasatospora kifunensis

MJM-341 (accession number: KCCM-10710P).

6. The anti-fungal composition according to claim 5, wherein the extract includes at least one of compounds selected from the group consisting of the compounds of formula Ia and the formula Ib as an active ingredient: [formula Ia]

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[formula Ib]

7. The anti-fungal composition according to claim 4 or 5, wherein the composition is administered to animals or plants.

8. The anti-fungal composition according to claim 4 or 5, wherein the fungus is pathogenic fungus selected from the group consisting of Candida albicans, Cryptococcus neoformans, Aspergillus niger and Rhizoctonia solani.

9. Kitasatospora kifunensis MJM-341 comprising 16s rRNA sequence represented by SEQ. ID. No.l (accession number: KCCM-10710P).

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