JP6650630B2 - Ergosterol-modified carrier, method for screening candidate antifungal agent, and method for purifying raw material of antifungal agent - Google Patents

Description

  The present disclosure relates to a technique for screening candidate substances for antifungal agents and a technique for purifying raw materials for antifungal agents.

  Novel antifungal agents have been developed by screening candidate substances for antifungal agents from substances produced by microorganisms (see Non-Patent Document 1). For example, amphotericin B, which is one of antifungal agents, is extracted from a culture solution of actinomycetes and used as a therapeutic agent for deep mycosis.

Helvetica Chimica Acta, 1981, 64, 1752-1765

  The above-mentioned amphotericin B binds to ergosterol, which is a main component of the fungal cell membrane, and has an action of destroying the fungus by forming pores in the cell membrane. However, amphotericin B also acts on cholesterol, which is a main component of the cell membrane of animals, and may cause serious side effects such as nephrotoxicity. Under such circumstances, development of a novel antifungal agent replacing amphotericin B has been desired.

  An object of one aspect of the present disclosure is to provide a technique for screening a candidate substance of an antifungal agent and a technique for purifying a raw material of an antifungal agent.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, succeeded in screening candidate antifungal agents and purifying raw materials of antifungal agents using an ergosterol-modified carrier described below. Thus, the present disclosure has been completed.

That is, one embodiment of the present disclosure is an ergosterol-modified carrier, in which ergosterol is chemically modified on the carrier directly or via a linker.
Another embodiment of the present disclosure is a method for screening a candidate substance for an antifungal agent, which includes a first step, a second step, and a third step. In the first step, there is a possibility that an ergosterol-modified carrier in which ergosterol is chemically modified directly or via a linker, and a candidate antifungal agent having a property of being adsorbed to ergosterol may be contained. The sample is brought into contact with a test sample. In the second step, the candidate substance may be adsorbed by removing impurities not adsorbed to the ergosterol-modified carrier from the ergosterol-modified carrier and the test sample contacted in the first step. The ergosterol-modified carrier is selected. In the third step, a process of releasing the candidate substance from the ergosterol-modified carrier to which the candidate substance has been adsorbed is performed on the ergosterol-modified carrier selected in the second step to try to recover the candidate substance.

  Still another embodiment of the present disclosure is a method for purifying a raw material of an antifungal agent, which includes a first step, a second step, and a third step. In the first step, an ergosterol-modified carrier in which ergosterol is chemically modified on the carrier directly or via a linker, and a raw material before purification containing a raw material of an antifungal agent having a property of being adsorbed to ergosterol, Make contact. In the second step, the ergosterol-modified carrier and the unpurified raw material contacted in the first step are removed to remove impurities not adsorbed on the ergosterol-modified carrier, whereby the ergosterol adsorbed with the antifungal agent raw material is removed. Screen the modified carrier. In the third step, a treatment for releasing the antifungal agent raw material from the ergosterol-modified carrier to which the antifungal agent raw material is adsorbed is performed on the ergosterol-modified carrier selected in the second step, whereby the antifungal agent is removed. Collect the raw material of the agent.

  By using the screening method of the present disclosure, a substance adsorbed on ergosterol can be recovered from a test sample in which many substances can be mixed. The substance adsorbed on ergosterol may itself have antifungal activity, in which case the screened substance itself can be used as an antifungal agent. Further, even when the screened substance itself does not have remarkable antifungal activity, by adding a functional group such that the antifungal activity is expressed or improved while leaving the property of being absorbed by ergosterol. Can impart antifungal activity to the screened material. Therefore, a novel antifungal agent can be developed by using the screening method of the present disclosure.

  In particular, since the substance adsorbed on ergosterol can be recovered by using the screening method of the present disclosure, a substance having a stronger antifungal activity than an antifungal agent that inhibits ergosterol biosynthesis may be obtained. There is.

  In addition, by using the screening method of the present disclosure, impurities that have not been adsorbed to the ergosterol-modified carrier are removed, so that the screening can be performed simultaneously with the screening of candidate antifungal agents.

  In addition, by using the purification method of the present disclosure, a substance adsorbed on ergosterol can be purified and recovered. The substance adsorbed on ergosterol is a raw material of the antifungal agent for the above-mentioned reason. Therefore, an antifungal agent can be produced using the raw material purified by the purification method of the present disclosure.

FIG. 1A is a chromatogram of sample 1A. FIG. 1B is a chromatogram of Sample 1B. FIG. 1C is a chromatogram of Sample 1C. FIG. 1D is a chromatogram of Sample 1D. FIG. 2A is a chromatogram of Sample 2A. FIG. 2B is a chromatogram of Sample 2B. FIG. 2C is a chromatogram of Sample 2C. FIG. 2D is a chromatogram of Sample 2D. FIG. 3A is a chromatogram of a 50% ethanol culture solution extract and a methanol extract extracted using a culture solution obtained by culturing actinomycetes of strain K15-0257. FIG. 3B is a chromatogram of a 50% ethanol culture extract and a methanol extract extracted using a culture solution obtained by culturing actinomycetes of strain K15-0265.

1. Ergosterol-modified carrier The ergosterol-modified carrier of the present disclosure is one in which ergosterol is chemically modified on the carrier directly or via a linker.

  The carrier used in the present disclosure is not particularly limited, and examples thereof include silica gel, cellulose, agarose, dextran, and polyvinyl chloride. Here, examples of ergosterol-modified silica gel which is an ergosterol-modified carrier when the carrier is silica gel are shown in the following formulas (I) and (II).

The linker used in the present disclosure is an atom or an atomic group connecting between ergosterol and the carrier. The linker is not particularly limited as long as it does not impair the function of ergosterol of the ergosterol-modified carrier. The position of the linker is not particularly limited as long as the effect of ergosterol of the ergosterol-modified carrier is not impaired. As shown in the following formula (I), the linker of the ergosterol-modified silica gel may be -RS-. R is a hydrocarbon chain (e.g. _C n _{H 2n).} Examples of linkers ergosterol modified silica gel represented by Formula _{(I), -CH 2 -S -} , - C 2 H 4 -S -, - C 3 H 6 -S -, - C 4 H 8 -S -And the like. Further, as shown in the following formula (II), the linker of the ergosterol-modified silica gel may be -X-. X is an atom or an atomic group connecting between ergosterol and silica gel. Examples of linkers ergosterol modified silica gel represented by Formula _{(II), -C 3 H 6} -NH-CH 2 -CO -, - C 3 H 6 -O-CH 2 -CHOH -, - C 3 H _6- and the like.

2. Screening method of candidate antifungal agent The screening method of the present disclosure is a method of attempting to recover a candidate antifungal agent from a test sample by using an ergosterol-modified carrier in affinity chromatography.

  The antifungal agent in the present disclosure broadly means an agent having a fungicidal action or a growth inhibitory action on fungi. The antifungal agent of the present disclosure targets ergosterol, which is a main component of a fungal cell membrane, as a target molecule. Further, the antifungal agent of the present disclosure can be used as a therapeutic agent for mycosis or an agricultural chemical. Examples of the antifungal agent include, but are not particularly limited to, for example, a remedy for dermatophytosis infecting humans and pets, a remedy for fungal diseases such as candidiasis, and leishmaniasis caused by Leishmania protozoa. Therapeutic agents, pesticides for diseases derived from plant fungi (for example, downy mildew and powdery mildew), fungicides for suppressing the occurrence of mold around water in baths and the like can be mentioned.

  The test sample in the present disclosure refers to a sample used to evaluate whether or not a substance adsorbed to ergosterol is present in the test sample using the screening method of the present disclosure. Examples of test samples include, for example, extracts extracted from animals, plants and microorganisms (eg, lactic acid bacteria, yeasts, molds, actinomycetes, etc.), culture solutions collected after culturing microorganisms for a certain period of time, And a chemically synthesized solution. However, the test sample is not limited to those exemplified above. In particular, actinomycetes contain many bacteria that produce antibiotics, and thus, actinomycetes are highly likely to produce substances useful as antifungal agents. Therefore, it is preferable to use a culture solution collected after culturing actinomycetes for a certain period of time as a test sample.

The screening method of the present disclosure can be performed by performing the following steps (a1), (b1), and (c1).
(A1) An ergosterol-modified carrier in which ergosterol is chemically modified directly or via a linker, and a carrier that may contain a candidate substance of an antifungal agent having a property of being adsorbed to ergosterol. A first step of bringing the test sample into contact with the test sample;
(B1) Ergosterol which may have adsorbed a candidate substance by removing impurities not adsorbed on the ergosterol-modified carrier from the ergosterol-modified carrier and the test sample contacted in the first step Second step of selecting a modified carrier.
(C1) a third step of attempting to recover the candidate substance by applying a treatment for releasing the candidate substance from the ergosterol-modified carrier to which the candidate substance has been adsorbed to the ergosterol-modified carrier selected in the second step .

  As the step (a1) of the screening method of the present disclosure, for example, a step of contacting the ergosterol-modified carrier with the test sample by passing the test sample through a column packed with an ergosterol-modified carrier as a main component ( Column method), a step of adding an ergosterol-modified carrier to a test sample and stirring the mixture (batch method), and the like.

  As the step (b1) of the screening method of the present disclosure, for example, a step of washing the column with a washing solution (column method), the ergosterol-modified carrier and the test sample brought into contact in the step (a1) are centrifuged, and ergosterol is obtained. A step of recovering the modified carrier (batch method) and the like can be mentioned.

  In step (c1) of the screening method of the present disclosure, the candidate substance can be released from the ergosterol-modified carrier to which the candidate substance has been adsorbed, using a polar solvent. As the polar solvent, for example, alcohol solvents such as ethanol and methanol can be used. In addition, polar solvents such as acetonitrile and acetone can also be used.

Further, between the step (b1) and the step (c1), a step of washing the ergosterol-modified carrier selected in the step (b1) may be included.
When such a screening method is used for high performance liquid chromatography or the like, a candidate antifungal agent can be concentrated or purified from a test sample.

3. Purification Method of Antifungal Agent Raw Material The purification of the present disclosure is a method of recovering an antifungal agent raw material from a pre-refining raw material containing an antifungal agent having a property of being adsorbed to ergosterol.

  The raw material of the antifungal agent of the present disclosure is not particularly limited, and examples thereof include candidate antifungal agents screened using the screening method of the present disclosure, novel antifungal agents, and existing antifungal agents. No.

The purification method of the present disclosure can be performed by performing the following steps (a2), (b2), and (c2).
(A2) An ergosterol-modified carrier in which ergosterol is chemically modified directly or via a linker is brought into contact with a raw material before purification containing a raw material of an antifungal agent having a property of being adsorbed to ergosterol. First step.
(B2) The ergosterol-modified carrier to which the raw material of the antifungal agent has been adsorbed by removing impurities not adsorbed on the ergosterol-modified carrier from the ergosterol-modified carrier and the raw material before purification contacted in the first step A second step of sorting out.
(C2) A treatment for releasing the antifungal agent raw material from the ergosterol-modified carrier to which the antifungal agent material is adsorbed is performed on the ergosterol-modified carrier selected in the second step, whereby the antifungal agent Third step of collecting raw materials.

  As the step (a2) of the purification method of the present disclosure, for example, a step of contacting the ergosterol-modified carrier with the raw material before purification by passing the raw material before purification through a column packed with the ergosterol-modified carrier as a main component ( Column method), a step of adding an ergosterol-modified carrier to the raw material before purification and stirring the mixture (batch method), and the like.

  As the step (b2) of the screening method of the present disclosure, for example, a step of washing the column with a washing solution (column method), the ergosterol-modified carrier and the raw material before purification contacted in the step (a2) are centrifuged, and ergosterol is obtained. A step of recovering the modified carrier (batch method) and the like can be mentioned.

  In step (c2) of the screening method according to the present disclosure, the antifungal agent raw material can be released from the ergosterol-modified carrier to which the antifungal agent raw material has been adsorbed using a polar solvent. As the polar solvent, for example, alcohol solvents such as ethanol and methanol can be used. In addition, polar solvents such as acetonitrile and acetone can also be used.

Further, between the step (b2) and the step (c2), a step of washing the ergosterol-modified carrier selected in the step (b2) may be included.
Even if the ergosterol-modified carrier is used for high-performance liquid chromatography or the like, the raw material of the antifungal agent can be purified from the raw material before purification.

Hereinafter, the present disclosure will be specifically described, but the present disclosure is not construed as being limited by these examples.
[Example 1: Production example]
An ergosterol-modified silica gel represented by the formula (III) was produced as represented by the following chemical formula 3. The ergosterol-modified silica gel represented by the formula (III) is an example of the ergosterol-modified silica gel represented by the formula (I). The specific manufacturing method is as follows.

  20 g of spherical silica gel (Chromatorex MB100-75 / 200, manufactured by Fuji Silysia Chemical Ltd.) was weighed and placed in a 500 mL flask. 300 mL of toluene was added thereto, and 5.9 g of 3-mercaptopropyltrimethoxysilane was added thereto while stirring the slurry, and reacted under a nitrogen atmosphere reflux condition. After cooling, the solution was drained and washed with an organic solvent. It was dried at 80 ° C. to obtain 21.5 g of silica gel having a thiol group on the surface. Silica gel having a thiol group on the surface is represented by the formula (α).

  Further, 20 g of silica gel having a thiol group was weighed and placed in a 500 mL flask. 300 mL of 1,4-dioxane was added thereto, and while stirring the slurry, 4.0 g of ergosterol was added to completely dissolve the mixture. Then, 80 mg of azobisisobutyronitrile was added, and the mixture was heated at 85 ° C. in a nitrogen atmosphere. Stirred. After cooling, the slurry was filtered, washed with an organic solvent, drained, and dried at 80 ° C. to obtain 20.7 g of ergosterol-modified silica gel represented by the formula (III).

[Example 2: Production example]
An ergosterol-modified silica gel represented by the formula (III) was produced as represented by the following chemical formula 4. The specific manufacturing method is as follows.

  10 mmol of 3-mercaptopropyltrimethoxylane and 10 mmol of ergosterol were placed in a separable flask, and 200 mL of 1,4 dioxane was added and dissolved. 5% mol of AIBN (azobisisobutyronitrile) was added to ergosterol, and the mixture was heated to 85 ° C. with stirring to react, thereby obtaining a silane coupling agent modified with ergosterol. Ergosterol-modified silane coupling agents are shown in formula (β).

  Further, 20 g of spherical silica gel (Chromatorex MB100-75 / 200, manufactured by Fuji Silysia Chemical Ltd.) was added to the reaction solution, and a reflux reaction was performed to immobilize the silica gel. After the reaction, the solution was removed, washed with an organic solvent, and dried to obtain ergosterol-modified silica gel represented by the formula (III).

[Example 3: Production example]
An ergosterol-modified silica gel represented by the formula (IV) was produced as represented by the following chemical formula 5. The ergosterol-modified silica gel shown in the formula (IV) is an example of the ergosterol-modified silica gel shown in the above formula (II). The specific manufacturing method is as follows.

  Ergosterol (8.0 g, 20 mmol) was weighed into a 200 mL flask and dissolved in 60 mL of dry THF (tetrahydrofuran). The mixture was cooled on an ice bath under a nitrogen atmosphere, and 45 mmol of NaH (sodium hydride) was gradually added. After adding NaH, the mixture was stirred for 30 minutes, bromoacetyl bromide (6.05 g, 30 mmol) was diluted with 10 mL of dry THF, and added over 1 hour using a dropping funnel. After adding the whole amount, the reaction was further performed for 3 hours while cooling in an ice bath, and 1N hydrochloric acid was added to terminate the reaction. The THF in the reaction solution was removed with an evaporator, and the aqueous layer was extracted with ethyl acetate using a separating funnel. The organic layer was washed with a 5% sodium hydrogen carbonate solution, 1N hydrochloric acid, water, and saturated saline, and dried over sodium sulfate. The solvent was distilled off, and the residue was purified by recrystallization from ethyl acetate / hexane to give the compound represented by the formula (γ) as a pale yellow solid (yield 43%).

  The compound represented by the formula (γ) (3.131 g, 6.1 mmol) was weighed in a flask and dissolved in 200 mL of THF. 11 g of spherical silica gel (Chromatorex NH MB100-75 / 200, manufactured by Fuji Silysia Chemical Ltd.) was added, and the mixture was reacted under heating and reflux conditions with stirring. After reacting for 12 hours, the reaction solution was drained, washed with an organic solvent, and dried to obtain ergosterol-modified silica gel represented by the formula (IV). The yield is shown in Table 1 below.

  The carbon content of the ergosterol-modified silica gel represented by the formula (IV) was determined by elemental analysis (using an elemental analyzer “Vario EL III” (manufactured by Elementar)). The functional group content of the ergosterol-modified silica gel represented by the formula (IV) was calculated from the difference from the carbon content of the raw material silica gel (Chromatorex NH MB100-75 / 200).

  The amount of the functional group calculated from the carbon content and the molecular structure was smaller than the amount of the functional group of the raw material silica gel of 970 μmol / g. However, considering that the functional groups are sterically large, the amount of the functional groups bonded is appropriate.

[Example 4: Adsorption test]
Commercially available amphotericin B was dissolved in a small amount of DMSO (dimethyl sulfoxide) and adjusted to a concentration of 100 μg / mL with methanol. The adjusted solution was diluted 10 times with distilled water to prepare a solution having a concentration of 10 μg / mL. 50 mg of the ergosterol-modified silica gel (hereinafter referred to as ergosterol-modified silica gel (III)) obtained in Example 1 was placed in a 1.5 mL centrifuge tube, and a small amount of methanol was added thereto. In addition, the mixture was stirred. To this, 1 mL of an amphotericin B aqueous solution was added and stirred. After centrifugation, the supernatant was removed, 1 mL of distilled water was added, and the mixture was stirred, centrifuged, and the supernatant was collected. The supernatant was used as Sample 1B.

  Using 100 mg of ergosterol-modified silica gel (III) and 100 mg of unmodified silica gel in place of the above-mentioned 50 mg of ergosterol-modified silica gel (III), the supernatant was recovered in the same manner as above, and the supernatant was used as sample 1C. And Sample 1D.

The thus collected samples 1B, 1C, and 1D, and an aqueous solution of amphotericin B (sample 1A) having a concentration of 10 μg / mL were analyzed by high performance liquid chromatography (hereinafter, referred to as HPLC). HPLC conditions are as follows.
Apparatus: HITACHI LaChrom Ultra (manufactured by Hitachi High-Technologies Corporation)
Column: Inartsil ODS-4 (inner diameter 3.0 mm x length 250 mm, manufactured by GL Sciences Inc.)
Solvent conditions: solvent A: water / 0.1% formic acid, solvent B: methanol / 0.1% formic acid. The ratio of solvent B is continuously 5 to 100% (0 to 10 minutes) and 100% (10 minutes or more). Was changed to.
Flow rate: 0.5 mL / min Column temperature: 40 ° C
Detection: UV400nm
Sample injection volume: 5 μL
FIGS. 1A, 1B, 1C, and 1D show the results of HPLC analysis of Sample 1A, Sample 1B, Sample 1C, and Sample 1D, respectively.

  As shown in FIG. 1A, in Sample 1A not containing ergosterol-modified silica gel (III), a peak was observed around an elution time of 27 minutes. On the other hand, as shown in FIGS. 1B and 1C, in the case of Samples 1B and 1C containing ergosterol-modified silica gel (III), the peak area at an elution time of about 27 minutes was smaller than that of Sample 1A. From this, it was found that amphotericin B was adsorbed on ergosterol-modified silica gel (III).

  Furthermore, since the peak area around the elution time of 27 minutes shown in FIG. 1C was smaller than the peak area shown in FIG. 1B, depending on the weight of ergosterol-modified silica gel (III), amphotericin B was changed to ergosterol-modified silica gel. (III) was found to be adsorbed.

In addition, as shown in FIG. 1D, a peak was observed around an elution time of 27 minutes, indicating that amphotericin B was not adsorbed to the unmodified silica gel.
[Example 5: Adsorption test and dissolution test]
Streptomyces sp. Stored at Kitasato Life Science Institute. K04-0037 strain (Streptomyces sp. Strain K04-0037) was prepared by adding 0.5% glucose, 0.5% Corn step powder, 1.0% oatmeal, 1.0% Pharmamedia, 0.5% K ₂ HPO ₄ , 0 amphotericin B is produced when cultured in a culture solution containing _{.4% MgSO 4 · 7H 2 O} . Using this culture solution, an adsorption test and an elution test of amphotericin B on the ergosterol-modified silica gel (III) obtained in Example 1 were performed.

  The K04-0037 strain was cultured with shaking in the above culture solution at 27 ° C. for 6 days. 50% ethanol was added to the culture solution, and the mixture was vigorously stirred, and the supernatant after centrifugation, which was the 50% ethanol culture solution extract, was collected and designated as Sample 2A. 100 mg of ergosterol-modified silica gel (III) obtained in Example 1 was taken and dispersed in a 1.5 mL centrifuge tube by adding a small amount of methanol, and a small amount of distilled water was added and stirred. Here, 1 mL of a 50% ethanol culture extract was added and stirred. After centrifugation, the supernatant was recovered and used as sample 2B. The precipitated silica gel was washed with 1 mL of distilled water, centrifuged, and the supernatant was collected to obtain Sample 2C. 1 mL of methanol was added to the precipitated silica gel, and the mixture was stirred well, and the methanol extract, which was the supernatant after centrifugation, was collected to obtain Sample 2D.

The samples 2A, 2B, 2C and 2D thus collected were respectively analyzed by HPLC. HPLC conditions are as follows.
Apparatus: HITACHI LaChrom Ultra (manufactured by Hitachi High-Technologies Corporation)
Column: Monobis (3.3 mm inner diameter x 150 mm length, manufactured by Kyoto Monotech Co., Ltd.)
Solvent conditions: solvent A: water / 0.1% formic acid, solvent B: methanol / 0.1% formic acid. Was changed to
Flow rate: 0.5 mL / min Column temperature: 40 ° C
Detection: UV400nm
Sample injection volume: 5 μL
FIGS. 2A, 2B, 2C, and 2D show the results of HPLC analysis of Sample 2A, Sample 2B, Sample 2C, and Sample 2D, respectively.

  As shown in FIG. 2A, a peak was observed around an elution time of 27 minutes, indicating that amphotericin B was contained in the 50% ethanol culture solution extract (sample 2A).

  As shown in FIG. 2B, in the 50% ethanol culture solution extract (sample 2B) contacted with ergosterol-modified silica gel (III), a peak was observed around an elution time of 27 minutes. It was smaller than the peak area shown in 2A. Since amphotericin B was adsorbed to the ergosterol-modified silica gel (III) in a saturated state, excess amphotericin B not adsorbed was observed.

As shown in FIG. 2C, no peak was observed around the elution time of 27 minutes, and thus amphotericin B was not observed in the supernatant (sample 2C) of the washing solution with distilled water.
As shown in FIG. 2D, a peak was observed around an elution time of 27 minutes, and amphotericin B was confirmed in the methanol extract (sample 2D). Therefore, amphotericin B contained in the 50% ethanol culture extract (sample 2A) was adsorbed on ergosterose-modified silica gel (III) and could be eluted with methanol.

[Example 6: Screening]
Actinomycetes isolated from various environments were cultured in the culture solution used in Example 3, and an equal amount of ethanol was added thereto to prepare 20 samples of a 50% ethanol culture solution extract. 100 mg of ergosterol-modified silica gel (III) obtained in Example 1 was taken and dispersed in a 1.5 mL centrifuge tube by adding a small amount of methanol, and a small amount of distilled water was added and stirred. To this, 1 mL of a 50% ethanol culture extract was added and stirred. After centrifugation, the precipitated silica gel was washed with 1 mL of distilled water, centrifuged, and the supernatant was removed. 1 mL of methanol was added, and the mixture was stirred well and centrifuged to collect a supernatant methanol extract. The 50% ethanol culture extract and the methanol extract were analyzed by HPLC.

HPLC conditions are as follows.
Apparatus: HITACHI LaChrom Ultra (manufactured by Hitachi High-Technologies Corporation)
Column: Monobis (inner diameter 3.3 mm x length 150 mm, manufactured by Kyoto Monotech Co., Ltd.)
Solvent conditions: solvent A: water / 0.1% formic acid, solvent B: methanol / 0.1% formic acid. The ratio of solvent B is continuously 5 to 100% (0 to 10 minutes) and 100% (10 minutes or more). Was changed to.
Flow rate: 0.5 mL / min Column temperature: 40 ° C
Detection: UV400nm
Sample injection volume: 5 μL
FIG. 3A shows the results of HPLC analysis of a 50% ethanol culture solution extract (culture solution extract in the figure) and a methanol extract extracted using a culture solution obtained by culturing actinomycetes of K15-0257 strain. . FIG. 3B shows the results of HPLC analysis of a 50% ethanol culture solution extract (culture solution extract in the figure) and a methanol extract, which were extracted using a culture solution obtained by culturing actinomycetes of K15-0265 strain. .

  As shown in FIG. 3A, in the methanol extract extracted using the culture solution containing no substance adsorbed on the ergosterol-modified silica gel (III), no remarkable peak was observed at an elution time of 6 minutes. Was. On the other hand, as shown in FIG. 3B, a peak was confirmed at an elution time of 6 minutes in a methanol extract extracted using a culture solution containing a substance adsorbed on ergosterol-modified silica gel (III). .

  Further, a large peak was observed in the extract of the 50% ethanol culture solution at an elution time of about 10 minutes shown in FIG. 3A and about 3 minutes shown in FIG. 3B, but the methanol extract after the treatment with ergosterol-modified silica gel (III) was observed. , And only the substance adsorbed on ergosterol is eluted with methanol, so that purification is easy.

In fact, the substance with an elution time of 6 minutes shown in FIG. 3B was found to be Streptazolin (molecular weight 207, C ₁₁ H ₁₄ NO ₃ ). The antifungal activity is known as the biological activity of this substance, suggesting that there is activity expression due to affinity with ergosterol.

Claims (8)

An ergosterol-modified carrier in which ergosterol is chemically modified on the carrier directly or via a linker ,
The ergosterol-modified carrier, wherein the carrier is silica gel, agarose, or dextran. An ergosterol-modified carrier according to claim 1 ,
An ergosterol-modified carrier represented by the formula (I).
(However, in the formula (I), R represents a hydrocarbon chain.)

An ergosterol-modified carrier according to claim 1 ,
An ergosterol-modified carrier represented by the formula (II).
(In the formula (II), X represents an atom or an atomic group connecting ergosterol and silica gel.)

An ergosterol-modified carrier in which ergosterol is chemically modified on the carrier directly or via a linker, and a test sample which may contain a candidate antifungal agent having a property of being adsorbed to the ergosterol. And a first step of contacting
The candidate substance may be adsorbed by removing impurities not adsorbed to the ergosterol-modified carrier from the ergosterol-modified carrier and the test sample contacted in the first step. A second step of selecting the ergosterol-modified carrier,
The process of releasing the candidate substance from the ergosterol-modified carrier on which the candidate substance is adsorbed is performed on the ergosterol-modified carrier selected in the second step, thereby attempting to recover the candidate substance. A third step;
A method for screening a candidate substance for an antifungal agent, comprising: The screening method according to claim 4 , wherein
The screening method, wherein, in the first step, the test sample is passed through a column packed with the ergosterol-modified carrier as a main component, thereby bringing the ergosterol-modified carrier into contact with the test sample. The screening method according to claim 4 or claim 5 ,
In the third step, a screening method, wherein the candidate substance is released with methanol from the ergosterol-modified carrier to which the candidate substance has been adsorbed. The screening method according to any one of claims 4 to 6 , wherein
The screening method, wherein the test sample is a culture solution collected after culturing actinomycetes for a certain period of time. First contacting an ergosterol-modified carrier, in which ergosterol is chemically modified on the carrier directly or via a linker, with a raw material before purification containing a raw material of an antifungal agent having a property of being adsorbed to the ergosterol; Process and
By removing impurities not adsorbed to the ergosterol-modified carrier from the ergosterol-modified carrier and the raw material before purification contacted in the first step, the ergo to which the raw material of the antifungal agent has been adsorbed is removed. A second step of selecting a sterol-modified carrier,
By subjecting the ergosterol-modified carrier selected from the second step to release the antifungal agent from the ergosterol-modified carrier to which the antifungal agent has been adsorbed, A third step of recovering the fungicide material;
A method for purifying a raw material for an antifungal agent, comprising:

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