Screening method and production method for prostate cancer therapeutic agent

Abstract

To provide a technique that enables screening of components with prostate cancer therapeutic effect from microbial autolysates and also allows the recovery of such components with high recovery efficiency.SOLUTION: The present invention provides a screening method for a prostate cancer therapeutic agent comprising predetermined steps of autolysis reaction, target recovery, and culture.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for screening and a method for producing a therapeutic agent for prostate cancer. [Background technology]

[0002] Microbial metabolites have various functions and are used in the fields of food, medicine, and the like. One method for obtaining such metabolites is the autolysis of microorganisms (for example, Patent Document 1). Autolysis refers to the decomposition of microbial tissues by the action of enzymes produced by the microorganisms themselves after their death. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2002-355008 A Summary of the Invention [Problem to be solved by the invention]

[0004] In the course of searching for therapeutic agents for prostate cancer, the present inventors have found that useful components may be contained in autolysis products of microorganisms. On the other hand, the amount of useful components produced in the autolysis reaction products is very small, and a technology that can recover the useful components with high recovery efficiency is required.

[0005] The present invention has been made in consideration of the above-mentioned situation, and aims to provide a technology that can screen components having therapeutic effects on prostate cancer from the products of microbial autolysis reaction, and can recover such components with high recovery efficiency. [Means for solving the problem]

[0006] The present inventors have found that the above problems can be solved by subjecting the autolysis product of a microorganism to an extraction treatment using a combination of a specific solvent, and have thus completed the present invention. More specifically, the present invention provides the following.

[0007] (1) A method for screening a therapeutic agent for prostate cancer, comprising: The screening method comprises: an autolysis reaction step of obtaining a reactant by autolysis of a microorganism; a first recovery step of mixing the reactant and ethanol, centrifuging the mixture, and recovering a first supernatant; a second recovery step of mixing the first supernatant and butanol, centrifuging the mixture, and recovering a second supernatant; Culturing prostate cancer cells in the presence of the second supernatant; Including, In the culturing step, a first viable cell count is determined when the prostate cancer cells are cultured in the presence of the second supernatant, and a second viable cell count is determined when the prostate cancer cells are cultured under the same conditions as in the culturing step except that the prostate cancer cells are cultured in the absence of the second supernatant; When the first viable cell count relative to the second viable cell count is less than 90%, the second supernatant is obtained as a prostate cancer therapeutic agent. Screening methods.

[0008] (2) A method for producing a prostate cancer therapeutic agent, comprising the steps of: The manufacturing method comprises: an autolysis reaction step of obtaining a reactant by autolysis of a microorganism; a first recovery step of mixing the reactant and ethanol, centrifuging the mixture, and recovering a first supernatant; a second recovery step of mixing the first supernatant and butanol, centrifuging the mixture, and recovering a second supernatant; A manufacturing method comprising: Effect of the Invention

[0009] According to the present invention, a technique is provided that enables screening of components having therapeutic effects on prostate cancer from the products of microbial autolysis and enables recovery of such components with high recovery efficiency. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the results of HPLC analysis in an example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited thereto.

[0012] <Method of screening for prostate cancer therapeutic agents> The method for screening a therapeutic agent for prostate cancer according to the present invention (hereinafter also referred to as the "screening method of the present invention") comprises the following steps: · Autolysis reaction process in which reactants are obtained through the autolysis reaction of microorganisms A first recovery step of mixing the reactant obtained in the autolysis reaction step with ethanol, centrifuging the resulting mixture, and recovering a first supernatant. A second recovery step of mixing the first supernatant and butanol, centrifuging the mixture, and recovering a second supernatant. Culturing the prostate cancer cells in the presence of the second supernatant

[0013] It is known that metabolic products produced by microorganisms may contain useful components that have pharmacological effects on humans, etc. However, since the useful components contained in metabolites are often only present in trace amounts, identification of the useful components and their pharmacological actions requires large-scale cultivation and removal of contaminants, which are complicated procedures and require specialized equipment.

[0014] On the other hand, in the course of searching for therapeutic agents for prostate cancer, the present inventors have found that autolysis products, which are a type of metabolic product of microorganisms, may contain useful components. Therefore, the present inventors have investigated a simple and high-yield method for extracting useful components contained in the autolysis reaction product. As a result, it was found that useful components contained in the autolysis product can be easily obtained by subjecting the autolysis product to an extraction treatment with ethanol and butanol. Furthermore, it was found that the useful components obtained by such a treatment can have a prostate cancer treatment effect. Depending on the microorganism from which it is derived, the autolysis reaction product may contain almost no useful components, but by performing screening after the above-mentioned extraction process, the presence or absence of useful components can be easily determined.

[0015] Furthermore, the present inventors have unexpectedly found that useful components can be recovered with high recovery efficiency by subjecting the autolysis reaction product to ethanol extraction and then butanol extraction. In particular, it was a very unexpected finding that an extract obtained by extraction treatment with ethanol and butanol exhibited higher activity than the autolysis reaction product itself.

[0016] It was also found that when only ethanol extraction was performed on the autolysis product, the useful components could not be sufficiently recovered, and when only butanol extraction was performed, the recovery efficiency of the useful components was extremely low. From these findings, it was also found that if the order of ethanol extraction and butanol extraction was reversed, it was almost impossible to recover useful components.

[0017] In the present invention, the term "prostate cancer therapeutic effect" includes the effect of suppressing the progression of prostate cancer, the effect of curing prostate cancer, the effect of improving symptoms, the effect of maintaining the condition after cure, and the like, by inhibiting the growth and proliferation of prostate cancer cells.

[0018] In the present invention, the "(component) recovery efficiency" means the ratio of the extracted weight of the component (extracted weight / microbial pellet weight) to the amount of microorganisms (microbial pellet weight) subjected to the autolysis reaction. The recovery efficiency can be determined by the method shown in the Examples.

[0019] In the present invention, the term "high recovery efficiency (of a component)" encompasses, for example, a recovery efficiency determined by the method shown in the Examples of preferably 3% or more, more preferably 5% or more.

[0020] Each step of the screening method of the present invention will be described below.

[0021] (1) Self-digestion reaction process The autolysis reaction step is a step in which a microorganism undergoes an autolysis reaction to obtain a reaction product (autolysis reaction product).

[0022] In the present invention, the "reactant of the autolysis reaction of a microorganism" refers to a reactant (metabolite) obtained as a result of the decomposition action of enzymes produced by the microorganism itself. Such reactants include, for example, what is defined in the Food Chemicals Codex (FCC) as "autolyzed yeast."

[0023] (1-1) Types of microorganisms The microorganism in the present invention is not particularly limited as long as it causes an autolysis reaction, but preferably includes yeast, lactic acid bacteria, etc. The microorganism may be one type alone or two or more types in combination.

[0024] Examples of yeast fungi include those belonging to the genera Saccharomyces, Cyberlindnera, Debaryomyces, Candida, Yarrowia, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Williopsis, Galactomyces, Torulaspora, and Rhodotorula.

[0025] Examples of lactic acid bacteria include those belonging to the genera Lactobacillus, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, Streptococcus, and Bifidobacterium.

[0026] (1-2) Self-digestion reaction process The autolysis reaction can be carried out by any conventionally known process. Preferred examples are described below. As long as the autolysis step is carried out, some or all of the following steps may be carried out.

[0027] In addition, each of the following steps is preferably carried out at a pH close to neutral (for example, pH 5 or higher and 8 or lower) in order to avoid decomposition of useful components.

[0028] (1-2-1) Preculture step The pre-culture step is a step in which the microorganisms to be subjected to the autolysis reaction are grown in order to obtain a sufficient amount of reactants.

[0029] The conditions for the pre-culture step (medium, culture conditions, etc.) can be appropriately set depending on the type of microorganism, the amount of microorganism to be obtained, etc.

[0030] For the pre-culture step, the method shown in the Examples can be adopted.

[0031] (1-2-2) Pretreatment process The pretreatment step is a step of preparing the microorganism to be subjected to the autolysis reaction into a desired form.

[0032] In the pretreatment step, the microorganisms may be pelletized by centrifugation or the like, and then washed.

[0033] In the pretreatment step, the microorganisms may be pelleted by centrifugation or the like, and the pellets may be suspended in an appropriate solvent (culture medium, sterilized water, etc.).

[0034] The form of the microorganisms prepared in the pretreatment step is preferably a microbial liquid (a suspension of microorganisms).

[0035] The microorganisms obtained in the pretreatment step may be frozen and stored until they are subjected to the autolysis reaction.

[0036] The pretreatment step can employ the method shown in the Examples.

[0037] (1-2-3) Autolysis process The conditions for the autolysis step are not particularly limited, but preferably, the microorganisms placed in a medium (water, liquid medium, etc.) are subjected to any one of the following treatments. (i) Standing at 10 to 60°C (ii) Cultivation at 10 to 60°C with rotation at 10 to 30 rpm

[0038] The reaction time in the autolysis step can be appropriately set depending on the amount of microorganisms, etc., but is usually 16 hours or more. However, if the reaction time for the autolysis reaction is too long, there is a possibility that the useful components will decompose, so it is preferable to set the reaction time within 48 hours.

[0039] The type and amount of the medium in the autolysis step can be appropriately set depending on the type of microorganism, the amount of the reaction product to be obtained, etc.

[0040] The self-digestion step may employ the method shown in the Examples.

[0041] The autolysis process can be completed after a set reaction time has elapsed. However, it is preferable to confirm the lysis of the cell walls using a stereomicroscope to confirm that the autolysis reaction has progressed sufficiently and that an autolysis reaction product has been obtained.

[0042] (1-2-4) Process after the self-digestion process After the autolysis step is completed, the reaction product (liquid) is appropriately centrifuged, and the supernatant is collected as the autolysis reaction product.

[0043] After the autolysis step is complete, the microorganisms may be killed using a heat block or the like.

[0044] The resulting autolysis product may be frozen and stored until it is used in the subsequent steps.

[0045] (2) First recovery process The first recovery step is a step of mixing the reaction product obtained in the autolysis reaction step with ethanol, followed by centrifugation to recover a first supernatant.

[0046] In the present invention, "ethanol" includes any solution containing ethanol (C2H6O). However, from the viewpoint of increasing the extraction efficiency in the first recovery step, the ethanol is preferably 100% ethanol or an aqueous ethanol solution. When the ethanol is an aqueous ethanol solution, the ethanol concentration is not particularly limited as long as it is a sufficient amount to sufficiently recover useful components from the autolysis reaction product, but is preferably 50 vol% or more and 90 vol% or less, more preferably 60 vol% or more and 80 vol% or less, and most preferably 67 vol%.

[0047] The amount of ethanol is not particularly limited as long as it is sufficient to sufficiently recover useful components from the autolysis reaction product, but is preferably 0.5 to 5 times, more preferably 1.5 to 3 times, and most preferably 2 times the amount of the autolysis reaction product.

[0048] The reactants and ethanol are placed together in a container (such as a tube) and mixed appropriately before centrifuging. Before centrifuging, the mixture may be stirred by any means (such as a vortex mixer).

[0049] The conditions for centrifugation in the first recovery step are not particularly limited as long as the conditions allow a supernatant to be obtained. The rotation speed is preferably 10,000 rpm or more and 20,000 rpm or less. The centrifugation time is preferably from 5 minutes to 15 minutes. The centrifugation temperature is preferably 0°C or higher and 10°C or lower.

[0050] For the first recovery step, the method shown in the Examples can be adopted.

[0051] After centrifugation in the first recovery step, the resulting supernatant is recovered as a first supernatant, and the supernatant is subjected to a second recovery step. Before being subjected to the second recovery step, the first supernatant may be subjected to concentration or the like to reduce the amount of solvent (such as water).

[0052] (3) Second recovery process The second recovery step is a step of mixing the first supernatant with butanol, followed by centrifugation to recover a second supernatant.

[0053] In the present invention, "butanol" refers to butanol (CH 10 However, from the viewpoint of increasing the extraction efficiency in the second recovery step, the butanol is preferably 100% butanol or an aqueous butanol solution. When the butanol is an aqueous butanol solution, the butanol concentration is not particularly limited as long as it is a sufficient amount to sufficiently recover useful components from the first supernatant, but is preferably 30 vol% or more and 70 vol% or less, more preferably 40 vol% or more and 60 vol% or less, and most preferably 50 vol%.

[0054] The amount of butanol is not particularly limited as long as it is sufficient to sufficiently recover useful components from the first supernatant, but is preferably 0.3 to 3 times, more preferably 0.8 to 2 times, and most preferably the same amount (1 time) as the first supernatant.

[0055] The first supernatant and butanol are placed together in a container (such as a tube) and mixed appropriately, and if the mixture separates into two layers upon standing, the supernatant is recovered. If no clear separation is observed, centrifugation may be performed. The above mixing may be carried out by stirring using any means (such as a vortex mixer).

[0056] The conditions for centrifugation in the second recovery step are not particularly limited as long as the conditions allow a supernatant to be obtained. The rotation speed is preferably 10,000 rpm or more and 20,000 rpm or less. The centrifugation time is preferably from 5 minutes to 15 minutes. The centrifugation temperature is preferably 0°C or higher and 10°C or lower.

[0057] For the second recovery step, the method shown in the Examples can be adopted.

[0058] After centrifugation in the second recovery step, the resulting supernatant is recovered as a second supernatant and subjected to the culture step. Before being subjected to the culture step, the second supernatant may be subjected to concentration or the like to replace the solvent with a solvent other than butanol (such as an aqueous ethanol solution). In such a case, the solvent other than butanol is preferably an aqueous ethanol solution of 50 vol% to 100 vol%, more preferably an aqueous ethanol solution of 60 vol% to 80 vol%, and most preferably an aqueous ethanol solution of 67 vol%. Prior to the solvent replacement, the second supernatant may be powdered.

[0059] (4)Culture process The culturing step is a step of culturing the prostate cancer cells in the presence of the second supernatant. In this step, a first viable cell count is determined when the prostate cancer cells are cultured in the presence of a second supernatant, and a second viable cell count is determined when the prostate cancer cells are cultured under the same conditions as in the culture step, except that the prostate cancer cells are cultured in the absence of the second supernatant. Furthermore, the first viable cell count relative to the second viable cell count is determined, and based on these values, it is determined whether or not a component contained in the second supernatant is useful as a therapeutic agent for prostate cancer.

[0060] (4-1)Culture conditions The culture conditions in the culture step can be any conditions that allow the growth of prostate cancer cells, except that the culture is performed in the presence of the second supernatant. However, as described below, it is necessary to measure the number of viable prostate cancer cells at two points: at the start of the culture process and at the end of the culture process.

[0061] The prostate cancer cells used in the culturing step may be cell lines established from prostate cancer tissue derived from any animal (human or non-human mammal, etc.).

[0062] The prostate cancer cells are preferably derived from the same organism as the subject of treatment with the prostate cancer therapeutic agent. For example, when the subject of treatment with the prostate cancer therapeutic agent is a human, the prostate cancer cells used in the culture step are preferably human-derived cells (such as PC3 cells).

[0063] An example of a method for culturing in the presence of a second supernatant is a method in which the second supernatant is added to a growth medium for prostate cancer cells and then cultured. The amount of the second supernatant added is not particularly limited, but may be 1 vol % or more and 10 vol % or less relative to the growth medium.

[0064] The culture time in the culture step is not particularly limited, but from the viewpoint of accurately confirming the presence or absence and the degree of the therapeutic effect of the second supernatant on prostate cancer, it is preferably from 16 hours to 72 hours.

[0065] The culture step may be carried out according to the method described in the Examples.

[0066] (4-2) Measurement of viable cell count In order to determine the effectiveness of the second supernatant as a prostate cancer therapeutic agent, a "first viable cell count" is measured when prostate cancer cells are cultured in the presence of the second supernatant, and a "second viable cell count" is measured when prostate cancer cells are cultured under the same conditions as in the culture step except that they are cultured in the absence of the second supernatant, and the first viable cell count relative to the second viable cell count is determined.

[0067] In the present invention, the "first viable cell count" refers to the viable cell count of prostate cancer cells immediately after the start of the culture period in the culture step. "Immediately after the culture time has elapsed in the culture step" means the point in time at which the cells removed from the culture device are subjected to measurement of the viable cell count, and can be, for example, within 60 minutes after the culture time has elapsed.

[0068] In the present invention, the "second viable cell count" refers to the viable cell count of prostate cancer cells immediately after the passage of the culture time when cultured under the same conditions as the culture step, except that the culture is performed in the absence of the second supernatant.

[0069] The number of viable cells can be determined by visual inspection under a microscope or by any assay known in the art. Assays for determining the number of viable cells include cell viability assays (MTS assays) using commercially available kits.

[0070] (4-3) Determination of cell viability Based on the first viable cell count and the second viable cell count, the ratio of the first viable cell count to the second viable cell count (hereinafter also referred to as "cell viability") is determined using the following formula. Cell viability (%) = number of live cells in first group / number of live cells in second group x 100

[0071] If the cell viability is less than 90% (preferably less than 80%), it means that the second supernatant subjected to the culture step may be useful as a prostate cancer therapeutic agent. Therefore, the second supernatant is obtained as a prostate cancer therapeutic agent or a candidate thereof.

[0072] It should be noted that viable cells grow over time when cultured in an environment in which the cells can grow, i.e., in an environment in which the cells can grow, the number of viable cells after the culture step will be greater than the number of viable cells at the start of the culture step. Therefore, a cell viability rate of less than 90% means that the cell proliferation inhibitory effect is sufficiently high.

[0073] (5) Other processes After the culturing step, the second supernatant obtained as a prostate cancer therapeutic agent may be subjected to other steps required for formulation. Such steps include screening methods different from the screening method of the present invention, component analysis, toxicity tests, stability tests, and the like.

[0074] <Method of manufacturing the prostate cancer treatment agent> The present invention also encompasses a method for producing a prostate cancer therapeutic agent (hereinafter also referred to as "the production method of the present invention"), which comprises the above-mentioned autolysis reaction step, first recovery step, and second recovery step.

[0075] In the production method of the present invention, the second supernatant may be subjected to the above-mentioned culture step to confirm the cell viability. If the cell viability is less than 90% (preferably less than 80%), the second supernatant can be determined to be an effective prostate cancer therapeutic agent.

[0076] The configuration of the production method of the present invention can be the same as that of the screening method of the present invention.

[0077] The second supernatant may be formulated in any manner. EXAMPLES

[0078] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0079] <Test 1: Screening of prostate cancer therapeutic agents> A reaction product obtained by autolysis of a microorganism was prepared by the following method, and the proliferation activity of the reaction product on prostate cancer cells was evaluated.

[0080] (1) Preparation of microorganisms The present inventors isolated about 100 kinds of strains from samples collected at various locations (fields, forests, etc.) in Iwaki City, Fukushima Prefecture, Japan, and obtained microorganisms. Most of them were yeasts or lactic acid bacteria. In this test, lactic acid bacteria capable of growing under aerobic conditions were selected and subjected to each process.

[0081] (2) Self-digestion reaction process The reaction product was obtained by autolysis of the microorganisms in the following manner.

[0082] (2-1) Preculture step Each microorganism was placed in a 50 mL plastic tube together with 10 mL of liquid medium (pH 7.0 or so) and cultured at 120 rpm for 1-2 days at 30-37°C. After confirming bacterial growth, the culture was transferred to a 500 mL flask containing 100 mL of liquid medium and further cultured under the same conditions as above. Through this process, sufficient amounts of microorganisms were obtained for approximately 50 of the isolated strains to obtain reactants through autolysis.

[0083] In this step, YPD medium was used for culturing the yeast, and MRS medium was used for culturing the lactic acid bacteria.

[0084] (2-2) Pretreatment process After completion of the rotational culture, the bacterial liquid was transferred to a plastic tube and centrifuged at 3,200 rpm and 4° C., the supernatant was discarded, and the bacterial pellet was collected. Next, 10 to 15 mL of sterile water was added to the bacterial pellet, and the mixture was centrifuged at 3,200 rpm and 4° C. to wash the bacterial pellet.

[0085] The weight of the bacterial pellet was measured, and an equal to 5-fold amount of sterilized MilliQ water was added and suspended in the pellet. The pH was then adjusted to 5 to 8 using 2N sodium hydroxide to obtain a microbial liquid to be subjected to autolysis. The microbial liquid was frozen at -20°C for approximately 1 hour before the autolysis reaction.

[0086] (2-3) Self-digestion reaction of microbial liquid The microbial liquid was subjected to either (i) standing at 40 to 55° C. for 40 to 48 hours or (ii) rotation culture at 40 to 55° C. at 10 to 30 rpm for 40 to 48 hours in a liquid medium to carry out an autolysis reaction. The cell wall lysis of the resulting reaction product was confirmed using a stereomicroscope, and it was confirmed that the autolysis reaction had progressed sufficiently and that a reaction product was obtained by the autolysis reaction.

[0087] In this step, YPD medium was used for culturing the yeast, and MRS medium was used for culturing the lactic acid bacteria.

[0088] After the autolysis reaction was completed, the microbial liquid was transferred to a 1.5 mL Eppendorf tube and sterilized in a heat block at 98 to 100°C for 10 to 30 minutes. Next, the mixture was centrifuged at 14,000 rpm for 5 to 15 minutes at 4°C, and the supernatant was collected. Filter sterilization was performed in some cases. This procedure yielded a reaction product from the autolysis reaction. The weight of the reaction product (extraction weight) was also measured. The resulting reaction product was adjusted to a concentration of 10 to 100 mg / mL with sterilized MilliQ water and stored frozen.

[0089] (3) First recovery process The reaction product was mixed with a twice amount of ethanol (67 vol% aqueous ethanol solution), and then centrifuged at 14,000 rpm for 10 minutes at 4°C, and the first supernatant was collected.

[0090] (4) Second recovery process The first supernatant was mixed with an equal amount of butanol (50 vol% butanol aqueous solution), and then centrifuged at 14,000 rpm for 10 minutes at 4° C. to recover the second supernatant. The second supernatant was dried using a centrifugal concentrator, and then adjusted to a concentration of 10 to 200 mg / mL with a 67 vol% ethanol aqueous solution.

[0091] (5)Culture process A cell viability assay was performed using the second supernatant and a prostate cancer cell line (PC3 cells) according to the following method. PC3 cells are prostate cancer cells that grow independently of androgen, and are widely used in various experiments as castration-resistant prostate cancer cells.

[0092] (5-1) Preparation of PC3 cells PC3 cells were cultured in 10% FBS / RPMI / penicillin-streptomycin medium at 37°C in a CO2 incubator.

[0093] (5-2) Cell culture in the presence of the second supernatant First, PC3 cells were washed with PBS and then cultured in 0.25% trypsin-EDTA solution at 37° C. for 5 minutes. Next, PC3 cells were placed in 10% FBS / RPMI / penicillin-streptomycin medium, and the resulting cell suspension was transferred to a 96-well plate at 3.0–6.0 × 10 3 PC3 cells were placed at a cell volume (100 μl) of cells / well. Furthermore, the second supernatant (5 μl) was added to each well of the plate, and the plates were cultured in a CO 2 incubator at 37° C. for 43 hours. In addition, culture was also carried out under the same conditions as above, except that the second supernatant (5 μl) was not added.

[0094] (5-3) Cell viability assay (MTS assay) After the incubation was completed, the culture was collected and immediately subjected to an MTS assay using "Cell count kit-8" (Dojindo Laboratories). Specifically, the kit's reagent (10 μl) was added to each well and incubated in a CO2 incubator at 37°C for 3 hours, after which the number of viable cells was determined by measuring wavelengths of 450 nm and 620 nm using a microplate reader. The number of viable cells in the culture in the presence of the second supernatant corresponds to the "first viable cell count." The number of viable cells in the culture in the absence of the second supernatant corresponds to the "second viable cell count." The assay was repeated four times, and the mean and standard deviation were calculated. Significant differences were determined using Student t-test.

[0095] Based on the obtained results, the "first viable cell number" relative to the "second viable cell number" was calculated. Hereinafter, the first viable cell number relative to the second viable cell number is also referred to as the "cell viability."

[0096] (6) Results Of the approximately 50 reactions tested, 11 had cell viability below 90% (e.g., 50%), and therefore may be candidates for effective prostate cancer treatments. In addition, with reactants other than the above 11 types of reactants, the cell viability was approximately 150% or more (i.e., the proliferation of PC3 cells was not suppressed, and no therapeutic effect on prostate cancer was observed).

[0097] The above results show that the properties of the autolysis reaction products vary depending on the individual microorganism, and further, that by subjecting the autolysis reaction products to the screening method of the present invention, components effective in treating prostate cancer can be easily extracted.

[0098] <Test 2: Confirmation of the significance of each step in the screening method of the present invention> In the above Test 1, we focused on the reaction products in which the cell viability was less than 90%, and investigated the effects of the autolysis reaction and the presence or absence of a recovery step when preparing the reaction products on the results.

[0099] Specifically, in the above-mentioned Test 1, a microorganism (one type) from which a reaction product with a cell viability rate of less than 90% was derived was used, and a sample (reaction product from the autolysis reaction, or supernatant) was recovered without performing any of the autolysis reaction step, the first recovery step, and the second recovery step. Next, a culture step (culture using PC3 cells and MTS assay) was carried out in the presence of each sample.

[0100] The conditions for each step were the same as those in Test 1 above. The obtained samples were subjected to MTS assay and calculation of recovery efficiency, and were evaluated according to the following criteria. The results are shown in Table 1. Note that the lower the cell viability in the MTS assay, the higher the therapeutic effect on prostate cancer.

[0101] (1) Evaluation criteria for MTS assay A: Cell viability is less than 90%. B: Cell viability is 90% or more but less than 150%. C: Cell viability is 150% or higher.

[0102] (2) Evaluation criteria for recovery efficiency A: Recovery efficiency is 5% or more. B: Recovery efficiency is 2% or more and less than 5%. C: Recovery efficiency is 1% or more but less than 2%. D: Recovery efficiency is less than 1%.

[0103] The recovery efficiency was calculated based on the following definition. Recovery efficiency (%) = Extraction weight / Bacteria pellet weight * 100 "Extraction weight" means the weight of the reaction product specified in the section "(2-3) Autolysis reaction of microbial liquid" in Test 1. "Bacteria pellet weight" means the bacterial pellet weight specified in the section "(2-2) Pretreatment process" in Test 1.

[0104] [Table 1]

[0105] As shown in Table 1, it is evident that by carrying out a treatment that satisfies all of the requirements of the present invention, a fraction having an effective therapeutic effect on prostate cancer can be obtained with high recovery efficiency (Example).

[0106] In particular, as can be seen from a comparison between "Comparative Example 1" and "Example", it was an extremely surprising result that treatment with ethanol and butanol resulted in higher activity of the useful components than the autolysis reaction product itself. In addition, it was found that when only ethanol extraction was performed on the autolysis reaction product, the useful components could not be sufficiently recovered (Comparative Example 3).In addition, it was found that when only butanol extraction was performed, the recovery efficiency of the useful components was significantly low (Comparative Example 2).

[0107] <Test 3: Comparison with conventional methods> The method of the present invention uses ethanol or butanol as an extraction solvent, and therefore can be considered to be one form of a method for extracting fat-soluble components (such as lipids). On the other hand, as a conventional method for extracting lipids and the like, a method using methanol and chloroform is widely known. In this example, extraction was performed based on the above-mentioned conventional method and compared with the extraction that satisfies the requirements of the present invention. As the conventional method, the method of Bligh & Dyer et al. (Can. J. Biochem. Physiol. Vol. 37 (1959)) was used as a reference.

[0108] (1) Recovery of extract Bacterial pellets were obtained from the same microorganisms as in Test 2 by the same methods as in "(2-1) Pre-culture step" and "(2-2) Pre-treatment step" in Test 1 above. Next, 10 to 15 mL of sterile water was added to the bacterial pellet, and the mixture was centrifuged at 3,200 rpm and 4° C. to wash the bacterial pellet.

[0109] The weight of the bacterial pellet was measured, and an equal amount of sterilized MilliQ water (0.1 M acetic acid) was added to suspend the pellet. Methanol (5 times the weight of the bacterial pellet) and chloroform (2.5 times the weight of the bacterial pellet) were added to the obtained suspension, and the mixture was stirred. After stirring, the mixture was left at room temperature for 10 minutes. Next, chloroform (2.5 times the weight of the bacterial pellet) was added and stirred, and then sterilized MilliQ water (2.5 times the weight of the bacterial pellet) was added and further stirred. The resulting suspension was centrifuged at 3,200 rpm and 4° C., and the lower layer was collected and concentrated by centrifugation. The resulting concentrate was dissolved in a 67% aqueous methanol solution, and then centrifuged at 14,000 rpm for 10 minutes at 4° C. to collect the supernatant.

[0110] The obtained supernatant was subjected to the following high performance liquid chromatographic (HPLC) analysis.

[0111] (2)HPLC analysis The HPLC analysis was carried out as follows.

[0112] (2-1) Sample preparation The following three samples were prepared as samples. Each sample was dissolved in 90% methanol / pure water, and approximately 100 μg was used for HPLC analysis. First supernatant: the sample obtained in the "first recovery step" in the "Example" of Test 2 Second supernatant: the sample obtained in the "second recovery step" in the "Example" of Test 2 Third supernatant: Sample obtained in Test 3 (conventional method)

[0113] (2-2) Equipment used, etc. Reverse phase C18 column: "Triact-C18" (150 x 2.1 mm, 5 μm, 12 nm) YMC HPLC equipment: "Prominence" series, manufactured by Shimadzu Corporation Solvent (liquid A): HPLC grade pure water (0.1% formic acid) Solvent (liquid B): HPLC grade methanol Measurement wavelength: 260nm

[0114] (2-3) Elution conditions Elution was carried out at a flow rate of 0.2 mL / min in the following order: Solution A: 90%, Solution B: 10% for 5 minutes A gradient of 0% solution A and 100% solution B for 10 min Solution A: 0%, Solution B: 100% for 20 minutes Solution A: 90%, Solution B: 10% for 20 minutes

[0115] (3) Results The results of the HPLC analysis are shown in Figure 1. "A" indicates the results for the first supernatant, "B" the results for the second supernatant, and "C" the results for the third supernatant. The waveforms detected in each supernatant were different, indicating that the composition of each supernatant was different. This result also showed that different components can be extracted if the extraction conditions are different, even if the microorganisms are the same.

Claims

1. A method for screening a therapeutic agent for prostate cancer, comprising: The screening method comprises: an autolysis reaction step of obtaining a reactant by autolysis of a microorganism; a first recovery step of mixing the reactant and ethanol, centrifuging the mixture, and recovering a first supernatant; a second recovery step of mixing the first supernatant and butanol, centrifuging the mixture, and recovering a second supernatant; Culturing prostate cancer cells in the presence of the second supernatant; Including, In the culturing step, a first viable cell count is determined when the prostate cancer cells are cultured in the presence of the second supernatant, and a second viable cell count is determined when the prostate cancer cells are cultured under the same conditions as in the culturing step except that the prostate cancer cells are cultured in the absence of the second supernatant; When the first viable cell count relative to the second viable cell count is less than 90%, the second supernatant is obtained as a prostate cancer therapeutic agent. Screening methods.

2. A method for producing a prostate cancer therapeutic agent, comprising: The manufacturing method comprises: an autolysis reaction step of obtaining a reactant by autolysis of a microorganism; a first recovery step of mixing the reactant and ethanol, centrifuging the mixture, and recovering a first supernatant; a second recovery step of mixing the first supernatant and butanol, centrifuging the mixture, and recovering a second supernatant; A manufacturing method comprising:

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