Method for inhibiting growth of cancer cells using supercritical fluid-extracted product of agaricus blazei murrill

Abstract

To provide an agent for inhibiting the growth of cancer cells.SOLUTION: This agent comprises supercritical fluid-extracted product of Agaricus blazei murrill as an active ingredient.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to an agent for inhibiting cancer cell proliferation, and particularly to an agent for inhibiting cancer cell proliferation containing, as an active ingredient, a supercritical fluid extract of Agaricus blazei Murrill.

Background Art

[0002] Cancer is a major cause of death worldwide, and the mechanism by which cancer occurs has not yet been fully elucidated. It is already known that carcinogenesis or tumorigenesis is thought to be caused by the accumulation of exogenous or endogenous factors within cells, which leads to genetic abnormalities, errors in intracellular signal transduction pathways, loss of control over cell division, abnormal proliferation of cells, and the gradual formation of cancer cells.

[0003] Clinically, there are many drugs for treating cancer, such as cisplatin, doxorubicin, and paclitaxel. However, the cure rate of cancer by these drugs is quite limited. The main reasons include individual differences among patients, severe side effects associated with anticancer drugs, and drug resistance of cancer cells.

[0004] Therefore, engineers in this technical field are developing drugs that have anticancer activity without causing side effects by using active components that can be used for treating cancer derived from natural plants.

[0005] Agaricus blazei Murrill, AbM, Agaricus subrufescens (scientific name), Agaricus brasiliensis, or Agaricus rufotegulis, Japanese name: Nisemorinokasa, Kawariharatake, Hime Matsutake, is an edible and medicinal fungus of the genus Agaricus in the family Agaricaceae, and its main production areas are distributed in Brazil, Peru, and southern California in the United States, etc.

[0006] In previous studies, it has been demonstrated that Agaricus shows a wide range of pharmacological activities such as antioxidant, anti-inflammation, hypoglycemic, and hypolipidemic.

[0007] In addition, there are many studies on the application of Agaricus extracts in the pharmaceutical field. For example, in Non-Patent Document 1, it is described that the ethanol-water extract of Agaricus can inhibit the growth of cancer cells in nude mice bearing human promyelocytic leukemia cells (NB-4).

[0008] In Non-Patent Document 2, it has been discovered that the supercritical fluid extract of Agaricus has antimicrobial activity.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0010] At present, there is no literature disclosing that Agaricus supercritical fluid extract can be used for the treatment of cancer, and the idea of using Agaricus supercritical fluid extract for the treatment of cancer is not found. Among them, the inventor of the present application unexpectedly discovered that Agaricus supercritical fluid extract can be used for more effective suppression of cancer cell growth, and through intensive research, the present invention has been achieved.

Means for Solving the Problems

[0011] Therefore, the present invention provides an agent for suppressing cancer cell growth, which is characterized by containing Agaricus supercritical fluid extract as an active ingredient.

Effects of the Invention

[0012] Since the present invention contains Agaricus supercritical fluid extract as an active ingredient, it has the effect of suppressing the growth of cancer cells.

Brief Description of the Drawings

[0013]

Figure 1

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Figure 8

Mode for Carrying Out the Invention

[0014] First, it should be understood that even if some prior art documents are cited in this specification, in Taiwan or any country, the content of such prior art documents does not indicate common general knowledge in the field to which the present invention pertains.

[0015] Also, it should be understood that the term "comprising" means "including but not limited to", and the term "comprises" has a corresponding meaning.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. One of ordinary skill in the art will recognize, and be able to utilize, many methods and materials similar or equivalent to those described herein in the practice of this disclosure. Indeed, the disclosure is in no way limited to the methods and materials described.

[0017] The present invention provides a use of an Agaricus supercritical fluid extract, that is, a cancer cell growth inhibitor containing the Agaricus supercritical fluid extract as an active ingredient.

[0018] The Agaricus supercritical fluid extract is obtained by extracting Agaricus by supercritical fluid extraction.

[0019] As used herein, the terms “treating” or “treatment” mean preventing, reducing, alleviating, ameliorating, relieving or controlling one or more clinical signs in a disease or disorder, and lowering, stopping or reversing the progression of the severity in the condition or symptom being treated.

[0020] According to the present invention, the cancer cells are selected from the group consisting of lung adenocarcinoma cells, gastric adenocarcinoma cells, colorectal cancer cells, breast cancer cells, liver cancer cells, pancreatic cancer cells, prostate cancer cells, cervical cancer cells, and combinations thereof.

[0021] According to the present invention, the cancer cell growth inhibitor can contain, as an active ingredient, a mixture containing an Agaricus supercritical fluid extract and a substance selected from the group consisting of an ethanol extract of Curcuma longa, a Ganoderma lucidum supercritical fluid extract, and combinations thereof.

[0022] The ethanol extract of Curcuma longa is obtained by extracting Curcuma longa by ethanol extraction.

[0023] The Ganoderma lucidum supercritical fluid extract is obtained by extracting Ganoderma lucidum by supercritical fluid extraction.

[0024] In some embodiments, the cancer cell growth inhibitor contains, as an active ingredient, a mixture containing an Agaricus supercritical fluid extract and an ethanol extract of Curcuma longa.

[0025] When the cancer cell growth inhibitor contains, as an active ingredient, a mixture containing an Agaricus supercritical fluid extract and an ethanol extract of Curcuma longa, in some embodiments, the weight ratio of the Agaricus supercritical fluid extract to the ethanol extract of Curcuma longa is in the range of 1:1 to 1:5, and in some embodiments, the weight ratio of the Agaricus supercritical fluid extract to the ethanol extract of Curcuma longa is 1:1.

[0026] In some embodiments, the cancer cell growth inhibitor contains, as an active ingredient, a mixture comprising an Agaricus supercritical fluid extract and a Ganoderma supercritical fluid extract.

[0027] When the cancer cell growth inhibitor contains, as an active ingredient, a mixture comprising an Agaricus supercritical fluid extract and a Ganoderma supercritical fluid extract, in some embodiments, the weight ratio of the Agaricus supercritical fluid extract to the Ganoderma supercritical fluid extract is in the range of 1:1 to 1:5, and in some embodiments, the weight ratio of the Agaricus supercritical fluid extract to the Ganoderma supercritical fluid extract is 1:1.

[0028] In some embodiments, the cancer cell growth inhibitor contains, as an active ingredient, a mixture comprising an Agaricus supercritical fluid extract, a turmeric ethanol extract, and a Ganoderma supercritical fluid extract.

[0029] When the cancer cell growth inhibitor contains, as an active ingredient, a mixture comprising an Agaricus supercritical fluid extract, a turmeric ethanol extract, and a Ganoderma supercritical fluid extract, in some embodiments, the weight ratio of the Agaricus supercritical fluid extract to the turmeric ethanol extract to the Ganoderma supercritical fluid extract is in the range of 1:1:1 to 1:5:1, and in some embodiments, the weight ratio of the Agaricus supercritical fluid extract to the turmeric ethanol extract to the Ganoderma supercritical fluid extract is 1:1:1, and in some embodiments, the weight ratio of the Agaricus supercritical fluid extract to the turmeric ethanol extract to the Ganoderma supercritical fluid extract is 1:2:1.

[0030] The method of supercritical fluid extraction is within the scope of the specialized knowledge and routine skills of those skilled in the art, and reference can be made to, for example, the above-mentioned Mazzutti S. et al. (2012).

[0031] The operating conditions of the supercritical fluid extraction method vary depending on factors such as the type of supercritical fluid used, the extraction raw materials of Agaricus and Ganoderma, the treatment method, and the usage amount in order to obtain the most excellent extraction results. The selection of the operating conditions of the supercritical fluid extraction method can be routinely determined by those skilled in the art.

[0032] According to the present invention, the supercritical fluid can be supercritical CO2, supercritical H2O, supercritical methanol or supercritical ethanol. In some embodiments, the supercritical fluid is supercritical CO2.

[0033] According to the present invention, the extraction raw materials of Agaricus and Ganoderma lucidum can be fresh fruiting bodies or pre-processed fruiting bodies. The processing can be selected from the group consisting of drying treatment (such as freeze-drying treatment), heat treatment, grinding treatment, cutting treatment, pulverization treatment and combinations thereof. In some embodiments, the extraction raw material of Agaricus is a freeze-dried fruiting body. In some embodiments, the extraction raw material of Ganoderma lucidum is a freeze-dried fruiting body.

[0034] According to the present invention, the supercritical fluid extraction can be carried out for 30 to 960 minutes in an environment where the temperature is 40°C to 60°C and the pressure is 1071.33 psi to 4350 psi.

[0035] In some embodiments, the supercritical fluid extraction is carried out for 120 minutes in an environment where the temperature is 60°C and the pressure is 4350 psi.

[0036] According to the present invention, the method of ethanol extraction is within the scope of the specialized knowledge and daily skills of those skilled in the art. For example, reference can be made to Taiwan Patent Publication No. I748565.

[0037] The operating conditions of the ethanol extraction method vary depending on factors such as the extraction raw material of turmeric, the treatment method, and the usage ratio of ethanol to turmeric in order to obtain the best extraction results. The selection of the operating conditions of the extraction method using ethanol can be routinely determined by those skilled in the art.

[0038] According to the present invention, the extraction raw material of turmeric can be fresh rhizomes, fresh leaves, fresh flowers, fresh whole plants, or pre-processed rhizomes, pre-processed leaves, pre-processed flowers, and pre-processed whole plants. The processing can be selected from the group consisting of drying treatment (such as freeze-drying treatment), heat treatment, grinding treatment, shredding treatment, pulverization treatment, and combinations thereof. In some embodiments, the extraction raw material of turmeric is freeze-dried rhizomes.

[0039] According to the present invention, ethanol extraction is carried out for 15 to 150 minutes in an environment with a temperature of 10°C to 100°C.

[0040] In some embodiments, ethanol extraction is carried out for 30 minutes in an environment with a temperature of 25°C.

[0041] According to the present invention, the cancer cell growth inhibitor can be in a dosage form suitable for parenteral administration, oral administration, or topical administration.

[0042] According to the present invention, the cancer cell growth inhibitor can further contain a pharmaceutically acceptable carrier widely used in the drug manufacturing technology.

[0043] For example, pharmaceutically acceptable carriers can include at least one of the following reagents: solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrating agents, dispersing agents, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. The selection and quantity of these reagents are within the expertise and conventional technical scope of those having ordinary knowledge in the technical field of the present invention.

[0044] According to the present invention, the cancer cell growth inhibitor can be manufactured into a dosage form suitable for parenteral administration (including injections, for example, sterile aqueous solutions or dispersions) by techniques generally known to those with ordinary knowledge in the technical field of the present invention, and can be administered via one of the parenteral routes of intraperitoneal injection, intrapleural injection, intramuscular injection, intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection, intrathecal injection, intracranial injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration.

[0045] According to the present invention, the cancer cell growth inhibitor can be manufactured into a dosage form suitable for oral administration by techniques generally known to those with ordinary knowledge in the technical field of the present invention, and the dosage form suitable for oral administration includes, but is not limited to, sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0046] According to the present invention, the cancer cell growth inhibitor can be manufactured into an external preparation suitable for topical administration to the skin by a technique generally known to those having ordinary knowledge in the technical field of the present invention, and the external preparation includes, but is not limited to, emulsion, gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, serum, paste, foam, drop, suspension, salve, and bandage.

[0047] The present invention also provides a method for treating cancer, which comprises administering the above Ganoderma lucidum supercritical fluid extract or a cancer cell growth inhibitor containing the above Ganoderma lucidum supercritical fluid extract as an active ingredient to an individual in need of cancer treatment.

[0048] As used herein, the terms "administer" and "administration" are interchangeable and mean "introducing, providing or delivering a predetermined active ingredient to a subject by any suitable route to achieve the intended function and produce the expected effect".

[0049] As used herein, the term "subject" refers to any mammalian animal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, rats, etc.

[0050] According to the present invention, the dosage and dosing frequency of the above Ganoderma lucidum supercritical fluid extract or the cancer cell growth inhibitor containing the above Ganoderma lucidum supercritical fluid extract as an active ingredient can be varied depending on factors such as the disease or disorder to be treated, the severity thereof, the administration route, the age of the subject to be treated, the health condition, the response, and the like. Generally, according to the cancer cell growth inhibitor of the present invention, it can be administered either as a single dose or in divided doses over several times.

[0051] Detailed Description of Examples Hereinafter, examples of the present invention will be described. It should be understood that these examples are illustrative and explanatory, and should not be construed as limiting the present invention.

[0052] [Examples] General Experimental Materials: 1. Source and Culture of Cell Lines: The types and accession numbers of the cell lines used in the following examples are shown in Table 1 below. All of these cell lines were purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan.

[0053] Table 1: Types and Accession Numbers of Cell Lines (The depository institution is the above BCRC)

[0054]

Table 1

[0055] The culture of these 8 cell lines was carried out using the media shown in Table 2 below in an incubator with the culture conditions set at 37°C and 5% CO2. Thereafter, the medium was replaced with fresh medium approximately every 2 to 3 days.

[0056] When the cell density reached about 80% confluence, cell subculture was performed.

[0057] Table 2: Media used for culturing 8 cell lines

[0058]

Table 2

[0059] General experimental methods: 1. Statistical analysis: The experimental data obtained in the following examples are expressed as "mean ± standard error of the mean (SEM)".

[0060] All data were analyzed by Student's T - test and the differences between each group were evaluated.

[0061] When the result of the obtained statistical analysis is p < 0.05, it indicates statistical significance.

[0062] Example 1. Production of Agaricus supercritical fluid extract The fruiting bodies of Agaricus purchased from Taiwan LOHAS BIOTECH Development Corp. were washed with deionized water and then freeze - dried for 48 hours. Then, the freeze - dried Agaricus fruiting bodies were ground with a grinding machine (model number: TS10HS, manufacturer: Taiwan Tinso), and sieved with a sieve having a pore size of 0.38 mm to obtain Agaricus powder.

[0063] Then, about 9 kg of Agaricus powder was placed in the extraction vessel of a supercritical fluid extraction system (purchased from Supercritical Technologies Co., Ltd, Taiwan, model number: SE-201C). After introducing CO2 into the extraction vessel, supercritical fluid extraction (SFE) was carried out for 120 minutes in an environment with a temperature of 60 °C and a pressure of 4350 psi.

[0064] During the extraction process, CO2 passed through the extraction vessel at a flow rate of 2.5 L / min and entered the collection vessel at normal pressure, thereby obtaining a pasty Agaricus supercritical fluid extract in the collection vessel.

[0065] Example 2. High performance liquid chromatography (HPLC) analysis of Agaricus supercritical fluid extract To know the distribution of the main components of the Agaricus supercritical fluid extract of the present invention, the Agaricus supercritical fluid extract obtained in Example 1 was analyzed by HPLC.

[0066] Experimental materials: For HPLC analysis, the Agaricus supercritical fluid extract obtained in Example 1 was dissolved in isopropyl alcohol (IPA) to obtain a solution sample with a concentration of 0.2 g / mL. The following HPLC analysis was performed using this solution sample.

[0067] Experimental method: The HPLC analyzer used in this experiment is a high-performance liquid chromatography system (model number: Ultimate 3000, manufacturer: Thermo Fisher Scientific, USA), a pump (model number: HPG-3200BX, manufacturer: Thermo Fisher Scientific, USA), and a variable wavelength detector (model number: VWD-3400RS, manufacturer: Thermo Fisher Scientific, USA). The chromatography column is Hypersil GOLD aQ (manufacturer: Thermo Fisher Scientific, USA, Part no.: 25305-159270A) and has a size of 150 mm in length × 20 mm in inner diameter. The operating conditions of HPLC are shown in Table 3 below.

[0068] Table 3: Operating Conditions of HPLC

[0069]

Table 3

[0070] Results: Figure 1 is a diagram of the HPLC spectrum of the Agaricus supercritical fluid extract obtained in Example 1. According to Figure 1, six main peaks (labeled as peak a1 to peak a6 respectively) appeared in the Agaricus supercritical fluid extract of the present invention during the retention time period from 0 minute to 50 minutes.

[0071] Example 3. Evaluation of the Anticancer Effects of Different Extracts of Agaricus Experimental Materials: 1. Preparation of the test solution of the Agaricus ethanol extract: 30 g of the Agaricus powder obtained in Example 1 was added to 150 mL of ethanol with a concentration of 95% and mixed thoroughly. Then, ethanol extraction was carried out for 60 minutes in an environment with a temperature of 60°C, and the filtrate was obtained after filtration with filter paper having a pore size of 7 μm.

[0072] The filtrate was concentrated for 1 hour using a rotary evaporator (rotary evaporator, model number: N-1300, manufacturer: EYELA) to obtain a paste-like Agaricus ethanol extract.

[0073] Then, 100 mg of the Agaricus ethanol extract was dissolved in 1 mL of ethanol with a concentration of 95%, and then an appropriate amount of DMEM was added to obtain a test solution of the Agaricus ethanol extract with a concentration of 400 μg / mL (hereinafter referred to as "test solution E").

[0074] 2. Preparation of test solution of Agaricus water extract 30 g of the Agaricus powder obtained in Example 1 was added to 180 mL of deionized water and mixed well. After performing water extraction for 60 minutes in an environment with a temperature of 80°C, it was filtered through filter paper with a pore size of 7 μm to obtain filtrate 1.

[0075] 30 g of the Agaricus powder obtained in Example 1 was added to 180 mL of deionized water and mixed well. After performing water extraction for 60 minutes in an environment with a temperature of 4°C, it was filtered through filter paper with a pore size of 7 μm to obtain filtrate 2.

[0076] Using a rotary evaporator, each of filtrate 1 and filtrate 2 was concentrated for 2 hours and then freeze-dried for 48 hours to obtain two types of powdered Agaricus water extracts. The Agaricus water extract extracted in an environment of 80°C is referred to as water extract 1, and the Agaricus water extract extracted in an environment of 4°C is referred to as water extract 2.

[0077] Then, 100 mg of the water extract 1 was dissolved in 1 mL of deionized water, and then an appropriate amount of DMEM was added to obtain a test solution of the water extract 1 with a concentration of 400 μg / mL (hereinafter referred to as "test solution W1").

[0078] 100 mg of the water extract 2 was dissolved in 1 mL of deionized water, and then an appropriate amount of DMEM was added to obtain a test solution of the water extract 2 with a concentration of 400 μg / mL (hereinafter referred to as "test solution W2").

[0079] 3. Preparation of Test Solution of Agaricus Supercritical Fluid Extract Then, 100 mg of the Agaricus supercritical fluid extract obtained in Example 1 was dissolved in 1 mL of ethanol with a concentration of 95%, and then an appropriate amount of DMEM was added to obtain a test solution of the Agaricus supercritical fluid extract with a concentration of 400 μg / mL (hereinafter referred to as "test solution S").

[0080] Experimental Method: A549 cells, AGS cells, SW480 cells, and MDA-MB-231 cells that were subcultured according to the first item "Cell Line Source and Culture" of the above general experimental materials were grouped as shown in Table 4, and the cells of each group were inoculated into a 96-well culture plate containing 100 μL of the medium shown in Table 2 above and cultured in an incubator (37 °C, 5% CO2) for 18 hours.

[0081] Then, by adding fresh corresponding medium as shown in Table 2 above to each well of the 96-well cell culture plate, the cell cultures of each group were replaced with fresh medium. And as shown in Table 4, for each experimental group and control group, after adding an appropriate amount of the corresponding test solution so that the final concentration becomes 200 μg / mL, the cell cultures of each group were treated by culturing in an incubator (37 °C, 5% CO2) for 48 hours. Only the treatment of culturing the cells of each control group in an incubator (37 °C, 5% CO2) for 48 hours without adding the test solution was performed.

[0082] Table 4: Cells of Each Group and Test Solution for Treating the Cells

[0083]

Table 4

[0084] After processing each group, the liquid in each well was removed, washed with phosphate buffered saline (PBS), and then 100 μL of WST-1 reagent [water-soluble tetrazolium-1 reagent, manufacturer: Roche, Switzerland, Cat. No. 11644807001] was added, and the reaction was carried out in an incubator (37 °C, 5% CO2) for 1 hour.

[0085] Then, using an ELISA reader (model number: SPARK, manufacturer: TECAN, Switzerland) at a wavelength of 450 nm, the absorbance value (OD 450 ) of each well was measured. Also, an appropriate amount of medium was added to a 96-well cell culture plate, and the absorbance value was measured at a wavelength of 450 nm to obtain the background absorbance value (OD 450 ).

[0086] The cell viability (%) was calculated by substituting the obtained absorbance value (OD 450 ) into Equation 1 below.

[0087] Equation 1: A = (B - D) / (C - D) × 100 Where, A = cell viability (%), B = absorbance value (OD 450 ) of each measured group C = absorbance value (OD 450 ) of the measured control group D = background absorbance value (OD 450 ) The experimental data obtained according to the method described in Item 1, "Statistical Analysis" of the above general experimental method were analyzed. Results: Figure 2 shows the measured cell viability after A549 cells, AGS cells, SW480 cells, and MDA-MB-231 cells were treated with different extracts.

[0088] According to FIG. 2, for four types of cancer cell lines, namely A549 cells, AGS cells, SW480 cells, and MDA-MB-231 cells, the cell survival rate decreased to 0% in each experimental group. In each of Comparative Groups 1 to 4 (Comparative Group 1 includes Comparative Groups 1-1 to 3-1, Comparative Group 2 includes Comparative Groups 1-2 to 3-2, Comparative Group 3 includes Comparative Groups 1-3 to 3-3, and Comparative Group 4 includes Comparative Groups 1-4 to 3-4), no obvious decrease like that in each experimental group was observed in the cell survival rate compared with the cell survival rate of the corresponding control group. Furthermore, in Comparative Groups 2-2, 3-2, and 3-3, the cell survival rate was much higher than the cell survival rate of the corresponding control group.

[0089] According to the experimental results, it was clearly shown that the Ganoderma lucidum supercritical fluid extract of the present invention exhibited excellent cytotoxicity against lung adenocarcinoma cells, gastric adenocarcinoma cells, colorectal cancer cells, and breast cancer cells, and had a broad cancer cell growth inhibitory effect.

[0090] Example 4. Evaluation of the in vitro anti-cancer effect by the single use of Ganoderma lucidum supercritical fluid extract and the combined use with other extracts Experimental materials: 1. Preparation of freeze-dried powder of Curcuma longa ethanol extract: The Curcuma longa ethanol extract was prepared with reference to the method described in Taiwan Patent Publication No. I748565B. Briefly, 30 g of Curcuma longa rhizome powder was added to 150 mL of ethanol with a concentration of 95% and mixed thoroughly, and then stirring extraction was performed at a temperature of 25°C and a rotation speed of 280 rpm for 30 minutes. After filtration through a 250-mesh filter screen, the filtrate was taken and concentrated using the above rotary evaporator to remove ethanol in 2 hours. Then, ethanol was added again and mixed uniformly at 4°C, followed by freeze-drying for 48 hours to obtain the freeze-dried powder of Curcuma longa ethanol extract. 2. Preparation of Grifola frondosa supercritical fluid extract: Instead of using the fruiting bodies of Agaricus, the fruiting bodies of Hericium erinaceus produced in Zhongpu Township, Chiayi County, Taiwan were used. Also, the volume of the Hericium erinaceus powder was adjusted to 4 kg, and the temperature of supercritical fluid extraction (SFE) was set to 40 °C. Referring to the method described in Example 1 above, a paste-like supercritical fluid extract of Hericium erinaceus was produced. 3. Production of mixtures and mixture solutions containing different extracts The Agaricus supercritical fluid extract obtained in Example 1 above, the freeze-dried powder of the turmeric ethanol extract obtained in Items 1 and 2 of this Example 4, and the paste-like supercritical fluid extract of Hericium erinaceus were uniformly mixed at the weight ratios shown in Table 5 below to obtain four paste-like mixtures (i.e., Mixture 1 to Mixture 4) containing different extracts.

[0091] Table 5: Four mixtures containing different extracts

[0092]

Table 5

[0093] 100 mg of Mixture 3 was dissolved in 1 mL of ethanol with a concentration of 95%, and then an appropriate amount of DMEM was added to obtain Mixture Solution 3 with a concentration of 10,000 μg / mL. Also, 100 mg each of Mixture 1 to Mixture 3 was dissolved in 1 mL of ethanol with a concentration of 95%, and then an appropriate amount of DMEM was added to obtain Mixture Solutions 1 to 3 with respective concentrations of 400 μg / mL.

[0094] Experimental method: A. Comparison of the anti-cancer effects of the Agaricus supercritical fluid extract and Mixture Solution 3 The Mia Paca-2 cells, HeLa 229 cells, and PC-3 cells that were subcultured according to the content of Item 1 "Source and culture of cell lines" in the above general experimental materials were grouped as shown in Table 6, and the cells of each group were inoculated into a 96-well cell culture plate containing 100 μL of the medium shown in Table 2 above and cultured in an incubator (37 °C, 5% CO2) for 18 hours.

[0095] Then, by adding the corresponding fresh medium as shown in Table 2 above to each well of a 96-well cell culture plate, the cell cultures of each group were replaced with fresh medium. Then, as shown in Table 6, an appropriate amount of test solution S obtained in Item 3 of Example 3 or an appropriate amount of the above mixture solution 3 with a concentration of 10,000 μg / mL was added to each experimental group so as to obtain different final concentrations. After that, the cell cultures of each group were treated by culturing them in an incubator (37 °C, 5% CO2) for 48 hours. Only the treatment of culturing the cells of each control group in an incubator (37 °C, 5% CO2) for 48 hours without adding the test solution and the mixture solution was performed.

[0096] Table 6: Cells of each group and the solution for treating the cells

[0097]

Table 6

[0098] Then, as shown in Table 6, after treating each group, the following sulforhodamine B assay was performed using an In Vitro Toxicology Assay Kit (manufacturer: Sigma, Cat. No. TOX6-1KT).

[0099] 50 μL of trichloroacetic acid (TCA) solution was added to the cell cultures of each group and allowed to act for 1 hour in an environment at 4 °C to fix the cells.

[0100] Then, the liquid in each well was removed, washed with sterile water, and 50 μL of SRB reagent with a concentration of 0.4% was added. After allowing it to act at room temperature for 30 minutes, the liquid in each well was removed, washed with acetic acid with a concentration of 1%, and 100 μL of Tris-base with a concentration of 10 mM was added and mixed for 30 minutes using a digital orbital shaker (model number: SK-O180-S, manufacturer: DLAB).

[0101] After that, using the above ELISA reader at a wavelength of 565 nm, the absorbance value (OD 565 ) of each well was measured. Also, an appropriate amount of medium was added to a 96-well cell culture plate and the absorbance value was measured at a wavelength of 565 nm to obtain the background absorbance value (OD 565 ).

[0102] The cell viability (%) was calculated by substituting the obtained absorbance value (OD 565 ) into Equation 2 below.

[0103] Equation 2: E = (F - H) / (G - H) × 100 Where, E = cell viability (%), F = absorbance value (OD 565 ) of each group measured G = absorbance value (OD 565 ) of the measured control group H = background absorbance value (OD 565 ) The experimental data obtained according to the method described in Item 1, "Statistical Analysis" of the above general experimental method were analyzed.

[0104] B. Comparison of the anti-cancer effects of mixture solutions 1 to 3: A549 cells, AGS cells, HepG2 cells, SW480 cells, and MDA-MB-231 cells that were subcultured according to the content of item 1, "Cell line source and culture," in the above general experimental materials were grouped as shown in Table 7, and the cells of each group were inoculated into a 96-well cell culture plate containing 100 μL of the medium shown in Table 2 above and cultured in an incubator (37°C, 5% CO2) for 18 hours.

[0105] Then, the cell cultures of each group were replaced with fresh medium by adding fresh corresponding medium as shown in Table 2 above to each well of the 96-well cell culture plate. As shown in Table 7, for each experimental group, after adding an appropriate amount of the above mixture solution 1 to mixture solution 3 with a corresponding concentration of 400 μg / mL so that the final concentration was 200 μg / mL, the cell cultures of each group were treated by culturing in an incubator (37°C, 5% CO2) for 48 hours. The cells of each control group were only cultured in an incubator (37°C, 5% CO2) for 48 hours without adding the test solution and the mixture solution.

[0106] Table 7: Cells of each group and the mixture solution for treating the cells

[0107]

Table 7

[0108] After treating each group, according to the method described in Example 3 above, the cell viability of the cell cultures of each group was analyzed.

[0109] Results: A. Comparison of the anticancer effects of test solution S (agaricus supercritical fluid extract) and mixture solution 3: Figures 3 to 5 show the measured cell viability after MIA Paca-2 cells, HeLa 229 cells, and PC-3 cells were treated with test solution S or mixture solution 3 with a concentration of 10,000 μg / mL, respectively. According to FIGS. 3 to 5, for three types of cancer cell lines, namely MIA Paca-2 cells, HeLa 229 cells, and PC-3 cells, compared with the corresponding control groups, the cell survival rates of each of Experimental Groups 1 to 3 (Experimental Group 1 includes Experimental Groups 1-1 to 4-1, Experimental Group 2 includes Experimental Groups 1-2 to 4-2, and Experimental Group 3 includes Experimental Groups 1-3 to 4-3) decreased. In particular, the cell survival rates of Experimental Groups 3-1 to 3-3 and Experimental Groups 4-1 to 4-3 further decreased significantly with the increase in the concentration of the mixture [i.e., dose-dependent effect], and were all lower than the cell survival rates of the corresponding Experimental Groups 1-1 to 1-3 and the corresponding Experimental Groups 2-1 to 2-3.

[0110] According to the experimental results, it was revealed that the sole use of the Agaricus supercritical fluid extract of the present invention or the combined use with the ethanol extract of Curcuma longa and the supercritical fluid extract of Ganoderma mannentake both have an effective cancer cell growth inhibitory effect on pancreatic cancer cells, cervical cancer cells, and prostate cancer cells.

[0111] B. Comparison of the anti-cancer effects of Mixture Solutions 1 to 3 FIG. 6 shows the measured cell survival rates after A549 cells, AGS cells, HepG2 cells, SW480 cells, and MDA-MB-231 cells were each treated with one of Mixture Solutions 1 to 3 at a concentration of 400 μg / mL.

[0112] According to FIG. 6, for five types of cancer cell lines, namely A549 cells, AGS cells, HepG2 cells, SW480 cells, and MDA-MB-231 cells, compared with the corresponding control groups, the cell survival rates of each experimental group 1 to each experimental group 5 (experimental group 1 includes experimental groups 1-1 to 3-1, experimental group 2 includes experimental groups 1-2 to 3-2, experimental group 3 includes experimental groups 1-3 to 4-3, experimental group 4 includes experimental groups 1-4 to 3-4, and experimental group 5 includes experimental groups 1-5 to 3-5) were significantly decreased.

[0113] According to the experimental results, the combined use of the Agaricus supercritical fluid extract of the present invention and the turmeric ethanol extract or the combined use with the Ganoderma lucidum supercritical fluid extract, or the combined use of the Agaricus supercritical fluid extract, the turmeric ethanol extract, and the Ganoderma lucidum supercritical fluid extract of the present invention all clearly have an effective cancer cell growth inhibitory effect on lung adenocarcinoma cells, gastric adenocarcinoma cells, liver cancer cells, colorectal cancer cells, and breast cancer cells.

[0114] Example 5. Evaluation of the in vivo anti-cancer effect of the single use of the Agaricus supercritical fluid extract and its combined use with other extracts Experimental materials: 1. Experimental animals: The male Balb / cAnN.Cg-Foxnl nu / CrlNarl nude mice (5 weeks old, body weight about 19.61 ± 1.36 g) used in this example were purchased from the National Laboratory Animal Center, R.O.C. in Taiwan.

[0115] All experimental animals were housed in an animal room with independent air conditioning facilities that maintained a cycle of 12 hours of light and 12 hours of darkness, a room temperature of 22 ± 2 °C, and a relative humidity of 55 ± 15%, and water and feed were provided abundantly. After one week of breeding, they were used in the following experiments.

[0116] Regarding the breeding environment of experimental animals, the environment, and all experimental processes, they all comply with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) in Taiwan.

[0117] 2. A549 cell solution The A549 cells obtained in item 1 of the above general experimental materials were centrifuged at 5000 rpm and 25 °C for 5 minutes, and the obtained cell pellet was collected. Subsequently, 1 mL of fresh corresponding medium as shown in Table 2 above was added to suspend the cell pellet to obtain a suspension.

[0118] 10 μL of the suspension was diluted with 990 μL of PBS and adjusted to the desired cell concentration determined using plate counting medium to obtain an A549 cell solution with 1×10 7 cells.

[0119] 3. MIA Paca-2 cell solution The MIA Paca-2 cells obtained in item 1 of the above general experimental materials were centrifuged at 5000 rpm and 25 °C for 5 minutes, and the obtained cell pellet was collected. Subsequently, 1 mL of fresh corresponding medium as shown in Table 2 above was added to suspend the cell pellet to obtain a suspension.

[0120] 10 μL of the suspension was diluted with 990 μL of PBS and adjusted to the desired cell concentration determined using plate counting medium to obtain an MIA Paca-2 cell solution with 1×10 7 cells.

[0121] 4. Powdered mixture 3 With reference to the method described in Lombardo D. and Kiselev M.A. (2022), Pharmaceutics., doi: 10.3390 / pharmaceutics14030543, encapsulation of the paste-like mixture 3 obtained in Item 3 of Example 4 into liposomes was performed to obtain a powdery mixture 3.

[0122] Experimental method: A. Evaluation of anti-lung adenocarcinoma effect: 1×10 7 of the above A549 cell solution containing A549 cells was subcutaneously injected into a site near the dorsal side of the right hind limb of Balb / cAnN.Cg-Foxnl nu / CrlNarl nude mice. When the 14th day after the injection of A549 cells and the tumor diameter reached 5 mm to 6 mm, the nude mice were randomly divided into 6 groups (n = 8 for each group). The 6 groups included 1 control group and 5 experimental groups (i.e., experimental groups 1 to 5).

[0123] The nude mice in experimental groups 1 to 5 were given the paste-like agaricus supercritical fluid extract obtained in Example 1 and the paste-like mixtures 1 to 4 obtained in Item 3 of Example 4 at the daily doses shown in Table 8 below by oral gavage. The nude mice in each experimental group were administered once a day for a total of 35 days. The nude mice in each control group were not treated.

[0124] Table 8: Extracts or mixtures orally administered to nude mice in each group

[0125]

Table 8

[0126] Before the start of administration (i.e., on the 0th day), and at the end of the 7th, 14th, 21st, 28th, and 35th days after the start of administration, the tumor volume (mm3 ) was measured.

[0127] Then, the experimental data obtained according to the method described in Item 1, "Statistical Analysis" of the general experimental method were analyzed.

[0128] B. Evaluation of anti-pancreatic cancer effect: 1×10 7 of the above MIA Paca-2 cell solution containing MIA Paca-2 cells was subcutaneously injected into a site near the dorsal side of the right hind limb of Balb / cAnN.Cg-Foxnl nu / CrlNarl nude mice. And on the 14th day after the injection of MIA Paca-2 cells and when the diameter of the tumor reached 5 mm to 6 mm, the nude mice were randomly divided into 6 groups (n = 8 for each group). The 6 groups included 1 control group and 5 experimental groups (i.e., Experimental Group 1 to Experimental Group 5).

[0129] The nude mice in Experimental Group 1 to Experimental Group 5 were given the paste-like mixture 3 obtained in Item 3 of Example 4 and the powdery mixture 3 obtained in Item 4 of Example 5 at the daily dosages shown in Table 9 below by the oral gavage method.

[0130] The nude mice in each experimental group were administered once a day for a total of 35 days. The nude mice in each control group were not treated.

[0131] Table 9: Mixtures orally administered to nude mice in each group

[0132]

Table 9

[0133] Before the start of administration (i.e., Day 0), and at the end of Day 7, Day 14, Day 21, Day 28, and Day 35 after the start of administration, the tumor volume (mm 3 ) of the nude mice in each group was measured using a TM900 tumor measuring instrument manufactured by Peira.

[0134] Then, the experimental data obtained according to the method described in Item 1, "Statistical Analysis" of the general experimental method were analyzed. Results: A. Evaluation of the anti-lung adenocarcinoma effect: Figure 7 is a diagram showing the change over time in the volume of A549 tumors in nude mice of each group after administration.

[0135] According to Figure 7, the volume of A549 tumors in nude mice of the control group gradually increased over time. In contrast, the increase over time in the volume of A549 tumors in nude mice of Experimental Groups 1 to 5 was relatively slow, and particularly, the rate of increase in the volume of A549 tumors in nude mice of Experimental Groups 4 to 5 was the slowest.

[0136] According to the experimental results, the Agaricus supercritical fluid extract of the present invention has a significantly effective inhibitory effect on the growth of lung adenocarcinoma cells in vivo even when used alone. Furthermore, it has been clarified that when the Agaricus supercritical fluid extract of the present invention is used in combination with at least one of the turmeric ethanol extract and the Ganoderma lucidum supercritical fluid extract, the effect of suppressing the growth of cancer cells is further improved.

[0137] B. Evaluation of the anti-pancreatic cancer effect: Figure 8 is a diagram showing the change over time in the volume of MIA Paca-2 tumors in nude mice of each group after administration.

[0138] According to Figure 8, the volume of MIA Paca-2 tumors in nude mice of the control group gradually increased over time. In contrast, the increase over time in the volume of MIA Paca-2 tumors in nude mice of Experimental Groups 1 to 5 was relatively slow.

[0139] According to the experimental results, when the Agaricus supercritical fluid extract of the present invention is combined with the turmeric ethanol extract and the Ganoderma lucidum supercritical fluid extract to form a mixture, it has been clarified that the mixture has an effect of suppressing the growth of pancreatic cancer cells in vivo even in the form of a paste or powder.

[0140] Summarizing the above experimental results, the Agaricus supercritical fluid extract of the present invention can effectively treat various cancers (including lung adenocarcinoma, gastric adenocarcinoma, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, prostate cancer, and cervical cancer) even when used alone, and can also be used in combination with either the turmeric ethanol extract or the Ganoderma lucidum supercritical fluid extract, or even when used in combination with the turmeric ethanol extract and the Ganoderma lucidum supercritical fluid extract, the therapeutic effect is further improved.

[0141] Therefore, the Agaricus supercritical fluid extract of the present invention can be applied to pharmaceuticals for treating cancer and can be used as an active ingredient of a cancer cell growth inhibitor.

[0142] All patents and literature references cited herein, as well as the references described therein, are hereby incorporated by reference in their entirety into this specification. In case of conflict, the description including the definitions shall prevail.

[0143] The present invention has been described in connection with what are considered to be exemplary embodiments, but the present invention is not limited to the disclosed embodiments and is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to include all such modifications and equivalent arrangements.

[0144] The above embodiments are illustrative of the principles and effects of the present invention and do not limit the present invention. Those skilled in the art can make some changes and modifications to the above embodiments on the premise of not departing from the spirit and scope of the present invention. Therefore, all changes and modifications made by those skilled in the art on the premise of not departing from the gist of the present invention should also be included in the protection scope of the present invention.

Industrial Applicability

[0145] Since the cancer cell growth inhibitor of the present invention contains the Agaricus supercritical fluid extract as an active ingredient, it is suitable for suppressing the growth of cancer cells.

Claims

1. 1. A cancer cell proliferation inhibitor comprising an Agaricus supercritical fluid extract as an active ingredient.

2. The cancer cell proliferation inhibitor described in claim 1, characterized in that the cancer cells are selected from the group consisting of lung adenocarcinoma cells, gastric adenocarcinoma cells, colon cancer cells, breast cancer cells, liver cancer cells, pancreatic cancer cells, prostate cancer cells, cervical cancer cells, and combinations thereof.

3. The cancer cell proliferation inhibitor described in claim 1, characterized in that it contains as an active ingredient a mixture containing the Agaricus supercritical fluid extract and one selected from the group consisting of a turmeric ethanol extract, a Ganoderma lucidum supercritical fluid extract, and combinations thereof.

4. The cancer cell proliferation inhibitor according to claim 3, characterized in that it contains as an active ingredient a mixture containing the Agaricus supercritical fluid extract, the Turmeric ethanol extract, and the Ganoderma lucidum supercritical fluid extract.

5. The cancer cell proliferation inhibitor according to claim 1, which is in a dosage form for parenteral administration, oral administration or topical administration.

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