Method for using placenta extract to produce a preparation for inhibiting normal cell proliferation in human ovarian cancer cells and method for producing a preparation for inhibiting normal cell proliferation in human ovarian cancer cells

Abstract

To provide a novel method of using a placenta extract, focusing on suppressing the growth of cancer cells.SOLUTION: The use of a placenta extract intends to suppress the growth of cancer cells. The use of a placenta extract also intends to provide the placenta extract as an active ingredient in a pharmaceutical preparation that suppresses the proliferation of cancer cells while allowing normal cells to proliferate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a placenta extract For the manufacture of a drug for inhibiting normal cell proliferation and human ovarian cancer cell proliferation How to use and a method for producing a preparation for inhibiting normal cell proliferation and human ovarian cancer cell proliferation Regarding 。 [Background technology]

[0002] Recent advances in medical technology and the widespread availability of nutritious diets have enabled humans to achieve longer lifespans. However, this increased lifespan has also led to an increase in the incidence of cancer. Therefore, there is a demand for components (or materials) that can be ingested through pharmaceuticals or daily diet and that have the effect of inhibiting the proliferation of cancer cells.

[0003] Meanwhile, mammalian placenta (placenta) and its extracts have been attracting attention from men and women of all ages as new materials that can improve both health and beauty (see, for example, Patent Document 1).

[0004] Patent Document 1 discloses a placenta extract powder-containing food product characterized by comprising 50-70% by mass of placenta extract powder extracted from pig placenta, 20-30% by mass of brewer's yeast, 5-10% by mass of chicken egg skin powder, and 5-10% by mass of rice straw powder.

[0005] The placenta extract powder-containing food disclosed in Patent Document 1 can provide a food that has excellent storage stability by suppressing the solidification of the placenta extract powder, and is also expected to have the effects of the placenta extract powder (for example, physical condition regulating effects in menopausal disorders and whitening effects). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-158214 Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, placenta has excellent effects in both health and beauty. It has also been reported to have the effect of reducing side effects caused by other treatments.

[0008] One example of such a report is "Placenta Extract Effective in Recovering from Radiation Damage (by Tsuneo Kata, Asahi Shimbun, September 25, 1982)." This article reports that mice exposed to radiation levels that would have killed them within 20 days could survive for 200 days simply by being injected with placenta extract.

[0009] One of the well-known effects of placenta is its ability to reduce the side effects of radiation-induced cancer treatment. This effect is thought to be due to the synergistic action of multiple substances contained in placenta to repair genes damaged by radiation.

[0010] However, the direct effect of placenta on cancerous cells remained unknown. Through extensive research, the present inventors have demonstrated that placenta extract has the effect of suppressing the proliferation of cancer cells.

[0011] The present invention provides a new method for using placenta extract, focusing on the inhibition of cancer cell proliferation. [Means for solving the problem]

[0012] In the present invention, (1) Use of a placenta extract as an active ingredient in a pharmaceutical product for inhibiting the proliferation of normal cell proliferation and human ovarian cancer cell proliferation, which inhibits the proliferation of human ovarian cancer cell proliferation while promoting the proliferation of normal cells, for the manufacture of the pharmaceutical product for inhibiting the proliferation of normal cell proliferation and human ovarian cancer cell proliferation, wherein the placenta extract is an extract obtained by extracting from freeze-dried powder of horse placenta using dimethyl sulfoxide as an extraction solvent. It was decided that.

[0014] In addition, in the present invention, ( 2 ) A method for manufacturing a pharmaceutical for inhibiting normal cell proliferation and human ovarian cancer cell proliferation, which contains placenta extract as an active ingredient for inhibiting the proliferation of human ovarian cancer cells while promoting the proliferation of normal cells, wherein the placenta extract is an extract extracted from freeze-dried powder of horse placenta using dimethyl sulfoxide as an extraction solvent. [Effects of the Invention]

[0018] According to the present invention , human ovary The use of placenta extracts as a means of inhibiting the growth of cancer cells can be presented as an option.

[0019] In addition, while normal cells are proliferating, human ovary Since the placenta extract is used as an active ingredient in a preparation that inhibits the proliferation of cancer cells, a new method of using the placenta extract can be provided. In particular, the placenta extract has a similar effect to normal cells. human ovary Uses that produce opposing effects on cancer cells and manufacturing method can be provided. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 is an explanatory diagram showing the test results of a normal cell (CCD841) proliferation test. [Figure 2] FIG. 1 is an explanatory diagram showing the test results of a colon cancer cell (HCT-116) proliferation test. [Figure 3] FIG. 1 is an explanatory diagram showing the test results of a cervical cancer cell (HeLa) proliferation test. [Figure 4] FIG. 1 is an explanatory diagram showing the test results of a neuroblastoma (SH-SY5Y) proliferation test. [Figure 5] FIG. 1 is an explanatory diagram showing the test results of a human astrocytoma cell (1321N1) proliferation test. [Figure 6] FIG. 1 is an explanatory diagram showing the test results of a human breast cancer cell (MDA-MB-453) proliferation test. [Figure 7] FIG. 1 is an explanatory diagram showing the test results of a human hepatoma cell (HuH-7) proliferation test. [Figure 8] FIG. 1 is an explanatory diagram showing the test results of a human ovarian cancer cell (OVK18) proliferation test. [Figure 9] FIG. 1 is an explanatory diagram showing the test results of a human pancreatic cancer cell (KLM-1) proliferation test. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to a method for using placenta extract, and more particularly to a novel method for using placenta extract to inhibit the proliferation of cancer cells by pharmaceuticals or functional foods.

[0026] Placenta extract is an extract of mammalian placenta (placenta), and when extraction is performed using a solvent, the placenta extract contains the extract or a dried product of the extract.

[0027] The placenta that is the raw material from which the placenta extract is extracted is not particularly limited, and placentas from various mammals including pigs, horses, and humans can be used.

[0028] In addition, the use in this embodiment refers to use as a raw material for a preparation used for the purpose of suppressing the proliferation of cancer cells. Examples of the preparation include pharmaceuticals and functional foods.

[0029] Pharmaceuticals are intended to be used for the diagnosis, treatment, or prevention of disease, and include, for example, prescription drugs prescribed by doctors at hospitals and over-the-counter (OTC) drugs sold at pharmacies or drug stores.

[0030] The form of placenta extract used as a pharmaceutical is not particularly limited, but may be, for example, an oral preparation that acts by oral administration, an external preparation that acts from outside the body, or an injection that acts by being placed directly under the skin or into a blood vessel.

[0031] Furthermore, functional foods are a concept that encompasses all foods that do not contain pharmaceutical ingredients and are believed to contribute to maintaining and promoting health, and include, for example, nutritional supplements, health supplements, nutritionally adjusted foods, as well as general foods such as so-called supplements, and health functional foods such as foods for specified health uses, foods with nutrient functions, and foods with functional claims.

[0032] The above explanations of pharmaceuticals and functional foods are merely examples of equivalent products to aid in the understanding of the present invention, and the interpretation of each term is not limited to these listed products, etc. However, the applicant may limit the present invention to these products, etc. when filing a patent for this application.

[0033] In this way, by using placenta extract to suppress the proliferation of cancer cells in pharmaceuticals and functional foods, it is possible to present the option of employing placenta extract as a means of suppressing the proliferation of cancer cells.

[0034] The present invention also provides a method for using placenta extract as an active ingredient in a formulation that inhibits the growth of cancer cells while promoting the growth of normal cells.

[0035] Cancer cells refer to cells that have acquired the ability to continue growing (abnormal proliferation) due to DNA mutations. Examples include colon cancer cells, cervical cancer cells, neuroblastoma, human astrocytoma cells, human breast cancer cells, human liver cancer cells, human ovarian cancer cells, and human pancreatic cancer cells, which are used in the experiments described below.

[0036] Normal cells are healthy cells that constitute a living body or are cultured in vitro and have not become cancerous. For example, normal colon cells are used in the experiments described below.

[0037] The active ingredient is a component that exhibits the effect of suppressing the proliferation of cancer cells. For example, if the formulation is a pharmaceutical product, it is an active ingredient, and if it is a functional food product, it is a functional ingredient.

[0038] The active ingredient may be a placenta extract used as it is, or may be a component purified from the placenta extract.

[0039] The placenta extract may be composed of a group of components that are soluble in an aqueous solvent containing water as the main component, i.e., the placenta extract may be an extract obtained by extraction with an aqueous solvent.

[0040] Here, the aqueous solvent is a solvent whose main component is water, and includes not only water itself but also solvents containing water at a ratio of, for example, 50 w / w% or more. The components other than water that make up the aqueous solvent can be, for example, solid components (powder, etc.) that are soluble in water, or liquids that are miscible with water, such as alcohol.

[0041] The placenta extract may be composed of a group of components that are soluble in a non-aqueous specific solvent selected from dimethyl sulfoxide, ethanol, methanol, and hexane, or a mixture of two or more of these. In other words, the placenta extract may be an extract extracted with a non-aqueous specific solvent.

[0042] The solvent may also be a mixed solvent of an aqueous solvent and a non-aqueous specific solvent, i.e., a solvent containing less than 50 w / w% water, with the remainder being the non-aqueous specific solvent, a solid (powder, etc.) component that is soluble in the mixed solvent to be prepared, or a liquid that is miscible with water, such as alcohol.

[0043] The components purified from placenta extract can be components that exhibit the effect of suppressing the proliferation of cancer cells, and can also be components that exhibit the effect of suppressing the proliferation of cancer cells and promoting the proliferation of normal cells at the same time.

[0044] In this way, by using placenta extract as an active ingredient in a formulation that inhibits the growth of cancer cells while promoting the growth of normal cells, a method of use can be provided that produces opposing effects on normal cells and cancer cells.

[0045] Furthermore, the method of using the placenta extract according to the present application has been clarified by the inventors through in vitro experiments, and exhibits different effects depending on the concentration of the placenta extract and the type of cancer cells exposed to the placenta extract.

[0046] Specifically, in the method of using placenta extract, the placenta extract is an extract extracted with an aqueous solvent containing water as the main component, and can be characterized in that a single dose of the formulation contains an amount that can produce an effect in vivo that is equivalent to the growth inhibitory effect exerted when the cancer cells cultured in vitro are exposed to the extract at a concentration of more than 0 μg / mL and not more than 400 μg / mL.

[0047] According to this method of using placenta extract, by adjusting the concentration of the placenta extract, it is possible to provide a method of using placenta extract that suppresses the growth of cancer cells while promoting the growth of normal cells.

[0048] Furthermore, in the method for using the placenta extract, the placenta extract is an extract extracted with an aqueous solvent containing water as the main component, and can be characterized in that a single dose of the formulation contains an amount that can produce an effect in vivo that is equivalent to the growth inhibitory effect exerted when the cancer cells cultured in vitro are exposed to the extract at a concentration of more than 400 μg / mL.

[0049] According to this method of using placenta extract, by adjusting the concentration of the placenta extract, it is possible to provide a method of using placenta extract that suppresses the proliferation of cancer cells without affecting the proliferation of normal cells.

[0050] Furthermore, the method for using the placenta extract can be characterized in that the placenta extract is used on colon cancer cells, human liver cancer cells, or human pancreatic cancer cells.

[0051] According to such a method for using the placenta extract, the proliferation of colon cancer cells, human liver cancer cells or human pancreatic cancer cells can be strongly inhibited in a concentration-dependent manner.

[0052] Furthermore, a method for using the placenta extract can be characterized in that the placenta extract is used on cervical cancer cells, neuroblastoma, human astrocytoma cells, human breast cancer cells, or ovarian cancer cells.

[0053] According to this method of using placenta extract, it is possible to provide a method of using placenta extract that inhibits the proliferation of cervical cancer cells, neuroblastoma cells, human astrocytoma cells, human breast cancer cells, or ovarian cancer cells to approximately the same extent regardless of concentration.

[0054] The method of use according to this embodiment will be further explained below with reference to experimental results. Note that, although the following describes an example in which a placenta extract extracted from horse placenta (hereinafter referred to as horse placenta extract) is used, the origin of placenta is not limited to horse.

[0055] [1. Preparation of placenta extract] Due to the recent increase in cancer incidence, there is a demand for ingredients (or materials) that can suppress the growth of cancer cells in pharmaceuticals and functional foods. Placenta has attracted attention as a new material that can promote both human health and beauty, but its direct effect on cancer cells remains unknown.

[0056] In this experiment, horse placenta powder, a freeze-dried powder of horse placenta, was used as the placenta extract.

[0057] First, 100 mg of horse placenta powder was weighed into a 1.5 ml tube, and 1 ml of extraction solvent was added and stirred at room temperature for 30 minutes. The extraction solvents used were sterilized ultrapure water as an aqueous solvent and dimethyl sulfoxide (hereinafter also referred to as DMSO) as a non-aqueous specific solvent.

[0058] After stirring, the tube is left at room temperature, and the obtained supernatant is called equine placenta extract.This equine placenta extract is prepared by adding 1 mL of extraction solvent to 100 mg of placenta powder, and is called 100 mg / ml sample.In the following tests, the diluted sample prepared by appropriately diluting the 100 mg / ml sample is called 1 mg / ml sample, for example, if the sample is diluted 100 times, it is called 1 mg / ml sample, and if the sample is diluted 10,000 times, it is called 10 μ g / ml sample.

[0059] [2. Cell proliferation test 1] In this test, normal cells or cancer cells were co-cultured with the sample, and the cell viability was measured.

[0060] In this study, normal colon cells (CCD841) were used as normal cells, and colon cancer cells (HCT-116), cervical cancer cells (HeLa), neuroblastoma cells (SH-SY5Y), and human astrocytoma cells (1321N1) were used as cancer cells.

[0061] Cervical cancer cells, human astrocytoma cells, and neuroblastoma cells were cultured in Dulbecco's Modified Eagle Medium (D-MEM), normal cells in Eagle's Minimal Essential Medium (E-MEM), and colon cancer cells in McCoy's 5A Modified Medium, supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin (SP).

[0062] Then, 1.0 x 105 100 μL of cells / mL were seeded and cultured overnight in a CO 2 incubator (37°C, 5% CO 2 ).

[0063] After overnight incubation, the medium was removed and 100 μL of liquid medium containing 0.5% FBS and 1% SP was added. 0.5 μL of sample solution was added to each well of the liquid medium, and the cells were incubated in a CO2 incubator (37°C, 5% CO2) for 72 hours.

[0064] The cell viability was measured by the MTT method. For the MTT method, the medium was removed from the plate after 72 hours of culture, and 100 μL of MTT staining solution (5 mg / mL in medium) was added to each well, followed by static culture at 5% CO2 and 37°C for 4 hours.

[0065] After 4 hours of static culture, the MTT staining solution was removed, 100 μL of hydrochloric acid-isopropanol solution was added to each well, and the plate was left at room temperature for 4 hours in the dark.

[0066] Thereafter, the absorbance at 570 nm was measured using a microplate reader and used as an index of cell viability.

[0067] Figure 1 is a graph showing cell viability relative to a control of normal cells, where Figure 1(a) shows the cell viability of a placenta extract extracted with an aqueous solvent (hereinafter also referred to as an aqueous placenta extract), and Figure 1(b) shows the cell viability of a placenta extract extracted with a specific non-aqueous solvent (hereinafter referred to as a non-aqueous placenta extract).

[0068] As shown in Figure 1(a), when the aqueous placenta extract was tested at 10μg / mL, 100μg / mL, and 400μg / mL samples, the 10μg / mL sample showed a cell proliferation promoting effect of approximately 20%, and the 100μg / mL sample showed a cell proliferation promoting effect of approximately 15%. In contrast, the 400μg / mL sample showed no significant cell proliferation promoting effect compared to the control.

[0069] This revealed that placenta aqueous extract promotes the proliferation of normal cells at relatively low concentrations (10 μg / mL, 100 μg / mL), but does not affect the proliferation of normal cells at relatively high concentrations (400 μg / mL).

[0070] In other words, it is thought that the aqueous placenta extract has a proliferation-promoting effect when exposed to normal cells at concentrations exceeding 0 μg / L and up to 400 μg / mL, but does not affect proliferation when exposed to normal cells at concentrations of 400 μg / mL or higher.

[0071] In contrast, when the diluted samples of the non-aqueous placenta extract were examined, a cell proliferation promoting effect of approximately 20% was observed at all concentrations examined, as shown in Figure 1(b).

[0072] From this, it was considered that the non-aqueous placenta extract has an effect of promoting cell proliferation of normal cells at a substantially constant rate regardless of the concentration.

[0073] Furthermore, taking into account the results for these aqueous and non-aqueous placenta extracts, it is interesting to note that the non-aqueous placenta extract promoted the proliferation of normal cells equally at all sample concentrations, whereas the aqueous placenta extract promoted the proliferation of normal cells more strongly at lower sample concentrations.

[0074] Figure 2 is a graph showing the cell viability of colon cancer cells relative to the control, where Figure 2(a) shows the cell viability of the aqueous placenta extract, and Figure 2(b) shows the cell viability of the non-aqueous placenta extract.

[0075] As shown in Figure 2(a), when the aqueous placenta extract was examined at 10 μg / mL, 100 μg / mL, and 400 μg / mL samples, the 100 μg / mL sample showed a cell proliferation inhibitory effect of approximately 20%, and the 400 μg / mL sample showed a cell proliferation inhibitory effect of approximately 70%.

[0076] This demonstrates that the aqueous placenta extract has a strong inhibitory effect on the proliferation of colon cancer cells in a concentration-dependent manner.

[0077] In contrast, when the diluted samples of the placenta non-aqueous extract were examined, as shown in Figure 2(b), a cell proliferation inhibitory effect of approximately 40% was observed in the 100 μg / mL sample and approximately 60% in the 400 μg / mL sample.

[0078] Furthermore, taking into account the results for these aqueous and non-aqueous placenta extracts, it can be said that both aqueous placenta extracts and non-aqueous placenta extracts exhibit strong inhibitory effects on the proliferation of colon cancer cells in a concentration-dependent manner.

[0079] Here, when comparing the results of placenta aqueous extract in normal cells and colon cancer cells, it is believed that the placenta aqueous extract promotes the growth of normal cells and suppresses the growth of colon cancer cells in the 100 μg / mL sample, and suppresses the growth of colon cancer cells in the 400 μg / mL sample without affecting the growth of normal cells.

[0080] Furthermore, when comparing the results of placenta non-aqueous extracts in normal cells and colon cancer cells, it is believed that concentrations of 100 μg / mL or higher promote the proliferation of normal cells at a roughly constant rate and suppress the proliferation of colon cancer cells at a roughly constant rate.

[0081] Figure 3 is a graph showing the cell viability of cervical cancer cells relative to the control, where Figure 3(a) shows the cell viability of the aqueous placenta extract, and Figure 3(b) shows the cell viability of the non-aqueous placenta extract.

[0082] As shown in Figure 3(a), when the aqueous placenta extract was examined at 10 μg / mL, 100 μg / mL, and 400 μg / mL samples, a cell proliferation inhibitory effect of approximately 30% was observed in the 100 μg / mL and 400 μg / mL samples.

[0083] This demonstrates that the aqueous placenta extract exhibits a substantially constant inhibitory effect on the proliferation of cervical cancer cells at a certain concentration or higher.

[0084] In contrast, when the diluted samples of the non-aqueous placenta extract were examined, as shown in Figure 3(b), a cell proliferation inhibitory effect of approximately 30% was observed in the 100 μg / mL and 400 μg / mL samples.

[0085] Furthermore, taking into account the results for these aqueous and non-aqueous placenta extracts, it can be said that both aqueous placenta extracts and non-aqueous placenta extracts exhibit a substantially consistent inhibitory effect on the proliferation of cervical cancer cells at concentrations of 100 μg / mL or higher.

[0086] Here, comparing the results of placenta aqueous extract on normal cells and cervical cancer cells, it appears that the placenta aqueous extract promotes the proliferation of normal cells and inhibits the proliferation of cervical cancer cells in the 100 μg / mL sample, and inhibits the proliferation of cervical cancer cells without affecting the proliferation of normal cells in the 400 μg / mL sample.

[0087] Furthermore, when comparing the results of the placenta non-aqueous extract on normal cells and cervical cancer cells, it is believed that a concentration of 100 μg / mL or higher will promote the growth of normal cells at a roughly constant rate and suppress the growth of cervical cancer cells at a roughly constant rate.

[0088] Figure 4 is a graph showing cell viability relative to the control of neuroblastoma, where Figure 4(a) shows the cell viability of an aqueous placenta extract, and Figure 4(b) shows the cell viability of a non-aqueous placenta extract.

[0089] As shown in Figure 4(a), when the aqueous placenta extract was examined at 10 μg / mL, 100 μg / mL, and 400 μg / mL samples, a cell proliferation inhibitory effect of approximately 20% was observed at all concentrations examined.

[0090] This demonstrates that the aqueous placenta extract exhibits a substantially constant inhibitory effect on the proliferation of neuroblastoma at a certain concentration or higher.

[0091] In contrast, when the diluted samples of the placenta non-aqueous extract were examined, as shown in Figure 4(b), a cell proliferation inhibitory effect of approximately 10% was observed at 10 μg / mL and 100 μg / mL, and approximately 90% at 400 μg / mL.

[0092] Furthermore, based on the results of these aqueous and non-aqueous placenta extracts, it can be said that both aqueous and non-aqueous placenta extracts exhibit a certain inhibitory effect on the proliferation of neuroblastoma. In particular, a significantly strong cell proliferation inhibitory effect was confirmed at 400 μg / mL of the non-aqueous placenta extract.

[0093] Here, comparing the results of placenta aqueous extract in normal cells and neuroblastoma cells, it appears that the placenta aqueous extract promotes the proliferation of normal cells and inhibits the proliferation of neuroblastoma in the 10 μg / mL and 100 μg / mL samples, and inhibits the proliferation of neuroblastoma in the 400 μg / mL sample without affecting the proliferation of normal cells.

[0094] In addition, when comparing the results of the placenta non-aqueous extract in normal cells and neuroblastoma, it is considered that it promotes the proliferation of normal cells and inhibits the proliferation of neuroblastoma cells at all sample concentrations tested. In particular, at 400 μg / mL of the placenta non-aqueous extract, there is no difference in the proliferation of normal cells compared to other sample concentrations, but it is considered to have a strong effect of inhibiting the proliferation of neuroblastoma.

[0095] Figure 5 is a graph showing the cell viability of human astrocytoma cells relative to the control, where Figure 5(a) shows the cell viability of an aqueous placenta extract, and Figure 5(b) shows the cell viability of a non-aqueous placenta extract.

[0096] As shown in Figure 5(a), when the aqueous placenta extract was examined using 10 μg / mL, 100 μg / mL, and 400 μg / mL samples, a cell proliferation inhibitory effect of approximately 20% was observed in the 100 μg / mL and 400 μg / mL samples.

[0097] This demonstrates that an aqueous placenta extract exhibits a substantially constant inhibitory effect on the proliferation of human astrocytoma cells at a certain concentration or higher.

[0098] In contrast, when the diluted samples of the non-aqueous placenta extract were examined, as shown in Figure 5(b), a cell proliferation inhibitory effect of approximately 10% was observed at all concentrations examined.

[0099] This demonstrates that the non-aqueous placenta extract exhibits an inhibitory effect on the proliferation of human astrocytoma cells to approximately the same extent regardless of the sample concentration.

[0100] Furthermore, taking into account the results for these aqueous and non-aqueous placenta extracts, it was confirmed that aqueous placenta extracts and non-aqueous placenta extracts exhibit a substantially consistent inhibitory effect on the proliferation of human astrocytoma cells at sample concentrations of 100 μg / mL or higher.

[0101] Here, comparing the results of placenta aqueous extract in normal cells and human astrocytoma cells, it appears that the placenta aqueous solvent promotes the proliferation of normal cells and inhibits the proliferation of neuroblastoma in the 100 μg / mL sample, and inhibits the proliferation of neuroblastoma in the 400 μg / mL sample without affecting the proliferation of normal cells.

[0102] Furthermore, when comparing the results of the placenta non-aqueous extract on normal cells and human astrocytoma cells, it was found that it promoted the proliferation of normal cells at all sample concentrations tested. In particular, the cell proliferation inhibition effect of each sample concentration on human astrocytoma cells was almost constant.

[0103] Based on the results of this test, when placenta aqueous extract is used in an amount that can produce an effect in the body that is equivalent to the growth inhibitory effect produced when in vitro-cultured colon cancer cells, cervical cancer cells, neuroblastoma or human astrocytoma cells are exposed to a concentration of more than 0 μg / mL and not more than 400 μg / mL of placenta aqueous extract, it can provide an active ingredient in a formulation that inhibits the growth of colon cancer cells, cervical cancer cells, neuroblastoma or human astrocytoma cells while allowing normal cells to grow.

[0104] Furthermore, when the aqueous placenta extract is used in an amount that can produce an effect in vivo that is equivalent to the growth inhibitory effect produced when in vitro-cultured colon cancer cells, cervical cancer cells, neuroblastoma or human astrocytoma cells are exposed to the aqueous placenta extract at a concentration of more than 400 μg / mL, the aqueous placenta extract can provide an active ingredient in a formulation that inhibits the growth of colon cancer cells, cervical cancer cells, neuroblastoma or human astrocytoma cells without affecting the growth of normal cells.

[0105] On the other hand, when the placenta non-aqueous extract is used in an amount that can produce an effect in the body that is equivalent to the growth inhibitory effect produced when in vitro-cultured colon cancer cells, cervical cancer cells, neuroblastoma or human astrocytoma cells are exposed to the placenta non-aqueous extract at a concentration of more than 0 μg / mL, it can provide an active ingredient in a formulation that inhibits the growth of colon cancer cells, cervical cancer cells, neuroblastoma or human astrocytoma cells while allowing normal cells to grow.

[0106] Furthermore, when used on colon cancer cells, the placenta extract can inhibit proliferation in a concentration-dependent manner, and when used on cervical cancer cells, neuroblastoma cells, or human astrocytoma cells, it can inhibit proliferation at a substantially constant level above a certain concentration.

[0107] [3. Cell proliferation test 2] In this test, cancer cells and the sample were co-cultured and the cell viability was measured.

[0108] In this study, human breast cancer cells (MDA-MB-453), human liver cancer cells (HuH-7), human ovarian cancer cells (OVK18), and human pancreatic cancer cells (KLM-1) were used.

[0109] For cell culture, human breast cancer cells, human liver cancer cells, and human pancreatic cancer cells were pre-cultured using RPMI-1640 (containing 1% SP and 10% FBS), and human ovarian cancer cells were pre-cultured using MEMα (containing 1% SP and 10% FBS).

[0110] Then, 0.5 x 10 4 The plates were seeded at a concentration of 1000 cells / well and cultured overnight in a CO2 incubator (37°C, 5% CO2). Human breast cancer cells (MDA-MB-453) were cultured overnight in a sealed environment at 37°C and 0% CO2.

[0111] After overnight incubation, the medium was removed and replaced with serum-free medium containing the sample solution (containing 1% SP), and the cells were cultured for 24 hours at 37°C and 5% CO2 (MDA-MB-453: 37°C, 0% CO2). The sample solution was added to both aqueous and non-aqueous systems to final concentrations of 5, 50, 100, and 500 μg / mL.

[0112] The cell viability was measured by the MTT method. For the MTT method, the medium was removed from the plate after 24 hours of culture, and 100 μL of serum-free medium (containing 1% SP) without sample solution was added to each well. Next, 20 μL of MTT staining solution (5 mg / mL in phosphate-buffered saline (hereinafter also referred to as PBS)) was added to each well, and the plates were left to stand in a CO2 incubator for 4 hours. Human breast cancer cells were also cultured in the same manner at 37°C and 0% CO2 for 4 hours.

[0113] After 4 hours of static culture, the serum-free medium was removed, 100 μL of hydrochloric acid-isopropanol solution was added to each well, and the formazan was completely dissolved by pipetting. The absorbance at 570 nm was then measured using a microplate reader, and the cell viability was calculated.

[0114] Figure 6 is a graph showing the cell viability of human breast cancer cells relative to the control, where Figure 6(a) shows the cell viability in an aqueous placenta solvent and Figure 6(b) shows the cell viability in a non-aqueous placenta extract.

[0115] As shown in Figure 6(a), when the aqueous placenta extract was tested at 5μg / mL, 50μg / mL, 100μg / mL, and 500μg / mL samples, a cell proliferation inhibitory effect was observed at all concentrations tested. In particular, a cell proliferation inhibitory effect of approximately 60% was observed at concentrations of 50μg / mL or higher.

[0116] This demonstrates that the aqueous placenta extract exhibits a substantially constant inhibitory effect on the proliferation of human breast cancer cells at a certain concentration or higher.

[0117] In contrast, when the diluted samples of the non-aqueous placenta extract were examined, the cell proliferation inhibitory effect was observed at all concentrations tested, as shown in Figure 6(b). In particular, a cell proliferation inhibitory effect of approximately 60% was observed at concentrations of 50 μg / mL or higher.

[0118] Furthermore, taking into account the results for these aqueous and non-aqueous placenta extracts, it can be said that both aqueous placenta extracts and non-aqueous placenta extracts inhibit the proliferation of human breast cancer cells, and that at concentrations of 50 μg / mL or higher, each exhibits a substantially consistent inhibitory effect.

[0119] Here, when comparing the results of the placenta aqueous extract on human breast cancer cells in this test with those on normal cells tested in Cell Proliferation Test 1, it appears that at low concentrations (10 μg / mL sample), the placenta aqueous extract inhibits the proliferation of human breast cancer cells while promoting the proliferation of normal cells, and at concentrations of 400 μg / mL or higher, it inhibits the proliferation of human breast cancer cells without affecting the proliferation of normal cells.

[0120] Furthermore, when comparing the results of the placenta non-aqueous extract on human breast cancer cells in this test with those on normal cells tested in Cell Proliferation Test 1, it is believed that a placenta non-aqueous extract at a concentration of 10μg / mL or higher will inhibit the proliferation of human breast cancer cells while promoting the proliferation of normal cells. In particular, a concentration of 50μg / mL or higher is thought to inhibit the proliferation of human breast cancer cells by approximately 60% and promote the proliferation of normal cells by approximately 20%.

[0121] Figure 7 is a graph showing the cell viability of human liver cancer cells relative to the control, where Figure 7(a) shows the cell viability in an aqueous placenta solvent, and Figure 7(b) shows the cell viability in a non-aqueous placenta extract.

[0122] As shown in Figure 7(a), when the aqueous placenta extract was tested at 5μg / mL, 50μg / mL, 100μg / mL, and 500μg / mL samples, a cell proliferation inhibitory effect was observed at all concentrations tested. In particular, a strong inhibitory effect on the proliferation of human liver cancer cells was observed, depending on the sample concentration.

[0123] This demonstrates that the aqueous placenta extract has a strong inhibitory effect on the proliferation of human liver cancer cells in a concentration-dependent manner.

[0124] In contrast, when the diluted samples of the non-aqueous placenta extract were examined, the cell proliferation inhibitory effect was observed at all concentrations tested, as shown in Figure 7(b). In particular, a cell proliferation inhibitory effect of approximately 60% was observed at concentrations of 50 μg / mL or higher.

[0125] Furthermore, based on the results of these aqueous and non-aqueous placenta extracts, it can be said that both aqueous placenta extracts and non-aqueous placenta extracts exhibit an inhibitory effect on the proliferation of human liver cancer cells. In particular, it can be said that the inhibitory effect increases depending on the sample concentration.

[0126] Here, when comparing the results of the placenta aqueous extract on human liver cancer cells in this test with those on normal cells tested in Cell Proliferation Test 1, it is believed that at low concentrations (10 μg / mL sample), the placenta aqueous extract inhibits the proliferation of human liver cancer cells while promoting the proliferation of normal cells, and at concentrations of 400 μg / mL or higher, it inhibits the proliferation of human liver cancer cells without affecting the proliferation of normal cells.

[0127] Furthermore, when comparing the results of the placenta non-aqueous extract on human liver cancer cells in this test with those on normal cells tested in Cell Proliferation Test 1, it is believed that the placenta non-aqueous extract, at a concentration of 10μg / mL or more, inhibits the proliferation of human liver cancer cells while promoting the proliferation of normal cells. In particular, at a concentration of 50μg / mL or more, it is believed to inhibit the proliferation of human breast cancer cells by approximately 60% and promote the proliferation of normal cells by approximately 20%.

[0128] Figure 8 is a graph showing the cell viability of human ovarian cancer cells relative to the control, where Figure 8(a) shows the cell viability in an aqueous placenta solvent and Figure 8(b) shows the cell viability in a non-aqueous placenta extract.

[0129] As shown in Figure 8(a), when the aqueous placenta extract was examined at 5 μg / mL, 50 μg / mL, 100 μg / mL, and 500 μg / mL samples, a cell proliferation inhibitory effect of approximately 20% was observed at all concentrations examined.

[0130] This demonstrates that aqueous placenta extracts exhibit a substantially constant inhibitory effect on the proliferation of human ovarian cancer cells at concentrations of 5 μg / mL or more.

[0131] In contrast, when the diluted samples of the non-aqueous placenta extract were examined, the cell proliferation inhibitory effect was observed at all concentrations tested, as shown in Figure 8(b). In particular, a cell proliferation inhibitory effect of approximately 30% was observed at concentrations of 50 μg / mL or higher.

[0132] Furthermore, based on the results of these aqueous and non-aqueous placenta extracts, it can be said that both the aqueous placenta extract and the non-aqueous placenta extract exhibit an inhibitory effect on the proliferation of human ovarian cancer cells. In particular, it can be said that at a predetermined sample concentration or higher, each extract can achieve a substantially constant cell proliferation inhibitory effect.

[0133] Here, when comparing the results of the placenta aqueous extract on human ovarian cancer cells in this test with those on normal cells tested in Cell Proliferation Test 1, it is believed that at low concentrations (10 μg / mL sample), the placenta aqueous extract inhibits the proliferation of human ovarian cancer cells while promoting the proliferation of normal cells, and at concentrations of 400 μg / mL or higher, it inhibits the proliferation of human ovarian cancer cells without affecting the proliferation of normal cells.

[0134] Furthermore, when comparing the results of the placenta non-aqueous extract on human ovarian cancer cells in this test with those on normal cells tested in Cell Proliferation Test 1, it is believed that the placenta non-aqueous extract, at a concentration of 10μg / mL or more, inhibits the proliferation of human ovarian cancer cells while promoting the proliferation of normal cells. In particular, at a concentration of 50μg / mL or more, it is believed that the proliferation of human ovarian cancer cells is inhibited by approximately 20% and the proliferation of normal cells is promoted by approximately 20%.

[0135] Figure 9 is a graph showing the cell viability of human pancreatic cancer cells relative to the control, where Figure 9(a) shows the cell viability in an aqueous placenta solvent and Figure 9(b) shows the cell viability in a non-aqueous placenta extract.

[0136] As shown in Figure 9(a), when the aqueous placenta extract was examined using 5 μg / mL, 50 μg / mL, 100 μg / mL, and 500 μg / mL samples, a cell proliferation inhibitory effect of approximately 30% was observed in the 5 μg / mL sample and approximately 60% in the 500 μg / mL sample.

[0137] This demonstrates that the aqueous placenta extract has a strong inhibitory effect on the proliferation of human pancreatic cancer cells in a concentration-dependent manner.

[0138] In contrast, when the non-aqueous placenta extract was examined for each diluted sample, it was found to have an inhibitory effect on cell proliferation at all concentrations tested, as shown in Figure 9(b). In particular, at concentrations of 50 μg / mL or higher, it was found to have an inhibitory effect on cell proliferation of approximately 60%.

[0139] Furthermore, taking into account the results for these aqueous and non-aqueous placenta extracts, it was confirmed that aqueous placenta extracts and non-aqueous placenta extracts exhibited inhibitory effects on human pancreatic cancer cells at all sample concentrations tested.

[0140] Here, when comparing the results of the placenta aqueous extract on human pancreatic cancer cells in this test with those on normal cells tested in Cell Proliferation Test 1, it appears that at low concentrations (10 μg / mL sample), the placenta aqueous extract inhibits the proliferation of human pancreatic cancer cells while promoting the proliferation of normal cells, and at concentrations of 400 μg / mL or higher, it inhibits the proliferation of human pancreatic cancer cells without affecting the proliferation of normal cells.

[0141] Furthermore, when comparing the results of the placenta non-aqueous extract on human pancreatic cancer cells in this test with those on normal cells tested in Cell Proliferation Test 1, it is believed that the placenta non-aqueous extract inhibits the proliferation of human pancreatic cancer cells and promotes the proliferation of normal cells at concentrations of 10μg / mL or more. In particular, at concentrations of 50μg / mL or more, it is believed to inhibit the proliferation of human breast cancer cells by approximately 60% and promote the proliferation of normal cells by approximately 20%.

[0142] Based on the results of this test, placenta aqueous extract and non-aqueous extract can provide active ingredients in formulations that inhibit the proliferation of human breast cancer cells, human liver cancer cells, human ovarian cancer cells, or human pancreatic cancer cells, if used in amounts that can produce an effect in the body that is equivalent to the growth inhibitory effect produced when the placenta aqueous extract is exposed to in vitro-cultured human breast cancer cells, human liver cancer cells, human ovarian cancer cells, or human pancreatic cancer cells.

[0143] Furthermore, considering the results of promoting the proliferation of normal cells shown in Cell Proliferation Test 1, it can be said that the placenta aqueous extract can provide an active ingredient in a formulation that inhibits the proliferation of human breast cancer cells, human liver cancer cells, human ovarian cancer cells, or human pancreatic cancer cells while promoting the proliferation of normal cells, if it is used in an amount that can produce an effect in the body that is equivalent to the proliferation inhibitory effect produced when in vitro-cultured human breast cancer cells, human liver cancer cells, human ovarian cancer cells, or human pancreatic cancer cells are exposed to the placenta aqueous extract at a concentration of more than 0 μg / L and not more than 500 μg / L.

[0144] Furthermore, when the aqueous placenta extract is used in an amount that can produce an effect in vivo that is equivalent to the growth inhibitory effect produced when in vitro-cultured human breast cancer cells, human liver cancer cells, human ovarian cancer cells, or human pancreatic cancer cells are exposed to the aqueous placenta extract at a concentration of more than 500 μg / L, it can also be said that the aqueous placenta extract can provide an active ingredient in a formulation that inhibits the growth of human breast cancer cells, human liver cancer cells, human ovarian cancer cells, or human pancreatic cancer cells without affecting the growth of normal cells.

[0145] On the other hand, when the placenta non-aqueous extract is used in an amount that can produce an effect in vivo that is equivalent to the growth inhibitory effect produced when in vitro-cultured human breast cancer cells, human liver cancer cells, human ovarian cancer cells, or human pancreatic cancer cells are exposed to the placenta non-aqueous extract at a concentration of more than 0 μg / L, the placenta non-aqueous extract can provide an active ingredient in a formulation that inhibits the growth of human breast cancer cells, human liver cancer cells, human ovarian cancer cells, or human pancreatic cancer cells while allowing normal cells to grow.

[0146] Furthermore, when used on colon cancer cells, the placenta extract can inhibit proliferation in a concentration-dependent manner, and when used on cervical cancer cells, neuroblastoma cells, or human astrocytoma cells, it can inhibit proliferation at a substantially constant level above a certain concentration.

[0147] [4. Summary] As shown in the results of Cell Proliferation Test 1 (Figures 2 to 5), placenta extract has the effect of inhibiting the proliferation of colon cancer cells, cervical cancer cells, neuroblastoma cells, and human astrocytoma cells. Also, as shown in the results of Cell Proliferation Test 2 (Figures 6 to 9), placenta extract has the effect of inhibiting the proliferation of human breast cancer cells, human liver cancer cells, human ovarian cancer cells, and human pancreatic cancer cells.

[0148] Therefore, the placenta extract has the effect of inhibiting the proliferation of cancer cells. In other words, it can be said that the placenta extract can be used for the purpose of inhibiting the proliferation of cancer cells.

[0149] Furthermore, as shown in the results of Cell Proliferation Test 1 (Figure 1), placenta extract has the effect of promoting the proliferation of normal cells.

[0150] Therefore, the placenta extract has the effect of inhibiting the proliferation of cancer cells while promoting the proliferation of normal cells. In other words, the placenta extract can be used as an active ingredient in a preparation that inhibits the proliferation of cancer cells while promoting the proliferation of normal cells.

[0151] Furthermore, as shown in the results of Cell Test 1 (Figure 1), the aqueous placenta extract showed a cell proliferation promoting effect of approximately 20% in the 10μg / mL sample, approximately 15% in the 100μg / mL sample, and no significant promoting effect was observed in the 400μg / mL sample compared to the control. In other words, the aqueous placenta extract tends to promote cell proliferation at lower concentrations relative to normal cells, and has a tendency to have no effect on cell proliferation at higher concentrations.

[0152] Therefore, by adjusting the concentration of the placenta aqueous extract exposed to normal cells, the use of the placenta aqueous extract can provide a method of use that inhibits the proliferation of cancer cells while having different effects on normal cells.

[0153] Specifically, the placenta extract is extracted with an aqueous solvent containing water as the main component, and is characterized in that a single dose of the formulation contains an amount that can produce an effect in vivo that is equivalent to the growth inhibitory effect produced when cancer cells cultured in vitro are exposed to the extract at a concentration of more than 0 μg / mL and not more than 400 μg / mL, thereby enabling the growth of cancer cells to be inhibited while promoting the growth of normal cells.

[0154] Furthermore, the placenta extract is extracted with an aqueous solvent containing water as the main component, and is characterized in that a single dose of the preparation contains an amount that can produce an effect in vivo that is equivalent to the growth inhibitory effect exerted when cancer cells cultured in vitro are exposed to the extract at a concentration of more than 400 μg / mL, thereby making it possible to inhibit the growth of cancer cells without affecting the growth of normal cells.

[0155] Furthermore, placenta extract (especially aqueous placenta extract) has a concentration-dependent strong inhibitory effect on the proliferation of colon cancer cells, human liver cancer cells, and human pancreatic cancer cells, as shown in Cell Proliferation Test 1 (Figure 2) and Cell Proliferation Test 2 (Figures 7 and 9).

[0156] Therefore, by using the placenta extract on colon cancer cells, human liver cancer cells or human pancreatic cancer cells, the effect on the proliferation of colon cancer cells, human liver cancer cells or human pancreatic cancer cells can be adjusted depending on the concentration of the placenta extract.

[0157] Furthermore, placenta extract (especially aqueous placenta extract) has the effect of exhibiting a substantially constant inhibitory effect on cervical cancer cells, neuroblastoma, human astrocytoma cells, human breast cancer cells, and human ovarian cancer cells, regardless of concentration, as shown in Cell Proliferation Test 1 (Figures 3 to 5) and Cell Proliferation Test 2 (Figures 6 and 8).

[0158] Therefore, the use of placenta extract is characterized by its use on cervical cancer cells, neuroblastoma, human astrocytoma cells, human breast cancer cells, and human ovarian cancer cells, and as long as the placenta extract is at a predetermined concentration or higher, a substantially constant inhibitory effect can be obtained for each cell. The invention included in this application also has the following effects. According to the present invention, the placenta extract is used to inhibit the proliferation of cancer cells, which provides a new method of using the placenta extract and also provides an option of using the placenta extract as a means of inhibiting the proliferation of cancer cells. Furthermore, since the placenta extract is used as an active ingredient in a preparation that inhibits the proliferation of cancer cells while promoting the proliferation of normal cells, a new method of using the placenta extract can be provided, particularly a method of using the placenta extract that produces opposing effects on normal cells and cancer cells. Furthermore, the placenta extract is an extract extracted with an aqueous solvent containing water as the main component, and a single dose of the formulation contains an amount that can produce an effect in vivo that is equivalent to the growth inhibitory effect produced when the cancer cells cultured in vitro are exposed to the extract at a concentration of more than 0 μg / mL and not more than 400 μg / mL.Therefore, by adjusting the concentration of the placenta extract and using it, a method of using the placenta extract can be provided that inhibits the growth of cancer cells while promoting the growth of normal cells. Furthermore, the placenta extract is an extract extracted with an aqueous solvent containing water as the main component, and a single dose of the formulation contains an amount that can produce an effect in vivo that is equivalent to the growth inhibitory effect exerted when the cancer cells cultured in vitro are exposed to the extract at a concentration of more than 400 μg / mL.Therefore, by adjusting the concentration of the placenta extract and using it, a method of using the placenta extract to inhibit the growth of cancer cells without affecting the growth of normal cells can be provided. Furthermore, since the placenta extract is used on colon cancer cells, human liver cancer cells, or human pancreatic cancer cells, it is possible to provide a method for using the placenta extract to strongly inhibit the proliferation of colon cancer cells, human liver cancer cells, or human pancreatic cancer cells in a concentration-dependent manner. That is, when using the placenta, the effect on the proliferation of colon cancer cells, human liver cancer cells, or human pancreatic cancer cells can be adjusted depending on the concentration of the placenta extract. Furthermore, since the placenta extract is used on cervical cancer cells, neuroblastoma, human astrocytoma cells, human breast cancer cells, or ovarian cancer cells, it is possible to provide a method for using the placenta extract that exhibits a substantially constant inhibitory effect on cervical cancer cells, neuroblastoma, human astrocytoma cells, human breast cancer cells, or ovarian cancer cells at a predetermined concentration or higher.

Claims

1. 1. Use of a placenta extract as an active ingredient in a pharmaceutical product for inhibiting the proliferation of normal proliferating human ovarian cancer cells, which inhibits the proliferation of human ovarian cancer cells while allowing normal cells to proliferate, for the manufacture of said pharmaceutical product for inhibiting the proliferation of normal proliferating human ovarian cancer cells, Use of placenta extract for the manufacture of a pharmaceutical for inhibiting normal cell proliferation and human ovarian cancer cell proliferation, characterized in that the placenta extract is an extract extracted from freeze-dried powder of horse placenta using dimethyl sulfoxide as an extraction solvent.

2. A method for producing a pharmaceutical product for inhibiting the proliferation of normal cells and human ovarian cancer cells, which contains a placenta extract as an active ingredient for inhibiting the proliferation of human ovarian cancer cells while proliferating normal cells, comprising: A method for producing a pharmaceutical for inhibiting normal cell proliferation and human ovarian cancer cell proliferation, characterized in that the placenta extract is an extract extracted from freeze-dried powder of horse placenta using dimethyl sulfoxide as an extraction solvent.

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