Pharmaceutical composition for inhibiting androgen receptor (AR) pathway activity and containing pomegranate seed extract, and method for producing pharmaceutical composition for inhibiting androgen receptor (AR) pathway activity

Abstract

The purpose of the present invention is to provide a composition having an action of inhibiting the androgen receptor (AR) pathway activity, and the present invention aims to effectively utilize discarded pomegranate seeds and add value thereto.　A pharmaceutical composition for inhibiting the androgen receptor (AR) pathway activity according to the present invention is characterized by containing a pomegranate seed extract. Furthermore, a preferred embodiment of the pharmaceutical composition for inhibiting the androgen receptor (AR) pathway activity according to the present invention is characterized in that the inhibition of the androgen receptor (AR) pathway activity is due to the inhibition of the transcription activity of the androgen receptor (AR). Furthermore, a preferred embodiment of the pharmaceutical composition for inhibiting the androgen receptor (AR) pathway activity according to the present invention is characterized in that the inhibition of the androgen receptor (AR) pathway activity is due to the inhibition of the androgen receptor (AR) protein expression.

Description

Pharmaceutical composition for suppressing the activity of the androgen receptor (AR) pathway containing pomegranate seed extract, and method for producing a pharmaceutical composition for suppressing the activity of the androgen receptor (AR) pathway

[0001] The present invention relates to a pharmaceutical composition for suppressing the activity of the androgen receptor (AR) pathway and a method for producing a pharmaceutical composition for suppressing the activity of the androgen receptor (AR) pathway, and particularly relates to a pharmaceutical composition for suppressing the activity of the androgen receptor (AR) pathway containing pomegranate seed extract and a method for producing a pharmaceutical composition for suppressing the activity of the androgen receptor (AR) pathway.

[0002] Pomegranate is a fruit in which the seeds account for a fairly large proportion of the whole fruit. In principle, however, the seeds remaining after juicing are discarded. As a rare example of using pomegranate seeds, there is a supplement proposal system using pomegranate seed extract, in which the proposed supplement calculation means outputs, as a proposed supplement, at least one of the group consisting of multivitamins, multiminerals, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin E, vitamin P, niacin, biotin, collagen, elastin, polyphenol, pantothenic acid, hyaluronic acid, acetylglucosamine, inositol, coenzyme Q10, α-carotene, β-carotene, soybean saponin, royal jelly, trehalose, folic acid, rose petal extract, pomegranate seed extract, black cohosh extract, soybean isoflavone, kudzu isoflavone squalene, brewer's yeast, silkworm extract, calcium, magnesium, copper, iron, zinc, manganese when the desired item information is about beauty. A supplement proposal system is known (Patent Document 1).

[0003] JP 2011-232989

[0004] However, at present, in many cases, pomegranate seeds are generally treated as waste, and other than the above patent document, they are not effectively utilized at present, and the effective utilization of waste is being reexamined.

[0005] Incidentally, androgens (also called male hormones) bind to androgen receptors, form dimers, enter the nucleus, and exert their function by binding to target genes and regulating their expression. Therefore, if the activity of the androgen receptor (AR) pathway can be regulated, it is thought that the proliferation of malignant cells can also be controlled by the activity of this pathway. On the other hand, benign prostatic hyperplasia (BPH), a disease affecting hormone-dependent cells, particularly prostate cells, is known to develop with age in men. It is estimated that 80% of elderly men develop it, and more than half of them complain of symptoms such as nocturia, urinary incontinence, and a feeling of incomplete bladder emptying. It is significantly related to the decline in quality of life for elderly men. BPH and prostate cancer are caused by an excessive increase in prostate cells, which proliferate in a male hormone-dependent manner. The enlarged prostate tissue compresses the urethra, causing symptoms such as a feeling of incomplete bladder emptying and frequent urination. Treatment for these diseases includes radiation therapy, surgery, and hormone therapy. Among these, hormone therapy is used as the first choice. This works by suppressing the proliferation of prostate cells through castration, which reduces the production of male hormones.

[0006] However, it is known that hormone-dependent cancer cells gradually transform into hormone-independent cancer cells. In this context, there is a need for the development of foods and pharmaceuticals that can prevent the onset of benign prostatic hyperplasia and prostate cancer, or slow their progression after onset.

[0007] Therefore, the object of the present invention is to provide a composition that has the effect of suppressing the activity of the androgen receptor (AR) pathway, with the aim of effectively utilizing and adding value to pomegranate seeds that would otherwise be discarded.

[0008] In order to achieve the above objective, the inventors diligently studied effective ways to utilize pomegranate seeds, and as a result, arrived at the present invention.

[0009] In other words, the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention is characterized by containing pomegranate seed extract.

[0010] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the inhibition of the activity of the androgen receptor (AR) pathway is characterized by the inhibition of the transcriptional activity of the androgen receptor (AR).

[0011] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the inhibition of the activity of the androgen receptor (AR) pathway is characterized by suppression of the expression level of the androgen receptor (AR) protein.

[0012] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the pharmaceutical composition is characterized by inhibiting the activity of the androgen receptor (AR) pathway in the presence of testosterone and / or dihydrotestosterone.

[0013] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the proliferation of hormone-dependent cells is suppressed by inhibiting the activity of the androgen receptor (AR) pathway.

[0014] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the hormone-dependent cells are characterized by being prostate cells.

[0015] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the prostate cells are characterized by being either male hormone-dependent or male hormone-independent cells.

[0016] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the male hormone-dependent cells are characterized by being LNCaP cells (Human Prostate Carcinoma cell line).

[0017] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the pomegranate seed extract is characterized by containing plant sterols.

[0018] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the plant sterol is characterized by being sitosterol.

[0019] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the content of the pomegranate seed extract is characterized by being 1 to 100 μg / mL relative to the total amount of the composition.

[0020] Furthermore, the quasi-drug of the present invention is characterized by containing the pharmaceutical composition of the present invention as an active ingredient.

[0021] The present invention provides a method for producing a pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway containing pomegranate seed extract, comprising the steps of: crushing pomegranate seeds to obtain a pulverized product, immersing the pulverized product in a solvent, and then separating the supernatant; obtaining pomegranate seed extract by separating the supernatant; and adjusting the obtained pomegranate seed extract to an effective amount.

[0022] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the solvent is characterized by being at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane, and water.

[0023] The pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention has the advantageous effect of enabling the effective utilization of pomegranate seeds, which were conventionally treated as waste. Furthermore, the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention has the advantageous effect of having the action of inhibiting the activity of the androgen receptor (AR) pathway.

[0024] Figure 1 shows the effect of pomegranate seed ethanol extract (PSE: Punica granatum Seed Etanol extract) on the viability of LNCaP cells. Figure 2 shows the digested results of the effect on cell viability when cell proliferation is induced in the presence of male hormones. Figure 3 shows the results of a comparative evaluation of hormone-dependent cell proliferation using the fluorescence intensity of firefly luciferase, with cultures treated with DHT alone and those treated with DHT and PSE as controls. Figure 4 shows the band results of AR and α-tubulin protein expression levels. Figure 5 shows the results of a comparative evaluation of AR protein expression levels, corrected for α-tubulin expression levels, with the treatment of DHT alone as a control.

[0025] The pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention is characterized by containing pomegranate seed extract. Pomegranate seed extract is an extract derived from pomegranate seeds. The pomegranate seed extract applicable to the present invention includes all pomegranate seed extracts as long as they are derived from pomegranate seeds.

[0026] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the inhibition of the activity of the androgen receptor (AR) pathway is characterized by the inhibition of the transcriptional activity of the androgen receptor (AR). The AR (androgen receptor) is present in cells, and when male hormones are secreted, the male hormones and ARs bind. For example, male hormones are converted to dihydrotestosterone (DHT) by the action of a reductase (5α-reductase) present in prostate cells, and this DHT binds to AR to form a DHT / AR complex. The DHT / AR complex translocates to the cell nucleus and binds to a specific DNA sequence (in this case, an androgen-responsive element called ARE), promoting the transcription of target genes (such as PSA and genes involved in cell proliferation). Because the reaction in this pathway causes prostate cells to proliferate, it is also called male hormone-dependent proliferation. In this invention, surprisingly, it was found that a pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway containing pomegranate seed extract suppresses the transcriptional activity of the androgen receptor (AR), and consequently suppresses the activity of the androgen receptor (AR) pathway, as shown in the examples described later.

[0027] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the inhibition of the activity of the androgen receptor (AR) pathway is characterized by the suppression of the expression level of the androgen receptor (AR) protein. As shown in the examples described later, regarding the effect of PSE on the transcriptional activity of AR, since an androgen response element (ARE) is incorporated, if a DHT / AR complex is formed, it binds to the ARE, and consequently, prostate cells proliferate. This is because, if only DHT is present, DHT / AR complexes are formed one after another, and proliferation does not stop, but when PSE is added, the DHT / AR complex is not formed, and proliferation is suppressed. Two reasons can be considered for this. The first is that the action of PSE may reduce the mRNA expression of AR, and the amount of subsequent protein synthesis may decrease (according to the central dogma, proteins are produced by translation from mRNA, which is genetic information). This is a consideration in the case where PSE reduces the amount of AR protein produced.

[0028] The second possibility is that while PSE does not affect the amount of AR protein produced, it may suppress the binding of DHT to the AR protein by promoting AR degradation. To elaborate on this AR degradation, the AR protein is unstable on its own and is stabilized by binding to DHT. However, if components of PSE bind to the DHT-binding pocket (binding site) of AR, structural stability is not guaranteed, and AR degradation is promoted. As shown in the examples described later, the results of the evaluation of AR protein expression levels show a decrease due to the action of one of these two hypotheses, but the second hypothesis is considered more likely. This is because the results showed that PSE did not affect the expression level of AR mRNA, meaning that it did not affect the amount of AR protein produced, and it is thought that AR was reduced by the action of a component that exhibits competitive inhibitory activity, as suggested by the second hypothesis.

[0029] Therefore, it is thought that PSE suppresses cancer cell proliferation by reducing the amount of AR protein, thereby reducing the amount of DHT / AR complex formed.

[0030] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the pharmaceutical composition is characterized by inhibiting the activity of the androgen receptor (AR) pathway in the presence of testosterone and / or dihydrotestosterone. Testosterone is a representative androgen secreted from the testes. In the prostate gland, one of the target organs, it undergoes reduction at the 5α position within the cell and, as 5α-dihydrotestosterone (DHT), binds to the androgen receptor (AR) and enters the nucleus, where it exerts its effects through gene activation. Androgens are male hormones, also known as male hormones. Dihydrotestosterone also binds to the androgen receptor and exerts androgenic effects. That is, activation of androgen signaling occurs, leading to the proliferation of prostate cells.

[0031] Therefore, the pharmaceutical composition of the present invention also has the effect of suppressing the proliferation of hormone-dependent cells, particularly prostate cells. Conventionally, in the presence of testosterone, it is converted to active testosterone by the action of reductase, and as 5α-dihydrotestosterone (DHT), it binds to the androgen receptor (AR) and enters the nucleus, exerting its effect through gene activation, and ultimately causing the proliferation of prostate cells. Surprisingly, however, in the present invention, it has been found that the presence of pomegranate seed extract can suppress the proliferation of hormone-dependent cells, and thus prostate cells, even in the presence of testosterone and / or dihydrotestosterone.

[0032] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway, and by extension, the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells, the proliferation of hormone-dependent cells is suppressed by inhibiting the activity of the androgen receptor (AR) pathway.

[0033] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway, and consequently, the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells (hereinafter also referred to as the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway, etc.), the hormone-dependent cells are characterized by being prostate cells. That is, in the present invention, the target cells are not particularly limited, but for example, hormone-dependent cells, such as prostate cells, can be mentioned.

[0034] Furthermore, in preferred embodiments of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway of the present invention, the prostate cells are characterized by being either male hormone-dependent or male hormone-independent cells.

[0035] Furthermore, in preferred embodiments of the present invention, such as the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway, the male hormone-dependent cells are characterized by being LNCaP cell lines (Human Prostate Carcinoma cell lines), from the viewpoint of mimicking the state of the prostate gland in vivo.

[0036] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway, and consequently, the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells, the androgen-independent cells are characterized by being the PC-3 cell line or the DU145 cell line. The PC-3 cell line and the DU145 cell line are cell lines derived from human prostate cancer, established from bone metastases and brain metastases, respectively. Since these are androgen-independent prostate cancer cells, they are established from cells that have acquired hormone resistance and become hormone-independent.

[0037] Generally, LNCaP cells are used in research as an androgen-dependent prostate cell line, and PC-3 and DU145 cells are used as androgen-independent cell lines. As examples of target cells, LNCaP cells can be used as a hormone-dependent cell line for prostate cancer cells, and PC-3 cells and DU145 cells can be used as hormone-independent cells.

[0038] Furthermore, in the present invention, there are several other established cell lines derived from prostate cancer cells, and for example, the VCaP cell line, the 22Rv1 cell line, the MDA-PCa 2a cell line, etc. may be used. The VCaP cell line is a cell line that exhibits androgen dependence similar to LNCaP cells, but has high AR expression levels and also has splicing variants with mutated AR. The 22Rv1 cell line is a cell line derived from LNCaP, which exhibits androgen dependence, but some clones also exhibit androgen independence. The MDA-PCa 2a cell line is a cell line that exhibits androgen dependence and proliferates at low concentrations of androgens, and is a cell line established from bone metastases. In this specification, in order to evaluate the target cells in the prostate, the LNCaP (Human Prostate Carcinoma cell line) cell line is mainly used as the reference, reflecting early hormone-dependent prostate cancer and from the viewpoint that AR is functional, but it is not intended to be limited to this.

[0039] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, the pharmaceutical composition is characterized in that it also inhibits the proliferation of hormone-dependent cells in the presence of testosterone and / or dihydrotestosterone.

[0040] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway, and consequently, the pharmaceutical composition for inhibiting the proliferation of hormone-mediated cells, the pomegranate seed extract is characterized by containing plant sterols that regulate androgen receptor (AR) signaling and suppress the abnormal proliferation of prostate cells, from the viewpoint of inhibiting the proliferation of prostate cells. Plant sterols, also called phytosterols, are a general term for alcohols with a steroid skeleton found in higher plants. Sitosterol, stigmasterol, and spinasterol are typical plant sterols, and they exist in plants either free or as fatty acid esters.

[0041] Further, in a preferred embodiment of the pharmaceutical composition for suppressing the activity of the androgen receptor (AR) pathway of the present invention, and thus the pharmaceutical composition for suppressing the growth of hormonal cells, sitosterol in the pomegranate seed extract can particularly suppress the action of 5α-reductase that converts testosterone (TP) into dihydrotestosterone (DHT). From this perspective, the plant sterol is characterized by being sitosterol.

[0042] In the present invention, among the components found in the pomegranate seed extract, plant sterols (especially sitosterol) have the effect of suppressing the action of 5α-reductase, which is an enzyme for converting testosterone → dihydrotestosterone (DHT). Since plant sterols suppress the synthesis of active DHT, it is considered that they can suppress the growth of hormonal cells, and thus prostate cells. From the examples described later, the growth inhibitory effect on prostate cancer cells was evaluated using testosterone and DHT, and the inhibitory effect was confirmed in both cases. Therefore, in the action evaluation using testosterone, the action of sitosterol may be involved, and the synthesis of DHT may be suppressed, resulting in the suppression of the growth of prostate cancer cells.

[0043] Therefore, in the present invention, since plant sterols such as sitosterol are contained in the pomegranate seed extract, it may be involved in suppressing the growth of prostate cancer cells by suppressing the conversion to DHT.

[0044] Further, in a preferred embodiment of the pharmaceutical composition for suppressing the activity of the androgen receptor (AR) pathway of the present invention, and thus the pharmaceutical composition for suppressing the growth of hormonal cells, from the perspective of the concentration range showing an effect, the content of the pomegranate seed extract is 1 to 100 μg / mL, more preferably 12.5 to 100 μg / mL, based on the total amount of the composition.

[0045] Further, the method for producing a pharmaceutical composition for suppressing the activity of the androgen receptor (AR) pathway of the present invention, and thus a pharmaceutical composition for suppressing the growth of hormonal cells, comprises a step of immersing a pulverized product obtained by pulverizing pomegranate seeds in a solvent and then separating the supernatant, a step of obtaining a pomegranate seed extract by separating the supernatant, and a step of adjusting the obtained pomegranate seed extract to an effective amount.

[0046] Further, in a preferred embodiment of the method for producing a pharmaceutical composition for suppressing the activity of the androgen receptor (AR) pathway of the present invention, and thus a pharmaceutical composition for suppressing the growth of prostate cells, the solvent is at least one selected from the group consisting of ethyl acetate, ethyl acetate - hexane, ethyl acetate - methanol, ethanol, methanol, hexane, and water. For example, shaking extraction can also be performed. In shaking extraction, for example, it can be extracted while being set on a rotator in a low-temperature chamber such as about 4°C and rotated. Further, in a preferred embodiment of the pharmaceutical composition of the present invention, from the perspective of assuming application to food, the solvent is ethanol, and it is characterized by obtaining a pomegranate seed ethanol extract extracted with ethanol.

[0047] Here, first, an example of a method for preparing a pomegranate seed extract will be described. First, pomegranate seeds are prepared. The pomegranate seeds are washed and dried as necessary. Drying is preferably performed sufficiently. This is for uniformly performing subsequent pulverization.

[0048] Next, the pomegranate seeds are pulverized. The method of pulverization is not particularly limited, and known pulverizers such as a ball mill, hammer mill, roller mill, rod mill, sample mill, stamp mill, disintegrator, mortar, and blender with a cooling device can be used. Since it is also conceivable that decomposition of the pomegranate seed composition occurs due to heat generation during pulverization, the pulverization time can be set to several seconds and repeated a dozen or so times.

[0049] Next, the pomegranate seeds are crushed to obtain a pulverized product, and then the pulverized product is immersed in various solvents. The solvent in this case is not particularly limited, and a solvent can be appropriately selected according to the desired effect. Furthermore, in a preferred embodiment of the method for producing pomegranate seed extract of the present invention, the solvent is characterized by being at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane, and water. Examples of solvents include both polar and nonpolar solvents such as ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water, hexane, ethyl acetate, chloroform, and acetone. Preferably, examples include ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, methanol, ethanol, and water.

[0050] Immersion can be carried out under gentle stirring. Various solutions are obtained by immersing the pulverized material in various solvents. Stirring is performed on each solution according to its state, and in some cases the solution may be left to stand. When stirring is performed, there are no particular limitations, but stirring can be continued for 10 to 48 hours, preferably about 1 day (24 hours).

[0051] Subsequently, pomegranate seed extract can be obtained by separating the supernatant. If necessary, the supernatant can be evaporated to dryness. Evaporation to dryness can be carried out using an evaporator on a warm bath at 20°C to 60°C, preferably 37°C to 40°C. By evaporating to dryness, the pomegranate seed extract can be stored for a long period of time.

[0052] The components contained in pomegranate seeds are separated according to their physical properties by extracting them using solvents with different polarities. Therefore, the types and amounts of components in pomegranate seed extract will differ depending on the solvent used.

[0053] Furthermore, the quasi-drug of the present invention is characterized by containing the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway, and consequently, the pharmaceutical composition for inhibiting the proliferation of prostate cells, as an active ingredient. The quasi-drug applicable to the present invention is not particularly limited as long as it contains the pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway, and consequently, the pharmaceutical composition for inhibiting the proliferation of prostate cells, as an active ingredient.

[0054] <Effective Amount> The composition according to the present invention is prepared in the form of an effective amount of pomegranate seed extract and an appropriate dosage form.

[0055] The dosage of pomegranate seed extract in the composition of the present invention can be changed depending on the patient's condition and its severity, the form of administration, the selected route of administration, and the number of doses per day.

[0056] The dosage of pomegranate seed extract in the composition of the present invention can be set to 1000 mg / kg / day when administered to mice, and in humans, a lower amount is preferable due to differences in sensitivity, etc.

[0057] Furthermore, the forms of administration can include oral medications (tablets, capsules, coated tablets, granules, solutions, syrups), and topical ointments and creams. From the perspective of skin hydration and barrier function, the target patients can be women, men, adults, and children alike.

[0058] The dosage form may include other conventional ingredients, such as stabilizers, sweeteners, colorants, and flavorings.

[0059] <Acute Toxicity Test> Regarding the components of pomegranate seed extract, since it contains linolenic acid, it is believed that no toxicity has been observed.

[0060] An embodiment of the present invention will be described below, but the present invention is not limited to the embodiment described below.

[0061] Example 1 First, we investigated the inhibitory effect of pomegranate seed extract on benign prostatic hyperplasia. The sample used was pomegranate (scientific name: Punica granatum), and the cells and culture information used are as follows.

[0062] <Cells used> LNCaP cells: androgen-sensitive human prostate cell line

[0063] <Culture Information> ・Charcoal dextran FBS (CDFBS): Steroid hormone-removed FBS This is to culture in an environment that eliminates the influence of endogenous hormones. FBS was treated with activated charcoal and dextran to remove the steroid hormones present in the FBS, and FBS was used.

[0064] • Phenol Red-Free RPMI1640 Medium: This is a culture medium used for culturing cells, and normally a red medium containing phenol red is used. However, because phenol red exhibits hormone-like effects, in experiments using hormones, as in this example, cells are cultured in a clear, phenol red-free medium.

[0065] As hormone-dependent cells, prostate cancer cells were used in the experiment. Specifically, the following experiment was conducted to investigate the inhibitory effect of pomegranate seed ethanol extract on the proliferation of prostate cancer cells. Pomegranate seeds were powdered using a mill, extracted by permeation stirring with 99.5% ethanol for one day, and the supernatant obtained by centrifugation was dried under reduced pressure to obtain pomegranate seed ethanol extract (PSE: Punica granatum Seed Etanol extract). LNCaP cells were placed in a 96-well plate at a rate of 1.0 × 10⁶ 5 Cells were seeded at a concentration of cells / mL and pre-cultured in phenol red-free RPMI1640 medium containing 5% CDFBS at 37°C under 5% CO2. After 24 hours of incubation, the cells were cultured for 72 hours in phenol red-free RPMI medium containing 5% CDFBS, which also contained pomegranate seed ethanol extract prepared at various concentrations and either 100 nM testosterone propionate (TP) or 10 nM dihydrotestosterone (DHT) as male hormones.

[0066] The pomegranate seed extract content is as follows: 50 g of pomegranate seeds were extracted from 500 mL of ethanol to obtain 3.2 g of pomegranate seed ethanol extract. The yield was 3.2 g, and the yield was 6.4%. Therefore, less than 10% of the pomegranate seed extract was obtained from 50 g of pomegranate seeds. This extract was redissolved and added to cells at concentrations ranging from 12.5 μg / mL to 50 or 100 μg / mL.

[0067] After culturing, the effect on cell viability was evaluated by adding WST-8 reagent and measuring the absorbance at 450 nm. Figure 1 shows the evaluation results of the effect of PSE on LNCaP cells without the addition of male hormones, and Figure 2 shows the evaluation results of the effect on cell viability when cell proliferation was induced by the addition of male hormones. Each value is shown as mean ± standard error. Significance testing was performed using Dunnett's test. The significance level is expressed as **p < 0.01 and ***p < 0.001.

[0068] As shown in Figure 1, PSE did not have any effect on LNCaP cell viability compared to the control group. As shown in Figure 2, PSE was shown to suppress the proliferation of androgen-dependent LNCaP cells.

[0069] Example 2 Next, a component analysis of PSE was performed by metabolome analysis. Metabolome analysis was performed to investigate the components contained in PSE. PSE was prepared in the same manner as in Example 1. This dried material was dissolved again in ethanol and used as a sample for analysis of metabolites contained in PSE by LC-MS and CE-MS at Human Metabolome Technologies, Inc. Table 1 shows the plant sterols that were identified as components.

[0070]

[0071] As shown in Table 1, PSE contains various plant sterols. Specifically, the various plant sterols in PSE were cycloartenol, lanosterol, desmosterol, sitosterol, stigmasterol, and β-sitosterol ferulate. Among the plant sterols, sitosterol is known to inhibit the activity of 5α-reductase, which converts testosterone to dihydrotestosterone (DHT). Therefore, it was found that the plant sterols contained in PSE may contribute to the improvement of benign prostatic hyperplasia by inhibiting 5α-reductase production and suppressing AR expression.

[0072] Example 3 Next, we investigated the effect of PSE on the transcriptional activity of AR. First, we created the luciferase reporter plasmid PGL4.14-TATA-ARE by incorporating the TATA sequence and androgen response element (ARE) into the PGL4.14 plasmid. 2.0 × 10⁶ LNCaP cells were placed in a 24-well plate. 5Cells were seeded at a concentration of cells / mL and pre-cultured in phenol red-free RPMI1640 medium containing 5% CDFBS at 37°C under 5% CO2. After 24 hours of culture, cells were transfected with PGL4.14-TATA-ARE using the Lipofectamin method and cultured for another 24 hours. After 24 hours of culture, cells were cultured for another 24 hours in phenol red-free RPMI medium containing PSE at various concentrations and 10 nM dihydrotestosterone (DHT). After culture, proteins were extracted from the cells and evaluated by correcting the fluorescence intensity of firefly luciferase to that of sea urchin using a Dual Luciferase Reporter assay. Cells cultured with only DHT added were used as a control for comparative evaluation. Each value is shown as mean ± standard error. Significance testing was performed using Dunnett's test. The significance level is expressed as ***p < 0.001.

[0073] The results in Figure 3 reveal that, compared to the control, PSE inhibits the transcriptional activity of AR, thereby suppressing the androgen-dependent proliferation of LNCaP cells.

[0074] In this example, firefly luciferase is used as the luminescent protein. This experiment can also be described as an experiment conducted in Example 1 to clarify whether PSE works by suppressing the AR-mediated reaction that inhibits cancer cell proliferation. Generally, luciferase assays are used to visualize firefly luminescence and evaluate gene expression and specific intracellular activities. In this case, since the firefly luminescence gene is produced simultaneously with the activation of the AR pathway, the activity of the target AR pathway can be numerically evaluated by assessing the intensity of the luminescence. Therefore, the luminescence intensity was stronger in the group with only DHT added compared to the group without DHT, and the luminescence intensity was suppressed when PSE was added, indicating that the activity of the AR pathway was inhibited. Here, if the luminescence intensity did not change even after adding PSE, it would suggest that it acts on a pathway other than the AR pathway to suppress cancer cell proliferation, so this is done to clearly clarify its involvement in the AR pathway.

[0075] Example 4 Next, the effect of PSE on the expression level of AR protein was investigated. LNCaP cells were placed in a 6 cm dish at a rate of 1.0 × 10⁶ 5 Cells were seeded at a concentration of cells / mL and pre-cultured in phenol red-free RPMI1640 medium containing 5% CDFBS at 37°C under 5% CO2. After 24 hours of culture, cells were cultured for 72 hours in phenol red-free RPMI medium containing 5% CDFBS and 10 nM dihydrotestosterone (DHT) at various concentrations. After culture, proteins were extracted from the cells, and the expression level of AR protein was evaluated by Western blotting. Flutamide, which exhibits competitive inhibitory activity with AR, was used as a positive control. Figure 4 shows the band results for AR and α-tubulin protein expression levels. Figure 5 shows the results of comparing AR protein expression levels, corrected for α-tubulin expression, with a control group treated with DHT only. Each value is shown as mean ± standard error. Significance testing was performed using Dunnett's test. The significance level is expressed as *p < 0.05.

[0076] The results shown in Figures 4 and 5 reveal that PSE suppresses the activation of the AR pathway by inhibiting the expression level of AR proteins.

[0077] Since proteins, in principle, follow a gene-mediated synthesis pathway known as the central dogma, suppressing AR expression itself could involve suppressing mRNA gene expression. Furthermore, proteins also have mechanisms for degradation after they are synthesized. AR, in particular, is unstable and binds to DHT for stabilization; however, the binding of PSE components to DHT could potentially contribute to the degradation of the AR protein.

[0078] Furthermore, it has been found that the expression level of AR increases when DHT is added compared to when no DHT is added. The exact mechanism for why the AR band is darker when only DHT is added compared to when nothing is added is not yet clear, but the following is considered possible: In this experiment, the amount of AR protein is measured, so an antibody that specifically reacts with AR is used to detect the protein band. In this case, both individual AR and AR bound to DHT are detected. Therefore, it is thought that the addition of DHT creates a DHT / AR complex, suppressing the degradation of AR, and thus the expression level appears darker. Supporting this, when the effect on gene expression levels was evaluated using another plant extract, the result was that there was no effect on gene expression levels regardless of whether DHT was added or not. Therefore, it is expected that the reason why the band is darker when DHT is added is because protein degradation is suppressed.

[0079] Furthermore, the mechanism of action of Flutamide is that it binds to the AR protein to which DHT binds, preventing DHT from binding. This is called competitive inhibition. The AR protein is an unstable protein on its own and can only be stabilized by forming a complex with DHT. Therefore, it is thought that the binding of Flutamide to AR compromises its structural stability, leading to a decrease in the amount of AR protein, as shown in the experimental results.

[0080] According to this invention, in addition to contributing to the medical industry, it has a high degree of social contribution, such as the effective utilization of waste, and has high utility in a wide range of applications.

Claims

1. A pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway, characterized by containing pomegranate seed extract.

2. The pharmaceutical composition for inhibiting activity according to claim 1, characterized in that the inhibition of the activity of the androgen receptor (AR) pathway is achieved by inhibiting the transcriptional activity of the androgen receptor (AR).

3. The pharmaceutical composition for inhibiting activity according to claim 1, characterized in that the inhibition of the activity of the androgen receptor (AR) pathway is achieved by suppressing the expression level of the androgen receptor (AR) protein.

4. The pharmaceutical composition for inhibiting activity according to claim 1, characterized in that it inhibits the activity of the androgen receptor (AR) pathway in the presence of testosterone and / or dihydrotestosterone.

5. The pharmaceutical composition for inhibiting activity according to claim 1 or 2, characterized in that the proliferation of hormone-dependent cells is suppressed by inhibiting the activity of the androgen receptor (AR) pathway.

6. The activity-inhibiting pharmaceutical composition according to claim 3, characterized in that the proliferation of hormone-dependent cells is suppressed by inhibiting the activity of the androgen receptor (AR) pathway.

7. The pharmaceutical composition for inhibiting activity according to claim 5, characterized in that the hormone-dependent cells are prostate cells.

8. The pharmaceutical composition for inhibiting activity according to claim 7, characterized in that the prostate cells are male hormone-dependent or non-dependent cells.

9. The pharmaceutical composition for inhibiting activity according to claim 8, characterized in that the androgen-dependent cells are LNCaP cells (Human Prostate Carcinoma cell line).

10. The pomegranate seed extract contains plant sterols, characterized in that it is the pharmaceutical composition for inhibiting activity according to claim 1.

11. The pharmaceutical composition for inhibiting activity according to claim 10, wherein the plant sterol is sitosterol.

12. The pharmaceutical composition for inhibiting activity according to claim 1, characterized in that the content of the pomegranate seed extract is 1 to 100 μg / mL relative to the total amount of the composition.

13. A quasi-drug characterized by containing the pharmaceutical composition described in claim 1 or 2 as an active ingredient.

14. A method for producing a pharmaceutical composition for inhibiting the activity of the androgen receptor (AR) pathway containing pomegranate seed extract, characterized by comprising the steps of: 1) immersing a pulverized material obtained by crushing pomegranate seeds in a solvent, and then separating the supernatant; 2) obtaining pomegranate seed extract by separating the supernatant; and 3) adjusting the obtained pomegranate seed extract to an effective amount.

15. The method according to claim 14, characterized in that the solvent is at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane, and water.

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