Pharmaceutical composition for inhibiting proliferation of hormone-dependent cells containing pomegranate seed extract, and method for producing pharmaceutical composition for inhibiting proliferation of hormone-dependent cells

Abstract

The purpose of the present invention is to provide a composition having an inhibitory action on the proliferation of hormone-dependent cells in order to make effective use of and add value to pomegranate seeds to be discarded.　A pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to the present invention is characterized by containing pomegranate seed extract. A preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to the present invention is characterized in that the hormone-dependent cells are prostate cells. A preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to the present invention is characterized in that the prostate cells are androgen-dependent or independent cells.

Description

Pharmaceutical composition for inhibiting the growth of hormone-dependent cells containing pomegranate seed extract, and method for producing the pharmaceutical composition for inhibiting the growth of hormone-dependent cells

[0001] The present invention relates to a pharmaceutical composition for inhibiting the growth of hormone-dependent cells and a method for producing the pharmaceutical composition for inhibiting the growth of hormone-dependent cells, and particularly relates to a pharmaceutical composition for inhibiting the growth of hormone-dependent cells containing pomegranate seed extract and a method for producing the pharmaceutical composition for inhibiting the growth of hormone-dependent cells.

[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 few examples of using pomegranate seeds, there is a supplement proposal system using pomegranate seed extract, in which the proposed supplement calculation means, when the desired item information is related to beauty, outputs 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 as a proposed supplement (Patent Document 1).

[0003] JP-A-2011-232989

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

[0005] 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 experience symptoms such as nocturia, urinary incontinence, and a feeling of incomplete bladder emptying. It significantly contributes to a decline in the quality of life (QOL) of 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, leading to symptoms such as a feeling of incomplete bladder emptying and frequent urination. Treatment options for these diseases include radiation therapy, surgery, and hormone therapy. Among these, hormone therapy is the first choice. This involves castration, which suppresses the production of male hormones and thus inhibits the proliferation of prostate cells.

[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 proliferation of hormone-dependent cells, 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 proliferation of hormone-dependent cells of the present invention is characterized by containing pomegranate seed extract.

[0010] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, the hormone-dependent cells are characterized by being prostate cells.

[0011] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, the prostate cells are characterized by being either male hormone-dependent or non-male hormone-dependent cells.

[0012] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, the male hormone-dependent cells are characterized by being an LNCaP (Human Prostate Carcinoma cell line) cell line.

[0013] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, the androgen-independent cells are characterized by being a PC-3 cell line or a DU145 cell line.

[0014] 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 by inhibiting the proliferation of hormone-dependent cells in the presence of testosterone and / or dihydrotestosterone.

[0015] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, the pomegranate seed extract is characterized by containing plant sterols.

[0016] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, the plant sterol is characterized by being sitosterol.

[0017] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells 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.

[0018] Furthermore, the quasi-drug of the present invention is characterized by containing the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention as an active ingredient.

[0019] Furthermore, the present invention provides a method for producing a pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells, comprising the steps of: crushing pomegranate seeds to obtain a pulverized product, immersing it 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.

[0020] Furthermore, in a preferred embodiment of the method for producing the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, the solvent is characterized in that it is at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane, and water.

[0021] The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention has the advantageous effect of making effective use of pomegranate seeds, which were conventionally treated as waste. Furthermore, the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention has the advantageous effect of having the effect of inhibiting the proliferation of hormone-dependent cells, such as prostate cells.

[0022] 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.

[0023] The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to 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.

[0024] Furthermore, the target cells are not particularly limited, but examples include hormone-dependent cells, such as prostate cells. In a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, the prostate cells are characterized by being either male hormone-dependent or non-male hormone-dependent cells.

[0025] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, the male hormone-dependent cells are characterized by being an LNCaP (Human Prostate Carcinoma cell line) cell line, from the viewpoint of mimicking the state of the prostate gland in vivo.

[0026] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention, 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, and were 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.

[0027] 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.

[0028] 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.

[0029] 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 by inhibiting the proliferation of hormone-dependent cells in the presence of testosterone and / or dihydrotestosterone.

[0030] The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells of the present invention has the effect of inhibiting the proliferation of hormone-dependent cells, particularly prostate cells. In the present invention, the effect of inhibiting the proliferation of hormone-dependent cells is also present in the presence of testosterone and / or dihydrotestosterone. Testosterone is a representative androgen secreted from the testes. In the prostate, 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), enters the nucleus, and 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] 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 androgen receptors (ARs) and enters the nucleus, exerting its effects 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-mediated cells, and consequently prostate cells, even in the presence of testosterone and / or dihydrotestosterone.

[0032] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-mediated cells of the present invention, the pomegranate seed extract is characterized by containing plant sterols, from the viewpoint of inhibiting the proliferation of hormone-mediated cells, such as prostate cells. Plant sterols, also known as phytosterols, are a general term for alcohols with a steroid skeleton found in higher plants. Sitosterol, stigmasterol, and spinasterol are typical plant sterols, which exist in plants either as free or as fatty acid esters.

[0033] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormone-mediated cells of the present invention, the plant sterol is characterized by being sitosterol, from the viewpoint that sitosterol in pomegranate seed extract can particularly inhibit the activity of 5α-reductase, which converts testosterone (TP) to dihydrotestosterone (DHT).

[0034] In this invention, it is not fully understood which of the components found in the pomegranate seed extract contributes to the suppression of hormone-mediated cells, such as prostate cancer cells. However, plant sterols (especially sitosterol) have the effect of inhibiting the activity of 5α-reductase, an enzyme that converts testosterone to dihydrotestosterone (DHT), and plant sterols also inhibit the synthesis of active DHT. Therefore, it is thought that they suppress the proliferation of hormone-mediated cells, and consequently, prostate cells. In the examples described later, the inhibitory effect on the proliferation of prostate cancer cells was evaluated using testosterone and DHT, and an inhibitory effect was confirmed in both cases. Therefore, in the evaluation of the effect of testosterone, it is possible that the effect of sitosterol was involved, suppressing the synthesis of DHT and thus inhibiting the proliferation of prostate cancer cells.

[0035] Therefore, in this invention, since pomegranate seed extract contains plant sterols such as sitosterol, it may be possible that these sterols are involved in suppressing the proliferation of prostate cancer cells by inhibiting their conversion to DHT.

[0036] Furthermore, in a preferred embodiment of the pharmaceutical composition for inhibiting the proliferation of hormonal cells of the present invention, the content of the pomegranate seed extract is characterized in that, from the viewpoint of the concentration range in which the effect is observed, it is 1 to 100 μg / mL, more preferably 12.5 to 100 μg / mL, relative to the total amount of the composition.

[0037] Furthermore, the present invention provides a method for producing a pharmaceutical composition for inhibiting the proliferation of hormone-mediated cells, comprising the steps of: immersing a pulverized material obtained by crushing pomegranate seeds 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.

[0038] Furthermore, in a preferred embodiment of the method for producing the pharmaceutical composition for inhibiting the proliferation of prostate cells 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. The earlier pomegranate seed extract can be obtained by shaking extraction. In shaking extraction, for example, the extraction can be performed by setting the mixture on a rotator and rotating it in a low-temperature room such as about 4°C. Furthermore, in a preferred embodiment of the pharmaceutical composition of the present invention, the solvent is characterized by being ethanol, from the viewpoint of considering its application to food products. The pomegranate seed ethanol extract extracted with ethanol is concentrated under reduced pressure to obtain pomegranate seed extract.

[0039] Here, we will first describe one example of a method for preparing pomegranate seed extract. First, prepare the pomegranate seeds. Wash and dry the pomegranate seeds as needed. It is preferable to dry them thoroughly, in order to ensure uniform grinding later.

[0040] Next, the pomegranate seeds are crushed. The crushing method is not particularly limited, and known crushing machines such as ball mills, hammer mills, roller mills, rod mills, sample mills, stamp mills, disintegrators, mortars, and blenders with cooling devices can be used. However, since the heat generated during crushing may cause decomposition of the pomegranate seed composition, the crushing time should be set to a few seconds and repeated more than ten times.

[0041] Next, after pulverizing the pomegranate seeds to obtain a pulverized product, the pulverized product is immersed in various solvents. The solvent in this case is not particularly limited, and the solvent can be appropriately set according to the desired effect. Further, in a preferred embodiment of the method for producing a pomegranate seed extract of the present invention, 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. As the solvent, polar and non - polar solvents such as ethyl acetate, ethyl acetate - hexane, ethyl acetate - methanol, ethanol, methanol, water, hexane, ethyl acetate, chloroform, and acetone can be mentioned. Preferably, ethyl acetate, ethyl acetate - hexane, ethyl acetate - methanol, methanol, ethanol, water, etc. can be mentioned.

[0042] The immersion can be carried out under gentle stirring. The pulverized product is immersed in various solvents to obtain various solutions. For the various solutions, stirring is carried out according to the state of the solution, and in some cases, the solution can be left as it is. When stirring, although not particularly limited, stirring can be continued for 10 hours to 48 hours, preferably for about 1 day (24 hours).

[0043] Thereafter, a pomegranate seed extract can be obtained by separating the supernatant. If necessary, the supernatant is evaporated to dryness. Evaporation to dryness can be carried out on a warm bath at 20°C to 60°C, preferably 37°C to 40°C, using an evaporator. By evaporating to dryness, the pomegranate seed extract can be stored for a long time.

[0044] Further, the quasi - drug of the present invention is characterized by containing the pharmaceutical composition for inhibiting the proliferation of prostate cells of the present invention 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 proliferation of prostate cells of the present invention as an active ingredient.

[0045] <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.

[0046] The dosage of pomegranate seed extract in the composition of the present invention can be changed according to the condition and severity of the patient to be administered, dosage form, selected administration route, number of administrations per day, etc.

[0047] When implementing in mice, the dosage of pomegranate seed extract in the composition of the present invention can be set at a dosage of 1000 mg / kg / day, and in humans, it is preferably a lower amount due to differences in sensitivity, etc.

[0048] In addition, dosage forms include oral preparations (tablets, capsules, coated tablets, granules, solutions, syrups), ointments and creams for application, etc. The patients to be administered can be targeted regardless of gender, male or female, adult or child, from the perspective of skin moisturization and barrier function.

[0049] The dosage form can include other conventional components, for example, stabilizers, sweeteners, colorants, flavorings, etc.

[0050] <Acute toxicity test> Regarding the components contained in pomegranate seed extract, since it also contains linolenic acid, it is considered that no toxicity is observed.

[0051] Hereinafter, an example of the present invention will be described, but the present invention is not construed as being limited to the following examples.

[0052] Example 1 First, the inhibitory effect of pomegranate seed extract on benign prostatic hyperplasia was investigated. The sample used was pomegranate (scientific name: Punica granatum), and the cells and culture information used are as follows.

[0053] <Cells used> LNCaP cells: Androgen-sensitive human prostate cell line

[0054] <Culture information> ・Charcoal dextran FBS (CDFBS): Steroid hormone-removed FBS This is for culturing in an environment where the influence of endogenous hormones is removed. FBS treated with activated carbon and dextran to remove the steroid hormones present in FBS was used.

[0055] • 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061]

[0062] 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.

[0063] 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 containing pomegranate seed extract for inhibiting the proliferation of hormone-dependent cells.

2. The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to claim 1, characterized in that the hormone-dependent cells are prostate cells.

3. The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to claim 2, characterized in that the prostate cells are either male hormone-dependent or non-male hormone-dependent cells.

4. The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to claim 3, characterized in that the androgen-dependent cells are LNCaP (Human Prostate Carcinoma cell line) cell lines.

5. The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to claim 3, characterized in that the androgen-independent cells are the PC-3 cell line or the DU145 cell line.

6. The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to claim 1 or 2, characterized in that the pharmaceutical composition inhibits the proliferation of hormone-dependent cells in the presence of testosterone and / or dihydrotestosterone.

7. The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to claim 1, characterized in that the pomegranate seed extract contains plant sterols.

8. The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to claim 7, wherein the plant sterol is sitosterol.

9. The pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells according to claim 1, characterized in that the pomegranate seed extract content is 1 to 100 μg / mL relative to the total amount of the composition.

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

11. A method for producing a pharmaceutical composition for inhibiting the proliferation of hormone-dependent cells containing pomegranate seed extract, characterized by comprising the steps of: 1) 12. The method according to claim 11, 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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