WO2023090542A1 - Pharmaceutical composition containing hot water extract of fruit of vaccinium bracteatum thunb. as active ingredient for preventing or treating benign prostatic hyperplasia - Google Patents

Abstract

The present invention relates to a composition for preventing or treating benign prostatic hyperplasia, the composition containing, as an active ingredient, an extract of the fruit of Vaccinium bracteatum thunb. The composition inhibits the in vitro generation of 5 alpha reductase, dihydrotestosterone, and androgen receptor (AR). In a benign prostatic hyperplasia rat model induced by testosterone, the composition inhibits prostate tissue enlargement, inhibits 5-alpha reductase generation in prostate hypertrophy tissue, relieves enlarged epithelial cells, and inhibits polyps, thus exhibiting an excellent benign prostatic hyperplasia prevention or inhibition effect in animal models.

Description

A pharmaceutical composition for preventing or treating prostatic hyperplasia comprising a hot water extract of the fruit of mosae tree (VACCINIUM BRACTEATUM THUNB.) as an active ingredient

The present invention relates to a composition for preventing and treating benign prostatic hyperplasia containing an extract of the fruit of moss japonica as an active ingredient. More specifically, it relates to a pharmaceutical composition for preventing or treating benign prostatic hyperplasia that can be safely used without toxicity and side effects by using the fruit of Vaccinium bracteatum Thunb., which is a natural raw material.

Benign prostatic hyperplasia (BPH) is a disease in which the volume of the prostate around the urethra increases for various reasons due to various causes, compresses the urethra, and as a result, symptoms such as decreased urine output occur.

Recently, in men, urinary frequency (more than 8 times/day), nocturia, urgency (strong and sudden need to urinate), urgency (difficulty urinating), retardation (delayed urine excretion), and intermittent urination (urine flow is interrupted). It is defined as a complaint of lower urinary tract symptoms, which collectively refers to symptoms indicating bladder emptying disorders, such as urinary incontinence and the need to exert force during urination. It is known that the cause of prostate enlargement is related to the male hormones testosterone and dihydrotestosterone (DHT).

Drug treatment for BPH is largely classified into alpha blockers and androgen inhibitors (5 alpha-reductase inhibitors). Alpha-blockers are drugs that lower pressure and tension in the prostate and urethra, and include terazosin, doxazosin, tamsulosin, and alfuzosin. Side effects such as disability have been reported. Androgen inhibitors are drugs that prevent enlargement of the prostate by inhibiting 5-alpha-reductase, and include finasteride and dutasteride. Side effects related to sexual function have been reported.

On the other hand, Vaccinium bracteatum Thunb. is a dicotyledonous angiosperm plant of the order Rhododendron, which grows in coastal mountains. The height is 1-3m, and the twigs are grayish brown to gray and almost hairless. The leaves are alternate, thick, elliptical or oblong, with a thick leathery texture. There are fine sawtooth on the edge, and there is a small dot under the backside. The flower blooms in June and is reddish-white, and about 10 bell-shaped flowers hang on the raceme hanging down and bracts remain. Fruits are berries, round, covered with white powder, about 6mm in diameter, ripe in October, and edible.

As the standard of living of modern people improves, interest in natural resources that have little side effects and can be collected from nature is increasing. In particular, efforts are being made to develop a substance that has few side effects and is helpful in preventing and recovering from diseases by studying the efficacy of natural products such as traditionally known mosae tree. Relevant information is lacking.

Provided is a composition for preventing or treating prostatic hyperplasia that can be safely used without toxicity and side effects by extracting an active ingredient from the fruit of the moss tree, which is a natural raw material.

The present invention includes a fruit extract of Vaccinium Bracteatum Thunb. as an active ingredient, and the extract is an extract available from any one selected from water, lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. Characterized in that It provides a functional food composition for preventing or treating benign prostatic hyperplasia or a pharmaceutical composition for preventing or improving benign prostatic hyperplasia.

The pharmaceutical composition for preventing or treating benign prostatic hyperplasia of the present invention may be provided by containing 0.1 to 2000 mg/kg/body weight/day of the extract of the moss fruit or 0.01 to 99.9% by weight of the composition. The pharmaceutical composition may be formulated in any one form selected from powders, granules, tablets, capsules, suspensions, emulsions, syrups, transdermal preparations, suppositories, or sterile injectable solutions. The functional food composition may be prepared in any one form selected from tablets and capsules, soft capsules, granules, and liquids.

The composition containing the hot-water extract of the fruit of the present invention has an activity of reducing testosterone-induced prostate (LNCaP) or prostate weight, suppressing 5-alpha-reductase-2 production, and reducing tissue polyps.

The present invention is a composition for preventing or treating prostatic hyperplasia using a mosae tree fruit extract as an active ingredient, using mosae tree, a domestic natural resource, and safely preventing or treating prostatic hyperplasia without toxicity or side effects, and as a health functional food composition. can be used

Figure 1 shows the powder and liquid phase of the extract of the fruit of Mosaenamu prepared in the present invention.

Figure 2 is a graph showing the survival rate of the prostate epithelial cell line (LNCaP) for the moss extract.

Figure 3 shows the effect on the contents of prostate-specific enzyme (5α-reductase 2), dihydrotestosterone (DHT), and prostate-specific antigen (PSA) according to the treatment of the robin fruit extract in the prostate epithelial cell line (LNCaP) by testosterone. It is a picture.

Figure 4 is a picture showing the androgen receptor (AR) protein expression according to testosterone-induced prostatic epithelial cell line (LNCaP) treatment with moss berry extract.

Figure 5 is a picture showing the effect of inhibiting prostate tissue hypertrophy according to the treatment of the moss berry extract in a testosterone-induced benign prostatic rat model.

Figure 6 is a picture showing the effect of inhibiting the content of 5-alpha reductase in prostate tissue according to the treatment of the moss berry extract in a rat model of prostatic hyperplasia induced by testosterone.

7 is a diagram showing a cross-sectional staining photograph of prostate tissue according to the treatment of a moss fruit extract in a testosterone-induced benign prostatic rat model.

The present invention relates to a composition for preventing or treating benign prostatic hyperplasia containing an extract of the fruit of moss japonica as an active ingredient, and the configuration and effects of the present invention will be described in more detail through specific examples and comparative examples.

1. Manufacture of mosae tree fruit extract

Mosae tree fruit extract was extracted by adding 50 L of distilled water to 5 kg of dried Moss tree fruit by removing moisture, and heating at 100 ° C. for 3 hours. Filtered, reduced pressure and concentrated. The concentrated hot-water extract was frozen and dried at -40 ° C to 40 ° C for 72 hours using a freeze dryer.

In the above method, 53.0 g (10.6%) of the hot water extract of the fruit of mosae tree was obtained and used as a sample for the following experiments related to the prevention or treatment of benign prostatic hyperplasia. Figure 1 shows the powder and liquid phase of the extract of the fruit of the moss tree prepared in the present invention.

2. Confirmation of cytotoxicity of the fruit extract of mosae tree

Prior to confirming the effect of the extract of the fruit extract of the present invention, a cytotoxicity test was performed to determine an appropriate concentration capable of producing an effect without exhibiting cytotoxicity. Figure 2 is a graph showing the survival rate of the prostate epithelial cell line (LNCaP) for the moss extract.

In order to confirm the cytotoxicity of the moss fruit extract, prostate cancer cells were treated with the extract and then the cell viability was confirmed. LNCaP cells (Korea Cell Line Bank 21740), a prostate cancer cell line, were cultured in RPMI-1640 (Gibco) medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin at 37°C and 5% CO ₂ conditions. .

The cultured cells were dispensed in a 96-well plate at a concentration of 1x10 ⁵ cell/well, cultured, and after 12 hours, the extract of the fruit of the moss tree prepared in the present invention was treated at a concentration of 0, 10, 50, and 100 μg/ml, respectively. . After culturing for 24 hours, 10 μL of EZCytox solution was added to each well, and toxicity was confirmed by measuring absorbance at 450 nm using a plate reader. As shown in FIG. 2, it was confirmed that the extract of the fruit of the moss tree had no cytotoxicity up to 50 μg/ml.

3. Confirmation of 5α-reductase, DHT and PSA contents of the fruit extract

Figure 3 shows the effect on the contents of prostate-specific enzyme (5α-reductase 2), dihydrotestosterone (DHT), and prostate-specific antigen (PSA) according to the treatment of the robin fruit extract in the prostate epithelial cell line (LNCaP) by testosterone. It is a picture.

After treating prostate cells with testosterone and extracts from the fruit of mosae tree, the contents of 5-alpha-reductase, dihydrotestosterone (DHT) and prostate-specific antigen (PSA) were checked. It was confirmed whether the extract was effective in prostatic hyperplasia disease. LNCaP cells were dispensed in a 6-well plate at a concentration of 1x10 ⁶ cells/well and cultured at 37°C.

After 24 hours, the cells were treated with 1 μM of testosterone, and simultaneously treated with the prepared hot-water extract at concentrations of 0, 5, 10, and 50 μg/ml, and then harvested after 24 hours of culture. As a positive control (BPH), only 1 μM of testosterone was treated without extract, and as a control, 10 μM of Finasteride, a treatment for prostatic hyperplasia, was treated with 1 μM of testosterone instead of the extract. The untreated group was used as a negative control group.

Phosphate-buffered saline pH7.4 (Gibco, USA) was added to the cells harvested after culture, and homogenization was performed with a Microcentrifuge Sample Pestle. The cell homogenate was centrifuged at 4℃ and 5,000 rpm for 5 minutes (Mikro 200R, Hettich, USA) to separate the supernatant, and 5-alpha reductase, dihydrotestosterone and prostate were separated using each ELISA KIT (CUSABIO, USA). Specific antigens were measured.

As shown in FIG. 3, it was confirmed that 5-alpha reductase activity was inhibited by 14% in the cell group treated with 50 μg/ml of the seedling fruit extract compared to the control group. In addition, it was confirmed that the inhibitory effect of dihydrotestosterone activity was excellent in the cell groups treated with 5, 10, and 50 μg/ml of the extract of the seedling fruit compared to the control group. On the other hand, the content of prostate-specific antigen in the cell groups treated with 10 and 50 μg/ml of the moss fruit extract tended to decrease, but no statistical significance was observed.

4. Confirmation of the Androgen Receptor (AR) Expression Inhibiting Effect of the Fruit Extract of Mosaic Tree

Figure 4 is a picture showing the results of testosterone-induced androgen receptor (AR) protein expression in prostate epithelial cell line (LNCaP) according to the treatment of moss berry extract. Prostate epithelial cells (LNCaP) were treated with testosterone and moss fruit extract, and the protein expression of androgen receptor (AR) was confirmed to confirm whether the moss fruit extract was effective in prostatic hyperplasia. LNCaP cells were dispensed in a 6-well plate at a concentration of 1x10 ⁶ and cultured at 37°C.

After 24 hours, the cells were treated with 1 μM of testosterone, and simultaneously treated with the prepared hot-water extract at concentrations of 0, 5, 10, and 50 μg/ml, and then harvested after 24 hours of culture. As a positive control group (BPH), only 1 μM of testosterone was treated without the extract, and as a control group (Fina), 10 μM of Finasteride, a treatment for prostatic hyperplasia, was treated with 1 μM of testosterone instead of the extract. The untreated group was used as a negative control group.

Cells harvested after culture are treated with RIPA buffer (50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate, with a protease inhibitor cocktail) to isolate the protein.

The separated protein was quantified using a protein assay kit (Bio-Rad protein assay kit, Bio-Rad, USA), and the quantified protein was subjected to SDS-PAGE on a 10% polyacrylamide gel, and the protein was transferred to a PVDF membrane. .

Thereafter, the PVDF membrane is reacted with 5% skim milk for about 1 hour to remove non-specific binding proteins on the surface, and the primary antibody, anti-AR antibody or anti-β-actin antibody, is used for one day at 4 ° C. reacted Thereafter, the secondary antibody was treated at room temperature for 1 hour, and the level of AR protein expression was confirmed using an ECL kit (Thermo scientific, USA). As shown in FIG. 4, it was confirmed that the AR protein expression inhibitory effect was remarkably superior to the control group in the cell group treated with 5, 10, or 50 μg/ml of the moss fruit extract.

5. Production of animals with benign prostatic hyperplasia and measurement of prostate weight

Figure 5 is a picture showing the effect of inhibiting prostate tissue hypertrophy according to treatment with hot water extract of moss tree fruit in a rat model of prostatic hyperplasia induced by testosterone. Male SD rats aged 9 weeks (weighing less than 330 g) were domesticated for 1 week, and then 3 mg/kg of testosterone propionate (TP) was dissolved in corn oil and subcutaneously injected for 4 weeks to induce prostate hyperplasia.

The hot-water extract of the fruit of mosae tree was orally administered at 50 and 100 mg/kg for 4 weeks, and finasteride (FI), a 5α-reductase inhibitor used for the treatment of benign prostatic hyperplasia, was orally administered at a dose of 5 mg/kg as a positive control group. After measuring the body weight a total of 5 times at intervals of one week from the start of the experiment, and sacrificing the rats the day after the last administration and treatment, each group (normal group; Sham, prostatic hyperplasia induced group (negative control group); BPH, prostatic hyperplasia induced group + finasteride administration group (positive control group); FI, benign prostatic hyperplasia group + moss fruit extract 50mg/kg administration group; VBF50, benign prostatic hyperplasia induction group + mosae tree fruit extract 100mg/kg administration group; prostates were removed from VBF100 and weighed The left prostate tissue was removed and 5α-reductase was measured in the tissue, and the prostate growth inhibitory effect (%) was calculated according to the following equation.

Prostatic hyperplasia inhibitory effect (%) = 100 - {(prostate weight in the seed-treated group or positive control group - normal prostate weight) * 100 / (negative control prostate weight - normal prostate weight)}

As shown in FIG. 5, it was confirmed that the size of the prostate was reduced in the groups (VBF50 and VBF100) administered with the mulberry fruit extract than in the group (BPH) in which prostatic hyperplasia was induced by TP.

Table 1 shows a table of prostate tissue weights according to the treatment of moss berry extract in a testosterone-induced benign prostatic rat model. As shown in Table 1, as a result of measuring the anti-hypertrophy effect of the prostate by orally administering the fruit extract of mosae tree to an animal model inducing benign prostatic hypertrophy, the prostate size was 0.30 ± 0.01 in the control group, 29.7% compared to 0.18 ± 0.02 in the normal group. has increased. Prostate hyperplasia growth inhibitory effect was 73.56% in the positive control group, and 20.84% and 28.75% respectively in the group administered with 50 or 100mg/kg of moss fruit, showing inhibitory effects in a concentration-dependent manner.

Table of prostate tissue weight following treatment with moss fruit extract in testosterone-induced benign prostatic hyperplasia rat model

group	Prostate weight (g/100BW)	Growth inhibitory effect (%)
Sham	0.18±0.02***	-
BPH	0.30±0.01	-
FI	0.21±0.02***	73.56
VBF50	0.27±0.02	20.84
VBF100	0.26±0.01*	28.75

6. Alpha-reductase measurement Figure 6 is a diagram showing the inhibitory effect of 5-alpha-reductase content in prostate tissue according to the treatment of the moss fruit extract in a rat model of prostatic hyperplasia induced by testosterone. The prostate tissue of the sacrificed rat was washed once with physiological saline and homogenized for 1 minute with a Wise Stir homogenizer (Daihan Scientific, Korea) after adding 4 times phosphate-buffered saline pH7.4 (Gibco, USA). Prostate tissue homogenate was centrifuged at 4°C and 5,000 rpm for 5 minutes (Mikro 200R, Hettich, USA) to separate the supernatant. measured.

As shown in FIG. 6, 62.79 ± 17.36, 617.45 ± 82.86, 232.8 ± 52.48, 446.0 ± 66.76, and 314.9 ± 67.47 in the normal group, negative control group, positive control group, and 50 and 100 mg / kg of mosae tree fruit, respectively. A significant decrease was shown in the positive control group and the group administered with 100 mg/kg of moss fruit.

6. Prostate histopathological observation

7 is a diagram showing a cross-sectional staining photograph of prostate tissue according to the treatment of a moss fruit extract in a testosterone-induced benign prostatic rat model. The right prostate tissues of the sacrificed rats were fixed in 10% neutral formalin, paraffin-embedded and sectioned, and stained with Hematoxylin-Eosin. Histological changes in the epithelial cells and stroma of the prostate were observed under an optical microscope.

As shown in FIG. 7, as a result of inducing prostatic hyperplasia, the prostate epithelial tissue became thicker and a number of polyps were observed in the negative control group (BPH) compared to the normal group, but in the group administered with 50 or 100 mg / kg of moss fruit, the epithelial tissue became thin and A decrease in polyps was also observed.

8. Manufacture of pharmaceuticals or health foods using extracts from the fruit of mosae tree

Pharmaceutical preparations or health foods for the prevention or treatment of prostatic hyperplasia using the fruit extract of moss tree as an active ingredient may be prepared in the form of tablets and capsules, soft capsules, granules, and liquids. According to another suitable embodiment, the pharmaceutical formulation or health food may be prepared as a beverage additive.

The pharmaceutical formulation or health food for preventing or treating prostatic hyperplasia is a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol such that the composition containing the mosae tree fruit extract as an active ingredient is contained in an amount of 0.01 to 99.9% by weight , It can be prepared by formulating into a transdermal preparation, a suppository or a sterile injectable solution.

In the case of a sterile injection solution, the pharmaceutical composition can be prepared by mixing 99.9 to 0.01% by weight of purified water or glucose with 0.01 to 99.9% by weight of the pharmaceutical composition, and in the case of capsules, freeze-drying the pharmaceutical composition to 0.01 to 99.9% by weight %, and can be prepared by mixing 99.9 to 0.01% by weight of vitamins and calcium.

The daily dosage of the pharmaceutical composition prepared above is provided in an amount of 10 to 2000 mg/kg body weight of the extract, and a pharmaceutical preparation for preventing or treating enlargement of the prostate or health containing 0.01 to 99.9% by weight of the pharmaceutical composition. can be made into food.

The present invention is a composition for preventing or inhibiting prostatic hyperplasia, which contains an extract of moss fruit as an active ingredient, and is useful for preventing or inhibiting prostatic hyperplasia in testosterone-induced prostate cells (LNCaP) and an enlarged prostate rat model, so it has industrial applicability. .

Claims (5)

1. A pharmaceutical composition for preventing or treating prostatic hyperplasia, comprising a hot water extract of the fruit of Vaccinium Bracteatum Thunb.
2. The pharmaceutical composition for preventing or treating prostatic hyperplasia according to claim 1, which comprises 0.01 to 99.9% by weight of the composition.
3. The method according to claim 1, wherein the composition is formulated in any one form selected from powders, granules, tablets, capsules, suspensions, emulsions, syrups, transdermal preparations, suppositories, or sterile injectable liquids. Prevention of prostatic hyperplasia, characterized in that or a pharmaceutical composition for treatment
4. Functional food composition for preventing or improving prostatic hyperplasia, characterized in that it contains a hot water extract of the fruit of mosae tree ( Vaccinium Bracteatum Thunb.) as an active ingredient
5. The functional food composition for preventing or improving prostatic hyperplasia according to claim 4, wherein the functional food composition is prepared in any one form selected from tablets and capsules, soft capsules, granules, and liquids.

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