KR102180876B1 - Novel use of Couroupita guianensis - Google Patents

Abstract

The present invention provides a new use of the cannonball tree. There is an advantage in that the present invention can effectively treat, ameliorate, inhibit and/or prevent an enlarged prostate.

Description

Novel use of Couroupita guianensis {Novel use of Couroupita guianensis}

The present invention relates to a cannonball tree, and more particularly, to a new use of the cannonball tree.

Benign prostatic hyperplasia (BPH) is one of the most common male urinary diseases, and refers to a condition in which the prostate around the urethra is abnormally enlarged, preventing the excretion of urine, resulting in various symptoms. It is known that one of the causes of prostate enlargement caused by prostate cell proliferation is due to the secretion of male hormones, since symptoms of prostatic hyperplasia usually occur only when the age of 50 years or older and male hormones are secreted normally during puberty.

The main male hormone involved in the growth of prostate tissue is DHT (dihydrotestosterone). DHT is testosterone converted by 5AR (5-alpha reductase). 5AR is a NADPH (nicotinamide adenine dinucleotide phosphate (reduced form)) dependent protein of microsomes, 3-oxo-5α-steroid 4-dehydrogenase, which reduces double bond steroids such as testosterone.

There are two types of 5AR in the body, 5AR1 (Type I, type 1) and 5AR2 (Type II, type 2). 5AR1 is mainly expressed in the liver, adrenal gland, and skin, and 5AR2 is mainly expressed in the prostate. 5AR2 is known to play an important role in the pathogenesis of prostatic hyperplasia. It is known that when 5AR2 is overexpressed and the concentration of DHT increases, a large amount of DHT binds to the androgen receptor (AR) and causes prostate enlargement. Therefore, finding an inhibitor of 5AR (especially 5AR2) is known to be important in the treatment of prostatic hypertrophy (Xingsheng Wang et.al., “Establishment of a Novel Model for Studying the Effects of Extracts of Chinese Herb Medicine on Human Type II). 5α-Reductase in Vitro ”, YAKUGAKU ZASSHI 130(9) 1207-1214, 2010, etc.}.

On the other hand, the cannonball tree ( Couroupita guianensis ) belongs to the Lecythidaceae family and is mainly distributed in tropical America, Madagascar, Australia, and tropical-subtropical Asia. Such cannonball trees are known to exhibit biological activity in insects, bacteria, fungi, and viruses {Yadav Anu et.al., “Repellency and toxicity of Couroupita guianensis leaf extract against Silverleaf Whitefly ( Bemisia tabaci )”, International Journal of Scientific and Research Publications, 5(4), 2015, etc.}.

Xingsheng Wang et.al., “Establishment of a Novel Model for Studying the Effects of Extracts of Chinese Herb Medicine on Human Type Ⅱ 5α-Reductase in Vitro”, YAKUGAKU ZASSHI 130(9) 1207-1214, 2010 Yadav Anu et.al., “Repellency and toxicity of Couroupita guianensis leaf extract against Silverleaf Whitefly (Bemisia tabaci)”, International Journal of Scientific and Research Publications, 5(4), 2015

The problem to be solved by the present invention is to provide a new use of the cannonball tree.

The subject of the present invention is not limited to the above-mentioned subject, and other subjects not mentioned will be clearly understood by those skilled in the art from the following description.

The inventors of the present invention have surprisingly found that the cannonball tree has an effect on prostatic hyperplasia, thereby completing the present invention.

The present invention provides a medical and/or food use for the treatment, prevention, amelioration and/or inhibition of prostatic hyperplasia of the cannonball tree. Treatment is meant to encompass improvement and alleviation of symptoms related to prostatic hyperplasia, and prevention is meant to encompass inhibition of development of a disease in the pre-disease stage.

In one embodiment, the present invention provides a pharmaceutical composition for the treatment or prevention of prostatic hyperplasia comprising cannonball tree as an active ingredient.

The cannonball tree ( Couroupita guianensis or Couroupita guianensis Aubl.) is a deciduous tree and its origin is a tropical plant that is a tropical rain forest such as South America and Central America.

The cannonball tree may be a whole plant or part of the cannonball tree, and may be, for example, one or more selected from leaves, flowers, fruits, seeds, branches, stems, and roots.

The cannonball tree may be at least one selected from a raw cannonball tree, a cannonball tree structure, or a cannonball tree solvent extract.

The solvent may be a polar solvent.

The solvent may be at least one selected from water, alcohol, or a mixed solvent thereof.

The alcohol may be a lower alcohol, and may be one or more selected from alcohols having 1 to 5 carbon atoms. More preferably, the alcohol may be at least one selected from methanol or ethanol.

The prostatic hyperplasia is the activity of 5-alpha reductase (5AR), expression of 5-alpha reductase, production of dihydrotestosterone (DHT), and expression of androgen receptor (AR). It may be by one or more selected from among.

At least one selected from the activity of the 5-alpha reductase, the production of dihydrotestosterone, and the expression of the androgen receptor may be performed in prostate cells.

At least one selected from the expression of the 5-alpha reductase and the production of dihydrotestosterone may be performed in prostate tissue.

The activity of the 5-alpha reductase may be an excess activity of the 5-alpha reductase in prostate cells.

Expression of the 5-alpha reductase may be overexpression of the 5-alpha reductase in prostate tissue.

The production of dihydrotestosterone may be an overproduction of the dihydrotestosterone in prostate cells or prostate tissues.

The overproduction of dihydrotestosterone may be caused by overactivity of 5-alpha reductase or overexpression of 5-alpha reductase.

The treatment or prevention is one selected from inhibition of prostate cell division, inhibition of prostate tissue enlargement, inhibition of 5-alpha reductase activity, inhibition of 5-alpha reductase expression, inhibition of dihydrotestosterone production, and inhibition of androgen receptor expression by the cannonball tree. It may be due to the above.

The 5-alpha reductase may be a 2-type 5-alpha reductase (5AR2 or 5AR Type II).

The composition of the present invention may contain 0.1 to 95% by weight of the active ingredient.

In addition, the present invention provides a food composition for improving or inhibiting prostatic hyperplasia comprising the cannonball tree as an active ingredient.

Unless otherwise stated, the food composition is applied in the same manner as long as the matters mentioned in the pharmaceutical composition of the present invention are not contradicted. The'improvement' is included in'treatment', and means that a condition or symptom is improved. The'inhibition' is included in'prevention' and means preventing the occurrence of a condition or symptom.

The food composition may be variously included in foods including beverages, and may be in the form of beverages, gums, teas, health functional foods, and the like, and the health functional foods may be formulated in formulations such as tablets and capsules. The term "health functional food" includes foods manufactured (including processing. The same applies hereinafter) using raw materials or ingredients that have functions useful for the human body according to the Korean Health Functional Food Act, and the term "functional" means It refers to obtaining useful effects for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body. The food composition may contain conventional food additives, and the food additives are chemical compounds such as ketones, glycine, sodium citrate, nicotinic acid, cinnamic acid, etc., natural pigments, licorice extract, crystalline cellulose, high color pigments, guar gum, etc. And mixed preparations such as additives, sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar coloring preparations. In addition, the food composition is meant to include food additives and the like.

The present invention also provides a method for treating, preventing, ameliorating or inhibiting prostatic hyperplasia, comprising administering cannonball tree to mammals including humans in need thereof.

The administered cannonball tree may be an effective amount of cannonball tree.

The cannonball tree may be included in the formulation.

The cannonball tree may be included in the composition.

The composition may be a food composition.

The composition may be a pharmaceutical composition.

In addition, the present invention provides a use for preparing a composition for the treatment, prevention, improvement or inhibition of prostatic hyperplasia of the cannonball tree.

The composition may include the cannonball tree as an active ingredient.

The composition may be a food composition.

The composition may be a pharmaceutical composition.

In addition, the present invention provides a use for preparing a preparation for the treatment or prevention of prostatic hyperplasia of the cannonball tree.

Unless otherwise stated, matters mentioned in the pharmaceutical composition, food composition, method, and use of the present invention are applied identically, unless contradictory to each other.

The cannonball tree or the composition may be administered orally or parenterally to mammals including humans, and the active ingredient may be formulated and administered with a pharmaceutically acceptable carrier. When formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, diluents or excipients can be used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the composition of the present invention, such as starch, calcium carbonate, sucrose, lactose, and / Or it can be prepared by adding gelatin or the like. In addition, lubricants such as magnesium and talc may be used. Liquid preparations for oral administration include suspensions, solvents, emulsions and syrups, and various additives in addition to water and liquid paraffin, which are commonly used simple diluents, such as wetting agents, sweeteners, fragrances and/or preservatives, etc. May be included. Formulations for parenteral administration include injectable solutions, suspensions, emulsions, lyophilized agents, nasal cleansers and suppositories. Injectable solutions, suspensions, and emulsions can be prepared by mixing water, non-aqueous solvents or suspensions with active ingredients, and non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, ethyl oleate. And injectable esters such as. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol and/or gelatin, and the like may be used. When administered parenterally, it can be administered by subcutaneous injection, intravenous injection, or intramuscular injection.

The cannonball tree included in the composition of the present invention or used in a use or method is an adult standard (60 kg) in a dose of 1 to 2000 mg, preferably 100 to 1000 mg, more preferably 500 to 1000 mg per day. Can be used. At this time, the cannonball tree may be preferably a cannonball tree solvent extract. Administration can be administered once or several times a day. However, the scope of the present invention is not limited by the dosage and frequency of administration.

The cannonball tree can be formulated by adding additives such as a pharmaceutically or food-acceptable carrier, excipient, or diluent, and for information on formulation, see Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA, etc. Reference can be made to the literature.

Accordingly, the composition of the present invention further includes a pharmaceutically acceptable additive or a food pharmaceutically acceptable additive, and may be composed of the active ingredient and the pharmaceutically acceptable additive or the food pharmaceutically acceptable additive.

According to the present invention, an enlarged prostate can be effectively treated, prevented, ameliorated and/or inhibited.

1 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of survival of the prostate cell line PC3 cells.
2 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of cell division of the prostate cell line PC3 cells.
3 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of 5AR activity in the prostate cell line PC3 cells.
4 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of AR expression in prostate cell line RWPE-1 cells.
5 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of prostate tissue hypertrophy in an animal model of prostatic hyperplasia.
6 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of 5AR expression in an animal model of prostatic hyperplasia.
7 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of DHT production in an animal model of prostatic hyperplasia.

Hereinafter, the advantages and features of the present invention, and a method of achieving them will become apparent with reference to the embodiments described later in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms different from each other, and only these embodiments make the disclosure of the present invention complete, and common knowledge in the technical field to which the present invention pertains. It is provided only to completely inform the scope of the invention to the possessor, and the invention is only defined by the scope of the claims.

Throughout the specification, “and/or” includes each and every combination of one or more of the recited elements.

The terms used in the present specification are for describing exemplary embodiments and are not intended to limit the present invention. In this specification, the singular form also includes the plural form unless specifically stated in the phrase. As used herein, “comprises” and/or “comprising” do not exclude the presence or addition of one or more other elements and/or steps to the mentioned elements and/or steps.

In this specification,'live cannonball tree' refers to a whole plant or part of a cannonball tree that has not undergone a separate drying process after harvesting, and'cannonball tree construction' means that a raw cannonball tree is dried. .

In addition, the term "cannonball tree solvent extract" refers to extracting at least one selected from raw cannonball tree or cannonball tree dry matter with a solvent. In this case,'Cannonball Tree Solvent Extract' has the same meaning as'Cannonball Tree Extract'.

Hereinafter, in Examples, it was confirmed that the cannonball tree exhibits an effect on prostatic hyperplasia using a prostate cell line and an experimental animal.

As for the reagents used in the examples, the highest grade available on the market was used, and those purchased from Sigma, etc. were used.

<Example 1> Confirmation of Use of Cannonball Tree I

1-1. Cannonball tree preparation

Cannonball tree structure (a dry product obtained by shading and pulverizing the leaves of the Cannonball tree collected in Bangladesh in 2017, sold in sales at Korea Research Institute of Bioscience and Biotechnology, and overseas biological material centers) 100% of 100%-MeOH (HPLC grade) ) Was added and ultrasonic extraction was performed at a temperature of 40°C. After ultrasonic extraction for 15 minutes, it was allowed to stand for 2 hours, and the same procedure was performed 10 times a day for 3 days to obtain an extract. Then, the extract was concentrated with a vacuum concentrator (Buchi R-220) to prepare a cannonball tree solvent extract.

1-2. Cell preparation

The prostate cell line PC3 cells were distributed and used from the Korea Cell Line Bank. Cells were 5%-CO ₂ in DMEM (Dulbecco's modified Eagle's medium) medium supplemented with 10% heat-inactivated FBS (fetal bovine serum), penicillin (P/S) and streptomycin, It was maintained at 37 ℃ culture conditions.

1-3. Prostate cell viability inhibition assay

In order to analyze the effect of cannonball tree on the inhibition of prostate cell survival, MTT{3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide} assay was used. MTT assay is an experimental method that can determine the effect of a test substance on cell viability. Using this MTT assay, the effect of cannonball tree on the inhibition of prostate cell survival was analyzed.

1.5x10 ⁵ cells/ml of cells prepared as in 1-2 are seeded on a 96-well plate at 100 ul each, and 24 hours later, the Cannonball tree solvent extract prepared as in 1-1 is added to DMSO (dimethyl sulfoxide). Dissolved and prepared at a concentration of 100 mg/ml, diluted with cell culture solution, and treated at a final concentration of 50 and 100 ug/ml for 24 hours. After adding 10 ul of MTT reagent at a concentration of 2 mg/ml to each well, it was further cultured for 1 hour, and after removing the culture medium, 100 ul of DMSO was added to extract MTT formazan. The amount of MTT formazan was quantified with a multi-well spectroscopy (Tecan, Infinite) at a test wavelength of 540 nm. The control group was treated in the same manner as the cannonball tree solvent extract treatment group, except that PBS (phosphate buffer saline) (10 ul) was treated instead of the cannonball tree solvent extract. From the quantitative results, cell viability (%) compared to the control was calculated, and the results are shown in FIG. 1.

1 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of survival of the prostate cell line PC3 cells. The x-axis represents the control group (CON) and the cannonball tree solvent extract (cannonball tree extract) treated group, and the y-axis represents the cell survival rate as a relative ratio (relative ratio%) to the control group.

As shown in Fig. 1, it can be seen that the cannonball tree does not have a significant effect on the survival of the prostate cell line at dose concentrations of 50 and 100 ug/ml. That is, it can be seen that the cannonball tree does not show toxicity to prostate cells.

1-4. Prostate cell division inhibitory ability assay

In order to analyze the effect of cannonball tree on inhibition of prostate cell division, BrdU (5-bromo-2'-deoxyuridine) incorporation assay was used. BrdU incorporation assay is a test method that detects newly synthesized DNA in proliferating cells with BrdU antibody, and is an experiment that can determine whether a test substance inhibits cell division. Using such a BrdU incorporation assay, the effect of cannonball tree on the inhibition of prostate cell division was analyzed.

2x10 ⁵ cells/ml of cells prepared as described in 1-2 are seeded in a 96-well plate at 100 ul each, and after 24 hours, 100 mg/ml of the Cannonball tree solvent extract prepared as in 1-1 is dissolved in DMSO. After preparation at the concentration, it was diluted with a cell culture solution and treated with a final concentration of 50 and 100 ug/ml. The degree of BrdU incorporation was measured using a kit (Abcam, ab126556). After 2 hours of extract treatment, 20 ul of BrdU label reagent was added and incubated for 24 hours. After completely removing the cell culture solution, 200 ul of a fixing solution was added and left at room temperature for 30 minutes, and then the fixing solution was removed and dried. After washing twice with PBS, 100 ul of anti-BrdU monoclonal Detector Antibody was added and left at room temperature for 1 hour. After removing and washing the solution, Peroxidase Goat Anti-Mouse IgG was added, left for 30 minutes, and washed. 100 ul of TMB (3,3',5,5'-Tetramethylbenzidine) peroxidase substrate was added and reacted at room temperature for 30 minutes under light shielding, and then 100 ul of stop solution was added to terminate the reaction. The degree of BrdU incorporation was measured with a multi-well spectroscopy (Tecan, Infinite) at a test wavelength of 450/540 nm. The control group was treated in the same manner as the Cannonball tree solvent extract treatment group, except that PBS (10 ul) was treated instead of the Cannonball tree solvent extract. From the quantitative results, the BrdU incorporation ratio (%) compared to the control was calculated, and the results are shown in FIG. 2.

2 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of division of the prostate cell line PC3 cells. The x-axis represents the control group (CON) and the Cannonball tree solvent extract (Cannonball tree extract) treated group, and the y-axis represents BrdU incorporation as a percentage (relative ratio%) compared to the control group.

As shown in Figure 2, it can be seen that the cannonball tree significantly inhibited the cell division of the prostate cell line. From these results, it can be seen that the cannonball tree is effective against prostatic hypertrophy by inhibiting prostate cell division. This is because cannonball trees can suppress prostatic hypertrophy, which causes the prostate to become enlarged by cell division.

From the above experimental results, it can be seen that the cannonball tree does not show toxicity to prostate cells and inhibits cell division, thus maintaining the normal function of the prostate and selectively inhibiting the enlargement of the prostate, which is particularly effective in prostatic hyperplasia. .

Accordingly, it can be seen that the present invention can provide a medical and/or food use for the treatment, prevention, improvement and/or inhibition of prostatic hyperplasia of the cannonball tree.

<Example 2> Confirmation of Use of Cannonball Tree II

5AR (5-alpha reductase) is the most important enzyme that mediates the conversion of testosterone to DHT (dihydrotestosterone). DHT is known to play the most important role in the growth of prostate tissue. Therefore, by confirming whether the cannonball tree inhibits DHT production, and by checking whether it inhibits the activity of 5AR (especially 5AR2, which is mainly expressed in the prostate), we tried to find out whether it exhibits an effect on prostatic hyperplasia.

2-1. Cannonball tree preparation

Cannonball tree solvent extract was prepared in the same manner as in Example 1-1.

2-2. 5AR activity inhibitory ability assay

2x10 ⁵ cells/ml of cells prepared as described in 1-2 were seeded on a 6-well plate and cultured with 60-70% confluence. After removing all the medium and replacing it with the Opti-MEM medium, 100 ng of 5AR2 plasmid (SRD5A2, OriGene, USA) per well was transfected for 24 hours. Thereafter, DMEM supplemented with 10% FBS was replaced, and testosterone (8.8 ug/ml) and Cannonball tree solvent extract (50, 100 ug/ml) were treated and further cultured for 24 hours. After that, the protein was extracted and the amount of DHT produced was quantified using a DHT assay kit (Mybiosource, #MBS266535). The control group was prepared in the same manner as the Cannonball tree solvent extract treatment group, except that PBS (10 ul) was treated instead of the Cannonball tree solvent extract. The positive control group was prepared in the same manner as the cannonball tree solvent extract treatment group, except that Dutasteride (20 uM) was treated instead of the cannonball tree solvent extract. The results are shown in FIG. 3.

3 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of 5AR activity in the prostate cell line PC3 cells. The x-axis represents the control group (CON), the positive control group (dutasteride) and the cannonball tree solvent extract-treated group (cannonball tree extract), and the y-axis represents the amount of DHT produced as a percentage (relative ratio%) compared to the control group.

As shown in FIG. 3, in the case of the positive control group, the amount of DHT produced by dutasteride showed a tendency to decrease, and in the case of the Cannonball tree solvent extract-treated group, the production of DHT was significantly reduced compared to the control group. . Therefore, it can be seen that the cannonball tree suppresses the generation of DHT. As a result, the cannonball tree inhibits the production of DHT, which plays the most important role in prostate tissue growth, thereby inhibiting prostate tissue growth, thereby exhibiting an effect on prostatic hypertrophy.

In addition, in the above experiment, since DHT generated excessively from prostate cells was mainly due to overexpression (or activation) of 5AR (especially 5AR2) in prostate cells, inhibition of DHT production by cannonballs was mainly 5AR (especially, It can be seen that it is due to the inhibition of the activity of 5AR2).

From these results, it can be seen that the cannonball tree significantly reduces the production of DHT, an active testosterone, by inhibiting the activity of 5AR (especially 5AR2), which is effective in prostatic hyperplasia. In particular, it can be seen that the activity of 5AR (especially 5AR2) made in prostate cells and/or for prostatic hypertrophy due to DHT production.

Accordingly, it can be seen that the present invention can provide a medical and/or food use for the treatment, prevention, improvement and/or inhibition of prostatic hyperplasia of the cannonball tree.

<Example 3> Confirmation of Use of Cannonball Tree III

Androgens, such as testosterone and dihydrotestosterone (DHT), bind to androgen receptors (AR) in the nuclei of prostate epithelium and strut cells. Dihydrotestosterone induces structural changes and dimerization by binding to androgen receptors, and consequently stimulates prostate-specific antigens (PSA) and other growth factors that induce prostate proliferation to induce prostate hypertrophy. As we age, sex hormones change and the concentration of testosterone in the blood decreases. However, in response to this, the activity of 5-alpha reductase in cells increases in order to maintain the balance of the endocrine system in the body, and accordingly, dihydrotestosterone in prostate cells increases, and the number of androgen receptors that respond to it in the cells is rather gradually. It shows an increasing pattern. Therefore, although testosterone decreases with age, the concentration of dihydrotestosterone in the prostate does not change significantly. On the other hand, it has been reported that relatively high levels of nuclear androgen receptors are expressed in thickened prostate tissue compared to normal prostate. Increasing the expression of the androgen receptor maintains the level of dihydrotestosterone in the prostate even if the total amount of androgens decreases. The normal androgen receptor is interpreted to be activated when the expression of the mRNA of the androgen receptor and the androgen receptor protein is increased even when the blood androgen level is low.Therefore, inhibition of androgen receptor protein expression can also be seen to mean inhibition of androgen receptor activity.

The concentration of dihydrotestosterone in the prostate is about twice that of testosterone, and in terms of its action on the prostate, it has an androgen titer that is about 5 times that of testosterone. In general, most androgen receptors in the prostate stably maintain a binding state with dihydrotestosterone, which has a higher affinity than testosterone, so inhibition of androgen receptor expression eventually induces inhibition of the binding of dihydrotestosterone and androgen receptors, and as a result, dihydrotestosterone and androgen It can be seen that inhibition of prostate proliferation induced by receptor binding leads to inhibition of prostatic hyperplasia.

Therefore, in the following, it was examined whether the cannonball tree can directly inhibit the expression of the androgen receptor, and it was attempted to find out whether the cannonball tree has an effect on prostatic hyperplasia.

3-1. Cannonball tree preparation

Cannonball Tree Dried Material (Dried and pulverized leaves of Cannonball Tree collected in Bangladesh in 2017 and powdered, sold at Korea Research Institute of Bioscience and Biotechnology, overseas biological material center) 100 g of 100%-MeOH (HPLC grade) ) Was added and ultrasonic extraction was performed at a temperature of 40°C. After ultrasonic extraction for 15 minutes, it was allowed to stand for 2 hours, and the same procedure was performed 10 times a day for 3 days to obtain an extract. Thereafter, a cannonball tree solvent extract obtained by concentrating the extract with a vacuum concentrator (Buchi R-220) (cannonball tree total methanol extract) was prepared. Cannonball tree solvent extract obtained by concentrating under reduced pressure (Cannonball tree total methanol extract) was suspended in distilled water, and the same amount of normal-hexane was added and left to stand in a fractionation funnel for 60 minutes. When the layers were completely divided, only the normal-hexane layer was Obtained as a first fraction. Further fractionation was performed in the same manner to obtain a second fraction and a third fraction, and the first fraction, the second fraction, and the third fraction were all mixed to obtain a normal-hexane fraction. In the distilled water suspension remaining after normal-hexane fractionation, chloroform fraction, ethyl acetate fraction, and normal-butanol fraction were sequentially performed in the same manner as the normal-hexane fractionation method, and chloroform A fraction, an ethyl acetate fraction, and a butanol fraction were obtained. Each fraction was concentrated under reduced pressure, powdered, and dissolved in DMSO to prepare a concentration of 100 mg/ml.

3-2. Cell preparation

Prostate cell line RWPE-1 cells were purchased from Korea Cell Line Bank and used. Cells were 5%-CO ₂ in DMEM (Dulbecco's modified Eagle's medium) medium supplemented with 10% heat-inactivated FBS (fetal bovine serum), penicillin (P/S) and streptomycin, It was maintained at 37 ℃ culture conditions.

3-3. AR expression analysis

4.5×10 ⁵ cells/ml of cells prepared as described in 3-2 were seeded into 6-well plates at 2 ml each, and cultured for 24 hours. After removing all of the medium and replacing it with DMEM containing no FBS, 0.5 uM of testosterone propionate (TP) was treated, and after 1 hour, the total methanol extract of Cannonball tree was treated at a concentration of 10 ug/ml. After 24 hours, proteins were extracted and western blotting was performed to analyze the expression level of intracellular androgen receptors using anti-AR antibody. The control group was treated in the same manner as the group treated with the total methanol extract of Cannonball tree except that testosterone propionate and the total methanol extract of Cannonball tree were not treated. Except for the Cannonball tree total methanol extract was treated in the same manner as the treatment group. In addition, the normal-hexane fraction treatment group, the chloroform fraction treatment group, the ethyl acetate fraction treatment group, and the butanol fraction treatment group were treated with normal-hexane fraction, chloroform fraction, ethyl acetate fraction, and butanol fraction, respectively, instead of the total methanol extract of Cannonball. Except for the Cannonball tree total methanol extract was treated in the same manner as the treatment group. The results are shown in FIG. 4.

4 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of AR expression in prostate cell line RWPE-1 cells. The x-axis is the control group (CON), the negative control group (TP), the cannonball tree total methanol extract treatment group (T), the normal-hexane fraction treatment group (H), the chloroform fraction treatment group (C), and the ethyl acetate fraction treatment group (E). , And the butanol fraction treatment group (B), and the y-axis represents the amount of androgen receptor protein expression (androgen receptor protein expression) as a relative ratio (relative ratio) to the negative control group (*** p<0.001; compared with the control group) ). In FIG. 4, the total methanol extract and fraction of Cannonball Trees refers to the total methanol extract treatment group of Cannonballs tree, normal-hexane fraction treatment group, chloroform fraction treatment group, ethyl acetate fraction treatment group, and butanol fraction treatment group.

As shown in FIG. 4, in the case of the negative control group, the amount of protein expression of the androgen receptor was significantly increased by the testosterone propionate treatment, and the total methanol extract treatment group, normal-hexane fraction treatment group, the chloroform fraction treatment group It can be seen that in the group, the ethyl acetate fraction treatment group, and the butanol fraction treatment group, the amount of protein expression of the androgen receptor was significantly reduced compared to the negative control group. Therefore, it can be seen that the cannonball tree directly inhibits the expression of androgen receptors in prostate cells. As a result, cannonball tree suppresses the expression of androgen receptors, thereby reducing the binding between DHT and androgen receptors, thereby inhibiting prostate proliferation, thereby exhibiting an effect on prostatic hyperplasia.

Accordingly, it can be seen that the present invention can provide a medical and/or food use for the treatment, prevention, improvement and/or inhibition of prostatic hyperplasia of the cannonball tree.

<Example 4> Confirmation of Use of Cannonball Tree IV

4-1. Cannonball tree preparation

100 ml of 50%-EtOH (HPLC grade) to 1 g of Cannonball Tree Dried (dry and pulverized leaves of Cannonball trees collected in Bangladesh in 2017 and powdered, sold at Korea Research Institute of Bioscience and Biotechnology, and overseas biological material centers) ) Was added, and ultrasonic extraction was performed at a temperature of 40°C. The extracted extract was repeated once 1 hour and 3 times, and concentrated with a vacuum concentrator (Buchi R-220) to prepare a Cannonball tree solvent extract.

4-2. Preparation of experimental animals

Experimental animals were 6 weeks old male Wistar rats. The breeding environment of the experimental animals was at 23±3 ℃, the relative humidity was 55±5%, and the contrast was about 12 hours, and the feed and water were freely ingested. Experimental animals were housed in 2 per breeding box, and weighing and bedding exchange were carried out once every 5 days and managed.

Experimental animals were castration to rule out the effects of intrinsic testosterone after a week of acclimatization. The experimental animal was anesthetized with isoflurane, laid the back side toward the operator, and fixed. The skin at the end of the scrotum was cut to cut the left and right testicles and epididymis, and the incision surface was sutured to perform surgery. All experimental animals were administered antibiotics (cefazolin) and analgesic (carprofen) before surgery to relieve inflammation and pain. Stabilization for 7 days after castration to heal the wound on the surgical site, and the following experiment was initiated. The body weight of the animals at the initiation time was to be an average of 200-250 g for each group, and the number of experimental animals per group was 8. During the experiment, other groups were arranged so that they were not in one breeding box, and water and feed were fed freely.

4-3. Experimental animal treatment

TP (testosterone propionate, testosterone propionate) was injected subcutaneously at 3 mg/kg body weight/day once a day for 6 weeks, and at the same time, the cannonball tree solvent extract was also 0.5%-CMC (carboxy methylcellulose). ) And administered orally at 100 mg/kg body weight/day. At this time, TP was used by dissolving in peanut oil so that the total injection volume was 100 μl based on the weight of the experimental animal measured every 3 days in order to minimize the error of the experiment. The positive control group was treated in the same manner as the Cannonball tree solvent extract treatment group, except that saw palmetto was diluted in peanut oil instead of the diluted Cannonball tree solvent extract and administered orally at 100 mg/kg body weight/day, and the control group was diluted Cannonball tree Instead of the solvent extract, 0.5%-CMC was orally administered at 10 ml/kg body weight/day, and treated in the same manner as in the Cannonball tree solvent extract treatment group.

4-4. Final weighing, autopsy, and sampling of experimental animals

After 6 weeks of experiment, the experimental animals were fasted for 16 hours and the final body weight was measured. Thereafter, the experimental animals were euthanized by cervical dislocation, the prostate (dorsal prostate and ventral prostate) and the bladder were removed, carefully separated, washed once with PBS, and then lightly drained.

4-5. PI analysis

In order to determine whether the cannonball tree inhibits prostate tissue hypertrophy, PI (Prostate index) was analyzed.

The weight of the prostate extracted in 4-4 was measured. From the measured prostate weight and the last weight measured in 4-4, PI was calculated by the following formula. The results are shown in FIG. 5.

PI = {Extracted prostate weight (g)/Last weight (g)} x 100

5 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of prostate tissue hypertrophy in an animal model of prostatic hyperplasia. The x-axis represents the control group, the cannonball tree solvent extract-treated group (cannonball tree extract), and the positive control group (saw palmetto), respectively, and the y-axis represents PI (Prostate weight/body weight*100) (*p<0.1; control and compare). In Figure 4, b.w. means body weight.

As shown in FIG. 5, in the case of the positive control group, it can be seen that the PI decreases due to saw palmetto, and in the case of the Cannonball tree solvent extract-treated group, the PI decreases significantly compared to the positive control group. From these results, it can be seen that the cannonball tree suppresses the enlargement of the prostate tissue.

4-6. 5AR and DHT analysis in prostate tissue

To confirm whether the cannonball tree suppresses the expression of 5AR (especially 5AR2, which is mainly expressed in the prostate) and suppresses DHT production, ELISA (Enzyme-Linked ImmunoSorbent Assay, enzyme immunoassay) was used. Specifically, ELISA was performed according to the manufacturer's instructions using a 5AR2 ELISA kit (Cusabio, #CSB-EL022654RA) and a DHT ELISA kit (Mybiosource, #MBS266535).

The prostate tissue extracted in step 4-4 was homogenized to obtain a homogenate, and the expression level of 5AR2 and the production amount of DHT were analyzed using an ELISA kit.

To measure the 5AR expression level, 50 μl of the homogenate was added to HRP-conjugated SRD5A2 (rat 3-oxo-5-alpha-steroid-4-dehydrogenase 2) and SRD5A2-specific in a 96-well microplate pre-coated with goat-anti-rabbit antibody. After mixing well with the antibody, shaking incubation was performed at 37 degrees Celsius for 1 hour. After incubation, the substrate solution was added and the reaction was stopped with sulfuric acid solution after 15 minutes, and the difference between samples was measured within 10 minutes at 450 nm. The measurement value was calculated using a standard graph and the unit was expressed in pg/mL. The results are shown in FIG. 6.

To measure the amount of DHT production, 50 μl of the homogenate was incubated with HRP-conjugated DHT and DHT-specific antibody in a 96 well microplate pre-coated with goat-anti-rabbit antibody for 1 hour at room temperature with shaking. After incubation, the substrate solution was added and the reaction was stopped with sulfuric acid solution after 15 minutes, and the difference between samples was measured within 10 minutes at 450 nm. The measurement value was calculated using a standard graph and the unit was expressed in pg/mL. The results are shown in FIG. 7.

6 is a graph showing the results of analyzing the effect of cannonball tree on the inhibition of 5AR expression in an animal model of prostatic hyperplasia, and FIG. 7 is a graph showing the result of analyzing the effect of cannonball tree on the inhibition of DHT production in an animal model of prostatic hyperplasia to be. The x-axis represents the control group, the cannonball tree solvent extract treatment group (cannonball tree extract), and the positive control group (saw palmetto), respectively, the y-axis of FIG. 6 represents the expression level of 5AR2, and the y-axis of FIG. 7 represents the amount of DHT production (* p<0.1; compared to control). In FIGS. 6 to 7, b.w. means body weight.

As shown in FIGS. 6 to 7, in the case of the cannonball tree solvent extract treatment group, the 5AR2 expression level and DHT production level were significantly reduced compared to the control group, and it can be seen that it is effective compared to the positive control group.

From these results, it can be seen that the cannonball tree suppresses 5AR2 expression and suppresses DHT production. Therefore, it can be seen that cannonball tree is effective in prostatic hyperplasia by inhibiting 5AR expression and DHT production.

As a result, it can be seen that the present invention can provide a medical and/or food use for the treatment, prevention, improvement and/or inhibition of prostatic hyperplasia of the cannonball tree.

In particular, such treatment, prevention, improvement and/or inhibition is specifically shown in the experimental results, inhibition of prostate cell division, inhibition of prostate tissue enlargement, inhibition of 5-alpha reductase activity, 5-alpha reductase Expression inhibition, dihydrotestosterone production, and androgen receptor expression inhibition.

<Preparation Example 1> Preparation of pharmaceutical composition

Cannonball tree solvent extract 100 mg, corn starch 100 mg, lactose 100 mg, and magnesium stearate 2 mg prepared in the same manner as in Example 4-1 were filled in a gelatin capsule to prepare a capsule.

<Production Example 2> Preparation of food composition

Cannonball tree solvent extract (10% by weight), liquid fructose (0.5% by weight), oligosaccharide (2% by weight), sugar (2% by weight), and salt (0.5% by weight) prepared in the same manner as in Example 4-1. A health drink was prepared by adding water to the balance, mixing it homogeneously, and sterilizing it instantaneously.

Claims (6)

A pharmaceutical composition for the treatment or prevention of prostatic hyperplasia comprising cannonball tree as an active ingredient. The method of claim 1,
The pharmaceutical composition of the cannonball tree is at least one selected from raw cannonball tree, cannonball tree dry matter, or cannonball tree solvent extract. The method of claim 2,
The solvent is at least one selected from water, alcohol, or a mixed solvent thereof. The method of claim 3,
The alcohol is at least one selected from alcohols having 1 to 5 carbon atoms. The method of claim 1,
The treatment or prevention is one selected from inhibition of prostate cell division, inhibition of prostate tissue enlargement, inhibition of 5-alpha reductase activity, inhibition of 5-alpha reductase expression, inhibition of dihydrotestosterone production, and inhibition of androgen receptor expression by the active ingredient. The pharmaceutical composition by the above. A food composition for improving or inhibiting prostatic hyperplasia, comprising cannonball tree as an active ingredient.

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