KR102223015B1 - Voratin isolated from Symbiodinium voratum and composition for preventing, improving or treating benign prostatic hyperplasia comprising the same as an effective ingredient - Google Patents

Abstract

The present invention relates to a boratin represented by the following formula (1). Thereby, finasteride or dutasteride can be replaced, it is highly soluble in water with a polarity, and can be extracted from natural ingredients, and 5AR2 (5α-reductase 2) in LNCap cells and PC3 cells of the prostate gland Type) can be effectively treated for prostatic hyperplasia.
[Formula 1]

Description

TECHNICAL FIELD [Voratin isolated from Symbiodinium voratum and composition for preventing, improving or treating benign prostatic hyperplasia comprising the same as an effective ingredient}

The present invention relates to a novel compound, boratin, and a composition for preventing, improving or treating prostatic hyperplasia comprising the same, and more particularly, to a novel compound extracted from the benthic Marine Dinoflagellate, boratin, a method for extracting the same, and It relates to a composition for preventing, improving or treating an enlarged prostate comprising the same as an active ingredient.

Prostatic hyperplasia was defined as a condition in which the flow of urine was reduced by obstructing the urinary tract under the bladder due to an enlarged prostate. , Urgency (difficulty holding up the urine), delayed urine (delayed urine output), interrupted urine (the flow of urine is interrupted), symptoms that indicate bladder discharge disorders, such as urination Prostate enlargement is defined as'appeal of'. The most important triggers of the occurrence of prostatic hyperplasia are increased age and male hormones, and histological enlargement of the prostate starts from 35 years of age, and enlargement of the prostate appears in 60% of men in their 60s and 90% of men in their 80s. 50% of the patients complain of dysuria due to enlarged prostate.

Drug treatments for prostatic hyperplasia are largely classified into alpha blockers and androgen inhibitors (5-alpha-reductase inhibitors). Alpha blockers are drugs that lower pressure and tension in the prostate urethra, and commercially available alpha blockers include terazosin, doxazosin, tamsulosin, and alfuzosin. However, since alpha blockers do not have the effect of reducing prostate enlargement, they are only temporary treatment methods, and side effects such as dizziness, lethargy, headache, and visual field disorder have been reported.

On the other hand, androgen inhibitors are drugs that prevent enlargement of the prostate by inhibiting 5α-reductase, which plays a key role in the process of enlargement of the prostate. As 5-alpha reductase inhibitors, finasteride and dutasteride are on the market. However, these drugs are nonpolar and do not dissolve in water, so the effect of rapid absorption may be slow, and sexual dysfunction has also been reported.

Accordingly, there is a demand for the development of a new prostatic hyperplasia treatment agent that exhibits polarity, is well soluble in water, is not toxic, and can be extracted from natural materials.

Korean Registered Patent Publication No. 10-1714370 Korean Patent Application Publication No. 10-2017-0014472

An object of the present invention is to be able to replace finasteride or dutasteride, and to be polarly soluble in water, extract from natural ingredients, and 5AR2 (5α-) in LNCap cells and PC3 cells of the prostate. reductase type 2), a novel compound that can prevent or delay the conversion of male hormone testosterone in target cells that respond to androgens to dihydrotestosterone (DHT) by 5α-reductase (5AR2). To provide.

Another object of the present invention is to provide a method for extracting boratin, a novel compound useful for the treatment of prostatic hyperplasia, from Symbiodinium voratum, a benthic Marine Dinoflagellate.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating prostatic hyperplasia comprising boratin, a novel compound extracted from Symbiodinium voratum, as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving prostatic hyperplasia comprising boratin, a novel compound extracted from Symbiodinium voratum, as an active ingredient.

According to an aspect of the present invention,

Boratin represented by the following formula (1) is provided.

[Formula 1]

The boratin can inhibit the expression of 5AR2 (5α-reductase type 2).

The 5AR2 may be expressed in at least one selected from LNCap cells and PC3 cells of the prostate.

According to another aspect of the present invention,

(a) preparing an extract by extracting the cultured Symbiodinium voratum with alcohol having 1 to 4 carbon atoms;

(b) fractionating the extract with a solvent in which water and butanol are mixed, and then re-fractionating the organic layer with a solvent in which an aqueous solution of hexane and methanol is mixed; And

(c) separating and obtaining boratin represented by the following formula (1) from the re-fractionated residue by chromatography.

[Formula 1]

Preferably, the alcohol having 1 to 4 carbon atoms in step (a) may be methanol.

Preferably, step (c),

Fractionating the residue with a step gradient solvent system of water and methanol on a reverse phase column, and then obtaining a solution eluted with 60% methanol and 40% water;

Fractionating the eluted solution into 5 fractions using Sephadex column chromatography (Sephadex LH20) to separate and obtain a second eluted solution; And

And purifying the second eluted solution by reverse phase liquid chromatography including an aqueous ethanol solution having a concentration gradient to obtain boratin.

According to another aspect of the present invention,

A composition for inhibiting the expression of 5AR2 (5α-reductase type 2) comprising boratin represented by the following formula (1) as an active ingredient is provided.

[Formula 1]

According to another aspect of the present invention,

There is provided a pharmaceutical composition for preventing or treating prostatic hyperplasia comprising boratin represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

According to another aspect of the present invention,

There is provided a food composition for preventing or improving prostatic hyperplasia comprising boratin represented by the following formula (1) as an active ingredient.

[Formula 1]

Boratin, a novel compound extracted from Symbiodinium voratum of the present invention, can replace finasteride or dutasteride, and is polarly soluble in water, and can be extracted from natural ingredients. The male hormone testosterone in target cells responding to androgen by inhibiting the expression of 5AR2 (5α-reductase type 2) in LNCap cells and PC3 cells of the prostate gland is dihydrotestosterone (DHT) by 5α-reductase (5AR2). ) It has the effect of preventing or delaying the conversion to treat an enlarged prostate.

^{1 is a 1} H NMR spectrum of a compound isolated according to Example 1.
^{2 is a 13} C NMR spectrum of a compound isolated according to Example 1.
3 shows the molecular structure of the compound of Example 1 according to COSY and TOCSY spectrum analysis.
Figure 4 shows the relative three-dimensional structure of the boratin compound determined from the pairing constant between hydrogens and the signal of the ROESY spectrum.
5 shows the results of cell viability analysis according to the MTT assay.
6 shows the results of measuring 5-alpha reductase inhibitory activity.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art may easily implement the present invention.

However, the following description is not intended to limit the present invention to a specific embodiment, and in describing the present invention, when it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted. .

The terms used herein are used only to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present application, terms such as "comprise" or "have" are intended to designate the existence of features, numbers, steps, actions, elements, or combinations thereof described in the specification, but one or more other features or It is to be understood that the possibility of the presence or addition of numbers, steps, actions, elements, or combinations thereof is not preliminarily excluded.

Hereinafter, a novel compound of the present invention, boratin, will be described.

Boratin, a novel compound of the present invention, is represented by the following formula (1).

[Formula 1]

The boratin is characterized by inhibiting the expression of 5AR2 (5α-reductase type 2).

The 5AR2 is characterized in that it is expressed in at least one selected from LNCap cells and PC3 cells of the prostate.

Hereinafter, a method for extracting boratin of the present invention will be described.

First, the cultured Symbiodinium voratum is extracted with alcohol having 1 to 4 carbon atoms to prepare an extract (step a).

The alcohol having 1 to 4 carbon atoms is preferably methanol.

Next, the extract is fractionated with a solvent in which water and butanol are mixed, and the organic layer is re-partitioned with a solvent in which an aqueous solution of hexane and methanol is mixed (step b).

Finally, the boratin represented by Chemical Formula 1 is separated and obtained from the re-fractionated residue by chromatography (step c).

It is preferable to perform this step in the following order.

First, the residue was fractionated on a reverse phase column with a step gradient solvent system of water and methanol, and then a solution eluted with 60% methanol and 40% water was obtained.

Next, the eluted solution was fractionated again into 5 fractions using Sephadex column chromatography (Sephadex LH20), and the second eluted solution was separated and obtained.

Thereafter, the second eluted solution is purified by reverse phase liquid chromatography containing an aqueous ethanol solution having a concentration gradient to obtain boratin.

Meanwhile, in the present specification, the term "included as an active ingredient" means including an amount sufficient to achieve the efficacy or activity of boratin. In one embodiment of the invention, the boratin in the composition of the invention is, for example, 0.001 mg/kg or more, preferably 0.1 mg/kg or more, more preferably 10 mg/kg or more, even more preferably 100 mg/kg or more, even more preferably 250 mg/kg or more, and most preferably 1 g/kg or more. Since boratin is a natural product and does not have side effects on the human body even if it is administered in an excessive amount, the upper limit of the quantity of boratin contained in the composition of the present invention can be selected and carried out by a person skilled in the art within an appropriate range.

The present invention provides a composition for inhibiting the expression of 5AR2 (5α-reductase type 2) comprising boratin represented by Chemical Formula 1 as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating prostatic hyperplasia comprising boratin represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, lubricants. Agents or flavoring agents may be used.

For administration, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients, and may be preferably formulated into a pharmaceutical composition.

The formulation form of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, trackcacanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

As acceptable pharmaceutical carriers in the composition formulated as a liquid solution, as sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injection formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Furthermore, it can be preferably formulated according to each disease or ingredient using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, and is usually skilled. The qualified physician can easily determine and prescribe an effective dosage for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10g/kg.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art Alternatively, it may be manufactured by placing it in a multi-capacity container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

The present invention provides a food composition for preventing or improving prostatic hyperplasia comprising boratin represented by Formula 1 as an active ingredient.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gums, ice creams, vitamin complexes, health supplements. Etc.

The food composition of the present invention may include not only boratin as an active ingredient, but also ingredients commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of drinks and beverages, in addition to the boratin of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included.

The present invention provides a health functional food comprising a food composition for preventing or improving prostatic hyperplasia comprising the boratin as an active ingredient. Health functional foods are foods prepared by adding boratin to food materials such as beverages, teas, spices, chewing gums, confectionery, etc., or by encapsulation, powdering, or suspension. However, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of boratin added in such a health functional food varies depending on the type of the target health functional food and cannot be uniformly defined, but it can be added within a range that does not impair the original taste of the food. It is in the range of 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, usually 0.1 to 100% by weight, preferably 0.5 to 80% by weight, may be added. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

Hereinafter, an embodiment according to the present invention will be described in detail.

[Example]

Example One

(One) Symbiodinium voratum Cultivation of

For the isolation and cultivation of Symbiodinium voratum, stony coral A. japonica was collected from Jeju Island, Korea in 2012. Samples were placed in plastic bags, stored in an ice box, and then transferred to the laboratory. Clonal culture of S. voratum (strain number HACCP DF-078) was established by separation of two consecutive single cells.

The bottle containing f/2 medium and S. voratum was refilled with freshly filtered seawater, covered with a lid, and placed on a shelf at 20°C under 50 μE/m²/s illumination. As the concentration of S. voratum increased, S. voratum was then transferred to 50, 125 and 500 ml polycarbonate (PC) bottles containing fresh f/2 seawater medium. When S. voratum-dense cultures (about 5,000 cells ㎖ ^-1 ) were obtained, they were transferred to a new 500 ㎖ PC bottle containing fresh f/2 seawater medium every 3 weeks. When the concentration of S. voratum was increased, the DNA nucleotide sequence of the cultured cells was analyzed, and after gene recognition, the morphology was analyzed when the amount of culture was sufficient.

The small subunit rDNA sequence of this species was identical to that of S. voratum. Next, the culture was transferred to a 2 L polycarbonate bottle and cultured, and then transferred back to a 20 L polycarbonate bottle. Cells were cultured in the quiescent phase of the growth curve after 3 weeks. The same process was repeated to obtain a volume of 300 L. This amount was again transferred to a 1 ton-capacity tubular incubator for large-scale cultivation. Cultivation was performed for 3 weeks in 22 ~ 25 ℃, salinity of about 30 psu, light intensity 50 ~ 100 μ E m ^-2 sec ^-1 , f/2-Si medium. Thereafter, S. voratum cells were obtained by continuous centrifugation (8,000 rpm, 6 hours) when the cell concentration reached about 300,000-350,000 cells ㎖ ^-1. The collected cells were stored in an ultra-low temperature freezer (-75° C.) and then moisture was removed with a freeze dryer (-85° C., 10 mTorr, 48 h).

(2) Boratin Extraction and separation of compounds

The obtained cells were extracted with 100% MeOH _{and fractionated with H 2} O and BuOH. The organic layer was re-partitioned with n-hexane and 85% MeOH aqueous solution. The aqueous layer (2.5 g) was treated with a reversed-phase column eluting stepwise from 50% to 100% MeOH in 10% MeOH increase to obtain 6 fractions (I to VI).

Among them, fraction II (60% MeOH: 40% H ₂ O) was divided into 5 fractions using a Sephadex LH20 column. The active fraction (II-2, 100 mg) was separated by reverse phase HPLC (column: Phenomenex C8, 250×10 mm, _{quantitative elution with a solvent from 45% MeOH + 55% H 2} O, flow rate: 1.0 ml/min). Most amino acids and voratin-containing fractions (5.3 mg) were obtained.

Then, the mixture containing boratin was purified by reverse-phase HPLC (column: Phenomenex Polar 250 × 4.6 mm; gradient elution from 50% MeOH to 75% for 20 minutes, flow rate: 0.6 ml/min) to obtain boratin. (4.1 mg).

[Experimental Example]

Experimental example 1: Identification of new compounds

The molecular weight of the compound of Example 1 was determined as ^{[M+H] +} = 388.2120 from a high-performance mass spectrometer and found to be _{C 22} H ₂₉ NO _5. ^{It was confirmed that the 3388 and 1593cm -1} signals observed from the infrared spectroscopy contained a hydroxy group and a carbonyl group.

In addition, ^{the 1} H and ¹³ C NMR spectrum analysis results of the compound of Example 1 measured with _{methanol-d 4} solvent are shown in Table 1 below, and the ¹ H NMR spectrum analysis results are shown in FIGS. 1 and ¹³ C NMR spectrum analysis. The results are shown in FIG. 2.

In the hydrogen NMR spectrum, three resonance lines, which are well-identified in the low-field region, and two resonance lines, which correspond to the doublet methyl hydrogen wire in the high magnetic field, were observed. In addition, in the carbon NMR spectrum, 2 methyl carbons, 7 methine carbons, 7 methylene carbons, and 4 carbons without hydrogen were observed. Two carbons that were not observed in the carbon NMR spectrum were observed, one each in the HSQC and HMBC spectra.

d _C d _H , mult( J ) One
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22 57.2, t
31.1, t
40.4, d
162.7, s
123.2, d
147.1, d
125.3, d
159.5, s
73.3, d
72.3, d
35.6, t
26.2, d
40.9, t
100.2, s
44.9, t
143.7, s
37.6, t
73.5, d ^*
178.1, s ^*
17.7, q
21.1, q
110.4, t a 4.71, dt(12.2, 9.5); b 4.99, ddd(12.2, 9.5, 2.5)
a 2.10, m; b 2.71, m
3.74, hex(7.8)

7.91, d(7.8)
8.48, t(7.8)
8.05, d(7.8)

4.86, d(9.1)
3.87, ddd(11.3, 9.1, 2.9)
a 1.76, ddd(13.2, 11.3, 5.9); b 1.93, dt(13.2, 2.9)
2.08, m
1.69, d(5.1)

a 2.13, d(13.7); b 2.19, d(13.7)

a 1.97, broad d (12.7); b 2.09, m
2.70, m

1.56, d(6.9)
1.26, d(7.3)
a 4.56, broad singlet; b 4.58, br s

Experimental example 2: Analysis of molecular structure

3 shows the molecular structure of the compound of Example 1 according to COSY and TOCSY spectrum analysis. According to this, in order to determine the structure through the connection between carbon and hydrogen, the COSY and TOCSY spectra were first analyzed, and as shown, the connection partial structures A to D with a given thick line could be constructed. The correlation of hydrogen is given as an arrow. In particular, it was possible to confirm the structure of the indolizium portion in which the carbon chain between the nitrogen atom in the pyridine ring and carbon 4 of the pyridine is connected. Next, the connection from partial structures B to D was made through chital carbon No. 14, methylene carbon No. 15, and non-hydrogen carbon No. 16. It was confirmed that hydrogen 13 in partial structure B was connected with the HMBC signal with carbon 14 and the HMBC signal with hydrogen 15 and carbon 14, and also the exomethylene hydrogen of 22 with carbon 15 and carbon 17. The HMBC signal demonstrates this connectivity.

The novel compound prepared according to Example 1 is given 9 degrees of unsaturation from the molecular formula, and among them, except for the previously described 7 degrees of unsaturation corresponding to the double bond, the indolizium functional group, and the carbonyl group, compound 1 from the other two It can be guessed that there are two ring types. In order to know the ring structure, deuterium-induced isotope shift was observed using CD ₃ OD and CD ₃ OH solvents. As a result, carbon NMR spectra obtained in both solvents appeared as large as 0.12 ppm in carbon 9, whereas For carbons 10 and 14, there was no significant difference in chemical shift values. From this result, it was confirmed that the two carbons 10 and 14 were connected by an ether ring. It can be assumed that the rest of the ring is composed of an ether ring with carbon 14 and carbon 18. It was confirmed by ROESY experiment that this is located close between hydrogen 13 and hydrogen 15, and hydrogen 10 and 18 in the molecular structure of #1. Therefore, it was confirmed that the boratin compound is a structure having a spiroketal group. Furthermore, the linkage of this functional group to the previously identified indolidium is made between carbon 9 and carbon 8 from the HMBC signal between hydrogen 9 and hydrogen 7 and 8.

The relative conformational structure of the boratin compound was determined from the pairing constant between hydrogens and the signal from the ROESY spectrum. As shown in FIG. 4, first, the trans orientation was given as 7.8 Hz between hydrogen 9 and hydrogen 10, and the spiral chital ring was axial in ROESY signals with hydrogen 13 and hydrogen 15, and hydrogen 10 and 18. -It had an axial shape, and the relative orientation of the indolizium and the spiral chital ring could be determined by the ROESY signal of hydrogen 22 and methyl hydrogen 20. Finally, the 21st methyl group connected to the 12th carbon could determine the direction of the 21st methyl group by the ROESY signal with the 10th hydrogen.

It can be seen from the molecular formula of the boratin compound that it is composed of a carboxylate anion instead of a carboxylic acid, which is supported by a ^{characteristic signal of 1593 cm -1 given in the infrared spectrum.}

Experimental example 3: Cytotoxicity assessment

The compound prepared according to Example 1 was dissolved in DMSO (final concentration 0.1%) and diluted in serum-free medium. Before analysis, cells were inoculated into 96-well plates (LNCap: 2×10 ⁵ cells/ml; PC3 cells: 3×10 ⁵ cells/ml 100 μl per well) and incubated for 24 hours. Thereafter, the cells were treated with boratin at concentrations of 1 and 10 μM for 24, 48, and 72 hours. The inhibitory activity of each compound on cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were incubated with 2 mg/ml MTT for 2 hours. Then, the supernatant was aspirated and 200 µl of DMSO was added to dissolve formazan. After all crystals were completely dissolved, the absorbance at 450 nm was measured in a micro plate reader. Data are expressed as the percentage of viable cells relative to the untreated control culture. Data are expressed as the average of three independent experiments.

The results of cell viability analysis according to the MTT assay are shown in FIG. 5. According to this, it was confirmed that the cell viability was not significantly reduced by the treatment with boratin, indicating that there was little cytotoxicity.

Experimental example 4: 5- alpha reductase Inhibitory activity measurement

LNCap and PC3 cells were purchased from a Korean cell line bank. Cells were maintained in DMEM containing 10% FBS containing penicillin (100 IU/ml) and streptomycin (10 mg/ml) at 5% CO _{2 atmosphere and 95% relative humidity, 37°C.}

LNCap and PC3 cells ^{were inoculated into 6-well plates with 3×10 5} cells, and then the compounds of Example 1 were treated with 1 μM and 10 μM, respectively, for 72 hours. 5-alpha reductase inhibitors Finasteride (Fina) and Dutasteride (Duta) were used as positive controls.

Cells were washed with ice-PBS and RIPA containing Protease Inhibitor Cocktail (Santa Cruz, CA) was extracted for 30 minutes on ice. The protein lysate was centrifuged at 13,000 x g for 30 minutes at 4°C. Dissolved protein (40 μg) was separated by SDS-PAGE (8-12%) at 100-120V and transferred to a PVDF membrane.

The membrane was blocked with 5% fat-free milk in PBST buffer for 1 hour at room temperature, and the membrane was incubated overnight at 4°C with the primary antibody. The membrane was washed 3 times with PBST buffer and incubated with the secondary antibody for 1 hour at room temperature. Bands were visualized using ECL (Thermo Scientific) and calibrated with the Chemidoc Imaging System (Bio-Rad, Hercules, CA).

Figure 6 shows the results of measuring 5-alpha reductase inhibitory activity according to Western blotting. According to this, the expression of 5AR2 in LNCap cells 72 hours after administration of the boratin compound was suppressed by 44% at a concentration of 10 μM compared to cells not administered. It has been shown that the inhibition of boratin is comparable to the positive control finasteride or dutasteride. In PC3 cells, the expression of 5AR2 was decreased by 81% at 1 μM and 44% at 10 μM compared to cells without the administration of boratin.

In the above, embodiments of the present invention have been described, but those of ordinary skill in the relevant technical field add, change, delete or add components within the scope not departing from the spirit of the present invention described in the claims. It will be possible to variously modify and change the present invention by the like, and it will be said that this is also included within the scope of the present invention.

Claims (9)

Boratin represented by the following formula (1).
[Formula 1]

The method of claim 1,
The boratin is boratin, characterized in that it inhibits the expression of 5AR2 (5α-reductase type 2). The method of claim 2,
The 5AR2 is boratin, characterized in that expressed in at least one selected from LNCap cells and PC3 cells of the prostate. (a) preparing an extract by extracting the cultured Symbiodinium voratum with alcohol having 1 to 4 carbon atoms;
(b) fractionating the extract with a solvent in which water and butanol are mixed, and then re-fractionating the organic layer with a solvent in which an aqueous solution of hexane and methanol is mixed; And
(c) separating and obtaining boratin represented by the following formula (1) from the re-fractionated residue by chromatography.
[Formula 1]

The method of claim 4,
The method of extracting boratin, characterized in that the alcohol having 1 to 4 carbon atoms in step (a) is methanol. The method of claim 4,
Step (c),
Fractionating the residue with a step gradient solvent system of water and methanol on a reverse phase column, and then obtaining a solution eluted with 60% methanol and 40% water;
Fractionating the eluted solution into 5 fractions using Sephadex column chromatography (Sephadex LH20) to separate and obtain a second eluted solution; And
Purifying the second eluted solution by reverse phase liquid chromatography containing an aqueous ethanol solution having a concentration gradient formed therein to obtain boratin. delete A pharmaceutical composition for preventing or treating prostatic hyperplasia comprising boratin represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

A food composition for preventing or improving prostatic hyperplasia comprising boratin represented by the following formula (1) as an active ingredient.
[Formula 1]

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