KR102296821B1 - Extract of 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 composition for preventing, alleviating or treating prostatic hypertrophy, including an extract of Symbiodinium voratum as an active ingredient. The composition can substitute for finasteride or dutasteride, is polar and can be dissolved well in water, can be extracted from a naturally occurring ingredient, and can treat prostatic hypertrophy effectively by inhibiting expression of 5AR2 (5α-reductase type 2), AR (Androgen receptor) and PCNA (proliferating cell nuclear antigen).

Description

An extract of Symbiodinium boratum and a composition for preventing, improving or treating benign prostatic hyperplasia comprising the same as an active ingredient TECHNICAL FIELD

The present invention relates to an extract of Symbiodinium voratum, which is a Benthic Marine Dinoflagellate, and a composition for preventing, improving or treating enlarged prostate comprising the same as an active ingredient.

BPH was defined as a condition in which the prostate enlarged and blocked the urinary tract at the lower part of the bladder, resulting in decreased urine flow. Lower urinary tract symptoms, which collectively refer to symptoms indicating bladder discharge disorders such as urgency (difficulty with urination), delayed urination (delayed discharge of urine), interrupted urination (interruption of the flow of urine), and a phenomenon that requires force during urination. Prostatic hyperplasia is defined as 'appeal of The most major triggers for the development of BPH are age increase and male hormones, and histological enlargement of the prostate starts at the age of 35, and 60% of men in their 60s and 90% of men in their 80s show an enlarged prostate. 50% of the patients complain of dysuria due to enlarged prostate.

Drug treatment for benign prostatic hyperplasia is largely divided into alpha blockers and androgen inhibitors (5-alpha reductase inhibitors). Alpha blockers are drugs that lower the pressure and tension of the prostatic urethra, and commercially available alpha blockers include terazosin, doxazosin, tamsulosin, and alfuzosin. However, alpha blockers have no effect on reducing the enlarged prostate, so they are only a temporary treatment method, and side effects such as dizziness, lethargy, headache, and visual field disturbance 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 commercially available. However, these drugs are non-polar and do not dissolve in water, so the effect of rapid absorption may be slow, and sexual dysfunction is also reported.

Accordingly, there is a demand for the development of a new therapeutic agent for benign prostatic hyperplasia that exhibits polarity, is soluble in water and has no toxicity, and can be extracted from natural materials.

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

An object of the present invention is a prostate comprising a Symbiodinium voratum extract capable of inhibiting AR (Androgen receptor), PCNA (proliferating cell nuclear antigen) and 5AR2 (5α-reductase type 2) activity as an active ingredient To provide a pharmaceutical composition for preventing or treating hypertrophy.

Another object of the present invention is a Symbiodinium voratum extract capable of inhibiting AR (Androgen receptor), PCNA (proliferating cell nuclear antigen) and 5AR2 (5α-reductase type 2) activity as an active ingredient. To provide a food composition for the prevention or improvement of benign prostatic hyperplasia comprising.

According to one aspect of the present invention,

There is provided a pharmaceutical composition for preventing or treating benign prostatic hyperplasia comprising an extract of Symbiodinium voratum as an active ingredient.

The extraction solvent of the Symbiodinium voratum extract may be water, an alcohol having 1 to 4 carbon atoms, or a mixture thereof.

The Symbiodinium voratum extract may have the ability to inhibit the expression of one or more selected from Androgen receptor (AR), proliferating cell nuclear antigen (PCNA), and 5AR2 (5α-reductase type 2).

The expression of at least one selected from the group consisting of androgen receptor (AR), proliferating cell nuclear antigen (PCNA), and 5α-reductase type 2 (5AR2) may be achieved in RWPE-1 cells of the prostate.

According to another aspect of the present invention,

There is provided a food composition for preventing or improving prostatic hyperplasia comprising an extract of Symbiodinium voratum as an active ingredient.

The extraction solvent of the Symbiodinium voratum extract may be water, an alcohol having 1 to 4 carbon atoms, or a mixture thereof.

The Symbiodinium voratum extract may have the ability to inhibit the expression of one or more selected from Androgen receptor (AR), proliferating cell nuclear antigen (PCNA), and 5AR2 (5α-reductase type 2).

The expression of at least one selected from the group consisting of androgen receptor (AR), proliferating cell nuclear antigen (PCNA), and 5α-reductase type 2 (5AR2) may be achieved in RWPE-1 cells of the prostate.

The composition comprising the extract of Symbiodinium voratum of the present invention as an active ingredient inhibits AR (Androgen receptor), PCNA (proliferating cell nuclear antigen) and 5AR2 (5α-reductase type 2) activity, thereby preventing enlarged prostate. It is effective in preventing, improving or treating, and can replace conventional therapeutic agents finasteride or dutasteride with natural ingredients.

1 and 2 are analysis results of 5AR2, PCNA, AR inhibitory activity of the extract of Symbiodinium boratum.
^{3 is a 1} H NMR spectrum of the compound isolated according to Example 1.
^{4 is a 13} C NMR spectrum of the compound isolated according to Example 1.
Figure 5 shows the molecular structure of the compound of Example 1 according to COZY and TOCSY spectrum analysis.
6 shows the relative three-dimensional structure of the boratin compound determined from the pairing constant between hydrogens and the signal of the ROESY spectrum.
7 shows the results of cell viability analysis according to the MTT assay.
8 shows the measurement results of the 5AR2 inhibitory activity of the novel compound boratin.

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 to which the present invention pertains can easily carry out the present invention.

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

The terminology used herein is only used to describe specific embodiments, and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, element, or combination thereof described in the specification exists, but is one or more other features or It should be understood that the existence or addition of numbers, steps, acts, elements, or combinations thereof, is not precluded in advance.

The pharmaceutical composition for preventing or treating benign prostatic hyperplasia of the present invention includes an extract of Symbiodinium voratum as an active ingredient.

It is preferable to extract the Symbiodinium boratum extract by drying and pulverizing the Symbiodinium boratum, and then adding it to a solvent of 1 to 30 times the dry weight, preferably 5 to 15 times the volume of the dry weight.

The solvent is preferably water, an alcohol having 1 to 4 carbon atoms, or a mixture thereof, more preferably hot water extraction or ethanol extraction, and still more preferably ethanol extraction.

After performing the extraction process, it is preferable to filter to remove impurities, and to concentrate the filtrate so that the solid content is 15% or more. More preferably, after concentration, the concentrate is powdered through freeze-drying, hot air drying, or drying by spraying method, but is not limited thereto.

The Symbiodinium voratum extract is characterized in that it has an expression inhibitory ability of one or more selected from Androgen receptor (AR), proliferating cell nuclear antigen (PCNA) and 5AR2 (5α-reductase type 2).

The expression of at least one selected from the group consisting of androgen receptor (AR), proliferating cell nuclear antigen (PCNA), and 5α-reductase type 2 (5AR2) may be achieved in RWPE-1 cells of the prostate.

As used herein, the term 'extract' also includes a fraction obtained by additionally fractionating the extract. That is, the Symbiodinium boratum extract includes not only those obtained by using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. In addition, the fraction obtained by passing the extract or fraction through an ultrafiltration membrane having a constant molecular weight cut-off value, and the fraction obtained through various purification methods additionally performed, such as separation by various chromatography, of the present invention Symbiodinium boratum contained in the extract.

In one embodiment, the Symbiodinium boratum extract may be a fraction obtained by refractionation of an organic solvent extract with a second organic solvent. Here, the organic solvent extract includes, in a broad sense, the oil fraction of the Symbiadinium boratum extract described above, the liquid fraction of the Symbiodinium boratum extract, and the wax fraction of the Symbiadinium boratum extract. of these means the liquid fraction of the Symbidinium boratum extract. Therefore, in one embodiment, the fraction obtained by refractionation of the organic extract with the second organic solvent may refer to a fraction obtained by refractionation of the liquid fraction of the Symbiodinium boratum extract with the second organic solvent. In another embodiment, the Symbiodinium boratum extract may be a fraction obtained by refractionation of a lower alcohol extract with a second organic solvent. In another embodiment, the Symbiodinium boratum extract may be a fraction obtained by refractionation of an ethanol extract with hexane.

As used herein, the term 'extract' includes not only the crude extract obtained by treating Symbiadinium boratum with an extraction solvent, but also the processed product of the Symbiodinium boratum extract. For example, the Symbiodinium boratum extract may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying.

In addition, the Symbiadinium boratum extract of the present invention is broadly meant to include a Symbiadinium boratum processed product, such as Symbiadinium boratum powder, formulated so that it can be administered to the human body. has Although the experiment was conducted with the Symbiadinium boratum extract in the present invention, it will be expected by those skilled in the art that the desired effect can be achieved even in the same form as the Symbiadinium boratum processed product.

Hereinafter, the 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 in that it inhibits the expression of 5AR2 (5α-reductase type 2).

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

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

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

This step is preferably performed in the following order.

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

Next, the eluted solution is fractionated again into 5 fractions using Sephadex column chromatography (Sephadex LH20) to obtain a second eluted solution.

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

On the other hand, in the present specification, the term 'comprising as an active ingredient' means including an amount sufficient to achieve the efficacy or activity of the Symbiodinium boratum extract or boratin. In one embodiment of the present invention, the Symbidinium boratum extract or boratin in the composition of the present invention is, for example, 0.001 mg/kg or more, preferably 0.1 mg/kg or more, more preferably 10 mg/kg or more. or more, more preferably 100 mg/kg or more, still more preferably 250 mg/kg or more, and most preferably 1 g/kg or more. Symbidinium boratum extract or boratine is a natural product, and since there is no side effect to the human body even when administered in excess, the upper quantitative limit of the Symbiadinium boratum extract or boratine contained in the composition of the present invention can be selected and implemented by those skilled in the art within an appropriate range. have.

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

The present invention provides a pharmaceutical composition for the prevention or treatment of benign 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 adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, and a lubricant. agent or flavoring agent, etc. may be used.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For formulation in the form of, for example, tablets or capsules, the active ingredient may be combined with an orally, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or required, suitable binders, lubricants, disintegrants and color-developers may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth 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.

In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, 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 bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

Furthermore, it can be preferably formulated according to each disease or component using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by 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, administration mode, age, weight, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient, and usually skilled A trained physician can readily determine and prescribe an effective dosage for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-10 g/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 performed by a person of ordinary skill in the art to which the present invention pertains. Alternatively, it may be prepared by being introduced into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

The present invention provides a food composition for preventing or improving prostatic hyperplasia comprising an extract of Symbiodinium voratum as an active ingredient.

Since the content of the Symbiadinium boratum extract is the same as the description in the pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia including the Symbiadinium boratum as an active ingredient, the details thereof will be referred to.

The present invention provides a food composition for the prevention or improvement of benign 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, sweets, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gums, ice cream, vitamin complexes, health supplements etc.

The food composition of the present invention may include not only the active ingredients, but also ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatine, 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 prepared as a drink or beverage, citric acid, high fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included in addition to boratin of the present invention.

The present invention provides a health functional food comprising a food composition for preventing or improving prostatic hyperplasia comprising the Symbidinium boratum extract or boratin as an active ingredient. A health functional food is a food prepared by adding Symbidinium boratum extract or boratine to food materials such as beverages, teas, spices, gum, and confectionery, or by encapsulating, powdering, or suspension. It means to bring a specific effect, but unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time 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 ingested on a daily basis. The amount of boratine added in such a health functional food varies depending on the type of health functional food and cannot be uniformly defined. 50% by weight, preferably in the range from 0.1 to 20% by weight. In addition, in the case of a health functional food in the form of pills, granules, tablets or capsules, it is usually added in an amount of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

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

[Example]

Example 1: Symbiodinium boratum (S. voratum) alcohol extract preparation

(1) Cultivation of Symbidinium boratum

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

Bottles containing f/2 medium and S. voratum were refilled with freshly filtered seawater, capped and placed on a shelf at 20°C under illumination of 50 µE/m2/s. 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 a high-density culture of S. voratum (about 5,000 cells ㎖ ^-1 ) was obtained, it was transferred to a new 500 ㎖ PC bottle containing fresh f/2 seawater medium about every 3 weeks. When the concentration of S. voratum increased, the DNA base sequence of the cultured cells was analyzed, and after gene identification, the morphology was analyzed when the culture amount was sufficient.

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

(2) Preparation of Symbidinium boratum alcohol extract

The Cymbidinium boratum cells obtained in (1) were mixed with 30% ethanol aqueous solution at a ratio of 1:10 (w/v) and extracted twice for 24 hours, then the filtered solution was combined and concentrated using a vacuum concentrator. and then freeze-dried to prepare a 30% ethanol extract of Symbiodinium boratum.

Example 2: Preparation of Symbidinium boratum alcohol extract

A Symbidinium boratum extract was prepared in the same manner as in Example 1, except that a 70% ethanol aqueous solution was used instead of a 30% ethanol aqueous solution.

Example 3: Preparation of Symbiodinium boratum hot water extract

에서 24 시간 동안 열수추출한 것을 제외하고는 실시예 1과 동일한 방법으로 심비어디니움 보라텀 추출물을 제조하였다.100% using water instead of 30% ethanol aqueous solution

A Symbidinium boratum extract was prepared in the same manner as in Example 1, except that hot water extraction was performed for 24 hours.

Example 4: Extraction and Isolation of Boratin Compound

In (1) of Example 1 The obtained Cymbidinium boratum cells were extracted with 100% MeOH _{and fractionated with H 2} O and BuOH. The organic layer was refractionated with n-hexane and 85% MeOH aqueous solution. The aqueous layer (2.5 g) was treated with a reverse phase column eluting stepwise from 50% to 100% MeOH in 10% MeOH increments to obtain 6 fractions (I-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 Х 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.

Thereafter, 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: Measurement of AR, PCNA, 5AR2 inhibitory activity of Symbiodinium boratum extract

에서 페니실린 (100 IU/㎖) 및 스트렙토 마이신 (10 mg/㎖)을 함유한 10 % FBS 함유 DMEM에서 유지시켰다.RWPE-1 cells were purchased from the Korean Cell Line Bank. Cells are in a 5% CO ₂ atmosphere and 95% relative humidity, 37

was maintained in DMEM containing 10% FBS containing penicillin (100 IU/ml) and streptomycin (10 mg/ml).

RWPE-1 cells were inoculated in a 6-well plate at 3 x 10 ⁵ cells/well, and then treated with 0.5 μM of testosterone propionate (TP). After 1 hour, the cymbidinium extracts of Examples 1, 2 and 3 were treated at concentrations of 0.1, 1 and 5 μM, respectively, for 24 hours. A 5AR2 inhibitor finasteride (finasteride) and dutasteride (dutasteride) 50 μM treatment group was used as a positive control.

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

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

AR (Androgen receptor), PCNA (proliferating cell nuclear antigen), and 5AR2 (gene SRD5A2) inhibitory activity measurement results according to Western blotting are shown in FIGS. 1 and 2 . According to this, it was observed that the expression of AR, PCNA and 5AR2 was significantly inhibited compared to the TP-treated group 24 hours after administration of the extracts of Examples 1 to 3 to RWPE-1 cells, respectively. The most excellent activity was observed when the 70% ethanol extract of Symbiadinium boratum of Example 2 was treated at a concentration of 5 μM, and the expression of AR, PCNA, and 5AR2 was 51% and 67%, respectively, compared to the cells treated with only TP. , decreased to 55% level.

Experimental Example 2: Identification of new compounds

The molecular weight of the boratin compound isolated according to Example 4 was measured as ^{[M+H] +} = 388.2120 from a high-performance mass spectrometer, and was found to be _{C 22} H ₂₉ NO _{5 .} 3388, 1593 cm ^-1 signals observed from infrared spectroscopy were confirmed to contain a hydroxyl group and a carbonyl group.

In addition, ¹ H and ¹³ C NMR spectrum analysis results for boratin measured with _{methanol-d 4} ^{solvent are shown in Table 1 below, 1} H NMR spectrum analysis results are shown in FIG. 3, and ¹³ C NMR spectrum analysis results is shown in FIG. 4 .

In the hydrogen NMR spectrum, three well-identified resonance lines in the low field region and two resonance lines corresponding to doublet methyl hydrogen lines were observed in the high magnetic field region. In the carbon NMR spectrum, 2 methyl carbons, 7 methine carbons, 7 methylene carbons, and 4 non-hydrogen carbons were observed. Two carbons not observed in the carbon NMR spectrum were observed, one each in the HSQC and HMBC spectra.

δ _C δ _H , multi ( J ) 12
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, br d(12.7); b 2.09, m
2.70, m

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

Experimental Example 3: Analysis of molecular structure

Figure 5 shows the molecular structure of the boratin compound prepared according to Example 4 according to COZY and TOCSY spectrum analysis. According to this, in order to determine the structure through the connection between carbon and hydrogen, the COZY and TOCSY spectra were first analyzed, and as shown in the figure, it was possible to compose structures A to D of the connection parts with thick lines, and the analysis of the HMBC spectrum showed that carbon and Hydrogen correlations are given as arrows. In particular, it was possible to confirm the structure of the indolidium moiety in which the carbon chain between the nitrogen atom in the pyridine ring and carbon 4 of the pyridine was connected. Next, the linkages from partial structures B to D were made through the chital carbon at position 14, the methylene carbon at position 15, and the non-hydrogen carbon at position 16. It was confirmed that the 13th hydrogen in partial structure B is linked by the HMBC signal with the 14th carbon and the 15th hydrogen with the HMBC signal between the 15th and 14th carbons. The HMBC signal demonstrates this connectivity.

The boratine compound prepared according to Example 4 has 9 degrees of unsaturation from the molecular formula, and among them, Compound 1 from the other two except for 7 degrees of unsaturation corresponding to the double bond, indolidium functional group, and carbonyl group as already described. It can be inferred that there are two ring forms. In order to know the ring structure, the deuterium-induced isotope shift phenomenon was _{observed using the CD 3} OD and CD ₃ OH solvents. As a result, the carbon NMR spectrum obtained from the two solvents was as large as 0.12 ppm at the 9th carbon, whereas There was no significant difference in chemical shift values for carbons 10 and 14. From this result, it was confirmed that both carbons 10 and 14 are connected by an ether ring. It can be deduced that the rest of the ring consists of an ether ring with carbons 14 and 18. It was confirmed by the ROESY experiment that it is located close between the 13th and 15th hydrogens and the 10th and 18th hydrogens in the molecular structure of No.1. Therefore, it was confirmed that the boratin compound has a structure having a spirochetal group. Furthermore, this functional group is formed between the 9th carbon and the 8th carbon from the HMBC signal between the 9th hydrogen and the 7th and 8th hydrogens.

6 shows the relative three-dimensional structure of the boratin compound determined from the pairing constant between hydrogens and the signal of the ROESY spectrum. The relative three-dimensional structure of the boratine compound was determined from the pairing constant between hydrogens and the signal of the ROESY spectrum. According to this, the trans orientation was first given at 7.8 Hz between the 9th and 10th hydrogens, and the spiral chital ring was axial- It was in an axial form, and the relative orientation of the indolidium and the spiral chital ring could be determined by the ROESY signal between hydrogen 22 and methyl hydrogen 20. Finally, the 21st methyl group connected to the 12th carbon was able to determine the direction of the 21st methyl group by the ROESY signal with the 10th hydrogen.

From the molecular formula of boratine compound, it can be confirmed that it consists of a carboxylate anion instead of a carboxylic acid, which is supported by the ^{characteristic signal 1593 cm -1 given in the infrared spectrum.}

Experimental Example 4: Cytotoxicity evaluation

The boratin compound prepared according to Example 4 was dissolved in DMSO (final concentration 0.1%) and diluted in a serum-free medium. Prior to analysis, cells were seeded in 96-well plates (LNCap: 2×10 ⁵ cells/ml; PC3 cells: 3 Х10 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 the formazan. After all crystals were completely dissolved, the absorbance at 450 nm was measured in a microplate reader. Data are expressed as percentage of viable cells relative to untreated control cultures. Data are expressed as the mean of three independent experiments.

The results of cell viability analysis according to the MTT assay are shown in FIG. 7 . According to this, it was confirmed that the cell viability was not significantly reduced by the boratin treatment, and there was almost no cytotoxicity.

Experimental Example 5: Measurement of 5AR2 inhibitory activity of boratin compound

에서 페니실린 (100 IU/㎖) 및 스트렙토 마이신 (10 mg/㎖)을 함유한 10 % FBS 함유 DMEM에서 유지시켰다.LNCap and PC3 cells were purchased from the Korean Cell Line Bank. Cells are in a 5% CO ₂ atmosphere and 95% relative humidity, 37

was maintained in DMEM containing 10% FBS containing penicillin (100 IU/ml) and streptomycin (10 mg/ml).

LNCap and PC3 cells were inoculated in a 6-well plate at 3 x 10 ⁵ cells/well, and then treated with 0.5 uM testosterone propionate (TP). After 1 hour, the compound of Example 1 was treated with 1 μM and 10 μM, respectively, for 24 hours. 5-alpha reductase inhibitors Finasteride (Fina) and Dutasteride (Duta) were used as positive controls.

Cells were washed with ice-PBS and extracted using RIPA containing Protease Inhibitor Cocktail (Santa Cruz, CA) for 30 min on ice. The protein lysate was centrifuged at 13,000 x g at 4 °C for 30 min. Dissolved protein (40 μg) was separated by SDS-PAGE (8-12 %) at 100-120 V and transferred to 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 primary antibody. Membranes were washed 3 times with PBST buffer and incubated with secondary antibody for 1 hour at room temperature. Bands were visualized using ECL (Thermo Scientific) and corrected with Chemidoc Imaging System (Bio-Rad, Hercules, CA).

The results of measuring 5AR2 inhibitory activity according to the Western blotting method are shown in FIG. 6 . According to this, the expression of 5AR2 was inhibited by 44% at a concentration of 10 μM in LNCap cells 72 hours after administration of boratin compound compared to cells not administered. It has been shown that the inhibition of boratin is comparable to that of the positive controls finasteride or dutasteride. The expression of 5AR2 in PC3 cells was found to be decreased by 81% at 1 μM and 44% at 10 μM compared to cells not administered with boratin.

In the above, although embodiments of the present invention have been described, those of ordinary skill in the art can add, change, delete or add components within the scope that does not depart from the spirit of the present invention described in the claims. It will be said that various modifications and changes of the present invention can be made by, and this is also included within the scope of the present invention.

Claims (8)

A pharmaceutical composition for preventing or treating benign prostatic hyperplasia, comprising an extract of Symbiodinium voratum as an active ingredient. According to claim 1,
The extraction solvent of the Symbiodinium voratum extract is water, an alcohol having 1 to 4 carbon atoms, or a pharmaceutical composition for preventing or treating benign prostatic hyperplasia, characterized in that it is a mixture thereof. According to claim 1,
The Symbiodinium voratum extract is AR (Androgen receptor), PCNA (proliferating cell nuclear antigen), and 5AR2 (5α-reductase type 2) prevention of prostatic hyperplasia, characterized in that it has the ability to inhibit the expression of one or more selected from or a therapeutic pharmaceutical composition. 4. The method of claim 3,
The AR (androgen receptor), PCNA (proliferating cell nuclear antigen), and 5AR2 (5α-reductase type 2) expression of one or more selected from the prostatic hypertrophy prevention or treatment pharmaceutical composition, characterized in that made in RWPE-1 cells of the prostate . A food composition for preventing or improving prostatic hyperplasia, comprising an extract of Symbiodinium voratum as an active ingredient. According to claim 1,
The extract solvent of the Symbiodinium voratum extract is water, an alcohol having 1 to 4 carbon atoms, or a food composition for preventing or improving prostatic hyperplasia, characterized in that it is a mixture thereof. According to claim 1,
The Symbiodinium voratum extract is AR (Androgen receptor), PCNA (proliferating cell nuclear antigen), and 5AR2 (5α-reductase type 2) prevention of prostatic hyperplasia, characterized in that it has the ability to inhibit the expression of one or more selected from or a food composition for improvement. 8. The method of claim 7,
The AR (androgen receptor), PCNA (proliferating cell nuclear antigen), and 5AR2 (5α-reductase type 2) expression of one or more selected from the prostatic hyperplasia prevention or improvement food composition, characterized in that made in RWPE-1 cells of the prostate .

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