KR20230087142A - Composition for preventing, improving or treating benign prostatic hyperplasia and alopecia comprising Voratin isolated from Symbiodinium voratum and as an effective ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating benign prostatic hyperplasia or alopecia, comprising boratin, a compound represented by Formula 1 below, or a pharmaceutically acceptable salt thereof as an active ingredient. As a result, it can replace finasteride or dutasteride, is polar, soluble in water, can be extracted from natural ingredients, 5AR2 (5α-reductase type 2), AR (Androgen receptor) And by inhibiting the expression of proliferating cell nuclear antigen (PCNA), benign prostatic hyperplasia or alopecia can be effectively treated.
[Formula 1]

Description

Composition for preventing, improving or treating benign prostatic hyperplasia and alopecia comprising Voratin isolated from Symbiodinium voratum and as an effective ingredient }

The present invention relates to a composition for preventing, improving or treating prostatic hyperplasia and alopecia, comprising boratin, a compound isolated from an extract of Symbiodinium voratum, a benthic marine dinoflagellate, as an active ingredient. .

Prostatic hyperplasia was defined as a condition in which the flow of urine was reduced due to the enlargement of the prostate gland, blocking the urinary tract at the lower part of the bladder. Lower urinary tract symptoms, which collectively refer to symptoms indicating bladder emptying disorders, such as urgency (difficulty in urinating), urinary urgency (delayed urine output), interrupted urine flow (urine flow is interrupted), and the need to strain while urinating. Prostate hyperplasia is defined as 'complaint of'.

The most important triggers for the occurrence of BPH are age increase and male hormones. Histological enlargement of the prostate begins at the age of 35, and enlargement of the prostate occurs in 50% of men in their 60s and 90% of men in their 80s. 50% of patients complain of dysuria due to prostate enlargement.

Drug treatment for BPH is largely classified into alpha blockers and androgen inhibitors (5-alpha reductase inhibitors). Alpha-blockers are drugs that lower pressure and tension in the prostate and urethra. 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 a temporary treatment method, and side effects such as dizziness, lethargy, headache, and visual disturbance have been reported.

In addition, 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. Finasteride and dutasteride are commercially available as 5-alpha reductase inhibitors. However, these drugs are non-polar and insoluble in water, so the effect of rapid absorption may be slow, and sexual dysfunction is also reported.

As interest in male appearance increases, interest in the treatment of alopecia is also increasing. Recently, many studies for the treatment of hair loss have been conducted, but it is still not known exactly what causes hair loss.

Currently, studies on many regulatory factors involved in hair loss and hair growth mechanisms are being actively conducted. In particular, it has been found that many kinds of cytokines and growth factors are involved in the morphogenesis and growth of hair follicles. For example, tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interferon-γ (Interferon-γ) γ, IFN-γ), Fibroblast Growth Factor-5 (FGF5), Hepatocyte Growth Factor (HGF), Vascular Epithelial Growth Factor (VEGF), Insulin-like Growth Factor- These include Insulin-like Growth Factor-I (IGF-I), Insulin-like Growth Factor-II (IGF-II), and Epithelial Growth Factor Receptor (EGFR). . TNF-α increases cell death in stromal cells and inhibits hair shaft elongation, and IL-1α and IL-1β also inhibit hair follicle growth. In addition, overexpression of IFN-γ causes hair loss, and FGF5 induces a catagen phase in rat hair follicles, while HGF promotes hair follicle growth. In addition, IGF-I and IGF-II promote the growth of hair follicles in a concentration-dependent manner when culturing hair follicles without insulin, and in the case of transgenic mice in which the EGF-receptor is removed, cell necrosis It has been reported that hair follicles disappear.

On the other hand, androgenetic alopecia (AGA), which is the most typical form of hair loss, is known to occur dependently on androgen hormones. Representative androgen hormones in mammals include testosterone and dihydrotestosterone, and dihydrotestosterone has the property of binding more strongly to the androgen receptor (AR) than testosterone. Studies have reported that hydrotestosterone appears higher. Dihydrotestosterone binds to androgen receptors and delays protein synthesis in hair follicle cells, thereby shortening the growth period of hair follicles and causing hair loss by atrophying hair follicles.

Testosterone is converted to dihydrotestosterone by 5α-reductase. 5α-reductase includes type 1 5α-reductase and type 2 5α-reductase. Type 1 5α-reductase is prominently expressed in sebaceous glands but not in hair follicles, and type 2 5α-reductase is found in beard and forehead hair and the outer root sheath of hair follicles, but not sebaceous glands and dermal papillae. Of the two forms, dihydrotestosterone, which is converted from testosterone by type 2 5α-reductase, results in gradual miniaturization of dermal papillae and hair follicles. It has also been reported that when dermal papilla cells are cultured by treatment with dihydrotestosterone, the amount of transforming growth factor-beta 2 (TGF-β2), which induces a catagen and inhibits hair follicle growth, increases. Finasteride, a type 2 5α-reductase activity inhibitor, can prevent hair loss by reducing dihydrotestosterone (DHT) in hair loss patients.

Finasteride and dutasteride, which are 5α reductase inhibitors, are known to be effective in treating hair loss by inhibiting the conversion of testosterone into dihydrotestosterone (DHT), an active metabolite, by 5α reductase, but these drugs lead to erectile dysfunction and decrease in libido. And it is known to cause side effects such as a decrease in semen volume. As such, although the currently used drug treatment is somewhat effective, there is a problem in that such side effects occur.

Accordingly, there is a demand for the development of new therapeutic agents for prostatic hyperplasia and alopecia that can be extracted from natural materials that are non-toxic and minimize side effects to the human body.

Korean Registered Patent Publication No. 10-1714370 Korean Registered Patent Publication No. 10-2114133

An object of the present invention is to obtain boratin, a compound isolated from Symbiodinium voratum extract, capable of inhibiting AR (Androgen receptor), PCNA (proliferating cell nuclear antigen) and 5AR2 (5α-reductase type 2) activities. It is to provide a pharmaceutical composition for preventing or treating prostatic hyperplasia or alopecia comprising as an active ingredient.

Another object of the present invention is a compound isolated from Symbiodinium voratum extract capable of inhibiting AR (Androgen receptor), PCNA (proliferating cell nuclear antigen) and 5AR2 (5α-reductase type 2) activity. It is to provide a food composition for preventing or improving prostatic hyperplasia or alopecia comprising phosphorus boratine as an active ingredient.

According to one aspect of the present invention,

A pharmaceutical composition for preventing or treating benign prostatic hyperplasia or alopecia comprising boratin or a pharmaceutically acceptable salt thereof represented by Formula 1 below as an active ingredient is provided.

[Formula 1]

The pharmaceutical composition for preventing or treating alopecia may be for inhibiting the expression of one or more selected from AR (Androgen receptor), 5AR2 (5α-reductase type 2) and PCNA (proliferating cell nuclear antigen).

Suppression of the expression of one or more types selected from among the Androgen receptor (AR) and 5α-reductase type 2 (5AR2) can be achieved in LNCap cells of the prostate.

Suppression of the expression of one or more selected from 5AR2 (5α-reductase type 2) and PCNA (proliferating cell nuclear antigen) can be achieved in LNCap cells of the prostate.

According to another aspect of the present invention,

Provided is a food composition for preventing or improving prostatic hyperplasia or alopecia, comprising boratin, a compound represented by Formula 1 below, and a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

According to another aspect of the present invention,

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

(b) obtaining a re-extract by re-extracting the extract with a butanol solvent; and

(c) isolating and obtaining boratin represented by the following formula (1) from the re-extract by chromatography; preventing or treating prostatic hyperplasia or alopecia comprising boratin as an active ingredient; A method for preparing a pharmaceutical composition for use is provided.

[Formula 1]

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

Step (c) is,

(c-1) fractionating the re-extract with a step gradient solvent system of water and methanol on a reverse phase column to obtain a solution eluted with 50% methanol and 50% water;

(c-2) fractionating the eluted solution into 4 fractions again using Sephadex column chromatography (Sephadex LH20) to separate the first eluted solution; and

(c-3) purifying the first eluted solution by reverse-phase liquid chromatography containing an aqueous solution of acetonitrile (ACN) having a concentration gradient to obtain boratin.

In step (c-2), the first eluted solution is obtained by separating the four fractions having a retention time (Rt) of 15 to 17 minutes using reverse phase liquid chromatography using a phenomenex polar column. can be obtained.

According to another aspect of the present invention,

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

(b) obtaining a re-extract by re-extracting the extract with a butanol solvent; and

(c) isolating and obtaining boratin represented by the following formula (1) from the re-extract using chromatography; to prevent or improve prostatic hyperplasia or alopecia comprising boratin as an active ingredient; A method for preparing a food composition for use is provided.

[Formula 1]

The composition containing boratin, which is a compound isolated from the Symbiodinium voratum extract of the present invention as an active ingredient, has AR (Androgen receptor), PCNA (proliferating cell nuclear antigen) and 5AR2 (5α-reductase type 2) activities It is effective in preventing, improving or treating prostatic hyperplasia by inhibiting, and can replace finasteride or dutasteride, which are conventional treatments, with natural ingredients.

1 is an HRESIMS analysis result of Experimental Example 1.
2 is a 1H NMR analysis result of Experimental Example 1.
3 is a 13C NMR analysis result of Experimental Example 1.
4 is a result of COZY NMR analysis of Experimental Example 1.
5 is a result of HSQC NMR analysis of Experimental Example 1.
6 is an HMBC NMR analysis result.
7 is a result of ROESY NMR analysis.
8 is a cytotoxicity analysis result according to Experimental Example 2.
9 is a result of measuring androgen receptor (AR) expression level (a) and 5AR2 expression level (b) upon boratine treatment in LNCap cells of Experimental Example 3.
10 is a result of measuring the 5AR2 expression level (a) and the PCNA expression level (b) upon boratine treatment in RWPE-1 cells of Experimental Example 3.

In the following, various aspects and various embodiments of the present invention are described in more detail.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention.

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

Terms used herein are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, the terms "comprise" or "having" are intended to indicate that there is a feature, number, step, operation, component, or combination thereof described in the specification, but one or more other features or It should be understood that the presence or addition of numbers, steps, operations, elements, or combinations thereof is not precluded.

Hereinafter, a pharmaceutical composition for preventing or treating prostatic hyperplasia or alopecia comprising boratin or a pharmaceutically acceptable salt thereof, which is a novel compound of the present invention, as an active ingredient will be described.

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

[Formula 1]

The pharmaceutical composition for preventing or treating prostatic hyperplasia and alopecia is characterized in that it is for inhibiting the expression of at least one selected from AR (Androgen receptor), 5AR2 (5α-reductase type 2) and PCNA (proliferating cell nuclear antigen).

Suppression of the expression of one or more types selected from among the Androgen receptor (AR) and 5α-reductase type 2 (5AR2) can be achieved in LNCap cells of the prostate.

Suppression of the expression of one or more selected from 5AR2 (5α-reductase type 2) and PCNA (proliferating cell nuclear antigen) can be achieved in LNCap cells of the prostate.

Boratin, a novel compound of the present invention, may be used for preventing or treating androgenetic alopecia, which is caused by a mechanism similar to prostatic hyperplasia.

Hereinafter, a method for preparing a pharmaceutical composition for preventing or treating benign prostatic hyperplasia or alopecia comprising the compound boratine represented by Formula 1 of the present invention as an active ingredient will be described.

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

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

Next, the extract is re-extracted with a butanol solvent to obtain a re-extract (step b).

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

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

First, the re-extract was fractionated with a step gradient solvent system of water and methanol on a reverse phase column, and then a solution eluted with 50% methanol and 50% water was obtained (c-1).

Thereafter, the eluted solution is re-fractionated into 4 fractions using Sephadex column chromatography (Sephadex LH20), and the first eluted solution is separated (c-2).

The first eluted solution can be obtained by separating the four fractions having a retention time (Rt) of 15 to 17 minutes using reverse phase liquid chromatography using a phenomenex polar column.

Next, boratin is obtained by purifying the first eluted solution by reverse phase liquid chromatography containing an aqueous solution of acetonitrile (ACN) in which a concentration gradient is formed (c-3).

Meanwhile, in the present specification, the term 'comprising as an active ingredient' means containing an amount sufficient to achieve the efficacy or activity of boratine. In one embodiment of the present invention, 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, 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 boratine is a natural product and does not have side effects on the human body even when administered in excess, the upper limit of the amount of boratin included in the composition of the present invention can be selected and implemented by those skilled in the art within an appropriate range.

The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the above active ingredients, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, and lubricants. agents or flavoring agents 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 above-described active ingredients 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 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 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 compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include 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 if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can 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 method, patient's age, body weight, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity, and is usually skilled in the art. A trained physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. 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 formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art, or Or it can be prepared by incorporating into a multi-dose 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 contain a dispersing agent or stabilizer.

The present invention provides a food composition for preventing or improving prostatic hyperplasia or alopecia, comprising boratin, a compound represented by Formula 1, and a pharmaceutically acceptable salt thereof as an active ingredient.

The content of boratin, which is the compound represented by Formula 1, is the same as the description of the pharmaceutical composition for preventing or treating prostatic hyperplasia or alopecia comprising Cymbiadinium boratum as an active ingredient, so for details, refer to that part. do.

In addition, the present invention provides a method for preparing a food composition for preventing or improving prostatic hyperplasia or alopecia comprising boratin represented by Formula 1 as an active ingredient.

A method for producing a food composition for preventing or improving prostatic hyperplasia or alopecia containing boratin represented by Formula 1 as an active ingredient is for preventing or treating prostatic hyperplasia or alopecia containing boratin represented by Formula 1 as an active ingredient. Since it is the same as the preparation method of the pharmaceutical composition, reference will be made to that part for details.

The food composition according to the present invention can be formulated in the same way 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, chewing gum, ice cream, vitamin complexes, and health supplements. etc.

The food composition of the present invention may include not only the above active ingredients, but also ingredients commonly added during food preparation, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings 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 such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, 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 corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included in addition to boratin according to the present invention.

The present invention provides a health functional food comprising a food composition for preventing or improving prostatic hyperplasia or alopecia comprising boratine as an active ingredient. Functional health food refers to food produced by adding boratin to food materials such as beverages, teas, spices, chewing gum, confectionery, etc., or by encapsulation, powdering, suspension, etc. 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 health functional foods cannot be uniformly defined depending on the type of target health functional food, but it can be added within a range that does not impair the original taste of the food, and is usually from 0.01 to 0.01 50% by weight, preferably in the range of 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range 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 pills, tablets, capsules or beverages.

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

[Example]

Example 1: Isolation of boratin from S. voratum extract

(1) Cultivation of Cymbiadinium boratem

For the isolation and cultivation of S. voratum, stony coral (Alveopora japonica) was collected from the waters of Jeju Island, Korea. Samples were placed in plastic bags, stored in an ice box, and transported to the laboratory. A clonal culture of S. voratum (strain number HACCP DF-078) was established by two consecutive 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 an illumination of 50 µE/m2/s. As the concentration of S. voratum increased, the 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 every 3 weeks. When the concentration of S. voratum increased, the DNA sequence of the cultured cells was analyzed, and after genetic identification, the morphology was analyzed when the culture amount was sufficient.

The rDNA sequence of the small subunit of this species was identical to that of S. voratum. Next, the culture was transferred to a 2 L polycarbonate bottle and incubated, then transferred back to a 20 L polycarbonate bottle. Cells were cultured at the stationary 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 tubular incubator with a capacity of 1 ton for large-scale cultivation. Cultivation was performed for 3 weeks at 22-25 °C, salinity of about 30 psu, light intensity of 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 ml ^-1 . The collected cells were stored in a cryogenic freezer (-75°C) and then dehydrated with a freeze dryer (-85°C, 10 mTorr, 48 h).

(2) Extraction and separation of boratine

in (1) The obtained Cymbiadinium boratum cells were extracted with MeOH, suspended with H ₂ O, and then extracted with hexane, chloroform, ethyl acetate and butanol solvents. The butanol extract was treated with a reverse phase column eluting stepwise from 50% to 100% MeOH in increments of 10% MeOH to give 6 fractions (MR1-MR6).

Of these, the MR1 (50% aqueous MeOH solution) fraction was chromatographed using a Sephadex LH20 column using a methanol solvent to obtain four fractions (M1 to M4).

Boratin (Rt = 16 min, 2.5 mg) was determined by reverse-phase HPLC (40% MeOH + 60% H ₂ O elution solvent (containing 2 mM ammonium formate and 50 mM formic acid)) with a phenomenex polar column (250 Х 10 mm, 5 μm). It was separated from MR1-M1 using the RI detector.

Next, boratine containing impurities was analyzed by reverse-phase HPLC (column: phenomenex C8, 250 x 4.6 mm; gradient elution at 10% ACN and 90% H ₂ O for 10 min followed by 30% ACN for 10 min; flow rate was 1.0 ml /min; 254 nm UV detector) to obtain 4.1 mg of boratin as a colorless powder.

IR λ _max 3388, 2923, 1598, and 1409 cm ^-1 ; UV (MeOH) λ _max 271 (ε 2425) nm;

[Experimental example]

Experimental Example 1: Confirmation of Boratin Separation and Structural Analysis

The results of analysis by HRESIMS (m/z 362.1963 [M + H] ⁺ ) for boratum, a compound isolated according to Example 1, are shown in FIG. It was confirmed that the chemical formula was C ₂₀ H ₂₇ NO ₅ .

Meanwhile, ¹ H NMR and ¹³ C NMR analysis results are shown in Table 1 below, and ¹ H NMR analysis graphs are shown in FIG. 2 and ¹³ C NMR analysis graphs are shown in FIG. 3, respectively.

(CD ₃ OD at 500MHz) _δC δ _H (J in Hz) 57.3 CH ₂ ^a 5.05, ddd (12.7, 9.1, 3.7) ^b 4.70, ddd (12.7, 8.1, 8.8) 31.4 CH ₂ ^a 2.74, dtd (12.9, 8.1, 3.7) ^b 2.02, dq (12.9, 8.8) 40.4, CH 3.78, tq (8.8, 7.1) 162.8, C 123.3, CH 7.95 d (8.1) 147.0, CH 8.52, t(8.1) 124.8, CH 8.03, d (8.1) 159.8, C. 73.0, CH 4.90, d (8.8) 76.5, CH 3.82, ddd (11.0, 8.8, 2.0) 37.8 CH ₂ ^a 2.16, br d (12.5) ^b 1.07, ddd (12.5, 12.5, 12.5) 27.4, CH 2.09 m 41.8 CH ₂ ^a 1.90, dd (12.7, 2.7) ^b 1.31, t (12.7) 108.8, C. 37.5 CH ₂ ^a 1.68 m ^b 1.66 m - 29.6 CH ₂ ^a 1.70 m ^b 1.57 m 79.1 CH ₂ 3.48, d d (9.1, 5.1) 178.3, C 18.2, CH ₃ 1.54, d (7.1) 22.5 CH ₃ 0.98, d (6.4) ^a downfield chemical shift; ^b upfield chemical shifts

In addition, the COZY NMR spectrum (at 500 MHz) for confirming the specific chemical structure of the bora term of Example 1 is shown in FIG. 4, and the HSQC NMR spectrum (at 500 MHz [Black: CH ₂ , Blue: CH + CH ₃ ]) is shown in FIG. 5, the HMBC NMR spectrum (at 500 MHz) is shown in FIG. 6, and the ROESY NMR spectrum (at 500 MHz) is shown in FIG. Boraterm was confirmed to be a compound represented by Chemical Formula 1.

[Formula 1]

Experimental Example 2: Cytotoxicity assay

RWPE-1 (normal human prostate epithelial cell line) and LNCap (human prostate adenocarcinoma cell line) were purchased from the American Type Culture Collection (Manassas, VA, United States). RWPE-1 cells were grown in keratinocyte serum-free medium (K-SFM; Gibco BRL, Grand Island, NY, USA) with 0.05 mg/ml bovine pituitary extract and 5 ng/ml epidermal growth factor and 1% (v/v) antibiotics. (100 U/mL penicillin and 100 μg/mL streptomycin) were supplemented and cultured. LNCap cells were cultured in RPMI1640 (Sigma-Aldrich Inc. P4333) containing 10% (v/v) FBS, 1% (v/v) antibiotics (100 U mL-1 penicillin and 100 μg mL-1 streptomycin). GenDEPOT, CM059-050).

The effect of boratine treatment in Example 1 on prostatic hyperplasia (BPH) was evaluated using RWPE-1 and LNCap human prostate cells cultured according to the above method treated with tettosterone propionate (TP). Before evaluating the antiBPH activity, the cytotoxicity of boratin 1-3 was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Specifically, cytotoxicity was assessed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide based on the reduction of MTT to formazan by cellular dehydrogenase. MTT) assay. RWPE-1 and LNCaP cells were dispensed in a 96-well plate at a density of 3 x 10 ⁴ cells/well and 1 x 10 ⁴ cells/well, respectively, and cultured for 24 hours. Then, cells were treated with various concentrations of each compound for 24 hours. MTT dye reagent was prepared at 2 mg/mL in distilled water and added to each well for 1 hour at 37°C. The insoluble formazan precipitate was dissolved in DMSO and absorbance was measured at 550 nm in a microplate reader (Infinite M200 Pro, TECAN).

As such, the results of measuring cytotoxicity are shown in FIG. 8 (***p < 0.001). (a) is the cytotoxicity result of boratin on RWPE-1 cells, and (b) is the cytotoxicity result of boratin on LNCap cells. According to this, it did not show significant cytotoxicity to RWPE-1 and LNCap at concentrations of 0.1-10 μM.

Experimental Example 3: AR, 5AR2, PCNA Expression Inhibition Analysis

Expressions of androgen receptor (AR) and 5AR2 (5α-reductase type 2) were evaluated in LNCap cells, and expressions of 5AR2 and proliferating cell nuclear antigen (PCNA) were evaluated in RWPE-1 cells. The specific experimental methods are as follows.

RWPE-1 and LNCaP cells were seeded in a 6-well plate at a density of 2×10 ⁵ cells/well and 6×10 ⁵ cells/well, respectively, and overnight. Cells were treated with 0.5 μM of testosterone propionate (TP) for 1 hour and then treated with 10 μM of finasteride at the indicated concentrations or boratine at various concentrations for an additional 24 hours. Cells were washed three times with cold-phosphate buffered saline (PBS), and cell lysates were extracted with lysis buffer containing protease inhibitor cocktail (Thermo Scientific, USA, Prod # 78425). Protein extracts were centrifuged at 13,000 rpm for 20 minutes at room temperature. Protein content of cell lysates was quantified by Bradford assay. Thirty micrograms of the obtained protein were separated on 8-10% SDS-PAGE at 100V and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 1 hour at room temperature. Anti-androgen receptor (5153T, Cell Signaling Technology), anti-PSA (5365S, Cell Signaling Technology), anti-SRD5A2 (sc-293232, Santa Cruz Biotechnology), anti-PCNA (13110S, Cell Signaling Technology), anti-β -Actin (4967S, Cell Signaling Technology) was used in 1% skim milk. Next, the membrane was incubated with each primary antibody overnight at 4°C. After washing three times with TBST buffer, immunoreactive bands were visualized using immunopure peroxidase-conjugated mouse anti-rabbit IgG or goat anti-mouse IgG (1:10000 dilution, Santa Cruz Biotechnology). Membranes were incubated with secondary antibodies for 1 hour at room temperature. Blots were washed 3 times with TBST buffer. Protein bands were visualized using ECL solution and calibrated using the Chemidoc Imaging System (Fusion FX5, Vilber Lourmat, France).

Dihydrotestosterone (DHT) is an active androgen that is converted from testosterone by the catalytic enzyme 5AR. DHT causes BPH by binding to the androgen receptor (AR) and stimulating the production of various growth factors. Thus, inhibition of 5AR2 expression or activity is generally considered to prevent and delay the progression of BPH.

PCNA plays essential roles in DNA replication, cell division and regulation of various cell cycles. A significant increase in PCNA gene expression was observed in prostate tissue from BPH rats. Upregulation of PSA secretion was also observed in BPH patients.

9 is a Western blot result for measuring androgen receptor (AR) expression level (a) and 5AR2 expression level (b) upon boratine treatment in LNCap cells (*p < 0.05, **p < 0.01 and ***p < 0.001 compared to TP-treated cells). According to this, it was confirmed that the expression of PSA induced by tettosterone propionate (TP) in LNCap cells was inhibited by boratin isolated according to Example 1. That is, compared to finasteride treatment, treatment with boratin significantly suppressed the increase in AR expression caused by TP. In addition, it was confirmed that the expression of 5AR2 was significantly suppressed in cells treated with boratin at a concentration of 10 μM.

10 is a Western blot result for measuring 5AR2 expression level (a) and PCNA expression level (b) upon boratine treatment in RWPE-1 cells (*p < 0.05, **p < 0.01 and ***p < 0.001 compared to TP-treated cells). According to this, 5AR2 expression was suppressed in RWPE-1 cells when boratine was treated, and in particular, it was confirmed that the effect of inhibiting PCNA expression was very strong.

Hereinafter, formulation examples of the composition containing the powder of the present invention will be described, but the present invention is not intended to limit them, but only to be specifically described.

Formulation Example 1. Preparation of powder

Boratin 500 mg of Example 1

Lactose 100 mg

Talc 10 mg

A powder is prepared by mixing the above ingredients and filling them in an airtight bag.

Formulation Example 2. Preparation of tablets

Boratine of Example 1 300 mg

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 3. Preparation of capsule formulation

Boratin of Example 1 200 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a conventional capsule preparation method.

Formulation Example 4. Preparation of Injections

Boratin of Example 1 600 mg

Mannitol 180 mg

Sterile Distilled Water for Injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

It is prepared with the above component content per 1 ampoule according to the conventional method for preparing injections.

Formulation Example 5. Preparation of liquid formulation

7.5 g of Boratine from Example 1

Isomerized sugar 10 g

5 g mannitol

Appropriate amount of purified water

According to the conventional liquid formulation manufacturing method, each component is dissolved in purified water, lemon flavor is added in an appropriate amount, the above components are mixed, and then purified water is added to adjust the total amount to 100g, and then filled into a brown bottle to be sterilized. to prepare a liquid.

Formulation Example 6. Preparation of granules

Boratin of Example 1 1,900 mg

Appropriate amount of vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

10 μg of biotin

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium Carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dibasic Calcium Phosphate 55 mg

Potassium citrate 90 mg

Calcium Carbonate 100 mg

Magnesium Chloride 24.8 mg

Although the composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for granules in a preferred embodiment, the mixing ratio may be arbitrarily modified, and after mixing the above ingredients according to a conventional granule manufacturing method, It can be prepared and used for preparing a health functional food composition according to a conventional method.

Although the embodiments of the present invention have been described above, those skilled in the art can add, change, delete, or add components within the scope not departing from the spirit of the present invention described in the claims. The present invention can be variously modified and changed by the like, and this will also be said to be included within the scope of the present invention.

Claims (10)

A pharmaceutical composition for preventing or treating benign prostatic hyperplasia or alopecia, comprising boratin, a compound represented by Formula 1, or a pharmaceutically acceptable salt thereof as an active ingredient;
[Formula 1]

According to claim 1,
The pharmaceutical composition for preventing or treating alopecia is for preventing or treating prostatic hyperplasia or alopecia, characterized in that it is for inhibiting the expression of one or more selected from AR (Androgen receptor), 5AR2 (5α-reductase type 2) and PCNA (proliferating cell nuclear antigen) pharmaceutical composition for use. According to claim 2,
The AR (Androgen receptor) and 5AR2 (5α-reductase type 2) inhibiting the expression of one or more selected from LNCap cells of the prostate, characterized in that the prostatic hyperplasia or alopecia prevention or treatment pharmaceutical composition. According to claim 2,
The 5AR2 (5α-reductase type 2) and PCNA (proliferating cell nuclear antigen) inhibiting the expression of one or more selected from the LNCap cells of the prostate, characterized in that the prostatic hyperplasia or alopecia prevention or treatment pharmaceutical composition. A food composition for preventing or improving prostatic hyperplasia or alopecia, comprising boratin, a compound represented by Formula 1, and a food-acceptable salt thereof as an active ingredient.
[Formula 1]

(a) obtaining an extract by extracting Symbiodinium voratum with an alcohol having 1 to 4 carbon atoms;
(b) obtaining a re-extract by re-extracting the extract with a butanol solvent; and
(c) isolating and obtaining boratin represented by the following formula (1) from the re-extract by chromatography; preventing or treating prostatic hyperplasia or alopecia comprising boratin as an active ingredient; Method for preparing a pharmaceutical composition for use;
[Formula 1]

According to claim 6,
Method for producing a pharmaceutical composition for preventing or treating prostatic hyperplasia or alopecia, characterized in that the alcohol having 1 to 4 carbon atoms in step (a) is methanol. According to claim 6,
Step (c) is,
(c-1) fractionating the re-extract with a step gradient solvent system of water and methanol on a reverse phase column to obtain a solution eluted with 50% methanol and 50% water;
(c-2) fractionating the eluted solution into 4 fractions again using Sephadex column chromatography (Sephadex LH20) to separate the first eluted solution; and
(c-3) purifying the first eluted solution by reverse-phase liquid chromatography containing an aqueous solution of acetonitrile (ACN) in which a concentration gradient is formed to obtain boratin; or Method for producing a pharmaceutical composition for preventing or treating alopecia. According to claim 8,
In step (c-2), the first eluted solution is obtained by separating the four fractions having a retention time (Rt) of 15 to 17 minutes using reverse phase liquid chromatography using a phenomenex polar column. A method for producing a pharmaceutical composition for preventing or treating prostatic hyperplasia or alopecia, characterized in that obtained. (a) obtaining an extract by extracting the cultured Symbiodinium voratum with alcohol having 1 to 4 carbon atoms;
(b) obtaining a re-extract by re-extracting the extract with a butanol solvent; and
(c) isolating and obtaining boratin represented by the following formula (1) from the re-extract using chromatography; to prevent or improve prostatic hyperplasia or alopecia comprising boratin as an active ingredient; Method for producing a food composition for use;
[Formula 1]

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