KR20210052377A - Composition for the prevention or treatment of prostate-related disease comprising Rhodiola sachalinensis root extract containing kaempferol and epicatechin gallate - Google Patents

Abstract

The present invention relates to a composition for preventing and treating benign prostatic hyperplasia comprising a Rhodiola sachalinensis root extract, in which the size and weight of prostate tissues are significantly reduced in an experimental animal model of prostatic hyperplasia by a pharmacological action of Kaempferol and Epicatechin gallate contained in Rhodiola extract, thereby exhibiting an effect of alleviating an epithelial cell thickness and vesicle area. Furthermore, the conversion of testosterone to DHT is improved by effectively reducing the expression of 5-AR, which is associated with induction of benign prostatic hyperplasia. Thus, the Rhodiola sachalinensis root extract containing Kaempferol and Epicatechin gallate at a concentration of 15 mg and 2 mg or more per kg of Rhodiola sachalinensis root extract of the present invention can be advantageously used as a pharmaceutical composition or a health functional food for the prevention and treatment of benign prostatic hyperplasia.

Description

Composition for the prevention or treatment of prostate-related disease comprising Rhodiola sachalinensis root extract containing kaempferol and epicatechin gallate}

The present invention relates to a composition for preventing or treating prostate-related diseases comprising a rhododendron extract containing camphorol and epicatechin gallate.

The prostate gland is a male appendix consisting of glandular and fibromyal tissue. Prostatitis, enlarged prostate, and prostate cancer are the most common diseases. Among them, the most common disease in men over 50 is enlarged prostate. Benign prostatic hyperplasia (BPH) is the increase in the volume of the prostate around the urethra and compresses the urethra, resulting in bladder storage symptoms such as frequent urination and urgency, delayed urination, interruption, and the need to give strength during urination. It is collectively referred to as a symptom indicative of impaired excretion, and the exact pathogenesis has not yet been identified, but not only is the change in sex hormones with aging, but it is also related to metabolic syndromes such as insulin resistance, abnormal lipid metabolism, high blood pressure, and abdominal obesity. Was reported (Castelli T et al. , 2016; Tikoo K et al. , 2017).

The main male hormone involved in prostate tissue growth is dihydrotestosterone (DHT), and as you age, about 90% of testosterone in the prostate is converted to dihydrotestosterone. The 5-alpha-reductase (5-AR) inhibitor, which is the most prescribed among the treatments for prostatic hyperplasia, does not affect testosterone, which acts as a male hormone, but selectively inhibits the production of dihydrotestosterone, thereby suppressing the anti-androgen activity. The enlarged prostate can be reduced to treat urination disorders. In addition, alpha blocker, a blood pressure drug, does not actually reduce the enlarged prostate, but it relaxes the muscle tissue of the prostate or bladder and has the effect of reducing the symptoms. However, these drugs must be taken continuously to improve symptoms, and side effects such as lowering of blood pressure and tachycardia and various sexual dysfunctions have been reported due to sympathetic nerve suppression. Accordingly, there is an increasing need for the development of natural materials that are effective for treating prostate disease without side effects, and research on this is being made (Korean Patent Laid-Open Patent No. 10-2012-0021281), but it is still insufficient.

Rhodiola sachalinensis A. Bor is a perennial medicinal plant belonging to the genus Pseudomonas phyllaceae stony flowers, and is distributed in the alpine regions of western and northern Asia. It is a plant with special adaptability that can survive at 1,700-2,300m above sea level, with low temperature, dryness, low oxygen, strong ultraviolet rays, and a large temperature difference between day and night. It is reported that it improves adaptive capacity and regulates physiological functions of living organisms to maintain normal life activities, and has high antioxidant activity. Rhododendron is used for edible and medicinal purposes, and has been used as a sedative, antipyretic, and astringent in the private sector.In oriental medicine, it is effective in tonic medicine, especially for senile heart atrophy and sound stomach. It is used as a medicine to treat weakness and postpartum. As a pharmacological ingredient, it is reported that there are 20 kinds of amino acids and 18 kinds of trace elements along with the main four ingredients such as salidroside, rosavin, rosin, and rosarin. have.

The prior technologies related to Hong Gyeong-cheon include skin whitening composition (Korean Patent No. 10-1661545), virus disease prevention and treatment (Korean Patent No. 10-0981296), diabetes prevention and treatment (Korean Patent No. 10-0179088). ), circulatory disease prevention and treatment (Korean Patent No. 10-0265385), liver fibrosis suppression and immune function enhancing composition (Korean Patent No. 10-0386809). However, there is no report that the extract of honggyeongcheon can be used as a composition for the treatment and prevention of prostatic hyperplasia and urination disorders resulting therefrom, and therefore, the main components in the extract of honggyeongcheon have not been identified.

The present inventors made diligent efforts to develop a therapeutic agent for prostate disease that can be administered for a long time due to low toxicity without causing side effects or drug resistance using natural extracts. As a result, Rhododendron extract is a natural material that can block the progression of prostatic hyperplasia. By confirming the possibility, the present invention was completed.

In addition, the present inventors described camphorol (Kaempferol; 3,5,7,4'-Tetrahydroxyflavone; 3'-Deoxyquercetin) and Epicatechin gallate (ECG) as active ingredients for reducing prostatic hyperplasia in the extract of Rhododendron. Epicatechin 3-O-gallate; Epicatechol 3-gallate] was isolated and identified, and its pharmacological efficacy was confirmed in more than 15 mg of camphorol and more than 2 mg of epicatechin gallate from the Rhododendron extract.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of prostatic hyperplasia comprising a rhododendron extract containing camphorol and epicatechin gallate.

Another object of the present invention is to provide a health functional food composition for preventing or improving prostatic hyperplasia, comprising a rhododendron extract containing camphorol and epicatechin gallate.

Another object of the present invention is to provide a quasi-drug composition for preventing or improving prostatic hyperplasia, comprising a rhododendron extract containing camphorol and epicatechin gallate.

Another object of the present invention is to provide a drinking water additive for preventing or improving prostatic hyperplasia, comprising a rhododendron extract containing camphorol and epicatechin gallate.

Another object of the present invention is to provide a method for preventing or treating an enlarged prostate comprising administering the above-described composition to animals other than humans.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating prostatic hyperplasia, comprising camphorol represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating prostatic hyperplasia, comprising epicatechin gallate represented by Chemical Formula 2 or a pharmaceutically acceptable salt thereof.

Preferably, another object of the present invention is camperol (Kaempferol) and epicatechin gallate (Epicatechin gallate); Or to provide a pharmaceutical composition for the prevention or treatment of prostatic hyperplasia, including their pharmaceutically acceptable salts thereof.

According to one aspect of the present invention, the present invention provides a composition for preventing or treating prostatic hyperplasia, comprising an extract of honggyeongcheon.

The present inventors made intensive research efforts to develop a therapeutic agent for prostatic hyperplasia without side effects and toxicity using natural extracts, and as a result of the honggyeongcheon extract, increasing the level of dihydrotestosterone (DHT), which is essential for the occurrence, growth, and maintenance of prostatic hyperplasia in an animal model of prostatic hyperplasia. It was found that by significantly inhibiting prostate hyperplasia, it is possible to cure prostatic hyperplasia.

According to one aspect of the present invention, the present invention provides a composition for preventing or treating prostatic hyperplasia, comprising a rhododendron extract comprising camphorol and epicatechin gallate.

The present inventors made intensive research efforts to develop a therapeutic agent for prostatic hyperplasia without side effects and toxicity using natural extracts, and as a result of the honggyeongcheon extract, increasing the level of dihydrotestosterone (DHT), which is essential for the occurrence, growth, and maintenance of prostatic hyperplasia in an animal model of prostatic hyperplasia. It was found that by significantly inhibiting prostate hyperplasia, it is possible to cure prostatic hyperplasia.

The composition of the present invention includes a rhododendron extract containing at least 15 mg/kg of camphorol and at least 2 mg/kg of epicatechin gallate as an active ingredient. The term'extract' used while referring to honggyeongcheon in this specification is formulated so that not only the extraction result obtained by treating the honggyeongcheon with an extraction solvent, but also the honggyeongcheon itself can be administered to animals or humans (e.g., powders, solutions, capsules, tablets, etc. ) It has the meaning of including the processed honggyeongcheon.

In the case of obtaining the honggyeongcheon extract used in the composition of the present invention by treating the honggyeongcheon with an extraction solvent, various extraction solvents may be used. Preferably, polar solvents or non-polar solvents may be used, and suitable as polar solvents are (i) water, (ii) alcohols (preferably, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal -Butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol), (iii) acetic acid, (iv) DMFO (dimethyl-formamide) and (v) DMSO (dimethyl sulfoxide) Can contain. Suitable non-polar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro It includes any one selected from the group consisting of methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF.

According to one embodiment of the present invention, the extract of the present invention is obtained by treating a honggyeongcheon with a solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.

According to an embodiment of the present invention, the honggyeongcheon extract may be extracted by a pneumatic extraction method or an ultrasonic extraction method.

The honggyeongcheon extract may be extracted from any one or more parts selected from the group consisting of leaves, stems, roots, fruits, and seeds.

As used herein, the term'extract' has the meaning commonly used as a crude extract in the art as described above, but broadly includes a fraction obtained by additionally fractionating the extract. That is, honggyeongcheon extract includes not only those obtained by using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (one prepared for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through the purification method are also included in the extract of honggyeongcheon of the present invention.

The rhododendron extract of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying.

In the present specification, the term'prostatic hyperplasia' refers to a disease occurring in the prostate, which is a male reproductive and urinary organ.

The term "prevention" used in the present invention may mean any action of inhibiting or delaying prostatic hyperplasia by administering the composition for preventing or improving prostatic hyperplasia according to the present invention to an individual.

The term "treatment" used in the present invention may mean any action to improve or benefit the symptoms of an enlarged prostate by administering the composition of the present invention to an individual suspected of having an enlarged prostate.

As used herein, the term "improvement" may mean any action that at least reduces the severity of a parameter related to a condition to be treated, for example, a symptom.

According to the present invention, the composition of the present invention treats, ameliorates and ameliorates an enlarged prostate by inducing a decrease in the activity of 5-alpha reductase. In the present invention, the term "5-alpha reductase" refers to an enzyme that converts testosterone, a type of male hormone, into dihydrotestosterone (DHT). 5-alpha-reductase inhibitors are known to prevent prostate growth as a treatment for prostate-related diseases such as enlarged prostate.

In an animal model of prostatic hyperplasia in which the rhododendron extract was induced by TP (testosterone propionate), the present inventors found that the rhododendron extract inhibits the increase in prostate weight, and increases the level of dihydro testosterone (DHT), which is essential for the occurrence, growth, and maintenance of prostatic hyperplasia. It was confirmed that it was significantly suppressed (Figs. 1 to 3).

In addition, the present inventors have separated and identified camperol and eticatechin galade, which are active ingredients having an effect on prostatic hyperplasia, from the extract of honggyeongcheon of the present invention, and camphorol isolated and identified from the extract of honggyeongcheon is 15mg or more, epicatechin gallate. The pharmacological effect was confirmed in the rate of 2 mg or more (FIGS. 4 to 22).

The pharmaceutical composition for preventing or treating prostatic hyperplasia according to the present invention may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier to be provided as a food, pharmaceutical, feed additive, and drinking water additive. The term “pharmaceutically acceptable carrier” as used herein may mean a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound.

The kind of the carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

In addition, if necessary, other conventional additives such as antioxidants, buffers and/or bacteriostatic agents can be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to Formulations, pills, capsules, granules, or tablets can be formulated and used.

The administration method of the pharmaceutical composition for preventing or treating prostatic hyperplasia according to the present invention is not particularly limited, and may be according to a method commonly used in the art. As a non-limiting example of the mode of administration, the composition may be administered orally or parenterally. The pharmaceutical composition for preventing, improving or treating prostatic hyperplasia of the present invention may be prepared in various formulations according to the intended administration method.

The pharmaceutical composition of the present invention can be used as a single formulation, and can be prepared and used as a combined formulation including additional drugs known to have an approved prostatic hyperplasia treatment effect, and formulated using a pharmaceutically acceptable carrier or excipient. It may be manufactured in a unit dosage form or may be prepared by placing it in a multi-dose container.

In another aspect, the present invention provides a method for preventing or treating an enlarged prostate comprising administering the pharmaceutical composition for preventing or treating an enlarged prostate to an individual. The term "individual" used in the present invention may mean all animals including humans who have developed or are likely to develop an enlarged prostate.

The prevention or treatment method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to an individual who has developed or is at risk of developing an enlarged prostate.

In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and generally in an amount of 0.001 to 1000 mg/kg, preferably 0.05 To 500 mg/kg, more preferably 0.1 to 100 mg/kg may be administered once or several times a day. However, for the purposes of the present invention, a suitable total daily use amount of the composition containing the extract may be determined by the treating physician within the range of correct medical judgment, and may be administered once or divided into several times. However, for the purposes of the present invention, a specific therapeutically effective amount for a specific patient is a specific composition, including the type and degree of reaction to be achieved, whether other agents are used in some cases, the patient's age, weight, general health condition, It is preferable to apply differently according to various factors including sex and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used with or concurrently with the specific composition, and similar factors well known in the medical field.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without causing side effects in consideration of all of the above factors, and can be easily determined by a person skilled in the art.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through a variety of routes.

The method of administering the pharmaceutical composition according to the present invention is not particularly limited, and may follow a method commonly used in the art. As a non-limiting example of the mode of administration, the composition may be administered orally or parenterally. The pharmaceutical composition according to the present invention can be prepared in various formulations according to the intended administration method.

The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or several times by dividing the dose.

According to another embodiment of the present invention, the present invention provides a health functional food composition for preventing or improving prostatic hyperplasia, comprising a rhododendron extract containing camphorol and epicatechin gallate.

The composition of the present invention includes a rhododendron extract containing at least 15 mg/kg of camphorol and at least 2 mg/kg of epicatechin gallate as an active ingredient. The term'extract' used while referring to honggyeongcheon in this specification is formulated so that not only the extraction result obtained by treating the honggyeongcheon with an extraction solvent, but also the honggyeongcheon itself can be administered to animals or humans (e.g., powders, solutions, capsules, tablets, etc. ) It has the meaning of including the processed honggyeongcheon.

The type of food is not particularly limited, and may include all foods in a conventional sense. Non-limiting examples of foods to which the above substances can be added include meat, sausages, bakery, chocolate, candy, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea. , Drinks, alcoholic beverages, and vitamin complexes. When the composition is used as a food additive, the composition may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

The food composition may include a food pharmaceutically acceptable carrier.

The food composition may be a health functional food. Functional food is the same term as food for special health use (FoSHU), and refers to foods with high medical and medical effects that have been processed to efficiently display bioregulatory functions in addition to nutritional supply. , The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc., in order to obtain a useful effect in preventing or improving prostatic hyperplasia.

At this time, the content of the extract contained in the food is not particularly limited thereto, but may be included in 0.01 to 100% by weight, more preferably 1 to 80% by weight based on the total weight of the food composition. When the food is a beverage, it may be included in a ratio of 1 to 30 g, preferably 3 to 20 g, based on 100 ml.

The health functional food can be prepared by a method commonly used in the art, and during the production, raw materials and ingredients commonly added in the art may be added to prepare it. In addition, unlike general drugs, food is used as a raw material, and there is no side effect that may occur when taking drugs for a long time, and portability may be excellent.

According to another embodiment of the present invention, the present invention provides a quasi-drug composition for preventing or improving prostatic hyperplasia, comprising a rhododendron extract containing camphorol and eticatechin galade. The composition of the present invention may be added to a quasi-drug composition for the purpose of preventing or treating an enlarged prostate.

The composition of the present invention includes a rhododendron extract containing at least 15 mg/kg of camphorol and at least 2 mg/kg of epicatechin gallate as an active ingredient. The term'extract' used while referring to honggyeongcheon in this specification is formulated so that not only the extraction result obtained by treating the honggyeongcheon with an extraction solvent, but also the honggyeongcheon itself can be administered to animals or humans (e.g., powders, solutions, capsules, tablets, etc. ) It has the meaning of including the processed honggyeongcheon.

In the present invention, the term "quasi-drug" refers to fibers, rubber products, or the like, which are used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals, weak or not directly acting on the human body, and Or non-machine and similar, as an article corresponding to one of the preparations used for sterilization, insecticide, and similar purposes for the prevention of infectious type, for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals. It may mean items that are not instruments, machines, or devices among the items used, and items other than those that are not instruments, machines, or devices among items used for the purpose of pharmacologically affecting the structure and function of humans or animals. In addition, the quasi-drug may include skin external preparations and personal hygiene products. Preferably, it may be a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, or an ointment, but is not limited thereto.

When the composition according to the present invention is used as a quasi-drug additive, the composition may be added as it is or may be used together with other quasi-drug or quasi-drug components, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the intended use.

According to another embodiment of the present invention, the present invention provides a drinking water additive for preventing or improving prostatic hyperplasia comprising the honggyeongcheon extract. The drinking water additive of the present invention may be used in a manner in which a composition containing the Honggyeongcheon extract is separately prepared in the form of a drinking water additive and mixed with drinking water, or directly added when preparing drinking water. The drinking water additive of the present invention may be in a liquid or dry state, and preferably may be in the form of a dried powder. A drying method for preparing the drinking water additive of the present invention in a dried powder form is not particularly limited, and a method commonly used in the art may be used. The drinking water additive of the present invention may further include other additives as needed. Non-limiting examples of the usable additives include binders, emulsifiers, preservatives, etc. added to prevent deterioration of the quality of drinking water; There are amino acids, vitamins, enzymes, probiotics, flavoring agents, non-protein nitrogen compounds, silicates, buffers, coloring agents, extracting agents or oligosaccharides added to increase the utility of feed or drinking water. It may contain additionally. These may be used alone or two or more may be added together.

The present invention can provide a pharmaceutical composition for the prevention or treatment of prostatic hyperplasia, comprising camphorol (Kaempferol) represented by the following Formula 1 or a pharmaceutically acceptable salt thereof:

[Formula 1]

.

.

The present invention can provide a pharmaceutical composition for preventing or treating prostatic hyperplasia, comprising epicatechin gallate represented by the following formula (2) or a pharmaceutically acceptable salt thereof:

[Formula 2]

.

.

The present invention is camphorol (Kaempferol) represented by the formula (1) and epicatechin gallate (Epicatechin gallate) represented by the formula (2); Or it may provide a pharmaceutical composition for the prevention or treatment of prostatic hyperplasia, including their pharmaceutically acceptable salts thereof.

As a result of the experiment, the inventors of the present application confirmed that the active ingredients of honggyeongcheon were camphorol and epicatechin gallate. Accordingly, prostate stromal cells and prostatic hyperplasia cells of Rhododendron, camphorol, and epicatechin gallate were treated to _{measure their IC 50} concentrations. Accordingly, it was found that the extract of honggyeongcheon was 24.8 ~ 45 ug/mL, the camphorol was 4.5 ~ 8.3 ug/ml, and the epicatechin gallate was 11.4 ~ 16.2 ug/ml. Therefore, the synergistic effect of camphorol and epicatechin gallate on the killing effect of prostate stromal cells and prostatic hyperplasia was further tested. As a result, the mixture was treated in a molar ratio of 7:3 compared to camphorol and picatechin gallate alone. When it showed the most significant killing effect (Fig. 23).

The composition containing the Rhododendron extract of the present invention as an active ingredient contains camphorol (Caempferol) and epicatechin gallate (epicatechin gallate) as indicator substances, and camphorol is 15 mg or more, and epicatechin gallate is 2 mg or more. By inhibiting the activity of the alpha-reductase enzyme, there is an effect that can effectively prevent, improve and treat an enlarged prostate. In addition, the composition comprising as an active ingredient a rhododendron extract containing more than 15 mg of camphorol and more than 2 mg of epicatechin gallate of the present invention is a natural drug, comprising food compositions and quasi-drug compositions for the prevention or improvement of prostate-related diseases. It can be applied to various products including. In addition, the rhododendron extract of the present invention does not show hepatotoxicity and nephrotoxicity as a result of animal experiments and is derived from natural products, so it can be safely and continuously used without causing serious irritation or harmful effects in the body in addition to the prevention and improvement of prostatic hyperplasia. have.

1 is a comparison of the size of the prostate of rats by extracting the prostate 4 weeks after the experiment in order to confirm the effect of the rhododendron extract on prostate size.
Figure 2 shows the effect of honggyeongcheon extract on the prostate index.
Figure 3 shows the results of measuring the concentrations of DHT and testosterone in prostate tissue and blood of honggyeongcheon extract using an ELAISA kit.
Figure 4 shows the separation and purification process of the compounds Rr-5-7 and Rr-5-8 having activity in prostatic hyperplasia apoptosis.
Figure 5 shows the ESI-mass spectrum (top: positive mode, bottom: negative mode) of the fraction Rr-5-7 isolated from the extract of honggyeongcheon having a prostatic hyperplasia cell killing effect.
^{Figure 6 shows the 1} H NMR spectrum of the fraction Rr-5-7 isolated from the extract of honggyeongcheon having a prostatic hyperplasia cell killing effect.
^{Figure 7 shows the 13} C NMR spectrum of the fraction Rr-5-7 isolated from the extract of honggyeongcheon having a prostatic hyperplasia cell killing effect.
^{Figure 8 shows the 1} H- ¹ H COSY spectrum of the fraction Rr-5-7 isolated from the extract of honggyeongcheon having a prostatic hyperplasia cell killing effect.
Figure 9 shows the ¹ H- ¹ H COSY honggyeongcheon separated fraction from the extract having the enlarged prostate cell killing effect by the spectrum analysis part structure of Rr-5-7 (thick line).
Figure 10 shows the HMQC spectrum of the fraction Rr-5-7 isolated from the honggyeongcheon extract having a prostatic hyperplasia cell killing effect.
Figure 11 shows the HMBC spectrum of the fraction Rr-5-7 isolated from the extract of honggyeongcheon having a prostatic hyperplasia cell killing effect.
Figure 12 shows the two-dimensional NMR correlations of fraction Rr-5-7 isolated from the extract of honggyeongcheon, which has apoptosis effect of prostatic hyperplasia.
^{Figure 13 shows the 1} H NMR and ¹³ C NMR peak assignments of fraction Rr-5-7 isolated from the extract of honggyeongcheon having a prostatic hyperplasia cell killing effect.
Figure 14 shows the ESI-mass spectrum (top: positive mode, bottom: negative mode) of the fraction Rr-5-8 isolated from the extract of honggyeongcheon having a prostatic hyperplasia cell killing effect.
^{Figure 15 shows the 1} H NMR spectrum of the fraction Rr-5-8 isolated from the extract of honggyeongcheon having a prostatic hyperplasia cell killing effect.
^{Figure 16 shows the 13} C NMR spectrum of the fraction Rr-5-8 isolated from honggyeongcheon extract having a prostatic hyperplasia cell killing effect.
^{Figure 17 shows the 1} H- ¹ H COSY spectrum of the fraction Rr-5-8 isolated from honggyeongcheon extract having a prostatic hyperplasia cell killing effect.
Figure 18 shows the ¹ H- ¹ a partial structure (a thick line) of the fractions separated from the Rr-5-8 honggyeongcheon extract having a BPH apoptotic effects analyzed by H COSY spectrum.
Figure 19 shows the HMQC spectrum of the fraction Rr-5-8 isolated from the extract of honggyeongcheon having a prostatic hyperplasia cell killing effect.
Figure 20 shows the HMBC spectrum of the fraction Rr-5-8 isolated from honggyeongcheon extract having a prostatic hyperplasia cell killing effect.
Figure 21 shows the two-dimensional NMR correlations of fraction Rr-5-8 isolated from the extract of honggyeongcheon, which has apoptosis effect of prostatic hyperplasia.
^{Figure 22 shows the 1} H NMR and ¹³ C NMR peak assignment of the fraction Rr-5-8 isolated from the extract of honggyeongcheon having a prostatic hyperplasia cell killing effect.
23 is a result of confirming the _{IC 50} concentrations of honggyeongcheon, camphorol, and epicatechin gallate treated on prostate stromal cells and prostatic hyperplasia cells.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Example 1. Preparation of honggyeongcheon extract

After cutting and crushing the roots of the well-dried Gosan honggyeongcheon, 100g of each powder was immersed in 1,000 mL of 50% by weight ethanol solution for 30 minutes, and then an ultrasonic extractor fixed at 50℃ (SJC-II-50L, GREENAPPLE, Inc. , China) for 1 hour and then extracted for 30 minutes at a frequency of 20 KHz at an output of 1,000 W. After extraction, the extract was centrifuged at 10,000 rpm, the filtrate (1) was separately taken, and the remaining residue was ultrasonically extracted under the same conditions, and centrifuged at 10,000 rpm to obtain a new filtrate (2). After mixing the filtrates (1) and (1), ethanol was removed using a vacuum concentrator, and then freeze-dried to obtain about 35-40 g of an ultrasonic extract powder.

Experimental Example 1. Confirmation of the effect of improving prostatic hyperplasia of rhododendron extract

1-1. Animal model of prostatic hyperplasia

After birth, divided into the normal control group, the prostatic hyperplasia inducing group, the prostatic hyperplasia inducing group administered honggyeongcheon extract (2.5, 5, 10 mg/kg), and the prostatic hyperplasia inducing group administered finasteride (1mg/kg) in the positive control group. Testosterone Propionate (TP, 3mg/kg, Samil Pharmaceutical, Korea) was administered subcutaneously daily for 4 weeks to 8 male rats at 10 weeks to induce prostatic hypertrophy. Rhododendron extract and finasteride were administered orally.

1-2. Separation of blood and prostate tissue

Four weeks after the experiment, all mice were fasted for 24 hours, inhaled anesthesia using isofuran/nitrogen/oxygen, blood was collected by laparotomy, and then the prostate was removed and the weight was measured. After removing blood with physiological saline, the extracted prostate was weighed after removing the remaining water and blood from the filter paper.

1-3. Prostate weight and weight-to-weight ratio measurement

The weight of the prostate was measured using an electronic scale after removing fat, foreign substances and water around the prostate. In addition, the ratio of prostate weight per body weight was calculated using the following formula.

Prostate weight ratio (%) = prostate weight × 100 / body weight

1-4. Measurement of testosterone concentration in blood

After the end of the experiment, the level of testosterone was measured from the isolated serum using an ELISA kit (Abcam, USA).

1-5. Statistical analysis

Statistical analysis was performed with ANOVA. ^* p<0.01, ^** p<0.001 were significantly different compared to the normal control group, ^# p<0.05, ^## p<0.01 compared to the prostatic hyperplasia-causing group. The case where there was a significant difference in time was considered.

<Experiment result>

Effect of Rhododendron extract on the prostate index

The prostate index was calculated by dividing the body weight (100g) by the prostate tissue weight (mg). As shown in FIG. 2, the prostatic hyperplasia-induced group increased the total prostate weight index compared to the normal control group, while the honggyeongcheon extract-administered group all decreased the total prostate weight index compared to the prostatic hyperplasia-induced group. Compared with the prostatic hyperplasia-inducing group, in the Finasteride-administered group, which is a positive control, the change in the prostate index could not be confirmed unlike the honggyeongcheon extract-administered group (FIG. 2).

Check the concentration of dihydrotestosterone (DHT) and testosterone in blood and prostate tissue

In order to examine the activity of 5-AR converting from testosterone to DHT, testosterone and DHT concentrations were measured. As a result, the concentration of DHT in the blood and prostate tissue in the prostatic hyperplasia-inducing group was significantly higher than that of the normal control group, but the DHT concentration in the honggyeongcheon extract-administered group was significantly lower than that of the prostatic hyperplasia-inducing group (Fig. 3). . However, the concentration of testosterone in prostate tissue and blood appeared opposite to the pattern of DHT concentration. It was detected low in the prostatic hyperplasia-causing group, whereas it was high in the rhododendron extract administration group. In other words, it is believed that the amount of testosterone converted to DHT decreased and recovered as the activity of the extract of honggyeongcheon decreased the expression of 5-AR and thus decreased.

Experimental Example 2. Identification of substances having activity in improving prostatic hyperplasia

2-1. Isolation and purification of active ingredients

Add 10 L of 70% methanol aqueous solution to 9.3 kg of honggyeongcheon extract powder, extract at 60° for 2 hours, filter to separate the filtrate (3), and add 10 L of 70% methanol aqueous solution to the remaining residue for repeated extraction. After filtering, the filtrate (4) was obtained. The filtrate (3) and the filtrate (4) were combined and concentrated under reduced pressure to remove methanol. The filtrate from which the solvent was removed was adsorbed on Diaion HP-20 resin and then eluted with 30%, 60%, and 100% methanol aqueous solutions in 4 L each.

The eluted fraction of 60% methanol, which shows the killing activity of prostatic hyperplasia cells, was concentrated under reduced pressure, and silica gel column chromatography was performed using the elution solvent chloroform-methanol (100:1→1:1, v/v, stepwise). After concentrating the chloroform-methanol (5:1, v/v) fraction (Rr-5, FIG. 4), Sephadex LH-20 column chromatography was performed using methanol. Each fraction was analyzed by HPLC and divided into a total of 14 groups (Rr-5-1 to Rr-5-14), and the killing activity of prostatic hyperplasia cells by each group was measured, and Rr-5-7 and Rr-5- Two active fractions of 8 were obtained (Fig. 4).

After concentrating the active fraction Rr-5-7, ODS MPLC was performed using a 60%→100% methanol aqueous solution as an elution solvent. As a result of analyzing each fraction by HPLC, fractions 6 and 7 with high purity were concentrated and named as compound Rr-5-7.

In addition, after concentrating the active fraction Rr-5-8, ODS MPLC was performed using a 20%→100% methanol aqueous solution as an elution solvent. As a result of analyzing each fraction by HPLC, fractions 5 to 8 with high purity were concentrated and named as compound Rr-5-8.

2-2. Identification of active ingredients

2-2-1. Chemical structure of active ingredient Rr-5-7

2-2-1-1. Mass spectrum measurement and analysis

ESI-mass spectrum was measured to reveal the molecular weight of the purified active compound Rr-5-7. That is a result of measuring the ESI-mass spectrum in positive mode [M + H] ⁺ a m / z was observed at 287.2, measurement results of ESI-mass spectrum in the negative mode [MH] ^- is observed in the m / z 285.3 It was found that the molecular weight was 286 (Fig. 5).

2-2-1-2. Measurement and interpretation of NMR spectrum

In order to investigate the chemical structure of the compound Rr-5-7, ¹ H NMR, ¹³ C NMR, ¹ H- ¹ H COSY, HMQC, and HMBC spectra were measured and analyzed by dissolving in _{CD 3 OD.}

2-2-1-2-1. ^One Measurement and interpretation of H NMR spectrum

Dissolved in CD ₃ ^{OD and measured 1} H NMR spectrum (Fig. 6), 8.06 (2H, d, J = 8.5 Hz), 6.90 (2H, d, J = 8.5 Hz) in 1,4-disubstituted benzene at ppm Two aromatic methine proton peaks were observed at the derived aromatic methine proton, 6.39 (1H, d, J = 2.0 Hz) and 6.19 (1H, d, J = 2.0 Hz) ppm. The 2.0 Hz coupling constant observed between 6.39 and 6.19 ppm was found to be meta-coupled to each other.

2-2-1-2-2. ¹³ Measurement and interpretation of C NMR spectrum

¹³ C NMR spectrum measurement results (Fig. 7), carbonyl carbon at 177.4 ppm, 165.8, 162.5, 160.6, 158.3, 148.0, oxygenated sp ² quaternary carbon at 137.1 ppm, 130.7 (X2), 116.3 (X2), 99.3, ^{Sp 2} methine carbon was observed at 94.5 ppm and sp ² quaternary carbon at 123.7 and 104.5 ppm. These characteristics indicated that this compound is a flavonoid compound. Thereafter the chemical structure of the compounds was identified by the measurement and analysis of the two-dimensional NMR spectrum including ^{1 H- 1 H COSY, HMQC,} HMBC.

2-2-1-2-3. ^One H- ^One H COSY spectrum measurement and analysis

^{As a result of measuring and analyzing 1} H-1 H COSY spectrum (FIG. 8), two substructures including 1,4-disubstituted benzene shown in FIG. 9 were identified.

2-2-1-2-4. Measurement and interpretation of HMQC spectrum and HMBC spectrum

As a result of measuring and analyzing the HMQC spectrum (FIG. 10), all observed proton-bearing carbons could be identified. As a result of measuring and analyzing the HMBC spectrum (Fig. 11), from 8.06 ppm of aromatic methine proton to 160.6, 148.0 ppm of oxygenated sp ² carbon, 6.90 ppm of aromatic methine proton to 123.7 ppm carbon, 6.57 ppm of aromatic methine proton From, a long-range correlation was observed for ^{sp 2} quaternary carbon at 163.2 and 153.9 ppm. In addition, long-range correlations were observed from 6.39 ppm of aromatic methine proton to 165.8, 158.3, 104.5, and 99.3 ppm of carbon and 6.19 ppm of aromatic methine proton to 165.8, 162.5, 104.5 and 94.5 ppm of carbon. The chemical structure of this compound was determined from the HMBC correlation, the chemical shift value of the remaining 177.4 ppm of carbonyl carbon, 137.1 ppm of quaternary carbon, and the molecular weight of 286 as shown in FIG. 12. As a result of searching the database based on the above chemical structure, the compound was identified as Kaempferol (same name: 3,5,7,4'-Tetrahydroxyflavone;3'-Deoxyquercetin; Kaemferol). ^{13 shows the assignment of 1} H NMR and ¹³ C NMR peaks of the compound Rr-5-7.

2-2-2. Chemical structure of active ingredient Rr-5-8

2-2-2-1. Mass spectrum measurement and analysis

ESI-mass spectrum was measured to reveal the molecular weight of the purified active compound Rr-5-8. That is a result of measuring the ESI-mass spectrum in positive mode [M + H] ⁺ a m / z was observed at 443.2, measurement results of ESI-mass spectrum in the negative mode [MH] ^- is observed in the m / z 441.1 It was found that the molecular weight was 442 (FIG. 14).

2-2-2-2. Measurement and interpretation of NMR spectrum

In order to identify the chemical structure of the compound Rr-5-8, it was dissolved in DMSO-d ₆ ^{and analyzed by measuring 1} H NMR, ¹³ C NMR, ¹ H- ¹ H COSY, HMQC, and HMBC spectrum.

2-2-2-2-1. ^One Measurement and interpretation of H NMR spectrum

Dissolved in DMSO- d ₆ ^{and measured 1} H NMR spectrum (Fig. 15), 6.73 (1H, dd, J = 8.5, 2.0 Hz), 6.64 (1H, d, J = 8.5 Hz), 6.85 (1H, d , J = 2.0 Hz) aromatic methine proton derived from 1,2,4-trisubstituted benzene at ppm, two aromatic methine protons superimposed on 6.81 (2H, s) ppm, 5.92 (1H, d, J = 2.0 Hz), Two aromatic methine proton peaks were observed at 5.82 (1H, d, J = 2.0 Hz) ppm. The 2.0 Hz coupling constant observed between 5.92 and 5.82 ppm was found to be meta-coupled to each other. In addition, two oxygenated methine at 5.34 and 5.01 ppm and one boiling methylene proton at 2.92 (1H, dd, J = 17.0, 4.5 Hz) and 2.67 (1H, br. d, J = 17.0 Hz) ppm were observed.

2-2-2-2-2. ¹³ Measurement and interpretation of C NMR spectrum

¹³ C NMR spectrum measurement results (Fig. 16), carbonyl carbon at 165.1 ppm, 156.4, 156.0, 155.5, 145.4 (X2), 144.6 (X2), oxygenated sp ² quaternary carbon at 138.5 ppm, 117.5, 115.0, 114.2, this 108.5 (X2), 95.5, to ^{94.3 ppm sp 2 methine carbon, 129.3} , 119.2, oxygenated methine ppm to 97.2, 25.6 ppm in the ^{sp 2 quaternary carbon, 76.4, 68.0} ppm to the methylene carbon was observed. These characteristics indicated that this compound is a flavonoid-based compound containing a gallate moiety. The chemical structure of this compound was identified by the measurement and analysis of the two-dimensional NMR spectrum including ^{1 H- 1 H COSY, HMQC,} HMBC.

2-2-2-2-3. ^One H- ^One H COSY spectrum measurement and analysis

^{As a result of measuring and analyzing 1} H-1 H COSY spectrum (FIG. 17), three partial structures including 1,2,4-trisubstituted benzene shown in FIG. 18 were identified.

2-2-2-2-4. Measurement and interpretation of HMQC spectrum and HMBC spectrum

As a result of measuring and analyzing the HMQC spectrum (FIG. 19), all observed proton-bearing carbons could be identified. In addition, as a result of measuring and analyzing the HMBC spectrum (Fig. 20), 144.6 ppm of oxygenated sp ² carbon and 76.4 ppm of methine carbon from 6.85 and 6.73 ppm of aromatic methine proton, and 144.6 ppm of oxygenated from 6.64 ppm of aromatic methine proton. sp ² carbon and 129.3 ppm carbon, 156.4, 156.0, 97.2, 94.3 ppm sp ² carbon from 5.92 ppm aromatic methine proton, 156.4, 155.5, 97.2, 95.5 ppm sp ² carbon from 5.82 ppm aromatic methine proton Long-range correlation was observed. In addition, long-range correlations were observed from 5.01 ppm of methine proton to 129.3, 117.5 and 114.2 ppm of carbon, and from 2.92/2.67 ppm of methylene proton to 155.5 and 97.2 ppm of carbon, revealing the catechin moiety. This catechin structure was estimated to be epicatechin from the proton coupling constants of 5.01 and 5.34 ppm. A long-range correlation was observed from 6.81 ppm of aromatic methine proton to 165.1 ppm of carbonyl carbon and 145.4 and 138.5 ppm of oxygenated sp ² carbon, and it was found that gallate moiety was formed from their chemical shift (FIG. 21). Therefore, it was determined that the gallate moiety of this compound was bound to epicatechin. Although the binding position of the gallate moiety could not be confirmed, the binding position of the gallate moiety was inferred from the chemical shift value of the stored oxygenated methine proton of 5.34 ppm, and thus the chemical structure of the present compound was determined as shown in FIG. 22. As a result of searching the database based on the above chemical structure, this compound was identified as epicatechin gallate (same name: Epicatechin 3-O-gallate; Epicatechol 3-gallate). Figure 22 shows the assignment (assignment) of ^{the 1} H NMR and ¹³ C NMR peaks of the compound Rr-5-8.

Experimental Example 3. The killing effect of Kaempferol and ECG on prostatic hyperplasia and prostate stromal cells

Prostatic hyperplasia cells (benign prostatic hyperplasia cells; BPH-1 (purchased ATCC)) and prostate stromal cells (WMPY-1; purchased ATCC) ^{were plated on a 96 well plate at 1×10 6} and incubated at 37°C for 24 hours. The extract, kampferol (Sigma aldrich), and ECG (Sigma aldrich) were dissolved in DMSO and treated by concentration, and the IC ₅₀ concentration was measured through MTT assay. As shown in Table 1 below, honggyeongcheon extract was 24.8 to 45 ug/mL, Kaempferol was 4.5 to 8.3 ug/ml, and ECG was 11.4 to 16.2 ug/ml.

Kaempferol and ECG were prepared at 10 μM, and Kaempferol and ECG were respectively mixed and treated at various molar ratios, and then Kaempferol and ECG were treated in prostatic hyperplasia cells and prostate stromal cells, and the IC ₅₀ concentration was measured. As a result, Kaempferol and ECG Compared to the single treatment, when treated at a molar ratio of 7:3, the killing effect was most significant (FIG. 23). This was significantly better at 7:3 compared to treatment alone, but the killing effect was superior to other molar ratios (5:5 or 3:7).

Enlarged prostate cells Prostate stromal cells Rhododendron extract (IC ₅₀ , ug/mL) 45 ± 3.4 24.8 ± 2.4 Kaempferol (IC ₅₀ , ug/mL) 8.3 ± 0.5 4.5 ± 1.2 ECG (IC ₅₀ , ug/mL) 11.4 ± 0.7 16.2 ± 0.9

Claims (11)

Camperol (Kaempferol) and epicatechin gallate (Epicatechin gallate) containing a rhododendron extract containing, a pharmaceutical composition for the prevention or treatment of prostatic hypertrophy. The pharmaceutical composition of claim 1, wherein the honggyeongcheon extract is extracted with any one solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof. The pharmaceutical composition of claim 1, wherein the honggyeongcheon extract is extracted by pneumatic extraction or ultrasonic extraction using hot water. The pharmaceutical composition of claim 1, wherein the honggyeongcheon extract is extracted from any one or more sites selected from the group consisting of leaves, stems, roots, fruits, and seeds. A health functional food composition for preventing or improving prostatic hyperplasia, comprising a rhododendron extract containing camphorol and epicatechin gallate. A quasi-drug composition for preventing or improving prostatic hyperplasia, comprising a rhododendron extract containing camphorol and epicatechin gallate. Drinking water additive for preventing or improving prostatic hyperplasia, comprising a rhododendron extract containing camphorol and epicatechin gallate. A method for preventing or treating an enlarged prostate comprising administering the composition of claim 1 to an animal other than a human. A pharmaceutical composition for the prevention or treatment of prostatic hyperplasia, comprising camphorol (Kaempferol) represented by the following Formula 1 or a pharmaceutically acceptable salt thereof:
[Formula 1]

. A pharmaceutical composition for preventing or treating prostatic hyperplasia, comprising epicatechin gallate represented by the following formula (2) or a pharmaceutically acceptable salt thereof:
[Formula 2]

Camperol represented by the following Formula 1 (Kaempferol) and epicatechin gallate represented by Formula 2 (epicatechin gallate); Or a pharmaceutical composition for the prevention or treatment of prostatic hyperplasia, comprising a pharmaceutically acceptable salt thereof:
[Formula 1]

,
[Formula 2]

.

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