KR102519649B1 - 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 the prevention and treatment of prostatic hyperplasia containing an extract of Rhodiola Rosea, in an experimental animal model inducing prostatic hyperplasia by the pharmacological action of kaempferol and epicatechin gallate contained in the Rhodiola extract. The size and weight of prostate tissue were significantly reduced, and the epithelial cell thickness and vesicular area were improved. In addition, the conversion of testosterone to DHT was improved by effectively reducing the expression of 5-AR related to the induction of BPH. Therefore, the rhodiola extract containing kaempferol and epicatechin gallate of the present invention at concentrations of 15 mg and 2 mg or more per kg of the rhodiola extract, respectively, can be usefully used as a pharmaceutical composition or 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 rhodiola extract containing kaempferol and epicatechin gallate.

The prostate is an accessory gonad in men composed of glandular tissue and fibromuscular tissue, and prostatitis, benign prostatic hyperplasia, and prostate cancer are the most common diseases. Benign prostatic hyperplasia (BPH) is caused by an increase in the volume of the prostate around the urethra, which compresses the urethra, resulting in bladder storage symptoms such as frequent urination and urgency, and symptoms of delayed urination, interrupted urination, and force during urination. It is a collective term for symptoms indicating an excretion disorder. Although the exact pathogenesis is not yet known, the main cause is the change in sex hormones due to aging, and it is also related to metabolic syndrome such as insulin resistance, abnormal lipid metabolism, high blood pressure, and abdominal obesity. have been reported (Castelli T et al. , 2016; Tikoo K et al. , 2017).

The main male hormone involved in the growth of prostate tissue is dihydrotestosterone (Dihydrotestosterone, DHT), and about 90% of testosterone is converted into dihydrotestosterone in the prostate with age. 5-alpha-reductase (5-AR) inhibitors, which are most frequently prescribed among prostatic hyperplasia treatments, have anti-androgen activity by selectively inhibiting the production of dihydrotestosterone without affecting testosterone, which represents the male hormone action. It can treat urinary disorders by shrinking the enlarged prostate. In addition, alpha blockers, which are blood pressure drugs, do not actually shrink the enlarged prostate, but have the effect of alleviating the symptoms by relaxing the muscle tissue of the prostate or bladder. However, these drugs must be taken continuously to improve symptoms, and side effects such as a drop in 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 effective for the treatment of prostate diseases without side effects, and research on this is being conducted (Korean Patent Publication No. 10-2012-0021281), but it is still insufficient.

Rhodiola sachalinensis A. Bor is a perennial medicinal plant belonging to the angiosperm family Crassulaceae 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 to 2,300 m above sea level where the temperature is low, dry, low in oxygen, strong ultraviolet rays shine, and the temperature difference between day and night is large. It is reported to improve the adaptability, regulate the physiological function of organisms, maintain life activities in a normal state, and have high antioxidant activity. Rhodiola rosea has been used for food and medicinal purposes, and has been used as a sedative, antipyretic, and astringent in the private sector. In oriental medicine, it is effective as a tonic, especially for the elderly, heart failure, yin and phlegm, and is used as a good drug for diabetes, anemia, gallbladder disease, general and physical fatigue, and nerves. It is used as a medicine for weakness and postpartum treatment. It is reported that there are 20 kinds of amino acids and 18 kinds of trace elements along with 4 main ingredients such as salidroside, rosavin, rosin, and rosarin as pharmacological ingredients. there is.

Prior art related to Rhodiola Rosea includes a skin whitening composition (Korean Patent No. 10-1661545), viral disease prevention and treatment (Korean Patent No. 10-0981296), and diabetes prevention and treatment (Korean Patent No. 10-0179088). ), prevention and treatment of circulatory diseases (Korean Patent No. 10-0265385), liver fibrosis inhibition and immune function enhancing composition (Korean Patent No. 10-0386809), and the like. However, there is no report that the rhodiola extract can be used as a composition for the treatment and prevention of benign prostatic hyperplasia and urination disorder resulting therefrom, and therefore, the main components of the rhodiola extract have not been identified.

The present inventors have made diligent efforts to develop a treatment for prostate disease that can be administered for a long period of time without causing side effects or drug resistance using natural extracts and has low toxicity. The present invention was completed by confirming the possibility.

In addition, the present inventors found that kaempferol; 3,5,7,4'-Tetrahydroxyflavone; 3'-Deoxyquercetin) and epicatechin gallate [Epicatechin gallate (ECG); Epicatechin 3-O-gallate; Epicatechol 3-gallate] was isolated and identified, and the pharmacological efficacy of kaempferol isolated from rhodiola extract was confirmed at 15 mg or more and epicatechin gallate at 2 mg or more.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating benign prostatic hyperplasia, comprising a rhodiola extract containing kaempferol 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 rhodiola extract containing kaempferol and epicatechin gallate.

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

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

Another object of the present invention is to provide a method for preventing or treating prostatic hyperplasia comprising administering the aforementioned composition to an animal other than human.

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

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

Preferably another object of the present invention is kaempferol and epicatechin gallate (Epicatechin gallate); Or to provide a pharmaceutical composition for preventing or treating benign prostatic hyperplasia, containing a pharmaceutically acceptable salt thereof.

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

As a result of diligent research efforts to develop a treatment for prostatic hyperplasia without side effects and toxicity using natural extracts, the present inventors found that rhodiola extract increases dihydrotestosterone (DHT) levels, which are essential for the development, growth, and maintenance of prostatic hyperplasia in an animal model of benign prostatic hyperplasia. By significantly inhibiting, it was found that prostate hyperplasia was treated.

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

As a result of diligent research efforts to develop a treatment for prostatic hyperplasia without side effects and toxicity using natural extracts, the present inventors found that rhodiola extract increases dihydrotestosterone (DHT) levels, which are essential for the development, growth, and maintenance of prostatic hyperplasia in an animal model of benign prostatic hyperplasia. By significantly inhibiting, it was found that prostate hyperplasia was treated.

The composition of the present invention includes a rhodiola extract containing 15 mg/kg or more of kaempferol and 2 mg/kg or more of epicatechin gallate as active ingredients. In this specification, the term 'extract' used while referring to Rhodiola is not only an extraction result obtained by treating Rhodiola with an extraction solvent, but also a formulation (eg, powder, solution, capsule, tablet, etc.) so that Rhodiola itself can be administered to animals or humans. ) has a meaning that also includes the Rhodiola workpiece.

When the rhodiola extract used in the composition of the present invention is obtained by treating rhodiola with an extraction solvent, various extraction solvents may be used. Preferably, polar solvents or non-polar solvents may be used, and suitable polar solvents include (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) any one selected from the group consisting of can include Suitable as non-polar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkanes, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Penten, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro and 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 rhodiola with a solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.

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

The rhodiola 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 a meaning commonly used in the art as a crude extract in the art as described above, but also includes fractions obtained by additional fractionation of the extract in a broad sense. That is, Rhodiola chrysanthemum extract includes not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through the purification method is also included in the rhodiola extract of the present invention.

Rhodiola extract of the present invention can be prepared in a powder state by additional processes such as distillation under reduced pressure and lyophilization or spray drying.

As used herein, the term 'prostatic hyperplasia' refers to a disease that occurs in the prostate, which is a male reproductive and urinary organ.

As used herein, the term "prevention" may refer to any action that inhibits or delays BPH by administering the composition for preventing or improving BPH according to the present invention to a subject.

As used herein, the term "treatment" may refer to any action that improves or benefits the symptoms of BPH by administering the composition of the present invention to a subject suspected of having BPH.

As used herein, the term "improvement" may refer to any activity that at least reduces a parameter associated with a condition to be treated, for example, the severity of a symptom.

According to the present invention, the composition of the present invention treats, ameliorates and alleviates prostatic hyperplasia by inducing a decrease in the activity of 5-alpha reductase. As used herein, 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 the growth of the prostate as a treatment for prostate-related diseases such as benign prostatic hyperplasia.

The present inventors found that rhodiola extract suppresses the increase in prostate weight in an animal model of prostatic hyperplasia induced by TP (testosterone propionate), and increases dihydrotestosterone (DHT) levels, which are essential for the development, growth, and maintenance of benign prostatic hyperplasia. Significant inhibition was confirmed (Figs. 1 to 3).

In addition, the present inventors separated and identified kaemphorol and epicatechin galade, which are active ingredients having an effect on prostatic hypertrophy, from the rhodiola extract of the present invention, and separated and identified kaemphorol from the rhodiola extract, at 15 mg or more, epicatechin gall. The pharmacological effect of the rate was confirmed at 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 and provided as food, medicine, feed additive, drinking water additive, and the like. As used herein, the term 'pharmaceutically acceptable carrier' may refer to a carrier or diluent that does not inhibit the biological activity and properties of the administered compound while not stimulating living organisms.

The type 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 may be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form main solutions such as aqueous solutions, suspensions, and emulsions. It may be formulated into a dosage form, pill, capsule, granule or tablet for use.

The administration method of the pharmaceutical composition for preventing or treating prostatic hyperplasia 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 administration method, the composition may be administered by oral administration or parenteral administration. The pharmaceutical composition for preventing, improving or treating prostatic hyperplasia of the present invention may be prepared in various formulations depending on the desired administration method.

The pharmaceutical composition of the present invention can be used as a single formulation, or can be prepared and used as a combination formulation by further including a drug known to have a recognized therapeutic effect on prostatic hyperplasia, and formulated using a pharmaceutically acceptable carrier or excipient. It can be prepared in unit dose form or introduced into a multi-dose container.

As another aspect, the present invention provides a method for preventing or treating benign prostatic hyperplasia comprising administering to a subject a pharmaceutical composition for preventing or treating benign prostatic hyperplasia. As used herein, the term "subject" may refer to all animals, including humans, who have developed or are likely to develop benign prostatic hyperplasia.

Specifically, the preventive or therapeutic method of the present invention may include administering the composition in a pharmaceutically effective amount to a subject having or at risk of developing benign prostatic hyperplasia.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and is generally in an amount of 0.001 to 1000 mg/kg, preferably 0.05 mg/kg. to 500 mg/kg, more preferably 0.1 to 100 mg/kg, once a day to several divided doses. However, for the purpose of the present invention, a suitable total daily amount of the composition containing the extract can be determined by a treating physician within the scope of correct medical judgment, and can be administered once or divided into several times. However, for purposes of the present invention, a specific therapeutically effective amount for a particular patient is determined by the type and extent of the response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, general state of health, It is preferable to apply differently according to various factors including gender and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently used with a 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 in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without causing side effects in consideration of all the above factors, and can be easily determined by those 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 various routes.

The administration method of 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 administration method, the composition may be administered by oral administration or parenteral administration. The pharmaceutical composition according to the present invention may be prepared in various formulations depending on the desired 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 administered 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 rhodiola extract containing kaempferol and epicatechin gallate.

The composition of the present invention includes a rhodiola extract containing 15 mg/kg or more of kaempferol and 2 mg/kg or more of epicatechin gallate as active ingredients. In this specification, the term 'extract' used while referring to Rhodiola is not only an extraction result obtained by treating Rhodiola with an extraction solvent, but also a formulation (eg, powder, solution, capsule, tablet, etc.) so that Rhodiola itself can be administered to animals or humans. ) has a meaning that also includes the Rhodiola workpiece.

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 substance can be added include meat, sausages, baked goods, chocolate, candy, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea , drinks, alcoholic beverages, and vitamin complexes. When using the composition as a food additive, the composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.

The food composition may include a food chemically 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 food with high medical and medical effects processed to efficiently display bioregulatory functions in addition to nutrient supply. , The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, and pills in order to obtain useful effects for preventing or improving prostatic hyperplasia.

At this time, the content of the extract included 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. If 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 can be prepared by adding raw materials and components commonly added in the art during the manufacture. In addition, unlike general drugs, there is an advantage in that there is no side effect that may occur when taking a drug for a long time by using food as a raw material, and it can be excellent in portability.

According to another embodiment of the present invention, the present invention provides a quasi-drug composition for preventing or improving prostatic hyperplasia, comprising a rhodiola extract containing kaempferol and eticathechin galade. The composition of the present invention may be added to a quasi-drug composition for the purpose of preventing or treating prostatic hyperplasia.

The composition of the present invention includes a rhodiola extract containing 15 mg/kg or more of kaempferol and 2 mg/kg or more of epicatechin gallate as active ingredients. In this specification, the term 'extract' used while referring to Rhodiola is not only an extraction result obtained by treating Rhodiola with an extraction solvent, but also a formulation (eg, powder, solution, capsule, tablet, etc.) so that Rhodiola itself can be administered to animals or humans. ) has a meaning that also includes the Rhodiola workpiece.

In the present invention, the term "quasi-drugs" refers to textiles, rubber products or similar products used for the purpose of treating, mitigating, treating or preventing diseases of humans or animals, products that have weak effects on the human body or do not directly act on the human body, and devices or non-machines and similar items, products falling under one of the agents used for sterilization, insecticidal, and similar purposes to prevent infectious diseases, for the purpose of diagnosing, treating, mitigating, treating, or preventing human or animal diseases It may refer to items that are not instruments, machines or devices among items used and items other than 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-drugs may include external skin preparations and personal hygiene products. Preferably, it may be a disinfectant cleaner, shower foam, gargreen, wet tissue, detergent soap, hand wash, or 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 used together with other quasi-drugs or quasi-drug ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use.

According to another embodiment of the present invention, the present invention provides a drinking water additive for preventing or improving prostatic hyperplasia containing the rhodiola extract. The drinking water additive of the present invention may be used in a way in which a composition containing the rhodiola extract is separately prepared in the form of a drinking water additive and mixed with drinking water or directly added during preparation of drinking water. The drinking water additive of the present invention may be in a liquid or dry state, preferably in a dried powder form. 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 if necessary. Non-limiting examples of the usable additives include binders, emulsifiers, and preservatives added to prevent deterioration of drinking water quality; There are amino acids, vitamins, enzymes, probiotics, flavors, non-protein nitrogen compounds, silicates, buffers, coloring agents, extractants, or oligosaccharides added to increase the effectiveness of feed or drinking water. can be further included. These may be used singly or two or more may be added together.

The present invention may provide a pharmaceutical composition for preventing or treating prostatic hyperplasia, comprising Kaempferol represented by Formula 1 below or a pharmaceutically acceptable salt thereof:

[Formula 1]

.

.

The present invention may provide a pharmaceutical composition for preventing or treating prostatic hyperplasia, comprising epicatechin gallate represented by Formula 2 below or a pharmaceutically acceptable salt thereof:

[Formula 2]

.

.

The present invention relates to kaempferol represented by Formula 1 below and epicatechin gallate represented by Formula 2 below; Or it may provide a pharmaceutical composition for preventing or treating prostatic hyperplasia, including a pharmaceutically acceptable salt thereof.

As a result of the experiment, the present inventors confirmed that the active ingredients of Rhodiola Rosea were kaempferol and epicatechin gallate. Accordingly, the prostatic stromal cells and prostatic hyperplasia cells were treated with Rhodiola agar, kaempferol and epicatechin gallate, and their IC ₅₀ concentrations were measured. Accordingly, it was confirmed that Rhodiola chrysanthemum extract was 24.8 ~ 45 ug/mL, kaempferol was 4.5 ~ 8.3 ug/ml, and epicatechin gallate was 11.4 ~ 16.2 ug/ml. Therefore, as a result of further experiments on the synergistic effect of kaempferol and epicatechin gallate in the killing effect of prostate stromal cells and prostatic hyperplasia cells, it was found that kaempferol and picatechin gallate were mixed at a molar ratio of 7:3 compared to treatment alone. showed the most significant killing effect at the time (FIG. 23).

The composition containing the Rhodiola Rosea extract of the present invention as an active ingredient contains 15 mg or more of kaempferol and 2 mg or more of epicatechin gallate with kaempferol and epicatechin gallate as indicators. By inhibiting the activity of alpha reductase, there is an effect of effectively preventing, improving, and treating benign prostatic hyperplasia. In addition, the composition containing Rhodiola chrysanthemum extract containing 15 mg or more of kaempferol and 2 mg or more of epicatechin gallate according to the present invention as an active ingredient is a natural medicine that is a food composition or a quasi-drug composition for preventing or improving prostate-related diseases. It can be applied to a variety of products, including In addition, the Rhodiola Rosea extract of the present invention does not show hepatotoxicity and renal toxicity as a result of animal experiments and is derived from natural products, so it can be safely and continuously used without causing severe stimulation or harmful effects in the body in addition to the efficacy of preventing and improving prostatic hyperplasia. there is.

Figure 1 is a comparison of the size of the prostate of the rat after 4 weeks of the experiment to confirm the effect of Rhodiola extract on the size of the prostate.
Figure 2 shows the effect of rhodiola extract on the prostate index.
Figure 3 shows the results of measuring the concentration of DHT and testosterone in the Rhodiola extract and prostate tissue and blood using an ELAISA kit.
Figure 4 shows the isolation and purification process of the compounds Rr-5-7 and Rr-5-8 having an activity on prostatic hyperplasia apoptosis.
Figure 5 shows the ESI-mass spectrum (top: positive mode, bottom: negative mode) of fraction Rr-5-7 isolated from rhodiola extract having prostatic hyperplasia cell killing effect.
Figure 6 shows the ¹ H NMR spectrum of the fraction Rr-5-7 isolated from Rhodiola extract having prostatic hyperplasia cell killing effect.
Figure 7 shows the ¹³ C NMR spectrum of the fraction Rr-5-7 isolated from the rhodiola extract having a prostatic hyperplasia cell killing effect.
Figure 8 shows the ¹ H- ¹ H COZY spectrum of the fraction Rr-5-7 isolated from the Rhodiola extract having a prostatic hyperplasia cell killing effect.
Figure 9 shows the partial structure (thick line) of the fraction Rr-5-7 isolated from Rhodiola extract having prostatic hyperplasia cell killing effect analyzed by ^1H - ^1H COZY spectrum.
Figure 10 shows the HMQC spectrum of the fraction Rr-5-7 isolated from the rhodiola extract having prostatic hyperplasia cell killing effect.
Figure 11 shows the HMBC spectrum of the fraction Rr-5-7 isolated from the rhodiola extract having a prostatic hyperplasia cell killing effect.
FIG. 12 shows two-dimensional NMR correlations of fractions Rr-5-7 isolated from rhodiola extract having prostatic hyperplasia cell killing effect.
Figure 13 shows ^{the 1} H NMR and ¹³ C NMR peak assignment of fraction Rr-5-7 isolated from Rhodiola extract having prostatic hyperplasia cell killing effect.
Figure 14 shows the ESI-mass spectrum (top: positive mode, bottom: negative mode) of fraction Rr-5-8 isolated from rhodiola extract having prostatic hyperplasia cell killing effect.
Figure 15 shows the ¹ H NMR spectrum of the fraction Rr-5-8 isolated from Rhodiola rosea extract having prostatic hyperplasia cell killing effect.
Figure 16 shows the ¹³ C NMR spectrum of the fraction Rr-5-8 isolated from the Rhodiola chrysanthemum extract having a prostatic hyperplasia cell killing effect.
Figure 17 shows the ¹ H- ¹ H COZY spectrum of the fraction Rr-5-8 isolated from Rhodiola extract having prostatic hyperplasia cell killing effect.
Figure 18 shows the partial structure (thick line) of the fraction Rr-5-8 isolated from Rhodiola extract having prostatic hyperplasia cell killing effect analyzed by ^1H - ^1H COZY spectrum.
Figure 19 shows the HMQC spectrum of the fraction Rr-5-8 isolated from Rhodiola extract having prostatic hyperplasia cell killing effect.
Figure 20 shows the HMBC spectrum of the fraction Rr-5-8 isolated from the Rhodiola extract having a prostatic hyperplasia cell killing effect.
FIG. 21 shows two-dimensional NMR correlations of fractions Rr-5-8 isolated from rhodiola extract having prostatic hyperplasia cell killing effect.
22 shows ^{the 1} H NMR and ¹³ C NMR peak assignments of fraction Rr-5-8 isolated from rhodiola extract having prostatic hyperplasia cell killing effect.
23 is a result of confirming the IC ₅₀ concentration of Rhodiola Rosea, kaempferol, and epicatechin gallate by treating prostate stromal cells and prostate hyperplasia cells.

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

Example 1. Preparation of rhodiola extract

The well-dried root of Rhodiola Rhodiola was sliced and ground, and 100 g of each powder was immersed in 1,000 mL of a 50% by weight ethanol solution for 30 minutes, followed by an ultrasonic extractor (SJC-II-50L, GREENAPPLE Co., Ltd.) fixed at 50 ° C. , 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, and the filtrate (1) was taken separately, 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 filtrate (1) and (1), ethanol was removed using a vacuum concentrator, and then freeze-dried to obtain about 35 to 40 g of ultrasonic extract powder.

Experimental Example 1. Check the effect of improving prostatic hyperplasia of Rhodiola extract

1-1. Prostatic hyperplasia animal model

It was divided into normal control group, benign prostatic hypertrophy group, benign prostatic hyperplasia induced group with rhodiola extract administered (2.5, 5, 10 mg/kg), and benign prostatic hyperplasia induced group with finasteride (1mg/kg) administered as a positive control group. Testosterone propionate (TP, 3 mg/kg, Samil Pharmaceutical, Korea) was administered subcutaneously daily to 8 male white papers at 10 weeks to induce prostatic hyperplasia, and rhodiola extract and finasteride were orally administered.

1-2. Isolation of blood and prostate tissue

After 4 weeks of the experiment, all rats were fasted for 24 hours, inhaled anesthetized using isofuran/nitrogen/oxygen, blood was collected by laparotomy, and then the prostate was removed and weighed. The extracted prostate was weighed after removing the blood with physiological saline and removing the remaining water and blood with filter paper.

1-3. Measurement of prostate weight and weight-to-weight ratio

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

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

1-4. Measurement of testosterone levels in the blood

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

1-5. statistical analysis

Statistical analysis was performed by ANOVA. ^* p<0.01, ^** p<0.001 are significantly different compared to the normal control group, ^# p<0.05, ^## p<0.01 are compared to the prostatic hyperplasia induced group. Cases with significant differences were considered.

<Experiment result>

Effect of rhodiola extract on prostate index

The prostate index was calculated by dividing body weight (100 g) by prostate tissue weight (mg). As shown in FIG. 2, the total prostate weight index was increased in the BPH-induced group compared to the normal control group, while the total prostate weight index was decreased in all Rhodiola extract-administered groups compared to the BPH-induced group. Compared to the prostatic hyperplasia induced group, in the finasteride-administered group, which is a positive control group, no change in prostate index was observed, unlike the rhodiola extract-administered group (FIG. 2).

Determination of concentrations of dihydrotestosterone (DHT) and testosterone in blood and prostate tissue

To examine the ability of 5-AR to convert testosterone to DHT, testosterone and DHT concentrations were measured. As a result, the DHT concentration in the blood and prostate tissue of the prostatic hyperplasia induced group was significantly higher than that of the normal control group, but the DHT concentration level of the rhodiola extract-treated group was significantly lower than that of the prostatic hyperplasia induced group (FIG. 3) . However, the concentration of testosterone in prostate tissue and blood appeared opposite to the pattern of DHT concentration. That is, it is thought that the Rhodiola extract reduced the expression of 5-AR, and as the activity was lowered, the amount of testosterone converted to DHT was reduced and recovered.

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

2-1. Separation and purification of active ingredients

Add 10 L of 70% methanol aqueous solution to 9.3 kg of Rhodiola extract powder, extract at 60 ° for 2 hours, filter to separate the filtrate (3), add 10 L of 70% methanol aqueous solution to the remaining residue, and repeat the extraction. After filtering, a filtrate (4) was obtained. After combining the filtrate (3) and the filtrate (4), methanol was removed by concentration under reduced pressure. The solvent-free filtrate was adsorbed on Diaion HP-20 resin and eluted with 4 L each of 30%, 60%, and 100% methanol aqueous solutions.

The 60% methanol elution fraction, which shows the apoptotic activity of BPH cells, was concentrated under reduced pressure, and silica gel column chromatography was performed using chloroform-methanol (100:1→1:1, v/v, stepwise) as an elution solvent. 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 ~ Rr-5-14), and the apoptotic activity for BPH cells in 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 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 having high purity were concentrated and named 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 interpretation

ESI-mass spectrum was measured to reveal the molecular weight of the purified active compound Rr-5-7. That is, as a result of measuring the ESI-mass spectrum in positive mode, [M+H] ⁺ was observed at m/z 287.2, and as a result of measuring ESI-mass spectrum in negative mode, [MH] ^- was observed at 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 identify the chemical structure of compound Rr-5-7, it was dissolved in CD ₃ OD and analyzed by measuring ¹ H NMR, ¹³ C NMR, ¹ H- ¹ H COSY, HMQC, and HMBC spectrum.

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

As a result of measuring ¹ H NMR spectrum by dissolving in CD ₃ OD (Fig. 6), 8.06 (2H, d, J = 8.5 Hz), 6.90 (2H, d, J = 8.5 Hz) ppm in 1,4-disubstituted benzene Two aromatic methine proton peaks were observed at 6.39 (1H, d, J = 2.0 Hz) and 6.19 (1H, d, J = 2.0 Hz) ppm. It was found that the coupling constant of 2.0 Hz observed between 6.39 and 6.19 ppm is meta-coupled with each other.

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

As a result of measuring ¹³ C NMR spectrum (FIG. 7), carbonyl carbon at 177.4 ppm, 165.8, 162.5, 160.6, 158.3, 148.0, and oxygenated sp ² quaternary carbon at 137.1 ppm, 130.7 (X2), 116.3 (X2), 99.3, Sp ² methine carbon at 94.5 ppm and sp ² quaternary carbon at 123.7 and 104.5 ppm were observed. These characteristics indicated that this compound was a flavonoid compound. After that, the chemical structure of this compound was identified by measurement and analysis of two-dimensional NMR spectrum such as ¹ H- ¹ H COSY, HMQC, and HMBC.

2-2-1-2-3. ^One H- ^One Measurement and interpretation of H COZY spectrum

As a result of measuring and analyzing the ^{1H- 1H} COZY spectrum (FIG. 8), two partial structures including 1,4-disubstituted benzene shown in FIG. 9 below 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 interpreting the HMBC spectrum (FIG. 11), from 8.06 ppm of aromatic methine proton to 160.6 and 148.0 ppm of oxygenated sp ² carbon, from 6.90 ppm of aromatic methine proton to 123.7 ppm of carbon, and 6.57 ppm of aromatic methine proton A long-range correlation was observed for sp ² quaternary carbon at 163.2 and 153.9 ppm from . In addition, a long-range correlation was observed from 6.39 ppm of aromatic methine proton to 165.8, 158.3, 104.5, 99.3 ppm of carbon, and from 6.19 ppm of aromatic methine proton to 165.8, 162.5, 104.5, 94.5 ppm of carbon. The chemical structure of this compound was determined as shown in FIG. 12 from these HMBC correlations, the remaining 177.4 ppm of carbonyl carbon, the chemical shift value of 137.1 ppm of quaternary carbon, and the molecular weight of 286. As a result of database search based on the above chemical structure, this 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 compound Rr-5-7.

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

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

ESI-mass spectrum was measured to reveal the molecular weight of the purified active compound Rr-5-8. That is, as a result of measuring the ESI-mass spectrum in positive mode, [M+H] ⁺ was observed at m/z 443.2, and as a result of measuring ESI-mass spectrum in negative mode, [MH] ^- was observed at 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 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

As a result of measuring ¹ H NMR spectrum after dissolving in DMSO- d ₆ (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) ppm, two aromatic methine protons, 5.92 (1H, d, J = 2.0 Hz), superimposed on 6.81 (2H, s) ppm, derived from 1,2,4-trisubstituted benzene Two aromatic methine proton peaks were observed at 5.82 (1H, d, J = 2.0 Hz) ppm. It was found that the coupling constant of 2.0 Hz observed between 5.92 and 5.82 ppm is meta-coupled with each other. Also, two oxygenated methines at 5.34 and 5.01 ppm and one non-equivalent 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

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

2-2-2-2-3. ^One H- ^One Measurement and interpretation of H COZY spectrum

As a result of measuring and analyzing the ^{1H- 1H} COZY 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 interpreting 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 protons and 144.6 ppm of oxygenated from 6.64 ppm of aromatic methine protons 156.4, 156.0, 97.2, 94.3 ppm of sp ² carbon and 129.3 ppm of carbon from 5.92 ppm of aromatic methine protons, and 156.4, 155.5, 97.2, 95.5 ppm of sp ² carbon from 5.82 ppm of ^aromatic methine protons A long-range correlation was observed. In addition, a long-range correlation was 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 the gallate moiety was formed from their chemical shift (FIG. 21). Therefore, it was determined that this compound had a gallate moiety bound to epicatechin. Although the bonding position of the gallate moiety could not be confirmed, the bonding position of the gallate moiety was inferred from the chemical shift value of the oxygenated methine proton stored at 5.34 ppm, and thus the chemical structure of this compound was determined as shown in FIG. 22. As a result of database search based on the above chemical structure, this compound was identified as epicatechin gallate (same name: Epicatechin 3-O-gallate; Epicatechol 3-gallate). 22 shows the assignment of ¹ H NMR and ¹³ C NMR peaks of compound Rr-5-8.

Experimental Example 3. Killing effect of Kaempferol and ECG on benign prostatic hyperplasia and prostate stromal cells

Benign prostatic hyperplasia cells (BPH-1 (purchased from ATCC)) and prostate stromal cells (WMPY-1; purchased from ATCC) were spread on a 96-well plate at 1x10 ⁶ and incubated at 37°C for 24 hours, then rhodiola. The extract, kampferol (Sigma aldrich), and ECG (Sigma aldrich) were dissolved in DMSO and treated for each concentration, and the IC ₅₀ concentration was measured through MTT assay. As shown in Table 1 below, Rhodiola 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 10uM, and Kaempferol and ECG were mixed and treated at various molar ratios. Then, prostatic hyperplasia cells and prostate stromal cells were treated with Kaempferol and ECG, and IC ₅₀ concentrations were measured. As a result, Kaempferol and ECG When treated with a molar ratio of 7:3 compared to single treatment, the most significant killing effect was shown (FIG. 23). Although 7:3 was significantly better than treatment alone, the killing effect was superior to other molar ratios (5:5 or 3:7).

benign prostatic cells prostate stromal cells Rhodiola 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)

Kaempferol represented by Chemical Formula 1 and epicatechin gallate represented by Chemical Formula 2; Or as a pharmaceutical composition for the prevention or treatment of prostatic hyperplasia, comprising a pharmaceutically acceptable salt thereof,
The kaempferol: epicatechin gallate was included in a molar ratio of 7:3,
The killing effect of BPH cells at the molar ratio is increased by 63% to 106% compared to the treatment with kaempferol or epicatechin gallate alone,
A pharmaceutical composition for preventing or treating prostatic hyperplasia, characterized in that the killing effect of prostate stromal cells at the molar ratio is increased by 38% to 86% compared to the treatment with kaempferol or epicatechin gallate alone:
[Formula 1]

,
[Formula 2]

delete Kaempferol represented by Chemical Formula 1 and epicatechin gallate represented by Chemical Formula 2; Or as a health functional food composition for preventing or improving prostatic hyperplasia, including pharmaceutically acceptable salts thereof,
The kaempferol: epicatechin gallate was included in a molar ratio of 7:3,
The killing effect of BPH cells at the molar ratio is increased by 63% to 106% compared to the treatment with kaempferol or epicatechin gallate alone,
A functional food composition for preventing or improving prostatic hyperplasia, characterized in that the killing effect of prostate stromal cells at the molar ratio is increased by 38% to 86% compared to the treatment with kaempferol or epicatechin gallate alone:
[Formula 1]

,
[Formula 2]

delete delete delete delete delete delete delete delete

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