KR20200133968A - Composition comprising extracts of saw palmetto and rhodiola sachalinensis for preventing or treating benign prostatic hyperplasia - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a food composition for preventing, alleviating or treating prostatic hyperplasia containing a Rhodiola rosea extract as an active component. A composition of the present invention not only has no side effects through establishment of manufacturing conditions of the Rhodiola rosea extract, but also significantly improves treatment effects of prostatic hyperplasia and exhibits stable efficacy, and thus exhibits a synergistic effect when used in combination with a Serenoa repens extract, thereby being able to be usefully used as an active component of a prostatic hyperplasia therapeutic agent, quasi-drugs, and the food composition.

Description

A composition for preventing, improving, or treating prostatic hypertrophy containing saw palmetto and rhododendron extract as active ingredients {COMPOSITION COMPRISING EXTRACTS OF SAW PALMETTO AND RHODIOLA SACHALINENSIS FOR PREVENTING OR TREATING BENIGN PROSTATIC HYPERPLASIA}

The present invention relates to the use of honggyeongcheon extract or honggyeongcheon and saw palmetto complex extract to prevent, improve or treat prostatic hypertrophy.

Benign prostatic hyperplasia (BPH) has been known as a very common disease in men after their 40s around the world after the clinical definition for it was established. Symptoms of BPH include lower urinary tract symptoms (LUTS), which are urinary disorders, including urgency, frequency, weak stream, nocturia, and cup due to insufficient urination. Symptoms such as incomplete emptying of urine appear. BPH is known to be caused by overproliferation of prostate epithelial cells and stromal cells surrounding the urethra. The cause of this is that testosterone decreases with age. Dihydrotestosterone (DHT), which is produced by conversion of testosterone by 5α-reductase (5-AR), is known to stimulate prostate cells to grow abnormally. The biggest discomfort caused by an enlarged prostate is that it is difficult to relieve fatigue due to the inability to maintain sufficient sleep at night, or to receive severe psychological stress due to frequent urination symptoms during outdoor activities.

To improve prostatic hyperplasia, 5-AR inhibitors or α-adrenergic receptor blockers have been prescribed in the form of pharmaceuticals. Several studies have used plant extracts such as tomato and black bokbunja extract to control the expression of DHT and 5-AR to prevent or treat prostatic hyperplasia. Among them, the extract of saw palmetto berry (SP) is a representative natural material and is widely used as a health supplement for improving prostatic hyperplasia ^[1] . In Korea, more than 30 companies are producing and selling related products as it has been recognized as a notified raw material for health functional food useful for prostate health. Early clinical studies on saw palmetto have shown that saw palmetto is effective for lower urinary tract symptoms caused by prostatic hyperplasia ^[2] . However, after Avins and Bent ^[3] reported that they did not find any scientific evidence that saw palmetto improves prostatic hyperplasia through their research, problems with the efficacy and toxicity of saw palmetto on prostatic hyperplasia have continued. Has emerged ^{[4, 5]} . In particular, although saw palmetto extract is a widely used dietary supplement, most studies on potential toxicity or side effects have been conducted for less than one year, so there is a need for more clear toxicity studies based on various population groups and study periods. Has been raised ^[6] . Recently, Avins et al. ^[7] reported that no serious toxicity was observed even when saw palmetto was ingested at a concentration of up to 960 mg per day for 18 months, but asymptomatic as well as analysis on the interaction between prescription drugs and other dietary supplements was still excluded. He mentioned that there is a need for research on side effects that may occur when taking saw palmetto extract for a long time. In Korea, since the issue of the efficacy of saw palmetto extract for improving prostatic hyperplasia was raised in 2015 through the Korean Society for Urinary Incontinence with Urination Disorders ^[8] , health functions were conducted by the Ministry of Food and Drug Safety in 2017 according to the Health Functional Food Act. Announced a food reevaluation plan. Despite the reports of negative research results on saw palmetto at home and abroad, saw palmetto extract is still sold well as a solution for men with lower urinary tract symptoms, so the cytotoxicity and prostate health of saw palmetto extract In addition to the scientific basis for the presence or absence of the improvement effect, there is a need to develop high-functional prostate health improvement-related materials derived from highly safe natural products in addition to saw palmetto extract.

On the other hand, Rhodiola sachalinensis is a perennial plant that grows in alpine regions over 2,000 m above sea level, and its roots are well dried and used as a material for alternative medicines. Honggyeongcheon is rich in phenylpropanoids, flavonoids, tannins, glycosides and organic acids ^[9] . The main ingredients of Honggyeongcheon include salidroside, rosavins and ρ-tyrosol, which are effective in improving depression, fatigue and cognitive dysfunction. It is known to have anti-inflammatory, anti-cancer, cardiovascular and neuroprotective effects ^[10] . Currently, extracts of Rhodiola sachalinensis (RS) is a noticeable raw material that can help improve fatigue, and various products are being developed, but the efficacy for improving prostate health has not been proven, so it has not been commercialized.

The inventors of the present invention confirmed that the extract of honggyeongcheon cures prostatic hyperplasia while researching the treatment for prostatic hyperplasia with less side effects, and after that, through continuous research, we established the manufacturing conditions for the extract of honggyeongcheon, which shows superior prostatic hypertrophy treatment effect. . Furthermore, the present invention was completed by confirming that the complex extract obtained by mixing the honggyeongcheon extract with the saw palmetto extract did not exhibit the cytotoxicity of the saw palmetto extract alone and at the same time synergistically induces the therapeutic effect of prostatic hyperplasia.

An object of the present invention is to provide a technology capable of treating an enlarged prostate without side effects.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above-described problems, the present invention provides a pharmaceutical composition for preventing or treating prostatic hyperplasia, containing an extract of Rhodiola sachalinensis as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of prostatic hyperplasia, comprising honggyeongcheon ( Rhodiola sachalinensis ) extract and saw palmetto extract as active ingredients.

According to an embodiment of the present invention, the honggyeongcheon extract may be prepared by immersing the honggyeongcheon powder in an ethanol solution and then ultrasonically extracting.

According to an embodiment of the present invention, the saw palmetto extract may be a saw palmetto fruit extract.

According to an embodiment of the present invention, the honggyeongcheon extract and saw palmetto extract may be included in a weight ratio of 1:2 to 2:1.

The present invention also provides a health functional food composition for preventing or improving prostatic hyperplasia, containing honggyeongcheon ( Rhodiola sachalinensis ) extract and saw palmetto extract as active ingredients.

Hereinafter, the present invention will be described in more detail.

As described above, saw palmetto extract is a representative natural material and is widely used as a health supplement for improving prostatic hyperplasia worldwide, but it has been reported that no scientific evidence that saw palmetto improves prostatic hyperplasia has been found. Since then, while issues regarding the efficacy and toxicity of saw palmetto on prostatic hyperplasia have been continuously raised, scientific evidence for the presence or absence of the cytotoxic and prostate health improvement effect of saw palmetto extract is needed, as well as saw palmetto extract. In addition, development of materials related to high functional prostate health improvement derived from natural products with high safety is continuously required.

Therefore, the present inventors, while searching for a safe natural product therapeutic agent in the body while effectively treating prostatic hyperplasia, the extract of Hong Gyeong-cheon alone did not show toxicity to testicular cells and normal prostate cells, and specifically kills prostatic hyperplasia cells, thereby reducing prostatic hyperplasia. It was confirmed that there is a preventive or therapeutic effect, and the conditions for producing honggyeongcheon extract that can maximize the treatment effect of prostatic hyperplasia were established.

In addition, it was confirmed that the extract of saw palmetto alone was surprisingly insignificant in killing prostatic hyperplasia cells, while having cytotoxicity to testis cells and normal prostate cells.

Furthermore, it was found that the mixed extract of saw palmetto extract and honggyeongcheon extract at a concentration of insignificant treatment effect for prostatic hyperplasia showed an elevated prostatic hyperplasia cell killing effect compared to each single extract, while the cytotoxicity to normal cells was significantly reduced. And completed the present invention.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of prostatic hyperplasia, comprising the extract of Rhodiola sachalinensis as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of prostatic hyperplasia, containing honggyeongcheon ( Rhodiola sachalinensis ) extract and saw palmetto extract as active ingredients.

In the present invention, the term "pharmaceutical composition" refers to a mixture in which other chemical components such as a diluent or a carrier are mixed with a composition comprising the extract of Rhododendron and saw palmetto of the present invention as active ingredients.

In the present invention, the term "treatment" refers to an approach to obtain beneficial or desirable clinical results. It may also mean increasing the survival rate compared to the expected survival rate without treatment. In the present invention, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include the disorder to be prevented as well as the treatment required for a disorder that has already occurred.

In the present invention, the term “synergistic” means that the effect generated when each ingredient is administered in combination (combination) is greater than the sum of the effects generated when administered alone as a single ingredient [Chou and Talalay , Adv. Enzyme. Regul., 22:27-55, 1984].

In the present invention, the term "honggyeongcheon" means that the roots of honggyeongcheon ( Rhodiola sachalinensis ) are dried. Hong Gyeong-cheon is known to be effective in improving depression, fatigue and cognitive dysfunction, as well as antioxidant, anti-inflammatory, anti-cancer, cardiovascular and neuroprotective effects.

In the present invention, the term “Saw Palmetto” means dried fruits of Saw Palmetto ( Serenoa repens ).

The honggyeongcheon and saw palmetto of the present invention may be purchased and used commercially, or may be directly harvested or cultivated in nature.

In a specific embodiment of the present invention, an extract extracted by ultrasonication after immersing honggyeongcheon powder in an ethanol solution was used.

In the present invention, the term "extract" refers to a liquid component obtained by immersing a target substance in various solvents and then extracting it at room temperature, low temperature, or warm state for a certain period of time, and removing the solvent from the liquid component. It means the result of obtained solid content etc. In addition, in addition to the resulting product, it can be comprehensively interpreted as including all of the diluted solution of the resultant, the concentrate thereof, the preparation thereof, and the purified product.

In a specific embodiment of the present invention, a complex extract obtained by mixing honggyeongcheon and saw palmetto extracts in a weight ratio of 1:2, 1:1, and 2:1, respectively, was treated on the prostatic hyperplasia cells to confirm the therapeutic effect of prostatic hyperplasia. As a result, as shown in FIG. 3, when the honggyeongcheon and saw palmetto extract were mixed and administered in a weight ratio of 1:2 to 2:1, it could be seen that a synergistic effect on prostatic hyperplasia cell death was exhibited. Specifically, the synergistic effect on prostatic hyperplasia cell death was confirmed in all complex extract administration groups.

The composition of the present invention is not toxic to testicular cells and normal prostate cells and thus has an additional advantage to the human body.

In a specific embodiment of the present invention, in order to confirm the human stability of the extracts of honggyeongcheon and saw palmetto, the cell viability was confirmed after treatment with these extracts on testis cells and normal prostate cells. As a result, it was confirmed that the survival rate of normal cells by saw palmetto was only 50-60% at the therapeutically effective concentration, so that the cytotoxicity was very high.In contrast, Rhododendron extract was effective against normal cells in all ranges with therapeutic effects. It was confirmed that it did not show cytotoxicity. In addition, when the saw palmetto extract and the rhododendron extract are mixed in a weight ratio of 4:1, 2:1, 1:1, and 1:2, in the testis cells, the saw palmetto extract and the rhododendron extract are mixed in a weight ratio of 1:2. It was confirmed that cytotoxicity was significantly suppressed in the mixed administration group, and in the normal prostate cells, it was confirmed that the cytotoxicity was significantly suppressed from the group administration of the saw palmetto extract and the rhododendron extract at a weight ratio of 1:1. I did.

Therefore, the composition according to the present invention can be safely used without side effects to the human body while increasing the therapeutic effect of prostatic hyperplasia.

The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical composition according to the present invention is formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. I can.

Carriers, excipients, and diluents that may be contained in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient for the compound, such as starch, calcium carbonate, sucrose or lactose. , Gelatin, etc. are mixed to prepare it. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. .

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

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 the effective dose level is the type and severity of the individual, age, sex, and type of infected virus. , Drug activity, sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The 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 side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention can be used alone or in combination with surgery, hormone therapy, drug therapy, and methods of using a biological response modifier for the prevention or treatment of prostatic hyperplasia.

The pharmaceutical composition may be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection.

In another aspect, the present invention provides a health functional food composition for preventing or improving prostatic hyperplasia, containing honggyeongcheon ( Rhodiola sachalinensis ) extract and saw palmetto extract as active ingredients.

The health functional foods include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, It may contain organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, it may contain natural fruit juices and pulp for the production of fruit juices and vegetable beverages. These components may be used independently or in combination. In addition, health functional foods may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin complex. I can.

The health beverage composition of the present invention is not particularly limited to the liquid component, except that it contains a mixture of honggyeongcheon and saw palmetto extract, and may contain various flavoring agents or natural carbohydrates as an additional component, such as a conventional beverage.

In addition, the above health functional food may additionally contain food additives, and the suitability as a “food additive” is determined by the general rules of the Food Additive Code approved by the Food and Drug Administration and the general test method, etc. It is judged according to the relevant standards and standards.

Items listed in the "Food Additives Code", for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as reduced pigment, licorice extract, crystalline cellulose, high cooling pigment, guar gum, etc., L -Mixed preparations such as sodium glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar coloring preparations, etc. are mentioned.

In the health functional food composition for preventing or improving prostatic hyperplasia, the description of honggyeongcheon and saw palmetto extract, its effects, and all related descriptions are the same as described above, so that the description is made to avoid excessive complexity of the present specification due to redundant description. Omit it.

The composition of the present invention not only has no side effects even when administered for a long period of time, but also has excellent and stable efficacy for treating prostatic hyperplasia, and exhibits a synergistic effect, especially when used in combination with saw palmetto extract, to prevent or treat prostatic hyperplasia, quasi-drugs, health functions. It can be usefully used as a food material.

1 is a result of confirming the therapeutic effect of prostatic hyperplasia of rhododendron extract or saw palmetto extract: A is a result of measuring cell viability after treatment of rhododendron or saw palmetto alone extract by concentration on prostatic hyperplasia cells, B is LDH activity Analysis results.
Figure 2 is a result of confirming the synergistic treatment effect of prostatic hypertrophy of the composite extract of honggyeongcheon and saw palmetto.
Figure 3 is a result of confirming the synergistic treatment effect of prostatic hyperplasia concentration-dependent honggyeongcheon extract: A is a result of measuring the cell viability after treating prostatic hyperplasia cells at different concentration ratios, B is Western Blot result, C is the ELISA measurement result.
Figure 4 is a result of confirming the cytotoxicity of the testis cells of honggyeongcheon extract or saw palmetto extract: A is a result of measuring the cell viability after treatment of the testis cells with the extract of Rhododendron or saw palmetto alone, B is LDH As a result of analyzing activity.
Figure 5 is a result of confirming the cytotoxicity of the prostate normal cells of the rhododendron extract or saw palmetto extract: A is the result of measuring the cell viability after treating the prostate normal cells with the extract of Rhodiolaris or saw palmetto alone by concentration, B Is the result of analysis of LDH activity.
6 is a result of confirming the cytotoxicity reduction effect of the composite extract of honggyeongcheon and saw palmetto on testis cells: A is a result of measuring the cell viability after treating the testis cells with the complex extract, B is a result of analyzing LDH activity , C is the result of measuring tosterone.
Figure 7 is a result of confirming the cytotoxicity reduction effect of the honggyeongcheon and saw palmetto complex extract on normal prostate cells: A is a result of confirming the increase in cell viability dependent on the concentration of the honggyeongcheon extract concentration, B is a result of a decrease in LDH activity dependent on the concentration of the honggyeongcheon extract Confirmation result.

Hereinafter, the present invention will be described in more detail based on Examples and Test Examples. However, the present invention is not limited by the following Examples or Test Examples.

[Example 1]

Establishment of manufacturing conditions for Honggyeongcheon extract

<1-1> Experimental method

High-temperature pneumatic extraction and ultrasonic extraction were compared in order to establish an extraction method for mass extraction of useful substances effective in improving prostatic hyperplasia from Rhododendron. At this time, the solvent for extraction was prepared separately in 30% by weight and 50% by weight by diluting ethanol in purified water, and then 100 g of each powder was immersed in each of 1,000 mL of ethanol solution. Pneumatic extraction was performed using a pneumatic extractor (DH 700, Daehan Mediaan, Korea) at a pressure of 100 Mpa for 12 hours at 50°C, followed by centrifugation at 10,000 rpm to separate the filtrate (1). After extraction, 1,000 mL of ethanol was added to the residue again, followed by pneumatic extraction at 50° C. for 24 hours and centrifuged at 10,000 rpm to obtain a new filtrate (2). After mixing the filtrates (1) and (2), ethanol was removed using a vacuum concentrator, and then freeze-dried to obtain a high-pressure extract powder. For ultrasonic extraction, 100g of each powder was immersed in 1,000 mL of 30% by weight and 50% by weight ethanol solution for 30 minutes, and then allowed to stand for 1 hour in an ultrasonic extractor (SJC-II-50L, GREENAPPLE, China) fixed at 50°C. Extracted for 30 minutes at a frequency of 20 KHz at the output of W. After extraction, the extract was centrifuged at 10,000 rpm, the filtrate (3) 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 (4). After mixing the filtrates (3) and (4), ethanol was removed using a vacuum concentrator, and then freeze-dried to obtain an ultrasonic extract powder.

<1-2> Confirmation of extraction yield

The 30% by weight ethanol extract and 50% by weight ethanol extract obtained from Honggyeongcheon were weighed, and the content of solids obtained from 100 g of the powder sample was calculated as a percentage (%) according to extraction conditions to calculate the extraction yield.

[Table 1]

Comparison of solid content extraction yield according to extraction method (p<0.05)

In the same sample, different English superscripts (a-b) of the value of the solid content extraction rate obtained from the difference in ethanol solution concentration according to the same extraction method indicate that there is statistical significance (p<0.05).

Other English superscripts (X-Y) of the solid content extraction rate values obtained according to the extraction method under the same ethanol solution conditions in the same sample indicate statistical significance (p<0.05).

The solid content extraction rate according to the extraction method in ethanol solvent conditions is shown in [Table 1]. The higher the ethanol content, the higher the extraction rate of the honggyeongcheon extract was under the ultrasonic extraction conditions than the high-temperature pneumatic extraction. Specifically, there was no statistical significance in pneumatic extraction, but it was found to slightly increase. On the other hand, in ultrasonic extraction, the extraction rate was 29.39% when the ethanol concentration was 30% by weight and 37.53% when the ethanol concentration was 50% by weight, indicating that about 8.19% more was extracted. When converted back to percentage, about 21.68% by weight Showed increased extraction efficiency. When the ethanol concentration was fixed at 50% by weight, the extraction rate of pneumatic extraction was 26.24%, and the extraction rate of ultrasonic extraction was 11.29% higher, and the extraction rate was increased by about 30.08% in terms of percentage.

<1-3> Confirmation of total polyphenol content

After preparing a dried sample at a concentration of 1.0 mg/mL, 1.8 mL of distilled water was added to 200 μL of the solution, 200 μL of 1N Folin-Ciocalteu's phe-nol reagent was added, and then left at room temperature for 5 minutes. 2 mL of 7% Na2CO3 was added to the mixture, and the mixture was allowed to stand at room temperature for 1 hour, and the absorbance was measured at 760 nm. The standard curve was prepared using gallic acid.

[Table 2]

Comparison of total polyphenol content in solids according to extraction method (p<0.05)

The total polyphenol content contained in the solid content according to the extraction method is shown in [Table 2]. The higher the ethanol content in Honggyeongcheon, the higher the total polyphenol content was in ultrasonic extraction conditions than in pneumatic extraction.

<1-4> DPPH radical scavenging ability

DPPH radical scavenging ability is obtained by dispensing 0.5 mL of 0.2 mM 2,2-diphenyl-2-picrylhydrazl (DPPH) solution to 0.5 mL of the extract (10.0 μg/mL), leaving it for 30 minutes, and absorbance at 517 nm (GENESYS 10 UV). Was measured. The control group was measured by the same method by adding a solvent instead of the sample. The positive control was tested by preparing Vitamin C at 10.0 μg/mL. In addition, in order to correct the absorbance value for the color of the sample itself, methanol was added instead of 0.2 nm DPPH to measure the absorbance. The antioxidant activity of the extract was expressed as a percentage based on the obtained value, and the closer the converted value to 100%, the higher the antioxidant activity was expressed.

[Table 3]

DPPH radical scavenging ability (p<0.05) at the same concentration (10.0μg/mL) of each extract according to the extraction method

DPPH radical scavenging ability values according to the extraction method in ethanol solvent conditions are shown in [Table 3]. The higher the ethanol content of Rongkyungcheon, the higher the DPPH radical scavenging ability in ultrasonic extraction than in pneumatic extraction. Specifically, when the ethanol concentration of honggyeongcheon powder was 30% by weight, pneumatic extraction was 57.28% and ultrasonic extraction was 72.32%, which was 15.04% (20.79% as a percentage).

<1-5> Toxicity to normal prostate cells

The cytotoxicity of the extract according to the extraction method to normal prostate cells was tested by MTT assay. MRC-5 fibroblasts per well and normal RWPE prostate cells were inoculated into a 96 well plate and cultured at 37°C for 24 hours to obtain a cell monolayer, and the extract diluted with a cell inoculation medium 6.25 ~ 100 ug/ mL was inoculated at 100 uL/well and cultured for 48 hours. After 48 hours, 100 uL of the culture solution was removed, 10 uL of MTT solution was added to each well, and the mixture was allowed to stand for 4 hours in an incubator at 37°C at 5% CO2. After the MTT solution was completely removed, formazan crystals formed in the cells were dissolved with 100 μL dimethyl sulfoxide (DMSO), and the absorbance was measured at 490 nm with an ELISA plate reader (BioTec Ex800, USA).

[Table 4]

Toxic effect of each extract (100 μg/mL) on normal cells according to the extraction method (p<0.05)

[Table 4] shows the cytotoxic effects of the two extracts according to the extraction method on normal cells. As a result of cytotoxicity in normal cell lines MRC-5 fibroblasts and RWPE prostate normal cells, rhododendron extract was not able to confirm cytotoxicity in both 30% by weight and 50% by weight of ethanol, an extract prepared by ultrasonic and pneumatic extraction. It was also found that there was no cytotoxicity as no apoptosis effect was found.

<1-6> Apoptosis effect of prostatic hyperplasia cells

The killing effect of the extract of Rhododendron rhododendron according to the extraction conditions on prostatic hyperplasia cells was tested by MTT (3-(4,5-dimethylthiazol)-2, 5-diphenyltetrasolium bromide) assay. BPH-1 cells at 1×10 ⁵ cells/well density in a 96-well culture plate containing 10% fetal bovine serum, 100 units/ml penicillin and 100 ug/ml streptomycin in RPMI 1620 medium at 37°C, 5% After 1 day incubation in a CO ₂ environment, it was replaced with RPMI 1640 medium to which serum was not added, and each extract was added for each concentration and cultured for 24 hours. MTT solution (5mg/ml, Sigma, USA) was added at 20ul per well, reacted at 37°C for 4 hours, the MTT solution was removed, and DMSO was added at 100ul per well. After dissolving the formazan derivative at room temperature, absorbance was measured at 490 nm.

[Table 5]

Killing effect of prostatic hyperplasia epidermal cells (BPH-1) at the same concentration (100 μg/mL) of each extract according to the extraction method (p<0.05)

The killing effect of the extract of honggyeongcheon according to the extraction method on prostatic hyperplasia cells was measured. As shown in [Table 5], as a result of cytotoxicity in PC3 prostatic hyperplasia cells, the positive control group administered with finesteride showed a cell viability of 66.25%, and it could be confirmed that there was a cell apoptosis effect in the extract sample of Honggyeongcheon. When the ethanol concentration was high and the ultrasonic extraction method was used, the killing effect of prostatic hyperplasia cells was higher.

In conclusion, in the test to develop the conditions for extracting honggyeongcheon according to the present invention, it was found that the ultrasonic extraction method extracts more substances useful for improving prostatic hyperplasia than the pneumatic extraction method.

[Example 2]

Preparation of Rhododendron and Saw Palmetto Complex Extract

<2-1> Preparation of Honggyeongcheon extract

The honggyeongcheon extract was prepared by applying the optimal extraction conditions of the honggyeongcheon extract identified in Example 1. Specifically, Hong Gyeongcheon was provided by Yanbian University in China as a product from Tibet Autonomous Region, China. After making a well-dried rhododendron into powder, 100g of the powder is immersed in 1 L of 50% ethanol solution for 60 minutes, and then repeated twice at 30℃ for 30 minutes using an ultrasonic extractor (SJC-Ⅱ-50L, GREENAPPLE, Uhan, China). After extraction, centrifugation was performed at 5,000 rpm for 20 minutes at 4°C, and the supernatant was taken and lyophilized. The freeze-dried rhododendron extract powder was vacuum-packed and stored in a refrigerator to be used in the following experiment.

<2-2> Preparation of saw palmetto extract

The saw palmetto extract used in the present invention was purchased from K company.

<2-3> Preparation of complex extract

A composite extract was prepared by mixing the honggyeongcheon extract prepared above and the saw palmetto extract, and then used in the experiment.

[Experiment method]

Cell culture

Prostate normal cells (RWPE) and BPH cells (BPH-1) used in this experiment were distributed from Hanyang University College of Medicine, and TM3 testis cells were distributed from Korea Cell Line Bank (KCLB, Seoul). Cell culture was performed by adding 10% fetal bovine serum (FBS) 100 unit/mL penicillin and 100 μg/mL streptomycin to DMEM (Dulbecco Modified Eagle Medium) and RPMI-1640 medium, and maintaining 95% humidity. It was cultured in a 37°C, 5% CO ₂ incubator (MCO-170AIC-PK, Panasonic, Sakata, Japan). Subculture was performed while the cells were grown to about 90% of the floor area.

MTT analysis

In each cell line, MTT (Tetrazolium, Sigma) analysis was performed to confirm the cell viability for RS and SP. Dispense 100 μL of each cell line into a 96-well plate at a concentration of 110 ⁵ cells/mL, incubate in a 37°C, 5% CO ₂ incubator for 24 hours, and treat the samples at a concentration of 6.25 ~ 100 μg/mL, respectively. Then, it was incubated for 24 hours. 10 μL of MTT solution dissolved in PBS buffer solution at 5 μg/mL to each well was added and reacted for 1 hour again. After confirming the formation of formazan, the medium was completely removed, and 100 μL of DMSO (Dimethyl Sulfoxide) was added to dissolve the formazan formed at the bottom of the well, and then, using an ELISA reader (SPECTRA MAX 190, Molecular Devices, Califonia, USA). Absorbance was measured at 450 nm to show the cell viability when the control cells cultured without treatment with each extract were 100%.

Lactate dehydrogenase (LDH) activity assay

LDH was measured using a CytoTox detection kit (Takara, Shuzo Co., Ltd, Otsu, Japan). The positive control group was defined as the non-sampled group, and the negative control group was defined as not treated with both cells and samples, and the relative LDH production was measured.

DNA fragmentation analysis

Cell death detection ELISA plus kit (Roche Molecular Biochemicals, Mannheim, Germany) was used to quantify apoptotic-specific DNA fragmentation. After some amount of SP and RS were treated on the cells, the lysate supernatant was recovered. After transferring 20 uL of the supernatant to a streptavidin-coated plate, 80 uL of immune-reagent was added to each microwell and reacted at room temperature for 2 hours. Each well was washed with an incubation buffer, and after reacting for 20 to 30 minutes by adding a substrate solution, absorbance was measured at 405 nm.

Measurement of testosterone concentration in cells

Intracellular testosterone concentration was measured using an ELISA kit (Enzo, Farmingdale, NY, USA). After the cultivation was completed, 100 uL of the medium was transferred to a goat anti-mouse IgG microtiter plate, and then incubated for 1 hour after addition of a testosterone antibody. After washing, the reaction was stopped after adding a stop solution, and absorbance was measured at 405 nm.

Western blot analysis

Each cell was dispensed into a 6-well plate so that the number of cells was 4×10 ⁵ cells/well, cultured with 80% confluence, and then treated with SP and RS for 24 hours. After incubation, the wells were washed twice with PBS of pH 7.4, and cells were collected. Cell lysate was prepared using lysis buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1% Triton X-100, 1 M DTT), and the supernatant obtained by centrifugation at 13,000 rpm for 15 minutes at 4°C. Was used as the total protein extract. Protein was quantified by Bio-Radprotein assay (Bio-Rad Laboratories, Hercules, CA, USA), and 100 μg of protein and sample buffer (60 mM Tris-HCl; pH 6.8, 2% SDS, 25% glycerol, 14.4 mM 2- mercaptaethanol, 0.1% bromphenol blue) was mixed, boiled at 100°C for 10 minutes, cooled, and electrophoresed. The acrylamide gel containing the separated protein was transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA) by electroblotting, and then placed in PBS-T (0.1% Tween 20 in PBS) containing 5% skim milk. Gourd was cultured at room temperature for about 1 hour to block non-specific proteins and washed with PBS-T for about 15 minutes. The prepared membrane was treated with the primary antibody for 2 hours or more at room temperature or over night at 4°C, washed with PBS-T, and reacted at room temperature for about 1 hour using a secondary antibody suitable for the treated primary antibody. After washing again with PBS-T and applying an enhanced chemiluminescence (ECL) solution (Amersham Life Science Corp, Arlington Heights, IL, USA), the expression amount of a specific protein was confirmed (Amersham Imager 680, GE Healthcare BioScience, Uppsala, Sweden). ). The antibodies used in this experiment were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and the peroxidase-labeled donkey anti-rabbit immunoglobulin peroxidase-labeled sheep anti-mouse immunoglobulin used as the secondary antibody was purchased from Amersham Corp.

Statistical processing

The results obtained in each experiment were expressed as mean ± standard deviation (SD), and the significant difference between the data was p<0.05 level using Student's t-test and Duncan's multiple range test, one-way analysis of variance (ANOVA). Verified in.

[Experiment result]

1. Confirmation of the effect of improving prostatic hyperplasia by extract of honggyeongcheon or saw palmetto alone

<1-1> Confirmation of the effect of honggyeongcheon extract on improving prostatic hyperplasia

The effect of improving prostatic hypertrophy of honggyeongcheon alone extract prepared under the manufacturing conditions established in the present invention was confirmed through MTT analysis for the cell viability of the honggyeongcheon extract in prostatic hyperplasia cells BPH-1.

As a result, as shown in [Fig. 1], as a result of treating BPH-1 prostatic hyperplasia cells with honggyeongcheon extract, the survival rate of prostatic hyperplasia cells decreased in a concentration-dependent manner, thereby confirming the effect of improving prostatic hypertrophy of the honggyeongcheon extract.

This is consistent with the results of Example <1-6>, and referring to [Table 5], the survival rate of prostatic hyperplasia cells treated with the honggyeongcheon extract prepared under the production conditions of the present invention was 55.52%, indicating that it was positive. It can be seen that the cell survival rate of the control group administered with finesteride is 66.25%, which is superior to that of the positive control group. In addition, LDH activity seems to increase somewhat in a concentration-dependent manner, but since it is not significant, it is thought to be irrelevant to the decrease in cell viability by the extract of honggyeongcheon.

<1-2> Confirmation of the effect of saw palmetto extract on the treatment of prostatic hyperplasia

In order to confirm the effect of improving prostatic hyperplasia of the extract of saw palmetto alone, the extract of saw palmetto alone was treated with BPH-1 in prostatic hyperplasia cells at a concentration of 6.25 to 100 ug/mL, and then the cell viability was confirmed through MTT analysis.

As a result, as shown in [Fig. 1], there was no change in the treatment concentration of saw palmetto extract from 6.25 ug/mL to 50 ug/mL concentration, and about 41% of cells at 100 ug/mL concentration The survival rate was confirmed. However, unlike the Rhododendron extract, the LDH activity at 100 ug/mL was also reduced by about 0.5 times compared to the control, which is thought to be a decrease in LDH activity due to a decrease in cell viability.

2. Synergistic prostatic hyperplasia treatment effect of Rhododendron and saw palmetto extract

<2-1> Confirmation of the synergistic therapeutic effect of the complex extract on prostatic hyperplasia cells

In this experiment, the treatment effect of the complex extract obtained by mixing saw palmetto extract and rhododendron extract was confirmed. First, referring to the experimental results of the single extract, saw palmetto extract and rhododendron extract were prepared respectively at concentrations confirmed to have no or insignificant effect on prostate treatment, and then mixed to prepare a composite extract. Thereafter, the prostatic hyperplasia cells BPH-1 were treated with saw palmetto alone extract, honggyeongcheon alone extract, and complex extract, respectively, and then the cell viability was measured through MTT analysis.

As a result, as shown in the following [Table 6] and [FIG. 2], when 50 ug/ml of saw palmetto extract was treated alone, the prostatic hyperplasia cell survival rate exceeded 95%, and the treatment effect was not remarkable, 25 The group administered with ug/ml of honggyeongcheon extract alone had a slightly more prostatic hyperplasia cell killing effect than the control group, but this was not significant. On the other hand, in the case of the combined extract of saw palmetto and honggyeongcheon, it was confirmed that the prostatic hyperplasia cell survival rate was significantly decreased compared to the control group and the single administration group, and the treatment effect of prostatic hyperplasia was significantly increased.

[Table 6]

Based on the results obtained in the above experiment, the results of comparing the theoretical value calculated by the Colby's equation and the measured value in order to additionally verify the synergy effect by the mixing treatment are shown in Table 7 below. At this time, the therapeutic effect of prostatic hyperplasia is shown in the following [Table 7] by converting the number of prostatic hyperplasia cells killed by the extract treatment into a percentage, and the calculated composite calculated by substituting the measured percentage of the single extract administration group into the following Colby formula The percentage predicted value of the extract is also shown in [Table 7].

E = (A + B)-(A×B / 100)

In the above formula, A is the drug effect of the active ingredient A, B is the drug effect of the active ingredient B, and E is the predicted drug effect when ingredient A and ingredient B (A+B) are mixed as a predicted value.

[Table 7]

As a result, in the case of the combined extract of saw palmetto and rhododendron, as shown in [Table 7], the actual value of the prostatic hyperplasia treatment effect was significantly higher at 34.3% than the theoretical value of 23.65%, and the effect was synergistically improved compared to the active ingredient applied alone. It can be seen that it represents.

These results indicate that the composite extract exhibits a synergistic effect in the treatment effect of prostatic hyperplasia compared to the case of using each extract alone, so that the amount of use of each of the Rhododendron extract or the cow palmetto extract can be reduced. It is suggested that the amount of used saw palmetto extract can be reduced.

<2-2> Confirmation of synergistic prostatic hyperplasia treatment effect according to concentration treatment of honggyeongcheon extract

In this experiment, in the combination treatment of saw palmetto extract and honggyeongcheon extract, the synergistic prostatic hyperplasia treatment effect was confirmed by varying the concentration of the honggyeongcheon extract. First, the Saw Palmetto extract was treated in the same manner as 50 ug/ml in prostatic hyperplasia cells BPH-1, and 25, 50, and 100 ug/ml of honggyeongcheon extract were treated, respectively, and then cell viability was measured through MTT analysis. In addition, the expression rate of the regulatory protein involved in apoptosis was measured through Western blot, and the DNA fragmentation concentration was measured using an ELISA kit.

As a result, cell viability decreased in a concentration-dependent manner when treated in combination with honggyeongcheon extract compared to treatment with saw palmetto extract alone, and cell viability was about 57 when combined treatment of saw palmetto extract and honggyeongcheon extract in a ratio of 1:2. It was confirmed that the largest decrease in% was observed (FIG. 3A).

Next, as a result of examining the expression of proteins related to apoptosis, the extract of honggyeongcheon increased the expression of Bax protein, which promotes apoptosis in a concentration-dependent manner, and reduced the expression of Bcl2 protein, which inhibits apoptosis. It was confirmed that the expression of the PCNA protein was decreased (Fig. 3B). At this time, the DNA fragmentation concentration, which is a typical phenomenon of apoptosis, was also significantly increased (Fig. 3C).

3. Confirmation of cytotoxicity of extract of honggyeongcheon or saw palmetto alone

In order to confirm whether the rhododendron extract or saw palmetto extract exhibits cytotoxicity in testis cells and normal prostate cells, TM3 testis cells and normal prostate cells (RWPE) were treated with each single extract by concentration, and then cell viability through MTT analysis. Was confirmed.

<3-1> Confirmation of apoptosis effect on testis cells

In order to confirm the cytotoxicity of the saw palmetto extract to testis cells, the saw palmetto extract was treated at a concentration of 6.25, 12.5, 25, 50, and 100 μg/mL, and then MTT assay was performed to measure the cell viability.

As a result, as shown in [Fig. 4], when the saw palmetto extract was treated at a concentration of 6.25, 12.5, 25, 50, and 100 ug/mL, the cell viability of 91, 82, 74, 68, and 61% were respectively shown. It was confirmed that the cell viability was dependently decreased (FIG. 4A). In addition, it was confirmed that the LDH activity increased according to the increase in cell damage caused by the saw palmetto extract treatment (Fig. 4B).

However, it was confirmed that there was no cytotoxicity unlike saw palmetto extract alone, since treatment with the extract of honggyeongcheon did not affect the cell viability and LDH activity of testis cells.

From the above results, it was possible to confirm the cytotoxicity ^(15-17) of the saw palmetto extract, and it was also confirmed that the honggyeongcheon extract has an effect of reducing the cytotoxicity derived from the saw palmetto extract.

<3-2> Confirmation of apoptosis effect on normal prostate cells

In the same manner as described above, the presence or absence of cytotoxicity of saw palmetto and honggyeongcheon alone extract against normal prostate cells was confirmed.

As a result, it was confirmed that there was no cytotoxicity, unlike saw palmetto alone, when treatment with the extract of honggyeongcheon as shown in FIG. 5 did not affect the cell viability and LDH activity of testis cells.

However, the Saw Palmetto extract decreased the cell viability in a concentration-dependent manner, as in the results shown in testis cells, showing 74% and 61% cell viability at concentrations of 50 ug/mL and 100 ug/mL, respectively. As cell damage increased, LDH activity also increased significantly from 1.37 to 1.5 times at 50 ug/mL and 100 ug/mL concentrations, respectively, compared to the control group.

From the above results, it can be seen that the saw palmetto extract has very high cytotoxicity to normal cells at a concentration that has an effect of improving prostatic hyperplasia.

4. Confirmation of the synergistic cytotoxic inhibitory effect of the complex extract of honggyeongcheon and saw palmetto

<4-1> Confirmation of synergistic apoptosis inhibition effect on testis cells

As it was confirmed that single treatment of saw palmetto extract induces concentration-dependent cytotoxicity in testis cells, it was confirmed that the combined extract of honggyeongcheon and saw palmetto could inhibit cytotoxicity. And, since testis cells are the main source of testosterone that promotes sperm formation, the effect of the extract of honggyeongcheon and saw palmetto complex on the concentration of testosterone in cells was confirmed.

As a result, as shown in [Fig. 6A], cell viability was significantly increased when the complex extract was treated with the same amount than when the saw palmetto extract was treated alone, and LDH activity was significantly increased by the increase in cell viability. Decreased to.

In addition, the concentration of intracellular testosterone decreased in a concentration-dependent manner at 50 ug/mL and 100 ug/mL of saw palmetto extract, resulting in 2.4 and 1.2 pg/mL, respectively, whereas when combined with Rhododendron extract, it gradually increased and increased to 100 ug/mL. The mL concentration was also higher than that of the control group (Fig. 6B). In other words, it is thought that the testosterone content increased by suppressing apoptosis due to cytotoxicity when the complex extract was treated compared to the saw palmetto extract alone treatment group.

<4-2> Confirmation of synergistic apoptosis inhibitory effect on normal prostate cells

As it was confirmed that concentration-dependent cytotoxicity was induced in normal prostate cells by treatment with saw palmetto extract alone, it was confirmed that the combined extract of honggyeongcheon and saw palmetto could inhibit cytotoxicity.

As a result, as shown in [Fig. 7], it was found that the survival rate of normal prostate cells increased and the LDH activity decreased as the concentration of the honggyeongcheon extract in the complex extract increased in the normal prostate cells, similar to the inhibitory effect shown in testis cells. .

[references]

[1] Wilt TJ, Ishani A, Stark G. 1998. Saw palmetto extracts for treatment of benign prostatic hyperplasia. JAMA 280: 1604-1609.

[2] Tacklind J, MacDonald R, Rutks I. 2009. Serenoa repens for benign prostatic hyperplasia. Cochrane Database Syst Rev 2: 1423.

[3] Avins AL, Bent S. 2006. Saw palmetto and lower urinary tract symptoms: what is the latest evidence? Curr Urol Reports 7: 260-265.

[4] Bent S, Kane C, Shinohara K. 2006. Saw palmetto for benign prostatic hyperplasia. N Engl J Med 354: 557-566.

[5] Avins AL, Bent S, Staccone S. 2008. A detailed safety assessment of a saw palmetto extract. Complement Ther Med 2008:147-154.

[6] Barry MJ, Meleth S, Lee JY, et al. 2011. Effect of increasing doses of saw palmetto extract on lower urinary tract symptoms: a randomized trial. JAMA 306: 1344-1351.

[7] Avins AL, Lee JY, Meyers CM, Barry MJ. 2013. Safety and toxicity of Saw palmetto in the complementary and alternative medicine for urological symptoms (CAMUS) trial. J Urol 189: 1415-1420.

[8] Food Safety Information Service. 2017. Research on data research on functional raw materials for re-evaluation.

[9] Ming DS, Hillhouse BJ, Guns ES, Eberding A, Xie S, Vimalanathan S, Towers GH. 2005. Bioactive compounds from Rhodiola rosea (Crassulaceae). Phytother Res 19: 740-743.

[10] Panossian A, Wikman G, Sarris J. 2010. Rosen root (Rhodiola rosea): traditional use, chemical composition, pharmacology and clinical efficacy. Phytomedicine 17: 481-493.

Claims (8)

Rhodiola sachalinensis ( Rhodiola sachalinensis ) A pharmaceutical composition for the prevention or treatment of prostatic hyperplasia containing an extract as an active ingredient.
Rhodiola sachalinensis ( Rhodiola sachalinensis ) A pharmaceutical composition for the prevention or treatment of prostatic hyperplasia containing an extract and saw palmetto extract as active ingredients.
The method according to claim 1 or 2,
The honggyeongcheon extract is a pharmaceutical composition, characterized in that prepared by ultrasonic extraction after immersing the honggyeongcheon powder in an ethanol solution.
The method of claim 2,
The pharmaceutical composition, characterized in that the combination of the honggyeongcheon extract and saw palmetto extract increases the prostatic hyperplasia cell killing effect.
The method of claim 2,
Pharmaceutical composition, characterized in that the combination of the honggyeongcheon extract and saw palmetto extract reduce toxicity to testicular cells and normal prostate cells.
The method of claim 2,
The saw palmetto extract is a pharmaceutical composition, characterized in that the saw palmetto fruit extract.
The method of claim 2,
The honggyeongcheon extract and saw palmetto extract will be contained in a weight ratio of 1:2 to 2:1, pharmaceutical composition.
Hong Gyeongcheon ( Rhodiola sachalinensis ) extract and saw palmetto extract as an active ingredient for preventing or improving prostatic hyperplasia health functional food composition.

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