WO2015111832A1 - Composition for preventing or treating prostate-related diseases, containing poncirus trifoliate extract - Google Patents

Abstract

The present invention relates to: a pharmaceutical composition for preventing or treating prostate-related diseases, containing a Poncirus trifoliata extract or a fraction thereof as an active ingredient; a method for preventing or treating prostate-related diseases by using the composition; and a food composition, a feed composition and a quasi-drug composition for preventing or alleviating prostate-related diseases, containing a Poncirus trifoliata extract.

Description

A composition for the prevention or treatment of prostate related diseases, including fruit juice extract

The present invention provides a pharmaceutical composition for the prevention or treatment of prostate-related diseases comprising the fruit juice extract or fractions thereof as an active ingredient, a method for preventing or treating prostate-related diseases using the composition, a prostate-related disease comprising the fruit juice extract or fractions thereof. It relates to a food composition, a feed composition, and a quasi-drug composition for prevention or improvement.

The prostate gland is composed of glands and fibromuscular tissues, and the male gonads may have water bumps or stones, but prostatitis, prostatic hyperplasia, and prostate cancer are the most common diseases. Prostate-related diseases in Koreans are characterized by more prostatitis than Westerners, the incidence of prostate cancer is very low, and the prostate hypertrophy is gradually increasing due to an increase in the elderly population. Urinary tract disease in patients 50 years and older is most common in the order of enlarged prostate, prostate cancer, and prostatitis.

First, prostatic hyperplasia is associated with bladder storage and delayed urination (such as urinary urgency, urinary urge to urinate more than eight times a day, night urination, and strong and sudden urinary in men over 50 years of age. It is the name of testosterone that is a male hormone by 5 alpha-reductase, such as urine coming out by moxibustion), severed urine (breaking up of urine), and urination during urination. Dihydrotestosterone (DHT), which is converted from (testosterone, T), is known to be involved in prostatic hypertrophy (Urology. 2003 Apr; 61 (4 Suppl 1): 2-7.). Recently, the number of prostate hyperplasia patients in Korea has increased sharply from 45,897 people in 2006 to 84,2069 in 2011, which is 83.5%. In particular, the number of patients with prostatic hyperplasia is 2 to 5 males 60 years old or older, and over 80 years old, more than half complain of enlarged prostate symptoms. The number of patients with prostatic hyperplasia is expected to increase in the future due to the rush into the aging society and the improvement of the rate of receipt. Therefore, the demand for the prevention and treatment of prostatic hyperplasia is expected to increase continuously.

Prostatitis is also a disease that accounts for about 3-12% of all urological patients, but is most common in urological patients under 50 years of age. In particular, chronic pelvic pain, the most prominent symptom of prostatitis, is known to be more severe than suffering from prostatic hyperplasia or sexual dysfunction, and it is common in active young age groups and has an adverse effect on quality of life. However, it is difficult to diagnose and treat because the cause is not clear and there are no characteristic findings in symptoms or tests. The effects of dihydrotestosterone (DHT), which is converted from the male hormone testosterone (T) by 5 alpha-reductase, have been reported on the prostatitis (The Journal of Urology, Volume 170, Issue 6, Part 6). 1, December 2003, Pages 2486-2489)

Prostate cancer is a malignant tumor that occurs in the prostate, and prostate cancer is also known to be highly related to the conversion of testosterone to dihydrotestosterone by 5 alpha-reductase (J Steroid Biochem Mol Biol. 2002 Nov; 82 (4-5): 393-400.). In the West, prostate cancer is the most common cancer among men and has a high incidence. However, gastric prostate cancer is difficult to detect early because there is no specific symptom.In many cases, hospitals are visited only after the growth of cancer tissues causes urination, or prostate cancer has spread to other organs such as bones and symptoms such as bone pain. . Therefore, there is a growing demand for prophylactic and therapeutic agents for prostate cancer.

As such, the number of patients with prostate-related diseases, including prostatic hyperplasia, prostatitis, and prostate cancer, has steadily increased with the entry into an aging society, and consumers' interest in the prevention and treatment of the prostate has increased. Efforts have been made to find an effective treatment for prostate-related diseases.As a result, a composition containing fruit extract as a natural medicine reduces the activity of 5-alpha-reductase, which converts testosterone, a male hormone, to dihydrotestosterone, to prevent prostate-related diseases. And it was confirmed that the treatment can be completed the present invention.

One object of the present invention to provide a pharmaceutical composition for the prevention or treatment of prostate-related diseases, including as an active ingredient Ponciri Fructus extract or fractions thereof.

It is another object of the present invention to provide a method for preventing or treating a prostate-related disease, comprising administering the pharmaceutical composition for preventing or treating the prostate-related disease to an individual in need thereof.

It is another object of the present invention to provide a food composition for preventing or ameliorating prostate-related diseases comprising a fruit-fat extract or a fraction thereof as an active ingredient.

It is another object of the present invention to provide a feed composition for the prevention or improvement of prostate-related diseases comprising the fruiting extract or a fraction thereof as an active ingredient.

It is another object of the present invention to provide a quasi-drug composition for the prevention or improvement of prostate-related diseases comprising the fruit fruit extract or a fraction thereof as an active ingredient.

As one embodiment for achieving the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of prostate-related diseases comprising a fruit extract or a fraction thereof as an active ingredient.

As used herein, the term "pile" refers to the unripe fruit of the Pancirus trifoliata Rafinesque, a deciduous broad-leaved ornamental belonging to the Rutaceae family. It is a round shape with a diameter of 3 cm. The flowering period of the tanza trees is from May to May, the fruiting season is from September to November, and the harvest time is from September to October. It is known to grow wild on islands such as Jeju Island and Gadeok Island in the south of Korea, and it is cultivated in Gyeongju and South-South of Korea. Ripe fruits are called crust, and crust and crust have different effects and are used for other purposes. In addition, according to the Food and Drug Administration, Jisil is known to exhibit anti-inflammatory effects, efficacy on gastritis and gastric ulcers, effects on metabolic osteoporosis diseases, and anti-cancer activity, but is not known for the treatment of prostate-related diseases. First identified by the In the present invention, the fruit can be purchased commercially, or can be used collected or cultivated in nature.

In the present invention, the term "extract" means the extract of the fruit. The extract is a low alcohol having 1 (C1) to 4 (C4) carbon atoms, such as water, methanol, ethanol, etc., in volumes of about 5 to 30 times the dry weight, preferably about 10 to 20 times the dry weight. Eluting with a polar solvent such as or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, the extraction temperature is 20 ℃ to 100 ℃, preferably 60 ℃ to 100 ℃, extraction period is about It may be an extract extracted by using an extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction, filtration extraction, or ultrasonic extraction for 1 hour to 4 days, but the extract may exhibit a prophylactic or therapeutic effect of a prostate related disease of the present invention. However, the present invention is not limited thereto, and may include any extract, a diluent or concentrate of the extract, a dried product obtained by drying the extract, or all of these modifiers or purified products.

For example, the extract may be included in an amount of 0.01 to 100% by weight, more preferably 1 to 80% by weight, based on the total weight of the pharmaceutical composition, respectively.

As used herein, the term "fraction" refers to the result obtained by performing fractionation to separate a specific component or a specific group of components from a mixture comprising several different components.

In the present invention, the fractionation method for obtaining the fraction is not particularly limited, and may be performed according to a method commonly used in the art. Solvent fractionation by treatment of various solvents, ultrafiltration fractionation through passage of ultrafiltration membranes with constant molecular weight cut-off values, and various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) Chromatography), and combinations thereof. In one embodiment of the present invention was used to obtain a fraction from the extract by treating a predetermined solvent to the extract obtained by extracting the fruit. The kind of solvent used to obtain the fraction in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvents include polar solvents such as water and alcohols; And nonpolar solvents such as hexane, ethyl acetate, chloroform, dichloromethane, and the like. These may be used alone or in combination of two or more thereof. In the case of using alcohol in the fractionation solvent, alcohols of C1 to C4 may be preferably used.

For example, the fraction may be prepared by fractionating the fruit extract with a solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, hexane (hexane), ethyl acetate, and a mixed solvent thereof. As another example, the fraction may be prepared by fractionating the fruit extract with water, hexane, ethyl acetate, or butanol. The present inventors confirmed that the ethyl acetate extract of the fruit-derived extract significantly inhibits the activity of 5 alpha-reductase in a concentration-dependent manner, and thus has an excellent therapeutic effect in prostate-related diseases (FIGS. 5 and 6).

The fractions may each comprise 0.01 to 100% by weight, more preferably 1 to 80% by weight relative to the total weight of the pharmaceutical composition.

The composition comprising the fruit juice extract of the present invention can be used for the prevention or treatment of prostate-related diseases by using the activity of reducing the 5 alpha-reductase. In the present invention, the term "5 alpha reductase" refers to an enzyme that converts testosterone, a type of male hormone, to dihydrotestosterone (DHT), and the enzymes include type 1 and type 2. 5 alpha-reductase inhibitors are known to treat prostate-related diseases such as enlarged prostate, which is known to prevent the growth of the prostate.

The present inventors confirmed that the fruit juice extract has an effect of significantly reducing the activity of 5 alpha-reductase, which is highly related to the induction of prostate-related diseases such as prostatitis, prostatic hyperplasia, and prostate cancer (Experimental Example 1 and FIG. 1).

In addition, in hypertrophic animal models induced by testosterone propionate (TP), the fibrin extract inhibits the increase in prostate weight and significantly inhibits the increase in DHT (dihydro testosterone) levels essential for the development, growth and maintenance of the prostate gland. The proliferation of prostate epithelial cells was very suppressed. In particular, this effect was confirmed to show the effect of the degree to reach the finasteride that is currently used as a treatment for prostate-related diseases (Experimental Example 2, Figures 2 to 4).

As used herein, the term "prostate-related disease" means a disease occurring in the prostate gland, which is a reproductive and urinary organ of a male, and for example, one selected from the group consisting of prostatic hyperplasia, prostatitis, prostate cancer, prostate abscess, and prostate calcification The above diseases may be included, but are not limited thereto and may include all of various diseases caused by prostate problems. Prostate abscess is a disease in which bacteria penetrate into the prostate through the urethra and rapidly form necrotic pouches following acute inflammation, creating necrotic pouches. When a disease develops, a sudden high fever and chills appear. You can't have urinary tract symptoms and you'll feel extreme urination. Prostate calcification is a disease caused by the sinking of lime stones in the prostate. The absence means that too much constituent in the body fluids, including urine is not liquefied all means that the remaining calcium, oxalate, phosphate crystallized.

As used herein, the term "prevention" may refer to any action that inhibits or delays the development of a prostate-related disease by administering to a subject a pharmaceutical composition for preventing or treating a prostate-related disease according to the present invention.

As used herein, the term "treatment" may mean any action that improves or benefits the symptoms of a prostate-related disease by administering the composition of the present invention to a subject suspected of developing a prostate-related disease.

As used herein, the term "improvement" may mean any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.

The pharmaceutical composition for preventing or treating prostate-related diseases according to the present invention may further include a pharmaceutically acceptable carrier, and may be formulated together with the carrier to provide food, medicine, feed additives, and drinking water additives. . As used herein, the term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not interfere with the biological activity and properties of the compound to be administered without stimulating the organism.

The kind of the carrier usable in the present invention is not particularly limited, and any carrier can be used as long as it is a conventionally used and pharmaceutically acceptable carrier in the art. 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 thereof.

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, and the like may be additionally added to provide a solution such as an aqueous solution, a suspension, an emulsion or the like. It may be formulated into a use formulation, pills, capsules, granules or tablets.

The mode of administration of the pharmaceutical composition for preventing or treating prostate-related diseases according to the present invention is not particularly limited, and may be in accordance with methods commonly used in the art. As a non-limiting example of the mode of administration, the composition may be administered by oral or parenteral administration. The pharmaceutical composition for preventing, ameliorating or treating the prostate-related disease of the present invention may be prepared in various formulations according to the desired administration method.

The pharmaceutical composition of the present invention may also be used as a single agent, and may be prepared and used in a combination formulation, further including a drug known to have a recognized prostate-related disease treatment effect, and formulated using a pharmaceutically acceptable carrier or excipient. It may be prepared in unit dose form or incorporated into a multi-dose container.

As another aspect, the present invention provides a method for preventing or treating a prostate-related disease comprising administering to the individual a pharmaceutical composition for preventing or treating the prostate-related disease. As used herein, the term "individual" may mean any animal, including humans, who have or are likely to develop a prostate-related disease.

The method of preventing or treating the present invention may specifically include administering the composition in a pharmaceutically effective amount to a subject having or at risk of developing a prostate-related disease.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, generally in an amount of 0.001 to 1000 mg / kg, preferably 0.05 The amount of to 200 mg / kg, more preferably 0.1 to 100 mg / kg may be administered once to several times daily. However, for the purposes of the present invention, a suitable total daily usage of the composition comprising the extract may be determined by the practitioner within the correct medical judgment and may be administered once or in divided doses. However, for the purposes of the present invention, the specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health of the patient, It is desirable to apply differently depending on various factors and similar factors well known in the medical field, including sex and diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or co-specific with the specific composition.

The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can achieve the maximum effect in a minimum amount without causing side effects, and can be easily determined by those skilled in the art.

As used herein, the term "administration" refers to introducing the pharmaceutical composition of the present invention to a patient in any suitable manner, the route of administration of the composition of the present invention being oral or parenteral as long as it can reach the target tissue. Administration can be via a variety of routes.

The mode of administration of the pharmaceutical composition according to the present invention is not particularly limited, and may be in accordance with methods commonly used in the art. As a non-limiting example of the mode of administration, the composition may be administered by oral or parenteral administration. The pharmaceutical compositions according to the invention may be prepared in various formulations depending on the desired mode of administration.

The frequency of administration of the composition of the present invention is not particularly limited, but may be administered once a day or several times in divided doses.

As another aspect, the present invention provides a food composition for preventing or ameliorating prostate related diseases.

Since the fruit has been used for a long time as a natural material and has been proved to be safe, it can be prepared and consumed in the form of a food that can prevent or improve prostate-related diseases.

The kind of the food is not particularly limited, and may include all foods in a general sense. Non-limiting examples of foods to which the substance may be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, drinks, tea , A drink, an alcoholic beverage, and a vitamin complex. When the composition is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method.

The food composition may comprise a food 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 means foods that have high medical effects and medical effects that are processed so that their bioregulatory functions are efficiently displayed in addition to nutrition. In addition, the food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. in order to obtain useful effects in preventing or improving prostate related diseases.

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

The health functional food can be prepared by a method commonly used in the art, and the preparation can be prepared by adding the raw materials and ingredients commonly added in the art. In addition, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug with food as a raw material, can be excellent in portability.

As another aspect, the present invention provides a feed composition for the prevention or amelioration of prostate-related diseases.

The feed composition may include a feed additive. The feed additive of the present invention corresponds to a feed supplement in the Feed Control Act.

As used herein, the term "feed" may refer to any natural or artificial diet, one meal, or the like, or a component of the one meal, for the animal to eat, ingest, and digest.

The kind of the feed is not particularly limited, and may be used a feed commonly used in the art. Non-limiting examples of the feed include, but are not limited to, vegetable feeds such as cereals, fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; And animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single cell proteins, zooplankton or foods. These may be used alone or in combination of two or more thereof.

As another aspect, the present invention provides a quasi-drug composition for preventing or ameliorating prostate-related diseases. The composition of the present invention can be added to the quasi-drug composition for the purpose of preventing or treating prostate related diseases.

As used herein, the term "quasi drug" refers to a fiber, rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or animal, has a weak action on the human body or does not directly act on the human body, Or non-machinery and the like, or any of the agents used for sterilization, insecticide and similar purposes for the purpose of preventing infection, for the purpose of diagnosing, treating, reducing, treating or preventing human or animal diseases. It may mean an article other than an apparatus, machine or apparatus, and an article which is not an apparatus, machine or apparatus, which is used for the purpose of pharmacologically affecting the structure and function of a human or animal. In addition, the quasi-drug may include external skin preparations and personal hygiene products. Preferably, it may be, but is not limited to, a disinfectant cleaner, a shower foam, a gagreen, a wet tissue, a detergent soap, a hand wash, or an ointment.

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

In another aspect, the present invention provides a drinking water additive for preventing or ameliorating prostate-related diseases including the fibrin extract.

The drinking water additive of the present invention may be prepared by separately preparing the composition containing the fruit extract in the form of drinking water additives and mixing the drinking water, or may be used by adding directly in the preparation of drinking water.

The drinking water additive of the present invention may be in a liquid or dry state, preferably in the form of a dry powder. The drying method for preparing the drinking water additive of the present invention in the form of a dried powder is not particularly limited, and a method commonly used in the art may be used. The drinking water additive of the present invention may further include other additives as necessary. Non-limiting examples of the additives that can be used include binders, emulsifiers, preservatives, etc., added to prevent deterioration of drinking water quality; Amino acids, vitamins, enzymes, probiotics, flavors, nonprotein nitrogen compounds, silicates, buffers, colorants, extractants, or oligosaccharides are added to increase the usefulness of feed or drinking water. It can be included as. These may be used alone or two or more kinds may be added together.

Composition comprising the fruit extract of the present invention as an active ingredient inhibits the activity of the 5 alpha-reductase, there is an effect that can effectively prevent, improve, and treat prostate-related diseases such as enlarged prostate, prostatitis, prostate cancer.

In addition, the composition comprising the fruit extract of the present invention as an active ingredient may be applied to various products including a food composition, a composition for animal feed, a cosmetic composition, and a quasi-drug composition for the prevention or improvement of prostate-related diseases as a natural drug.

In addition, the composition of the present invention is derived from natural products and can be used safely without causing serious irritation or harmful effects in the body in addition to preventing, treating, and improving efficacy of prostate-related diseases.

Figure 1 shows the inhibitory effect of the activity of 5 alpha-reductase according to the concentration of the fruit extract.

Figure 2 shows the measurement of the prostate gland according to the fruit extract extract in the enlarged prostate animal model.

Figure 3 shows the measurement of the change in DHT (dihydro testosterone) according to the fruit extract extract in the prostatic hypertrophy-derived animal model.

Figure 4 shows the histopathological changes of the prostate gland according to the fruit extract in the prostatic hypertrophy-derived animal model.

NC shown in the figure is a normal group, BPH is a prostatic hyperplasia-induced group, Fin is a prostatic hyperplasia-induced group + finasteride administration group, GO-10 is a prostatic hypertrophy-induced group + fibrotic extract administration group.

Figure 5 shows the inhibitory effect of the activity of 5 alpha-reductase according to the concentration of the fraction of the fruit extract.

Figure 6 shows the measured prostate weight according to the treatment of the fraction of the fruiting extract in the prostatic hypertrophy-derived animal model (NC: normal group, BPH: prostatic hypertrophy induced group, Fin: prostatic hypertrophy induced group + finasteride administration group, Hexane: Prostatic hyperplasia + fibroblast hexane fraction, EA: prostatic hyperplasia + fibrosyl ethyl acetate fraction, BuOH: prostatic hyperplasia + fission butanol fraction, Water: prostatic hyperplasia + hypoglycemic fraction).

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are only for illustrating the present invention by way of example, and the scope of the present invention should not be construed as being limited by these Examples and Experimental Examples.

Example 1 Preparation of Fruit Extracts (Used in Experimental Example 1)

After purchasing and grinding a commercially available basement, 10 L of 70% ethanol was added to 1 kg of the ground product, and ultrasonic extraction was performed three times for 1 hour using an ultrasonic extractor. The ultrasonic extract was filtered using Whatman (46 x 57 cm) filter paper to remove insoluble materials, and then concentrated under reduced pressure at 40 ° C with a condenser equipped with a cooling condenser. In order to completely remove the solvent of the extract concentrated under reduced pressure, 1500 mL of purified water was added and suspended, and 152.2 g of an extract was obtained using a lyophilizer (yield: 15.22%).

Example 2 Preparation of Animal Experimental Fruit Extract (Used in Experimental Example 2)

After obtaining and grinding a commercially available fruit room, 100 L of 70% ethanol was added to 10 kg of the ground product. Thereafter, ultrasonic extraction was performed three times for 1 hour. The ultrasonic extract was filtered using Advantec No. 2 (110 mm) filter paper to remove insoluble materials, and then concentrated under reduced pressure at 60 ° C. using a condenser equipped with a cooling condenser. In order to completely remove the solvent of the extract concentrated under reduced pressure, 1000 mL of purified water was added and suspended, and 1368.6 g of an extract was obtained using a lyophilizer (yield 13.69%).

Example 3: Preparation of Fractions of Fruits Extract (Used in Experimental Examples 3 and 4)

3-1. Preparation of Fruity Hexane Fraction

1.3 kg of the fruit cell extract of Example 2 was suspended in 3.0 L of distilled water, and 4.5 L of hexane was added thereto, and the solvent was fractionated three times. The hexane fractions were obtained, concentrated under reduced pressure and lyophilized to give 62.5 g of hexane fractions (yield: 4.81%).

3-2. Manufacture of fruit fat ethyl acetate fraction (GO10-EA)

4.5 L of ethyl acetate was added to the remaining water fraction after the hexane fraction of Example 3-1, and the solvent fractionation was performed three times. Ethyl acetate fractions were obtained, concentrated under reduced pressure, and lyophilized to give 213.3 g of ethyl acetate fractions (yield: 16.41%).

3-3. Preparation of fruity butanol fraction (GO10-B)

4.5 L of butanol was added to the remaining water fraction after the ethyl acetate fraction of Example 3-2, and the solvent fractionation was performed three times. The butanol fractions were obtained, concentrated under reduced pressure and lyophilized to give 545.5 g of butanol fraction (yield: 41.96%).

3-4. Manufacture of fruit compartment water fraction (GO10-W)

The remaining water fraction after the butanol fraction of Example 3-3 was concentrated under reduced pressure and lyophilized to give 421.4 g of water fraction (yield: 32.42%).

Experimental Example 1 Confirmation of the Inhibitory Effect of 5-alpha Reductase by Fruit Extracts

5-alpha-reductase converts testosterone to dihydro testosterone, and the inhibition of 5-alpha-reductase activity is pointed to cause prostate-related diseases. Thus, in order to confirm whether the composition has a therapeutic effect on prostate-related diseases, the activity inhibitory effect of 5 alpha reductase, a representative disease treatment marker, was determined.

In order to confirm the inhibitory effect on the 5 alpha-reductase of the fruit extract prepared in Example 1, the change in the amount of NADPH used when the conversion from testosterone to dihydro testosterone was measured by the absorbance change 5 alpha The activity of the reductase was calculated. This was done by applying the method disclosed in the following literature (Methods Find Exp Clin Pharmacol. 1998; 20 (4): 283-7, Bioorg Med Chem Lett. 2011; 21 (11): 3439-42).

1-1. Preparation of Enzyme Composition Containing 5 Alpha Reductase Isolated from Rat Liver

12-week-old female SD rats (Central experimental animals, Korea) were anesthetized with dieth ether, and then dissected to extract the liver. Four-fold buffer solution (10 mM Tris-HCl, pH 7.0), 50 mM EDTA, 5 mM MgCl 2, 50 mM NaCl, 132 mM Sucrose, 0.004% 2-mercaptoethanol) were added and homogenized with a cell disruptor. The supernatant obtained by centrifugation (10,000ｘg) of the hepatocyte crushing solution was again ultracentrifuged (105,000ｘg) to obtain a microsomal fraction, which was suspended in the buffer solution and stored at -80 ° C.

1-2. Determination of Enzyme Inhibition Rate for 5-alpha Reductase

In order to confirm the effect of inhibiting the 5 alpha-reductase activity of the fruit fruit extract of the present invention, the absorbance change was measured three times for each of the following five groups having different concentrations of fruit fruit extract.

Group 1: 25 μg of the 5-alpha-reductase composition of Experimental Example 1-1 above

Group 2: a composition comprising 50 μg of testosterone (T0028, TCI, Japan) added to group 1

-Group 3: composition in which Group 2 was reacted with 60 μg / ml of the fruiting extract of Example 1

Group 4: a composition in which 70 μg / ml of the fruiting extract of Example 1 was reacted with group 2

Group 5: a composition in which Group 2 was reacted with 80 μg / ml of the fruiting extract of Example 1

50 μM of NADPH solution (N1630, Sigma-aldrich, USA) was added to groups 1-5. Then, the absorbance change was measured at 340 nm at 5 minute intervals using an ELISA measuring instrument (Benchmark Plus, Bio-Rad. USA) while reacting at 37 ° C. for 60 minutes.

Based on the measured change in absorbance, the activity of 5 alpha-reductase was calculated by the following equation, and the results are shown in FIG. 1 and Table 1 and Table 2.

(DELTA: OD340: Absorbance change at 340 nm per minute, A: total reaction volume (ml), B: enzyme liquid volume (ml), C: optical measurement length of the reaction vessel (cm). 6.22 is the molecular absorption coefficient of NADPH)

Statistical analysis was performed by ANOVA. ** P <0.01 is considered to be significantly different from Group 2 when #P <0.05 and ## P <0.01 are significant when compared to Group 1.

Table 1 Group 1 Group 2 Group 3 Group 4 Group 5 Active-1 2.53 4.47 4.34 3.71 3.34 Active-2 2.48 4.51 4.52 3.70 3.16 Active-3 2.71 4.74 4.43 3.97 3.11 Average 2.57 4.57 4.43 3.79 3.20 Standard Deviation 0.12 0.15 0.09 0.15 0.12

(Unit: mU / ml, U = unit)

TABLE 2 Group 1 Group 2 Group 3 Group 4 Group 5 Active (%)-1 -2.3 94.7 88.3 57.0 38.3 Active (%)-2 -4.5 97.0 97.3 56.4 29.3 Active (%)-3 6.8 108.3 92.8 69.9 27.1 Average 0.00 100.00 92.79 61.11 31.58 Standard Deviation 5.97 7.25 4.51 7.64 5.97

(unit: %)

Table 2 shows that for Group 1 and Group 2 without adding the fruit extract, the activity of Group 1 without testosterone, which is a substrate of 5 alpha-reductase based on the activity values of Table 1, was 0%, and 5 alpha-reductase. The activity of group 2 containing testosterone, which is a substrate, is converted to 100%. Therefore, in the absence of any inhibitor, the enzyme activity may be compared with the addition of the fruit extract based on 100% of the activity value of the 5 alpha-reductase activity against the testosterone as a substrate.

As described above, by measuring the activity (%) of the 5 alpha-reductase in the group 3 to 5 with the addition of different concentrations of the fruit extract to the group 2 composition, to know the effect of inhibiting the 5 alpha-reductase activity of the fruit extract according to the concentration saw. As a result, it was confirmed that the activity (%) of 5 alpha-reductase was inhibited rapidly from 100 to 92.79, 61.11 and 31.58 (%) in group 5 as the concentration of the fruit extract increased.

As described above, the fruit extract in the present invention is an inhibitor of 5-alpha-reductase, which converts testosterone (T) into dihydro testosterone (DHT) and has an effect of inhibiting 5-alpha-reductase, an important enzyme involved in prostate-related diseases. Appeared. In addition, it was confirmed that as the concentration of the fruit extract increased, the activity (%) of 5 alpha-reductase decreased rapidly, significantly inhibiting the action of 5-alpha-reductase. In particular, the fruit extract in the present invention was confirmed to have an excellent activity inhibitory effect of 5 alpha-reductase at a concentration of 0.1μg / ml or more and 90μg / ml or less.

1-3. Determination of Enzyme Inhibition Rate for 5-alpha Reductase (Control: Finasteride)

A positive control (finasteride) was used as a sample to compare the inhibitory effect of 5 alpha reductase. Finasteride is an inhibitor of type 2 alpha-reductase, which is now widely used in the art.

Based on the enzyme inhibition rate and the concentration of 50% inhibiting the activity of 5 alpha-reductase based on the enzyme measured in the groups 3 to 5 of Experimental Example 1-2 was calculated.

In addition, the enzyme inhibition rate and IC 50 of finasteride (F1293, Sigma-aldrich, USA) at 0.25 μM, 0.5 μM, and 1 μM concentrations were calculated by the same method.

The results are shown in Table 3 below. * P <0.05, ** P <0.01 is significant when compared with group 1, while # P <0.05 and ## P <0.01 are significant when comparing with group 2.

TABLE 3 sample density Enzyme Inhibition Rate (%) IC50 Basement room (μg / ml) 607080 7.21 ± 4.5 138.89 ± 7.6468.42 ± 5.97 73.92 ± 0.63 Finasteride (μM) 0.250.51 10.53 ± 2.6060.90 ± 15.3578.20 ± 12.42 0.60 ± 0.12

As can be seen from the results of Table 3, it was confirmed that the higher the concentration of the fruit extract, the activity inhibition rate (5%) of the 5 alpha-reductase was rapidly increased to significantly inhibit the action of the 5 alpha-reductase.

In addition, the IC50 of the fruit extract in the present invention was 73.92 ± 0.63 (μg / ml), the IC50 of finasteride, an inhibitor of 5-alpha-reductase widely used in the art is 0.60 ± 0.12 (μM). This can be seen that the 5 alpha-reductase activity inhibitory effect is excellent considering that the fruit extract is a natural extract.

These results support the fact that the fruit extract in the present invention inhibits the 5 alpha-reductase to prevent, treat, and improve the prostate-related diseases in which 5 alpha-reductase is important.

Experimental Example 2: Confirmation of the effect of prostatic hypertrophy in the animal model (extract)

2-1. Animal Model Production

Ten weeks-old male wistar rats (purchased central experiment animals, 6 per group) were purified for 1 week, and then TP (testosterone propionate) was injected subcutaneously at 3 mg / kg for 4 weeks to prepare a prostatic hypertrophy inducing group.

One hour before the injection of TP, the fruit extract of Example 2 was orally administered at 200 mg / kg for 4 weeks to prepare an experimental group. A positive control group was prepared by orally administering 10 mg / kg of finasteride, a 5α-reductase inhibitor used as a prostatic hypertrophy agent. In the solvent control, corn oil was injected subcutaneously.

2-2. Prostate weight measurement

After extracting the prostate gland from the animal model produced in Experimental Example 2-1, the weight thereof was measured and shown in FIG. 2 (NC: normal group, BPH: prostatic hypertrophy group, Fin: prostatic hypertrophy group + finasteride administration group, GO -10: prostatic hyperplasia-induced group plus the fruit extract administration group, the same below). Statistical analysis was performed by ANOVA. * P <0.05, ** P <0.01 is significant difference compared to normal group (NC), # P <0.05, ## P <0.01 is the trigger group (BPH ) And significant differences were considered.

As a result, as shown in FIG. 2, the prostate weight of the prostatic hypertrophy-inducing group (BPH) was more than doubled as compared to the normal group (NC), but the prostate weight was increased in the group (GO-10) to which the fibroblasts were administered. Significant inhibition was confirmed. The inhibitory effect appeared very similar to Finasteride (Fin), which is currently used as a therapeutic agent, suggesting that the effect of the fruiting extract is very good.

2-3. Measurement of DHT changes in the prostate

To measure changes in DHT (dihydrotestosterone), which is essential for the development, growth and maintenance of the prostate, proteins were isolated from prostate tissue isolated from sacrificed mice after 4 weeks of drug administration. Extraction buffer [composition: 50 mM Tris pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 1 mM PMFS, 10 μl / ml aprotinin, 1% Igapel 630 (Sigma Chem. Co. USA), 10 mM Sodium Fluoride (NaF), 0.5 mM ethylenediamine tetraacetic acid (EDTA), 0.1 mM ethylene glycol tetraacid tetraacetic acid (EGTA) and 0.5% sodium deoxycholate] were used to homogenize the prostate. The homogenate was centrifuged at 14000 rpm for 20 minutes and the supernatant and insoluble aggregates were separated.

Protein concentration of the isolated supernatant was measured using a Bio-Rad protein assay kit (Bio-Rad, USA). In order to measure the content of DHT in the supernatant, an ELISA kit (Dakara, Japan) that specifically reacts with DHT was used, and the content of each DHT was measured according to the manufacturer's method. The measured value was converted into relative units of protein quantitative value. Statistical analysis was performed by ANOVA. * P <0.05, ** P <0.01 is significant difference compared to normal group (NC), # P <0.05, ## P <0.01 is the trigger group (BPH ) And significant differences were considered.

As a result, as shown in FIG. 3, the DHT level of the prostatic hypertrophy-inducing group (BPH) was significantly increased compared to the normal group (NC), but the DHT level was increased in the group (GO-10) to which the fibroblasts were administered. Significant inhibition was confirmed. The inhibitory effect appeared very similar to Finasteride (Fin), which is currently used as a therapeutic agent, suggesting that the effect of the fruiting extract is very good.

2-4. Comparison of histopathological changes in the prostate

For histopathology, paraffin embedding was performed after fixation of the ventral prostate tissue with 10% neutral buffer-formalin for 24 hours in the group treated with the glandular extract (GO-10). . Embedding tissue was cut to 4 μm thickness to make sections and stained with hematoxylin and Eosin Y (ThermoShandon, USA). Immediately afterwards, the sections were encapsulated with the encapsulating solution, and the slides after the dyeing and encapsulation were examined under an optical microscope.

As a result, as shown in Figure 4, compared with the normal group (NC) was confirmed the result of the hyperproliferation of the height and number of epithelial cells in the prostatic hypertrophy-inducing group (BPH). However, the hyperproliferation of epithelial cells was significantly reduced in the group (GO-10) to which the fibroblasts were administered. In particular, this effect was found to be superior to Finasteride (Fin) which is currently used as a therapeutic agent, suggesting that the effect of the fruit extract is very excellent.

Experimental Example 3 Determination of Enzyme Activity of 5 Alpha Reductase Enzyme Extracts from Fruits

In order to confirm the inhibitory effect of 5-alpha-reductase activity on the fractions of the fruit fruit extract, the absorbance changes were measured three times in each of the following five groups with different concentrations of fruit fruit fractions by the method described in Experimental Examples 1-2. The activity of 5 alpha-reductase was calculated. Statistical analysis was performed by ANOVA. * P <0.05, ** P <0.01 is significant when compared with group 1, # P <0.05, ## P <0.01 is considered significant when compared with group 2. It was.

Group 1: 25 μg of the 5-alpha-reductase composition of Experimental Example 1-1 above

Group 2: a composition comprising 50 μg of testosterone (T0028, TCI, Japan) added to group 1

-Group 3: ethyl acetate fraction of Example 3-2 (GO10-EA), butanol fraction of Example 3-3 (GO10-B), water fraction of Example 3-4 (GO10-W), respectively, in Group 2 Was reacted at a concentration of 50 μg / mL

Group 4: a composition in which group 2 was reacted with GO10-EA, GO10-B, and GO10-W at a concentration of 100 μg / mL

Group 5: a composition in which group 2 was reacted with GO10-EA, GO10-B, and GO10-W at a concentration of 200 μg / mL

As a result, as shown in Figure 5, especially ethyl acetate fractions significantly inhibited the activity of the 5 alpha-reductase in a concentration-dependent, it was confirmed that exhibited a particularly significant inhibitory effect at a concentration of 200 μg / mL or more.

Experimental Example 4: Confirmation of the effect of prostate hypertrophy treatment in animal model (fraction)

4-1. Animal Model Production

Ten weeks-old male wistar rats (purchased central experiment animals, 6 per group) were purified for 1 week, and then TP (testosterone propionate) was injected subcutaneously at 3 mg / kg for 4 weeks to prepare a prostatic hypertrophy inducing group.

One hour before TP injection, each of the four fractions prepared in Example 3 was orally administered at 100 mg / kg for 4 weeks to prepare an experimental group. A positive control group was prepared by orally administering 10 mg / kg of finasteride, a 5α-reductase inhibitor used as a prostatic hypertrophy agent. In the solvent control, corn oil was injected subcutaneously.

Hereinafter, the produced animal model group is expressed as follows. NC: normal group, BPH: prostatic hyperplasia group, Fin: prostatic hyperplasia group + finasteride administration group, Hexane: prostatic hyperplasia group + fission hexane fraction (Example 3-1) administration group, EA: prostate hypertrophy group + hypochlorous ethyl Acetate fraction (Example 3-2) administration group, BuOH: prostatic hypertrophy-induced group + fission butanol fraction (Example 3-3) administration group, Water: prostate hypertrophy-induced group + fat chamber water fraction (Example 3-4) administration group.

4-2. Prostate weight measurement

After extracting the prostate gland from each group produced in Experimental Example 4-1, the weight thereof was measured and shown in FIG. 6. Statistical analysis was performed by ANOVA. * P <0.05 and ** P <0.01 were significantly different compared to normal group (NC). # P <0.05 and ## P <0.01 were the trigger group (BPH). ) And significant differences were considered.

As a result, as shown in Figure 6, compared with the normal group (NC), the prostate weight of the prostatic hypertrophy-inducing group (BPH) increased more than two times, in the case of the fraction of the fibrin extract significantly inhibited the increase in the prostate weight Confirmed. In particular, the effects of butanol fraction (BuOH) and water fraction (Water) was excellent. The inhibitory effect was found to be very similar to Finasteride (Fin), which is currently used as a therapeutic agent, suggesting that the effect of the fraction of the fruiting extract is very good.

As described above, the fruiting extract and its fractions not only significantly reduce the activity of 5-alpha-reductase, which is highly associated with the induction of prostate-related diseases such as prostatitis, prostatic hyperplasia, and prostate cancer, but also prostatic hypertrophy animal model induced by TP. Fruit extracts also inhibited the increase of prostate weight, significantly inhibited the increase of DHT (dihydrotestosterone) level, which is essential for the development, growth and maintenance of the prostate, and significantly inhibited the proliferation of prostate epithelial cells. In particular, it was confirmed that this effect is equivalent to finasteride that is currently used as a treatment for prostate-related diseases.

Therefore, fruit juice extract or fractions thereof can be effectively used for the prevention, treatment, and improvement of prostate-related diseases.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (9)

Phosphorus (Ponciri Fructus) extract or a fraction thereof as an active ingredient, a prophylactic or therapeutic pharmaceutical composition for the treatment of diseases. The method of claim 1, The fruit extract is prepared by extracting with a solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof. The method of claim 1, The fraction is prepared by fractionating the fruit extract with a solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, hexane (hexane), ethyl acetate, and mixed solvents thereof. The method of claim 1, Prevention or treatment of the prostate-related disease is made by reducing the activity of 5 alpha-reductase, pharmaceutical composition. The method of claim 1, The prostate-related disease is one or more diseases selected from the group consisting of prostatic hyperplasia, prostatitis, prostate cancer, prostate abscess, and prostate calcification. A method of preventing or treating a prostate related disease, comprising administering to a subject a pharmaceutical composition according to any one of claims 1 to 5. Food composition for the prevention or amelioration of prostate-related diseases comprising Posiliri Fructus extract or a fraction thereof as an active ingredient. The method of claim 7, wherein The food composition is a health functional food, food composition. Po quasi Fructus extract or a quasi-drug composition for the prevention or improvement of prostate-related diseases comprising a fraction thereof as an active ingredient.

Images