Composition for preventing or treating benign prostatic hyperplasia by using isoflavone-enhanced fraction of maclura tricuspidata fruit extract

Abstract

The present invention relates to a composition for preventing or treating benign prostatic hyperplasia, comprising, as an active ingredient, a fraction of a Maclura tricuspidata fruit extract, wherein the sum of the concentrations of alpinumisoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinumisoflavone in the fraction of a Maclura tricuspidata fruit extract is 60-90 w / w%.

Description

Composition for the prevention or treatment of benign prostatic hyperplasia using an isoflavone-fortified Cudrania tricuspidata fruit extract fraction

[0001] The present invention relates to a composition for the prevention or treatment of benign prostatic hyperplasia comprising an isoflavone-enriched Cudrania tricuspidata fruit extract fraction as an active ingredient.

[0002] With increasing interest in improving the quality of life due to the aging of the human population, there is a growing demand for treatment of Benign Prostatic Hyperplasia (BPH), one of the representative diseases affecting elderly men. BPH is a condition in which an enlarged prostate compresses the urethra, causing lower urinary tract symptoms such as frequent urination, a sensation of incomplete bladder emptying, and urinary urgency. Male hormones in the prostate play a significant role in the growth, development, and pathological state of the prostate through male hormone receptors. In particular, type 2 5-alpha reductase present in the prostate plays a crucial role in the development of BPH by converting male hormones into dihydrotestosterone (DHT), a more potent male hormone. Specifically, in patients deficient in type 2 5-alpha reductase, the prostate is very small and does not exhibit BPH.

[0003] Selective alpha-blockers (terazosin, doxazosin, tamsulosin) and 5-alpha-reductase inhibitors (finasteride, dutasteride) are mainly prescribed in clinical practice, but they have a fatal side effect called sexual dysfunction.

[0004] Recently, herbal therapies have been gaining prominence to complement the side effects and drawbacks of conventional synthetic drugs used to prevent and treat benign prostatic hyperplasia, and health functional foods for this purpose (e.g., product name Saw Palmetto) are also being sold.

[0005] Meanwhile, *Cudrania tricuspidata*, belonging to the Moraceae family, has been used in traditional herbal medicine. It is used to treat various diseases, and its efficacy in improving inflammation, gastritis, tumors, and liver cell damage is reported in traditional medical literature such as the *Donguibogam* and *Sikmulboncho*. In folk medicine, *Cudrania tricuspidata* has been used as a compound medicinal tea alongside various other herbal medicines, contributing to the prevention of inflammatory diseases and the maintenance of health due to its anti-inflammatory and antioxidant effects. Notably, no side effects have been reported with consistent consumption, and it is generally regarded as a plant that can be used safely.

[0006] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia, comprising a Cudrania tricuspidata fruit extract fraction as an active ingredient, wherein the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone in the Cudrania tricuspidata fruit extract fraction is 60 w / w% to 90 w / w%.

[0007] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0008] The present invention provides a pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia, comprising a Cudrania tricuspidata fruit extract fraction as an active ingredient, wherein the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone in the Cudrania tricuspidata fruit extract fraction is 60 w / w% to 90 w / w%.

[0009] The above Cudrania tricuspidata fruit extract fraction can be prepared by including the following steps: i) adsorbing a crude Cudrania tricuspidata fruit extract onto an adsorption resin; ii) washing the crude extract not adsorbed onto the adsorption resin with a washing solvent; and iii) adding a desorption solvent to the washed adsorption resin to desorb the extract.

[0010] In step i) above, the adsorption resin may be a styrene-based adsorption resin or an acrylic-based adsorption resin.

[0011] The styrene-based adsorption resin may be one or more selected from the group consisting of Diaion HP20 resin, D101 macroporous resin, Amberlite XAD-2, Amberlite XAD-4, and Amberlite XAD-1180, and the acrylic-based adsorption resin may be one or more selected from the group consisting of Amberlite XAD-7, Amberlite XAD-7HP, Amberlite XAD-8, and Amberlite XAD-2000.

[0012] In step ii) above, the washing solvent may be water or a mixed solvent of water and C1-C4 lower alcohols.

[0013] In step iii) above, the desorption solvent may be a C1 to C4 lower alcohol or acetone.

[0014] In one embodiment of the present invention, a health functional food composition for preventing or improving benign prostatic hyperplasia is provided, comprising a Cudrania tricuspidata fruit extract fraction as an active ingredient, wherein the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone in the Cudrania tricuspidata fruit extract fraction is 60 w / w% to 90 w / w%.

[0015] The above Cudrania tricuspidata fruit extract fraction can be prepared by including the following steps: i) adsorbing a crude Cudrania tricuspidata fruit extract onto an adsorption resin; ii) washing the crude extract not adsorbed onto the adsorption resin with a washing solvent; and iii) adding a desorption solvent to the washed adsorption resin to desorb the extract.

[0016] The isoflavone-enhanced Cudrania tricuspidata fruit extract fraction according to the present invention is prepared by adsorbing a crude Cudrania tricuspidata fruit extract onto an adsorption resin, washing the unadsorbed crude extract with a washing solvent, and then adding a desorption solvent to the washed adsorption resin to desorb it, and has the advantage of increasing the concentrations of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone compared to the crude Cudrania tricuspidata fruit extract.

[0017] Accordingly, the isoflavone-enriched Cudrania tricuspidata fruit extract fraction according to the present invention effectively inhibits cell proliferation of prostate epithelial cells and reduces the thickness of prostate epithelial tissue, thereby being effectively used for the prevention or treatment of benign prostatic hyperplasia.

[0018] Figure 1 is a graph showing the results of the analysis of active ingredients through UHPLC analysis of fractions at each step during manufacturing according to Example 1.

[0019] Figure 2 is a graph showing the results of the analysis of active ingredients through UHPLC analysis of fractions at each step during manufacturing according to Example 2.

[0020] Figure 3 is a graph analyzing the effect of treatment with the Cudrania tricuspidata fruit extract fraction prepared in Example 1 on RWPE-1 cells.

[0021] Figure 4 is a graph analyzing the effect of treatment with the Cudrania tricuspidata fruit extract fraction prepared in Example 1 on the cell proliferation rate of DHT-stimulated RWPE-1 cells.

[0022] Figure 5 is a graph analyzing the effect of the Cudrania tricuspidata fruit extract fraction prepared in Example 1 on the thickness of mouse prostate epithelial tissue.

[0023] While conducting research on *Maclura (Cudrania) tricuspidata*, the inventors confirmed that by preparing a *Maclura (Cudrania) fruit extract fraction by adsorbing a crude extract of *Maclura (Cudrania)* fruit onto an adsorption resin, washing the unadsorbed crude extract with a washing solvent, and then adding a desorption solvent to the washed adsorption resin to desorb the extract, the concentration of a component effective for the prevention or treatment of benign prostatic hyperplasia can be optimally increased, and thus completed the present invention.

[0024]

[0025] "Alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone" in this specification are all types of isoflavones or isoflavone-based substances.

[0026] In this specification, the term "prostate enlargement" broadly includes diseases in which an enlarged prostate compresses the urethra and causes lower urinary tract symptoms such as frequent urination, residual urine, and urgency (inability to hold urine).

[0027] In this specification, "prevention" refers to any act of delaying the onset of benign prostatic hyperplasia by administering the composition of the present invention, and "treatment" and "improvement" refer to any act of improving or beneficially changing the symptoms of benign prostatic hyperplasia by administering the composition of the present invention.

[0028]

[0029] The present invention will be described in detail below.

[0030]

[0031] Pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia

[0032]

[0033] The present invention provides a pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia, comprising a Cudrania tricuspidata fruit extract fraction as an active ingredient, wherein the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone in the Cudrania tricuspidata fruit extract fraction is 60 w / w% to 90 w / w%.

[0034] To this end, the Cudrania tricuspidata fruit extract fraction according to the present invention is enriched with isoflavones, and the Cudrania tricuspidata fruit extract fraction can be prepared by comprising the steps of: i) adsorbing a crude Cudrania tricuspidata fruit extract onto an adsorption resin; ii) washing the crude extract not adsorbed onto the adsorption resin with a washing solvent; and iii) adding a desorption solvent to the washed adsorption resin to desorb the extract.

[0035]

[0036] First, the pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia according to the present invention contains an isoflavone (or isoflavone-based substance)-enriched Cudrania tricuspidata fruit extract fraction as an active ingredient. In the Cudrania tricuspidata fruit extract fraction, the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone may be 60 w / w% to 90 w / w%, and preferably 80 w / w% to 90 w / w%, but is not limited thereto. Thus, the Cudrania tricuspidata fruit extract fraction effectively inhibits cell proliferation of prostate epithelial cells and reduces the thickness of prostate epithelial tissue, thereby being effectively used for the prevention or treatment of benign prostatic hyperplasia.

[0037] That is, the above Cudrania tricuspidata fruit extract fraction is fortified with isoflavones (or isoflavone-based substances), and is characterized by increasing the concentrations of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone compared to the above Cudrania tricuspidata fruit crude extract, preferably increasing them by at least three times. In particular, the above Cudrania tricuspidata fruit extract fraction may have increased the concentration of 6,8-diprenylgenistein by at least six times compared to the above Cudrania tricuspidata fruit crude extract.

[0038] For example, in the above-mentioned Cudrania tricuspidata fruit extract fraction, the concentration ratio of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone may be 1:1.5:0.5 to 1:2:1, and is preferably 1:1.5:1 to 1:2:1, but is not limited thereto. That is, the above-mentioned Cudrania tricuspidata fruit extract fraction is characterized by having a higher concentration of 6,8-diprenylgenistein compared to alpinum isoflavone or 4'-O-methylalpinum isoflavone.

[0039] For example, in the above Cudrania tricuspidata fruit extract fraction, the concentration of alpinum isoflavone may be 10 w / w% to 30 w / w% (e.g., 20 w / w% to 30 w / w%), the concentration of 6,8-diprenylgenistein may be 30 w / w% to 50 w / w% (e.g., 35 w / w% to 45 w / w%), and the concentration of 4'-O-methylalpinum isoflavone may be 10 w / w% to 30 w / w% (e.g., 15 w / w% to 25 w / w%).

[0040]

[0041] Next, the pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia according to the present invention contains an isoflavone (or isoflavone-based substance) enriched Cudrania tricuspidata fruit extract fraction as an active ingredient, wherein the Cudrania tricuspidata fruit extract fraction can be prepared by comprising: i) a step of adsorbing a crude extract of Cudrania tricuspidata fruit onto an adsorption resin; ii) a step of washing the crude extract not adsorbed onto the adsorption resin with a washing solvent; and iii) a step of desorbing the extract by adding a desorption solvent to the washed adsorption resin.

[0042]

[0043] i) Step of adsorbing crude extract of Cudrania tricuspidata fruit onto an adsorption resin

[0044] The above crude extract of Cudrania tricuspidata fruit can be extracted from Cudrania tricuspidata fruit using water, an organic solvent, or a mixture thereof as a solvent according to conventional methods, and it is preferable to extract using water, C1 to C4 lower alcohols, or acetone as a solvent, and more preferable to extract using methanol, ethanol, or acetone as a solvent, but is not limited thereto.

[0045] Specifically, the crude extract of Cudrania tricuspidata fruit is preferably prepared by adding water, an organic solvent, or a mixture thereof in an amount of 5 to 50 times the weight of the Cudrania tricuspidata fruit after chopping dried Cudrania tricuspidata fruit, and more preferably by adding 10 to 30 times the weight, but is not limited thereto. The extraction temperature is preferably 10°C to 150°C, and more preferably 20°C to 120°C, but is not limited thereto. The extraction time is preferably 1 hour to 20 hours, and more preferably 3 hours to 10 hours, but is not limited thereto. Cold maceration, ultrasonic extraction, or reflux cooling extraction methods may all be used as extraction methods, but are not limited thereto. The number of extractions is preferably 1 to 5 times, and more preferably 2 to 3 times, but is not limited thereto. Additionally, the crude extract of Cudrania tricuspidata fruit may be diluted, concentrated, or purified and dried after dilution or concentration for use.

[0046] By adsorbing the crude extract of the Cudrania tricuspidata fruit onto an adsorption resin, there is an advantage in effectively removing unnecessary polar components such as sugars. To achieve this, a synthetic adsorption resin may be used, and a styrene-based adsorption resin or an acrylic-based adsorption resin may be used. In this case, the styrene-based adsorption resin may be one or more selected from the group consisting of Diaion HP20 resin, D101 macroporous resin, Amberlite XAD-2, Amberlite XAD-4, and Amberlite XAD-1180, and the acrylic-based adsorption resin may be one or more selected from the group consisting of Amberlite XAD-7, Amberlite XAD-7HP, Amberlite XAD-8, and Amberlite XAD-2000.

[0047] Specifically, it is preferable to use a styrene-based adsorption resin that has non-polarity or weak polarity and is suitable for adsorbing non-polar components due to its large surface area and porous structure, but is not limited thereto. By doing so, unnecessary polar components such as sugars can be removed more effectively.

[0048]

[0049] ii) A step of washing the crude extract not adsorbed on the adsorption resin with a washing solvent.

[0050] The washing solvent is a polar solvent intended to optimize the concentration of active ingredients by removing sugars, etc., from the crude extract that are not adsorbed by the adsorption resin. It is preferable to use water or a mixed solvent of water and C1-C4 lower alcohols as the washing solvent, but is not limited thereto. When a mixed solvent of water and C1-C4 lower alcohols is used as the washing solvent, it is preferable that the concentration of C1-C4 lower alcohols in the mixed solvent be greater than 0 v / v% to 50 v / v%, but is not limited thereto. For example, by performing a first washing using water as the washing solvent and then performing an additional washing using a mixed solvent of water and C1-C4 lower alcohols, sugars, etc., from the crude extract that are not adsorbed by the adsorption resin can be removed more effectively.

[0051]

[0052] iii) A step of desorbing by adding a desorption solvent to the washed adsorption resin.

[0053] The above adsorption resin is in a state where it has been washed with the above washing solvent, and a Cudrania tricuspidata fruit extract fraction can be prepared (separated) by adding a desorption solvent to the washed adsorption resin to desorb (elute) the sample.

[0054] C1 to C4 lower alcohols or acetone may be used as the desorption solvent. This allows for the optimal separation of the concentration of components effective for the prevention or treatment of benign prostatic hyperplasia. For example, a Cudrania fruit extract fraction can be prepared by adding a C1 to C4 lower alcohol as the desorption solvent to perform primary desorption, and then adding acetone to the adsorbent resin that was desorbed in the primary step to perform additional desorption. If necessary, the use of C1 to C4 lower alcohols as the desorption solvent may be omitted, and the Cudrania fruit extract fraction can be prepared by using only acetone for desorption.

[0055]

[0056] The concentration of the above Cudrania tricuspidata fruit extract fraction may be 1 μg / mL to 100 μg / mL, preferably 10 μg / mL to 50 μg / mL, and more preferably 10 μg / mL to 20 μg / mL, but is not limited thereto. At this time, if the concentration is below the above range, there is a limitation in that it cannot effectively inhibit cell proliferation in prostate epithelial cells, and if the concentration exceeds the above range, there may be concerns regarding toxicity, including cytotoxicity.

[0057]

[0058] The above pharmaceutical compositions may each be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external formulations, suppositories, and sterile injectable solutions according to conventional methods, and may include suitable carriers, excipients, or diluents commonly used in the manufacture of pharmaceutical compositions for formulation.

[0059] The above-mentioned carrier, excipient, or diluent may include various compounds or mixtures such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0060] When formulating, it can be manufactured using diluents or excipients such as commonly used fillers, weights, binders, wetting agents, disintegrants, and surfactants.

[0061] A solid dosage form for oral administration can be prepared by mixing at least one excipient, such as starch, calcium bonate, sucrose or lactose, gelatin, etc., with the above-mentioned Cudrania tricuspidata fruit extract fraction. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0062] Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin.

[0063] Preparations for parenteral administration include sterile aqueous solutions, water-insoluble preparations, suspensions, emulsions, lyophilized preparations, and suppositories. As water-insoluble solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, glycerol gelatin, etc. may be used.

[0064] The preferred dosage of the above pharmaceutical composition varies depending on the patient's condition, body weight, severity of the disease, drug form, route of administration, and duration, but can be appropriately selected by a person skilled in the art. However, for a desirable effect, it may be administered at a dose of 0.001 to 2,000 mg / kg per day, preferably 0.1 to 1,000 mg / kg, and more preferably 10 to 500 mg / kg. The administration may be performed once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosage.

[0065] The above pharmaceutical composition may be administered to mammals, such as rats, mice, livestock, and humans, via various routes. All modes of administration may be, for example, orally, rectally or intravenously, intramuscularly, subcutaneously, intrathecally, or intracerebroventricularly.

[0066]

[0067] Health functional food composition for the prevention or improvement of benign prostatic hyperplasia

[0068]

[0069] The present invention provides a health functional food composition for preventing or improving benign prostatic hyperplasia, characterized in that it contains a Cudrania tricuspidata fruit extract fraction as an active ingredient, wherein the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone in the Cudrania tricuspidata fruit extract fraction is 60 w / w% to 90 w / w%.

[0070] To this end, the Cudrania tricuspidata fruit extract fraction according to the present invention is enriched with isoflavones, and the Cudrania tricuspidata fruit extract fraction can be prepared by comprising the steps of: i) adsorbing a crude Cudrania tricuspidata fruit extract onto an adsorption resin; ii) washing the crude extract not adsorbed onto the adsorption resin with a washing solvent; and iii) adding a desorption solvent to the washed adsorption resin to desorb the extract.

[0071]

[0072] The health functional food composition for preventing or improving benign prostatic hyperplasia according to the present invention contains an isoflavone (or isoflavone-based substance)-enhanced Cudrania tricuspidata fruit extract fraction as an active ingredient. Since the "Cudrania tricuspidata fruit extract fraction and the method of preparing the same" has been described above, a redundant explanation will be omitted.

[0073]

[0074] The concentration of the above Cudrania tricuspidata fruit extract fraction may be 1 μg / mL to 100 μg / mL, preferably 10 μg / mL to 50 μg / mL, and more preferably 10 μg / mL to 20 μg / mL, but is not limited thereto. At this time, if the concentration is below the above range, there is a limitation in that it cannot effectively inhibit cell proliferation in prostate epithelial cells, and if the concentration exceeds the above range, there may be concerns regarding toxicity, including cytotoxicity.

[0075]

[0076] In the above-mentioned health functional food composition, when the Cudrania tricuspidata fruit extract fraction is used as an additive to the health functional food, it may be added as is, used together with other foods or food ingredients, or used appropriately according to conventional methods. The mixing amount of the active ingredients can be appropriately determined according to each purpose of use, such as prevention, health, or treatment.

[0077] The formulation of the above health functional food composition can be in the form of powder, granule, pill, tablet, or capsule, as well as any form of general food or beverage.

[0078] There are no specific restrictions on the types of food mentioned above, and examples of food to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include all food in the conventional sense.

[0079] Generally, when manufacturing food or beverages, the above Cudrania tricuspidata fruit extract fraction may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of raw material. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range, and furthermore, since the present invention uses natural substances, there is no problem in terms of safety, so it may be used in an amount greater than the above range.

[0080] The beverage among the above health functional food compositions may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the above natural carbohydrates may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage.

[0081] In addition to the above, the above health functional food composition may contain various nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, the above health functional food composition may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. The proportion of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the above health functional food composition.

[0082]

[0083] Alternatively, the present invention provides a use for a composition for the prevention, treatment, or improvement of benign prostatic hyperplasia, characterized in that the composition comprises a Cudrania tricuspidata fruit extract fraction as an active ingredient, wherein the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone in the Cudrania tricuspidata fruit extract fraction is 60 w / w% to 90 w / w%.

[0084] Alternatively, the present invention provides a method for preventing, treating, or improving benign prostatic hyperplasia comprising the step of administering to an individual a composition characterized by including a Cudrania tricuspidata fruit extract fraction as an active ingredient, wherein the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone in the Cudrania tricuspidata fruit extract fraction is 60 w / w% to 90 w / w%. In this case, "individual" refers to a subject requiring treatment for a disease, and more specifically, refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0085]

[0086] As reviewed above, the isoflavone-enhanced Cudrania tricuspidata fruit extract fraction according to the present invention is prepared by adsorbing a crude Cudrania tricuspidata fruit extract onto an adsorption resin, washing the unadsorbed crude extract with a washing solvent, and then adding a desorption solvent to the washed adsorption resin to desorb it, and has the advantage of increasing the concentrations of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone compared to the crude Cudrania tricuspidata fruit extract.

[0087] Accordingly, the isoflavone-enriched Cudrania tricuspidata fruit extract fraction according to the present invention effectively inhibits cell proliferation of prostate epithelial cells and reduces the thickness of prostate epithelial tissue, thereby being effectively used for the prevention or treatment of benign prostatic hyperplasia.

[0088]

[0089] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0090]

[0091] [Example]

[0092] Comparative Example 1: Preparation of crude extract of Cudrania tricuspidata fruit

[0093] A crude extract was prepared by extracting Cudrania tricuspidata fruit at room temperature using 100% ethanol and then removing the solvent through a vacuum concentrator.

[0094]

[0095] Comparative Example 2: Preparation of crude extract of Cudrania tricuspidata fruit

[0096] A crude extract was prepared in the same manner as in Comparative Example 1, except that 100% methanol was used instead of 100% ethanol.

[0097]

[0098] Comparative Example 3: Preparation of crude extract of Cudrania tricuspidata fruit

[0099] A crude extract was prepared in the same manner as in Comparative Example 1, except that 100% acetone was used instead of 100% ethanol.

[0100]

[0101] Example 1: Preparation of Cudrania tricuspidata fruit extract fraction using Diaion HP-20 resin

[0102] 142.1 mg of crude extract of Cudrania tricuspidata prepared in Comparative Example 1 was adsorbed onto 1.4 g of Diaion HP-20 resin at room temperature. At this time, the crude extract that was not adsorbed was washed once with 400 mL of H2O washing solvent [H2O fraction: 59.9 mg (42.2%)], and then washed a second time with 200 mL of 50% methanol [50% MeOH fraction: 32.4 mg (22.8%)].

[0103] Subsequently, 200 mL of 100% methanol was added to the washed Diaion HP-20 resin and the MeOH fraction was prepared by primary desorption [MeOH fraction: 17.0 mg (12.0%)]. 300 mL of acetone was added to the primary desorbed Diaion HP-20 resin and the Acetone fraction was finally prepared by secondary desorption [Acetone fraction (or Enriched fraction): 25.1 mg (17.7%)].

[0104] The active ingredients were analyzed by UHPLC analysis on the fractions of each step under the conditions shown in Table 1 below, and the results are shown in Figure 1.

[0105]

[0106] UHPLC Analysis Conditions Instrument: Waters ACQUITY UPLC Column: AQUITY UPLC BEH C18 (100 × 2.1 mm × 1.7 µm) Mobile Phase: A: 0.1% formic acid in water B: Acetonitrile Flow rate 0.3 mL / min, Column temperature 35 ℃, Detector PDA 200-400 nm @ 254 nm, Injection volume 2.0 µL, Operating time 15.0 min, Injection concentration 2.0 mg / mL

[0107]

[0108] As shown in Figure 1, the MeOH fraction and Acetone fraction are similar in their active ingredients as a large amount of sugars and the like are removed through washing as a preliminary step, and in particular, it is confirmed that isoflavones are enhanced. Commonly, they are confirmed to contain alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone.

[0109]

[0110] Example 2: Preparation of Cudrania tricuspidata fruit extract fraction using D101 macroporous resin

[0111] 145.9 mg of crude extract of Cudrania tricuspidata prepared in Comparative Example 1 was adsorbed onto 1.4 g of D101 macroporous resin at room temperature. At this time, the unadsorbed crude extract was washed once with 400 mL of H2O washing solvent [H2O fraction: 43.7 mg (30.0%)], then washed a second time with 200 mL of 25% methanol [25% MeOH fraction: 29.3 mg (20.1%)], and then washed a third time with 200 mL of 50% methanol [50% MeOH fraction: 28.6 mg (19.6%)].

[0112] Subsequently, 200 mL of 100% methanol was added to the washed D101 macroporous resin and desorbed in the first step to produce the MeOH fraction [MeOH fraction: 12.7 mg (8.7%)]. 300 mL of acetone was added to the D101 macroporous resin desorbed in the first step and desorbed in the second step to produce the final Acetone fraction [Acetone fraction (or Enriched fraction): 20.7 mg (14.2%)].

[0113] The active ingredients were analyzed by UHPLC analysis under the conditions shown in Table 1 above for each step fraction, and the results are shown in Figure 2.

[0114] As shown in Fig. 2, similar to Example 1, the MeOH fraction and Acetone fraction are similar in their active ingredients as a large amount of sugars, etc., are removed through washing as a preliminary step, and in particular, isoflavones are confirmed to be enhanced. Commonly, they are confirmed to contain alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone.

[0115]

[0116] Experimental Example 1: Analysis of Active Component Concentration in Isoflavone-Fortified Cudrania tricuspidata Fruit Extract Fraction

[0117] For the crude extract of Cudrania tricuspidata fruit prepared in Comparative Example 1 (crude extracts ①, ②, ③) and the Cudrania tricuspidata fruit extract fraction prepared in Example 1 (enriched fractions ①, ②, ③), the concentrations of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone as active ingredients were quantified by UHPLC analysis under the conditions shown in Table 1, and the results are shown in Table 2.

[0118]

[0119] Active ingredient No. Area (μV*sec) Result (μg / ml) Concentration in Sample (w / w%) Area (μV*sec) Result (μg / ml) Concentration in Sample (w / w%) Area (μV*sec) Result (μg / ml) Concentration in Sample (w / w%) Crude Extract ① 20667610.5226.681936767.2086.261459345.2274.72 Crude Extract ② 20928510.7036.601948367.2806.141460585.2394.60 Crude Extract ③ 20475010.3896.561996507.5776.401455665.1924.66 Enriched fraction ①142773995.05523.752471455147.76941.121177041104.88019.58Enriched fraction ②140075493.18623.472384748142.41939.961188870106.02319.92Enriched fraction ③141512294.18123.712402708143.52740.261181406105.30119.80

[0120]

[0121] As shown in Table 2, it is confirmed that the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone in the samples of the Cudrania tricuspidata fruit extract fractions prepared in Example 1 (Enriched fractions ①, ②, ③) is significantly increased by at least about 3 times compared to the crude Cudrania tricuspidata fruit extract prepared in Comparative Example 1 (crude extracts ①, ②, ③). That is, the Cudrania tricuspidata fruit extract fractions (Enriched fractions ①, ②, ③) prepared in Example 1 were found to have a total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone in the sample of approximately 83 w / w% to approximately 84 w / w%. In particular, it can be seen that the concentration of 6,8-diprenylgenistein in the sample was significantly increased by at least six times.

[0122] Specifically, it is confirmed that the concentration of alpinum isoflavone in the Cudrania tricuspidata fruit extract fractions (Enriched fractions ①, ②, ③) prepared in Example 1 is 10 w / w% to 30 w / w% (specifically, 20 w / w% to 30 w / w%), the concentration of 6,8-diprenylgenistein is 30 w / w% to 50 w / w% (specifically, 35 w / w% to 45 w / w%), and the concentration of 4'-O-methylalpinum isoflavone is 10 w / w% to 30 w / w% (specifically, 15 w / w% to 25 w / w%).

[0123]

[0124] Experimental Example 2: Evaluation of the therapeutic efficacy of isoflavone-fortified Cudrania tricuspidata fruit extract fraction for benign prostatic hyperplasia

[0125] (1) Analysis of the effect of isoflavone-fortified Cudrania tricuspidata fruit extract fraction on the cell viability of prostate epithelial cells

[0126] Before evaluating whether the Cudrania tricuspidata fruit extract fraction inhibits cell proliferation in DHT-stimulated human prostate epithelial cell line RWPE-1, the effects of treatment with the crude Cudrania tricuspidata fruit extract prepared in Comparative Example 1 (crude extract) and treatment with the enriched fraction prepared in Example 1 (enriched fraction) on RWPE-1 cells were compared, and the results are shown in Figure 3.

[0127] As shown in Figure 3, when RWPE-1 cells were treated with crude extract and enriched fraction at a concentration of 0.20 μg / ml, it was confirmed that there was no significant effect on cell viability at any of the treatment concentrations. Therefore, based on these results, subsequent cell experiments were conducted at a concentration of 0.20 μg / ml.

[0128]

[0129] (2) Analysis of the effect of isoflavone-fortified Cudrania tricuspidata fruit extract fraction on the cell proliferation rate of prostate epithelial cells

[0130] Subsequently, the effects of treatment with the crude extract of Cudrania tricuspidata prepared in Comparative Example 1 (crude extract) and treatment with the enriched fraction of Cudrania tricuspidata prepared in Example 1 on the cell proliferation rate of DHT-stimulated RWPE-1 cells were compared, and the results are shown in Figure 4.

[0131] As shown in Figure 4, it was confirmed that the cell proliferation rate of RWPE-1 cells, which was significantly increased by DHT treatment, was effectively inhibited only by treatment with the enriched fraction at a concentration of 10-20 μg / ml. These results indicate that the enriched fraction effectively inhibits the cell proliferation of DHT-stimulated RWPE-1 cells, which can be seen as having a superior antiproliferative effect compared to the crude extract.

[0132]

[0133] (3) Analysis of the effect of isoflavone-fortified Cudrania tricuspidata fruit extract fraction on prostate epithelial tissue thickness

[0134] Six-week-old male ICR mice (35±2 g) were purchased from Samin Science (Chung-cheong bukdo, Republic of Korea) for the experiment. The experimental animals were housed according to the experimental animal guidelines established by the Animal Ethics Committee of Kyung Hee University. After an acclimatization period of one week prior to the start of the experiment, food and water were provided freely during the four weeks of the experiment. The animals were kept in an environment with light controlled at 12-hour intervals, maintaining a temperature of 22 ± 2°C and a humidity of 55 ± 9%.

[0135] Experimental animals were randomly assigned to a total of 5 groups, with 7 animals (n=7) in each group. (1) Control (CON) group, (2) Benign prostatic hyperplasia induction (BPH) group, (3) Finasteride 5 mg / kg administration (Fina) group, (4) 100 mg / kg administration of crude extract of Cudrania tricuspidata prepared in Comparative Example 1 (crude extract) group, and (5) 100 mg / kg administration of Cudrania tricuspidata extract fraction prepared in Example 1 (enriched fraction) group.

[0136] BPH was induced in experimental animals, excluding the CON group, by subcutaneously injecting 5 mg / kg of testosterone propionate (TP) for 4 weeks excluding weekends. Simultaneously with the induction of BPH, the positive control drug Fina (5 mg / kg), crude extract (100 mg / kg), and enriched fraction (100 mg / kg) were orally administered.

[0137] Prostate tissues obtained from each group were fixed with 4% paraformaldehyde and embedded in paraffin to obtain sections, and Hematoxylin-eosin staining (H&E staining) was performed to observe changes in the thickness of the prostate epithelial tissue using a light microscope, and the results are shown in Figure 5.

[0138] At this time, all values ​​were expressed as mean ± standard deviation (SD). Data were determined using one-way analysis of variance (ANOVA) and Dunnett's post hoc test, and a P value of 0.05 or less was considered statistically significant.

[0139] As shown in Figure 5, it was confirmed that the thickness of the prostate epithelial tissue, which was significantly increased in the BPH group compared to the CON group, decreased in the Fina group, crude extract group, and enriched fraction group. In particular, the enriched fraction group was confirmed to show a statistically significant effect in alleviating prostate enlargement compared to the crude extract group.

[0140]

[0141] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. Contains a Cudrania tricuspidata fruit extract fraction as an active ingredient, and A pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia, characterized in that, in the above-mentioned Cudrania tricuspidata fruit extract fraction, the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone is 60 w / w% to 90 w / w%.

2. In Paragraph 1, The above Cudrania tricuspidata fruit extract fraction is i) A step of adsorbing crude extract of Cudrania tricuspidata fruit onto an adsorption resin; ii) a step of washing the crude extract not adsorbed on the adsorption resin with a washing solvent; and iii) A pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia, characterized by being manufactured by including the step of adding a desorption solvent to the washed adsorption resin to desorb the adsorption.

3. In Paragraph 2, A pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia, characterized in that the adsorption resin in step i) above is a styrene-based adsorption resin or an acrylic-based adsorption resin.

4. In Paragraph 3, A pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia, characterized in that the styrene-based adsorption resin is one or more selected from the group consisting of Diaion HP20 resin, D101 macroporous resin, Amberlite XAD-2, Amberlite XAD-4, and Amberlite XAD-1180, and the acrylic-based adsorption resin is one or more selected from the group consisting of Amberlite XAD-7, Amberlite XAD-7HP, Amberlite XAD-8, and Amberlite XAD-2000.

5. In Paragraph 2, A pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia, characterized in that the washing solvent in step ii) above is water or a mixed solvent of water and C1-C4 lower alcohols.

6. In Paragraph 2, A pharmaceutical composition for the prevention or treatment of benign prostatic hyperplasia, characterized in that the desorption solvent in step iii) above is a C1-C4 lower alcohol or acetone.

7. Contains a Cudrania tricuspidata fruit extract fraction as an active ingredient, and A health functional food composition for preventing or improving benign prostatic hyperplasia, characterized in that, in the above-mentioned Cudrania tricuspidata fruit extract fraction, the total concentration of alpinum isoflavone, 6,8-diprenylgenistein, and 4'-O-methylalpinum isoflavone is 60 w / w% to 90 w / w%.

8. In Paragraph 7, The above Cudrania tricuspidata fruit extract fraction is i) A step of adsorbing crude extract of Cudrania tricuspidata fruit onto an adsorption resin; ii) a step of washing the crude extract not adsorbed on the adsorption resin with a washing solvent; and iii) A health functional food composition for preventing or improving benign prostatic hyperplasia, characterized by being manufactured by including the step of adding a desorption solvent to the washed adsorption resin to desorb the adsorption.

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