KR102332246B1 - Composition for preventing or treating for benign prostatic hyperplasia comprising mixture extract of schisandrae fructus and corni fructus and the method for manufacturing thereof - Google Patents

Abstract

Provided are a composition for preventing or treating benign prostatic hyperplasia containing a mixed extract of Schisandra chinensis and Cornus officinalis fruits, and a method for manufacturing the same. Specifically, the composition for preventing or treating benign prostatic hyperplasia containing a mixed extract of Schisandra chinensis and Cornus officinalis fruits is composed of a mixed extract of mixed raw materials in which Schisandra chinensis and Cornus officinalis fruits are mixed in a ratio of 1 : 1 to 1 : 2.

Description

Composition for the prevention or treatment of benign prostatic hyperplasia, comprising a mixed extract of Omizawa cornus oil, and a manufacturing method thereof

The present invention relates to a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of omizawa cornus oil, and a method for preparing the same, and more particularly, to an extraction method using a suitable extraction solvent and an optimal blending ratio of omizawa cornus oil. It relates to a composition for preventing or treating benign prostatic hyperplasia comprising a mixed extract of omija and cornus oil, which can effectively improve hypertrophy, and a method for preparing the same.

BENIGN PROSTATIC HYPERPLASIA is a condition in which the prostate, located below the bladder, enlarges excessively. As the enlarged prostate presses on the urethra passing through the prostate, the discharge of urine is hindered and various urination symptoms appear. Accordingly, BPH can lead to urination disorders such as nocturia, frequent urination, and incontinence, or diseases such as urinary tract infection and urinary tract obstruction, if appropriate management and treatment are not performed, thereby reducing the quality of life. BPH was a disease that mainly occurred in people over 60 years old, but due to the aging of the population and westernization, the number of new patients is increasing as BPH disease occurs frequently even in men in their 30s and 40s.

Lycopene, selenium, vitamin D, vitamin E, and phytoestrogens are known as good materials for the prostate, and the most used material among them is saw palmetto extract. Saw palmetto extract is a natural coconut extract, and all of it is imported from abroad, so there are problems such as supply and demand problems and an increase in manufacturing cost. is increasing

In addition, conventional foods containing saw palmetto extract may have side effects such as vomiting, constipation, gastrointestinal disorders, lethargy and decreased libido after ingestion, thereby reducing side effects and improving the prevention or treatment efficacy of enlarged prostate There is a demand for development of natural materials that can be used.

On the other hand, CORNI FRUCTUS (or CORNUS OFFICINALIS) refers to the fruit of a cornus tree, a shrub belonging to the Dogwood family, contains various organic acids, and has antibacterial and anti-inflammatory effects, so it has been used as a medicine since ancient times. Recently, as studies proving that cornus oil is effective in alleviating prostatic hyperplasia, various health foods using cornusflower have been released.

Specifically, prior art 1 (Republic of Korea Patent No. 10-2081156) discloses a composition for improving benign prostatic hyperplasia containing a cornflower extract and the like. However, cornus oil has an inherent astringent and strong sour taste along with a slight sweetness, so it is necessary to supplement the astringent and sour taste in order to increase consumer preference.

Schisandrae (SCHISANDRAE FRUCTUS (or, SCHIZANDRA)) refers to the fruit growing on the Schisandra tree, and 20 to 30 red grains form a cluster. Omija means sour, bitter, sweet, spicy, and salty, that is, it has five flavors, and it has its own taste and flavor. Omija contains abundant amounts of saponins, anthocyanins, flavonoids, and amino acids that have anti-inflammatory and antioxidant properties, and is known to be good for fatigue recovery, cold prevention, and male diseases. Therefore, it has been used in oriental medicine for a long time to treat prostate diseases along with bokbunja, guyeopcho, etc., and recently, as in Prior Art 2 (Korea Patent No. 10-1634779), for the treatment of enlarged prostate containing omija as an active ingredient Techniques for the composition are continuously being studied.

However, there is an increasing demand for the development of natural materials that can further enhance the prevention or treatment efficacy of benign prostatic hyperplasia, reduce side effects inherent in the material, and increase consumer preference by adding taste and flavor.

(Patent No. 0001) Republic of Korea Patent No. 10-2081156 (Patent No. 0002) Republic of Korea Patent No. 10-1634779

In order to solve the above problems, the present invention is to prevent enlarged prostate comprising a mixed extract of Omizawa cornus oil, which can more effectively improve prostatic hyperplasia by appropriately mixing and using Omizawa cornus oil, a natural material produced in Korea. Or to provide a therapeutic composition and a method for preparing the same.

In addition, the present invention can improve the taste and flavor by supplementing the astringent and sour taste inherent in cornus oil through the optimal blending ratio of omija and cornus oil, and can improve the problem of restricting large amounts of intake due to the warm nature of omija. An object of the present invention is to provide a composition for the prevention or treatment of benign prostatic hyperplasia, and a method for preparing the same, comprising a mixed extract of cornflower and cornflower oil.

In addition, the present invention can effectively extract the active ingredient of Omizawa cornus oil using an appropriate extraction solvent, and can improve product quality by reducing sediment. To provide a therapeutic composition and a method for preparing the same.

In order to achieve the above object, one aspect of the present invention is for prostate enlargement comprising a mixed extract of Omizawa cornus oil, characterized in that it consists of a mixed extract of mixed raw materials in which Omizawa cornus oil is mixed in a ratio of 1:1 to 1:2. A composition for prevention or treatment is provided.

In one embodiment of the present invention, the mixed extract is extracted by adding 100 parts by weight of an extraction solvent in which water and ethanol are mixed with respect to 10 parts by weight of the mixed raw material, and the concentration of ethanol in the extraction solvent is 40 to 60 wt%. characterized in that it is composed.

In one embodiment of the present invention, after 0.4 to 0.8 parts by weight of the enzyme is added to the beta-glucanase enzyme with respect to 100 parts by weight of the mixed extract, it is characterized in that the added enzyme is inactivated.

Another aspect of the present invention is the first step of preparing a mixed raw material in which Omizawa cornus oil is mixed in a ratio of 1:1 to 1:2, and an extraction solvent composed of water and ethanol is mixed with the mixed raw material to prepare a raw material solvent mixture A second step of forming, a third step of extracting the raw solvent mixture at a temperature of 45 to 55° C. for 2 to 6 hours to form a mixed extract, a fourth step of concentrating the mixed extract to have a concentration of 55 to 65 brix It provides a method for preparing a composition for preventing or treating benign prostatic hyperplasia, comprising a fifth step of filtering after sterilizing the concentrated mixed extract and sterilizing the concentrated extract.

In one embodiment of the present invention, in the second step, 100 parts by weight of the extraction solvent is mixed with respect to 10 parts by weight of the mixed raw material, and the concentration of ethanol in the extraction solvent is 40 to 60 wt%.

In one embodiment of the present invention, the fourth step includes the first step 4-1 of first concentrating the mixed extract to have a sugar content of 20 to 30 brix, and beta with respect to 100 parts by weight of the first concentrated mixed extract. Step 4-2 of reacting by adding 0.4 to 0.8 parts by weight of glucanase (β-glucanase), and heating the mixed extract reacted with the enzyme at a temperature of 90 to 100 ° C. for 5 to 20 minutes to inactivate the enzyme It is characterized in that it comprises the step 4-3 and the second step 4-4 of concentrating the mixed extract in which the enzyme is inactivated to have 55 to 65 brix.

The composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of Omizawa cornus oil of the present invention and a method for producing the same can more effectively improve prostatic hyperplasia using Omizawa cornus oil, a medicinal berry produced in Korea.

In addition, the composition for the prevention or treatment of benign prostatic hyperplasia, comprising the mixed extract of omija and cornus oil of the present invention, and a method for preparing the same can improve taste and flavor by supplementing the astringent and sour taste inherent in cornflower oil, and due to the warm nature of omija It is possible to improve the problem of limiting large amounts of intake.

In addition, the composition for the prevention or treatment of benign prostatic hyperplasia comprising the mixed extract of Schisandra oleifera of the present invention and the method for preparing the same can effectively extract the active ingredients of Omizawa cornus oil using an appropriate extraction solvent, and reduce the sediment Product quality can be improved.

However, the effects of the invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a flow chart for explaining a method for preparing a composition for preventing or treating benign prostatic hyperplasia comprising a mixed extract of omija and cornus oil according to an embodiment of the present invention.
Figure 2 is a flow chart showing a method for producing a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of Omiza and cornus oil including an enzyme reaction and an enzyme inactivation process according to another embodiment of the present invention.
3A is a chart showing the cell viability (cell viability) of LNCaP cells according to the concentration of DHT according to an embodiment of the present invention.
Figure 3B is a chart showing the degree of proliferation of LNCaP cells according to the treatment time of DHT according to an embodiment of the present invention.
3C is an image of observing the morphological change of LNCaP cells by DHT according to an embodiment of the present invention with a phase contrast microscope.
3D is a chart showing the degree of AR expression in LNCaP cells according to the concentration of DHT according to an embodiment of the present invention.
4A is a table showing the results of a cytotoxicity test (MTT assay), which is the effect of the hot water extract of Schisandrae Fructu on the cell viability of LNCaP cells according to an embodiment of the present invention.
FIG. 4B is a table showing the results of a cytotoxicity test (MTT assay), which is the effect of Corni Fructus hot water extract on the cell viability of LNCaP cells according to an embodiment of the present invention.
4C is a table showing the results of the cytotoxicity test (MTT assay), which is the effect of the 40% ethanol extract of Corni Fructus on the cell viability of LNCaP cells according to an embodiment of the present invention.
4D is a table showing the results of a cytotoxicity test (MTT assay), which is the effect of a 40% ethanol extract of Corni Fructus on the cell viability of LNCaP cells according to an embodiment of the present invention.
5A is Schisandra hot water extract (SF-water), Cornus officinalis hot water extract (CF-water), Schisandra 40% ethanol extract (SF-EtOH) and Cornus officinalis before DHT treatment on LNCaP cells according to an embodiment of the present invention. Table showing the degree of cell proliferation in the case of pretreatment with 40% ethanol extract (CF -EtOH).
FIG. 5B is a Schisandra hot water extract (SF-water), Cornus officinalis hot water extract (CF-water), Schisandra 40% ethanol extract (SF-EtOH) and Cornus officinalis before DHT treatment on LNCaP cells according to an embodiment of the present invention. A chart showing the expression changes of androgen-related genes (AR and PSA) and cell proliferation-related indicator genes (PCNA) in the case of pretreatment with 40% ethanol extract (CF-EtOH).
Figure 6A is a chart showing the degree of ROS generation by treating LNCaP cells according to an embodiment of the present invention with 100 nM DHT.
FIG. 6B shows Schisandra hot water extract (SF-water), Cornus officinalis hot water extract (CF-water), Schisandra 40% ethanol extract (SF-EtOH) and Cornus extract 40% ethanol in LNCaP cells according to an embodiment of the present invention. A table showing the degree of ROS generation after pretreatment (CF -EtOH) and treatment with 100 nM DHT.
7A is a mixture containing Schisandra (SF) 40% ethanol extract and Cornus officinalis (CF) 40% ethanol extract prepared in Examples 1 to 3 according to an embodiment of the present invention at respective mixing ratios. A chart showing the results of the cytotoxicity test (MTT assay) showing the effect on the cell viability of LNCaP cells.
7B is a mixture containing Schisandra (SF) 40% ethanol extract and Cornus officinalis (CF) 40% ethanol extract prepared in Examples 1 to 3 according to an embodiment of the present invention in respective mixing ratios. A chart showing the level of androgen-related gene expression induced by DHT in pretreated LNCaP cells.
8 is a 1:1 mixture of a 40% ethanol extract of Schisandra (SF) or Cornus officinalis (CF) and a 60% ethanol extract of Schisandra (SF) or Cornus officinalis (CF), Schisandra (SF) or Cornus officinalis (SF) LNCaP cells Table showing the results of the cytotoxicity test (MTT assay) showing the effect on the DHT-stimulated growth of.
FIG. 9 is a chart showing the effect of 40% and 60% ethanol extracts of Schisandra or Cornus officinalis, a 1:1 mixture of each, on AR expression in DHT-stimulated LNCaP cells.
FIG. 10A shows the effect of 40% and 60% ethanol extracts of Schisandra (SF) or Cornus officinalis (CF), a 1:1 mixture thereof, on PSA expression in DHT-stimulated LNCaP cells according to an embodiment of the present invention; A chart showing the results of an ELISA experiment.
FIG. 10B shows the effect of 40% and 60% ethanol extracts of Schisandra (SF) or Cornus officinalis (CF), a 1:1 mixture thereof, on PSA expression in DHT-stimulated LNCaP cells according to an embodiment of the present invention; A chart showing the degree of PSA production showing
11A is a chart showing the results of a flow cytometer showing the degree of ROS generation by treating LNCaP cells according to an embodiment of the present invention with 100 nM DHT.
11B shows the effect of 40% and 60% ethanol extracts of Schisandra (SF) or Cornus officinalis (CF), a 1:1 mixture thereof, on intracellular ROS production in DHT-stimulated LNCaP cells according to an embodiment of the present invention; A chart showing the results of a flow cytometer showing .
FIG. 11C shows the effect of 40% and 60% ethanol extracts of Schisandra (SF) or Cornus officinalis (CF), a 1:1 mixture thereof, on intracellular ROS production in DHT-stimulated LNCaP cells according to an embodiment of the present invention; An image showing the results of fluorescence microscopic observation.
12A is an image showing the composition prepared in Example 5, and FIG. 12B is an image showing the composition prepared in Example 8.

Hereinafter, a preferred embodiment of a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of omija and cornus oil according to the present invention and a method for preparing the same will be described in detail with reference to the accompanying drawings.

Composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of Omizawa Cornus officinalis

The present invention provides a composition for preventing or treating benign prostatic hyperplasia comprising a mixed extract of Omiza and Cornus officinalis. Specifically, the composition for the prevention or treatment of benign prostatic hyperplasia of the present invention may be composed of a mixed extract of a mixed raw material in which Omizawa cornflower oil is mixed in a ratio of 1:1 to 1:2. That is, the composition for the prevention or treatment of benign prostatic hyperplasia of the present invention comprises a mixed extract formed by mixing omizawa cornflower oil as a raw material in a ratio of 1:1 to 1:2 to form a mixed raw material, and extracting the mixed raw material it could be

That is, the present invention is composed by mixing Omizawa Cornus oil, which has the prevention or treatment effect of prostatic hyperplasia, in an appropriate ratio, thereby maintaining the intrinsic efficacy of the material of Omizawa Cornus oil and harmoniously harmonizing with each other through taste and flavor. It is possible to provide a composition for the prevention or treatment of benign prostatic hyperplasia that can improve.

When Omija and cornus oil composed of the above mixed raw material is mixed in a larger amount than cornus oil, not in a ratio of 1:1 to 1:2, when it is mixed in a larger amount, it lowers the blood sugar level when ingesting a large amount of Schisandra. Therefore, it can be used to a limited extent, and the unique efficacy, taste and flavor of cornflower oil may not harmoniously harmonize. In addition, when Omizawa cornus oil composed of the above mixed raw material is mixed in a larger amount than 1:2 ratio, rather than in a ratio of 1:1 to 1:2, the astringent taste and sour taste inherent to cornus oil are mixed. It may become strong and the overall taste and flavor may deteriorate, and it may be difficult to satisfy consumer preference.

In one embodiment of the present invention, the mixed extract is extracted by adding 100 parts by weight of an extraction solvent in which water and ethanol are mixed with respect to 10 parts by weight of the mixed raw material, and the concentration of ethanol in the extraction solvent is 40 to 60 wt%. may be configured. Specifically, the mixed extract may be composed of a mixed extract by performing ethanol extraction by adding an extraction solvent in which water and ethanol are mixed to a mixed raw material in which Omizawa cornflower oil is mixed in a ratio of 1:1 to 1:2. At this time, extraction may be performed by mixing 100 parts by weight of the extraction solvent with respect to 10 parts by weight of the mixed raw material.

As the mixed raw material and the extraction solvent are mixed in a ratio of 1:10 and extracted, the mixed extract is effectively extracted with the active ingredients contained in the omija and the cornus oil, so that the taste and flavor can be improved. It is possible to improve the quality of the composition for the prevention or treatment of benign prostatic hyperplasia, including the mixed extract of cornflower oil.

In addition, in detail, the concentration of the ethanol in the extraction solvent in which the water and ethanol are mixed may be comprised of 40 to 60 wt%. When the concentration of the ethanol in the extraction solvent is less than 40wt%, the active ingredients contained in Omizawa Cornus officinalis may not be easily extracted, so that the quality of the mixed extract may be lowered or the process yield may be lowered. In addition, when the concentration of the ethanol in the extraction solvent exceeds 60 wt%, the water content in the extraction solvent is reduced and the water-soluble active ingredients contained in Omizawa cornus oil are not extracted, so the quality, taste and flavor of the mixed extract may affect

In one embodiment of the present invention, after 0.4 to 0.8 parts by weight of the beta-glucanase enzyme is added with respect to 100 parts by weight of the mixed extract, the added enzyme may be inactivated. That is, the mixed extract contained in the composition for the prevention or treatment of benign prostatic hyperplasia including the mixed extract of Omiza and Cornus officinalis of the present invention undergoes an enzymatic reaction such as hydrolysis as the beta-glucanase enzyme is added. It may be that the precipitate in the mixed extract has been removed through the

Specifically, 0.4 to 0.8 parts by weight of the beta-glucanase enzyme may be added with respect to 100 parts by weight of the mixed extract. When less than 0.4 parts by weight of the beta-glucanase enzyme is added with respect to 100 parts by weight of the mixed extract, the enzyme reaction may not be sufficiently performed, so it may be difficult to remove the precipitate in the mixed extract. In addition, when the beta-glucanase enzyme is added in excess of 0.8 parts by weight with respect to 100 parts by weight of the mixed extract, the manufacturing cost may increase due to an increase in cost due to an increase in the amount of the enzyme added, within the above-described range It may be that the enzyme is added in

The enzyme added to the mixed extract may be to inactivate the enzyme after the enzyme reaction proceeds. The inactivation of the enzyme is to inactivate the enzyme by stopping the enzyme reaction, and may be carried out through appropriate temperature and heating. Through the inactivation of these enzymes, the mixed extract can maintain its original color and flavor for a long time.

As described above, the composition for the prevention or treatment of prostatic hyperplasia comprising the mixed extract of Omija and Cornus officinalis of the present invention uses a beta-glucanase enzyme to remove the precipitate formed in the composition, the mixed extract The purity of can be increased, it is possible to maintain the unique taste and flavor of the mixed material of the mixed extract, it is possible to improve the quality of the composition.

In addition, the composition for the prevention or treatment of benign prostatic hyperplasia comprising the mixed extract of omija and cornus oil of the present invention is a medicinal berry produced in Korea, using natural materials such as cornflower omija to improve the prevention or treatment effect of enlarged prostate. can In addition, the composition for the prevention or treatment of benign prostatic hyperplasia comprising the mixed extract of omija and cornus oil of the present invention can enhance the taste and flavor by supplementing the astringent and sour taste inherent in cornflower oil. problem can be improved.

Method for producing a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of Omizawa Cornus officinalis

Another aspect of the present invention provides a method for preparing a composition for the prevention or treatment of benign prostatic hyperplasia, comprising a mixed extract of Omija and Cornus officinalis.

1 is a flow chart showing a method for preparing a composition for preventing or treating benign prostatic hyperplasia, comprising a mixed extract of Schisandra and cornus oil according to an embodiment of the present invention.

Referring to Figure 1, the method for preparing a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of Omizawa cornus oil of the present invention prepares a mixed raw material obtained by mixing Omizawa cornus oil in a ratio of 1:1 to 1:2 a first step (S10), a second step (S20) of mixing an extraction solvent composed of water and ethanol with the mixed raw material to form a raw material solvent mixture (S20), and the raw material solvent mixture at a temperature of 45 to 55° C. for 2 hours to A third step (S30) of extracting for 6 hours to form a mixed extract, a fourth step (S40) of concentrating the mixed extract to have a concentration of 55 to 65 brix (S40) and sterilizing the concentrated mixed extract, followed by filtering It may include step 5 (S50).

As shown in FIG. 1 , S10 may be to prepare a mixed raw material in which Omizawa cornflower oil is mixed in a ratio of 1:1 to 1:2. Omizawa cornflower oil can be used as it is, or can be collected and dried. It may be to wash cornflower oil and omija, and mix omija and omija oil in a ratio of 1:1 to 1:2. It may be to put the mixed raw material mixed with cornflower oil and omija into a conventional extraction device. According to the embodiment, the cornus oil may be used by removing many toxic seeds.

In S10, when Omija and cornus oil composed of the mixed raw material are mixed in a larger amount than cornus oil, not in a ratio of 1:1 to 1:2, when mixed in a larger amount When ingesting a large amount of omija It can be used limitedly due to its properties, and the unique efficacy, taste and flavor of cornflower oil may not harmoniously harmonize. In addition, when Omizawa cornus oil composed of the above mixed raw material is mixed in a larger amount than 1:2 ratio, rather than in a ratio of 1:1 to 1:2, the astringent taste and sour taste inherent to cornus oil are mixed. It may become strong and the overall taste and flavor may deteriorate, and it may be difficult to satisfy consumer preference.

1, S20 may be to form a raw material solvent mixture by mixing the extraction solvent consisting of water and ethanol to the mixed raw material, that is, mixing the extraction solvent to extract the mixed raw material prepared in S10. As such, the extraction solvent may be an ethanol aqueous solution composed of water and ethanol. By using an aqueous ethanol solution as the extraction solvent, both the fat-soluble active ingredient and the water-soluble active ingredient contained in Omizawa Cornus officinalis can be easily extracted.

In one embodiment of the present invention, in the second step, 100 parts by weight of the extraction solvent is mixed with respect to 10 parts by weight of the mixed raw material, and the concentration of ethanol in the extraction solvent may be 40 to 60 wt%. Specifically, by mixing 100 parts by weight of the extraction solvent with respect to 10 parts by weight of the mixed raw material, it is possible to effectively extract the active ingredients contained in Cornus oleifera and Schisandra Schisandra. If less than 100 parts by weight of the extraction solvent is mixed with respect to 10 parts by weight of the mixed raw material, the extraction efficiency may be lowered and the amount of the extract may be reduced. In addition, when the extraction solvent is mixed in excess of 100 parts by weight with respect to 10 parts by weight of the mixed raw material, the concentration time increases in the subsequent concentration process, which may affect the process efficiency.

In addition, the concentration of the ethanol in the extraction solvent in which the water and ethanol are mixed may be composed of 40 to 60 wt%. When the concentration of the ethanol in the extraction solvent is less than 40wt%, the active ingredients contained in cornflower oil and omija are not easily extracted, so that the quality of the mixed extract may be lowered or the process yield may be lowered. In addition, when the concentration of the ethanol in the extraction solvent exceeds 60 wt%, the water content in the extraction solvent is reduced and the water-soluble active ingredients contained in cornflower oil and omija are not extracted, so the quality, taste and flavor of the mixed extract may affect

Referring to FIG. 1 , S30 may be to form a mixed extract by extracting the raw material solvent mixture at a temperature of 45 to 55° C. for 2 to 6 hours. Specifically, the S30 extracts the active ingredient contained in the mixed raw material contained in the raw solvent mixture by performing extraction using an extraction solvent that is a 40% ethanol aqueous solution to a 60% ethanol aqueous solution to extract the unique taste, flavor and It may be to form a mixed extract having a flavor. The step S30 may be to use a conventional extraction device used for extracting fruit, fruits, vegetables, etc., such as common berries.

Specifically, in the present invention, when extracting the raw material solvent mixture, the extraction is performed at a relatively low temperature for 2 to 6 hours at a temperature of 45 to 55 ° C. It can prevent the back from being denatured or destroyed. More specifically, the extraction temperature may be 50 ℃, the extraction time may be 4 hours.

When the temperature at which the raw material solvent mixture is extracted is carried out at less than 45° C., the nutrients and active ingredients contained in Omizawa cornflower oil are not easily extracted, and the extraction efficiency may be lowered. In addition, when the temperature at which the raw material solvent mixture is extracted is carried out at a temperature exceeding 55° C., the extracted active ingredient may be denatured or destroyed, and the unique taste and flavor of the mixed solvent may be lost together with the extraction solvent. may evaporate.

In addition, when the extraction time of the raw material solvent mixture is less than 2 hours, it is difficult to sufficiently extract the intrinsic components of the raw material mixture, and thus the quality may be deteriorated. In addition, when the extraction time of the raw material solvent mixture exceeds 4 hours, a part of the extracted mixed extract may be evaporated, and the extraction efficiency may be lowered.

Referring to FIG. 1 , S40 may be to concentrate the mixed extract to have a sugar content of 55 to 65 brix. By concentrating the mixed extract extracted in S30 to have a target sugar content, it can be carried out using a conventional concentrator. In one embodiment, the concentration temperature may be carried out at 50 to 60 ℃, it may be concentrated under reduced pressure until the mixed extract has a sugar content of 55 to 65 brix as measured by a sugar content meter.

Figure 2 is a flow chart showing a method for producing a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of omija and cornus oil including an enzyme reaction process and an enzyme inactivation process according to another embodiment of the present invention.

Referring to FIG. 2 , in another embodiment of the present invention, the fourth step includes a first step 4-1 of first concentrating the mixed extract to have a sugar content of 20 to 30 brix, and the first concentrated mixed extract Step 4-2 of reacting by adding 0.4 to 0.8 parts by weight of beta-glucanase with respect to 100 parts by weight, and heating the mixed extract reacted with the enzyme at a temperature of 90 to 100° C. for 5 to 20 minutes The 4-3 step of inactivating the enzyme and the 4-4 step of second concentrating the mixed extract in which the enzyme has been deactivated to have 55 to 65 brix may be included. That is, in another embodiment of the present invention, by adding an enzyme to the concentrated mixed extract and performing a reaction with the enzyme, the precipitate formed in the mixed extract can be decomposed and easily removed. Through this, it is possible to increase the production yield and improve the quality of the product when applied to the product.

Specifically, referring to FIG. 2 , S41 may be a primary concentration of the mixed extract extracted in S30 to have a sugar content of 20 to 30 brix. Through this, the ethanol in the mixed extract extracted in S30 can be evaporated and removed, and as the ethanol is removed, the enzymatic reaction described later can be smoothly performed.

Referring to FIG. 2 , the S42 may be reacted by adding 0.4 to 0.8 parts by weight of a beta-glucanase enzyme with respect to 100 parts by weight of the first concentrated mixed extract. The enzyme may be added by adjusting an appropriate amount according to the pH or active ingredient extraction amount, which may vary for each material of the raw material, Omija and Cornus officinalis. The beta-glucanase enzyme is known as a glycolytic enzyme, and commercially available ones may be used in the present invention. By adding the beta-glucanase (β-glucanase) enzyme to the first concentrated mixed extract, it is possible to decompose the extract components precipitated in the first concentrated mixed extract to remove the precipitate. In one embodiment, for a smooth enzymatic reaction in S42, S42 is performed at a temperature of 40 to 60° C. at which the beta-glucanase enzyme can be smoothly activated for about 4 to 5 hours. can

When the beta-glucanase enzyme is added in an amount of less than 0.4 parts by weight with respect to 100 parts by weight of the first concentrated mixed extract in S42, the enzyme reaction is not sufficiently performed, thereby inhibiting the production of precipitates in the mixed extract It can be difficult to do. In addition, when the beta-glucanase enzyme is added in excess of 0.8 parts by weight with respect to 100 parts by weight of the first concentrated mixed extract in S42, the manufacturing cost may increase due to the increase in cost due to the increase in the amount of the enzyme added. have.

Referring to FIG. 2 , S43 may be to inactivate the enzyme by heating the mixed extract reacted with the enzyme at a temperature of 90 to 100° C. for 5 to 20 minutes. That is, the enzyme in the mixed extract is inactivated by heating the mixed extract reacted with the enzyme after the enzyme reaction is sufficiently advanced using the beta-glucanase enzyme in S42. Through this enzyme inactivation process, the mixed extract can maintain its own color and flavor. In addition, when the composition of the present invention is used as a product such as food or medicine, it is possible to maintain the original state of the composition without change in formulation or quality.

When the enzyme is deactivated in S43, if it is carried out at a temperature of less than 90° C. or the process time is less than 5 minutes, the enzyme injected into the mixed extract is not completely deactivated, so that the taste, flavor and quality of the composition may be reduced. In addition, when the enzyme inactivation in step S43 is carried out at a temperature exceeding 100° C. or the process time exceeds 20 minutes, thermal denaturation of the active ingredients contained in the mixed extract and the unique taste and flavor of cornus oil and omija are evaporated. can be

Referring to FIG. 2 , S44 may be a secondary concentration of the enzyme-inactivated mixed extract to have 55 to 65brix. Through this, it is possible to prepare a mixed extract of Omizawa cornus oil having an appropriate sugar content. The concentration process may be performed the same or similar to the concentration process in S40 and S41 described above.

Referring to FIG. 1 , S50 may be sterilizing the concentrated mixed extract and then filtering. Preservability can be improved by sterilizing the concentrated mixed extract, and the sterilization process may be performed through a conventional sterilization apparatus. Thereafter, the purity and quality of the composition of the present invention can be improved by filtering out precipitates or foreign substances remaining in the concentrated mixed extract through a filtration process.

As described above, the method for preparing a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of Schisandra oleracea of the present invention can effectively extract the active ingredient of Omizawa cornus oil using an appropriate extraction solvent, and remove the precipitate. can be reduced to improve product quality.

Hereinafter, the contents of the experiment using the composition for the prevention or treatment of benign prostatic hyperplasia, including the mixed extract of omija and cornflower oil, and the preparation method thereof will be described in detail.

I. Experiment on the effect of mixed extract of Omizawa Cornus officinalis on enlarged prostate

This experiment was conducted to compare the effects of the mixed extract of Schisandra oleifera on the promotion of proliferation of LNCaP prostate cells and increase in the expression of prostatic hyperplasia inducing factors by 5α-dihydrotestosterone.

1. Preparation of test materials and test methods

Schisandra (SF) hot water extract used in this study, Cornus officinalis (CF) hot water extract, Schisandra (SF) 40% ethanol (EtOH) extract, Cornus oil (CF) 40% ethanol (EtOH) extract, Schisandra Mixture of (SF) and Cornus officinalis (CF) (1:1) 40% ethanol extract, Mixture of Schisandra (SF) and Cornus officinalis (CF) (1:2) 40% Ethanol extract, Schisandra (SF) and Cornus officinalis (CF) A mixture (2:1) of 40% ethanol extract and a mixture of (1:1) 60% ethanol extract of Schisandra (SF) and cornus oil (CF) were prepared through the following Examples and Comparative Examples.

[Comparative Example 1]

A raw material solvent mixture obtained by adding 100 parts by weight of water to 10 parts by weight of the dried omija raw material was put into an extractor, and hot water extraction was performed at 50° C. for 4 hours. The hot water extract was placed in a concentrator and concentrated to have a sugar content of 60 brix. After sterilizing the concentrated extract, it was filtered to prepare a Schisandra hot water extract.

[Comparative Example 2]

A raw material solvent mixture obtained by adding 100 parts by weight of water to 10 parts by weight of the dried cornus oil raw material was put into an extractor, and hot water extraction was performed at 50° C. for 4 hours. The hot water extract was placed in a concentrator and concentrated to have a sugar content of 60 brix. After sterilizing the concentrated extract, it was filtered to prepare an extract of hot water from Cornus officinalis.

[Comparative Example 3]

A raw material solvent mixture obtained by adding 100 parts by weight of a 40% ethanol aqueous solution to 10 parts by weight of the dried omija raw material was put into an extractor and extracted at 50° C. for 4 hours. The extract was placed in a concentrator and concentrated to have a sugar content of 60brix. After sterilizing the concentrated extract, it was filtered to prepare a Schisandra 40% ethanol extract.

[Comparative Example 4]

A raw material solvent mixture in which 100 parts by weight of a 40% ethanol aqueous solution was added to 10 parts by weight of the dried cornus oil raw material was put into an extractor, and the mixture was extracted at 50° C. for 4 hours. The extract was placed in a concentrator and concentrated to have a sugar content of 60brix. After sterilizing the concentrated extract, it was filtered to prepare a 40% ethanol extract of cornflower oil.

[Example 1]

A mixed raw material was prepared by mixing dried cornus oil raw material and dried omija raw material in a ratio of 1:1. The raw material solvent mixture obtained by adding 100 parts by weight of 40% ethanol aqueous solution to 10 parts by weight of the mixed raw material was put into an extractor and extracted at 50° C. for 4 hours to form a mixed extract. The mixed extract was placed in a concentrator and concentrated to have a sugar content of 60brix. After sterilizing the concentrated extract, it was filtered to prepare a 40% ethanol-extracted mixture of Omizawa Cornus officinalis mixed in a 1:1 ratio.

[Example 2]

A mixed raw material was prepared by mixing dried cornus oil raw material and dried omija raw material in a ratio of 1:1. A raw material solvent mixture obtained by adding 100 parts by weight of 40% ethanol aqueous solution to 10 parts by weight of the mixed raw material was put into an extractor and extracted at 50° C. for 4 hours to form a mixed extract. The mixed extract was placed in a concentrator and concentrated to have a sugar content of 60brix. After sterilizing the concentrated extract, it was filtered to prepare a 40% ethanol-extracted mixture of Omizawa cornus oil mixed in 1:2 ratio.

[Example 3]

A mixed raw material was prepared by mixing dried cornus oil raw material and dried omija raw material in a ratio of 1:1. A raw material solvent mixture obtained by adding 100 parts by weight of a 40% ethanol aqueous solution to 10 parts by weight of the mixed raw material was put into an extractor and extracted at 50° C. for 4 hours to form a mixed extract. The mixed extract was placed in a concentrator and concentrated to have a sugar content of 60brix. After sterilizing the concentrated extract, it was filtered to prepare a 40% ethanol-extracted mixture of Omizawa cornus oil mixed in a 2: 1 ratio.

[Example 4]

A mixed raw material was prepared by mixing dried cornus oil raw material and dried omija raw material in a ratio of 1:1. A raw material solvent mixture obtained by adding 100 parts by weight of a 60% ethanol aqueous solution to 10 parts by weight of the mixed raw material was put into an extractor and extracted at 50° C. for 4 hours to form a mixed extract. The mixed extract was placed in a concentrator and concentrated to have a sugar content of 60brix. After sterilizing the concentrated extract, it was filtered to prepare a 60% ethanol-extracted mixture of Omija cornflower oil mixed in 1:1 ratio.

The composition of the mixed extracts of Comparative Examples 1 to 4 and Examples 1 to 4 is summarized in Table 1 below.

division Raw material extraction solvent Mixing status and mixing ratio Comparative Example 1 Schisandra water do not mix Comparative Example 2 corn milk water do not mix Comparative Example 3 Schisandra 40% ethanol solution do not mix Comparative Example 4 corn milk 40% ethanol solution do not mix Example 1 Schisandra and Cornflower Oil 40% ethanol solution Schisandra: Cornus milk = 1:1 mixture Example 2 Schisandra and Cornflower Oil 40% ethanol solution Schisandra: Mixed with Cornus officinalis = 1:2 Example 3 Schisandra and Cornflower Oil 40% ethanol solution Schisandra : Mixed with Cornus officinalis = 2:1 Example 4 Schisandra and Cornflower Oil 60% ethanol solution Schisandra: Cornus milk = 1:1 mixture

1) cell culture

Human prostate cells (LNCaP) used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). LNCaP cells were obtained by adding 10% bovine calf serum (BCS, WELGENE) and 100 unit/ml penicillin/streptomycin (P/S) to Roswell Park Memorial Institute Medium (RPMI-1640, WELGENE, Daegu, Republic of Korea) at 37°C. Incubated under 5% CO _{2 .}

2) Preparation of the drug

The extract used in this study was prepared by the present applicant as in Comparative Examples 1 to 4 and Examples 1 to 4 described above. 5α-Dihydrotestosterone (DHT) was purchased from Sigma-Aldrich Chemical Co. (St. Lousi, MO, USA) and used in the experiment. Each extract was used after making a stock solution at 200 mg/mL and appropriately diluted and mixed according to the experiment.

3) Measurement of cell viability using MTT assay

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT; Invitrogen, Carlsbad, CA, USA) to evaluate cell proliferation by DHT and cytotoxicity of Schisandra and Cornus officinalis extract ) assay was performed. First, in order to check the cytotoxicity of the drug itself, 7×10 ⁴ LNCaP cells per well were dispensed in a 6 well plate and stabilized for 24 hours.

After that, after replacing with a cell culture medium containing 100 unit/ml P/S, culture for 24 hours, and DHT treatment time using a medium containing 5% FBS and 100 unit/mL P/S in the medium Previously, the extract was treated with a drug by concentration, and cultured for 48 hours. After adding 0.5 mg/ml of MTT to each well, it was reacted for 2 hours at _{37° C. 5% CO 2 in an incubator.} After removing the medium, dimethyl sulfoxide (DMSO, Sigma-Aldrich Chemical Co.) was added to dissolve all the generated formazan, and then 200 μL each was transferred to a 96-well plate using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Absorbance values were measured at 540 nm. Cytotoxicity for each substance was expressed in terms of percentage based on the control value.

4) Measurement of cell proliferation rate using a hemocytometer

To check the cell proliferation rate by DHT treatment, 7×10 ⁴ LNCaP cells per well were dispensed in a 6 well plate and stabilized for 24 hours. Then, after replacing with a medium containing 100 unit/ml P/S, cultured for 24 hours, and then treated with DHT by concentration using a medium containing 5% FBS and 100 unit/mL P/S. After 48 hours, the cells were collected by centrifugation and washed once with phosphate buffered saline (PBS). The number of living cells was counted using an inverted microscope (Carl Zeiss, Gottingen, Germany).

5) Western blot analysis

The extract was treated 1 hour before DHT treatment, and cells exposed to DHT for 48 hours were harvested and then centrifuged at 3000 rpm for 5 minutes. After removing the supernatant, 1X lysis buffer [25 mM Tris-Cl (pH 7.5), 250 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM phenymethylsulfonyl fluoride (PMSF), 5 mM dithiothreitol (DTT)] was added to the pellet. After reacting at ℃ 30 minutes, centrifugation was performed at 14,000 rpm and 4℃ for 30 minutes to obtain a supernatant. Protein concentration was quantified using Bio-Rad protein quantification reagent (Bio-Rad, Hercules, CA, USA), and the same amount of Laemilni sample buffer (Bio-Rad) was mixed to prepare a protein sample.

에서 over night시킨 다음, 1x PBS-T (PBS with Tween 20)로 10분간 3번 세척하고 적정 2차 항체를 사용하여 상온에서 1시간 30분 반응시킨 후 1x PBS-T로 세척 후 Chemi-smart(Vilber Lourmat, France)를 이용하여 조사 대상 단백질의 발현 정도를 분석하였다. Electrophoresis was performed using sodium dodecyl sulphate (SDS)-polyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). After that, 5% skim milk was treated for 30 minutes and the primary antibody was treated for 4

After over night in 1x PBS-T (PBS with Tween 20), washed 3 times for 10 minutes, reacted for 1 hour and 30 minutes at room temperature using appropriate secondary antibody, washed with 1x PBS-T, and then washed with Chemi-smart ( Vilber Lourmat, France) was used to analyze the expression level of the target protein.

6) Measurement of changes in the production of reactive oxygen species

2',7'-dichlorodihydrofluorescein diacetate (DCF-DA; Invitrogen) staining method was used to measure changes in reactive oxygen species (ROS) production in LNCaP cells. In order to check the efficacy of the extract against ROS generated by DHT, LNCaP cells were dispensed in a 6 well plate and _{cultured for 24 hours in an incubator at 37°C 5% CO 2} , the extract was treated 1 hour before, and DHT was incubated for an appropriate time. processed. After reacting 10 μM of DCF-DA in an _{incubator at 37 ° C. 5% CO 2} for 20 minutes, the change in ROS generation was evaluated using a flow cytometer (Accuri Cytomerers, Inc., Ann Arbor, MI, USA).

7) Human Prostate Specific Antigen (PSA) ELISA analysis

The culture solution of cells treated with DHT and extract was collected and centrifuged at 2000 rpm for 5 minutes to obtain a supernatant. The supernatant and the standard solution were transferred to a 96-well plate to which the antibody was bound, and reacted at 37°C for 2 hours, and 1x Biotinylated detection antibody was added to the same amount of the supernatant and the standard solution and reacted at 37°C for 1 hour. After washing 5 times with 1x wash buffer, 1x HRP-avidin solution was put in a 96 well plate, reacted at 37° C. for 1 hour, and washed 5 times with wash buffer. After that, the TMB substrate solution was put in a 96-well plate and reacted for 15 to 30 minutes, a stop solution was added, and absorbance was measured at 450 nm with an ELISA reader.

Each sample value was calculated by substituting it into the standard curve, and the standard used for PSA measurement, 100x biotinylated detection antibody, 100x HRP-avidin, biotinylated detection antibody diluent, HRP-avidin diluent, wash buffer, TMB substrate, and stop solution were human Those provided in the KLK ₃ /PSA ELISA kit (LifeSpan Biosciences, Inc., Seattle, WA, USA) were used.

8) Statistical processing

Statistical analysis was performed using one-way ANOVA of GraphPad Prism® version 5.0 (Graphpad Inc., San Diego, CA, USA), and post-test with Tukey's test. .

2. Experimental results

1) Establishment of an in vitro DHT-induced prostatic hypertrophy model using LNCaP cells

Figure 3A is a chart showing the cell viability (cell ciability) of LNCaP cells according to the concentration of DHT according to an embodiment of the present invention, Figure 3B is the treatment time of DHT according to an embodiment of the present invention is a diagram showing the degree of proliferation of LNCaP cells according to the present invention, and FIG. 3C is an image of observing the morphological change of LNCaP cells by DHT according to an embodiment of the present invention with a phase contrast microscope, and FIG. 3D is a chart showing the degree of AR expression in LNCaP cells according to the concentration of DHT according to an embodiment of the present invention.

Specifically, cells from A to 3D of FIG. 3 ^{were inoculated in a 6 well plate at 7x10 5} cells/ml and treated with DHT at the indicated concentration for 24 or 48 hours. The cell viability of FIG. 3A and the viable cell number of FIG. 3B were measured by metabolic dye-based MTT assay and trypan blue assay, respectively. Data are expressed as mean ± standard deviation (n=3). Statistical analysis was performed using analysis of variance between groups (ANOVA-Tukey's pst-hoc test).

Compared to control, * is p<0.05, *** is p<0.001. am.

3C is an image showing the morphological change of LNCaP cells as a result of observing the morphological change of LNCaP cells with a phase-contrast microscope. After treatment with DHT of the indicated concentration in FIG. 3D for 48 hours, the cells were lysed, and then the same amount of cell lysate was separated from the SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Cellular proteins were visualized using the indicated antibody and ECL detection system, and actin was used as an internal control.

3A to 3D, in order to investigate the effect of DHT on the proliferation of LNCaP cells, DHT was treated for each appropriate concentration and cultured for an appropriate time, followed by MTT assay and hemocytometer counting. As shown in FIG. 3A, when cultured for 24 hours after DHT treatment, there was no significant difference in cell viability, but when cultured for 48 hours, there was a significant difference in the DHT 50nM treatment group, and further increased when 100nM DHT treatment was performed. appeared to be In addition, as can be seen from the results shown in FIG. 3B , as a result of confirming the degree of proliferation of LNCaP cells by treatment with DHT for 48 hours, it can be seen that the proliferation promotion phenomenon was significantly increased at 100 nM of DHT. As a result of confirming the effect of DHT inducing survival and proliferation promotion on the morphology of LNCaP cells, the overall cell density was increased when DHT was treated for 48 hours as shown in FIG. 3C . Testosterone is converted to DHT by 5-alpha reductase in the process of inducing enlargement of the prostate. do.

Therefore, in order to confirm whether the in vitro prostate enlargement model was properly established in LNCaP cells, AR expression was confirmed after 48 hours of treatment with DHT for each concentration. It increased in a concentration-dependent manner.

2) Effect of Schisandra and Cornus officinalis hot water extract and 40% ethanol extract on the proliferation of LNCaP cells

FIG. 4A is a diagram showing the results of a cytotoxicity test (MTT assay), which is the effect of Schisandrae Fructu hot water extract on the cell viability of LNCaP cells according to an embodiment of the present invention, FIG. 4B is It is a table showing the results of the cytotoxicity test (MTT assay), which is the effect of Corni Fructus hot water extract on the cell viability of LNCaP cells according to an embodiment of the present invention, FIG. 4C is an embodiment of the present invention It is a table showing the results of the cytotoxicity test (MTT assay), which is the effect of the 40% ethanol extract of Corni Fructus on the cell viability of LNCaP cells. A chart showing the results of the cytotoxicity test (MTT assay), which is the effect of Corni Fructus 40% ethanol extract on the cell viability of LNCaP cells.

4A to 4D, cells were each treated with the above-mentioned extracts for 48 hours. Cell viability was then measured by MTT assay. Data are expressed as mean ± standard deviation (n=3). Statistical analysis was performed using analysis of variance between groups (ANOVA-Tukey's post-hoc test). ** means p < 0.01, ns means not significant compared to control.

As shown in FIGS. 4A to 4D, cytotoxicity was investigated for each extract in NLCaP cells. According to the MTT assay result, as shown in FIG. 4A, the hot water extract of Schisandrae Fructu somewhat inhibited the proliferation of NLCaP cells at 350 μg/ml.

4B to 4D, Corni Fructus hot water extract (Comparative Example 2) and Schisandrae Fructu 40% ethanol extract (Comparative Example 3) and Corni Fructus 40% ethanol extract ( Comparative Example 4) did not show cytotoxicity up to a treatment concentration of 350 μg/ml. Therefore, based on these results, 250 μg/ml, which is a concentration that does not show NLCaP cytotoxicity in the same extracts of the four extracts, was set, and subsequent experiments were carried out.

3) Effect of Schisandra and Cornus officinalis hot water extract on the promotion of proliferation of LNCaP cells and expression of androgen receptor-related genes by DHT treatment

5A is Schisandra hot water extract (SF-water), Cornus officinalis hot water extract (CF-water), Schisandra 40% ethanol extract (SF-EtOH) and Cornus officinalis before DHT treatment on LNCaP cells according to an embodiment of the present invention. It is a chart showing the degree of cell proliferation in the case of pretreatment with 40% ethanol extract (CF-EtOH), and FIG. 5B is a Schisandra hot water extract (SF-water) before DHT treatment on LNCaP cells according to an embodiment of the present invention. ), androgen-related genes (AR and PSA) and cell proliferation in the case of pre-treatment with Cornus officinalis (CF-water), Schisandra 40% ethanol extract (SF-EtOH) and Cornus officinalis 40% ethanol extract (CF-EtOH) It is a chart showing the expression change of the indicator gene (PCNA).

Specifically, in FIGS. 5A to 5B, the cells were pretreated with the extract for 1 hour before 100nM DHT treatment for 48 hours. Cell viability in FIG. 5A was measured by MTT assay, and data were expressed as mean±standard deviation (n=4). Statistical analysis was performed using analysis of variance between groups (ANOVA-Tukey's post-hoc test). ** is p <0.01 versus control group, # is p <0.05, ## is p < 0.01 versus DHT treatment group. In FIG. 5B , the expression levels of AR, AR co-activators (ARA70 and SRC1) and PCNA were determined by Western blotting using whole cell lysates. Equivalent protein loading was confirmed with actin as an internal control.

As can be seen from the result of FIG. 5A, as a result of pretreatment of each extract at a concentration of 250 μg/ml without cytotoxicity 1 hour before, Schisandra (SF) hot water extract improves cell proliferation promotion by DHT couldn't do it However, Cornus officinalis (CF) hot water, Schisandra (SF) 40% ethanol (EtOH) extract, and Cornus officinalis (CF) 40% ethanol (EtOH) extract improved cell proliferation promotion by DHT.

Referring to FIG. 5B, under the same conditions, each extract confirmed the expression changes of androgen-related genes (AR and PSA) and cell proliferation-related indicator genes (PCNA). As a result, when 100 nM of DHT was treated alone for 48 hours, AR , PSA and PCNA expression increased, and although the Schisandra extract inhibited the expression of AR, but had no significant effect on the expression of PSA and PCNA, the Sansuyu hot water extract suppressed the expression of AR and PSA.

However, Schisandra 40% ethanol extract and Cornus officinalis 40% ethanol extract suppressed the expression of AR, PSA and PCNA increased by DHT. According to these results, Schisandra 40% ethanol extract and Cornus officinalis 40% ethanol extract extracted using 40% ethanol aqueous solution of Schisandra hot water extract and Cornus officinalis has a significant effect on the inhibition of proliferation of LNCaP cells and the suppression of the expression of genes related to prostate enlargement. was able to confirm

4) Effect of Schisandra and Cornus officinalis hot water extract on ROS generation by DHT treatment in LNCaP cells

6A is a chart showing the degree of ROS generation by treating LNCaP cells with 100 nM DHT according to an embodiment of the present invention, and FIG. -water), Cornus officinalis hydrothermal extract (CF-water), Schisandra 40% ethanol extract (SF-EtOH), and Cornus officinalis 40% ethanol extract (CF-EtOH) were pretreated and treated with 100nM DHT to show the degree of ROS generation. am.

Specifically, FIG. 6A shows the results of treating LNCaP cells with 100 nM DHT. In FIG. 6B, cells were pre-treated with the extracts prepared in 250 μg/ml Comparative Examples 1 to 4 for 1 hour, and then treated with 100 nM DHT for 1 hour. Cells were collected for flow cytometry and stained with DCF-DA dye. Intracellular ROS production was determined by counting the percentage of DCF-DA stained cells.

It has been reported that excessive generation of ROS induces hyperproliferation mediated by androgen growth signals in an in vitro model of human prostate hypertrophy. Therefore, in this study, it was confirmed whether DHT was involved in ROS generation in LNCaP cells, and as shown in FIG. 6A , ROS production increased significantly from 30 minutes after 100 nM DHT treatment, and at 1 hour, 10 compared to the control group. It increased more than twice and then gradually decreased.

Based on this, in order to check whether the extracts prepared in Comparative Examples 1 to 4 can inhibit the ROS generated by DHT, NAC and Schisandra (SF) hot water extract, which are known as scavenge of ROS 1 hour before DHT treatment, and cornus oil (CF) hot water extract, Schisandra (SF) 40% ethanol extract, and Cornus officinalis (CF) 40% ethanol extract were treated. According to the results shown in FIG. 6B, ROS increased to 25% when 100 nM DHT was treated alone for 1 hour. was restored to the control level at 1.2% by pretreatment with NAC, 12.6% for Schisandra (SF) hot water extract, 4.4% for Cornus officinalis (CF) hot water extract, 9.5% for Schisandra (SF) 40% ethanol extract, and Cornus officinalis ( CF) 40% ethanol extract was found to be reduced to 1.8%, respectively. That is, the relative inhibitory effect of DHT-induced ROS production in NLCaP cells was found in the hot water extract of Cornus oleifera (SF) rather than the hot water extract, and the 40% ethanol extract of Schisandra (SF) and 40% ethanol of Cornus officinalis (CF) rather than the hot water extract. It was found that the extract was more effective.

5) Comparison of the efficacy of the mixture of Schisandra 40% ethanol extract and Cornus officinalis 40% ethanol extract

7A is a mixture containing Schisandra (SF) 40% ethanol extract and Cornus officinalis (CF) 40% ethanol extract prepared in Examples 1 to 3 according to an embodiment of the present invention at respective mixing ratios. It is a chart showing the results of the cytotoxicity test (MTT assay) showing the effect on the cell viability of LNCaP cells, B of FIG. 7 is Schisandra produced in Examples 1 to 3 according to an embodiment of the present invention ( A table showing the level of androgen-related gene expression induced by DHT in LNCaP cells pretreated with a mixture containing 40% ethanol extract (SF) and 40% ethanol extract (CF) in each mixing ratio.

7A, the cells were treated with a mixture containing the 40% ethanol extract of Schisandra 40% ethanol extract and the 40% ethanol extract of cornflower oil prepared in Examples 1 to 3 at 1:1, 2:1 and 1:2 for 48 hours. . Cell viability was then measured by MTT assay. Data are expressed as mean ± standard deviation (n=3). Statistical analysis was performed using analysis of variance between groups (ANOVA-Tukey's post-hoc test). *** is p < 0.0001 (compared to control), # is p < 0.05, ## is p < 0.01, and ### means p < 0.001 vs. DHT treatment group.

7A to 7B are results of examining the expression of prostatic hyperplasia induction related genes according to different treatment mixing ratios of 40% ethanol extract having excellent antioxidant efficacy. To this end, the ratios of Schisandra 40% ethanol extract and Cornflower 40% ethanol extract were mixed in 1:1, 2:1, and 1:2 ratios, respectively, and the effect of the mixture on DHT-induced cell proliferation and androgen-related gene expression inhibition was confirmed. did. When a mixture of Schisandra 40% ethanol extract and Cornflower 40% ethanol extract in 1:1, 2:1, and 1:2 ratios was treated at 250 μg/ml, respectively, no significant toxicity was observed on LNCaP cells.

Referring to FIG. 7A, looking at the phenomenon of promoting LNCaP cell proliferation induced by DHT, the mixture of Schisandra 40% ethanol extract and Cornus officinalis 40% ethanol extract mixed in 2:1 is also reduced, but 1:1 ratio and 1:2 The mixture of Schisandra 40% ethanol extract and Cornus officinalis 40% ethanol extract mixed in the ratio decreased more significantly. In addition, referring to FIG. 7B, DHT-induced androgen-related gene expression is also mixed at a 1:1 ratio and 1:2 ratio compared to a mixture of Schisandra 40% ethanol extract and Cornflower 40% ethanol extract mixed at a 2:1 ratio. The mixture of 40% ethanol extract of Schisandra Schisandra and 40% ethanol extract of cornflower oil further decreased the expression of AR, ARA70 and PSA increased by DHT.

6) Comparison of improvement efficacy of 40% ethanol extract and 60% ethanol extract of Schisandra and Cornus officinalis for promoting cell proliferation of LNCaP cells by DHT

8 is a 1:1 mixture of a 40% ethanol extract of Schisandra (SF) or Cornus officinalis (CF) and a 60% ethanol extract of Schisandra (SF) or Cornus officinalis (CF), Schisandra (SF) or Cornus officinalis (SF) LNCaP cells It is a chart showing the results of the cytotoxicity test (MTT assay) showing the effect on the growth of DHT stimulation.

In Figure 8, the cells were treated with a 1: 1 mixture of 40% ethanol extract of Schisandra (SF) or Cornus officinalis (CF) and 60% ethanol extract of Schisandra (SF) or Cornus officinalis (CF) for 1 hour for 48 hours. preprocessed. Cell viability was then measured by MTT assay. Data are expressed as mean ± standard deviation (n = 3). Statistical analysis was performed using analysis of variance between groups (ANOVA-Tukey's post-hoc test). *** is p < 0.0001 (relative to control), # is p < 0.05, ## is p < 0.01, and ### is p < 0.001 versus DHT treatment group.

According to the previous results (FIGS. 4 and 5), compared to the hot-water extract of Schisandra (SF) or Cornus officinalis (CF), the 40% ethanol extract of Schisandra (SF) or Cornus officinalis (CF) resulted in DHT-induced cell proliferation and androgen-related gene expression. And it was found that there was a difference in efficacy depending on the extraction method as it appeared more effectively in the inhibition of ROS generation. Based on this, a 60% ethanol extract and a 40% ethanol extract were compared to determine the difference according to the change in the ethanol concentration in the ethanol extraction process of Schisandra and Cornus officinalis.

For this purpose, a mixture of Schisandra 40% ethanol extract, Cornusian oil 40% ethanol extract, Schisandra 60% ethanol extract and Cornusian oil 60% ethanol extract, Schisandra 40% ethanol extract, and Cornus oil 40% ethanol extract in a 1:1 ratio, and Schisandra 60% LNCaP cells were treated with a mixture of ethanol extract and 60% ethanol extract of cornflower oil in a 1:1 ratio.

According to the MTT assay results shown in FIG. 8, not only the 40% ethanol extract of Schisandra (SF) and Cornus officinalis (CF), but also the mixture of 60% ethanol extract and 1:1 ratio did not show significant cytotoxicity in LNCaP cells. And, when 250 μg/ml of a mixture of 40% ethanol extract, 60% ethanol extract, Schisandra and cornflower oil was treated, all of the DHT-induced LNCap cell proliferation promotion was significantly reduced. However, the 150μg/ml treatment showed a significant reduction effect only in the mixture of 60% ethanol extract of cornflower oil, 1:1 ratio of omija and 60% ethanol extract of Cornus officinalis. Based on these results, it is judged that the efficacy of the 60% ethanol extract in improving the DHT-induced cell proliferation promotion is higher than that of the 40% ethanol extract.

7) Comparison of the efficacy of 40% and 60% ethanol extracts of Omizawa Cornus officinalis on the increase of androgen receptor-related gene expression by DHT in LNCaP cells

FIG. 9 is a chart showing the effect of 40% and 60% ethanol extracts of Schisandra or Cornus officinalis, a 1:1 mixture of each, on AR expression in DHT-stimulated LNCaP cells.

The following is a comparison of the efficacy of six different mixtures of Schisandra and Cornus officinalis extracts on DHT-induced androgen-associated genes (AR and PSA) and cell proliferation-associated indicator genes (PCNA) increase in LNCaP cells. Fig. According to the Western blot analysis results shown in Fig. 7, the expression of AR increased by the treatment with DHT was the most decreased in the treatment group of the 60% ethanol extract in a 1:1 ratio, and the expression of ARA70 was largely decreased by the six extracts. However, among them, the 60% ethanol extract formulation group at a ratio of 1:1 showed the greatest reduction effect. PCNA expression was decreased when the 60% ethanol extract was treated rather than the 40% ethanol extract, and decreased in the order of cornflower oil, omija, and a mixture of 1:1 ratio. Through this, it can be seen that among the six complex extracts, a 60% ethanol extract complex in a ratio of 1:1 is the most effective for inhibiting the expression of AR, ARA70 and PCNA.

8) Comparison of efficacy of 40% and 60% ethanol extracts of Omizawa Cornus officinalis on DHT-induced increase in PSA expression in LNCaP cells

FIG. 10A shows the effect of 40% and 60% ethanol extracts of Schisandra (SF) or Cornus officinalis (CF), a 1:1 mixture thereof, on PSA expression in DHT-stimulated LNCaP cells according to an embodiment of the present invention; is a chart showing the results of the ELISA experiment, and FIG. 10B is a 40% and 60% ethanol extract of Schisandra (SF) or Cornus officinalis (CF) according to an embodiment of the present invention, and a 1:1 mixture thereof is DHT It is a chart showing the degree of PSA production showing the effect on PSA expression in LNCaP cells stimulated with .

In FIG. 10A , the cell culture supernatant of PSA was investigated by ELISA. Data are expressed as mean ± standard deviation (n=3). Statistical analysis was performed using analysis of variance between groups (ANOVA-Tukey's post-hoc test). *** is P < 0.001 versus control, # is p < 0.05, and ### is p < 0.001 versus DHT group. 10B shows the expression level of PSA was determined by Western blotting. Actin was used as an internal control.

10A to 10B, the efficacy of 40% and 60% ethanol extracts of Schisandra (SF) and Cornus officinalis (CF) on the production of PSA induced by DHT in LNCaP cells was compared. As can be seen from the ELISA results presented in A of FIG. 10, when LNCaP cells were treated with DHT alone, increased PSA production was achieved by mixing Schisandra (SF) and Cornus officinalis (CF) in a 1:1 ratio of 60% ethanol extract mixture 150 μg/ ml treatment showed a decreasing effect, and when treated at 250 μg/ml, a mixture of 40% ethanol extract in a 1:1 ratio and 60% ethanol extract of Schisandra (SF) and Cornus officinalis (CF) inhibited the production of PSA. showed a reducing effect. In particular, it was confirmed that PSA production was most significantly inhibited when 250 μg/ml of a 60% ethanol extract mixture mixed with Schisandra (SF) and Cornus officinalis (CF) in a 1:1 ratio was treated.

9) Comparison of efficacy of 40% and 60% ethanol extracts of Omizawa Cornus officinalis on ROS production by DHT treatment in LNCaP cells

11A is a diagram showing the results of a flow cytometer showing the degree of ROS generation by treating LNCaP cells with 100 nM DHT according to an embodiment of the present invention, and FIG. 11B is an embodiment of the present invention Flow cytometer results showing the effect of 40% and 60% ethanol extracts of Schisandra (SF) or Cornus asiatica (CF), a 1:1 mixture thereof, on intracellular ROS production in DHT-stimulated LNCaP cells according to It is a chart, and FIG. 11C is a 40% and 60% ethanol extract of Schisandra (SF) or Cornus officinalis (CF) according to an embodiment of the present invention, and a 1:1 mixture thereof is used for intracellular ROS in DHT-stimulated LNCaP cells. It is an image showing the result of fluorescence microscopic observation showing the effect on the production.

11A shows that LNCaP cells were treated with 100 nM DHT for different times. 11B shows cells were pretreated with 250 μg/ml 40% and 60% ethanol extract of SF or CF, 1:1 mixture for 1 hour, and then treated with 100 nM DHT for 1 hour. Cells were collected and stained with DCF-DA dye for flow cytometry. Intracellular ROS production was determined by counting the percentage of DCF-DA stained cells. 11C, the stained cells were photographed with fluorescence using a fluorescence microscope.

11, 40% ethanol extract, 60% ethanol of Schisandra (SF) and Cornus officinalis (CF) on the accumulation of ROS generated by DHT treatment in LNCaP cells in order to compare the antioxidant activity of the ethanol extract of S. omija and Cornus officinalis. The effect of a mixture of extract and 40% 60% ethanol extraction 1:1 ratio was investigated. According to the flow cytometry analysis results of A and in FIG. 11, ROS production increased to 55.4% when treated with DHT alone was about 20% by the 40% ethanol extract of Schisandra (SF), and the 40% ethanol extract of Cornus officinalis (CF) at 1% level. was reduced to 5% by a mixture of 40% ethanol extract in a 1:1 ratio. Also, the 60% ethanol extract of Schisandra (SF) was reduced to 10.8%, the 60% ethanol extract of Cornus officinalis (CF) was reduced to 0.2%, and the 1:1 ratio of 60% ethanol extraction was reduced to 1.1% for the mixture. In addition, the results obtained by fluorescence microscopy also showed a similar tendency to inhibition of ROS generation (FIG. 11C). Therefore, it was confirmed that the DHT-induced ROS generation showed a more improved effect in the 60% ethanol extract than in the 40%, and it was confirmed that the mixture of 1:1 ratio showed a more improved effect compared to the effect of the ethanol extract alone of Schisandra.

3. Analysis of experimental results

1) Comparison of efficacy evaluation of hot water extract and 40% ethanol extract

In an in vitro prostate enlargement model using LNCaP cells, we evaluated the improvement of prostate enlargement with hot water extracts of Schisandra and oleracea and 40% ethanol extracts. To this end, an effective concentration capable of not exhibiting cytotoxicity to LNCaP cells and at the same time improving LNCaP cell proliferation promotion (prostate hypertrophy) induced by DHT was set ( FIG. 4 ). As a result, when the hot water extract and 40% ethanol extract were treated at 250 μg/ml, the hot water extract of Cornus officinalis, and the 40% ethanol extract of Schisandra and cornflower oil inhibited the proliferation of LNCaP cells, androgen-related genes (AR and PSA) and cell proliferation-related indicators. The inhibitory efficacy of gene (PCNA) expression was confirmed (FIG. 5). In addition, as a result of evaluating the effect on DHT-induced ROS production, all four extracts significantly reduced the DHT-induced ROS production, and the 40% ethanol extract had a higher ROS production inhibitory effect than the hot-water extract. (Fig. 6).

2) Setting the optimal mixing ratio of Schisandra and Cornus officinalis extract

The ability to improve prostate enlargement in an in vitro prostate enlargement model using NLCaP cells was evaluated by mixing the ratio of 40% ethanol extracts of Schisandra Schisandra and Cornus officinalis at 1:1, 2:1, and 1:2, respectively. The stimulation of cell proliferation induced by DHT was significantly reduced by 1:1, 2:1, and 1:2 mixtures of cornflower oil and omija, 1:1 and 1:2 compared to the mixture of 2:1 ratios. There was a higher significant difference in the mixture of ratios. In addition, the increase in androgen-related genes (AR, PSA, and ARA70) induced by DHT was reduced by a mixture of Schisandra and Cornus officinalis in a ratio of 1:1 and 1:2 ( FIG. 7 ).

3) Comparison of efficacy evaluation of 40% ethanol extract and 60% ethanol extract

To evaluate the ability of a 1:1 mixture of 40% ethanol extract, 60% ethanol extract, 40% ethanol extraction, and 60% ethanol extraction of Schisandra and Cornus officinalis in the LNCaP prostate enlargement model induced by DHT, a total of 6 methods Extracts and combinations of DHT-induced LNCaP cells were treated with various concentrations. At a concentration of 150 μg/ml, the DHT-induced cell proliferation was inhibited by a mixture of 60% ethanol Schisandra Schisandra extract, cornflower oil, and an ethanol 60% extract of Schisandra Schisandra. At 250 μg/ml treatment concentration, 6 types of Both the extract and the combination inhibited cell proliferation. However, compared to the 40% ethanol extract, the 60% ethanol extract had a proliferation inhibitory effect even at a lower concentration, and compared to the 60% ethanol extract of the Omija and Cornus officinalis extract, it was more There was a high significant difference.

Next, the improvement ability of AR, PSA and PCNA expression increase and PSA production and expression increase in LNCaP cells stimulated with DHT was evaluated. Overall, the reduction effect was higher in the 60% ethanol extract than in the 40% ethanol extract , showed the greatest effect in a 60% extract mixture in a 1:1 ratio ( FIGS. 8 to 10 ). As a result of evaluating the inhibitory ability of ROS generation in DHT-treated LNCaP cells, the ROS inhibitory efficacy was higher in the 60% ethanol extract and the 1:1 ratio than the 40% ethanol extract and the 1:1 ratio mixture, and Schisandra 60 The inhibitory ability of the % ethanol extract was found to have a greater effect when the 60% ethanol extract of Cornus officinalis was mixed (FIG. 11).

In conclusion, a 1:1 mixture of 60% ethanolic extracts of Schisandra and Cornus officinalis was used in a DHT-induced NLCaP prostatic hypertrophy model with a 1:1 mixture of 40% ethanol extract of Schisandra and Cornus officinalis and 40% ethanol extract of Schisandra and Cornus officinalis and 60% of Schisandra and Cornus officinalis. It was confirmed that the improvement of proliferation promotion was significantly higher than that of the ethanol extract.

II. Experiment on the presence of sediment in the composition according to the enzymatic reaction process in the preparation of the mixed extract of Omizawa Cornus officinalis

The following experiment evaluates whether there is sediment in the composition according to the enzymatic reaction process in the preparation method of the composition for the prevention or treatment of benign prostatic hyperplasia comprising the mixed extract of omija and cornus oil of the present invention and the appearance characteristics related to the color of the composition. .

It was checked by visually confirming whether there was a precipitate in the composition and the appearance characteristics related to the color of the composition. If there is sediment, it is indicated by ○, if the amount of sediment is large, it is indicated by ◎, and if there is no sediment, it is indicated by X.

[Example 5]

A mixed raw material was prepared by mixing dried cornus oil raw material and dried omija raw material in a ratio of 1:1. A raw material solvent mixture obtained by adding 100 parts by weight of a 60% ethanol aqueous solution to 10 parts by weight of the mixed raw material was put into an extractor, and a mixed extract was formed at 50° C. for 4 hours. The extract was placed in a concentrator and concentrated to have a sugar content of 60brix. After sterilizing the concentrated extract, it was filtered to prepare a 60% ethanol-extracted mixture of Omija cornflower oil mixed in 1:1 ratio.

[Example 6]

A mixed raw material was prepared by mixing dried cornus oil raw material and dried omija raw material in a ratio of 1:1. A raw material solvent mixture obtained by adding 100 parts by weight of a 60% ethanol aqueous solution to 10 parts by weight of the mixed raw material was put into an extractor and extracted at 50° C. for 4 hours to form a mixed extract. The mixed extract was placed in a concentrator and concentrated to have a sugar content of 25brix. After reacting by adding 0.01 parts by weight of beta-glucanase enzyme to 100 parts by weight of the first concentrated mixed extract, the enzyme was inactivated by heating at 100° C. for 10 minutes. It was put in a concentrator and re-concentrated to have a sugar content of 60 brix. After sterilizing the concentrated mixed extract, it was filtered to prepare a 60% ethanol-extracted mixture of Omija cornflower oil mixed in 1:1 ratio.

[Example 7]

A mixed raw material was prepared by mixing dried cornus oil raw material and dried omija raw material in a ratio of 1:1. A raw material solvent mixture obtained by adding 100 parts by weight of a 60% ethanol aqueous solution to 10 parts by weight of the mixed raw material was put into an extractor and extracted at 50° C. for 4 hours to form a mixed extract. The mixed extract was placed in a concentrator and concentrated to have a sugar content of 25brix. 0.2 parts by weight of a beta-glucanase enzyme was added and reacted with respect to 100 parts by weight of the first-concentrated mixed extract, and then heated at 100° C. for 10 minutes to inactivate the enzyme. It was put in a concentrator and re-concentrated to have a sugar content of 60 brix. After sterilizing the concentrated mixed extract, it was filtered to prepare a 60% ethanol-extracted mixture of Omija cornflower oil mixed in 1:1 ratio.

[Example 8]

A mixed raw material was prepared by mixing dried cornus oil raw material and dried omija raw material in a ratio of 1:1. A raw material solvent mixture obtained by adding 100 parts by weight of a 60% ethanol aqueous solution to 10 parts by weight of the mixed raw material was put into an extractor and extracted at 50° C. for 4 hours to form a mixed extract. The mixed extract was placed in a concentrator and concentrated to have a sugar content of 25brix. After reacting by adding 0.4 parts by weight of beta-glucanase enzyme to 100 parts by weight of the first-concentrated mixed extract, the enzyme was inactivated by heating at 100° C. for 10 minutes. It was put in a concentrator and re-concentrated to have a sugar content of 60 brix. After sterilizing the concentrated mixed extract, it was filtered to prepare a 60% ethanol-extracted mixture of Omija cornflower oil mixed in 1:1 ratio.

[Example 9]

A mixed raw material was prepared by mixing dried cornus oil raw material and dried omija raw material in a ratio of 1:1. A raw material solvent mixture obtained by adding 100 parts by weight of a 60% ethanol aqueous solution to 10 parts by weight of the mixed raw material was put into an extractor and extracted at 50° C. for 4 hours to form a mixed extract. The mixed extract was placed in a concentrator and concentrated to have a sugar content of 25brix. 0.8 parts by weight of a beta-glucanase enzyme was added and reacted with respect to 100 parts by weight of the first-concentrated mixed extract, and then heated at 100° C. for 10 minutes to inactivate the enzyme. It was put in a concentrator and re-concentrated to have a sugar content of 60 brix. After sterilizing the concentrated mixed extract, it was filtered to prepare a 60% ethanol-extracted mixture of Omija cornflower oil mixed in 1:1 ratio.

[Example 10]

A mixed raw material was prepared by mixing dried cornus oil raw material and dried omija raw material in a ratio of 1:1. A raw material solvent mixture obtained by adding 100 parts by weight of a 60% ethanol aqueous solution to 10 parts by weight of the mixed raw material was put into an extractor and extracted at 50° C. for 4 hours to form a mixed extract. The mixed extract was placed in a concentrator and concentrated to have a sugar content of 25brix. After reacting by adding 1.0 parts by weight of beta-glucanase enzyme to 100 parts by weight of the first-concentrated mixed extract, the enzyme was inactivated by heating at 100° C. for 10 minutes. It was put in a concentrator and re-concentrated to have a sugar content of 60 brix. After sterilizing the concentrated mixed extract, it was filtered to prepare a 60% ethanol-extracted mixture of Omija cornflower oil mixed in 1:1 ratio.

The amount of beta-glucanase enzyme added to the composition prepared in Experimental Example 2 is shown in Table 2 below. Example 5 is a composition prepared without performing an enzymatic process.

Category (unit) Diluted mixed extract (parts by weight) Enzyme addition amount (parts by weight) Example 5 not diluted not added Example 6 100 0.01 Example 7 100 0.2 Example 8 100 0.4 Example 9 100 0.8 Example 10 100 1.0

The results of precipitation and sensory evaluation of the compositions for the prevention or treatment of benign prostatic hyperplasia prepared in Examples 5 to 10 performed in Experimental Example 2 are shown in Table 3 below.

division whether sediment Color-Related Appearance Features Example 5 ◎ The extract in liquid form forms the upper and middle layers, and some precipitates are dispersed in the upper and middle layers, giving it a pale red color.
In the lower layer, 7 to 10 parts by weight are accumulated based on 100 parts by weight of the total composition. Example 6 ○ The extract in liquid form forms the upper and middle layers, and some precipitates are dispersed in the upper and middle layers, giving it a pale red color.
In the lower layer, 3 to 5 parts by weight are accumulated based on 100 parts by weight of the total composition. Example 7 X In the extract in liquid form, precipitates with small particle sizes are dispersed and have a pale red color. Example 8 X The extract in liquid form has a bright, vivid red color. Example 9 X The extract in liquid form has a dark red color. Example 10 X The extract in liquid form has a dark red color.

(○: with a precipitate, ◎: with a lot of precipitate, X: without a precipitate) Referring to Table 3, as a result of the experiment, a precipitate was observed only in Example 5 in which the enzyme process was not performed and in Example 6 in which a very small amount of the enzyme was added.

12A is an image showing the composition prepared in Example 5, and FIG. 12B is an image showing the composition prepared in Example 8.

12A , in Example 5, it was confirmed that a large amount of the precipitate was accumulated in the lower layer with the naked eye, and the upper middle layer also showed a pale red color while becoming opaque by the dispersed precipitate.

In Example 6, the sediment accumulated in the lower layer was reduced, but it was possible to confirm the sediment accumulated in the lower layer. This can be considered that the enzymatic reaction was performed, but as the enzyme was added in a small amount, the enzymatic reaction did not occur sufficiently, and some precipitates remained.

In Example 7, no precipitate was accumulated in the lower layer, but the composition exhibited an opaque pale red color, indicating that precipitates having a small particle size were dispersed in the composition.

In Example 8, no precipitate was observed, and the color of the composition was bright and vivid red, indicating that almost no precipitate dispersed in the composition remained.

In Example 9, no precipitate was observed, and the color of the composition showed a red color close to dark red, and there was no precipitate dispersed in the composition, so that the turbidity of the entire composition was lowered It is judged that the color of

In Example 10, the same external characteristics as in Example 9 were observed. This is, when 0.8 parts by weight of the beta-glucanase enzyme is added in Example 9, the precipitate can be completely removed, and in Example 10, 1.0 parts by weight by increasing the beta-glucanase enzyme addition amount Even if added, it may mean that there is no change in physical properties, including removal of sediment, externally. That is, 0.4 to 0.8 parts by weight of a beta-glucanase enzyme can be added to 100 parts by weight of the diluted mixed extract to properly remove the precipitate in the mixed extract, and within the above range, the raw material of Omizawa cornus oil It can be added by adjusting the amount of beta-glucanase enzyme added according to the varying pH or the amount of active ingredient.

As described above, the method for preparing a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of cornus oil and omija of the present invention is beta-glucanase enzyme 0.4 to 100 parts by weight of the diluted mixed extract as described above. After reacting by adding 0.8 parts by weight, by performing a process of inactivating the enzyme, the precipitate can be effectively removed and the unique color of the Omizawa Cornus officinalis mixed extract can be clearly displayed, thereby improving the quality of the composition.

As such, those skilled in the art to which the present invention pertains will understand that the above-described technical configuration of the present invention may be implemented in other specific forms without changing the technical spirit or essential features of the present invention.

Therefore, the embodiments described above are to be understood as illustrative and not restrictive in all respects, and the scope of the present invention is indicated by the following claims rather than the above detailed description, the meaning and scope of the claims, and All changes or modifications derived from the concept of equivalents thereof should be construed as being included in the scope of the present invention.

Claims (6)

It is composed of a mixed extract of mixed raw materials in which Omizawa Cornus Oil is mixed in a weight ratio of 1:1,
The mixed extract is
It is extracted by adding 100 parts by weight of an extraction solvent in which water and ethanol are mixed with respect to 10 parts by weight of the mixed raw material,
The concentration of ethanol in the extraction solvent is composed of 60 wt%,
The mixed extract is a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of Omija and Cornus officinalis, characterized in that it is formed by extraction at 50° C. for 4 hours. delete The method of claim 1,
After 0.4 to 0.8 parts by weight of a beta-glucanase enzyme is added with respect to 100 parts by weight of the mixed extract, the added enzyme is inactivated. A composition for prophylaxis or treatment. A first step of preparing a mixed raw material mixed with Omizawa cornflower oil in a weight ratio of 1:1;
a second step of mixing an extraction solvent composed of water and ethanol with the mixed raw material to form a raw material solvent mixture;
a third step of extracting the raw solvent mixture at a temperature of 50° C. for 4 hours to form a mixed extract;
a fourth step of concentrating the mixed extract to have a concentration of 55 to 65 brix; and
After sterilizing the concentrated mixed extract, a fifth step of filtering;
The mixed extract is
It is extracted by adding 100 parts by weight of an extraction solvent in which water and ethanol are mixed with respect to 10 parts by weight of the mixed raw material,
A method of producing a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of Omizawa cornus oil, characterized in that the concentration of ethanol in the extraction solvent is comprised of 60 wt%. delete 5. The method of claim 4,
The fourth step is
Step 4-1 of first concentrating the mixed extract to have a sugar content of 20 to 30 brix;
Step 4-2 of reacting by adding 0.4 to 0.8 parts by weight of beta-glucanase with respect to 100 parts by weight of the first concentrated mixed extract;
Step 4-3 of inactivating the enzyme by heating the mixed extract reacted with the enzyme at a temperature of 90 to 100° C. for 5 to 20 minutes;
Method of producing a composition for the prevention or treatment of benign prostatic hyperplasia comprising a mixed extract of Omizawa cornus oil, characterized in that it comprises a 4-4 step of secondary concentration of the enzyme-inactivated mixed extract to have 55 to 65 brix.

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