WO2016144125A2 - Novel use of solanum nigrum l. extract - Google Patents

Abstract

The present invention provides a novel use of a Solanum nigrum L. extract. The present invention has an advantage of effectively treating, alleviating and/or preventing endometriosis or a complication thereof.

Description

New Uses of Yonggyu Extract

The present invention relates to a yonggyu extract, and more particularly to a novel use of the yonggyu extract.

Endometriosis is a very common gynecological disorder that occurs in 3-10% of women of childbearing age and 25-35% of infertile women, and the endometrial tissue abnormally proliferates in places other than the uterus (ovary, abdominal cavity, intestine, bladder). Disease.

Endometriosis is a symptom of dysmenorrhoea, dyspareunia, and various pains (eg, lung involvement: pneumothorax, hemothorax / ureteral involvement: ureter obstruction, hematuria), 30-50% of patients. Has been identified as the cause of infertility.

To date, there is no fundamental treatment for endometriosis other than endometrial removal through surgery, which may limit the pregnancy and the recurrence rate is high.

Current medical treatments include conservative therapies using painkillers and anti-inflammatory drugs to relieve pain, and hormone therapy to control the menstrual cycle using danazol, progesterone, and GnRH.

In addition, long-term use of these hormones causes a number of side effects (increase in weight, water accumulation, fatigue, acne, oily skin, grandmothers, atrophic vaginitis, hot flashes, muscle spasms, unstable emotional states, hepatotoxicity) The relapse rate for treatment is also known to be very high.

Endometriosis is not fatal, but it is a disease that causes discomfort in daily life due to extreme pain and even infertility in severe cases, and is a disease that must be seriously recognized and must be overcome in women's quality of life and pregnancy problems. So far, there is no prevention and treatment.

Therefore, there is an urgent need to develop a new concept of endometriosis prevention and treatment, which has low side effects, rather than invasive surgery, simple analgesia, and artificial menstrual cycle control, especially for women of childbearing age.

On the other hand, Yonggyu extract is known to have an AIDS therapeutic effect (Korean Patent Publication No. 1020060034178).

[Preceding technical literature]

[Patent Documents]

(Patent Document 1) Republic of Korea Patent Publication No. 1020060034178, April 21, 2006, Summary

The problem to be solved by the present invention is to provide a novel use of the extract.

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

The inventors have surprisingly found that Yonggyu extract has an effect on the treatment, improvement and / or prevention of endometriosis or its complications and completed the present invention.

The present invention provides a medicament and / or food use for treating, ameliorating and / or preventing endometriosis or complications thereof.

The present invention also provides a pharmaceutical composition for treating or preventing endometriosis or its complications, comprising the extract of Yonggyu as an active ingredient.

The yonggyu (Solani Nigri Herba) is listed in the Korean Pharmacopoeia (Herbal Medicine) standard collection, may be the ground portion of the solarum nigrum (Solanum nigrum Linne (branch family Solanaceae)). The ground portion includes stems and leaves, and may include flowers.

The pharmaceutical composition may further include a pharmaceutically acceptable additive, and may be composed of the yonggyu extract and the additive.

The endometriosis or complications thereof may be due to one or more overexpression selected from MMP-2, MMP-9, COX-2, TNF-alpha (α), or MCP-1.

The complication may be one or more selected from the group consisting of pelvic inflammatory disease, pelvic adhesion, ovarian cyst, uterine myoma, ectopic pregnancy and infertility.

The yonggyu extract is a solvent extract extracted from the yonggyu as a solvent, the solvent may be at least one selected from water, alcohol, or a mixture thereof.

The alcohol may be a lower alcohol having 1 to 5 carbon atoms.

The solvent may be 70% by volume ethanol.

The Yonggyu extract may be a butanol fraction extract obtained by fractionating 70% by volume of ethanol extract with 70% by volume of ethanol, butanol.

The butanol fraction extract (A) preparing a 70 volume% ethanol extract extracted with 70 volume% ethanol; (B) adding water to the 70% by volume ethanol extract of step (A); (C) fractionating 70 vol% ethanol extract added with water of step (B) with n-hexane; (D) removing the n-hexane fraction of step (C) and fraction-extracting the remaining aqueous layer with dichloromethane; (E) removing the dichloromethane fraction of step (D) and fractionating the remaining aqueous layer with ethyl acetate; And (F) may be a fraction extract obtained by removing the ethyl acetate fraction of step (E) and fraction extraction of the remaining aqueous layer with butanol.

The treatment or prevention may be by inhibiting adhesion or migration of endometriosis cells.

The attachment or migration inhibition may be by inhibition of expression of one or more selected from MMP-2 or MMP-9.

The treatment or prevention may be due to one or more selected from inhibition of inflammation and pain related enzyme expression or inhibition of inflammatory cytokine expression.

The present invention also provides a food composition for improving or preventing endometriosis or its complications, comprising the extract of Yonggyu as an active ingredient.

Unless otherwise stated, the food composition is equally applicable unless the contradictions mentioned in the pharmaceutical composition of the present invention are contradictory. The term 'improvement' is included in the 'treatment' and means that the condition or symptom is improved.

The food composition may be included in a variety of foods, including beverages, may be in the form of beverages, gum, tea, health functional foods, etc. The health functional foods may be formulated in the form of tablets, capsules and the like. The health functional food refers to a food manufactured using a raw material or ingredient having functional functionality useful for the human body according to Korean Health Functional Food Act No. 12669 (including processing. The following is referred to). By means of nutrient control or physiological effects on the structure and function of the human body to obtain useful effects for health purposes. The food composition may include a conventional food additive, the food additive is a natural chemical such as ketones, glycine, sodium citrate, nicotinic acid, cinnamon acid, navy, licorice extract, crystalline cellulose, high pigment, guar gum And mixed preparations such as additives, sodium L-glutamate preparations, noodle addition alkalis, preservatives, and tar dyes.

The present invention also provides a treatment, or prophylaxis, of endometriosis or a complication thereof, comprising administering the yonggyu extract to a mammal, including a human being in need thereof, and the treatment of endometriosis or a complication thereof. Or for the preparation of prophylactic formulations. The administered yonggyu extract may be an effective amount of yonggyu extract. The present invention also provides a extract of yonggyu for use in the treatment, improvement or prevention of endometriosis or complications thereof.

Unless otherwise stated, the statements mentioned in the pharmaceutical compositions of the present invention apply equally to the methods, extracts, and uses of the present invention unless contradictory.

The Yonggyu extract or composition can be administered orally or parenterally to mammals, including humans, and can be administered by formulating an active ingredient in combination with a pharmaceutically acceptable additive. When formulated, additives commonly used may be carriers, fillers, extenders, binders, wetting agents, disintegrants, surfactants, diluents, excipients and the like other than the active ingredient. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, and And / or by adding gelatin or the like. Lubricants such as magnesium and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions and syrups, and various excipients such as wetting agents, sweeteners, fragrances and / or preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. May be included. Formulations for parenteral administration include injectable solutions, suspensions, emulsions, lyophilizers, nasal washes and suppositories. Injectable solutions, suspensions and emulsions can be prepared by mixing water, non-aqueous solvents or suspending solvents with the active ingredient. As non-aqueous solvents and suspending solvents, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and ethyl oleate Injectable esters such as and the like. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol and / or gelatin may be used. Parenteral administration may be by subcutaneous injection, intravenous injection or intramuscular injection.

Yonggyu extract contained in the composition of the present invention or used in the use and method is a dose of 0.0001 to 1000 mg / kg, preferably 0.0001 to 100 mg / kg, more preferably 0.001 to 10 mg / kg per day, based on an adult woman Can be used as Administration can be administered once a day or in divided doses. However, the scope of the present invention is not limited by the dose and frequency of administration.

The composition of the present invention may contain 0.1 to 99.9% by weight of the active ingredient relative to the total weight of the composition.

The Yonggyu extract can be formulated by adding a pharmaceutically or food-acceptable carrier, excipient or diluent, etc. For formulation, see Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA, etc. Reference may be made.

The present invention can effectively treat, ameliorate and / or prevent endometriosis or complications thereof.

Figure 1 is a view showing the results of the MMP-2 expression inhibitory effect confirmation experiment of Yonggyu alcohol extract.

Figure 2 is a view showing the results of confirming the inhibitory effect of MMP-9 expression of Yonggyu alcohol extract.

Figure 3 is a diagram showing the results of confirming the inhibitory effect of COX-2 expression of Yonggyu alcohol extract.

Figure 4 is a view showing the results of experiments confirming the inhibitory effect of TNF alpha expression of Yonggyu alcohol extract.

Figure 5 is a view showing the results of the MCP-1 expression inhibitory effect confirmation experiment of Yonggyu alcohol extract.

Figure 6 is a view showing the results of experiments confirming the effect of endometriosis cell adhesion and implantation inhibition of the alcoholic alcohol extract.

Figure 7 is a diagram showing the results of experiments confirming the inhibitory effect of endometriosis cell migration of the alcoholic extract of Yonggyu.

8 is a view showing the results of the effect confirmation experiment in the intraperitoneal administration model of Yonggyu alcohol extract.

Figure 9 is a diagram showing the results of effect verification experiments in the surgical induction model of Yonggyu alcohol extract.

10 is a view showing the results of the MMP-2 expression inhibitory effect confirmation experiment of Yonggyu butanol fraction extract.

Figure 11 is a view showing the results of the MMP-9 expression inhibitory effect confirmed by the extract of Yonggyu butanol fraction.

Figure 12 is a view showing the results of the COX-2 expression inhibitory effect confirmed by the extract of Yonggyu butanol fraction.

Figure 13 is a view showing the results of the test TNF alpha expression inhibition effect of Yonggyu butanol fraction extract.

14 is a view showing the results of the MCP-1 expression inhibitory effect confirmation experiment of Yonggyu butanol fraction extract.

15 is a diagram showing the results of experiments confirming the effect of endometriosis cell adhesion and implantation inhibition of the extract of Yonggyu butanol fraction.

Figure 16 is a diagram showing the results of confirming the inhibitory effect of MMP-2 expression of Yonggyu water extract.

Figure 17 is a diagram showing the results of confirming the inhibitory effect of COX-2 expression of Yonggyu water extract.

18 is a diagram showing the results of experiments confirming the inhibitory effect of TNF alpha expression of Yonggyu water extract.

19 is a view showing the results of confirming the inhibitory effect of MCP-1 expression of Yonggyu water extract.

Hereinafter, the present invention will be described in more detail with reference to Examples, Preparation Examples and Drawings, but the following Examples and Preparation Examples are only for illustrating the present invention, and the contents of the present invention are given in the following Examples, Preparation Examples and Drawings. It is not limited by.

Changes in the expression of cell migration and adhesion-related enzymes {MMP-2 (matrix metallopeptidase-2) and / or matrix metallopeptidase-9 (MMP-9)}, inflammation and pain related enzymes {COX-2 (cyclooxygenase-) characteristic of endometriosis 2)} change in expression, inflammatory cytokine {TNF-alpha (tumor necrosis factor alpha) and / or expression of Monocyte chemotactic protein-1 (MCP-1)}, endometriosis cell adhesion and implantation change, endometriosis cell migration change By confirming the effect of Yonggyu extract on the back, it was confirmed in the following method that Yonggyu extract has the effect of treating, improving or preventing endometriosis or its complications.

The endometriosis model cells described in the following examples were prepared with a 12Z cell line, human immortalized endometriotic epithelial cells, developed using active endometriosis tissue of an endometriosis woman (Johann-Wolfgang-Goethe-). Dr. Universitaet (Germany) Offered by Starzinski-Powitz}. These cells have been shown to be similar to the molecular biology of known active endometotric tissues (Banu et al., 2008), and have been used as a new in vitro model for the molecular biology of endometriosis. Cells were 37 degrees Celsius, 5% carbon dioxide-95 in RPMI 1640 and DMEM / F12 medium (10% fetal bovine serum, 100 U / ml penicillin G, 100 g / ml streptomycin added; Life Technology, Grand Island, New York) % Was maintained in air conditions.

Example 1 Confirmation of Treatment, Improvement or Prevention of Endometriosis or Complications of Yonggyu Alcohol Extract

1-1. Preparation of Yonggyu Alcohol Extract

Dried yonggyu (Solanum nigrum, production area: Yeongcheon, Gyeongsangbuk-do) was purchased at Gyeongdong market, South Korea, and yonggyu outpost was pulverized to 400um particle size. 100 liters of a 70% (v / v) ethanol aqueous solution was added to 10 kg of the prepared solu- tion, and the filtrate was immersed at room temperature for 24 hours, and the extract was repeated once in the same manner. The extracted filtrate was concentrated under reduced pressure at 60 degrees Celsius, and the concentrated solution was lyophilized to obtain 1.5 kg of powdered 70% ethanol extract.

1-2. Confirmation of inhibitory effect on enzyme expression related to cell migration and adhesion

To determine whether 70% by volume ethanol extract inhibits the mRNA expression of proteolytic enzymes MMP-2 and MMP-9, which play an important role in the migration and infiltration of endometriosis cells, real-time PCR is performed. It was.

Specifically, after treatment with 25 ug / mL, 50 ug / mL, or 100 ug / mL of 70% by volume ethanol extract for 12 hours (12 pit) epithelial endometriotic cells (12Z), TRIzol (Invitrogen Canada, Burlington, ON, Canada) was used to extract the total RNA, followed by reverse transcription polymerase chain reaction (RT-PCR). Specifically, using a first-strand cDNA as a template, using the Thermal Cycler Dice Real Time PCR System (Takara, Japan) denaturation for 5 seconds at 95 degrees Celsius, annealing for 10 seconds at 57 degrees Celsius, 72 degrees Celsius SYBR Green real-time PCR was performed by repeating 45 times under the condition of performing extension for 20 seconds in the figure. The MMP-2, MMP-9 and calibration GAPDH primers used in the SYBR Green real-time PCR are shown in Table 1 below, and the average of Ct values of MMP-2 and MMP-9 is triplelicate. Obtained by measurement, corrected by the Ct value of GAPDH. In addition, the yonggyu extract untreated group as a control (control; CON), except that the treatment was the same as the yonggyu extract treated group. As a result, in the 12Z cells of endometriosis cells, 70% ethanol extract inhibited the mRNA expression of endogenous MMP-2 and MMP-9 which is a representative protein expressed in endometriosis and plays an important role in cell migration and infiltration. It confirmed (refer FIG. 1, FIG. 2).

TABLE 1

Figure 1 is a view showing the results of the MMP-2 expression inhibitory effect confirmed experiment of Yonggyu alcohol extract, Figure 2 is a road showing the results of MMP-9 expression inhibitory effect confirmed by the alcoholic extract, in the x-axis of Figures 1 and 2 con represents a control group, 25 represents a 70 μg% ethanol extract 25ug / mL administration group, the y-axis of Figure 1 shows the relative MMP2 mRNA expression (Relative MMP2 mRNA expression), the y-axis of Figure 2 relative MMP9 mRNA expression (Relative MMP9 mRNA expression). As shown in the figure, 70% by volume ethanol extract can be seen that effectively inhibits the expression of MMP-2 and MMP-9, even at a concentration of 25ug / mL.

1-3. Inhibition of enzyme expression related to inflammation and pain

Real-time PCR was performed to determine the effect of 70% by volume ethanol extract on the mRNA expression of COX-2, which is known to be associated with endometriosis pain. Specifically, erythrocytes were treated with 25% ug / mL, 50 ug / mL, or 100 ug / mL, respectively, for 24 hours in 12% (epithelial endometriotic cells) of endometriosis cells, followed by TRIzol (Invitrogen Canada, Burlington, ON, Canada) was used to extract the total RNA, and reverse transcription polymerase chain reaction (RT-PCR) was performed. Specifically, using a first-strand cDNA as a template, using a Thermal Cycler Dice Real Time PCR System (Takara, Japan) denaturation for 5 seconds at 95 degrees Celsius, annealing for 10 seconds at 57 degrees Celsius, 72 degrees Celsius SYBR Green real-time PCR was performed by repeating 45 times under the condition of performing extension for 20 seconds in the figure. The COX-2 primers used for the SYBR Green real-time PCR and the GAPDH primers for correction are shown in Table 2 below, and the average of Ct values of COX-2 is obtained by triplelicate measurement. Corrected by the Ct value. In addition, the yonggyu extract untreated group as a control (control; CON), except that the treatment was the same as the yonggyu extract treated group.

TABLE 2

As a result, it was confirmed in the endometriosis cells 12Z cells that 70% ethanol extract inhibits the mRNA expression of COX-2, a pain-related characteristic enzyme of endometriosis cells (see FIG. 3).

Figure 3 is a road showing the results of the COX-2 expression inhibitory effect confirmed by Yonggyu alcohol extract, con on the x-axis of Figure 3 is a control, 25 is a 70wt% ethanol extract 25ug / mL administration group, y of Figure 3 The axis shows the relative COX2 mRNA expression. As shown in the figure, it can be seen that the 70% by volume ethanol extract effectively inhibits COX-2 expression even at a concentration of 25ug / mL.

1-4. Inhibition of inflammatory cytokine expression

Real-time PCR was performed to determine whether 70% by volume ethanol extract affects inflammatory cytokine mRNA expression, which is known to be overexpressed in endometriosis cells.

Specifically, erythrocytes were treated with 25% ug / mL, 50 ug / mL, or 100 ug / mL, respectively, for 24 hours in 12% (epithelial endometriotic cells) of endometriosis cells, followed by TRIzol (Invitrogen Canada, Burlington, ON, Canada) was used to extract the total RNA, and reverse transcription polymerase chain reaction (RT-PCR) was performed. Specifically, using a first-strand cDNA as a template, using a Thermal Cycler Dice Real Time PCR System (Takara, Japan) denaturation for 5 seconds at 95 degrees Celsius, annealing for 10 seconds at 57 degrees Celsius, 72 degrees Celsius SYBR Green real-time PCR was performed by repeating 45 times under the condition of performing extension for 20 seconds in the figure. The TNF-a primer, MCP-1 primer, and calibration GAPDH primers used in the SYBR Green real-time PCR are shown in Table 3 below, and the average of Ct values of TNF-a and MCP-1 is tripletlicate ( triplicate) and corrected by the Ct value of GAPDH. In addition, the yonggyu extract untreated group as a control (control; CON), except that the treatment was the same as the yonggyu extract treated group.

TABLE 3

As a result, it was confirmed in the endometriosis cells 12Z cells that the 70% ethanol extract inhibits the mRNA expression of inflammatory cytokines TNF-alpha and MCP-1 of endometriosis cells (see Figs. 4 and 5).

Figure 4 is a view showing the results of the test TNF-alpha expression inhibitory effect of Yonggyu alcohol extract, Figure 5 is a road showing the results of MCP-1 expression inhibitory effect confirmed by Yonggyu alcohol extract, in the x-axis of Figures 4 and 5 con denotes the control group, 25 denotes the administration volume of the 70% by volume ethanol extract 25ug / mL, the y-axis of Figure 4 shows the relative TNF-alpha mRNA expression, the y-axis of Figure 5 is the relative MCP-1 mRNA Expression (Relative MCP-1 mRNA expression). As shown in the figure, it can be seen that the 70% by volume ethanol extract effectively inhibits TNF-alpha and MCP-1 expression even at a concentration of 25ug / mL.

1-5. Confirmation of endometriosis cell adhesion and inhibitory effect

Attachment assay was repeated three times to confirm the effect of Yonggyu 70% ethanol extract on the attachment and implantation of endometriosis cells.

Specifically, Met-5A, a human peritoneal methothelial cells, was used to determine whether yonggyu 70% ethanol extract inhibited endometriosis cells, which are characteristic of endometriosis, from attaching and implanting to organs peritoneal peritoneal cavity. Cells (American Type Culture Collection (ATCC)) were treated with 10% FBS, 100 U / ml penicillin G and 100 g / ml streptomycin (Life Technologies, Grand Island, NY, USA), and 400 nM hydrocortisone (Sigma Chemical). Co, St. Louis, Mo., USA) was incubated in a 5% CO ₂ incubator in 199 medium. The Met-5A cells were dispensed into 96-well plates. Cell tracker green DMEM / F12 containing 50 ug / mL, 100 ug / mL, or 200 ug / mL of 70% ethanol extract of 12Z cells (2 X 10 ³ / well), an endometriosis cell line labeled with CMFDA. After mixing with the medium, it was added to previously dispensed Met-5A cells. The mixed cells were incubated in a 5% CO ₂ -incubator at 37 degrees Celsius for one hour, then the plates were inverted and washed with a phosphate-buffered solution containing calcium and magnesium. Attached cells were analyzed at a wavelength of 490 nm, with the calibration control set at 100% and the percentage of attached cells calculated. In addition, the yonggyu extract untreated group was treated as a control (control; CON), the same as the yonggyu extract treated group except for the extract treatment.

As a result, it was confirmed that endometriosis cells 12Z treated with 70% ethanol extract were inhibited from adhering to Met-5A cells surrounding the outside of the peritoneum and the peritoneal organs (see FIG. 6).

Figure 6 is a road showing the results of endometriosis cell adhesion and anti-implantation inhibitory effect of Yonggyu alcohol extract, in the x-axis of Figure 6 is the control group, 50ug / ml is 70ug% ethanol extract 50ug / mL administration group, The y-axis of FIG. 6 shows the ratio of attachment cells (Optical Density (OD) ratio,%}). As shown in the figure, it can be seen that the 70% by volume ethanol extract effectively inhibits cell adhesion even at a concentration of 50ug / mL.

1-6. Confirmation of inhibitory effect on endometriosis cell migration

Transwell-migration assay was repeated three times to confirm the effect of Yonggyu 70% ethanol extract on the migration of endometriosis cells. Specifically, after dispensing 12Z cells, which are endometriosis cells, into an upper part of a transwell plate in a 24-well-transwell plate (8 um pore size), respectively, 25 ug / mL, 50 ug / mL, and 100 ug / mL of 70% ethanol extract was treated. After treatment with 70% ethanol extract, cells transferred to the membrane on the outer side of the transwell were fixed with methanol and stained with 0.5% crystal violet for 10 minutes. The cells moved to the outer surface of the transwell were observed and counted under a microscope of 200 X magnification and graphed. In addition, the yonggyu extract untreated group as a control (control; CON), except that the treatment was the same as the yonggyu extract treated group. As a result, it was confirmed that the migration of endometriosis cells 12Z treated with 70% ethanol extract of Yonggyu was significantly inhibited (see FIG. 7).

7 is a road showing the results of experiments to confirm the effect of inhibiting endometriosis cell migration of the alcoholic alcohol extract, on the x-axis of Figure 7 con is a control, 25ug / ml is a 70ug% ethanol extract 25ug / mL administration group, Figure 7 The y-axis represents the percentage of migration. As shown in the figure, it can be seen that the 70% by volume ethanol extract effectively inhibits cell adhesion even at a concentration of 25ug / mL.

1-7. Confirmation of effect on intraperitoneal injection induction model

The evaluation was performed to confirm the effect of Yonggyu 70% ethanol extract on Intraperitoneal injection endometriosis induction model.

Induction of endometriosis was induced by extracting the uterus to be implanted in a donor mouse and then sculpted into the abdominal cavity of the recipient mouse, drug administration is shown in [Table 4] below, 70% ethanol extract 125 mg / kg, 250 mg / kg and 500 mg / kg were orally administered for 5 weeks, respectively. The abdominal cavity was opened at the expense of endometriosis-induced animal models to determine the number of implantations in the peritoneum and intraperitoneal (small intestine, large intestine, stomach, liver, bladder, ovary, etc.).

TABLE 4

In addition, the vehicle (Vehicle) administration group was treated in the same manner as the Yonggyu extract administration group except that 200ul of 10% Tween 80 instead of the extract. In addition, the positive control was treated in the same manner as the Yonggyu extract administration group, except that 0.3mg / kg dienogest instead of Yonggyu extract.

As a result, it was confirmed that 70% ethanol extract of Yonggyu had a significant inhibitory effect on endometriosis tissue formation (see FIG. 8).

8 is a road showing the results of experiments confirmed the effect of the ingestion model of the alcoholic extract of Yonggyu, vehicle on the x-axis of Figure 8 vehicle administration, dienogest dienogest administration group, Yonggyu 125 is 70% by volume ethanol extract 125mg / kg The administration group is shown, and the y-axis of FIG. 8 represents the number of implants. As shown in the figure, it can be seen that 70% by volume of ethanol extract, even at 125 mg / kg, effectively exhibited an endometriosis tissue formation inhibitory effect.

1-8. Confirmation of effect in Surgical induced model

The evaluation was performed to confirm the effect of Yonggyu 70% ethanol extract on Surgical induced endometriosis induction model.

Induction of endometriosis was induced by implanting endometrial cells by cutting the left uterus of SD rat (female) to 7 mm length and suturing both ends with silkam so that the endometrial part was in contact with the peritoneum. 5], and 100 mg / kg, 300 mg / kg, and 500 mg / kg of 70% ethanol extract were orally administered for 4 weeks. The implant size (inflammation site size) in the peritoneum where the endometriosis cells were transplanted by abdominal ablation was sacrificed at the expense of an endometriosis induced animal model.

TABLE 5

In addition, vehicle (Vehicle) administration group was treated in the same manner as Yonggyu extract administration group, except that the solvent for dissolving the extract instead of the extract in proportion to the body weight. The extract was dissolved in 10% Tween 80. In addition, a positive control (Positive control) was treated in the same manner as the Yonggyu extract administration group, except that 0.3mg / kg dienogest instead of Yonggyu extract.

As a result, it was confirmed that the 70% ethanol extract of Yonggyu had an inhibitory effect on endometriosis tissue formation (see FIG. 9).

Figure 9 is a road showing the results of experiments confirming the effect of the surgical extract of Yonggyu alcohol extract, vehicle on the x-axis of Figure 9 vehicle administration vehicle, positive control group dienogest administration group, Yonggyu 100mg / kg Yonggyu extract 100mg / kg administration group The y-axis represents the implant size. As shown in the figure, 70% by volume of ethanol extract, even 100mg / kg, it can be seen that effectively shows the effect of inhibiting endometriosis tissue formation.

From these results, the effect of treating, improving or preventing endometriosis or its complications of the yonggyu alcohol extract can be confirmed.

Example 2 Confirmation of Treatment, Improvement, or Prevention of Endometriosis and Complications of Yonggyu Butanol Fraction Extract

2-1. Preparation of Yonggyu Butanol Fraction Extract

Yonggyu butanol fraction extract was prepared by dividing into normal hexane fraction, dichloromethane fraction, ethyl acetate fraction, butanol fraction, and water layer through a systematic fraction of 1 kg of 70% ethanol extract prepared in the same manner as in 1-1. . Specifically, 4 L of water was added to 1 kg of the powdered extract, and the alcohol extract to which water was added was fractionated with 20 L of n-hexane, the n-hexane fraction was taken separately, and the remaining aqueous layer was fractionated with 20 L of dichloromethane to dichloromethane fraction. Separately, the remaining aqueous layer was fractionated with 40 liters of ethyl acetate, the ethyl acetate fraction was separated separately, and the remaining aqueous layer was fractionated with 40 liters of butanol to obtain a butanol fraction, and the butanol fraction obtained by this procedure was concentrated under reduced pressure, The concentrated solution was lyophilized to obtain 141 g of butanol fraction extract in powdered 70% ethanol extract. In addition, the water layer remaining after the butanol fraction extraction was obtained separately.

2-2. Confirmation of inhibitory effect on enzyme expression related to cell migration and adhesion

Instead of 25 ug / mL, 50 ug / mL, or 100 ug / mL of 70% by volume ethanol extract, 0.5-1 ug / mL, 1 ug / mL, or 2 ug / mL of 2-1. Except for the same operation as in Example 1-2.

As a result, the endometriosis cell is a representative protein expressed in endometriosis and the butanol fraction extract of 70% ethanol extract inhibits the mRNA expression of endogenous MMP-2 and MMP-9 which plays an important role in cell migration and infiltration. It was confirmed in the phosphorus 12Z cells (see Fig. 10, 11).

Figure 10 is a view showing the results of the MMP-2 expression inhibitory effect confirmed by Yonggyu butanol fraction extract, Figure 11 is a road showing the results of the MMP-9 expression inhibitory effect confirmed by Yonggyu butanol fraction extract, Figures 10 and 11 x In the axis, con denotes the control group, 0.5 denotes the 0.5ug / mL administration group of the silica gel butanol fraction extract, and the y-axis of FIG. 10 represents the relative levels of MMP-2 mRNA, and the y-axis of FIG. Relative levels of MMP-9 mRNA are shown. As shown in the figure, it can be seen that the extract of butyol butanol extract effectively inhibits MMP-2 and MMP-9 expression even at a concentration of 0.5 ug / mL.

2-3. Inhibition of enzyme expression related to inflammation and pain

Instead of 25 ug / mL, 50 ug / mL, or 100 ug / mL of 70% ethanol extract, except that 0.5 ug / mL, 1 ug / mL, or 2 ug / mL of 2-1. It carried out similarly to Example 1-3.

As a result, it was confirmed in 12Z cells that are endometriosis cells that the extract of Yong-butanol fraction inhibits the mRNA expression of COX-2, a pain-related characteristic enzyme of endometriosis cells (see FIG. 12).

Figure 12 is a road showing the results of the COX-2 expression inhibitory effect of the extract of the yonggyu butanol fraction extract, in the x-axis of Figure 12 con represents the control group, 0.5 represents the yonggyu butanol fraction extract 0.5ug / mL administration group, y-axis is COX- Relative levels of 2 mRNA (Relative levels of COX-2 mRNA) are shown. As shown in the figure, it can be seen that the extract of butyol butanol extract effectively inhibits COX-2 expression even at a concentration of 0.5 ug / mL.

2-4. Inhibition of inflammatory cytokine expression

Instead of 25 ug / mL, 50 ug / mL, or 100 ug / mL of 70% ethanol extract, except that 0.5 ug / mL, 1 ug / mL, or 2 ug / mL of 2-1. It carried out similarly to Example 1-4.

As a result, it was confirmed in 12Z cells that are endometriosis cells that the extracts of Yonggyu butanol fraction inhibit mRNA expression of inflammatory cytokines TNF-alpha and MCP-1 of endometriosis cells (see FIGS. 13 and 14).

Figure 13 is a view showing the results of the test TNF-alpha expression inhibitory effect of Yonggyu butanol fraction extract, Figure 14 is a road showing the results of MCP-1 expression inhibition test of Yonggyu butanol fraction extract, Figure 13 and 14 x In the axis, con represents the control group, 0.5 represents the 0.5ug / mL administration group of Yonggyu butanol fraction extract, the y axis of FIG. 13 represents the relative levels of TNF-a mRNA, and the y axis of FIG. Relative levels of MCP-1 mRNA are shown. As shown in the figure, it can be seen that the extract of Yonggyu butanol fraction effectively inhibits TNF-alpha and MCP-1 expression even at a concentration of 0.5 ug / mL.

2-5. Confirmation of endometriosis cell adhesion and inhibitory effect

Instead of 50 ug / mL, 100 ug / mL, or 200 ug / mL of 70% ethanol extract, 0.5-1 g / mL, 1 ug / mL, or 2 ug / mL of 2-1. Except for the same procedure as in Example 1-5.

As a result, it was confirmed that endometriosis cells 12Z treated with the extract of Butyol butanol fraction extract inhibited adhesion to Met-5A cells surrounding the outside of the peritoneum and the peritoneal organs (see FIG. 15).

Figure 15 is a road showing the results of confirming the effect of endometriosis cell adhesion and implantation inhibitory effect of the extract of Yonggyu butanol fraction, in the x-axis of Figure 15 is the control group, 1 represents the 1ug / mL administration group of the extract of Yonggyu butanol fraction, y-axis The ratio of Attachment cells (Optical Density (OD) ratio,%) is shown. As shown in the figure, it can be seen that the extract of Yonggyubutanol fraction effectively inhibits cell adhesion even at low concentrations.

From these results, it can be confirmed that the extract of Yonggyu butanol fraction has the effect of treating, improving or preventing endometriosis or its complications.

<Example 3> Confirmation of the treatment, improvement or prevention of endometriosis and complications of Yonggyu fraction extract

3-1. Preparation of Yonggyu fraction extract

The remaining water layer after extracting the normal hexane fraction, dichloromethane fraction, ethyl acetate fraction, and butanol fraction, obtained by the same method as the fraction extraction method of 1-2. Hexane fraction extract, dichloromethane fraction extract, ethyl acetate fraction extract, and water layer extract were obtained in powder form 105g, 16g, 36g, 477g, respectively.

3-2. Confirmation of inhibitory effect on enzyme expression related to cell migration and adhesion

In the same manner as in Example 2-2, except that the fraction extract described in Table 6 or Table 7 was used instead of the extract of Butyol fraction.

The results are shown in Table 6 and Table 7, Table 6 is the results for MMP-2, Table 7 shows the results for MMP-9.

TABLE 6

TABLE 7

As a result, it can be seen that each fraction extract inhibits MMP-2 and / or MMP-9 expression.

3-3. Inhibition of enzyme expression related to inflammation and pain

The same procedure as in Example 2-3 was carried out, except that the fraction extract of Table 8 was used instead of the extract of Butyol fraction.

The results are shown in Table 8. As a result, it can be seen that each fraction extract inhibits the mRNA expression of COX-2, which is a characteristic enzyme related to pain in endometriosis cells.

TABLE 8

3-4. Inhibition of inflammatory cytokine expression

The same procedure as in Example 2-4. Was carried out except that the fraction extract of Table 9 or Table 10 was used instead of the extract of Butyol fraction.

The results are shown in Table 9 and Table 10, Table 9 is the results for TNF-alpha, the results for MCP-1 in Table 10. As a result, it can be seen that each fraction extract inhibits the mRNA expression of the inflammation-related cytokines TNF-alpha and MCP-1 of endometriosis cells.

TABLE 9

TABLE 10

From these results, it can be confirmed that Yonggyu fraction extract is effective in the treatment, improvement or prevention of endometriosis and complications.

Example 4 Confirmation of the Treatment, Improvement or Prevention of Endometriosis and Complications of Yonggyu Water Extract

4-1. Preparation of Yonggyu Water Extract

Dried Yonggyu (Solanum nigrum, production area: Yeongcheon, Gyeongsangbuk-do) was purchased at Gyeongdong Market (South Korea) and pulverized. 1 liter of distilled water was added to 500 g of the prepared silicon broth and extracted using a water bath for 3 hours. The extract was filtered using filter paper and lyophilized for 48 hours to obtain a powdered red water extract.

4-2. Confirmation of inhibitory effect on enzyme expression related to cell migration and adhesion

In the same manner as in Example 1-2, except that 100 ug / mL of the water extract of 4-1. Was used instead of 25 ug / mL, 50 ug / mL, or 100 ug / mL of the 70% by volume ethanol extract. Was carried out.

As a result, it was confirmed in the 12Z cells, endometriosis cells, that Yong-Su extract inhibited the mRNA expression of endogenous MMP-2, which is a representative protein expressed in endometriosis and plays an important role in cell migration and infiltration (see FIG. 16). ).

Figure 16 is a road showing the results of the MMP-2 expression inhibitory effect of Yonggyuk water extract, in the x-axis of Figure 16 is the control group, 100ug / ml is 100ug / mL administration group of the water-based water extract, y-axis is MMP-2 Relative levels of MMP-2 mRNA are shown. As shown in the figure, it can be seen that yonggyu water extract effectively inhibits MMP-2 expression.

4-3. Inhibition of enzyme expression related to inflammation and pain

In the same manner as in Example 1-3, except that 100 ug / mL of the water extract of 4-1. Was used instead of 25 ug / mL, 50 ug / mL, or 100 ug / mL of the 70% ethanol extract. It was.

As a result, it was confirmed in the 12Z cells as endometriosis cells that Yong-Su Water Extract inhibited the mRNA expression of COX-2, a pain-related characteristic enzyme of endometriosis cells (see FIG. 17).

Figure 17 is a road showing the results of the inhibitory effect of COX-2 expression of Yonggyu water extract, on the x-axis of Figure 17 con represents a control, 100ug / ml represents a 100ug / mL administration group, y-axis is COX-2 Relative levels of COX-2 mRNA are shown. As shown in the figure, it can be seen that the yonggyu water extract effectively inhibits COX-2 expression.

4-4. Inhibition of inflammatory cytokine expression

In the same manner as in Example 1-4, except that 100 ug / mL of the water extract of 4-1. Was used instead of 25 ug / mL, 50 ug / mL, or 100 ug / mL of the 70% ethanol extract. It was.

As a result, it was confirmed in the 12Z cells that are endometriosis cells that the yonggyu water extract inhibits the mRNA expression of inflammatory endokines TNF-alpha and MCP-1 of endometriosis cells (see Figs. 18 and 19).

18 is a view showing the results of the TNF-alpha expression inhibitory effect confirmed by Yonggyu water extract, Figure 19 is a road showing the results of MCP-1 expression inhibitory effect confirmed by Yonggyu water extract, in the x-axis of Figure 18 and 19 con represents a control group, 100ug / ml represents a 100ug / mL administration group yonggyu water extract, y-axis in Figure 18 represents the relative levels of TNF-alpha mRNA, y-axis in Figure 19 is MCP- Relative levels of 1 MCP-1 mRNA. As shown in the figure, it can be seen that the water extract effectively inhibits TNF-alpha and MCP-1 expression.

From these results, it can be confirmed that the yonggyu water extract is effective in the treatment, improvement or prevention of endometriosis and complications.

Thus, Yonggyu extract is endometriosis or its complications by inhibiting cell migration and adhesion related enzyme expression, inhibiting inflammation and pain related enzyme expression, inhibiting inflammatory cytokine expression, endometriosis cell migration, endometriosis cell adhesion and / or implantation inhibition, etc. It can be seen that there is a treatment, improvement and / or prophylactic effect of.

Preparation Example 1 Preparation of Pharmaceutical Composition

A capsule was prepared by filling a gelatin capsule with 300 mg of yonggyu extract, corn starch 100 mg, lactose 100 mg, and magnesium stearate 2 mg prepared in the same manner as in any of the methods described in Examples 1-1 to 4-1.

Preparation Example 2 Preparation of Food Composition

Yonggyu extract (4% by weight), liquid fructose (0.5% by weight), oligosaccharide (2% by weight), sugar (2% by weight) prepared in the same manner as in any of the methods described in Examples 1-1 to 4-1. ), And salt (0.5% by weight) was added water to adjust the balance, and then homogeneously blended to instant sterilization to prepare a healthy beverage.

Since the present invention can effectively treat, ameliorate and / or prevent endometriosis or complications thereof, there is industrial applicability.

Claims (14)

Pharmaceutical composition for the treatment or prevention of endometriosis or its complications, comprising the extract of Yonggyu as an active ingredient. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable additive, the pharmaceutical composition comprising the extract and the additive. The pharmaceutical composition of claim 1, wherein the endometriosis or complications thereof are due to one or more overexpression selected from MMP-2, MMP-9, COX-2, TNFalpha, or MCP-1. The pharmaceutical composition of claim 1, wherein the complication is at least one selected from the group consisting of pelvic inflammatory disease, pelvic adhesion, ovarian cyst, uterine myoma, ectopic pregnancy and infertility. The pharmaceutical composition of claim 1, wherein the Yonggyu extract is a solvent extract obtained by extracting the Yonggyu, as a solvent, wherein the solvent is at least one selected from water, alcohol, or a mixture thereof. The pharmaceutical composition of claim 5, wherein the solvent is 70 volume% ethanol. The pharmaceutical composition of claim 1, wherein the extract of Yonggyu is a butanol fraction extract obtained by fractionating 70% by volume of ethanol extracted with 70% by volume of ethanol with butanol. The method of claim 7, wherein The butanol fraction extract is (A) preparing a 70% by volume ethanol extract extracted with 70% by volume ethanol; (B) adding water to the 70% by volume ethanol extract of step (A); (C) fractionating 70 vol% ethanol extract added with water of step (B) with n-hexane; (D) removing the n-hexane fraction of step (C) and fraction-extracting the remaining aqueous layer with dichloromethane; (E) removing the dichloromethane fraction of step (D) and fractionating the remaining aqueous layer with ethyl acetate; And (F) The pharmaceutical composition as a fraction extract obtained by removing the ethyl acetate fraction of step (E) and fractional extraction of the remaining aqueous layer with butanol. The pharmaceutical composition of claim 1, wherein the treatment or prevention is by inhibiting adhesion or migration of endometriosis cells. The pharmaceutical composition of claim 9, wherein the inhibition of adhesion or migration is by inhibition of expression of one or more selected from MMP-2 or MMP-9. The pharmaceutical composition of claim 1, wherein the treatment or prevention is by at least one selected from inhibition of inflammation and pain related enzyme expression or inhibition of inflammatory cytokine expression. Food composition for endometriosis or its complications, or prevention thereof, comprising the extract of Yonggyu as an active ingredient. Use for the preparation of a formulation for the treatment or prevention of endometriosis or complications thereof. A method of treating, or preventing, endometriosis or a complication thereof, comprising administering a extract of a yonggyu to a mammal, including a human, in need thereof.

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