KR20200003772A - Composition for preventing or treating ischemic diseases comprising extract of Tozan as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating ischemic diseases comprising an extract of Cuscuta japonica seeds as an active ingredient and, more specifically, to a health functional food composition, a pharmaceutical composition and a quasi-drug composition for preventing or alleviating ischemic diseases comprising an extract of Cuscuta japonica seeds or fractions thereof as an active ingredient, or a method for treating ischemic diseases using the pharmaceutical composition. The extract of Cuscuta japonica seeds according to the present invention is effective in improving blood stream of an ischemic animal model, promoting wound healing, reducing inflammation and promoting angiogenesis for the composition of the present invention to be usefully used for preventing, alleviating or treating ischemic diseases.

Description

Composition for preventing or treating ischemic diseases comprising extract of Tozan as an active ingredient

The present invention relates to a composition for the prevention or treatment of ischemic diseases comprising the earth and sand extract as an active ingredient, specifically, a health functional food composition for the prevention or improvement of the ischemic disease comprising the earth and sand extract or fractions thereof as an active ingredient; Pharmaceutical compositions; Quasi-drug compositions; Or to a method for treating ischemic disease using the pharmaceutical composition.

Ischemic disease is a blood circulation disorder caused by decreased blood inflow, and is caused by various factors such as aging, trauma, complications, menopause, and the like. Hematogenous disorders cause aerobic metabolic disorders, reduced ATP production, and eventually lead to dysfunction or necrosis of cells, which can lead to permanent damage to tissues when a series of processes called ischemic shock cascades are triggered. The method is being developed.

For example, a composition for the prevention and treatment of ischemic diseases containing nogin as an active ingredient (Korean Patent Publication No. 10-2016-0086193), a composition for the prevention or treatment of ischemic diseases including liposomes carrying VEGF-derived peptides (Korean Patent Publication No. 10-2016-0141068), a composition for treating or preventing ischemic diseases including lycopene (Korean Patent Publication No. 10-1293882) and the like has been developed.

On the other hand, Tosa is a seed of Saengsam, also called Saesam Seed or Geumsacho. In Dongbogam, the Sasa has been shown to improve vibrancy, revitalize, and have an effect on back pain and diabetes. In addition, the effects of the earth and sand, such as improving the strength of the kidneys, strengthening the kidneys, strengthening the bone function, diuretic effect, tongue effect, night blindness improvement, etc., is widely used as a herbal medicine related to the disease. However, there is no known therapeutic effect on the ischemic disease of the soil dead.

Against this background, the present inventors have made diligent research efforts to develop a substance for treating ischemic disease. As a result, we have found that Tosa extract improves lower limb blood flow, promotes wound healing and reduces inflammation in animal models in which ovaries are excised and lower limb ischemia is developed. By promoting angiogenesis and confirming that the ischemic disease can be improved / treated, the present invention was completed.

One object of the present invention is to provide a dietary supplement for the prevention or improvement of ischemic diseases comprising the earth and sand extract or fractions thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of ischemic disease comprising the earth and sand extract or fractions thereof as an active ingredient.

Yet another object of the present invention is to provide a method of treating ischemic disease comprising administering the pharmaceutical composition to a subject.

Another object of the present invention to provide a quasi-drug composition for the prevention or improvement of ischemic disease comprising the earth and sand extract or fractions thereof as an active ingredient.

In order to achieve the above object, one embodiment of the present invention provides a nutraceutical composition for the prevention or improvement of ischemic diseases comprising the earth and sand extract or fractions thereof as an active ingredient.

In the present invention, the tosa extract extracts the ovaries are excised and the blood vessels of the lower limbs ligation to improve the blood flow of the lower limbs in the animal model caused ischemic disease, promote wound healing, reduce inflammation and promote angiogenesis, thereby preventing ischemic disease, It has been confirmed that it can be usefully used to improve or treat.

As used herein, the term "earth and sand" refers to the seed of Cuscuta japonica , a perennial vine plant, belonging to the family Asteraceae. Tosa is known as a medicine that mainly protects the liver and kidneys, brightens the eyes, helps yang, and strengthens the kidney function. However, no effect on ischemic disease is known and was first identified by the inventors. In the present invention, the earth and sand may be purchased commercially, or may be used collected or grown in nature.

As used herein, the term "extract" refers to an extract, such as an extract obtained by extracting and treating the earth and sand, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a modifier or purified product of the extract, or a mixture thereof. And extracts of all formulations that can be formed using extracts.

The method of extracting the earth and sand is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method, hot water extraction method, ultrasonic extraction method, filtration method, reflux extraction method, and the like, these may be performed alone or in combination of two or more methods.

In the present invention, the kind of extraction solvent used to extract the earth and sand is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent may include water, alcohols having 1 to 4 carbon atoms or mixed solvents thereof, and these may be used alone or in combination of one or more thereof.

Specifically, the extraction solvent may be water or ethanol, but is not limited thereto.

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

In the present invention, the fractionation method for obtaining the fraction is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed through an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatography (size, charge, hydrophobicity). Or chromatographed fractions), and combinations thereof, which are prepared for separation according to affinity. Specifically, a method of obtaining a fraction from the extract by treating a predetermined solvent with an extract obtained by extracting the earth and sand of the present invention.

The kind of the fractionation solvent used to obtain the fraction in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvents include polar solvents such as water and alcohols having 1 to 4 carbon atoms; Nonpolar solvents such as hexane and ethyl acetate; Or a mixed solvent thereof. These may be used alone or in combination of one or more, but are not limited thereto.

In addition, the extract or fraction may be prepared and used in the form of a dry powder after extraction, but is not limited thereto.

As used herein, the term “ischemic disease” refers to a disease that is symptomatic by the stopping of the blood stream (ischemia), which ultimately involves irreversible damage to cells and tissues. By stopping blood flow, the amount of oxygen supplied to tissues or cells is reduced, which causes cell death, thereby degrading the function of tissues and organs. Thus, ischemic diseases are generally treated with mechanisms that increase the flow of blood flow or regenerate / angine blood vessels. The ischemic disease includes various sub-conditions, and specifically, cerebral ischemia, cardiac ischemia, diabetic vascular heart disease, heart failure, myocardial hypertrophy, retinal ischemia, ischemic colitis, ischemic acute renal failure, stroke, brain trauma, neonatal hypoxia, and lower extremity vessels. It may be an ischemic disease or an extremity ischemic disease, and more specifically, it may be a lower limb vascular ischemic disease or an extremity ischemic disease, but is not limited thereto.

In addition, the ischemic disease may be caused by menopause.

The term "menopausal" is also referred to as the period of menopause or menopause, which is manifested by the loss of female reproductive function. In menopause, the function of the ovary decreases due to aging, resulting in fewer female hormones, resulting in hormonal imbalance, which causes various abnormal symptoms. Typically, glucose or lipid metabolism disorders such as obesity due to menopause; Bone homeostasis disorders such as osteoporosis; Energy metabolic disorders such as flushing; Or blood flow disorders such as ischemic diseases.

For the purpose of the present invention, the Tosa extract may be to induce the migration or proliferation of vascular endothelial cells, to promote the formation of vascular endothelial cells, or to increase blood flow.

Inducing / promoting the movement or proliferation of vascular endothelial cells, the formation of blood vessels of vascular endothelial cells, an increase in blood flow, etc. is a mechanism that is actively used in the treatment of ischemic diseases. Tosaja extract exhibiting such effects is useful for the treatment of ischemic diseases. Can be used.

In one specific embodiment of the present invention, for animal models in which the ovaries are excised and the blood vessels of the lower extremities are ligation resulting in ischemic disease, the extract of Tosa extract improves blood flow in the lower extremities, promotes the production of peripheral blood vessels, and promotes wound healing-related cytology. It was found to increase the expression of caine, decrease the expression of inflammatory cytokines, and promote vascular endothelial cell migration and tube formation (FIGS. 1-6).

This suggests that the composition of the present invention comprising the above-mentioned tosa extract can be usefully used for the prevention, improvement or treatment of ischemic diseases.

The term "functional food" of the present invention is the same term as a food for special health use (FOSHU), and a medicine and a medical effect processed to efficiently display a bioregulatory function in addition to nutrition. Means high food. In the present invention, the health functional food is compatible with health food or health supplement food.

The health functional food composition of the present invention can be ingested on a daily basis, and unlike general medicines, there is no side effect that can occur when taking long-term use of natural medicines as raw materials, for the purpose of preventing or improving ischemic disease. It can be very useful.

As used herein, the term "prevention" means any action of inhibiting or delaying symptoms by administration of a composition comprising the above-mentioned tosa extract or a fraction thereof.

As used herein, the term " improvement " means any action that reduces a parameter, such as the extent of symptoms, associated with the condition treated with the administration of the food composition.

The health functional food may be prepared to be formulated to be selected from the group consisting of pills, powders, granules, acupuncture, tablets, capsules and beverages in order to obtain a useful effect in the prevention or improvement of ischemic diseases. There is no restriction | limiting in particular in the kind of food which can add the composition containing the earth and sand extract of this invention, or its fraction, For example, there are various drinks, gum, tea, a vitamin complex, a dietary supplement, etc.

The health functional food composition may add other ingredients that do not interfere with the preventive or improving effect of the health functional food, the type is not particularly limited. For example, various herbal extracts, food acceptable additives, natural carbohydrates, and the like may be contained as additional ingredients, such as conventional foods.

In addition, the health functional food composition may further comprise a food acceptable carrier, which can be used by those skilled in the art as appropriate. For example, various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters , Stabilizers, preservatives, glycerin, alcohols, carbonation agents used in the carbonated beverage, and the like, but the type is not limited by the above examples.

In addition, the health functional food composition may include additional ingredients that are commonly used in food compositions to improve the smell, taste, time and the like. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu). It may also contain amino acids such as lysine, tryptophan, cysteine, valine and the like.

In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), fungicides (bleaching powder and highly bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisol (BHA), butylhydroxytoluene ( BHT), etc.), colorants (such as tar pigments), colorants (such as sodium nitrite, sodium nitrite), bleach (sodium sulfite), seasonings (such as MSG glutamate), sweeteners (ducin, cyclate, saccharin, sodium, etc.) Food additives such as flavors (vanillin, lactones, etc.), swelling agents (alum, potassium D-tartrate, etc.), reinforcing agents, emulsifiers, thickeners (pigments), coatings, gum herbicides, foam inhibitors, solvents, improvers, etc. ) Can be added.

Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of ischemic disease comprising the earth and sand extract or fractions thereof as an active ingredient.

At this time, the definition of "soil," "extract", "fraction", "ischemic disease" and "prevention" are as described above.

As used herein, the term "treatment" refers to any action in which pain is alleviated or ameliorated or beneficially altered by administration of a composition comprising the earth and sand extract.

The pharmaceutical composition of the present invention may include 0.001 to 80, specifically 0.001 to 70, more specifically 0.001 to 60% by weight based on the total weight of the composition of the earth and sand extract or fractions thereof, but is not limited thereto.

In addition, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition, the carrier may comprise a non-naturally occuring carrier (carrier) have.

Specific examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used, but is not limited thereto.

In addition, the pharmaceutical composition may be a tablet, a pill, a powder, a granule, a capsule, a suspension, a solution, an emulsion, a syrup, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilizer and a suppository, respectively, according to a conventional method. It may have any one formulation selected from the group consisting of, and the formulation may be in various forms, oral or parenteral. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used, but is not limited thereto.

As a specific example, a solid preparation for oral administration may be used in tablets, pills, powders, granules, capsules, etc., the solid preparation may be at least one excipient, for example, starch, calcium carbonate, sucrose (lactose) or lactose ( lactose), gelatin and the like can be used. In addition, a lubricant such as magnesium stearate, talc, and the like may be used in addition to the simple excipient, but is not limited thereto.

In addition, as a liquid preparation for oral administration, suspending agents, liquid solutions, emulsions, syrups, etc. may be used. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, and preservatives, may be used. And the like can be used. For parenteral administration, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations or suppositories may be used. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. may be used, but is not limited thereto.

In addition, as a base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. may be used, but is not limited thereto.

Another aspect of the invention provides a method of treating ischemic disease comprising administering the pharmaceutical composition to a subject.

At this time, the definition of "ischemic disease" and "treatment" is as described above.

As used herein, the term “administration” means introducing a composition comprising said earthworm extract or fractions thereof into a subject in a suitable manner.

As used herein, the term "individual" means all animals, including rats, mice, domestic animals, and the like, including humans, in which ischemic diseases may be caused or may be caused.

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

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type and severity of the subject, severity, age, sex, activity of the drug, Sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In addition, single or multiple administrations can be made. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

In addition, the pharmaceutical composition may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally, or topically) according to a desired method, and the dosage is based on the condition and weight of the patient, and the degree of disease. Depending on the drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

As a specific example, the pharmaceutical composition may generally be administered in an amount of 0.001 to 1000 mg / kg, more specifically 0.05 to 200 mg / kg, most specifically 0.1 to 100 mg / kg once a day or several times. Preferred dosages may be appropriately selected by those skilled in the art depending on the condition and weight of the individual, the severity of the disease, the form of the drug, the route of administration and the duration.

Another aspect of the present invention provides a quasi-drug composition for the prevention or improvement of ischemic disease comprising the earth and sand extract or fractions thereof as an active ingredient.

As used herein, the term "quasi drug" refers to articles that have a lesser action than pharmaceuticals among articles used for the purpose of diagnosing, treating, ameliorating, alleviating, treating, or preventing a disease of a human or animal. For example, quasi-drug products under the Pharmaceutical Affairs Law exclude products used for the purpose of medicines, and include products used for the treatment or prevention of diseases of humans / animals, and products with slight or no direct action on the human body.

When the earth and sand extract according to the present invention or a fraction thereof is used as an quasi-drug additive, the composition may be added as it is, or used with other quasi-drug or quasi-drug components, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).

In addition, the quasi-drug composition is not particularly limited, but may be prepared in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels or gels.

Tosa extract according to the present invention exhibits the effect of improving the blood flow of the ischemic animal model, promote wound healing, reduce inflammation and promote angiogenesis, the composition of the present invention comprising the same for the prevention, improvement or treatment of ischemic diseases It can be usefully used.

1 is a graph showing the blood flow improvement effect in the lower limb ischemia model of the earth and sand extract, and relates to the ischemia / non-ischemia ratio for the blood flow. At this time, OH is the ischemic animal model is not administered tosa extract, OH + tosa 150 is the ischemic animal model is administered 150mg / kg tosa extract, OH + Tosa 450 is the ischemic animal model administered 450mg / kg tosa extract OVX means ovarian resection and HLI means limb ischemic ligation. * Is also P <0.05: vs. -7d, a is P <0.05: OH vs. OH + Tosa 150, b is P <0.05: OH vs. OH + tosa 450, and c is P <0.05: OH + tosa 150 vs. OH + Tosa 450 is shown.
Figure 2 is a graph showing the peripheral blood vessel production promoting effect of the Tosa extract, it relates to the density of peripheral blood vessels. At this time, OH is the ischemic animal model is not administered tosa extract, OH + tosa 150 is the ischemic animal model is administered 150mg / kg tosa extract, OH + Tosa 450 is the ischemic animal model administered 450mg / kg tosa extract Means. Also, a is P <0.05: vs. OH, and b are P <0.05: OH vs. OH + Tosa 150 is shown.
Figure 3 is an image and graph showing the effect of increased expression of wound healing-related cytokines (CXCL12, CD54) of Tosa extract, relates to the cytokine array results. At this time, the vehicle (vehicle) is the ischemic animal model (control group) is not administered tosa extract, the tosaja 150 is the ischemic animal model is administered 150mg / kg tosaja extract, the Tosa 450 is administered 450mg / kg tosaja extract Mean ischemic animal model. Also, a is P <0.05: vs. Vehicle, and b was P <0.05: vs. Tosa 150 is shown.
4 is a graph showing the effect of reducing the expression of inflammatory cytokines (TNF-α, IL-6, IL-1b) of Tosa extract. At this time, the vehicle is the ischemic animal model (control group) that is not administered tosa extract, the tosaja 150 ischaemia animal model is administered 150mg / kg tosaja extract, the tosaja 450 is ischaemia is administered 450mg / kg tosasa extract Means an animal model. Also, a is P <0.05: vs. Vehicle, and b was P <0.05: vs. Tosa 150 is shown.
5 is a graph showing the vascular endothelial cell migration promoting effect of the Tosa extract, relates to the results of the migration assay (migration assay). At this time, Con is a control compound / extract not treated vascular endothelial cells, VEGF 50 ng / ㎖ is a positive control group 1, E ₂ 1 nM treated with VEGF 50 ng / ㎖ vascular endothelial cells E ₂ 1 nM This means the treated positive control group 2. In addition, a is P <0.01, and b is P <0.001: vs. Control group.
6 is a graph showing the effect of promoting the vascular endothelial tube formation of the earth and sand extract. In this case, Con is a control group that is not treated with compound / extract in vascular endothelial cells, VEGF 50 ng / ml is positive control group 1 treated with VEGF 50 ng / ml in vascular endothelial cells, Velbe 1 pM is 1 pM of vinblastine ( Vinblastine) treated positive control group 2, E ₂ 1 nM refers to the positive control group 3 treated with E ₂ 1 nM in vascular endothelial cells. In addition, a is P <0.05, and b is P <0.01: vs. Control group.

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

Example 1 Preparation of Tosa Extract

In order to prepare the earth and sand extract, 10 times the water or ethanol of the earth and sand was added, and reflux extraction treatment was carried out to obtain the earth and sand extract. The tosa extract No. After filtering with 2 filter paper (0.8 μm) filter, tosa extract was prepared by concentration and lyophilization using a rotary evaporator (rotary evaporator).

Example 2. Confirmation of the blood flow improvement effect of the Tosa extract

In order to confirm the improvement / treatment effect of Tosa extract on ischemic disease, the effect of improving blood flow in the lower limb ischemia model of Tosa extract was confirmed.

Specifically, ovariectomy (OVX) was performed at 7 weeks of age after receiving a 6-week-old female C57BL / 6 mouse. Thereafter, the ischemic model was completed by ligating the blood vessels (artery, vein) of the left hind-limb (HLI) area at 8 weeks of age for the ovarian-resected mice. This experiment was reviewed by the Korean Institute of Oriental Medicine, Deliberation of Experimental Animal Ethics (17-028), and the breeding and maintenance of experimental animal was performed at the temperature of 23 ± 1 ℃ and humidity of 50 ± in accordance with the animal care and use regulations of the Korean Institute of Oriental Medicine. The animals were housed in individually ventilated cages (IVCs) in a constantly maintained nursery with a 12-hour contrast cycle. Tosa were diluted in 0.1% CMC (Carboxymethyl cellulose) and administered orally in the morning at a concentration of 150 mg / kg or 450 mg / kg, respectively. After the establishment of the ischemic model, blood flow in the lower extremity was measured using a laser Doppler imaging device for 14 days, and the ischemic / non-ischemic ratio was compared with that of the right lower extremity, a simulated surgical control. Blood flow rate was calculated.

As a result, as shown in Figure 1, the blood flow rate immediately after the lower limb ischemia (blood flow rate) was confirmed that significantly reduced in all control and experimental groups compared to before the induction of lower limb ischemia (-7 days). On the other hand, in the experimental group treated with low to high concentrations of Tosa extract, it was confirmed that blood flow increased significantly after 14 days of induction of lower limb ischemia.

Through the above results, it was found that the Tosa extract significantly increases the blood flow reduced by ischemia of the lower limb, and thus may be useful for preventing, improving or treating ischemic disease.

Example 3 Confirmation of Peripheral Blood Vessel Promoting Effect of Tosa Extract

In order to confirm the improvement / treatment effect of Tosa extract on ischemic disease, the effect of promoting the peripheral blood vessel generation in the ischemic model of tosa extract was confirmed.

Specifically, immunohistochemical staining was performed on the thigh and calf muscles of the left lower extremity in which lower limb ischemia was induced using CD31 (cluster of differentiation 31) used as a biomarker of vascular endothelial cells. The tissue was cut into paraffin sections to a thickness of 5 μm and deparaffinized using xylene. At high concentrations, low concentrations of ethanol were used after finally hydrating in tap water. Abcam's anti-CD31 (rabbit polyclonal to CD31) was used as the antibody, and the number of unit areas (mm ² ) was counted by counting the number of peripheral blood vessels (capillary) based on the final stained positive cells (brown color). Converted to.

As a result, as can be seen in Figure 2, when treated with sesame seed extract was significantly increased peripheral blood vessel formation of the resected lower limb ischemia model, it was confirmed that the concentration-dependent increase.

Through the above results, it was found that the Tosa extract significantly increases the production of peripheral blood vessels, and thus may be useful for preventing, improving or treating ischemic disease.

Example 4 Confirmation of Increasing Effect of Wound Healing Cytokine Expression in Tosa Extract

In order to confirm the improvement / treatment effect of Tosa extract on ischemic disease, the effect of increasing the expression of cytokines related to wound healing of Tosa extract was confirmed.

Specifically, Tosa extract was administered to the animal model subjected to lower limb ischemia after ovariectomy for 3 weeks at 150 mg / kg / day and 450 mg / kg / day, respectively, and cytokine expression levels of CXCL12 and CD54 were measured for the serum obtained from the animal model. It was confirmed. R & D SYSTEMS kit (Proteome Profiler, Mouse Cytokine Array Panel A, ARY006) was used, and after the serum was dispensed, the expression changes of each cytokine were confirmed through the density of the spots.

As a result, as shown in Figure 3, when treated with Tosa extract, the amount of CXCL12, CD54 cytokine expression of the resected lower limb ischemia model significantly increased, which was confirmed to increase in a concentration-dependent manner.

Through the above results, it was found that the Tosa extract increases the expression of cytokines involved in wound healing, and thus may be useful for preventing, improving or treating ischemic disease.

Example 5. Confirmation of Inflammatory Cytokine Expression Reduction Effect of Tosa Extract

In order to confirm the improvement / treatment effect of Tosa extract on ischemic disease, the effect of increasing the expression of inflammatory cytokines of Tosa extract was confirmed.

Specifically, Tosa extract was administered to the animal model subjected to lower limb ischemia for 3 weeks at 150 mg / kg / day and 450 mg / kg / day after ovarian resection, and the lower leg thigh and calf muscle of the left lower limb where the ischemia was induced in the animal model. Total RNA was extracted from the affected area of. Using 10 g of cDNA synthesized by reverse transcription of the RNA as a template, cytokines (TNF-α, IL-6, IL-1b) related to the inflammatory response were amplified by PCR.

As a result, as shown in Figure 4, when treated with Tosa extract, the amount of TNF-α, IL-6, IL-1b cytokine expression of the ovarian excised lower limb ischemia model is significantly reduced, which is concentration-dependently reduced It was confirmed.

From the results, it was found that the Tosa extract can be useful for the prevention, improvement or treatment of ischemic diseases because it reduces the expression of cytokines causing inflammation.

Example 6. Confirmation of vascular endothelial cell migration promoting effect

In order to confirm the improvement / treatment effect of Tosa extract against ischemic disease, the effect of promoting the vascular endothelial cell migration was confirmed.

Specifically, 3 × 10 ³ cells of vascular endothelial cells (HUVAC) were dispensed into the upper wells of the transwell, and the seedling extracts were added to the serum free EGM2 culture medium. Cells were incubated for 6 hours by addition of 10 and 100 μg / ml. At this time, the control group was set as a cell not treated with the compound or extract, the positive control group 1 treated with 50 ng / ml VEGF, the positive control group 2 treated with 1 nM E ₂ cells. The migrated vascular endothelial cells were stained by H & E staining to compare the effect of Tosa extract of the present invention on vascular endothelial cell migration.

As a result, as shown in Figure 5, it was confirmed that when treated with tosa extract is significantly promoted the movement of vascular endothelial cells compared to the control group, which was confirmed that the concentration-dependent increase. In addition, it was confirmed that when treated with 100 ㎍ / ㎖ of Tosa extract showed a similar effect to the treatment of the positive control group E ₂ .

Through the above results, it was found that the tosa extract may be useful for the prevention, improvement or treatment of ischemic diseases because it promotes angiogenesis by promoting the movement of vascular endothelial cells.

Example 7 Confirmation of Tube Formation Promoting Effect of Tosa Extract

In order to confirm the improvement / treatment effect of Tosa extract on ischemic disease, the effect of promoting the formation of tuberculosis according to the tube formation promoting effect of Tosa extract was confirmed.

Specifically, vascular endothelial cells (HUVAC) were dispensed in a number of 3 × 10 ⁵ into a 24-well plate coated with Matrigel using an extracellular matrix (ECM). Then, tube formation of vascular endothelial cells was induced at 37 ° C. for 24 hours using EGM2 medium containing 1% FBS (fetal bovine serum). After 24 hours, the microscope was observed and photographed, and tube formation ability was measured using angiogenesis analyzer of Image J program and compared with the control group. In this case, as a control, cells not treated with the compound or extract, cells treated with 50 ng / ml VEGF as positive control group 1, cells treated with 1 pM Vinblastine (Velbeine) as positive control group 2, and Positive control group 3 was set to cells treated with 1 nM of E ₂ .

As a result, as shown in Figure 6, it was confirmed that when treated with Tosa extract significantly increased the tube formation degree of vascular endothelial cells compared to the control group was not treated, it was confirmed that the concentration-dependent increase. In addition, the tube formation-promoting effect of the earth and sand extract as described above is superior to vinblastine even at a low concentration of 1 ㎍ / ㎖, and when treated with 100 ㎍ / ㎖ showed a similar effect to the treatment of the positive control E ₂ It was confirmed.

Through the above results, it was found that the Tosa extract can be useful for the prevention, improvement or treatment of ischemic diseases because it promotes angiogenesis by promoting tube formation of vascular endothelial cells.

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

Claims (9)

Health functional food composition for the treatment, prevention or improvement of ischemic disease comprising the earth and sand extract or fractions thereof as an active ingredient.
The dietary supplement composition of claim 1, wherein the extract is prepared by extracting the earth and sand with one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.
The method of claim 1, wherein the fraction is prepared by fractionating the earth and sand extract with a solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, hexane (Hexane), ethyl acetate (Ethyl acetate) and a mixed solvent thereof That, health functional food composition.
The health functional food composition of claim 1, wherein the ischemic disease is a lower limb vascular ischemic disease or an extremity ischemic disease.
The dietary supplement composition of claim 1, wherein the ischemic disease is caused by menopause.
The dietary supplement composition of claim 1, wherein the tosa extract extracts induces the migration or proliferation of vascular endothelial cells, promotes the formation of vascular endothelial cells, or increases blood flow.
Pharmaceutical composition for the prevention or treatment of ischemic diseases comprising the earth and sand extract or fractions thereof as an active ingredient.
A method of treating ischemic disease, comprising administering the pharmaceutical composition of claim 7 to a subject other than a human.
Quasi-drug composition for the prevention or improvement of ischemic disease comprising the earth and sand extract or fractions thereof as an active ingredient.

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