KR102028334B1 - Composition for preventing, improving or treating angiogenesis-dependent disorder comprising extract of medicinal herbal mixture as effective component - Google Patents

Abstract

The present invention relates to a composition for the prevention, improvement or treatment of angiogenic dependent diseases comprising a mixture of herbal extracts as an active ingredient, and in detail, a mixed extract of peony, cheonggung, taxa, tangui, bokyeong and baekchul as an active ingredient. It relates to a composition for preventing, ameliorating or treating angiogenic dependent diseases. The active ingredient of the present invention promotes estradiol, a female hormone reduced by menopause, and in particular, promotes the production and migration of vascular endothelial cells. Therefore, it can be utilized as a pharmaceutical composition or health functional food composition for the prevention, improvement or treatment of angiogenic dependent diseases.

Description

Composition for preventing, improving or treating angiogenesis-dependent disorder comprising extract of medicinal herbal mixture as effective component

The present invention relates to a composition for the prevention, improvement or treatment of angiogenic dependent diseases comprising a herbal extract as an active ingredient.

Angiogenesis refers to the process by which new blood vessels are formed, which rarely occurs in normal living conditions, but is an essential process in embryogenesis, corpus luteum formation or wound healing. The angiogenesis process is generally caused by stimulation of angiogenesis factors, resulting in the formation of lumen due to the degradation of the basal membrane, proliferation, proliferation, and differentiation of vascular endothelial cells due to proteases. Consists of being generated. Angiogenesis processes are known to be tightly controlled by several types of promoters and inhibitors, including growth factors, cytokines, lipid metabolites and latent fragments of hemostatic proteins.

On the other hand, menopausal (climacteric) is a type of endocrine syndrome that refers to a transition period in which the gradual and gradual decrease in ovarian function causes a decrease or loss in physiological and sexual function. Menopause comes. During menopause, acute symptoms may occur due to hormonal changes such as decreased estrogen production, elevated follicular stimulating hormone (FSH) and luteininzing hormone (LH). That is, as an initial symptom, vasomotor symptom such as hot flush and night sweat and ischemic disease due to menopause may appear. As a result, menopausal syndrome causes numbness of the hands and feet, and causes local anemia.

Angelicae gigantis Radix, on the other hand, uses the roots of Angelica gigas Nakai. In China, Angelica sinensis (Oliv.) Diels (中國 當歸) is used, and in Japan, Angelica acutiloba (Sieb. & Zuc.) Kitagawa is used. Angelica is mainly contained coumarin decursin (decursin), decursinol (decursinol), decursinol angelate (decursinol angelate) and the like. Decusin and decosinol have sedation, analgesic, myrrhosis, anti-tumor, and brain cell protection, and are known to be effective in treating blood vessels due to platelet aggregation inhibition. In oriental medicine, Angelica is known to be used for anemia, gynecology, and postpartum recovery of pregnant women. In addition, it is known to be effective for severe cough and swelling. In addition, Dongguk not only strengthens the uterus, but also regulates menstruation to remove wastes such as fish and blood, improves coldness and poor color, helps recovery from postpartum and postpartum, and promotes bowel movements. This cold symptoms can be improved.

Peony Root is a perennial plant of the Buttercup family, which includes Radix Paeoniae Alba and Radix Paeoniae Rubra. White peony and red peony are determined by the presence or absence of a peel. Peeled peony and peeled are called peony. In addition to antipyretics, earl pill has the effects of contraction inhibitory effect on gastric and intestinal smooth muscle and uterine smooth muscle, prevention of atherosclerosis, blood pressure lowering and blood flow improvement for vascular disease. This effect of peony extract appears to be due to antioxidant effect, platelet aggregation inhibitory effect, hyperlipidemia prevention, and the like. In addition, glycosides, which are part of the constituents of peony, have been reported to be effective in the treatment of cerebral infarction.

Atractylodis Rhizoma Alba causes the rooting of perennial plant inserts belonging to the Asteraceae family, and is a fragrant conditional agent that is often used in prescription medicine for the supplementation of the spleen and for the supplementation of the spleen. Action, hypoglycemic effect, lowering blood pressure, diuretic effect is effective.

Poor cocos (Poria cocos) is parasitic in the roots of the tree, the size of the fungal nucleus (菌核) is 10 ~ 30cm and white is called the white baekbokyeong (白 茯 笭), red is called the red bokbokyeong (赤 茯 笭). It is an herbal medicine and is effective in tonic, diuretic and soothing. It is used for kidney disease, cystitis or urethritis.

The uterus is a biologically active substance present in the uterus and tetramethylpyrazine was first isolated in 1957 and has been widely used in the treatment of angina pectoris and cerebral ischemic syndromes in China since the 1970s. Wang et al. (1979) reported that tetramethylpyrazine phosphate had positive inotropic and chronotropic effects in the anesthetized dog's heart and had a direct relaxation effect on peripheral blood vessels. Tetramethylpyrazine is also known to increase coronary blood flow in dog experiments, has a potent relaxation effect in both the mind and body circulation, and relaxes in the lungs.

Also known as Alismataceae (澤瀉) is soetae herbs, and the scientific name Alisma canaliculatum . It is a pure water aquatic plant with no main stem and short root stems to form tubers and many beard roots. It lives in wetlands like paddy fields and paddy fields. It grows rarely throughout Korea and is distributed rarely. In oriental medicine, tubers are dried and used as medicinal herbs. Weakness is cold and cold, and it has the effect of Isu (利 水), branch (지), and jigal (止渴). Urinary deficiency, swelling swelling, nephritis, cystitis, urethritis, pregnant edema, each, diarrhea, bun, diabetes, hypertension, fat, liver, etc. As a pharmacological action, antioxidant action, diuretic action, antiallergic action and the like have been reported.

On the other hand, as a technology related to Dongguk Paksan, functional food containing a herbal composition having an improvement function of female menopausal symptoms is known in Korean Patent No. 0419121, but the angiogenesis dependency including the herbal medicine mixed extract of the present invention as an active ingredient There is no disclosure of a composition for preventing, ameliorating or treating a disease.

The present invention is derived by the above requirements, the present invention relates to a composition for the prevention, improvement or treatment of angiogenesis-dependent diseases comprising the herbal extract as an active ingredient, the herbal extract is a menopause induced In an animal model, the present invention was completed by enhancing the production of estradiol, promoting the production and migration of vascular endothelial cells, and affecting the expression of ERK, Akt and eNOS proteins associated therewith.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of angiogenesis-dependent diseases comprising a mixed extract of peony, cheonggung, taxa, Angelica, bokyeong and baekchul as an active ingredient.

In addition, the present invention provides a health functional food composition for the prevention or improvement of angiogenesis-dependent diseases, including a mixed extract of peony, cheonggung, taxa, Angelica, bokyeong, and baekchul as an active ingredient.

The present invention relates to a composition for the prevention, improvement or treatment of angiogenic dependent diseases comprising a mixture of herbal extracts as an active ingredient, and in detail, a mixed extract of peony, cheonggung, taxa, tangui, bokyeong and baekchul as an active ingredient. It relates to a composition for preventing, ameliorating or treating angiogenic dependent diseases. The active ingredient of the present invention can be used as a composition for preventing, improving or treating angiogenesis-dependent diseases by promoting estradiol, a female hormone reduced by menopausal age, and in particular, by promoting the production and migration of vascular endothelial cells.

1 is a result confirming the content of 17-β estradiol contained in platelets of normal female mice (Control) and ovarian ablation mice (OVX). ** means statistically significant decrease in 17-β estradiol in ovarian excised mice (OVX) compared to control, p <0.01.
Figure 2 is a result of confirming the change in blood flow according to the administration of the medicinal herb extract of the present invention in ovarian extraction and lower limb ischemia animal model. (A) is an image showing the blood flow measured by laser doppler perfusion imaging (LDPI), (B) is a graph quantitatively showing the blood flow. OVX-HLI is the lower limb ischemia group after ovarian extraction, OVX-HLI-SV is the simvastatin feed group after ovarian extraction and lower limb ischemia induction, and OVX-HLI-DJ is the mixed extract of the present invention after ovarian extraction and lower limb ischemia induction Feed benefit group.
Figure 3 is a result confirming the capillary blood formation and the ratio of capillary / muscle according to the administration of the herbal medicine mixture extract of the present invention. (A) Immunohistochemical staining results for the anti-CD31 antibody, (B) is a graph showing the ratio of capillary / muscle, ** is a simvastin or mixed extract of the present invention after ischemic induction after ovarian extraction Feed fed group showed statistically significant increase in capillary / muscle ratio compared to ovarian extraction group.
4 is a result of confirming the movement of vascular endothelial cells, (A) is confirmed by H & E staining the migrated cells, (B) is a graph showing this quantitatively.
5 is a result confirming the expression levels of ERK, p-ERK, Akt, p-Akt and eNOS in the lung muscle and calf muscle tissue according to the administration of the herbal extracts of the present invention.

The present invention relates to a pharmaceutical composition for the prevention or treatment of angiogenesis-dependent diseases comprising a mixed extract of peony, cheonggung, taxa, Angelica, bokyeong, and baekchul as an active ingredient.

The angiogenesis-dependent disease is preferably at least one selected from ischemia due to menopause, diabetic foot ulcer, ischemic stroke, ischemia and cerebrovascular dementia, but is not limited thereto. It is characterized by symptoms of sprains.

The mixed extract may be prepared by a method comprising the following steps, but is not limited thereto:

(1) extracting by adding an extraction solvent to the mixed Chinese medicine of Peony, Cheonggung, Taxa, Angelica, Fuling, and Baekchul;

(2) filtering the extract of step (1); And

(3) concentrating and drying the filtered extract of step (2) to produce an extract.

The extraction solvent in step (1) is preferably selected from water, C ₁ ~ C ₄ lower alcohol or a mixture thereof, more preferably water, but is not limited thereto. In the above production method, the extraction method may use all conventional methods known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction. The extraction solvent is preferably extracted by adding 1 to 20 times the weight of the mixed herbal medicine (Peak, Cheongung, Taxa, Angelica, Bokyeong and Baekchul), more preferably 5 to 15 times, and even more preferably 10 Pear is to add. Extraction temperature is preferably 60 ~ 100 ℃ but is not limited thereto. In addition, the extraction time is preferably 1 to 48 hours, more preferably 1 to 24 hours, most preferably 3 hours is not limited thereto. In the above method, the concentration in step (3) is preferably, but not limited to, using a vacuum rotary concentrator or a vacuum rotary evaporator. In addition, drying is preferably carried out under reduced pressure drying, vacuum drying, boiling drying, spray drying or lyophilization, more preferably freeze drying but not limited thereto.

The mixing ratio of Peony, Cheonggung, Taxa, Angelica, Bokyeong, and Baekchul is a weight ratio of 1 to 6: 1 to 4: 1 to 4: 0.5 to 2: 0.5 to 2: 0.5 to 2 is preferably a mixing ratio, More preferably, the weight ratio is 3: 2: 2: 1: 1: 1, but is not limited thereto.

The mixed extract is characterized in that to promote the proliferation of vascular endothelial cells, the ability of vascular endothelial cells or angiogenesis, the angiogenesis is characterized in that it promotes angiogenesis by activating ERK and Akt-eNOS signal .

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when parenteral administration, it is preferable to select external or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection methods. Preferably it is used for external skin.

The pharmaceutical compositions of the present invention may be formulated using diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may comprise at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. 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, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, “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 means the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

The dosage of the composition of the present invention can be used in a variety of ranges depending on the weight, age, sex, health, diet, time of administration, administration method, excretion rate and severity of the patient.

The present invention relates to a health functional food composition for the prevention or improvement of angiogenesis-dependent diseases comprising a mixed extract of peony, cheonggung, taxa, Angelica, bokyeong, and baekchul as an active ingredient.

The angiogenic dependent disease is preferably at least one selected from ischemia due to menopause, diabetic foot ulcer, ischemic stroke, ischemia and cerebrovascular dementia.

Health functional food composition for the prevention or improvement of angiogenesis-dependent diseases comprising a mixed extract of peony, cheonggung, taxa, Angelica, bokyeong, baekryeok of the present invention as an active ingredient powder, granules, pills, tablets, capsules, candy, syrup And it can be prepared by any one selected from the beverage, or by adding as a component of the food, can be appropriately prepared according to a conventional method.

Examples of foods to which the mixed extract of Peony, Cheongung, Taxa, Angelica, Bokryeong, and Baekchul of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gum, Dairy products, including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes can be any one of the forms selected, and includes all of the health functional food in a conventional sense.

The dietary supplement includes various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alkonic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. Others may contain pulp for the production of natural fruit juices and vegetable drinks. These components can be used independently or in combination.

The dietary supplement composition of the present invention may contain various flavors or natural carbohydrates as additional ingredients. The natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

Experimental Method and Materials

1. Experimental Animal

The rats used for the experiment were C57BL / 6J, 7 weeks old, and were purchased from Orient Bio (Seongnam, Gyeonggi-do), and were reared without restriction for 1 week for adaptation at the Experimental Animal Research Center, Korea Institute of Oriental Medicine. Sterilized feed and water were supplied in a sterile breeding machine, and used for the experiment after an adaptation period of 12 hours / 12 hours (light / dark) lighting conditions for a week. Females weighed 18-24 g. This experiment was conducted after approval of the Animal Experimental Ethics Committee of Korea Institute of Oriental Medicine (14-76).

2. Surgical Treatment

(1) Ovariectomized model

General anesthesia was performed by administering 60 mg / kg of ketamine (Ketamine HCl) in rat abdominal cavity. The animal was lying in a prone position and disinfected with alcohol at the dorsal incision, and then the muscle layer was dissected. The adipose tissue was pulled to bind the ovary connected to the end with a non-hygroscopic suture (5-0) and removed. Both ovaries were removed to remove both ovaries and then sutured with hygroscopic sutures (5-0). Animal models include Sham group, Ovarian extraction group (OVX), Ovarian ischemia-induced leg ovary (OVX-HLI), Ovarian extraction and simvastatin 10 mg / kg / day feed group after induction of ischemia. OVX-HLI-SV), ovarian extraction and lower limb ischemia were classified into 200mg / kg / day feed group (OVX-HLI-DJ) of the present invention, and the ovarian extraction group was recovered one week after ovarian extraction. Had

(2) Hindlimb ischemia model (HLI model)

General anesthesia was performed by administering 60 mg / kg of ketamine (Ketamine HCl) in rat abdominal cavity. The lower extremity of the right lower extremity was cut in about 2 cm to identify blood vessels, and then the arteries and veins were separated from the lower part of the inferior epigastric arter to the midpoint of the superficial femoral artery.

(3) Determination of estradiol concentration in blood

In order to measure the concentration of estradiol, a female hormone in the blood, 10 days after ovarian extraction, blood was collected from the abdominal vein of the control group and the ovarian extraction group (OVX) that did not extract the ovary, and then plasma was separated. Enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) was measured using.

(4) Laser Doppler Perfusion Imaging

Preoperative, 1, 3, 7, 14, 21, and 28 days after ligation surgery, anesthetized the experimental animals with ketamine, remove hairs, and check blood flow with laser Doppler (Periscam, Sweden) in both lower extremities. Measured. The blood flow rate of the operated limb is expressed as the ratio with the blood flow rate of the nonischemic limb.

(5) Organization Preparation

60 mg / kg of ketamine (Ketamine HCl) intraperitoneally in rat abdominal anesthesia and sacrificed. For histological study, the muscle and gastronemius muscles of the thighs were extracted and fixed in paraformaldehyde (PFA) at 28 days after ligation. The extracted muscle and gastronemius muscles were frozen in liquid nitrogen and stored at -80 ° C until the experiment.

(6) Immunohistochemical staining and reading (Immunohistopathologic studies)

After making paraffin embedding tissue, 5μm-thick sections were attached to the glass slide, paraffin was removed, and after hydration, soaked in EDTA solution for 10 minutes to expose the hidden antigen. The mixture was placed at room temperature for 30 minutes, treated with 3% hydrogen peroxide for 10 minutes to inhibit endogenous peroxidase, and washed with distilled water. The subsequent process was performed using LSAB kit (DAKO). Two primary antibodies, CD31 (PECAM-1, BD Biosciences), were treated with a dilution of 1: 200, respectively, and reacted at room temperature for 2 hours, and washed with TBS (Tris-buffered saline). After 30 minutes at room temperature with a secondary antibody, washed with TBS and reacted with peroxidase-labeled streptavidin (stretavidin) for 30 minutes at room temperature, and then developed with diaminobenzidine for 10 minutes at room temperature. CD31 immunochemical tissue staining was performed by selecting three sites with the highest vascular density and counting the number of blood vessels that were positive in the optical microscope 200-fold field of view.

(7) Western blot analysis

Experimental animal muscle tissue was dissolved in RIPA buffer [50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet-P40, deoxycholic acid, 0.1% sodium dodecyl sulfate]. Cellular solution was obtained from the supernatant after centrifugation at 13,000g for 15 minutes at 4 ℃. Cytoplasmic solution (20 μg protein) was electrophoresed on 10% SDS-PAGE and the protein was transferred to nitrocellulose membrane. Membrane was washed twice with PBS-Tween containing 5% skim milk, and PBS-Tween containing the antibody against the target protein was added thereto and reacted for 2 hours. . After washing the membrane twice with PBS-Tween solution, a secondary antibody suitable for the primary antibody was added and reacted at room temperature for 2 hours. The membrane was washed three times with PBS-Tween and then photosensitive using an ECL system.

(8) statistics

Statistical comparison was performed using the GraphPad PRISM statistical package (ver 2.00, Graphpad software inc., San Diego, USA). The comparison with the control group and the experimental group was post-validated by ANOVA followed by Tuckey's post hoc test. Each value was expressed as mean ± standard deviation, and the test significance was based on the p value <0.05.

Example  1. Peony, Cheongung, Taxi, Angelica, Fuling and Whiteness  Mixed extract ( DJ Manufacturing

At a weight ratio of 3: 2: 2: 1: 1: 1, 10-15 times of water is added to the mixture of Peony, Cheongung, Taxa, Angelica, Bokryeong, and Baekchul, and the mixture is refluxed at 100 ° C for 3 hours to mix An extract was obtained. The obtained mixed extract was filtered under reduced pressure with a 0.4 μm filter, concentrated in a vacuum rotary concentrator, and lyophilized using a freeze dryer.

Example  2. Ovarian Extraction Menopausal Guidance Model

As a result of confirming whether the concentration of estradiol in blood was significantly reduced after inducing menopause through ovarian extraction, as shown in FIG. 1, the estradiol concentration of estradiol in the menopausal induction animal model (OVX) compared to the normal group (Control) The concentration was statistically significantly reduced.

Example  3. Femoral artery and vein By ligation  Establishment of Peripheral Vascular Disorder Animal Model Blocking Blood Flow

After ligating the femoral artery and vein of one leg in C57BL / 6J mice, an animal model system of lower limb ischemic disease was constructed in which blood vessels were cut out to block blood flow.

Example  4. Peony, celestial arch, taxa, donkey, ghost of the present invention and Whiteness  Changes in Indicators by Administration of Mixed Extract (DJ)

Indicator change by administration of the mixed extract of the present invention Sham OVX OVX-HLI OVX-HLI-SV OVX-HLI-DJ Number of animals 7 6 7 7 7 Weight (g) 21.29 ± 0.95 ^a 24.17 ± 1.17 ^c 22.14 ± 0.69 ^{b, c} 23.43 ± 0.79 ^{b, c} 24.14 ± 1.68 ^c Liver weight (g) 1.10 ± 0.09 ^a 1.24 ± 0.07 ^b 1.09 ± 0.05 ^a 1.05 ± 0.09 ^a 1.03 ± 0.07 ^a Height weight (g) 0.12 ± 0.01 ^a 0.12 ± 0.01 ^a 0.11 ± 0.01 ^a 0.11 ± 0.01 ^a 0.12 ± 0.01 ^a

As disclosed in Table 1, the mixed extract of the present invention showed a weight, liver, and kidney weight almost similar to that of the ovary extraction model, and there was little weight gain, but it was almost significant compared to the ovarian extraction wheat ischemia induction model. There was no difference.

Example  5. Measurement of Blood Flow Change by Administration of the Mixed Extract of the Present Invention

Before surgery, on day 1 (day 1), day 3 (day 3), day 7 (day 7), day 14 (day 14), day 21 (day 21) and day 28 (day 28), LDPI ( Blood flow was measured by laser doppler perfusion imaging.

As a result, as shown in Figure 2, the positive control group simvastatin (Limvastatin) administration group was ligated, and after 3 days showed a difference in blood flow compared to the control group HLI-OVX group, compared to the control group in the mixed extract administration group of the present invention Increased blood flow, especially 14 days after ligation.

Example  6. Confirmation of changes in capillary density by immunochemical tissue staining

 By administering the mixed extract of the present invention, in order to observe the changes in capillaries in the femoral region, the density of the capillaries was compared by measuring the expression level after immunohistochemical staining using CD31, a specific expression factor of the vascular endothelial cells. It was.

As a result, the expression of CD31 in the positive control group (OVX-HLI-SV) and the mixed extract administration group (OVX-HLI-DJ) of the present invention was increased compared to the CD31 expression in the control group (OVX-HLI) ligated after ovarian extraction. It was confirmed.

After 28 days of administration of the mixed extract of the present invention, the ratio of capillary / muscle was confirmed. As a result, 0.54 ± 0.03, 0.66 ± 0.05, 0.63 ± 0.05 were measured in the control group, the positive control group, and the mixed extract administration group of the present invention, respectively, and the capillary / muscle ratio was significantly increased in the positive control group and the mixed extract administration group of the present invention. It was confirmed that the increase (Fig. 3).

Example  7. Effect on vascular endothelial cell migration

The lower surface of the transwell plate filter with an 8 μm pore size for the assay for chemotactic motility of human umbilical vein cells (HUVEC) is 2% gelatin. coated with gelatin, HUVEC cells were trypsin-enzymatically and suspended to a final 1 × 10 ⁶ cell / ml concentration. The mixed extract of the present invention was mixed with the 250 μl suspension and loaded into the upper well to move for 6 hours. Thereafter, H & E staining was performed to confirm the degree of migration of vascular endothelial cells. As a result, as shown in FIG. It was confirmed.

Example  8. In vascular endothelial cells ERK , Akt  And eNOS Impact on

Proliferation, migration or morphological changes of endothelial cells by angiogenic factors are induced through activation of various signaling pathways. Phosphorylation of ERK, Akt and eNOS is known as important mediators of this signaling pathway. Therefore, in Example 8, to determine the effect of the mixed extract of the present invention on the phosphorylation regulation of eNOS, ERK, and Akt, Western blot was performed using specific antibodies that recognize the respective phosphorylation forms.

As a result, as shown in FIG. 5, the expression of eNOS which is closely related to angiogenesis is increased in the adductor muscle where the blood flow recovery is most observed after the ischemic induction by administration of the mixed extract of the present invention. It was.

Claims (8)

delete delete delete delete delete delete Prevention or improvement of numbness or tingling of the hands or feet by menopause comprising water mixed extract of Peony, Cheongung, Taxa, Angelica, Bokryeong, and Baekchul, mixed at a weight ratio of 3: 2: 2: 1: 1: 1 Functional food composition for use. delete

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