KR20110121848A - Angiogenesis inducing agent comprising the fractions from the extracts of patrinia villosa juss. as an active ingredient - Google Patents

Abstract

PURPOSE: An angiogenesis promoting agent containing a fraction of Patrinia villosa extract is provided to remarkably promote endothelial cell proliferation and to reduce tissue necrosis. CONSTITUTION: An angiogenesis promoting agent contains a fraction as an active ingredient, which is prepared by fractioning Patrinia villosa extract with an organic solvent. The Patrinia villosa extract is isolated using water, low alcohol of C1-C4, or mixture solvent thereof. The low alcohol is ethanol or methanol. The promoting agent is used for treating ischemia, chronic ulcer, ischemic stroke, rheumatoid arthritis, psoriasis, ulcer, artery scleroma, myocardial infarction, angina or vascular dementia. A health food for promoting angiogenesis contains the fraction as an active ingredient.

Description

Angiogenesis inducing agent comprising the fractions from the extracts of Patrinia villosa Juss. as an active ingredient}

The present invention is Patrinia villosa ) angiogenesis-promoting agent containing a fraction of the extract as an active ingredient.

New blood vessels are tightly regulated by new blood vessels formed from existing vasculature. Angiogenesis plays an important role in a wide range of pathophysiological processes such as wound healing, menstrual restoration, hematopoiesis and tumor growth (Folkman J and Shing Y (1992) Angiogenesis. J Biol Chem 267 (16): 10931-10934; Kieran MW et al., (2003) Angiogenesis inhibitors and hypoxia. Nat Med 9 (9): 1104; author reply 1104-1105). During neovascularization, endothelial cells break down the basement membrane, migrate around the intercellular matrix, proliferate to produce new blood vessels, differentiate into adjacent coronary spurts, and soon form a functional capillary loop. These cellular phenomena are mediated by various intracellular signaling pathways, in which focal adhesion kinase (FAK) is known to be involved. The FAK has been reported to play an important role in endothelial cell migration and survival (Avraham HK et al., (2003) J Biol Chem 278 (38): 36661-36668). Deletion of FAK in endothelial cells in vitro (in in vitro ) decreases tubulation, cell survival, proliferation, and migration (Shen TL et al., (2005) J Cell Biol 169 (6): 941-952), overexpression of FAK in endothelial cells promotes neovascularization induced by wounds as well as neovascularization induced by ischemia (Peng X et al., (2004) ) Cardiovascular research 64 (3): 421-430).

Patrinia villosa is a herb commonly found in East Asia and northeastern North America. In oriental medicine, it has been used for hundreds of years for inflammation, abdominal pain after birth, and so on. In oriental medicine, Yulmu ( Coix) lachryma - jobi ), rich man ( Aconitum The medicinal herb called "Eijinbupajangsan" consisting of carmichaeli ) and teggal was used almost exclusively until the surgical treatment for acute appendicitis. Dukgal's qualitative effect is that according to the original record, mainly to lower the fever, detoxify, decontaminate, remove postpartum blood and cure abdominal pain. In addition to the anti-inflammatory effects of tuggal, various compounds included in teggal have been reported to have anti-cancer effects against various cancer cell lines (Chiu LC et al., (2006) J Ethnopharmacol 105 (1-2): 263-268 Zhang T et al., (2008) Phytother Res 22 (5): 640-645). However, in vivo still extracts of turmeric extract or fractions thereof in In vivo ) activity and signaling mechanisms for its biological activity have been rarely reported.

Therefore, the present inventors have studied to discover a substance for promoting neovascularization derived from natural products having high therapeutic activity and low side effects, the fractions isolated from the extract of the extract is endothelial cell proliferation and migration, the formation of tubular structure and endothelial cells Promotes germination and in vivo through animal experiments ( in vivo) been significantly reduced tissue necrosis of limb ischemia model and which showed a remarkably excellent angiogenic activity relative to ttukgal extract fraction a said ttukgal acetate fraction with an organic solvent may be useful in promoting neovascularization The present invention was completed by revealing.

An object of the present invention is Patrinia villosa ) to provide angiogenesis-promoting agent containing the fraction separated from the extract as an active ingredient.

In order to achieve the above object, the present invention provides a neovascular accelerator containing a fraction obtained by distilling turmeric extract with an organic solvent as an active ingredient.

In addition, the present invention provides a nutraceutical promoting health functional food containing a fraction of the turmeric extract as an organic solvent fraction as an active ingredient.

Fractions isolated from the extract of Dunggal of the present invention promote the proliferation and migration of endothelial cells, formation of tubular structures and endothelial cell germination through activation of FAK signaling pathway, and in vivo ( in In vivo ) limb ischemia model significantly reduces the probability of tissue necrosis, and the neovascularization activity of the fractions from turmeric extract was significantly higher than that of turmeric extract. It can be usefully used as an effective drug for the treatment of neovascular dependent diseases such as vascular disease or wound healing.

Figure 1 is a diagram showing the manufacturing process of the fraction of turmeric extract.
FIG. 2 shows the in vitro of Turmeric extract (PVE) for cultured HUVEC ( in In vitro ):
(A): cell proliferation measured via WST-1 analysis;
(B): migrated cell measurement by counting of cells in scratch line, microscope magnification × 200, Scale bar = 300 μm;
(C): tube formation of cells; And
(D): Aortic ring endothelial germination phenomenon induced by turmeric extract, scored from 0 (minimum positive) to 5 (best positive).
Figure 3 shows the relationship of FAK in neovascular signal induced by turmeric extract:
(A): Western blot analysis; And
(B): Induction of HUVEC proliferation of turmeric extract, inhibited by FAK inhibitor (SC203950).
Figure 4 shows the in vivo ( in intact extract of turmeric) in mouse hind limb ischemia model In vivo ) effect.
(A): Perfusion picture immediately after surgery (left) and limb shape for measuring necrosis 3 days after surgery;
(B): probability of necrosis of mice treated with PBS, VEGF, turmeric extract or fractions thereof; And
(C): Necrosis probability of mice treated with PBS, VEGF, Turmeric extract or fractions thereof, depending on perfusion rate. Low perfusion rate: 0-30% / min; Intermediate perfusion rate: 30-100% / min; High perfusion rate: <100% / min. Bonferroni's post hoc test: *, p = 0.01. **, p <0.001 (ANOVA F 2,18 = 23.232, p <0.001).
FIG. 5 is a diagram showing the biological activity of the fractions isolated from turmeric extract (n-hexane fraction: 10.3 mg / l, ethyl acetate fraction: 7.1 mg / l, n-butanol fraction: 5.5 mg / l and water fraction: 25.2 Mg / l).
(A): cell viability;
(B): cell migration;
(C): tube formation analysis;
(D): aortic ring endothelial cells grown; And
(E): Western blot analysis of molecules related to neovascular signals over tungal fraction or VEGF treatment time.

Hereinafter, the present invention will be described in detail.

The present invention is Patrinia villosa ) provides an angiogenesis-promoting agent containing the fraction obtained by fractionating the extract with an organic solvent as an active ingredient.

Preferably, the fraction is n-hexane fraction, ethyl acetate fraction, n-butanol fraction or water fraction obtained by systematic fractionation of turmeric extract with n-hexane, ethyl acetate, n-butanol and water, and ethyl acetate fraction. More preferably, but is not limited thereto.

The neovascular promoter is preferably for angiogenesis-dependent diseases selected from the group consisting of ischemia, chronic ulcer, wound healing, burn, ischemic stroke, rheumatoid arthritis, psoriasis, ulcer, arteriosclerosis, myocardial infarction, angina pectoris and cerebrovascular dementia. It is not limited to this.

The turmeric extract is preferably prepared by a manufacturing method comprising the following steps, but not always limited thereto:

1) extracting by adding an extraction solvent to the turmeric;

2) cooling the extract of step 1) and filtering; And

3) drying the filtered extract of step 2) under reduced pressure.

In the above method, the turmeric in step 1) can be used without limitation, such as cultivated or commercially available. The turquoise can use all of the outpost, leaves, stems, or roots.

The extraction solvent is preferably water, alcohol or a mixture thereof. It is preferable to use C ₁ to C ₄ lower alcohols as the alcohol, ethanol or methanol is preferably used as the lower alcohol, and more preferably water, but is not limited thereto. As the extraction method, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used, and the extraction may be preferably performed one to five times by the hot water extraction method, and more preferably three times of extraction. . The extraction solvent is preferably extracted by adding 5 to 15 times to the weight of dried turquoise, more preferably by adding 10 times. The extraction temperature is preferably 40 to 80 ° C and more preferably 50 to 60 ° C, but is not limited thereto. In addition, the extraction time is preferably 1 to 5 hours, more preferably 2 hours is not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. In addition, drying may be used under reduced pressure drying, vacuum drying, boiling drying, spray drying or lyophilization, preferably lyophilization, but is not limited thereto.

In a specific embodiment of the invention, the turquoise was washed with water and then dried in the shade and at room temperature. The dried turmeric was put in an extraction container, and extracted with extraction solvent at 50 ~ 60 ℃ for 2 hours. After obtaining turmeric extract, the solids were removed using a filter paper and the like, and the suspension was centrifuged to filter the supernatant under reduced pressure. The extract was concentrated under reduced pressure and lyophilized to prepare turmeric extract.

The fraction of the turmeric extract is preferably prepared by a manufacturing method including the step of preparing a fraction by adding an organic solvent to the prepared turmeric extract, but is not limited thereto.

The fraction may be obtained by repeating the fractionation process from 1 to 5 times, preferably 3 times from the turmeric extract, and preferably concentrated under reduced pressure after the fraction, but is not limited thereto.

In a specific embodiment of the present invention, the turmeric extract is suspended in distilled water, and then branched in order of n-hexane, ethyl acetate, n-butanol, and water, which are solvents having different polarities, and each solvent fraction is concentrated under reduced pressure. N-hexane fractions, ethyl acetate fractions, n-butanol fractions and water fractions were prepared from turmeric extract.

In order to determine the in vitro effect of fractions of the extract of Dunggal extract on neovascularization, we measured the proliferation and migration of human umbilical vascular endothelial cells (HUVECs) and ex vivo ( ex. In vivo ) neovascular activity was measured by matrigel analysis, and the formation of mouse aortic ring endothelial germination (Sprouting) activity was confirmed. As a result, HUVEC treated with fractions isolated from turmeric extract showed significantly increased cell proliferation and migration compared to untreated control group (A and B in FIG. 5), which was more proliferated and migrated than turmeric extract. Was found to significantly promote (A and B in FIG. 2, A and B in FIG. 5). In addition, fractions of turmeric extract have been found to promote endothelial tube formation and endothelial germination in the aortic rings. In particular, the activity of the ethyl acetate fraction in the n-hexane, ethyl acetate, n-butanol and water fractions is remarkably enhanced. High (C and D in FIG. 5).

The inventors confirmed the phosphorylation of molecules involved in VEGFR2 and / or FAK signaling pathways by Western blot analysis in order to confirm the effect of fractions isolated from turmeric extract on intracellular signaling pathways associated with neovascularization. As a result, the ethyl acetate fraction of turmeric extract was found to induce the phosphorylation of focal adhesion kinase (FAK) and Akt (FIG. 5E). At this time, the turmeric ethyl acetate fraction has been shown to promote the phosphorylation of FAK and Akt in a shorter time (within 10 minutes) than the extract (A of Figure 3), the ethyl acetate fraction thereof compared to the turmeric extract The effect on blood vessel-related signaling pathways was excellent. In addition, when treated with a FAK inhibitor, it was shown that the effect of promoting the proliferation of HUVEC by turmeric extract (FIG. 3B). Thus, it can be seen that the neovascularization activity of the fractions of the turmeric extract is through the activation of the FAK signaling pathway, which is known to act on neovascular processes such as endothelial cell migration and proliferation.

The inventors of the present invention, based on the confirmed neovascular activity of the fractions isolated from the extract Dunggal in vitro, in vivo ( in In vivo ), in order to confirm neovascular activity, a fraction of the turmeric extract was administered to the mouse hind limb ischemia model to determine the probability of tissue necrosis. At this time, a fraction of 2.5 g / L Tegall extract was injected intramuscularly at a dose of 20 μl, which was determined according to the ratio of the extract to the body weight of the mouse. Typically, about 1 to 3% of turmeric extract is known to be absorbed into the body in a single dose (3.75 g) of traditional prodrugs, so mice with a body weight of about 20 g were injected at the above dosages. As a result, it was confirmed that in the case of the mice administered the ethyl acetate fraction of turmeric extract, the probability of tissue necrosis was significantly reduced compared to the control group administered with phosphate buffered saline (PBS). In particular, Dunggal ethyl acetate fraction was found to significantly reduce the probability of tissue necrosis compared to the extract (see Figure 4).

Therefore, in vitro of the fractions separated from the turmeric extract of the present invention ( in vitro) and in vivo (in In vivo ) since the neovascularization activity was confirmed, the fraction obtained by extracting the turmeric extract with an organic solvent may be usefully used as an active ingredient of an angiogenesis promoter.

The neovascular promoters of the present invention may be used in the form of oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.

Solid form preparations for oral administration include powders, granules, tablets, capsules, soft capsules, and the like. Oral liquid preparations include suspensions, solvents, 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. . Formulations for parenteral administration include powders, granules, tablets, capsules, sterile aqueous solutions, solutions, non-aqueous solutions, suspensions, emulsions, syrups, suppositories, aerosols, etc. It may be used in the form of a formulation, and preferably, an external skin pharmaceutical composition of cream, gel, patch, spray, ointment, warning agent, lotion agent, linen agent, pasta agent or cataplasma agent may be prepared and used. It is not limited to this. Non-aqueous solvents and suspending agents may be used as vegetable oils such as propylene glycol pharmaceutical polyethylene glycol pharmaceutical olive oil, injectable esters such as ethyl oleate, etc. As a base of suppositories, witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The neovascular promoter according to the present invention may further include a carrier, an excipient, a disintegrant, a sweetener, a lubricant, a flavoring agent, and a diluent which are commonly used. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The disintegrants include sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, chitosan, guar gum, low-substituted hydroxypropyl cellulose, magnesium aluminum silicate, and polyacryline Potassium and the like.

In addition, the neovascular promoter according to the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, Calcium hydrogen phosphate, lactose, mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, Aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included 0.1 to 90 parts by weight based on the neovascular promoter.

The neovascular promoter of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the above components. The accelerator of the present invention may comprise 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the fraction of the present invention relative to the total weight of the promoter.

In addition, the neovascular promoter of the present invention may be administered orally or parenterally, and when parenteral administration is selected for external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. It is desirable to.

The dosage of the neovascular promoter of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and severity of the disease of the patient, and the daily dosage is from turmeric extract The amount of the separated fraction is 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and may be administered 1 to 6 times a day.

The neovascular promoter of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of neovascular dependent diseases.

In another aspect, the present invention provides a method for promoting neovascularization comprising administering to the subject a fraction of a pharmaceutically effective amount of turmeric extract with an organic solvent.

The subject is a vertebrate and preferably a mammal, more preferably an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, cat, and most preferably an ape-like animal such as a chimpanzee or gorilla.

The method of administration may be administered orally or parenterally, intraperitoneal, rectal, subcutaneous, intravenous, intramuscular, intrauterine dural, intracerebroventricular or intrathoracic It can be administered by injection.

The pharmaceutically effective amount is 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, but is not limited thereto. Dosage may vary depending on the weight, age, sex, health status, diet, duration of administration, method of administration, elimination rate, severity of disease, and the like of a particular patient.

The neovascularization is preferably involved in a disease selected from the group consisting of ischemia, chronic ulcer, wound healing, burn, ischemic stroke, rheumatoid arthritis, psoriasis, ulcer, arteriosclerosis, myocardial infarction, angina pectoris and cerebrovascular dementia It doesn't work.

Fractions isolated from the extract of Dunggal of the present invention significantly promote endothelial cell proliferation, endothelial cell migration, formation of capillary-like structures, and endothelial germination compared to extracts, which are essential for neovascularization and stimulate FAK signals involved in neovascularization. activated, and a living body as compared with the case of administration of ttukgal extract (in In vivo ), it was confirmed that significantly reduced tissue necrosis of the limb ischemia model, fractions of the turmeric extract with an organic solvent can be useful for promoting neovascularization.

In addition, it provides a dietary supplement for neovascularization containing the fractions of the turmeric extract with an organic solvent as an active ingredient.

The health functional food is preferably for neovascular dependent diseases selected from the group consisting of ischemia, chronic ulcers, wound healing, burns, ischemic stroke, rheumatoid arthritis, psoriasis, ulcers, arteriosclerosis, myocardial infarction, angina pectoris and cerebrovascular dementia. It is not limited to this.

The fraction separated from the turmeric extract of the present invention can be added as it is or used with other food or food ingredients, and can be suitably used according to conventional methods.

There is no particular limitation on the kind of food. Examples of foods to which the fractions of the turmeric extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea , Drinks, alcoholic beverages and vitamin complexes, and includes all of the health food in the usual sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the fractions of the extract of Dunggal of the present invention are various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin , Alcohols, carbonating agents used in carbonated drinks, and the like. Others may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Fractions isolated from the extract of Dunggal of the present invention significantly promote endothelial cell proliferation, endothelial cell migration, formation of capillary-like structures, and endothelial germination compared to extracts, which are essential for neovascularization and stimulate FAK signals involved in neovascularization. In vivo ( in In vivo ), it was confirmed that the tissue necrosis of the limb ischemia model was significantly reduced compared to the extract, and the fraction of the turmeric extract with the organic solvent may be usefully used for the functional food for promoting neovascularization.

Hereinafter, the present invention will be described in detail by way of examples, comparative examples and production examples.

However, the following Examples, Comparative Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Comparative Examples and Preparation Examples.

< Comparative example  1> Pot  Preparation of Extract

600 g of Patrinia villosa , purchased from OmniHub (Daegu, Korea), was extracted by heating in distilled water for 2 hours. The extract was filtered and lyophilized under low pressure using a rotary vacuum concentrator to yield 8.8% (52.8 / 600 g; w / w) yield of dried water extract (PVE).

< Example  1> Pot  Extract Fraction  Produce

Phylogenous water extract obtained in <Comparative Example 1> was systematically fractionated, n-hexane (n-Hexane) (19.51%; 10.3 / 52.8 g; w / w), ethyl acetate (EtOAc) (13.4%; 7.1 / 52.8 g; w / w), n-butanol (n-BuOH) (10.61%; 5.6 / 52.8 g; w / w), and water (47.73%; 25.2 / 52.8 g; w / w) fractions were obtained, respectively. (FIG. 1).

< Example  2> Pot  Extract Fraction  Effect on Endothelial Cell Proliferation

Human umbilical vascular endothelial cells (HUVECs) have been previously described (Choi DY et al., (2005) Biol Pharm Obtained from fresh umbilical cord according to the method described in Bull 28 (12): 2248-2252) and incubated in EGM ^™ -2 complete medium (Cambrex, East Rutherford, NJ, USA). Upon saturation, cells were harvested with trypsin-EDTA solution. All experiments were carried out using HUVECs passaged 3 to 6 times. To analyze the cell proliferation effect of fractions of Dunggal extract, 96-well dish containing 100 μl of HUVEC supplemented with 10% fetal bovine serum (Gibco, Invitrogen, Carlsbad, Calif., USA) Nunc, Roskilde, Denmark) were dispensed at a density of 5 × 10 ³ cells per well. Cells were maintained in serum-free medium for 12 hours and then treated or untreated with turmeric extract or fractions thereof, or further incubated for 72 hours under 25 mg / L VEGF (R & D Systems, Minneapolis, MN, USA). 10 μl of WST-1 solution (Boehringer, Mannheim, Germany) was then added to each well and the extent of cell proliferation was measured at 450 nm with an ELISA reader (Bio-Rad Laboratories, CA, USA).

As a result, in the case of cells cultured by treating the fractions of turmeric extract, the survival of HUVEC was significantly increased as compared with the control group (FIG. 5A). This was found to be higher than in cells treated with Dunggal extract as a whole (A of FIG. 2 and A of FIG. 5).

< Example  3> Pot  Extract Fraction  Effect on Cell Migration

HUVEC cells were dispensed into 6-well dishes containing 2 ml of DMEM medium with 10% FBS and incubated for 12 hours at 37 ° C. in a humid environment of 5% CO ₂ . Cells were maintained in serum-free medium for 24 hours, then treated or untreated with turmeric extract, or treated with one of four fractions (n-hexane, ethyl acetate, n-butanol, and water fractions), or 25 μg / Incubated under 1 VEGF. Then one straight line was drawn on the culture dish using the micro tip. 14 hours after the bottom was scratched, the image was confirmed by a microscope, and the unilateral movement was measured by counting the cells entering the scratched area. Each measurement was repeated four times and four images of all scratches were obtained.

As a result, the fraction of turmeric extract significantly improved the migration of HUVEC cells (FIG. 5B). In particular, in the fractions, the cells treated with the ethyl ethyl acetate fraction were found to promote the movement of cells more than about 4 times more than the cells treated with the other fractions, respectively. A lot of movement was shown (B of FIG. 2 and B of FIG. 5).

 < Example  4> Pot  Extract Fraction  Cell tube ( tube ) Effect on formation

Tube formation activity of HUVEC by fractions of Turmeric extract was performed according to previously reported methods (Kim HZ et al., (2009) Biochim Biophys Acta 1793 (5): 772-780). Briefly, growth factor reduced Matrigel (Becton Dickinson Labware, Lincoln Park, NJ, USA) was dissolved at 4 ° C. and 200 μl of Matrigel was used to prepare pre-chilled 6-well plates. Used to coat. The plates were then incubated for 30 minutes at 37 ° C. in a humid environment of 5% CO 2. Turmeric extract, fractions thereof, or VEGF were added to the Matrigel layer at a density of 1 × 10 ⁵ cell counts / well at various concentrations. The tube formed on the Matrigel layer was counted 3 hours after incubation.

As a result, the fraction of Dunggal extract significantly improved the tube forming activity of HUVEC cells compared to the control. In particular, it was confirmed that the ethyl acetate fraction of the fraction has the highest pipe formation promoting effect (Fig. 5 C).

< Example  5> Pot  Extract Fraction  Effects on Aortic Ring Endothelial Cells

A previously reported method of separating descending aorta from C57 / BL6 mice (Orient Bio Inc., Seoul, Korea) (Min JK et al., (2004) Cancer According to Res 64 (2): 644-651), the slices were sliced into aortic rings (diameter> 1.5 mm; thickness> 1.5 mm). The aortic rings were placed in 4-well dishes coated with 100 μl of growth factor reduced Matrigel, covered with 30 μl of the same Matrigel and sealed, and 1 ml of DMEM containing 10% FBS was added to each well. Added. Samples of the aortic rings were incubated for 7 hours with or without various concentrations of turmeric extract, fractions thereof, or VEGF. Then, microscopic images were obtained for analysis of endothelial cells grown from the aortic rings.

As a result, endothelial cell germination was observed at a significantly higher level than the control group around the edges of the mouse aortic rings treated with the fractions of the turmeric extract. In addition, the ethyl acetate fraction of the fraction was the highest effect (Fig. 5 D).

< Example  6> Pot  Extract Fraction  Topical attachment kinase ( focal adhesion kinase , FAK) effect on phosphorylation

HUVEC cells were aliquoted into 6-well dishes and cultured for 24 hours under treatment with or without treatment of turmeric extract, fractions thereof or VEGF to confirm intracellular signaling mechanisms for neovascularization activity of turmeric extract. The cells contain a phosphatase inhibitor It was lysed with M-PER lysis buffer (Pierce, Rockford, IL, USA) and total cell lysates were monoclonal antibodies specific for phospho-FAK and total FAK (Cell Signaling Tech, Beverly, MA, USA) and HRP bound secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for Western blot analysis.

As a result, the cells treated with Dunggal extract promoted time-dependent phosphorylation of FAK (tyrosine 576/577 residues) and delayed phosphorylation of Akt (FIG. 3A). In addition, when the FAK inhibitor SC203950 was treated, the proliferation of HUVEC induced by turmeric extract was significantly suppressed (FIG. 3B). On the other hand, in the treatment of the fraction of Dunggal extract, both phosphorylation of FAK (576/577) and Akt was induced, and it was confirmed that phosphorylation of the protein was induced 10 minutes after the treatment with ethyl acetate fraction (FIG. 5). E). Thus, it can be seen that the fractions of Dunggal extract showed a faster FAK signal activation effect than when the extract was treated (A of FIG. 3).

< Example  7> in vivo for ischemia model in vivo ) Pot  Extract Fraction  effect

<7-1> Preparation of mouse hind limb ischemia model

BalB / cAnNCriBgi-nu nude male mice used in the experiment were purchased from Charles River Japan, Inc. (Yokohama, Japan). All mice were used 7-8 weeks old (15-20 g). Hind limb ischemia under ketamine-xylazine anesthesia Induced by ligation of the right femoral artery and vein. Animal management and testing procedures were conducted with the approval of the Animal Management Committee of Korea Advanced Institute of Science and Technology. Mice were divided into four groups with PBS (20 μl; 0.9% NaCl, 0.1% BSA) as a control, or turmeric extract or fractions thereof (20 μl; 2.5 g / L), or VEGF (20) for therapeutic neovascularization. A saline solution containing [mu] l; 130 mg / l) was injected intramuscularly. Continuous indocyanine green (ICG) perfusion imaging was performed immediately after surgery and 3 days after surgery (Kang Y et al., (2009) PLoS One 4 (1): e4275) (A in FIG. 4).

<7-2> NIR  Fluorescence imaging

As previously reported (Kang Y et al., (2009) PLoS One 4 (1): e4275), a custom optical system was used for NIR fluorescence imaging. Briefly, these systems include CCD digital cameras with custom 830 nm bandpass filters (Asahi Spectra USA, Torrance, CA, USA) and 760 nm light emitting diode arrays (SMC760; Marubeni America, Sunnyvale, CA, USA). PIXIS 1024; Princeton Instruments, Princeton, NJ, USA). For time-continuous ICG imaging, ICG (400 mmol / L 0.1 mL; Sigma, St. Louis, MO, USA) was injected in a single intravenous injection into the tail vein of mice anesthetized with ketazine-xylazine. ICG fluorescence serial images were obtained in the dark for 10 minutes at intervals of 1 second immediately after tail vein injection, and a customized computerized program was used to confirm perfusion map and necrosis probability. The partial perfusion rate of the ischemic limb was measured immediately after surgery, and the necrotic site in the ischemic limb was analyzed 3 days after the surgery. The number of extremity sites given was about 300-380. The necrosis probability of ischemic tissue was graphed using the correlation between regional perfusion rate (mean perfusion rate of 2 × 2 pixels) and the corresponding regional necrosis (measured three days after surgery). Necrosis probability was calculated based on data from about 3500 sites of PBS treated mice and about 2800 sites of VEGF treated mice or turmeric extracts or fractions treated mice. Each data is shown as mean ± SEM. Statistical comparisons were made using ANOVA and Bonferroni's multiple comparison test method. For analysis of tissue perfusion in the mouse hind limb ischemia model, the statistical significance of limb perfusion rate was measured by ANOVA and Bonferroni's post-hoc test for three or four groups. Statistically different samples compared to untreated controls are indicated with an asterisk unless otherwise noted (*, P <0.05; **, P <0.01; ***, P < 0.001).

As a result, in the tissues with low initial perfusion rate, each group administered with Dunggal extract, fractions thereof, VEGF or PBS control showed little difference in the probability of death, and significantly different in the probability of tissue death for the site with medium perfusion rate. It was confirmed that, compared to the PBS administration group, the fraction administration group of turmeric extract sharply reduced the probability of death (FIG. 4). In particular, in the case of moderate perfusion rate, turmeric ethyl acetate fraction was found to have a significantly lower probability of necrosis than turmeric extract. Thus, the recovery of ischemic tissue is different depending on the initial perfusion rate, it was confirmed that the recovery of ischemic tissue is difficult in tissue less than 30% per minute, which is 5% or less of normal perfusion rate. On the other hand, in tissues with intermediate perfusion rate (30-100% per minute), there was a significant difference in the probability of necrosis between the groups, and the group treated with the fraction of turmeric extract significantly reduced the rate of necrosis. It was found that ischemic tissues were reperfused by concomitant growth as a result.

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

<1-1> Powder  Produce

2 g of the ethyl acetate fraction of Example 1

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of Tablet

100 mg of the ethyl acetate fraction of Example 1

Corn starch 100 mg

100 mg of milk

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of the ethyl acetate fraction of Example 1

Corn starch 100 mg

100 mg of milk

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

&Lt; 1-4 >

1 g of the ethyl acetate fraction of <Example 1>

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.

<1-5> Preparation of granules

150 mg of the ethyl acetate fraction of <Example 1>

Soybean Extract 50mg

Glucose 200 mg

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

< Manufacturing example  2> Manufacture of food

Foods containing the fraction of turmeric extract of the present invention were prepared as follows.

<2-1> Production of flour food

0.5-5.0 parts by weight of the n-hexane fraction of <Example 1> was added to the flour, and bread, cake, cookies, crackers, and noodles were prepared using the mixture.

<2-2> soup  And juicy ( gravies Manufacturing

0.1-5.0 parts by weight of the n-hexane fraction of <Example 1> was added to soups and broth to prepare meat products for health promotion, soups and noodles for noodles.

<2-3> ground Beef  Produce

10 parts by weight of the n-butanol fraction of <Example 1> was added to the ground beef to prepare a ground beef for health promotion.

<2-4> dairy products ( dairy products Manufacturing

5-10 parts by weight of the n-butanol fraction of <Example 1> was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The ethyl acetate fraction of <Example 1> was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by spraying and drying with a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the ethyl acetate fractions of <Example 1> prepared above were prepared by combining the following ratios:

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Ethyl acetate fraction of Example 1 (3 parts by weight),

Ganoderma lucidum (0.5 parts by weight), and

Sulfur (0.5 part by weight).

< Manufacturing example  3> Manufacturing of beverage

<3-1> Health drink  Produce

Homogeneously blends 5 g of ethyl acetate fraction of <Example 1> with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%) After the instant sterilization it was prepared by packaging in a small packaging container such as glass bottles, plastic bottles.

<3-2> Preparation of Vegetable Juice

5 g of the n-hexane fraction of <Example 1> was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<3-3> Preparation of Fruit Juice

1 g of the n-hexane fraction of <Example 1> was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

< Manufacturing example  4> Preparation of feed additive

The present inventors prepared the feed additive with the composition as follows using the ethyl acetate fraction of <Example 1> as an active ingredient.

<The composition of the feed additive>

Ethyl acetate fraction of <Example 1>: 0.1 to 20% by weight,

Lipase: 0.001 to 0.01% by weight,

Tertiary calcium phosphate: 1-20% by weight,

Vitamin E: 0.01-0.1% by weight,

Enzyme powder: 1 to 10% by weight,

Lactic acid bacteria: 0.1 to 10% by weight,

Bacillus culture medium: 0.01 to 10% by weight, and

Glucose: 20 to 90% by weight.

Claims (10)

Patrinia villosa ) Angiogenesis-promoting agent containing the fraction obtained by fractionating the extract with an organic solvent as an active ingredient.
The method of claim 1, wherein the extract is water, C ₁ An neovascular promoter characterized by being extracted with a lower alcohol of C ₄ or a mixed solvent thereof.
The neovascular promoter according to claim 2, wherein the lower alcohol is ethanol or methanol.
According to claim 1, wherein the fraction is n-hexane fraction, ethyl acetate fraction, n-butanol fraction, n-butanol fraction or water fraction obtained by sequentially sifting the turmeric extract with n-hexane, ethyl acetate, n-butanol and water New blood vessel promoter
The neovascular promoter according to claim 4, wherein the fraction is an ethyl acetate fraction.
The neovascular promoter according to claim 1, wherein the neovascular promoter is selected from the group consisting of ischemia, chronic ulcer, wound healing, burn, ischemic stroke, rheumatoid arthritis, psoriasis, ulcer, arteriosclerosis, myocardial infarction, angina pectoris and cerebrovascular dementia Angiogenesis promoter, characterized in that for dependent diseases.
A health functional food for the promotion of neovascularization, containing the fraction obtained by distilling turmeric extract into an organic solvent.
The method of claim 7, wherein the fraction is n-hexane fraction, ethyl acetate fraction, n-butanol fraction or n-butanol fraction obtained by systematically fractionating turmeric extract with n-hexane, ethyl acetate, n-butanol and water New dietary supplements for health functional foods.
According to claim 8, wherein the fraction is an neovascular promoting health functional food, characterized in that the ethyl acetate fraction.
The dietary supplement for the neovascularization of claim 7 is an ischemia, chronic ulcer, wound healing, burn, ischemic stroke, rheumatoid arthritis, psoriasis, ulcer, arteriosclerosis, myocardial infarction, angina pectoris and cerebrovascular dementia The neovascularization promoting health functional food, characterized in that for the selected neovascular dependent disease.

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