KR101880515B1 - Composition for preventing, improving or treating ischemic disease comprising extract of Tetragonia tetragonoides as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating ischemic diseases comprising a Tetragonia tetragonoides extract as an active ingredient. The Tetragonia tetragonoides extract promotes migration and proliferation of vascular endothelial cells and promotes formation of vascular endothelial cells to increase blood flow. Therefore, the Tetragonia tetragonoides extract may be used as a pharmaceutical composition or a health functional food composition for preventing, ameliorating or treating ischemic diseases through an angiogenesis promoter action.

Description

[TECHNICAL FIELD] The present invention relates to a composition for preventing, ameliorating or treating an ischemic disease comprising an extract of Tetragonia tetragonoides as an active ingredient.

The present invention relates to a composition for preventing, ameliorating or treating an ischemic disease comprising an effective amount of a first-time extract as an active ingredient.

Ischemia refers to a state of reduced blood supply to organs, tissues or regions and ultimately leads to necrosis of cells and tissues, irreversible damage. Therefore, peripheral blood circulation, especially in blood circulation, can be considered to be very important for formation and expansion of capillary blood vessels. That is, the increase of blood flow in a blood vessel necessarily requires formation and expansion of blood vessels.

In addition, angiogenesis is a series of processes in which new blood vessels are formed in the existing blood vessels. In the blood vessels, migration of vascular endothelial cells and invasion through the extracellular matrix (ECM) invasion, proliferation, and differentiation into blood vessels (tube generation). The angiogenesis of the physiological state can occur for a short time according to the development of the fetus, female menstruation and local oxygen deficiency. Therapeutic angiogenesis is utilized because ischemic diseases or fractures do not supply blood flow smoothly.

On the other hand, Seonjangcho is a perennial plant that grows in the sandy seaside of the sea. The stem grows or stands diagonally. Its length is 40 ~ 50cm. Leaves are alternate and thick and petiole is about 2cm. Blade is egg-shaped triangle or oval, 4 ~ 6cm long, 3 ~ 5cm wide. The flowers hang 1-2 on the leaf axil from the beginning of April to the end of November, and the calyx tubers are shallowly divided into 4 to 5 branches, the surface is green and the inside is yellow. There are no petals, the fruit is nuts, and there are thorn like projections on the upper part. The young leaves eat herbs and use the outposts as medicines. It is native to southern Korea, Ulleungdo and Jeju Island, and is distributed in Japan, China, South Asia, Australia, North America and South America. It is used for the treatment of gastroenteritis, gastric ulcer, stomach cancer, uterine cancer, swelling of the skin, swelling caused by infestation by the oil hole, and so on. The outpost contains abundant iron, calcium, vitamin A and various vitamin B.

As a conventional technology for the use of the medicinal herb extract of the present invention, Korean Patent No. 1108885 discloses a technique relating to an antidiabetic agent having antidiabetic and blood cholesterol inhibitory activity, and Korean Patent No. 0483183, Extracts and pharmaceutical compositions containing them are known, but a composition for preventing, ameliorating or treating ischemic diseases containing the extract of the present invention as an active ingredient is not known.

The present invention provides a composition for preventing, ameliorating or treating ischemic diseases comprising the extract of Opadonthus edulis as an active ingredient. The present invention relates to a composition for preventing, improving or treating ischemic diseases, , Production and formation of blood, thereby enhancing blood flow volume, thereby completing the present invention.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating an ischemic disease comprising an extract of Opium japonica as an active ingredient.

The present invention also provides a health functional food composition for preventing or ameliorating an ischemic disease comprising an effective amount of a first-time extract as an active ingredient.

The present invention relates to a composition for preventing, ameliorating or treating an ischemic disease comprising an effective amount of a first-time extract as an active ingredient. Since the first-time extract of the present invention induces the migration, formation and formation of vascular endothelial cells, It can be used for medicines or health functional foods for prevention, improvement or treatment of diseases.

FIG. 1 shows a result of a laser Doppler image analysis of the improvement of the bleeding disorder of the present invention.
FIG. 2 shows the results of confirming the proliferation rate of vascular endothelial cells according to the concentration of the first-time extract of the present invention.

The present invention relates to a pharmaceutical composition for the prevention or treatment of an ischemic disease comprising an effective amount of a first-time extract as an active ingredient.

The ischemic diseases are preferably at least one selected from the group consisting of lower limb ischemic diseases, limb ischemic diseases, ischemic cerebrovascular diseases, ischemic heart diseases, and ischemia due to necrosis caused by scars and burns, and the above ischemic ischemic diseases or limb ischemic diseases The disease is characterized by menopausal age, but is not limited thereto.

Ischemia due to menopausal period may include cases in which blood circulation is not good, such as hand or foot numbness, palpitation.

The progeny extract can be prepared by a method including, but not limited to, the following steps:

(1) extracting by adding an extraction solvent in advance;

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

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

In step (1), the extraction solvent is preferably selected from water, a C ₁ -C ₄ lower alcohol or a mixture thereof, more preferably ethanol, even more preferably 70% (v / v) ethanol But is not limited thereto.

In the above production method, any conventional method known in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used. The extraction solvent is preferably added by 1 to 20 times the dry weight of the first step, more preferably 5 to 15 times, and further preferably 10 times by weight. The extraction temperature is preferably 4 to 50 ° C, but is not limited thereto. The extraction time is preferably 10 to 100 hours, but is not limited thereto. In the above method, the concentration of the step (3) is preferably, but not limited to, using a vacuum rotary condenser or a vacuum rotary evaporator. In addition, drying is preferably carried out under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, more preferably freeze drying or the like.

The progeny extract of the present invention is characterized by inducing migration and proliferation of vascular endothelial cells and promoting formation of vascular endothelial cells to increase blood flow.

In addition to the herbal extract, the composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and it is preferable to use an intravenous or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebral injection method during parenteral administration.

The pharmaceutical composition of the present invention can be prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

The composition according to the present 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 the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention may be varied depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.

The present invention also relates to a health functional food composition for preventing or ameliorating an ischemic disease comprising an effective amount of a first-time extract as an active ingredient.

Preferably, the ischemic disease is at least one selected from the group consisting of lower limb ischemic diseases, limb ischemic diseases, ischemic cerebrovascular diseases and ischemic heart diseases, but is not limited thereto.

The health functional food composition for preventing or ameliorating an ischemic disease comprising the extract of the present invention as an active ingredient may be prepared from any one selected from powder, granule, ring, tablet, capsule, candy, syrup and beverage, And can be suitably prepared according to a conventional method.

Examples of the food to which the extract of the present invention can be added include a dairy product including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, A tea, a drink, an alcoholic beverage, and a vitamin complex, all of which include health functional foods in a conventional sense.

The health functional foods include various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, alkynic acid and its salts, , pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices and vegetable drinks. These components may be used independently or in combination.

The health functional food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient. The natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, 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.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  One. Early spring  Preparation of extract

 In the present invention, the fresh shoots used in the present invention were collected or used in Jeju Island, South Korea. The dried seedlings were added with 10 times 70% (v / v) ethanol, and leached at room temperature for 48 hours. An extract was obtained. The obtained first-time extract was filtered with a 0.4 mu m filter, concentrated by a vacuum rotary condenser and lyophilized.

Example  2. Early spring  Analysis of blood flow changes by administration of extracts

Female C57BL / 6 mice were fed 6 weeks of age, followed by a week of fertilization, and then ovariectomized at 7 weeks of age. At the age of 8 weeks, left hind-limb blood vessels (arteries, veins) in two areas were ligated and separated to complete the ischemic model.

This experiment was conducted by Korea Institute of Oriental Medicine (16-018), and the breeding and maintenance of experimental animals were conducted at 23 ± 1 ℃ and 50 ± 2 ℃, respectively, based on the animal care and use regulations of Korea Institute of Oriental Medicine. 10%, and 12 hours of light cycle, and maintained in an IVC (individually ventilated cage).

For the treatment of benignant control I (simvastatin; SV), positive control II (estrogen, E ₂ ), and prosodic extract, samples were included in the diets of AIN-76A for freezing.

Blood flow of the left lower limb was measured using a laser Doppler imaging device for 28 days and compared with that of the right lower limb as a simulated surgical control, an ischemic / non-ischemic ratio (blood flow rate).

As a result, the blood flow rate after lower limb ischemia was significantly decreased in all groups compared with before ischemia induction (-7 days).

In contrast to the control group (OH), which was treated with ovariectomy (OVX) after ischemia (HLI), there was a significant difference in the blood flow rate between the control group I and the high- The effect was observed. After 14 days and 28 days after induction of ischemic injury, significant effects were observed in both positive control (I, II) and nonsteroidal extract administration groups (Table 1 and Fig. 1) compared to negative control vehicle.

The results of the laser Doppler image analysis for the improvement of the hemostasis disorder of the present invention group
Before surgery On the day of surgery After surgery -7 days 0 days 7 days 14 days 28th vehicle (OH) 1.01 + 0.04 0.21 ± 0.05 ^* 0.40 0.12 0.50 0.07 0.64 ± 0.08 Positive control I
(OH + SV; 20 mg / kg / day) 0.99 ± 0.03 0.25 0.06 ^* 0.61 + 0.07 ^a 0.70 ± 0.11 ^a 0.90 + 0.06 ^a Positive control II
(OH + E ₂ ; 100 μg / kg / day) 1.02 + 0.02 0.29 0.08 ^* 0.47 ± 0.08 ^b 0.78 ± 0.09 ^a 0.85 + 0.08 ^a TTG 150
(OH + TTG; 150 mg / kg / day) 1.00 + 0.02 0.21 0.04 ^* 0.47 ± 0.07 ^b 0.62 ± 0.06 ^ac 0.83 0.07 ^a TTG 450
(OH + TTG; 450 mg / kg / day) 0.99 + 0.04 0.22 0.04 ^* 0.55 + 0.10 ^a 0.63 + 0.09 ^ac 0.87 ± 0.09 ^a

* p < Before surgery, ^a p <0.05 vs. Vehicle, ^b p < Positive I, ^c P < Positive II.

Example  3. Identification of non-distal peripheral angiogenesis

Immunohistochemical staining was performed on left lower limb thigh and calf muscle induced by lower limb ischemia using CD31 (cluster of differentiation 31), which is a biomarker of vascular endothelial cells. The tissues were sliced to a thickness of 5 mu m with paraffin sections, and deparaffinized with xylene. Low concentration of ethanol was used at high concentration and finally water was used in tap water. Antibody used is a number of Abcam Inc. Anti-CD31 (rabbit polyclonal to CD31 ) a was used, based on the final staining positive cell (brown color), peripheral blood vessel (capillary) counted per unit area (mm ²⁾ the number of Respectively.

As a result, it was confirmed that peripheral blood vessel formation was statistically significantly increased in the case of the treatment with the progeny extract as shown in Table 2. [

Identification of Peripheral Angiogenesis by the Treatment of Pseudomonas aeruginosa Extract group Negative control
(OH) Positive control I
(OH + SV) Positive control II
(OH + E ₂₎ 150 seconds
(OH + TTG150) 450 seconds
(OH + TTG450) Peripheral blood vessel distribution (mm ² ) 251.8 ± 15.2 419.6 ± 50.8 ^a 406.3 ± 27.6 ^a 342.9 ± 13.8 ^abc 407.1 ± 21.6 ^ad

^a p < Vehicle, ^b p < Positive I, ^c P < Positive II, ^d P < TTG  150

Example  4. Early spring  Identification of vascular endothelial cell proliferation by treatment of extract

The vascular endothelial cells were attached to a 96-well plate at 4 × 10 ³ cells / well and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. After incubation for 24 hours, 10 μl of EZ-cytox (Biomax. Korea) was added to the culture solution. After 4 hours, the absorbance was measured at 450 nm , And the measured value was compared with the control group to calculate the proliferation rate of the cells.

As a result, as shown in the following Table 3 and FIG. 2, the cell proliferation rate was statistically significant in the case of treatment with 5, 50, and 100 / / ㎖ of the pre-treatment extracts compared with the cells not treated with the TTG Respectively.

The proliferation rate of vascular endothelial cells TTG
(占 퐂 / ml) 0 One 2 5 50 100 Growth rate
(%) 100 ± 6.52 101.94 + - 4.65 100.52 + - 3.54 126.01 + 4.02 ^a 137.46 ± 2.93 ^a 144.14 + - 5.44 ^a

^a p < 0.001 vs . non - treated cell

Example  5. Early spring  Effect of Extracts on Vascular Endothelial Cell Migration

To the upper well of the transwell, 3 × 10 ³ HUVAC cells and serum-free EGM2 medium were added with 10 and 50 μg / ml, respectively, 50 ng / ml VEGF treated group, 1 nM E2 treated group, and 10 μM simvastatin treated group for 6 hours. The migrated vascular endothelial cells were stained with H & E staining method, and the effect of the progeny extract of the present invention on the vascular endothelial cell migration was compared with the control group and the migration rate.

As a result, as shown in Table 4, it was confirmed that the addition of 10 and 50 μg / ml of Puerariae Radix extract significantly increased the migration speed statistically compared to the control group without any treatment (Normal).

Vascular endothelial cell migration (migration) group Normal VEGF
(50 ng / ml) E ₂
(1 nM) Simvastatin
(10 [mu] M) Early spring
(10 [mu] g / ml) Early spring
(50 mu g / ml) No. of migrated cells 1 ± 0.82 122.33 + 4.92 ^a 105.33 ± 4.11 ^a 58.33 ± 3.68 ^a 61 ± 5.72 ^a 106.33 + 4.19 ^{a, b}

^a p < Normal, ^b  p < Simvastatin

Example  6. Early spring  Effect of Extract on the Differentiation of Vascular Endothelial Cells

The vascular endothelial cells were divided into 3 × 10 ⁵ Matrigel-coated 24-well plates with extracellular matrix (ECM) in EGM2 medium containing 1% fetal bovine serum. Tube formation was induced. Twelve hours later, the cells were stained with Calsein AM, observed and photographed with a microscope and the tube branching interval was measured using the Image J program's Angiogenesis Analyzer and compared with the control group.

As a result, as shown in Table 5, it was confirmed that the tube formation rate was statistically significantly increased when 10 and 50 μg / ml of the first extracts were added, respectively, as compared with the control (Normal) without any treatment.

Tube-forming ability of vascular endothelial cells group Normal Bin blastin
(1pM) VEGF
(50 ng / ml) E ₂
(1 nM) Simvastatin
(10 [mu] M) Early spring
(10 [mu] g / ml) Early spring
(50 mu g / ml) Branching interval (%) 100 ± 4.96 96.38 + - 4.51 261.58 ± 26.32 ^c 194.07 ± 19.63 ^b 93.80 ± 9.40 176.96 ± 9.40 ^{a, d} 222.39 + - 1.56 ^{c, e}

^a p < 0.05,  ^b  p < 0.01 ^c p < Normal, ^d  p < 0.05,  ^e  p < Simvastatin

Claims (7)

A pharmaceutical composition for preventing or treating a lower limb ischemic disease or a limb ischemic disease comprising an effective amount of a first-time herb extract as an active ingredient. delete The pharmaceutical composition according to claim 1, wherein the lower limb ischemic disease or the limb ischemic disease is caused by menopausal period, wherein the composition is for preventing or treating limb ischemic diseases or limb ischemic diseases. The method of claim 1, wherein the extraction solvent of the tetragonia tetragonioides extract is water, C ₁ ~ C ₄ lower alcohol, or not, characterized in that mixtures of these vascular disease or ischemic limb distal prevention or treatment a pharmaceutical composition for ischemic diseases. 2. The method according to claim 1, wherein the pre-emergence herbal extract induces migration and proliferation of vascular endothelial cells and promotes formation of vascular endothelial cells to thereby increase blood flow. &Lt; / RTI &gt; Wherein the composition comprises an effective amount of a crude extract of the present invention as an active ingredient, for preventing or ameliorating a vascular ischemic disease or a limb ischemic disease. delete

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