KR101485107B1 - Composition for blood circulation comprising Gastrodia elata, Siegesbeckeae glabrescens, and Saposhnikoviae divaricata Schiskin extracts - Google Patents

Abstract

In the present invention, provided is a composition for circulating blood including extracts of Gastrodia elata, Sigesbeckia glabrescens Makino and Saposhnikovia divaricata Schiskin. The extract mixture of Gastrodia elata, Sigesbeckia glabrescens Makino and Saposhnikovia divaricata Schiskin can be used for treating or preventing diseases related to blood circulation or similar diseases. For example, a functional food composition for promoting health like improving thrombosis includes Gastrodia elata, Sigesbeckia glabrescens Makino and Saposhnikovia divaricata Schiskin extracts as active ingredients and acceptable carriers in food and thus is allowed to efficiently use the same.

Description

[0001] The present invention relates to a composition for improving blood circulation comprising Gastrodia elata, Siegesbeckeae glabrescens, and Saposhnikoviae divaricata Schiskin extracts,

The present invention relates to a composition for improving blood circulation comprising an extract of Angelica keiskei koidz.

Diseases related to thrombosis, which accounts for about 39% of all deaths in the world, have recently been increasing in Korea due to westernization of diet, excessive stress, and aging population.

The world's antithrombotic and anticoagulant markets are expected to reach $ 24.4 billion by 2015, and sales should gradually increase until 2022. In particular, the incidence, prevalence, and number of diagnoses in developing countries such as China and India are expected to contribute to sales growth. The introduction of new oral anticoagulants, which are superior in terms of safety and efficacy, and pharmaceuticals and medicines with drug delivery methods that facilitate easy prescription and compliance are expected to replace existing treatments.

It is anticipated that products for the treatment of antiplatelet agents, heparins, fibrinolytics, vitamin K antagonists, direct thrombin inhibitors and direct factor Xa inhibitors will be actively marketed in the international market in 2012.

Streptokinase, urokinase, and tissue plasminogen activator (t-PA) are known to be currently used as thrombolytic enzymes, and mainly plasminogen in vivo is called plasmin Lt; RTI ID = 0.0 > a < / RTI >

Such plasminogen activators are classified as allergies, fever, local hemorrhages, short half-lives and expensive drugs, and thus they are limited to thrombosis treatment agents. In addition, the proteolytic enzymes include brinase, papain, ochrase, trypsin, and plasmin. However, when injected by intravenous injection, they destroy the thrombus formation factors due to random protein breakdown, . Streptokinase and europinease are exogenous enzymes that convert plasminogen to plasmin. They contain side effects such as fever, allergies, local hemorrhage and hypotension, and they are classified as medicines that are very expensive. And is not used for prevention of thrombus formation in the body.

Therefore, it is urgent to develop a safe and inexpensive blood circulation improving agent separated from various natural resources.

1. Korean Patent Publication No. 10-2006-0005805 (Jan. 18, 2006)

The inventors of the present invention have conducted intensive studies to search for and develop a functional substance in natural products. As a result, they found that a mixed extract of Chunma, scarlet and windshield has not only side effects but also blood circulation improving function. The present invention has been accomplished.

Accordingly, an object of the present invention is to provide a composition for improving circulation, which comprises a mixed extract of chymus, sclerotium and windshield as an active ingredient, and comprises a pharmaceutically acceptable carrier.

INDUSTRIAL APPLICABILITY As described above, the mixed extract of Ganoderma lucidum, Heliothermia and Windshield according to the present invention can be effectively used for the treatment and prevention of diseases or similar diseases requiring blood circulation improvement.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of measurement of electron donating ability against DPPH radicals,
2 is a graph showing the activity of antioxidant enzymes,
FIGS. 3A to 3E are graphs showing concentration-dependent effects on the platelet aggregation of the chromatin, spots,
FIG. 4 is a graph showing platelet aggregation inhibition ratios of the chimma, spearmint, windshield extract,
FIG. 5 is a graph showing the results of measurement of the electron donating ability of DPPH radical according to the concentration of GNC-5,
6 is a graph showing the results of measuring the thrombus weight by treating the mixed extract of Chunma,
7 is a graph showing the flow of blood flow in an animal model of carotid thrombosis,
8 is a graph showing the antioxidative activity of an animal model deprived of chronic cerebral perfusion,
9 is a graph showing the activity of glutathione reductase activity measured,
FIG. 10 is a graph showing the electroencephalogram before and after the administration of the extract,
11 is a graph showing a result of underwater maze learning.

In one aspect, the present invention provides a composition for improving circulation, which comprises a mixed extract of chymus, europium, and windshield as an active ingredient, and comprises a pharmaceutically acceptable carrier.

Among the compositions of the present invention, Gastrodia elata refers to the roots of Gastrodia elata Blume, a perennial herb that belongs to the Orchidaceae family, and includes red roots, They were also called other names such as Hama, Shingo, and Jeongpungcho. Because there is no chlorophyll in Chunma, photosynthesis of nutrients by using carbon assimilation is impossible, and it grows by supplying nutrition with convenient mingling with mulberry mulberry (Amillaria mellea) because there is no root. Chunma is mainly distributed in Korea, China, Japan, Taiwan and it grows in the forest of valley with many humus. It is 60 ~ 100 ㎝ in size and has leafless potato stalks. Bulb stem is long oval, 10 ~ 18 ㎝ in length and 3.5 ㎝ in diameter. The stem is reddish brown with small leaves. Leaf-like leaves are membranous, with a margin and the lower part surrounding the stem. Flowers are yellowish brown, bloom in June-July, and 3 foreign perianths are combined. It looks like a distorted ball, the top part is divided into 3 pieces, and there are two pieces of fire-proofed inside. It is an antiparasitic plant that grows in symbiosis with other mushrooms in oak root.

In addition, Siegesbeckeae glabrescens is a plant called Compositee which lives in Jeju and southern parts of Korea, Japan, Manchuria, China and Taiwan. It is an annual plant which is widely grown in mountains and fields. It blooms around August. It is said that the scent of grass is similar to pigs, and because it has a spicy and spicy taste, it is also called a celebration. When young sprouts are soaked in water, the bitter taste disappears. It is also called "vise" because it is eaten with oil and salt. The stem is straight and cylindrical, 35-100 cm tall, brownish purple, with fine hairs but without hair and branches. Leaves of the central part are long and petiole triangle, length is 4 ~ 15 ㎝, width is 3.5 ~ 11 ㎝. It has short hairs on both sides and irregular sawtooth on the edge. As it goes up, the leaf becomes smaller and it is long oval or linear. The flower blooms in yellow, and the tip of the branch and stem is head-shaped flower, which is composed of a lot of small flowers with no peduncle at the end of the peduncle. The fruit is an aphrodisiac, with an egg length of 2 ㎜ upside down. It has four fascinating lines and sticks to other objects.

The main efficacy is the efficacy of anti-inflammatory, anti-inflammatory and anti-inflammatory effects of rheumatoid arthritis, hypertension, malaria, insomnia, paralysis due to paralysis, swelling, rash, skin itching and eczema. It has recently been reported to have antiallergic and contraceptive effects. Diterpenodis, siegesbeckioside, siegebeckiol and siegesbeckic acid are known to be active ingredients.

On the other hand, Saposhnikoviae divaricata Schiskin is a perennial plant belonging to Umbelliferae, which is a perennial herb called jinhwa wind, mountain wind wind, screen wind herb, mountain windbreak tree, It is widely distributed in Korea, China, Vietnam and Mongolia. It is a 3 - year - old herb with a height of 60 ~ 100 ㎝ and a stem diameter of 1.5 ㎝. It is dark green with dark purple cell stripes, with many branches and no hairs. The surface of leaves is dark green, the back side is green, and the flesh is thick and glossy. In June-July, peduncles of branches and trunks are split into umbrella-shaped mountains. On the mountain, 20 ~ 30 small peduncles were divided and bloomed in a double-sided form with a small scattering. The long, rounded fruit is dark brown and contains about 1,000 seeds, weighing about 4 grams.

It is known that the main effect of windshield by oriental medicine is to treat sweating, paralysis, analgesic, diuretic, paralysis, limb paralysis, shinhae, gerundam, toothache, neuralgia and rheumatism.

The mixed extracts of dried chestnut, squid, and windshield according to the present invention were prepared by mixing these materials in a predetermined ratio (for example, a ratio of 1: 1: 1 to 2: 1: 1) After the addition of a reflux condenser, the mixture was warmed in a water bath at 95-100 ° C for 12 hours to obtain an extract. The extract was cooled to about 50 ° C and filtered with a layer of gauze to obtain a supernatant. The same extraction and filtration operations were repeated three times. The supernatants were combined, concentrated under reduced pressure using a rotary evaporator, dissolved in a small amount of distilled water, and the finally obtained extract solution was lyophilized at -80 ° C to obtain a powder form .

Alternatively, the chewing gum, scarlet, and windshield extracts may be thoroughly cleaned, dried, and then placed in a container. The ingredients and water are mixed in a weight ratio of 1: 1 to 5 parts by weight and placed in a autoclave autoclave. Extraction at a temperature of 2 to 5 hours, filtration and concentration.

The chimma, scarlet and windshield extracts according to the present invention are natural plant extracts and can be used as food and therapeutic agents for the treatment and prevention of diseases requiring improvement of blood circulation because they exhibit not only toxicity but also high antioxidative activity.

Accordingly, the present invention provides, in a further aspect thereof, a composition for improving circulation comprising, as an active ingredient, a chewing gum, an excipient and a windshield extract, and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention can be used as a medicament for improving blood circulation and a person skilled in the art can easily identify individuals who are suspected of suffering from such diseases, conditions and abnormalities using standard diagnostic techniques. Such diseases include, but are not limited to, brain diseases such as cerebral thrombosis, cerebral embolism, pulmonary diseases such as obstructive pulmonary disease, pulmonary infarction, deep vein thrombosis, gait disorder, blood flow obstructive anemia, arteriosclerotic ganglion, neuralgia, , Cardiovascular diseases such as angina pectoris, ischemic heart disease, myocardial infarction and the like, and the composition of the present invention may be particularly preferably used for the diseases And those skilled in the art can easily identify individuals who are presumed to suffer from such diseases, conditions and disorders.

The pharmaceutical composition of the present invention can be formulated in combination with a known pharmaceutical carrier. Generally, the active ingredient is mixed with a pharmaceutically acceptable liquid or solid carrier and, if necessary, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, , Granules, powders, powders, capsules, and the like, liquid solutions such as liquid solutions, suspensions, and emulsions. It may also be a dried product which can be made into a liquid phase by the addition of a suitable carrier before use.

The pharmaceutical composition of the present invention may be administered by any of the parenteral preparations such as oral agents, injections, and spot-applying agents.

The pharmaceutical carrier can be selected in accordance with the above dosage form and formulation, and in the case of an oral preparation, for example, starch, lactose, white sugar, mannitol, carboxymethyl cellulose, corn starch, inorganic salt and the like are used. In the preparation of an oral preparation, a binder, a disintegrant, a surfactant, a lubricant, a flowability promoter, a mating agent, a colorant, a perfume, and the like may be further blended.

On the other hand, in the case of the parenteral preparation, the composition containing the protease, which is the active ingredient of the present invention, is dissolved in distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, soybean oil, Propylene glycol, polyethylene glycol and the like, and if necessary, adding a bactericide, stabilizer, isotonizing agent, an anhydrous agent and the like.

The pharmaceutical composition of the present invention is administered by an appropriate route according to the form of the preparation. The method of administration is not particularly limited, and it can be administered by the contents, external application and injection. The injection can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, and the like.

The dose of the pharmaceutical composition of the present invention is appropriately set in accordance with the form of the pharmaceutical composition, the administration method, the purpose of use, and the age, body weight, and symptoms of the patient to which the active ingredient is administered. The amount is, for example, 10 μg to 10 mg / kg per adult day. However, those skilled in the art will appreciate that the pharmacokinetic properties of a particular reagent and its mode and route of administration; Age, health, weight; It will be understood that the dosage varies depending on known factors such as the nature and extent of the symptoms, the type of concurrent treatment, the frequency of treatment and the desired effect.

In addition, the extract of Chunma, Helium, and Windshield of the present invention can be provided as a health functional food for improving blood circulation.

The formulation of the food composition according to the present invention can be formulated in the form of a drink such as an oral preparation, a pouch, or a drink, according to a conventional method in the art.

The dosage of the food composition of the present invention is appropriately set in accordance with the form of the composition, the method of administration, the purpose of use, and the age, weight and symptom of the individual to which the composition is applied. Capsules from 20mg to 100mg per 450mg can be taken as an adult dose of 6 capsules per day. Of course, since the dosage varies depending on various conditions, the amount may be less than the above range, or may be more than the above range.

The food composition according to the present invention may contain conventional additives or additives, or sweeteners such as licorice, bean curd powder, wolfberry extract, St. John's Wort, selenium yeast, vitamin C, citric acid, nicotinic acid, One or more ingredients selected from the group consisting of sodium, aspartame, saccharin, pectin, maltitol, sorbitol, xylitol, guar gum, skim milk powder and oligosaccharide may be added to increase taste and aesthetics. They are suitably used in an amount of about 50 to 80% based on the total weight of the composition of the present invention.

<Examples>

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples.

The chewing gum, scorpion and windblown used in the following examples were purchased from a herbal cooperative association in Hwasun-gun, Jeollanam-do.

Example  1: Chungma single extract ( GSS -1)

5 kg of 80% ethanol was added to 1 kg of dried chitin. After the reflux condenser was installed, it was warmed in a water bath at 95-100 ° C for 12 hours. The extract was cooled to about 50 ° C and filtered with several layers of gauze to take the supernatant. The extraction and filtration operations were repeated three times, and the supernatants were combined and concentrated under reduced pressure using a rotary evaporator. The resulting extract solution was lyophilized at -80 ° C to obtain 217 g of a powdered Chionma single extract (GSS-1), which was used as a sample.

Example  2: Recruitment  Single extract ( GSS -2)

To 1 kg of dried squares was added 5,000 ml of 80% ethanol. After the reflux condenser was installed, it was warmed in a water bath at 95-100 ° C for 12 hours. The extract was cooled to about 50 ° C and filtered with several layers of gauze to take the supernatant. The extraction and filtration operations were repeated three times, and the supernatants were combined and concentrated under reduced pressure using a rotary evaporator. The resulting extract was lyophilized at -80 ° C to obtain 155.8 g of a single extract (GSS-2) in powder form, which was used as a sample.

Example  3: windshield single extract ( GSS -3)

To 1 kg of dried windshield was added 5,000 ml of 80% ethanol. After the reflux condenser was installed, it was warmed in a water bath at 95-100 ° C for 12 hours. The extract was cooled to about 50 ° C and filtered with several layers of gauze to take the supernatant. The extraction and filtration operations were repeated three times, and the supernatants were combined and concentrated under reduced pressure using a rotary evaporator. The resulting extract was lyophilized at -80 ° C to obtain 185.0 g of a powdery windshield extract (GSS-3), which was used as a sample

Example  4: Recruitment , Windshield mixed extract ( GSS -4)

Dried chestnut, squirting, and windshield were mixed at a ratio of 1: 1: 1, and 5 kg of 80% ethanol was added to a total of 1 kg. After the reflux condenser was installed, it was warmed in a water bath at 95-100 ° C for 12 hours. The extract was cooled to about 50 ° C and filtered with several layers of gauze to take the supernatant. The extraction and filtration operations were repeated three times, and the supernatants were combined and concentrated under reduced pressure using a rotary evaporator. The resulting extract solution was lyophilized at -80 ° C to obtain 185.5 g of powdery mixed extract (GSS-4), which was used as a sample.

Example  5: Recruitment , Windshield mixed extract ( GSS -4)

Dried chestnut, squid, and wind were mixed at a ratio of 2: 1: 1, and 5 kg of 80% ethanol was added to 1 kg total. After the reflux condenser was installed, it was warmed for 12 hours in a water bath at 95-100 ° C. The extract was cooled to about 50 ° C and filtered with several layers of gauze to take the supernatant. The extraction and filtration operations were repeated three times, and the supernatants were combined and concentrated under reduced pressure using a rotary evaporator. The resulting extract solution was lyophilized at -80 ° C. to obtain 176.9 g of powdery mixed extract (GSS-4), which was used as a sample.

Example  6: Pharmaceutical formulation

Form of formulation (tablets)

Mixed extract (Example 4) 10 mg

Lactose 60 mg

Starch 15 mg

Magnesium stearate 5 mg

Crystalline cellulose 15 mg

One embodiment of the present invention is the use of the blood circulation improving agent as an example of an effective ingredient of the tablet as described above. Tablets may be prepared by conventional methods and one preferred embodiment is in the form of tablets or soft capsules with conventional enteric coatings (e. G., Hydroxypropylmethylcellulose phthalate), sugar coating or coatings.

Example  5: Manufacture of pouches

1 g of licorice as an additive was added to 100 g of the mixed extract obtained in Example 4 and vacuum packed in a conventional manner to prepare a pouch.

Example  6: Manufacture of drinking water

20 g of citric acid, 17 g of aspartame and 30 g of vitamin C were added to 100 g of the mixed extract obtained in Example 5, and the remaining was added with purified water to prepare 10 L of drinking water and pasteurized. As a result, the beverage of the present invention obtained favorable responses to conventional beverage products in terms of mouth feel, taste, and overall evaluation.

Test Example  1: General composition analysis

General components were analyzed according to the AOAC method. The content of crude protein was calculated by multiplying the nitrogen content measured by Kjeldahl method by the nitrogen factor of 6.25. The results are shown in Table 1.

division Crude protein Chunma 6.15% Spots 4.65% Wind wind 7.69%

From the above results, the crude protein content of the samples was 6.15%, 4.65%, and 7.69%, respectively.

Test Example  2: Analysis of organic acids

To 1 g of the lyophilized sample, 50 ml of distilled water was added and the mixture was refluxed at 120 ° C for 1 hour, extracted with 50 ml of water, and centrifuged (3,000 rpm, 30 min) ), ^{Purified with} Sep-Pak C18, and analyzed by HPLC using a 0.45 ㎛ membrane filter (Millipore Co., USA). The analysis conditions are shown in Table 2, and the content was calculated by the external standard method. The results are shown in Table 3 (mg).

Item Analysis condition Device Agilent Technologies 1200 Series column Agilent Zorbax SB-Aq (4.6 x 150 mm, 5 [mu] m) menstruum 20 mM NaHPO ₄ : ACN (99: 1) Column temperature 30 ℃ wavelength 210 nm Flow rate 1.0 ml / min Injection capacity 5 μl

division Oxalic acid Malic acid Citric acid Succinic acid Total content Chunma 125.39 1,428.15 1,746.38 1,514.01 4,813.93 Spots  14.70  832.40 405.68  350.50 1,603.28 Wind wind  15.06 708.40 380.68   308.20 1,412.34

As can be seen from Table 3, oxalic acid, malic acid, citric acid and succinic acid were detected as main organic acids, and the total content of organic acids of Chunma was 4813.94 ㎎%, which was higher than that of 1,603.28 ㎎% and 1,412.34 ㎎% Respectively. Among the four organic acids detected, citric acid was the highest at 1746.38 ㎎%, malic acid was 832.43 ㎎% and malic acid was 708.40 ㎎%.

Test Example  3: Free sugar analysis

Free sugar components were analyzed by the method of Wilson et al., And free sugar extraction was treated as shown in Fig. 17. Was added to 80% ethanol 20㎖ to 1g sample was refluxed for 1 hour at 120 ℃, with 50 ㎖ jeongyonghan then centrifuged (3,000 rpm, 30 min) and was purified by C18 Sep-Pak ^ㄾ then 0.45 ㎛ membrane filter (Millipore Co., USA), and the filtrate was analyzed by HPLC. The content was calculated by the external standard method, and the HPLC conditions are shown in Table 4. The results are shown in Table 5 (mg).

Item Analysis condition Device Agilent Technologies 1200 Series column Agilent Zorbax SB-Aq (4.6 x 150 mm, 5 [mu] m) menstruum 85% acetonitrile Column temperature 30 ℃ Flow rate 1.4 ml / min Injection capacity 5 μl

division Fructose Glucose Sucrose Total content A thousand words 3.56 4.75 3.07 11.38 Succulent 0.40 0.90 0.00 1.30 Wind 10.30 8.93 0.00 1.23

From Table 5, the main sugars were fructose, glucose and sucrose. The content of free sugars was found to be 11.38 ㎎% and 1.41 ㎎%, respectively. The contents of fructose were 10.30 ㎎% and 3.57 ㎎%, respectively, and that of glucose was 4.75 ㎎%, 0.90 ㎎% and 8.93 ㎎%, respectively. The content of sucrose was 3.07 ㎎ %.

Test Example  4: Analysis of constituent amino acids

0.1 g of the sample was placed in a test tube, 10 ml of a 6N-HCl solution was added thereto, and the end of the test tube was melted to make an ampule and sealed. The filtrate obtained by hydrolysis at 110 ° C for 24 hours was filtered through filter paper and 100 ml of methanol was used. The filtrate was concentrated at 50 ° C to completely evaporate hydrochloric acid and methanol, and then diluted with 5 ml of 20 mM HCl. After filtration with 0.45 ㎛ membrane filter, a certain amount of the filtrate was subjected to derivatization using AccQ-Tag reagent, and the constituent amino acids were analyzed by HPLC. The analysis conditions are shown in Table 6. The results are shown in Table 7 (mg).

Item Analysis condition Device Agilent Technologies 1200 Series Detector Agilent Technologies 1200 Series FLD column AccQ-Tag ^™ (Waters Co., 150 mm L x 3.9 mm ID) Buffer A: AccQ-Tag Eluent A (acetate-phosphate buffer)
B: AccQ-Tag Eluent B (60% acetonitrile) Column temperature 30 ℃ Flow rate 1.0 ml / min Injection capacity 10 μl

division A thousand words Succulent Wind Non - essential Aspartic acid 406.93 900.55 1,208.50 Serine 252.07 470.60 600.60 Glutamic acid 530.72 740.50 1,108.47 Glycine 382.96 460.55 960.57 Arginine 276.07 400.06 1,100.06 Alanine 233.54 540.50 560.54 Proline 189.96 435.60 920.58 Tyrosine 0.00 86.40 176.50 Essential Valine 246.04 320.50 382.67 Methionine 0.00 140.40 136.40 Lysine 261.4 485.70 683.11 Isoleucine 164.37 370.30 600.30 Leucine 225.48 680.12 900.12 Threonine 129.99 135.00 353.00 Histidine 328.75 430.40 550.40 Phenylalaine 167.66 186.50 175.50 content 3,795.94 6,783.68 9,691.42

The total amino acid content of the samples was 3795.94 ㎎%, 6,783.68 ㎎% and 9,791.42 ㎎%, respectively. The major amino acids were as follows: glutamic acid, 530.72 ㎎%, Aspartic acid and glycine. Glutamic acid and leucine were the major constituent amino acids. Aspartic acid and glutamic acid were the main constituent amino acids. Aspartic acid and glutamic acid were the major constituent amino acids, respectively.

Test Example  5: inorganic component analysis

The inorganic components were analyzed by pretreatment by the dry decomposition method and the analysis conditions are shown in Table 8. Concentrate 1 g of sample at 600 ° C to obtain a white ash. 10 ml of concentrated hydrochloric acid, diluted twice with ash, was added to the filtrate and evaporated to dryness in a water bath. 10 ml of diluted hydrochloric acid was added, diluted with distilled water to 100 ml, and the filtrate was used as an analytical sample. The quantitative determination of each inorganic component was carried out using an atomic absorption spectrophotometer (Perkin Elmer AAnalyst 400). Standard concentrations of each element were adjusted to 0.1, 0.5 and 1.0 ppm, and standard calibration curves were prepared and analyzed. The results are shown in Table 9 (mg).

Item Analysis condition Device Atomic Absorption Spectrophotometer
(Perkin Elmer Analyst 400) Flow rate C ₂ H ₂ , 2.0 ml / min Oxidant rate Air, 10.0 ml / min Wavelength (nm) K: 766.49, Mg: 285.51, Na: 589.00, Ca: 422.67

division A thousand words Succulent Wind K 649.23 134.38 140.50 Ca 60.45 42.20 30.50 Na 14.47 8.23 11.40 Ma 72.25 96.49 65.30 CD 0.00 0.00 0.00 Cr 0.00 0.00 0.00 Cu 0.00 0.00 0.00 content 796.4 281.3 247.7

As a result, total inorganic components were found to be 796.40 ㎎%, 281.3 ㎎%, 247.7 ㎎%, and 247.7 ㎎%, respectively, in Chunma, . The major inorganic components of the samples were as follows; K was 649.23 ㎎% and Ca and Mg were the major inorganic components. The content of K was 134.38 ㎎%, Ca and Mg were the major inorganic components. The content of K was 140.50 ㎎% and Mg was the highest at 65.30 ㎎%.

Test Example  6: DPPH solution Using Antioxidant ability  Measure

To investigate the antioxidant activity of the sample, free radical scavenging activity, one of the antioxidative activities, was measured according to the method of Blois. DPPH (16 mg) was dissolved in 100 ml of ethanol, mixed with 100 ml of distilled water, filtered through a filter paper, 0.5 ml of the sample was mixed with 2.5 ml of the filtrate, and the mixture was reacted at room temperature for 10 minutes. To measure the amount of DPPH remaining, the change in absorbance was measured at 528 nm using a microplate reader (Molecular Device, Sunnyvale, CA, USA). Ascorbic acid was used as a positive control.

The antioxidant activity of DPPH radicals was measured to determine the antioxidative activity of the extracts of Chunma, Scutellariae Radix, Windward monoculture and mixed extract (Example 4). The results are shown in Fig. 1 is a graph showing the results of measurement of the electron donating ability against the DPPH radical.

As a result, DPPH radical scavenging activity of chimma, scarlet, windblown mono - extract and mixed extract showed DPPH radical scavenging effect compared to ascorbic acid in each sample. It was confirmed that the free radical scavenging activity of GSS-4 was superior to that of the control group.

Test Example  7: Measurement of antioxidant enzyme activity

Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) are three enzymes important for cell defense against ROS-mediated cellular damage. Antioxidant defense systems in the body, including SOD, CAT and GPx, can be damaged by the aging process. The resulting damage can even lead to mutations in genetic material that can lead to disruption of biological processes and death of cells and the development of cancer. Thus, the effects of oxidative stress and the progression of degenerative diseases and aging can be prevented.

Superoxide dismutase (SOD) activity was measured by the method of inhibiting autoxidation of epinephrine to adrenochromine to confirm the antioxidative effect of the extract of Chunma, ) Activity was measured by the method of Karmazyn et al., Which measures the amount of oxygen generated upon decomposition of H ₂ O ₂ by this enzyme with an oxygen monitor. Glutathione peroxidase (GPx) activity was measured by Paglia and Valentine methods. The results are shown in Fig. 2 is a graph showing antioxidant enzyme activity.

The antioxidant enzymes SOD (superoxide dismutase), CAT (catalase), and GPx (glutathione peroxidase) were found to have a SOD value of 7.20 ± 0.50 U / ㎎ protein, CAT 10.05 ± 0.40 U / ㎎ protein and GPx 2.12 ± 0.30 U / ㎎ / protein. In GSS-2, SOD showed 7.32 ± 0.50 U / ㎎ / protein, CAT 9.83 ± 0.50 U / ㎎ / protein and GPx 2.30 ± 0.20 U / protein, CAT 9.86 ± 0.50 U / ㎎ / protein and GPx 2.56 ± 0.26 U / ㎎ / protein, respectively. In GSS-4, SOD 9.33 ± 0.68 U / ㎎ / protein and CAT 10.48 ± 0.84 U / GPx 3.05 ± 0.45 U / ㎎ / protein. GSS-5 was expressed as SOD 8.27 ± 0.33 U / ㎎ / protein, CAT 10.09 ± 0.60 U / ㎎ / protein and GPx 3.19 ± 0.32 U / ㎎ / protein.

As a result, the mixed extracts of Chunma, Euphoria, and windshield showed free radical scavenging ability and antioxidant enzyme activity. Especially, mixed extracts of 80% ethanol, which was prepared with the ratio of 2: 1: GSS-5 was found to have excellent free radical scavenging activity and antioxidant enzyme activity, and the use of GSS-5 as a functional material containing active ingredients for blood purification and antioxidant efficacy was confirmed.

Test Example  8: Platelet aggregation inhibition In vitro  Efficacy test

(1) Experimental animals

Sprague-Dawley (SD) male rats weighing about 250 ± 50 g (8 weeks old, male, multiscience) were used in this experiment. The temperature of the breeding room was 25 ± 1 ℃ and the humidity was 55 ± 10%. The illumination was adjusted to a 12-hour light-dark cycle to maintain constant environmental conditions in the animal laboratory for the entire period of the study. Water and solid feed (Samyang Corporation, Korea) were freely available. Breeding and treatment of all experimental animals used in this experiment were conducted according to the Ethical Guidelines of the Animal Experimental Ethics Committee.

(2) Preparation of blood

Anesthetized gas mixed with 70% N ₂ O and 28.5% O ₂ gas and 2.0% enflurane (Choongwae Pharma, Korea) was placed in an anesthesia chamber (Royal medical, Korea) After inhalation anesthesia was used, the animals were fixed to the operating table, and general inhalation anesthesia was maintained. Anticoagulants were collected through cardiac puncture using plastic syringes with anticoagulation (3.8% sodium citrate) at a ratio of 1: 9 (v / v) to blood. The collected blood was centrifuged at 1,100 rpm for 10 minutes, and the supernatant was collected to obtain platelet-rich plasma (PRP). Platelet counts of PRP were measured using a Coulter counter (Coulter electronics, USA) and adjusted to 3 x 10 ⁵ / μl. The remaining material layer was centrifuged at 3,000 rpm for 5 minutes and the supernatant was collected to obtain platelet poor plasma (PPP).

(3) Measurement of platelet aggregation inhibitory activity

In order to investigate the inhibitory effect of platelet aggregation on platelet aggregation, an optical method using platelet-rich plasma using Aggrego-meter (Chrono Log, Havertown, PA, USA) And the degree of change in light transmission. 450 μl of blood was added to the plastic cuvettes and reacted at 37 ° C for about 10 minutes. 20 μl of various concentrations of the samples were added to stir bars and reacted at 1,200 rpm with stirring. After confirming the baseline for 2 minutes, 7 ㎕ collagen (1 ㎎ / ㎖) was added to induce platelet aggregation and measured for up to 10 minutes. The final concentration of the sample of Example 5 was prepared by dissolving it in 10% (v / v) DMSO (dimethyl sulfoxide) to a concentration of 5, 10 and 25 mg / ml, Platelet aggregation inhibitory activity was measured. Platelet aggregation inhibition activity was determined by the percentage of aggregation (%) induced by collagen (A) and the aggregation (%) after treatment of natural material (GSS-5) ), And the induced aggregation (%) after treatment with chimma, squamous cell, and windshield mixed extract was expressed as the inhibition rate (%) according to the following equation as the experimental group (C).

x 100
A: Control aggregation(%)
B: Experimental aggregation(%)Inhibition (%) =

x 100
A: Control aggregation (%)
B: Experimental aggregation (%)

In order to examine the inhibitory effect of the mixed extract on the platelet aggregation reaction, coagulation was induced by collagen. 3A to 3E are graphs showing concentration-dependent effects on platelet aggregation of chromatin, spots, and windshield.

The coagulation rate of the extracts were determined to be 66.90 ± 2.08%, 50.20 ± 2.11%, and 41.29 ± 2.02%, respectively, at the concentration of 5 ㎎ / ㎖, 10 ㎎ / ㎖ and 25 ㎎ / ㎖. The coagulation rate was measured as 69.30 ± 3.00%, 60.25 ± 2.05%, and 48.20 ± 2.01%, respectively, by treating the extract with 5 ㎎ / ㎖, 10 ㎎ / ㎖ and 25 ㎎ / ㎖. The coagulability of the extracts was estimated to be 67.52 ± 2.03%, 58.06 ± 1.85% and 41.20 ± 1.41%, respectively, after treatment with 5 ㎎ / ㎖, 10 ㎎ / ㎖ and 25 ㎎ /

The agglutination rate was measured as 68.50 ± 1.75%, 45.20 ± 1.35%, and 10.0%, respectively, after treatment with 5 mg / ml, 10 mg / ml and 25 mg / 35.00 ± 1.90% respectively. One-way ANOVA showed statistically significant difference (p <.001), and in the post-test, there was a significant difference between the groups in terms of 10 ㎎ / ㎖ and 25 ㎎ / (P <.001), respectively.

The agglutination rate was 61.75 ± 1.50%, 42.60 ± 1.45%, and 60.60 ± 1.50%, respectively, after treatment with 5, 10, and 25 mg / 33.20 ± 1.95%. One-way ANOVA showed statistically significant differences (p <.001), and in the post-test, there was a significant difference in all concentration levels compared to the control group (P <.001), respectively.

In order to examine the inhibitory effect of the mixed extract on the platelet aggregation reaction, coagulation was induced by collagen. FIG. 4 is a graph showing platelet aggregation inhibition rates of the chimma, spearmint, windshield extract, and mixed extract thereof. The concentration of 5 mg / ㎖, 10 ㎎ / ㎖ and 25 ㎎ / ㎖ resulted in 12.68 ± 2.25 (%) of the total cohesion (%) of control, %, 35.37 ± 1.46%, and 50.96 ± 2.02%, respectively. At the concentration of 5 ㎎ / ㎖, 10 ㎎ / ㎖ and 25 ㎎ / ㎖, the extracts from the flounder showed a tendency to increase the coagulation inhibition rate as the concentration increased to 9.20 ± 2.4%, 27.60 ± 2.16% and 48.24 ± 1.84%. In addition, the coagulation inhibition rate tended to increase as the concentration of the extracts increased from 23.5 ± 2.4%, 32.40 ± 2.16% and 54.20 ± 1.74% at concentrations of 5 ㎎ / ㎖, 10 ㎎ / ㎖ and 25 ㎎ / ㎖, respectively.

The inhibition rate of cohesion on the control of maximum cohesion (%) of control mixture of chama, scarlet and windblown mixture (1: 1: 1) was 13.65 ± 2.28 at 5 ㎎ / ㎖, 10 ㎎ / ㎖ and 25 ㎎ / , 21.50 ± 2.40%, 41.20 ± 2.50%, 49.80% at the concentrations of 5 ㎎ / ㎖, 10 ㎎ / ㎖ and 25 ㎎ / ㎖ for the mixed extracts (2: And 1.58%, respectively, as the concentration increased.

(4) DPPH solution Using Antioxidant ability  Measure

Free radical scavenging activity, one of the antioxidant activities, was measured by Blois method in order to examine the antioxidant ability of natural materials. Dissolve 16 mg of DPPH in 100 ml of ethanol and mix with 100 ml of distilled water. Filter 2.5 ml of this filtrate with physiological saline solution (5 mg / ml, 10 mg / ml, 25 mg / And 0.5 ml of the mixture was mixed and reacted at room temperature for 10 minutes. To measure the amount of DPPH remaining, the change in absorbance was measured at 528 nm using a microplate reader (Molecular Device, Sunnyvale, CA, USA). As a positive control, ascorbic acid was used.

In order to measure the antioxidative activity of the natural extract according to the present invention, the electron donating ability to DPPH radical was measured and the results are shown in FIG. FIG. 5 is a graph showing the results of measurement of electron donating ability against DPPH radical according to the concentration of GNC-5, GNC-5, and GNC-5.

In each concentration, DPPH radical scavenging effect was shown in a concentration dependent manner compared with Ascorbic acid. The highest eradication effect was 25 ㎎ / ㎖, which is the highest concentration, compared to the control group. The results of measurement of the electron donating ability according to the concentration of the mixed extract of Ganoderma lucidum, Heliothermia, and windshield were shown in FIG. DPPH radical scavenging effect was shown in a concentration dependent manner compared to ascorbic acid at each concentration.

Test Example  9: Thrombogenic inhibition In vivo  Efficacy test

(1) Treatment of test animals

The test animals were treated in the same manner as in Test 8 and were divided into four groups as a control group in which no treatment was carried out to detect the thrombogenic inhibitory effect, 100 ㎎ / ㎏ of the mixed extract, 300 ㎎ / ㎏, 500 ㎎ / Respectively. Group Ⅰ which received 100 ㎎ / ㎏, Group Ⅱ which received 300 ㎎ / ㎏, Group Ⅲ which received 500 ㎎ / ㎏, and control group which had no treatment for blood flow. Respectively. Twelve rats were divided into groups, and the extracts of the above examples were orally administered once a day for five times a week for three weeks at the same time.

(2) FeCl ₃ Using Carotid artery thrombosis  Induced animal model

Carotid artery thrombosis was induced using FeCl ₃ to construct an animal model of carotid thrombosis. Anesthetized gas mixed with 70% N ₂ O and 28.5% O ₂ gas and 2.0% enflurane (Choongwae Pharma, Korea) was placed in an anesthesia chamber (Royal medical, Korea) After anesthesia with suction, it was fixed to the operating table and continued inhalation general anesthesia. The right common carotid artery (CCA) was identified and excised. Doppler flow prove was placed in the carotid artery and blood flow was monitored through monitor. The filter paper (2 mm square) was applied to the blood vessels and 35% FeCl ₃ was added for 3 minutes. After 3 minutes, the filter paper was removed and the carotid artery was washed and observed for 30 minutes. After a certain amount of time, the thrombus was formed. After 30 minutes, the thrombus was carefully removed from the thrombus site and the weight was measured.

(3) Blood flow measurement

100 μl of blood was collected from each group, and the time required for blood to pass through the capillary was measured by a micro channel array flow analyzer KH-6 (MC Lab, Japan) after anticoagulation with heparin.

(4) Carotid artery thrombosis  Results of thrombus weighing in induced animal models

To investigate the antithrombotic efficacy, an experimental animal model of carotid thrombosis was made using FeCl ₃ and the thrombus weight was measured. The results are shown in Fig. FIG. 6 is a graph showing the result of measuring the thrombus weight by treating a mixed extract of chymoma, sclerotinia, and windshield. The thrombus weight was measured to be 0.71 ± 0.15 ㎎ / ㎜, 0.68 ± 0.12 ㎎ / ㎜, 0.49 ± 0.10 ㎎ / ㎜ after treatment of 100%, 300 ㎎ / ㎏ and 500 ㎎ / ㎎ / ㎜. One-way ANOVA showed statistically significant difference (p <.01) between the groups. In the post-test, 100 ㎎ / ㎏ group showed statistical significance (P <.01) and 400 ㎎ / ㎏ administration group (p <.01), respectively.

(5) Blood flow improvement effect

To investigate the antithrombotic efficacy, an experimental animal model of carotid thrombosis was made using FeCl ₃ and its effect on blood flow was examined. The results are shown in Fig. 7 is a graph showing the flow of blood flow in an animal model of carotid thrombosis. Group Ⅰ: Mixed extract (2: 1: 1) 100 ㎎ / ㎏, Group Ⅱ: Mixed extract (2: 1: 1) 300 ㎎ / .

Blood was collected from each group and the time for blood to pass through 25 μl, 50 μl, 75 μl, and 100 μl of capillary was measured using a micro channel array. One-way ANOVA was used to determine the significance of each group. The results were statistically significant at 50 ㎕ (p <.05), 75 ㎕ (p <.05), and 100 ㎕ (p <.001) (P <.05) compared to the control group, and 75 ㎕ compared to the control group, the Ⅱ group (p <.05) (p <.01), group Ⅲ (p <.001) and group Ⅲ (p <.05) were significantly different in group Ⅲ (p <.05) 001) showed a significant difference.

Test Example  10: Antioxidant efficacy In vivo  black

(1) Preparation of sample

Sprague-Dawley (SD) male rats weighing about 250 ± 50 g (8 weeks, male, multiple sciences) were used as animal models for each condition. Respectively.

(2) chronic Decreased cerebral perfusion  Animal model ( Chronic cerebral hypoperfusion model )

Chronic cerebral hypoperfusion was induced by bilateral common carotid occlusion (2VO). Anesthetized gas mixed with 70% N ₂ O and 28.5% O ₂ gas and 2.0% enflurane (Choongwae Pharma, Korea) was placed in an anesthesia chamber (Royal medical, Korea) After inhalation anesthesia was used, the animals were fixed to the operating table, and general inhalation anesthesia was maintained. After maintaining a constant temperature using a rectal thermometer and a thermal pad, the common carotid artery (CCA) was exposed and the silk suture (6-0, Korea, Korea). After the operation, the surgical site was sutured, disinfected and injected with an anti - inflammatory agent.

(3) antioxidant enzyme Glutathione peroxidase  Active measurement

Glutathione peroxidase activity was assayed by measuring the activity of H ₂ O ₂ , 1 mM GSH, glutathione reductase (2 IU), 0.2 mM NADPH and enzyme source in 0.1 M Tris · HCl buffer (pH 7.2) And the amount of NADPH consumed to reduce GSSG produced during the reaction for 5 minutes at 25 ° C was measured at 340 nm to calculate its activity. The activity of the enzyme was expressed in nmole by the amount of NADPH oxidized at 1 mg of protein per minute.

Activity of antioxidant enzymes was measured to investigate the antioxidant efficacy of animal models with reduced cerebral perfusion. The experimental animals were divided into two groups: control group (n = 12), control group without chronic treatment (n = 12), natural substance (chimma, 5) 300 ㎎ / ㎏ of mixed extract (GSS - 5) of group Ⅰ (n = 12) administered with 100 ㎎ / ㎏ and natural substance (chimma, Group Ⅲ (n = 12) treated with 500 ㎎ / ㎏ of mixed extracts (GSS-5) (group Ⅱ, n = 12) 12). For each group, the samples were orally administered at the same time, 5 times a day once a day, and 1 ml each day for 3 weeks.

The results are shown in Fig. 8 is a graph showing antioxidative activity of an animal model deprived of chronic cerebral perfusion. NADPH oxidase / min / ㎎ protein in Group Ⅱ, 32.55 ± 2.54 nM NADPH oxidase / min / ㎎ protein in Group Ⅱ, and 31.60 ± 2.54 nM protein in Group Ⅲ ± 3.54 nM NADPH oxidised / min / mg protein respectively.

One-way ANOVA was used to determine the significance of each group (p <0.05). In post-test, the difference was statistically significant between the control group and the experimental group Ⅰ, the experimental group Ⅱ, and the experimental group Ⅲ (P <0.5).

(4) Antioxidant enzyme Glutathione reductase  Active measurement

Glutathione reductase activity was determined by the method of Paglia and Valentine in GSSG (0.1 mM Tris buffer, pH 8.0). 27 mM EDTA, 6 mM NADPH and an enzyme source, and the amount of NADPH consumed to reduce GSSG produced during reaction at 25 ° C for 5 minutes was measured at 340 nm to calculate the chelation. The activity of the enzyme was expressed in nmole by the amount of NADPH oxidized at 1 mg of protein per minute.

The results are shown in Fig. 9 is a graph showing the activity of glutathione reductase activity measured. NADPH oxidase / min / ㎎ protein, Group Ⅱ 23.46 ± 2.50 nM NADPH oxidase / min / ㎎ protein, Group Ⅱ 32.68 ± 3.56 nM, NADPH oxidase / min / ㎎ protein and Group Ⅲ 32.36 ± 4.54 nM NADPH oxidised / min / mg protein, respectively.

One-way ANOVA showed statistically significant difference (p <0.001) between the groups. In the post-test, there was a significant difference between the control group and the experimental group Ⅰ, the experimental group Ⅱ, and the experimental group Ⅲ (P <0.5).

(5) EEG measurement

After puncturing with pentobarbital (25 ㎎ / ㎏, IP) and urethane (0.5 g / ㎏, IP), drill holes were made on the bilateral frontal bone (2.5 mm in front and 2.5 mm in the lateral direction) 1.5 mm) was used as a recording electrode for EEG, and two fine screws were placed on the interparietal bone of the cerebellum as a reference and ground electrode. The connecting pin connected to the screw was placed in a 2 x 2 grid and the dental cement was applied and fixed on top of the two cages. The EEG signal was connected to a bio-potential amplifier (CyberAmp 380, Axon Instruments, Inc., USA, AM System, Inc., USA), amplified (10,000 times) and filtered (0.1-60 Hz) using an A / D converter (Asan Instruments, Inc., USA), and data acquisition program was used for numerical conversion at a rate of 200 Hz. Animals were placed in a plastic box (40 cm x 50 cm x 25 cm) After being accustomed to the recording environment, the brain waves were recorded. EEG was recorded for 20-30 minutes after the administration of each extract. The EEG frequency band was divided into delta 1-4, theta 4-8, alpha 8-13, beta L 13-21, beta H 21-30, gamma 30-50, and total (1-50 Hz).

The results are shown in Fig. FIG. 10 is a graph showing the electroencephalogram before and after the administration of the extract. FIG. The power and total power of delta, theta alpha, beta and gamma band were observed after administration of mixed extract of Chunma, (P <.05), beta-H and gamma-band power were significantly increased in the GSS-5 group compared with the control group But there was no significant effect.

(6) Cognitive function evaluation

The water tank used as an underwater maze was filled with water of 30 ㎝ in height with a diameter of 180 ㎝ and a height of 50 ㎝ with a temperature of 22 ± 2 ℃. A video camera was installed at the top of the water tank to keep the spatial cues constant. A circular transparent acrylic cylinder with a diameter of 10 cm is installed and placed 1.5 cm lower than the water surface. The underwater labyrinth is divided into four equal quadrants and divided into NE, NW, SE and SW. Among them, an escape pad is placed in the center of the northeastern quadrant, Respectively.

After 10 days recovery period after model induction, labyrinth learning started. The experiment started with the animal's gaze facing the wall of the aquarium so that the inside of the aquarium was not visible and the animal was picked up from a central quadrant wall. Four times a day training was carried out and after 3 weeks' final enrollment, an underwater maze test was performed. The acquisition test measures the time taken to ascend to the escape within 180 seconds from the time of arrival, and the time to stay in the quadrant where there was escaping during free swimming within 180 seconds from receipt was measured.

The results are shown in Fig. FIG. 11 is a graphed diagram showing the results of underwater labyrinth learning. FIG. (P <.001) in the experimental group Ⅰ, 28.55 ą 9.65 sec in the experimental group Ⅱ (p <.001), and the experimental group Ⅰ Ⅲ was 36.40 ± 6.82 seconds (p <.01) compared with the control group.

12 shows the results of the grasping procedure in the underwater labyrinth learning. In the underwater labyrinthine learning, the group Ⅰ was 3.90 ± 0.45 sec, the group Ⅱ was 4.90 ± 0.65 sec, the group Ⅲ was 4.75 ± 0.40 sec, and the control group was 3.25 ± 0.45 sec. Compared with 0.67 second (p <.05).

Claims (5)

A pharmaceutical composition for the prevention or treatment of thrombosis, which comprises an extract of Angelica keiskei koidz.
The method according to claim 1,
Wherein the extract of Chunmei, Heliothermia and Windshield is obtained by extracting with water, ethanol or a mixed solvent thereof.
The method according to claim 1,
Wherein the extract of chymus, lycopene and windshield is obtained by extracting with a mixed solvent of water and ethanol.
A health functional food composition for the prevention of thrombosis, which comprises an effective ingredient of an extract of Angelica keiskei koidz.
5. The method of claim 4,
Wherein said composition is provided in the form of an oral, pouch, drink, or beverage.

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