KR20150105718A - Pharmaceutical composition for the prevention and treatment of thromboembolism comprising mixture extracts of phellunus baumii and salvia miltiorrhiza - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of thromboembolism including a mixed extract of Phellinus baumii and Salvia miltiorrhiza as an active ingredient. The composition exhibits a significant therapeutic effect in animal models with derived pulmonary thromboembolism, and significantly suppresses the COX expression involved in inflammation and blood flow improvement. More specifically, the extract prepared by mixing Phellinus baumii and Salvia miltiorrhiza at the ratio of 5:1 exhibits an excellent therapeutic effect compared to Phellinus baumii or Salvia miltiorrhiza. Accordingly, the mixed extract of Phellinus baumii and Salvia miltiorrhiza can be used as the composition for preventing and treating thromboembolism.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for prevention and treatment of embolism containing an extract of Sambrook,

The present invention relates to a method for producing a mushroom baumii) and Salvia (Salvia The present invention relates to a pharmaceutical composition for preventing and treating thromboembolism containing an extract of miltiorrhiza as an active ingredient.

Venous thromboembolism (VTE) is called deep vein thrombosis (DVT) or pulmonary thromboembolism and is generally preventable disease. Venous thromboembolism occurs in one to two out of a thousand persons per year in industrialized countries. Deep vein thrombosis occurs not only in the lower half of the body, but also in the arm, brain sinus, and intestine. Deep vein thrombosis of the calf veins results in small thrombosis and fewer long-term sequelae. On the other hand, proximal deep-vein thrombosis often induces pulmonary embolism in the scab, thigh, and iliac vein, often causing post-thrombotic syndrome (Tienan Zhu, Isabelle Martinez and Joseph Emmerich, Arterioscler Thromb Vasc Biol. 2009; 29: 298-310) .

Pulmonary thromboembolism, also called deep-vein thrombosis, occurs when the thrombus generated in the deep vein of the body falls off the vein wall, and travels through the right atrium and right ventricle to the blood vessels of the lungs, ≪ / RTI > Pulmonary embolism is relatively easy to treat if it is found only at the right time, but it is very difficult to diagnose it because of the ambiguous and uncharacteristic early symptoms of dyspnea, syncope, cough and hemoptysis. In the United States, at least 630,000 cases The third cause of death is considered to be the cause of death (Korea Patent Publication No. 2012-0056312). Damage to blood vessels can result in damage to vascular endothelial cells and activation of platelets. Vasoconstrictor factors such as Thromboxane A2 (TXA2), endothelin and thrombin are secreted to cause contraction of vascular smooth muscle cells and contraction of blood vessels. In addition, endothelial cell damage leads to vasoconstriction and platelet aggregation when nitric oxide (NO) secretion decreases. Pulmonary embolism is caused by blood vessel damage caused by these factors, resulting in accumulation of venous thrombosis. These thromboses fall and obstruct the pulmonary arteries, leading to diseases and deaths due to pulmonary embolism.

BACKGROUND ART Cyclooxygenase (hereinafter abbreviated as 'COX') is an enzyme involved in biosynthesis of prostaglandins (abbreviated as 'PGs') from arachidonic acid (abbreviated as AA) (COX-1 and COX-2) in vivo (Cell, 83, 345, 1995). COX-1 is a constitutive enzyme that is expressed in normal state to protect the body's homeostasis such as protection of the gastrointestinal tract and control of renal function. COX-2 is an inducible enzyme, (J. Biol. Chem., 271, 33157, 1996) is known to increase intracellular expression by mitogens or cytokines during the immune response. Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the COX-2 enzyme and cause inflammatory analgesia, but also unnecessarily inhibit the COX-1 enzyme, leading to gastrointestinal bleeding and renal toxicity. USA, 91, 3228 (1994), Proc. Natl. Acad. Sci. USA, 91, 12013 (1994)). Therefore, studies on a substance capable of selectively inhibiting COX-2 have been actively conducted (WO 9606840; Bioorg. Med. Chem. Lett. 5, 2377, 1995; Ann. Report. Med. 1997). Recently, celebrex or vioxx has been developed as a drug that selectively inhibits COX-2 and alleviates side effects such as gastrointestinal bleeding.

Phellinus linteus ) is also known as woody mud mushroom, and in Dongbokgok, it is recorded in the name of 湯 虫 (桑 木 耳). It has a diameter of 6 to 12 cm and a thickness of 2 to 10 cm and has various shapes such as a semicircular shape, a flat shape, a round mountain shape, and a horseshoe shape. The dark brown hairs on the surface are short and densely packed, then disappear and grow to become crisp. It has a dark brown-brown ring groove, and the back and the back are divided. The edges are bright yellow, the lower side is tan, and the flesh is yellowish brown. The spores are pale yellowish brown and ball shaped. It is a perennial wood spruce that is overlaid on mulberry trees. It looks like clumps of clay in the early days. After it is grown, it is called "tree tongue" because it is shaped like a stump on a tree stump. The polysaccharides contained in the mushroom are known to exhibit anticancer activity, immunological activity, and antitumor effect by activating the immune system. Thus far, many studies have been conducted in Korea, Japan, and China. The polysaccharide component has been shown to be effective for anti - cancer and immunity enhancement, and it is cultivated in Korea as a valuable medicinal product in large quantities. For medicinal purposes, the moon is yellow or light yellow, and is characterized by lack of flavor and aroma. It is easy to eat with a light taste, and it grows in Korea, Japan, Australia and North America.

Salvia miltiorrhiza of the Labiatae is a perennial herb that is naturally grown or cultivated in Northeast China. Dansam contains phenanthraquinone pigments: tanshinone I dark brown (I), tanshinine II red (II), cryptotanshinone yellow (III), and tanshinol ) I and II are known. However, the pharmacological action of these ingredients is not yet known. It has been known that the aqueous solution of Dansamp is slightly inhibitory to various skin fungi. However, the effect of Dansamp and Situ mushroom mixed extract on pulmonary embolism It is not known.

Accordingly, the inventors of the present invention have conducted research to develop embolism prevention and therapeutic agents, and found that a mixed extract of a mixture of a mushroom and a dandelion has significant paralysis improvement effect, a reduction in thrombus formation and a vascular damage protection effect in a pulmonary embolism-induced rat In particular, the present inventors found that the combination of the mushroom extract and Panax ginseng mixture showed remarkable therapeutic effect of pulmonary embolism in comparison with Dansamp or Situ mushroom, The present invention has been accomplished on the basis of the fact that it can be effectively used as an effective ingredient of a composition for preventing and treating embolism.

The object of the present invention is to provide a method for the production of phellinus to provide a baumii) and Salvia miltiorrhiza embolism (thromboembolism), and blood circulation improved preventive and therapeutic composition containing (Salvia miltiorrhiza) mixture extract as an active ingredient.

In order to achieve the above object, the present invention Phellinus (Phellunus baumii) and Salvia (Salvia The present invention provides a pharmaceutical composition for preventing and treating thromboembolism containing an extract of miltiorrhiza as an active ingredient.

In addition, the present invention provides a health food for prevention and improvement of embolism containing an extract of Phellodendron mushroom and Panax ginseng as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for improving blood circulation containing an extract of Phellinus linteus and a mixture of Panax ginseng as an active ingredient.

In addition, the present invention provides a health food for improving blood circulation containing an extract of a mixture of Pseudomonas externa and Panax ginseng as an active ingredient.

In addition, the present invention provides a pharmaceutical composition exclusively for hematopoietic stem cells containing the mushroom and radish-mixed extract as an active ingredient.

In addition, the present invention provides an anti-diabetic health food containing an extract of Phellinus linteus and Panax ginseng as an active ingredient.

The mushroom of the present invention ( Phellunus baumii) and Salvia (Salvia miltiorrhiza ) showed significant therapeutic effect on pulmonary embolism in pulmonary thromboembolism induction animal model and remarkably inhibited the expression of COX involved in blood circulation improvement and inflammation. Especially, It was confirmed that the extracts of the present invention showed excellent therapeutic effect of pulmonary embolism in comparison with that of Dansamp or Phellinus linteus, and thus it was confirmed that the above-mentioned mixed extract of Phellinus linteus and Panax ginseng can be effectively used as a composition for the prevention or treatment of thromboembolism.

FIG. 1 is a graph showing the effect of a mixture of a mushroom and a radish on a lung lesion in an embolism-induced lung tissue.
FIG. 2 is a graph showing the effect of a mixture of a mushroom and a radish on arterial thickness changes by inducing embolization and then separating the artery.
FIG. 3 is a graph showing the effect of the combination of a mushroom and a radish extract on the degree of expression of COX-1 and COX-2 protein in embryo-induced rats by western blotting:
(A) shows the degree of COX-1 and COX-2 protein expression by Western blot analysis, (B) is a graph showing quantitation of COX-1 protein expression in (A) Of COX-2 < / RTI >
Saline: saline solution;
AS: Aspirin group;
PB: Situation mushroom;
SM: Danshen.
FIG. 4 is a graph showing the effect of mixed mushroom and monascus extract on the degree of expression of TXA2 and PGE2 in embryo-induced rats.
(A) shows the expression level of TXA2, and (B) shows the expression level of PGE2.
Saline: saline solution;
AS: Aspirin group;
PB: Situation mushroom;
SM: Danshen.
5 is a graph showing the measurement of nitric oxide produced by PCA in vascular endothelial cells:
(A) shows the nitric oxide production as green fluorescence and (B) shows the nitric oxide production as a graph using DAF-2.
6 is a graph showing the measurement of nitric oxide produced by T2A in vascular endothelial cells:
(A) shows the nitric oxide production as green fluorescence and (B) shows the nitric oxide production as a graph using DAF-2.
FIG. 7 is a graph showing the measurement of nitric oxide produced by vortex endothelial cells from a mixture of Matsutake mushroom: Dansam = 5: 1 ratio;
(A) shows the nitric oxide production as green fluorescence and (B) shows the nitric oxide production as a graph using DAF-2.
FIG. 8 is a graph showing the effect of T2A, PCA, and matsutake mushroom on the phosphorylation of eNOS in vascular endothelial cells, and the effect of 5: 1 ratio mixture extracts.
PB: Situation mushroom;
SM: Danshen.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing a mushroom baumii) and Salvia (Salvia The present invention provides a pharmaceutical composition for preventing and treating thromboembolism containing an extract of miltiorrhiza as an active ingredient.

It is preferable, but not limited, that the Phellinus linteus extract and Panax ginseng mixture extract are produced by a manufacturing method comprising the following steps.

1) extracting the mushroom and ginseng with an extraction solvent;

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

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

In the above method, the extraction solvent in step 1) is preferably extracted with water, C1 to C2 lower alcohol or a mixture thereof, and the lower alcohol is preferably ethanol or methanol, but is not limited thereto . The extraction solvent is preferably 0.1 to 10 times, more preferably 0.3 to 5 times, based on the dried mushroom and dried mushroom, but is not limited thereto. The extraction temperature is preferably 20 to 70 DEG C, but is not limited thereto. In addition, the extraction time may be 12 to 48 hours, but is not limited thereto.

In this method, the vacuum concentration of step 3) may be by using a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. The drying may be, but not limited to, vacuum drying, vacuum drying, boiling, spray drying or lyophilization.

The mixed extract is preferably mixed at a weight ratio of 1: 1 to 10: 1, more preferably at a weight ratio of 10: 3 to 10: 1, more preferably at a weight ratio of 5: 1 Most preferably, but not limited thereto.

The mixed extract may be extracted after mixing the mushroom and the ginseng, and the extract obtained by extracting the mushroom and the ginseng may be mixed and used, but the present invention is not limited thereto.

The embolism is preferably pulmonary thromboembolism, but is not limited thereto.

In a specific example of the present invention, the inventors of the present invention prepared a mushroom extract and a ginseng extract in order to confirm the effect of preventing and treating embolism of a mushroom and a radish mixture extract in an experimental animal, (See Table 1), and the effect of collagen and epinephrine on the pulmonary embryogenesis induced rats (Table 2).

In addition, the inventors of the present invention, in order to confirm the pulmonary histological changes of the experimental animals, peeled off the lung tissues to make slides, and as a result, the increase of epilepsy and thrombus formation in the mushroom and monascus mixed extract- In particular, it was found that the thrombus formation was less in the 5: 1 mixed extract group than in the 5: 4 mixed extract group (see FIG. 1) As a result of checking the slides, the normal thickness was maintained similar to the arterial thickness of the normal control group in the case of the mixed mushroom and radix extract extracts with different ratios. Particularly, in the mixed extract ratio 5: 4 ratio, And the thickness of the blood vessels was observed. Thus, it was confirmed that the 5: 1 group of the mixed extract had a greater protective effect on vascular damage due to collagen and epinephrine than the 5: 4 group (see FIG. 2).

In order to examine the degree of expression of COX-1 and COX-2 in the mononuclear cells and the degree of formation of TXA2 and PGE2 in plasma, the present inventors extracted blood from the abdominal artery of rats, 1 expression was statistically significant at the 5: 1 ratio of mixed extracts, and COX-2 expression was similar to that of normal control with 5: 1 ratio of mixed extracts 3). In TXA2, the ratio of mixed extracts was significantly lower in both groups than in the pulmonary embolism control group. PGE2 showed a statistically significant difference in the 5: 1 group compared to the mixed extract 5: 4 group (See FIG. 4).

In addition, the present inventors cultured vascular endothelial cells to confirm the effect of preventing and treating embolism of mushroom and radish mixed extracts in cells. As a result, PCA, a state mushroom indicator substance, The expression of NO was increased and the expression of extracellular nitric oxide was also increased (see FIG. 5). T2A, which is a Dansamp index, had no effect on nitric oxide production regardless of its concentration (see FIG. 6) As shown in FIG. 7, the degree of phosphorylation of eNOS, a vasodilating substance, was confirmed by PCA and T2A as compared with the control group There was no difference, but it was confirmed that more than 3 times eNOS phosphorylation occurred in the 5: 1 ratio mixed extract (see FIG. 8). The results showed that the 5: 1 mixed extract of Sasa mushroom and Panax ginseng showed a remarkable effect compared to the effects of Sasa mushroom and Panax ginseng.

Therefore, the mixed extract of Phellinus linteus and Panax ginseng of the present invention exhibits a therapeutic effect of significant pulmonary embolism in a pulmonary embolism induction animal model and remarkably inhibits COX expression which is involved in blood circulation improvement and inflammation, Of the extracts of the present invention showed excellent therapeutic effect of pulmonary embolism in comparison with the mushroom or dansamp, and thus it was confirmed that the mixture of the mushroom and the dansamp ginseng can be effectively used as a pharmaceutical composition for the prevention or treatment of embolism.

The composition of the present invention can be administered orally or parenterally and can be administered in various formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, It is prepared.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient such as starch, calcium carbonate, sucrose, lactose And gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included in addition to water and liquid paraffin, which are commonly used simple diluents. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol and gelatin can be used.

The composition of the present invention may be administered parenterally or by subcutaneous injection, intravenous injection, or intramuscular injection.

In addition, the present invention provides a health food for prevention and improvement of embolism containing an extract of Phellodendron mushroom and Panax ginseng as an active ingredient.

The mixed extract is preferably mixed at a weight ratio of 1: 1 to 10: 1, more preferably at a weight ratio of 10: 3 to 10: 1, more preferably at a weight ratio of 5: 1 Most preferably, but not limited thereto.

The mixed extract of Phellinus linteus and Panax ginseng of the present invention exhibits a therapeutic effect of significant pulmonary embolism in a pulmonary embolism induction animal model and remarkably suppresses the expression of COX which is involved in blood circulation improvement and inflammation and in particular, It was confirmed that the mixed extracts showed an excellent therapeutic effect of pulmonary embolism compared with that of Dansamp or Phellinus linteus, and thus it was confirmed that the combination of the mushroom and Panax ginseng can be useful as a health food for preventing or improving embolism.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

When the mixture of the mushroom and radish mixed extract of the present invention is used as a food additive, the extract can be used as it is, or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the amount of the compound in the health food may be 0.1 to 90 parts by weight of the total food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Natural carbohydrates include 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. As the sweetening agent, natural flavors such as tau Martin and stevia extract, synthetic flavors such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.

In addition to the above, the mushroom and ragwort mixed extract of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, Glycerin, alcohols, carbonating agents used in carbonated beverages, and the like. In addition, the mushroom and radish mixed extract of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination.

In addition, the present invention provides a pharmaceutical composition for improving blood circulation containing an extract of Phellinus linteus and a mixture of Panax ginseng as an active ingredient.

The mixed extract is preferably mixed at a weight ratio of 1: 1 to 10: 1, more preferably at a weight ratio of 10: 3 to 10: 1, more preferably at a weight ratio of 5: 1 Most preferably, but not limited thereto.

The mixed extract of Phellinus linteus and Panax ginseng of the present invention exhibits a therapeutic effect of significant pulmonary embolism in a pulmonary embolism induction animal model and remarkably suppresses the expression of COX which is involved in blood circulation improvement and inflammation and in particular, It was confirmed that the mixed extracts showed an excellent therapeutic effect of pulmonary embolism compared with that of Dansamp or Phellinus linteus. Therefore, it was confirmed that the combined extracts of Phellinus linteus and Panax ginseng can be used as a pharmaceutical composition for improving blood circulation.

In addition, the present invention provides a health food for improving blood circulation containing an extract of a mixture of Pseudomonas externa and Panax ginseng as an active ingredient.

The mixed extract is preferably mixed at a weight ratio of 1: 1 to 10: 1, more preferably at a weight ratio of 10: 3 to 10: 1, more preferably at a weight ratio of 5: 1 Most preferably, but not limited thereto.

The mixed extract of Phellinus linteus and Panax ginseng of the present invention exhibits a therapeutic effect of significant pulmonary embolism in a pulmonary embolism induction animal model and remarkably suppresses the expression of COX which is involved in blood circulation improvement and inflammation and in particular, It was confirmed that the mixed extracts showed a superior therapeutic effect of pulmonary embolism compared with that of dansamp or mushroom, so that the mushroom and danshang mixed extracts could be useful as a health food for improving blood circulation.

In addition, the present invention provides a pharmaceutical composition exclusively for hematopoietic stem cells containing the mushroom and radish-mixed extract as an active ingredient.

The mixed extract is preferably mixed at a weight ratio of 1: 1 to 10: 1, more preferably at a weight ratio of 10: 3 to 10: 1, more preferably at a weight ratio of 5: 1 Most preferably, but not limited thereto.

The mixed extract of Phellinus linteus and Panax ginseng of the present invention exhibits a therapeutic effect of significant pulmonary embolism in a pulmonary embolism induction animal model and remarkably suppresses the expression of COX which is involved in blood circulation improvement and inflammation and in particular, It was confirmed that the mixed extracts showed a superior therapeutic effect of pulmonary embolism compared with the control of Panax ginseng or Phellinus linteus. Therefore, it was confirmed that the combination of Panax ginseng and Panax ginseng can be used as a pharmaceutical composition exclusive for hematopoiesis.

In addition, the present invention provides an anti-blood-only health food containing the mushroom and the mixed extract of Panax ginseng as an active ingredient.

The mixed extract is preferably mixed at a weight ratio of 1: 1 to 10: 1, more preferably at a weight ratio of 10: 3 to 10: 1, more preferably at a weight ratio of 5: 1 Most preferably, but not limited thereto.

The mixed extract of Phellinus linteus and Panax ginseng of the present invention exhibits a therapeutic effect of significant pulmonary embolism in a pulmonary embolism induction animal model and remarkably suppresses the expression of COX which is involved in blood circulation improvement and inflammation and in particular, It was confirmed that the mixed extracts showed an excellent therapeutic effect of pulmonary embolism compared with that of Panax ginseng or Panax ginseng, so that the combination of Panax ginseng and Panax ginseng can be useful as a health food for anti - thrombosis.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

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

< Example  1> Situation mushroom ( Phellunus baumii ) Preparation of extract

The ethanol extract of Phellinus linteus used in the present invention was prepared.

Specifically, 10 kg of mushroom (Doowon Agricultural Products, Gyeonggi-Do, Korea) was added to the extraction tank by washing and drying, and 80% ethanol was added at 5 times the weight of the mushroom and refluxed for 3 hours at 60 ° C. In the extraction process, the extract was filtered with a paper filter paper, 80% ethanol was added to the filtrate again, and the extract was extracted at 60 ° C for 3 hours. The extract was filtered using paper filter paper. The filtrate was concentrated under reduced pressure at 50 DEG C or lower.

< Example  2> Sansam ( Salvia miltiorrhiza ) Preparation of extract

Ethanol extract used in the present invention was prepared.

Specifically, Dansamp (NutraMax, China) was washed and dried, and 10 kg of the extract was put into an extraction tank, and the extract of Panax ginseng was prepared in the same manner as in <Example 1>.

< Example  3> Situ mushroom and Sansam  Preparation of mixed extracts

In order to prepare the mixture of the mushroom and the radish, the mushroom extract and the radish extract prepared in Examples 1 and 2 were mixed at a weight ratio of 5: 4 or 5: 1, respectively, 5: 4 mixed extracts and 5: 1 mixed extracts of Panax ginseng and Panax ginseng were prepared.

< Example  4> Situation in laboratory animals Mushrooms Sansam  Embolism of mixed extracts thromboembolism ) Prevention and treatment effect confirmation

<4-1> Preparation of experimental animals

The mushroom and radish extract prepared by the methods of Example 1 and Example 2 were mixed and administered to experimental animals to confirm the effect of preventing and treating embolism.

Specifically, the experimental animals were male Sprague-dawley (aged 5 weeks old) (purchased from a central laboratory animal) male forty weeks. The animals were fed one by one in stainless steel wastes, and were allowed to eat free food and water. The animals were kept at a temperature of 23 ± 1 ° C and humidity of 50 ± 5%, and the illumination period was constant at 12-hour intervals. The experimental animals were adapted to the laboratory environment for 1 week and then divided into 5 groups of 8 animals by the nude method. The experimental group and each experimental group were set as shown in Table 1 below.

Experimental design Experimental group Administration extract Normal group
(Sham injection) Normal group Administration of physiological saline Induction of pulmonary embolism
(Collagen, injection of epinephrine)
Control group Administration of physiological saline Aspirin group (AS) Administration of aspirin (AS) 150 mg / kg b.w / day Condition mushroom (PB): Radish (SM) = 5: 4 (500 mg / kg b.w / day + 400 mg / kg b.w / day) Condition mushroom (PB): Radish (SM) = 5: 1 500 mg / kg b.w./day + ginseng (SM) 100 mg / kg b.w / day

<4-2> Pulmonary embolism Pulmonary thromboembolism ) Induction of paralysis to protect the effect of symptoms

The test substances in Table 1 were administered once a day for 7 days orally. After oral administration of the test substance to rats fasted for 12 hours on the last day, collagen (Chrono-log) and epinephrine (15 μg collagen + 3 μg epinephrine / rat) was injected into the tail vein for 15 min. After the pulmonary arterial infarction or recovery from paralysis was observed, pulmonary embolism thromboembolism) was confirmed.

Specifically, when a mixed solution of collagen and epinephrine is injected into rats, acute postmenopausal color due to thrombus formation is observed, and paralysis continues for 15 minutes or more through general paralysis. As shown in Table 2, in the control group in which pulmonary embolism was induced by collagen and epinephrine compared with the normal control group, 5 out of 8 mice showed paralysis and showed only 37.5% protection effect. In the aspirin group, only 2 out of 8 animals showed paralysis and 75% protection effect. In the mixed extract group, 62.5% protection effect against pulmonary embolism was observed in both 5: 4 or 5: 1 ratio of mushroom: Respectively. As shown in Table 2, there was a statistically significant increase in paralytic symptoms in the control group in which pulmonary embolism was induced compared to the normal control group (Table 2).

Effect of Mixed Extracts of Mushrooms and Danams on Pulmonary Embolism in Rats Dose (mg / kg) No.paralyzed / No.tested Protection (%) Normal control group Saline 0/8 100 Control group Saline 5/8 37.5 Aspirin group 150 2/8 75 Situ mushroom + ginseng 500 + 400 3/8 62.5 Situ mushroom + ginseng 500 + 100 3/8 62.5

<4-3> Identification of pulmonary histological changes

In order to confirm the pulmonary histological changes of the experimental animals prepared in [Table 1], the lung tissues were detached and fixed in 10% formalin for 24 hours or longer, and paraffin blocks were made and cut into 5 μm to make slides. Pulmonary tissue slides were stained with hematoxylin and eosin for hematoxylin-eosin staining (H & E staining) and pathological examination was performed using an optical microscope.

As shown in FIG. 1, the collagen and epinephrine were administered to the lungs (control group) in the control group, as shown in FIG. 1. As a result, Intravenous thrombosis was observed with increased epilepsy. On the other hand, the aspirin group showed a significant decrease in thrombosis and a substantial increase in pulmonary function compared to the control group. Similar to the aspirin group, the increase of epilepsy and the thrombus formation were decreased in the mixed mushroom and radix extract group with different ratios compared to the control group. In particular, it was confirmed that the thrombus formation was less in the 5: 1 mixed extract group than in the 5: 4 mixed extract group (Fig. 1).

<4-4> Confirmation of changes in arterial histological thickness

In order to confirm the arterial histological thickness change of the experimental animals prepared in [Table 1], the arteries were separated and frozen at -70 ° C using an OCT compound (Sakura Finetek USA Inc.) cryostat microtome (Leica) at -20 ° C to a thickness of 5 μm. Arterial tissue slides were stained with hematoxylin and eosin and hematoxylin - eosin staining was performed to observe changes in arterial thickness using an optical microscope.

As shown in FIG. 2, when the collagen and epinephrine were injected into the lung lesion group (control group), the interval between the elastic fibers in the blood vessels was significantly larger than that in the normal control group It was confirmed that the thickness of the blood vessel wall was thickened. On the other hand, in the rats with mixed mushroom and radix extracts of different ratios, the normal thickness was maintained similar to the arterial thickness of the normal control group. Especially, in the mixed extract ratio 5: 4 group, the interval between the elastic fibers in the blood vessels was widened and the blood vessels became thicker than the mixed extract ratio 5: 1 group. Therefore, it was confirmed that the protective effect against collateral and epinephrine-induced vascular injury was greater in the mixed extract ratio 5: 1 group than in the 5: 4 group (Fig. 2).

<4-5> Mononuclear  cell( MNC , mononuclear cell) COX -One, COX -2 expression confirmation

COX (Cyclooxygenase) is an enzyme that converts arachidonic acid into prostaglandin (PG). It is involved in cell proliferation and activation of carcinogens. There are two types of COX-1 and COX-2. COX-1 is present in most tissues and maintains the homeostasis of the body by producing PG and is involved in blood flow maintenance, cell differentiation, and mucus production. Here, inhibition of COX-1 inhibits the production of platelet aggregation factor TXA2, which results in antiplatelet activity. COX-2 is mainly involved in PG production in inflamed areas. COX-2 is derived from cytokines in the presence of inflammation, and PGE2 plays an important role as a mediator of inflammation and pain.

The present inventors confirmed the expression levels of COX-1 and COX-2 protein in monocytes and the degree of formation of TXA2 and PGE2 in plasma when they induced embolism with collagen and epinephrine.

Specifically, blood was collected from the abdominal artery of the rats. In order to prevent blood clotting, the cells were immersed in EDTA (ethylene diamine tetra acetate) tube, centrifuged and the plasma was separated and mononuclear cells were separated using Histopaque 1077 (Sigma) and stored at -70 ° C. Respectively.

Mononuclear cells were lysed at 1 × 10 ⁵ cells / ml and proteins were extracted. BSA (bovine serum albumin, Bradford) method was used for protein determination. 30 ug of the quantified protein sample was separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to PVDF (polyvinylidene difluoride) membrane (Bio-Rad). The PVDF membrane was blocked overnight at -4 ° C with 0.1% TBST (Tris-buffered saline) containing 5% BSA. After washing with TBST, the cells were washed with 5% BSA solution containing primary antibody (COX-1, COX-2 (1: 200 dilution; Santa Cruz), β-actin (1: 1000 dilution; Santa Cruz) After the reaction, the cells were washed 3 times with TBST for 5 minutes and reacted with the secondary antibody for 2 hours at room temperature. After the secondary antibody reaction, the cells were washed in TBST and visualized with ECL system (INTRON biotechnology). Protein visualization and quantitative analysis were performed using the imaging equipment ChemiDoc XRS + (Bio-Rad).

The expression level of COX-1 and COX-2 was measured by Western blot analysis and shown in FIG. 3, and quantified and shown in a graph.

As a result, as shown in FIG. 3, the expression of COX-1 was 2.7-fold higher in the control group than in the control group, which was statistically significantly higher than that of the normal control group. Respectively. On the other hand, the 5: 1 ratio of mixed extracts showed statistical significance in expression level similar to that of normal control.

In addition, COX-2 expression was significantly increased by 4.3-fold in the control group that induced pulmonary embolism compared with the normal control group. The ratio of 5: 4 of mixed extract increased 3.7-fold, but the expression of aspirin was similar to that of normal control (FIG. 3).

<4-6> TXA2 , PGE2  Confirm creation

In the control group, which showed pulmonary embolism, COX-1 increased the amount of TXA2 produced by the increase of COX-1. Therefore, the ratio of TXA2 and PGE2 Were measured.

As a result, as shown in Fig. 4, the mixed extract ratio in TXA2 was significantly lower in both groups than in the pulmonary embolism control group, but there was no difference between the two groups. However, PGE2 in relation to COX-2 was found to be lower in the mixed extracts than in the control group that induced pulmonary embolism. In comparison with the mixed extract 5: 4 group, statistically significant (Fig. 4).

< Example  5> Situation in cells Mushroom and Sansam  Prevention and treatment of embolism of mixed extracts

The optimized umbilical vein endothelial (HUVEC) ratio of 5: 1 and the respective indicator (PCA (Protocatechuic acid), T2A (Tanshinone 2A) cells, and human vascular endothelial cells), the expression of nitric oxide (NO), which is a substance produced in vascular endothelial cells, was changed in a concentration-dependent manner both inside and outside of the cells. Then, Western blotting was performed to measure vasodilator eNOS (endothelial nitric oxidase synthase) expression.

<5-1> Culture of vascular endothelial cells

Vascular endothelial cells were obtained from the American Type Culture Collection and cultured at 37 ° C, 5% CO _{2 ,} 95% air. DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS (Fetal bovine serum, Gibco) and 1% penicillin-streptomycin-neomycin (Gibco) was used.

<5-2> Confirmation of nitric oxide production

The cells were incubated at a concentration of 1 × 10 ⁵ cells / well in a 24-well plate and then the cells were incubated at 90% or higher. The extracellular nitrogen oxides were starvated for 2 hours, treated with the sample for concentration, incubated at 37 ° C for 10 minutes with light blocking. After that, A23182 was treated, followed by incubation at 37 ° C for 5 minutes. After dispensing in a 96-well plate, 0.1 uM DAF-2 was treated and incubated at 37 DEG C for 30 minutes with light shielding, and then measured with a fluorescence detector (wavelength 495-515 nm; Thermo). The cells were incubated for 2 hours at 37 ° C for 30 minutes. The cells were treated with DAF-2DA (diaminofluorescein-2-diacetate) for 2 hours. After washing with HBSS (Hank's Balanced Salt Solution), the sample is treated at the concentration and incubated at 37 ° C for 10 min. The cells were incubated at 4 ° C for 5 minutes to fix the cells to the wells with 2% paraformaldehyde, and then the fluorescence was observed with a microscope (Nikon).

As a result, as shown in Fig. 5, Fig. 6 and Fig. 7, PCA, a state mushroom indicator substance, increased intracellular nitric oxide expression in a concentration-dependent manner at a concentration higher than 25 ug / ml, (Fig. 5). T2A, which is a Dansamp index, showed no effect on nitric oxide production regardless of the concentration (Fig. 6) 1 ratio, the nitric oxide production was increased in both the inside and outside of the cell in a concentration-dependent manner as the concentration increased (Fig. 7).

<5-3> eNOS Confirmation of expression of

The cells were incubated at a density of 1 × 10 ⁶ cells / ml on a 100-mm plate, and then the experiment was carried out when 100% cells were filled. The samples were incubated at 37 ° C for 10 min. After washing, the supernatant was collected by centrifugation. The supernatant was removed and the remaining pellet was dissolved to extract proteins. Protein quantification was performed by the BSA method. 30 ug of the quantified protein sample was separated by 10% SDS-PAGE and transferred to PVDF membrane. The PVDF membrane was blocked with 0.1% TBST containing 5% skimmed milk for 1 hour. After overnight incubation at -4 ° C in a 5% BSA solution containing primary antibody (eNOS, phospho-eNOS (1: 1000 dilution; Cell Signaling) and β-actin (1: 1000 dilution; Santa Cruz) After washing three times for 5 minutes with TBST, the cells were reacted with the secondary antibody for 1 hour and 30 minutes at room temperature. After the secondary antibody reaction, the cells were washed in TBST and visualized with ECL system (INTRON biotechnology). Protein visualization and quantitative analysis were performed using the imaging equipment ChemiDoc XRS + (Bio-Rad).

As a result, as shown in FIG. 8, the degree of phosphorylation of eNOS at a ratio of 5: 1 ratio of PCA, DSA, Phellinus linteus and Panax ginseng to PCA and T2A Showed no difference compared to the control group, but at the mixed extract ratio of 5: 1, it was confirmed that eNOS phosphorylation more than 3 times occurred (FIG. 8).

Therefore, it was confirmed that the ratio of 5: 1 mixed extract of Sasa mushroom and Panax ginseng had a remarkable effect compared with the effect of each of mushroom and danshen.

< Manufacturing example  1> Preparation of pharmaceutical preparations

<1-1> Sanje  Produce

2 g &lt; RTI ID = 0.0 &gt;

Lactose 1 g

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

<1-2> Preparation of tablets

100 mg of the mushroom and water extract of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of 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 mushroom and water extract of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

1 g of the mushroom and radish mixed extract of the present invention

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

150 mg of the mushroom and water extract of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

< Manufacturing example  2> Manufacture of health food

<2-1> Production of flour food

0.5-5.0 parts by weight of the mixture of the mushroom and the radish of the present invention was added to wheat flour, and the mixture was used to prepare bread, cake, cookies, crackers and noodles.

<2-2> soup  And juicy ( gravies )

0.1 to 5.0 parts by weight of the mixed extract of Phellinus linteus and Panax ginseng according to the present invention was added to the soup and the juice to prepare a health improvement meat product, noodle soup and juice.

<2-3> Ground Beef  Produce

10 parts by weight of the mixture of the mushroom and radish extract of the present invention was added to ground beef to prepare ground beef for health promotion.

<2-4> Dairy products ( dairy products )

5 to 10 parts by weight of a mixture of the mushroom and radish sprouts of the present invention was added to milk, and the milk was used to prepare various dairy products such as butter and ice cream.

<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 mixture of the mushroom and the radish extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and dried in a hot air drier, and pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The grains, seeds, and the mixed mushroom and radish extract of the present invention were prepared in the following proportions.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 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)

(3 parts by weight) of the mushroom and rape seed mixture of the present invention,

(0.5 part by weight),

(0.5 parts by weight)

< Manufacturing example  3> Manufacturing of beverage

<3-1> Health drink  Produce

5 g of the mixture of the mushroom and ginseng mixed extract of the present invention was homogeneously mixed with a supplementary ingredient such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5% After sterilization, they were packaged in small containers such as glass bottles and plastic bottles.

<3-2> Preparation of vegetable juice

Vegetable juice was prepared by adding 5 g of the mixed mushroom and radish extract of the present invention to 1,000 ml of tomato or carrot juice.

<3-3> Production of fruit juice

Fruit juice was prepared by adding 1 g of the mixed mushroom and water extract of the present invention to 1,000 ml of apple or grape juice.

Claims (9)

Phellunus baumii) and Salvia (Salvia miltiorrhiza ) mixed extract as an active ingredient for the prevention and treatment of thromboembolism.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the mixed extract is obtained by mixing a mushroom and a decanter at a weight ratio of 1: 1 to 10: 1.
The pharmaceutical composition according to claim 1, wherein the mixed extract is extracted with water, a C ₁ to C ₂ lower alcohol or a mixture thereof.
A health food for prevention and improvement of embolism containing mushroom and radix mixed extract as an active ingredient.
[Claim 5] The health food according to claim 4, wherein the mixed extract is a mixture of a mushroom and a ginseng at a weight ratio of 1: 1 to 10: 1.
A pharmaceutical composition for improving blood circulation comprising a combination of mushroom and radish extract as an active ingredient.
A health food for improving blood circulation which contains a combination of mushroom and radish extract as an active ingredient.
A pharmaceutical composition exclusively for hematopoietic stem cells, which comprises a combination of a mushroom and a radish mixed extract as an active ingredient.
Exclusive health food containing only mushrooms and radish blended extract as an active ingredient.

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