Preparation of phellinus igniarius extract and application of phellinus igniarius extract in products for improving and / or treating myocardial infarction

Abstract

The invention provides preparation of a phellinus igniarius extract and application of the phellinus igniarius extract in a product for improving and / or treating myocardial infarction, and belongs to the technical field of medicines. The invention provides an application of a phellinus igniarius extract in preparation of a product for improving and / or treating myocardial infarction. The product comprises any one or more than two of medicine, health food and functional food. In the invention, the phellinus igniarius extract can obviously reduce myocardial infarction area, improve cardiac dysfunction, improve myocardial hypertrophy symptom, reduce myocardial cell apoptosis, inhibit inflammatory response, inhibit myocardial cell fibrosis and improve oxidative stress level, so that the effect of efficiently improving and / or treating acute myocardial infarction is achieved; the death rate of acute myocardial infarction is reduced. The result of the embodiment shows that the phellinus igniarius alcohol extract and the phellinus igniarius extract can achieve a better effect in improving and / or treating acute myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the preparation of a phellinus igniarius extract and its application in products for improving and / or treating myocardial infarction. Background Art

[0002] Acute myocardial infarction (AMI) is acute ischemic and hypoxic myocardial injury caused by coronary artery thrombosis or vasospasm, with clinical manifestations of chest pain, dyspnea, dizziness, nausea, sweating, etc. Therefore, AMI is defined as a "four highs and one more" type of cardiovascular disease with high morbidity, high mortality, high disability rate, high recurrence rate and multiple cumulative organs, and has become the most important cardiovascular disease that endangers human life and health.

[0003] The local ischemia and hypoxia caused by AMI will lead to the death of a large number of myocardial cells, affecting the normal structure and function of the heart. Then the heart will enter the self-repair stage. First, a strong aseptic inflammatory response will be induced in the infarct and downstream areas to remove dead / damaged cardiac cells and extracellular matrix. This stage is called the acute inflammatory phase. After the inflammatory phase, the heart will enter the post-inflammatory repair phase. Fibroblasts proliferate and begin to synthesize collagen to form scar tissue. This scar tissue does not have the elasticity and contractile function of normal myocardium, but helps to maintain structural integrity. However, due to the non-renewability of myocardial cells, the heart repair process usually cannot completely restore the existing myocardial structure and function. In addition, excessive activity, prolonged time or spatial expansion of the reactions involved in any of the above stages will lead to additional damage, including the loss of myocardial cells, inhibition of left ventricular contractile function, enhanced matrix degradation, etc., which will eventually lead to enlargement of the heart chamber, excessive tissue necrosis, destruction of the integrity of the ventricular wall, heart rupture, and even when scarring is formed, the degree of fibrosis will increase far beyond the initial infarction lesion area, and the prognosis will be even worse. It is known that thrombosis caused by rupture of coronary atherosclerotic plaques is the direct cause of AMI, but the molecular mechanism of how AMI leads to severe myocardial structural and functional damage is still not fully understood.

[0004] Phellinus igniarius, also known as mulberry, is a very precious large medicinal fungus. It belongs to the genus Phellinus of the family Pseudocercocephala in taxonomy. Phellinus igniarius tastes slightly bitter and can benefit the five internal organs, soften hard masses, detoxify, stop bleeding, activate blood circulation, activate the stomach and stop diarrhea. It is used by the people to treat gonorrhea, metrorrhagia, leukorrhea, etc. Phellinus igniarius contains a variety of active ingredients such as polysaccharides, polyphenols, flavonoids, triterpenes, alkaloids, steroids and other compounds. Pharmacological studies have shown that Phellinus igniarius has a variety of activities, such as anti-tumor, antiviral, hypoglycemic, hypolipidemic, immune-enhancing, liver protection, acute kidney repair and antibacterial effects. There is no relevant research on the function of Phellinus igniarius in improving and / or treating acute myocardial infarction. Summary of the invention

[0005] In view of the defects in the prior art, the object of the present invention is to provide an application of Phellinus igniarius extract in the preparation of products for improving and / or treating myocardial infarction.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The present invention provides an application of a mulberry linterus extract in preparing a product for improving and / or treating myocardial infarction; the product comprises any one or more of medicines, health foods and functional foods.

[0008] Preferably, the improvement and / or treatment of myocardial infarction includes any one or two or more of the following (1) to (9):

[0009] (1) Significantly reduce the area of ​​myocardial infarction;

[0010] (2) Effectively reduce mortality;

[0011] (3) Improve cardiac dysfunction;

[0012] (4) Improve symptoms of myocardial hypertrophy;

[0013] (5) Reduce cardiomyocyte apoptosis;

[0014] (6) Inhibit inflammatory response;

[0015] (7) Inhibit cardiomyocyte fibrosis;

[0016] (8) Improve the body’s oxidative stress level;

[0017] (9) Inhibit changes in serum LDH and / or CK levels.

[0018] Preferably, the active ingredients of the Phellinus igniarius extract include: phenolic acids, flavonoids, terpenes, alkaloids, steroids, lignin and coumarins.

[0019] Preferably, the Phellinus linteus extract includes Phellinus linteus alcohol extract and / or Phellinus linteus extract.

[0020] Preferably, the active ingredients of the mulberry linterus alcohol extract include ferulic acid, erythritol acid N, daidzein, Rhein-8-O-(6'-O-acetyl)glucoside and akebia phenylethanol glycoside B; the active ingredients of the mulberry linterus extract include dihydroquercetin and chalcone.

[0021] Preferably, the active substances of the mulberry linden alcohol extract and the mulberry linden extract also include caffeic acid, mulberry linden J, milkvine, Inoscavin A, dihydrokaempferol, mulberry linden A, gallic acid, vanillic acid, p-coumaric acid, mulberry linden D, mulberry linden A, philligridin B, mulberry linden I, purpurogenol, mulberry linden C, baicalin, protocatechuic aldehyde, 7-oxy-methyleriocin and mulberry linden G.

[0022] Preferably, the preparation method of the mulberry linterus ethanol extract comprises:

[0023] The mulberry ignia medicinal material block is mixed with an ethanol solution and subjected to reflux extraction to obtain a mulberry ignia alcohol extract;

[0024] The mulberry linseed alcohol extract is dried to obtain the mulberry linseed alcohol extract.

[0025] Preferably, the volume fraction of ethanol in the ethanol solution is 70% to 95%; the mass volume ratio of the Phellinus igniarius medicinal material block to the ethanol solution is (1 to 3) g: (10 to 30) mL.

[0026] Preferably, the temperature of the reflux extraction is 60-80° C.; the time of the reflux extraction is 2-4 h.

[0027] Preferably, the preparation method of the Phellinus linteus extract comprises:

[0028] The Phellinus igniarius medicinal material block is mixed with the ethanol solution and subjected to ultrasonic treatment to obtain the Phellinus igniarius extract;

[0029] The Phellinus linteus extract is extracted with ethyl acetate and then dried to obtain the Phellinus linteus extract.

[0030] Preferably, the volume fraction of ethanol in the ethanol solution is 70% to 95%; the mass volume ratio of the Phellinus igniarius medicinal material block and the ethanol solution when mixed is 1g: (10 to 20) mL; the power of the ultrasonic treatment is 150 to 200W; the temperature of the ultrasonic treatment is 50 to 70°C; and the time of the ultrasonic treatment is 1 to 3h.

[0031] Preferably, the dosage form of the drug includes any one or more of powder, oral solution, honey pills and granules.

[0032] Beneficial effects of the present invention

[0033] The present invention provides an application of a mulberry linterus extract in the preparation of a product for improving and / or treating myocardial infarction; the product includes any one or more of a medicine, a health food, and a functional food. In the present invention, the mulberry linterus extract can significantly reduce the area of ​​myocardial infarction, improve cardiac dysfunction, improve symptoms of myocardial hypertrophy, reduce myocardial cell apoptosis, inhibit inflammatory response, inhibit myocardial cell fibrosis, and improve the body's oxidative stress level, thereby achieving the effect of efficiently improving and / or treating acute myocardial infarction and reducing the mortality rate of acute myocardial infarction. The present invention shows through the results of the examples that both the mulberry linterus ethanol extract and the mulberry linterus extract can achieve good results in improving and / or treating acute myocardial infarction. Compared with the mulberry linterus extract, the mulberry linterus ethanol extract has a better effect in improving and / or treating acute myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0035] Figure 1 Normal electrocardiogram of mice before modeling;

[0036] Figure 2 ST-segment elevation electrocardiogram after acute myocardial infarction mouse model;

[0037] Figure 3 This is a graph showing the survival rate of mice in each group in Application Example 1;

[0038] Figure 4 This is a diagram showing typical left ventricular M-mode echocardiograms and related parameter analysis results of the parasternal short-axis view of each group of mice in Application Example 1;

[0039] Figure 5 This is the result of TTC staining of each group of mice in Application Example 1 and the percentage of myocardial infarction area in mice;

[0040] Figure 6 This is a graph showing the test results of serum biochemical index lactate dehydrogenase (LDH) in each group of mice in Application Example 1;

[0041] Figure 7 This is a graph showing the results of the detection of the content of creatine kinase (CK), a biochemical indicator in the serum of each group of mice in Application Example 1;

[0042] Figure 8 This is a diagram of the whole heart of each group of mice in Application Example 1 and the results of heart weight analysis;

[0043] Fig. 9The results of H&E staining of heart tissues of mice in each group in Application Example 1;

[0044] Fig.10 This is the result of evaluating the cross-sectional area of ​​cardiomyocytes by WGA staining in each group of mice in Application Example 1;

[0045] Fig.11 This is the result of TUNEL apoptosis staining of mice in each group in Application Example 1;

[0046] Fig.12 This is a graph showing the results of myocardial inflammation detection in each group of mice in Application Example 1;

[0047] Fig.13 This is the test result of inflammatory factors TNF-ɑ, IL-10 and IL-6 in each group of mice in Application Example 1;

[0048] Fig.14 This is a diagram showing Masson's trichrome staining and picrosirius red staining and analysis results of mice in each group in Application Example 1;

[0049] Fig.15 This is a graph showing the results of immunohistochemistry and Western-blot testing of the expression levels of myocardial fibrosis-related markers in each group of mice in Application Example 1;

[0050] Fig.16 This is a graph showing the results of detecting the expression levels of myocardial oxidative stress-related markers in each group of mice in Application Example 1. DETAILED DESCRIPTION

[0051] The present invention provides an application of a mulberry linterus extract in the preparation of a product for improving and / or treating myocardial infarction; the product includes any one or more of medicines, health foods and functional foods. In the present invention, the improvement and / or treatment of acute myocardial infarction includes any one or more of the following (1) to (9): (1) significantly reducing the area of ​​myocardial infarction; (2) effectively reducing mortality; (3) improving cardiac dysfunction; (4) improving symptoms of myocardial hypertrophy; (5) reducing myocardial cell apoptosis; (6) inhibiting inflammatory response; (7) inhibiting myocardial cell fibrosis; (8) improving the body's oxidative stress level; (9) inhibiting changes in the content of LDH and / or CK in serum. In the present invention, the dosage form of the drug may include any one or more of powder, oral liquid, honey pill and granule.

[0052] As an optional embodiment of the present invention, the myocardial infarction may include acute myocardial infarction.

[0053] In the present invention, the Phellinus igniarius extract can significantly reduce the myocardial infarction area caused by acute myocardial infarction. The present invention shows through the example results that the mulberry linterus ethanol extract and the mulberry linterus extract as the mulberry linterus extract can significantly reduce the myocardial infarction area of ​​the acute myocardial infarction mouse model.

[0054] In the present invention, the mulberry linterus extract can significantly reduce the mortality rate of acute myocardial infarction. The present invention shows through the example results that both the mulberry linterus ethanol extract and the mulberry linterus extract as mulberry linterus extract can reduce the mortality rate of the acute myocardial infarction mouse model, among which the mulberry linterus ethanol extract can more significantly reduce the mortality rate of the acute myocardial infarction mouse model.

[0055] In the present invention, the Phellinus igniarius extract can significantly improve cardiac dysfunction caused by acute myocardial infarction. The present invention shows through the results of the examples that the mulberry igniarius alcohol extract and the mulberry igniarius extract as mulberry igniarius extract can significantly improve the contractile function of the heart in an acute myocardial infarction mouse model, reverse the changes in echocardiographic parameters in an acute myocardial infarction mouse model, and achieve the effect of improving cardiac dysfunction.

[0056] In the present invention, the Phellinus igniarius extract can significantly improve the symptoms of myocardial hypertrophy caused by acute myocardial infarction. The present invention shows through the results of the examples that the mulberry igniarius ethanol extract and the mulberry igniarius extract as mulberry igniarius extract can significantly reduce inflammatory cell infiltration, reduce myocardial tissue damage, reduce heart weight, and reduce the cross-sectional area of ​​myocardial cells, thereby significantly improving the symptoms of myocardial hypertrophy caused by acute myocardial infarction.

[0057] In the present invention, the Phellinus igniarius extract can significantly reduce the apoptosis of myocardial cells caused by acute myocardial infarction. The implementation results of the present invention show that the ethanol extract of Phellinus igniarius and the extract of Phellinus igniarius as the extract of Phellinus igniarius can significantly reduce the number of apoptotic myocardial cells in the acute myocardial infarction mouse model.

[0058] In the present invention, the mulberry linterus extract can significantly inhibit the inflammatory response caused by acute myocardial infarction. The present invention shows through the results of the examples that the mulberry linterus ethanol extract and the mulberry linterus extract as mulberry linterus extracts can effectively regulate the levels of inflammatory factors in the serum and myocardial tissue of the acute myocardial infarction mouse model, and have the effect of inhibiting the inflammatory response.

[0059] In the present invention, the mulberry linterus extract can significantly inhibit myocardial cell fibrosis caused by acute myocardial infarction. The present invention shows through the example results that the mulberry linterus ethanol extract and the mulberry linterus extract as mulberry linterus extract can significantly inhibit the increase in the expression level of myocardial fibrosis-related proteins and inhibit myocardial cell fibrosis.

[0060] In the present invention, the mulberry linterus extract can significantly improve the body's oxidative stress level. The present invention shows through the results of the examples that the mulberry linterus ethanol extract and the mulberry linterus extract as mulberry linterus extracts can significantly inhibit the increase in the expression of oxidative stress proteins in an acute myocardial infarction mouse model, thereby improving the body's oxidative stress level.

[0061] In the present invention, the Phellinus linteus extract can significantly inhibit the changes in the content of LDH and / or CK in serum. The present invention shows through the results of the examples that the mulberry linteus ethanol extract and the mulberry linteus extract as mulberry linteus extract can significantly reduce the content of LDH and / or CK in the serum of the acute myocardial infarction mouse model.

[0062] In the present invention, the mulberry linterus extract can significantly reduce the area of ​​myocardial infarction, improve cardiac dysfunction, improve symptoms of myocardial hypertrophy, reduce myocardial cell apoptosis, inhibit inflammatory response, inhibit myocardial cell fibrosis, and improve the body's oxidative stress level, thereby achieving the effect of efficiently improving and / or treating acute myocardial infarction and reducing the mortality rate of acute myocardial infarction. The present invention shows through the results of the examples that the mulberry linterus ethanol extract and the mulberry linterus extract can achieve good results in improving and / or treating acute myocardial infarction. Compared with the mulberry linterus extract, the mulberry linterus ethanol extract has a better effect in improving and / or treating acute myocardial infarction.

[0063] In the present invention, the active ingredients of the Phellini extract include substances such as mulberry linteric acid, flavonoids, terpenes, alkaloids, steroids, lignin and coumarin. In the present invention, the Phellini extract includes mulberry linteric alcohol extract and / or mulberry linteric extract. In the present invention, the main active ingredients in the mulberry alcohol extract and the mulberry extract that exert their medicinal effects include: caffeic acid, mulberry linterin J (Phelligridin J), milk tree alkaloids (Hispidin), InoscavinA, dihydrokaempferol, mulberry linterin A (Hispolon), gallic acid, vanillic acid, p-coumaric acid, mulberry linterin D (PhelligridinD), mulberry linterin A (PhelligridinA), Phelligridin B, mulberry linterin I (Phelligridin I), porphyrin, mulberry linterin C (Phelligridin C), baicalin, protocatechuic aldehyde, 7-oxy-methyl eriocin and mulberry linterin G (Phelligridin G) and other compounds. In the present invention, the active ingredients of the mulberry linterus alcohol extract include ferulic acid, lucidenic acid N, daidzein, rhein-8-O-(6'-acetyl) glucoside and desrhamnosylisoacteoside; while the active ingredients of the mulberry linterus extract do not contain ferulic acid, lucidenic acid N, daidzein, rhein-8-O-(6'-O-acetyl) glucoside and desrhamnosylisoacteoside. In the present invention, the active ingredients of the mulberry linterus extract include dihydroquercetin and chalcone, while the active ingredients of the mulberry linterus alcohol extract do not contain dihydroquercetin and chalcone. The present invention does not specifically limit the source of the mulberry linterus extract, and any conventional mulberry linterus extract in the art can be used. The present invention does not specifically limit the source of the mulberry linterus used to prepare the mulberry linterus extract, and any conventional mulberry linterus in the art can be used. As an optional embodiment of the present invention, the mulberry linterus can be any one or more of mulberry mulberry linterus, poplar mulberry linterus and wild horse mulberry linterus. In the present invention, the mulberry linterus extract can also be prepared by itself. The present invention does not specifically limit the preparation method of the mulberry linterus extract, and the preparation method of the conventional mulberry linterus extract in the art can be adopted. As an optional embodiment of the present invention, the mulberry linterus extract can be mulberry linterus alcohol extract and / or mulberry linterus extract.

[0064] As an optional embodiment of the present invention, the preparation method of the mulberry linterus ethanol extract may include:

[0065] The mulberry ignia medicinal material block is mixed with an ethanol solution and subjected to reflux extraction to obtain a mulberry ignia alcohol extract;

[0066] The mulberry linseed alcohol extract is dried to obtain the mulberry linseed alcohol extract.

[0067] The invention mixes the Phellinus igniarius medicinal material block with an ethanol solution and then performs reflux extraction to obtain the Phellinus igniarius alcohol extract.

[0068] When the present invention uses Phellinus igniarius to prepare the mulberry igniarius alcohol extract, it is preferred to first cut Phellinus igniarius into small pieces to obtain mulberry igniarius medicinal material blocks. In the present invention, the size of the mulberry igniarius medicinal material blocks can be 1 to 5 cm, or 1 to 3 cm. After obtaining the mulberry igniarius medicinal material blocks, the present invention mixes the mulberry igniarius medicinal material blocks with an ethanol solution. As an optional embodiment of the present invention, the volume fraction of ethanol in the ethanol solution can be 70% to 95%, or 70%. The mass volume ratio of the mulberry igniarius medicinal material blocks to the ethanol solution can be (1 to 3) g: (10 to 30) mL, or 1 g: 20 mL. After obtaining a mixed system of mulberry igniarius and ethanol aqueous solution, the present invention performs reflux extraction. As an optional embodiment of the present invention, the temperature of the reflux extraction can be 60 to 80° C., or 70° C.; the time of the reflux extraction can be 2 to 4 hours, or 3 hours. After the reflux extraction is completed, the present invention obtains the mulberry igniarius alcohol extract.

[0069] After obtaining the mulberry ignia alcohol extract, the present invention dries the mulberry ignia alcohol extract to obtain the mulberry ignia alcohol extract.

[0070] The present invention does not specifically limit the drying method, and any conventional drying method in the art can be used. As an optional embodiment of the present invention, the drying method includes rotary evaporation concentration drying and vacuum freeze drying. In the present invention, the temperature of the rotary evaporation concentration drying can be 50-80°C, or 50-60°C; the speed of the rotary evaporation concentration drying can be 50-100 rpm, or 80 rpm; the vacuum degree of the rotary evaporation concentration drying is 0.03MPa. The present invention uses rotary evaporation concentration drying to dry the mulberry ignia alcohol extract to be free of alcohol taste. After the rotary evaporation concentration drying is completed, the present invention performs vacuum freeze drying. As an optional embodiment, the temperature of the vacuum freeze drying can be -20°C; the time of the vacuum freeze drying can be 24h. As an optional embodiment of the present invention, after the vacuum freeze drying is completed, the present invention can grind the dried product into fine powder to obtain the mulberry ignia alcohol extract.

[0071] As an optional embodiment of the present invention, the preparation method of the Phellinus linteus extract may include:

[0072] The Phellinus igniarius medicinal material block is mixed with the ethanol solution and subjected to ultrasonic treatment to obtain the Phellinus igniarius extract;

[0073] The Phellinus linteus extract is extracted with ethyl acetate and then dried to obtain the Phellinus linteus extract.

[0074] The invention mixes the Phellinus igniarius medicinal material block with an ethanol solution and performs ultrasonic treatment to obtain the Phellinus igniarius extract.

[0075] When the present invention uses Phellinus igniarius to prepare the Phellinus igniarius extract, it is preferred to first cut Phellinus igniarius into small pieces to obtain Phellinus igniarius medicinal material blocks. In the present invention, the size of the Phellinus igniarius medicinal material blocks can be 1 to 5 cm, or 1 to 3 cm. After obtaining the Phellinus igniarius medicinal material blocks, the present invention mixes the Phellinus igniarius medicinal material blocks with an ethanol solution. As an optional embodiment of the present invention, the volume fraction of ethanol in the ethanol solution can be 70% to 95%; the mass volume ratio of the Phellinus igniarius medicinal material blocks and the ethanol solution when mixed can be 1g: (10 to 20) mL. As an optional embodiment of the present invention, the power of the ultrasonic treatment can be 150 to 200W, or 160W; the temperature of the ultrasonic treatment can be 50 to 70°C, or 50°C; the time of the ultrasonic treatment can be 1 to 3h, or 1h. As an optional embodiment of the present invention, the number of ultrasonic treatments can be 2 times. As an optional embodiment of the present invention, the step of performing two ultrasonic treatments of the present invention may include: after mixing the Phellinus igniarius medicinal material block with an ethanol solution, performing a first ultrasonic treatment to obtain a first extract and a medicinal residue; after mixing the obtained medicinal residue with an ethanol solution, performing a second ultrasonic treatment to obtain a second extract; combining the first extract and the second extract to obtain a Phellinus igniarius extract. As an optional embodiment of the present invention, when performing the first ultrasonic treatment, the volume fraction of ethanol in the ethanol solution may be 95%, and the mass volume ratio of the Phellinus igniarius medicinal material block to the ethanol solution may be 1g:10mL; when performing the second ultrasonic treatment, the volume fraction of ethanol in the ethanol solution may be 70%; the amount of ethanol solution added to the obtained medicinal residue is preferably added based on the mass of the Phellinus igniarius medicinal material block; the volume mass ratio of the amount of ethanol solution added to the medicinal residue to the Phellinus igniarius medicinal material block is 20mL:1g.

[0076] After obtaining the Phellinus linteus extract, the present invention extracts the Phellinus linteus extract with ethyl acetate and then dries it to obtain the Phellinus linteus extract.

[0077] As an optional embodiment of the present invention, when extracting the Phellinus linteus extract, the volume ratio of the Phellinus linteus extract to ethyl acetate can be 2:1. As an optional embodiment of the present invention, the present invention can obtain 60 mL of Phellinus linteus extract concentrate by rotary evaporation of the Phellinus linteus extract, and then extract the 60 mL of Phellinus linteus extract concentrate with 30 mL of ethyl acetate. As an optional embodiment of the present invention, the number of extractions can be 3 times. After the extraction of the present invention is completed, the obtained extract is dried to obtain the Phellinus linteus extract. The drying method includes rotary evaporation concentration drying and vacuum freeze drying. In the present invention, the temperature of the rotary evaporation concentration drying can be 50-80°C, or 50-60°C; the speed of the rotary evaporation concentration drying can be 50-100 rpm, or 80 rpm; the vacuum degree of the rotary evaporation concentration drying is 0.03MPa. The present invention adopts rotary evaporation concentration drying until there is no alcohol taste. After the rotary evaporation concentration drying is completed, the present invention performs vacuum freeze drying. As an optional embodiment, the temperature of the vacuum freeze drying can be -20°C; the time of the vacuum freeze drying can be 24h. As an optional embodiment of the present invention, after vacuum freeze drying, the present invention can grind the dried product into fine powder to obtain the Phellinus igniarius extract.

[0078] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0079] In the following implementation of the present invention, the effect of the Phellinus linteus extract is verified by taking the Phellinus linteus as an example.

[0080] Example 1

[0081] Poplar Phellinus Lindl Extract

[0082] 1. Preparation of Morinda tinctoria ethanol extract

[0083] Cut Phellinus igniarius into small pieces, generally with a size of 1 to 3 cm, to obtain Phellinus igniarius medicinal material blocks, take 30 g of Phellinus igniarius medicinal material blocks and place them in a round-bottom flask, add 600 mL of ethanol solution with a volume percentage of 70% ethanol according to the mass volume ratio of medicinal material to reagent of 1 g:20 mL, and reflux extraction is carried out at 70°C for 3 hours, then filter the extract, and evaporate it on a rotary evaporator (water bath temperature of 60°C, speed of 80 rpm, vacuum degree of about 0.03 MPa) until there is no alcohol smell, take it out, and then perform vacuum freeze drying at a temperature of -20°C for 24 hours. After the vacuum freeze drying is completed, grind the obtained dry material into fine powder, weigh and record it, and obtain the Phellinus igniarius alcohol extract.

[0084] 2. Preparation of Phellinus linteus Extract

[0085] Cut Phellinus igniarius into small pieces, generally 1-3 cm in size, to obtain Phellinus igniarius medicinal material blocks. Take 60g Phellinus igniarius medicinal material blocks, add 600mL of ethanol solution with a volume percentage of 95% ethanol according to the mass volume ratio of medicinal materials to reagents of 1g:10mL, ultrasonic power 160W, ultrasonic temperature 50℃ for 1h, filter the filtrate, add 1200mL of ethanol solution with a volume percentage of 70% ethanol according to the mass volume ratio of medicinal materials to reagents of 1g:20mL, and then ultrasonic power 160W, ultrasonic temperature 50℃ for 1h. After the ultrasonic is completed, filter the filtrate. Combine the two filtrates, and use a rotary evaporator for rotary evaporation. The rotary evaporation is carried out under the conditions of water bath temperature 60℃, speed 80 rpm, and vacuum degree of about 0.03MPa. Rotary evaporation is carried out until there is no alcohol taste. Adjust the residual liquid volume of rotary evaporation to be fixed within 60mL as much as possible. If it exceeds 60mL, continue rotary evaporation. If it is less than 60mL, use a small amount of distilled water to keep the volume within 60mL. The remaining liquid was then transferred to a separatory funnel for extraction, 30 mL of ethyl acetate was taken each time, and extraction was performed three times. The ethyl acetate extracts were combined and vacuum freeze-dried. The vacuum freeze-drying temperature was -20°C and the vacuum freeze-drying time was 24 h. After the vacuum freeze-drying was completed, the obtained dry material was ground into fine powder, weighed and recorded to obtain the Phellinus igniarius extract.

[0086] Example 2

[0087] Analysis of components of ethanol extract of Phellinus igniarius and extract of Phellinus igniarius

[0088] 1. Sample Processing

[0089] The mulberry linterus alcohol extract and mulberry linterus extract prepared in Example 1 were placed in a freeze dryer (Scientz-100F) and vacuum freeze-dried for 63 hours to obtain further freeze-dried mulberry linterus alcohol extract and mulberry linterus extract, namely, mulberry linterus alcohol extract freeze-dried powder and mulberry linterus extract freeze-dried powder.

[0090] The lyophilized powder of mulberry linterus alcohol extract and the lyophilized powder of mulberry linterus extract were ground into powder using a grinder (MM 400, Retsch) (30 Hz, 1.5 min); the lyophilized powder of mulberry linterus alcohol extract and the lyophilized powder of mulberry linterus extract were weighed respectively using an electronic balance (MS105DM), that is, 50 mg of sample powder was weighed respectively, and 1200 μL of -20°C pre-cooled 70% methanol water internal standard extract was added (if less than 50 mg, 1200 μL of extractant was added per 50 mg of sample). The internal standard (L-2-chlorophenylalanine) extract was prepared by dissolving 1 mg of the standard in 1 mL of 70% methanol water to prepare a 1000 μg / mL standard stock solution, and the 1000 μg / mL stock solution was further diluted with 70% methanol to prepare a 250 μg / mL internal standard solution.

[0091] The extract solution containing the internal standard was vortexed once every 30 minutes, each time for 30 seconds, for a total of 6 times; after centrifugation (speed 12000rpm, 3min), the supernatant was aspirated, the sample was filtered with a microporous filter membrane (0.22μmpore size), and stored in an injection bottle for UPLC-MS / MS analysis.

[0092] 2. Liquid phase conditions

[0093] Chromatographic column: Agilent SB-C18 1.8μm, 2.1mm*100mm; mobile phase: phase A is ultrapure water (with 0.1% formic acid added), phase B is acetonitrile (with 0.1% formic acid added); elution gradient: 0.00min B phase ratio is 5%, within 9.00min, the B phase ratio linearly increases to 95%, and maintains at 95% for 1min, 10.00-11.10min, the B phase ratio is reduced to 5%, and balanced at 5% for 14min; flow rate 0.35mL / min; column temperature 40℃; injection volume 2μL.

[0094] 3. Mass spectrometry conditions

[0095] The temperature of the electrospray ionization (ESI) source was 500°C; the ion spray voltage (IS) was 5500V (positive ion mode) / -4500V (negative ion mode); the ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) were set to 50, 60 and 25 psi, respectively, and the collision-induced ionization parameter was set to high. The QQQ scan used the MRM mode, and the collision gas (nitrogen) was set to medium. The DP and CE of each MRM ion pair were completed by further optimization of the declustering potential (DP) and collision energy (CE). A specific set of MRM ion pairs was monitored in each period according to the metabolites eluted in each period.

[0096] The active ingredients of the alcohol extract of Morinda tinctoria obtained by testing include: caffeic acid, morphine J (Phelligridin J), milk tree alkaloids (Hispidin), Inoscavin A, dihydrokaempferol, Morinol A (Hispolon), gallic acid, vanillic acid, p-coumaric acid, Morin D (Phelligridin D), Morin A (Phelligridin A), Phelligridin B, Morin I (Phelligridin I), porphyrin, Morin C (Phelligridin C), baicalin, protocatechuic aldehyde, 7-oxy-methyl eriocin, Morin G (Phelligridin G), ferulic acid, lucidic acid N, daidzein, rhein-8-O-(6'-acetyl) glucoside and desrhamnosylisoacteoside.

[0097] The active ingredients of Phelligridin extract include: Caffeic acid, Phelligridin J, Hispidin, Inoscavin A, Dihydrokaempferol, Hispolon, Gallic acid, Vanillic acid, p-coumaric acid, Phelligridin D, Phelligridin A, Phelligridin B, Phelligridin I, Porphyrin, Phelligridin C, Baicalin, Protocatechuic aldehyde, 7-O-methyleriocin, Phelligridin G, Dihydroquercetin and Chalcone.

[0098] Among them, ferulic acid, lucidenic acid N, daidzein, rhein-8-O-(6'-acetyl) glucoside and desrhamnosylisoacteoside are unique to the mulberry linterus ethanol extract; dihydroquercetin and chalcone are unique to the mulberry linterus extract.

[0099] Application Example 1

[0100] 1. Cardiac ultrasound function testing method

[0101] Cardiac ultrasound function tests were performed on mice in each group before surgery and on the 28th day after surgery. The specific procedures were as follows:

[0102] The mouse chest and abdomen were depilated, and the isoflurane gas concentration in the anesthesia machine was adjusted to 2% to anesthetize the mouse. After the induction of anesthesia was completed, the maintenance anesthesia concentration was set to 1% to 1.5%.

[0103] After applying the coupling agent, place the probe parallel to the midline of the mouse on its left chest, with the tip of the probe facing the head of the mouse. At this time, turn the tip of the probe 45° to the left to make the probe parallel to the long axis of the mouse's left ventricle. The image obtained is the parasternal long axis section of the left ventricle.

[0104] On the basis of the long axis, the probe head is rotated about 90° to the right, and the probe is moved and tilted regularly to display the level of the papillary muscles. The image obtained is the short-axis papillary muscle slice of the left ventricle.

[0105] Left ventricular function test: 8 points were selected, namely, the upper wall of the ventricular septum at end diastole, the lower wall of the ventricular septum at end diastole, the upper edge of the free wall at end diastole, the lower edge of the free wall at end diastole, the upper wall of the ventricular septum at end systole, the lower wall of the ventricular septum at end systole, the upper edge of the free wall at end systole, and the lower edge of the free wall at end systole for measurement to obtain the ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at end diastole (LVIDd), left ventricular internal diameter at end systole (LVIDs), left ventricular wall thickness at end diastole (LVPWd), left ventricular wall thickness at end systole (LVPWs), left ventricular end volume at end diastole (LVEDV), and left ventricular end volume (LVESV).

[0106] Before the operation, the experimental mice were tested for cardiac ultrasound function to confirm that the mice had normal cardiac function and to exclude mice with possible abnormal cardiac function. The following experiments were performed using mice with normal cardiac function.

[0107] 2. Establishment of animal model of acute myocardial infarction, referred to as myocardial infarction model

[0108] Preparation and anesthesia of mice were performed in the same manner as described above for cardiac ultrasound functional testing.

[0109] Surgery: The acute myocardial infarction model was established in C57BL / 6J mice by ligating the left anterior descending coronary artery. The specific steps are as follows:

[0110] Open chest: Find the most obvious position of the mouse's apical beat, make a 1 cm incision on the left side of the sternum along the direction of the mouse's head, use hemostat and tweezers to bluntly separate the pectoralis major and pectoralis minor muscles layer by layer, quickly open the chest cavity from the third and fourth intercostal spaces, rotate the hemostat about 45° to the left to open the intercostal space, and use the heartbeat rhythm to make the heart pop out automatically.

[0111] Find the ligation position: find the approximate position, gently scrape the heart with a 6-0 ligation needle, and you can clearly see the left coronary artery, which is different from the surrounding color. The specific ligation position is 1 to 2 mm away from the root at the junction of the lower edge of the left atrial appendage and the pulmonary artery cone. Use a 6-0 suture to quickly pass through and ligate. The ligation width and depth should be appropriate. The ligation is successful when the anterior wall of the left ventricle turns cyanotic or pale to the naked eye.

[0112] Suturing the chest: After ligation, check for bleeding, gently return the heart to the chest, and quickly squeeze out the air to avoid pneumothorax. Remove the anesthesia mask, wait for spontaneous breathing to resume, and suture the muscles and skin with 4-0 sutures.

[0113] Awakening: After surgery, place the mouse on a heating blanket and wait for it to wake up. After regaining mobility, put it back into the cage.

[0114] 3. Experimental Setup

[0115] 1) Treatment of the drugs used in the test: The mulberry linterus alcohol extract of Example 1 was dissolved in a sodium carboxymethylcellulose solution to obtain a mulberry linterus alcohol extract suspension; the mulberry linterus extract of Example 1 was dissolved in a sodium carboxymethylcellulose solution to obtain a mulberry linterus extract suspension. The mass concentration of the mulberry linterus alcohol extract in the mulberry linterus alcohol extract suspension was 8.0 mg / mL, and the mass concentration of the mulberry linterus extract in the mulberry linterus extract suspension was 8.0 mg / mL. The mass concentration of sodium carboxymethylcellulose in the sodium carboxymethylcellulose solution was 0.5%.

[0116] 2) 6-8 week old C57BL / 6J male mice (SPF grade) were selected as the research subjects. Eight of them were randomly selected as the sham control group, recorded as the sham group. The sham control group underwent thoracotomy using the method in step 2, but no ligation was performed. The remaining mice were used to establish an animal model of myocardial infarction using the method in step 2 above. After confirming that the model was successful, the 63 model mice were randomly divided into three experimental groups, with 21 mice in each experimental group. The three experimental groups were respectively used as the model group, recorded as the MI group; the model group + mulberry igniare extract group, recorded as the MI+AE group; the model group + mulberry igniare extract group, recorded as the MI+EX group.

[0117] Among them, after the model was successfully established in the MI+EX group, mice were gavaged 2 hours after ligation, and then gavaged every day. The daily dose of mulberry ignia ethanol extract per mouse was 100 mg kg -1 ;

[0118] After the MI+AE group mice were successfully modeled, they were gavaged 2 hours after ligation, and then gavaged every day. The daily dose of Phellinus igniarius extract for each mouse was 100 mg kg -1 ;

[0119] After the MI group mice were successfully modeled, they were gavaged with the same volume of sodium carboxymethylcellulose solution every day;

[0120] The sham group was intragastrically administered with the same volume of sodium carboxymethylcellulose solution every day.

[0121] The above-mentioned administration method was used for continuous gavage administration for 28 days. The death of mice in each group was observed and recorded within 28 days. The cardiac function of surviving mice was detected, and the levels of serum biochemical indicators lactate dehydrogenase (LDH) and creatine kinase (CK) were detected. After 28 days of gavage administration, 2% isoflurane was used to anesthetize the heart to perform echocardiography. The heart function of mice was detected by echocardiography. Mice were anesthetized with isoflurane, and serum and heart tissue were obtained.

[0122] 3) Before and after modeling, the mice were tested for electrocardiogram. The results of electrocardiogram before and after modeling are as follows: Figures 1-2 As shown. Figure 1 Normal electrocardiogram of mice before modeling; Figure 2 Electrocardiogram of ST segment elevation after modeling in mice.

[0123] Depend on Figures 1-2 It was found that before ligation of the left anterior descending coronary artery of mice, the electrocardiogram showed that the ST segment was not elevated; after ligation of the left anterior descending coronary artery of mice, the electrocardiogram showed that the ST segment was significantly elevated, and the absolute value of the elevation was ≥0.1mV, indicating that the mouse model was successfully established.

[0124] 4) Observe and record the death of mice in each group within 28 days after intragastric administration, and record the survival rate of mice. Figure 3 shown.

[0125] like Figure 3 As shown, no mice died in the Sham group within 28 days of oral administration, that is, the survival rate was 100%; 8 mice died in the MI group, and the survival rate was 62%, which was statistically different from the Sham group (P < 0.05); 6 mice died in the MI+EX group, and 2 mice died in the MI+AE group. The survival rates of the above treatment groups were 71% and 90%, respectively, and the differences were statistically significant compared with the MI group (P < 0.05). The cause of death of mice in the MI group may be related to the large infarct area or complications such as malignant arrhythmia. The administration of mulberry ignia ethanol extract can effectively reduce the mortality rate of mice after myocardial infarction.

[0126] 5) At the two time points of oral administration and 28 days, cardiac ultrasound function tests were performed on the surviving mice in each group using the method in step 1 above. The typical left ventricular M-mode echocardiograms and related parameter analysis results of the mice in each group are shown in the figure. Figure 4 . Figure 4 In the middle, # indicates P < 0.05 compared with the control (Sham); * indicates P < 0.05 compared with the model group (MI). The same applies below.

[0127] Depend on Figure 4 The results showed that compared with the Sham group, the LVAWd and LVAWs of the mice in the MI group were significantly increased, and the EF and FS were significantly decreased (P<0.05), but the LVIDd and LVIDs were significantly increased (P<0.05); compared with the model group, the EF and FS of the mice in the MI+AE group were improved (P<0.05). This suggests that the mulberry ignia extract can improve the contractile function of the heart of mice after myocardial infarction, and it was found that the changes in the echocardiographic parameters of the mice treated with the mulberry ignia extract were reversed, indicating that the mulberry ignia extract has an improving effect on myocardial dysfunction.

[0128] 6) 28 days after intragastric administration, the myocardial infarction area of ​​surviving mice was detected by TTC (2,3,5-triphenyltetrazolium chloride) staining.

[0129] The TTC staining method was used to detect the proportion of myocardial infarction area in each group of mice. The percentage of myocardial infarction area in mice is shown in Table 1; the staining results and the percentage of myocardial infarction area in mice are shown in Figure 5 shown.

[0130] Table 1 Myocardial infarction area percentage in mice (mean)

[0131] Group Myocardial infarction area (%) Sham Group 0 MI group 57.75±4.99 MI+EX Group 34.33±2.08 MI+AE group 34.33±0.57

[0132] 28 days after the left anterior descending branch of the mouse coronary artery was ligated, the color of the heart turned white. Figure 5 TTC staining showed that the myocardium of mice after surgery was divided into red and white, red for normal myocardium and white for necrotic myocardium. After calculation, the myocardial infarction area of ​​mice in the model group was 57.75%, the myocardial infarction area of ​​mice gavaged with mulberry linoleum alcohol extract was 34.33%, and the myocardial infarction area of ​​mice gavaged with mulberry linoleum extract was 34.33%. This shows that mulberry linoleum alcohol extract and mulberry linoleum extract can significantly reduce the area of ​​myocardial infarction in mice.

[0133] 7) 28 days after intragastric administration, the serum biochemical indexes lactate dehydrogenase (LDH) and creatine kinase (CK) were detected.

[0134] 28 days after intragastric administration, the mice in each group were anesthetized with isoflurane and blood was collected. The blood was centrifuged at 3000 r / min at 4°C for 20 min, and the supernatant was collected and stored at -80°C. The serum biochemical indicators lactate dehydrogenase (LDH) and creatine kinase (CK) were detected using a biochemical analyzer.

[0135] The results of serum biochemical indexes lactate dehydrogenase (LDH) and creatine kinase (CK) in each group of mice were as follows: Figures 6-7 As shown. Among them, Figure 6 The results of serum biochemical index lactate dehydrogenase (LDH) test of mice in each group; Figure 7 Figure 2 is the detection result of serum biochemical index creatine kinase (CK) content in each group of mice.

[0136] Depend on Figures 6-7 It was found that the serum LDH content of the sham group, MI group, MI+EX group, and MI+AX group was 358±38.64U / L, 510±75.98U / L, 369.4±47U / L, and 361±43.79U / L, respectively; the serum CK content was 542±80.35U / L, 1031.5±228.23U / L, 906.25±200.79U / L, and 605±96.96U / L, respectively. Compared with the sham group, the serum LDH content of the mice in the MI group was statistically different (P<0.05). Compared with the MI group, the serum LDH content of the mice in the MI+EX group and the MI+AX group could be reduced (P<0.05). Compared with the sham group, the serum CK content of the mice in the MI group was significantly increased (P<0.05). Compared with the MI group, the MI+EX group and the MI+AE group could reduce the serum CK content of mice, but only the MI+AE group had a statistically significant difference (P<0.05). Morinda tinctoria ethanol extract can inhibit the serum LDH and CK content of mice after myocardial infarction.

[0137] 8) Effect of Phellinus igniarius extract on myocardial hypertrophy after myocardial infarction

[0138] (1) 28 days after intragastric administration, the body weight and tibia length of the surviving mice in each group were measured. Then, the surviving mice in each group were anesthetized and their hearts were removed and weighed. Figure 8 As shown in A; the ratio of heart weight to body weight of mice in each group Figure 8 As shown in B; the ratio of heart weight to tibia length of mice in each group Figure 8 As shown in C. Figure 8 HW / BW is the ratio of heart weight to mouse body weight; HW / TL is the ratio of heart weight to tibia length.

[0139] Depend on Figure 8 The results showed that the heart of the MI group was significantly larger than that of the Sham group, and the cardiac hypertrophy index was significantly increased compared with the body weight and tibia length (P<0.05). After drug treatment, there was no significant difference between the MI+EX group and the MI group, and the cardiac hypertrophy index was significantly improved in the MI+AE group.

[0140] After anesthetizing the surviving mice in each group, the hearts were taken out, a part of which was fixed in 4% paraformaldehyde, and the rest was passed through liquid nitrogen and stored at -80°C for future use.

[0141] (2) Paraffin embedding

[0142] After the heart tissue is taken out, it is immediately immersed in 4% paraformaldehyde for fixation for more than 24 hours.

[0143] Place the tissue in a labeled tissue embedding box and rinse under running water for 1 hour.

[0144] Dehydration: Dehydrate in the following order: 75% ethanol (75% ethanol refers to an ethanol aqueous solution with an ethanol volume fraction of 75%, the same below) for 60 min; 85% ethanol for 60 min; 95% ethanol for 60 min; 100% I ethanol for 30 min; 100% II ethanol for 30 min; 100% III ethanol for 30 min; 100% IV ethanol for 30 min.

[0145] Dehydration and transparentization: After the heart tissue is dehydrated, it is treated in a reagent with a volume ratio of xylene to ethanol of 1:1 for 30 minutes; then treated in xylene I for 30 minutes, then treated in xylene II for 30 minutes; finally treated in xylene III for 30 minutes.

[0146] Paraffin immersion: Soak the deethanol-transparent heart in soft wax for 2 hours and then transfer to hard wax for 2 hours.

[0147] Embedding: Add a layer of melted hard wax to the bottom of the disposable embedding plate, clamp the heart tissue soaked in hard wax, place it with the cut surface facing down on the bottom of the embedding plate, place it on the embedding table to allow it to solidify slightly, place the preheated embedding frame on the embedding plate, continue to add melted wax to the upper edge of the embedding frame, place it on a frozen ice plate, wait for it to completely solidify, gently peel off the embedding plate, and check for cracks.

[0148] Storage: Store the embedded wax blocks at 4°C.

[0149] (3) Paraffin section

[0150] Fix the embedding block on the specimen stage so that the bottom section of the embedding block is parallel to the cross section of the specimen clamp. Push the knife stage to the appropriate position so that the slicer and the tissue section form an angle of about 5°, and the upper and lower edges of the embedding block are parallel to the knife edge.

[0151] Trimming: Adjust the slice thickness on the micro-motion device. Start with 20μm thickness and adjust the slice thickness to 4μm when the tissue is cut.

[0152] Slicing: Turn the wheel with your right hand, hold the brush in your left hand to pick up the slices under the blade, and drag the cut wax strip. When the required slices are complete in the wax strip, stop rotating.

[0153] Spreading: Use tweezers to gently pick up the cut wax tape and transfer it to room temperature water. Then use a glass slide to transfer the intact film to a spreader in a 45°C constant temperature water bath.

[0154] Pick up the slices: After the tissue is fully unfolded, pick it up with a labeled slide, place it vertically on a slice rack, place it in an oven at 37°C overnight, and then collect the slices for later use.

[0155] (4) H&E (Hematoxylin-eosin) staining

[0156] Drying: Place the cut paraffin sections into the section rack and bake in an oven at 60°C for 2 hours.

[0157] Dewaxing: Dewaxing in xylene twice, 10 min each time.

[0158] Rehydration: follow the order of 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol, 3 minutes for each gradient, and finally soak in distilled water for 2 minutes.

[0159] Staining: circle the stained tissue with an immunohistochemistry pen, add hematoxylin stain solution, cover the tissue and stain the cell nucleus for 7 minutes, and wash off the floating color with distilled water; differentiate with differentiation solution for 1 minute, add tap water to soak and wash twice, each time for 5 minutes; stain with eosin stain solution for 2 minutes, and pour off the excess stain solution.

[0160] Dehydration: Soak in 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol I for 2 seconds each, and soak in 100% ethanol II for 1 minute.

[0161] Transparency: Soak in xylene twice, 1 min each time.

[0162] Sealing: Dry the slices after being transparentized with xylene, add neutral gum, and seal with a clean cover glass. Dry them in a fume hood by self-heating and observe them under a microscope.

[0163] The results of H&E staining of the heart tissues of mice in each group are shown in Fig. 9 shown.

[0164] Depend on Fig. 9 The results showed that the cardiomyocytes in the Sham group were neatly arranged, the cytoplasm was uniform and rich, and the interstitium was normal. In the MI group, some cardiomyocytes had missing nuclei and vacuolar changes in the cells. The cardiomyocytes in the infarcted area were disorderly arranged and had unclear boundaries, and a large number of inflammatory cells were infiltrated. In contrast, the myocardial tissue damage in the MI+EX and MI+AE groups was significantly reduced, with only a small amount of inflammatory cells infiltrating. Phellinus igniarius extract can effectively reduce the pathological damage caused by myocardial infarction in mice, and the alcohol extract is more effective than the extract.

[0165] (5) WGA staining

[0166] WGA staining was used to assess the cross-sectional area (CSA) of cardiomyocytes as follows:

[0167] 1) Drying, dewaxing and rehydration are the same as H&E staining.

[0168] 2) Antigen repair: Place tissue sections in citrate repair solution (pH 6.0) and perform antigen repair in a pressure cooker. After the water boils, count for 5 minutes. After taking out, cool naturally to room temperature and wash with water twice, each time for 5 minutes.

[0169] 3) Add WGA staining solution: Prepare WGA-AF488 staining solution (diluted with PBS 1:100), add 30 μL of WGA-AF488 staining solution to each tissue, and place in a wet box at 4°C overnight.

[0170] 4) Rewarming: Take out the slices on the next day and let them stand at room temperature or 37°C for 30 minutes to rewarm. Then discard the primary antibody and rinse with PBS three times, 5 minutes each time.

[0171] 5) DAPI nuclear staining: Add DAPI dropwise onto the tissue and let stand for 5 to 10 minutes.

[0172] 6) Add tissue autofluorescence quencher onto the tissue and let stand for 5 to 10 minutes.

[0173] 7) Sealing: Seal the slides with anti-fluorescence quenching sealing solution and take photos under a fluorescence microscope.

[0174] The cross-sectional area of ​​myocardial cells was evaluated by WGA staining in each group of mice. Fig.10 .

[0175] Depend on Fig.10 The results showed that the CSA of the Sham group was significantly lower than that of the MI group (P<0.05). Compared with the MI group, the inhibitory effects of MI+EX and MI+AE groups on cardiomyocyte hypertrophy were stronger. This showed that mulberry linterus ethanol extract and mulberry linterus extract could inhibit myocardial hypertrophy after myocardial infarction.

[0176] 9) Effect of Phellinus igniarius extract on cardiomyocyte apoptosis after myocardial infarction

[0177] (1) TUNEL apoptosis staining

[0178] The drying, dewaxing and rehydration of the slides were the same as those for H&E staining.

[0179] Membrane permeabilization: Add 20 μg / mL DNase-free proteinase K and allow to act at room temperature for 30 minutes to permeabilize the cell membrane and nuclear membrane to facilitate the entry of the TUNEL probe into the cell nucleus and ensure adequate staining.

[0180] Washing: Wash 3 times with PBS, 5 min each time.

[0181] Prepare TUNEL detection solution: TdT enzyme and fluorescent labeling solution are prepared in a ratio of 1:9. Each sample requires 50 μL. Prepare the detection solution according to the sample volume, mix thoroughly, and store in a dark place.

[0182] Incubation and staining: Place each sample in a humidified chamber, add 50 μL of the prepared detection solution to cover the tissue, and incubate at 37°C in the dark for 6 minutes.

[0183] Washing: Wash 3 times with PBS, 5 min each time.

[0184] Nuclear staining: add appropriate amount of DAPI to each sample and stain for 10 minutes.

[0185] Washing: Wash 3 times with PBS, 5 min each time.

[0186] Sealing and observation: Seal the slides with anti-fluorescence quenching sealing solution and observe under a microscope.

[0187] (2) Cell apoptosis is the main factor affecting mortality and infarct size after myocardial infarction. Fig.11 In the TUNEL colorimetric method, normal cardiomyocytes are stained blue, while apoptotic cardiomyocytes are stained red. After merging with DAPI, they become bright pink, which is used for statistical analysis to determine the apoptosis rate of cells.

[0188] The statistical results of apoptosis rate of mouse cells in each group are as follows Fig.11 shown.

[0189] Depend on Fig.11 It was found that compared with the Sham group, the apoptosis rate of cardiomyocytes in the MI group was increased (P<0.05); compared with the MI group, the number of apoptotic cardiomyocytes in the MI+EX and MI+AE groups was reduced after oral administration (P<0.05); mulberry ignia ethanol extract and mulberry ignia extract significantly inhibited the apoptosis of cardiomyocytes in mice after myocardial infarction.

[0190] 10) Effect of Phellinus igniarius extract on myocardial inflammation after myocardial infarction

[0191] An appropriate amount of serum samples were taken from mice in each group 28 days after oral administration, and the levels of IL-6, IL-10 and TNF-α in the serum of mice in each group were detected using a microplate reader according to the instructions of the IL-6 (Mouse IL-6 ELISA Kit Beyotime Catalog No.: PI326), IL-10 (Mouse Interleukin 10 (IL-10) ELISA Kit Bioswamp Catalog No.: MU30055), and TNF-α (Mouse TNF-α ELISA Kit Beyotime Catalog No.: PT512) ELISA kits.

[0192] Immunohistochemical staining was used to detect the infiltration of macrophages (F4 / 80) in mouse myocardial tissue. The specific steps are as follows:

[0193] Drying, dewaxing and rehydration were the same as for H&E staining.

[0194] High-temperature antigen repair in a water bath: Place a small container containing 0.01M sodium citrate buffer solution in a large container containing a certain amount of tap water, and heat to boil on an induction cooker. When the temperature in the small container reaches 92℃-98℃, place the slices in the sodium citrate buffer and time for 20 minutes, then remove from the electric stove, cool naturally at room temperature for 30 minutes, and wash twice with PBS, 5 minutes each time.

[0195] Block endogenous peroxidase: Place the sections in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes. Place the slides in PBS and wash them three times on a horizontal shaker for 5 minutes each time.

[0196] Blocking: Place the slices in a wet box, circle the tissue with an immunohistochemistry pen, add 5% BSA solution inside the circle, and block in a 37°C incubator for 30 minutes.

[0197] Incubate with primary antibody: Gently shake off the blocking solution, place the slices flat in a humidified box, add the primary antibody prepared in a certain ratio (F4 / 801:900), and place the humidified box in a 4°C refrigerator overnight.

[0198] Rewarming: Place the wet box at room temperature for 1 hour, then wash 3 times with PBS, 5 minutes each time.

[0199] Incubate with secondary antibody: After the slices are dried, add the prepared horseradish peroxidase-labeled secondary antibody staining solution in the circle and incubate at 37°C for 30 minutes, then wash with PBS three times, 5 minutes each time.

[0200] DAB color development: Add the prepared DAB color development solution into the circle, observe the staining degree under a microscope to control the color development time, the positive part is brown-yellow, and the color development is terminated by rinsing with tap water.

[0201] Counterstaining of cell nuclei: add hematoxylin staining solution for 10 seconds and rinse with tap water.

[0202] Dehydrated and mounted slides were stained with H&E.

[0203] Effects of Phellinus igniarius extract on myocardial inflammation after myocardial infarction Figures 12-13 shown.

[0204] Depend on Figures 12-13It was found that compared with the Sham group, the levels of pro-inflammatory factors IL-6, TNF-α and IL-10 in the serum of mice in the MI group were significantly increased (all P<0.05); at the same time, compared with the MI group, the above inflammatory factors in the serum of the MI+EX and MI+AE groups were significantly reduced (all P<0.05). The results showed that the extract of Phellinus igniarius played an inhibitory role in inflammatory response by regulating the levels of inflammatory factors in the serum of mice after MI. Compared with the Sham group, the level of F4 / 80 in the myocardial tissue of mice in the MI group was significantly increased (all P<0.05), however, the level of F4 / 80 in the myocardial tissue of mice in the MI+EX and MI+AE groups was significantly lower than that in the MI group (all P<0.05). Compared with the MI group, the levels of anti-inflammatory cytokines in the myocardial tissue of mice in the MI+EX group were significantly increased, and the difference was statistically significant (all P<0.05). This result suggests that the extract of Phellinus igniarius can inhibit the infiltration of inflammatory factors in myocardial tissue and inhibit the inflammatory response.

[0205] 11) Effect of Phellinus igniarius extract on myocardial cell fibrosis after myocardial infarction

[0206] (1) Masson's trichrome staining

[0207] Drying, sectioning and rehydration were the same as for H&E staining.

[0208] Staining: Use the prepared Weigert iron hematoxylin stain (WeigertA and WeigertB mixed in equal amounts) to stain the nucleus for 5 minutes, and wash with distilled water; differentiate with acidic ethanol differentiation solution for 15 seconds, and wash with water; return to blue with Masson blueing solution for 5 minutes, and wash with distilled water for 1 minute; stain with Ponceau fuchsin stain for 10 minutes; add weak acid working solution for 1 minute to make the color clearer and brighter; differentiate with phosphomolybdic acid aqueous solution until the collagen fibers are light red and the fibers are red, usually 1 to 2 minutes; wash with weak acid working solution for 1 minute and then directly put into aniline blue stain for 20 seconds, and continue to wash with weak acid working solution for 1 minute.

[0209] The dehydration and sealing steps are the same as those for H&E staining.

[0210] (2) Sirius red staining

[0211] Drying, sectioning and rehydration were the same as for H&E staining.

[0212] Staining: circle the stained tissue with an immunohistochemistry pen, add Sirius red staining solution and stain for 30 minutes; rinse with running water slightly, then stain the nucleus with hematoxylin staining solution for 8 minutes, and rinse with running water for 10 minutes.

[0213] The dehydration and sealing steps are the same as those for H&E staining.

[0214] (3) Masson's trichrome staining and picrosirius red staining and analysis results Fig.14 shown.

[0215] The proliferation of cardiac fibroblasts and excessive deposition of extracellular matrix are characteristic changes of myocardial fibrosis after myocardial infarction. Masson staining and Sirius red staining are commonly used detection methods to evaluate myocardial fibrosis. This experiment evaluated the deposition of collagen fibers after myocardial infarction in mice by Masson staining and Sirius red staining. The results of Masson staining showed that compared with the Sham group, the normal myofibroblast tissue (red) in the MI group was significantly reduced, and collagen fibers (blue) were deposited instead, but the administration of Phellinus igniarius extract could effectively inhibit the generation of collagen fibers caused by myocardial infarction and improve myocardial fibrosis after myocardial infarction in mice. Similarly, Sirius red staining also reflected similar results to Masson staining, that is, compared with the Sham group, the expression of collagen fibers (red) in the MI group was significantly increased, and the expression of collagen fiber content was significantly reduced after the administration of Phellinus igniarius extract, indicating that Phellinus igniarius extract can effectively inhibit myocardial fibrosis after myocardial infarction in mice.

[0216] 12) Evaluation of the expression levels of myocardial fibrosis-related markers by immunohistochemistry

[0217] (1) Immunohistochemical staining

[0218] The drying, dewaxing and rehydration of the slides were the same as those for H&E staining.

[0219] Antigen repair in a water bath at high temperature: Place a container containing 0.01M sodium citrate buffer solution in a large container containing a certain amount of tap water, and heat it to boil on an induction cooker. When the temperature in the small container reaches 92°C to 98°C, place the slices in the sodium citrate buffer solution and time it for 20 minutes. Then remove it from the electric stove, cool it naturally at room temperature for 30 minutes, and wash it twice with PBS, 5 minutes each time.

[0220] Block endogenous peroxidase: Place the sections in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes. Place the slides in PBS and wash them three times on a horizontal shaker, each time for 5 minutes.

[0221] Blocking: Place the slices in a wet box, circle the tissue with an immunohistochemistry pen, add 5% BSA solution inside the circle, and block in a 37°C incubator for 30 minutes.

[0222] Incubate with primary antibody: Gently shake off the blocking solution, place the slices flat in a humidified box, add the primary antibody prepared in a certain proportion, and place the humidified box in a 4°C refrigerator overnight.

[0223] Rewarming: Place the wet box at room temperature for 1 hour, then wash 3 times with PBS, 5 minutes each time.

[0224] Incubate with secondary antibody: After the slices are dried, add the prepared horseradish peroxidase-labeled secondary antibody staining solution in the circle and incubate at 37°C for 30 minutes, then wash with PBS three times, 5 minutes each time.

[0225] DAB color development: Add the prepared DAB color development solution into the circle, observe the staining degree under a microscope to control the color development time, the positive part is brown-yellow, and the color development is terminated by rinsing with tap water.

[0226] Counterstaining of cell nuclei: add hematoxylin staining solution for 10 seconds and rinse with tap water.

[0227] The dehydration and sealing steps are the same as those for H&E staining.

[0228] The configuration ratio of primary antibodies is: Col-1 (Collagen Ⅰ) 1:1000, α-SMA 1:500.

[0229] The expression levels of myocardial fibrosis-related markers were evaluated by immunohistochemistry, and the results were as follows: Fig.15 shown.

[0230] Depend on Fig.15 The results showed that compared with the Sham group, the expression levels of myocardial fibrosis-related proteins in the MI group were significantly increased, as manifested by the increase in the expression levels of Collagen Ⅰ (Col-1) and α-SMA, while the mulberry ignia extract inhibited the increase in the expression of the above proteins, among which the mulberry ignia alcohol extract had a better inhibitory effect.

[0231] 13) Effects of Phellinus igniarius extract on oxidative stress after myocardial infarction

[0232] The expression levels of myocardial oxidative stress-related markers were evaluated by immunohistochemistry, and the results were as follows: Fig.16 shown.

[0233] Depend on Fig.16 It was found that compared with the Sham group, the expression level of myocardial oxidative stress-related proteins in the MI group increased significantly, as shown by the increase in the expression levels of 8-OHdG and CaMKⅡ, and the expression of 8-OHdG and CaMKⅡ in the MI+EX and MI+AE groups decreased significantly compared with the MI group, indicating that the extract of Phellinus igniarius inhibited the increase in the expression of oxidative stress proteins.

[0234] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An application of a Phellinus igniarius extract in the preparation of a product for improving and / or treating myocardial infarction; the product comprises any one or more of a medicine, a health food and a functional food.

2. The application according to claim 1, characterized in that: The improvement and / or treatment of myocardial infarction includes any one or two or more of the following (1) to (9): (1) Significantly reduce the area of ​​myocardial infarction; (2) Effectively reduce mortality; (3) Improve cardiac dysfunction; (4) Improve symptoms of myocardial hypertrophy; (5) Reduce cardiomyocyte apoptosis; (6) Inhibit inflammatory response; (7) Inhibit cardiomyocyte fibrosis; (8) Improve the body’s oxidative stress level; (9) Inhibit changes in serum LDH and / or CK levels.

3. The application according to claim 1, characterized in that: The active ingredients of the mulberry linterus extract include mulberry linterus phenolic acids, flavonoids, terpenes, alkaloids, steroids, lignin and coumarin.

4. The use according to claim 1, characterized in that: The mulberry linterus extract comprises mulberry linterus alcohol extract and / or mulberry linterus extract.

5. The use according to claim 1, characterized in that: The active ingredients of the mulberry linterus alcohol extract include ferulic acid, erythritol acid N, daidzein, rhein-8-O-(6'-acetyl) glucoside and akebia phenylethanol glycoside B; the active ingredients of the mulberry linterus extract include dihydroquercetin and chalcone.

6. The use according to claim 5, characterized in that: The active substances of the mulberry linden alcohol extract and the mulberry linden extract also include caffeic acid, mulberry linden J, milk tree alkaloids, inoscavin A, dihydrokaempferol, mulberry lindenol A, gallic acid, vanillic acid, p-coumaric acid, mulberry linden D, mulberry linden A, philligridin B, mulberry linden I, purpurogenol, mulberry linden C, baicalein, protocatechuic aldehyde, 7-oxy-methyleriocin and mulberry linden G.

7. The use according to claim 4, characterized in that: The preparation method of the mulberry linterus ethanol extract comprises: The mulberry ignia medicinal material block is mixed with an ethanol solution and subjected to reflux extraction to obtain a mulberry ignia alcohol extract; The mulberry linseed alcohol extract is dried to obtain the mulberry linseed alcohol extract.

8. The use according to claim 7, characterized in that: The volume fraction of ethanol in the ethanol solution is 70% to 95%; the mass volume ratio of the Phellinus igniarius medicinal material block to the ethanol solution is (1 to 3) g: (10 to 30) mL; the temperature of the reflux extraction is 60 to 80° C.; and the time of the reflux extraction is 2 to 4 hours.

9. The use according to claim 4, characterized in that: The preparation method of the Phellinus linteus extract comprises: The Phellinus igniarius medicinal material block is mixed with the ethanol solution and subjected to ultrasonic treatment to obtain the Phellinus igniarius extract; The Phellinus linteus extract is extracted with ethyl acetate and then dried to obtain the Phellinus linteus extract.

10. The use according to claim 9, characterized in that: The volume fraction of ethanol in the ethanol solution is 70% to 95%; the mass volume ratio of the Phellinus igniarius medicinal material block and the ethanol solution when mixed is 1g: (10 to 20) mL; the power of the ultrasonic treatment is 150 to 200W; the temperature of the ultrasonic treatment is 50 to 70°C; and the time of the ultrasonic treatment is 1 to 3h.

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