Preparation method and application of Yixin pill

Abstract

The present application relates to the technical field of medicine, in particular to a preparation method and application of Yixin pill. The preparation method comprises the following steps: crushing red ginseng, safflower, Fupian (black Shunpian) and Sanqi, and performing complex enzyme hydrolysis, inactivation, to obtain extract A; crushing toad venom and pearl powder, mixing with ox horn tip powder, performing papain hydrolysis, inactivation, to obtain extract B; combining extract A and extract B, adding beta-glucosidase for hydrolysis, inactivation, filtration, concentrating and drying the filtrate, to obtain extract C; fully dissolving artificial cow-bezoar, artificial musk, borneol and benzoin, adding sodium alginate solution and chitosan solution, stirring, freeze-drying, to obtain component D; mixing extract C and component D, adding starch, stirring, pill-making, drying, activated carbon coating, polishing, to obtain Yixin pill. The Yixin pill prepared by the preparation method has better effects of reducing blood pressure and blood lipid than Yixin pill prepared by the pharmacopoeia method and other comparative examples.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for preparing and applying a heart-nourishing pill. Background Technology

[0002] Cardiovascular diseases mainly include coronary heart disease, angina pectoris, and myocardial infarction, and are characterized by symptoms such as palpitations, shortness of breath, chest tightness, and chest pain. They are diseases that cause ischemia in the heart and other tissues throughout the body. Among them, coronary heart disease is caused by atherosclerosis of the coronary arteries and their branches due to various risk factors, and is mainly characterized by myocardial ischemia and impaired cardiac pumping function.

[0003] Currently, Western medicines for treating cardiovascular diseases mainly include nitrates, beta-blockers, and calcium channel blockers. While these drugs can alleviate symptoms to some extent, they only temporarily improve the condition and cannot fundamentally cure the disease. Furthermore, long-term use may cause adverse reactions such as allergies and gastrointestinal discomfort. Traditional Chinese medicine believes that the pathogenesis of cardiovascular diseases is mainly due to imbalance of Yin and Yang, and disordered Qi flow leading to stagnation of heart blood and obstruction of the heart vessels, resulting in pain due to obstruction. However, existing traditional Chinese medicine preparations, such as Compound Danshen Tablets and Yindan Xinnaotong Soft Capsules, have shortcomings such as incomplete drug use, low efficacy, and slow onset of action, and often require combination with multiple drugs.

[0004] While traditional Chinese medicine (TCM) offers advantages in treating cardiovascular diseases through multiple pathways, multiple targets, dynamic holistic treatment, and fewer toxic side effects, the potency of a single TCM herb is lower than that of Western medicine. Chinese invention patent application CN117244027A discloses a pharmaceutical composition for treating cardiovascular diseases and its preparation method. This composition consists of colon-targeting hydrogel, trumpet creeper, *Erigeron breviscapus*, *Ligusticum chuanxiong*, *Sparganium stoloniferum*, *Citrus medica*, *Citrus medica*, *Peucedanum praeruptorum*, and lecithin. The composition uses a hydrogel to adhere the TCM extracts to the colonic surface, allowing for prolonged retention and increasing the concentration and absorption of the extracts, thereby enhancing bioavailability. However, this patent still has the problem of not fully considering the pharmacological activity, safety, and stability of the herbs when selecting their extracts. The Pharmacopoeia of the People's Republic of China, Volume I, discloses the prescription composition and preparation method of Yixin Pill, which has the effects of invigorating qi and warming yang, promoting blood circulation and relieving pain. However, because the medicine contains precious medicinal materials such as red ginseng, artificial musk, artificial bezoar, pearl, and Panax notoginseng, as well as special medicinal materials such as aconite (black aconite slices), toad venom, and artificial musk (which have both precious and controlled attributes), it is necessary to improve its preparation process based on previous experiments, further fully extract the active ingredients in the raw materials, reduce resource waste, and further improve the efficacy of Yixin Pill. Summary of the Invention

[0005] In view of the prior art, the present invention provides a preparation method and application of Yixin Pill. The method involves compound enzymatic hydrolysis of red ginseng, safflower, aconite root (black aconite root slices), and Panax notoginseng powder; enzymatic hydrolysis of toad venom, pearl powder, and ox horn tip powder using papain; and adsorption of artificial bezoar, artificial musk, borneol, and benzoin using sodium alginate solution and chitosan solution. After freeze-drying, the resulting composition is obtained by mixing all the components. This method fully extracts the active ingredients from the raw materials, reduces resource waste, and further improves the efficacy of Yixin Pill.

[0006] This invention is achieved through the following technical solutions:

[0007] The first aspect of this invention relates to a method for preparing a Yixin Pill, wherein the Yixin Pill is prepared from the following raw materials in parts by weight: 870-890 parts red ginseng, 340-360 parts artificial bezoar, 50-60 parts artificial musk, 200-210 parts pearl, 370-390 parts Panax notoginseng, 170-180 parts borneol, 170-180 parts benzoin, 140-150 parts toad venom, 350-360 parts aconite (black aconite slices), 290-300 parts ox horn tip powder, and 50-60 parts safflower. The preparation method includes the following steps:

[0008] S1: Crush red ginseng, safflower, aconite (black aconite slices) and Panax notoginseng, add water, hydrolyze with compound enzyme, inactivate, and obtain extract A;

[0009] S2: Toad venom and pearl powder are mixed with ox horn powder, water is added, papain is used to hydrolyze and inactivate the powder, and the mixture is filtered to obtain extract B;

[0010] S3: Combine extract A and extract B, add β-glucosidase for enzymatic hydrolysis, inactivate, filter, concentrate and dry the filtrate to obtain extract C;

[0011] S4: Artificial bezoar, artificial musk, borneol, and benzoin are mixed, and then an ethanol aqueous solution and PEG400 are added. After the mixture is fully dissolved, a mixed solution consisting of sodium alginate solution and chitosan solution is added, stirred, and freeze-dried to obtain component D.

[0012] S5: Mix extract C and component D, add starch, stir well, form into pellets, dry, coat with activated charcoal, and polish to obtain the final product.

[0013] Preferably, the complex enzyme in step S1 is cellulase and pectinase.

[0014] More preferably, the mass ratio of cellulase to pectinase is 3-6:2-4.

[0015] Preferably, in step S1, the pH is adjusted to 5.0-6.5 before enzymatic hydrolysis, the amount of compound enzyme is 0.1-1% of the mass of the raw material to be hydrolyzed, the hydrolysis temperature is 30-50℃, and the hydrolysis time is 1-3h.

[0016] Preferably, in step S2, the pH is adjusted to 6.5-7.5 before enzymatic hydrolysis, the amount of papain used is 1-3% of the mass of the raw material to be hydrolyzed, the hydrolysis temperature is 40-50℃, and the hydrolysis time is 3-5h.

[0017] Preferably, in step S3, the pH is adjusted to 5-6.5 before enzymatic hydrolysis, the amount of β-glucosidase is 0.2-0.8% of the mass of the raw material to be hydrolyzed, the hydrolysis temperature is 45-60℃, and the hydrolysis time is 1-3h.

[0018] Preferably, in step S4, the volume percentage of ethanol in the aqueous ethanol solution is 75%-85%, and the mass ratio of the aqueous ethanol solution to PEG400 is 5-8:1.

[0019] Preferably, in the mixed solution described in step S4, the sodium alginate solution has a mass-to-volume ratio of 1-3%, and the chitosan solution is prepared by dissolving 1-2g of chitosan in 100mL of 1% acetic acid aqueous solution. The mixed solution can be obtained by mixing the sodium alginate solution and the chitosan solution at a volume ratio of 1:1.

[0020] Preferably, the weight ratio of the mixed solution in step S4 to the total mass of the artificial bezoar, artificial musk, borneol, and benzoin in that step is 1-3:1.

[0021] The second aspect of the present invention relates to the use of the Yixin Pill prepared by the above preparation method in the preparation of antihypertensive and / or lipid-lowering drugs.

[0022] Preferably, the dosage form of the drug is tablets, pills, granules or capsules.

[0023] The beneficial effects of this invention are as follows: By performing compound enzymatic hydrolysis on red ginseng, safflower, aconite root (black aconite slices), and Panax notoginseng powder; by using papain to hydrolyze toad venom, pearl powder, and ox horn tip powder; and by adsorbing artificial bezoar, artificial musk, borneol, and benzoin with sodium alginate solution and chitosan solution, followed by freeze-drying, the effective components of the raw materials are fully extracted through the above-mentioned improved preparation process, reducing resource waste and further enhancing the efficacy of Yixin Pill. The Yixin Pill prepared by the method of this invention has superior effects in lowering blood pressure and blood lipids compared to Yixin Pill prepared by the pharmacopoeia method and other comparative examples. Detailed Implementation

[0024] To clarify the purpose, technical solution, and advantages of this invention, the following detailed description is provided in conjunction with specific embodiments. To facilitate understanding of the technical means, creative features, and achieved objectives and effects of this invention, the following detailed description further clarifies the invention in conjunction with specific embodiments. However, these embodiments are merely preferred embodiments and not exhaustive. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used is conventional equipment, and the equipment materials used in each embodiment are the same. This invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in this invention are all commercially available products in this technical field, with a purity of 98% or higher.

[0025] In the embodiments and comparative examples of this invention, Yixin Pill is prepared from the following raw materials in parts by weight: 882g red ginseng, 294g ox horn powder, 147g toad venom, 176g borneol, 59g safflower, 353g artificial bezoar, 353g aconite (black aconite slices), 59g artificial musk, 382g Panax notoginseng, 176g benzoin, and 206g pearl.

[0026] Example 1:

[0027] A method for preparing Yixin Pill, comprising the following steps:

[0028] S1: Grind red ginseng, safflower, aconite (black aconite slices) and Panax notoginseng into fine powder, add 3 times the amount of water, adjust the pH to 6, add 0.5% of the mass of the raw material to be enzymatically hydrolyzed, a compound enzyme composed of cellulase and pectinase (mass ratio of 5:3) for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 40℃, the enzymatic hydrolysis time is 2h, inactivation, to obtain extract A;

[0029] S2: Toad venom and pearl powder are ground into fine powder, mixed with ox horn powder, and 3 times the amount of water is added. Papain at 1.5% of the mass of the raw material to be enzymatically hydrolyzed is added for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40℃ and the enzymatic hydrolysis time is 2h. After inactivation, extract B is obtained.

[0030] S3: Combine extract A and extract B, adjust pH to 6, add 0.5% β-glucosidase (by weight of the raw material to be enzymatically hydrolyzed) for enzymatic hydrolysis, inactivate, filter, concentrate and dry the filtrate to obtain extract C;

[0031] S4: Artificial bezoar, artificial musk, borneol, and benzoin are mixed and then a solvent consisting of 80% ethanol aqueous solution and PEG400 (mass ratio of the two is 6:1) is added until fully dissolved. Then, a mixed solution consisting of sodium alginate solution and chitosan solution is added in a volume ratio of 2% each, with a volume ratio of 1:1. The mixture is stirred, freeze-dried, and component D is obtained.

[0032] S5: Mix extract C and component D, add starch, stir well, form into pellets, dry, coat with activated charcoal, and polish to obtain the final product.

[0033] Example 2:

[0034] A method for preparing Yixin Pill, comprising the following steps:

[0035] S1: Grind red ginseng, safflower, aconite (black aconite slices) and Panax notoginseng into fine powder, add 3 times the amount of water, adjust the pH to 6, add 1% of the mass of the raw material to be enzymatically hydrolyzed with a compound enzyme composed of cellulase and pectinase (mass ratio of 3:4) for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 40℃, the enzymatic hydrolysis time is 2h, and the enzyme is inactivated to obtain extract A.

[0036] S2: Toad venom and pearl powder are ground into fine powder, mixed with ox horn powder, 3 times the amount of water is added, and 3% of the mass of the raw material to be enzymatically hydrolyzed is added for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40℃ and the enzymatic hydrolysis time is 2h. After inactivation, extract B is obtained.

[0037] S3: Combine extract A and extract B, adjust pH to 6, add 0.3% β-glucosidase (by weight of the raw material to be enzymatically hydrolyzed) for enzymatic hydrolysis, inactivate, filter, concentrate and dry the filtrate to obtain extract C;

[0038] S4: Artificial bezoar, artificial musk, borneol, and benzoin are mixed and then a solvent consisting of 80% ethanol aqueous solution and PEG400 (mass ratio of the two is 6:1) is added until fully dissolved. Then, a mixed solution consisting of sodium alginate solution and chitosan solution is added in a volume ratio of 2% each, with a volume ratio of 1:1. The mixture is stirred, freeze-dried, and component D is obtained.

[0039] S5: Mix extract C and component D, add starch, stir well, form into pellets, dry, coat with activated charcoal, and polish to obtain the final product.

[0040] The difference from Example 1 lies in the slightly different enzymatic hydrolysis conditions in steps S1-S3. Specifically, in step S1, the mass ratio of cellulase to pectinase in the compound enzyme is 3:4, and the amount of compound enzyme added is 1% of the mass of the raw material to be hydrolyzed; in step S2, the amount of papain added is 3% of the mass of the raw material to be hydrolyzed; and in step S3, the amount of β-glucosidase added is 0.3% of the mass of the raw material to be hydrolyzed. The rest is the same as in Example 1.

[0041] Example 3:

[0042] A method for preparing Yixin Pill, comprising the following steps:

[0043] S1: Grind red ginseng, safflower, aconite (black aconite slices) and Panax notoginseng into fine powder, add 3 times the amount of water, adjust the pH to 6, add 0.3% of the mass of the raw material to be enzymatically hydrolyzed with a complex enzyme composed of cellulase and pectinase (mass ratio of 3:1) for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 40℃, the enzymatic hydrolysis time is 2h, and the enzyme is inactivated to obtain extract A.

[0044] S2: Toad venom and pearl powder are ground into fine powder, mixed with ox horn powder, 3 times the amount of water is added, and 1% of the mass of the raw material to be enzymatically hydrolyzed is added for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40℃ and the enzymatic hydrolysis time is 2h. After inactivation, extract B is obtained.

[0045] S3: Combine extract A and extract B, adjust pH to 6, add 0.8% β-glucosidase (by weight of the raw material to be enzymatically hydrolyzed) for enzymatic hydrolysis, inactivate the enzyme, filter, concentrate and dry the filtrate to obtain extract C;

[0046] S4: Artificial bezoar, artificial musk, borneol, and benzoin are mixed and then a solvent consisting of 80% ethanol aqueous solution and PEG400 (mass ratio of 6:1) is added until fully dissolved. Then, a mixed solution consisting of sodium alginate solution and chitosan solution is added in a volume ratio of 2% each, with a volume ratio of 1:1. The mixture is stirred, freeze-dried, and component D is obtained.

[0047] S5: Mix extract C and component D, add starch, stir well, form into pellets, dry, coat with activated charcoal, and polish to obtain the final product.

[0048] The difference from Example 1 lies in the slightly different enzymatic hydrolysis conditions in steps S1-S3. Specifically, in step S1, the mass ratio of cellulase to pectinase in the compound enzyme is 3:1, and the amount of the compound enzyme is 0.3% of the mass of the raw material to be hydrolyzed; in step S2, the amount of papain is 1% of the mass of the raw material to be hydrolyzed; and in step S3, the amount of β-glucosidase is 0.8% of the mass of the raw material to be hydrolyzed. The rest is the same as in Example 1.

[0049] Comparative Example 1:

[0050] The difference from Example 1 is that in step S1, no compound enzyme is used for enzymatic hydrolysis; only 0.5% of the mass of the raw material to be hydrolyzed is added for enzymatic hydrolysis. The rest is the same as in Example 1.

[0051] Comparative Example 2:

[0052] The difference from Example 1 is that in step S1, no compound enzyme is used for enzymatic hydrolysis; only 0.5% of the mass of the raw material to be hydrolyzed is added for enzymatic hydrolysis. The rest is the same as in Example 1.

[0053] Comparative Example 3:

[0054] The difference from Example 1 is that a compound enzyme is used for enzymatic hydrolysis in step S2. Specifically, 1.5% of the mass of the raw material to be hydrolyzed is added to carry out enzymatic hydrolysis using a compound enzyme composed of papain and trypsin (mass ratio of 1:1). The rest is the same as in Example 1.

[0055] Comparative Example 4:

[0056] The difference from Example 1 is that β-glucosidase is not used in step S3. Instead, extracts A and B are directly filtered, the filtrate is concentrated and dried to obtain extract C.

[0057] Comparative Example 5:

[0058] The difference from Example 1 lies in step S4, which specifically involves: grinding borneol into a fine powder, mixing it with artificial bezoar and artificial musk to obtain a mixed powder; dissolving benzoin in 75% ethanol and mixing it with the mixed powder to obtain component D. The rest is the same as in Example 1.

[0059] Comparative Example 6:

[0060] The medicine was prepared according to the preparation method of Yixin Pill in Part I of the Pharmacopoeia of the People's Republic of China. Specifically, the above eleven ingredients were as follows: red ginseng, safflower, toad venom, and aconite (black aconite slices) were crushed into coarse powder, soaked in 60% ethanol for 24 hours, percolated, the percolate was collected, the ethanol was recovered, and concentrated into a thick paste; pearl and Panax notoginseng were crushed into fine powder; borneol was finely ground, mixed with ox horn powder, artificial bezoar, and artificial musk, and sieved; benzoin was dissolved in 75% ethanol, mixed with the above thick paste and powder, 300g of starch was added, stirred well, and made into a soft material. 100,000 pills were made, dried below 60℃, coated with activated carbon, and polished to obtain the final product.

[0061] Experimental Example 1:

[0062] The Yixin Pills from Examples 1-3 and Comparative Examples 1-6 were dissolved in distilled water to prepare suspensions for later use. TC, TG, HDL-C, and LDL-C reagent kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.; high-fat diet (formula: 10% lard, 2% cholesterol, 0.2% propylthiouracil, 0.5% pig bile salts, 87.3% basal feed).

[0063] Healthy male SD rats, weighing 180-200g, were selected and acclimatized for 5 days with free access to food and water. After the acclimatization period, they were randomly divided into a blank control group, a high-fat model group, Examples 1-3 groups, and Comparative Examples 1-6 groups, with 10 rats in each group. Except for the blank control group, which was fed a normal diet, all other groups were fed a high-fat diet. Examples 1-3 and Comparative Examples 1-6 were administered Yixin Pill by gavage at a dose of 50 mg / kg bw, with a gavage volume of 10 mL / kg, once daily for 28 consecutive days. The blank control group was administered the same dose of distilled water by gavage. Blood was collected from the abdominal aorta of the rats 24 hours after the last administration to measure serum TC, TG, HDL-C, and LDL-C levels. One-way ANOVA was performed using SPSS 13.0 software, and statistically significant differences were found (P < 0.05). The results are shown in Table 1.

[0064] Table 1: Effects of rat serum TC, TG, HDL-C, and LDL-C levels

[0065]

[0066] Note: Compared with the blank control group # P < 0.05 ## P < 0.01; compared with the high-fat model group, *P < 0.05, **P < 0.01; compared with Example 1, there were no significant differences between the drug administration groups. & P < 0.05 && P < 0.01.

[0067] As can be seen from the results in the above table, the contents of TC, TG, and LDL-C in the blood of rats in the high-fat model group were significantly increased compared with the blank control group (P < 0.05 or P < 0.01), and the content of HDL-C was significantly decreased compared with the blank control group (P < 0.05), indicating that feeding with high-fat diet can cause hyperlipidemia. After administration, the contents of TC, TG, and LDL-C in the blood of rats in each administration group were significantly decreased compared with the model group, and the content of HDL-C was significantly increased compared with the model group, showing that the drugs in Examples 1-3 and Comparative Examples 1-6 all had a positive regulatory effect on the blood lipids of hyperlipidemic rats, but the positive regulatory effect of the examples was significantly better than that of each comparative example. It shows that the Yixin Pills obtained by the preparation method of the present invention have a more significant positive regulatory effect on the blood lipid-related components of hyperlipidemic rats. Especially in the effect of increasing the content of HDL-C in the serum of hyperlipidemic rats, the effect of Example 1 of the present invention was significantly different from that of Comparative Example 6 prepared by the pharmacopoeia method (P < 0.01).

[0068] Experimental Example 2: Pharmacodynamic test on spontaneously hypertensive rats

[0069] The Yixin Pills of the groups of Examples 1-3 and the groups of Comparative Examples 1-6 were dissolved in distilled water to prepare a suspension for standby.

[0070] Experimental animals: Spontaneously hypertensive rats (SHR), male, 64 weeks old, clean grade, body weight (310 ± 10) g, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (License number: SCXK (Beijing) 2020-0003); WKY rats, male, 64 weeks old, clean grade, body weight (310 ± 10) g, provided by the Animal Experiment Center of Central South University (Medical Animal No. 2021-010). The animals were acclimated in this laboratory for 1 week before the experiment.

[0071] Grouping: (1) Normal group: 10 WKY rats at 64 weeks old, continuously intragastrically administered with normal saline for 8 weeks; (2) Aging group: 10 SHR rats at 64 weeks old, continuously intragastrically administered with normal saline for 8 weeks; (3) Treatment group: divided into the groups of Examples 1-3 and the groups of Comparative Examples 1-6, with 10 SHR rats at 64 weeks old in each group, continuously intragastrically administered with the corresponding Yixin Pills for 8 weeks, the dose was 50 mg / kg bw, the intragastric volume was 10 mL / kg, once a day.

[0072] Index detection:

[0073] (1) Blood pressure measurement in rats: After administration, the blood pressure of rats in each group was measured using a conscious small animal blood pressure measuring device. Specifically, the instrument was turned on and preheated for about 15 minutes to calibrate the pressure signal. The rats were then placed in the fixing box according to their weight and placed in the animal fixing frame. The rat tail was inserted into the tail near the base through the pressure sleeve. At this time, the rat tail should have passed through the pulse sensor and been inserted into the heating tube in the tail heater, so that the rat tail was just above the "pulse signal sensing plate" of the pulse sensor. The pressure plate of the rat tail was adjusted to make the sensing plate tight. Apply pressure to the caudal artery below the rat's tail. After the rat's pulse stabilizes, measure its blood pressure. When the pressure sleeve is applied to a pressure exceeding the systolic pressure, blood flow to the caudal artery is blocked, and the pulse disappears. Then, gradually reduce the applied pressure. When the applied pressure is reduced to the systolic pressure, the pulse begins to appear; this is the systolic pressure (SAP). As the applied pressure continues to decrease, the pulse amplitude continues to increase. When the applied pressure is reduced to the diastolic pressure, the pressure sleeve on the caudal artery is removed, and the pulse amplitude reaches its maximum value; this is the diastolic pressure (DAP). The specific values ​​are shown in the table below.

[0074] Table 2: Effects on blood pressure in rats (mean ± SD)

[0075]

[0076] Compared with the normal group, ## P < 0.01; compared with the aging group, *P < 0.05, **P < 0.01; compared with Example 1, there were no significant differences between the drug administration groups. & P < 0.05 && P < 0.01.

[0077] Based on the above results, it can be seen that the drug in the treatment group has a lowering effect on both systolic and diastolic blood pressure in the aging group rats. However, compared with Example 1, the Yixin Pill prepared by the method provided by the present invention is more effective in lowering blood pressure in spontaneously hypertensive model rats than the other comparisons.

[0078] (2) Effects on Hcy and SIRT1 expression

[0079] On the second day after drug administration, 5 mL of fasting venous blood was collected from rats using aseptic aspiration vessels, and serum was separated by centrifugation. Serum homocysteine ​​(Hcy) levels were monitored using chemiluminescence immunoassay. The aging protein SIRT1 was detected using immunofluorescence histological assay. Paraffin sections were first dewaxed to water, repaired, and washed with distilled water. Normal serum was used for blocking for 10 minutes, the serum was discarded, and no washing was performed. The primary antibody (SIRT1) working solution was added and incubated for 30-60 minutes at 37°C overnight (approximately 16 hours) at 4°C. Then, PBS was washed for 5 minutes three times. CY3-labeled goat anti-rabbit and FITC-labeled goat anti-mouse immunofluorescence secondary antibodies (1:400, Shanghai Beyotime Biotechnology Co., Ltd.) were added, and the mixture was protected from light for 1 hour. The sections were then washed with PBS 3 times per minute (3 × 3 min), followed by DAPI addition. The sections were protected from light for 10 minutes, washed with PBS 3 times per minute (3 × 3 min), and then mounted with an anti-fluorescence quenching mounting medium under an optical microscope. PBS was used as a negative control instead of the immunofluorescence primary antibody.

[0080] The results are shown in Table 3.

[0081] Table 3: Effects on Hcy and SIRT1 expression (mean ± SD)

[0082]

[0083] Compared with the normal group, ## P < 0.01; compared with the aging group, *P < 0.05, **P < 0.01; compared with Example 1, there were no significant differences between the drug administration groups. & P < 0.05.

[0084] Compared with the normal group, the serum homocysteine ​​(Hcy) level in the aging group rats was significantly increased (P < 0.01). Compared with the aging group, the homocysteine ​​level in the treatment group was significantly decreased (P < 0.05, P < 0.01). Furthermore, compared with the group in Example 1, the comparative groups 1-6 were less effective in reducing homocysteine ​​levels than the groups in the examples (P < 0.05). The Yixin Pill prepared by the method provided in this invention is more effective in correcting homocysteine ​​levels in a spontaneously hypertensive rat vascular aging model.

[0085] Compared with the normal group, SIRT1 expression was significantly increased in the aging group (P<0.01); compared with the aging group, SIRT1 expression was significantly decreased in the treatment group, with statistically significant differences (P<0.05, P<0.01); and compared with the Example 1 group, the comparative groups were less effective in reducing SIRT1 expression than the Example 1 group, with statistically significant differences (P<0.05). This suggests that the Yixin Pill prepared by the method provided in this invention has a better effect on inhibiting the expression of the vascular aging-related protein SIRT1 in spontaneously hypertensive aging rats.

[0086] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a heart-nourishing pill, wherein the heart-nourishing pill is prepared from the following raw materials in parts by weight: 870-890 parts red ginseng, 340-360 parts artificial bezoar, 50-60 parts artificial musk, 200-210 parts pearl, 370-390 parts Panax notoginseng, 170-180 parts borneol, 170-180 parts benzoin, 140-150 parts toad venom, 350-360 parts black aconite, 290-300 parts ox horn powder, and 50-60 parts safflower, characterized in that, The preparation method includes the following preparation steps: S1: Pulverize red ginseng, safflower, black aconite slices and Panax notoginseng, hydrolyze with compound enzyme, inactivate, and obtain extract A; S2: Toad venom and pearl powder are mixed with horn tip powder, and then hydrolyzed and inactivated by papain to obtain extract B; S3: Combine extract A and extract B, add β-glucosidase for enzymatic hydrolysis, inactivate, filter, concentrate and dry the filtrate to obtain extract C; S4: Artificial bezoar, artificial musk, borneol, and benzoin are mixed, and then ethanol aqueous solution and PEG400 are added. After being fully dissolved, a mixture of sodium alginate solution and chitosan solution is added, stirred, and freeze-dried to obtain component D. S5: Mix extract C and component D, add starch, stir well, form into pellets, dry, coat with activated carbon, and polish to obtain the final product; in The complex enzyme in step S1 is cellulase and pectinase, and the mass ratio of cellulase to pectinase is 3-6:2-4.

2. The preparation method according to claim 1, characterized in that, In step S1, the pH is adjusted to 5.0-6.5 before enzymatic hydrolysis, the amount of compound enzyme is 0.1-1% of the mass of the raw material to be hydrolyzed, the hydrolysis temperature is 30-50℃, and the hydrolysis time is 1-3h.

3. The preparation method according to claim 1, characterized in that, In step S2, the pH is adjusted to 6.5-7.5 before enzymatic hydrolysis, the amount of papain used is 1-3% of the mass of the raw material to be hydrolyzed, the hydrolysis temperature is 40-50℃, and the hydrolysis time is 3-5h.

4. The preparation method according to claim 1, characterized in that, In step S3, the pH is adjusted to 5-6.5 before enzymatic hydrolysis, the amount of β-glucosidase is 0.2-0.8% of the mass of the raw material to be hydrolyzed, the hydrolysis temperature is 45-60℃, and the hydrolysis time is 1-3h.

5. The preparation method according to claim 1, characterized in that, In step S4, the volume percentage of ethanol in the aqueous ethanol solution is 75%-85%, and the mass ratio of the aqueous ethanol solution to PEG400 is 5-8:

1.

6. The preparation method according to claim 1, characterized in that, In the mixed solution described in step S4, the sodium alginate solution has a mass-to-volume ratio of 1-3%, and the chitosan solution is prepared by dissolving 1-2g of chitosan in 100mL of 1% acetic acid aqueous solution. The mixed solution is obtained by mixing the sodium alginate solution and the chitosan solution at a volume ratio of 1:

1.

7. The preparation method according to claim 1, characterized in that, The weight ratio of the mixed solution in step S4 to the total mass of the artificial bezoar, artificial musk, borneol, and benzoin in this step is 1-3:

1.

8. The use of Yixin Pill prepared according to any one of claims 1-7 in the preparation of antihypertensive and / or lipid-lowering drugs, characterized in that, The dosage form of the drug is tablets, pills, granules or capsules.

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