Ginseng antler traditional Chinese medicine preparation formula and experimental method for influence of ginseng antler traditional Chinese medicine preparation formula on bradyarrhythmia

Abstract

The invention discloses a ginseng and antler formula traditional Chinese medicine preparation formula which comprises 9-11 parts by mass of ginseng, 6-8 parts by mass of antler, 3-5 parts by mass of radix aconiti carmichaeli, 6-8 parts by mass of angelica sinensis, 1-3 parts by mass of cassia twig, 1-3 parts by mass of salvia miltiorrhiza, 1-2 parts by mass of radix paeoniae alba, 1-2 parts by mass of eucommia ulmoides and 2-3 parts by mass of pseudo-ginseng, and relates to the technical field of ginseng and antler formulas. According to the ginseng antler traditional Chinese medicine formula and the experimental method for the influence of the ginseng antler traditional Chinese medicine formula on bradyarrhythmia, the medicine formula has the effects of warming and tonifying heart and kidney, promoting blood circulation to remove blood stasis and nourishing yin to restore pulse; and through the method, the influence condition of the ginseng antler prescription on the bradyarrhythmia can be more accurately obtained, the experiment efficiency is improved, and the research on the ginseng antler prescription is accelerated.

Description

Technical Field

[0001] The invention relates to the technical field of ginseng and antler prescriptions, and in particular to a ginseng and antler prescription Chinese medicine formula and an experimental method for its effect on bradyarrhythmia. Background Art

[0002] The main effects of Shenrong prescription are warming and nourishing the heart and kidney, promoting blood circulation and removing blood stasis, and nourishing yin and restoring pulse. This prescription shows good effects in the prevention and treatment of bradyarrhythmia, but its internal mechanism is not yet clear and lacks relevant experimental proof. In this experiment, SD male rats were used as research subjects. The bradyarrhythmia rat model was prepared by intragastric administration of propranolol hydrochloride 30 mg·kg-1·d-1 and cold stimulation method for 14 consecutive days. After the successful modeling on the 15th day of the experiment, propranolol solution (15 mg·kg-1·d-1) was continued to be administered to maintain the stability of the model and drug intervention was started. The low, medium and high dose groups of Shenrong prescription were intragastricly administered with different concentrations of Chinese medicine solution, and the atropine group was intragastricly administered with atropine solution every day. After 4 weeks, samples were collected to detect the corresponding indicators. The pathological sections of the rat sinoatrial node and surrounding tissues were observed. The levels of cAMP, cGMP and Na+-K+-ATPase were measured by ELISA, and the cAMP / cGMP ratio was calculated. Real-time fluorescence quantitative PCR reaction and immunoblotting experiments were performed on the sinoatrial node and surrounding tissues. After obtaining the experimental data, statistical analysis was performed to draw conclusions and reveal the targets and pathways of the ginseng and antler prescription in the treatment of BA. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides a Chinese medicinal formula of ginseng and antler prescription and an experimental method for its effect on bradyarrhythmia, thereby solving the above-mentioned problems.

[0004] To achieve the above objectives, the present invention is implemented through the following technical scheme: the Chinese medicine formula of Shenrong Fang includes 9-11 parts by mass of ginseng, 6-8 parts by mass of deer antler, 3-5 parts by mass of aconite, 6-8 parts by mass of angelica, 1-3 parts by mass of cinnamon twig, 1-3 parts by mass of salvia miltiorrhiza, 1-2 parts by mass of white peony root, 1-2 parts by mass of eucommia and 2-3 parts by mass of Panax notoginseng.

[0005] Preferably, the Chinese medicinal formula of Shenrong Fang includes 10 parts by mass of ginseng, 7 parts by mass of deer antler, 4 parts by mass of aconite, 7 parts by mass of angelica, 2 parts by mass of cinnamon twig, 2 parts by mass of salvia miltiorrhiza, 1.5 parts by mass of white peony root, 1.5 parts by mass of Eucommia ulmoides and 2.5 parts by mass of Panax notoginseng.

[0006] The experimental method for the effect of the Chinese medicine formula of Shenrong Fang on bradyarrhythmia consists of the following steps:

[0007] S1. Select 6-8 week old SD male rats with a body weight range of 180g-210g, feed with ordinary feed and pure water, and keep the ambient temperature at 22-24℃ and humidity at 40±10%;

[0008] S2. 90 SD male rats were randomly divided into a blank group, a model group, a low-dose, medium-dose, and high-dose Shenrong Fang group, and an atropine group, with 15 rats in each group. The electrocardiogram of each rat was measured before and after the experiment, and the body weight was weighed and the heart yang deficiency score was evaluated every day. After the experiment, 3 rats from each group were taken to measure the content of the corresponding protein in the sinoatrial node tissue.

[0009] S3 and SD male rats were fed adaptively for 7 days. The blank group was gavaged with pure water, and the other groups were gavaged with 30 mg·kg-1·d-1 propranolol hydrochloride solution, combined with cold stimulation method, to prepare BA rat model for 14 consecutive days.

[0010] S4. 14 days before modeling, take a sufficient amount of propranolol sustained-release tablets, crush them with a mortar, pour them into a centrifuge tube, calculate the volume of distilled water and add it to the tube to prepare a solution with a drug concentration of 3 mg / ml; after the modeling is successfully established on the 15th day of the experiment, change the drug concentration to 1.5 mg / ml as the maintenance dose of the BA model; take a sufficient amount of atropine tablets, crush them with a mortar, pour them into a centrifuge tube, and then add distilled water to dissolve them, and finally prepare a drug concentration of 0.3 mg / ml; add ginseng, pilose antler, aconite, angelica, cassia twig, It is composed of 9 Chinese medicinal materials including salvia miltiorrhiza, white peony root, eucommia bark and panax notoginseng. The pieces are soaked for 1 hour. Ginseng, pilose antler, aconite root and panax notoginseng are put into a casserole and decocted for 1 hour. The remaining medicines are then poured in and decocted over high heat for 1 hour. Then, the mixture is decocted over low heat for 2 hours. The mixture is filtered through gauze for 5 times and then decocted and concentrated. Finally, the concentration of the Chinese medicine in the low-dose group of Shenrong Fang is 0.5535 g crude drug / ml, the concentration of the Chinese medicine in the medium-dose group of Shenrong Fang is 1.107 g crude drug / ml, and the concentration of the Chinese medicine in the high-dose group of Shenrong Fang is 2.214 g crude drug / ml.

[0011] S5. The volume of liquid for rat gavage was calculated based on 10 mL / kg / d of liquid. Except for the blank group rats which were gavaged with distilled water, the model rats in the other groups were gavaged with the above-prepared propranolol hydrochloride solution with a drug concentration of 3 mg / ml. After 14 days, the blank group and the model group were gavaged with pure water, the atropine group was gavaged with the prepared atropine solution, and the Chinese medicine group was gavaged with the prepared low-dose, medium-dose and high-dose Chinese medicine solution of Shenrong Fang respectively; weigh and observe the general condition every day;

[0012] S6. Western Blot sampling method: After 14 days of drug intervention, the rats were examined for electrocardiograms and samples were collected. First, the rats were anesthetized intraperitoneally with 2% pentobarbital solution at a dose of 30 mg / kg. After complete anesthesia, the electrocardiogram was collected using the BL420F biofunctional experimental system. Then, the abdominal and thoracic cavities were cut open to expose the internal organs. The abdominal aorta of the rats was peeled off with a glass needle. After exposing a sufficient length of the artery, a blood collection needle was used to slowly puncture. After bleeding, the needle tail was inserted into a vacuum blood collection tube to collect blood, and the tube was left to stand for 30 minutes. After centrifugation using a centrifuge, the supernatant in the tube was taken and stored in a new centrifuge tube, and then placed in a -80°C refrigerator for standby use.

[0013] S7. The observation conditions mainly include the changes in weight, food intake, coat color, activity sensitivity and mental state of the rats; observe the changes in the rats before and after the experiment, record the weight and other physical signs, and count the survival of the rats in each group after the experiment; at the same time, calculate the Yang deficiency sign score of the rats, measure the electrocardiogram of the rats, determine the levels of cAMP, cGMP and Na+-K+-ATPase in the serum by ELISA, detect the expression of HCN4, β-catenin, CX45, CX43, and Cyclin D1 proteins in the sinoatrial node and surrounding tissues by Western Blot, detect the expression levels of related genes in tissues by qRT-PCR, observe the pathological changes of the sinoatrial node and surrounding tissues by HE and Masson staining, and detect the contents of HCN4, β-catenin, CX45, CX43, and Cyclin D1 in the sinoatrial node and surrounding tissues of each group of rats by immunohistochemistry;

[0014] S8. Measurement data were expressed as mean ± standard deviation x ± s, and GraphPad Prism 8.0.1 and SPSS 27.0 software were used for statistical analysis of data. One-way analysis of variance (ANOVA) was used for comparison among multiple groups. When the variances were equal, the LSD method was used for testing, and when the variances were unequal, the Dunnetts' T3 method was used for testing. P < 0.05 indicated statistical difference, and P < 0.01 indicated extremely significant difference.

[0015] Preferably, after successful modeling on the 15th day of the experiment in S3, 15 mg·kg-1·d-1 of propranolol solution is continued to be administered to maintain the stability of the model and start drug intervention.

[0016] Preferably, in S6, the rat sternum is cut with scissors, and then the heart is peeled off, residual blood is washed with saline, and then the moisture is absorbed with absorbent paper, and they are wrapped in tin foil respectively, each group is marked, and stored in a liquid nitrogen tank; another batch of rat heart tissues are taken and placed in a centrifuge tube, and then a 4% paraformaldehyde solution is poured into the tissue to fix the tissue for subsequent pathological tissue sectioning and detection.

[0017] The present invention provides a Chinese medicine formula of Shenrong Fang and an experimental method for its effect on bradyarrhythmia. The invention has the following beneficial effects: the Shenrong Fang is prepared by using a formula of ginseng, pilose antler, aconite root, angelica root, cassia twig, salvia miltiorrhiza, white peony root, eucommia bark and Panax notoginseng, and has the functions of warming and nourishing the heart and kidney, promoting blood circulation and removing blood stasis, and nourishing yin and restoring pulse; and the method can more accurately obtain the effect of Shenrong Fang on bradyarrhythmia, improve experimental efficiency, and accelerate the research on Shenrong Fang. DETAILED DESCRIPTION

[0018] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0019] Embodiment 1

[0020] The invention provides a technical solution: a traditional Chinese medicine formula of Shenrong Fang, comprising 9 parts by mass of ginseng, 6 parts by mass of pilose antler, 3 parts by mass of aconite, 6 parts by mass of angelica, 1 part by mass of cassia twig, 1 part by mass of salvia miltiorrhiza, 1 part by mass of white peony root, 1 part by mass of eucommia bark and 2 parts by mass of notoginseng.

[0021] The experimental method for the effect of the Chinese medicine formula of Shenrong Fang on bradyarrhythmia consists of the following steps:

[0022] S1. Select 6-week-old SD male rats with a body weight range of 180g, feed with ordinary feed and pure water, and keep the ambient temperature at 22℃ and humidity at 40±10%;

[0023] S2. 90 SD male rats were randomly divided into a blank group, a model group, a low-dose, medium-dose, and high-dose Shenrong Fang group, and an atropine group, with 15 rats in each group. The electrocardiogram of each rat was measured before and after the experiment, and the body weight was weighed and the heart yang deficiency score was evaluated every day. After the experiment, 3 rats from each group were taken to measure the content of the corresponding protein in the sinoatrial node tissue.

[0024] S3 and SD male rats were adaptively fed for 7 days. The blank group was gavaged with pure water, and the other groups were gavaged with 30 mg·kg-1·d-1 propranolol hydrochloride solution, combined with cold stimulation method, to prepare BA rat model for 14 consecutive days. After the model was successfully established on the 15th day of the experiment in S3, 15 mg·kg-1·d-1 propranolol solution was continued to be given to maintain the stability of the model and start drug intervention.

[0025] S4. 14 days before modeling, take a sufficient amount of propranolol sustained-release tablets, crush them with a mortar, pour them into a centrifuge tube, calculate the volume of distilled water and add it to the tube to prepare a solution with a drug concentration of 3 mg / ml; after the modeling is successfully established on the 15th day of the experiment, change the drug concentration to 1.5 mg / ml as the maintenance dose of the BA model; take a sufficient amount of atropine tablets, crush them with a mortar, pour them into a centrifuge tube, and then add distilled water to dissolve them, and finally prepare a drug concentration of 0.3 mg / ml; add ginseng, pilose antler, aconite, angelica, cassia twig, It is composed of 9 Chinese medicinal materials including salvia miltiorrhiza, white peony root, eucommia bark and panax notoginseng. The pieces are soaked for 1 hour. Ginseng, pilose antler, aconite root and panax notoginseng are put into a casserole and decocted for 1 hour. The remaining medicines are then poured in and decocted over high heat for 1 hour. Then, the mixture is decocted over low heat for 2 hours. The mixture is filtered through gauze for 5 times and then decocted and concentrated. Finally, the concentration of the Chinese medicine in the low-dose group of Shenrong Fang is 0.5535 g crude drug / ml, the concentration of the Chinese medicine in the medium-dose group of Shenrong Fang is 1.107 g crude drug / ml, and the concentration of the Chinese medicine in the high-dose group of Shenrong Fang is 2.214 g crude drug / ml.

[0026] S5. The volume of liquid for rat gavage was calculated based on 10 mL / kg / d of liquid. Except for the blank group rats which were gavaged with distilled water, the model rats in the other groups were gavaged with the above-prepared propranolol hydrochloride solution with a drug concentration of 3 mg / ml. After 14 days, the blank group and the model group were gavaged with pure water, the atropine group was gavaged with the prepared atropine solution, and the Chinese medicine group was gavaged with the prepared low-dose, medium-dose and high-dose Chinese medicine solution of Shenrong Fang respectively; weigh and observe the general condition every day;

[0027] S6, through Western Blot sampling method: After 14 days of drug intervention, the rats were examined for electrocardiograms and samples were collected. First, the rats were anesthetized intraperitoneally with 2% pentobarbital solution at a dosage of 30 mg / kg. After complete anesthesia, the electrocardiogram was collected using the BL420F biofunctional experimental system, and then the abdominal and thoracic cavities were cut open to expose the internal organs. The abdominal aorta of the rats was peeled off with a glass needle. After exposing a sufficient length of the artery, the blood collection needle was slowly punctured. After bleeding, the needle tail was inserted into a vacuum blood collection tube to collect blood, and the blood was allowed to stand for 30 minutes. After centrifugation using a centrifuge, the supernatant in the tube was taken and stored in a new centrifuge tube, and then placed in a -80°C refrigerator for standby use; in S6, the rat sternum was cut with scissors, and then the heart was peeled off. The residual blood was washed with physiological saline, and then the moisture was absorbed with absorbent paper, and the tissues were wrapped in tin foil respectively, each group was marked, and stored in a liquid nitrogen tank; another batch of rat heart tissues were taken and placed in a centrifuge tube, and then 4% paraformaldehyde solution was poured into the tissue to fix the tissue for subsequent pathological tissue sections and detection.

[0028] S7. The observation conditions mainly include the changes in weight, food intake, coat color, activity sensitivity and mental state of the rats; observe the changes in the rats before and after the experiment, record the weight and other physical signs, and count the survival of the rats in each group after the experiment; at the same time, calculate the Yang deficiency sign score of the rats, measure the electrocardiogram of the rats, determine the levels of cAMP, cGMP and Na+-K+-ATPase in the serum by ELISA, detect the expression of HCN4, β-catenin, CX45, CX43, and Cyclin D1 proteins in the sinoatrial node and surrounding tissues by Western Blot, detect the expression levels of related genes in tissues by qRT-PCR, observe the pathological changes of the sinoatrial node and surrounding tissues by HE and Masson staining, and detect the contents of HCN4, β-catenin, CX45, CX43, and Cyclin D1 in the sinoatrial node and surrounding tissues of each group of rats by immunohistochemistry;

[0029] S8. Measurement data were expressed as mean ± standard deviation x ± s, and GraphPad Prism 8.0.1 and SPSS 27.0 software were used for statistical analysis of data. One-way analysis of variance (ANOVA) was used for comparison among multiple groups. When the variances were equal, the LSD method was used for testing, and when the variances were unequal, the Dunnetts' T3 method was used for testing. P < 0.05 indicated statistical difference, and P < 0.01 indicated extremely significant difference.

[0030] Embodiment 2

[0031] The invention provides a technical solution: a traditional Chinese medicine formula of Shenrong Fang, comprising 10 parts by mass of ginseng, 7 parts by mass of pilose antler, 4 parts by mass of aconite, 7 parts by mass of angelica, 2 parts by mass of cassia twig, 2 parts by mass of salvia miltiorrhiza, 1.5 parts by mass of white peony root, 1.5 parts by mass of eucommia bark and 2.5 parts by mass of notoginseng.

[0032] The experimental method for the effect of the Chinese medicine formula of Shenrong Fang on bradyarrhythmia consists of the following steps:

[0033] S1. Select 8-week-old SD male rats with a body weight range of 210g, feed with ordinary feed and pure water, and keep the ambient temperature at 24℃ and humidity at 40±10%;

[0034] S2. 90 SD male rats were randomly divided into a blank group, a model group, a low-dose, medium-dose, and high-dose Shenrong Fang group, and an atropine group, with 15 rats in each group. The electrocardiogram of each rat was measured before and after the experiment, and the body weight was weighed and the heart yang deficiency score was evaluated every day. After the experiment, 3 rats from each group were taken to measure the content of the corresponding protein in the sinoatrial node tissue.

[0035] S3 and SD male rats were adaptively fed for 7 days. The blank group was gavaged with pure water, and the other groups were gavaged with 30 mg·kg-1·d-1 propranolol hydrochloride solution, combined with cold stimulation method, to prepare BA rat model for 14 consecutive days. After the model was successfully established on the 15th day of the experiment in S3, 15 mg·kg-1·d-1 propranolol solution was continued to be given to maintain the stability of the model and start drug intervention.

[0036] S4. 14 days before modeling, take a sufficient amount of propranolol sustained-release tablets, crush them with a mortar, pour them into a centrifuge tube, calculate the volume of distilled water and add it to the tube to prepare a solution with a drug concentration of 3 mg / ml; after the modeling is successfully established on the 15th day of the experiment, change the drug concentration to 1.5 mg / ml as the maintenance dose of the BA model; take a sufficient amount of atropine tablets, crush them with a mortar, pour them into a centrifuge tube, and then add distilled water to dissolve them, and finally prepare a drug concentration of 0.3 mg / ml; add ginseng, pilose antler, aconite, angelica, cassia twig, It is composed of 9 Chinese medicinal materials including salvia miltiorrhiza, white peony root, eucommia bark and panax notoginseng. The pieces are soaked for 1 hour. Ginseng, pilose antler, aconite root and panax notoginseng are put into a casserole and decocted for 1 hour. The remaining medicines are then poured in and decocted over high heat for 1 hour. Then, the mixture is decocted over low heat for 2 hours. The mixture is filtered through gauze for 5 times and then decocted and concentrated. Finally, the concentration of the Chinese medicine in the low-dose group of Shenrong Fang is 0.5535 g crude drug / ml, the concentration of the Chinese medicine in the medium-dose group of Shenrong Fang is 1.107 g crude drug / ml, and the concentration of the Chinese medicine in the high-dose group of Shenrong Fang is 2.214 g crude drug / ml.

[0037] S5. The volume of liquid for rat gavage was calculated based on 10 mL / kg / d of liquid. Except for the blank group rats which were gavaged with distilled water, the model rats in the other groups were gavaged with the above-prepared propranolol hydrochloride solution with a drug concentration of 3 mg / ml. After 14 days, the blank group and the model group were gavaged with pure water, the atropine group was gavaged with the prepared atropine solution, and the Chinese medicine group was gavaged with the prepared low-dose, medium-dose and high-dose Chinese medicine solution of Shenrong Fang respectively; weigh and observe the general condition every day;

[0038] S6, through Western Blot sampling method: After 14 days of drug intervention, the rats were examined for electrocardiograms and samples were collected. First, the rats were anesthetized intraperitoneally with 2% pentobarbital solution at a dosage of 30 mg / kg. After complete anesthesia, the electrocardiogram was collected using the BL420F biofunctional experimental system, and then the abdominal and thoracic cavities were cut open to expose the internal organs. The abdominal aorta of the rats was peeled off with a glass needle. After exposing a sufficient length of the artery, the blood collection needle was slowly punctured. After bleeding, the needle tail was inserted into a vacuum blood collection tube to collect blood, and the blood was allowed to stand for 30 minutes. After centrifugation using a centrifuge, the supernatant in the tube was taken and stored in a new centrifuge tube, and then placed in a -80°C refrigerator for standby use; in S6, the rat sternum was cut with scissors, and then the heart was peeled off. The residual blood was washed with physiological saline, and then the moisture was absorbed with absorbent paper, and the tissues were wrapped in tin foil respectively, each group was marked, and stored in a liquid nitrogen tank; another batch of rat heart tissues were taken and placed in a centrifuge tube, and then 4% paraformaldehyde solution was poured into the tissue to fix the tissue for subsequent pathological tissue sections and detection.

[0039] S7. The observation conditions mainly include the changes in weight, food intake, coat color, activity sensitivity and mental state of the rats; observe the changes in the rats before and after the experiment, record the weight and other physical signs, and count the survival of the rats in each group after the experiment; at the same time, calculate the Yang deficiency sign score of the rats, measure the electrocardiogram of the rats, determine the levels of cAMP, cGMP and Na+-K+-ATPase in the serum by ELISA, detect the expression of HCN4, β-catenin, CX45, CX43, and Cyclin D1 proteins in the sinoatrial node and surrounding tissues by Western Blot, detect the expression levels of related genes in tissues by qRT-PCR, observe the pathological changes of the sinoatrial node and surrounding tissues by HE and Masson staining, and detect the contents of HCN4, β-catenin, CX45, CX43, and Cyclin D1 in the sinoatrial node and surrounding tissues of each group of rats by immunohistochemistry;

[0040] S8. Measurement data were expressed as mean ± standard deviation x ± s, and GraphPad Prism 8.0.1 and SPSS 27.0 software were used for statistical analysis of data. One-way analysis of variance (ANOVA) was used for comparison among multiple groups. When the variances were equal, the LSD method was used for testing, and when the variances were unequal, the Dunnetts' T3 method was used for testing. P < 0.05 indicated statistical difference, and P < 0.01 indicated extremely significant difference.

[0041] Embodiment 3

[0042] The invention provides a technical solution: a traditional Chinese medicine formula of Shenrong Fang, comprising 11 parts by weight of ginseng, 8 parts by weight of pilose antler, 5 parts by weight of aconite, 8 parts by weight of angelica, 3 parts by weight of cassia twig, 3 parts by weight of salvia miltiorrhiza, 2 parts by weight of white peony root, 2 parts by weight of eucommia bark and 3 parts by weight of notoginseng.

[0043] The experimental method for the effect of the Chinese medicine formula of Shenrong Fang on bradyarrhythmia consists of the following steps:

[0044] S1. Select 6-week-old SD male rats with a body weight range of 180g, feed with ordinary feed and pure water, and keep the ambient temperature at 22℃ and humidity at 40±10%;

[0045] S2. 90 SD male rats were randomly divided into a blank group, a model group, a low-dose, medium-dose, and high-dose Shenrong Fang group, and an atropine group, with 15 rats in each group. The electrocardiogram of each rat was measured before and after the experiment, and the body weight was weighed and the heart yang deficiency score was evaluated every day. After the experiment, 3 rats from each group were taken to measure the content of the corresponding protein in the sinoatrial node tissue.

[0046] S3 and SD male rats were adaptively fed for 7 days. The blank group was gavaged with pure water, and the other groups were gavaged with 30 mg·kg-1·d-1 propranolol hydrochloride solution, combined with cold stimulation method, to prepare BA rat model for 14 consecutive days. After the model was successfully established on the 15th day of the experiment in S3, 15 mg·kg-1·d-1 propranolol solution was continued to be given to maintain the stability of the model and start drug intervention.

[0047] S4. 14 days before modeling, take a sufficient amount of propranolol sustained-release tablets, crush them with a mortar, pour them into a centrifuge tube, calculate the volume of distilled water and add it to the tube to prepare a solution with a drug concentration of 3 mg / ml; after the modeling is successfully established on the 15th day of the experiment, change the drug concentration to 1.5 mg / ml as the maintenance dose of the BA model; take a sufficient amount of atropine tablets, crush them with a mortar, pour them into a centrifuge tube, and then add distilled water to dissolve them, and finally prepare a drug concentration of 0.3 mg / ml; add ginseng, pilose antler, aconite, angelica, cassia twig, It is composed of 9 Chinese medicinal materials including salvia miltiorrhiza, white peony root, eucommia bark and panax notoginseng. The pieces are soaked for 1 hour. Ginseng, pilose antler, aconite root and panax notoginseng are put into a casserole and decocted for 1 hour. The remaining medicines are then poured in and decocted over high heat for 1 hour. Then, the mixture is decocted over low heat for 2 hours. The mixture is filtered through gauze for 5 times and then decocted and concentrated. Finally, the concentration of the Chinese medicine in the low-dose group of Shenrong Fang is 0.5535 g crude drug / ml, the concentration of the Chinese medicine in the medium-dose group of Shenrong Fang is 1.107 g crude drug / ml, and the concentration of the Chinese medicine in the high-dose group of Shenrong Fang is 2.214 g crude drug / ml.

[0048] S5. The volume of liquid for rat gavage was calculated based on 10 mL / kg / d of liquid. Except for the blank group rats which were gavaged with distilled water, the model rats in the other groups were gavaged with the above-prepared propranolol hydrochloride solution with a drug concentration of 3 mg / ml. After 14 days, the blank group and the model group were gavaged with pure water, the atropine group was gavaged with the prepared atropine solution, and the Chinese medicine group was gavaged with the prepared low-dose, medium-dose and high-dose Chinese medicine solution of Shenrong Fang respectively; weigh and observe the general condition every day;

[0049] S6, through Western Blot sampling method: After 14 days of drug intervention, the rats were examined for electrocardiograms and samples were collected. First, the rats were anesthetized intraperitoneally with 2% pentobarbital solution at a dosage of 30 mg / kg. After complete anesthesia, the electrocardiogram was collected using the BL420F biofunctional experimental system, and then the abdominal and thoracic cavities were cut open to expose the internal organs. The abdominal aorta of the rats was peeled off with a glass needle. After exposing a sufficient length of the artery, the blood collection needle was slowly punctured. After bleeding, the needle tail was inserted into a vacuum blood collection tube to collect blood, and the blood was allowed to stand for 30 minutes. After centrifugation using a centrifuge, the supernatant in the tube was taken and stored in a new centrifuge tube, and then placed in a -80°C refrigerator for standby use; in S6, the rat sternum was cut with scissors, and then the heart was peeled off. The residual blood was washed with physiological saline, and then the moisture was absorbed with absorbent paper, and the tissues were wrapped in tin foil respectively, each group was marked, and stored in a liquid nitrogen tank; another batch of rat heart tissues were taken and placed in a centrifuge tube, and then 4% paraformaldehyde solution was poured into the tissue to fix the tissue for subsequent pathological tissue sections and detection.

[0050] S7. The observation conditions mainly include the changes in weight, food intake, coat color, activity sensitivity and mental state of the rats; observe the changes in the rats before and after the experiment, record the weight and other physical signs, and count the survival of the rats in each group after the experiment; at the same time, calculate the Yang deficiency sign score of the rats, measure the electrocardiogram of the rats, determine the levels of cAMP, cGMP and Na+-K+-ATPase in the serum by ELISA, detect the expression of HCN4, β-catenin, CX45, CX43, and Cyclin D1 proteins in the sinoatrial node and surrounding tissues by Western Blot, detect the expression levels of related genes in tissues by qRT-PCR, observe the pathological changes of the sinoatrial node and surrounding tissues by HE and Masson staining, and detect the contents of HCN4, β-catenin, CX45, CX43, and Cyclin D1 in the sinoatrial node and surrounding tissues of each group of rats by immunohistochemistry;

[0051] S8. Measurement data were expressed as mean ± standard deviation x ± s, and GraphPad Prism 8.0.1 and SPSS 27.0 software were used for statistical analysis of data. One-way analysis of variance (ANOVA) was used for comparison among multiple groups. When the variances were equal, the LSD method was used for testing, and when the variances were unequal, the Dunnetts' T3 method was used for testing. P < 0.05 indicated statistical difference, and P < 0.01 indicated extremely significant difference.

[0052] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions. The sentence "includes an element defined by ... does not exclude the existence of other identical elements in the process, method, article or device including the element".

[0053] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Chinese medicinal formula of Shenrong Fang, characterized in that: The composition comprises 9-11 parts by weight of ginseng, 6-8 parts by weight of pilose antler, 3-5 parts by weight of aconite, 6-8 parts by weight of angelica, 1-3 parts by weight of cassia twig, 1-3 parts by weight of salvia miltiorrhiza, 1-2 parts by weight of white peony root, 1-2 parts by weight of eucommia bark and 2-3 parts by weight of Panax notoginseng.

2. The Chinese medicinal formula of Shenrong Fang according to claim 1, characterized in that: The composition includes 10 parts by mass of ginseng, 7 parts by mass of pilose antler, 4 parts by mass of aconite root, 7 parts by mass of angelica root, 2 parts by mass of cinnamon twig, 2 parts by mass of salvia miltiorrhiza, 1.5 parts by mass of white peony root, 1.5 parts by mass of eucommia bark and 2.5 parts by mass of Panax notoginseng.

3. The experimental method of the effect of the Chinese medicine formula of Shenrong Fang on bradyarrhythmia is characterized in that: It consists of the following steps: S1. Select 6-8 week old SD male rats with a body weight range of 180g-210g, feed with ordinary feed and pure water, and keep the ambient temperature at 22-24℃ and humidity at 40±10%; S2. 90 SD male rats were randomly divided into a blank group, a model group, a low-dose, medium-dose, and high-dose Shenrong Fang group, and an atropine group, with 15 rats in each group. The electrocardiogram of each rat was measured before and after the experiment, and the body weight was weighed and the heart yang deficiency score was evaluated every day. After the experiment, 3 rats from each group were taken to measure the content of the corresponding protein in the sinoatrial node tissue. S3 and SD male rats were fed adaptively for 7 days. The blank group was gavaged with pure water, and the other groups were gavaged with 30 mg·kg-1·d-1 propranolol hydrochloride solution, combined with cold stimulation method, to prepare BA rat model for 14 consecutive days. S4. 14 days before modeling, take a sufficient amount of propranolol sustained-release tablets, crush them with a mortar, pour them into a centrifuge tube, calculate the volume of distilled water and add it to the tube to prepare a solution with a drug concentration of 3 mg / ml; after the modeling is successful on the 15th day of the experiment, the drug concentration is changed to 1.5 mg / ml as the maintenance dose of the BA model; take a sufficient amount of atropine tablets, crush them with a mortar, pour them into a centrifuge tube, and then add distilled water to dissolve them, and finally prepare a drug concentration of 0.3 mg / ml; 9 Chinese medicinal materials including ginseng, pilose antler, aconite, angelica, cinnamon twig, salvia miltiorrhiza, white peony root, eucommia bark and Panax notoginseng are soaked for 1 hour, ginseng, pilose antler, aconite and Panax notoginseng are put into a casserole and decocted for 1 hour, then the remaining drugs are poured in and decocted over high heat for 1 hour, then decocted over low heat for 2 hours. h, filtered through gauze for 5 times, then boiled and concentrated, and finally boiled into a medicinal solution with a concentration of 0.5535 g crude drug / ml in the low-dose group of Shenrong Fang, 1.107 g crude drug / ml in the medium-dose group of Shenrong Fang, and 2.214 g crude drug / ml in the high-dose group of Shenrong Fang; S5. The volume of liquid for rat gavage was calculated based on 10 mL / kg / d of liquid. Except for the blank group rats which were gavaged with distilled water, the model rats in the other groups were gavaged with the above-prepared propranolol hydrochloride solution with a drug concentration of 3 mg / ml. After 14 days, the blank group and the model group were gavaged with pure water, the atropine group was gavaged with the prepared atropine solution, and the Chinese medicine group was gavaged with the prepared low-dose, medium-dose and high-dose Chinese medicine solution of Shenrong Fang respectively; weigh and observe the general condition every day; S6. Western Blot sampling method: After 14 days of drug intervention, the rats were examined for electrocardiograms and samples were collected. First, the rats were anesthetized intraperitoneally with 2% pentobarbital solution at a dose of 30 mg / kg. After complete anesthesia, the electrocardiogram was collected using the BL420F biofunctional experimental system. Then, the abdominal and thoracic cavities were cut open to expose the internal organs. The abdominal aorta of the rats was peeled off with a glass needle. After exposing a sufficient length of the artery, a blood collection needle was used to slowly puncture. After bleeding, the needle tail was inserted into a vacuum blood collection tube to collect blood, and the tube was left to stand for 30 minutes. After centrifugation using a centrifuge, the supernatant in the tube was taken and stored in a new centrifuge tube, and then placed in a -80°C refrigerator for later use. S7. The observation conditions mainly include the changes in weight, food intake, coat color, activity sensitivity and mental state of the rats; observe the changes in the rats before and after the experiment, record the weight and other physical signs, and count the survival of the rats in each group after the experiment; at the same time, calculate the yang deficiency sign score of the rats, measure the electrocardiogram of the rats, determine the levels of cAMP, cGMP and Na+-K+-ATPase in the serum by ELISA, detect the expression of HCN4, β-catenin, CX45, CX43, and CyclinD1 proteins in the sinoatrial node and surrounding tissues by Western Blot, detect the expression levels of related genes in tissues by qRT-PCR, observe the pathological changes of the sinoatrial node and surrounding tissues by HE and Masson staining, and detect the contents of HCN4, β-catenin, CX45, CX43, and Cyclin D1 in the sinoatrial node and surrounding tissues of each group of rats by immunohistochemistry; S8. Measurement data were expressed as mean ± standard deviation, and GraphPad Prism 8.0.1 and SPSS 27.0 software were used for statistical analysis of data. One-way analysis of variance (ANOVA) was used for comparison among multiple groups. When the variances were equal, the LSD method was used for testing, and when the variances were unequal, the Dunnetts' T3 method was used for testing. P < 0.05 indicated statistically significant differences, and P < 0.01 indicated extremely significant differences.

4. The experimental method for the effect of the Shenrong Fang Chinese medicine formula on bradyarrhythmia according to claim 3, characterized in that: After the model was successfully established on the 15th day of the experiment in S3, 15 mg·kg-1·d-1 of propranolol solution was continued to be administered to maintain the stability of the model and start drug intervention.

5. The experimental method for the effect of the Shenrong Fang Chinese medicine formula on bradyarrhythmia according to claim 3, characterized in that: In S6, the rat's sternum was cut with scissors, and then the heart was removed. The residual blood was washed with saline, and the moisture was absorbed with absorbent paper. The tissues were wrapped in tin foil, each group was marked, and stored in a liquid nitrogen tank. Another batch of rat heart tissues were placed in a centrifuge tube, and then 4% paraformaldehyde solution was poured into it to fix the tissue for subsequent pathological tissue sectioning and testing.