CN109771486B - Chinese medicinal compound preparation for treating infantile viral myocarditis and preparation process thereof - Google Patents
Chinese medicinal compound preparation for treating infantile viral myocarditis and preparation process thereof Download PDFInfo
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Abstract
The invention relates to a traditional Chinese medicine compound preparation for treating infantile viral myocarditis and a preparation process thereof, belonging to the technical field of medical application of effective parts of traditional Chinese medicines. The technical scheme is as follows: pulverizing radix Panacis Quinquefolii into coarse particles, adding ethanol, heating and reflux-extracting for 3 times, concentrating the filtrate with rotary evaporator, extracting the concentrated filtrate with petroleum ether and water saturated n-butanol for 3 times, collecting supernatant, recovering n-butanol from the supernatant with rotary evaporator, evaporating the rest medicinal liquid in water bath, concentrating, and drying in drying chamber to obtain radix Panacis Quinquefolii total saponin; the preparation method of radix astragali total saponin, cortex moutan total saponin and semen Vaccariae total saponin is the same as above; the preparation ratio of each component is as follows: the preparation mass ratio of the American ginseng total saponin, the astragalus root total saponin, the moutan bark total saponin and the cowherb seed total saponin is 6: 6: 4: 1. the raw material medicine has wide source and low cost; the medicine effect is obvious, and the medicine is safe and nontoxic; the dosage is small, and the medicine is convenient for children to take; the oral medicine is convenient for storage.
Description
Technical Field
The invention relates to a traditional Chinese medicine compound preparation for treating infantile viral myocarditis and a preparation process thereof, in particular to application of Coxsackie Virus B (CVB) in enteroviruses in myocardial tissue damage and myocardial cell apoptosis, and belongs to the technical field of medical application of effective parts of traditional Chinese medicines.
Background
Viral Myocarditis (VMC) refers to a localized or diffuse acute or chronic inflammatory disease of the myocardium caused by Viral infection, and is an infectious myocardial disease. The VMC is a wide range of sick people, most of which are induced by enteroviruses and respiratory viruses in preschool children and school childrenAnd (4) sending. Coxsackievirus A group, Coxsackievirus B group (CVB), echovirus and poliovirus are common myocarditis-causing pathogenic viruses, wherein the CVB is the most main pathogen and accounts for about 30-50%, and comprises six serotypes, wherein B3The virus has the strongest pathogenicity, is easy to cause severe cases and death cases, and is the main pathogenic virus of the children with viral myocarditis in China. After human beings are infected with CVB, myocarditis occurs in about 5-7%, and some cases can develop Dilated Cardiomyopathy (DCM), so that sequelae such as cardiac insufficiency and the like are left, and the serious cases need heart transplantation. In the forensic case of sudden death, the sudden death rate of VMC and DCM is second only to coronary heart disease and hypertension, and is third in sudden cardiac death. In recent years, the incidence of VMC in children and teenagers is on a remarkable rising trend, accounts for 10% -44% of the sudden death of the teenagers, becomes an important cause of the sudden death of the children and the teenagers, and causes wide attention in the medical field.
The VMC treatment medicine has limited types, and the treatment medicine specially aiming at the infantile viral myocarditis is lacking, so that the development of a new treatment medicine is urgently needed. Western medicine treatment mainly aims at symptomatic treatment of viral infection and myocardial inflammation, but the effect is not ideal, adverse reactions with different degrees are frequent, and the price is high. Although a plurality of compounds are recorded in the traditional Chinese medicine literature to treat the disease, the clinical application dosage is large, and particularly, the defects of large dosage and difficult application of the traditional Chinese medicine for children exist, and the mechanism of protecting the cardiac muscle needs to be further researched and verified, so the research on the application of the effective traditional Chinese medicine components to the treatment of the intractable diseases is particularly urgent. The combination of ancient experience and modern pharmacological research results, and the screening of a novel, high-efficiency and nontoxic traditional Chinese medicine consisting of effective active ingredients for treating the infantile viral myocarditis is urgent.
No specific disease name in the traditional Chinese medicine corresponds to the viral myocarditis, and if the patient is infected by an acute disease, the patient can be treated by warm diseases; if arrhythmia is the main category, it is in the category of palpitation and severe palpitation; for chest oppression and chest pain, it is usually treated by chest impediment, and it is also related to heart water, sweating syndrome, consumptive disease and sudden death. The traditional understanding of the pathogenesis of the disease in traditional Chinese medicine is caused by external affection of wind-heat, damp-heat and pathogenic toxin. In recent years, with further enrichment and accumulation of clinical experience, Chinese medical researchers have provided a plurality of new ideas and insights, such as accumulation of blood stasis, accumulation of turbid phlegm, weakness of spleen and stomach and the like can cause the disease, and the pathogenesis theory of viral myocarditis is enriched. Western medicine considers that immune injury and apoptosis are decisive factors of virus-induced cell injury. Besides the direct myocardial dysfunction and cell structure destruction caused by virus infection, Fas/FasL pathway, Caspase family, Bcl-2 family and the like can be activated to start the apoptosis of myocardial cells.
With the rapid development of molecular biology, the research on pathogenesis of VMC is advanced to a certain extent, but no specific medicine exists in the aspect of treatment so far, and a treatment medicine specially aiming at the infantile viral myocarditis is lacked. Western medicine treatment mainly aims at symptomatic treatment of viral infection and myocardial inflammation, the clinical existing symptomatic treatment medicines can be used for the limited pediatric patients, and at present, free radical scavengers are clinically applied, such as intravenous or oral vitamin C, coenzyme Q10, vitamin Bco, ATP, inosine, cyclic adenosine monophosphate, cytochrome C, glucocorticoid and the like, but the treatment effect is not ideal, adverse reactions with different degrees are frequent, and the price is high. Although a plurality of compounds are recorded in the traditional Chinese medicine literature to treat the disease, the traditional Chinese medicine literature has poor curative effect and large clinical application dosage, particularly has the defects of large dosage and difficult application of the traditional Chinese medicine for children, and the mechanism of protecting the cardiac muscle needs to be further researched and verified, so the research on the application of the effective traditional Chinese medicine components to the treatment of the intractable disease is particularly urgent. We think that the combination of ancient experience and modern pharmacological research results screens new, high-efficiency and nontoxic novel traditional Chinese medicine consisting of effective active ingredients for treating infantile viral myocarditis is urgent.
Disclosure of Invention
The invention aims to provide a traditional Chinese medicine compound preparation for treating infantile viral myocarditis and a preparation process thereof, and the traditional Chinese medicine effective part group is used for treating the infantile viral myocarditis, in particular CVB3The induced myocardial cell injury and apoptosis have obvious technical effects and solve the problems in the background technology.
The technical scheme of the invention is as follows:
a traditional Chinese medicine compound preparation for treating infantile viral myocarditis is prepared from the following raw material medicines: american ginseng total saponin, astragalus root total saponin, moutan bark total saponin and cowherb seed total saponin, wherein the mass ratio of the raw material medicines is 6: 6: 4: 1.
a preparation process of a traditional Chinese medicine compound preparation for treating infantile viral myocarditis comprises the following process steps:
the method comprises the following steps: preparation of the active ingredient
Pulverizing radix Panacis Quinquefolii into coarse particles, adding ethanol, heating and reflux-extracting for 3 times, concentrating the filtrate with rotary evaporator, extracting the concentrated filtrate with petroleum ether and water saturated n-butanol for 3 times, collecting supernatant, recovering n-butanol from the supernatant with rotary evaporator, evaporating the rest medicinal liquid in water bath, concentrating, and drying in drying chamber to obtain radix Panacis Quinquefolii total saponin; the preparation method of radix astragali total saponin, cortex moutan total saponin and semen Vaccariae total saponin is the same as above;
step two: the mass ratio of each component
The preparation ratio of each component is as follows: the preparation mass ratio of the American ginseng total saponin, the astragalus root total saponin, the moutan bark total saponin and the cowherb seed total saponin is 6: 6: 4: 1.
the invention is further illustrated in four sections below. A first part: preparation of effective components of Chinese medicinal materials. A second part: the proportion and usage amount of each effective part are allocated. And a third part: the pharmacodynamic action of the effective part group is verified on an in vivo animal model and an in vitro cell model. The fourth part: the traditional Chinese medicine effective part group has the advantages and beneficial effects of treating the infantile viral myocarditis.
A first part: and (3) preparing an effective component.
Pulverizing radix Panacis Quinquefolii into coarse particles, adding ethanol, heating and reflux-extracting for 3 times, concentrating the filtrate with rotary evaporator, extracting the concentrated filtrate with petroleum ether and water saturated n-butanol for 3 times, collecting supernatant, recovering n-butanol from the supernatant with rotary evaporator, evaporating the residual medicinal liquid in water bath, and drying in drying chamber to obtain radix Panacis Quinquefolii total saponin; the preparation method of radix astragali total saponin, cortex moutan total saponin, and semen Vaccariae total saponin is the same as above.
A second part: the proportion and the usage amount of the components are prepared.
Repeated in vivo and in vitro drug effect screening experiments show that the components with exact curative effect are found. The mass ratio of each component is as follows: the preparation proportion of the American ginseng total saponin, the astragalus root total saponin, the moutan bark total saponin and the cowherb seed total saponin is 6: 6: 4: 1. the dosage is as follows: the clinically recommended dose is 5mg/kg (about 300 mg/d). The medication method comprises the following steps: is administered orally. The course of treatment is as follows: the preparation is recommended to be used for 1-2 weeks, namely 7-14 days, and can be used alone or used together with other medicines.
And a third part: the pharmacodynamic action of the compound preparation is verified on an in vivo animal model and an in vitro cell model.
An in vivo animal model; adopting 0.2mL of 0.8-time ID for aseptic injection in the abdominal cavity50CVB of3A VMC mouse model prepared by the method of virus liquid is provided with a high-dose group, a medium-dose group and a low-dose group of a traditional Chinese medicine compound preparation, a ribavirin group, a normal control group and a model control group, and after the groups are given with corresponding medicines for 14 days, the heart function, myocardial injury markers, namely creatine kinase isoenzyme (CK-MB), myocardial troponin (cTnI and cTnT), pathological changes (HE staining) and myocardial infarction area are detected. Experimental results show that after the traditional Chinese medicine compound preparation is administered, the cardiac function index of a mouse is obviously improved, the contents of CK-MB, cTnI and cTnT are reduced, and the pathological injury and myocardial infarction area are reduced. The data show that the traditional Chinese medicine compound preparation can improve the structure and the function of the cardiac muscle of the VMC mouse, and the effect of the traditional Chinese medicine compound preparation can be realized by relieving the damage of cardiac muscle cells, improving the myocardial ischemia and the function thereof and improving the hypoxia tolerance.
An in vitro cell model; separating and culturing rat primary myocardial cell, and determining the maximum nontoxic concentration (TC) of the drug0). Taking the culture plate with grown monolayer cells, inoculating the culture plate with CVB3Adsorbing the virus liquid in an incubator for 1h, and adding TC0The following 4 concentrations of test drugs were combined with a virus control group, and the effect of the drugs on CVB was determined by cytopathic effect assay (CPE)350% inhibition concentrationDegree (IC)50) And a Therapeutic Index (TI); the TdT mediated dUTP nick end labeling technology (TUNEL method) is used for measuring the apoptosis rate of the myocardial cells; and detecting the expression conditions of the Bax and Bcl-2 proteins by using a protein immunoblotting method (Western-blot). The experimental result shows that the medicine is used for treating enterovirus CVB3The strain has obvious inhibition effect on myocardial cytopathy, the apoptosis index of myocardial cells is reduced, after the medicament intervenes in the infected myocardial cells, the Bcl-2 protein expression is increased, the Bax protein expression is reduced, and the Bax/Bcl-2 ratio is reduced. The data show that the medicine has obvious pharmacodynamic action on inhibiting apoptosis in an external experiment, and the mechanism of the medicine is probably related to inhibiting the proliferation of viruses in cells, up-regulating Bcl-2 expression and down-regulating Bax expression.
The fourth part: the traditional Chinese medicine active ingredient group has the advantages and beneficial effects of treating the infantile viral myositis.
The pediatric viral myocarditis still has no specific treatment medicine so far, mainly aims at treating viral infection and myocardial inflammation, has limited clinical symptomatic treatment medicines, and urgently needs to develop a new treatment medicine. The invention discovers that the effective part group of the traditional Chinese medicine can be used for treating the infantile viral myocarditis, particularly myocardial tissue and myocardial cell injury caused by virus, has obvious technical effect and is worthy of popularization and application.
The invention has the following positive effects: the raw material medicine has wide source and low cost; the medicine effect is obvious, and the medicine is safe and nontoxic; the dosage is small, and the medicine is convenient for children to take; the oral medicine is convenient for storage.
Drawings
FIG. 1 is a flow chart of the preparation of total saponins of the present invention;
FIG. 2 is a diagram showing the effect of the effective fraction group of the present invention on the cardiac function of VMC mice (
±s，n=10）；
FIG. 3 is a diagram showing the effect of the effective fraction group of the present invention on CK-MB activity in serum of VMC mice (
±s，n=10）；
FIG. 4 is a diagram showing the effect of the effective fraction group of the present invention on the activity of cTnI and cTnT in the serum of VMC mouse (
±s，n=10）；
FIG. 5 is a schematic diagram showing the effect of the effective fraction group of the present invention on the pathological morphology of the myocardium of VMC mice-normal group (HE staining, X200);
FIG. 6 is a schematic diagram of a model group (HE staining, X200) showing the effect of the effective fraction group of the present invention on the pathological morphology of the myocardium of VMC mice;
FIG. 7 is a schematic diagram showing the effect of the effective fraction group of the present invention on the pathological morphology of the myocardium of VMC mice-saponin low dose group (HE staining, X200);
FIG. 8 shows the effect of the effective fraction group of the present invention on the pathological morphology of the myocardium of VMC mice-ribavirin group (HE staining, X200);
FIG. 9 shows the effect of the effective fraction group of the present invention on the pathological morphology of the myocardium of VMC mice-the medium saponin dose group (HE staining, X200);
FIG. 10 shows the effect of the effective fraction group of the present invention on the pathological morphology of the myocardium of VMC mice-saponin high dose group (HE staining, X200);
FIG. 11 shows the effect of the effective fraction group of the present invention on the myocardial infarction area of VMC mice (
±s，n=10）；
FIG. 12 shows the effect of the effective fractions of the present invention on the inhibition of cardiomyocyte apoptosis (i.e., 1-5 doses of drug: 0 (virus control), 9.77, 19.54, 39.07, and 78.13 ug/mL);
FIG. 13 shows the effect of the effective fraction group of the present invention on the expression of apoptosis factors Bcl-2 and Bax (note: 1-4 doses of drugs: 0 (virus control group), 19.54, 39.07, 78.13 ug/mL).
Detailed Description
The invention is further described with reference to the following figures and examples:
a traditional Chinese medicine compound preparation for treating infantile viral myocarditis is prepared from the following raw material medicines: american ginseng total saponin, astragalus root total saponin, moutan bark total saponin and cowherb seed total saponin, wherein the mass ratio of the raw material medicines is 6: 6: 4: 1.
a preparation process of a traditional Chinese medicine compound preparation for treating infantile viral myocarditis comprises the following process steps:
the method comprises the following steps: preparation of the active ingredient
Pulverizing radix Panacis Quinquefolii into coarse particles, adding ethanol, heating and reflux-extracting for 3 times, concentrating the filtrate with rotary evaporator, extracting the concentrated filtrate with petroleum ether and water saturated n-butanol for 3 times, collecting supernatant, recovering n-butanol from the supernatant with rotary evaporator, evaporating the rest medicinal liquid in water bath, concentrating, and drying in drying chamber to obtain radix Panacis Quinquefolii total saponin; the preparation method of radix astragali total saponin, cortex moutan total saponin and semen Vaccariae total saponin is the same as above.
Step two: the mass ratio of each component
The preparation ratio of each component is as follows: the preparation mass ratio of the American ginseng total saponin, the astragalus root total saponin, the moutan bark total saponin and the cowherb seed total saponin is 6: 6: 4: 1.
the specific experimental scheme is as follows:
a first part: preparation of effective fractions.
Crushing American ginseng into coarse particles, adding 80% ethanol in an amount which is 6-8 times that of the coarse particles, heating and refluxing for 3 times, extracting for 2 hours each time, concentrating an extracting solution by using a rotary evaporator, recovering the ethanol, extracting concentrated solution by using petroleum ether and water saturated n-butyl alcohol for 3 times in an isovolumetric manner respectively, taking supernate, combining the extracted 3 times of supernate, recovering the n-butyl alcohol by using the rotary evaporator, evaporating the residual liquid medicine after recovery into paste by using a 70 ℃ water bath, and then drying in a 70 ℃ blast drier to constant weight to obtain the American ginseng total saponins. The preparation methods of the total saponins of radix astragali, total saponins of cortex moutan and total saponins of semen Vaccariae are the same as above, and the preparation process is shown in figure 1. The material objects of the traditional Chinese medicine decoction pieces are consistent with the names, and the quality of the traditional Chinese medicine decoction pieces accords with the standard of pharmacopoeia of the people's republic of China 2015 edition.
A second part: the proportion and the usage amount of the components are prepared.
Repeated in vivo and in vitro drug effect screening experiments show that the components with exact curative effect are found. In vitro experiments, the compound preparation is observed to CoxB by adopting a cytopathic effect method (CPE)3Strain, CoxB4、CoxB5Cytopathic inhibitory effect; in vitro test on enterovirus CoxB3Infected BABL/c mice were administered both prophylactically and therapeutically and the severity and number of deaths, mortality, protection from death and life extension of the suckling mice were observed. The pharmacodynamic test result shows that the mass ratio of the American ginseng total saponin, the astragalus root total saponin, the moutan bark total saponin and the cowherb seed total saponin prepared in each effective part is 6: 6: 4: the drug effect is best when 1. Therefore, the compound preparation has the configuration mass ratio of the American ginseng total saponin, the astragalus root total saponin, the moutan bark total saponin and the cowherb seed total saponin of each effective part as 6: 6: 4: 1. the dosage is as follows: the clinically recommended dose is 5mg/kg (about 300 mg/d). The medication method comprises the following steps: is administered orally. The course of treatment is as follows: the preparation is recommended to be used for 1-2 weeks, namely 7-14 days, and can be used alone or used together with other medicines.
And a third part: the pharmacodynamic action of the effective fractions of the traditional Chinese medicine is verified on an in vivo animal model and an in vitro cell model.
1. In vivo animal experiments
1.1 Experimental materials
1.1.1 Experimental animals Male BALB \ c mouse, at 3 weeks, SPF grade, half male and female, all have no abnormality through electrocardiogram test (the selection standard: mouse is II-lead electrocardiogram under the waking state, ST segment is close to isoelectric line, T wave is not low level and can be selected). License number: SCXK (Jing) 2014-: 11401300076233, available from Experimental animals Breeding Ltd, Jinmuyang, Beijing.
1.1.2 Virus strains Coxsackie B3Continuously propagating family virus (Nacy strain) on Hep-2 cell for 3 generations, repeatedly freezing and thawing for 3 times after cytopathic effect reaches ++ - +++ (75% -100% of cells are diseased), centrifuging at 2000rpm for 5min, collecting supernatant, and storing in-80 deg.C refrigerator (CVB) of Chinese institute of traditional Chinese medicine institute3Both virus strains and Hep-2 cells were purchased from the institute for viruses, institute of preventive medicine, academy of sciences, china).
1.1.3 drugs and test drugs (i.e., the invention), prepared in the institutional laboratory of traditional Chinese medicine, university of North China; ribavirin granules, Sichuan Baili pharmaceutical responsibility Co., Ltd, lot number H51023508. The CK-MB determination kit is purchased from Nanjing to build a bioengineering institute; cTnI, cTnT were purchased from Bgaer, Germany.
1.1.4 high-speed medicine pulverizer (model: WK-1000A), sincere mechanical Limited of Qingzhou; an electric temperature-adjusting electric heating jacket (model: 98-1-B), Tester instruments Co., Ltd., Tianjin; a rotary evaporator (model: RE-5205), Shanghai Yanglong Biochemical apparatus factory; an electrothermal blowing dry box (model: GZX-9070 MBE), Shanghai Bocheng industries, Inc. medical equipment factory; electrocardiographs (model: ECG-6511), Shanghai medical electronics factories; digital display constant temperature water bath (model: HH-6), national electric appliances Limited; Bio-Rad3550 model automatic enzyme-linked immunoassay instrument, Bio-Rad corporation, USA; model BX63 optical microscope, Olympus, japan.
1.2 Experimental methods
1.2.1 CVB3Viral mouse in vivo TD50The virus was assayed at 10-fold dilutions to 8 titers, 10 each0、10-1、10-2、10-3、10-4、10-5、10-6And 10-7Doubling virus stock solution, selecting 10 male BALB/c mice for each gradient, diluting virus with DMEM, and performing intraperitoneal inoculation on CVB on the first day of experiment3Virus, 0.2 mL/mouse, observing for 14 days, recording death number of mice in each virus titer group, applying Reed-Muench method to count results, and calculating CVB3LD of virus50Is 10-6.2。
1.2.2 determination of viral infection amount CVB3Diluting the virus with normal saline to 0.5-4 times LD 5080 BALB/c mice were selected, 10 mice per dilution, 0.2 mL/mouse, and infected by intraperitoneal injection. Animals were observed daily for 15 consecutive days for mortality. Animals did not die within 15 days after infection but were pickedThe concentration at which the myocardial zymogram has changed significantly compared with the normal control group is the infection amount at the time of the experiment. The experimental results show that: 0.8 times LD50When infected, the mice did not die, but the myocardial zymogram had changed significantly compared to the control group, so it was determined as the infection amount at the time of the experiment.
1.2.3 viral myocarditis animal model building method modeling by intraperitoneal injection of CVB3A method of producing a virus. After the mice were raised in the laboratory for one week, 20 mice were randomly selected as normal control groups, and 0.2ml of undiluted Hep-2 cell frozen and thawed solution was aseptically injected into the abdominal cavity. Intraperitoneal sterile injection of 0.2mL and 0.8 LD to other mice50CVB of3A virus. Quilt CVB3Mice infected with myocarditis have a series of symptoms such as emaciation, sparse and lusterless fur, hypomotility, anorexia, shivering and the like, and the elevation of the activity of serum myocardial enzyme and the change of myocardial pathology are used as indexes for successful model building.
1.2.4 animal grouping and dosing schedules BALB/c mice with no abnormal electrocardiogram were selected from 60 animals, half of the animals were male and female, and randomly divided into 6 groups: normal control group, model control group, positive drug control group (ribavirin 50 mg/kg) and saponin high, medium and low dose groups, each group containing 10. And (3) performing intragastric administration on the normal control group and the model control group by using distilled water, and performing intragastric administration on the other groups by using corresponding medicaments, wherein the intragastric administration is started 2 hours after the intraperitoneal injection in each group, each group is administered for 1 time every day, the administration is continued for 14 days, and the volume is 20 mL/kg.
1.2.5 administration dosage design the administration dosage of the effective part group saponin high-dosage group is 10mg/kg, and each mg contains 16g of crude drug (equivalent to 2 times of the clinical adult dosage of 60 kg); the administration dosage of the effective part group saponin middle agent group is 5mg/kg, and each mg contains 16g of crude drug (equivalent to the equivalent time of the clinical adult dosage of 60 kg); the administration dosage of the effective part group saponin low dose group is 2.5g/kg, and each mg contains 16g of crude drug (equivalent to the equivalent time of the clinical adult dosage of 60 kg).
1.2.6 Experimental period, taking materials and treating specimen of mice Abdominal infection CVB3After 2 hours, each group was drenched with the corresponding drug, and the normal control group and the model control group were drenched with distilled water for 1 time a day for 14 days. On the 15 th day after the administration, the test animals were aseptically operated to pick up the eyeballs and blood,separating serum (storing in refrigerator at-20 deg.C), quickly dislocating cervical vertebra of mouse, killing, cutting open thoracic cavity, cutting heart, fixing and storing in 10% formaldehyde solution, and HE staining.
1.2.7 detection of indices cardiac function (cardiac ejection fraction EF, left ventricular off-axis shortening rate FS), myocardial injury markers creatine kinase isoenzyme (CK-MB) and cardiac troponin (cTnI, cTnT), pathomorphological changes (HE staining), myocardial infarction area, viral load in myocardial tissue.
1.2.8 statistical processing experimental data, Excel table database, SPSS13.0 statistical software package for analysis, and data measurement
S, single-factor analysis of variance for comparisons between groups, t-test for comparisons between groups, toP<0.05 indicates that the difference is statistically significant.
1.3 results of the experiment
1.3.1 the influence of the effective part group of the traditional Chinese medicine on the cardiac function of VMC mice is obviously reduced in model groups such as EF and FS; the compound preparation has obvious improvement on cardiac function indexes of various medicine groups, and has obvious difference compared with a model groupP<0.05), the middle dose group has better effect on improving cardiac function than other dose groups. The results are shown in FIG. 2.
1.3.2 the effect of the effective part group of the traditional Chinese medicine on the activity of CK-MB, cTnI and cTnT in the serum of a VMC mouse, the CK-MB in the serum of a model group mouse is increased, and the cTnI and cTnT of myocardial tissues are reduced; compared with the model group, the three indexes of the compound preparation are obviously improved, wherein the high and medium dosage groups have statistical difference with the model group (P<0.01). The results are shown in FIGS. 3 and 4.
1.3.3 the effect of the effective part group of the traditional Chinese medicine on the pathological morphology of the cardiac muscle of the VMC mouse, the myocardial tissue of the mouse in a normal control group is arranged orderly, the structure is complete, the cell nucleus is obvious and is circular or similar to circular, the small blood vessels among the cardiac muscle are rich, and the cardiac muscle interstitium is not abnormally changed. Most of the cardiac muscles of the model control group have extensive fusion lesions, disordered cell arrangement, sheet necrosis and interstitial edema, and have obvious cardiac muscle dissolution, rupture or gap widening, small perivascular inflammatory reactions in cardiac muscle tissues are obvious, and are mainly infiltrated by lymphocytes and monocytes, and the lesions are concentrated on subendocardial muscle layers and the periphery of blood vessels. The low-dose group myocardial tissue spot-sheet focal necrosis range and perivascular inflammatory reaction of the traditional Chinese medicine compound preparation are reduced compared with a model group, but more obvious pathological changes such as myocardial cell swelling, dissolution, fracture and the like are still seen. The ribavirin group and the traditional Chinese medicine compound preparation have higher degree of pathological changes than the model group, the pathological change range is further reduced, most myocardial cells are arranged neatly, myocardial cell swelling, myocardial lysis and inflammatory infiltration are relieved, and pathological changes tend to heal. The results are shown in FIGS. 5 to 10.
1.3.4 influence of the effective fraction group of the Chinese medicine on the myocardial infarction area of the VMC mice the degree of myocardial necrosis was reflected by measuring the percentage of the myocardial infarction part in the myocardial tissue section to the whole myocardial area (degree of myocardial necrosis = total number of points in the necrotizing foci/total number of points falling on the whole myocardial section x 100%). Compared with the normal group, the model control group and each medicine group have obvious myocardial necrosis (P<0.01); compared with the model group, the myocardial infarction area of each dose group of the Chinese herbal compound preparation is obviously reduced, and the statistical difference is (P<0.01). The results are shown in FIG. 11.
1.3.5 this group of effective parts of Chinese medicine is on CVB3After the medicine is administered therapeutically to the mice with the viral myocarditis, the viral load in the heart tissues of the mice with the viral myocarditis can be obviously reduced, and the mice with the viral myocarditis have significant difference compared with a model control group (the medicine has the advantages of obvious effect on the viral load of the heart of the mice with the viral myocarditis, and the effect of reducing the viral load of the heart of the mice with theP<0.05), the inhibition rate is 56.39%. The results are shown in Table 1.
TABLE 1 Pair of CVBs3Effect of viral load on Heart tissues of mice with pathogenic and toxic myocarditis
Note: compared with the model control group,*P<0.05，**P<0.01
2. in vitro cell experiments
2.1 materials of the experiment
2.1.1 animal Wistar rat, 3 days , SPF grade, purchased from beijing weitonglihua laboratory animal technology llc, license No.: SCXK (Kyoto) 2016-.
2.1.2 cells and viral strain human laryngeal carcinoma epithelial cells (Hep-2), purchased from the cell center of the basic institute of Kyoho medical university, China. CVB3、CVB4、CVB5The strain is purchased from American type culture Collection, is routinely passaged in ABSL-2 laboratory of Chinese institute of traditional Chinese medicine of Chinese academy of sciences, and is preserved at-80 ℃ for later use.
2.1.3 medicine and its preparation method the effective parts group of the traditional Chinese medicine related to the invention is provided by the institute of traditional Chinese medicine preparation department of North China university. The traditional Chinese medicine decoction pieces are purchased from the outpatient service of subsidiary hospitals of North China university of science and technology, and the quality of the decoction pieces conforms to the standard of pharmacopoeia of the people's republic of China. The medicine is prepared into 10mg/mL mother solution by PBS, filtered and sterilized for later use, and diluted according to the needed times during the experiment.
2.1.4 reagents and instruments PBS phosphate buffer, 0.25% pancreatin, DMEM-H medium, fetal bovine serum (Gibco, USA); double antibody (Hyclore Corp.); TUNEL kit (Roche, germany); rabbit anti-rat Bcl-2, Bax polyclonal antibody (Cell Signaling Technology, usa); beta-actin, goat anti-rabbit horseradish peroxidase marker enzyme (warham bosch de). Biosafety cabinets (model a2 MSC1.2, Thermo corporation, usa); CO 22Incubator (Thermo-371, Thermo corporation, usa); inverted fluorescence microscope (BX 51, Olympus, japan); ultra-low temperature refrigerator (ULT Freezer, Thermo Electron Corporation).
2.2 Experimental methods
2.2.1 isolation and culture of Primary cardiomyocytes 6 Wistar rats, after sacrifice, Disinfection of the chest and abdomen of the suckling rats with 75% alcohol, cutting open the chest, taking the heart, removing the cardiac envelope and other tissues, cutting off the ventricles, washing 3 times with Han K's solution, cutting to 1-2 mm3Transferring the fragment of myocardial tissue into a centrifuge tube, adding 8mL of 0.25% trypsin, digesting in a 37 deg.C constant temperature shaker for 50min, adding 2mL of a solution containingTerminating digestion of culture solution containing 10% serum, centrifuging, removing supernatant, adding culture solution to suspend cells, filtering with 200 mesh filter screen, inoculating cell solution into culture flask, incubating in incubator, changing solution after 24 hr, and culturing conventionally.
2.2.2 drug toxicity test drug was diluted 1: 2-1: 256 times (original concentration 10 mg/mL) in culture medium, added to a monolayer of cardiomyocytes growth plate at 100. mu.L/well, and each dilution was repeated 4 times, while setting normal cell control. The plates were placed at 37 ℃ in 5% CO2Culturing in incubator, observing cytopathic condition under inverted microscope every day, and determining the lowest dilution factor of cell without obvious pathological changes as maximum nontoxic concentration (TC)0) 50% cytotoxic concentration (TC) was calculated by the Reed-Muench method50）。
2.2.3 inhibition of viral cardiomyopathy culture plates grown in monolayers were inoculated with CVB3Virus liquid (100 times TCID)50) 100 mu L/hole, adsorbing in incubator for 1h, adding total saponin of KANG KEJIAO Capsule with concentration of 4 concentrations below maximum nontoxic concentration, 100 mu L/hole, and setting virus control group. The cytopathic condition was observed daily and the test results were recorded when the viral control group became ++++ cellular. 50% Inhibitory Concentration (IC) calculated as Reed-Muench50) And Therapeutic Index (TI), TI = TC50/IC50。
Cytopathic effect is judged according to grade 6 standard-: the cells grow normally and no disease is generated; + -: cytopathic effects are less than 10% of the entire monolayer; +: cytopathic effects account for less than about 25% of the entire monolayer of cells; ++: cytopathic effects comprise less than about 50% of the total monolayer of cells; +++: cytopathic effects account for less than about 75% of the total monolayer of cells; ++++: cytopathic effects account for more than about 75% of the total monolayer of cells.
2.2.4 apoptosis Rate of cardiomyocytes 4% paraformaldehyde fixed cells, permeabilized with 1% Triton X-100 for 2min, rinsed with PBS, added with labeled reaction solution containing TdT, DIG-dUTP, incubated at 37 deg.C for 1H, rinsed with PBS, 3% H2O2Sealing, developing with DAB for 5min, counterstaining with hematoxylin, dehydrating, and sealing. Under microscope observation, the brown particles in cytoplasm or nucleus areApoptotic cells. 3 sections were taken for each group, 10 fields (40 ×) were collected for each section, apoptotic cells were counted and the Apoptosis Index (AI) was calculated.
AI (%) = number of apoptotic nuclei/number of total counted nuclei × 100%
2.2.5 expressing Bcl-2 and Bax proteins in cardiomyocytes to take cardiomyocytes, adding 200uL RIPA buffer solution, uniformly mixing, then placing on ice to crack for 40min, centrifuging at 10000rpm multiplied by 15min, extracting proteins after homogenizing, carrying out SDS-PAGE after quantitative denaturation, transferring membranes, sealing, adding Bax and Bcl-2 antibodies (1: 1000), incubating overnight at 4 ℃, adding secondary antibodies after membrane washing, incubating for 2h at 37 ℃, washing membranes, incubating by ECL developer, photographing, and analyzing the gray value of protein bands.
2.2.6 statistical processing the experimental data, after Excel table library building and arrangement, using SPSS13.0 statistical software package to analyze, and measuring data to
S, single-factor analysis of variance for comparisons between groups, t-test for comparisons between groups, toP<0.05 indicates that the difference is statistically significant.
2.3 results of the experiment
2.3.1 test of toxicity of effective fractions of the Chinese medicine on myocardial cells of rats0Is 78.13 ug.mL-1，TC50Is 269.52ug-1. The results are shown in Table 2.
2.3.2 inhibition of Virus induced myocardial cytopathy by the effective fractions of the Chinese medicine3、CVB4、CVB5The strain has obvious inhibition effect on myocardial cell cytopathy and IC thereof50Respectively 46.67, 67.76 and 67.76 ug.mL-1TI was 5.78, 3.98, respectively. The results are shown in tables 3 and 4.
2.3.3 inhibiting effect of the effective part group of the Chinese medicine on virus-induced cytopathic effect the medicine can be used for in vitro inhibiting enterovirus CVB3、CVB4、CVB5The strain has obvious inhibition effect on myocardial cell cytopathy and IC thereof50Respectively 46.67, 67.76 and 67.76 ug.mL-1TI was 5.98, 3.98, respectively. The results are shown in tables 5 and 6.
2.3.4 the inhibiting effect of the effective part group of the traditional Chinese medicine on the myocardial cell apoptosis is that the myocardial cell apoptosis index of a virus group is 32 percent, the myocardial cell apoptosis index after the medicine intervenes is obviously reduced, wherein, the inhibiting effect of the cell apoptosis of the dose groups of 19.54, 39.07 and 78.13ug/mL is especially obvious, and the obvious difference is that (the inhibiting effect of the effective part group of the traditional Chinese medicine on the myocardial cell apoptosis is obvious compared with the virus group)P<0.05). Within the effective drug concentration range, the higher the drug concentration, the lower the apoptosis index. The results are shown in FIG. 12.
2.3.5 the effect of the effective part group of the traditional Chinese medicine on the expression of apoptosis factors Bcl-2 and Bax protein on the expression of virus contrast group cardiac muscle cell Bcl-2 protein is reduced, the expression of Bax protein is increased, and the ratio of Bcl-2/Bax is reduced; after different doses of the traditional Chinese medicine preparation are added, the expression of the BCl-2 of the cardiac muscle cells is increased, and the expression of Bax is reduced; the Bcl-2 protein expression is increased and the Bax protein expression is reduced in the three dose groups compared with the virus control group (P<0.05) The ratio of Bcl-2/Bax gradually decreases as the dosage of the test drug decreases. The results are shown in FIG. 13.
3. Comprehensive discussion of experimental data
In vivo animal experiments: adopting 0.2mL of 0.8-time ID for aseptic injection in the abdominal cavity50CVB of3A viral myocarditis mouse model is prepared by the method of virus liquid, wherein a high-dose group, a medium-dose group and a low-dose group of traditional Chinese medicine, a ribavirin group, a normal control group and a model control group are arranged, and after the groups are given with corresponding medicines for 14 days, cardiac function, myocardial injury markers creatine kinase isoenzyme (CK-MB), myocardial troponin (cTnI and cTnT), pathological changes (HE staining) and myocardial infarction area are detected. Experimental results show that after the traditional Chinese medicine effective part group is administered, the cardiac function index of a mouse is obviously improved, the contents of CK-MB, cTnI and cTnT are reduced, and the pathological injury and myocardial infarction area are reduced. The data show that the medicine can improve the structure and the function of the myocardium of mice with viral myocarditis, and the effect of the medicine can be realized by relieving myocardial cell injury, improving myocardial ischemia and function and improving hypoxia tolerance.
In vitro cell experiments: separating and culturing rat primary myocardial cell, and determining the maximum nontoxic concentration (TC) of the drug0). Taking the culture plate with grown monolayer cells, inoculating the culture plate with CVB3Adsorbing the virus liquid in an incubator for 1h, and adding TC0The following 4 concentrations of test drugs were combined with a virus control group, and the effect of the drugs on CVB was determined by cytopathic effect assay (CPE)3 50% Inhibitory Concentration (IC)50) And a Therapeutic Index (TI); the TdT mediated dUTP nick end labeling technology (TUNEL method) is used for measuring the apoptosis rate of the myocardial cells; and detecting the expression conditions of the Bax and Bcl-2 proteins by using a protein immunoblotting method (Western-blot). The experimental result shows that the medicine is used for treating enterovirus CVB3The strain has obvious inhibition effect on myocardial cytopathy, the apoptosis index of myocardial cells is reduced, after the medicament intervenes in the infected myocardial cells, the Bcl-2 protein expression is increased, the Bax protein expression is reduced, and the Bax/Bcl-2 ratio is reduced. The data show that the medicine has obvious effect on inhibiting apoptosis in an external experiment, and the mechanism of the medicine is probably related to inhibiting the proliferation of viruses in cells and inhibiting the proliferation of the viruses in the cellsUp-regulation of Bcl-2 expression and down-regulation of Bax expression.
Experimental data section of the invention: application of CVB in animal models3The strain-infected BALB/c suckling mouse successfully prepares a mouse viral myocarditis disease model, and the pharmacodynamic action of the traditional Chinese medicine effective part group on the viral myocarditis model mouse is evaluated through therapeutic administration; on the cell model, the CVB of the effective part group of the traditional Chinese medicine is observed3、CVB4、CVB5The antiviral effects of three common pathogenic viral strains of viral myocarditis provide experimental basis for clinical medication of the invention, and meanwhile, methodology reference is provided for evaluation of the drug effect of viral myocarditis.
The fourth part: the traditional Chinese medicine effective part group has the advantages and beneficial effects of treating the infantile viral myocarditis
The invention has obvious advantages and beneficial effects on treating the infantile viral myocarditis. Firstly, the composition and the preparation process of the effective parts of the traditional Chinese medicine are simple. The invention only consists of four effective fractions of American ginseng root total saponin, astragalus root total saponin, moutan bark root total saponin and cowherb seed total saponin. The preparation method is a mode of extracting by organic solvent after alcohol extraction. Secondly, the raw material medicine has wide source and low price. The raw material medicines of the medicine, namely the American ginseng, the astragalus, the salvia miltiorrhiza and the cowherb seed, have wide sources, and can reduce the social and economic burdens of patients. Thirdly, the medicine has obvious effect, and is safe and nontoxic. The in vivo and in vitro efficacy tests prove that the medicament can improve the structure and the function of the myocardium of mice with viral myocarditis and has good protection effect on myocardial cell injury caused by viruses. The long-term toxicity test of rats indicates that the clinically planned dosage of the invention is safe and has no obvious toxic or side effect. Fourthly, the medicine is convenient for children to take and store. The invention is a new preparation composed of effective parts of traditional Chinese medicine, has small clinical application dosage, is convenient for children patients to take, and has good stability and convenient storage because the characters of the effective components of the medicine are powder.
The variety of therapeutic drugs for infantile viral myocarditis is limited, and development of new therapeutic drugs is urgently needed. Western medicine treatment mainly aims at symptomatic treatment of viral infection and myocardial inflammation, but the effect is not ideal, adverse reactions with different degrees are frequent, and the price is high. Although a plurality of compounds are recorded in the traditional Chinese medicine literature to treat the disease, the clinical application dosage is large, and particularly, the defects of large dosage and difficult application of the traditional Chinese medicine for children exist, and the mechanism of protecting the cardiac muscle needs to be further researched and verified, so the research on the application of the effective traditional Chinese medicine components to the treatment of the intractable diseases is particularly urgent. The combination of ancient experience and modern pharmacological research results, and the screening of new, highly effective and nontoxic novel traditional Chinese medicine consisting of effective active ingredients for treating viral myocarditis is urgent. The invention can be used for treating infantile myocardial damage caused by viruses and has an unexpected technical effect.
So far, no literature report exists about the application of the effective part group of the traditional Chinese medicine in the infantile viral myocarditis, and the traditional Chinese medicine has the advantages and beneficial effects of treating the myocardial cell injury caused by viruses, and comprises the following steps:
1. the composition and the preparation process are simple. The invention only consists of four effective parts, namely American ginseng root total saponin, astragalus root total saponin, moutan bark root total saponin and cowherb seed total saponin. The preparation method is a mode of extracting by organic solvent after alcohol extraction.
2. The raw material medicine has wide source and low price. The raw material medicines of the traditional Chinese medicine effective part group, namely the American ginseng, the astragalus, the salvia miltiorrhiza and the cowherb seed, have wide sources, and have lower price compared with the conventional western medicines for treating the infantile viral myocarditis, such as interferon, coenzyme Q10, penicillin and the like. In addition, the preparation process of the effective part group of the traditional Chinese medicine is simple, and complex instruments and equipment are not needed. The whole medicine has low cost and can reduce the social and economic burden of patients.
3. Has obvious curative effect, high safety and no toxicity. The invention proves that the effective part group of the traditional Chinese medicine is in myocardial cell injury caused by virus, in particular CVB (CVB)3Induced myocardial cell injury, and CVB3The obvious drug effect is shown in the process of triggering apoptosis. The medicine can improve the structure and function of mouse myocardium with viral myocarditis, and has good protection effect on myocardial cell injury caused by virus. The traditional Chinese medicine compound preparation is used for injecting the stomach of the SD rat for 26 weeks, the non-toxic reaction dose is 200mg/kg, and the preparation is equivalent to the clinical applicationThe dosage is 40 times of the dosage, SD rats do not show obvious toxic reaction, which indicates that the clinically planned dosage of the invention is safe and has no obvious toxic or side effect.
4. The medicine dose is small, and the medicine is convenient for children to take. The traditional Chinese medicine compound has the defects of large dosage and difficult application for children patients due to large clinical application dosage, and is a novel preparation consisting of the effective parts of the traditional Chinese medicine, so that the clinical application dosage is greatly reduced, and the traditional Chinese medicine compound is convenient for the children patients to take.
5. Convenient for administration and storage. The effective components of the Chinese medicine are orally taken medicine and are convenient to take. The effective components of the medicine are powder, so the medicine has good stability, and is convenient to store by using solid or syrup preparations such as tablets, granules, capsules, dripping pills, syrup and the like.
Claims (2)
1. A Chinese medicinal compound preparation for treating pediatric myocarditis caused by Coxsackie group B virus is characterized by being prepared from the following raw material medicaments: american ginseng total saponin, astragalus root total saponin, moutan bark total saponin and cowherb seed total saponin, wherein the mass ratio of the raw material medicines is 6: 6: 4: 1.
2. a preparation process of a traditional Chinese medicine compound preparation for treating pediatric myocarditis caused by Coxsackie group B viruses is characterized by comprising the following process steps:
the method comprises the following steps: preparation of the active ingredient
Pulverizing radix Panacis Quinquefolii into coarse particles, adding ethanol, heating and reflux-extracting for 3 times, concentrating the filtrate with rotary evaporator, extracting the concentrated filtrate with petroleum ether and water saturated n-butanol for 3 times, collecting supernatant, recovering n-butanol from the supernatant with rotary evaporator, evaporating the rest medicinal liquid in water bath, concentrating, and drying in drying chamber to obtain radix Panacis Quinquefolii total saponin; the preparation method of radix astragali total saponin, cortex moutan total saponin and semen Vaccariae total saponin is the same as above;
step two: the mass ratio of each component
The preparation ratio of each component is as follows: the preparation mass ratio of the American ginseng total saponin, the astragalus root total saponin, the moutan bark total saponin and the cowherb seed total saponin is 6: 6: 4: 1.
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